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Immunohistochemistry on Limited Tissue Samples: Do’S and Don’Ts

Immunohistochemistry on Limited Tissue Samples: Do’S and Don’Ts

3/31/2016

Disclosures

• I fretted over this . . . Immunohistochemistry on Limited Tissue Samples: Do’s and Don’ts

Andrew M Bellizzi, M.D. Department of Pathology University of Iowa Hospitals and Clinics andrew‐[email protected]

Disclosures

• Last night I was sleepless . . . in Seattle

1 3/31/2016

Disclosures

• Otherwise, I have nothing to disclose

Goals Outline

• To make an accurate, specific diagnosis with as • Competing interests: other ancillary studies few immunostains as possible • Technical aspects of immunocytochemistry • Enemies of the state: non‐specific immunos • To provide (prognostic and) predictive info • Next‐generation immunohistochemistry • To avoid less useful immunostains • Favorite markers/panels • To perform clinically valid immunohistochemistry – Carcinoma of unknown origin – Lung vs. • To triage tissue for other ancillary studies – – Solid pancreatic tumors – – Lymphoma

2 3/31/2016

63‐year‐old woman with liver, lung, and adrenal masses; presumed lung primary based on FNA of liver lesion 4‐months prior; EGFR/ALK/ROS1 wild‐type; progression on platinum‐ based chemotherapy; biopsy for cancer mutation profiling

TTF‐1

3 3/31/2016

Taher, Thanks for raising this issue in the setting of our increasingly engaging in this era of personalized medicine. This was my case, as staff pathologist. I ordered the TTF‐1 because I believed it was necessary to the diagnosis. In fact, in this case, it was negative. I had already ordered it BEFORE your call to the hot seat. So, there was no communication breakdown. I was aware of the prior diagnosis. I read your note while I was evaluating the glass slides. I was aware that the primary driver of this biopsy was to perform NGS. There was actually much more tumor present in the immunostained slide than on the initial H&E, fortunately. I was pessimistic of the prospects for NGS success based on the initial H&E. I was acutely sensitive of the purpose of this biopsy, which is why I limited myself to ONE critical immunostain. Taking one 5 micron section off the tissue block (for IHC) DOES NOT compromise the ability to perform molecular, especially if the sections for molecular are taken simultaneously. The anatomic pathologist is first and foremost responsible that a correct diagnosis is rendered. So, in this case I have to reasonably reassure myself that this is, in fact, metastatic lung adenocarcinoma. In this instance, both purposes should be served. Your note on the consult sheet WAS CRITICAL. If you hadn't made this comment, I would have performed a more extensive immunopanel. As always, communication is key. I would be happy to discuss this issue at greater length or in person. Best, Andrew

Tension Between Diagnosis and Predictive Ancillary Tests in FNA/Small Biopsy Testing is Increasing Tumor Type Standard Applications Extended Applications Breast Cancer ER, PR, HER2 Esophagus/Gastric AdCA HER2 • Use of IHC in pre‐op lung cytologic specimens Oropharygeal SCC and p16 or HPV ISH Head and Neck SCC of Study Period Diagnosis of AdCA Diagnosis of SCC Occult Origin IHC Frequency IHC Frequency Lung Adenocarcinoma EGFR,NGS ALK ROS1, PD‐L1 2000‐2004 14% (22/156) 11% (5/46) RET, MET, LKB1, PTEN 2005‐2010 86% (134/156) 89% (41/46) Colon Cancer KRAS, NRAS, BRAF, MSI PIK3CA, PTEN, EGFR CN Mesothelioma p16 FISH • >600% increase in IHC utilization Thyroid Aspirate MultigeneBRAF, KRAS Pancreatic Aspirate Cyst fluid analysis KRAS, DNA content Melanoma BRAF KIT Sarcoma FISH or RT/PCR (forPanel specific diagnosis) Hematolymphoid Flow cytometry, IgH/TCR gene rearrangements, FISH (for diagnosis or prognosis) Ocque R, Tochigi N, Ohori P, Dacic S. Am J Clin Pathol 2011;136:81‐7.

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Aisner DL, Sams SB. Diagn Cytopathol 2012;40:511‐24.

What’s Old is New

Gailey MP, et al. Cancer Cytopathol 2015. 123;30‐9. Knoepp SM, Roh MH. Cancer Cytopathol 2013. 121:120‐8. Snow AM, et al. BMC Clin Pathol 2014. 14;30. Hunt JL, et al. Diagn Cytopathol 1998;18:377‐80.

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Lessons Learned

• Do consider every biopsy of a tumor to be a potential molecular diagnostics specimen • Do consider cutting unstained sections up‐front for molecular testing, if ordering IHC • Do communicate with your clinical colleagues about priorities for the specimen • Do not give up hope if you’ve exhausted a specimen: you may be able to recover DNA from routinely processed material

FNA of a Mural‐Based Gastric Mass Cell block KIT KIT and DOG1 Intensity by FNA Method

• Cell blocks of 52 GIST, 24 LMS, 10 other • No significant difference in number of cells in EUS vs CT cellblocks • EUS‐FNA: FNA collected into CytoLyt (methanol‐based DOG1 KIT on subsequent resection fixative) • CT‐FNA: FNA collected into RPMI • plasma and thrombin added to produce cell block, fixed in 10% formalin, processed for paraffin embedding

Hwang DG, et al. Am J Clin Pathol 2011;135:448‐53.

