M. Papotti, M. VolanteUniversità di Torino

M. Papotti, M. VolanteUniversità 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

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 TUMORSNE 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 & SynonymsGlossary & Synonyms

Neuroendocrine tumor classification

MENU

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 BENIGNTHE MALIGNANTTHE CLINICALLY AGGRESSIVE

Incidence,

Localisation,

Endocrine function,

Symptoms,

Treatment,

Behavior,

Survival

ClinicalMorphological

Hormonal

Ki67

Gene expression

• Single or multiple

• Well demarcated or invasive

• Size 0.5-15 cm.

• Cystic appearance

• Lymph node mets

• Distant mets

• Single or multiple

• Well demarcated or invasive

• Size 0.5-15 cm.

• Cystic appearance

• Lymph node mets

• Distant mets

1 cm

Gastric micro-carcinoids

insulinoma

Macro

Clinical

MorphologicalHormonal

Ki67 Gene expression

Tumor architectureMicro

Gross features and structure are not enough for the purpose of identifying homogeneous groups with clinical relevance

Gross features and structure are not enough for the purpose of identifying homogeneous groups with clinical relevance

Clinical

MorphologicalHormonal

Ki67 Gene expression

THE BENIGN

THE MALIGNA

NT

THE CLINICAL

LY AGGRESS

IVE

19941994

20002000

CLASSIFICATION PROBLEMSCLASSIFICATION PROBLEMS

2000 WHO CLASSIFICATION2000 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 Depth of invasioninvasion

• Size of tumorSize of tumor• AngioinvasionAngioinvasion• High grade High grade

cellular atypiacellular 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

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 2005 16:1374

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 BEHAVIORBenign

UncertainExtrapancreatic growth no noAngioinvasion no yesSize >=2 cm no yesMitoses >2 /10HPF no yesKi67 >2% no yes

BENIGN vs UNCERTAIN BEHAVIORBenign

UncertainExtrapancreatic growth no noAngioinvasion no yesSize >=2 cm no yesMitoses >2 /10HPF no yesKi67 >2% no yes

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

LYMPHNODE LIVERLOCAL LOCAL INVASIONINVASION

FOREGUT NET CLASSIFICATIONFOREGUT NET CLASSIFICATION

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

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

Difficulties in Difficulties in identifying the identifying the intermediate intermediate

entities entities

Difficulties in Difficulties in identifying the identifying the intermediate intermediate

entities entities

TC SCLC

LCNECAC

SPECTRUM OF PULMONARY NETS

Indolent clinical behaviorIndolent clinical behavior Aggressive clinical behaviorAggressive clinical behaviorIndolent clinical behaviorIndolent clinical behavior

Aggressive clinical behaviorAggressive clinical behavior

TCTC AC AC SCLC SCLCTCTC AC AC SCLC SCLC

Significantly Significantly different survivaldifferent survival

Significantly Significantly different survivaldifferent survival

Arrigoni et al 1972Arrigoni et al 1972Arrigoni et al 1972Arrigoni et al 1972

SPECTRUM OF PULMONARY NETS

<2 mitoses 3-10 mitoses >10 mitoses small cells<2 mitoses 3-10 mitoses >10 mitoses small cellsno necrosis or necrosis (necrosis) (necrosis)no necrosis or necrosis (necrosis) (necrosis)<2 mitoses 3-10 mitoses >10 mitoses small cells<2 mitoses 3-10 mitoses >10 mitoses small cellsno necrosis or necrosis (necrosis) (necrosis)no necrosis or necrosis (necrosis) (necrosis)

TCTC ACAC LCNECLCNEC SCLC SCLCTCTC ACAC LCNECLCNEC SCLC SCLC

Significantly Significantly different survival different survival p<0.0001p<0.0001

Significantly Significantly different survival different survival p<0.0001p<0.0001

Significantly Significantly different survival different survival p<0.0001p<0.0001

Significantly Significantly different survival different survival p<0.0001p<0.0001 NO significantly NO significantly

different survivaldifferent survival NO significantly NO significantly different survivaldifferent survival

Travis et al

Am J Surg Pathol 22,934,1998

SPECTRUM OF PULMONARY NETS

TCTC ACAC LCNECLCNEC SCLC SCLCTCTC ACAC LCNECLCNEC SCLC SCLC

TCTC ACAC LCNEC LCNECTCTC ACAC LCNEC LCNEC

SCLCSCLC SCLCSCLC

NO significantly NO significantly different survivaldifferent survival NO significantly NO significantly different survivaldifferent survival

Asamura et al JCO 24:70,2006

SPECTRUM OF PULMONARY NETS

Biological behaviourBiological behaviour

Degree of differentiationDegree of differentiation

LUNGLUNG

GEPGEP

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.WELL DIFF.

