Systematic review on inmunotherapy for POLE ultramutated and MSI-H advanced endometrial carcinoma

Héctor Villa Martínez TITOR: Regueiro García, Benito COTITORA: Villanueva Silva, María José Departamento titor: Microbioloxía (CHUVI) Departamento cotitora: Oncoloxía (CHUVI) Curso académico: 2019-2020 Convocatoria: 1ª AUTOR: HÉCTOR VILLA MARTÍNEZ TITOR: Regueiro García, Benito COTITORA: Villanueva Silva, María José Departamento titor: Microbioloxía (CHUVI) Departamento cotitora: Oncoloxía (CHUVI) Curso académico: 2019-2020 Convocatoria: 1ª AUTOR: HÉCTOR VILLA MARTÍNEZ Héctor Villa Martínez ABSTRACT Key Words: Endometrial cancer, POLE ultramutated, microsatellite instability (MSI), Mismatch-Repair Deficiency, immunotherapy, checkpoint inhibitors. BACKGROUND: Recent advances in molecular biology have enabled identification of microsatellite instability high (MSI-H), DNA polymerase epsilon mutation (POLE) as two of four endometrial carcinoma (EC) subtypes, plausibly amenable to treatment with immune checkpoint inhibitors (ICI). OBTECTIVE: To perform a systematic review of all available evidence with ICI in MSIH and POLE advanced EC subtypes to assess efficacy and tolerability. MATERIAL AND METHODS: An electronic search of clinical trials with MSI-H and POLE advanced EC, published as a research article or in abstract form between 2010-2020, was performed. No language restrictions were applied. A predefined PROPERO-LIKE was designed in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Population was defined as POLE, mismatch repair deficiency (MMRd), MSI-H advanced EC; intervention as ICI, anti-PD1, Anti PDL1, PD1/PDL1 axis, immunotherapy, clinical trial. Response rate (RR), clinical benefit rate (CBR) progression free survival (PFS) and overall survival (OS), where available, as well as adverse events were assessed. RESULTS: The search identified 573 records. After screening phase and eligibility process, eleven phase I or II trials were included for final analysis, with 9 monotherapy trials (140 MSI-H patients in total, 1 POLE, 113 MSI-H/MMRd evaluable) and 2 in combination therapy trials (9 MSI-H/MMRd patients, 0 POLE). A pooled analysis in monotherapy MMRd/MSI-H patients yielded 48% RR (10% complete response; 38% partial response, some long lasting), with 57% CBR. Lack of results on PFS or OS in most trials preclude a pooled analysis on survival. Adverse events were as expected. CONCLUSION: ICI showed promising activity in MSI-H advanced EC, some of them long lasting. PFS and OS results are pending. Future investigation is needed to establish the role of these agents in monotherapy and/or in combination. Héctor Villa Martínez 2 INDEX 1. Background 2. Objective 3. Material and methods 4. Search strategy 5. Trial selection 6. Data extraction 7. Results 7.1. Monotherapies 7.2. Combination therapies 7.3. Side effects associated to immune checkpoint inhibitor therapy 8. Discussion 9. Conclusion 10. References 11. Appendix 11.1. PROSPERO-LIKE Protocol Immunotherapy in endometrial carcinoma 3 BACKGROUND Uterine cancer is the most common gynecologic cancer in developed countries, of which endometrial carcinomas account for >90% of cases (1). Most cases are detected at an early stage (2). With a median age at diagnosis of 60 years, and closely related to elevated body mass index, five-year survival rate is over 95% in those tumors confined to the uterus at diagnose (1), which they account for 85% of cases, plummeting to 17% in patients with distant metastatic disease (1). Figure 1. SEER 18 2010-2016, All Races, Female Summary Stage 2000 In 1983 a first endometrial carcinoma subtype classification, based on clinical and hormonal features, was proposed by Bokhman, distinguishing between type I endometrioid (well and poorly differentiated) and type II (including serous, clear cell, carcinosarcoma, dedifferentiated and undifferentiated subtypes) (3). Recent advances in molecular biology, mainly in genome-wide analyses, have revealed a wide range of genomic alterations in endometrial carcinomas, providing valuable insight into the pathogenesis of these tumors. As thus, The Cancer Genome Atlas classification delineated in 2013 four subgroups groups of endometrial cancer (EC) as follows: microsatellite instability high (MSI-H), DNA polymerase epsilon (POLE), copy number high, and copy number low (4). Carcinomas with a hotspot mutation of the catalytic unit DNA polymerase epsilon (POLE), the one in charge of nuclear DNA replication and repair, display an ultramutated sequence (5). This sequence is recognizable to the immune system, and confers a good prognosis, among high-risk early endometrial cancer (6)(7)(5)(8). Likewise, MSI high tumors are hypermutated due to their inability to repair replicational mutations related to alteration of MLH1, MSH2, MSH6, PMS2 genes, developing characteristic microsatellite repeats which make these tumors immunologically active and susceptible to treatment with immunotherapy (IT) (9)(10). Héctor Villa Martínez 4 Figure 2. TCGA MOLECULAR CLASSIFICATION Kandoth C. et al. Nature 2013: 497, 67-73 Most endometrial cancers (EC) are detected at early stages and cured by local treatments, either surgery, radiotherapy or both. Upon recurrence, the prognostic is dismal, with no cure possible with current systemic treatments and short median survival time is expected (5). Even though the two higher mutational subtypes, (POLE)-ultra-mutated and microsatellite instability-hyper-mutated (MSI-H), usually convey a better prognosis, life expectancy is short upon recurrence. Previous studies have evidenced that MSI-H and POLE endometrial carcinoma exhibit a high mutational rate and neoantigen load, increased tumor infiltrating lymphocytes (TILs) as well as high expression of PDL1, rationale for a potential activity of checkpoint inhibitors (11) (12) (13). Neoantigens are presented to inactive T-cells by antigen-presenting-cells (APC, such as dendritic cells or macrophages) through interaction of the major histocompatibility complex (MHC) and T-cell receptors, the so-called priming phase, i.e. primary signal for T-cell activation. To fully achieve lymphocyte activation, another costimulatory signal is required, through interaction between B7 on APCs and CD28 on T-cells (14). In order to preclude indefinite T-cell activation (which would otherwise implicate enhance autoimmunity), a coinhibitory signal, known as Checkpoint 1, stops lymphocyte stimulus, once adequate activation is achieved. As such, CTLA-4 is produced in T-cells and transported to the cell surface in a proportional way to antigen stimulation; it binds to B7 with greater affinity than CD28, resulting in specific T-cell inactivation (checkpoint 1)(15). Once activated, activated T-cells move to peripheral tissues to eradicate neoantigenharboring cells, i.e. tumor cells, the so-called effector phase. Mission accomplished, activated T cells undergo apoptosis through programmed cell death receptor (PD-1) activation (Checkpoint 2, at peripheral tissue level). Tumor cells can express PDL-1 (PD-1 ligand) to prevent T-cell attack (15). Immunotherapy in endometrial carcinoma 5 Immune checkpoint blockage through CTLA-4 (checkpoint 1 inhibitors) or PD1/PD1-L1 (checkpoint 2 inhibitors), by means of monoclonal antibodies, increase cytotoxic T-cell activity, resulting in both, increased tumor response and immune-related adverse events. Pembrolizumab, a checkpoint 2, anti-PD1 inhibitor has been proved to have clinical utility in MSI tumors regardless tumor type, granting the first Food and Drug Administration (FDA) drug approval relying on molecular diagnosis alone, based on NCT0187651 trial (16). In endometrial cancer, with MSI high tumors prevalence being as high as 40%, according to The Cancer Genome Atlas data (only 2% in serous tumors), this indication could be of the upmost importance, even though acknowledging the fact that only two endometrial MSI High where included in the aforementioned trial (6). Figure 3. NEOANTIGEN RELEASE BY MSI-H TUMOR OF AGNOSTIC ORIGIN OBJECTIVE To perform a systematic review in order to identify and summarize all available evidence with immune checkpoints inhibitors in both mismatch deficient highly mutated and POLE-ultramutated advanced endometrial carcinoma, to assess efficacy and tolerability. Héctor Villa Martínez 6 MATERIAL AND METHODS Inclusion criteria were clinical trials with highly mutated and ultramutated endometrial carcinoma, published as a research article or in abstract form between 2010 and 2019. No language restrictions were applied (Table 1). A predefined protocol PROSPERO-like was designed and subsequently followed in accordance with the PRISMA guideline for systematic review (see protocol on Appendix 1). Population was defined as Pole-ultramutated, mismatch deficient, highly mutated or MSI-H endometrial carcinoma. Intervention was defined as checkpoint inhibitor, anti-PD1, Anti PDL1. PD1/PDL1 axis, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, immunotherapy, in clinical trial. Pooled response rate (RR) and clinical benefit rate (CBR) were estimated as outcomes as were progression free survival (PFS) and overall survival (OS), duration of response (DOR) and time to response (TTR), where available as well as adverse events. SEARCH STRATEGY An comprehensive