Karen Howarth

Array painting reveals a high frequency of balanced translocations in breast cancer cell lines that break in cancer-relevant genes.

Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1 Mb resolution on bacterial artificial chromosome (BAC) arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2 kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1 Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Second, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300 and FOXP4. Two gene fusions were demonstrated, TAX1BP1–AHCY and RIF1–PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer.

Tandem duplication of chromosomal segments is common in ovarian and breast cancer genomes

The application of paired‐end next generation sequencing approaches has made it possible to systematically characterize rearrangements of the cancer genome to base‐pair level. Utilizing this approach, we report the first detailed analysis of ovarian cancer rearrangements, comparing high‐grade serous and clear cell cancers, and these histotypes with other solid cancers. Somatic rearrangements were systematically characterized in eight high‐grade serous and five clear cell ovarian cancer genomes and we report here the identification of > 600 somatic rearrangements. Recurrent rearrangements of the transcriptional regulator gene, TSHZ3, were found in three of eight serous cases. Comparison to breast, pancreatic and prostate cancer genomes revealed that a subset of ovarian cancers share a marked tandem duplication phenotype with triple‐negative breast cancers. The tandem duplication phenotype was not linked to BRCA1/2 mutation, suggesting that other common mechanisms or carcinogenic exposures are operative. High‐grade serous cancers arising in women with germline BRCA1 or BRCA2 mutation showed a high frequency of small chromosomal deletions. These findings indicate that BRCA1/2 germline mutation may contribute to widespread structural change and that other undefined mechanism(s), which are potentially shared with triple‐negative breast cancer, promote tandem chromosomal duplications that sculpt the ovarian cancer genome. Copyright

Adaptation to mTOR kinase inhibitors by amplification of eIF4E to maintain cap-dependent translation.

ABSTRACT 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.

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.

Contributions to drug resistance in glioblastoma derived from malignant cells in the sub-ependymal zone

Glioblastoma, the most common and aggressive adult brain tumor, is characterized by extreme phenotypic diversity and treatment failure. Through fluorescence-guided resection, we identified fluorescent tissue in the sub-ependymal zone (SEZ) of patients with glioblastoma. Histologic analysis and genomic characterization revealed that the SEZ harbors malignant cells with tumor-initiating capacity, analogous to cells isolated from the fluorescent tumor mass (T). We observed resistance to supramaximal chemotherapy doses along with differential patterns of drug response between T and SEZ in the same tumor. Our results reveal novel insights into glioblastoma growth dynamics, with implications for understanding and limiting treatment resistance.

The Relative Timing of Mutations in a Breast Cancer Genome

Many tumors have highly rearranged genomes, but a major unknown is the relative importance and timing of genome rearrangements compared to sequence-level mutation. Chromosome instability might arise early, be a late event contributing little to cancer development, or happen as a single catastrophic event. Another unknown is which of the point mutations and rearrangements are selected. To address these questions we show, using the breast cancer cell line HCC1187 as a model, that we can reconstruct the likely history of a breast cancer genome. We assembled probably the most complete map to date of a cancer genome, by combining molecular cytogenetic analysis with sequence data. In particular, we assigned most sequence-level mutations to individual chromosomes by sequencing of flow sorted chromosomes. The parent of origin of each chromosome was assigned from SNP arrays. We were then able to classify most of the mutations as earlier or later according to whether they occurred before or after a landmark event in the evolution of the genome, endoreduplication (duplication of its entire genome). Genome rearrangements and sequence-level mutations were fairly evenly divided earlier and later, suggesting that genetic instability was relatively constant throughout the life of this tumor, and chromosome instability was not a late event. Mutations that caused chromosome instability would be in the earlier set. Strikingly, the great majority of inactivating mutations and in-frame gene fusions happened earlier. The non-random timing of some of the mutations may be evidence that they were selected.

Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes

BackgroundIt 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.ResultsWe 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.ConclusionsThis 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.

Tracking evolution of aromatase inhibitor resistance with circulating tumour DNA analysis in metastatic breast cancer

Background Selection of resistance mutations may play a major role in the development of endocrine resistance. ESR1 mutations are rare in primary breast cancer but have high prevalence in patients treated with aromatase inhibitors (AI) for advanced breast cancer. We investigated the evolution of genetic resistance to the first-line AI therapy using sequential ctDNA sampling in patients with advanced breast cancer. Patients and methods Eighty-three patients on the first-line AI therapy for metastatic breast cancer were enrolled in a prospective study. Plasma samples were collected every 3 months to disease progression and ctDNA analysed by digital droplet PCR and enhanced tagged-amplicon sequencing (eTAm-Seq). Mutations identified in progression samples by sequencing were tracked back through samples before progression to study the evolution of mutations on therapy. The frequency of novel mutations was validated in an independent cohort of available baseline plasma samples in the Study of Faslodex versus Exemestane with or without Arimidex (SoFEA) trial, which enrolled patients with prior sensitivity to AI. Results Of the 39 patients who progressed on the first-line AI, 56.4% (22/39) had ESR1 mutations detectable at progression, which were polyclonal in 40.9% (9/22) patients. In serial tracking, ESR1 mutations were detectable median 6.7 months (95% confidence interval 3.7-NA) before clinical progression. Utilising eTAm-Seq ctDNA sequencing of progression plasma, ESR1 mutations were demonstrated to be sub-clonal in 72.2% (13/18) patients. Mutations in RAS genes were identified in 15.4% (6/39) of progressing patients (4 KRAS, 1 HRAS, 1 NRAS). In SoFEA, KRAS mutations were detected in 21.2% (24/113) patients although there was no evidence that KRAS mutation status was prognostic for progression free or overall survival. Conclusions Cancers progressing on the first-line AI show high levels of genetic heterogeneity, with frequent sub-clonal mutations. Sub-clonal KRAS mutations are found at high frequency. The genetic diversity of AI resistant cancers may limit subsequent targeted therapy approaches.

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.

Are breast cancers driven by fusion genes

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.