Testing molecular biomarkers in endometrial cancer : POLE, L1CAM, p53, MMR

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1 2018 년대한부인종양학회제 33 차춘계학술대회 Testing molecular biomarkers in endometrial cancer : POLE, L1CAM, p53, MMR Jin Hwa Hong Department of Obstetrics and Gynecology Guro Hospital, College of Medicine, Korea University

2 Historical/Pathologic classification of endometrial cancer (Dualistic model) USEFUL, BUT limited by substantial heterogeneity within each type and significant overlap between Type I and II TP53 mutations >90% of SC, but also in 11% of EEC Gynecol Oncol 1983;15:10-7

3 Flaws of current pathologic classification and risk stratification Significant inter-observer variability in high-grade tumors (G3 EEC and SC) 56 high-grade ECs reviewed by 3 expert pathologists 62.5% concordance rate regarding subtype diagnosis Overall kappa statistics for FIGO grade : moderate level ( ) Int J Gynecol Pathol 2016;35:16-24 Highly subjective and poor reproducibility, imprecise estimation of risk of recurrence or death Limitation of current major risk stratification systems RSS YEAR No. of patients Criteria included PORTEC Histologic type, grade, MI, age GOG Histologic type, grade, MI, FIGO stage, age SEPAL Histologic type, grade, MI, FIGO stage, LVSI ESMO Histologic type, grade, MI, FIGO stage ESMO modified Histologic type, grade, MI, LVSI ESMO-ESGO-ESTRO Histologic type, grade, MI, FIGO stage, LVSI Compare accuracy in stratifying patients according to the risk of recurrence and LN metastasis Poor-to-moderate discrimination for recurrence and LN metastasis Over-treatment or under-treatment in real-world practice Limit individualized treatment More reproducible, objective, biologically informative system needed

New genomic era : molecular classification of EM CA (TCGA) 373 EC endometrioid (n=307), serous (n=53), mixed endometrioid and serous (n=13) POLE-ultramutated (7%) MSI-hypermutated (28%) CN low (39%)

4 New genomic era : molecular classification of EM CA (TCGA) 373 EC endometrioid (n=307), serous (n=53), mixed endometrioid and serous (n=13) POLE-ultramutated (7%) MSI-hypermutated (28%) CN low (39%) CN high (26%) Histotype Mostly endometrioid Mostly endometrioid Endometrioid Mostly serous, but endometrioid (26%) Grade High High Mostly low High Mutation rate Very high High Moderate Low Frequent mutations PTEN (94%), PIK3CA (71%), PIK3R1 (65%), ARID1A (76%), KRAS (53%), ARID5B (47%) PTEN (88%), RPL22 (33%), KRAS (35%), PIK3CA (54%), PIK3R1 (40%), ARID1A (37%) PTEN (77%), CTNNB1 (52%), PIK3CA (53%), PIK3R1 (33%), ARID1A (42%) Prognosis Favorable Intermediate Intermediate Poor TP53 (92%), PPP2R1A (22%), PIK3CA (47%) Clinical significance of TCGA study Subset of EC contain distinct pattern of SCNAs and mutations that do not correlate with pathology-based risk stratification Nature 2013;497:67-73

5 Controversy of G3 EEC type I or II?, prognosis similar to USC or more favorable?, optimal treatment? Grade 3 EEC (n=381) POLE (12.9%), MMRd (36.2%), p53 abn (20.7%), no abnormality (30.2%) Grade 3 EEC Stage I, Grade 3 EEC Grade 3 EEC Stage I, Grade 3 EEC Grade 3 EEC mixture of molecular subtypes of EM CA, NOT homogeneous entity Am J Surg Pathol 2018;42:561-8

POLE mutation Ultramutated POLE subgroup novel finding from TCGA POLE : Major catalytic subunit of DNA polymerase epsilon involved in nuclear DNA replication and repair POLE exonuclease domain

6 POLE mutation Ultramutated POLE subgroup novel finding from TCGA POLE : Major catalytic subunit of DNA polymerase epsilon involved in nuclear DNA replication and repair POLE exonuclease domain mutations (EDM) mostly found in 3 hotspot regions with V411L, P286R, S459F Increased replication error rates and resultant ultramutator phenotype Clinical significance Mostly FIGO stage I and G3 EEC Marked intratumoral heterogeneity Morphological ambiguity mimicking serous carcinoma Rich in tumor infiltrating lymphocytes G3 EEC with abundant TILs Serous-like features with tumor giant cells POLE ultramutated subgroup favorable outcome even with higher grade Methodology Sanger sequencing or panel-based NGS

7 Targeted POLE sequencing in EC from PORTEC-1 and -2 trials (n=788) POLE mutation rate : 6.1% POLE mutant EC (vs POLE wt) : more G3, less LVSI and MI <50% POLE-mutant EC : excellent prognosis independent of other known prognostic variables may help reduce overtreatment JNCI 2014;107(1):402

8 Retrospective cohort (N=406) for target sequencing of POLE EDM POLE EDM rate : 9.6% (82% endometrioid type, 49% - G3 EEC) PFS & DSS POLE EDM >> POLE-wt in both total and grade 3 only (even with high risk features high grade, LVSI, non-endometrioid) Higher PFS and DSS in patients with POLE EDM than POLE wt regardless of adjuvant treatment Clin Cancer Res 2016;22(12):

PCR amplification and Sanger sequencing for POLE mutation in 131 EC (POLE EDM rate : 8.5%) Improved overall survival in POLE mutated EC vs POLE-wt EC (P=0.

9 PCR amplification and Sanger sequencing for POLE mutation in 131 EC (POLE EDM rate : 8.5%) Improved overall survival in POLE mutated EC vs POLE-wt EC (P=0.04) Increased resistance to carboplatin in POLE mut vs POLE wt (p=0.004) PD-1 + CD8 + tumor infiltrating lymphocytes count significantly higher in POLE mut vs POLE wt (125 vs 15, P=0.017) PD-1 + CD4 + tumor infiltrating lymphocytes count significantly higher in POLE mut vs POLE wt (51 vs 10, P=0.017) Favorable prognosis of POLE mutated EC is not secondary to higher chemosensitivity but likely linked to enhanced immunogenicity Gynecol Oncol 2017;144:146-52

MMR (Mismatch repair) MMR a process that in normal cells permits the recognition and repair of genetic mismatches generated during replication.

10 MMR (Mismatch repair) MMR a process that in normal cells permits the recognition and repair of genetic mismatches generated during replication. Microsatellite instability (MSI) - condition caused by a deficient MMR system (expansion or reduction in length of repetitive DNA sequences in tumor DNA) d/t methylation of promoter region (MLH1) d/t genetic mechanism (germline or somatic mutations in MMR genes) MMR gene MLH1, MSH2, MSH6, PMS2 Defective MMR d/t pathogenic germline variants in MMR genes Lynch syndrome (20-25% of MSI-H EC) Determination of MMR-d in EC Identification of EC caused by Lynch syndrome Potential candidate for immunotherapy Inconsistent finding regarding clinicopathological features MMR-d favorable prognosis (Asia) vs aggressive nature (West) Methodology MSI assay and IHC staining of MMR proteins MSI EEC with peritumoral and tumor infiltrating lymphocytes

11 MMRd and MSI testing recommendations NCCN clinical practice guidelines in Oncology (NCCN guidelines) for Colon cancer, Rectal cancer, and Uterine Neoplasms include MMR or MSI testing - NCCN Guidelines for Colon cancer: Universal MMR or MSI testing is recommended for all patients with a personal history of colon or rectal cancer to identify individuals with Lynch syndrome, to inform use of immunotherapy in patients with metastatic disease and to inform decisions for patients with stage II disease. - NCCN Guidelines for Uterine neoplasms: Universal testing of endometrial tumors for MMR gene Ref) NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines ) for Colon Cancer and Uterine Neoplasms V

