Low Mutation Rate Research Articles

Activation of DNA Damage Tolerance Pathways May Improve Immunotherapy of Mesothelioma.

Simple SummaryA critical step in the success of immunotherapy is the presentation of tumor-derived peptides by the major histocompatibility complex I (MHC-I) of tumor cells. These neoantigens are potentially immunogenic and trigger immune responses orchestrated by cytotoxic cells. In malignant mesothelioma (MM), tumor development is nevertheless characterized by a low mutation rate despite major structural chromosomal rearrangements driving oncogenesis. In this paper, we propose a paradigm based on the mechanisms of the DNA damage tolerance (DDT) pathways to increase the frequency of non-synonymous mutations. The idea is to transiently activate the error-prone DDT in order to generate neoantigens while preserving a fully competent antitumor immune response.Immunotherapy based on two checkpoint inhibitors (ICI), programmed cell death 1 (PD-1, Nivolumab) and cytotoxic T-lymphocyte 4 (CTLA-4, Ipilimumab), has provided a significant improvement in overall survival for malignant mesothelioma (MM). Despite this major breakthrough, the median overall survival of patients treated with the two ICIs only reached 18.1 months vs. 14 months in standard chemotherapy. With an objective response rate of 40%, only a subset of patients benefits from immunotherapy. A critical step in the success of immunotherapy is the presentation of tumor-derived peptides by the major histocompatibility complex I (MHC-I) of tumor cells. These neoantigens are potentially immunogenic and trigger immune responses orchestrated by cytotoxic cells. In MM, tumor development is nevertheless characterized by a low mutation rate despite major structural chromosomal rearrangements driving oncogenesis (BAP1, NF2, CDKN2AB). In this opinion, we propose to investigate an approach based on the mechanisms of the DNA damage tolerance (DDT) pathways to increase the frequency of non-synonymous mutations. The idea is to transiently activate the error-prone DDT in order to generate neoantigens while preserving a fully competent antitumor immune response.

Detection of MYD88 L265P Mutation By Next Generation Deep Sequencing in Peripheral Blood Mononuclear Cells of Waldenstrom Macroguloblinemia and IgM Monoclonal Gammopathy of Undetermined Significance

Introduction: Whole genome sequencing has recently revealed MYD88 L265P somatic mutations in Waldenstrom macroguloblinemia (WM) tumor cells. Allele-specific polymerase chain reaction (AS-PCR) using bone marrow (BM) aspirate and peripheral blood (PB) is the most frequently used technique to detect the specific mutation. In search for a non-invasive, simple, and more sensitive detection of MYD88 L265P mutation, we performed next generation deep sequencing (NGS) technique using unselected peripheral blood mononuclear cells (PBMCs) collected from WM and IgM monoclonal gammopathy of undetermined significance (IgM MGUS) patients. The main purpose of this study is to assess the clinical implication of MYD88 L265P mutation burden detected by NGS technique using unselected PBMCs.Patients and methods: The study was approved from the Institutional Review Board. After informed consent was obtained in accordance with the Declaration of Helsinki, PBMCs were collected from 38 patients with WM and 20 patients with IgM MGUS. The median time from diagnosis to PBMC collection was 2.2 years (range, 0-25 years). Twenty-nine (76.3%) of WM patients were previously treated. The overall response (CR+PR+MR) rate at the time of analysis was 75.8% (22/29). Genomic DNA was extracted from unselected PBMCs and 300 ng of DNA was used for NGS analysis. Variation rates of MYD88 at the nucleotide position corresponding to L265P were measured with an Illumina MiSeq DNA sequencer by molecular barcoding technology essentially as previously reported (Nature Protocols 12, 664:2017). PCR products were sequenced on a MiSeq DNA sequencer with a Reagent kit v2. for 300 cycles. Serial dilution assessment was carried out to investigate the sensitivity of the method by analyzing the DNA of BM sample from a MYD88 mutation positive patient diluted in DNA from a healthy control.Results: Dilution assessment demonstrated that NGS technique was detectable to a sensitivity of 0.02%, ×5 higher than previously reported sensitivity by AS-PCR. MYD88 L265P mutation was detected from 6/9 (66.7%), 8/29 (27.6%), and 11/20 (55%) in patients with untreated WM, previously treated WM, and IgM MGUS, respectively. Among the patients who were positive forMYD88 L265P mutations, the median percentage of mutant allele relative to WT was 3.0% (0.14-32.3%), 0.24% (0.02-33.8%), and 0.55% (0.06-2.85%), in untreated WM, previously treated WM, and IgM MGUS, respectively. Patients with Untreated WM and those with lymphadenopathy showed a trend toward higher mutant allele burden in PBMCs, but the difference was not statistically significant. Among 26 previously treated WM patients, those with at least minimal response showed significantly lower detection rate of MYD88 L265P mutation (13.6 vs. 75.0%, P=0.02) and mutant allele burden (median, 0.00% vs. 0.23%, P=0.01), compared to patients with less than minimal response.Conclusion: This study is the first to report NGS analysis of MYD88 L265P mutation using unselected PBMCs of WM and IgM MGUS patients. The sensitivity for MYD88 L265P mutation in unselected PBMCs from IgM MGUS patients was comparable with that detected by AS-PCR using CD19-selected PB cells (Leukemia, 2014;28:1698-704). Although its specificity is in need for further investigations, NGS technique successfully detected the mutation carried by minimal tumor cells included in unselected PBMCs with high sensitivity as AS-PCR using CD19-selected PB cells. The mutation detection rate of previously treated WM patients was lower than that of untreated patients in our study and previously reported cohort; however, this in turn may potentially indicate that NGS detection of MYD88 L265P mutation in PBMC be clinically utilized in assessing treatment response by monitoring tumor burden during patients' clinical course. DisclosuresNo relevant conflicts of interest to declare.

