Immunotherapy Efficacy in the Initial Lines of Treatment in Advanced Upper Gastrointestinal Malignancies: A Systematic Review of the Literature

Abstract Background The therapeutic role of immune checkpoint inhibitors (ICIs) has represented the cutting edge of clinical research in upper gastrointestinal (GI) malignancies, with these agents now included in the armamentarium of treatment options for advanced gastric and esophageal cancers. Methods We performed a systematic literature review and pooled analysis to map out the currently available robust clinical evidence for the use of ICIs in upper GI cancers. Immunotherapy (IO), either as monotherapy or in combination with chemotherapy, and its role in first-line, maintenance, and second-line settings, as well as in specific clinical and biological subgroups, were critically appraised. All statistical tests were 2-sided. Results ICIs, in combination with chemotherapy, have provided statistically significant overall survival benefit in the first-line setting in gastric and gastro-esophageal adenocarcinomas (hazard ratio [HR] = 0.83, 95% confidence interval [CI] = 0.76 to 0.90, P < .001; based on 4 studies) and esophageal squamous cell carcinoma (HR = 0.72, 95% CI = 0.64 to 0.81, P < .001; based on 3 studies), albeit with heterogeneous efficacy according to biomarker expression. Patients with esophageal squamous cell carcinoma, and in particular high programmed cell death ligand-1 expression, derive survival benefit when treated with IO in the second-line setting (HR = 0.74, 95% CI = 0.68 to 0.82, P < .001; for any level of programmed cell death ligand-1 expression). Clinical trials interrogating the combination of IO with chemotherapy in second-line treatment should be seriously considered in upper GI adenocarcinomas. The role of maintenance IO after initial disease control is still unclear and cannot be recommended. Impressive response rates and survival benefit from IO have been reported in patients with microsatellite instability-high tumors (HR = 0.33, 95% CI = 0.19 to 0.57, P < .001), and this warrants further prospective biomarker-driven studies. Conclusions IO is changing the treatment landscape in upper GI malignancies. The rapidly developing evidence in the field needs to be critically appraised while further validation of the existing information from ongoing trials is awaited.

2 [15]. Nonetheless the NIVO1-IPI3 combinational immunotherapy arm was closed before patients enrolled and the study continue as phase III, two arms RCT, randomizing in 1:1 parallel assignment [24]. The exact randomization modality was actually not described in formal sources [15,24] at the time of this review, and the only data available from this study are from the trial presentation at last 2020 ESMO conference and ClinicalTrials.gov [15,24] .Overall 1581 patients were recruited from 176 centers globally since October 2016 [15,24], 782 of those (out of 789 allocated) received the experimental chemo-immunotherapy %) in the control arm did not receive the allocated treatment [24], nonetheless survivals outcomes were analyzed in ITT [15]. It should be noted that the 60% of the patients enrolled in the study (955/1581) were with PD-L1 CPS equal or greater than 5 [15]. For the CHEKMATE 648 (NCT03143153) trial, a randomized, open-label phase 3 study, three arm study, patients were randomized between combinational immunotherapy (nivolumab 3 mg/kg every two weeks + ipilimumab 1mg/kg every six weeks) vs. combinational chemo-immunotherapy with nivolumab 240mg every two weeks plus chemo (fluorouracil + cisplatin every four weeks) vs. chemotherapy alone as the first-line treatment for advanced esophagal squamous cell carcinoma (ESCC). Randomization with parallel assignment was performed, nonetheless, the exact randomization modality is actually not described in formal sources [17,25] as the study is still formally unpublished and the only data available from this study are from the trial presentation at last 2021 ASCO conference and ClinicalTrials.gov [17,25] . Overall 970 patients were recruited from 27 countries globally from 29 th June 2017 [17,25]. 321 received the experimental chemo-immunotherapy treatment and 324, chemotherapy only. Survival outcomes were analyzed in ITT [17]. The 49% of the patients enrolled in the study were with PD-L1 CPS equal or greater than 1 [17].
For the ESCORT-1 st (NCT03691090) trial [18,26], a randomized double-blind, placebo-controlled 1:1, phase 3, two arms study. Patients were randomized to receive camrelizumab 200 mg or placebo, both combined with up to 6 cycles of paclitaxel (175 mg/m 2 ) and cisplatin (75 mg/m 2 ). All were given intravenously every three weeks as the first-line treatment for advanced esophagal squamous cell carcinoma (ESCC). The exact randomization modality is actually not described in formal sources [18,26], as the study is still formally unpublished and the only data available from this study are from the trial presentation at last 2021 ASCO conference and ClinicalTrials.gov [18,26]. Overall 596 patients were recruited from Chinese Hospitals from the 3 rd of December 2018 to the 12 th May 2020, 298 of those received the experimental chemo-immunotherapy treatment and 297chemotherapy only [18]. Survival outcomes were analyzed in ITT. The 49% of the patients enrolled in the study were with PD-L1 CPS equal or greater than 1 [18]. . b) Studies overview: Although the model of randomization was described in two of the six analyzed studies [4,22], withdrawals were described in the five studies [4,13,[15][16][17]. All studies were randomized phase III trials, including OS and PFS in their primary outcomes, and relative analyses were performed in ITT [4,13,[15][16][17][18]. Only one study enrolled patients with PD-L1 CPS of one or greater [4]. Thereafter data for the overall patients population (independently of PD-L1 CPS expression) are available only for 5 trials [13,[15][16][17][18]. In the interventional arm, two studies used pembrolizumab [4,16], three nivolumab [13,15,17] and one camrelizumab [18]. In five studies the chemotherapy regimen used included a platinum compound (cisplatin or oxaliplatin) plus a fluoropyrimidine agent (5-FU, capecitabine, tegafurgimeracil-oteracil) [4,13,[15][16][17][18]. One study used paclitaxel plus cisplatin as the chemotherapy regimen [18]. No anthracyclines were used [4,13,[15][16][17][18]. Overall 5363 were enrolled and randomized across six trials, of those 4801 patients were eligible for cumulative analysis as they were randomized to combined chemo-immunotherapy with checkpoint inhibitors (n = 2400) vs. chemotherapy alone (n=2401). Overall 61 patients did not receive allocated treatment, 19 in experimental arms and 42 in controls arms. Since survival outcomes (OS and PFS) were analyzed in the intention-to-treat method, 4802 patients were eligible for analysis of the primary outcomes (2400 in the chemoimmunotherapy arm and 2402 in the standard chemotherapy arm). Primary outcome of the pooled analyses was to assess the cumulative actually available randomized evidence for overall survival and progression free survival for the use of 1 st line immunotherapy in the treatment of esophago-gastric cancer when compared to standard 1 st line chemotherapy. Secondary outcome was to assess whether the stratification by PDL1 status, histology and tumor location may have any effect on the calculated comprehensive randomized evidence for overall survival and progression free survival. Comprehensive analysis for response rate was also part of the secondary outcome.

Maintenance treatment:
Calculation of cumulative available randomized evidence (Pooled Analysis) for the use of maintenance immunotherapy compared to no maintenance

a) Descriptive analyses
The systematic review of literature, identified overall two randomized trials comparing the use of maintenance immunotherapy vs no maintenance IO in Upper Gastrointestinal Tract Malignancies [10,11].
Data presented at ASCO 2021 identified one more maintenance trial with PFS analysis [14].
The first trial (NCT01585987), was a phase II open -label randomized trial, evaluating the use of an anti-CTLA4 antibody immunotherapy, ipilimumab (investigational arm) versus best supportive care (BSC) as maintenance treatment among patients with advanced / metastatic gastric or gastroesophageal junction cancer who achieved at least stable disease with first line chemotherapy with a platinum plus fluoropyrimidine regimen [10]. Ipilimumab was administered as 10mg/kg every 3 weeks for four doses, then 10 mg/kg every 12 weeks for up to three years, while BSC could include also the continuation of fluoropyrimidine until progression or toxicity. Between July 2012 and July 2014 a total of 143 patients with gastric or gastroesophageal junction from 12 countries across ASIA, Europe and North America were enrolled; 114 of those were randomized by 1:1 assignment: 57 in the investigational arm and 57 in the control arm, however only 51/57 in the BSC arm received the assigned management. Cross over was not allowed. Database locks occurred on June 2014 due to trial cessation on a preplanned interim analysis. Primacy outcome was the evaluation of immune-related Progression Free Survival (irPFS) as per assessment of a blinded independent review committee (IRC) according to immune related response criteria (irRC). Overall survival analysis was part of secondary outcomes [10].
The JAVELIN Gastric 100 trial (NCT02625610), was an open-label, parallel assignment, multicenter, randomized phase III trial. All patients received the same first-line induction therapy for up to 12 weeks with one of three regimens: A) oxaliplatin 85 mg/m 2 intravenously (IV) and leucovorin 200 mg/m 2 (or equivalent levoleucovorin dose), followed by fluorouracil (FU) 2,600 mg/m 2 by continuous infusion over 24 hours on day 1, every 2 weeks; B) oxaliplatin 85 mg/m 2 IV and leucovorin 400 mg/m 2 (or equivalent levoleucovorin dose), followed by FU 400 mg/m 2 IV on day 1 and FU 2,400 mg/m 2 by continuous infusion over 46 to 48 hours on days 1 to 2, every 2 weeks; C) or oxaliplatin 130 mg/m 2 IV on day 1 and capecitabine 1,000 mg/m 2 orally twice daily for 2 weeks, followed by a 1-week rest period, every 3 weeks [11]. Patients without progressive disease (PD) per RECIST (version 1.1) after induction chemotherapy, confirmed by an independent radiologist, were randomly assigned 1:1 to either maintenance therapy with avelumab 10 mg/kg IV every 2 weeks or continuation of the same chemotherapy. All patients received best supportive care (BSC). The primary end point was OS (time from random assignment to death resulting from any cause). OS was assessed in all randomly assigned patients and in randomly assigned patients with PD-L1-positive tumors (PD-L1 protein expression in ≥ 1% of tumor cells). PD-L1 status was assessed centrally at baseline using the PD-L1 immunohistochemical (IHC) 73-10 performance evaluation-only assay (Agilent Technologies/Dako, Carpinteria, CA). Study was global recruiting patients from Australia, Asia, North America and Europe (178 sites in 17 countries). Between December 31-2015, and November 29 -2017, 805 patients were enrolled, 499 of those achieved disease control in the 12-week induction phase and were randomly assigned to Avelumab maintenance (n = 249) or continue chemotherapy (n = 250). Fifty four patients (30 in the interventional arm and 24 in the chemotherapy arm) were PD-L1-positive (expression in ≥ 1% of tumor cells). In the chemotherapy arm, seven patients were considered unsuitable for further chemotherapy and received BSC only [11].

b) Studies overview:
Overall 1010 patients were enrolled in the two maintenance trials, and 818 patients were randomized in maintenance line trials and eligible for intention-to-treat analysis (ITT analysis), 411 in the immunotherapy arm and 407 in the standard maintenance arm.
All identified trials analyzed only adenocarcinoma histology and described withdrawals in detail while no study described in detail the randomization modality. [10,11,14,27,28]. All trials analyzed were multicenter [10,11,14].The first study was a phase II trial immunerelated progression free survival (irPFS) as primary outcome [10]], while the second study was a phase III study with primary outcome the evaluation of overall survival as in all randomly assigned patients and in those with PD-L1-positive tumors [11]. The third study was an adaptive phase II 5 arm trial [14].
