Chronic lymphocytic leukemia (CLL) and diffuse large B-

Transcription

1 Clinical Prognostic Biomarkers in Chronic Lymphocytic Leukemia and Diffuse Large B-Cell Lymphoma Elizabeth C. Chastain, MD; Eric J. Duncavage, MD Context. Diffuse large B-cell lymphoma and chronic lymphocytic leukemia are 2 of the most common B-cell lymphomas in adults. Both diffuse large B-cell lymphoma and chronic lymphocytic leukemia share heterogeneous outcomes, and the use of prognostic biomarkers to better stratify risk in these patients has now become commonplace. Objective. To review chronic lymphocytic leukemia and diffuse large B-cell lymphoma biomarkers commonly used in the clinical laboratory, which can be divided into the following 3 main groups by testing methodology: chromosomal based (including fluorescence in situ hybridization and cytogenetics), expression based (including immunohistochemistry and flow cytometry), and DNA based (including gene sequencing for somatic mutations and IGVH mutational status). Data Sources. Review of recent literature. Conclusions. In chronic lymphocytic leukemia, important biomarkers include expression of CD38 and ZAP-70, IGVH mutational status, somatic mutations in TP53 and NOTCH1, and abnormalities in chromosomes 11, 12, 13q, and 17. In diffuse large B-cell lymphoma, important biomarkers include chromosomal rearrangement of BCL2, BCL6, and MYC and expression of CD5, BCL2, and CD43, as well as somatic mutations in TP53 and BCL6. (Arch Pathol Lab Med. 2015;139: ; doi: / arpa ra) Chronic lymphocytic leukemia (CLL) and diffuse large B- cell lymphoma (DLBCL) are 2 of the most common B- cell malignancies in adults, affecting almost and more than patients, respectively, in the United States each year. 1 Overall survival for both lymphoma subtypes can vary widely, and lymphoma biomarkers have an important role in informed treatment decisions. Such biomarkers often offer improved risk stratification over clinical based staging systems, including the CLL staging systems by Rai et al 2 and by Binet et al 3 and the modified Ann Arbor staging system in DLBCL. 4 Commonly used prognostic lymphoma biomarkers can be generally divided into 2 categories involving either genotypic, DNA-level changes or phenotypic, expression-level changes. In the clinical laboratory, genotypic biomarkers are generally measured using fluorescence in situ hybridization (FISH) for the detection of chromosomal additions, deletions, or both or polymerase chain reaction based methods for the determination of immunoglobulin rearrangement status. Phenotypic expression markers are generally measured by flow cytometry when fresh tissue is available or by immunohistochemistry on formalin-fixed, paraffin-embedded tissue. Herein, we will review prognostic DLBCL and CLL biomarkers that are frequently used in the clinical laboratory. B-CELL CLL AND SMALL LYMPHOCYTIC LYMPHOMA Chronic lymphocytic leukemia is the most common leukemia in the Western world, accounting for up to 30% of all adult leukemias, 5 and is characterized by proliferation of clonal mature B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Diagnosis is established by demonstrating a peripheral blood lymphocytosis of /L or higher for at least 3 months, review of the blood smear morphology, and immunophenotyping of circulating lymphocytes. Chronic lymphocytic leukemia is a widely heterogeneous disease with regard to clinical presentation, progression, and treatment response and remains incurable with conventional chemotherapy. Chronic lymphocytic leukemia and small lymphocytic lymphoma biomarkers relevant to the clinical laboratory are summarized in Table 1. CHROMOSOMAL ABNORMALITIES AND COPY NUMBER ALTERATIONS Recurrent CLL-associated chromosomal copy number alterations are present in approximately 80% of cases and are readily identified by interphase FISH or array-based methods. 6,7 The most frequently identified abnormalities are listed below. Accepted for publication March 27, Published as an Early Online Release September 23, From the Department of Pathology and Immunology, Washington University, St Louis, Missouri. 13q14 Deletion The authors have no relevant financial interest in the products or The interstitial deletion of the long arm of chromosome companies described in this article. Reprints: Eric Duncavage, MD, Department of Pathology and 13 is detected in approximately 50% of CLL cases with Immunology, Washington University, 660 S Euclid Ave, Campus Box abnormal FISH results, making it the most common 8118, St Louis, MO ( [email protected]). cytogenetic abnormality. 6 This alteration is often found in 602 Arch Pathol Lab Med Vol 139, May 2015 CLL and DLBCL Biomarkers Chastain & Duncavage

2 Table 1. Biomarkers Associated With Chronic Lymphocytic Leukemia Abnormality Prognosis Detection Method Chromosomal abnormalities 13q del Favorable FISH, array-based þ12 Likely poor methods, karyotype 11q del Poor 17p del Poor Protein expression ZAP-70 Poor Flow cytometry CD38 Poor IGVH mutational status Unmutated Poor Sequencing Hypermutation Favorable VH3-21 use Poor Recurrent somatic gene mutations TP53 Poor Sequencing NOTCH1 Poor Abbreviation: FISH, fluorescence in situ hybridization. patients also harboring mutated immunoglobulin heavychain variable (IGVH) region genes, a subset associated with favorable outcomes (see IGVH Mutational Status ). Eighty percent of cases show monoallelic loss, with the remaining 20% showing biallelic deletion as determined by high-resolution single-nucleotide polymorphism array profiling. 8 The 13q14 deletion appears to involve a critical region containing the DLEU2 gene, encoding mir15a and mir16-1, which act to increase expression of the antiapoptotic gene BCL2, among other targets. 9 Deletion of 13q14 in the absence of other recurrent cytogenetic findings confers the best prognosis of all chromosomal changes; however, when deletion 13q14 is associated with additional recurrent FISH, the favorable prognostic outcome is ameliorated. Trisomy 12 Less than 20% of CLL cases with chromosomal abnormalities harbor trisomy 12. This alteration has not been extensively studied, and the exact molecular mechanism is uncertain. In isolation, trisomy 12 may indicate somewhat shorter survival than in patients without a recurrent cytogenetic abnormality. 10 There appears to be an association between Notch homolog 1, translocation associated (NOTCH1) somatic mutations and trisomy 12 (see NOTCH1 Mutations ). 11q Deletion Deletion of 11q occurs in 10% to 20% of patients with CLL having abnormal FISH results. Most cases include deletion at 11q22.3 of the ataxia-telangectasia mutated (ATM) gene locus. 11 The ATM protein product is an upstream regulator responsible for the activation of the p53 tumor suppressor. Loss of ATM function results in defects in DNA repair, with possible accumulation of additional genomic lesions and prosurvival and progrowth pathway activation. Patients with this deletion tend to be younger, have significant lymphadenopathy with rapid clinical disease progression, and demonstrate somewhat shorter survival compared with those without del11q. 12,13 17p Deletion Five to ten percent of CLL cases with copy number changes will have deletion of the short arm of chromosome 17 and are associated with allelic loss of the TP53 gene, resulting in reduced cycle control and DNA damage repair. The 17p deletions are frequently biallelic (see TP53 Mutations ), and some cases may demonstrate duplication of the remaining allele, resulting in copy-neutral loss of heterozygosity. 11,14 It should be noted that copy-neutral loss heterozygosity in the setting of point mutations will result in likely dysfunctional p53 with a normal interpretation of FISH. 15 Deletion of 17p confers the shortest survival time among all subsets of CLL with cytogenetic abnormalities and is an independent adverse prognostic factor. 6 f-chain ASSOCIATED PROTEIN KINASE 70 AND CD38 PROTEIN EXPRESSION Immunophenotypic evaluation by flow cytometry of f- chain associated protein kinase 70 (ZAP-70) and CD38 may be used to predict the clinical course of CLL and is frequently part of standard diagnostic panels. ZAP-70 is a cytoplasmic kinase in the T-cell receptor signaling complex (not expressed on normal B lymphocytes) that is expressed in CLL cells. 16 CD38 is a surface cyclic adenosine diphosphate ribose glycoprotein that functions to support many B-cell interactions and has a role in differentiation. 17 Expression of one or both of these proteins generally indicates a worse prognosis. Both markers are used as surrogates for IGVH mutational status and are independent prognostic factors. 18,19 Concordance between ZAP-70 expression (typically.20% of cells) and IGVH mutational status ranges from 80% to 95%, with significantly higher ZAP-70 expression in CLL cases having unmutated IGVH status compared with mutated cases. 20,21 In the absence of high-risk cytogenetic findings, ZAP-70 expression and IGVH mutational status are strongly correlated. 22 Findings have indicated that elevated CD38 expression (usually.30% of CLL cells) is correlated with the mutational status of IGVH genes; however, up to one-third of patients may show discordant results. 23 CD38 expression is correlated with other clinical adverse prognostic factors, including advanced disease stage and disease progression. 24,25 Discordance between ZAP-70 and CD38 expression is observed in 30% to 40% of patients and is associated with outcomes intermediate between concordant-positive and concordantnegative cases. 18 IGVH MUTATIONAL STATUS Immunoglobulin gene alterations are a normal process during the development and differentiation of B lymphocytes, including somatic mutations with the variable region of the heavy chain. Two subsets of CLL can be determined based on the presence or absence (unmutated,.98% homology with germline) of somatic hypermutation by molecular sequencing methods. These 2 subsets differ in many clinical and prognostic aspects. Patients with CLL cells containing IGVH with somatic hypermutation are likely to have lower Binet and Rai clinical stage disease, del13q, and the absence of CD38 and ZAP-70 expression, all of which are associated with a favorable prognosis. 17,19 In contrast, those with unmutated IGVH tend to have more advanced disease stages and more aggressive clinical course and tend to acquire high-risk cytogenetic alterations and be Arch Pathol Lab Med Vol 139, May 2015 CLL and DLBCL Biomarkers Chastain & Duncavage 603

3 associated with CD38 and ZAP-70 expression. Survival in these patients is significantly shorter, irrespective of the disease stage. The specific variable region VH3-21 is an exception in that, irrespective of the IGVH mutational status, findings indicate that rearrangement of this region is associated with an adverse prognosis. 26 clinical stage, and trisomy 12, all related to shorter overall survival and progression-free survival than in patients without NOTCH1 mutations. In addition, these studies have documented an association between the presence of a NOTCH1 mutation and transformation to DLBCL (Richter syndrome). RECURRENT SOMATIC GENE MUTATIONS The emergence of clinical next-generation sequencing (NGS) technology has led to a more comprehensive evaluation of somatic mutations in many malignancies, including CLL. Next-generation sequencing provides simultaneous analysis of numerous genes with high throughput and increased sensitivity. Whole-exome investigations of large cohorts of CLL show a very heterogeneous landscape of somatic mutations, with only a few genes mutated in 10% to 15% of cases and a large number of genes mutated at lower frequencies (2% 5%). 27 TP53 and NOTCH1 gene mutations fall in the former category and are described in further detail below. TP53 Mutations TP53 is a tumor suppressor gene and is the most commonly mutated gene in cancer; however, the TP53 mutation rate is lower in hematologic malignancies compared with that in solid tumors. At initial diagnosis, 5% to 15% of CLL cases have at least 1 somatic mutation in TP53, and the incidence increases (up to 40%) in patients with prior treatment, indicating clonal evolution. 13,28 TP53 mutations are most frequently located within the DNAbinding domain (typically exons 5 9), with missense variants being the most common and the remainder being indels, nonsense, or splice-site variants. 28 These alterations result in a truncated or altered protein structure, leading to partial or absent protein function. To date, most laboratories use FISH for evaluation of large-scale TP53 loss, but 25% to 45% of patients may harbor mutations without allelic loss. 29 This demonstrates the increasing importance of complete evaluation by sequencing methods. New guidelines by the European Research Initiative on CLL 30 recommend testing for both allelic loss by FISH and mutational testing by sequencing. All studies agree that TP53 mutations in CLL result in a poor prognosis, with almost all patients requiring therapy and many cases being refractory to treatment. 13,31,32 NOTCH1 Mutations The NOTCH1 gene encodes a transmembrane protein that functions as a ligand-activated transcription factor, regulating cell differentiation, proliferation, and apoptosis, and has been shown to have an important role in normal T-cell development. 33 Most mutations occur within exon 34 in the transcription activation domain (TAD) and the rich in proline (P), glutamic acid (E), serene (S), and threonine (T) domain (PEST). 34,35 The most common variant is a 2-base pair frameshift deletion within the PEST domain (p.p2515rfs*4), resulting in overexpression of a truncated and more stable protein. Mutations in these domains upregulate the NOTCH1 signaling pathwayandseemtobeassociatedwithamoreaggressive disease course in hematologic malignancies. 36 Studies 37,38 have shown that NOTCH1 mutations in CLL identify a subgroup more frequently associated with unmutated IGVH, high expression of ZAP-70 and CD38, advanced DIFFUSE LARGE B-CELL LYMPHOMA Diffuse large B-cell lymphoma is an aggressive lymphoma that has been partially subdivided into distinct entities based on morphology and molecular and immunophenotypic characteristics. However, most cases remain heterogeneous, without features of a distinct subdivision, and are referred to as DLBCL, not otherwise specified. Most DLBCL cases arise de novo, but DLBCL may transform from a lower-grade lymphoma such as CLL or follicular lymphoma. Diagnosis is established by complete or partial architectural effacement of lymphoid tissues by a medium to large cell infiltrate. Response to treatment is highly variable, with some patients achieving complete clinical remission, while others fail multiple therapeutic regimens and succumb to disease progression. In contrast to CLL, little consensus exists regarding the most clinically relevant DLBCL biomarkers, which is reflected in the variability of DLCBL biomarker testing across clinical laboratories. 38 Diffuse large B-cell lymphoma biomarkers relevant to the clinical laboratory are summarized in Table 2. ANTIGEN EXPRESSION CD5 CD5 expression as measured by flow cytometry or immunohistochemical staining is present in approximately 10% of DLBCL cases, 39 and most cases are suspected transformations of CD5 þ low-grade lymphoma. While expression itself is not an independent prognostic indicator, it is more commonly associated with adverse clinical prognostic features, including advanced age, extranodal involvement at presentation, and higher-stage disease at diagnosis. 40,41 BCL2 Approximately 50% of DLBCLs express BCL2 by immunohistochemistry due to a BCL2 rearrangement, nuclear factor jb activation, or 18q21 amplification. 42 Studies 43,44 have shown increased BCL2 expression to be highly relevant and significantly associated with decreased event-free and overall survival. The observed frequency of high BCL2 expression is increased in nodal DLBCL compared with extranodal DLBCL. 45 Ki-67 The Ki-67 proliferation index expression is highly variable, generally ranging from 20% to 80% and occasionally reaching 80% to 100%. 46,47 The prognostic influence is controversial, with some studies 48,49 demonstrating that a higher proliferation index is associated with a poor prognosis, while other studies 46,50 show that expression is associated with a good prognosis. EBV Epstein-Barr virus (EBV) encoded RNA positivity by in situ hybridization is associated with specific subclassifications of DLBCL, including EBV-positive DLBCL of the elderly, plasmablastic lymphoma, and primary effusion lymphoma. It is often seen in cases of immune dysregula- 604 Arch Pathol Lab Med Vol 139, May 2015 CLL and DLBCL Biomarkers Chastain & Duncavage

4 Table 2. Biomarkers Associated With Diffuse Large B-Cell Lymphoma Abnormality Prognosis Detection Method Antigen expression CD5 Poor Flow cytometry, IHC BCL2 Poor IHC CD43 Poor Flow cytometry, IHC Ki-67 mitotic index Variable IHC EBV Poor ISH Cell of origin GCB variant Favorable IHC Non GCB/ABC variant Poor Chromosomal abnormalities BCL2 rearrangement Neutral FISH BCL6 rearrangement Favorable MYC rearrangement Poor Double hit (MYC þ BCL2) Poor Recurrent somatic gene mutations TP53 Poor Sequencing BCL6 Favorable Abbreviations: ABC, activation of peripheral blood B cell; EBV, Epstein- Barr virus; FISH, fluorescence in situ hybridization; GCB, germinal center B cell; IHC, immunohistochemistry; ISH, in situ hybridization. tion or immunosuppression. These malignancies typically follow aggressive clinical courses; therefore, EBV expression is associated with a poor prognosis. 51,52 CD43 CD43 (sialophorin) is a type I transmembrane glycoprotein that is expressed primarily on the surface of T cells, monocytes, and granulocytes. CD43 is expressed in approximately 20% of DLBCLs, and expression of CD43 has been correlated with a reduced overall and event-free overall survival compared with CD43-negative DLBCL. 53,54 CELL-OF-ORIGIN CLASSIFICATION Perhaps the most controversial DLBCL biomarkers involve so-called cell-of-origin testing. Gene expression profiling investigations have successfully identified 2 prognostically distinct molecular subgroups of DLBCL. 55 The first group demonstrated an expression pattern similar to that of normal germinal center B cells (GCB variant), and the second group showed a pattern consistent with activation of peripheral blood B cells (ABC variant). No specific morphologic features are correlated with either subtype, and the GCB variant showed significantly better overall and event-free survival compared with the ABC variant. Gene expression profiling microarray studies are typically not used in the clinical laboratory because of technical challenges associated with collecting fresh tissue for RNA extraction and limiting batch effects in cases run individually; however, numerous immunohistochemical algorithms that serve as surrogates for gene expression profiling have been developed and may be used for classification of DLBCL into GCB and non-gcb or ABC variants. 56 The algorithm by Hans et al 44 (Figure, A) is probably one of the most commonly used and relies on the presence of CD10 and Bcl-6 expression in the absence of MUM1 expression to classify a GCB variant. In the absence of CD10, lack of Bcl-6 expression or Bcl-6 expression with MUM1 positivity indicates a non-gcb variant. Other commonly used immunohistochemical cell-of-origin classification schemes include the algorithm by Choi et al 57 (Figure, B), in which 2 to 4 immunohistochemical stains are evaluated (including GCET1, MUM1, CD10, BCL6, and FOXP1), and a tally algorithm 56 (Figure, C), which uses 4 to 5 immunohistochemical stains (including CD10, GCET1, MUM1, FOXP1, and LMO2). 56 Concordance between these immunohistochemical algorithms and gene expression profiling is variable, ranging from 74% to 93%. While most of these algorithms were designed before the use of rituximab, studies 58,59 indicate that the prognostic importance remains significant even with Commonly used cell-of-origin classification algorithms in diffuse large B-cell lymphoma (DLBCL). A, Hans algorithm. B, Choi algorithm. C, Tally algorithm, in which all 4 stains are performed on all cases and scored. If the numbers of germinal center B-cell positive and activation of peripheral blood B-cell positive immunohistochemistry (IHC) stains are equal, an LMO2 stain is run as a tiebreaker. Arch Pathol Lab Med Vol 139, May 2015 CLL and DLBCL Biomarkers Chastain & Duncavage 605

5 the addition of this monoclonal antibody to standard chemotherapeutic regimens. CHROMOSOMAL ABNORMALITIES BCL2 Rearrangement Approximately 20% of DLBCL cases harbor a BCL2 rearrangement due to t(14;18)(q32;q21) translocation. This same translocation is often identified in follicular lymphoma, and the presence of BCL2 rearrangement in DLBCL may indicate transformation from a follicular lymphoma or may represent a de novo process. Some studies 42,60 have indicated no prognostic significance of BCL2 rearrangement in DLBCL, although it tends to be associated with the GCB subtype; however, other investigators have described shorter disease-free survival or decreased response to therapy in patients with BCL2 rearrangement. 61 BCL6 Rearrangement BCL6 rearrangement (3q27) occurs in approximately 30% of DLBCL cases. Series have indicated a lack of BCL6 rearrangement to be associated with an inferior outcome. 60,62 However, other investigators found that those having non Ig/BCL6 fusion reportedly experience a worse outcome compared with those having Ig/BCL6 fusion. 63 MYC Rearrangement The hallmark of Burkitt lymphoma is the presence of a MYC rearrangement (8q24); however, approximately 10% of DLBCL cases will contain a MYC rearrangement. In the case of DLBCL, the partner is more often a nonimmunoglobulin gene. 64 Detection of a MYC rearrangement is associated with poor outcomes. 60,62 Double-Hit DLBCL Double-hit DLBCL describes a subset of cases that show evidence of both a MYC rearrangement and a concurrent IGH-BCL2 rearrangement. Typically, double-hit DLBCL has distinct morphologic findings, including an increased mitotic rate and an appearance similar to that of Burkitt lymphoma. Double-hit DLBCL is characterized by a more aggressive clinical course and reduced overall survival compared with non double-hit DLBCL RECURRENT SOMATIC GENE MUTATIONS BCL6 Mutations Approximately 70% of DLBCL cases contain somatic mutations of BCL6; these are independent of BCL6 rearrangement. 68 Mutations indicate that the cell has been through the germinal center, and expression secondary to mutations inhibits apoptosis and differentiation, leading to cell proliferation. The presence of these somatic mutations is suggested to be a favorable prognostic marker. 69 TP53 Mutations TP53 mutations are present in approximately 20% of DLBCLs. The role that TP53 mutations have in the pathogenesis of DLBCL is not entirely clear, but it is hypothesized to be associated with histologic transformation from low-grade lymphomas in some cases. 70,71 Similar to mutations described above in CLL, mutations of TP53 in DLBCL are also associated with a poor prognosis and with unfavorable complete remission rates. 72 CONCLUSION The lymphoma biomarkers included in this review represent commonly used assays with established prognostic/diagnostic significance. However, given the rapid pace of discovery in sequencing-based lymphoma studies coupled with the growth of sequencing-based diagnostics in the clinical laboratory, a greater number of lymphoma biomarkers is anticipated to become available in the coming years. Such lymphoma biomarkers will have to be carefully evaluated to establish independent prognostic and diagnostic significance beyond the currently used assays. This study was supported by the Department of Pathology and Immunology, Washington University. References 1. Division of Cancer Control and Population Sciences, National Cancer Institute. Surveillance, Epidemiology, and End Results Program: SEER data, Accessed January 15, Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS. Clinical staging of chronic lymphocytic leukemia. Blood. 1975;46(2): Binet JL, Auquier A, Dighiero G, et al. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer. 1981;48(1): Lister TA, Crowther D, Sutcliffe SB, et al. Report of a committee convened to discuss the evaluation and staging of patients with Hodgkin s disease: Cotswolds meeting [published correction appears in J Clin Oncol. 1990;8(9): 1602]. J Clin Oncol. 1989;7(11): Swerdlow SH, Campo E, Harris NL, Jaffe ES, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. International Agency for Research on Cancer: Lyon, France; Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343(26): Malek SN. The biology and clinical significance of acquired genomic copy number aberrations and recurrent gene mutations in chronic lymphocytic leukemia. Oncogene. 2013;32(23): Ouillette P, Collins R, Shakhan S, et al. The prognostic significance of various 13q14 deletions in chronic lymphocytic leukemia. Clin Cancer Res. 2011;17(21): Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and downregulation of micro- RNA genes mir15 and mir16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2002;99(24): Chiorazzi N. Implications of new prognostic markers in chronic lymphocytic leukemia. Hematology Am Soc Hematol Educ Program. 2012; 2012: Ouillette P, Collins R, Shakhan S, et al. Acquired genomic copy number aberrations and survival in chronic lymphocytic leukemia. Blood. 2011;118(11): Neilson JR, Auer R, White D, et al. Deletions at 11q identify a subset of patients with typical CLL who show consistent disease progression and reduced survival. Leukemia. 1997;11(11): Shanafelt TD, Witzig TE, Fink SR, et al. Prospective evaluation of clonal evolution during long-term follow-up of patients with untreated early-stage chronic lymphocytic leukemia. J Clin Oncol. 2006;24(28): Malcikova J, Smardova J, Rocnova L, et al. Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival, and response to DNA damage. Blood. 2009;114(26): Saddler C, Ouillette P, Kujawski L, et al. Comprehensive biomarker and genomic analysis identifies p53 status as the major determinant of response to MDM2 inhibitors in chronic lymphocytic leukemia. Blood. 2008;111(3): Crespo M, Bosch F, Villamor N, et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med. 2003;348(18): Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med. 2005;352(8): Del Principe MI, Del Poeta G, Buccisano F, et al. Clinical significance of ZAP-70 protein expression in B-cell chronic lymphocytic leukemia. Blood. 2006; 108(3): Rassenti LZ, Jain S, Keating MJ, et al. Relative value of ZAP-70, CD38, and immunoglobulin mutation status in predicting aggressive disease in chronic lymphocytic leukemia. Blood. 2008;112(5): Rassenti LZ, Huynh L, Toy TL, et al. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med. 2004;351(9): Wiestner A, Rosenwald A, Barry TS, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood. 2003; 101(12): Arch Pathol Lab Med Vol 139, May 2015 CLL and DLBCL Biomarkers Chastain & Duncavage

6 22. Kröber A, Bloehdorn J, Hafner S, et al. Additional genetic high-risk features such as 11q deletion, 17p deletion, and V3-21 usage characterize discordance of ZAP-70 and VH mutation status in chronic lymphocytic leukemia. J Clin Oncol. 2006;24(6): Lin K, Sherrington PD, Dennis M, Matrai Z, Cawley JC, Pettitt AR. Relationship between p53 dysfunction, CD38 expression, and IgV H mutation in chronic lymphocytic leukemia. Blood. 2002;100(4): Matrai Z. CD38 as a prognostic marker in CLL. Hematology. 2005;10(1): Boonstra JG, van Lom K, Langerak AW, et al. CD38 as a prognostic factor in B cell chronic lymphocytic leukaemia (B-CLL): comparison of three approaches to analyze its expression. Cytometry B Clin Cytom. 2006;70(3): Tobin G, Thunberg U, Johnson A, et al. Somatically mutated Ig V H 3 21 genes characterize a new subset of chronic lymphocytic leukemia. Blood. 2002; 99(6): Quesada V, Conde L, Villamor N, et al. Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia. Nat Genet. 2011;44(1): Zenz T, Eichhorst B, Busch R, et al. TP53 mutation and survival in chronic lymphocytic leukemia. J Clin Oncol. 2010;28(29): Hallek M, Cheson BD, Catovsky D, et al; International Workshop on Chronic Lymphocytic Leukemia. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute Working Group 1996 guidelines [published correction appears in Blood. 2008;112(13): 5259]. Blood. 2008;111(12): Pospisilova S, Gonzalez D, Malcikova J, et al; European Research Initiative on CLL (ERIC). ERIC recommendations on TP53 mutation analysis in chronic lymphocytic leukemia. Leukemia. 2012;26(7): Stilgenbauer S, Sander S, Bullinger L, et al. Clonal evolution in chronic lymphocytic leukemia: acquisition of high-risk genomic aberrations associated with unmutated VH, resistance to therapy, and short survival. Haematologica. 2007;92(9): Trbusek M, Malcikova J, Mayer J. Selection of new TP53 mutations by therapy in chronic lymphocytic leukemia. Leuk Res. 2011;35(7): Ferrando AA. The role of NOTCH1 signaling in T-ALL. Hematology Am Soc Hematol Educ Program. 2009: Fabbri G, Rasi S, Rossi D, et al. Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation. J Exp Med. 2011;208(7): Puente XS, Pinyol M, Quesada V, et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature. 2011; 475(7354): Weng AP, Ferrando AA, Lee W, et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science. 2004;306(5694): Rossi D, Rasi S, Fabbri G, et al. Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia. Blood. 2012;119(2): Villamor N, Conde L, Martínez-Trillos A, et al. NOTCH1 mutations identify a genetic subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor outcome. Leukemia. 2013;27(5): Yamaguchi M, Seto M, Okamoto M, et al. De novo CD5 þ diffuse large B- cell lymphoma: a clinicopathologic study of 109 patients. Blood. 2002;99(3): Harada S, Suzuki R, Uehira K, et al. Molecular and immunological dissection of diffuse large B cell lymphoma: CD5 þ, and CD5 with CD10 þ groups may constitute clinically relevant subtypes. Leukemia. 1999;13(9): Yamaguchi M, Ohno T, Oka K, et al. De novo CD5-positive diffuse large B- cell lymphoma: clinical characteristics and therapeutic outcome. Br J Haematol. 1999;105(4): Gascoyne RD, Adomat SA, Krajewski S, et al. Prognostic significance of Bcl-2 protein expression and Bcl-2 gene rearrangement in diffuse aggressive non- Hodgkin s lymphoma. Blood. 1997;90(1): Barrans SL, Carter I, Owen RG, et al. Germinal center phenotype and bcl-2 expression combined with the International Prognostic Index improves patient risk stratification in diffuse large B-cell lymphoma. Blood. 2002;99(4): Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103(1): Villuendas R, Piris MA, Orradre JL, Mollejo M, Rodriguez R, Morente M. Different bcl-2 protein expression in high-grade B-cell lymphomas derived from lymph node or mucosa-associated lymphoid tissue. Am J Pathol. 1991;139(5): Hall PA, Richards MA, Gregory WM, d Ardenne AJ, Lister TA, Stansfeld AG. The prognostic value of Ki67 immunostaining in non-hodgkin s lymphoma. J Pathol. 1988;154(3): Kreipe H, Wacker HH, Heidebrecht HJ, et al. Determination of the growth fraction in non-hodgkin s lymphomas by monoclonal antibody Ki-S5 directed against a formalin-resistant epitope of the Ki-67 antigen. Am J Pathol. 1993; 142(6): Miller TP, Grogan TM, Dahlberg S, et al. Prognostic significance of the Ki- 67 associated proliferative antigen in aggressive non-hodgkin s lymphomas: a prospective Southwest Oncology Group trial. Blood. 1994;83(6): Slymen DJ, Miller TP, Lippman SM, et al. Immunobiologic factors predictive of clinical outcome in diffuse large-cell lymphoma. J Clin Oncol. 1990;8(6): Wilson WH, Grossbard ML, Pittaluga S, et al. Dose-adjusted EPOCH chemotherapy for untreated large B-cell lymphomas: a pharmacodynamic approach with high efficacy. Blood. 2002;99(8): Oyama T, Yamamoto K, Asano N, et al. Age-related EBV-associated B-cell lymphoproliferative disorders constitute a distinct clinicopathologic group: a study of 96 patients. Clin Cancer Res. 2007;13(17): Dojcinov SD, Venkataraman G, Pittaluga S, et al. Age-related EBVassociated lymphoproliferative disorders in the Western population: a spectrum of reactive lymphoid hyperplasia and lymphoma. Blood. 2011;117(18): Lai R, Weiss LM, Chang KL, Arber DA. Frequency of CD43 expression in non-hodgkin lymphoma: a survey of 742 cases and further characterization of rare CD43 þ follicular lymphomas. Am J Clin Pathol. 1999;111(4): Mitrovic Z, Ilic I, Nola M, et al. CD43 expression is an adverse prognostic factor in diffuse large B-cell lymphoma. Clin Lymphoma Myeloma. 2009;9(2): Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B- cell lymphoma identified by gene expression profiling. Nature. 2000;403(6769): Meyer PN, Fu K, Greiner TC, et al. Immunohistochemical methods for predicting cell of origin and survival in patients with diffuse large B-cell lymphoma treated with rituximab. J Clin Oncol. 2011;29(2): Choi WW, Weisenburger DD, Greiner TC, et al. A new immunostain algorithm classifies diffuse large B-cell lymphoma into molecular subtypes with high accuracy. Clin Cancer Res. 2009;15(17): Fu K, Weisenburger DD, Choi WW, et al. Addition of rituximab to standard chemotherapy improves the survival of both the germinal center B-cell like and non germinal center B-cell like subtypes of diffuse large B-cell lymphoma. J Clin Oncol. 2008;26(28): Malumbres R, Chen J, Tibshirani R, et al. Paraffin-based 6-gene model predicts outcome in diffuse large B-cell lymphoma patients treated with R-CHOP. Blood. 2008;111(12): Kramer MH, Hermans J, Wijburg E, et al. Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma. Blood. 1998; 92(9): Tang SC, Visser L, Hepperle B, Hanson J, Poppema S. Clinical significance of bcl-2-mbr gene rearrangement and protein expression in diffuse large-cell non-hodgkin s lymphoma: an analysis of 83 cases. J Clin Oncol. 1994;12(1): Vitolo U, Gaidano G, Botto B, et al. Rearrangements of bcl-6, bcl-2, c-myc and 6q deletion in B-diffuse large-cell lymphoma: clinical relevance in 71 patients. Ann Oncol. 1998;9(1): Akasaka T, Ueda C, Kurata M, et al. Nonimmunoglobulin (non-ig)/bcl6 gene fusion in diffuse large B-cell lymphoma results in worse prognosis than Ig/ BCL6. Blood. 2000;96(8): Hummel M, Bentink S, Berger H, et al. A biologic definition of Burkitt s lymphoma from transcriptional and genomic profiling. N Engl J Med. 2006; 354(23): Niitsu N, Okamoto M, Miura I, Hirano M. Clinical features and prognosis of de novo diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC translocations. Leukemia. 2009;23(4): Aukema SM, Siebert R, Schuuring E, et al. Double-hit B-cell lymphomas. Blood. 2011;117(8): Li S, Lin P, Fayad LE, et al. B-cell lymphomas with MYC/8q24 rearrangements and IGH@BCL2/t(14;18)(q32;q21): an aggressive disease with heterogeneous histology, germinal center B-cell immunophenotype and poor outcome. Mod Pathol. 2012;25(1): Capello D, Vitolo U, Pasqualucci L, et al. Distribution and pattern of BCL-6 mutations throughout the spectrum of B-cell neoplasia. Blood. 2000;95(2): Vitolo U, Botto B, Capello D, et al. Point mutations of the BCL-6 gene: clinical and prognostic correlation in B-diffuse large cell lymphoma. Leukemia. 2002;16(2): Lo Coco F, Gaidano G, Louie DC, Offit K, Chaganti RS, Dalla-Favera R. p53 Mutations are associated with histologic transformation of follicular lymphoma. Blood. 1993;82(8): Sander CA, Yano T, Clark HM, et al. p53 Mutation is associated with progression in follicular lymphomas. Blood. 1993;82(7): Ichikawa A, Kinoshita T, Watanabe T, et al. Mutations of the p53 gene as a prognostic factor in aggressive B-cell lymphoma. N Engl J Med. 1997;337(8): Arch Pathol Lab Med Vol 139, May 2015 CLL and DLBCL Biomarkers Chastain & Duncavage 607