Open access Original research Ascites and resistance to immune J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. checkpoint inhibition in dMMR/MSI-­H metastatic colorectal and gastric cancers Giovanni Fucà ‍ ‍ ,1 Romain Cohen ‍ ‍ ,2 Sara Lonardi ‍ ‍ ,3 Kohei Shitara,4 Maria Elena Elez,5 Marwan Fakih,6 Joseph Chao ‍ ‍ ,6 Samuel J Klempner,7,8 Matthew Emmett,7,8 Priya Jayachandran,9 Francesca Bergamo,10 Marc Díez García,5 Giacomo Mazzoli,1 Leonardo Provenzano,1 Raphael Colle,2 Magali Svrcek,11 Margherita Ambrosini,1 Giovanni Randon,1 Aakash Tushar Shah,12 Massimiliano Salati,13 Elisabetta Fenocchio,14 Lisa Salvatore,15 Keigo Chida,4 Akihito Kawazoe,4 Veronica Conca,16,17 Giuseppe Curigliano,18,19 Francesca Corti,1 Chiara Cremolini,16,17 Michael Overman,20 Thierry Andre ‍ ‍ ,2 Filippo Pietrantonio ‍ ‍ 1 To cite: Fucà G, Cohen R, ABSTRACT considered as a peculiar subgroup with highly unfavorable Lonardi S, et al. Ascites Background Despite unprecedented benefit from outcomes to current ICI-­based therapies. Novel strategies and resistance to immune immune checkpoint inhibitors (ICIs) in patients with to target the immune-­suppressive niche in malignant checkpoint inhibition in dMMR/ effusions should be investigated, as well as next-­ mismatch repair deficient (dMMR)/microsatellite instability MSI-­H metastatic colorectal high (MSI-­H) advanced gastrointestinal cancers, a relevant generation ICIs or intraperitoneal approaches. and gastric cancers. Journal for ImmunoTherapy of Cancer proportion of patients shows primary resistance or short-­ 2022;10:e004001. doi:10.1136/ term disease control. Since malignant effusions represent jitc-2021-004001 an immune-­suppressed niche, we investigated whether INTRODUCTION peritoneal involvement with or without ascites is a poor Although mismatch repair deficient (dMMR) ► Additional supplemental prognostic factor in patients with dMMR/MSI-­H metastatic and/or microsatellite instability high material is published online only. colorectal cancer (mCRC) and gastric cancer (mGC) (MSI-­H) advanced gastrointestinal cancers To view, please visit the journal receiving ICIs. are relatively uncommon (about 4% of both online (http://​dx.d​ oi.​org/​10.​ Methods We conducted a global multicohort study at metastatic colorectal cancer (mCRC) and 1136/j​ itc-​2021-0​ 04001). Tertiary Cancer Centers and collected clinic-­pathological gastric cancer (mGC)), immune checkpoint data from a cohort of patients with dMMR/MSI-­H inhibitors (ICIs) have reliably demonstrated GF and RC are joint first authors. mCRC treated with anti-­PD-­(L)1 ±anti-­CTLA-­4 agents durable responses and unprecedented at 12 institutions (developing set). A cohort of patients survival outcomes in this patients’ population TA and FP are joint senior with dMMR/MSI-­high mGC treated with anti-­PD-­1 authors. independent from the tumor site of origin.1 agents±chemotherapy at five institutions was used as ICIs have become a new standard of care for Accepted 06 January 2022 validating dataset. Results The mCRC cohort included 502 patients. After patients with dMMR/MSI-­ H cancers, and a median follow-­up of 31.2 months, patients without their use is nowadays supported by random- peritoneal metastases and those with peritoneal ized clinical trials (RCTs).2–4 Nevertheless, metastases and no ascites had similar outcomes (adjusted about 30% of patients with MSI/dMMR HR (aHR) 1.15, 95% CI 0.85 to 1.56 for progression-­free advanced mCRC display intrinsic resistance survival (PFS); aHR 0.96, 95% CI 0.65 to 1.42 for overall to anti-­PD(L)-­1 monotherapy both in phase survival (OS)), whereas inferior outcomes were observed II proof-­ of-­ concept trials conducted in in patients with peritoneal metastases and ascites (aHR pretreated patients,5 6 and in the Keynote-­177 © Author(s) (or their 2.90, 95% CI 1.70 to 4.94; aHR 3.33, 95% CI 1.88 to 5.91) phase III first-­line trial.2 A similar percentage employer(s)) 2022. Re-­use compared with patients without peritoneal involvement. of primary resistance was reported in a non-­ permitted under CC BY-­NC. No The mGC cohort included 59 patients. After a median colorectal cohort and in post hoc analyses of commercial re-­use. See rights follow-­up of 17.4 months, inferior PFS and OS were and permissions. Published by RCT in mGC.7–9 reported in patients with peritoneal metastases and ascites BMJ. Of note, the occurrence of early progres- (aHR 3.83, 95% CI 1.68 to 8.72; aHR 3.44, 95% CI 1.39 to For numbered affiliations see 8.53, respectively), but not in patients with only peritoneal sive disease (PD) at first disease reassessment end of article. metastases (aHR 1.87, 95% CI 0.64 to 5.46; aHR 2.15, may be explained by the phenomenon of 95% CI 0.64 to 7.27) when compared with patients pseudoprogression,10 or by the misdiagnosis Correspondence to Dr Filippo Pietrantonio; without peritoneal involvement. of dMMR/MSI-­ H status.11 However, several ​filippo.