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TP53-disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long-term response to ibrutinib. We hereby report that ibrutinib-induced in vitro apoptosis and proliferation inhibition were significantly lower in TP53-mutated (TP53-M) CLL cells compared to TP53 wild-type cells. Contrariwise, venetoclax effectively killed TP53-M cells. Gene expression profile analysis of TP53-M cells revealed a downmodulation of B-cell receptor (BCR)-related genes and an upmodulation of genes with anti-apoptotic/pro-survival activity, suggesting that the survival and proliferation of TP53-M cells are less dependent on the BCR pathway. These observations further support the use of drug combinations for the optimal management of TP53-M CLL patients., (© 2018 British Society for Haematology and John Wiley & Sons Ltd.)

Background: Chronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patients requiring immediate treatment, others show an initial indolent phase followed by progression and others do not progress for decades. The latter two subgroups usually display mutated IGHV genes and a favorable FISH profile., Patients and Methods: Patients with absence of disease progression for over 10 years (10-34) from diagnosis were defined as ultra-stable CLL (US-CLL). Forty US-CLL underwent extensive characterization including whole exome sequencing (WES), ultra-deep sequencing and copy number aberration (CNA) analysis to define their unexplored genetic landscape. Microarray analysis, comparing US-CLL with non-US-CLL with similar immunogenetic features (mutated IGHV/favorable FISH), was also carried out to recognize US-CLL at diagnosis., Results: WES was carried out in 20 US-CLL and 84 non-silent somatic mutations in 78 genes were found. When re-tested in a validation cohort of 20 further US-CLL, no recurrent lesion was identified. No clonal mutations of NOTCH1, BIRC3, SF3B1 and TP53 were found, including ATM and other potential progression driving mutations. CNA analysis identified 31 lesions, none with known poor prognostic impact. No novel recurrent lesion was identified: most cases showed no lesions (38%) or an isolated del(13q) (31%). The expression of 6 genes, selected from a gene expression profile analysis by microarray and quantified by droplet digital PCR on a cohort of 79 CLL (58 US-CLL and 21 non-US-CLL), allowed to build a decision-tree capable of recognizing at diagnosis US-CLL patients., Conclusions: The genetic landscape of US-CLL is characterized by the absence of known unfavorable driver mutations/CNA and of novel recurrent genetic lesions. Among CLL patients with favorable immunogenetics, a decision-tree based on the expression of 6 genes may identify at diagnosis patients who are likely to maintain an indolent disease for decades.

The activity and safety of a regimen combining lenalidomide with fludarabine and cyclophosphamide (FC) was investigated in patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). Treatment consisted of six monthly courses of the FC regimen combined with 14 days of lenalidomide given at the starting dose of 2.5 mg during course 1. The maximum tolerated dose of lenalidomide was 5 mg. Forty patients were assessed for response, 66% were IGHV unmutated, 45% showed deletion 11q or 17p. The overall response and complete remission rates were 62.5% and 22.5%, respectively, the median progression-free and overall survival (OS) were 19 and 45 months, respectively. Grade 3-4 granulocytopenia was observed in 65% of cases, severe infections in 7.5%, the lenalidomide-related toxicity was mild. In conclusion, the results of this study demonstrate that low-dose lenalidomide associated with the FC schedule is an effective treatment for R/R patients with CLL, associated with an acceptable safety profile.

