Phase II Study Evaluating 2 Dosing Schedules of Oral Foretinib (GSK1363089), cMET/VEGFR2 Inhibitor,in Patients with Metastatic Gastric Cancer

Purpose

The receptors for hepatocyte and vascular endothelial cell growth factors (MET and VEGFR2, respectively) are critical oncogenic mediators in gastric adenocarcinoma. The purpose is to examine the safety and efficacy of foretinib, an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR2 receptors, for the treatment of metastatic gastric adenocarcinoma.

Patients and Methods

Foretinib safety and tolerability, and objective response rate (ORR) were evaluated in patients using intermittent (240 mg/day, for 5 days every 2 weeks) or daily (80 mg/day) dosing schedules. Thirty evaluable patients were required to achieve alpha = 0.10 and beta = 0.2 to test the alternative hypothesis that single-agent foretinib would result in an ORR of ≥25%. Up to 10 additional patients could be enrolled to ensure at least eight with MET amplification. Correlative studies included tumor MET amplification, MET signaling, pharmacokinetics and plasma biomarkers of foretinib activity.

Results

From March 2007 until October 2009, 74 patients were enrolled; 74% male; median age, 61 years (range, 25–88); 93% had received prior therapy. Best response was stable disease (SD) in 10 (23%) patients receiving intermittent dosing and five (20%) receiving daily dosing; SD duration was 1.9–7.2 months (median 3.2 months). Of 67 patients with tumor samples, 3 had MET amplification, one of whom had SD. Treatment-related adverse events occurred in 91% of patients. Rates of hypertension (35% vs. 15%) and elevated aspartate aminotransferase (23% vs. 8%) were higher with intermittent dosing. In both patients with high baseline tumor phospho-MET (pMET), the pMET:total MET protein ratio decreased with foretinib treatment.

Conclusion

These results indicate that few gastric carcinomas are driven solely by MET and VEGFR2, and underscore the diverse molecular oncogenesis of this disease. Despite evidence of MET inhibition by foretinib, single-agent foretinib lacked efficacy in unselected patients with metastatic gastric cancer.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00725712","term_id":"NCT00725712"}}NCT00725712     

CBL Is Frequently Altered in Lung Cancers: Its Relationship to Mutations in MET and EGFR Tyrosine Kinases

Background

Non-small cell lung cancer (NSCLC) is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET), and functionality in NSCLC.

Methods and Findings

Using archival formalin-fixed paraffin embedded (FFPE) extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively) transfected in NSCLC cell lines, there was increased cell viability and cell motility.

Conclusions

Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.     

Prognostic Significance of MET Amplification and Expression in Gastric Cancer: A Systematic Review with Meta-Analysis

Background and Aims

MET, the hepatocyte growth factor receptor, is a receptor tyrosine kinase overexpressed and activated in a subset of gastric cancer. Several studies investigated the relationship between MET amplification and expression with the clinical outcome in patients with gastric cancer, but yielded conflicting results. We performed a systematic review and meta-analysis to determine the influence of MET amplification and expression on prognosis in gastric cancer.

Methods

MEDLINE and EMBASE were searched for studies that explored the association between MET amplification and expression with survival in patients with gastric cancer up to 1 April, 2013. Data of individual hazard ratios (HRs) and 95% confidence intervals (CIs) for meta-analyses were extracted from the publications and combined in pooled HRs.

Results

Fourteen studies involving 2,258 patients with gastric cancer were included. It was suggested that MET overexpression had an unfavorable impact on survival of patients with gastric cancer, with HRs (95% CIs) of 2.57 (95% CI: 1.97–3.35) overall, 2.82 (95% CI: 1.86–4.27) among studies using amplification for measure scale of MET and 2.42 (95% CI: 1.66–3.54) for expression. The magnitude of association was reduced whereas remained statistically significant in high quality studies or in larger sample size studies and corresponding HRs were 2.18(1.76, 2.70) and 2.35(1.93, 2.87), respectively, without significant heterogeneity.

Conclusion

The findings from present study indicated that higher MET gene amplification and expression in gastric cancer was an indicator of poor prognosis.     

Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes

Background

The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.

Results

We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations.

Conclusions

Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users.     

MET and AKT genetic influence on facial emotion perception

Background

Facial emotion perception is a major social skill, but its molecular signal pathway remains unclear. The MET/AKT cascade affects neurodevelopment in general populations and face recognition in patients with autism. This study explores the possible role of MET/AKT cascade in facial emotion perception.

Methods

One hundred and eighty two unrelated healthy volunteers (82 men and 100 women) were recruited. Four single nucleotide polymorphisms (SNP) of MET (rs2237717, rs41735, rs42336, and rs1858830) and AKT rs1130233 were genotyped and tested for their effects on facial emotion perception. Facial emotion perception was assessed by the face task of Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT). Thorough neurocognitive functions were also assessed.