​pietrantonio@​ Conclusions Patients with dMMR/MSI-­H gastrointestinal promising studies investigated the associ- istitutotumori.​mi.​it cancers with peritoneal metastases and ascites should be ation of primary resistance with specific Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 1 Open access biomarkers, including a relatively lower tumor muta- RAS mutational status and BRAF mutational status. For J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. tional burden,12 13 dMMR/MSI-­ H heterogeneity,13 and patients with dMMR/MSI-­H mGC the following disease-­ elevated systemic inflammation as assessed by blood-­ specific data were also retrieved when available: primary based parameters.14 site of origin and histology. Malignant ascites is a common clinical complication in The presence of peritoneal involvement and ascites was patients with peritoneal metastases from gastrointestinal assessed by means of conventional imaging techniques cancers. Despite the well-­ established poor prognostic (ie, abdominal CT scan or MRI) at each participating effect of both peritoneal involvement and ascites in center. The presence of metastatic peritoneal involve- patients with several types of advanced cancers, malignant ment was coded as a polytomous categorical variable ascites is also characterized by a pervasive immunosup- encompassing three values: no peritoneal metastases, pressive microenvironment.15 16 Indeed, serous cavities peritoneal metastases without ascites, peritoneal metas- are an immunologic niche due to a variety of immunosup- tases with ascites. Patients with evidence of peritoneal pressive networks, such as those driven by cavity-­resident metastases with ascites were included in the ‘ascites’ macrophages with Tim-­4 overexpression.17 Drawing from group, whereas patients with no peritoneal metastases or these considerations, we hypothesized that patients with peritoneal metastases without ascites were included in the MSI-­H cancers and coexisting malignant ascites may have ‘no ascites’ group. poorer outcomes and high rates of primary resistance to ICI treatment. To examine this hypothesis, we assembled Statistical analyses a large multinational cohort of patients with dMMR/ PFS was defined as the time from the first dose of ICIs MSI-­H mCRC or GC receiving ICIs to investigate the administration to PD or death from any cause, whichever prognostic role of peritoneal involvement with or without occurred first. OS was defined as the time from the first ascites. dose of ICIs administration to death from any cause. To examine baseline differences between groups, Pearson’s χ2 test or Fisher’s exact test were used, as appropriate. To METHODS examine baseline differences according to the metastatic Patients’ population peritoneal involvement coded as a polytomous categor- This was a retrospective, multicentric, international study ical variable, pairwise Fisher’s exact test was used. Survival that included patients with advanced dMMR/MSI-­H CRC analyses were performed using the Kaplan-­Meier method or GC treated with ICI-­based therapy from June 2014 to and Cox proportional hazards regression. Univariable May 2021. We assembled two cohorts of dMMR/MSI-­H Cox regression analysis was used to assess the effect of gastrointestinal cancers consisting of mCRC (developing different baseline factors on PFS and OS. Variables signifi- set or cohort 1) and mGC (validating set or cohort 2) cantly associated with survival outcomes at the univariable taking advantage of the electronic medical records of the analysis were then fitted in a multivariable Cox propor- participating centers. Inclusion criteria were: (1) patho- tional hazards regression model to identify independent logically/cytologically confirmed diagnosis of dMMR/ predictors of OS and PFS. HRs with the corresponding MSI-­H CRC or GC, with dMMR/MSI-­H status confirmed 95% CIs were provided for Cox’s proportional hazards by local institutional testing per international guide- regression models. All statistical tests were two tailed, and lines18; (2) treatment with at least one cycle of ICI-­based a p<0.05 was considered statistically significant. Statistical therapy for advanced disease within clinical trials or per analyses were performed using R software (V.3.5.0) and R clinical practice; (3) age ≥18 years. Cohort 1 contained Studio (V.1.1.447). patients with dMMR/MSI-­H mCRC treated with anti-­PD-­ (L)1±anti CTLA-­4 agents at 12 institutions worldwide. Cohort 2 contained patients with dMMR/MSI-­H mGC treated with anti-­PD-­1±chemotherapy at five institutions RESULTS worldwide. Information about clinical outcomes of ICI-­ Patients’ characteristics based treatment (ie, progression-­free survival (PFS) and Cohort 1 included 502 patients with dMMR/MSI-­H mCRC overall survival (OS)) as well as the following demo- from 12 institutions (online supplemental table 1). About graphic and clinico-­ pathological data (baseline to the 60% of the patients included in cohort 1 received the ICI-­ initiation of the ICI-­ based treatment) were retrieved based treatment within clinical trials. Clinico-­pathological for all the patients included: age, sex, Eastern Cooper- and treatment characteristics are reported in table 1, ative Oncology Group (ECOG) Performance Status both overall and according to the presence or absence (PS), primary tumor resection, presence of synchronous of ascites. Briefly, peritoneal involvement without ascites metastases, number and localization of metastases, pres- was present in 172 patients (34.3%), whereas ascites was ence of peritoneal involvement and ascites, information present in 25 patients (5.0%). The presence of ascites about prior systemic treatment for metastatic disease, was associated with poorer PS and RAS wild-­type/BRAF type of ICI-­based treatment. For patients with dMMR/ mutated status. Online supplemental table 2 shows the MSI-­ H mCRC the following disease-­ specific data were pairwise analysis of all characteristics according to the also retrieved when available: primary tumor sidedness, three subgroups of patients with peritoneal involvement 2 Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. Table 1 Patients and disease characteristics in the developing set (Cohort 1: dMMR/MSI-­high mCRC), overall and according to the presence or absence of ascites Total (N=502) No ascites (N=477) Ascites (N=25) Characteristics N (%) N (%) N (%) P value Sex 0.511  Female 229 (45.6) 216 (45.3) 13 (52.0)  Male 273 (54.4) 261 (54.7) 12 (48.0) Age 0.301  <70 378 (75.3) 357 (74.8) 21 (84.0)  ≥70 124 (24.7) 120 (25.2) 4 (16.0) ECOG PS 0.005  0 237 (47.2) 232 (48.6) 5 (20.0)  ≥1 265 (52.8) 245 (51.4) 20 (80.0) Primary tumor resection >0.999  No 90 (17.9) 86 (18.0) 4 (16.0)  Yes 412 (82.1) 391 (81.9) 21 (84.0) Primary tumor sidedness 0.267  Left 172 (34.3) 166 (34.8) 6 (24.0)  Right 330 (65.7) 311 (65.2) 19 (76.0)  NA RAS mutational status 0.048  All wild type 317 (63.1) 296 (62.1) 21 (84.0)  RAS mutated 165 (32.9) 161 (33.7) 4 (16.0)  NA 20 (4.0) 20 (4.2) 0 (0.0) BRAF mutational status 0.054  All wild-­type 351 (69.9) 337 (70.6) 14 (56.0) BRAF mutated   132 (26.3) 121 (25.4) 11 (44.0)  NA 19 (3.8) 19 (4.0) 0 (0.0) Synchronous metastases 0.203  No 242 (48.2) 233 (48.9) 9 (36)  Yes 258 (51.4) 242 (50.7) 16 (64)  NA 2 (0.4) 2 (0.4) 0 (0) Liver metastases 0.246  No 306 (61.0) 288 (60.4) 18 (72.0)  Yes 196 (39.0) 189 (39.6) 7 (28.0) Lung metastases 0.595  No 412 (82.1) 390 (81.8) 22 (88.0)  Yes 90 (17.9) 87 (18.2) 3 (12.0) Lymphnodal metastases 0.521  No 210 (41.8) 198 (41.5) 12 (48.0)  Yes 292 (58.2) 279 (58.5) 13 (52.0) Bone metastases 0.320  No 479 (95.4) 456 (95.6) 23 (92.0)  Yes 23 (4.6) 21 (4.4) 2 (8.0) No of metastatic sites 0.076  1 227 (45.2) 220 (46.1) 7 (28.0)  ≥2 275 (54.8) 257 (53.9) 18 (72.0) Continued Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 3 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. Table 1 Continued Total (N=502) No ascites (N=477) Ascites (N=25) Characteristics N (%) N (%) N (%) P value Prior treatment for metastatic disease 0.195  No 96 (19.1) 94 (19.7) 2 (8.0)  Yes 406 (80.9) 383 (80.3) 23 (92.0) ICI regimen 0.525  a-­PD(L)−1 332 (66.1) 314 (65.8) 18 (72.0)  a-­PD-­1+a-­CTLA-­4 170 (33.9) 163 (34.2) 7 (8.0) P-­values marked with bold indicate statistically significant p-­values. dMMR, mismatch repair deficient; ECOG, Eastern Cooperative Oncology Group; ICI, immune checkpoint inhibitor; mCRC, metastatic colorectal cancer; MSI-­H, microsatellite instability high; NA, not available; PS, Performance Status. with or without ascites and those without peritoneal 5.8 months and 6.6%; figure 1C,D). Patients without peri- involvement. toneal metastases had similar PFS and OS to those with Cohort 2 included 59 patients with dMMR/MSI-­high peritoneal metastases and no ascites, regardless of the mGC from 5 institutions (online supplemental table 3). specific ICI approach (online supplemental figure 2). About 30% of the patients included in cohort 2 received In cohort 2, the median follow-­ up was 17.4 month the ICI-­based treatment within clinical trials. Clinico-­ (IQR: 11.8–51.3). Online supplemental figure 3A,B shows pathological and treatment characteristics are reported PFS and OS in the overall population. The presence of in table 2, both overall and according to the presence ascites was significantly associated with shorter PFS and or absence of ascites. Peritoneal involvement without OS (p<0.001 and p=0.006; figure 2A,B). Patients without ascites was present in 11 patients (18.6%), whereas ascites peritoneal metastases had similar PFS and OS to those was present in 17 patients (28.8%). Of note, ascites was with peritoneal metastases and no ascites (2-­ year PFS significantly associated with poorer PS, non-­ resected rate: 50.2% vs 61.4%; HR 1.48, 95% CI 0.55 to 4.02; 2-­year primary tumor, synchronous presentation of metastases OS rate: 56.5% vs 60.6%; HR 1.69, 95% CI 0.55 to 5.18), and >1 sites of metastases. whereas worse PFS and OS were restricted to patients with peritoneal metastases and ascites (2-­year PFS rate: 17.7%; Survival outcomes according to the presence of peritoneal HR 4.57, 95% CI 2.07 to 10.09; 2-­year OS rate: 26.5%, HR involvement and ascites 4.18, 95% CI 1.70 to 10.26). Table 4 shows the univariable In cohort 1, the median follow-­up time was 31.2 months and multivariable models for PFS and OS: presence of (IQR 15.6–46.0). Online supplemental figure 1A,B shows ascites had an independent effect on survival outcomes PFS and OS in the overall population. The presence of (adjusted HR for PFS: 3.83 (95% CI 1.68 to 8.72); for OS: ascites was significantly associated with shorter PFS and 3.44 (95% CI 1.39 to 8.53)). OS (both p<0.001; figure 1A,B). Patients without perito- neal metastases had similar PFS and OS to those with peri- toneal metastases and no ascites (2-­year PFS rate: 62.6% vs 60.3%; HR 1.08, 95% CI 0.80 to 1.46; 2 year OS rate: DISCUSSION 73.2% vs 75.3%; HR 0.92, 95% CI 0.63 to 1.33), whereas In this study, the presence of ascites, but not peritoneal poorer PFS and OS were restricted to patients with peri- involvement without ascites, was associated with extremely toneal metastases and ascites (2-­year PFS rate: 30.4%; HR poor survival outcomes in the largest reported cohort of 2.80, 95% CI 1.65 to 4.75; 2-­year OS rate: 29.7%, HR 3.58, patients with dMMR/MSI-­ H mCRC and mGC treated 95% CI 2.06 to 6.22). table 3 shows the univariable and with ICIs. The results were consistent in both a very large multivariable models for PFS and OS: presence of ascites primary analysis dataset of mCRC and an external valida- had an independent effect on both survival outcomes tion set of patients with dMMR/MSI-­H mGC. Despite the (adjusted HR for PFS: 2.90 (95% CI 1.70 to 4.94); for OS: association of ascites with worse performance status and 3.33 (95% CI 1.88 to 5.91)), as well as ECOG PS, presence other unfavorable features (such as RAS wild-­type/BRAF of lung metastases, prior treatment for advanced disease mutated status in mCRC, and synchronous presentation/ and ICI treatment type (anti-­PD(L)-­1 monotherapy or non-­resected primary tumor in mGC), the presence CTLA-­ anti-­ 4 combination). We then investigated the of ascites was independently associated with both PFS impact of specific ICI approaches (combination with and OS, and notably demonstrated a similar HR >3.0 CTLA-­4 vs PD-­1 monotherapy) and observed that shorter in both dMMR/MSI-­ H mCRC and dMMR/MSI mGC. PFS and OS outcomes were restricted to patients with We acknowledge that the lack of independent, central ascites receiving anti-­PD(L)-­1 monotherapy (median and imaging revision is a limitation of our study, since mild 2-­year PFS: 1.6 months and 6.9%; median and 2-­year OS: amounts of ascites may not be reported systematically. 4 Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. Table 2 Patients and disease characteristics in the validating set (Cohort 2: dMMR/MSI-­high mGC), overall and according to the presence or absence of ascites Total (N=59) No ascites (N=42) Ascites (N=17) Characteristics N (%) N (%) N (%) P value Sex 0.840  Female 22 (37.3) 16 (38.1) 6 (35.3)  Male 37 (62.7) 26 (61.9) 11 (64.7) Age 0.304  <70 32 (54.2) 21 (50.0) 11 (64.7)  ≥70 27 (45.8) 21 (50.0) 6 (35.3) ECOG PS 0.047  0 18 (30.5) 16 (38.1) 2 (11.8)  ≥1 41 (69.5) 26 (61.9) 15 (88.2) Primary tumor resection 0.029  No 32 (54.2) 19 (45.2) 13 (76.5)  Yes 27 (45.8) 23 (54.8) 4 (23.5) Primary site of origin 0.662  Gastroesophageal junction (GEJ) 6 (10.2) 5 (11.9) 1 (5.9)  Gastric 53 (89.8) 37 (88.1) 16 (94.1) Histology 0.195  Intestinal 43 (72.9) 33 (78.6) 10 (58.8)  Diffuse/other 16 (27.1) 9 (21.4) 7 (41.2) Synchronous metastases 0.033  No 23 (39.0) 20 (47.6) 3 (17.6)  Yes 36 (61.0) 22 (52.3) 14 (82.4) Liver metastases 0.310  No 45 (76.3) 30 (71.4) 15 (88.2)  Yes 14 (23.7) 12 (28.6) 2 (11.8) Lymphnodal metastases >0.999  No 13 (22.0) 9 (21.4) 4 (23.5)  Yes 46 (78.0) 33 (78.6) 13 (76.5) Lung metastases >0.999  No 49 (83.1) 35 (83.3) 14 (82.4)  Yes 10 (16.9) 7 (16.7) 3 (17.6) Bone metastases 0.620  No 54 (91.5) 39 (92.9) 15 (88.2)  Yes 5 (8.5) 3 (7.1) 2 (11.8) No of metastatic sites 0.013  1 17 (28.8) 16 (38.1) 1 (5.9)  ≥2 42 (71.2) 26 (61.9) 16 (94.1) Prior treatment for metastatic disease >0.999  No 7 (11.9) 5 (11.9) 2 (11.8)  Yes 52 (88.1) 37 (88.1) 15 (88.2) ICI regimen 0.308  a-­PD-­1 54 (91.5) 37 (88.1) 17 (100)  a-­PD-­1+chemotherapy 5 (8.5) 5 (11.9) 0 (0) P-­values marked with bold indicate statistically significant p-­values. dMMR, mismatch repair deficient; ECOG, Eastern Cooperative Oncology Group; ICIs, immune checkpoint inhibitors; mGC, metastatic gastric cancer; MSI, microsatellite instability; PS, Performance Status. Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 5 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. Figure 1 Kaplan-­Meier curves for progression-­free survival (A) and overall survival (B) in the subgroups of dMMR/MSI-­H mCRC patients without peritoneal metastases, with peritoneal metastases and no ascites or with peritoneal metastases and ascites; Kaplan-­Meier curves for progression-­free survival (C) and overall survival (D) of patients dMMR/MSI-­H mCRC according to the presence of ascites and the type of ICI regimen. dMMR, mismatch repair deficient; ICI, immune checkpoint inhibitors; mCRC, metastatic colorectal cancer; MSI-­H, microsatellite instability high; NA, not available; OS, overall survival; PFS, progression-­free survival. However, the presence of peritoneal involvement and The frequency of malignant ascites was much higher ascites was assessed at each center by expert radiolo- in patients with mGC (28.8%), consistent with the higher gists, ensuring a high quality of the radiology reports. frequency of peritoneal involvement in this tumor type, The reproducibility of our results confirms the agnostic compared with those with mCRC (5%).19 Despite the role of MSI as a predictive biomarker for the efficacy of potential generalizability of our results to all patients ICIs and establish the role of malignant ascites as a poor with MSI-­high advanced solid tumors, caution should prognostic factor for dMMR/MSI-­H mCRC treated with be adopted in specific tumor types with extremely low anti-­PD(L)-­1 agents or dMMR/MSI-­H mGC treated with frequency of dMMR/MSI-­ H—such as cholangiocarci- anti-­PD-­1 agents±chemotherapy independently from the noma and pancreatic cancers. In fact, non-­randomized anatomic tumor site of origin. trial cohorts showed lower than expected response rates 6 Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. Table 3 Univariable and multivariable Cox proportional hazards regression models for PFS and OS in the developing set (Cohort 1: dMMR/MSI-­high mCRC) PFS OS Univariable Multivariable Univariable Multivariable Characteristics HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value Sex 0.673 0.644  Female Ref Ref  Male 1.06 (0.80 to 1.41) 1.08 (0.77 to 1.52) Age (years) 0.723 0.588  <70 Ref Ref  ≥70 1.061 (0.77 to 1.47) 1.11 (0.76 to 1.63) ECOG S 0.001 0.003 <0.001 0.001  0 Ref Ref Ref Ref  ≥1 1.608 (1.20 to 2.15) 1.56 (1.16 to 2.08) 1.79 (1.30 to 2.48) 1.78 (1.25 to 2.55) Primary tumor 0.802 0.317 resection  No Ref Ref  Yes 1.05 (0.72 to 1.54) 1.281 (0.79 to 2.08) Primary tumor 0.917 0.342 sidedness  Left Ref Ref  Right 1.02 (0.75 to 1.37) 0.85 (0.60 to 1.20) RAS mutational 0.724 0.459 status  All wild-­type Ref Ref RAS mutated   0.95 (0.70 to 1.28) 0.87 (0.61 to 1.25) BRAF mutational 0.196 0.151 status  All wild-­type Ref Ref BRAF mutated 1.23 (0.90 to 1.67)   1.303 (0.91 to 1.87) Synchronous 0.737 0.460 metastases  No Ref Ref  Yes 1.05 (0.79 to 1.39) 1.14 (0.81 to 1.59) Liver metastases 0.221 0.105  No Ref Ref  Yes 1.20 (0.90 to 1.59) 1.32 (0.94 to 1.85) Lung metastases <0.001 <0.001 0.001 0.023  No Ref Ref Ref Ref  Yes 1.812 (1.31 to 2.50) 1.79 (1.29 to 2.48) 1.83 (1.26 to 2.66) 1.60 (1.06 to 2.40) Lymphnodal 0.249 0.885 metastases  No Ref Ref  Yes 0.85 (0.64 to 1.13) 1.03 (0.73 to 1.45) Bone metastases 0.085 0.011 0.078  No Ref Ref Ref  Yes 1.67 (0.93 to 3.01) 2.22 (1.20 to 4.12) 1.77 (0.94 to 3.35) No of metastatic 0.078 0.013 0.356 sites  1 Ref Ref Ref  ≥2 1.30 (0.97 to 1.74) 1.56 (1.10 to 2.22) 1.21 (0.81 to 