CD23 Receptor Positivity Has No Prognostic Implication in Chronic Lymphocytic Leukemia.

Abstract Abstract 4397 BACKGROUND Chronic Lymphocytic Leukemia (CLL) phenotypically expresses CD23, although the percentage of positive cells measured by flow cytometry is variable. We sought to analyze whether the percent of CD23 positive cells in the CLL clone correlates with time to treat (TTT), overall survival (OS) and prognostic markers CD38, ZAP-70, and IGHV mutation status. METHODS We retrospectively analyzed the flow cytometry data of 332 CLL patients on the gated population of cells that were CD5 and CD19 positive. Percentage positivity for CD23, CD38, and ZAP-70 was noted. CD38 and ZAP-70 were considered positive at cut-offs of >= 30% and >=20%, respectively. CD23 was considered negative at <30% and positive at >= 30%. IGHV sequence was determined from cDNA and then compared to germline to assess mutation status using IMGT/V-QUEST. The distributions of time from diagnosis until start of treatment and overall survival were stratified by CD23 positivity, estimated using the product limit method, and compared using the log rank test. Those who had expired without treatment or were alive and not treated at this time point were censored in the TTT analysis. Those who were still alive were censored in the OS analysis. Associations of CD23 positivity with IGHV mutation status, ZAP-70, and CD38 positivity were examined using the chi-square test. RESULTS Out of 332 patients, 25 had diminished CD23 expression (<30%) whereas 307 had normal CD23 expression (>30%). There was no difference in time until start of treatment or overall survival based on CD23 %positivity. CD23 %positivity showed no associations with IGHV mutation status, ZAP-70 or CD38 positivity. CONCLUSION CD23 percent positivity has no prognostic significance in CLL. There is no correlation between CD23 percent positivity and poor prognostic markers such as CD38, ZAP-70, or IGHV mutation status. Disclosures: No relevant conflicts of interest to declare.

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Absolute Monocyte Count and Serum CD14 As Prognostic Markers In Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v122.21.5297.5297 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5297-5297

Author(s):

