Prediction of response to immune checkpoint blockade in patients with metastatic colorectal cancer with microsatellite instability.

BACKGROUND: Mismatch repair-deficient (dMMR) tumors displaying microsatellite instability (MSI) represent a paradigm for the success of immune checkpoint inhibitor (ICI)-based immunotherapy, particularly in patients with metastatic colorectal cancer (mCRC). However, a proportion of patients with dMMR/MSI mCRC exhibit resistance to ICI. Identification of tools predicting MSI mCRC patient response to ICI is required for the design of future strategies further improving this therapy. PATIENTS AND METHODS: We combined high-throughput DNA and RNA sequencing of tumors from 116 patients with MSI mCRC treated with anti-programmed cell death protein 1 ± anti-cytotoxic T-lymphocyte-associated protein 4 of the NIPICOL phase II trial (C1, NCT03350126, discovery set) and the ImmunoMSI prospective cohort (C2, validation set). The DNA/RNA predictors whose status was significantly associated with ICI status of response in C1 were subsequently validated in C2. Primary endpoint was progression-free survival by immune RECIST (iRECIST) (iPFS). RESULTS: Analyses showed no impact of previously suggested DNA/RNA indicators of resistance to ICI, e.g. MSIsensor score, tumor mutational burden, or specific cellular and molecular tumoral contingents. By contrast, iPFS under ICI was shown in C1 and C2 to depend both on a multiplex MSI signature involving the mutations of 19 microsatellites hazard ratio cohort C2 (HRC2) = 3.63; 95% confidence interval (CI) 1.65-7.99; P = 1.4 × 10-3] and the expression of a set of 182 RNA markers with a non-epithelial transforming growth factor beta (TGFB)-related desmoplastic orientation (HRC2 = 1.75; 95% CI 1.03-2.98; P = 0.035). Both DNA and RNA signatures were independently predictive of iPFS. CONCLUSIONS: iPFS in patients with MSI mCRC can be predicted by simply analyzing the mutational status of DNA microsatellite-containing genes in epithelial tumor cells together with non-epithelial TGFB-related desmoplastic RNA markers.

Somatic mutational landscape of extracranial arteriovenous malformations and phenotypic correlations.

BACKGROUND: Somatic genetic variants may be the cause of extracranial arteriovenous malformations, but few studies have explored these genetic anomalies, and no genotype-phenotype correlations have been identified. OBJECTIVES: The aim of the study was to characterize the somatic genetic landscape of extracranial arteriovenous malformations and correlate these findings with the phenotypic characteristics of these lesions. METHODS: This study included twenty-three patients with extracranial arteriovenous malformations that were confirmed clinically and treated by surgical resection, and for whom frozen tissue samples were available. Targeted next-generation sequencing analysis of tissues was performed using a gene panel that included vascular disease-related genes and tumour-related genes. RESULTS: We identified a pathogenic variant in 18 out of 23 samples (78.3%). Pathogenic variants were mainly located in MAP2K1 (n = 7) and KRAS (n = 6), and more rarely in BRAF (n = 2) and RASA1 (n = 3). KRAS variants were significantly (P < 0.005) associated with severe extended facial arteriovenous malformations, for which relapse after surgical resection is frequently observed, while MAP2K1 variants were significantly (P < 0.005) associated with less severe, limited arteriovenous malformations located on the lips. CONCLUSIONS: Our study highlights a high prevalence of pathogenic somatic variants, predominantly in MAP2K1 and KRAS, in extracranial arteriovenous malformations. In addition, our study identifies for the first time a correlation between the genotype, clinical severity and angiographic characteristics of extracranial arteriovenous malformations. The RAS/MAPK variants identified in this study are known to be associated with malignant tumours for which targeted therapies have already been developed. Thus, identification of these somatic variants could lead to new therapeutic options to improve the management of patients with extracranial arteriovenous malformations.

Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) syndrome is a childhood cancer predisposition syndrome involving biallelic germline mutations of MMR genes, poorly recognised by clinicians so far. METHODS: Retrospective review of all 31 patients with CMMRD diagnosed in French genetics laboratories in order to describe the characteristics, treatment and outcome of the malignancies and biological diagnostic data. RESULTS: 67 tumours were diagnosed in 31 patients, 25 (37%) Lynch syndrome-associated malignancies, 22 (33%) brain tumours, 17 (25%) haematological malignancies and 3 (5%) sarcomas. The median age of onset of the first tumour was 6.9 years (1.2-33.5). Overall, 22 patients died, 9 (41%) due to the primary tumour. Median survival after the diagnosis of the primary tumour was 27 months (0.26-213.2). Failure rate seemed to be higher than expected especially for T-cell non-Hodgkin's lymphoma (progression/relapse in 6/12 patients). A familial history of Lynch syndrome was identified in 6/23 families, and consanguinity in 9/23 families. PMS2 mutations (n=18) were more frequent than other mutations (MSH6 (n=6), MLH1 (n=4) and MSH2 (n=3)). CONCLUSIONS: In conclusion, this unselected series of patients confirms the extreme severity of this syndrome with a high mortality rate mostly related to multiple childhood cancers, and highlights the need for its early detection in order to adapt treatment and surveillance.

Genome sequencing identify chromosome 9 inversions disrupting ENG in 2 unrelated HHT families.

Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber disease, is a dominant inherited vascular disorder. The clinical diagnosis is based on the Curaçao criteria and pathogenic variants in the ENG and ACVRL1 genes are responsible for most cases of HHT. Four families with a negative targeted gene panel and selected by a multidisciplinary team were selected and whole-genome sequencing was performed according to the recommendations of the French National Plan for Genomic Medicine. Structural variations were confirmed by standard molecular cytogenetic analysis (FISH). In two families with a definite diagnosis of HHT, we identified two different paracentric inversions of chromosome 9, both disrupting the ENG gene. These inversions are considered as pathogenic and causative for the HHT phenotype of the patients. This is the first time structural variations are reported to cause HHT. As such balanced events are often missed by exon-based sequencing (panel, exome), structural variations may be an under-recognized cause of HHT. Genome sequencing for the detection of these events could be suggested for patients with a definite diagnosis of HHT and in whom no causative pathogenic variant was identified.

Germline RAD51C mutations in ovarian cancer susceptibility.

Several genes might explain BRCA1/2 negative breast and ovarian family cases. Deleterious mutations in few genes involved in the Fanconi complex are responsible for Fanconi anemia at the homozygous state and breast cancer (BC) susceptibility at the heterozygous state (BRCA2, PALB2, BRIP1). RAD51C plays an important role in the double-strand break repair pathway and a biallelic missense mutation in the RAD51C gene was found in a Fanconi anemia-like disorder. Subsequently, six monoallelic pathogenic mutations were identified after screening 480 BRCA1/2 negative breast and ovarian cancer (BC/OC) pedigrees. Several reports were unsuccessful to replicate these results. To investigate whether germline mutations in RAD51C are associated with an increased risk of developing BC/OC, we screened, by Sanger sequencing of the coding sequence, 117 index cases of breast and ovarian families from French or European origin, and negative for BRCA1/2 mutations. In our study, we found 3 pathogenic mutations among 117 families screened which corresponds to a 2.6% frequency. Our results confirm that RAD51C is a susceptibility gene for ovarian and BC and that this gene should be screened for mutations in families with multiple BC/OC.

Rare germline large rearrangements in the BRCA1/2 genes and eight candidate genes in 472 patients with breast cancer predisposition.

Hereditary breast cancers account for up to 5-10 % of breast cancers and a majority are related to the BRCA1 and BRCA2 genes. However, many families with breast cancer predisposition do not carry any known mutations for BRCA1 and BRCA2 genes. We explored the incidence of rare large rearrangements in the coding, noncoding and flanking regions of BRCA1/2 and in eight other candidate genes--CHEK2, BARD1, ATM, RAD50, RAD51, BRIP1, RAP80 and PALB2. A dedicated zoom-in CGH-array was applied to screen for rearrangements in 472 unrelated French individuals from breast-ovarian cancer families that were being followed in eight French oncogenetic laboratories. No new rearrangement was found neither in the genomic regions of BRCA1/2 nor in candidate genes, except for the CHEK2 and BARD1 genes. Three heterozygous deletions were detected in the 5' and 3' flanking regions of BRCA1. One large deletion introducing a frameshift was identified in the CHEK2 gene in two families and one heterozygous deletion was detected within an intron of BARD1. The study demonstrates the usefulness of CGH-array in routine genetic analysis and, aside from the CHEK2 rearrangements, indicates there is a very low incidence of large rearrangements in BRCA1/2 and in the other eight candidate genes in families already explored for BRCA1/2 mutations. Finally, next-generation sequencing should bring new information about point mutations in intronic and flanking regions and also medium size rearrangements.

Unexplained polyposis: a challenge for geneticists, pathologists and gastroenterologists.

Two main colorectal polyposis syndromes have been described, familial adenomatous polyposis and MUTYH-associated polyposis syndromes. Some polyposis remains unexplained: 20% of adenomatous polyposis and serrated polyposis. The aim of this study was to evaluate in a cohort of patients with unexplained polyposis whether a genetic defect could be detected. Individuals presenting polyposis with more than 40 adenomas or more than 20 serrated polyps (hyperplastic, sessile serrated and mixed), without causative mutation identified, were included. Complementary explorations on APC or MUTYH were performed: search for APC mosaicism, splicing-affecting mutations, large genomic rearrangement of MUTYH. Four genes of Wnt pathway (AXIN2, PPP2R1B, WIF1, SFRP1) and two genes of transforming growth factor-ß (TGF-ß) pathway (SMAD4, BMPR1A) were screened for germline mutation. Twenty-five patients had an unexplained adenomatous polyposis (familial or sporadic). Five pathogenic mutations were found: four in APC gene (with one case of mosaicism) and one in BMPR1A gene. The exploration of APC mosaicism was better performed from adenoma DNA with high-resolution melting. The screening of the candidate genes did not find any causative mutation. Thirteen individuals had an unexplained serrated polyposis and a frameshift on SMAD4 gene was identified. All mutations were identified in familial cases of polyposis. After new pathological examination, both BMPR1A and SMAD4 cases were found to be associated with a juvenile polyposis while the polyposis was initially described as adenomatous or undetermined. In 17% (6/38) of the patients the causative mutation of the polyposis was identified. Genetic causes were heterogeneous. Sporadic polyposis patients must be considered as potential APC mosaicism. The histological classification of polyposis is strongly important in direct genetic exploration.

ACVRL1 germinal mosaic with two mutant alleles in hereditary hemorrhagic telangiectasia associated with pulmonary arterial hypertension.

Germline mutations in genes encoding members of the transforming growth factor-ß (TGF-ß)/bone morphogenetic protein (BMP) superfamily are causal for two hereditary vascular disorders, hereditary hemorrhagic telangiectasia (HHT) and heritable pulmonary arterial hypertension (PAH). When the two diseases coexist, activin A receptor type II-like kinase-1 (ACVRL1) gene mutations are usually identified. We report a remarkable ACVRL1 germinal and somatic mosaicism characterized by the presence of two distinct mutant alleles and a non-mutant ACVRL1 allele in a woman diagnosed with PAH at the age 40. She also met the Curaçao diagnostic criteria for HHT based on additional findings of telangiectases, epistaxis and arteriovenous malformations. Mutation analysis of ACVRL1 identified two adjacent heterozygous deleterious mutations within exon 10: c.1388del (p.Gly463fsX2) and c.1390del (p.Leu464X) in a region enriched by mutation-associated DNA motifs. The mother transmitted the c.1388del to one child and the c.1390del to two children confirming germinal mosaicism. Allele-specific polymerase chain reaction analysis showed that c.1388del is the predominant mutation in lymphocytes of the index case. Haplotype analysis revealed that both mutant alleles have a common chromosomal origin which is distinct from that of the mother's non-mutant ACVRL1 allele. These distinct mutant alleles in tissues and germline could have arisen by DNA structure-mediated events occurring in the early stages of the mother's embryogenesis, prior to the segregation of her germline, which ultimately led to the independent transmission of each allele. These highlight the complexity of genomic events occurring during early embryogenesis and the consequences of mutational mosaicism upon pathogenic variability.

Frequent mutation in North African patients with MUTYH-associated polyposis.

MUTYH-associated polyposis (MAP) has been characterized as an autosomal recessive disease predisposing to a variable number of colorectal adenomas with a high risk of cancer. Numerous studies have indicated that two missense mutations (Y179C and G396D) account for about 80% of MUTYH allelic variants in Europeans. Ethnic and geographic differences in the mutation spectrum have been observed. The aim of this study was to report mutations in patients from North Africa, determine the incidence of the c.1227_1228dup mutation in our cohort of MUTYH patients and to evaluate the existence of a founder effect. Within a group of 36 families with MAP, 11 were shown to have a homozygous c.1227_1228dup mutation. These families came from Algeria (n = 5), Tunisia (n = 4), Morocco (n = 1) and Portugal (n = 1). Probands belonging to families of North African origin showed a significantly higher frequency of c.1227_1228dup (78.6% vs 4.5%, p < 0.0001). Haplotype analyses were performed using 10 microsatellite markers surrounding the MUTYH gene spanning a region of 4.4 cM. We identified a common haplotype of at least 1.3 cM in all families suggesting a founder effect for this mutation.

Discordance between immunochemistry of mismatch repair proteins and molecular testing of microsatellite instability in colorectal cancer.

BACKGROUND: DNA mismatch repair system deficiency (dMMR) is found in 15% of colorectal cancers (CRCs). Two methods are used to determine dMMR, immunohistochemistry (IHC) of MMR proteins and molecular testing of microsatellite instability (MSI). Only studies with a low number of patients have reported rates of discordance between these two methods, ranging from 1% to 10%. MATERIALS AND METHODS: Overall, 3228 consecutive patients with CRCs from two centers were included. Molecular testing was carried out using the Pentaplex panel and IHC evaluated four (MLH1, MSH2, MSH6, and PMS2; cohort 1; n = 1085) or two MMR proteins (MLH1 and MSH2; cohort 2; n = 2143). The primary endpoint was the rate of discordance between MSI and MMR IHC tests. RESULTS: Fifty-one discordant cases (1.6%) were initially observed. Twenty-nine out of 51 discordant cases were related to IHC misclassifications. In cohort 1, after re-reading IHC and/or carrying out new IHC, 16 discordant cases were reclassified as nondiscordant. In cohort 2, after the addition of MSH6/PMS2 IHC and re-examination, 13 were reclassified as nondiscordant. In addition, 10 misclassifications of molecular tests were identified. Finally, only 12 discordant cases (0.4%) remained: 5 were proficient MMR/MSI and 7 were dMMR/microsatellite stable. CONCLUSIONS: Our study confirmed the high degree of concordance between MSI and MMR IHC tests. Discordant cases must be reviewed, and if needed, tests must be repeated and analyzed by an expert team.

The TP53 Arg72Pro and MDM2 309G>T polymorphisms are not associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers.

BACKGROUND: The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T>G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance. METHODS: To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T>G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework. RESULTS: No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR)=1.01, 95% confidence interval (CI): 0.93-1.10, P(trend)=0.77; MDM2: HR=0.96, 95%CI: 0.84-1.09, P(trend)=0.54) or for BRCA2 mutation carriers (TP53: HR=0.99, 95%CI: 0.87-1.12, P(trend)=0.83; MDM2: HR=0.98, 95%CI: 0.80-1.21, P(trend)=0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association. CONCLUSION: There was no evidence that TP53 Arg72Pro or MDM2 309T>G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers.

The contribution of germline rearrangements to the spectrum of BRCA2 mutations.

BACKGROUND: Few germline BRCA2 rearrangements have been described compared with the large number of germline rearrangements reported in the BRCA1 gene. However, some BRCA2 rearrangements have been reported in families that included at least one case of male breast cancer. OBJECTIVE: To estimate the contribution of large genomic rearrangements to the spectrum of BRCA2 defects. METHODS: Quantitative multiplex PCR of short fluorescent fragments (QMPSF) was used to screen the BRCA2 gene for germline rearrangements in highly selected families. QMPSF was previously used to detect heterozygous deletions/duplications in many genes including BRCA1 and BRCA2. RESULTS: We selected a subgroup of 194 high risk families with four or more breast cancers with an average age at diagnosis of < or = 50 years, who were recruited through 14 genetic counselling centres in France and one centre in Switzerland. BRCA2 mutations were detected in 18.6% (36 index cases) and BRCA1 mutations in 12.4% (24 index cases) of these families. Of the 134 BRCA1/2 negative index cases in this subgroup, 120 were screened for large rearrangements of BRCA2 using QMPSF. Novel and distinct BRCA2 deletions were detected in three families and their boundaries were determined. We found that genomic rearrangements represent 7.7% (95% confidence interval 0% to 16%) of the BRCA2 mutation spectrum. CONCLUSION: The molecular diagnosis of breast cancer predisposition should include screening for BRCA2 rearrangements, at least in families with a high probability of BRCA2 defects.

Detection of plasma tumor DNA in head and neck squamous cell carcinoma by microsatellite typing and p53 mutation analysis.

Recent arguments have suggested that tumor DNA in cancer patients could be found in plasma, but different points remain unclear. Using a series of 117 head and neck squamous cell carcinoma tumors, our goals for this study were: (a) to quantify the amount of plasma DNA; (b) to evaluate the presence of plasma tumor DNA; and (c) to analyze the clinical relevance of tests based on plasma DNA analyses. Low levels of plasma DNA were found in most samples, but all were successfully amplified. Two different methods were used to detect tumor-specific genetic alterations: (a) microsatellite instability at UT5085 with an established sensitivity of 1:500; and (b) p53 mutation screening. Of the 117 tumors typed at UT5085, 65 demonstrated bandshifts (55%). Plasma and tumor DNA a showed similar alteration in only one case among these samples, and the prevalence of tumor DNA in plasma was estimated to be <2% using microsatellite analysis. Tumor DNA was detected in plasma at a higher prevalence (2 of 11 cases) when using p53 mutant allele-specific amplification. These results showed that in plasma, tumor DNA is largely diluted by normal DNA. By comparison with previously published studies, the prevalence of microsatellite alterations in plasma in this series of head and neck squamous cell carcinomas is very low, despite the fact that a large series of tumors was analyzed. To explain this discrepancy, we analyzed the possibility of PCR artifacts as suspected by the presence of loss of heterozygosity in two plasma DNA samples without a similar tumor DNA alteration. When DNA concentrations were under the threshold of detection (<100 ng/ml), we demonstrated that PCR artifacts could occur at random, and, if misinterpreted, these false genetic alterations could artificially enhance the frequency of plasma DNA alterations. This may have been suspected in previously published series, but it has never been discussed before. Microsatellite analysis on plasma DNA is difficult to interpret and can frequently be misleading. Plasma DNA should be analyzed with very sensitive and specific methods such as mutant allele-specific amplification, which excludes artifacts but requires specific optimization that is probably not compatible with routine and clinical use.

p53 gene status as a predictor of tumor response to induction chemotherapy of patients with locoregionally advanced squamous cell carcinomas of the head and neck.

PURPOSE: To determine whether p53 gene status predicts tumor responses to platinum- and fluorouracil-based induction chemotherapy in locoregionally advanced squamous cell carcinomas of the head and neck. PATIENTS AND METHODS: Tumor responses of 105 patients were measured at the primary tumor site. Objective response and major response were defined by a 50% and 80% reduction in tumor size, respectively. All coding parts of p53 gene were directly sequenced. p53 expression in tumor cells was determined by immunohistochemistry. Human papillomavirus infection was detected by polymerase chain reaction. Odd ratios were adjusted by stepwise logistic regression analysis. RESULTS: p53 mutations, p53 expression, and tumor stage were sufficient to explain the variation in tumor responses to chemotherapy in multivariate models. p53 mutation was the only variable to significantly predict objective response (odds ratio, 0. 23; 95% confidence interval, 0.10 to 0.57; P =.002) and was the strongest predictor of major response (odds ratio, 0.29; 95% confidence interval, 0.11 to 0.74; P =.006). p53 expression (odds ratio, 0.39; 95% confidence interval, 0.16 to 0.98) and tumor stage (odds ratio, 0.31; 95% confidence interval, 0.10 to 0.96) also predicted major response. Specific mutations (contact mutations) accounted for much of the reduction in the risk of major response associated with overall mutations. In complementary analyses, p53 expression was weakly predictive of major response in the subgroup with wild-type p53, and p53 mutations also predicted histologic response. CONCLUSION: p53 gene mutations are strongly associated with a poor risk of both objective and major responses to chemotherapy. Contact mutations are associated with the lowest risk of major response to chemotherapy.

Shear stress induces angiotensin converting enzyme expression in cultured smooth muscle cells: possible involvement of bFGF.

OBJECTIVE: Hemodynamic stresses are considered to be important regulators of gene expression in vascular cells. In this study, we have investigated the role of shear stress on ACE expression in cultured rat vascular cells, and focused on the regulation of ACE expression in smooth muscle cells. METHODS: Rat aortic endothelial cells, smooth muscle cells and fibroblasts isolated from Wistar rats were submitted to shear stress using a laminar shear flow parallel chamber. RESULTS: A 10 dynes/cm2 shear rate for 24 h increased ACE activity in the three vascular cell types (x 2.14 in endothelial cells, x 2.9 in smooth muscle cells, x 3.33 in fibroblasts). This induction was blocked by a 24 h pre-incubation with a translation blocker (10(-4)M cycloheximide) showing the role of protein neosynthesis. Therefore the study was focused on smooth muscle cells and we demonstrated that the increase in ACE activity was due to an elevation in ACE mRNA level in response to a 10 dynes/cm2 shear stress for 24 h. This induction was dependent on the shear intensity (P < 0.0001). Six hours of a 15 dynes/cm2 shear stress showed no effect on ACE activity or mRNA expression. In contrast, the same duration of shear significantly increased bFGF mRNA level (x 3.7). Conversely, bFGF dose dependently increased ACE mRNA expression and activity in smooth muscle cells. This result suggests that bFGF could be one of the potential inductors of ACE expression in the stressed smooth muscle cells. CONCLUSIONS: Mechanical stress increases ACE expression in vascular cells. bFGF could be one of the potential factors involved in this activation. This phenomenon could participate in the role of ACE activity in vascular wall remodeling.

Effect of pneumoperitoneum on circulating tumor DNA.

BACKGROUND: Release and circulation of tumor DNA could be favored by surgery. No data is available for the effect of laparoscopy on this phenomenon. MATERIAL AND METHODS: The aim of this study was to assess the impact of CO2 laparoscopy on circulating tumor DNA. Two xenografts of ovarian cancer were obtained by intraperitoneal inoculation (IP) of IGR-OV1 or NIH:OVCAR-3 cells in nude rats. CO2 laparoscopy (L), gasless laparoscopy (GL), midline laparotomy (ML) or general anesthesia as a control (C) were randomly carried out when the tumor graft was present in the peritoneal cavity. A sterile blood sample was taken in each case as soon as the experiment was completed. DNA was subsequently extracted and amplified (PCR, primers HLA GH 26 and HLA GH 27 specific for human DNA). In each model, we compared the influence of each surgical approach on circulating tumor DNA. Statistics were performed with the Wilcoxon test and Fisher exact test. 1: RESULTS: Eighteen rats were included in each group. Our protocol could detect an amount of tumor DNA equivalent to 10 cells/ml of blood. This technique was specific. Circulating tumor DNA was frequently observed in the IGR-OV1 model (45 to 50%), without significant difference between groups (p=0.6). In the NIH: OVCAR-3 model, the detection rate ranged from 22% (control group) to 64% (gasless group); but the overall comparison between the four groups was not significant (p = 0.2). CONCLUSION: In this experimental trial, CO2 laparoscopy had no deleterious effect on circulating tumor DNA. Biologic characteristics of tumors could also play a role.

Deep vein thrombosis during enoxaparin prophylactic treatment in a young pregnant woman homozygous for factor V Leiden and heterozygous for the G127-->a mutation in the thrombomodulin gene.

Variability of thrombotic disease among individuals homozygous for factor V Leiden has been described. It has been shown that some thrombotic patients carry an additional genetic risk factor such as protein C, protein S, antithrombin deficiency or the G20210A mutation on the prothrombin gene. The occurrence of a deep vein thrombosis during enoxaparin prophylactic treatment in a pregnant woman homozygous for factor V Leiden, without other known prothrombotic genetic factors, led us to investigate her thrombomodulin gene. We found that the patient was heterozygous for the previously described G127-->A mutation, which results in an Ala25-->Thr substitution. Furthermore, for this patient, the allelic combination at the 1418 polymorphic site was C/T, which predicts an Ala455-->Val replacement. Although larger studies are required, this case report suggests that thrombomodulin gene mutations could be an additional genetic risk factor for thrombosis in carriers of the factor V Leiden mutation.

[Gene therapy in oncology: applications to lung cancer]. / Thérapie génique en oncologie: applications au cancer du poumon.

Gene therapy defines a new therapeutic avenue whose site of action is at the level of the gene itself; viral vectors (adenovirus, retrovirus, herpes virus) or non-viral (liposomes, plasmids) enable the transfer of a fraction of DNA (transgenic) to the target itself. In this review, we present recently acquired data on the mechanisms of oncogenesis and anti-tumor immunity which have enabled the application of several therapeutic strategies in oncology; the transfer of gene(s), coding for cytokines or for coactivation factors in order to develop active immunotherapy; the transfer of suicides genes; the transfer of multidrug resistance gene (MDR1); the transfer of tumor suppressor genes or of cDNA coding for antisense oligonucleotides in order to correct genomic anomalies which are responsible for the malign phenotype. The development of gene therapy demands the resolution of a number of technical difficulties such as vectorisation, targeting, and the expression of the stability of the trans-gene. Phase 1 trials in man have established the innocuity of certain vectors and have confirmed the expression of trans-genes (marker genes). Compared to monogenic hereditary diseases, the "molecular heterogenetic" of bronchial tumours, the consequence of the instability of the genome and the diversity of amplified oncogenes are a major difficulty. In addition, each one of these approaches prevents limiting factors: for example the exclusive targeting of malign cells is an indispensable pre-requisite for the transfer of suicide genes and in the same way the expression the tumour in antigens is the pre-requisite for the development of active immunotherapy. We report the overall results of applied trials for pulmonary carcinomas on murine models and present their applications which are underway in men.

[Mutation in the MSH2 gene in Muir-Torre syndrome]. / Mutation du gène MSH2 dans le syndrome de Muir-Torre.

BACKGROUND: The Muir-Torre syndrome is an autosomal dominant hereditary condition predisposing to cancer. It is characterized by skin tumors associated with adenocarcinoma of the colon or other neoplasias observed in the context of hereditary non-polyposis colorectal cancer (HNPCC). The Muir-Torre syndrome is also characterized by the frequent presence of multiple colonic polyps and the relatively moderate aggressivity of the tumors. CASE REPORT: We studied a family with Muir-Torre syndrome. We sequenced the exons of the hMSH2 gene in this family and identified heterozygous germinal mutation by G insert at position 2427 (2427insG). This mutation changes the lecture phase producing a premature codon stop. DISCUSSION: Our study confirms the predominant responsibility of the hMSH2 gene in Tuir-Torre syndrome. This clinical case and data reported in the literature demonstrate the importance of searching for a history of non-polyposis colorectal cancer in patients and relatives and the unstable genome characteristic of these tumors found in sebaceous tumors or keratoacanthomas. Sequencing the hMSH2 gene should be a priority when clinical features, history and laboratory tests suggest Muir-Torre syndrome.

[Molecular analysis of genetic predispositions to breast cancer]. / Analyse moléculaire des prédispositions génétiques au cancer du sein.

The molecular diagnosis of genetic abnormalities responsible for genetic predisposition to breast cancer is made difficult by the large size of the genes and the diversity of gene mutations found within these genes. The molecular diagnosis of responsible mutations requires the implementation of particular analytical methods, for which we give two examples, the protein truncation test and the direct sequence analysis of the cDNA. Results obtained with these two methods demonstrate the interest of studying the sequence of messenger RNA expressed by predisposing genes. The study also describes an abnormal splicing and two rearrangements responsible for genetic predisposition to breast cancer.