| Abstract|| |
Background: The distinction between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) is not always easy, so much so that the WHO 2008 Blue Book has incorporated a provisional category of "B-cell lymphoma, unclassifiable with features intermediate between DLBCL and BL." One of the immunohistochemical (IHC) markers used at times to differentiate between the two is IHC expression of BCL2, which was initially believed to be consistently negative in BL. Later it was recognized that weak BCL2 expression is still compatible with the diagnosis of BL. To verify or otherwise this current view this study was undertaken. Materials and Methods: We retrieved 39 confirmed cases of BL, in both children and adults. All these cases had typical morphology, IHC profile, and Mib-1 index that are typical of BL. All these cases were then stained with a monoclonal antibody against BCL2 oncoprotein, using 2-step Envision system. Results: Out of 39 cases, 31 cases (79.4%) were completely negative for BCL2, whereas 5 (12.8%) were weak focal positive. However, another 4 (10.2%) cases did show strong diffuse cytoplasmic staining for BCL2. Fluorescent in-Situ hybridization (FISH) for t(14:18) was optimally done on 6 out of 9 cases. All these 4 cases were from adults with 3 out of 4 arising in the parotid region. Two out of 4 cases also showed t(8:14) on FISH. Conclusions: It was concluded that contrary to the common belief, strong BCL2 IHC expression is possible in typical BL in adults and cannot be absolutely relied upon to distinguish between BL and DLBCL.
Keywords: BCL2, Burkitt lymphoma, immunohistochemical, Distinguishing between diffuse large B-cell lymphoma
|How to cite this article:|
Pervez S, Raza M Q, Mirza A, Pal A. Strong BCL2 expression in Burkitt lymphoma is not uncommon in adults. Indian J Pathol Microbiol 2011;54:290-3
|How to cite this URL:|
Pervez S, Raza M Q, Mirza A, Pal A. Strong BCL2 expression in Burkitt lymphoma is not uncommon in adults. Indian J Pathol Microbiol [serial online] 2011 [cited 2013 Dec 5];54:290-3. Available from: http://www.ijpmonline.org/text.asp?2011/54/2/290/81599
| Introduction|| |
Distinguishing between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) is not always easy. Consequently, it proves to be a challenge to distinguish between the two on a day-to-day basis, especially when it comes to choosing appropriate treatment options for the affected adult patients.
The morphologic and immunohistochemical (IHC) criteria currently used to distinguish between the two include cell size, monomorphism, and Mib-1 index. However, some DLBCL cases may very closely mimic BL properties; and they may have a proliferation index close to 100% and a BL-like (BLL) monomorphic population. In such cases, one of the useful immunostains that has been used in the past is BCL2 IHC expression.
Most studies have shown that BL does not express BCL2 protein on IHC.  However, a few recent studies reported occasional weak expression  in otherwise Classical BL. The latest WHO Blue Book confirms that weak IHC BCl2 expression may be seen in Classical BL. ,
It therefore appears that the experiences are varied, and for good reasons. Many questions abound, such as what is true BL vs BLL, is it endemic or sporadic type, in a child or adult, what is the definition of weak expression, is it number of cells stained or intensity of staining, and so on. This study was therefore undertaken to further understand this puzzle in our patients, who were both children and adults diagnosed with BL.
| Materials and Methods|| |
A total of 39 cases diagnosed and treated as BL, over a period of 18 months (June 2007-June 2008), were retrieved from the archives of the "Histopathology Section" AKUH, Karachi, a major referral center for the whole country. All these cases already had an extensive IHC workup as per the WHO guidelines. The data, apart from the patient's demographic and clinical details, included information regarding LCA, CD20, CD79a, CD10, CD3, Tdt, BCL6, and Mib-1. Six out of 9 BCL2-positive cases were also subjected to FISH for t(8:14). These included 2 cases that showed strong BCL2 staining. On the other 2 cases, FISH testing was unsuccessful due to technical reasons.
All cases were reviewed by a senior hematopathologist for adequacy, morphology, and IHC workup. Relevant blocks were retrieved and 4-μm sections were cut. All the sections were stained with a monoclonal antibody against BCL2 oncoprotein (clone 124, Dako, Denmark) using 2-step Envision technique. Briefly, the retrieved tissue samples were first fixed in 10% buffered formalin and embedded in paraffin after routine processing. The sections were then cut and mounted on positively charged superfrost slides and dewaxed in an oven for 1 h at 60°C. Following this, the sections were cleared in xylene and rehydrated in graded ethanol. For epitope retrieval, the tissue sections were heated in Tris-EDTA (pH 9.0) for 16 min at 450 W in a scientific microwave oven. Once the epitopes were retrieved, the sections were cooled down gradually to room temperature and washed with running tap water. Endogenous peroxidase was blocked by incubating the tissue in 3% hydrogen peroxide at room temperature for 10 min and washing with distilled water. The tissue samples were then incubated with a primary antibody in a dilution of 1:20 for 30 min at room temperature. After incubation with primary antibody, immune detection was performed with the 2-step polylabeling method, as mentioned earlier, using the Envision system (Dako, Denmark) with diaminobenzidine (DAB) chromogen as a substrate. After DAB, the slides were counterstained by hematoxylin and finally were mounted with Distyrene-Plasticizer-Xylene.
Final preparations were scored as follows (only cytoplasmic staining was observed and scored):
Negative: No staining observed
Weak: 1%-25% tumor cells stained
Moderate: 26%-75% tumor cells stained
Strong: >75% tumor cells stained
Intensity of Staining was also taken into account as follows:
Weak: Light brown staining at higher magnifications only (20× and 40×) Strong: Dark brown staining visible even at low magnifications (4× and 10×)
FISH testing on whole nuclei extracted from paraffin-embedded tissue
The IgH/Myc Dual Fusion Probe (Vysis, Abbot, USA) is used for gene amplification. Between 5 and 10 sections of 35-μm thick were cut, dewaxed in xylene at 37°C, and rehydrated through graded alcohols. Following 2 changes of distilled water, the sections were incubated in prewarmed digestion buffer (0.1 mol/L Tris; 0.07 mol/L NaCl; 0.1% NP-40; pH 7.4) at 37°C for 30 min. The digestion buffer was replaced with fresh, prewarmed buffer containing 0.05% Protease, and the sections were agitated for 1 h at 37°C.
The digested nuclei were then harvested by pipetting the supernatant nuclear suspension from the undigested tissue, and centrifuged at 1800 × g. The harvested nuclei were washed once in phosphate-buffered saline and once in 3:1 methanol acetic acid (MAA).The nuclei were then resuspended in MAA, and the nuclear suspension was dropped by pipette onto 3-aminopropyltriethoxy silane-coated slides. The slides were then fixed in MAA for 30 min and incubated in 2× standard saline citrate (SSC) at 37°C for 1 h. Following dehydration through graded alcohols, the probe was applied in hybridization buffer, and a cover slip sealed on using rubber cement. The slides were incubated in hybridization buffer at 90°C for 20 min, and then allowed to cool back to 37°C. Using a Vysis Hybrite machine, denaturation was carried out at 75°C for 5 min and hybridization at 37°C for 16 h.
Post-hybridization wash consisted of 2 min in pre-warmed 0.4× SSC/0.3× NP-40 at 70°C; followed by 5 min in 2× SSC/0.1× NP-40 at room temperature. 4-6-diamino-2-phenylindole. DAPI counter stain (10 μL) was applied and cover slip was placed. The slides were cooled down before screening using a fluorescent light microscope.
The fusion DNA probe hybridizes to the band 14q32 (IGH) in spectrum Green, band 8q24 (MYC) in Spectrum red. In the interphase nuclei of normal cells, the probe generally appears as 4 distinct signals (2 each red and green). In the interphase nuclei of t(8;14) cells, fusion signals plus 2, 1 red and 1 green, signals are present to indicate the occurrence of the translocation .
| Results|| |
Out of a total of 39 cases of BL [Figure 1]a and b stained with IHC BCL2, a total of 9 cases (23.1%) were found to be positive for BCL2. Out of these 9 cases positive for BCL2, 5 cases (12.8%) were weak/focal positive for BCL2 [Figure 1]c, whereas the remaining 4 (10.3%) cases showed strong and diffuse BCL2 positivity [Figure 1]d. Of the 39 cases, FISH for t(8:14) was done on 6 cases, including 4 showing focal BCL2 positivity and 2 cases that showed strong BCL2 positivity. Four out of 6 cases showed evidence of t(8:14) translocation, and these included 2 of the 4 cases showing strong BCL2 positivity [Figure 2]a and b.
|Figure 1: (a) H and E-stained section of a case of Burkitt lymphoma (note typical morphology with starry sky appearance); (b) a higher magnification of the same as in (a); (c) Bcl2 immunostaining on a case of BL (note focal weak cytoplasmic staining); and (d) Bcl2 immunostaining on a case of BL (note strong diffuse cytoplasmic staining)|
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|Figure 2: (a) FISH for t(8:14) translocation (negative cases showing 2 green (IgH) and 2 red (MYC, 8q24) signals); and (b): positive cases showing fusion signals|
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Of these 39 cases, 20 (51.3%) were categorized as children considering 15 years of age as the cutoff. The remaining 19 cases (48.7%) were categorized as adults. The mean age of children and adults was 8.3 and 42.1 years, respectively, whereas the median age of children and adults was 8 and 40 years, respectively [Table 1] and [Table 2].
Among the children, only 2 of the 20 cases (10%) showed weak/focal BCL2 expression [Figure 1]c and none showed strong expression. In contrast, 7 of the 19 cases (36.8%) in the adults category expressed BCL2; 3 out of 7 (42.8%) positive cases showed a weak/focal expression, whereas the remaining 4 (57.2%) showed strong/diffuse expression [Figure 1]d; [Table 1] and [Table 2]. These 4 cases were carefully reviewed again. All these cases did show typical morphologic and IHC profile of "Classical BL." They all showed uniform medium-sized neoplastic lymphoid cells with multiple small fine nucleoli and finely dispersed chromatin. The features of "atypical BL," such as unusually prominent nucleoli, variation in size of cells, or prominent pleomorphism, were absent. The results thus suggest that increasing age is significantly associated with increased frequency of IHC BCL2 expression, including strong/diffuse expression.
Another interesting point was that 3 out of 4 (75%) strong BCL2 cases were seen at the extranodal parotid region.
| Discussion|| |
BL is the most aggressive B-cell non-Hodgkin's lymphoma characterized by a high mitotic index, high tumor cell apoptosis, c-MYC gene translocations on chromosome 8, and the expression of surface IgM, monotypic kappa, or lambda light chain, CD19, CD20, CD10, and BCL6.  The disease is broadly categorized into 3 types:  endemic, with a high incidence in Africa and strong association with the Epstein-Barr Virus (EBV); sporadic, which affects the population worldwide and occurs more often in children and adolescents than in adults, with about 30% EBV association; and immunodeficiency associated.  Although diagnosis of endemic BL using conventional methods is relatively simple due to a typical clinical presentation, distinguishing sporadic BL from DLBCL via conventional pathologic methods is not always straightforward. ,, In this scenario, BCL2 expression is often taken into account.  As per the WHO Blue Book, BCL2 expression is negative in classical BL, with a revision in recent WHO criteria, allowing weak BCL2 expression still compatible with BL diagnosis.  This distinction is of critical importance as the treatment regimen for BL comprises rigorous high-dose chemotherapy, including intrathecal administration. The treatment for DLBCL, however, is less intensive, using CHOP-which comprises cyclophosphamide, doxorubicin, vincristine, and prednisone-along with monoclonal CD20 antibody (rituximab) in most cases.  As BL progresses swiftly and can lead to death in a few days, a quick diagnosis is essential to begin treatment of BL patients as soon as possible. However, this also means that a misdiagnosis of DLBCL in a BL patient has its consequences, as the prescribed treatment for DLBCL may be ineffective in BL and vice versa. This is more pertinent in adults because most patients in the pediatric age group with a diagnosis of DLBCL are still treated with BL protocols.
It is of interest to note that these cases of overlap, like the disease itself, occur less often in children and young adults,  suggesting more conformity and hence ease of diagnosis in these patients. As BCL2 is used as a marker to differentiate between DLBCL and BL, the results of our investigations detract from its reliability as a diagnostic marker. Of the 39 confirmed BL cases, 23.1% of patients had positive BCL2 expression. Of these, the expression of BCL2 was weak or focal in 5 cases, whereas the remaining 4 demonstrated diffuse strong BCL2 positivity, giving a frequency of expression of strong BCL2 positivity of 9.8%. It may also be noted that of these 4 adult cases, in 3 the site of lymphoma being the parotid area. This finding is significant as it contradicts the current WHO criteria of negative or weak expression, that there may be many BL cases being misdiagnosed as DLBCL due to reliance on this as one of the criteria. Strong BCL2 expression contradicts the classical pathogenesis of BL itself, as BCL2 is an antiapoptotic gene, which could affect the high rate of apoptosis otherwise seen in BL. This may have implications on the treatment and management of the disease in such patients. However, it is noteworthy that the current WHO classification, being well aware of this caveat, has included a provisional category of "DLBCL with features intermediate between DLBCL and BL". 
In conclusion, this study shows that strong BCL2 expression is possible in up to 10% of cases of otherwise classic BL, particularly in adults where the expression may be at times strong and diffuse. Therefore, BCL2 expression is not necessarily useful to make this distinction, and, as such, calls for further research into the matter and revision of the current WHO diagnostic criteria.
| Acknowledgment|| |
We thank Dr. Tariq Moatter for his help in FISH Testing and Ms. Shamsha Punjwani for her secretarial help.
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Department of Pathology and Microbiology, Aga Khan University Hospital, Stadium Road P.O. Box 3500, Karachi 74800
[Figure 1], [Figure 2]
[Table 1], [Table 2]