Low CD49d expression and long telomere identify a chronic lymphocytic leukemia subset with highly favourable outcome
Authors: Davide Rossi, Chiara Lobetti Bodoni, Antonella Zucchetto, Silvia Rasi, Lorenzo De Paoli, Marco Fangazio, Francesca Maria Rossi, Marco Ladetto, Valter Gattei, Gianluca Gaidano
Journal: American Journal of Hematology (2010)

CD49d expression and telomere length (TL) represent novel prognostic markers for chronic lymphocytic leukemia (CLL) [1-7]. The prognostic information carried by CD49d expression and TL in CLL has its rationale in the disease biology. CD49d is an adhesion molecule mediating cell-to-cell and cell-to-extracellular matrix interactions [8]. In CLL cells, CD49d transmits prosurvival/antiapoptotic signals from the tumor microenvironment to tumor cells [9]. Telomeres ensure genetic stability and regulate critical cellular functions, including proliferation and senescence [10]. In CLL, short TL associates with a fast proliferative history of the leukemic cells [1, 11, 12]. Both CD49d expression and short TL have been associated with increased genetic instability of the CLL clone [3, 13]. From a clinical standpoint, a peculiar feature shared by CD49d expression and short TL is the association with CLL proliferation markers, including expression of CD38, high lactate dehydrogenase (LDH), high β-2-microglobulin, short time to lymphocyte doubling, and short time to progression to a more advanced stage [1-7]. Here, we tested whether the concomitant presence of high CD49d expression and short TL in the same CLL patient may help refine disease stratification for treatment prediction in patients that presented in early to intermediate Binet stage (Binet A and B) and that are candidate to watch and wait as initial management. The study is based on a consecutive series of 128 newly diagnosed CLL presenting in Binet stage A or B (Table I). CLL diagnosis and management were based on International Workshop on Chronic Lymphocytic Leukemia (IWCLL) guidelines [14, 15]. By flow cytometry, CD49d expression ≥30% was documented in 49 of 128 (38.3%) cases. By Southern blot, median TL was 5955 bp (25th–75th percentiles: 4554–6998 bp). By applying the best cut off point of TL for CLL prognostication [4], 40 of 128 (31.3%) CLL harbored a short TL ≤5000 bp. When treated either as continuous or categorical variables, CD49d expression and TL do not show colinearity (Pearson R = −0.156, P = 0.078; χ2 P = 0.148, respectively), and therefore do not behave as reciprocal surrogates. After a median follow-up of 81 months, 64 of 128 patients were treated, accounting for a median treatment-free survival (TFS) of 86.4 months. By univariate analysis, CLL harboring CD49d expression ≥30% at diagnosis were characterized by a significantly higher risk of treatment requirement (P = 0.002) compared with cases with CD49d expression <30% (P = 0.002; Table II and Fig. 1A). Also, CLL harboring TL ≤5000 bp at diagnosis were characterized by a significantly higher risk of treatment requirement compared with cases with TL >5000 bp (P = 0.001; Table II and Fig. 1B). Treatment-free survival (TFS) according to CD49d expression (Panel A), telomere length (TL) (Panel B), and CD49d expression and TL in combination (Panel C). By multivariate analysis penalized by Firth's bias correction, CD49d expression ≥30% (HR: 2.10; 95% confidence interval (CI): 1.18–3.73; P = 0.011) and TL ≤5000 bp (HR: 1.91; 95% CI: 1.09–3.35; P = 0.023) were selected as independent predictors of TFS in this CLL cohort, along with IGHV homology ≥98% (P = 0.025), Binet stage B (P < 0.001), and absolute lymphocyte count >20 × 109/L (P = 0.005; Table II). To test whether the association between CD49d expression and TL has an impact on CLL outcome, patients were grouped into the following categories: (i) CD49dlow/TLlong CLL (CD49d <30%/TL >5000 bp; 58 of 128, 45.3%); (ii) CD49dhigh/TLshort CLL (CD49d ≥ 30%/TL ≤5000bp; 30/128, 23.4%); and (iii) discordant cases (CD49dlow/TLshort; 21 of 128, 16.4%; and CD49dhigh/TLlong; 19 of 128, 14.8%). Bivariate analysis revealed that the combined usage of CD49d expression and TL allowed to segregate CLL cases projected to require treatment from CLL with an indolent course. In fact, CD49dhigh/TLshort CLL and discordant cases were characterized by a TFS significantly shorter than that of CD49dlow/TLlong CLL (P < 0.05 in all pairwise comparisons; Fig. 1C). Our results document that: (i) CD49d expression and TL exert a complementary effect in driving prognosis of early stage CLL; (ii) early stage CLL harboring the CD49dlow/TLlong profile are characterized by a very favorable prognosis. The concomitant assessment of CD49d expression and TL may help refine further the prognostic stratification of early stage CLL and, in particular, may serve the scope of identifying highly stable patients. The complementary action of CD49d and TL in driving CLL prognosis may stem from the biological effects exerted by these two markers on the CLL clone. In fact, the microenvironmental prosurvival signals provided by CD49d expression, and the genetic instability and proliferating propensity associated with short TL may both confer biological aggressiveness to CLL [1-13]. According to this model, CLL cases with both low CD49d expression and long telomeres (CD49dlow/TLlong CLL) may be less exposed to deleterious microenvironmental stimuli and genetic instability, and, consequently, at reduced risk of clinical progression and more prone to maintain clinical stability. The study was based on a consecutive series of 128 previously untreated CLL who presented in Binet stage A or B for initial evaluation at the Division of Hematology of the Amedeo Avogadro University of Eastern Piedmont from June 1996 through June 2008. Median follow-up of alive patients, calculated by inverted censoring method, was 81 months. Patients provided written informed consent in accordance with local institutional review board requirements and Declaration of Helsinki. CLL diagnosis and management were based on IWCLL guidelines [14]. The following biological variables were analyzed on fresh or cryopreserved peripheral blood mononuclear cells collected at CLL diagnosis before treatment: (i) IGHV gene homology to germline; (ii) FISH karyotype; (iii) TP53 mutations; (iv) CD49d, CD38, and ZAP70 expression; and (v) TL. In all samples, the fraction of CD19+/CD5+ cells was ≥70% (median: 83%; range: 73–90%). Expression of CD49d was analyzed by three-color immunofluorescence by combining phycoerithrin (PE)-conjugated antiCD49d mAbs with Peridinin-Chlorophyll-Protein-Cyanine-5.5 (PerCP-Cy5.5)-conjugated antiCD19 mAbs and fluorescein isothyocyanate-conjugated antiCD5 mAbs [5, 7]. Expression data were reported as percent of CD5+CD19+ CLL cells displaying specific fluorescence intensity >98–99% of the same cell population stained with control Ig. CD38 and ZAP70 were analyzed as reported [5]. The 30% cut-off value for CD49d expression was selected based on maximally selected rank statistics results and corresponded to a value previously validated in the literature [5]. Cut-off points of 30% and 20% were utilized to define positivity for CD38 and ZAP70, respectively [5]. Peak TL was determined by Southern blot analysis [16]. Peak TL was preferred to mean TL because this approach allows accurate measurement of TL in the presence of up to 30% contaminating non-neoplastic cells, as documented by extensive cell dilution studies [16]. Briefly, 2 μg of genomic DNA were digested by mixing HinfI and RsaI (Roche Diagnostics, Mannheim, Germany). Telomere restriction fragments were separated by 0.8% agarose gel electrophoresis. Gels were transferred to a positively charged nylon membrane (Roche Diagnostic Mannheim, Germany) and UV cross linked. Hybridization and detection were performed using the TeloTAGGG Telomere Length Assay Kit (Roche Diagnostics, Mannheim, Germany). Membranes were scanned and analyzed with Kodak Digital Science 1D Software (Scientific Imaging Systems, New Haven, CT). The best cut-off point of TL for CLL prognostication has been previously identified as 5000 bp [6]. TFS was measured from diagnosis to first-line treatment, death, or last follow-up. The accepted indications to initiate treatment were based on IWCLL criteria [14]. Categorical variables were compared by χ2 test. Correlation between continuous variables was assessed by measuring the Spearman's correlation coefficient. Survival was analyzed by Kaplan-Meier method. Univariate and multivariate Cox models were used to verify the independent prognostic power of each parameter. To account for possible monotone likelihood, the Firth's bias correction method was applied to Cox regression when several highly predictive covariates were introduced in the analysis. Model minimization was performed by stepwise backward elimination. All statistical tests were two-sided. Statistical significance was defined as P < 0.05. The analysis was performed with SPSS software v.17.0 (Chicago, IL). Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Davide Rossi*, Chiara Lobetti Bodoni , Antonella Zucchetto , Silvia Rasi*, Lorenzo De Paoli*, Marco Fangazio*, Francesca Maria Rossi , Marco Ladetto , Valter Gattei , Gianluca Gaidano*, * Division of Hematology, Department of Clinical and Experimental Medicine and BRMA, Amedeo Avogadro University of Eastern Piedmont and Azienda Ospedaliero-Universitaria Maggiore della Carità, Novara, Italy, Division of Hematology, Department of Experimental Medicine and Oncology, University of Turin, Italy, Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano, Italy.