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Tumour DNA sequencing is essential for precision medicine since it guides therapeutic decisions but also fosters the identification of patients who may benefit from germline testing. Notwithstanding, the tumour-to-germline testing workflow presents a few caveats. The low sensitivity for indels at loci with sequences of identical bases (homopolymers) of ion semiconductor-based sequencing techniques represents a well-known limitation, but the prevalence of indels overlooked by these techniques in high-risk populations has not been investigated. In our study, we addressed this issue at the homopolymeric regions of BRCA1/2 in a retrospectively selected cohort of 157 patients affected with high-grade ovarian cancer and negative at tumour testing by ION Torrent sequencing. Variant allele frequency (VAF) of indels at each of the 29 investigated homopolymers was systematically revised with the IGV software. Thresholds to discriminate putative germline variants were defined by scaling the VAF to a normal distribution and calculating the outliers that exceeded the mean + 3 median-adjusted deviations of a control population. Sanger sequencing of the outliers confirmed the occurrence of only one of the five putative indels in both tumour and blood from a patient with a family history of breast cancer. Our results indicated that the prevalence of homopolymeric indels overlooked by ion semiconductor techniques is seemingly low. A careful evaluation of clinical and family history data would further help minimise this technique-bound limitation, highlighting cases in which a deeper look at these regions would be recommended., (© 2023. The Author(s).)

Biliary tract cancers (BTCs) have poor prognosis and limited therapeutic options. The impact of O 6 -methylguanine-DNA methyltransferase (MGMT) inactivation in advanced BTC patients is not established. We investigated the prevalence, prognostic, and predictive impact of MGMT inactivation in two multicenter cohorts. MGMT inactivation was assessed through PCR and immunohistochemistry (IHC) in an Italian cohort; the results were then externally validated using RNA sequencing (RNA-seq) data from the BTC subcohort of the Molecularly Aided Stratification for Tumor Eradication Research (MASTER) precision oncology program of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium. Among 164 Italian cases, 18% presented MGMT promoter hypermethylation (> 14%) and 73% had negative MGMT protein expression. Both were associated with worse overall survival (OS; HR 2.31; P < 0.001 and HR 1.99, P = 0.012, respectively). In the MASTER cohort, patients with lower MGMT mRNA expression showed significantly poorer OS (median OS [mOS] 20.4 vs 31.7 months, unadjusted HR 1.89; P = 0.043). Our results suggest that MGMT inactivation is a frequent epigenetic alteration in BTC, with a significant prognostic impact, and provide the rationale to explore DNA-damaging agents in MGMT-inactivated BTCs., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5 + B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5 + B cells, nor in specific B-cell subpopulations or the CD5 + cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bagnara, Colombo, Reverberi, Matis, Massara, Cardente, Ubezio, Agostini, Agnelli, Neri, Cardillo, Vergani, Ghiotto, Mazzarello, Morabito, Cutrona, Ferrarini and Fais.)

Tumour testing of the BRCA1/2 genes is routinely performed in patients with different cancer histological subtypes. To accurately identify patients with tumour-detected germline pathogenic variants (PVs) is a relevant issue currently under investigation. This study aims at evaluating the performance of the tumour-to-germline diagnostic flowchart model defined at our Institutional Molecular Tumour Board (MTB). Results from tumour BRCA sequencing of 641 consecutive unselected cancer patients were discussed during weekly MTB meetings with the early involvement of clinical geneticists for appropriate referral to genetic counselling. The overall tumour detection rate of BRCA1/2 PVs was 8.7% (56/641), ranging from 24.4% (31/127) in high-grade ovarian cancer to 3.9% (12/304) in tumours not associated with germline BRCA1/2 PVs. Thirty-seven patients with PVs (66%) were evaluated by a clinical geneticist, and in 24 of them (64.9%), germline testing confirmed the presence of the PV in blood. Nine of these patients (37.5%) were not eligible for germline testing according to the criteria in use at our institution. Cascade testing was subsequently performed on 18 relatives. The tumour-to-germline diagnostic pipeline, developed in the framework of our institutional MTB, compared with guideline-based germline testing following genetic counselling, proved to be effective in identifying a higher number of germline BRCA PVs carriers., Competing Interests: FB: Consultant Advisory Board: Roche, EMD Serono, NMS Nerviano Medical Science, Sanofi, MSD, Novartis, Incyte, BMS, Menarini. Speaker: BMS, Healthcare Research & Pharmacoepidemiology, Merck Group, ACCMED, Nadirex, MSD, Pfizer, Servier, Sanofi, Roche, AMGEN, Incyte, Dephaforum. Principal Investigator for Novartis, F.Hoffmann-LaRoche Ltd, BMS, Ignyta Operating INC, Merck Sharp & Dohme Spa, Kymab, Pfizer, Tesaro, MSD, MedImmune LCC, Exelixis Inc., LOXO Oncology Incorporated, DAICHI SANKIO Dev. Limited, Basilea Pharmaceutica International AG, Janssen-Cilag International NV, Merck KGAA MN: Travel expenses from Celgene, speaker honorarium from Accademia della Medicina and consultant honoraria from EMD Serono, Basilea Pharmaceutica, Incyte and MSD Italia. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Azzollini, Vingiani, Agnelli, Tamborini, Perrone, Conca, Capone, Busico, Peissel, Rosina, Ducceschi, Mantiero, Lopez, Raspagliesi, Niger, Duca, Damian, Proto, de Braud, Pruneri and Manoukian.)

Aim: Diagnostic laboratories are progressively introducing next-generation sequencing (NGS) technologies in the routine workflow to meet the increasing clinical need for comprehensive molecular characterization in cancer patients for diagnosis and precision medicine, including fusion-transcripts detection. Nevertheless, the low quality of messenger RNA (mRNA) extracted from formalin-fixed paraffin-embedded (FFPE) samples may affect the transition from traditional single-gene testing approaches [like fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), or polymerase chain reaction (PCR)] to NGS. The present study is aimed at assessing the overall accuracy of RNA fusion transcripts detection by NGS analysis in FFPE samples in real-world diagnostics., Methods: Herein, NGS data from 190 soft tissue tumors (STTs) and carcinoma cases, discussed in the context of the institutional Molecular Tumor Board, are reported and analyzed by FusionPlex © Solid tumor kit through the manufacturer's pipeline and by two well-known fast and accurate open-source tools [Arriba (ARR) and spliced transcripts alignment to reference (STAR)-fusion (SFU)]., Results: The combination of FusionPlex © Solid tumor with ArcherDX ® Analysis suite (ADx) analysis package has been proven to be sensitive and specific in STT samples, while partial loss of sensitivity has been found in carcinoma specimens., Conclusions: Albeit ARR and SFU showed lower sensitivity, the use of additional fusion-detection tools can contribute to reinforcing or extending the output obtained by ADx, particularly in the case of low-quality input data. Overall, our results sustain the clinical use of NGS for the detection of fusion transcripts in FFPE material., Competing Interests: The authors declare that they have no conflicts of interest., (© The Author(s) 2022.)

We previously published the results of a pilot study showing that vaccination with tumor-loaded dendritic cells (DCs) induced both T and B cell response and produced clinical benefit in the absence of toxicity in patients with relapsed, indolent non-Hodgkin lymphoma (iNHL). The purpose of the present short report is to provide a 15-year follow-up of our study and to expand the biomarker analysis previously performed. The long-term follow-up highlighted the absence of particular or delayed toxicity and the benefit of active immunization with DCs loaded with autologous, heat-shocked and UV-C treated tumor cells in relapsed iNHL (5-year and 10-year progression-free survival (PFS) rates: 55.6% and 33.3%, respectively; 10-year overall survival (OS) rate: 83.3%). Female patients experienced a better PFS (p=0.016) and a trend towards a better OS (p=0.185) compared with male patients. Of note, we observed a non-negligible fraction of patients (22%) who experienced a long-lasting complete response. In a targeted gene expression profiling of pre-treatment tumor biopsies in 11 patients with available formalin-fixed, paraffin-embedded tissue, we observed that KIT , ATG12 , TNFRSF10C , PBK , ITGA2 , GATA3 , CLU , NCAM1 , SYT17 and LTK were differentially expressed in patients with responder versus non-responder tumors. The characterization of peripheral monocytic cells in a subgroup of 14 patients with available baseline blood samples showed a higher frequency of the subset of CD14 ++ CD16 + cells (intermediate monocytes) in patients with responding tumors. Since in patients with relapsed iNHL the available therapeutic options are often incapable of inducing a long-lasting complete remission and can be sometimes characterized by intolerable toxicity, we think that the encouraging results of our long-term follow-up analysis represent a stimulus to further investigate the role of active vaccination in this specific setting and in earlier lines of therapy and to explore novel combinatorial strategies encompassing other innovative immunotherapy agents, such as immune-checkpoint inhibitors., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

