TYPING AND TERMINOLOGY: TIPS ON KEY DIAGNOSIS IN OVARY, FALLOPIAN TUBE AND

ENDOMETRIUM

W Glenn McCluggage

Belfast, Northern Ireland

GYNAECOLOGICAL FAMILIAL CANCER SYNDROMES

• BRCA1/2- fallopian tube/ ovary • Lynch- endometrium, ovary • Peutz Jeghers- cervical “gastric-type” carcinoma; ovarian

sex cord-stromal tumours (SCTAT) • DICER1- Sertoli-Leydig, cervical embryonal

rhabdomyosarcoma • SMARCA4- ovarian small cell carcinoma hypercalcaemic

type • Cowden’s syndrome – endometrium • Hereditary leiomyomatosis/ RCC syndrome • Tuberose sclerosis- PEComas

GYNAECOLOGICAL FAMILIAL CANCER SYNDROMES

• BRCA1/2- ovarian/ tubal high grade serous carcinoma (HGSC) only; uterine serous- controversial (probably not)

• Lynch- endometrium (probably endometrioid, often unusual morphology; dedifferentiated endometrioid; undifferentiated carcinoma); ovary (endometrioid and clear cell)

• PATHOLOGIST HAS KEY ROLE IN POSSIBLE IDENTIFICATION OF SUCH SYNDROMES BUT MUST MAKE CORRECT DIAGNOSIS

OVARIAN EPITHELIAL CARCINOMA (WHO 2014)

• low grade serous

• high grade serous (70%, >80% of advance stage cases)

• mucinous

• endometrioid

• clear cell

• Brenner

• seromucinous

• undifferentiated

REPRODUCIBILITY OF OVARIAN CARCINOMA TYPING

• Brugghe et al (IJGC, 1995) - 61%

• Bertelsen et al (IJGC, 1993) - 72% (serous and endometrioid); 86% (mucinous); 100% (clear cell)

• Baak et al (AQCH, 1986) - significant variation

• Cramer et al (APLM, 1987) – suboptimal

• Lund et al (APMIS, 1991) – 68%

• Sakamoto et al (Gynecol Oncol, 1994) -53%

• ICON 5- poor

RECENT STUDY ON REPRODUCIBILITY OF OVARIAN CARCINOMA TYPING

• Koebel et al, Am J Surg Pathol 2010;34;984-93.

• excellent agreeement

• participants had training in modern criteria

• important for subtype specific ovarian cancer treatments

• important for identifying familial predisposition

• CONTRAST WITH UTERINE CARCINOMA

• good marker- WT1

OVARIAN HIGH GRADE SEROUS CARCINOMA

PSEUDOENDOMETRIOID

WT1 in PSEUDOENDOMETRIOID AREAS IN HIGH GRADE SEROUS CARCINOMA

CLEAR CELL CHANGE IN SEROUS CARCINOMA (SOMETIMES POSTCHEMOTHERAPY)

WT1 IN TRANSITIONAL-LIKE AREAS IN HIGH GRADE SEROUS CARCINOMA

MIXED OVARIAN CARCINOMAS

• rare

• most commonly reported are mixed serous/ endometrioid; mixed serous/clear cell (mostly variants of high grade serous carcinoma- doubtful if these combinations exist)

• occasionally get mixed endometrioid/ clear cell (association with endometriosis)

• occasionally others

CLEAR CELL CARCINOMA

HNF1 beta

NAPSIN A IN CLEAR CELL CARCINOMA

“OVARIAN” SEROUS CARCINOMA (OSC)

• two distinct tumour types (called low grade and high grade OSC)

• not two grades of same neoplasm

• different neoplasms with different underlying pathogenesis, molecular events, behaviour, prognosis

• high grade much more common than low grade (approx 20:1)

• use instead of traditional grading schemes

PATHOGENESIS (LOW GRADE SEROUS)

• low grade arise from pre-existing benign and borderline tumour (probably not all cases)

• micropapillary variant of serous borderline may be intermediate stage in development of low grade serous carcinoma

• well-defined adenoma-carcinoma sequence

PATHOGENESIS (HIGH GRADE SEROUS)

• thought to arise directly from ovarian surface epithelium or epithelium of cortical inclusion cysts or epithelium of distal fallopian tube

• precursor lesion is serous tubal intraepithelial carcinoma (STIC)

