AmpliSeqTM Cancer Hotspot Panel V2 Research Articles

Molecular profiling of solid tumors by next-generation sequencing: an experience from a clinical laboratory.

Personalized targeted therapies have transformed management of several solid tumors. Timely and accurate detection of clinically relevant genetic variants in tumor is central to the implementation of molecular targeted therapies. To facilitate precise molecular testing in solid tumors, targeted next-generation sequencing (NGS) assays have emerged as a valuable tool. In this study, we provide an overview of the technical validation, diagnostic yields, and spectrum of variants observed in 3,164 solid tumor samples that were tested as part of the standard clinical diagnostic assessment in an academic healthcare institution over a period of 2 years. The Ion Ampliseq™ Cancer Hotspot Panel v2 assay (ThermoFisher) that targets ~2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes was validated, and a total of 3,164 tumor DNA samples were tested in 2 years. A total of 500 tumor samples were tested by the comprehensive panel containing all the 50 genes. Other samples, including 1,375 lung cancer, 692 colon cancer, 462 melanoma, and 135 brain cancer, were tested by tumor-specific targeted subpanels including a few clinically actionable genes. Of 3,164 patient samples, 2,016 (63.7%) tested positive for at least one clinically relevant variant. Of 500 samples tested by a comprehensive panel, 290 had a clinically relevant variant with TP53, KRAS, and PIK3CA being the most frequently mutated genes. The diagnostic yields in major tumor types were as follows: breast (58.4%), colorectal (77.6%), lung (60.4%), pancreatic (84.6%), endometrial (72.4%), ovary (57.1%), and thyroid (73.9%). Tumor-specific targeted subpanels also demonstrated high diagnostic yields: lung (69%), colon (61.2%), melanoma (69.7%), and brain (20.7%). Co-occurrence of mutations in more than one gene was frequently observed. The findings of our study demonstrate the feasibility of integrating an NGS-based gene panel screen as part of a standard diagnostic protocol for solid tumor assessment. High diagnostic rates enable significant clinical impact including improved diagnosis, prognosis, and clinical management in patients with solid tumors.

Detection of genetic mutations in patients with breast cancer from Saudi Arabia using Ion AmpliSeq™ Cancer Hotspot Panel v.2.0.

Next-Generation Sequencing allows for quick and precise sequencing of multiple genes concurrently. Recently, this technology has been employed for the identification of novel gene mutations responsible for disease manifestation among breast cancer (BC) patients, the most common type of cancer amongst Arabian women, and the major cause of disease-associated death in women worldwide. Genomic DNA was extracted from the peripheral blood of 32 Saudi Arabian BC patients with histologically confirmed invasive BC stages I-III and IV, as well from 32 healthy Saudi Arabian women using a QIAamp® DNA Mini Kit. The isolated DNA was quantified using a Qubit™ dsDNA BR Assay Kit with a Qubit 2.0 Fluorometer. Ion semiconductor sequencing technology with an Ion S5 System and AmpliSeq™ Cancer Hotspot Panel v2 were utilized to analyze ~2,800 mutations described in the Catalogue of Somatic Mutations in Cancer from 50 oncogenes and tumor suppressor genes. Ion Reporter Software v.5.6 was used to evaluate the genomic alterations in all the samples after alignment to the hg19 human reference genome. The results showed that out of the 50 genes, 26 mutations, including 17 (65%) missense point mutations (single nucleotide variants), and 9 (35%) frameshift (insertion/deletion) mutations, were identified in 11 genes across the cohort in 61 samples (95%). Mutations were predominantly focused on two genes, PIK3CA and TP53, in the BC genomes of the sample set. PIK3CA mutation, c.1173A>G located in exon 9, was identified in 15 patients (46.9%). The TP53 mutations detected were a missense mutation (c.215C>G) in 26 patients (86.70%) and 1 frameshift mutation (c.215_216insG) in 1 patient (3.33%), located within exon 3 and 5, respectively. This study revealed specific mutation profiles for every BC patient, Thus, the results showed that Ion Torrent DNA Sequencing technology may be a possible diagnostic and prognostic method for developing personalized therapy based on the patient's individual BC genome.

PTEN, ATM, IDH1 mutations and MAPK pathway activation as modulators of PFS and OS in patients treated by first line EGFR TKI, an ancillary study of the French Cooperative Thoracic Intergroup (IFCT) Biomarkers France project

ObjectivesTumor mutation screening is standard of care for patients with stage IV NSCLC. Since a couple of years, widespread NGS approaches used in routine diagnostics to detect driver mutations such as EGFR, KRAS, BRAF or MET allows the identification of other alterations that could modulated the intensity or duration of response to targeted therapies. The prevalence of co-occurring alterations that could affect response or prognosis as not been largely analyzed in clinical settings and large cohorts of patients. Thanks to the IFCT program “Biomarkers France“, a collection of samples and data at a nation-wide level was available to test the impact of co-mutations on first line EGFR TKI in patients with EGFR mutated cancers. Materials and methodsTargeted NGS was assessed on available (n = 208) samples using the Ion AmpliSeq™ Cancer Hotspot Panel v2 to screen for mutations in 50 different cancer genes. ResultsThis study showed that PTEN inactivating mutations, ATM alterations, IDH1 mutations and complex EGFR mutations were predictors of short PFS in patients with a stage 4 lung adenocarcinoma receiving first line EGFR TKI and that PTEN, ATM, IDH1 and KRAS mutations as well as alterations in the MAPK pathway were related to shorter OS. ConclusionThese findings may lead to new treatment options in patients with unfavorable genotypes to optimize first line responses.