FLT3 Mutation in Acute Myeloid Leukemia: Primary or Secondary Mutation?

The receptor tyrosine kinase FLT3 gene is commonly mutated in acute myeloid leukemia (AML) and its mutation is associated with significantly more aggressive disease and poor outcome. However, in the co-presence of NPM1 mutation, the disease is less aggressive and is associated with better outcome. FLT3 inhibitor midostaurin (Rydapt; Novartis Pharmaceuticals, Inc.) was recently approved for patients with FLT3 mutations. While the presence ofa FLT3 mutation is commonly associated with AML,it can develop in less acute myeloid neoplasms, such as MDS and CMML, and implies transformation into more acute phase. In this context, the FLT3 mutation is typically detected in a subclone of the original chronic myeloid clone. The prevalence of subclonal FLT3 mutations is unknown, as is the clinical relevance of subclonal FLT3, especially when treated with FLT3 inhibitors. We used variant allele frequency (VAF) and next generation sequencing (NGS) to determine if FLT3 mutations are founding or secondary mutations and to characterize the co-mutational background in large number of consecutive patients who presented with a clinical impression of myeloid neoplasm.Methods: A total of 6390 consecutive bone marrow aspirate samples or peripheral blood samples were submitted with clinical impression of AML, MDS, or MPN between January 2014 and mid 2017 were tested for mutations in myeloid genes using NGS. We used the TruSight Myeloid panel (Illumina, San Diego, CA) for detecting missense mutations and fragment length analysis (FLA) for detecting internal tandem repeat (ITD) in FLT3 . DNA was extracted from samples using the QIAamp DNA Mini Kit. This NGS testing covers mutations in 54 myeloid-related genes. The average depth of sequencing was 10,000x.Results: A total of 2670 (42%) of the samples showed a mutation in one or more of the tested genes. Of the samples with mutations, FLT3 ITD and kinase domain (KD) mutations were detected in 313 (12%) patients. Of these patients, 208 (66.5%) had ITD and 117 (37.4%) had KD mutations. Twelve patients (3.8%) had both ITD and KD mutations. NPM1 mutation was detected in 138 (44%) of the patients. Most of FLT3 -KD mutations were in codon 835, but mutations in codons 836, 839, 842 and 845 were also found. Additionally, missense mutations in codons 576, 592, and 593 at the site of ITD were detected. To distinguish a FLT3 mutation as a founding mutation rather than a secondary mutation, we used a lower VAF of 10% as a cut-off. Using this approach, we found that 27% (115/313) of patients had secondary FLT3 mutations. Of these subclonal FLT3 mutants, NPM1 co-mutation was detected in 61 (53%) patients, but was not statistically significant (P=0.06). Patients with FLT3 mutation as a subclone had a significantly higher number of co-mutated genes (P-value <0.0001). This group of patients had higher (P≤0.01) mutations in TET2, DNMT3A, NRAS, IDH2, EZH2, and BCOR, consistent with an MDS/CMML background. On the other hand, patients with both FLT3 and NPM1 mutations had a significantly lower number of co-mutant genes (P<0.0001) and particularly lower (P≤0.01) mutation rate in U2AF1, SF3B1, SRSF2, RUNX1, IDH1, BCOR, and AXL1 .Conclusion: FLT3 mutations are frequently (27%) a secondary mutation in a subclone of the neoplastic founding mutations. This appears to be more common when FLT3 mutation is developing in a background of MDS/CMML. The co-presence of an NPM1 mutation is more commonly associated with fewer co-mutations in MDS-related genes. This data suggests that in addition to detecting ITD by FLA, using NGS for profiling mutations in FLT3 -KD and other myeloid genes should be considered whenever FLT3 mutations are evaluated. Further, the clinical decision for combination therapy should consider the entire mutation profile and not only mutations in FLT3 . [Display omitted] DisclosuresMa:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Sudarsanam:NeoGenomics: Employment. Jiang:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Hummel:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Clinical Subtypes, Molecular Features and Treatment Strategies of Aggressive NK-Cell Leukemia: A Multicenter Analysis of 113 Patients

Aggressive NK-cell leukemia (ANKL) represents a rare form of NK cell neoplasm and a distinct disease entity within the continuous spectrum of EBV-T/NK-LPDs. This disease is more prevalent among people from Asia and Central and South America. The median overall survival (OS) is less than two months, irrespective of treatments. Due to the rarity of ANKL, the clinical features and underlying pathogenesis are still lack of understanding.To date, less than 350 cases of ANKL have been described in English literature worldwide. Most of these cases were sporadic case reports or studies with cohort scales no more than 40 patients. From October 2003 to July 2016, we conducted a retrospective study with 113 confirmed ANKL patients enrolled in 10 clinical centers located in different geographic regions across China. This study was approved by the institutional review board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All cases were centrally reviewed by three hematopathologists and three hematologists.All the patients were of the Han nationality living in the mainland China and had no history of CAEBV, SMBA or other EBV-T/NK-LPDs. Sudden fever, pancytopenia, hepatosplenomegaly, hypofibrinogenemia, hyperferritinemia and elevated lactate dehydrogenase (LDH) levels with dismal survival had characterized in these patients. The median age of the entire cohort was 37 years. The median overall survival was 55 days and 1-year survival rate was 4.42% (5/113). OS was estimated from the onset of disease to the date of death or the end of the study.A subacute clinical course was demonstrated in 18 patients (15.93%, 18/113). Prior to the fulminant onset, they manifested infectious mononucleosis (IM)-like symptoms for more than three months (median 4 months, range: 3-15 months). Female predominance (P = 0.007), alleviated hyperferritinemia (P < 0.001), transaminitis (ALT, P = 0.009) and hypofibrinogenemia (P = 0.038) and a marked survival advantage (P <0.001) were noted at diagnosis in these patients, irrespective of whether patients administered with allo-HSCT were excluded or not. Notably, even if the period of prodromal phases were not included in the estimation of the OS of these patients, this marked survival advantage could still be observed when patients received allo-HSCT were excluded (P = 0.042). All these data suggested that patients with prolonged prodromal phases represent a clinical subtype of ANKL which differed with the others. Therefore, we propose to define ANKL with IM-like symptoms lasting for more than 3 months as “subacute ANKL”, while the others who had acute clinical course as “classic ANKL”.To clarify the underlying pathogenesis, genes of interest were screened by Ion Torrent AmpliSeq™. The panel contained 18 candidate genes identified in our previous WGS analysis of 8 ANKL patients, including transcriptional factors (TP53 and MYC), JAK-STAT pathway genes (JAK2, JAK3, STAT3, STAT5A, STAT5B and STAT6), other signaling pathway genes (PIK3CB, NFKB1, NFKBIA, MAP3K13, MAPK10, NRAS and FGFR1) and epigenetic regulators (PRDM9, CREBBP and TET2). Our results showed TP53 gene had a significant lower mutation rate in subacute ANKL than that in classic ANKL (P =0.038). Except for that, the other gene mutation patterns including genes in JAK-STAT pathway, were similar between the two groups. Especially, TP53 mutations were not found in patients of subacute ANKL subtype, while enriched in 13 classic ANKL patients (44.83%, 13/29). In addition, most of the detected TP53 missense mutations (61.54%, 8/13) were located in the DNA binding domain.Survival analysis revealed L-asparaginase, methotrexate and dexamethasone (AspaMetDex) was an effective induction regimen with no therapy-related mortality. The complete remission rate and overall response rate in newly diagnosed patients was 30.77% (4/13) and 76.92% (10/13), respectively. Allo-HSCT was the only curative treatment with 2-year survival rate of 43.86% (3/7). Multivariate analysis showed serum LDH level, clinical subtype and administration of L-asparagine based chemotherapy or allo-HSCT were factors independently predictive for survival.In conclusion, these data depicted the clinical characteristics and molecular features of ANKL. It also provided insights into the treatments and outcomes of this deadly type of leukemia. Future prospective studies or clinical trials hold great promise for ANKL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Utility of FOXL2 mutation testing in differential diagnosis of adult granulosa cell tumour

Background: Adult granulosa cell tumour (AGCT) is a low-grade malignant sex cord-stromal tumour accounting for approximately 5% of malignant ovarian tumours, with a peak incidence occurring at 50–55 years. The majority of cases demonstrate distinctive clinical and morphologic features, and up to 97% of AGCT cases carry a recurrent, somatic missense mutation in codon 134 of the transcription factor, FOXL2 (C134W). Whilst FOXL2 mutation testing may not be required in cases with classic morphology and immunophenotype (inhibin, calretinin, CD99 positive), FOXL2 mutation testing aids diagnosis in problematic cases. The differential diagnosis may include other sex cord-stromal tumours, such as cellular fibroma, thecoma, and juvenile granulosa cell tumour, all of which have a very low rate of FOXL2 mutation. Whilst immunohistochemistry is useful in confirming sex cord-stromal tumour type, immunophenotype does not distinguish between tumours within this category. Aims: We explore the histologic spectrum of AGCT and other sex cord-stromal tumours; and correlate the findings with FOXL2 mutation testing. Methods: FOXL2 mutation testing was performed initially using targeted PCR and Sanger sequencing (Hudson Institute) or subsequently by next-generation sequencing using the Illumina Trusight Tumour 26 kit (Austin Health). Results: Cases discussed comprise three AGCTs confirmed with FOXL2 mutation: two with atypical pathological features (1 luteinised, 1 extensively necrotic), and one with atypical clinical behaviour; and two cases of sex cord-stromal tumour where absence of FOXL2 mutation supported diagnosis (cellular fibromas).

A targeted ancestry informative InDels panel on capillary electrophoresis for ancestry inference in Asian populations.

CE is the primary methodology used in forensic DNA typing. Alleles of commonly used types of genetic markers could be separated and detected via CE based on dye color and migration time. Insertion/deletion (InDel) is an ideal genetic marker for forensic DNA analysis due to their abundance in the human genome, low mutation rate, availability of their allele types via CE, and elimination of stutter peaks. Moreover, InDels could be used as ancestry informative markers since allele frequencies of InDels is different among geographically separated populations. Several ancestry informative insertion/deletion panels have been established based on CE platform to achieve the intercontinental populations distinction. However, improvements to differentiate intracontinental populations is few. In this study, 21 InDels with fixation index (FST ) > 0.15were selected and assembled into one ancestry informative insertion/deletion panel. Using well-designed primers, those 21 InDels could be amplified successfully and genotyped on the CE platform accurately and completely. The panel showed a large FST distance distinction among the ten Asian populations. Using clustering analysis, ten Asian populations were classified into three subgroups: East Asian, Southeast Asian, and South Asian subgroups. To evaluate the panel's capability in ancestry inference, a validation experiment was undertaken with 319 individuals from four geographically separated populations in China. Four Chinese populations were classified into different ancestry subgroups and 81.8% test individuals' ancestry could be inferred correctly. Our result showed that development of high ancestry informative InDels panel based on CE platform is a potential for individual ancestry inference among intracontinental populations.

How does feedback from phage infections influence the evolution of phase variation in Campylobacter?

Campylobacter jejuni (C. jejuni) causes gastroenteritis following the consumption of contaminated poultry meat, resulting in a large health and economic burden worldwide. Phage therapy is a promising technique for eradicating C. jejuni from poultry flocks and chicken carcasses. However, C. jejuni can resist infections by some phages through stochastic, phase-variable ON/OFF switching of the phage receptors mediated by simple sequence repeats (SSR). While selection strength and exposure time influence the evolution of SSR-mediated phase variation (PV), phages offer a more complex evolutionary environment as phage replication depends on having a permissive host organism. Here, we build and explore several continuous culture bacteria-phage computational models, each analysing different phase-variable scenarios calibrated to the experimental SSR rates of C. jejuni loci and replication parameters for the F336 phage. We simulate the evolution of PV rates via the adaptive dynamics framework for varying levels of selective pressures that act on the phage-resistant state. Our results indicate that growth reducing counter-selection on a single PV locus results in the stable maintenance of the phage, while compensatory selection between bacterial states affects the evolutionary stable mutation rates (i.e. very high and very low mutation rates are evolutionarily disadvantageous), whereas, in the absence of either selective pressure the evolution of PV rates results in mutation rates below the basal values. Contrastingly, a biologically-relevant model with two phase-variable loci resulted in phage extinction and locking of the bacteria into a phage-resistant state suggesting that another counter-selective pressure is required, instance, the use of a distinct phage whose receptor is an F336-phage-resistant state. We conclude that a delicate balance between counter-selection and phage-attack can result in both the evolution of phase-variable phage receptors and persistence of PV-receptor-specific phage.

Haplogroup Prediction Using Y-Chromosomal Short Tandem Repeats in the General Population of Bosnia and Herzegovina.

Human Y-chromosomal haplogroups are an important tool used in population genetics and forensic genetics. A conventional method used for Y haplogroup assignment is based on a set of Y-single nucleotide polymorphism (SNP) markers deployed, which exploits the low mutation rate nature of these markers. Y chromosome haplogroups can be successfully predicted from Y-short tandem repeat (STR) markers using different software packages, and this method gained much attention recently due to its labor-, time-, and cost-effectiveness. The present study was based on the analysis of a total of 480 adult male buccal swab samples collected from different regions of Bosnia and Herzegovina. Y haplogroup prediction was performed using Whit Athey’s Haplogroup Predictor, based on haplotype data on 23 Y-STR markers contained within the PowerPlex® Y23 kit. The results revealed the existence of 14 different haplogroups, with I2a, R1a, and E1b1b being the most prevalent with frequencies of 43.13, 14.79, and 14.58%, respectively. Compared to the previously published studies on Bosnian-Herzegovinian population based on Y-SNP and Y-STR data, this study represents an upgrade of molecular genetic data with a significantly larger number of samples, thus offering more accurate results and higher probability of detecting rare haplogroups.

Integrative modeling of transmitted and de novo variants identifies novel risk genes for congenital heart disease.

Whole-exome sequencing (WES) studies have identified multiple genes enriched for de novo mutations (DNMs) in congenital heart disease (CHD) probands. However, risk gene identification based on DNMs alone remains statistically challenging due to heterogenous etiology of CHD and low mutation rate in each gene. In this manuscript, we introduce a hierarchical Bayesian framework for gene-level association test which jointly analyzes de novo and rare transmitted variants. Through integrative modeling of multiple types of genetic variants, gene-level annotations, and reference data from large population cohorts, our method accurately characterizes the expected frequencies of both de novo and transmitted variants and shows improved statistical power compared to analyses based on DNMs only. Applied to WES data of 2,645 CHD proband-parent trios, our method identified 15 significant genes, half of which are novel, leading to new insights into the genetic bases of CHD. These results showcase the power of integrative analysis of transmitted and de novo variants for disease gene discovery.

Genetic profiles of familial late-onset Alzheimer's disease in China: The Shanghai FLOAD study

Compared with early-onset familial AD (FAD), the heritability of most familial late-onset Alzheimer's disease (FLOAD) cases still remains unclear. However, there are few reported genetic profiles of FLOAD to date. In the present study, targeted sequencing of selected candidate genes was conducted for each of 90 probands with FLOAD and 101 unrelated matched normal controls among Chinese Han population. Results show a significantly lower rate of mutation in APP and PSENs, and APOE ε4 genetic risk is higher for FLOAD. Among the Chinese FLOAD population, the most frequent variant was CR1 rs116806486 [5.6%, 95% CI (1.8%, 12.5%)], followed by coding variants of TREM2 (4.4%, 95%CI (1.2%, 10.9%)) and novel mutations of ACE [3.3%, 95%CI (0.7%, 9.4%)]. Next, we found that novel pathogenic mutations in ACE including frame-shift and nonsense mutations were in association with FLOAD regardless of APOE ε4 status. Evidence from the Alzheimer's disease Neuroimaging Initiative (ADNI) database also supported this finding in different ethnicities. Results of in vitro analysis suggest that frame-shift and nonsense mutations in ACE may be involved in LOAD through decreased ACE protein levels without affecting direct processing of APP.

MO063DESCRIPTION OF GENETIC VARIANTS IN A COHORT OF TOLVAPTAN ADPKD PATIENTS

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal cystic disease. It is genetically heterogeneous: 72-75% of ADPKD cases are related to mutations in the PKD1, 15-18 % to PKD2 and the remaining 7–10% of affected are genetically unresolved (GUR). Recent years, new drugs have emerged as promising agents that may retard the progression of ADPKD, such as Tolvaptan. In Italy Tolvaptan is available since 2016 and commonly used since 2017 in ADPKD patients, which fulfill the criteria of “rapid disease progression”, according to the European recommendations. High intra-interfamilial variability in pedigrees was observed, despite the same germ-line mutation. This could be explained by other clinical or genetic factors (environmental, modifier genes, etc), that may affect disease severity. The aim of the study is to describe the genetic variants in a cohort of Tolvaptan ADPKD patients (pts) referral to Renal Genetic Disease Ambulatory of Nephrology Department. Method Patients with ADPKD and in Tolvaptan treatment were enrolled. Diagnosis of ADPKD was made upon the revised Ravine’s criteria and Eligibility Criteria for Tolvaptan was made upon Italian indication for Tolvaptan prescription according to Italian Medicine Agency (AIFA) and European Medicines Agency (EMA). We performed genetic analysis (PKD1, PKD2 and PKHD1 genes) to identify mutations by NGS capture-based target enrichment kit (Sophia Genetic™), sequencing on Illumina MySeq Platform® and Sanger Sequencing on 3500 Series Genetic Analyzer (Applied Biosystems™). Results Eighteen pts [median age 46 (IQR 39-48) yrs ], 12 male, were included in the analysis. We manage to perform genetic analysis in all pts. Genetic analysis was essential in 4 patients without family history for Tolvaptan eligibility. Sixteen pts (88,9%) have mutations in PKD1, confirming what is already known from the literature for rapid progressor subjects; 2 pts are characterized by PKD2 mutations, both truncating. In only one pt, concomitant with a PKD1 mutation, also a PKHD1 mutation was found. In order to better characterize the cohort it was decided to subdivide the pts into 3 groups, by gene involvement and mutation type: 1st group: 12 Subjects with truncated PKD1 mutation (66.7%). In 7 pts (58,3%) the mutations are within exons (5 and from exon 11 to 15 inclusive) that encode for Immunoglobulin-like repeats or PKD domain of Polycystin 1 (PC1). 2nd group: 4 Subjects with non-truncated PKD1 mutations (22.2%). 3 pts (75.0%) are characterized by missense variants, as previous studies highlighted (a higher percentage of missense mutations in subjects with non-truncating mutations). In 2 pts (50%) the mutations are within exons (2 and 6) that encode for C-type lectin domain (CTLs) di PC1 and typical domain of extracellular protein. 3rd group: 2 Subjects with a PKD2 mutation (11.1%), both truncating. These data confirmed the lower mutation rate of PKD2 compared to PKD1 and highlighted an effective prevalence of truncation mutations in rapid disease progressors as previous reported. Conclusion Although our cohort of patients is small, we manage to perform genetic analysis in all pts reaching a detection rate of 100%. In 9 of 16 pts (56,3%) with PKD1 mutation the presence of mutations in exons coding PKD domain in PC1 or Immunoglobulin-like repeats or typical domain of extracellular protein allows us to hypothesize that the resulting alteration of the polycystin-mediated cell recognition and communication processes play a crucial role in the pathogenesis of ADPKD.

Multimodal analysis of cell-free DNA whole-genome sequencing for pediatric cancers with low mutational burden

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers.

Plastid NDH Pseudogenization and Gene Loss in a Recently Derived Lineage from the Largest Hemiparasitic Plant Genus Pedicularis (Orobanchaceae).

The plastid genome (plastome) is highly conserved in both gene order and content and has a lower mutation rate than the nuclear genome. However, the plastome is more variable in heterotrophic plants. To date, most such studies have investigated just a few species or only holoheterotrophic groups, and few have examined plastome evolution in recently derived lineages at an early stage of transition from autotrophy to heterotrophy. In this study, we investigated the evolutionary dynamics of plastomes in the monophyletic and recently derived Pedicularis sect. Cyathophora (Orobanchaceae). We obtained 22 new plastomes, 13 from the six recognized species of section Cyathophora, six from hemiparasitic relatives and three from autotrophic relatives. Comparative analyses of gene content, plastome structure and selection pressure showed dramatic differences among species in section Cyathophora and in Pedicularis as a whole. In comparison with autotrophic relatives and other Pedicularis spp., we found that the inverted repeat (IR) region in section Cyathophora had expansions to the small single-copy region, with a large expansion event and two independent contraction events. Moreover, NA(D)H dehydrogenase, accD and ccsA have lost function multiple times, with the function of accD being replaced by nuclear copies of an accD-like gene in Pedicularis spp. The ccsA and ndhG genes may have evolved under selection in association with IR expansion/contraction events. This study is the first to report high plastome variation in a recently derived lineage of hemiparasitic plants and therefore provides evidence for plastome evolution in the transition from autotrophy to heterotrophy.

Base-substitution mutation rate across the nuclear genome of Alpheus snapping shrimp and the timing of isolation by the Isthmus of Panama

BackgroundThe formation of the Isthmus of Panama and final closure of the Central American Seaway (CAS) provides an independent calibration point for examining the rate of DNA substitutions. This vicariant event has been widely used to estimate the substitution rate across mitochondrial genomes and to date evolutionary events in other taxonomic groups. Nuclear sequence data is increasingly being used to complement mitochondrial datasets for phylogenetic and evolutionary investigations; these studies would benefit from information regarding the rate and pattern of DNA substitutions derived from the nuclear genome.ResultsTo estimate the genome-wide neutral mutation rate (µ), genotype-by-sequencing (GBS) datasets were generated for three transisthmian species pairs in Alpheus snapping shrimp. A range of bioinformatic filtering parameters were evaluated in order to minimize potential bias in mutation rate estimates that may result from SNP filtering. Using a Bayesian coalescent approach (G-PhoCS) applied to 44,960 GBS loci, we estimated µ to be 2.64E−9 substitutions/site/year, when calibrated with the closure of the CAS at 3 Ma. Post-divergence gene flow was detected in one species pair. Failure to account for this post-split migration inflates our substitution rate estimates, emphasizing the importance of demographic methods that can accommodate gene flow.ConclusionsResults from our study, both parameter estimates and bioinformatic explorations, have broad-ranging implications for phylogeographic studies in other non-model taxa using reduced representation datasets. Our best estimate of µ that accounts for coalescent and demographic processes is remarkably similar to experimentally derived mutation rates in model arthropod systems. These results contradicted recent suggestions that the closure of the Isthmus was completed much earlier (around 10 Ma), as mutation rates based on an early calibration resulted in uncharacteristically low genomic mutation rates. Also, stricter filtering parameters resulted in biased datasets that generated lower mutation rate estimates and influenced demographic parameters, serving as a cautionary tale for the adherence to conservative bioinformatic strategies when generating reduced-representation datasets at the species level. To our knowledge this is the first use of transisthmian species pairs to calibrate the rate of molecular evolution from GBS data.