• doesn’t arise from borderline tumour

MORPHOLOGY

• classification as high grade or low grade serous is reproducible (MD Anderson system)

• distinction based mainly on nuclear atypia (not on architecture) in worst area of tumour BUT

• low grade serous – uniform nuclei with mild atypia; < 12 mitoses per 10 HPFs; usually approximately 2/10 HPFs; no necrosis or multinucleation

• high grade serous – moderate to marked atypia; >12 mitoses per 10 HPFs; often necrosis and multinucleate cells

high grade serous

MOLECULAR (LOW GRADE SEROUS)

• KRAS or BRAF mutations in approx 2/3 (early in evolution – mutations found in benign and borderline tumours; identical mutations in borderline and malignant areas in same tumour)

• KRAS and BRAF mutations are mutually exclusive; equivalent effect on tumorigenesis

• no Tp53 mutations/abnormalities

MOLECULAR (HIGH GRADE SEROUS)

• Tp53 mutations (early in evolution-seen in early microscopic tumours eg BRCA1/2) (p53 dysfunction found in almost 100% high grade serous using stringent methods)

• BRCA1/2 abnormalities (germline or somatic mutations or hypermethylation)

• no BRAF and only occasional KRAS mutations

DISTINCTION BETWEEN LOW GRADE AND HIGH GRADE SEROUS CA

• Usually straightforward

• Occasionally difficult

• Rarely get admixtures (transformation)

• Only reliable marker is p53

p53

• p53 immunohistochemistry- lot of confusion • only consider positive/significant if diffuse strong nuclear

immunoreactivity (75-80% cells suggested- associated with missence mutation)

• p53 null consistent with serous carcinoma (different type of mutation (nonsense) or deletion resulting in truncated protein which is not detected by immunohistochemistry)

• most normal tissues and tumours exhibit focal, weak, heterogenous staining (“wild-type” staining) (usually <50%)

• DON’T REPORT AS POSITIVE OR NEGATIVE- REPORT AS “WILD-TYPE” or “MUTATION-TYPE”

p53- “all or nothing staining of diagnostic importance”

mutation type wild type

mutation

type

p53

• high grade serous carcinomas diffusely positive or totally (“flat”) negative- “mutation-type” staining

• low grade serous, clear cell, mucinous and most low grade endometrioid exhibit “wild-type” staining (some high grade endometrioid exhibit “mutation-type” staining but these are rare)

• OCCASIONAL EXCEPTIONS

HIGH GRADE SEROUS MIMICKING LOW GRADE

p53

LOW GRADE OSC WITH “BIGGER” NUCLEI

p53

TRANSFORMATION LOW GRADE INTO HIGH GRADE

• rare (AJSP 2012; 36; 368-375)

• can be overdiagnosed (bigger nuclei in low grade serous)

• can transform to HGSC, anaplastic carcinoma, carcinosarcoma

• p53 may not be reliable in such cases (may not be p53 mutated)

LOW GRADE TRANSFORMING INTO HIGH GRADE

p16 IN OVARIAN/ TUBAL (and uterine) HIGH GRADE SEROUS CARCINOMA

• Diffusely positive in most cases

• Low grade serous, endometrioid, clear cell- focally positive

• HGSC- approximately 2/3 homogeneous; 1/3 heterogeneous

• TMA- 115 HGSC (49 heterogenous; 63 homogenous)

• In stage I- III cases, homogeneous p16 worse prognosis (OS- significantly better with heterogenous staining- p=0.0367)

negative

heterogenous/ focal

homogenous/ diffuse

OVARIAN/ TUBAL CARCINOMAS ASSOCIATED WITH BRCA ABNORMALITIES

• Only HGSC (probably) (older studies- some endometrioid and clear cell- probably misdiagnosed)

• About 30- 50% of all HGSCs have BRCA abnormalities (germline (15%) or somatic mutations; promotor methylation)

• Better prognosis

• More chemosensitive; PARP inhibitors

• Predictive and prognostic significance

USCAP 2015

• Multi-institutional study- Belfast, Vancouver, Barts and the London

• 117 tubal/ ovarian neoplasms (borderline/ malignant) in patients with germline BRCA1/ 2 mutation

• Slides reviewed

• 106 of 117 (91%) HGSC (some had been originally called high grade endometrioid)

