M. Papotti, M. Volante Università di Torino Tumori neuroendocrini Il ruolo dell’anatomia patologica Corso AME Milano, 25 maggio 2007

Caro Mauro, …. Riguardo alla tua relazione, considerato il titolo, avremmo piacere che tu affrontassi nel tempo che hai a disposizione 20 min la classificazione del neuroendocrino, i limiti della citologia in rapporto alle scelte terapeutiche, il discorso dei recettori e della loro utilità in pratica clinica. considera che sarà un corso AME (…) con un taglio decisamente pratico e questo anche per quanto riguarda l'anatomia patologica. Invito del dr R. Cozzi

Medullary thyroid carcinoma Pheochromocytoma/Paraganglioma Parathyroid adenoma/carcinoma Pituitary adenoma/carcinoma Different diagnostic criteria and classification for

neuroendocrine tumor classification MENU - History, Definition & Glossary - Pathological classification  pure NE tumors  mixed NE/exocrine tumors - Further characterisation - History, Definition & Glossary - Pathological classification  pure NE tumors  mixed NE/exocrine tumors - Further characterisation

History 1907 “carcinoid” term Oberndorfer 1930 carcinoid syndrome Cassidy 1940/50 “helle zellen” Feyrter 1952 serotonin discovery Erspamer & Asero 1963 fore-, mid-, hind-gut carcinoids Williams & Sandler 1965/70 APUD concept Pearse 1980 diffuse NE system WHO 1994 “neuroendocrine tumor” replaces carcinoid Capella et al 2000 classification of endocrine tumors WHO Solcia et al History 1907 “carcinoid” term Oberndorfer 1930 carcinoid syndrome Cassidy 1940/50 “helle zellen” Feyrter 1952 serotonin discovery Erspamer & Asero 1963 fore-, mid-, hind-gut carcinoids Williams & Sandler 1965/70 APUD concept Pearse 1980 diffuse NE system WHO 1994 “neuroendocrine tumor” replaces carcinoid Capella et al 2000 classification of endocrine tumors WHO Solcia et al

History 1907 “Karzinoid” term Siegfried Oberndorfer History 1907 “Karzinoid” term Siegfried Oberndorfer 6 ileal tumors, slowly growing, no metastases

NE TUMORS carcinoid, malignant carcinoid apudoma islet cell tumor adenoma / microadenoma vs carcinoma Kultchisky cell tumor / carcinoma endocrine carcinoma endocrine neoplasm hormone…-oma (insulinoma, gastrinoma,..) pancreatic (A/B/D/PP)-cell tumor carcinoid, malignant carcinoid apudoma islet cell tumor adenoma / microadenoma vs carcinoma Kultchisky cell tumor / carcinoma endocrine carcinoma endocrine neoplasm hormone…-oma (insulinoma, gastrinoma,..) pancreatic (A/B/D/PP)-cell tumor Glossary & Synonyms

Neuroendocrine tumor classification MENU - History, Definition & Glossary - Pathological classification  pure NE tumors  mixed NE/exocrine tumors - Further characterisation - History, Definition & Glossary - Pathological classification  pure NE tumors  mixed NE/exocrine tumors - Further characterisation

WHICH CLASSIFICATION CRITERION ???? Clinical Morphological Hormonal Ki67 Gene expression THE BENIGN THE MALIGNANT THE CLINICALLY AGGRESSIVE

Incidence, Localisation, Endocrine function, Symptoms, Treatment, Behavior, Survival Clinical Morphological Hormonal Ki67 Gene expression

Single or multiple Well demarcated or invasive Size cm. Cystic appearance Lymph node mets Distant mets Single or multiple Well demarcated or invasive Size cm. Cystic appearance Lymph node mets Distant mets 1 cm Gastric micro- carcinoids insulinoma Macro Clinical Morphological Hormonal Ki67 Gene expression

Tumor architecture Micro

Gross features and structure are not enough for the purpose of identifying homogeneous groups with clinical relevance Clinical Morphological Hormonal Ki67 Gene expression THE BENIGN THE MALIGNANT THE CLINICALLY AGGRESSIVE

CLASSIFICATION PROBLEMS

2000 WHO CLASSIFICATION Introduces terms tumor/carcinoma (vs carcinoid) ….LUNG EXCLUDED Replaces neuroendocrine with endocrine Incorporates hormonally active tumors ( morpho-functional approach ) Recognizes mixed exo-endocrine tumors Incorporates information on tumor grade Introduces criteria of malignancy (angioinvasion, Ki67 pancreas, only) Introduces terms tumor/carcinoma (vs carcinoid) ….LUNG EXCLUDED Replaces neuroendocrine with endocrine Incorporates hormonally active tumors ( morpho-functional approach ) Recognizes mixed exo-endocrine tumors Incorporates information on tumor grade Introduces criteria of malignancy (angioinvasion, Ki67 pancreas, only)