12 MSI / MMR test in Korea 1. MSI 검사 (PCR) 명칭 : 비유전성유전자검사 중합효소연쇄반응 확장 폴리아크릴아마이드겔전기영동 [STR Markers] / 진단검사의학과전문의등판독. 코드번호 : C / 분류번호 : 나 583 나 (2) 적응증 : 유전성비용종성대장암위험군의선별진단, 산발성대장암및위암의예후판단및치료방법선택에정보제공 수가 : 295,503 원 ( 상급종합병원가산료포함 ) 2. MMR 검사 (IHC) 명칭 : 면역조직 ( 세포 ) 화학검사 [ 종목당 ] 코드번호 : C5575 / 분류번호 : 나 557 적응증 : 세포나조직에특정단백이존재하는지를알아보기위함 수가 : 57,616원 ( 상급종합병원가산료포함 ) Test Reagents to targets Result indicative of MSI-H IHC Abs to: MLH1/MSH2/MSH6/PMS2 Any 1 (or more) of 4 proteins absent PCR PCR probes to: BAT25, BAT26, NR21, NR24, Mono27 or BAT25, BAT26, Di 5S346, Di 2S123, Di 17S250 2 of 5 loci differ in size from corresponding normal loci

001) Discordant cases (n=41, 6%) - Subclonal loss of MMR expression (n=18) - MSS or MSI-L with loss of MMR expression (n=20) - MSI-L or MSI-H with

13 696 ECs from PORTEC-1 and -2 trials Compare MSI (pentaplex panel) and MMR protein (MLH1, PMS2, MSH2, MSH6) IHC Frequency of MSS, MSI-H, MSI-L : 74%, 24%, 2% Concordance between MSI and IHC analysis 94% (655/696, kappa = 0.854, p<0.001) Discordant cases (n=41, 6%) - Subclonal loss of MMR expression (n=18) - MSS or MSI-L with loss of MMR expression (n=20) - MSI-L or MSI-H with retained MMR expression (n=3) Advantage of MMR protein IHC over MSI analysis - Lower cost - Easy availability (FFPE, universal protocol for IHC) - Identification of genes mutated - Can be performed in any laboratories Ann Oncol 2017;28:96-102

POLE and MSI as predictive marker for efficacy of immune check point inhibitors Howitt et al (JAMA Oncol 2015) - Retrospective IHC analysis on 63 ECs specimens - 1 st study to confirm the association

14 POLE and MSI as predictive marker for efficacy of immune check point inhibitors Howitt et al (JAMA Oncol 2015) - Retrospective IHC analysis on 63 ECs specimens - 1 st study to confirm the association between POLE/MSI and neoantigen load/lymphocyte count Predicted neoantigen load proportional to mutation load (POLE > MSI > MSS) TIL count POLE & MSI > MSS POLE-mutated and MSI EC : excellent candidate for immunotherapies targeting PD-1 pathway

Phase 2 in 41 metastatic carcinoma with or without MMR-deficiency Pembrolizumab 10mg/kg IV every 2 wks 1 ST study to evaluate the efficacy of immune checkpoint inhibitor in EC Response to PD-1

15 Phase 2 in 41 metastatic carcinoma with or without MMR-deficiency Pembrolizumab 10mg/kg IV every 2 wks 1 ST study to evaluate the efficacy of immune checkpoint inhibitor in EC Response to PD-1 blockade irorr and irpfs 40% and 78% in MMR-def colorectal cancer (vs 0% and 11% in MMR-prof) MMR-def tumor > MMR-prof irorr and irpfs 71% and 67% in MMR-def non-colorectal cancer (2 ECs with MSI-H included 1 CR and 1 PR) NEJM 2015;372(26):

Frequency of POLE EDM and MSI-H in recurrent EC : 2% (POLE) and 24% (MSI) Patient #1 Mixed EC (clear/endometrioid), FIGO IIIA Heavily pretreated state chemotx (cisplatindoxorubicin-paclitaxel,

infiltrate Weak PD-L1 expression in 5% of tumor cells Nivolumab 3mg/kg every 2 weeks Partial response on CT scan Patient #2 UPSC, FIGO IIIC2 Pre-treatment 3M post-treatment 7M post-treatment Heavily

16 Frequency of POLE EDM and MSI-H in recurrent EC : 2% (POLE) and 24% (MSI) Patient #1 Mixed EC (clear/endometrioid), FIGO IIIA Heavily pretreated state chemotx (cisplatindoxorubicin-paclitaxel, paclitaxel-carbo, single pacliataxel, RTx, 2ndary debulking, HTx (megace), Target Tx (cediranib, bevacizumab) NGS & MSI test POLE mut & MSS tumor Moderate amount of peri- and intratumoral T cell infiltrate Weak PD-L1 expression in 5% of tumor cells Nivolumab 3mg/kg every 2 weeks Partial response on CT scan Patient #2 UPSC, FIGO IIIC2 Pre-treatment 3M post-treatment 7M post-treatment Heavily pretreated state chemotx (docetaxelcarbo, doxorubicin single, dd paclitaxel), RTx NGS & MSI test MSI-high tumor based on MSH6 mutation Partial response on CT scan (at 3M after treatment continued at 9M) p53 wild-type, moderate amount of peritumoral T cell infiltrate, PD-L1 expression in 20% of peri- and intratumor T cell, not in tumor cell Nivolumab 3mg/kg every 2 weeks Clin Cancer Res 2016;22(23):5682-7

Unresectable or metastatic, MSI-H or MMR-d solid tumors that progressed following prior treatment 2017. 5. 23.

17 Unresectable or metastatic, MSI-H or MMR-d solid tumors that progressed following prior treatment KEYNOTE-016/-164/-012/-028/-158 STUDY (n=149/ec=14) Pembrolizumab : 200mg Q3W or 10mg/kg Q2W ORR: 39.6% (CR: 7.4%, PR: 32.2%, CRC: 36%, Other cancer: 46%)

18 Pembrolizumab (Keytruda ) 사전신청요법 암질환심의위원회에서신청기관에국한하여인정된요법 참고 : 새로이인정된요법의경우파란색으로표시하였음. 요법코드암종세부암종항암화학요법투여대상 4002 위암 - pembrolizumab 진행성위선암또는위식도접합부선암 3 차이상 4003 비호지킨림프종 NK/T-cell lymphoma pembrolizumab 이전요법에불응하는 NK/T-cell lymphoma 2 차이상 4006 직결장암 - pembrolizumab 4008 연부조직육종 - pembrolizumab 4010 비호지킨림프종 (PMLBCL) primary mediastinal B cell lymphoma pembrolizumab 4012 기타암흉선암 pembrolizumab 4013 기타암 - pembrolizumab 4014 간담도암담도 / 담낭암 pembrolizumab 4015 자궁경부암 - pembrolizumab 4016 기타암 악성흉막중피종악성복막중피종 pembrolizumab 4017 두경부암침샘암 pembrolizumab 4019 두경부암비인두암 pembrolizumab MMR-d(Mismatch repair - deficient) 또는 MSI-H(Microsatellite Instability- High) 직결장암 18세이상절제불가능한재발성또는전이성 UPS(undifferentiated pleomorphic sarco ma), DDLPS(dedifferentiated liposarcoma) 만 18세이상재발성및불응성의 primary mediastinal large B cell lymphoma 백금기반항암요법에진행한 invasive thymoma, thymic carcinoma MMR-d(Mismatch repair - deficient) 또는 MSI-H(Microsatellite Instability- High) 고형암 ( 직결장암제외 ) 표준치료에실패하였거나표준치료가적합하지않은 PD-L1-positive 진행성담도암, 담낭암 ( 바터팽대부암제외 ) 진행성자궁경부편평상피세포암 (advanced cervical squamous cell cancer) pemetrexed/platinum 요법에진행한악성중피종 ( 흉막 / 복막 ) 18세이상의절제불가능한전이성침샘암환자 (ECOG 0-1, PD-L1 >1% 을모두만족하는경우 ) 편평상피세포암을제외한비인두암 (PD-L1 positive인경우 ) 투여단계 3 차이상 2 차이상 2 차이상 2 차이상 2 차이상 2 차이상 2 차이상 2 차이상 2 차이상 2 차이상 2018 년 4 월 16 일현재총 289 개요법인정중 투여요법급여상세사항기타사항 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 고식적요법 (palliative) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약값전액본인부담 (100/100) 약가 : 1 회 (2 vials) 투여기준 5,692,220 원.

