Mitotic Spindle Assembly Inhibition Through Aurora A (MLN8237) Plus Vincristine Is Synthetic Lethal and Synergizes with Rituximab As a Curative Therapy in Aggressive B-Cell Non-Hodgkin Lymphoma

Abstract Abstract 2721 Most aggressive B-cell non-Hodgkin lymphomas (B-NHL) are not curable with current chemo-immunotherapy combinations. Synthetic lethal interactions with novel agents may yield effective combination therapies with minimal toxicity for aggressive B-NHL. Auroras (A and B) are a family of mitotic oncogenic serine/threonine kinases intimately involved in high fidelity regulation of cell division. Aberrant over-expression of Auroras leads to genetic instability, polyploidy, tumor initiation and progression. Over-expression of Auroras in aggressive B-NHL promotes resistance to microtubule targeted agents (MTA, taxanes and vinca alkaloids). Si-RNA knockdown or pharmacologic inhibition of Aurora with MLN8237 [M], an ATP-site small molecule inhibitor, leads to enhanced sensitivity of B-NHL cells to MTAs. We hypothesized that promotion of microtubule de-polymerization with vincristine [V] would be more effective in synergizing with M than a microtubule polymerizing agent docetaxel [D]. We demonstrated that M plus V is synergistic while M plus D is additive in B-NHL cell culture models. Further, the addition of rituximab [R] enhanced apoptosis of B-NHL cells treated with MV or MD therapy. Mouse xenograft models of mantle cell lymphoma show modest single agent activity for M, R, D and V with tumor growth inhibition (TGI) of ∼10–15% (p=0.01). Of the doublets, MV caused tumor regression while MD and MR caused TGI (∼55–60%) and (∼20–25% (p=0.001) respectively. Although MV caused tumor regression, mice relapsed after 2 weeks of stopping therapy. In contrast, MVR had no relapses 120 days after stopping therapy, while MDR led to TGI of ∼85% (p=0.001). Kaplan-Meier analysis of overall survival showed that the mice treated with MV and MVR had a statistically significant improvement in overall survival when compared with the control (p<0.0001) or MR (p=0.0043). Gene expression profiling (human HG-U133A) and confirmatory Western blotting of harvested tumors at the end of treatment (3 weeks) confirmed reactivation of cell cycle regulatory genes including PCNA, Aurora B, cyclin B1 and cyclin D1, in MV versus MVR. Moreover, MVR therapy continues to inhibit Aurora B by repressing genes regulating mitotic sister chromatid segregation by repressed gene expression analyzed by Gene Ontology. Thus, addition of R to MV represents a novel therapeutic strategy that warrants clinical trial evaluation in aggressive B-NHL [Funded by the Lymphoma SPORE 1 P50 CA 130805 01A1]. Disclosures: No relevant conflicts of interest to declare.

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Gene Expression Profiling of Persistent Polyclonal B-Cell Lymphocytosis Using Whole Genome Microarrays.

Blood ◽

10.1182/blood.v108.11.4289.4289 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4289-4289

Author(s):

Charles H. Lawrie ◽

Xavier Troussard ◽

Hossein Mossafa ◽

Chris Hatton

Keyword(s):

Gene Expression ◽

T Cell ◽

B Cell ◽

Elevated Serum ◽

Constitutive Expression ◽

Splenic Marginal Zone Lymphoma ◽

Whole Genome ◽

Normal Lymphocyte ◽

Over Expression ◽

Lymphocyte Populations

Abstract Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare B-cell disorder characterised by elevated serum levels of polyclonal IgM and persistent lymphocytosis of often bi-nucleated B-cells. The molecular basis of this disorder however remains poorly understood. We therefore used whole genome expression microarrays (Affymetrix U133 plus2.0) to compare the gene expression profile of 14 cases of PPBL with normal lymphocyte populations (6 B cell (CD19+) and 3 T cell (CD3+) samples). PPBL patients (10 female, 4 male) had a median age of 43 (range 28–63) and all were smokers with an average lymphocyte count of 5.34 x 107/ml (range 3.7–9) at time of presentation. Eight of these cases had +i(3q) and eight cases exhibited PCC, four cases displayed both chromosomal abnormalities. Normal lymphocyte populations were obtained from two pools of 12 individuals and individual donors. Microarray data were first RMA pre-processed and then expression levels of patient samples’ genes compared with levels of both normal B cell and T cell samples. ANOVA (P<0.01) was used to identify differentially expressed genes. Thirty-six genes were found to be up-regulated and 157 genes were down-regulated in all patient samples by at least 2-fold. Components of the AP-1 transcription complex (FOS, FOSB and JUN) were found to be highly up-regulated in all PPBL samples with an average of 7.2, 7.9 and 31.6 fold expression respectively. The cell cycle regulatory molecule CDC27 was also found to be up-regulated in all patient samples (8.8-fold). Down-regulated genes of interest included the B-cell specific transcription factor BACH2 (3-fold), tumor-suppressor SMAD4 (3.6-fold) and apoptotic genes DRAK1 (3-fold) and CAS (5-fold). These data are currently being validated by quantitative RT-PCR. We propose that over-expression of AP-1 may have significance in PPBL as increased expression of components of AP-1 have previously been linked with lymphoproliferative disorders including Hodgkin’s lymphoma and splenic marginal zone lymphoma. The underlying reason for over-expression of AP-1 in PPBL remains unknown but two characteristic features of this disease could provide clues; firstly AP-1 up-regulation has been demonstrated to be tobacco-induced in animal models and secondly the LMP1 protein of EBV can result in constitutive expression of the AP-1 complex.

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Outcome of Adults with Acute Lymphoblastic Leukemia Treated with a Pediatric-Inspired Therapy: A Single Institution Experience

Blood ◽

10.1182/blood.v116.21.4337.4337 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4337-4337

Author(s):

Guillermo J. Ruiz-Delgado ◽

Julio Macias-Gallardo ◽

Julia Lutz-Presno ◽

Maryel Montes-Montiel ◽

Guillermo J. Ruiz-Arg\)elles

Keyword(s):

Overall Survival ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Institution ◽

B Cell Malignancies ◽

Secondary Diabetes ◽

Free Survival ◽

Results Of Treatment ◽

Better Than

Abstract Abstract 4337 The results of treatment of adults with ALL remain unsatisfactory. Pediatric-inspired treatments seem to be related with better outcomes. Eighty adult ALL patients were prospectively treated in a single institution in a 16-year period with a schedule based on the St. Jude's TOTAL XI pediatric protocol employing vincristine, prednisone, asparaginase, daunorubicin, etoposide, cytarabine, methotrexate, mercaptopurine and triple intratecal therapy. Median age was 31 years (range 18 – 86); 92% were B-cell malignancies and 14% were Ph1 (+). Ten patients did not complete the first course of chemotherapy and 4 exited early. 44 of 66 patents (67%) achieved a complete remission; relapses presented in 57%. The median probability of overall survival (OS) was 28 months, whereas the 144-month OS was 27%. The median probability of leukemia-free survival (LFS) was 28 months, and the 144-month LFS 35%. Ph1 (+) patients did worse than Ph1-negative and T-cell leukemias did better than B-cell ones. Concerning toxicity, eight patients had toxic deaths (12%), two developed acute pancreatitis and one secondary diabetes. This pediatric-inspired therapy rendered better results than those obtained in similar socioeconomic circumstances using adult-oriented treatments; tolerance was acceptable and costs were low since it employs affordable drugs and can be delivered as outpatients. Disclosures: No relevant conflicts of interest to declare.

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Characterization of lincRNA BALIR-6 in MLL rearranged B-lymphoblastic leukemia

Blood ◽

10.1182/blood.v122.21.3730.3730 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3730-3730

Author(s):

Norma Iris Rodriguez-Malave ◽

Weihong Yan ◽

Giuseppe Basso ◽

Martina Pigazzi ◽

Dinesh S. Rao

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Microarray Data ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Regulate Gene Expression ◽

In Cis ◽

Regulate Gene

Abstract A new class of non-coding RNA, known as long intergenic non-coding RNAs (lincRNAs), has only recently been described. These lincRNAs have been found to play a role in various molecular processes within the cell including gene regulation, acting as sinks for microRNAs, and regulating splicing, implicating them in development and oncogenic processes. B lymphoblastic leukemia (B acute lymphoblastic leukemia; B-ALL), a malignancy of precursor B-cells, harbors mutations and translocations that result in a dysregulated gene expression. Interestingly, dysregulated expression of lincRNAs has been found in various cancers, but has not yet been described in B-ALL. Recently, we completed a gene expression profiling study in human B-ALL samples, which showed differential lincRNA expression in samples with particular cytogenetic abnormalities. This led us to hypothesize that lincRNAs may be related to disease pathogenesis. Here, we describe a promising lincRNA from our microarray data designated B-ALL associated long intergenic RNA 6 (BALIR-6). Expression of BALIR-6 is highest in patient samples carrying the MLL rearrangement (n=16; when compared to patients with TEL-AML1-translocated, n=39; E2A-PBX1-translocated, n=8; BCR-ABL-translocated, n=3; and cytogenetically normal cases, n=56; 1-way ANOVA p<0.0001) and showed significant variance in the expression level based on the immunophenotype (1-way ANOVA p=0.0004). BALIR-6 is located on chromosome 3p24.3 in humans, and exists in a syntenic gene block in with neighboring genes SATB1 and TBC1D5, and is conserved in mammals. Rapid Amplification of cDNA Ends (RACE) uncovered multiple transcript isoforms; from these, three were cloned out and sequenced, corresponding to the genomic locus as predicted. In B-ALL cell lines, BALIR-6 expression was highest in RS411 cells, which carry the MLL rearrangement, when compared to other B-ALL cell lines. This suggests that the cell lines may show a similar expression pattern to human B-ALL samples. To study the functional role of BALIR-6 we utilized siRNA in a mmu-miR-155 expression cassette to knockdown the transcript. In RS411 cells we observed a reduction in proliferation by MTS assay. Additionally, we observed an increase Sub-G0 cells and a decrease in G2-M phase cells by propidium iodide staining, suggesting an increase in apoptosis. Conversely, overexpression of BALIR-6 in a mouse pre-B cell line (70Z/3), leads to an increase in proliferation. Interestingly, during normal B cell development, BALIR-6 is dynamically expressed, with high expression in pre-B cells and subsequent downregulation, suggesting that a normal role during development is being hijacked in patients with B-ALL. Mechanistically, a few recent studies have described that lincRNAs can regulate gene expression in cis. To explore whether BALIR 6 regulates surrounding genes in cis, we analyzed microarray data of MLL rearranged B-ALL samples, finding that expression of BALIR-6 correlates with expression of surrounding genes SATB1 and TBC1D5. Interestingly for SATB1, this correlation is also seen in human B cell developmental stages. Altering BALIR-6 expression by siRNA mediated knockdown or overexpression causes an effect on the expression of surrounding genes SATB1 and TBC1D5. Previous findings have shown that dysregulated SATB1 has been seen in a variety of malignancies, suggesting a mechanism for how BALIR-6 may produce the changes in cell growth and apoptosis described above. Altogether, these results identify a novel and interesting RNA transcript with the potential to regulate gene expression and pathogenesis in B-ALL with MLL rearrangement, suggesting novel diagnostic, prognostic, and therapeutic implications. Disclosures: No relevant conflicts of interest to declare.

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Defining The Role Of Microrna-146a In B Cell Lymphomagenesis

Blood ◽

10.1182/blood.v122.21.3805.3805 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3805-3805

Author(s):

Jorge Contreras ◽

Jayanth Kumar Palanichamy ◽

Tiffany Tran ◽

Dinesh S. Rao

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Expression Patterns ◽

Significant Proportion ◽

B Cell Lymphoma ◽

Gene Expression Patterns

Abstract Diffuse large B cell lymphoma (DLBCL) is one of the most common Non-Hodgkin lymphomas among adults. It is a heterogeneous disease characterized by multiple mutations and translocations. Gene expression profiling studies have revealed several characteristic gene expression patterns, with two main patterns emerging, namely Germinal Center(GC) type, and Activated B Cell (ABC) type. ABC-type DLBCL shows gene expression patterns that resemble activated B-cells, with increased expression of anti-apoptotic, and pro-proliferative genes. Critically, upregulation of the NF-κB the pathway is a hallmark of ABC-type DLBCL and has been shown to be necessary for survival, and is caused by several different mutations at different levels within the pathway. Recent work has revealed the critical importance of a new class of small RNA molecules, namely microRNAs, in gene regulation. Of these, microRNA-146a (miR-146a) was discovered as an NF-κB induced microRNA that plays a role as a negative feedback regulator of this pathway by targeting adaptor proteins. To further characterize miR-146a, mice deficient for this miRNA were created, and were found to develop lymphadenopathy, splenomegaly, and myeloid proliferation. As expected, immune cells in these mice have an upregulated NF-κB pathway and many of the phenotypes can be ameliorated by inhibition of the NF-κB pathway. Importantly, a significant proportion of the animals develop B-cell lymphoma at older ages. In this study, we examined the role of miR-146a in the development of malignancy in B-cells. To accelerate the role of miR-146a in tumor formation we overlaid the miR-146a deficient allele onto the Eμ-Myc like mouse model. Eμ-Myc mice develop tumors on average by 14weeks of age. The transgenic status of animals was verified by genotyping, RNA and protein expression analyses. miR-146a sufficient and deficient animals on the Eμ-Myc background were followed for tumor latency by peripheral blood analysis and careful physical examination. Based on approved humane criteria for animal discomfort, animals were sacrificed and hematopoietic tissue was harvested for analysis. Mice deficient for miR-146a had a statistically reduced survival in comparison with miR-146a sufficient animals with a p-value of .0098 (Kaplan Meir survival analysis). Complete Blood Count of animals at time of death revealed an increase leukemia presentation in the miR-146a deficient background. FACS analysis of tumor tissue from both groups revealed an increase in the number of IgM positive tumors in the miR-146a-deficient background indicating skewing towards more mature B cell neoplasms when miR-146a is lacking. Lineage analysis of tumors verified them to be of B cell origin although a subset of miR-146a sufficient tumors had higher numbers of infiltrating myeloid cells compared to deficient animals. Furthermore, histologic analysis of hematopoietic organs showed that while infiltration remained similar in kidneys and liver, more spleens in the miR-146a deficient background tended to be less involved. Our extensive histopathologic and immunophenotypic analyses indicate that miR-146a deficiency drives a more aggressive malignant phenotype in the B-cell lineage. In keeping with this, our profiling studies of human DLBCL suggest that a subset of DLBCL show decreased expression of miR-146a. We are currently examining the status of NF-κB in the murine tumors and using high throughput sequencing approaches to delineate gene expression differences between miR-146a sufficient and deficient tumors. We anticipate the discovery of novel gene targets of miR-146a and expect that these studies will lead to improved diagnostic and therapeutic options for patients of B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

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Clinical Monoclonal B Lymphocytosis (cMBL), Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL): Diagnostic Criteria, Features At Diagnosis and Natural History

Blood ◽

10.1182/blood.v122.21.5273.5273 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5273-5273

Author(s):

Rodrigo Santacruz ◽

Julio Delgado ◽

Tycho Baumann ◽

Maria Rozman ◽

Martha Aymerich ◽

...

Keyword(s):

Overall Survival ◽

Multivariate Analysis ◽

B Cells ◽

B Cell ◽

Cell Count ◽

Diagnostic Criteria ◽

Conflicts Of Interest ◽

Consistent Difference ◽

Transformation Rate ◽

Mutational Status

Abstract Introduction CLL, SLL and cMBL are considered to be part of the same spectrum of clonal expansions of CD5+ B cells. Clinically, the transition from one to another of these forms over time is a well recognized event. Diagnostic criteria to separate these disorders have been proposed (IWCLL, 2008; WHO, 2008). Aim To compare presenting and evolving features of three groups of patients with cMBL, Rai 0 CLL or SLL and to ascertain the usefulness of current diagnostic criteria for these disorders. Patients and Methods Retrospective study of clinical, biologic and evolving characteristics of patients diagnosed with cMBL, Rai 0 CLL or SLL according to current criteria (CLL: ≥5x109 clonal B cells/L in peripheral blood; SLL <5x109/L clonal B cells with lymphadenopathy, organomegaly, cytopenia or disease-related symptoms; cMBL <5x109 clonal B cells with no signs or symptoms). Results Baseline characteristics of the patients are shown in the Table. Median age was 68 years (range, 24-94) and 57% of patients were males. Out of 1,093 patients, 79 had cMBL (7.2%), 522 Rai 0 CLL (48%) and 94 SLL (8.6%). Overall, adverse biomarkers such as high LDH (p<0.001), high B2M (p<0.001), increased ZAP70 (p<0.001), high CD38 (p<0.001), unmutated IGHV status (p=0.002), +12 (p=0.02) and 11q- (p=0.01) were significantly more frequent in SLL. In subgroup analyses, the only difference between cMBL and Rai 0 CLL was a higher proportion of cases with mutated IGHV in cMBL (p=0.008). Furthermore, when SLL was compared to Rai I to IV CLL no differences were observed (data not shown). The actuarial risk for transformation from cMBL or SLL to CLL was 4.2% and 4.4 % per year (p=0.5), respectively. Median TTFT was significantly shorter in the SLL group (12 m.) than in Rai 0 CLL (174 m.) or cMBL (244 m.) (p<0.001). Median overall survival was also significantly shorter for SLL (94 m.) compared with Rai 0 CLL and cMBL (153 and 157 m., respectively) (p=0.028). Multivariate analysis of 695 patients (cMBL/Rai 0-CLL/SLL) revealed four variables independently correlated with shorter TTFT: diagnosis of SLL vs. Rai 0 CLL vs. cMBL (HR 2.28; p=0.008), high ZAP70 (HR 4.08; p<0.001), high CD38 (HR 4.68; p=0.001) and increased serum B2M levels (HR 1.54; p = 0.031). Importantly, however, when the multivariate analysis was restricted to patients with cMBL and Rai 0 CLL, variables correlated with TTFT were the clonal B-cell count (HR 3.76; p=0.01), ZAP70 (HR 3.31; p<0.001), and CD38 (HR 4.61; p=0.02). FISH cytogenetics, IGHV mutational status,NOTCH1 or SF3B1 mutations were not included in the analysis because of missing data. Conclusions Disparities in biologic and clinical features of cMBL, CLL and SLL mainly reflect the different tumor burden in this spectrum of CD5+ monoclonal B cell disorders. In this study, the transformation rate from either cMBL or SLL to CLL was around 4% per year. As a result of differences in tumor mass, the need for therapy was shorter in SLL than in Rai 0 CLL and cMBL, and the overall survival poorer. Biologically, the only consistent difference between cMBL and Rai O CLL was a higher proportion of mutated IGHV in cMBL. Clinically, however, no differences in median survival were observed. Moreover, the clonal B cell count was the most reliable predictor of disease outcome in both cMBL and Rai 0 CLL. Therefore, the distinction between cMBL and Rai 0 CLL seems hardly justified. Disclosures: No relevant conflicts of interest to declare.

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Erfe-Encoding FAM132B in Congenital Dyserythropoietic Anemia Type II

Blood ◽

10.1182/blood.v126.23.535.535 ◽

2015 ◽

Vol 126 (23) ◽

pp. 535-535

Author(s):

Roberta Russo ◽

Immacolata Andolfo ◽

Luigia De Falco ◽

Francesco Manna ◽

Antonella Gambale ◽

...

Keyword(s):

Gene Expression ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Transferrin Saturation ◽

Inverse Correlation ◽

Type Ii ◽

Ineffective Erythropoiesis ◽

Congenital Dyserythropoietic Anemia ◽

Over Expression

Abstract Recessive mutations in SEC23B gene cause congenital dyserythropoietic anemia type II (CDAII), a rare hereditary disorder hallmarked by ineffective erythropoiesis, iron overload, and reduced expression of hepatic hormone hepcidin (Iolascon, 2013). The most recently described hepcidin regulator is the erythroblast-derived hormone erythroferrone (ERFE), a member of TNF-α superfamily that specifically inhibits hepcidin production in experimental models (Kautz, 2014). However, the function of ERFE in humans remains to be investigated. To determine whether dysregulation of ERFE expression is associated with ineffective erythropoiesis and iron-loading in CDAII, we studied the ERFE-encoding FAM132B gene expression in 48 SEC23B-related CDAII patients and 29 age and gender matched healthy controls (HCs). Twelve new cases and four novel SEC23B mutations were described. Samples were obtained after informed consent, according to the Declaration of Helsinki. Genomic DNA, mutational screening, RNA isolation, cDNA preparation, and qRT-PCR were performed as previously described (Russo, 2013). All patients were young adults (17.0±2.5 years at diagnosis), with increased serum ferritin (395.4±67.6 ng/mL) and transferrin saturation (71.9±5.4 %). We observed a statistically significant overexpression of FAM132B gene in peripheral blood mononuclear cells from CDAII patients (9.09±0.08) compared to HCs (8.32±0.12, p<0.0001). A similar trend was obtained when evaluating FAM132B expression in reticulocytes from a subset of patients and HCs. Of note, a statistically significant correlation between peripheral blood and reticulocyte FAM132B expression from the same patients was observed (Spearman ρ= 0.78, p=0.02). Although the role of ERFE in peripheral blood is still unknown, our observations suggested that the evaluation of FAM132B mRNA in peripheral blood is a reliable and easy-to-measure marker of ERFE levels. When we divided CDAII patients into two sub-groups accordingly to FAM132B gene expression, we observed a statistically significant reduction in hemoglobin (Hb) level in the high-FAM132B subset (8.6±0.4 g/dL) respect to low-FAM132B one (10.1±0.5 g/dL, p=0.02). Of note, the expression level of FAM132B did not correlate with the transfusion regimen. The higher amount of ERFE reflects the increased iron demand for Hb production as well as the expanding abnormal erythropoiesis, as attested by the increased RDW and sTfR (although not significant) in high-FAM132B patients. This in turn leads to reduced hepcidin in high-FAM132B group (4.2±1.8 nM) compared to low-FAM132B one (5.9±1.8 nM, p=0.05), resulting in augmented iron delivery to the erythron. Although the iron balance data do not differ significantly between the two groups, a tendency to decreased hepcidin/ferritin ratio and increased transferrin saturation was observed in high-FAM132B patients. Thus, FAM132B overexpression seems to contribute to the inappropriate suppression of hepcidin with subsequent hemosiderosis observed in CDAII. Consistent with our previous studies, we observed a reduced SEC23B expression in our patients compared to HC. Indeed, FAM132B and SEC23B gene expression exhibited an inverse correlation (Spearman ρ=-0.36, p=0.01). We confirmed the ex vivo data about inverse correlation between FAM132B and SEC23B expression observed in our patients by establishing K562 SEC23B-silenced cells. To knockdown SEC23B gene expression in K562 cells two different pGIPZ Lentiviral shRNAmir for SEC23B (shSEC23B-70/-74) were used. We observed a higher expression of FAM132B at 5 days of erythroid differentiation in K562 SEC23B-silenced cell compared to not-silenced ones. Conversely, SEC23B expression was lower in both shSEC23B compared to sh-CTR at 2 and 5 days of differentiation. Although the mechanisms of hemin-induced differentiation are quite different from EPO-induced ones, we can hypothesize that FAM132B over-expression is related to the maturative arrest and the subsequent increased number of erythroid precursors. This study provides the first analysis on ERFE regulation in humans. Our data suggest that ERFE over-expression in CDAII patients is the result of both physiological and pathological mechanisms leading to hepcidin suppression in condition of dyserythropoiesis. Nevertheless, it seems that ERFE cannot be the main erythroid regulator of hepcidin suppression, at least in CDAII patients. Disclosures No relevant conflicts of interest to declare.

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CD44 over Expression Caused Drug Resistance in Activated B Cell-like Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v128.22.5316.5316 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5316-5316

Author(s):

Qinjun Zhou ◽

Yongqiang Wei ◽

Xiaolei Wei ◽

Qi Wei ◽

Ru Feng

Keyword(s):

Drug Resistance ◽

B Cell ◽

Growth Regulation ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Annexin V ◽

B Cell Lymphoma ◽

Over Expression ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract CD44, a transmembrane glycocoptotein, involved in tumor cell survival, migration , invasion, metastasis and prognosis of many cancers. This study was performed to investigate the effects of CD44 over expression on the biological function and the chemotherapeutic sensitivity of ABC-DLBCL. The full length CD44 cDNA was cloned into pEASY-T vector and then transfected into activated B cell-like diffuse large B-cell lymphoma cell line OCI-ly3. QPCR and western blot confirmed that the CD44 expression was up-regulated in both mRNA and protein levels in CD44-transfected OCI-ly3 cells. The cell proliferation of OCI-ly3-CD44 cells was significantly faster than that in OCI-ly3-GFP cells and the OCI-ly3 cells (p<0.001). Annexin V-APC/PI staining results showed that the apoptosis ratio wasn't difference among three groups (p=0.676). OCI-ly3-CD44 cells showed significantly higher migration ratio than OCI-ly3-GFP cells and the OCI-ly3 cells(p=0.031). The IC50 of doxorubicin in OCI-ly3-CD44 cells (0.348±0.072uM) was higher than that in OCI-ly3-GFP (0.348±0.072uM ) and OCI-ly3cells(0.138±0.029uM ) ( P<0.001). After treatment with doxorubicin for 24h, the OCI-ly3-CD44 cells showed lower apoptotic ratio than OCI-ly3-GFP and OCI-ly3 cells ( (17.5±1.33)% VS. (41.7±9.91) %, (41.8±7.23)%), P<0.001). In conclusion, CD44 plays a key role in cell growth regulation and chemo-resistance and is a potential target to overcome drug resistance and improve prognosis in DLBCL. Disclosures No relevant conflicts of interest to declare.

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Gene Expression Analysis Uncovers Similarity and Differences Among Burkitt Lymphoma Subtypes

Blood ◽

10.1182/blood.v116.21.2494.2494 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2494-2494

Author(s):

Pier Paolo Piccaluga ◽

Giulia De Falco ◽

Manjunat Kustagi ◽

Anna Gazzola ◽

Annalisa Astolfi ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

B Cell ◽

Burkitt Lymphoma ◽

Who Classification ◽

Cell Cycle Control ◽

B Cell Lymphomas ◽

Over Expression

Abstract Abstract 2494 Background. Burkitt lymphoma (BL) is currently listed in the WHO classification of lymphoid tumors as a single genetic and morphological entity with variation in clinical presentation. In particular, three clinical subsets of BL are recognized: endemic (eBL), sporadic (sBL) and immunodeficiency associated (ID-BL). Each affects different populations and can present with different features. So far, possible differences in their gene expression profiles (GEP) have not been investigated. In this study we aimed to 1) assess whether BL subtypes present with differences in their GEP; 2) investigate the relationship of the different BL subtypes with the non-neoplastic cellular counterparts; 3) Identify genes and programs specifically deregulated in BLs and possibly contributing to the malignant phenotype. Methods. We studied by GEP 128 cases of B-cell derived malignancies and 20 samples of normal B-cell subpopulations GEP analysis. In particular, we included 40 BLs (13 eBLs, 21 sBLs 6 HIV-BLs), 40 follicular lymphomas, 10 chronic lymphocytic leukemias, 10 GCB-type diffuse large B-cell lymphomas, 10 ABC-type DLBCL, 5 primary mediastinal B-cell lymphomas, 13 HIV-related DLBCL, as well as 10 germinal center (GC), 5 naïve and 5 memory cells samples. GEP results were confirmed by dividing BL cases into training and test subgroups. In addition, as further validation, we performed immunohistochemistry (IHC) on tissue microarrays containing 85 BL cases as well as functional assays in vitro and in vivo, by focusing on the role of RBL2, a tumor suppressor gene involved in cell cycle control and mutated in eBL. Specifically, we used cell transfection and shRNAs (for mimicking MYC over-expression and RBL2 silencing), soft agar and invasion capability assays, and xenografted mouse models. Results. First, we found that BLs constitute a unique molecular entity, with a relatively homogeneous GEP, distinct from other B-cell malignancies. Indeed, by unsupervised analysis all BLs clearly clustered apart of other lymphomas. However, by supervised analysis, we found that BL subtypes presented slight differences in their GEPs. Particularly, eBLs and ID-BLs appeared to be almost identical, diverging from sBLs. Specifically, they varied for genes involved in cell cycle control, BCR-signaling, and TNF/NFKB-pathways. Of note, eBLs and ID-BLs on one hand, and sBLs on the other (roughly corresponding to EBV+ vs. EBV− cases) also differed for genes target of mi-R127a, which is altered in EBV+ cases as a direct consequence of viral integration. To further investigate cell cycle regulation in BLs, we inferred a network of RBL2-depending genes by reverse engineering, by uncovering possible RBL2 transcriptional targets. Interestingly, we found that eBL and sBL diverged for genes belonging to such network. Notably, we provided evidences that RBL2 can cooperate with MYC in inducing a neoplastic phenotype in vitro and in vivo. In particular, lymphoblastoid cells engineered to carry both MYC over-expression and RBL2 silencing presented with increased colony formation and matrix invasion capabilities, and higher efficiency in inducing tumor formation in nude mice if compared to single transfectants (MYC+ or RBL2−). Moreover, as the present WHO classification does not definitely identify the counterpart of eBL, we compared BLs GEP to those of normal B-cells. We found that all BL subtypes were intimately related to GC cells (by showing an early stage GC differentiation arrest), differing from them for molecules specially involved in cell proliferation, immune response, and signal transduction. Finally, as further validation of GEP, we studied by IHC the expression of SPARC and CYR61, two molecules involved in human tumorigenesis. Indeed, they turned out to be consistently expressed by neoplastic elements in all instances, as indicated by GEP analysis. Conclusions. Our study provided substantial insights on the pathobiology of BLs, by offering novel evidences which may be relevant for its classification and possibly future treatment. Disclosures: No relevant conflicts of interest to declare.

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Inotuzumab Ozogamicin (Ino) May Overcome the Impact of Philadelphia Chromosome (Ph)-like Phenotype in Adult Patients (pts) with Relapsed/Refractory (R/R) Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood-2019-126940 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1641-1641 ◽

Cited By ~ 1

Author(s):

Elias Jabbour ◽

Kathryn G. Roberts ◽

Koji Sasaki ◽

Yaqi Zhao ◽

Chunxu Qu ◽

...

Keyword(s):

Gene Expression ◽

Overall Survival ◽

Stem Cell ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Advisory Committees ◽

The Impact

Background: Ino showed significant activity in phase II trials in pts with R/R ALL, that was subsequently confirmed in Phase III trial where Ino demonstrated higher response rates and superior overall survival vs standard of care chemotherapy (SOC) in adults with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (R/R ALL).Ph-like or BCR-ABL1-like ALL possesses a gene expression profile similar to that of BCR-ABL1 ALL but lacks the BCR-ABL1 fusion protein. It is characterized by increased expression of hematopoietic stem-cell genes, deletion of B-cell lineage genes and kinase-activating alterations. Ph-like ALL is associated with refractoriness to standard induction/consolidation chemotherapy and poor prognosis. Aim: To evaluate the outcomes of pts with R/R Ph-like ALL treated in phase II trial with Ino monotherapy. Methods: We performed an integrated analysis of whole genome sequencing (to identify sequence mutations, structural variations and DNA copy number alterations), and transcriptome sequencing (RNAseq; to quantify gene expression, determine Ph-like gene expression profile and identify fusions) on 53 patients' samples treated with Ino between June 2010 and September 2012. Results: Fifty-three evaluable pts with R/R ALL with stored baseline samples were analyzed. Pts characteristics are summarized in Table 1. Median age was 50 years. Ino was given as Salvage 1, Salvage 2, and Salvage 3 and beyond in 20 (38%), 18 (34%), and 15 (28%) pts, respectively. Figure 1 reflects the different genomic subgroups identified among 53 evaluable pts. Ph-like gene signature was found in 12 pts (22.6%). Among these 12 pts, 6 had IGH-CRLF2, 2 IGH-EPOR, 1 SNX2-ABL1, and 3 had no fusions identified. The overall response rates (ORR) were 54% [complete remission (CR) 20%, CR with partial hematologic recovery (CRh) 32%, and marrow CR (CRi) 2%]. Among pts with morphologic remission, 46% and 82% achieved minimal residual disease (MRD) negativity at CR and at any time, respectively. The ORR for pts with Ph-like ALL, Ph-positive ALL, ALL with KMT2A, and others were 58% (CR=25%; CRh=33%), 42% (CR=8%; CRh=33%), 57% (CR=14%; CRh=29%; CRi=14%), and 56% (CR=26%; CRh=30%), respectively. The respective overall MRD negativity rates were 71%, 100%, 75%, and 83% (Table 1). The median follow-up was 60 months. The median event-free (EFS) and overall survival (OS) were 3.3 and 5.4 months, respectively. There was no difference in EFS and OS between the subgroups analyzed (P=0.464; P=0.824). The median EFS and OS were 4.5 and 4.5 months for pts with Ph-like, 3.1 and 7.2 months for those with Ph-positive ALL, 2.8 and 4.4 months for those with KMT2A, and 2.2 and 4.6 months for others (Table 1). 21 (40%) pts had subsequent allogeneic stem cell transplant; 6 (50%), 3 (25%), 4 (57%), and 8 (36%) in each subgroup, respectively. The rate of VOD was 3 (6%) with no difference among different subgroups. Conclusion: The current analysis suggest that Ino therapy may overcome the impact of Ph-like phenotype in pts with ALL. Confirmation of these findings in a larger cohort and in frontline ALL patients is needed. Disclosures Jabbour: Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding. Sasaki:Pfizer: Consultancy; Otsuka: Honoraria. Jain:Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ravandi:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xencor: Consultancy, Research Funding; Macrogenix: Consultancy, Research Funding; Menarini Ricerche: Research Funding; Selvita: Research Funding; Cyclacel LTD: Research Funding. Short:AstraZeneca: Consultancy; Takeda Oncology: Consultancy, Research Funding; Amgen: Honoraria. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Konopleva:Cellectis: Research Funding; Agios: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Ascentage: Research Funding; Eli Lilly: Research Funding; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Kisoji: Consultancy, Honoraria; Ablynx: Research Funding; Genentech: Honoraria, Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Astra Zeneca: Research Funding. Mullighan:Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel. Kantarjian:Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Research Funding; Amgen: Honoraria, Research Funding; Immunogen: Research Funding; AbbVie: Honoraria, Research Funding; Astex: Research Funding; BMS: Research Funding; Cyclacel: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Honoraria, Research Funding; Jazz Pharma: Research Funding; Takeda: Honoraria.

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Clinicopathologic Comparison of Plasmablastic Lymphoma in HIV-Positive Versus HIV-Negative Patients: Single-Center Experience

Blood ◽

10.1182/blood.v128.22.1864.1864 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1864-1864 ◽

Cited By ~ 1

Author(s):

Jule Vasquez ◽

Jorge Huamanchumo ◽

Shirley Quintana

Keyword(s):

Overall Survival ◽

Hiv Infection ◽

B Cell ◽

Conflicts Of Interest ◽

Plasmablastic Lymphoma ◽

Aggressive Lymphoma ◽

Cancer Center ◽

Hiv Positive ◽

Hiv Patients ◽

Hiv Negative

Abstract Background: Plasmablastic lymphoma (PBL) is an aggressive lymphoma associated mainly to HIV infection, although cases in immunocompetent patients are described as well. Objective: To describe the prevalence, the clinicopathologic features and determine the overall survival of lymphoma patients according human immunodeficiency virus (HIV) status. Methods: We reviewed the pathology database at Instituto Nacional de Enfermedades Neoplasicas (INEN), the leading cancer center of Peru from 2005 to 2014. 6218 cases were lymphomas, 5031 cases were Non-Hodgkin lymphoma and 3905 cases were B-cell lineage. 22 met diagnosis of PBL, 4 patients were excluded (1 prior treatment, 1 synchronous malignancy and 2 incomplete medical records). Finally, we had 18 cases for evaluation. Survival curves were estimated by Kaplan Meier. Statistical analysis was based on SPSS Program version 22. Results: The prevalence of PBL was 0.004% of all Non-Hodgkin lymphomas and 0.005% of B-cell lymphomas. 13 of 18 cases (72.2%) were HIV-positive patients (PBL-HIV+). The median age for PBL-HIV+ was 37 years (range 22-67 years) and 58 years (range 53-74 years) for HIV-negative patients (PBL-HIV-).The extra-oral primary was the most frequent primary site in both groups. The advanced stage was 80% in PBL-HIV- patients. Presence of B-symptoms and Ki-67>80% were greater in PBL-HIV- patients. CHOP or CHOP-like regimen was the common treatment in both groups, only one patient received DA-EPOCH (PBL-HIV+ group). HAART-naïve patients were 77%. The median OS time was 43 months (range 1-84 months) in PBL-HIV+ patients and 13 months (range 0-15 months) in PBL-VIH- patients, the 5-yrs-OS was 26.9% y 0% respectively. Conclusions: Plasmablastic lymphoma is a rare lymphoma, either associated or not to HIV infection. Advanced and aggressive disease is a distinctive feature in both lymphomas. The PBL-HIV- has a worse prognosis with shorter overall survival compared to the PBL-HIV+ patients. Disclosures No relevant conflicts of interest to declare.

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