Fusions involving the oncogenic gene RET have been observed in thyroid and lung cancers. Here we report RET gene alterations, including amplification, missense mutations, known fusions, novel fusions, and rearrangements in breast cancer. Their frequency, oncogenic potential, and actionability in breast cancer are described. Two out of eight RET fusions (NCOA4-RET and a novel RASGEF1A-RET fusion) and RET amplification were functionally characterized and shown to activate RET kinase and drive signaling through MAPK and PI3K pathways. These fusions and RET amplification can induce transformation of non-tumorigenic cells, support xenograft tumor formation, and render sensitivity to RET inhibition. An index case of metastatic breast cancer progressing on HER2-targeted therapy was found to have the NCOA4-RET fusion. Subsequent treatment with the RET inhibitor cabozantinib led to a rapid clinical and radiographic response. RET alterations, identified by genomic profiling, are promising therapeutic targets and are present in a subset of breast cancers.

BackgroundIn humans, imaging of tumors provides rapid, accurate assessment of tumor growth and location. In laboratory animals, however, the imaging of spontaneously occurring tumors continues to pose many technical and logistical problems. Recently a mouse model was generated in which a chimeric protein consisting of HIF-1α oxygen-dependent degradation domain (ODD) fused to luciferase was ubiquitously expressed in all tissues. Hypoxic stress leads to the accumulation of ODD-luciferase in the tissues of this mouse model which can be identified by non-invasive bioluminescence measurement. Since solid tumors often contain hypoxic regions, we performed proof-of-principle experiments testing whether this transgenic mouse model may be used as a universal platform for non-invasive imaging analysis of spontaneous solid tumors.Methods and Materials ODD-luciferase transgenic mice were bred with MMTV-neu/beclin1+/− mice. Upon injection of luciferin, bioluminescent background of normal tissues in the transgenic mice and bioluminescent signals from spontaneously mammary carcinomas were measured non-invasively with an IVIS Spectrum imaging station. Tumor volumes were measured manually and the histology of tumor tissues was analyzed.ConclusionOur results show that spontaneous mammary tumors in ODD-luciferase transgenic mice generate substantial bioluminescent signals, which are clearly discernable from background tissue luminescence. Moreover, we demonstrate a strong quantitative correlation between the bioluminescent tumor contour and the volume of palpable tumors. We further demonstrate that shrinkage of the volume of spontaneous tumors in response to chemotherapeutic treatment can be determined quantitatively using this system. Finally, we show that the growth and development of spontaneous tumors can be monitored longitudinally over several weeks. Thus, our results suggest that this model could potentially provide a practical, reliable, and cost-effective non-invasive quantitative method for imaging spontaneous solid tumors in mice.

Background Biomarkers predicting tumor response are important to emerging targeted therapeutics. Complimentary methods to assess and understand genetic changes and heterogeneity within only few cancer cells in tissue will be a valuable addition for assessment of tumors such as prostate cancer that often have insufficient tumor for next generation sequencing in a single biopsy core. Methods Using confocal microscopy to identify cell-to-cell relationships in situ, we studied the most common gene rearrangement in prostate cancer (TMPRSS2 and ERG) and the tumor suppressor CHD1 in 56 patients who underwent radical prostatectomy. Contingency tables and the chi-square test were used to determine associations between mutation status of the genes and tumor phenotype. Results Wild type ERG was found in 22 of 56 patients; ERG copy number was increased in 10/56, and ERG rearrangements confirmed in 24/56 patients. In 24 patients with ERG rearrangements, the mechanisms of rearrangement were heterogeneous, with deletion in 14/24, a split event in 7/24, and both deletions and split events in the same tumor focus in 3/24 patients. Overall, 14/45 (31.1%) of patients had CHD1 deletion, with the majority of patients with CHD1 deletions (13/14) correlating with ERG-rearrangement negative status (p< 0.01). Conclusions These results demonstrate the ability of confocal microscopy and FISH to identify the cell-to-cell differences in common gene fusions such as TMPRSS2–ERG that may arise independently within the same tumor focus. These data support the need to study complimentary approaches to assess genetic changes that may stratify therapy based on predicted sensitivities.

Background: Prostate cancer (PCa) phenotypes vary from indolent to aggressive. Molecular subtyping may be useful in predicting aggressive cancers and directing therapy. One such subtype involving deletions of chromodomain helicase DNA binding protein 1 ( CHD1), a tumor suppressor gene, are found in 10–26% of PCa tumors. In this study, we evaluate the functional cellular effects that follow CHD1 deletion. Methods: CHD1 was knocked out (KO) in the non-tumorigenic, human papillomavirus 16 (HPV16)-immortalized prostate epithelial cell line, RWPE-1, using CRISPR/Cas9. In vitro assays such as T7 endonuclease assay, western blot, and sequencing were undertaken to characterize the CHD1 KO clones. Morphologic and functional assays for cell adhesion and viability were performed. To study expression of extracellular matrix (ECM) and adhesion molecules, a real-time (RT) profiler assay was performed using RWPE-1 parental, non-target cells (NT2) and CHD1 KO cells. Result: Compared to parental RWPE-1 and non-target cells (NT2), the CHD1 KO cells had a smaller, rounder morphology and were less adherent under routine culture conditions. Compared to parental cells, CHD1 KO cells showed a reduction in ECM and adhesion molecules as well as a greater proportion of viable suspension cells when cultured on standard tissue culture plates and on plates coated with laminin, fibronectin or collagen I. CHD1 KO cells showed a decrease in the expression of secreted protein acidic and rich in cysteine (SPARC), matrix metalloproteinase 2 (MMP2), integrin subunit alpha 2 (ITGA2), integrin subunit alpha 5 (ITGA5), integrin subunit alpha 6 (ITGA6), fibronectin (FN1), laminin subunit beta-3 precursor (LAMB3), collagen, tenascin and vitronectin as compared to parental and NT2 cells. Conclusion: These data suggest that in erythroblast transformation specific (ETS) fusion-negative, phosphatase and tensin homolog ( PTEN) wildtype PCa, deletion of CHD1 alters cell-cell and cell-matrix adhesion dynamics, suggesting an important role for CHD1 in the development and progression of PCa.

Prostate cancer (PCa) develops into lethal disease in about 10% of men diagnosed with this malignancy. Common genomic changes are seen in PCa such as alterations in CDKN1B, RB1, TP53, PTEN, NKX3-1, MYC and androgen receptor. Oncogenic fusions involving the Ets family are seen in 50-70% PCa patients, the most common of which is TMPRSS2-ERG. TMPRSS2-ERG influences cell migration, particularly when PTEN is co-deleted or PIK3CA activating mutations are present. However, in the absence of ERG fusions, molecular drivers of the PCa phenotype are less clear but include alterations in SPOP (6-15% of PCa), SPINK1 (10%), MAP3K7 (18-38%) and CHD1 (15-27%). CHD1 (Chromo-domain Helicase DNA Binding Protein-1) is a chromatin remodeling factor with many roles including homologous recombination mediated DNA repair of double strand breaks and maintenance of genomic stability and therefore it is hypothesized to cause the predominant intrachromosomal rearrangements observed in Ets fusion negative tumors. This study was undertaken to evaluate the cellular effects that follow CHD1 deletion that could aid PCa initiation and progression. CHD1 was knocked out in the non tumorigenic, HPV16 immortalised prostate epithelial cell line, RWPE-1, using CRISPR/Cas9. Compared to parental RWPE-1 and non target cells (NT2), the CHD1 KO cells had a smaller and rounder morphology and were less adherent to tissue culture plates. To study expression of extracellular matrix and adhesion molecules, RT profiler assay was performed using RWPE-1 parental and CHD1 KO cells. CHD1 KO cells showed a decrease in the expression of SPARC, MMP2, ITGA2, ITGA5, ITGA6, FN1, LAMB3, collagen, tenascin and vitronectin as compared to parentals and NT2. Reduction in extracellular matrix and adhesion molecules and altered morphology of CHD1 KO cells as compared to parentals and NT2 cells suggest that loss of CHD1 reduces cell-cell and cell-matrix adhesion. These results suggest that in the absence of PTEN loss in Ets negative tumors, deletion of CHD1 gene could change cell adhesion dynamics ascribing a new role for CHD1 in the development and progression of PCa. Citation Format: Aparna Kareddula, Whitney Petrosky, Irina Tereshchenko, Daniel Medina, Hana Aviv, Eric Singer, Robert S. DiPaola, Kim M. Hirshfield. CHD1 as regulator of cell adhesion in Ets fusion negative prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5883. doi:10.1158/1538-7445.AM2017-5883