Abstract
Chronic lymphocytic leukaemia (CLL) is characterized by expression of CD5 on clonal B cells, and is partly driven by activated B-cell receptor (BCR) signalling. While CD5 is known to be a negative regulator of BCR signalling, it is unknown if variability in CD5 expression exists among patients and whether CLL cell CD5 expression affects CLL clinical outcomes. We assessed the extent to which CD5 expression is correlated with clinical outcomes, and whether this information adds to currently used prognostic markers. We evaluated CD5 expression from 1275 blood samples, established prognostic markers and time to event data from 423 CLL patients followed at the Duke University and Durham VA Medical Centers. CD5 median fluorescence intensity (MFI) was largely stable over time in individual patients, but ranged between 0Á5 and 760 in the entire cohort. Lower CD5 MFI was significantly associated with a shorter time to first therapy. CD5 MFI, combined with established clinical and molecular prognostic markers, significantly improved risk-stratification. CD5 may affect disease outcomes by suppressing signalling through the BCR. Thus, a strategy to modulate CLL cell CD5 expression or function could be a therapeutic approach in CLL.
Summary
Chronic lymphocytic leukaemia (CLL) is characterized by expression of CD5 on clonal B cells, and is partly driven by activated B-cell receptor (BCR) signalling. While CD5 is known to be a negative regulator of BCR signalling, it is unknown if variability in CD5 expression exists among patients and whether CLL cell CD5 expression affects CLL clinical outcomes. We assessed the extent to which CD5 expression is correlated with clinical outcomes, and whether this information adds to currently used prognostic markers. We evaluated CD5 expression from 1275 blood samples, established prognostic markers and time to event data from 423 CLL patients followed at the Duke University and Durham VA Medical Centers. CD5 median fluorescence intensity (MFI) was largely stable over time in individual patients, but ranged between 0Á5 and 760 in the entire cohort. Lower CD5 MFI was significantly associated with a shorter time to first therapy. CD5 MFI, combined with established clinical and molecular prognostic markers, significantly improved risk-stratification. CD5 may affect disease outcomes by suppressing signalling through the BCR. Thus, a strategy to modulate CLL cell CD5 expression or function could be a therapeutic approach in CLL.
Summary
Keywords: chronic lymphocytic leukaemia, CD5, prognosis, B-cell receptor signalling, biomarker.
Summary
Overactive and ligand independent B-cell receptor (BCR) signalling and subsequent heightened signal transduction are a hallmark of oncogenesis in chronic lymphocytic leukaemia (CLL) (Iacovelli et al, 2015; Ten Hacken & Burger, 2016) . In CLL, an incurable B-cell malignancy with a range of clinical aggressiveness, increased signalling through the BCR pathway has been the rationale for targeted therapeutics, including ibrutinib (Herman et al, 2011) and idelalisib (Herman et al, 2010) . In addition, CD38 and ZAP70, two proteins that modulate BCR signalling, are currently used for prognostication and risk stratification at the time of CLL diagnosis (Damle et al, 1999; Chen et al, 2002; Wiestner et al, 2003; Malavasi et al, 2011) . The existing research suggests that other molecules that are part of the BCR pathway may also be prognostically or therapeutically important.
Summary
CLL malignant B lymphocytes are characterized by their expression of CD5, a protein typically found on normal T lymphocytes and B1a B cells. CD5 has been demonstrated to be a negative regulator of BCR signalling in CLL cells or T-cell receptor signalling in normal T cells (Gary-Gouy et al, 2000 Soldevila et al, 2011) . In clinical practice, CD5 expression on CLL lymphocytes, measured by flow cytometry, is reported as either 'positive' or 'negative.' The level of CD5 expression is not typically taken into consideration for diagnostic or prognostic purposes.
Summary
Given the role of CD5 in BCR signalling, we reviewed CD5 protein expression in CLL lymphocytes over time from patients who are part of the CLL cohort at the Duke University and Durham VA Medical Centers. We sought to determine the prognostic significance of CD5 expression in terms of time to first therapy (TTT) and overall survival (OS), as well as to evaluate CD5 expression together with established CLL prognostic markers.
Methods

Patients
Patients with a diagnosis of CLL who were evaluated at the Duke University and Durham VA Medical Centers were enrolled in Institutional Review Board-approved protocols at their respective institutions to collect clinical data and blood samples. Data obtained included date of diagnosis, Rai stage (categorized as 0, 1 or 2, and 3 or 4), treatment history, gender, age at diagnosis, immunoglobulin heavy chain variable region (IGHV) gene mutation status, ZAP70 status (>20% considered positive), CD38 status (>30% considered positive), cytogenetic aberrations by interphase fluorescence in situ hybridization (FISH) testing, and CLL International Prognostic Index (IPI) score.
