ALBERT

Description: Introduction. Chronic lymphocytic leukemia (CLL) with 11q deletion has been associated to a poor prognosis, but the clinical course of patients carrying this lesion is variable. This aberration, most often monoallelic, is present in 10-17% of newly diagnosed CLL and in 20-30% of patients with progressive or chemorefractory disease. The minimal deleted region (MDR) (2-3 Mbp) is located on the 11q22.3-q23.1 region and includes ATM. Moreover, 30-40% of 11q- CLL have also an inactivating ATM mutation on the other allele. The deleted region on 11q can also include BIRC3, a gene that is often deleted or mutated in advanced/chemorefractory stages of the disease. Although BIRC3 disruption has been associated to a poor prognosis, its prognostic implications in addition to ATM deletion are not well defined. The aim of this study was to perform a copy number aberration (CNA) and gene sequencing analyses on a cohort of CLL patients with 11q- in order to identify subgroups with potential prognostic relevance based on: i) the inclusion of BIRC3 in the deleted region; ii) the presence of BIRC3 mutation; iii) the presence of other CNAs. Methods. The study has included 55 untreated CLL patients followed at our Institution or enrolled in GIMEMA clinical trials (2003-2013). Genomic DNA was extracted from peripheral blood samples. CNA analysis was performed by genomic hybridization on the CytoScan HD array (Affymetrix), which contains more than 2.6 x 106 markers for copy number analysis and 750.000 SNPs. Data were analyzed using both Partek Genomics Suite and ChAS (Chromosome Analysis Suite, Affymetrix) software. The resulting CNAs were verified by visual examination of the plotted copy number profiles. BIRC3 mutations (exons 6-9) were evaluated by Sanger sequencing. Time to first treatment (TFT) was calculated from the date of diagnosis to the date of first therapy or last follow-up; progression-free survival (PFS) from the date of first therapy to the date of progression, death or last follow-up. Results. Baseline characteristics of the 55 cases were as follows: median age at diagnosis 59 years (range 39-84), male gender in 81.8% of patients, progressive disease in 62%. All patients showed 11q- by FISH (median 80%, range 25-99% of nuclei); germline IGHV were present in 96.4% of cases; TP53 deletion in 1 case and TP53 mutation in none; NOTCH1 mutation in 4/40 cases; SF3B1 mutation in 5/40 (all mutually exclusive with only 1 case having both SF3B1 and BIRC3 mutations). By CytoScan HD array, the size of 11q- was very variable, ranging from 0.36 Mbp to 65.14 Mbp; the MDR was located on 11q22.3 region, encompassing 4 genes (ACAT1, ATM, CUL5, NPAT). BIRC3 was included in the deleted region in 45/55 cases (81.8%) and was mutated in 4/54 (7.5%), being always deleted on the other allele. Beside 11q-, 51 cases (92.7%) showed several additional CNAs (average 4.9, range 1-14 per patient), with 5 recurrent lesions: 2p gain in 11 cases, del4(p15.2) in 6, del19(p13.3) in 6, 8q gain in 5 and del4(q22.1) in 4. BIRC3 deletion was not associated to the number of additional CNAs nor to specific CNA. After a follow-up of 59.6 months (range 7.4-229.7), 40 of 47 evaluable patients have received treatment (median TFT 15.8 months, range 0-167). BIRC3 deleted cases (n=37) showed a TFT not significantly different from WT cases (n=10). Conversely, BIRC3 mutation was associated to a shorter TFT (p 3 CNAs larger than 5 Mb (n=14) or 〉10 CNAs (n=5), or the presence of 2p gain, del4(p15.2), del19(p13.3) or 8q gain. So far, 22 patients have been evaluated for PFS after first-line therapy (median 28.7 months). BIRC3 deleted cases (n=17) were not associated to a shorter PFS compared to WT cases (n=5), in line with the results from Rose-Zerilli et al (Haematologica 2014). Conclusions. Among CLL with 11q-: 1) BIRC3 deletion involves more than 80% of cases, whilst the mutation is rare (7.5%); 2) BIRC3 deletion is not associated to a higher genomic complexity; 3) BIRC3 deletion does not seem to influence TFT or PFS of 11q- CLL; 4) BIRC3 mutation is strongly associated to a short TFT; 5) BIRC3 biallelic lesions can be associated to a marked hyperleucocytosis at diagnosis and immediate need of treatment. Disclosures No relevant conflicts of interest to declare.