RNA-sequencing could be nowadays considered the gold standard to study the coding and noncoding transcriptome. The great advantage of high-throughput sequencing in the characterization and quantification of long noncoding RNA (lncRNA) resides in its capability to capture the complexity of lncRNA transcripts configuration patterns, even in the presence of several alternative isoforms, with superior accuracy and discovery power compared to other technologies such as microarrays or PCR-based methods. In this chapter, we provide a protocol for lncRNA analysis using through high-throughput sequencing, indicating the main difficulties in the annotation pipeline and showing how an accurate evaluation of the procedure can help to minimize biased observations.

Despite the fact that next-generation sequencing approaches, in particular RNA sequencing, provide deep genome-wide expression data that allow both careful annotations/mapping of long noncoding RNA (lncRNA) molecules and de-novo sequencing, lncRNA expression studies by microarray is a still cost-effective procedure that could allow to have a landscape of the most characterized lncRNA species. However, microarray design does not always correctly address the overlap between coding and noncoding samples to discriminate between the original transcript source. In order to overcome this issue, in this chapter we present a bioinformatics pipeline that enables accurate annotation of GeneChip ® microarrays, to date the most commonly adopted among the commercial solutions. Overall, this approach holds two main advantages, a gain in specificity of transcript detection and the adaptability to the whole panel of GeneChip ® arrays.

The landscape of structural variants (SVs) in multiple myeloma remains poorly understood. Here, we performed comprehensive analysis of SVs in a large cohort of 752 multiple myeloma patients by low coverage long-insert whole genome sequencing. We identified 68 SV hotspots involving 17 new candidate driver genes, including the therapeutic targets BCMA ( TNFRSF17 ), SLAMF and MCL1. Catastrophic complex rearrangements termed chromothripsis were present in 24% of patients and independently associated with poor clinical outcomes. Templated insertions were the second most frequent complex event (19%), mostly involved in super-enhancer hijacking and activation of oncogenes such as CCND1 and MYC . Importantly, in 31% of patients two or more seemingly independent putative driver events were caused by a single structural event, demonstrating that the complex genomic landscape of multiple myeloma can be acquired through few key events during tumor evolutionary history. Overall, this study reveals the critical role of SVs in multiple myeloma pathogenesis., Competing Interests: Conflict of interest statement No conflict of interests to declare.

Tyrosine kinases have been implicated in promoting tumorigenesis of several human cancers. Exploiting these vulnerabilities has been shown to be an effective anti-tumor strategy as demonstrated for example by the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, for treatment of various blood cancers. Here, we characterize a new multiple kinase inhibitor, ARQ531, and evaluate its mechanism of action in preclinical models of acute myeloid leukemia. Treatment with ARQ531, by producing global signaling pathway deregulation, resulted in impaired cell cycle progression and survival in a large panel of leukemia cell lines and patient-derived tumor cells, regardless of the specific genetic background and/or the presence of bone marrow stromal cells. RNA-seq analysis revealed that ARQ531 constrained tumor cell proliferation and survival through Bruton's tyrosine kinase and transcriptional program dysregulation, with proteasome-mediated MYB degradation and depletion of short-lived proteins that are crucial for tumor growth and survival, including ERK, MYC and MCL1. Finally, ARQ531 treatment was effective in a patient-derived leukemia mouse model with significant impairment of tumor progression and survival, at tolerated doses. These data justify the clinical development of ARQ531 as a promising targeted agent for the treatment of patients with acute myeloid leukemia.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still open questions. Herein, we investigated the functional significance of the oncogenic lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in MM. Our study demonstrates that NEAT1 expression level is higher in MM than in the majority of hematological malignancies. NEAT1 silencing by novel LNA-gapmeR antisense oligonucleotide inhibits MM cell proliferation and triggers apoptosis in vitro and in vivo murine MM model as well. By transcriptome analyses, we found that NEAT1 targeting downregulates genes involved in DNA repair processes including the Homologous Recombination pathway, which in turn results in massive DNA damage. These findings may explain the synergistic impact on apoptosis observed in MM cell lines co-treated with inhibitors of both NEAT1 and PARP. The translational significance of NEAT1 targeting is further underlined by its synergistic effects with the most common drugs administered for MM treatment, including bortezomib, carfilzomib, and melphalan. Overall, NEAT1 silencing is associated with a chemo-sensitizing effect of both conventional and novel therapies, and its targeting could therefore represent a promising strategy for novel anti-MM therapeutic options.

Purpose: Existing data do not explain the reason why some individuals homozygous for the hypomorphic FECH allele develop erythropoietic protoporphyria (EPP) while the majority are completely asymptomatic. This study aims to identify novel possible genetic variants contributing to this variable phenotype., Methods: High-throughput resequencing of the FECH gene, qualitative analysis of RNA, and quantitative DNA methylation examination were performed on a cohort of 72 subjects., Results: A novel deep intronic variant was found in four homozygous carriers developing a clinically overt disease. We demonstrate that this genetic variant leads to the insertion of a pseudo-exon containing a stop codon in the mature FECH transcript by the abolition of an exonic splicing silencer site and the concurrent institution of a new methylated CpG dinucleotide. Moreover, we show that the hypomorphic FECH allele is linked to a single haplotype of about 20 kb in size that encompasses three noncoding variants that were previously associated with expression quantitative trait loci (eQTLs)., Conclusion: This study confirms that intronic variants could explain the variability in the clinical manifestations of EPP. Moreover, it supports the hypothesis that the control of the FECH gene expression can be mediated through a methylation-dependent modulation of the precursor messenger RNA (pre-mRNA) splicing pattern.

Microarray analysis of the multiple myeloma (MM) miRNome has unraveled the differential expression of miRNAs in cytogenetic subgroups, their involvement in the tumor biology and their effectiveness in prognostic models. Herein, the small RNA transcriptional landscape in MM has been investigated exploiting the possibilities offered by small RNA-seq, including accurate quantification of known mature species, discovery and characterization of isomiRs, and miRNA-offset RNAs (moRNAs). Matched small RNA-seq and miRNA GeneChip ® microarray expression profiles were obtained in a representative panel of 30 primary MM tumors, fully characterized for genomic aberrations and mutations. RNA-seq and microarray gave concordant estimations of known species. Enhanced analysis of RNA-seq data with the miR&moRe pipeline led to the characterization of 655 known and 17 new mature miRNAs and of 74 moRNAs expressed in the considered cohort, 5 of which (moR-150-3p, moR-24-2-5p, moR-421-5p, moR-21-5p, and moR-6724-5p) at high level. Ectopic expression of miR-135a-3p in t(4;14) patients, upregulation of moR-150-3p and moR-21-5p in t(14;16)/t(14;20) samples, and of moR-6724-1-5p in patients overexpressing CCND1 were uncovered and validated by qRT-PCR. Overall, RNA-seq offered a more complete overview of small non-coding RNA in MM tumors, indicating specific moRNAs that demand further investigations to explore their role in MM biology.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM.

