[Show abstract][Hide abstract] ABSTRACT: Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.
Proceedings of the National Academy of Sciences 10/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults. We have analyzed exome sequencing data from 127 individuals with CLL and Sanger sequencing data from 214 additional affected individuals, identifying recurrent somatic mutations in POT1 (encoding protection of telomeres 1) in 3.5% of the cases, with the frequency reaching 9% when only individuals without IGHV@ mutations were considered. POT1 encodes a component of the shelterin complex and is the first member of this telomeric structure found to be mutated in human cancer. Somatic mutation of POT1 primarily occurs in gene regions encoding the two oligonucleotide-/oligosaccharide-binding (OB) folds and affects key residues required to bind telomeric DNA. POT1-mutated CLL cells have numerous telomeric and chromosomal abnormalities that suggest that POT1 mutations favor the acquisition of the malignant features of CLL cells. The identification of POT1 as a new frequently mutated gene in CLL may facilitate novel approaches for the clinical management of this disease.
[Show abstract][Hide abstract] ABSTRACT: NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.Leukemia advance online publication, 8 January 2013; doi:10.1038/leu.2012.357.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 12/2012; · 10.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have extensively characterized the DNA methylomes of 139 patients with chronic lymphocytic leukemia (CLL) with mutated or unmutated IGHV and of several mature B-cell subpopulations through the use of whole-genome bisulfite sequencing and high-density microarrays. The two molecular subtypes of CLL have differing DNA methylomes that seem to represent epigenetic imprints from distinct normal B-cell subpopulations. DNA hypomethylation in the gene body, targeting mostly enhancer sites, was the most frequent difference between naive and memory B cells and between the two molecular subtypes of CLL and normal B cells. Although DNA methylation and gene expression were poorly correlated, we identified gene-body CpG dinucleotides whose methylation was positively or negatively associated with expression. We have also recognized a DNA methylation signature that distinguishes new clinico-biological subtypes of CLL. We propose an epigenomic scenario in which differential methylation in the gene body may have functional and clinical implications in leukemogenesis.
[Show abstract][Hide abstract] ABSTRACT: Conde L, Vilaseca I, Alós L, Bernal-Sprekelsen M, Cardesa A & Nadal A (2012) Histopathology Methylthioadenosine phosphorylase inactivation depends on gene deletion in laryngeal squamous cell carcinoma Aims: Methylthioadenosine phosphorylase (MTAP) is an essential enzyme for the methionine and adenosine salvage pathway in normal cells, frequently inactivated in many different human cancers. MTAP status could be important for tumour cell sensitivity to adjuvant chemotherapy. To our knowledge, there have been no reports to date on MTAP status in laryngeal carcinoma. Methods and results: A series of 31 laryngeal squamous cell carcinomas was investigated for MTAP mRNA expression using reverse transcription and quantitative polymerase chain reaction (qPCR), as well as for MTAP gene deletion and/or promoter hypermethylation using qPCR and methylation-specific PCR, respectively. Low MTAP mRNA expression was found in 32% of cases, and was associated with MTAP gene deletion (in 70%; P < 0.001) but not with MTAP promoter hypermethylation, indicating that, in this tumour, gene deletion is the main mechanism for MTAP inactivation. Neither low mRNA expression nor gene deletion was associated with any of the clinicopathological parameters investigated. Conclusion: Given the significance of MTAP status for cell sensitivity to different chemotherapeutic regimens, our results suggest that determination of MTAP inactivation should be taken into consideration in managing laryngeal squamous cell carcinomas.
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in Western countries. Chromosomal abnormalities commonly found using conventional cytogenetics and FISH are del(11)(q22-23), trisomy 12, del(13)(q14), and del(17)(p13). Trisomy 12 is the most frequent numerical abnormality in CLL. It can appear isolated or associated with other chromosomal aberrations, including t(14;18)(q32;q21) and trisomy 18. The aim of this study was to determine whether CLL patients with isolated trisomy 12 or associated with other chromosomal alterations have different clinico-pathological features, including a different distribution NOTCH1 mutation. Patients were classified into four groups: Group 1, isolated trisomy 12 (n=14); Group 2, trisomy 12 plus trisomy 18 (n=4); Group 3, trisomy 12 plus t(14;18) (n=8); and Group 4: patients with trisomy 12 plus other abnormalities not involving BCL2 (n=28). The Binet stage and expression of ZAP70 were significantly different among cytogenetic groups. NOTCH1 mutations were detected in 6/12 (50%) patients from Group 1, 4/25 (16%) patients from Group 4, and in no patient from groups 2 and 3 (P=0.020). Patients in Group 2 had a more rapid disease progression (median Treatment-free Survival 2 months) as against patients from Groups 1 (50 months), 3 (69 months), or 4 (68 months; P=0.001). These findings indicate that the distribution of NOTCH1 mutations in CLL with trisomy 12 is heterogeneous and that the presence of additional chromosomal abnormalities such as trisomy 18 could change the prognosis of these patients.
Genes Chromosomes and Cancer 05/2012; 51(9):881-9. · 3.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we perform whole-exome sequencing of samples from 105 individuals with chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in Western countries. We found 1,246 somatic mutations potentially affecting gene function and identified 78 genes with predicted functional alterations in more than one tumor sample. Among these genes, SF3B1, encoding a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), is somatically mutated in 9.7% of affected individuals. Further analysis in 279 individuals with CLL showed that SF3B1 mutations were associated with faster disease progression and poor overall survival. This work provides the first comprehensive catalog of somatic mutations in CLL with relevant clinical correlates and defines a large set of new genes that may drive the development of this common form of leukemia. The results reinforce the idea that targeting several well-known genetic pathways, including mRNA splicing, could be useful in the treatment of CLL and other malignancies.
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukaemia (CLL), the most frequent leuk-aemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution 1,2 . Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes 3,4 . The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene func-tion. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immu-noglobulins. The patterns of somatic mutation, supported by func-tional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our know-ledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the iden-tification of clinically relevant mutations in cancer. To gain insights into the molecular alterations that cause CLL, we performed whole-genome sequencing of four cases representative of different forms of the disease: two cases, CLL1 and CLL2, with no mutations in the immunoglobulin genes (IGHV-unmutated) and two cases, CLL3 and CLL4, with mutations in these genes (IGHV-mutated) (Supplementary Table 1 and Supplementary Information). We used a combination of whole-genome sequencing and exome sequencing, as well as long-insert paired-end libraries, to detect variants in chromo-somal structure (Supplementary Fig. 1 and Supplementary Tables 2–5). We obtained more than 99.7% concordance between whole-genome sequencing calls and genotyping data, indicating that the coverage and parameters used were sufficient to detect most of the sequence variants in these samples (Supplementary Information). We detected about 1,000 somatic mutations per tumour in non-repetitive regions (Fig. 1a, Supplementary Fig. 2 and Supplementary Table 6). These numbers of somatic mutations were lower than the numbers in mela-noma and lung carcinoma 5,6 , but in agreement with previous estimates of less than one mutation per megabase (Mb) for leukaemias 7 . The most common substitution was the transition G.A/C.T, usually occurring in a CpG context (Fig. 1b and Supplementary Fig. 2). We also detected marked differences in the mutation pattern between CLL samples and these differences were associated with tumour subtype (Fig. 1b). Thus, IGHV-mutated cases showed a higher proportion of A.C/T.G muta-tions than cases with unmutated IGHV (16 6 0.2% versus 6.2 6 0.1%). The base preceding the adenine in A to C transversions showed an over-representation of thymine, when compared to the prevalence expected from its representation in non-repetitive sequences in the wild-type genome (P , 0.001, Fig. 1c), and there were fewer A to C substitutions at GpA dinucleotides than would be expected by chance (P , 0.001). These differences between CLL subtypes might reflect the molecular mechanisms implicated in their respective development. The pattern and context of mutations are consistent with their being introduced by the error-prone polymerase g during somatic hypermutation in immunoglobulin genes 8 . This indicates that polymerase g could con-tribute to the high frequency of A . T to C . G transversions in cases with IGHV-mutated. It also extends the differences observed between these two CLL subtypes to the genomic level. We classified the somatic mutations into three different classes according to their potential functional effect (Supplementary Informa-tion). We also searched for small insertions and deletions (indels) in coding regions: we found and validated five somatic indels, which caused frameshifts in protein-coding regions (Supplementary Table 7).
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.
[Show abstract][Hide abstract] ABSTRACT: To determine whether early development of carcinoma in young patients could be explained by an alternative pathway such as microsatellite instability or whether it follows the classical tumor suppressor pathway characterized by loss of heterozygosity.
Microsatellite instability, loss of heterozygosity, and multiple mismatch repair, p16, p53, and p63 protein expression were analyzed in a series of 18 young patients with laryngeal cancer.
Only 2 of the 18 cases showed low microsatellite instability, whereas 9 of 17 presented loss of heterozygosity in at least one of the markers tested. All cases retained multiple mismatch repair protein expression. The p53, p16, and p63 expression profiles were consistent with the classical tumor suppressor pathway.
Laryngeal carcinoma in young patients develops through the classical tumor suppressor pathway.
Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 04/2011; 33(2):111-8. · 0.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There are no effective antifibrotic therapies for patients with liver diseases. We performed an experimental and translational study to investigate whether ghrelin, an orexigenic hormone with pleiotropic properties, modulates liver fibrogenesis. Recombinant ghrelin was administered to rats with chronic (bile duct ligation) and acute (carbon tetrachloride) liver injury. Hepatic gene expression was analyzed by way of microarray analysis and quantitative polymerase chain reaction. The hepatic response to chronic injury was also evaluated in wild-type and ghrelin-deficient mice. Primary human hepatic stellate cells were used to study the effects of ghrelin in vitro. Ghrelin hepatic gene expression and serum levels were assessed in patients with chronic liver diseases. Ghrelin gene polymorphisms were analyzed in patients with chronic hepatitis C. Recombinant ghrelin treatment reduced the fibrogenic response, decreased liver injury and myofibroblast accumulation, and attenuated the altered gene expression profile in bile duct-ligated rats. Moreover, ghrelin reduced the fibrogenic properties of hepatic stellate cells. Ghrelin also protected rats from acute liver injury and reduced the extent of oxidative stress and inflammation. Ghrelin-deficient mice developed exacerbated hepatic fibrosis and liver damage after chronic injury. In patients with chronic liver diseases, ghrelin serum levels decreased in those with advanced fibrosis, and ghrelin gene hepatic expression correlated with expression of fibrogenic genes. In patients with chronic hepatitis C, polymorphisms of the ghrelin gene (-994CT and -604GA) influenced the progression of liver fibrosis. Conclusion: Ghrelin exerts antifibrotic effects in the liver and may represent a novel antifibrotic therapy.
[Show abstract][Hide abstract] ABSTRACT: CDK4 is involved in the control of G1-S phase transition as a part of the CCND1/CDK4 complexes. CCNDI and CDK4 gene alterations have been implicated in the development of different tumors. CCND1 has been associated with progression in laryngeal carcinomas. CDK4 protein overexpression was described associated to CCND 1 overexpression in these tumors. However, the mechanisms implicated were not known. We analyzed CDK4 gene alterations and mRNA expression in a series of carcinomas of the larynx, and the results were compared to CCND1 expression and clinicopathological characteristics of the patients. CDK4 mRNA was overexpressed in 42 out of 60 tumors (70%) associated with CCND1 mRNA overexpression because 15 out of 16 cases with high CCND1 levels showed simultaneous increased levels of CDK4 mRNA (p = 0.023) and 12 (87%) of the tumors overexpressing both genes were in stage 4. No CDK4 gene amplifications, rearrangements, or mutations were detected in any of the tumors, including 24 overexpressed cases. These findings confirm that CDK4 overexpression is a frequent phenomenon in laryngeal carcinomas, which occurs at the transcriptional level but is related neither to gene amplification nor to gene mutation, and suggest that cooperation with CCND1 may be involved in the progression of laryngeal tumors.
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 03/2007; 450(2):161-7. · 2.68 Impact Factor