Description: The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 〉 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 〉 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.

Description: The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1–positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Δ4-7) and by removal of exons 2 through 7 (Δ2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Δ4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Δ2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

Description: During the years of 2005 to 2008, the MILE (Microarray Innovations in LEukemia) study research program was performed in 11 laboratories across three continents: 7 from the European Leukemia Network (ELN, WP13), 3 from the US and 1 in Singapore. The first stage was designed as biomarker discovery phase to generate whole-genome gene expression profiles (GEP) from recognized categories of clinically relevant leukemias and myelodysplastic syndromes (MDS). These were C1: mature B-ALL with t(8;14), C2: pro-B-ALL with t(11q23)/MLL, C3: c-ALL/pre-B-ALL with t(9;22), C4: T-ALL, C5: ALL with t(12;21), C6: ALL with t(1;19), C7: ALL with hyperdiploid karyotype, C8: c-ALL/pre-B-ALL without specific genetic abnormalities, C9: AML with t(8;21), C10: AML with t(15;17), C11: AML with inv(16)/t(16;16), C12: AML with t(11q23)/MLL, C13: AML with normal karyotype or other abnormalities, C14: AML with complex aberrant karyotype, C15: CLL, C16: CML, C17: MDS, and C18: non-leukemic and healthy bone marrow samples as controls and were compared to conventional diagnostic assays (“Gold Standard”). Data from the completed MILE Stage I included 2143 retrospectively collected adult and pediatric samples tested with HG-U133 Plus 2.0 microarrays (Affymetrix). In total only 47 analyses (2.1%) failed technical quality criteria. Cross-validation accuracy (average of three 30-fold cross-validations) of the final 2096 MILE Stage I samples was 92.1% concordant with the center-specific “Gold Standard” diagnosis (average call rate 99.4%). In nine classes the sensitivity was ≥94.3%: C2, C3, C4, C5, C9, C10, C11, C15, and C16. Lower sensitivities were observed for C7, C8, C14, and C17; which can largely be explained by the biological heterogeneity and non-standardized “Gold Standard” definitions for these entities. Yet, it is notable that all these classes showed specificities above 98.1%. In order to assess the clinical utility of microarray-based diagnostics a prospective Stage II was subsequently performed using a customized microarray representing 1480 probe sets. Overall, 1156 high quality GEP have been generated in MILE Stage II and represent an independent and blinded test set for the algorithms developed. A focused classification scheme aimed at accurately addressing only acute leukemias resulted in a 95.5% median sensitivity and a 99.5% median specificity for the 14 classes included in the classifier (C1 – C14, n=696). Lower accuracies were observed for the interface of C7–C8 in ALL, as well as C12 and C14 in AML. Interestingly, during the process of discrepant results analyses, it was observed that for 7.5% (n=52) of acute leukemias microarray results were correctly diagnosing samples as compared to the initial “Gold Standard” diagnoses entered into the study database, either because of erroneous entries into case report forms (24%) or subsequent re-testing of left-over material following the suggested diagnosis from the microarray (76%). In addition, predicted accuracies for CLL, CML and MDS in Stage II were 99.2%, 95.2%, and 81.5%, respectively. In conclusion, the MILE research study confirms in a final cohort of 3252 patients that microarrays accurately classify acute and chronic leukemia samples into known diagnostic and prognostic sub-categories. This final report underlines that the standardized method of gene expression profiling with low technical failure rate and simplified standard operating procedures may improve current “Gold Standards” as an adjunct to conventional diagnostic algorithms and potentially offers a reliable diagnostic/prognostic tool for many patients who don’t have access to a state-of-the-art “Gold Standard” workup. Our gene expression database, intended to be submitted to the public domain, will further contribute to research that aims to elucidate the molecular understanding of leukemias.

