Chromosome 6q deletion correlates with poor prognosis and low relative expression of FOXO3 in chronic lymphocytic leukemia patients
2017; Wiley; Volume: 92; Issue: 10 Linguagem: Inglês
10.1002/ajh.24852
ISSN1096-8652
AutoresMarie Jarošová, Martina Hrubá, Alexandra Oltová, Karla Plevová, Lenka Krůzová, Eva Kriegová, Regina Fillerová, Eva Koriťáková, Michael Doubek, Daniel Lysák, Vít Procházka, Marek Mráz, Karel Indrák, Tomáš Papajík,
Tópico(s)Lymphoma Diagnosis and Treatment
ResumoAmerican Journal of HematologyVolume 92, Issue 10 p. E604-E607 E-ONLY ARTICLESFree Access Chromosome 6q deletion correlates with poor prognosis and low relative expression of FOXO3 in chronic lymphocytic leukemia patients Marie Jarosova, Corresponding Author Marie Jarosova jarosova.marie@fnbrno.cz orcid.org/0000-0002-2194-3433 Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech RepublicCorrespondence Marie Jarosova, Center of Molecular Biology and Gene Therapy, Department of Internal Medicine, Hematology-Oncology,University Hospital and Faculty of Medicine, Masaryk University Brno, Cernopolni 202/9, 61300 Brno, CZ. Email: jarosova.marie@fnbrno.czSearch for more papers by this authorMartina Hruba, Martina Hruba Department of Medical Genetics, University Hospital Pilsen, Czech RepublicSearch for more papers by this authorAlexandra Oltova, Alexandra Oltova Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech RepublicSearch for more papers by this authorKarla Plevova, Karla Plevova Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech Republic Central European Institute of Technology, Masaryk University Brno, Czech RepublicSearch for more papers by this authorLenka Kruzova, Lenka Kruzova Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorEva Kriegova, Eva Kriegova Department of Immunology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorRegina Fillerova, Regina Fillerova Department of Immunology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorEva Koritakova, Eva Koritakova Department of Biostatistics and Analysis, Faculty of Medicine, Masaryk University Brno, Czech RepublicSearch for more papers by this authorMichael Doubek, Michael Doubek Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech RepublicSearch for more papers by this authorDaniel Lysak, Daniel Lysak Department of Hemato-Oncology, University Hospital Pilsen, Czech RepublicSearch for more papers by this authorVit Prochazka, Vit Prochazka Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorMarek Mraz, Marek Mraz Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech Republic Central European Institute of Technology, Masaryk University Brno, Czech RepublicSearch for more papers by this authorKarel Indrak, Karel Indrak Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorTomas Papajik, Tomas Papajik Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this author Marie Jarosova, Corresponding Author Marie Jarosova jarosova.marie@fnbrno.cz orcid.org/0000-0002-2194-3433 Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech RepublicCorrespondence Marie Jarosova, Center of Molecular Biology and Gene Therapy, Department of Internal Medicine, Hematology-Oncology,University Hospital and Faculty of Medicine, Masaryk University Brno, Cernopolni 202/9, 61300 Brno, CZ. Email: jarosova.marie@fnbrno.czSearch for more papers by this authorMartina Hruba, Martina Hruba Department of Medical Genetics, University Hospital Pilsen, Czech RepublicSearch for more papers by this authorAlexandra Oltova, Alexandra Oltova Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech RepublicSearch for more papers by this authorKarla Plevova, Karla Plevova Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech Republic Central European Institute of Technology, Masaryk University Brno, Czech RepublicSearch for more papers by this authorLenka Kruzova, Lenka Kruzova Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorEva Kriegova, Eva Kriegova Department of Immunology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorRegina Fillerova, Regina Fillerova Department of Immunology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorEva Koritakova, Eva Koritakova Department of Biostatistics and Analysis, Faculty of Medicine, Masaryk University Brno, Czech RepublicSearch for more papers by this authorMichael Doubek, Michael Doubek Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech RepublicSearch for more papers by this authorDaniel Lysak, Daniel Lysak Department of Hemato-Oncology, University Hospital Pilsen, Czech RepublicSearch for more papers by this authorVit Prochazka, Vit Prochazka Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorMarek Mraz, Marek Mraz Department of Internal Medicine, Hematology-Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Czech Republic Central European Institute of Technology, Masaryk University Brno, Czech RepublicSearch for more papers by this authorKarel Indrak, Karel Indrak Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this authorTomas Papajik, Tomas Papajik Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Czech RepublicSearch for more papers by this author First published: 11 July 2017 https://doi.org/10.1002/ajh.24852Citations: 8 Funding information: AZV ČR VES, Grant/Award Number: 15-31834A (KP) and 16-29622A (MM) of the Ministry of Health, Czech Republic AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Detection of genetic changes has improved the current risk stratification in chronic lymphocytic leukemia (CLL).1, 2 Among the known recurrent chromosomal abnormalities,1 6q deletion is less frequent and controversy surrounding its prognostic significance still remains.3, 4 The study aimed at a genetic analysis of a group of CLL patients with chromosome 6q deletion for determination of its prognostic significance, extent, the minimal deleted region (MDR) using array comparative genomic hybridization (arrayCGH), and finally to dermine the relative expression of candidate genes located therein. Peripheral blood and/or bone marrow samples from 1158 CLL patients diagnosed and treated in three Czech hematology centers (Brno, Olomouc and Pilsen) were examined in 2001–2016 using conventional cytogenetics (CG) and FISH. The patient characteristics are listed in Supporting Information Table S1. Chromosome 6q deletion was found by CG (53) and FISH (38) in a total of 91 (7.9%) patients. Complete data and material for further analysis were available for only 70 patients, including 42 patients examined at CLL diagnosis and 28 treated patients examined in the course of the disease (Table 1). Table 1. Characteristics of patients with 6q deletion (N = 70) Characteristics Patients with 6q deletion Sex - males, N (%) 53 (75.7%) Age, med (min - max) 63 (34–87) Binet staging (A/B/C/UNK), N (%) 24 (34.3%)/23 (32.9%)/19 (27.1%)/4 (5.7%) Mutation status IVGH (mutated/unmutated/both), N (%) 4 (5.7%)/65 (92.9%)/1 (1.4%) Treated in time of examination, N (%) 28 (40.0%) Deceased, N (%) 28 (40.0%) Follow up of living patients, med (min - max) 4.7 (0.4–15.7) Follow up of all patients, med (min - max) 4.8 (0.1–15.7) FISH (changes/complex changes/solo del 6q), N (%) 29 (41.4%)/17 (24.3%)/24 (34.3%) CG + FISH (changes/complex changes/solo del 6q), N (%) 21 (30.0%)/39 (55.7%)/10 (14.3%) ATM deletion, N (%) 14 (20.0%) p53 deletion, N (%) 17 (24.3%) 13q deletion, N (%) 30 (42.9%) Trisomy 12, N (%) 2 (2.9%) N-number, CG- conventional cytogenetics, UNK- unclassified, med-median,complex changes-finding of three or more changes. Deletion 6q was confirmed as a single aberration in 10 patients, together with one additional aberration in 21 and as part of a complex karyotype in 39 patients. Evaluation of the frequency of other changes using a FISH CLL panel1 showed that 13q deletion was the most frequent change regardless of the number of changes, occurring in 30/70 (42.8%) patients. TP53 deletion was present in 17 (24.3%) patients, with 13 of them having a complex karyotype. The results concerning the presence of additional changes identified by CG and FISH are shown in Supporting Information Figure S1. The extent of 6q deletion was mapped using arrayCGH. As shown in Supporting Information Figure S2, the range of 6q deletion was very heterogeneous. Analysis showed a “large deletion” covering the entire arm of chromosome 6 (7 patients) and an “intermediate” deletion, a deletion always affecting the 6q telomeric region but not the 6q12 centromeric region (12 patients); deletions other than the two were classified as “interstitial” (51 patients). Given the group size, statistical analysis of the clinical significance of the extent of various deletion types was not conducted. ArrayCGH determined the MDR as a deletion in the region 107.7–108.7 Mb in the band 6q21 containing the genes SCML4, SEC63, OSTM1, NR2E1, SNX3, LACE1, and FOXO3 (Supporting Information Figure S2). To determine the impact of the deletion on the expression of genes located in the MDR, their relative expression was analyzed using quantitative RT-PCR with the TaqMan assays (Supporting Information Methods). We analyzed 17 patients with 6q deletion, 30 CLL patients without 6q deletion and 19 healthy controls. Low relative mRNA expression of FOXO3, LACE1, SNX3, and SCML4 was observed in CLL patients with chromosome 6q deletion as compared to those without the deletion and healthy controls (Figure 1). Expression profiling of FOXO3, a negative regulator of cell cycle and/or apoptosis, revealed lower levels of FOXO3 transcripts in CLL patients with 6q deletion than in those without 6q deletion (P = .03) and healthy subjects (P < .0001).This was completed by examination of NF-kB relative expression, a gene regulated by FOXO3, confirming its high expression (Figure 1). We hypothesized that FOXO3 is the candidate tumor suppressor gene located at the 6q21 region since survival of normal and malignant B cells is largely dependent on signals from the PI3K-Akt-FOXO pathway. The PI3 kinase pathway contributes to cell survival in part through the activation of Akt and subsequent phosphorylation of the FOXO factors, which inhibits transcriptional functions of these pro-apoptotic genes.5 Additionally, pro-apoptotic FOXO3 competes for binding sites in DNA with the anti-apoptotic transcription factor FOXP1, associated with aggressiveness of CLL and B cell lymphoma.6, 7 Therefore, we analyzed the prognostic role of FOXO3 mRNA levels in an independent cohort of 94 CLL patients (the 6q region status was not examined in them). We divided the cohort by median FOXO3 expression and performed a Kaplan-Meier survival analysis. Compared with cases having low-level expression of FOXO3, those with high-level expression had a significantly shorter median OS (13.1 vs 19.8 years, HR: 2.1 [CI 1.01–4.4]) (Supporting Information Graph S1). Furthermore, we evaluated some of the available clinical and laboratory parameters such as Binet stage, IGHV mutation status and treatment. As many as 92.9% patients with 6q deletion had unmutated IGHV status. We did not show any relationship with Binet stage or treatment at the time of examination (Supporting Information Table S2) or a statistically significant difference in the OS between patients with single 6q aberrations and those with 6q deletion and additional changes (Supporting Information Graph S2). Statistical analysis of the OS of patients with 6q deletions and all examined patients without 6q deletion showed a statistically significant difference, with the OS being shorter in patients with 6q deletions (Supporting Information Graph S3). The graph of the OS of 791 patients (with available FISH data) stratified according to the most common chromosomal changes such as deletions of 13q, 11q, 17p, and +12 shows poor prognosis of patients with 6q deletion (Figure 1B). Figure 1Open in figure viewerPowerPoint Distribution of relative mRNA expression (ratio target gene/reference PGK1 gene) of LACE1, SEC63, NR2E1, SCML4, OSTM1, SNX3, FOXO3 and NFkB1 in peripheral blood of patients with CLL without 6q deletion (CLL), CLL with 6q deletion (CLL-DEL), and healthy controls (Controls). Group means are indicated by horizontal bars, error bars indicate 95CI. B. Overall survical according to chromosomal aberrations (N=791) We confirmed that 6q deletion in CLL patients indicates poor prognosis and is related to lower expression of the FOXO3 gene which can become a candidate gene for targeted treatment. The FOXO3 gene belongs to a family of transcription factors characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through the expression of genes necessary for cell death. Restoring the activity of FOXO3 promotes tumor cell death.8, 9 Overall, our data support the notion that homeostatic chemokines contribute to CLL resistance to cell death through inactivation of the transcription factor FOXO3, which may represent a novel therapeutic target in this hematopoietic malignancy.10 In conclusion, presented here are results of the so far largest group of CLL patients with chromosome 6q deletion, confirming that most frequently, the deletion is part of complex karyotypes and is associated with unfavorable prognosis. Low expression of the FOXO3 gene in the MDR is additional evidence of the tumor suppressive role of the gene in the 6q21 region and its role in CLL pathogenesis. ACKNOWLEDGMENTS The authors would like to thank M. Holzerova, PhD and H. Urbankova, PhD for assistance with routine FISH and BAC arrayCGH analysis, Dr. R. Urbanova and Dr. P. Turcsanyi for outpatient care, Dr. M. Divoka for isolating DNA and RNA and Mgr. T. Necasova for figure preparation. CONFLICT OF INTERESTS The authors declare no potential conflict of interest. AUTHOR CONTRIBUTIONS MJ designed the study, collected and analyzed the data, interpreted the results and wrote the paper. LK, AO, MH and KP performed FISH and arrayCGH analyses and contributed to the interpretation of the results. EK4 and RF performed expression analysis. VP, TP, MD, DL and KI treated the patients, collected their material and provided clinical information. EK5 performed statistical analysis. MM performed molecular analysis of FOXO3. All authors contributed to editing the manuscript. Supporting Information Additional Supporting Information may be found online in the supporting information tab for this article. Filename Description ajh24852-sup-0001-suppfig1.docx25.8 KB Supporting Information Figure 1. ajh24852-sup-0002-suppfig2.tif197.6 KB Supporting Information Figure 2. ajh24852-sup-0003-suppgraph1.doc71 KB Supporting Information Graph 1. ajh24852-sup-0004-suppgraph2.doc354 KB Supporting Information Graph 2. ajh24852-sup-0005-suppgraph3.tif47.2 KB Supporting Information Graph 3. ajh24852-sup-0006-supptable1.doc38.5 KB Supporting Information Table 1. ajh24852-sup-0007-supptable2.doc31.5 KB Supporting Information Table 2. ajh24852-sup-0008-suppinfo.docx14.6 KB Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Döhner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000; 343(26): 1910– 1916. 2Landau DA, Tausch E, Taylor-Weiner AN, et al. Mutations driving CLL and their evolution in progression and relapse. Nature. 2015; 526(7574): 525– 530. 3Rodríguez-Vicente AE, Díaz MG, Hernández-Rivas JM. Chronic lymphocytic leukemia: a clinical and molecular heterogeneous disease. Cancer Genet. 2013; 206(3): 49– 62. 4Wang DM, Miao KR, Fan L, et al. Intermediate prognosis of 6q deletion in chronic lymphocytic leukemia. Leuk Lymphoma. 2011; 52(2): 230– 237. 5Zhang X, Tang N, Hadden TJ, Rishi AK. Akt, FoxO and regulation of apoptosis. Biochim Biophys Acta. 2011; 1813(11): 1978– 1986. 6van Boxtel R, Gomez-Puerto C, Mokry M, et al. FOXP1 acts through a negative feedback loop to suppress FOXO-induced apoptosis. Cell Death Differ. 2013; 20(9): 1219– 1229. 7Mraz M, Chen L, Rassenti LZ, et al. miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1. Blood. 2014; 124(1): 84– 95. 8Essafi A, Fernández de Mattos S, Hassen YA, et al. Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells. Oncogene. 2005; 24(14): 2317– 2329. 9Sunters A, Fernández de Mattos S, Stahl M, et al. FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem. 2003; 278(50): 49795– 49805. 10Tichini M, Essafi M, Jeandel PY, Cassuto JP, Deckert M, Bernard A. Homeostatic chemokines increase survival of B-chronic lymphocytic leukemia cells through inactivation of transcription factor FOXO3a. Oncogene. 2007; 26(50): 7081– 7091. Citing Literature Volume92, Issue10October 2017Pages E604-E607 FiguresReferencesRelatedInformation
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