Gene Expression Profile Reveals a Unique Pattern of Protein Kinases in Chronic Lymphocytic Leukemia (CLL): Potential Role of the Second Generation Protein Kinase Inhibitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3117-3117
Author(s):  
Simona Tavolaro ◽  
Sabina Chiaretti ◽  
Monica Messina ◽  
Nadia Peragine ◽  
Roberta Maggio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kinase Inhibitors ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Mutational Status ◽  
Healthy Donors ◽  
Igvh Mutational Status ◽  
B Lineage

Abstract Purpose. CLL is a malignancy of mature B cells with an heterogeneous clinical course. In order to identify novel therapeutic targets and given the role of protein kinase (PK) deregulation in cancer development and the availability of specific PK inhibitors, oligonucleotide arrays were used to determine if CLL patients exhibit a specific PK pattern. The results were validated by Q-PCR, and the in vitro efficacy of a dual specific inhibitor, dasatinib (Bristol-Myers Squibb, Princeton, NJ), was tested. Methods. The gene expression profile of 44 CLL and 137 acute lymphocytic leukemia (ALL) patients was evaluated using the HG U133 Plus 2.0 Affymetrix arrays. Two additional sets of CLL (49 cases) were utilized as test sets: 505 PK genes were used for all analyses, which included unsupervised clustering, Analysis of Variance (ANOVA) and t-test analysis. To validate the gene expression data, a Q-PCR approach was used on 14 CLL, 6 B-lineage ALL, 3 T-ALL and CD19+ enriched normal B cells, isolated from peripheral blood of 3 healthy donors. Finally, to evaluate the in vitro effects of dasatinib on primary CLL cells, the MTT assay was performed on 21 untreated CLL samples following 72 and 96 hours of drug exposure. Results. Unsupervised analysis on CLL samples and different ALL subgroups showed a very homogeneous PK gene profile in CLL. ANOVA corroborated these results. Among the PK that proved overexpressed in CLL, we identified 16 PK genes highly expressed in all 3 CLL sets analyzed. For these genes, Q-PCR analysis showed that CLL cases display a significantly higher expression level, when compared to B-lineage ALL, T-ALL and, more importantly, to CD19+ cells from healthy controls. PK expression was also analyzed within different CLL subclasses, subdivided on the basis of the IgVH mutational status, as well as CD38 and ZAP-70 expression. The comparison among these groups did not show a specific PK signature associated with biologic features; similar results were obtained by Q-PCR analysis. To validate the gene expression profiling and Q-PCR findings, and to test the efficacy of dasatinib, functional in vitro experiments were performed on primary CLL cells. Treatment with 1μM dasatinib induced an overall viability reduction of 40% after 96 hours in the CLL samples analyzed; no significant differences were associated with the IgVH mutational status. These findings indicate that this inhibitor is functionally active on CLL cells, without however reaching marked levels of cytotoxicity when used as a single agent. Conclusions. Our results highlight a very homogeneous PK signature in CLL, independently of specific biologically-defined prognostic subgroups. A set of these PKs is more highly expressed in CLL cells than in CD19+ cells from healthy donors. Dasatinib treatment induces a ∼40% viability reduction after 96 hours exposure. Overall, these results suggest that PK inhibitors, namely dual kinase inhibitors, may have a role in the management of CLL patients particularly in combination with other therapeutic agents. * ST and SC equally contributed to the study

Heat Shock Protein 90 (Hsp90) Activity Status Is An Independent Prognostic Marker in Chronic Lymphocytic Leukemia (CLL) and Correlates with IgVH Mutational Status, ZAP-70 Expression and Time to First Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2544-2544
Author(s):  
Januario E. Castro ◽  
Juan S. Barajas-Gamboa ◽  
Julio A. Diaz-Perez ◽  
Lina M. Ariza-Serrano ◽  
Johanna Melo-Cardenas ◽  
...  
Keyword(s):  
Disease Progression ◽  
Progressive Disease ◽  
Prognostic Markers ◽  
Hsp90 Inhibitor ◽  
Lymphocytic Leukemia ◽  
Independent Prognostic Marker ◽  
In Vitro Sensitivity ◽  
Mutational Status ◽  
Igvh Mutational Status