Description: The role of Vγ9Vδ2 T cells in chronic lymphocytic leukemia (CLL) is unexplored, although these cells have a natural inclination to react against B-cell malignancies. Proliferation induced by zoledronic acid was used as a surrogate of γδ TCR-dependent stimulation to functionally interrogate Vγ9Vδ2 T cells in 106 untreated CLL patients. This assay permitted the identification of responder and low-responder (LR) patients. The LR status was associated with greater baseline counts of Vγ9Vδ2 T cells and to the expansion of the effector memory and terminally differentiated effector memory subsets. The tumor immunoglobulin heavy chain variable region was more frequently unmutated in CLL cells of LR patients, and the mevalonate pathway, which generates Vγ9Vδ2 TCR ligands, was more active in unmutated CLL cells. In addition, greater numbers of circulating regulatory T cells were detected in LR patients. In multivariate analysis, the LR condition was an independent predictor of shorter time-to-first treatment. Accordingly, the time-to-first treatment was significantly shorter in patients with greater baseline numbers of total Vγ9Vδ2 T cells and effector memory and terminally differentiated effector memory subpopulations. These results unveil a clinically relevant in vivo relationship between the mevalonate pathway activity of CLL cells and dys-functional Vγ9Vδ2 T cells.

Description: Among all cytogenetic subtypes of ALL, the BCR-ABL1 fusion gene, which is causative of chronic myeloid leukemia (CML), defines the subgroup of ALL with the worst prognosis. The reasons of the aggressive nature of BCR-ABL1-positive ALL have not yet been completely understood. To identify, at the submicroscopic level, oncogenic lesions that cooperate with BCR-ABL1 to induce ALL and blastic transformation of CML, we used an integrated molecular approach including high resolution genomic analysis of copy number alterations by single nucleotide polymorphism (SNP) arrays (500K, Affymetrix Inc., Santa Clara, CA, USA), genomic amplification, cloning, deep sequencing, gene expression profiling (GEP) and analysis of epigenetic changes to study 97 de novo BCR-ABL1- positive ALL adult patients (pts). High level amplification and homozygous deletions were identified in all pts, with deletions outnumbering amplification almost 3:1. The most frequent somatic copy number alteration was deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros identified in 59/97 pts (61%). Ikaros is required for the earliest stages of lymphoid lineage commitment and acts as tumor suppressor in mice. The IKZF1 deletions were predominantly mono-allelic (64% vs 36%) and were limited to the gene in all cases, identifying IKZF1 as the genetic target. Real-time quantitative PCR and FISH analysis confirmed SNP results. We characterized and mapped all breakpoints to recognize that two major deletions occur in BCR-ABL1-positive ALL: type A characterized by loss of exons 4–7 (37/97, 38%) and due to breakpoints occurring in the region spanning from 50380371 to 50380388 and from 50431123 to 50431167 in introns 3 and 7, respectively; type B characterized by removal of exons 2–7 (18/97, 19%) and due to a variable pattern of breakpoints in introns 1 (from 50317112 to 50317936) and 7. A variable number of nucleotides (patient-specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of both deletions correlated with the expression of a dominant-negative isoform Ik6 with cytoplasmic localization and oncogenic activity, and of an aberrant transcript containing only exons 1 and 8, respectively. Interestingly, 2 pts with a homozygosis deletion showed simultaneously both type A and B deletions. In other 2 pts, the deletion involved the whole IKZF1 gene. The IKZF1 alteration was not a frequent event across different leukemias, since it was identified also at the progression of CML to lymphoid blast crisis (66%), but never in myeloid blast crisis CML (n=10), chronic phase CML (n=30) and acute myeloid leukemia (n=28). Known DNA sequences and structural features were mapped along the breakpoint cluster regions including heptamer recombination signal sequences recognized by the RAG enzymes during V(D)J recombination and activation-induced cytidine deaminase (AID) consensus motifs (DGYW/WRCH), suggesting that IKZF1 deletions could arise from aberrant RAG and/or AID-mediated recombination. GEP analysis of pts with IKZF1 deletion vs wild-type pts identified a unique signature characterized by a down-regulation of genes involved in pre-B-cell differentiation (e.g. VPREB1, VPREB3, IGLL3), demonstrating that genomic IKZF1 alterations have a strong impact on trascriptoma and contribute to a block of B-cell differentiation. For 83 out of 97 BCR-ABL1 ALL pts correlation between molecular data and clinical outcome was available. Univariate analysis showed that the IKZF1 deletion is a negative prognostic marker influencing the cumulative incidence of relapse (10.1 months for pts with deletion vs 56.1 months for wild-type pts, p=0.0103) and disease-free survival (DFS) (10.1 months vs 32.1 months, respectively, p=0.0229). The negative prognostic impact of the IKZF1 deletion on DFS was also confirmed by multivariate analysis (p=0.0445). In conclusion, deletion of IKZF1 resulting in either haploinsuffciency and in the expression of the dominant negative isoforms is an important event in the development of BCR-ABL1 B-progenitor ALL which significantly influences clinical outcome.

Description: Acute lymphoblastic leukemia (ALL) is characterized by the presence of specific rearrangements associated with distinct gene expression signatures defined by high density microarray. Rearrangements of MLL, E2A/PBX1, BCR/ABL and TEL/AML1 are examples of this phenomenon, which also provide insights into the mechanisms of malignant transformation in these cases. Nevertheless, many cases of ALL do not carry known molecular abnormalities and do not exhibit unique gene expression profiles. In these cases, the mechanisms of malignant transformation remain unknown. Our goal was to develop an integrated genetic approach to discover new mechanisms of transformation in ALL and to define patterns of expression associated with these genetic abnormalities. Gene expression profiles were determined in B-lineage ALL from 95 adult patients using Affymetrix U95Av2 GeneChips. Within this group, high density SNP analysis was performed on 16 paired samples of normal and leukemia cells from the same patient using Affymetrix Mapping 10K Arrays. SNP analysis of normal and ALL DNA based on chromosome copy number interference revealed the presence of several regions of amplification, the most frequent involving chromosome X (3 samples, plus 1 Klinefelter syndrome, minimal amplified region: Xp22.31 to Xp11.1) and chromosome 21 (3 samples plus 1 Down syndrome, minimal amplified regions 21q223.13 to 21q22.2). Further analysis showed a frequent occurrence of LOH on chromosome 3, 6, and 21. Remarkably, this analysis revealed the presence of LOH in a portion of chromosome 9p (9p13.3 to 9p24.3) in 25% of cases. FISH analysis confirmed the presence of a homozygous deletion at 9p21 in 2 of these samples, whereas the 2 remaining cases showed a normal copy number, suggesting that in these 2 patients a deletion followed by duplication occurred. Analysis of gene expression based on genes located in this deleted region identified two genes with reduced expression: tropomyosin 2, a putative tumor suppressor gene and a transcript whith an unknown function. Analysis of gene expression in these cases revealed differential expression of 124 genes: of these, only 25 were highly expressed in the “9p LOH cases”. The use of this gene set in a heat map of all the cases without molecular abnormalities revealed a cluster that included 10 cases. 5 have known 9p abnormalities involving p15 and/or p16, 2 have 9p LOH by SNP analysis and 2 did not have sufficient material for further analysis. Given these results, we evaluated to what extent there was an overlap between these two phenomena. This revealed 34 transcripts that were consistently selected, suggesting that these genes represent a unique signature reflecting leukemic transformation involving the 9p region. These results demonstrate that integrated analysis of gene expression and SNP-based comparison of leukemia and normal cells can identify previously unknown groups of tumors with distinct genetic profiles. Further studies can now be undertaken to better define the mechanisms of transformation in these leukemias.

