Establishing a definitive pathologic diagnosis in surgical

SPECIAL ARTICLE Best Practices in the Application of Immunohistochemistry in Urologic Pathology Report From the International Society of Urological Pathology Consensus Conference Mahul B. Amin, MD,* Jonathan I. Epstein, MD,w Thomas M. Ulbright, MD,z Peter A. Humphrey, MD, PhD,y Lars Egevad, MD,8 Rodolfo Montironi, MD,z David Grignon, MD,z Kiril Trpkov, MD,# Antonio Lopez-Beltran, MD,** Ming Zhou, MD,ww Pedram Argani, MD,w Brett Delahunt, MD,zz Daniel M. Berney, MD,yy John R. Srigley, MD,88 Satish K. Tickoo, MD,zz and Victor E. Reuter, MDzz Abstract: Members of the International Society of Urological Pathology (ISUP) participated in a half-day consensus conference to discuss guidelines and recommendations regarding best practice approaches to use of immunohistochemistry (IHC) in differential diagnostic situations in urologic pathology, including bladder, prostate, testis and, kidney lesions. Four working groups, selected by the ISUP leadership, identified several highinterest topics based on common or relevant challenging diagnostic situations and proposed best practice recommendations, which were discussed by the membership. The overall summary of the discussions and the consensus opinion forms the basis of a series of articles, one for each organ site. This Special Article summarizes the overall recommendations made by the four From the *Department of Pathology & Laboratory Medicine, Cedars- Sinai Medical Center, Los Angeles, CA; wdepartment of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD; zdepartment of Pathology, Indiana University School of Medicine, Indianapolis, IN; ydepartment of Pathology & Immunology, Washington University School of Medicine, St Louis, MO; wwdepartment of Pathology, New York University Medical Center; zzdepartment of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY; 8Department of Pathology-Oncology, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden; zsection of Pathological Anatomy, Polytechnic University of the Marche Region School of Medicine, United Hospitals, Ancona, Italy; #Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB; 88Trillium Health Partners, Mississauga and McMaster University, Hamilton, ON, Canada; **Unit of Anatomic Pathology, Department of Surgery Universidad de Cordoba, Cordoba, Spain; zzdepartment of Pathology and Molecular Medicine, Wellington School of Medicine and Health Sciences, Wellington, New Zealand; and yydepartment of Cellular Pathology, Centre for Molecular Oncology, Queen Mary University of London, London, UK. M.B.A and J.I.E. are joint first authors. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Mahul B. Amin, MD, Department of Pathology & Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Suite 8707, Los Angeles, CA 90048 (e-mail: mahul.amin@ cshs.org). Copyright r 2014 by Lippincott Williams & Wilkins working groups. It is anticipated that this ISUP effort will be valuable to the entire practicing community in the appropriate use of IHC in diagnostic urologic pathology. Key Words: ISUP recommendations, immunohistochemistry, best practices, summary, prostate adenocarcinoma, urothelial carcinoma, renal cell carcinoma, germ cell tumors, sex cord-stromal tumors (Am J Surg Pathol 2014;38:1017 1022) Establishing a definitive pathologic diagnosis in surgical pathology involves integration of clinical information, gross pathology, routine microscopic findings, and ancillary studies, including immunohistochemistry (IHC). In recent years, IHC has markedly informed our practice, adding objectivity to our diagnoses. There has been an explosion of literature promulgating various new antibodies and their potential utility in diagnostic pathology. However, as with any technique, IHC has its share of pitfalls and disadvantages (clone selection, titration, validation, false positives, false negatives, etc.). Besides technical and interpretation-related (subcellular localization and pattern) deficiencies, a recurring problem remains antibody overuse that is, use of a disproportionate number of antibodies without a judiciously selected IHC panel. An important prerequisite in contemporary health care is efficient stewardship of resources and testing. The Executive Committee of the International Society of Urological Pathology (ISUP) undertook a proactive approach, with a goal to provide the best practice recommendations in the application of IHC in the diagnostic urologic pathology. Our aim was to establish guidelines and recommendations that would benefit the entire practicing community in the appropriate use of IHC in diagnostic urologic pathology. To achieve this objective, ISUP conducted a half day conference on Sunday, March 3, 2013, at the 102nd United States and Canadian Academy of Pathology (USCAP) in Baltimore. Am J Surg Pathol Volume 38, Number 8, August 2014 www.ajsp.com 1017

Amin et al Am J Surg Pathol Volume 38, Number 8, August 2014 PROCESS FOR DEVELOPING THE ISUP RECOMMENDATIONS FOR BEST PRACTICES Four working groups were identified to propose best practices recommendations for the prostate, kidney, bladder, and testis organ sites. For each organ site, working group members identified and selected several high-interest topics on the basis of a particular common or relevant challenging diagnostic situation (eg, workup of atypical small acinar proliferations in the prostate or role of IHC in subtyping of germ cell tumors) or a pattern-based differential diagnostic approach (eg, IHC panel for spindle cell lesions of the bladder or IHC in the workup of renal tumors with predominantly clear cells). The proposed recommendations, on the basis of the literature review, personal professional experience, and discussions among the working group members, were presented and discussed at the conference and were subject to a vote by the ISUP members attending the conference. The consensus recommendations were accepted on the basis of a majority vote by the ISUP members attending the conference. If there were minor differences of opinion, the consensus recommendations were modified on the basis of the discussion. If no consensus was established, due to lack of published information or major differences in experience, these issues were noted as having no consensus. The moderators of the consensus conference were M.B.A. and J.I.E. The prostate working group comprised J.I.E. (Lead) and P.A.H., L.E., and R.M. The urinary bladder group consisted of M.B.A. (Lead) and D.G., K.T., and A.L.-B. The kidney working group was led by V.E.R. with P.A., B.D., and M.Z. as members of the renal IHC group. The testis working group comprised T.M.U. (Lead) and D.M.B., J.R.S., and S.K.T. B.D. recorded the minutes for the conference. TOWARD BEST PRACTICE APPROACHES IN THE APPLICATION OF IHC IN UROLOGIC PATHOLOGY Several of the working group members have made numerous observations in their clinical and consultation practices, which point to some prevalent flaws in the integration of IHC as a diagnostic adjunct. Although consult experiences are inherently skewed toward difficult or controversial cases, several recurring themes observed among such cases are noteworthy and broadly applicable irrespective of the specific diagnostic challenge to be resolved by IHC. These issues are summarized in Table 1 and are likely to be applicable to most topics covered by the various workgroups of the Consensus Conference (and, for that matter, for all surgical pathology). The optimal best practice approach is to construct an appropriate, well-balanced IHC panel on the basis of a specific differential diagnostic consideration. In keeping with this general philosophy, therefore, the approach varied between the 4 workgroups toward diagnostically challenging differentials for their organ sites. This is reflected in the overall organization and presentation of the recommendations in the TABLE 1. General for a Best Practice Approach in the Application of IHC in Urologic Pathology Observations Best Practice Approach Tendency to apply a wide array of antibodies (almost indiscriminate) to arrive at a diagnosis Serious misdiagnoses are made by inappropriate use of IHC or by incomplete knowledge of range of antibody expression Unexpected immunoreactions frequently occur Lack of attention to pattern of staining Lack of attention to controls Suboptimal blocks selected for study or incorrect areas of a section are evaluated Overall overreliance on IHC staining to arrive at the final diagnosis Construct a well-balanced IHC panel on the basis of the specific differential diagnostic situation: Do not use antibodies singly, preferably include antibodies that are expected to be positive and negative for the case Panels need to be adjusted as novel markers are reported Continuously consider discarding older antibodies, as newer more valuable antibodies are reported More is not necessarily better. If you have no idea about the diagnosis, do not stain it. Be continuously updated with the literature regarding the range of expression utility of antibodies constantly changes as new studies are published and new markers are introduced There is no perfect antibody. Sensitivity and specificity is not inherent to an antibody but varies on the basis of the diagnostic situation Antibody positivity is valuable only in the context of appropriate cellular localization (nuclear, cytoplasmic, membranous) Attention to both external and internal controls is imperative Misinterpretation may occur As different pathologies have different staining reactions Different grades or patterns may have different staining reactions Selection of the appropriate block/s containing lesional areas pertinent to the differential diagnosis is absolutely critical Diagnose every case after review of IHC only in the context of morphology and the clinical situation 4 manuscripts generated as part of each workgroup s preconference, consensus conference, and postconference deliberations. These manuscripts are published in the online-only format of this month s issue. 