Predictive Biomarkers of Response to Venetoclax in Combination with Cobimetinib in Relapsed/Refractory Multiple Myeloma (RRMM)

Predictive Biomarkers of Response to Venetoclax in Combination with Cobimetinib in Relapsed/Refractory Multiple Myeloma (RRMM)

Relevance and actionable mutational spectrum in oral squamous cell carcinoma.

Screening for lesions in the oral cavity is critical for early diagnosis of oral squamous cell carcinoma (OSCC). Targeted next generation sequencing-based (NGS) mutation analysis of cancer driver genes becomes a reality for personalized medicine and cancer therapeutics. In the present study, we have performed a targeted NGS-based mutation analysis of 50 known oncogenes and tumor suppressor genes in clinically diagnosed potentially malignant lesions and tissues of OSCC. NGS-based analysis of DNA obtained from biopsies of histopathologically confirmed cases of potentially malignant lesions and OSCC specimens were performed using Ion AmpliSeq™ Cancer Hotspot Panel V2 using the Ion Proton™ Sequencer System, followed by data analysis using Ion Reporter™ and Torrent Suite™ software. NGS analysis indicated a total of 69 mutations present in 25 genes in potentially malignant lesions and OSCC specimens. We identified recurrent mutations in known OSCC driver genes ATM (11%), TP53 (55%), HRAS (16%), SMAD4 (13%), PIK3CA (16%), and ERBB4 (11%) in potentially malignant lesions and OSCC specimens. Driver mutation analysis identified recurrent TP53 and HRAS driver mutations in our OSCC specimens. Data generated from our study may enable an application of targeted NGS analysis of driver mutations for better therapeutic choice and improved outcomes for OSCC subjects when combined with clinical diagnosis.

Genomic alterations in STK11 can predict clinical outcomes in cervical cancer patients

ObjectiveCervical cancer is the fourth most common cause of cancer-related deaths in Asian women, due to its poor prognosis. This study aimed to decipher genomic alteration profiles of a cohort of Japanese cervical cancer patients to understand why certain patients benefited from molecular targeted therapies and their prognostic significance. MethodsDuring 2008–2018, 154 cervical cancer patients underwent a potentially curative resection procedure at the National Cancer Center Hospital. Genomic DNA samples were analyzed using Ion AmpliSeq™ Cancer Hotspot Panel v2. Alterations in the copy number of PIK3CA, ERBB2, PTEN, and STK11 were detected using the TaqMan assay. HPV-positive results were confirmed by genomic testing and in situ hybridization assay. ResultsThe frequency of genomic alterations in PIK3CA (36%), STK11 (16%), PTEN (11%), TP53 (11%), and KRAS (8%) was >5%. KRAS mutations were preferentially detected in patients with adenocarcinomas, and the frequency of PIK3CA mutations in patients with squamous cell carcinomas was higher than that in patients with other histological cancer types. HPV-positive results were observed in 139/154 (90.3%) patients, and TP53 mutants were detected in HPV-negative specimens. In this study, the overall survival of patients with genomic alterations in STK11 was worse than in patients with wild–type STK11 (hazard ratio = 10.6, P = 0.0079) and TCGA dataset (hazard ratio = 2.46, P = 0.029). ConclusionsMore than one-third of Japanese cervical cancer patients exhibit mutations targeted by molecular targeted therapies. We have proposed the prognostic value of STK11 genomic alterations.

Cervical cancer patients that respond to chemoradiation therapy display an intense tumor infiltrating immune profile before treatment

Cervical cancer (CC) is a major cause of death and suffering to women globally with 570,000 new cases in 2017. It disproportionately affects those living in resource-constrained countries such as Brazil, with 90% of the deaths from CC happening in low and middle-income countries. Early detection is still the best strategy for improving response to therapy and survival and cases detected in advanced stages show variable response rates to the standard chemoradiation therapy protocols. Both the genetic landscape and the immune status of patients can dramatically affect cancer progression and response to therapy, as well as disease recurrence. Here we performed a comprehensive sequencing analysis using the cancer gene panel - Ion AmpliSeq™ Cancer hotspot Panel V2 CHPv2, as well as determined the immune infiltrate composition of a group of locally advanced CC patients with the goal of identifying genetic and immune characteristics associated with a clinical response to therapy. The expression levels of CD68+ tumor-associated macrophages (TAMs) and CD8+ tumor-infiltrating lymphocytes (TILs), as well as the immune checkpoint molecules PD-1, PD-L1 and PD-L2 in stroma and in tumor regions were analyzed by immunohistochemistry (IHC). The HPV infection status with high-risk strains was also determined. Twenty-one samples from patients with squamous cell carcinoma segregated into responder (11) and non-responder (10) groups according to standard chemoradiation therapy response were studied. Our findings indicate that responder patients showed an increase of an inflammatory tumor microenvironment as indicated by higher numbers of CD8+ and PD-L2+ TILs, as well as higher expression of PD-L1 immunoreactive area, as compared to the non-responder group. Additionally, our results demonstrate a correlation between the number of gene mutations and PD-L2+ TILs in the responder group. The genes PIK3CA and KDR/VEGFR were the most mutated genes, corroborating past findings. Together, these findings indicate an inflammatory tumor microenvironment present in patients that will respond to future chemoradiation treatment as compared to those that will not. This points to possible future predictors of response to therapy in CC patients.

