Evaluation of CD160 and CD200 Expression as Differentiating Markers between Chronic Lymphocytic Leukemia and Other Mature B-Cell Neoplasms.

Background: The present work aimed to investigate the expression of CD160/ CD200 in CLL and other mature B-cell neoplasms (MBN) and their use as an additional diagnostic tool for differentiating CLL from other MBN. Materials and Methods: Using flow cytometry, we detected the expression of CD160 &CD200 on B-cells from 30 CLL patients, 30 other MBN patients in addition to 20 controls. CDs160/200 measurements were determined as a percentage expression (≥20% was considered positive) and as a ratio of the mean ﬂuorescence intensities (MFIR) of leukemic cells/controls and were considered positive when the ratios were ≥2 and 20, respectively. Results: 90% and 100% of the CLL group expressed CDs160/200 in comparison to 60% and 63.3% of other MBN (p=0.007, p<0.001), respectively. By MFIR, 96.7% and 50% of our CLL group expressed CDs160/200 in comparison to 76.7% and 30% of other MBN, respectively. CDs160/ 200 were not expressed on the controls. Positive co-expression of CD160 and CD200 was found in 90% of the CLL cases, 60% of HCL patients and only in 40% of B-NHL. However, double negative expression of both markers was found only in 24% of the B-NHL patients. Conclusion : CD160 with CD200 can be used as additional diagnostic markers to the available routine panel to differentiate between B-CLL and other non-specified B-NHL patients.


INTRODUCTION
B-cell chronic lymphoproliferative disorders (B-CLPD) include a heterogeneous group of disease entities arising from clonal proliferation of mature B-lymphocytes 1 . B-CLPD is now most often diagnosed by flow cytometric immunophenotyping that identifies a clonal light-chain restricted population expressing Bcell markers in the blood or BM 2 . However, a final diagnosis cannot be done in all patients with B-CLPD by these methods 3 . Clinical features of CLL at presentation are heterogeneous, and a definitive diagnosis is based on the combination of peripheral blood Bcell lymphocytosis (≥5×10 9 /L), characteristic morphology, and immunophenotype. Matutes flow cytometry score is particularly useful for differentiating between CLL and other MBN 4,5 CLL classically has score of 4 or 5. However, some CLL cases may have an atypical immunophenotype and/or morphological features resulting in diagnostic confusion 6 . CD160 is an Ig-like activating natural killer (NK) cell receptor 7,8 .The CD160 gene is on chromosome 1q42.3 9 , expressed on most circulating NK cells and on a subset of circulating cytotoxic T cells, but not on B cells 7,8 . Binding of CD160 to both classical and non-classical major histocompatibility complex class I enhances NK and CD8 + cytotoxic-T lymphocytes functions [10][11][12][13] , as well as cytokine production, including IFN-ᵞ TNF-á, and IL6 11,12 . Recent work has demonstrated CD160 expression in malignant B cells 14 . CD200, is a type I glycoprotein that is expressed on thymocytes, activated T cells, B cells, dendritic cells, endothelial cells, and neurons but not on NK cells 15,16 . The CD200 gene is mapped to chromosome 3q13.2 17,18 . CD200 interacts with the CD200 receptor, which is confined to antigen presenting cells of myeloid origin and a subset of T cells 15 , resulting in immunosuppressive functions 19 . CD200 expression was reported in CLL versus negative expression in MCL. The expression of CD200 was also reported in HCL, multiple myeloma, lymphoblastic lymphoma/leukemia, lymphoplasmacytic lymphoma, acute myeloid leukemias, and other non-hematologic malignancies 20 .
Recently, it has been reported that CDs160/200 were expressed in most cases of CLL 21,22 . In the absence of histological and/or cytogenetics/molecular additional explorations, the addition of CDs160/200 expression detection in atypical B-CLPD proliferative syndromes of uncertain diagnosis could help to reach a definitive conclusion and better orientate patients toward the most appropriate therapy. In addition Human CD160 APC-conjugated Antibody, clone 688327 and Human CD200 percpconjugated Antibody, clone 325516 from R&D systems-Biotechne, USA were added to our panel and analyzed for both patients and controls. Peripheral blood cells were processed for investigation of CD160 and CD200 expression using a combination of CD19, CD160, CD200 and CD5. 100μls of EDTA anti-coagulated PB were added to a falcon tube, and then, 10 μls of CD160/CD200/CD19/CD5 were added, mixed well with a vortex mixer and incubated for 10 minutes in the dark at room temperature. After incubation, the cells were washed twice with PBS, and centrifuged at 2000 rpm for 2 minutes each at room temperature, then two mls lysing solution were added to each sample, mixed gently and incubated for 10 minutes at room temperature in the dark, then the tubes were centrifuged for two minutes at 2000 rpm and the supernatant was discarded leaving approximately 50 µls of fluid, the cells were then washed twice with PBS. The supernatant was gently aspirated and discarded; leaving approximately 50µLs of fluid, the pellet was resuspended in 0.3 ml PBS for flow cytometric analysis. A negative control tube was submitted to all of the previous steps except the staining step. This was done to compensate for nonspecific background of auto-fluorescence and to distinguish between fluorescent positive and fluorescent negative cell populations. Data were acquired on BD FACSCalibur instrument. A four-color flow cytometer using CellQuest Pro software, 1x10 4 events were acquired. On interpretation of the CD160/ CD200 tube, a gate was set around the CD19 positive cells against side scatter and then we detected the percent of cells positive for CD5/19/160 and CD5/19/200 in both percentage and MFI if the cases were positive for CD5, and if the cases were negative for CD5; CD19/160 and CD19/200 percent and MFI were detected (supplementary Figure.). Fluorescence intensity was measured using a logarithmic scale with signal intensity ranging from 10 0 to 10 4 arbitrary units. A ratio of MFI of cells incubated with anti CDs160/200 mAbs against the negative control was established. The positivity threshold of these ratios for B-cell was set at 2 and 20, respectively. Alternately, positivities for CDs160/200 were defined as at least 20% positive cells above the negative background fluorescence. Consistency in fluorescence was verified over the course of the study using Calibrite* beads (cat no: 340486; Becton Dickinson and company BD Biosciences San Jose, USA) gated according to the procedure recommended by the manufacturer. Bone marrow aspiration or trephine biopsy was performed in patients, if necessary.

Statistical analysis
Data were fed into the computer and analyzed using IBMSPSS software package version 20.0. (Armonk, NY: IBM Corp). Significance of the obtained results was judged at the 5% level. Receiver operating characteristic (ROC) curves analysis was used to determine the predictive value of CD160/CD200.

RESULTS
Demographic data, Rai clinical staging system, clinical and laboratory data are presented in (Table 1).  Fig.1A). As regards the CD160 MFIR, a statistical significance was found between CLL group and B-NHL group (p=0.048). No statistically significant difference was detected in HCL group (p=0.057) (Table 2, Fig.1A). CD200 % expression was positive in 100% of CLL patients versus 56% of B-NHL and 100% of HCL patients, thus showing statistical significance in B-NHL patients (p<0.001) and no statistically significant difference was found in HCL ( Table 2, Fig.1B). As regards the CD200 MFIR, a statistical significance was found between CLL group and B-NHL group (p=0.048) and no statistically significant difference was detected in HCL group (p=1.000) (Table 2, Fig.1B). On classifying our patients, according to the coexpression of CD160/CD200, we relied only on the percentage positivity rather than the MFI ratio depending on the results revealed by our ROC curves as regards the sensitivity, specificity and AUC which were much more superior in the percentage rather than MFI ratio (sensitivity= 86.67%, 70%, specificity=80%, 63.33%, AUC=0.940, 0.724 respectively) ( Fig. 2A, Fig. 2B).  Regarding co-expression of CDs160/200, it was double positive in 90% of CLL patients compared to 40% and 60% of B-NHL and HCL patients, respectively. While double negative expression of both markers was found only in 24% of B-NHL patients. Moreover, no double negative was seen in CLL and HCL patients (Fig.3A, Figure.3B).  Regarding matutes scoring, 24 (80%) of CLL patients were 4-5 score and 25 (83.3%) of MBN were 0-2 score. The remaining 11 cases were scored 3 . All the 6 CLL cases which scored 3 were positively co-expressing CDs160/200 by % and MFIR. On the other hand, only 1 from the 5 B-NHL cases that were score 3 was positive for CD160% and MFIR, while the other 4 cases were double negative by % and MFIR. A correlation study was done between CDs160/CD200 expression and different laboratory and clinical parameters, including hemoglobin level, platelet count, total leucocytic count, the absolute lymphocytic count, Rai staging system and matutes score and no statistically significant correlation was found between any of these parameters (p>0.05). On studying the correlation between CD160 and CD200 as regards both % expression and MFIR, no significant correlation was detected. Regarding CD160, we found that 88.3 %( 53/60) of our cases were either double positive in percentage expression and ratio or double negative in percentage expression and ratio (results are consistent) and only 11.7 % (7/60) were positive by ratio and negative by percentage. No cases were positive by percent and had negative MFIR. On studying CD200, we found that only 41.5%(25/60) of our cases showed consistent positivity or negativity to both percentage expression and MFIR, while 58.5%(35/60) of the cases showed discordant results were 50% of the cases were positive by percentage and negative by ratio and 8.5% were positive by ratio and negative by percentage. From the above findings and the fact that the MFIR was more consistent with the % expression in CD160 only rather than the CD200 and depending on our findings regarding the ROC curves sensitivity and specificity, we had to rely on the % expression in detecting the positivity rather than the MFIR (Table 2).

DISCUSSION
Our study aimed to evaluate CDs160/CD200 expression in CLPD that is presented in leukemic phase which can be misdiagnosed as CLL when atypical clinical, morphological, histochemical, immunophenotypic and/or molecular presentation are affected. In our study, 90% of patients with CLL had positive CD160 expression on B-cell clones by percentage versus 60% B-NHL and 60% HCL, while by MFIR 96.7% of patients with CLL had positive CD160 B-cell clones versus 80% B-NHL and 60% HCL. In agreement with our study, Farren TW et al. 21 found that CD160 expression was detected in 98% of CLL cases, 100% of HCL, and 16% of other B-LPD cases. They reported that the restriction of CD160 expression to malignant B cells indicates that it is a tumor-specific antigen and an attractive target for the assessment of minimal residual disease in CD160+ B-LPDs. Lesesve JF et al. 16 20 Stated that CD200 was expressed in all CLL patients (100%), and showed higher statistical significance when compared to B-NHL (P<0.001). Also, CD200 was expressed in the 2(100%) cases of HCL. They concluded that the high expression of CD200 in CLL and HCL could open the option for targeted immune (anti-CD200) therapy. Poongodi R et al. 26 reported that CD200 expression was seen in 100% of CLL and HCL patients. On the contrary, CD200 was not expressed on other CLPD except 2 cases (1 MCL and 1FL). Regarding patients with a Matutes score 3, 6 patients with a diagnosis of CLL had an atypical immunophenotype. All these patients coexpressed CD160 and CD200 by percentage and by MFIR. 5 patients with B-NHL presented with a Matutes score 3. Only one case expressed CD160 by percentage and by MFIR, but none of them had both CDs160/ 200 ratios or percentage expression above defined thresholds; this shows the power of using both markers co-expression in discriminating CLL and other CLPD cases with score 3. In Lesesve JF et al. 16 , 3 patients with CLL had an atypical immunophenotype. The leukemic cell of these patients expressed CDs160/CD200 with a MFIR >2, ≥20, respectively. Thus, the 3 showed positive co-expression. 15 patients with non-CLL B-cell neoplasms had score 3 and none of them had positive co-expression of CDs160/200. Interestingly, we found that co-expression of CDs160/CD200 was found in 90% of CLL and in 60% of HCL compared to 40% of other B-NHL patients; moreover, 24% of B-NHL patients showed double negative expression of both markers. According to the above presented data and other studies in the literature, CD160 and CD200 are sensitive markers for CLL. This finding recommends using CD160 in combination with CD200 to increase their sensitivity as an additional diagnostic tool to differentiate between CLL and B-NHL. Moreover, they can be used to confirm the diagnosis of HCL that may lack any of CD11c, CD25 or CD103.

CONCLUSION
Flow cytometric expression of CD160 in combination with CD200 can be used as additional diagnostic markers in the routine panel to differentiate between B-CLL and B-NHL.

CONFLICT OF INTERESTS
The authors declare that they have no conflict of interests.