C-myc Status Research Articles

Expression and Clinical Significance of Ki-67, CD10, BCL6, MUM1, c-MYC, and EBV in Diffuse Large B Cell Lymphoma Patients.

Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) in adults. Although studies regarding the association between the expression of Ki-67, CD10, BCL6, and MUM1 proteins, as well as c-MYC amplification and EBV status with clinicopathologic characteristics have rapidly progressed, their co-expression and prognostic role remain unsatisfactory. Therefore, this study aimed to investigate the association between the expression of all markers and clinicopathologic features and their prognostic value in DLBCL. Also, the co-expression of markers was investigated. The protein expression levels and prognostic significance of Ki-67, CD10, BCL6, and MUM1 were investigated with clinical follow-up in a total of 53 DLBCL specimens (including germinal center B [GCB] and activated B cell [ABC] subtypes) as well as adjacent normal samples using immunohistochemistry (IHC). Besides, the clinical significance and prognostic value of c-MYC and EBV status were also evaluated through chromogenic in situ hybridization (CISH), and their correlation with other markers was also assessed. The results demonstrated a positive correlation between CD10 and BCL6 expression, with both markers being associated with the GCB subtype ( P< 0.001 and P =0.001, respectively). Besides, we observe a statistically significant association between MUM1 protein expression and clinicopathologic type ( P< 0.005) as well as a positive association between c-MYC and recurrence ( P =0.028). Our survival analysis showed that patients who had responded to R-CHOP treatment had better overall survival (OS) and progression-free survival (PFS) than those who did not. Collectively, this study's results add these markers' value to the existing clinical understanding of DLBCL. However, further investigations are needed to explore markers' prognostic and biological roles in DLBCL patients.

Targeting BCL2 Overcomes Resistance and Augments Response to Aurora Kinase B Inhibition by AZD2811 in Small Cell Lung Cancer.

Therapeutic resistance to frontline therapy develops rapidly in small cell lung cancer (SCLC). Treatment options are also limited by the lack of targetable driver mutations. Therefore, there is an unmet need for developing better therapeutic strategies and biomarkers of response. Aurora kinase B (AURKB) inhibition exploits an inherent genomic vulnerability in SCLC and is a promising therapeutic approach. Here, we identify biomarkers of response and develop rational combinations with AURKB inhibition to improve treatment efficacy. Selective AURKB inhibitor AZD2811 was profiled in a large panel of SCLC cell lines (n = 57) and patient-derived xenograft (PDX) models. Proteomic and transcriptomic profiles were analyzed to identify candidate biomarkers of response and resistance. Effects on polyploidy, DNA damage, and apoptosis were measured by flow cytometry and Western blotting. Rational drug combinations were validated in SCLC cell lines and PDX models. AZD2811 showed potent growth inhibitory activity in a subset of SCLC, often characterized by, but not limited to, high cMYC expression. Importantly, high BCL2 expression predicted resistance to AURKB inhibitor response in SCLC, independent of cMYC status. AZD2811-induced DNA damage and apoptosis were suppressed by high BCL2 levels, while combining AZD2811 with a BCL2 inhibitor significantly sensitized resistant models. In vivo, sustained tumor growth reduction and regression was achieved even with intermittent dosing of AZD2811 and venetoclax, an FDA-approved BCL2 inhibitor. BCL2 inhibition overcomes intrinsic resistance and enhances sensitivity to AURKB inhibition in SCLC preclinical models.

Angiosarcoma of the visceral organs: A morphological, immunohistochemical, and C-MYC status analysis.

Visceral angiosarcomas are rare malignant tumors with poor prognosis. The pathogenesis remains unclear. Herein, we describe the morphological, immunohistochemical, and C-MYC status of a series of visceral angiosarcomas. We evaluated the clinicopathologic and C-MYC status of visceral angiosarcomas (n=12) and compared them to a control series of angiosarcomas arising in cutaneous (n=15) and soft tissue structures (n=15). Clinical follow-up data were obtained for all patients and exhibited high metastasis and mortality rates. Malignant endothelial cells displayed a range of morphological features including nonepithelioid, epithelioid, and mixed features, forming vasoformative (n=6), solid (n=4) or mixed (n=2) architectures. Epithelioid morphology was present in 4/12 tumors. Mitoses ranged from 3 to 60 per 10 high-power fields. Necrosis was observed in 10/12 tumors. By immunohistochemistry, all angiosarcomas expressed at least 2 markers of endothelial differentiation, including CD31, CD34, vWF, ERG, and Fli-1. Eight cases of C-MYC amplification and 5 cases of C-MYC translocation were detected. Our data showed that visceral angiosarcoma is more common in women, and the clinical presentations of patient age and tumor size were significantly different between the study and control groups. No significant difference in staining between the visceral angiosarcoma and control groups was observed for endothelial markers, while different C-MYC statuses were detected.

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.

Differentiated approach in the treatment of diffuse large B-cell lymphoma according to c-MYC and BCL2 status

Background. The treatment results of patients with diffuse large B-cell lymphoma (DLBCL) with c-MYC proto-oncogene and BCL2 protein positivity according to the CHOP-21 ± R scheme remain unsatisfactory, which dictates the need to intensify the therapy regimen. Due to the fact that most authors combine DH (double hit) lymphomas and DE (double expressor) lymphomas for analysis, these data do not allow forming an accurate idea of the independent significance of the phenomenon of activated transcription factor c-MYC and BCL2 protein in patients with DLBCL and the results of treatment specifically in this group. Some researchers believe that the presence of c-MYC gene aberration and BCL2 protein expression are associated with negative clinical characteristics. Nevertheless, the clinical features of this subgroup of DLBCL remain poorly understood.The objective of the study was to analyze the incidence of c-MYC gene aberrations, BCL2 expression in DLBCL patients and to evaluate the effectiveness of intensive R + Hyper-CVAD chemotherapy regimen in this group of patients.Materials and methods. We analyzed the data of 80 patients aged 18–65 years with a newly diagnosed DLBCL for the period from 2018 to 2020. The diagnosis was established by histological and immunohistochemical examination. The tumor stages (II–IV) were assessed according to the Ann Arbor classification; the patient’s somatic status – according to the ECOG scale with a total of no more than 3 points. To detect c-MYC gene aberrations in the tumor tissue, fluorescent in situ hybridization was performed; to determine the BCL2 protein expression and Ki-67 proliferative index – an immunohistochemical study was performed. Patients with identified c-MYC gene aberrations received treatment, which included 8 courses of polychemotherapy according to the R + Hyper-CVAD scheme and high-dose chemotherapy according to the BFR scheme with autologous hematopoietic stem cell transplantation. Patients with DLBCL, in whom c-MYC gene aberration was not detected, were assigned to the group of intermediate and low malignancy. They received a polychemotherapy regimen according to the R-CHOP-21 scheme (up to 8 courses).Results and conclusion. For the first time in Kazakhstan, we studied the incidence of c-MYC proto-oncogene aberrations, BCL2 protein expression and proliferative activity (Ki-67) in DLBCL patients, which made it possible to identify groups of highly aggressive tumor and intermediate, low malignancy, apply personalized polychemotherapy programs and evaluate the immediate treatment efficacy. This approach made it possible to increase treatment effectiveness in the group of patients with highly aggressive DLBCL and to achieve comparable results with therapy in patients with a more favorable prognosis.

The green tea polyphenol EGCG is differentially associated with telomeric regulation in normal human fibroblasts versus cancer cells

Introduction: Topical investigations have demonstrated that oxidative stress and inflammation play key roles in biological aging and determine incidence and course of age-related diseases. Lifestyle and environmental factors hugely impact epigenetic regulation and DNA stability with telomere attrition and epigenetic instability providing a potential record of the cumulative burden of endogenous and exogenous oxidative noxae. Certain physiologically active plant components exhibit antioxidative activities affecting epigenetic regulation of inflammation response and DNA repair.Methods: Against this background, the present study investigated green tea polyphenol epigallocatechin gallate (EGCG) in the context of telomere regulation in Caco-2 colorectal adenocarcinoma cells vs. ES-1 primary skin fibroblasts. Cell lines were treated with 20 and 200 µM EGCG for 36, 72 and 144 hours, respectively. Telomerase activity, relative telomere length as well as methylation status of hTERT and c-Myc from different culture conditions were assessed. Malondialdehyde (MDA) served as a surrogate marker of potential pro-oxidative effects of EGCG in a physiologically relevant tissue model.Results: EGCG incubation was associated with telomere shortening and decreased telomerase activity in Caco-2 cells, and relatively longer telomeres along with increased methylation of six 5'—C—phosphate—G—3' (CpG) sites in the promoter region of human Telomerase Reverse Transcriptase (hTERT) in fibroblasts. At low concentrations, EGCG significantly decreased oxidative damage to lipids in Caco-2 cells and attenuated H2O2 induced oxidation at higher concentrations.Conclusion: These results suggest differential EGCG-mediated telomeric modulation in cancer vs. primary cells and a specific antioxidant activity of EGCG against oxidative damage to lipids in abnormal cells.Keywords: Caco-2, epigallocatechin gallate, telomeres, hTERT, DNA methylation, telomerase, oxidative stress, malondialdehyde

Detection of Post-translational Modifications on MYC.

Detection of post-translational modifications in c-Myc is an invaluable tool in assessing Myc status, particularly in cancer. However, it can be challenging to detect these modifications. The evaluation of phosphorylation status of c-Myc can also be challenging with the current commercially available phosphorylation sensitive antibodies. Here, we describe protocols for the immunoprecipitation of endogenous c-Myc to probe for phosphorylation status, as well as the detection of ubiquitination and SUMOylation on c-Myc. We will also discuss the challenges of detecting phosphorylated c-Myc in formalin-fixed paraffin-embedded tissues by immunofluorescence and describe a protocol using a new rat monoclonal antibody we have generated suitable for this purpose.

MS09.02 Clincal and Molecular Biomarkers for Selection of Sclc Patients Candidate to Immunecheckpoint Blockade

Immunotherapy has dramatically altered the treatment options available to patients with lung cancer. In the past year, small cell lung cancer (SCLC) fully joined the immunotherapy era with approvals by the US Federal Drug Administration (FDA) for three separate immune checkpoint inhibitors – atezolizumab for frontline therapy in patients with extensive stage SCLC (ES-SCLC) (in combination with platinum-etoposide chemotherapy) and nivolumab and pembrolizumab (each as monotherapy) for relapsed SCLC. The combination of nivolumab plus ipilimumab also demonstrated durable activity in a subset of patients treated on the Phase 1/2 CheckMate032 trial, with an overall response rate of 22% and a 2 year overall survival rate of 26%.1 The addition of atezolizumab to carboplatin and etoposide as a new standard of care was based on results from the Phase 3 IMpower133 trial.2 In that randomized trial, the addition of atezolizumab to carboplatin-etoposide, followed by atezolizumab maintenance, led to improvements in both progression free survival (PFS) (4.3 months in the placebo control arm versus 5.2 months in patients receiving atezolizumab) and overall survival (OS) (10.3 months with placebo versus 12.3 months with atezolizumab). Additional randomized trials testing other immune checkpoint inhibitors in combination with standard platinum-etoposide chemotherapy are ongoing, with clinical findings expected in the next several months. This includes the phase 3 trial of durvalumab plus platinum-etoposide (CASPIAN), which has now been reported to show improved overall survival (OS) with the addition of durvalumab at a planned interim analysis (press release). Despite these landmark approvals for immune checkpoint inhibitors in ES-SCLC, a large number of patients with SCLC do not appear to receive clinic benefit with the currently available inhibitors of PD-1/PD-L1 and/or CTLA-4. Furthermore, there are not yet established biomarkers for identifying those patients with SCLC who are likely to respond. As with non-small cell lung cancer, immunohistochemistry (IHC) for PD-L1 levels and tumor mutation burden (TMB) are both candidate biomarkers.3,4 However, neither of these have been prospectively validated to date in SCLC and there may be important differences in their performance depending on how testing is done (e.g., variation between antibodies, scoring methods/cutoffs, or technical differences between molecular platforms). Recently, new combinations of targeted therapies together with immune checkpoint inhibitors (such as inhibitors of DNA damage response (DDR) such as PARP1 or Chk1 to enhance STING pathway activation) have demonstrated promise in preclinical studies of SCLC and are being translated into the clinic for further investigation.5 In addition, other new immunotherapeutic approaches are being tested in ongoing trials. Examples of these include studies of chimeric antigen receptor T-cells (CAR-T) and bi-specific T-cell engagers (BiTE molecules) targeting the notch inhibitor ligand DLL3 for patients with relapsed SCLC. In this context, additional biomarkers related to specific combinations of targeted and immune-therapies and/or new classes of immunotherapy (e.g., SLFN11 levels for PARP inhibitors; cMyc status for Chk1 inhibitors; markers of STING pathway activation; or DLL3 expression levels) may emerge as additional biomarkers relevant to immune responses. Finally, a better understanding of tumor and immune environment heterogeneity between patients – as well as intra-tumoral heterogeneity – will lead to more effective strategies for matching patients to specific immunotherapies and overcoming immunotherapy resistance. REFERENCES 1. Della Corte CM, Gay CM, Byers LA. Beyond chemotherapy: Emerging biomarkers and therapies as small cell lung cancer enters the immune checkpoint era. Cancer 2019;125:496-8. 2. Horn L, Mansfield AS, Szczesna A, et al. First-Line Atezolizumab plus Chemotherapy in Extensive-Stage Small-Cell Lung Cancer. N Engl J Med 2018;379:2220-9. 3. Antonia SJ, Lopez-Martin JA, Bendell J, et al. Nivolumab alone and nivolumab plus ipilimumab in recurrent small-cell lung cancer (CheckMate 032): a multicentre, open-label, phase 1/2 trial. Lancet Oncol 2016;17:883-95. 4. Hellmann MD, Callahan MK, Awad MM, et al. Tumor Mutational Burden and Efficacy of Nivolumab Monotherapy and in Combination with Ipilimumab in Small-Cell Lung Cancer. Cancer Cell 2018;33:853-61 e4. 5. Sen T, Rodriguez BL, Chen L, et al. Targeting DNA Damage Response Promotes Antitumor Immunity through STING-Mediated T-cell Activation in Small Cell Lung Cancer. Cancer Discov 2019;9:646-61. Immunotherapy, biomarkers, small cell lung cancer (SCLC)

Impact of Double- or Triple-Hit Pathology on Rates and Durability of Radiation Therapy Response Among Patients With Relapsed or Refractory Large B-Cell Lymphoma

PurposeDouble-hit lymphomas and triple-hit lymphomas (DHL/THL), also known as high-grade B-cell lymphoma with MYC and BCL2 or BCL6 rearrangements, are associated with chemoresistance and inferior survival. However, whether radiation therapy (RT) efficacy is altered in DHL/THL is less well characterized. Among patients with relapsed or refractory large B-cell lymphoma (R/R LBCL), we compared rates and durability of response between patients with and without DHL/THL. Methods and MaterialsWe retrospectively reviewed consecutive R/R LBCL patients who were irradiated at a single institution from January 2008 to June 2017. Patients in whom c-MYC rearranged status was known were evaluated for response to RT, in-field control, progression-free, and overall survival. ResultsAmong 245 irradiated patients with LBCL, 41 patients with confirmed c-MYC status were treated for R/R disease (14 DHL/THL, 27 non-DHL/THL) and formed our cohort. Compared with non-DHL/THL, more DHL/THL patients had progressive disease at RT (71% vs 48%), had larger gross tumor volumes (GTV; median 696 mL vs 117 mL), and were treated with palliative intent (71% vs 41%). Despite similar RT doses (median 35 Gy), radiographic complete response rate was lower among DHL/THL patients: 14.3% versus 64.7% (P = .01). With a median 2 years of follow-up, one in-field failure was observed in each group. DHL/THL patients had inferior progression-free survival (7% vs 46%; P = .02) and overall survival (14% vs 68%; P = .03) at 6 months. ConclusionsR/R LBCL is responsive to RT, although rates of response are lower among DHL/THL patients. Given poor survival after RT, in-field control was hard to evaluate in this cohort. Larger cohorts are required to better elucidate whether differences in response rates are driven by larger disease burden at RT versus tumor biology. These findings are of increasing pertinence in light of use of RT as bridging therapy to cellular immunotherapies.

Impact of Double/Triple Hit Lymphoma on Radiation Therapy Efficacy Among Patients with Relapsed/Refractory Diffuse Large B Cell Lymphoma

Double/triple-hit lymphomas (DHL/THL), recently recognized in the WHO 2017 as high grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement, are associated with chemoresistance, relapsed/refractory (R/R) disease, and worse survival. While radiation therapy (RT) is an effective treatment for R/R diffuse large B-cell lymphoma (DLBCL), its efficacy for DHL/THL is unknown. As RT efficacy may be associated with chemosensitivity, we hypothesized that R/R DLBCL patients with DHL/THL may have lower rates of response to RT compared to non-DHL/THL patients. We retrospectively reviewed patients with R/R DLBCL irradiated at a single institution between 1/2008 and 6/2017. Patients without confirmed c-MYC translocation status (our institution’s standard for DHL/THL screening) were excluded. Among patients irradiated to gross disease, radiographic assessment of RT response within the RT portal was categorized as complete/partial response (CR/PR), stable disease (SD), and progressive disease (PD). Local control (LC), distant failure (DF), progression-free (PFS) and overall survival (OS) were calculated with Kaplan Meier methods. Among 244 irradiated patients with DLBCL, 80 patients had confirmed c-MYC status. Forty-nine had R/R disease (15 DHL/THL, 34 non-DHL/THL) and formed our cohort (Table). Compared with non-DHL/THL patients, DHL/THL patients were more likely male, have germinal center B-cell like (GCB) cell of origin, and less likely to receive curative-intent RT, despite receiving a similar dose of RT. Most (90%) were irradiated to gross disease. With a median follow up of 184 days, over half (59.5%) achieved a CR/PR to RT, which was higher among non-DHL/THL versus DHL/THL patients: 70.8% vs 33.3%. Notably, 60% of DHL/THL patients had SD after RT. One patient with non-DHL/THL progressed within the RT field. DHL/THL patients had worse DF (79% vs 45%; p=0.001), PFS (20% vs 44%; p=0.021), and OS (20% vs 65%; p=0.081) at 6 months. Acute toxicity was grade 3 (dermatitis, hematologic, nausea, dysphagia) in 10.2% and grade 4 (tumor lysis syndrome) in 2%. RT appears to be a well-tolerated and effective treatment for R/R DLBCL patients. While DHL/THL patients had lower response rates to RT, none had PD within the RT field. These findings may be driven, in part, by the short survival of DHL/THL patients after RT and require confirmation in other cohorts.Abstract 255; TableSummary of clinical and treatment characteristicsAll (N=49)Non DHL/THL (N=34)DHL/THL (N=15)Median age at diagnosis, years Male sex Primary chemo-refractory GCB subtype Median # chemo before RT (range) % stem cell transplant59.1 67.3% 44.9% 51.3% 2 (1-6) 42.9%58.9 61.8% 47.1% 41.2% 2 (1-6) 41.2%59.7 80% 40.0% 86.7% 2 (1-4) 46.7%Curative (vs palliative)RT intentRT to gross (vs microscopic) diseaseMedian RT dose (range)46.9% 90% 35 Gy (4-57)52.9% 88.2% 34.6 Gy (4-57)33% 93.3% 36 Gy (20-45) Open table in a new tab

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.

