MYC Gene Copy Number Research Articles

Clinicopathologic Characteristics of MYC Copy Number Amplification in Breast Cancer.

Introduction. MYC overexpression is a known phenomenon in breast cancer. This study investigates the correlation of MYC gene copy number amplification and MYC protein overexpression with coexisting genetic abnormalities and associated clinicopathologic features in breast cancer patients. Methods. The study analyzed data from 81 patients with localized or metastatic breast cancers using targeted next-generation sequencing and MYC immunohistochemical studies, along with pathological and clinical data. Results. Applying the criteria of MYC/chromosome 8 ratio ≥5, MYC copy number amplified tumors (n = 11, 14%) were associated with invasive ductal carcinoma (91% vs 68%, P = .048), poorly differentiated (grade 3, 64% vs 30%, P = .032), mitotically active (Nottingham mitotic score 3, 71% vs 20%, P = .004), estrogen receptor (ER)-negative (45% vs 12%, P = .008), and triple-negative (56% vs 12%, P = .013) compared to MYC non-amplified tumors. Among MYC-amplified breast cancer patients, those with triple-negative status showed significantly shorter disease-free survival time than non-triple negative MYC-amplified patients (median survival month: 25.5 vs 127.6, P = .049). MYC amplification is significantly associated with TP53 mutation (P = .007). The majority (10 of 11; 91%) of MYC-amplified tumors showed positive c-MYC immunostaining. Conclusion. Breast cancers with MYC copy number amplication display distinct clinicopathologic characteristics indicative of more aggressive behavior.

Clinicopathological analysis of diffuse large B-cell lymphoma using molecular biomarkers: a retrospective analysis from 7 Hungarian centers.

The clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy. This is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression. A total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4-62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS. In our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.

MYC copy number and mRNA expression in chronic lymphocytic leukemia patients exposed to ionizing radiation due to the Chornobyl NPP accident.

To investigate the frequency ofMYCgene copy number amplification in IR-exposed CLL patients and relate the findings to theMYCmRNA levels, the presence of unfavourable prognosis mutations (TP53, SF3B1, NOTCH1), and patient`s outcome. The analysis of MYC copy number was carried out by real-time quantitative polymerase chain reaction (PCR) in 70IR-exposed CLL patients. The MYC mRNA expression was measured by real-time quantitative reverse transcription PCR. Increased MYC gene copy number was present in 5.7% of cases. There was a statistically significant association between increased MYC copy number and increased MYC mRNA (p<0.014). Additionally, somatic deletion in MYC locus was found in one patient. Most of patients (80%) with detected MYC aberrations were previously untreated, suggesting that these lesions might occur early in the course of the disease. The MYC aberrations were found mutually exclusive with high risk TP53and SF3B1mutations, while one case was identified, where MYC amplification and NOTCH1mutation coincided simultaneously. Regarding clinical outcome, the MYC aberrations were associated with a shorter time to first treatment (3vs 25months, p = 0.008) as well as reduced overall survival (60vs 139months). Our data suggest that MYC aberrations might be an early event in IR-related CLL and contribute to aggressive disease development in the absence of high risk TP53and SF3B1mutations.

MYC aberrations in AC and PDA

Introduction: The aim of our study was investigation of the presence of MYC amplification in PDA and carcinoma of the vater. Method: 74 cases of PDA, 15 cases of cancer of the vater and seven cases of control pancreatic tissue were included in the study. We examined the expression of MUC1, 2 and 5AC types by immunohistochemical analyses in PDA and carcinoma of the vater. Dual-color interphase fluorescent in situ hybridization (FISH) was performed on 4-μm thick sections of formalin-fixed paraffin-embedded (FFPE) tumors. Results: Most of the PDA have been reported as - MUC1 + / MUC5AC + and accounted for 42% (31/74), the smallest was a group with the intestinal mucin phenotype (MUC2 +) and only 7% (5/74). The group with the gastric phenotype (MUC5AC +) was - 15% (11/74). Carcinoma of the vater in most cases have been reported as MUC2 + and accounted for 80% (12/15), 20% (3/15) of cases the tumor presented as MUC1 + /MUC5AC +. In normal ducts of all 7 control cases the tested loci had balanced (diploid) profile. FISH analysis of PDA revealed of MYC gene copy number in 21/74 (28%) cases. Eight of the 74 pancreatic tumors (10,8%) revealed MYC gain in association with an increased chromosome 8 copy number. Conclusion: MYC gene amplification should be considered as a marker of a more aggressive behavior in cancers biliary pancreaticduodenal region. Diploid profile MYC gene in carcinomas of the vater indicates a more favorable prognosis compared with ductal adenocarcinomas of the pancreas.

