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Introduction: EGFR T790M is the most common mechanism of acquired resistance to EGFR TKIs in pts with EGFR-mutant lung cancer. AZD9291 is an irreversible, mutant-selective EGFR TKI developed to have potency against both sensiziting EGFR mutations and T790M. In the ongoing phase I study of AZD9291 (AURA, NCT01802632), the response rate in pts with T790M-positive lung cancer was >60%. The molecular mechanism underlying acquired resistance to AZD9291 is not known. Methods & Results: To explore for mechanisms of resistance to AZD9291, we studied cfDNA extracted from pretreatment and post-progression plasma collected on AURA.Next-generation sequencing (NGS) of cfDNA was first performed on an exploratory cohort of 7 pts. All exons of a 20 gene panel (including EGFR) underwent PCR amplification and NGS using an Illumina HiSeq. In 1 pt, NGS of progression plasma identified a new EGFR C797S mutation in exon 20, not present in pretreatment plasma. Stable expression of C797S in Ba/F3 cells induced a >100-fold increase in IC50 to AZD9291 compared to EGFR activating and T790M mutations alone. To validate the plasma NGS, digital droplet PCR (ddPCR) assays were developed to detect key EGFR mutations including C797S. 15 T790M-positive cases were identified with progression plasma available for analysis. Serial plasma ddPCR showed that both the EGFR activating and T790M mutation levels decreased with AZD9291 treament and increased at progression, with 3 molecular subtypes of resistance apparent. In 6 pts (40%), C797S was detected in addition to T790M; NGS of resistance biopsies from 2 of these pts confirmed presence of acquired C797S. In 5 pts (33%), T790M was detected without evidence of C797S. Intriguingly, in 4 pts (27%), the T790M levels became undetectable with treatment despite high levels of the EGFR activating mutation at progression, suggesting overgrowth of a competing non-T790M resistance mechanism. Further NGS of progression plasma revealed additional evidence of the genomic heterogeneity of resistance. Individual sequencing reads indicate that C797S and T790M can occur either in cis or in trans (i.e. on competing resistant alleles). In the 2 pts with tumor NGS demonstrating C797S, plasma NGS identified both the DNA alteration seen in tumor as well as a second DNA alteration encoding for C797S. Conclusion: Using complementary assays for genomic analyses of cfDNA, we identified 3 molecular subtypes of acquired resistance to AZD9291, including an EGFR C797S mutation never before reported in pts. Due to the key role of the C797 residue in drug binding, C797S is expected to induce resistance to all irreversible EGFR TKIs currently in clinical development. Plasma NGS revealed substantial genomic heterogeneity and highlights the need for combination therapies to effectively prevent or treat drug resistance in cancer. Citation Format: Geoffrey R. Oxnard, Kenneth S. Thress, Cloud P. Paweletz, Enriqueta Felip, Byoung Chul Cho, Daniel Stetson, Brian Dougherty, Zhongwu Lai, Aleksandra Morkovets, Ana Vivancos, Yanan Kuang, Dalia Ercan, Mireille Cantarini, J Carl Barrett, Pasi A. Janne. Analysis of cell-free plasma DNA (cfDNA) identifies 3 molecular subtypes of acquired resistance to AZD9291, a novel EGFR tyrosine kinase inhibitor (TKI), in patients (pts) with advanced lung cancer. [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 LB-123. doi:10.1158/1538-7445.AM2015-LB-123

Somatic mutations in BRAF are predictive of the clinical efficacy of BRAF and/or MEK inhibitors for advanced melanoma. Resistance to treatment is observed for most patients despite a high response rate to kinase inhibitors. While repeated tumor biopsy is the gold standard for evaluating genetic changes following treatment, this is not generally feasible. The analysis of circulating melanoma cells (CMCs) has the potential to be a non-invasive means of interrogating the biology underlying response and resistance. Here we report on the isolation, detection, and characterization of CMCs from patients using size filtration followed by immunoflourescence and/or fluorescent in situ hybridization (FISH) analyses. Blood from a cohort of 20 patients with advanced melanoma was collected in an initial proof of concept experiment. CMCs were identified in 14 out of 20 melanoma blood samples (70%) with a range of 2.5-99 CMCs per 3mL of blood by immunofluorescence (IF) using a cocktail of 4 melanoma specific antibodies (HMB45, MART-1, Tyrosinase and MiTF). Cells that appeared tumor like by microscopy, but that did not stain for CD45 or the cocktail of melanoma specific antibodies were also detected. To further characterize these cells, FISH was performed with a set of 4 melanoma-specific probes (RREB1, MYB, Centromere 6, CCND1). In a cohort of 13 patients with melanoma specimens that had paired blood specimens, CMCs were detected in all 13 specimens ranging from 7.2-76 CMCs per 3mL of blood with more identification by FISH in 10 out 13 samples. Additionally, cells that were double negative by IF were observed to be positive by FISH. CMCs are detectable in the majority of patients with advanced melanoma. Use of IF and FISH can help detect and characterize CMCs. These tools will be useful in characterizing treatment related changes in CMCs. Citation Format: Masahiko Yanagita, Jason J. Luke, Cloud P. Paweletz, Elena Ivanova, Paul T. Kirschmeier, Stephen F. Hodi, Pasi A. Jänne. Isolation and characterization of circulating melanoma cells by size filtration and fluorescent in situ hybridization. [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 1598. doi:10.1158/1538-7445.AM2015-1598

High grade serous ovarian carcinoma (HGSOC) is the most common form of ovarian cancer; effective therapeutics for HGSOC remains elusive and is major unmet medical need. Standard treatment for these patients is surgery followed by platinum- based therapy. While the majority of patients exhibit an initial response to this standard therapy, many develop disease recurrence. In order to advance therapeutic development we have established a panel of low passage, well characterized, orthotopic, luciferized HGSOC patient derived xenografts (PDXs) that accurately represent the disease characteristics and are amenable to assess efficacy evaluations of novel therapeutics. Ovarian PDXs were established by implanting mice intraperitoneally with fresh human ovarian cancer cells purified from operative or paracentesis samples under an IRB-approved protocol. Implanted mice were sacrificed after showing signs of abdominal distension and ascites development. Fresh mouse ascites-derived ovarian cancer cells were luciferized ex vivo by lentivirus based methodologies to allow non-invasive methods of tumor burden measurement. Luciferized PDX models were further expanded, banked and utilized for drug efficacy and biomarker evaluation studies. DNA from patient material and matched established PDX models was extracted and subjected to CNV on the Affymetrix CytoscanTM HD microarray platform and BROCA cancer risk panel targeted sequencing. Data from these analyses indicated that the PDXs are molecularly diverse and faithfully maintain the genetic alterations and copy number variation profiles of the patient tumors. Consistent with clinical disease, these orthotopic PDX models exhibit diffusely disseminated peritoneal disease with tissue infiltration to the omentum, ovaries and pancreas, along with ascites and abdominal distention. Immunohistochemical analysis of PDX tumor tissues infiltrated in major organs for ovarian specific PAX8, p53 and WT1 markers confirmed the high grade serous type of ovarian tumors. Orthotopic PDXs have been validated and are shown to be robust and amenable for drug efficacy evaluations by Bioluminescent Imaging. Their response to standard carboplatin mirrors the response in the patients from which they were derived. Surrogate biomarkers such as plasma CA-125 measured by BioScale AMMP method and circulating human cfDNA by LINE-1 qPCR have been validated and qualified in these models to support secondary methods of tumor burden evaluation. Conclusion: A platform of HGSOC PDX models that retain histologic and molecular characteristics of primary tumors that are expected to be predictive of clinical outcomes and amenable for efficacy testing of novel therapeutics is established. These models are being utilized for hypothesis driven combination efficacy and biomarker studies for translation to the clinic. Citation Format: Sangeetha S. Palakurthi, Joyce F. Liu, Qing Zeng, Shan Zhou, Wei Huang, Elena Ivanova, Cloud Paweletz, John R. Murgo, Justin Evangelista, Melissa Buttimer, Jennifer Curtis, Huiying Piao, Prafulla Gokhale, Colin Pritchard, Jessie M. English, Paul Kirschmeier, Kwok-Kin Wong, Ursula A. Matulonis, Ronny Drapkin. Establishment, characterization, and clinical correlation of a platform of ovarian patient-derived xenograft (PDX) models. [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 1471. doi:10.1158/1538-7445.AM2015-1471

11068 Background: Genotype directed therapy is now standard of care for advanced NSCLC pts. However, adequate tumor tissue for comprehensive genotyping remains a challenge. Recent research suggests that CTC capture or cfDNA analysis allows for non-invasive diagnosis and monitoring of treatment. This prospective trial was designed to quantify the predictive value of CTC and cfDNA analyses of EGFR-mutant NSCLC pts treated with first-line erlotinib. Methods: TKI naive EGFR mutant NSCLC pts were enrolled in a phase II trial of erlotinib treatment. Paired blood for cfDNA and CTC analysis was collected at baseline prior to therapy and every 2 months during follow ups. Plasma genotyping was performed by ddPCR for EGFR19del, L858R, T790M while CTCs were isolated by CellSearch and analyzed by IF and MET-FISH. Repeat biopsies at progression were performed when feasible. Results: Between 2/10 and 1/15, 60 EGFRmutant pts (L858R:17, 19del:38, other:5) were enrolled. As of 1/15, 44 patients have discontinued therapy (3...

11089 Background: Plasma genotyping of cell-free DNA (cfDNA) has the potential to allow for noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies....

1080 Background: Data suggests that MET expression and activation are important for initiation and progression of TNBC. We evaluated the efficacy of cabozantinib (XL184), a novel inhibitor of multi...

Detection of minor, genetically distinct subpopulations within tumors is a key challenge in cancer genomics. Here we report STAR-FISH (specific-to-allele PCR-FISH), a novel method for the combined detection of single-nucleotide and copy number alterations in single cells in intact archived tissues. Using this method, we assessed the clinical impact of changes in the frequency and topology of PIK3CA mutation and HER2 (ERBB2) amplification within HER2-positive breast cancer during neoadjuvant therapy. We found that these two genetic events are not always present in the same cells. Chemotherapy selects for PIK3CA-mutant cells, a minor subpopulation in nearly all treatment-naive samples, and modulates genetic diversity within tumors. Treatment-associated changes in the spatial distribution of cellular genetic diversity correlated with poor long-term outcome following adjuvant therapy with trastuzumab. Our findings support the use of in situ single cell-based methods in cancer genomics and imply that chemotherapy before HER2-targeted therapy may promote treatment resistance.

In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry (Pollard, H. B., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210-2226). In the present work we compared the IB3-1 cells with IB3-1/S9 daughter cells repaired by gene transfer with AAV-(wild type)CFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [(35)S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFkappaB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that approximately 30% of the 51-member hybrid high abundance CF proteome interacts with the NFkappaB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine.

Here we describe the use of SELDI-MS to detect dose-dependent peptide changes in plasma from mice treated with vehicle or rosiglitazone at one of two doses (10 and 30 mg/kg). SELDI features differentiating spectra from the three conditions were found and used to train classifiers. Samples treated with vehicle could be reliably distinguished from samples treated with either dose, but samples treated with the different doses could not be reliably distinguished from one another. We conclude that while SELDI-TOF mass spectra can be used to distinguish treated from untreated samples, the reproducibility and information content of SELDI-TOF are currently not sufficient as a pharmacodynamic readout to distinguish between mice treated with 10 or 30 mg/kg of rosiglitazone. This raises more general questions about whether SELDI's sensitivity is sufficient for detecting dose-dependent changes in plasma.

AT-rich DNA elements play an important role in regulating cell-specific gene expression. One of the AT-rich DNA binding proteins, SATB1 is a novel type of transcription factor that regulates gene expression in the hematopoietic lineage through chromatin modification. Using DNA-affinity purification followed by mass spectrometry we identified and isolated a related protein, SATB2 from the developing rat cerebral cortex. SATB2 shows homology to SATB1 and the rat protein is practically identical to the mouse and human SATB2. Using competitive EMSA, we show that recombinant SATB2 protein binds with high affinity and specificity to AT-rich dsDNA. Using RT-PCR, Western analysis and immunohistochemistry we demonstrate that SATB2 expression is restricted to a subset of postmitotic, differentiating neurons in the rat neocortex at ages E16 and P4. We suggest that similar to its homologue SATB1, SATB2 is also involved in regulating gene expression through altering chromatin structure in differentiating cortical neurons.

Lysing of a complete biopsy sample results in a mixture of desired and undesired proteins, reflecting the originating cell types. Therefore microdissecting tissue material is mandatory prior to sample lysis and all downstream applications of protein analysis (proteomics). The two most important dissecting methods for bladder tissue specimens are manual microdissection and laser microdissection. Sample transfer can further be separated into manual laser pressure catapulting (LPC) and laser capture microdissection (LCM). One of the possible downstream applications of protein analysis is surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry. The small quantities of tissue obtained by microdissection are sufficient for use in the SELDI technique.

Here we describe and characterize a small serine/threonine kinase (SSTK) which consists solely of the N- and C-lobes of a protein kinase catalytic domain. SSTK protein is highly conserved among mammals, and no close homologues were found in the genomes of nonmammalian organisms. SSTK specifically interacts with HSP90-1beta, HSC70, and HSP70 proteins, and this association appears to be required for SSTK kinase activity. The SSTK transcript was most abundant in human and mouse testes but was also detected in all human tissues tested. In the mouse testis, SSTK protein was localized to the heads of elongating spermatids. Targeted deletion of the SSTK gene in mice resulted in male sterility due to profound impairment in motility and morphology of spermatozoa. A defect in DNA condensation in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins. SSTK phosphorylated histones H1, H2A, H2AX, and H3 but not H2B or H4 or transition protein 1 in vitro. These results demonstrate that SSTK is required for proper postmeiotic chromatin remodeling and male fertility. Abnormal sperm chromatin condensation is common in sterile men, and our results may provide insight into the molecular mechanisms underlying certain human infertility disorders.

Lysing of a complete biopsy sample results in a mixture of desired and undesired proteins, reflecting the originating cell types. Therefore microdissecting tissue material is mandatory prior to sample lysis and all downstream applications of protein analysis (proteomics). The two most important dissecting methods for bladder tissue specimens are manual microdissection and laser microdissection. Sample transfer can further be separated into manual laser pressure catapulting (LPC) and laser capture microdissection (LCM). One of the possible downstream applications of protein analysis is surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry. The small quantities of tissue obtained by microdissection are sufficient for use in the SELDI technique.

The field of proteomics holds promise for the discovery of new biomarkers for the early detection and diagnosis of disease, molecular targets for therapy and markers for therapeutic efficacy and toxicity. A variety of proteomics approaches may be used to address these goals. Two-dimensional gel electrophoresis (2D-PAGE) is the cornerstone of many discovery-based proteomics studies. Technologies such as laser capture microdissection (LCM) and highly sensitive MS methods are currently being used together to identify greater numbers of lower abundance proteins that are differentially expressed between defined cell populations. Newer technologies such as reverse phase protein arrays will enable the identification and profiling of target pathways in small biopsy specimens. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) analysis enables the high throughput characterization of lysates from very few tumor cells or body fluids and may be best suited for diagnosis and monitoring of disease. Such technologies are expected to supplement our arsenal of mRNA-based assays, and we believe that in the future, entire cellular networks and not just a single deregulated protein will be the target of therapeutics and that we will soon be able to monitor the status of these pathways in diseased cells before, during and after therapy.

Clinical proteomics is an exciting new subdiscipline of proteomics that involves bedside application of proteomic technologies. A new and potentially revolutionary technology and approach for early disease detection, surveillance, and monitoring is proteomic pattern diagnostics. Using this approach, high throughput mass spectrometry generates a proteomic fingerprint of a given body fluid, such as serum or nipple fluid aspirants (NAF), in less than 30 s. This information archive is then used by new types of bioinformatic pattern recognition algorithms to identify patterns of protein changes that can discriminate cancer from healthy and unaffected individuals. This entire process can take place in less than a minute and requires only a droplet of blood, NAF, or ductal lavage washings. The new concept that is introduced by this platform is that the underlying identities of the proteins that comprise the patterns are not known and do not need to be known; the pattern itself becomes the diagnostic.

As we emerge into the post-genome era, proteomics finds itself as the driving force field as we translate the nucleic acid information archive into understanding how the cell actually works and how disease processes operate. Even so, the traditionally held view of proteomics as simply cataloging and developing lists of the cellular protein repertoire of a cell are now changing, especially in the sub-discipline of clinical proteomics. The most relevant information archive to clinical applications and drug development involves the elucidation of the information flow of the cell; the “software” of protein pathway networks and circuitry. The deranged circuitry of the cell as the drug target itself as well as the effect of the drug on not just the target, but also the entire network, is what we now are striving towards. Clinical proteomics, as a new and most exciting sub-discipline of proteomics, involves the bench-to-bedside clinical application of proteomic tools. Unlike the genome, there are potentially thousands of proteomes: each cell type has its own unique proteome. Moreover, each cell type can alter its proteome depending on the unique tissue microenvironment in which it resides, giving rise to multiple permutations of a single proteome. Since there is no polymerase chain reaction equivalent to proteomics- identifying and discovering the “wiring diagram” of a human diseased cell in a biopsy specimen remains a daunting challenge. New micro-proteomic technologies are being and still need to be developed to drill down into the proteomes of clinically relevant material. Cancer, as a model disease, provides a fertile environment to study the application of proteomics at the bedside. The promise of clinical proteomics and the new technologies that are developed is that we will detect cancer earlier through discovery of biomarkers, we will discover the next generation of targets and imaging biomarkers, and we can then apply this knowledge to patient-tailored therapy.

The field of proteomics holds promise for the discovery of new biomarkers for the early detection and diagnosis of disease, molecular targets for therapy and markers for therapeutic efficacy and toxicity. A variety of proteomics approaches may be used to address these goals. Two-dimensional gel electrophoresis (2D-PAGE) is the cornerstone of many discovery-based proteomics studies. Technologies such as laser capture microdissection (LCM) and highly sensitive MS methods are currently being used together to identify greater numbers of lower abundance proteins that are differentially expressed between defined cell populations. Newer technologies such as reverse phase protein arrays will enable the identification and profiling of target pathways in small biopsy specimens. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) analysis enables the high throughput characterization of lysates from very few tumor cells or body fluids and may be best suited for diagnosis and monitoring of disease. Such technologies are expected to supplement our arsenal of mRNA-based assays, and we believe that in the future, entire cellular networks and not just a single deregulated protein will be the target of therapeutics and that we will soon be able to monitor the status of these pathways in diseased cells before, during and after therapy.

High grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy in the United States. It is the most common form of ovarian cancer but early detection and effective therapeutics remain elusive and are a major unmet medical need. The current standard of care for ovarian cancer is platinum based therapy, without consideration of histological subtypes. While many patients exhibit an initial response to this standard therapy, few achieve long term remissions or cures. A major limitation in advancing therapeutic development has been a lack of experimental animal models that accurately represent the spectrum of HGSOC, are amenable to assess efficacy of novel therapeutics, are predictive of patient responses in the clinic and can identify patient subsets that will benefit from specific targeted therapeutics. To address this gap, we generated a platform of clinically relevant, early passage, orthotopic HGSOC patient-derived xenograft (PDX) models. These models were established by implanting mice intraperitoneally with fresh human ovarian cancer cells purified from operative or paracentesis samples under an IRB-approved protocol. Implanted mice were sacrificed after showing signs of abdominal distension and ascites development. Fresh mouse ascites-derived ovarian cancer cells were luciferized ex vivo by sophisticated lentivirus based methodologies to allow for non-invasive methods of tumor burden measurement. Luciferized PDX models were further expanded, banked and utilized for drug efficacy and biomarker evaluation studies. Surrogate biomarkers such as plasma CA-125 measured by BioScale AMMP method and circulating human cfDNA by LINE-1 qPCR have been validated and qualified in these models to support secondary methods of tumor burden evaluation. A diverse library of clinically-annotated, HGSOC PDX models has been established that encompass both treatment naïve and treatment refractory HGSOC. Consistent with clinical disease, these orthotopic PDX models exhibit diffusely disseminated peritoneal disease with tissue infiltration to the omentum, ovaries, pancreas and spleen; along with distended abdomens full with ascites. Immunohistochemistry for ovarian specific markers including PAX8 and CK7 were analyzed in these tissues and shown to maintain fidelity to patient material through serial passages in mice. In vivo drug sensitivity studies with platinum and platinum/taxane doublet regimens recapitulate response as expected in HGSOC and, in a model of chemotherapy-resistant disease, demonstrate expected resistance as predicted by the clinical history. A clinically relevant HGSOC PDX model platform, amenable for testing efficacy of novel therapeutics and delineating responder ID has been established. Data will be presented with ongoing additional characterization of this platform. Citation Format: Sangeetha S. Palakurthi, Joyce F. Liu, Qing Zeng, Shan Zhou, Elena Ivanova, Cloud Paweletz, John Murgo, Justin Evangelista, Jennifer Curtis, Huiying Piao, Prafulla Gokhale, Jessie M. English, Paul Kirschmeier, Kwok-Kin Wong, Ursula A. Matulonis, Ronny Drapkin. Patient derived xenograft model platform of high grade serous ovarian cancer supporting discovery of targeted therapies and biomarkers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1202. doi:10.1158/1538-7445.AM2014-1202

Tumor genotyping has become standard in the care of non-small cell lung cancer (NSCLC), colorectal cancer, and melanoma because of its power in guiding the personalization of treatment with molecularly targeted therapies. A major limitation of cancer genotyping is the availability of pre- and post-treatment tumor biopsy specimens for accurate assessment of cancer biology. In addition, serial tumor biopsies from NSCLC patients at time of disease response or progression are not always desirable from the patient perspective and feasibility can be limited due to accessible tumor tissue and cost. The analysis of circulating cell-free DNA (cfDNA) has demonstrated potential to be a non-invasive means of interrogating the biology underlying response and resistance. Here we report the detection, quantification and monitoring of EGFR mutations by droplet digital PCR in cfDNA on a prospective clinical trial of EGFR-mutant NSCLC patients on erlotinib. Droplet digital PCR, a novel technology that emulsifies DNA input into ∼20,000 consistently sized droplets, each individually genotyped, represents a promising approach for non-invasive quantification of specific tumor mutations. EGFR L858R, Del(19) and T790M were serially measured in over 300 samples from 40 lung cancer patients treated on erlotinib. Complete plasma response was seen in 8 cases; in 6, rising levels of inhibitor sensitizing EGFR Del(19) and L858R and acquired EGFR T790M mutations were detected up to 4 months prior to objective radiographic progression. An analysis of patients that progressed showed that 53% had tumor-genotype-confirmed EGFR sensitizing mutations in their plasma at diagnosis. Four of these patients had a tumor rebiopsy adequate for EGFR genotyping at progression, and EGFR T790M was confirmed in each. This contrasts to the same analysis on circulating tumor cells (CTC), collected simultaneously: No CTCs were identified at diagnosis and only 22% of patients had any detectable CTCs at progression. Our results strongly suggest that cfDNA genotyping has clinical utility in the molecular assessment of patients at diagnosis, and providing molecular understanding of patient's tumor evolution, in real time, by serial monitoring for resistance mutations. Citation Format: Cloud P. Paweletz, Geoffrey R. Oxnard, Yanan Kuang, Allison O'Connell, Masahiko Yanagita, Melissa M. Messineo, Paul Kirschmeier, Jessie M. English, David M. Jackman, Pasi A. Jänne. Serial monitoring of EGFR mutations in plasma and matched tissue from EGFR mutant non-small cell lung cancer patients on erlotinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 939. doi:10.1158/1538-7445.AM2014-939

The identification of oncogenic driver mutations in cancer has led to the rapid rise of genotype-directed treatments such as EGFR and BRAF kinase inhibitors. Standard tumor biopsy remains a cumbersome and morbid procedure for patients, leading to a growing interest in noninvasive plasma genotyping approaches. Circulating tumor cells are of interest; however, the processing of specimens is complicated and time consuming. By comparison, cell-free DNA (cfDNA) genotyping has the potential to be convenient and relatively simple to process in a short time period. Several technologies are under development for cfDNA analysis, such as allele-specific polymerase chain reaction (PCR), coamplification at Lower Denaturation temperatures (COLD) PCR, emulsion PCR, and massively parallel sequencing. Broad clinical validity will need to be established for different assays, and clinical utility will need to be evaluated within prospective trials to determine which assays will best predict the efficacy of therapy and patient outcomes. In addition, assay standardization will be critical prior to widespread use in routine clinical practice. The Cell Free DNA Working Group, under the sponsorship of Transgenomic, was convened to evaluate the molecular assays in development and provide recommendations for application and interpretation of these tests in the context of future clinical research. The consensus commentary of the Cell Free DNA Working Group for the use of cfDNA plasma genotyping assays is presented here, including future steps in the development of these technologies.

1106 Background: Data suggests that MET expression and activation is important for initiation and progression of triple-negative breast cancer. Tivantinib (ARQ 197) is an orally administered agent ...

The widespread use of serum markers during cancer screenings has led to the belief that there may be tumor markers yet to be discovered that offer better specificity and sensitivity than prostate-specific antigen (PSA). Proteomics, the analysis and characterization of global protein modifications, will add to our understanding of gene function and aid in biomarker and/or therapeutic target discovery. In the past, most proteomic studies were either performed using tumor cell lines or homogenized bulk tissue. Unfortunately, these approaches may not accurately reflect molecular events that take place in the actual ductal epithelium that change as a consequence of the malignant process. This report describes alternative proteomic-based approaches aimed at the identification of protein markers in the actual premalignant and frankly malignant epithelium.

This unit presents a collection of protocols for the microisolation, manipulation, and amplification of the RNA content of microdissected cells. Even though emphasis in these protocols is given for microdissected cells, these protocols have successfully been used for bulk tissue (i.e., less than 10 microg).

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3'-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1(Ser410) and p-PDK1(Thr513) are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1(Ser241)), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.

There has been increasing interest in developing cancer therapies targeting PI3K pathway nodes. However, inhibition of a single node in PI3K pathway such as mTOR by rapamycin analogues results in compensatory activation of survival signaling pathway such as AKT and thereby limiting monotherapy activity. Therefore, in most tumor types, multipathway inhibition, guided by an in-depth understanding of feedback and crosstalk between pathways, may be required for tumor regression. It is well known that that mTOR inhibition upregulates pAKT via the S6K-IRS2 negative feedback loop. Inhibiting both mTOR and IGF1R may therefore ablate such feedback upregulation and lead to clinical response. However, it is unclear which tumor types will respond to the combination in the clinic. Here, we present preclinical and molecular profiling data that supports LumB breast cancer as a potential indication for MK-8669/MK-0646 (mTOR/IGF1 R inhibitor combination). When a panel of over 60 breast cancer cell lines was treated with MK-8669, ER+ cell lines and HER2+ cell lines were clearly more responsive than the triple negative ones. Meanwhile, the status of the MAPK pathway, as captured by a RAS gene expression signature, correlated with resistance to MK-8669 across the panel. Moreover, MK-8669 treatment unregulated multiple key nodes of the IGF1R pathway in the breast cell line panel, especially IRS2, whose upregulation correlated with the response to MK-8669. Furthermore, IGF1R, whose mRNA level correlates with the response to MK-0646 (IGF1 Ri) in multiple internal and external studies, is the highest in a subset of LumB tumors, suggesting a potential dependency on the IGF1R pathway in those tumors. Finally, a comparison between the gene expression profiles of breast cancer cell lines and tumors revealed that the ER+ breast cancer cell lines only represented the LumB, not LumA, subtype of human breast tumors. Taken together, these findings allow us to hypothesize that LumB breast cancer may be enriched in responders to the MK-0646/MK-8669 combination. In 2009, Merck initiated a phase I trial of MK-8669/MK-0646 in which clinical response was observed in LumB breast cancer patients. A phase II clinical trial has been initiated to test the hypothesis retrospectively. This talk is also presented as Poster A52. Citation Information: Clin Cancer Res 2010;16(14 Suppl):PR3.

The human genome encodes some 500 protein kinases and an estimated twenty percent of drugs currently in development target this enzyme class. Because kinase inhibitors frequently bind to the conserved ATP-binding pocket, in vitro kinase selectivity profiling of drug candidates are an integral part of the small-molecule drug discovery process. Likewise, the co-development of pharmacodynamic (PD) biomarkers is critical to drug development as they link drug-target inhibition in cells with biological activity. As such PD biomarkers are essential for the interpretation of in vivo PK/PD/efficacy studies, which ultimately inform medicinal chemistry. Here, we present a universal mass-spectrometry-based approached to discover kinase drug-target engagement biomarkers using the phosphoinositide dependent protein kinase 1 (PDK1) as an example. Western blot analysis using a commercial available phosphosite-specific PDK1 antibody (Ser241) revealed that classical, ATP-competitive PDK1 inhibitors do not dynamically impact PDK1 T-loop phosphorylation in cells, despite cell biochemical inhibition of phospho-AKT(Thr308), a bona fide PDK1 substrate phosphorylation site. However, because receptor tyrosine kinases (RTKs), lipid kinases and other pathways often signal via AKT, the monitoring of phospho-AKT as a PD biomarker for PDK1 is not sufficient for delineating the specificity of small-molecule inhibitors and ascertain the degree of PDK1 target engagement in cells. Here, we used stable isotope labeling by amino acids (SILAC) in cell culture combined with immunoprecipitation of PDK1 protein from cells for quantitative mass-spectrometry-based identification of phosphorylation sites modulated by PDK1 inhibitor treatment. We mapped 10 Serine/Threonine phosphorylation sites on PDK1 including several novel phosphorylation sites. Of these, two PDK1 phosphorylation sites (Ser410 and Thr513) were modulated by pharmacological PDK1 inhibition and the degree of dephosphorylation correlated with inhibitor potency in PDK1 enzymatic assays. By contrast, pharmacological inhibition of PDK1 did not quantitatively modulate PDK1 T-loop phosphorylation (Ser241) consistent with the initial western blot analysis. Altogether, our study provides proof-of-concept for identifying novel kinase target engagement PD biomarkers for PDK1, complementing pathway biomarkers such p-AKT and p-S6RP, which are modulated broadly by PI3K-pathway targeting agents including inhibitors of PI3K/mTOR and RTKs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4953.

In order to facilitate the development of HGF/ c-MET inhibitors, we undertook a genome-wide analysis to identify a “c-MET activation signature” that could be used to identify tumors that are potentially dependent on HGF / c-MET signaling and therefore more responsive to HGF/ c-MET inhibition. We first developed a c-MET co-expression signature by identifying genes that correlate with the mRNA expression of c-MET across internal panels of kidney, lung, and colorectal tumors. Many genes correlated with the expression of c-MET, including the epidermal growth factor receptor (EGFR). In order to assess the biology of genes co-expressed with cMET, we performed pathway enrichment analysis to identify pathways or pre-existing signatures that were enriched in this cMET co-expression signature more than we would expect by chance. Our previously reported RAS signature (Loboda et al, 2009 AACR annual meeting) was the top pathway enrichment, indicating a link between genes involved in RAS signaling and cMET expression across tumors. To further assess this relationship, we tested the correlation between c-MET and our RAS signature across independent cohorts of breast and lung tumors, including primary and metastatic samples. A strong correlation between c-MET expression and our RAS signature was present in all cases and was conserved in metastatic tumors. We then assessed the relationship between c-MET and RAS activation using reverse-phase protein arrays measuring approximately 70 proteins across a panel of 89 lung cancer cell lines. Four proteins were identified that significantly correlated positively with the RAS signature: total ERBB4, pMEK, pERK, and pMET. The finding that pMET correlated with RAS signature in addition to pMEK and pERK supports the connection between c-MET signaling and RAS activation, as this relationship is observed on the phospho-protein as well as mRNA levels. Taken together, these data suggest that cMET can serve as a driver of RAS signaling. To test this, we performed a xenograft study in which GTL-16 xenografts (MET amplified gastric cancer cell line) were treated with a novel small molecule inhibitor of c-MET (MK-8033). Animals were treated with vehicle or with a single dose of 10, 33, 66, 100, or 200 mpk MK-8033. At each dose, we collected samples for profiling at 2, 8, and 12 hours post-dose. MK-8033 treatment caused a dose-and time-dependent inhibition of the RAS signature at all three time points, and the level of RAS signature inhibition was related to efficacy. These data support the hypothesis that cMET is a driver of RAS signaling in some tumor contexts and suggest that basal levels of RAS pathway activation may be an important determinant of response to HGF / c-MET inhibition in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 107.

Protein phosphorylation plays a key role in cell signaling and pathophysiological alterations in protein kinases and phosphatases contribute to human diseases. We used stable isotope labeling by amino acids (SILAC) in cell culture combined with phospho-antibody based enrichment for quantitative mass-spectrometry-based identification of differentially phosphorylated proteins in response to small-molecule PI3K-pathway inhibition. We quantified over 500 non-redundant serine/threonine phosphopeptides (NSTPs) containing either the AKT-, MAPK-substrate, or PDK1-docking motif. Of these NSTPs, 71 phosphoproteins were modulated by inhibitors targeting either AKT, PDK1 or PI3K/mTOR and a common set of 11 were modulated by all three drugs. Bioinformatics analysis of the regulated phosphoproteins identified core components of the canonical PI3K pathway and showed enrichment in adaptor and scaffolding molecules involved in cell polarity (PAK4), cytoskeletal reorganization (Filamin, Stathmin, Dynamin) vesicle transport (LARP1, VPS13D, SLC20A1), protein translation (S6RP and PRAS40), and transcription (EIF4BP-1). These results have guided the rational selection of antibodies for biomarker and patient stratification assays and a newly developed phosphor-specific antibody against PRAS40T246 shows specificity for oncogenic PI3K-pathway activation in PTEN-deficient mouse prostate tumor tissue by immunohistochemistry. In addition, the phospho-PRAS40T246 biomarker was evaluated across a panel of 67 breast and 96 lung cancer cell lines and in triple negative human breast tumor tissue. In these datasets, phopspho-PRAS40T246 positively correlates with phospho-AKTS473, but not PTEN protein expression. As such, we have positioned PRAS40T246 as a clinically relevant biomarker for the identification of PI3K-pathway activated tumors to enable individualized cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5560.

Among the many types of molecular mechanisms that are currently understood, none is more important for the control of cellular faith than the network of protein kinases and phosphatases. The importance of understanding these signaling networks is furthermore highlighted by the fact that virtually all major pharmaceutical companies have significant projects focused on targeted interference against oncogenic kinases as potential anti-tumor therapies. Yet, a system's wide analysis of signaling phosphorylations by these inhibitors in in vivo (i.e. tissue or xenograft models) is cost prohibitive and rarely performed. The use of mass spectrometry is a powerful approach to study complex mixtures, and some techniques have used either chemical or metabolical isotopic labeling strategies to quantify protein differences between cohorts. While these techniques have been shown to be useful and are routinely applied for cell culture systems, they do not extend readily to in vivo studies as these require either combining of samples or are dependent on the duty cycle of the mass spectrometer. Here we report a generally applicable proteomic approach to identify global protein phosphorylation changes in in-vivo systems. This label-free discovery platform, named phospho-differential mass spectrometry (phospho-dMS) does not require complex mixing or pooling strategies or isotope labeling, and instead identifies statistically significant changes in full scan mass spectrometry data. Hence, in-vivo samples can be analyzed individually, allowing the use of longitudinal designs and within-subject data analysis. We demonstrate phospho-dMS on 27 breast tumor tissues samples (3 concentrations, 3 time points, 3 mice each) excised from a PTEN-LOF mouse model. We quantified in excess of 2000 high confidence phosphorylation events. The most prominent protein modules inhibited by compound treatment (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4561.

Epidermal growth factor receptor (EGFR) is expressed in a majority of non-small cell lung carcinomas (NSCLC) and it has been an attractive target for the development of therapeutic agents. The small-molecule EGFR tyrosine kinase inhibitors (TKI), including gefitinib and erlotinib, are currently used in the treatment of late stage NSCLC. Activating mutations in the oncogene KRAS has been associated with resistance to EGFR targeted therapy. Cross-talk between EGFR and insulin like growth factor receptor (IGF1R) has been reported. MK-0646 (dalotuzumab), a monoclonal antibody targeting IGF1R is currently being developed for the treatment of various cancers. Pre-clinical studies suggest that response to EGFR inhibitors can be substantially increased by combining with an IGF1R inhibitor such as MK-0646, a hypothesis currently being tested in clinical studies. In this study, sensitivity to MK-0646 or erlotinib or their combination was compared in a panel of NSCLC cell lines harboring wild type or activating mutations in KRAS. Erlotinib sensitivity was observed in 3/26 (12%) cell lines, all of which harbored wild type KRAS. In contrast, MK-0646 sensitivity was observed in 4/26 (15%), two of which harbor activating mutations in RAS. Sensitivity to erlotinib/MK-0646 combination was observed in 11/26 (42%) cell lines. Activating mutations in KRAS did not confer resistance to MK-0646 treatment. To identify predictive biomarkers that would enrich responders to MK-0646 therapy, gene expression profiling and reverse phase protein array were performed in this panel of NSCLC cell lines. Analysis of reverse-phase protein arrays identified IGF1R levels as a predictive marker of response to MK-0646. In contrast, ERBB3 levels and a gene signature of epithelial mesenchymal transition (EMT) were predictive of sensitivity to erlotinib, but not response to MK-0646. Sensitivity to MK-0646 was observed in primary human tumors propagated in mice or cell line xenografts harboring activating mutations in KRAS. The activity of MK-0646 in KRAS mutant NSCLC models is associated with inhibition of the PI3K pathway and subsequent down-regulation of the “Growth factor associated gene-expression signature” (GFS). The parallel RAS-MAPK pathway was not significantly modulated by MK-0646 treatment. The results suggest that patients with high levels of IGF1R expression, independent of KRAS status, will benefit from MK-0646 therapy. Growing number of studies suggest that EGFR inhibitors is not efficacious in patients harboring tumors with activating mutations in KRAS. MK-0646 therapy may overcome this resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-303.

We have tested PI3K and MAPK targeting compounds in a panel of 69 breast cancer cell lines. Following the molecular stratification of breast cancer tumors, we have used ER and HER2 biomarkers to stratify cell lines into HER2 positive (HER2+), ER positive (ER+), and triple negative (TN; ER-, PR- and HER2-) subgroups. Both gene expression and response to compounds showed significant differences among the subtypes. Response to mTOR and AKT inhibition was the greatest in ER+ and HER2+ groups (86%, 85% for mTORi and 64%, 77% for AKTi response rates, respectively), whereas TN cell lines showed preferential sensitivity to MEK inhibition (30%). The combination of AKT plus MEK and mTOR plus MEK inhibitors showed synergy in TN tumors (56% and 80% response rates, respectively), particularly in the epithelial subgroup. We believe that RAS pathway deregulation, as reported by a gene expression signature of RAS activation, is the key latent variable that explains the difference in responses. This conclusion is based on pathway activation differences between subtypes. We observe that the TN group has both elevated PI3K and MAPK signaling, evident by high levels of RAS, pERK, and pMEK signatures, whereas the ER+/GGI+ group only has high levels of PI3K signaling. Thus, mTOR, AKT, or MEK alone is insufficient to inhibit the TN lines, whereas the combos are very effective. Finally, we have assessed the prevalence of the EMT and RAS signatures across subtypes of breast tumors. Similar to cell lines, the RAS signature is markedly higher in TN compared to ER+/GGI+ and, to a lesser extent, compared to HER2+ tumors. The EMT signature on the other hand is not significantly different among subtypes. This would suggest that the response patterns observed in ER+/GGI+ and TN/E subtypes of this breast panel will be applicable to a substantial subpopulation of breast tumors (∼38%). Importantly, these breast subtypes represent the largest unmet need and are a high priority for the clinical oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1986.

There has been increasing interest in developing cancer therapies targeting PI3K pathway nodes. Unfortunately, the number of tumor types responding to PI3K pathway inhibitors as monotherapy has been limited, since most cancer cells accumulate genetic alterations that result in the constitutive activation of a complex network of proliferation and survival signals. As such, the inhibition of a single survival/proliferation pathway or node within this broader network may be circumvented by constitutive activation (or feedback activation) of an alternative survival pathway, and thus be insufficient for inducing a clinical response. Therefore, in most tumor types, dual pathway inhibition, guided by an in-depth understanding of feedback and cross-talk between pathways, may be required to induce tumor regression. Here we present a nonclinical study exploring mutual feedback activation of MAPK and PI3K pathways. Inhibition of the PI3K pathway by MK-2206 (AKT inhibitor) or MK-8669 (mTOR inhibitor) resulted in feedback activation of MAPK pathway nodes in subsets of lung cell lines. Similarly, inhibition of the MAPK pathway by a MEK inhibitor resulted in feedback activation of the PI3K pathway. Notably, a lack of such feedback activation was associated with sensitivity to the mono inhibitors. The strength of the feedback activation correlated with changes of several receptor tyrosine kinases, suggesting them as a part of the feedback mechanism. Finally, combined inhibition of both MAPK and PI3K pathways synergistically inhibited the feedback activation of both pathways and led to improved in vitro efficacy. Taken together, our findings identify mutual feedback activation of the PI3K and MAPK pathways in response to specific inhibitors and underscore the importance of combined therapeutic approaches with inhibitors to both pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4151.

The hydroxamic acid vorinostat (SAHA) is an HDAC inhibitor that induces differentiation, growth arrest and/or apoptosis of malignant cells both in vitro and in vivo, and is currently being tested in the clinic for a variety of indications. In particular, vorinostat has demonstrated an overall response rate of approximately 30% in advanced cutaneous T-cell lymphoma (CTCL). The purpose of this study was to identify biomarkers that predict response to vorinostat in CTCL using preclinical model systems based upon gene expression profiling and pathogenesis of lymphoma. Herein we report the results of our evaluation of the STAT (signal transducer and activator of transcription) signaling pathway. The relative level of mRNA and protein expression as well as activation status of several members of this family have been evaluated in lymphoid cell lines with diverse vorinostat sensitivity (proliferation/viability IC50 range: 0.3–14 mM). Among them, the more responsive CTCL cells lines HH and HUT102 exhibited an IC50 ≃ 0.8 mM. These cells underwent apoptosis in response to vorinostat as assessed by TUNEL assay, in contrast to Hut78 and MJ cells (IC50 > 2 mM) that did not show signs of DNA fragmentation upon incubation in vorinostat-containing media for 48 h. Our results indicate that in lymphoma cell lines elevated protein levels and persistent activation of STAT1, 3 and 5 correlate with resistance to vorinostat. Immunofluorescence microscopy studies revealed that STAT proteins preferentially localize to the nuclear compartment in cells with impaired vorinostat response, consistent with the expected distribution for this group of transcription factors in the active state. Simultaneous treatment with a pan-JAK inhibitor (JAK inhibitor I; Calbiochem) resulted in an additive antiproliferative effect consistent with a survival, antiapoptotic role for STAT proteins in the response to vorinostat treatment. Immunohistochemical analysis of STAT1 in skin biopsies isolated from CTCL patients (N = 49) enrolled in the vorinostat Phase IIb clinical trial showed that malignant T cells were positively stained in approximately half of the samples (21 positively stained; 25 negatively stained; 3 poor quality specimens), and in those cases a relationship between nuclear accumulation of STAT1 and lack of response to treatment exists (p=0.007

The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence.

SummaryBackground MET expression and activation appear to be important for initiation and progression of triple-negative breast cancer. Tivantinib (ARQ 197) is an orally administered agent that targets MET, although recent preclinical data suggests the agent may have mechanisms of action that are independent of MET signaling. We conducted a phase 2 study of tivantinib monotherapy in patients with metastatic triple-negative breast cancer. Methods Patients with metastatic triple-negative breast cancer who had received 1 to 3 prior lines of chemotherapy in the metastatic setting were enrolled into this two-stage, single arm phase 2 study. Treatment consisted of twice daily oral dosing of tivantinib (360 mg po bid) during a 21-day cycle. Patients underwent restaging scans at 6 weeks, and then every 9 weeks. Tumor biomarkers that might predict response to tivantinib were explored. Results 22 patients were enrolled. The overall response rate was 5 % (95 % CI 0–25 %) and the 6-month progression-free survival (PFS) was 5 % (95 % CI 0–25 %), with one patient achieving a partial response (PR). Toxicity was minimal with only 5 grade ≥3 adverse events (one grade 3 anemia, one grade 3 fatigue, and 3 patients with grade 3/4 neutropenia). Conclusion This study represents the first evaluation of tivantinib for the treatment of metastatic triple-negative breast cancer. These results suggest that single agent tivantinib is well tolerated, but did not meet prespecified statistical targets for efficacy.