Jacob Austin-Breneman

Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients.

Good bacteria help fight cancer Resident gut bacteria can affect patient responses to cancer immunotherapy (see the Perspective by Jobin). Routy et al. show that antibiotic consumption is associated with poor response to immunotherapeutic PD-1 blockade. They profiled samples from patients with lung and kidney cancers and found that nonresponding patients had low levels of the bacterium Akkermansia muciniphila. Oral supplementation of the bacteria to antibiotic-treated mice restored the response to immunotherapy. Matson et al. and Gopalakrishnan et al. studied melanoma patients receiving PD-1 blockade and found a greater abundance of “good” bacteria in the guts of responding patients. Nonresponders had an imbalance in gut flora composition, which correlated with impaired immune cell activity. Thus, maintaining healthy gut flora could help patients combat cancer. Science, this issue p. 91, p. 104, p. 97; see also p. 32 Gut bacteria influence patient response to cancer therapy. Preclinical mouse models suggest that the gut microbiome modulates tumor response to checkpoint blockade immunotherapy; however, this has not been well-characterized in human cancer patients. Here we examined the oral and gut microbiome of melanoma patients undergoing anti–programmed cell death 1 protein (PD-1) immunotherapy (n = 112). Significant differences were observed in the diversity and composition of the patient gut microbiome of responders versus nonresponders. Analysis of patient fecal microbiome samples (n = 43, 30 responders, 13 nonresponders) showed significantly higher alpha diversity (P < 0.01) and relative abundance of bacteria of the Ruminococcaceae family (P < 0.01) in responding patients. Metagenomic studies revealed functional differences in gut bacteria in responders, including enrichment of anabolic pathways. Immune profiling suggested enhanced systemic and antitumor immunity in responding patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplants from responding patients. Together, these data have important implications for the treatment of melanoma patients with immune checkpoint inhibitors.

Analysis of Immune Signatures in Longitudinal Tumor Samples Yields Insight into Biomarkers of Response and Mechanisms of Resistance to Immune Checkpoint Blockade

UNLABELLED Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) blockade (n = 53) followed by programmed death-1 (PD-1) blockade at progression (n = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified. SIGNIFICANCE These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. Cancer Discov; 6(8); 827-37. ©2016 AACR.See related commentary by Teng et al., p. 818This article is highlighted in the In This Issue feature, p. 803.

Integrated molecular analysis of tumor biopsies on sequential CTLA-4 and PD-1 blockade reveals markers of response and resistance

Profiling of melanoma patients treated with checkpoint blockade reveals TCR clonality and copy number loss as correlates of therapeutic response. Checking on checkpoint inhibitors Immune checkpoint blockade has greatly improved the success of treatment in melanoma and other tumor types, but it is expensive and does not work for all patients. To optimize the likelihood of therapeutic success and reduce the risks and expense of unnecessary treatment, it would be helpful to find biomarkers that can predict treatment response. Roh et al. studied patients treated with sequential checkpoint inhibitors targeting CTLA-4 and then PD-1. In these patients, the authors discovered that a more clonal T cell population specifically correlates with response to PD-1 blockade, but not CTLA-4, which may help identify the best candidates for this treatment. In addition, increased frequency of gene copy number loss was correlated with decreased responsiveness to either therapy. Immune checkpoint blockade produces clinical benefit in many patients. However, better biomarkers of response are still needed, and mechanisms of resistance remain incompletely understood. To address this, we recently studied a cohort of melanoma patients treated with sequential checkpoint blockade against cytotoxic T lymphocyte antigen–4 (CTLA-4) followed by programmed death receptor–1 (PD-1) and identified immune markers of response and resistance. Building on these studies, we performed deep molecular profiling including T cell receptor sequencing and whole-exome sequencing within the same cohort and demonstrated that a more clonal T cell repertoire was predictive of response to PD-1 but not CTLA-4 blockade. Analysis of CNAs identified a higher burden of copy number loss in nonresponders to CTLA-4 and PD-1 blockade and found that it was associated with decreased expression of genes in immune-related pathways. The effect of mutational load and burden of copy number loss on response was nonredundant, suggesting the potential utility of a combinatorial biomarker to optimize patient care with checkpoint blockade therapy.

