Tumor Mutation Profiles Research Articles

Cervical small cell neuroendocrine tumor mutation profiles via whole exome sequencing.

Cervical small cell neuroendocrine tumors (CSCNETs) are rare, aggressive neuroendocrine tumors (NETs). Reliable diagnostic and prognostic CSCNET markers are lacking, making diagnosis and prognosis prediction difficult, and treatment strategies limited. Here we provide mutation profiles for five tumor-normal paired CSCNETs using whole exome sequencing (WES). We expanded our assessment of frequently mutated genes to include publicly available data from 55 small intestine neuroendocrine tumors, 10 pancreatic neuroendocrine tumors, 42 small cell lung cancers, six NET cell lines, and 188 cervical cancers, along with our five CSCNETs. We identified 1,968 somatic mutations, including 1,710 missense, 106 nonsense, 144 splice site, 4 lncRNA, 3 nonstop, and 1 start codon mutation. We assigned functions to the 114 most frequently mutated genes based on gene ontology. ATRX, ERBB4, and genes in the Akt/mTOR pathway were most frequently mutated. Positive cytoplasmic ERBB4 immunohistochemical staining was detected in all CSCNET tumors tested, but not in adjacent normal tissues. To our knowledge, this study is the first to utilize WES in matched CSCNET and normal tissues to identify somatic mutations. Further studies will improve our understanding of how ATRX and ERBB4 mutations and AKT/mTOR signaling promote CSCNET tumorigenesis, and may be leveraged in novel anti-cancer treatment strategies.

Comparison of Clinically Relevant Mutation Profiles Between Preoperative Biopsy and Corresponding Surgically Resected Specimens in Japanese Patients With Non–Small-cell Lung Cancer by Amplicon-based Massively Parallel Sequencing

BackgroundAmplicon-based massively parallel sequencing (MPS) is an effective platform for identifying clinically actionable mutations across many genes in limited amounts of tissue. Most lung cancers are diagnosed and staged using small tissue samples obtained by transbronchial biopsy (TBB). To determine whether the mutations in TBB specimens detected by amplicon-based MPS reflect those present in the tumors, we compared the mutational profiles of preoperative TBB specimens and corresponding surgically resected specimens. Patients and MethodsFresh-frozen primary tumor specimens from non–small-cell lung cancer patients (n = 46) obtained preoperatively by TBB and during surgical resection were analyzed. The concordance of mutations detected by amplicon-based MPS in the 2 sample types was investigated, and the allele frequency of the mutations common to both specimens from the same patient was determined. ResultsAn initial assessment of DNA quantity revealed that 46% of the TBB specimens (21 of 46) had less than the lower limit for amplicon-based MPS. These 21 TBB specimens were consequently omitted from the analysis. Of the 29 mutations detected in the TBB and/or surgically resected specimens from 25 patients, 23 were present in both samples, for a concordance rate of 79%. ConclusionAmplicon-based MPS with TBB specimens approximately reflects clinically relevant tumor mutation profiles. However, the rate of TBB specimens with sufficient DNA quantity for amplicon-based MPS was only around 50%. Therefore, surgically resected specimens have a valuable role in exploratory and comprehensive genomic profiling.

PD-L1 expression in colorectal cancer is associated with microsatellite instability, BRAF mutation, medullary morphology and cytotoxic tumor-infiltrating lymphocytes

AbstractProgrammed cell death 1 (PD-1) and its ligand (PD-L1) are key suppressors of the cytotoxic immune response. PD-L1 expression on tumor cells may be induced by the immune microenvironment, resulting in immune escape (adaptive immune resistance), and an adverse prognosis in many malignancies. In colorectal carcinoma the response to PD-1/PD-L1 inhibition is correlated with microsatellite instability. However, little is known about the clinicopathologic, molecular, and prognostic characteristics of colorectal carcinoma with PD-L1 expression. We performed immunohistochemistry for PD-L1 on 181 cases of colorectal carcinoma with known microsatellite instability and mutational status, and correlated PD-L1 expression with clinicopathologic features including tumor-infiltrating lymphocyte burden/immunophenotype, tumor mutational profile, and disease-specific survival. PD-L1 was expressed in tumors from 16 patients (9%) who were more often older (P=0.006) and female (P=0.035), with tumors exhibiting a larger size (P=0.013), but lower stage (P<0.001). PD-L1 expression was associated with increased CD8 and TBET-positive tumor-infiltrating lymphocytes, medullary phenotype, poor differentiation, microsatellite instability, BRAF mutation (P<0.001 for each), and a lower frequency of KRAS mutation (P=0.012). On multivariate analysis, PD-L1 expression was associated with medullary morphology and frequent CD8-positive tumor-infiltrating lymphocytes, suggesting adaptive immune resistance. PD-L1 positivity was not predictive of survival in the entire cohort, but it was associated with a lower disease-specific survival within the microsatellite-instability high cohort. PD-L1 expression in colorectal carcinoma is associated with clinicopathologic and molecular features of the serrated pathway of colorectal carcinogenesis, and is associated with a worse outcome within microsatellite-unstable tumors. These findings support the role of PD-L1 expression in providing normally immunogenic colorectal carcinoma a means of immune evasion and a more aggressive biology, provide a potential mechanistic explanation for the favorable response of microsatellite-unstable colorectal carcinoma to PD-1/PD-L1 pathway blockade, and suggest potential predictive and prognostic roles of PD-L1 immunohistochemistry in colorectal carcinoma.

Abstract 5291: OncoMD: A powerful genomics data analysis and interpretation platform for cancer discovery research

Abstract Cancer cells gain fitness advantage over normal cells by accumulating mutations over time. Next Generation DNA sequencing technologies have made the detection of genetic alterations in tumor genomes affordable. Powerful data analysis and interpretation tools enable extraction of disease-relevant information from diverse genomics data to guide diagnosis and treatment decisions. We have built a robust cancer analytics platform OncoMD that combines tumor mutation profiles, expression signatures, copy number variations, epigenetic alterations and drug sensitivity to create a holistic view of human cancer enabling discovery of new targets for therapy and prognosis. We have analyzed the rich mutational data captured in OncoMD to identify potential T-cell neo-epitopes in different cancers and examined their prevalence in the context of the tumor microenvironment. The presence of T-cell neo-epitopes alone is not sufficient to sensitize tumors to immunotherapy drugs and requires cooperation from the tumor microenvironment. Although mutational burden was a strong predictor of T-cell neo-epitopes in our analysis, other genetic alterations such as overexpression, splicing, gene-fusions and insertions/deletions can produce altered peptides exhibiting high affinity binding to HLA class I and II molecules in cancers with low burden of missense mutations. We determined the number of potential T-cell neo-epitopes after classifying tumors based on their epithelial, stromal and immune cell content using gene expression signatures. We observed that the normalized T-cell neo-epitope burden was strongly anti-correlated with the T-cell content of the tumors, compared to their epithelial or stromal content. Together, our findings suggest that tumors enriched in T-cells may be subjected to immunoediting processes and carry relatively fewer T-cell neo-epitopes to avoid elimination. These tumors are likely to respond to immunotherapy treatments. Citation Format: Ravi Gupta, Kiran V. Paul, Kartik Kumaramangalam, Sam Santhosh, Amitabha Chaudhuri. OncoMD: A powerful genomics data analysis and interpretation platform for cancer discovery research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5291.

Abstract 2753: Landscape of somatic mutations in DNA repair genes in prostate cancer

Abstract Prostate Cancer (PCa) is a genetic disease that is characterized by multiple genomic alterations. Studies have shown that there is increasing associations between DNA repair genes and PCa. Previously we have reported somatic mutations in Polb and Polk enzymes that are critical for base excision repair and translesion DNA synthesis respectively. In the present study we identified frequently mutated genes that are involved in DNA repair pathway by sequencing all exons of DNA repair pathway genes (129). A total of 57 tumors with matched non tumor tissue was used. Tumors include 24 Caucasians, 21 African Americans (AA). PCa tumors that were ≥6 gleason score and ≥50% of viable tumor cells were only included. All the PCa were procured from Tulane University (TU) Hospital, New Orleans and ethical approvals were obtained from the TU Local Research Ethical Committee. Genomic DNA from tumors was using agilent SureSelect kit was customized to enrich the target that contained all exons of 129 genes of DNA repair pathways. Samples were sent for sequencing by Illumina HiSeq 2000. We validated experimentally somatic mutations in genes that were either affected by somatic non-synonymous or synonymous mutations with a frequency significantly greater than background rates (P&amp;lt;0.05). We considered a somatic mutation to be validated if the targeted sequencing confirmed the presence of the mutation in tumor and it was not found in the matched normal sample. Overall we identified 875 somatic mutations in AA, in which 414 were non-synonymous, 427 synonymous, 14 were stop-gain and 1 was frameshift mutation. AA demonstrated 52 somatic mutations/tumor and Caucasians exhibited 46 somatic mutations/tumor. In terms of Nucleotide excision repair (NER), Mismatch repair (MMR) and Base excision repair (BER) we identified variation in NER, MMR and BER pathways in AA [highest mutation in NER (mean-66) followed by MMR (mean-47) and then BER pathways (mean-37)] whereas our report shows that MMR, BER and NER equally mutated in Caucasians. Furthermore analysis of distribution of mutations between low grade (6) and high-grade Gleason scores (8 or &amp;gt;8) in AA and Caucasians revealed that 12 mutations per tumor for Gleason score 6, versus 22.3 mutations for Gleason 8, 9 or 10. Due to the limitation of samples with gleason score 6 in both races we could not draw a trend individually. Analysis of mutation spectra across the DNA repair genes revealed that C&amp;gt; T transitions (27%) markedly higher in AA PCa followed by T &amp;gt; C transitions (24%). Interestingly in Caucasians PCa C &amp;gt; T transitions (26.8%) were higher including marked increase of C &amp;gt; A (17%) and T &amp;gt; G (17%) transversions. Understanding the mutational changes at the individual patient level could enable personalized treatment and patient selection based on tumor mutational profile for clinical drug testing might be critical for development of new treatments strategies. Support PC094628, and NIH grant 8 P20 GM103518 Citation Format: Santosh Yadav, Muralidharan Anbalagan, Melody Baddoo, Erik Flemington, Krzysztof Moroz, Kathleen Hering-Smith, Nick Makridakis. Landscape of somatic mutations in DNA repair genes in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2753.

LINE-1 hypomethylation and mutational status in cutaneous melanomas

Epigenetic dysregulation is an important emerging hallmark of cutaneous melanoma development. The global loss of DNA methylation in gene-poor regions and transposable DNA elements of cancer cells contributes to increased...

Liquid biopsies for liquid tumors: emerging potential of circulating free nucleic acid evaluation for the management of hematologic malignancies.

Circulating free nucleic acids; cell free DNA and circulating micro-RNA, are found in the plasma of patients with hematologic and solid malignancies at levels higher than that of healthy individuals. In patients with hematologic malignancy cell free DNA reflects the underlying tumor mutational profile, whilst micro-RNAs reflect genetic interference mechanisms within a tumor and potentially the surrounding microenvironment and immune effector cells. These circulating nucleic acids offer a potentially simple, non-invasive, repeatable analysis that can aid in diagnosis, prognosis and therapeutic decisions in cancer treatment.

Prognosis and Clinicopathologic Features of Patients With Advanced Stage Isocitrate Dehydrogenase (IDH) Mutant and IDH Wild-Type Intrahepatic Cholangiocarcinoma.

Conflicting data exist regarding the prognostic impact of the isocitrate dehydrogenase (IDH) mutation in intrahepatic cholangiocarcinoma (ICC), and limited data exist in patients with advanced-stage disease. Similarly, the clinical phenotype of patients with advanced IDH mutant (IDHm) ICC has not been characterized. In this study, we report the correlation of IDH mutation status with prognosis and clinicopathologic features in patients with advanced ICC. Patients with histologically confirmed advanced ICC who underwent tumor mutational profiling as a routine part of their care between 2009 and 2014 were evaluated. Clinical and pathological data were collected by retrospective chart review for patients with IDHm versus IDH wild-type (IDHwt) ICC. Pretreatment tumor volume was calculated on computed tomography or magnetic resonance imaging. Of the 104 patients with ICC who were evaluated, 30 (28.8%) had an IDH mutation (25.0% IDH1, 3.8% IDH2). The median overall survival did not differ significantly between IDHm and IDHwt patients (15.0 vs. 20.1 months, respectively; p = .17). The pretreatment serum carbohydrate antigen 19-9 (CA19-9) level in IDHm and IDHwt patients was 34.5 and 118.0 U/mL, respectively (p = .04). Age at diagnosis, sex, histologic grade, and pattern of metastasis did not differ significantly by IDH mutation status. The IDH mutation was not associated with prognosis in patients with advanced ICC. The clinical phenotypes of advanced IDHm and IDHwt ICC were similar, but patients with IDHm ICC had a lower median serum CA19-9 level at presentation. Previous studies assessing the prognostic impact of the isocitrate dehydrogenase (IDH) gene mutation in intrahepatic cholangiocarcinoma (ICC) mainly focused on patients with early-stage disease who have undergone resection. These studies offer conflicting results. The target population for clinical trials of IDH inhibitors is patients with unresectable or metastatic disease, and the current study is the first to focus on the prognosis and clinical phenotype of this population and reports on the largest cohort of patients with advanced IDH mutant ICC to date. The finding that the IDH mutation lacks prognostic significance in advanced ICC is preliminary and needs to be confirmed prospectively in a larger study.

Abstract 929: Tumor genotyping in the phase III GRID study of regorafenib vs placebo in tyrosine kinase inhibitor (TKI)-refractory GIST: Detection of KIT mutations in circulating tumor DNA comparing digital PCR and massive parallel sequencing

Abstract Background: The GRID study demonstrated that regorafenib provides a significant improvement in progression-free survival (HR 0.27; p&amp;lt;0.0001) compared with placebo in patients with advanced gastrointestinal stromal tumors (GIST) following failure of at least imatinib and sunitinib. Mutations in KIT play an important role in GIST and identification of the specific KIT mutations that each GIST patient harbors could have important therapeutic implications. However, determining tumor genotype in TKI-refractory GIST has proven challenging due to intra and inter-tumoral genetic heterogeneity and the difficulty associated with obtaining fresh tumor biopsies. To overcome this limitation, tumor genotyping in GRID was performed using baseline plasma as a source of circulating tumor-derived DNA. We previously performed genotyping using BEAMing (a mutation-specific technology) and have now extended this analysis using Safe-SeqS (Kinde et al. 2011 PNAS 108:9530) a targeted NGS-based platform aiming to detect previously undetectable cKIT mutations. Methods: 91 plasma samples from patients enrolled in the Ph 3 study (GRID) for which BEAMing data (Jeffers et al 2013 JCO 31:10503) were subjected to Safe-SeqS covering cKit Exon 8 to Exon 18. Results: In 6 of 32 samples reported to be cKIT wildtype by BEAMing, mutations were identified by SafeSeqS. The detection of primary KIT exon 9 mutations showed a high degree of concordance among the two mutation-detection methods evaluated. Secondary / resistance hotspot mutations were also readily detected by both methods, although a greater number of such mutations were detected by Safe-SeqS than by BEAMing. The localization of the additional mutations detected by Safe-SeqS in known mutational hotspots supports their legitimacy. Safe-SeqS also detected KIT mutations for which BEAMing assays had not been developed, whereas in 17 samples a mutation for which a BEAMing assay was available was not detectable by Safe SeqS. In 58% (10/17) of samples, the mutant allele frequency found by BEAMing was close to the detection limit of this platform (&amp;lt;0.05). The cKITM541L mutation in Exon10 was found in 25% (23/91) of the samples. Conclusion: Our data support the use of Safe-SeqS as a sensitive and specific “liquid biopsy” method for non-invasive tumor genotyping of patients with GIST, enabling the identification of known and novel tumor-associated mutations using circulating DNA. These results confirm and extend the genotypic heterogeneity that had previously been identified in GRID circulating DNA samples by BEAMing. The comprehensive tumor mutational profiles generated by Safe-SeqS will be used to evaluate potential correlations between tumor genotype and clinical outcome. Citation Format: Michael Jeffers, Henrik Seidel, Susanne Schwenke, Joachim Reischl, Mark Rutstein, Christian Kappeler, Iris Kuss, Michael Teufel. Tumor genotyping in the phase III GRID study of regorafenib vs placebo in tyrosine kinase inhibitor (TKI)-refractory GIST: Detection of KIT mutations in circulating tumor DNA comparing digital PCR and massive parallel sequencing. [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 929. doi:10.1158/1538-7445.AM2015-929

Abstract 222: PathLink: Leveraging clinical informatics to empower translational pathology by connecting biospecimens to outcomes

Abstract Biorepositories are valuable resources to the cancer research community and arguably invaluable if they include detailed longitudinal health information such as personal risk factors, disease progression, and treatment outcomes. However, manual review of patient records is comparatively inefficient (expensive, time-intensive, and less reliable) than data extraction from the electronic health record (EHR). Using Research Data Network funds granted from the Patient Centered Outcomes Research Institute (PCORI), PathLink is being developed as an institution-wide biorepository linked to Vanderbilt's EHR data warehouses, the Synthetic and Research Derivatives, and utilizing pathological specimens that exist or would otherwise be discarded, without duplicating existing biorepositories and other core resources. We are cataloging samples from clinical and research collections and creating a sustainable and prospective link to structured health information. PathLink will de-identify samples on-demand, permitting translational investigators real-time sample access while also preserving tissue for future clinical assays. The EHR derivatives already include fundamental review features and standardized data extraction mechanisms that can be easily leveraged for tissue-based research projects. Projects proposing a de-identified research approach can also be crosslinked to our germline genetic biobank of &amp;gt;200,000 subjects and will have lower barriers to access through a streamlined regulatory mechanism. Proof-of-concept studies underway include the banking of tissues with molecular genetic pathology testing (n = 3642), a large subset already annotated with SNaPshot tumor mutation profiling. Macrodissected tumor cells were collected from cases accessioned from 2010 to 2013 and stored as a tumor lysate reserve (B-tubes) pending successful completion of clinical assays. After extraction and quality testing of genomic material, PathLink was seeded with the transfer of these remnant samples to the biorepository. Pilot studies suggest that more than 75% of B-tubes will contain sufficient genomic material of high quality for targeted gene sequencing (&amp;gt;250ng, n = 120). We will digitally scan corresponding tissue sections and create tissue microarrays from cases with adequate tissue. In conclusion, PathLink is a new concept in biobanking, minimizing time and cost, and enabling more efficient translation of tissue-based discoveries from bench to clinical care and back again. A unique feature is that much of the collection is a virtual catalog of partner biorepositories, and most of the sample processing is executed by existing core facilities, minimizing overhead while also increasing interdepartmental collaboration. By July 2015, we expect to have catalogued at least 300,000 pathological specimens and have 3,000 tumor DNA samples ready for distribution to approved translational projects. Citation Format: Jennifer M. Giltnane, Jana Shirey-Rice, Jodell Linder, Erica Bowton, James Cowan, Xiaoming Wang, Jon Scherdin, Melissa Basford, Kimberly Dahlman, Joseph Roland, Kerry Wiles, Cynthia Vnencak-Jones, Kay Washington, Jill Pulley. PathLink: Leveraging clinical informatics to empower translational pathology by connecting biospecimens to outcomes. [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 222. doi:10.1158/1538-7445.AM2015-222

How to Identify the Right Patients for the Right Treatment in Metastatic Colorectal Cancer (mCRC)

A major challenge in metastatic colorectal cancer (mCRC) is the identification of specific biomarkers that are likely to predict which patients will benefit from a specific treatment, especially in recent years, that available options have substantially increased with the introduction of novel targeted therapies. To this date, a number of studies have shown that a tumor mutational profile influences outcome in mCRC patients treated with an anti-epidermal growth factor receptor (anti-EGFR)-targeting agent and should, therefore, be used to guide treatment decision. Despite our undeniable progress toward a more personalized treatment approach, we are not yet there. Prognostic and predictive markers have been identified, but their influence in distinct patient populations has not been fully elucidated. Reality seems to be more complex; clinical performance status of the patients and existing comorbidities and our treatment goal, alongside the tumor and patient mutational profile and molecular signature, will guide our initial and subsequent treatment choices.

Abstract 1886: Highly sensitive detection of circulating tumor DNA in plasma as a biomarker of colorectal cancer

Abstract Circulating tumor DNA (ctDNA) from the blood of cancer patients is a promising biomarker of certain types of colorectal cancer, with potential use in screening, treatment selection, therapy response monitoring, minimal residual disease detection, and surveillance. We present results demonstrating that the OnTarget ctDNA detection assay can be used as an indicator of the presence of genomic variants associated with colorectal cancer in patients prior to treatment. The study includes three cohorts of individuals: young normal controls, age-matched normal controls, and colorectal cancer patients whose blood was drawn prior to treatment or surgery, and whose diagnosis confirms the presence of a colorectal tumor. Tissue from the primary tumor biopsy was also collected and analyzed with the OnTarget assay. Using Boreal Genomics' OnTarget 46-mutation panel, we find high correlation between the tumor mutational profile and the plasma mutation profile even for early stage patients demonstrating high sensitivity (88% plasma-tissue concordance to date) of the assay. By applying the same assay in the normal population we demonstrate extremely low false positive rates, highlighting the potential of the assay as a tool for therapy monitoring, surveillance, or screening. The OnTarget assay is capable of single molecule sensitivity, specificity to ≤0.01% mutation abundance versus wild-type, and multiplexing of over 100 mutations in a single test from a single plasma sample. The OnTarget technology employs a combination of Next Generation DNA Sequencing and a proprietary target selection process that removes wild-type DNA prior to amplification to greatly improve signal to noise and reduce the error rate of sequencing. We present an overview of the technology and results from an ongoing study to demonstrate the potential clinical utility of ctDNA as an effective biomarker to guide management of colorectal cancer. Citation Format: Matthew Wiggin, Jaryn Perkins, Laura Mai, Valentina Vysotskaia, Andre Marziali. Highly sensitive detection of circulating tumor DNA in plasma as a biomarker of colorectal cancer. [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 1886. doi:10.1158/1538-7445.AM2014-1886

Abstract 4808: Aflibercept is highly active in clinically relevant, patient derived xenografts of colorectal cancer

Abstract Aflibercept (ziv-aflibercept in USA), a new fusion protein, targets vascular endothelial growth factor (VEGF)-A, VEGF-B, and placental growth factor (PlGF). Aflibercept + FOLFIRI is approved for treating metastatic colorectal cancer (mCRC) that is resistant to or has progressed following an oxaliplatin containing regimen. We investigated the pharmacological activity of aflibercept in CRC patient derived xenografts (PDX), most metastases-derived, and characterized PDX molecular and phenotypic properties. In 48 CRC PDX, Whole Exome Sequencing revealed: 85% APC, 79% TP53, 51% KRAS, 3% NRAS, 25% PIK3CA, 11% PTEN and 8% BRAF mutants. There was no significant association between angiogenic factors, tumor mutation profiles, and response to aflibercept. In tumors, protein levels of human (h) VEGF-A were significantly higher than mouse (m) VEGF-A, hPlGF, and mPlGF (Chiron et al, ESMO, 2013). Affymetrix probes detected VEGF-A and PIGF but not VEGF-B mRNA in most PDX. There was no significant association between expression of angiogenic factors and response to aflibercept. Histological sections of CRC tumors from PDX, xenografts created from cell lines, and patients’ tumor specimens indicated that stromal involvement in human tumors was recapitulated in PDX but not in tumors from cell lines. PlGF expression was analyzed by immunohistochemistry on colon cancer tissue arrays. A significant number of colon cancer specimens were positive for PlGF, expressed in tumor cells and stroma. Aflibercept resulted in anti-tumor activity in 47/48 CRC PDX models and complete tumor stasis in 31/48 models. Efficacy data from 5 PDX models responsive to aflibercept and CRC standard of care (SOC) drugs, all administered as monotherapies, are below. The robust activity of aflibercept in CRC PDX models that are sensitive to SOC drugs suggest that CRC patients would benefit from aflibercept treatment, independently of mutational status or expression of angiogenic factors. In Vivo Sensitivity Profile (optimal Tumor/Control (%))CRC PDX ModelPrior TreatmentsAflibercept 25 mg/kg, SC, 2x/wk5-FU100 mg/kg, IP, 1x/wkIrinotecan 50 mg/kg, IV, 1x/wkOxaliplatin 10 mg/kg, IP, Q2WCetuximab30 mg/kg, IV,1x/wkKRAS StatusCFX 1991Not available-2.531.51.856.342.1MutantCFX 2032Not available3.147.76.760.535.7MutantCFX 2048Not available-10.326.221.350.12.6Wild-typeCFX 20655-FU, leucovorin-11.920.614.471.82.2Wild-typeCFX 2067None6.253.045.158.914.2Wild-type Citation Format: Rebecca G. Bagley, Zakia Amalou, Marielle Chiron, Vincent Vuaroqueaux, Anne Caron, Nina Baltes, Heinz H. Fiebig. Aflibercept is highly active in clinically relevant, patient derived xenografts of colorectal cancer. [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 4808. doi:10.1158/1538-7445.AM2014-4808

Abstract 2985: In vivo evaluation of regorafenib and liposomal-doxorubicin in a panel of low passage uterine START-PDX tumor models

Abstract Background: Uterine and ovary cancers account for a majority of gynecologic cancer-related deaths in Western countries. While initial cure rates are high, recurrent disease is often chemoresistant with few treatment options. Regorafenib is a recently approved multi-tyrosine kinase targeting VEGFR2 and Tie2. As Tie2 has been implicated in the pathogenesis of uterine cancers, we evaluated regorafenib as a potential therapy in our panel of uterine PDX models and compared activity to the approved therapy liposomal-doxorubicin. Methods: PDX uterine models were established in immune-deficient mice from primary or metastatic patient tissue and once established were confirmed by histologic comparative analysis with patient tumor tissue, linked with patient treatment and outcome data and characterized for activating mutations and known amplifications. Drug sensitivity studies were performed in each model evaluating daily oral regorafenib or weekly intravenous liposomal-doxorubicin versus control; group sizes were n=2-3 for control and n=1 for treatment arms. Study endpoints included a set day following treatment initiation with tumor growth inhibition or delay and regression reported at study completion. Results: For this project we evaluated twenty-five uterine models. Statistically significant (p&amp;lt;0.05) regorafenib activity was reported in a subset of evaluated endometrial models; however, activity was not limited to a particular tumor subtype or mutation profile. Liposomal-doxorubicin also demonstrated differential but significant tumor growth inhibition towards models in the evaluated panel. Conclusion: We have screened preclinical activity of regorafenib and liposomal-doxorubicin towards a panel of uterine START-PDX models and identified sensitive and resistant models. Additional genomic and characterization studies are underway to better understand activity of regorafenib with the goal of identifying a sensitivity profile for patient stratification. Citation Format: Justin Meade, Michael J. Wick, Teresa Vaught, Lizette Gamez, Roger Chavez, Anthony Tolcher, Drew Rasco, Amita Patnaik, Allan J. White, Kyriakos Papadopoulos. In vivo evaluation of regorafenib and liposomal-doxorubicin in a panel of low passage uterine START-PDX tumor models. [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 2985. doi:10.1158/1538-7445.AM2014-2985

Abstract 3573: Comprehensive non-invasive tumor sequencing: High fidelity sequencing of tumor-derived circulating cell-free DNA across 300 cancer patients

Abstract Background: Analysis of circulating tumor nucleic acids by massively parallel DNA sequencing has created an exciting new tool for capturing an accurate, more complete and real-time picture of a patient's tumor profile throughout the body and the course of disease. However, the widespread use of this biomarker has been limited by the high-quality false positives and non-systematic distortion present in current NGS assays, especially where the tumor-derived fraction is low (&amp;lt;1%). These limitations have heretofore impeded routine clinical use of circulating DNA in oncology. Methods: Our differentiated sequencing assay, Digital Sequencing Technology, has enabled robust, ultra-sensitive and ultra-specific detection of rare genomic abnormalities in cell-free circulating DNA. Digital Sequencing relies on enabling single-molecule sequencing using current next-gen sequencers. Our assay is able to eliminate most of the errors and distortion introduced by these sequencers and produce near-perfect representations of all rare variants. Results: We have shown that in sequencing 0.1% cancer cell line titrated samples across a broad 80kbp cancer panel, standard Illumina sequencing generates several high-quality false positives with allele frequencies of 0.05-5%, while our novel Digital Sequencing Technology results in high sensitivity and completely error-free detection of rare variants. Furthermore, we have applied our technology to analyze rare tumor-derived cell-free DNA from more than 300 cancer patient plasma samples collected at multiple sites (various US and international locations) and across different cancer types. We investigated the concordance of tumor mutation profiles derived from circulating cell-free DNA with those derived from matched tumor biopsy samples in colorectal, breast, NSCLC, prostate and melanoma cancers across patients (Stage II-IV). We also studied the presence of de novo mutations (SNV and CNV) in these cancer patient plasma samples. In general, the range of tumor DNA in plasma was determined to be between 0.03%-85.9% in these studies. This comprehensive dataset illustrates the broad power of cell-free DNA analysis as a diagnostic biomarker in cancer patients. Conclusion: The present work shows the strong potential clinical impact of Digital Sequencing in analysis of circulating cell-free tumor-derived nucleic acids, thereby allowing researchers and clinicians to comprehensively and non-invasively monitor the genetic dimension of cancer throughout the body. Citation Format: Stefanie A. Mortimer, Dragan Sebisanovic, Gangwu Mei, Benjamin Schiller, Lai Mun Siew, Aubrey Zapanta, Helmy Eltoukhy, AmirAli Talasaz. Comprehensive non-invasive tumor sequencing: High fidelity sequencing of tumor-derived circulating cell-free DNA across 300 cancer patients. [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 3573. doi:10.1158/1538-7445.AM2014-3573

Next generation sequencing and tumor mutation profiling: are we ready for routine use in the oncology clinic?

Next generation sequencing (NGS) coupled with sophisticated bioinformatics tools yields an unprecedented amount of information regarding tumor genetics, with the potential to reveal insights into tumor behavior. NGS and other multiplex genomic assays are rapidly spilling from the laboratory into the clinic through numerous commercial and academic entities. This raises the important question as to whether we are ready to use these data in clinical decision-making. While genetic lesions are clearly targeted by a new generation of biological cancer therapies, and certain regulatory approvals are actually coupled to single gene assays, we still do not know if the vast information on other genomic alterations is worth the added cost, or even worse, the inappropriate and unproven assignment of patients to treatment with an unapproved drug carrying potentially serious side effects. On the other hand, the trend toward a precision medicine pathway is clearly accelerating, and clinical trials validating pathway-driven personalized cancer therapeutics will be necessary in both the community and academic settings. Lower cost and wider availability of NGS now raises a debate over the merit of routine tumor genome-wide analysis.

Approaches to integrating germline and tumor genomic data in cancer research.

Cancer is characterized by a diversity of genetic and epigenetic alterations occurring in both the germline and somatic (tumor) genomes. Hundreds of germline variants associated with cancer risk have been identified, and large amounts of data identifying mutations in the tumor genome that participate in tumorigenesis have been generated. Increasingly, these two genomes are being explored jointly to better understand how cancer risk alleles contribute to carcinogenesis and whether they influence development of specific tumor types or mutation profiles. To understand how data from germline risk studies and tumor genome profiling is being integrated, we reviewed 160 articles describing research that incorporated data from both genomes, published between January 2009 and December 2012, and summarized the current state of the field. We identified three principle types of research questions being addressed using these data: (i) use of tumor data to determine the putative function of germline risk variants; (ii) identification and analysis of relationships between host genetic background and particular tumor mutations or types; and (iii) use of tumor molecular profiling data to reduce genetic heterogeneity or refine phenotypes for germline association studies. We also found descriptive studies that compared germline and tumor genomic variation in a gene or gene family, and papers describing research methods, data sources, or analytical tools. We identified a large set of tools and data resources that can be used to analyze and integrate data from both genomes. Finally, we discuss opportunities and challenges for cancer research that integrates germline and tumor genomics data.

Circulating tumor DNA as a highly specific diagnostic marker for colorectal cancer.

e22126 Background: Circulating tumor DNA (ctDNA) from cancer patient plasma is a promising biomarker with applications in screening, treatment selection, therapy response monitoring, minimal residual disease detection, and surveillance. We investigate the value of ctDNA as a biomarker in colorectal cancer to augment clinical decision-making. Methods: We present a ctDNA detection assay simultaneously capable of single molecule sensitivity, specificity to 0.01%– 0.001% mutation abundance versus wild-type, and multiplexing over 100 mutations from a single plasma sample. The employs a proprietary target selection process that removes wild-type DNA prior to library construction to greatly reduce the error rate of DNA sequencing. Using this assay, we analysed plasma from three cohorts of individuals including normal controls (n=13), age-matched normal controls (n=17), and colorectal cancer patients (n=17) across all stages of disease whose blood was drawn prior to treatment or surgery, and whose diagnosis confirms the presence of a tumor. Tissue from the primary tumor biopsy was also collected and analysed with the OnTarget assay. Results: We find a high correlation between the tumor mutational profile and the plasma mutation profile even for early stage patients demonstrating high sensitivity (92% plasma-tissue concordance over all patients) and exceptional specificity (100%: no false positives in normal cohorts). Additionally, mutations deemed from clinical evidence to originate from distant metastatic sites are detected in the plasma, highlighting the promise of the liquid biopsy approach for application in clinical care. We present concordance, sensitivity, and specificity results from the colorectal cancer patient cohort and the cohort of healthy individuals. Conclusions: Initial assay results from 30 healthy individuals and 17 CRC patients indicate that OnTarget detection of ctDNA provides an exceptionally specific assay for the presence of colorectal tumors and may have utility as a biomarker to guide colorectal cancer management across early-stage and metastatic disease.

Measurement of circulating tumor DNA as a cancer biomarker in gastrointestinal malignancies using a novel next-generation sequencing method.

217 Background: Circulating tumor DNA (ctDNA) holds promise as a highly specific cancer biomarker. The presence of mutant tumor-derived DNA fragments in the blood provides an opportunity to non-invasively assess tumor mutation profiles and to quantify changes in tumor DNA levels over time. Methods: After obtaining informed consent, plasma samples were collected prospectively at multiple time points in a cohort of patients (pts) with various gastrointestinal (GI) malignancies in the locally advanced, metastatic and adjuvant settings. Hotspot regions of genes known to be commonly mutated in GI tumors were amplified by multiplexed PCR, and the resultant amplicons were subjected to next-generation ultra-deep sequencing. Suppression of sequencer and PCR errors allowed mutations to be identified and quantified with a sensitivity of approximately 1 variant in 5,000 molecules. Sample-specific barcoding allowed simultaneous analysis of up to 96 samples. Results: 29 out of 74 available samples from 17 pts were analyzed for the presence of ctDNA. 3 pts had KRAS mutations exclusively. 2 pts had mutations in two of the evaluated genes: KRAS plus PIK3CA and KRAS plus TP53. 2 pts with measurable mutant ctDNA had samples analyzed at multiple time points. One of these patients with locally advanced pancreatic cancer initially had a KRAS G12D mutation which disappeared after treatment with chemo-radiation therapy, but then he developed significantly rising levels of a different KRAS G12L mutation just preceding the diagnosis of liver metastases by imaging. A second pt with metastatic colon cancer had high levels of ctDNA prior to treatment that decreased dramatically after initiation of mFOLFOX and bevacizumab therapy. Conclusions: Our initial analysis indicates that our technique is able to quantify multiple mutations at very low copy numbers accurately in this cohort of pts with GI cancers. Moreover, we were able to monitor changes in mutation type and quantity using this non-invasive “liquid biopsy” approach. Analysis of remaining samples is ongoing, and additional longitudinal data will be presented.

Tumor Plasticity Driven by Spatial Micro-heterogeneity in the Tumor Microenvironment Contributes to Therapy Resistance

Metastases account for over 90% of tumor related deaths. The classic anticancer inhibitors of cellular proliferation are also cytotoxic to host cells thus limiting effective doses and invariably leading to the development of drug resistance. Recent literature demonstrate some tumors harbor mutations like the EGFR-activating mutations in lung cancer or the BRAF V600E mutation in melanoma that can be targeted with specific inhibitors with promising clinical outcomes [1,2]. Even in such cases therapy is not guaranteed for all patients as tumors cease to respond after several rounds of drug treatment either because the tumor acquires resistance due to new mutations that bypass the blocked pathway or because resistant low-frequency sub clones become dominant. The fact that no treatment eliminates all tumor cells even though the re-emerging tumors are still sensitive to the treating drugs suggests that a fraction of the tumor cells are able to evade treatment through mechanisms as yet unclear. Factors contributing to therapy resistance include intra-tumor heterogeneity (genetic and phenotypic), tumor plasticity and spatial heterogeneity in the tumor microenvironment. Genetic intra-tumor heterogeneity arises from accumulating tumor mutations as tumor progresses. Most of the mutations, as in normal evolution, would be expected to be neutral or deleterious to the tumor cell. Some mutations however confer survival advantages as in the examples above [1,2], while other mutations may confer resistance to particular treatments. The EGFR T790M mutation for example confers resistance to EGFR tyrosine kinase inhibitors [3]. Establishing tumor mutation profiles enables better targeting of treatments.