electronically search was performed in PubMed, Embase, Clinicaltrials.Gov, Web of Science for articles as well as ASCO and ESMO meeting database for abstracts reporting the use of immunotherapy in advanced/recurrent endometrial carcinoma (Table 1), using a combination of broad terms related to endometrial carcinoma and immunotherapy, checkpoint inhibitors, POLE*, MSI*, clinical trial. A thorough manual search in the reference section of the retrieved articles as well as in the appendix was also conducted. Inclusion criteria were clinical trials trial with both subtypes (POLE and MSI) between January 2010 and March 2020. Trial selection was performed by author of the present graduation work with double check with the librarian, supervised by the cotutor. There was no restriction about the language used in the publications. TRIAL SELECTION Clinical trials were selected on the basis of advanced endometrial carcinoma focusing on or including POLE mutated and/or MSH-High subgroups, treated with immunotherapy directed to PD1 and PD-L1. Therefore, the terms applied in the search include checkpoint inhibitors, POLE, MSI-H, recurrent or advanced endometrial carcinoma, immunotherapy, clinical trial, PD1, PDL1, Pembrolizumab, Nivolumab, Atezolizumab, Avelumab, Durvalumab. No restriction on type or number previous line was applied. Ongoing studied with published results were included as well. Apart from monotherapy trials, combination therapies studies were included, although evaluated in a separate section. In addition, endometrial carcinomas whose molecular characteristics were mismatch repair deficient (MMRd) were considered as highly mutated (MSI-H) and finally included. Immunotherapy in endometrial carcinoma 7 Table 1. Search criteria for the selection process Exclusion criteria were: (i) patients with no advanced/recurrent endometrial carcinoma, (ii) clinical trials with no POLE or MSI subtype tumors reported, (iii) studies that matched different databases, (iv) completed trials with no published results, (v) ongoing trials with no published results, (vi) phase III trials, (vii) case reports, narrative reviews, editorials, news articles, commentaries or letters. In the event of multiple publications reporting the same trial, only the most recent data were considered. DATA EXTRACTION The studies retrieved during the search were screened for relevance. Those defined as being potentially eligible were fully evaluated to find out if they met the requirements for inclusion criteria. They were accepted or rejected based on the predefined inclusion and exclusion criteria. For each included study the following information was extracted: number of patients enrolled, design, type of trial, age, stage, performance status, sample size, treatment type, type of immunotherapy delivered, other drugs if applied, median follow-up, response type according to RECIST/iRECIST criteria (17) (complete response, partial response, stable disease, progression), overall response rate (ORR, including partial response plus complete response), clinical benefit rate (complete response plus partial response plus long lasting stable disease), time to progression (TTR, defined as the time elapsed from the first cycle to response demonstration), duration of the response (DOR, defined as the time elapsed from the first response documentation of partial or complete response to progression, last follow-up or death, Type of study Immunotherapy Clinical trial Condition or domain being studied Advanced/recurrent endometrial carcinoma Participants/Population Adult population (>18 years of age) included in population-based studies Timeline criteria Studies published between January 2010 and March 2020 Linguistic criteria Any language Héctor Villa Martínez 8 whichever came first), progression free survival (PFS), defined as the time elapsed from the first cycle to progression, last follow-up or death, whichever came first, overall survival (OS), defined as the time elapsed from the inclusion in the study to death or last follow-up), and toxicity. Adverse Events (AEs) were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0. RESULTS A search on three major medical and scientific databases articles and oncology meeting abstracts yielded 573 records, through database searching. After identification, 502 were rejected, 481 based on title, and 21 for being case reports, narrative reviews, editorials or letters. Of the 71 remaining for screening, 47 were excluded either based on abstract (20), because of being an ongoing trial without efficacy results available (22), or for duplication (5). Twentyfive trials were assessed for eligibility, of which 14 were rejected because no on mutated or ultramutated endometrial subtype were included, leaving a total of 11 phase I or II trials on immune checkpoint inhibitors dealing with advanced endometrial carcinoma and reporting data on survival (overall or progression-free survival), tumor response, or adverse events, meeting the inclusion criteria and included in this systematic review for final analysis (Figure 4). In 2015, Le and colleagues published a phase II study showing, for the first time, efficacy of immune checkpoint blockade with pembrolizumab in different treatment-refractory progressive cancer types with a mismatch-repair deficiency (MMRd), as compared with mismatch-repair proficient (MMRp) colorectal patients (16). Coprimary end points were the 20-week immune-related progression-free survival rate and the immune-related objective response rate. The MMR proficient group showed no response as compared with 40% objective response rate in the MMRd colorectal group (95% CI 12-74) and 71% (95% CI 29-96). Amongst MMRd patient non-colorectal cases (7 evaluable for response/9 recruited), 2 were endometrial carcinoma showing 1 complete response and 1 partial response by RECIST (Appendix 2 of the trial publication). Median time to response was 12 weeks in the latter group. Moreover, after a median follow up of 21 weeks, median PFS was 5.4 months, with median OS not reached in MMR deficient (MMRd) non colorectal cancer patients, two of which were endometrial cancer patients (2/7) (16). Following those results, on May 23 2017, the Food and Drug Administration (FDA) granted accelerated approval to pembrolizumab for tumor-agnostic treatment, i.e. regardless of solid tumor tissue/site of origin, provided they are metastatic or unresectable, they express microsatellite instability-high (MSI-H) or DNA mismatch repair deficiency (MMRd), and they have progressed to at least one previous line and have no satisfactory treatment alternative [FDA home page]. Based on previously reported programmed death ligand 1 (PDL-1) positivity as a plausible predictor of response to immune checkpoint inhibitors in solid tumors (16), a multicohort phase Ib basket trial was launched (KEYNOTE-028) to evaluate safety and efficacy of pembrolizumab in advanced solid tumors with PDL1 positivity, the results from the advanced endometrial cancer cohort being released in 2017 (18). Skilled patients were treated with Immunotherapy in endometrial carcinoma 15 endpoint, overall population type, MSI-H/POLE cohort, and results, above all ORR, and PFS and OS, when available. Table 3 displays the retrieved results on efficacy in terms of response within MSIH/MMRd EC population included in the 11 trials phase I/II prospective trials with monotherapy immune checkpoint inhibitors resulting in an overall response rate (ORR, Complete response + Partial response, by RECIST 1.1) of 48% in MSI-H/MMRd population. AntipPD-1 therapy resulted in an overall response rate of 53.85% (35/65) in MSI-H/ MMRd cohort. AntiPDL1 therapy resulted in an overall response rate of 39.6% (19/48). With a total study population of 141 patients (140 MSI-H/MMRd and 1 POLE), and 113 MSI-H assessed for response per RECIST 1.1, a pooled analyses was performed for response rate (Figure 4) as well as for clinical benefit rate (Figure 5) in MSI-H endometrial cancer included in the 11 aforementioned checkpoint inhibitors monotherapy trials. COMBINATION THERAPIES Makker et al. presented at 2018 ASCO annual meeting the results of a multicentre, openlabel, single-arm, phase I/II trial (NCT02501096) in patients with advanced metastatic endometrial cancer (with a maximum of 2 previous lines in the phase II part), irrespective of microsatellite instability (MSI) or mismatch repair (MMR) status, treated with pembrolizumab in combination with lenvatinib, a multikinase inhibitor with antiangiogenic activity. Pembrolizumab 200 mg was administered intravenously every 3 weeks, and 20 mg oral lenvatinib daily. The results were later published in The Lancet Oncology in 2019 (28). The primary outcome was the objective response rate at week 24 according to irRECIST. Secondary endpoints were OS, DOR and PFS assessed by investigators. The median follow-up time was 13.3 months. The study enrolled 54 patients, the most common subtypes were endometroid and serous carcinoma. 