Analytical Evaluation of an NGS Testing Method for Routine Molecular Diagnostics on Melanoma Formalin-Fixed, Paraffin-Embedded Tumor-Derived DNA.

Next Generation Sequencing (NGS) is a promising tool for the improvement of tumor molecular profiling in view of the identification of a personalized treatment in oncologic patients. To verify the potentiality of a targeted NGS (Ion AmpliSeq™ Cancer Hotspot Panel v2), selected melanoma samples (n = 21) were retrospectively analyzed on S5 platform in order to compare NGS performance with the conventional techniques adopted in our routine clinical setting (Sequenom MassARRAY system, Sanger sequencing, allele-specific real-time PCR). The capability in the identification of rare and low-frequency mutations in the main genes involved in melanoma (BRAF and NRAS genes) was verified and integrated with the results deriving from other oncogenes and tumor suppressor genes. The analytical evaluation was carried out by the analysis of DNA derived from control cell lines and FFPE (Formalin-Fixed, Paraffin-Embedded) samples to verify that the achieved resolution of uncommon mutations and low-frequency variants was suitable to meet the technical and clinical requests. Our results demonstrate that the amplicon-based NGS approach can reach the sensitivity proper of the allele-specific assays together with the high specificity of a sequencing method. An overall concordance among the tested methods was observed in the identification of classical and uncommon mutations. The assessment of the quality parameters and the comparison with the orthogonal methods suggest that the NGS method could be implemented in the clinical setting for melanoma molecular characterization.

Abstract 439: Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells

Abstract Background. RareCyte has developed platform technology for visual identification and single cell retrieval of rare cells in blood, including circulating tumor cells (CTCs). There is increasing interest in mutational analysis of circulating tumor cells (CTCs) as a liquid biopsy application. Because of the minuscule amount of DNA present in a single cell (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as elevated error rates that can lead to false positive mutations. Amplicon-based next generation sequencing (abNGS) is a high-throughput method which enables genetic confirmation of malignancy and discovery of de novo pathogenic mutations. Here we present a method for performance of single cell abNGS on model CTCs without prior WGA using a commercially available pan-cancer hotspot panel. Methods. A549 lung cancer cells as model CTCs (mCTCs) were spiked into whole blood, which was processed by AccuCyte® separation onto slides. After formalin fixation, multi-parameter immunofluorescence and automated imaging (CyteFinder®) were used to identify CTCs - visualized as nucleated cells expressing epithelial markers (cytokeratin or EpCAM) and not expressing white blood cell markers. mCTCs were mechanically retrieved by CytePicker® into PCR tubes and either amplified by WGA (PicoPLEX®) or lysed in a PCR-compatible lysis buffer. WGA products or cell lysates were used as template for the AmpliSeq™ Cancer HotSpot Panel v2 for Illumina® library preparation; additional PCR cycles were added during target amplification to compensate for low DNA input in the non-WGA samples. Libraries were sequenced on an Illumina MiSeq and analyzed using the BaseSpace bioinformatics suite. Results. Single mCTCs that underwent amplicon-based NGS library prep direct from cell lysate (non-WGA) displayed increased uniformity of coverage with decreased target dropout when compared to WGA cells. Median read depth increased 7-fold with the non-WGA method. On average, 8 of 15 variants present in bulk A549 genomic DNA were observed in single mCTCs sequenced after WGA, while 12 out of 15 were observed with the non-WGA method. Additionally, the false positive error frequency of non-WGA samples was < 5% of the WGA samples. The non-WGA method was applied to CTCs identified in blood from a prostate cancer patient and confirmed presence of PTEN and TP53 mutations identified by cell-free DNA analysis. Conclusions. Amplicon-based targeted single-cell sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. Citation Format: Nolan G. Ericson, Arturo B. Ramirez, Alisa C. Clein, Celestia S. Higano, Daniel E. Sabath, Eric P. Kaldjian. Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 439.

