New classifications of gynecologic cancers

Frédérique Penault-Llorca MD, PhD

Centre Jean PERRIN – 58 Rue Montalembert - 63011 CLERMONT-FERRAND CEDEX

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LINKS OF INTEREST

• AbbVie, AstraZeneca, Bayer, BMS, Jannsen, Lilly, MSD, Merck Lifa, Novartis, Pfizer, Puma,Roche, Sanofi, Takeda, honoraria and/or research grants in the field of biomarkers

• MEDSCAPE, PEER VOICE, Publiclin, Edimarkauthorships for educational materials

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Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012

Int J Cancer. 2015 Mar 1;136(5):E359-86.

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CERVICAL CANCER

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Squamous cell carcinoma of the cervix is almost always associated with HPV infection: multi-stage

carcinogenesis of > 10-15 years

From PA Just - Correspondances en Onco-Théranostic - Vol. VII - n° 4; 2018

• HPV infection in basal cells - expression of "early" viral proteins ( basal cell)/ "late" ( superf cells)

• High-grade lesions: expansion in height, decreased production of virions• Carcinoma: integration to tumor DNA stop production of virions• Frequent regressions of low grade lesions

Normal cervico-vaginal junction

Low grade intraepithelial

lesion

High grade intraepithelial

lesion

Invasive carcinoma

Viral DNA Episomal

Integrated

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Therapeutic consequences

• HPV infection:– Vaccination +++– Expression of VEGF (via E7 action) antiangiogenic– PI3KCA pathway activation: AKT / MTOR (60% EC and

74% ADK) and tumor suppressor pathway TGFβ (41% EC and 26% ADK) mTOR / AKT inhibitors?

– Tabacco is a co-carcinogen prevention

• Immunotherapies? PD-L1 non reliable biomarker• In absence HPV infection

– Poorly known, would present preferential activation of Wnt / β-catenin and Sonic Hedgehog pathways

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ENDOMETRIAL CARCINOMA

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First of all: familial forms

• Familial Endometrial Carcinomas– Lynch II (HNPCC)

• Mutations in DNA repair genes

• Endometrial carcinoma 15yrs earlier (46yrs old)

• Cumulated risk: 40 to 70%

• Family history should be explored for all endometrial K

• Screening and prophylactic hysterectomy

– Cowden• PTEN Mutation

• Risk X 3 à 5 : cumulated risk: 28%5t

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Endometrial carcinoma Lynch

• < 50 yrs– Multifocal

– Uterine lower segment

– High grade endometrioid

– Heterogeneous tumors

• Non endometrioid < 60 yrs

• Lymphocytic stroma

• Invasive tumors, embolies

• Synchronous T ovaries/endometrium: CCC

Screening by IHC MSH1, MLH2, MLH6, PMS2 when endometrial carcinoma is diagnosed in women under 60yrs regardless family history

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Endometrial carcinoma <40yrs

• Endometroid G1-2 (80%)

• Stage I-II (90%)

• Good prognosis: 6% of mortality

• Synchronous T ovary/endometrium (13%): endometroid

• 16% MSI-H phenotype : worse prognosis(mortality 23% vs 6%)

• Don’t forget the other causes: polycystic ovariessyndroma and granulosa cell tumor

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IT WAS « ALMOST » SIMPLE

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« ENDOCRINE » CLASSIFICATION

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Type Ihormono dependent

• 50 – 59yrs

• Hyperestrogeny• 66 yrs (post menoposal)

• Absence of hyperestrogeny

Type II

non hormono dependent

Endometrial carcinoma

70-75%

5-10%

1-5%

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• 77 to 80%

• Endometrioid

• Hyperplasia of endometrium

• Precursor : atypical hyperplasia

• 10 to 23%

• Serous and clear cell

• Atrophic endometrium

• precursor : EIC

Intraepithelial carcinoma EIC

Type I Type II

Endometrial carcinomaBokhman

Bokhman, based on the pathology of Russian women in the 70’s, 80’sOther histotypes are excluded including Lynch