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 642: Comparison between in situ hybridization and digital polymerase chain reaction methods for detecting c-MYC gene copy number gain in tissue and cell-free plasma samples of colorectal cancer patients

Abstract Introduction: We focused on the utility of the droplet digital polymerase chain reaction (ddPCR) for detecting c-MYC gene copy number (GCN) gain in cell-free plasma and tumor tissue of CRC patients. Methods: c-MYC GCN was determined by using dual-color silver in situ hybridization (SISH) and ddPCR in retrospective cohort of 193 CRC patients (cohort 1) and prospective cohort of 64 CRC patients (cohort 2). Additionally, c-MYC GCN was analyzed by ddPCR method in cell-free plasma samples of cohort 2. Results: In cohort 1, c-MYC GCN gain, defined as mean c-MYC copies/nucleus ≥ 4.0 in SISH analysis, was observed in 34 (17.5%), and c-MYC GCN gain by ddPCR method in 7 (3.6%). c-MYC GCN by SISH was significantly correlated with ddPCR results (ρ= 0.532, P &amp;lt; 0.001). Forty cases (20.7%) showed intratumoral genetic heterogeneity, but it did not affect the concordance between SISH and ddPCR results (P &amp;gt; 0.05). c-MYC GCN gain by both SISH and ddPCR methods had the worst prognosis (P = 0.001). In cohort 2, c-MYC GCN by SISH was significantly associated with tissue ddPCR (ρ=0.349, P = 0.005), but not with plasma ddPCR (P = 0.620). Intratumoral genetic and regional heterogeneity of c-MYC status have been found in 29 (45.3%) and 8 (12.7%). c-MYC status in plasma was associated with tissue ddPCR, but borderline statistical significance (ρ= 0.246, P = 0.050). Conclusions: Our findings suggested that results by SISH and ddPCR methods were discordant. Therefore, detecting method should be carefully selected to determine c-MYC GCN status. Citation Format: Soo Hyun Seo, Kyu Sang Lee, Soo Kyung Nam, Kyoung Un Park, Heung-Kwon Oh, Duck-Woo Kim, Sung-Bum Kang, Hye Seung Lee. Comparison between in situ hybridization and digital polymerase chain reaction methods for detecting c-MYC gene copy number gain in tissue and cell-free plasma samples of colorectal cancer patients [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 642.

Primary Failure Diffuse Large B Cell Lymphoma: Early Autologous or Donor Hematopoietic Cell Transplantation Not Effective in Patients with Ultra-High Risk Features

Introduction: Modern chemoimmunotherapy cures the majority of patients (pts) with newly diagnosed diffuse large B cell lymphomas (DLBCL). The paradigm for pts who relapse has been salvage chemotherapy followed by autologous hematopoietic cell transplantation (auto-HCT) when chemosensitive disease. This approach is often also applied to pts with primary treatment failure (PTF), although this group is heterogeneous and outcomes of HCT in this population have not been well described.Methods: Fifteen US academic medical centers contributed pts to the REgistry of diFfuse large B cell lymphoma with prImary treatmeNt failurE (REFINE) collaboration. REFINE retrospectively captured patient, disease and treatment characteristics and response as assessed by treating center. Eligible pts were ≥ 18 years diagnosed with DLBCL during 2008-2015, who received upfront therapy including anthracycline and CD20-directed antibody and developed one of 3 patterns of PTF: relapse < 6 months following CR (early relapse- ER); only partial remission (PR) or stable disease (SD) with upfront therapy (residual disease-RD); progressive disease (PD) while receiving upfront therapy (primary progression-PP). Pts with HIV infection, primary CNS lymphoma or lymphoma transforming from a more indolent histology were excluded.Results: Among 331 cases included in REFINE, patterns of PTF were ER in 95 (28.7%), RD in 92 (27.8%) and PP in 144 (43.5%) pts. Salvage therapy was administered to 94.6% of pts. We analyzed the 157 (47.4%) PTF pts who subsequently underwent HCT, 132 auto-HCT and 33 allo-HCT (8 after failure of auto-HCT) (Table). Nearly half of pts were in CR at transplant, after 1-2 lines of salvage therapy. Among all pts receiving auto-HCT, 2-year progression-free survival (PFS) from time of transplant was 38.4% (95% C.I. 29.6-47.2%) and overall survival (OS) 54.9% (95% C.I. 44.9-64.9%). Two-year OS from auto-HCT was affected by pattern of PTF: 71.3% for ER, 55.0% for RD and 41.7% for PP (P=0.05). OS at 2-years was 23.7% for auto-HCT pts with germinal center B (GCB)type,c-myc(+) (by FISH) tumors vs. 66.7% for GCB, c-myc (-), vs. 67.8% for non-germinal center (NGC) (P=0.01). Auto-HCT pts with intermediate-high/high NCCN-IPI at time of PTF had 2-year OS of 28.6% vs. 66.0% for intermediate-low and 80.9% for low risk (P<0.001). In multivariate analysis, PP pattern, c-myc(+) status and intermediate-high/high NCCN-IPI at time of PTF, hereforth called ultra-high-risk (UHR) fetures, negatively affected survival. Auto-HCT pts with no UHR features had 2-year OS of 74.3%(95% C.I. 60.0-88.6%)vs. 59.6% (95% C.I. 44.5-74.7%)for pts with 1 vs. 10.7% (95% C.I. 0-24.4%)for pts with 2-3 features (Figure, P<0.001). Among pts who underwent allo- HCT, 2-year PFS and OS were 20.5% (95% C.I. 6.4-34.6%)and 32.7% (95% C.I. 13.1-52.3%)respectively. Failure of allo-HCT was primarily due to disease progression, with only one death occuing due to HCT complications.Conclusions: Pts with DLBCL experiencing PTF and 2 or more UHF features have dismal outcome after auto-HCT and should be targeted for experimental modalities of cellular therapy. Outcomes of allo-HCT in DLBCL with PTF are poor, due to rapid and fatal relapses. [Display omitted] [Display omitted] DisclosuresCosta:Sanofi: Honoraria, Research Funding. Reddy:INFINITY: Membership on an entity's Board of Directors or advisory committees; KITE: 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. Karmali:Celgene: Speakers Bureau. Chavez:Janssen: Speakers Bureau. Calzada:Seattle Genetics: Research Funding. Barta:Janssen: Honoraria, Speakers Bureau; Celgene, Merck, Seattle Genetics: Research Funding. Flowers:Pharmacyclics, LLC, an AbbVie Company: Research Funding; Mayo Clinic: Research Funding; Acerta: Research Funding; Roche: Consultancy, Research Funding; TG Therapeutics: Research Funding; ECOG: Research Funding; Genentech: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; NIH: Research Funding; Infinity: Research Funding; Millenium/Takeda: Research Funding; AbbVie: Research Funding. Fenske:Seatle Genetics: Honoraria; Pharmacyclics: Honoraria; Celgene: Honoraria; Millennium/Takeda: Research Funding. Cohen:Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding. Hamadani:Takeda: Research Funding.

Long Non-coding RNA HOTAIR Expression in Diffuse Large B-Cell Lymphoma: In Relation to Polycomb Repressive Complex Pathway Proteins and H3K27 Trimethylation.

BackgroundA long non-coding RNA hox transcript antisense intergenic RNA (HOTAIR) is involved in epigenetic regulation through chromatin remodeling by recruiting polycomb repressive complex 2 (PRC2) proteins (EZH2, SUZ12, and EED) that induce histone H3 trimethylation at lysine 27 (H3K27me3). Deregulation of c-MYC and interaction between c-MYC and EZH2 are well known in lymphomagenesis; however, little is known about the expression status of HOTAIR in diffuse large B-cell lymphomas (DLBCLs).MethodsThe expression status of PRC2 (EZH2, SUZ12, and EED), H3K27me3, c-MYC, and BCL2 was analyzed using immunohistochemistry (n = 231), and HOTAIR was investigated by a quantification real-time polymerase chain reaction method (n = 164) in DLBCLs.ResultsThe present study confirmed the positive correlation among PRC2 proteins, H3K27me3, and c-MYC in DLBCLs. Expression level of HOTAIR was also positively correlated to EZH2 (p < .05, respectively). Between c-MYC and HOTAIR, and between c- MYC/BCL2 co-expression and HOTAIR, however, negative correlation was observed in DLBCLs (p < .05, respectively). High level of H3K27me3 was determined as an independent prognostic marker in poor overall survival (hazard ratio, 2.0; p = .023) of DLBCL patients. High expression of HOTAIR, however, was associated with favorable overall survival (p = .004) in the univariate analysis, but the impact was not significant in the multivariate analysis. The favorable outcome of DLBCL with HOTAIR high expression levels may be related to the negative correlation with c- MYC expression or c-MYC/BCL2 co-expression.ConclusionsHOTAIR expression could be one of possible mechanisms for inducing H3K27me3 via EZH2-related PRC2 activation, and induced H3K27me3 may be strongly related to aggressive DLBCLs which show poor patient outcome.

C-MYC Copy-Number Gain Is an Independent Prognostic Factor in Patients with Colorectal Cancer.

BackgroundThe aim of this study was to determine the incidence and clinicopathological significance of c-MYC gene copy-number (GCN) gain in patients with primary colorectal cancer (CRC).MethodsThe c-MYC GCN was investigated in 367 consecutive CRC patients (cohort 1) by using dual-color silver in situ hybridization. Additionally, to evaluate regional heterogeneity, we examined CRC tissue from 3 sites including the primary cancer, distant metastasis, and lymph-node metastasis in 152 advanced CRC patients (cohort 2). KRAS exons 2 and 3 were investigated for mutations.ResultsIn cohort 1, c-MYC gene amplification, defined by a c-MYC:centromere of chromosome 8 ratio ≥ 2.0, was detected in 31 (8.4%) of 367 patients. A c-MYC GCN gain, defined by ≥ 4.0 c-MYC copies/nucleus, was found in 63 (17.2%) patients and was associated with poor prognosis (P = 0.015). Multivariate Cox regression analysis showed that the hazard ratio for c-MYC GCN gain was 2.35 (95% confidence interval, 1.453–3.802; P < 0.001). In a subgroup of stage II-III CRC patients, c-MYC GCN gain was significantly associated with poor prognosis by univariate (P = 0.034) and multivariate (P = 0.040) analyses. c-MYC protein overexpression was observed in 201 (54.8%) out of 367 patients and weakly correlated with c-MYC GCN gain (ρ, 0.211). In cohort 2, the c-MYC genetic status was heterogenous in advanced CRC patients. Discordance between GCN gain in the primary tumor and either distant or lymph-node metastasis was 25.7% and 30.4%, respectively. A similar frequency for c-MYC GCN gain and amplification was observed in CRC patients with both wild-type and mutated KRAS.Conclusions c-MYC GCN gain was an independent factor for poor prognosis in consecutive CRC patients and in the stage II-III subgroup. Our findings indicate that the status of c-MYC may be helpful in predicting the patients’ outcome and for managing CRC patients.

Assessment of global and gene-specific DNA methylation in rat liver and kidney in response to non-genotoxic carcinogen exposure

Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC–MS/MS in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity.

Abstract P6-05-27: p53, c-Myc and EGFR protein interactions confer worse prognosis in patients with triple negative breast cancer (TNBC)

Abstract Background - aim: TNBC is a heterogeneous disease comprising different molecular subtypes that are mostly defined by gene expression profiling and are related to patient outcome. Herein, we investigated the prognostic significance of molecular subtype-related, surrogate protein markers. Methods: Immunohistochemistry was applied on tissue microarrays containing a series of 330 centrally assessed TNBC from early high-risk breast cancer patients treated with anthracyclines and/or taxanes. The following markers were examined (in parentheses: cut-offs for low/high and negative/positive): Ki67 (14%); p53 (10%); c-Myc (10%); P-cadherin (10%); E-cadherin (10%); Claudin-7 (H-score median: 105); CK5 (1%); EGFR (1%); androgen receptor (AR [1%]). Results: The majority of tumors (269/327, 81.5%) were grade-III ductal carcinomas of no special type. Tumors were frequently positive for Ki67 (239/314, 76.1%), E-cadherin (193/271, 71.2%), P-cadherin (249/302, 82.5%) and expressed markers of the basal phenotype (CK5+ and/or EGFR+, 268/316, 84.8%). Grade-III, Ki67-high carcinomas and tumours positive for E- and P-cadherin were more prevalent among those of basal phenotype (Fisher's exact p&amp;lt;0.001). Tumors expressing low levels of Claudin-7 were infrequently positive for AR and EGFR proteins (p = 0.015 and p = 0.003, respectively). AR positivity was more frequent among CK5 negative, grade I-II, and Ki67-low tumors (p = 0.021, p = 0.05 and p = 0.011 respectively). When examined individually, none of these markers was related to patient outcome. An interaction was noticed between p53 and c-Myc immunopositivity (Wald's p = 0.028 for disease-free [DFS] and p = 0.005 for overall survival [OS]). Among patients with c-Myc negative tumors, patients with p53 positive tumors (n = 63) had shorter median DFS (46.8 months) and OS (56 months) (HR 1.9; 95%CI 1.0-3.9; p = 0.049 and HR 2.28; 95%CI 1.0-4.8; p = 0.031, respectively) as compared to those with p53 negative tumors (n = 56; median DFS 69 months, OS 70 months). Similarly, in the absence of p53, c-Myc expression correlated with shorter median OS (44.5 months) (HR 2.1; 95%CI 1.0-4.5; p = 0.038) but showed only a trend for shorter DFS as compared to c-Myc negative tumors. p53 protein status was a discriminating factor for EGFR negative tumors (Wald's p = 0.052), whereby p53 positive tumors (n = 54) were associated with reduced DFS as compared to p53 negative tumors (n = 68; median DFS: 47 vs. 68.8 months; HR 2.1; 95%CI 1.1-4.0; p = 0.025). Accordingly, EGFR immunopositivity conferred a trend for shorter DFS within p53 negative cases. Conclusions: In the current study, it was not possible to assign prognostic significance to individual proteins that have been introduced as surrogate markers for gene expression-based molecular subtypes in TNBC. By contrast, significant interactions involving the status of p53 and c-Myc or EGFR were observed. It seems that aberrant p53 adversely influences tumor behaviour in the absence of c-Myc and EGFR, while the opposite is also effective but to a lesser extent. These findings are hypothesis generating and need to be pursued in sufficiently powered studies for validating the apparently mutually exclusive adverse effects of p53, c-Myc and EGFR on tumor behaviour. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-27.

TP53 and C-MYC Co-Alterations – A Hallmark of Oral Cancer Progression / SIMULTANA ALTERACIJA TP53 I C-MYC GENA – OBELEŽJE PROGRESIJE ORALNIH KARCINOMA

Summary Background: Head and neck squamous cell carcinoma, including oral cancer, is the sixth most common cancer worldwide. Despite advances in surgery and treatment, the 5-year survival rate has not improved significantly. There- fore, reliable molecular markers for oral cancer progression are badly needed. Methods: We conducted a copy number analysis to esti- mate amplification status of c-myc, cycD1 and EGFR onco- genes, mutational PCR-SSCP analysis to determine activa- tion of H-ras oncogene and inactivation of TP55 tumour suppressor gene and methylation specific PCR analysis to evaluate hypermethylation of p16 and MGMT genes. Results: c-myc oncogene was amplified in 56.7%, cycD1 in 20% and EGFR in 16.7% of Oral Squamous Cell Carci- noma (OSCC) cases while H-ras was activated in 33.3% of samples. Amplification of c-myc was significantly associat- ed with the tumour grade 2. Interestingly, EGFR and H-ras alterations were mutually exclusive. p16 and MGMT were inactivated by hypermethylation in 30% and 13.3% of cases. Co-alteration of cycD1 and p16 were not observed in any of the analyzed samples. TP53 was inactivated in 56.7% of samples and was significantly associated with progression of OSCC, grade 2 and stage 2. Moreover, TP55 and c-myc oncogene were simultaneously altered in grade 2 OSCC. Conclusions: The most promising marker of OSCC pro- gression remains the TP53 tumour suppressor, which is the most frequently mutated gene in oral cancers. Since there is synergism between TP55 and c-myc, it seems that co- alteration of these two genes could be also a good marker of OSCC progression from gradel to grade 2 tumours.

A Re-Emerging Marker for Prognosis in Hepatocellular Carcinoma: The Add-Value of FISHing c-myc Gene for Early Relapse

Hepatocellular carcinoma is one leading cause of cancer-related death and surgical resection is still one of the major curative therapies. Recently, there has been a major effort to find mechanisms involved in carcinogenesis and early relapse. c-myc gene abnormality is found in hepatocarcinogenesis. Our aim was to analyze the role of c-myc as prognostic factor in terms of overall survival and disease-free survival and to investigate if c-myc may be an important target for therapy. We studied sixty-five hepatocellular carcinomas submitted to surgical resection with curative intent. Size, macro-microvascular invasion, necrosis, number of nodules, grading and serum alfa-fetoprotein level were registered for all cases. We evaluated the c-myc aberrations by using break-apart FISH probes. Probes specific for the centromeric part of chromosome 8 and for the locus specific c-myc gene (8q24) were used to assess disomy, gains of chromosomes (polysomy due to polyploidy) and amplification. c-myc gene amplification was scored as 8q24/CEP8 > 2. Statistical analysis for disease-free survival and overall survival were performed. At molecular level, c-myc was amplified in 19% of hepatocellular carcinoma, whereas showed gains in 55% and set wild in 26% of cases. The 1- and 3-year disease-free survival and overall survival for disomic, polysomic and amplified groups were significantly different (p=0.020 and p=.018 respectively). Multivariate analysis verified that the AFP and c-myc status (amplified vs. not amplified) were significant prognostic factors for overall patients survival. c-myc gene amplification is significantly correlated with disease-free survival and overall survival in patients with hepatocellular carcinoma after surgical resection and this model identifies patients with risk of early relapse (≤12 months). We suggest that c-myc assessment may be introduced in the clinical practice for improving prognostication (high and low risk of relapse) routinely and may have be proposed as biomarker of efficacy to anti-c-myc targeted drugs in clinical trials.