easy to perform, inexpensive method than TP53 mutation analysis p53 IHC - Surrogate

19 p53 In TCGA frequent TP53 mutation in copy-number high group Increased TP53 somatic mutation in uterine serous carcinoma (as HGSOC) Poor clinical outcome p53 IHC quick, easy to perform, inexpensive method than TP53 mutation analysis p53 IHC - Surrogate marker for TP53 mutation? YES - Favorable accuracy (sensitivity 0.96, specificity 1.00)

20 L1CAM (L1 cell adhesion molecule) 200 to 220 kda membrane glycoprotein of immunoglobulin superfamily, involved in neurogenesis Expressed in variety of tumors, associated with poor clinical outcome (tumor cell motility - invasion, metastasis) Expert Opin Biol Ther 2008;8(11):

21 ENITEC collaboration study (Br J Cancer 2016) L1CAM exp 10% in stage I EEC, 18% in advanced stage EEC, 75% in non-eec Associated with advanced stage, higher grade, non-eec, LVSI, LN metastasis, distant recurrences Strong predictor of reduced survival for EEC, not non-eec Analysis of TCGA (GO 2016) High L1CAM advanced stage, higher grade, serous histology, positive cytology, positive pelvic and para-aortic LN Independent prognostic factor for reduced OS Association with p53 expression in high-risk EC (Mod Pathol 2016) Significant association between p53-mutant tumors and diffuse L1CAM expression in total cohort and non-eec cohort L1CAM : not a TP53 mutation proxy p53-independent activating pathway such as β-catenin/wnt signaling and TGF-β1 L1CAM expression in curettage sample (Br J Cancer 2017) Highly significant correlation of L1CAM expression between in curettage and final hysterectomy specimen L1CAM expression in curettage LN metastasis

22 Application of TCGA into real world practice TCGA is advantageous in more accurate risk-stratification, BUT not readily applicable in the real world practice Limitations Too costly, complex and unsuitable for widespread implementation into routine clinical practice Exome sequencing Whole genome sequencing RNA sequencing mirna sequencing Reverse phase protein arrays DNA methylation Copy number analysis Using fresh frozen tumor specimens (rather than FFPE) Post-TCGA, more pragmatic approaches that can be readily applied to current pathology practice ProMisE/Vancouver model Leiden/TransPORTEC model

23 ProMisE/Vancouver group molecular classification (Proactive Molecular Risk classification tool for Endometrial cancers) Patients 143 ECs FFPE materials from Vancouver General Hospital cases banked in OVCARE tissue bank (all histotypes, stages and grades included) Aim to design simple, lower cost, molecular-based classification methodologies that can recover TCGA subtypes combined analysis with ESMO clinical risk groups or pathological parameters 1. POLE mutation status by sequencing (with or without PTEN) 2. MSI status by MMR IHC (MSI assay switched to MMR IHC) 3. CN status by p53 IHC (FISH, p53 IHC, TP53 sequencing) Total (N=143) MMR IHC abn 41 (29%) POLE EDM 12 (9%) p53 wt 63 (45%) p53 abn 25 (18%) Age (Mean) Stage II/III/IV 41% 0 19% 44% Grade 3 41% 42% 16% 84% Histology Endometrioid (85%) Endometrioid (92%) Endometrioid (97%) Serous/Mixed (60%) Positive LVSI 61% 48% 19% 56% Positive LN 24% 0 3% 24% Adj. RTx 59% 42% 27% 64% Br J Cancer 2015;113:

) prompt screening of LS referral for hereditary cancer center 25% of Low or intermediate risk group : p53 abn group

24 ProMisE/Vancouver group molecular classification (Proactive Molecular Risk classification tool for Endometrial cancers) Improved ability to discriminate EC outcomes when both traditional and molecular tools are used MMR IHC (1 st ) prompt screening of LS referral for hereditary cancer center 25% of Low or intermediate risk group : p53 abn group risk of under-treatment Almost 50% of POLE : High-risk group CTx or RTx under standard clinical care Br J Cancer 2015;113:

25 FFPE blocks from 57 patients Cohort subdivided into 4 predefined groups according to ProMisE model (MMR-D, POLE EDM, p53 wt, p53 abn) VS. Cohen s kappa (histotype and grade) = 0.44 and 0.7 Concordance between preoperative curettage and hysterectomy : molecular alteration is superior to grade and histotype Gynecol Oncol 2016;143:46-53

26 Leiden/TransPORTEC molecular classification 947 early-stage EECs FFPE samples from PORTEC-1 and -2 (n=614 with high-intermediate risk features) Aim to validate prognostic significance of molecular classification tool to confirm efficacy of integration of clinicopathologic and molecular risk factors Total (N=834) P53-mut 74 (9%) MSI 219 (26%) POLE-mut 49 (6%) NSMP 492 (59%) Age <60 7 (9.5%) 35 (16%) 19 (38.8%) 77 (15.7%) Grade 3 26 (35.1%) 36 (16.4%) 13 (26.6%) 35 (7.1%) MI>50% 39 (52.7%) 148 (67.6%) 24 (49%) 372 (75.6%) Substantial LVSI 4 (5.4%) 19 (8.9%) 0 14 (2.9%) Treatment Observation 17 (23%) 63 (28.8%) 16 (32.7%) 145 (29.5%) EBRT 38 (51.3%) 113 (51.6%) 25 (51%) 233 (47.4%) VBT 19 (25.7%) 43 (19.6%) 8 (16.3%) 114 (23.1%) L1 CAM : p53-mut > NSMP > POLE-mut > MSI β-catenin (CTNNB1 mut): NSMP > MSI > p53-mut > POLE-mut 27 (3%) tumors - >1 alterations (p53, MSI or POLE) POLE with p53 no recurrence MSI with POLE or p53 or both 5 recurrences Clin Cancer Res 2016;22(16):

15% 50% 35% Improved risk assessment by integrating POLE,

More objective variable (mutational status) Identification of

for LS Unsolved issues Prospective confirmation Omission of RTx

27 15% 50% 35% Improved risk assessment by integrating POLE, L1CAM, MSI, p53, CTNNB1 with histopathologic factors Advantages More objective variable (mutational status) Identification of patients with more favorable prognosis Facilitate prescreening for LS Unsolved issues Prospective confirmation Omission of RTx in favorable prognosis group? Which subgroup is more sensitive to RTx?

28 Study begin : June 2016 Study end : June 2025

29 POLE, CTNNB1 : Sanger sequencing L1CAM, p53, MMR : IHC

30 Conclusions Paradigm shift in EC pathology practice Conventional Pathological classification Genomic/proteomic/ transcriptomic characterization (TCGA) New pragmatic methods (Integration of molecular classification into conventional pathological classification) Tailoring adjuvant treatment (reduce under- or over-treatment) Form the basis for future prospective studies Impact in the pre-operative setting (high concordance between curettage and hysterectomy specimen) Extent of surgery lymphadenectomy : can be omitted when high-grade EEC with POLE mutation aggressive surgery including lymphadenectomy and omentectomy: p53-aberrant EC Urgency of surgery young women with POLE mutation or p53-wt EC : hormone therapy and delay hysterectomy Impact on adjuvant treatment Mainly in high-intermediate risk EC (PORTEC-4a) Cost-effective analysis (cost of molecular testing vs. saving cost of adjuvant RTx) Impact on surveillance Tailored surveillance p53-aberrant EC : more close observation than POLE mutated EC Impact in recurrent disease Immune checkpoint inhibitor in MMR-d and POLE mutated EC

31 Thank you for your attention!

Case presentation 04/13/2017. Genomic/morphological classification of endometrial carcinoma

Case presentation 04/13/2017. Genomic/morphological classification of endometrial carcinoma Genomic/morphological classification of endometrial carcinoma Robert A. Soslow, MD soslowr@mskcc.org architecture.about.com Case presentation 49 year old woman with vaginal bleeding Underwent endometrial