Distinct clinical patterns and immune infiltrates are observed at time of progression on targeted therapy versus immune checkpoint blockade for melanoma

ABSTRACT We have made major advances in the treatment of melanoma through the use of targeted therapy and immune checkpoint blockade; however, clinicians are posed with therapeutic dilemmas regarding timing and sequence of therapy. There is a growing appreciation of the impact of antitumor immune responses to these therapies, and we performed studies to test the hypothesis that clinical patterns and immune infiltrates differ at progression on these treatments. We observed rapid clinical progression kinetics in patients on targeted therapy compared to immune checkpoint blockade. To gain insight into possible immune mechanisms behind these differences, we performed deep immune profiling in tumors of patients on therapy. We demonstrated low CD8+ T-cell infiltrate on targeted therapy and high CD8+ T-cell infiltrate on immune checkpoint blockade at clinical progression. These data have important implications, and suggest that antitumor immune responses should be assessed when considering therapeutic options for patients with melanoma.

Does It MEK a Difference? Understanding Immune Effects of Targeted Therapy

BRAF inhibitor (BRAFi) treatment enhances antitumor immunity, but is associated with increased intratumoral PD-L1 expression. MEK inhibitors (MEKi) may alter T-cell function; however, recent studies demonstrate preserved T-cell infiltrate during treatment with BRAFi/MEKi. These data have important implications for combining BRAFi/MEKi and checkpoint blockade in the treatment of melanoma. Clin Cancer Res; 21(14); 3102–4. ©2015 AACR. See related article by Kakavand et al., p. 3140

Genomic and immune heterogeneity are associated with differential responses to therapy in melanoma

Appreciation for genomic and immune heterogeneity in cancer has grown though the relationship of these factors to treatment response has not been thoroughly elucidated. To better understand this, we studied a large cohort of melanoma patients treated with targeted therapy or immune checkpoint blockade (n = 60). Heterogeneity in therapeutic responses via radiologic assessment was observed in the majority of patients. Synchronous melanoma metastases were analyzed via deep genomic and immune profiling, and revealed substantial genomic and immune heterogeneity in all patients studied, with considerable diversity in T cell frequency, and few shared T cell clones (<8% on average) across the cohort. Variables related to treatment response were identified via these approaches and through novel radiomic assessment. These data yield insight into differential therapeutic responses to targeted therapy and immune checkpoint blockade in melanoma, and have key translational implications in the age of precision medicine.Melanoma: Tumor differences within a patient may explain heterogeneous responsesPatients with metastatic melanoma display molecular and immune differences across tumor sites associated with differential drug responses. A team led by Jennifer Wargo from the University of Texas MD Anderson Cancer Center, Houston, USA, studied the radiological responses of 60 patients with metastatic melanoma, half of whom received targeted drug therapy and half of whom received an immune checkpoint inhibitor. The majority (83%) showed differences in responses across metastases. The group then profiled tumors in a subset, and found molecular and immune heterogeneity in different tumors within the same patient. Heterogeneity in mutational and immune profiles within tumors from individual patients could explain differences in treatment response. Knowing this, the authors emphasize the importance of acquiring biopsies from more than one tumor site in order to best tailor therapies to the features of metastatic cancer.

Molecular and immune heterogeneity in synchronous melanoma metastases.

Despite recent advances in the treatment of metastatic melanoma through targeted and immunotherapy, the majority of patients do not achieve a durable response. Research efforts to better understand responses are underway, and numerous molecular mechanisms of resistance to targeted therapy have been identified. There is a growing appreciation of genomic heterogeneity as a contributor to resistance to therapy, although immune heterogeneity has not been well characterized. The goal of the present study is to better understand genomic and immune heterogeneity in synchronous metastases within melanoma patients, with the potential to identify actionable strategies to overcome resistance. In this study, we prospectively evaluated 36 tumors from 16 melanoma patients (n=5 treatment-naive, n=6 targeted therapy, n=5 immunotherapy). Distinct synchronous metastases were evaluated by whole exome sequencing and NanoString analysis and showed up to 36% tumor-specific mutations as well as significant differences in expression of immune pathway effectors. Accordingly, we performed immune profiling by flow cytometry and immunohistochemistry demonstrating significant immune heterogeneity between synchronous melanoma tumors in all patients, most notably in the CD4+ and CD8+ T cell compartment. Deep TCR sequencing data revealed that T cell populations infiltrating synchronous metastases presented different specificities, with less than 10% of T cell clones shared between 2 tumors in the same patient. Additionally, the NetMHC 3.4 algorithm revealed that 10-30% of predicted neoantigens were unique to individual tumors and that over 10% of these presented high HLA-binding affinity. Together, these data suggest significant genomic and immune heterogeneity between synchronous metastases in melanoma patients – not only in the setting of therapy but also prior to its initiation. This has important clinical implications, and could help explain variable responses to therapy, however this hypothesis must be tested carefully in a larger data set. Nonetheless, these findings may have significant implications for the treatment of melanoma and other cancers.

