Best Practice in Lymphoma Diagnosis and Reporting Specific disease appendix

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1 Best Practice in Lymphoma Diagnosis and Reporting Specific disease appendix This is a companion to the guideline on best practice in Lymphoma diagnosis and reporting that can also be found on the BCSH website. British Committee for Standards in Haematology Royal College of Pathologists Address for correspondence: BCSH Administrator British Society for Haematology 100 White Lion Street London N1 9PF [email protected] Writing group: Parker A 1, Bain B 2, Devereux S 3, Gatter K 4, Jack A 5, Matutes E 6, Rooney N 7, Ross F 8, Wilkins B 9, Wotherspoon A 10, Ramsay A. 11 Guideline update and changes: This guideline is an updated version of the 2008 guideline, revised to take into account the 2008 WHO guidelines as they apply to the lymphoproliferative disorders. There have been no changes to the sections on histiocytic and dendritic cell neoplasms. The pathology of posttransplant lymphoproliferative disorder is dealt with in the BCSH guideline on this topic. This appendix does not include all diagnoses. Review date: April 2012 (first published in April 2010). Disclaimer While the advice and information in these guidelines is believed to be true and accurate at the time of going to press, neither the authors, the British Society for Haematology nor the publishers accept any legal responsibility for the content of these guidelines. 1 Anne Parker, (BCSH Lead), Consultant Haematologist, Glasgow Royal Infirmary, Glasgow 2 Barbara Bain, Consultant Haematologist, St Marys Hospital, Paddington 3 Steve Devereux, Consultant Haematologist, Kings College Hospital, London, 4 Kevin Gatter, Professor of Pathology, John Radcliffe Hospital, Oxford 5 Andrew Jack, Consultant Histopathologist, Leeds General Hospital, Leeds 6 Estella Matutes, Consultant Haematologist, The Royal Marsden, London 7 Nick Rooney, Consultant Histopathologist, Southmead Hospital, Bristol 8 Fiona Ross, Consultant Clinical Scientist, Wessex Regional Genetics Laboratory, Salisbury, 9 Bridget Wilkins, Consultant Histopathologist, Royal Victoria Infirmary, Newcastle upon Tyne 10 Andrew Wotherspoon, Consultant Histopathologist, The Royal Marsden, London, 11 Alan Ramsay, Consultant Histopathologist (RCPath Lead), University College Hospital, London

2 Index for Disease Specific Appendix Mature B Cell neoplasms 1. Chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma (SLL) (p02) 2. B-cell prolymphocytic leukaemia (B-PLL) (p11) 3. Lymphoplasmacytic lymphoma / Waldenström macroglobulinaemia (p16) 4. Splenic B-cell marginal zone lymphoma (p20) 5. Hairy cell leukaemia (p27) 6. Plasma cell myeloma and other plasma cell neoplasms (p32) 7. Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (p43) 8. Nodal marginal zone lymphoma (p48) 9. Follicular Lymphoma (p52) 10. Mantle cell lymphoma (p57) 11. Diffuse large B cell lymphoma (p63) a. Mediastinal B cell lymphoma b. Plasmablastic lymphomat-cell/ histiocyte-rich large B cell lymphoma c. ALK positive DLBCL d. Intravascular lymphoma e. Primary effusion lymphoma 12. Non-endemic Burkitt lymphoma (p75) Mature T- and NK-cell neoplasms 13. Extranodal NK/T cell lymphoma, nasal type (p81) 14. Enteropathy-associated T-cell lymphoma (p85) 15. Hepatosplenic T-cell lymphoma (p88) 16. Angioimmunoblastic lymphoma (p92) 17. Anaplastic large cell lymphoma, ALK-positive and ALK-negative (p96) 18. Peripheral T-cell lymphoma, not otherwise specified (p101) 19. T-lymphoblastic leukaemia/ lymphoma (p106) 20. T-cell prolymphocytic leukaemia (p110) 21. T-cell large granular lymphocytic leukaemia (p115) 22. Aggressive natural killer-cell leukaemia (p120) 23. Adult T-cell luekaemia/ lymphoma (p124) Hodgkin Lymphoma 24. Classical Hodgkin Lymphoma (p129) 25. Nodular lymphocyte predominant Hodgkin lymphoma (p133) Histiocytic and Dendritic cell neoplasms 26. Histiocytic Sarcoma (p137) 27. Langerhans cell histiocytosis (p140) 28. Langerhans Cell Sarcoma (p144) 29. Interdigitating dendritic cell sarcoma/ tumour (p147) 30. Follicular dendritic cell sarcoma/ tumour (p150) 31. Dendritic cell tumour not otherwise specified (p 153)

3 1 Chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma (SLL) 1.1 Disease and Clinical Features This is a clonal disorder of mature B lymphocytes with a variable clinical course in which there is accumulation of neoplastic cells in the bone marrow, peripheral blood and secondary lymphoid tissues. The clinical features result from immunological dysfunction, lymph node and organ infiltration and, later in the course of the disease, bone marrow failure. 1.2 Peripheral blood Peripheral blood lymphocyte count > 5 x 10 9 /l in CLL Peripheral blood lymphocyte count may be < 5.0 x 10 9 /l, these cases being referred to as monoclonal B lymphocytosis with CLL phenotype or, if there is significant tissue infiltration, as small lymphocytic lymphoma. Typical morphology: Small lymphocytes, scanty cytoplasm, clumped chromatin, indistinct or absent nucleoli

4 1.3 Bone marrow Examination of the bone marrow is not routinely required in CLL but may be indicated in the following situations:- where there is diagnostic difficulty, as a prognostic indicator, to document the response to therapy, to assess haemopoietic reserve (particularly when autoimmune cytopenia is suspected) or as a research investigation. Bone marrow aspirate morphology is similar to that of the peripheral blood Trephine biopsy histology typically reveals mixed nodular and interstitial infiltration of intertrabecular spaces, rarely diffuse ( packed marrow pattern) with small lymphocytes. Nodules may be based on proliferation centres containing para-immunoblasts (histological equivalents of prolymphocytes) but only rarely on pre-existing, non-neoplastic follicles. In untreated patients, paratrabecular infiltration is almost never found. 1.4 Lymph node & spleen Lymph node biopsy is seldom necessary in CLL. However, it may be performed if there is persistent enlargement of a single lymph node group after therapy or when high grade transformation is suspected. The usual context for lymph node biopsy is in diagnosis of small lymphocytic lymphoma in the absence of features of CLL. The usual pattern is of diffuse effacement of lymph node structure with variable numbers of proliferation centres (sometimes called pseudofollicles). Transformation to a diffuse, high grade B-cell lymphoma (Richter s transformation) and Hodgkin lymphoma may occur. EBV has been implicated in some of these cases. As a result of immunosuppression, high grade lymphoma can also develop in B lymphocytes that are not part of the neoplastic clone. 1.5 Immunophenotype The typical immunophenotype of CLL is given below in table 1.1. Additional antigens such as CD38 (Damle, et al 1999) and ZAP70 (Crespo, et al 2003, Orchard, et al 2004, Rassenti, et al 2004, Wiestner, et al 2003) have been shown to have prognostic significance in B-CLL but are not required for diagnosis.

5 Similarly, demonstration of p53 dysfunction by FACS immunophenotyping following in vitro exposure of cells to ionising radiation is prognostically valuable although not currently essential for diagnosis (Lin, et al 2002, Carter, et al 2004). Table 1.1 CLL immunophenotype scoring system (Delgado, et al 2003) Marker Score points 1 0 Smlg Weak Strong CD5 Positive CD23 Positive FMC7 Positive CD22 or CD79b Weak Strong Score = or > 4 in 92% of cases of CLL If <= 3 then exclude mantle cell lymphoma by one or both of: (i) Immunostaining of bone marrow trephine sections or lymph node for nuclear cyclin D1 (ii) FISH for t(11:14) using blood, BM or lymph node. Other lymphoproliferative disorders such as marginal zone, follicular and lymphoplasmacytic lymphomas can usually be distinguished by their immunophenotype. In problematic cases, lymph node or other tissue biopsy may be appropriate if the distinction is clinically important. 1.6 Cytogenetics & molecular genetics Conventional cytogenetic studies are frequently not informative because of a failure of CLL cells to divide in vitro. FISH studies are useful in differentiating mantle cell lymphoma from CLL. Perform FISH for t(11;14) with specific CCND1/IGH probes or CCND1 breakapart probe in all cases of CLL with atypical morphology or a score of 3 or less. If

6 a bone marrow trephine biopsy or lymph node specimen is available, immunohistochemistry for cyclin D1 should also be performed. Cytogenetic abnormalities have been shown to correlate with prognosis; deletion of 13q14 being associated with a good prognosis, trisomy 12 and deletion of 6q with an intermediate and deletion of 17p (TP53) and 11q (ATM)with a poor prognosis (Dohner, et al 2000). The loss of TP53 is strongly associated with poor response to alkylating agents and purine analogues. (Stilgenbauer and Dohner 2002) However, finding these abnormalities by FISH does not provide any diagnostic information as all are common in many subtypes of lymphoma. 1.7 Prognostic markers Clinical factors Staging systems based on the extent of lymphadenopathy and the presence of anaemia and/or thrombocytopenia (excluding that with an auto-immune basis) are the simplest and best validated predictors of outcome in CLL. These systems do not predict disease progression in early stage disease; however those with progressive stage A disease have a similar outcome to stage B patients. Males with CLL generally have a worse prognosis than females regardless of clinical stage (Catovsky, et al 1989). Table 1.2 Binet staging system for CLL (Binet et al. 1981) Stag Features % Median survival e (months) A < 3 lymphoid areas* B = or > 3 lymphoid areas C Haemoglobin < 100 g/l or platelets < x 10 9 /l *The five lymphoid areas comprise unilateral or bilateral cervical, axillary and inguinal lymphadenopathy, hepatomegaly and splenomegaly Biological markers

7 In addition to easily measurable clinical variables, a number of other biomarkers correlate with outcome in CLL and these are summarised below (adapted from Oscier, et al 2004) Table 1.3. Biological variables that correlate with prognosis in CLL Factor Outcome Reference Trephine biopsy Diffuse pattern worse than nondiffuse (Montserrat, et al 1996) Lymphocyte doubling time <12 months worse than >12 months (Montserrat, et al 1986) Serum markers High lactate dehydrogenase, 2 (Di Raimondo, et al microglobulin, soluble thymidine kinase, soluble CD23 worse 2001, Hallek, et al 1999, Knauf, et al 1997, Schwarzmeier, et al 2002) CD38 >20-30% CD38+ve worse (Damle, et al 1999) Genetic del 17p worse than del 11q worse (Dohner, et al 2000) abnormalities than trisomy 12 worse than del 13q. del 17p resistant to therapy except corticosteroids and alemtuzumab IG V H status Unmutated immunoglobulin variable genes (>98% germline) (Damle, et al 1999, Hamblin, et al 1999) worse Using the threshold of 98% to divide mutated from unmutated cases, patients with Vh3.21 fall into the mutated group but nevertheless have a poor prognosis (Tobin, et al 2002). P53 expression Poor response to chemotherapy, response to Alemtuzumab (Cordone, et al 1998, Lozanski, et al 2004)

8 ZAP 70 Correlates (partially) with unmutated IG V H status (Crespo, et al 2003, Orchard, et al 2004, Rassenti, et al 2004, Wiestner, et al 2003) 1.8 Pitfalls Cytology in peripheral blood and aspirated marrow is variable and not a reliable guide to diagnosis. Atypical morphological features include presence of >10% but <55% prolymphocytes (also called CLL-PL) and >15% lymphoplasmacytic cells +/- cells with cleft nuclei (Bennett, et al 1989). Immunophenotyping is occasionally atypical, with absence of expression of CD5 or CD23 (rarely both). In such atypical variants, marrow trephine biopsy or lymph node histology may be helpful and is recommended; immunohistological phenotyping can reveal weak expression or a proportion of cells expressing CD5/CD Differential diagnosis The main differential diagnosis is between other types of non-hodgkin lymphoma with blood overspill. These include the following Mantle cell lymphoma B-cell prolymphocytic leukaemias Follicular lymphoma Marginal zone lymphomas Recommendations Recommendations for diagnosis of CLL Diagnostic criteria Peripheral blood lymphocyte count > 5 x 10 9 /l Peripheral blood immunophenotype score = or > 4 Histology demonstrating CD5/CD23 if no blood involvement. Level of evidence Grade C level IV

9 Cases with score < 4 should have FISH for t(11:14) and cyclin D1 immunohistochemistry to exclude MCL. Cases requiring treatment should have FISH or functional studies done for investigation of TP53 mutations 1.11 References Bennett, J.M., Catovsky, D., Daniel, M.T., Flandrin, G., Galton, D.A., Gralnick, H.R. & Sultan, C. (1989) Proposals for the classification of chronic (mature) B and T lymphoid leukaemias. French-American-British (FAB) Cooperative Group. J Clin Pathol, 42, Binet, J.L., Auquier, A., Dighiero, G., Chastang, C., Piguet, H., Goasguen, J., Vaugier, G., Potron, G., Colona, P., Oberling, F., Thomas, M., Tchernia, G., Jacquillat, C., Boivin, P., Lesty, C., Duault, M.T., Monconduit, M., Belabbes, S. & Gremy, F. (1981) A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer, 48, Carter, A., Lin, K., Sherrington, P.D., Pettitt, A.R. (2004) Detection of p53 dysfunction by flow cytometry in chronic lymphocytic leukaemia. Br J Haematol, 127, Catovsky, D., Fooks, J. & Richards, S. (1989) Prognostic factors in chronic lymphocytic leukaemia: the importance of age, sex and response to treatment in survival. A report from the MRC CLL 1 trial. MRC Working Party on Leukaemia in Adults. Br J Haematol, 72, Crespo, M., Bosch, F., Villamor, N., Bellosillo, B., Colomer, D., Rozman, M., Marce, S., Lopez-Guillermo, A., Campo, E. & Montserrat, E. (2003) ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med, 348, Cordone, I., Masi, S., Mauro, F.R., Soddu, S., Morsilli, O., Valentini, T., Vegna, M.L., Guglielmi, C., Mancini, F., Giuliacci, S., Sacchi, A., Mandelli, F. & Foa, R. (1998) p53 Expression in B-Cell Chronic Lymphocytic Leukemia: A Marker of Disease Progression and Poor Prognosis. Blood, 91, Damle, R.N., Wasil, T., Fais, F., Ghiotto, F., Valetto, A., Allen, S.L., Buchbinder, A., Budman, D., Dittmar, K., Kolitz, J., Lichtman, S.M., Schulman, P., Vinciguerra, V.P., Rai, K.R., Ferrarini, M. & Chiorazzi, N. (1999) Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood, 94,

10 Delgado, J., Matutes, E., Morilla, A.M., Morilla, R.M., Owusu-Ankomah, K.A., Rafiq-Mohammed, F., del Giudice, I. & Catovsky, D. (2003) Diagnostic significance of CD20 and FMC7 expression in B-cell disorders. Am J Clin Pathol, 120, Di Raimondo, F., Giustolisi, R., Lerner, S., Cacciola, E., O'Brien, S., Kantarjian, H. & Keating, M.J. (2001) Retrospective study of the prognostic role of serum thymidine kinase level in CLL patients with active disease treated with fludarabine. Ann Oncol, 12, Dohner, H., Stilgenbauer, S., Benner, A., Leupolt, E., Krober, A., Bullinger, L., Dohner, K., Bentz, M. & Lichter, P. (2000) Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med, 343, Hallek, M., Langenmayer, I., Nerl, C., Knauf, W., Dietzfelbinger, H., Adorf, D., Ostwald, M., Busch, R., Kuhn-Hallek, I., Thiel, E. & Emmerich, B. (1999) Elevated serum thymidine kinase levels identify a subgroup at high risk of disease progression in early, nonsmoldering chronic lymphocytic leukemia. Blood, 93, Hamblin, T.J., Davis, Z., Gardiner, A., Oscier, D.G. & Stevenson, F.K. (1999) Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood, 94, Knauf, W.U., Ehlers, B., Mohr, B., Thiel, E., Langenmayer, I., Hallek, M., Emmerich, B., Adorf, D., Nerl, C. & Zwingers, T. (1997) Prognostic impact of the serum levels of soluble CD23 in B-cell chronic lymphocytic leukemia. Blood, 89, Lin, K., Sherrington, P.D., Dennis, M., Matrai, Z., Cawley, J.C., Pettitt, A.R. (2002) Relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation in chronic lymphocytic leukemia. Blood, 100,: (Erratum in: Blood 2002, 100, 2291.) Lozanski, G., Heerema, N.A., Flinn, I.W., Smith, L., Harbison, J., Webb, J., Moran, M., Lucas, M., Lin, T., Hackbarth, M.L., Proffitt, J.H., Lucas, D., Grever, M.R. & Byrd, J.C. (2004) Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions. Blood, 103, Montserrat, E., Sanchez-Bisono, J., Vinolas, N. & Rozman, C. (1986) Lymphocyte doubling time in chronic lymphocytic leukaemia: analysis of its prognostic significance. Br J Haematol, 62, Montserrat, E., Villamor, N., Reverter, J.C., Brugues, R.M., Tassies, D., Bosch, F., Aguilar, J.L., Vives-Corrons, J.L., Rozman, M. & Rozma, C. (1996) Bone marrow assessment in B-cell chronic lymphocytic leukaemia: aspirate or biopsy? A comparative study in 258 patients. Br J Haematol, 93, Orchard, J.A., Ibbotson, R.E., Davis, Z., Wiestner, A., Rosenwald, A., Thomas, P.W., Hamblin, T.J., Staudt, L.M. & Oscier, D.G. (2004) ZAP-70 expression and prognosis in chronic lymphocytic leukaemia. Lancet, 363,

11 Oscier, D., Fegan, C., Hillmen, P., Illidge, T., Johnson, S., Maguire, P., Matutes, E. & Milligan, D. (2004) Guidelines on the diagnosis and management of chronic lymphocytic leukaemia. British Journal of Haematology, 125, Rassenti, L.Z., Huynh, L., Toy, T.L., Chen, L., Keating, M.J., Gribben, J.G., Neuberg, D.S., Flinn, I.W., Rai, K.R., Byrd, J.C., Kay, N.E., Greaves, A., Weiss, A. & Kipps, T.J. (2004) ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med, 351, Schwarzmeier, J.D., Shehata, M., Hilgarth, M., Marschitz, I., Louda, N., Hubmann, R. & Greil, R. (2002) The role of soluble CD23 in distinguishing stable and progressive forms of B-chronic lymphocytic leukemia. Leuk Lymphoma, 43, Stilgenbauer, S. & Dohner, H. (2002) Campath-1H-Induced Complete Remission of Chronic Lymphocytic Leukemia despite p53 Gene Mutation and Resistance to Chemotherapy. N Engl J Med, 347, Tobin, G., Thunberg, U., Johnson, A., Thorn, I., Soderberg, O., Hultdin, M., Botling, J., Enblad, G., Sallstrom, J., Sundstrom, C., Roos, G. & Rosenquist, R. (2002) Somatically mutated Ig V(H)3-21 genes characterize a new subset of chronic lymphocytic leukemia. Blood, 99, Wiestner, A., Rosenwald, A., Barry, T.S., Wright, G., Davis, R.E., Henrickson, S.E., Zhao, H., Ibbotson, R.E., Orchard, J.A., Davis, Z., Stetler-Stevenson, M., Raffeld, M., Arthur, D.C., Marti, G.E., Wilson, W.H., Hamblin, T.J., Oscier, D.G. & Staudt, L.M. (2003) ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood, 101,

12 2 B-cell prolymphocytic leukaemia (B-PLL) 2.1 Definition and clinical features B-PLL is a rare clonal disorder of mature B cells with prolymphocytic morphology characterised by high peripheral blood white count, little lymphadenopathy and marked splenomegaly. Patients are generally elderly; there is a male predominance and often a more aggressive course than in CLL. About 20% of cases of B-PLL have been reported to have a t(11;14) translocation but these probably represent a leukaemic variant of mantle cell lymphoma. (Ruchlemer, et al 2004) 2.2 Peripheral blood Total white cell count is usually >100 x 10 9 /l, although it may be lower in patients with slowly progressive disease. Anaemia and thrombocytopenia may be present due to hypersplenism and/or bone marrow failure. Prolymphocytes comprise > 55% of circulating cells. These are intermediate sized cells with relatively small volume, mildly basophilic cytoplasm and round nuclei with moderately condensed chromatin and a prominent nucleolus. 2.3 Bone marrow Bone marrow aspirate shows heavy infiltration by prolymphocytes. Bone marrow trephine biopsy histology shows a diffuse intertrabecular or mixed diffuse and interstitial infiltrate of small to medium-sized lymphoid cells, with prominent single nucleoli, corresponding closely with the cytological appearances. Nuclear cyclin D1 staining is seen in some cases in association with the t(11;14) abnormality, emphasising the possibility that they are variants of, or closely related to, MCL. Immunohistochemistry for CD5 and CD23 yields varying results. 2.4 Lymph node & spleen Lymphadenopathy is not prominent, but nodes show diffuse infiltration by prolymphocytes. Proliferation centres (pseudofollicles) are generally not seen.

13 Histology of the spleen shows infiltration of both red and white pulp with prolymphocytes. 2.5 Immunophenotype Table 2.1 Recommended Panel for diagnosis of B-PLL Marker SmIg CD5 CD23 FMC7 CD22 or CD79b CD20 Cyclin D1 Reaction Strong Positive in 33% of cases Usually negative Strong Strong Strong 2.6 Cytogenetics & molecular genetics Cytogenetic abnormalities appear to be similar to those in CLL (Lens, et al 1997, Lens, et al), but prognostic associations have not been proven. Early reports suggest that about 20% of cases of possible B-PLL have a t(11;14), but these probably represent a leukaemic variant of mantle cell lymphoma (Ruchlemer, et al 2004). Abnormalities of the TP53 gene are found in more than 50% of cases (Lens, et al 1997). 2.7 Prognostic factors Advanced age, the presence of anaemia and TP53 abnormalities predict a poor prognosis (Hercher, et al 2001). 2.8 Pitfalls Cases of diffuse high grade B-cell lymphoma may present with bone marrow and peripheral blood involvement. The morphology and immunophenotype may be

14 similar to B-PLL and differentiation may require correlation with the clinical picture and tissue biopsy findings. There may be clinical and cytomorphological overlap with either CLL or mantle cell lymphoma, making immunophenotyping essential and genetic investigations advisable in all cases. If cyclin D1 expression is identified this should be considered to be mantle cell lymphoma. The immunophenotype may be variant and should be interpreted with caution. 2.9 Differential diagnosis Leukaemic and blastoid variants of mantle cell lymphoma may be confused with B-PLL in peripheral blood and lymph node respectively. Atypical CLL (CLL-PL) can also be confused with B-PLL Recommendations Recommendations for diagnosis of B-PLL Diagnostic criteria Lymphocytosis with more than 55% prolymphocytes Level of evidence Grade C level IV Immunophenotype SmIgM/D strong, CD20 strong, CD22/CD79b strong, FMC7+, CD5+ in 33% patients. CD23 usually negative. Immunohistochemistry for Cyclin D1 in all cases to exclude MCL and/or FISH for t(11;14) References Hercher, C., Robain, M., Davi, F., Garand, R., Flandrin, G., Valensi, F., Vandeputte, H., Albert, A., Maynadie, M., Troussard, X., Simon, G.H., Lespinasse, J., Portefaix, G. & Merle-Beral, H. (2001) A multicentric study of 41 cases of B-prolymphocytic leukemia: two evolutive forms. Leuk Lymphoma, 42, Lens, D., De Schouwer, P.J., Hamoudi, R.A., Abdul-Rauf, M., Farahat, N., Matutes, E., Crook, T., Dyer, M.J. & Catovsky, D. (1997) p53 abnormalities in B-cell prolymphocytic leukemia. Blood, 89, Lens, D., Matutes, E., Catovsky, D. & Coignet, L.J. (2000) Frequent deletions at 11q23 and 13q14 in B cell prolymphocytic leukemia (B-PLL). Leukemia, 14,

15 Ruchlemer, R., Parry-Jones, N., Brito-Babapulle, V., Attolico, I., Wotherspoon, A.C., Matutes, E. & Catovsky, D. (2004) B-prolymphocytic leukaemia with t(11;14) revisited: a splenomegalic form of mantle cell lymphoma evolving with leukaemia. Br J Haematol, 125,

16 3 Lymphoplasmacytic lymphoma / Waldenström macroglobulinaemia 3.1 Definition and clinical features Lymphoplasmacytic lymphoma (LPL) is a slowly progressive, clonal disorder of mature B cells, with features of plasma cell differentiation. Paraproteinaemia (usually IgM) is common, and may give rise to hyperviscosity. In some patients this can be associated with peripheral neuropathy. Splenomegaly is frequent but not usually massive and lymphadenopathy, when present, is not usually prominent. WM is the term used to describe cases of LPL in which there is an IgM paraprotein, which may be associated with hyperviscosity. 3.2 Peripheral blood Peripheral blood films typically show marked rouleaux formation and variable numbers of small lymphocytes, lymphoplasmacytoid cells and plasma cells, the latter rarely present in high numbers. Anaemia, neutropenia and thrombocytopenia may occur because of hypersplenism and/or bone marrow failure. 3.3 Bone marrow Bone marrow aspirate morphology is similar to that found in peripheral blood. Trephine biopsy histology shows irregular nodular and paratrabecular infiltrates, with or without additional diffuse interstitial infiltration. Intrasinusoidal infiltration is uncommon, in contrast with splenic marginal zone and mantle cell lymphomas. Plasma cells may contain PAS-positive inclusions of immunoglobulin, which may appear in the cytoplasm (Russell bodies) or indenting the nucleus (Dutcher bodies). The proportions of lymphocytes, lymphoplasmacytoid cells and plasma cells vary widely. There may also be scattered larger blast cells but no true paraimmunoblasts or proliferation centres. Accompanying reactive mast cells are often abundant (Wilkins, et al 2001). 3.4 Lymph node

17 The lymph nodes contain diffuse or vaguely nodular infiltrates of mixed lymphoid cells encompassing the spectrum described above. Absence of neoplastic follicles, expanded marginal zones or infiltrates of monocytoid B cells is important in differentiating lymphoplasmacytic lymphoma from other types of small B-cell lymphoma (Nathwani, et al 2001). 3.5 Immunophenotype Table 3.1 Recommended Panel for Diagnosis of Lymphoplasmacytic Lymphoma Marker Reaction SmIg Positive. Usually IgM+IgD+/- rarely IgG or IgA CD5 (rare atypical cases +) CD10 CD23 (rare atypical cases +) CD19, CD20, Positive CD22, CD79a Positive CD25, CD27 Positive CD103, CD138 BCL2, PAX5 Positive BCL6 3.6 Cytogenetics & molecular genetics It has been reported that a t(9;14)(p13;q32) involving PAX5 and IGH occurs in about 50% of LPL cases (Iida, et al 1996) but no confirmation from other groups has been found. Waldenström macroglobulinemia cases do not usually have translocations involving IGH but frequently have a deletion of 6q (Schop and Fonseca 2003). This is seen in many types of lymphoma and has neither diagnostic nor prognostic value in this group. In cases with an atypical immunophenotype, FISH to exclude t(11;14) and t(14;18) may be helpful. 3.7 Prognostic features A variety of clinical and laboratory parameters have been correlated with a poor prognosis including: advanced age, male sex, weight loss, the presence of one

18 or more cytopenias, raised beta 2 microglobulin level, high IgM paraprotein level and low serum albumin (Johnson, et al 2006). 3.8 Pitfalls Cases with little evidence of plasma cell differentiation may be confused with other small B-cell lymphomas; immunophenotyping can exclude CLL, MCL and follicular lymphoma but not splenic or extranodal marginal zone lymphomas (Berger et al 2005). 3.9 Differential diagnosis CLL Follicular lymphoma Plasma cell myeloma Splenic marginal zone lymphoma/slvl Node-based and extranodal (MALT-type) marginal zone B cell lymphoma Mantle cell lymphoma 3.10 Recommendations Recommendations for diagnosis of Lymphoplasmacytic Lymphoma Diagnostic criteria Level of evidence IgM (or other) monoclonal gammopathy of any Grade C level IV concentration Bone marrow infiltration by small lymphocytes showing plasmacytoid and/or mature plasma cell differentiation Nodular, interstitial or paratrabecular pattern of bone marrow infiltration Immunophenotype: Sm IgM+ CD5- CD10- CD19+ CD20+ CD22+ CD23- CD25+ CD27+ FMC7+ CD103- CD138-. Additional investigations

19 Plasma viscosity, tests of renal and hepatic function Direct antiglobulin test, cold agglutinin titre and cryoglobulins Beta 2 microglobulin 3.11 References Berger, F., Traverse-Glehen, A., Felman, P., Callet-Bauchu, E., Baseggio, L., Gazzo, S., Thieblemont, C., Ffrench, M., Magaud, J.P., Salles, G., Coiffer, B. (2005) Clinicopathologic features of Waldenstrom's macroglobulinemia and marginal zone lymphoma: are they distinct or the same entity? Clin Lymphoma, 5, Iida, S., Rao, P.H., Nallasivam, P., Hibshoosh, H., Butler, M., Louie, D.C., Dyomin, V., Ohno, H., Chaganti, R.S. & Dalla-Favera, R. (1996) The t(9;14)(p13;q32) chromosomal translocation associated with lymphoplasmacytoid lymphoma involves the PAX-5 gene. Blood, 88, Johnson, S.A., Birchall, J., Luckie, C., Oscier, D.G. & Owen, R.G. (2006) Guidelines on the management of Waldenstrom macroglobulinaemia. Br J Haematol, 132, Nathwani, B.N., Harris, N.L., Weisneberger, D., Isaacson, P.G., Piris, M.A., Berger, F., Muller-Hermelink, H.K. & Swedlow, S.H. (2001) Follicular Lymphoma. In: Pathology & Genetics: Tumours of haematopoietic and lymphoid tissues (ed. by E.S. Jaffe, N.L. Harris, H. Stein & J.W. Vardiman), pp IARC Press, Lyon. Schop, R.F. & Fonseca, R. (2003) Genetics and cytogenetics of Waldenstrom's macroglobulinemia. Semin Oncol, 30, Wilkins, B., Buchan, S., Webster, J. & Jones, D. (2001) Tryptase-positive mast cells accompany lymphocytic as well as lymphoplasmacytic lymphoma infiltrates in bone marrow trephine biopsies. Histopathology, 39,

20 4 Splenic B-cell marginal zone lymphoma 4.1 Definition and Clinical features Splenic B-cell marginal zone lymphoma (SMZL) is a slowly progressive, clonal disorder of mature B lymphocytes, in which there is usually marked splenomegaly, little lymphadenopathy and, in about 30% of cases, a low level (often IgG) paraproteinaemia. When abnormal cells are found in the peripheral blood or bone marrow, the term splenic lymphoma with villous lymphocytes (SLVL) is often used. Transformation to high-grade non-hodgkin lymphoma occurs in about 10% of cases (Oscier, et al 2005; Iannitto, et al 2004, Dogan & Isaacson, 2003, Audouin, et al 2003, Parry-Jones, et al 2003, Thieblemont,et al 2003, Chacon, et al 2002, Catovsky & Matutes 1999). 4.2 Peripheral blood Variable, but frequently low numbers of small to medium sized mature lymphoid cells with faintly basophilic cytoplasm, moderately clumped chromatin and indistinct nucleoli are found in the peripheral blood. Some have plasmacytoid features and polar cytoplasmic villous projections. Pancytopenia due to hypersplenism and/or bone marrow infiltration may be present. Selective monocytopenia is not a feature of this disorder. 4.3 Bone marrow In contrast to classical hairy cell leukaemia, the bone marrow is usually easy to aspirate. Infiltration may be minor, and the cytological features of the abnormal cells are similar to those of the peripheral blood. Bone marrow trephine biopsy appearances vary from subtle, interstitial and/or intrasinusoidal infiltration (Franco, et al 2001, Franco, et al 2004), that may not be visible with standard histological stains, to very extensive nodular and paratrabecular infiltration. If nodular or paratrabecular infiltrates are present, it is exceptional not to find interstitial infiltration in addition. Nodules in some cases strongly resemble reactive lymphoid follicles and may show germinal centre formation. These are believed to represent colonisation of pre-existing nonneoplastic follicles by the lymphoma cells. The latter are usually small

21 lymphocyte-like, but they may be more heterogeneous, with evidence of plasmacytoid differentiation, although the number of mature plasma cells is usually low (Van Huyen, et al 2000). Para-immunoblasts are not seen, and the number of accompanying reactive mast cells in the background stroma is not increased. Histological features in patients whose disease presents as SLVL in the peripheral blood do not differ significantly from those found in SMZL (Hammer, et al 1996, Pawade, et al 1995, Isaacson, et al 1994, Schmid, et al 1992). 4.4 Lymph node & spleen Peripheral lymph nodes are not usually enlarged in SMZL, and consequently lymph node biopsy for diagnosis is rare. However, splenic hilar lymph nodes are often involved, with a diffuse or vaguely nodular infiltrate of cells identical to those found in the spleen. A marginal zone distribution around preserved nonneoplastic follicles is rarely seen (Isaacson, et al 1994). Most cases of SMZL, but not all (Rosso, et al 1995), have moderate to massive splenic enlargement at the time of splenectomy, which does not correlate with the presence or extent of peripheral blood involvement. Disease in the spleen predominantly involves the white pulp, causing expansion of white pulp nodules with a biphasic pattern. Germinal centres are atrophic or absent, and individual white pulp nodules have a central component of small B lymphocytes resembling those of the normal mantle zone. This central nodule is surrounded by a variably enlarged marginal zone containing plasmacytoid cells and scattered larger blast cells, some with immunoblastic features. It remains a point of controversy whether a very high content of the latter indicates SMZL that has progressed to higher grade disease requiring a more aggressive approach to treatment (Cualing, et al 2000, Lloret, et al 1999). The splenic red pulp shows involvement by SMZL to a highly variable extent. In many cases, this takes the form of multiple pericapillary satellite nodules of heterogeneous plasmacytoid marginal zone B cells accompanied by small clusters of epithelioid macrophages. In other cases, particularly those with large numbers of circulating neoplastic cells, there is extensive diffuse red pulp

22 infiltration, involving sinusoidal lumens as well as cords. Rare examples of entirely diffuse small B-cell lymphomas of the spleen have been described, with no or minimal nodular involvement of the white pulp (Traverse-Glehen et al 2008). The relationship of such cases to variant cases of HCL remains unproven (Mollejo, et al 2002).and the 2008 WHO classification recognises them as distinct, although still provisional, entities separate from SMZL (Piris et al 2008) 4.5 Immunophenotype Table 4.1 Recommended Panel for the diagnosis of Splenic B-cell Marginal Zone Lymphoma Marker Reaction SmIg Strong, IgM and IgD CD5, CD10, CD23 Usually negative CD19, CD20, FMC7 Positive CD22 & CD79b Positive CD11c Commonly positive CD25 Often negative CD43, CD103, CD123 Cyclin D1 4.6 Cytogenetics & molecular genetics Loss of 7q21-32 has been reported in up to 40% of cases of SMZL (Mateo, et al 1999, Oscier, et al 1996). Cases with t(11;14) and cyclin D1 expression probably represent mantle cell lymphoma. Other described abnormalities include trisomy 3, deletion 13q and deletions of TP Prognostic factors None described 4.8 Pitfalls The histological pattern of marginal zone involvement in the spleen may be mimicked by other small B-cell lymphomas. Intrasinusoidal infiltration is bone marrow is not specific for SMZL/SLVL (Schenka et al 2003, Costes et al 2002). 4.9 Differential diagnosis

23 This includes lymphoplasmacytic lymphoma, CLL, hairy cell leukaemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukaemia variant and mantle cell lymphoma (Pittalunga, et al 1996). SMZL is differentiated from these disorders by the presence of characteristic polar villous lymphocytes in the peripheral blood, the immunophenotype, splenic histology and absence of the t(11;14) translocation demonstrated by FISH Recommendations Recommendations for diagnosis of Splenic B-cell Marginal Zone Lymphoma Diagnostic criteria Level of evidence Presence of polar villous lymphocytes in peripheral Grade C level IV blood Histology of bone marrow or spleen Immunophenotype SmIgM & IgD positive, CD19, CD20, FMC7, CD79a, CD79b and CD11c positive. CD25 positive in 25% of cases. CD5 moderate to weak positive in 20% of cases. CD103, CD123, CD43, CD10 and cyclin D1 negative Immunohistochemistry for cyclin D1 in all cases to exclude MCL and/or FISH for t(11;14). References Audouin, J., Le Tourneau, A., Molina, T., Camilleri-Broet, S., Adida, C., Comperat, E., Benattar, L., Delmer, A., Devidas, A., Rio, B., Diebold, J. (2003) Patterns of bone marrow involvement in 58 patients presenting primary splenic marginal zone lymphoma with or without circulating villous lymphocytes. Br J Haematol,122, Catovsky, D., Matutes, E. (1999) Splenic lymphoma with circulating villous lymphocytes/splenic marginal-zone lymphoma. Semin Hematol, 36, Chacon, J.I., Mollejo, M., Munoz, E., Algara, P., Mateo, M., Lopez, L., Andrade, J., Carbonero, I.G., Martinez, B., Piris, M.A., Cruz, M.A. (2002) Splenic

24 marginal zone lymphoma: clinical characteristics and prognostic factors in a series of 60 patients. Blood, 100, Costes, V., Duchayne, E., Taib, J., Delfour, C., Rousset, T., Baldet, P., Delsol, G., Brousset, P. (2002) Intrasinusoidal bone marrow infiltration: a common growth pattern for different lymphoma subtypes. Br J Haematol, 119, Cualing, H., Steele, P., Zellner, D. (2000) Blastic transformation of splenic marginal zone B-cell lymphoma. Arch Pathol Lab Med, 124, Dogan, A., Isaacson, P.G. (2003) Splenic marginal zone lymphoma. Semin Diagn Pathol, 20, Franco, V., Florena, A.M., Stella, M., Rizzo, A., Iannitto, E., Quintini, G., Campesi, G. (2001) Splenectomy influences bone marrow infiltration in patients with splenic marginal zone cell lymphoma with or without villous lymphocytes. Cancer, 91, Franco, V., Florena, A.M., Ascani, S., Paulli, M., Salvato, M., Pileri, S.A. (2004) CD27 distinguishes two phases in bone marrow infiltration of splenic marginal zone lymphoma. Histopathology, 44, Hammer, R.D., Glick, A.D., Greer, J.P., Collins, R.D., Cousar, J.B. (1996) Splenic marginal zone lymphoma. A distinct B-cell neoplasm. Am J Surg Pathol, 20, Iannitto, E., Ambrosetti, A., Ammatuna, E., Colosio, M., Florena, A.M., Tripodo, C., Minardi, V., Calvaruso, G., Mitra, M.E., Pizzolo, G., Menestrina, F., Franco, V. (2004) Splenic marginal zone lymphoma with or without villous lymphocytes. Hematologic findings and outcomes in a series of 57 patients. Cancer, 101, Isaacson, P.G., Matutes, E., Burke, M. & Catovsky, D. (1994) The histopathology of splenic lymphoma with villous lymphocytes. Blood, 84, Lloret, E., Mollejo, M., Mateo, M.S., Villuendas, R., Algara, P., Martinez, P., Piris, M.A. (1999) Splenic marginal zone lymphoma with increased number of blasts: an aggressive variant? Hum Pathol, 30, Mateo, M., Mollejo, M., Villuendas, R., Algara, P., Sanchez-Beato, M., Martinez, P. & Piris, M.A. (1999) 7q31-32 allelic loss is a frequent finding in splenic marginal zone lymphoma. Am J Pathol, 154, Mollejo, M., Algara, P., Mateo, M.S., Sanchez-Beato, M., Lloret, E., Medina, M.T., Piris, M.A. (2002) Splenic small B-cell lymphoma with predominant red pulp involvement: a diffuse variant of splenic marginal zone lymphoma? Histopathology, 40, Oscier, D.G., Gardiner, A. & Mould, S. (1996) Structural abnormalities of chromosome 7q in chronic lymphoproliferative disorders. Cancer Genet Cytogenet, 92,

25 Oscier, D., Owen, R., Johnson, S. (2005) Splenic marginal zone lymphoma. Blood Rev, 19, Parry-Jones, N., Matutes, E., Gruszka-Westwood, A.M., Swansbury, G.J., Wotherspoon, A.C., Catovsky, D. (2003) Prognostic features of splenic lymphoma with villous lymphocytes: a report on 129 patients. Br J Haematol, 120, Pawade, J., Wilkins, B.S., Wright, D.H. (1995) Low-grade B-cell lymphomas of the splenic marginal zone: a clinicopathological and immunohistochemical study of 14 cases. Histopathology, 27, Piris M, Foucar K, Mollejo M, Campo E, Falini B. (2008) Splenic B-cell leukaemia/lymphoma, unclassifiable. In: Swerdlow SH, Campo C, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds) WHO classification of tumours of haematopoietic and lymphoid tissues, IARC, Lyon

26 Pittaluga, S., Verhoef, G., Criel, A., Wlodarska, I., Dierlamm,J., Mecucci,C., Van den Berghe, H., De Wolf-Peeters, C.. (1996) "Small" B-cell non-hodgkin's lymphomas with splenomegaly at presentation are either mantle cell lymphoma or marginal zone cell lymphoma. A study based on histology, cytology, immunohistochemistry, and cytogenetic analysis. Am J Surg Pathol, 20, Rosso, R., Neiman, R.S., Paulli, M., Boveri, E., Kindl, S., Magrini, U., Barosi, G. (1995) Splenic marginal zone cell lymphoma: report of an indolent variant without massive splenomegaly presumably representing an early phase of the disease. Hum Pathol, 26, Schenka, A.A., Gascoyne, R.D., Duchayne, E., Delsol, G., Brousset, P. (2003) Prominent intrasinusoidal infiltration of the bone marrow by mantle cell lymphoma. Hum Pathol, 34, Schmid, C., Kirkham, N., Diss, T., Isaacson, P.G. (1992) Splenic marginal zone cell lymphoma. Am J Surg Pathol, 16, Traverse-Glehen A, Baseggio L, Callet-Bauchu E, Morel D, Gazzo S, Ffrench M, Verney A, Rolland D, Thieblemont C, Magaud J-P, Salles G, Coiffier B, Berger F, Felman P (2008). Splenic red pulp lymphoma with numerous basophilic villous lymphocytes: a distinct clinicopathologic and molecular entity? Blood, 111, Thieblemont, C., Felman, P., Callet-Bauchu, E., Traverse-Glehen, A., Salles, G., Berger, F., Coiffier, B. (2003) Splenic marginal-zone lymphoma: a distinct clinical and pathological entity. Lancet Oncol, 4, Van Huyen JP, Molina T, Delmer A, Audouin J, Le Tourneau A, Zittoun R, Bernadou A, Diebold J. (2000) Splenic marginal zone lymphoma with or without plasmacytic differentiation. Am J Surg Pathol, 24,

27 5 Hairy cell leukaemia 5.1 Definition and clinical features A rare chronic lymphoproliferative disorder characterised by marked splenomegaly, minimal lymphadenopathy and the presence of hairy lymphocytes in the peripheral blood and bone marrow (Bethel & Sharpe 2003). Patients may present with symptoms relating to pancytopenia, atypical infection or splenomegaly (Allsup & Cawley 2004, Allsup & Cawley 2002). 5.2 Peripheral blood Typical patients present with a mild macrocytosis, pancytopenia and monocytopenia. Hairy cells are often infrequent; they are medium sized with relatively abundant cytoplasm and numerous fine hair-like projections. The nuclei are usually oval or slightly indented with moderately clumped chromatin and absent or very inconspicuous nucleoli. In hairy cell leukaemia variant, the WBC is high with abnormal villous cells that have more prominent nucleoli. Bone marrow Bone marrow is often difficult to aspirate (dry-tap) in HCL due to the presence of fibrosis. In bone marrow trephine biopsy specimens, haemopoietic components are patchily or completely replaced by a monotonous infiltrate of small-to-mediumsized lymphoid cells that appear spaced out because of their relatively abundant pale cytoplasm. Their nuclei are oval to kidney-shaped. Accompanying stromal features are diffuse, fine reticulin fibrosis or, rarely, overt collagen fibrosis or even osteosclerosis. Sinusoids may appear to gape open because of the increased reticulin. There is usually pronounced red cell leakage into the marrow stroma and an increase in numbers of scattered reactive mast cells; sometimes there is also a reactive plasmacytosislymph node & spleen Lymph nodes are rarely sampled for histology in HCL. Appearances resemble those in the spleen. The spleen is usually, but not always, significantly enlarged by the time of clinical presentation of HCL. Splenic structure is effaced by a monotonous infiltrate with

28 cytological features as above so that the red and white pulp are not clearly distinguishable. White pulp structures are usually very severely atrophic, to the point of near or complete invisibility and the architecture of red pulp cords and sinusoids is extensively disrupted by the infiltrate, sometimes creating peliosislike areas in the tissue, congested with red cells. Interstitial red cell leakage may also be prominent, as in bone marrow. 5.3 Immunophenotype The gene expression profile of the tumour cells in classical HCL is that of an activated memory B lymphocyte (Basso, et al 2004) and the immunophenotype broadly reflects this (Matutes 2006). IG V H genes are mutated and, although most cases express IgG, multiple heavy chain genes have been detected in some (Forconi, et al 2004). Table 5.1 Recommended Panel for diagnosis of HCL Marker Reaction Tartrate-resistant acid phosphatse Positive SmIg Strong. IgG CD5, CD10, CD23 Usually negative CD19, CD20, CD22, FMC7 Positive CD79b Often negative CD11c, CD25, CD103, CD123 Positive Cyclin D1 50% positive on immunohistochemistry CD72 (DBA.44) Positive Cytochemistry for tartrate-resistant acid phosphatase (TRAP) has been superseded by its detection by immunohistochemistry (Hoyer & Li 1997) and by immunophenotyping using the above markers (Went et al 2005, Del Giudice et al 2004, Bethel et al 2003, Cordone et al 1995). Cytogenetics & molecular genetics No consistent abnormalities are described. 5.4 Prognostic factors

29 No specific abnormalities are described 5.5 Pitfalls In some cases, the presence of macrocytosis, pancytopenia and bone marrow fibrosis may lead to confusion with myelodysplastic syndrome, aplastic anaemia or myelofibrosis. Rare patients have florid marrow osteosclerosis. Differential diagnosis The main differential diagnoses include SMZL (CD103 and CD123 are usually negative), splenic diffuse red pulp small B-cell lymphoma Hairy Cell Leukaemia Variant HCL variant is now thought to be a distinct entity from HCL. Cases of HCL variant may have similar bone marrow morphology to classical HCL or have morphological features less readily distinguishable from lymphocytic, prolymphocytic, lymphoplasmacytic or marginal zone lymphoma (Ya-In et al 2005, Zinzani et al 1990). As in bone marrow, splenic histology in HCL variant overlaps considerably with that of other small B cell lymphomas. The following are helpful in distinguishing hairy cell leukaemia from the HCL variant. Monocytopenia and macrocytosis are not features of hairy cell leukaemia variant (Cessna et al 2005, Matutes et al 2003, Dunphy & Petruska 1996, Sainati et al 1990). Tartrate-resistant acid phosphatase is not expressed in cases of HCL variant and whilst CD103 is often positive, CD123 and CD25 are usually negative (Del Giudice, et al 2004) Recommendations Recommendations for the diagnosis of Hairy Cell Leukaemia Diagnostic criteria Level of evidence Peripheral blood film shows characteristic hairy cells Grade C level IV (but numbers may be very low)

30 Bone marrow biopsy with immunohistochemistry on tissue sections: CD20, DBA44, tartrate-resistant acid phosphatase Flow cytometry on PB or BM cell suspensions with a panel of McAbs CD19, CD20, CD22, SmIg, CD11c, CD25, CD103, CD References Allsup, D.J., Cawley, J.C. (2002) The diagnosis and treatment of hairy-cell leukaemia. Blood Rev, 16, Allsup, D.J., Cawley, J.C. (2004) Diagnosis, biology and treatment of hairy-cell leukaemia. Clin Exp Med, 4, Basso, K., Liso, A., Tiacci, E., Benedetti, R., Pulsoni, A., Foa, R., Di Raimondo, F., Ambrosetti, A., Califano, A., Klein, U., Dalla Favera, R. & Falini, B. (2004) Gene expression profiling of hairy cell leukemia reveals a phenotype related to memory B cells with altered expression of chemokine and adhesion receptors. J Exp Med, 199, Bethel, K.J., Sharpe, R.W. (2003) Pathology of hairy-cell leukaemia. Best Pract Res Clin Haematol, 16, Cessna, M.H., Hartung, L., Tripp, S., Perkins, S.L., Bahler, D.W. (2005 ) Hairy cell leukemia variant: fact or fiction. Am J Clin Pathol, 123, Cordone, I., Annino, L., Masi, S., Pescarmona, E., Rahimi, S., Ferrari, A., Giubilei, E., Pignoloni, P., Faraggiana, T., Mandelli, F. (1995) Diagnostic relevance of peripheral blood immunocytochemistry in hairy cell leukaemia. J Clin Pathol, 48, Del Giudice, I., Matutes, E., Morilla, R., Morilla, A., Owusu-Ankomah, K., Rafiq, F., A'Hern, R., Delgado, J., Bazerbashi, M.B. & Catovsky, D. (2004) The diagnostic value of CD123 in B-cell disorders with hairy or villous lymphocytes. Haematologica, 89, Dunphy, C.H., Petruska, P.J. (1996) Atypical prolymphocytic variant of hairy-cell leukemia: case report and review of the literature. Am J Hematol, 53, Forconi, F., Sahota, S.S., Raspadori, D., Ippoliti, M., Babbage, G., Lauria, F. & Stevenson, F.K. (2004) Hairy cell leukemia: at the crossroad of somatic mutation and isotype switch. Blood, 104, Matutes E, Wotherspoon A, Catovsky D. (2003) The variant form of hairy-cell leukaemia. Best Pract Res Clin Haematol, 16,

31 Matutes, E. (2006) Immunophenotyping and differential diagnosis of hairy cell leukemia. Hematol Oncol Clin North Am, 20, Sainati, L., Matutes, E., Mulligan, S., de Oliveira, M.P., Rani, S., Lampert, I.A., Catovsky, D. (1990) A variant form of hairy cell leukemia resistant to alpha-interferon: clinical and phenotypic characteristics of 17 patients. Blood, 76, Went, P.T., Zimpfer, A., Pehrs, A.C., Sabattini, E., Pileri, S.A., Maurer, R., Terracciano, L., Tzankov, A., Sauter, G., Dirnhofer, S. (2005) High specificity of combined TRAP and DBA.44 expression for hairy cell leukemia. Am J Surg Pathol, 29, Ya-In, C., Brandwein, J., Pantalony, D., Chang, H. (2005) Hairy cell leukemia variant with features of intrasinusoidal bone marrow involvement. Arch Pathol Lab Med, 129, Zinzani, P.L., Lauria, F., Buzzi, M., Raspadori, D., Gugliotta, L., Bocchia, M., Macchi, S., Algeri, R., Tura, S. (1990) Hairy cell leukemia variant: a morphologic, immunologic and clinical study of 7 cases. Haematologica, 75,

32 6 Plasma cell myeloma and other plasma cell neoplasms 6.1 Definition and clinical features These are malignancies of mature plasma cells characterised by the presence of intact or fragments of monoclonal immunoglobulin in the blood and/or urine, a plasma cell infiltrate in the bone marrow and/or plasma cell tumour(s) in other tissues. Clinical features result from the associated immunodeficiency, the effects of the tumour on bone with lytic lesions, fractures and hypercalcaemia and the effects of the abnormal protein, which include renal impairment and hyperviscosity. 6.2 Peripheral blood The blood film often shows background staining and rouleaux formation due to the paraprotein, pancytopenia when there is heavy bone marrow infiltration and, rarely, circulating plasma cells or plasmablasts. When more than 2 x 10 9 /l plasma cells/blasts are found in the blood the term plasma cell leukaemia is used (Garcia-Sanz et al 1999). 6.3 Bone marrow The bone marrow aspirate shows infiltration with plasma cells which may have atypical cytological features including multinuclearity and cytoplasmic inclusions; there may be variable numbers of plasmablasts (Grogan 2003, Milla et al 2001). Infiltration may be patchy and the percentage of plasma cells variable. In true solitary or multifocal osseous plasmacytomas, unless the iliac crest is the lesional site, trephine biopsy from this usual position will show no plasmacytosis and have no demonstrable clonal population of plasma cells. Light chain restriction is the usual method for convenient demonstration of monoclonality. In trephine biopsy sections, this can be performed by in situ hybridisation for light chain messenger RNA (mrna) or by immunohistochemistry. Since the marrow stroma may show a high background staining for immunoglobulin, light chain immunohistochemistry requires the use of optimised methods in an experienced

33 laboratory. The use of polyclonal antisera for immunohistochemistry may also give non-specific staining. With regard to histological features in bone marrow trephine biopsy specimens, the WHO classification considers only density of plasma cell infiltration as a contributory criterion in distinguishing between monoclonal gammopathy of uncertain significance (MGUS) and early/smouldering plasma cell myeloma. This fails to acknowledge the contribution that recognition of abnormal plasma cell cytology, subtle abnormal patterns of infiltration, particularly inter-fatty space infiltration, and an abnormal immunophenotype can make to the assessment of minor plasma cell infiltrates as being neoplastic (Grogan 2003). Also, no recognition is given to the sensitivity of in situ hybridisation (ISH) as a tool for indicating light chain restriction as evidence supporting monoclonality, particularly in cases with a reversed kappa-to-lambda ratio. However, the clinical significance of applying a label of early/smouldering plasma cell myeloma rather than MGUS remains uncertain (Rosinol et al 2003, Cesana et al 2002, Van de Donk et al 2001). Angiogenesis accompanying myelomatous infiltration of bone marrow may be of prognostic significance (Bhati et al 2006, Vacca et al 2006). The normal ratio of plasma cells is 2-4 kappa-expressing cells to each lambda positive cell; a ratio of 8 (or more) kappa to 1 lambda strongly suggests a monoclonal kappa-positive population and can be readily visualised by ISH with a plasmacytosis of approximately 5% in trephine sections. Conversely, a ratio of even 4 lambda-expressing cells to 1 kappa-positive one indicates the presence of a monoclonal lambda-positive plasma cell population. Plasma cell cytology is more likely to be atypical or blastic in examples with a relatively high burden of neoplastic cells in the marrow. Extensive, aggressive plasma cell neoplasms may have highly pleomorphic cytology and may mimic high grade NHL, acute leukaemias or solid non-haemopoietic neoplasms. Even within the length of a modest trephine biopsy core, infiltration may not be uniform in extent or distribution; at its extreme, this variation can produce single or multiple localised tumourlets. Depending on the size and number of the latter, and on the extent of any accompanying diffuse plasmacytosis, they may be

34 regarded as either multiple plasmacytomas or components of disseminated plasma cell myeloma. They are probably best regarded as the latter, and use of the term plasmacytoma(s) restricted to radiographically evident lesions, because of the potential for localised radiotherapy as effective treatment for the latter in many cases. Localised plasmacytomas tend to consist of solid monotonous masses of mature-appearing plasma cells. If the cytology is more aggressive and plasmablastic or pleomorphic, caution should be exercised before excluding a more disseminated plasma cell neoplasm. Osteosclerotic variants of plasma cell myeloma also occur and plasma cell neoplasms may be accompanied by intracellular or extracellular immunoglobulin deposition, or by deposition of light chain-derived amyloid, which may be vascular or interstitial or both. 6.4 Immunophenotype Table 6.1 Recommended Panel for the diagnosis of Plasma Cell Myeloma Marker Immunofluorescence Immunohistochemistry CD45 Usually negative CD19 Usually weak positive CD20 (5-10% positive associated with t(11;14) expression) CD79a Highly variable CD138 Positive Positive MUM1/IRF4 N/A Positive CD56 Usually positive usually positive CD38 Positive Positive Cyto Ig light light chain restriction light chain restriction chain κ, λ (Ely and Knowles 2002, Mateo Manzanera, et al 2005, Sezer, et al 2001) The use of flow cytometry for the detection of minimal residual disease shows promise in assessing response to therapy and appears to be more sensitive than immunofiaxtion (Rawstron, et al 2002, San Miguel, et al 2002).

35 6.5 Cytogenetics & molecular genetics These are not essential for diagnosis. Conventional cytogenetic analysis is frequently unsuccessful but it appears that the detection of deletions of 13q by metaphase analysis may have prognostic value, along with t(4;14) and deletion of TP53 by FISH (Stewart and Fonseca 2005). The t(11:14) abnormality is found in 10-15% of cases, resulting in over-expression of cyclin D Prognostic factors It is only recently that adequate genetic analysis of plasma cell neoplasms has been possible, and new potential prognostic markers are still being reported. As treatment is also changing, caution should be applied when interpreting FISH results. Many of these markers can also be seen in MGUS and do not appear to predict progression A number of laboratory and clinical parameters have been shown to predict prognosis. These reflect the underlying biology of the disorder and the disease burden. Staging systems have been devised to risk stratify patients. Table 6.2 Prognostic factors for Plasma Cell Myeloma Factors Predictor of poor outcome Cytogenetics del(13q) in metaphases, t(4;14) and deletion p53 by FISH Haematology Low haemoglobin, heavy plasma cell infiltrate, plasmablastic morphology Biochemistry Abnormal renal function, hypercalcaemia, low serum albumin, raised serum beta2 microglobulin Immunology High paraprotein concentration, Radiology Presence of lytic bone lesions The new international scoring system shown below is now recommended instead of other systems (Greipp, et al 2003) Table 6.3 International prognostic scoring system for Plasma Cell Myeloma Stage Factor Median survival

36 (months) 1 Serum β2 microglobulin < 3.5 mg/ml and 62 albumin >35g/l 2 Neither 1 nor Serum β2 microglobulin > 5.5 mg/ml Other variants of plasma cell neoplasia Variants include MGUS, asymptomatic/smouldering myeloma (Ng et al 2006, Perez-Andres et al 2005, Ely & Knowles 2002, Sezer et al 2001, Milla et al 2001) and solitary plasmacytoma of bone. Plasma cell neoplasia also manifests as extraosseous plasmacytoma (Kremer et al 2005, Menke et al 2001), POEMS syndrome/castleman disease and the immunoglobulin deposition disorders such as primary amyloidosis and heavy and light chain deposition diseases POEMS syndrome This is a rare variant of plasma cell myeloma in which sclerotic bone lesions are found in conjunction with variable Polyneuropathy (demyelinating sensorimotor type), Organomegaly (hepatosplenomegaly), Endocrinopathy (gynaecomastia, testicular atrophy and diabetes mellitus), Monoclonal gammopathy and Skin changes (hyperpigmentation and hypertrichosis). In some cases this syndrome is seen in multicentric Castleman disease and in some patients there appears to be a relationship with human herpesvirus 8 infection. Bone marrow trephine biopsy histology, if lesional tissue is included, shows single or multiple osteosclerotic plasmacytomas. Background marrow has no significant plasmacytosis. The immunoglobulin produced is usually IgG or IgA with a marked predominance of lambda light chain. Lymph node histology is that of the plasma cell variant of Castleman disease Primary amyloidosis In this condition there is deposition of a fibrillary protein in the liver, kidneys, heart, gastrointestinal tract, peripheral nerves and other tissues resulting in organ impairment (See Diagnosis is dependent on demonstration of amyloid protein and exclusion of

37 secondary (non-immunoglobulin) amyloidosis. The protein binds Congo and Sirius Red dyes with apple-green birefringence when viewed under polarised light. Although considered part of the spectrum of plasma cell neoplasia, clinical features arising as a result of protein deposition predominate and are manifest at a low tumour burden such as that found in MGUS. The light chain type in primary amyloidosis is usually lambda, presumably reflecting easier conversion of this protein, compared with kappa, into beta-pleated sheet structures Light and heavy chain deposition disorders. These may present with features resembling primary amyloidosis but the protein is non-fibrillary as seen by electron microscopy and does not bind Congo Red/Sirius Red. Abnormal protein comprising heavy or light chains (or both) is deposited in tissues such as the heart and liver leading to organ dysfunction. As with primary amyloidosis, the associated tumour burden is usually low. In light chain deposition disease, the light chain type is usually kappa and neoplastic cells, if identifiable, are usually plasma cells. Heavy chain diseases (mu and gamma) usually affect older individuals and involve lymph nodes, marrow, spleen and peripheral blood, with varying lymphocytic, lymphoplasmacytoid and mature plasma cell morphology in cellular components. Occasional examples appear to be variants of diffuse large B-cell lymphoma. Alpha chain disease differs in presenting typically with small intestine and mesenteric lymph node involvement causing malabsorption in younger individuals. The small bowel in alpha chain disease has histological features resembling those seen in extranodal marginal zone B-cell lymphomas of MALT type. 6.8 Pitfalls Extreme pleomorphism in some cases of plasma cell myeloma may lead to histological confusion with metastatic solid malignancies such as poorly differentiated carcinoma or melanoma. Rarely, the pleomorphic cells mimic atypical megakaryocytes and cause confusion with myelodysplastic or myeloproliferative states. Reactive plasmacytosis may be impossible to distinguish from very low levels of bone marrow involvement by a plasma cell neoplasm.