• Others- 4 low grade serous, 1 serous borderline, 1 endometrioid ca ovary, 1 endometrioid ca tube, 2 clear cell, 1 carcinosarcoma, 1 dysgerminoma

• Others may be coincidental- molecular analysis underway to investigate

MORPHOLOGY OF BRCA ASSOCIATED HIGH GRADE SEROUS CARCINOMAS

• SET pattern (Solid, pseudoEndometrioid, Transitional)

• Higher mitoses; more geographical necrosis • ? Increased tumour intraepithelial T lymphocytes • Different patterns of metastatic disease

(“pushing” rather than “infiltrative”) • Similarities to breast carcinomas in BRCA • BRCA1 immunohistochemistry- some correlation

with BRCA status (loss of staining) but not perfect and difficult marker to get to work

OVARIAN EPITHELIAL CARCINOMA

• “Ovarian cancer” = 5 distinct diseases

• HGSC: Major histotype

• Traditionally considered to be ovarian in origin

• New insights into pathogenesis of ‘ovarian’ HGSC have emerged over the last decade

• Paradigm shift

EXTRAPELVIC HIGH GRADE SEROUS CARCINOMA- SITE OF ORIGIN

• FIGO 2014- same staging system (ovary, tube, peritoneum, undesignated)

• FIGO 2014 and WHO 2014- no recommendations regarding designating site of origin

• WHO- the decision as to primary site should be pragmatic, based on experience and professional judgement

• DOMINANT MASS THEORY TRADITIONALLY USED (ovary designated as primary site in most cases)

• implications:- epidemiology, tumour incidence/mortality, cancer registries, entry into clinical trials

• different viewpoints- STIC/ in situ criteria; dominant mass criteria

Ovarian? Peritoneal? Tubal? Undesignated? …. CHAOS!

INCIDENTAL SPORADIC HIGH GRADE SEROUS CARCINOMA

• established that incidental tumours in patients with BRCA1/2 mutation are of tubal origin (prophylactic BSOs)

• 3 papers recently published- unsuspected STIC/ HGSC incidentally detected

• Proves that tubal disease is not secondary

• PROVES that sporadic extrauterine HGSC of tubal origin (FINAL PIECE OF EVIDENCE)

Am J Surg Pathol, 2014

Study Total

cases

Cases

with

STIC

Invasive

HGSC

in tube

Invasive

HGSC in

ovary

Organ-

confined

Disease

(tube OR

ovary)

Organ-

confined:

tube

Organ-

confined:

ovary

Rabban,

2014

4 4 3 1 3 3 0

Morrison,

2014

22 22 6 1 21 21 0

Gilks, 2014 21 20 12 2 18 18 0

Total 47 47 22 4 43 43 0

Summary of findings of incidental HGSC in a non-prophylactic setting

PROPOSAL FOR DESIGNATING SITE OF ORIGIN OF HGSC (Histopathology 2014; 65; 149-154 )

• extensive examination of tube (SEE-FIM)

• any STIC or mucosal serous ca in tube- tubal origin

• if fallopian tube or fimbria not identified (obliterated by mass)- tubal origin

• ovarian primary if tumour in ovary and nothing in mucosa of tube (STIC or invasive)

• primary peritoneal- nothing in tube or ovary (vanishingly rare- will likely disappear) (MODIFICATION OF PAPER)

• USING THESE CRITERIA- approximately 80% tubal primaries

• undesignated- very small proportion

Implications for specimen handling

• SEE-FIM protocol ESSENTIAL for identifying STIC/early tubal involvement

Application of site assignment to a case series

ASSESSMENT OF A NEW SYSTEM FOR PRIMARY SITE ASSIGNMENT IN HIGH-GRADE SEROUS CARCINOMA OF FALLOPIAN TUBE, OVARY AND PERITONEUM

Naveena Singh1, C. Blake Gilks2, Nafisa Wilkinson3, W. Glenn McCluggage4 1Department of Cellular Pathology, Barts Health NHS Trust, London, United Kingdom; 2Department of Pathology, Vancouver General Hospital and University of British Columbia, Vancouver, Canada; 3Department of Histopathology, St James’s Hospital, Leeds, United Kingdom; 4Department of Pathology, Belfast Health and Social Care Trust, Belfast, United Kingdom.

Histopathology 2015, accepted for publication

RETROSPECTIVE (n=151) PROSPECTIVE (n=111)

Primary

site

T O P U T O P U

Chemo

naive

63

(79%)

16

(20%)

0

(0%)

1

(1%)

44

(83%)

9

(17%)

0

(0%)

0

(0%)

Post-

NACT

48

(68%)

16

(22%)

7

(10%)

0

(0%)

44

(76%)

7

(12%)

4

(7%)

3

(5%)

Singh et al, 2015

Basis for tubal assignment in 44 chemonaive cases

Criterion Number (%)

STIC only 5 (11%)

Invasive mucosal +/- STIC 26 (59%)

Entire tube or part of tube incorporated in mass

13 (30%)

Total 44

Implications of Proposal

• Consistency and uniformity in reporting and in message to patients

• Tumour registry data

• Cases which never undergo surgery (diagnosed on core biopsy only) – default primary site should be fallopian tube

• NEED TO INTRODUCE WIDELY- RCPath DATASETS, GYNAE ONCOLOGISTS

ICCR (International Collaboration on Cancer Reporting)

• group to consolidate various cancer datasets

worldwide (pathology colleges of UK, USA, Canada, Australasia, ESP)

• endometrial cancer dataset already developed

• ovarian/ fallopian tube/ primary peritoneal cancer dataset now developed (on ICCR website; paper in press in Modern Pathology)

• ADOPTION OF SITE ASSIGNMENT CRITERIA DESCRIBED

Russell Vang

Blaise Clarke

Blake Gilks

Colin Stewart

Xavier Matias-Guiu

Ben Davidson Glenn McCluggage

Harry Hollema

Yoshiki Mikami

Jonathan Lederman

PROBLEMS WITH TYPING OF UTERINE CARCINOMAS

• papillary variants of endometrioid carcinoma; glandular and solid variants of serous carcinoma- DO NOT USE TERM PAPILLARY SEROUS CARCINOMA

• mixed type 1 and type 2 (mixed carcinomas PROBABLY more common than in ovary - may arise secondary to p53 mutation in type 1 tumour)

• significant interobserver variability in distinction between serous, clear cell, grade 3 endometrioid, undifferentiated, carcinosarcoma (“high grade” endometrial carcinomas)

• LOT OF PROBLEMS IN TYPING ENDOMETRIAL CARCINOMAS

PAPILLARY VARIANTS OF ENDOMETRIOID CARCINOMA

IMMUNOHISTOCHEMISTRY- SEROUS VERSUS “LOW GRADE” ENDOMETRIOID

• use a panel

• interpret along with morphology

• overlap in some cases

IMMUNOHISTOCHEMISTRY

• p53- MOST USEFUL IF INTERPRET CORRECTLY

• ER

• p16

• HMGA2 (expressed more commonly in serous than endometrioid)

• PTEN (lost in many low grade endometrioid carcinomas; preserved in serous carcinomas)

• IMP3 (expressed more commonly in serous than endometrioid)

CLASSIC IMMUNOPHENOTYPE

• low grade endometrioid- ER diffuse +ve, p53 wild-type, p16 patchy

• serous- ER-ve, p53 mutation-type, p16 diffuse +ve

BUT CAN BE OVERLAP

“SUBTLE” SEROUS CARCINOMA

ER

p53

p16

ER

• classically low grade endometrioid carcinoma diffusely positive and serous carcinoma negative

• reality- many/most serous carcinomas are ER positive (new antibodies/retrieval methods; different spectrum of serous carcinomas)

MIXED TYPE 1 AND TYPE 2 (USUALLY ENDOMETRIOID AND SEROUS)

• 2 types- at least one of which is type 2 carcinoma

• WHO 2014- minimum percentage arbitrarily set at 5%

• Report any amount of type 2 tumour

• type 2 may evolve from type 1 cancer via p53 mutation (dedifferentiation/ progression)

• NEED TO SEE 2 DISTINCT MORPHOLOGICAL AREAS WITH DISTINCT IMMUNOPHENOTYPES

TRUE MIXED SEROUS-ENDOMETRIOID

p53

CHANGES ON SURFACE OF UTERINE ADENOCARCINOMA

• endometrioid or mucinous type

• especially likely to be sampled on biopsy

• micropapillary or microglandular architecture

• may look like papillary syncytial metaplasia

• may look like cervical microglandular hyperplasia (get subnuclear vacuolation in this) (caution before diagnosing cervical MGH in endometrial biopsy in postmenopausal woman)

