Protein Expression and Methylation Status of the CKI p21, p15 and p16 in Adult Acute Lymphoblastic Leukemia (ALL) Patients: Prognostic Implications.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1843-1843
Author(s):  
Chiara Gregorj ◽  
Fabiana De Cave ◽  
Maria Teresa Petrucci ◽  
Maria R. Ricciardi ◽  
Maria C. Scerpa ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Induction Treatment ◽  
Gene Methylation ◽  
Aberrant Expression ◽  
Normal Peripheral Blood ◽  
P16 Protein

Abstract Cyclin-dependent kinase inhibitors (CKI) regulate cell division resulting aberrantly expressed in many types of cancer. Alterations of CKI have been reported in acute leukemia, as the result of gene promoter methylation. Despite the common frequency of these alterations, little has been reported on the role of CKI aberrant protein expression and results are less clear, especially in acute lymphoblastic leukemia (ALL). The aim of this study was to analyze p21, p15 and p16 protein expression and their gene methylation status in primary cells from adult ALL cases enrolled in the LAL2000 GIMEMA protocol. Normal peripheral blood lymphocytes (PBL) and 91 primary samples from untreated ALL patients were evaluated in this study. The p21, p15 and p16 protein expression was analyzed by Western blot using the specifically MoAbs. The CKI gene methylation status was investigated using a widely accepted method based on bisulfite modification of DNA, followed by the use of the methylation-specific PCR assay (MSP). This assay was further validated in vitro by SSI methylase. Normal PBL from 10 healthy donors, as described, did not expressed all CKIs and resulted unmethylated. The p21 expression was found in 28/91 cases (30.8%); in contrast, samples were found constantly unmethylated. The p15 expression was found in 44/85 cases (51.8%) and its gene methylated in 41.7%; a significant correlation was found between absence of protein expression and gene methylation (P=0.040). The p16 resulted never expressed in adult ALL, while its promoter was found methylated in 8/42 cases (19.1%). A significant association (P=0.037) was observed between p21 expression and immunophenotype; in fact, 3/24 (12.5%) T-ALL and 24/65 (36.9%) B-lineage ALL expressed this protein. The p16 methylation was associated with T-ALL (P=0.082). Achievement of CR was not influenced by single protein expression, nor by gene methylation status. However, the co-expression of p15 and p21 was associated with failure to induction treatment; in fact, only 6/67 (9%) of patients co-expressing p15 and p21 achieved CR (P=0.021). In summary, in adult ALL p21 is not methylated and p16 is never found expressed, and CR achievement is adversely affected by the co-expression of p21 and p15. In conclusion, we report that in addition to CKI methylation, aberrant expression of CKI, namely p21 and p15, is associated with poor outcome in adult ALL, suggesting that chemotherapy resistance may be promoted in these cases by cell cycle arrest and/or abnormal survival.

The Methylation Status of p21 Is Not Relevant in Adult Acute Lymphoblastic Leukemia Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4552-4552
Author(s):  
Chiara Gregorj ◽  
Fabiana De Cave ◽  
Maria R. Ricciardi ◽  
Maria C. Scerpa ◽  
Cristina M. Precupanu ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Induction Therapy ◽  
Lymphoblastic Leukemia ◽  
Raji Cell ◽  
Methylation Status ◽  
Philadelphia Chromosome ◽  
Normal Peripheral Blood

Abstract Methylation of CpG islands in the 5′ gene region is associated with transcriptional silencing of gene expression. The hypermethylation of tumor suppressor genes has been described in various tumor tissues, as in gastric and pancreatic cancer, as well as in acute myeloid leukemia, suggesting its potential role in tumorigenesis. Among the three members of the Kip/Cip family of cyclin dependent kinase inhibitors (CKI) p21, p27 and p57, little is known of their methylation status in hematological malignancies and contrasting studies have been reported on the role of p21 hypermethylation in the pathogenesis of acute lymphoblastic leukemia (ALL). The aim of our study was to analyze in primary blasts from adult ALL enrolled in the GIMEMA protocols 0496 and LAL2000 the methylation status of p21, defining in addition its protein expression by Western blot using the monoclonal antibody p21-WAF1 (Santa Cruz, CA). Primary samples from 81 untreated ALL patients were processed using a widely accepted method based on bisulfite modification of DNA, followed by the use of methylation-specific PCR assay (MSP). The human lymphoblastic cell lines (Jurkat, RPMI8866 and CEM), the myeloid cell line OCI-AML3 and normal peripheral blood lymphocytes (PBL) from 10 healthy donors were characterized by a consistent p21 promoter unmethylation (negative controls). In contrast, it was weakly methylated in the Raji cell line and strongly methylated in the Rael (Burkitt’s lymphoma) cell line (positive controls). This assay was further validate in vitro by SsI methylase. In the present study we analyzed 54 B-lineage ALLs, 25 T-ALLs and 2 biphenothypic leukemias; the mean WBC value at diagnosis was 125.6x109/L and 20 samples were Philadelphia chromosome positive. 71/81 of patients studied for p21 methylation were evaluated for response: 53 (74.6%) achieved complete remission (CR) after induction therapy, 8 (11.3%) patients were resistance and 10 (14.1%) died during induction therapy. DNA methylation was not observed in any of the adult ALL patients. p21 protein expression was found in OCI-AML3, Raji and RPMI8866 cell lines, while resulted negative in the Jurkat cell line and in normal PBL. Preliminary results obtained in the ALL samples showed that this protein was expressed in 8/29 (27.6%) cases. In summary, we demonstrated in a large number of primary ALL cases studied at presentation that the p21 gene is not methylated in this population and therefore that the status of p21 methylation does not play a role in the pathogenesis of adult ALL.

scholarly journals CD20 up-regulation in pediatric B-cell precursor acute lymphoblastic leukemia during induction treatment: setting the stage for anti-CD20 directed immunotherapy

Blood ◽  
2008 ◽  
Vol 112 (10) ◽  
pp. 3982-3988 ◽  
Author(s):  
Michael N. Dworzak ◽  
Angela Schumich ◽  
Dieter Printz ◽  
Ulrike Pötschger ◽  
Zvenyslava Husak ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Induction Treatment ◽  
Cell Precursor ◽  
Anti Cd20 ◽  
The Impact

Abstract CD20 is expressed in approximately one- half of pediatric acute lymphoblastic leukemia (ALL) cases with B-cell precursor (BCP) origin. We observed that it is occasionally up-regulated during treatment. To understand the impact of this on the potential effectiveness of anti-CD20 immunotherapy, we studied 237 CD10+ pediatric BCP-ALL patients with Berlin-Frankfurt-Munster (BFM)–type therapy. We analyzed CD20 expression changes from diagnosis to end-induction, focusing on sample pairs with more than or equal to 0.1% residual leukemic blasts, and assessed complement-induced cytotoxicity by CD20-targeting with rituximab in vitro. CD20-positivity significantly increased from 45% in initial samples to 81% at end-induction (day 15, 71%). The levels of expression also increased; 52% of cases at end-induction had at least 90% CD20pos leukemic cells, as opposed to 5% at diagnosis (day 15, 20%). CD20 up-regulation was frequent in high-risk patients, patients with high minimal residual disease at end-induction, and patients who suffered later from relapse, but not in TEL/AML1 cases. Notably, up-regulation occurred in viable cells sustaining chemotherapy. In vitro, CD20 up-regulation significantly enhanced rituximab cytotoxicity and could be elicited on prednisolone incubation. In conclusion, CD20 up-regulation is frequently induced in BCP-ALL during induction, and this translates into an acquired state of higher sensitivity to rituximab. This study was registered at http://www.clinicaltrials.gov as #NCT00430118.

Protein Expression of p15 and p21 Plays an Unfavorable Prognostic Role in Adult Acute Lymphoblastic Leukemia (ALL) Patients Independently of Their Gene Promoter Methylation Status.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2802-2802
Author(s):  
Fabiana De Cave ◽  
Maria Teresa Petrucci ◽  
Chiara Gregorj ◽  
Maria Rosaria Ricciardi ◽  
Samantha Decandia ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Protein Expression ◽  
Promoter Methylation ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Gene Promoter ◽  
Prognostic Role ◽  
P21 Protein ◽  
Promoter Methylation Status ◽  
P16 Protein

Abstract Epigenetic silencing of tumor suppressor (TS) genes is a hallmark in human leukemias, particularly through DNA methylation. Cyclin-dependent kinase inhibitors (CKI) are, among other genes, frequently found methylated in their promoter region. This epigenetic modification has been described also in acute lymphoblastic leukemia (ALL). However, the relationship between aberrant DNA methylation and protein expression of TS genes has not yet been extensively evaluated in adult ALL series. The aim of this study was to analyze in primary cells from newly diagnosed adult ALL patients, uniformly treated according to the LAL2000 GIMEMA protocol, the promoter methylation status of p73, p21, p15 and p16, evaluating in addition the p21, p15 and p16 protein expression. The DNA methylation status of promoter regions was investigated, according to cell availability, using a widely accepted method based on bisulfite modification of DNA, followed by methylation-specific PCR assay (MSP). Protein expression was evaluated by Western blot. Normal peripheral blood lymphocytes, as already described, resulted unmethylated for p73, p21, p15 and p16, and did not express the p21, p15 and p16 proteins. In ALL patients, in contrast, only the p21 promoter region was found constantly unmethylated. The p15, p16 and p73 promoter genes were found methylated in 15/37 (40.5%), 8/43 (18.6%) and 9/36 (25%) patients, respectively. Only 2/23 cases (8.6%) resulted simultaneously methylated for p15, p16 and p73. The p21 and p15 protein expression was found in 28/85 (32.9%) and 44/85 cases (51.8%), respectively. The p16 protein, in contrast, was never expressed. The p16 methylation was associated with the T-ALL (P=0.005) phenotype and with higher white blood cell (WBC) counts (P=0.027). Resistance to spontaneous induction of apoptosis was significantly associated with p21 protein expression (P=0.019) and its co-expression with p15 (P=0.049). Achievement of CR was not influenced by gene methylation status, nor by single protein expression. Interestingly, the co-expression of p15 and p21 was associated with failure to induction treatment: only 6/63 (9.5%) patients co-expressing p15 and p21 obtained a CR (P=0.027). Multivariate analysis confirmed the unfavorable role of this protein co-expression (P=0.059) on CR achievement. In contrast, once patients achieved remission, p21 protein expression was associated with a prolonged DFS, as confirmed by multivariate analysis for DFS (P=0.039). In conclusion, p15 and p21 protein expression plays an unfavorable prognostic role in adult ALL patients independently of the p73, p21, p15 and p16 gene promoter methylation status.

Hypermethylation Status of Cancer-Associated Genes in Pediatric Acute Lymphoblastic Leukemia (ALL): Incidence and Potential Implications in Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4422-4422
Author(s):  
Omar L Caban ◽  
Aimee Pons ◽  
Nilka J. Barrios ◽  
Adriana Baez
Keyword(s):  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Induction Treatment ◽  
Aberrant Methylation ◽  
Gene Methylation ◽  
Mlh1 Gene ◽  
Methylation Index ◽  
Frequent Event ◽  
Number Of Genes

Abstract Abstract 4422 Introduction Acute Lymphoblastic Leukemia (ALL) is the most common malignancy diagnosed in children, representing nearly one third of all pediatric cancers. Aberrant methylation of CpG island of the promoter region of genes causes gene silencing and could be critical in the initiation and progression ALL. Patients and Methods The study included a group of 10 de novo pediatric cases of ALL and 18 healthy control children. The cases were treated according to the University Children Hospital protocol and followed-up for 28 days. In the present study we assessed the methylation frequency of p14ARF, p15CDKN2B, p16CDKN2A, MLH1, CTNNB1, and APAF1 genes before cases started therapy (Day 0) and after completing the induction phase at Day 28. DNA was extracted from peripheral blood cells from cases and controls. Methylation status was performed using the MethyLight technique. The methylation index (MI; ratio between the number of genes methylated and the number of genes analyzed) was calculated and results were correlated to minimal residual disease (MRD) status of the cases. Results The gene most frequently methylated was p15CDKN2B (90% of cases on Day 0). Frequencies of p16CDKN2A, MLH1, CTNNB1 gene methylation were 80%, 70%, and 10% respectively. A coexistence of p15CDKN2B, p16CDKN2A and MLH1 gene methylation was observed. No patient showed methylation in p14ARF and APAF1 genes. The methylation index ranged from 0 to 0.67 with a median of 0.5. After induction therapy was completed (Day 28) the most frequently methylated gene was p15CDKN2B (80% of cases on Day 28). Frequencies of MLH1, p16CDKN2A, p14ARF and CTNNB1 gene methylation were 40%, 30%, 20% and 10% respectively. No patient showed methylation of the APAF1 genes on Day 28. The methylation index ranged from 0 to 0.67 with a median of 0.17 on Day 28. We found that after the induction treatment methylation of p15CDKN2B, p16CDKN2A, and MLH1 is a frequent event. Interestingly methylation of p14ARF was detected after induction. No significant differences in the frequencies of gene methylation and presence of minimal residual disease between patients with and without aberrant methylation, respectively, were found. We found that the methylation status was not associated with patient age at diagnosis, sex, FAB classification and cytogenetic changes. Conclusions Our findings suggest that aberrant methylation of p15CDKN2B gene is a frequent event in this pathology. The relationship between methylation of p15CDKN2B and leukemia has been reported previously. It appears that methylation of p15CDKN2B is an early event in ALL and continue to be present even after patients completed the induction treatment. Simultaneously, our data would confirm that, in our cohort, the methylation of p16CDKN2A and MLH1 gene promoters is a frequent event in pediatric ALL. However, to assess the impact of promoter methylation of these tumor suppressor genes on disease prognosis longer follow-up and a larger patient population is warranted. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽  
1978 ◽  
Vol 52 (4) ◽  
pp. 712-718 ◽  
Author(s):  
SD Smith ◽  
EM Uyeki ◽  
JT Lowman
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Lymphoid Cells ◽  
Assay System ◽  
Leukemic Cells ◽  
Specific Cell ◽  
Specific Cell Surface

Abstract An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

scholarly journals Low molecular weight B cell growth factor induces proliferation of human B cell precursor acute lymphoblastic leukemias

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 132-138 ◽  
Author(s):  
B Wormann ◽  
SR Mehta ◽  
AL Maizel ◽  
TW LeBien
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Growth Factor ◽  
Cell Growth ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Cell Precursor ◽  
B Cell Growth Factor ◽  
B Cell Precursors

Experiments were conducted to determine the effect of low mol wt B cell growth factor (L-BCGF) on B cell precursor acute lymphoblastic leukemia (ALL). L-BCGF induced a significant increase in 3H-TdR incorporation in 28 of 37 bone marrow aspirates from patients with B cell precursor ALL, with stimulation indices ranging from 2 to 129. Fluorescence-activated cell sorting confirmed that in five of seven patients the common acute lymphoblastic leukemia antigen (CALLA)/CD10 positive leukemic cells were responding directly to L-BCGF. L-BCGF was capable of inducing, in some patients, an increase in absolute viable cells and could also induce colony formation in vitro. The response of B cell precursor ALL was not attributable to beta IL 1, IL 2, or gamma interferon. These results indicate that the majority of B cell precursor ALL undergo a proliferative response to L-BCGF, suggesting a regulatory role for this lymphokine in the growth of B cell precursors.

scholarly journals Pharmacogenomic markers of glucocorticoid response in the initial phase of remission induction therapy in childhood acute lymphoblastic leukemia

2018 ◽  
Vol 52 (3) ◽  
pp. 296-306 ◽  
Author(s):  
Vladimir Gasic ◽  
Branka Zukic ◽  
Biljana Stankovic ◽  
Dragana Janic ◽  
Lidija Dokmanovic ◽  
...  
Keyword(s):  
Retrospective Study ◽  
Acute Lymphoblastic Leukemia ◽  
Peripheral Blood ◽  
Initial Phase ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Remission Induction ◽  
Induction Treatment ◽  
Glucocorticoid Response ◽  
Blast Count

AbstractBackgroundResponse to glucocorticoid (GC) monotherapy in the initial phase of remission induction treatment in childhood acute lymphoblastic leukemia (ALL) represents important biomarker of prognosis and outcome. We aimed to study variants in several pharmacogenes (NR3C1,GSTsandABCB1) that could contribute to improvement of GC response through personalization of GC therapy.MethodsRetrospective study enrolling 122 ALL patients was carried out to analyze variants ofNR3C1(rs33389, rs33388 and rs6198),GSTT1(null genotype),GSTM1(null genotype),GSTP1(rs1695 and rs1138272) andABCB1(rs1128503, rs2032582 and rs1045642) genes using PCR-based methodology. The marker of GC response was blast count per microliter of peripheral blood on treatment day 8. We carried out analysis in which cut-off value for GC response was 1000 (according to Berlin-Frankfurt-Munster [BFM] protocol), as well as 100 or 0 blasts per microliter.ResultsCarriers of rareNR3C1rs6198 GG genotype were more likely to have blast count over 1000, than the non-carriers (p = 0.030).NR3C1CAA (rs33389-rs33388-rs6198) haplotype was associated with blast number below 1000 (p = 0.030).GSTP1GC haplotype carriers were more likely to have blast number below 1000 (p = 0.036), below 100 (p = 0.028) and to be blast negative (p = 0.054), whileGSTP1GT haplotype and rs1138272 T allele carriers were more likely to be blasts positive (p = 0.034 and p = 0.024, respectively).ABCB1CGT (rs1128503-rs2032582-rs1045642) haplotype carriers were more likely to be blast positive (p = 0.018).ConclusionsOur results have shown thatNR3C1rs6198 variant andGSTP1rs1695-rs1138272 haplotype are the most promising pharmacogenomic markers of GC response in ALL patients.

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