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The staining of cytologic We This Might Be So . . . preparations with a battery of antibodies to the various cell • Surgical Pathologists • Cytopathologists may request components or products is very order IHC on FFPE tissue IHC on: costly and rarely rewarding . . . – Direct Smears The results of ICC vary according • Air‐dried to the batch of antibodies and – Unfixed – Post‐fixed the technical skills of the • Alcohol‐fixed laboratories, the interpretation – Unstained – Stained of the results is not always easy, – Destained and the problem with – Cytospins borderline positive stains is – Monolayer Preparations often perplexing. – Cell Blocks

Koss LG. The future of cytology. The Wachtel lecture for 1988. Acta Cytol 1990;34:1‐9.

Cell Block Cell Blocks: Advantages and Disadvantages

• Mandelbaum FS. • Advantages Diagnosis of malignant – Processed similarly to surgical pathology material tumors by paraffin – Facilitates multiple sections sections of centrifuged exudates. J Lab Clin – Easy to store Med. 1917; 2:580. • Disadvantages – Not amenable to on‐site adequacy assessment – May be pauci/acellular • 10.6% of 246 lung/thoracic FNA’s (Rafael OC, et al 2014) • 57% of 76 consecutive EBUS FNA’s (Knoepp, Roh 2013)

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Performance in Other Cytology Potential Sources of Pre‐Analytic Variation Specimen Types • Delay in fixation • Type, concentration, pH of fixative • Fixation time • Tissue Processing • Paraffin impregnation (paraffin melting point) • Block/slide storage

Engel KB, Moore HM. Arch Pathol Lab Med 2011;135:537‐43.

Potential Sources of Analytic Variation Optimization and Validation

• Antigen retrieval • Optimization: determination of provisional – Yes or no assay conditions, which is most often involves – Enzyme or heat‐induced staining a single case or small number of cases • For heat‐induced: buffer, pH, heat source at varying assay conditions • Primary antibody dilution • Validation: testing and appropriate tissue set • Duration of primary antibody incubation to determine analytic sensitivity and • Detection chemistry (ABC, polymer‐based) specificity, to reasonably assure that the test performs as expected

Goldsmith J. CAP Today Q&A. July 13, 2013. Goldsmith J. CAP Today Q&A. July 13, 2013. Fitzgibbons PL, et al. Arch Pathol Lab Med 2014;138:1432‐43.

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Recommendation

• Tissue should be fixed in 10% neutral‐pH, phosphate‐buffered formalin for a minimum of 8 hours. Non‐formalin‐based fixatives and or alternative fixation methodologies are strongly discouraged in regard to IHC, in large part because performance data are limited and extrapolation from formalin‐fixed data is unreliable.

Goldstein NS, et al. Appl Immunohistochem Mol Morphol 2007;15:124‐33.

Recommendation Antigen Retrieval

• Antigen retrieval (AR) is presumed to “restore” • Air‐dried smears theoretically should require the antigenicity after the formalin fixation. (less or) no AR The parameters of an AR protocol must be • Alcohol‐fixed smears (coagulation, rather than balanced to match the unique length and type cross‐linking, fixation) theoretically should of tissue fixation of the individual laboratory require (less or) no AR and the characteristics of the individual • Over‐AR produces high‐background staining antibody. and strong edge staining • Under‐AR produces false negative IHC

9 3/31/2016

Recommendations

• Laboratories must validate all IHC tests before placing them into clinical service • For initial analytic validation of nonpredictive factor assays, laboratories should test a minimum of 10 positive and 10 negative tissues

Fitzgibbons PL, et al. Arch Pathol Lab Med 2014;138:1432‐43.

Recommendations Controls

• “If IHC is regularly done on cytologic specimens that • “Unless the laboratory has a large bank of are not processed in the same manner as the tissues used for assay validation (eg, alcohol‐fixed cell blocks, similarly prepared cytology material for air‐dried smears, formalin‐postfixed specimens), positive and negative IC controls (non‐ laboratories should test a sufficient number of such formalin‐fixed cytology specimen by the exact cases to ensure that assays consistently achieve expected results . . . The strength of evidence was same preparation method as the current inadequate to address the criteria and number of sample being tested), then criteria for having samples needed for validation with cytology “proper controls” is not met and any specimens. If an assay has not been fully validated on interpretation of IC results should be suspect.” cytologic specimens, laboratories may include a disclaimer in their report that results should be interpreted with caution.” Fowler LJ, Lachar WA. Arch Pathol Lab Med 2008;132:372‐83.

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Controls Multiplexing

• “In our experience, unstained direct smears in • Double‐staining, triple‐staining, etc. high quantities can be prepared using • Performing a second IHC on a previously IHC‐ centrifuged cellular material for effusion stained slide (if negative) specimens and these can be used for positive control ICC reactions.”

Knoepp SM, Roh MH. Cancer Cytopathol 2013. 121:120‐8. Dabbs DJ, Wang X. Diagn Cytopathol 1998;18:166‐9.

Lessons Learned

• Do perform ICC on cell blocks, if possible • Do examine cell block technique if “ICC performing suboptimally • Do consider including cytology specimens in clinical IHC validations • Do include similarly processed positive controls on ICC runs • Do not perform ICC on other cytology specimen types unless the procedure has been specifically optimized for them (esp. antibody dilution, antigen retrieval)

11 3/31/2016

Myths

• CA125 is specific for Müllerian origin • CA19‐9 is specific for pancreatic origin • CK19 is specific for pancreatobiliary origin • MOC‐31 is an adenocarcinoma marker • RCC is specific for renal origin • Vimentin is specific for sarcoma

CA125 Expression Tumor Site % Positive CA19‐9 Expression (adenocarcinoma unless otherwise noted) (at ≥10% cells staining with ≥ moderate intensity)

Ovary 98 Tumor Site (n) % Positive (adenocarcinoma unless otherwise noted) Endometrium 90 (26) 85 Cervix 53 Breast, ductal (119) 48 Pancreas 82 Breast, lobular (10) 10 63 Lung (35) 69 Lung 56 Stomach (39) 56 51 Colon (25) 76 Head and Neck (SCC) 40 Ovary (29) 41 Thyroid 39 Kidney (45) 27 Breast 27 Mesothelioma <<1 Lung (SCC) 23 Melanoma, lymphoma, sarcoma, 0 neuroendocrine, hepatocellular Gremel G, et al. Histopathology 2014;64:293‐305. Kaufmann O, et al. Histopathology 1996;29:233‐40.

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CK19 Expression Tumor Site % Positive (adenocarcinoma unless otherwise noted) (at ≥10% cells staining with ≥ moderate intensity) Cholangiocarcinoma 100 Pancreas 100 Gallbladder 100 Lung 100 Ovary 98 Breast 98 Colon 98 Stomach 98 Urothelial 95 Cervix 93 Lung (SCC) 91 Melanoma, lymphoma, hepatocellular 0

Gremel G, et al. Histopathology 2014;64:293‐305. MOC‐31

Dx Algorithm: Poorly Differentiated MOC‐31 Expression Carcinoma in the Liver

Tumor Type (n) % Positive IHC menu for this

Squamous cell carcinoma (110) 41 HCC vs. ICC vs. application: Mesothelioma (10) 0 Perform Hep Par 1, MOC‐31 HCC Markers: (10) 20 (alternatively Arginase‐1, Claudin‐4) ‐ Hep Par 1 Neuroendocrine carcinoma (5) 60 ‐ GPC3 Lung adenocarcinoma (6) 100 ‐ pCEA/CD10 Colon adenocarcinoma (6) 100 Hep Par 1 (+)/ Hep Par 1 (‐)/ (canalicular) Urothelial carcinoma (11) 55 MOC‐31 (+) MOC‐31 (‐) ‐ Arginase‐1 Neuroendocrine tumor, midgut (5) 100  HCC  ICC or Metastasis Neuroendocrine tumor, pancreas (5) 80 Non‐HCC Markers: ‐ MOC‐31 ‐ Claudin‐4

Savage EC, Gailey MP, Bellizzi AM. Abstract at 2014 USCAP Annual Meeting.

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Hepatocellular carcinoma

73‐year‐old man with liver lesion, subtotally necrotic Hep Par 1 MOC‐31

Large right retroperitoneal tumor

14 3/31/2016

13 cm tumor spanning the kidney and adrenal, expresses CD10, melan A, and inhibin

PAX8 vs PAX2 Expression

Tumor Type PAX8 (% Positive) PAX2 (% Positive) Clear cell 97 95 Papillary 100 76 Chromophobe 88 56 Collecting duct 71 43

Serous 98 55 Endometrioid 94 25 Clear cell 100 19 Transitional 67 11

Thyroid 91 0

Ozcan A, et al. Am J Surg Pathol 2011;35:1837‐47. Ozcan A, et al. Arch Pathol Lab Med 2012;136:1541‐51. PAX8 Laury AR, et al. Am J Surg Pathol 2011;35:816‐26.

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Endometrioid Endometrial vs. Endocervical AdCA Endometrioid Endocervical Endometrial (n=26: ER, vim, CEA) (n=30: ER, vim, CEA) (n=23: p16) Melanoma Burkitt lymphoma (n=29: p16) ER 93% 38% Vimentin 97% 8% CEA 70% 96% (usually in squamous foci) p16 97% (any staining) 96% (any staining) 27% (≥50% cells stain) 96% (100% cells stain) 10% (100% cells stain)

McCluggage WG, et al. Int J Gynecol Pathol. 2002 Jan;21(1):11‐5. Vimentin Vimentin McCluggage WG, Jenkins D. Int J Gynecol Pathol. 2003 Jul;22(3):231‐5.

