Tumor and immune remodeling following radiotherapy in human renal cell carcinoma

Background Studies evaluating peripheral patient samples show radiation can modulate immune responses, yet the biological changes in human tumors particularly at the cellular level remain largely unknown. Here, we address how radiation treatment shapes the immune compartment and interactions with cancer cells within renal cell carcinoma (RCC) patient tumors.Methods To identify how radiation shaped the immune compartment and potential immune interactions with tumor cells we evaluated RCC tumors from patients treated only with nephrectomy or with radiation followed by nephrectomy. Spectral flow cytometry using a 35-marker panel was performed on cell suspensions to evaluate protein expression within immune subsets. To reveal how radiation alters programming of immune populations and interactions with tumor cells, we examined transcriptional changes by single-cell RNA sequencing (scRNAseq).Results Spectral flow cytometry analysis revealed increased levels of early-activated as well as effector programmed cell death protein-1 (PD-1)+ CD8 T-cell subsets within irradiated tumors. Following quality control, scRNAseq of tumor samples from nephrectomy-only or radiation followed by nephrectomy-treated patients generated an atlas containing 34,626 total cells. Transcriptional analysis revealed increased transition from stem-like T-cell populations to effector T cells in irradiated tumors. Interferon (IFN) pathways, that are central to radiation-induced immunogenicity, were enriched in irradiated lymphoid, myeloid, and cancer cell populations. Focused cancer cell analysis showed enhanced antigen presentation and increased predicted TRAIL-mediated and IFN-mediated interactions between tumor cells and the same effector T-cell subsets increased by radiation. TRAIL and IFN pathways enriched in irradiated tumors were associated with survival in patients treated with immunotherapy.Conclusions These findings identify the source of IFN enrichment within irradiated RCC and reveal heightened levels of PD-1+ CD8+ T-cell subsets and increased probability of interactions with tumor cells following standalone radiation treatment. This study provides a window into the irradiated tumor-immune microenvironment of patients and rationale for treatment combinations

Systemic infusion of TLR3-ligand and IFN-α in patients with breast cancer reprograms local tumor microenvironments for selective CTL influx

Background Presence of cytotoxic T lymphocytes (CTL) in the tumor microenvironment (TME) predicts the effectiveness of cancer immunotherapies. The ability of toll-like receptor 3 (TLR3) ligands, interferons (IFNs) and COX2 inhibitors to synergistically induce CTL-attracting chemokines (but not regulatory T cell (Treg)-attractants) in the TME, but not in healthy tissues, observed in our preclinical studies, suggested that their systemic application can reprogram local TMEs.Methods Six evaluable patients (33–69 years) with metastatic triple-negative breast cancer received six doses of systemic chemokine-modulating (CKM) regimen composed of TLR3 ligand (rintatolimod; 200 mg; intravenous), IFN-α2b (20 MU/m2; intravenous) and COX2 inhibitor (celecoxib; 2×200 mg; oral) over 2 weeks. The predetermined primary endpoint was the intratumoral change in the expression of CTL marker, CD8α, in the post-CKM versus pre-CKM tumor biopsies. Patients received follow-up pembrolizumab (200 mg, intravenously, every 3 weeks), starting 3–8 days after completion of CKM.Results Post-CKM biopsies showed selectively increased CTL markers CD8α (average 10.2-fold, median 5.5-fold, p=0.034) and granzyme B (GZMB; 6.1-fold, median 5.8-fold, p=0.02), but not FOXP3 (Treg marker) relative to HPRT1 expression, resulting in the increases in average CD8α/FOXP3 ratio and GZMB/FOXP3 ratio. CKM increased intratumoral CTL-attractants CCL5 and CXCL10, but not Treg-attractants CCL22 or CXCL12. In contrast, CD8+ T cells and their CXCR3+ subset showed transient decreases in blood. One clinical response (breast tumor autoamputation) and three stable diseases were observed. The patient with clinical response remains disease free, with a follow-up of 46 months as of data cut-off.Conclusions Short-term systemic CKM selectively increases CTL numbers and CTL/Treg ratios in the TME, while transiently decreasing CTL numbers in the blood. Transient effects of CKM suggest that its simultaneous application with checkpoint blockade and other forms of immunotherapy may be needed for optimal outcomes

Evaluation of multiple myeloma measurable residual disease by high sensitivity flow cytometry: An international harmonized approach for data analysis

Background Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression-free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter-laboratory variation in MM MRD reporting. Methods Seventeen participants from 13 countries performed blinded analysis of the same eight de-identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re-analyzed for MRD and other bone marrow cells (Stage 2). Inter-laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3). Results In Stage 1, participants agreed on MRD+/MRD- status 89% and 68% of the time respectively. Inter-observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD- status improved to 74%. Better consistency enumerating all parameters among operators resulted in near-unanimous agreement on sample adequacy. Conclusion Uniform flow cytometry data analysis substantially reduced inter-laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint

A phase 1/2 clinical trial of invariant natural killer T cell therapy in moderate-severe acute respiratory distress syndrome

Abstract Invariant natural killer T (iNKT) cells, a unique T cell population, lend themselves for use as adoptive therapy due to diverse roles in orchestrating immune responses. Originally developed for use in cancer, agenT-797 is a donor-unrestricted allogeneic ex vivo expanded iNKT cell therapy. We conducted an open-label study in virally induced acute respiratory distress syndrome (ARDS) caused by the severe acute respiratory syndrome-2 virus (trial registration NCT04582201). Here we show that agenT-797 rescues exhausted T cells and rapidly activates both innate and adaptive immunity. In 21 ventilated patients including 5 individuals receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO), there are no dose-limiting toxicities. We observe an anti-inflammatory systemic cytokine response and infused iNKT cells are persistent during follow-up, inducing only transient donor-specific antibodies. Clinical signals of associated survival and prevention of secondary infections are evident. Cellular therapy using off-the-shelf iNKT cells is safe, can be rapidly scaled and is associated with an anti-inflammatory response. The safety and therapeutic potential of iNKT cells across diseases including infections and cancer, warrants randomized-controlled trials

A phase 1/2 clinical trial of invariant natural killer T cell therapy in moderate-severe acute respiratory distress syndrome

Invariant natural killer T (iNKT) cells, a unique T cell population, lend themselves for use as adoptive therapy due to diverse roles in orchestrating immune responses. Originally developed for use in cancer, agenT-797 is a donor-unrestricted allogeneic ex vivo expanded iNKT cell therapy. We conducted an open-label study in virally induced acute respiratory distress syndrome (ARDS) caused by the severe acute respiratory syndrome-2 virus (trial registration NCT04582201). Here we show that agenT-797 rescues exhausted T cells and rapidly activates both innate and adaptive immunity. In 21 ventilated patients including 5 individuals receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO), there are no dose-limiting toxicities. We observe an anti-inflammatory systemic cytokine response and infused iNKT cells are persistent during follow-up, inducing only transient donor-specific antibodies. Clinical signals of associated survival and prevention of secondary infections are evident. Cellular therapy using off-the-shelf iNKT cells is safe, can be rapidly scaled and is associated with an anti-inflammatory response. The safety and therapeutic potential of iNKT cells across diseases including infections and cancer, warrants randomized-controlled trials.</p

No Evidence That Genetic Variation in the Myeloid-Derived Suppressor Cell Pathway Influences Ovarian Cancer Survival.

Background: The precise mechanism by which the immune system is adversely affected in cancer patients remains poorly understood, but the accumulation of immunosuppressive/protumorigenic myeloid-derived suppressor cells (MDSCs) is thought to be a prominent mechanism contributing to immunologic tolerance of malignant cells in epithelial ovarian cancer (EOC). To this end, we hypothesized genetic variation in MDSC pathway genes would be associated with survival after EOC diagnoses.Methods: We measured the hazard of death due to EOC within 10 years of diagnosis, overall and by invasive subtype, attributable to SNPs in 24 genes relevant in the MDSC pathway in 10,751 women diagnosed with invasive EOC. Versatile Gene-based Association Study and the admixture likelihood method were used to test gene and pathway associations with survival.Results: We did not identify individual SNPs that were significantly associated with survival after correction for multiple testing (P &lt; 3.5 × 10-5), nor did we identify significant associations between the MDSC pathway overall, or the 24 individual genes and EOC survival.Conclusions: In this well-powered analysis, we observed no evidence that inherited variations in MDSC-associated SNPs, individual genes, or the collective genetic pathway contributed to EOC survival outcomes.Impact: Common inherited variation in genes relevant to MDSCs was not associated with survival in women diagnosed with invasive EOC. Cancer Epidemiol Biomarkers Prev; 26(3); 420-4. ©2016 AACR

No evidence that genetic variation in the myeloid-derived suppressor cell pathway influences ovarian cancer survival

The precise mechanism by which the immune system is adversely affected in cancer patients remains poorly understood, but the accumulation of immune suppressive/pro-tumorigenic myeloid-derived suppressor cells (MDSCs) is thought to be one prominent mechanism contributing to immunologic tolerance of malignant cells in epithelial ovarian cancer (EOC). To this end, we hypothesized genetic variation in MDSC pathway genes would be associated with survival after EOC diagnoses.status: publishe

No evidence that genetic variation in the myeloid-derived suppressor cell pathway influences ovarian cancer survival

Background: The precise mechanism by which the immune system is adversely affected in cancer patients remains poorly understood, but the accumulation of immune suppressive/pro-tumorigenic myeloid-derived suppressor cells (MDSCs) is thought to be one prominent mechanism contributing to immunologic tolerance of malignant cells in epithelial ovarian cancer (EOC). To this end, we hypothesized genetic variation in MDSC pathway genes would be associated with survival after EOC diagnoses. Methods: We measured the hazard of death due to EOC within 10 years of diagnosis, overall and by invasive subtype, attributable to SNPs in 24 genes relevant in the MDSC pathway in 10,751 women diagnosed with invasive EOC. Versatile Gene-based Association study (VEGAS) and the Admixture Likelihood method (AML), were used to test gene and pathway associations with survival. Results: We did not identify individual SNPs that were significantly associated with survival after correction for multiple testing (p&lt;3.5 x 10-5), nor did we identify significant associations between the MDSC pathway overall, or the 24 individual genes and EOC survival. Conclusions: In this well-powered analysis, we observed no evidence that inherited variations in MDSC-associated SNPs, individual genes, or the collective genetic pathway contributed to EOC survival outcomes. Impact: Common inherited variation in genes relevant to MDSCs were not associated with survival in women diagnosed with invasive EOC

No Evidence That Genetic Variation in the Myeloid-Derived Suppressor Cell Pathway Influences Ovarian Cancer Survival.

Background: The precise mechanism by which the immune system is adversely affected in cancer patients remains poorly understood, but the accumulation of immunosuppressive/protumorigenic myeloid-derived suppressor cells (MDSCs) is thought to be a prominent mechanism contributing to immunologic tolerance of malignant cells in epithelial ovarian cancer (EOC). To this end, we hypothesized genetic variation in MDSC pathway genes would be associated with survival after EOC diagnoses.Methods: We measured the hazard of death due to EOC within 10 years of diagnosis, overall and by invasive subtype, attributable to SNPs in 24 genes relevant in the MDSC pathway in 10,751 women diagnosed with invasive EOC. Versatile Gene-based Association Study and the admixture likelihood method were used to test gene and pathway associations with survival.Results: We did not identify individual SNPs that were significantly associated with survival after correction for multiple testing (P &lt; 3.5 × 10-5), nor did we identify significant associations between the MDSC pathway overall, or the 24 individual genes and EOC survival.Conclusions: In this well-powered analysis, we observed no evidence that inherited variations in MDSC-associated SNPs, individual genes, or the collective genetic pathway contributed to EOC survival outcomes.Impact: Common inherited variation in genes relevant to MDSCs was not associated with survival in women diagnosed with invasive EOC. Cancer Epidemiol Biomarkers Prev; 26(3); 420-4. ©2016 AACR