Giacomo Tuana

Biological and clinical relevance of miRNA expression signatures in primary plasma cell leukemia

Purpose: Primary plasma cell leukemia (pPCL) is a rare and very aggressive form of plasma cell dyscrasia. To date, no information on microRNA (miRNA) expression in pPCL has been reported. This study aimed at investigating the involvement of miRNAs in pPCL and their possible relationship with higher tumor aggressiveness. Experimental design: Global miRNA expression profiles were analyzed in highly purified malignant plasma cells from 18 pPCL untreated patients included in a prospective clinical trial. MiRNA expression patterns were evaluated in comparison with a representative series of multiple myeloma patients, in relation to the most recurrent chromosomal abnormalities (as assessed by fluorescence in situ hybridization and single-nucleotide polymorphism-array analysis), and in association with clinical outcome. MiRNA expression was also integrated with gene expression profiles in pPCL and multiple myeloma samples. Results: We identified a series of deregulated miRNAs in pPCL (42 upregulated and 41 downregulated) in comparison with multiple myeloma. Some of them, on the basis of their reported functions and putative target genes computed by integrative analysis, might have a role in the pathobiology of pPCL. As regards chromosomal aberrations, the expression of some miRNAs mapped to hotspot altered regions was associated with DNA copy number of the corresponding loci. Finally, 4 miRNA (miR-497, miR-106b, miR-181a*, and miR-181b) were identified as having expression levels that correlated with treatment response, and 4 (miR-92a, miR-330-3p, miR-22, and miR-146a) with clinical outcome. Conclusions: Overall, our study provides insights into the possible contribution of miRNAs in the pathogenesis of pPCL and suggests targets for future therapeutic investigations. Clin Cancer Res; 19(12); 3130–42. ©2013 AACR.

The Reconstruction of Transcriptional Networks Reveals Critical Genes with Implications for Clinical Outcome of Multiple Myeloma

Purpose: The combined use of microarray technologies and bioinformatics analysis has improved our understanding of biological complexity of multiple myeloma (MM). In contrast, the application of the same technology in the attempt to predict clinical outcome has been less successful with the identification of heterogeneous molecular signatures. Herein, we have reconstructed gene regulatory networks in a panel of 1,883 samples from MM patients derived from publicly available gene expression sets, to allow the identification of robust and reproducible signatures associated with poor prognosis across independent data sets. Experimental Design: Gene regulatory networks were reconstructed by using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) and microarray data from seven MM data sets. Critical analysis of network components was applied to identify genes playing an essential role in transcriptional networks, which are conserved between data sets. Results: Network critical analysis revealed that (i) CCND1 and CCND2 were the most critical genes; (ii) CCND2, AIF1, and BLNK had the largest number of connections shared among the data sets; (iii) robust gene signatures with prognostic power were derived from the most critical transcripts and from shared primary neighbors of the most connected nodes. Specifically, a critical-gene model, comprising FAM53B, KIF21B, WHSC1, and TMPO, and a neighbor-gene model, comprising BLNK shared neighbors CSGALNACT1 and SLC7A7, predicted survival in all data sets with follow-up information. Conclusions: The reconstruction of gene regulatory networks in a large panel of MM tumors defined robust and reproducible signatures with prognostic importance, and may lead to identify novel molecular mechanisms central to MM biology. Clin Cancer Res; 17(23); 7402–12. ©2011 AACR.

Clinical Monoclonal B lymphocytosis versus Rai 0 Chronic Lymphocytic Leukemia: a Comparison of Cellular, Cytogenetic, Molecular, and Clinical Features

Purpose: To investigate the incidence and clinical relevance of classic and new prognostic markers, IGHV gene mutational status, and chromosomal abnormalities in clinical monoclonal B lymphocytosis (cMBL) compared with Rai stage 0 chronic lymphocytic leukemia (Rai0-CLL). Experimental Design: A group of 136 patients with cMBL and a group of 216 Rai0-CLL cases were investigated prospectively. Results: IGHV-mutated cases were significantly more frequent among cMBLs (P = 0.005), whereas the distribution of CD38 and ZAP-70 positive cases, of patients with NOTCH1 and SF3B1 mutations or exhibiting the major CLL cytogenetic abnormalities, was similar in the two groups. Moreover, no significant differences were found either in IGHV/IGHD/IGHJ gene usage or in the overall prevalence of stereotyped IGHV gene sequences. Cells from cMBL and Rai0-CLL exhibited similar gene and microRNA (miRNA) signatures; in addition, when grouped according to the IGHV mutational status, IGHV-unmutated cases showed different transcriptional signatures compared with IGHV-mutated patients, irrespective of the cMBL or Rai0-CLL classification. cMBL diagnosis per se was predictive of longer progression-free survival. Conclusions: Our study based on a prospective series of patients indicates that no major differences exist between the circulating cells from cMBL and Rai0-CLL, at least based on a comparison of the markers used in the study. This possibly suggests that the two conditions mainly differ in the initial size of the monoclonal cell population, which may influence the subsequent timing of clonal expansion and clinical manifestations. Clin Cancer Res; 19(21); 5890–900. ©2013 AACR.

Genome‐wide analysis of primary plasma cell leukemia identifies recurrent imbalances associated with changes in transcriptional profiles

Primary plasma cell leukemia (pPCL) is a rare, yet aggressive form of de novo plasma cell tumor, distinct from secondary PCL (sPCL) which represents a leukemic transformation of pre‐existing multiple myeloma (MM). Herein, we performed a comprehensive molecular analysis of a prospective series of pPCLs by means of FISH, single nucleotide polymorphism (SNP) array and gene expression profiling (GEP). IGH@ translocations were identified in 87% of pPCL cases, with prevalence of t(11;14) (40%) and t(14;16) (30.5%), whereas the most frequent numerical alterations involved 1p (38%), 1q (48%), 6q (29%), 8p (42%), 13q (74%), 14q (71%), 16q (53%), and 17p (35%). We identified a minimal biallelic deletion (1.5 Mb) in 8p21.2 encompassing the PPP2R2A gene, belonging to a family of putative tumor suppressors and found to be significantly down‐regulated in deleted cases. Mutations of TP53 were identified in four cases, all but one associated with a monoallelic deletion of the gene, whereas activating mutations of the BRAF oncogene occurred in one case and were absent in N‐ and K‐RAS. To evaluate the influence of allelic imbalances in transcriptional expression we performed an integrated genomic analysis with GEP data, showing a significant dosage effect of genes involved in transcription, translation, methyltransferase activity, apoptosis as well as Wnt and NF‐kB signaling pathways. Overall, we provide a compendium of genomic alterations in a prospective series of pPCLs which may contribute to improve our understanding of the pathogenesis of this aggressive form of plasma cell dyscrasia and the mechanisms of tumor progression in MM. Am. J. Hematol. 2013.

Next‐generation sequencing study finds an excess of rare, coding single‐nucleotide variants of ADAMTS13 in patients with deep vein thrombosis

The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Rare single‐nucleotide variants (SNVs) of the coding areas of hemostatic genes may explain part of this missing heritability. The ADAMTS13 and VWF genes encode two interconnected proteins with fundamental hemostatic functions, the disruption of which may result in thrombosis.

The expression pattern of small nucleolar and small Cajal body-specific RNAs characterizes distinct molecular subtypes of multiple myeloma.

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs involved in the maturation of other RNA molecules and generally located in the introns of host genes. It is now emerging that altered sno/scaRNAs expression may have a pathological role in cancer. This study elucidates the patterns of sno/scaRNAs expression in multiple myeloma (MM) by profiling purified malignant plasma cells from 55 MMs, 8 secondary plasma cell leukemias (sPCLs) and 4 normal controls. Overall, a global sno/scaRNAs downregulation was found in MMs and, even more, in sPCLs compared with normal plasma cells. Whereas SCARNA22 resulted the only sno/scaRNA characterizing the translocation/cyclin D4 (TC4) MM, TC2 group displayed a distinct sno/scaRNA signature overexpressing members of SNORD115 and SNORD116 families located in a region finely regulated by an imprinting center at 15q11, which, however, resulted overall hypomethylated in MMs independently of the SNORD115 and SNORD116 expression levels. Finally, integrative analyses with available gene expression and genome-wide data revealed the occurrence of significant sno/scaRNAs/host genes co-expression and the putative influence of allelic imbalances on specific snoRNAs expression. Our data extend the current view of sno/scaRNAs deregulation in cancer and add novel information to the bio-molecular complexity of plasma cell dyscrasias.