Of note, in the first study an anti-CTLA4 antibody (ipilimumab) was used as checkpoint inhibitor [10], while in the second and third study an anti PD-L1 antibodies (avelumab and durvalumab respectively) was used [11,14]. Thereafter cumulative results in this subsetting are not stringent, pertains only the overall general involvement of cheek-point inhibitors molecules in the maintenance treatment of upper GI tumor, but did not give additional information on the performance of each targetable checkpoint-pathway. No taxanes or anthracyclines was used in first line chemotherapy regimens. No study used atezolizumab, nivolumab, or pembrolizumab as a maintenance treatment in the investigational arm [10,11,14].
Primary outcome of the pooled analyses was to assess the cumulative actually available randomized evidence for overall survival and progression free survival for the use of maintenance check point inhibitor immunotherapy in the treatment of esophago-gastric cancer when compared to standard chemotherapy alone. Stratification by histology was not possible since from inclusion criteria all patients should have had adenocarcinoma histology. Comprehensive analyses for additional response rate were part of our secondary outcomes. Since we are dealing with maintenance studies, we define additional responses the additional objective responses achieved after the randomization for the maintenance treatment after the first line regimen (induction regimen).

Second line treatment:\
Calculation of cumulative available randomized evidence (Pooled Analysis) for the use of immunotherapy compared to standard chemotherapy c) Descriptive analysis The systematic review of literature identified overall five randomized trials comparing the use of immunotherapy vs. standard chemotherapy in second line management of Upper Gastrointestinal Tract carcinomas [5][6][7][8]19]. Data presented at ASCO 2021 congress identified one more trial [20].
The ESCORT trial (NCT03099382) was an open label phase III randomized trial. Patients with advanced/metastatic esophageal squamous cell carcinoma were recruited from 43 hospitals in China between May 2017 to July, 2018 [8], and randomized to receive either camrelizumab200mg (every two weeks OR the investigators' choice chemotherapy (Docetaxel 75 mg/m² on day 1 of each 3week cycle, OR irinotecan 180 mg/m², on day 1 of each 2-week cycle) as second line treatment for the disease. Randomization assigned (1:1) was done using the Randomization and Trial Supply Management system (RTSM system; Bioknow, Beijing, China) via central block randomization method. The randomization sequences were generated by a randomization specialist of the sponsor in SAS (version 9.4). The sponsor's study team was masked to treatment allocation. The primary endpoint was overall survival. In total 457 patients were randomized, 229 in the experimental arm and 228 in the control arm, nonetheless only 228 and 220 received the allocated regimens (6 consent withdrawn, 2 inclusion criteria violation, 1 other reason) [8]. The ATTRACTION-3trial (NCT02569242) was an open-label randomized phase 3 trial. Patients were recruited from 90centers in Asia, Europe and North America (Denmark, Germany, Italy, Japan, South Korea, Taiwan, the UK, and the USA) [6]. Between January 2016, and May 2017,419 patients affected by advanced esophageal squamous cell carcinoma were randomly (1:1) assigned to receive second line treatment either with nivolumab 240 mg every 2 weeks (n =210), or investigator's choice of chemotherapy (paclitaxel 100 mg/m2 once per week for 6 weeks followed by 1 week off OR docetaxel 75 mg/m2 every 3 weeks) n=209. Of those 1 patients per arm did not receive the allocated regimen because of death (investigational arm) and decision withdrawal (chemotherapy arm) [6]. Randomization was done using an interactive web response system with a block size of four; an authorized vendor generate the sequentially numbered containers to ensure random allocation, and to assign patients to study treatments; and the web registration system ensured that the container sequence was concealed until the treatment allocation was completed. The primary endpoint was overall survival [6].. The KEYNOTE181 trial (NCT03933449), was a global, open-label randomized phase 3 trial. Patients with advanced or metastatic esophageal cancer that progressed after one line of prior therapy were recruited from154 centers in 32 countries between December 2015 and June 2017 [5]. Both squamous and adenocarcinoma histology were allowed.
