Minor Mutations Research Articles

Characterization of the dynamics of human cytomegalovirus resistance to antiviral drugs by ultra-deep sequencing

Prophylactic or preemptive treatment strategies are required to prevent human cytomegalovirus (CMV) infections in transplant recipients. However, treatment failure occurs when CMV resistant-associated variants (RAVs) are selected. Although the diversity of CMV is lower than that of RNA viruses, CMV appears to show some genetic instability, with possible minor emerging resistance that may be undetectable by Sanger sequencing. We aimed to examine CMV-resistance mutations over time by ultra-deep sequencing (UDS) and Sanger sequencing in a kidney transplant recipient experiencing CMV infection. This patient showed a transient response to three different antiviral drugs (valganciclovir, foscarnet, and maribavir) and four episodes of CMV resistance over two years. The full-length UL97 (2.3kpb) and partial UL54 (2.4kpb) CMV genes were studied by UDS and Sanger sequencing and linkage mutations calculated to determine RAVs. We detected four major and five minor resistance mutations. Minor resistant variants (2–20%) were detected by UDS, whereas major resistance substitutions (>20%) were identified by both UDS and Sanger method. We detected cross-resistance to three drugs, despite high CMV loads, suggesting that the fitness of the viral mutants was not impaired. In conclusion, CMV showed complex dynamic of resistance under antiviral drug pressure, as described for highly variable viruses. The emergence of successive RAVs constitutes a clinically challenging complication and contributes to the difficulty of therapeutic management of patients.

Routine drug resistance testing in HIV-1 proviral DNA, using an automated next- generation sequencing assay

BackgroundHIV-1 DNA genotypic drug resistance testing is increasingly performed to guide treatment switching or simplification in controlled patients. The Sentosa NGS platform is a fully automated system marketed for drug resistance testing on HIV-1 RNA samples. ObjectivesThe aim of this study was to evaluate this automated NGS solution for routine resistance genotypic resistance testing in proviral HIV-1 DNA. Study designSanger sequencing (SS) of the reverse transcriptase (RT), protease (PR) and integrase (IN) genes was performed using the French ANRS protocol. NGS was performed retrospectively on frozen samples, using the Sentosa platform combined with the Sentosa SQ HIV genotyping Assay. ResultsA total of 77 samples were run once using NGS. A successful sequencing of the three HIV-1 genes (RT, PR, IN) was obtained for 45 samples. The number of cumulated RAMs was 179, 185 and 219 with SS, NGS 20% and NGS 10% respectively; however most of them were minor mutations in the PR region. The mutation detection rate was similar between SS and NGS 20%. Several discordances were observed between both methods in the RT and PR regions, mainly due to the use of different DNA extracts, and hypermutation. ConclusionsHIV-1 DNA genotypic resistance testing can be performed with the Sentosa platform. Few technical optimizations are still needed to include the extraction step and to improve the sequencing efficiency.

486P - Phase II study of low-dose afatinib maintenance treatment for patients with EGFR-mutated non-small cell lung cancer (NJLCG1601)

BackgroundAfatinib is a 2nd generation EGFR-TKI and has been a standard first line treatment for patients (pts) with advanced NSCLC with EGFR mutations. Afatinib has good efficiency, whereas afatinib has severe adverse events (AEs) and often lead to discontinuation of treatment or a dose reduction. NJLCG1601 was conducted to evaluate the efficacy of low-dose afatinib maintenance treatment. MethodsEGFR-TKI-naïve stage III / IV / recurrent NSCLC pts harboring EGFR mutations (exon 19 deletion or exon 21 L858R or minor mutation) were enrolled. Pts received afatinib 40mg orally once a day. When prescribed AEs (grade ≧ 2 in CACAE ver 4.0) or unacceptable AEs occurred, a dose of afatinib was reduced from 40mg to 30mg, 30mg to 20mg. The treatment was continued until disease progression, occurrence of intolerable severe AEs, or withdrawal of consent. The primary endpoint was 1-year progression free survival (PFS) rate. Secondary endpoints were PFS, overall response rate (ORR), the incidence of AEs, and the incidence of grade ≧ 3 AEs. Assuming a threshold 1-year PFS rate of 42% and an expected 1-year PFS rate of 63%, a total of 26 pts was required to have 90% power at a two-tailed alpha of 0.05. This is a subset analysis of patients with adenocarcinoma in NJLCG1601 study (Trial Identifier, UMIN000020688). ResultsBetween Apr 2016 and Sep 2017, 30 pts were enrolled. Among 30 patients, 28 patients (93%) were adenocarcinoma. Pts characteristics were as follows: median ages, 70 years (range 46-79 years); proportion of males, 43%; clinical stage, IV 61% and post-op 39%; EGFR mutation types, exon 19 deletion 54%, ex21 L858R 36%, and minor mutations 11%. 1-year PFS rate was 53.6% (95% CI, 33.8-69.8), median PFS was 13.0 months, and ORR was 78.6%. Dose reductions occurred in 86% (24/28). Incidence of all cause grade ≧ 3 was 60% which is smaller than the frequency of Japanese subset in LUX-Lung 3. ILD was occurred in 3 patients and 1 patients died. Treatment-related deaths were observed in 2 pts. ConclusionsLow-dose afatinib maintenance treatment reduced treatment-related adverse events without detracting from the therapeutic efficacy. Legal entity responsible for the studyUMIN000020688. FundingHas not received any funding. DisclosureA. Nakamura: Honoraria (self): MSD; Honoraria (self): Chugai; Honoraria (self): AstraZeneca. S. Inoue: Honoraria (self): AstraZeneca; Honoraria (self): Eli Lilly; Honoraria (self): MSD; Honoraria (self): Otsuka Pharmaceutical; Honoraria (self), research funding: Kyorin Pharmaceutical; Honoraria (self): Ono Pharmaceutical; Honoraria (self): GlaxoSmithKline; Honoraria (self): Daiichi Sankyo Company; Honoraria (self): Taiho Pharmaceutical; Honoraria (self): Chugai Pharmaceutical; Honoraria (self): Teijin Pharma; Honoraria (self), research funding: Novartis Pharma; Honoraria (self): Pfizer Inc; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Meiji Seika Pharma. S. Sugawara: Honoraria (self): AstraZeneca; Honoraria (self): Chugai Pharma; Honoraria (self): Nippon Boehringer Ingelheim; Honoraria (self): Taiho Pharmaceutical; Honoraria (self): Pfizer; Honoraria (self): Eli Lilly; Honoraria (self): Novartis; Honoraria (self): Kyowa Hakko Kirin; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Ono Pharmaceutical; Honoraria (self): MSD. All other authors have declared no conflicts of interest.

Accessory mutations balance the marginal stability of the HIV-1 protease in drug resistance.

The HIV-1 protease is a major target of inhibitor drugs in AIDS therapies. The therapies are impaired by mutations of the HIV-1 protease that can lead to resistance to protease inhibitors. These mutations are classified into major mutations, which usually occur first and clearly reduce the susceptibility to protease inhibitors, and minor, accessory mutations that occur later and individually do not substantially affect the susceptibility to inhibitors. Major mutations are predominantly located in the active site of the HIV-1 protease and can directly interfere with inhibitor binding. Minor mutations, in contrast, are typically located distal to the active site. A central question is how these distal mutations contribute to resistance development. In this article, we present a systematic computational investigation of stability changes caused by major and minor mutations of the HIV-1 protease. As most small single-domain proteins, the HIV-1 protease is only marginally stable. Mutations that destabilize the folded, active state of the protease therefore can shift the conformational equilibrium towards the unfolded, inactive state. We find that the most frequent major mutations destabilize the HIV-1 protease, whereas roughly half of the frequent minor mutations are stabilizing. An analysis of protease sequences from patients in treatment indicates that the stabilizing minor mutations are frequently correlated with destabilizing major mutations, and that highly resistant HIV-1 proteases exhibit significant fractions of stabilizing mutations. Our results thus indicate a central role of minor mutations in balancing the marginal stability of the protease against the destabilization induced by the most frequent major mutations.