Expression of hsa-miR-9 and MYC Copy Number Variation in Hereditary Diffuse Gastric Cancer.

Approximately, 15-50% of families affected by hereditary diffuse gastric cancer (HDGC) exhibit CDH1 germline mutations. CDH1 gene encodes E-cadherin, protein essential to the cell-cell contact of gastric epithelium. Studies have shown that hsa-miR-9 participates in this protein downregulation. Moreover, MYC is responsible for the transcription of hsa-miR-9-3. In the present study, hsa-miR-9 expression and MYC copy number variation were investigated to elucidate the hsa-miR-9 role in HDGC. Tumor samples were obtained from nine individuals with HDGC history belonging to four Brazilian families. Then, relative quantification of hsa-miR-9 expression and MYC gene copy number variation analysis were performed by real-time PCR. In all the samples, an overexpression of hsa-miR-9 and an increased MYC copy number (≥3 copies) were observed. hsa-miR-9 acts as an oncomiR in HDGC. In addition, we suggest that hsa-miR-9 acts as second event in individuals with HDGC carrying CDH1 gene germinline mutations.

Open-Label, Phase 2 Study to Evaluate the Efficacy and Safety of CUDC-907 in Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma, Including Patients with MYC Alterations

Patients (pts) with relapsed and/or refractory (RR) MYC-altered diffuse large B-Cell lymphoma (DLBCL) respond poorly to available therapeutic options. Due to acquired chemoresistance, these patients are often ineligible for or relapse following autologous stem cell transplantation (ASCT), and do not respond well to additional cytotoxic therapy. Our previous translational studies have demonstrated that combined pharmacologic inhibition of HDAC and PI3K signaling, via CUDC-907, effectively suppresses MYC activity through transcriptional and translational mechanisms. In MYC-dependent DLBCL and NUT midline carcinoma cell lines, CUDC-907 treatment has demonstrated dose-dependent decreases in MYC protein expression and a more potent inhibition of MYC expression than various HDAC and PI3K inhibitors, either alone or in combination. CUDC-907 has also shown greater in vitro and in vivo activity than single-target HDAC or PI3K inhibitors, especially in MYC-altered DLBCL models. In a phase 1 study, RR MYC-altered DLBCL pts achieved objective, durable responses while treated with CUDC-907, including complete responses (CRs). As of 15 March 2016, among the 5 response evaluable pts with RR MYC-altered DLBCL, 4 (80%) achieved objective responses (3 CRs and 1 partial response [PR]). All 3 CRs observed occurred in subjects with MYC gene copy number gain. In addition, to support our diagnostic plan, we evaluated MYC in a panel of DLBCL samples to better understand the prevalence of alterations. This ongoing phase 2 study is designed to further examine the efficacy of CUDC-907 in RR DLBCL pts with MYC-altered disease.Up to 200 pts with RR DLBCL, including those who have MYC-altered disease per central testing, will be enrolled to receive either CUDC-907 alone or in combination with rituximab. Within each treatment cohort (n= ~60), pts will be stratified based on MYC status as determined by central testing: MYC translocation by FISH (Groups A and D) versus MYC copy number gain by FISH or ≥40% of cells scored as MYC protein expression positive by immunohistochemistry (Groups B and E). Pts with negative testing for MYC-altered disease will still be eligible for treatment (Groups C and F). Other key eligibility criteria include: Age ≥ 18 years, ECOG performance status score ≤ 2, measurable disease on CT imaging, at least 2 but no more than 4 prior lines of therapy for DLBCL which may include ASCT, and confirmed availability of adequate viable tissue (fresh or most recent archival) for central testing. Pts with transformed follicular lymphoma will be eligible. However, other B-cell lymphomas including B- cell lymphoma unclassifiable with features intermediate between DLBCL and Burkitt lymphoma and Burkitt lymphoma are not eligible.All pts will take 60 mg of CUDC-907 orally once a day on a 5 days on/2 days off schedule in 21-day cycles. Pts assigned to the combination arm will also receive rituximab 375 mg/m2 IV on Day 1 of each cycle for the first 6 cycles. Response will be evaluated every other cycle per Cheson criteria by central review and survival assessments will occur every 12 weeks.The primary endpoint is objective response rate (ORR); secondary endpoints include duration of response (DOR), disease control rate (DCR), progression free survival (PFS), median and 6-month PFS, and overall survival (OS). Other disease associated biomarkers, including BCL-2 and BCL-6, will also be evaluated. Clinical trial information: NCT02674750. DisclosuresHafeez:Curis, Inc.: Employment. Gharavi:Curis: Employment. Chander:Curis Inc.: Employment. Ma:Curis Inc: Employment. Oki:Novartis: Research Funding.

