Resistance characterization of ledipasvir and velpatasvir in hepatitis C virus genotype 4.

HCV genotype 4 (GT4) has often been overlooked in drug development, even though it infects ~20 million people worldwide. Ledipasvir/sofosbuvir and sofosbuvir/velpatasvir were highly efficacious in GT4 HCV-infected patients from GS-US-337-1119 and GS-US-342-1138. Here, we characterize the resistance profile of ledipasvir (LDV) and velpatasvir (VEL) in patients with GT4 HCV infection. NS5A deep-sequencing was performed for 454 patients infected with HCV GT4 at baseline, including 44 patients enrolled in GS-US-337-1119 and 116 patients enrolled in GS-US-342-1138, and at relapse for patients with virologic failure. LDV and VEL susceptibilities of 56 patient isolates were determined. In GS-US-337-1119, SVR12 rates were 100% for all subtypes except 4b and 4r. Phenotypic assessment of 56 HCV NS5A patient isolates from various GT4 subtypes indicated that LDV had high potency for the common subtypes 4a/d, and subtypes 4c/f/k/l/m/n/o/p/r/t despite the presence of resistance-associated substitutions (RASs). For the rare GT4b, LDV median EC50 was higher, but with a broad range of individual values. Importantly, all GT4b isolates tested had 2-4 NS5A RASs, some including Y93H. Similarly, the 2 GT4r infected patients who had virologic relapse had rare triple RASs. Reversion of these substitutions to the consensus residue significantly increased LDV susceptibility. In GS-US-342-1138, all patients achieved SVR12, regardless of their subtype or presence of RASs. In vitro data confirmed that VEL is potent against all GT4 isolates tested. LDV and VEL are potent antiviral drugs, estimated to be effective against >95% and >99%, respectively, of GT4 HCV isolates.

Resistance Analyses of Japanese Hepatitis C-Infected Patients Receiving Sofosbuvir or Ledipasvir/Sofosbuvir Containing Regimens in Phase 3 Studies.

High rates of sustained virologic response (SVR) has been achieved in Japanese patients with chronic hepatitis C virus (HCV) genotype (GT)1 and GT2 infection treated with ledipasvir/sofosbuvir (LDV/SOF) ±ribavirin (RBV) and SOF+RBV, respectively. We evaluated the effect of baseline HCV NS5A and NS5B resistance-associated variants (RAVs) on treatment outcome and characterized variants at virologic failure. Baseline deep sequencing for NS5A and NS5B genes was performed for all GT1 patients. Deep sequencing of NS5A (GT1 only) and NS5B (GT1 and GT2) was performed for patients who failed treatment or discontinued early with detectable HCV RNA (i.e., >25 IU/mL). In patients with HCV GT1 infection, 22.3% (GT1a: 2/11; GT1b: 74/330) had ≥1 baseline NS5A RAV. The most frequent NS5A RAVs in GT1b were Y93H (17.9%, 59/330) and L31M (2.4%, 8/330). Despite the presence of NS5A RAVs at baseline, 100% and 97% of patients achieved SVR12, compared with 100% and 99% for those with no NS5A RAVs with LDV/SOF and LDV/SOF+RBV, respectively. All patients with NS5B RAVs at baseline achieved SVR12. Of the 153 patients with GT2 infection (GT2a 60.1%, GT2b 39.9%), 3.3% (5/153) experienced viral relapse. No S282T or other NS5B RAVs were detected at baseline or relapse; no change in susceptibility to SOF or RBV was observed at relapse. In conclusion, LDV/SOF and SOF+RBV demonstrate a high barrier to resistance in Japanese patients with HCV GT1 and GT2 infection. The presence of baseline NS5A RAVs did not impact treatment outcome in GT1 Japanese patients treated with LDV/SOF for 12 weeks.

GS-9857 in patients with chronic hepatitis C virus genotype 1-4 infection: a randomized, double-blind, dose-ranging phase 1 study.

GS-9857, an inhibitor of the hepatitis C virus (HCV) nonstructural protein (NS) 3/4A, demonstrates potent activity against HCV genotypes 1-6 and improved coverage against commonly encountered NS3 resistance-associated variants (RAVs). In this study, the safety, tolerability, antiviral activity and pharmacokinetics (PK) of GS-9857 were evaluated in patients with chronic HCV genotype 1-4 infection. Patients with genotype 1-4 infection received placebo or once-daily GS-9857 at doses ranging from 50 to 300 mg for 3 days under fasting conditions. GS-9857 was well tolerated; all reported adverse events (AEs) were mild or moderate in severity. Diarrhoea and headache were the most commonly reported AEs. Grade 3 or 4 laboratory abnormalities were observed in 17% of patients receiving GS-9857; there were no Grade 3 or 4 abnormalities in alanine aminotransferase, aspartate aminotransferase or alkaline phosphatase levels. GS-9857 demonstrated potent antiviral activity in patients with chronic HCV infection, achieving mean and median maximum reductions in HCV RNA of ≥3 log10 IU/mL following administration of a 100-mg dose in patients with HCV genotype 1a, 1b, 2, 3 or 4 infection. The antiviral activity of GS-9857 was unaffected by the presence of pretreatment NS3 RAVs. In patients with genotype 1-4 infection, GS-9857 exhibited linear PK and was associated with a median half-life of 29-42 h, supporting once-daily dosing. Thus, the tolerability, efficacy and pharmacokinetic profile of GS-9857 support its further evaluation for treatment of patients with chronic HCV infection.

Evolution of the HCV viral population from a patient with S282T detected at relapse after sofosbuvir monotherapy.

Clinical phase II/III studies of the nucleotide analogue HCV NS5B inhibitor sofosbuvir (SOF) have demonstrated high efficacy in HCV-infected patients in combination therapy. To date, resistance to SOF (S282T in NS5B) has rarely been detected in patients. In this study, we investigated the evolution of S282T viral variants detected in one HCV genotype 2b-infected patient who relapsed following 12 weeks of SOF monotherapy. Deep sequencing of the NS5B gene was performed on longitudinal plasma samples at baseline, days 2 and 3 on SOF, and longitudinal samples post-SOF treatment through week 48. Intrapatient HCV evolution was analysed by maximum-likelihood phylogenetic analysis. Deep sequencing analysis revealed a low level pre-existence of S282T at 0.05% of viral sequences (4/7755 reads) at baseline and 0.03% (6/23 415 reads) at day 2 on SOF. Viral relapse was detected at week 4 post-treatment where 99.8% of the viral population harboured S282T. Follow-up analysis determined that S282T levels diminished post-treatment reaching undetectable levels 24-48 weeks post-SOF. Phylogenetic analysis together with the persistence of unique post-treatment mutations in all post-SOF samples suggested that growth of wild type resulted from reversion of the S282T mutant to a wild type and not outgrowth of the baseline wild-type population. Our data suggest that a very low level of pre-existing S282T at baseline in this patient was enriched and transiently detected following SOF monotherapy. Despite relapse with drug resistance to SOF, this patient was successfully retreated with SOF plus ribavirin for 12 weeks and is now cured from HCV infection.

Lack of referral for genetic counseling and testing in BRCA1/2 and Lynch syndromes: a nationwide study based on 240,134 consultations and 134,652 genetic tests.

Based on nationwide data from the French national cancer institute (INCa), we analyzed the evolution of cancer genetics consultations and testing over time, and the uptake of targeted tests in relatives of families with BRCA1/2 or MMR genes mutation. Genetic testing and consultations for familial high-risk individuals are exclusively funded and monitored by the INCa in France. All nationwide cancer genetics centers reported annually standardized parameters of activity from 2003 to 2011. The analysis included a total of 240,134 consultations and 134,652 genetic tests enabling to identify 32,494 mutation carriers. Referral for hereditary breast and ovarian cancer (HBOC) or colorectal cancer predisposition syndromes represented 59 % (141,639) and 23.2 % (55,698) consultations, respectively. From 2003 to 2011, we found a dramatic and steady increase of tests performed for BRCA1/2 (from 2,095 to 7,393 tests/year, P < 0.0001) but not for MMR genes (from 1,144 to 1,635/year, P = NS). The overall percentage of deleterious mutations identified in the probands tested was 13.8 and 20.9 % in HBOC and Lynch syndromes, respectively. Pooled analysis for BRCA1/2 and Lynch syndrome tests showed an inverse relationship between the percentage of mutation detected and the absolute number of tests performed over the time (overall Cochran-Armitage test for trend: P < 0.001). In families with BRCA1/2 or MMR identified mutations, there was an average number of 2.94 and 3.28 relatives performing targeted tests, respectively. This nationwide study shows a lack of referral and genetic testing in Lynch as compared to HBOC syndromes. Only a third of relatives of a proband with a predisposing mutation performed a targeted test. Enhanced information about benefit of genetic testing should be given to clinicians and patients for Lynch syndrome and relatives of a proband carrying an identified predisposing mutation.

French professionals in genetic counselor careers.

The profession of genetic counseling in France was recognized in 2004, based on the recommendations of a mandate commissioned by the Health Minister to explore the medical demographics of France. The report predicted a shortage of health professionals in the field of genetics, particularly in light of the rapid development of molecular testing. Development of the profession was supported by a legal framework, and today 107 genetic counselors have graduated from the specific educational program which awards the Professional Master's Degree of Human Pathology, entitled Master in Genetic Counseling and Predictive Medicine. Here we will trace the development of the profession in France and review the demographic characteristics of the students and genetic counselors practicing the profession today.

[Genetic testing in the context of the revision of the French law on bioethics]. / Les tests génétiques à l'heure de la deuxième révision des lois de bioéthique.

This article focuses on six questions raised by genetic testing in human: (1) the use of genetic tests, (2) information given to relatives of patients affected with genetic disorders, (3) prenatal and preimplantatory diagnosis for late onset genetic diseases and the use of pangenomic tests in prenatal diagnosis, (4) direct-to-consumer genetic testing, (5) population screening in the age of genomic medicine and (6) incidental findings when genetic testing are used.

Subjective interpretation of inconclusive BRCA1/2 cancer genetic test results and transmission of information to the relatives.

OBJECTIVE: BRCA1/2 gene mutations are not frequently identified in breast or ovarian cancer patients who are the first members of their family to be tested. Little is known about how probands interpret and cope with these results, which are generally referred to as 'inconclusive'. The aim of this study was to describe subjective understanding by women with cancer in response to an inconclusive BRCA1/2 test, describing the difficulties or non-difficulties they encountered about the transmission of information to their family. METHODS: A cohort of 30 women with breast/ovarian cancer were followed for a period of up to 2 years after delivery of their inconclusive genetic test results. Self-administered questionnaires with closed and open questions were distributed. A qualitative analysis of open-ended questions is presented here. RESULTS: These women's reactions to inconclusive results were of three kinds. The majority (n=14) were still uncertain about their carrier status, which is an adequate medical interpretation of the results, while others (n=9) took their inconclusive results to mean that they were definitely not carriers, and the women in the last group (n=7) were convinced that they were actually carriers. There was some overlap between these perceptions and actual genetic risk. CONCLUSIONS: The transmission of information to the family was found to differ qualitatively across the three groups and more difficulties in this respect were expressed by those who were uncertain about their carrier status.

Turcot syndrome confirmed with molecular analysis.

Turcot syndrome is clinically characterized by the occurrence of primary brain tumor and colorectal tumor and has, in previous reports, been shown associated with germline mutations in the genes APC, MLH1, MHS6, and PMS2. To date, only few families have been documented by molecular analysis. We report two new families with Turcot syndrome to illustrate and review its characteristics and facilitate diagnosis. Molecular analysis revealed two germline mutations, one in the MLH1 gene and one in MSH2. The latter has never been describe in the literature. Personal and familial relevant anamnestic data from patients with glioma might aid in the diagnosis of genetic disorders. The subsequent molecular characterization may contribute to the appropriate care of affected patients and asymptomatic gene 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.

Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations.

BACKGROUND: We have previously demonstrated that breast cancers associated with inherited BRCA1 and BRCA2 gene mutations differ from each other in their histopathologic appearances and that each of these types differs from breast cancers in patients unselected for family history (i.e., sporadic cancers). We have now conducted a more detailed examination of cytologic and architectural features of these tumors. METHODS: Specimens of tumor tissue (5-microm-thick sections) were examined independently by two pathologists, who were unaware of the case or control subject status, for the presence of cell mitosis, lymphocytic infiltration, continuous pushing margins, and solid sheets of cancer cells; cell nuclei, cell nucleoli, cell necrosis, and cell borders were also evaluated. The resulting data were combined with previously available information on tumor type and tumor grade and further evaluated by multifactorial analysis. All statistical tests are two-sided. RESULTS: Cancers associated with BRCA1 mutations exhibited higher mitotic counts (P = .001), a greater proportion of the tumor with a continuous pushing margin (P<.0001), and more lymphocytic infiltration (P = .002) than sporadic (i.e., control) cancers. Cancers associated with BRCA2 mutations exhibited a higher score for tubule formation (fewer tubules) (P = .0002), a higher proportion of the tumor perimeter with a continuous pushing margin (P<.0001), and a lower mitotic count (P = .003) than control cancers. CONCLUSIONS: Our study has identified key features of the histologic phenotypes of breast cancers in carriers of mutant BRCA1 and BRCA2 genes. This information may improve the classification of breast cancers in individuals with a family history of the disease and may ultimately aid in the clinical management of patients.

Major improvement in the efficacy of BRCA1 mutation screening using morphoclinical features of breast cancer.

A family history of breast and/or ovarian cancer is the main criterion used in screening BRCA1 gene carriers. However, ascertaining a patient's family history is a difficult task, which significantly restricts the use of this parameter in clinical practice. Alternative individual criteria that can be used to identity BRCA1 gene carriers would, therefore, be of great value. In this context, it was recently established that BRCA1-associated breast cancers (BRCA1-BCs) show a specific morphoclinical pattern. In multivariate analyses, the two most discriminant morphoclinical parameters available for establishing the BRCA1 status, in addition to an early age at onset, are estrogen receptor negativity (ER-) and poor tumor differentiation (TD3). Here we tested the efficacy of these two morphological parameters as BRCA1 mutation indicators and investigated their economic impact, in a population-based survey on a series of women who developed invasive breast cancer by the age of 35 years, regardless of their family history. A high rate of 28.6% of BRCA1 mutations was found to have occurred in the group of tumors with both ER- and TD3 versus only 3.6% in tumors with other profiles (P = 0.007; odds ratio, 10.8). When the sole criterion used was early onset by the age of 35 years, the mutation rate was found to be 8.6%. The resulting cost of testing only women with ER- and TD3 tumors worked out at 30% that of testing the whole population of women with cancer by the age of 35 years, and the sensitivity was found to be of 66%. Lastly, the family history of ER- and TD3 cases with a BRCA1 mutation was investigated retrospectively, and none of these cases was found to have a particularly extensive family history of breast and/or ovarian cancer. The use of these morphological features of BRCA1-BCs that are currently typed in clinical practice, therefore, provides a helpful and cost-effective tool for those making decisions about genetic screening. This strategy makes it possible to identify gene carriers who would be overlooked using current criteria.

Genome-wide search for loss of heterozygosity shows extensive genetic diversity of human breast carcinomas.

Deletions of genomic regions involving tumor suppressor genes are thought to be important in the initiation and progression of breast cancer. We conducted a genome-wide search for deleted regions in a series of 75 human breast carcinomas by studying the allelic patterns of 184 microsatellite markers distributed over all chromosomes and looking for loss of heterozygosity (LOH). We identified 56 regions of consistent LOH. Strikingly, every tumor had a different set of deletions. To study this complexity, we applied a phylogenetic-like type of analysis. Each region was involved in a certain proportion of tumors, ranging from 20 to 62%; the most frequently involved regions were on chromosome arms 8p, 11q, 16q, and 17p. There was a correlation (P = 0.005) between the level of LOH and the size of the tumors. Tumors with a high level of LOH were also highly proliferative and had a high mitotic index.

Germ line mutation at BRCA1 affects the histoprognostic grade in hereditary breast cancer.

Histoprognostic grade is a determinant parameter to select the initial therapeutic strategy in breast cancer. Our aim was to analyze the grade repartition in BRCA1-associated breast cancer (BC) and to explore the possible connections between grade and the BRCA1 gene function. We first compared 27 BRCA1-associated BCs from 14 families with 4,461 cases from an administrative district registry and 242 cases from a hospital-based registry, matching for grade and constitutive elements, and then considered their repartition in families. We observed a prevalence of grade 3 (P < 0.0001) in BRCA1-associated BC. This was attributed mainly to nuclear polymorphism (P < 0.0001), mitotic activity (P < 0.0001), and tubular differentiation (P = 0.0004), implying that BRCA1-associated BCs are highly proliferating tumors. Moreover, it is suggested that grade segregates as a genetic trait within families (P = 0.0015), and this was attributed to the mitotic index segregation only (P = 0.0005). Therefore grade, through its components, could be interpreted as the morphological translation of the BRCA1 germ line mutation. Thus, a genotype-phenotype correlation may exist between the type of mutation and the aggressiveness of the disease. These findings are bound to have an important impact in the care management of hereditary breast cancer at the individual and at the familial level and in the comprehensive approach of breast cancer development.

Truncation at conserved terminal regions of BRCA1 protein is associated with highly proliferating hereditary breast cancers.

The existence of two subgroups of BRCA1-associated breast cancer (BC) families has been recently posited: the first with highly proliferating tumors, and the second composed of cases with a low proliferation rate. Our aim was to test whether the proliferation rate of BRCA1-associated breast cancers was affected by the site of the germ line mutation in the BRCA1 gene. We analyzed the distribution of the mitotic index, a histoprognostic grade component shown to segregate in families, matching for germ line mutation location in a series of 28 breast cancers from 20 kindreds. We observed a prevalence of highly proliferating tumors when the mutation occurs in the two terminal conserved domains of the BRCA1 protein, ie., in the amino and carboxyl termini (P = 0.0024). Our data provide evidence for a genotype-phenotype correlation and along with their strong conservation during evolution argue for the importance of these two regions in the control of mammary cell growth.

Mutations at BRCA1: the medullary breast carcinoma revisited.

BRCA1-associated breast cancers (BRCA1-BCs) frequently harbor a high histoprognostic grade, p53 alterations, and estrogen receptor negativity. Although these parameters predict a poor outlook, the overall survival in BRCA1-BCs is equivalent to or even better than that in sporadic cases. These features are reminiscent of what is observed for breast carcinoma of the medullary type, a high-grade tumor with a particular favorable course. To explore a possible relationship between this phenotype and BRCA1 mutations, we first compared 32 BRCA1-BCs and 200 consecutive cases of breast cancer without familial history for the prevalence of typical medullary breast carcinoma (TMC) using the criteria given by Ridolfi et al. [R. Ridolfi et al, Cancer (Phila.), 40: 1365-1385, 1977]. Second, we searched for BRCA1 mutations in a set of 18 cases of TMC, using denaturing gradient gel electrophoresis and Cleavase fragment length polymorphism scanning. Six of 32 (19%) BRCA1-BCs were of the TMC type, compared to 0 of 200 controls (P < 0.0001). Among the 18 TMCs, 2 BRCA1 nonsense mutations were found. This corresponds to almost 7 times the contribution of BRCA1 mutations in the general population. Two additional missense mutations were identified. Together, these results suggest that, although TMC and BRCA1-BCs are not strictly coincidental, an important connection between the two populations does exist.

Familial hematological malignancies: ASXL1 gene investigation.

PURPOSE: Familial aggregation among patients with several hematological malignancies has been revealed. This emphasizes the importance of genetic factors. Only few genes predisposing to familial hematological malignancies have been reported until now due to the low occurrence. We have described in previous study PRF1 and CEBPA variants that might contribute to the background of genetic factors, which encourage us to extend our investigations to other cooperating genes. The aim of this study is to determine whether germline additional sex combs-like 1 (ASXL1) gene mutations may be involved? METHODS/PATIENTS: In this study, we investigated the candidate gene ASXL1 by direct sequencing in 88 unrelated Tunisian and French families with aggregated hematological malignancies. RESULTS: We report a new p.Arg402Gln germline missense substitution in two related Tunisian patients which has not been previously described. We identified here this variant for the first time in non-Hodgkin lymphoma. The p.Arg402Gln variant was not found in 200 control chromosomes. In silico analysis has predicted potential deleterious effect on ASXL1 protein. CONCLUSIONS: From an extended candidate genes analyzed in the field of familial hematological malignancies, ASXL1 might be involved. This variant should be considered since a potential damaging effect was predicted by in silico analysis, with a view to develop functional assay in order to investigate the biological assessment.

Highly favorable outcome in BRCA-mutated metastatic breast cancer patients receiving high-dose chemotherapy and autologous hematopoietic stem cell transplantation.

Breast cancer carrying BRCA mutation may be highly sensitive to DNA-damaging agents. We hypothesized a better outcome for BRCA-mutated (BRCA(mut)) metastatic breast cancer (MBC) patients receiving high-dose chemotherapy and autologous hematopoietic stem cell transplantation (HDC AHSCT) versus unaffected BRCA (BRCA wild type; (BRCA(wt))) or patients without documented BRCA mutation (BRCA untested (BRCA(ut))). All female patients treated for MBC with AHSCT at Institut Paoli-Calmettes between 2003 and 2012 were included. BRCA(mut) and BRCA(wt) patients were identified from our institutional genetic database. Overall survival (OS) was the primary end point. A total of 235 patients were included. In all, 15 patients were BRCA(mut), 62 BRCA(wt) and 149 BRCA(ut). In multivariate analyses, the BRCA(mut) status was an independent prognostic factor for OS (hazard ratio (HR): 3.08, 95% confidence interval (CI): 1.10-8.64, P=0.0326) and PFS (HR: 2.52, 95% CI :1.29-4.91, P=0.0069). In this large series of MBC receiving HDC AHSCT, we report a highly favorable survival outcome in the subset of patients with documented germline BRCA mutations.

Two germ-line mutations affecting the same nucleotide at codon 257 of p53 gene, a rare site for mutations.

Codon 257 of the p53 gene is an extremely rare target for somatic mutations (accounting for only two of 1600 published mutations). We report here two constitutional mutations both affecting the second nucleotide of codon 257. A thymine to adenine transversion resulting in an amino acid change from leucine to glutamine was found in one proband who developed multiple independent malignant tumors (osteosarcoma, phyllodes tumor, soft-tissue sarcoma). Her mother died of early-onset breast cancer. In the other case, a deletion resulting in a frameshift in the C-terminal coding region of p53 was found in a woman who was diagnosed with breast cancer at age 34. This woman belongs to a family with features of Li-Fraumeni syndrome. In both cases, the p53 mutations identified in the proband was found in other members of the family. Codon 257, even if rarely mutated in somatic cells, may thus be an important target for germ-line mutations.