|Year : 2009 | Volume
| Issue : 4 | Page : 254-262
Characterization of genetic lesions in apoptosis-regulating and proliferation control genes in diffuse large B-cell non-Hodgkin's lymphoma
Shahid Pervez1, Muhammd I Nasir1, Tariq Moatter1, Adeeb Ahsan1, Amna Haq2, Tariq Siddiqui2
1 Department of Pathology and Microbiology, Aga Khan University Medical Centre, Karachi, Pakistan
2 Department of Medicine, Aga Khan University Medical Centre, Karachi, Pakistan
|Date of Web Publication||11-Feb-2010|
Department of Pathology and Microbiology, Aga Khan University Medical Centre, PO Box 3500, Stadium Road, Karachi
Source of Support: Research grant from University Research Council (URC), grant ID 1WL 707-02-421, Conflict of Interest: None
Background : This study was conducted to analyze the frequency, expression patterns, and the impact of individual proteins BCL2, BCL6, and p53 on overall survival (OS) in adult, diffuse large B-cell lymphoma (DLBCL) patients. BCL2 gene was further investigated for potential alterations at the DNA level and correlated with OS.
Materials and Methods : A total of 117 adult well-characterized DLBCL cases were included. The panel of antibodies comprised CD45, CD20, CD79a, CD3, BCL2, BCL6, and p53. PCR was also employed to correlate the events at the DNA level in BCL2.
Results : The mean and median ages were 47.74 and 49 with a M:F ratio of 2.07:1. The incidence of BCL2, BCL6, and p53 expression was observed in 64.10%, 37.60%, and 52.13% of cases, respectively. Amplifiable quality DNA was available from 90 cases. BCL2/IGH translocation was found in 35/90 patients (38.88%) with 24 cases showing BCL2 (MBR)/IGH and 11 cases BCL2 (mcr)/IGH translocation. No association between BCL2 overexpression and BCL2 /IGH translocation was seen. Clinical data were available for 52 patients treated by CHOP therapy. It was found that patients with p53 overexpression had decreased overall survival (P = 0.0004) whereas BCL2, BCL6 expression, and BCL2/IGH translocation had no impact on overall survival.
Conclusion : Our data suggest that simple p53 protein expression by IHC at the time of diagnosis may help to identify high-risk patients, who may benefit with more aggressive and newer treatments in addition to standard CHOP.
Keywords: BCL2, BCL6, p53, BCL2/IGH translocation, DLBCL, overall survival
|How to cite this article:|
Pervez S, Nasir MI, Moatter T, Ahsan A, Haq A, Siddiqui T. Characterization of genetic lesions in apoptosis-regulating and proliferation control genes in diffuse large B-cell non-Hodgkin's lymphoma. J Can Res Ther 2009;5:254-62
|How to cite this URL:|
Pervez S, Nasir MI, Moatter T, Ahsan A, Haq A, Siddiqui T. Characterization of genetic lesions in apoptosis-regulating and proliferation control genes in diffuse large B-cell non-Hodgkin's lymphoma. J Can Res Ther [serial online] 2009 [cited 2021 Jun 13];5:254-62. Available from: https://www.cancerjournal.net/text.asp?2009/5/4/254/59901
| > Introduction|| |
Diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS), is the most common non-Hodgkin's lymphoma (NHL) subtype, as defined by the World Health Organization (WHO) classification of lymphoid neoplasms.  DLBCL accounts for about 25-30% of adult NHL, in western countries, with a higher rate in developing countries. In Pakistan, the prevalence of DLBCL approaches epidemic proportions.  DLBCL is among the few aggressive tumor types that are curable with chemotherapy alone. DLBCL patients with similar histologic types show marked differences in probabilities of survival. An overall cure rate varies from 50% in advanced and 70% in the localized stage. , During the last decade, most studies dealing with the heterogeneity of DLBCL have focused on protein expression or molecular alterations and observed a potential prognostic value of these alterations.
The Bcl6 proto-oncogene has been originally identified because of its involvement in chromosomal translocation affecting band 3q7 in B-lineage diffuse large-cell lymphoma.  BCL6 rearrangements (chromosomal translocations involving the BCL6 5'-noncoding region in 3q27) occur at a frequency of 20-40% in DLBCL and 6-14% in follicular lymphoma (FL). ,, The prognostic significance of 3q27 translocation and BCL6 expression has been analyzed in different series. BCL6 rearrangement has been described as an independent marker of favorable clinical outcome in DLBCL. 
The second translocation implicated in DLBCL is the antiapoptotic BCL2 gene. The BCL2 gene was originally discovered by virtue of its involvement in the (14; 18) (q32; q21) translocation. ,, In the t(14; 18), the BCL2 gene on 18q21 is juxtaposed with the IgH gene on 14q32, resulting in overexpression of the structurally intact and functional BCL2 protein. The breakpoints on chromosome 14 are tightly clustered, occurring immediately 5` to the IgH joining regions. The majority of breakpoints on chromosome 18 are also tightly clustered. Two well-known clusters are the major breakpoint cluster region (MBR) and the minor breakpoint cluster region (MCR). ,
Expression of the BCL20 protein is independent of the translocation, as evidenced by its expression in a number of normal tissues, as well as a spectrum of lymphoproliferative disorders without a t(14; 18).  A high level of the BCL20 protein confers a survival advantage to B cells by inhibiting apoptosis and more generally may block a common cell death pathway induced by chemotherapy, conferring clinical drug resistance on cells overexpressing BCL20 protein. ,
The p53 tumor suppressor gene is probably the most frequently mutated gene in human cancers.  p53 mutations and the stabilization of the p53 protein are associated with more aggressive clinical behavior and treatment resistance in all histologic types of low-growth-fraction lymphomas. The contribution of p53 inactivation to the aggressive phenotype has also been observed at the clinical level in DLBCL, in which p53 overexpression or mutation has been reported as being associated with lower survival probability. ,
NHL is a very common malignancy in Pakistan. ,,, Recently, we  have reported 76% prevalence of DLBCL among all B-NHL cases reported in a major referral center for lymphoma diagnosis in Pakistan. This is the highest in the region ever reported. The present study was undertaken to investigate the expression of cell cycle and apoptosis-regulating proteins in our population with DLBCL both at expression and genetic level and to evaluate the impact of these proteins on overall survival (OS) in our patient population.
| > Materials and Methods|| |
Cases of DLBCL diagnosed between January 1995 and December 2002 were identified using Systematized Nomenclature of Medicine (SNOMED) code system from the central record database of the histopathology section. One hundred and seventeen (117) adult patients diagnosed with DLBCL and for whom adequate material (paraffin-embedded blocks) was available were selected for the present study. According to the history provided for each patient, none of them have had HIV/AIDS.
Histology and immunohistochemistry
The diagnosis of DLBCL was based on the criteria established in the WHO classification. Histologic slides were reviewed in all cases. All cases were immunophenotyped. The panel of antibodies included antibodies against the following antigens: CD45, CD20, CD79a, CD3, BCL2, p53, and Bcl6 [Table 1]. Immunohistochemistry was performed with DAKO Envision+ System, HRP (DAB) (DAKO, Denmark). Immunohistochemical analysis was performed on all 119 cases using formalin-fixed, paraffin-embedded tissue sections. Slides for CD45, CD20, CD79a, CD45R, and CD3 were treated with 10 mmol/l Tris buffer, 1 mmol/l EDTA, pH 9.0. Pretreatment was carried out by placing slides in a microwave oven for 14 min at a power of 450 watts. For BCL2, Bcl6, and p53, antigen retrieval was achieved by immersion of sections in 0.1 mM citrate buffer, pH 6.0, followed by three cycles in a microwave oven for 5 min each at a power of 450 watts. Optimum dilutions of all antibodies [Table 1] were made in a normal swine serum (DAKO, Denmark). All test and control slides (both positive and negative) were incubated with primary antibodies in a humidifying chamber while 0.01× PBS was applied to negative slides. The bound antibody was detected using the DAKO Envision System peroxidase technique, with diaminobenzidine (DAB) as chromogen, according to the manufacturer's instruction. Counterstaining was done with Harris hematoxylin. Sections were dehydrated, cleared with a xylene phenol mixture, and then mounted with DPX mounting media.
The expression was scored semiquantitatively under 40× magnification of Olympus BX51 light microscope. The results were scored in categories (−ive = no staining, +ive = ≤10% of neoplastic cells exhibited immunohistochemical staining in nucleus and/or cytoplasm, ++ = >10% and ≤ 50%, and +++ ³ 50%).
DNA extraction from paraffin-embedded tissue chips
Genomic DNA was extracted from all specimens. Cut sections from each case were deparaffinized and DNA was extracted by the modified DNAzol method.  Briefly after deparaffinization, the sample tissue was treated with a 350 µl lysis buffer (400 mM Tris-HCl, 60 mM EDTA, 150 mM NaCl, 1% SDS; final pH 8.0), and homogenized. Eighteen microliter (18 µl) proteinase K (10 mg/ml) was added to the homogenate and tubes were left overnight at 55°C with intermittent shaking. Later samples were centrifuged at 2500-3000 rpm for 5 min and the supernatants were transferred in a fresh tube containing 650 µl DNAzol, mixed well, and were kept at room temperature for 5 min. Later 500 µl of 100% ethanol was added to precipitate DNA. DNA was obtained by spinning the samples at 14,000 rpm for 10 min, pallet were washed with 70% ethanol and dried pallet to remove any traces of ethanol. Finally, the pallet was resuspended in 50 µl of DNase-free water. Four microliter (4 µl) stock was used to quantitate the DNA by taking O.D. at 260 nm and 280 nm.
Control fragment amplification by PCR
A 240 bp fragment of the β-globin gene was used to assess the presence of amplifiable quality. The primer sequences for β-globin gene polymerase chain reaction (PCR) were 5´-GA AAA TAG ACC AAT AGG CAG-3´ (GH 21) and 5´-ACA CAA CTG TGT TCA CTA GC-3´ (PCO3). PCR was performed in a 50 µl final volume containing 500 ng DNA, 0.2 mM dNTPs, 75 pM of each primer, 1.5 units of Taq polymerase in a 1× PCR buffer with 1.5 mM MgCl 2 . PCR was performed using a Perkin Elmer 9600 thermal cycler, with denaturation done at 94°C for one cycle of 2 min, then 30 cycles of denaturation 94°C for 90 s, annealing at 55°C for 90 s, extension at 72°C for 90 s, and a final extension at 72°C for 5 min. The amplified products were analyzed in a 2% agarose (Life Technologies, USA) gel by electrophoresis and visualized by staining with ethidium bromide under UV light. Cases that failed to amplify the b-globin fragment when tested at three different dilutions were considered failures. A negative control (blank) and a positive control (DNA extracted from blood) were included in each run.
PCR analysis for BCL2/IgH
Cases with β-globin amplification by PCR were then amplified by a nested PCR assay to check for any potential gene rearrangement in MBR and MCR locus of the BCL2 gene.
MBR-type BCL2/IgH0 nested PCR
MBR PCR was carried out in a final volume of 50 µl according to the protocol described by Barker et al.,  with some minor modifications. The reaction mixture contained 500 ng template DNA, 0.1 µM oligonucleotide primers, 200 µM of each dNTPs, and 1.5 units of Taq polymerase (Advance Biotechnologies, USA) in PCR buffer (500 mM KCl, 100 mM Tris-HCl pH 9.0, 1.0% Triton-X100, 1.5 mM MgCl 2 ). Amplification was performed in a thermal cycler 9700 (Perkin Elmer, USA). A 5 µl aliquot of the first PCR was used as a DNA template for the 30 cycles second round of PCR. The amplified products was analysed by 2% agarose (Life Technologies, USA) gel electrophoresis containing ethidium bromide. The gel was visualized with UV light and amplification reactions containing a discrete band in the range of 140-250 base pairs were considered positive. A negative control (blank) and a positive control (DNA from a known case of follicular lymphoma, positive for MBR BCL2/IgH) were included in each run. Cases with amplifiable DNA that were negative after two attempts with the BCL2 primers were considered BCL2 negative.
mcr type BCL2/IgH nested PCR
Nested PCR amplification for mcr/ IgH was carried out in a final volume of 50 µl according to the protocol described by Gribben et al,  with some minor modifications. The reaction mixture included 500 ng template DNA, 0.1 µM oligonucleotide primers, 200 µM of each dNTPs, and 1.5 units of Taq polymerase (Advance Biotechnologies, USA) in a PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1.0% Triton-X100, 1.5 mM MgCl 2 ). Amplification was performed in a thermal cycler 9700 (Perkin Elmer, USA). A 5 l aliquot of the first PCR was used as a DNA template for the second round of PCR of 30 cycles. The amplified products were analyzed by 2% agarose gel electrophoresis containing ethidium bromide. The gels were visualized with UV light to detect the discrete band. A negative control (blank) and a positive control (DNA from a known case of follicular lymphoma, positive for MCR BCL2/IgH) were included in each run. Cases with amplifiable DNA that was negative after two attempts with the BCL2 primers were considered BCL2 negative.
Univariate and survival analyses were carried out using SPSS (Statistical Package for Social Sciences) software, version 13.
The variables recorded and analyzed were age, gender, treatment, primary site (nodal vs. extranodal), BCL20 protein (neg. vs. pos.), BCL20 protein (nodal vs. extranodal), occurrence of BCL2/IgH translocation (neg. vs. pos.), occurrence of BCL2/IgH translocation (nodal vs. extranodal), occurrence of BCL2/IgH translocation ( BCL2 protein neg. vs. BCL2 protein pos.), BCL2 protein pos. ( BCL2/IgH translocation pos. vs. BCL2/IgH translocation neg.), BCL2 protein neg., ( BCL2/IgH translocation pos. vs. BCL2/IgH translocation negative), p53 protein (neg. vs. pos.), BCL6 protein (neg. vs. pos.), and p53 protein (nodal vs. extranodal).
Out of 117 cases, clinical data were available for 52 (inpatients) cases diagnosed and treated at our hospital by six to eight cycles of standard CHOP therapy alone. These 52 patients were grouped as those with no or low BCL2, Bcl6, and p53 expression (negative or 1+ expression) and high BCL2, Bcl6, and p53 expression (2+, 3+). The clinical follow-up ranged from 1 to 390 weeks (median 119 weeks). Follow-up time was counted until death or last evaluation. Seven patients died of lymphoma, and three died of other causes. OS was calculated from the date of diagnosis until the patient's death or last contact. OS curves were calculated by the method of Kaplan and Meier for each prognostic factor BCL2, Bcl6, p53 protein expression, and BCL2/IgH gene rearrangement.
| > Results|| |
There were 117 cases with adult DLBCL (age > 15 years) [Figure 1]. Among these 117 patients, there were 79 males (67.5%) and 38 females (32.47%), with a male-to-female ratio of 2.07:1. The calculated mean and median ages were 47.74 and 49 with the age range of 15-83 years.
Fifty-nine patients (50.42%) presented with the primary extranodal site, and fifty-six patients (47.86%) had a primary nodal DLBCL. For two patients (1.70%), the primary site of disease was unknown. The distribution according to extranodal sites was as follows: GIT (22 cases; 18.80%); oral cavity and adjacent (17 cases; 14.52%); CNS (5 cases; 4.27%); pharynx (3 cases; 2.56%); bone (3 cases; 2.56%); skin (3 cases; 2.56%); spleen (2 cases; 1.68%) and breast, testis, prostate, and thyroid gland (1 each).
Among nodal DLBCL, the distribution of nodal sites was as follows: head and neck (27 cases; 23.07%); lymph node, NOS (11 cases; 9.40%); extremities (12 cases; 10.25%); intra-abdominal (5 cases; 4.27%), and multiple regions (1 case).
Overall BCL2 expression
BCL2 expression by IHC was observed in 75/117 patients' tumors (64.10%) [Figure 2] and [Table 2]. There was no significant difference in expression rates between nodal lymphoma cases (37 of 56, 66.07%) versus extranodal lymphoma cases (37 of 59, 62.71%) (P = NS).
BCL2 expression and survival
Immunostaining for BCL2 was carried out on all 52 cases for which follow-up was available. Altogether, the BCL2 Expression was observed in 32/52 cases (61.5%). There were 23 patients with a negative (0) or weak positive (1+) BCL2 expression while in 29 patients, a high (2+ and 3+) expression was noted. There was no significant difference in expression rates between nodal lymphoma cases (17 of 23, 73.91%) versus extranodal lymphoma cases (15 of 29, 51.72%) (P-value = 0.183). Among 23 patients with a negative (0) or low (1+) BCL2 expression, 3 died. The average estimate of survival in this group was 284 weeks (95% confidence interval 177-392.6 weeks); on the other hand, among 29 patients with a high (2+ and 3+) BCL2 expression, 7 died. The average estimate of survival in this group was 265 weeks (95% confidence interval 199.6-330.8 weeks). The difference between the two groups (negative or 1+ BCL2 expression vs. 2+ and 3+ BCL2 expression) was nonsignificant (P = 0.7) [Table 3].
Occurrence of BCL2/IgH translocations
An amplifiable quality of DNA was extracted from 90 cases as determined by amplification of the b-globin gene fragment. BCL2/IgH translocation was found in 35/90 patients (38.88%) [Table 4]. Among 35 cases, there were 24 cases with BCL2 MBR/ IgH and 11 cases with BCL2 mcr/ IgH translocation [Figure 3] and [Figure 4]. There was no significant difference in the occurrence of BCL2/IgH translocations between nodal lymphoma cases (16 cases; six BCL2 mcr/ IgH , 11 BCL2 MBR/ IgH) versus extranodal lymphoma cases (17 cases; 5 BCL2 mcr/ IgH, 11 BCL2 MBR/ IgH) (P = NS). Two cases, in which the primary site was unknown, showed BCL2 MBR/ IgH translocation.
The relationship between the translocation and BCL2 protein expression is shown in [Table 5]. Of the BCL2/IgH-positive patients, 65.72% were also BCL2 protein positive while of the BCL2/IgH-negative patients, 67.27% of the cases expressed BCL2 protein. In DLBCL positive for BCL2/IgH translocation, the most common pattern of expression was BCL2+/bcl6−/p53+, which was observed in 10 out of 35 cases (28.57%).
Occurrence of BCL2/IgH translocation and survival
An amplifiable quality of DNA was extracted from 45 out of 52 cases with proper follow-up. BCL2/IgH gene rearrangement was observed in 16 of 45 patients. A higher tendency of occurrence of BCL2/IgH translocation was seen in nodal lymphoma cases (10 of 21, 47.61%) as compared to extranodal lymphoma cases (6 of 24, 25%) (P-value = 0.20).
Among 16 BCL2/IgH-positive cases, 1 patient died. The average estimate of survival in this group was 266.1 weeks (95% confidence interval 176.9-355.3 weeks); on the other hand, among 18 BCL2/IgH-negative patients, 4 patients died. The average estimate of survival in this group was 339.1 weeks (95% confidence interval 269-409.2 weeks). The difference between the two groups, BCL2/IgH positive versus BCL2/IgH negative, was nonsignificant (P = 0.12) [Table 3].
Overall Bcl6 expression
Bcl6 immunohistochemical expression was found in 44/117 patients (26.49%) [Table 2] and [Figure 5]. There was no significant difference in expression rates between nodal lymphoma cases (23 of 56, 41.07%) versus extranodal lymphoma cases (20 of 59, 33.89%) (P = NS).
Bcl6 expression and survival
Immunostaining for Bcl6 was performed on all 52 inpatient cases. Altogether BCL6 expression was observed in 22/52 patients (42.30%). There was no significant difference in expression rates between nodal lymphoma cases (13 of 23, 56.52%) versus extranodal lymphoma cases (9 of 29, 31.03%) (P-value = 0.12).
There were 35 patients with negative (0) or low (1+) Bcl6 expression while in 17 patients, a high (2+ and 3+) expression was observed. Among 35 patients with negative (0) or low (1+) Bcl60 expression, 7 died. The average estimate of survival in this group was 252.7 weeks (95% confidence interval 182.4-322.9 weeks); on the other hand, among 17 patients with a high (2+ and 3+) Bcl6 expression, 3 died. The average estimate of survival in this group was 311.2 weeks (95% confidence interval 233.3-389.1 weeks). The difference between the two groups (negative or 1+ Bcl60 expression vs. 2+ and 3+ Bcl6 expression) was nonsignificant (P = 0.7) [Table 3].
Overall p53 expression
p53 nuclear expression was observed in 61/117 patients (29.91%) [Table 2] and [Figure 6]. There was no significant difference in expression rates between nodal lymphoma cases (29 of 56, 51.78%) versus extranodal lymphoma cases (32 of 59, 54.23%) (P = NS). [Table 2] gives breakup of nodal versus extranodal p53-positive and -negative cases with site/localization.
p53 expression and survival
Immunostaining for p53 was performed on all 52 inpatient cases. Twenty-two cases (38.46%) were p53 positive. Altogether, p53 expression was observed in 30/52 patients (57.69%). There was no significant difference in expression rates between nodal lymphoma cases (17 of 23, 73.91%) versus extranodal lymphoma cases (13 of 29, 44.82%) (P-value = 0.07). There were 34 patients with a negative (0) or low (1+) p53 expression while in 18 patients, a high (2+ and 3+) p53 expression was observed. Among 34 patients with a low p53 expression, 2 died. The average estimate of survival in this group was 340.1 weeks (95% confidence interval 293.7-386.5 weeks); on the other hand, among 18 patients with a high p53 expression, 8 died. The average estimate of survival in this group was 184.9 weeks (95% confidence interval 95-274.9 weeks). The difference between the two groups (negative or 1+ p53 expression vs. 2+ and 3+ p53 expression) was highly significant (P = 0.0004) [Table 3] and [Figure 7].
| > Discussion|| |
There is considerable evidence to suggest that there are clinically and biologically distinct subgroups of DLBCL. It is now also clear that the differences in the clinical features and treatment responses of DLBCL are due to a marked molecular heterogeneity that underlies the aggressiveness of the disease and tumor progression. ,, It has been shown that resistance to apoptosis is an important factor in lymphomatogensis. ,, Much emphasis therefore has been placed on changes in apoptosis-regulating and proliferation control genes such as BCL2, Bcl6, and p53.
BCL2 is a proto-oncogene that promotes B-cell survival via inhibition of apoptosis and confers chemotherapy resistance.  BCL2 protein has been found to be expressed in a variable proportion (24-79%) of DLBCLs ,,, more frequently in nodal than in extranodal tumors. ,,, In the current study, the incidence of BCL20 expression was observed in 75/117 patients (64.10%) with no significant association with OS in a subset of patients on whom follow-up was available and who were treated with six to eight cycles of standard chop therapy alone.
The prognostic impact of BCL2 protein expression is evaluated in multiple large-scale clinical trials and is found to be significant. In a recent large study among 128 patients diagnosed with de novo DLBCL, Colomo et al. observed BCL2 protein expression in 59% of the cases; it was concluded that the expression of BCL2 was associated with poor OS. In another large series, Kramer et al. studied 156 patients with de novo DLBCL and concluded that a high BCL2 protein expression has adverse effects on both OS (P = 0.008) and DFS (P = 0.01). Similarly, Gascoyne et al.  in their study on 116 patients of DLBCL observed positive BCL2 protein expression in 28 of 116 (24%) patients. It was concluded that BCL2 expression correlated with decreased 8-year OS (34% v 60%), P < 0.01. BCL2 protein expression was found to be a strong significant predictor of OS and DFS in DLBCLs. Sohn et al. in Korea observed high levels of BCL2 expression in 24/94(26.4%) patients with de novo DLBCL. However, there was no significant difference in the OS (P = 0.0682) between the BCL2 -positive (n = 24) and BCL2 -negative (n = 67) groups. In our series, survival and follow-up data were available for 52 patients. Cases were grouped as negative (0) or weak positive (1+) BCL2 expression versus high (2+ and 3+) BCL2 expression. There was no significant difference in OS between the two groups (P = 0.7)
We also performed Kaplan-Meier analysis to determine the impact of BCL2/IgH translocation on OS in DLBCL patients. An amplifiable quality of DNA was available from 45 out of 52 cases with proper follow-up. BCL2/IgH gene rearrangement was observed in 16 of 45 patients. A higher tendency of occurrence of BCL2/IgH translocation was seen in nodal lymphoma cases (10 of 21, 47.61%) as compared to extranodal lymphoma cases (6 of 24, 25%). However, the impact of BCL2/IgH positive versus BCL2/IgH negative expression on OS was nonsignificant (P = 0.12).
Bcl6 is another proto-oncogene evaluated in our study. The expression of Bcl6 has been found in the majority of DLBCLs, ranging from 48% to 76% in various series, including both nodal and extranodal sites. ,,, In our series, Bcl6 immunohistochemical expression was found in 44/117 patients (37.60%), which is lower than reported elsewhere. Immunohistochemical studies of Bcl6 expression and its relationship to outcome in DLBCL are growing in number. Weng et al. in Beijing studied Bcl6 expression in 41 patients of DLBCL, it was observed that the expression of Bcl6 protein was related with better prognosis (P = 0.0049). Similarly Hans et al. evaluated 152 cases of DLBCL. Bcl6 protein expression (cases were considered positive if 30% or more of the tumor cells stained positive) was observed in 56% of the patients. It was found that the expression of Bcl6 was associated with better OS (P <0.001). In another study, Berglund et al. evaluated 161 DLBCL patients. Positive staining for Bcl6 was observed in 48% (78/161) cases (cut-off >30 cells staining). It was concluded that the expression of Bcl6 (P = 0.00003) was associated with better OS. However, in a large series of 128 cases using same technique of IHC, Colmo et al. observed that Bcl6 although highly expressed (72% positivity, cut-off 25%) was not found to have any impact on OS. We also evaluated the impact of Bcl6 expression on OS in our inpatient population. The difference between the two groups (negative or 1+ Bcl6 0 expression vs. (++, +++ Bcl6 expression) was nonsignificant (P = 0.7).
p53 overexpression has been observed in DLBCLs but with variable frequencies ranging from 18% to 56.5%. ,,,, In our series using clone DO-7, p53 nuclear expression was observed in 61/117 patients (52.13%), which is among few of higher expressions reported.
We also performed Kaplan-Meier analysis to determine the impact of p53 overexpression on overall survival in our 52 DLBCL inpatients. It was observed that p53 protein overexpression was associated with decreased overall survival (184.9 weeks in 2+, 3+ p53 expression in patients vs. 340.1 weeks in negative or 1+ p53 expression in patients) and a highly significant P-value (P = 0.0004).
Several studies have investigated the significance of p53 in DLBCL using a variety of methods, including IHC, loss of heterozygosity analysis, single-strand conformational polymorphisim analysis, and direct sequencing. It has been found that p53 immunoreactivity, which may or may not correlate with p53 mutation, is an adverse independent prognostic factor in DLBCL. In a Japanese study, Zhang et al. assessed a panel of surface markers, and proliferating, suppressor and oncogenic proteins including p53 in 158 DLBCL patients. IHC was employed. It was observed that a high p53 expression and a low BCL6 expression were associated with a shorter DFS. In another study conducted in Brazil, Pagnano et al.  evaluated the expression of apoptosis-regulating proteins in DLBCL patients in relation with clinical outcome. IHC was employed. p53 nuclear overexpression was observed in 9/32 patients (28%). It was found that patients with negative or low p53 expression had a better OS (P = 0.0045). Ichikawa et al. also reported the poor prognostic value of p53 gene mutations in a series of 102 patients with aggressive B-cell lymphoma, including 59 DLBCL patients. PCR-SSCP and DNA sequencing techniques were used for the analysis. Overall survival was significantly lower among patients with p53 mutations than among patients with wild-type p53 gene.
However, in few studies no significant correlation was observed. Sohn et al. in Korea evaluated the impact of p53 expression on OS in a series of 94 patients with de novo DLBCL. IHC was employed to study the p53 overexpression. p53 expression was observed in 21 patients (22.6%).There was no significant difference in the OS (P = 0.1741) between the p53-positive and p53-negative groups. Colomo et al. in a joint study of University of Barcelona, and University of Perugia, Italy, studied the relationship of p53 and its impact on outcome in 128 cases of DLBCL. p53 overexpression was observed in 28% cases. No significant difference was found in terms of response or OS with respect to p53 expression. Weng et al. and Llanos et al.  also reported similar findings.
This study is the first ever from Pakistan looking at the genetic level of DLBCL by using state-of-the-art methodology including molecular characterization by PCR, besides morphologic and immunophenotypic analysis. This study suggests that the prevalence of DLBCL in Pakistan is occurring in younger patients with a median age of 47.7 years and with a clear male preponderance. Though it is established elsewhere that increased incidence rates of NHL in the younger age group are attributed to its association with AIDS,  Pakistan is identified as a low-risk population for HIV infection. The prevalence of HIV/AIDS is limited to sporadic cases of Pakistanis (and their sexual partners) who have traveled abroad, or overseas contract workers and injection drug users. ,, In our patients, no overt immunodeficiency/history of HIV was present.
In recent years, the dissection of DLBCL into germinal center (GC) and postgerminal center phenotype (PGC) is becoming common as it is rather well established that GC phenotype has a significant survival advantage over PGC phenotype using standard CHOP therapy. Bcl6 and CD10 are usually used as a marker of GC phenotype while negativity of the above with expression of MUM1 as marker for PGC phenotype. The current literature also suggests that if anti-CD20 (rituximab) is added to CHOP, the difference in survival between the two phenotypes may be narrowed. However, in developing countries like Pakistan where affordability is still a core issue with per capita income of less than US$ 1000, no health insurance, and about 40% population still living below poverty line, newer drugs like anti-CD20 may at best be afforded by a lucky few. Under these circumstances, it still remains highly desirable to stratify DLBCL, as DLBCL remains a major problem in the adult population, luckily with a chance of cure in a significant number with this diagnosis. As it is well established that p53 alteration is usually a very common event happening at the time of transformation of low-grade B lymphomas into DLBCL and many studies including ours suggest its impact on OS, it may be another candidate to be included in the work-up to stratify DLBCL into a good, bad, or ugly kind of exercise.
| > Conclusion|| |
In our series, p53 overexpression was observed in 52.13% of cases, which is among few of the highest reported cases elsewhere. In this study, p53 protein expression was found to have a significant impact on poor OS (P = 0.0004). The other well-established marker BCL2 was also found to be expressed in most of the cases, although not found to be significantly associated with OS. BCL2/IgH studies by PCR revealed that a good percentage of our cases constitute this translocation as our results (38.9%) are among the few showing high occurrence. Bcl6 protein expression also did not show any significant impact on OS in our patients. Our data suggest that simple p53 protein expression by IHC at the time of diagnosis may help to identify high-risk patients, who may benefit with more aggressive and newer treatments in addition to standard CHOP therapy.
| > Acknowledgment|| |
This study was supported by a research grant from University Research Council (URC), grant ID 1WL 707-02-421.
| > References|| |
|1.||Stein H, Warnke RA, Chan WC, Jaffe ES, Chan JK, Gatter KC, et al. Diffuse Large B-Cell Lymphoma, not otherwise specified in WHO classification of tumours of haematopoietic and lymphoid tissues. 4 th ed. Lyon, France: IARC Press; 2008. p. 233-37. |
|2.||Abid MB, Nasim F, Anwar K, Pervez S. Diffuse large B cell lymphoma in Pakistan: An emerging epidemic? Asian Pac J Cancer Prev 2005;6:531-4. |
|3.||Miller TP, Dahlberg S, Cassady JR, Adelstein DJ, Spier CM, Grogan TM, et al. Chemotherapy alone compared with chemotherapy plus radiotherapy for localized intermediate- and high-grade non-Hodgkin′s lymphoma. N Engl J Med 1998;339:21-6. |
|4.||Fisher RI, Gaynor ER, Dahlberg S, Oren MM, Grogan Tm, Mize EM, et al Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin′s lymphoma. N Engl J Med 1993;328:1002-6. |
|5.||Baron BW, Nucifora G, McCabe N, Espinosa R, Le Beau MM, McKeithan TW, et al. Identification of the gene associated with the recurring chromosomal translocations t(3;14)(q27;q32) and t(3;22)(q27;q11) in B-cell lymphomas. Proc Nat Acad Sci USA 1993;90:5262-6. |
|6.||Bastard C, Deweindt C, Kerckaert JP, Lenormand B, Rossi A, Pezzella F, et al LAZ3 rearrangements in non-Hodgkin′s lymphoma: Correlation with histology, immunophenotype, karyotype, and clinical outcome in 217 patients. Blood 1994;83:2423-7. |
|7.||Offit K, Lo Coco F, Louie DC, Parsa NZ, Leung D, Portlock C, et al Rearrangement of the bcl-6 gene as a prognostic marker in diffuse large-cell lymphoma. N Engl J Med 1994;331:74-80. |
|8.||Lo Coco F, Ye BH, Lista F, Corradini P, Offit K, Knowles DM, et al. Rearrangements of the BCL6 gene in diffuse large cell non-Hodgkin′s lymphoma. Blood 1994;83:1757-9. |
|9.||Tsujimoto Y, Cossman J, Jaffe E, Crocl CM. Involvement of the Bcl-2 gene in human follicular lymphoma. Science 1985;228:1440-3. |
|10.||Bakhshi A, Jensen JP, Goldman P, Wright JJ, McBride OW, Epstein AL, et al. Cloning the chromosomal breakpoint of t(14; 18) human lymphomas: Clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 1985;41:899-906. |
|11.||Cleary ML, Sklar J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally active locus on chromosome18. Proc Natl Acad Sci USA 1985;82:7439-43. |
|12.||Pezzella F, Tse AG, Cordell JL, Purford KA, Gatter KC, Mason DY, et al. Expression of the bcl-2 oncogene protein is not specific for the 14;18 chromosomal translocation. Am J Pathol 1990;137:225-32. |
|13.||Miyashita T, Reed JC. Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 1993;8:151-7. |
|14.||Miyashita T, Reed JC. Bcl-2 gene transfer increases relative resistance of S49.1 and WEHI7.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs. Cancer Res 1992;52:5407-11. |
|15.||Newcomb EW. P53 gene mutations in lymphoid diseases and their possible relevance to drug resistance. Leuk Lymph 1995;17:211-21. |
|16.||Ichikawa A, Kinoshita T, Watanabe T, Kato H, Nagai H, Mura Kami Y, et al Mutations of the p53 gene as a prognostic factor in aggressive B-cell lymphoma. N Engl J Med 1997;337:529-34. |
|17.||Piris MA, Pezzella F, Martinez-Montero JC, Orradre JL, Villuendas R, Sanchez Beato M, et al. p53 and bcl-2 expression in high-grade B-cell lymphomas: Correlation with survival time. Br J Cancer 1994;69:337-41. |
|18.||Cancer Registry and clinical Data Management (CRCDM), Shaukat Khanum Memorial Cancer Hospital and Research Canter (SKMCH and RC) - ( www.shaukatkhanum.org.pk ). Report based on cancer cases registered at SKMCH and RC from December 1994 to December 2008 and in 2008. Released June, 2009. |
|19.||Bhurgri Y, Pervez S, Bhurgri A, Faridi N, Usman A, Kazi LA, et al. Increasing incidence of non-Hodgkin′s lymphoma in Karachi, 1995-2002. Asian Pac J Cancer Prev 2005;6:364-9. |
|20.||Aziz Z, Sana S, Saeed S, Akram M. Institution based tumor registry from Punjab: Five year data based analysis. J Pak Med Assoc 2003;53:350-3. |
|21.||Bhurgri Y, Hasan SH, Pervez S, Kayani N, Hussainy AS, Muzaffar S, et al. Large scale pathology-based cancer data: A reflection of population-based cancer data. Pathol Oncol Res 2002;8:62-7. |
|22.||Chomczynski P, Mackey K, Drews R, Wilfinger W. A reagent for the rapid isolation of genomic DNA. Bio Techniques 1995;22:550-3. |
|23.||Baker R, Worth CA, Peiper SC. Cytometric detection of DNA amplified with fluorescent primers: Applications to analysis of clonal bcl-2 and Ig H gene rearrangements in malignant lymphomas. Blood 1994;83:1079-85. |
|24.||Gribben JG, Freedman A, Woo SD, Blake K, Shu RS, Freeman G, et al. All advanced stage non-Hodgkin′s lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment. Blood 1991;78:3275-80. |
|25.||Gascoyne RD, Krajewska M, Krajewski S, Connors JM, Reed JC. Prognostic significance of Bax protein expression in diffuse aggressive non-Hodgkin′s lymphoma. Blood 1997;90:3173-8. |
|26.||Hermine O, Haioun C, Lepage E, d′Agay MF, Briere J, Lavignac C, et al Prognostic significance of bcl-2 protein expression in aggressive non- Hodgkin′s lymphoma. Groupe d′Etude des Lymphomes de l′Adulte (GELA). Blood 1996;87:265-72. |
|27.||Hill ME, MacLennan KA, Cunningham DC, Vaughan Hudson B, Burke M, Clarke P, et al. Prognostic significance of BCL-2 expression and bcl-2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin′s lymphoma: A British National Lymphoma Investigation Study. Blood 1996;88:1046-51. |
|28.||Sander CA, Yano T, Clark HM, Harris C, Longo DL, Jaffe ES, et al. p53 mutation is associated with progression in follicular lymphomas. Blood 1993;82:1994-2004. |
|29.||Xu Y, McKenna RW, Molberg KH, Kroft SH. Clinicopathologic analysis of CD10+ and CD10- diffuse large B-cell lymphoma: Identification of a high-risk subset with coexpression of CD10 and bcl-2. Am J Clin Pathol 2001;116:183-90. |
|30.||King BE, Chen C, Locker J, Kant J, Okuyama K, Falini B, et al Immunophenotypic and genotypic markers of follicular center cell neoplasia in diffuse large B-cell lymphomas. Mod Pathol 2000;13:1219-31. |
|31.||Fang JM, Finn WG, Hussong JW, Goolsby CL, Cubborn AR, Variakojis D. CD10 antigen expression correlates with the t(14;18)(q32;q21) major breakpoint region in diffuse large B-cell lymphoma. Mod Pathol 1999;12:295-300. |
|32.||Villuendas R, Piris MA, Orradre JL, Mollejo M, Rodriguez R, Morente M. Different bcl-2 protein expression in high-grade B-cell lymphomas derived from lymph node or mucosa-associated lymphoid tissue. Am J Pathol 1991;139:989-93. |
|33.||Skinnider BF, Horsman DE, Dupuis B, Gascogyne RD. Bcl-6 and Bcl-2 protein expression in diffuse large B-cell lymphoma and follicular lymphoma: Correlation with 3q27 and 18q21 chromosomal abnormalities. Hum Pathol 1999;30:803-8. |
|34.||Rantanen S, Monni O, Joensuu H, Franssilak, Knuvtilas. Causes and consequences of BCL2 over expression in diffuse large B-cell lymphoma. Leuk Lymph 2001;42:1089-98. |
|35.||Colomo L, Lopez-Guillermo A, Perales M, Rives S, Martinez A, Bosch F, et al. Clinical impact of the differentiation profile assessed by immunophenotyping in patients with diffuse large B-cell lymphoma. Blood 2003;101:78-84 |
|36.||Kramer MH, Hermans J, Wijburg E, Phillippo K, Geelen E, Vankrieken JH, et al. Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma. Blood 1998;92:3152-62. |
|37.||Sohn SK, Jung JT, Kim DH, Kwak EK, Park T, Shin DG, et al. Prognostic significance of bcl-2, bax, and p53 expression in diffuse large B-cell lymphoma. Am J Hematol 2003;73:101-7. |
|38.||Weng Y, Gao ZF, Liu K, Zhang WJ, Ke XY, Li M. Factors affecting the prognosis of diffuse large B-cell lymphoma in Chinese. Zhonghua Nei Ke Za Zhi 2005;44:681-3. |
|39.||Kwon MS, Go JH, Choi JS, Lee SS, Ko YH, Rlee JC, et al Critical evaluation of Bcl-6 protein expression in diffuse large B-cell lymphoma of the stomach and small intestine. Am J Surg Pathol 2003;27:790-8. |
|40.||Hans CP, Weisenburger DD, Greiner TC, Gaseoyne RD, Delabief J, Ott G, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 2004;103:275-82. |
|41.||Berglund M, Thunberg U, Amini RM, Book M, Roos G, Erianson M, et al. Evaluation of immunophenotype in diffuse large B-cell lymphoma and its impact on prognosis. Mod Pathol 2005;18:1113-20. |
|42.||Pagnano KB, Silva MD, Vassallo J, Aranh FJ, Saad ST. Apoptosis-regulating proteins and prognosis in diffuse large B cell non-Hodgkin′s lymphomas. Acta Haematol 2002;107:29-34. |
|43.||Llanos M, Alvarez-Arguelles H, Aleman R, Oramas J, Diaz-Floresl, Batista N. Prognostic significance of Ki-67 nuclear proliferative antigen, bcl-2 protein, and p53 expression in follicular and diffuse large B-cell lymphoma. Med Oncol 2001;18:15-22. |
|44.||Zhang A, Ohshima K, Sato K, Kanda M, Suzumiya J, Shimazaki K, et al. Prognostic clinicopathologic factors, including immunologic expression in diffuse large B-cell lymphomas. Pathol Int 1999;49:1043-52. |
|45.||Gail MH, Pluda JM, Rabkin CS, Bigger RJ, Goedert JJ, Horm JW, et al. Projections of the incidence of non-Hodgkin′s lymphoma related to acquired immunodeficiency syndrome. J Natl Cancer Inst 1991;83:695-701. |
|46.||Baqi S, Kayani N, Khan JA. Epidemiology and clinical profile of HIV / AIDS in Pakistan. Trop Doct 1999;29:144-8. |
|47.||Rehman N, Emmanuel F, Akhtar S. HIV transmission among drug users in Larkana, Pakistan. Trop Doct 2007;37:58-9. |
|48.||Achakzai M, Kassi M, Kasi PM. Seroprevalences and co-infections of HIV, hepatitis C virus and hepatitis B virus in injecting drug users in Quetta, Pakistan. Trop Doct 2007;37:43-5. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]