• may be mistaken for component of serous carcinoma and result in overdiagnosis of mixed tumour

CONFUSION WITH SEROUS/ CLEAR CELL CARCINOMA COMPONENT

ER

p53 Mutation in Endometrial Adenocarcinomas

• early ubiquitous (sentinel event) in serous carcinomas

• late event in endometrioid adenocarcinomas (associated with tumour progression)

• can be useful- serous (all aberrant); endometrioid (sometimes only aberrant in “solid” areas)

• Tp53 mutation definitional of serous carcinoma

• don’t need p53 staining in classic cases

• if p53 staining not aberrant- probably not serous carcinoma

UTERINE CLEAR CELL CARCINOMA

• very uncommon (beware before make diagnosis)

• GET CLEAR CELLS IN OTHER TUMOUR TYPES (serous, endometrioid, carcinosarcoma, undifferentiated) (SIGNIFICANT INTEROBSERVER VARIABILITY)

ENDOMETRIAL CLEAR CELL CA

ER p53

HNF1

beta

IMMUNOHISTOCHEMISTRY OF CLEAR CELL CARCINOMA

• variable (? because of different criteria)

• usually ER negative and p53 wild-type

• hepatocyte nuclear factor 1 beta/ Napsin A

SECRETORY VARIANT OF ENDOMETRIOID CARCINOMA

SEROUS WITH CLEAR CELL FEATURES

p53

p16

UNDIFFERENTIATED ENDOMETRIAL CARCINOMA

• category included in WHO classification

• diagnosis often missed

• definition- “a tumour composed of medium or large cells with complete absence of glandular or squamous differentiation and with absence or minimal (<10%) neuroendocrine differentiation”

• DYSCOHESIVE TUMOUR CELLS

UNDIFFERENTIATED ENDOMETRIAL CARCINOMA

• specific histological diagnosis • pathologists often reluctant to make diagnosis • distinguish from grade 3 endometrioid

adenocarcinoma • may be associated with low grade endometrioid

adenocarcinoma (dedifferentiated endometrioid adenocarcinoma or mixed endometrioid and undifferentiated carcinoma)

• similar tumours in ovary • undifferentiated/dedifferentiated element may be

seen in recurrence or metastasis • extremely poor prognosis

EMA

IMMUNOHISTOCHEMISTRY

• usually positive for keratins and EMA but staining may be very focal, although strong

• EMA staining more consistent than cytokeratins

• minor neuroendocrine marker positivity not uncommon

• ER usually negative

dedifferentiated endometrioid adenocarcinoma

DIFFERENTIAL DIAGNOSIS

• grade 2 or 3 endometrioid adenocarcinoma

• undifferentiated sarcoma (some may have epithelioid appearance) (value of EMA)

• solid variant of serous ca

• carcinosarcoma

• small cell or large cell neuroendocrine carcinoma (minor degree of neuroendocrine marker positivity allowable)

• malignant lymphoma, plasmacytoma

• PNET

• epithelioid sarcoma, rhabdomyosarcoma

DIFFERENTIAL DIAGNOSIS- grade 3 endometrioid adenocarcinoma

Glands- not in undiff Ca Squamous elements- not in undiff Ca

DIFFERENTIAL DIAGNOSIS- carcinosarcoma

2 elements and sharp demarcation may suggest carcinosarcoma

Epithelial component is low grade in dedifferentiated ca and high grade in carcinosarcoma

Immunohistochemistry (cytokeratins and EMA)

MMR ABNORMALITIES

• ? dedifferentiated/ undifferentiated carcinomas more common with MMR abnormalities, including HNPCC/Lynch syndrome

• also LUS location, tumour infiltrating lymphocytes, Crohn’s like lymphoid aggregates, tumour heterogeneity, synchronous ovarian clear cell carcinoma

• often get loss of MLH1/PMS2- can be Lynch or secondary to MLH1 methylation

MMR IMMUNOHISTOCHEMISTRY

MLH1 PMS2

WHY IS IT SO DIFFICULT TO TYPE ENDOMETRIAL CARCINOMAS?

• mixed tumours (probably more common than in ovary but ? being overdiagnosed)

• no good marker (WT1 in ovary)

• lot of endometrial carcinomas exhibit microsatellite instability- prone to mutations with different clones emerging

• ISGyP working groups set up to provide recommendations