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Immune checkpoint inhibitors (ICI) widely improved the treatment of solid and hematologic malignancies. Yet, a remarkable proportion of patients receiving ICI develop immune related adverse events (irAEs) which are difficult to define as treatment-related. This underlines the need to develop a biomarker to guide irAE diagnosis. We developed a novel flow cytometry assay combining measurement of anti-PD-1 (programmed cell death protein-1) occupancy and evaluation of remaining PD-1 receptor availability with anti-IgG4 PE and anti-PD-1 BV421. We prospectively collected blood and biological fluids samples from patients treated by IgG4 anti-PD-1 therapy (nivolumab or pembrolizumab), with (n=18) or without (n=12) current irAE. We analyzed PD-1+ and IgG4+ staining pattern and MFI values of these parameters on CD4 and CD8 T cells, and IgG4+/PD-1+ MFI ratios are calculated. A higher mean fluorescence intensity IgG4+/PD-1+ ratio was measured on peripheral CD4+ T cells of irAE cases, when compared to controls (p=0.003). ICI-related toxicity is therefore associated with increased therapeutic antibody occupancy of PD-1 receptors on CD4+ T cells. Furthermore, in one case of ICI-related pneumonitis, binding of therapeutic antibody was stronger on lung CD4+ T cell than in blood. In another case of ICI-related encephalitis, the PD-1 receptor occupancy was total on CSF CD4 T cells, but only partial on peripherical CD4 T cells. Our results suggest that flow cytometry monitoring of ICI occupancy can be used in patients treated with monoclonal ICI to guide irAE diagnosis.

BackgroundSquamous carcinoma of the anal canal (SCAC) is a human papillomavirus (HPV)-driven cancer with poor prognosis in locally advanced or recurrent settings. Carboplatin–paclitaxel is the preferred first-line regimen for unresectable locally advanced or metastatic SCAC, with the reported median progression-free survival (PFS) and overall survival (OS) of 8.1 and 20.0 months, respectively. Immune checkpoint blockade (ICB) demonstrates improved survival in HPV-driven cervical and head and neck cancers. Retifanlimab (INCMGA00012) is an investigational humanized, hinge-stabilized, immunoglobulin G4κ monoclonal antibody targeting programmed cell death-1 (PD-1), with characteristics common to the ICB class. In POD1UM-202, retifanlimab showed substantial clinical activity and an expected safety profile in patients with advanced SCAC who progressed on platinum-based chemotherapy. Based on these encouraging results, POD1UM-303/InterAACT 2 (NCT04472429), a phase III, double-blind, randomized, multiregional study, investigates the addition of retifanlimab to the standard of care (SOC) carboplatin–paclitaxel in patients with inoperable locally recurrent or metastatic SCAC not previously treated with systemic chemotherapy.Methods and analysisPatients ≥18 years with inoperable locally recurrent or metastatic SCAC, measurable disease per RECIST v1.1, and no prior systemic chemotherapy or PD-(L)1-directed therapy will be enrolled and stratified by PD-L1 expression, region, and extent of disease. Patients with well-controlled human immunodeficiency virus infection are eligible. Planned enrollment is approximately 300 patients worldwide, with a 1:1 randomization to retifanlimab or placebo. Patients will receive up to six induction cycles (24 weeks) of carboplatin (area-under-the-curve 5 on day 1) and paclitaxel (80 mg/m2 on days 1, 8, and 15) every 28 days per SOC. Concurrently, retifanlimab 500 mg or placebo will be administered intravenously in a blinded fashion on day 1 of each 28-day cycle for up to 13 cycles (1 year) in the absence of unacceptable toxicity, disease progression, withdrawal of consent, loss to follow-up, or premature discontinuation. Crossover to open-label retifanlimab will be allowed for patients assigned to placebo upon verification of progression by blinded independent central radiographic review (BICR). The primary study endpoint is PFS per RECIST v1.1 by BICR. Secondary endpoints are OS, objective response rate, duration of response, disease control rate, safety, and retifanlimab pharmacokinetics. The study is currently recruiting.Clinical Trial Registrationhttps://clinicaltrials.gov/ct2/show/NCT04472429; https://clinicaltrialsregister.eu/ctr-search/search?query=2020-000826-24

Cell-free DNA (cfDNA) analysis is a minimally invasive method that can be used to detect genomic abnormalities by directly testing a blood sample. This method is particularly useful for immunosuppressed patients, who are at high risk of complications from tissue biopsy. The cfDNA tumor fraction (TF) varies greatly across cancer type and between patients. Thus, the detection of molecular alterations is highly dependent on the circulating TF. In our study, we aimed to calculate the TF and characterize the copy number aberration (CNA) profile of cfDNA from patients with rare malignancies occurring in immunosuppressed environments or immune-privileged sites. To accomplish this, we recruited 36 patients: 19 patients with non-Hodgkin lymphoma (NHL) who were either human immunodeficiency virus (HIV)-positive or organ transplant recipients, 5 HIV-positive lung cancer patients, and 12 patients with glioma. cfDNA was extracted from the patients’ plasma and sequenced using low-coverage whole genome sequencing (LC-WGS). The cfDNA TF was then calculated using the ichorCNA bioinformatic algorithm, based on the CNA profile. In parallel, we performed whole exome sequencing of patient tumor tissue and cfDNA samples with detectable TFs. We detected a cfDNA TF in 29% of immune-suppressed patients (one patient with lung cancer and six with systemic NHL), with a TF range from 8 to 70%. In these patients, the events detected in the CNA profile of cfDNA are well-known events associated with NHL and lung cancer. Moreover, cfDNA CNA profile correlated with the CNA profile of matched tumor tissue. No tumor-derived cfDNA was detected in the glioma patients. Our study shows that tumor genetic content is detectable in cfDNA from immunosuppressed patients with advanced NHL or lung cancer. LC-WGS is a time- and cost-effective method that can help select an appropriate strategy for performing extensive molecular analysis of cfDNA. This technique also enables characterization of CNAs in cfDNA when sufficient tumor content is available. Hence, this approach can be used to collect useful molecular information that is relevant to patient care.