Description: Abstract 12 Background: Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism (SNP) microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, so far, the occurrence of PAX5 alterations and their clinical correlation has not been investigated in adults with BCR-ABL1-positive ALL. Aim: To characterize the rearrangements on 9p involving PAX5 and their clinical significance in adult BCR-ABL1-positive ALL. Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results: By SNP array analysis we identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was hemizygous. Four patterns of PAX5 deletion were observed: 1) focal deletion involving only the PAX5 gene in one case (3%); 2) deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); 3) broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); 4) deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in either PAX5 haploinsufficiency or expression of PAX5 alleles with impaired DNA-binding or transcriptional activation activity. In one patient the deletion of PAX5 involved only a subset of exons (exons 2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used, demonstrating that genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Furthermore, both normal and deleted PAX5 subgroups were investigated for point mutations by sequencing analysis but we did not found nucleotide substitutions, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. However, when we investigated the association of PAX5 deletions with clinical outcome, no association with PAX5 status was detected (overall survival: p=0.3294; disease free-survival,DFS: p=0.9249 and cumulative incidence of relapse, CIR: p=0.945), suggesting that PAX5 deletions are not associated with outcome. Twenty-three patients (26%) had deletion of both PAX5 and IKZF1, encoding for the transcription factor Ikaros. We assessed the contribution of both PAX5 and IKZF1 alterations on clinical outcome finding out that the occurrence of both normal PAX5 and IKZF1 loss is associated with a shorter DFS (p=0.04) and higher CIR cumulative incidence of relapse in comparison to patients with both normal IKZF1 and PAX5 (p = 0.009). These results were confirmed by multivariate analysis. Conclusion: PAX5 deletions are frequent events in adult BCR-ABL1 positive ALL and are often associated with IKZF1 loss. The genomic status of both PAX5 and IKZF1 can be useful to identify at diagnosis groups of patients with worse prognosis. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Disclosures: No relevant conflicts of interest to declare.

Description: Background. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of relatively mature B cells and by a very variable clinical course. This clinical heterogeneity is sustained by different biologic parameters, such as the mutational status of the immunoglobulin variable genes (IgVH), CD38 and ZAP-70 expression. In order to investigate the potential role of protein kinase (PK) inhibitors in CLL, we evaluated the gene expression profile of 1324 probesets annotated as PK using the HGU133 Plus2.0 Affymetrix arrays. Methods. We evaluated 44 CLL and 137 acute lymphocytic leukemia (ALL) patients. Two additional sets of CLL (49 cases) were utilized to validate the results obtained. Probesets identified as PK genes were used for all the analyses, namely unsupervised clustering, Analysis of Variance (ANOVA) and t-test analysis. ANOVA was performed using a p-value of ≤0.001: further selection was performed retaining only those probesets whose mean expression level was ≥300 in at least one group and showed a fold change difference of ≥1.5 across all groups. Finally, to specifically identify genes differentially expressed between different subclasses of CLL, a t-test was applied: probesets were required to have a p-value ≤0.05 and a fold change〉1.5. Results. Unsupervised analysis, performed on CLL samples and different ALL subgroups, highlighted in CLL a unique and very homogeneous pattern characterized by the overexpression of a large set of PK; these results were further confirmed by ANOVA. Moreover, we identified 16 PK genes that were highly expressed in all 3 CLL sets analyzed. These genes codify for proteins with tyrosine kinase activity (SYK, LYN, BLK, LCK, JAK1, CSK and FGR), serin-threonin kinase activity (PIM2, PFTK1, TLK1, MAP4K1, PDPK1, PRKCB1 and STK10) or both (GRK6 and WEE1). Some of the selected genes are members of important protein kinase families, involved in cellular signaling, such as Src kinases (SFK), MAPK and JAK kinase family. PK expression was also analyzed in different CLL subclasses, subdivided according to different prognostic factors; in particular, we compared IgVH mutated vs unmutated patients, CD38+ vs CD38- cases and, finally, ZAP-70+ vs ZAP-70- patients in the 3 experimental CLL sets. Comparison between IgVH mutated vs unmutated cases highlighted a differential expression of ZAP-70 in all the 3 sets analyzed. Contrariwise, no PK was associated with the other prognostic parameters. Thus, these analyses did not show a specific signature associated with the abovementioned biologic features, suggesting that PK overexpression is specific of the disease itself rather than of CLL subclasses. Conclusions. Our results show that CLL is characterized by a very peculiar PK signature and identify some potential molecular targets. Moreover, our findings indicate that a common mechanism of PK-mediated deregulation is operational in CLL cells, independently of other prognostic factors. Based on these pre-clinical data, we propose that second generation PK inhibitors may have a role in the management of all CLL patients.

Description: Background. CLL is the most common leukemia in the western world and is characterized by the accumulation of relatively mature B cells. From a clinical point of view, CLL patients display a variable course. This clinical heterogeneity is sustained by different biologic parameters, such as the immunoglobulin variable gene (IgVH) mutational status, ZAP-70 expression and specific cytogenetic alterations. Even though extensive molecular characterizations of CLL cells have been performed, a candidate gene responsible for the disease is still lacking. Thus, there is a compelling need to identify other mechanisms that may play a role in CLL. miRs are an abundant class of small non-coding RNAs which modulate the expression of their target mRNAs at the post-transcriptional and/or translational level. So far, 462 miRs have been identified in the human genome: many are involved in cancer, acting either as oncogenes or tumor suppressors. Methods. In order to define the potential role of miRs in CLL, the expression of these small RNAs was evaluated by cloning, as well as by quantitative Real-Time PCR (qRT-PCR) in a total of 56 CLL patients: 9 cases were evaluated by cloning, 46 by qRT-PCR and 4 by both methods; peripheral blood B lymphocytes and cord blood cells enriched for CD19+ were used as controls. Results. By cloning, it was possible to identify roughly 20 miRs that contribute to more than 90% of the expressed miRs in CLL and healthy donors. Furthermore, we observed a significant upregulation (at least 3 fold) of miR-21, miR-155 and miR-150 in CLL patients compared to healthy donors. To validate these findings, we next measured the expression of 22 of the most expressed miRNAs in 51 CLL patients and in 7 healthy donors (5 CD19+ peripheral blood lymphocytes and 2 CD5+ cord blood cells) by qRT-PCR: this approach confirmed the differential expression of miR-21, miR-155 and miR-150 expression between leukemic and normal cells, and identified 2 additional miRs, namely miR-92 and miR-222, that are downregulated in the controls. Unsupervised clustering based on qRT-PCR results allowed to subdivide the cases analyzed into 3 major subgroups: the first comprised all the 5 controls, the second a set of patients that had a strong downregulation of miR-15 and miR-16, and the third was inclusive of all the remaining cases. It is of worth noting that the patients who showed a downregulation of miR-15 and miR-16 had a del13q14 in homozygosis, in line with previous reports. Finally, a supervised approach highlighted a differential expression of miR-150, miR-29bc and miR-223 between IgVH mutated and unmutated; consistently with this finding, miR-150 was also differentially expressed between ZAP-70+ and ZAP-70− cases. Conclusions. Our study indicates that 3 miRs are strongly and homogeneously upregulated in CLL patients, suggesting that they may play an important role in disease initiation and/or progression. Finally, a small set of miRs is distinctive of prognostic subgroups. Further investigations on these small RNAs is ongoing, to specifically define their role in CLL.