RESUMEN
Programmed death-ligand 1 (PD-L1) expression on tumor cells (TCs) by immunohistochemistry is rapidly gaining importance as a diagnostic for the selection or stratification of patients with non-small cell lung cancer (NSCLC) most likely to respond to single-agent checkpoint inhibitors. However, at least two distinct patterns of PD-L1 expression have been observed with potential biological and clinical relevance in NSCLC: expression on TC or on tumor-infiltrating immune cells (ICs). We investigated the molecular and cellular characteristics associated with PD-L1 expression in these distinct cell compartments in 4,549 cases of NSCLC. PD-L1 expression on IC was more prevalent and likely reflected IFN-γ-induced adaptive regulation accompanied by increased tumor-infiltrating lymphocytes and effector T cells. High PD-L1 expression on TC, however, reflected an epigenetic dysregulation of the PD-L1 gene and was associated with a distinct histology described by poor immune infiltration, sclerotic/desmoplastic stroma, and mesenchymal molecular features. Importantly, durable clinical responses to atezolizumab (anti-PD-L1) were observed in patients with tumors expressing high PD-L1 levels on either TC alone [40% objective response rate (ORR)] or IC alone (22% ORR). Thus, PD-L1 expression on TC or IC can independently attenuate anticancer immunity and emphasizes the functional importance of IC in regulating the antitumor T cell response.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígeno B7-H1/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Neoplasias Pulmonares/inmunología , Anticuerpos Monoclonales Humanizados , Humanos , Inmunohistoquímica/métodos , Neoplasias Pulmonares/tratamiento farmacológico , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Masculino , Persona de Mediana Edad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
KRAS- and BRAF-mutant tumors are often dependent on MAPK signaling for proliferation and survival and thus sensitive to MAPK pathway inhibitors. However, clinical studies have shown that MEK inhibitors are not uniformly effective in these cancers indicating that mutational status of these oncogenes does not accurately capture MAPK pathway activity. A number of transcripts are regulated by this pathway and are recurrently identified in genome-based MAPK transcriptional signatures. To test whether the transcriptional output of only 10 of these targets could quantify MAPK pathway activity with potential predictive or prognostic clinical utility, we created a MAPK Pathway Activity Score (MPAS) derived from aggregated gene expression. In vitro, MPAS predicted sensitivity to MAPK inhibitors in multiple cell lines, comparable to or better than larger genome-based statistical models. Bridging in vitro studies and clinical samples, median MPAS from a given tumor type correlated with cobimetinib (MEK inhibitor) sensitivity of cancer cell lines originating from the same tissue type. Retrospective analyses of clinical datasets showed that MPAS was associated with the sensitivity of melanomas to vemurafenib (HR: 0.596) and negatively prognostic of overall or progression-free survival in both adjuvant and metastatic CRC (HR: 1.5 and 1.4), adrenal cancer (HR: 1.7), and HER2+ breast cancer (HR: 1.6). MPAS thus demonstrates potential clinical utility that warrants further exploration.
RESUMEN
In the age of personalized medicine stratifying tumors into molecularly defined subtypes associated with distinctive clinical behaviors and predictable responses to therapies holds tremendous value. Towards this end, we developed a custom microfluidics-based bladder cancer gene expression panel for characterization of archival clinical samples. In silico analysis indicated that the content of our panel was capable of accurately segregating bladder cancers from several public datasets into the clinically relevant basal and luminal subtypes. On a technical level, our bladder cancer panel yielded robust and reproducible results when analyzing formalin-fixed, paraffin-embedded (FFPE) tissues. We applied our panel in the analysis of a novel set of 204 FFPE samples that included non-muscle invasive bladder cancers (NMIBCs), muscle invasive disease (MIBCs), and bladder cancer metastases (METs). We found NMIBCs to be mostly luminal-like, MIBCs to include both luminal- and basal-like types, and METs to be predominantly of a basal-like transcriptional profile. Mutational analysis confirmed the expected enrichment of FGFR3 mutations in luminal samples, and, consistently, FGFR3 IHC showed high protein expression levels of the receptor in these tumors. Our bladder cancer panel enables basal/luminal characterization of FFPE tissues and with further development could be used for stratification of bladder cancer samples in the clinic.
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Bancos de Muestras Biológicas , Regulación Neoplásica de la Expresión Génica , Microfluídica/métodos , Transcripción Genética , Neoplasias de la Vejiga Urinaria/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Simulación por Computador , Femenino , Formaldehído , Genes Relacionados con las Neoplasias , Humanos , Masculino , Persona de Mediana Edad , Adhesión en Parafina , Reproducibilidad de los Resultados , Fijación del Tejido , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Mutations in ESR1 have been associated with resistance to aromatase inhibitor (AI) therapy in patients with ER+ metastatic breast cancer. Little is known of the impact of these mutations in patients receiving selective oestrogen receptor degrader (SERD) therapy. In this study, hotspot mutations in ESR1 and PIK3CA from ctDNA were assayed in clinical trial samples from ER+ metastatic breast cancer patients randomized either to the SERD fulvestrant or fulvestrant plus a pan-PI3K inhibitor. ESR1 mutations are present in 37% of baseline samples and are enriched in patients with luminal A and PIK3CA-mutated tumours. ESR1 mutations are often polyclonal and longitudinal analysis shows distinct clones exhibiting divergent behaviour over time. ESR1 mutation allele frequency does not show a consistent pattern of increases during fulvestrant treatment, and progression-free survival is not different in patients with ESR1 mutations compared with wild-type patients. ESR1 mutations are not associated with clinical resistance to fulvestrant in this study.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Estradiol/análogos & derivados , Antagonistas del Receptor de Estrógeno/farmacología , Receptor alfa de Estrógeno/genética , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mama/patología , Neoplasias de la Mama/sangre , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/genética , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Supervivencia sin Enfermedad , Estradiol/farmacología , Estradiol/uso terapéutico , Antagonistas del Receptor de Estrógeno/uso terapéutico , Receptor alfa de Estrógeno/antagonistas & inhibidores , Estrógenos/metabolismo , Femenino , Fulvestrant , Humanos , Indazoles/farmacología , Indazoles/uso terapéutico , Persona de Mediana Edad , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéuticoRESUMEN
Activation of the PI3K pathway occurs commonly in a wide variety of cancers. Experience with other successful targeted agents suggests that clinical resistance is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN-deficient cancers. We generated cell lines resistant to the pan-PI3K inhibitor GDC-0941 from parental PTEN-null breast cancer cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines that was recurrent in cancer patients. Stable expression of mutant PIK3CB variants conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. Furthermore, we show that the p110ß D1067Y mutant was highly activated and induced PIP3 levels at the cell membrane, subsequently promoting the localization and activation of AKT and PDK1 at the membrane and driving PI3K signaling to a level that could withstand treatment with proximal inhibitors. Finally, we demonstrate that the PIK3CB D1067Y mutant behaved as an oncogene and transformed normal cells, an activity that was enhanced by PTEN depletion. Collectively, these novel preclinical and clinical findings implicate the acquisition of activating PIK3CB D1067 mutations as an important event underlying the resistance of cancer cells to selective PI3K inhibitors.
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Neoplasias de la Mama/tratamiento farmacológico , Mutación , Inhibidores de las Quinasa Fosfoinosítidos-3 , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Resistencia a Antineoplásicos , Femenino , Humanos , Fosfohidrolasa PTEN/deficiencia , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/fisiologíaRESUMEN
Breast cancer is a heterogeneous disease and patients are managed clinically based on ER, PR, HER2 expression, and key risk factors. We sought to characterize the molecular landscape of high-risk breast cancer patients enrolled onto an adjuvant chemotherapy study to understand how disease subsets and tumor immune status impact survival. DNA and RNA were extracted from 861 breast cancer samples from patients enrolled onto the United States Oncology trial 01062. Samples were characterized using multiplex gene expression, copy number, and qPCR mutation assays. HR+ patients with a PIK3CA mutant tumor had a favorable disease-free survival (DFS; HR 0.66, P=0.05), however, the prognostic effect was specific to luminal A patients (Luminal A: HR 0.67, P=0.1; Luminal B: HR 1.01, P=0.98). Molecular subtyping of triple-negative breast cancers (TNBCs) suggested that the mesenchymal subtype had the worst DFS, whereas the immunomodulatory subtype had the best DFS. Profiling of immunologic genes revealed that TNBC tumors (n=280) displaying an activated T-cell signature had a longer DFS following adjuvant chemotherapy (HR 0.59, P=0.04), while a distinct set of immune genes was associated with DFS in HR+ cancers. Utilizing a discovery approach, we identified genes associated with a high risk of recurrence in HR+ patients, which were validated in an independent data set. Molecular classification based on PAM50 and TNBC subtyping stratified clinical high-risk patients into distinct prognostic subsets. Patients with high expression of immune-related genes showed superior DFS in both HR+ and TNBC. These results may inform patient management and drug development in early breast cancer.
RESUMEN
Breast cancers are categorized into three subtypes based on protein expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/ERBB2). Patients enroll onto experimental clinical trials based on ER, PR, and HER2 status and, as receptor status is prognostic and defines treatment regimens, central receptor confirmation is critical for interpreting results from these trials. Patients enrolling onto experimental clinical trials in the metastatic setting often have limited available archival tissue that might better be used for comprehensive molecular profiling rather than slide-intensive reconfirmation of receptor status. We developed a Random Forests-based algorithm using a training set of 158 samples with centrally confirmed IHC status, and subsequently validated this algorithm on multiple test sets with known, locally determined IHC status. We observed a strong correlation between target mRNA expression and IHC assays for HER2 and ER, achieving an overall accuracy of 97 and 96%, respectively. For determining PR status, which had the highest discordance between central and local IHC, incorporation of expression of co-regulated genes in a multivariate approach added predictive value, outperforming the single, target gene approach by a 10% margin in overall accuracy. Our results suggest that multiplexed qRT-PCR profiling of ESR1, PGR, and ERBB2 mRNA, along with several other subtype associated genes, can effectively confirm breast cancer subtype, thereby conserving tumor sections and enabling additional biomarker data to be obtained from patients enrolled onto experimental clinical trials.
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Algoritmos , Biomarcadores de Tumor/genética , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/metabolismo , ARN Neoplásico/genética , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Ensayos Clínicos Fase III como Asunto , Femenino , Estudios de Seguimiento , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Límite de Detección , Estudios Multicéntricos como Asunto , Análisis Multivariante , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , Curva ROC , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptor ErbB-2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de SupervivenciaRESUMEN
Lung cancer remains the primary cause of cancer-related deaths worldwide. Improved tools for early detection and therapeutic stratification would be expected to increase the survival rate for this disease. Alterations in the molecular pathways that drive lung cancer, which include epigenetic modifications, may provide biomarkers to help address this major unmet clinical need. Epigenetic changes, which are defined as heritable changes in gene expression that do not alter the primary DNA sequence, are one of the hallmarks of cancer, and prevalent in all types of cancer. These modifications represent a rich source of biomarkers that have the potential to be implemented in clinical practice. This perspective describes recent advances in the discovery of epigenetic biomarkers in lung cancer, specifically those that result in the methylation of DNA at CpG sites. We discuss one approach for methylation-based biomarker assay development that describes the discovery at a genome-scale level, which addresses some of the practical considerations for design of assays that can be implemented in the clinic. We emphasize that an integrated technological approach will enable the development of clinically useful DNA methylation-based biomarker assays. While this article focuses on current literature and primary research findings in lung cancer, the principles we describe here apply to the discovery and development of epigenetic biomarkers for other types of cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Epigenómica/métodos , Histonas/fisiología , Neoplasias Pulmonares/genética , ARN no Traducido/genética , Islas de CpG , Epigénesis Genética , Epigenómica/instrumentación , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Reproducibilidad de los ResultadosRESUMEN
PURPOSE: The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. METHODS: We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. RESULTS: Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. CONCLUSION: AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.
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Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , ADN de Neoplasias/análisis , Genes Relacionados con las Neoplasias/genética , Secuencia de Bases , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Análisis Mutacional de ADN/métodos , Formaldehído , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación/genética , Parafina , Fosfatidilinositol 3-Quinasas/genética , Análisis de Secuencia de ADN , Adhesión del Tejido , Fijación del TejidoRESUMEN
Patients with newly diagnosed, early stage estrogen receptor positive (ER+) breast cancer often show disease free survival in excess of five years following surgery and systemic adjuvant therapy. An important question is whether diagnostic tumor tissue from the primary lesion offers an accurate molecular portrait of the cancer post recurrence and thus may be used for predictive diagnostic purposes for patients with relapsed, metastatic disease. As the class I phosphatidylinositol 3' kinase (PI3K) pathway is frequently activated in ER+ breast cancer and has been linked to acquired resistance to hormonal therapy, we hypothesized pathway status could evolve over time and treatment. Biomarker analyses were conducted on matched, asynchronous primary and metastatic tumors from 77 patients with ER+ breast cancer. We examined whether PIK3CA and AKT1 alterations or PTEN and Ki67 levels showed differences between primary and metastatic samples. We also sought to look more broadly at gene expression markers reflective of proliferation, molecular subtype, and key receptors and signaling pathways using an mRNA analysis platform developed on the Fluidigm BioMark™ microfluidics system to measure the relative expression of 90 breast cancer related genes in formalin-fixed paraffin-embedded (FFPE) tissue. Application of this panel of biomarker assays to matched tumor pairs showed a high concordance between primary and metastatic tissue, with generally few changes in mutation status, proliferative markers, or gene expression between matched samples. The collection of assays described here has been optimized for FFPE tissue and may have utility in exploratory analyses to identify patient subsets responsive to targeted therapies.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Metaboloma , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Microfluídica , Mutación/genética , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , Células Tumorales CultivadasRESUMEN
PURPOSE: Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples. EXPERIMENTAL DESIGN: We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer. RESULTS: We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA. CONCLUSIONS: MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Mutación , Neoplasias/genética , Proteínas Supresoras de Tumor/genética , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Fosfatidilinositol 3-Quinasa Clase Ia , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Receptores ErbB/genética , Técnicas de Genotipaje , Humanos , Células MCF-7 , Neoplasias/metabolismo , Neoplasias/patología , Fosfohidrolasa PTEN/genética , Adhesión en Parafina , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Reproducibilidad de los Resultados , Proteínas Supresoras de Tumor/metabolismo , Proteínas ras/genéticaRESUMEN
PURPOSE: The oncogenic PI3K/Akt/mTOR pathway is an attractive therapeutic target in cancer. However, it is unknown whether the pathway blockade required for tumor growth inhibition is clinically achievable. Therefore, we conducted pharmacodynamic studies with GDC-0068, an ATP competitive, selective Akt1/2/3 inhibitor, in preclinical models and in patients treated with this compound. EXPERIMENTAL DESIGN: We used a reverse phase protein array (RPPA) platform to identify a biomarker set indicative of Akt inhibition in cell lines and human-tumor xenografts, and correlated the degree of pathway inhibition with antitumor activity. Akt pathway activity was measured using this biomarker set in pre- and post-dose tumor biopsies from patients treated with GDC-0068 in the dose escalation clinical trial. RESULTS: The set of biomarkers of Akt inhibition is composed of 10 phosphoproteins, including Akt and PRAS40, and is modulated in a dose-dependent fashion, both in vitro and in vivo. In human-tumor xenografts, this dose dependency significantly correlated with tumor growth inhibition. Tumor biopsies from patients treated with GDC-0068 at clinically achievable doses attained a degree of biomarker inhibition that correlated with tumor growth inhibition in preclinical models. In these clinical samples, compensatory feedback activation of ERK and HER3 was observed, consistent with preclinical observations. CONCLUSION: This study identified a set of biomarkers of Akt inhibition that can be used in the clinical setting to assess target engagement. Here, it was used to show that robust Akt inhibition in tumors from patients treated with GDC-0068 is achievable, supporting the clinical development of this compound in defined patient populations.
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Proteína Oncogénica v-akt/antagonistas & inhibidores , Piperazinas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirimidinas/administración & dosificación , Transducción de Señal/genética , Animales , Humanos , Ratones , Proteína Oncogénica v-akt/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The objective of this study was to evaluate circulating hepatocyte growth factor (cHGF) as a pharmacodynamic biomarker of Met inhibition for onartuzumab (MetMAb, OA5D5v2) in a phase I trial in patients with advanced cancers and a phase II trial in non-small cell lung cancer (NSCLC). The phase I study was a dose escalation trial with onartuzumab administered i.v. once every three weeks. The phase II study was a randomized two-arm trial in which onartuzumab or placebo was administered in combination with erlotinib in 137 patients with second and third line (2/3L) NSCLC. cHGF levels were evaluated by ELISA at multiple time points over the treatment period. Onartuzumab administration resulted in an acute and sustained rise in cHGF in both the phase I and phase II studies. Elevation in cHGF was independent of dose or drug exposure and was restricted to onartuzumab treatment. Neither higher baseline nor elevated change in cHGF levels upon treatment could simply be attributed to tumor burden or number of liver metastasis. We have shown that elevated cHGF can consistently and reproducibly be measured as a pharmacodynamic biomarker of onartuzumab activity. The elevation in cHGF is independent of tumor type, dose administered, or dose duration. Although these studies were not powered to directly address the contribution of cHGF as a predictive, on-treatment, circulating biomarker, these data suggest that measurement of cHGF in future expanded studies is warranted.