Abstract 1705: Genetic analysis of thymoma and thymic carcinoma

Abstract [Background] Thymic epidermal tumors (thymoma and thymic cancer) are rare, and its genetic profiles are unclear. Molecular targeted therapy of thymic tumors has not been established. Clarifying a molecular alteration and to identify the novel targets for molecular targeted therapy is important. So, we conducted the next generation sequencing (NGS) analysis with surgically resected thymic tumor tissues. [Methods] From July 2013 to October 2018, thirty patients who were resected thymic epidermal tumors in our hospital were enrolled in this study. We got written informed consent from patients, and NGS analysis of extracted DNA sample was performed in 24 patients to the present time. DNA were extracted from fresh frozen surgically resected tissues (tumors and paired normal tissues) and DNA amplicon sequencing was performed with a custom panel of 53 cancer-related genes based on Ion AmpliSeq™ Cancer Hotspot Panel v2 comprising major oncogenes and tumor suppressor genes (including GTF2I). Sequencing was carried out with an Ion Torrent PGM™. Sequencing data were analyzed using Ion Reporter™. In addition, we collected the patient’s information (age, sex, Masaoka-Koga staging, World Health Organization (WHO) histologic classification, tumor size, and complication such as myasthenia gravis and pure red cell aplasia) from medical records. The study was approved by the Institutional Review Board (IRB) of the Nagasaki University Hospital. [Results] A total of 30 patients were enrolled in this study. The median age was 62 years old (range, 34-84 years old), patients characteristics were 9 male and 21 female, WHO histologic classification (type A, AB, B1, B2, B3, thymic carcinoma) was 1, 11, 4, 10, 2, 2, and Masaoka-Koga staging I, II, III, IV were 12, 6, 9, 3, respectively. Median tumor size was 47mm (range 11-110 mm). Moreover, complications were 7 myasthenia gravis, 4 acetylcholine receptor antibody-positive (do not confirm diagnossis of myasthenia gravis), 1 pure red cell aplasia, and 1 agranulocytosis. In NGS sequencing, the nonsynonymous mutations of HRAS and NRAS (HRAS Q61R, HRAS G13R, and NRAS Q61K) was detected in three patients. These RAS gene mutations were reported to be pathogenic in various malignancies. Low frequently DNMT3A mutation was detected in the other two patients, however this mutation were not validated in other methods. No genetic alterations were detected in the rest 19 patients. [Conclusion] The frequency of genetic alterations in thymic epidermal tumors was very low in this study, however, active mutations of RAS oncogene were detected in three patients. Although the RAS are still not established as treatment targets, it should be one of the interacting molecules. Further investigation is required to establish new therapeutic strategy according to the genetic alteration in thymic tumors. Citation Format: Hiroyuki Yamaguchi, Hiroshi Gyotoku, Hirokazu Taniguchi, Daisuke Sasaki, Midori Shimada, Yosuke Dotsu, Hiroaki Senju, Norihito Kaku, Takaya Ikeda, Minoru Fukuda, Katsunori Yanagihara, Hiroshi Mukae. Genetic analysis of thymoma and thymic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1705.

Abstract 4889: Comparison of tumor mutational burden using the Ion Oncomine™ TML and FoundationOne™ assays with routine clinical FFPE tissue samples to predict durable clinical benefit in lung cancer and melanoma patients - a multivariate analysis integrating PD-L1 and CD8+ evaluation

Abstract Background and objective: Tumor mutational burden (TMB) has emerged in recent clinical trials as a novel biomarker for the stratification of non-small cell lung cancer (NSCLC) patients treated with anti-PD1/PD-L1 antibodies. TMB in trials has been mainly assessed using the FoundationOne™ test (Foundation Medicine, Cambridge, MA, USA), which requires samples to be sent to the authorized testing center for the analysis. Therefore, our aim was to compare an inhouse assessment of TMB using the Ion Oncomine™ TML (Thermo Fisher Scientific, Waltham, MA, USA) and the FoundationOne™ tests. Furthermore, we evaluated PD-L1 expression in tumor cells and the level of tumor infiltrating CD8+ lymphocytes by immunohistochemistry to find the best combination of predictive biomarkers. Materials and Methods: Late stage lung adenocarcinoma (51) and melanoma (25) patients with first- or second-line treatment with checkpoint inhibitors were included prospectively and selected randomly. Tissue FFPE sections were sent out for TMB and genetic assessment using the FoundationOne™ test, and parallel in-house analysis using the Ion Oncomine™ TML assay and the Ion AmpliSeq™ Cancer Hotspot Panel v2. IHC for PD-L1 (22C3 pharmDx assay; Dako Agilent, Santa Clara, CA), in addition to CD8+ lymphocyte expression analyses (clone SP57; Roche Ventana, Tucson, AZ) were performed. Results: TMB was assessed by the Ion Oncomine™ TML assay in comparison to FoundationOne™, demonstrating a high correlation in melanoma (R² = 0.95), but a lower correlation in NSCLC patients (R² = 0.88). Prediction of a durable clinical response by receiver operating characteristic (ROC) was comparable between Oncomine™ TML (AUC = 0.70) and FoundationOne™ (AUC = 0.74) and also similar to the CD8+ score (AUC = 0.76) and PD-L1 expression (AUC = 0.72). Combining different biomarkers did not improve the predictive value. In addition, detection of actionable mutations exhibited a good correlation between the different gene panels. TMB analysis using the Ion Oncomine™ TML assay was initially ineffective as elevated deamination rates from formalin fixed samples interfered with the analysis but this could be successfully overcome with bioinformatic filtering and UDG repair. Stringent sample requirements for FoundationOne™ testing prevented the analysis in 22% and 8% of lung cancer and melanoma samples, respectively. Conclusion: TMB can be assessed using the Ion Oncomine™ TML assay since its predictive value is comparable to that obtained with the FoundationOne™ test. Combining different biomarkers did not improve the prediction of clinical responses in this series with a limited number of patients. Updated clinical data will be presented during the congress. Citation Format: Simon Heeke, Jonathan Benzaquen, Elodie Long-Mira, Benoit Audelan, Virginie Lespinet, Olivier Bordone, Salomé Lalvé, Katia Zahaf, Michel Poudenx, Pierre-Michel Dugourd, Madleen Chassang, Thierry Passeron, Hervé Delingette, Charles-Hugo Marquette, Véronique Hofman, Marius Ilié, Paul Hofman. Comparison of tumor mutational burden using the Ion Oncomine™ TML and FoundationOne™ assays with routine clinical FFPE tissue samples to predict durable clinical benefit in lung cancer and melanoma patients - a multivariate analysis integrating PD-L1 and CD8+ evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4889.

Abstract 4603: Clinical application and potential usefulness of targeted next-generation sequencing on resected pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) carries a dismal prognosis. Virtually all PDA are characterized by mutation of four driver genes: KRAS, TP53, CDKN2A and SMAD4 and a long tail of rarely mutated genes. Application of targeted next-generation sequencing (NGS) has entered clinical routine for colon, lung and other cancers. Among patients with resected PDA, usefulness, applicability and prognostic significance of NGS results are still a matter of debate. Aims: To evaluate: 1) the alterations of the 4 main driver genes and patient outcomes after resection 2) the usefulness of targeted NGS in finding targetable alterations. Methods: We analyzed DNA alterations in FFPE tumors among 279 patients with curatively resected PDA who were treated at 4 Academic Hospitals (Franco-Belgian consortium). Sequencing libraries were prepared using a 50 genes panel (Ion AmpliSeqTM Cancer HotSpot Panel v2, Life Technologies). Sequencing was performed on an Ion ProtonTM System using an Ion PITM Sequencing 200 Kit and an Ion PITM Chip Kit v3 (Life Technologies). Associations of driver gene alterations with disease-free survival (DFS) and overall survival (OS) were evaluated using Cox proportional hazards regression with estimation of hazard ratios (HRs) and 95% CIs and adjusted for age, sex, tumor characteristics and institution. Results: Of the 279 patients analyzed, 163 (58.4%) were men and 116 (41.6%) were women, with a median age of 64.59 (36.9-87.5) years. KRAS, TP53, CDKN2A and SMAD 4 were mutated in 246, 193, 45 and 44 patients respectively. Patients with KRAS mutant tumors did not have worse DFS (median [95% CI], 12.2 [11.35-14.6] months) and OS (23.9 [21.1-30.1] months) compared to patients with KRAS wild-type tumors (DFS, 14.3 [9.64-19.4] months; OS, 31.8 [19.4-53.5] months). The mutational status of TP53 or SMAD4 was not associated with DFS or OS as well. CDKN2A mutations were associated with a lower OS (mutation: 20.5 [14.6-33.0] versus wild-type 26.5 [21.9-33.3] months OS, log-rank <1% HR 1.65 [1.124-2.436]) but not to a lower DFS. Patients had slightly worse DFS and OS if they had a greater number of altered driver genes. Compared with patients with 0 to 2 altered genes, those with 3 to 4 altered genes had worse DFS (HR, 1.377 [95% CI, 1-1.89; P=.05]) and OS (HR, 1.476 [95% CI, 1.04-2.09; P= .028]). For some patients (n=27, 9.68%), we found mutations that could be targeted (i.e PTEN, STK11, GNAS, PIK3CA, FLT3, BRAF, IDH1/2, RET, FGFR3, KIT, AKT1). Conclusions: Analysis of the four main driver gene alterations introduces weak prognostic information with little added clinical value. However, application of NGS in resected PDA is quite feasible and is able to find targetable mutations. KRAS and CDKN2A deserve future attention for targeting specific mutations Citation Format: Francesco Puleo, Rémy Nicolle, Yuna Blum, Jérôme Cros, Nabila Elarouci, Denis Franchimont, Jacques Devière, Aurélien de Reyniès, Pierre Laurent-Puig, Jean-Baptiste Bachet, Raphaël Maréchal, Jean-Luc Van Laethem. Clinical application and potential usefulness of targeted next-generation sequencing on resected pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4603.

Comprehensive molecular and phenotypic profiling of end-stage IV cancer identifies treatable targets and improves survival in individual patients

Background: Comprehensive profiling of patients’ tumor tissue in cases with end-stage IV cancer was conducted to identify mutations, proteins and activated cancer pathways potentially eligible for targeted therapies. Data evaluation included test results, clinical data, clinical trials and longitudinal follow-up data. Methods: Tissue collection and processing was conducted under highly standardized and controlled conditions to preserve the expression profile in tissue as it appears in the human body. Samples that passed the quality control (tumor content ≥ 50%) were analyzed using the following techniques: immunohistochemistry (IHC), next-generation sequencing (NGS) and simple western charge-based assays (NanoPro 1000 system). Expression of the major receptor proteins and phosphorylation of the signaling proteins ERK1/2, MEK1/2 and AKT of the MAP kinase pathway and the PI3K/AKT/mTOR-pathway were measured to infer targets on the (phospho-) proteomic level. On the genomic level DNA sequencing of “hotspot” regions covering 50 oncogenes and tumor suppressor genes was conducted using the Ion AmpliSeqTM Cancer Hotspot Panel v2. Results: Analyses of 82 patients were performed and 27 patients met the determined evaluation criteria. These patients were divided into two groups. In one group (n = 13) targets for available therapeutics could be identified and patients were treated accordingly. In the other group (n = 14) targets could not be found or were not regarded as useful by the oncologist. These patients were treated with conventional, palliative therapies. The 6-month follow-up revealed a better outcome when patients were treated according to an identified target. 46% of patients responded to targeted treatment. 15% of patients had a complete remission (CR), 8% had a stable disease (SD) and another 23% had a partial remission (PR). 38% progressed under targeted therapy and 15% died. In contrast, only 21% responded under conventional, palliative therapy by showing a stable disease, 64% of patients progressed (PD) and 14% died. No complete or partial remission was observed in patients treated by conventional approaches. Conclusions: A comprehensive molecular and phenotypic tumor characterization can identify in individual patients, suffering from highly advanced cancer, targeted therapies that impact cancer growth and lead in single cases even to a complete remission. Legal entity responsible for the study: IndivuTest GmbH Funding: IndivuTest GmbH Disclosure (FOR ALL LISTED AUTHORS): A. Samsen, S.Schneider, S. Von Der Heyde, W. Saeger, B. Grebenstein and Hartmut Juhl are employees of IndivuTest GmbH. The authors declare no other competing financial interests. The study was conducted in Hamburg, Germany, and reviewed and approved by the competent ethics review committee of the Medical Association of Hamburg, Germany under reference number PV5035. Prior to this study, all patients gave their written informed consent.

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA).

Isolation and genotyping of circulating tumor cells (CTCs) is gaining an increasing interest by clinical researchers in oncology not only for investigative purposes, but also for concrete application in clinical practice in terms of diagnosis, prognosis and decision treatment with targeted therapies. For the mutational analysis of single CTCs, the most advanced biotechnology methodology currently available includes the combination of whole genome amplification (WGA) followed by next-generation sequencing (NGS). However, the sequence of these molecular techniques is time-consuming and may also favor operator-dependent errors, related to the procedures themselves that, as in the case of the WGA technique, might affect downstream molecular analyses. A preliminary approach of molecular analysis by NGS on a model of CTCs without previous WGA procedural step was performed. We set-up an artificial sample obtained by spiking the SK-MEL-28 melanoma cell line in normal donor peripheral whole blood. Melanoma cells were first enriched using an AutoMACS® (Miltenyi) cell separator and then isolated as single and pooled CTCs by DEPArray™ System (Silicon Biosystems). NGS analysis, using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies) with the Ion Torrent PGM™ system (Life Technologies), was performed on the SK-MEL-28 cell pellet, a single CTC previously processed with WGA and on 1, 2, 4 and 8 recovered CTCs without WGA pre-amplification. NGS directly carried out on CTCs without WGA showed the same mutations identified in SK-MEL-28 cell line pellet, with a considerable efficiency and avoiding the errors induced by the WGA procedure. We identified a cost-effective, time-saving and reliable methodological approach that could improve the analytical accuracy of the liquid biopsy and appears promising in studying CTCs from cancer patients for both research and clinical purposes.

Evaluation of a novel approach to circulating tumor cell isolation for cancer gene panel analysis in patients with breast cancer.

Liquid biopsy isolation of circulating tumor cells (CTCs) allows the genomic analysis of CTCs, which is useful in the determination of personalized cancer therapy. In the present study, CTCs from patients with breast cancer were enriched and successfully analyzed using cancer gene panel analysis. Blood samples from 11 patients with breast cancer were collected and CTCs enriched for using size-based filtration. The enriched CTCs were analyzed using immunofluorescence staining with antibodies directed against epithelial cell adhesion molecule (EpCAM) and cluster of differentiation 45. The genomic DNA of CTCs was extracted, amplified and 50 genes screened for mutations using the Ion AmpliSeq™ Cancer Hotspot Panel v2. EpCAM staining detected CTCs in 10/11 patients and the average CTC count was 3.9 in 5 ml blood. The average purity of enriched CTCs was 14.2±29.4% and the average amount of amplified DNA was 28.6±11.9 µg. Catalogue Of Somatic Mutations In Cancer mutations were detected in the CTCs and included IDH2, TP53, NRAS, IDH1, PDGFRA, HRAS, STK11, EGFR, PTEN, MLH1, PIK3CA, CDKN2A, KIT and SMARCB1. In conclusion, a novel size-based filtration approach for the isolation of CTCs was evaluated and successfully applied for the genomic analysis of CTCs from patients with breast cancer.

899P - Defining the genomic landscape of vulvar squamous cell carcinoma (VSCC) using next generation sequencing: the role of HPV infection

Surgery for vulvar squamous cell carcinoma (VSCC) is associated with physical and psychological morbidity. Targeted therapies have not influenced outcomes, in part because the genomic landscape of VSCC remains incompletely understood and is complicated by two etiologies: HPV-dependent and HPV-independent disease. Next-generation sequencing (NGS) was used to identify somatic mutations by HPV status (+/-). We conducted a retrospective review of patients (pts) seen at the Ottawa Hospital, Ottawa, Canada between 2000-2012 for VSCC. Clinical and pathological data was collected and pts with adequate tumor for HPV and molecular analysis were identified. HPV status was determined by polymerase chain reaction (PCR) with primers targeting the L1 region of HPV DNA. The Ion AmpliSeq™ Cancer Hotspot Panel v2 is being used to examine the presence of 50 known mutations. Forty-seven pts with VSCC were included. 22/47 (47%) were HPV+ and 25/47 (53%) were HPV-. Median age at diagnosis was 60 years (IQR 49-76) and 69 years (IQR 58-77), for HPV+ and HPV-, respectively. Disease stage by HPV status (+ vs -) was: I (15 vs 16), II (2 vs 0), III (2 vs 9) and IV (3 vs 0). Molecular data is available on 31 pts (17 HPV + , 14 HPV-). Among HPV+ pts, 15/17 had ≥ 1 mutation. Mutational frequencies among HPV+ pts were: TP53 7/17, PIK3CA 6/17, KDR 5/17, KIT: 3/17, FGFR3 3/17, and one mutation each of PTEN, CTNNB1, APC, KRAS, ERBB4, ATM, SMARCB1, FLT3, CDK2A. Among the HPV- pts, 12/14 had ≥ 1 mutation. Mutational frequencies among HPV- pts were: TP53 8/14, HRAS 3/14, CDKN2A 2/14, PI3KCA 2/14, KDR 1/14 and GNA11 1/14. VSCC is characterized by a high mutation rate and a high prevalence of HPV infection. HPV-dependent and HPV-independent disease have unique mutational profiles. The high prevalence of “actionable” mutations supports the need for trials of targeted therapies. Molecular data on the full study cohort (n = 47) will be presented.

Abstract 3962: Circulating tumor DNA (ctDNA) as an alternative to tumor biopsies for precision oncology: The Jules Bordet Institute experience

Abstract Background In the era of precision medicine, data obtained from sequencing the cancer genome of a patient can be integrated into clinical decision-making. Liquid biopsy using ctDNA is a non-invasive tool, which can circumvent unfeasible or unrepresentative tumor biopsies and allow continuous monitoring of the disease thereby contributing to rational drug delivery. Experimental procedures Precision-f is a feasibility molecular screening study that enrolled 34 patients with metastatic melanoma, non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Results from targeted gene sequencing (TGS) of metastatic biopsies were presented at AACR 2015. The patients had a blood draw concomitantly with the metastatic biopsy for plasma isolation. Similar to biopsies, somatic mutations (substitutions, small insertions and deletions) were assessed using the Ion AmpliSeq™ Cancer Hotspot Panel v2, designed to amplify 207 amplicons covering 2800 COSMIC mutations from the 50 most commonly reported oncogenes and tumor suppressor genes with a sensitivity of 1%. All mutations identified in the biopsy and/or plasma samples using TGS are currently being validated using Droplet Digital PCR (ddPCR) technology. Results Evaluable ctDNA-derived sequencing data were available for 32 patients. The sequencing results between biopsies and plasma were fully concordant in 63% (20/32) of the patients. TGS of ctDNA failed to detect clinically relevant mutations (EGFR, PIK3CA, KRAS and BRAF) identified in the metastatic biopsies in 19% (6/32) of the patients. In contrast, biologically relevant mutations (STK11, KRAS, PIK3CA and SMO) not detected in tumor biopsies were found in the ctDNA in 9% (3/32) of the patients. Interestingly, in one CRC patient a KRAS G13D mutation still potentially associated with benefit to anti-EGFR therapy was detected only in the tumor biopsy whereas a KRAS A146T mutation mediating resistance to anti-EGFR therapy was detected solely in ctDNA. Tumor biopsies and plasma are currently being tested using ddPCR to validate the findings. The results of these analyses will be presented at the meeting. Conclusion Although technical challenges remain, ctDNA profiling appears to be a promising tool for precision oncology by allowing the identification of genomic alterations associated with drug resistance and tumor progression. Citation Format: Philippe G. Aftimos, Marion Maetens, Françoise Rothé, David Brown, Yacine Barèche, Alexandre Irrthum, Thierry Berghmans, Joseph Kerger, Alain Hendlisz, Michail Ignatiadis, Martine Piccart, Christos Sotiriou, Ahmad Awada. Circulating tumor DNA (ctDNA) as an alternative to tumor biopsies for precision oncology: The Jules Bordet Institute experience. [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 3962.

Abstract 3968: Comparison of NGS and digital PCR for the detection of point mutations in breast tumor and plasma samples

Abstract Background Over the past years, tumor derived alterations in plasma has emerged as a promising cancer biomarker. The clinical applications envisioned with liquid biopsies range from monitoring of treatment response to disease progression and acquired resistance. However, the analysis of circulating tumor DNA (ctDNA) is challenging owing to the small fraction of tumor specific DNA relative to background levels of non-tumor derived DNA and therefore requires highly specific and sensitive techniques. Experimental procedures The Ion AmpliSeqTM Cancer Hotspot Panel v2, covering 2,800 COSMIC mutations from 50 cancer genes was used to analyze 31 primary tumor and metastatic lesions along with 19 matched plasma samples from 13 breast cancer (BC) patients receiving neoadjuvant treatment and 6 advanced stage metastatic BC patients. Digital PCR (dPCR) experiments were set up using mutation-specific assays (Biorad) for the detection of 13 hotspot mutations in PIK3CA, TP53, PTEN, AKT1, CDKN2A and STK11 genes previously identified in the solid tumor samples using next generation sequencing (NGS). Results The specificity and sensitivity of the mutation-specific dPCR assays were tested in dilution curves using synthetic oligonucleotides and tumor samples bearing the mutation of interest. Mutations could be robustly detected in plasma samples using dPCR with a sensitivity as low as 0.1% and a sensitivity of 0.5% using NGS. All mutations previously identified using NGS were validated using dPCR in tumor (n = 27) as well as in plasma samples (n = 5). Moreover, dPCR identified 4 additional mutations in 4 plasma samples in the neoadjuvant setting and 4 in the metastatic setting, 2 in tumor and 2 in plasma samples. The mutant allelic frequencies (MAF) of these newly identified mutations were very low (range 0.2%-1.8%) close to the detection limit of the technology. Through the use of dPCR, the detection rate of plasma ctDNA increased from 8% using NGS to 38% in the neoadjuvant setting and from 67% using NGS to 100% in the metastatic setting thereby highlighting the greater sensitivity of the dPCR technology. The MAF assessed by NGS and dPCR were highly correlated both in the neoadjuvant and metastatic (Spearman ρ = 0.84 and ρ = 0.97) settings, a higher concordance between NGS and dPCR being observed when the MAF are high. Conclusions The use of dPCR allows the detection of low-frequency tumor-specific mutations in plasma with a greater sensitivity compared to NGS providing the high accuracy and sensitivity essential for liquid biopsies. Citation Format: Françoise Rothé, Ghizlane Rouas, Michelle Coureau, Marion Maetens, David Brown, Christos Sotiriou, Michail Ignatiadis. Comparison of NGS and digital PCR for the detection of point mutations in breast tumor and plasma samples. [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 3968.

Next-Generation Sequencing in Salivary Gland Basal Cell Adenocarcinoma and Basal Cell Adenoma.

Basal cell adenoma and basal cell adenocarcinoma represent basaloid salivary gland neoplasms that show cyto-morphologic similarity but differ at the histologic level by their invasive qualities, as adenocarcinomas show invasion beyond their capsule, a finding not seen in the adenomas. Due to the low incidence of these tumors, the molecular mechanism underlying their pathogenesis is poorly understood. We sought to further delineate these neoplasms through mutation profiling by targeted next-generation sequencing (NGS). Twenty cases (basal cell adenocarcinoma=10; basal cell adenoma=10) were retrospectively selected from a previous analysis. NGS was performed using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA). The data was analyzed using the Ion Torrent Suite Software (Life Technologies) followed by a laboratory-developed pipeline. One of eight cases of basal cell adenocarcinoma had a mutation, which was an activating mutation in PIK3CA (c.3140A>G, p.H1047R). No mutations were detected in the remaining basal cell adenocarcinomas. In the basal cell adenomas, the CTNNB1 p.I35T mutation was identified in three of nine (3/9) cases. A missense mutation in the ATM gene (c.2572T>C, p.F858L) was seen in a basal cell adenoma with an allele frequency of 53%, raising the possibility of a germline mutation. The overall findings suggest that although there is cytomorphologic similarity, differences exist between these two tumors at the histologic and genetic level. Although the numbers of cases are limited, the aberrations in genes affecting different signaling pathways in the basal cell adenocarcinoma versus the basal cell adenomas suggest that basal cell adenocarcinoma likely arises de novo and not from basal cell adenoma.

Molecular Heterogeneity of Ewing Sarcoma as Detected by Ion Torrent Sequencing.

Ewing sarcoma (ES) is the second most common malignant bone and soft tissue tumor in children and adolescents. Despite advances in comprehensive treatment, patients with ES metastases still suffer poor outcomes, thus, emphasizing the need for detailed genetic profiles of ES patients to identify suitable molecular biomarkers for improved prognosis and development of effective and targeted therapies. In this study, the next generation sequencing Ion AmpliSeq™ Cancer Hotspot Panel v2 was used to identify cancer-related gene mutations in the tissue samples from 20 ES patients. This platform targeted 207 amplicons of 2800 loci in 50 cancer-related genes. Among the 20 tissue specimens, 62 nonsynonymous hotspot mutations were identified in 26 cancer-related genes, revealing the molecular heterogeneity of ES. Among these, five novel mutations in cancer-related genes (KDR, STK11, MLH1, KRAS, and PTPN11) were detected in ES, and these mutations were confirmed with traditional Sanger sequencing. ES patients with KDR, STK11, and MLH1 mutations had higher Ki-67 proliferation indices than the ES patients lacking such mutations. Notably, more than half of the ES patients harbored one or two possible ‘druggable’ mutations that have been previously linked to a clinical cancer treatment option. Our results provided the foundation to not only elucidate possible mechanisms involved in ES pathogenesis but also indicated the utility of Ion Torrent sequencing as a sensitive and cost-effective tool to screen key oncogenes and tumor suppressors in order to develop personalized therapy for ES patients.