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.

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.

A Pilot Feasibility Study of Yttrium-90 Liver Radioembolization Followed by Durvalumab and Tremelimumab in Patients with Microsatellite Stable Colorectal Cancer Liver Metastases.

LESSONS LEARNED: Radioembolization with yttrium-90 resin microspheres can be combined safely with full doses of durvalumab and tremelimumab in patients with metastatic colorectal cancer. Regional radioembolization with yttrium-90 resin microspheres did not result in any hepatic or extrahepatic responses to a combination of durvalumab and tremelimumab. The lack of immunomodulatory responses to yttrium-90 on biopsies before and after treatment rules out a potential role for this strategy in converting a "cold tumor" into an "inflamed," immune responsive tumor. BACKGROUND: PD-1 inhibitors have been ineffective in microsatellite stable (MSS) metastatic colorectal cancer (CRC). Preclinical models suggest that radiation therapy may sensitize MSS CRC to PD-1 blockade. METHODS: Patients with MSS metastatic CRC with liver-predominant disease who progressed following at least one prior line of treatment were treated with yttrium-90 (Y90) radioembolization to the liver (SIR-Spheres; Sirtex, Woburn, MA) followed 2-3 weeks later by the combination of durvalumab and tremelimumab. A Simon two-stage design was implemented, with a planned expansion to 18 patients if at least one response was noted in the first nine patients. RESULTS: Nine patients enrolled in the first stage of the study, all with progressive disease (PD) during or after their first two cycles of treatment. Per preplanned design, the study was closed because of futility. No treatment-related grade 3 or greater toxicities were recorded. Correlative studies with tumor biopsies showed low levels of tumor-infiltrating lymphocyte (TIL) infiltration in tumor cancer islands before and after Y90 radioembolization. CONCLUSION: Y90 radioembolization can be added safely to durvalumab and tremelimumab but did not promote tumor-directed immune responses against liver-metastasized MSS CRC.

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR.

Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.

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.

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.

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.

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.

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.

Metabolic Checkpoint of Immune Cells in Melanoma Brain Metastases.

Metabolic features of both cancer cells and immune cells shift with nutrient availability in the tumor microenvironment, resulting in differential effects on antitumor immune function. The work of Fischer and colleagues demonstrates that increased oxidative phosphorylation in brain metastases in patients with melanoma is a key regulator of intracranial immune surveillance and that inhibition of oxidative phosphorylation could reduce the incidence of intracranial brain metastases in a murine model of melanoma.See related article by Fischer et al., p. 628.

Synchronous recurrence of concurrent colon adenocarcinoma and dedifferentiated liposarcoma.

A 62-year-old man presented with concurrent sigmoid colon adenocarcinoma and small bowel mesenteric dedifferentiated liposarcoma. Following surgical resection of the colon cancer, complete excision of the mesenteric sarcoma and adjuvant folinic acid, fluorouracil and oxaliplatin (FOLFOX) chemotherapy, the patient demonstrated no radiological evidence of disease for more than 2 years. The patient then developed synchronous recurrence of both cancers: the colon cancer metastasised to the liver and a pelvic lymph node, and the liposarcoma recurred in the original location. The patient underwent additional chemotherapy with complete response of the metastatic colon cancer and stable disease for the liposarcoma. The recurrent mesenteric tumour was subsequently resected. Although concurrent cancers have been reported, this unique case of synchronous recurrence raises interesting hypotheses regarding host-tumour interaction and immune surveillance.

Resident memory CD8+ T cells within cancer islands mediate survival in breast cancer patients.

CD8+ tumor-infiltrating lymphocytes (TILs) correlate with relapse-free survival (RFS) in most cancer types, including breast cancer. However, subset composition, functional status, and spatial location of CD8+ TILs in relation to RFS in human breast tumors remain unclear. Spatial tissue analysis via quantitative immunofluorescence showed that infiltration of CD8+ T cells into cancer islands was more significantly associated with RFS than CD8+ T cell infiltration into either tumor stroma or total tumor. Localization into cancer islands within tumors is mediated by expression of the integrin CD103, which is a marker for tissue-resident memory T cells (TRMs). Analysis of fresh tumor samples revealed that CD8+ TRMs are functionally similar to other CD8+ TILs, suggesting that the basis of their protective effect is their spatial distribution rather than functional differences. Indeed, CD103+ TRMs, as compared with CD103-CD8+ TILs, are enriched within cancer islands, and CD8+ TRM proximity to cancer cells drives the association of CD8+ TIL densities with RFS. Together, these findings reveal the importance of cancer island-localized CD8+ TRMs in surveillance of the breast tumor microenvironment and as a critical determinant of RFS in patients with breast cancer.

Human breast tumor-infiltrating CD8+ T cells retain polyfunctionality despite PD-1 expression.

Functional CD8+ T cells in human tumors play a clear role in clinical prognosis and response to immunotherapeutic interventions. PD-1 expression in T cells involved in chronic infections and tumors such as melanoma often correlates with a state of T-cell exhaustion. Here we interrogate CD8+ tumor-infiltrating lymphocytes (TILs) from human breast and melanoma tumors to explore their functional state. Despite expression of exhaustion hallmarks, such as PD-1 expression, human breast tumor CD8+ TILs retain robust capacity for production of effector cytokines and degranulation capacity. In contrast, melanoma CD8+ TILs display dramatic reduction of cytokine production and degranulation capacity. We show that CD8+ TILs from human breast tumors can potently kill cancer cells via bi-specific antibodies. Our data demonstrate that CD8+ TILs in human breast tumors retain polyfunctionality, despite PD-1 expression, and suggest that they may be harnessed for effective immunotherapies.