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The mechanistic target of rapamycin (mTOR) protein kinase coordinates responses to nutrients and growth factors and is an anti-cancer drug target. To anticipate how cells will respond and adapt to chronic mTOR complex (mTORC)1 and mTORC2 inhibition, we have generated SW620 colon cancer cells with acquired resistance to the ATP-competitive mTOR kinase inhibitor AZD8055 (SW620:8055R). AZD8055 inhibited mTORC1 and mTORC2 signalling and caused a switch from cap-dependent to internal ribosome entry site (IRES)-dependent translation in parental SW620 cells. In contrast, SW620:8055R cells exhibited a loss of S6K signalling, an increase in expression of the eukaryotic translation initiation factor eIF4E and increased cap-dependent mRNA translation. As a result, the expression of CCND1 and MCL1, proteins encoded by eIF4E-sensitive and cap-dependent transcripts, was refractory to AZD8055 in SW620:8055R cells. RNAi-mediated knockdown of eIF4E reversed acquired resistance to AZD8055 in SW620:8055R cells; furthermore, increased expression of eIF4E was sufficient to reduce sensitivity to AZD8055 in a heterologous cell system. Finally, although the combination of MEK1/2 inhibitors with mTOR inhibitors is an attractive rational drug combination, SW620:8055R cells were actually cross-resistant to the MEK1/2 inhibitor selumetinib (AZD6244). These results exemplify the convergence of ERK1/2 and mTOR signalling at eIF4E, and the key role of eIF4E downstream of mTOR in maintaining cell proliferation. They also have important implications for therapeutic strategies based around mTOR and the MEK1/2–ERK1/2 pathway.

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.

11533Background: Effective and non-invasive methods for actionable molecular alterations in NSCLC patients (pts) are urgently needed. Molecular profiling is often limited by access to sufficient tu...

For many years, it was assumed that gene fusions were a type of mutation confined largely to leukemias and sarcomas. However, fusion genes are now known to be important in several epithelial cancers and a number have been described in breast cancers. In the December 2011 issue of Nature Medicine, Robinson and colleagues reported many more gene fusions -including the first recurrent fusion, SEC16A-NOTCH1 - in breast cancers. Several genes, including members of the MAST (microtubule-associated serine threonine) kinase and Notch gene families, are fused more than once. This finding supports an emerging story that most breast cancers express a number of fusion genes.

Reciprocal chromosome translocations are often not exactly reciprocal. Most familiar are deletions at the breakpoints, up to megabases in extent. We describe here the opposite phenomenon—duplication of tens or hundreds of kilobases at the breakpoint junction, so that the same sequence is present on both products of a translocation. When the products of the translocation are mapped on the genome, they overlap. We report several of these “overlapping-breakpoint” duplications in breast cancer cell lines HCC1187, HCC1806, and DU4475. These lines also had deletions and essentially balanced translocations. In HCC1187 and HCC1806, we identified five cases of duplication ranging between 46 kb and 200 kb, with the partner chromosome showing deletions between 29 bp and 31 Mb. DU4475 had a duplication of at least 200 kb. Breakpoints were mapped using array painting, i.e., hybridization of chromosomes isolated by flow cytometry to custom oligonucleotide microarrays. Duplications were verified by fluorescent in situ hybridization (FISH), PCR on isolated chromosomes, and cloning of breakpoints. We propose that these duplications are the counterpart of deletions and that they are produced at a replication bubble, comprising two replication forks with the duplicated sequence in between. Both copies of the duplicated sequence would go to one daughter cell, on different products of the translocation, while the other daughter cell would show deletion. These duplications may have been overlooked because they may be missed by FISH and array-CGH and may be interpreted as insertions by paired-end sequencing. Such duplications may therefore be quite frequent.

Rearrangements of the genome can be detected by microarray methods and massively parallel sequencing, which identify copy-number alterations and breakpoint junctions, but these techniques are poorly suited to reconstructing the long-range organization of rearranged chromosomes, for example, to distinguish between translocations and insertions. The single-DNA-molecule technique HAPPY mapping is a method for mapping normal genomes that should be able to analyse genome rearrangements, i.e. deviations from a known genome map, to assemble rearrangements into a long-range map. We applied HAPPY mapping to cancer cell lines to show that it could identify rearrangement of genomic segments, even in the presence of normal copies of the genome. We could distinguish a simple interstitial deletion from a copy-number loss at an inversion junction, and detect a known translocation. We could determine whether junctions detected by sequencing were on the same chromosome, by measuring their linkage to each other, and hence map the rearrangement. Finally, we mapped an uncharacterized reciprocal translocation in the T-47D breast cancer cell line to about 2 kb and hence cloned the translocation junctions. We conclude that HAPPY mapping is a versatile tool for determining the structure of rearrangements in the human genome.

It has been recognised for some time that reciprocal chromosome translocation breakpoints are often complex. Most familiar are net deletions of material at the breakpoints, up to megabases in extent. We describe here the opposite phenomenondduplication of tens of kilobases of sequence so that the same sequence is present on both products of a translocation and the breakpoints overlap. Breakpoints from three breast cancer cell lines were determined by array painting. In array painting, chromosomes are isolated by flow cytometry and hybridized to DNA microarrays. This showed eighteen approximately balanced translocations. In six cases, all breakpoint junctions were mapped to 2-kb resolution using high-resolution array painting, FISH with fosmids and PCR on sorted chromosomes. A total of four translocations, in two of the cell lines, showed overlap of breakpoints, ranging in size from 46 kb to 200 kb. By SNP analysis, the duplicated sequences derived from the same chromosome. Of the remaining twelve translocations mapped to lower resolution, two reciprocal translocations showed large overlapping breakpoints of around 1.5 Mb. In most cases, knowledge of the overlap was necessary for correct interpretation of gene targets. Several of the overlapping breakpoints broke genes, and a fusion was formed between CTCF and SCUBE2. These overlaps would generally be missed by conventional FISH analysis. We propose that these duplicated sequences could arise through errors at replication, possibly by the production of stalled replication ‘bubbles’, rather than single replication forks. If translocations form during replication, the gains we have seen could be the counterpart of the previously described losses of sequence: of the two daughter cells formed, one would show gain and the other loss at the breakpoint(s). IDENTIFYING PROGNOSTIC MARKERS FOR HIGH GRADE NON-MUSCLE INVASIVE BLADDER CANCER

Introduction BRAF is a confirmed therapeutic target in non–small cell lung cancer (NSCLC), as the BRAF inhibitor dabrafenib, in combination with the MEK inhibitor trametinib, is approved for the treatment of NSCLC harbouring BRAF V600E mutation. Scant evidence is available concerning the mechanisms of resistance to BRAF/MEK inhibitors in BRAFV600E NSCLC. Patients and methods Patients with BRAFV600E NSCLC with acquired resistance to BRAF/MEK inhibitors were included in the institutional, prospective MATCH-R (from "Matching Resistance") trial and underwent tumour and liquid biopsies at the moment of radiological progression. Extensive molecular analyses were performed, including targeted next-generation sequencing (NGS), whole-exome sequencing (WES), RNA sequencing and comparative genomic hybridisation (CGH) array. Results Of the 11 patients included, eight had progressed on dabrafenib-trametinib combination, two on dabrafenib monotherapy and one on vemurafenib (BRAF inhibitor). Complete molecular analyses were available for seven patients, whereas an additional case had only targeted NGS and CGH array data. Among these eight patients, acquired molecular events potentially responsible for resistance were detected in three who progressed on dabrafenib-trametinib combination, that is, MEK1 K57N, RAS viral (v-ras) oncogene homolog (NRAS) Q61R and rat sarcoma viral oncogene homolog (KRAS) Q61R mutations. One patient progressing on dabrafenib monotherapy developed a PTEN frameshift mutation. No molecular hints addressing resistance emerged in the remaining four patients with analyses performed. Tumour mutational burden, evaluated by WES in seven patients, was low (median = 2.06 mutations/megabase, range = 1.57–3.75 mut/Mb). Conclusions Novel resistance mechanisms to BRAF/MEK inhibitors in BRAFV600E NSCLC were identified, pointing out the recurring involvement of the MAPK pathway and guiding the development of new treatment strategies.

Introduction: In patients with oncogene-addicted NSCLC and isolated central nervous system progression (iCNS), tissue biopsy is challenging, and the clinical utility of plasma liquid biopsy (i.e., circulating tumor DNA [ctDNA]) is unknown.Methods: Patients with advanced NSCLC with known baseline genomic alteration (GA) (EGFR, ALK, BRAF, KRAS, HER2, ROS1, MET, PIK3CA, STK11, TP53) on tissue were divided into three groups on the basis of their disease progression pattern: iCNS, extra-CNS only (noCNS), or both (cCNS). All patients with available plasma ctDNA were included and were analyzed by next-generation sequencing InVisionFirst-Lung. ctDNA was considered positive if at least one GA was detected. Cell-free tumor DNA was analyzed in cerebrospinal fluid when available.Results: Out of 517 patients screened, 247 were included: 54 had iCNS, 99 had noCNS, and 94 had cCNS progressive disease (64, 128, and 110 ctDNA samples, respectively). CtDNA was positive in 52% iCNS versus 84% in noCNS and 92% in cCNS (p Conclusions: Although tagged amplicon-based nex-tgeneration sequencing has high detection rates of GA in plasma ctDNA in patients with NSCLC with extra-CNS disease, detection rate of GAs (52%) is lower in the subset of patients with iCNS disease. Complementary tests such as cerebrospinal fluid cell-free DNA may be useful. Further evidence would be beneficial to understand the genomic landscape in patients with NSCLC and iCNS. (C) 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Background It has recently emerged that common epithelial cancers such as breast cancers have fusion genes like those in leukaemias. In a representative breast cancer cell line, ZR-75-30, we searched for fusion genes, by analysing genome rearrangements. Results We first analysed rearrangements of the ZR-75-30 genome, to around 10kb resolution, by molecular cytogenetic approaches, combining array painting and array CGH. We then compared this map with genomic junctions determined by paired-end sequencing. Most of the breakpoints found by array painting and array CGH were identified in the paired end sequencing—55% of the unamplified breakpoints and 97% of the amplified breakpoints (as these are represented by more sequence reads). From this analysis we identified 9 expressed fusion genes: APPBP2-PHF20L1, BCAS3-HOXB9, COL14A1-SKAP1, TAOK1-PCGF2, TIAM1-NRIP1, TIMM23-ARHGAP32, TRPS1-LASP1, USP32-CCDC49 and ZMYM4-OPRD1. We also determined the genomic junctions of a further three expressed fusion genes that had been described by others, BCAS3-ERBB2, DDX5-DEPDC6/DEPTOR and PLEC1-ENPP2. Of this total of 12 expressed fusion genes, 9 were in the coamplification. Due to the sensitivity of the technologies used, we estimate these 12 fusion genes to be around two-thirds of the true total. Many of the fusions seem likely to be driver mutations. For example, PHF20L1, BCAS3, TAOK1, PCGF2, and TRPS1 are fused in other breast cancers. HOXB9 and PHF20L1 are members of gene families that are fused in other neoplasms. Several of the other genes are relevant to cancer—in addition to ERBB2, SKAP1 is an adaptor for Src, DEPTOR regulates the mTOR pathway and NRIP1 is an estrogen-receptor coregulator. Conclusions This is the first structural analysis of a breast cancer genome that combines classical molecular cytogenetic approaches with sequencing. Paired-end sequencing was able to detect almost all breakpoints, where there was adequate read depth. It supports the view that gene breakage and gene fusion are important classes of mutation in breast cancer, with a typical breast cancer expressing many fusion genes.