Working with Human Tissues for Translational Cancer Research.

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Abstract 1301: Inter- and intra-tumoral immune and genomic heterogeneity in patients with metastatic melanoma

There have been significant advances in the treatment of metastatic melanoma via the use of targeted and immunotherapy, however a significant proportion of patients still progress on treatment. Intense research efforts to better understand resistance are underway, and multiple molecular resistance mechanisms to targeted therapy have been identified. Appreciation of intratumoral genetic heterogeneity as a contributor to resistance to therapy is growing, although immune heterogeneity in multiple solid tumors within a same patient has been less well characterized. The goal of the present study is to better understand the leukocyte infiltrate in multiple melanoma tumors within the same patient at the time of disease progression, with the potential to identify actionable strategies to overcome resistance to therapy. In this study, we prospectively evaluated 8 tumors from 4 patients on either targeted or immunotherapy. Tissues were evaluated by polychromatic flow cytometry, complemented with immunohistochemical (IHC) analysis of tissue sections. Specifically, we utilized 5 distinct flow cytometry panels comprising 35 antibodies and an 11-marker IHC antibody panel to better understand the intratumoral leukocyte composition as well as presence of immune checkpoint molecules PD-1 and PD-L1. Results demonstrate significant immune heterogeneity between different melanoma tumors within the same patient in the majority of patients studied. Of note, there were significant differences in CD4+, CD8+, and regulatory T cells (p Citation Format: Alexandre Reuben, Zachary A. Cooper, Whijae Roh, Yu Cao, Jacob Austin-Breneman, Hong Jiang, Rodabe N. Amaria, Pei-Ling Chen, Michael T. Tetzlaff, Lynda Chin, Andrew Futreal, Michael A. Davies, Jennifer A. Wargo. Inter- and intra-tumoral immune and genomic heterogeneity in patients with metastatic melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1301. doi:10.1158/1538-7445.AM2015-1301

Raising the bar: optimizing combinations of targeted therapy and immunotherapy

Major breakthroughs have arisen in the treatment of melanoma and other cancers through the use of targeted and immunotherapy. Therapies targeting the BRAF V600 mutation, such as vemurafenib and dabrafenib, were FDA-approved in 2011 and 2013, following demonstration of rapid, marked response in a majority of patients expressing the BRAF V600 mutation and a survival benefit over then standard-of-care therapy with dacarbazine (1,2). However, the vast majority of responding patients eventually relapse, most often within only 6-12 months of treatment initiation (3,4). Another form of immunotherapy, immune checkpoint blockade, exploits a tumor-deployed immune escape mechanism through which tumors impede the immune response by binding checkpoint molecules which serve as brakes, specifically on T lymphocytes. Such therapies involving monoclonal blocking antibodies against cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) were approved in 2011 and 2014, respectively. Though these treatments are associated with responses in fewer patients (20-35%) (5,6) than treatment with targeted therapy, responses are often durable (7) with a significant proportion of patients achieving durable disease control. Unfortunately, many patients do not derive benefit from these forms of therapy (1,2,5,6), and more therapeutic options are needed. Another form of therapy that has been studied extensively is adoptive cell therapy (ACT), and involves the isolation and expansion of antigen-specific lymphocytes from tumor (tumor infiltrating lymphocytes-TIL) (8) or peripheral blood (9) from patients with melanoma (and other cancer types). This form of therapy is associated with responses in approximately 50% of metastatic melanoma patients (10), though its use has been limited by the technical expertise involved in isolation and expansion of these cells, as well as the infrastructure required for this therapeutic approach (11).