Results

Regarding MET rs2237717, individuals with the CT genotype performed better in facial emotion perception than those with TT (p = 0.016 by ANOVA, 0.018 by general linear regression model [GLM] to control for age, gender, and education duration), and showed no difference with those with CC. Carriers with the most common MET CGA haplotype (frequency = 50.5%) performed better than non-carriers of CGA in facial emotion perception (p = 0.018, df = 1, F = 5.69, p = 0.009 by GLM). In MET rs2237717/AKT rs1130233 interaction, the C carrier/G carrier group showed better facial emotion perception than those with the TT/AA genotype (p = 0.035 by ANOVA, 0.015 by GLM), even when neurocognitive functions were controlled (p = 0.046 by GLM).

Conclusions

To our knowledge, this is the first study to suggest that genetic factors can affect performance of facial emotion perception. The findings indicate that MET variances and MET/AKT interaction may affect facial emotion perception, implicating that the MET/AKT cascade plays a significant role in facial emotion perception. Further replication studies are needed.     

Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method

Purpose

The analysis of MET gene copy number (CN) has been considered to be a potential biomarker to predict the response to MET-targeted therapies in various cancers. However, the current standard methods to determine MET CN are SNP 6.0 in the genomic DNA of cancer cell lines and fluorescence in situ hybridization (FISH) in tumor models, respectively, which are costly and require advanced technical skills and result in relatively subjective judgments. Therefore, we employed a novel method, droplet digital PCR (ddPCR), to determine the MET gene copy number with high accuracy and precision.

Methods

The genomic DNA of cancer cell lines or tumor models were tested and compared with the MET gene CN and MET/CEN-7 ratio determined by SNP 6.0 and FISH, respectively.

Results

In cell lines, the linear association of the MET CN detected by ddPCR and SNP 6.0 is strong (Pearson correlation = 0.867). In tumor models, the MET CN detected by ddPCR was significantly different between the MET gene amplification and non-amplification groups according to FISH (mean: 15.4 vs 2.1; P = 0.044). Given that MET gene amplification is defined as MET CN >5.5 by ddPCR, the concordance rate between ddPCR and FISH was 98.0%, and Cohen''s kappa coefficient was 0.760 (95% CI, 0.498–1.000; P <0.001).

Conclusions

The results demonstrated that the ddPCR method has the potential to quantify the MET gene copy number with high precision and accuracy as compared with the results from SNP 6.0 and FISH in cancer cell lines and tumor samples, respectively.     

Replication of the association of a MET variant with autism in a Chinese Han population

Background

Autism is a common, severe and highly heritable neurodevelopmental disorder in children, affecting up to 100 children per 10,000. The MET gene has been regarded as a promising candidate gene for this disorder because it is located within a replicated linkage interval, is involved in pathways affecting the development of the cerebral cortex and cerebellum in ways relevant to autism patients, and has shown significant association signals in previous studies.

Principal Findings

Here, we present new ASD patient and control samples from Heilongjiang, China and use them in a case-control and family-based replication study of two MET variants. One SNP, rs38845, was successfully replicated in a case-control association study, but failed to replicate in a family-based study, possibly due to small sample size. The other SNP, rs1858830, failed to replicate in both case-control and family-based studies.

Conclusions

This is the first attempt to replicate associations in Chinese autism samples, and our result provides evidence that MET variants may be relevant to autism susceptibility in the Chinese Han population.     

Impact of two myostatin (MSTN) mutations on weight gain and lamb carcass classification in Norwegian White Sheep (Ovis aries)

Background

Our aim was to estimate the effect of two myostatin (MSTN) mutations in Norwegian White Sheep, one of which is close to fixation in the Texel breed.

Methods

The impact of two known MSTN mutations was examined in a field experiment with Norwegian White Sheep. The joint effect of the two MSTN mutations on live weight gain and weaning weight was studied on 644 lambs. Carcass weight gain from birth to slaughter, carcass weight, carcass conformation and carcass fat classes were calculated in a subset of 508 lambs. All analyses were carried out with a univariate linear animal model.

Results

The most significant impact of both mutations was on conformation and fat classes. The largest difference between the genotype groups was between the wild type for both mutations and the homozygotes for the c.960delG mutation. Compared to the wild types, these mutants obtained a conformation score 5.1 classes higher and a fat score 3.0 classes lower, both on a 15-point scale.

Conclusions

Both mutations reduced fatness and increased muscle mass, although the effect of the frameshift mutation (c.960delG) was more important as compared to the 3''-UTR mutation (c.2360G>A). Lambs homozygous for the c.960delG mutation grew more slowly than those with other MSTN genotypes, but had the least fat and the largest muscle mass. Only c.960delG showed dominance effects.     