Overall, 628 patients were randomly assigned 1:1 to pembrolizumab 200 mg every 3 weeks vs. investigator's choice of standard of care chemotherapy (paclitaxel 80-100 mg/m2 on days 1, 8, and 15 of each 28-day cycle, OR docetaxel 75 mg/m2 on day 1 of each 21-day cycle, OR irinotecan 180 mg/m2 on day 1 of each 14-day cycle [5]. Among those 314 patients were randomized to the pembrolizumab arm and 314 to chemotherapy arm; 4 patients in the chemotherapy arm did not receive the allocated treatment. and The study had three primary end points: overall survival in patients with PD-L1 CPS >10, in patients with squamous cell carcinoma, and in all patients. The KEYNOTE 061 trial (NCT02370498), was a global randomized, open-label, phase 3 study. Patients with advanced gastric or gastroesophageal junction cancer that progressed after one line of prior therapy were recruited from 148 centers in 30 countries between June 2015 and July 2016 [7]. Only adenocarcinoma histology was allowed. Overall, 592 patients were randomly assigned 1:1 using a central interactive voice-response and integrated web-response system to receive pembrolizumab 200 mg every 3 weeks (n = 296) or to paclitaxel 80 mg/m2 intravenously on days 1, 8, and 15 of 4-week cycles (n = 296).Of those only 294 / 296 in the experimental arm and 276/296 received the allocated treatment. Reasons of not receiving allocated treatment were not described. The allocation schedule was generated by the system vendor using a computerized random list generator. Primary endpoints were overall survival in patients with a PD-L1 CPS of 1 or higher. The KEYNOTE 063 trial (NCT03019588) was an Asian, multicenter, open-label randomized phase 3 trial; patients with advanced gastric or gastro-esophageal junction adenocarcinoma that progressed after one line of prior therapy should have been recruited from 36 centers in China, Korea, Taiwan and Malaysia from February 2017 Nonetheless, the study was prematurely stopped after the presentation of the results from the KEYNOTE-061 where not survival benefit of pembrolizumab vs. paclitaxel was demonstrated [7], Overall 94 patients were recruited and 1:1randomized to receive pembrolizumab 200 mg every 3 weeks (n = 47) or to standard dose paclitaxel [19]. Primary endpoints were both overall survival and PFS [29]. Only data from an abstract presentation to ASCO 2020 were available for this study [19]. The RATIONALE 302 trial (NCT03430843) [20] is a global randomized phase 3 study involving 132 sites in 10 countries. Adults with histologically confirmed advanced/unresectable or metastatic ESCC whose disease progressed following prior systemic therapy, entered the study [20,30]. Overall, 512 patients were 1:1 randomized to receive Tislelizumab 200 mg intravenously every 3 weeks (n=256) or investigator-chosen standard chemotherapy -paclitaxel, docetaxel, or irinotecan-(n=256) and treated until disease progression, unacceptable toxicity, or withdrawal. Primary endpoints was overall survival in all randomized patients [20]. Only data from an abstract presentation to ASCO 2021 were available for this study [20,30] d) Studies overview: All six trials analyzed were multicenter phase 3 randomized controlled trials, including OS in their primary outcomes, and relative analysis was performed with intention-to-treat (ITT) method [5][6][7][8]19,20]. Four of these were global [5][6][7]20], one Asian [19] and one was a national Chinese study [8]. All studies described the mode of randomization [5][6][7][8]19,20], nonetheless the exact randomization process was correctly described only three trials [6][7][8]. Accordingly, the role of funding sources was described in all completed trials in their full text publication [5][6][7][8], but not in the Asian trials that was prematurely interrupted and for which we have only an abstract presentation at last ASCO and some details from the NCT03019588 registration [19]. Correct withdrawals description was available only in two trials [6,8]. In one trial, 7% of the patients in the chemotherapy arm did not receive the allocated treatment [7]. Quality of life assessment part of the study outcomes only in two studies [6,8]. All six trials compared IO monotherapy vs. standard chemotherapy as second line treatment [5][6][7][8]19,20]. Four studies included patients with esophageal carcinomas [5,6,8]; three of them only with squamous histology [6,8]. The other two trials enrolled patients with G/GEJ adenocarcinomas [7,19]. In the interventional arms, one study used Camrelizumab [8], one Nivolumab [6], one Tislelizumab [20] and 3 Pembrolizumab [5,7,19] while as chemotherapy comparator a taxane based regimen (paclitaxel or docetaxel) was used in 5 studies [5][6][7][8]19], irinotecan based regimen was feasible in three trials [5,8,20].
Overall 2702 were enrolled and randomized across five trials and were eligible for cumulative analyzes since randomized to immunotherapy with checkpoint inhibitors (n = 1352) vs. chemotherapy alone (n=1350). Of those, 2651 were treated with the allocated regimens: 1348 (99.7%) in the experimental arms and 1303 (96.5%) in the control arms. Probability for receiving the treatment was statistically lower in the control arm (OR 12.1558, 95% CI: 4.3672 -33.8349, z = 4.782, p < 0001). Overall 51 patients did not received allocated treatment, 4 in experimental arms and 47 in controls arms. Since survival outcomes (OS and PFS) were analyzed in ITT methodology, 2702 patients were eligible for analyses of our primary outcomes; 1348 in the immunotherapy arms and 1303 in the standard chemotherapy arms. Primary outcome of the pooled analyses was to assess the cumulative actually available randomized evidence for overall survival and progression free survival for the use of 2 st line immunotherapy in the treatment of esophago-gastric cancer when compared to standard 2 st line chemotherapy. Secondary outcome was to assess whether the stratification by PD-L1 status, histology and MSI may have any effect on the calculated comprehensive randomized evidence for overall survival and progression free survival. Comprehensive analyses for response rate were also part of our secondary outcome.

Supplementary Tables
Supplementary Table 1        A)1st line, all studies included; B) 1st line, only patients with PD-L1 CPS ≥ 1% were included; C) 1st line, only patients with PD-L1 CPS ≥ 5 or 10% were included; D) 1st line, patients with any PD-L1 CPS were included (i.e. Keynote 062 reporting only patients with CPS >1 was excluded); E) 1st line, patients with adenocarcinomas were included; F) 1st line, patients with squamous cell carcinomas were included The Inverse-Variance (IV) statistical method was applied for calculation of pooled Hazard Ratios (HRs). The Fixed Effects (FE) model was adopted to estimate the pooled ratios. A) all studies included; B) patients with any PD-L1 CPS were included (i.e. Checkmate 649 and Keynote 062 were excluded); C) patients with PD-L1 CPS ≥ 5% or 10% were included.
The Mantel-Haenszel (MH) statistical method was applied for calculation of pooled odds ratios. No heterogeneity was detected, so the Fixed Effects (FE) model was adopted to estimate the pooled ratios. The Inverse-Variance (IV) statistical method was applied for calculation of pooled Hazard Ratio (HR). No heterogeneity was detected, so the Fixed Effects (FE) model was adopted to estimate the pooled ratio.
Abbreviations: IV: Inverse-Variance; HR: Hazard Ratio; CI: confidence intervals; FE: Fixed Effects Supplementary Figure 6. Pooled Hazard Ratio for Progression Free Survival of studies evaluating maintenance with immune checkpoint inhibitors in upper gastrointestinal malignancies. The error bars represent the 95% confidence intervals. A 2-sided p value < .05 was considered significant.
The Inverse-Variance (IV) statistical method was applied for calculation of pooled Hazard Ratio (HR). Heterogeneity was detected, so the Random Effects (RE) model was adopted to estimate the pooled ratio. The 95% confidence intervals (CIs) were used for the analysis.