Results of immunopanel favor endometrial origin Lessons Learned

• Do not extrapolate the results of differential‐ specific markers beyond the differential (e.g., CK19, MOC‐31) Uterine cervical mass ER, PR • Do not order CA125, CA19‐9, RCC (No No Never) • Vimentin??? If you must . . . sparingly . . . But please don’t tell me that you did . . .

Vimentin p16

16 3/31/2016

Gene Expression Profiling Comparing Next‐Generation Immunohistochemistry Urothelial, Kidney, and Prostate Cancer • Mine developmental biology and molecular genetic literature to find: – Lineage‐restricted transcription factors – Markers identified by gene expression profiling – Protein correlates of molecular genetic events • Bottom line: our markers keep getting better

Higgins JPT, et al. Am J Surg Pathol. 2007 May;31(5):673‐80.

Historically, diagnostic armamentarium geared toward Primacy of lineage‐restricted transcription factors cytoplasmic or membranous differentiation markers; reduced sensitivity in poorly differentiated tumors

Breast cancer Mammaglobin GCDFP‐15 GATA‐3: highly expressed, regardless of differentiation

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Regional Differential Diagnosis Diagnosis of Broad Tumor Class

• Brain (carcinoma, melanoma, lymphoma, glioma) • Carcinoma (broad spectrum keratins, EMA [aka MUC1]) • Lymph node (lymphoma vs. other) – Supraclavicular (anything), Periaortic (germ cell tumor) • Melanoma (S‐100, SOX10, melan‐A, HMB‐45, MiTF, BRAF) • Mediastinum (lung, lung, lung, thymic [KIT,CD5], germ cell) • Lymphoma (CD45; CD43, CD79a, MUM1, ALK1, CD30 if LCA‐) • Visceral organ (1° vs metastasis) • Sarcoma (CD34, MDM2/CDK4 – DDLPS , add’l based on morph.) – Lung (AdCA vs SCC vs metastasis) • Mesothelioma (WT‐1, calretinin, CK5/6, D2‐40) – Liver (HCC vs pancreatobiliary vs other) • Germ cell tumor (SALL4, PLAP) • Pleural effusion (AdCA vs mesothelioma) • /Ovary (1° surface epithelial vs metastatic GI) • Non‐epithelial neuroendocrine (CG, SYN) • Retroperitoneum (well and dedifferentiated ) • Somatic (sarcoma vs. metastasis) • Bone (blastic‐prostate, breast; lytic‐RCC, thyroid; mixed‐lung)

Diagnosis of Carcinoma Type Which Screening Keratin Should I Use? Clone 12345678911 1 1 1 1 1 1 1 1 0 1 2 3 4 5 6 7 8 9 Carcinoma AE1/AE3 XXXXXX X X XXX X (cohesive, keratin and/or EMA+) OSCAR XX XX MAK‐6 X XXX XX MNF116 XX X X Adenocarcinoma Squamo‐ Neuroendocrine Large polygonal cell CAM5.2 XX (gland forming/ transitional (CK7/CK20‐) (CG and/or SYN+) mucin producing) –see (p63, p40, KL1 XX XXXX X X XXX next algorithm CK5/6+) NET NEC 34βE12 XX XX Ki‐67 Ki‐67 ≤20% >20% • In general, any of these are acceptable SCC CK7 var. • It’s not the number of keratins, per se, but rather the affinity GATA3‐ ? UC HCC CK7/CK20+ Hep Par 1 + (e.g., CAM5.2 vs AE1/AE3 in HCC/RCC) GATA3+ GPC3+ AdCC RCC Mel‐A+ • Stratified epithelia: K1‐6, 9‐17 PAX8+ Inhibin+ SYN+ • Simple epithelia: 7, 8, 18, 19, 20 SF1+ Ordóñez NG. Hum Pathol. 2013 Jul;44(7):1195‐215.

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Coordinate Expression of CK7/CK20 Cancer Epidemiology and Morphology‐ Based Site of Origin Generator Site (tumor) CK7 CK20 Most Morphology Most Morphology Prostate, HCC, AdCC, RCC Common Common -- Primary Sites Primary Sites in Men in Women Lung, Breast, Müllerian, (Rank Order) (Rank Order) +- Prostate Characteristic nuclear Breast Variable Pancreatobiliary (PB), Upper GI (UGI) (25%) features regardless of (26%) (AdCA) Gleason grade: (AdCA) Bladder, UGI, PB, Mucinous Ovarian, monomorphous and Colon (esp. Rectum), Occ. Lung ++ prominent nucleoli Lung (15%) Variable (AdCA) Lung (14%) Variable (AdCA) (AdCA, SCC, (AdCA, SCC, Colon, Merkel cell, Occ. UGI NEC) NEC) -+ Colorectum Characteristic Colorectum Characteristic (10%) cytoarchitectural (10%) cytoarchitectural (AdCA) features: “tall, dark, (AdCA) features: “tall, dark, and dirty” and dirty”

IHC to Assign AdCA Site of Origin: Lung Adenocarcinoma vs Ranked from Most to Least Ordered (2011) Squamous Cell Carcinoma • CDX2 (enteric differentiation) Marker AdCA SCC • TTF‐1 (lung, thyroid) Transcription (% positive) (% positive) • ER and PR (breast, Müllerian) factors in bold CK7 100 68 • PAX8 (kidney, Müllerian, thyroid) • p53 and WT‐1 (serous carcinoma, latter also exp. by mesothelioma) TTF‐186 21 • Napsin A (lung, papillary RCC) p63 31 100 • PSA and PSAP (prostate) CK5/6 21 100 • GCDFP‐15 and mammaglobin (breast) • Thyroglobulin (thyroid) • p63, CK5/6 have a specificity problem ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ • TTF‐1 has a sensitivity problem • GATA‐3 (breast; also urothelial) • NKX3.1 (prostate) Ocque R, Tochigi N, Ohori P, Dacic S. Am J Clin Pathol 2011;136:81‐7.

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p40 (ΔNp63) Napsin A

Marker Lung AdCA SCC (% positive) (% positive) Napsin A 87 (n=303) 3 (n=200) Marker AdCA SCC Large Cell Lymphoma TTF‐1 64 (n=94) 2 (n=94) (% positive) (% positive) (% positive) p63 31 100 54 • p40 3* 100 0 Lower rate of TTF‐1+ AdCA than literature (75‐85%) • * Each 1‐5% cells staining Napsin A frequently expressed by RCC (esp. papillary) and clear cell carcinoma • TTF‐1+/p40‐ AdCA • TTF‐1‐/p40‐ AdCA

Bishop JA, et al. Mod Pathol 2012;25:405‐15. • TTF‐1‐/p40+ SCC Turner BM, et al. Arch Pathol Lab Med 2012;136:163‐71.

Soft Tissue: IHC to Assign Lineage Soft Tissue Markers Validated at

Line of Differentiation Characteristic Historically Useful Immunostains U of Iowa in Last 3‐Years Adipocytic Fatty S‐100 Name Diagnostic Application Fibroblastic/myofibroblastic Spindled; light pink SMA, CD34, ALK Beta‐catenin Desmoid (70%), solid‐pseudopapillary neoplasm, So‐called fibrohistiocytic Shrinking group (e.g., TSGCT) None especially pancreatoblastoma; (50‐90%), fetal‐type lung AdCA, colonic adenoma/AdCA Spindled; brightly eosinophilic SMA, desmin MDM2 and CDK4 Well and dedifferentiated liposarcoma Pericytic Spindled; “circumferential SMA (MDM2 FISH) perivascular growth pattern” TLE1 ; Pitfalls: weaker staining in other , Skeletal muscle Rhabdomyo(sarco)ma Desmin, myogenin frequently positive in carcinomas Vascular Vascular channels CD34, CD31, Factor VIII MUC4 Low‐grade fibromyxoid sarcoma, sclerosing epithelioid Chondro‐osseous Cartilage, osteoid S‐100 (75%); also broadly expressed by epithelia Interstitial cell of Cajal GIST KIT, CD34 ERG Vascular tumors,prostate cancer (50%); occ. myeloid leukemias Nerve sheath Spindle cell; wavy nuclei S‐100, GFAP and Ewing sarcoma (10%) “Uncertain” “Distinctive” tumor types (e.g., Keratins, EMA, S‐100, STAT6 (NAB2‐STAT6 translocation) synovial sarcoma, epithelioid (TFE3, HMB‐45, WT‐1) HHV8 Kaposi sarcoma, primary effusion lymphoma, multicentric sarcoma, PEComa) Castleman disease, plasmablastic lymphoma arising in Undifferentiated/unclassified Formerly MFH Diagnosis of exclusion plasmablastic lymphoma

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Soft Tissue Markers Validated at U of Iowa in Last 3‐Years Name Diagnostic Application

SOX10 Melanoma, MPNST (50%), , myoepithelial differentiation; superior in sensitivity and specificity to S‐100 INI1 Epithelioid sarcoma (90%), malignant rhabdoid tumors of soft tissue, kidney, CNS (>95%), medullary carcinoma of the kidney, Burkitt lymphoma other INI1‐deficient tumors (some epithelioid MPNST, DLBCL myoepithelial CA of soft tissue, extraskeletal myxoid chondrosarc.)

c‐Myc c‐Myc

CD20 CD10 Cyclin Other Useful Tumor Type CD79a CD5 CD43 Bcl‐2 Bcl‐6 D1 Markers/Notes PAX5 Advantages of ICC over Flow Cytometry Tumors composed of small cells Extranodal marginal zone lymphoma of Kappa/lambda light Occ. chain restriction (occ.); • Hodgkin lymphoma mucosa‐associated + ‐‐(30%) ‐ Var. keratins to highlight lymphoid tissues lymphoepithelial lesions • Burkitt lymphoma vs DLBCL, GC Type (MALT lymphoma) – BL: c‐Myc+, Bcl‐2‐ Ki‐67 proliferation index Mantle cell >40‐60% associated with – DLBCL: c‐Myc‐, Bcl‐2+ or ‐ lymphoma + ‐ ++++poor prognosis; rarely CD5 or cyclin D1‐

Higher grade tumors more likely to show Follicular lymphoma ++‐‐‐+ aberrant IHC (e.g., CD10‐ or CD43+)

Chronic lymphocytic leukemia/small CD23+ lymphocytic + ‐ ++‐ + lymphoma

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CD20 CD10 Cyclin Other Useful Tumor Type CD79a CD5 CD43 Bcl‐2 Hematolymphoid Markers Validated at Bcl‐6 D1 Markers/Notes PAX5 Tumors composed of intermediate/large cells U of Iowa in Last 2‐Years CD10 Var. Diffuse large B‐cell (30‐60%) + Rare (10%) Occ. (25%) ‐ Var. MUM1 (35‐65%) Name Diagnostic Application lymphoma Bcl‐6+ (60‐90%) Tryptase Mast cells Ki‐67 proliferation index Burkitt lymphoma ++‐ + ‐‐approaches 100%; TdT‐; c‐Myc+; CD163 Monocyte‐macrophage lineage marker (more specific than CD68) Bcl‐2 occ. weak +

CD20/PAX5‐ EBV EBER (60‐75%); CD45‐; c‐MYC Burkitt lymphoma and c‐MYC activated DLBCL Plasmablastic + ‐ + ‐ + MUM1/CD138/CD38+; lymphoma CD79a+ EMA/CD30 var.; (50‐85%) association with HIV SOX11 Mantle cell lymphoma (w/ emphasis on identifying CycD1‐ cases) Granulocytic ‐‐‐+ ‐‐CD68/KIT/CD99/ CD34/TdT Var. Sarcoma

CD20 Var. CD10+ (60%) B‐lymphoblastic (25‐50%) TdT (95%), CD34/CD99+; ‐ + (67%) ‐ + CD13/CD33 Occ. lymphoma Bcl‐6‐ CD79a/PAX5+

CD20/ PAX5‐ TdT (95%), CD3/CD99+; T‐lymphoblastic CD10 Var. CD4/CD8 double+ (70%); CD1a Var. CD79a + (90%) ‐ + (67%); CD34 Var.; CD13/CD33 lymphoma Bcl‐6‐ Rare (5‐10%) Occ.

Lessons Learned Thank you!!! • Do perform a limited panel of IHC to assign tumor type/site of origin based on clinical and morphologic differential (“big 3” + appropriate “next‐gen” markers) • Do not equate p63+ with SCC; do consider p40 • Do consider using “next‐gen” markers to make specific sarcoma and lymphoma diagnoses

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U of Iowa Development Queue Diagnosis of Broad Tumor Class Name Diagnostic Application Androgen receptor Sebaceous CA, salivary duct CA, prostate CA; Pitfall: low specificity Arginase Hepatocellular CA; Pitfall: occ. stains AdCA Claudin‐4 Broad‐spectrum epithelial marker not expressed by hepatocytes/meso’s Clusterin Follicular dendritic cell sarcoma, ALCL, tenosynovial GCT, non‐ileal NET D2‐40 Mesothelioma (inc. sarc.), lymphatic endothelium, seminoma, others Glutamine synthetase Focal nodular , BCAT‐HA, HCC vs. B9 liver: Pitfall: other CAs + Mammaglobin Breast CA;surrogate for ETV6 FISH in mammary analogue secretory CA NKI‐C3 (CD63) Cellular neurothekeoma; + in many other tumors inc. melanoma NUT NUT midline carcinoma (undifferentiated CA with abrupt keratinization) p40 (ΔNp63) Squamous cell carcinoma (superior spec. to p63); basal cells and myoep’s SATB2 Colon AdCA; osteoblastic differentiation; rectal NET SDHB SDHB‐deficient GIST, paraganglioma/pheochromocytoma, other SMAD4 Pancreatic ductal AdCA (50% demonstrate loss); midgut NET (40%) SF1 Adrenal cortical , sex‐cord stromal tumors

Morphologic “Boxes”

• Cohesive, poorly cohesive, dyscohesive

• Spindle cell (sarcoma, sarcomatoid carcinoma) Pleomorphic and High‐Grade are • Pleomorphic (anything) NOT Synonymous • Round cell (lymphoma, sarcoma) • Epithelioid (carcinoma, melanoma)

• Monomorphous or pleomorphic

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Clear cell sarcoma Melanoma

CDX2 heterogenous: UGI, Monomorphous Pleomorphic CDX2 homogenous: LGI mucinous ovarian, PB (20%)

NET Site of Origin Algorithm

CK20 weak, patchy: UGI, CK20 diffuse, strong: LGI mucinous ovarian, PB (≤40%) Maxwell JE, Sherman SK, Stashek KM, O’Dorisio TM, Bellizzi AM, Howe JR. Surgery. 2014 Dec;156(6):1359‐66.

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Midgut NETs have a characteristic histologic Pancreatic NETs are of diverse histologic appearance and nearly always express CDX2 appearance and nearly always express ISL1

Transcription Factor “Infidelity” in NEC Frequently Expressed Transcription Factors (%) Merkel Cell Small Cell Extrapulmonary Carcinoma Lung Carcinoma Visceral NEC (n=40) (n=24) (n=19) FLI1 90 88 78 Merkel cell carcinoma Small cell lung carcinoma GATA3 24 8 40 Islet 1 97 88 58 Myc 29 25 61 PAX6 78 29 26 PLAG1 58 79 72 SATB2 63 42 89 SOX2 100 96 89 TTF1 59644 CK20+/TTF‐1‐ TTF‐1+/CK20‐ NECs expressed a median of 8 TFs (range 0‐18) out of 38 examined (≥90% Merkel cell carcinoma (≥90% SCLC, 45% visceral NEC) Czeczok TW, Gailey MP, Hornick JL, Bellizzi AM. Mod Pathol. 2014 Feb;27(S2):152A.

25 3/31/2016

76‐year‐old man with past tobacco use and asbestos exposure p/w pleural effusion; at thoracotomy diaphragm encased by nodular Calretinin, WT‐1, CK5/6, fibrous tissue; multiple plaque like areas throughout pleural cavity Pan‐keratin MOC‐31, Ber‐EP4, STAT6

Mesothelioma‐Marker Expression in Sarcomatoid Mesothelioma

AE1/AE3 75% (18/24) CAM5.2 96% (23/24) MNF‐116 100% (21/21) Calretinin 25% (6/24) WT‐1 33% (8/24) D2‐40 100% (24/24)

All of these were prospectively dx’d as pancreatic neuroendocrine tumor . . . Chirieac LR, et al. Am J Cancer Res. 2011;1(1):14‐24. only one of them is

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Immunophenotype of Cellular Epithelioid Cellular Epithelioid Neoplasms of the Pancreas Neoplasms of the Pancreas • Solid neoplasms composed predominantly of neoplastic elements with little stroma PNET SPN ACC PB – Pancreatic neuroendocrine tumor (PNET) Broad Spectrum + 70% + + – Solid‐pseudopapillary neoplasm (SPN) Keratins – Acinar cell carcinoma (ACC) Synaptophysin 95% 20‐70% Rare cells 80% – Pancreatoblastoma (PB) • On cytology: dyshesive, monomorphic Chromogranin 90% ‐ Rare cells 80% – SPN: 3 of 6 misdiagnosed as PNET (Bardales et al ‘04) – ACC: 2 of 4 misdiagnosed as PNET (Stelow et al ‘06) Trypsin Rare cells ‐ 95% 95% – ACC: 14/29 misdiagnosed, 5 as PNET, 5 as ductal AdCA β‐catenin <5% >95% 10% >90% (Sigel et al ‘13)

BCAT

Core biopsy from a 40 cm retroperitoneal tumor demonstrates undifferentiated neoplasm composed of sheets of epithelioid cells. After performing 17 immunostains a diagnosis of “malignant neoplasm” Trypsin BCAT Trypsin indeterminate for sarcoma, carcinoma, or lymphoma was rendered.

27 3/31/2016

Most consistent with dedifferentiated liposarcoma Resection of similar case

Well‐differentiated component

MDM2, CDK4 Abrupt transition Dedifferentiated component

Keratin and/or EMA‐positivity do not assure a diagnosis of carcinoma

Potential Pitfall #1

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Leiomyosarcoma Smooth muscle actin, desmin+ Keratin AE1/AE3+: keratin, EMA expression in 30‐40% LMS

EMA+, AE1/AE3+, GATA‐3 focal + p63, CK5/6, ER, PR, TTF‐1, WT‐1, CD31, S‐100, HMB45 all ‐ 33‐year‐old woman with 1‐year h/o R hip pain; large SQ mass Conclusion: Favor metastatic carcinoma, ? breast or urothelial

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63‐year‐old man with increasing hip pain x 1 month; INI1: absent expression  epithelioid sarcoma, proximal‐type proximal femur lesion with soft tissue extension

Undifferentiated Malignant Neoplasm with Osteoclast‐like Giant Cells • Undifferentiated/anaplastic carcinoma – Keratin AE1/AE3 – CDX2, PAX8, TTF‐1 • Osteosarcoma – SATB2 • – Desmin, SMA, caldesmon

Keratin AE1/AE3+ (desmin, SMA ‐) Conclusion: Favor undifferentiated carcinoma

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Musculoskeletal radiologist sug. presence of chondroid matrix (MRI) Abrupt transition from WD chondrosarcoma to undifferentiated Subsequent femoral head resection for pathologic fracture neoplasm  dedifferentiated chondrosarcoma

