Unmutated Chronic Lymphocytic Leukemia Cases Research Articles

Abstract 3482: METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression

Abstract RNA splicing dysregulation is a hallmark of cancers and underlies the onset and progression of diseases. Chronic lymphocytic leukemia (CLL) is one of the most common adult leukemia in western countries. Spliceosome mutations occur in ~20% of patients. However, the mechanism for splicing defects in spliceosome unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis of primary CLL samples, we discovered proteins involved in RNA splicing are post-transcriptionally upregulated. Coupled with clinical annotation, we found spliceosome protein abundance is an independent risk factor and associated with poor prognosis. Splice variants found in CLL are highly overlapped with those driven by high spliceosome abundance but not splicing factor mutations, indicating high spliceosome abundance contributes to genetic lesion-independent splicing defects. To identify potential regulators for spliceosome, we proteome-widely analyzed the proteins that highly correlated with splicing factors expression. Analysis of 113 CLL samples has consistently identified METTL3 upregulation with positive correlation with 77.6% of detected splicing factors. METTL3 is an RNA methyltransferase that modifies N6-methyladenosine (m6A) on mRNA and regulates the translation of m6A-installed transcripts. m6A level on mRNA is increased in CLL cells with differential m6A highly enriched on splicing related transcripts. Moreover, high METTL3 expression in CLL is also associated with poor clinical outcomes. These results suggested that METTL3 translationally controls splicing factors through m6A and plays a role in CLL progression. Toward this end, we demonstrated that METTL3 is essential for CLL growth in both in vitro and in vivo studies. Knock out (KO) and pharmaceutical inhibition of METTL3 decreased cell growth and splicing factor expression. Overexpression of wildtype but not catalytic mutant METTL3 restored both defects in METTL3 KO cells, indicating that the regulation of splicing factor is m6A-dependent. To dissect the underlying mechanism, we performed an integrated Ribo-seq, RNA-seq, and MeRIP-seq on CLL cells with or without METTL3. KO of METTL3 decreased overall translation efficiency (TE) with RNA splicing as the most significantly affected pathway. Splicing factors with reduced TE displayed either hypo-m6A at stop codon region or hyper-m6A at CDS regions upon METTL3 KO, as direct or indirect targets of METTL3. Moreover, we found that m6A at stop codon and CDS regions regulates splicing factor translation via ribosome recycling and ribosome pausing, respectively. Taken together, our results uncovered a novel regulatory axis for METTL3 that controls splicing factor translation and contributes to CLL progression. Our study highlights a post-transcriptional layer of m6A modification as a major contributor to genetic lesion-independent splicing defects in CLL. Citation Format: Yiming Wu, Meiling Jin, Mike Fernandez, Kevyn Hart, Aijun Liao, Stacey M. Fernandes, Tinisha McDonald, Zhenhua Chen, Daniel Röth, Lucy Ghoda, Guido Marcucci, Markus Kalkum, Raju K. Pillai, Alexey V. Danilov, Jianjun Chen, Jennifer R. Brown, Steven T. Rosen, Tanya Siddiqi, Lili Wang. METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3482.

A practical guide for mutational signature analysis in hematological malignancies

Analysis of mutational signatures is becoming routine in cancer genomics, with implications for pathogenesis, classification, prognosis, and even treatment decisions. However, the field lacks a consensus on analysis and result interpretation. Using whole-genome sequencing of multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and acute myeloid leukemia, we compare the performance of public signature analysis tools. We describe caveats and pitfalls of de novo signature extraction and fitting approaches, reporting on common inaccuracies: erroneous signature assignment, identification of localized hyper-mutational processes, overcalling of signatures. We provide reproducible solutions to solve these issues and use orthogonal approaches to validate our results. We show how a comprehensive mutational signature analysis may provide relevant biological insights, reporting evidence of c-AID activity among unmutated CLL cases or the absence of BRCA1/BRCA2-mediated homologous recombination deficiency in a MM cohort. Finally, we propose a general analysis framework to ensure production of accurate and reproducible mutational signature data.

Targeting IRAK4 Disrupts Inflammatory Pathways and Delays Tumor Development in Chronic Lymphocytic Leukemia

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and the innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediatedby the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 5% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Gene expression profiling of 423 CLL cases, assessed by the computational GSEA analysis tool, identified enrichment of gene sets related to cytokines and inflammation in the subgroup carrying mutations in MYD88. Accordingly, CLL cells from individuals with MYD88 mutations showed higher p65 NF-κB activity and secreted higher basal levels of CCL2, CCL3 and CCL4 cytokines compared with unmutated cases. To assess the effects of this pathway in CLL, we stimulated these cells with a TLR agonists mix, containing Pam3CSK4, HKLM, FSL1 and ODN2006, which was able to induce cytokines secretion, increase in NF-KB activity and STAT3 signaling as well as cells proliferation and migration. Here, we study the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. ND2158 was able to efficiently decrease all TLR effects seen with the mix like STAT3 signaling and the secretion of CCL2, CCL3, CCL4, TNFα, IL1β, IL10 and IL6 cytokines (assessed by Luminex). NF-κB signaling was also modulated by the drug, as observed by the downregulation of IκBα phosphorylation protein level, p65 translocation to the nucleus and decreased p65 DNA binding activity. ND2158 also impaired CLL proliferation (checked by carboxyfluoresceinsuccinimidyl ester staining) and migration towards CXCL12 in MYD88-mutated and -unmutated CLL cases.ND2158 also induced a selective dose-dependent cytotoxic effect in CLL cells, without differences regarding their MYD88 mutational status. To further validate this results, we checked the effect of ND2158 in splenocytes from leukemic Eμ-TCL1 mice and similar antitumor effects were seen.In addition to the direct impact on tumor cells, we analyzed whether ND2158 also affects other immune cells that also signal via MYD88/IRAK4. Monocytes, broadly known for their importance in tumor supporting, were also modulated by TLR agonists, and a reduction in activation markers and cytokines secretion was achieved after ND2158 treatment.In vivo studies in Eμ-TCL1 leukemic mice showed a significant reduction in tumor load in CLL-affected organs (peripheral blood, spleen, lymph node and peritoneal cavity) associated with reduced PD-L1 expression levels in mice treated with 100 mg/kg/day of the IRAK4 inhibitor. A decrease in spleen weight and size was also detected after the treatment. Corroborating the in vitro studies,ND2158 also modulated tumor-supporting monocytes. The effect of ND2158 was also assessed in CD8+ effector T cells, where a low proliferative (Ki-67+ percentage), exhausted phenotype (increase in TIGIT, CD244, CD160 and LAG3 markers) and less functional population (increase in the immune checkpoint protein PD-1) was found.Based on our in vitro data, it seems that ND2158 targets both MYD88 mutated and unmutated CLL cases to a similar degree. Since CLL progression in the TCL1 mouse model has not been reported to be driven by an activating MYD88 mutation, our in vivo results support the notion that IRAK4 inhibition might also be effective in CLL patients with an unmutated MYD88 status. To overcome the potential negative impact of IRAK4 inhibition on T cells, the combination treatment with drugs that improve T cell function and avoid their rapid exhaustion should be considered. As we observed increased expression of several inhibitory receptors on CD8+ T cells in ND2158-treated mice, including PD-1 and TIGIT, blocking these receptors with antibodies might be a successful strategy to overcome loss of T cell function and improve therapy outcome.In summary, our data show the importance of TLR signaling in CLL development and suggest IRAK4 as a novel treatment target for CLL that targets both the malignant cells and the tumor-supportive inflammatory milieu. DisclosuresNo relevant conflicts of interest to declare.

Low Frequency Of IGHV4-39/IGHD6-13/IGHJ5 Rearrangement With Stereotyped HCDR3 (subset #8) In Russian and Ukrainian Chronic Lymphocytic Leukemia Patients

The leukemia cells of unrelated patients with chronic lymphocytic leukemia (CLL) display a restricted repertoire of immunoglobulin gene rearrangements and very similar structure of B-cell receptor (stereotyped BCR). Numerous different subsets of stereotyped BCRs were identified. Some associations between specific BCRs subsets, clinical course of CLL, and molecular abnormalities were found. Namely, short overall survival, accumulation of SF3B1 mutations in subset #2, high risk for Richter's transformation, trisomy 12, NOTCH1 mutations in subset #8, increased frequency of NOTCH1 mutations in subset #1 were revealed (Rossi et al., 2009, 2013; Streffoed et al., 2013).The aim of this study was to evaluate distribution of different stereotyped BCR subsets in Russian and Ukrainian CLL cohort with special attention on prognostic unfavorable subsets. MethodsImmunoglobulin heavy chain variable (IGHV) genes of 969 CLL patients (564 from Russia and 405 from Ukraine) were amplified according to BIOMED-2 rules, sequenced and analyzed with IMGT and IgBlast databases. Additionally, NOTCH1 c.7544_7545delCT mutations were analyzed in 301 patients (randomly collected and in the prognostic unfavorable subsets) by amplification refractory mutation system (ARMS) PCR as proposed by Rossi D. et al., 2012. In 19 cases NOTCH1 mutational status was confirmed by direct DNA Sanger sequencing. ResultsJoined Russian-Ukrainian CLL cohort was presented mainly by unmutated (UM) IGHV gene cases (64.2%). The distribution of major clusters of stereotyped BCR did not differ from those presented in the literature (comparison was made separately for cases with mutated (M) and UM IGHV genes, taking into account the differences in the mutational status of IGHV genes between our CLL cohort and CLL cohorts from European countries and USA), except for subset #8, which was found in one case only (0.16% of UM IGHV gene cases). At the same time, subset #8 (IGHV4-39/IGHD6-13/IGHJ5 rearrangement) belongs to the group of “major” subsets in CLL (Agathangelidis et al., 2012), and its frequency varies from 0.9% to 2% among cases with UM IGHV genes in the most of presented CLL cohorts (Murray et al., 2008; Messmer et al., 2009; Maura et al., 2011). Frequency of subset #2 had tendency to be lower among Russian CLL patients (1.4% of M CLL cases), but not among Ukrainian patients (4.5% of M CLL cases) comparing with European CLL cohorts.NOTCH1 c.7544_7545delCT was identified in 27 out of 301 patients (9.0%), which was in accordance with other CLL studies. Twenty five cases with NOTCH1 mutations had UM IGHV genes and 2 cases expressed M IGHV3-21 gene (both subset #2). As well, majority of NOTCH1 mutated cases (17/27, 63%) showed stereotyped BCRs, which belonged to the following subsets: #1 (2 of 12), #2 (2 of 7), #3 (1 of 6), #7 (2 of 10), #9 (2 of 10), #25 (1 of 2), #59 (1 of 3) (subset's designation according Murray et al., 2008), UA/ref2 (1 of 2), UA/ref11 (2 of 2) (subset's designation according Bilous et al., 2010), or having a homology with the CLL sequences of the public CLL database. No NOTCH1 mutations were found in 11 IGHV4-39-used cases, including one case from subset #8. In 10 of 27 (37.0%) NOTCH1 mutated cases IgHV1-69 gene was used, while among wild type NOTCH1 patients IgHV1-69 was presented in 65/274 (23.7%) cases (p=0.12). ConclusionThe distribution of the major subsets of stereotyped BCRs in CLL patients from Russia and Ukraine (eastern slavic countries) was similar to the western European countries and USA, except for low frequency of subset #8, associated with unfavorable clinical course. Our preliminary data suggested that NOTCH1 c.7544_7545delCT mutation is typical for UM CLL cases with stereotyped BCRs, but no clear association with specific subsets was found in the absence of subset #8. Disclosures:No relevant conflicts of interest to declare.

Methylation status regulates lipoprotein lipase expression in chronic lymphocytic leukemia

Among different prognostic factors in chronic lymphocytic leukemia (CLL), we previously demonstrated that lipoprotein lipase (LPL) is associated with an unmutated immunoglobulin profile and clinical poor outcome. Despite the usefulness of LPL for CLL prognosis, its functional role and the molecular mechanism regulating its expression are still open questions. Interaction of CLL B-cells with the tissue microenvironment favors disease progression by promoting malignant B-cell growth. Since tissue methylation can be altered by environmental factors, we investigated the methylation status of the LPL gene and the possibility that overexpression could be associated with microenvironment signals. Our results show that a demethylated state of the LPL gene is responsible for its anomalous expression in unmutated CLL cases and that this expression is dependent on microenvironment signals. Overall, this work proposes that an epigenetic mechanism, triggered by the microenvironment, regulates LPL expression in CLL disease.

Abstract 1033: Distinct response of chronic lymphocytic leukemia cells to PI-3Kδ inhibitor GS-1101 is related to MAPK signaling and likely associated with IGHV mutation.

Abstract Background: PI3Kδ is expressed and regulates survival and proliferation of malignant B-cells. The PI-3Kδ inhibitor, GS-1101, has promising therapeutic efficacy in chronic lymphocytic leukemia (CLL) through inhibiting B-cell receptor signaling, PI3K signaling and chemokine networks. It is currently in clinical trials either as monotherapy or in combination with others therapies. Here we report the exploration of the signaling molecules which are related to distinct response of CLL cells to GS-1101. Methods: Cells isolated from peripheral blood of CLL patients were treated with GS-1101 and analyzed by flow cytometry based apoptotic assay. Human Phospho-Kinase Array (R&D Systems) was performed with CLL cells which had distinct response to GS-1101. RNA or genomic DNA was isolated and analyzed for IGHV somatic hypermutation (InVivoScribe Technologies). Sequences containing more than 2% deviation from the germline sequence were considered as somatically hypermutated. Results: To date, 20 CLL cases have been tested for their in vitro response to GS-1101. In comparison to the control group, GS1101-exposed CLL cells displayed a wide range of apoptotic cells (3 to 59%). 3 cases with high levels apoptosis in response to GS-1101 were subjected to Human Phospho-Kinase Array screening. These showed down regulation of p-ERK1/2(T202/Y204), p-P38α(T180/Y182), MEK1/2 (S218/S222), among tested 46 kinases’ phosphorylation sites, upon GS-1101 exposure. Two of the three cases also showed down-regulation of p-STAT5(Y699). In contrast, no alteration or minor up-regulation of the above 4 targets was observed in a GS-1101 non-responsive case. Currently we are performing immunoblotting assay to evaluate/confirm the alteration of above targets in multiple CLL cases and preliminary p-P38 expression data is consistent with that of array assay. 15 of the tested 20 CLL samples had high quality of IGHV sequence data from both directions which cover the entire variable region of IGH (FR1, CDR1, FR2, CDR2 and FR3). Nine cases were IGHV unmutated with a mean of GS1101 caused apoptotic induction of 25.3% (range 5-59%); while the mean of apoptotic induction of the 6 cases with somatic hypermutation was 12.5% (range 3 - 32%). The p value of the unmutated verse mutated group was 0.046 (T-test). Conclusion: Our preliminary studies suggest that GS-1101-induced apoptosis is associated with alterations in the MAPK signaling pathway. Combination of GS-1101 with MAPK inhibitor may be rational for further synergy study. GS-1101 may have differential activity against IGHV unmutated CLL cases compared to IGHV mutated ones. Ongoing studies with additional cases will further evaluate this observation. Profiling CLL cells for IGHV mutation status might be a strategy to predict CLL patients’ response to GS-1101 in clinical trials. Citation Format: Xiaoxian Zhao, Juraj Bodo, Eric D. Hsi. Distinct response of chronic lymphocytic leukemia cells to PI-3Kδ inhibitor GS-1101 is related to MAPK signaling and likely associated with IGHV mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1033. doi:10.1158/1538-7445.AM2013-1033

CD72 Expression Patterns in ZAP70 Positive and Negative Chronic Lymphocytic Leukemia: Potential Regulatory Role in BCR Signalling

Introduction: Absence of mutations in IgVH genes or higher number of ZAP70+ cells (as a surrogate marker) in chronic lymphocytic leukemia (CLL) B-cells defines a patient group with a poorer clinical course. These features relate to the role of BCR signalling in the proliferation and survival of CLL B-cells, and establish a link between these markers and the biology of CLL prognostic subgroups. The identification of additional players in this context may help to better understand the molecular basis of this disease and contribute to develop new therapeutic approaches. A search for genes potentially related to BCR signalling, when comparing mutated and unmutated CLL cases using serial analysis of gene expression, revealed a 4-fold increase of CD72 tags in unmutated samples, a specific B cell surface glycoprotein known to transmit both positive and negative signals in BCR signalling.Objective: This finding lead us to explore the potential role of CD72 on BCR signalling in distinct CLL prognostic subgroups, as defined by ZAP70 expression.Methods: Percentage of ZAP70+ and CD72+ cells were evaluated by flow cytometry on gated CD19+CD5+ cells in 25 CLL samples. Positive cases for ZAP70 and CD72 were defined using a cut-off of 35% and 40% positive cells, respectively. Real time PCR was used to quantify the expression levels of 3 genes related to proliferation and survival, RELB, Beta-Catenin (CTNNB1) and AKT1, on 16 CD19+ enriched (purity > 90%) CLL samples.Results: Samples were classified as 11 ZAP70+ and 14 ZAP70−. Median percentage of CD72+ cells in ZAP70+ was significantly higher than for ZAP70− cases (82% compared to 39%, respectively, P=0.0029). Furthermore, percentages of CD72 and ZAP70 were positively correlated (r=0.5930 and P=0.0009). Interestingly, ZAP70+ cases were restricted to CD72+ cases (n=11, CD72+ZAP70+ [+/+]), whereas six CD72+ cases were ZAP70− (ZAP70−CD72+ [−/+]). Finally, there were 8 cases CD72−ZAP70− [−/−]. No differences among these 3 groups were observed in regard to laboratory parameters (white blood cells, total lymphocytes, lymphocyte percentage, haemoglobin, haematocrit and platelet number). Despite the reduced number of samples analysed (6 +/+, 6 −/− and 4 −/+), transcripts for RELB (P<0.05), CTNNB1 (P<0.05), and AKT1(P=0.057) were expressed at higher levels in ZAP70+CD72+ than in ZAP70−CD72+ samples. Additionally, the transcripts were expressed at higher levels in ZAP70−CD72− than in ZAP70−CD72+ samples, and this difference was statistically significant (P<0.05) for CTNB1 and AKT1, but not for RELB (P=0.054).Conclusion: Our data indicate that higher percentages of ZAP70+ cells are associated with higher expression levels of transcripts related to proliferation and survival of CLL B-cells. In the absence of ZAP70 expression, CD72 may act as a negative regulator of the BCR pathway, as indicated by the lowest levels of transcripts on ZAP70−CD72+ cases.

The Receptor for Hyaluronic Acid Mediated Motility (RHAMM): Characterization as an Immunotherapeutical Target in Chronic Lymphocytic Leukemia (CLL) and First Results of RHAMM-Derived Peptide Vaccination Trial.

Background and Aims: The increased expression of receptor of hyaluronic acid mediated motility (RHAMM) was noted in CLL. The defective expression of RHAMM-exon4 splice variant that correlated with chromosomal instability was found to bear negative prognostic value in patients with multiple myeloma. Therefore, here we characterized expression of RHAMM with its splice variants and correlated it with prognosis in CLL. Moreover, based on our previous results we initiated a RHAMM-derived R3 peptide vaccination study for patients with B-CLL in early stages of disease.Methods: Messenger RNA expression of RHAMM/CD168 was assessed by quantitative RT-PCR in blood samples from 65 B-CLL patients, at the time of diagnosis. The splice variant expression was measured using fluorescence capillary electrophoresis. Three hundred mcg RHAMM R3 peptide (ILSLELMKL) emulsified with the incomplete Freund's adjuvant (ISA-51) day 3, as well as GM-CSF days 1–5, was administrated four times subcutaneously at a biweekly interval.Results: The real-time RT-PCR results revealed higher RHAMM/TBP expression rates in patients with unmutated IgVH status compared with mutated CLL cases (0.047 vs. 0.029, p=0.012). Both splice variants RHAMMFL as well as RHAMM-exon4 showed significantly higher expression in unmutated CLL cases. Increased expression of RHAMM/TBP was noted in advanced stages of disease. A tendency towards higher RHAMM/TBP expression ratios was observed in B-CLL cases with del11q. CLL patients with a RHAMM/TBP ratio >0.028 showed a significantly shorter median treatment-free survival (TFS) (16 vs. 38 months, p=0.0019). In a bivariate analysis, patients with VH mutated status and lower amounts of RHAMM presented longer TFS compared to unmutated cases with a high RHAMM expression (110 vs. 8 months, respectively). An enhanced RHAMM expression was observed in bone marrow samples of 5 B-CLL patients compared with the results obtained in blood (RHAMM/TBP 0.102 vs 0.032). Similar tendency to higher RHAMM/TBP ratios was observed after CD40L stimulation (RHAMM/TBP 0.025 vs.0.113). In five patients who completed all four doses of R3 peptide vaccine no toxicity related to vaccine was observed. Reduction of leukemic cells was noted in 3 patients but no clinical responses meeting NCI criteria was observed. Immunological responses were observed in patients who achieved hematological improvement. Conclusion: RHAMM/CD168 is a novel LAA in B-CLL patients, and its expression levels are correlated with the clinical course of the disease. RHAMM expression might indicate the proliferative potential of leukemic cells. First results of peptide vaccination using RHAMM-derived peptide showed limited reduction of tumor burden.

Immunoglobulin (Ig) Variable Gene Usage Identifies a Subset of Unmutated Chronic Lymphocytic Leukemia (CLL) Patients Expressing 51p1-Like alleles and a Particularly Unfavorable Prognostic Outcome.

Great interest has recently focused on a biased use of the immunoglobulin (Ig) heavy chain variable region genes (VH genes) and on a preferential light chain (VL gene) usage in patients with chronic lymphocytic leukemia (CLL). VH / VL bias maybe correlated with a possible response to a common antigen epitope. To further investigate the Ig VH gene usage in CLL, DNA was extracted from peripheral blood mononuclear cells of 137 consecutive unrelated and untreated CLL cases. PCR amplification was performed using VH family specific and consensus JH primers, followed by automated sequencing. The sequences were aligned to Ig sequences from the GenBank and V-BASE databases. The VH family gene usage was as follows: VH1 19.7%, VH2 4.4%, VH3 49.6%, VH4 21.9%, VH5 2.2% and VH6 2.2%. Mutated VH genes (>2% mutations) were found in 58.4% of the cases analyzed and unmutated VH genes (<2% mutations) were detected in 41.6% of samples. Within the VH1 gene family, 88.9% of cases were unmutated and the most frequently expressed alleles were related to 51p1 (a major VH1-69 allele), that accounted for 10.2% of all VH rearranged genes and for 51.9% of rearranged VH1 genes. Among all patients expressing unmutated 51p1-like alleles, a preferential use of certain D segments was observed: D2-2 accounted for 28% of cases, followed by D3-3 and D3-16 that accounted for 21.4% and 14.3%, respectively; D1-20, D3-10, D3-22, D5-18 and D6-6 were less frequently expressed (1 case each). The most frequently used JH gene segment was JH6 (64.3%), whereas JH4 and JH3 accounted for 21.4% and 14.3%, respectively. All patients expressing 51p1-like alleles showed an identical CDR2 region. The CDR3 region was characterized by similarities in a subset of cases: the amino acid motifs DIVVVPAA(I/M) and GGXYDY(I/V)WGSYRPNDAFDI were the most common and accounted each for 14.3% of CLL samples that expressed 51p1-like alleles: 79% of all patients expressing 51p1-like alleles had a long CDR3 region of 19.4 ± 2.9 codons. In contrast to what has been observed in other diseases, e.g. mixed cryoglobulinemia and non-Hodgkin lymphoma, all VH1-69 expressing CLL cases analyzed were not associated with HCV infection. The overall median survival for this subset of patients was 40 months, considerably shorter than the reported median survival of unmutated CLL cases; furthermore, to date 36% of patients have succumbed of their disease. The results of this study confirm, on the one hand, that restricted CDR3 features and a biased VH gene repertoire are suggestive of a role of antigen selection operational in this disease and have allowed, on the other hand, to identify a subset of patients with a particularly unfavorable survival compared to the overall population of unmutated CLL cases.

Chronic lymphocytic leukemia patients with highly stable and indolent disease show distinctive phenotypic and genotypic features

AbstractDifferent biologic features have been associated with a more or less aggressive clinical course in chronic lymphocytic leukemia (CLL). In the present study, 20 patients with highly stable CLL observed at a single institution over a period of 10 to 23 years and who never required treatment were extensively characterized. The aim was to identify a distinct and reproducible biologic profile associated with disease stability that may be used to recognize at presentation CLL patients who are likely to have a very benign clinical course and for whom treatment is not indicated. The results obtained indicate that numerous parameters are closely associated with disease stability: a typical CLL morphology and immunophenotype, the lack of expression of the CD38 antigen, the mutated immunoglobulin (Ig) heavy (H) chain variable (V) pattern, the absence of p53 mutations, a CD4/CD8 ratio more than 1, the lack of 17p and 11q deletions and of complex karyotypic aberrations, and the occurrence of the 13q14 deletion. No case displayed the VH3-21 gene, linked in mutated CLL with a poor outcome. In addition, the VH1-69 gene associated with unmutated CLL cases was never detected. These biologic features were coupled with an indolent clinical course characterized by an unmodified clinical stage over time, and by lack of autoimmune phenomena and of major infections requiring parental antibiotics. At a time when aggressive therapeutic strategies are always more frequently used in the management of CLL, the distinctive features of patients with long-lived stable disease should be prospectively identified at presentation.