Abstract Abstract 2544 The clinical presentation of chronic lymphocytic leukemia (CLL) is variable and includes at least two distinct clinical subtypes: indolent or progressive disease. Several prognostic markers such as the mutational status of the IgVH genes or ZAP-70 expression identify patients that will have more aggressive clinical courses. We have previously reported that ZAP-70 expression in CLL requires the support and stability provided by the Hsp90 activated complex (Hsp90-AC) and that Hsp90 inhibitors induce apoptosis in CLL cells preferentially in patients with high–risk/rapid progressive disease. Hsp90 requires the conformation of a multimeric complex with other co-chaperones (Hop, p23) to become active and function as a chaperone for mutated, over-expressed, misfolded or constitutively activated proteins. However, to measure Hsp90-AC activity is challenging and requires protein assays that are semi-quantitative and not always can provide a functional read out. To address this problem we designed a test to measure Hsp90-AC by taking advantage of the higher avidity of this complex to bind Hsp90 inhibitors such as 17-AAG. We hypothesize that Hsp90-AC activity is an independent prognostic marker of disease progression in CLL and to test this we measured the ability of 17-AAG to inhibit Hsp90-AC and promote apoptosis in vitro in a cohort of previously untreated CLL patients. This evaluation was performed in samples collected at the time of diagnosis and the results were correlated with the time from diagnosis to first treatment (t-Tx1) as well as the presence of other known prognostic markers. Ninety-five previously untreated CLL patients were included in this cohort. In vitro sensitivity/apoptosis to Hsp90-AC inhibition using 17-AAG (1μg/ml) was measured by flow cytometry using DiOC6 and PI after 48 hours of incubation. Only samples with more than 60% viability were included and we selected a cut-point of ≥ 53% induction of apoptosis to determine if a sample was sensitive or not to Hsp90-AC inhibition (This cut-point was selected using a ROC analysis for optimal diagnostic performance). We found that 37 patients (39%) were sensitive to 17-AAG. These patient had a median time from diagnosis to first treatment (t-Tx1) of 7 years compared to 4.2 years in patients that were resistant to 17-AAG (p=0.04). In addition, 37 patients (39%) had unmutated IgVH genes with a median t-Tx1 of 8.7 years compared to 4.2 years in patients with mutated IgVH genes (p=0.02) and 41 patients (43%) had high levels of ZAP-70 expression (>20% by flow cytometry) and this group of patients had a median t-Tx1 of 7 years vs. 4.2 years in ZAP-70 negative patients (p=0.03). We evaluated the association between the degree of Hsp90-AC inhibition (17-AGG sensitivity) and IgVH mutational status and found a strong correlation between these two variables with shorter t-Tx1 in patients that were IgVH unmutated and sensitive to Hsp90 inhibitor (p=0.0003). We found a similar strong correlation between ZAP-70 expression and sensitivity to Hsp90-AC inhibitor (p=0.003). In our patient cohort, we saw that IgVH, ZAP-70 and Hsp90 inhibitor sensitivity are independent prognostic markers in CLL (Cox regression, p=0.002). We found that the sensibility and specificity of ZAP-70 test for high-risk CLL (sensitivity 81.1% [95% IC 67–95%] and specificity 77.6%[95% IC 65–90%]) was increased when the Hsp90-AC inhibitor sensitivity test was added (sensitivity 87% [95% IC 71–100%] and specificity 91%[95% IC 79–100%]). In conclusion, in vitro inhibition of Hsp90-AC in CLL using 17-AAG is an independent prognostic maker and strong predictor of the need for treatment. Hsp90-AC inhibition showed a high correlation with IgVH mutational status and ZAP-70 expression. Additionally, patients sensitive to 17-AAG whose leukemic cells also highly express ZAP-70 appear to have the worst prognosis with the shortest t-Tx1. Our data suggest that in vitro sensitivity to 17-AAG at the time of diagnosis serves as a quantitative surrogate marker for Hsp90-AC activity and correlates with disease progression in CLL. This is the first time that such correlation has been established in a cohort of cancer patients and shows the relevance of the Hsp90 chaperone system in cancer biology and disease progression. Disclosures: No relevant conflicts of interest to declare.