CD5 measurement
Blood was obtained from CLL patients attending the Duke University or the Durham VA Medical Centers. CLL B lymphocytes were purified using negative selection (RosetteSep, StemCell Technologies, Vancouver, Canada) as previously described (Weinberg et al, 2007) . CD5 expression was measured by immunophenotyping the purified CLL cells using flow cytometry (PE anti-human CD5, BD #555353, Clone UCHT2; BD Pharmingen, San Jose, CA, USA), as previously described (Weinberg et al, 2007) , from January 2011 to March 2017. CD5 values (relative fluorescence intensity) reported are from median fluorescence intensity (MFI) of CD5 minus MFI of the isotype control, determined with FlowJo software (FlowJo, Ashland, Oregon, USA).
Statistical evaluation
The primary outcome was TTT, which was defined as time from date of CLL diagnosis to date of first therapy or date of last follow-up if untreated. OS was defined as time from date of CLL diagnosis to date of death or date of last follow-up if alive.
Statistical evaluation
CD5 MFI was evaluated in longitudinal measurements from individual CLL patients for change over time. The median values and the interquartile ranges were determined for each patient. Because of low variation of CD5 expression from longitudinal samples collected from CLL patients, the mean of CD5 MFI for each patient was used for purposes of prognostic analyses. In these prognostic analyses, the term CD5 MFI is used to represent the mean of the CLL cell CD5 values for each patient.
Statistical evaluation
Prognostic analyses were conducted using Cox proportional hazards models, as described before (Friedman et al, 2016) . Hazard ratios (HR), 95% confidence intervals (CI), and P values are reported. Analyses were based on all available patients with complete baseline data. Missingness of baseline covariates was considered non-informative. CD5 MFI for each patient was evaluated after log10 transformation, as a continuous variable or dichotomized (high versus low) at the median value. CD38, ZAP70, IGHV and FISH statuses were treated as binary variables (reference values were CD38À, ZAP70À, IGHV mutated, and FISH negative for 17p and/or 11q deletions, respectively). Unfavourable FISH was defined as the presence of 11q and/or 17p deletion, whereas favourable FISH was defined as the absence of these findings (Dohner et al, 2000) . Rai stage at diagnosis was simplified as low risk (stage 0), intermediate risk (stages 1 and 2) and high risk (stages 3 and 4). We used Cox proportional hazards models to investigate associations between survival times (OS and TTT) and CD5 MFI (continuous variable) and/or other prognostic markers.
Statistical evaluation
To determine if CD5 MFI added information to established prognostic markers, we used analysis of deviance to compare multivariate Cox proportional hazards models. A multivariate Cox proportional hazards model including CD5 MFI with CD38, ZAP70, IGHV and FISH status was compared to a nested model lacking CD5 MFI. As an illustration of the contribution of CD5 MFI, hazard ratio (HR), 95% confidence interval (CI) and P values are reported for all the variables in the full model.
Statistical evaluation
We visualized the association of CD5 MFI dichotomized at the median and time-to-event outcomes in Kaplan-Meier (KM) plots. These analyses were not used for model-building purposes. Pearson's correlation method was used to evaluate associations between continuous variables. Wilcoxon rank sum test was used to compare CD5 MFI values between groups of CLL patients.
Statistical evaluation
Statistical analyses and figure generation were performed using the R software environment for statistical computing and graphics (R Core Team, 2016) , with ggplot2, forestplot, and survival extension packages. P values and CI estimates were not adjusted for multiple testing.
Results

CLL patient characteristics
We evaluated data collected from CLL patients at Duke University and the Durham VA Medical Centers (n = 423). Demographic, clinical, and molecular prognostic factors are outlined in Table I . This cohort of patients includes a range of patient risk subgroups, including high risk patients with unmutated IGHV (34%) and unfavourable cytogenetics of 11q and/or 17p deletion (14%).
CD5 expression on malignant lymphocytes varies among CLL patients
While CLL B lymphocytes are defined by the presence of CD5 expression, we noted that the degree of CD5 protein expression was not uniform among patients in the Duke/ Durham VA cohort. We evaluated CD5 expression by MFI on 1275 blood samples, with one to 13 samples obtained longitudinally per patient between January 2011 and March 2017. We observed that CD5 was relatively stable in longitudinal samples from individual patients over time. As seen in Fig 1A, the median CD5 MFI for each patient ranged between 0Á5 and 760 (relative fluorescence intensity units). Representative flow cytometry plots are shown in Fig 1B. Our evaluation of CD5 measurements over time demonstrated that for most patients, CD5 MFI measurements were relatively stable (Fig 2A) . Five CLL patients had more than three CD5 MFI measurements spanning the period before and after initial therapy for CLL. Initial therapy was chlorambucil (n = 2), rituximab (n = 1), fludarabine, cyclophosphamide and rituximab (n = 1) and pentostatin, cyclophosphamide and ofatumumab (n = 1). Comparing before therapy and after therapy values indicated that CD5 expression did not significantly change over time in these patients (Fig 2B) . These data indicate that a range of CD5 expression is found in CLL patients, but for each patient, there is relative stability of CD5 expression over time.
CD5 expression on malignant lymphocytes varies among CLL patients
CD5 expression was not significantly different in CLL patients grouped by established molecular prognostic factors, such as IGHV mutation status, CD38 and unfavourable interphase cytogenetics by FISH (Dohner et al, 2000) , but was higher in ZAP70 positive samples compared with ZAP70 negative samples (233Á3 vs. 186Á1; P = 0Á027). In addition, African American CLL patients had lower CD5 expression on their CLL cells than White CLL patients (147Á4 vs. 227, P = 0Á030).
CD5 expression on malignant lymphocytes varies among CLL patients
Higher CLL cell CD5 expression is associated with superior clinical outcomes Elevated CD5 MFI evaluated as a continuous variable was significantly associated with a longer TTT (HR 0Á67; 95% CI 0Á48-0Á95; P = 0Á025) but not OS. High CD5 MFI (dichotomized at the median per-patient CD5 MFI, 216Á08) was significantly associated with longer TTT (Fig 3A; HR 0Á67; 95% CI 0Á50-0Á90; P = 0Á008) and associated with improved OS, although this was not significant (Fig 3B; HR  0Á59 ; 95% CI 0Á33-1Á04, P = 0Á068). The low number of events (deaths) in this group of patients who were alive and providing samples over the six-year period probably reduced the power to detect a significant impact of CD5 expression on OS. In the high CD5 MFI group, median TTT was 13Á0 years, compared to 5Á8 years in the low CD5 MFI group. Likewise, in the high CD5 MFI group, median OS was not reached, whereas in the low CD5 MFI group, the median OS was 27Á3 years.
CD5 expression on malignant lymphocytes varies among CLL patients
Elevated CD5 expression adds to risk-stratification when combined with established prognostic markers In this cohort of CLL patients, established prognostic factors (Rai stage, CD38, ZAP70, IGHV, interphase cytogenetics and CLL IPI) as well as low CD5 MFI were associated with inferior clinical outcomes, specifically TTT (Table II) . Because all patients in this group were alive and providing samples in the recent past, the impact of these established factors on OS appears limited (although they are significantly associated with both TTT and OS in the entire Duke/Durham VA CLL cohort; data not shown). Given that low expression of CD5 is associated with inferior clinical outcomes, we evaluated whether CD5 expression could further risk-stratify patient groups defined by established prognostic markers. We found that CD5 MFI assigned CLL patients into more defined risk groups, particularly stratifying those in favourable risk groups (Fig 4) . Similar results were found when CD5 MFI was evaluated as a dichotomized variable. For example, among CLL patients with low CD38 expression, low CD5 MFI (below the median per-patient CD5 MFI) identified patients with shorter TTT (HR 0Á62, 95% CI 0Á44-0Á87, P = 0Á005). The same effect was seen in CLL patients with low ZAP70 expression (HR 0Á62, 95% CI 0Á39-0Á97, P = 0Á038), mutated IGHV (HR 0Á56, 95% CI 0Á34-0Á94, P = 0Á027) and favourable FISH (HR 0Á60, 95% CI 0Á41-0Á89, P = 0Á012). These results suggest that, when combined with other prognostic markers, low CD5 expression identifies CLL patients at risk for more rapid progression and need for treatment, particularly in patients otherwise defined as having favourable risk. In a multivariate Cox proportional hazards model, low CD5 MFI continued to be significantly associated with inferior TTT in a model incorporating the molecular prognostic markers CD38, ZAP70, IGHV and interphase cytogenetics by FISH (Table III , P = 0Á012). There was a significant difference between a full model containing CD5 MFI and established molecular prognostic markers to a reduced model containing established molecular prognostic markers alone (analysis of deviance, P = 0Á018). This indicates that in this cohort, CD5 MFI adds important prognostic information about TTT to commonly used prognostic markers.
Discussion
We demonstrate here that CD5 expression is not uniform among CLL patients, but is relatively static over time for each individual CLL patient. Higher CD5 expression is associated with improved clinical outcomes, specifically longer time to therapy. Even within the context of established clinical and molecular prognostic markers, CD5 MFI adds important risk-stratification information.
Discussion
CD5 expression is a defining feature of CLL. However, several case series have reviewed 'CD5-negative CLL,' a B-cell lymphoproliferative disorder with similar outcomes to typical CLL (Sheikh et al, 2002; Romano et al, 2015; Demir et al, 2017) . These reports did not specify the degree of CD5 'negativity,' so it is not clear if these patients are similar to CLL (Falchi et al, 2013) , although the underlying reason for this is unknown (Coombs et al, 2012) . Our current work demonstrates that African Americans with CLL had lower CD5 expression than Whites with CLL, suggesting that at least part of the differences in clinical outcomes may be related to CD5 expression and downstream signalling.
Discussion
CD5 appears to dampen BCR signalling in CLL cells through co-localization of phosphorylated CD5 with SHP1 at the cell membrane (Tibaldi et al, 2011) . This in vitro work raises the possibility that there may be an inverse linear correlation between CD5 expression and BCR signalling in CLL cells, which may explain why patients with low CD5-expressing CLL have inferior clinical outcomes. Interestingly, we did not find a significant correlation between immunoglobulin isotype (presence of specific light or heavy chain surface expression) and CD5 expression (data not shown), suggesting that any effect of CD5 on BCR signalling does not depend on a specific immunoglobulin isotype.
Discussion
Others have demonstrated that CD5 expression is positively correlated with CD20 expression in circulating CLL cells, and surface CD5 expression appears to increase initially and then subsequently fall after starting ibrutinib (Pavlasova et al, 2016) . Thus, CD5 expression could modulate responses to anti-CD20 antibodies (rituximab, ofatumumab, obinutuzumab) and/or Bruton tyrosine kinase inhibitors (ibrutinib, acalabrutinib). Additional research is required to evaluate these hypotheses.
Discussion
Our results, from a well-characterized CLL cohort with 6 years of immunophenotyping data, potentially have important implications for CLL patients treated and followed at other centres. Our immunophenotyping assays have been consistent over time but have not been regularly calibrated as would be done in a CLIA (Clinical Laboratory Improvement Amendments) certified laboratory. However, errors in CD5 measurement or inter-batch variability between flow cytometry runs would be expected to reduce the chance of finding a significant association between CD5 expression and clinical outcomes. In addition, our analysis focused on measurements of CD5 expression from CLL cells enriched by negative selection, a process not typically performed in clinical settings. However, because we found that CD5 MFI in negatively selected CLL cells is highly correlated with CD5 MFI in a gated CD5+CD19+ population from whole blood immunophenotyping (P < 0Á0001, correlation coefficient r = 0Á958), we do not believe that cell preparation techniques significantly affect the results obtained. The results outlined in our report need to be confirmed with data from other institutions and from whole blood immunophenotyping. (events) 403 (185) 67 (44) 323 (138) 179 (102) 211 (80) 145 (99) 200 (62) 60 (43) 230 ( Forest plot of hazard ratios (HR; black squares) and confidence intervals (horizontal lines) of the association between CD5 MFI (continuous variable) and time to first treatment (TTT) in molecularly defined prognostic groups. HR less than 1 indicates higher CD5 MFI is associated with longer TTT, whereas HR above 1 indicates higher CD5 MFI is associated with shorter TTT. CD5 and CD38 have a significant P interaction of 0Á021, but all other interactions were not significant. FISH, fluorescence in situ hybridization; M, mutated; MFI, median fluorescence intensity; UM, unmutated. Our report, that lower CD5 expression is associated with shorter time to therapy in CLL, suggests that CD5 may modulate disease aggressiveness through its role as a negative regulator of BCR signalling. This would add to existing evidence that modulators of BCR signalling, such as CD38 and ZAP70, affect CLL cell biology and disease aggressiveness. CD5 is currently measured at diagnosis through flow cytometry, and CD5 quantification could be reviewed by clinicians as part of evaluating prognostic information. Together with the possibility that CD5 might influence responses to CLL therapeutics, our findings could have significant implications for the care of CLL patients.

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