Tumor Phosphatidylinositol-3-Kinase Signaling and Development of Metastatic Disease in Locally Advanced Rectal Cancer

Background

Recognizing EGFR as key orchestrator of the metastatic process in colorectal cancer, but also the substantial heterogeneity of responses to anti-EGFR therapy, we examined the pattern of composite tumor kinase activities governed by EGFR-mediated signaling that might be implicated in development of metastatic disease.

Patients and Methods

Point mutations in KRAS, BRAF, and PIK3CA and ERBB2 amplification were determined in primary tumors from 63 patients with locally advanced rectal cancer scheduled for radical treatment. Using peptide arrays with tyrosine kinase substrates, ex vivo phosphopeptide profiles were generated from the same baseline tumor samples and correlated to metastasis-free survival.

Results

Unsupervised clustering analysis of the resulting phosphorylation of 102 array substrates defined two tumor classes, both consisting of cases with and without KRAS/BRAF mutations. The smaller cluster group of patients, with tumors generating high ex vivo phosphorylation of phosphatidylinositol-3-kinase-related substrates, had a particularly aggressive disease course, with almost a half of patients developing metastatic disease within one year of follow-up.

Conclusion

High phosphatidylinositol-3-kinase-mediated signaling activity of the primary tumor, rather than KRAS/BRAF mutation status, was identified as a hallmark of poor metastasis-free survival in patients with locally advanced rectal cancer undergoing radical treatment of the pelvic cavity.     

Whole Exome Analysis Identifies Frequent CNGA1 Mutations in Japanese Population with Autosomal Recessive Retinitis Pigmentosa

Objective

The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population.

Methods

In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed.

Results

Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation.

Conclusions

This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.     

Exome Sequencing of 47 Chinese Families with Cone-Rod Dystrophy: Mutations in 25 Known Causative Genes

Objective

The goal of this study was to identify mutations in 25 known causative genes in 47 unrelated Chinese families with cone-rod dystrophy (CORD).

Methods

Forty-seven probands from unrelated families with CORD were recruited. Genomic DNA prepared from leukocytes was analyzed by whole exome sequencing. Variants in the 25 genes were selected and then validated by Sanger sequencing.

Results

Fourteen potential pathogenic mutations, including nine novel and five known, were identified in 10 of the 47 families (21.28%). Homozygous, compound heterozygous, and hemizygous mutations were detected in three, four, or three families, respectively. The 14 mutations in the 10 families were distributed among CNGB3 (three families), PDE6C (two families), ABCA4 (one family), RPGRIP1 (one family), RPGR (two families), and CACNA1F (one family).

Conclusions

This study provides a brief view on mutation spectrum of the 25 genes in a Chinese cohort with CORD. Identification of novel mutations enriched our understanding of variations in these genes and their associated phenotypes. To our knowledge, this is the first systemic exome-sequencing analysis of all of the 25 CORD-associated genes.     

MET Gene Copy Number Predicts Worse Overall Survival in Patients with Non-Small Cell Lung Cancer (NSCLC); A Systematic Review and Meta-Analysis

Objectives

MET is a receptor present in the membrane of NSCLC cells and is known to promote cell proliferation, survival and migration. MET gene copy number is a common genetic alteration and inhibition o MET emerges as a promising targeted therapy in NSCLC. Here we aim to combine in a meta-analysis, data on the effect of high MET gene copy number on the overall survival of patients with resected NSCLC.

Methods

Two independent investigators applied parallel search strategies with the terms “MET AND lung cancer”, “MET AND NSCLC”, “MET gene copy number AND prognosis” in PubMed through January 2014. We selected the studies that investigated the association of MET gene copy number with survival, in patients who received surgery.

Results

Among 1096 titles that were identified in the initial search, we retrieved 9 studies on retrospective cohorts with adequate retrievable data regarding the prognostic impact of MET gene copy number on the survival of patients with NSCLC. Out of those, 6 used FISH and the remaining 3 used RT PCR to assess the MET gene copy number in the primary tumor. We calculated the I² statistic to assess heterogeneity (I² = 72%). MET gene copy number predicted worse overall survival when all studies were combined in a random effects model (HR = 1.78, 95% CI 1.22–2.60). When only the studies that had at least 50% of adenocarcinoma patients in their populations were included, the effect was significant (five studies, HR 1.55, 95% CI 1.23–1.94). This was not true when we included only the studies with no more than 50% of the patients having adenocarcinoma histology (four studies HR 2.18, 95% CI 0.97–4.90).

Conclusions

Higher MET gene copy number in the primary tumor at the time of diagnosis predicts worse outcome in patients with NSCLC. This prognostic impact may be adenocarcinoma histology specific.     

