Zachary Boyd

In vivo Antitumor Activity of MEK and Phosphatidylinositol 3-Kinase Inhibitors in Basal-Like Breast Cancer Models

Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy for this disease. We investigated the role of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors as targeted therapies for basal-like breast cancer. Experimental Design: We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor xenograft models. Results: We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity to a selective inhibitor of MEK compared with models representative of other breast cancer subtypes. We also showed that loss of PTEN is a negative predictor of response to MEK inhibition, that treatment with a selective MEK inhibitor caused up-regulation of PI3K pathway signaling, and that dual blockade of both PI3K and MEK/extracellular signal–regulated kinase signaling synergized to potently impair the growth of basal-like breast cancer models in vitro and in vivo. Conclusions: Our studies suggest that single-agent MEK inhibition is a promising therapeutic modality for basal-like breast cancers with intact PTEN, and also provide a basis for rational combination of MEK and PI3K inhibitors in basal-like cancers with both intact and deleted PTEN.

TRPS1 Targeting by miR-221/222 Promotes the Epithelial-to-Mesenchymal Transition in Breast Cancer

The microRNAs miR-221 and miR-222 promote a phenotype associated with metastasis and are found in a clinically aggressive form of breast cancer. Parsing Breast Cancer Subtype with MicroRNAs MicroRNAs (miRNAs), short noncoding RNAs that bind to and silence target mRNAs, have emerged as playing crucial regulatory roles not only in normal cellular processes but also in pathological conditions, such as cancer. Stinson et al. analyzed miRNA expression in different types of human breast cancer and found that miR-221 and miR-222 (miR-221/222) abundance was increased in the clinically aggressive basal-like subtype compared to the less aggressive luminal subtype. They determined that signaling through the epidermal growth factor receptor (EGFR)–RAS–extracellular signal–regulated kinase (ERK) pathway increased miR-221/222 transcription, and they defined a transcriptional regulatory pathway through which miR-221/222 promoted a phenotype associated with cancer cell invasion and metastasis. Their data suggest that combining inhibition of the EGFR-RAS-ERK pathway with standard chemotherapy could, by limiting miR-221/222 production, provide a strategy to combat metastasis in the basal-like subtype of breast cancer. The basal-like subtype of breast cancer has an aggressive clinical behavior compared to that of the luminal subtype. We identified the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype–specific miRNAs and showed that expression of miR-221/222 decreased expression of epithelial-specific genes and increased expression of mesenchymal-specific genes, and increased cell migration and invasion in a manner characteristic of the epithelial-to-mesenchymal transition (EMT). The transcription factor FOSL1 (also known as Fra-1), which is found in basal-like breast cancers but not in the luminal subtype, stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of the epidermal growth factor receptor (EGFR) or MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. Furthermore, miR-221/222–mediated reduction in E-cadherin abundance depended on their targeting the 3′ untranslated region of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1), which inhibited EMT by decreasing ZEB2 (zinc finger E-box–binding homeobox2) expression. We conclude that by promoting EMT, miR-221/222 may contribute to the more aggressive clinical behavior of basal-like breast cancers.

miR-221/222 Targeting of Trichorhinophalangeal 1 (TRPS1) Promotes Epithelial-to-Mesenchymal Transition in Breast Cancer

MicroRNAs miR-221 and miR-222 are associated with a clinically aggressive form of breast cancer and promote epithelial-to-mesenchymal transition. Compared with the luminal subtype, the basal-like subtype of breast cancer has an aggressive clinical behavior, but the reasons for this difference between the two subtypes are poorly understood. We identified microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs that decrease expression of epithelial-specific genes and increase expression of mesenchymal-specific genes. In addition, expression of these miRNAs increased cell migration and invasion, which collectively are characteristics of the epithelial-to-mesenchymal transition (EMT). The basal-like transcription factor FOSL1 (also known as Fra-1) directly stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. The miR-221/222–mediated reduction in E-cadherin abundance depended on their targeting of the 3′ untranslated region (3′UTR) of TRPS1 (trichorhinophalangeal syndrome type 1), which is a member of the GATA family of transcriptional repressors. TRPS1 inhibited EMT by directly repressing expression of ZEB2 (Zinc finger E-box–binding homeobox 2). Therefore, miR-221/222 may contribute to the aggressive clinical behavior of basal-like breast cancers.

Proteomic analysis of breast cancer molecular subtypes and biomarkers of response to targeted kinase inhibitors using reverse-phase protein microarrays

Although breast cancer molecular subtypes have been extensively defined by means of gene expression profiling over the past decade, little is known, at the proteomic level, as to how signaling pathways are differentially activated and serve to control proliferation in different breast cancer subtypes. We used reverse-phase protein arrays to examine phosphorylation status of 100 proteins in a panel of 30 breast cancer cell lines and showed distinct pathway activation differences between different subtypes that are not obvious from previous gene expression studies. We also show that basal levels of phosphorylation of key signaling nodes may have diagnostic utility in predicting response to selective inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. Finally, we show that reverse-phase protein arrays allow the parallel analysis of multiple pharmacodynamic biomarkers of response to targeted kinase inhibitors and that inhibitors of epidermal growth factor receptor and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase result in compensatory up-regulation of the phosphatidylinositol 3-kinase/Akt signaling pathway. [Mol Cancer Ther 2008;7(12):3695–706]

A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses

Molecular profiling of human cancer is complicated by both stromal contamination and cellular heterogeneity within samples from tumor biopsies. In this study, we developed a tissue-processing protocol using mechanical dissociation and flow cytometric sorting that resulted in the respective enrichment of stromal and tumor fractions from frozen pancreatic adenocarcinoma samples. Molecular profiling of DNA from the sorted populations using high-density single nucleotide polymorphism arrays revealed widespread chromosomal loss of heterozygosity in tumor fractions but not in either the stromal fraction or unsorted tissue specimens from the same sample. Similarly, a combination of KRAS mutations and chromosomal copy number changes at key pancreatic cancer loci, such as CDK2NA and TP53, was detected in a substantial proportion of the tumor fractions but not in matched stromal fractions from the same sample. This approach to tissue processing could greatly expand the amount of archived tissue that is available for molecular profiling of human cancer and enable a more accurate diagnosis of genetic alterations in patient samples.

Current Status of Companion and Complementary Diagnostics

US Food and Drug Administration (FDA)‐approved diagnostic assays play an increasingly common role in managing patients to prolong lifespan while also enhancing quality of life. Diagnostic assays can be essential for the safe and effective use of therapeutics (companion diagnostic), or may inform on improving the benefit/risk ratio without restricting drug access (complementary diagnostic). This tutorial reviews strategic considerations for drug and assay development resulting in FDA‐approved companion or complementary diagnostic status.

Current Status of Companion and Complementary Diagnostics: Strategic Considerations for Development and Launch

US Food and Drug Administration (FDA)‐approved diagnostic assays play an increasingly common role in managing patients to prolong lifespan while also enhancing quality of life. Diagnostic assays can be essential for the safe and effective use of therapeutics (companion diagnostic), or may inform on improving the benefit/risk ratio without restricting drug access (complementary diagnostic). This tutorial reviews strategic considerations for drug and assay development resulting in FDA‐approved companion or complementary diagnostic status.

Immune biomarkers associated with clinical benefit from atezolizumab (MPDL3280a; anti-PD-L1) in advanced urothelial bladder cancer (UBC)

Meeting abstracts Atezolizumab (anti-PD-L1) has demonstrated robust clinical activity in UBC [[1][1]]. Elevated PD-L1 expression on tumor-infiltrating immune cells (IC) is associated with increased clinical efficacy; however, the contribution of other immune biomarkers is unknown. In this study, we

Abstract B001: Development of a PD-L1 companion diagnostic IHC assay (SP142) for atezolizumab

Background: Understanding of immune tolerance mechanisms of cancer has prompted the development of cancer immunotherapies such as atezolizumab (anti-PD-L1, MPDL3280A). Robust, durable responses have been observed, leading to Breakthrough status designation by the FDA for atezolizumab for previously treated NSCLC and bladder cancer (UBC) patients. Roche/Ventana have developed a companion diagnostic (CDx) for atezoliziumab using a robust immunohistochemistry (IHC) assay and antibody clone (SP142), optimized to detect PD-L1 expression in both tumor cells (TC) and tumor-infiltrating immune cells (IC). Here we describe the characteristics of the SP142 assay, PD-L1 expression patterns observed by immunohistochemistry in TC and IC compartments, and their association with clinical efficacy for NSCLC and UBC patients. Methods: The anti-human PD-L1 rabbit monoclonal antibody SP142 was optimized for detection of both TC and IC expression of PD-L1 with the Ventana OptiView DAB IHC Detection Kit on the automated BenchMark ULTRA platform. The VENTANA PD-L1 (SP142) CDx assay was validated for use in formalin-fixed, paraffin-embedded samples of NSCLC and UBC in a series of studies addressing sensitivity, specificity, robustness, and precision. Formalin fixed, paraffin embedded tumor specimens were obtained from patients prescreened and/or enrolled into Genentech Ph I study PCD4989g, and PD-L1 expression was measured using the SP142 assay and PCR gene expression assays. Results: The SP142 assay met pre-defined acceptance criteria for TC and IC assessment of PD-L1 expression in NSCLC and UBC tumors, including >90% inter-reader concordance between pathologist readers. Rapid and durable responses were observed in the Ph I study (PCD4989g), and correlated with PD-L1 expression patterns observed by immunohistochemistry in TC and IC. In the same Ph I study (PCD4989g), PD-L1 expression as measured by PCR did not demonstrate the same predictive value as observed for IHC. Conclusions: The PD-L1 IHC (SP142) CDx is a robust assay to measure PD-L1 expression in both IC and TC. Further, the high reproducibility of results between pathologists shows that the scoring algorithms in UBC and NSCLC are precise, reproducible, and practical in the diagnostic setting. The results indicate that favorable atezolizumab efficacy for UBC is strongly associated with higher IC levels as assessed by the sensitive and specific PD-L1 IHC (SP142) CDx assay. In NSCLC, favorable atezolizumab efficacy is associated with tumors expressing either high TC or high IC levels by the PD-L1 IHC (SP142) CDx assay. These data support the predictive value of tumor PD-L1 levels by IHC for NSCLC and UBC patients receiving atezoliziumab. Citation Format: Zachary S. Boyd, Dustin Smith, Brian Baker, Bharathi Vennapusa, Hartmut Koeppen, Marcin Kowanetz, Sanjeev Mariathasan, Jean-Marie Bruey, Howard Mackey, Gregg Fine, Simonetta Mocci, Priti Hegde, J. Andrew Williams, Ian McCaffery. Development of a PD-L1 companion diagnostic IHC assay (SP142) for atezolizumab. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B001.

Myeloid cell biology and inhibition of anti-tumor immune responses by MPDL3280A in urothelial bladder cancer

Treatment options for metastatic urothelial bladder cancer (UBC) are limited. Mutational complexity is known to be high in UBC and may correlate with increased immunogenicity. MPDL3280A, a human PD-L1 monoclonal antibody containing an engineered Fc-domain designed to promote a Th1-driven response, has demonstrated a RECIST response rate of 43% in diagnostically selected, pretreated patients with UBC. A total of 68 patients (67 with efficacy evaluable) were enrolled in the UBC cohort of the Phase I study; 45% were PD-L1 IHC diagnostic positive as defined by expression of PD-L1 on ≥ 5% of tumor-infiltrating immune cells. In the prescreened UBC population, the prevalence of PD-L1-positive patients was 27%. Comprehensive gene expression analyses of UBC tumors were conducted to interrogate the tumor immune microenvironment in PD-L1-positive tumors and to identify potential mechanisms associated with response or resistance to MPDL3280A. In this study, PD-L1-positive tumors exhibited a high prevalence of gene expression markers associated with T-effector cells (Teff), including perforin, IFNγ, CD8A, granzyme B, granzyme A and EOMES. Additionally, a low baseline signature of genes associated with myeloid cell markers, including IL1B and IL8, appeared to be statistically significantly associated (P<0.01) with MPDL3280A response, suggesting a potential role for myeloid biology in resistance to MPDL3280A treatment in UBC. Tumor burden markers, including CA-125, CA19-9 and human chorionic gonadotropin (HCG), have been associated with chemotherapy response markers in UBC. A marked decrease in these markers, including CEA, CA19-9, CA-125 and HCG, was observed with MPDL3280A response after 1 treatment cycle, potentially enabling an on-treatment monitoring alternative for response to therapy. Similarly, evaluation of cytokines on treatment identified markers, including IL-6 and IL-10, elevated as early as Cycle 2 only in patients without response to MPDL3280A. These circulating cytokines and tumor-associated gene signatures suggest potential mechanisms associated with resistance and response to MPDL3280A in UBC and provide a rationale for informed combination strategies to further improve treatment benefit in this indication.