Early macrophage response to obesity encompasses Interferon Regulatory Factor 5 regulated mitochondrial architecture remodelling.

Adipose tissue macrophages (ATM) adapt to changes in their energetic microenvironment. Caloric excess, in a range from transient to diet-induced obesity, could result in the transition of ATMs from highly oxidative and protective to highly inflammatory and metabolically deleterious. Here, we demonstrate that Interferon Regulatory Factor 5 (IRF5) is a key regulator of macrophage oxidative capacity in response to caloric excess. ATMs from mice with genetic-deficiency of Irf5 are characterised by increased oxidative respiration and mitochondrial membrane potential. Transient inhibition of IRF5 activity leads to a similar respiratory phenotype as genomic deletion, and is reversible by reconstitution of IRF5 expression. We find that the highly oxidative nature of Irf5-deficient macrophages results from transcriptional de-repression of the mitochondrial matrix component Growth Hormone Inducible Transmembrane Protein (GHITM) gene. The Irf5-deficiency-associated high oxygen consumption could be alleviated by experimental suppression of Ghitm expression. ATMs and monocytes from patients with obesity or with type-2 diabetes retain the reciprocal regulatory relationship between Irf5 and Ghitm. Thus, our study provides insights into the mechanism of how the inflammatory transcription factor IRF5 controls physiological adaptation to diet-induced obesity via regulating mitochondrial architecture in macrophages.

Prognostic value of tumor mutational burden in patients with oral cavity squamous cell carcinoma treated with upfront surgery.

BACKGROUND: Oral cavity is the most prevalent site of head and neck squamous cell carcinomas (HNSCCs). Most often diagnosed at a locally advanced stage, treatment is multimodal with surgery as the cornerstone. The aim of this study was to explore the molecular landscape of a homogenous cohort of oral cavity squamous cell carcinomas (OCSCCs), and to assess the prognostic value of tumor mutational burden (TMB), along with classical molecular and clinical parameters. PATIENTS AND METHODS: One hundred and fifty-one consecutive patients with OCSCC treated with upfront surgery at the Institut Curie were analyzed. Sequencing of tumor DNA from frozen specimens was carried out using an in-house targeted next-generation sequencing panel (571 genes). The impact of molecular alterations and TMB on disease-free survival (DFS) and overall survival (OS) was evaluated in univariate and multivariate analyses. RESULTS: Pathological tumor stage, extranodal spread, vascular emboli, and perineural invasion were associated with both DFS and OS. TP53 was the most mutated gene (71%). Other frequent molecular alterations included the TERT promoter (50%), CDKN2A (25%), FAT1 (17%), PIK3CA (14%), and NOTCH1 (15%) genes. Transforming growth factor-ß pathway alterations (4%) were associated with poor OS (P = 0.01) and DFS (P = 0.02) in univariate and multivariate analyses. High TMB was associated with prolonged OS (P = 0.01 and P = 0.02, in the highest 10% and 20% TMB values, respectively), but not with DFS. Correlation of TMB with OS remained significant in multivariate analysis (P = 0.01 and P = 0.005 in the highest 10% and 20% TMB values, respectively). Pathological tumor stage combined with high TMB was associated with good prognosis. CONCLUSION: Our results suggest that a high TMB is associated with a favorable prognosis in patients with OCSCC treated with upfront surgery.

Identification of homozygous deletions at chromosome 16q23 in aflatoxin B1 exposed hepatocellular carcinoma.

Loss of heterozygosity (LOH) represents the most frequent genetic alteration observed in hepatocellular carcinoma (HCC). Chromosome 16q is of particular interest as it exhibits LOH in 29% of HCC tumors and is frequently lost in breast, prostate, ovarian and gastric carcinomas. We genotyped 157 HCC tumors for 17 microsatellite markers distributed on chromosome 16q and determined a common region of LOH localized between the markers D16S518 and D16S504. By refining the boundaries of two interstitial LOH and two homozygous deletions, the critical region was delimited to 180 kb between D16S3096 and D16S3029. This region is located in intron 8 of the WWOX/FOR gene, but a search for mutations in all coding exons of this gene in 27 HCC tumors and cell lines did not reveal any tumor somatic alterations. Furthermore, by RT-PCR, no abnormal transcripts of this WWOX/FOR gene was detected in nine HCC cell lines. Finally, analysis of the p53 gene mutations with the clinical parameters of all tumors revealed that the two homozygous deletions have occurred in tumors presenting a R249S mutation. Our data revealed a relationship between chromosome 16q homozygous deletions and R249S p53 mutations in tumors where the patient had been exposed to aflatoxin B1 (P=0.002). These results are consistent with a role of aflatoxin B1 in the instability of chromosome 16q at the fragile site FRA16D. However, the nature of the specific gene that is altered during hepatocarcinogenesis remains to be elucidated.

Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis.

BACKGROUND & AIMS: To evaluate how characterization of genetic alterations can help in the elucidation of liver carcinogenesis pathways, 137 tumors were analyzed. METHODS: High-density allelotype, p53, Axin1, and beta-catenin gene mutations were determined. Alterations were analyzed according to clinical parameters. RESULTS: Tumors could be divided into 2 groups according to chromosome stability status. In the first group, demonstrating a chromosome stability, beta-catenin mutation associated with chromosome 8p losses were frequently found as the single genetic alterations. beta-catenin mutations were associated with large tumor size and with negative hepatitis B virus status. In the second group, demonstrating a chromosome instability, the most frequent allelic losses were on chromosome 1p, 4q, 6q, 9p, 13q, 16p, 16q, and 17p; Axin1 and p53 were frequently mutated. All of these alterations, except losses on 6q and 9p, were associated with hepatitis B virus infection. P53 mutations, 17p, 13q losses, and a high value of the fractional allelic loss index were associated with poor differentiated tumors, independently of risk factors. Finally, in the whole series, chromosome 9p and 6q losses were associated with poor prognosis. CONCLUSIONS: Two main pathways defined by genetic alterations show different risk factors and clinical characteristics. Furthermore, loss of chromosome 9p or 6q is an independent prognostic indicator.

Molecular characterization of germline NF2 gene rearrangements.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease that causes a predisposition to nervous system tumors. Deleterious point mutations have been found in about 55% of NF2 patients, and large genomic deletions account for approximately 33% of NF2 gene alterations. The majority of these deletions are larger than 50 kb, with a breakpoint usually lying outside the NF2 gene. We identified two cases of intragenic deletion with loss of 1.5 and 40 kb, respectively. In both cases, one boundary of the deletion was located in or at the proximity of an SVA sequence in NF2 intron 4. No sequence identity longer than 5 bases and no signal of specific recombination have been evidenced on either side of the deletion breakpoints. These observations are compatible with a nonhomologous recombination being responsible for the genomic deletions. In a third case, a paracentric inversion of chromosome 22 was found. This chromosomal rearrangement breaks the NF2 gene in two parts and carries the first NF2 exon in a juxta-centromeric position. The variability in position of the deletions and the observation of a new chromosomal rearrangement in the NF2 gene underscore the importance of FISH analysis in the molecular diagnosis of NF2.

Beta-catenin mutations in hepatocellular carcinoma correlate with a low rate of loss of heterozygosity.

To determine the frequency of Wnt/Wingless beta catenin pathway alteration in human hepatocellular carcinoma, a beta catenin and APC gene mutation screening was performed in a series of 119 tumors. An activating beta catenin mutation in exon 3 was found in 18% of the cases. Among tumors lacking beta catenin mutation, no APC mutation has been evidenced in a subset of 30 cases tested. The correlation between beta catenin mutation status and chromosome segment deletions was studied on a set of 48 hyperploid tumors. Chromosome 1p, 4q and 16p deletions were significantly associated with the absence of beta catenin mutation (P<0.05). Furthermore the Fractional Allelic Loss was significantly smaller in the beta catenin mutated tumors than in the non-mutated tumors (0.12 versus 022). Taken together, these results suggest, the existence of two carcinogenesis mechanisms. The first mechanism implies a beta catenin activating mutation associated with a low rate of loss of heterozygosity. The second mechanism, operating in a context of chromosomal instability, would involve tumor suppressor genes.

Characterisation of 16 polymorphic markers in the NF2 gene: application to hemizygosity detection.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to nervous system tumors. Point mutations are evidenced in about 50% of the NF2 patients and large genomic deletions account for approximately 33% of the NF2 gene alterations. To facilitate the deletion screening, 16 polymorphic markers were identified in the NF2 genomic sequence enabling an hemizygosity test in familial studies.

[Semi-automated quantitative method for detecting the loss of heterozygosity at the long arm of chromosome 4 in hepatocellular carcinoma]. / Détection quantitative semi-automatique des pertes d'hétérozygotie sur le bras long du chromosome 4 dans les carcinomes hépatocellulaires.

OBJECTIVES: Chromosomal deletions are the most frequent genetic alterations observed in hepatocellular carcinoma. Loss of heterozygosity on chromosome 4q has been observed in 40% of hepatocellular carcinomas suggesting the presence of a tumor suppressor gene which has not yet been identified. METHODOLOGY: We developed a semi-automated quantitative genotyping method which allowed us to characterize 119 hepatocellular carcinomas with 22 fluorescent microsatellite markers distributed on chromosome 4q. RESULTS: 4q loss was observed in 40% of cases. Among these deletions, 19 cases of partial or interstitial loss made it possible to define two common minimal regions of deletion of 25.1 and 37.6 centimorgans localized between markers D4S414 and D4S430 and between markers D4S3033 and D4S408, respectively. CONCLUSION: This work represents the first step towards the identification and characterization of new genes involved in hepatic carcinogenesis.

NF2 gene in neurofibromatosis type 2 patients.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to nervous system tumors. The schwannomin (also termed merlin) protein encoded by the NF2 gene shows a close relationship to the family of cytoskeleton-to-membrane proteins linkers ERM (ezrin-radixin-moesin proteins). Even though penetrance of the disease is >95% and no genetic heterogeneity has been described, point mutations in the NF2 gene have been observed in only 34-66% of the screened NF2 patients, depending on the series. In order to generate tools that would enable an exhaustive alteration screening for the NF2 gene, we have deduced its entire genomic sequence. This knowledge has provided the delineation of a mutation screening strategy which, when applied to a series of 19 NF2 patients, has revealed a high recurrence of large deletions in the gene and has raised the efficiency of mutation detection in NF2 patients to 84% of the cases in this series. The remaining three patients who express two functional NF2 alleles are all sporadic cases, an observation compatible with the presence of mosaicism for NF2 mutation.

Chromosome translocation based on illegitimate recombination in human tumors.

Recurrent chromosome translocations in nonhematological tumors are restricted to specific subtypes, and their mechanism is currently unknown. Analysis of the sequence data of 113 interchromosomal junctions derived from 77 Ewing's tumors carrying the characteristic t(11;22) translocation indicate that, in this tumor, translocations are initiated independently on each chromosome in regions that lack site specific recombination signal. Local sequence duplications, deletions, and, most importantly, inversions that are diagnostic of DNA hairpin formation indicate that, at the breakpoint, single-stranded DNA ends are processed individually before interchromosomal joining. Taken together, these observations suggest that chromosome translocations in Ewing's tumors are mediated through a genuine illegitimate recombination mechanism.

Identification of new members of the Gas2 and Ras families in the 22q12 chromosome region.

Monitoring of loss of heterozygosity in human tumors has suggested that the 22q12 region may contain another tumor suppressor gene(s) in addition to the NF2 gene. The genomic sequencing of a 40-kb region bounded by the EWS and BAM22 genes and containing a CpG-rich region has identified two new genes in the q12 region of chromosome 22. One of them, GAR22, is closely related to mouse Gas2, a gene isolated as being negatively regulated by serum and growth factors and exhibiting a high expression in growth arrested mouse fibroblasts. The other, RRP22, belongs to the Ras superfamily, in which it defines a new subgroup. Its expression appears to be strictly limited to the central nervous system. Growth-arrest-specific and Ras-related genes are likely candidates to be involved in tumorigenic processes. Although no mutation was observed in a small set of meningiomas and schwannomas, alteration of these new genes should now be searched in other tumors with frequent allelic losses on chromosome 22 not associated with NF2 mutation.