Daphne R. Friedman ◽

Kathleen K. Harnden ◽

Youwei Chen ◽

Alicia D. Volkheimer ◽

J. Brice Weinberg

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Risk Stratification ◽

Clinical Outcomes ◽

Prognostic Markers ◽

Prognostic Significance ◽

Hazard Ratio ◽

Lymphocytic Leukemia ◽

Monocyte Count ◽

Interphase Cytogenetics ◽

Mutation Status

Abstract Introduction Although chronic lymphocytic leukemia (CLL) is a generally indolent malignancy, there is a spectrum of disease aggressiveness. Clinical and molecular prognostic markers are helpful for the clinician and for the patient, in terms of disease management and life planning. Additional prognostic markers can help with further risk stratification, especially for CLL patients with “low-risk” disease. The prognostic value of absolute monocyte count (AMC) has been evaluated in various malignancies, including CLL where elevated AMC at diagnosis was shown to be associated with rapid time to first therapy (TTT), and in one series, inferior overall survival (OS). The mechanism by which elevated AMC is associated with worse treatment free survival is not known. However, CD14, which is secreted by monocytes, improves in vitro CLL cell survival, and is found at high levels in the serum of CLL patients. We hypothesized that elevated AMC at the time of CLL diagnosis is associated with inferior survival and that elevated serum CD14 is associated with high AMC and worse survival. Methods CLL patients followed at the Duke University and Durham VA Medical Centers and enrolled in an IRB approved protocol to collect clinical data and blood samples were evaluated. We selected patients for whom AMC was measured between three months prior to diagnosis to three months after diagnosis. We evaluated the correlation between AMC and TTT and OS, with AMC as a continuous and as a dichotomized variable. We also assessed the prognostic capability of AMC in relation to other clinical and molecular prognostic markers, such as Rai stage, race, interphase cytogenetics by FISH, CD38 and ZAP70 expression, and IGHV mutation status. We measured serum CD14 levels using an ELISA assay, and evaluated the correlation between CD14 levels and clinical outcomes or AMC. Cox proportional hazard models were used to evaluate time to event outcomes, Wilcoxon rank sum test and Kruskal-Wallis rank sum test were used to compare AMC to other prognostic markers, and Pearson’s correlation test was used to compare continuous variables. Results From a cohort of over 600 CLL patients, we selected 222 patients with AMC measured ± three months from the date of diagnosis. AMC ranged from 0 to 7.63 cells/mL. With a median follow up of 5.2 years (range 0.1 – 18.2), 102 patients (46%) had been treated, and 59 patients (27%) died. This was not significantly different from the entire cohort. Higher AMC was significantly correlated with shorter TTT (p = 0.002, hazard ratio 1.37, 95% CI 1.12 – 1.68) and inferior OS (p = 0.017, hazard ratio 1.39, 95% CI 1.06 – 1.83). There was no significant difference in AMC in patients stratified by Rai stage, race, interphase cytogenetics, CD38 or ZAP70 expression, or IGHV mutation status. When combined with molecular prognostic markers (IGHV mutation status, CD38 and ZAP70 expression, and interphase cytogenetics) in multivariate models, AMC retained significant prognostic power for TTT and OS. The serum soluble CD14 levels were measured in CLL patients from this cohort, with a mean CD14 level of 2.3 ug/mL. The prognostic significance of serum CD14 and correlation with AMC will be presented. Conclusions Absolute monocyte count at the time of CLL diagnosis is associated with inferior clinical outcomes – both TTT and OS. These results confirm and extend other reports evaluating the prognostic significance of circulating monocytes in CLL. Our evaluation of serum CD14, a monocyte-derived secreted protein that promotes CLL cell viability, in concert with AMC may provide a possible explanation for the associations identified in this cohort of patients. As an easily measured clinical marker, AMC can be readily used and/or combined with other prognostic markers to improve risk stratification and patient counseling at the time of diagnosis. Disclosures: No relevant conflicts of interest to declare.

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FCRL2 Expression Is Predictive of IgVH Mutation Status and Clinical Progression in Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v110.11.488.488 ◽

2007 ◽

Vol 110 (11) ◽

pp. 488-488 ◽

Cited By ~ 1

Author(s):

Fu Jun Li ◽

Yufeng Li ◽

Mikail A. Shakhmatov ◽

Elena Kashentseva ◽

Jiongru Wu ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Prognostic Markers ◽

Clinical Course ◽

Univariate Analysis ◽

Prognostic Significance ◽

Surface Expression ◽

Lymphocytic Leukemia ◽

Clinical Progression ◽

Mutation Status

Abstract B cell chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy of two subtypes that can be stratified according to the mutation status of the immunoglobulin heavy chain variable region gene (IgVH). Because the clinical course of this disease differs dramatically between these subgroups, accurately identifying indolent patients (mutated-MT IgVH) from aggressive ones (unmutated-UM IgVH) is of increasing importance. CD38 and ZAP-70 expression are both considered useful prognostic markers in CLL and are utilized by most clinical laboratories. Although they are associated with Ig mutation status and predictive of clinical course, both proteins have some limitations and demonstrate variable discordance with IgVH mutation status. Thus, the proper diagnostic use of these indicators and their optimal detection remains under investigation. The identification of additional diagnostic and prognostic markers would not only be helpful for subgrouping CLL patients and choosing appropriate treatment options, but could also be informative for understanding the cellular origins and biological defects underlying this lymphoproliferative disorder. Recent characterization of five human Fc receptor-like (FCRL1–5) molecules possessing tyrosine-based activating and/or inhibitory potential that are expressed by distinct B cell subpopulations, suggests they could be useful diagnostic, prognostic and/or therapeutic markers in B lineage malignancies. To determine their prognostic potential in CLL, FCRL1–5 surface expression was evaluated with a panel of specific monoclonal antibodies by flow cytometry and compared with currently established markers of prognosis including IgVH mutation status, as well as CD38 surface expression, ZAP-70 cytoplasmic expression, and clinical parameters from 107 CLL cases. Our results demonstrate that FCRL1–3, and FCRL5 are expressed by CLL cells, but FCRL4 is not. Except for FCRL1, the expression of FCRL2, FCRL3, and FCRL5 was found at significantly higher levels on CLL cells than the polyclonal CD19+ B cell population in normal individuals. Univariate analysis revealed that all four FCRLs, as well as CD38 and ZAP-70, were significantly associated with IgVH mutation status. However multivariate logistical analysis confirmed that only FCRL2 and ZAP-70 maintain predictive value. Furthermore, significant negative correlations between FCRL2 and ZAP-70 expression (r=0.60, P<0.0001), and FCRL2 and CD38 expression (r=0.49, P<0.0001) were identified. Strikingly, FCRL2 provided excellent sensitivity and specificity, demonstrating 94.4% concordance with IgVH mutation compared to 77.6% for CD38 and 80.4% for ZAP-70. In a subset of patients for which clinical data were initially available, log-rank analysis revealed that similar to IgVH mutation status, FCRL2 can predict disease progression; the median time to first therapy was 13 years for FCRL2 high expressers compared to 4 years for FCRL2 low individuals. Our data indicate that FCRL2 is a novel prognostic marker of IgVH mutation status and clinical progression that may complement the clinical prognostic significance of CD38 and ZAP-70, and further improve therapeutic strategies for patients afflicted with CLL.

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Mutation Status and Immunoglobulin Gene Rearrangements in Patients from Northwest and Central Region of Spain with Chronic Lymphocytic Leukemia

BioMed Research International ◽

10.1155/2014/257517 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

I. González-Gascón y Marín ◽

J. A. Hernández ◽

A. Martín ◽

M. Alcoceba ◽

M. E. Sarasquete ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Central Region ◽

Lymphocytic Leukemia ◽

Gene Rearrangements ◽

Immunoglobulin Gene ◽

Mutation Status ◽

Almost All ◽

Time To First Treatment ◽

Immunoglobulin Gene Rearrangements

The aim of this study was to investigate the frequency and mutation status of the immunoglobulin heavy variable chain (IGHV) in a cohort of 224 patients from northwest and central region of Spain diagnosed with chronic lymphocytic leukemia (CLL), and to correlate it with cytogenetic abnormalities, overall survival (OS) and time to first treatment (TTFT). 125 patients had mutated IGHV, while 99 had unmutated IGHV. The most frequently used IGHV family was IGHV3, followed by IGHV1 and IGHV4. The regions IGHV3-30, IGHV1-69, IGHV3-23, and IGHV4-34 were the most commonly used. Only 3.1% of the patients belonged to the subfamily IGHV3-21 and we failed to demonstrate a worse clinical outcome in this subgroup. The IGHV4 family appeared more frequently with mutated pattern, similar to IGHV3-23 and IGHV3-74. By contrast, IGHV1-69 was expressed at a higher frequency in unmutated CLL patients. All the cases from IGHV3-11 and almost all from IGHV5-51 subfamily belonged to the group of unmutated CLL.

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Abnormal Free Light Chain Ratios in Chronic Lymphocytic Leukemia: A New Prognostic Factor?.

Blood ◽

10.1182/blood.v114.22.1237.1237 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1237-1237 ◽

Cited By ~ 2

Author(s):

Johannes Matschke ◽

Lewin Eisele ◽

Ludger Sellmann ◽

Ulrich Duehrsen ◽

Jan Duerig ◽

...

Keyword(s):

Prognostic Factor ◽

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Risk Groups ◽

Typical Feature ◽

Lymphocytic Leukemia ◽

Al Amyloidosis ◽

Abnormal Ratio

Abstract Abstract 1237 Poster Board I-259 Introduction Free light chains (FLC) have prognostic significance in monoclonal gammopathy of undetermined significance, solitary plasmocytoma of bone, smouldering myeloma, multiple myeloma, Waldenstroms macroglobulinaemia and AL amyloidosis. Although monoclonal protein secretion is a typical feature of plasma cell dyscrasias, it can also be detected in other B cell malignancies including chronic lymphocytic leukemia (CLL). Recent data suggest a significant correlation between abnormal ratio of FLC and outcome. Therefore, we investigated FLC in a large cohort of 120 patients in order to assess the role of FLC in CLL. Methods and Results Plasma samples which had been previously cryopreserved and collected at the time before the initiation of therapy or six months after finishing therapy were used. The levels of FLC were assessed using nephelometric immunoassays (The Binding Site) and quantified nephelometrically with the BNII analyser. A normal FLC range (κlγ) was defined as 0.26-1.65. Moreover, in all cases we evaluated the M band on immunofixation (IF). Abnormal FLC ratios were found in 71 patients (59%) whereas the IF was positive in only 32 cases (27%). In 48 cases the FLC ratio was positive while IF was negative and in only 9 cases the IF was positive while the FLC ratio was normal. In total, 23 patients had both a positive IF and an abnormal FLC ratio. Patients with an abnormal FLC ratio for γ had a significantly shorter treatment-free survival (TFS) than patients with an abnormal ratio for κ or with a normal FLC ratio (median TFS: 34 versus 78 versus 109 months, p=0.042). Evaluation of several disease characteristics in association with FLC of the patients' B-CLL cells showed no significant differences for FLC in the different risk groups (ZAP-70 status, CD38 status, cytogenetics and Binet stage) suggesting no correlation of the FLC with these already established adverse prognostic factors. Conclusion FLC can be detected in a substantial fraction of patients with CLL and the FLC technique improves detection of M-proteins. Moreover, an abnormal FLC ratio is associated with worse outcome, particularly those with a low abnormal FLC ratio. Evaluation of the prognostic significance of abnormal FLC in a larger cohort is currently under way. This data will be presented at the meeting. Future studies are warranted to elucidate the role of FLC as biomarkers of disease and as a prognostic factor for response. Disclosures No relevant conflicts of interest to declare.

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Plasma Circulating Caspase-3 Index as a Biomarker in Patients with Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.4381.4381 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4381-4381

Author(s):

Jean-Marie Bruey ◽

Zeev Estrov ◽

Hagop Kantarjian ◽

Susan O'Brien ◽

Michael Keating ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Normal Control ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Performance Status ◽

