C-myc Amplification Research Articles

Ibrutinib in c-MYC and HER2 Amplified Oesophagogastric Carcinoma: Results of the Proof-of-Concept iMYC Study.

Oesophagogastric (OG) cancer is a highly lethal disease requiring novel treatment options. c-MYC and/or HER-2 amplified oesophageal cancer models have demonstrated sensitivity to BTK inhibition with ibrutinib. We evaluated the safety and efficacy of ibrutinib in patients with c-MYC and/or HER2 amplified pre-treated advanced OG cancer. c-MYC and HER2 amplification status were determined by FISH. The primary endpoint was overall response rate (ORR). Secondary endpoints were disease control rate (DC) at 8 weeks, safety, progression-free survival (PFS) and overall survival (OS). Eleven patients were enrolled. Eight patients had c-MYC amplified tumours, six were HER2 amplified and three were c-MYC and HER2 co-amplified. Grade ≥ 3 adverse events were fever, neutropenia, and vomiting. Grade ≥ 3 gastrointestinal haemorrhage occurred in three patients and was fatal in two cases. Among seven evaluable patients, three patients (43%) achieved a best response of SD at 8 weeks. No PR or CR was observed. Disease control was achieved for 32 weeks in one patient with a dual c-MYC and HER2 highly co-amplified tumour. The median PFS and OS were 1.5 (95% CI: 0.8–5.1) and 5.1 (95% CI: 0.8–14.5) months, respectively. Ibrutinib had limited clinical efficacy in patients with c-MYC and/or HER2 amplified OG cancer. Unexpected gastrointestinal bleeding was observed in 3 out of 8 treated patients which was considered a new safety finding for ibrutinib in this population.

Evidence for ABL Amplification in Multiple Myeloma and Therapeutic Implications.

Background Cytogenetic abnormalities are considered initiating events in the pathogenesis of multiple myeloma (MM) and are assumed to be of clinical significance. Methods Fluorescence in situ hybridization (FISH) was used to analyze chromosomal architecture in 101 patients with MM. We evaluated overall patient survival and assessed the cytotoxicity of imatinib against MM cells using a CCK8 assay. Results ABL gene amplification was detected in 67 patients (66.3%). However, ABL gene amplification was not associated with clinical features, cytogenetic abnormalities (c-Myc amplification, IGH rearrangement, RB1 deletion, p53 deletion, or 1q21 amplification), or overall survival. ABL amplification in MM cell lines (LP-1 and U266) was revealed by FISH. Furthermore, the ABL protein was easily detectable in MM cell lines and some tumor cells by western blotting. A CCK8 assay indicated limited cytotoxicity of imatinib against MM cells. Conclusions Our study firstly discussed ABL gene amplification was prevalent in MM cells, and we believe that the ABL gene would potentially be a useful target in the treatment of combination strategy for MM with ABL amplification in the future.

From the Editor’s Desk...

From the Editor’s Desk...

MiR-212-3p functions as a tumor suppressor gene in group 3 medulloblastoma via targeting nuclear factor I/B (NFIB)

Haploinsufficiency of chromosome 17p and c-Myc amplification distinguish group 3 medulloblastomas which are associated with early metastasis, rapid recurrence, and swift mortality. Tumor suppressor genes on this locus have not been adequately characterized. We elucidated the role of miR-212-3p in the pathophysiology of group 3 tumors. First, we learned that miR-212-3p undergoes epigenetic silencing by histone modifications in group 3 tumors. Restoring its expression reduced cancer cell proliferation, migration, colony formation, and wound healing in vitro and attenuated tumor burden and improved survival in vivo. MiR-212-3p also triggered c-Myc destabilization and degradation, leading to elevated apoptosis. We then isolated an oncogenic target of miR-212-3p, i.e. NFIB, a nuclear transcription factor implicated in metastasis and recurrence in various cancers. Increased expression of NFIB was confirmed in group 3 tumors and associated with poor survival. NFIB silencing reduced cancer cell proliferation, migration, and invasion. Concurrently, reduced medullosphere formation and stem cell markers (Nanog, Oct4, Sox2, CD133) were noted. These results substantiate the tumor-suppressive role of miR-212-3p in group 3 MB and identify a novel oncogenic target implicated in metastasis and tumor recurrence.