Chronic lymphocytic leukaemia (CLL) is uncommon in Chinese population and its biology, genetics and treatment outcome in Chinese patients have not been comprehensively investigated. In this study, we studied the clinicopathological features and outcome of 212 Chinese patients with newly diagnosed CLL in Hong Kong and Singapore. The median age at diagnosis was 64 years. The majority of patients presented with early-stage disease (Binet stage A, 56.1%). Del(13)(q14) was the most frequent abnormality (41.7%) detected by fluorescence in situ hybridization (FISH) analysis. Del(17p) and TP53 gene mutations were detected in 7.8% and 8.2% of patients, respectively. MYD88 mutations were found at a higher frequency (11.5%) than expected. CLL with unmutated variable region of the immunoglobulin heavy chain genes (IGHV) occurred in only 31.2% of cases, and was associated with advanced-stage disease (p <0.01) and adverse FISH abnormalities (p<0.01). With a median follow-up of 39 months, the median overall survival (OS) was 108 months. The presence of del(17p) or TP53 mutations was associated with a significantly shorter time to first treatment and an inferior OS (p <0.01). Unmutated IGHV was also associated with a significantly shorter time to treatment (p <0.01). Among patients who required treatment, the median OS and progression-free survival (PFS) were 107 and 23 months, respectively. The presence of del(17p) was associated with a significantly inferior OS and PFS (p <0.01). In summary, Chinese CLL patients had similar genetic aberrations at diagnosis compared with those of Western populations. FISH abnormalities are major factors affecting outcome.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the Western world, whereas in Asia the incidence is about 10 times lower. The basis for this ethnic and geographic variation is currently unknown. The aim of this study was to characterize IGHVDJ rearrangements and stereotype of the HCDR3 region in a series of 623 Chinese CLL, in order to identify possible differences in immunoglobulin gene usage and their potential pathogenetic implications. Chinese CLL were compared to 789 Italian CLL. Chinese patients showed a higher proportion of mutated IGHV and a more frequent usage of IGHV3-7, IGHV3-74, IGHV4-39 and IGHV4-59 genes. A significantly lower usage of IGHV1-69 and IGHV1-2 was documented, with comparable IGHV3-21 frequency (3% Chinese vs 3.8% Italian CLL). The proportion of known stereotyped receptors was significantly lower in Chinese (19.7%) than in Italian CLL (25.8%), despite a significantly higher frequency of subset #8 (p= 0.0001). Moreover, new paired clusters were identified among Chinese cases. Overall, these data support a potential different antigenic exposure between Eastern and Western CLL.

Whole exome sequencing and copy number aberration (CNA) analysis were performed on cells taken from peripheral blood (PB) and lymph nodes (LN) of patients with chronic lymphocytic leukaemia (CLL). Of 64 non-silent somatic mutations, 54 (84·4%) were clonal in both compartments, 3 (4·7%) were PB-specific and 7 (10·9%) were LN-specific. Most of the LN- or PB-specific mutations were subclonal in the other corresponding compartment (variant frequency 0·5-5·3%). Of 41 CNAs, 27 (65·8%) were shared by both compartments and 7 (17·1%) were LN- or PB-specific. Overall, 6 of 9 cases (66·7%) showed genomic differences between the compartments. At subsequent relapse, Case 10, with 6 LN-specific lesions, and Case 100, with 6 LN-specific and 8 PB-specific lesions, showed, in the PB, the clonal expansion of LN-derived lesions with an adverse impact: SF3B1 mutation, BIRC3 deletion, del8(p23·3-p11·1), del9(p24·3-p13·1) and gain 2(p25·3-p14). CLL shows an intra-patient clonal heterogeneity according to the disease compartment, with both LN and PB-specific mutations/CNAs. The LN microenvironment might contribute to the clonal selection of unfavourable lesions, as LN-derived mutations/CNAs can appear in the PB at relapse., (© 2015 John Wiley & Sons Ltd.)

To clarify whether karyotype aberrations (KA) involving regions not covered by the standard fluorescence in situ hybridization (FISH) panel have independent prognostic relevance, we evaluated KA by conventional cytogenetics in a learning cohort (LC; n = 166) and a validation cohort (VC; n = 250) of untreated chronic lymphocytic leukemia (CLL) patients. In the VC, novel mitogens were used to improve metaphase generation and TP53, NOTCH1, and SF3B1 mutations were assessed. KA undetected by FISH were found in 35 and 35% of the cases in the LC and VC, respectively. In addition to FISH, KA allowed reclassification of 23 and 26% of cases in the LC and VC, respectively, into a higher cytogenetic risk group. By multivariate analysis, both in the LC and VC, KA other than isolated 13q deletion correlated with a shorter time to first treatment (TFT; P < 0.001 and 0.003, respectively), while a complex karyotype predicted a worse overall survival (OS, P = 0.015 and 0.010, respectively). In the VC, where a comprehensive biologic assessment was performed, a shorter TFT was also predicted by stage (P < 0.001), IGHV mutational status (P = 0.05), and del(17p)/TP53 mutations (P = 0.033) while stage (P = 0.023) and del(17p)/TP53 mutations (P = 0.024) independently predicted a shorter OS. FISH results did not independently impact on TFT and OS, in the LC and VC cohorts; this was also the case for NOTCH1 and SF3B1 mutations in the VC. We suggest that in CLL, conventional karyotyping with novel mitogens could be more effective than FISH for the detection of KA allowing for a more precise refinement of prognosis., (© 2015 Wiley Periodicals, Inc.)

To assess the involvement of microRNAs (miRNAs) in B-cell receptor (BCR) stimulation, we first evaluated miRNA profiling following IgM cross-linking in chronic lymphocytic leukemia (CLL) cells and in normal B lymphocytes. Second, we combined miRNA and gene expression data to identify putative miRNA functional networks. miRNA profiling showed distinctive patterns of regulation after stimulation in leukemic versus normal B lymphocytes and identified a differential responsiveness to BCR engagement in CLL subgroups according to the immunoglobulin heavy chain variable region mutational status and clinical outcome. The most significantly modulated miRNAs in stimulated CLL are miR-132 and miR-212. Notably, these miRNAs appeared regulated in progressive but not in stable CLL. Accordingly, gene profiling showed a significant transcriptional response to stimulation exclusively in progressive CLL. Based on these findings, we combined miRNA and gene expression data to investigate miR-132 and miR-212 candidate interactions in this CLL subgroup. Correlation analysis pointed to a link between these miRNAs and RB/E2F and TP53 cascades with proproliferative effects, as corroborated by functional analyses. Finally, basal levels of miR-132 and miR-212 were measured in an independent cohort of 20 unstimulated CLL cases and both showed lower expression in progressive compared to stable patients, suggesting an association between the expression of these molecules and disease prognosis. Overall, our results support a model involving miR-132 and miR-212 upregulation in sustaining disease progression in CLL. These miRNAs may therefore provide new valuable strategies for therapeutic intervention., (© 2015 Wiley Periodicals, Inc.)

In chronic lymphocytic leukemia, NOTCH1, SF3B1, BIRC3 and TP53 disruptions are recurrent and affect survival. To define their incidence and clinical impact in patients undergoing first-line treatment, we evaluated 163 cases enrolled in the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) LLC0405 protocol (fludarabine plus alemtuzumab or fludarabine plus cyclophosphamide), for young patients, or in the ML21445 protocol (chlorambucil plus rituximab), for elderly patients. NOTCH1, SF3B1, BIRC3 and TP53 disruptions were detected in 15.9%, 12.2%, 8.6% and 10.4% of cases. NOTCH1 mutations correlated with a shorter treatment-free interval (p = 0.058), an unmutated immunoglobulin heavy variable gene (IGHV) status (p < 0.0001), CD38 and ZAP-70 expression (p = 0.0025 and 0.026, respectively) and trisomy 12 (p = 0.0028), SF3B1 mutations with an unmutated IGHV status (p = 0.02), and BIRC3 disruptions with an unmutated IGHV configuration (p = 0.01) and 11q deletion (p < 0.0001). NOTCH1 and SF3B1 did not appear to impact on overall response, while an inferior response was observed for BIRC3- and TP53-disrupted cases in the LLC0405 and ML21445 protocols, respectively. Progression-free survival, evaluable in the LLC0405 protocol - not affected by NOTCH1, SF3B1 and TP53 - appeared inferior for BIRC3 disruption. NOTCH1 and SF3B1 mutations may be overcome by aggressive regimens, while BIRC3 might impact on outcome also in intensive regimens.

Minimal residual disease (MRD) is becoming increasingly important in chronic lymphocytic leukaemia (CLL) as treatment strategies are progressively improving. The primary objective of this study was to compare the applicability of three different flow cytometric approaches: basic 4-colour analysis, European Research Initiative in CLL (ERIC) consensus method and 8-colour analysis. Secondly, we investigated the sensitivity and specificity of flow cytometry (FC) compared to molecular analyses for MRD detection. A total of 462 CLL samples were evaluated by basic FC; in 143, ERIC consensus method was also performed and all three FC methodologies were applied in a subgroup of 10 cases. No discordance in defining MRD-positive/negative samples was observed between the FC methods; within positive samples, the ERIC consensus method and 8-colour analysis showed the most accurate results. MRD was analysed by FC and polymerase chain reaction (PCR) in 243 cases: concordant results were obtained in 199/243 samples (81·9%); 42/243 were FC-/PCR+. Overall, the sensitivity and specificity of FC compared to PCR was 96·5% and 77·2%, respectively. Both FC and PCR proved suitable for the detection of MRD and prediction of progression-free survival, which was significantly reduced in MRD-positive patients, regardless of the methodology. These results offer the rationale for a strategy to monitor MRD in CLL patients., (© 2014 John Wiley & Sons Ltd.)

In a phase II trial, we evaluated chlorambucil and rituximab (CLB-R) as first-line induction treatment with or without R as maintenance for elderly chronic lymphocytic leukemia (CLL) patients. Treatment consisted of eight 28-day cycles of CLB (8 mg/m(2) /day, days 1-7) and R (day 1 of cycle 3, 375 mg/m(2) ; cycles 4-8, 500 mg/m(2) ). Responders were randomized to 12 8-week doses of R (375 mg/m(2) ) or observation. As per intention-to-treat analysis, 82.4% (95% CI, 74.25-90.46%) of 85 patients achieved an overall response (OR), 16.5% a complete response (CR), 2.4% a CR with incomplete bone marrow recovery. The OR was similar across Binet stages (A 86.4%, B 81.6%, and C 78.6%) and age categories (60-64 years, 92.3%; 65-69, 85.2%; 70-74, 75.0%; ≥75, 81.0%). CLB-R was well tolerated. After a median follow-up of 34.2 months, the median progression-free survival (PFS) was 34.7 months (95% CI, 33.1-39.5). TP53 abnormalities, complex karyotype, and low CD20 gene expression predicted lack of response; SF3B1 mutation and BIRC3 disruption low CR rates. IGHV mutations significantly predicted PFS. R maintenance tended towards a better PFS than observation and was safe and most beneficial for patients in partial response and for unmutated IGHV cases. CLB-R represents a promising option for elderly CLL patients., (Copyright © 2014 Wiley Periodicals, Inc.)

Fludarabine refractoriness (FR) represents an unsolved clinical problem of chronic lymphocytic leukemia (CLL) management. Although next-generation sequencing studies have led to the identification of a number of genes frequently mutated in FR-CLL, a comprehensive evaluation of the FR-CLL genome has not been reported. Toward this end, we studied 10 FR-CLLs by combining whole-exome sequencing and copy number aberration (CNA) analysis, which showed an average of 16.3 somatic mutations and 4 CNAs per sample. Screening of recurrently mutated genes in 48 additional FR-CLLs revealed that ~70% of FR-CLLs carry ≥1 mutation in genes previously associated with CLL clinical course, including TP53 (27.5%), NOTCH1 (24.1%), SF3B1 (18.9%), and BIRC3 (15.5%). In addition, this analysis showed that 10.3% of FR-CLL cases display mutations of the FAT1 gene, which encodes for a cadherin-like protein that negatively regulates Wnt signaling, consistent with a tumor suppressor role. The frequency of FAT1-mutated cases was significantly higher in FR-CLL than in unselected CLLs at diagnosis (10.3% vs 1.1%, P = .004), suggesting a role in the development of a high-risk phenotype. These findings have general implications for the mechanisms leading to FR and point to Wnt signaling as a potential therapeutic target in FR-CLL.

In 45, ≤ 60 years old patients with CLL and an adverse biologic profile, a front-line treatment with Fludarabine and Campath (Alemtuzumab(®)) was given. The overall response rate was 75.5%, the complete response rate (CR) 24.4% with the lowest CR rates, 16.7% and 8.3%, in 11q and 17p deleted cases. The 3-year progression-free survival (PFS) and overall survival were 42.5% and 79.9%, respectively. PFS was significantly influenced by CLL duration, beta2-microglobulin, and improved by post-remissional stem cell transplantation. Front-line fludarabine and alemtuzumab showed a manageable safety profile and evidence of a benefit in a small series of CLL patients with adverse biologic features., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor (BCR) belonging to subset #1 (IGHV1-5-7/ IGKV1-39) display a poor outcome. To characterize their genetic and genomic features and BCR function, we selected 20 subset #1 CLL from a series of 579 cases. Subset #1 CLL, all showing unmutated IGHV, were associated with the presence of del(11q) (50%) in comparison with unmutated CLL, unmutated stereotyped CLL other than subset #1 and with cases using the same IGHV genes but a heterogeneous VH CDR3 (non-subset #1 CLL). There were no distinctive features regarding CD38, ZAP-70, and TP53 disruption. NOTCH1, SF3B1, and BIRC3 were mutated in 15%, 0%, and 5% of cases, respectively, while BIRC3 was deleted in 22% of cases. Microarray unsupervised analysis on 80 unmutated/mutated/stereotyped/non-stereotyped CLL showed a tight clustering of subset #1 cases. Their genomic signature exhibited several differentially expressed transcripts involved in BCR signal transduction, apoptosis regulation, cell proliferation, and oxidative processes, regardless of del(11q). Accordingly, BCR ligation with anti-IgM revealed a significant higher proliferation of subset #1 versus unmutated non-subset #1 CLL, both at baseline and after 24–48 hr stimulation. Subset #1 CLL represent a paradigmatic example of the direct link between BCR structure, function, and patients prognosis.

Gene profile and functional changes upon IgD cross-linking were evaluated in chronic lymphocytic leukemia (CLL). Microarrays highlighted responsiveness to IgD in all cases, independently of clinico-biological characteristics. Stimulated samples exhibited the down-regulation of transcripts of B-cell receptor signaling and cell-adhesion at 24h and the up-modulation of differentiation and apoptosis genes at 48 h. A significant increase in apoptosis upon ligation was also documented. Furthermore, comparison between IgD and IgM stimulation displayed a differential transcriptional/functional response. In conclusion, CLL respond to IgD displaying expression changes and cell-death enhancement, indicating the apoptosis induction via-IgD as an alternative approach for CLL management., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We analyzed TP53 mutations in 483 chronic lymphocytic leukemia patients at different phases of the disease and found a higher incidence of mutations at the later phases and a distinctive mutation profile in each phase. p53 function evaluated by immunoblotting and flow cytometry after cell irradiation was impaired in 28 of 109 cases. Three phenotypically different dysfunctions were observed: type I, associated with heterozygous missense TP53 mutations (typically present at diagnosis) and partially resistant to radiation-induced killing; types II and III, with a higher incidence of microdeletions, nonsense mutations and bi-allelic TP53 defects (common in progressive and chemoresistant cases) and a complete radioresistance. Furthermore, in 4 of 28 patients, all chemoresistant, we found p53 dysfunctions without TP53 mutations. In chronic lymphocytic leukemia patients, a disease phase-specific variability in the p53 mutation profile and function takes place, and both analyses could be useful to guide treatment choices.

The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

Trisomy 12, the third most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), confers an intermediate prognosis. In our cohort of 104 untreated patients carrying +12, NOTCH1 mutations occurred in 24% of cases and were associated to unmutated IGHV genes (P=0.003) and +12 as a sole cytogenetic abnormality (P=0.008). NOTCH1 mutations in +12 CLL associated with an approximately 2.4 fold increase in the risk of death, a significant shortening of survival (P<0.01) and proved to be an independent predictor of survival in multivariate analysis. Analogous to +12 CLL with TP53 disruption or del(11q), NOTCH1 mutations in +12 CLL conferred a significantly worse survival compared to that of +12 CLL with del(13q) or +12 only. The overrepresentation of cell cycle/proliferation related genes of +12 CLL with NOTCH1 mutations suggests the biological contribution of NOTCH1 mutations to determine a poor outcome. NOTCH1 mutations refine the intermediate prognosis of +12 CLL.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease characterized by defects in the DNA damage response and apoptosis. Among the factors involved in these pathways, we focused on the enzyme poly(ADP-ribose) polymerase 1 (PARP1) and on its substrate Che-1 by evaluating their basal expression and functional changes upon irradiation (IR). Microarray experiments were performed on 98 untreated CLL cases. Next, freshly isolated primary cells from 21 untreated patients were analyzed for in vitro response to irradiation through Western blot, PARP activity assay, Annexin-V analysis, and PARP1 basal expression by quantitative polymerase chain reaction. Microarray analysis showed that PARP1 and CHE1 were constitutively expressed in CLL and had a high degree of correlation with each other and with TP53. PARP1 and TP53 downmodulation was associated with worse clinical outcomes, especially in TP53-mutated cases. Next, CLL samples from 21 untreated patients were classified as responders and nonresponders based on IR-induced PARP1 cleavage. Notably, while responder samples were characterized by Che-1 and p53 induction at 8 hours and reduction at 24 hours post-IR, nonresponders included both samples with p53 dysfunctions and cases with a normal IR-induced Che-1 and/or p53 response. Finally, we observed that PARP1 was downregulated in nonresponder vs responder samples and that its basal expression was positively correlated with PARP1 cleavage after IR. In conclusion, we showed that reduced expression of PARP1 is associated with an impairment of CLL responsiveness to cell death., (Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease., Design and Methods: Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval., Results: Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations., Conclusions: ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval.

A comprehensive panel of clinical-biological parameters was prospectively evaluated at presentation in 112 patients with chronic lymphocytic leukemia (<65 years), to predict the risk of progression in early stage disease. Eighty-one percent were in Binet stage A, 19% in stages B/C. Treatment-free survival was evaluated as the time from diagnosis to first treatment, death or last follow up. In univariate analysis, advanced stage, hemoglobin, platelets, white blood cell, leukemic lymphocyte count, raised beta 2-microglobulin and LDH, unmutated immunoglobulin variable region genes, CD38, del(17p), del(11q) and +12, were significantly associated with a short treatment-free survival; the T/leukemic lymphocyte ratio was associated with a better outcome. Multivariate analysis of treatment-free survival in stage A patients selected a high white blood cell count and unmutated immunoglobulin variable region genes as unfavorable prognostic factors and a high T/leukemic lymphocyte ratio as a favorable one. At diagnosis, these parameters independently predict the risk of progression in stage A chronic lymphocytic leukemia patients.

Given that TP53 alterations predict prognosis and response to therapy in chronic lymphocytic leukemia (CLL), screening for TP53 mutations has an increasing role in patient management. TP53 direct sequencing is a time-consuming method, while the AmpliChip p53 Research Test is a novel non time-consuming microarray-based resequencing assay and queries Exons 2-11. We evaluated the impact of TP53 mutations on clinical outcome by analyzing 98 untreated CLL using the AmpliChip p53 Research Test and direct sequencing and performed microarrays analysis on TP53 mutated and/or deleted cases. The AmpliChip p53 Research Test detected 17 mutations in 14 patients (17.3%); a significant association between TP53 mutations and del(17p) was recorded. From a clinical standpoint, a higher percentage of mutation was found in CLL with unfavorable outcome (17.2% vs. 7.1% in progressive vs. stable cases). Detection of TP53 mutations by the AmpliChip p53 Research Test was associated with a significantly worse survival (P = 0.0002). Comparison of the array and direct sequencing tests showed that the p53 Research Test detected more mutations, although it failed to identify two microdeletions. Finally, microarrays analysis showed a more distinctive signature associated with del(17p) than with TP53 mutations, likely due to a concomitant gene dosage effect. The AmpliChip p53 Research Test is a straightforward method that bears prognostic value. This study confirms a high percentage of TP53 mutations in CLL with unfavorable outcome and a significant association between TP53 aberrations and del(17p). Finally, specific gene expression profiles are recognized for TP53 alterations., (Copyright © 2011 Wiley-Liss, Inc.)

To investigate the role of protein kinases (PKs) in chronic lymphocytic leukemia (CLL), we performed gene expression profile on 505 PK genes. Comparison between CLL with acute lymphocytic leukemia (ALL) patients highlighted an homogeneous up-modulation of several PKs in CLL, 16 also overexpressed in two additional CLL cohorts. Q-PCR analysis confirmed these findings. No differences were observed in the main prognostic subclasses, indicating that PK overexpression is specific of the disease itself. Tests in vitro showed that Dasatinib partially reduced CLL cells viability, mostly in IGHV germline patients. These findings suggest that treatment with second generation tyrosine kinase (TK) inhibitors may represent an attractive therapeutic strategy for CLL patients., (Copyright 2009 Elsevier Ltd. All rights reserved.)

In chronic lymphocytic leukemia (CLL), spontaneous regressions are an exceptional phenomenon, whose biologic features are unknown. We describe 9 CLL patients who underwent a spontaneous clinical regression over an 11-year follow-up, despite a residual neoplastic clone detected by flow cytometry. CD38 and ZAP-70 were negative in all cases. Immunoglobulin heavy chain variable region (IgVH) genes, mutated in all 7 evaluable patients, were restricted to the VH3 family in 6, with the usage of V(H)3-30 gene in 2. The light chain variable region genes were mutated in 6 of 8 cases, with the use of V(kappa)4-1 gene in 3. Microarray analysis of CLL cells showed a distinctive genomic profile with an overrepresentation of BCR-related and ribosomal genes, regulators of signal transduction and transcription. The number of activated T lymphocytes expressing IFN-gamma, TNF-alpha, and IL-4 was similar between CLL in spontaneous regression and healthy persons. In conclusion, spontaneous clinical regressions can occur in CLL despite the persistence of the neoplastic clone, and the biologic features include negative CD38 and ZAP-70, mutated V(H)3-30 and V(kappa)4-1 genes. The peculiar gene profile suggests that BCR signaling may play an important role in this scenario as the most significant feature of the leukemic clone in regression.

Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgV(H) mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G(1) phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG(0/1) cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgV(H) M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.