Surrogate Marker ◽

Quantitative Measure ◽

Lymphocytic Leukemia ◽

Mutation Status ◽

Control Subjects

Abstract Abstract 4381 Because of the accumulation of lymphocytes and relative paucity of proliferating cells characteristic of B-cell chronic lymphocytic leukemia (CLL), defective apoptosis has been proposed as a key step in the pathogenesis of this disease. Activity levels of the proapoptotic enzyme caspase-3 have been used as a simple, quantitative measure of ongoing apoptosis in various cancers. Here we explored the clinical value of assessing apoptosis levels in patients with CLL, using caspase-3 activity in plasma as a surrogate marker for apoptosis. The study included 194 patients with CLL and 96 normal control subjects. Caspase-3 activity was measured in plasma samples by incubation with the substrate DEVD. Circulating caspase-3 activity was detectable in the plasma of all CLL patients and normal control subjects, but was significantly (P=0.005) lower in CLL patients (median=7.49; range=4.2-19.68 pmol/min/μL) than in controls (median=8.27; range=4.54-34.30 pmol/min/ul. Absolute levels of caspase-3 levels in plasma in CLL did not correlate with any of the laboratory variables examined (WBC, platelets, HGB, B2M), Rai stage, or performance status. To assess the extent of apoptosis in relevance to level of the disease or tumor load, the caspase-3 index was calculated by normalizing plasma caspase-3 activity to the number of circulating lymphocytes in peripheral blood. The circulating caspase-3 index correlated negatively with bone marrow cellularity (P<0.001), spleen size (P= 0.002), and number of sites of enlarged lymph nodes (P<0.001). Interestingly, the circulating caspase-3 index correlated positively with Rai stage (P=0.03, Kruskal-Wallis) but not IgVH mutation status (P=0.74) or performance status (p=0.72). More importantly, higher circulating caspase-3 index values (>8 pmol/min/1000 lymphocytes/ul) were significantly associated with poor overall survival (P=0.005). However, in multivariate analysis incorporating caspase-3 index along with beta-2 microglobulin level and IgVH mutation status showed that caspase-3 was not predictor of survival (p=0.7). In conclusion, apoptosis as determined using plasma caspase-3 activity is low in CLL. However, high circulating caspase-3 activity index values appear to reflect more aggressive disease. Further studies are needed to explore the possibility that this circulating caspase-3 index reflects the proportion of cells that are transformed into larger cells, with consequently higher proliferation and apoptosis rates, in patients with CLL. Disclosures: No relevant conflicts of interest to declare.

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Increased CD39 Expression on CD4+ T-Lymphocytes Has Clinical and Prognostic Significance in Chronic Lymphocytic Leukemia,

Blood ◽

10.1182/blood.v118.21.3895.3895 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3895-3895

Author(s):

Yair Herishanu ◽

Inbal Hazan-Hallevi ◽

Sigi Kay ◽

Varda Deutsch ◽

Aaron Polliack ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Anti Inflammatory

Abstract Abstract 3895 Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes pro-inflammatory extra-cellular ATP, generates anti-inflammatory adenosine and also protects regulatory T cells from ATP-induced cell death. In this study we investigated the clinical significance of CD39 expression on CD4+T-cells in 45 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4+CD39+ lymphocytes were increased in the peripheral blood of patients with CLL (4.6%±2.28 vs. 17.3%±12.49, respectively, p=0.004), and correlated with advanced stage of disease (9.72%±5.76, 18.15%±12.03 and 25.90%±16.34, of CD4+ lymphocytes, in patients with Rai stages 0, 1+2 and 3+4, respectively, p=0.019). CD4+CD39+ cells were also higher in patients with CLL who needed therapeutic intervention (untreated; 12.99%±10.63 vs treated; 22.21%±12.88, p=0.01) and in those who were ZAP70+ or had b2-microglobulin levels>3g/L. There were more CD4+CD39+ lymphocytes in the bone marrow compartment (22.25%±16.16) than in the peripheral blood (16.60%±15.84, p=0.009). In-vitro studies showed that CD39 can be induced on CD4+cells by exposure to ATP or indirectly, following B-cell receptor (BCR) engagement (CD4+CD39+ lymphocytes increased by 1.56 fold, in the BCR engaged samples compared to their paired controls; 20.27%±11.3 vs. 13%±9.42, respectively, p=0.0006). Conclusions: Increased CD39 expression on CD4+ T-lymphocytes in CLL associates with an aggressive disease. This may reflect the ability of the leukemic cells to suppress the surrounding immune environment, and contribute to a poorer prognosis. CD39+ may also serve as a future target for the development of novel therapies with immune modulating anti–tumor agents in CLL. Disclosures: No relevant conflicts of interest to declare.

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CD49d Is The Strongest Flow Cytometry-Based Predictor Of Overall Survival In Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v122.21.672.672 ◽

2013 ◽

Vol 122 (21) ◽

pp. 672-672 ◽

Cited By ~ 1

Author(s):

Pietro Bulian ◽

Tait D Shanafelt ◽

Chris Fegan ◽

Antonella Zucchetto ◽

Lilla Cro ◽

...

Keyword(s):

Flow Cytometry ◽

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Research Funding ◽

Early Stage ◽

Cox Model ◽

Univariate Analysis ◽

Lymphocytic Leukemia ◽

Bivariate Analysis ◽

The Impact

Abstract Purpose Although CD49d is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL), definitive validation evidence is lacking. A worldwide multi-center analysis was performed using published and unpublished CLL series to evaluate the impact of CD49d as overall survival (OS) and treatment free survival (TFS) predictor. Patients and Methods A training/validation strategy was chosen to find the optimal CD49d cut-off. The hazard ratio (HR) for death and treatment imposed by CD49d was estimated by pooled analysis of 2,972 CLL, and Cox analysis stratified by center and stage was used to adjust for confounding variables. The importance of CD49d over other flow cytometry-based prognosticators (CD38, ZAP-70) was ranked by recursive partitioning. Results Patients with ≥30% of neoplastic cells expressing CD49d were considered CD49d+. The decrease in OS at 5 and 10-years among CD49d+ cases was 7% and 23% (decrease in TFS 26% and 25% respectively). The pooled HR of CD49d for OS was 2.5 (2.3 for TFS) in univariate analysis. This HR remained significant and of similar magnitude (HR=2.0) in a Cox model adjusted for clinical and biological prognosticators. Hierarchical trees including all cases, or restricted to early stage or patients ≤65 years, always selected CD49d as the most important flow-cytometry-based biomarker, with negligible additional prognostic information added by CD38 or ZAP-70. Consistently, by bivariate analysis, CD49d reliably identified patients' subsets with poorer outcome independent of CD38 and ZAP-70. Conclusions In this analysis of ∼3000 patients, CD49d emerged as the strongest flow cytometry-based predictor of OS and TFS in CLL. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Burger:Pharmacyclics: Research Funding.

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del(17p13.1) in Chronic Lymphocytic Leukemia Confers Poor Prognosis Even at Low Percentage Involvement and Increases Proportionately with Increase in Clonal Involvement.

Blood ◽

10.1182/blood.v110.11.2073.2073 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2073-2073 ◽

Cited By ~ 1

Author(s):

Leslie A. Andritsos ◽

Jeffrey Jones ◽

Gerard Lozanski ◽

Thomas S. Lin ◽

Kristie A. Blum ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Proportional Hazards ◽

Chromosomal Abnormalities ◽

Prognostic Significance ◽

Continuous Variable ◽

Cox Proportional Hazards ◽

Lymphocytic Leukemia ◽

Fish Analysis ◽

Time To Progression

Abstract Background: The del(17p13.1) abnormality has been shown to have great prognostic significance in chronic lymphocytic leukemia (CLL), predicting a shorter time to progression and decreased overall survival when compared with other chromosomal abnormalities and a normal karyotype. We sought to assess the significance of low percentage del 17p in untreated patients with CLL. Patients and Methods: Patients with B-cell CLL who had received no prior therapy were eligible for evaluation. At the time of initial visit, baseline variables were collected including patient demographics, Rai stage, peripheral blood immunophenotyping, routine cytogenetic evaluation and FISH analysis (described below), and beta-2-microglobulin. Patients were followed for time to progressing (defined as need for first therapy) and overall survival. Cytogenetic and FISH analyses were performed according to standard laboratory methods. Probes for FISH were D12Z1 (12 centromere), TP53 del(17p13.1), del(11q22.3) and D13S319 del(13q14), (all from Abbott Molecular) and were used according to manufacturer directions. For each sample, 200 nuclei were analyzed per probe, 100 nuclei by each of two analysts. The association between percentage del(17p13.1) and time to progression, defined as time from FISH to initiation of treatment, was explored using Cox proportional hazards regression. For purposes of analysis, percentage del(17p13.1) was alternately considered as both a continuous and dichotomous variable (various cut points at 5% increments). Results: From August 2001 to February 2006, 54 untreated patients with CLL were found on initial evaluation to have del(17p13.1) ≥ 5% of nuclei analyzed. The median age was 61 (range 45–90), with 36 males and 18 females. At the time of initial FISH analysis, 14 patients had Rai stage 0 disease, 22 had Rai stage 1 disease, 10 had Rai stage 2 disease, and 8 had Rai stages 3 or 4 disease. 50 patients were evalualuable for time to progression and overall survival. Considered as a continuous variable, del(17p13.1) percent positive was associated with a significantly increased hazard for shorter time to progression. For each 10% increase in del(17p13.1) percentage, there was a 20% increase in the hazard for shorter time to disease progression [HR 1.20, 95%CI(1.05, 1.37), p=0.007]. Using alternative cutpoints for a positive test, del(17p13.1) percentage was associated with an increased hazard for TTP at all percentages greater than 10, an association that became statistically significant at 25%. Conclusion: del(17p13.1) in CLL predicts for shorter time to first treatment even at lower percentages, with the hazard ratio increasing with increasing percentage. Further study in a larger patient sample is warranted to determine whether del17p should be considered significant even at lower percentages not currently defined as “positive.” Figure Figure

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Prognostic Stratification of Patients with Chronic Lymphocytic Leukemia Based on Quantitative Flow Cytometry Evaluation of ZAP-70 Versus Homology of IgVH Plot.

Blood ◽

10.1182/blood.v112.11.2081.2081 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2081-2081

Author(s):

Marti n Klabusay ◽

Viera Kuhrova ◽

Viera Hrabcakova ◽

Petr Coupek ◽

Jiri Mayer

Keyword(s):

Flow Cytometry ◽

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Technical Problems ◽

Mutation Status ◽

New Approach ◽

Prognostic Stratification ◽

Group A ◽

Group B ◽

Quantitative Flow

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is the most common type of leukemia in the adult population. It has a heterogeneous behavior and variable prognosis. While some patients experience indolent disease requiring no therapy for many years, others demonstrate a more aggressive type unresponsive to therapy. An accurate prognostic stratification is essential for optimizing the therapeutic strategy. Many prognostic factors are presently known, but their relationships and significance are often not clearly understood. Several markers have been identified, including IgVH mutation status and ZAP-70. Mutation of IgVH (less than 98% homology with the germline sequence) is correlated with better prognosis. Expression of ZAP-70, which is a cytoplasmic ζ-associated tyrosine-kinase essential for T-cell receptor signal transduction, is associated with more rapid disease progression and shorter survival. However, its detection by flow cytometry has many technical difficulties, resulting in high interlaboratory variability. The expression of intracellular ZAP-70 in 217 patients with diagnosis of B-CLL was determined using a new approach: quantitative flow cytometry on CD5+19+ tumor cells. Other laboratory and clinical parameters were evaluated, including gender, age, type and number of therapies, CD38 expression, cytogenetics and mutation status of IgVH. The expressions of ZAP-70 and CD38 were measured in molecules of equivalent soluble fluorochrome (MESF units). The results were correlated with mutation status of IgVH. Patients were divided into two groups by cluster analysis in a plot of IgVH homology versus ZAP-70 MESF (left panel). Overall survival curves for these groups are shown (right panel). It could follow that patients above the diagonal (triangles, group B) in the IgHV versus MESF plot have significantly poorer prognoses (p=0.0001) than do patients below this diagonal (circles, group A). Expression of CD38 above 15000 MESF showed worse prognosis for patients in the group B (p=0.035). Only three of 61 patients with chromosomal aberrations associated with poor prognosis (del11q and del17p) were found in the group A, but all of them had del13q present, as well. The authors introduced a new approach for evaluating ZAP-70 expression in B-CLL cells using quantitative flow cytometry that is easily standardized and not burdened with technical problems. Plotting IgVH homology (%) versus ZAP-70 quantitative expression (MESF) could clearly divide patients according to their prognoses. Figure Figure

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The Earliest Activation Marker CD69 Is An Independent and Strong Progression Indicator in B-Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.2359.2359 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2359-2359

Author(s):

Giovanni Del Poeta ◽

Maria Ilaria Del Principe ◽

Cristina Simotti ◽

Francesco Buccisano ◽

Luca Maurillo ◽

...

Keyword(s):

Flow Cytometry ◽

Multivariate Analysis ◽

Prognostic Factor ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Significant Prognostic Factor ◽

Therapeutic Decisions ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abstract 2359 Poster Board II-336 B-cell chronic lymphocytic leukemia (B-CLL) exhibits features of activated and antigen-experienced B-lymphocytes and CD69 overexpression resembles B cells at an earlier and greater state of activation (Damle, 2002 and 2007). Moreover, the recent in vitro generation of anti-CD69 monoclonal antibodies able to inhibit either tumor growth or enhance NK cell function (Esplugues, 2005) prompt us to extensively evaluate CD69 antigen in our B-CLL cases. The primary endpoints of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD69; 2) to assess the additive prognostic value of CD69 and ZAP-70 and finally 3) to confirm CD69 as an independent prognostic factor. We investigated 401 patients (pts), median age 65 years, 219 males and 182 females. With regard to modified Rai stages, 120 pts had a low stage, 263 an intermediate stage and 18 a high stage. ZAP-70 and CD69 were determined by multicolor flow cytometry, fixing the cut-off values > 20% and >30%, respectively. ZAP-70+ and CD69+ pts were 166/401 (41%) and 108/401 (27%), respectively. CD69 lower than 30% was significantly associated with low Rai stage (105/120; P<0.0001), lymphocyte doubling time >12 months (258/332; P=0.00001), beta-2 microglobulin (B-2M) <2.2 mg/dl (181/223; P=0.00004) and soluble CD23 <70 U/ml (196/240; P<0.00001). There were significant correlations between lower CD69 and IgVH gene mutated status (283 total cases, 150/210; P=0.0001) or low risk (normal or 13q-) FISH cytogenetics (296 total cases, 149/213; P<0.00001). Equally, a strict association was found between lower CD69 and lower ZAP-70 (185/293; P=0.0003). With regard to clinical outcome, both a shorter PFS and OS were observed in ZAP-70+ pts (6% vs 56% at 12 years and 30% vs 95% at 16 years; P<0.00001) as well as in CD69+ pts (9% vs 49% at 14 years, P<0.00001 and 49% vs 75% at 16 years; P=0.00002). Noteworthy, ZAP-70 and CD69 showed additive prognostic properties, since ZAP-70 <20% plus CD69 <30% identified a B-CLL subset at better prognosis with regard to PFS (65% vs 2% at 12 years; P<0.00001, Figure) and OS (97% vs 35% at 14 years; P<0.00001). The two discordant subsets (CD69+ZAP-70 negative and CD69-ZAP-70+) showed an intermediate outcome (Figure). In multivariate analysis of PFS, CD69 (P=0.001) and ZAP-70 (P=0.005) together with cytogenetics and B-2M were confirmed to be independent prognostic factors. On the other hand, with regard to multivariate analysis of OS, age > or < 60 years resulted to be the most significant prognostic factor (P=0.004) followed by CD38 (P=0.01), IgVH status (P=0.02) and ZAP-70 (P=0.04). In conclusion, CD69 antigen, determined by flow cytometry, should be considered a novel important prognostic parameter in B-CLL and its easy and rapid laboratory determination may allow us to identify early progressive pts in order to take timely therapeutic decisions. Disclosures: No relevant conflicts of interest to declare.

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