53% had received two o more treatment lines (43% and 13%, respectively). Regarding microsatellite status, 4 patients (8%) were MSI-H, 45 cases (85%) were MSS and 4 (8%), unknown. (28) After evaluation, 53 patients were assessed, 21 patients responded, accounting for an ORR 39.6% (CI 95%: 26.5-54.0) at week 24, including 1 complete response and 20 partial responses assessed by investigators. After independent review, 3 complete responses (5.7%) and 22 partial response (41.5%) were identified ORR (47.5%). Twenty-two patients had stable disease (35.8%), 5 progressed (9.4%) and 4 (7.5%) were unknown/not assessable Héctor Villa Martínez Trial, references Trial type Agent Primary endpoint Secondary endpoint Overall population type MSI-H/POLE EC Results Le et al Phase II, MMR status stratification. 3 cohorts: A: colon MMRp; B: colon MMRd C: non-colon MMRd Pembrolizumab IrPFS, IrORR OS, ORR, DCR Colon MMR stratification and noncolon MMRd (2 EC) 2 MSI-H ORR: 100% Otte et al/Keynote 0-28 Multicohort phase Ib basket trial Pembrolizumab ORR PFS, OS, DOR PDL-1 positive advanced solid tumors (24 EC) 1 POLE (Retrospectively assessed in responders, 3/23) ORR;100% (CI 95%), PFS 14 months + Fader et al. Multicohort phase II basket trial Pembrolizumab ORR NA MMRd solid tumors 9 MMRd ORR: 56 % (CI 95%), OS at 12 months: 89%, OS NR Tamura et al. Multicohort phase II basket trial Nivolumab ORR PFS, OS, DOR Uterine corpus cancer, cervical cancer, STS 2 MSI-H Retrospectively assessed in 8/23 (2MSI-H; 6MSS) ORR 100% (CI 80%); OS6: 100 % Azad et al. Z1D subprotocol of NCIMATCH (EAY131) Multicohort phase II basket trial Nivolumab ORR PFS6, time to progression, toxicity MMRd non-colorectal cancer (13 EC) 13 MMRd 11 MMRd evaluable ORR: 45% (CI 90%), F. Liu et al. Multicohort phase Ia basket trial Atezolizumab Safety and tolerability ORR, PFS, DOR. Exploratory: OS, Biomarkers Solid or hematologic malignancies 1 MSI-H Biomarkers: PDL-1, MSI status, TMB ORR 100% (CI 95%); OS 9.5 months (CI 95%), Konstantinopoulos et al. Phase II umbrella trial Avelumab PFS6 DOR PFS, OS TRAEs, IrORR EC 2 COHORTS MMRd/POLE-MMRp 15 MMRd 0 POLE ORR: 26.7 % (CI 95%); CBR: 40% PFS6: 40% (CI 95%) Antill et al. PHAEDRA trial Phase II umbrella trial Durvalumab ORR IRECIST DCR16w IrAE MMRd or MMRp EC 35 MMRd ORR: 40% (CI 95%), DCR16w: 60% (CI 95%) Oaknin et al. Multicohort phase I-II basket trial TSR-042 (Dostarlimab) ORR, DOR (Part 2B) ORR, DOR, disease control rate, IrORR Solid tumors Part 2B: EC Cohort A1: MMRd/MSI-H Cohort A2: MMRp/MSS 65 MSI-H ORR: 48.8% (CI 95%), CBR: clinical benefit rate; DCR16w: Disease control rate at 16 weeks; Duration of response; EC: endometrial cancer; IrAE: immune related adverse event; IrORR: Immune related objective response rate; IrPFS: Immune related progression free survival MMRd: Mismatch repair deficiency; MMRp: Mismatch repair proficient; MSI-H: Microsatellite instability high; NR: not reached; DOR: ORR: Objective response rate; OS: Overall survival; OS6: Overall survival at 6 months; PFS 6: PFS at 6 months; PFS: Progression free survival; STS: Soft tissue sarcoma; TRAEs: Treatment related adverse events. Table 2. Clinical trial including MSH-I/MMRd patients treated with checkpoint inhibitors 2 monotherapy Héctor Villa Martínez Table 3. POOLED ANALYSIS IN MSI-H EC WITH CHECKPOINT INHIBITORS IN THE ASSESSED POPULATION Among the 4 MSI-H patients, 3 had stable disease, 2 of them ongoing at 30 and 90 weeks, and one progressed, accounting for an ORR of 75% (28). Median progression-free survival was 7.4 months (95%, CI: 5.0-Not estimable). DOR of at least 6 months was present in 11 patients 79.3 % (95% CI, 48.5-92.9), and 8 patients maintained response at 12 months, 79.3 % (95% CI) by independent review. As far as treatment related adverse events, are concerned, fifty patients (94%) experienced some grade of AE, with 36 cases presenting grade 3 treatment-related adverse events (68%) and no grade 4 were registered. Five patients (9%) discontinued the study because of TRAEs, 2 because of grade 3 acute renal failure (one of them associated with grade 2 ischemic colitis), one grade 3 hypertransaminasemia and 1 due to the only grade 5 toxicity in the study, an intracranial hemorrhage damned related to treatment (28). Trial, references Agent MSI-H/ POLE cohort ORR CR PR SD LG PD CBR Le et al Pembrolizumab 2 MSI-H 100% (2/2) 1 1 - - - Keynote 028 Pembrolizumab* 1 POLE 100% (1/1) - 1 - - - Fader et al Pembrolizumab* 9 MMRd 56 % (5/9) 1 4 3 (3) 1 Tamura et al Nivolumab* 2 MSI-H 100% (2/2) - 2 - - - Azad et al Nivolumab* 11 MMRd 55% (6/11) 2 4 1 - 4 F. Liu et al Atezolizumab ‡ 1 MSI-H 100% (1/1) - 1 - - - Konstantinopoulo s et al Avelumab ‡ 15 MMRd (12 evaluated) 33% (4/12) 1 3 4 (2) 4 Antill et al Durvalumab ‡ 35 MMRd 40% (14/35) 4 10 7 (7) 14 Oaknin et al TSR-042* 65 MMRd; (41 evaluated) 48.8% (20/41) 2 18 6 - 15 Total 1 POLE 140 MSIH/MMRd (113 with results available) ORR MSI-H: 54/113: 47.79% 11 (9.7 3%) 43 (38.05%) 22 (19,47%) (12) (10,62%) 38 (33.63%) 62/113 (58.4%) *: Anti-PD1 therapy; ‡: Anti PD-L1 therapy; CBR: clinical benefit rate (CR+PR + LG); CR: Complete response; LG: long lasting stable disease; MMRd: Mismatch repair deficiency; MSI-H: Microsatellite instability high; ORR: Objective response rate; PD: Progression disease; PR: Partial response; SD: Stable disease. Héctor Villa Martínez 18 Figure 4. POOLED ANALYSIS OF RESPONSE RATE IN MSI-H EC WITH CHECKPOINT INHIBITORS-2 MONOTHERAPY Figure 5. POOLED ANALYSIS OF CLINICAL BENEFIT RATE IN MSI-H EC WITH CHECKPOINT INHIBITORS-2 MONOTHERAPY 10% 38% 19% 34% Complete reponse Partial response Stable disease Progression disease 10% 38% 10% 9% 34% Complete response Partial response Long lasting stable disease Stable diase Progression disease Immunotherapy in endometrial carcinoma 19 Overall, the most frequent adverse events were hypertension (58%), fatigue (55%), diarrhea (51%), and hypothyroidism (47%) (28). Rubinstein et al presented preliminary results of a randomized phase II trial of Durvalumab (D), a PDL1 inhibitor, with or without Tremelimumab (T), a cytotoxic T-lymphocyteassociated protein 4 (CTLA-4) inhibitor in advanced endometrial carcinoma and endometrial carcinosarcoma (ClinicalTrials.gov number, NCT03277482) at 2019 ASCO annual meeting. Patients were randomly assigned to receive D 1500 mg intravenously (IV) every 4 weeks in monotherapy or in association with T 75 mg IV every 4 weeks for 4 cycles, followed by D1500 mg intravenously every 4 weeks (29). The primary outcome was objective response rate (ORR) by RECIST 1.1. Fifty-six patients (28 per arm) were enrolled at planned interim analysis, with two patients excluded per evaluation (one on each arm) due to early death. There were 15 patients with endometrioid histology, 15 with carcinosarcoma, 14 with serous subtype and 12 patients with another histology (29). Most tumors were MSS (48 patients, 86%). There were 5 MSI-H cases (9%), and the remaining 3 (5%) were unknown. In the monotherapy group, 3 patients obtained partial response (2 MSS, 1 MSI-H) and 1 patient had a complete response (MSS), with an ORR of 14.8% (CI: 6,6-100%). The median PFS in the monotherapy arm was 7.6 weeks and PFS at 24 weeks 13.3% (CI 6.1-100%). Median duration of response (DOR) was 16 weeks. (29) In the combination treatment arm, 2 patients obtained CR (1: MSI-H, 1: MSS) and 1 achieved PR (MSS). The ORR was 11.1% (CI: 4.2-100%). Median PFS was 8.1 weeks, and PFS at 24 weeks was 18.5% (CI 10.1-100%). DOR was 8 weeks. Thus, of the 5 MSI-H patients, one obtained a partial response (1/5, 20%) and another achieved a complete response (1/5, 20%), accounting for an ORR in the MSI-H population of 40% (2/5). Grade 3 and 4 TRAEs were more common in the combination arm than in the monotherapy arm, namely 7% vs 32% and 4% vs 11%, respectively. Two patients discontinued treatment due to a TRAE. Most common AEs related to treatment were diarrhea (20%) fatigue (23%), nausea (14%), pruritis (11%) and vomiting (13%) (29) Table 4. Clinical trial including MSH-I/MMRd patients treated with combination therapies Trial, references Trial type Agent Primary endpoint Overall population type MSIH/POLE EC Results Makker et al Multicohort phase I/II basket trial Pembrolizumab + Lenvatinib ORR Advanced EC 45 MSS, 4 MSI-H 75% (CI 95%), ORR Rubinstein et al Phase II umbrella trial Durvalumab + Tremelimumab ORR Persistent or recurrent EC 48 MSS, 5 MSI-H 40% (CI 90%), ORR CI: confidence interval; EC: endometrial carcinoma; MSI-H: Microsatellite instability high; MSS: Microsatellite stable; ORR: objective response rate; Héctor Villa Martínez 20 SIDE EFFECTS ASSOCITED WITH IMMUNE CHECKPOINT INHIBITOR THERAPY Immune checkpoint blockade has shown good benefit in the treatment of many types of cancer. It works blocking intrinsic down-regulators of immunity like CTLA-4 or programmed cell death (PD-1) or its ligand (PDL-1). These drugs increase the activity of the immune system. Immune checkpoint blockade can produce inflammatory side effects, witch of them are usually denominated immune-related adverse events. This side effects can occur in any organ system but gastrointestinal tract, skin, endocrine glands and liver are most commonly involved. (30). Although it is less frequent, cardiovascular system, central nervous system, pulmonary and hematologic systems can also be affected (30). Immune checkpoint inhibitors are not directed only to tumor-specific T cell, they can also activate non-tumor-specific responses because of the antigens expressed on non-tumor tissue. It would be an undesirable response. IrAE can occur in a variety of organs. Even though