6q deletion is frequent but unrelated to patient prognosis in breast cancer

BackgroundDeletions involving the long arm of chromosome 6 have been reported to occur in breast cancer, but little is known about the clinical relevance of this alteration.MethodsWe made use of a pre-existing tissue microarray with 2197 breast cancers and employed a 6q15/centromere 6 dual-labeling probe for fluorescence in situ (FISH) analysisResultsHeterozygous 6q15 deletions were found in 202 (18%) of 1099 interpretable cancers, including 19% of 804 cancers of no special type (NST), 3% of 29 lobular cancers, 7% of 41 cribriform cancers, and 28% of 18 cancers with papillary features. Homozygous deletions were not detected. In the largest subset of NST tumors, 6q15 deletions were significantly linked to advanced tumor stage and high grade (p < 0.0001 each). 6q deletions were also associated with estrogen receptor negativity (p = 0.0182), high Ki67 proliferation index (p < 0.0001), amplifications of HER2 (p = 0.0159), CCND1 (p = 0.0069), and cMYC (p = 0.0411), as well as deletions of PTEN (p = 0.0003), 8p21 (p < 0.0001), and 9p21 (p = 0.0179). However, 6q15 deletion was unrelated to patient survival in all cancers, in NST cancers, or in subsets of cancers defined by the presence or absence of lymph-node metastases.ConclusionOur data demonstrate that 6q deletion is a frequent event in breast cancer that is statistically linked to unfavorable tumor phenotype and features of genomic instability. The absence of any prognostic impact argues against a clinical applicability of 6q15 deletion testing in breast cancer patients.

Therapeutic Potential of the Cyclin-Dependent Kinase Inhibitor Flavopiridol on c-Myc Overexpressing Esophageal Cancer.

Tumors with elevated c-Myc expression often exhibit a highly aggressive phenotype, and c-Myc amplification has been shown to be frequent in esophageal cancer. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. We therefore investigated whether exploiting elevated c-Myc expression is effective in treating esophageal cancer with the CDK inhibitor flavopiridol. We found frequent overexpression of c-Myc in human esophageal cancer cell lines and tissues. c-Myc overexpression correlated with accelerated esophageal cancer subcutaneous xenograft tumor growth. Esophageal cancer cells with elevated c-Myc expression were found preferentially more sensitive to induction of apoptosis by the CDK inhibition flavopiridol compared to esophageal cancer cells with lower c-Myc expression. In addition, we observed that flavopiridol alone or in combination with the chemotherapeutic agent nanoparticle albumin-bound paclitaxel (NPT) or in combinations with the targeted agent BMS-754807 significantly inhibited esophageal cancer cell proliferation and subcutaneous xenograft tumor growth while significantly enhancing overall mice survival. These results indicate that aggressive esophageal cancer cells with elevated c-Myc expression are sensitive to the CDK inhibitor flavopiridol, and that flavopiridol alone or in combination can be a potential therapy for c-Myc overexpressing esophageal cancer.

Cytotoxic effects and tolerability of gemcitabine and axitinib in a xenograft model for c-myc amplified medulloblastoma

Medulloblastoma is the most common high-grade brain tumor in childhood. Medulloblastomas with c-myc amplification, classified as group 3, are the most aggressive among the four disease subtypes resulting in a 5-year overall survival of just above 50%. Despite current intensive therapy regimens, patients suffering from group 3 medulloblastoma urgently require new therapeutic options. Using a recently established c-myc amplified human medulloblastoma cell line, we performed an in-vitro-drug screen with single and combinatorial drugs that are either already clinically approved or agents in the advanced stage of clinical development. Candidate drugs were identified in vitro and then evaluated in vivo. Tumor growth was closely monitored by BLI. Vessel development was assessed by 3D light-sheet-fluorescence-microscopy. We identified the combination of gemcitabine and axitinib to be highly cytotoxic, requiring only low picomolar concentrations when used in combination. In the orthotopic model, gemcitabine and axitinib showed efficacy in terms of tumor control and survival. In both models, gemcitabine and axitinib were better tolerated than the standard regimen comprising of cisplatin and etoposide phosphate. 3D light-sheet-fluorescence-microscopy of intact tumors revealed thinning and rarefication of tumor vessels, providing one explanation for reduced tumor growth. Thus, the combination of the two drugs gemcitabine and axitinib has favorable effects on preventing tumor progression in an orthotopic group 3 medulloblastoma xenograft model while exhibiting a favorable toxicity profile. The combination merits further exploration as a new approach to treat high-risk group 3 medulloblastoma.

