Tissue Transglutaminase (TG2) is a potential therapeutic target in the treatment of chemoresistant breast cancer

Tissue transglutaminase (TG2) has been suggested to be a key player in the progression and metastasis of chemoresistant breast cancer. One of the foremost survival signalling pathways implicated in causing drug resistance in breast cancer is the constitutive activation of NFκB (Nuclear Factor -kappa B) induced by TG2. This study provides a mechanism by which TG2 constitutively activates NFκB which in turn confers chemoresistance to breast cancer cells against doxorubicin. Breast cancer cell lines with varying expression levels of TG2 as well as TG2 null breast cancer cells transfected with TG2 were used as the major cell models for this study. This study made use of cell permeable and impermeable TG2 inhibitors, specific TG2 and Rel A/ p65 targeting siRNA, TG2 functional blocking antibodies, IKK inhibitors and a specific targeting peptide against Rel A/p65 to investigate the pathway of activation involved in the constitutive activation of NFκB by TG2 leading to drug resistance. Crucial to the activation of Rel A/p65 and drug resistance in the breast cancer cells is the interaction between the complex of IκBα and Rel A/p65 with TG2 which results in the dimerization of Rel A/p65 and polymerization of IκBα. The association of TG2 with the IκBα-NFκB complex was determined to be independent of IKKα/β function. The polymerized IκBα is degraded in the cytoplasm by the μ-calpain pathway which allows the cross linked Rel A/ p65 dimers to translocate into the nucleus. Using R283 and ZDON (cell permeable TG2 activity inhibitors) and specific TG2 targeting siRNA, the Rel A/ p65 dimer formation could be inhibited. Co-immunoprecipitation studies confirmed that the phosphorylation of the Rel A/p65 dimers at the Ser536 residue by IKKε took place in the cell nucleus. Importantly, this study also investigated the transcriptional regulation of the TGM2 gene by the pSer536 Rel A/ p65 dimer and the importance of this TG2-NFκB feedback loop in conferring drug resistance to breast cancer cells. This data provides evidence that TG2 could be a key therapeutic target in the treatment of chemoresistant breast cancer.

De novo methylation of tumor suppressor gene p16/INK4a is a frequent finding in multiple myeloma patients at diagnosis.

The p16 gene competes with cyclin D for binding to CDK4/CDK6 and therefore inhibits CDK4/6 complex kinase activity, resulting in dephosphorylation of pRb and related G1 growth arrest. Inactivation of this gene has been involved in a variety of tumors by different mechanisms: homozygous/hemyzygous deletions, point mutations and methylation of a 5' CpG island into exon E1alpha of the p16 gene. Homozygous deletions have been rarely found in multiple myeloma (MM) and no point mutations have been reported. Two recent studies have reported a high prevalence of methylation in the exon E1alpha of the p16 gene, but included only a small number of cases. We have analyzed the methylation pattern of exon E1alpha of the p16 gene in 101 untreated MM and five primary plasma cell leukemias (PCL). A PCR assay, relying on the inability of some restriction enzymes to digest methylated sequences, was used to analyze the methylation status. Southern blot analysis was used to confirm these results. Forty-one of 101 MM patients (40.5%) as well as four of the five (80%) primary PCL patients had shown methylation of the exon E1alpha. Our study confirms that hypermethylation of the p16 gene is a frequent event in MM. Leukemia (2000) 14, 183-187.

Genomic Signatures and Antiviral Drug Susceptibility Profile of A(H1N1)pdm09

Background: Genetic changes in influenza surface and internal genes can alter viral fitness and virulence. Mutation trend analysis and antiviral drug susceptibility profiling of A(H1N1)pdm09 viruses is essential for risk assessment of emergent strains and disease management. Objective: To profile genomic signatures and antiviral drug resistance of A(H1N1)pdm09 viruses and to discuss the potential role of mutated residues in human host adaptation and virulence. Study design: A(H1N1)pdm09 viruses circulating in Portugal during pandemic and post-pandemic periods and 2009/2010 season. Viruses were isolated in MDCK-SIAT1 cell culture and subjected to mutation analysis of surface and internal proteins, and to antiviral drug susceptibility profiling. Results: The A(H1N1)pdm09 strains circulating during the epidemic period in Portugal were resistant to amantadine. The majority of the strains were found to be susceptible to oseltamivir and zanamivir, with five outliers to neuraminidase inhibitors (NAIs) identified. Specific mutation patterns were detected within the functional domains of internal proteins PB2, PB1, PA, NP, NS1, M1 and NS2/NEP, which were common to all isolates and also some cluster-specific. Discussion: Modification of viral genome transcription, replication and apoptosis kinetics, changes in antigenicity and antiviral drug susceptibility are known determinants of virulence. We report several point mutations with putative roles in viral fitness and virulence, and discuss their potential to result in more virulent phenotypes. Monitoring of specific mutations and genetic patterns in influenza viral genes is essential for risk assessing emergent strains, disease epidemiology and public health implications.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon Region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.

Prevalence and resistance mutations of non-B HIV-1 subtypes among immigrants in Southern Spain along the decade 2000-2010.

Most of the non-B HIV-1 subtypes are predominant in Sub-Saharan Africa and India although they have been found worldwide. In the last decade, immigration from these areas has increased considerably in Spain. The objective of this study was to evaluate the prevalence of non-B subtypes circulating in a cohort of HIV-1-infected immigrants in Seville, Southern Spain and to identify drug resistance-associated mutations. METHODS: Complete protease and first 220 codons of the reverse transcriptase coding regions were amplified and sequenced by population sequencing. HIV-1 subtypes were determined using Stanford University Drug Resistance Database, and phylogenetic analysis was performed comparing multiple reported sequences. Drug resistance mutations were defined according to the International AIDS Society-USA. RESULTS: From 2000 to 2010 a total of 1,089 newly diagnosed HIV-1-infected patients were enrolled in our cohort. Of these, 121 were immigrants, of which 98 had ethical approval and informed consent to include in our study. Twenty-nine immigrants (29/98, 29.6%) were infected with non-B subtypes, of which 15/29 (51.7%) were CRF02-AG, mostly from Sub-Saharan Africa, and 2/29 (6.9%) were CRF01-AE from Eastern Europe. A, C, F, J and G subtypes from Eastern Europe, Central-South America and Sub-Saharan Africa were also present. Some others harboured recombinant forms CRF02-AG/CRF01-AE, CRF2-AG/G and F/B, B/C, and K/G, in PR and RT-coding regions. Patients infected with non-B subtypes showed a high frequency of minor protease inhibitor resistance mutations, M36I, L63P, and K20R/I. Only one patient, CRF02_AG, showed major resistance mutation L90M. Major RT inhibitor resistance mutations K70R and A98G were present in one patient with subtype G, L100I in one patient with CRF01_AE, and K103N in another patient with CRF01_AE. Three patients had other mutations such as V118I, E138A and V90I. CONCLUSIONS: The circulation of non-B subtypes has significantly increased in Southern Spain during the last decade, with 29.6% prevalence, in association with demographic changes among immigrants. This could be an issue in the treatment and management of these patients. Resistance mutations have been detected in these patients with a prevalence of 7% among treatment-naïve patients compared with the 21% detected among patients under HAART or during treatment interruption.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different "hot spots," were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

Polymorphic mutations associated with the emergence of the multinucleoside/tide resistance mutations 69 insertion and Q151M.

BACKGROUND: We hypothesized that polymorphic mutations exist that are associated with the emergence of the multinucleoside resistance mutations (MNR), 69 insertion and Q151M. METHODS: The Swiss HIV Cohort Study was screened, and the frequencies of polymorphic mutations in HIV-1 (subtype B) were compared between patients detected with the 69 insertion (n = 17), Q151M (n = 29), ≥2 thymidine analogue mutations (TAM) 1 (n = 400) or ≥2 TAM 2 (n = 249). Logistic regressions adjusted for the antiretroviral treatment history were performed to analyze the association of the polymorphic mutations with MNR. RESULTS: The 69 insertion and TAM 1 were strongly associated and occurred in 94.1% (16 of 17) together. The 69 insertion seemed to emerge as a consequence of the TAM 1 pathway (median years until detection: 6.8 compared with 4.4 for ≥2 TAM 1, P Wilcoxon = 0.009). Frequencies of 8 polymorphic mutations (K43E, V60I, S68G, S162C, T165I, I202V, R211K, F214L) were significantly different between groups. Logistic regression showed that F214L and V60I were associated with the emergence of Q151M/TAM 2 opposed to 69 insertion/TAM 1. S68G, T165I, and I202V were associated with Q151M instead of TAM 2. CONCLUSIONS: Besides antiretroviral therapy, polymorphic mutations may contribute to the emergence of specific MNR mutations.

Monitoring of malaria parasite resistance to chloroquine and sulphadoxine-pyrimethamine in the Solomon Islands by DNA microarray technology.

BACKGROUND: Little information is available on resistance to anti-malarial drugs in the Solomon Islands (SI). The analysis of single nucleotide polymorphisms (SNPs) in drug resistance associated parasite genes is a potential alternative to classical time- and resource-consuming in vivo studies to monitor drug resistance. Mutations in pfmdr1 and pfcrt were shown to indicate chloroquine (CQ) resistance, mutations in pfdhfr and pfdhps indicate sulphadoxine-pyrimethamine (SP) resistance, and mutations in pfATPase6 indicate resistance to artemisinin derivatives. METHODS: The relationship between the rate of treatment failure among 25 symptomatic Plasmodium falciparum-infected patients presenting at the clinic and the pattern of resistance-associated SNPs in P. falciparum infecting 76 asymptomatic individuals from the surrounding population was investigated. The study was conducted in the SI in 2004. Patients presenting at a local clinic with microscopically confirmed P. falciparum malaria were recruited and treated with CQ+SP. Rates of treatment failure were estimated during a 28-day follow-up period. In parallel, a DNA microarray technology was used to analyse mutations associated with CQ, SP, and artemisinin derivative resistance among samples from the asymptomatic community. Mutation and haplotype frequencies were determined, as well as the multiplicity of infection. RESULTS: The in vivo study showed an efficacy of 88% for CQ+SP to treat P. falciparum infections. DNA microarray analyses indicated a low diversity in the parasite population with one major haplotype present in 98.7% of the cases. It was composed of fixed mutations at position 86 in pfmdr1, positions 72, 75, 76, 220, 326 and 356 in pfcrt, and positions 59 and 108 in pfdhfr. No mutation was observed in pfdhps or in pfATPase6. The mean multiplicity of infection was 1.39. CONCLUSION: This work provides the first insight into drug resistance markers of P. falciparum in the SI. The obtained results indicated the presence of a very homogenous P. falciparum population circulating in the community. Although CQ+SP could still clear most infections, seven fixed mutations associated with CQ resistance and two fixed mutations related to SP resistance were observed. Whether the absence of mutations in pfATPase6 indicates the efficacy of artemisinin derivatives remains to be proven.

Predictors for the emergence of the 2 multi-nucleoside/nucleotide resistance mutations 69 insertion and Q151M and their impact on clinical outcome in the Swiss HIV cohort study.

The 69 insertion and Q151M mutations are multi-nucleoside/nucleotide resistance mutations (MNR). The prevalence among 4078 antiretroviral therapy (ART)-experienced individuals was <1.3%. Combined ART fully prevented MNR in subtype B infections. Case-control studies were performed to identify risk factors. Control subjects were patients with ≥ 3 thymidine-analogue mutations. The 69 insertion study (27 control subjects, 14 case patients) identified didanosine exposure as a risk (odds ratio, 5.0 per year; P = .019), whereas the Q151M study (which included 44 control subjects and 25 case patients) detected no associations. Following detection, individuals with Q151M tended to have lower suppression rates and higher mortality rates, relative to control subjects. Additional studies are needed to verify these findings in non-subtype B infections.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Role of Magnesium and its mitochondrial transporter MRS2 in the modulation of drug-induced apoptosis leading to multidrug resistance phenotype

Magnesium is an essential element for many biological processes crucial for cell life and proliferation. Growing evidences point out a role for this cation in the apoptotic process and in developing multi drug resistance (MDR) phenotype. The first part of this study aimed to highlight the involvement of the mitochondrial magnesium channel MRS2 in modulating drug-induced apoptosis. We generated an appropriate transgenic cellular system to regulate expression of MRS2 protein. The cells were then exposed to two different apoptotic agents commonly used in chemotherapy. The obtained results showed that cells overexpressing MRS2 channel are less responsiveness to pharmacological insults, looking more resistant to the induced apoptosis. Moreover, in normal condition, MRS2 overexpression induces higher magnesium uptake into isolated mitochondria respect to control cells correlating with an increment of total intracellular magnesium concentration. In the second part of this research we investigated whether magnesium intracellular content and compartmentalization could be used as a signature to discriminate MDR tumour cells from their sensitive counterparts. As MDR model we choose colon carcinoma cell line sensitive and resistant to doxorubicin. We exploited a standard-less approach providing a complete characterization of whole single-cells by combining X-Ray Fluorescence Microscopy , Atomic Force Microscopy and Scanning Transmission X-ray Microscopy. This method allows the quantification of the intracellular spatial distribution and total concentration of magnesium in whole dehydrated cells. The measurements, carried out in 27 single cells, revealed a different magnesium pattern for both concentration and distribution of the element in the two cellular strains. These results were then confirmed by quantifying the total amount of intracellular magnesium in a large populations of cells by using DCHQ5 probe and traditional fluorimetric technique.

The Antibiotic Resistance and Prescribing in European Children Project: A Neonatal and Pediatric Antimicrobial Web-based Point Prevalence Survey in 73 Hospitals Worldwide

BACKGROUND: The neonatal and pediatric antimicrobial point prevalence survey (PPS) of the Antibiotic Resistance and Prescribing in European Children project (http://www.arpecproject.eu/) aims to standardize a method for surveillance of antimicrobial use in children and neonates admitted to the hospital within Europe. This article describes the audit criteria used and reports overall country-specific proportions of antimicrobial use. An analytical review presents methodologies on antimicrobial use.

METHODS: A 1-day PPS on antimicrobial use in hospitalized children was organized in September 2011, using a previously validated and standardized method. The survey included all inpatient pediatric and neonatal beds and identified all children receiving an antimicrobial treatment on the day of survey. Mandatory data were age, gender, (birth) weight, underlying diagnosis, antimicrobial agent, dose and indication for treatment. Data were entered through a web-based system for data-entry and reporting, based on the WebPPS program developed for the European Surveillance of Antimicrobial Consumption project.

RESULTS: There were 2760 and 1565 pediatric versus 1154 and 589 neonatal inpatients reported among 50 European (n = 14 countries) and 23 non-European hospitals (n = 9 countries), respectively. Overall, antibiotic pediatric and neonatal use was significantly higher in non-European (43.8%; 95% confidence interval [CI]: 41.3-46.3% and 39.4%; 95% CI: 35.5-43.4%) compared with that in European hospitals (35.4; 95% CI: 33.6-37.2% and 21.8%; 95% CI: 19.4-24.2%). Proportions of antibiotic use were highest in hematology/oncology wards (61.3%; 95% CI: 56.2-66.4%) and pediatric intensive care units (55.8%; 95% CI: 50.3-61.3%).

CONCLUSIONS: An Antibiotic Resistance and Prescribing in European Children standardized web-based method for a 1-day PPS was successfully developed and conducted in 73 hospitals worldwide. It offers a simple, feasible and sustainable way of data collection that can be used globally.