C-myc Amplification Research Articles

Mebendazole induces apoptosis via C-MYC inactivation in malignant ascites cell line (AGP01)

The objective of study was to examine the role of MBZ on malignant ascites cells and the involvement of C-MYC. Comet assay was used to assess the genotoxic effects of MBZ in AGP01 cells and human lymphocytes; differential staining by ethidium bromide and acridine orange, caspase 3/7 and flow cytometry assay was done to access the mechanisms of apoptosis and cell cycle analysis of MBZ in AGP01 cells. C-MYC amplification, C-MYC mRNA and C-MYC protein expression were evaluated by FISH, RT-qPCR and Western blotting, respectively. In addition, cytotoxicity of MBZ was evaluated in AGP01 and AGP01 shRNA MYC by MTT. MBZ significantly increased the damage index and no produced in human lymphocytes. MBZ caused remarkable cell cycle arrest in G0/G1 and G2/M phases at 0.5μM and 1.0 μM, respectively and induced significantly apoptosis in higher concentrations. Additionally, MBZ (0.5 μM and 1.0 μM) increased caspase 3 and 7 activities. MBZ decreased signals, C-MYC mRNA and C-MYC protein expression in AGP01 cells. MBZ induced lower cell viability in AGP01 cells compared AGP01 shRNA MYC in the same concentration. Therefore, our results show the evidence of C-MYC gene as one of the pathways by which MBZ induces cell death in gastric cancer cells.

Abstract P1-15-22: Clinicopathological significance of diversity index for c-MYC copy number variation after primary systemic therapy in breast cancer

Abstract Background: Chemotherapy can alter tumor cell populations by exerting selection pressure. This study was performed to evaluate the change in Shannon diversity index for c-MYC copy number variation after primary systemic therapy (PST) and its clinical implications in breast cancer patients treated with PST. Materials and Methods: One hundred and forty-four breast cancer patients who had residual disease after anthracycline- or anthracycline and taxane-based PST were included in the study. Associations between Shannon index for c-MYC copy number variation in pre- and post-PST breast cancer samples and clinicopathologic features of tumors as well as patient survival were analyzed. Results: Among the 119 patients with both pre- and post-PST samples available for comparison, 17 (14.3%) underwent change in c-MYC amplification status and 40 (33.6%) in c-MYC copy number gain status with most cases showing positive to negative conversion. In the whole group, Shannon index for c-MYC copy number variation was decreased in post-PST specimens compared to pre-PST specimens. Especially, the chemo-responsive group showed a more significant decrease in Shannon index after PST. However, there was no difference in diversity indices between pre- and post-PST specimens in the chemo-resistant group. c-MYC copy number gain and high Shannon indices for c-MYC copy number variation in both pre- and post-PST specimens were associated with adverse clinicopathologic features of breast cancer. In survival analyses, high Shannon indices for c-MYC copy number variation in post-PST samples as well as pre-PST samples were revealed as an independent prognostic factor for decreased disease-free survival. Upon subgroup analysis according to hormone receptor status, high Shannon indices before and after PST were associated with adverse clinical outcome in hormone receptor-positive group but not in hormone receptor-negative subgroup. Conclusions: These results suggest that a change in Shannon index for c-MYC copy number variation after PST reflects chemo-responsiveness and that Shannon index after PST can be used as a prognostic factor in breast cancer patients who receive PST. Citation Format: Kim G, Chung YR, Kim HJ, Kim M, Ahn S, Park SY. Clinicopathological significance of diversity index for c-MYC copy number variation after primary systemic therapy in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-15-22.

Mutational landscape of primary, metastatic, and recurrent ovarian cancer reveals c-MYC gains as potential target for BET inhibitors

Ovarian cancer remains the most lethal gynecologic malignancy. We analyzed the mutational landscape of 64 primary, 41 metastatic, and 17 recurrent fresh-frozen tumors from 77 patients along with matched normal DNA, by whole-exome sequencing (WES). We also sequenced 13 pairs of synchronous bilateral ovarian cancer (SBOC) to evaluate the evolutionary history. Lastly, to search for therapeutic targets, we evaluated the activity of the Bromodomain and Extra-Terminal motif (BET) inhibitor GS-626510 on primary tumors and xenografts harboring c-MYC amplifications. In line with previous studies, the large majority of germline and somatic mutations were found in BRCA1/2 (21%) and TP53 (86%) genes, respectively. Among mutations in known cancer driver genes, 77% were transmitted from primary tumors to metastatic tumors, and 80% from primary to recurrent tumors, indicating that driver mutations are commonly retained during ovarian cancer evolution. Importantly, the number, mutation spectra, and signatures in matched primary-metastatic tumors were extremely similar, suggesting transcoelomic metastases as an early dissemination process using preexisting metastatic ability rather than an evolution model. Similarly, comparison of SBOC showed extensive sharing of somatic mutations, unequivocally indicating a common ancestry in all cases. Among the 17 patients with matched tumors, four patients gained PIK3CA amplifications and two patients gained c-MYC amplifications in the recurrent tumors, with no loss of amplification or gain of deletions. Primary cell lines and xenografts derived from chemotherapy-resistant tumors demonstrated sensitivity to JQ1 and GS-626510 (P = 0.01), suggesting that oral BET inhibitors represent a class of personalized therapeutics in patients harboring recurrent/chemotherapy-resistant disease.

Clinical implications of changes in the diversity of c-MYC copy number variation after neoadjuvant chemotherapy in breast cancer

Chemotherapy can alter the makeup of a tumor cell population by exerting selection pressure. We examined the change in Shannon index, a mathematical diversity measure used in ecology, for c-MYC copy number variation (CNV) after neoadjuvant chemotherapy and evaluated its clinical significance in breast cancer. Associations between Shannon indices for c-MYC CNV in pre- and post-neoadjuvant chemotherapy breast cancer samples and clinicopathologic features of tumors as well as patient survival were analyzed in 144 patients. A change in c-MYC amplification and copy number gain status was found in 14.3% and 33.6% with most cases showing positive to negative conversion. The chemo-sensitive group showed a significant decrease in Shannon index after neoadjuvant chemotherapy. However, there was no difference in diversity indices between pre- and post-neoadjuvant chemotherapy specimens in the chemo-resistant group. In survival analyses, high Shannon indices for c-MYC CNV in post-neoadjuvant chemotherapy samples as well as those in pre-neoadjuvant chemotherapy samples were revealed as independent prognostic factors for poor disease-free survival not only in the whole group but also in the chemo-resistant subgroup. These findings suggest that a change in Shannon index for c-MYC CNV after neoadjuvant chemotherapy reflects chemo-responsiveness and that Shannon indices after neoadjuvant chemotherapy have a prognostic value in breast cancer patients who receive neoadjuvant chemotherapy.

Prognostic Significance of c-MYC Amplification in Esophageal Squamous Cell Carcinoma

We investigated the frequency of c-MYC amplification in esophageal squamous cell carcinoma (ESCC), including both stage I to II and III to IVa disease, and evaluated the correlation of c-MYC amplification with clinicopathologic variables and outcome. In 259 ESCCs resected at Zhongshan Hospital, Fudan University, from January 2007 to November 2010, c-MYC amplification was analyzed by using tissue microarray, with fluorescence in situ hybridization assay. c-MYC gene amplification was found in 43.2% (112 of 259) of patients with ESCC. Significant differences were found between c-MYC amplification and patient age(p= 0.009) and lymph node metastasis (p= 0.046). The median follow-up period was 33 months (range: 4to102 months). A survival difference was found betweenpatients with different c-MYC status. Among 112 patients with c-MYC amplification, a significantly poorer prognosis was observed, with a median disease-free survival (DFS) and overall survival (OS) of 24.0 and 31.0 months compared with 48.0 and 48.0 months, respectively, for patients without c-MYC amplification (p= 0.011 and 0.018). On univariate and multivariate analysis, site, clinical stage, lymph node metastasis, adjuvant therapy, and c-MYC amplification were associated with DFS and OS. When patients were divided into stage I to II and stage III to IV subgroups, c-MYC amplification tended to associate with poorer survival but without statistical difference (p > 0.05). c-MYC amplification was associated with age and lymph node metastasis and was an independent poor-prognostic factor for DFS and OS in the full cohort of patients with ESCC.

83P - Comparing clonality between components of combined hepatocellular carcinoma and cholangiocarcinoma by targeted sequencing

Background: Combined hepatocellular-cholangiocarcinoma (cHCC-CC) is a very rare tumor. It has two different components (hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC)) in a single mass. Although several studies have determined genetic characteristics of cHCC-CC, Next-generation sequencing (NGS) data for comparing clonality of cHCC-CC are currently unavailable. Methods: We selected four cHCC-CC cases and micro-dissected HCC, CC, and normal components separately from each case. DNA and RNA were isolated from each sample and sequenced by Oncomine Comprehensive Panel (OCP) interrogating 143 cancer genes using Ion S5 XL sequence platform. Genetic features of HCC and CC from each patient were compared. Results: All cases successfully produced NGS data. Two cases demonstrated different mutations between HCC and CC components (biclone) while two cases shared the same mutations between two components (monoclone). SNPs of TP53 (4/4) and PTEN (1/4) and gene amplifications of MET (1/4), c-MYC (1/4), and CDK6 (1/4) were found in CC component. In HCC component, SNPs of TP53 (3/4), PTEN (1/4) and CTNNB1 (1/4) and CCND1 amplification (1/4) were detected. Two biclonal cases showed histologically distinguished border between HCC and CC components with or without intermediate cell foci. Two monoclonal cases showed histologically ambiguous border between HCC and CC components with more intermingled pattern than biclonal cases. Conclusions: We compared genetic compositions of HCC and CC components and matched clonality with histologic feature in cHCC-CC using targeted sequencing. Two (50%) of four cases had different clones between HCC and CC components with more distinguished histologic features than monoclonal cases. Considering such heterogeneity, partly sequencing is recommended for cHCC-CC, especially in those who have histologically distinguished HCC and CC components regardless of the presence of intermediate component. Legal entity responsible for the study: Nara Yoon. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Detection of CCND1 , C-MYC , and FGFR1 amplification using modified SYBR Green qPCR and FISH in breast cancer

The aims of this study were to detect CCND1 , C-MYC , and FGFR1 amplification using qPCR, confirmation with FISH, and to further assess their clinicopathological relevance. Thirty-five breast tumor samples were analyzed for amplification of the selected genes using modified SYBR Green qPCR. The accuracy of the qPCR was assessed by FISH as a gold-standard method. CCND1 , C-MYC , and FGFR1 amplifications were observed in 34.28%, 28.57%, and 17.14% of the 35 samples, respectively. qPCR results were significantly confirmed by FISH and qPCR and FISH showed excellent correlation (P = 0.000). CCND1 amplification with tumor stage (P = 0.044), positive metastatic status (P = 0.042), positive family history (P = 0.042), and C-MYC status (P = 0.005); C-MYC amplification with tumor size (P = 0.021), tumor grade (P = 0.018), tumor stage (P = 0.032), and FGFR1 status (P < 0.000); and FGFR1 amplification with tumor size (P = 0.041) and positive ER status (P = 0.042) were statistically associated. Our findings revealed that the applied qPCR approach could precisely quantify the relative gene copy number. More studies with a larger sample size are suggested to confirm the clinicopathological value of CCND1 , C-MYC , and FGFR1 amplification.

Abstract 1352: Inactivation of factors of DNA double-strand break repair by homologous recombination or non-homologous end-joining leads to frequent catastrophic genomic events in murine and human tumors

Abstract Chromothripsis and chromoanasynthesis are two forms of genomic instability leading to complex genomic rearrangements that affect one or very few chromosomes. These one-off catastrophic events play a role in numerous tumor entities as well as in some congenital diseases. The availability of murine models recapitulating both phenomena would substantially facilitate the investigation of the mechanistic aspects underlying catastrophic genomic events. Homologous recombination repair (HR) and canonical Non-Homologous-End-Joining (cNHEJ) represent the two major processes for DNA double-strand break repair in mammalian cells. Conditional inactivation of key factors of either of these two pathways, such as Brca2 for HR and Xrcc4 or Lig4 for cNHEJ in nestin-expressing or Emx1-expressing murine neural progenitor cells leads to medulloblastomas and gliomas in a p53-deficient background. We showed by whole-genome sequencing that these tumors frequently display chromothripsis or chromoanasynthesis (33 to 73% of the analyzed tumors, n= 27) and that catastrophic rearrangements drive tumor development. FISH analysis identified a link between chromoanasynthesis and increased numerical and structural aberrations and with the presence of marker chromosomes. In addition, amplifications of c-Myc and n-Myc likely facilitate catastrophic events. Detailed analysis of the microhomologies at the breakpoint junctions on the chromosomes affected by complex genomic rearrangements identified cNHEJ and alternative end-joining as likely repair processes involved in chromothripsis and chromoanasynthesis. Treatment of cells derived from the mouse tumors with inhibitors of HR and/or alternative end-joining (e.g. RAD51 and PARP inhibitors, respectively) in combination with DNA damage revealed the dependence of these tumor cells on specific repair processes and showed that these DNA repair deficiencies can be utilized for synthetic lethality approaches. Comparison of the mouse tumors with whole-genome sequencing data from human medulloblastomas (n=68) and gliomas (n=32) identified an association between chromothripsis and deficiencies in repair processes, by analyzing copy-number level aberrations affecting repair factors and mutational signatures of DNA double-strand break repair defects. This link between DNA repair deficiency and chromothripsis was further confirmed in additional tumor entities such as breast cancer (n=356) and melanoma (n=69). In analogy to the clinical use of PARP inhibitors in the context of BRCA-deficient breast cancer, our findings point towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements. Citation Format: Manasi Ratnaparkhe, John Wong, Pei-Chi Wei, Mario Hlevnjak, Paul Northcott, David T. Jones, Marcel Kool, Anna Jauch, Agata Pastorczak, Andrey Korshunov, Rajiv Kumar, Susanna M. Downing, Stefan M. Pfister, Marc Zapatka, Peter J. McKinnon, Frederick W. Alt, Peter Lichter, Aurelie Ernst. Inactivation of factors of DNA double-strand break repair by homologous recombination or non-homologous end-joining leads to frequent catastrophic genomic events in murine and human tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1352.

Combination therapy with protein kinase inhibitor H89 and Tetrandrine elicits enhanced synergistic antitumor efficacy

BackgroundTetrandrine, a bisbenzylisoquinoline alkaloid that was isolated from the medicinal plant Stephania tetrandrine S. Moore, was recently identified as a novel chemotherapy drug. Tetrandrine exhibited a potential antitumor effect on multiple types of cancer. Notably, an enhanced therapeutic efficacy was identified when tetrandrine was combined with a molecularly targeted agent. H89 is a potent inhibitor of protein kinase A and is an isoquinoline sulfonamide.MethodsThe effects of H89 combined with tetrandrine were investigated in vitro with respect to cell viability, apoptosis and autophagy, and synergy was assessed by calculation of the combination index. The mechanism was examined by western blot, flow cytometry and fluorescence microscopy. This combination was also evaluated in a mouse xenograft model; tumor growth and tumor lysates were analyzed, and a TUNEL assay was performed.ResultsCombined treatment with H89 and tetrandrine exerts a mostly synergistic anti-tumor effect on human cancer cells in vitro and in vivo while sparing normal cells. Mechanistically, the combined therapy significantly induced cancer cell apoptosis and autophagy, which were mediated by ROS regulated PKA and ERK signaling. Moreover, Mcl-1 and c-Myc were shown to play a critical role in H89/tetrandrine combined treatment. Mcl-1 ectopic expression significantly diminished H89/tetrandrine sensitivity and amplified c-Myc sensitized cancer cells in the combined treatment.ConclusionOur findings demonstrate that the combination of tetrandrine and H89 exhibits an enhanced therapeutic effect and may become a promising therapeutic strategy for cancer patients. They also indicate a significant clinical application of tetrandrine in the treatment of human cancer. Moreover, the combination of H89/tetrandrine provides new selectively targeted therapeutic strategies for patients with c-Myc amplification.

Distinct Histopathologic and Molecular Alterations in Inflammatory Bowel Disease-Associated Intestinal Adenocarcinoma: c-MYC Amplification is Common and Associated with Mucinous/Signet Ring Cell Differentiation.

Chronic idiopathic inflammatory bowel disease (IBD) is a significant risk factor for the development of intestinal adenocarcinoma. The underlying molecular alterations in IBD-associated intestinal adenocarcinoma remain largely unknown. We compared the clinicopathologic and molecular features of 35 patients with 47 IBD-associated intestinal adenocarcinomas with a consecutive series of 451 patients with sporadic colorectal carcinoma identified at our institution and published data on sporadic colorectal carcinoma. c-MYC amplification was the most frequent molecular alteration identified in 33% of IBD-associated intestinal adenocarcinoma that is a significantly higher frequency than in sporadic colorectal carcinoma (8%) (P = 0.0001). Compared to sporadic colorectal carcinoma, IBD-associated intestinal adenocarcinomas more frequently demonstrated mucinous differentiation (60% vs 25%, P < 0.001) and signet ring cell differentiation (28% vs 4%, P < 0.001). Mucinous and signet ring cell differentiation were significantly associated with the presence of c-MYC amplification (both with P < 0.05). HER2 positivity (11%), KRAS exon 2 or 3 mutation (10%), and IDH1 mutation (7%) were less commonly observed in IBD-associated intestinal adenocarcinoma. There was an association between poor survival and HER2 status with 3 of 4 patients having HER2-positive adenocarcinoma dead of disease at last clinical follow-up; however, no statistically significant survival effect was identified for any of the molecular alterations identified. We demonstrate that IBD-associated intestinal adenocarcinomas have a high frequency of c-MYC amplification that is associated with mucinous and signet ring cell differentiation. Many of the identified molecular alterations have potential therapeutic relevance, including HER2 amplification, IDH1 mutation, and low frequency KRAS mutation.

C-MYC amplification and c-myc protein expression in pancreatic acinar cell carcinomas. New insights into the molecular signature of these rare cancers.

The molecular alterations of pancreatic acinar cell carcinomas (ACCs) and mixed acinar-neuroendocrine carcinomas (MANECs) are not completely understood, and the possible role of c-MYC amplification in tumor development, progression, and prognosis is not known. We have investigated c-MYC gene amplification in a series of 35 ACCs and 4 MANECs to evaluate its frequency and a possible prognostic role. Gene amplification was investigated using interphasic fluorescence in situ hybridization analysis simultaneously hybridizing c-MYC and the centromere of chromosome 8 probes. Protein expression was immunohistochemically investigated using a specific monoclonal anti-c-myc antibody. Twenty cases had clones with different polysomies of chromosome 8 in absence of c-MYC amplification, and 5 cases had one amplified clone and other clones with chromosome 8 polysomy, while the remaining 14 cases were diploid for chromosome 8 and lacked c-MYC amplification. All MANECs showed c-MYC amplification and/or polysomy which were observed in 54% pure ACCs. Six cases (15.3%) showed nuclear immunoreactivity for c-myc, but only 4/39 cases showed simultaneous c-MYC amplification/polysomy and nuclear protein expression. c-myc immunoreactivity as well as c-MYC amplification and/or chromosome 8 polysomy was not statistically associated with prognosis. Our study demonstrates that a subset of ACCs shows c-MYC alterations including gene amplification and chromosome 8 polysomy. Although they are not associated with a different prognostic signature, the fact that these alterations are present in all MANECs suggests a role in the acinar-neuroendocrine differentiation possibly involved in the pathogenesis of MANECs.

Targeting c-Myc: JQ1 as a promising option for c-Myc-amplified esophageal squamous cell carcinoma

c-Myc amplification-induced cell cycle dysregulation is a common cause for esophageal squamous cell carcinoma (ESCC), but no approved targeted drug is available so far. The bromodomain inhibitor JQ1, which targets c-Myc, exerts anti-tumor activity in multiple cancers. However, the role of JQ1 in ESCC remains unknown. In this study, we reported that JQ1 had potent anti-proliferative effects on ESCC cells in both time- and dose-dependent manners by inducing cell cycle arrest at G1 phase, cell apoptosis, and the mesenchymal-epithelial transition. Follow-up studies revealed that both c-Myc/cyclin/Rb and PI3K/AKT signaling pathways were inactivated by JQ1, as indicated by the downregulation of c-Myc, cyclin A/E, and phosphorylated Rb, AKT and S6. Tumor suppression induced by JQ1 in c-Myc amplified or highly expressed xenografts was higher than that in xenografts with low expression, suggesting its potential role in prediction. In conclusion, targeting c-Myc by JQ1 could cause significant tumor suppression in ESCC both in vitro and in vivo. Also, c-Myc amplification or high expression might serve as a potential biomarker and provide a promising therapeutic option for ESCC.

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-β-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

Medulloblastoma with transitional features between Group 3 and Group 4 is associated with good prognosis

Medulloblastoma, the most common malignant pediatric brain tumor, is a heterogeneous disease, with the existence of at least four molecular types: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 and Group 4 tumors. The latter two groups, which can be identified by an application of multi-gene expression or methylation profiling, show sometimes ambiguous categorization and are still classified for diagnostic reason as non-SHH/non-WNT medulloblastomas in updated WHO 2016 classification. In order to better characterize non-SHH/non-WNT tumors, we applied the method based on the Nanostring nCounter Technology, using the 26 genes codeset in 68 uniformly treated medulloblastoma patients. This allowed for identification of tumors, which shared common Group 3 and Group 4 gene signatures. We recognized three transcriptional groups within non-WNT/non-SHH tumors: Group 3, Group 4 and the Intermediate 3/4 Group. Group 3, in line with previously published results, showed poor prognosis with survival rate < 40%, frequent metastases, large cell/anaplastic pathology and presence of tumors with MYCC amplification. This is in contrast to patients from the Intermediate 3/4 Group who showed the best survival rate (100%). Overall and progression free survival were better for this group than for Group 3 (p = 0.001, for both) and Group 4 (p = 0.064 and p = 0.066, respectively). Our work supports the view that within the non-WNT/non-SHH tumors different risk groups exist and that the current two groups classifier may be not sufficient for proper clinical categorization of individual patients.

Reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells.

Fallopian tube secretory epithelial cells (FTSECs) have been suggested to be the source of high-grade serous ovarian carcinoma (HGSOC). Although several genetic alterations are known to be involved in HGSOC development, the minimal requirements remain unclear. We aimed to identify oncogenic mutations indispensable for HGSOC development in a stepwise model, using immortalized FTSECs. FTSECs were isolated from clinical samples and immortalized by overexpression of cyclin D1, CDK4R24C, and hTERT. Oncogenic mutations in the p53, c-Myc, and RAS/PI3K pathways were mimicked by lentiviral transduction. We found two distinct patterns of gene alteration essential for HGSOC development: p53/KRAS/AKT and p53/KRAS/c-Myc. Dominant-negative p53, alone or combined with oncogenic KRAS (KRASV12), constitutively active AKT (CA-AKT), and c-Myc, did not induce tumorigenesis in immortalized cells; however, overexpression of CA-AKT or c-Myc, along with dominant-negative p53 and KRASV12, conferred tumorigenic potential. Transformed FTSECs formed tumors in nude mice that were grossly, histologically, and immunohistochemically similar to human HGSOCs. Interestingly, mice harboring tumors with c-Myc amplifications displayed extensive metastases, consistent with the increased dissemination in their human counterparts. Thus, aberrant p53/KRASV12/c-Myc or p53/KRASV12/PI3K-AKT signaling was the minimum requirement for FTSEC carcinogenesis. The model based on this evidence could shed light on the early stages of HGSOC development.

Genetic alterations in benign prostatic hyperplasia patients.

Background: Benign prostate hyperplasia (BPH) is a classical age-related disease of the prostate, present in 20% of men at the age of 40 years with progression to 70% by the age of 60 years. BPH is associated with various lower urinary tract symptoms, which affect their day-to-day life.Materials and methods: Our objective was to evaluate the association between HER-2/neu, c-myc, p53, and clinicopathological variables in 45 patients diagnosed with benign prostatic hyperplasia using fluorescence in situ hybridization (FISH). The patients underwent transurethral prostate resection to address their primary urological problem. All patients were evaluated by use of a comprehensive medical history and rectal digital examination. The preoperative evaluation also included serum prostate specific antigen (PSA) measurement and ultrasonographic measurement of prostate volume. Results: The mean (± standard deviation) age of the 45 patients was 69.65 ± 8.97 years. The mean PSA value of the patients was 9.25 ± 5.12 ng/mL. The mean prostate volume was 65.46 ± 11.43 mL. Amplification of HER-2/neu was seen in 4/45 (8.9%) cases and amplification of c-myc was seen in 5 of 45 (11.1%) cases; both genes were not associated with adverse clinicopathological variables. Deletion of p53 was seen in 20/45 (44.4%) cases. p53 gene was significantly associated with a severe AUASI (American Urological Association Symptom Index) score.Conclusion: In this study, we discussed important genetic markers in benign prostatic hyperplasia patients which may, in the future, be used as markers for diagnosis and prognosis, as well as targets for therapeutic intervention.

PDTM-46. TARGETING METABOLIC ADAPTATION IN MYC DRIVEN PEDIATRIC NEURONAL TUMORS

BACKGROUND/OBJECTIVES MYC family proteins are implicated in many human cancers, but their therapeutic targeting has proven challenging. MYCN and c-MYC amplification in childhood neuroblastoma (NB) and medulloblastoma (MB) are associated with aggressive disease and high mortality. Novel and effective therapeutic strategies are therefore urgently needed for these tumors. MYC-driven oncogenic transformation impairs cell survival under nutrient deprivation (ND), a characteristic stress condition within the tumor microenvironment. We recently identified eukaryotic Elongation Factor 2 Kinase (eEF2K) as a pivotal mediator of the adaptive response of tumor cells to ND. We therefore hypothesized that eEF2K facilitates the adaptation of MYCN/MYC amplified NB/MB to ND, and that inhibiting this pathway can impair tumor progression.

Aligning digital CD8+ scoring and targeted next-generation sequencing with programmed death ligand 1 expression: a pragmatic approach in early-stage squamous cell lung carcinoma.

To study programmed death ligand 1 (PD-L1) expression, tumour-infiltrating T lymphocytes (TILs) and the molecular context in patients with early-stage squamous cell lung carcinomas (SCCs). The study included samples from 40 patients (discovery cohort) and 29 patients (validation cohort) diagnosed with early-stage SCC. PD-L1 immunohistochemistry (IHC) was performed with three commercially available clones (E1L3N, SP263 and SP142). CD8+ TILs were scored with a digital algorithm. All tumours were analysed with targeted next-generation sequencing (NGS). Additionally, TP53 mutations were investigated with direct sequencing. In both cohorts, we observed a significant association between CD8+ TILs density and high PD-L1 IHC expression in tumour cells (TCs). Furthermore, high SP142 PD-L1 expression in immune cells (ICs) was also associated significantly with CD8+ TILs density. Therefore, CD8+ TILs density discriminated between patients with high versus low PD-L1 IHC expression with excellent sensitivity and specificity. Interestingly, the highest percentages of PD-L1-positive TCs with the three antibodies were found in samples with cyclin-dependent kinase 6 (CDK6) amplification, with high amplification of proto-oncogene C-Myc (CMYC) or with cyclin D1-PI3 kinase subunit alpha (CCND1-PIK3CA) co-amplification. High SP142 PD-L1 IHC expression in ICs showed a non-significant correlation with TP53 mutations. Conversely, most cases with fibroblast growth factor receptor 1 (FGFR1) amplification were negative for all PD-L1 clones. Our preliminary results support the use of digital CD8+ TILs scoring and targeted NGS alongside PD-L1 expression. The approach presented herein could help define patients with SCCs candidates to immune checkpoints inhibitors.

Genotyping tumour DNA in cerebrospinal fluid and plasma of a HER2-positive breast cancer patient with brain metastases

BackgroundCentral nervous system (CNS) involvement contributes to significant morbidity and mortality in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (mBC) and represents a major challenge for clinicians. Liquid biopsy of cerebrospinal fluid (CSF)-derived circulating tumour DNA (ctDNA) harbours clinically relevant genomic alterations in patients with CNS metastases and could be effective in tracking tumour evolution.MethodsIn a HER2-positive mBC patient with brain metastases, we applied droplet digital PCR (ddPCR) and next-generation whole exome sequencing (WES) analysis to measure ctDNA dynamic changes in CSF and plasma collected during treatment.ResultsBaseline CSF-derived ctDNA analysis revealed TP53 and PIK3CA mutations as well as ERBB2 and cMYC amplification. Post-treatment ctDNA analysis showed decreased markers level in plasma, consistent with extra-CNS disease control, while increased in the CSF, confirming poor treatment benefit in the CNS.DiscussionAnalysis of ctDNA in the CSF of HER2-positive mBC is feasible and could represent a useful companion for clinical management of brain metastases.

Effects of cell cycle regulatory genes on breast cancer neo-adjuvant chemotherapy by M-FISH

Objective: The dysregulation of cell cycle could influence cell proliferation, differentiation and response to medicine. The purpose of this study is to explore the correlation between cell cycle regulatory genes and breast cancer neo-adjuvant chemotherapy (NAC) in patients with local advanced breast cancer, and thus to find some predictors of NAC to provide guidance for clinical treatment. Methods: Ninety five cases of local advanced breast cancer were collected, which were treated with NAC of TAC (Taxanes/Anthracycline/Cyclophosphamide) regimen. Multi-gene fluorescence in situ hybridization (M-FISH) was used to study the correlation between copy number variations of cell cycle regulatory genes (c-myc, Mdm2, CCND1, CHEK2, Rb1, p53, p16, p21) and clinical effect of NAC. Results: In the effective group, there were 18 cases of c-Myc amplification and 52 cases of no amplification. There were 11 cases of CCND1 amplification and 59 cases of no amplification; 12 cases of p53 deletion and 58 cases without deletion; 11 cases of p16 deletion and 59 cases without deletion. In the ineffective group, there were 12 cases of c-Myc amplification and 13 cases of no amplification; CCND1 amplification in 9 cases, no amplification in 16 cases; p53 deletion in 10 cases, no deletion in 15 cases; p16 deletion in 10 cases and no deletion in 15 cases. The copy number of the above four genes was statistically different between the two groups. C-Myc gene amplification (P=0.040), CCND1 gene amplification (P=0.033), p53 gene deletion (P=0.020) and p16 gene deletion (P=0.012) were significant correlation with poor effect of NAC. Among them, the effect of NAC in patients with two or more genes copy number variations of c-Myc, CCND1, p53, p16 were poorer than that in patients with one or not gene copy number variations (P=0.000). In addition, the copy number variations of CCND1 (P=0.049), c-Myc (P=0.049), and p16 (P=0.008) were correlated with neo-adjuvant chemotherapeutic effect respectively in Luminal breast cancer, Her-2 positive breast cancer, and triple-negative breast cancer. Conclusion: These results indicated that the copy number variations of c-Myc, CCND1, p53, and p16 in patients were significant correlation with poor effect of NAC.