Keratin‐Positive Soft Tissue Tumors EMA‐Positive Soft Tissue Tumors

• Chondroid • Epithelioid MPNST • Pleomorphic liposarcoma • Solitary circumscribed neuroma • Pleomorphic liposarcoma • Ectopic hamartomatous thymoma • Calcifying aponeurotic • Meningioma • Desmoplastic fibroblastoma • Ossifying fibromyxoid tumor • Lipofibromatosis • Hybrid nerve sheath tumor • Solitary fibrous tumor • Myoepithelial tumors of soft tissue • Dermatofibrosarcoma protuberans • Acral fibromyxoma • Inflammatory myofibroblastic tumor • Synovial sarcoma • Solitary fibrous tumor • Angiomatoid fibrous • Myxoinflammatory fibroblastic sarcoma • Epithelioid sarcoma • Low‐grade fibromyxoid sarcoma • Myoepithelial tumors of soft tissue • Leiomyosarcoma • Desmoplastic small round cell tumor • Sclerosing epithelioid fibrosarcoma • Synovial sarcoma • • Extrarenal rhabdoid tumor • Leiomyosarcoma • Epithelioid sarcoma • Schwannoma (cross‐reactivity with GFAP) • Undifferentiated/unclassified sarcoma • Pleomorphic rhabdomyosarcoma • Desmoplastic small round cell tumor • Epithelioid hemangioma • Chondroblastoma • Epithelioid hemangioma • Extrarenal rhabdoid tumor • Pseudomyogenic hemangioendothelioma • Dedifferentiated chondrosarcoma • Epithelioid hemangioendothelioma • Undifferentiated/unclassified sarcoma • Epithelioid hemangioendothelioma • Conventional osteosarcoma • Angiosarcoma • Conventional osteosarcoma • Angiosarcoma • Ewing sarcoma • Neurofibroma • Chordoma • Gastrointestinal stromal tumor • Chordoma • Perineurioma • Epithelioid hemangioma • Sclerosing perineurioma • Adamantinoma • Dermal nerve sheath • Adamantinoma • Dermal nerve sheath myxoma • Osteofibrous dysplasia

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Dr. Bellizzi, what kind of S‐100‐positive carcinoma is this?

78‐year‐old man p/w word‐finding difficulty: L temporal lobe mass Pan‐keratin S‐100

S‐100 Expression in Adenocarcinoma

Primary Tumors Metastatic Tumors Salivary gland 80% (n=15) 75% (n=4) Lung 7% (n=27) 12% (n=25) Breast 60% (n=20) 62% (n=8) Esophagus 0% (n=8) 0% (n=2) Stomach 20% (n=10) 25% (n=8) Gallbladder 0% (n=1) 0% (n=1) Melan A (A103) HMB‐45 Colorectum 25% (n=28) 23% (n=13) Pancreas 0% (n=8) 0% (n=5) Conclusion: Kidney 65% (n=23) 66% (n=3) metastatic melanoma Endometrium 78% (n=36) 64% (n=14) Ovary 84% (n=24) 87% (n=22) Prostate 0% (n=27) 0% (n=8) BRAF mutation testing: Unknown origin 22% (n=9) wild‐type Total 43% (n=228) 39% (n=122) Herrera GA, et al. Am J Clin Pathol. 1988 Feb;89(2):168‐76. MiTF

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Keratin‐Positivity in Melanoma Keratin‐Positivity in Lymphoma

Primary Recurrent or 1 cutaneous metastatic • Anaplastic large cell lymphoma (5/18; 28%) melanoma melanoma • 13/866 (1.5%) hematolymphoid tumors in TMA2 (n=62) (n=22) – 5/18 (28%) mantle cell lymphomas Vimentin 100% 100% S‐100 95% 95% • Otherwise, case reports and small series NSE 87% 77% HMB45 97% 64% NKI‐C3 97% 95% Cytokeratin 0% 23% (KL1, CAM5.2, 35βH11)

1. Gustmann C, et al. Am J Pathol. 1991 Jun;138(6):1413‐22. Achilles E, Schröder S. Pathologe. 1994 Aug;15(4):235‐41. 2. Adams H, et al. Pathol Res Pract. 2008;204(8):569‐73.

EMA‐positivity in Hematolymphoid Neoplasms

• Anaplastic large cell lymphoma (50‐95%) • Plasma cell neoplasms (most) Beware of LCA‐negative • Diffuse large B‐cell lymphoma hematolymphoid neoplasms – TC/HR, ALK+, plasmablastic, primary effusion lymphoma • T‐cell lymphoma (20%) Potential Pitfall #2 • Nodular lymphocyte‐predominant Hodgkin (L&H) • Follicular dendritic cell sarcoma (var.)

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LCA‐Negative Hematolymphoid Neoplasms

• Acute megakaryoblastic leukemia • B‐cell acute lymphoblastic leukemia/lymphoma • ALK‐positive large B‐cell lymphoma – EMA+, CK‐/+; CD20‐, CD79a‐ • Plasmablastic lymphoma (EBER usually +) – EMA+/‐; CD20‐, PAX5‐ • Anaplastic large cell lymphoma, ALK‐positive (some) • Classical Hodgkin lymphoma • Follicular dendritic cell sarcoma

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