Abstract 975: Antitumor effect of cyclin- dependent kinase inhibitor flavopiridol in c-Myc overexpressing esophageal cancer

Abstract Tumors with elevated c-Myc expression often exhibit highly aggressive phenotype and c-Myc amplification has been shown to be frequent in esophageal cancer. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. We therefore investigated whether exploiting a synthetic-lethal approach dependent on elevated c-Myc signaling is effective in treating esophageal cancer with a CDK inhibitor flavopiridol. We found frequent overexpression of c-Myc in esophageal cancer cell lines and tissues. c-Myc overexpression correlated with accelerated esophageal cancer subcutaneous xenograft tumor growth without association with improved overall survival in esophageal cancer patients. c-Myc expression was enhanced by Wnt signaling activator (6-bromoindirubin-3′ -oxime (BIO) or pcDNA3-cMyc and c-Myc expression was depleted by c-Myc siRNAs. Interestingly, esophageal cancer cells with elevated c-Myc expression were preferentially more sensitive to induction of apoptosis by CDK inhibition flavopiridol compared to esophageal cancer cells with lower c-Myc expression. In addition, we observed that flavopiridol alone or in combinations with chemotherapeutic agent nanoparticle albumin-bound paclitaxel (NPT) or in combinations with targeted agent BMS-754807 significantly inhibited esophageal cancer cell proliferation and subcutaneous xenograft tumor growth with significantly enhancing overall mice survival. These results indicate that aggressive esophageal cancer cells with elevated c-Myc expression are sensitive to CDK inhibitor flavopiridol and flavopiridol alone or in combination with cytotoxic or targeted agents can be a potential therapy for c-Myc overexpressing esophageal cancer. Citation Format: Md Sazzad Hassan, Niranjan Awasthi, Jun Li, Margaret A. Schwarz, Urs von Holzen. Antitumor effect of cyclin- dependent kinase inhibitor flavopiridol in c-Myc overexpressing esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 975.

Rituximab with Dose-Adjusted EPOCH (R-DA-EPOCH) As First-Line Treatment in Patients with Double-Hit Lymphoma: Preliminary Results in 35 Consecutive Patients from Two Institutions

▪Introduction: Patients with aggressive B cell lymphomas carrying concomitant c-Myc and Bcl-2 and/or Bcl-6 gene rearrangement (DHL/THL) have a poor outcome when treated with standard R-CHOP regimen. Better results were reported in patients treated with more intensive programs, currently used in highly proliferative diseases such as Burkitt lymphoma. In particular, R-DA-EPOCH obtained the best result in a retrospective study comparing different intensive regimens (Oki et al. BJH 2014). We report the experience with this program of two hematologic centres in Northern Italy in patients with dysregulation of c-Myc gene associated with Bcl-2 and/or Bcl-6 abnormalities.Methods: From January 2014, all consecutive fit patients aged less than 80 years, with diffuse large B cell lymphoma (DLBCL) or lymphoma with intermediate features between DLBCL and Burkitt (BCLU), including 10 patients with histological features suggesting transformation from follicular lymphoma, and showing DHL or THL by fluorescent in situ hybridization (FISH), were treated with R-DA-EPOCH and central nervous system prophylaxis with intrathecal methotrexate +/- cytarabine . Patients with c-Myc amplification (more than 4 signals) plus Bcl-2 and/or Bcl-6 gene rearrangement or amplification were also included (AMPL). Pre-treatment with one cycle of R-CHOP was allowed in patients in need of urgent treatment, pending the results of FISH analysis. Autologous stem cell transplantation (ASCT) was planned in one of the two centres for patients who reached at least a partial remission after six R-DA-EPOCH courses. Cell of origin (COO) was defined by immunohistochemistry (IHC) according to Hans's algorithm.Results: Thirty-five patients (30 DLBCL and 5 BCLU) were treated. They received a median of 6 courses of R-DA-EPOCH (range 1-6) administered either to inpatients or to outpatients using a single portable infusion pump. Six patients were pre-treated with R-CHOP and 10 patients received consolidation with ASCT. According to FISH analysis, 18 cases were DHL, 6 THL and 11 AMPL. The median age of the whole group was 63 years (range 34-79). Twenty-nine patients were male (83%). Thirty-two patients (91%) had Ann-Arbor stage III/IV and 25 (71%) had high-intermediate/high risk score according to International Prognostic Index (IPI), with extranodal presentation in 74%, mainly in bone and gastrointestinal tract. According to Hans's algorithm, 85% of patients were of germinal center COO. By IHC double expression of myc (>40%) and of bcl-2 protein (>50%) was reported in 75% of patients. Median lymphoma cells proliferation fraction assessed by Ki-67 IHC was 80% (range 35-100%). Three patients died of infectious complications during chemotherapy. The overall response rate for the entire cohort was 71%. Of seven refractory patients, five have died of lymphoma, one is disease free after allogeneic transplantation and one is still on treatment. Among 25 patients who achieved a response to front-line therapy, one died of suicide and 24 are still in remission after a median of 15 months. The 1-year PFS and OS rates were 69% and 73%, respectively. There was no significant association of PFS or OS with age, COO, type of histology, transformation from FL, extranodal involvement, Ki-67. THL had significant worse survival comparing to DHL and AMPL (20% vs 88% vs 68% p 0.03) (Fig.1).Conclusions: These results confirm R-DA-EPOCH as a feasible program in unselected fit patients up to the age of 80. Short-term efficacy was high, considering the poor prognosis of double or triple-hit aggressive B-cell lymphoma. Few data are available to define the role of consolidative ASCT and its utility is still unclear. Treatment response seems to favourably impact on patient survival. A longer follow-up on larger number of patients is ongoing to confirm these promising results. [Display omitted] DisclosuresCorradini:Takeda: Honoraria; Sanofi: Honoraria; Novartis: Honoraria; Roche: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Gilead: Honoraria. Rossi:Teva: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.

