IgVH Gene Mutational Status Research Articles

Sex Chromosome Loss May Represent a Disease-Associated Clonal Population in Chronic Lymphocytic Leukemia (CLL),

Abstract 3534Sex chromosome loss is observed in the healthy population and increases with advancing age. It is also observed in hematological malignancies with variable clinical value. Its significance as a disease marker in CLL is not clearly defined. We undertook this study to evaluate whether Y or X loss is an age- and/or a CLL-associated abnormality, since it has not yet been reported.Out of 198 patients with stage B/C CLL analyzed by karyotype (K), 6 (3%) exhibited a loss of sex chromosome (Sutton et al, blood 2011). We also identified 14/745 (2%) additional patients in our CLL-database (2002–11). All patients had a Matutes score ≥ 4. K was performed on peripheral blood using TPA until 2005, and then CPG + IL2. FISH analyses were carried out to detect trisomy 12 and 11q22 (ATM), 13q14, 17p13 (TP53), X/Y deletions. IGVH gene mutational status was analyzed by PCR with a sequence homology cutoff of 98%. B- and T-cells were sorted by flow cytometry from peripheral blood of 5 patients (purity ≥ 94%) using CD19+ and CD3+ respectively. Polynuclear and mononuclear cells were enriched by Ficoll (purity ≥ 99%) for 4 patients.There were 13 males and 7 females (n =20). At the time of the K, there were 11 Binet stage A and 9 stage B, and median age was 64 (36–78). Regarding IGVH status, 9/12 (75%) cases were mutated. We observed 13 -Y and 7 -X, chromosome loss being the sole abnormality in 10 (50%) cases. The percentage of cells with -X or -Y ranged between 3 to 20/20 mitoses. K was complex (≥ 3 abnormalities) in 4 cases. When associated with another chromosomal abnormality, -X or -Y was the primary change in 2 cases, in the same clone in 7 cases, and as a sub-clone change in one case. FISH identified 16/20 (80%) del13q, 4/20 (20%) tri12, 3/20 (15%) delATM, 2/20 (10%) delTP53. Among del13q, there were 8 monoallelic, 7 concomitant mono- and biallelic, and 1 biallelic deletion. Sex chromosome loss was confirmed by interphase FISH in 14 patients, with percentage of loss between 5 to 84% (median: 68%). Co-hybridization with two probes including the X/Y probe was performed for 13 patients. The two analyzed abnormalities were present in independent clones in 4 cases, in the same cells in 2 cases; -X or -Y appeared as the primary change in 4 cases, and as a sub-clone change in 3 cases. The analyses of polynuclear and mononuclear populations in 4 patients showed a maximum of 2% of polynuclear cells with X or Y loss, whereas the mononuclear cells exhibited a significant higher loss frequency (range 6–87%, p =.03). The analyses of B- and T-cells in 5 samples showed a significant increase in losses frequency in B-cells (range 88–96%) compared to T-cells (2%, 2%, 3%, 4%, 6%) (p =.008).The incidence of -X in peripheral blood lymphocytes is about 3% of the cells in healthy women aged 16–50, rising to 5% in women aged over 85 years. The incidence of Y loss in men is distinctly lower, with a frequency less than 1% of the cells before 85 years (Wodja et al, J Appl Genet, 2003). The median of cells with sex chromosome loss in our series is 68%, with only 2 patients with low percentage: a 66-year old female (5%) and a 68-year old male (6%). However these percentages are higher than those observed in healthy people. Moreover, we never observed the loss of a sex chromosome in more than 2% in polynuclear cells of any of the patients, indicating that sex chromosome loss in CLL is associated with the tumor cells. It is not clear whether the low proportion of loss of sex chromosome in CD3+ cells that exceed in one case only slightly our laboratory threshold of 5% is due to contamination. Sex chromosome loss may occur during K evolution, as well as the acquisition of additional abnormalities may occur in cells with primary sex chromosome loss. Even if loss appears through an age-related effect in elderly cells, our data support an oncogenic property of this abnormality. One patient with –Y as the sole aberration in CLL, has developed a myelodysplastic syndrome upon treatment, with complex karyotype without -Y, suggesting that Y loss had been associated to CLL. In addition, del13q, especially when biallelic, appears to be closely associated to the loss of sex chromosome, as compared to our database (del13q: 16/20 (80%) vs 141/279 (50%), p =.01; biallelic del13q: 8/16 (50%) vs 24/141(17%), p =1×10−5).In conclusion, sex chromosome loss has to be considered as a clonal abnormality in CLL, is significantly associated with biallelic 13q deletion, and may participate to oncogenic transformation. Disclosures:No relevant conflicts of interest to declare.

Morphological Atypia Represents Favorable Prognosis of Chronic Lymphocytic Leukemia in Japanese Patients

Abstract 4594 Background:B-chronic lymphocytic leukemia (B-CLL) is rare in Japan. Therefore, only a few data are available on the clinical characteristics of B-CLL for Japanese patients (pts). We reported that the rate of atypical CLL appeared to be higher in Japanese pts than that of Caucasians. Patients and Methods:This nation-wide study was performed from Oct. 2003 to March 2008, and was supported by Japanese Elderly Leukemia and Lymphoma Study Group. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Boards of the core members’ hospitals. The pts with CLL and CLL-like disease were registered. Precursor leukemia and adult T-cell leukemia/lymphoma were excluded. The smears of peripheral blood and bone marrow of all the registered pts were examined by the Diagnosis Committee by using the 4th edition of WHO classification and FAB classification. FAB classification defines typical CLL as prolymphocytes≤10% and atypical CLL including CLL/PLL as 11%≤prolymphocyte<55% and CLL mixed as prolymphocytes<10% and pleomorphic lymphocyte>15%. Mantle cell lymphoma was excluded by means of cytogenetics, fluorescent in situ hybridization of CCND1-IGH, and immunohistochemistry of CCND1. Results:One hundred and thirty-one evaluable pts were registered. There were 96 pts with B-CLL, 2 SLL, 1 B-PLL, 3 hairy cell leukemia, 16 leukemic phase of other B-cell lymphomas. Thirteen pts had NK or T cell leukemia/lymphoma with 7 T-LGL, 4 T-PLL, 1 NK-LGL, and 1 Sezary syndrome. Among B-CLL, there were 62 pts with typical morphology (typical CLL), 18 with atypical morphology (atypical CLL), 16 with intermediate morphology between typical and atypical CLL (intermediate CLL). The differences among 3 subgroups are shown in Table. No differences were found between the 3 morphological subgroups for age, sex, stage (modified Rai and Binet classification), hemoglobin level, platelet counts, the presence of hepatomegaly, splenomegaly and lympoadenopathy, ECOG-PS and the rate of treatment requirement. However, the incidence of CD5 and CD23 expression was higher in typical CLL than in atypical/intermediate morphology groups (P<0.001), i.e. 7% of the pts with atypical CLL showed co-expression of CD5 and CD23. The overall survival (OS) of typical CLL pts was lower than that of intermediate or atypical CLL pts (Figure). Conclusion:Morphological atypia was more frequent in Japanese B-CLL pts, partly because of the low incidence of typical CLL. The atypical/intermediate CLLs were associated with atypical immunophenotype (absence of CD5 and/or CD23) and favorable prognosis. Prospective study is ongoing to evaluate other prognositic factors including the implication of IgVH gene mutation status and ZAP-70 expression in Japan.TableTypicalIntermediateAtypicalPPatients No.621618Age, median6765670.50Sex (male/female)1.301.292.60.47CD20+98.4%100%88.9%0.16CD5+98.4%81.30%77.8%0.003CD5+23+86.5%50.0%7.0%<0.001No treatment45%50%61%0.49Outcome(died)29.8%0%12.5%0.018 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Relevance of IgVH Gene Somatic Hypermutation and Interphase Cytogenetics in Lymphomatous Presentation of Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are regarded as the same entity, with SLL restricted to tissue cases featuring no leukemic phase. In this study, the authors evaluate a group of SLL cases for cytogenetic abnormalities and IgVH gene mutational status to illicit differences between CLL and SLL. IgVH gene polymerase chain reaction amplification and subsequent sequencing were preformed on formalin-fixed, paraffin-embedded archival tissue of 44 patients (SLL n = 34 or CLL n = 10). Cytogenetic data, CD38, and ZAP-70 expression were also evaluated for these cases. The data indicate that 9/34 (26%) SLL cases have somatic hypermutation >2%, which is less than the CLL group where 40% were mutated (4/10). Cytogenetic abnormalities were seen in 58% of the SLL cases with many showing abnormalities associated with favorable to intermediate prognosis. The authors' attempt to compare CLL with SLL with regards to cytogenetic and IgVH mutational status shows no statistically significant difference.