Clinical and Pathologic Correlation of Increased MYC Gene Copy Number in Diffuse Large B-Cell Lymphoma.

Only a few studies have investigated the presence of increased MYC gene copy number (ICN) as a prognostic indicator in patients with diffuse large B-cell lymphoma (DLBCL), and the results have been variable. We compared overall survival in patients with ICN to MYC-negative patients and investigated the prognostic significance of increased MYC gene copy number. Two groups, those with MYC ICN (n= 33) and those with no MYC aberrations (n= 43), identified by fluorescence in-situ hybridization DNA probes for the MYC region at 8q24, were compared for survival (1-9 years), MYC immunohistochemical (IHC) protein expression, and treatment protocol. Comparison of cases of DLBCL with MYC ICN to those with no MYC aberration demonstrated no significant difference in survival (P= .58). Additionally, no difference in survival was found between patients with increased MYC protein expression (IHC MYC≥ 40%) compared to those with IHC MYC< 40% (P= .5). Comparison of Ki-67 proliferation rates, stratified into low and high groups, did not achieve statistical significance (P= .67). Patients with MYC ICN showed a slightly increased MYC protein expression (P > .05). Importantly, the majority of patients in both groups (79% of patients with ICN and 81% of patients with no MYC aberrations) were treated with rituximab-based therapies. No significant difference in survival was found between patients with DLBCL with MYC ICN and patients with no MYC aberrations (P= .58).

Abstract B24: Targeting ERK-MYC axis in pancreatic cancer

Abstract Pancreatic cancer (PCa) is a devastating malignancy with almost 90% lethality of all stages. In PCa, constitutively activated mutated KRAS induces persistent ERK signaling which stabilizes the oncogenic MYC protein. With very limited efficacy of chemotherapy, novel treatment options are urgently need for PCa. Here, we used patient-derived PCa cells and matched PCa tissue explants (PDX) to search for cytotoxic anti-PCa activity among the existing 867 drugs. Only a minority of tested agents were confirmed as active in vivo on patient-matched PDX models. From a short list of the agents with activity in vivo, we focused on triptolide (TLD), an irreversible inhibitor of ERCC3. In pilot testing of triptolide efficacy in a “xenopatient” trial of 8 PCa PDX models, we found that TLD was highly effective against a minority of tumors which achieved complete regression lasting for 3-6 weeks, while other models only showed growth delay. Genomic profiling of highly sensitive PDX models unfirmly demonstrated MYC-amplification. We tested TLD activity in vitro using PDX-matched patient-derived PCa cell lines. Nanomolar TLD elicited rapid (1-4 hours) and profound depletion of MYC oncoprotein in MYC-amplified PCa cell lines, but in PCa cells with normal MYC gene copy number. Furthermore, TLD-resistant PCa cells and PDX tumors exhibited rapid increase of phosphorylated ERK and its downstream targets including stable phospho-MYC protein. This activity of TLD against non-phosphorylated forms of MYC protein suggests interdependence of ERCC3 and MYC in cancer. Expression of ERCC3 was MYC-dependent in a Tet-OFF MYC cell line model. Conversely, silencing of ERCC3 suppressed MYC level. The KRAS-&amp;gt;ERK pathway is critical for initiation and progression of PCa. Attempts to disrupt this signaling axis by blocking MEK activity produces only transient cytostatic effect followed by rapid reactivation of the pathway mediated by MYC. Our results further confirm this dependency of PCa on the integrity of the activated KRAS-&amp;gt;ERK-&amp;gt;MYC signaling axis, and that pharmacological blockade of ERCC3 can be a highly effective strategy against PCa, and in other recalcitrant carcinomas with MYC-amplification. We thus provide a useful biomarker for future clinical development of TLD analogs such as Minnelide which is currently being tested in a Phase I clinical trial. Citation Format: Natalia Beglyarova, Eugenia Banina, Natalya Skobeleva, Vladimir Khazak, Igor Astsaturov. Targeting ERK-MYC axis in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B24.

MYC and human telomerase gene (TERC) copy number gain in early-stage non-small cell lung cancer.

We investigated the frequency of MYC and TERC increased gene copy number (GCN) in early-stage non-small cell lung cancer (NSCLC) and evaluated the correlation of these genomic imbalances with clinicopathologic parameters and outcome. Tumor tissues were obtained from 113 resected NSCLCs. MYC and TERC GCNs were tested by fluorescence in situ hybridization (FISH) according to the University of Colorado Cancer Center (UCCC) criteria and based on the receiver operating characteristic (ROC) classification. When UCCC criteria were applied, 41 (36%) cases for MYC and 41 (36%) cases for TERC were considered FISH-positive. MYC and TERC concurrent FISH-positive was observed in 12 cases (11%): 2 (17%) cases with gene amplification and 10 (83%) with high polysomy. By using the ROC analysis, high MYC (mean ≥ 2.83 copies/cell) and TERC (mean ≥ 2.65 copies/cell) GCNs were observed in 60 (53.1%) cases and 58 (51.3%) cases, respectively. High TERC GCN was associated with squamous cell carcinoma (SCC) histology (P=0.001). In univariate analysis, increased MYC GCN was associated with shorter overall survival (P=0.032 [UCCC criteria] or P=0.02 [ROC classification]), whereas high TERC GCN showed no association. In multivariate analysis including stage and age, high MYC GCN remained significantly associated with worse overall survival using both the UCCC criteria (P=0.02) and the ROC classification (P=0.008). Our results confirm MYC as frequently amplified in early-stage NSCLC and increased MYC GCN as a strong predictor of worse survival. Increased TERC GCN does not have prognostic impact but has strong association with squamous histology.

Abstract 2351: MYC gene copy number determine MYC expression and sensitivity to a MYC-inhibitor in multiple myeloma cells

Abstract Background The role of MYC in multiple myeloma has been under debate, but evidence now point to an important role for this oncogene in disease initiation, progression and maintenance. The key role of MYC in maintenance of tumor cell survival makes MYC a potential target for therapy. Acting as a transcription factor, MYC binds to its obligate partner MAX. Small-molecule inhibitors that target MYC-MAX heterodimerization prevent DNA binding and hence the transactivation of MYC target genes. Purpose We have previously shown that in many myeloma cell clones inhibition of MYC activity leads to apoptosis, but to varying degrees. The relation between the cells' response to inhibition of MYC, and levels of MYC gene, RNA and/or protein has not been thoroughly investigated earlier. Thus, we wanted to explore this using a panel of myeloma cell lines and primary myeloma cells. Methods A panel of myeloma cell lines was treated with the MYC-MAX inhibitor 10058-F4 before assessment of relative cell growth using CellTiter Glo, that measures the cells' ATP content. To determine MYC expression we analyzed DNA copy numbers using PCR, mRNA content using Nanostring technology and protein levels using Western Blotting. To screen primary myeloma cells for sensitivity to MYC-inhibition, we used our newly developed method for screening of cell viability in cocultures with bone marrow stromal cells. Results Myeloma cell lines showed a great variation in number of MYC gene copy number as well as MYC mRNA and protein levels. Interestingly, there was a good correlation between DNA copy numbers and mRNA levels, indicating that in general the MYC gene copy number is determining for the level of MYC mRNA in myeloma cells. Moreover, protein levels also showed a relatively high degree of correlation with MYC mRNA. The relative sensitivity for MYC-inhibition for each cell clone was compared with MYC expression. We could not see a clear correlation between sensitivity to MYC-inhibition and MYC expression in cell lines. However, cell lines with high MYC gene copy number and MYC expression tended to be among the most sensitive cell lines to MYC-MAX inhibition. We also tested a number of primary myeloma cells for sensitivity to the MYC-MAX-inhibitor in a cell viability assay in the presence of stromal cells. The presence of pooled patient bone marrow stromal cells (BMSC) did not affect the sensitivity of patient cells to the 10058-F4 MYC-MAX-inhibitor. Results on the correlation of MYC expression and sensitivity of primary cells to inhibition of MYC activity will be presented. Citation Format: Toril Holien, Kristine Misund, Glenn Buene, Oddrun E. Olsen, Katarzyna A. Baranowska, Anders Waage, Anders Sundan. MYC gene copy number determine MYC expression and sensitivity to a MYC-inhibitor in multiple myeloma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2351. doi:10.1158/1538-7445.AM2014-2351

EGFR and MYC gene copy number aberrations are more common in squamous cell carcinoma than keratoacanthoma: a FISH study

Epidermal growth factor receptor (EGFR) and MYC genomic aberrations have been described in cutaneous squamous cell carcinoma (SCC) but have not been widely investigated in keratoacanthoma (KA). EGFR and MYC were evaluated by fluorescence in situ hybridization and immunohistochemistry in 8 verrucae, 19 involuting KA (IKA), 23 classic KA (CKA), 6 atypical KA (AKA) and 19 SCC. Increased EGFR gene copy number was seen in 9 of 23 CKA and 14 of 19 SCC (p = 0.03). Increased MYC gene copy number was observed in 7 of 23 CKA and 17 of 19 SCC (p = 0.0001). MYC gene amplification was more common in SCC than CKA (p = 0.005), while EGFR gene amplification was rare and not significant. MYC protein overexpression was identified in 6 of 23 CKA and 14 of 19 SCC (p = 0.005). There was no statistical difference in EGFR protein overexpression in SCC and CKA (p = 0.06). EGFR and MYC aberrations were rare in IKA. AKA showed EGFR and MYC anomalies at an incidence intermediate between CKA and SCC. EGFR and MYC gene copy number aberrations are more common in SCC than KA. The incidence of aberrations parallels the degree of cytologic atypia in KA.

Prediction of outcome in patients with low‐grade squamous intraepithelial lesions by fluorescence in situ hybridization analysis of human papillomavirus, TERC, and MYC

Cytology is an excellent method with which to diagnose preinvasive lesions of the uterine cervix, but it suffers from limited specificity for clinically significant lesions. Supplementary methods might predict the natural course of the detected lesions. The objective of the current study was to test whether a multicolor fluorescence in situ hybridization (FISH) assay might help to stratify abnormal results of Papanicolaou tests. A total of 219 liquid-based cytology specimens of low-grade squamous intraepithelial lesions (LSIL), 49 atypical squamous cells of undetermined significance (ASCUS) specimens, 52 high-grade squamous intraepithelial lesion (HSIL) specimens, and 50 normal samples were assessed by FISH with probes for the human papillomavirus (HPV), MYC, and telomerase RNA component (TERC). Subtyping of HPV by polymerase chain reaction (PCR) was performed in a subset of cases (n=206). There was a significant correlation found between HPV detection by FISH and PCR (P<.0001). In patients with LSILs, the presence of HPV detected by FISH was significantly associated with disease progression (P<.0001). An increased MYC and/or TERC gene copy number (>2 signals in>10% of cells) prevailed in 43% of ASCUS specimens and was more frequent in HSIL (85%) than in LSIL (33%) (HSIL vs LSIL: P<.0001). Increased TERC gene copy number was significantly correlated with progression of LSIL (P<.01; odds ratio, 7.44; area under the receiver operating characteristic curve, 0.73; positive predictive value, 0.30; negative predictive value, 0.94) CONCLUSIONS: The detection of HPV by FISH analysis is feasible in liquid-based cytology and is significantly correlated with HPV analysis by PCR. The analysis of TERC gene copy number may be useful for risk stratification in patients with LSIL.