Combination of BR101801 and Venetoclax Demonstrates Synergistic Activity in DLBCL Cell Lines Harboring Double Hit and Double Expressor Alterations

Introduction: Double hit DLBCL with concurrent rearrangement of c-Myc and BCL-2 as well as double expressor DLBCL with concomitant overexpression of c-Myc and BCL-2 are characterized to have very poor outcomes in the following standard R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy as well as short survival in progressing clinical courses. In DLBCL patients, about 20% to 30% of patients have shown the dual c-Myc and BCL-2 aberrance. Thus, several new drugs are being developed to achieve a durable response in double hit and double expressor DLBCL. The phase I study in patients with RR DLBCL using Venetoclax monotherapy which is a BCL-2 inhibitor, showed a disappointing objective response [CR:4/34(12%), PR:2/34(6%)]. This result shows that a combined approach involving a c-Myc controlling agent and BCL-2 inhibitor may be required to overcome the poor response of RR DLBCL, especially double hit and double expressor DLBCL. BR101801 is a first-in-class, oral, dual target inhibitor of DNA-PK and PI3Kδ. The combined inhibition of dual targets tightly regulates the protein stability of c-Myc independent of genetic alterations such as translocation, amplification, and overexpression. The purpose of this study is to investigate the functional significance of BR101801 on the growth inhibition and c-Myc regulation in double hit and double expressor DLBCL cell lines.Methods: Westernblot of protein expression levels was analyzed with the human DLBCL cell lines that were treated with BR101801, PI3Kδ-specific inhibitors (Idelalisib, TGR-1202) and DNA-PK inhibitor (NU-7441) or DMSO for 24 hr. After treating SU-DHL-4 cell with cyclohexamide (CHX, 20 ug/ml), and BR101801 (0.5 μM) alone or in combination, the lysates was analyzed with the westernblot. Cells (5x103/well) were incubated with various concentrations of compounds and the cell viability was determined after 72 hr by the WST-8 assay. Cell lines were classified as c-Myc translocation (DOHH2, SU-DHL-6, SU-DHL-10, OCI-LY-18), c-Myc amplification (SU-DHL-4, OCI-LY-3), and c-Myc expressor (U2932). Cell lines additionally showed BCL-2 alteration such as translocation (DOHH2, SU-DHL-4, SU-DHL-6, OCI-LY-18) and amplification (OCI-LY-3, U2932).Results: BR101801 showed potent inhibitory activity on c-Myc protein stability whereas no activity was found in Idelalisib, TGR-1202, or NU7441 (Figure 1A; SU-SHL-4, 1B; SU-DHL-6). Using CHX to block de novo protein synthesis on SU-DHL-4 cells, the increased rate of c-Myc degradation by GSK3β regulation was observed (Figure 1C). SU-DHL-10 cell lines harboring c-Myc alterations exhibited no sensitivity to the PI3Kδ inhibitor such as Idelalisib (IC50≒4.4 μM) and TGR-1202 (IC50>10 μM) and DNA-PK inhibitor such as NU7441 (IC50≒1.1 μM). However, the combination of Idelalisib and NU7441 resulted in 200-fold (IC50≒ 0.02 uM) growth inhibition (Figure 1D), which was comparable to the effect of BR101801. SU-DHL-6 cells showed low sensitivity to Venetoclax alone with IC50≒0.16 μM. However, the IC50 value of Venetoclax at 0.1, 1, 10 nM of BR101801 was decreased to 0.12 uM, 0.003 uM and < 0.001 uM (11 pM), respectively in a dose-dependent manner (Figure 1E).Conclusions: In this study, the rationale of co-targeting of PI3Kδ and DNA-PK in c-Myc regulation and growth inhibition were demonstrated. The dual targets of DNA-PK and PI3Kδ were essential to control the protein stability of c-Myc in DLBCL cell lines harboring various c-Myc alterations. In conclusion, the potent effect of BR101801 with Venetoclax in DLBCL cells harboring alterations on both c-Myc and BCL-2 suggests a mechanistic rationale as a breakthrough treatment for RR DLBCL patients. To ensure translation to humans, the study on the efficacy of BR101801 in primary cells isolated from double hit and double expressor as well as RR DLBCL patients is currently conducted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MYCL1 Amplification and Expression of L-Myc and c-Myc in Surgically Resected Small-Cell Lung Carcinoma.

Purpose: The Myc family, especially C-MYC and MYCL1, has been found involved in small-cell lung carcinoma (SCLC). Identification of the frequency of C-MYC and MYCL1 expression among SCLC patients may help to identify potential targets for therapeutic intervention. Our aim was to detect MYCL1 amplification, L-Myc and c-Myc expression, and investigate clinicopathological characteristics and survival status in patients with surgically resected SCLC. Methods: MYCL1 amplification was detected using fluorescence in situ hybridization (FISH), while L-Myc and c-Myc protein expressions were determined using immunohistochemistry (IHC) in the primary tumors of 46 resected SCLC patients. Results: Among the 46 evaluated specimens, MYCL1 amplification was identified in 3/46 cases (6.5%). One of the positive cases was MYCL1 gene amplification combined with fusion. 3/46 (6.5%) was positive for L-myc protein expression, and 4/46 (8.7%) was positive for c-Myc protein expression. Conclusion: Our study firstly multidimensional explored the expression of MYCL1 amplification, L-Myc and c-Myc protein and investigated clinicopathological characteristics and survival status in patients with surgically resected SCLC, which makes a contribution to subsequent research and therapeutic strategies.

BIOL-07. MIR-212 FUNCTIONS AS A TUMOR SUPPRESSOR GENE IN GROUP 3 MEDULLOBLASTOMA VIA TARGETING NUCLEAR FACTOR I/B (NFIB)

Medulloblastoma (MB), the most frequent malignant pediatric brain tumor is subdivided into four primary subgroups, i.e. wingless-type (WNT), sonic hedgehog (SHH), group 3, and group 4. Haploinsufficiency of chromosome 17p13.3 and c-myc amplification distinguish high-risk group 3 tumors, which are associated with rapid metastasis, recurrence and early mortality. We sought to identify the role of miR-212, which resides on chromosome 17p13.3, in the pathophysiology of group 3 MB. RNA expression analyses revealed dramatically reduced levels of miR-212 in group 3 tumors and cell lines mainly through epigenetic silencing via histone modifications (deacetylation). Restoring in vitro miR-212 expression reduced tumor cell proliferation, colony formation, wound healing, migration and invasion with decreased p-AKT and p-ERK levels in group 3 MB cell lines. Interestingly, a shift in differential c-myc phosphorylation (from serine-62 to threonine-58) was also discovered with miR-212 expression, resulting in reduced total c-myc levels, concurrent with elevated cellular apoptosis. In turn, pro-apoptotic binding partners of c-myc, i.e. Bin-1 and P19ARF, were upregulated in these cells. These findings were recapitulated in stable inducible miR-212 expressing tumor cells. Using a combination of transcriptomic data and a dual luciferase assay, we isolated an important oncogenic target of miR-212, i.e, NFIB, a nuclear transcription factor implicated in metastasis and recurrence. Increased expression of NFIB was confirmed in group 3 tumors, with poor survival shown in high NFIB-expressing patients. As prior, transient NFIB silencing in vitro reduced not only tumor cell proliferation, colony formation, wound healing, migration and invasion, but also medullosphere formation along with decreased expression of stem cell markers (Nanog, Oct4, Sox2, CD133), confirming its role in tumor recurrence possibly via augmenting tumor stemness. Taken together, these results substantiate the tumor suppressive role of miR-212 in group 3 MB and provide a potential new oncogenic target implicated in tumor recurrence, NFIB.

Globo H expression in metastatic colorectal cancer (CRC).

3527 Background: Globo H is a carbohydrate antigen that is highly expressed on the cell surface of epithelial cancers but not in normal tissue, and has been reported to correlate with poor prognosis. An attractive therapeutic target, Globo H-targeted agents are being tested in early clinical trials (e.g., OBI-833, a Globo H antigen conjugated to a mutated diphtheria toxin with potential antineoplastic activities, and OBI-999, an antibody-drug conjugate (ADC) consisting of a Globo H monoclonal antibody with a synthetic antineoplastic agent). We aim to describe the molecular features associated with Globo H expression in CRC. Methods: A total of 7,604 CRC tumors were tested by Caris Life Sciences (Phoenix, AZ) by NextGen DNA and RNA sequencing. The expression of β3GalT5, FUT-1 and FUT-2 were evaluated as surrogates for Globo H expression as they are the key enzymes in its biosynthesis. An average z-score of the 3 genes (GloboH) and of β3GalT5 (B3) alone were calculated; tumors with top quartile z-scores were considered expression-high (Q4) and bottom quartile, expression-low (Q1). QuantiSEQ was used to assess immune cell infiltration in the tumor microenvironment (TME). Statistical significance was determined using chi-square/Fisher-Exact and adjusted for multiple comparisons (q&lt;0.05). Consensus molecular subtype (CMS) was developed using RNA seq data. Results: When the 3 genes were considered, GloboH-H tumors showed higher prevalence of CMS1 and CMS4 (23.8% vs. 12%; 38.7% vs. 29.4%) and lower prevalence of CMS2 (40% vs. 18.7%) compared to GloboH-L. Similar patterns of CMS distribution were seen for B3 alone. B3-H tumors were significantly more frequently TMB-H (&gt;=10) (11.4% vs. 8.3%), PD-L1 positive (5.7% vs. 3.4%) and MSI-H/dMMR (8.3% vs. 5.5%). Strong positive associations were seen with mutations in BRAF, KRAS, RSPO3 fusion, and cMYC amplification with B3 alone and GloboH (all q&lt;0.05). Anti-tumor CD4+ T cells and NK cells were increased in the TME with increased expression of GloboH and B3 (q&lt;0.05). However, immune suppressive neutrophils and Tregs were also increased. Dendritic cells were negatively associated with B3 expression while endothelial cells and fibroblasts showed a positive association with GloboH and B3. Conclusions: The association with TMB-H, MSI-H, and PD-L1 status suggests that in some tumors Globo H may be a promising target for combination therapy with immune checkpoint inhibition. The association with different cell populations suggests manipulating the cellular balance in the TME as an approach to improve the efficacy of treatment. NK cell checkpoint inhibitors are in clinical trials and might be utilized in high Globo H cancers; treatments inducing DCs in tumors have been shown to enhance responses to BRAF and PD-L1 blockade and might be applicable in the context of Globo H immunotherapy to overcome Treg immune suppression. Anti-Globo H vaccines and ADCs might be particularly effective in BRAF and KRAS-mutant CRC patients.

Association of TP53 mutation with decreased prevalence of MSI-high, RAS and PI3KCA mutations in metastatic colorectal cancer.

e15578 Background: TP53 tumor suppressor gene is mutated in approximately 50% of colorectal cancer (CRC). How TP53 mutations are associated with the prevalence of the other common genomic alterations such as RAS (KRAS/NRAS), BRAF, PI3KCA, as well as microsatellite stability (MSI) is not clear. Methods: We investigated the impact of TP53 mutations on other common genomic alterations and survival in patients with metastatic CRC using the NGS data within Kaiser Permanente Northern California (KPNC). Results: From November 2017 to January 2021, genomic profiling was performed on 1056 patients with metastatic CRC, of whom 740 patients harbored a TP53 mutation (TP53mut) and 316 patients had wild-type TP53 (TP53wt). We found that median overall survival (OS) was similar between the TP53wt and TP53mut patients (50.1 vs 47.5 months, p = 0.9), however, the percent with a Ras mutation was significantly higher in patients with TP53wt compared to TP53mut (63.2 vs 45.2%, p = 0.0001). Interestingly, the percent with MSI-high was also significantly higher in TP53wt than TP53mut patients (11.1 vs 1.4%, p = 0.0001), however, the response rate of the MSI-high patients to immune checkpoint inhibitor (ICI) was similar (40 vs 37.5%). In addition, a significantly higher percent of patients with TP53wt had PI3KCA mutations and a significantly lower percent had c-Myc amplifications compared to patients with TP53mut (PI3KCA, 32 vs 10.7%, p = 0.0001; c-Myc, 1.26 vs 4.6%, p = 0.008). There was no significant difference in the percent of BRAF mutations between the two patient populations (6.2 vs 9.8%). A significantly higher percent of patients with TP53wt and a PI3KCA mutation had a Ras mutation compared to patients with TP53mut and a PI3KCA mutation (81.2 vs 57%, p = 0.0004). However, TP53 mutation status was not significantly associated with the OS of patients with either a Ras, or BRAF, or PI3KCA mutation, or combination of Ras and PI3KCA mutations. Conclusions: TP53 mutation is associated with decreased prevalence of Ras, PI3KCA mutation and MSI-high in patients with metastatic CRC, however, without impacting the OS or response rate to ICI.

Novel Glutamine Antagonist JHU395 Suppresses MYC-Driven Medulloblastoma Growth and Induces Apoptosis.

Medulloblastoma is the most common malignant pediatric brain tumor. Amplification of c-MYC is a hallmark of a subset of poor-prognosis medulloblastoma. MYC upregulates glutamine metabolism across many types of cancer. We modified the naturally occurring glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) by adding 2 promoeities to increase its lipophilicity and brain penetration creating the prodrug isopropyl 6-diazo-5-oxo-2-(((phenyl (pivaloyloxy) methoxy) - carbonyl) amino) hexanoate, termed JHU395. This prodrug was shown to have a 10-fold improved CSF-to-plasma ratio and brain-to-plasma ratio relative to DON. We hypothesized that JHU395 would have superior cell penetration compared with DON and would effectively and more potently kill MYC-expressing medulloblastoma. JHU395 treatment caused decreased growth and increased apoptosis in multiple human high-MYC medulloblastoma cell lines at lower concentrations than DON. Parenteral administration of JHU395 in Nu/Nu mice led to the accumulation of micromolar concentrations of DON in brain. Treatment of mice bearing orthotopic xenografts of human MYC-amplified medulloblastoma with JHU395 increased median survival from 26 to 45 days compared with vehicle control mice (p < 0.001 by log-rank test). These data provide preclinical justification for the ongoing development and testing of brain-targeted DON prodrugs for use in medulloblastoma.

Collision of Epstein\u2013Barr virus-positive and -negative gastric cancer, diagnosed by molecular analysis: a case report

BackgroundEpstein–Barr virus (EBV)-positive gastric carcinoma (GC) is defined by the proliferation of GC cells with EBV infection. The co-existence of EBV-positive and -negative components in a single GC is rare. We report a case of GC with the co-existence of EBV-positive and EBV-negative components, in which we performed—for the first time—various molecular analyses to elucidate their histogenesis.Case presentationAn 81-year-old man was diagnosed with GC based on the results of endoscopy and a pathological examination of the biopsy specimen. Systemic chemotherapy was performed, since lymph node and lung metastases were diagnosed based on computed tomography. Total gastrectomy and lymph node dissection were performed after chemotherapy, after confirming that the size of the metastatic lymph nodes had decreased and that the lung metastasis had disappeared. Grossly, a type 3 tumor was located in the middle posterior part of the stomach body. At the cut section, the tumor consisted of a white and solid part on the anal side of the tumor and a flat and elevated part on the oral side. Histologically, the former part consisted of GC with lymphoid stroma and the latter part was composed of poorly differentiated adenocarcinoma without prominent lymphocytic infiltration. The two histopathological components were clearly separated from each other. On EBV-encoded small RNA (EBER)-in situ hybridization (ISH), the part with the lymphoid stroma component was positive, while the other part was negative. Immunohistochemistry revealed that both components showed the overexpression of p53. Sequencing of TP53 using DNA extracted from the two components was conducted, and revealed different patterns. Targeted next generation sequencing revealed MYC amplification in the EBV-positive component of the tumor and HER2 amplification in the EBV-negative part. Immunohistochemistry revealed that the EBV-positive part was C-MYC( +)/HER2(−) and the EBV-negative part was C-MYC(−)/HER2( +). Correspondingly, chromogenic ISH and dual-color ISH showed amplification of C-MYC and no amplification of HER2 in the EBV-positive part, and no amplification of C-MYC and amplification of HER2 in the EBV-negative part.ConclusionWe presented a case of collision of two different GCs composed of EBER-ISH ( +)/C-MYC ( +) and EBER-ISH (−)/HER2 ( +) cells.

Distinct functions of transforming growth factor-\u03b2 signaling in c-MYC driven hepatocellular carcinoma initiation and progression

Dysregulation of transforming growth factor-beta (TGFβ) signaling has been implicated in liver carcinogenesis with both tumor promoting and inhibiting activities. Activation of the c-MYC protooncogene is another critical genetic event in hepatocellular carcinoma (HCC). However, the precise functional crosstalk between c-MYC and TGFβ signaling pathways remains unclear. In the present investigation, we investigated the expression of TGFβ signaling in c-MYC amplified human HCC samples as well as the mechanisms whereby TGFβ modulates c-Myc driven hepatocarcinogenesis during initiation and progression. We found that several TGFβ target genes are overexpressed in human HCCs with c-MYC amplification. In vivo, activation of TGFβ1 impaired c-Myc murine HCC initiation, whereas inhibition of TGFβ pathway accelerated this process. In contrast, overexpression of TGFβ1 enhanced c-Myc HCC progression by promoting tumor cell metastasis. Mechanistically, activation of TGFβ promoted tumor microenvironment reprogramming rather than inducing epithelial-to-mesenchymal transition during HCC progression. Moreover, we identified PMEPA1 as a potential TGFβ1 target. Altogether, our data underline the divergent roles of TGFβ signaling during c-MYC induced HCC initiation and progression.

Thrombospondin-1 mimetics are promising novel therapeutics for MYC-associated medulloblastoma.

BackgroundMedulloblastoma (MB) comprises four subtypes of which group 3 MB are the most aggressive. Although overall survival for MB has improved, the outcome of group 3 MB remains dismal. C-MYC (MYC) amplification or MYC overexpression which characterizes group 3 MB is a strong negative prognostic factor and is frequently associated with metastases and relapses. We previously reported that MYC expression alone promotes highly aggressive MB phenotypes, in part via repression of thrombospondin-1 (TSP-1), a potent tumor suppressor.MethodsIn this study, we examined the potential role of TSP-1 and TSP-1 peptidomimetic ABT-898 in MYC-amplified human MB cell lines and two distinct murine models of MYC-driven group 3 MBs.ResultsWe found that TSP-1 reconstitution diminished metastases and prolonged survival in orthotopic xenografts and promoted chemo- and radio-sensitivity via AKT signaling. Furthermore, we demonstrate that ABT-898 can recapitulate the effects of TSP-1 expression in MB cells in vitro and specifically induced apoptosis in murine group 3 MB tumor cells.ConclusionOur data underscore the importance of TSP-1 as a critical tumor suppressor in MB and highlight TSP-1 peptidomimetics as promising novel therapeutics for the most lethal subtype of MB.

Chromogenic In Situ Hybridization (CISH) as a Method for Detection of C-Myc Amplification in Formalin-Fixed Paraffin-Embedded Tumor Tissue: An Update.

In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations, or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE ) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40× magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology, and the slides can be stored for a long time.

Increased Number of Chromosomal Imbalances and High-Level DNA Amplifications in Mantle Cell Lymphoma Are Associated With Blastoid Variants

AbstractMantle cell lymphomas (MCLs) are characterized by 11q13 chromosomal translocations and cyclin D1 overexpression. The secondary genetic and molecular events involved in the progression of these tumors are not well known. In this study, we have analyzed 45 MCLs (32 typical and 13 blastoid variants) by comparative genomic hybridization (CGH). To identify the possible genes included in the abnormal chromosome regions, selected cases were analyzed for P53, P16INK4a, RB, C-MYC, N-MYC, BCL2, BCL6, CDK4, and BMI-1 gene alterations. The most frequent imbalances detected by CGH were gains of chromosomes 3q (49%), 7p (27%), 8q (22%), 12q (20%), 18q (18%), and 9q34 (16%) and losses of chromosomes 13 (44%), 6q (27%), 1p (24%), 11q14-q23 (22%), 10p14-p15 (18%), 17p (16%), and 9p (16%). High-level DNA amplifications were identified in 11 different regions of the genome, predominantly in 3q27-q29 (13%), 18q23 (9%), and Xq28 (7%). The CGH analysis allowed the identification of regional consensus areas in most of the frequently involved chromosomes. Chromosome gains (P = .02) and losses (P = .01) and DNA amplifications (P = .015) were significantly higher in blastoid variants. The significant differences between blastoid and typical tumors were gains of 3q, 7p, and 12q, and losses of 17p. CGH losses of 17p correlated with P53 gene deletions and mutations. Similarly, gains of 12q and high-level DNA amplifications of 10p12-p13 were associated with CDK4 and BMI-1 gene amplifications, respectively. One of 2 cases with 8q24 amplification showed C-MYC amplification by Southern blot. Alterations in 2p, 3q, 13, and 18q were not associated with N-MYC, BCL6, RB, orBCL2 alterations, respectively, suggesting that other genes may be the targets of these genetic abnormalities in MCLs. Increased number of gains (0 v 1-4 v >4 gains per case) (P = .002), gains of 3q (P = .02), gains of 12q (P = .03), and losses of 9p (P = .003) were significantly associated with a shorter survival of the patients. These results indicate that an increased number of chromosome imbalances are associated with blastoid variants of MCLs and may have prognostic significance.