Sequential SNP6.0 Microarray Analysis In Rai Stage 0 CLL Patients with Either Stable or Progressive Identifies Genomic Evolution In Both Cohorts

AbstractAbstract 1696Sequential karyotypic and/or FISH analysis in patients with CLL show evidence of genomic evolution in 10 – 20% of cases. Common secondary abnormalities include loss of the second allele of 13q14, additional trisomies or translocations involving the immunoglobulin gene loci in patients with trisomy 12 and complex karyotypic abnormalities in patients with loss and/or mutation of the p53 and ATM genes. However the clinical significance of genomic evolution in patients presenting with early stage disease remains uncertain. Nor is it clear whether the use of high resolution genomic arrays can detect additional clinically relevant abnormalities in this cohort of patients. To address these issues we performed sequential genomic studies using the affymetrix SNP 6.0 platform (Affymetrix, Santa Clara, CA), G-banding and FISH in 29 patients presenting with either stage A0 CLL or MBL in whom IGVH gene mutational status and CD38 and ZAP 70 expression were known. In each case, Partek Genomic Suite (Partek Incorporated, St Louis, MS) was used to identify acquired copy number alterations (CNAs) and copy number neutral LOH events (CNNLOH) by comparing the disease presentation (PT) and subsequent (FU) SNP tumor profiles to each other and to matched germ-line DNA extracted from buccal cells. Patients were subdivided into those that subsequently exhibited stable disease for a minimum of 5 years (n=9) with a median time between sampling of 8 years (range 2 – 15) or those that developed progressive disease (n=20) with a median time between sampling of 4 years (range 1 – 10). 8 patients with progressive disease were treated prior to the follow up sample. All stable cases and 13/20 with progressive disease had mutated IGHV genes. The concordance between G-banding, FISH and SNP array data was >99%. In each case, a more complex genome was present at FU. The acquisition of additional CNAs was not associated with IgVH mutation status, ZAP-70, CD38 positivity or whether a patient had been treated. When these data were stratified for whether a patient subsequently exhibited stable or progressive disease, the following observations were made. Firstly, there was no difference in the acquisition of small CNAs (<1Mb) between stable or progressive patients. However, the mean number of additional large CNAs (>1Mb) per patient and the total genomic size of these additional changes were greater in progressive cases; stable and progressive cases exhibited a mean of 0.67 and 1.1 additional CNAs at FU and a mean total imbalance of 2.33Mb and 16.9Mb, respectively. In this limited series these differences did not reach statistical significance. Interestingly, the frequency of copy number variations (CNV, annotated in the Database of Genomic Variation; http://projects.tcag.ca/variation/) was higher in cases that went on to develop progressive disease (P=0.002). The size of established recurrent CNAs also changed between time-points. Most notably, several cases exhibited larger 13q14 deletions at FU. Two patients with deletion or CNNLOH of 17p displayed a more complex genome at FU, characterized by multiple CNAs larger than 1Mb. One patient acquired CNNLOH on 17p at FU in addition to five additional CNAs (>1Mb). Conversely, the degree of complexity did not increase in a patient with an ATM deletion. Six cases without an apparent abnormality of ATM or TP53 showed complex genomes at PT and additional CNAs (mean 2.8) at FU. A series of novel, recurrent CNAs were observed exclusively in FU samples, including duplications of 13q and 15q observed in 38% and 53% of patients, respectively. In conclusion, using high resolution microarray analysis we show genomic evolution in early stage patients with predominantly good risk biomarkers who pursued either an indolent or progressive course. However, our data suggests that larger acquired CNAs may be more prognostically relevant than smaller regions. We show preliminary data implicating CNVs in the pathogenesis of CLL and we have also identified a series of novel acquired regions of CNAs that may harbor cancer genes which it is hoped will contribute to our understanding of the pathophysiology of CLL and/or have clinical relevance. Disclosures:No relevant conflicts of interest to declare.

The Kinase Inhibitor, PCI-32765, Demonstrates Activity In Chronic Lymphocytic Leukemia Cells Independent of Microenvironmental Survival Signals

AbstractAbstract 1385Bruton tyrosine kinase (Btk), a member of the Tec family kinases, has a well-characterized role in B-cell antigen receptor (BCR) signaling and B-cell activation. Btk is activate by Src family kinases which lead to downstream activation of essential cell survival pathways such as NF-κB and MAP kinase. Although Btk is expressed in multiple hematopoietic cells, the primary defect in Btk knockout mice is B-cell specific, suggesting a more selective B-cell function consistent with its role in the BCR signaling pathway. Btk was identified in an unbiased screen as an essential signaling kinase for survival of certain lymphomas including diffuse large B-cell lymphoma, but no information is available about its role in CLL cell survival. Inhibition of Btk by PCI-32765, a selective irreversible Btk inhibitor, has demonstrated promising clinical activity in an ongoing phase 1 study in B-cell non-Hodgkin lymphoma. We therefore hypothesized that inhibition of Btk would induce cytotoxicity in B-CLL cells. BTK protein expression was observed in CLL cells, but not NK or T-cells as previously reported. Surprisingly, significant variability in BTK expression was noted among different CLL patient samples. Treatment of primary CLL cells with PCI-32765 at concentrations ranging from 0.1–10μM resulted in significant cell killing compared to untreated control. At a 10μM concentration, PCI-32765 resulted in a median 73% viable cells at 48 hours as detected by annexin V/propidium iodide flow cytometry (n=55). Although again, considerable variability was observed among patient samples (range 93.5–5.5%). We found no correlation between response to PCI-32765 and prognostic factors such as interphase cytogenetics or IgVH gene mutational status, suggesting a potential clinical benefit to even high-risk CLL patients. To determine the selectivity of PCI-32765, we evaluated cytotoxicity of lymphocytes derived from healthy volunteers. At 10μM, PCI-32765 resulted in a median 90% viable normal B-cells with little variability (n=5), showing substantially lower cytotoxicity in normal B-cells relative to CLL tumor cells. The cytotoxicity observed with PCI-32765 in CLL cells was accompanied by PARP cleavage and the induction of caspase 3 activity, and PCI-32765-mediated cell death was blocked by the pan-caspase inhibitor z-VAD-fmk (n=3). These results indicate that PCI-32765 is dependent on caspase activation for its ability to induce apoptosis in CLL cells. Further studies to determine the apoptotic mechanism showed that PCI-32765 treatment (10μM) partially reversed CD40L- or CpG-induced phosphorylation of ERK1/2 and also caused an increase in endoplasmic reticulum-derived intracellular calcium flux in CLL cells (n=8). Microenvironmental factors such as CD40L, TNF-α, IL-6 and BAFF can induce activation of CLL and/or decrease spontaneous apoptosis. Treatment of CLL cells with PCI-32765 prevented the protective anti-apoptotic effect promoted by these cytokines, suggesting that this agent can overcome the protection induced by microenvironmental stimuli. Similarly, co-culture of CLL cells on the human stromal cell line HS-5 significantly decreases the spontaneous apoptosis of CLL cells. PCI-32765 treatment was able to overcome this stromal protection, and induced apoptosis comparable to cells cultured in suspension (median cytotoxicity 86% vs. 84%, respectively; 10μM PCI-32765 at 48 hr, n=5). This suggests that the cytotoxic effect elicited by PCI-32765 will not be significantly diminished by the presence of an in vivo microenvironment. Taken together, our results suggest that PCI-32765 acts directly on CLL cells to induce apoptosis and helps to block microenvironment-derived protection. These investigations demonstrate the efficacy of targeting Btk in CLL and provide preclinical validation for ongoing Phase 1/2 clinical trials for the treatment of this disease.This work is supported by the Leukemia and Lymphoma Society, D. Warren Brown Foundation and The OSU Leukemia SPORE grant funded by the NCI. Disclosures:Buggy:Pharmacyclics, Inc.: Employment. Hamdy:Pharmacyclics, Inc.: Employment.

Research progress on prognostic markers of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) remains the most common adult leukemia. The recent progress on research of molecular and cellular genetics of CLL promotes the development of the diagnosis, treatment and prognosis for CLL patients. IGVH gene mutation status is the most important prognostic marker for CLL patients. Zeta-chain-associated protein kinase (ZAP-70) can be used as a surrogate marker for IGVH mutation status. CD38 is a type II transmembrane glycoprotein promoting B cell activation and proliferation, which can improve the survival of CLL cells and enhance their proliferation, so it also can be used as an independent prognostic indicator for CLL. Chromosome aberrations are found in more than 80% of CLL cases. The most frequent abnormalities are losses of chromosomal material, with deletions in band 13q14 being the most common. The most common gains of chromosomal material are trisomies 12q. Human leukocyte antigen G (HLA-G) is a non-classical HLA-I gene. Increased expression of HLA-G leads to the malignant progression of CLL, significantly shortens survival, indicating HLA-G might serve as a prognostic marker in CLL. Toll-like receptors (TLA) are important component of natural immunity. The combination of TLR agonists and release chemotherapy, monoclonal antibodies and tumor vaccines would bring a breakthrough for the treatment of CLL.

AID protein expression in chronic lymphocytic leukemia/small lymphocytic lymphoma is associated with poor prognosis and complex genetic alterations

The biological behavior of chronic lymphocytic leukemia and small lymphocytic lymphoma is unpredictable. Nonetheless, non-mutated IgVH gene rearrangement, ATM (11q22–23) and p53 (17p13) deletion are recognized as unfavorable prognosticators in chronic lymphocytic leukemia. The mRNA expression of activation-induced cytidine deaminase (AID), an enzyme indispensable for somatic hypermutation processes, was claimed to be predictive of non-mutated chronic lymphocytic leukemia cells in blood. Here, we evaluated AID protein expression compared with known molecular and immunohistochemical prognostic indicators in 71 chronic lymphocytic leukemia/small lymphocytic lymphoma patients using a tissue microarray approach. We found AID heterogeneously expressed in tumor cells as shown by colocalization analysis for CD5 and CD23. Ki-67 positive paraimmunoblasts of the proliferation centers displayed the highest expression. This observation is reflected by a significant association of AID positivity with a high proliferation rate (P=0.012). ATM deletion was detected in 10% (6/63) of patients and p53 deletion in 19% (13/67) of patients. Moreover, both ATM (P=0.002) and p53 deletion (P=0.004) were significantly associated with AID. IgVH gene mutation was seen in 45% (27/60) of patients. Twenty-five percent (17/69) of patients with AID-positive chronic lymphocytic leukemia/small lymphocytic lymphoma displayed a shorter survival than AID-negative chronic lymphocytic leukemia/small lymphocytic lymphoma patients (61 vs 130 months, P=0.001). Although there was a trend, we could not show an association with the IgVH gene mutation status. Taken together, our study shows that AID expression is an indicator of an unfavorable prognosis in chronic lymphocytic leukemia/small lymphocytic lymphoma patients, although it is not a surrogate marker for the IgVH status. Furthermore, the microenvironment of proliferation centers seems to influence AID regulation and might be an initiating factor in its transformation.

Melanoma and Non-Melanoma Skin Cancer in Patients with Chronic Lymphocytic Leukemia.

Abstract 1268Poster Board I-290 PurposeThere are reports of an increased risk of skin cancers in patients with B-Chronic Lymphocytic Leukemia (CLL). These skin cancers include basal cell and squamous cell carcinoma. This analysis was performed to more completely define the prevalence of skin cancers in patients in the Mayo Clinic Rochester CLL database and to look for contributing factors. MethodsThe Mayo Clinic Rochester CLL Database includes all patients with a diagnosis of CLL since January 1995 seen in the Division of Hematology and who have signed institutional review board approved consents for research. For this study, 2240 patients were analyzed to compare differences in characteristics between CLL patients with and without skin cancer. Chi-square statistics were used to compare qualitative variables (age categories, gender, referral status, ALC categories, CD38, ZAP-70, IgVH gene mutation status, FISH categories, Rai stage), and t-tests were used for quantitative variables (age at diagnosis, ALC values). Overall survival (OS) and time to first treatment (TFT) analyses were performed with results being displayed using Kaplan-Meier curves and p-values calculated using a log-rank test. Prevalence of melanoma among CLL patients was compared to the age-adjusted prevalence of melanoma in individuals in the Iowa SEER registry. ResultsMedian follow-up for the 2240 patients diagnosed between 1/1/1995 and 8/11/2009 was 4.6 years. In aggregate, 293 (13.1%) patients were found to have non-melanoma skin cancer (squamous cell carcinoma or basal cell carcinoma) cancer. The diagnosis of non-melanoma skin cancer occurred before the CLL diagnosis in 39% and at or after the diagnosis of CLL in 61%. There were 57 (2.5%) cases of melanoma in association with CLL. The diagnosis of melanoma occurred before the CLL diagnosis in 38% and at or after the diagnosis of CLL in 62%.The prevalence of non-melanoma skin cancer and melanoma skin cancer were both higher in non-referred (geographically regional) CLL patients than referred CLL patients (16.6% vs. 11.4%, p<0.001 for non melanoma; 2.0% vs. 3.6%, p=0.03 for melanoma). The prevalence of melanoma in CLL patients was higher than that of age-adjusted prevalence for individuals in the Iowa SEER registry (2.5% vs. 0.03%; p<0.001).We next evaluated the relationship between CLL patients with skin cancer and demographic characteristics, prognostic parameters, and CLL related treatment. The risk of non-melanoma skin cancer in CLL was found to be associated with age (median age at diagnosis with skin cancer 67.6 vs. without skin cancer 63.3, p<0.001) and with sex (males 15.1% vs females 8.9%, p <0.001). There was no statistical significant difference in frequency of non-melanoma skin cancer associated with absolute lymphocyte count (ALC), Rai stage, CD 38, Zap 70, IgVH gene mutation status, FISH, or treatment history. The risk of melanoma in CLL was found to be associated with age at diagnosis (median age with melanoma 69.9 vs. without melanoma 63.8, p=0.002) and CD38 (positive 1.3% vs negative 3.1%, p=0.03). There was no statistically significant difference associated with gender, ALC, Rai stage, Zap 70, IgVH gene mutation status, FISH, or treatment history.Since the presence of skin cancer could be a marker of immune dysregulation we hypothesized skin cancer may be associated with clinical outcome of CLL. Accordingly, we evaluated the relationship between non-melanoma skin cancer and melanoma skin cancer and TFT and OS. Contrary to our hypothesis, TFT (median 6.0 years vs. 4.9; p=0.04) and OS (median 10.8 years vs. 9.7; p=0.02) of patients with non-melanoma skin cancer were both longer than those without non-melanoma skin cancer. No differences in TFT (p=0.06) or OS (p=0.66) were observed among patients with melanoma compared to those without melanoma. ConclusionsWe find in our cohort of CLL patients a higher prevalence of melanoma than the general population. Risk of melanoma and non-melanoma skin cancer among patients with CLL does not appear to be related to CLL characteristics, with the exception of CD 38, or CLL outcome. The diagnosis of melanoma and non-melanoma skin cancer in patients with CLL does not appear to be a risk factor for either CLL specific outcomes (TFT) or shorter survival. DisclosuresKay:Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding. Zent:Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding.

The Cyclin Dependent Kinase Inhibitor SCH 727965 Demonstrates Promising Pre-Clinical and Early Clinical Activity in Chronic Lymphocytic Leukemia.

Abstract 886Patients with chronic lymphocytic leukemia (CLL) having del(17p13.1), complex karyotype, or fludarabine-refractory disease have few therapeutic options. The cyclin-dependent kinase (CDK) inhibitor flavopiridol (Alvocidib) has been demonstrated to be very effective in genetically high-risk and fludarabine-refractory CLL cases, with a response rate of approximately 50%. We pursued pre-clinical and now early clinical studies of SCH 727965. SCH 727965 is a selective inhibitor of CDK 1, 2, 5 and 9 (IC50 of < 5nM) that shows in vitro and in vivo anti-tumor activity in a variety of pre-clinical models. Notably, SCH 727965 has an improved therapeutic index over other CDK inhibitors such as flavopiridol. A solid tumor phase I study with SCH 727965 administered by 2-hour IV infusion on days 1, 8 and 15 of a 28-day cycle has been completed, resulting in a recommended phase II dose of 12 mg/m2. Our pre-clinical studies with SCH 727965 demonstrate potent induction of apoptosis in CLL patient cells following a 2-hour incubation, with an LC50 of 240 nM. This effect is independent of prior treatment status, IgVH gene mutational status, and high-risk cytogenetic abnormalities. Importantly, minimal cytotoxicity toward normal T cells is observed. Similar to other CDK inhibitors in CLL, SCH 727965 promotes rapid down-regulation of the pro-survival gene MCL1 at both the mRNA and protein levels. Stimulation of CLL cells with CD40L mimics the lymph node microenvironment and increases resistance of CLL cells to drug-induced apoptosis (Smit et al., Blood 109:1660-68, 2007). However, co-treatment of CLL cells with SCH 727965 plus CD40L does not abrogate the apoptotic effect of SCH 727965. Based upon these promising data, a disease-specific phase I study of SCH 727965 has been initiated in previously treated CLL patients. This trial started at a dose level below the phase II dose established in the solid tumor study, due to the previous observation of hyperacute tumor lysis in CLL with other CDK inhibitors. SCH 727965 is administered by 2-hour IV infusion on days 1, 8 and 15 of a 28-day cycle. Dosing cohorts are: 5 mg/m2, 7 mg/m2, 10 mg/m2, and 14 mg/m2. Further dose-escalation is allowed, depending on the frequency of dose limiting toxicities observed. To date, we have completed enrollment to cohorts 1 and 2 of the study, with ten patients enrolled. Clinical features of the patients include a median age of 58 (range 43 to 73), 60% Rai stage 3 or 4, and 60% with high-risk genomic features (del17p13, del11q22, or more than two abnormalities). These patients received a median of six (range 1 to 12) prior therapies, and 90% received prior fludarabine therapy. Biochemical evidence of tumor lysis (increased potassium, phosphate, or LDH) post-treatment has been observed in three patients. Common toxicities observed include: nausea, vomiting, diarrhea and fatigue. One patient with pre-treatment neutropenia developed fatal bacterial sepsis during cycle 1 of therapy. Three patients have discontinued therapy due to progressive disease after cycle one, including one patient who died from CLL disease. Two patients discontinued therapy with stable disease. Four patients remain on therapy; one in the lowest dose cohort has demonstrated a partial response by IWCLL 2008 criteria. The other three patients currently are early in the course of planned therapy. The clinical observation of rapid improvement in palpable adenopathy after SCH 727965 treatment is consistent with the laboratory observation of continued sensitivity of CD40L-exposed CLL cells to SCH 727965. Pharmacodynamic studies demonstrate down-modulation of MCL1 in a subset of patients. These data provide promising pre-clinical, and now early clinical, evidence of the potential therapeutic benefit of SCH 727965 in CLL. Dose escalation of SCH 727965 continues and updated results will be presented. This work was supported by the D. Warren Brown Foundation and Specialized Center of Research from the Leukemia and Lymphoma Society. Disclosures:Small:Schering Plough: Employment. Statkevich:Schering Plough: Employment. Bannerji:Schering-Plough: Employment.

Overexpression of the Protein Tyrosine Phosphatase Lyp Reduces the Responsiveness of Chronic Lymphocytic Leukemia B-Cells to B-Cell Receptor Ligation.

Abstract 800CLL B cells display many features that suggest a role for antigen stimulation in the development and progression of the disease. These include the expression of stereotyped B-cell receptors (BCRs), the association between IgVH gene mutation status and prognosis, and the gene-expression profile of antigen-stimulated B cells. In addition, CLL B cells have other BCR-related features that distinguish them from normal B lymphocytes, such as lower levels of surface Ig, less efficient BCR signal transduction and increased basal activity of the proximal BCR signaling molecules Lyn and Syk. We have now investigated whether any of these features are related to aberrant expression or function of the phosphatases SHP-1, SHP-2 and Lyp (PTPN22), which regulate the amplitude and duration of the BCR signal by dephosphorylating various components of the BCR signal transduction unit. These phosphatases are also interesting because mutated or polymorphic variants have been linked to various malignant or autoimmune diseases.We started our study by performing nucleotide sequence analysis of the complete coding region of SHP1, SHP2 and Lyp in 8, 21 and 29 CLL B cell samples, respectively. Overall, only two mutations were identified (an R527C substitution in SHP2 and a Q456E substitution in Lyp, each in a single patient), suggesting that these phosphatases are infrequently mutated in CLL. The previously reported Lyp polymorphisms R620W and R263Q were observed in 2 additional cases. We next investigated expression of these phosphatases in purified CLL and normal B cells by immunoblotting. Expression of SHP1 and SHP2 was relatively uniform in the different CLL B-cells samples (n=42) and was not different from normal B cells (n=4). In contrast, expression of Lyp was markedly higher in most CLL samples, with 35 of the 49 investigated cases exhibiting 2 to more than 10 fold higher levels than normal B cells (n=5) (CLL, mean Lyp levels 4.7, SD +/−3.7; normal B cells, mean Lyp levels 0.9, SD +/−0.1, P=0.022). The mean Lyp levels were somewhat higher in U-CLL than M-CLL (6.0 vs. 3.9) and ZAP-70-positive than ZAP-70-negative cases (5.6 vs. 4.7), but these differences were not statistically significant. Analysis of Lyp expression in various lymphoma B-cell lines (n=9) also did not reveal significant differences with respect to normal B-cells, suggesting that Lyp overexpression is a specific feature of CLL.To determine what are the consequences of Lyp overexpression on BCR signaling, we downregulated Lyp in primary CLL B-cells by RNA interference and investigated activation of BCR signaling molecules following sIgM crosslinking. Downregulation of Lyp resulted in a substantial increase in BCR-induced phosphorylation of Lyn (Y397), Syk (Y352), BLNK (Y84) and ERK (T202/Y204), suggesting that overexpression of this phosphatase may be at least partially responsible for the lower BCR signaling capacity of CLL B-cells.Since Lyp expression can be induced in resting T cells by activation with anti-CD3, we investigated whether BCR stimulation will have a similar effect on CLL B-cells. A two-fold increase in Lyp levels was observed after 24 hours of sustained BCR stimulation with immobilized anti-IgM, whereas transient stimulation with soluble anti-IgM resulted in a 20% decrease in Lyp levels. These effects were specific for Lyp, since no such changes were observed in the expression of SHP1 and SHP2.In summary, this study shows that CLL B-cells specifically overexpress the phosphatase Lyp, and important negative regulator of BCR signaling that has been implicated in the pathogenesis of several common autoimmune diseases. Given the observation that Lyp can be induced by sustained BCR engagement and in view of recent findings that Lyp is also overexpressed in anergic B cells, these data further support the notion that CLL cells are continuously exposed to (auto)antigen in vivo. Disclosures:No relevant conflicts of interest to declare.

Quantification of Recent Bone Marrow B-Cell and Thymic T-Cell Emigrants in Chemotherapy Naïve Patients with Chronic Lymphocytic Leukemia.

Abstract 2353Poster Board II-330 BackgroundChronic lymphocytic leukemia (CLL) results in accumulation of mature, malignant, monoclonal B cells in blood, lymph nodes, spleen, liver, and bone marrow. Patients with CLL have fundamental defects in both humoral and cell-mediated immunity that significantly impact on its clinical course. It is still not known if these immune abnormalities include a decrease of new B- and T-cell mobilization from their production sites. During early lymphocyte development and differentiation, occurring in bone marrow and in thymus, B- and T-cells undergo rearrangements of B-cell and T-cell receptor genes whereby specific chromosomal sequences are excised to produce episomal DNA products identified respectively as T-cell receptor excisional circles (TRECs) and Kappa-deleting recombination excision circles (KRECs). Being stable circular fragments of DNA, TRECs and KRECs do not replicate during the mitotic process required for cell proliferation and, therefore, they are diluted out by cell divisions and are lost when the cells die. Since T-cells leaving the thymus are 70% TRECs+ and KRECs are present randomly in about 50% of neo-produced normal B-cells, the quantification of TRECs and KRECs allows a good estimation of the thymic and bone marrow output. AimTo investigate the extent of neo-synthesis of normal B and T cells in untreated patients with CLL. Patients and MethodsTwelve previously untreated CLL patients were enrolled in this study. M:F ratio was 5:1; median age was 66; 7 patients had mutated IgVH genes, while 5 patients had unmutated IgVH genes; all patients were stage A Binet; FISH analysis including del(13q), del(11q), del(17p) and trisomy 12 detected 13q deletion in 6 patients while the other 6 patients presented no abnormalities. TRECs and KRECs were measured in mononuclear cells isolated from peripheral blood by duplex quantitative Real-Time PCR. This new assay, based on the use of a standard curve prepared with a plasmid containing fragments of TRECs, KRECs and of a reference gene, allows to quantify neo-produced B and T lymphocytes. T-cell subpopulations were determined by flow cytometry as follows: recent thymic emigrants (RTE) as cells with CD4+CD45RA+CCR7+CD31+ phenotype, regulatory T cells (Treg), as CD4+CD25int/highCD127low, RTE-Treg as Treg expressing CCR7+CD31+CD25int/high markers, effector memory (TEM) and central memory (TCM) T cells as lymphocytes displaying CD4+CD45RA-CCR7- and CD4+CD45RA-CCR7+ phenotype. ResultsThe overall number of CD4+ lymphocytes was increased in chemotherapy-naïve patients with CLL (1585/μl vs 953/μl; p=0.02). TRECs were significantly higher in CLL patients compared to age-matched healthy controls (2.9/ml vs 1.4/ml; p=0.04), while the proportion of RTE was lower (24.9% vs 32.4%; p=0.02). No significant differences were observed in the percentage of Treg and RTE-Treg as well as of TCM. On the contrary, the percentage of TEM was higher (22.4% vs 10.4%; p=0.01) in CLL patients. The number of KRECs was lower in CLL patients than in controls (3.8/ml vs 7.4/ml; p=0.004). No correlation between IgVH gene mutational status, Ig levels and KRECs was found. ConclusionsThe neo-synthesis of normal T, with the exception of Treg, and B cells is reduced in patients with CLL compared to controls, even in a good prognosis, chemotherapy-naïve subset. The increased number of TRECs+ cells and TEM together with low RTE could be ascribed to the increased number of circulating CD4+ lymphocytes which do not appear to be recently mobilized from the thymus (CD31- cells) or to undergo peripheral expansion, but to accumulate in the peripheral blood. The low number of KRECs compared to normal controls could be interpreted as a consequence of the abnormal leukemic B-cell expansion which may impair normal B cell neo-synthesis. The level of KRECs did not seem to correlate to the mutational status of IgVH genes or the Ig level but a larger number of patients is needed to confirm these preliminary data and to establish whether the analysis of TRECs and KRECs may help clarify the complex immune abnormalities in CLL. Disclosures:No relevant conflicts of interest to declare.

Impact of IgVH Gene Mutational Status and VH Family Usage on Chromosomal Aberrations Detected by High-Resolution Array-CGH in B-CLL.

Abstract 1246Poster Board I-268B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a variable clinical course. Genomic aberrations as detected by FISH analysis and the mutational status of the IgVH genes have been found to have prognostic clinical significance with unmutated IgVH genes associated with clinically more aggressive disease. Previous studies have suggested that unmutated IgVH B-CLL is associated with increased chromosomal instability and increased chromosomal aberrations leading to accelerated disease progression. We recently performed a high-throughput, high-resolution array-based comparative genomic hybridization (aCGH) in a cohort of B-CLL patients. Fifty-eight patients have been analyzed by using the Sureprint G3 microarray (one million probes, Agilent). Our working hypothesis was that we would see increased genomic aberrations and instability in the unmutated IgVH group. The total number of copy number aberrations and total megabases (Mb) deleted or gained per genome were used as a surrogate marker for genomic instability. Variations in chromosomal aberrations in various VH families were also evaluated. B-CLL with unmutated IgVH genes utilized members of the VH1, VH3, VH4 and VH5 families with VH1-69 the most prevalent. B-CLL with mutated IgVH genes utilized VH families VH1, VH2, VH3, VH4, and VH6. Considerable variation was seen in the number of genomic abnormalities between cases. Overall, 315 abnormalities have been identified (236 deletions and 79 gains), with a median of 4 copy-number abnormalities per patient (range 0-32). In patients with IgVH mutational status available the median number of chromosomal aberrations in the mutated group was 5 (range 1-17) and unmutated group was 4 (range 0-20). Copy number deletions were significantly more common than chromosomal gains for the total group and in both the unmutated and mutated IgVH gene groups. Mean number of megabases (Mb) of DNA deleted in the unmutated IgVH group was 17.80Mb (standard deviation 24.17 Mb, median 6.63 Mb, range 0 - 90.55Mb) and 40.65 Mb in the mutated IgVH group (standard deviation 86.04 Mb, median 15.35Mb, range 0 – 307.57Mb). Mean number of Mb gained in the unmutated group was 37.74Mb (standard deviation 57.58Mb, median 0.44Mb, range 0 – 132.47Mb) and 32.99Mb (standard deviation 59.76Mb, median 0.14Mb, range 0 – 172.50Mb) in the mutated group. Nine patients had B-CLL with VH1-69 genes and 8 of them were unmutated. Median number of chromosomal deletions was 2 and gains also 2 (ranges 0 – 8 deletions and 0 – 4 gains) for this group. Similar findings were seen for the other VH families. These results initially suggest that total copy number variations do not account for the observed clinical differences between mutated and unmutated IgVH B-CLL. The catalogue of potentially clinically relevant chromosomal abnormalities found in this aCGH study is being currently evaluated. DisclosuresZent:Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Fonseca:BMS: Consultancy; Amgen: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

Characterization of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in Shanghai, China: Molecular and cytogenetic characteristics, IgV gene restriction and hypermutation patterns

The clinical, cytogenetic and molecular features of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), a disease previously considered to be rare in Asia, were examined in consecutive series of 70 cases diagnosed by our laboratory over a 30-month period. Clonal abnormalities were observed in 80% of CLL/SLL cases using a combination of conventional cytogenetic and fluorescence in situ hybridization (FISH) analysis. Those involving 14q32/IGH were the most frequent (24 cases), followed by trisomy 12 and 11q abnormalities. IgVH gene usage was non-random with over-representation of VH4-34, VH3-23 and a previously unreported increase in VH3-48 gene use. Somatic hypermutation (SHM) of IgVH germline sequences was observed in 56.5% of cases with stereotyped patterns of SHM observed in VH4-34 heavy chain complimentary-determining (HCDR1) and framework region CFR2 sequences. These findings in a Chinese population suggest subtle geographical differences in IgVH gene usage while the remarkably specific pattern of SHM suggest that a relatively limited set of antigens may be involved in the development of this disease worldwide. IgVH gene mutation status was a significant predictor of initial survival in CLL/SLL. However, an influence of karyotype on prognosis was not observed.

CD69 as a Surrogate Marker for IgVH Gene Mutation Status in Chronic Lymphocytic Leukaemia (CLL)

Chronic lymphocytic leukemia (CLL) is characterized by a heterogeneous clinical course where some patients have an indolent disease and survive for decades while others have a rapidly progressive disease with short survival. Clinical staging according to Rai or Binet are useful in predicting survival. However, in early stages a subset of patients progress while others remain stable. CLL can be subdivided into two groups based on IgVH gene mutation status, and it has been shown that patients with unmutated IgVH genes have poorer survival irrespective of clinical stage. A major drawback using mutation status as a prognostic marker is that it is expensive and time consuming. Therefore, there is a need for surrogate markers that can be analyzed in routine laboratories.We analyzed the expression of CD69, CD38 and ZAP-70 in CD19+ cells from CLL patients (n=28) and healthy donors (n=10) using flow cytometry. The gene expression of ZAP-70 was also analyzed by real-time PCR. CD69 was higher expressed in B cells from CLL patients compared with controls (35±31% and 2.6±1.8% CD69+/CD19+ cells respectively, P=0.001). Patients with unmutated IgVH genes (UM CLL) had a higher percentage of CD69+ cells compared with patients with mutatated IgVH (M CLL; 70±24% vs. 18±12%, P=0.00076). Furthermore, there was a strong concordance, even better than CD38 and ZAP-70, between IgVH mutation status and expression of CD69 (96%). Thus, we suggest that CD69 expression can be used as a surrogate marker for mutation status. Furthermore, this analysis is reliable and inexpensive. [Display omitted]

Interactome-Based Molecular Prognosis of Chronic Lymphocytic Leukemia

The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous. Several prognostic factors have been identified that can stratify patients into groups that differ in their relative tedancy for disease progression and/or survival. Microarray studies have highlighted differences in mRNA levels found between such CLL subgroups. We hypothesize that gene expression profiling might define a repertoire of transcriptional activity contributing to or resulting from the dynamic evolution of CLL cells. To evaluate for this, we profiled approximately 200 CLL patients (of >95% CD19+CD5+ peripheral blood mononuclear cells in each sample) on mRNA expression microarrays using Affymetrix HG-U133 plus 2 GeneChips. We first sought to develop an expression-based prognosis that assigns patients to “aggressive” (high-risk) or “indolent” (low-risk) groups based on their gene expression correlated to the treatment-free survival from the date of sample collection. Each of the ~22,000 genes was scored by the Cox metric, which measures the correlation between its gene expression level and treatment-free survival in a designated training set. Unsupervised 2-means clustering technique was then used to separate the training samples into two risk groups based on the similarity of their mRNA expression of the top Cox-scored genes. Patients in an independent test set were assigned to the “aggressive” or “indolent” groups based on their expression similarity to that of the training samples. The two risk groups defined by the gene signature displayed significantly different behaviors with respect to treatment-free survival (Fig. 1); however, neither of the two commonly-used prognostic factors, IgVH gene mutational status or leukemia-cell expression of ZAP-70 protein, could segregate these subgroups to the same degree of statistical significance. To achieve better prediction performance based upon biological-defendable models, we further adopted the network-based classification scheme we previously developed for predicting metastasis potential of breast cancers. The network-based approach identified prognostic markers not as individual genes but as subnetworks extracted from molecular interaction databases. Gene expression profiles from CLL patients were mapped to a large human molecular interaction network, consisting of 49,419 interactions (including protein-protein and protein-DNA interactions) among 9,795 genes/proteins, compiled from high-throughput screenings and curation of previous measurements reported in the literature. A search over this network was performed to identify prognostic subnetworks that could be used to predict treatment-free survival. Specifically, each subnetwork was scored by a vector of activities across all patients, where the activity for a given patient is a function of the expression levels of its member genes. A subnetwork's prognostic power was computed as the uni-variate Cox score between the activity vector and the patient's treatment-free survival. The resulting ~200 prognostic subnetworks identify new putative cancer markers and provide an array of “small-scale” models charting the molecular mechanisms correlated with CLL progression, e.g. subnetworks detailing interactions between proteins participating in Wnt signaling, Notch signaling, or cell death. Moreover, our network-based classification achieves higher accuracy in predicting duration of treatment-free survival in newly diagnosed patients than identified uniparameter prognostic markers or standard gene-expression array analyses. Thus, our network-based approach integrating protein interactions with CLL expression profiles leads to increased classification accuracy and, simultaneously, provides a view of the biological processes underlying cancer progression. [Display omitted]

Integrating Newer with Traditional Prognostic Factors in Evaluating Patients with CLL Receiving Frontline Chemoimmunotherapy.

Prognostic factors are important for evaluating patients (pts) with CLL, owing to heterogeneity in response to treatment and clinical course. The goal of this analysis was to evaluate the newer prognostic factors in relation to traditional ones in previously untreated pts receiving frontline chemoimmunotherapy. Traditional prognostic factors include age, Rai stage, absolute lymphocyte count (ALC), lymphocyte doubling time, and many others. Newer prognostic factors, include IgVH gene mutation status (MS), chromosome abnormalities by FISH, and leukemia cell expression of ZAP70. Finally, the clinical endpoint is important in considering and evaluating prognostic factors, since significant independent factors can be different for response to treatment, time to progression (TTP), and overall survival (OS).We evaluate 488 previously untreated pts who had an NCI-WG '96 indication for treatment and received frontline chemoimmunotherapy (CIT). Treatment was with a fludarabinebased CIT regimen: FCR (n=300), FCMR (n=30), FCR3 (n=65), FCR+GM-CSF (n=45), or CFAR (n=48). The CR rate, TTP, and OS were not significantly different between the CIT regimens; median follow-up times varied. The overall estimated median follow-up time was 62mo, TTP was 76mo, and OS was not reached. An extensive list of pretreatment characteristics were evaluated in univariable analyses as traditional factors including age, Rai stage, beta-2 microglobulin (β2M), CD38, and others, as well as newer prognostic factors including chromosome abnormalities by FISH, IgVH MS and ZAP70 expression by immunohistochemistry. Clinical endpoints included complete remission (CR), TTP, and OS. Multiple factors were significant (p<.05) and varied according to clinical endpoint. Next, we evaluated these significant factors in multivariable (MV) analyses to identify the significant (p<.05) independent factors for each endpoint. The rank order importance for each independent factor identified in the MV model with current follow-up is shown (Table 1).Table 1 Multivariable ModelCRTTPOS# Pts in Model483301483# Events351101106CharacteristicRank-Order Significance in MV Modelβ2M1-1Hemoglobin2--Age-22CD38 (30% cut point)-3-IgVH MS-1-We evaluated the newer prognostic factors (IgVH MS, del 17p, and ZAP70) separately in each final MV model to assess significant (p<.05) independent correlations (Table 2).Table 2 Significance of Factor in MV ModelCharacteristicCRTTPOSDel 17pp<.05p<.05p<.05IgVH MSNot Sig.p<.05Not Sig.ZAP70Not Sig.p<.05p<.05When including del 17p in models for TTP and OS, age and IgVH MS were no longer significant. Continued follow-up may identify additional important factors for time-toevent endpoints. Nomograms will be developed for each endpoint and full analyses will be shown. These analyses enable us to generate expectations and help in evaluating efficacy of new regimens for patients going on frontline CIT. Age, β2M, and del 17p appear to be the most important independent factors for OS in previously untreated patients who receive frontline CIT. Considering the newer prognostic factors, β2M continues to remain a major, independent, significant prognostic factor.

