Smrati Jain

Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

t(14;18)-negative follicular lymphomas are associated with a high frequency of BCL6 rearrangement at the alternative breakpoint region

A frequent chromosomal translocation in mature B-cell non-Hodgkin lymphoma affects band 3q27 and results in the deregulation of the B-cell lymphoma 6 (BCL6) gene. Two breakpoint clusters have been described thus far, the major breakpoint region (MBR) and an alternative breakpoint region (ABR) that is located 245–285 kb 5′ to BCL6. Translocation at the MBR predominates in diffuse large B-cell lymphoma, whereas translocation at the ABR is reported to be frequently associated with grade 3B follicular lymphoma. However, translocation at the ABR has not been studied in a large series of follicular lymphomas, particularly t(14;18)-negative follicular lymphomas. Therefore, we studied BLC6 rearrangements at the MBR and ABR by using break-apart fluorescence in situ hybridization (FISH) probes in 142 cases of follicular lymphomas, including 63 t(14;18)-negative and 79 t(14;18)-positive cases. Conventional cytogenetic (karyotype) analysis was also performed in 58 of the 63 t(14;18)-negative cases. BCL6 rearrangement was found in 26% of t(14;18)-negative and 19% of t(14;18)-positive follicular lymphoma. t(14;18)-negative cases showed a high frequency of rearrangement at the ABR (12%) with an ABR/MBR ratio of 0.86, compared with only 5% with an ABR/MBR ratio of 0.36 in the t(14;18)-positive cases. BCL6 rearrangements were found in all grades of follicular lymphoma but were most frequent in grade 3 t(14;18)-negative follicular lymphoma (60%). FISH analysis had a higher sensitivity for detecting BCL6 rearrangements than conventional cytogenetics. In conclusion, BCL6 rearrangements occur at a similar frequency in t(14;18)-negative follicular lymphoma and diffuse large B-cell lymphoma. However, t(14;18)-negative follicular lymphoma appears to have a higher frequency of rearrangement at the ABR compared with t(14;18)-positive follicular lymphoma and diffuse large B-cell lymphoma. Therefore, it is important to perform FISH analysis with ABR to determine possible involvement of BCL6 rearrangement in follicular lymphoma, especially in t(14;18)-negative cases.

Alteration/deficiency in activation-3 (Ada3) plays a critical role in maintaining genomic stability.

Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3−/− cells. Notably, Ada3−/− cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G₁ or G₂ phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3fl/fl and Ada3−/− cells confirmed higher residual DNA damage in Ada3−/− cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability.

Isolated MYC cytogenetic abnormalities in diffuse large B-cell lymphoma do not predict an adverse clinical outcome

In this study, we investigated the significance of MYC, BCL2 and BCL6 gene abnormalities in a cohort of 205 diffuse large B-cell lymphoma (DLBCL) patients studied by conventional and/or fluorescence in situ hybridization cytogenetic analysis. Combining these methods, 172 cases (84%) were classified as MYC−, 17 (8%) were MYC+/BCL2−/BCL6−, and 16 (8%) were double/triple-hit lymphomas (i.e. MYC+/BCL2+, MYC+/BCL6+, or MYC+/BCL2+/BCL6+). We found a significant difference in event-free survival (EFS) among the three groups (p = 0.02), with the double/triple-hit group having the worst EFS. Patients who were MYC+, but BCL2− and BCL6−, had the best EFS. We conclude that patients with MYC+ DLBCL, but without BCL2 or BCL6 abnormalities, do not have a worse outcome when compared to those who are MYC−. However, patients with double/triple-hit DLBCL have a very poor outcome and should be treated with aggressive or novel therapies.

Risk factors for non‐Hodgkin lymphoma subtypes defined by histology and t(14;18) in a population‐based case‐control study

The t(14;18) chromosomal translocation is the most common cytogenetic abnormality in non‐Hodgkin lymphoma (NHL), occurring in 70–90% of follicular lymphomas (FL) and 30–50% of diffuse large B‐cell lymphomas (DLBCL). Previous t(14;18)‐NHL studies have not evaluated risk factors for NHL defined by both t(14;18) status and histology. In this population‐based case‐control study, t(14;18) status was determined in DLBCL cases using fluorescence in situ hybridization on paraffin‐embedded tumor sections. Polytomous logistic regression was used to evaluate the association between a wide variety of exposures and t(14;18)‐positive (N = 109) and ‐negative DLBCL (N = 125) and FL (N = 318), adjusting for sex, age, race, and study center. Taller height, more lifetime surgeries, and PCB180 exposure were associated with t(14;18)‐positivity. Taller individuals (third tertile vs. first tertile) had elevated risks of t(14;18)‐positive DLBCL (odds ratio [OR] = 1.8, 95% confidence interval [CI] 1.1–3.0) and FL (OR = 1.4, 95%CI 1.0–1.9) but not t(14;18)‐negative DLBCL. Similar patterns were seen for individuals with more lifetime surgeries (13+ vs. 0–12 surgeries; t(14;18)‐positive DLBCL OR = 1.4, 95%CI 0.7–2.7; FL OR = 1.6, 95%CI 1.1–2.5) and individuals exposed to PCB180 greater than 20.8 ng/g (t(14;18)‐positive DLBCL OR = 1.3, 95%CI 0.6–2.9; FL OR = 1.7, 95%CI 1.0–2.8). In contrast, termite treatment and high alpha‐chlordane levels were associated with t(14;18)‐negative DLBCL only, suggesting that these exposures do not act through t(14;18). Our findings suggest that putative associations between NHL and height, surgeries, and PCB180 may be t(14;18)‐mediated and provide support for case‐subtyping based on molecular and histologic subtypes. Future efforts should focus on pooling data to confirm and extend previous research on risk factors for t(14;18)‐NHL subtypes.

Disruption of chromosomal locus 1p36 differentially modulates TAp73 and ΔNp73 expression in follicular lymphoma

Abstract The TP73 gene is located at the chromosome 1p36 locus that is commonly disrupted or deleted in follicular lymphoma (FL) with poor prognosis. Therefore, we analyzed the expression of the pro-apoptotic TAp73 and anti-apoptotic ΔNp73 isoforms in cases of FL with normal or abnormal 1p36. We observed a significant increase in ΔNp73 expression and ΔNp73:TAp73 ratio, lower expression of cleaved caspase-3 and a higher frequency of Ki-67 and proliferating cell nuclear antigen (PCNA) positive cells in cases of FL with abnormal 1p36. A negative correlation between the ΔNp73:TAp73 ratio and cleaved caspase-3 expression, and a positive correlation between ΔNp73 expression and Ki-67 or PCNA, were observed. The expression of TAp73 and its pro-apoptotic transcriptional targets BIM. PUMA and NOXA were significantly lower in FL compared to reactive follicular hyperplasia. Together, our data demonstrate that 1p36 disruption is associated with increased ΔNp73 expression, decreased apoptosis and increased proliferation in FL.

Abstract 312: Disruption of chromosomal locus 1p36 differentially modulates TAp73 and ΔNp73 expression and aggressiveness in non-Hodgkin's lymphoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Consistent cytogenetic aberrations and the consequent molecular alterations have considerably facilitated the classification of non-Hodgkins lymphoma (NHL). A cytogenetic abnormality involving chromosome region 1p36 is frequently detected in NHL cases and is associated with a poor prognosis and high risk of transformation from an indolent to a more aggressive NHL subtype. However, the molecular events mediating the biological consequences of this cytogenetic abnormality are poorly understood. TP73 is one of the most distally located candidate genes at the 1p36 locus, and is likely to be disrupted or deleted. Furthermore, we have observed frequent loss of heterozygosity in the P73 locus using fluorescence in situ hybridization (FISH) in NHL cases with the 1p36 abnormality. TP73 gene function is controlled by harmony between two isoforms with opposing functions, the full length TAp73 protein (pro-apoptotic) and the NH2-terminally truncated ΔNp73 protein (anti-apoptotic through antagonizing both TAp73 and p53). To determine whether 1p36 abnormalities alter p73 expression, we measured the mRNA expression of TAp73 and ΔNp73 by real-time PCR in diagnostic specimens of cytogenetically characterized NHL cases with and without 1p36 disruption. We observed a down-regulation of TAp73 in NHL cases with and without 1p36 disruption. However, there was a significant increase in the ΔNp73 expression (p<0.01) resulting in a significantly up-regulated (p<0.01) ΔNp73:TAp73 ratio among NHL cases harboring 1p36 abnormalities compared to NHL cases without 1p36 abnormalities. To evaluate the functional significance of this imbalance, we performed immmunohistochemical analysis on diagnostic biopsy specimens from NHL cases with and without 1p36 abnormalities using TAp73, ΔNp73, cleaved caspase 3 (an apoptosis marker), and PCNA (a proliferation marker) antibodies on a tissue microarray. We observed a statistically-significant negative correlation between the ΔNp73:TAp73 ratio and the apoptosis marker cleaved caspase 3 (r=−0.614; p=0.015); and a significant positive correlation between ΔNp73 and the proliferation marker PCNA (r=0.532; p=0.041), indicating increased proliferation and decreased apoptosis with up-regulated ΔNp73. Additionally, we observed a significant positive correlation between TAp73 and cleaved caspase 3 (r=0.772; p=0.001), thus associating the TAp73 isoform with apoptosis. Together, our data demonstrates that 1p36 disruption leads to differential regulation of p73 gene expression, resulting in an imbalance in TP73 isoform expression (gain of ΔNp73 and loss of TAp73), with diminished apoptosis and increased proliferation, thus explaining the prognostic effect of 1p36 disruption. These findings provide new insight into the differential regulation of p73 gene expression that is mediated by this cytogenetic abnormality. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 312. doi:10.1158/1538-7445.AM2011-312

Abstract 222: Differential p73 isoform expression profile in non-Hodgkin's lymphoma with 1p36 chromosomal abnormality modulates angiogenic phenotype

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Alteration of tumor suppressor gene function often leads to imbalance in expression of angiogenic and angiostatic factors facilitating disease progression. TP73, a member of the p53 tumor suppressor gene family, regulates many important cellular pathways and has been shown to be abnormally expressed in non-Hodgkins lymphoma (NHL). TP73 gene function is controlled by harmony between two isoforms with opposing functions, the full length TAp73 protein (pro-apoptotic) and the NH2-terminally truncated ΔNp73 protein (anti-apoptotic through antagonizing both TAp73 and p53). We have observed differential ΔNp73 up-regulation and increase in ΔNp73:TAp73 expression ratio among NHL cases with 1p36 chromosomal disruption as compared to NHL cases without 1p36 disruption, which is associated with aggressiveness. To decipher whether deregulated p73 isoforms expression modulates the angiogenic potential in NHL we examined the mRNA expression of angiogenic factors (VEGF-A, FGF-2, and angiogenic chemokine CXCL-8) and angiostatic factors (TSP-1 and Endostatin) in NHL cases with and without chromosome 1p36 abnormality by real-time PCR. We observed higher levels of expression of angiogenic factors (VEGF-A, FGF-2, and CXCL-8) and lower levels of angiostatic factors (TSP-1 and Endostatin) in NHL cases with 1p36 abnormality as compared to NHL cases without 1p36 abnormality. Next, we performed a non-parametric bivariate correlation analysis (Kendalls tau_b and Spearmans rho) to examine whether ΔNp73:TAp73 expression ratio correlates with the expression of angiogenic and angiostatic factors. We observed a significant correlation between ΔNp73:TAp73 expression ratio with the expression of VEGF-A (r=0.900, p<0.001), FGF-2 (r=0.620, p<0.05) and TSP-1 (r=−0.873, p<0.001). To further elucidate the relationship between differential p73 isoforms expression and the expression pattern of angiogenic and angiostatic factors, we stably transfected an NHL cell line with 1p36 disruption, Granta-519 (TAp73 non expressor) with a wild type TAp73 mammalian expression vector. We observed an up-regulation of TSP-1 and Endostatin expression among Granta-519 cells transfected with TAp73 vectors compared to isogenic control vector transfected cells. Our data highlights the molecular and functional consequences of the differential p73 isoforms expression pattern caused by 1p36 chromosomal disruption on angiogenic phenotype in NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 222. doi:10.1158/1538-7445.AM2011-222