Abstract
To evaluate the utility of CD43 and CD200 in differentiating chronic lymphocytic leukemia (CLL) from other mature B-cell neoplasms. This was a cross-sectional study on patients diagnosed with B-cell neoplasms on flowcytometry. The median fluorescence intensity (MFI) of CD43, CD200 expressing neoplastic B-cells were compared between the CLL and non-CLL B-cell neoplasms followed by receiver operating characreristic curve (ROC) analysis. In addition, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CD43 and CD200 in diagnosing CLL were analysed. A total of 137 patients were included. The CLL group consisted 87 patients and non-CLL group consisted 50 patients. The Mann–Whitney U test showed significant CD43 expression (U = 997.5, Z= − 5.265, p < 0.001) and CD200 expression (U = 932.0, Z = − 5.5, p < 0.01) in CLL patients compared to non-CLL patients. The area under the curve were 0.771 and 0.786 for MFI of CD43 and CD200 in differentiating CLL from non-CLL group respectively. The optimal cut-off of MFI for CD43 and CD200 were 1323 and 1775 respectively. The sensitivity, specificity, PPV and NPV of CD43 in diagnosing CLL cases were 97.7%, 66%, 83.3% and 94.2% respectively. The sensitivity, specificity, PPV and NPV of CD200 in diagnosing CLL cases were 100%, 32%, 71.9% and 100% respectively. CD43 and CD200 are useful markers in differentiating CLL from other mature B-cell neoplasms with higher MFI expression of both markers found in CLL.
Keywords: CD43, CD200, CLL, Flowcytometry, Mature B-cell neoplasms
Introduction
Mature B-cell neoplasms are a group of non- Hodgkin lymphomas where there is sustained accumulation of monoclonal B-cells. As per the upcoming 5th edition of the WHO classification of lymphoid neoplasms the mature B-cell neoplasms group consists of 12 entities that includes (1) Pre-neoplastic and small lymphocytic proliferations (monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia/ small lymphocytic lymphoma (CLL/SLL)), (2) Splenic B-cell lymphomas and leukemias, (3) Lymphoplasmacytic lymphoma (LPL), (4) Marginal zone lymphoma (MZL), (5) Follicular lymphoma (FL), (6) Cutaneous follicular centre lymphoma, (7) Mantle cell lymphoma (MCL), (8) Transformations of indolent B-cell lymphomas, (9) Large B-cell lymphomas, (10) Burkitt lymphoma (11) KSHV/HHV-8 associated B-cell lymphoid proliferations and lymphomas, (12) Lymphoid proliferations and lymphomas associated with immune deficiency and dysregulation [1].
The diagnosis of CLL relies on morphology and immunophenotype. Morphologically, the cells are small mature appearing lymphoid cells with ‘soccer ball’ like chromatin and scanty cytoplasm. Immunophenotyping, which is done with flowcytometry, characteristically co-express CD5 and CD23 in addition to B-cell antigens like CD19, CD20. The intensity of CD20, CD79b, surface immunoglobulins are characteristically low when compared with normal B-cells [2]. However, there are certain shortcomings with usage of limited panel of markers in diagnosing mature B-cell neoplasm. This is because there is overlap with certain markers between the entities, e.g. CD5 positivity in MCL and also not all cases of CLL express this typical immunophenotypic profile. Atypical CLL patients may express bright surface immunoglobulin/ CD23 negativity or FMC7 positivity and can be mistakenly diagnosed as MCL which may have different prognostic and therapeutic implication than CLL [3]. This dilemma can be solved by performing cytogenetics/ fluorescence in-situ hybridization (FISH) for t(11;14) which can be detected in 97% MCL cases [4]. However, FISH is a costly investigation and may not be performed for all patients.
Hence there was an interest in development of markers which can differentiate CLL from other mature B-cell neoplasms- CD43, CD200 were one among them. CD200 was useful in differentiating CLL from other mature lymphoid neoplasms and also was useful in diagnosing atypical CLL [5–7]. CD200 is bright positive in CLL and HCL but are negative in MCL and dim positive in other splenic lymphomas [7]. CD43 is usually expressed in CLL but not in follicular lymphoma [8]. Hence, the combination of CD43 and CD200 can help in diagnosis of CLL in borderline cases. This is also reflected in the harmonization project for deciding marker panel for diagnosis of CLL- CD19, CD5, CD20, CD23, kappa and lambda were considered “required” markers and CD43, CD79b, CD81, CD200, CD10, ROR1 were considered as “recommended” markers to refine diagnosis in borderline cases [9]. Fewer studies have been done to evaluate the utility of CD43 and CD200 in distinguishing CLL from other mature B-cell neoplasms [8, 10, 11]. The aim of this study is to evaluate the utility of CD43 and CD200 expression in differentiating CLL from other mature B-cell lymphomas.
Materials and Methods
The study was done in Department of Hematology, of a tertiary care centre in North India after obtaining approval from the ethics committee (IECPG-609/28.10.2021) for waiver of consent since it was an observational study and does not affect the treatment or disease outcome. It was a cross-sectional study from August 2020 to December 2022. Consecutive treatment naïve patients who visited outpatient department with suspicion of lymphoproliferative disorders and were diagnosed with B-cell neoplasms on flowcytometry were included in the study.
Peripheral blood was collected up to the recommended volume as per the ethylenediamine tetraacetic acid (EDTA) vial used for flowcytometry analysis. The sample is initially screened using the lymphoid screening tube recommended by Euroflow consortium. This 8-colour 12-marker tube assesses the B-cell clonality along with CD5, CD38 expression on B-cells and in addition determines the lymphocyte subset percentages. The BD OneFlow™ LST (Lymphoid Screening Tube) was used for the same. The antibody panel consisted of CD45 V500-C (2D1), CD19 PE-Cy7 (SJ25-C1), CD20 V450 (L27), Anti-Lambda FITC (1-155-2), Anti-Kappa PE (TB28-2), CD38 APC-H7 (HB7), CD3 APC (SK7), CD4 V450 (SK3), CD8 FITC (SK1), CD5 PerCP-Cy5.5 (L17F12), Anti-TCR-1 PE-Cy7 (11F2), CD56 PE (MY31). The sample was processed on the same day by stain-lyse-wash technique.
Antibody panel and gating strategy: In addition to the LST tube, another tube consisting CD23 FITC (EBVCS-5), CD200 PE (MRC OX-104), CD79b PERCP-Cy5.5 (CB3-1), CD19 PE-Cy7 (SJ25C1), CD10 APC (HI10a), CD43 APC-H7 (1G10), CD20 V450 (L27), CD45 V500-C (2D1) were used for all cases. All antibodies were sourced from BD Biosciences, San Jose, California. Peripheral blood was processed as per stain-lyse-wash protocol and at least one lakh events were acquired on BD FACS Canto II™ (BD Biosciences, San Jose, California). CS&T™ beads and OneFlow™ Setup beads were run daily. Compensation was done monthly using the OneFlow™ Setup beads and OneFlow™ FC beads.
The median fluorescence intensity (MFI) of CD43, CD200 expressing neoplastic B-cells were observed and compared between the CLL and non-CLL B-cell neoplasms. The demographic details of the patients were noted from the request forms.
Statistical analysis was performed using SPSS software version 20 (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.). The MFI of CLL and non-CLL patients were compared using Mann–Whitney U test and ROC curve analysis was done to evaluate the cut off for MFI in differentiating CLL from non-CLL cases. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CD43 and CD200 in diagnosing CLL were analysed.
Results
A total of 137 patients were included in the study which included 82 male patients and 55 female patients (Male:Female = 1.4:1). Age of the patients ranged from 24 years to 86 years with mean of 59.2 years and standard deviation of 11.8. The diagnosis of 137 patients is described in Table 1.
Table 1.
Diagnosis of the 137 mature B-cell neoplasms included in the study
| Diagnosis | Number of cases (%) |
|---|---|
| MBL | 5 (3.6) |
| CLL | 80 (58.4) |
| Atypical CLL, FL, progression in CLL | 2 each (1.5) |
| CD5- CD10- lymphomas | 30 (21.9) |
| HCL, MCL | 7 each (5.1) |
| WM, DLBCL | 1 each (0.7) |
| Total | 137 |
MBL, Monoclonal B-cell lymphocytosis; CLL, Chronic lymphocytic leukemia; FL, Follicular lymphoma; HCL, Hairy cell leukemia; MCL, Mantle cell lymphoma; WM, Waldenstörm macroglobulinemia; DLBCL, Diffuse large B-cell lymphoma
The diagnosis was grouped as CLL immunophenotype (n − 87) which included the 5 cases of MBL, 2 atypical CLL in addition to CLL cases (Fig. 1) and non-CLL immunophenotype (n − 50) that included all other cases. The MFI of CD43 and CD200 for each case of CLL and non-CLL immunophenotype group was recorded and one-sample Kolmogorov-Smirnov test was performed and found that the data distribution was not normal. Hence, Mann–Whitney U test was performed to compare the MFI of CD43 and CD200 between CLL and non-CLL immunophenotype group. The Mann–Whitney test showed U = 997.50, Z= − 5.26, p < 0.0001 for MFI of CD43 and U = 932.0, Z = − 5.55, p < 0.0001 for MFI of CD200 in differentiating CLL from non-CLL cases.
Fig. 1.
CD43 and CD200 expression among other markers in a patient of CLL
The median CD43 and CD200 expression is statistically significant between CLL and non-CLL immunophenotype groups (p < 0.001). Following the significant expression of CD43 and CD200 in CLL group of patients, receiver operating characteristic curve (ROC) was generated to derive a cut off value of MFI to differentiate CLL from non-CLL group. The area under the curve for the MFI of CD43 and CD200 were 0.771 and 0.786 respectively. The optimal cut-off of MFI for CD43 from our study was found to be 1323 and for CD200 the MFI was 1775.
Finally, the sensitivity, specificity, PPV and NPV of CD43 and CD200 in differentiating CLL from non-CLL cases were performed. The details are described in Table 2. The sensitivity of CD43 in diagnosing CLL cases was 97.7% with specificity of 66%. The PPV was 83.3% and NPV was 94.2%. The sensitivity of CD200 in diagnosing CLL cases was 100% with specificity of 32%. The PPV was 71.9% and NPV was 100%. In addition, the sensitivity and specificity of CD43 and CD200 co-expression was also performed. The details are described in Table 3. The sensitivity of co-expression of CD43 and CD200 in diagnosing CLL was 97.7% with specificity of 74%. The PPV was 86.7% and NPV was 94.8%.
Table 2.
CD43 and CD200 expression in CLL and non-CLL cases
| CLL cases | Non-CLL cases | Total | |
|---|---|---|---|
| CD43 positive | 85 | 17 | 102 |
| CD43 negative | 2 | 33 | 35 |
| Total | 87 | 50 | 137 |
| CD200 positive | 87 | 34 | 121 |
| CD200 negative | 0 | 16 | 16 |
| Total | 87 | 50 | 137 |
Table 3.
CD43 and CD200 co-expression in CLL and non-CLL cases
| CLL cases | Non-CLL cases | Total | |
|---|---|---|---|
| CD200 and CD43 positive | 85 | 13 | 98 |
| CD200 and/ or CD43 negative | 2 | 37 | 39 |
| Total | 87 | 50 |
Discussion
In this study, we evaluated the utility of CD43, CD200 in differentiating CLL from non-CLL cases. A total of 137 patients of mature B-cell neoplasms were studied. They were divided into CLL (n-87) and non-CLL (n-50) cases. The mean age at diagnosis was 59.15 with standard deviation (SD) of 11.8. The diagnosis of CLL requires morphology and immunophenotyping. Typical cases of CLL show mature appearing lymphoid cell with clumped soccer ball like chromatin and scanty cytoplasm. The clonal leukemic B-cells co-express CD5, CD23 with dim expression of CD20, CD79b, CD22, SIgM/sIgD and lacks CD10 and FMC7 expression. However the marker profile of CLL may show atypical features such as negative CD5/ CD23 and bright SIg/CD79b or positive FMC7 [12]. Therefore, relying on limited panel of markers may result in misdiagnosis of atypical CLL as MCL or other lymphomas. In our study out of the 87 cases of CLL, 1 (1.1%) was negative for CD5 and 14 (16.1%) were either dim/ heterogenous positive compared to 72(82.7%) bright CD5 positive cases. 9 cases (10.3%) were negative for CD23, 10 cases (11.5%) were either dim/ heterogenous positive and 68 cases (78.2%) were bright positive for CD23. Among the non-CLL cases, CD5 and CD23 were negative in all HCL patients. In MCL patients, CD5 was bright positive in 6/7 (85.7%) cases and dim positive in 1/7 (14.3%) case and CD23 was uniformly negative in all 7 (100%) cases. In a study by Ho et al., 28 cases were diagnosed as MCL on flowcytometry but 32% (9/28) cases lacked t(11;14) in FISH analysis and were harbouring cytogenetic abnormalities commonly found in CLL [3]. This shows the overlapping features of atypical CLL with MCL and the requirement of FISH for final diagnosis. However, not all patients may be subjected to FISH studies. This need for new markers were addressed in the European Research Initiative on CLL (ERIC) and European Society for Clinical Cell Analysis (ESCCA) harmonization project wherein the “required” markers for diagnosis of CLL includes CD19, CD5, CD20, CD23, kappa and lambda. The “recommend” markers include CD43, CD79b, CD81, CD200, CD10, ROR1 [9]. The utility of CD43 and CD200 in diagnosis of CLL are discussed in various studies [8, 10, 11].
The primary aim of the study was to identify whether the MFI values of CD43 and CD200 between CLL and non-CLL cases were significantly different. The Mann–Whitney U test showed significant CD43 expression (U = 997.5, Z= − 5.265, p < 0.001) and CD200 expression (U = 932.0, Z = − 5.5, p < 0.01) in CLL compared to non-CLL patients. In a study by Challagundla et al., CD200 expression was brighter in CLL than normal B-cells and MCL cases were usually dim or negative. All 119 cases of CLL were positive for CD200 and only 2 had MFI of less than 1000 [13]. Next, we tried to determine the optimal cut-off for the MFI of CD43 and CD200 in differentiating CLL and non-CLL using curve analysis. The area under the curve for CD43, CD200 were 0.771 and 0.786 respectively and the optimal MFI cut-off was found to be 1323 and 1775 respectively. To our knowledge, no such studies evaluating the MFI cut off for CD43 and CD200 in differentiating CLL and non-CLL cases are published in peer reviewed journals.
Finally, the sensitivity and specificity, PPV and NPV of CD43 and CD200 for diagnosing CLL were evaluated. Although CD43 and CD200 are not used for diagnosis of CLL as standalone markers, this might be useful in borderline cases to predict the diagnosis. CD43 was positive in 85/87 cases of CLL (97.7%) and 17/50 non-CLL cases (34%). The sensitivity was 97.7% and specificity was 66%. The PPV and NPV were 83.3% and 94.2% respectively. CD200 was positive in all 87 cases of CLL (100%) and 34/50(68%) of non-CLL cases. The sensitivity was 100% and specificity was 32%. The low specificity is probably due to the HCL and CD5, CD10 negative splenic lymphoma cases. The PPV and NPV were 71.9% and 100% respectively. A summary of the studies done on CD43, CD200 expression in B-cell neoplasms is given in Table 4. In a study by Subramanian et al., the sensitivity and specificity for co-expression of CD43 and CD200 were analysed and was present in all 28 CLL cases and were absent in none of the CLL cases. Among the non-CLL cases excepting HCL and MCL, 1/32 showed co-expression of CD43 and CD200 and were absent in 31/32 cases. The sensitivity and specificity were 100% and 96.88% with PPV, NPV of 96.55% and 100% respectively [10]. In our study, the sensitivity of co-expression of CD43 and CD200 in diagnosing CLL was 97.7% with specificity of 74%. The PPV was 86.7% and NPV was 94.8%. Among the HCL cases, CD43 was negative in 5/7 (71.4%) and dim/ heterogenous in 2/7 (28.6%) whereas CD200 was uniformly positive in all 7 patients of HCL (100%). In MCL cases, CD43 was negative in 3/7 (42.9%), dim and bright positive in 2/7 each (28.6%), CD200 was negative in 6/7 cases (85.7%) and positive in 1/7 cases (14.3%). The MCL case with CD200 positivity was diagnosed because of CD5 positivity and CD23 negativity. Whether the case will fit into atypical CLL rather than MCL needs to be evaluated further since CD200 is mostly negative in MCL cases except for few cases of leukemic non-nodal MCL [13–15].
Table 4.
Summary of studies on CD43 and CD200 expression in B-cell neoplasms
| Study (year) | Result |
|---|---|
| Subramanian [10] | The sensitivity, specificity, PPV, NPV of Co-expression of CD43 and CD200 in diagnosing CLL were 100%, 96.88%, 96.55% and 100% respectively |
| Falay [11] | CD43 expression was found in 95.7% patients with atypical CLL and 98.3% of typical CLL. CD200 was moderate to strong positive in 95.8% of CLL patients |
| Hoffmann et al. [8] | Sensitivity of CD43 and CD200 expression was 100% and specificity was 94.7% |
| Present study | Sensitivity, specificity, PPV and NPV of co-expression of CD43 and CD200 in diagnosing CLL were 97.7%, 74%, 86.7% and 94.8% respectively |
PPV, Positive predictive value; NPV,Negative predictive value; CLL, Chronic lymphocytic leukemia
The limitations in our study are lack of FISH/ cytogenetics in borderline cases to correlate with the expression of CD43 and CD200.
Conclusion
CD43 and CD200 are useful markers in differentiating CLL from other mature B-cell neoplasms with higher MFI expression of both markers found in CLL. The sensitivity, specificity for CD43 in diagnosing CLL was found to be 97.7%, 66% compared to sensitivity, specificity of 100%, 32% for CD200 respectively. The sensitivity of co-expression of CD43 and CD200 in diagnosing CLL was 97.7% with specificity of 74%.
Funding
The study was not funded by any organization.
Declarations
Conflict of interest
No conflict of interest.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Alaggio R, Amador C, Anagnostopoulos I, Attygalle AD, Araujo IB, de Berti O. The 5th edition of the World Health Organization classification of haematolymphoid tumours: lymphoid neoplasms. Leukemia. 2022;36:1720–1748. doi: 10.1038/s41375-022-01620-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, et al. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood. 2018;131:2745–2760. doi: 10.1182/blood-2017-09-806398. [DOI] [PubMed] [Google Scholar]
- 3.Ho AK, Hill S, Preobrazhensky SN, Miller ME, Chen Z, Bahler DW. Small B-cell neoplasms with typical Mantle cell lymphoma immunophenotypes often include chronic lymphocytic leukemias. Am J Clin Pathol. 2009;131:27–32. doi: 10.1309/AJCPPAG4VR4IPGHZ. [DOI] [PubMed] [Google Scholar]
- 4.Remstein ED, Kurtin PJ, Buño I, Bailey RJ, Proffitt J, Wyatt WA, et al. Diagnostic utility of fluorescence in situ hybridization in mantle-cell Lymphoma. Br J Haematol. 2000;110:856–862. doi: 10.1046/j.1365-2141.2000.02303.x. [DOI] [PubMed] [Google Scholar]
- 5.Mora A, Bosch R, Cuellar C, Vicente EP, Blanco L, Martino R, et al. CD200 is a useful marker in the diagnosis of chronic lymphocytic Leukemia. Cytometry B Clin Cytom. 2019;96:143–148. doi: 10.1002/cyto.b.21722. [DOI] [PubMed] [Google Scholar]
- 0.Ting YS, Smith SBC, Brown DA, Dodds AJ, Fay KC, Ma DDF, et al. CD200 is a useful diagnostic marker for identifying atypical chronic lymphocytic Leukemia by flow cytometry. Int J Lab Hematol. 2018;40:533–539. doi: 10.1111/ijlh.12857. [DOI] [PubMed] [Google Scholar]
- 7.Sandes AF, Chauffaille M, de Oliveira L, Maekawa CRMC, Tamashiro Y, Takao N. CD200 has an important role in the differential diagnosis of mature B-cell neoplasms by multiparameter flow cytometry. Cytometry B Clin Cytom. 2014;86:98–105. doi: 10.1002/cytob.21128. [DOI] [PubMed] [Google Scholar]
- 8.Hoffmann J, Rother M, Kaiser U, Thrun MC, Wilhelm C, Gruen A, et al. Determination of CD43 and CD200 surface expression improves accuracy of B-cell lymphoma immunophenotyping. Cytometry B Clin Cytom. 2020;98:476–482. doi: 10.1002/cyto.b.21936. [DOI] [PubMed] [Google Scholar]
- 9.Rawstron AC, Kreuzer KA, Soosapilla A, Spacek M, Stehlikova O, Gambell P, et al. Reproducible diagnosis of chronic lymphocytic Leukemia by flow cytometry: an European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) harmonisation project. Cytometry B Clin Cytom. 2018;94:121–128. doi: 10.1002/cyto.b.21595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Subramanian PG, Gadage VS, Kumar A, Badrinath Y, Ghogale S, Shet T, et al. Diagnostic utility of CD200 and CD43 co-expression by flow cytometry in differentiating chronic lymphocytic Lymphoma from other of mature B cell non Hodgkin’s Lymphoma. Blood. 2010;116:4615. doi: 10.1182/blood.V116.21.4615.4615. [DOI] [Google Scholar]
- 11.Falay M, Afacan Öztürk B, Güneş K, Kalpakçı Y, Dağdaş S, Ceran F, et al. The role of CD200 and CD43 expression in Differential diagnosis between chronic lymphocytic Leukemia and mantle cell lymphoma. Turk J Hematol. 2018;35:94–98. doi: 10.4274/tjh.2017.0085. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Campo E, Harris NL. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: International Agency for Research on Cancer; 2017. p. 586. [Google Scholar]
- 13.Challagundla P, Medeiros LJ, Kanagal-Shamanna R, Miranda RN, Jorgensen JL. Differential expression of CD200 in B-cell neoplasms by flow cytometry can assist in diagnosis, subclassification, and bone marrow staging. Am J Clin Pathol. 2014;142:837–844. doi: 10.1309/AJCPBV9ELXC0ECVL. [DOI] [PubMed] [Google Scholar]
- 14.Palumbo GA, Parrinello N, Fargione G, Cardillo K, Chiarenza A, Berretta S, et al. CD200 expression may help in differential diagnosis between mantle cell Lymphoma and B-cell chronic lymphocytic Leukemia. Leuk Res. 2009;33:1212–1216. doi: 10.1016/j.leukres.2009.01.017. [DOI] [PubMed] [Google Scholar]
- 15.Hu Z, Sun Y, Schlette EJ, Tang G, Li S, Xu J, et al. CD200 expression in mantle cell lymphoma identifies a unique subgroup of patients with frequent IGHV mutations, absence of SOX11 expression, and an indolent clinical course. Mod Pathol. 2018;31:327–336. doi: 10.1038/modpathol.2017.135. [DOI] [PubMed] [Google Scholar]