Insight into the pathogenesis of chronic lymphocytic leukemia (CLL) through analysis of IgVH gene usage and mutation status in familial CLL

AbstractTo address the proposition that familial B-cell chronic lymphocytic leukemia (CLL) may exhibit a more restricted phenotype than sporadic CLL with respect to immunoglobulin gene usage or ontogenic development, we compared immunoglobulin (Ig) heavy chain variable region (VH) gene usage and IgVH mutation status in 327 patients with CLL from 214 families with 724 patients with sporadic cases. The frequency of mutated CLL was higher in familial CLL (P < .001), and there was evidence of intrafamilial concordance in mutation status (P < .001). The repertoire and frequency of IgVH usage was, however, not significantly different between familial and sporadic CLL. Furthermore, IgVH usage was not correlated between affected members of the same family. These observations provide evidence that familial CLL is essentially indistinguishable from sporadic CLL, favoring a genetic basis to disease development in general rather than a simple environmental etiology.

The significance of soluble HLA-G plasma levels as well as messenger HLA-G for B-cell chronic lymphocytic leukemia (B-CLL)

The immunosuppression accompanies B-cell chronic lymphocytic leukemia (B-CLL) but might be also responsible for disease progression by enabling CLL cells to escape from the immunosurveillance. Some particles involved in the regulation of an immune system might represent prognostic value for B-CLL. Recently we found no correlation between HLA-G on messenger and protein level, suggesting that HLA-G is released in soluble form. To confront this hypothesis we characterized soluble HLA-G (sHLA-G) by the prognostic factors in the first cohort of 34 CLL patients. No correlation was observed between sHLA-G levels in ZAP-70(+) and ZAP-70(−) CLL as well as in CD38(+) CLL and CD38(−) CLL patients. Next, we wondered whether gene expression of HLA-G, which represent the whole HLA-G pool in the cell, posses prognostic value for CLL. In the second cohort of 41 CLL patients we assessed messenger levels of HLA-G by the strongest prognostic factors in CLL including cytogenetics, IgVH mutational status, ZAP-70 as well as CD38. No changes of HLA-G expression levels were found in different CLL groups characterized by IgVH gene mutational status, ZAP-70 as well as CD38. We observed no differences in expression of HLA-G in various cytogenetic groups of CLL including del17p, del13q, del11q, +8q, +3q, del14q and del6q when compared to those with normal karyotype or with 12+. Both, mRNA expression of HLA-G and levels of its soluble form in plasma bring no additional prognostic value for B-CLL patients.

Distinctive IgVH Gene Segments Usage and Mutation Status in Chinese Patients with Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by the relentless accumulation of monoclonal B cells with the appearance of small mature lymphocytes and a characteristic CD5 and CD19 co-expression immunophenotype. The incidence of CLL in Asian countries is lower than that in the Western ones, where CLL is the most common leukemia. To evaluate the frequency and mutation status of immunoglobulin (Ig) variable heavy chain gene (IgVH) expression in Chinese patients with CLL. We investigated IgVH gene segments usage and mutation status by multiplex RT-PCR in 52 CLL patients, and analyzed the relationship between IgVH somatic mutation status and the expression of CD38, ZAP-70 and CLLU1. 38 patients had mutated IgVH, and 14 had unmutated IgVH. The most frequently expressed VH gene family was found to be VH3 (46.2%) followed by VH4 (40.4%), VH1 (5.8%), VH2 (5.8%) and VH7 (1.9%), with no expression of VH5 and VH6 gene families. VH1-69 and VH3-21 which commonly overused in Western CLL weren't detected in our cohort. The frequency of IgVH gene families indicates significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. IgVH gene mutation status was significantly associated with the expression of CD38 and CLLU1. The expression of them may be simple and reliable surrogates for the identification of IgVH mutations.VH gene family usage and mutation statusVH familynMutated VH geneUnmutated VH geneVH1330VH2321VH324195VH421165VH5000VH6000VH7101

The Proliferative Response to CpG ODN Stimulation Predicts Disease Progression in B-Cell Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) B-cells from certain patients exhibit marked proliferation following stimulation with unmethylated CpG-oligodeoxynucleotides (CpG-ODN). In a recent study we observed that CpG-ODN induced proliferation occurs primarily in leukemic B-cells from patients with progressive disease and unmutated immunoglobulin (Ig) VH genes. To further investigate whether the proliferative response following CpG-ODN stimulation can be used as a prognostic marker in CLL, we evaluated progression-free survival (PFS) and treatment-free survival (TFS) in a larger series of well characterized patients that had been followed for an extended period. In addition, we correlated the proliferative response with other prognostic factors, including Ig VH gene mutation status, ZAP-70 expression and the presence of “high risk” cytogenetic abnormalities. Analysis of 3H thymidine incorporation in purified CLL B-cells stimulated for 72 hours with CpG-ODN 2006 revealed a proliferative response in 23 (40%) of the 58 evaluated cases. Eighteen (78%) of the proliferating cases had unmutated and 5 (22%) had mutated (M) Ig VH genes, whereas among the 34 non-proliferating cases with available Ig VH gene data 8 (24%) had unmutated and 26 (76%) mutated Ig VH genes (P=<0.001 by chi-square test). ZAP-70 was expressed by 16 of the proliferating (70%) and 14 of the non-proliferating cases (40%), but this difference was not statistically significant (P = 0.053). The high-risk risk cytogenetic abnormalities del(17p13.1) and del(11q22.3) were detected in 6 of the 21 evaluated proliferating (29%) and in 3 of the 30 evaluated non-proliferating cases (10%) (P = 0.136). Median PFS was 25 months in the proliferating and 72 months in the non-proliferating subset (P=0.001). Similarly, median TFS was significantly shorter in the proliferating (30 months) than in the non-proliferating subset (114 months, P=0.001). In summary, these data confirm that the proliferative response to CpG-ODN stimulation is strongly correlated with Ig VH gene mutation status, but does not depend on ZAP-70 expression. Although high-risk cytogenetic abnormalities were more frequent in the proliferating subset, the difference was not significant. Cases with a positive proliferative response had significantly shorter PFS and TFS, indicating that the proliferative capacity of the leukemic cells is an important determinant of the clinical course in CLL. Elucidation of the mechanisms that underlie the different proliferative capacity of CLL B-cells should provide additional insight in the pathogenesis of CLL and may lead to the identification of novel and clinically useful prognostic markers.