Non-synonymous Point Mutations Research Articles

The Possible Influence of Non-synonymous Point Mutations within the FimA Adhesin of Non-typhoidal Salmonella (NTS) Isolates in the Process of Host Adaptation.

Non-typhoidal Salmonella (NTS) remains a global pathogen that affects a wide range of animal species. We analyzed a large number of NTS isolates of different host origins, including Salmonella Heidelberg (n = 80, avian), S. Dublin (50, bovine), S. Typhimurium var 5- (n = 40, porcine), S. 4,5,12,:i:- (n = 40, porcine), S. Cerro (n = 16, bovine), and S. Montevideo (n = 14, bovine), using virulence profiling of the bcfC, mgtC, ssaC, invE, pefA, stn, sopB, and siiE virulence-associated genes, a biofilm production assay, pulsed field gel electrophoresis, and the full-length sequencing of the fimA (adhesin) and iroN (receptor) genes. We determined a key amino acid substitution, A169 (i.e., threonine changed to alanine at position 169), in the FimA protein that changed ligand affinity of FimA toward N-acetyl-D-glucosamine. This finding clearly indicates the important role of non-synonymous single nucleotide polymorphism (nsSNPs) in adhesin functionality that may impact the host tropism of NTS. This nsSNP was found in S. Heidelberg and S. Cerro isolates. Although this was not the case for the IroN receptor, the phylogeny of this receptor and different host origins of NTS isolates were positively correlated, suggesting existence of specific host immune selective pressures on this unique receptor in S. enterica. We found that pefA, a gene encoding major fimbrial subunit, was the most-segregative virulence factor. It was associated with S. Heidelberg, S. Typhimurium var 5- and S. 4,5,12,:i:- but not with the rest of NTS strains. Further, we observed a significantly higher frequency of non-biofilm producers among NTS strains that do not carry pefA (42.5%) compared to S. Heidelberg (2.5%) and S. Typhimurium var 5- (7.5%) and S. 4,5,12,:i:- (0%). This study provides new insights into the host adaptation of avian and mammalian NTS isolates that are based on the bacterial antigens FimA and IroN as well as the interrelationships between host adaptation, overall genetic relatedness, and virulence potential in these NTS isolates.

Assessing the spectrum of germline variation in Fanconi anemia genes among patients with head and neck carcinoma before age 50.

Patients with Fanconi anemia (FA) have an increased risk for head and neck squamous cell carcinoma (HNSCC). The authors sought to determine the prevalence of undiagnosed FA and FA carriers among patients with HNSCC as well as an age cutoff for FA genetic screening. Germline DNA samples from 417 patients with HNSCC aged <50 years were screened for sequence variants by targeted next-generation sequencing of the entire length of 16 FA genes. The sequence revealed 194 FA gene variants in 185 patients (44%). The variant spectrum was comprised of 183 nonsynonymous point mutations, 9 indels, 1 large deletion, and 1 synonymous variant that was predicted to effect splicing. One hundred eight patients (26%) had at least 1 rare variant that was predicted to be damaging, and 57 (14%) had at least 1 rare variant that was predicted to be damaging and had been previously reported. Fifteen patients carried 2 rare variants or an X-linked variant in an FA gene. Overall, an age cutoff for FA screening was not identified among young patients with HNSCC, because there were no significant differences in mutation rates when patients were stratified by age, tumor site, ethnicity, smoking status, or human papillomavirus status. However, an increased burden, or mutation load, of FA gene variants was observed in carriers of the genes FA complementation group D2 (FANCD2), FANCE, and FANCL in the HNSCC patient cohort relative to the 1000 Genomes population. FA germline functional variants offer a novel area of study in HNSCC tumorigenesis. FANCE and FANCL, which are components of the core complex, are known to be responsible for the recruitment and ubiquitination, respectively, of FANCD2, a critical step in the FA DNA repair pathway. In the current cohort, the increased mutation load of FANCD2, FANCE, and FANCL variants among younger patients with HNSCC indicates the importance of the FA pathway in HNSCC. Cancer 2017;123:3943-54. © 2017 American Cancer Society.