Clinical-Biological Characterization of Variant B Chronic Lymphocytic Leukemia, Characterized by a Mantle Cell Lymphoma-Like Immunophenotype, t(11;14)(q13;q32) Negative.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1259-1259
Author(s):  
Andrea Ferrario ◽  
Lilla Cro ◽  
Nadia Zucal ◽  
Marta Lionetti ◽  
Francesco Bertoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Mantle Cell Lymphoma ◽  
Statistical Difference ◽  
Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Mutational Status ◽  
Mantle Cell ◽  
Igvh Mutational Status

Abstract Abstract 1259 Poster Board I-282 We describe the clinical-biological features of 63 cases of variant B chronic lymphocytic leukemia (v-B-CLL), characterized by a mantle cell lymphoma-like immunophenotype, atypical cytomorphology in absence of t(11;14)(q13;q32) in FISH analysis. A historical series of 130 B-CLL was used as comparison. The v-B-CLL were significantly different from the B-CLL in terms of the following clinico-hematological variables: age <70 yrs (p <.001), lymphocytosis <20 × 109/ (p <.001), lymphocyte doubling time < 12 months (p = .02), high serum β2-microglobulin levels (p <.001), and splenomegaly (p = .002). Considering immunophenotipic features, CD38 and CD49d expression were significantly more expressed in v-B-CLL than B-CLL (p <.001); whereas, no statistical difference was observed for ZAP-70 reactivity. Considering the IgVH mutation status, there were more patients mutated in the v-B-CLL group than in the B-CLL group (p = .001). Other significant differences were found about the frequency of the recurrent chromosome alterations, evaluated by means of FISH analysis: trisomy 12 was more frequent in v-B-CLL ( p<.001), while del13q14, considered as a single alteration, was more frequent in B-CLL (p=.008). Gene expression profiling of a panel of 9 v-B-CLL compared with 60 B-CLL samples indicated that the variant group is characterized by a specific molecular pattern of gene expression. In particular we observed the upregulation of tumor protein D52 (TPD52), and that of 6 genes (AFF1, GMPS, PICALM, JUN, REL, RAC2) known to be protooncogenes. Furthermore, we found that upregulated genes with apoptosis related functions (IL-7, HSP90B1, NOTCH2, BECN1, ANXA4, MCL1) are all negative regulators of apoptosis. Microarray analysis revealed that various genes (TRIM38, EEF1D, CASP1, MALT1, RHOH0) involved in the I-kB kinase/NF-kB cascade of the canonical NF-kB signaling pathway, are furtherly upregulated in v-B-CLL. Furthermore, among the genes found differentially expressed in SAM analysis, we observed also the upregulation of CD1c (according to the surface expression of this antigen), OSBPL3 and ITGA4. After a median follow-up of 55 months (range 4-196) and 60 months (range 6-180), 25/42 (59%) v-CLL and 55/93 (59 %) CLL pts were treated. TTT was significant different between 2 groups when the IgVH mutational status was considered (p= .006). Median OS of v-CLL subset was 112 months vs 171 months of CLL subset. When the IgVH mutational status was considered, mutated cases showed a worse OS even if a statistical difference was not observed (p= 0.062). In conclusion, our study identifies, on the basis of a defined CFM-FISH diagnostic approach, a variant form of B-CLL that shows peculiar biological and clinical features that should be considered in the future clinical and prognostic studies. The inclusion of this form in B-CLL study could alter the interpretation of results, especially related to biological markers. Disclosures No relevant conflicts of interest to declare.

scholarly journals In-Vitro Drug Sensitivity Screening in Chronic Lymphocytic Leukemia (CLL) Primary Patient Samples Identifies Drug Candidates for Precision Cancer Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4676-4676
Author(s):  
Deepak Balaji balaji Thimiri Govinda Raj ◽  
Andrea Cremaschi ◽  
Sigrid Strand Skånland ◽  
Alexandra Gade ◽  
Fredrik H. Schjesvold ◽  
...  
Keyword(s):  
Research Funding ◽  
Drug Sensitivity ◽  
Kinase Inhibitors ◽  
Proteasome Inhibitors ◽  
Lymphocytic Leukemia ◽  
Mutation Status ◽  
Drug Candidates ◽  
Healthy Donors ◽  
Screening Platform

Abstract Introduction Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults and is currently considered incurable. Although current treatment regimens prolong life for patients, CLL eventually relapses. Efficient therapies may require a personalized approach that combines targeting cancer cells and the tumor microenvironment by restoring the patient's own anti-tumor immunity. However, a major limitation is that no efficient approach exists to identify the most effective drugs for each patient and cancer stage. With the aim to support future introduction of individualized treatment for patients, we assessed the sensitivity of CLL patient samples to several drug candidates using our in vitro functional drug sensitivity screening platform. Methods We have established novel in vitro culture settings that mimic the CLL tumor microenvironment and allow proliferation of CLL cells for 5 days. Using our unique method, we performed drug screening on 26 patient samples and 10 healthy donors against a customized, annotated library of 516 drugs including kinase inhibitors, proteasome inhibitors, B-cell pathway inhibitors and several other approved drug classes. Primary patient samples were cultured in 384 well-plates with the presence of individual drugs over a concentration range over 5 logs. Drug sensitivity was assessed using CellTiter-Glo® luminescent cell viability assay and CellTox™ green cytotoxicity assay on day 5. Drug Sensitivity Score (DSS) was then calculated for each drug using the IC50 value, slope and area under the curve (AUC). DSSs for CLL patient samples were next compared with DSS of healthy donors for the full patient sample cohort screened so far to generate a selective DSS (sDSS = DSSpatient - DSShealthy) for each patient. Drugs which have sDSS >5 were considered clearly more effective for patient samples in the in vitro test system. CLL samples were assessed for sDSS using our screening data and we ranked all the drugs by their score. Results In order to find drug candidates for targeted therapies in CLL patients, we performed in vitro drug sensitivity screening on 13 IgVH unmutated and 13 IgVH mutated CLL patient samples, as well as 10 healthy donors (due to the lower number of cells healthy donors were pooled into two samples of 5 donors each). Our in vitro assay showed that proteasome inhibitors, kinase inhibitors and several approved CLL drug candidates were considered sensitive in the majority of patient samples. This included venetoclax, the Bcl-2 inhibitor ABT-737, doxorubicin, acalabrutinib, other kinase inhibitors (sunitinib, volasertib, trametinib, copanlisib) and proteasome inhibitors (carfilzomib, bortezomib). Selective drug sensitivity scores of the top 5 drugs in all patient samples (73 drugs in total) are shown in the heatmap (Figure 1). Venetoclax showed a higher sDSS score in 10 of the 20 patients with an average sDSS score of 22.3 followed by ABT-737 (Bcl-2 inhibitor) with an average sDSS score of 19.7. By performing hierarchical clustering analysis (Euclidean distance, Ward linkage method), we observed unsupervised clustering of patient samples irrespective of the IgVH mutation status. We are currently expanding the analysis by classifying the patient samples by age, sex and mutation status. Conclusion Our novel CLL culture method that allows cell proliferation along with our established functional in-vitro drug sensitivity screening platform enabled us to screen a number of patient samples and evaluate the sensitivity of a library of approved drugs and investigational drug candidates for CLL. Our analysis shows that several drugs may be effective for CLL and can be tested in drug combinations in order to identify synergistic effects. As a future perspective, we want to combine machine learning strategies with the experimental drug screening strategies to identify drug combinations and validate drug candidates by xenografting and in precision medicine clinical trials. Figure 1: Selective Drug Sensitivity Screening (sDSS) score for top 3 drugs (44 drugs) for 20 CLL patient samples. Green label is IgVH mutated CLL patient samples and blue label is IgVH unmutated patient samples. Disclosures Schjesvold: Oncopeptides: Consultancy; Abbvie: Honoraria; Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Adaptive: Consultancy; Bayer: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding.

scholarly journals MiR-181b in Chronic Lymphocytic Leukemia B Cells Is Regulated By Cellular Interaction with CD4+ T Cells and Increases the CTL Toxicity Versus the Leukemic Clone

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4134-4134
Author(s):  
Mirco di Marco ◽  
Serena Veschi ◽  
Rosa Visone ◽  
Giuseppe Leone ◽  
Paola Lanuti ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cd4 T Cells ◽  
Lymphocytic Leukemia ◽  
Healthy Donors ◽  
Leukemic Clone

Abstract Clinical progression of chronic lymphocytic leukemia (CLL) is characterized by gradual reduction of the ratio T/B cells, along with immune cell dysfunction due, at least in part, to T cell defects, such as decreased expression of CD40L and reduced signaling via the TCR CD3. This compromise the ability of T cells to respond and to eliminate leukemic cell from CLL patients. Enhanced activation of either allogenic or autologous T cells can drive the death of CLL cells in vitro and in human subjects. Changes in microRNAs expression also characterize clinical progression of CLL with a strong decrease of miR-181b/a and miR-130a associated with the more aggressive phase of the disease. The miR-181b targets anti-apoptotic proteins, such as BCL-2 and MCL1 and its expression correlates with those protein levels in CLL. In this study we demonstrate that the expression of those microRNAs in CLL-B cells, are regulated by T cells. We co-cultured allogenic pure CLL-B cells with either activated (CD2, CD3 and CD28 antibodies, used to mimic antigen-presenting cells) or not activated CD4+ T cells from healthy donors. We observed a significant increase of mir-181b/a and miR-130a expression in CLL B-cells after co-culture with activated CD4+ T cells in 8 out of 11 cases. A significant increase of these miRs was also determined in purified CLL B-cells after 4 days activation of peripheral blood mononuclear cells (PBMCs) from CLL patients, even if in minor rate. By the use of specific antibodies, co-culture with Hela CD40 expressing cells and transwell experiments, we established that this effect is a T/B contact-dependent signaling mediated through CD40L-CD40 interaction. We determine that increased expression of the 3 miRs occurs at the transcriptional level. Since the expression of miR-181b showed the most significant variation in previous experiments it was selected for further analyses. We next investigated the in vivo role of the miR-181b in highly immunodeficient mice. The CLL cell line, MEC-01, infected with either the LV-miR-181b_coGFP or the LV-CTRL_coGFP was intravenously inoculated in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were sacrificed after 4 weeks and assayed for percentage of GFP+ cells in bone marrow and spleen compartments. The miR-181b did not show any specific effect into the leukemic clone. However when the same cells were inoculated in an environment hosting mature T cells, miR-181b consistently influences the death of leukemic cells (Fig 1B), suggesting that T cells are required to potentiate the apoptotic role of this miRNA. To explain what we observed in vivo, we mixed in vitro MEC-01 infected with either the LV-miR-181b or the LV-CTRL and CD8+ T cells from healthy donors. After few hours of contact T cells showed stronger cytotoxic effect on MEC-01 carrying miR-181b as compared to the control. Mixed lymphocyte reaction CD40L-activated CLL and T cells is used to generate effector CTLs. Therefore we grew T cell with CD40L-activated MEC-01 in which the expression of miR-181b was either shut down by lentiviral vector or unchanged as control. After one week, we monitored by cytofluorimetry the CD38 surface marker on T cells since its expression has been associated with more active CTLs and, by ELISA, the release of IL-10, the inhibitor of the potent inducer of CTLs INF-g. We demonstrate that activated MEC-01 with higher expression of miR-181b leads to an increase of the cell number expressing CD38 and this was accompanied by a reduced release of IL-10 from B cells through down-regulation of c-FOS, which we show to be target of the miR-181b and to promote the transcription of the IL-10. In conclusion, our data suggest a role of the miR-181b in the immune response against CLL-B cells. We show that an efficient activation of CD4+ T cells through CD3-complex pathway and a right CD40L-CD40 interaction lead to a significant increase of the some miRNAs deregulated over the progression of chronic lymphocytic leukemia, namely miR-181b. This miRNA potentiates the cytotoxicity of T cells favoring the killing of the leukemic clone. Disclosures No relevant conflicts of interest to declare.

The Aberrantly Expressed Long Noncoding RNA, TRERNA1, Predicts for Aggressive Disease in Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2911-2911
Author(s):  
Cecelia R Miller ◽  
Amy S. Ruppert ◽  
Kevin Coombes ◽  
Amy M. Lehman ◽  
James S. Blachly ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Chronic Lymphocytic Leukemia ◽  
Dna Damage Response ◽  
Lymphocytic Leukemia ◽  
High Expression ◽  
Aggressive Disease ◽  
Damage Response

Abstract Long noncoding RNAs (lncRNAs) have emerged as important regulators of gene expression, and dysregulated expression of lncRNAs has been reported in many cancers. One of the most documented ways by which lncRNAs can contribute to cancer aggressiveness is by altering gene expression through associations with chromatin modifying proteins. However, the vast majority of lncRNAs dysregulated in cancer remain to be functionally characterized. Specifically, there have been few studies investigating the role of lncRNAs in chronic lymphocytic leukemia (CLL). Numerous studies have been published documenting the role of micro RNAs (miRs) in CLL, indicating that non-coding RNAs can play a significant role in this disease. Therefore we hypothesize that dysregulated lncRNA expression in CLL contributes to aggressive disease. We performed microarray analysis using Arraystar Human LncRNA Array v2.0, a platform that analyzes over 30,000 lncRNA transcripts in addition to 30,000 coding transcripts. We found that many lncRNAs are aberrantly expressed in CLL compared to healthy donor B cells. We identified the lncRNA treRNA (TRERNA1) as overexpressed in CLL cells (p = .0014). treRNA has been previously described to have enhancer-like function (Ørom et al., 2010) as well as translational regulatory functions (Gummireddy et al., 2013). It has been reported as overexpressed in breast cancer lymph-node metastases and colon cancer (Gummireddy et al., 2013). In addition to expressing spliced treRNA, CLL cells contain a transcript that retains the intron between the two coding exons due to insufficient splicing. Therefore we investigated the prognostic significance of both spliced and retained intron treRNA (ri-treRNA) in subsequent studies. We obtained 144 well-characterized CLL patient samples from the CLL Research Consortium (CRC) and measured transcript expression by quantitative reverse transcription PCR (qRT-PCR). We separated patients into high or low expressers of treRNA relative to the overall median expression and found that patients with higher expression of treRNA have significantly shorter time to treatment (p = .04). High expression of treRNA also correlates with the poor prognostic indicators unmutated IGHV (p < .0001) and low ZAP70 methylation (p <.001). We validated the correlation with unmutated IGHV in a second cohort of 147 previously untreated CLL samples collected prior to starting therapy in a clinical trial (E2997; Grever et al., 2007) comparing fludarabine to the combination of fludarabine plus cyclophosphamide (FC). We found that high expression (relative to the median) of spliced, but not ri-treRNA, independently predicts for shorter progression free survival in patients receiving FC (HR 3.14, 95% CI 1.61-6.14, p < .001). Since the data from this clinical study suggests a role for treRNA in mediating DNA damage response, we established a stable retroviral system to further study this observation in vitro. We used the CLL cell line established in our lab (OSUCLL; Hertlein et al., 2013) to express empty vector or treRNA. Expression of treRNA does not alter viability, proliferation, or migration. However, OSUCLL expressing treRNA display modest resistance to FC treatment. This correlates with less induction of the DNA damage indicator, γH2AX, as well as the DNA damage response protein, TP53, although these changes were not statistically significant. Consistent with the clinical data, ri-treRNA did not show a differential response to in vitro FC treatment. In summary, we have identified a lncRNA in CLL which may play a role in DNA damage response, and serve as a biomarker predictive of aggressive disease. Disclosures Flinn: Celgene Corporation: Research Funding.

A Genomic Strategy To Refine Prognosis and Predict Response to Therapy in Chronic Lymphocytic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3096-3096
Author(s):  
Daphne R. Friedman ◽  
J. Brice Weinberg ◽  
Anil Potti ◽  
Alicia D. Volkheimer ◽  
Karen M. Bond ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Doubling Time ◽  
Cross Validation ◽  
Response To Therapy ◽  
Lymphocytic Leukemia ◽  
Expression Data ◽  
Mutational Status ◽  
Igvh Mutational Status ◽  
Leave One Out

Abstract Chronic Lymphocytic Leukemia (CLL) is notable for variation in aggressiveness of disease. Many patients with low-risk disease at diagnosis can be followed expectantly, while others rapidly require therapy. While a number of factors aid in determining prognosis, they have no role in predicting response to chemotherapy. We collected clinical data from a cohort of 220 patients with CLL followed at Duke University and the Durham VA Medical Centers. We assessed factors such as stage, leukocyte doubling time, IgVH mutational status, CD38 and ZAP-70 expression, cytogenetics, and time to treatment. We isolated RNA from purified CD5+, CD19+ leukemia cell samples from low and intermediate risk CLL patients, assessed the RNA expression with Affymetrix U133 Plus 2.0 GeneChips, and analyzed differential gene expression using previously developed methods of Bayesian binary regression. In a group of seventy-nine CLL patients who either eventually required (n = 45) or did not (n = 34) require therapy, CD38 status, ZAP70 expression, and cytogenetics were not statistically different (p > 0.5) while IgVH mutational status and leukocyte doubling time were different (p < 0.003). Of the patients treated, 69% were treated with chlorambucil, 51% with fludarabine, 31% with cyclophosphamide, and 49% with rituximab. Using genomic expression data, we developed a 100 gene expression signature that correlated with the need for eventual therapy. The accuracy of this signature was 94% using the leave-one-out cross validation method. Further validation using published data is currently being conducted, and data will be presented. Genes associated with the need for treatment include cell cycle regulators (such as E2F2 and CDK6), inhibitors of apoptosis (such as BAG1), and apolipoprotein B. In addition, using techniques described previously (Potti A et al, Nature Medicine, 2006, 12(11):1294), gene expression data coupled with either in vitro drug sensitivity in the NCI-60 panel of cell lines or in vivo drug response data were used to generate genomic models predictive of sensitivity to drugs commonly used in CLL including fludarabine, cyclophosphamide, and rituximab with accuracies on leave-one-out cross validation of 93%, 96%, and 79% respectively. These predictive models are currently being applied to gene expression data from leukemia samples of CLL patients to predict response to therapy (data to be presented). Thus, a genomic approach can be used to determine which CLL patients will require treatment. Importantly, models of chemosensitivity may predict response to therapy, and thus may ultimately help target the appropriate treatment for each patient.

scholarly journals OP0045 NLRP12 INVOLVES IN THE LUPUS WITH POSITIVE INTERFERON SIGNATURE

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 23.2-24
Author(s):  
Y. P. Tsao ◽  
F. Y. Tseng ◽  
C. W. Chao ◽  
M. H. Chen ◽  
S. T. Chen
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Animal Models ◽  
Nucleic Acids ◽  
Disease Activity ◽  
Interferon Signature ◽  
Healthy Donors ◽  
Healthy Control ◽  
Lupus Disease Activity

Background:Systemic lupus erythematous (SLE) is a systemic autoimmune disease with diverse etiological factors. It was recognized that interferon (IFN) signature involved in the progress of SLE. NLRP12 (NOD-like receptor family (NLR) pyrin domain containing 12) is a pyrin containing NLR protein that we had linked its new biological function to the cross-regulation of Toll like receptor (TLRs) and Rig-I like receptor (RIG-I) pathways. NLPR12 acts as an innate immune check-point in regulating type I IFNs expression during TLRs and RIG-I activation. The importance of NLRP12 in lupus disease activity remained to be elucidated.Objectives:To clarify the role of NLRP12 in regulating the interferon signature.Methods:Peripheral blood mononuclear cells (PBMCs) were collected from SLE patients and healthy donors for analysis of NLRP12 and IFN-α gene expression by RT-QPCR. PBMCs were applied for Chromatin immuneprecipitation (ChIP) assay and electrical mobility shift assay (EMSA) to determine the putative transcription factor that regulates NLRP12 expression. An involvement of epigenetic regulation of NLRP12 expression in SLE patients was also analyzed. Bone marrow derived dendritic cells (BMDCs) were collected from wild type mouse and Nlrp12 knocked-out mice. Another CD14+ monocytes were isolated from 10 cases of lupus patients and 8 cases of healthy control, following by stimulating different type of nucleic acids, and IFN-α and IL-6 were measured with ELISA assay. CD14+ monocytes in lupus patients were also pre-treated with IFNAR2 antibody for further nucleic acid stimulation. Two mice models were applied for evaluation the role of Nlrp12: intraperitoneal injection of TMPD (2,6,10,14-tetramethylpentadecane, or pristane) in C57BL/6 mice and Faslpr mice. Both models were conducted with and without Nlrp12 knockout.Results:NLRP12 expression was significantly lower in PBMC isolated from SLE patients compared to healthy donors. The inverse correlation was observed in NLRP12 and IFNA gene expression as well as NLRP12 expression and amount of double-stranded DNA autoantibody in SLE patients. NLRP12 expression showed negative correlations with IFN-α treatment, as well as herpes simplex virus-1 (HSV-1) infection. Results from ChIP and EMSA analysis indicated a potential transcription factor 1 (TF-1) regulating NLRP12 promoter activity. TF-1 lead to transcriptional suppression of NLRP12 in SLE PBMC, and it was gradually induced after IFN treatment. Recruitment of TF-1 to NLRP12 promoter in SLE PBMC compared to the healthy PBMC was detected, and increased when treating with IFN. Human CD14+ monocytes collected from lupus and healthy control stimulating with different type of nucleic acids revealing significant increasing level of IFN-α and IL-6 in lupus patients. Among animal models, both pristine induced mice and Faslpr mice revealed increasing autoantibodies production and severity of glomerulonephritis in Nlrp12-/- group in comparison with Nlrp12+/+ ones, indicating the role of NLRP12 in maintaining positive interferon signature as well as disease activity.Conclusion:Expression level of NLRP1.2 has been demonstrated to be a biomarker of disease activity in SLE patients. The NLRP12 was involved in the interferon signature, which was also negatively regulated by TF-1. Both clinical samples and animal models revealed NLRP12 in maintaining the positive interferon signature, indicating the possible role of exacerbating factor for lupus disease activity.Disclosure of Interests:None declared

scholarly journals Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia

2001 ◽  
Vol 194 (11) ◽  
pp. 1639-1648 ◽  
Author(s):  
Andreas Rosenwald ◽  
Ash A. Alizadeh ◽  
George Widhopf ◽  
Richard Simon ◽  
R. Eric Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Germ Line ◽  
Gene Expression Signature ◽  
Lymphocytic Leukemia ◽  
Common Mechanism ◽  
Human Leukemia ◽  
Mutational Status ◽  
Cell Chronic Lymphocytic Leukemia

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

Distinct gene expression patterns in chronic lymphocytic leukemia defined by usage of specific VH genes

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 2090-2093 ◽  
Author(s):  
Dirk Kienle ◽  
Axel Benner ◽  
Alexander Kröber ◽  
Dirk Winkler ◽  
Daniel Mertens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Expression Of Genes ◽  
Vh Genes ◽  
Signaling Activation

The mutation status and usage of specific VH genes such as V3-21 and V1-69 are potentially independent pathogenic and prognostic factors in chronic lymphocytic leukemia (CLL). To investigate the role of antigenic stimulation, we analyzed the expression of genes involved in B-cell receptor (BCR) signaling/activation, cell cycle, and apoptosis control in CLL using these specific VH genes compared to VH mutated (VH-MUT) and VH unmutated (VH-UM) CLL not using these VH genes. V3-21 cases showed characteristic expression differences compared to VH-MUT (up: ZAP70 [or ZAP-70]; down: CCND2, P27) and VH-UM (down: PI3K, CCND2, P27, CDK4, BAX) involving several BCR-related genes. Similarly, there was a marked difference between VH unmutated cases using the V1-69 gene and VH-UM (up: FOS; down: BLNK, SYK, CDK4, TP53). Therefore, usage of specific VH genes appears to have a strong influence on the gene expression pattern pointing to antigen recognition and ongoing BCR stimulation as a pathogenic factor in these CLL subgroups.

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