Autosomal Recessive Transmission of MYBPC3 Mutation Results in Malignant Phenotype of Hypertrophic Cardiomyopathy

Background

Hypertrophic cardiomyopathy (HCM) due to mutations in genes encoding sarcomere proteins is most commonly inherited as an autosomal dominant trait. Since nearly 50% of HCM cases occur in the absence of a family history, a recessive inheritance pattern may be involved.

Methods

A pedigree was identified with suspected autosomal recessive transmission of HCM. Twenty-six HCM-related genes were comprehensively screened for mutations in the proband with targeted second generation sequencing, and the identified mutation was confirmed with bi-directional Sanger sequencing in all family members and 376 healthy controls.

Results

A novel missense mutation (c.1469G>T, p.Gly490Val) in exon 17 of MYBPC3 was identified. Two siblings with HCM were homozygous for this mutation, whereas other family members were either heterozygous or wild type. Clinical evaluation showed that both homozygotes manifested a typical HCM presentation, but none of others, including 5 adult heterozygous mutation carriers up to 71 years of age, had any clinical evidence of HCM.

Conclusions

Our data identified a MYBPC3 mutation in HCM, which appeared autosomal recessively inherited in this family. The absence of a family history of clinical HCM may be due to not only a de novo mutation, but also recessive mutations that failed to produce a clinical phenotype in heterozygous family members. Therefore, consideration of recessive mutations leading to HCM is essential for risk stratification and genetic counseling.     

Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

Background

The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines.

Methodology/Principal Findings

Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import.

Conclusion/Significance

This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.     

Novel Secondary Somatic Mutations in Ewing's Sarcoma and Desmoplastic Small Round Cell Tumors

Background

Ewing''s sarcoma (ES) and desmoplastic small round cell tumors (DSRCT) are small round blue cell tumors driven by an N-terminal containing EWS translocation. Very few somatic mutations have been reported in ES, and none have been identified in DSRCT. The aim of this study is to explore potential actionable mutations in ES and DSRCT.

Methodology

Twenty eight patients with ES or DSRCT had tumor tissue available that could be analyzed by one of the following methods: 1) Next-generation exome sequencing platform; 2) Multiplex PCR/Mass Spectroscopy; 3) Polymerase chain reaction (PCR)-based single- gene mutation screening; 4) Sanger sequencing; 5) Morphoproteomics.

Principal Findings

Novel somatic mutations were identified in four out of 18 patients with advanced ES and two of 10 patients with advanced DSRCT (six out of 28 (21.4%));KRAS (n = 1), PTPRD (n = 1), GRB10 (n = 2), MET (n = 2) and PIK3CA (n = 1). One patient with both PTPRD and GRB10 mutations and one with a GRB10 mutation achieved a complete remission (CR) on an Insulin like growth factor 1 receptor (IGF1R) inhibitor based treatment. One patient, who achieved a partial remission (PR) with IGF1R inhibitor treatment, but later developed resistance, demonstrated a KRAS mutation in the post-treatment resistant tumor, but not in the pre-treatment tumor suggesting that the RAF/RAS/MEK pathway was activated with progression.

Conclusions

We have reported several different mutations in advanced ES and DSRCT that have direct implications for molecularly-directed targeted therapy. Our technology agnostic approach provides an initial mutational roadmap used in the path towards individualized combination therapy.     

Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore

Background

Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region.

Methods

Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed.

Results

Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations.

Conclusions

Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.     

Detection of epidermal growth factor receptor mutations in formalin fixed paraffin embedded biopsies in Malaysian non-small cell lung cancer patients

Background

Somatic mutations of the epidermal growth factor receptor (EGFR) are reportedly associated with various responses in non-small cell lung cancer (NSCLC) patients receiving the anti-EGFR agents. Detection of the mutation therefore plays an important role in therapeutic decision making. The aim of this study was to detect EGFR mutations in formalin fixed paraffin embedded (FFPE) samples using both Scorpion ARMS and high resolution melt (HRM) assay, and to compare the sensitivity of these methods.

Results

All of the mutations were found in adenocarcinoma, except one that was in squamous cell carcinoma. The mutation rate was 45.7% (221/484). Complex mutations were also observed, wherein 8 tumours carried 2 mutations and 1 tumour carried 3 mutations.

Conclusions

Both methods detected EGFR mutations in FFPE samples. HRM assays gave more EGFR positive results compared to Scorpion ARMS.     

Whole genome characterization of the mechanisms of daptomycin resistance in clinical and laboratory derived isolates of Staphylococcus aureus

Background

Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus.

Methods

Whole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates.

Results

On average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol.

Conclusion

Point mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus.