3/23/2017. Differentiation: Differentiation: Immunohistochemistry. Well Differentiated vs. Poorly Differentiated Neuroendocrine Neoplasms

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1 Disclosure Statement When Immunostains Can Get You Into Trouble (and how they can help you out): Neuroendocrine Neoplasms Arthur Purdy Stout Society March 5, 2017 Dr. Klimstra receives royalty payments from Up To Date and the American Registry of Pathology PET CT David S. Klimstra, MD Chairman, Department of Pathology James Ewing Alumni Chair of Pathology Attending Pathologist Memorial Sloan Kettering Cancer Center Professor of Pathology and Laboratory Medicine Weill Cornell Medical College Neuroendocrine Neoplasms Diverse but related groups of tumors Lung, thymus, pancreas, GI tract, other sites Characteristic pathologic features Immunohistochemical evidence of neuroendocrine differentiation (chromogranin / synaptophysin / CD56) Differentiation: Extent of resemblance of the cells of a neoplasm to their normal cellular counterparts Usually closely linked to grade (for NETs) Range of biological aggressiveness Can be either well differentiated tumors or poorly differentiated carcinomas Differentiation: Immunohistochemistry A Synaptophysin Well Differentiated vs. Poorly Differentiated Neuroendocrine Neoplasms Two different families Both share neuroendocrine differentiation Can be difficult to distinguish Fundamentally different Cell of origin Relationship to non-ne neoplasia Genetic background Clinical aggressiveness Treatment CarcinoidTumor Small Cell Carcinoma 1

2 Well Differentiated Classification of Pulmonary Neuroendocrine Neoplasms Low Grade Carcinoid Tumor Intermediate Grade Atypical Carcinoid Tumor WHO 2010 Grading of GEP-NETs Grade Mitoses Ki-67 Index G1 < 2 / 10 H.P.F. < 3% G / 10 H.P.F. 3-20% Poorly Differentiated High Grade Small Cell Carcinoma Large Cell Neuroendocrine Carcinoma G3 > 20 / 10 H.P.F. > 20% Poorly Differentiated (High Grade ) Neuroendocrine Carcinoma Pancreatic NETs: Overall Survival by Grade Rindi et al., J Natl Cancer Inst 2012; 104: 764 Terminology for Neuroendocrine Neoplasms: WHO 2010/2015 Well Differentiated NETs Well differentiated NET (pancreas, GI tract, etc.) Carcinoid tumor / atypical carcinoid tumor (lung, thymus) Poorly Differentiated NECs Small cell carcinoma Large cell neuroendocrine carcinoma Mixed neuroendocrine carcinoma (with component of adenocarcinoma, squamous cell carcinoma, etc.) Use of Immunohistochemistry in Neuroendocrine Neoplasms Recognition of Neuroendocrine Differentiation Diagnosis (recognition of neuroendocrine differentiation) Delineation of primary site PET-CT PET-CT Well Differentiated Neuroendocrine (Carcinoid) Tumor Determination of grade, classification, prognosis 2

3 Recognition of Neuroendocrine Differentiation PET-CT Poorly Differentiated Neuroendocrine Carcinoma Recognition of Neuroendocrine Differentiation: Immunohistochemical Markers Conventional markers A Synaptophysin CD56 (neural cell adhesion molecular / NCAM) Neuron specific enolase (NSE) CD57 / Leu7 PET-CT PGP9.5 Novel markers Synaptic vesicle protein 2 (SV2) Achaete-scute complex homolog (MASH1) Insulinoma-associated protein 1 (INSM1) Neuroendocrine secretory protein 55 (NESP55) Sensitivity of A Sensitivity of Synaptophysin % Negative (n) Pulmonary carcinoid tumor 3% (368) Duodenal NET 7% (61) Ileal NET 4% (51) Pancreatic NET 17% (108) Thymic carcinoid tumor 19% (95) Pheochromocytoma 1% (182) Pulmonary small cell carcinoma 57% (596) Pulmonary large cell NE carcinoma 37% (252) % Negative (n) Pulmonary carcinoid tumor 2% (333) Pulmonary atypical carcinoid tumor 10% (115) Rectal NET 4% (28) Ileal NET 2% (58) Pancreatic NET 1% (75) Thymic carcinoid tumor 21% (101) Pheochromocytoma 2% (188) Pulmonary small cell carcinoma 25% (97) Pulmonary large cell NE carcinoma 17% (268) Source: Immunoquery Source: Immunoquery Small Bowel Tumor with Mesenteric Deposits Tumor positive with somatostatin receptor scintigraphy Synaptophysin CD56 3