Abstract 4838: Optimization of prostate cancer organoid culture methods to grow PDX and human biopsy tissue

Abstract A major obstacle in the field of prostate cancer research is the limited capacity to culture prostate cancer-derived cells in vitro. There are relatively few established prostate cancer cell lines in use today. Those that are available are amenable to in vitro drug assays and mechanistic studies, but do not well-represent the genetic diversity of the disease, nor do they accurately predict in vivo response. Recently, organoid culture techniques have been developed for prostate cancer by the laboratories of Hans Clevers and Charles Sawyers that have increased our ability to grow metastatic tumor-derived prostate cancer cells in vitro. This represents a great step forward, but we are still restricted by the limited availability of biopsy tissue and the relatively low success rate (~20%) in establishing new organoid lines. The LuCaP series of patient-derived xenografts (PDXs) represents a well-characterized set of prostate cancer specimens that is genetically diverse, reflective of a range of treatment histories, and available to the research community. We have modified the organoid culture technique based upon systematic comparisons and analysis for organoid growth of the LuCaP PDXs. Our modified conditions represent an improvement upon the original growth conditions. We have successfully cultured 21 out of 24 LuCaPs attempted, for at least one generation, and 16 out of 24 are able to grow long-term over several generations. This includes 3 of the newest LuCaPs (167, 170.2, and 189.3) that we have genomically characterized. Comparison of the PDX and various organoid generations showed significant conservation of genomic changes ranging from 75-90% for nonsynonymous point mutations. The modified organoid culture method immediately and significantly increases the number of prostate cancer lines that are available by making the LuCaP PDXs accessible to in vitro culture. In addition to the PDXs, our method has improved the success for culturing metastatic samples grown from biopsies. We show here that 2 out of 3 patient-derived organoid lines that we have established, grow well in the modified conditions but poorly in the original culture conditions, the third grows equally well in both conditions. Citation Format: Mike L. Beshiri, Crystal Tran, Adam G. Sowalsky, Holly M. Nguyen, Supreet Agarwal, Caitlin M. Tice, Eva Corey, Kathy Kelly. Optimization of prostate cancer organoid culture methods to grow PDX and human biopsy tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4838. doi:10.1158/1538-7445.AM2017-4838

Molecular genetics of aggressive B-cell lymphoma.

Molecular genetics of aggressive B-cell lymphoma.

Caprine and ovine Greek dairy products: The official German method generates false-positive results due to κ-casein gene polymorphism

Caseins are widely used for species identification of dairy products. Isoelectric focusing (IEF) of para-κ-casein peptide is used as the official German method for the differentiation between caprine (isoform A) and ovine (isoform B) dairy products, based on their different isoelectric points. The discrimination between Greek goat and ewe dairy products using IEF has, however, been shown to be problematic because of the existence of the ewe isoform in milk from Greek indigenous dairy goats. This could be due to nucleotide polymorphisms within the goat κ-casein gene of Greek indigenous breeds, which alter the isoelectric point of the para-κ-casein peptide and lead to false positive results. Previous DNA analysis of the goat κ-casein gene has shown high levels of polymorphism; however, no such information is available for Greek indigenous dairy goats. Therefore, 87 indigenous dairy goats were sequenced at exon IV of κ-casein gene. In total, 9 polymorphic sites were detected. Three nonsynonymous point mutations were identified, which change the isoelectric point of the goat para-κ-casein peptide so that it appears identical to that of the ewe peptide. Ten composite genotypes were reconstructed and 6 of them included the problematic point mutations. For the verification of genetic results, IEF was carried out. Both goat and ewe patterns appeared in the problematic genotypes. The frequency of these genotypes could be characterized as moderate (0.23) to high (0.60) within Greek indigenous breeds. However, this is not an issue restricted to Greece, as such genotypes have been detected in various non-Greek goat breeds. In conclusion, IEF based on the official German method is certainly inappropriate for ovine and caprine discrimination concerning Greek dairy goat products, and consequently a new method should be established.

The importance of mRNA structure in determining the pathogenicity of synonymous and non-synonymous mutations in haemophilia.

Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled insight into genotype-phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non-synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half-life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated. To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease-associated mutations in haemophilia. Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease-associated and synonymous vs. non-synonymous point mutations in F8, F9 and a third X-linked gene, DMD (dystrophin). In F8 and DMD, disease-associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101-151 nucleotide fragment length appears to be a feasible range for structuring future studies. mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease-associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications.

Retinoic Acid Receptor Alpha Expression Drives Cell-Cycle Progression and Is Associated with Increased Sensitivity to Retinoids in Peripheral T-Cell Lymphoma

Background: Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with marked clinical, pathological, and molecular heterogeneity. Outcomes following standard therapy generally are poor; however, few candidate therapeutic targets have been identified for precision medicine approaches. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to natural or synthetic retinoids. Retinoids have been used successfully to treat acute promyelocytic leukemia and some cutaneous T-cell lymphomas (CTCLs). However, the function of RARA and the action of retinoids in PTCL have not been defined.Methods:Based on identification of a PTCL patient with a non-synonymous point mutation, RARA R394Q, identified in the Mayo Clinic Center for Individualized Medicine, we sought to characterize the role of RARA in PTCL cells. To investigate the role of wild-type and mutant RARA, we constructed expression vectors containing either wild-type RARA or RARA R394Q coding sequences, and also used siRNAs targeting RARA to study the role of native RARA expression. Cell lines derived from post-thymic T-cell malignancies were used for in vitro studies, including HuT78 and Mac-1 (both derived from circulating tumor cells from CTCL patients) and Karpas 299 (from an ALK-positive anaplastic large cell lymphoma). Following RARA overexpression or knockdown, we measured cell growth, cell cycle regulation, and sensitivity to synthetic retinoids. In addition, RNA sequencing and pathway analysis were performed to profile the transcriptomic response to retinoids in malignant T cells.Results:In two RARAlow cell lines, Karpas 299 and HuT78, overexpression of wild-type RARA or RARA R394Q significantly increased cell growth (p<0.001), with a greater increase observed from mutant versus wild-type RARA in Karpas 299 (136% of control versus 122%; p=0.04). Accordingly, knockdown of wild-type RARA in the RARAhigh cell line, Mac-1, resulted in a 22% inhibition of cell growth (p=0.0002). This inhibition specifically was associated with G1 cell cycle arrest (120% of control; p=0.004) and decreased protein expression of the G1-S-associated cyclin-dependent kinases, CDK2, CDK4, and CDK6. These kinases were up-regulated by overexpression of RARA in RARAlow HuT78 cells. The relatively RARA-specific retinoid, AM80 (tamibarotene), and the less specific retinoid, all-trans retinoic acid (ATRA), resulted in RARA protein degradation, cell growth inhibition that was both dose-dependent and proportional to baseline RARA expression, G1 arrest, and CDK protein up-regulation. Gene-set enrichment analysis (GSEA) of transcriptome data confirmed that genes down-regulated by AM80 were highly enriched for regulators of cell cycle and particularly G1-S transition. Finally, overexpressing RARA in RARAlow Karpas 299 and HuT78 cell lines significantly increased the ability of AM80 to inhibit CDK2/4/6 expression and cell growth (16% to 23% greater growth inhibition than control; p<0.05).Conclusions:RARA drives cyclin-dependent kinase expression and G1-S transition in malignant T cells, and promotes cell growth. These functions may be enhanced by specific RARA gene mutations. Synthetic retinoids inhibit these functions in a dose-dependent fashion, and are most effective in cells with high RARA expression. These data suggest RARA as a candidate therapeutic target in some PTCL patients. DisclosuresNowakowski:Celgene: Research Funding; Morphosys: Research Funding; Bayer: Consultancy, Research Funding.

A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster

Spinosad, a widely used and economically important insecticide, targets the nicotinic acetylcholine receptor (nAChRs) of the insect nervous system. Several studies have associated loss of function mutations in the insect nAChR α6 subunit with resistance to spinosad, and in the process identified this particular subunit as the specific target site. More recently a single non-synonymous point mutation, that does not result in loss of function, was identified in spinosad resistant strains of three insect species that results in an amino acid substitution (G275E) of the nAChR α6 subunit. The causal role of this mutation has been called into question as, to date, functional evidence proving its involvement in resistance has been limited to the study of vertebrate receptors. Here we use the CRISPR/Cas9 gene editing platform to introduce the G275E mutation into the nAChR α6 subunit of Drosophila melanogaster. Reverse transcriptase-PCR and sequencing confirmed the presence of the mutation in Dα6 transcripts of mutant flies and verified that it does not disrupt the normal splicing of the two exons in close vicinity to the mutation site. A marked decrease in sensitivity to spinosad (66-fold) was observed in flies with the mutation compared to flies of the same genetic background minus the mutation, clearly demonstrating the functional role of this amino acid substitution in resistance to spinosad. Although the resistance levels observed are 4.7-fold lower than exhibited by a fly strain with a null mutation of Dα6, they are nevertheless predicated to be sufficient to result in resistance to spinosad at recommended field rates. Reciprocal crossings with susceptible fly strains followed by spinosad bioassays revealed G275E is inherited as an incompletely recessive trait, thus resembling the mode of inheritance described for this mutation in the western flower thrips, Frankliniella occidentalis. This study both resolves a debate on the functional significance of a target-site mutation and provides an example of how recent advances in genome editing can be harnessed to study insecticide resistance.

Clonality analysis of multifocal papillary thyroid carcinoma by using genetic profiles.

Papillary thyroid carcinoma (PTC) is the most common adult thyroid malignancy and often presents with multiple anatomically distinct foci within the thyroid, known as multifocal papillary thyroid carcinoma (MPTC). The widespread application of the next-generation sequencing technologies in cancer genomics research provides novel insights into determining the clonal relationship between multiple tumours within the same thyroid gland. For eight MPTC patients, we performed whole-exome sequencing and targeted region sequencing to identify the non-synonymous point mutations and gene rearrangements of distinct and spatially separated tumour foci. Among these eight MPTCs, completely discordant mutational spectra were observed in the distinct cancerous nodules of patients MPTC1 and 5, suggesting that these nodules originated from independent precursors. In another three cases (MPTC2, 6, and 8), the distinct MPTC foci of these patients had no other shared mutations except BRAF V600E, also indicating likely independent origins. Two patients (MPTC3 and 4) shared almost identical mutational spectra amongst their separate tumour nodules, suggesting a common clonal origin. MPTC patient 7 had seven cancer foci, of which two foci shared 66.7% of mutations, while the remaining cancer foci displayed no common non-synonymous mutations, indicating that MPTC7 has multiple independent origins accompanied by intraglandular disease dissemination. In this study, we found that 75% of MPTC cases arose as independent tumours, which supports the field cancerization hypothesis describing multiple malignant lesions. MPTC may also arise from intrathyroidal metastases from a single malignant clone, as well as multiple independent origins accompanied by intrathyroidal metastasis.

Molecular Basis for Resistance to Fluazifop-P-Butyl in Itchgrass (Rottboellia cochinchinensis) from Costa Rica

Rottboellia cochinchinensis is an annual grass weed species known as itchgrass, or "caminadora" in America´s Spanish speaking countries, and has become a major and troublesome weed in several crops. The application of fluazifop-P-butyl at recommended rates (125 g a.i. ha-1) was observed to be failing to control itchgrass in a field in San José, Upala county, Alajuela province, Costa Rica. Plants from the putative resistant R. cochinchinensis population survived fluazifop-P-butyl when treated with 250 g a.i. ha-1 (2X label rate) at the three- to four-leaf stage under greenhouse conditions. PCR amplification and sequencing of partial carboxyl transferase domain (CT) of the acetyl-CoA carboxylase (ACCase) gene were used to determine the molecular mechanism of resistance. A single non-synonymous point mutation from TGG (susceptible plants) to TGC (putative resistant plants) that leads to a Trp-2027-Cys substitution was found. This Trp-2027-Cys mutation is known to confer resistance to all aryloxyphenoxyproprionate (APP) herbicides to which fluazifop-P-butyl belongs. To the best of our knowledge, this is the first report of fluazifop-P-butyl resistance and a mutation at position 2027 for a Costa Rican R. cochinchinensis population.

Targeted next-generation sequencing of locally advanced squamous cell carcinomas of the head and neck reveals druggable targets for improving adjuvant chemoradiation

BackgroundDespite clear differences in clinical presentation and outcome, squamous cell carcinomas of the head and neck (SCCHN) arising from human papilloma virus (HPV) infection or heavy tobacco/alcohol consumption are treated equally. Next-generation sequencing is expected to reveal novel targets for more individualised treatment. Patients and methodsTumour specimens from 208 patients with locally advanced squamous cell carcinoma of the hypopharynx, oropharynx or oral cavity, all uniformly treated with adjuvant cisplatin-based chemoradiation, were included. A customised panel covering 211 exons from 45 genes frequently altered in SCCHN was used for detection of non-synonymous point and frameshift mutations. Mutations were correlated with HPV status and treatment outcome. ResultsMutational profiles and HPV status were successfully established for 179 cases. HPV– tumours showed an increased frequency of alterations in tumour suppressor genes compared to HPV+ cases (TP53 67% versus 4%, CDKN2A 18% versus 0%). Conversely, HPV+ carcinomas were enriched for activating mutations in driver genes compared to HPV– cases (PIK3CA 30% versus 12%, KRAS 6% versus 1%, and NRAS 4% versus 0%). Hotspot TP53 missense mutations in HPV– carcinomas correlated with an increased risk of locoregional recurrence (hazard ratio [HR] 4.3, 95% confidence interval [CI] 1.5–12.1, P=0.006) and death (HR 2.2, 95% CI 1.1–4.4, P=0.021). In HPV+ SCCHN, driver gene mutations were associated per trend with a higher risk of death (HR 3.9, 95% CI 0.7–21.1, P=0.11). ConclusionsDistinct mutation profiles in HPV– and HPV+ SCCHN identify subgroups with poor outcome after adjuvant chemoradiation. Mutant p53 and the phosphoinositide 3-kinase pathway were identified as potential druggable targets for subgroup-specific treatment optimisation.

Multiple Mutations on the Second Acetylcholinesterase Gene Associated With Dimethoate Resistance in the Melon Aphid, Aphis gossypii (Hemiptera: Aphididae).