Microscopic features of malignancy Depth of invasion Depth of invasion Size of tumor Size of tumor Angioinvasion Angioinvasion High grade cellular atypia High grade cellular atypia GI NETS

3 TIE SYSTEM for GI NETs

Invasion of surrounding tissues Size of tumor (>6 cm is suspected) High grade cellular atypia Focal or diffuse necrosis High mitotic index Blood or lymph vessel & nerve invasion Ki67 > 2% Invasion of surrounding tissues Size of tumor (>6 cm is suspected) High grade cellular atypia Focal or diffuse necrosis High mitotic index Blood or lymph vessel & nerve invasion Ki67 > 2% Microscopic features of malignancy PANCREATIC NETs

3 TIE SYSTEM for pancreatic NETs

Does this system fit with the clinical practice? Is it easily applicable? Problem of other non-carcinoid NETs (eg malignant pheochromocytoma, MTC, parathyroid carcinoma, Merkel cell carcinoma, etc…) E. Bajetta, L. Catena, G. Procopio, E. Bichisao, L. Ferrari, S. Della Torre, S. De Dosso, S. Iacobelli, R. Buzzoni, L. Mariani and J. Rosai Is the new WHO classification of neuroendocrine tumours useful for selecting an appropriate treatment? Ann Oncol :1374

Does this system fit with the clinical practice? Is it easily applicable? Benign vs uncertain behaviour in well differentiated pancreatic NETs Appendiceal NETs (small & invasive?) Mixed endocrine – exocrine tumors Use morphology or Ki67 first? The new TNM system for foregut NETs Extra GEP: pulmonary NETs  especially intermediate grades

BENIGN vs UNCERTAIN BEHAVIOR BenignUncertain Extrapancreatic growthnono Angioinvasionnoyes Size >=2 cmnoyes Mitoses >2 /10HPF noyes Ki67 >2%noyes BENIGN vs UNCERTAIN BEHAVIOR BenignUncertain Extrapancreatic growthnono Angioinvasionnoyes Size >=2 cmnoyes Mitoses >2 /10HPF noyes Ki67 >2%noyes

Resembles WD NET (benign), but in general: size >3 cm, mitoses >2, Ki67 >5% but above all: unequivocal signs of malignancy must be present Resembles WD NET (benign), but in general: size >3 cm, mitoses >2, Ki67 >5% but above all: unequivocal signs of malignancy must be present LYMPHNODELIVER LOCAL INVASION

FOREGUT NET CLASSIFICATION Recent proposal of a staging system for fore- gut NETs and improvement of grading system using mitoses + Ki67

Difficulties in identifying the intermediate entities TCSCLC LCNEC AC SPECTRUM OF PULMONARY NETS

Indolent clinical behavior Aggressive clinical behavior Indolent clinical behavior Aggressive clinical behavior TC AC SCLC Significantly different survival Arrigoni et al 1972 SPECTRUM OF PULMONARY NETS

10 mitoses small cells 10 mitoses small cells no necrosis or necrosis (necrosis) (necrosis) 10 mitoses small cells 10 mitoses small cells no necrosis or necrosis (necrosis) (necrosis) TCACLCNEC SCLC Significantly different survival p< NO significantly different survival Travis et al Am J Surg Pathol 22,934,1998 SPECTRUM OF PULMONARY NETS

TCACLCNEC SCLC TCAC LCNEC SCLC SCLC NO significantly different survival Asamura et al JCO 24:70,2006 SPECTRUM OF PULMONARY NETS

Biological behaviour Degree of differentiation LUNG GEP TYPICAL CARCINOID ATYPICAL CARCINOID SMALL CELL/ LARGE CELL NE CARCINOMA WELL DIFF. NE TUMOR benign/borderline WELL DIFF. NE CARCINOMA POORLY DIFF. NE CARCINOMA (small/large cell) WELL DIFF. HIGH GRADE POORLY DIFF. LOW GRADE MEEC

Neuroendocrine tumor classification MENU - History, Definition & Glossary - Pathological classification  pure NE tumors  mixed NE/exocrine tumors - Further characterisation - History, Definition & Glossary - Pathological classification  pure NE tumors  mixed NE/exocrine tumors - Further characterisation

2000 WHO CLASSIFICATION CATEGORIES RECOGNIZED 1 well differentiated endocrine tumor 2 well differentiated endocrine carcinoma 3 poorly differentiated endocrine carcinoma 4 mixed exocrine-endocrine tumor (5 tumor-like lesions) CATEGORIES RECOGNIZED 1 well differentiated endocrine tumor 2 well differentiated endocrine carcinoma 3 poorly differentiated endocrine carcinoma 4 mixed exocrine-endocrine tumor (5 tumor-like lesions)

CGA Exocrine Endocrine

NE differentiation in colon cancer CGA mucin 1995

pure non-NE ca. focal NE non-NE ca. Non-NE carcinomas with NE differentiation Breast (WHO2004) Lung (WHO2004) GI tract (WHO2003) Prostate (WHO2003) BREAST and COLORECTAL CANCERS …..ANY BIOLOGICAL and/or CLINICAL SIGNIFICANCE?? STOMACH & PROSTATE CANCERS YES NO Y/N ? NSCLC

>5% NE+ cells

Am J Surg Pathol August 2006 LCNEC of the stomach are significantly more aggressive than conventional ADC. ADC-NED have also a worse prognosis than conventional ADC (borderline significance)

CGA increase during progression Reduced survival of CGA+ cases

July 1996 Nov 1997 Oct 1999 May 2000 Rectal polyp, cancerised Rectum resection Local recurrence Pelvic recurrence Stable disease Radical Surgery ChTx 1RTChTx 2 April 2007 ADC stage B1 CgA ChTx 1: 5-FU + folic acid (6x) RT: 50 Grey ChTx 2: oxaliplatin + 5-FU + folic acid (12x) CASE REPORT FNAB Oncologia Prof Dogliotti dr Tampellini

pure NE tum. pure non-NE ca. focal non-NE focal NE 100% 30%0% mixed (collision) NE tum. non-NE ca. WDET - TC WDEC - AC PDEC – SCLC/LCNEC Mixed NE/non-NE carcinomas Non-NE carcinomas with NE differentiation Breast (WHO2004) Lung (WHO2004) GI tract (WHO2003) Prostate (WHO2003) WHO 2000 Endocrine tumors WHO 2000 Endocrine WHO 2004 lung NE non-NE 30% mixed (intermingled)

Biological behaviour Degree of differentiation LUNG GEP TYPICAL CARCINOID ATYPICAL CARCINOID SMALL CELL/ LARGE CELL NE CARCINOMA WELL DIFF. NE TUMOR benign/borderline WELL DIFF. NE CARCINOMA POORLY DIFF. NE CARCINOMA (small/large cell) WELL DIFF. HIGH GRADE POORLY DIFF. LOW GRADE Mixed Endo- Exocrine Ca

LUNG GEP TYPICAL CARCINOID ATYPICAL CARCINOID SMALL CELL/ LARGE CELL NE CARCINOMA WELL DIFF. NE TUMOR benign/borderline WELL DIFF. NE CARCINOMA POORLY DIFF. NE CARCINOMA ( small/large cell ) HOW TO BREAK THE ARROW? GEP LUNG MEEC Light microscopy + ……??

Neuroendocrine tumor classification MENU - History, Definition & Glossary - Pathological classification pure NE tumors mixed NE/exocrine tumors - Further characterisation - History, Definition & Glossary - Pathological classification pure NE tumors mixed NE/exocrine tumors - Further characterisation

Further characterisation hormonal Ki 67 receptors molecular Further characterisation hormonal Ki 67 receptors molecular THE BENIGN THE MALIGNANT THE CLINICALLY AGGRESSIVE

MARKERSMARKERS ChromograninsSynaptophysinN-CAM Cytoker. 34E12 NSE, VMAT PGP9.5NeurofilamentsChromograninsSynaptophysinN-CAM Cytoker. 34E12 NSE, VMAT PGP9.5Neurofilaments Hormones: Hormones: calcitonin, bombesin, insulin, glucagon, somatostatin, gastrin, PP, VIP, ACTH, serotonin, … NEW: secretagogin [Virch Arch 449, 402, Oct 2006] NEW: ghrelin, cortistatin, obestatin, secretagogin [Virch Arch 449, 402, Oct 2006] Hormones: Hormones: calcitonin, bombesin, insulin, glucagon, somatostatin, gastrin, PP, VIP, ACTH, serotonin, … NEW: secretagogin [Virch Arch 449, 402, Oct 2006] NEW: ghrelin, cortistatin, obestatin, secretagogin [Virch Arch 449, 402, Oct 2006] CGA SSTR2

Further characterisation Ki 67 proliferation index Further characterisation Ki 67 proliferation index LOW HIGH

KI-67: DIAGNOSTIC USE 2000

TTP P = Brizzi et al, 2007 (submitted) KI-67: PROGNOSTIC USE Prognostic value if groups are homogeneous (eg all WDCA or PDCA). Otherwise possible bias due to tumor differentiation P = KI-67 in WD NE carcinoma OS

10 to 15% cut-off levels according to therapy modalities KI-67: USE for Tx STRATEGY

SEQUENTIAL STEPS in the DIAGNOSTIC PROCESS of the PATHOLOGIST 1 Use morphology (histopathology + markers) to enter each NET case into homogeneous groups (eg WHO classification criteria) 2 Within each group, analyse known (eg Ki67) or new prognostic markers 3 Analyse molecules useful for targeted therapy, if requested

SOMATOSTATIN RECEPTORS (SSTR) The pathologist has to search for SSTR presence in a tumor (YES/NO) and, if YES, provide information on SSTR subtype PROBLEM 1: What to search for? selective SRIF analogs for single SSTR subtypes vs universal analog for all SSTR subtypes selective SRIF analogs for single SSTR subtypes vs universal analog for all SSTR subtypes PROBLEM 2: How to identify SSTRs?

Accessible to all laboratoriesAccessible to all laboratories Low costLow cost Commercial antibodies [??]Commercial antibodies [??] Applicable on archival tissuesApplicable on archival tissues Applicable on biopsies and FNAsApplicable on biopsies and FNAs Identifies SSTR type (1-5) [??]Identifies SSTR type (1-5) [??] Need to standardize interpretationNeed to standardize interpretation Accessible to all laboratoriesAccessible to all laboratories Low costLow cost Commercial antibodies [??]Commercial antibodies [??] Applicable on archival tissuesApplicable on archival tissues Applicable on biopsies and FNAsApplicable on biopsies and FNAs Identifies SSTR type (1-5) [??]Identifies SSTR type (1-5) [??] Need to standardize interpretationNeed to standardize interpretation How to identify SSTRs? 3 Immunohistochemistry 1 Autoradiography 2 RT-PCR / ISH 1 Autoradiography 2 RT-PCR / ISH sst2

predominant membrane staining for SSTR2 Which SSTR antibodies for IHC ??

Correlation SSTR-IHC with Octreotide scintigraphy Surgical samples (lung) 88% (22/25 cases) sst2sst2 bronchial biopsy Surgical specimen Main goal of SSTR localisation: provide information correlated with clinical data

Collaborative project on SSTR2A IHC in correlation with in vivo data 107 cases of NET with variable degrees of differentiation and different locations, all with Octreoscan and/or information on clinical response to SS analogues (from Universities of Turin, Varese and Naples) 107 cases of NET with variable degrees of differentiation and different locations, all with Octreoscan and/or information on clinical response to SS analogues (from Universities of Turin, Varese and Naples) Complete clinico- pathological data Complete clinico- pathological data IHC for SSTR2A using 3 different commercial polyclonal Abs IHC for SSTR2A using 3 different commercial polyclonal Abs

Proposed IHC score for SSTR2 ( based on staining pattern) Case 2187A/2187B NE tumor …2.5+? 1+ or +/-

Score 0 (Negative) SUBCELLULAR PATTERN EXTENSION OF POSITIVE TUMOR CELL POPULATION Score 1Score 2Score 3 Pure cytoplasmic Membranous incomplete Membranous circumferential (Absent) 1-100%<50%>50% CONCORDANCE WITH OCTREOSCAN DATA 50% 54%87%94%

Correlation SSTR2 IHC with in vivo data 107 cases = 70 WD NET/CA 18 PD NE CA 9 MTC 10 others Concordance IHC/Octreoscan: 77% (82/107 cases) Concordance IHC/SS analogue response: 81% (21/26 cases) IHC score: 0 totally negative 1 cytoplasmic + 2 focal membrane + 3 diffuse membrane IHC+/OS+ 21 IHC-/OS- (19/25 discrepant cases being IHC-/Octreoscan+)

Thank you!!! University of Turin at San Luigi Hospital, Orbassano

NSG NSG+MUC MUC Pure NE cell Amphicrine cell Pure non-NE cell (i.e. mucinous)

sst3sst3 LCNEC SqCa sst in endothelia, necrosis and intra- tumoral lymphocytes

SUMMARY OF OBSERVED STAINING PATTERNS IN sst IHC sst2: predominant membrane (cytoplasmic possible, if weaker than membrane ) sst3: cytoplasmic (with membrane increase) sst5: membrane and cytoplasmic

sst2A IHC expression in 71 NE tumors of the lung Typical carcinoid (24) score 0/1 4 score 2/3 20 (83%) score 2/3 20 (83%) Atypical carcinoid (20) score 0/1 8 score 2/3 12 (60%) LCNEC (17) score 0/1 7 score 2/3 10 (59%) SCLC (10) score 0/1 4 score 2/3 6 (60%)