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Anticuerpos Monoclonales/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Factor de Crecimiento de Hepatocito/sangre , Neoplasias Pulmonares/tratamiento farmacológico , Células Neoplásicas Circulantes/metabolismo , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/patología , Clorhidrato de Erlotinib , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento de Hepatocito/biosíntesis , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/patología , Masculino , Estadificación de Neoplasias , Proteínas Proto-Oncogénicas c-met/biosíntesis , Proteínas Proto-Oncogénicas c-met/genética , Quinazolinas/administración & dosificaciónRESUMEN
BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. METHODS: We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. RESULTS: The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. CONCLUSIONS: We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.
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Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Inhibidores Enzimáticos/farmacología , Citometría de Flujo/métodos , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Biomarcadores de Tumor/metabolismo , Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Indazoles/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Sulfonamidas/farmacologíaRESUMEN
PURPOSE: Tumors with oncogenic dependencies on the HER family of receptor tyrosine kinases (RTKs) often respond well to targeted inhibition. Our previous work suggested that many cell lines derived from squamous cell carcinomas of the head and neck (SCCHNs) depend on autocrine signaling driven by HER2/3 dimerization and high-level co-expression of HRG. Additionally, results from a Phase I trial of MEHD7495A, a dual-action antibody that blocks ligand binding to EGFR and HER3, suggest that high-level HRG expression was associated with clinical response in SCCHN patients. Here we explore the hypothesis that high-level HRG expression defines a subpopulation of SCCHNs with activated HER3. EXPERIMENTAL DESIGN: qRT-PCR expression profiling was performed on >750 tumors of diverse origin, including >150 therapy-naïve, primary, and recurrent SCCHNs. Activated HER3, defined by immunoprecipitation of phospho-HER3, was compared to HRG expression in SCCHN samples. Paracrine versus autocrine expression was evaluated using RNA-in situ hybridization. RESULTS: SCCHN tumors express the highest levels of HRG compared to a diverse collection of other tumor types. We show that high HRG expression is associated with activated HER3, whereas low HRG expression is associated with low HER3 activation in SCCHN tumors. Furthermore, HRG expression is higher in recurrent SCCHN compared to patient-matched therapy naïve specimens. CONCLUSIONS: HRG expression levels define a biologically distinct subset of SCCHN patients. We propose that high-level expression of HRG is associated with constitutive activation of HER3 in SCCHN and thus defines an actionable biomarker for interventions targeting HER3.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Neurregulina-1/metabolismo , Receptor ErbB-3/metabolismo , Biomarcadores de Tumor , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Expresión Génica , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Humanos , Inmunohistoquímica , Neurregulina-1/genética , Receptor ErbB-3/genética , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
PURPOSE: Cancer of the ovary confers the worst prognosis among women with gynecological malignancies, primarily because most ovarian cancers are diagnosed at late stage. Hence, there is a substantial need to develop new diagnostic biomarkers to enable detection of ovarian cancer at earlier stages, which would confer better prognosis. In addition, the identification of druggable targets is of substantial interest to find new therapeutic strategies for ovarian cancer. METHODS: The expression of 22,500 genes in a series of 67 serous papillary carcinomas was compared with 9 crudely enriched normal ovarian tissue samples by RNA hybridization on oligonucleotide microarrays. Multiple genes with near-uniformly expression were elevated in carcinomas of varying grade and malignant potential, including several previously described genes (e.g., MUC-1, CD9, CD24, claudin 3, and mesothelin). We performed immunohistochemical staining with antibodies against several of the proteins encoded by differentially expressed genes in an independent cohort of 71 cases of paraffin-embedded ovarian cancer samples. RESULTS: We found striking differences in EpCAM (p < 0.005), CD9 (p < 0.001), MUC-1 (p < 0.001), and claudin 3 proteins (p < 0.001) but not for mesothelin (p > 0.05) using the Mann-Whitney U test. CONCLUSIONS: Protein expression of a majority of the differentially expressed genes tested was found to be elevated in ovarian carcinomas and, as such, define potential new biomarkers or targets.
Asunto(s)
Cistadenocarcinoma Seroso/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/genética , Ovario/metabolismo , Análisis por Conglomerados , Cistadenocarcinoma Seroso/metabolismo , Femenino , Humanos , Neoplasias Ováricas/metabolismoRESUMEN
PURPOSE: Class 1 phosphatidylinositol 3-kinase (PI3K) plays a major role in cell proliferation and survival in a wide variety of human cancers. Here, we investigated biomarker strategies for PI3K pathway inhibitors in non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Molecular profiling for candidate PI3K predictive biomarkers was conducted on a collection of NSCLC tumor samples. Assays included comparative genomic hybridization, reverse-transcription polymerase chain reaction gene expression, mutation detection for PIK3CA and other oncogenes, PTEN immunohistochemistry, and FISH for PIK3CA copy number. In addition, a panel of NSCLC cell lines characterized for alterations in the PI3K pathway was screened with PI3K and dual PI3K/mTOR inhibitors to assess the preclinical predictive value of candidate biomarkers. RESULTS: PIK3CA amplification was detected in 37% of squamous tumors and 5% of adenocarcinomas, whereas PIK3CA mutations were found in 9% of squamous and 0% of adenocarcinomas. Total loss of PTEN immunostaining was found in 21% of squamous tumors and 4% of adenocarcinomas. Cell lines harboring pathway alterations (receptor tyrosine kinase activation, PI3K mutation or amplification, and PTEN loss) were exquisitely sensitive to the PI3K inhibitor GDC-0941. A dual PI3K/mTOR inhibitor had broader activity across the cell line panel and in tumor xenografts. The combination of GDC-0941 with paclitaxel, erlotinib, or a mitogen-activated protein-extracellular signal-regulated kinase inhibitor had greater effects on cell viability than PI3K inhibition alone. CONCLUSIONS: Candidate biomarkers for PI3K inhibitors have predictive value in preclinical models and show histology-specific alterations in primary tumors, suggesting that distinct biomarker strategies may be required in squamous compared with nonsquamous NSCLC patient populations.
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Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Indazoles/farmacología , Neoplasias Pulmonares/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Pirimidinas/farmacología , Sulfonamidas/farmacología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Sinergismo Farmacológico , Clorhidrato de Erlotinib , Femenino , Amplificación de Genes , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Fosfohidrolasa PTEN/metabolismo , Paclitaxel/farmacología , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinazolinas/farmacología , Transducción de Señal , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite early detection and reduced risk of death, prostate cancer still remains the second leading cause of cancer death in American men. There is currently no cure for advanced prostate cancer. The multistage, stochastic and highly heterogeneous nature of prostate cancer, coupled with genetic and epigenetic alterations that occur during disease progression and response to therapy, represent fundamental challenges in our quest to understand and control this complex and prevalent disease. Recent advances in drug development and breakthroughs in omics technologies have renewed our efforts to identify novel biomarkers for prostate cancer prognosis, prediction, and therapeutic response monitoring. In this perspective article, we overview the current status and highlight future prospects of biomarkers for prostate cancer, a disease that affects millions of men worldwide.
RESUMEN
PURPOSE: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in patients with epithelial cancers. Less is known about CTCs as surrogate endpoints or their use for predictive biomarker evaluation. This study investigated the utility of CTC enumeration and characterization using the CellSearch platform, as well as mutation detection in circulating tumor DNA (ctDNA), in patients with advanced non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Forty-one patients were enrolled in a single-arm phase II clinical trial of erlotinib and pertuzumab. Peripheral blood was analyzed for CTC enumeration, EGFR expression in CTCs, and detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC levels were correlated with 2[18F]fluoro-2-deoxy-D-glucose-positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging and survival endpoints. RESULTS: CTCs were detected (≥ 1 CTC) at baseline in 78% of patients. Greater sensitivity for mutation detection was observed in ctDNA than in CTCs and detected mutations were strongly concordant with mutation status in matched tumor. Higher baseline CTC counts were associated with response to treatment by Response Evaluation Criteria in Solid Tumors (RECIST, P = 0.009) and decreased CTC counts upon treatment were associated with FDG-PET and RECIST response (P = 0.014 and P = 0.019) and longer progression-free survival (P = 0.050). CONCLUSION: These data provide evidence of a correlation between decreases in CTC counts and radiographic response by either FDG-PET or RECIST in patients with advanced NSCLC. These findings require prospective validation but suggest a potential role for using CTC decreases as an early indication of response to therapy and ctDNA for real-time assessment of mutation status from blood.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN de Neoplasias/sangre , Neoplasias Pulmonares/tratamiento farmacológico , Células Neoplásicas Circulantes , Anticuerpos Monoclonales Humanizados/administración & dosificación , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico por imagen , Supervivencia sin Enfermedad , Determinación de Punto Final , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Femenino , Genotipo , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/genética , Masculino , Mutación , Tomografía de Emisión de Positrones , Quinazolinas/administración & dosificaciónRESUMEN
The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor-resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients.