Genetic Variation and Regulation of MICA Alters Natural Killer Cell-Mediated Immunosurveillance in Early-Onset Colorectal Cancer.

The incidence of colorectal cancer (CRC) among individuals under age 50, or early-onset CRC (EOCRC), has been rising over the past few decades for unclear reasons, and the etiology of the disease remains largely unknown. Known genetic risk factors do not explain this increase, pointing to possible environmental and as-yet unidentified genetic contributors and their interactions. Previous research linked genetic variation on chromosome 6 to increased CRC risk. This region harbors multiple immune genes, including the gene encoding Major Histocompatibility Complex (MHC) class I polypeptide-related sequence A (MICA). MICA is a polygenic ligand for the Natural Killer Group 2D receptor (NKG2D), a receptor expressed on Natural Killer (NK) cells and other lymphocytes. Given that intra-tumoral NK cell infiltration correlates with favorable CRC outcomes, we hypothesized that germline genetic variation in MICA could influence CRC risk. In a discovery set of 40,125 cases and controls, we show that the minor G allele at Chr6:31373718C>G (hg19) is associated with increased risk for CRC (odds ratio (OR) = 1.09, 95% confidence interval (CI) 1.04 - 1.15, p = 0.0009). The effect is stronger in EOCRC (OR = 1.26, 95% CI 1.08 - 1.44, p = 0.0023) than in those 50 and over (OR = 1.07, 95% CI 1.02 - 1.13; p = 0.012) (Ratio of ORs = 1.32, 95% CI 1.14 - 1.52, p = 0.0002). In an independent validation set of 77,983 cases and controls, the adjusted interaction by age-of-onset was significant at OR = 1.15 (95% CI 1.03 - 1.34, p = 0.0150) with a higher risk in EOCRC. Expression quantitative trait locus analysis in normal colonic epithelia showed that MICA RNA expression decreases linearly with each additional copy of the minor G allele (p = 3.345 × 10e-18). Bulk RNA analysis of the tumor immune microenvironment revealed that tumors from patients with CG or GG genotypes have lower resting and activated NK cell infiltration as compared to tumors from patients with CC genotype. Multiplex immunofluorescence analysis demonstrated that patients with a G allele (i.e. CG or GG genotype, but not CC genotype) have a statistically significant decrease in the number of NK cells in tumor compared to adjacent normal colonic mucosa. Taken together, population-based epidemiologic, molecular, genetic, cellular and immunologic evidence demonstrate that MICA genotype is associated with increased risk of EOCRC and reduced number of NK cells in colorectal tumors, suggesting that patients with a G allele have altered NK cell-mediated immunosurveillance. These novel findings suggest that EOCRC may have a previously unrecognized innate immune-mediated etiology which merits further investigation.

Tumor draining lymph nodes connected to cold triple-negative breast cancers are characterized by Th2-associated microenvironment.

Tumor draining lymph nodes (TDLN) represent a key component of the tumor-immunity cycle. There are few studies describing how TDLNs impact lymphocyte infiltration into tumors. Here we directly compare tumor-free TDLNs draining "cold" and "hot" human triple negative breast cancers (TDLNCold and TDLNHot). Using machine-learning-based self-correlation analysis of immune gene expression, we find unbalanced intranodal regulations within TDLNCold. Two gene pairs (TBX21/GATA3-CXCR1) with opposite correlations suggest preferential priming of T helper 2 (Th2) cells by mature dendritic cells (DC) within TDLNCold. This is validated by multiplex immunofluorescent staining, identifying more mature-DC-Th2 spatial clusters within TDLNCold versus TDLNHot. Associated with this Th2 priming preference, more IL4 producing mast cells (MC) are found within sinus regions of TDLNCold. Downstream, Th2-associated fibrotic TME is found in paired cold tumors with increased Th2/T-helper-1-cell (Th1) ratio, upregulated fibrosis growth factors, and stromal enrichment of cancer associated fibroblasts. These findings are further confirmed in a validation cohort and public genomic data. Our results reveal a potential role of IL4+ MCs within TDLNs, associated with Th2 polarization and reduced immune infiltration into tumors.

Organ-Specific Immune Setpoints Underlie Divergent Immune Profiles across Metastatic Sites in Breast Cancer.