1.80) Continued Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 7 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. Table 3 Continued PFS OS Univariable Multivariable Univariable Multivariable Characteristics HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value Prior systemic 0.001 0.015 0.007 0.0563 treatment for metastatic disease  No Ref Ref Ref Ref  Yes 2.061 (1.31 to 3.24) 1.76 (1.12 to 2.79) 2.13 (1.23 to 3.70) 1.73 (0.99 to 3.03) ICI regimen <0.001 <0.001 <0.001 <0.001  a-­PD(L)−1 Ref Ref Ref Ref  a-­PD-­1+a-­ 0.46 (0.33 to 0.64) 0.46 (0.33 to 0.64) 0.41 (0.27 to 0.62) 0.41 (0.27 to 0.62) CTLA-­4 Peritoneal <0.001 0.002 <0.001 0.001 metastases  No Ref Ref Ref Ref  Yes, without 1.08 (0.80 to 1.46) 1.15 (0.85 to 1.56) 0.92 (0.63 to 1.33) 0.96 (0.65 to 1.42) ascites  Yes, with 2.80 (1.65 to 4.75) 2.90 (1.70 to 4.94) 3.58 (2.06 to 6.22) 3.33 (1.88 to 5.91) ascites P-­values marked with bold indicate statistically significant p-­values. dMMR, mismatch repair deficient; ECOG, Eastern Cooperative Oncology Group; ICIs, immune checkpoint inhibitors; mCRC, metastatic colorectal cancer; MSI, microsatellite instability; OS, overall survival; PFS, progression-­free survival; PS, Performance Status. Figure 2 Kaplan-­Meier curves for progression-­free survival (A) and overall survival (B) in the subgroups of dMMR/MSI-­H mGC patients without peritoneal metastases, with peritoneal metastases and no ascites or with peritoneal metastases and ascites. dMMR, mismatch repair deficient; mGC, metastatic gastric cancer; MSI-­H, microsatellite instability high; NA, not available; OS, overall survival; PFS, progression-­free survival. 8 Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 Table 4 Univariable and multivariable Cox proportional hazards regression models for PFS and OS in the validating set (Cohort 2: dMMR/MSI-­high mGC) PFS OS Univariable Multivariable Univariable Multivariable Characteristics HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value Sex 0.643 0.870  Female Ref Ref  Male 0.84 (0.41 to 1.73) 0.94 (0.41 to 2.11) Age 0.695 0.483  <70 Ref Ref  ≥70 0.87 (0.44 to 1.73) 0.76 (0.35 to 1.65) Eastern Cooperative Oncology Group 0.018 0.410 0.022 0.301 (ECOG) Performance Status (PS)  0 Ref Ref Ref Ref  ≥1 2.94 (1.20 to 7.18) 1.54 (0.55 to 4.31) 3.49 (1.20 to 10.2) 1.84 (0.58 to 5.88) Primary tumor resection 0.003 0.133 0.009 0.066  No Ref Ref Ref Ref  Yes 0.30 (0.13 to 0.66) 0.46 (0.17 to 1.27) 0.30 (0.12 to 0.74) 0.37 (0.13 to 1.07) Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 Primary site of origin 0.353 0.419  Gastroesophageal junction (GEJ) Ref Ref  Gastric 0.60 (0.21 to 1.75) 0.60 (0.18 to 2.05) Histology 0.506 0.575  Intestinal Ref Ref  Diffuse/other 1.29 (0.61 to 2.71) 1.27 (0.56 to 2.93) Synchronous metastases 0.460 0.855  No Ref Ref  Yes 1.33 (0.63 to 2.81) 0.93 (0.42 to 2.06) Liver metastases 0.294 0.201  No Ref Ref  Yes 0.60 (0.23 to 1.56) 0.46 (0.14 to 1.52) Lymphnodal metastases 0.767 0.947  No Ref Ref  Yes 1.14 (0.49 to 2.62) 0.97 (0.39 to 2.42) Lung metastases 0.789 0.802  No Ref Ref Continued Open access 9 J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. 10 Open access Table 4 Continued PFS OS Univariable Multivariable Univariable Multivariable Characteristics HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value  Yes 0.88 (0.34 to 2.28) 0.87 (0.30 to 2.53) Bone metastases 0.039 0.065 0.057  No Ref Ref Ref  Yes 3.13 (1.06 to 9.27) 2.94 (0.94 to 9.20) 3.36 (0.98 to 11.64) No of metastatic sites 0.175 0.165  1 Ref Ref  ≥2 1.74 (0.78 to 3.87) 1.92 (0.76 to 4.80) Prior treatment for metastatic disease 0.792 0.719  No Ref Ref  Yes 0.87 (0.30 to 2.48) 0.80 (0.24 to 2.68) ICI regimen 0.722 0.908  a-­PD-­1 Ref Ref  a-­PD-­1 +chemotherapy 0.77 (0.18 to 3.24) 1.10 (0.27 to 4.63) Peritoneal metastases <0.001 0.005 0.006 0.023  No Ref Ref Ref Ref  Yes, without ascites 1.48 (0.55 to 4.02) 1.87 (0.64 to 5.46) 1.69 (0.55 to 5.18) 2.15 (0.64 to 7.27)  Yes, with ascites 4.57 (2.07 to 10.09) 3.83 (1.68 to 8.72) 4.18 (1.70 to 10.26) 3.44 (1.39 to 8.53) P-­values marked with bold indicate statistically significant p-­values. dMMR, mismatch repair deficient; ICIs, immune checkpoint inhibitors; mGC, metastatic gastric cancer; MSI, microsatellite instability; OS, overall survival; PFS, progression-­free survival. Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. Open access to PD-­ 1 blockade in these specific cancers, consistent of T cells, explaining (at least in part) why we observed J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. with a highly immune-­ suppressive tumor microenvi- extremely good outcomes in mCRC patients with ascites ronment that may counterbalance the positive effect of treated with the combination of anti-­CTLA-­4 plus anti-­PD-­1 hypermutation.7 combination. Despite the greater efficacy reported with Our study cannot clarify if malignant ascites is just a poor dual immune checkpoint blockade in non-­ randomized prognostic factor (which is well established for patients studies and in our series, the potential benefit of adding anti-­ with advanced GI cancers20 21) or if it is predictive of resis- CTLA-­4 agents to an anti-­PD(L)-­1 backbone in patients with tance to current ICI-­based strategies. However, several dMMR/MSI-­H mCRC and malignant ascites should be inter- works highlighted the immunosuppressive environment preted with caution considering the low number of patients of serous human cavities and malignant effusions.15–17 22–25 included in this analysis. Notably our results are aligned Contrary to the more intuitive theory that peritoneum with the observation that dual checkpoint inhibition yielded and pleura could represent an immune-­excluded milieu relatively better outcomes in patients with poor prognostic because of the difficulty of immune system to penetrate features such as those with elevated systemic inflammation the effusion fluids, recent studies supported the idea of and poorer PS.14 26–28 Based on these considerations, the use serous cavities as an immune-­enriched environment with of more aggressive therapies such as dual CTLA-­4/PD-­1 inhi- high concentration of immune-­ suppressive cells such bition and chemoimmunotherapy in patients with dMMR/ as macrophages, myeloid-­derived suppressive cells and MSI-­H advanced GI cancers and ascites may be warranted. T regulatory cells.15–17 Also, a recent work showed that Additional novel strategies including intraperitoneal delivery cavity-­resident Tim-­4+ macrophages populate the pleural of immunomodulatory agents and exploration of check- and peritoneal microenvironment and can induce an points enriched in peritoneal metastases (eg, VISTA, LAG3, immune-­ suppressed microenvironment by impairing and TIM3) are worthy of further exploration.29–31 CD8+ T cells proliferation.17 In this context, dual Tim-­4/ Prospective validation of the potential predictive role of PD-­1 blockade was synergic in murine models.17 In ascites with regard to the intensification of ICI-­based regi- attempt to clinically validate these results, the authors mens may be possible from post-­hoc analyses of ongoing analyzed a monocentre cohort of 61 patients with MSI-­ randomized trials, for instance, the ongoing COMMIT high mCRC treated with ICIs, reporting worse outcomes trial investigating FOLFOX/bevacizumab/atezolizumab vs in those with peritoneal metastases, with unadjusted HRs atezolizumab (NCT02997228) or the ongoing CA209-­8HW for PFS and OS of 2.69 and 3.59, respectively. However, trial with ipilimumab–nivolumab versus nivolumab across given the relatively low prevalence of ascites in their multiple treatment lines (NCT04008030). The same concept cohort of mCRC, it was not possible to investigate the may be applied to patients with dMMR/MSI-­H mGC. In fact, prognostic impact of peritoneal metastases in a multivari- although the outcomes of these subjects were not different able model and according to the presence or absence of when receiving anti-­PD-­1 alone or with chemotherapy,4 8 32 malignant effusion. the addition of chemotherapy to ICI should be further inves- Recently, the immune-­ suppressed microenvironment tigated in the relevant subgroup of patients with dMMR/ of ascites was demonstrated also in patients with mGC, MSI-­H mGC and malignant ascites. irrespective of MMR status.22 Consistent with these find- In conclusion, patients with dMMR/MSI-­ H mCRC or ings, similar data were found in other immune-­sensitive mGC with peritoneal metastases and ascites have inferior tumors.23 24 In a multicentre retrospective series, patients outcomes with ICI therapy and should be viewed as a clinical with metastatic non-­small cell lung cancer and pleural subgroup requiring particular attention. Importantly, peri- effusion treated with anti-­PD(L)-­1 agents showed poorer toneal involvement without ascites may not impart the same survival outcomes and higher early death rates, even in unfavorable outcomes and should not be regarded as a clin- the subgroup with high PD-­L1 expression.25 ical factor associated with failure of ICIs. Our work has several potential implications for clinical practice. First, a subset of patients may sometimes develop Author affiliations 1 Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, ascites as a late-­stage complication of peritoneal metastases. Milan, Italy Based on the recent results of the Keynote-­ 177 first-­ line 2 Sorbonne Université, Department of Medical Oncology, Hôpital Saint-­Antoine, AP-­ mCRC trial,2 and considering that our study showed that no HP and INSERM, Unité Mixte de Recherche Scientifique 938, Centre de Recherche prior treatment for advanced disease was independently asso- Saint-­Antoine, Equipe Instabilité des Microsatellites et Cancer, Equipe labellisée par ciated with good outcomes, ICIs should be offered as early as la Ligue Nationale contre le Cancer, Paris, France 3 Medical Oncology 3 and Medical Oncology 1, Istituto Oncologico Veneto IOV-­IRCSS, possible in patients with peritoneal metastases and hopefully Padua, Italy prior to the development of malignant ascites. Second, we 4 Department of Gastroenterology and Gastrointestinal Oncology, National Cancer showed that patients with ascites treated with anti-­PD(L)-­1 Center Hospital East, Kashiwa, Japan monotherapy had worse outcomes and almost all were dead 5 Department of Medical Oncology, Vall d'Hebron Barcelona Hospital Campus, by the 2-­year time point, whereas no PFS and OS events were Vall d'Hebron Institute of Oncology (VHIO), Universitat Autonoma de Barcelona, Barcelona, Spain reported in patients treated with anti-­CTLA-­4 plus anti-­PD-­1 6 Department of Medical Oncology and Therapeutic Research, City of Hope combination. The peculiar immune microenvironment of Comprehensive Cancer Center, Duarte, California, USA the serous cavities in the presence of ascites might confer a 7 Mass General Cancer Center and Department of Medicine, Massachusetts General particular susceptibility to anti-­ CTLA-­4-­ mediated priming Hospital, Boston, Massachusetts, USA Fucà G, et al. J Immunother Cancer 2022;10:e004001. doi:10.1136/jitc-2021-004001 11 Open access 8 Harvard Medical School, Boston, Massachusetts, USA of the translations (including but not limited to local regulations, clinical guidelines, J Immunother Cancer: first published as 10.1136/jitc-2021-004001 on 2 February 2022. Downloaded from http://jitc.bmj.com/ on April 1, 2022 by guest. Protected by copyright. 9 Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of terminology, drug names and drug dosages), and is not responsible for any error Medicine, University of Southern California, Los Angeles, California, USA and/or omissions arising from translation and adaptation or otherwise. 10 Medical Oncology 1, Istituto Oncologico Veneto IOV-­IRCSS, Padua, Italy Open access This is an open access article distributed in accordance with the 11 Sorbonne Université, Department of Pathology, Hôpital Saint-­Antoine, AP-­HP, and Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which INSERM, Unité Mixte de Recherche Scientifique 938, Centre de Recherche Saint-­ permits others to distribute, remix, adapt, build upon this work non-­commercially, Antoine, Equipe Instabilité des Microsatellites et Cancer, Equipe labellisée par la and license their derivative works on different terms, provided the original work is Ligue Nationale contre le Cancer, Paris, France properly cited, appropriate credit is given, any changes made indicated, and the use 12 Baylor College of Medicine, Houston, Texas, USA is non-­commercial. See http://creativecommons.org/licenses/by-nc/4.0/. 13 Division of Oncology, Department of Oncology and Hematology, University Hospital Author note TA and FP are joint last authors. of Modena, PhD Clinical and Experimental Medicine (CEM), University of Modena and Reggio Emilia, Modena, Italy ORCID iDs 14 Multidisciplinary Outpatient Oncology Clinic, Candiolo Cancer Institute FPO-­IRCCS, Giovanni Fucà http://orcid.org/0000-0002-1560-2253 Candiolo, Italy Romain Cohen http://orcid.org/0000-0001-9602-5162 15 Department of Medical Oncology, Fondazione Policlinico Universitario Agostino Sara Lonardi http://orcid.org/0000-0002-7593-8138 Gemelli IRCCS, Rome, Italy Joseph Chao http://orcid.org/0000-0002-1809-504X 16 Unit of Medical Oncology 2, Azienda Ospedaliero-­Universitaria Pisana, Pisa, Italy Thierry Andre http://orcid.org/0000-0002-5103-7095 17 Department of Translational Research and New Technologies in Medicine and Filippo Pietrantonio http://orcid.org/0000-0002-8530-8420 Surgery, University of Pisa, Pisa, Italy 18 European Institute of Oncology (IEO), IRCCS, Milan, Italy 19 Department of Oncology and Hemato-­oncology, University of Milan, Milan, Italy REFERENCES 20 Department of Gastrointestinal Oncology, University of Texas MD Anderson Cancer 1 Le DT, Durham JN, Smith KN, et al. 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