MYC gene copy number (GCN) and sensitivity to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC).

e21018 Background: Monoclonal antibodies against Epidermal Growth Factor Receptor (EGFR) demonstrated efficacy in mCRC patients without mutations in the KRAS gene. Previous data in breast and lung cancer suggested that MYC GCN affects sensitivity to anti-EGFR agents. Methods: This retrospective study was conducted in a cohort of 303 mCRC patients treated with cetuximab/panitumumab, either alone (N=24) or in combination with chemotherapy (N=279). MYC GCN was assessed by fluorescence in situ hybridization (FISH) in primary colorectal cancer tissue samples. Results: In the study population response rate (RR) was 28%, median progression-free survival (PFS) 5.6 months and median overall survival (OS) 11.8 months. MYC was successfully evaluated in 298 cases and was amplified in 17 patients (5.7%). Individuals with MYC amplification showed a trend for a lower RR (7.7% versus 28.9%, p=0.12), shorter PFS (3.0 months versus 5.8 months, p=0.22) and shorter OS (11.3 months versus 12.6 months, p=0.15) than patients with non-amplified tumors. A Receiver Operating Characteristic (ROC) analysis was also performed in order to verify whether a better MYC GCN cut-off could discriminate between anti-EGFR sensitive and resistant cases. In this analysis, patients with mean MYC GCN ≥2.53 (N=199, 66.8%) had a significantly higher RR (32.2% versus 19.1%, p=0.02), a longer PFS (5.8 months versus 4.5 months, p=0.06) and a longer OS (12.3 months versus 11.4 months, p=0.59) than patients with mean GCN &lt;2.53 (N=99, 33.2%). In order to assess the potential confounding effect of KRAS and BRAF status, we further analyzed the outcome of the 81 KRAS/BRAF wild-type patients according to MYC GCN. In this subset, a trend for improved RR (42% versus 20%, p=0.05), PFS (8.6 months versus 4.2 months, p=0.16) and OS (13.5 months versus 9.7 months, p=0.51) favored MYC FISH GCN ≥2.73 patients. Conclusions: MYC amplification is a rare event in mCRC, not significantly reducing sensitivity to anti-EGFR agents. A trend for better outcome was observed in presence of increased MYC GCN.

Abstract P2-09-08: c-MYC (MYC) Protein Expression and Associations with Trastuzumab Benefit in Early-Stage, HER2+ Breast Cancer in Context of the NCCTG Adjuvant Trial, N9831

Abstract Previous findings suggested that patients (pts) with copy number alterations of MYC (8q24) and centromere 8 (CEN 8) in the setting of HER2-positive breast cancer may be associated with improved outcome to adjuvant trastuzumab. Our tissue microarray (TMA) data suggested that alternate cutpoints for MYC copy number anomalies [of 802 pts, those with MYC:CEN8 ratio ≥1.3 or &amp;lt;1.3 &amp; polysomy 8 (N=658; 82%) had a hazard ratio (HR) of 0.44 (P&amp;lt;0.001) and those with MYC copy number ≥2.5 (N=669; 84%) had a HR of 0.42 (P&amp;lt;0.001)] were potentially predictive of benefit from adjuvant trastuzumab when administered concurrently with chemotherapy in the NCCTG N9831 HER2+ phase III trial. Purpose: We were interested in determining whether MYC is the target protein responsible for the suggested benefit from trastuzumab. We evaluated the association between MYC protein expression and disease-free survival (DFS) of pts randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) using N9831 TMAs. Patients/Methods: This analysis included 1220 pts from Arms A, B, and C. The percentage of strong (3+) nuclear staining was determined in TMA sections containing 3 cores per block using immunohistochemistry (IHC; clone 9E10, Sigma-Aldrich, St. Louis, MO). A patient's tumor was considered positive (MYC+) if the maximum nuclear 3+ staining percentage across cores was &amp;gt;30% a priori. Median follow-up was 5.8 years. Results: Of 1220 pts with completed IHC analyses, 557 (46%) were MYC+. MYC+ was associated with a higher rate of hormone receptor positivity (58% vs 48%, chi-sq P&amp;lt;0.001) and higher rate of nodal positivity (92% vs 84%, chi-sq P&amp;lt;0.001) but not associated with age, race, tumor histology, tumor grade, or tumor size. In comparing DFS between arms C and A, MYC+ and MYC-pts had HRs of 0.46 (p=0.002; A: 187 pts, 57 events, C: 161 pts, 25 events) and 0.70 (p=0.09; A: 213 pts, 59 events, C: 208 pts, 38 events), respectively (interaction p=0.20). In comparing DFS between arms B and A, MYC+ and MYC-pts had HRs of 0.74 (p=0.11; B: 209 pts, 52 events) and 0.81 (p=0.29; B: 242 pts, 53 events), respectively (interaction p=0.80) (Figure). In addition, pts with nuclear MYC staining of 3+ in 0-9%, 10-30%, 31-60%, and 61-100% of cells had HRs (C vs A) of 0.98 (95% CI 0.53-1.82), 0.54 (95% CI 0.30-0.97), 0.55 (95% CI 0.27-1.12), and 0.37 (95% CI 0.19-0.75). Conclusions: In our analysis of 1220 pts, we observed that MYC protein expression positively correlated with hormone receptor positivity and nodal status. We observed a trend towards greater trastuzumab benefit among MYC+ as compared to MYC-pts. A rising trend in trastuzumab benefit was observed with increasing MYC protein expression. Analyses including pts with whole sections and outcome by MYC protein and gene copy number will be presented. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-08.

