Currently, there is no characteristic microRNA (miRNA) expression pattern in Epstein-Barr virus+ diffuse large B-cell lymphoma of the elderly (EBV+DLBCLe). This study aims to characterize a signature profile and identify miRNAs that can be used as biomarkers and alternative therapeutic targets for EBV+DLBCLe. Seventy-one DLBCL patients aged 50 years and older were included and four EBV+ and four EBV– samples were analyzed in two miRNA array platforms (pilot study). A larger multicenter cohort (29 EBV+DLBCLe and 65 EBV–DLBCL patients) was used to validate the results by real-time polymerase chain reaction. In the pilot study, 9% of DLBCL were EBV+DLBCLe by in situ hybridization. In multicenter study, EBV+DLBCLe group showed a predominance of non-germinal center B-cell origin. Overall survival duration of EBV+DLBCLe was significantly inferior to that of EBV–DLBCL patients. We found 10 deregulated miRNAs in the two groups, but only seven were statistically different. We confirmed overexpression of hsa-miR-126, hsa-miR-146a, hsa-miR-146b, hsa-miR-150, and hsa-miR-222 and underexpression of hsa-miR-151 in EBV+DLBCLe cases compared to EBV–DLBCL cases. Hsa-miR-146b and hsa-miR-222 showed high specificity for identifying EBV+DLBCLe. The present study proposed a miRNA signature for EBV+DLBCLe and our findings suggest that hsa-miR-146b and hsa-miR-222 could be biomarkers and therapeutic targets.
Despite advances in treatment, 30% of diffuse large B-cell lymphoma (DLBCL) cases are refractory or relapse after chemoimmunotherapy. Currently, the relationship between angiogenesis and angiomiRs in DLBCL is unknown. We classified 84 DLBCL cases according to stromal signatures and evaluated the expression of pro- and antiangiomiRs in paraffin embedded tissues of DLBCL and correlated them with microvascular density (MVD). 40% of cases were classified as stromal-1, 50% as stromal-2 and 10% were not classified. We observed increased expression of proangiomiRs Let-7f, miR-17, miR-18a, miR-19b, miR-126, miR-130a, miR-210, miR-296 and miR-378 in 14%, 57%, 30%, 45%, 12%, 12%, 56%, 58% and 48% of the cases, respectively. Among antiangiomiRs we found decreased expression of miR-16, miR-20b, miR-92a, miR-221 and miR-328 in, respectively, 27%, 71%, 2%, 44% and 11%. We found association between increased expression of proangiomiRs miR-126 and miR-130a and antiangiomiR miR-328 and the subtype non-GCB. We found higher levels of the antiangiomiRs miR-16, miR-221 and miR-328 in patients with low MVD and stromal-1 signature. IPI and CD34 confirmed independent impact on survival of the study group. None of the above angiomiRs showed significance as biomarker in an independent serum samples cohort of patients and controls. In conclusion, we confirmed association between antiangiomiRs miR-16, miR-221 and miR-328 and stromal-1 signature. Four angiomiRs emerged as potential therapeutic targets: proangiomiRs miR-17, miR-210 and miR-296 and antiangiomiR miR-20b. Although the four microRNAs seem to be important in DLBCL pathogenesis, they were not predictive of DLBCL onset or relapse in the serum independent cohort.
Background EBV+ diffuse large B-cell lymphoma of the elderly (EBV+DLBCLe) is considered a provisional entity in the latest World Health Organization classification. It affects individuals older than 50 years without prior documented immunodeficiency.This disorder has unfavorable clinical course even after the advent of immunotherapy associated to anthracycline-based chemotherapy. It is linked to Epstein-Barr virus and the physiopathology is related to the presence of the virus itself, senescence and immunological deterioration. Currently there is not a characteristic pattern of expression of microRNAs in EBV+DLBCLe. Aims To characterize a signature profile for this new entity and to explore microRNAs as biomarkers and potential alternative therapeutic targets for EBV+DLBCLe. Methods 124 cases of patients of DLBCL were treated at Hospital Sao Paulo UNIFESP/EPM between 2000 to 2010 and had paraffin blocks available for immunohistochemical and molecular analyses. Seventy-one of 124 patients were more than 50 years and were potential candidates to be considered EBV+DLBCLe (pilot study). In situ hybridization was used for EBV detection (EBER1, Invitrogen) in a tissue microarray slide. Total RNA was obtained from tumor slides using Recover All Total Nucleic Acid Isolation kit (Applied Biosystems). We obtained cDNAs using Megaplex Pools for microRNA Expression (Applied Biosystems). The cDNA was inserted into two platforms containing 384 human microRNA each (Taqman Low Density Arrays) on 7900 Real Time PCR Systems (Applied Biosystems). Data analyses were made in mathematical-statistical environment “R”. The normalization method 2-deltaCt was performed using the endogenous RNU48, and it was identified as the most stable among samples by software Normfinder. It was also used RNU6 recommended by the manufacturer, in a comparative way. MicroRNAs differentially expressed in EBV+ group compared to EBV negative were identified by means of nonparametric tests rank products (RankProd) and Wilcoxon rank-sum (R-Stats). We considered differentially expressed microRNAs which average fold change above or below 1.5.After, real-time quantitative PCR was performed through 7500 Real time PCR Systems (Applied Biosystems) using TaqMan Small RNA kit assays and normalized with RNU48. Results 8.5% of cases of DLBCL were considered EBV+DLBCLe after ISH for EBV. 53.1% of the pilot study were considered GCB and 43.9% non-GCB according to Hans et al. algorithm (2004); 73.7% were classified as worse prognosis (groups 3 and 4) according to Salles et. al(2011) model combining IPI and immunohistochemical markers (bcl-2 and Ki67).We selected four of EBV+ and four of EBV negative samples matched by age, gender, stage and IPI to be analyzed in the PCR platforms. We found 10 deregulated microRNAs among the two groups. However, only seven microRNAs achieved statistically significant differences and would be the start point of a microRNA signature profile proposal to be validated in a larger multicentric cohort (total of 29 EBV+DLBCLe versus 65 DLBCL). Among them let-7g, miR-126, miR-146a, miR-146b, miR-150 and miR-155 were overexpressed in EBV+DLBCLe comparing to EBV-negative DLBCL whereas miR-151 was underexpressed. After validation in 29 EBV+DLBCLe (including 23 new cases) and 65 EBV negative cases we confirmed overexpression of miR-126 in 75.8% (median 2.14 vs 0.14,p< 0.0001), miR-146a in 62% (median 1.94 vs 0.49, p = 0.0035) ,miR-146b in 51.7% (median 1.51 vs 0.11 , p< 0.0001),miR-150 in 96.5 % (median 20.54 vs 2.56,p< 0.0001) and miR-222 in 23,8% of cases (median 0.67 vs 0.08,p< 0.0001,Mann-Whitney) and also confirmed underexpression of miR-151 in 96% of EBV+DLBCL cases. Although miR-222 was overexpressed in ¼ of the cases, it showed high speficity (98%) and positive predictive value (83%), Area Under the Curve= 0.87774, when EBV+ when compared to EBV negative cases. Summary /Conclusion The merit of the present study is to propose a microRNA signature for a recently described disease and to highlight miR-222 as a possible biomarker and therapeutic target for EBV+DLBCLe. The main routes deregulated are NF-KappaB and PI3K-AKT pathway, being PTEN a target of the overexpressed miR-222. Thus, the findings suggest that antagomiRs for miR-222,that are being tested in some types of cancer, could be also used as adjuvant therapy to R-CHOP in EBV+DLBCL. (Supported by FAPESP 2010/17668-6). Disclosures: No relevant conflicts of interest to declare.
Background: B-cell development is a complex biological process that initiates in bone marrow and matures in lymph nodes. These hierarchical stages are tightly regulated by transcription factors and microRNAs (miRNAs). B-cells are prone to genetic alteration during these maturation stages due to DNA breaks required for B-cell receptor re-arrangement and additional changes that occur during the germinal center (GC)-reaction for class switch recombination and somatic hypermutation. The unwarranted genomic changes resulting from these processes can lead to B-cell malignancies with varied clinical and pathological characteristics. Of the lymphoma subtypes derived from mature B-cells, a subset of diffuse large B-cell lymphoma (DLBCL), primary mediastinal large cell lymphoma (PMBL), follicular lymphoma (FL) and Burkitt lymphoma (BL) are putatively derived from GC B-cells, with varied morphological, pathological and clinical characteristics. These tumor are characterized by distinct genetic abnormalities,[ t(14;18) in GCB-DLCBL, and FL or t(8;14)] in BL, and mutational profile. We postulate miRNA expression signatures, may also explain the wide-ranging clinico-pathological characteristics of these GC B- cell derived tumors. Methods: We performed a meta-analysis of miRNA profiles of DLBCL, BL, PBML, FL and EBV+DLBCL performed by us or others. (Iqbal 2015; 2012; Leich 2011; Andrade 2014). We also included lymphomas derived from naïve B-cells (MCL, SLL) and post GC B-cells (activated B cells like ABC-DLBCL), EBV (+) DLBCL, normal B-cell subsets and cell lines of distinct B-cell lineages. The majority of the cases have been characterized by gene expression profiling (GEP) with corresponding pathological clinical characteristics in above studies. The data was obtained using Taqman® human microRNA array (Applied Biosystems, CA) containing 380 miRNAs and analyzed using BRB-array Tools. Results In our initial miRNA expression profile of 249 lymphomas including GCB-DLBCL (n=34), ABC-DLBCL(n= 29), PMBL(n= 9), BL(n= 33), FL (n= 32), EBV+DLBCL (n=8); MCL(n=30) and SLL(n=12)showed distinct hierarchical clusters enriched with GEP defined subgroup, with few interspersed cases from other subgroups suggesting that miRNAs can complement and delineate GEP defined molecular subgroups, an observation consistent with earlier findings (Iqbal 2015). When we examined miRNAs, that are specifically associated with GC B- derived lymphomas, we observed a subset of miRNA including miR-146a, miR-142-3p, miR-17, miR19b, and miR106a were present at higher levels (top 5% in abundance by CT values) in GC B lymphomas. Of these, somatic mutations in miR142 have been recently reported in GC derived lymphomas (Alyssa, Leukemia 2016). These miRNAs also represented the most abundant miRNAs in centroblast, suggesting lineage specificity of miRNA signature in these lymphomas. However, miR222 highly expressed in centroblast was not expressed at higher levels in GCB-DLBCL and BL. A number of miRNAs specifically abundant in distinct lymphomas included (BL: miR-20a, miR-16), (GCB-DLBCL: miR-223, miR-24) and (FL: miR222). MiR-150 was highly expressed in NHLs putatively derived from naive B-cells like MCL or SLL, but absent from GC-derived NHLs. Consistent with this, miR-150 was present in naive B-cells, but not in centroblast, suggesting that miR-150 may have discrete function during early B-cell differentiation and late maturation stages. Within GC derived lymphoma, we examined miRNA profiles in clinically aggressive (BL); indolent lymphomas (FL) and intermediate aggressive (GCB-DLBCL) and their association with normal B-cell subsets, and indeed a number of miRNAs (ex. miR-150) were associated with indolent lymphomas, whereas a subset of miRNAs was present in aggressive lymphomas (ex. miR-16), suggesting these miRNAs may contribute the distinct clinical characteristics. Though we have identified unique miRNA profiles in these GC derived NHLs, the target genes of these miRNAs remains to be identified. Conclusion: We identified microRNAs that were differentially expressed between GC-derived DLBCL and other lymphomas, which may help explain distinguishing features of these lymphomas. Disclosures No relevant conflicts of interest to declare.
1570 Introduction: MicroRNAs (miRNAs) are a class of endogenous short non-coding RNAs that control gene expression by acting on target mRNAs for promoting either their degradation or translational repression. Some of them have involvement in regulating various aspects of angiogenesis, including proliferation, migration and morphogenesis of endothelial cells, which are important in regulating cardiovascular development and cancer. The term “angiomiR” is used to define the miRNAs that control angiogenesis. They are classified into pro-angiomiRs, those that promote angiogenesis, and anti-angiomiRs, those that inhibit angiogenesis. The identification of angiomiRs as the key to regulating angiogenesis has opened new paths in the treatment of vascular and oncology diseases. Aims: This study aims to analyze the expression angiomiRs in diffuse large B-cell lymphoma (DLBCL) and to correlate them with clinical and histological features to identify possible biomarkers and prognostic factors. Patients and Methods: We studied 93 samples of de novo DLBCL diagnosed between 2000 and 2010. All the cases were HIV-negative. MicroRNAs were obtained from paraffin embedded tumor samples using RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues (Applied Biosystems). Four angiomiRs (miR-378, miR-296, miR-210 and miR-126) were analyzed. RNU44 and U18 were used as endogenous controls for quantitative PCR (TaqMan®Small RNA Assays). We set a threshold of a 1.5-fold difference in angiomiRs expression compared to controls (palatine tonsil). Results: miR-378, miR-296, miR-210 and miR-126 overexpression were observed in 43%, 47%, 22% and 5%, respectively. Considering that miR-378 and miR-296 were frequently overexpressed in DLBCL, we further analyzed the following variables: age (<=60 versus >60 years), Ann Arbor Staging System (I-II versus III-IV), International Prognostic Index (0–2 versus 3–5), DLBCL classification (NOS versus subtypes), tumor origin according to Hans (2004) algorithm (GCB versus non-GCB). We observed higher median miR-296 expression in DLBCL classified as stage III-IV (p = 0.0415, Mann-Whitney). For the other variables, were did not find any statistically significant difference between groups. Conclusions: miR-296, that directly decreased the levels of hepatocyte-growth factor regulated tyrosine kinase substrate (HGS) and indirectly upregulate VEGFR2 and PDGFRβ, was overexpressed in almost 50% of de novo DLBCL and was associated with advanced stage disease. Our study brings new information about the role of microRNA importance in DLBCL development and can be explored as prognostic and therapeutic target for patients suffering from this prevalent malignancy (Supported by FAPESP 2010/17668-6). Disclosures: No relevant conflicts of interest to declare.
The identification of subtypes of DLBCL defined by expression of genes related to tumor stroma (Lenz et al, 2008) may determine new paths in the treatment of these diseases using anti-angiogenic drugs. The relationship of these genes with the pro- and antiangiomiRs is still unknown. Aims 1) to classify DLBCL cases according to the signature stromal-1 and stromal-2 using tissue microarray (TMA) and immunohistochemistry; 2) to evaluate the expression of pro- and antiangiomiRs in paraffin embedded tissues affected by DLBCL and correlate them with the signatures of the tumor stroma. Methods 111 DLBCL (NOS and variants, all HIV negative) admitted to the Hospital São Paulo between 2000 and 2010 were included in this study. Expression of CD68 (stromal-1 marker) was classified into four (0-3) levels. To analyze the expression of CD34 (MVD, stromal-2), we conducted a manual count of microvessels in the entire field of TMA. We performed miRNA extraction from paraffin samples using RecoverAll ™ Total Nucleic Acid Isolation Kit for FFPE Tissues (Applied Biosystems). Real-time quantitative PCR was performed using TaqMan® Small RNA kit Assays and normalized with U18 and RNU44. Pro-angiomirs Let-7f, miR-92a, miR-130a, miR-210, miR-296 and miR-378 and anti-angiomiRs miR-16, miR-20b, miR-221 and miR-328 were selected for analyses and were considered differentially expressed when tumor samples levels were 1.2 times higher or lower than normal samples. Results Stromal-1 cases had high expression of CD68 (scores 2-3) and low expression of CD34 (quartiles I-II) (32.6% of cases), and as stromal-2 cases had low expression of CD68 (scores 0-1) and high expression of CD34 (quartiles III-IV) (11.6% of cases). The 40 cases that had high expression of both CD68 and CD34 (42.1%) were additionally considered as stromal-2, due to high MVD scores. Cases of low expression of CD68 and CD34 (13.7% of cases) were not scored. We observed a statistically significant relationship between the median expression of CD34 and Ann Arbor stage (I-II versus III-IV), with a predominance of high MVD expression in patients with advanced stage disease (III-IV). The median of vessels in stages I-II was 82.5 (range 9 to 240) and in stages III-IV was 111 (range 12-297) (p = 0.0276, Mann-Whitney). For the marker CD68 (stromal-1) and for the other variables, there were no statistically significant associations between groups. 101 of 111 initial cases were considered suitable for analysis of microRNAs. We observed overexpression of angiomiRs miR-92a, miR-296, miR-210, miR-378, Let-7f and miR-130a in 100%, 56.5%, 55,5%, 48.5%, 14.9% and 13.9% of cases, respectively. Among antiangiomiRs we found underexpression of miR-20b, miR-221, miR-16 and miR-328 in respectively 65.7%, 45.5%, 27.7% and 12.9%. We also observed a correlation between overexpression of miR-20b and low MVD (p = 0.0225, Mann-Whitney) and overexpression of miR-221 and low MVD (p = 0.0339, Mann-Whitney), suggesting a correlation between increased antiangiomiRs expression and low angiogenesis profile. For other variables, there were no statistically significant differences between groups. Conclusion Stromal-2 signature was found in 53.7% of cases. The higher MVD in cases of DLBCL with advanced stage (Ann Arbor III-IV) shows a possible association between angiogenesis and more aggressive/disseminated disease, being a possible target for antiangiogenic therapy in cases that do not achieve a complete response with R-CHOP. Two angiomiRs emerge as potential new targets: proangiomiR miR-92a, overexpressed in 100% of DLBCL, and antiangiomiR miR-20b, underexpressed in 65.7% of cases. Our study provides new information about the importance of the role of microRNAs in the development of DLBCL, which can be exploited as a therapeutic target for patients with this malignant disease of high prevalence. (Supported by FAPESP 2010/17668-6). Disclosures: No relevant conflicts of interest to declare.
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