Multiple myeloma (MM) is a clonal proliferation of bone marrow plasma cells characterized by highly heterogeneous genetic background and clinical course, whose pathogenesis remains largely unknown. Long ncRNAs (lncRNAs) are a large class of non-protein-coding RNA, involved in many physiological cellular and genomic processes as well as in carcinogenesis and tumor evolution. Although still in its infancy, the role of lncRNAs in MM is progressively expanding. Besides studies on selected candidates, lncRNAs expression at genome-wide transcriptome level is confined to microarray technologies, thus investigating a limited collection of transcripts. In the present study investigating a cohort of 30 MM patients, a deep RNA-sequencing analysis overwhelmed previous array studies and allowed the most accurate definition of lncRNA transcripts structure and expression, ultimately providing a comprehensive catalogue of lncRNAs specifically associated with the main MM molecular subgroups and genetic alterations. Despite the small number of analyzed samples, the high accuracy of RNA-sequencing approach for complex transcriptome processing led to the identification of 391 deregulated lncRNAs, 67% of which were also detectable and validated by whole-transcript microarrays. In addition, we identified a list of lncRNAs, with potential relevance in MM, co-expressed and in close proximity to genes that might undergo a cis-regulatory relationship.

The identification of overexpressed miRNAs in multiple myeloma (MM) has progressively added a further level of complexity to MM biology. miRNA and gene expression profiles of two large representative MM datasets, available from retrospective and prospective series and encompassing a total of 249 patients at diagnosis, were analyzed by means of in silico integrative genomics methods, based on MAGIA2 and Micrographite computational procedures. We first identified relevant miRNA/transcription factors/target gene regulation circuits in the disease and linked them to biological processes. Members of the miR-99b/let-7e/miR-125a cluster, or of its paralog, upregulated in t(4;14), were connected with the specific transcription factors PBX1 and CEBPA and several target genes. These results were validated in two additional independent plasma cell tumor datasets. Then, we reconstructed a non-redundant miRNA-gene regulatory network in MM, linking miRNAs, such as let-7g, miR-19a, mirR-20a, mir-21, miR-29 family, miR-34 family, miR-125b, miR-155, miR-221 to pathways associated with MM subtypes, in particular the ErbB, the Hippo, and the Acute myeloid leukemia associated pathways.

Anaplastic large-cell lymphoma (ALCL) is a clinical and biological heterogeneous disease that includes systemic anaplastic lymphoma kinase (ALK)-positive and ALK-negative entities. To discover biomarkers and/or genes involved in ALK-negative ALCL pathogenesis, we applied the cancer outlier profile analysis algorithm to a gene expression profiling data set including 249 cases of T-cell non-Hodgkin lymphoma and normal T cells. Ectopic coexpression of ERBB4 and COL29A1 genes was detected in 24% of ALK-negative ALCL patients. RNA sequencing and 5' RNA ligase-mediated rapid amplification of complementary DNA ends identified 2 novel ERBB4-truncated transcripts displaying intronic transcription start sites. By luciferase assays, we defined that the expression of ERBB4-aberrant transcripts is promoted by endogenous intronic long terminal repeats. ERBB4 expression was confirmed at the protein level by western blot analysis and immunohistochemistry. Lastly, we demonstrated that ERBB4-truncated forms show oncogenic potentials and that ERBB4 pharmacologic inhibition partially controls ALCL cell growth and disease progression in an ERBB4-positive patient-derived tumorgraft model. In conclusion, we identified a new subclass of ALK-negative ALCL characterized by aberrant expression of ERBB4-truncated transcripts carrying intronic 5' untranslated regions., (© 2016 by The American Society of Hematology.)

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., (Copyright © 2012 Wiley Periodicals, Inc.)

The development of autoimmune hemolytic anemia (AIHA) in patients with chronic lymphocytic leukemia (CLL) is associated with specific biological features. The occurrence of AIHA was hereby investigated in a retrospective series of 585 CLL patients with available immunoglobulin heavy chain variable (IGHV) gene status. AIHA occurred in 73 patients and was significantly associated with an IGHV unmutated (UM) status (P < 0.0001) and unfavorable [del(17)(p13) and del(11)(q23)] cytogenetic lesions (P < 0.0001). Stereotyped HCDR3 sequences were identified in 29.6% of cases and were similarly represented among patients developing or not AIHA; notably, subset #3 was associated with a significantly higher risk of AIHA than the other patients (P = 0.004). Multivariate analysis showed that UM IGHV, del(17)(p13) and del(11)(q23), but not stereotyped subset #3, were the strongest independent variables associated with AIHA. Based on these findings, we generated a biological risk score for AIHA development according to the presence of none (low risk), one (intermediated risk), or two (high risk) of the independent risk factors. Overall, our data indicate that UM IGHV status and/or unfavorable cytogenetic lesions are associated with the risk of developing secondary AIHA in CLL patients and suggest a possible role of specific stereotyped B-cell receptor subsets in a proportion of cases., (Copyright © 2012 Wiley Periodicals, Inc.)

Recent studies have described chromosome 2p gain as a recurrent lesion in chronic lymphocytic leukemia (CLL). We investigated the 2p gain and its relationship with common prognostic biomarkers in a prospective series of 69 clinical monoclonal B-cell lymphocytosis (cMBL) and 218 early stage (Binet A) CLL patients. The 2p gain was detected by FISH in 17 patients (6%, 16 CLL, and 1 cMBL) and further characterized by single nucleotide polymorphism-array. Overall, unfavorable cytogenetic deletions, i.e., del(11)(q23) and del(17)(p13) (P = 0.002), were significantly more frequent in 2p gain cases, as well as unmutated status of IGHV (P < 1 × 10(-4) ) and CD38 (P < 1 × 10(-4) ) and ZAP-70 positive expression (P = 0.003). Furthermore, 2p gain patients had significantly higher utilization of stereotyped B-cell receptors compared with 2p negative patients (P = 0.009), and the incidence of stereotyped subset #1 in 2p gain patients was significantly higher than that found in the remaining CLLs (P = 0.031). Transcriptional profiling analysis identified several genes significantly upregulated in 2p gain CLLs, most of which mapped to 2p. Among these, NCOA1 and ROCK2 are known for their involvement in tumor progression in several human cancers, whereas among those located in different chromosomes, CAV1 at 7q31.1 has been recently identified to play a critical role in CLL progression. Thus, 2p gain can be present since the early stages of the disease, particularly in those cases characterized by other poor prognosis markers. The finding of genes upregulated in the cells with 2p gain provides new insights to define the pathogenic role of this lesion., (Copyright © 2012 Wiley Periodicals, Inc.)

Homeobox (HOX) gene transcription factors are frequently deregulated in hematologic malignancies and involved in leukemogenic transformation [1]. Moreover, their overexpression has been associated with tumoral-induced neoangiogenesis in solid cancer [2]. The expression and the role of these genes have not yet been completely elucidated in multiple myeloma (MM). Recently, we reported that a small fraction of MM patients shows a HOXB7 overexpression as compared with normal samples and that HOXB7 expression correlates with bone marrow angiogenesis and the production of the proangiogenic factors by MM cells [3]. Other authors previously reported that HOXA cluster genes are expressed in a small fraction of MM patients [4]. Herein, we extended our previous evidences with the evaluation of the expression level of HOXB7 and the other gene family members in a large number of primary MM cells in relationship with the different molecular subgroups of MM and the presence of specific chromosome translocations. We found that HOXB7 and other genes of HOX family have a preferential distribution based on the characteristics of molecular MM subtypes based on the translocations/cyclins (TC) classification, suggesting a potential relationship between HOX genes expression, angiogenesis, and molecular features of MM patients.

Network analysis has emerged as a powerful approach to understand complex phenomena and organization in social, technological and biological systems. In particular, it is increasingly recognized the role played by the topology of cellular networks, the intricate web of interactions among genes, proteins and other molecules regulating cell activity, in unveiling the biological mechanisms underlying the physiological states of living organisms. In this study, critical analysis of network components has been applied to inspect the transcriptional and post-transcriptional regulatory networks reconstructed from mRNA and microRNA expression data of multiple myeloma (MM) samples. Specifically, the importance of a gene as a putative regulatory element has been assessed calculating the drop in the network performance caused by its deactivation instead of quantifying its degree of connectivity. The application of critical analysis to transcriptional and post-transcriptional regulatory networks allowed inferring novel regulatory relations potentially functional in multiple myeloma.

To investigate the patterns of genetic lesions in a panel of 23 human multiple myeloma cell lines (HMCLs), we made a genomic integrative analysis involving FISH, and both gene expression and genome-wide profiling approaches. The expression profiles of the genes targeted by the main IGH translocations showed that the WHSC1/MMSET gene involved in t(4;14)(p16;q32) was expressed at different levels in all of the HMCLs, and that the expression of the MAF gene was not restricted to the HMCLs carrying t(14;16)(q32;q23). Supervised analyses identified a limited number of genes specifically associated with t(4;14) and involved in different biological processes. The signature related to MAF/MAFB expression included the known MAF target genes CCND2 and ITGB7, as well as genes controlling cell shape and cell adhesion. Genome-wide DNA profiling allowed the identification of a gain on chromosome arm 1q in 88% of the analyzed cell lines, together with recurrent gains on 8q, 18q, 7q, and 20q; the most frequent deletions affected 1p, 13q, 17p, and 14q; and almost all of the cell lines presented LOH on chromosome 13. Two hundred and twenty-two genes were found to be simultaneously overexpressed and amplified in our panel, including the BCL2 locus at 18q21.33. Our data further support the evidence of the genomic complexity of multiple myeloma and reinforce the role of an integrated genomic approach in improving our understanding of the molecular pathogenesis of the disease. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat., (Copyright (c) 2006 Wiley-Liss, Inc.)

Background and Objectives: The chromosome 13 deletion (Delta13) is one of the most frequent chromosomal alterations in multiple myeloma (MM). Delta13 is associated with an unfavorable prognosis, although there is increasing agreement that its prognostic relevance must be related to the ploidy status and the presence of different chromosomal translocations. The aim of this study was to provide a comprehensive analysis of the transcriptional features of Delta13 in MM., Design and Methods: Highly purified plasma cells from 80 newly diagnosed MM patients were characterized by means of fluorescence in situ hybridization (FISH) and high-density oligonucleotide microarray for gene expression profiling and chromosomal alterations., Results: We identified 67 differentially expressed genes in the patients with and without the chromosome 13 deletion, all of which were downregulated in the cases with Delta13: 44 mapped along the whole chromosome 13, seven on chromosome 11 and three on chromosome 19. Functional analyses of the selected genes indicated their involvement in protein biosynthesis, ubiquitination and transcriptional regulation. An integrative genomic approach based on regional analyses of the gene expression data identified distinct chromosomal regions whose global expression modulation could differentiate Delta13-positive cases, in particular the upregulation of 1q21-1q42 and the downregulation of 19p and almost the entire chromosome 11. FISH analyses confirmed the close relationship between Delta13-positivity and the presence of extra copies of 1q21-1q42 (p=6 x 10(-4)) or the absence of chromosome 11 and 19 trisomy (p=5 x 10(-4))., Interpretation and Conclusions: Our results indicate that distinct types of chromosomal aberrations are closely related to the transcriptional profiles of Delta13-positive cases, suggesting that the contribution of Delta13 to the malignancy should be considered together with associated abnormalities.

Purpose: The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM). A recently proposed classification grouped MM patients into five classes on the basis of their cyclin D expression profiles and the presence of the main translocations involving the immunoglobulin heavy chain locus (IGH) at 14q32. In this study, we provide a molecular characterization of the identified translocations/cyclins (TC) groups., Materials and Methods: The gene expression profiles of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes and identify their transcriptional fingerprints. The cyclin D expression data were validated by means of real-time quantitative polymerase chain reaction analysis; fluorescence in situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion., Results: Class-prediction analysis identified 112 probe sets as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. The TC2 group, which showed extra copies of the CCND1 locus and no IGH translocations or the chromosome 13q deletion, was characterized by the overexpression of genes involved in protein biosynthesis at the translational level. A meta-analysis of published data sets validated the identified gene expression signatures., Conclusion: Our data contribute to the understanding of the molecular and biologic features of distinct MM subtypes. The identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.