1 4 Whereas this Special Article provides a broad overview and summary of the ISUP recommendations made in all 4 manuscripts, each separate manuscript (bladder, prostate, kidney, and testis) contains more detailed discussion and context, references, tables, and illustrations. Summary of the Bladder Workgroup The bladder group discussed 5 topics. 1 1018 www.ajsp.com r 2014 Lippincott Williams & Wilkins

Am J Surg Pathol Volume 38, Number 8, August 2014 Best Practices in IHC Applications in Urologic Pathology (A) Confirmation of urothelial differentiation at a metastatic site or in a bladder primary in a tumor with unusual histology There is no ideal marker or established panel to confirm urothelial differentiation. On the basis of the differential diagnostic consideration, positivity for GATA3, CK20, p63, and either HMWCK or CK5/6 is of value in proving urothelial differentiation in the appropriate morphologic and clinical context. Uroplakin III may be used as a second-line marker. Markers with potential utility but needing additional validation include S100P and Uroplakin II. There is limited utility for CK7 and thrombomodulin; a CK7-negative tumor is less likely to be a urothelial carcinoma (UCa). (B) Distinction of reactive atypia from carcinoma in situ It was the consensus that morphology remains the gold standard in this differential diagnosis and that, at best, the IHC panel of CK20/p53/ CD44(s) has potential utility but is variably used and has limitations. The immunostaining pattern must be interpreted with strict morphologic correlation as overreliance on IHC may be misleading, particularly in the posttreatment setting. IHC has no role in the distinction of dysplasia versus carcinoma in situ and in the grading of papillary UCa. (C) Role of IHC in staging of bladder cancer In a subset of cases, depending on the clinical and histologic context, broad-spectrum cytokeratins (to identify early or obscured invasion) and desmin (distinction of muscle from desmoplasia and to highlight muscle contours for subclassification) may have a distinct role in staging UCa. Limited experience and conflicting data preclude recommendation of use of smoothelin or vimentin for subclassifying the type of muscle at this time. (D) Distinction between spindle cell lesions of the bladder Depending on the situation, in a purely spindled proliferation and in limited specimens, if IHC is resorted to, we recommend an IHC panel of 6 markers including ALK-1, SMA, desmin, cytokeratin (AE1/AE3), and p63 with either of HMCK or CK5/6. (E) Prognostic and predictive markers in current clinical practice Currently, there are no prognostic IHC or molecular studies that are recommended to be routinely performed on biopsy or resection specimens. Summary of the Prostate Workgroup The prostate group covered 9 topics: (A) IHC panel for the diagnosis of limited adenocarcinoma in needle biopsies (i) Workup of atypical biopsies in general: Either HMWCK (34BE12 or CK5/6 or others) or p63 or a combination of the 2 with AMACR either in a double or triple cocktail is recommended for the workup of small foci of atypical glands suspicious for adenocarcinoma of the prostate. ERG is optional as it is present in only 40% to 50% of prostate cancers and also positive in high-grade prostatic intraepithelial neoplasia. In the setting of obvious carcinoma, there is no justification to perform basal cell staining and staining for AMACR. In the setting of obviously benign glands, there is no justification to perform basal cell staining and staining for AMACR. (ii) Workup of atypical foci with definite cancer in other parts: (a) Gleason score 3+4 = 7 or higher-grade cancer on at least 1 part. Is workup of other parts with atypical foci suspicious for Gleason score 3+3 = 6 cancer indicated? ISUP Recommendation: Unlikely to change clinical treatment so not recommended. (b) Gleason score 4+3 or 4+4 = 8 cancer on at least 1 part. Is workup of other parts with atypical small foci suspicious for the same-grade cancer indicated? ISUP Recommendation: The extent of high-grade cancer could affect clinical treatment such that workup of other atypical possible high-grade cancer foci is justified. (c) Gleason score 4+3 or higher-grade cancer on at least 1 part. Is workup of other parts for cribriform intraductal carcinoma as against diagnosing Gleason pattern 4 indicated, if doing so does not change the overall grade? ISUP Recommendation: Given that intraductal carcinoma in the vast majority of cases is considered an extension of high-grade cancer into prostatic ducts and acini, it is not recommended in the setting of definitive invasive high-grade cancer that workup of additional cribriform lesions be pursued. (d) Gleason score 3+3 on at least 1 part. Is workup of other parts for possible small foci of 3+3 = 6 indicated? ISUP Recommendation: The number of positive cores and/or their location could possibly affect subsequent therapy in terms of suitability for active surveillance or focal therapy, such that unless one knows with certainty that it would not affect therapy, it is justified to perform an IHC workup of additional atypical foci. (B) Poorly differentiated prostate adenocarcinoma versus UCa Option to use PSA as a first test to identify prostate adenocarcinoma (PCa) and GATA3 to identify UCa. r 2014 Lippincott Williams & Wilkins www.ajsp.com 1019

Amin et al Am J Surg Pathol Volume 38, Number 8, August 2014 If GATA3 not is available, then HMWCK and p63 can be used. If the tumor is PSA positive with intense staining and HMWCK and p63 negative, the findings are diagnostic of PCa. If the tumor is equivocal/weak/negative for PSA and negative/focal for p63 and HMWCK, then P501S, NKX3.1, and GATA3 has to be used. Some experts also include PAP in this second round of staining. If the tumor is negative for PSA and diffusely strongly positive for p63 and HMWCK, the findings are diagnostic of UCa. If the tumor is negative for PSA and moderately to strongly positive for GATA3, the findings are diagnostic of UCa. Laboratories should be encouraged to use GATA3 for UCa and add P501S and NKX3.1 as prostate markers in addition to PSA, p63, and HMWCK. If GATA3, p501s, and NKX3.1 are not available in equivocal cases, they should be sent out for consultation to laboratories with these antibodies. The prostate workgroup discussed 7 other topics, which included: (C) high-grade prostatic adenocarcinoma versus bladder adenocarcinoma; (D) prostatic small cell carcinoma versus high-grade prostatic adenocarcinoma; (E) metastatic carcinoma of unknown primary rule out PCa; (F) nonspecific granulomatous prostatitis/xanthoma versus high-grade PCa; (G) adult prostate sarcoma versus sarcomatoid carcinoma of prostate; (H) colorectal adenocarcinoma versus high-grade PCa; and (I) prognostic IHC markers. Numerous tables outline the advantages and disadvantages of commonly used antibodies on the basis of the applicable ISUP recommendations for each specific diagnostic situation. These are outlined in detail in the online version of the manuscript. 2 Summary of the Testis Workgroup The testis workgroup discussed 6 topics: To resolve these differential diagnostic situations, the workgroup has presented 9 algorithms as tables with specific antibodies within each algorithm to help resolve challenging scenarios within these headings. These are outlined in detail with illustrations in the online version of the manuscript. 3 (A) The differential diagnosis of germ cell tumors A general approach to the subtyping of germ cell tumors can be achieved therefore by a limited panel consisting of antibodies directed against OCT4, CD117, CD30, and Glypican 3 (GPC3), although this may be further reduced if the light microscopic morphology is also limited to a more restricted differential. For instance, the differential diagnosis of seminoma versus embryonal carcinoma could be effectively evaluated in most cases by a 2-stain panel of CD117 and CD30. (B) The differential diagnosis of sex cord-stromal tumor and germ cell tumor A reasonable and efficient panel for the differential diagnosis of sex cord-stromal tumor versus germ cell tumor consists of SALL4, a-inhibin, and calretinin. In a laboratory lacking SALL4, an alternative, although somewhat more complex, algorithm can be devised to assist with this differential diagnosis using a panel of OCT4, GPC3, a-inhibin, and calretinin. (C) The differential diagnosis of lymphoma and germ cell tumor An efficient initial panel for the differential diagnosis of germ cell tumor and large cell lymphoma of the testis consists of SALL4, CD45, CD20, and CD3 or, in the absence of SALL4, consists of