|Year : 2012 | Volume
| Issue : 2 | Page : 129-134
Molecular detection of 6q deletion in Egyptian patients with B-cell chronic lymphoproliferative disorders
Manal M. Welson1, Dalia G. Amin1, Esam H. Elnoshokaty2
1 Department of Clinical and Chemical Pathology, Faculty of Medicine, National Cancer Institute, Cairo University, Cairo, Egypt
2 Department of Clinical and Chemical Pathology, National Cancer Institute, Cairo University, Cairo, Egypt
|Date of Submission||20-Feb-2012|
|Date of Acceptance||12-Mar-2012|
|Date of Web Publication||23-Jun-2014|
Dalia G. Amin
Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Cairo
Source of Support: None, Conflict of Interest: None
Deletions of the long arm of chromosome 6 (6q) are among the most frequent chromosome aberrations in multiple human tumors, including acute lymphocytic leukemia and non-Hodgkin’s lymphoma (NHL). The accurate characterization of the 6q deletions is of clinical significance in lymphoid malignancies because they are related to the prognosis. The aim of this study was to evaluate the frequency of 6q21 deletion in B-cell chronic lymphoproliferative disorders (BCLPDs) and to evaluate the association of this deletion with other prognostic criteria.
Patients and methods
A fluorescence in-situ hybridization probe set on chromosome 6q21 was used to evaluate 6q21 deletion in 63 patients with different BCLPDs, they were 30 patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), 21 patients with NHL, four patients with Waldenström macroglobulinemia, and eight patients with multiple myeloma.
6q21 deletion was detected in 20% of CLL/SLL (6/30), (40%) of diffuse large B-cell lymphoma (2/6), 33.3% of follicular lymphoma (2/6), and 20% of mantle cell lymphoma (1/5), 25% of multiple myeloma (2/8), and in 50% of Waldenström macroglobulinemia (2/4) patients. None of the two patients with splenic lymphoma or the three patients with unclassifiable B-NHL showed 6q21 deletion. The median percentage of clonal lymphoid cells with deletion (6q21) was 45% (33–88%). CLL/SLL patients with 6q21 deletion had a significantly lower hemoglobin concentration (P=0.006); a significantly higher mean age (P=0.009); percent of peripheral blood prolymphocytes (P=0.001); and level of serum lactate dehydrogenase (P=0.009) compared with those without the deletion. All CLL/SLL patients (100%) with 6q deletion were allocated to stage C and stages III and IV of Binet and Rai staging systems, respectively, whereas only (58.3%) of those without 6q deletion were allocated to the same advanced stages; however, this was not statistically significant. Among NHL patients, 6q deletion was associated with older age (>60 years, P=0.01), lower hemoglobin concentration (P=0.003), higher level of β2-microglobulin (β2M) and lactate dehydrogenase (P=0.01 and 0.00, respectively), and higher incidence of splenomegaly (P=0.003). All NHL patients with 6q deletion were stratified into a high-risk group according to the International Prognostic Index, whereas those without 6q deletion, approximately 44%, were classified as a high-risk group (P=0.02). Staging of NHL patients according to the Ann Arbor system showed no significant difference between patients with 6q deletion and those without.
On the basis of the high frequency of deletion (6q) in BCLPDs, we suggest a pathogenetic role of this deletion in BCLPDs. Our results support previous studies that have shown an impact of 6q deletion on prognosis in subtypes of BCLPDs.
Keywords: B-cell chronic lymphoproliferative disorders, fluorescence in-situ hybridization, 6q
|How to cite this article:|
Welson MM, Amin DG, Elnoshokaty EH. Molecular detection of 6q deletion in Egyptian patients with B-cell chronic lymphoproliferative disorders. Egypt J Haematol 2012;37:129-34
|How to cite this URL:|
Welson MM, Amin DG, Elnoshokaty EH. Molecular detection of 6q deletion in Egyptian patients with B-cell chronic lymphoproliferative disorders. Egypt J Haematol [serial online] 2012 [cited 2021 Sep 18];37:129-34. Available from: http://www.ehj.eg.net/text.asp?2012/37/2/129/135067
| Introduction|| |
B-cell chronic lymphoproliferative disorders (BCLPDs) include neoplastic proliferations of peripheral B-cells; they represent a spectrum of diseases with a broad morphological, clinical, and biological diversity 1.
BCLPDs vary widely in their clinical course and presentation. These diseases range from more common, clinically indolent processes to the more aggressive disorders presenting with significant clinical problems. Survival times also vary widely, with the more aggressive disorders typically associated with shorter (1–3 years) survival as opposed to longer (5–10 years or more) survival in the clinically indolent processes 1. Accurate classification and evaluation of prognosis are essential in the clinical management of patients with BCLPDs 2.
Nonrandom chromosomal deletions reflect loss of genetic material and suggest the presence of tumor suppressor genes that are inactivated during the process of malignant transformation. Deletions of chromosome 6q are found in many types of cancer, including melanoma, prostate cancer, fibroadenomas, and carcinoma of the breast and other sites. Chromosome 6q deletions are also commonly found in lymphoid malignancies such as acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma (NHL), multiple myeloma (MM), mantle zone lymphoma, and Waldenström’s macroglobulinemia (WM). 6q deletions may appear to be terminal or interstitial 2,3.
Karyotype analysis is the standard method for the diagnosis of chromosome aberrations in hematological malignancies; however, small deletions and subtle translocations (as the case with 6q deletion) are difficult or impossible to detect using conventional banding methods. The development of fluorescence in-situ hybridization (FISH) technology has improved the ability to detect 6q deletion accurately. Moreover, FISH allows analysis of both metaphase (proliferating) and interphase (nonproliferating) cells and can therefore be used to monitor minimal residual disease and early relapses 4.
Two distinct regions of minimal cytogenetic deletion have been identified at 6q16–q23 and 6q25–q27 in ALL and NHL. Recent studies at the molecular level have refined the minimal common deletions in 6q21 and in 6q27 5,6.
The pattern of 6q deletions in hematological malignancies might partly depend on the subtype and the immunophenotype of the malignant cells. Knowledge of 6q deletion patterns might be helpful in the differentiation of subtypes of these diseases 7.
Lawce and Olson 3 have reported that, in childhood B-cell and T-cell ALL, a deletion of 6q is the hallmark of a neutral prognosis; in adult ALL, it indicates a favorable prognosis, but in CLL, B-cell small lymphocytic lymphoma (SLL), WM, and MM, it has a poor prognosis.
As the deletion of 6q has prognostic implications and may be used to monitor residual disease, we considered it important to study this abnormality in BCLPDs for a better understanding of the mechanism of tumorigenesis and the molecular categorization of these diseases in order to refine treatment modalities.
Aim of the work
The study aimed to determine the frequency of 6q21 deletion in BCLPDs and to evaluate the association of this deletion with other prognostic criteria.
| Patients and methods|| |
The present study was carried out on 63 patients with different BCLPDs; they were subclassified as follows: 30 patients (47.6%) had CLL/SLL, 21 patients (33.3%) had NHL, four patients (6.3%) had WM, and eight patients (12.7%) had MM. These patients were referred from Kasr El-Aini Hospital, Cairo University, and the National Cancer Institute for diagnosis in the Clinical Pathology Department, Cairo University, during the period from June 2010 to November 2011. Ten age-matched and sex-matched healthy volunteers were included as a control group. Informed consent was obtained from each participant. The study was approved by the ethics committee of Cairo University.
Diagnosis was established according to standard morphologic, immunophenotypic [on peripheral blood (PB) or bone marrow (BM) sample], cytogenetic, and histopathologic (BM trephine biopsy and/or lymph nodes) analyses. The clinical and laboratory data of the patient group are summarized in [Table 1].
6q deletion was evaluated using FISH on interphase cells derived from 24-h harvests of unstimulated PB or BM cultures.
The mononuclear cell layer was separated from fresh PB or BM samples using Ficoll hypaque density gradient centrifugation. Mononuclear cells were cultured for 24 h (short-term cell culture) without phytohemagglutinin. Five milliliters of RPMI 1640 medium supplemented with 15% of fetal calf serum and 1% L-glutamine was added to each culture tube. Colcemid was added at a final concentration of 0.05 μg/ml 60 min before harvesting. Cells were treated with 0.075 mol/l KCl hypotonic solution at 37°C for 15 min, followed by three changes of 3 : 1 methanol : glacial acetic acid fixative solution. The cells were finally resuspended in this fixative solution. The cells suspended in fixative solution were spread on slides. The slides were placed in a 2× SSC (1×SSC=150 mmol/l sodium chloride and 15 mmol/l sodium citrate solution) bath at 37°C for 30 min and then dehydrated in 70, 80, and 100% ethanol. After air-drying the slides, FISH procedures were performed as described before 8, using a locus-specific identifier, dual color probe ON 6q21/E6 (Ref: KBI-10105, Lot: 00030638; Kereatech, Amsterdam, Netherlands), which hybridizes to band 6q21 (red) and to the centromere locus (spectrum green) of human chromosome 6, according to the manufacturer’s instructions. In brief, 10 µm of the probe was applied, the slide was covered with a glass cover temporarily fixed with fixogum, codenaturation was carried out at 80°C for 2.5 min on a hot plate, then probe hybridization for 12–20 h at 37°C in a humidified chamber, the fixogum was removed, the slide was washed for 2 min at room temperature (RT) in wash buffer II, 2 min in 72°C in wash buffer I, 1 min at RT in wash buffer II, and then 1 min each in 70, 85, and 100% ethanol, and air dried at RT. Slides were counterstained and mounted with DAPI.
Images were viewed under a fluorescent microscope with the proper filter set: DAPI/red/green were used to visualize the fluorescent signals. At least 200 nonoverlapping nuclei were analyzed blindly by at least two observers [Figure 1]. The pictures were captured using an Olympus BX60 microscope (Olympus America Inc., Center Valley, Pannsylvania, USA) equipped with a compulog IMAC-CCD S30 (MetaSystem, Belmont, Massachusetts, USA) camera module and the in-situ imaging system (ISIS 2) software version 2.5 (MetaSystem, Belmont, Massachusetts, USA).
|Figure 1: Fluorescence in-situ hybridization for chromosome 6: bottom arrow, four signals of an interphase nucleus (normal): two green signals (internal control) represent the centromere of chromosome 6 and two red signals represent the 6q21region; top arrows, two interphase cells with three signals: two green (internal control) and one red signal, representing the deletion of 6q21 (bottom arrow).|
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The cutoff of positivity at 5% was determined using published studies on the basis of an extensive control analysis 9, and this was validated using normal PB specimens from control participants.
Data were statistically described in terms of mean±SD, median and range, or frequencies (number of cases) and percentages when appropriate. Comparison of numerical variables between the study groups was carried out using the Student t-test for independent samples when normally distributed and the Mann–Whitney U-test for independent samples when not normally distributed. For comparison of categorical data, the χ2-test was performed. The exact test was used instead when the expected frequency was less than 5. P values less than 0.05 were considered statistically significant. All statistical calculations were carried out using computer programs SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, Illinois, USA) version 15 for Microsoft Windows.
| Results|| |
Interphase-FISH with the 6q21 probe detected hemizygous 6q deletions in 20% of CLL/SLL (6/30), 23.8% of NHL (5/21), 25% of MM (2/8), and in 50% of WM (2/4) patients. The median percentage of clonal lymphoid cells with deletion (6q21) was 45 % (33–88%). The total frequency of 6q deletion among the 63 patients with BCLPDs was 23.8% (15/63). Neither homozygous deletions nor monosomy 6 were detected in this cohort [Table 2].
|Table 2: Comparison of chronic lymphocytic leukemia/small lymphocytic lymphoma patients with 6q deletion and those without the deletion in terms of different clinical and laboratory data|
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Among NHL patients, the highest percentage of 6q deletion was reported in the diffuse large B-cell lymphoma (DLBCL) category (40%) (2/6); the frequency of 6q deletion was 33.3% (2/6) in follicular lymphoma (FL) and 20% (1/5) in mantle cell lymphoma (MCL). None of the two patients with splenic lymphoma (SL) or the three patients with unclassifiable B-NHL showed 6q deletion.
In the CLL/SLL group, patients with 6q deletion had a significantly lower hemoglobin (Hb) concentration (P=0.006), and a significantly higher mean age (P=0.009); percent of PB prolymphocytes (P=0.001); and level of serum lactate dehydrogenase (LDH) (P=0.009) compared with patients without the deletion, whereas no significant difference was found between patients with 6q deletion and those without when they were compared in terms of other clinical and hematological data such as incidence of splenomegaly, hepatomegaly, and lymphadenopathy, male/female ratio, the total leukocyte count (TLC), platelet count, percent of PB lymphocytes, percent of BM lymphocytes, and the absolute lymphocyte count.
All CLL/SLL patients (100%) with 6q deletion were allocated to stage C and stages III and IV of the Binet and Rai staging systems, respectively, whereas only 58% (14/24) of those without 6q deletion were allocated to the same advanced stages; however, this comparison did not show a statistical significance.
A comparative study, of NHL patients with 6q deletion and NHL patients with no 6q deletion, showed that 6q deletion was associated with older age (>60 years, P=0.01), lower Hb concentration (P=0.003), higher level of β2-microglobulin (β2M) and LDH (P=0.01 and 0.00 respectively), and higher incidence of splenomegaly (P=0.003), and these associations were statistically significant. However, no significant difference was found between the same two groups when they were compared in terms of the incidence of hepatomegaly or lymphadenopathy, male/female ratio, TLC, platelet count, and percent of PB or BM lymphoma cells.
All NHL patients with 6q deletion were stratified into the high-risk group according to the International Prognostic Index 1, whereas of those without 6q deletion, 44% (7/16) were classified into the high-risk group and this difference was statistically significant (P=0.02). Staging of NHL patients according to the Ann Arbor system showed no significant difference between patients with 6q deletion and those without as the entire former group was allocated to stages II and III whereas around 81% of the latter group was allocated to the same two stages.
In the MM group, the two patients with 6q deletion had a β2M level of 3.9 and 4.3 mg/l and a monoclonal immunoglobulin level of 9 and 10 g/l; as for MM patients with no deletion, they had a median β2M level of 2.8 and a range of 2.6–3.1 mg/l and a median monoclonal immunoglobulin level of 3.95 and a range of 3–8 g/l. Because of the small number of patients in this group, statistical analysis could not be performed.
| Discussion|| |
The association between losses in the 6q region and the appearance of different lymphoid malignancies suggests that this region contains tumor suppressor gene(s) whose loss of function contributes to lymphoma transformation or progression but definitive evidence for their role has not yet been established.
On the basis of molecular genetic analysis of several subtypes of malignant lymphomas, at least two independent critical regions of commonly deleted segments, one at 6q21–q23 and one at 6q25–q27, have been identified 10. In CLL/SLL, deletions of 6q21–q23 were identified 11, and the proximal location of the minimally deleted region was identified as a critical deletion region spanning markers D6S283 to D6S270 on chromosome band 6q21 in 6% of CLL cases 12.
FISH probe mapping on 6q21 and 6q27 confirmed that all deletions could be detected with the 6q21 probe, whereas only one-third of the patients also showed 6q27 deletions 12. Zhang et al. 13 identified a 4–5 Mb minimal deletion region in band 6q21 in a variety of lymphomas and lymphoid malignancies.
Previous studies on NHL have shown that 6q21 deletions are associated with a poor prognosis 10, 14, 15. Recently, Lawce and Olson 3 reported that 6q deletion in CLL/SLL, WM, and MM has a poor prognosis.
As the deletion of 6q has prognostic implications in NHL and other BCLPDs, establishing their presence is very important from a clinical point of view.
Most of our cells failed to enter into the metaphase and the count was carried on interphase cells. Interphase-FISH with the 6q21 probe detected hemizygous 6q deletions in 20% of CLL/SLL (6/30).
In agreement, a similar frequency (26%) of 6q deletion was reported among SLL/CLL patients 11. Cuneo and Castoldi 16 reported that deletions/translocations involving 6q21–23 were detected in 15–25% of SLL/CLL patients.
A much lower frequency (7%) was reported by Stilgenbauer et al. 12 for 6q deletions in SLL/CLL patients 17. On reviewing their study, we found that the majority (63%, 189/300) of CLL patients who were included in that study had early-stage disease (Binet A). In contrast, only 6.7% (2/33) of the patients included in our study were staged as Binet A, with the majority (66.7%, 14/33) staged as Binet C, and as 6q deletions have been reported to be among the most commonly acquired secondary chromosome aberrations in CLL patients 17, 18, this may partially explain the higher frequency that was found in the current study.
In rank order of incidence, we found 6q deletion in 50% of WM, 40% of DLBCL, 33.3% of FL, 25% of MM, and 20% of MCL. None of the two patients with SL or the three patients with unclassifiable B-NHL showed 6q deletion.
In agreement with our results for WM, Schop et al. 19 reported a frequency between 42 and 63% for 6q deletion (as the former frequency considered patients with deletion in greater than 25% of cells whereas the latter considered the deletion in >10% of clonal cells), in a more recent study by the same author, a frequency of 55% was reported for 6q deletion in another cohort of WM 20. In contrast, the frequency of 6q deletion in WM was reported to be 38% 21, consistent with the frequency (34%) reported by Ocio et al. 22 reviewing their study, they reported a jump of this frequency (34%) to be 54% when cytoplasmic immunoglobulin M-FISH was used.
Our study was in agreement with that of Honma et al. 23 and Beà and Campo 24, who found 6q deletion in 38 and 40% of DLBCL patients, respectively. In contrast to our findings, Pasqualucci et al. 25 reported that the BLIMP1 gene (the locus that lies on chromosome 6q21–q22.1) is inactivated by structural alterations in 24% of DLBCL. They applied the FISH technique on formalin-fixed paraffin or frozen archival tissues using a very high cutoff of greater than 50% for the diagnosis of cases as deleted, whereas our reported frequency was based on the application of the FISH technique on interphase cells using a cutoff of 5% for positivity, which may explain the discrepancy in these results.
We reported a frequency of 6q deletion of 33.3% in FL, 25% in MM, and 20% in MCL. Similar frequencies (30, 29%) were reported in FL by Cuneo et al. 26 and Chen et al. 27, respectively. Higher frequencies for 6q deletion (31 and 37%) in MCL were reported by Honma et al. 23 and Beà and Campo 24, respectively. Our frequency was estimated in five MCL patients; the small number of patients could explain this variability. In agreement with our results, Avet-Loiseau et al. 28 found 6q deletion in 20 and 21% of MM patients, respectively. The 2 patients with SL in our patient group did not show 6q deletion. Watkins et al. 29 found 6q deletion in 16% of SL patients.
We assessed status of the 6q deletion in the context of clinical information in our study, which showed that CLL/SLL patients with 6q deletion had a significantly lower Hb concentration, and a significantly higher mean age; percent of PB prolymphocytes; and level of LDH compared with patients without the deletion. No significant difference was found between the same two groups when they were compared in terms of the incidence of hepatomegaly or lymphadenopathy, male/female ratio, TLC, platelet count, and percent of PB or BM clonal cells.
In agreement, Offit et al. 11 and Cuneo and Castoldi, 16 showed a correlation between 6q deletion in CLL/SLL and leukemic involvement by atypical larger forms with the morphologic appearance of prolymphocytes. Offit et al. 11 also reported no difference in the leukocyte count or proportion with extranodal disease between the two groups.
In contrast, Cuneo et al. 30 observed that patients with 6q deletion showed a higher TLC count and frequent splenomegaly; they also reported an association with a shorter period of treatment free and overall survival. Reviewing their study, Cuneo and colleagues included patients with CLL/prolymphocytic leukemia in their analysis; these patients usually have a markedly elevated TLC, splenomegaly as a constant finding, and a higher incidence of 6q deletion 31,32. It is worth noting here that all our CLL patients had typical CLL with less than 10% prolymphocytes.
In the current study, all the CLL/SLL patients with 6q deletion were in the advanced disease stage (Binet stage C and Rai stages III and IV), whereas only 58% (14/24) of those without 6q deletion were allocated to the same advanced stages; however, this comparison did not show a statistical significance. In agreement, Offit et al. 11 found no significant difference in proportion with early-stage disease between CLL patients with or without 6q deletion.
An association between 6q deletion in NHL and older age (>60 years), lower Hb concentration, higher level of β2M and LDH, and higher incidence of splenomegaly was found in the present study. According to the International Prognostic Index, a significantly higher percentage of NHL patients with 6q deletions were scored as high risk in comparison to those without the deletion.
In the MM group, the two patients with 6q deletion tended to have a higher β2M and monoclonal immunoglobulin level; however, the small sample size in this group did not allow a statistical analysis. Ocio et al. 33 found an association between 6q deletion and higher levels of β2M and paraprotein in WM patients.
It is assumed that one or more tumor suppressor genes are localized in the deleted regions of 6q. Three regions of minimal molecular deletions (RMD) in 6q have been detected in NHL. These RMDs were reported to be associated with certain pathological subtypes of NHL, namely, RMD1 at 6q25–27, RMD2 at 6q21, and RMD3 at 6q23 10,11.
Several candidate genes of interest have been localized to 6q21 including BLIMP1, FOXO2, CD24, cyclin C gene, and the AF6q21 gene 34–37. BLIMP1, in particular, is an attractive tumor suppressor candidate gene owing to its well-established role as a master gene regulator for B-lymphocytic cell proliferation and differentiation 38. Partial or whole losses in this master regulatory gene may in turn result in different functional capabilities for BLIMP1, and as such, may differentially influence the predilection for B-cell malignancies 39. Lin et al. 40 also reported the FOXO3A gene, known to be involved in lymphocyte proliferation and apoptosis, as a target for 6q deletion.
In conclusion, interphase-FISH represents a powerful, rapid, quantitative, and sensitive tool for monitoring 6q21 deletion. We suggest an important pathogenetic role of 6q deletion in BCLPDs on the basis of its high frequency in this histologic subset. Our results are in agreement with previous studies that have shown an impact of 6q deletion on prognosis in subtypes of BCLPDs.
As cytogenetic aberrations can be used as a marker for minimal residual disease in different hematologic malignancies, monitoring of 6q deletion may also aid the prediction of relapse in BCLPDs, but this should be validated by other studies with a large cohort of patients in the follow-up period.
Moreover, collaborative investigations with a larger sample size and additional 6q probes will provide better insights into the prognostic relevance of 6q deletions in BCLPDs. The identification of novel chromosomal abnormalities that may impact prognosis would be of particular interest for a patient-adapted risk therapeutic strategy.
| Acknowledgements|| |
This work was funded by grants from the Faculty of Medicine, Cairo University, Cairo, Egypt.
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[Table 1], [Table 2]