P1.09-03 Clinicopathological Characteristics for NSCLC Harboring EGFR Exon 20 Insertion

For the majority of EGFR-mutant NSCLC patients harboring exon 19 deletion or exon 21 L858R, EGFR-TKIs are key therapies for outstanding anti-tumor effects. However, for the population harboring minor mutations, clinical benefit of EGFR-TKIs are controversial. Specifically, exon 20 insertion is acknowledged as a poor prognostic mutation type compared to other minor mutations. There are few reports describing clinicopathological characteristics harboring exon 20 insertion NCSLC. We retrospectively analyzed patients harboring EGFR exon 20 insertion at our institution over 3 years. Clinical testing for the detection of EGFR mutations was cobas® EGFR Mutation Test v2. We evaluated pathological features for diagnostic specimens or re-biopsy samples, CT or PET images for the detection of primary lesions or metastatic locations. A total of 213 EGFR-mutant adenocarcinomas were reviewed for the study and screened. Of these, 19 were positive for exon 20 insertion (8.9%). Of 19 cases, 13 displayed advanced stage while 6 cases were classified as early stage. Compound major mutation was detected in 26.3% (3 cases were exon 19 deletion; 2 cases were exon 21 L858R). In advanced cases, the location of primary lesions was found mainly in the subpleural domain (84.6%), and pleural dissemination was confirmed in 77%. The majority of cases included pleural effusion. Furthermore, 70% contained mucinous type pathologically in advanced cases. In total, more than half of the cases showed well-differentiated subtypes pathologically, which was contrary to a past report1). Contrary to a past report, EGFR exon 20 insertion in adenocarcinoma often showed pathologically mucinous and well-differentiated subtypes. For clinical characteristics, especially in advanced stage, pleural dissemination and effusion were observed with high frequency, which might be due to the primary lesion often located in the subpleural domain. 1) Mol Cancer Ther; 2012(2); 220–9

In vivo passage of Salmonella Typhimurium results in minor mutations in the bacterial genome and increases in vitro invasiveness

Eggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of Salmonella outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used Salmonella typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the Salmonella genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either Salmonella Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of Salmonella Mbandaka. Salmonella shedding in both single and co-infected birds was variable over the course of the 16-week experiment. Salmonella Typhimurium and Salmonella Mbandaka were identified in feces of co-infected birds. Salmonella colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in Salmonella Typhimurium strains isolated from co-infected birds. Isolates of Salmonella Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance.

Transmitted drug resistance mutations and trends of HIV-1 subtypes in treatment-naïve patients: A single-centre experience.

Transmitted drug resistance (TDR) and HIV-1 genetic diversity may affect treatment efficacy and clinical outcomes. Here we describe the circulating viral subtypes and estimate the prevalence of drug resistance among antiretroviral therapy (ART)-naïve patients attending Sapienza University Hospital (Rome, Italy) from 2006-2017. Genotypic resistance testing (GRT) was performed on 668 ART-naïve patients for integrase (n = 52), protease and reverse transcriptase (n = 668) sequences. Twenty-one different HIV-1 subtypes and circulating recombinant forms (CRFs) were identified. Subtype B was the most common (67.1%), followed by CRF02_AG (8.4%), and subtypes C and F (both 6.0%). A significantly increase in the proportion of non-B strains (P < 0.001) and the rate of non-Italian patients was observed over time. The overall prevalence of TDR was 9.4% (NRTI, 4.2%; NNRTI, 5.8%; and PI, 1.0%) and was higher in subtype B strains. Transmitted INSTI mutations (Q148H and G140S) responsible for high-level resistance to raltegravir and elvitegravir and intermediate resistance to dolutegravir and bictegravir were found, for the first time, in two individuals. Minor or accessory INSTI mutations were detected in 17.3% of patients. No significant decrease in the prevalence of TDR was documented over time. The significant increase in non-B subtypes suggests that the molecular epidemiology of HIV-1 is changing. Detection of a major INSTI mutation in two ART-naïve patients highlights the importance of performing GRT before commencing treatment. This finding and the lack of a significant reduction in TDRs underline the importance of continuous surveillance of resistance mutations.

Antiviral activity of HIV-1 integrase strand-transfer inhibitors against mutants with integrase resistance-associated mutations and their frequency in treatment-naïve individuals.

The development of resistance to human immunodeficiency virus 1 (HIV-1) integrase strand-transfer inhibitors (INSTI) has been documented; however, knowledge of the impact of pre-existing integrase (IN) mutations on INSTI resistance (INSTI-R) is still evolving. The frequency of HIV-1 IN mutations in 2177 treatment-naïve subjects was investigated, along with the INSTI susceptibility of site-directed mutant viruses containing major and minor INSTI-R mutations. Total 6 of 39 minor INSTI-R mutations (M50I, S119P/G/T/R, and E157Q) were found in >1% of IN-treatment-naïve subjects with no impact on INSTI susceptibility. When each combined with major INSTI-R mutation, M50I, S119P, and E157Q led to decreased susceptibility to elvitegravir but remained sensitive to dolutegravir and bictegravir.

The impact of SOCS1 mutations in diffuse large B-cell lymphoma.

SummaryMutations in SOCS1 are frequent in primary mediastinal B‐cell lymphoma and classical Hodgkin lymphoma. In the latter, SOCS1 mutations affect the length of the encoded protein (major mutations) and are associated with shorter patient survival. Two independent studies examined the prognostic impact of SOCS1 mutations in diffuse large B‐cell lymphoma (DLBCL) and showed differing results. This may be due to the small number of included patients, the heterogeneity of patients’ demographics and the distinct treatment schemes in these studies. To overcome the size limitations of these previous studies, we assessed SOCS1 mutations in the RICOVER‐60 cohort. The cohort uniformly consists of elderly patients (aged 61–80 years) treated with the CHOP‐14 scheme (cyclophosphamide, hydroxydaunorubicin, vincristine, prednisolone at 14‐day intervals) with or without an additional rituximab treatment. Patient outcomes were analysed with regard to overall SOCS1 mutation frequency, major and minor mutations and a novel impact‐based classifier – against the treatment modalities. Patients harbouring putative pathogenic SOCS1 mutations showed significant reduced overall survival within the CHOP plus rituximab group. Hence, putative pathogenic SOCS1 mutations seem to efface the beneficial effect of the therapeutic CD20 antibody. Comparing published data of whole exome and transcriptome sequencing of a large DLBCL cohort confirmed that predicted deleterious SOCS1 mutations forecast pre‐eminent survival in early onset DLBCL.

Parallel Evolution of HIV-1 in a Long-Term Experiment.

One of the most intriguing puzzles in biology is the degree to which evolution is repeatable. The repeatability of evolution, or parallel evolution, has been studied in a variety of model systems, but has rarely been investigated with clinically relevant viruses. To investigate parallel evolution of HIV-1, we passaged two replicate HIV-1 populations for almost 1 year in each of two human T-cell lines. For each of the four evolution lines, we determined the genetic composition of the viral population at nine time points by deep sequencing the entire genome. Mutations that were carried by the majority of the viral population accumulated continuously over 1 year in each evolution line. Many majority mutations appeared in more than one evolution line, that is, our experiments showed an extreme degree of parallel evolution. In one of the evolution lines, 62% of the majority mutations also occur in another line. The parallelism impairs our ability to reconstruct the evolutionary history by phylogenetic methods. We show that one can infer the correct phylogenetic topology by including minority mutations in our analysis. We also find that mutation diversity at the beginning of the experiment is predictive of the frequency of majority mutations at the end of the experiment.

Abstract 3917: Double PIK3CA mutations in cis enhance oncogene activation and sensitivity to PI3K alpha inhibitors in breast cancer

Abstract Activating mutations in PIK3CA, the gene coding for the catalytic subunit (p110α) of phosphoinositide 3-kinase (PI3K), are the most frequent oncogenic alterations in all cancers, including estrogen receptor-positive (ER+) breast cancer. There are many distinct oncogenic PIK3CA mutations including major hotspot mutations (e.g. E542K, E545K, H1047R) which are predictive of response to PI3Kα inhibitors. While responses to PI3Kα inhibitors can be variable, some patients with single hotspot mutant tumors derive a deep and prolonged clinical benefit. In a comprehensive analysis of the genomic data available in breast cancer, we observed double PIK3CA mutations and investigated their potential biological relevance and potential correlation with sensitivity to PI3Kα inhibitors. We detected double PIK3CA mutations in 10-15% of all PIK3CA-mutant cancers including breast cancer. Double PIK3CA mutations in breast cancer are defined by a pattern of co-expression of a major hotspot mutation associated with a recurrent second-site PIK3CA mutation (‘minor mutation’). We have found that these double PIK3CA mutations are compound, that is in cis on the same allele, using single molecule real time sequencing (SMRT-seq) of fresh breast tumor samples. Double compound PIK3CA mutations result in increased PI3K activity and downstream signaling compared to single hotspot mutants in nontransformed cells and in ER+ breast cancer cells. Double compound mutations also increase cell proliferation and xenograft growth compared to single hotspot mutants. Biochemical experiments using recombinant PI3K compound mutant protein complexes reveal a combination mechanism of action, with simultaneous PI3K destabilization and increased membrane binding compared to single hotspot mutants, leading to increased kinase activity. Importantly, these compound mutations predict for increased sensitivity to PI3Kα inhibitors compared to single hotspot mutants in both preclinical models and also in patients with breast cancer. Together, our data support that double compound PIK3CA mutations enhance the oncogenicity of single hotspot PIK3CA mutations and this increased dependency results in increased sensitivity to PI3Kα inhibitors compared to single hotspot mutations. We propose that double compound activating mutations are an additional mechanism of oncogenic transformation in addition to other known mechanisms such as gene amplification, point mutation, or gene translocation. We speculate that double compound mutant PIK3CA may function as a clinical biomarker of increased sensitivity to PI3K-directed targeted therapies and may improve the therapeutic window of PI3K inhibitors in ER+ breast cancer and other PIK3CA-mutant tumor histologies. Citation Format: Neil Vasan, Pedram Razavi, Jared Johnson, Hong Shao, Hardik Shah, Alesia Antoine, Erik Ladewig, Alexander Gorelick, Eneda Toska, Guotai Xu, Abiha Kazmi, Matthew T. Chang, Barry S. Taylor, Maura N. Dickler, Komal Jhaveri, Raul Rabadan, Ed Reznik, Melissa L. Smith, Robert Sebra, Lewis C. Cantley, Maurizio Scaltriti, Jose Baselga. Double PIK3CA mutations in cis enhance oncogene activation and sensitivity to PI3K alpha inhibitors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3917.

First Case Report of an Unusual Echis genus (Squamata: Ophidia: Viperidae) Body Pattern Design in Iran.

Three families of venomous snakes exist in Iran including Viperidae, Elapidae, and Hydrophidae. Viperidae family is the only family with a widespread distribution. Saw-scaled vipers are important poisonous snakes in Asia and Africa. This name is given to this snake due to the presence of obliquely keeled and serrated lateral body scales. Distribution of this genera is mostly reported in the central and southern regions of Iran. This genus has four main clades: the Echis carinatus, E. coloratus, E. ocellatus, and E. pyramidum. Design pattern in Echis species plays an important role in camouflage and variety of habitat. In the present report, we investigated a specimen from the eastern region of Iran; we examined 25 specimens of Echis that were collected from the eastern region of our country. Among them, only one specimen with a different pattern was found compared with the other 24 specimens by surveying meristic, mensural, and design pattern characters using valid key identifiers. The similarities between the specific Echis with a different pattern and other 24 specimens were also studied and compared. The results of this investigation clearly showed that although the pattern of the lateral white line and block on dorsal body of the specific Echis snake was different, since the meristic and mensural characters were similar to other Echis snakes it can be concluded that this specimen is not a different species; the difference in these patterns may be due to a minor genetic mutation of that specimen. It is the first case report of Echis carinatus sochureki Stemmler, 1969 from Iran with a different pattern.

Identification of H1N1 influenza virus-derived T-cell epitopes and the contribution in the cross-reactivities to avian influenza virus

Influenza viruses, such as 2009 pandemic A(H1N1) influenza virus (2009 pH1N1) and avian influenza A (H7N9) virus (H7N9), which spread across the species, pose a great threat to human health and cause socioeconomic losses. Since 2009, circulation of 2009 pH1N1 has become a seasonal influenza virus. The sustaining epidemics have resulted in certain T-cell immune level among healthy populations. T-cell epitopes are mainly derived from conserved internal proteins of influenza virus compared with B-cell epitopes, influenza virus-specific cross-reactive CD4+ and CD8+ T-cells broadly exist among the population, providing protection from subsequent infections by heterotypic viruses. Cytotoxic T lymphocytes (CTLs) specific for influenza A viruses mostly target internal, nonglycosylated proteins including M1, which are enriched with immunodominant CTL epitopes and markedly conserved among diverse strains compared to HA and NA. In comparison to M1 protein of 2009 pH1N1, the H7N9 M1 has 4 segments with clustering substitutions. In this study, we defined the cross-reactivity immunity between 2009 pH1N1 and H7N9 and identified four T-cell epitopes: H1-M42 (LMEWLKTR), H1-M102 (KLKREITFHGAK), H1-M202s (AMEVANQTR) and H1-M244 (MGVQMQRFK) in the mutant segments in 2009 pH1N1. T-cell responses were investigated using freshly isolated PBMCs from individuals ( n = 16) through IFN-γ ELISPOT with these peptides derived from influenza virus M1 protein that are not conserved between 2009 pH1N1 and H7N9. We investigated the baseline of pre-existing immunity targeting M1 mutant segments of avian H7N9 influenza virus compared to 2009 pH1N1 among a healthy population. There was a certain level of T-cell immunity to H7N9 in the healthy population, but still weaker than that to 2009 pH1N1. Peptides derived from H7N9 which had mutations induced significant lower T-cell responses. The limited pre-existing T-cell immunity against H7N9 in healthy populations was partially contributed by H7N9 amino acid mutations in novel identified epitopes. Furthermore, we found that H1-M102 and H1-M244 could bind with HLA-A*1101 stably after renaturation in vitro , while the binding of H1-M42 and H1-M202s with HLA-A*3303 were relatively weak. Combined refolding and functional studies based on T-cell epitopes derived from influenza virus illustrated that minor mutation of an epitope can lead to a profound effect on the antigenicity of the peptide, which may also influence both HLA binding and TCR docking. Our study on T-cell immunity against influenza viruses provides an important reference for understanding the preexisting immune responses to avian influenza virus, and benefits the development of universal influenza vaccine. At the same time, considering that a few amino acid mutations will change the immunogenicity of the peptide completely, the study of antigenic variability of influenza virus major T cell immunogens such as M1, NP and PB1 are crucial in the development of universal vaccines.

Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier

BackgroundMycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens.ResultsWe show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association.ConclusionWe show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.

Application of amplicon-based targeted sequencing with the molecular barcoding system to detect uncommon minor EGFR mutations in patients with treatment-na\xefve lung adenocarcinoma

BackgroundIn lung cancer, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitizing mutations co-existing with rare minor EGFR mutations are known as compound mutations. These minor EGFR mutations can lead to acquired resistance after EGFR tyrosine kinase inhibitor treatment, so determining the mutation status of patients is important. However, using amplicon-based targeted deep sequencing based on next-generation sequencing to characterize mutations is prone to sequencing error. We therefore assessed the benefit of incorporating molecular barcoding with high-throughput sequencing to investigate genomic heterogeneity in treatment-naïve patients who have undergone resection of their non-small cell lung cancer (NSCLC) EGFR mutations.MethodsWe performed amplicon-based targeted sequencing with the molecular barcoding system (MBS) to detect major common EGFR mutations and uncommon minor mutations at a 0.5% allele frequency in fresh–frozen lung cancer samples.ResultsProfiles of the common mutations of EGFR identified by MBS corresponded with the results of clinical testing in 63 (98.4%) out of 64 cases. Uncommon mutations of EGFR were detected in seven cases (10.9%). Among the three types of major EGFR mutations, patients with the G719X mutation had a significantly higher incidence of compound mutations than those with the L858R mutation or exon 19 deletion (p = 0.0052). This was validated in an independent cohort from the Cancer Genome Atlas dataset (p = 0.018).ConclusionsOur findings demonstrate the feasibility of using the MBS to establish an accurate NSCLC patient genotype. This work will help understand the molecular basis of EGFR compound mutations in NSCLC, and could aid the development of new treatment modalities.

Characterization of Rosa ‘Harison’s Yellow’ clones using SNP markers

Rosa ‘Harison’s Yellow’ is a historic old garden rose that originated in the United States c. 1824. It was found in the garden of George Folliot Harison, Esq. in New York City and was introduced by local nurserymen by 1830. ‘Harison’s Yellow’ has bright yellow, loosely double blooms borne for approximately a month in spring, tolerates a wide range of climatic conditions, and is easily propagated from rhizomes and root suckers. Its phenotype suggests it is a hybrid involving R. spinosissima and R. foetida. ‘Harison’s Yellow’ can be found on old United States farmsteads and in stopping places along routes taken by settlers as they traveled west and southwest in the 1800s. Nine clones of ‘Harison’s Yellow’ were obtained for comparison. They were supplied by botanical gardens, commercial nurseries, and individuals, some of whom have had the rose in their family for generations. Six of the nine samples were received as own-root plants which we determined to be triploid. Phenotypic characteristics of all the plants were very similar. The nine ‘Harison’s Yellow’ selections, plus eight additional roses used as controls, were analyzed using the WagRhSNP Axiom® rose SNP array in order to learn whether the ‘Harison’s Yellow’ roses were the same genotype. Out of the 24,512 polymorphic SNPs among all the roses analyzed, only 167 SNPs were polymorphic among the ‘Harison’s Yellow’ samples. All ‘Harison’s Yellow’ selections could be distinguished from each other. Rosa foetida ‘Austrian Copper’ was the most similar to the ‘Harison’s Yellow’ roses with 4,466 polymorphic SNPs separating them. The SNP data suggest that these nine ‘Harison’s Yellow’ samples, and surprisingly one additional single flowered selection (sold as ‘Yellow Spin’), are likely derived from the same ortet, with minor detectable mutations among the ramets that have accumulated since ‘Harison’s Yellow’ entered commerce.

Analysis of KRAS, NRAS and BRAF mutational profile by combination of in-tube hybridization and universal tag-microarray in tumor tissue and plasma of colorectal cancer patients.

Microarray technology fails in detecting point mutations present in a small fraction of cells from heterogeneous tissue samples or in plasma in a background of wild-type cell-free circulating tumor DNA (ctDNA). The aim of this study is to overcome the lack of sensitivity and specificity of current microarray approaches introducing a rapid and sensitive microarray-based assay for the multiplex detection of minority mutations of oncogenes (KRAS, NRAS and BRAF) with relevant diagnostics implications in tissue biopsies and plasma samples in metastatic colorectal cancer patients. In our approach, either wild-type or mutated PCR fragments are hybridized in solution, in a temperature gradient, with a set of reporters with a 5' domain, complementary to the target sequences and a 3' domain complementary to a surface immobilized probe. Upon specific hybridization in solution, which occurs specifically thanks to the temperature gradients, wild-type and mutated samples are captured at specific location on the surface by hybridization of the 3’ reporter domain with its complementary immobilized probe sequence. The most common mutations in KRAS, NRAS and BRAF genes were detected in less than 90 minutes in tissue biopsies and plasma samples of metastatic colorectal cancer patients. Moreover, the method was able to reveal mutant alleles representing less than 0,3% of total DNA. We demonstrated detection limits superior to those provided by many current technologies in the detection of RAS and BRAF gene superfamily mutations, a level of sensitivity compatible with the analysis of cell free circulating tumor DNA in liquid biopsy.

Miliary lung metastases from non-small cell lung cancer with Exon 20 insertion: A dismal prognostic entity: A case report.

Miliary lung metastases have been reported to be frequently observed in nοn-small cell lung cancer (NSCLC) with major EGFR mutations, which consist of exon 19 deletion and exon 21 L858R. However, it remains undetermined whether NSCLC with minor EGFR mutations possesses characteristics similar to those with major EGFR mutations. In the present study, two cases of miliary lung metastases from NSCLC with exon 20 insertion were reported. Both the patients visited our hospital because of cough and/or dyspnea and were treated with chemotherapeutic agents, including platinum-doublet regimen. In addition, 1 patient received afatinib during the clinical course. However, all therapeutic regimens did not result in the desired outcome, and the respiratory condition rapidly deteriorated. Both the patients succumbed to disease within 3 months from the beginning of the 1st-line treatment due to disease progression. To conclude, chest physicians should be aware that miliary lung metastases could develop in NSCLC patients with exon 20 insertion and present a dismal prognosis.

P3.01-45 Multifactorial Gene Alterations in EGFR Bypass Pathway are Induced by Afatinib in T790M-Mutant NSCLC Resistant to Osmertinib

The 3rd generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) was developed to target the EGFR T790M resistance mutation in non-small cell lung cancer patients resistant to 1st or 2nd generation EGFR-TKIs. Although some mechanisms of acquired resistance to 3rd generation EGFR-TKI such as EGFR C797S mutation have been reported, the effect and resistant mechanisms to afatinib followed by osmertinib has not been well-known. Nine patients with EGFR T790M-mutant NSCLC resistance to 3rd generation EGFR-TKI were enrolled in this study and treated with afatinib. Plasma samples were collected before treatment, 4 weeks after treatment, and at disease progression. Mutation profile and tumor mutation burden (TMB) in plasma cell free DNA (cfDNA) were analyzed by CAPP-seq. The objective response rate and median progression-free survival of afatinib were 0% and 2.0 months, respectively. At the time of 4 weeks after treatment, four patients developed disease progression and five patients showed stable disease. A total of 36 somatic mutations or amplification were detected in plasma cfDNA before afatinib treatment; EGFR activating mutations in 8 patients, T790M mutation in 4, TP53 mutations in 6, PIK3CA mutations in 3, BRAF mutations in 3, MET amplification in 3, CTNNB1 mutations in 2, ERBB2 mutations in 2, C797S mutation in 1, SMAD4 mutation in 1, EGFR minor mutation in 1, KRAS mutation in 1, and APC mutation in one patient. EGFR C797S mutation in cfDNA was detected during afatinib treatment in two cases. In the patients having stable disease at 4 weeks after treatment, mutant allele frequency and TMB tended to decline once, and then increased in association with disease progression. Detection of mutant allele frequency and TMB of ctDNA by CAPP-seq could monitor the effectiveness and resistance to afatinib. Resistant mechanisms to afatinib might be characterized by multifactorial bypass pathway activation.

A Surveillance on Protease Inhibitor Resistance-Associated Mutations Among Iranian HIV-1 Patients

Background: Drug resistance is emerging as one of the greatest challenges in the development of effective treatment for HIV infection. The importance of clinical studies in this field stems from the world wide growing of treatment drug-resistant mutations. Objectives: This study was performed to determine the HIV subtype and the resistance mutations to the protease inhibitors in both untreated HIV patients and patients under treatment with protease inhibitors (PIs) in Iran. Methods: The study was conducted on two groups of participants. The first group consisted of 25 HIV patients who did not receive any antiretroviral treatment. The second group included 25 HIV patients who have being treated with a combination of protease inhibitors. After genome extraction, a nested polymerase chain reaction was performed to amplify the protease gene. Upon confirmation using electrophoresis, the amplicons or PCR products were sequenced and analyzed to determine the drug resistance mutations as well as the viral subtypes. Results: No mutations were found in the first group; however, 32% of the samples in the second group had PI related drug-resistance mutations. The major mutations were V82A and M46I, which were seen in 12% of the samples, while the minor mutation F53L was seen in 16% of the samples. The subtype analysis showed that 94% of the samples were subtype CRF35_AD, and 6% were of defined as subtype A. Conclusions: The present study reports updates on the mutations related to protease resistance in Iranian HIV patients receiving treatment. Our data, as well as existing reports, support the need for the optimization of treatment to prevent emergence of resistant viruses and a search for new antiretroviral drug candidates for HIV patients.