The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is an important cosmopolitan and extremely polyphagous species capable of causing direct and indirect damage to various crops. Insecticide resistance in melon aphids is of particular concern. To determine the basis of resistance, organophosphate (OP)-resistant strains of A. gossypii were obtained by continuous selection with dimethoate in the laboratory, and resistance mechanisms were investigated along with susceptible strains. Three resistant strains LKR-1, LKR-2, and LKR-3 exhibiting 270-, 243-, and 210-fold resistance obtained after 30 generations of selection with dimethoate, respectively, were utilized in this study. The role of acetylcholinesterase (AChE), a target enzyme for OPs and carbamates (CMs), was investigated. AChE enzyme assay revealed that there was no significant change in the activities of AChE in resistant and susceptible strains. However, AChE inhibitory assay showed that 50% of the enzyme activity in resistant strains was inhibited at significantly higher concentration of dimethoate (131.87, 158.65, and 99.29 µmolL(−1)) as compared with susceptible strains (1.75 and 2.01 µmolL(−1)), indicating AChE insensitivity owing to altered AChE. Molecular diagnostic tool polymerase chain reaction-restriction fragment length polymorphism revealed the existence of two consistent non-synonymous point mutations, single-nucleotide polymorphism, viz., A302S (equivalent to A201 in Torpedo californica Ayres) and S431F (equivalent to F331 in T. californica), in the AChE gene Ace2 of resistant strains. Further, cloning and sequencing of a partial fragment of Ace2 (897 bp) gene from susceptible and resistant strains revealed an additional novel mutation G221A in resistant strains, LKR-1 and LKR-2. Susceptible Ace2 genes shared 99.6 and 98.9% identity at the nucleic acid and amino acid levels with resistant ones, respectively. Functional analysis of these point mutations was assessed by in silico docking studies using the modeled wild-type and naturally mutated AChE2. Computational analysis showed that the conformational changes in AChE2 active site due to structural gene substitutions (A302S, S431F, and G221A) significantly reduced the level of ligand (OP-dimethoate, omethoate, and CM-pirimicarb) binding, suggesting that they are potentially associated with resistance development. These results unambiguously suggested that multiple mutations located in the enzyme active site are responsible for AChE insensitivity to dimethoate and are likely the molecular basis for dimethoate resistance in these selected field populations of A. gossypii.

Abstract 05: Genetic heterogeneity in rectal cancer - Identification of subpopulations of tumor cells resistant to neoadjuvant CRT

Abstract One of the benefits of neoadjuvant chemotherapy and radiation therapy (CRT) for the management of rectal cancer is tumor downstaging that can ultimately lead to complete tumor regression (known as complete pathological response - pCR). In a previous study using PET/CT imaging to assess tumor response to CRT, 50% of rectal cancers showed a continuous decrease in metabolic activity (estimated by standard uptake value measurements) between 6 and 12 weeks from CRT completion. However the remaining 50% of the cases showed increased metabolic activity within that period. We reasoned that the increased metabolic activity observed in these later patients could be determined by the clonal expansion of a genetically distinct subpopulation of tumor cells resistant to CRT. To address this question we performed exome sequencing and mutation detection analysis on normal tissue, primary tumor and residual tumor from 7 patients with rectal cancer that exhibited an increase in metabolic activity after CRT. Overall, about 20Gb of unambiguously mapped sequences were generated for each sample resulting in an average fold-coverage of 30X. Captured sequences mapped to the reference human genome were then used for the detection of SNPs and non-synonymous somatic point mutations in all three samples from each patient. Approximately 30,000 single nucleotide variants (SNVs) were identified in each sample and most of these SNVs were common to all samples. As expected, the majority of these common inherited variants (96%) have already been described in dbSNP. To identify non-synonymous somatic mutations occurring in primary and residual tumor samples, we excluded those present in the normal sample and those already described as a known SNP in dbSNP resulting in a mean of 243 SNVs per patient. Noteworthy, a significant number of non-synonymous somatic mutations were exclusively found in the primary and residual tumor samples of each patient, 30 and 32 SNVs on average respectively, revealing a high degree of tumor genetic heterogeneity. Furthermore, we were able to identify non-synonymous somatic mutations that were presented in both samples (mean of 107 SNVs), and for these mutations we determined the mutant allele frequency (number of reads representing the mutation/total number of reads covering the mutated base) in the primary and the residual tumor. We then searched for mutations with significantly different allele frequencies in the two samples, as these mutations would likely represent genetically distinct subpopulations of tumor cells selected during CRT. We were able to identify, on average, 4 SNVs enriched in primary tumor and 37 SNVs enriched in residual tumor per patient. In conclusion, based on exome sequencing of rectal tumors that exhibit incomplete response to CRT and have increased tumor metabolism we were able to identify non-synonymous mutations that may be associated with specific mechanisms of resistance or sensitivity to CRT. Functional analysis of these mutations and mutated genes will be presented, providing new insights into the molecular events influencing response to neoadjuvant CRT in rectal cancer. Citation Format: Fabiana Bettoni, Elisa R. Donnard, Bruna R. S. Correa, Paula F. Asprino, Fernanda C. Koyama, Natália M. Felício, Bruna H. Hessel, Pedro A. F. Galante, Anamaria A. Camargo, Angelita Habr-Gama, Rodrigo O. Perez. Genetic heterogeneity in rectal cancer - Identification of subpopulations of tumor cells resistant to neoadjuvant CRT. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 05.

Genomic Correlates to the Newly Proposed Grading Prognostic Groups for Prostate Cancer

Recommendations by the International Society of Urologic Pathology and 2016 World Health Organization blue book propose the use of a five-tiered prostate cancer (PCa) grading system. The five prognostic grade groupings (PGGs) ranging from 1 to 5 are defined as Gleason grades ≤6, 3+4, 4+3, 8, and >8, respectively. Recent work suggests that each group is associated with a distinct risk of biochemical PCa recurrence. In this study, we sought genomic support for PGGs using whole-exome and whole-genome sequencing data for 426 clinically localized PCas treated by radical prostatectomy. After adjustment for tumor purity for the sequencing data, we observed a significant frequency increase in genomic amplifications and deletions (p=0.013) and in nonsynonymous point mutations (p=0.008) with increasing risk group. Interestingly, PGG1 (low risk) was entirely haploid, whereas PGG2–5 exhibited increasing polyploidy frequency. Principal component analysis of genomic profiles revealed that PGG1, PGG2, and PGG3 represent distinct classes, but PGG4 and PGG5 exhibit genomic similarity. Together, these observations for the largest PCa genomic data set to date provide support for increasing genomic alterations with increasing PGG. This is the first genomic correlation of the PGG system. Future work will need to explore the clinical utility of PGGs in prospective studies with long-term follow-up. Patient summaryGleason grading for prostate cancer provides important information for guiding clinical care. A new proposal by leading pathologists favors translating Gleason grades into five risk categories. In this study, a comprehensive analysis of the largest genomic data set on prostate cancer to date, we demonstrate molecular support for this new five-tiered system.

Phenotypic- and Genotypic-Resistance Detection for Adaptive Resistance Management in Tetranychus urticae Koch.

Rapid resistance detection is necessary for the adaptive management of acaricide-resistant populations of Tetranychus urticae. Detection of phenotypic and genotypic resistance was conducted by employing residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods, respectively. RCV was useful for detecting the acaricide resistance levels of T. urticae, particularly for on-site resistance detection; however, it was only applicable for rapid-acting acaricides (12 out of 19 tested acaricides). QS was effective for determining the frequencies of resistance alleles on a population basis, which corresponded to 12 nonsynonymous point mutations associated with target-site resistance to five types of acaricides [organophosphates (monocrotophos, pirimiphos-methyl, dimethoate and chlorpyrifos), pyrethroids (fenpropathrin and bifenthrin), abamectin, bifenazate and etoxazole]. Most field-collected mites exhibited high levels of multiple resistance, as determined by RCV and QS data, suggesting the seriousness of their current acaricide resistance status in rose cultivation areas in Korea. The correlation analyses revealed moderate to high levels of positive relationships between the resistance allele frequencies and the actual resistance levels in only five of the acaricides evaluated, which limits the general application of allele frequency as a direct indicator for estimating actual resistance levels. Nevertheless, the resistance allele frequency data alone allowed for the evaluation of the genetic resistance potential and background of test mite populations. The combined use of RCV and QS provides basic information on resistance levels, which is essential for choosing appropriate acaricides for the management of resistant T. urticae.

TumorTracer: a method to identify the tissue of origin from the somatic mutations of a tumor specimen

BackgroundA substantial proportion of cancer cases present with a metastatic tumor and require further testing to determine the primary site; many of these are never fully diagnosed and remain cancer of unknown primary origin (CUP). It has been previously demonstrated that the somatic point mutations detected in a tumor can be used to identify its site of origin with limited accuracy. We hypothesized that higher accuracy could be achieved by a classification algorithm based on the following feature sets: 1) the number of nonsynonymous point mutations in a set of 232 specific cancer-associated genes, 2) frequencies of the 96 classes of single-nucleotide substitution determined by the flanking bases, and 3) copy number profiles, if available.MethodsWe used publicly available somatic mutation data from the COSMIC database to train random forest classifiers to distinguish among those tissues of origin for which sufficient data was available. We selected feature sets using cross-validation and then derived two final classifiers (with or without copy number profiles) using 80 % of the available tumors. We evaluated the accuracy using the remaining 20 %. For further validation, we assessed accuracy of the without-copy-number classifier on three independent data sets: 1669 newly available public tumors of various types, a cohort of 91 breast metastases, and a set of 24 specimens from 9 lung cancer patients subjected to multiregion sequencing.ResultsThe cross-validation accuracy was highest when all three types of information were used. On the left-out COSMIC data not used for training, we achieved a classification accuracy of 85 % across 6 primary sites (with copy numbers), and 69 % across 10 primary sites (without copy numbers). Importantly, a derived confidence score could distinguish tumors that could be identified with 95 % accuracy (32 %/75 % of tumors with/without copy numbers) from those that were less certain. Accuracy in the independent data sets was 46 %, 53 % and 89 % respectively, similar to the accuracy expected from the training data.ConclusionsIdentification of primary site from point mutation and/or copy number data may be accurate enough to aid clinical diagnosis of cancers of unknown primary origin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0130-0) contains supplementary material, which is available to authorized users.

Evolutionary Transition from Pathogenicity to Commensalism: Global Regulator Mutations Mediate Fitness Gains through Virulence Attenuation.

Symbiotic interactions are indispensable for metazoan function, but their origin and evolution remain elusive. We use a controlled evolution experiment to demonstrate the emergence of novel commensal interactions between Pseudomonas aeruginosa, an initially pathogenic bacterium, and a metazoan host, Caenorhabditis elegans. We show that commensalism evolves through loss of virulence, because it provides bacteria with a double fitness advantage: Increased within-host fitness and a larger host population to infect. Commensalism arises irrespective of host immune status, as the adaptive path in immunocompromised C. elegans knockouts does not differ from that in wild type. Dissection of temporal dynamics of genomic adaptation for 125 bacterial populations reveals highly parallel evolution of incipient commensalism across independent biological replicates. Adaptation is mainly achieved through frame shift mutations in the global regulator lasR and nonsynonymous point mutations in the polymerase gene rpoB that arise early in evolution. Genetic knockouts of lasR not only corroborate its role in virulence attenuation but also show that further mutations are necessary for the fully commensal phenotype. The evolutionary transition from pathogenicity to commensalism as we observe here is facilitated by mutations in global regulators such as lasR, because few genetic changes cause pleiotropic effects across the genome with large phenotypic effects. Finally, we found that nucleotide diversity increased more quickly in bacteria adapting to immunocompromised hosts than in those adapting to immunocompetent hosts. Nevertheless, the outcome of evolution was comparable across host types. Commensalism can thus evolve independently of host immune state solely as a side-effect of bacterial adaptation to novel hosts.

Prevalence and mechanism of triazole resistance in Aspergillus fumigatus in a referral chest hospital in Delhi, India and an update of the situation in Asia.

Aspergillus fumigatus causes varied clinical syndromes ranging from colonization to deep infections. The mainstay of therapy of Aspergillus diseases is triazoles but several studies globally highlighted variable prevalence of triazole resistance, which hampers the management of aspergillosis. We studied the prevalence of resistance in clinical A. fumigatus isolates during 4 years in a referral Chest Hospital in Delhi, India and reviewed the scenario in Asia and the Middle East. Aspergillus species (n = 2117) were screened with selective plates for azole resistance. The isolates included 45.4% A. flavus, followed by 32.4% A. fumigatus, 15.6% Aspergillus species and 6.6% A. terreus. Azole resistance was found in only 12 (1.7%) A. fumigatus isolates. These triazole resistant A. fumigatus (TRAF) isolates were subjected to (a) calmodulin and β tubulin gene sequencing (b) in vitro antifungal susceptibility testing against triazoles using CLSI M38-A2 (c) sequencing of cyp51A gene and real-time PCR assay for detection of mutations and (d) microsatellite typing of the resistant isolates. TRAF harbored TR34/L98H mutation in 10 (83.3%) isolates with a pan-azole resistant phenotype. Among the remaining two TRAF isolates, one had G54E and the other had three non-synonymous point mutations. The majority of patients were diagnosed as invasive aspergillosis followed by allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. The Indian TR34/L98H isolates had a unique genotype and were distinct from the Chinese, Middle East, and European TR34/L98H strains. This resistance mechanism has been linked to the use of fungicide azoles in agricultural practices in Europe as it has been mainly reported from azole naïve patients. Reports published from Asia demonstrate the same environmental resistance mechanism in A. fumigatus isolates from two highly populated countries in Asia, i.e., China and India and also from the neighboring Middle East.

Babesia divergens apical membrane antigen-1 (BdAMA-1): A poorly polymorphic protein that induces a weak and late immune response

Babesiosis is an important veterinary and zoonotic tick borne disease caused by the hemoprotozoan Babesia spp. which infects red blood cell of its vertebrate host. In order to control the infection, vaccination that targets molecules involved in the invasion process of red blood cells could provide a good alternative to chemotherapy. Among these molecules, Apical Membrane Antigen-1 (AMA-1) has been described as an excellent vaccine candidate in Plasmodium spp. In this paper, we have investigated AMA-1 of Babesia divergens (BdAMA-1) as vaccine candidate by evaluating its polymorphism and by studying the humoral response against BdAMA-1 of sheep experimentally infected with B. divergens. Polymorphism of BdAMA-1 was investigated by sequencing the corresponding gene of 9 B. divergens isolates from different geographical areas in France. Two Bdama-1 haplotypes (A and B) could be defined based on 2 non-synonymous point mutations. In silico prediction of linear epitopes revealed that the antigenicity of the 2 haplotypes is very similar. Antibody production against the extracellular domain of BdAMA-1 is weak and late, between 1 and 5 months after the inoculation of parasites. Both IgG1 and IgG2 are components of the anti-BdAMA-1 response. These results indicate that while BdAMA-1 may not be an immuno-dominant antigen, it could induce a mixed type 1 and type 2 immune response. In light of these results, the potential of BdAMA-1 as vaccine candidate is discussed.

Abstract P2-03-08: Mutational landscape of metaplastic breast carcinomas

Abstract Introduction: Metaplastic breast carcinoma (MBC) is an aggressive histologic type of breast cancer, which preferentially displays a triple-negative phenotype. These tumors are characterized by the presence of malignant cells exhibiting differentiation towards squamous epithelium or mesenchymal elements, including spindle, chondroid, osseous and rhabdoid differentiation. Unlike other rare histologic types of breast cancer such as adenoid cystic and secretory carcinomas, which are underpinned by the MYB-NFIB and ETV6-NTRK3 fusion genes respectively, pathognomonic genetic alterations have not been identified in MBC. It has been suggested, however, that the frequency of PIK3CA somatic mutations would be significantly higher in MBCs than in other forms of triple-negative disease. Here we sought to characterize the mutational landscape of MBCs by means of high-depth whole exome sequencing analysis. Material and Methods: Twenty-one triple-negative MBCs were retrieved from the authors’ institutions. Representative sections from frozen blocks were microdissected to ensure tumor cell content greater than 50%. DNA samples extracted from microdissected tumor and matched peripheral blood leukocytes were subjected to high-depth (250x) whole exome sequencing on an Illumina GAIIx or HiSeq2000. Somatic point mutations were called using MuTect and somatic insertions and deletions (indels) were called using Strelka, Varscan2 and Haplotype Caller. Potentially pathogenic mutations were predicted using computational algorithms including PolyPhen-2, Mutation Taster, Mutation Assessor, CHASM and FATHMM. Significantly mutated genes were identified using MutSigCV. Pathway and network enrichment analysis of mutations was performed with Ingenuity Pathway Analysis and HOTNET. The genomic landscape of MBCs was compared with that of triple-negative breast cancers (TNBCs) analyzed as part of The Cancer Genome Atlas project. Results: A mean of 135 somatic non-synonymous point mutations and indels were identified per MBC. The most frequently mutated gene was TP53, found in 12/21 cases (57%), and the only significantly mutated gene as defined by MutSigCV (q&amp;lt;0.01). The repertoire of somatic mutations found in MBCs was qualitatively similar to that of TNBCs of no special type, and recurrently mutated genes were altered at similar frequencies in MBCs and TNBCs of no special type. When somatic mutations were annotated in pathways and networks, MBCs were found to have potentially pathogenic mutations affecting genes directly related to the PI3K pathway, including pathogenic non-synonymous mutations affecting PIK3CA, PIK3R1, PIK3R2, PIK3C2B, PIK3C2G and PTEN, significantly more frequently than TNBCs of no special type (10 out of 21 MBCs vs. 11 out of 62 TNBCs; Fisher's exact test p-value=0.0099). Conclusion: The majority (57%) of MBCs harbored non-synonymous mutations affecting TP53. While the frequencies of mutations affecting recurrently mutated genes in MBCs are similar to those found in other forms of TNBCs, MBCs significantly more frequently harbor mutations affecting PI3K pathway-related genes than TNBCs of no special type. Citation Format: Charlotte KY Ng, Britta Weigelt, Salvatore Piscuoglio, Y Hannah Wen, Maria R De Filippo, Luciano G Martelotto, Rachael Natrajan, Raymond Lim, Edi Brogi, Larry Norton, Anne Vincent-Salomon, Jorge S Reis-Filho. Mutational landscape of metaplastic breast carcinomas [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-08.