Nilotinib in KIT-driven advanced melanoma: Results from the phase II single-arm NICAM trial.

Mucosal (MM) and acral melanomas (AM) are rare melanoma subtypes of unmet clinical need; 15%-20% harbor KIT mutations potentially targeted by small-molecule inhibitors, but none yet approved in melanoma. This multicenter, single-arm Phase II trial (NICAM) investigates nilotinib safety and activity in KIT mutated metastatic MM and AM. KIT mutations are identified in 39/219 screened patients (18%); of 29/39 treated, 26 are evaluable for primary analysis. Six patients were alive and progression free at 6 months (local radiology review, 25%); 5/26 (19%) had objective response at 12 weeks; median OS was 7.7 months; ddPCR assay correctly identifies KIT alterations in circulating tumor DNA (ctDNA) in 16/17 patients. Nilotinib is active in KIT-mutant AM and MM, comparable to other KIT inhibitors, with demonstrable activity in nonhotspot KIT mutations, supporting broadening of KIT evaluation in AM and MM. Our results endorse further investigations of nilotinib for the treatment of KIT-mutated melanoma. This clinical trial was registered with ISRCTN (ISRCTN39058880) and EudraCT (2009-012945-49).

EuroClonality-NGS Recommendations for Evaluation of B-Cell Clonality Analysis by Next-Generation Sequencing: A Structured Approach with the DEPART Algorithm.

Next-generation sequencing (NGS)-based clonality analysis allows in-depth assessment of the clonal composition of a sample with high sensitivity for detecting small clones. Within the EuroClonality-NGS Working Group, a protocol for NGS Ig clonality analysis was developed and validated previously. This NGS-based approach was designed to generate small amplicons, making it suitable for samples with suboptimal DNA quality, especially material derived from formalin-fixed, paraffin-embedded tissue. Using expert assessment of NGS Ig clonality results as a reference, a structured algorithmic approach to the assessment of NGS-amplicon-based B-cell clonality analysis was developed. A structured approach with the Detection of clonality through Evaluation of sample quality and assessment of Pattern, Abundance and RaTio (DEPART) algorithm was proposed, which consecutively evaluates sample quality, the pattern of the clonotypes present, the abundance of the most dominant clonotypes, and the ratio between the dominant clonotypes and the background to evaluate the different Ig gene targets. Specific issues with respect to evaluation of the various Ig targets as well as the integration of results of individual targets into a molecular clonality conclusion are discussed and illustrated with case examples. Finally, the importance of interpretation of NGS-based clonality results in clinical and histopathologic contexts is discussed. It is expected that these recommendations will have clinical utility to facilitate proper evaluation of clonality assessment.

Redefining the high-grade B cell lymphoma with double/triple rearrangements of MYC and BCL2/BCL6 genes. Learning from a case report.

We report a patient initially diagnosed with a triple hit high-grade B cell lymphoma (HGBL-TH), in which further morphologic, immunohistochemical, and next-generation sequencing studies of subsequent specimens disclosed it to be a germinal center diffuse large B cell lymphoma (GC-DLBCL) with BCL2/BCL6 gene translocations, PVT1-deletion, and gain of MYC genes evolving from a previous follicular lymphoma. However, fluorescence in situ hybridization (FISH) studies with the break-apart probe for MYC gene showed a fusion and two separated signals (red and green, respectively) leading to the interpretation of MYC gene translocation and a false diagnosis of a TH-lymphoma, according to the recent WHO classification. Nevertheless, PVT1 deletion plus MYC gain/amplification has been described as a cause of the double-hi transcription profile. These data highlight the need for new criteria to identify these highly aggressive lymphomas.

FGF7-FGFR2 autocrine signaling increases growth and chemoresistance of fusion-positive rhabdomyosarcomas.

Rhabdomyosarcomas are aggressive pediatric soft-tissue sarcomas and include high-risk PAX3-FOXO1 fusion-gene-positive cases. Fibroblast growth factor receptor 4 (FGFR4) is known to contribute to rhabdomyosarcoma progression; here, we sought to investigate the involvement and potential for therapeutic targeting of other FGFRs in this disease. Cell-based screening of FGFR inhibitors with potential for clinical repurposing (NVP-BGJ398, nintedanib, dovitinib, and ponatinib) revealed greater sensitivity of fusion-gene-positive versus fusion-gene-negative rhabdomyosarcoma cell lines and was shown to be correlated with high expression of FGFR2 and its specific ligand, FGF7. Furthermore, patient samples exhibit higher mRNA levels of FGFR2 and FGF7 in fusion-gene-positive versus fusion-gene-negative rhabdomyosarcomas. Sustained intracellular mitogen-activated protein kinase (MAPK) activity and FGF7 secretion into culture media during serum starvation of PAX3-FOXO1 rhabdomyosarcoma cells together with decreased cell viability after genetic silencing of FGFR2 or FGF7 was in keeping with a novel FGF7-FGFR2 autocrine loop. FGFR inhibition with NVP-BGJ398 reduced viability and was synergistic with SN38, the active metabolite of irinotecan. In vivo, NVP-BGJ398 abrogated xenograft growth and warrants further investigation in combination with irinotecan as a therapeutic strategy for fusion-gene-positive rhabdomyosarcomas.

Inactivating NF1 Mutations Are Enriched in Advanced Breast Cancer and Contribute to Endocrine Therapy Resistance.

PURPOSE: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. EXPERIMENTAL DESIGN: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. RESULTS: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. CONCLUSIONS: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

Analysis of KRAS, NRAS, BRAF, PIK3CA and TP53 mutations in a large prospective series of locally advanced rectal cancer patients.

Little information is available on the clinical significance of cancer-related genes such as KRAS, NRAS, BRAF, PIK3CA and TP53 in nonmetastatic rectal cancer. We investigated mutations of these genes in a large prospective series of locally advanced rectal cancer (LARC) patients who were recruited into two phase II trials. Mutational analyses were performed with diagnostically validated methods including polymerase chain reaction, capillary electrophoresis single-strand conformational analysis, Sanger sequencing and next-generation sequencing. Associations between single or multiple gene mutations and clinicopathological characteristics and treatment outcomes were explored. Of these 269, 210 (78%) patients were assessable. Mutations of KRAS, NRAS, BRAF, PIK3CA and TP53 occurred in 43, 9, 4, 9 and 60% of patients, respectively. Concordance between paired biopsy and resection specimens was 82% for KRAS, 95% for NRAS, 99% for BRAF, 96% for PIK3CA and 63% for TP53. TP53 mutations were associated with extramural venous invasion on baseline MRI (78% vs. 65%, p = 0.04), poor pathological tumour regression (23% vs. 36%, p = 0.05) and a trend toward a worse 5-year progression-free survival (PFS; 60% vs. 74%, HR 1.59, p = 0.06). Patients with tumours harbouring mutation of TP53 and either KRAS or NRAS (32%) had a worse 5-year PFS than those with TP53/KRAS/NRAS wild-type tumours (54% vs. 72%, HR 1.75, p = 0.02). In univariate analysis, BRAF mutation predicted poor 5-year overall survival only among patients treated without cetuximab (20% vs. 73%, HR 3.29, p = 0.03). This is one of the largest biomarker studies in a prospective, largely homogeneous, LARC population. Our findings are hypothesis generating and require validation in independent series.

NUQA: Estimating Cancer Spatial and Temporal Heterogeneity and Evolution through Alignment-Free Methods.

Longitudinal next-generation sequencing of cancer patient samples has enhanced our understanding of the evolution and progression of various cancers. As a result, and due to our increasing knowledge of heterogeneity, such sampling is becoming increasingly common in research and clinical trial sample collections. Traditionally, the evolutionary analysis of these cohorts involves the use of an aligner followed by subsequent stringent downstream analyses. However, this can lead to large levels of information loss due to the vast mutational landscape that characterizes tumor samples. Here, we propose an alignment-free approach for sequence comparison-a well-established approach in a range of biological applications including typical phylogenetic classification. Such methods could be used to compare information collated in raw sequence files to allow an unsupervised assessment of the evolutionary trajectory of patient genomic profiles. In order to highlight this utility in cancer research we have applied our alignment-free approach using a previously established metric, Jensen-Shannon divergence, and a metric novel to this area, Hellinger distance, to two longitudinal cancer patient cohorts in glioma and clear cell renal cell carcinoma using our software, NUQA. We hypothesize that this approach has the potential to reveal novel information about the heterogeneity and evolutionary trajectory of spatiotemporal tumor samples, potentially revealing early events in tumorigenesis and the origins of metastases and recurrences. Key words: alignment-free, Hellinger distance, exome-seq, evolution, phylogenetics, longitudinal.

Investigating the potential clinical benefit of Selumetinib in resensitising advanced iodine refractory differentiated thyroid cancer to radioiodine therapy (SEL-I-METRY): protocol for a multicentre UK single arm phase II trial.

BACKGROUND: Thyroid cancer is the most common endocrine malignancy. Some advanced disease is, or becomes, resistant to radioactive iodine therapy (refractory disease); this holds poor prognosis of 10% 10-year overall survival. Whilst Sorafenib and Lenvatinib are now licenced for the treatment of progressive iodine refractory thyroid cancer, these treatments require continuing treatment and can be associated with significant toxicity. Evidence from a pilot study has demonstrated feasibility of Selumetinib to allow the reintroduction of I-131 therapy; this larger, multicentre study is required to demonstrate the broader clinical impact of this approach before progression to a confirmatory trial. METHODS: SEL-I-METRY is a UK, single-arm, multi-centre, two-stage phase II trial. Participants with locally advanced or metastatic differentiated thyroid cancer with at least one measureable lesion and iodine refractory disease will be recruited from eight NHS Hospitals and treated with four-weeks of oral Selumetinib and assessed for sufficient I-123 uptake (defined as any uptake in a lesion with no previous uptake or 30% or greater increase in uptake). Those with sufficient uptake will be treated with I-131 and followed for clinical outcomes. Radiation absorbed doses will be predicted from I-123 SPECT/CT and verified from scans following the therapy. Sixty patients will be recruited to assess the primary objective of whether the treatment schedule leads to increased progression-free survival compared to historical control data. DISCUSSION: The SEL-I-METRY trial will investigate the effect of Selumetinib followed by I-131 therapy on progression-free survival in radioiodine refractory patients with differentiated thyroid cancer showing increased radioiodine uptake following initial treatment with Selumetinib. In addition, information on toxicity and dosimetry will be collected. This study presents an unprecedented opportunity to investigate the role of lesional dosimetry in molecular radiotherapy, leading to greater personalisation of therapy. To date this has been a neglected area of research. The findings of this trial will be useful to healthcare professionals and patients alike to determine whether further study of this agent is warranted. It is hoped that the development of the infrastructure to deliver a multicentre trial involving molecular radiotherapy dosimetry will lead to further trials in this field. TRIAL REGISTRATION: SEL-I-METRY is registered under ISRCTN17468602 , 02/12/2015.

Somatic cancer genetics in the UK: real-world data from phase I of the Cancer Research UK Stratified Medicine Programme.

INTRODUCTION: Phase I of the Cancer Research UK Stratified Medicine Programme (SMP1) was designed to roll out molecular pathology testing nationwide at the point of cancer diagnosis, as well as facilitate an infrastructure where surplus cancer tissue could be used for research. It offered a non-trial setting to examine common UK cancer genetics in a real-world context. METHODS: A total of 26 sites in England, Wales and Scotland, recruited samples from 7814 patients for genetic examination between 2011 and 2013. Tumour types involved were breast, colorectal, lung, prostate, ovarian cancer and malignant melanoma. Centralised molecular testing of surplus material from resections or biopsies of primary/metastatic tissue was performed, with samples examined for 3-5 genetic alterations deemed to be of key interest in site-specific cancers by the National Cancer Research Institute Clinical Study groups. RESULTS: 10 754 patients (98% of those approached) consented to participate, from which 7814 tumour samples were genetically analysed. In total, 53% had at least one genetic aberration detected. From 1885 patients with lung cancer, KRAS mutation was noted to be highly prevalent in adenocarcinoma (37%). In breast cancer (1873 patients), there was a striking contrast in TP53 mutation incidence between patients with ductal cancer (27.3%) and lobular cancer (3.4%). Vast inter-tumour heterogeneity of colorectal cancer (1550 patients) was observed, including myriad double and triple combinations of genetic aberrations. Significant losses of important clinical information included smoking status in lung cancer and loss of distinction between low-grade and high-grade serous ovarian cancers. CONCLUSION: Nationwide molecular pathology testing in a non-trial setting is feasible. The experience with SMP1 has been used to inform ongoing CRUK flagship programmes such as the CRUK National Lung MATRIX trial and TRACERx.

Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing.

BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies. METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction. RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing. CONCLUSIONS: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution. CLINICALTRIALSGOV IDENTIFIER: NCT02112357.

Detection of EGFR Variants in Plasma: A Multilaboratory Comparison of a Real-Time PCR EGFR Mutation Test in Europe.

Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of a real-time PCR EGFR mutation test (cobas EGFR Mutation Test v2) to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per milliliter. The circulating tumor DNA was extracted by a cell-free circulating DNA sample preparation kit (cobas cfDNA Sample Preparation Kit), followed by duplicate analysis with the real-time PCR EGFR mutation test (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. All specificities were 98.8% to 100.0%. Coefficients of variation indicated good intralaboratory and interlaboratory repeatability and reproducibility but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the predefined copy number and the observed semiquantitative index values, which reflect the samples' plasma mutation load. This study demonstrates an overall robust performance of the real-time PCR EGFR mutation test kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes.

Treatment and Survival Outcome of BRAF-Mutated Metastatic Colorectal Cancer: A Retrospective Matched Case-Control Study.

BACKGROUND: Somatic v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutation, present in approximately 10% of metastatic colorectal cancer (mCRC) cases, is associated with poor prognosis. Patient outcome outside of clinical trials has only been reported in small series. We report real-world data on treatment and survival for BRAF-mutated (MT) patients at a single tertiary center, compared with a matched BRAF wild type (WT) control group. PATIENTS AND METHODS: All colorectal cancer patients tested for BRAF mutation, from October 2010 to November 2014 were identified. BRAF-MT mCRC cases were compared with an age and sex-matched BRAF-WT control group. Clinicopathological data were collected and survival calculated using the Kaplan-Meier method and comparisons made using Cox regression. RESULTS: Forty-three of 503 patients (8.5%) tested had BRAF-MT mCRC and were compared with 88 BRAF-WT controls. Median overall survival (mOS) was 18.2 months for BRAF-MT and 41.1 months for BRAF-WT mCRC patients (hazard ratio, 2.74; 95% confidence interval, 1.60-4.70; P < .001). Progression-free survival for BRAF-MT and WT patients, respectively, was: 8.1 months versus 9.2 months (P = .571) first-line, 5.5 months versus 8.3 months (P = .074) second-line, and 1.8 months versus 5.6 months (P = .074) third-line. Treatment using sequential fluoropyrimidine-based doublet chemotherapy was similar between both groups. Anti-epidermal growth factor receptor (EGFR) therapy was mainly given third-line with progressive disease in 90% (n = 9 of 10) of BRAF-MT patients at first restaging. CONCLUSION: In this case-control study, the poor mOS of BRAF-MT mCRC was associated with reduced treatment benefit beyond first-line. Sequential doublet chemotherapy remains a reasonable option in appropriately selected patients. BRAF-MT patients did not benefit from anti-EGFR therapy in this study. Recruitment to clinical trials is recommended to improve outcomes in BRAF-MT mCRC.

Molecular profiling of colorectal pulmonary metastases and primary tumours: implications for targeted treatment.

This study aimed to molecularly characterise colorectal pulmonary metastases (PM) and investigate whether their molecular profiles were concordant with those of the primary tumour. Clinical data and archival formalin fixed paraffin embedded tissue samples were retrospectively collected from patients who underwent ≥ 1 pulmonary metastasectomies for colorectal cancer between 1997-2012. Primary tumour and metastatic samples were analysed using a targeted capture sequencing panel of 46 cancer-associated genes. The 5-year progression-free and overall survival rates for the 81 patients in this study were 32% (95% CI 22-42%) and 77% (95% CI 66-85%) respectively. Fifty-four patients had samples available from ≥ 1 PM, and sequencing data were successfully obtained from 33 PM from 24 patients. The most frequently mutated genes were APC (71%), KRAS (58%) and TP53 (46%). Seventy-three percent of the 15 patients with matched primary and PM samples and 6 of the 7 patients (86%) with data from ≥ 2 PM had concordant molecular profiles. The concordance for KRAS and NRAS was 100%. At our institutions, patients with resectable colorectal PM had a favourable prognosis. RAS mutations were commonly detected in PM and the molecular profiles of colorectal PM were highly concordant with the primary tumour.

A Validation Study for the Use of ROS1 Immunohistochemical Staining in Screening for ROS1 Translocations in Lung Cancer.

INTRODUCTION: The presence of ROS proto-oncogene 1, receptor tyrosine kinase gene (ROS1) rearrangements in lung cancers confers sensitivity to ROS kinase inhibitors, including crizotinib. However, they are rare abnormalities (in ∼1% of non-small cell lung carcinomas) that are typically identified by fluorescence in situ hybridization (FISH), and so screening using immunohistochemical (IHC) staining would be both cost- and time-efficient. METHODS: A cohort of lung tumors negative for other common mutations related to targeted therapies were screened to assess the sensitivity and specificity of IHC staining in detecting ROS1 gene rearrangements, enriched by four other cases first identified by FISH. A review of published data was also undertaken. RESULTS: IHC staining was 100% sensitive (95% confidence interval: 48-100) and 83% specific (95% confidence interval: 86-100) overall when an h-score higher than 100 was used. Patients with ROS1 gene rearrangements were younger and typically never-smokers, with the tumors all being adenocarcinomas with higher-grade architectural features and focal signet ring morphologic features (two of five). Four patients treated with crizotinib showed a partial response, with three also showing a partial response to pemetrexed. Three of four patients remain alive at 13, 27, and 31 months, respectively. CONCLUSION: IHC staining can be used to screen for ROS1 gene rearrangements, with patients herein showing a response to crizotinib. Patients with tumors that test positive according to IHC staining but negative according to FISH were also identified, which may have implications for treatment selection.

Focused molecular analysis of small cell lung cancer: feasibility in routine clinical practice.

BACKGROUND: There is an urgent need to identify molecular signatures in small cell lung cancer (SCLC) that may select patients who are likely to respond to molecularly targeted therapies. In this study, we investigate the feasibility of undertaking focused molecular analyses on routine diagnostic biopsies in patients with SCLC. METHODS: A series of histopathologically confirmed formalin-fixed, paraffin-embedded SCLC specimens were analysed for epidermal growth factor receptors (EGFR), KRAS, NRAS and BRAF mutations, ALK gene rearrangements and MET amplification. EGFR and KRAS mutation testing was evaluated using real time polymerase chain reaction (RT-PCR cobas(®)), BRAF and NRAS mutations using multiplex PCR and capillary electrophoresis-single strand conformation analysis, and ALK and MET aberrations with fluorescent in situ hybridization. All genetic aberrations detected were validated independently. RESULTS: A total of 105 patients diagnosed with SCLC between July 1990 and September 2006 were included. 60 (57 %) patients had suitable tumour tissue for molecular testing. 25 patients were successfully evaluated for all six pre-defined molecular aberrations. Eleven patients failed all molecular analysis. No mutations in EGFR, KRAS and NRAS were detected, and no ALK gene rearrangements or MET gene amplifications were identified. A V600E substitution in BRAF was detected in a Caucasian male smoker diagnosed with SCLC with squamoid and glandular features. CONCLUSION: The paucity of patients with sufficient tumour tissue, quality of DNA extracted and low frequency of aberrations detected indicate that alternative molecular characterisation approaches are necessary, such as the use of circulating plasma DNA in patients with SCLC.

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.

FcγRIIa and FcγRIIIa polymorphisms and cetuximab benefit in the microscopic disease.

PURPOSE: FcγR polymorphisms have been reported to enhance the immune-mediated effects of cetuximab in metastatic colorectal cancer. There are no data on the relationship between these polymorphisms and cetuximab in the early-stage setting. We performed a pharmacogenomic analysis of EXPERT-C, a randomized phase II trial of neoadjuvant CAPOX followed by chemoradiotherapy, surgery, and adjuvant CAPOX±cetuximab in high-risk, locally advanced rectal cancer. EXPERIMENTAL DESIGN: FcγRIIa-H131R and FcγRIIIa-V158F polymorphisms were analyzed on DNA from peripheral blood samples. Kaplan-Meier method and Cox regression analysis were used to calculate survival estimates and compare treatment arms. RESULTS: Genotyping was successfully performed in 105 of 164 (64%) patients (CAPOX=54, CAPOX-C=51). No deviation from the Hardy-Weinberg equilibrium or association of these polymorphisms with tumor RAS status was observed. FcγRIIa-131R (HR, 0.38; P=0.058) and FcγRIIIa-158F alleles (HR, 0.21; P=0.007) predicted improved progression-free survival (PFS) in patients treated with cetuximab. In the CAPOX-C arm, carriers of both 131R and 158F alleles had a statistically significant improvement in PFS (5 years: 78.4%; HR, 0.22; P=0.002) and overall survival (OS; 5 years: 86.4%; HR, 0.24; P=0.018) when compared with patients homozygous for 131H and/or 158V (5-year PFS: 35.7%; 5-year OS: 57.1%). An interaction between cetuximab benefit and 131R and 158F alleles was found for PFS (P=0.017) and remained significant after adjusting for prognostic variables (P=0.003). CONCLUSION: This is the first study investigating FcγRIIa and FcγRIIIa polymorphisms in patients with early-stage colorectal cancer treated with cetuximab. We showed an increased clinical benefit from cetuximab in the presence of 131R and 158F alleles.

Next-generation sequencing: a change of paradigm in molecular diagnostic validation.

Next-generation sequencing (NGS) is beginning to show its full potential for diagnostic and therapeutic applications. In particular, it is enunciating its capacity to contribute to a molecular taxonomy of cancer, to be used as a standard approach for diagnostic mutation detection, and to open new treatment options that are not exclusively organ-specific. If this is the case, how much validation is necessary and what should be the validation strategy, when bringing NGS into the diagnostic/clinical practice? This validation strategy should address key issues such as: what is the overall extent of the validation? Should essential indicators of test performance such as sensitivity of specificity be calculated for every target or sample type? Should bioinformatic interpretation approaches be validated with the same rigour? What is a competitive clinical turnaround time for a NGS-based test, and when does it become a cost-effective testing proposition? While we address these and other related topics in this commentary, we also suggest that a single set of international guidelines for the validation and use of NGS technology in routine diagnostics may allow us all to make a much more effective use of resources.

The genomic landscape of oesophagogastric junctional adenocarcinoma.

The incidence of oesophagogastric junctional (OGJ) adenocarcinoma is rising rapidly in western countries, in contrast to the declining frequency of distal gastric carcinoma. Treatment options for adenocarcinomas involving the oesophagogastric junction are limited and the overall prognosis is extremely poor. To determine the genomic landscape of OGJ adenocarcinoma, exomes of eight tumours and matched germline DNA were subjected to massively parallel DNA sequencing. Microsatellite instability was observed in three tumours which coincided with an elevated number of somatic mutations. In total, 117 genes were identified that had predicted coding alterations in more than one tumour. Potentially actionable coding mutations were identified in 67 of these genes, including those in CR2, HGF , FGFR4, and ESRRB. Twenty-nine genes harbouring somatic coding mutations and copy number changes in the MSS OGJ dataset are also known to be altered with similar predicted functional consequence in other tumour types. Compared with the published mutational profile of gastric cancers, 49% (57/117) of recurrently mutated genes were unique to OGJ tumours. TP53, SYNE1, and ARID1A were amongst the most frequently mutated genes in a larger OGJ cohort. Our study provides an insight into the mutational landscape of OGJ adenocarcinomas and confirms that this is a highly mutated and heterogeneous disease. Furthermore, we have uncovered somatic mutations in therapeutically relevant genes which may represent candidate drug targets.