Targetable Gene Fusions Research Articles

Abstract 4793: Rapid pan-cancer identification of previously unidentified fusion genes to enable novel targeted therapeutics

Abstract Gene fusions or rearrangements are widely recognized as a significant player in driving tumorigenesis. Several key findings implicating fusion events have been reported in hematological malignancies (e.g. BCR-ABL in CML) and more recently in solid tumors (e.g. EML4-ALK in NSCLC). Next-generation sequencing, particularly RNA-seq, has rapidly led to discoveries of novel recurrent gene fusions in a wide variety of tumors (e.g. lung, breast, bladder, etc.). While the overall frequency of specific fusion events may be considered low, they have demonstrated significant value in guiding or developing treatment options and improving clinical outcomes. The advent of large-scale public sequencing efforts such as The Cancer Genome Atlas (TCGA) provide a tremendous opportunity in implementing creative analysis approaches and expanding the therapeutic opportunities of targeting gene fusions in cancer. In this study, we describe a comprehensive survey of fusion events of a select number of genes identified across various tumor types in TCGA. The computational demands of gene fusion discovery using RNA-seq data generated across thousands of tumors pose a serious challenge in pan-cancer analyses of clinically relevant gene fusions. To address this problem, we have developed an analytical pipeline to query genes of interest for 5′ or 3′ rearrangements that drastically lowers computational cost and increases throughput, compared to existing fusion analysis pipelines. This efficiency is primarily achieved by restricting the “alignment search space” of sequenced transcript reads to a fraction of the full read count, thereby enabling tangible gains in speed without sacrificing specificity or sensitivity. We applied this approach to search for fusion events involving well characterized genes in approximately 5700 paired-end RNA-seq tumor samples from 20 different cancer types sequenced by the TCGA project, completing the analysis in around 2 months on a modest hardware setup. Our results were validated against reported findings of prevalence in the respective cancer types (e.g. ETS-family gene fusions in prostate cancer and EML4-ALK fusions in lung cancer). As a striking result, we were able to identify several novel fusion partners for known fused oncogenes. Furthermore, the newly identified and also previously known fusion genes were discovered in novel tumor types, thus expanding the fusion landscape of well-known genes. For example, some of the 27 NTRK fusion genes found were observed in other indications than previously reported. In summary, we have successfully executed a valid functional and efficient analysis pipeline to reveal oncogenic rearrangements that play a key role in the initiation and progression of cancer. Furthermore, these events molecularly define clinical subsets of disease and as such, can guide personalized targeted therapies. Citation Format: Henrik Edgren, Kalle Ojala, Anja Ruusulehto, Gopi Ganji. Rapid pan-cancer identification of previously unidentified fusion genes to enable novel targeted therapeutics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4793. doi:10.1158/1538-7445.AM2015-4793

Unique Genetic and Survival Characteristics of Invasive Mucinous Adenocarcinoma of the Lung

Invasive mucinous adenocarcinoma is a unique histologic subtype of lung cancer, and our knowledge of its genetic and clinical characteristics is rapidly evolving. Here, we present next- generation sequencing analysis of nucleotide variant and fusion events along with clinical follow-up in a series of lung mucinous adenocarcinoma. We collected 72 mucinous adenocarcinomas from the United States and Korea. All had been previously assessed for KRAS and EGFR mutations. For KRAS wild-type cases (n = 30), we performed deep targeted next-generation sequencing for gene fusions and nucleotide variants and correlated survival and other clinical features. As expected, KRAS mutations were the most common alteration found (63% of cases); however, the distribution of nucleotide position alterations was more similar to that observed in gastrointestinal tumors than other lung tumors. Within the KRAS-negative cases, we found numerous potentially targetable gene fusions and mutations, including CD74-NRG1, VAMP2-NRG1, TRIM4-BRAF, TPM3-NTRK1, and EML4-ALK gene fusions and ERBB2, BRAF, and PIK3CA mutations. Unexpectedly, we found only two cases with TP53 mutation, which is much lower than observed in lung adenocarcinomas in general. The overall mutation burden was low in histologically confirmed mucinous adenocarcinomas from the public The Cancer Genome Atlas exome data set, regardless of smoking history, suggesting a link between TP53 status and mutation burden in mucinous tumors. There was no significant difference for recurrence-free survival between stage-matched mucinous and nonmucinous adenocarcinomas. It was notable that all recurrence sites were in the lungs for completely resected cases. Our data suggest that mucinous adenocarcinoma is typified by (1) frequent KRAS mutations and a growing list of gene fusions, but rare TP53 mutations, (2) a low mutation burden overall, and (3) a recurrence-free survival similar to stage-matched nonmucinous tumors, with recurrences limited to the lungs.

Abstract P4-04-07: Heterogeneous gene fusions detected by RNASeq show enrichment of insulin signaling pathway genes in breast cancer

Abstract Background: Recent discoveries of recurrent and targetable gene fusions in breast cancer suggest the need to characterize the functional significance of such genomic aberrations within larger cohorts. We quantify fusion transcript expression in patient samples using RNASeq and evaluate their functional significance using biological pathway enrichment analysis. Methods: We sequenced transcriptomes of core biopsy RNA from 97 breast tumors obtained from brief-exposure preoperative clinical trials BrUOG 211A/211B. HER2- patients were treated with brief exposure to bevacizumab (B) or nab-paclitaxel (nP) followed by treatment with B/nP/carboplatin while HER2+ patients received brief exposure to trastuzumab (T) or nP followed by T/nP/carboplatin. Paired-end sequencing on 55 baseline biopsies and 42 post-exposure biopsies using amplified total RNA yielded 55 million reads on average per sample. We assigned RNASeq-based PAM50 subtypes for each of the samples using standard methodology. Fusion transcript abundance was evaluated using two independent pipelines, TopHat and deFuse, due to their complementary strategies in fusion detection. We eliminated fusions of genes with their respective pseudogenes as likely false positives arising due to alignment artifacts. TopHat fusion calls with total supporting reads ≥10 and deFuse calls with probability of fusion ≥0.7 were considered reliable. Results: We identified high confidence gene fusions, detected by both TopHat and deFuse, in 30 of the 55 baseline biopsies (54.4%), with 3.3 fusions on average per sample and a maximum of 10. Fusions were predominantly associated with chromosomal aberrations (75%), with putative deletions responsible for 32% of fusions and translocations responsible for 43%. We find a high level of fusion transcript heterogeneity within breast cancers, detecting a total of 80 fusions across the 30 samples with only three fusions recurrent in two samples with high expression in each: MDN1-GAS5 in two basal breast cancers, KRAS-GRIP1 and ITPR2-CCDC91 in two LumB cancers. Several cancer-related genes were found to be fusion partners: AKT3-SMYD3, CREB1-PPP1R1C, FLOT2-TOP2A and FOXC1-ARID1B. Pathway analysis of the fusion genes at baseline revealed enrichment of proteasome (p = 0.000752), tight junction (p = 0.027), insulin signaling (p = 0.0284) and melanogenesis (p = 0.05) pathways after multiple testing correction (FDR≤0.25). We looked for modulation of gene fusions upon brief exposure to therapy in 18 patients and found a majority of the baseline fusion transcripts to be present post-brief exposure in 44% of the patients, irrespective of therapy regimen. Conclusions: We find that gene fusions in breast cancer are highly heterogeneous but are enriched with cancer-related pathway genes. This is the first study to report a novel gene-lincRNA fusion transcript (MDN1-GAS5). We are currently validating the fusion calls using qRT-PCR. The heterogeneity of detected fusions suggests that multiple mechanisms could underlie the selective advantage of tumor cells expressing fusion transcripts. The brief-exposure preoperative paradigm provides a unique opportunity to evaluate modulation of fusion transcripts that can shed light on their functional importance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-07.

Identification of Targetable FGFR Gene Fusions in Diverse Cancers

Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2, including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Because of the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts, which incorporate transcriptome analysis for gene fusions, are poised to identify rare, targetable FGFR fusions across diverse cancer types.

Mast and Notch Fusions Drive a Subset of Breast Cancers

Abstract Major finding: MAST kinase and Notch gene rearrangements are present in 5%–7% of breast cancers. Approach: Breast cancer cell lines and tumors were analyzed by paired-end transcriptome sequencing. Impact: Identification of targetable gene fusions could benefit a subset of breast cancer patients.

Functionally recurrent rearrangements of the MAST kinase and Notch gene families in breast cancer

Breast cancer is a heterogeneous disease, exhibiting a wide range of molecular aberrations and clinical outcomes. Here we employed paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers harbor an array of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving microtubule associated serine-threonine kinase (MAST) and Notch family genes. Both MAST and Notch family gene fusions exerted significant phenotypic effects in breast epithelial cells. Breast cancer lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and over-expression of MAST1 or MAST2 gene fusions had a proliferative effect both in vitro and in vivo. These findings illustrate that recurrent gene rearrangements play significant roles in subsets of carcinomas and suggest that transcriptome sequencing may serve to identify patients with rare, actionable gene fusions.

Rearrangements of the RAF kinase pathway in prostate cancer, gastric cancer and melanoma

Although recurrent gene fusions involving erythroblastosis virus E26 transformation-specific (ETS) family transcription factors are common in prostate cancer, their products are considered 'undruggable' by conventional approaches. Recently, rare targetable gene fusions involving the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, have been identified in 1-5% of lung cancers, suggesting that similar rare gene fusions may occur in other common epithelial cancers, including prostate cancer. Here we used paired-end transcriptome sequencing to screen ETS rearrangement-negative prostate cancers for targetable gene fusions and identified the SLC45A3-BRAF (solute carrier family 45, member 3-v-raf murine sarcoma viral oncogene homolog B1) and ESRP1-RAF1 (epithelial splicing regulatory protein-1-v-raf-1 murine leukemia viral oncogene homolog-1) gene fusions. Expression of SLC45A3-BRAF or ESRP1-RAF1 in prostate cells induced a neoplastic phenotype that was sensitive to RAF and mitogen-activated protein kinase kinase (MAP2K1) inhibitors. Screening a large cohort of patients, we found that, although rare, recurrent rearrangements in the RAF pathway tend to occur in advanced prostate cancers, gastric cancers and melanoma. Taken together, our results emphasize the key role of RAF family gene rearrangements in cancer, suggest that RAF and MEK inhibitors may be useful in a subset of gene fusion-harboring solid tumors and demonstrate that sequencing of tumor transcriptomes and genomes may lead to the identification of rare targetable fusions across cancer types.