MYC Protein Overexpression 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.

MYC overexpression in natural killer cell lymphoma: prognostic and therapeutic implications.

The current clinical management of extranodal natural killer (NK)/T-cell lymphoma (ENKTL) primarily depends on conventional chemotherapy and radiotherapy, underscoring the need for innovative therapeutic strategies. This study explores the clinical significance and therapeutic implication of c-MYC (MYC) in ENKTL. Initially, we identified MYC protein overexpression in approximately 75% of cases within a large cohort of 111 patients. MYC overexpression was strongly correlated with lymphoma cell proliferation and poor clinical outcomes. Intriguingly, integrating MYC expression into the prognostic index of NK cells lymphoma with Epstein-Barr virus (PINK-E) prognostic model significantly enhanced its predictive power. Subsequently, we implemented MYC knockdown in NK malignancy cell lines with MYC overexpression, resulting in significant viability reduction. RNA sequencing used to determine MYC function revealed a high overlap with canonical MYC-regulated genes and enrichment in metabolism and cell cycle regulation. Integrative analysis of the RNA-sequencing data upon MYC knockdown with gene expression profiles of primary ENKTL cases identified a subset of genes closely associated with MYC overexpression. Among these, CDK4 emerged as a potential therapeutic target, and its inhibition not only abrogated MYC function but also decreased MYC expression in NK malignancy cells. Furthermore, the clinical-grade CDK4/6 inhibitor palbociclib exhibited a potent anti-tumor effect in xenograft mouse models, especially when combined with gemcitabine. In summary, our study firmly establishes MYC as an oncogene with prognostic significance in ENKTL and highlights CDK4 inhibition as a promising therapeutic strategy for treating ENKTL with MYC overexpression.

Do Double-Expressor High-Grade B-Cell Lymphomas Really Need Intensified Treatment? A Report from the Real-Life Series of High-Grade B-Cell Lymphomas Treated with Different Therapeutic Protocols at the Institute of Oncology Ljubljana.

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements are known for their aggressive clinical course and so are the ones with MYC and BCL2 protein overexpression. The optimal therapy for these lymphomas remains to be elucidated. A retrospective analysis of all diffuse large B-cell lymphomas and high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements diagnosed between 2017 and 2021 at the Institute of Oncology Ljubljana, Slovenia, has been performed. Only patients with double-expressor lymphoma (DEL), double-hit lymphoma (DHL), or triple-hit lymphoma (THL) were included. Demographic and clinical parameters were assessed, as well as progression-free survival (PFS) and overall survival (OS). In total, 161 cases out of 309 (161/309; 52,1%) were classified as DEL. Sixteen patients had DHL, MYC/BCL2 rearrangement was observed in eleven patients, and MYC/BCL6 rearrangement was observed in five patients. Five patients were diagnosed with THL. Out of 154 patients (according to inclusion/exclusion criteria) included in further evaluation, one-hundred and thirty-five patients had double-expressor lymphoma (DEL), sixteen patients had DHL, and three patients had THL. In total, 169 patients were treated with R-CHOP, 10 with R-CHOP and intermediate-dose methotrexate, 19 with R-DA-EPOCH, and 16 with other regimens. The median follow-up was 22 months. The 5-year OS for the whole DEL group was 57.1% (95% CI 45.9-68.3%) and the 5-year PFS was 76.5% (95% CI 72.6-80.4%). The log-rank test disclosed no differences in survival between treatment groups (p = 0.712) while the high-risk international prognostic index (IPI) carried a significantly higher risk of death (HR 7.68, 95% CI 2.32-25.49, p = 0.001). The 5-year OS for DHL patients was 32.4% (95% CI 16.6-48.2%) while all three TH patients were deceased or lost to follow-up. Our analyses of real-life data disclose that the R-CHOP protocol with CNS prophylaxis is a successful and curative treatment for a substantial proportion of DEL patients.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Burkitt lymphoma (BL) is a rare but highly aggressive B-cell non-Hodgkin lymphoma. BL exhibits a characteristic immunophenotype that is positive for pan-B cell markers and CD10. The genetic hallmark of BL is the translocation of the MYC oncogene under the regulation of an immunoglobulin (IG) heavy or light chain enhancer, resulting in MYC protein overexpression. Notably, MYC translocation by itself is not sufficient for BL oncogenesis and a variety of cancer genes are recurrently mutated. BL can be divided epidemiologically but also, more recently, genetically based on driver mutations including DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Despite high survival rates, pediatric BL patients suffer from long-term side effects and relapses are usually fatal. In order to develop more targeted and less toxic therapies, a better understanding is needed of the ethology of the disease. Therefore, our aim is to characterise the cell-of-origin (COO) and dissect the life history of BL subtypes, by pinpointing when during tumorigenesis somatic mutations play a role and by identifying the rate-limiting steps of malignant transformation. Method: We collected single-cell suspensions of BL patient samples, including lymph node biopsies, bone marrow aspirates, as well as ascites and pleural fluid. To perform flow cytometry, single cells were stained with DAPI and a panel of antibodies: CD10/CD45/CD19 or CD10/CD3/CD20. Live B cells were separated with the DAPI-CD45+ CD19+ or DAPI-CD3-CD20+ phenotype. Subsequently, CD10+ B cells and CD10- B cells were sorted in 96-well plates (Figure 1). To confirm the presence or absence of the IG::MYC translocation a PCR was perfomed using patient-specific primers that flank the translocation locus. Whole genome amplification was carried out on the DNA of single cells using the Primary Template-directed Amplification (PTA) technique that was subsequently whole genome sequenced. Results: From a bone marrow sample of a 4-year-old female BL patient we found that 1% of immune cells were CD10+ B cells, while 17% of B cells were negative for the CD10 marker (Figure 2A). In the lymph node sample from the same patient, we found that 87% of B cells were CD10+ and 9% were CD10- (Figure 2B). PCR confirmed the MYC translocation in 2/6 CD10+ and 0/6 CD10- bone marrow-derived B cells (Figure 3). Keywords: Genomics, Epigenomics, and Other -Omics, Non-Hodgkin (Pediatric, Adolescent, and Young Adult), Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

MYC amplification in angiosarcoma depends on etiological/clinical subgroups – Diagnostic and prognostic value

Angiosarcomas (AS) are rare and heterogeneous malignant vascular tumors with a dismal prognosis. We undertook a retrospective study of AS cases with the intent of elucidating the prevalence of MYC amplification amongst different etiological/clinical subgroups and identifying MYC's potential as a prognostic biomarker.Retrospective collection of data and tumor samples from 110 patients diagnosed with AS in the Netherlands was conducted. Histopathological review and investigation of MYC gene amplification and MYC protein overexpression (using fluorescent in situ hybridization and immunohistochemistry, respectively) was performed.MYC amplification was identified in 50.9 % of cases and predominantly present in secondary AS (sAS) (especially radiotherapy-associated and Stewart-Treves AS, both 92.9 %). Within the primary AS (pAS) subgroup, skin non-UV-associated AS (69.2 %) and primary breast AS (29.4 %) demonstrated MYC amplification. MYC protein overexpression was observed in 47.7 % of all tumors with an overall sensitivity and specificity of 75 % and 81 % respectively. There was no significant difference in median overall survival (OS) between patients with MYC and non-MYC amplified AS. Patients with AS displaying MYC protein overexpression had significantly shorter OS than those without (p = 0.028).Although MYC amplification is characteristic for secondary radiotherapy-associated and Stewart-Treves AS, it is not exclusive to these groups and can also be found in a subset of pAS. Overall concordance of FISH and IHC is rather poor. Only primary breast AS showed 100 % concordance. Therefore MYC expression as surrogate marker should be used with caution. The role of MYC/MYC as a prognostic indicator is undefined, however resulting tailored treatment options could be considered.

Abstract 1111: MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers

Abstract Targeted therapy of ROS1 fusion-driven non-small cell lung cancer (NSCLC) with the tyrosine kinase inhibitor (TKI), crizotinib, has achieved notable clinical success. Despite these promising results, resistance to therapy poses a significant challenge. 47-92% of crizotinib-resistant tumors have a wild-type ROS1 kinase domain suggesting that concurrent aberrations in bypass signaling pathways contribute to sustained cancer growth even in the presence of ROS1 inhibition. Bypass resistance pathways are not well-characterized in ROS1 fusion-driven cancers; hence there is a dearth of secondary therapeutic strategies. To identify recurrent genomic alterations that co-occur with ROS1 fusions and could potentially impinge on sensitivity to crizotinib and other emerging ROS1 TKIs, we interrogated MSK-IMPACT datasets and found that 10% of patients with ROS1-fusion have concurrent MYC copy number amplification (CNA). We hypothesized that c-Myc overexpression compensates for the loss of ROS1 signaling due to ROS1 TKI treatment. Here we dissected the role of c-Myc overexpression in diminishing ROS1 TKI sensitivity using two models: 1) a MYC-amplified and crizotinib-resistant patient-derived ROS1-fusion NSCLC cell line (LUAD0006) and 2) a CRISPR-Cas9 engineered human bronchial epithelial EZR-ROS1 cell line (HBEC-ER) exhibiting sensitivity to ROS1 TKI. We validated LUAD0006 ROS1 TKI-resistance using cell viability assays and confirmed MYC CNA and protein overexpression with fluorescence in situ hybridization and immunoblotting, respectively. Live cell imaging assays revealed that ROS1-TKI or c-Myc knockdown via shRNA alone were insufficient to inhibit LUAD0006 cell growth; however combined ablation of ROS1 kinase activity and c-Myc expression significantly suppressed cell proliferation. Gain of function experiments using HBEC-ER revealed that these cells acquire functional resistance to multiple ROS1 TKIs upon expression of a degradation-resistant c-Myc T58A mutant. Notably, immunoblotting confirmed 50-fold overexpression of c-Myc T58A relative to endogenous c-Myc in unmodified cells. We also performed RNA sequencing and reverse-phase protein array (RPPA) analysis of LUAD0006 cells treated with or without crizotinib and found that targets related to cell cycle, apoptosis, and metabolism were differentially expressed. Finally, we found that CDK4/6 and BET bromodomain inhibitors re-sensitized LUAD0006 cells to crizotinib, demonstrating the importance of c-Myc to the survival of these TKI-resistant cells. Taken together, we demonstrate that CNA or post-translational stabilization that leads to c-Myc overexpression confers resistance to crizotinib and other emerging ROS1 TKIs. These patients will likely benefit from a combination therapeutic strategy that targets both ROS1 and c-Myc. Citation Format: Sudarshan R. Iyer, Igor Odintsov, Adam J. Schoenfeld, Hiroki Sato, Evan Siau, Alexander Drilon, Gregory J. Riely, Romel Somwar, Marc Ladanyi, Monika A. Davare. MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers [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 1111.

Mutations in the Phosphoinositide 3-Kinase Pathway (PI3K) Are Associated with Poor Overall Survival (OS) in Patients with Diffuse Large B-Cell Lymphoma Treated with R-CHOP

Introduction:Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease that is characterized by recurrent translocations and somatic mutations. Prognosis has been associated with clinical presentation, cell-of-origin, and genetic aberrations. The aim of this study was to determine whether somatic mutations are associated with OS in patients with DLBCL who have been treated with R-CHOP therapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone).Methods:We identified 354 patients between 2000-2016 at City of Hope and University of Manitoba with a diagnosis of de novo DLBCL who were treated with R-CHOP therapy. Patients with primary mediastinal large B-cell lymphoma, primary CNS B-cell lymphoma, and those with HIV or immunodeficiency were excluded. A custom targeted panel with 334 genes which included the most frequently mutated genes in B-cell lymphoma was used. Library prep with paired end 100 bp sequencing and 6-10 million reads/case was performed on an Illumina HiSeq 2500. Cases were evaluated by immunohistochemistry (IHC) for cell-of-origin by the Hans algorithm, as well as MYC (>40%) and BCL-2 (>50%) protein overexpression. Fluorescence in situ hybridization analysis also was performed using BCL2/IGH, BCL6/IGH, and MYC probes. Pathway analysis was performed using Gene Ontology and Ingenuity Pathway Analysis (IPA). Overall survival estimates for 5-year survival were estimated using Kaplan Meier methods. Both univariate and multivariable hazard ratios were calculated by Cox regression with two-sided p-values presented. The final multivariable model includes significant terms controlling for patient International Prognostic Index (IPI) score.Results:To date, 215 patients (median age 59 years, Male:Female ratio of 1.44:1) had complete clinical information and tissue available for sequencing. Between the two centers, there were no significant differences in patient demographics, International Prognostic Index (IPI) score, or clinical recurrence status. Germinal center B-cell origin (GCB) type DLBCL was more common (57%) compared to non-GCB type (43%). The most frequently mutated genes were KMT2D (29%), CREBBP (20%), TP53 (20%), TET2 (16%), B2M (14%), and MYD88 (13%), as well as 14 other genes (>10% each). There were 24 (11%) double- or triple-hit lymphomas (MYC, BCL2, and or BCL6) and 35 (16%) double-protein expressors (MYC and BCL2). Mutations in the phosphoinositide 3-kinase/Akt/mTOR pathway (5%, NF1, PIK3CD) (5-yr OS 50% vs 74%, HR = 6.3, p=0.0009), B-cell differentiation genes (13%, BCL6, PRDM1) (5-yr OS 47% vs 77%, HR = 3.1, p=0.0015), ubiquitination pathway (7%, KLHL14, UBR5) (5-yr OS 48% vs 75%, HR = 2.5, p=0.018), and histone and histone modifier genes (31%, HIST1H2BC, KMT2D) (5-yr OS 58% vs 79%, HR = 2.1, p=0.012) were associated with poor OS in DLBCL, independent of IPI score.Conclusion:Targeted sequencing is effective for identifying known mutations associated with the pathogenesis of DLBCL. In this study, mutations involving the PI3K/Akt/mTOR pathway were associated with the worst outcome in patients treated with R-CHOP therapy. Identifying mutations in major pathways may help with prognostication in this heterogenous disease, and provide information useful for targeted therapy. DisclosuresHerrera:Immune Design: Research Funding; Genentech: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Seattle Genetics: Research Funding; BMS: Consultancy, Research Funding; Merck: Consultancy, Research Funding. Pillai:Trillium Therapeutics: Research Funding. Nademanee:seattle Genetics: Speakers Bureau.

Aggressive B-cell Lymphoma with MYC/TP53 Dual Alterations Displays Distinct Clinicopathobiological Features and Response to Novel Targeted Agents.

Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2-targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. IMPLICATIONS: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.

COO, MYC / BCL2 Status and R-IPI Define a Group of Poor Prognosis DLBCL Patients in a Real-World Clinical Setting

BACKGROUND In DLBCL, and mainly in advanced-stage, the presence of an activated B-cell phenotype or a non-GCB phenotype, and the coexpression of Myc and Bcl2 protein by IHC, are usually considered to be associated with inferior outcomes. Some recent studies however found that non-GCB COO did not have prognostic impact in limited stage DLBCL patients. Further, in real life patient management, the clinical relevance of the assessment of the COO, and of Myc and Bcl2 protein overexpression (DE), is still not fully clarified. The aim of the research was to evaluate in a real-life context the prognostic impact of the COO and of DE status together with R-IPI. With this purpose we have therefore evaluated 142 DLBCL patients in stage I/IV by PET-CT. METHODS This is a retrospective multicenter study which recruited 142 DLBC patients with PET-CT-defined stage I/IV treated with R-CHOP or R-CHOP like between 2010 and 2016 in Modena, Rome and Pisa (Italy) and Haifa (Israel). Eligible patients had a morphologic diagnosis of DLBCL. All clinical data were recorded at diagnosis and during follow up including response assessment and survival outcome. COO was determined on FFPE diagnostic specimens either by IHC applying Hans algorithm, or at RNA level with Lymph2Cx assay by Nanostring technology. Expression of Myc and Bcl2 to define DE status was evaluated by IHC considering Myc and Bcl2 positive ≥40% and ≥50%, respectively. PET-CT imaging was interpreted locally utilizing Deauville criteria. The end points of the study were to determine the impact of COO, Myc/Bcl2 status and R-IPI on PFS and OS. PFS and OS were calculated using the Kaplan-Meyer method with 95 confidence interval (95C%CI), comparison between groups were made with logrank test. The effect of covariates was estimate by means of Cox PH regression, as hazard ratio (HR) with 95%CI. Statistical tests with P<0.10 were considered clinically interesting. RESULTS We identified 142 patients with morphological diagnosis of DLBCL and with PET-CT-defined Stage I/IV treated with R-CHOP or CHOP-like regimens between 2011 and 2018. Patients with evidence of histologic transformation from indolent lymphoma, and double-hit high grade lymphoma were excluded. Median age was 63 yrs, 55% were more than 65 yrs old, 64% had stage III/IV, and 38% had R-IPI 3-5. Bcl2 and Myc were positive in 65% and 20%, respectively. Double expression of Bcl2 and Myc was found in 20% of cases. By Lympho2Cx, COO subtypes were ABC, CGB and Unclassified in 37 %, 46 % and 16 %, respectively. We observed a good concordance between COO determined by Lympho2Cx and Hans algorithm (0.718). Analysis of association between COO subtype by Lympho2Cx and baseline characteristics showed significant association of ABC subtype with B symptoms, R-IPI 3-5, BCL2+, but not with Myc+ , end of treatment PET+, and Response. After a median follow up of 49 months, 3 yrs and 5 yrs PFS and OS were 65% and 59%, respectively in PFS and 80% and 73 % in OS. By univariable analysis we observed significant differences between R-IPI 3-5 vs 0-2, stage III-IV vs I-II, and Bcl2 + vs -. GCB and Unclassified subtypes, Myc - and DE - showed better PFS and OS in comparison with ABC subtype, Myc + and DE +, but the differences were not statistically significant. Forty-seven of the 142 patients underwent SNC prophylaxis with MTX IT or IV. While in patients who received consolidation treatment no difference in PFS or OS between GCB and ABC were observed, patients with GCB subtype showed better PFS and OS in comparison with ABC patients (P=0.05). Based on the results of the univariable analysis, we have assembled a Score from 0 to 3 with R-IPI 3-5 (weight 1), Bcl2 + (weight 1) and ABC subtype (weight 1). The differences in 5 yrs PFS and OS between Score 0, 1, 2-3 were statistically significant (Fig 1). Discussion Although COO is considered a relevant prognostic factor, several studies have produced conflicting results. Our retrospective study conducted on a relatively small group of cases shows that patients with GCB and Unclassified subtypes and DE have a moderately improvement of survival outcome. However, a score based on ABC subtype, Bcl2 overexpression and a high R-IPI score is able to identify a group of poor prognosis patients. As determination of COO by Lympho2Cx is rapid to perform and potentially applicable to clinical practice as well as the determination of Bcl2 and Myc protein expression by IHC, we believe that the use of the Score is useful in patient management in the real life context. Figure 1 Disclosures No relevant conflicts of interest to declare.

Molecular Background of Double Protein Expressor B-Cell Lymphomas

Introduction: Concomitant deregulation of MYC and BCL2, whether at the genomic or protein level, comprises clinically a significant biological high-risk feature in aggressive B-cell lymphomas. Yet, the molecular background of these lymphomas is not fully understood. We sought to comprehensively characterize BCL2 and MYC aberrations and to identify clinically feasible determinants of the recent genomic subtypes and clinical outcome in diffuse large B-cell lymphoma (DLBCL). Patients and Methods: We analyzed BCL2, BCL6 and MYC rearrangements and corresponding protein expression in the context of somatic exome-wide driver gene alterations, copy number annotations, transcriptome and clinical data in 181 patients with primary DLBCL. Results: We found that germinal center B-cell (GCB) and activated B-cell (ABC)-like DLBCLs utilized different mechanisms to deregulate MYC and BCL2 expression. While the structural variations of BCL2 were subtype-specific and specifically increased antiapoptotic BCL2 expression, MYC deregulation was associated with gene expression signatures including biosynthetic pathways and telomerase activity and depletion of JAK-STAT, cytokine and immune cell signatures. Genomic dissection of MYC translocations and protein overexpression revealed associations with other lymphoma drivers, of which we highlight a novel interplay between MYC deregulation and TP53 alterations. Double protein expression (DPE) arose from different molecular backgrounds in a subtype-dependent manner. In GCB-DLBCL, concurrent alterations of MYC and BCL2 loci gave rise to the majority of DPE DLBCLs. Importantly, we recognized more GCB lymphomas with double hit -like clinical behavior when BCL2 copy number alterations were considered. In the non-GCB DLBCLs in turn, concurrent alterations were rare and clinical behavior of DPE was dependent on the molecular context. Interestingly, BN2/C1 -like non-GCB lymphomas with BCL6 translocations had excellent outcomes regardless of the DPE status. Overall, DPE DLBCL defined a prognostic entity, which was independent of International Prognostic Index (IPI) and cell-of-origin, and together with TP53 alterations had synergistic dismal impact on survival. Conclusion: Our findings elucidate the pathogenesis and modern biological determinants of high-risk DLBCL and reveal subtype-specific and clinically feasible improvements to risk-stratification and predictors of outcome. Disclosures Leppa: Celgene: Consultancy; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding.

Lymphoid Neoplasms With Plasmablastic Differentiation: A Comprehensive Review and Diagnostic Approaches.

Plasmablastic neoplasms encompass several entities including plasmablastic lymphoma, plasmablastic plasmacytoma/multiple myeloma, primary effusion lymphoma and its extracavitary variant, anaplastic lymphoma kinase-positive large B-cell lymphoma, and Kaposi sarcoma-associated herpesvirus/human herpesvirus 8 (HHV8)-positive diffuse large B-cell lymphoma, not otherwise specified. Morphologically, the tumor cells are large with eccentrically located nuclei, prominent nucleoli, and basophilic/amphophilic cytoplasm. Immunophenotypically, the tumor cells express plasma cell-related antigens including CD38, CD138, interferon regulatory factor-4 (IRF4)/MUM1, PR domain zinc finger protein-1 (PRDM1), and/or X-box binding protein-1 (XBP1), with frequent loss of CD20. These tumors are diagnostically challenging for general pathologists due to their overlapping morphology and immunophenotype, and due to their rarity, and particularly so when clinical and radiologic information is insufficient. We also discuss HHV8-negative effusion-based lymphoma due to its overlapping features with primary effusion lymphoma. In this review, we focus on the useful diagnostic markers and pertinent molecular findings in these distinct entities and propose a practical diagnostic algorithm using anaplastic lymphoma kinase, HHV8, in situ hybridization for Epstein-Barr virus-encoded small RNA, immunoglobulin M, light chain stains, and clinicoradiologic criteria to avoid misdiagnosis. At the molecular level, MYC protein overexpression with or without MYC rearrangement and PRDM1-inactivating mutations or deletions are noted in a subset of such tumors, especially in plasmablastic lymphoma. Prognosis in these entities is dismal with conventional CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy. Therefore, novel target therapies, such as anti-CD30 agents, and/or immune blockade therapy, are potential treatment options in the future.

PF529 PROGNOSTIC CORRELATION OF CELL‐OF‐ORIGIN AND MYC, BCL‐2 AND BCL‐6 STATUS IN HIV‐ASSOCIATED DIFFUSE LARGE B‐CELL LYMPHOMAS

Background:Diffuse Large B‐cell lymphoma (DLBCL) can be classified by gene expression profiling (GEP) into 3 subtypes according to the putative Cell‐Of‐Origin (COO): the germinal center B‐cell type (GCB), the activated B‐cell type (ABC) and the unclassifiable type (UC). Immuno‐histochemistry (IHC) algorithms, being Hans's the mostly used, and NanoString (NS) 20‐genes assay can surrogate adequately GEP in COO assignment. In DLBCL HIV negative population treated with rituximab‐CHOP, GCB type is associated with a better outcome, while gene rearrangements of MYC plus BCL‐2 and/or BCL‐6 detected by fluorescence in situ hybridization (FISH) confer a worse prognosis. In HIV positive DLBCL, limited data are available on COO subtypes and genes rearrangements incidences and on their prognostic significance.Aims:To evaluate in HIV‐associated DLBCL: the proportion of COO subtypes defined by IHC and NS assay and the concordance of these methods; the prognostic impact of COO and of MYC, BCL‐2 and BCL‐6 status.Methods:We retrospectively analyzed 66 HIV positive patients (pts) with newly diagnosed DLBCL from 2000 to 2016. Histological samples were centrally reviewed for diagnosis confirmation, COO assignment according to Hans and MYC, BCL‐2 and BCL‐6 protein expression. NS Lymph2Cx assay was applied for COO identification and FISH using break‐a‐part probes for MYC, BCL‐2 and BCL‐6 gene analysis.Results:Pts characteristics at DLBCL diagnosis were: 73% male, median age 45 yrs (range: 28–83), 80% advanced stage, 34% high‐intermediate or high IPI score and median CD4+ cell count 188/microL (range: 8–1172). An anthracycline‐containing first‐line chemotherapy was delivered to 97% of pts with the addition of rituximab in 86%. By IHC COO assigned was GCB in 31 (47%) and non‐GCB in 35 (53%) cases. COO by NS was determined in 60 pts: 34 pts were GCB (57%), 11 ABC (18%) and 15 UC (25%). Hans algorithm and NS assay concordantly assigned COO subtypes in 80% of cases (Cohen's kappa = 0.604; p < 0.0005). After a median follow‐up of 50 months, 2‐yrs progression‐free survival (PFS) and overall survival (OS) of the entire series were 56% and 61%, respectively. No statistically significant survival difference was found according to IHC and NS COO assignment, although a trend for a superior OS in GCB subtype defined by NS was observed (2‐yrs OS: 71% vs 53%, p = 0.078). MYC, BCL‐2, BCL‐6 proteins expression and gene rearrangements are summarized in Table 1. BCL‐2 overexpression was detected in 22 pts (34%), was not sustained by BCL‐2 rearrangement and was associated with an inferior OS (2‐yrs OS 48% vs 67%, p = 0.047, HR: 2.04). MYC protein overexpression and MYC, BCL‐2, BCL‐6 gene rearrangements, both single or combined, did not impact survival. Stage IV and elevated LDH were associated with inferior PFS (p = 0.024 and p = 0.017) and OS (p = 0.014 and p = 0.027). A baseline CD4+ count >200/microL had a protective role for both progression and death (2‐yrs PFS: 63% vs 42%, p = 0.013, HR: 0.40; 2‐yrs OS: 72% vs 42%, p = 0.002, HR: 0.29). At multivariate analysis, only CD4+ count >200/microL was associated with superior PFS and OS.Summary/Conclusion:In HIV‐associated DLBCL we found an high concordance between Hans algorithm and NS in the COO subtypes allocation, with subgroups distribution by NS similar to what described in the HIV negative population. The trend for superior outcome in GCB type by NS needs to be confirmed in a larger cohort, as well as the prognostic role of MYC, BCL‐2, BCL‐6 status. DLBCL's adverse clinical features and severe immundeficiency correlated with a dismal prognosis, as expected.image

MYC Pathway Activation Is Frequently Observed in Treatment-Naive CLL and Defines a Subgroup with Particular Benefit from the Addition of Rituximab to Chemotherapy

MYC Pathway Activation Is Frequently Observed in Treatment-Naive CLL and Defines a Subgroup with Particular Benefit from the Addition of Rituximab to Chemotherapy

Prognostication of Diffuse Large B-Cell Lymphoma By Lympho2Cx Assay and BCL2 and MYC Expression: Application in a Real Life Context of 154 Patients

BACKGROUND: About one third of patients with diffuse large B-cell lymphoma (DLBCL) eventually die from their disease. Thus, it is extremely important to identify powerful and reliable predictive and prognostic markers. In real life patient management, the clinical relevance of the assessment of the Cell of Origin (COO), of MYC and BCL2 protein overexpression, is still not fully clarified. The aim of the study is to evaluate the predictive and prognostic value of COO with Lympho2Cx assay by Nanostring technology and BCL2 and MYC overexpression by immunohistochemistry (IHC), in a real-life context.METHODS: This is a retrospective multicenter study which recruited 154 DLBCL patients treated with R-containing regimen between June 2010 and December 2016 in Modena, Rome (Italy) and Haifa (Israel). All clinical data were recorded at diagnosis and during follow up, including response assessment and survival outcome. COO was determined on formalin-fixed paraffin-embedded diagnostic specimens either by IHC using a panel of antibodies which included CD10, BCL6, MUM1 (i.e. Hans algorithm), or at RNA level with Lymph2Cx assay by Nanostring technology. Expression of MYC and BCL2 was evaluated by IHC. Event free survival (EFS) is defined as the time from diagnosis to the time of last follow-up, or to one of the following events: any response other than complete remission (CR) at the end of therapy or death from any cause. It is assessed by Kaplan-Meier estimates and groups of risk are compared using the log rank test.RESULT: We have currently evaluated the data of 60% (95 out of the 154) of the patients enrolled in the study (Tab 1). After a median follow up of 49 months, EFS is 63% (95CI 51-72%).In univariate analysis, the variables with the greatest impact on the response and on EFS are absolute granulocyte count and BCL2 expression (MYC ongoing). Patients with low IPI showed better survival in comparison with patients with high IPI, but the difference is not statistically significant. By IHC non-GCB subtype was more common than GCB (56% vs. 44%); by Nanostring ABC, GCB and unclassified subtypes were 33%, 50% and 17%, respectively, and K statistics was 0,647, showing a substantial agreement between the results obtained by IHC and by Lympho2Cx assay. No statistically significant differences were observed in EFS among ABC, GCB and unclassified subtypes. However, BCL2 protein overexpression in ABC subtype is associated with shorter EFS.CONCLUSION: Early retrospective studies showed a survival advantage for GCB-type disease. Clinical trial data evaluating the impact of COO determined by GEP on prognosis have shown inconsistent results with 2 studies observing inferior survival of ABC subtype and 2 German studies showing no significant differences in PFS or OS among COO subtypes. Determination of COO by Lympho2Cx is attractive as it is relatively easy and rapid to perform and potentially applicable to clinical practice. However, these preliminary results do not support, at this point, its use as prognostic factor in clinical practice. The analysis of the remaining 59 patients will help to clarify the role Lympho2Cx assay in the context of real life.Table 1. Patients baseline characteristics. DisclosuresTadmor:ABBVIE: Consultancy; ROCHE: Research Funding; JNJ: Consultancy; NOVARTIS: Consultancy; PFIEZER: Consultancy.

Myc protein overexpression is a feature of progression and adverse prognosis in multiple myeloma.

Prognostic and predictive markers in multiple myeloma are continuously explored because of the heterogeneity of the tumor biology. Myc protein is the final product from activating MYC oncogene, but the prognostic impact in multiple myeloma is not well described. In a population-based cohort of 194 untreated, newly diagnosed patients with multiple myeloma, we assessed myc protein expression using CD138/myc immunohistochemical double stain and collected clinicopathological data. Cases with myc protein expression ≥40% (mycHIGH ) had a median overall survival of 11months compared to 48months in cases of myc protein expression <40% (mycLOW ) (P<0.01). MycHIGH was significantly correlated to R-ISS, high proliferation index, high percentage of plasma cell in bone marrow, plasmablastic morphology, high calcium level, and abnormal karyotype. In multivariate survival analyses, mycHIGH was independently associated with inferior overall survival with a hazard ratio of 2.5. Our results indicate myc protein overexpression to be associated with advanced multiple myeloma and poor prognosis.

Immune Characteristics of Chinese Diffuse Large B-Cell Lymphoma Patients: Implications for Cancer Immunotherapies

Immunotherapeutic agents have demonstrated encouraging signs of clinical utility in non-Hodgkin lymphoma. The goal of this study is to analyze the immune characteristics of Chinese patients with diffuse large B-cell lymphoma (DLBCL) to inform the development of immunotherapies in this patient population. Tumor samples from 211 DLBCL patients were analyzed for cell of origin (COO) and immune characteristics using the NanoString platform as well as MYC protein expression through immunohistochemistry. Lower incidence of the germinal center B-cell (GCB) subtype (93/211, 44.1%) was observed in this cohort. Compared to the GCB subtype, the activated B-cell (ABC) subtype was associated with significantly increased expression of multiple pro-inflammatory gene signatures and decreased expression of anti-inflammatory gene signatures. Instead of affecting the pro-inflammatory genes, MYC protein overexpression showed a negative correlation with the expression of T-cell receptor (TCR) and T regulatory genes as well as the OX40 gene. Regardless of COO, higher PD-L1 or IDO1 gene expression correlated with increased expression of T effector and Interferon-γ gene signatures while the expression of multiple oncogenes including ACTR3B, ERBB2, AKT2 and SMARCD1 was down-regulated. Our findings may thus be helpful in guiding further development of immunotherapies for the different subsets of Chinese DLBCL patients.

Prognostic significances of overexpression MYC and/or BCL2 in R-CHOP-treated diffuse large B-cell lymphoma: A Systematic review and meta-analysis

Numerous studies have investigated the prognostic values of MYC and/or BCL2 protein overexpression in diffuse large B-cell lymphoma (DLBCL). However, the results still demonstrate discrepancies among different studies. We aimed to do a systematic review and meta-analysis on the relationships between overexpression MYC and/or BCL2 and DLBCLs treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). This study followed the guidelines of PRISMA and Cochrane handbook. The hazard ratios (HRs) for overall survival (OS) were pooled to estimate the main effect size. Twenty studies recruited a total of 5576 patients were available for this meta-analysis. The results showed that MYC (HR = 1.96, 95%CI (confidence interval) = 1.69–2.27)without heterogeneity(I2 = 17.2%, P = 0.280), BCL2 (HR = 1.65, 95%CI = 1.43–1.89, I2 = 20.7%, P = 0.234) protein overexpression, and co-overexpression (HR = 2.58, 95%CI = 2.19–3.04, I2 = 17.2%, P = 0.275) had a poor prognosis in R-CHOP treated DLBCL patients, respectively. The current analysis indicated that MYC and/or BCL2 protein overexpression, and particularly co-overexpression was related to short overall survival in R-CHOP treated DLBCL patients, showing that application of the two new biomarkers can help to better stratify DLBCL patients and guide targeted treatment.

MYC Overexpression at the Protein and mRNA Level and Cancer Outcomes among Men Treated with Radical Prostatectomy for Prostate Cancer.

Background: The proto-oncogene MYC is implicated in prostate cancer progression. Whether MYC tumor expression at the protein or mRNA level is associated with poorer prognosis has not been well studied.Methods: We conducted a cohort study including 634 men from the Physicians' Health Study and Health Professionals Follow-up Study treated with radical prostatectomy for prostate cancer in 1983-2004 and followed up for a median of 13.7 years. MYC protein expression was evaluated using IHC, and we used Cox regression to calculate HRs and 95% confidence intervals (CIs) of its association with lethal prostate cancer (distant metastases/prostate cancer-related death). We assessed the association between MYC mRNA expression and lethal prostate cancer in a case-control study, including 113 lethal cases and 291 indolent controls.Results: MYC nuclear protein expression was present in 97% of tumors. MYC protein expression was positively correlated with tumor proliferation rate (r = 0.37; P < 0.001) and negatively correlated with apoptotic count (r = -0.17; P < 0.001). There were no significant associations between MYC protein expression and stage, grade, or PSA level at diagnosis. The multivariable HR for lethal prostate cancer among men in the top versus bottom quartile of MYC protein expression was 1.09 (95% CI, 0.50-2.35). There was no significant association between MYC mRNA expression and lethal prostate cancer.Conclusions: Neither MYC protein overexpression nor MYC mRNA overexpression are strong prognostic markers in men treated with radical prostatectomy for prostate cancer.Impact: This is the largest study to examine the prognostic role of MYC protein and mRNA expression in prostate cancer. Cancer Epidemiol Biomarkers Prev; 27(2); 201-7. ©2017 AACR.

CHK1 Inhibition in Small-Cell Lung Cancer Produces Single-Agent Activity in Biomarker-Defined Disease Subsets and Combination Activity with Cisplatin or Olaparib

Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo, LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC. Cancer Res; 77(14); 3870-84. ©2017 AACR.

Prognostic impact of MYC protein expression in central nervous system diffuse large B-cell lymphoma: comparison with MYC rearrangement and MYC mRNA expression

The prognostic role of MYC has been well documented in non-central nervous system diffuse large B-cell lymphoma; however, it remains controversial in central nervous system diffuse large B-cell lymphoma. To investigate the prognostic value of MYC, we analyzed the MYC protein expression by immunohistochemistry, mRNA expression by RNA in situ hybridization, and gene status by fluorescence in situ hybridization in 74 cases of central nervous system diffuse large B-cell lymphoma. Moreover, we examined the correlation between MYC translocation, mRNA expression, and protein expression. The mean percentage of MYC immunopositive cells was 49%. Using a 44% cutoff value, 49 (66%) cases showed MYC protein overexpression. The result of mRNA in situ hybridization using the RNA scope technology was obtained using the H-scoring system; the median value was 34.2. Using the cutoff value of 63.5, 16 (22%) cases showed MYC mRNA overexpression. MYC gene rearrangement was detected in five out of 68 (7%) cases. MYC translocation showed no statistically significant correlation with mRNA expression; however, all MYC translocation-positive cases showed MYC protein overexpression, with a higher mean percentage of MYC protein expression than that of translocation-negative cases (78 vs 48%, P=0.001). The level of MYC mRNA expression was moderately correlated with the level of MYC protein expression (P<0.001). The mean percentage of MYC protein expression in the high MYC mRNA group was higher than that in the low MYC mRNA group (70 vs 47%, P<0.001). A univariate analysis showed that age over 60 years, Eastern Cooperative Oncology Group (ECOG) performance status ≥2 and MYC protein overexpression were significantly associated with an increased risk of death. MYC translocation and MYC mRNA expression had no prognostic significance. On multivariate analysis, MYC protein overexpression and ECOG score retained prognostic significance.