the clinical benefits of immune checkpoint inhibitor therapy, intolerable adverse effects can happen during the treatment. In single agent trials, the incidence of any grade adverse event is described to range from 15 to 90%. The rate of severe adverse events demanding withdrawal of treatment is 0.5-13%. (31). From all studies included in this systematic review, toxicities associated with each treatment have been extracted, as specified in the protocol. Eight single agent trials have been included in this systematic review (two studies of pembrolizumab, two of nivolumab, one of atezolizumab, one of durvalumab and an anti-PD-1 named TSR-042). Figures 6-11 show most common side effects and grades reported in the different trials grouped by agents, when available. Each diagram shows the most frequent adverse effects, as well the most severe, reported in the trials and grouped by drug. Immunotherapy in endometrial carcinoma 21 Figure 6. Side effects by frequency and grades, reported in pembrolizumab trials Figure 7. Side effects by frequency and grades, reported in nivolumab trials 0 1 2 3 4 5 Allergic rinithis Diarrhea Abdominal pain Pruritus Fatigue Linphopenia Anemia Asthenia 29% 24% 24% 21% 21% 20% 11% 4% 0 1 2 3 4 5 36% 17% 17% 13% 9% 9% 4% 4% Héctor Villa Martínez 22 Figure 8. Side effects by frequency and grades, reported in atezolizumab trial. Figure 9. Side effects by frequency and grades, reported in avelumab trial 0 1 2 3 4 5 Diarrhea Fatigue Colitis Rash Diarrhea 20% 13% 6.7% 6.7% 6.7% 0 1 2 3 4 5 35.5% 16.1% 12.9% 12.9% 9.7% 9.7% 6.5% 6.5% Immunotherapy in endometrial carcinoma 23 Figure 10. Side effects by frequency and grades, reported in dostarlimab, TSR 042 trial Figure 11. Side effects by frequency and grades, reported Pembrolizumab + Lenvatinib trial 0 1 2 3 4 5 14.4% 12.8& 12.0% 1.6% 1.6% 1.6% 0 1 2 3 4 5 49% 47% 43% 25% 34% 2% 1.8% Héctor Villa Martínez 24 DISCUSSION Firstly, described in familial Lynch syndrome, mismatch repair deficiency (MMRd), results from germline mutations located in one of four MMR genes (MLH1, MSH2, MSH6, or PMS2). Less frequently, it is caused by deletions in the vicinity of the MSH2 gene on chromosome 2p21, at the epithelial cell adhesion molecule–locus (EpCAM) (32). Since then, other genetic causes resulting in MMRd have been identified, mainly inherited mutations in DNA damage repair (DDR) genes such as the ATM mutation found in one response reporter in the Liu et al. study (22). In addition to germline mutation, somatic sporadic causes of dMMR/MSI-H tumors have also been recognized, mainly in colorectal cancer (CRC), at approximately a 15% prevalence, while acknowledging its existence at a lower prevalence through many common cancer types, including endometrial cancer (33). In these somatic non-germline cases, MMRd usually results from epigenetic hypermethylation of the MLH1 promoter or, unfrequently due to either somatic structural rearrangements or homozygous somatic mutations in an MMR gene. As a failure to repair mutations in short repetitive DNA sequences, the so-called microsatellites, Both, genetic or somatic causes of loss of function in a MMR gene give rise to hypermutation and high microsatellite instability (MSI-H) (33). Many patients across the retrieved trial in this systematic review presented MMRd with no associated mutations and plausibly due to somatic mutations. Mismatch repair pathways are so important identifying and repairing mismatched bases during DNA replication in normal and cancer cells. Defects in DNA mismatch repair proteins and following microsatellite instability-high causes the accumulation of mutation in cancer related genes. It supposes the generation of neoantigens that will stimulate the anti-tumor response of the host (34). In 2017, following the results of Pembrolizumab efficacy, with an ORR of 39.6%, with 78% of responses lasting over 6 months in refractory MMRd tumors (16). The FDA granted accelerated drug approval to a pembrolizumab for any advanced o recurrent solid tumor regardless of tissue or origin of the tumor, i.e. the first tumor-agnostic treatment authorization, provided they are dMMR/MSI-H tumors, with no satisfactory therapeutic options after first line (35). Apart from anecdotal cases reports showing activity of Checkpoint inhibitors in advanced/recurrent endometrial carcinoma (36), 11 phase I-II trials having reported preliminary results including some or focusing in MMRd/MSI-H, have being identified across this systematic review, showing meaningful long-term immunotherapy-related responses in dMMR or MSI-H EC. However, aside from Le and colleagues’ trial, focused on MMRd colorectal and noncolorectal tumors, which gave raised to FDA approval, only 5 monotherapy trials were specifically designed to evaluate the activity of immune-checkpoint monotherapy in MMRd tumors, with only three of them with specific endometrial cancer cohorts. In an additional trial, MMRd was assessed as a biomarker, along with PDL-1 and TMB, as a predefined exploratory endpoint. In the remaining two mono-immunotherapy trials MSI status was assessed Immunotherapy in endometrial carcinoma 31 36. Santin AD, Bellone S, Buza N, Choi J, Schwartz PE, Schlessinger J, et al. Regression of chemotherapy-resistant Polymerase ε (POLE) ultra-mutated and MSH6 hyper-mutated endometrial tumors with nivolumab. Clin Cancer Res Off J Am Assoc Cancer Res. 2016; 1;22(23):5682–7. 37. Matulonis UA, Shapira-Frommer R, Santin AD, Lisyanskaya AS, Pignata S, Vergote I, et al. Antitumor activity and safety of pembrolizumab in patients with advanced recurrent ovarian cancer: results from the phaseII KEYNOTE-100 study. Ann Oncol Off J Eur Soc Med Oncol. 2019 01;30(7):1080–7. 38. Coffelt SB, de Visser KE. Immune-mediated mechanisms influencing the efficacy of anticancer therapies. Trends Immunol. 2015; 36(4):198–216. 39. Ding L, Kim H-J, Wang Q, Kearns M, Jiang T, Ohlson CE, et al. PARP Inhibition Elicits STING-Dependent Antitumor Immunity in Brca1-Deficient Ovarian Cancer. Cell Rep. 2018 11;25(11):2972-2980.e5. 40. Galluzzi L, Buqué A, Kepp O, Zitvogel L, Kroemer G. Immunological Effects of Conventional Chemotherapy and Targeted Anticancer Agents. Cancer Cell. 2015; 14;28(6):690–714. 41. Zitvogel L, Galluzzi L, Smyth MJ, Kroemer G. Mechanism of action of conventional and targeted anticancer therapies: reinstating immunosurveillance. Immunity. 2013; 25;39(1):74–88. Héctor Villa Martínez 32 APPENDIX Prospero like protocol according to PRISMA guidelines ADMINISTRATIVE INFORMATION Tittle Systematic review on immunotherapy for POLE ultramutated and MSI-H advanced endometrial carcinoma. Registration The protocol of this systematic review will not be recorded. Authors Héctor Villa Martínez supervised by the cotutor, María José Villanueva Silva. INTRODUCCTION Rationale Recent advances in molecular biology have revealed a wide range of genomic alterations in endometrial cancers. The Cancer Genome Atlas classification delineated four subgroups of EC: microsatellite instability high (MSI-H), DNA polymerase epsilon (POLE), copy number high and copy number low. Hotspot mutation of the catalytic unit DNA polymerase epsilon (POLE) display and ultramutated sequence that is recognizable to the immune system and confers a good prognosis. MSI-H tumors are hypermutated because of their inability to repair replicational mutations, which make these tumors immunologically active to treatment with immunotherapy. Objectives To perform a systematic review to summarize all available evidence with checkpoint inhibitors in highly and ultramutated endometrial cancer subtypes to asses efficacy and tolerability. METHODS Eligibility criteria Inclusion criteria will be phase I trial and phase II trial in both subtypes (POLE and MSI-H) between January 2012 and March 2020, in advanced/recurrent endometrial carcinoma. There will be no restrictions on the language used in the publications. Information sources An electronically search will be performed in PubMed, Embase, Clinicaltrials.gov, Web of Science for articles as well as ASCO and ESMO meeting database for abstracts. Search strategy Search strategy will include a combination of broad terms related to endometrial carcinoma and immunotherapy, checkpoint inhibitors, POLE, MSI-H, clinical trial, PD1, PD-L1, Pembrolizumab, Nivolumab, Atezolizumab, Avelumab, Durvalumab. Exclusion criteria Exclusion criteria will be: (i) no advanced endometria carcinoma, (ii) clinical trials with no POLE or MSI-H subtype tumors reported, (iii) studies that matched different databases, (iv) completed trials with no published results, (v) ongoing clinical trials with no published results (vi) phase III trials, (vii) Immunotherapy in endometrial carcinoma 33 case reports, narrative reviews, editorials, news articles, commentaries or letters. DATA Data items For each included study we will extract the following data: number of patients included, design, type of trial, age, stage, performance status, simple size, treatment type, type or immunotherapy, other drugs if applied, primary outcome, secondary outcomes, median follow-up, overall response rate (ORR), including partial response (PR) and complete response (CR), progression free survival (PFS), overall survival (OS), duration of response (DOR), time to response (TTR) and toxicity.