Small nucleolar RNAs as new biomarkers in chronic lymphocytic leukemia

BackgroundSmall nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs are non-coding RNAs involved in the maturation of other RNA molecules. Alterations of sno/scaRNA expression may play a role in cancerogenesis. This study elucidates the patterns of sno/scaRNA expression in 211 chronic lymphocytic leukemia (CLL) patients (Binet stage A) also in comparison with those of different normal B-cell subsets.MethodsThe patterns of sno/scaRNA expression in highly purified CD19+ B-cells of 211 CLL patients and in 18 normal B-cell samples - 6 from peripheral blood, and 12 from tonsils (4 germinal center, 2 marginal zone, 3 switched memory and 3 naïve B-cells) - were analyzed on the Affymetrix GeneChip® Human Gene 1.0 ST array.ResultsCLLs display a sno/scaRNAs expression profile similar to normal memory, naïve and marginal-zone B-cells, with the exception of a few down-regulated transcripts (SNORA31, -6, -62, and -71C). Our analyses also suggest some heterogeneity in the pattern of sno/scaRNAs expression which is apparently unrelated to the major biological (ZAP-70 and CD38), molecular (IGHV mutation) and cytogenetic markers. Moreover, we found that SNORA70F was significantly down-regulated in poor prognostic subgroups and this phenomenon was associated with the down-regulation of its host gene COBLL1. Finally, we generated an independent model based on SNORA74A and SNORD116-18 expression, which appears to distinguish two different prognostic CLL groups.ConclusionsThese data extend the view of sno/scaRNAs deregulation in cancer and may contribute to discover novel biomarkers associated with the disease and potentially useful to predict the clinical outcome of early stage CLL patients.

Relevance of Stereotyped B-Cell Receptors in the Context of the Molecular, Cytogenetic and Clinical Features of Chronic Lymphocytic Leukemia

Highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), are expressed in a recurrent fraction of patients affected by chronic lymphocytic leukemia (CLL). We investigated the IGHV status of 1131 productive IG rearrangements from a panel of 1126 CLL patients from a multicenter Italian study group, and correlated the presence and class of HCDR3 stereotyped subsets with the major cytogenetic alterations evaluated by FISH, molecular prognostic factors, and the time to first treatment (TTFT) of patients with early stage disease (Binet A). Stereotyped HCDR3 sequences were found in 357 cases (31.7%), 231 of which (64.7%) were unmutated. In addition to the previously described subsets, 31 new putative stereotypes subsets were identified. Significant associations between different stereotyped HCDR3 sequences and molecular prognostic factors, such as CD38 and ZAP-70 expression, IGHV mutational status and genomic abnormalities were found. In particular, deletion of 17p13 was significantly represented in stereotype subset #1. Notably, subset #1 was significantly correlated with a substantially reduced TTFT compared to other CLL groups showing unmutated IGHV, ZAP-70 or CD38 positivity and unfavorable cytogenetic lesions including del(17)(p13). Moreover, subset #2 was strongly associated with deletion of 13q14, subsets #8 and #10 with trisomy 12, whereas subset #4 was characterized by the prevalent absence of the common cytogenetic abnormalities. Our data from a large and representative panel of CLL patients indicate that particular stereotyped HCDR3 sequences are associated with specific cytogenetic lesions and a distinct clinical outcome.

Immune Thrombocytopenia in Patients with Chronic Lymphocytic Leukemia Is Associated with Stereotyped B-cell Receptors

Purpose: To assess biologic features related to the development of immune thrombocytopenia (ITP) in patients with chronic lymphocytic leukemia (CLL). Experimental Design: We retrospectively analyzed 463 patients with CLL with available immunoglobulin heavy-chain variable (IGHV) gene status and B-cell receptor (BCR) configuration [heavy-chain complementary-determining region 3 (HCDR3)], of whom thirty-six developed ITP, according to previously defined criteria. Most of them had available cytogenetic analysis. Results: We observed a significant association between ITP occurrence and IGHV unmutated gene status (P < 0.0001), unfavorable cytogenetic lesions (P = 0.005), and stereotyped HCDR3 (P = 0.006). The more frequent stereotyped HCDR3 subsets were #1 (IGHV1-5-7/IGHD6-19/IGHJ4; 16 of 16 unmutated) and #7 (IGHV1-69 or IGHV3-30/IGHD3-3/IGHJ6; 13 of 13 unmutated), both being significantly more represented among patients developing ITP (P = 0.003 and P = 0.001, respectively). Moreover, restricting the analysis to unmutated patients, subset #7 confirmed its independent significant association with the occurrence of ITP (P = 0.013). Both unmutated IGHV mutational status, del(11)(q23) and stereotyped BCR were significantly associated with shorter time to ITP development (P < 0.0001, P = 0.02, and P = 0.005, respectively) than other patients. Conclusion: Our data suggest that patients with CLL and peculiar BCR conformations are at higher risk of developing secondary ITP and that stereotyped BCR may be involved in the pathogenesis of this complication. Clin Cancer Res; 18(7); 1870–8. ©2012 AACR.

The HDAC inhibitor Givinostat modulates the hematopoietic transcription factors NFE2 and C-MYB in JAK2V617F myeloproliferative neoplasm cells

We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)(V617F) myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2(V617F) MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2(V617F) cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2(V617F) cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34(+) cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription.