Andrew Lawson

Activation of the ERK/MAPK pathway: a signature genetic defect in posterior fossa pilocytic astrocytomas

We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549–BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto‐inhibitory domains of BRAF and RAF1, which are replaced in‐frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low‐grade gliomas. Copyright

MAPK pathway activation and the origins of pediatric low-grade astrocytomas

Low‐grade astrocytomas (LGAs) are the most common type of brain tumor in children. Until recently, very little was known about the underlying biology and molecular genetics of these tumors. However, within the past year a number of studies have shown that the MAPK pathway is constitutively activated in a high proportion of LGAs. Several genetic aberrations which generate this deregulation of the MAPK pathway have been identified, most notably gene fusions between KIAA1549 and BRAF. In this review we summarize these findings, discuss how these gene fusions may arise and consider possible implications for diagnosis and treatment. J. Cell. Physiol. 222: 509–514, 2010.

RAF gene fusions are specific to pilocytic astrocytoma in a broad paediatric brain tumour cohort

Brain tumours are the most common solid tumour in children and are the primary cause of cancer-related death in children and young adults [4, 6]. The most prevalent childhood brain tumours are low-grade gliomas, specifically pilocytic astrocytomas (PAs, WHO Grade I) [1]. PAs are slow-growing tumours which are often cystic, and may occur sporadically or in association with the genetic disorder Neurofibromatosis type 1. Several recent studies including our own have identified novel KIAA1549–BRAF and SRGAP3–RAF1 gene fusions in the majority of PAs tested [3, 7, 8, 12]. In these fusions, the N-terminal autoinhibitory domains of the RAF proteins are replaced by those of KIAA1549 or SRGAP3, resulting in constitutive activation of the ERK/MAPK pathway. A recent study has suggested that the KIAA1549–BRAF fusion is more common in PAs originating in the cerebellum [5]. In low-grade glioma without RAF gene fusions there is increasing evidence for activation of the ERK/MAPK pathway through alternative mechanisms, such as point mutation of KRAS or BRAF [2, 11, 13]. Despite the high frequency of RAF gene fusions in PAs, they have not been investigated in other types of paediatric brain tumours. In this study, we screened a new cohort of 74 paediatric brain tumours, with a range of different pathologies, for all known KIAA1549–BRAF and SRGAP3–RAF1 fusion variants. Access to tumours and clinical data was in accordance with Local Research Ethics Committee (LREC) regulations: Great Ormond Street Hospital LREC reference number 05/Q0508/153. Tumours were classified by diagnostic criteria defined by the World Health Organization (WHO) [10]. Total RNA was extracted from fresh frozen tissue samples using the miRNeasy mini kit (Qiagen, Crawley, UK) and reverse transcribed using the SuperScript First-Strand cDNA synthesis system (Invitrogen, Carlsbad, CA). KIAA1549–BRAF fusions were detected using previously described primers and techniques [3]. The primers used for detecting SRGAP3–RAF1 fusions were 50-TGG CAGTAACCTCATCACCA-30 (located in SRGAP3 exon 10) and 50-GGTTGGGTCGACAACCTTTA-30 (located in RAF1 exon 11). All fusions identified by PCR were confirmed by direct sequencing on a 3100 Genetic Analyzer capillary sequencer (Applied Biosystems, Foster City, CA). Electronic supplementary material The online version of this article (doi:10.1007/s00401-010-0693-y) contains supplementary material, which is available to authorized users.

RAF gene fusion breakpoints in pediatric brain tumors are characterized by significant enrichment of sequence microhomology

Gene fusions involving members of the RAF family of protein kinases have recently been identified as characteristic aberrations of low-grade astrocytomas, the most common tumors of the central nervous system in children. While it has been shown that these fusions cause constitutive activation of the ERK/MAPK pathway, very little is known about their formation. Here, we present a detailed analysis of RAF gene fusion breakpoints from a well-characterized cohort of 43 low-grade astrocytomas. Our findings show that the rearrangements that generate these RAF gene fusions may be simple or complex and that both inserted nucleotides and microhomology are common at the DNA breakpoints. Furthermore, we identify novel enrichment of microhomologous sequences in the regions immediately flanking the breakpoints. We thus provide evidence that the tandem duplications responsible for these fusions are generated by microhomology-mediated break-induced replication (MMBIR). Although MMBIR has previously been implicated in the pathogenesis of other diseases and the evolution of eukaryotic genomes, we demonstrate here that the proposed details of MMBIR are consistent with a recurrent rearrangement in cancer. Our analysis of repetitive elements, Z-DNA and sequence motifs in the fusion partners identified significant enrichment of the human minisatellite conserved sequence/χ-like element at one side of the breakpoint. Therefore, in addition to furthering our understanding of low-grade astrocytomas, this study provides insights into the molecular mechanistic details of MMBIR and the sequence of events that occur in the formation of genomic rearrangements.

Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA.

Background Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by the T790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib when T790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advanced EGFR-mutant NSCLC patients. Material and methods ctDNA T790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48 EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. Progressing T790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage of T790M positive in ctDNA. Results The ctDNA T790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4–NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. Conclusion(s) ctDNA from liquid biopsy can be used as a surrogate marker for T790M in tumour tissue.

Molecular analysis of pediatric brain tumors identifies microRNAs in pilocytic astrocytomas that target the MAPK and NF-κB pathways

IntroductionPilocytic astrocytomas are slow-growing tumors that usually occur in the cerebellum or in the midline along the hypothalamic/optic pathways. The most common genetic alterations in pilocytic astrocytomas activate the ERK/MAPK signal transduction pathway, which is a major driver of proliferation but is also believed to induce senescence in these tumors. Here, we have conducted a detailed investigation of microRNA and gene expression, together with pathway analysis, to improve our understanding of the regulatory mechanisms in pilocytic astrocytomas.ResultsPilocytic astrocytomas were found to have distinctive microRNA and gene expression profiles compared to normal brain tissue and a selection of other pediatric brain tumors. Several microRNAs found to be up-regulated in pilocytic astrocytomas are predicted to target the ERK/MAPK and NF-κB signaling pathways as well as genes involved in senescence-associated inflammation and cell cycle control. Furthermore, IGFBP7 and CEBPB, which are transcriptional inducers of the senescence-associated secretory phenotype (SASP), were also up-regulated together with the markers of senescence and inflammation, CDKN1A (p21), CDKN2A (p16) and IL1B.ConclusionThese findings provide further evidence of a senescent phenotype in pilocytic astrocytomas. In addition, they suggest that the ERK/MAPK pathway, which is considered the major driver of these tumors, is regulated not only by genetic aberrations but also by microRNAs.

Abstract 3639: Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay

Circulating tumor DNA (ctDNA) is becoming established as a tool to supplement conventional biopsies for molecular characterization and monitoring of solid cancers, especially for cancers where tumor tissue is difficult to obtain or is only available at limiting quantity. This requires reliable identification, in patient plasma, of tumor-specific DNA alterations that in some cases may be present as a small fraction of the total cell-free DNA molecules. To overcome these technical challenges, we have developed an enhanced platform for tagged-amplicon deep sequencing (TAm-Seq™). Using a combination of efficient library preparation and statistically-based analysis algorithms, this platform can be used to sequence, identify and quantify cancer mutations across a gene panel including both cancer hotspots, as well as entire coding regions of selected genes. This poster will present validated performance specifications of this multi-gene ctDNA sequencing assay. To perform analytical validation, we used reference standards and plasma DNA controls to demonstrate the sensitivity, specificity and quantitative accuracy of this ctDNA analysis platform. We found that our workflow, using 4 mL input plasma, yields very high sensitivity for variants that are present at allele fraction 0.25% or higher in plasma, and retains substantial sensitivity at allele fractions as low as 0.1%. Using dilution mixtures of well-characterised reference samples, we show that the assay accurately quantifies allele fractions with precision predominantly limited by stochastic sampling. Analysis of plasma samples from control individuals demonstrates a low false positive rate. The assay also detects DNA amplifications (including in ERBB2, MYC, KRAS, EGFR, MET, FGFR1, FGFR2) when the ctDNA are sufficiently high. Together, these data demonstrate the analytical validity and robustness of the TAm-Seq assay and support its use as a basis for clinical applications. Citation Format: Davina Gale, Vincent Plagnol, Andrew Lawson, Michelle Pugh, Sarah Smalley, Karen Howarth, Mikidache Madi, Bradley Durham, Vasudev Kumanduri, Kitty Lo, James Clark, Emma Green, Nitzan Rosenfeld, Tim Forshew. Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay. [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 3639.

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA

Introduction Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. Materials and methods We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. Results The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. Conclusions These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.

Abstract 3192: Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer patients lacking tissue samples

Introduction: Approximately 30% of patients with an adenocarcinoma of the lung have an actionable driver mutation. Further understanding the molecular mechanisms of acquired resistance to targeted therapies provides key information for determining subsequent treatment options. Access to tumor tissue to perform either the initial molecular profile or at the point of acquired resistance, however, is often limited. Circulating tumor DNA (ctDNA) can be used as a minimally invasive method for the detection and quantification of molecular abnormalities. We performed a prospective study to assess molecular alterations in the ctDNA of NSCLC patients in whom the initial molecular profile or profile at acquired resistance was unknown due to lack of tumor tissue biopsy or insufficient cellularity in the biopsy. Methods: Plasma samples were collected from 52 pre-treated advanced NSCLC patients at the Gustave Roussy. DNA was extracted from TM assay covering regions from 35 cancer-related genes. Sequences were generated using Illumina sequencing. We also analysed plasma taken following treatments prescribed after the original molecular profile detected using plasma ctDNA. Results: From July 2015 to October 2015, 52 patients were included (63% female, 37% never-smoker, 95% diagnosed with an adenocarcinoma subtype, 95% with stage IV disease, and 54% had EGFR mutant tumors of which 68% had mutations in exon 19 and 32% had mutations in exon 21). ctDNA profiling was successfully performed for all patients, and mutations were detected in 38 of 52 patients. The median number of mutations detected in plasma samples was 1. Within the EGFR mutant subpopulation, T790M mutations were identified including 8 acquired cases (with a concomitant C797S mutation in 1 case) and 1 primary T790M mutation. Of these patients, 5 started personalised treatment with AZD9291 based on the results of ctDNA analysis. In the other 18 patients with EGFR mutant tumors, no acquired mutations associated with resistance were detected. Other results encompassed: 2 plasma samples with EGFR mutation exon 18 (G719A, G719C) leading to initiation of afatinib in one case, 1 case with EGFR mutation exon 21 (L861Q), 1 patients with ERBB2 exon 20 insertion, 3 KRAS mutant detected in plasma (G12C, G12S, G12F), 2 STK11 mutant samples, and 1 patient with a MET mutation (exon 14) who subsequently started crizotinib. Conclusions: ctDNA analysis with Inivata9s enhanced TAm-Seq™ provides an alternative method of ‘liquid biopsy’ for obtaining molecular profile of mutations present in NSCLC patients in the absence of an invasive tissue biopsy. Liquid biopsy identified cancer mutations in 73% of the study population, and 18% of those patients subsequently received treatment tailored to the plasma ctDNA detected mutations. An update on the analysis of 75 patients will be presented during the conference. Citation Format: Jordi Remon, Jean Charles Soria, David Planchard, Cecile Jovelet, Chloe Pannet, Ludovic Lacroix, Annas Gazzah, Andrew Lawson, Sarah Smalley, Kenth Howarth, David Gale, Emma Green, Vincent Plagnol, Nitzan Rosenfeld, Ken Oulassen, Nathalie Chaput, Benjamin Besse. Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer patients lacking tissue 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 3192.

Abstract B7: Assessing the clinical applications of ctDNA in patients with advanced stage metastatic cancer using our enhanced TAm-Seq platform

We have used Inivata9s enhanced tagged-amplicon deep sequencing (TAm-Seq™) platform to harness the power of circulating tumor DNA (ctDNA) to accurately identify low level mutations in multiple cancers, monitor the progression of disease and track patient response to therapy. We have developed an improved TAm-Seq platform, which enables detection of point mutations and indels in ctDNA with high levels of sensitivity and specificity. Our standardised TAm-Seq platform has a rapid turnaround time and is performed on plasma obtained from blood collected in a single 9mL standard EDTA tube. The TAm-Seq assay is able to analyze regions of interest (either hotspots or entire coding regions) in 35 distinct genes in the human genome with such sensitivity as to detect mutations at an allele frequency less than 1% with unpublished data demonstrating sensitivity as low as 0.1%. We performed longitudinal analyses on a series of case studies in different types of cancer to determine molecular profiles and monitor treatment response. Patient A is a stage IV NSCLC patient with bone metastases. The patient was unresponsive to initial treatment, with a suspected T790M EGFR mutation. Patient B is a BRAF V600E-positive cerebral anaplastic xanthoastrocytoma patient with first-line treatment of surgery and radiotherapy, presenting at relapse with rare multiple extra-cranial (lymph node and bone) metastases. An initial cohort of non-small cell lung cancer (NSCLC) patients, including the Patient A case study, had blood drawn at 2 time points (Day 1 and Day 21) which were analysed using TAm-Seq demonstrating clinical response correlation to reduction in mutant alleles between pre- and post-treatment. In addition to this, Patient A was confirmed by TAm-Seq as having the suspected T790M EGFR mutation at a 0.57% frequency. As tumor biopsy tissue was unavailable for testing resistant mutations in this instance, the detection of the T790M mutation by TAm-Seq enabled the patient to become eligible to receive AZD9291 off-label by special permission. The patient went on to show a complete metabolic response on PET scan. Patient B, previously presenting with a BRAF V600E mutant xanthoastrocytoma, was initiated off-label vemurafenib with ctDNA analysis detecting mutation frequency at 3.45% prior to treatment and 2.16% following 2 weeks of treatment. Follow-up samples have been collected at multiple time points for patient A and B with testing in progress. Additional case studies are in progress and all data will be presented. This work demonstrates the potential utility of using ctDNA to identify resistance mutations and monitor treatment response of a tumor when a conventional biopsy is unobtainable. It highlights the importance of high sensitivity mutation detection and analyzing multiple genomic regions, as such a high proportion of ctDNA mutations in our data were present at an allele frequency of Citation Format: Sarah K. Smalley, Davina Gale, Andrew RJ Lawson, Jordi Remon-Masip, Esperanza Perez, Michelle Pugh, Karen Howarth, Mehdi Touat, Jean-Charles Soria, Antoine Hollebecque, Benjamin Besse, Tim Forshew, Vincent Plagnol, Ludovic Lacroix, Nitzan Rosenfeld. Assessing the clinical applications of ctDNA in patients with advanced stage metastatic cancer using our enhanced TAm-Seq platform. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B7.