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Murali, Lancet Oncol 2014

HISTO-MOLECULAR SUBTYPES OF ENDOMETRIAL CANCER

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« MOLECULAR» CLASSIFICATION

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POLE MSILow copy

number MSSHigh copy

number serous

8% 40%28% 24%

Type I Type II

Endometrioid Serous & endo

I,II, III I,II, III I,II III

Histologic subtypes

Grade

Molecularsubtypes

Frequency

Bokmansubtypes

Alteredcopy nbr

Mutation rate

PI3K alterations

KRAS alterations

P53 mut

Pronostic

35% 1%5% >90%

Poor IntermediateGood

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TCGA endometroid carcinoma

ASCO® 2013 –Pashtan I, abstr. 5511 actualisé

Nature 2013;497:67-73

POLE

(ultramuted)

MSI

(hypermuted)

Copy number low

( endometroid )

Copy number high

(Non endometroid)

POLE (ultramuted)

Log rank p = 0,02

MSI (hypermuted)

Copy number low

Copy number high

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POLE MSILow copy

number MSSHigh copy

number serous

8% 40%28% 24%

Type I Type II

Endometrioid Serous & endo

I,II, III I,II, III I,II III

Histologic subtypes

Grade

Molecularsubtypes

Frequency

Bokmansubtypes

Alteredcopy nbr

Mutation rate

PI3K alterations

KRAS alterations

P53 mut

Pronostic

35% 1%5% >90%

Poor IntermediateGood

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RATIONAL FOR IO

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Parp inhibitors and endometrial carcinoma:

strong rational

• Defect in homologous recombination in serous tumors (type2)

– TCGA Group 4

– Similar evolution to high serous ov Ca

– BRCA : mutations in ~ 10% but probably underestimated

• Activity of Parp inhibitors in case of Pten inactivation or Arid1A alterations

– TCGA group 2 and 3

Leary, Current opinion Oncol 2016, Prurdle, Modern Pathol 2017

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Group 3 MSS

P53 wild type

Group 4P53 mutated

Group 2 MSI

Group 1POLE

mutated

PolEsequencing

IHC MMRMSH6, PMS2, MLH1, MSH2

IHC P53

ENDOMETRIAL K

POLE mutated MSI profile P53 mut 0 or>75%

Non mutated MMR Non mutated

Copy number high

Copy number low

Not routinelyperformed nowadays

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Group 3 MSS

P53 wild type

Group 4P53 mutated

Group 2 MSI

Group 1POLE

mutated

PolEsequencing

IHC MMRMSH6, PMS2, MLH1, MSH2

IHC P53

ENDOMETRIAL K

POLE mutated MSI profile P53 mut 0 or>75%

Non mutated MMR Non mutated

Copy number high

Copy number low

Immuno?Parp Inh?

Immuno?

Parp Inh?

Immuno?

Parp inh?

Other?

Combo Immuno-Parp?5t

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Conclusion

• Uterine carcinoma : determination of the proper histologic subtype is of paramount importance for treatment strategies

• Grade 3 endometrial carcinoma of good prognosis (POLE mutation) not routine yet

• Emerging potential targets

• Familial forms: systematic LYNCH screening for tumors before 60yrs old (IHC)

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OVARIAN EPITHELIAL TUMORS

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Ovarian cancer mortality

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Ovarian Epithelial Tumors

• Serous 50%

(transitional cell carcinoma) rare

• Endometrioid 20%

• Mucinous (intestinal) 15%

• Sero-mucinous (endocervical) rare

• Clear cell 5%

• Brenner 5%

• Indifferenciated 5-7%

WHO, 2014

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Risk factors for EOC

• Predispositions: 5-10%

– Hereditary monogenic

– Multifactorial

• Susceptibilities:10-30%

– Hereditary multigenic

– Multifactorial

• Sporadic: 60 - 85%

– Multifactorial 5t

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Possible genetic context

Family aggregations of ovarian cancer (frequent):- breast cancers (man-woman) / ovaryBRCA genes- colon / endometrium / ovaries (Lynch) MMR genes- ovaries onlyBRCA & MMR + other genes ...

- ≥ 2 ovarian cancers- 1 ovarian cancer <60 years

Hereditary diseases with associated clinical signs (rare): associated genes• Non-epithelial tumorsPeutz-Jegher, Swyer, familial gonadal neoplasia, Carney ...• Epithelial tumorsGorlin, A.T., Maffucci, Gardner, Werner, SLF, Cowden, Bloom ...

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First of all: familial forms

Genetic risk: 3 principal syndromes

• Ovarian cancer site specific:

•familial: >3 cases of OvCa, Mean age 49 yrs old,

•RR 40 if BRCA1 and 12% if BRCA2

•BRCA1:Mutation 75% of the cases

•Breast/ovaries syndrome:

•familial: >3 cases of breast Ca and > 2 OvCa, Mean Age= 52 yrs,

•RR X 50%=> RR 60% if BRCA1 vs RR 30% if BRCA2

•Mutation BRCA 1/2: 95% of the cases

Ford et al, 1994 Dauplat et al, 1998

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First of all: familial forms

Genetic risk: 3 principal syndromes

• Lynch (HNPCC Hereditary non-polyposis colorectal carcinoma)

•familial: >3 cases of CRC (one before 50 yrs), endomerium, biliary tract, urinary tract, ovaries, Mean age: 45 yrs, RR 10%, Microsatellite instability: MSH1, MSH2, PMS1, PMS2, MLH1 (MSH2: RR 38 MLH1: RR20 MSH6: RR1)

•Other Syndromes :

•Peutz Jegher Syndrome: STK11 (ch19) mutation; Prevalence: 1/10.000, hamartomatous Polyposis of the digestive tract, CRC (60%), Breast (32-54%), ovaries (20%), uterus (10%), pancreas

Lynch et al, 1986 Chabbert-Buffet et al, 2012

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IT WAS « ALMOST » SIMPLE

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Ovarian Oncogenesis Kurman 2008

• 25%• Stade I• Slow evolution• Mutations BRAF, KRAS, PTEN,

b catenin• Precursor :

– Endometriosis– Borderline T

• Endometrioid• Serous low grade• Mucinous• Clear cell carcinoma

• 75%

• > stade I

• Rapid evolution

• P53 Mutations

• Genetic instability

• De novo (but …STIC)

• High grade serous

• Undifferenciated carcinoma

• Carcinosarcoma (MMMT)

Type I Type II

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Dysplasie tubaire Dysplasie

ovarienne

60% ? 40% ?

A dual origin?

Crum et al, 2010

Tube dysplasia Ovariandysplasia

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Ovarian tumors linked to endometriosis

ARID 1A - ARID 1A +

ARID 1A –

20% of

endometrioid

ovarian

endometrioma

Endometriosis is precursor for

CCC, endometroid and seromucinous

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BUT….

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OVARIAN CANCER

HISTO-MOLECULAR SUBTYPE

90% 10%

Banerjee S & Kaye SB, Clin Cancer Res 2013

5 DIFFERENT EPITHELIAL OCs (EOCs)

Prat J, Virchow’s Archive 2012

high-grade serous (HGSOC) low-grade serous (LGSOC) mucinous endometrioidclear cell

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Banerjee S , and Kaye S B Clin Cancer Res 2013

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48

Parp Inhibitors Platinium salts

HER2

BRAF inhibitorsTZB ?Double inhibition : PI3K & MEK inhibitors

PI3KmTOR ?

ChimioresistanceMEK inhibitors?TZB? (HER2 amplification)

Inhibitors

of ARID1A

Loss of expression of

BAF250a*

fromWiegand K et al J Pathol 2011

Mc Conechy M et al J Pathol 2012

TP53 mutation Cromosomal instability

Inactivationof BRCA 1/2(Mutation or hypermethylation)

High-grade serous

Clear cell

Endometrioid

Mucinous

KRASMutation

KRASBRAFERBB2PIK3CAMutation

ARIDIAPI3KCAZNF217PPP2RIAMutation

CTNNBIPTENPIK3CAPPP2RIAMutation

Type II

Type I

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FOCUS ON BRCANESS IN HIGHGRADE SEROUS CARCINOMAS

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Low grade: Extremely rare

High grade: Common

Serous carcinomas

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• BRCA mutation testing must be proposed

– To all patients with high grade, undifferentiated, carcinosarcoma or clear cell ovarian (or primary peritoneal) serous or endometrioid cancer.

–Whatever the age or family history

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How to look for BRCA mutations

Germinal: hereditary

family implication

On blood or sputum

Somatic: acquired tumor

alterations

no family implications

test on the tumor

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Context

• Tumor Type: high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer

• Analysis of germline BRCA1/2 (blood) at the initial diagnosis of the tumor after oncogenetics consultation

• Analysis of somatic BRCA1/2 (the tumor): at the first platinosensitive relapse if BRCA1/2germinal mutation negative (~7% extra positive cases) or if not tested at baseline

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Somatic BRCA testing

Requirements

• Adequate quantity of tumor material (FFPE)

• Control of pre-analytical steps (optimal quality)

• NGS (Next Generation Sequencing) with external quality control

• Timing => Results: 2 to 3 weeks

Major limitations

• Tissue is the issue!

– Quantity of tissue available

– Quality of tissue available

• Complexity of NGS data analysis

• Validation of the results (Sanger)

• Turnaround time (costs if non-pooled tests)

FFPE: formalin-fixed, paraffin-embedded

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MOLECULAR TESTING ACCURACY DEPENDS UPON PATHOLOGISTS….AND SURGEONS5t

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Type of samples

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QUANTITY

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Type of samples

Mc Cluggage Mod Pathol 2015

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• Initial diagnosis: OK but beware of necrosis•Optimal and quick

fixation

Peritoneal biopsies => guidelines for surgeons

• 10 biopsies

• 5-10mm

• Beware of electrocoagulation

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Examples of unsuitable samplesMany samples do not have enough tumor

Tiny biopsies despite huge peritoneal

mets

Early discussion with surgeons or pathologists would have led to different specimens

Low tumor content (post neoadjuvant) or diffuse necrosis

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Examples of unsuitable samplesMany samples do not have enough tumor

Low tumor residual content or pCR

Post neoadjuvant

Early discussion with surgeons or pathologists would have led to different specimens

pCR: pathological complete response

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Example of adequate samplesTumor content and surface area are the key!

Cellularity > 20%

Surgical specimen Many small biopsies with dense tumor

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ANALYTICAL

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NGS for BRCA1/2

Goals

• Exhaustive coverage of BRCA1/2 genes

• Acceptable sensitivity

– Germinal: 30X

– Somatic: 100 to 300X

• Capture has improved a lot the screening!

Limits

• BRCA1/2: large genes, no hot spots

• Diversity of mutations (SNP, ins/del, RGT)

• Quantity of material

• Quality of samples and design

• Detection of large rearrangments (10% BRCA1, 2% BRCA2)

Del: deletion; ins: insertion; NGS: next generation sequencing; SNP: single-nucleotide polymorphism; RGT:

rearrangement

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POST-ANALYTICAL: INTERPRETATION

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Clinical report

• Suitability of tumor sample

• Target analysed: examined genes (i.e. BRCA1or BRCA2)

• Region of interest: the regions covered for each gene (e.g. coding region only or intronicand exonic regions)

• Overall results: either pathogenic/deleterious variant present or absent

• Mutation details (when present)5t

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Clinical report cont’

• Reference sequence

• Summary/interpretation:

– Pathogenic or likely pathogenic classification of the identified variants

– Non-pathogenic variants should not be reported, (laboratory may keep a record)

– Variants of unknown significance should be reported separately and clearly indicate the lack of sufficient clinical or biological evidence

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CONCLUSION

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Conclusion

• Ovarian carcinoma : determination of the proper histologic subtype is of paramount importance for treatment strategies (exclude metastasis for bilateral non serous tumors)

Always question the pathologist in case of bilateral non serous carcinoma

• Specific mutations for granulosa cell tumors (FOXL2) and Sertoli cell tumors (DICER)

• Familial forms5t

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Conclusion

• The spectrum of ovarian carcinoma shows heterogeneous disease with different biologyand putative targets

• Emerging potential targets (BRCA, PIK3CA, ARID1A, HER2…) + immunotherapy

• With the advent of parp inhibitors, the pathologists’role in the management of high grade serous tumors has changed

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EVERY women with a epithelial invasive non mucinous

ovarian cancer should be tested for BRCA 1 &2

Every women <60 years with an endometrial cancer

should be tested for MSI

Important take home messages

Time as come to incorporate genetics to GYN-oncology

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