HIGH GRADEHIGH GRADE

POORLY DIFF.POORLY DIFF.

LOW GRADELOW GRADE

MEEC

Neuroendocrine tumor classification

MENU

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 RECOGNIZED1 well differentiated endocrine

tumor2 well differentiated endocrine

carcinoma3 poorly differentiated endocrine

carcinoma• 4 mixed exocrine-endocrine tumor

• (5 tumor-like lesions)

CATEGORIES RECOGNIZED1 well differentiated endocrine

tumor2 well differentiated endocrine

carcinoma3 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 BREAST and COLORECTAL COLORECTAL CANCERSCANCERS

……..ANY BIOLOGICAL ..ANY BIOLOGICAL and/or CLINICAL and/or CLINICAL SIGNIFICANCE??SIGNIFICANCE??

STOMACH & STOMACH & PROSTATE PROSTATE CANCERSCANCERS

YES

NO

Y/N ? NSCLCNSCLC

>5% NE+ cells

Am J Surg Pathol August 2006LCNEC 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 1 RT ChTx 2

April 2007

ADC stage B1

CgA CgA 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%

100%

30% 0%

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-NE30%

mixed (intermingled)

Biological behaviourBiological behaviour

Degree of differentiationDegree of differentiation

LUNGLUNG

GEPGEP

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.WELL DIFF.

HIGH GRADEHIGH GRADE

POORLY DIFF.POORLY DIFF.

LOW GRADELOW GRADE

Mixed Endo-

Exocrine Ca

LUNGLUNG

GEPGEP

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?

GEPGEP

LUNGLUNG

MEEC

Light microscopy + ……??

Neuroendocrine tumor classification

MENU

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 BENIGNTHE MALIGNANTTHE CLINICALLY AGGRESSIVE

MARKERSMARKERSMARKERSMARKERS ChromograninsChromograninsSynaptophysinSynaptophysin

N-CAMN-CAMCytoker. 34Cytoker. 34E12E12

NSE, VMATNSE, VMATPGP9.5PGP9.5

NeurofilamentsNeurofilaments

ChromograninsChromograninsSynaptophysinSynaptophysin

N-CAMN-CAMCytoker. 34Cytoker. 34E12E12

NSE, VMATNSE, VMATPGP9.5PGP9.5

NeurofilamentsNeurofilaments

Hormones: Hormones: calcitonin, bombesin, insulin,

glucagon, somatostatin, gastrin, PP, VIP, ACTH,

serotonin, …NEW: NEW: ghrelin, cortistatin, obestatin, secretagogin secretagogin

[Virch Arch 449, 402, Oct 2006][Virch Arch 449, 402, Oct 2006]

Hormones: Hormones: calcitonin, bombesin, insulin,

glucagon, somatostatin, gastrin, PP, VIP, ACTH,

serotonin, …NEW: NEW: ghrelin, cortistatin, obestatin, secretagogin secretagogin

[Virch Arch 449, 402, Oct 2006][Virch Arch 449, 402, Oct 2006]CGA

SSTR2

Further characterisation• Ki 67 proliferation index

Further characterisation• Ki 67 proliferation index

LOWLOW HIGHHIGH

KI-67:KI-67:DIAGNOSTIC USEDIAGNOSTIC USE

2000

TTP

P = 0.043

Brizzi et al, 2007 (submitted)Brizzi et al, 2007 (submitted)

KI-67: KI-67: PROGNOSTIC PROGNOSTIC USEUSE

Prognostic value if groups are homogeneous (eg all WDCA or PDCA). Otherwise possible bias due to tumor differentiation

0 10 20 30 40 50 60 70 80 90 100 110

Months

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Cu

mu

lative Pro

po

rtion

Su

rviving

Ki67<5% Ki67>5%

P = 0.017

KI-67 in WD NE KI-67 in WD NE carcinomacarcinoma

OS OS

10 to 15% cut-o

ff

10 to 15% cut-o

ff

levels according to

levels according to

therapy modaliti

es

therapy modaliti

es

KI-67: USE for Tx STRATEGYKI-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) SOMATOSTATIN RECEPTORS (SSTR) The pathologist has to search for SSTR presence in a tumor (YES/NO) and, if YES, provide information on SSTR subtype

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?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?PROBLEM 2: How to identify SSTRs?

•Accessible to all laboratoriesAccessible to all laboratories•Low costLow cost•Commercial antibodies Commercial antibodies [??][??]•Applicable on archival tissues Applicable 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 tissues Applicable 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

HowHow to identify SSTRs? to identify SSTRs?HowHow to identify SSTRs? to identify SSTRs?

3 Immunohistochemistry3 Immunohistochemistry3 Immunohistochemistry3 Immunohistochemistry

1 Autoradiography 1 Autoradiography

2 RT-PCR / ISH2 RT-PCR / ISH

1 Autoradiography 1 Autoradiography

2 RT-PCR / ISH2 RT-PCR / ISH

sst2

predominant membrane staining for SSTR2predominant membrane staining for SSTR2

Which SSTR antibodies for IHC ??

Which SSTR antibodies for IHC ??

Correlation SSTR-IHC with Octreotide scintigraphyCorrelation SSTR-IHC with Octreotide scintigraphy

Surgical samples (lung)88% (22/25 cases)

sst2sst2sst2sst2

bronchiabronchial biopsyl biopsy

bronchiabronchial biopsyl biopsy

Surgical Surgical specimespecime

nn

Surgical Surgical specimespecime

nn

Main goal of SSTR localisation: provide information correlated with clinical data

Main goal of SSTR localisation: provide information correlated with clinical data

Collaborative project on SSTR2A IHC Collaborative project on SSTR2A IHC in correlation with in correlation with in vivoin vivo data dataCollaborative project on SSTR2A IHC Collaborative project on SSTR2A IHC in correlation with in correlation with in vivoin vivo data data

107 cases of NET with variable degrees of 107 cases of NET with variable degrees of differentiation and different locations, all with differentiation and different locations, all with Octreoscan and/or information on clinical Octreoscan and/or information on clinical response to SS analogues (from Universities of response to SS analogues (from Universities of Turin, Varese and Naples)Turin, Varese and Naples)

Complete clinico-Complete clinico-pathological datapathological data

IHC for SSTR2A IHC for SSTR2A using 3 different using 3 different commercial commercial polyclonal Abs polyclonal Abs

Proposed IHC score for SSTR2 (based on staining pattern)

Proposed IHC score for SSTR2 (based on staining pattern)

Case 2187A/2187BCase 2187A/2187B

NE tumorNE tumor

2+2+

2+2+

3+3+

…2.5+?…2.5+?

1+ or +/-1+ or +/-

Score 0

(Negative)

SUBCELLULAR PATTERN

EXTENSION OF POSITIVE TUMOR CELL POPULATION

Score 1 Score 2 Score 3

Pure cytoplasmic

Membranous incomplete

Membranous circumferential

(Absent) 1-100% <50% >50%

CONCORDANCE WITH OCTREOSCAN DATA

50% 54% 87% 94%

Correlation SSTR2 IHC Correlation SSTR2 IHC with with in vivoin vivo data dataCorrelation SSTR2 IHC Correlation SSTR2 IHC with with in vivoin vivo data data

107 cases =107 cases =70 WD NET/CA70 WD NET/CA18 PD NE CA18 PD NE CA9 MTC9 MTC10 others10 others

Concordance IHC/Octreoscan:Concordance IHC/Octreoscan: 77%77% (82/107 (82/107

cases)cases)

Concordance IHC/SS analogue response:Concordance IHC/SS analogue response: 81%81% (21/26 cases)(21/26 cases)

IHC score:IHC score:0 totally negative0 totally negative1 cytoplasmic +1 cytoplasmic +2 focal membrane +2 focal membrane +3 diffuse membrane +3 diffuse membrane +

--

++

61 IHC+/OS+21 IHC-/OS-

(19/25 discrepant cases (19/25 discrepant cases being IHC-/Octreoscan+)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)

sst3sst3sst3sst3 LCNEC

SqCa

sst in endothelia, necrosis and intra-tumoral lymphocytes

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 sst2A IHC expression in 71 NE tumors of the lungtumors of the lung

sst2A IHC expression in 71 NE sst2A IHC expression in 71 NE tumors of the lungtumors of the lung

Typical carcinoidTypical carcinoid (24)(24) score 0/1score 0/1 44 score 2/3score 2/3 2020 (83%)(83%)

Atypical carcinoidAtypical carcinoid (20)(20) score 0/1score 0/1 88score 2/3score 2/3 1212 (60%)(60%)

LCNECLCNEC (17)(17) score 0/1score 0/1 77score 2/3score 2/3 1010 (59%)(59%)

SCLCSCLC (10)(10) score 0/1score 0/1 44score 2/3score 2/3 66 (60%)(60%)