4 Specificity of and Synaptophysin % % Synaptophysin Positive (n) Positive (n) Breast ductal carcinoma 2% (287) Breast colloid carcinoma 18% (112) 41% (105) Pulmonary adenocarcinoma 2% (689) 11% (689) Pulmonary squamous cell carcinoma 2% (586) 4% (584) GIST 1% (88) Adrenal cortical carcinoma 2% (81) 63% (269) Renal cell carcinoma 2% (379) Clear cell sarcoma 25% (59) Melanoma 11% (114) Source: Immunoquery Specificity of CD56 for Neuroendocrine Neoplasms % Positive (n) Lung adenocarcinoma 3% (639) Lung squamous cell carcinoma 9% (520) Renal cell carcinoma 17% (455) Pancreatic solid pseudopapillary neoplasm 98% (152) Adrenal cortical carcinoma 88% (49) Melanoma 7% (130) Adult granulosa cell tumor 100% (40) Synovial sarcoma 51% (68) Rhabdomyosarcoma 76% (34) Granular cell tumor 95% (58) Glioma 36% (148) Dendritic cell tumor 94% (164) Nk T-cell lymphoma 74% (267) Chloroma 27% (62) Source: Immunoquery Immunohistochemical Staining for the Diagnosis of Well Differentiated Neuroendocrine (Carcinoid) Tumors Sensitivity For most primary sites, chromogranin and synaptophysin are highly sensitive When used in combination, ~95% positive Specificity Certain specific non-ne neoplasms stain predictably PET-CT 2-5% idiosyncratic staining of other neoplasms Is it necessary? Specific differential diagnoses Metastatic disease What about histologically typical primary tumors? (Am J Surg Pathol 2010;34: ) Are immunohistochemical stains for general neuroendocrine markers mandated as necessary in all cases? Agree strongly 23.53% 4 Agree with minor reservation 11.76% 2 Agree with major reservation 0% 0 Disagree with minor reservation (disagree mildly) 11.76% 2 Disagree with major reservation (disagree moderately) 23.53% 4 Disagree strongly 29.41% 5 Totals 100% 17 NO AGREEMENT Immunohistochemical Staining for the Diagnosis of Poorly Differentiated Neuroendocrine Carcinomas Small cell carcinoma NOT mandated when classic morphologic findings are present Consider ruling out alternatives (e.g., basaloid squamous cell carcinoma, spindle cell carcinoid tumor, primitive neuroectodermal tumor, etc.) or synaptophysin positive in ~75% of cases Large cell neuroendocrine carcinoma NE marker expression required for diagnosis Must be positive in 100% of cases (by definition) Which makers? Thoracic vs. gastroenteropancreatic How strongly / diffusely positive? WHO 2015: The diagnosis of LCNEC requires immunohistochemistry for confirmation of neuroendocrine differentiation. In decreasing order of frequency, NCAM/CD56 stains % of LCNEC cases, followed by chromogranin A in 80 85%, and synaptophysin in 50 60%. NCAM/CD56 needs a note of caution because of its lower specificity for neuroendocrine differentiation in lung cancer, but it is the mostsensitivemarkerin the appropriate morphological context of a neuroendocrine neoplasm. Aand synaptophysin are the most reliable stains for diagnostic accuracy in distinguishing LCNEC from non-neuroendocrine tumours, and one positive marker is enough if the staining is clear-cut. Large Cell Neuroendocrine Carcinoma Chr Syn CD56 4

5 Large Cell Neuroendocrine Carcinoma Chr Syn CD56 Large Cell Lung Carcinoma Large Cell Carcinoma with Neuroendocrine Morphology Large Cell Undifferentiated Carcinoma Large Cell Neuroendocrine Carcinoma Large Cell Carcinoma with Neuroendocrine Morphology Large Cell Carcinoma with Neuroendocrine Differentiation Chr, Synapto, CD56 Large Cell Carcinoma with Neuroendocrine Differentiation Genomic subgroups in LCNEC Gene alterations typical of: SCLC Adeno SCLC SCLC/SqCC TP53 78% Number of altered genes per case RB1 40% KRAS 22% STK11 40% MYCL 7% MYCN 2% IRS2 4% SOX2 11% FGFR1 4% PTEN 4% MEN1 SCLC like NSCLC like (predominantly adeno like) Carcinoidlike Loss total loss total Gain amp Mutation total mut Loss Gain Missense mutation Truncating mutation Loss by IHC/WT gene Rekhtman et al., Clin Cancer Res2016; 22:

6 3/23/2017 Mixed Adenocarcinoma Neuroendocrine Carcinoma Combined Neuroendocrine Carcinomas At least 30% of both neuroendocrine and non-neuroendocrine components Adenocarcinoma most common ( MANEC ) Also squamous, pancreatic acinar, other exocrine types Neuroendocrine component usually poorly differentiated; small cell carcinoma or LCNEC Lung, colon, pancreas, gallbladder, etc. Various combinations Biphasic Waxing and waning Amphicrine Aggressive biology; evolving genomic data; treatment as small cell carcinoma (?) Mixed Adenocarcinoma Neuroendocrine Carcinoma Synaptophysin Pancreas Mixed Acinar Neuroendocrine Carcinoma Chymotrypsin Adenocarcinoma with Neuroendocrine Differentiation Morphologically adenocarcinoma Neuroendocrine component <30% Neuroendocrine differentiation detected incidentally Focal NE differentiation: no prognostic impact Role of IHC for NE markers??? 6

7 Synaptophysin Synaptophysin Neuroendocrine Differentiation in Carcinomas: Treatment Implications Small cell carcinoma (lung or extrapulmonary) Platinum + etoposide Large cell neuroendocrine carcinoma Commonly treated like small cell carcinoma Few compelling studies; no randomized trials Carcinoma with neuroendocrine morphology / differentiation / features / minor elements / etc. Who knows???? Neuroendocrine Neoplasms: Determination of Grade, Classification, and Prognosis WHO 2010 Grading of GEP-NETs Grade Mitoses Ki-67 Index G1 < 2 / 10 H.P.F. < 3% ENETS/WHO Grading of GEP-NETs: Provisions Count mitoses in 50 high power fields Assess Ki67 based on counting 2000 (500) cells Assess Ki67 in hot spots If mitotic rate and Ki67 are discordant, assign higher grade G / 10 H.P.F. 3-20% G3 > 20 / 10 H.P.F. > 20% Poorly Differentiated (High Grade ) Neuroendocrine Carcinoma Ki67 7

8 Ki67 Labeling Index of NETs Digital Image Analysis for Ki67 Quantification Strong predictor of prognosis Correlates well with mitotic index Sharp separation of well and poorly differentiated neuroendocrine neoplasms Methods of Assessment Manual counting (2000 cells per ENETS) Eyeballed estimate Digital image analysis Ki67% = 1.7 Consistency of Ki67 Determination by Digital Image Analysis, Manual Cell Counting, and Eyeballed Estimate Determining the Ki67 Labeling Index of NETs: How We Do It Image Analysis vs. Manual Counting Image Analysis vs. Eyeballed Estimate (Mean of 20 observers) Eyeballed Estimate Interobserver (n=20) Intraclass Correlation (ICC) 95% Confidence Interval Tang et al. Am J Surg Pathol 2012; 36: Courtesy of Dr. Laura H. Tang Ki67: variation in labeling intensity My count of Ki67 positive cells in this highlighted area is? A. <3 B. 4-6 C D. >10 8

9 Ki67 Ki67 Heterogeneity in PanNETs Inst. 1 Inst. 2 Inst. 3 Heterogeneity of Ki67 Labeling in NETs: Impact on Prognostic Significance of Grading Heterogeneity of Ki67 Labeling in NETs: Impact on Prognostic Significance of Grading 45 resected hepatic metastases of WD NETs Virtual biopsy TMA Yang et al., Am J Surg Pathol 2011; 35: Ki67 on virtual biopsies and on whole sections 47% of cases with G1 vs. G2 heterogeneity Define grade based on highest Ki67 on whole sections: G2 identified in 48% of core biopsies (3 cores) G2 identified in 35% of core biopsies (1 core) Predictive value of G1 on core biopsy: 65% (3 cores); 59% (1 core) Ki67 and Mitotic Rate Discordance in PanNETs Ki67 and Mitotic Rate Discordance in PanNETs 297 WD PanNETs with Ki 67 data ( ) 36% discordance 264 Mitotic G1 33 Mitotic G2 Mitotic rate: <1 per 10 hpf (G1) Ki-67: 15% positive (G2) 165 Ki 67 G1 99 Ki 67 G2 McCall et al., Am J Surg Pathol 2013; 37: Ki 67 G1 25 Ki 67 G2 9

10 Ki67 and Mitotic Rate Discordance in PanNETs 264 Mitotic G1 165 Ki 67 G1 99 Ki 67 G2 McCall et al., Am J Surg Pathol 2013; 37: WD PanNETs with Ki 67 data ( ) 36% discordance 33 Mitotic G2 8 Ki 67 G1 25 Ki 67 G2 Ki-67 G2/mitotic G1 PanNETs have decreased overall survival Percentage Surviving K1M1 K2M Survival in Years p < 0.01 Well Differentiated PanNET What about G2 / G3 discordance?? (well differentiated tumor vs. poorly differentiated carcinoma) Mitotic rate = 8 / 10 HPF Mitotic rate = 12 / 10 HPF Ki67 = 45% Ki67 = 55% Poorly Differentiated Neuroendocrine Carcinoma Progression of Low Grade to High Grade Neuroendocrine Tumor Ki67 Mitoses <1/10 HPF Mitoses 13/10 HPF 10

11 Mixed Ductal Neuroendocrine Carcinoma of Pancreas Ki67 = 2% G1 Ki67 = 45% G3 Tang et al., Clin Cancer Res 2016; 22: 1011 Genetics of Neuroendocrine Neoplasms of the Pancreas Gene Small Cell Large Cell NEC W.D. PanNET Ductal ACa Small Cell Lung CA KRAS 25% 33% 0% >90% 0 10% CDKN2A 11% 50% 0% 80 95% 0 10% TP53 100% 90% 4% 75% 80% SMAD4 0% 10% 0% 55% 0% RB1 89% 50% 0% 13% 90% DAXX/ATRX 0% 0% 43% 0% MEN1 0% 0% 44% 0% 0% mtor genes 15% 1% Predictive and prognostic factors for treatment and survival in 305 patients with advanced gastrointestinal neuroendocrine carcinoma (WHO G3) Reviewed clinical data on advanced stage G3 NECs, Ki67 > 20% 252 patients received chemotherapy (platinum-based) Median survival = 11 mos. Response rate = 31% Stable disease rate = 33% Ki67 < 55% predicted a lower response rate (15% vs 42%, p < 0.001) Ki67 < 55% predicted a better survival (14 vs 10 months, P < 0.001) Yachida et al., Am J Surg Pathol 2012; 36: 173 Jiao et al., Science 2011; 331: 1199 Sorbye et al., Ann Oncol 2013; 24: Conclusion: Survival of High Grade Neuroendocrine Neoplasms of the Pancreas Some G3 NETs with Ki % may be well differentiated biologically!! ( Well Differentiated NET with an Elevated Proliferative Rate or Well Differentiated NET, G3 ) Basturk et al., Am J SurgPathol 2015; 39:

12 WD NE Tumor PD NE Carcinoma Grading of Pancreatic Neuroendocrine Neoplasms (WHO 2017) Stable Disease Grade Progression NE Tumor Lower Grade High Grade Carcinoma NE Carcinoma High Grade Rapid Disease Disease Progression Progression, Death Two Pathways to the Development of High Grade (G3) NE Neoplasms Well differentiated NE tumor* Grade Mitoses Ki-67 Index G1 <2 / 10 HPF </= 2% G / 10 HPF 3-20% G3** >20 / 10 HPF >20% *Organoid architecture, well differentiated cytology, absence of non-neuroendocrine carcinoma components, may have components of G1 or G2, usually strong immunoexpression of general NE markers **mitoses usually <20/HPF; Ki 67 >20% but usually <50% Poorly differentiated NE carcinoma* Grade Mitoses Ki-67 Index G3** >20 / 10 HPF >20% *Small cell carcinoma and large cell NE carcinoma; less organoid architecture, classic cytology of small cell and large cell NE CA, absence of G1 or G2 NE components, may have non-neuroendocrine carcinoma components, less diffuse immunoexpression of general NE markers **mitoses >20/10 HPF; Ki67 >20% and usually >50% G1 G2 G3 G3 How to distinguish G3 NEC (esp. large cell NE carcinoma) from G3 NET? WDNET PDNEC Ki67% Large Cell NEC Pancreatic G3 NE Neoplasms G3 NET How to distinguish G3 NEC (esp. large cell NE carcinoma) from G3 NET? Clinical clues History of well differentiated NET? Octreotide scan positive? FDG-PET positive? Morphologic clues Lower grade component? Non-neuroendocrine component? Mitotic rate? Molecular clues Status of TP53, RB1, DAXX, ATRX, MEN1 12

13 Well Differentiated PanNETs (G1-3) Exhibit a Different Molecular Phenotype from Poorly Differentiated NECs (G3) PD-NEC PD-NEC WD-NET TP53 RB1 DAXX / ATRX MEN1 p53 Rb WD PanNET 4% 0 43% 44% PD PanNEC 56% 72% 0 0 p53 Rb DAXX Jiao et al. Science 2011; 331: 1199 Yachida et al., Am J Surg Pathol 2012; 36: 173 Tang et al., Am J Surg Pathol 2016; 40: 1192 Classification of 33 High Grade Pancreatic Neuroendocrine Neoplasms by Secondary Evidence Immunohistochemical Initial Consensus Abnormalities Other Histologic Components Confirmed Classification G1/G2 WD-NET WD-NET WD-NET WD-NET DAXX G1/G2 WD-NET WD-NET WD-NET ATRX G1/G2 WD-NET WD-NET WD-NET G1/G2 WD-NET WD-NET Disease Specific Survival of High Grade (G3) Pancreatic Neuroendocrine Neoplasms WD-NET DAXX G1/G2 WD-NET WD-NET WD-NET G1/G2 WD-NET WD-NET Ambiguous G1/G2 WD-NET WD-NET /33 (58%) of high grade (G3) pancreatic NE neoplasms were morphologically ambiguous Ambiguous G1/G2 WD-NET WD-NET Ambiguous DAXX G1/G2 WD-NET WD-NET Ambiguous ATRX G1/G2 WD-NET WD-NET Ambiguous DAXX G1/G2 WD-NET WD-NET Ambiguous G1/G2 WD-NET WD-NET Ambiguous ATRX WD-NET Ambiguous DAXX G1/G2 WD-NET WD-NET Ambiguous DAXX G1/G2 WD-NET WD-NET Ambiguous G1/G2 WD-NET WD-NET Ambiguous G1/G2 WD-NET WD-NET Ambiguous G1/G2 WD-NET WD-NET Ambiguous G1/G2 WD-NET WD-NET Ambiguous p53/rb PD-NEC Ambiguous p53/smad4 Ductal adenocarcinoma PD-NEC Ambiguous p53/rb PD-NEC 18/19 (95%) morphologically ambiguous high grade pancreatic NEneoplasms successfully classified Percent survival WD-NET PD-NEC p< (N=20) (N=12) Ambiguous p53/rb PD-NEC Ambiguous p53 PD-NEC Ambiguous Undetermined PD-NEC-LCC DAXX G1/G2 WD-NET WD-NET PD-NEC-LCC Rb PD-NEC PD-NEC-LCC Ductal adenocarcinoma PD-NEC PD-NEC-SCC p53 Ductal adenocarcinoma PD-NEC PD-NEC-SCC Rb PD-NEC Months PD-NEC-SCC p53/rb Ductal adenocarcinoma PD-NEC PD-NEC Rb PD-NEC Tang et al., Am J Surg Pathol 2016; 40: 1192 PD-NEC p53 PD-NEC Tang et al., Am J Surg Pathol 2016; 40: 1192 Sequencing of Pancreatic Neuroendocrine Neoplasms at MSKCC p53 p53 13

14 Distinction of G3 NEC from G3 NET: Practical Issues Primary site Pancreas Most common DAXX/ATRX, MEN1 Other GI / pulmonary NETs WD G3 NETs uncommon Formal WHO classification pending p53, Rb, associated exocrine elements for PD Morphology for WD Role of Ki67 >50% = usually PD NEC <50% = either WD NET or PD NEC Role of mitotic rate <20 per 10 HPF = WD NET >20 per 10 HPF = PD NEC Use of Immunohistochemistry in Neuroendocrine Neoplasms: Conclusions IHC is needed for neuroendocrine neoplasm diagnosis, classification, and grading Limitations exist in interpretation and significance Exercise pragmatism, not nihilism IHC is just one tool in the diagnostic arsenal use morphology, clinical findings, molecular data, and common sense! 14