OCT4, GPC3, AE1/AE3, CD45, CD20, and CD3. (D) The differential diagnosis of metastatic carcinoma and germ cell tumor An efficient and concise initial IHC panel for the differential diagnosis of germ cell tumor versus high-grade carcinoma consists of SALL4, OCT4, and epithelial membrane antigen (EMA) or, alternatively, OCT4, GPC3, EMA, and CK7. If the diagnosis of metastatic carcinoma is supported, appropriate additional immunohistochemistry to determine the primary should be performed based on clinical information and morphology. (E) The differential diagnosis of choriocarcinoma and seminoma with syncytiotrophoblast cells This distinction can be accomplished with OCT4, which is uniformly positive in seminoma cells and negative in trophoblast cells. (F) The differential diagnosis of intratubular germ cell neoplasia and atypical intratubular germ cells OCT4 is the preferred marker because of its uniform sensitivity for IGCNU. Placental alkaline phosphatase and podoplanin are good alternative choices with a similar immunoreactivity profile. Summary of the Kidney Workgroup The kidney workgroup discussed 3 overarching topics. As there are several differential diagnostic situations, entities, and patterns in the classification of primary renal neoplasms, the workgroup has provided numerous tables in their manuscript to outline staining reactions and to support their overall recommendations. 4 (A) Markers to establish the diagnosis of renal neoplasm (site of origin) PAX8 is the most useful IHC marker for establishing the diagnosis of metastatic renal cell carcinoma (RCC). One can use IHC for other markers such as ER, CDX2, PSA, TTF-1, GATA3, and 1020 www.ajsp.com r 2014 Lippincott Williams & Wilkins

Am J Surg Pathol Volume 38, Number 8, August 2014 Best Practices in IHC Applications in Urologic Pathology p63 to help exclude other carcinomas, including those that also may label for PAX8. The other IHC markers currently in common practice (CD10, RCC marker antigen, Ksp-cadherin) are supportive of metastatic RCC but usually not indicated or useful. (B) Markers to aid in the classification of primary renal neoplasms (i) Tumors predominantly composed of clear cells Clear cell RCC can be distinguished from chromophobe RCC using a panel that includes CAIX, CD117, and CK7. Clear cell RCC can be distinguished from clear cell papillary RCC using a panel that includes CAIX, CK7, and racemase (AMACR). Qualitative differences in CAIX staining between the 2 entities is important to evaluate. Clear cell RCC can be distinguished from epithelioid angiomyolipoma using a panel that includes CAIX and cathepsin-k. We suggest including a wide-spectrum cytokeratin (AE1/AE3) or EMA to document the presence or absence of epithelial lineage. If the laboratory does not have cathepsin-k, the melanomaassociated markers HMB-45 and MART-1 can be used. Clear cell RCC can be distinguished from MiTF-TFE translocation associated carcinomas using a panel of antibodies that include cathepsin-k, TFE3, and TFEB. If TFE3 and TFEB are inconclusive (only weak or patchy nuclear staining or high background), fluorescence in situ hybridization assays should be performed. Epithelioid angiomyolipoma can be distinguished from MiTF-TFE translocation associated carcinoma by nuclear expression of PAX8 in the latter and total absence of CAIX and epithelial differentiation (AE1/ AE3, EMA) in the former. (ii) Tumors with a significant papillary component Special attention should be paid to areas of the tumor that transition from low grade to high grade. Proper prosecting can help select key diagnostic areas. A panel that includes CAIX, CK7, and racemase (AMACR) can discriminate between clear cell RCC with papillary areas and papillary RCC. This same panel can be used to distinguish clear cell RCC from true papillary RCC and clear cell papillary RCC. Type 2 papillary RCC is likely a heterogenous group of tumors that usually stain for racemase but may exhibit variable results with CK7, including total lack of staining. Before this diagnosis is rendered, other specific entities should be considered in the differential diagnosis. MiTF/TFE translocation associated carcinomas may have a prominent papillary component. Cathepsin-K shows diffuse immunoreactivity in most variants except t(x;17), whereas epithelial markers are usually negative. Antibodies directed toward the TFE3 and TFEB proteins are highly specific but somewhat unreliable, so break-apart fluorescence in situ hybridization probes directed against these gene fusions are better. A subset of these tumors will exhibit immunoreactivity with melanocytic markers (HMB-45 and MART-1). (iii) Tumors with extensive cytoplasmic eosinophilia Distinguishing oncocytoma from an eosinophilic variant of chromophobe RCC is the most common diagnostic challenge for which close attention to nuclear cytology and cytoplasmic features can be supplemented by performing IHC staining for CK7. Laboratories that have Ksp-cadherin may find this marker useful, although qualitative rather than quantitative differences in staining must be taken into account. S100A1 is potentially a very useful marker, which needs further validation. Oncocytic papillary RCC is a rare diagnosis that does not require IHC for proper classification. Oncocytic AML will express melanocytic markers and cathepsin-k. (iv) Tumors with a predominant sarcomatoid pattern of growth Careful prosecting and attention to areas of morphologic transition can aid in establishing a proper diagnosis and choosing the most appropriate section(s) to perform ancillary studies. Vimentin and PAX8 are unlikely to contribute significantly to the diagnosis, although PAX8 can narrow down the diagnosis to either renal or urothelial origin, ruling out metastatic disease. In combination with other markers, CAIX may help in classifying a tumor as a clear cell RCC, but interpretation must be performed carefully. Traditional markers of urothelial lineage but not PAX8 can help identify a sarcomatoid UCa. If sarcoma enters in the differential diagnosis, the possibility of leiomyosarcoma and dedifferentiated liposarcoma can be ruled out by performing appropriate staining studies. (v) Tumors with morphology suggestive of the distal nephron origin collecting duct carcinoma (CDC) and renal medullary carcinoma (RMC) CDC, on the basis of limited data, can be distinguished from RMC using a panel that includes INI-1 and OCT4, taking into consideration the clinical history of sickle cell trait. Cytokeratin 34bE12 may be expressed in CDC but not in RMC. UCa can be distinguished from both CDC and RMC using a panel that includes GATA3 and p63 (clone 4A4). (vi) RCC, unclassified type This category should only been used when all efforts to place the tumor into a distinct entity have been exhausted or when the immunophenotype is entirely at odds with the morphologic impression. r 2014 Lippincott Williams & Wilkins www.ajsp.com 1021

Amin et al Am J Surg Pathol Volume 38, Number 8, August 2014 The antibody panel used will depend on the entities being considered in the differential diagnosis. (C) Prognostic and predictive markers in renal neoplasms There are no markers or sets of markers ready for routine clinical use. Novel markers have been recently described that are associated with highrisk pathologic features and disease progression. However, these should not be used routinely until further clinical and technical validation is performed. Summary The proceedings, outcome, and recommendations of the 4 workgroups with specific ISUP recommendations form the basis of the 4 manuscripts accompanying this editorial, which are published in the online version of the manuscript. The aim is to provide relevant and succinct information of practical value to surgical pathologists to assist in their routine daily practice. For the pathologists in the United States, these recommendations have additional significance given the recent stance by the Centers for Medicare and Medicaid Services (CMS), the major payor for medical services, to reduce reimbursement for IHC. Such developments call for further scrutiny by all of us to achieve greater efficiency through implementation of best practice approaches to IHC in our continuous quest to arrive at precise and correct diagnoses for our patients. REFERENCES 1. Amin MB, Trpkov K, Lopez-Beltran A, et al. Best practices recommendations in the application of immunohistochemistry in the bladder lesions report from the International Society of Urologic Pathology Consensus Conference. Am J Surg Pathol. 2014;38:e20 e34. 2. Epstein JI, Egevad L, Humphrey PA, et al. Best practices recommendations in the application of immunohistochemistry in the prostate report from the International Society of Urologic Pathology Consensus Conference. Am J Surg Pathol. 2014;38:e6 e9. 3. Reuter VE, Argani P, Zhou M, et al. Best practices recommendations in the application of immunohistochemistry in the kidney tumors report from the International Society of Urologic Pathology Consensus Conference. Am J Surg Pathol. 2014;38:e35 e49. 4. Ulbright TM, Tickoo SK, Berney DM, et al. Best practices recommendations in the application of immunohistochemistry in testicular tumors report from the International Society of Urological Pathology Consensus Conference. AmJSurgPathol. 2014;38:e50 e59. 1022 www.ajsp.com r 2014 Lippincott Williams & Wilkins