PURPOSE:Anti-PD-L1 antibodies are associated with responses in <25% of patients with metastatic human papillomavirus-associated malignancies. VEGF signaling causes immune evasion and immune suppression within the tumor. We evaluated the anti-PD-L1 antibody atezolizumab and anti-VEGF antibody bevacizumab for patients with unresectable, advanced anal cancer. PATIENTS AND METHODS:For this phase II study, participants with previously treated, immunotherapy-naïve anal cancer received atezolizumab (1,200 mg) and bevacizumab (15 mg/kg) intravenously every 21 days. Responses were evaluated every 9 weeks (RECIST version 1.1). The primary endpoint was the best radiographic response. Median survival was estimated by Kaplan-Meier and compared for selected biomarkers (including paired pre- and on-treatment biopsies) using a log-rank test. RESULTS:Among 20 participants, the overall response rate was 11% [95% confidence interval (CI): 1.2-32]. Median progression-free survival and overall survival were 4.1 months (95% CI, 2.6-not assessable) and 11.6 months (95% CI, 9.5-20), respectively. One grade 5 bevacizumab-related bowel perforation occurred. Analyses of 16 paired biopsies linked increases in IFN-γ (P = 0.03) and inflammatory response (P = 0.02) gene expression signatures with prolonged progression-free survival, as did increases in CD3+CD8+PD1+ (P = 0.02) cells and decreases in CD3+FoxP3+ cells (P = 0.04) from 10 paired biopsies with multiplex immunofluorescence. A subgroup of anal cancers characterized by the SBS31 "prior-platinum" signature demonstrated shorter median overall survival (HR, 6.3; 95% CI, 1.2-32; P = 0.01). CONCLUSIONS:Atezolizumab and bevacizumab demonstrate activity similar to anti-PD-1 antibodies alone for unresectable anal cancer. Our translational data identify undescribed chromosomal and transcriptomic biomarkers associated with survival for metastatic anal cancer. These correlative findings warrant confirmation and further validation in larger, prospective immunotherapy trials for advanced anal cancer.

Gallbladder carcinoma is a deadly disease with a poor prognosis, and recent clinical data suggest only a modest benefit of PD1/PDL1 inhibitors in this disease. Optimizing immunotherapeutic approaches will require a detailed understanding of the immunogenomic landscape of this disease worldwide. We combined targeted next-generation sequencing and immunohistochemistry to create detailed immunogenomic landscapes from 2 cohorts of gallbladder cancer cases from the United States (n = 60) and Chile (n = 62). Mutations in TP53, SMAD4, KRAS, PIK3CA, ARID2, ARID1A, ATM, FBXW7, ERBB2, and NF1 were found in both the US and Chilean primary cohorts, as well as amplifications in ERBB2, CCNE1, MDM2/CDK4, and CCND1. Despite similar mutation profiles, the immune profiles were distinct, with the Latin American cohort having higher densities of biomarkers associated with CD4+ T cells and PD-1 but lower densities of CD68+ macrophages compared with the North American cohort. Clustering and correlation analyses suggest novel immune subgroups and clinical associations independently of any specific mutations. Additionally, supported by multiplexed single-cell imaging technology, we identified low CD4 and high V-domain Ig suppressor of T cell activation as a candidate biomarker pair of poor outcomes. In summary, our findings highlight the importance of sensitivity to geographic location when considering therapeutic developments and pave a path for further immune investigations of this understudied disease.

Background: The prognosis for patients with AML and MDS who relapse after allogeneic stem cell transplant (allo-SCT) is dismal. AML is susceptible to immunotherapy, as evidenced by the success of allo-SCT. Enhancing graft-versus-leukemia effect of allo-SCT is a major area of focus. We investigated combination immune checkpoint inhibition (ICI) using ipilimumab (Ipi) and nivolumab (Nivo) in a phase I dose escalation trial enrolling AML/MDS patients who relapsed after allo-SCT. Patients and Methods: For AML, evidence of relapse included bone marrow blast count ≥5% and/or presence of extramedullary disease and/or presence of marrow measurable residual disease (MRD) by multicolor flow cytometry. For MDS, evidence of relapse included appearance of dysplastic changes in the marrow, with or without increase in marrow blast count. Patients, ≥18 years-old, with a matched or haploidentical related donor, or 8/8 HLA matched unrelated donor (MUD) received Nivo (1mg/Kg) monotherapy administered on Day 1 and 15 of a 4 week cycle; ipilimumab (1mg/Kg) monotherapy administered on day 1 of a 3 week cycle; or combination Nivo (1 or 3 mg/Kg) + Ipi (1 or 3 mg/Kg), with Nivo and Ipi administered on day 1, followed by Nivo on days 15 and 29 of a 6 week cycle. Drug(s) were started > 42 days after stem cell infusion. Graft versus host disease (GVHD) prophylaxis was post-transplant cyclophosphamide 50 mg/kg on day +3 and +4, and tacrolimus ± MMF. Patients with history of >grade 2 GVHD were excluded. The primary outcome was to determine maximum tolerated dose (MTD) and dose limiting toxicity of monotherapy and combination Ipi + Nivo. ClinicalTrials.gov: NCT03600155. Results: 29 patients, 10 females and 19 males, with a median age of 59 (27-76) years were enrolled between April 2019 and May 2023. Of the 29 patients, 20 (69%) had AML, 7 (24%) had MDS, and 2 patients (7%) had CMML. Twenty-six (90%) patients had marrow disease only, 1 patient (3%) had extramedullary disease, and 2 patients (7%) had involvement of both. Twenty-two (76%) patients with AML and MDS had morphologic relapse, and 7 (24%) AML patients had MRD by flow cytometry. Five patients (33%) had a history of grade I GVHD and 10 (67%) patients had a history of grade II GVHD; in all cases, symptomatic GVHD resolution was achieved prior to study initiation. The median time from administration of SCT to study drug infusion was 179 days (range, 54-987). Seventeen (59%) patients were on GVHD prophylaxis during the study. Twenty-seven patients (93%) had received post SCT cyclophosphamide. Four patients (14%) developed GVHD on study. Of the 4 patients who developed GVHD, 2 (50%), 1 (25%) and 1(25%) patients developed grade II (skin and upper GI [UGI]), III (UGI and lower GI [LGI]) and IV (LGI) GVHD, respectively. Twenty-nine (100%), 28 (97%), 29 (100%), 16 (55%), and 7 (24%) patients experienced grades I, II, III, IV and V toxicities, respectively. Typical adverse events associated with ICI included: pneumonitis (n=3; 10%), 2 (7%) grade II and 1 (3%) grade III; colitis (n=3; 10%), 1 (3%) grade II, 1 (3%) grade III and 1 (3%) grade IV; hepatitis (n=6; 21%), 2 (7%) grade 1, 2 (7%) grade II, 1 (3%) grade III and 1 (3%) grade IV; endocrinopathy (n=3; 10%), 1 (3%) grade I, 1 (3%) grade II, and 1 (3%) grade III; dermatitis (n=4; 14%), 1(3%) grade 1, and 3 (10%) grade III. Dose limiting toxicities were noted at combination nivo 1 mg/Kg + Ipi 3 mg/Kg, mandating Ipi dose reduction to 1mg/Kg. During the study period, median follow-up was 65 days (range, 9-277 days); 13 patients experienced disease progression, GVHD, or death. Median PFS, estimated using Kaplan-Meier method, was 109 days (95%CI: 48 - NA days). Nine (31%) patients achieved stable disease or remission: 4 patients on Nivo 1 mg/Kg arm, 2 patients on Ipi 1 mg/Kg arm, 2 patients on Nivo + Ipi 1 mg/Kg arm, and 1 patient on the Nivo 3 mg/Kg + Ipi 1 mg/Kg arm. Patients with MDS had a longer PFS (HR = 0.15; CI, 0.02-1.18; p=0.03) vs. patients with AML. Of patients with extramedullary disease, 1 patient had CR of skin lesions and the second patient had a mixed response; both patients received Nivo 1mg/Kg + Ipi 1mg/Kg arm. Comprehensive immune profiling using CyTOF enabled the identification of distinct stem cell subsets and myeloid and lymphoid populations in bone marrow aspirates and circulating blood. Conclusion: This study met its primary objective of determining the MTD of the combination regimen with encouraging safety and efficacy outcomes. Our results warrant further study of this regimen.

<p>Clinical and pathological features of triple negative patient cohort used for TMA.</p>

<p>Plasmids construction; Cell proliferation assay; Migration assay and invasion assay; Transient transfection and luciferase reporter assay; Flow cytometry; Animal experiments; Case Selection, Tissue Microarray (TMA) Construction and analysis</p>

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.