Immune composition within the tumor microenvironment (TME) plays a central role in the propensity of cancer cells to metastasize and respond to therapy. Previous studies have suggested that the metastatic TME is immune-suppressed. However, limited accessibility to multiple metastatic sites within patients has made assessing the immune TME difficult in the context of multiorgan metastases. We utilized a rapid postmortem tissue collection protocol to assess the immune composition of numerous sites of breast cancer metastasis and paired tumor-free tissues. Metastases had comparable immune cell densities and compositions to paired tumor-free tissues of the same organ type. In contrast, immune cell densities in both metastatic and tumor-free tissues differed significantly between organ types, with lung immune infiltration being consistently greater than that in the liver. These immune profiling results were consistent between flow cytometry and multiplex immunofluorescence-based spatial analysis. Furthermore, we found that granulocytes were the predominant tumor-infiltrating immune cells in lung and liver metastases, and these granulocytes comprised most PD-L1-expressing cells in many tissue sites. We also identified distinct potential mechanisms of immunosuppression in lung and liver metastases, with the lung having increased expression of PD-L1+ antigen-presenting cells and the liver having higher numbers of activated regulatory T cells and HLA-DRlow monocytes. Together, these results demonstrate that the immune contexture of metastases is dictated by organ type and that immunotherapy strategies may benefit from unique tailoring to the tissue-specific features of the immune TME.

PSCA-CAR T cell therapy in metastatic castration-resistant prostate cancer: a phase 1 trial.

Despite recent therapeutic advances, metastatic castration-resistant prostate cancer (mCRPC) remains lethal. Chimeric antigen receptor (CAR) T cell therapies have demonstrated durable remissions in hematological malignancies. We report results from a phase 1, first-in-human study of prostate stem cell antigen (PSCA)-directed CAR T cells in men with mCRPC. The starting dose level (DL) was 100 million (M) CAR T cells without lymphodepletion (LD), followed by incorporation of LD. The primary end points were safety and dose-limiting toxicities (DLTs). No DLTs were observed at DL1, with a DLT of grade 3 cystitis encountered at DL2, resulting in addition of a new cohort using a reduced LD regimen + 100 M CAR T cells (DL3). No DLTs were observed in DL3. Cytokine release syndrome of grade 1 or 2 occurred in 5 of 14 treated patients. Prostate-specific antigen declines (>30%) occurred in 4 of 14 patients, as well as radiographic improvements. Dynamic changes indicating activation of peripheral blood endogenous and CAR T cell subsets, TCR repertoire diversity and changes in the tumor immune microenvironment were observed in a subset of patients. Limited persistence of CAR T cells was observed beyond 28 days post-infusion. These results support future clinical studies to optimize dosing and combination strategies to improve durable therapeutic outcomes. ClinicalTrials.gov identifier NCT03873805 .

Tumor heterogeneity and clinically invisible micrometastases in metastatic breast cancer-a call for enhanced surveillance strategies.

The biology of metastatic breast cancer (MBC) is understudied, primarily due to the difficulty of procuring multiple samples from patients with oligometastatic breast cancer. We developed a rapid postmortem tissue procurement program that allows the collection and analysis of numerous metastatic lesions, subclinical locations, and potential pre-metastatic niches that fall within this scope. We conducted a rapid postmortem tissue collection study on 9 patients with MBC. Patients and their families consented to donate tissues immediately after death in an IRB-approved study. Various disease subtypes, progression histories, organ involvement, and final causes of death are reported. In patients with hormone receptor-positive (HR+) disease, estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki-67 expression were heterogeneous across metastatic lesions within individual patients. Disease phenotype at the end of life trended toward complete loss of HR expression. Nearly all (n = 7) patients exhibited extensive tumor involvement of additional organs that had not been previously diagnosed clinically and were not retrospectively visible on recent imaging. Of these seven individuals, three included organs uncommonly associated with MBC: kidney, spleen, pancreas, and ovary. Finally, we identified clinically undetectable micrometastases in several organs uncommonly involved in MBC. Our findings raise several clinically relevant questions regarding the mechanisms of metastatic progression. Insights from this study argue for better surveillance strategies for monitoring MBC. We highlight the need to capture more accurate biomarker information in the context of heterogeneous disease and urge the consideration of treatment strategies that combine multiple targeted therapies.

STING signalling compensates for low tumour mutation burden to drive anti-tumour immunity.

BACKGROUND: While mutation-derived neoantigens are well recognized in generating anti-tumour T cell response, increasing evidences highlight the complex association between tumour mutation burden (TMB) and tumour infiltrating lymphocytes (TILs). The exploration of non-TMB determinants of active immune response could improve the prognosis prediction and provide guidance for current immunotherapy. METHODS: The transcriptomic and whole exome sequence data in The Cancer Genome Atlas were used to examine the relationship between TMB and exhausted CD8+ T cells (Tex), as an indicator of tumour antigen-specific T cells across nine major cancer types. Computational clustering analysis was performed on 4510 tumours to identify different immune profiles. NanoString gene expression analysis and single cell RNA-seq analysis using fresh human breast cancer were performed for finding validation. FINDINGS: TMB was found to be poorly correlated with active immune response in various cancer types. Patient clustering analysis revealed a group of tumours with abundant Tex but low TMB. In those tumours, we observed significantly higher expression of the stimulator of interferon genes (STING) signalling. Dendritic cells, particularly those of BATF3+ lineage, were also found to be essential for accumulation of Tex within tumours. Mechanistically, loss of genomic and cellular integrity, marked by decreased DNA damage repair, defective replication stress response, and increased apoptosis were shown to drive STING activation. INTERPRETATION: These results highlight that TMB alone does not fully predict tumour immune profiles, with STING signalling compensating for low TMB in non-hypermutated tumours to enhance anti-tumour immunity. Translating these results, STING agonists may benefit patients with non-hypermutated tumours. STING activation may serve as an additional biomarker to predict response to immune checkpoint blockades alongside TMB. Our research also unravelled the interplay between genomic instability and STING activation, informing potential combined chemotherapy targeting the axis of genomic integrity and immunotherapy. FUNDING: City of Hope Christopher Family Endowed Innovation Fund for Alzheimer's Disease and Breast Cancer Research in honor of Vineta Christopher; Breast Cancer Alliance Early Career Investigator Award; National Cancer Institute of the National Institutes of Health under award number R01CA256989 and R01CA240392.

Immunotherapy response in microsatellite stable metastatic colorectal cancer is influenced by site of metastases.

BACKGROUND: Prior studies indicate that colorectal cancer patients with liver metastases did not benefit from regorafenib, nivolumab (REGONIVO) or regorafenib, ipilimumab, nivolumab (RIN) treatments, while those without liver metastases showed significant response. This study explores the impact of metastatic sites on treatment outcomes. METHODS: Chemotherapy-refractory colorectal cancer patients treated with REGONIVO or RIN were evaluated, focusing on 2-month organ-specific response, ORR, PFS and OS based on metastatic sites. RESULTS: Of the 96 patients analyzed (58 REGONIVO, 38 RIN), liver or peritoneal metastases led to poor outcomes, with 0 % ORR, and median PFS of 2.0 and 1.5 months respectively. In contrast, lung-only metastases had an ORR of 56.3 % and a PFS of 14 months. The presence of concurrent LN or other extrahepatic metastatic disease in patients with lung metastatic disease diminished but did not prohibit responses. The 2-month response assessment revealed activity in the lungs, soft tissues, and distant lymph nodes. CONCLUSIONS: REGONIVO and RIN were most active in lung-only metastases. Liver and peritoneal metastases were resistant. Future checkpoint inhibitor trials in MSS colorectal cancer should stratify patients based on metastatic locations.

CF33-hNIS-anti-PD-L1 oncolytic virus followed by trastuzumab-deruxtecan in a patient with metastatic triple negative breast cancer: a case study.

Prognosis of metastatic triple negative breast cancer (mTNBC) remains poor despite recent advances in therapeutic options. Trastuzumab deruxtecan (T-DXd) has shown promising efficacy in patients with human epidermal growth factor receptor 2 (HER2)-low breast cancer, which is defined by immunohistochemistry (IHC) 1+ or 2+ and lack of HER2 amplification by fluorescence in situ hybridization (FISH) testing. The purpose of the study is to evaluate the safety and initial evidence of efficacy of intratumoral administration of CF33-hNIS-anti-PD-L1 (CHECKvacc) against mTNBC. Oncolytic virus CHECKvacc intratumoral injection is currently undergoing investigation in patients with mTNBC as a single agent (NCT05081492). The patient was enrolled on the clinical trial CHECKvacc for the Treatment of Metastatic Triple Negative Breast Cancer, received a single dose of CHECKvacc, and discontinued the study due to lack of immediate response. We report a case of a patient with mTNBC who was heavily pretreated and presented with extensive dermal metastasis. Two dermal metastasis biopsies in 2021 showed HER2 0 by IHC. The patient received a single dose of CHECKvacc and discontinued the study due to lack of immediate response. Twenty-five days later, the patient received treatment with T-DXd, and her tumor regressed significantly. The patient's disease-free survival was 10 months (December 2021-October 2022). The sequential treatment with intratumoral injection of CHECKvacc followed by T-DXd may have significant clinical activity in select patients with heavily pretreated mTNBC. ClinicalTrials.gov NCT05081492.

Peritumoral Immune-suppressive Mechanisms Impede Intratumoral Lymphocyte Infiltration into Colorectal Cancer Liver versus Lung Metastases.

Patients with microsatellite stable (MSS) colorectal cancer with liver metastases are resistant to immune checkpoint inhibitor (ICI) therapy, while about one-third of patients with colorectal cancer without liver metastases, particularly those with lung-only metastases, respond to ICI. We analyzed primary colorectal cancer tumors and major metastatic sites (liver, lung, peritoneal) using multiplex immunofluorescence and whole-slide spatial analyses to identify variations in immune contexture and regional localization within the tumor microenvironment. While levels of T and B cells within peritumoral regions were similar, their levels were significantly lower within the tumor core of liver and peritoneal metastases compared with lung metastases. In contrast, antigen-presenting cells (APC) and APC-T cell interactions were more abundant in all regions of lung metastases. We also identified an abundance of lymphoid aggregates throughout lung metastases, but these were present only within peritumoral regions of liver and peritoneal metastases. Larger lymphoid aggregates consistent with features of tertiary lymphoid structures were observed within or adjacent to primary tumors, but not metastatic lesions. Our findings were validated using NanoString GeoMx DSP, which further showed that liver metastases had higher expression of immune-suppressive markers, while lung metastases showed higher proinflammatory activity and T-cell activation markers. Peritoneal metastases demonstrated higher expression of cancer-associated fibroblast-related proteins and upregulated PD-1/PD-L1 signaling molecules. Our results demonstrate that functional status and spatial distribution of immune cells vary significantly across different metastatic sites. These findings suggest that metastatic site-dependent immune contexture may underlie discordant responses to ICI therapy in patients with MSS colorectal cancer. SIGNIFICANCE: Our results demonstrate that functional status and spatial distribution of immune cells vary significantly across different metastatic sites in MSS colorectal cancer. These findings suggest that metastatic site-dependent immune contexture may underlie discordant responses to ICI therapy in patients with MSS colorectal cancer.

Immunogenicity and efficacy of pembrolizumab and doxorubicin in a phase I trial for patients with metastatic triple-negative breast cancer.

Currently there is a limited understanding for the optimal combination of immune checkpoint inhibitor and chemotherapy for patients with metastatic triple-negative breast cancer (mTNBC). Here we evaluate the safety, efficacy, and immunogenicity of a phase I trial for patients with mTNBC treated with pembrolizumab plus doxorubicin. Patients without prior anthracycline use and 0-2 lines of prior systemic chemotherapies received pembrolizumab and doxorubicin every 3 weeks for 6 cycles followed by pembrolizumab maintenance until disease progression or intolerance. The primary objectives were safety and objective response rate per RECIST 1.1. Best responses included one complete response (CR), five partial responses (PR), two stable disease (SD), and one progression of disease (PD). Overall response rate was 67% (95% CI 13.7%, 78.8%) and clinical benefit rate at 6 months was 56% (95% CI 21.2%, 86.3%). Median PFS was 5.2 months (95% CI 4.7, NA); median OS was 15.6 months (95% CI 13.3, NA). Grade 3-4 AEs per CTCAE 4.0 were neutropenia n = 4/10 (40%), leukopenia n = 2/10 (20%), lymphopenia n = 2/10 (20%), fatigue n = 2/10 (20%), and oral mucositis n = 1/10 (10%). Immune correlates showed increased frequencies of circulating CD3 + T cells (p = 0.03) from pre-treatment to cycle 2 day 1 (C2D1). An expansion of a proliferative exhausted-like PD-1 + CD8 + T cell population was identified in 8/9 patients, and exhausted CD8 + T cells were significantly expanded from pre-treatment to C2D1 in the patient with CR (p = 0.01). In summary, anthracycline-naïve patients with mTNBC treated with the combination of pembrolizumab and doxorubicin showed an encouraging response rate and robust T cell response dynamics.Trial registration: NCT02648477.

Pembrolizumab plus vorinostat induces responses in patients with Hodgkin lymphoma refractory to prior PD-1 blockade.

This phase 1 study evaluated the addition of vorinostat to pembrolizumab in patients with relapsed/refractory (RR) classical Hodgkin lymphoma (cHL), diffuse large B-cell lymphoma, and follicular lymphoma. We report the results in cases of cHL. Adult patients with RR cHL who had received ≥1 prior lines of therapy and were ineligible for transplantation were treated in a dose-escalation cohort with 2 dose levels (DLs) and then on an expansion cohort at the recommended phase 2 dose (RP2D) in 21-day cycles. Vorinostat 100 mg twice a day (DL1) and 200 mg twice a day (DL2) was administered orally from days 1 to 5 and 8 to 12; all patients received pembrolizumab 200 mg IV every 3 weeks. The primary end point was safety and determination of RP2D. In total, 32 patients with cHL were enrolled, including 30 at DL2 (RP2D); 78% had received prior anti-programmed cell death 1 (anti-PD-1) therapy, and 56% were PD-1 refractory. Grade ≥3 adverse events (AEs) included hypertension (9%), neutropenia (9%), hypophosphatemia (9%), thrombocytopenia (6%), and lymphopenia (6%). Immune-related AEs included grade 1 or 2 thyroiditis (13%), grade 1 rash (6%), and grade 3 esophagitis/duodenitis (3%). The overall response rate (ORR) was 72% and complete response (CR) rate was 34%. Patients refractory to prior PD-1 blockade (n = 18) had ORR and CR rates of 56% and 11%, respectively. Pembrolizumab and vorinostat was well tolerated with a high ORR rate in RR cHL including in anti-PD-1-refractory disease. This trial was registered at www.clinicaltrials.gov as #NCT03150329.

Targeting STAT3 in tumor-associated antigen-presenting cells as a strategy for kidney and bladder cancer immunotherapy.

Introduction: Immune checkpoint blockade (ICB) improved clinical outcomes in renal and bladder cancer patients, but the response rates remain limited especially in metastatic disease. While STAT3 transcription factor is well-known master regulator of tumor immune evasion, little is known about the role of STAT3 in the resistance of renal or bladder cancers to immunotherapy. Methods: To better understand immune alterations associated with ICB resistance, we assessed blood biomarkers in renal cancer patients classified as responders or non-responders to first line nivolumab/ipilimumab immunotherapy. Results: We observed that non-responders showed elevated levels of proinflammatory mediators, such as IL-1RA, IL-6, IL-8 and to lesser extent IL-10, which are associated with STAT3 activation and tumor immunosuppression. In addition, we found STAT3 activation primarily in circulating myeloid immune cells such as tolerogenic MDSCs. To assess whether STAT3 inhibition within these cell subsets can promote antitumor immune responses and/or enhance sensitivity to ICB in vivo, we used an original antisense oligonucleotide (ASO) strategy for myeloid-cell selective STAT3 knockdown (CpG-STAT3ASO). Our results in syngeneic models of renal and bladder cancers in mice demonstrated potent antitumor activity of CpG-STAT3ASO alone in contrast to PD1 blockade alone in both models. The CpG-STAT3ASO/anti-PD1 combination improved therapeutic efficacy especially against bladder tumors. Therapeutic efficacy correlated with activation of dendritic cells (DCs) and M1 macrophages in the tumor microenvironment, reduced percentages of regulatory T cells (Tregs) and the expansion of CD8 T cells in both tumor models. Discussion/Conclusion: Our study underscores the potential of using myeloid-cell targeted CpG-STAT3 inhibitors for genitourinary cancer therapy to disrupt tolerogenic signaling, restore immune cell activity and sensitivity to immune checkpoint inhibitors and/or T cell-based immunotherapies.

Genomic Markers of CDK 4/6 Inhibitor Resistance in Hormone Receptor Positive Metastatic Breast Cancer.

Cyclin-dependent kinase 4/6 inhibitors are the standard of care for hormone receptor-positive metastatic breast cancer. This retrospective study reports on genomic biomarkers of CDK 4/6i resistance utilizing genomic data acquired through routine clinical practice. Patients with HR+ MBC treated with palbociclib, ribociclib, or abemaciclib and antiestrogen therapy were identified. Patients were grouped into early (<6 months); intermediate (6−24 months for 0−1 lines; 6−9 months for ≥2 lines); or late progressors (>24 months for 0−1 lines; >9 months PFS for ≥2 lines). NGS and RNA sequencing data were analyzed in association with PFS, and survival analysis was stratified by prior lines of chemotherapy. A total of 795 patients with HR+ MBC treated with CDK 4/6i were identified. Of these, 144 (18%) patients had genomic data and 29 (3.6%) had RNA data. Among the 109 patients who received CDK4/6i as 1st- or 2nd-line therapy, 17 genes showed associations with PFS (p-value ≤ 0.15 and HR ≥ 1.5 or HR < 0.5). Whole transcriptome RNAseq was analyzed for 24/109 (22%) patients with 0−1 prior lines of therapy and 56 genes associated with PFS (HR ≥ 4 or HR ≤ 0.25 and FDR ≤ 0.15). In this retrospective analysis, genomic biomarkers including FGFR1 amplification, PTEN loss, and DNA repair pathway gene mutations showed significant associations with shorter PFS for patients receiving CDK4/6 inhibitor therapy.

Tumor-infiltrating exhausted CD8+ T cells dictate reduced survival in premenopausal estrogen receptor-positive breast cancer.

CD8+ tumor-infiltrating lymphocytes (TILs) are associated with improved survival in triple-negative breast cancer (TNBC) yet have no association with survival in estrogen receptor-positive (ER+) BC. The basis for these contrasting findings remains elusive. We identified subsets of BC tumors infiltrated by CD8+ T cells with characteristic features of exhausted T cells (TEX). Tumors with abundant CD8+ TEX exhibited a distinct tumor microenvironment marked by amplified interferon-γ signaling-related pathways and higher programmed death ligand 1 expression. Paradoxically, higher levels of tumor-infiltrating CD8+ TEX associated with decreased overall survival of patients with ER+ BC but not patients with TNBC. Moreover, high tumor expression of a CD8+ TEX signature identified dramatically reduced survival in premenopausal, but not postmenopausal, patients with ER+ BC. Finally, we demonstrated the value of a tumor TEX signature score in identifying high-risk premenopausal ER+ BC patients among those with intermediate Oncotype DX Breast Recurrence Scores. Our data highlight the complex relationship between CD8+ TILs, interferon-γ signaling, and ER status in BC patient survival. This work identifies tumor-infiltrating CD8+ TEX as a key feature of reduced survival outcomes in premenopausal patients with early-stage ER+ BC.

Phase I/II trial of palbociclib, pembrolizumab and letrozole in patients with hormone receptor-positive metastatic breast cancer.

BACKGROUND: CDK4/6 inhibitors modulate immune response in breast cancer. This phase I/II trial was designed to test the safety and efficacy of palbociclib, pembrolizumab and letrozole in women with hormone receptor positive (HR+) human epidermal growth factor receptor 2 negative (HER2-) metastatic breast cancer (MBC). PATIENTS AND METHODS: Women with stage IV HR+ HER2- MBC were enrolled and treated with palbociclib, pembrolizumab and letrozole. Primary end-points were safety, tolerability and efficacy. RESULTS: Between November 2016 and July 2020, 23 patients were enrolled with 20 evaluable for response, including 4 patients in cohort 1 and 16 patients in cohort 2. Cohort 1 median age was 48 years (33-70) and cohort 2 median age was 55 (37-75). Cohort 1 closed early due to limited accrual. Grade III-IV adverse events were neutropenia (83%), leucopaenia (65%), thrombocytopenia (17%) and elevated liver enzymes (17%). In cohort 1, 50% achieved a partial response (PR) and 50% had stable disease (SD). In cohort 2, 31% achieved complete response (CR), 25% had PR and 31% had SD by Response Evaluation Criteria in Solid Tumours version 1.1. Median progression-free survival was 25.2 months (95% confidence interval [CI] 5.3, not reached) and median overall survival was 36.9 months (95% CI 36.9, not reached) in cohort 2 with a median follow-up of 24.8 months (95% CI 17.1, not reached). A correlative immune biomarker analysis was published separately. CONCLUSION: The combination of palbociclib, pembrolizumab and letrozole is well tolerated, and a complete response rate of 31% was identified in HR+ MBC patients who received this combination as front-line therapy. Confirmatory trials are required to better understand the immune-priming effects of CDK4/6 inhibitors.

Pre-existing effector T-cell levels and augmented myeloid cell composition denote response to CDK4/6 inhibitor palbociclib and pembrolizumab in hormone receptor-positive metastatic breast cancer.

BACKGROUND: Single-agent pembrolizumab treatment of hormone receptor-positive metastatic breast cancer (MBC) has demonstrated modest clinical responses. Little is known about potential biomarkers or mechanisms of response to immune checkpoint inhibitors (ICIs) in patients with HR+ MBC. The present study presents novel immune correlates of clinical responses to combined treatment with CDK4/6i and ICI. METHODS: A combined analysis of two independent phase I clinical trials treating patients with HR+ MBC was performed. Patients treated with the combination of the CDK4/6i palbociclib+the ICI pembrolizumab+the aromatase inhibitor (AI) letrozole (palbo+pembro+AI) were compared with patients treated with pembrolizumab+AI (pembro+AI). Peripheral blood mononuclear cells collected at pretreatment, 3 weeks (cycle 2 day 1) and 9 weeks (cycle 4 day 1) were characterized by high-parameter flow cytometry to assess baseline immune subset composition and longitudinal changes in response to therapy. RESULTS: In the peripheral blood, higher pretreatment frequencies of effector memory CD45RA+ CD8+ T cells and effector memory CD4+ T cells were observed in responders to palbo+pembro+AI. In contrast, this was not observed in pembro+AI-treated patients. We further characterized T-cell subsets of effector-like killer cell lectin-like receptor subfamily G member 1 (KLRG1+) ICOS+ CD4+ T cells and KLRG1+ CD45RA+ CD8+ T cells as baseline biomarkers of response. In comparison, pretreatment levels of tumor-infiltrating lymphocyte, tumor mutation burden, tumor programmed death-ligand 1 expression, and overall immune composition did not associate with clinical responses. Over the course of treatment, significant shifts in myeloid cell composition and phenotype were observed in palbo+pembro+AI-treated patients, but not in those treated with pembro+AI. We identified increased fractions of type 1 conventional dendritic cells (cDC1s) within circulating dendritic cells and decreased classical monocytes (cMO) within circulating monocytes only in patients treated with palbociclib. We also demonstrated that in palbociclib-treated patients, cDC1 and cMO displayed increased CD83 and human leukocyte antigen-DR isotype (HLA-DR) expression, respectively, suggesting increased maturation and antigen presentation capacity. CONCLUSIONS: Pre-existing circulating effector CD8+ and CD4+ T cells and dynamic modulation of circulating myeloid cell composition denote response to combined pembrolizumab and palbociclib therapy for patients with HR+ MBC. TRIAL REGISTRATION NUMBER: NCT02778685 and NCI02648477.

Dissecting Response to Cancer Immunotherapy by Applying Bayesian Network Analysis to Flow Cytometry Data.

Cancer immunotherapy, specifically immune checkpoint blockade, has been found to be effective in the treatment of metastatic cancers. However, only a subset of patients achieve clinical responses. Elucidating pretreatment biomarkers predictive of sustained clinical response is a major research priority. Another research priority is evaluating changes in the immune system before and after treatment in responders vs. nonresponders. Our group has been studying immune networks as an accurate reflection of the global immune state. Flow cytometry (FACS, fluorescence-activated cell sorting) data characterizing immune cell panels in peripheral blood mononuclear cells (PBMC) from gastroesophageal adenocarcinoma (GEA) patients were used to analyze changes in immune networks in this setting. Here, we describe a novel computational pipeline to perform secondary analyses of FACS data using systems biology/machine learning techniques and concepts. The pipeline is centered around comparative Bayesian network analyses of immune networks and is capable of detecting strong signals that conventional methods (such as FlowJo manual gating) might miss. Future studies are planned to validate and follow up the immune biomarkers (and combinations/interactions thereof) associated with clinical responses identified with this computational pipeline.

A Phase II Clinical Trial of Pembrolizumab and Enobosarm in Patients with Androgen Receptor-Positive Metastatic Triple-Negative Breast Cancer.

LESSONS LEARNED: The combination of enobosarm and pembrolizumab was well tolerated and showed a modest clinical benefit rate of 25% at 16 weeks. Future trials investigating androgen receptor-targeted therapy in combination with immune checkpoint inhibitors are warranted. BACKGROUND: Luminal androgen receptor is a distinct molecular subtype of triple-negative breast cancer (TNBC) defined by overexpression of androgen receptor (AR). AR-targeted therapy has shown modest activity in AR-positive (AR+) TNBC. Enobosarm (GTx-024) is a nonsteroidal selective androgen receptor modulator (SARM) that demonstrates preclinical and clinical activity in AR+ breast cancer. The current study was designed to explore the safety and efficacy of the combination of enobosarm and pembrolizumab in patients with AR+ metastatic TNBC (mTNBC). METHODS: This study was an open-label phase II study for AR+ (≥10%, 1+ by immunohistochemistry [IHC]) mTNBC. Eligible patients received pembrolizumab 200 mg intravenous (IV) every 3 weeks and enobosarm 18 mg oral daily. The primary objective was to evaluate the safety of enobosarm plus pembrolizumab and determine the response rate. Peripheral blood, tumor biopsies, and stool samples were collected for correlative analysis. RESULTS: The trial was stopped early because of the withdrawal of GTx-024 drug supply. Eighteen patients were enrolled, and 16 were evaluable for responses. Median age was 64 (range 36-81) years. The combination was well tolerated, with only a few grade 3 adverse events: one dry skin, one diarrhea, and one musculoskeletal ache. The responses were 1 of 16 (6%) complete response (CR), 1 of 16 (6%) partial response (PR), 2 of 16 (13%) stable disease (SD), and 12 of 16 (75%) progressive disease (PD). Response rate (RR) was 2 of 16 (13%). Clinical benefit rate (CBR) at 16 weeks was 4 of 16 (25%). Median follow-up was 24.9 months (95% confidence interval [CI], 17.5-30.9). Progression-free survival (PFS) was 2.6 months (95% CI, 1.9-3.1) and overall survival (OS) was 25.5 months (95% CI, 10.4-not reached [NR]). CONCLUSION: The combination of enobosarm and pembrolizumab was well tolerated, with a modest clinical benefit rate of 25% at 16 weeks in heavily pretreated AR+ TNBC without preselected programmed death ligand-1 (PD-L1). Future clinical trials combining AR-targeted therapy with immune checkpoint inhibitor (ICI) for AR+ TNBC warrant investigation.

Mature Dendritic Cells May Promote High-Avidity Tuning of Vaccine T Cell Responses.

Therapeutic vaccines can elicit tumor-specific cytotoxic T lymphocytes (CTLs), but durable reductions in tumor burden require vaccines that stimulate high-avidity CTLs. Recent advances in immunotherapy responses have led to renewed interest in vaccine approaches, including dendritic cell vaccine strategies. However, dendritic cell requirements for vaccines that generate potent anti-tumor T-cell responses are unclear. Here we use mathematical modeling to show that, counterintuitively, increasing levels of immature dendritic cells may lead to selective expansion of high-avidity CTLs. This finding is in contrast with traditional dendritic cell vaccine approaches that have sought to harness ex vivo generated mature dendritic cells. We show that the injection of vaccine antigens in the context of increased numbers of immature dendritic cells results in a decreased overall peptide:MHC complex load that favors high-avidity CTL activation and expansion. Overall, our results provide a firm basis for further development of this approach, both alone and in combination with other immunotherapies such as checkpoint blockade.

Arhgap25 Deficiency Leads to Decreased Numbers of Peripheral Blood B Cells and Defective Germinal Center Reactions.

Rho family GTPases are critical for normal B cell development and function, and their activity is regulated by a large and complex network of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, the role of GAPs in B cell development is poorly understood. In this study, we show that the novel Rac-GAP ARHGAP25 is important for B cell development in mice in a CXCR4-dependent manner. We show that Arhgap25 deficiency in mice leads to a significant decrease in peripheral blood B cell numbers as well as defects in mature B cell differentiation. Arhgap25-/- B cells respond to Ag stimulation in vitro and in vivo but have impaired germinal center formation and decreased IgG1 class switching. Additionally, Arhgap25-/- B cells show evidence of increased baseline motility and augmented chemotaxis to CXCL12. Taken together, these studies demonstrate an important role for Arhgap25 in peripheral B cell development and Ag response.