Increased MYC gene copy number correlates with increased mRNA levels in diffuse large B-cell lymphoma

Translocations involving the MYC gene and increased MYC mRNA levels are associated with poor outcome in diffuse large B-cell lymphoma. However, the presence of increased MYC gene copy number and/or polysomy of chromosome 8 have not been previously described. Utilizing dual color chromogenic in situ hybridization, we investigated MYC gene copy and chromosome 8 centromere numbers in 52 cases of diffuse large B-cell lymphoma. Cases were divided into those with "increased" or "not increased" MYC gene copy number for comparison with MYC mRNA levels, Ki-67 values, and survival. Increased MYC gene copy number was present in 38% of cases. Overall, the average MYC mRNA level was 2398 (range, 342 - 9783) and the percentage of nuclei positive for Ki-67 was 57.5% (range, 20-87%). Within the group with increased MYC copy number, the MYC mRNA values ranged from 816 to 5912 (average, 2843) and the Ki-67 values ranged from 23% to 83% (average, 57%). Within the group with not increased MYC copy number, MYC mRNA values ranged from 342 to 9783 (average, 2118) and the Ki-67 values ranged from 20% to 87% (average, 58%). There was a statistically significant relationship between increased MYC gene copy number and increased MYC mRNA (P=0.034) and a trend toward a relationship between increased mRNA and higher Ki-67 values. This is the first report that low level copy number increases are common in diffuse large B-cell lymphoma and that these changes correlate with MYC mRNA in a statistically significant manner. MYC copy number changes are an additional possible molecular mechanism that may result in increased mRNA and, likely, high proliferation and poor outcome.

Relationship of NKX3.1 and MYC Gene Copy Number Ratio and DNA Hypomethylation to Prostate Carcinoma Stage

Relationship of NKX3.1 and MYC Gene Copy Number Ratio and DNA Hypomethylation to Prostate Carcinoma Stage

MYC amplification in breast cancer: a chromogenic in situ hybridisation study

Aims: To analyse the correlation between MYC amplification and various clinicopathological features and outcome in a cohort of 245 patients with invasive breast carcinoma treated with surgery followed by anthracycline-based...

Relationship of NKX3.1 and MYC Gene Copy Number Ratio and DNA Hypomethylation to Prostate Carcinoma Stage

ObjectiveHigh stage prostate cancers have been reported to frequently harbor chromosome 8 alterations and hypomethylation of LINE-1 retrotransposons. The potential of these parameters for molecular staging of prostate carcinoma was investigated. MethodsHigh molecular weight DNA was extracted from 63 carcinoma tissues (22 pT2, 38 pT3, 3 pT4). Chromosome 8 alterations were followed by determining the ratio of NKX3.1 (at 8p21) to MYC (at 8q24) gene copy numbers (NKX3.1:MYC ratio) using a new real-time PCR technique. LINE-1 hypomethylation was quantified by Southern blot analysis. ResultsIn 42 carcinomas NKX3.1 copy numbers were altered, with decreases in 32 cases. Copy numbers of MYC were increased in 38 cases and diminished in four. The NKX3.1:MYC ratio was altered in 45 specimens, with a decrease in all but two. NKX3.1 loss was associated with tumor stage (p<0.03) and MYC gain with Gleason score (p<0.03). The NKX3.1:MYC ratio was highly significantly associated with tumor stage (p<0.002), displaying 66% sensitivity and 87% specificity. LINE-1 hypomethylation was related (p<0.004) to tumor stage, but exhibited lower sensitivity (59%) and specificity (77%). ConclusionA straightforward PCR technique detecting chromosome 8 alterations might be useful to predict which prostate cancers are organ-confined while determination of hypomethylation appears to be somewhat less well suited.

