| Abstract|| |
Background: Chronic lymphoproliferative disorders (CLPDs) are heterogeneous group of disorders with variable clinical presentations and outcomes. Therefore, accurate classification is crucial for treatment planning. At present, flow cytometry immunophenotyping (FCM-IPT) is a useful tool for diagnosing these diseases. However, overlapping immunophenotypes do exist. Recently, differential expression of CD200 and variation in number of CD20 antibody bound per cell (ABC) in different CLPDs has been reported. Materials and Methods: Seventy-seven CLPD cases were analyzed by FCM-IPT for CD200 expression, and Quantibrite bead was used to calculate CD20 ABC. Results: Variability in CD200 expression can help in the differentiation of chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL) from other CLPDs. CD200 was brightly expressed in 100% CLL cases, having homogenous bright (2+) intensity. On the contrary, CD200 was uniformly negative in all Mantle cell lymphoma cases except 1, in which the intensity was dim, and the mean fluorescence intensity was significantly lower than CLL. Furthermore, all HCL cases showed bright expression of CD200, thereby making it useful in differentiation from other CLPD with villous lymphocytes. Evaluation of CD20 ABC showed that it differs among various CLPD and was significantly lowest in CLL and highest in HCL both on peripheral blood and bone marrow samples. Conclusion: Our results support the fact that CD200 can be added to routine CLPD panel as it is useful in subcategorizing them. However, inclusion of CD20 ABC to routine panel does not seem plausible but may be done for difficult diagnostic cases or where anti-CD20 therapy is planned.
Keywords: CD20 antibody binding capacity, CD200, chronic lymphoproliferative disorders
|How to cite this article:|
Poongodi R, Varma N, Naseem S, Parveen B, Varma S. Utility of CD200 expression and CD20 antibody binding capacity in differentiating chronic lymphocytic leukemia from other chronic lymphoproliferative disorders. Indian J Pathol Microbiol 2018;61:50-7
|How to cite this URL:|
Poongodi R, Varma N, Naseem S, Parveen B, Varma S. Utility of CD200 expression and CD20 antibody binding capacity in differentiating chronic lymphocytic leukemia from other chronic lymphoproliferative disorders. Indian J Pathol Microbiol [serial online] 2018 [cited 2021 Oct 28];61:50-7. Available from: https://www.ijpmonline.org/text.asp?2018/61/1/50/228166
| Introduction|| |
B-lineage chronic lymphoproliferative disorders (CLPDs) represent clonal proliferations of mature B lymphocytes. The clinical presentations and natural histories of CLPDs are extremely heterogeneous and their outcome differs widely in terms of survival. Different therapeutic approaches are needed in different forms of disorders therefore categorizing various disease entities correctly is essential.
Characterization of CLPDs by immunophenotyping (IPT) has become an important and widely used method in hematology. Matutes et al. proposed a scoring system for diagnosing chronic lymphocytic leukemia (CLL) which is the most common type of B-CLPD and its based on the evaluation of 5 markers CD5, CD23, FMC-7, CD22/79b, and surface immunoglobulin (sIg). Scores in CLL usually range from 3 to 5 and for non-CLL cases from 0 to 2. Therefore, diagnosing CLL is easy when it exhibits the characteristic immunophenotype (CD5+, CD23+, FMC-7-, CD22/79b-, dim sIg). However, problem arises when dealing with noncharacteristic immunophenotypes with Matutes et al. score ≤3. The atypical immunophenotypes commonly encountered are cases of CLL lacking CD5 or CD23 expression or showing strong positivity for sIg. In a similar way, difficulty arises in differentiating between CLL and cases of Mantle cell lymphoma (MCL) when MCL cases show aberrant expression of CD23 and expression of CD5 by cases other than CLL and MCL. Hence, clinicians still require information from other techniques such as immunohistochemistry (IHC) for cyclin D1 positivity and/or karyotype or fluorescence in situ hybridization analysis for the diagnosis of MCL and excluding CLL. However, the genetic techniques are costly and are not available in all the centers. On the other hand, flowcytometry (FCM) analysis is already being done for diagnosis, and testing for additional markers will not only save time and be economical but has added advantage of using the same diagnostic sample.
The previous studies have reported CD200 to have a differential expression in CLPD, with a positive expression in CLL and hairy cell leukemia (HCL) a negative expression in MCL, thereby aiding in the differentiation of CLL from MCL.,, CD200 was initially described as the OX-2 tumor antigen. It is a type I membrane-associated glycoprotein and is a member of the immunoglobulin superfamily. It is expressed on a variety of cell types, including myeloid cells, endothelium, ovarian cells, placental trophoblasts, and neurons. Besides, CLPD and CD200 expression has been reported in other hematological malignancies, including multiple myeloma, acute myeloid leukemias, and acute lymphoblastic lymphoma/leukemia.,,
In this study, we, thus, planned to evaluate the expression of CD200 in various CLPDs and the utility of its expression in differentiating them.
In addition, we also planned to do CD20 quantitation in all cases of CLPDs and evaluate its expression levels and its utility in the differentiation of different CLPDs. Quantitation of CD20 indicates the number of CD20 molecules per B-lymphoid cell. Studies have shown that it varies in different CLPDs, being lowest in CLL, higher in follicular lymphoma (FL), MCL, splenic marginal zone lymphoma (SMZL), and diffuse large B-cell lymphoma (DLBCL) and highest in HCL.
In this study, we have evaluated whether CD200 expression and quantitation of CD20 antibody bound per cell (ABC) by FCM can help in categorizing various CLPDs, and in particular, differentiating CLL from other CLPDs. To the best of our knowledge, such a study has not been undertaken in a developing country like India before.
| Materials and Methods|| |
Cases were diagnosed according to 2008 World Health Organization classification based on clinical, morphological, immunophenotypic data. Matutes et al. score was calculated in all cases.
IPT was performed on peripheral blood (PB) and/or bone marrow (BM) samples, using combination of fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), peridinin-chlorophyll-protein complex (Per-CP), APC-H7, etc. tagged monoclonal antibodies (MoAb) for the characterization of cases. The routine panel in our department of hematology for the diagnosis of CLPDs on PB or BM includes MoAb for CD19, CD5, CD23, CD10, CD22, CD20, FMC-7, CD79b, kappa, lambda, CD11c, CD25, CD103, CD123, CD38, CD3, CD4, and CD8. In addition, CD200 was tested in all cases. Staining was done using lyse-wash technique.
At least 10,000 events were acquired. Data acquisition was performed on the FCM (FACS Canto II, BD Biosciences, San Jose). A particular CD marker was taken as positive if it was expressed in >20% cells. CD200 expression was evaluated semi-quantitatively by comparison with the negative control tube (unstained cells) and designated as 1+, if there was <1 log shift in mean fluorescence intensity (MFI) compared with negative control; 2+, if there was 1–2 log shift in MFI, and 3+, if there was >2 log shift in MFI.
Method for quantification of CD20
For CD20 ABC quantitation, 4 set of precalibrated Quantibrite PE beads and Quantibrite CD20 PE antibody (Becton Dickinson, San Jose) were used. Quantibrite CD20 PE antibody is produced to have 1 fluorochrome per antibody.
Method of staining for CD20 antibody bound per cell
For each sample, 3 tubes were prepared, tube 1 containing only CD45 APC, tube 2 containing CD45 APC and CD19 FITC, and tube 3 containing CD45 APC, CD19 FITC, and CD20 Quantibrite PE, for sequential gating. Before the acquisition of sample for ABC, first 7-color bead cytometer setup tube was run in BD FACS CANTO software followed by Quantibrite, and then the samples tubes in FACS DIVA software. Quantibrite beads, which are a set of 4 precalibrated beads, were run first for standardization before the acquisition of patients/control samples.
Calculation of phycoerythrin molecules per cell of the patients and controls
On a statistics Spreadsheet, the geometric means from the histogram statistics view for the four beads were entered. Then, the lot specific PE molecules per bead, provided in the Quantibrite PE kit box were entered. Log10 for the Quantibrite CD20 PE geometric mean and the PE molecules per bead was calculated. Then, a linear regression of log10 PE molecules per bead was plotted against log10 fluorescence, using the equation y = mx + c (where y equals log10 fluorescence and x equals log10 PE molecules per bead). The ABC for the unknown sample population was determined by substituting log Quantibrite CD20 PE geometric mean of the sample population in the equation. Hence, log-ABC was obtained in this way. ABC was determined by taking anti-log of log-ABC.
Data collected as per pro forma was analyzed with the appropriate statistical tests carried out using Statistical Package for the Social Sciences version 22. For normally distributed data, means of two groups were compared using student t-test. Comparison between >2 groups was performed using ANOVA test. For skewed data, Mann–Whitney test was applied for two groups. For >2 groups Kruskal–Wallis test was applied. Qualitative or categorical variables were described as frequencies and proportions. Proportions were compared using Chi-square or Fisher's exact test. Correlation and association were performed using the Spearman's correlation test. The P < 0.05 was considered to be statistically significant.
| Results|| |
A total of 77 CLPD cases were analyzed by FCM. Out of the 77 CLPD cases, CLL accounted for the majority of cases, comprising of 54 (70.1%) cases. Other CLPD cases in our study included 6 (7.8%) cases of MCL; 5 of (6.5%) HCL; 3 (3.9%) each of DLBCL unclassified (UNCLA) B cell lymphomas; 2 (2.6%) each of FL, SMZL; and 1 each of small lymphocytic lymphoma (SLL) and (Lymphoplasmacytic lymphoma [LPL]).
Flow cytometric analysis of CD200
In this study, CD200 expression was seen in all 54 cases of CLL (expression on 61.6%–99.7% of CD19-positive cells, mean 94%). The expression pattern was of homogenous bright (2+) intensity in the majority of cases and was seen in 38 (70.4%) cases. Conversely, CD200 was uniformly negative in all cases of MCL except 1 case, which showed positive CD200 expression in 48.1% of CD19-positive cells with 1+ intensity. Thus, in contrast to CLL, the only MCL which expressed CD200 had a dim intensity and lower percentage of positive cells. The mean MFI of the CLL cases was 1296 (range 286–4048), and it was significantly higher than CD200 positive MCL case, where the MFI was 472, and this was statistically significant (P = <0.001).
All cases of HCL in our study were positive for CD200 and showed the highest MFI (mean 3845.2; range 629–13258). Expression of CD200 was also noted in 2 (100%) cases of SMZL; however, the mean MFI and percentage of CD200 positive cells were lower than HCL, but this was not statistically significant (P = 0.25). Of the 2 FL cases, 1 (50%) case showed positive CD200 expression on 82.6% of CD19-positive cells with 1+ intensity and an MFI of 837. CD200 was negative in 2 (66.6%) cases of DLBCL, and the only positive case showed dim 1+ intensity in 79.5% of CD19-positive cells with an MFI of 436. On comparing CD200 MFI with intensity, we found a significant positive correlation (P< 0.001). Hence, increase in MFI showed a proportionate increase in intensity.
The details of CD200 expression in various CLPD is outlined in [Table 1]. Few flow cytometric dot plots of representative cases are shown in [Figure 1].
|Figure 1: Flow cytometric dot plots of (a) a case of chronic lymphocytic leukemia with CD200 positivity; (b) a case of atypical chronic lymphocytic leukemia with negative CD23 showing CD200 positivity; (c) a case of mantle cell lymphoma with negative CD200; (d) a case of atypical mantle cell lymphoma with CD23 positivity and CD200 is negative; (e) a case of hairy cell leukemia with CD200 positivity; and (f) a case of splenic marginal zone lymphoma with CD200 positivity|
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CD20 expression in chronic lymphoproliferative disorders cases
CD20 expression was seen in all the CLPD cases except in 1 case of CLL.
Comparison of CD20 antibody bound per cell of peripheral blood lymphoid cells of chronic lymphoproliferative disorders cases
CD20 ABC of PB and BM lymphoid cells was analyzed all the cases. Highest CD20 ABC was noted in LPL with a mean of 150660 followed by HCL with a mean of 95603.13 (range 22438.8–179060.6); higher in DLBCL (mean 66867.22; range 13129.6–145881), UNCLA lymphomas (mean 61806.56; range 46238.1–88715.6), SMZL (mean 53912.11; range 30199.5–77624.7), MCL (mean 44010.02; range 14092.9–72110.7); lower in SLL (20892.96), FL (mean 20896.83; range 14060.5–27733.2); and it was found to be lowest in CLL with a mean of 16567.83 (range 986.2–103514.2).
The highest mean CD20 ABC of HCL in comparison to the lowest mean CD20 ABC of CLL was statistically significant (P< 0.001). However, we did not find any statistically significant differences among the CD20 ABC of LPL, MCL, DLBCL, SMZL, SLL, and B-cell lymphomas UNCLA (P > 0.05).
CD20 ABC of lymphoid cells was also performed in 36 normal controls. Both PB and BM samples were analyzed for CD20 ABC in 9 controls and the remaining 27 control we performed CD20 ABC for PB lymphoid cells only. A higher CD20 ABC compared to normal controls was seen in HCL and LPL cases only. CLL and other remaining CLPD cases showed CD20 ABC lower than normal controls. Statistical analysis showed that the higher CD20 ABC of normal B lymphocytes in comparison to CLL lymphocytes was statistically significant (P = 0.001).
CD20 antibody bound per cell of bone marrow lymphoid cells of chronic lymphoproliferative disorders cases
The mean CD20 ABC of BM lymphoid cells in CLPD cases in our study was 41351.08 (range 2630.9–225943.6), and it was higher when compared to mean CD20 ABC of PB which was 28496.14 (range 986.2–179060.6). CD20 ABC of BM was highest in LPL (mean 168655.30), followed by HCL (mean 116913.53; range 30408.9–225943.6) and DLBCL (mean 92776.97; range 34197.9–151356); higher in MCL (mean 57400.10; range 29040.2–89330.5), FL (mean 52033.17; range 23713.7–80352.6), SMZL (mean 50958.08; range 23933.2–77983); lower in SLL (mean 9855.53); and CLL (mean 12463.08; range 2630.9–77983).
Similar to the findings of CD20 ABC of PB, the highest mean CD20 ABC of HCL in comparison to the lowest mean CD20 ABC of CLL was statistically significant (P< 0.001). We did not find any statistically significant differences between the CD20 ABC of LPL, MCL, DLBCL, SMZL, SLL, and UNCLA B cell lymphomas in BM samples (P > 0.05).
The CD20 ABC for BM lymphoid cells was studied in 9 controls, and the mean value was 48294.24 (range 13152.2–93756.2). It was found that the CD20 ABC values were lower than controls in CLL, SLL, and B-cell lymphomas UNCLA. In remaining CLPD cases, CD20 ABC was higher than controls. However, a statistically significant difference was found only between higher CD20 ABC of HCL compared to the controls (P = 0.020).
No statistically significant difference was observed in comparing CD20 ABC between PB and BM lymphoid cells for CLL and other CLPD cases.
The findings of CD 20 ABC of PB and BM are summarized in [Table 2] and [Table 3], respectively. Sequential gating for CD20 ABC with geometric mean is depicted in [Figure 2] and [Figure 3] for a case of HCL and CLL, respectively.
|Figure 2: Flow cytometric plots showing sequential gating for CD20 antibody bound per cell with geometric mean in a case of hairy cell leukemia|
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|Figure 3: Flow cytometric plots showing sequential gating for CD20 antibody bound per cell with geometric mean in a case of chronic lymphocytic leukemia|
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| Discussion|| |
In this study, we evaluated the utility of CD200 expression and CD20 quantification by FCM in categorizing various CLPDs and have addressed the question of whether CD200 expression and quantitation of CD20 can be used to differentiate various CLPDs and be used in the routine flow cytometry panel.
From the results of the present study, we have shown that CD200 can help in the differentiation of CLL and HCL from other CLPDs. CD200 expression was brightly expressed in 100% CLL cases, 61.6%–99.7% of cells were positive with a homogenous bright (2+) intensity in the majority of the cases. On the contrary, CD200 was uniformly negative in cases of MCL except 1 case which showed positive CD200 expression in 48.1% cells; however, the intensity was dim, and the MFI was significantly lower than CLL. Overall, the difference of CD200 expression between CLL and MCL was found to be statistically significant (P< 0.001). Hence, more number of CD200-positive cells with homogenous bright intensity is specific for CLL cases. A study done by El Desoukey et al., Palumbo et al., and Alapat et al. showed similar findings where 100% of CLL cases showed CD200 expression.,, McWhirter et al. have reported that B cells from all 87 CLL patients in their study exhibited 1.6–5.4 fold cell surface upregulation of CD200 relative to normal B cells.
All cases of HCL in our study brightly expressed CD200 (mean percentage of positive cells, 73.16; range, 46.7–98.4) and had the highest MFI (mean, 3845.2; range, 629–13258) compared to other CLPDs. Furthermore, Brunetti et al. and El Desoukey et al. reported bright CD200 positivity on all hairy cells from all HCL patients studied, with a large amount of this antigen on neoplastic cells. Dorfman and Shahsafaei confirmed CD200 expression in HCL by IHC analysis.
Our results showed positive but dim expression of CD200 in both the cases of SMZL, 1 case each of FL and DLBCL. A recent study done by Sandes et al. also found a similar expression on SMZL but negative expression on FL and DLBCL cases. However, few earlier reports have demonstrated negative expression in SMZL cases., Although different results obtained in different studies with regards to SMZL, the study conducted by Sandes et al. had more number of SMZL cases and the CD200 positivity occurring in SMZL appears to be significant; hence, more cases are needed to assess the pattern of CD200 positivity in these lymphomas. CD200 was negative or dimly expressed, in CD10 negative CLPDs and being not useful to distinguish FL from DLBCL and also not useful for the diagnosis of the CD5-negative/CD10-negative neoplasms. These results were comparable to the data reported by the Euroflow Consortium that demonstrated a varied pattern of CD200 expression in cases of CD10 negative DLBCL and MZL.
CD20 antibody bound per cell
We found that CD20 ABC of the lymphoid cells differs among various subtypes and it was significantly lowest in CLL and highest in HCL both on PB and BM samples (P< 0.001). Ginaldi et al. were the first to report CD20 ABC to be significantly lower in CLL and highest in HCL. However, no significant statistical difference was observed between the CD20 ABC of B-cell PLL, MZL, and SMZL by them. Similar finding was also reported by Huh et al., Olejniczak et al., and Prevodnik et al. who compared the CD20 expressions in CLL, HCL, DLBCL, FL, SMZL, and MCL.,, These studies have shown CD20 ABC in CLL cells to be significantly lower than in other B-cell lymphomas as demonstrated in our study as well. No significant statistical differences seen among other CLPD groups as shown in the previous studies. We also observed that the mean CD20 ABC of BM lymphoid cells of MCL (57.4 × 103), HCL (116.9 × 103), DLBCL (92.7 × 103), SMZL (50.9 × 103), LPL (168.6 × 103), and FL (52 × 103) of our cases were higher than controls (48.29 × 103) with the exception of CLL (12.4 × 103), SLL (9.85 × 103), and UNCLA B-cell lymphomas (35.7 × 103). However, we found a statistically significant difference only between higher mean CD20 ABC of HCL compared to the controls (P = 0.020). Whereas, in PB, a lower mean CD20 ABC of lymphoid cells was found in CLPD cases with the exception of HCL (95 × 103) and LPL (150.6 × 103) compared to normal B (76 × 103) lymphocytes in controls; however, the difference was not statistically significant (P = 1). The lower mean CD20 ABC of CLL in comparison to normal B lymphocytes was statistically significant (P = 0.001).
Ginaldi et al. also showed CD20 ABC of CLL to be significantly lower than control in PB samples which is in agreement with our study. However, their study had higher mean CD20 ABC on MCL, SMZL, and HCL compared to controls. However, in our study, we found similar results only in BM samples, whereas CD20 ABC of PB showed significantly higher value only in cases of HCL. However, these differences could be due to the small sample size of non-CLL cases in our study.
On comparing the PB and BM CD20 ABC values, in our study, no statistically significant differences were found among MCL, SMZL, and FL and as reported in the previous study by Huh et al. However, with regards to CLL they have reported a significantly higher number of CD20 molecules per cell in PB than in BM samples in CLL cases. Although our result regarding difference of CD20 ABC of PB and BM was not statistically significant, but a higher mean CD20 ABC was seen in PB compared to BM.
The finding of highest CD20 ABC of HCL in our study was consistent with the results of previous studies.,,, Although, the previous studies and our study on CD20 quantitation have shown that highest CD20 ABC is seen in HCL and lowest in CLL, the values of CD20 ABC have been variable in different studies. This may be due to the different methodologies and microbeads used in different studies.
In our study, we also found that the levels of CD20 are consistently lower in CLL compared to MCL cells. Even though this finding is not statistically significant, the consistently lower CD20 ABC of CLL compared to MCL could be confirmed by including more cases. Furthermore, because a similar finding has been previously reported too. This finding is important because CLL and MCL which are consistently positive for CD5 at times can show atypical immunophenotypic features such as CD23-positive MCL cases  and CD23-negative CLL cases  which we also encountered in our study. In such cases higher CD20 ABC of lymphoid cells with a homogenous bright expression of CD20 in CD5-positive cells would suggest the possibility of MCL rather than CLL. The finding of very high levels of CD20 ABC and MFI in HCL compared to cases of SMZL can be used in the differential diagnosis of these entities. It is because these diseases mimic each other and both have a similar clinical picture of splenomegaly without lymph node involvement and have circulating lymphoid cells with villous projections. This finding was in agreement with the study by Ginaldi et al. However, this needs to be validated on larger number of cases. The single case of LPL which we had in our study showed a high CD20 ABC in both PB, BM, and the levels are comparable with what we obtained in HCL. This was in contrast to the finding of Huh et al. who reported lower CD20 ABC in LPL compared with other CLPDs such as SMZL, MCL, and HCL. However, both the findings are not statistically significant, and the sample size was less in both the studies and larger studies have to be done on the entities to know the true mean values in these cases, because the diagnostic distinction between LPL and SMZL is at times difficult among the differentiated B-cell lymphoproliferative disorders.
| Conclusion|| |
Thus, in this study, we found that CD200 expression and CD20 quantitation aided in differentiating various CLPDs, especially CLL from MCL. All cases of CLL show CD200 positivity and low CD20ABC, compared to MCL where CD200 is predominantly negative, and CD20 ABC is high. In addition, among all CLPD, HCL had a distinct characteristic pattern for CD200 expression and CD20ABC, being brightest and highest for both.
However, more number of cases with all subtypes of CLPD needs to be studied to further substantiate our findings. Only a handful of studies are present, and to the best of our knowledge, this will be the first study from developing country, like India, to deal with the immunophenotypic features of CLPDs using CD200 and CD20 ABC together, and also comparing CD20 ABC on PB and BM samples simultaneously.
Clinical practice points
From our results, we would suggest that CD200 can be added to the routine CLPD panel.
Although, CD20ABC is useful in differentiating CLPD, presently adding it to the routine panel does not seem practical; however, it may be done in difficult diagnostic cases where anti-CD20 therapy is planned.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Department of Hematology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh - 160 012
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]