38 Vascular amyloidosis involving bone marrow cannot be ruled out if vessels of sufficient size (arterioles, venules or larger) are not included in the tissue, which is commonly the case. 6.9 Differential diagnosis The presence of a reactive non-clonal plasmocytosis can usually be excluded by the demonstration of a monoclonal plasma cell population by flow cytometry or by ISH/ immunocytochemistry applied to trephine biopsy sections. It may be difficult to distinguish plasma cell myeloma with blastic or highly pleomorphic cytology and the plasmablastic variant of diffuse large B cell lymphoma (Folk et al 2006). Immunophenotyping may overlap in such cases. In rare cases of body cavity-based plasmablastic diffuse large B-cell lymphoma the presence of HIV and tumour site may be helpful (Dong et al 2005); HHV8 is also almost universally present. In cases not associated with HIV there may be a background of therapeutic immunosuppression Recommendations Recommendations for the diagnosis of Plasma Cell Neoplasia Diagnostic criteria Level of evidence Typical morphology Grade C level IV Immunophenotype; Cyto Ig light chain restricted, CD38+, CD138+ There are 2 sets of internationally accepted diagnostic criteria for the diagnosis of plasma cell myeloma. WHO Criteria for the diagnosis of Plasma Cell Myeloma Major criteria* Bone marrow plasmacytosis (> 30% plasma cells) Minor criteria* Bone marrow plasmacytosis of 10-30%

39 Paraprotein present: serum IgG paraprotein more than 35 g/l or IgA paraprotein more than 20 g/l; urinary Bence-Jones protein more than 1 g/24 hours Plasmacytoma on biopsy Paraprotein present but at lower concentration Lytic bone lesions Reduced normal immunoglobulins: IgG < 6 g/l, IgA < 1 g/l, IgM < 0.5 g/l * diagnosis of other than smouldering myeloma requires a symptomatic patient with progressive disease; one major and two minor criteria or three minor criteria, including the first two listed, must be met. International Myeloma Working Group criteria for the diagnosis of monoclonal gammopathy of undetermined significance, asymptomatic (smouldering) myeloma and symptomatic myeloma (Durie, et al 2003) Monoclonal gammopathy of undetermined significance Serum paraprotein less than 30 g/l Asymptomatic (smouldering) myeloma Serum paraprotein at least AND/OR Symptomatic* myeloma Paraprotein in serum or urine Bone marrow clonal plasma cells less than 10% and low level infiltration in trephine biopsy specimen No evidence of other B- lineage lymphoproliferative disorder No related organ or tissue impairment such as bone lesions, lightchain associated amyloidosis, Bone marrow clonal plasma cells at least 10% No related organ damage or tissue impairment Bone marrow clonal plasma cells or plasmacytoma Related organ damage or tissue impairment

40 paraprotein-associated neurological damage, hypercalcaemia, renal impairment, anaemia, symptomatic hyperviscosity, more than two bacterial infections in 12 months * Includes asymptomatic patients who have evidence of organ damage 6.11 References Cesana, C., Klersy, C., Barbarano, L., Nosari, A.M., Crugnola, M., Pungolino, E., Gargantini, L., Granata, S., Valentini, M., Morra, E. (2002) Prognostic factors for malignant transformation in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. J Clin Oncol, 20, Dong, H.Y., Scadden, D.T., de Leval, L., Tang, Z., Isaacson, P.G., Harris, N.L. (2005) Plasmablastic lymphoma in HIV-positive patients: an aggressive Epstein-Barr virus-associated extramedullary plasmacytic neoplasm. Am J Surg Pathol, 29, Durie, B., Kyle, R., Belch, A., Bensinger, W., Blade, J., Boccadoro, M., Child, J., Comenzo, R., Djulbegovic, B., Fantl, D., Gahrton, G., Harousseau, J., Hungria, V., Joshua, D., Ludwig, H., Mehta, J., Morales, A., Morgan, G., Nouel, A., Oken, M., Powles, R., Roodman, D., San Miguel, J., Shimizu, K., Singhal, S., Sirohi, B., Sonneveld, P., Tricot, G. & Van Ness, B. (2003) Myeloma management guidelines: a consensus report from the Scientific Advisors of the International Myeloma Foundation. Hematology Journal, 4, Ely, S.A. & Knowles, D.M. (2002) Expression of CD56/Neural Cell Adhesion Molecule Correlates with the Presence of Lytic Bone Lesions in Multiple Myeloma and Distinguishes Myeloma from Monoclonal Gammopathy of Undetermined Significance and Lymphomas with Plasmacytoid Differentiation. Am J Pathol, 160, Folk, G.S., Abbondanzo, S.L., Childers, E.L., Foss, R.D. (2006) Plasmablastic lymphoma: a clinicopathologic correlation. Ann Diagn Pathol, 10, Garcia-Sanz, R., Orfao, A., Gonzalez, M., Tabernero, M.D., Blade, J., Moro, M.J., Fernandez-Calvo, J., Sanz, M.A., Perez-Simon, J.A., Rasillo, A., Miguel, J.F. (1999) Primary plasma cell leukemia: clinical, immunophenotypic, DNA ploidy, and cytogenetic characteristics. Blood, 93, Greipp, P., San Miguel, J., Fonseca, R., Avet-Loiseau, H., Jacobson, J., Rasmussen, E., Crowley, J. & Durie, B. (2003) Development of an

41 international prognostic index (IPI) for myeloma: report of the international myeloma working group. Hematology Journal, 4, S42. Grogan, T. (2003) Plasma cell myeloma marrow diagnosis including morphologic and phenotypic features. Semin Diagn Pathol., 20, Kremer, M., Ott, G., Nathrath, M., Specht, K., Stecker, K., Alexiou, C., Quintanilla-Martinez, L., Fend, F. (2005) Primary extramedullary plasmacytoma and multiple myeloma: phenotypic differences revealed by immunohistochemical analysis. J Pathol, 205, Mateo Manzanera, G., San Miguel Izquierdo, J. & Orfao de Matos, A. (2005) Immunophenotyping of plasma cells in multiple myeloma. Methods Mol Med., 113, Menke, D.M., Horny, H.P., Griesser, H., Tiemann, M., Katzmann, J.A., Kaiserling, E., Parwaresch, R., Kyle, R.A. (2001) Primary lymph node plasmacytomas (plasmacytic lymphomas). Am J Clin Pathol, 115, Milla, F., Oriol, A., Aguilar, J., Aventin, A., Ayats, R., Alonso, E., Domingo, A., Feliu, E., Florensa, L., Lopez, A., Perez-Vila, E., Rozman, M., Sanchez, C., Vallespi, T., Woessner, S. (2001) Usefulness and reproducibility of cytomorphologic evaluations to differentiate myeloma from monoclonal gammopathies of unknown significance. Am J Clin Pathol, 115, Ng, A.P., Wei, A., Bhurani, D., Chapple, P., Feleppa, F., Juneja, S. (2006) The sensitivity of CD138 immunostaining of bone marrow trephine specimens for quantifying marrow involvement in MGUS and myeloma, including samples with a low percentage of plasma cells. Haematologica, 91, Perez-Andres, M., Almeida, J., Martin-Ayuso, M., Moro, M.J., Martin-Nunez, G., Galende, J., Borrego, D., Rodriguez, M.J., Ortega, F., Hernandez, J., Moreno, I., Dominguez, M., Mateo, G., San Miguel, J.F., Orfao, A.; Spanish Network on multiple myeloma (G03/136); the Spanish Network of Cancer Research Centers (C03/10). (2005) Clonal plasma cells from monoclonal gammopathy of undetermined significance, multiple myeloma and plasma cell leukemia show different expression profiles of molecules involved in the interaction with the immunological bone marrow microenvironment. Leukemia, 19, Rawstron, A.C., Davies, F.E., DasGupta, R., Ashcroft, A.J., Patmore, R., Drayson, M.T., Owen, R.G., Jack, A.S., Child, J.A. & Morgan, G.J. (2002) Flow cytometric disease monitoring in multiple myeloma: the relationship between normal and neoplastic plasma cells predicts outcome after transplantation. Blood, 100, Rosinol, L., Blade, J., Esteve, J., Aymerich, M., Rozman, M., Montoto, S., Gine, E., Nadal, E., Filella, X., Queralt, R., Carrio, A., Montserrat, E. (2003) Smoldering multiple myeloma: natural history and recognition of an evolving type. Br J Haematol, 123,

42 San Miguel, J.F., Almeida, J., Mateo, G., Blade, J., Lopez-Berges, C., Caballero, D., Hernandez, J., Moro, M.J., Fernandez-Calvo, J., Diaz-Mediavilla, J., Palomera, L. & Orfao, A. (2002) Immunophenotypic evaluation of the plasma cell compartment in multiple myeloma: a tool for comparing the efficacy of different treatment strategies and predicting outcome. Blood, 99, Sezer, O., Heider, U., Zavrski, I. & Possinger, K. (2001) Differentiation of monoclonal gammopathy of undetermined significance and multiple myeloma using flow cytometric characteristics of plasma cells. Haematologica, 86, Stewart, A.K. & Fonseca, R. (2005) Prognostic and therapeutic significance of myeloma genetics and gene expression profiling. J Clin Oncol, 23, Van De Donk, N., De Weerdt, O., Eurelings, M., Bloem, A., Lokhorst, H. (2001) Malignant transformation of monoclonal gammopathy of undetermined significance: cumulative incidence and prognostic factors. Leuk Lymphoma, 42,

43 7 Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) 7.1 Definition and clinical features This lymphoma involves extranodal lymphoid tissue, shows infiltration of the marginal zones of B-cell follicles and extends into interfollicular regions. The infiltrating cells are derived from post-germinal centre marginal zone B cells. MALT lymphomas tend to arise in extranodal lymphoid tissue that has developed as part of an inflammatory response. It is classically associated with Helicobacter pylori infection in gastric MALT lymphoma (up to 90% of cases), but is frequently found in other extra-nodal sites such as other areas of the GI tract, skin and lungs as well as with underlying autoimmune conditions such as Hashimoto s thyroiditis (20% of thyroid MALT lymphoma (Zinzani, et al 1999)), lacrimal gland inflammation and Sjögren s syndrome (20% of salivary gland lymphomas) (Zinzani, et al 1999). Ocular MALT lymphomas have been associated in some instances with Chlamydia psittaci, Campylobacter jejuni and Borrelia burgdorferi infection. 7.2 Peripheral blood The peripheral blood can show features of iron deficiency anaemia with hypochromia and microcytosis due to chronic bleeding from gastritis, cytopenias due to marrow infiltration are rare. A paraprotein is not uncommon in stage IV disease and has a strong correlation with bone marrow involvement (Asatiani E., et al 2004). A peripheral blood lymphocytosis is a rare finding, but occasionally abnormal lymphoid cells can be seen. These are small to medium sized cells with abundant weakly basophilic cytoplasm with occasional small projections. 7.3 Bone marrow The aspirate morphology is similar to that seen in the peripheral blood. The trephine biopsy may be infiltrated and this is of variable pattern (nodular, interstitial or diffuse) with small monocytoid/centrocyte-like B cells. The reported

44 incidence of involvement varies in non-gastric MALT lymphoma from 15 to 44% (Thieblemont, et al 2000, Zinzani, et al 1999, Zucca, et al 2003). 7.4 Lymph node and extranodal sites In the extranodal tissue there is proliferation of centrocyte- like/monocytoid cells extending around follicles into the surrounding tissue. There may be large cells scattered within the infiltrate and lymphoepithelial lesions can be seen. Many of the infiltrating lymphocytes show plasma cell differentiation. Lymph nodes can become involved due to disease spread and, in this situation, the cells in the infiltrate are similar to those seen at the extranodal site, usually with a marginal zone or nodular growth pattern. 7.5 Immunophenotype Table 7.1 Recommended Panel for diagnosis of Extranodal marginal zone lymphoma of MALT Immunofluorescence Immunohistochemistry SmIg light chain κ, λ Weak to moderate light chain restriction Sometimes demonstrable light chain restriction; more often kappa than lambda CD19/CD20 Moderate Positive CD79a/CD79b Moderate Positive CD22 Moderate NA CD23 FMC7 Weak NA CD10, CD5 CD43 to weak Usually negative (positive in up to 30%) BCL2 NA Positive BCL6 NA Ki67 NA Low CD21 NA Highlights disrupted networks of FDC

45 Cyclin D1 NA CD138 NA Highlights plasma cell component BCL10 NA Nuclear staining in cases with t(1;14) and t(11;18) 7.6 Cytogenetics and molecular genetics A number of translocations are associated with MALT lymphomas. FISH can be done with a MALT1 break-apart probe for detection of t(11;18)(q21;q21) (Auer, et al 1997, Remstein, et al 2000)or t(14;18)(q32;q21) (Streubel, et al 2003). The t(14;18)(q32;q21) appears to be commonest in non-gastric forms whilst t(3;14)(p14.1;q32) (Streubel, et al 2005) is found more commonly in orbit and thyroid and not seen in gastric MALT. The t(1;14)(p22;q32) is also characteristic of this subgroup but requires full cytogenetic analysis to identify it. 7.7 Prognostic and therapeutic markers Gastric MALT lymphomas with t(11;18)(q21;q21) are said to be resistant to Helicobacter pylori eradication, but not to chemotherapy (Liu, et al, 2004) 7.8 Pitfalls The circulating lymphoma cells in the peripheral blood can be mistaken for hairy cells if assessed by morphology alone, due to the monocytoid appearance of circulating cells, which can have cytoplasmic projections. Immunophenotyping should therefore be carried out. The morphology seen from fine needle aspirates can make it difficult to distinguish this lymphoma from a reactive lymph node FNA specimen (Matsushima, et al 1999). 7.9 Differential diagnosis It is important to distinguish MALT lymphomas from mantle cell NHL and follicular NHL which can have similar cytological appearances. Diagnosis is aided by demonstrating absence of cyclin D1, CD5 and CD10 expression, plus absence of t(14;18) and t(11;14) in MALT lymphoma.

46 Reactive conditions, for example Helicobacter-associated gastritis, Hashimoto s thyroiditis, Sjogren s disease in salivary glands, can have very similar appearances to extra-nodal MALT lymphoma Recommendations Recommendations for diagnosis of Extranodal Marginal Zone Lymphoma of MALT Diagnostic criteria Level of evidence Immunophenotype: weak to moderate clonal SmIg, Grade C level IV CD19/CD20 positive, CD22/ CD79b moderate, CD23 negative, CD5 negative, CD10 negative, BCL2 positive, BCL6 negative, cyclin D1 negative 7.11 References Asatiani, E., Cohen, P., Ozdemirli, M., Kessler, C.M., Mavromatis, B. & Cheson, B.D. (2004) Monoclonal gammopathy in extranodal marginal zone lymphoma (ENMZL) correlates with advanced disease and bone marrow involvement. American Journal of Hematology, 77, Auer, I.A., Gascoyne, R.D., Conners, J.M., Cotter, F.E., Greiner, T.C., Sanger, W.G. & Horsman, D.E. (1997) t(11;18)(q21;q21) is the most common translocation in MALT lymphomas. Ann Oncol, 8, Liu, H., Ye, H., Ruskone-Fourmestraux, A., De Jong, D., Pileri, S., Thiede, C., Lavergne, A., Boot, H., Caletti, G., Wundisch, T., Molina, T., Taal, B., Elena, S., Thomas, T., Zinzani, P., Neubauer, A., Stolte, M., Hamoudi, R., Dogan, A., Isaacson, P. & Du, M. (2002) T(11;18) is a marker for all stage gastric MALT lymphomas that will not respond to H. pylori eradication. Gastroenterology, 122, Matsushima, A.Y., Hamele-Bena, D. & Osborne, B.M. (1999) Fine-needle aspiration biopsy findings in marginal zone B cell lymphoma. Diagnostic Cytopathology, 20, Remstein, E.D., James, C.D. & Kurtin, P.J. (2000) Incidence and Subtype Specificity of API2-MALT1 Fusion Translocations in Extranodal, Nodal, and Splenic Marginal Zone Lymphomas. Am J Pathol, 156, Streubel, B., Lamprecht, A., Dierlamm, J., Cerroni, L., Stolte, M., Ott, G., Raderer, M. & Chott, A. (2003) T(14;18)(q32;q21) involving IGH and MALT1 is a frequent chromosomal aberration in MALT lymphoma. Blood, 101,

47 Streubel, B., Vinatzer, U., Lamprecht, A., Raderer, M. & Chott, A. (2005) T(3;14)(p14.1;q32) involving IGH and FOXP1 is a novel recurrent chromosomal aberration in MALT lymphoma. 19, Streubel, B., Ye, H., Du, M., Isaacson, P., Chott, A. & Raderer, M. (2004) Translocation t(11;18)(q21;q21) is not predictive of response to chemotherapy with 2CdA in patients with gastric MALT lymphoma. Oncology, 66, Thieblemont, C., Berger, F., Dumontet, C., Moullet, I., Bouafia, F., Felman, P., Salles, G. & Coiffier, B. (2000) Mucosa-associated lymphoid tissue lymphoma is a disseminated disease in one third of 158 patients analyzed. Blood, 95, Zinzani, P.L., Magagnoli, M., Galieni, P., Martelli, M., Poletti, V., Zaja, F., Molica, S., Zaccaria, A., Cantonetti, A.M., Gentilini, P., Guardigni, L., Gherlinzoni, F., Ribersani, M., Bendandi, M., Albertini, P. & Tura, S. (1999) Nongastrointestinal Low-Grade Mucosa-Associated Lymphoid Tissue Lymphoma: Analysis of 75 Patients. J Clin Oncol, 17, Zucca, E., Conconi, A., Pedrinis, E., Cortelazzo, S., Motta, T., Gospodarowicz, M.K., Patterson, B.J., Ferreri, A.J.M., Ponzoni, M., Devizzi, L., Giardini, R., Pinotti, G., Capella, C., Zinzani, P.L., Pileri, S., Lopez-Guillermo, A., Campo, E., Ambrosetti, A., Baldini, L. & Cavalli, F. (2003) Nongastric marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue. Blood, 101,

48 8 Nodal Marginal Zone Lymphoma 8.1 Definition and clinical features The malignant population is derived from nodal marginal zone (monocytoid) B cells. The lymph nodes have similar morphology to that of extranodal MALT-type and splenic marginal zone lymphomas, with involvement of the lymph node marginal zones, but without extranodal involvement The clinical features are similar to those of small lymphocytic or follicular lymphoma with lymphadenopathy and occasionally splenomegaly % of cases are said to transform to DLBCL. 8.2 Peripheral blood Circulating lymphoma cells are unusual, (Berger, et al 2000) but, when present, are small lymphocytes with scanty cytoplasm and an irregular nuclear outline. The nuclear chromatin has fine strands with small clumps (Oertel, et al 2002). 8.3 Bone marrow Evidence of marrow infiltration on aspirate morphology is occasionally seen with features as in the peripheral blood. The lymphoid infiltrate seen in trephine biopsy sections consists of a mixture of monocytoid B cells and small lymphocytes and can have a variety of patterns. Involvement is relatively common (between 30 and 60%) (Conconi, et al 2001, Nathwani, et al 1999). 8.4 Lymph node The infiltrate can be peritrabecular, subcapsular or perifollicular with collections of pale cells with interspersed large cells. In addition there are frequently small numbers of neutrophils and plasma cells within the infiltrate. The appearances can be divided into two types: MALT-like and splenic marginal zone lymphomalike 8.5 Immunophenotype Table 8.1 Recommended Panel for diagnosis of Nodal Marginal Zone lymphoma

49 Immunofluorescence Immunohistochemistry SmIg light chain κ or λ Weak to moderate Rarely weakly demonstrable light chain restriction CD19/CD20 Strong Positive CD79a/CD79b Strong Positive CD22 Moderate N/A CD23 FMC7 Weak N/A CD10, CD5 BCL2 N/A Positive BCL6 N/A Ki67 N/A Low MUM1/IRF4 N/A CD21 N/A Marginal zones contain FDC meshwork Cyclin D1 N/A Cytoplasmic κ or λ Weak (light chain Light chain restriction restriction) 8.6 Cytogenetics and molecular genetics Detectable cytogenetic changes are extremely unusual, with the classic t(11;18)(q21;q21) of the extranodal form rarely seen in nodal marginal zone lymphoma (Rosenwald, et al 1999) 8.7 Prognostic and therapeutic markers No specific prognostic markers are currently identified 8.8 Pitfalls The lymph node may show follicular colonisation (Kojima, et al 2005), mimicking follicular NHL. 8.9 Differential diagnosis It is possible to distinguish nodal marginal zone lymphoma from marginal zone/monocytoid B-cell hyperplasia as BCL2 is not expressed by monocytoid B-

50 cells in reactive proliferations. Follicular lymphoma (with a marginal zone pattern) can be distinguished by the presence of CD10 and t(14;18), and mantle cell lymphoma by the presence of CD5 with cyclin D1 or t(11:14). It is important to be given an accurate history and examination findings to exclude secondary nodal involvement by extranodal marginal zone lymphoma of MALT Recommendations Recommendations for diagnosis of Nodal Marginal Zone Lymphoma Diagnostic criteria Level of evidence No evidence of extra-nodal involvement Grade C level IV Immunophenotype SmIgM/D strong, CD20 strong, CD22/CD79b strong, FMC7 moderate, CD5 negative, cyclin D1 negative. CD23 usually negative, BCL2 positive, BCL6 negative Immunohistochemistry for cyclin D1 in all cases to exclude MCL and/or FISH for t(11;14) References Berger, F., Felman, P., Thieblemont, C., Pradier, T., Baseggio, L., Bryon, P.-A., Salles, G., Callet-Bauchu, E. & Coiffier, B. (2000) Non-MALT marginal zone B-cell lymphomas: a description of clinical presentation and outcome in 124 patients. Blood, 95, Conconi, A., Bertoni, F., Pedrinis, E., Motta, T., Roggero, E., Luminari, S., Capella, C., Bonato, M., Cavalli, F. & Zucca, E. (2001) Nodal marginal zone B-cell lymphomas may arise from different subsets of marginal zone B lymphocytes. Blood, 98, Kojima, M., Nakamura, S., Murase, T., Motoori, T., Murayama, K., Iijima, M., Itoh, H., Sakata, N. & Masawa, N. (2005) Follicular colonization of nodal marginal-zone B-cell lymphoma resembling follicular lymphoma: report of 6 cases. Int J Surg Pathol, 13, Nathwani, B.N., Anderson, J.R., Armitage, J.O., Cavalli, F., Diebold, J., Drachenberg, M.R., Harris, N.L., MacLennan, K.A., Muller-Hermelink, H.K., Ullrich, F.A. & Weisenburger, D.D. (1999) Marginal Zone B-Cell Lymphoma: A Clinical Comparison of Nodal and Mucosa-Associated Lymphoid Tissue Types. J Clin Oncol, 17,

51 Oertel, J., Kingree, D., Bsuemann, C., Stein, H. & Dorken, B. (2002) Morphologic diagnosis of leukaemic B-lymphoproliferative disorders and the role of cyclin D1 expression. J Cancer Res Clin Oncol, 128, Rosenwald, A., Ott, G., Stilgenbauer, S., Kalla, J., Bredt, M., Katzenberger, T., Greiner, A., Ott, M.M., Gawin, B., Dohner, H. & Muller-Hermelink, H.K. (1999) Exclusive Detection of the t(11;18)(q21;q21) in Extranodal Marginal Zone B Cell Lymphomas (MZBL) of MALT Type in Contrast to other MZBL and Extranodal Large B Cell Lymphomas. Am J Pathol, 155,

52 9 Follicular lymphoma 9.1 Definition and clinical features This a lymphoma derived from germinal centre B cells composed of a variable mixture of centroblasts and centrocytes. It tends to present with generalised lymphadenopathy (rarely stage 1 or 2) and has a relapsing remitting course, but can transform to a more aggressive lymphoma. 9.2 Peripheral blood If there is marrow infiltration, anaemia and thrombocytopenia can be present to varying degrees. The white count can be normal or raised with abnormal cells found in some patients. Follicular lymphoma cells are frequently small with a thin rim of cytoplasm with a deep nuclear cleft or some nuclear irregularity, but can be larger and pleomorphic with nucleoli seen in some cells. If the disease undergoes transformation there may be circulating large cells with reticular chromatin, a peripheral nucleolus and variable amount of cytoplasm. 9.3 Bone marrow The bone marrow aspirate this may be normal due to the patchy nature of the disease. The infiltrating cells, if present, are similar to the cells seen in the peripheral blood. However, the marrow is said to be involved in up to 45% of cases (Armitage and Weisenburger 1998) and this can be identified in trephine biopsy sections as either a focal paratrabecular or random focal infiltration. More rarely the infiltrate is interstitial but it can be diffuse ( packed marrow pattern) if the infiltrate is particularly extensive. The predominant cell is the small cleaved centrocyte, but there can be a centroblastic component in addition. The reticulin stain is increased in infiltrated areas 9.4 Lymph node This shows loss of zoning of the follicle centres, reduced or absent germinal centre tingible body macrophages and an indistinct border to the follicle centres with poor mantle zone formation. Frequently there is an interfollicular infiltrate of mainly centrocytic cells (not considered to be a diffuse component), but there

53 may be a confluent diffuse component (it is important to report proportion of diffuse versus follicular, and note whether the cytology matches the follicular component or represents transformation to DLBCL). Table 9.1 Histological Grading of Follicular Lymphoma Grade No. of centroblasts per high power Cell type field as average of 10 high power fields Grade Grade Grade 3 A >15 Mixed centrocytes & centroblasts Grade 3B >15 Pure nodules of centroblasts 9.5 Other tissues Effusions can occur which may contain a lymphoid population. The clonality of the lymphoid cells can be demonstrated using flow cytometry immunophenotyping for light chain expression by immunofluorescence 9.6 Immunophenotype Table 9.2 Recommended Panel for diagnosis of Follicular Lymphoma Immunofluorescenc Immunohistochemistry e SmIg light chain κ, λ Moderate (light chain restriction) May be demonstrable in some cases SmIg heavy chain D, M, N/A Positive G CD19/CD20 Strong Positive CD79a/ CD79b/CD22 Strong Positive CD23 or positive Usually negative FMC7 Strong NA CD10 Moderate to strong Positive CD5

54 BCL2 N/A Positive BCL6 N/A Positive Ki67 N/A Variable. May highlight lack of zoning MUM1/IRF4 N/A (may be positive in higher grade examples) CD21 N/A To assess follicular and diffuse areas Cyclin D1 N/A 9.7 Cytogenetics and molecular genetics Full cytogenetic analysis is not essential as PCR/ FISH can be used to identify t(14;18)(q32;q21) in peripheral blood, marrow or lymph node. Variant translocations are rare, but do occur, so it may be best to use a break-apart probe rather than the BCL2/IgH combination. It is worth considering the use of FISH for BCL6 break-apart in t(14;18) negative follicular lymphoma which is histologically grade Prognostic and therapeutic markers The Follicular Lymphoma International Prognosis Index (FLIPI) score is important in determining prognosis. There are 5 factors: age (> 60 years vs 60 years), Ann Arbor stage (III-IV vs I-II), hemoglobin level (< 120 g/l vs 120 g/l), number of nodal areas (> 4 vs 4), and serum LDH level (above normal vs normal or below). Three risk groups are defined: low risk (0-1 adverse factor, 36% of patients), intermediate risk (2 factors, 37% of patients, hazard ratio [HR] of 2.3), and poor risk ( 3 adverse factors, 27% of patients, HR = 4.3). (Solal-Celigny, et al 2004) A diffuse component in grade 3 may confer a worse prognosis (Ott, et al 2002, Weisenburger, et al 2000) compared with other types of grade Pitfalls BCL2 staining may be negative in follicular lymphoma. This is found most frequently in grade 3 lesions (25% of cases) (Schraders, et al 2005). There are many variants with different patterns of the cellular organisation (e.g., floral and

55 reverse patterns), cellular morphology (e.g. marginal zone, signet ring, plasma cell differentiation) or extracellular components (e.g., sclerosis, hyaline material). The immunophenotype can also vary in that CD10 may be negative, CD43 may be positive, CD23 may be positive and, with flow cytometry only, CD5 can be positive in up to 25% of cases (Sanchez, et al 2002). Pure diffuse follicular lymphoma is very rare in lymph nodes although it is occasionally seen at relapse. It should be remembered that needle biopsy cores are not ideal for a meaningful assessment of architecture unless they are very large specimens Differential diagnosis It is extremely important to distinguish follicular lymphoma from reactive follicular hyperplasia and immunophenotyping is the best method to exclude this diagnosis (West, et al 2002). It can be difficult in some cases to distinguish follicular from mantle cell lymphoma, small lymphocytic lymphoma/cll with prominent pseudofollicles/proliferation centres, marginal zone B-cell lymphomas, nodular lymphocyte predominant Hodgkin lymphoma and MALT lymphoma at extranodal sites. It is therefore important to assess using a combination of morphology and immunophenotype plus, where necessary, genetic markers Recommendations Diagnostic criteria Immunophenotype - CD5 negative, CD19/CD20 positive, CD22 positive, CD23 negative, CD10 positive, cyclin D1 negative, BCL2 positive, BCL6 positive FISH/PCR for t(14;18) only required if atypical immunophenotype, Standard cytogenetics are not required. Level of evidence Grade C Level IV 9.12 References Armitage, J.O. & Weisenburger, D.D. (1998) New approach to classifying non- Hodgkin's lymphomas: clinical features of the major histologic subtypes.

56 Non-Hodgkin's Lymphoma Classification Project. J Clin Oncol, 16, Ott, G., Katzenberger, T., Lohr, A., Kindelberger, S., Rudiger, T., Wilhelm, M., Kalla, J., Rosenwald, A., Muller, J.G., Ott, M.M.& Muller-Hermelink, H.K. (2002) Cytomorphologic, immunohistochemical, and cytogenetic profiles of follicular lymphoma: 2 types of follicular lymphoma grade 3. Blood, 99, Sanchez, M.l., Almeida, J., Vidriales, B., Lopez - Berges, M.C., Garcia- Marcos, M.A., Moro, M.J., Corrales, A., Calmuntia, M.j., San Miguel, J.F. & Orfao, A. (2002) Incidence of phenotypic aberrations in a series of 467 patients with B chronic lymphoproliferative disorders: a basis for the design of specific four-colour stainings to be used for minimal residual disease investigation. Leukemia, 16, Schraders, M., de Jong, D., Kluin, P., Groenen, P. & van Krieken, H. (2005) Lack of Bcl-2 expression in follicular lymphoma may be caused by mutations in the BCL2 gene or by absence of the t(14;18) translocation. The Journal of Pathology, 205, Solal-Celigny, P., Roy, P., Colombat, P., White, J., Armitage, J.O., Arranz-Saez, R., Au, W.Y., Bellei, M., Brice, P., Caballero, D., Coiffier, B., Conde- Garcia, E., Doyen, C., Federico, M., Fisher, R.I., Garcia-Conde, J.F., Guglielmi, C., Hagenbeek, A., Haioun, C., LeBlanc, M., Lister, A.T., Lopez-Guillermo, A., McLaughlin, P., Milpied, N., Morel, P., Mounier, N., Proctor, S.J., Rohatiner, A., Smith, P., Soubeyran, P., Tilly, H., Vitolo, U., Zinzani, P.-L., Zucca, E. & Montserrat, E. (2004) Follicular Lymphoma International Prognostic Index. Blood, 104, Weisenburger, D., Gascoyne, R., Bierman, P., Shenkier, T., Horsman, D., Lynch, J. & al., e. (2000) Clinical significance of the t(14;18) and BCL2 overexpression in follicular large cell lymphoma. Leuk Lymphoma, 36, West, R., Warnke, R. & Natkunam, Y. (2002) The usefulness of immunohistochemistry in the diagnosis of follicular lymphoma in bone marrow biopsy specimens. Am J Clin Pathol., 117,

57 10 Mantle cell lymphoma 10.1 Definition and clinical features This lymphoma is derived from the naïve pre-germinal centre B-cells of the follicle mantle. It occurs more commonly in men, and tends to present with extensive generalized lymphadenopathy, often with liver, spleen and bone marrow involvement (Bosch, et al 1998). Extranodal involvement is not uncommon, particularly of the gastrointestinal tract and Waldeyer s ring. Mantle cell lymphoma tends to have a poor prognosis apart from in patients with limited disease i.e. stage I and IIa disease. (Leitch, et al 2003) 10.2 Peripheral blood There can be cytopenias due to marrow infiltration and there may be circulating lymphoma cells visible, with abnormal cells found in approximately 20-40% of patients (Argatoff, et al 1997, Cohen, et al 1998) but, despite the presence of these cells, the total white count is usually normal.(orchard, et al 2003, Pittaluga, et al 1996) However, the presentation can be with a frank leukaemic phase (Matutes, et al 2004) The abnormal cells are pleomorphic with variable size and amount of cytoplasm. The nucleus can be cleft or indented, nucleoli are common and there is lack of chromatin clumping (Wong, et al 1999) Bone marrow The bone marrow aspirate will often show evidence of marrow involvement with an abnormal population that has features similar to those seen in the peripheral blood. (Cohen, et al 1998) Trephine biopsy in the vast majority of patients, bone marrow involvement is evident in trephine biopsy sections (Argatoff, et al 1997),(Cohen, et al 1998). The infiltrate is of variable pattern, is composed of small to medium sized lymphoid cells with variable nuclear shape, often indented or cleft, and is associated with an increase in reticulin. (Wasman, et al 1996). Intrasinusiodal infiltration may be prominent Lymph node

58 Involved lymph nodes have a monomorphic infiltrate of small to intermediate sized lymphoid cells with (variably) irregular nuclear contours. The nuclear chromatin is dispersed and there are small indistinct eosinophilic nucleoli. There should be no proliferation centres or transformed cells (centroblastic or immunoblastic cells) seen. The infiltrate has a variable appearance either a mantle zone, nodular (obvious or vague) or diffuse growth pattern containing hyalinised small vessels and scattered epithelioid cells. There are blastoid variants of mantle cell lymphoma which are subdivided into classic (resembling lymphoblasts with dispersed chromatin and high mitotic rate) or pleomorphic (cells with large cleaved nuclei and prominent nucleoli) Immunophenotype Table 10.1 Recommended Panel for diagnosis of Mantle Cell Lymphoma Immunofluorescence Immunohistochemistry SmIg light chain κ, λ Moderate to strong Rarely demonstrable (light chain restriction) SmIg heavy chain N/A Usually IgM. Co-expression of IgD in 60% CD19/CD20 Strong Positive CD79a/ CD79b Strong Positive CD22 Strong N/A CD23 FMC7 N/A CD10 Usually CD5 Positive Positive BCL2 N/A Positive BCL6 N/A Ki67 N/A Low to moderate (except blatoid variants) MUM1/IRF4 N/A CD21 N/A Highlights residual FDC

59 meshworks Cyclin D1 N/A Positive (often heterogeneous) 10.6 Cytogenetics and molecular genetics FISH is the best technique to detect t(11;14)(q13;q32) in the majority of cases. Variants do occur, but are very rare, so it may be better to use a CCND1 breakapart probe than specific CCND1/IGH probes. However, the best method to aid diagnosis is to look for over-expression of cyclin D1 mrna as this is diagnostic of mantle cell lymphoma. Blastoid variants of mantle cell lymphoma may have additional mutations. (Bea, et al 1999) 10.7 Prognostic and therapeutic markers Indicators of poorer prognosis include peripheral blood involvement (Argatoff, et al 1997, Samaha, et al 1998) and a high mitotic rate.(argatoff, et al 1997, Bosch, et al 1998) The anti-cd20 antibody, rituximab, is currently undergoing clinical trials in this setting and has been shown to increase the incidence of molecular remission although this has not so far led to an increase in disease-free survival.(howard, et al 2002) Therefore an assessment of CD20 expression should be made Pitfalls There are very small numbers of cases (up to 5%) which are CD5 negative (Argatoff, et al 1997, Matutes, et al 2004), a few cases are weakly CD10 positive (Argatoff, et al 1997) and a few cases are CD23 positive (up to 10%) (Dorfman and Pinkus 1994, Gong, et al 2001, Matutes, et al 2004, Yin, et al 2002). In addition, very occasional cases have been found to be cyclin D1 negative (Yatabe, et al 2000),(Fu, et al 2005) and it should be remembered that Cyclin D1 staining is not restricted to mantle cell lymphoma. (Bosch, et al 1994, Ives Aguilera, et al 1998) Morphologically the small cell variant may resemble small lymphocytic lymphoma/b-cll.

60 10.9 Differential diagnosis It is important to distinguish mantle cell from reactive lymphadenopathy with broad mantle zones in reactive node. The following can appear similar morphologically but can be distinguished using the appropriate immunophenotyping panel and molecular techniques when needed; Follicular lymphoma CLL Marginal zone B-cell lymphomas (of splenic, nodal and MALT types) Nodular lymphocyte predominant Hodgkin lymphoma Large cell lymphomas in blastoid variants Lymphoblastic leukaemia/lymphoma in the blastoid variants B-PLL Recommendations Diagnostic criteria Immunophenotype - CD5 positive, CD19/CD20 positive, CD22 positive, CD23 negative, CD10 negative, cyclin D1 positive, BCL2 positive, BCL6 negative Over-expression of cyclin D1 mrna or positive by FISH for t(11;14)(q13;q32) Level of evidence Grade C Level IV References Argatoff, L.H., Connors, J.M., Klasa, R.J., Horsman, D.E. & Gascoyne, R.D. (1997) Mantle Cell Lymphoma: A Clinicopathologic Study of 80 Cases. Blood, 89, Bea, S., Ribas, M., Hernandez, J.M., Bosch, F., Pinyol, M., Hernandez, L., Garcia, J.L., Flores, T., Gonzalez, M., Lopez-Guillermo, A., Piris, M.A., Cardesa, A., Montserrat, E., Miro, R. & Campo, E. (1999) Increased Number of Chromosomal Imbalances and High-Level DNA Amplifications

61 in Mantle Cell Lymphoma Are Associated With Blastoid Variants. Blood, 93, Bosch, F., Jares, P., Campo, E., Lopez-Guillermo, A., Piris, M.A., Villamor, N., Tassies, D., Jaffe, E.S., Montserrat, E. & Rozman, C. (1994) PRAD- 1/cyclin D1 gene overexpression in chronic lymphoproliferative disorders: a highly specific marker of mantle cell lymphoma. Blood, 84, Bosch, F., Lopez-Guillermo, A., Campo, E., Ribera, J., Conde, E., Piris, M., Vallespi, T., Woessner, S. & Montserrat, E. (1998) Mantle cell lymphoma: presenting features, response to therapy, and prognostic factors. Cancer, 82, Cohen, Kurtin, Donovan & Hanson (1998) Bone marrow and peripheral blood involvement in mantle cell lymphoma. British Journal of Haematology, 101, Dorfman, D. & Pinkus, G. (1994) Distinction between small lymphocytic and mantle cell lymphoma by immunoreactivity for CD23. Mod Pathol., 7, Fu, K., Weisenburger, D.D., Greiner, T.C., Dave, S., Wright, G., Rosenwald, A., Chiorazzi, M., Iqbal, J., Gesk, S., Siebert, R., De Jong, D., Jaffe, E.S., Wilson, W.H., Delabie, J., Ott, G., Dave, B.J., Sanger, W.G., Smith, L.M., Rimsza, L., Braziel, R.M., Muller-Hermelink, H.K., Campo, E., Gascoyne, R.D., Staudt, L.M., Chan, W.C. & for the Lymphoma/Leukemia Molecular Profiling, P. (2005) Cyclin D1-negative mantle cell lymphoma: a clinicopathologic study based on gene expression profiling. Blood, 106, Gong, J., Lagoo, A., Peters, D., Horvatinovich, J., Benz, P. & Buckley, P. (2001) Value of CD23 determination by flow cytometry in differentiating mantle cell lymphoma from chronic lymphocytic leukemia/small lymphocytic lymphoma. Am J Clinical Pathology, 116, Howard, O.M., Gribben, J.G., Neuberg, D.S., Grossbard, M., Poor, C., Janicek, M.J. & Shipp, M.A. (2002) Rituximab and CHOP Induction Therapy for Newly Diagnosed Mantle-Cell Lymphoma: Molecular Complete Responses Are Not Predictive of Progression-Free Survival. J Clin Oncol, 20, Ives Aguilera, N.S., Bijwaard, K.E., Duncan, B., Krafft, A.E., Chu, W.-S., Abbondanzo, S.L., Lichy, J.H. & Taubenberger, J.K. (1998) Differential Expression of Cyclin D1 in Mantle Cell Lymphoma and Other Non- Hodgkin's Lymphomas. Am J Pathol, 153, Leitch, H.A., Gascoyne, R.D., Chhanabhai, M., Voss, N.J., Klasa, R. & Connors, J.M. (2003) Limited-stage mantle-cell lymphoma. Ann Oncol, 14, Matutes, E., Parry-Jones, N., Brito-Babapulle, V., Wotherspoon, A., Morilla, R., Atkinson, S., Elnenaei, M.O., Jain, P., Giustolisi, G.M., A'Hern, R.P. &

62 Catovsky, D. (2004) The leukemic presentation of mantle-cell lymphoma: disease features and prognostic factors in 58 patients. Leuk Lymphoma, 45, Orchard, J., Garand, R., Davis, Z., Babbage, G., Sahota, S., Matutes, E., Catovsky, D., Thomas, P.W., Avet-Loiseau, H. & Oscier, D. (2003) A subset of t(11;14) lymphoma with mantle cell features displays mutated IgVH genes and includes patients with good prognosis, nonnodal disease. Blood, 101, Pittaluga, S., Verhoef, G., Criel, A., Maes, A., Nuyts, J., Boogaerts, M. & De Wolf Peeters, C. (1996) Prognostic significance of bone marrow trephine and peripheral blood smears in 55 patients with mantle cell lymphoma. Leuk Lymphoma., 21, Samaha, H., Dumontet, C., Ketterer, N., Moullet, I., Thieblemont, C., Bouafia, F., Callet-Bauchu, E., Felman, P., Berger, F., Salles, G. & Coiffier, B. (1998) Mantle cell lymphoma: a retrospective study of 121 cases. Leukemia, 12, Wasman, J., Rosenthal, N. & Farhi, D. (1996) Mantle cell lymphoma. Morphologic findings in bone marrow involvement. Am J Clin Pathol., 106, Wong, K.-F., Chan, J.K.C., So, J.C.C. & Yu, P.-H. (1999) Mantle cell lymphoma in leukemic phase. Cancer, 86, Yatabe, Y., Suzuki, R., Tobinai, K., Matsuno, Y., Ichinohasama, R., Okamoto, M., Yamaguchi, M., Tamaru, J.-i., Uike, N., Hashimoto, Y., Morishima, Y., Suchi, T., Seto, M. & Nakamura, S. (2000) Significance of cyclin D1 overexpression for the diagnosis of mantle cell lymphoma: a clinicopathologic comparison of cyclin D1-positive MCL and cyclin D1- negative MCL-like B-cell lymphoma. Blood, 95, Yin, X., Robert, W.M., Sheryl, L.A. & Steven, H.K. (2002) Comparison of Immunophenotypes of Small B-Cell Neoplasms in Primary Lymph Node and Concurrent Blood or Marrow Samples. American Journal of Clinical Pathology, 118,

63 11 Diffuse large B-cell lymphomas 11.1 Definition and clinical features Diffuse large B-cell lymphomas (DLBCL) are a group of B-cell malignancies that are linked by aggressive clinical behaviour and the potential to be cured by anthracycline-containing combination chemotherapy. Within this category there is considerable heterogeneity of cellular features, site of origin and clinical outcome. DLBCL present as a rapidly growing mass in any lymph node group or nonlymphoid organ. The clinical stage, bulk of disease and extent of systemic symptoms vary between patients and these parameters form the basis of the International Prognostic Index (IPI), currently the most widely used tool for predicting clinical outcome. In most cases the tumour will respond rapidly to CHOP-R (cyclophosphamide, doxorubicin, vincristine, prednisolone, rituximab) type chemotherapy and around 50% of patients will be cured by primary therapy. (Coiffier, et al 2002) However, there are a number of poor prognosis variants, associated with progressive or refractory disease. These collectively account for about 15-20% of cases. These are detailed below Peripheral blood A small minority of patients present with an overt lymphocytosis and an additional small group have small numbers of circulating tumour cells in the presence of a normal lymphocyte count. Diagnosis is based on flow cytometry using the same criteria as for other organ sites. The diagnosis should always be confirmed by examination of a tissue specimen Bone marrow Less than 5% of patients with DLBCL present with primary bone marrow disease and a further 5-10% have marrow involvement diagnosed during staging investigations. In all cases a marrow aspirate for flow cytometry and a trephine biopsy are required. Morphological examination of an aspirate specimen alone is not

64 adequate for staging patients with DLBCL and an adequate trephine biopsy is required in all cases. Involvement of the bone marrow is not predictable from the extent of nodal disease, and the presence/absence of marrow disease is essential for the determination of stage and the IPI. In addition a significant minority of patients with DLBCL will have evidence of an underlying indolent lymphoma in the bone marrow. The pattern of marrow involvement varies from isolated nodules of tumour to diffuse marrow replacement, which may be associated with fibrosis. In most cases it is not possible to show clonal identity with the primary tumour and this finding should not be taken as direct evidence of transformed disease. Staging bone marrow biopsy specimens should be reported as: Normal Involved by DLBCL Involved by indolent lymphoma which has clonal or phenotypic identity with the presenting DLBCL. If identical re-classify primary tumour as transformed. Involved by CLL or indolent lymphoma of non-identical phenotype; report as of uncertain significance 11.4 Lymph node or extranodal sites The key morphological features of DLBCL are that there are partial or complete effacement of nodal architecture or infiltration of normal tissues at an extranodal site by a relatively monomorphic population of large B-lymphoid cells. The cytomorphology of DLBCL is highly variable and does not have proven clinical value as a means of sub-classification. Sub-division and prognostic assessment is based on the immunophenotype and chromosomal abnormalities in conjunction with the IPI. DLBCL may arise by transformation of an underlying indolent lymphoma, most commonly follicular lymphoma. The immunophenotype of the DLBCL and the underlying indolent component is usually the same. In some cases the indolent

65 lymphoma may not have been clinically apparent prior to the diagnosis of the DLBCL Immunophenotype Table 11.1 Recommended Panel for diagnosis of DLBCL Immunofluorescence Immunohistochemistry SmIg light chain κ or λ Strong N/A SmIg heavy chain D, N/A M > G (germinal centre type) M, G CD19/CD20 Strong Positive CD79a/CD79b Strong Positive CD22 Strong Positive CD23 FMC7 Strong N/A CD10 Dependent on Dependent on subtype subtype CD5 BCL2 N/A Variable BCL6 N/A Dependent on subtype MUM-1/IRF4 N/A Dependent on subtype Ki67 N/A >30% p53 N/A Variably positive CD45 N/A Dependent on subtype Cyclin D1 N/A PAX5 N/A Positive IRF4 N/A FOXP1 Depends of subtype GCET Depends on subtype CD138 Positive N/A (plasmablastic variant) CD56 Positive plasmablastic Positive (plasmablastic variant) variant) Cytoplasmic κ or λ Positive Positive (plasmablastic variant) (plasmablastic variant) p63 (antibody VS38c) N/A Positive (plasmablastic variant) 11.6 Subtypes The sub-classification of DLBCL into germinal centre and post germinal centre type is based on the results of gene expression microarray studies (Alizadeh, et al 2000, Hans, et al 2004, Rosenwald, et al 2003).The categories described in these studies can be readily replicated using flow cytometry and immunohistochemistry (Barrans, et al 2002, Hans, et al 2004).. However, in R-

66 CHOP treated patients there is little evidence that classification by immunocytochemistry is prognosticallly relevant. A revision of the criteria has been proposed that incorporate GCET and FOXP1 and several gene expression algorithms are available for use in CHOP-R treated patients. (Choi, et al 2009, Jais, et al 2008, Malumbres, et al 2008) Mediastinal large B-cell lymphoma This normally presents as an isolated mediastinal mass often with signs of superior vena cava obstruction. However, there can be other sites of disease, which are normally extra-nodal. The immunophenotype is post germinal centre with CD30 positive, MUM1/IRF4 positive and NFkB p65 expression. CD10 and CD5 are negative (Calvo, et al 2004, Feuerhake, et al 2005, Savage, et al 2003) Plasmablastic lymphoma This is most commonly found in the mouth and is associated with HIV infection (Colomo, et al 2004). The plasma cell morphology may not be readily apparent on microscopy. Non HIV patients with plasmablastic lymphoma may present with nodal, soft tissue or marrow disease. In some cases the distinction from myeloma may be problematic. The immunophenotype can be difficult due to down-regulation of the B antigens with weak/ absent CD19 and CD20, PAX5 negative, CD138 positive, CD79 variable, MUM1/IRF4 positive, CD56 positive (most), CD45 negative (usually) with a few cases expressing ALK1. (Onciu, et al 2003, Vega, et al 2005) T-cell/histiocyte-rich large B-cell lymphoma This is clinically distinct entity typically characterised by spleen and bone marrow disease. The diagnosis is based on a scattered population of atypical large B- cells in a background containing a mixed population of reactive T-cells and histiocytes. The B-cell immunophenotype is variable. The major differential diagnosis includes nodular lymphocyte predominant Hodgkin lymphoma, particularly where there are large numbers of neoplastic cells present. This appears to an aggressive variant of DLBCL. (Tsirigotis, et al 2001) ALK -positive DLBCL

67 This is a rare variant of DLBCL characterised by an ALK fusion protein and a plasma cell immunophenotype often with IgA expression, loss of CD45, CD20 and CD79a which can lead to difficulty in differential diagnosis with T-cell lymphomas and some non-haematological tumours. This is a poor prognostic group.(laurent, et al 2009) Intravascular Large B-cell lymphoma In this lymphoma, the neoplastic B-cell population is predominately confined to the lumen of blood vessels and is found predominantly in extranodal sites with absence of a mass lesion. The cells are CD19/20 positive, CD22 positive, CD79a/ CD79b positive and PAX5 positive and can have either germinal centre or post-germinal centre phenotype Primary effusion lymphoma (PEL) This rare form of DLBCL is seen in the setting of HIV infection (Carbone & Gloghini, 2005). The lymphoma typically presents as a pleural, pericardial or peritoneal effusion (Das, 2006) without an accompanying tumour mass. However extracavitary examples are recorded in which the lymphoma does form a solid mass, usually in soft tissue or the gastro-intestinal tract (Teruya-Feldstein, 2005). The tumour cells may have anaplastic, immunoblastic or plasmablastic features, and can show loss of CD20 expression with positive staining for CD138 or VS38c. CD45 is positive in the majority of cases. Cytoplasmic immunoglobulin may be detected, and the tumour cells often show CD30 and EMA positivity. The neoplastic cells of PEL invariably show infection with HHV8/KSHV, and EBV is also present in the majority of cases (Colomo, et al 2004, Hamoudi, 2004, Schulz, 1998, Schulz, 2006). Primary effusion lymphoma is typically highly aggressive and is commonly refractory to therapy (Jones, et al 1998). Where there is no tumour mass the diagnosis relies on cytological assessment of the cells, which should include HHV8 and EBV staining. Solid variants have been described and occur occasionally without any evidence of underlying immunocompromise Other rare/ provisional entities DLBCL of the leg

68 EBV associated DLBCL in the elderly (Park, et al 2007) Large B-cell lymphoma arising in HHV8-associated Castleman disease B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Hodgkin lymphoma. This is an important problem in differential diagnosis. The exact criteria are not fully established but typically these cases are CD30 postive with morphological features of HL but a complete B-cell phenotype Cytogenetics and molecular genetics No defining cytogenetic abnormalities are required for the diagnosis of DLBCL. The routine use of metaphase cytogenetics is not warranted in the diagnosis and staging of DLBCL. Cases with germinal centre phenotype (BCL2 negative), approaching 100% Ki67 and deregulation of p53 (p53+p21-) need to be investigated for MYC rearrangements to exclude Burkitt lymphoma.(mead, et al 2008) MYC rearrangement in the context of other abnormalities is indicative of transformed /highly aggressive disease. (Savage, et al 2009) CCND1/IgH FISH may be helpful in proving mantle cell lymphoma origin and should be undertaken in all CD5+ DLBCL in conjunction with Cyclin D1 expression Adverse prognostic features. The prognosis of DLBCL is assessed using the international prognostic index. A modification for CHOP-R treated patients has been proposed.(sehn, et al 2007) Most biological factors have lost significance in the era of CHOP-R including BCL2 expression. However, rearrangements of MYC particularly in association with t(14;18) remains a strong prognostic factor. (Johnson, et al 2009, Savage, et al 2009, Tomita, et al 2009). In additional EBV associated and ALK rearranged tumours appear to have a limited response to CHOP-R Pitfalls

69 Around 10% of DLBCL express CD5; the significance of this finding is uncertain except in so far as it may raise a suspicion of underlying CLL or mantle cell lymphoma. The possibility that these may represent transformed CLL or mantle cell lymphoma should be assessed by examination of the bone marrow and further immunophenotypic or cytogenetic studies Plasmablastic lymphoma may not be readily identified with down-regulation of the B-cell phenotype leading to under-recognition of this entity and there is a potential for misdiagnosis of a non-haematological malignancy. Mediastinal B-cell lymphoma and classical Hodgkin lymphoma can have similar appearances, but the CD30 expression is weak and the cells are also CD45 positive unlike those of classical Hodgkin lymphoma Differential Diagnosis Non-haematological malignancies Acute leukaemia Burkitt lymphoma Peripheral T-cell lymphomas Plasmacytomas/myeloma Hodgkin lymphoma classical and nodular lymphocyte predominant types EBV-induced proliferations in the context of primary infection, immunodeficiency or both Recommendations Recommendations for the diagnosis of DLBCL, its subtypes and other large B cell lymphomas Diagnostic criteria Level of evidence Basic Immunophenotype - a peripheral B-cell Grade C Level IV phenotype using CD19, CD20, CD79a or PAX-5 immunocytochemistry or CD19, CD20 and surface immunoglobulin/cd79b by flow cytometry.

70 Mediastinal large B-cell lymphoma activated B-cell phenotype - CD30 positive, MUM1/IRF4 positive and NFkB p65 expression Ki67 fraction should exceed 30%. Plasmablastic lymphoma phenotype - weak/absent CD19 and CD20, PAX5 negative, CD138 positive, CD79 variable, MUM1/IRF4 positive, CD56 positive (most), CD45 negative (usually) with a few cases expressing ALK1 Classical cytogenetics are NOT required Grade C Level IV Grade C Level IV Grade C Level IV Grade C Level IV References Alizadeh, A.A., Eisen, M.B., Davis, R.E., Ma, C., Lossos, I.S., Rosenwald, A., Boldrick, J.C., Sabet, H., Tran, T., Yu, X., Powell, J.I., Yang, L., Marti, G.E., Moore, T., Hudson, J., Lu, L., Lewis, D.B., Tibshirani, R., Sherlock, G., Chan, W.C., Greiner, T.C., Weisenburger, D.D., Armitage, J.O., Warnke, R., Levy, R., Wilson, W., Grever, M.R., Byrd, J.C., Botstein, D., Brown, P.O. & Staudt, L.M. (2000) Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature, 403, Barrans, S.L., Carter, I., Owen, R.G., Davies, F.E., Patmore, R.D., Haynes, A.P., Morgan, G.J. & Jack, A.S. (2002) Germinal center phenotype and bcl-2 expression combined with the International Prognostic Index improves patient risk stratification in diffuse large B-cell lymphoma. Blood, 99, Carbone, A., Gloghini, A. (2005) AIDS-related lymphomas: from pathogenesis to pathology. Br J Haematol, 130, Calvo, K.R., Traverse-Glehen, A., Pittaluga, S. & Jaffe, E.S. (2004) Molecular profiling provides evidence of primary mediastinal large B-cell lymphoma as a distinct entity related to classic Hodgkin lymphoma: implications for mediastinal gray zone lymphomas as an intermediate form of B-cell lymphoma. Adv Anat Pathol, 11, Choi, W.W., Weisenburger, D.D., Greiner, T.C., Piris, M.A., Banham, A.H., Delabie, J., Braziel, R.M., Geng, H., Iqbal, J., Lenz, G., Vose, J.M., Hans, C.P., Fu, K., Smith, L.M., Li, M., Liu, Z., Gascoyne, R.D., Rosenwald, A., Ott, G., Rimsza, L.M., Campo, E., Jaffe, E.S., Jaye, D.L., Staudt, L.M. & Chan, W.C. (2009) A new immunostain algorithm classifies diffuse large B-cell lymphoma into molecular subtypes with high accuracy. Clin Cancer Res, 15,

71 Coiffier, B., Lepage, E., Briere, J., Herbrecht, R., Tilly, H., Bouabdallah, R., Morel, P., Van Den Neste, E., Salles, G., Gaulard, P., Reyes, F., Lederlin, P. & Gisselbrecht, C. (2002) CHOP Chemotherapy plus Rituximab Compared with CHOP Alone in Elderly Patients with Diffuse Large-B-Cell Lymphoma. N Engl J Med, 346, Colomo, L., Loong, F., Rives, S., Pittaluga, S., Martinez, A., Lopez-Guillermo, A., Ojanguren, J., Romagosa, V., Jaffe, E.S. & Campo, E. (2004) Diffuse large B-cell lymphomas with plasmablastic differentiation represent a heterogeneous group of disease entities. Am J Surg Pathol, 28, Das, D.K. (2006) Serous effusions in malignant lymphomas: a review. Diagn Cytopathol, 34, Feuerhake, F., Kutok, J.L., Monti, S., Chen, W., LaCasce, A.S., Cattoretti, G., Kurtin, P., Pinkus, G.S., de Leval, L., Harris, N.L., Savage, K.J., Neuberg, D., Habermann, T.M., Dalla-Favera, R., Golub, T.R., Aster, J.C. & Shipp, M.A. (2005) NF(Kretz-Rommel, et al)b activity, function, and target-gene signatures in primary mediastinal large B-cell lymphoma and diffuse large B-cell lymphoma subtypes. Blood, 106, Go, J. & Yang, W. (2001) Expressions of p53 and p21 in primary gastric lymphomas. J Korean Med Sci., 16, Hamoudi, R., Diss, T.C., Oksenhendler, E., Pan, L., Carbone, A., Ascoli, V., Boshoff, C., Isaacson, P., Du, M.Q. (2004) Distinct cellular origins of primary effusion lymphoma with and without EBV infection. Leuk Res,28, Hans, C.P., Weisenburger, D.D., Greiner, T.C., Gascoyne, R.D., Delabie, J., Ott, G., Muller-Hermelink, H.K., Campo, E., Braziel, R.M., Jaffe, E.S., Pan, Z., Farinha, P., Smith, L.M., Falini, B., Banham, A.H., Rosenwald, A., Staudt, L.M., Connors, J.M., Armitage, J.O. & Chan, W.C. (2004) Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood, 103, Hermine, O., Haioun, C., Lepage, E., d'agay, M.F., Briere, J., Lavignac, C., Fillet, G., Salles, G., Marolleau, J.P., Diebold, J., Reyas, F. & Gaulard, P. (1996) Prognostic significance of bcl-2 protein expression in aggressive non- Hodgkin's lymphoma. Groupe d'etude des Lymphomes de l'adulte (GELA). Blood, 87, Hill, M.E., MacLennan, K.A., Cunningham, D.C., Vaughan Hudson, B., Burke, M., Clarke, P., Di Stefano, F., Anderson, L., Vaughan Hudson, G., Mason, D., Selby, P. & Linch, D.C. (1996) Prognostic significance of BCL-2 expression and bcl-2 major breakpoint region rearrangement in diffuse large cell non-hodgkin's lymphoma: a British National Lymphoma Investigation Study. Blood, 88, Ichikawa, A., Kinoshita, T., Watanabe, T., Kato, H., Nagai, H., Tsushita, K., Saito, H. & Hotta, T. (1997) Mutations of the p53 Gene as a Prognostic Factor in Aggressive B-Cell Lymphoma. N Engl J Med, 337,

72 Jais, J.P., Haioun, C., Molina, T.J., Rickman, D.S., de Reynies, A., Berger, F., Gisselbrecht, C., Briere, J., Reyes, F., Gaulard, P., Feugier, P., Labouyrie, E., Tilly, H., Bastard, C., Coiffier, B., Salles, G. & Leroy, K. (2008) The expression of 16 genes related to the cell of origin and immune response predicts survival in elderly patients with diffuse large B-cell lymphoma treated with CHOP and rituximab. Leukemia, 22, Johnson, N.A., Savage, K.J., Ludkovski, O., Ben-Neriah, S., Woods, R., Steidl, C., Dyer, M.J.S., Siebert, R., Kuruvilla, J., Klasa, R., Connors, J.M., Gascoyne, R.D. & Horsman, D.E. (2009) Lymphomas with concurrent BCL2 and MYC translocations: the critical factors associated with survival. Blood, blood Jones, D., Ballestas, M.E., Kaye, K.M., Gulizia, J.M., Winters, G.L., Fletcher, J., Scadden, D.T., Aster, J.C. (1998) Primary-effusion lymphoma and Kaposi's sarcoma in a cardiac-transplant recipient. N Engl J Med, 339, Kretz-Rommel, A., Qin, F., Dakappagari, N., Torensma, R., Faas, S., Wu, D. & Bowdish, K.S. (2007) In vivo targeting of antigens to human dendritic cells through DC-SIGN elicits stimulatory immune responses and inhibits tumor growth in grafted mouse models. J Immunother (1997), 30, Laurent, C., Do, C., Gascoyne, R.D., Lamant, L., Ysebaert, L., Laurent, G., Delsol, G. & Brousset, P. (2009) Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma: a rare clinicopathologic entity with poor prognosis. J Clin Oncol, 27, Malumbres, R., Chen, J., Tibshirani, R., Johnson, N.A., Sehn, L.H., Natkunam, Y., Briones, J., Advani, R., Connors, J.M., Byrne, G.E., Levy, R., Gascoyne, R.D. & Lossos, I.S. (2008) Paraffin-based 6-gene model predicts outcome in diffuse large B-cell lymphoma patients treated with R- CHOP. Blood, 111, Mead, G.M., Barrans, S.L., Qian, W., Walewski, J., Radford, J.A., Wolf, M., Clawson, S.M., Stenning, S.P., Yule, C.L. & Jack, A.S. (2008) A prospective clinicopathologic study of dose-modified CODOX-M/IVAC in patients with sporadic Burkitt lymphoma defined using cytogenetic and immunophenotypic criteria (MRC/NCRI LY10 trial). Blood, 112, Monti, S., Savage, K.J., Kutok, J.L., Feuerhake, F., Kurtin, P., Mihm, M., Wu, B., Pasqualucci, L., Neuberg, D., Aguiar, R.C.T., Cin, P.D., Ladd, C., Pinkus, G.S., Salles, G., Harris, N.L., Dalla-Favera, R., Habermann, T.M., Aster, J.C., Golub, T.R. & Shipp, M.A. (2005) Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. Blood, 105, Onciu, M., Behm, F.G., Downing, J.R., Shurtleff, S.A., Raimondi, S.C., Ma, Z., Morris, S.W., Kennedy, W., Jones, S.C. & Sandlund, J.T. (2003) ALK-

73 positive plasmablastic B-cell lymphoma with expression of the NPM-ALK fusion transcript: report of 2 cases. Blood, 102, Park, S., Lee, J., Ko, Y.H., Han, A., Jun, H.J., Lee, S.C., Hwang, I.G., Park, Y.H., Ahn, J.S., Jung, C.W., Kim, K., Ahn, Y.C., Kang, W.K., Park, K. & Kim, W.S. (2007) The impact of Epstein-Barr virus status on clinical outcome in diffuse large B-cell lymphoma. Blood, 110, Rosenwald, A., Wright, G., Leroy, K., Yu, X., Gaulard, P., Gascoyne, R.D., Chan, W.C., Zhao, T., Haioun, C., Greiner, T.C., Weisenburger, D.D., Lynch, J.C., Vose, J., Armitage, J.O., Smeland, E.B., Kvaloy, S., Holte, H., Delabie, J., Campo, E., Montserrat, E., Lopez-Guillermo, A., Ott, G., Muller-Hermelink, H.K., Connors, J.M., Braziel, R., Grogan, T.M., Fisher, R.I., Miller, T.P., LeBlanc, M., Chiorazzi, M., Zhao, H., Yang, L., Powell, J., Wilson, W.H., Jaffe, E.S., Simon, R., Klausner, R.D. & Staudt, L.M. (2003) Molecular Diagnosis of Primary Mediastinal B Cell Lymphoma Identifies a Clinically Favorable Subgroup of Diffuse Large B Cell Lymphoma Related to Hodgkin Lymphoma. J. Exp. Med., 198, Savage, K.J., Monti, S., Kutok, J.L., Cattoretti, G., Neuberg, D., de Leval, L., Kurtin, P., Cin, P.D., Ladd, C., Feuerhake, F., Aguiar, R.C.T., Li, S., Salles, G., Berger, F., Jing, W., Pinkus, G.S., Habermann, T., Dalla- Favera, R., Harris, N.L., Aster, J.C., Golub, T.R. & Shipp, M.A. (2003) The molecular signature of mediastinal large B-cell lymphoma differs from that of other diffuse large B-cell lymphomas and shares features with classical Hodgkin lymphoma. Blood, 102, Savage, K.J., Johnson, N.A., Ben-Neriah, S., Connors, J.M., Sehn, L.H., Farinha, P., Horsman, D.E. & Gascoyne, R.D. (2009) MYC gene re-arrangements are associated with a poor prognosis in diffuse large B-cell lymphoma patients treated with R-CHOP chemotherapy. Blood. Sehn, L.H., Berry, B., Chhanabhai, M., Fitzgerald, C., Gill, K., Hoskins, P., Klasa, R., Savage, K.J., Shenkier, T., Sutherland, J., Gascoyne, R.D. & Connors, J.M. (2007) The revised International Prognostic Index (R-IPI) is a better predictor of outcome than the standard IPI for patients with diffuse large B- cell lymphoma treated with R-CHOP. Blood, 109, Schulz, T.F. (1998) Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8). J Gen Virol, 79, Schulz, T.F. (2006) The pleiotropic effects of Kaposi's sarcoma herpesvirus. J Pathol, 208, Tsirigotis, P., Economopoulos, T., Rontogianni, D., Dervenoulas, J., Papageorgiou, E., Bollas, G., Mantzios, G., Kalantzis, D., Koumarianou, A. & Raptis, S. (2001) T-cell-rich B-cell lymphoma. Analysis of clinical features, response to treatment, survival and comparison with diffuse large B-cell lymphoma. Oncology, 61, Teruya-Feldstein, J. (2005) Diffuse large B-cell lymphomas with plasmablastic differentiation. Curr Oncol Rep, 7,

74 Tomita, N., Tokunaka, M., Nakamura, N., Takeuchi, K., Koike, J., Motomura, S., Miyamoto, K., Kikuchi, A., Hyo, R., Yakushijin, Y., Masaki, Y., Fujii, S., Hayashi, T., Ishigatsubo, Y. & Miura, I. (2009) Clinicopathological features of lymphoma/leukemia patients carrying both BCL2 and MYC translocations. Haematologica, 94, Vega, F., Chang, C.C., Medeiros, L.J., Udden, M.M., Cho-Vega, J.H., Lau, C.C., Finch, C.J., Vilchez, R.A., McGregor, D. & Jorgensen, J.L. (2005) Plasmablastic lymphomas and plasmablastic plasma cell myelomas have nearly identical immunophenotypic profiles. Mod Pathol, 18, Villuendas, R., Pezzella, F., Gatter, K., Algara, P., Sanchez-Beato, M., MartÍnez, P., MartÍnez, J.C., Munoz, K., Garcia, P., Sanchez, L., Kocialkowsky, S., Campo, E., Orradre, J.L. & Piris, M.A. (1997) p21waf1/cip1 and MDM2 expression in non-hodgkin's lymphoma and their relationship to p53 status: a p53+, MDM2-, p21-immunophenotype associated with missense p53 mutations. The Journal of Pathology, 181,

75 12 Non-endemic Burkitt lymphoma 12.1 Disease definition & clinical features The concept of Burkitt lymphoma arose from the study of Epstein-Barr virus (EBV) and malaria associated tumours in African children and the recognition that morphologically similar tumours occurred in non-malaria areas and in HIVinfected individuals.(jaffe, et al 1999) The concept underlying the non-endemic, non-hiv associated tumours has changed in recent years to include a group of aggressive germinal centre derived B-cell malignancies which have an activating re-arrangement of MYC as a single abnormality. This group is characterised as being poorly responsive to CHOP, but potentially curable by more intensive chemotherapy, such as CODOX-M/IVAC. (Mead, et al 2002, Thomas, et al 1999, Wang, et al 2003, Mead, et al 2008)) Burkitt lymphoma occurs at a younger median age than diffuse large B-cell lymphoma (DLBCL) with a strong male predominace, and typically presents with a rapidly growing abdominal mass. There is a much higher incidence of central nervous system (CNS) disease and bone marrow involvement than in DLBCL. A small subset of patients has an acute leukaemic presentation. In the past these patients were classified as B-ALL or ALL L3. This is now obsolete and the term Burkitt lymphoma should be used for all patients Peripheral blood Patients with marrow involvement will frequently have involvement of the peripheral blood and in a small number of patients a significant lymphocytosis is the presenting feature. There may be cytopenias due to infiltration causing marrow failure. The circulating cells have the appearance of large lymphoblasts with basophilic cytoplasm, many containing small vacuoles Bone marrow Marrow involvement, when present, is usually an extensive diffuse infiltrate. Classical blast morphology is well described and the features seen in the trephine biopsy are similar to those in other tissue biopsies. Morphological

76 features are not reliable and should not be used to make a diagnosis without adequate immunophenotyping and cytogenetic or FISH analysis Lymph node or extranodal sites The majority of patients present with abdominal masses and it is often difficult to be certain whether the tumour is arising in nodes or at an extranodal site. The tumour consists of a monomorphic population of large lymphoid cells, which are highly proliferative and show evidence of apoptosis. Unlike many cases of DLBCL, a reactive T-cell population is usually not present but there is a signficant macrophage component giving rise to a starry sky appearance. However, morphological features are not a reliable guide to the diagnosis and are very susceptible to variations in fixation. The CNS is involved in around 20% of patients. The diagnosis of CNS involvement using a CSF specimen requires that, if the peripheral blood is involved, it is free of red cells (which are indicative of peripheral blood contamination of the specimen) and that a population of CD19+ clonal B-cells is detectable by flow cytometry Immunophenotype Burkitt lymphoma cells undergo apoptosis and fresh tissue or EDTAanticoagulated specimens may undergo rapid deterioration. Some caution in interpreting results may be required if the specimen is more than a few hours old when it reaches the laboratory. Table 12.1 Recommended Panel for diagnosis of Burkitt lymphoma Immunofluorescence Immunohistochemistry SmIg light chain κ or λ Strong N/A CD19/CD20 Strong Positive CD79a/CD79b Strong Positive CD22 Strong Positive CD23 FMC7 Strong N/A CD10 Positive or negative Positive CD5 BCL2 N/A BCL6 N/A Positive (Dave, et al 2005, Dogan, et al 2000a, Nakamura,

77 et al 2002) MUM1 N/A Ki67 N/A Close to 100% CD45 Strong Positive Cyclin D1 p53 N/A Positive (Klumb, et al 2001, Lindstrom and Wiman 2002, Wilda, et al 2004) p21 (WAF1) N/A TdT CD Cytogenetics & molecular genetics The presence of a MYC re-arrangement must be demonstrated in every case by metaphase cytogenetics or interphase FISH. However, neither MYC break-apart nor MYC/IGH probes are adequate to find all MYC rearrangements. It is suggested that MYC/IGH are carried out where MYC break-apart, BCL2 and BCL6 are normal. In all cases absence of BCL2 and BCL6 rearrangements must be established. (Hecht and Aster 2000, Zhang, et al 2007)) A distinctive gene expression profile is associated with Burkitt lymphoma that allows a clear distinction from other types of aggressive B-cell malignancy. This profile is found in highly proliferative germinal centre type tumours that have MYC rearrangement in absence of other chromosomal translocations or complex karyotypic abnormalities. Morphological correlation with this profile is poor. (Dave et al 2006, Hummel et al 2006) 12.7 Prognostic tests There are no known specific prognostic factors other than age and performance status Pitfalls Cell and tissue samples are extremely susceptible to in vitro changes giving rise to poor quality specimens and fixation, which can affect morphology, immunophenotyping and cytogenetics. This is particularly important when assessing Ki67 expression, which should be approaching 100% and uniformly

78 strongly expressed, but account needs to be taken of degenerated cells, which will appear negative. DLBCL can have MYC rearrangements and morphology is unreliable. It is important therefore to have the correct phenotype. (Nakamura, et al 2002) 12.9 Differential Diagnosis DLBCL with MYC rearrangement. This may occur in transformed follicular lymphoma or as a de novo tumour. These are included within the WHO category of B-cell lymphoma with features intermediate between DLBCL and Burkitt lymphoma. The presence of cytogenetic abnormalities other than MYC rearrangements is very helpful in these cases.. Other HIV-associated lymphomas Acute lymphoblastic leukaemia; this should not be a problem if immunophenotyping is carried out as above Recommendations Recommendations for the diagnosis of non-endemic Burkitt Lymphoma Diagnostic criteria Level of evidence Interphase FISH/metaphase cytogenetics MUST Grade C level IV demonstrate MYC rearrangement No detection of BCL2 or BCL6 rearrangements Immunophenotype ; CD34, TdT, CD21, BCL2 negative, CD19, CD10, BCL6, p53 positive, Ki-67 strongly positive References Dave, S.S., Fu, K., Wright, G., Lam, L., Greiner, T.C., Weisenberger, D.D., Kluin, P.M., Boerma, E., Rosenwald, A., Ott, G., Muller-Hermelink, H.K., Gascoyne, R.D., Delabie, J., Rimsza, L.M., Braziel, R.M., Grogan, T.M., Campo, E., Jaffe, E.S., Vose, J., Armitage, J.O., Connors, J.M., Smeland, E.B., Kvaloy, S., Holte, H., Miller, T.P., Fisher, R.I., Montserrat, E., Wilson, W.H., Bahl, M., Zhao, H., Yang, L., Powell, J., Simon, R., Chan, W.C.,

79 Staudt, L.M. & Leukemia Lymphoma Molecular Profiling, Project. (2006) Molecular Diagnosis of Burkitt s lymphoma. N Engl J Med, 354, Dogan, A., Bagdi, E., Munson, P. & Isaacson, P. (2000) CD10 and BCL-6 expression in paraffin sections of normal lymphoid tissue and B-cell lymphomas.. Am J Surg Pathol, 24, Hecht, J.L. & Aster, J.C. (2000) Molecular Biology of Burkitt's Lymphoma. J Clin Oncol, 18, Hummel, M., Bentink, S., Berger, H., Klapper, W., Wessendorf, S., Barth, T.F.E., Bernd, H.-W., Cogliatti, S.B., Dierlamm, J., Feller, A.C., Hansmann, M.-L., Haralambieva, E., Harder, L., Hasenclever, D., Kuhn, M., Lenze, D., Lichter, P., Martin-Subero, J.I., Moller, P., Muller-Hermelink, H.-K., Ott, G., Parwaresch, R.M., Pott, C., Rosenwald, A., Rosolowski, M., Schwaenen, C., Sturzenhofecker, B., Szczepanowski, M., Trautmann, H., Wacker, H.- H., Spang, R., Loeffler, M., Trumper, L., Stein, H., Siebert, R. & the Molecular Mechanisms in Malignant Lymphomas Network Project of the Deutsche, K. (2006) A Biologic Definition of Burkitt's Lymphoma from Transcriptional and Genomic Profiling. N Engl J Med, 354, Jaffe, E.S., Diebold, J., Harris, N.L., Muller-Hermelink, H.-K., Flandrin, G. & Vardiman, J.W. (1999) Burkitt's Lymphoma: A Single Disease With Multiple Variants. Blood, 93, Karsan, A., Gascoyne, R.D., Coupland, R.W., Shepherd, J.D., Phillips, G.L. & Horsman, D.E. (1993) Combination of t(14;18) and a Burkitt's type translocation in B-cell malignancies. Leuk Lymphoma, 10, Klumb, C.E., de Resende, L.M., Tajara, E.H., Bertelli, E.C., Rumjanek, V.M. & Maia, R.C. (2001) p53 gene analysis in childhood B non-hodgkin's lymphoma. Sao Paulo Med J, 119, Lindstrom, M.S. & Wiman, K.G. (2002) Role of genetic and epigenetic changes in Burkitt lymphoma. Semin Cancer Biol, 12, Macpherson, N., Lesack, D., Klasa, R., Horsman, D., Connors, J.M., Barnett, M. & Gascoyne, R.D. (1999) Small Noncleaved, Non-Burkitt's (Burkitt-Like) Lymphoma: Cytogenetics Predict Outcome and Reflect Clinical Presentation. J Clin Oncol, 17, Mead, G.M., Sydes, M.R., Walewski, J., Grigg, A., Hatton, C.S., Norbert, P., Guarnaccia, C., Lewis, M.S., McKendrick, J., Stenning, S.P. & Wright, D. (2002) An international evaluation of CODOX-M and CODOX-M alternating with IVAC in adult Burkitt's lymphoma: results of United Kingdom Lymphoma Group LY06 study. Ann Oncol, 13, Mead, G.M., Barrans, S.L., Qian, W., Walewski, J., Radford, J.A., Wolf, M., Clawson, S.M., Stenning, S.P., Yule, C.L. & Jack, A.S. (2008) A prospective clinicopathologic study of dose-modified CODOX-M/IVAC in patients with sporadic Burkitt lymphoma defined using cytogenetic and

80 immunophenotypic criteria (MRC/NCRI LY10 trial). Blood, 112, Nakamura, N., Nakamine, H., Tamaru, J., Nakamura, S., Yoshino, T., Ohshima, K. & Abe, M. (2002) The distinction between Burkitt lymphoma and diffuse large B-Cell lymphoma with c-myc rearrangement. Mod Pathol, 15, Thomas, D.A., Cortes, J., O'Brien, S., Pierce, S., Faderl, S., Albitar, M., Hagemeister, F.B., Cabanillas, F.F., Murphy, S., Keating, M.J. & Kantarjian, H. (1999) Hyper-CVAD Program in Burkitt's-Type Adult Acute Lymphoblastic Leukemia. J Clin Oncol, 17, Wang, E.S., Straus, D.J., Teruya-Feldstein, J., Qin, J., Portlock, C., Moskowitz, C., Goy, A., Hedrick, E., Zelenetz, A.D. & Noy, A. (2003) Intensive chemotherapy with cyclophosphamide, doxorubicin, high-dose methotrexate/ifosfamide, etoposide, and high-dose cytarabine (CODOX- M/IVAC) for human immunodeficiency virus-associated Burkitt lymphoma. Cancer, 98, Wilda, M., Bruch, J., Harder, L., Rawer, D., Reiter, A., Borkhardt, A. & Woessmann, W. (2004) Inactivation of the ARF-MDM-2-p53 pathway in sporadic Burkitt's lymphoma in children. Leukemia, 18, Zhang, T.C., Zhou, X.Y., Yu, B.H., Zhang, T.M. & Shi, D.R. (2007) [Effects of AKT protein kinase activation on biologic behavior of diffuse large B cell lymphoma cells]. Zhonghua Bing Li Xue Za Zhi, 36,

81 13 Extranodal NK/T cell lymphoma, nasal type 13.1 Definition and clinical features This is a malignant NK/T lymphoid neoplasm that usually presents with mucosal ulceration and tissue necrosis involving the nasal cavity and adjacent tissues. The neoplasm affects adults, more commonly males, and has a distinct racial/geographical distribution. It is prevalent in populations originating from East Asia, Mexico and Central/South America and is rare in Western countries (Kwong, 2005). The usual site of the lymphoma is the rhinopharynx hence the designation of nasal type, although a lymphoma with similar morphology can be seen at other extranodal sites including skin and gastrointestinal tract (Ko, et al 2004). In these extra-nasal cases there is occasional involvement of the bone marrow or peripheral blood, and there may be some overlap with aggressive NK-cell leukaemia. A haemophagocytic syndrome has been described in a few patients. EBV seems to play a key pathogenetic role and is found in the neoplastic cells from the majority of patients, whether of Asian or Western descent. Occasional cases have been described in immunosuppressed patients Peripheral blood The blood is rarely involved, although cases with an associated haemophagocytic syndrome may show cytopenias Bone marrow The bone marrow aspirate is usually normal, but shows rare involvement in advanced and extra-nasopharyngeal disease. Bone marrow trephine biopsy is also usually normal, showing rare involvement in advanced and extra-nasopharyngeal disease Lymph node, spleen and other tissues The condition is primarily extranodal, and neither lymph nodes nor spleen are usually involved. Most cases are diagnosed from biopsies of nasal, nasopharyngeal or pharyngeal mucosa. The diagnosis is based primarily on the

82 histological features. There is ulceration and extensive necrosis of involved mucosa, and diffuse infiltration by lymphoid cells. The infiltrating cells are of variable size and show nuclear irregularity, pale cytoplasm, with or without cytoplasmic granulation (best visualized in tissue imprints) and a small or undetectable nucleolus. The neoplastic cells commonly show an angiocentric arrangement and there is destruction of blood vessel walls leading to the tissue ulceration and necrosis. There is often a prominent inflammatory background surrounding the lymphoid infiltrate, and the extent of this inflammation may make the diagnosis difficult, particularly in superficial biopsies Immunophenotype Table 13.1 Recommended Panel for the diagnosis of extranodal NK/T cell lymphoma, nasal type CD2 CD3 CD3-epsilon CD4 CD5 CD7 CD8 TCRβ CD16 CD43 CD45 CD45R0 CD56 CD57 EBV (EBER ISH) Granzyme Perforin Immunocytochemistry Positive Usually negative Positive (cytoplasmic) Positive (a few cases are positive) Positive Positive Positive Positive Positive Positive Positive

83 TIA-1 CD20 CD79a TdT (Lu, et al 2004) Positive 13.6 Cytogenetics and molecular genetics PCR for TCR rearrangement does not usually show evidence of clonality. Del(6)(q21q25) or isochromosome(6)(p10) are frequent, but not diagnostic (Siu, et al 2002). EBV is present in an episomal fashion and hence, useful to establish or confirm clonality Prognostic factors The overall prognosis is poor. No genetic or immunophenotypic markers have been shown to predict outcome (Ohshima, et al 2002) Pitfalls None known 13.9 Differential diagnosis Reactive inflammatory lesions (including vasculitides) Peripheral T-cell lymphoma unspecified Squamous cell carcinoma Recommendations Recommendations for the diagnosis of extranodal NK/T cell lymphoma, nasal type Diagnostic criteria Ulceration and necrosis of nasal or sinus mucosa. Angiocentric infiltrate of atypical lymphoid cells. Level of evidence Grade C level IV Immunophenotype; CD2+, CD56+, cytotoxic markers+, EBV+, cytoplasmic CD3 epsilon +, surface CD3-, CD References

84 Ko, Y.H., Cho, E.Y., Kim, J.E., Lee, S.S., Huh, J.R., Chang, H.K., Yang, W.I., Kim, C.W., Kim, S.W. & Ree, H.J. (2004) NK and NK-like T-cell lymphoma in extranasal sites: a comparative clinicopathological study according to site and EBV status. Histopathology, 44, Kwong, Y-L. (2005) Natural killer-cell malignancies: diagnosis and treatment. Leukemia, 19, Lu, D., Lin, C.N., Chuang, S.S., Hwang, W.S. & Huang, W.T. (2004) T-cell and NK/T-cell lymphomas in southern Taiwan: a study of 72 cases in a single institute. Leukemia & Lymphoma, 45, Ohshima, K., Liu, Q., Koga, T., Suzumiya, J. & Kikuchi, M. (2002) Classification of cell lineage and anatomical site, and prognosis of extranodal T-cell lymphoma -- natural killer cell, cytotoxic T lymphocyte, and non-nk/ctl types. Virchows Archiv, 440, Siu, L.L., Chan, J.K. & Kwong, Y.L. (2002) Natural killer cell malignancies: clinicopathologic and molecular features. Histology and Histopathology, 17,

85 14 Enteropathy-associated T-cell lymphoma 14.1 Definition and clinical features Enteropathy-associated T-cell lymphoma (EATL) is a malignant intestinal lymphoid neoplasm usually composed of large pleomorphic cells. The tumour is thought to arise from transformed intra-epithelial T-lymphocytes and shows an association with coeliac disease (Foss, et al 2000). Most intestinal lymphomas are B-cell derived and only a minority is T-cell. Enteropathy-associated T-cell lymphoma is uncommon and predominantly or exclusively affects the small intestine (jejunum and ileum). There is evidence for an association with coeliac disease on the basis that the latter may precede the lymphoma manifestations and/or the two diseases may be diagnosed concomitantly (Brousse & Meijer, 2005). In addition, most patients with EATL have an HLA haplotype characteristic of coeliac disease. EBV may be involved in cases originating from South and Central America Peripheral blood The peripheral blood is usually normal Bone marrow The bone marrow is usually not involved, and both aspirate and trephine biopsies are negative 14.4 Lymph node, spleen and other tissues Nodal involvement is uncommon. Most cases are diagnosed from intestinal biopsy or resection specimens. The intestine shows multiple areas of mucosal ulceration often with one or more exophytic ulcerated tumour masses. Microscopically there is an infiltrate composed of medium-sized to large atypical lymphocytes with round nuclei, prominent nucleoli and abundant pale cytoplasm. Eosinophils can be present and some cases show multinucleated anaplastic tumour cells. The adjacent bowel may show the features of enteropathy such as villous atrophy and crypt hyperplasia (Daum, et al 2001) Immunophenotype

86 Table 14.1 Recommended Panel for the diagnosis of enteropathyassociated intestinal T-cell lymphoma CD2 CD3 CD4 CD5 CD7 CD8 CD56 EBV (EBER ISH) Granzyme Perforin TIA-1 CD30 CD246 (ALK1) CD20 CD79a Immunohistochemistry Positive Positive Positive (a positive subset exists) (some cases positive) Positive Positive Positive Variably positive (Daum, et al 1997) 14.6 Cytogenetics and molecular genetics The HLA DQA1*0501 DQB1* 0201 haplotype characteristic of coeliac disease is seen in EATL. TCRB and TCRG are rearranged Prognostic factors There is no evidence that immunophenotype, histology and/or genetics have a prognostic impact in this lymphoma Pitfalls None Known 14.9 Differential diagnosis

87 Extranodal NK/T-cell lymphoma, nasal-type involving the gastrointestinal tract Peripheral T-cell lymphoma, not otherwise specified B-cell lymphomas such as mantle cell lymphoma and diffuse large B-cell lymphoma ATLL affecting the gastrointestinal tract Reactive inflammatory changes seen in patients with coeliac disease Recommendations Recommendations for the diagnosis of enteropathy-associated intestinal T-cell lymphoma Diagnostic criteria Ulceration of the bowel with or without evidence of enteropathy. Ulcers are associated with an infiltrate of medium to large lymphoid cells. Level of evidence Grade C level IV Immunophenotype; CD3+, CD30+ (at least focally), cytotoxic markers+, CD4-, CD5-, CD References Brousse, N. & Meijer, J.W. (2005) Malignant complications of coeliac disease. Best Practice & Research. Clinical Gastroenterology, 19, Daum, S., Foss, H.D., Anagnostopoulos, I., Dederke, B., Demel, G., Araujo, I., Riecken, E.O. & Stein, H. (1997) Expression of cytotoxic molecules in intestinal T-cell lymphomas. The German Study Group on Intestinal Non- Hodgkin Lymphoma. Journal of Pathology, 182, Daum, S., Weiss, D., Hummel, M., Ullrich, R., Heise, W., Stein, H., Riecken, E.O., Foss, H.D. & Intestinal Lymphoma Study Group. (2001) Frequency of clonal intraepithelial T lymphocyte proliferations in enteropathy-type intestinal T cell lymphoma, coeliac disease, and refractory sprue. Gut, 49, Foss, H.D., Coupland, S.E. & Stein, H. (2000) Clinico-pathologic forms of peripheral T-and NK-cell lymphomas. Pathology, 21,

88 15 Hepatosplenic T-cell lymphoma 15.1 Definition and Clinical features Hepatosplenic T-cell lymphoma is a systemic lymphoid neoplasm derived from cytotoxic T-cells. The lymphoma cells show prominent infiltration of sinusoidal spaces in liver, spleen and bone marrow. In most cases there is a clonal proliferation of T-cells bearing gamma/delta TCR chains, but there are rare examples that show alpha/beta TCR chains (Suarez, et al 2000). The main clinical manifestations are hepatosplenomegaly without lymphadenopathy, and cytopenias, the latter due to both bone marrow infiltration and hypersplenism. The clinical course is aggressive (Weidmann, 2000). The aetiology is unknown, but gamma-delta T-cell lymphomas are more common following solid organ transplant, and it has been suggested that chronic antigenic stimulation in an immunocompromised patient may have a pathogenic role. The clinical course is aggressive with a median survival of less than 2 years Peripheral blood Cytopenias are common. A few cases show circulating lymphoma cells of medium size with abundant pale cytoplasm Bone marrow The bone marrow aspirate may show infiltration by cells with similar morphology to those in the blood. The tumour cells are often arranged in clusters or clumps simulating metastasis of a solid tumour. In some cases the marrow shows a bizarre reactive pattern in which it is difficult to detect the few abnormal T cells present. Bone marrow trephine biopsy specimens show a lymphoid infiltration with medium-sized lymphocytes, but the cells may be difficult to detect without immunohistochemistry. The infiltrate is predominantly intrasinusoidal, as in spleen and liver, and the diagnosis can be made on the marrow when these tissues are not available for diagnosis Lymph node, spleen and other tissues

89 Lymph node involvement is rare. The sinusoids of the liver and spleen show a monomorphic infiltrate of small to medium-sized lymphocytes (Taguchi, et al 2004). The cells have a round nucleus with condensed chromatin and pale cytoplasm, and the same infiltrating cells are seen in the bone marrow (see above) Immunophenotype Table 15.1 Recommended Panel for the diagnosis of hepatosplenic T-cell lymphoma Immunohistochemistry CD2 Positive CD3 Positive CD4 CD5 CD7 Positive or negative CD8 (a few cases are positive) CD56 Positive EBV (EBER ISH) Granzyme Perforin (a few cases are positive) TIA-1 Positive CD20 CD79a (Kanavaros, et al 2000, Belhadj, et al 2003, Wei, et al 2005)) 15.6 Cytogenetics and molecular genetics PCR studies show TCRG clonality; TCRB may or may not be rearranged. A minority of cases are of TCRαβ lineage. An iso(7q) has been reported in virtually all cases investigated cytogenetically (Belhadj, et al 2003). This is frequently accompanied by trisomy 8. Cytogenetics is particularly helpful where the diagnosis is being made from bone marrow. In situ hybridisation analysis does not show the presence of EBV.

90 15.7 Prognostic factors None known Pitfalls Cases with an abnormal reactive pattern in the bone marrow can be incorrectly interpreted as myeloid or erythroid disease. The presence of iso(7q) is useful in this setting; the presence of trisomy 8 and abnormal reactive morphology should NOT be interpreted as suggesting a myeloid neoplasm (Wlodarska, et al 2002) Differential diagnosis Peripheral T-cell lymphoma, not otherwise specified T-cell large granular lymphocyte leukaemia The variant (αβ) form should be distinguished from; Mycosis fungoides Extranodal NK/T cell lymphoma nasal type EATL Recommendations Recommendations for the diagnosis of hepatosplenic T-cell lymphoma Diagnostic criteria Sinusoidal infiltration of liver, spleen or bone marrow by small to medium-sized lymphocytes. Level of evidence Grade C level IV Immunophenotype; CD3+, CD56+, TIA1+, TCRδ1+, CD5-, CD References Belhadj, K., Reyes, F., Farcet, J.P., Tilly, H., Bastard, C., Angonin, R., Deconinck, E., Charlotte, F., Leblond, V., Labouyrie, E., Lederlin, P., Emile, J.F., Delmas-Marsalet, B., Arnulf, B., Zafrani, E.S. & Gaulard, P. (2003) Hepatosplenic gammadelta T-cell lymphoma is a rare clinicopathologic entity with poor outcome: report on a series of 21 patients. Blood, 102, Kanavaros, P., Boulland, M.L., Petit, B., Arnulf, B. & Gaulard, P. (2000) Expression of cytotoxic proteins in peripheral T-cell and natural killer-cell (NK) lymphomas: association with extranodal site, NK or Tgammadelta

91 phenotype, anaplastic morphology and CD30 expression. Leukemia & Lymphoma, 38, Suarez, F., Wlodarska, I., Rigal-Huguet, F., Mempel, M., Martin-Garcia, N., Farcet, J.P., Delsol, G. & Gaulard, P. (2000) Hepatosplenic alphabeta T- cell lymphoma: an unusual case with clinical, histologic, and cytogenetic features of gammadelta hepatosplenic T-cell lymphoma. American Journal of Surgical Pathology, 24, Taguchi, A., Miyazaki, M., Sakuragi, S., Shinohara, K., Kamei, T. & Inoue, Y. (2004) Gamma/delta T cell lymphoma. Internal Medicine, 43, Wei, S.Z., Liu, T.H., Wang, D.T., Cao, J.L., Luo, Y.F. & Liang, Z.Y. (2005) Hepatosplenic gammadelta T-cell lymphoma. World Journal of Gastroenterology, 11, Weidmann, E. (2000) Hepatosplenic T cell lymphoma. A review on 45 cases since the first report describing the disease as a distinct lymphoma entity in Leukemia, 14, Wlodarska, I., Martin-Garcia, N., Achten, R., De Wolf-Peeters, C., Pauwels, P., Tulliez, M., de Mascarel, A., Briere, J., Patey, M., Hagemeijer, A. & Gaulard, P. (2002) Fluorescence in situ hybridization study of chromosome 7 aberrations in hepatosplenic T-cell lymphoma: isochromosome 7q as a common abnormality accumulating in forms with features of cytologic progression. Genes, Chromosomes & Cancer, 33,

92 16 Angioimmunoblastic T-cell lymphoma 16.1 Disease definition and Clinical features Angioimmunoblastic T-cell lymphoma is a peripheral T-cell neoplasm that combines systemic symptoms with a polymorphous infiltrate in lymph nodes. This lymphoma mainly affects adults, who often present with advanced disease. Systemic symptoms, lymphadenopathy, hepatosplenomegaly, skin rash and autoimmune phenomena or immune dysfunction are common (Ferry, 2002). There is no evidence for a distinct geographical or racial distribution. The presence of EBV has been frequently documented, but it is found in the B cells rather than in the T cells. There have been documented cases where an oligoclonal pattern of T-cell proliferation has preceded the development of lymphoma. Overall the prognosis is poor with a median survival of around 3 years. Patients die either from progressive disease or complications related to the immune disturbance Peripheral blood Hypo- or hypergammaglobulinaemia and autoimmune haemolytic anaemia are not uncommon. A leukaemic phase is exceedingly rare Bone marrow The bone marrow aspirate is hypercellular, with an increase in granulocytic precursors, eosinophilia and increased numbers of plasma cells. Involvement by lymphoma cells of medium size with a moderate amount of cytoplasm is present in some cases. Bone marrow trephine sections commonly show involvement at the time of presentation. The tissue is hypercellular and shows lymphoid infiltration with no distinct pattern of involvement. An accompanying reactive cellular component, as in the lymph nodes (see below), is often present Lymph node, spleen and other tissues The characteristic histology is seen in lymph nodes, and nodal biopsy is required for the diagnosis. The nodal architecture is effaced by a polymorphous infiltrate of small to medium-sized lymphocytes usually centred on the

93 paracortex. In most cases a proportion of the infiltrating cells show clear cytoplasm. Large immunoblasts and Reed-Sternberg-like cells may be present in small numbers. There is a background, mixed reactive population composed of small lymphocytes, eosinophils, plasma cells, prominent blood vessels with conspicuous endothelial cells, histiocytes and a network of follicular dendritic cells (shown by immunostaining) (Luzzatto, et al 2005). The germinal centres are atrophic or burnt out in the majority of cases, but can be hyperplastic (15% of cases) (Dogan, et al 2003) Immunophenotype Table 16.1 Recommended Panel for the diagnosis of angioimmunoblastic T-cell lymphoma Immmunohistochemistry CD2 Positive* CD3 Positive* CD4 Positive* CD5 Positive* CD7 Positive* CD8 Positive* CD10 Positive in neoplastic T-cells in ~50% of cases (Attygale, et al 2002) PD1, CXCL13, ICOS Markers of follicular helper T-cells, positive in the majority of cases (Rodriguez-Justo, et al 2009) EBV (EBER ISH) Positive in reactive B-cells CD21/CD23 Expanded FDC network CD246 (ALK1) CD20 (positive in reactive B-cells) CD79a (positive in reactive B-cells) * Loss of expression of some pan-t-cell markers is not uncommon. There is usually a predominance of CD4+ cells over CD8+ cells (Lee, et al 2003).

94 16.6 Cytogenetics and molecular genetics There is no cytogenetic signature for this disease. Clonal abnormalities with related and unrelated clones have been documented. The most common include extra copies of chromosomes 3, 5, and X and various structural abnormalities. There is TCR gene rearrangement in over two thirds of cases and in a few (approximately 10%) the IGH gene is also rearranged (Vergier, et al 2002). The presence of EBV has been documented in this lymphoma, but it appears that the infected cells are the B cells and not the clonal T-cell population (Luzzatto, et al 2005) Prognostic factors None known Pitfalls The morphology is not always diagnostic, and the reactive cellular population can make it difficult to determine if the lesion is of B-cell or T-cell lineage. Secondary EBV-positive B-cell lymphomas arise in some cases, probably due to immunosuppression, and the histology in these patients shows a combination of angioimmunoblastic T-cell lymphoma with a B-cell proliferation resembling a post-transplant lymphoproliferative disorder Differential diagnosis T-cell/histiocyte-rich diffuse large B-cell lymphoma Peripheral T-cell lymphoma, not otherwise specified Reactive paracortical hyperplasia Recommendations Recommendations for the diagnosis of angioimmunoblastic T-cell lymphoma Diagnostic criteria Lymph node showing architectural effacement with a polymorphous infiltrate of atypical lymphocytes and vascular proliferation. Level of evidence Grade C level IV

95 Immunophenotype; atypical T cells are CD3+ and PD1+, CXCL13+ & ICOS+ (most cases). Approximately 50% are CD10+. There is a expansion of the FDC network with CD21 or CD23, and a background population of EBV-positive B cells References Attygalle, A., Al-Jehani, R., Diss, T.C., Munson, P., Liu, H., Du, MQ., Isaacson, P.G. & Dogan, A. (2002) Neoplastic T cells in angioimmunoblastic T-cell lymphoma express CD10. Blood, 99, Dogan, A., Attygalle, A.D. & Kyriakou, C. (2003) Angioimmunoblastic T-cell lymphoma. British Journal of Haematology, 121, Ferry, J.A. (2002) Angioimmunoblastic T-cell lymphoma. Advances in Anatomic Pathology, 9, Lee, S.S., Rudiger, T., Odenwald, T., Roth, S., Starostik, P. & Muller-Hermelink, H.K. (2003) Angioimmunoblastic T cell lymphoma is derived from mature T-helper cells with varying expression and loss of detectable CD4. International Journal of Cancer, 103, Luzzatto, F., Pruneri, G., Benini, E., Manzotti, M., Laszlo, D., Martinelli, G. & Viale, G. (2005) Angioimmunoblastic T-cell lymphoma with hyperplastic germinal centres and a high content of EBV-infected large B-cells carrying IgH chain gene monoclonal rearrangement. Histopathology, 46, Rodriguez-Justo, M., Attygalle, A.D., Munson, P., Roncador, G., Marafioti, T. & Piris M.A. (2009) Angioimmunoblastic T-cell lymphoma with hyperplastic germinal centres: a neoplasia with origin in the outer zone of the germinal centre? Clinicopathological and immunohistochemical study of 10 cases with follicular T-cell markers. Modern Pathology, 22, Vergier, B., Dubus, P., Kutschmar, A., Parrens, M., Ferrer, J., de Mascarel, A. & Merlio, J.P. (2002) Combined analysis of T cell receptor gamma and immunoglobulin heavy chain gene rearrangements at the single-cell level in lymphomas with dual genotype. Journal of Pathology, 198,

96 17 Anaplastic large cell lymphoma, ALK-positive and ALKnegative 17.1 Definition and clinical features Anaplastic large cell lymphomas (ALCL) are neoplasms of activated large mature T cells that express CD30 and cytotoxic granule-associated antigens. The majority of cases also show a genetic alteration leading to over-expression of the ALK (anaplastic lymphoma kinase) gene, and these have been classified as a distinct entitiy (ALCL, ALK+ve) in the 2008 revision of the WHO classification (Delsol et al., 2008). Examples of systemic ALCL lacking ALK expression are grouped into a separate entity (ALCL, ALK-ve) (Mason et al., 2008). Clinical studies addressing differences between ALK+ve and ALK-ve ALCL are limited; they show a wide age distribution and a male predominance. ALCL, ALK+ve makes up 10-30% of childhood lymphomas (Jaffe, 2004), and the ALK-ve form has a peak incidence in the elderly. Most patients manifest with B symptoms and advanced stage disease and a substantial proportion have extranodal involvement such as skin, bone or lung. The prototypic disease is systemic primary ALCL, ALK+ve; a morphologically and phenotypically (but not biologically) similar anaplastic large cell lymphoma, ALK-ve, arises as a primary cutaneous neoplasm (Stein, et al 2000) Peripheral blood This is usually not involved Bone marrow The bone marrow aspirate rarely shows involvement. Bone marrow trephine biopsy shows involvement in 10-30% of cases. Involvement may be subtle and undetectable without immunostaining; there is often little stromal reaction and infiltrates maybe confined to sinusoids or even consist of scattered single cells Lymph node, spleen and other tissues Involved nodes show characteristic histology but, where ALCL is extranodal, the diagnosis can be made from biopsies of other involved sites. Diagnosis is

97 dependent upon the identification of cells with typical morphology in both ALK+ve and ALK-ve types. Lymph nodes show either diffuse effacement of the architecture or partial involvement with a sinusoidal and/or perivascular pattern. The most common histological pattern consists of an infiltrate of large pleomorphic cells with prominent cytoplasm. These characteristic or hallmark cells have a kidney-shaped nucleus with dispersed chromatin and a prominent nucleolus. The cytoplasm is abundant and there is often an identifiable eosinophilic Golgi region close to the nucleus. The WHO recognises small cell and lymphohistiocytic variants, and the published literature describes other morphological forms of ALCL (Benharroch, et al 1998, Falini, et al 1998). The lymphohistiocytic variant shows an admixture of neoplastic cells and histiocytes, with haemophagocytosis in some cases. The small cell variant is composed of smaller pleomorphic cells, and can lack classical large pleomorphic and hallmark cells. Immunohistochemistry is critical in the diagnosis of ALCL variants Immunophenotype Table 17.1 Recommended Panel for the diagnosis of anaplastic large cell lymphoma, ALK-positive and ALK-negative. CD2 CD3 CD4 CD5 CD7 CD8 TCRβ CD30 CD15 CD246 (ALK1) Immunohistochemistry Positive (rare positive cases) Positive (rare positive cases) Positive or negative (rare positive cases) (some cases positive) Positive Positive or negative, defining the different types

98 ALKc EMA EBV (EBER ISH) Granzyme Perforin TIA-1 CD20 CD79a PAX5 (Falini, 2001) Positive in a subset of cases Positive Positive Positive Positive 17.6 Cytogenetics & molecular genetics Molecular analysis shows a monoclonal configuration of the TCR genes in greater than 90% of cases, whether or not the cells express T-cell antigens. IGH is not rearranged. The translocation t(2;5) that involves ALK and the nucleophosmin gene, NPM, on chromosome 5 (Pulford, et al 2004) can be detected by both RT-PCR and FISH as well as conventional cytogenetics. However there are numerous variant forms of the translocation involving ALK and other partner chromosomes (Falini, et al 1999) and so the only reliable detection method is by FISH using a break-apart strategy with probes located on either side of ALK Prognostic factors Within the mature T-cell malignancies, ALCL patients have a relatively favourable prognosis. The disease is usually chemosensitive, even in relapse (which is seen in 30% of patients). ALK expression is the most important pathological prognostic factor. Patients with ALCL, ALK+ve have a higher 5- year survival rate than those with ALCL, ALK-ve. Primary cutaneous ALCL has a better prognosis than the systemic forms Pitfalls The small cell variant can be difficult to recognise, and negative CD3 staining can lead to diagnostic uncertainty. Anaplastic morphology and CD30 expression

99 can be seen as part of disease progression or at presentation in other subtypes of T- or B-cell lymphomas Differential diagnosis Hodgkin lymphoma T-cell/histiocyte-rich diffuse large B-cell lymphoma ALK-positive large B-cell lymphoma Secondary ALCL Peripheral T-cell lymphoma, not otherwise specified (particularly with the small cell variant of ALCL) Non-haemopoietic tumours Recommendations Recommendations for the diagnosis of anaplastic large cell lymphoma Diagnostic criteria Nodal or extranodal infiltrate composed of large pleomorphic cells. Sinusoidal or perivascular distribution in lymph nodes. Hallmark cells. Immunophenotype; CD30+, cytotoxic markers positive, most cases also positive for CD2 and EMA. CD246 (ALK1) or ALKc expression is characteristic in younger patients, and defines the ALK-positive type. CD15 and B-cell markers negative. TCR rearrangement and FISH for ALK translocation in difficult cases References Level of evidence Grade C level IV Benharroch, D., Meguerian-Bedoyan, Z., Lamant, L., Amin, C., Brugieres, L., Terrier-Lacombe, M.J., Haralambieva, E., Pulford, K., Pileri, S., Morris, S.W., Mason, D.Y. & Delsol, G. (1998) ALK-positive lymphoma: a single disease with a broad spectrum of morphology. Blood, 91,

100 Delsol G, Falini B, Müller-Hermelink HK, Campo E. Anaplastic large cell lymphoma (ALCL),ALK-positive. In: Swerdlow SH, Campo C, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds) 2008 WHO classification of tumours of haematopoietic and lymphoid tissues, IARC, Lyon Falini, B., Bigerna, B., Fizzotti, M., Pulford, K., Pileri, S.A., Delsol, G., Carbone, A., Paulli, M., Magrini, U., Menestrina, F., Giardini, R., Pilotti, S., Mezzelani, A., Ugolini, B., Billi, M., Pucciarini, A., Pacini, R., Pelicci, P.G. & Flenghi, L. (1998) ALK expression defines a distinct group of T/null lymphomas ("ALK lymphomas") with a wide morphological spectrum. American Journal of Pathology, 153, Falini, B., Pulford, K., Pucciarini, A., Carbone, A., De Wolf-Peeters, C., Cordell, J., Fizzotti, M., Santucci, A., Pelicci, P.G., Pileri, S., Campo, E., Ott, G., Delsol, G. & Mason, D.Y. (1999) Lymphomas expressing ALK fusion protein(s) other than NPM-ALK. Blood, 94, Falini, B. (2001) Anaplastic large cell lymphoma: pathological, molecular and clinical features. British Journal of Haematology, 114, Jaffe, E.S. (2004) Mature T-cell and NK-cell lymphomas in the pediatric age group. American Journal of Clinical Pathology, 122, S110-S121. Mason DY, Harris NL, Delsol G, Stein H, Campo E, Kinney MC, Jaffe ES Falini B. Anaplastic large cell lymphoma, ALK-negative. In: Swerdlow SH, Campo C, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds) 2008 WHO classification of tumours of haematopoietic and lymphoid tissues, IARC, Lyon Pulford, K., Lamant, L., Espinos, E., Jiang, Q., Xue, L., Turturro, F., Delsol, G. & Morris, S.W. (2004) The emerging normal and disease-related roles of anaplastic lymphoma kinase. Cellular and Molecular Life Sciences, 61, Stein, H., Foss, H.D., Durkop, H., Marafioti, T., Delsol, G., Pulford, K., Pileri, S. & Falini, B. (2000) CD30(+) anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features. Blood, 96,

101 18 Peripheral T-cell lymphoma, not otherwise specified 18.1 Definition and clinical features This category includes the mature/post-thymic T-cell neoplasms that do not fit into one of the recognisable subtypes of T-cell lymphoma. Multiple morphological subtypes are included in this group, which accounts for up to half of the peripheral T-cell lymphomas seen in Western countries. Most patients are adults, but children can be affected. The usual presentation is as a generalized disease with organomegaly and bone marrow infiltration. Constitutional symptoms are common. These tumours are predominantly nodal, but there may be involvement of the peripheral blood and extranodal sites (Ichimura, et al 2003). This WHO category includes several conditions that have been recognised as distinct entities in other classifications, including T-zone lymphoma, Lennert s lymphoma, pleomorphic mixed medium and large cell T- cell lymphoma, and T-immunoblastic lymphoma. There is no evidence that these represent distinct clinicopathological diseases, and their morphological identification shows a high degree of intra- and inter-observer variability. There is no evidence for a distinct geographical clustering (Au, et al 2005) or for the involvement of viruses such as EBV in the pathogenesis of these tumours Peripheral blood A leukaemic picture is seen in some cases. The circulating lymphoma cells are of variable size and show different nuclear shapes and degree of chromatin condensation; in some cases the cells may have the features of immunoblasts Bone marrow Bone marrow aspirates can contain cells with similar morphology to those seen in the blood. Although marrow involvement is a feature of leukaemic cases, it is not restricted to this group. Bone marrow trephine biopsy commonly shows involvement at presentation and the infiltration varies from subtle to diffuse and widespread. It is very common to encounter an accompanying hyperplastic and/or dysplastic haemopoietic reaction. There may also be haemophagocytosis, sometimes life-threatening.

102 18.4 Lymph node, spleen and other tissues Lymph node biopsy is usually required for the diagnosis, but when the disease is extra-nodal, or where nodes are not accessible the diagnosis may be made on other tissues such as bone marrow or liver. Affected lymph nodes show a diffuse infiltrate with a wide range of histological appearances. The infiltrate is composed of irregular cells of variable size ranging from rare cases composed predominantly of small cells through to medium or large cells that can have hyperchromatic or vesicular nuclei with prominent nucleoli. There may be a few Reed-Sternberg-like cells, prominent high endothelial venules, and a mixed inflammatory background. There are two notable histological variants: the T- zone variant, in which there is preferential infiltration of the interfollicular region of the node, and the lymphoepithelioid cell variant, in which numerous epithelioid histiocytes are present. There is no evidence, however, that these variants represent distinct disease entities Immunophenotype Table 18.1 Recommended Panel for the diagnosis of peripheral T-cell lymphoma, not otherwise specified CD2 CD3 CD4 CD5 CD7 CD8 EBV (EBER ISH) Granzyme Perforin TIA-1 Immunohistochemistry Positive* Positive* Positive* Positive* Positive* * (may be positive in reactive B cells) (occasional positives) (Asano, et al 2005)

103 CD30 Positive in occasional cases CD246 (ALK1) CD20 (occasional aberrant positive cases) CD79a * Pan-T cell markers CD2, CD3, CD5 and CD7 show variable expression; aberrant phenotypes with loss of one or more of these antigens are common (Nakamura, et al 1993) Cytogenetics and molecular genetics Most cases of peripheral T-cell lymphoma, unspecified will show TCR genes with a rearranged configuration. There is no specific karyotypic abnormality that characterizes this lymphoma; complex numerical and structural translocations are common and trisomy 3 has been reported in a proportion of cases, particularly in the lymphoepithelioid cell variant Prognostic & therapeutic markers The prognosis is poor (Arrowsmith, et al 2003). It has been suggested that the few cases with EBV positivity have a worse outcome but this needs to be substantiated in further studies (Zuccca & Zinzani, 2005) Pitfalls CD4 or CD7 expression by itself does not indicate the T-cell nature of the tumour, as myeloid sarcoma (granulocytic sarcoma) may be CD4 or CD7 positive, the former particularly in cases with monocytic differentiation. Peripheral T-cell lymphoma may show aberrant expression of pan B-cell markers such as CD20 or CD79a Differential diagnosis Reactive T-cell proliferations Other (well defined) T-cell lymphomas and mature T-cell leukaemias. This includes anaplastic large cell lymphomas, angioimmunoblastic lymphoma, HTLV-1+ adult T-cell leukaemia/lymphoma in its

104 lymphomatous form. Also T-cell prolymphocytic leukaemia in rare cases of peripheral T-cell lymphoma, not otherwise specified with a leukaemic phase NK-cell derived neoplasms T-cell/histiocyte-rich large B-cell lymphoma Myeloid sarcoma Recommendations Recommendations for the diagnosis of peripheral T-cell lymphoma, not otherwise specified Diagnostic criteria A destructive nodal or extranodal infiltrate of lymphoid cells with variable morphology and a polymorphic appearance. Expression of at least one pan T-cell marker (CD3, CD5, CD2 or CD7) and a CD4+/CD8- phenotype in most cases. Myeloid and B-cell markers should be negative, but occasional cases show aberrant expression of CD References Level of evidence Grade C level IV Arrowsmith, E.R., Macon, W.R., Kinney, M.C., Stein, R.S., Goodman, S.A., Morgan, D.S., Flexner, J.M., Cousar, J.B., Jagasia, M.H., McCurley, T.L. & Greer, J.P. (2003) Peripheral T-cell lymphomas: clinical features and prognostic factors of 92 cases defined by the revised European American lymphoma classification. Leukemia & Lymphoma, 44, Asano, N., Suzuki, R., Kagami, Y., Ishida, F., Kitamura, K., Fukutani, H., Morishima, Y., Takeuchi, K. & Nakamura, S. (2005) Clinicopathologic and prognostic significance of cytotoxic molecule expression in nodal peripheral T-cell lymphoma, unspecified. The American Journal of Surgical Pathology, 29, Au, W.Y., Ma, S.Y., Chim, C.S., Choy, C., Loong, F., Lie, AK., Lam, CC., Leung, A.Y., Tse, E., Yau, C.C., Liang, R. & Kwong, Y.L. (2005) Clinicopathologic features and treatment outcome of mature T-cell and natural killer-cell lymphomas diagnosed according to the World Health Organization

105 classification scheme: a single center experience of 10 years. Annals of Oncology, 16, Ichimura, K., Kagami, Y., Suzuki, R., Kojima, M., Yoshino, T., Ohshima, K., Koike, K., Kondo, E., Taji, H., Ogura, M., Morishima, Y., Akagi, T., Takahashi, T. & Nakamura, S. (2003) Phenotypic analysis of peripheral T/NK cell lymphoma: study of 408 Japanese cases with special reference to their anatomical sites. Pathology International, 53, Nakamura, S., Koshikawa, T., Koike, K., Kitoh, K., Suzuki, H., Oyama, A., Motoori, T., Kojima, M., Ogura, M. & Kurita, S. (1993) Phenotypic analysis of peripheral T cell lymphoma among the Japanese. Acta Pathologica Japonica, 43, Zucca, E. & Zinzani, P.L. (2005) Understanding the group of peripheral T-cell lymphomas, unspecified. Current Hematology Reports, 4,

106 19 T-lymphoblastic leukaemia/ lymphoma (T-ALL/T-LbLy) 19.1 Definition & Clinical features T-LbLy and T-ALL are neoplasms of precursor (thymic) T- lymphoid cells; T-ALL is considered to be the leukaemic form of T-LbLy. The majority of patients are young men who present with features identical to those seen in other acute lymphoid leukaemias (i.e. bone marrow failure); in addition, these patients may have a mediastinal mass, widespread lymphadenopathy, hepatomegaly, splenomegaly and effusions Peripheral blood The haemoglobin levels and platelet count, in contrast to B-lineage ALL, may be normal or slightly decreased. In T-ALL, the white blood cell count may be very high with circulating blasts while it is normal in T-LbLy; there may be eosinophilia. The circulating blasts are small to medium sized (L1 or L2 of the FAB morphology) with scanty agranular cytoplasm and a variable degree of chromatin condensation, with or without a small nucleolus; the nuclear outline is often, but not always, irregular Bone marrow This is consistently involved in T-ALL. The aspirate shows a variable degree of infiltration by blasts with similar morphology to those on the peripheral blood; there may be an increase in eosinophils and myeloid hyperplasia. Trephine biopsy shows infiltration, ranging from interstitial to diffuse, by lymphoblasts with stippled chromatin. There may be an increase in eosinophils Lymph node & other organs The nodal architecture is usually diffusely replaced by lymphoblasts, but rarely infiltration may be confined to the paracortical areas. The morphology can give a starry-sky like appearance. Histology of a mediastinal mass will show features similar to those seen in the lymph nodes. Effusions and cerebrospinal fluid (CSF) may contain neoplastic cells and involvement can be demonstrated by immunophenotyping.

107 19.5 Immunophenotype Table 19.1 Recommended Panel for the diagnosis of T-lymphoblastic leukaemia/lymphoma Immunofluorescence Immunohistochemistry CD1a Positive or negative Positive or negative CD2 Usually positive Usually positive CD3 Cytoplasmic positive Positive but often membrane negative CD3-epsilon Positive CD4 Positive or negative Positive or negative CD5 Usually positive Usually positive CD7 Positive Positive CD8 Positive or negative Positive or negative TCRβ Positive or negative Positive or negative Some cases are TCRγ positive and others are negative with TCRβ and TCRγ CD10 May be positive (20% Positive (30% cases) cases) CD45 Positive Positive CD56 Usually negative TdT Positive Positive CD19 CD20 CD79a Can be expressed Can be expressed CD22 N/A CD15 Usually negative Usually negative Myeloperoxidase/CD Cytogenetics and molecular genetics Cytogenetic abnormalities involve genes that code for the TCR chains and transcription factors. The most common aberrant findings are a cryptic deletion of 1q32 that involves the TAL1 gene and a cryptic translocation t(5;14)(q35;q32) that leads to the upregulation of the HOX11L2 gene. A few patients with T-LbLy have the t(8;13) and some of these latter patients may develop acute myeloid leukaemia/myeloid sarcoma. (Inhorn et al, 1995)

108 TCRG and/or TCRB are rearranged (Pilozzi et al, 1999) 19.7 Prognostic and therapeutic markers There are no specific markers identified, and with modern treatment schedules the prognosis of T-ALL is similar to that of B-cell lineage ALL. Preliminary studies suggest that microarray gene profiling may help to delineate two prognostic groups within T-ALL in adults (Chiaretti et al, 2004) Pitfalls The blasts may express some B cell markers; CD79a can be weakly expressed and CD10 is positive in about 20% of cases. TdT can be down-regulated (weak or negative) in 5-10% of cases and some cases co-express myeloid associated antigens such as CD33 and CD13. (Bene et al, 1995; Pilozzi et al, 1998; Hashimoto et al, 2002) 19.9 Differential diagnosis Mature (post-thymic) T-cell leukaemias and lymphomas B-lymphoblastic leukaemia/lymphoma Poorly differentiated acute myeloid leukaemia and acute megakaryoblastic leukaemia Mixed phenotype acute leukaemias Haematogones (physiological B-lineage blast cells Recommendations Recommendations for the diagnosis of T-lymphoblastic leukaemia/lymphoma Diagnostic criteria Level of evidence Immunophenotype; CD2, cytcd3, CD5, CD7, TdT Grade C level IV positive, MPO, CD19, CD20 negative FISH and molecular genetics are not required for diagnosis References

109 Bene, M.C., Castoldi, G., Knapp, W., Ludwig, W.D., Matutes, E., Orfao, A. & van't Veer, M.B. (European Group for the Immunological Characterisation of Leukemias). (1995) Proposals for the immunological classification of acute leukemias. Leukemia, 9, Chiaretti, S., Li, X., Gentleman, R., Vitale, A., Vignetti, M., Mandelli, F., Ritz, J. & Foa, R. (2004) Gene expression profile of adult T-cell acute lymphocytic leukemia identifies distinct subsets of patients with different response to therapy and survival. Blood, 103, Hashimoto, M., Yamashita, Y. & Mori, N. (2002) Immunohistochemical detection of CD79a expression in precursor T cell lymphoblastic lymphoma/leukaemias. Journal of Pathology, 197, Inhorn, R.C., Aster, J.C., Roach, S.A., Slapak, C.A., Soiffer, R., Tantravahi, R. & Stone, R.M. (1995) A syndrome of lymphoblastic lymphoma, eosinophilia and myeloid hyperplasia/malignancy associated with t(8;13)(p11;q11): description of a distinctive clinicopathologic entity. Blood, 85, Okuda, T., Fisher, R. & Downing, J.R. (1996) Molecular diagnostics in pediatric acute lymphoblastic leukemia. Molecular diagnostics, 1, Pilozzi, E., Pulford, K., Jones, M., Muller-Hermelink, H.K., Falini, B., Ralfkiaer, E., Pileri, S., Pezzella, F., de Wolf-Peeters, C., Arber, D., Stein, H., Mason, D. & Gatter, K.(1998) Co-expression of CD79a (JCB117) and CD3 by lymphoblastic lymphoma. Journal Pathology, 186, Pilozzi, E., Muller-Hermelink, H.K., Falini, B., Wolf-Peeters, C., Fidler, C., Gatter, K. &Wainscoat, J. (1999) Gene rearrangements in T-cell lymphoblastic lymphoma. Journal Pathology, 188, Pui, C-H. & Evans, W.E. (2006) Treatment of acute lymphoblastic leukaemia. New England Journal Medicine, 354,

110 20 T-cell prolymphocytic leukaemia (T-PLL) 20.1 Definition & clinical features This is a mature/post-thymic T-cell leukaemia of small to medium sized lymphocytes involving blood, bone marrow and lymphoid tissues. T-PLL affects adults and often manifests with high lymphocyte count, splenomegaly and, in about 25% of patients, skin involvement. A minority of patients are asymptomatic and have a smouldering form. (Matutes et al, 1991; Garand et al, 1998) There is no evidence of involvement of HTLV-I. (Pawson et al, 1997) T-PLL has been documented in patients with ataxia telangiectasia and the AT mutated (ATM) gene is thought to be involved in its pathogenesis Peripheral blood The white cell count is raised with an absolute lymphocytosis. The circulating lymphocytes are medium sized with condensed nuclear chromatin, a single nucleolus and a deeply irregular basophilic cytoplasm (typical form of T-PLL). Two morphological variants have been documented and are recognized by the WHO: the small-cell (small sized lymphocytes with small or no visible nucleolus) and cerebriform (highly convoluted nucleus) variants. These two cell variants have similar immunophenotypic and molecular genetics to the typical form Bone marrow The marrow is always involved and the aspirate shows variable infiltration by lymphocytes with similar morphology to the circulating cells. The pattern of infiltration in the trephine biopsy sections is either diffuse or mixed (nodular plus heavy interstitial) with increased reticulin staining Lymph node, spleen & other sites Histology from these sites is rarely available as the diagnosis is based on blood and marrow features. The infiltration is diffuse or predominantly in the paracortical areas; residual unaffected follicles may be present. The spleen histology shows red pulp infiltration and often involvement of the white cell pulp and the spleen capsule. The skin histology shows dermal infiltration with

111 lymphocytes arranged in clusters around the skin appendages without epidermotropism (Maket et al, 1994) Immunophenotype Table 18.1 Recommended Panel for the diagnosis of T-PLL Immunofluorescence Immunohistochemistry CD2 Positive Positive CD3 Positive or weak (20% Positive of cases are membrane CD3 negative) CD4 Positive Positive CD5 Positive Positive CD7 Positive Positive CD8 Usually negative Usually negative TCRB Positive (80% cases) Positive CD10 CD45 Positive Positive CD56 CD57 CD19/CD20/CD79a Cells from 25% of cases are CD4+/CD8+ and from 10% are CD8+ CD4- All cases are CD1a and TdT negative and CD52 is strongly expressed 20.6 Cytogenetics and molecular genetics The most common abnormalities (80% of cases) involve chromosome 14 at 14q11 and 14q32.1 bands, often inv(14)(q11q32), and abnormalities of chromosome 8 such as trisomy 8, iso(8q) or t(8;8). (Brito Babapulle et al, 1987; 1997) FISH may reveal 11q23 (ATM) deletion and deletions of 12p13. Oncogenes shown to be involved in the pathogenesis of T-PLL are: TCL1A and TCL1B (14q32), MTCP1 (Xq28) and ATM (11q23) (Stilgenbauer et al, 1997;

112 Stern et al, 1993; Griffi et al, 1998; Yullie et al, 1998). TCRB and/or TCRG are rearranged Prognostic and Therapeutic Markers Patients with smouldering disease appear to fare better. CD52 is strongly expressed (Ginaldi et al, 1998) and the CD52 (Campath-1H) antibody is used as first line therapy Pitfalls Some cases can lack of expression of membrane CD3 and TCRβ and a minority are CD Differential Diagnosis Precursor T-cell neoplasms B-PLL Other mature/peripheral T cell lymphomas Cutaneous T-cell lymphomas (Sézary syndrome) in the cerebriform T- PLL variant Recommendations Recommendations for the diagnosis of T-PLL Diagnostic criteria Morphology: prolymphocytes Level of evidence Grade C level IV Immunophenotype; CD2+, CD3+, CD4+ or CD4+/CD8+, CD7+, CD43+, anti-tcr+, CD8-/+, TdT- Cytogenetics and molecular genetics are not required for diagnosis, but maybe helpful in smouldering T-PLL References

113 Brito Babapulle, V., Pomfret, M., Matutes, E. & Catovsky, D. (1987) Cytogenetic studies on prolymphocytic leukemia. II. T cell prolymphocytic leukemia. Blood, 70, Brito Babapulle, V. & Catovsky, D. (1991) Inversions and tandem translocations involving chromosome 14q11 and 14q32 in T prolymphocytic leukemia and T cell leukemias in patients with ataxia telangiectasia. Cancer Genetics and Cytogenetics, 55, 1-9. Brito Babapulle, V., Maljaie, S.H., Matutes, E. Hedges. M., Yullie. M. & Catovsky, D. (1997) Relationship of T leukaemias with cerebriform nuclei to T prolymphocytic leukaemia: a cytogenetic analysis with in situ hybridization. British Journal of Haematolog, 96, Brito Babapulle, V., Baou, M., Matutes, E., Morilla, R., Atkinson, S. & Catovsky, D. (2001) Deletions of D13S25, D13S319 and RB 1 mapping to 13q14.3 in T cell prolymphocytic leukaemia. British Journal of Haematology 114, Dearden, C.E., Matutes, E., Cazin, B., Tjonnfjord, G.E., Parreira, A., Nomdedeu, B., Leoni, P., Clark, F.J., Rassam, S.M.B., Roques, T., Ketterer, N., Brito- Babapulle, V., Dyer, M.J.S. & Catovsky, D. (2001) High remission rate in T-cell prolymphocytic leukaemia with CAMPATH-1H. Blood. 98, Garand, R., Goasguen, J., Brizard, A., Buisine, J., Charpentier, A., Claisse, J.F., Duchayne, E., Lagrange, M., Segonds, C., Troussard, X. & Flandrin, G. (1998) Indolent course as a relatively frequent presentation in T prolymphocytic leukaemia. British Journal of Haematology, 103, Ginaldi, L., De Martinis, M., Matutes, E., Farahat, N., Morilla, R., Dyer, M.J.S. & Catovsky, D. (1998) Levels of expression of CD52 in normal and leukaemic B and T cells; correlation with in vivo therapeutic responses to Campath -1H. Leukemia Research 22, Gritti, C., Dastot, H., Soulier, J., Janin, A., Daniel, M.T., Madani, A., Grimber, G., Briand, P., Sigaux, F. & Stern, M.H. (1998) Transgenic mice for MTCP1 develop T cell prolymphocytic leukemia. Blood, 92, Mallett, R.B., Matutes, E., Catovsky, D., MacLennan, K., Mortimer, P.S. & Holden, C.A. (1994) Cutaneous infiltration in T-cell prolymphocytic leukaemia. British Journal of Dermatology, 132, Matutes, E., Brito-Babapulle, V., Swansbury, J., Ellis, J., Morilla, R., Dearden, C., Sempere, A. & Catovsky, D. (1991) Clinical and laboratory features of 78 cases of T-prolymphocytic leukemia. Blood 78, Matutes, E. (1998) T-cell prolymphocytic leukemia. Cancer Control, 5, Matutes, E., Brito-Babapulle, V., Dearden, C., Yuille, M.R. & Catovsky, D. (2001) Prolymphocytic leukemia of B and T cell types: biology and therapy. Chapter 24 in: Chronic Lymphoid leukemias, 2nd Edition. Editor B Cheson; Marcel Dekker, Inc, New York, pp

114 Matutes, E. (2003). Chronic T-cell lymphoproliferative disorders. Reviews in Clinical and Experimental Hematology, 6, (2003) Pawson, R., Schulz, T.F., Matutes, E. & Catovsky, D. (1997) The human T-cell lymphotropic viruses types I/II are not involved in T-prolymphocytic leukemia and large granular lymphocytic leukemia. Leukemia 11, Pekarsky, U., Hallas, C. & Croce, C.M. (2001) Molecular basis of mature T-cell leukemia. Journal of the American Medical Association, 286, Pekarsky, Y., Hallas, C., Isobe, M., Russo, G. & Croce, C.M. (1999) Abnormalities at 14q32.1 in T cell malignancies involve two oncogenes. Proceedings of the National Academy of Sciences USA, 96, Stern, M.H., Soulier, J., Rosenzwajg, M., Nakahara, K., Canki-Klain, N., Aurias, A., Sigaux, F. & Kirsch, I.R. (1993) MTCP-1: a novel gene on the human chromosome Xq28 translocated on the T cell receptor alpha/delta locus in mature T cell proliferations. Oncogene 8, Salomon-Nguyen, F., Brizard, F., Le Coniat, M., Radford, I., Berger, R. & Brizard, A. (1998) Abnormalities of the short arm of chromosome 12 in T cell prolymphocytic leukaemia. Leukemia 12, Stilgenbauer, S., Schaffner, C., Litterst, A., Liebisch, P., Gilad, S., Bar-Shira, A., James, M.R., Lichter, P. & Dohner, H. (1997) Biallelic mutations in the ATM gene in T prolymphocytic leukemia. Nature Medicine 3, Taylor, A.M., Metcalfe, J.A., Thick, M. & Mak, Y.F. (1996) Leukaemia and lymphoma in ataxia telangiectasia. Blood 87, Tuset, E., Matutes, E., Brito-Babapulle, V., Morilla, R. & Catovsky, D. (2001) Immunophenotyping changes and loss of CD 52 expression in two patients with relapsed T cell prolymphocytic leukaemia. Leukemia Lymphoma, 42, Yullie, M.A., Coignet, L.J., Abraham, S.M., Yaqub, F., Luo, L., Matutes E., Brito- Babapulle, V., Vorechovsky, I., Dyer, M.J. & Catovsky, D. (1998) ATM is usually rearranged in T-cell prolymphocytic leukaemia. Oncogene, 16,

115 21 T-cell large granular lymphocytic leukaemia (T-cell LGL leukaemia) 21.1 Definition & clinical features This is an indolent T-cell disorder that is characterized by the expansion of neoplastic large granular lymphocytes (LGL). The pathogenesis is unknown, although it may well relate to an immune dysfunction. There is no evidence for the involvement of viruses. The disease mainly, but not exclusively, affects adults, who are asymptomatic or present with symptoms resulting from cytopenias, particularly neutropenia, and/or with incidentally discovered neutropenia or mild lymphocytosis. Lymphadenopathy is very rare, whilst a moderate splenomegaly is present in about 50% of patients. It is prevalent in patients with rheumatoid arthritis and autoantibodies to different tissue components are detected in up to 25% of patients. (Lamy and Loughran, 2003; Oshimi et al, 1993; Starkebaum et al, 1997) The course is chronic and transformation into a high grade lymphoma is exceedingly rare (Matutes et al, 2001) Peripheral blood Cytopenias are a common finding. The lymphocyte count may be normal or mildly increased with the predominant cell being a large granular lymphocyte (abundant cytoplasm containing azurophilic granules and an eccentric nucleus with condensed chromatin and no visible or small nucleolus). There is variability of nuclear shapes ranging from regular to irregular outlines and of the prominence of the azurophilic granules Bone marrow The bone marrow is normo- to hypercellular often with normal haemopoietic reserve. The aspirate may show a mild increase in LGL often with a good haemopoietic reserve, although there may be maturation arrest in the myeloid series. In trephine biopsy sections there is a subtle lymphoid interstitial infiltration with or without reactive nodular lymphoid aggregates (CD20+ B cells plus CD4+ cells).

116 (Morice et al, 2002) An intrasinusoidal component may be present and apoptotic figures are not rare. The infiltration may be missed unless immunohistochemical stains are performed. (Evans et al, 2000) 21.4 Lymph node & spleen Lymph nodes are rarely available for examination. Spleen histology shows an expansion of the red pulp without invasion of the white pulp, which often shows hyperplasia of germinal centres with expansion of the mantle zone.(osuji et al, 2005) 21.5 Immunophenotype Table 22.1 Recommended Panel for the diagnosis of T-cell LGL leukaemia Immunofluorescence Immunohistochemistry CD2 Positive Positive CD3 Positive Positive CD4 Usually negative Usually negative CD5 Positive or negative Positive or negative CD7 Positive or negative Positive or negative CD8 Positive Positive TCRβ Positive CD10 CD16 Positive Positive CD45R0 CD56 Often negative Often negative CD57 Positive Sometimes positive CD52 Positive N/A TdT CD19 CD20 CD79a CD22 N/A EMA Usually negative EBV- EBER (ISH) Usually negative Perforin Positive Positive TIA-1 Positive Positive Granzyme B Positive Positive Myeloperoxidase/CD68 CD158 (KIRs) Positive or negative N/A In a few cases (<15%), LGLs are either CD4+ or CD4+/CD8+ and cells from these cases often co-express NK associated markers. In a minority of patients

117 the LGL express TCRγδ instead of the more usual TCRαβ. (Lamy and Loughran, 1999; 2003) CD52 is expressed in LGL (Osuji et al, 2005) 21.6 Cytogenetics and molecular genetics No distinct chromosomal abnormality has been documented, but DNA analysis by PCR/Southern-blot will show a clonal rearrangement of TCRB or TCRG. Clonality can also be demonstrated with immunostaining with monoclonal antibodies against the variable regions of the TCRβ chain or by the expression of specific KIRs (CD158). (Lima et al, 2001; Langerak et al, 2001; Morice et al, 2003; Epling-Burnette et al, 2004) 21.7 Prognostic and therapeutic markers CD56+ cases may have a more aggressive clinical course (Gentile et al, 1994) 21.8 Pitfalls TCRβ negative whilst TCRγ positive CD4 expression or coexpression or lack of expression of CD4/CD Differential diagnosis Reactive T-cell LGL proliferations (i.e., viral infections, post-splenectomy) Other mature T-cell neoplasms such as smouldering T-PLL Peripheral T-cell lymphomas, essentially hepatosplenic T-cell lymphoma Natural killer (NK) cell proliferations Recommendations Recommendations for the diagnosis of T-LGL leukaemia Diagnostic criteria Level of evidence Morphology: Large granular lymphocytes Grade C level IV Immunophenotype; CD2, CD3, CD, CD57, CD16 positive

118 T-cell clonality should be established by DNA analysis of the TCR genes. An alternative is immunophenotyping with monoclonal antibodies against the TCR variable regions of the B chain References Epling-Burnette, P.K., Painter, J.S., Chaurasia, P., Bai, F., Wei, S. & Djeu J.Y (2004) Dysregulated NK receptor expression in patients with lymphoproliferative disease of granular lymphocytes. Blood, 103, Evans, H.L., Burks, E., Viswanatha, D. et al. (2000). Utility of immunohistochemistry in bone marrow evaluation of T lineage large granular lymphocyte leukemia. Human Pathology, 31, Foucar, K., Matutes, E. & Catovsky, D. (2004) T-Cell large granular lymphocytic leukemia, T-cell prolymphocytic leukaemia and aggressive natural-killer cell leukemia/lymphoma. In: Non-Hodgkins Lymphomas. (PM Mauch, JO Armitage, B Coiffier, R Dalla-Favera, NL Harris Editors) Lippincott Williams & Wilkins. Philadelphia, pp Gentile, T.C., Uner, A.H., Hutchison. R.E., Wright. J., Ben-Ezra. J., Russell. E.C. & Loughran, T.P. Jr. (1994) CD3+, CD56+ aggressive variant of large granular lymphocyte leukemia. Blood, 84, Handgretinger, R., Geiselhart, A., Moris, A., Grau, R., Teuffel, D., Bethge, W., Kanz, L. & Fisch, P. (1999) Pure red cell aplasia associated with clonal expansion of granular lymphocytes expressing killer cell inhibitory receptors. New England Journal of Medicine, 340, Langerak, A.W., van den Beemd, R., Wolvers-Tettero, I.L., Boor, P.P., van Lochen, E.G., Hooijkaas, H. & van Dongen, J.J. (2001) Molecular and flow cytometric analysis of the Vbeta repertoire for clonality assessment in mature TCR alpha/beta T-cell proliferations. Blood, 98, Lamy, T., Loughran, T.P Jr. (1999) Current concepts: large granular lymphocyte leukemia. Blood Reviews, 13, Lamy, T., Loughran, T.P Jr. (2003) Clinical features of large granular lymphocyte leukemia. Seminars in Hematology, 40, Lima, M., Almeida, J., Santos, A.H., dos Anjos Teixeira, M., Alguero, M.C., Queiros, M.L., Balanzategui, A., Justica, B., Gonzalez, M., San Miguel, J.F. & Orfao, A. (2001) Immunophenotypic analysis of the TCR-Vbeta repertoire in 98 persistent expansions of CD3(+)/TCR-alpha-beta (+) large granular lymphocytes: utility in assessing clonality and insights in the pathogenesis of the disease. American Journal Pathology, 54,

119 Matutes, E., Wotherspoon, A.C., Parker, N.E., Osuji, N., Isaacson, P.G. & Catovsky, D. (2001) Transformation of T-cell large granular lymphocyte leukaemia into a high-grade large T-cell lymphoma. British Journal of Haematology, 115, Matutes, E., Coelho, E., Aguado, M.J., Morilla, R., Crawford, A., Owusu- Ankomah, K. & Catovsky, D. (1996) Expression of TIA-1 and TIA-2 in T cell malignancies and T cell lymphocytosis. Journal of Clinical Pathology, 49, Morice, W.G., Kurtin, P.J., Tefferi, A. & Hanson, C.A. (2002) Distinct bone marrow findings in T-cell granular lymphocytic leukemia revealed by paraffin section immunoperoxidase stains for CD8, TIA-1 and granzyme B. Blood, 99, Morice, W.G., Kurtin, P.J., Leibson, P.J, Tefferi, A. & Hanson C.A. (2003) Demonstration of aberrant T-cell and natural killer-cell antigen expression in all cases of granular lymphocytic leukaemia. British Journal of Haematology, 120, Melenhorst, J.J., Sorbara, L., Kirby, M., Hensel, N.F. & Barrett A.J. (2001) Large granular lymphocyte leukaemia is characterized by a clonal T cell receptor rearrangement in boht memory and effector CD8+ lymphocyte populations. British Journal of Haematology, 112, Oshimi, K., Yamada, O., Kaneko, T., Nishinarita, S., Iizuka, Y., Urabe, A., Inamori, T., Asano, S., Takahashi, S. & Hattori, M. (1993) Laboratory findings and clinical courses of 33 patients with granular lymphocyte proliferative disorders. Leukemia, 7, Osuji, N., Matutes, E., Catovsky, D., Lampert, I. & Wotherspoon, A. (2005). Histopathology of the spleen in T-cell large granular lymphocyte leukemia and T-cell prolymphocytic leukemia. A comparative review. American Journal of Surgical Pathology, 29, Osuji, N., del Guidice, I., Matutes, E., Morilla, A., Owusu-Ankomah, K., Morilla, R., Dunlop, A. & Catovsky, D. (2005) CD52 expression in T-cell lerge granular lymphocyte leukemia-implications for treatment with alemtuzumab. Leukemia Lymphoma, 46, Semenzato, G., Zambello, R., Starkebaum, G., Oshim,i K. & Loughran, T.P. Jr. (1997) The lymphoproliferative disease of granular lymphocytes: updated criteria for diagnosis. Blood, 89, Starkebaum, G., Loughran, T.P. Jr., Gaur, L.K., Davis, P. & Nepom, B.S. (1997).Immunogenetic similarities between patients with Felty's syndrome and those with clonal expansions of large granular lymphocytes in rheumatoid arthritis. Arthritis and Rheumatism, 40,

120 22 Aggressive natural killer (NK)-cell leukaemia 22.1 Definition & clinical features A rare neoplasm characterized by the proliferation of natural killer (NK) cells. There is an overlap between aggressive NK-cell leukaemia and extranodal NK/Tcell lymphoma, nasal type. The WHO underlines that aggressive NK-cell leukaemia might represent the leukaemic manifestation of the latter. It is more frequent in East Asia and has a strong association to EBV. Patients are young adults. Coagulopathy, multiorgan failure and haemophagocytic syndrome have been documented. The clinical course is aggressive. (Imamura et al, 1990; Hart et al, 1992; Chan et al, 1997; Sun et al, 1993). The possibility that there is an indolent form of NK-cell LGL leukaemia with similar features to T-cell LGL has been considered by the WHO in the discussion of aggressive NK-cell leukaemia Peripheral blood Cytopenias and a leukaemic picture are common. The circulating cells are medium sized with relatively abundant cytoplasm with or without granulation; the nuclear shape varies from round to slightly irregular, chromatin condensation is variable and a small nucleolus may or not be present. The cells in chronic NK-LGL are similar to the LGL of T-cell LGL leukaemia 22.3 Bone marrow The aspirate often shows infiltration by cells with similar morphology to those in the peripheral blood. It is also possible to see reactive histiocytes and haemophagocytosis in some cases. Trephine biopsy shows a variable pattern of infiltration ranging from subtle to diffuse. Apoptotic cells and necrosis are common; angioinvasion may be present 22.4 Lymph node & other organs There is an interstitial to diffuse infiltration by neoplastic cells in the organs affected 22.5 Immunophenotype

121 Table 23.1 recommended Panel for diagnosis of aggressive NK-cell leukaemia Immunofluorescence Immunohistochemistry CD2 Positive Positive CD3 CD3-epsilon Positive CD4 CD5 CD7 Usually negative Usually negative CD8 TCRβ CD16 Positive or negative Variable CD56 Positive Positive CD57 Usually negative Usually negative TdT CD19 CD20 CD22 N/A CD15 EMA Positive EBV- EBER (ISH) Positive TIA-1 Positive or negative Positive Granzyme B Positive Positive Myeloperoxidase/CD68 CD158 (KIRs) Positive or negative N/A Note: TdT may be weakly expressed Cytogenetics and molecular genetics There is no unique chromosomal abnormality associated with this neoplasm. Complex abnormalities, particularly involving chromosome 6, have been described; however, the latter is also seen in plasmacytoid dendritic cell leukaemias. TCRB and TCRG are in germ line configuration. EBV, when present, shows a clonal (episomal) pattern of integration Prognostic tests There are no specific markers for prognosis. Chronic NK-LGL may have a clinical course similar to that of T-cell LGL 22.8 Pitfalls The weak expression of TdT and occasionally of CD103 can cause confusion with other forms of leukaemia

122 22.9 Differential diagnosis Acute lymphoblastic leukaemia (ALL), in particular T-cell ALL Acute myeloid leukaemias Blastic plasmacytoid dendritic cell neoplasm T-cell LGL (in the indolent/chronic form of NK-LGL) Recommendations Recommendations for the diagnosis of aggressive NK-cell leukaemia Diagnostic criteria Immunophenotype; CD2+, CD3epsilon+, CD56+, CD5-, CD4-, CD8-, CD11b+/-, CD16+/-, CD57-/+, EBER+, TCR in germ line configuration References Level of evidence Grade C level IV Bayerl, M.G., Rakozy, C.K., Mohamed, A.N., Vo, T.D., Long, M., Eilender, D. & Palutke, M. (2002). Blastic natural killer cell lymphoma/leukemia: a report of seven cases. American Journal of Clinical Pathology, 117, Chan, J.K., Sin, V.C., Wong, K.F., Ng, C.S., Tsang, W.Y., Chan, C.H., Cheung, M.M. & Lau, W.H. (1997) Non nasal Lymphoma Expressing the Natural Killer Cell Marker CD56: A Clinicopathologic Study of 49 Cases of an Uncommon Aggressive Neoplasm. Blood, 89, Foucar, K., Matutes, E. & Catovsky, D. (2004) T-Cell large granular lymphocytic leukemia, T-cell prolymphocytic leukaemia and aggressive natural-killer cell leukemia/lymphoma. In: Non-Hodgkins Lymphomas. (PM Mauch, JO Armitage, B Coiffier, R Dalla-Favera, NL Harris Editors) Lippincott Williams & Wilkins. Philadelphia, pp Hart, D.N., Baker, B.W., Inglis, M.J., Nimmo, J.C., Starling, G.C., Deacon, E., Rowe, M., Beard, M.E. (1992). Epstein Barr viral DNA in acute large granular lymphocyte (natural killer) leukemic cells. Blood, 79, Imamura, N., Kusunoki, Y., Kawa-Ha, K., Yumura, K., Hara, J., Oda, K., Abe, K., Dohy, H., Inada, T., Kajihara, H. et al.(1990) Aggressive natural killer cell leukaemia/lymphoma: report of four cases and review of the literature. Possible existence of a new clinical entity originating from the third lineage of lymphoid cells. British Journal of Haematology, 75,

123 Koita, H., Suzumiya, J., Ohshima, K., Takeshita, M., Kimura, N., Kikuchi, M. & Koono, M. (1997) Lymphoblastic lymphoma expressing natural killer cell phenotype with involvement of the mediastinum and nasal cavity. American Journal of Surgical Pathology, 21, Matutes, E. & Osuji, N. (2004). Clinical and morphological features of natural killer (NK) cell disorders. Haematologica, 89 (supp 1), Mori, K.L., Egashira, M. & Oshimi, K. (2001). Differentiation stage of natural killer cell lineage lymphoproliferative disorders based on phenotypic analysis. British Journal of Haematology, 115, Nakamura, S., Koshikawa, T., Yatabe, Y. & Suchi, T. (1998) Lymphoblastic lymphoma expressing CD56 and TdT. American Journal of Surgical Pathology, 22, Nakamura, M.C. Natural killer cells and their role in disease. (2002). Laboratory Medicine, 33, Natkunam, Y., Warnke, R.A., Zahnder, J.L. & Cornbleet, P.J. (1999) Aggressive natural killer like T cell malignancy with leukemic presentation following solid organ transplantation. American Journal of Clinical Pathology, 21, Ohno, T., Kanoh, T., Arita, Y., Kuribayashi, K., Masuda, T., Horiguchi, Y., Taniwaki, M., Nosaka, T. & Hatanaka, M. (1988) Fulminant clonal expansion of large granular lymphocytes. Characterization of their morphology, phenotype, genotype, and function. Cancer, 62, Sun, T., Brody, J., Susin, M., Marino, J., Teichberg, S., Koduru, P., Hall, W.W., Urmacher, C. & Hajdu, S.I. (1993) Aggressive natural killer cell lymphoma/leukemia. A recently recognized clinicopathologic entity. American Journal of Surgical Pathology, 17, Suzuki, R., Nakamura, S., Suzumiya, J., Ichimura, K., Ichikawa, M., Ogata, K., Kura, Y., Aikawa, K., Teshima, H., Sako, M., Kojima, H., Nishio, M., Yoshino, T., Sugimori, H., Kawa, K. & Oshimi, K; NK-cell tumor study. (2005) Blastic natural killer cell lymphoma/leukemia (CD56-positive blastic tumor): prognostication and categorization according to anatomic sites of involvement. Cancer, 104,

124 23 Adult T cell leukaemia/lymphoma 23.1 Definition & clinical features Adult T-cell leukaemia/lymphoma (ATLL) is a peripheral T-cell neoplasm of pleomorphic activated T lymphocytes aetiologically linked to the human retrovirus HTLV-1. ATLL and HTLV-1 are prevalent in Japan, the Caribbean, certain regions of South-America and Africa and in immigrants from these countries to other regions. In the UK, ATLL is mainly, but not exclusively, seen in first or second generation immigrants of Afro-Caribbean descent (Matutes and Catovsky, 1998).The disease manifests in 75% of cases with leukaemia and in the remaining as a pure lymphomatous form. (Shimoyama et al, 1991) Usually the disease is disseminated, although chronic and smouldering forms have been recognized. Hypercalcaemia is common. A few cases present with isolated extranodal disease Peripheral blood The incidence of anaemia and thrombocytopenia is variable. In the leukaemic forms, the white cell count is raised with circulating atypical cells. The picture is pleomorphic with the predominant cell being a medium size lymphocyte with condensed chromatin and a polylobated nucleus (flower cell); nucleoli are not visible and when identified are small. The cytoplasm is medium in amount or scanty and is agranular. A few blast-like or immunoblast-like cells may be present. In the lymphomatous form of ATLL, the peripheral blood is not involved Bone marrow The aspirate may show infiltration by lymphocytes with the same morphological features as the cells in the blood. Trephine biopsy histology may or not show lymphoid infiltration with variable degree of involvement. Even in the leukaemic forms of ATLL, the infiltration may be very subtle or patchy. The pattern of infiltration ranges from interstitial to diffuse. A distinct feature in some cases with hypercalcaemia is the presence of marked osteoclastic activity with bone resorption.

125 23.4 Lymph node and other tissues The infiltration in lymph nodes is often diffuse, with the paracortical area being expanded by an infiltrate of lymphocytes of different size and nuclear shape; as in the peripheral blood, pleomorphism is often present. There may be some immunoblast-like and Reed-Sternberg-like cells (Duggan, et al, 1988). The latter appear to be non-neoplastic EBV-infected cells, thought to be secondary to the immunodeficiency resulting from the HTLV-1 infection. Overall, the histological picture of ATLL is not distinctive and the histological features may be indistinguishable from those of other peripheral T-cell lymphomas (Lennert et al, 1984; Oshimi et al, 1998). Skin histology will show lymphoid infiltrates in the dermis; epidermotropism is present in up to a half of the cases with a pattern indistinguishable from that of mycosis fungoides and Sézary syndrome Immunophenotype Table 24.1 Recommended Panel for the diagnosis of ATLL Immunofluorescence Immunohistochemistry CD2 Positive Positive CD3 Positive weak Positive CD4 Positive Positive CD5 Positive Positive CD7 CD8 CD25 Positive Positive TCRβ Positive (weak) N/A CD10 CD16 CD56 CD57 TdT CD19 or CD20

126 CD30 N/A Positive or negative in large cells CD15 N/A ALKc N/A EMA N/A EBV- EBER (ISH) N/A TIA-1 Granzyme B N/A Note: Strong expression of CD25 in almost all cases. T-cell activation markers (HLA-DR and CD38) may be positive. Rarely the phenotype is CD4+/CD8+ or cells lack expression of these two antigens 23.6 Cytogenetics and molecular genetics Cytogenetic analysis often shows a complex karyotype, particularly in the leukaemic forms, but so far no distinct abnormality has been documented in ATLL. The TCR genes are consistently clonally rearranged. Virtually all ATLL cases have a clonal or oligoclonal integration of HTLV-1 by Southern blot analysis. (Yamaguchi, 1994) PCR analysis with probes specific to the various fragments of HTLV-1 is an alternative that can be applied in both blood and tissue sections. This investigation is not usually required for the diagnosis. It has a role in patients who are seronegative for HTLV-1 but in whom there is a strong suspicion of ATLL and in cases of smouldering ATLL, to distinguish such patients from a healthy carrier of the virus. There is an overwhelming literature regarding the mechanisms of HTLV-1 oncogenesis. (Iwanaga et al, 2001) This seems to be complex but particularly mediated by the HTLV-1 tax gene through dysregulation of a variety of cellular (onco) genes in the lymphocytes by a trans-activation mechanism Prognostic and therapeutic markers Clinical forms, age, elevation of lactate dehydrogenase and the presence of hypercalcaemia are the main prognostic factors 23.8 Pitfalls

127 CD30 expression in the large cells can raise the possibility of classical Hodgkin lymphoma but the morphology and immunophenotype should distinguish between ATLL and chl. There can be unusual phenotypes in which cells are are CD8+ or have either co-expression or complete absence of both CD4 and CD Differential diagnosis Peripheral T-cell lymphoma, not otherwise specified Mycosis fungoides/sézary syndrome in the cutaneous ATLL forms CD30+ T-cell lymphomas Post-thymic T-cell leukaemias such as T-PLL Healthy HTLV-1 carriers in the case of evolving smouldering ATLL Recommendations Recommendations for the diagnosis of ATLL Diagnostic criteria HTLV-1 positive Histology of the lymph node is not needed for diagnosis in cases with a leukaemic form of ATLL in whom cytology, immunophenotyping and HTLV-I serology are typical of the disease Level of evidence Grade B, level IIa Grade C level IV Immunophenotype: CD2+, CD3+, CD4+, CD8-, CD25+, CD References Duggan, D., Ehrlich, G., Davey, F., Kwok, S., Sninsky, J., Goldberg, J., Baltrucki, L. & Poiesz, B.J. (1988) HTLV-I induced lymphoma mimicking Hodgkin s disease. Diagnosis by polymerase chain reaction amplification of specific HTLV-I sequences in tumor DNA. Blood, 71, Iwanaga, R., Ohtani, K., Hayashi, T. & Nakamura, M. (2001) Molecular mechanisms of cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I. Oncogene, 20, Lennert, K., Kikuchi, M., Sato, E., Suchi, T., Stansfeld, A.G., Feller, A.C., Hansmann, M.L., Muller-Hermelink H.K. & Godde-Salz, E. (1984) HTLV

128 positive and negative T-cell lymphomas. Morphological and immunohistochemical differences between European and HTLV-positive Japanese T-cell lymphomas. International Journal of Cancer, 35, Mahieux, R. & Gessain, A. (2003) HTLV-1 and associated adult T-cell leukemia/lymphoma. Reviews of Clinical and Experimental Hematology, 7, Matutes, E. & Catovsky, D. (1998) Adult T cell leukaemia lymphoma. Chapter 18 in: Leukaemia (3rd Edition) (Ed. J.A. Whittaker) Blackwell Scientific Publications, Oxford Ohshima, K., Suzumiya, J., Sato, K., Kanda, M., Sugihara, M., Haraoka, S., Takeshita, M. & Kikuchi, M. (1998) Nodal T-cell lymphoma in an HTLV-1 endemic area; proviral HTLV-1 DNA, histological classification and clinical evaluation. British Journal of Haematology, 101, Shimoyama, M. (1991) Diagnostic criteria and classification of clinical subtypes of adult T-cell leukemia-lymphoma: a report from the Lymphoma Study group ( ). British Journal of Haematology, 79, Tobinai, K., Watanabe, T. & Jaffe, E.S. (2004) Human T-cell leukemia virus type I associated adult T-cell leukemia lymphoma. In: Non-Hodgkins Lymphomas. (PM Mauch, JO Armitage, B Coiffier, R Dalla-Favera, NL Harris Editors) Lippincott Williams & Wilkins. Philadelphia, chapter 18 (pp: )

129 24 Classical Hodgkin lymphoma 24.1 Definition & clinical features Classical Hodgkin lymphomas usually arise in lymph nodes, typically in young adults. The infiltrated tissue has a relatively small number of scattered large mononucleated and multinucleated tumour cells (designated Hodgkin and Reed- Sternberg cells (HRS cells)) residing in an abundant heterogeneous mixture of non-neoplastic inflammatory and accessory cells (Jaffe, et al 2001). There are four subtypes of classical Hodgkin lymphoma known as Nodular sclerosis Mixed cellularity Lymphocyte-rich Lymphocyte-depleted Hodgkin lymphoma typically presents in one or more lymph nodes, but may be disseminated to other lymph nodes or to liver, spleen, mediastinum and bone marrow. The disease is staged according to the Ann Arbor staging classification (Lister, et al 1989) Peripheral blood Non-specific haematological abnormalities are common, e.g. anaemia of chronic disease, neutrophilia, eosinophilia, lymphopenia Bone marrow Bone marrow involvement at presentation is uncommon (especially now that most cases previously diagnosed as lymphocyte-depleted Hodgkin lymphoma has been reclassified as non-hodgkin lymphoma). Hodgkin lymphoma in the bone marrow is more frequent in HIV-positive patients and at relapse. Usually the abnormal cells are not aspirable so that histology is vital in making the diagnosis of marrow involvement Lymph node

130 All subtypes possess classical HRS cells, although the prototypical cells may be rare in some cases Nodular sclerosis classical Hodgkin lymphoma This is distinguished by prominent nodules, dense fibrotic bands and a thickened capsule. Sometimes, depending on fixation, HRS cells within the areas of fibrosis show retraction of their cytoplasmic membranes and appear to lie within an empty space or lacuna (so-called lacunar cells) Mixed cellularity classical Hodgkin lymphoma This is characterised by scattered HRS cells in a diffuse or at most vaguely nodular background. The capsule is not thickened. The lymph node architecture is generally effaced though occasional patients have an interfollicular infiltration with good preservation of non-neoplastic follicles Lymphocyte-rich classical Hodgkin lymphoma This is usually characterised by only a few scattered HRS cells, which are often associated with the mantle zones of residual reactive germinal centres Lymphocyte-depleted classical Hodgkin lymphoma This is the rarest variant of classical Hodgkin lymphoma. It is characterised by a diffuse population of HRS cells without the accompanying mixed cell infiltrate of the other variants. Many if not most of those cases diagnosed before immunophenotyping was available have been reclassified as non-hodgkin lymphoma. The diagnosis should only be made if all of the parameters for Hodgkin lymphoma, especially the immunophenotype, are supportive. In experienced hands a confident diagnosis of classical Hodgkin lymphoma may be possible on the basis of fine needle aspiration cytology, although most haematopathologists would recommend a follow up biopsy for subtyping and immunophenotyping Other tissues Effusions occur rarely; they may contain a lymphoid population with occasional HRS cells.

131 24.6 Immunophenotype: This is determined by the immunostaining profile of the HRS cells not the associated stromal and inflammatory cells (Carbone, et al 2002, Garcia-Cosio, et al 2004, Jaffe, et al 2001). Table 22.1 Recommended Panel for the diagnosis of classical Hodgkin lymphoma Marker Immunohistochemistry CD2/CD3 CD20 Weak -/+ CD79a Weak -/+ CD45 CD30 Positive CD15 Variable +/- CD246 (ALK1) EMA Weak -/+ EBV-LMP1/ EBER (ISH) Weak -/+ OCT2 BOB1 J chain MUM1/IRF4 Positive PAX5 Positive CD10 BCL6 CD75 CD Cytogenetics & molecular genetics There are no consistent genetic changes Prognostic & therapeutic markers

132 Subtyping shows a small difference with the order from best to worst being lymphocyte rich, nodular sclerosis, mixed cellularity and lymphocyte deleted Pitfalls It is important to assess CD15 staining carefully expression is frequently focal and may be negative (25% of cases) Differential Diagnosis Diffuse large B-cell lymphoma T-cell/histiocyte-rich large B-cell lymphoma Nodular lymphocyte predominant Hodgkin lymphoma (MUM1/IRF4 variable) ALK-negative anaplastic large cell lymphoma (PAX5 -) Recommendations Recommendations for the diagnosis of classical Hodgkin lymphoma Diagnostic criteria Immunophenotype; CD30, CD15, MUM1/IRF4 positive CD20, CD79a weak Cytogenetics are not required References Level of evidence Grade C level IV Carbone, A., Gloghini, A., Aldinucci, D., Gattei, V., Dalla-Favera, R. & Gaidano, G. (2002) Expression pattern of MUM1/IRF4 in the spectrum of pathology of Hodgkin's disease. Br J Haematol, 117, Garcia-Cosio, M., Santon, A., Martin, P., Camarasa, N., Montalban, C., Garcia, J.F. & Bellas, C. (2004) Analysis of transcription factor OCT.1, OCT.2 and BOB.1 expression using tissue arrays in classical Hodgkin's lymphoma. Mod Pathol, 17, Jaffe, E.S., Harris, N.L., Stein, H. & Vardiman, J.W. (eds.) (2001) Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon. Lister, T.A., Crowther, D., Sutcliffe, S.B., Glatstein, E., Canellos, G.P., Young, R.C., Rosenberg, S.A., Coltman, C.A. & Tubiana, M. (1989) Report of a committee convened to discuss the evaluation and staging of patients with Hodgkin's disease: Cotswolds meeting. J Clin Oncol, 7,

133 25 Nodular lymphocyte predominant Hodgkin lymphoma 25.1 Definition & clinical features Nodular lymphocyte predominant Hodgkin lymphoma is a monoclonal B-cell neoplasm, characterised by a nodular or nodular and diffuse, polymorphous proliferation of scattered large neoplastic B cells known as popcorn or L&H cells (lymphocytic and/or histiocytic Reed Sternberg like cells) or LP (lymphocyte predominant) cells. Most of these cells reside in or around follicular structures resembling progressively transformed germinal centres. This condition typically presents with localised lymphadenopathy. Disseminated disease at presentation is rare but becomes more frequent with progression. The disease develops slowly with periods of remission and relapse, but may be cured by therapy at any stage. The disease is rarely fatal apart from the infrequent cases of transformation into a diffuse large B-cell lymphoma (Huang, et al 2003) Peripheral blood There is no effect on peripheral blood 25.3 Bone marrow Bone marrow involvement is rare but when present is usually paratrabecular, resembling follicular lymphoma in the marrow (Khoury, et al 2004) Lymph node The lymph node is typically replaced by variably sized nodules that have many features in common with reactive progressively transformed germinal centres. Many of these may be associated with small epithelioid granulomas. The diagnosis rests on finding typical L&H (popcorn) cells both within and around these nodules. These cells are typically surrounded by a corona of reactive T cells. Variant histology is common, including examples with a more diffuse or T- cell-rich pattern Immunophenotype

134 This is determined by the immunostaining profile of the L&H cells not the associated reactive lymphoid cells in or around the nodules (Dogan, et al 2000, Jaffe, et al 2001). Table 26.1 Recommended panel for diagnosis of Nodular Lymphocyte Predominant Hodgkin Lymphoma Marker Immunocytochemistry CD2/CD3 CD20 Positive CD79a Positive CD45 Positive CD30 CD15 CD246 (ALK1) EMA Weak EBV-LMP1/ EBER (ISH) OCT2 Positive BOB1 Positive J chain Positive MUM1/IRF4 PAX5 Positive CD10 BCL6 Positive CD75 Positive/ CD Cytogenetics & molecular genetics There are no reliable tests that aid diagnosis 25.7 Prognostic and therapeutic tests

135 An accurate diagnosis is important as the disease itself has a very good prognosis 25.8 Pitfalls It is easy to over interpret apparently focally involved nodes Differential Diagnosis T-cell/histiocyte-rich large B-cell lymphoma (Boudova, et al 2003) Follicular lymphoma Lymphocyte-rich classical Hodgkin lymphoma Reactive follicular hyperplasia especially with progressive transformation of germinal centres Recommendations Recommendations for the diagnosis of Nodular Lymphocyte Predominant Hodgkin Lymphoma Diagnostic criteria Immunophenotype; CD20, CD79a, BCL6 positive CD30, CD15, MUM1/IRF4, CD10 negative Cytogenetics are not required References Level of evidence Grade C level IV Boudova, L., Torlakovic, E., Delabie, J., Reimer, P., Pfistner, B., Wiedenmann, S., Diehl, V., Muller-Hermelink, H.K. & Rudiger, T. (2003) Nodular lymphocyte-predominant Hodgkin lymphoma with nodules resembling T- cell/histiocyte-rich B-cell lymphoma: differential diagnosis between nodular lymphocyte-predominant Hodgkin lymphoma and T-cell/histiocyte-rich B- cell lymphoma. Blood, 102, Dogan, A., Bagdi, E., Munson, P. & Isaacson, P.G. (2000) CD10 and BCL-6 expression in paraffin sections of normal lymphoid tissue and B-cell lymphomas. Am J Surg Pathol, 24, Huang, J.Z., Weisenburger, D.D., Vose, J.M., Greiner, T.C., Aoun, P., Chan, W.C., Lynch, J.C., Bierman, P.J. & Armitage, J.O. (2003) Diffuse large B- cell lymphoma arising in nodular lymphocyte predominant hodgkin lymphoma. A report of 21 cases from the Nebraska Lymphoma Study Group. Leuk Lymphoma, 44,

136 Jaffe, E.S., Harris, N.L., Stein, H. & Vardiman, J.W. (eds.) (2001) Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon. Khoury, J.D., Jones, D., Yared, M.A., Manning, J.T., Jr., Abruzzo, L.V., Hagemeister, F.B. & Medeiros, L.J. (2004) Bone marrow involvement in patients with nodular lymphocyte predominant Hodgkin lymphoma. Am J Surg Pathol, 28,

137 26 Histiocytic sarcoma 26.1 Definition & clinical features A malignant proliferation of cells showing features resembling mature tissue histiocytes. This rare tumour usually presents as a solitary mass and can arise in lymph node, skin or other extra-nodal sites. Some patients show a systemic presentation with widespread disease at multiple sites ( malignant histiocytosis ). Systemic symptoms such as fever and weight loss are common, as are hepatosplenomegaly and pancytopenia (van der Walk & Meijer 1985, Schmidt, 1994) Peripheral blood This is usually normal 26.3 Bone marrow The aspirate is usually normal. Trephine biopsy may show involvement in advanced cases Lymph node One third of cases present with lymph node involvement, and nodes show an effaced architecture with diffuse sheets of large cells. The latter are pleomorphic and range from round to spindle cell shaped. Multinucleate forms may be present and there may be haemophagocytosis by the tumour cells Immunophenotype Table 27.1 Recommended Panel for diagnosis of Histiocytic Sarcoma Marker Immunohistochemistry CD1a CD3 CD4 (some weak positive) CD11c Weak/focal positive CD45 Positive CD163 Positive (Vos et al 2005)

138 CD20 CD79a CD30 HLA-DR S100 CD68 Myeloperoxidase Lysozyme Weak/focal positive Weak/focal positive Positive variable strength Weak/focal positive 26.6 Cytogenetics & molecular genetics There is no cytogenetic signature for this disease, and both TCR and IGH genes show a germline configuration (Sun, et al 2003) Prognostic & therapeutic markers There are no specific prognostic markers 26.8 Pitfalls: It is important to establish that there is negative staining for cytokeratins and melanoma markers. The morphology of histiocytic sarcoma can resemble anaplastic large cell lymphoma or diffuse large B-cell lymphoma, and these should be excluded immunohistochemically. Nodal or soft tissue deposits of monocytic cells many be seen in the rare form of myeloid sarcoma associated with acute monoblastic leukaemia ( monoblastic sarcoma ) Differential diagnosis Anaplastic large cell lymphoma Diffuse large B-cell lymphoma Recommendations Recommendations for the diagnosis of Histiocytic Sarcoma Diagnostic criteria Level of evidence

139 Nodal or extranodal infiltration by sheets of pleomorphic cells with occasional multinucleate giant cells. Some cases show haemophagocytosis. Immunophenotype; CD68, lysozyme, CD11c positive. CD20, CD3, CD1a, EMA, CD30. Cytogenetics are not required Grade C level IV References Schmidt, D. (1994) Monocyte/macrophage system and malignancies. Medical and Pediatric Oncology, 23, Sun, W., Nordberg, M.L. & Fowler, M.R. (2003) Histiocytic sarcoma involving the central nervous system: clinical, immunohistochemical, and molecular genetic studies of a case with review of the literature. American Journal of Surgical Pathology, 27, van der Valk, P. & Meijer, C.J. (1985) Histiocytic sarcoma. Clinical picture, morphology, markers, differential diagnosis. Pathology Annual, 20, Pt2;1-28. Vos, J.A., Abbondanzo, S.L., Barekman, C.L., Andriko, J.W., Miettinen, M. & Aguilera, N.S. Histiocytic sarcoma: a study of five cases including the histiocyte marker CD163. (2005) Modern Pathology, 18,

140 27 Langerhans cell histiocytosis (LCH) 27.1 Definition & clinical features This is a dendritic cell neoplasm in which the proliferating cells show the morphological and phenotypic features of Langerhans cells. The age range of affected patients is broad, but most cases arise in childhood (Arico, 2004). There is a male predilection, and LCH can be seen in association with lymphomas, both Hodgkin and non-hodgkin types (Christie, et al 2006). The clinical patterns of LCH have been divided into three groups, but there is a degree of overlap. Unifocal cases (eosinophil granuloma) usually involve bone, most commonly the skull, femur, pelvis or ribs. Multifocal single-system cases (Hand-Schuller-Christian disease) show involvement of multiple sites in one organ system (usually bone) and in multi-focal multi-system cases (Letterer-Siwe disease) there is involvement of multiple organ systems including bone, skin, liver spleen and lymph nodes. The overall prognosis is generally related to the extent of the disease (Hicks & Flaitz, 2005, Stockschlaeder & Sucker, 2006) Peripheral Blood There may be pancytopenia due to hypersplenism Bone marrow The aspirate may be infiltrated with a population of Langerhans cells as well as macrophages with an increase in eosinophils. Haemophagocytosis can be seen in some cases. The cells are irregular in shape as are the nuclei, which have finely clumped chromatin and faint nucleoli. The cytoplasm can contain azurophilic granules and is weakly basophilic. Ttrephine biopsy sections can show either clusters or sheets of cells, the nuclei of which have a distorted, twisted appearance. The infiltrate also has large numbers of accompanying macrophages and eosinophils Lymph node The majority of cases are extranodal, but nodal disease can be seen. There is an infiltrate of pale cells with grooved or folded nuclei, thin nuclear membranes and abundant eosinophilic cytoplasm. There is a variable associated population of

141 eosinophils and macrophage-derived giant cells. Electron microscopy may be useful in difficult cases, where Birbeck granules may be identified Immunophenotype Table 25.1 Recommended Panel for the diagnosis of Langerhans Cell Histiocytosis Marker Immunohistochemistry CD1a Positive CD3 CD4 Weak/focal positive CD45 Positive CD20 CD79a HLA-DR Weak/focal positive S100 Positive CD207 (langerin) Positive (Chikwava & Jaffe 2004) Factor XIIIa CD68 Weak/focal positive CD163 Weak/focal positive (Nguyen et al 2005) Myeloperoxidase Lysozyme Positive (40% of cases) PNA (peanut agglutinin) staining may also be positive. (Pileri, et al 2002) 27.6 Cytogenetics & molecular genetics There is no cytogenetic signature for this disease, and both TCR and IGH genes show a germline configuration Prognostic and therapeutic markers There are no specific prognostic or therapeutic markers for this diagnosis 27.8 Pitfalls

142 No specific features 27.9 Differential diagnosis Non-Langerhans histiocytoses and monoblastic leukaemias Recommendations Recommendations for the diagnosis of Langerhans Cell Histiocytosis Diagnostic criteria Immunophenotype. S100, CD1a positive, negative for myeloid, T, B and NK lineage markers Cells with Langerhans morphology References Level of evidence Grade C level IV Arico, M. (2004) Langerhans cell histiocytosis in adults: more questions than answers? European Journal of Cancer, 40, Chikwava, K. & Jaffe, R. (2004) Langerin (CD207) staining in normal pediatric tissues, reactive lymph nodes, and childhood histiocytic disorders. Pediatric and Developmental Pathology, 7, Christie, L.J., Evans, A.T., Bray, S.E., Smith, M.E., Kernohan, N.M., Levison, D.A. & Goodlad, J.R. (2006) Lesions resembling Langerhans cell histiocytosis in association with other lymphoproliferative disorders: a reactive or neoplastic phenomenon? Human Pathology, 37, Hicks, J. & Flaitz, C.M. (2005) Langerhans cell histiocytosis: current insights in a molecular age with emphasis on clinical oral and maxillofacial pathology practice. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics, 100, S Nguyen, T.T., Schwartz, E.J., West, R.B., Warnke, R.A., Arber, D.A. & Natkunam, Y. Expression of CD163 (hemoglobin scavenger receptor) in normal tissues, lymphomas, carcinomas, and sarcomas is largely restricted to the monocyte/macrophage lineage. (2005) American Journal of Surgical Pathology, 29, Pileri, S.A., Grogan, T.M., Harris, N.L., Banks, P., Campo, E., Chan, J.K., Favera, R.D., Delsol, G., De Wolf-Peeters, C., Falini, B., Gascoyne, R.D., Gaulard, P., Gatter, K.C., Isaacson, P.G., Jaffe, E.S., Kluin, P., Knowles, D.M., Mason, D.Y., Mori, S., Muller-Hermelink, H.K., Piris, M.A., Ralfkiaer, E., Stein, H., Su, I.J., Warnke, R.A. & Weiss, L.M. (2002) Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases. Histopathology, 41, 1-29.

143 Stockschlaeder, M. & Sucker, C. (2006) Adult Langerhans cell histiocytosis. European Journal of Haematology, 76,

144 28 Langerhans cell sarcoma 28.1 Definition & clinical features A neoplastic proliferation of Langerhans cells that show overtly malignant histological features. Langerhans cell sarcoma is extremely rare. There is a wide age range and a female predominance has been reported. The condition shows multi-organ involvement with disease affecting lymph nodes, liver, spleen, lung and bone. The prognosis is poor with an overall survival of 50% Peripheral blood There may be pancytopenia due to hypersplenism Bone marrow There are no specific features 28.4 Lymph node and other tissues Histology is essential for the diagnosis. There is an infiltrate of large pleomorphic cells with nuclear atypia, a high mitotic rate, and prominent nucleoli (Ben Ezra, et al 1991). Some morphological features of Langerhans cells may be present nuclear grooves are occasionally seen Immunophenotype Table 26.1 Recommended Panel for the Diagnosis of Langerhans Sarcoma Marker Immunohistochemistry CD1a Weak/focal positive CD3 CD20 CD79a S100 Weak/focal positive CD207 (langerin) Weak/focal positive CD68 Weak/focal positive Lysozyme Weak/focal positive CD31 Positive (Ferringer, et al 2006)

145 CD56 Positive in some cases (Kawase, et al 2005) PLAP Positive (Hage, et al 1993) Ki % 28.6 Cytogenetics & molecular genetics There is no cytogenetic signature for this disease, and both TCR and IGH genes show a germline configuration Prognostic and therapeutic markers: There are no specific prognostic or therapeutic markers for this diagnosis 28.8 Pitfalls There are no specific features 28.9 Differential diagnosis Langerhans cell sarcoma needs to be differentiated from other, non-langerhans, histiocytoses and anaplastic neoplasms, including carcinomas, sarcomas and melanomas Recommendations Recommendations for the diagnosis of Langerhans Cell Sarcoma Diagnostic criteria Immunophenotype; S100, CD1a positive, negative for myeloid, T, B and NK lineage markers Cells with Langerhans morphology References Level of evidence Grade C level IV Ben Ezra, J., Bailey, J., Azumi, N., Delsol, G., Stroup, R., Sheibani, K. & Rappaport, H. (1991) Malignant histiocytosis X. A distinct clinico pathological entity. Cancer, 68, Ferringer, T., Banks, P.M. & Metcalf, J.S. (2006) Langerhans Cell Sarcoma. Am J Dermatopathology, 28,

146 Hage, C., Willman, C., Favara, B. & Isaacson, P. (1993) Langerhans' cell histiocytosis (histiocytosis X): immunophenotype and growth fraction. Hum Pathol., 24, Kawase, T., Hamazaki, M., Ogura, M., Kawase, Y., Murayama, T., Mori, Y., Nagai, H., Tateno, M., Oyama, T., Kamiya, Y., Taji, H., Kagami, Y., Naoe, T., Takahashi, T., Morishima, Y. & Nakamura, S. (2005) CD56/NCAMpositive Langerhans cell sarcoma: a clinicopathologic study of 4 cases. Int J Hematol, 81,

147 29 Interdigitating dendritic cell sarcoma/tumour 29.1 Definition & clinical features A neoplastic proliferation of spindle shaped or ovoid cells showing features of interdigitating reticulum cells. This lesion most commonly presents with solitary lymph node involvement, but can be seen in skin or at other extra-nodal sites (Banner, et al 1997). The patient usually complains of an asymptomatic mass. The prognosis is generally good - local resection can be curative (Fonseca, et al 1998) Peripheral blood This not usually involved Bone marrow This not usually involved Lymph node Lymph nodes show a paracortical infiltrate in which the cells have a fascicular pattern with whorls and storiform areas. The cells are rounded and have ovoid or spindled nuclei, distinct nucleoli and abundant eosinophilic cytoplasm. The cytological grade of the lesion is variable, hence the designation of sarcoma or tumour. There are frequently admixed lymphocytes and occasionally plasma cells within the tumour. Electron microscopy may be of value (Pillay, et al 2004) Immunophenotype Table 27.1 Recommended Panel for diagnosis of interdigitating dendritic cell sarcoma/ tumour Marker Immunohistochemistry CD1a CD3 CD45 CD20 CD79a CD21

148 CD30 S100 CD68 Myeloperoxidase Positive Weak positive (Pileri, et al 2002, Jayaram, et al 2005) 29.6 Cytogenetics & molecular genetics There is no cytogenetic signature for this disease, and both TCR and IGH genes show a germline configuration Prognostic and therapeutic markers There are no specific prognostic or therapeutic markers 29.8 Pitfalls There are no specific features 29.9 Differential diagnosis There are no specific features Recommendations Recommendations for the diagnosis of interdigitating dendritic cell sarcoma/ tumour Diagnostic criteria Immunophenotype; S100 positive and CD1a negative, no lineage staining for B-cells, T-cells, and NK cells. Nodal infiltrate of round cells with abundant cytoplasm forming fascicles and whorls References Level of evidence Grade C level IV Banner, B., Beauchamp, M.L., Liepman, M. & Woda, B.A. (1997) Interdigitating reticulum-cell sarcoma of the intestine: a case report and review of the literature. Diagnostic Cytopathology, 17, Fonseca, R., Yamakawa, M., Nakamura, S., van Heerde, P., Miettinen, M., Shek, T.W., Myhre Jensen, O., Rousselet, M.C. & Tefferi, A. (1998) Follicular

149 dendritic cell sarcoma and interdigitating reticulum cell sarcoma: a review. American Journal of Hematology, 59, Jayaram, G., Mun, K.S., Elsayed, E.M. & Sangkar, J.V. (2005) Interdigitating dendritic reticulum cell sarcoma: cytologic, histologic and immunocytochemical features. Diagnostic Cytopathology, 33, Pileri, S.A., Grogan, T.M., Harris, N.L., Banks, P., Campo, E., Chan, J.K., Favera, R.D., Delsol, G., De Wolf-Peeters, C., Falini, B., Gascoyne, R.D., Gaulard, P., Gatter, K.C., Isaacson, P.G., Jaffe, E.S., Kluin, P., Knowles, D.M., Mason, D.Y., Mori, S., Muller-Hermelink, H.K., Piris, M.A., Ralfkiaer, E., Stein, H., Su, I.J., Warnke, R.A. & Weiss, L.M. (2002) Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases. Histopathology, 41, Pillay, K., Solomon, R., Daubenton, J.D. & Sinclair-Smith, C.C. (2004) Interdigitating dendritic cell sarcoma: a report of four paediatric cases and review of the literature. Histopathology, 44,

150 30 Follicular dendritic cell sarcoma/tumour 30.1 Definition & clinical features A neoplastic proliferation of spindle shaped or ovoid cells showing features of follicular dendritic cells. Patients present with a slow-growing painless mass. The mass involves lymph nodes in two-thirds of the cases, and the cervical region is the most common site. Axillary, mediastinal, mesenteric and retroperitoneal lymph nodes may also be affected and extra-nodal sites can be involved (Biddle, et al 2002, Satoh, et al 2003). There is an association with hyaline-vascular Castleman disease. The behaviour is usually indolent and treatment is by surgical excision. Local recurrence occurs in 50% of cases and metastases are seen in 25% of patients (Fonseca, et al 1998, Hynes, et al 2005) Peripheral blood There are no specific identifying features 30.3 Bone marrow There are no specific identifying features 30.4 Lymph node: There is a nodal infiltrate composed of spindle or ovoid cells forming fascicles, whorls and storiform areas. The nuclei have a granular appearance with small nucleoli and the cytoplasm is eosinophilic. Occasional multinucleate giant cells or epithelioid cells are present. There are admixed small lymphocytes and some cases show foci of necrosis. Electron microscopy may be helpful Immunophenotype: Table 28.1 Recommended Panel for diagnosis of follicular dendritic cell sarcoma/ tumour Marker Immunohistochemistry CD1a CD3 CD20 CD79a

151 CD21 CD23 CD35 CD207 (langerin) Desmoplakin Fascin CD68 Myeloperoxidase Positive Positive Positive Positive in some cases Positive Positive Positive in some cases (Perez-Ordonez & Rosai, 1998, Pileri, et al 2002) 30.6 Cytogenetics & molecular genetics There are no specific cytogenetics changes and the TCR and IGH genes are germline Prognostic and therapeutic markers There are no specific prognostic or therapeutic markers for this diagnosis 30.8 Pitfalls CD20 is occasionally positive in this tumour Differential Diagnosis There are no specific features Recommendations Recommendations for the diagnosis of follicular dendritic cell sarcoma/ tumour Diagnostic criteria Immunophenotype; CD21+, CD23+, desmoplakin+, negative for myeloid, T, B and NK lineage markers Fascicular infiltrate of spindle or ovoid cells References Level of evidence Grade C level IV Biddle, D.A., Ro, J.Y., Yoon, G.S., Yong, Y.W., Ayala, A.G., Ordonez, N.G., & Ro J. (2002) Extranodal follicular dendritic cell sarcoma of the head and neck

152 region: three new cases, with a review of the literature. Modern Pathology, 15, Fonseca, R., Yamakawa, M., Nakamura, S., van Heerde, P., Miettinen, M., Shek T.W, Myhre Jensen, O, Rousselet, M.C, Tefferi, A. (1998) Follicular dendritic cell sarcoma and interdigitating reticulum cell sarcoma: a review. American Journal of Hematology, 59, Hynes, B., Lock, M., Lubynski, R., & Hynes, B. (2005) Follicular dendritic cell sarcoma: case report and review of current management principles. Journal of Otolaryngology, 34, Perez-Ordonez, B. & Rosai, J. (1998) Follicular dendritic cell tumor: review of the entity. Seminars in Diagnostic Pathology, 15, Pileri, S.A., Grogan, T.M., Harris, N.L., Banks, P., Campo, E., Chan, J.K., Favera, R.D., Delsol, G., De Wolf-Peeters, C., Falini, B., Gascoyne, R.D., Gaulard, P., Gatter, K.C., Isaacson, P.G., Jaffe, E.S., Kluin, P., Knowles, D.M., Mason, D.Y., Mori, S., Muller-Hermelink, H.K., Piris, M.A., Ralfkiaer, E., Stein, H., Su, I.J., Warnke, R.A. & Weiss, L.M. (2002) Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases. Histopathology, 41, Satoh, K., Hibi, G., Yamamoto, Y., Urano, M., Kuroda, M. & Nakamur,a S. (2003) Follicular dendritic cell tumor in the oro-pharyngeal region: report of a case and a review of the literature. Oral Oncology, 39,

153 31 Dendritic cell sarcoma, not otherwise specified 31.1 Definition & clinical features This category includes dendritic cell neoplasms that do not fall into one of the well-defined categories. The clinical behaviour of these rare lesions has not been well defined, and the diagnosis is largely one of exclusion (Rosenberg, et al 2001, Martins, et al 2004) Peripheral blood There are no documented disease specific features 31.3 Bone marrow There are no documented disease-specific features 31.4 Lymph node A malignant neoplasm with some histological and phenotypic features of dendritic cells, but no specific features indicating a follicular dendritic cell, interdigitating reticulum cell or Langerhans cell origin.(dillon, et al 2002, Trierveiler Martins, et al 2004) The histological features of this rare neoplasm are not defined. Electron microscopy may be helpful Immunophenotype: Table 29.1 Recommended Panel for diagnosis of Dendritic Cell Sarcoma Marker Immunohistochemistry CD1a Positive in some cases CD3 CD20 CD79a S100 Positive in some cases Myeloperoxidase 31.6 Cytogenetics & molecular genetics There are no specific cytogenetic changes and the TCR and IGH genes are germline Prognostic and therapeutic Markers

154 There are no specific prognostic or therapeutic markers for this diagnosis 31.8 Pitfalls There are no specific features 31.9 Differential Diagnosis Langerhans cell and non-langerhans cell histiocytoses Other dendritic cell neoplasms Recommendations Recommendations for the diagnosis of dendritic cell sarcoma, unclassified Diagnostic criteria Immunophenotype; CD1a & S100 positive at least focally, CD21, CD23 and desmoplakin negative negative for myeloid, T, B and NK lineage markers References Level of evidence Grade C level IV Dillon, K.M., Hill, C.M., Cameron, C.H.S., Attanoos, R.L. & McCluggage, W.G. (2002) Mediastinal mixed dendritic cell sarcoma with hybrid features. J Clin Pathol, 55, Martins, MT., Witzel, AL., Sugaya, N.N., Kowalski, L.P. & de Araujo, V.C. (2004) Dendritic cell sarcoma of the oral cavity. Oral Oncology, 40, Rosenberg, A.S. & Morgan, M.B. (2001) Cutaneous indeterminate cell histiocytosis: a new spindle cell variant resembling dendritic cell sarcoma. J Cutan Pathol, 28, Trierveiler Martins, M., Lusvarghi Witzel, A., Nobuo Sugaya, N., Paulo Kowalski, L. & Cavalcanti de Araujo, V. (2004) Dendritic cell sarcoma of the oral cavity. Oral Oncology, 40,