Application of a Modified Real-Time PCR Technique for Relative Gene Copy Number Quantification to the Determination of the Relationship between NKX3.1 Loss and MYC Gain in Prostate Cancer

Gene amplifications and deletions play an important role in the pathogenesis of solid tumors, including prostate cancer. Real-time PCR is a powerful tool for quantitative DNA analysis, particularly when starting quantities of tumor tissue are minimal (1)(2)(3)(4)(5). In the present study, we describe a modification of the 2−ΔΔCT method, which recently was shown to be suitable for relative gene expression analyses (6). We used our technique to analyze prostate cancer cell lines and tissue samples to determine the relationship between homeodomain-containing transcription factor 3.1 (NKX3.1) and MYC gene copy number alterations in this tumor type. The specimens analyzed included (a) blood samples from healthy donors; (b) the prostate cancer cell lines DU145 (ATCC no. HTB-81) and PC3 (ATCC no. CRL-1435) and their derived sublines DU145MN1, PC3-N, and PC3-125-1L (7); (c) the colorectal cell line COLO320DM (ATCC no. CCL-220), which harbors a high-level MYC amplification; and (d) primary prostate adenocarcinoma samples obtained after radical prostatectomy from previously untreated patients. The specimens were histologically verified, and samples were taken as described previously (8). We extracted DNA from the snap-frozen prostate carcinoma samples, blood samples, and cell lines, using the Blood and Cell Culture DNA Midi Kit (Qiagen). We performed quantitative real-time PCR using the LightCycler™ system (Roche Diagnostics) with the FastStart DNA Master SYBR Green I LightCycler Kit (Roche Diagnostics). PCRs were run in duplicate with 20-μL reaction volumes containing 1× SYBR Green I PCR Buffer Mix, 5 mM MgCl2, 0.5 μM each primer, 1× FastStart Taq DNA Polymerase (Roche Diagnostics), and 50 ng of genomic DNA. The cycling conditions are given in Table 1⇓ . The hot start PCR method was applied to prevent incomplete DNA denaturation as discussed by Wilhelm et al. (9). Melting curve analysis was performed …

Molecular cytogenetic characterization of proximal-type epithelioid sarcoma.

Proximal-type epithelioid sarcoma is a recently described soft-tissue tumor that is distinguished from conventional-type epithelioid sarcoma by a far more aggressive clinical course, frequent location in the proximal anatomic regions, and variable rhabdoid morphology. Because of their rarity and peculiar morphology, proximal-type epithelioid sarcomas frequently pose serious diagnostic dilemmas, being easily misdiagnosed as a variety of other malignant neoplasms. To date, the information available on the genetic alterations associated with this tumor entity has been confined to single conventional cytogenetic reports. In this article, we present the results of a conventional and molecular cytogenetic analysis of six proximal-type epithelioid sarcomas. Spectral karyotyping analysis of these cases deciphered the characteristics of several marker chromosomes and complex translocations, leading to the recognition of recurrent rearrangements. The most frequently involved chromosome arm was 22q, and the identification of two cases with a similar translocation, t(10;22), suggests a role for one or more genes on chromosome 22 in the pathogenesis of this tumor and provides an opportunity for finely mapping the translocation-associated breakpoints. Chromosome arm 8q gain was also a frequent event and correlated with gain of MYC gene copy number, as demonstrated by fluorescence in situ hybridization. A review of both cases reported in the literature and those presented in this study reinforced the involvement of chromosomes 8 and 22 and also indicated frequent rearrangements of chromosomes 7, 14, 18, and 20.