Joshua M. Lang

Augmenting Antitumor Immune Responses with Epigenetic Modifying Agents

Epigenetic silencing of immune-related genes is a striking feature of the cancer genome that occurs in the process of tumorigenesis. This phenomena impacts antigen processing and antigen presentation by tumor cells and facilitates evasion of immunosurveillance. Further modulation of the tumor microenvironment by altered expression of immunosuppressive cytokines impairs antigen-presenting cells and cytolytic T-cell function. The potential reversal of immunosuppression by epigenetic modulation is therefore a promising and versatile therapeutic approach to reinstate endogenous immune recognition and tumor lysis. Pre-clinical studies have identified multiple elements of the immune system that can be modulated by epigenetic mechanisms and result in improved antigen presentation, effector T-cell function, and breakdown of suppressor mechanisms. Recent clinical studies are utilizing epigenetic therapies prior to, or in combination with, immune therapies to improve clinical outcomes.

Circulating Tumor Cells: Getting More from Less

Continued technologic innovation may soon permit the interrogation of rare circulating tumor cells to understand tumor biology, guide drug development, and personalize treatment for cancer patients. Recent insights into circulating tumor cells (CTCs) have been driven by numerous technological innovations aimed at isolating, purifying, and analyzing these rare cells. However, the information density within these cells has yet to be truly accessed and exploited for patient benefit. A device reported by Issadore et al. in this issue of Science Translational Medicine proposes a highly sensitive methodology that may both extend CTC capture to a broader patient population and provide greater understanding of biological targets for personalized medical therapies.

A negative selection methodology using a microfluidic platform for the isolation and enumeration of circulating tumor cells

Circulating tumor cells (CTCs) exist in the peripheral blood stream of metastatic cancer patients at rates of approximately 1 CTC per billion background cells. In order to capture and analyze this rare cell population, various techniques exist that range from antibody-based surface marker positive selection to methods that use physical properties of CTCs to negatively exclude background cells from a CTC population. However, methods to capture cells for functional downstream analyses are limited due to inaccessibility of the captured sample or labeling techniques that may be prohibitive to cell function. Here, we present a negative selection method that leverages a Microfluidic Cell Concentrator (MCC) to allow collection and analysis of this rare cell population without needing cell adhesion or other labeling techniques to keep the cells within the chamber. Because the MCC is designed to allow collection and analysis of non-adherent cell populations, multiple staining steps can be applied in parallel to a given CTC population without losing any of the population. The ability of the MCC for patient sample processing of CTCs for enumeration was demonstrated with five patient samples, revealing an average of 0.31 CTCs/mL. The technique was compared to a previously published method - the ELISPOT - that showed similar CTC levels among the five patient samples tested. Because the MCC method does not use positive selection, the method can be applied across a variety of tumor types with no changes to the process.

Selective Nucleic Acid Removal via Exclusion (SNARE): Capturing mRNA and DNA from a Single Sample

The path from gene (DNA) to gene product (RNA or protein) is the foundation of genotype giving rise to phenotype. Comparison of genomic analyses (DNA) with paired transcriptomic studies (mRNA) is critical to evaluating the pathogenic processes that give rise to human disease. The ability to analyze both DNA and mRNA from the same sample is not only important for biologic interrogation but also to minimize variance (e.g., sample loss) unrelated to the biology. Existing methods for RNA and DNA purification from a single sample are typically time-consuming and labor intensive or require large sample sizes to split for separate RNA and DNA extraction procedures. Thus, there is a need for more efficient and cost-effective methods to purify both RNA and DNA from a single sample. To address this need, we have developed a technique, termed SNARE (Selective Nucleic Acid Removal via Exclusion), that uses pinned oil interfaces to simultaneous purify mRNA and DNA from a single sample. A unique advantage of SNARE is the elimination of dilutive wash and centrifugation processes that are fundamental to conventional methods where sample is typically discarded. This minimizes loss and maximizes recovery by allowing nondilutive reinterrogation of the sample. We demonstrate that SNARE is more sensitive than commercially available kits, robustly and repeatably achieving mRNA and DNA purification from extremely low numbers of cells for downstream analyses. In addition to sensitivity, SNARE is fast, easy to use, and cost-effective and requires no laboratory infrastructure or hazardous chemicals. We demonstrate the clinical utility of the SNARE with prostate cancer circulating tumor cells to demonstrate its ability to perform both genomic and transcriptomic interrogation on rare cell populations that would be difficult to achieve with any current method.

Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance

Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies. Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate. Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3′ terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31–12.2; P = 0.02). Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704–15. ©2017 AACR.

Integrated Analysis of Multiple Biomarkers from Circulating Tumor Cells Enabled by Exclusion-Based Analyte Isolation.

Purpose: There is a critical clinical need for new predictive and pharmacodynamic biomarkers that evaluate pathway activity in patients treated with targeted therapies. A microscale platform known as VERSA (versatile exclusion-based rare sample analysis) was developed to integrate readouts across protein, mRNA, and DNA in circulating tumor cells (CTC) for a comprehensive analysis of the androgen receptor (AR) signaling pathway. Experimental Design: Utilizing exclusion-based sample preparation principles, a handheld chip was developed to perform CTC capture, enumeration, quantification, and subcellular localization of proteins and extraction of mRNA and DNA. This technology was validated across integrated endpoints in cell lines and a cohort of patients with castrate-resistant prostate cancer (CRPC) treated with AR-targeted therapies and chemotherapies. Results: The VERSA was validated in cell lines to analyze AR protein expression, nuclear localization, and gene expression targets. When applied to a cohort of patients, radiographic progression was predicted by the presence of multiple AR splice variants and activity in the canonical AR signaling pathway. AR protein expression and nuclear localization identified phenotypic heterogeneity. Next-generation sequencing with the FoundationOne panel detected copy number changes and point mutations. Longitudinal analysis of CTCs identified acquisition of multiple AR variants during targeted treatments and chemotherapy. Conclusions: Complex mechanisms of resistance to AR-targeted therapies, across RNA, DNA, and protein endpoints, exist in patients with CRPC and can be quantified in CTCs. Interrogation of the AR signaling pathway revealed distinct patterns relevant to tumor progression and can serve as pharmacodynamic biomarkers for targeted therapies. Clin Cancer Res; 23(3); 746–56. ©2016 AACR.

High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1

Background Expression of programmed-death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs) may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM) positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT)). Methods To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP) technology. Results Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33–100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples. Conclusions Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria, thus improving the specificity of CTC identification and the accuracy of biomarker evaluation.

Prioritization of cancer antigens: keeping the target in sight

Evaluation of: Cheever MA, Allison JP, Ferris AS et al. The prioritization of cancer antigens: a National Cancer Institute pilot project for the acceleration of translational research. Clin. Cancer Res. 15(17), 5323–5337 (2009). Amid an evolving landscape of anticancer agents, numerous approaches to elicit tumor-specific immune responses are under active investigation. In broad terms, these approaches have traditionally included passive and active immunotherapies, whereby cells, antibodies or cytokines with antitumor activity are delivered to the recipient, or are used to elicit antitumor immunity in the host. More recently, immune-modulatory approaches, including the use of T-cell checkpoint inhibitors or agents that can affect T-cell or antigen-presenting cell function, have been studied. Most clinical trials with individual immune agents, and vaccines in particular, have demonstrated limited success despite demonstrations of biological activity. It has thus become apparent that successful immunotherapy approaches to the treatment of cancer will probably require combinations of treatment modalities. This presents a challenge, however, given the multitude of vaccine approaches currently being tested and the even greater number of vaccine target antigens being evaluated. In a recent report by the National Cancer Institute Translational Research Working Group, Cheever and colleagues attempt to tackle this problem by developing weighted criteria by which to both rank and prioritize tumor vaccine antigens. This article attempts to provide some background for the importance of this effort, discusses the methods used and potential biases introduced, and attempts to put this in context with other investigations in the tumor immunology field.

Multidisciplinary intervention of early, lethal metastatic prostate cancer: Report from the 2015 Coffey-Holden Prostate Cancer Academy Meeting

The 2015 Coffey‐Holden Prostate Cancer Academy Meeting, themed: “Multidisciplinary Intervention of Early, Lethal Metastatic Prostate Cancer,” was held in La Jolla, California from June 25 to 28, 2015.

A Randomized Phase II Trial Evaluating Different Schedules of Zoledronic Acid on Bone Mineral Density in Patients With Prostate Cancer Beginning Androgen Deprivation Therapy

OBJECTIVE To assess the effects of timing and schedule of zoledronic acid (ZA) administration on bone mineral density (BMD) in patients beginning androgen deprivation therapy (ADT) for the treatment of recurrent prostate cancer. PATIENTS AND METHODS In this randomized, 3-arm trial, we evaluated changes in BMD after 3 different ZA administration schedules in men with recurrent prostate cancer who were beginning ADT. Forty-four patients were enrolled and randomized to receive a single dose of ZA given 1 week before beginning ADT (arm 1), a single dose of ZA given 6 months after beginning ADT (arm 2), or monthly administration of ZA starting 6 months after beginning ADT, for a total of 6 doses (arm 3). RESULTS Patients who received ZA before ADT had a significant improvement in BMD at the total proximal femur and trochanter after 6 months compared with the other groups. In addition, only patients in the arm that received multiple doses improved lumbar spine BMD while on ADT, with these findings persisting to 24 months. However, this group also experienced more grade 1 adverse events. CONCLUSIONS Analysis of these data suggests that ZA administration before initiation of ADT was superior to treatment 6 months after starting ADT in maintaining BMD. In addition, monthly ZA administration can increase BMD above baseline but is associated with more adverse events. Further study is needed to examine whether the timing and frequency of ZA therapy in patients on ADT can reduce fracture risk.