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1 Abstracts and References Soft Tissue Pathology Professor Cyril Fisher, Royal Marsden Hospital Learning points CD34 and CK positivity coexist in epithelioid sarcoma and epithelioid endothelial tumours. INI1 is negative in epithelioid sarcoma, malignant rhabdoid tumour, and some epithelioid MPNST. All other tumours with epithelioid morphology are positive. TLE is a highly sensitive but not wholly specific marker for all types of synovial sarcoma. H-caldesmon is negative in myofibroblastic lesions and positive in smooth muscle tumours, GIST and PEComa. ALK is positive in only up to 55% of inflammatory myofibroblastic tumours Mdm2 and cdk4 are useful for distinguishing atypical lipomatous tumours from lipomas and normal fat, and dedifferentiated liposarcoma from other pleomorphic sarcomas. Some antibodies are useful in diagnosis of one tumour type, and others more so as part of a panel since they are expressed in a variety of neoplasms. CD34 is of value in distinguishing between two diagnoses in several categories of soft tissue tumours including dermatofibrosarcoma vs. dermatofibroma, solitary fibrous tumour vs. synovial sarcoma, and epithelioid sarcoma vs. carcinoma. It is rarely expressed in carcinoma so that in combination with cytokeratin it is helpful for diagnosis of epithelioid sarcoma and epithelioid endothelial tumours. INI1 is the protein product of a gene on chromosome 22q11.2 and is negative in tumours in which the gene is mutated or deleted, notably epithelioid sarcoma and malignant rhabdoid tumour. Since normal tissues and other neoplasms express this in nuclei, it is an example of absence of immunoreactivity being of diagnostic use. TLE1 is positive in nuclei of synovial sarcoma for which it is highly sensitive but is also positive in some nerve sheath tumours and some solitary fibrous tumours. H-caldesmon is negative in myofibroblastic tumours and reactive lesions (e.g. nodular fasciitis) and therefore helps to distinguish the latter from smooth muscle neoplasms. Myopericytic lesions are usually SMA-and h- caldesmon-positive but desmin negative. Immunoreactivity for ALK is found in up to 55% of inflammatory myofibroblastic tumours, and particularly in childhood intra-abdominal and pulmonary lesions. ALK-positive inflammatory myofibroblastic tumours tend to recur but not metastasise, and ALK-negative ones are more aggressive. Mdm2 and cdk4 are useful for distinguishing atypical lipomatous tumours from lipomas and normal fat, and dedifferentiated liposarcoma from other pleomorphic sarcomas. Immunohistochemical panels should be tailored to address specific diagnostic problems. References 1. Fisher C. Epithelioid sarcoma of Enzinger. Adv Anat Pathol 2006;13: Modena P, Lualdi E, Facchinetti F et al. SMARCB1/INI1 tumor suppressor gene is frequently inactivated in epithelioid sarcomas. Cancer Res 2005;65: Terry J, Saito T, Subramanian S et al. TLE1 as a diagnostic immunohistochemical marker for synovial sarcoma emerging from gene expression profiling studies. Am J Surg Pathol 2007;31: Coffin CM, Hornick JL, Fletcher CD. Inflammatory myofibroblastic tumor: comparison of clinicopathologic, histologic, and immunohistochemical features including ALK expression in atypical and aggressive cases. Am J Surg Pathol 2007;31: Ceballos KM, Nielsen GP, Selig MK, O'Connell, JX. Is anti-h-caldesmon useful for distinguishing smooth muscle and myofibroblastic tumors? An immunohistochemical study. Am J Clin Pathol 2000;114: Binh MB, Sastre-Garau X, Guillou L et al. MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing well-differentiated and dedifferentiated liposarcoma

2 Dermatopathology Dr Kevin Hollowood, Oxford Learning points Melan-A and HMB-45 are usually not expressed in desmoplastic melanoma. The presence of S100-positive spindle cells in scars is a diagnostic pitfall in the diagnosis of desmoplastic melanoma. Despite a burgeoning literature on putative specific cytokeratin profiles, morphology remains more important than immunohistochemistry in the diagnosis and classification of most cutaneous adnexal tumours An antibody panel including p63, D2-40 and cytokeratin 5/6 is useful in distinguishing primary skin tumours from cutaneous metastases. An antibody panel of cytokeratin 20, neurofilaments, TTF-1 and MASH-1 distinguishes Merkel cell carcinoma from metastatic small cell carcinoma. Immunohistochemistry for the mismatch repair proteins MSH-2, MLH-1 and MSH in sebaceous tumours is a good screening tool for the Muir-Torre syndrome. The main role of immunohistochemistry in dermatopathology is in the diagnosis of immunobullous disease, cutaneous lymphomas and non-lymphomatous neoplasia. This presentation addresses the role of a number of common antibodies in the diagnosis of a variety of epithelial, melanocytic and cutaneous soft tissue neoplasms and considers the potential of two newer antibodies in the management of sebaceous tumours. Whilst emphasising the typical staining patterns of common tumours and the diagnostic utility of antibody panels in the assessment of malignant spindle cell tumours, poorly differentiated carcinomas, clear cell tumours and neuroendocrine neoplasia, it will also highlight important caveats and potential diagnostic pitfalls. Specifically, S100, melan-a and HMB-45 use in demonstrating melanocytic differentiation, their utility (or lack of it) in distinguishing benign and malignant melanocytic tumours and their expression in non-melanocytic tumours is considered. The diagnostic utility of p63 and cytokeratin 20 in some epithelial neoplasms and the use of EMA, CD34 and CD10 in a variety of epithelial and soft tissue neoplasms is discussed. Lastly the use of the new antibody adipophyllin in the diagnosis of sebaceous neoplasia and the potential application of mis-match repair protein immunohistochemistry in the management of patients presenting with cutaneous sebaceous tumours is considered. References 1. Chu P, Weiss L. Skin Tumors. In: Chu P, Weiss L (eds.). Modern Immunohistochemistry. Cambridge: Cambridge University Press, 2009; Wasserman J, Maddox J, Racz M, Petronic-Rosic V. Update on immunohistochemical methods relevant to dermatopathology. Arch Pathol Lab Med 2009; 133: Ohsie SJ, Sarantopoulos P, Cochran AJ, Binder SW. Immunohistochemical characteristics of melanoma. J Cutan Pathol 2008; 35: Folpe AL, Cooper K. Best practices in diagnostic immunohistochemistry. Pleomorphic cutaneous spindle cell tumors. Arch Pathol Lab Med 2007; 131: Tardio JC. CD-34 reactive tumors of the skin. An updated review of an ever-growing list of lesions. J Cutan Pathol 2009; 36: Eisen DB, Michael DJ. Sebaceous lesions and their associated syndromes: Part II. J Am Acad Dermatol 2009; 61:

3 My favourite antibodies: Lymphoreticular Disease Dr Nick Rooney Pathology Sciences Bristol In lymphoreticular disease the first decision to make is whether to use flow cytometry immunohistochemistry or FISH. All have their role, the key to using immunostains to make a diagnosis is understanding what each antibody is telling you. The choice of immunostain is determined by the differential diagnosis that confronts you. This lecture will cover the use of immunostaining in the following areas: 1. Loss of architecture 2. Architecture preserved but cells have an abnormal phenotype for location 3. Other cells present 4. Abnormal phenotype or gene expression 5. Demonstration of translocations The phenotype for location is illustrated below and can be found in : Nodal B-Cell Lymphoma and Hodgkin Lymphoma; current WHO classification and differential diagnostic dilemmas. Rooney N, Foster L, Byers R. Diagnostic Histopathology Jan 2010 ALL Follicular Lymphoma NLPHL CLLu CLLm DLBCL chl MZL PBL The minimum criteria for making a diagnosis of lymphoma is reviewed in Best Practice in Lymphoma Diagnosis and Reporting. Parker A, Baines B, Devereux S et al jointly produced by the British Committee for Standards in Haematology and the Royal College of Pathologists and available on the college website. In summary, my favourites 1. Architecture CD23 S100 Ki67 2. Cells PAX5 CD30 CD2 3. Abnormal expression CD10 CD5 CD23 OCT2/BOB1 Ki67 4. Gene expression BCL2 Cyclin D1 5. Treatment target CD20 6. FISH t(myc), t(bcl6), t(14;18)

4 Gastroentestinal pathology Professor Marco Novelli, UCL Learning points HP immunostaining can be very useful in post treatment biopsies. Pancytokeratin +/- synaptophysin immunostaining is useful to confirm small areas of poorly differentiated gastric adenocarcinoma. Cytokeratin +/- CDX2 immunostaining is useful in determining site of origin of carcinomas, but is not infallible. CD117 and DOG1 are the immunostains of choice in the diagnosis of GISTs. Mismatch repair immunostaining should be considered in colorectal/ovarian adenocarcinomas in middle-age patients. The vast majority of gastrointestinal histopathological diagnoses can be safely made on H&E stained sections. Immunohistochemistry plays a limited, but crucial role in tissue diagnosis and patient management. In benign gastrointestinal conditions immunostaining is useful in demonstrating occult organisms such as the bacteria Helicobacter Pylori or viruses CMV, Adenovirus, HHV8 and EBV. In tumour diagnosis immunostaining can be especially useful in confirming an epithelial origin for a tumour, localising the primary site of origin of a tumour, and in making a diagnosis in stromal neoplasms of the GI tract. Mismatch repair protein immunostaining may also be useful both in assessing the risk of familial cancer and in directing patient treatment. An understanding of when, and where, immunostaining may be of value is an important attribute for the gastrointestinal histopathologist. Respiratory pathology Dr Richard Attanoos, Cardiff Learning points Immunohistochemical panels are adjuncts to the histological diagnosis. They should not be interpreted without correlation with the H & E section. Immunohistochemistry plays an important role in the diagnosis of neoplastic and nonneoplastic epithelioid and spindle cell proliferations. It is, however, important to remember that antibody panels will vary to reflect the most likely differential diagnoses. This will be determined by serosal site (pleural, peritoneal, other), morphological phenotype (benign versus malignant, epithelioid, biphasic, sarcomatoid, small cell, anaplastic) and laboratory experience. There is no single immunohistochemical panel to cover all diagnostic mesothelial problems. For each diagnostic dilemma seek to identify at least 2 markers which will label the pathological entities under consideration in the differential diagnosis. Check that each antibody has been robustly validated in the literature. Check that the marker works well in your own laboratory and check controls. If results are conflicting, expand the panel. Caution is advised when comparing immunohistochemical results in the literature there is lack of standardisation. Different antibody clones, commercial sources, antibody dilutions, retrieval methods, incubation times provide technical bias. Different laboratories interpret the threshold cut point between negativity / positivity differently and some do not pay close attention to marker distribution. New markers emerge all the time initial claims reporting high specificity and sensitivity are common but always require substantiation before acceptance into everyday practice panels. Common immunohistochemical pitfalls in the diagnosis of mesothelioma include an eagerness to over interpret weak, focal immunopositivity in small pleural needle cores

5 (>10% available cells should be positive) and to lack awareness of recognised false positive reactions (especially with epithelial markers, often <10% cells positive). In pulmonary pathology, one area dominates in its demands for immunohistochemistry pleural biopsy interpretation. Specimens range from blind closed needle cores through to open thoracotomy pleural biopsies. Most are performed to investigate recurrent effusions and / or radiologically detectable pleural thickening in which there is a suspicion of malignancy (mesothelioma). The diagnosis of malignant pleural mesothelioma is regarded as problematic because there is a recognised mimicry of a variety of other lesions, neoplastic and non-neoplastic. The diagnostic dilemmas may be summarised according to the morphological characteristics on H & E stain. These are: malignant epithelioid mesothelioma versus carcinoma and other epithelioid cancers, malignant sarcomatoid mesothelioma versus sarcomatoid carcinoma or sarcoma, benign versus malignant mesothelial proliferations (reactive mesothelial hyperplasia versus epithelioid mesothelioma and fibrous pleurisy versus desmoplastic mesothelioma). Irrespective of the diagnostic problems facing the surgical pathologist, immunohistochemistry plays a central role in their resolution. In pleural biopsies, the accurate diagnosis of a mesothelial proliferation as neoplastic or not has a central role on patient management (medical and legal). It is important not to over call the diagnosis of mesothelioma. At least 2 markers should be used which label the pathological entities under consideration in the differential diagnosis. In the 2009 Consensus statement from the International Mesothelioma Interest Group (1) the following marker(s) were identified as very useful in each pathological scenario. 1. Epithelioid mesothelioma versus Pulmonary adenocarcinoma or Other Mesothelial markers: Calretinin and cytokeratin 5/6 Carcinoma markers: MOC -31 and TTF 1 Mesothelial markers: Calretinin can be demonstrated in virtually all epithelioid subtype mesotheliomas. Polyclonal antibodies show a nuclear and cytoplasmic distribution. The marker does not distinguish between reactive and neoplastic mesothelium (this is a potential pitfall with subserosal spindle cells being positive). Up to 10% pulmonary adenocarcinomas may be positive with focal (<10%) cytoplasmic expression. Cytokeratin 5/6 is expressed in 80% epithelioid mesotheliomas and up to 19% lung adenocarcinomas. (Squamous carcinomas are positive use p63).alternate mesothelial markers: WT1, and Podoplanin (D2 40) Carcinoma markers: MOC 31expressed in 95% lung adenocarcinomas with up to 10% mesotheliomas showing focal positive in mesothelioma. Thyroid transcription factor 1 (TTF 1) shows nuclear expression in up to 85% lung adenocarcinomas with no recorded positivity in mesothelioma. Alternate carcinoma markers: Monoclonal CEA, BG8 (Lewis antigen)(2) Pancytokeratin is useful in distinguishing mesothelioma /carcinoma from some other epithelioid tumours. Both epithelioid mesothelioma and carcinoma are virtually all cytokeratin positive negativity points towards another tumour (melanoma, epithelioid haemangioendothelioma, lymphoma (anaplastic large cell lymphoma). 2. Malignant sarcomatoid mesothelioma versus sarcomatoid carcinoma or sarcoma Cytokeratins - AE1/3, cam 5.2, CK 7 Mesothelial markers Calretinin and D2 40 (WT 1 and CK 5/6 no good) 3. Benign versus malignant mesothelial proliferations Reactive mesothelial hyperplasia versus epithelioid mesothelioma Reactive mesothelium marker: Desmin Neoplastic mesothelium marker: EMA, p53. Alternate GLUT - 1

6 EMA (diffuse membranous expression) in 80% mesotheliomas with up to 20% reactive mesothelial cells showing focal membranous staining. Carcinoma cells show cytoplasmic expression.p53 shows nuclear expression in ~50% mesotheliomas but is not seen in reactive mesothelium. Desmin seen in 85% reactive epithelioid mesothelial cells is also seen in 10% epithelioid mesotheliomas. Fibrous pleurisy versus desmoplastic mesothelioma Cytokeratin most desmoplastic mesotheliomas express cytokeratin. This may be focal. Cytokeratin is useful to examine the cellular edge of the spindle cell proliferation and highlights infiltration into fat and lung, if present. In contrast fibrous pleurisy has a defined flat non infiltrative margin. References 1. Husain AN, Colby TV, Ordonez NG, et al. Guidelines for Pathologic Diagnosis of Malignant mesothelioma: A consensus statement from the International Mesothelioma Interest Group. (2009). Arc. Patho Lab Med. 133: King J, Thatcher N, Pickering C, et al. Sensitivity and specificity of immunohistochemical markers used in the diagnosis of epithelioid mesothelioma: a detailed systematic analysis using published data. (2006) Histopathology 48: Gynaecological Pathology Professor Glenn McCluggage, Belfast Immunohistochemistry plays an important role in various diagnostic scenarios in gynaecological pathology. However, immunohistochemistry is sometimes overdone and it is always to be remembered that immunohistochemistry is an adjunct technique and an aid to careful morphological examination. Since no antibody is totally specific for any given tumour and since unexpected positive and negative staining reactions may occur, panels of markers should always be used and these panels should be carefully focused depending on the differential diagnosis under consideration. In many diagnostic scenarios, especially in the examination of an unusual ovarian neoplasm, judicious sampling is sometimes more useful than immunostains. However, when carefully used, immunohistochemistry can be extremely useful and is paramount in diagnosis in many cases. Markers discussed in this talk are:- 1. WT1 - useful as a marker of ovarian, peritoneal and tubal serous carcinomas. Most endometrioid adenocarcinomas are negative, as are most, but not all, uterine serous carcinomas. Other useful applications include the diagnosis of mesothelioma, ovarian small cell carcinoma of hypercalcaemic type and desmoplastic small round cell tumour. 2. CK7,CK20,CA125,CA19.9,CEA,CDX2- useful as a panel in distinguishing between an ovarian adenocarcinoma and a metastasis, especially from the colorectum and to a lesser extent the upper gastrointestinal tract. Many primary ovarian mucinous neoplasms express the intestinal markers listed, limiting their diagnostic utility in distinguishing between a primary ovarian mucinous neoplasm and a metastasis from the upper gastrointestinal tract, including the pancreas and biliary tree. 3. ER and PR- useful in confirmation that a neoplasm, either epithelial or mesenchymal, is of gynaecological origin. Many gynaecological adenocarcinomas are positive, even high grade serous carcinomas (ovary more commonly than uterus). May also be useful in a therapeutic sense in predicting a response to hormone manipulation; for example endometrial stromal sarcoma and aggressive angiomyxoma. 4. p16 - useful in the cervix as a surrogate marker of the presence of high risk HPV. Useful diagnostic applications include the distinction between CGIN and TEM, the distinction between an endometrial and an endocervical adenocarcinoma and helping to confirm a metastatic cervical adenocarcinoma in the ovary. Some neoplasms, for example high grade serous carcinomas of the uterus and ovary and uterine leiomyosarcomas, diffusely express

7 p16 due to non-hpv related mechanisms. p16 may be useful in the distinction between a uterine leiomyosarcoma and mimics such as symplastic leiomyoma. It may also be useful in the distinction between a uterine serous carcinoma (usually diffusely positive) and an endometrioid adenocarcinoma (usually negative or focally positive). 5. p63- a useful marker of squamous lineage in a cervical carcinoma. Most cervical squamous carcinomas exhibit diffuse nuclear immunoreactivity while most adenocarcinomas and neuroendocrine carcinomas are negative, although there are some exceptions. 6. inhibin, calretinin, CD56- useful markers of an ovarian sex cord-stromal tumour and of similar primary uterine neoplasms (uterine tumour resembling ovarian sex cord tumour). 7. EMA- useful in the distinction between an ovarian endometrioid adenocarcinoma with sex cord-like elements (EMA positive) and an ovarian sex cord-stromal tumour (EMA negative) (cytokeratins are of limited value in this regard). Useful in diagnosis of undifferentiated endometrial carcinoma and distinction from undifferentiated sarcoma. 8. TTF1 and thyroglobulin- useful in confirmation of unusual malignant thyroid neoplasms in the ovary and in unusual variants of struma ovarii, for example clear cell, oxyphil and cystic variants. Some gynaecological adenocarcinomas and neuroendocrine neoplasms are also TTF1 positive, sometimes diffusely so. 9. p53- most high grade serous carcinomas in the female genital tract are positive. Useful in the distinction between a uterine serous and endometrioid adenocarcinoma. Useful in picking up early tubal serous carcinomas and tubal intraepithelial carcinoma, for example in prophylactic salpingo-oophorectomy specimens in BRCA1 and BRCA2 patients. 10. desmin, SMA, h-caldesmon, CD10- useful as part of a panel in the distinction between a uterine smooth muscle and an endometrial stromal neoplasm. CD10 is a very non-specific marker and many neoplastic types may be positive. References 1 McCluggage WG. A critical appraisal of the value of immunohistochemistry in diagnosis of uterine neoplasms. A critical appraisal of the value of immunohistochemistry in diagnosis of uterine neoplasms. Adv Anat Pathol 2004;11; McCluggage WG. Immunohistochemical and functional biomarkers of value in female genital tract lesions. Int J Gynecol Pathol 2006;25; McCluggage WG, Young RH. Immunohistochemistry as a diagnostic aid in the evaluation of ovarian tumors. Immunohistochemistry as a diagnostic aid in the evaluation of ovarian tumors. Semin Diagn Pathol 2005;22; McCluggage WG. Recent advances in immunohistochemistry in gynaecological pathology. Histopathology 2002;40; Mittal K, Soslow R, McCluggage WG. Application of immunohistochemistry to gynecologic pathology. Arch Pathol Lab Med 2008;132; Breast pathology Professor Ian Ellis, Nottingham Demonstration of inferred clonality / presence of a uniform cell type using ER and cytokeratin stains are useful in classification of epithelial proliferations Myoepithelial stains are helpful in distinguishing invasive versus non invasive lesions Myoepithelial stains are helpful in distinguishing papillary carcinoma in situ from benign papilloma Spindle cell lesion diagnosis requires immunophenotyping Sub classification of invasive breast cancer can be assisted by Immunocytochemistry, for example diagnosis of invasive micropapillary carcinoma using EMA and the recognition of basal like and HER2 subclasses requires phenotyping

8 Predictive testing of breast cancer form hormone receptors and HER protein requires high quality standardised and validated IHC assays References 1. Putti et al. Breast Pathology practice. Histopathology 2005, 47, Breast Pathology. Eds O Malley FP and Pinder SE. Churchill Livingstone Elsevier, Ellis IO Pinder SE and Lee AHS. Breast Pathology. In Diagnostic Histopathology of Tumours Third Edition. Ed. Fletcher CDM. Churchill Livingstone Elsevier, Pathology Reporting of Breast Disease. NHSBSP and RCPath. A Joint Document Incorporating the Third Edition of the NHS Breast Screening Programme s Guidelines for Pathology Reporting in Breast Cancer Screening and the Second Edition of The Royal College of Pathologists Minimum Dataset for Breast Cancer Histopathology. NHSBSP Publication No 58, Walker RA, Bartlett J, Dowsett M, Ellis IO, Hanby A, Jasani B, Miller K, Pinder S. HER2 testing in the UK - further update to recommendations. J Clin Pathol Jul;61(7): Please see additional sheets at back of the pack Urological pathology Dr Ken Grigor, Edinburgh Learning Points Renal tumours should be stained with a panel of antibodies to confirm origin from renal parenchyma, and to determine the subtype. Transitional cell and squamous cell carcinomas of the bladder express different cytokeratins Primary and metastatic prostate cancers stain for PSA, and invasive carcinomas lack basal cells demonstrable by immunocytochemistry Testicular tumours are normally diagnosed on conventional H&E staining although immunocytochemistry may be useful Immunohistochemistry is often essential in the diagnosis of tumours of the Kidney, Bladder, Prostate and Testis. Renal Tumours: Unlike most carcinomas, renal cell carcinomas (RCC) usually stain for both pancytokeratin and vimentin, and this is helpful to determine the origin of metastatic carcinomas when the site of the primary in not apparent. The main subtypes of RCC (clear cell, chromophil/papillary, chromophobe, oncocytoma) can be differentiated using a panel of antibodies including CD10, EMA, AMACR and C-kit. Staining for CK7 can be used if the tumour is poorly differentiated and may be a collecting duct carcinoma, or transitional cell carcinoma of the renal pelvis. Bladder tumours: Transitional cell carcinomas are almost always positive for CK7, and negative for CK14. They are reported to be positive for CK20, but frequently staining for this antigen is only patchy, or negative. Squamous cell carcinomas are CK14 positive, and negative for CK7 & CK20. Carcinoma in situ (CIS) of the bladder shows full thickness positive staining for CK20, but this pattern may also be seen in lesser severities of dysplasia, and also in reactive changes due to inflammation. CK20 staining must not be considered diagnostic of CIS. Adenocarcinoma of Prostate: Carcinoma of the prostate (CaP) stains for prostatic specific antigen (PSA) which is useful to determine the origin of metastatic carcinomas. Poorly differentiated carcinomas in the prostate may arise from the glandular epithelium, large prostatic ducts, or urothelium: these can be differentiated by staining for PSA, prostatic acid phosphatase, and CK7. The

9 diagnosis of malignancy in prostate biopsies can be confirmed by demonstrating lack of basal cells which show cytoplasmic staining for high molecular weight cytokeratin such as 34βE12, and nuclear staining for P63. Positive cytoplasmic staining for AMACR is seen in malignant prostatic epithelium including invasive adenocarcinoma and prostatic intraepithelial neoplasia (PIN). Germ cell tumours of the testis: Seminoma and teratoma/nonseminomatous germ cell tumour (NSGCT) can normally be differentiated on conventional H&E staining, but may be confirmed by staining for placental alkaline phosphatase (PLAP), pancytokeratin, human chorionic gonadotrophin (hcg), alphafetoprotein (AFP), C-kit, and Oct3/4. The proliferation index as determined by staining for Ki67 is related to the prognosis of seminoma. Please see spreadsheet overleaf

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