Functional differentiation analysis of duplicated mlpha gene in Oujiang color common carp ( Cyprinus carpio var. color ) on colour formation

Melanophilin (mlph) encodes a subfamily of Rab effector proteins which is an important gene for the transportation of mature melanosomes. Duplicated mlpha gene (mlpha1 and mlpha2) of Oujiang color common carp (Cyprinus carpio var. color) was firstly identified in the present study, which includes an open reading frame (ORF) length of 1713 bp and 1536 bp, encoding 570 and 511 amino acids respectively. Spatial and temporal expression, in situ hybridization and disruption experiments were conducted to explore the functional differentiation of mlpha (mlpha1 and mlpha2) during the melanin transport process of Oujiang color common carp. Distinct expression pattern of mlpha1 and mlpha2 were identified, and the expression level of mlpha1 in red skin of whole red with black patch (RB-R) and white skin of whole white with black patch (WB-W) was significantly lower than their corresponding black skin of RB and WB (RB-B and WB-B). On the contrary, the expression level of mlpha2 in RB-R and WB-W was significantly higher than their corresponding black skin (RB-B and WB-B). Mlpha2 was highly expressed in the “Whole red” (WR) skin, and mlpha1 was lowly expressed in the WR. Meanwhile, abundant positive ISH hybridization signals of mlpha2 were identified in 5 days post-hatch larvae (5dph) of WR. The high mutation rate of mlpha1 and mlpha2 genes resulted in a significant dilution phenotype, while the low mutation rate led to partial skin to appear discoloration phenomenon of Oujiang color common carp (RB and WB). The findings indicated that mlpha is an indispensable functional gene that may participate in the transportation of pigments and functional differentiation may exist between mlpha1 and mlpha2 after duplication. Mlpha1 may be mainly involved in the transportation of melanin, and mlpha2 may also participate in the transportation of other pigments.

Integrated DNA and RNA sequencing to reveal early drivers of metastasis in gastric cancer.

e16096 Background: Gastric cancer (GC) is the second cause of cancer-related death and metastasis is an important cause of death. Considering difficulties in searching for metastatic driver mutations, we tried a novel strategy to identify early drivers in primary gastric tumors. In this study, we conducted an integrative genomic analysis on GC patients and identified early drivers that lead to metastases. Methods: Tumors and matched normal tissues from 400 Chinese GC patients were characterized by whole-exome sequencing (WES), transcriptome sequencing and targeted exome sequencing (TES), especially the comparative analysis results between higher metastatic potential (HMP) group with T1 stage and lymph-node metastases, and lower metastatic potential (LMP) group without lymph-nodes or distant metastases. Functional experiments on candidate driver mutations were applied in GC cell lines to explore their roles in metastasis and immune escape. Results: HMP group presented higher mutation load and heterogeneity, enrichment in immunosuppressive signaling, lower tumor purity, more stromal and immune cell infiltration than LMP group. We identified 262 differentially expressed mRNA, lncRNA and miRNA between HMP and LMP group (with more than twice proportion of 2-fold increased or decreased tumor than normal tissue in HMP than LMP group, and with significantly 2-fold higher or lower in HMP tumors than LMP tumors). 168 candidate prometastatic mutations were found by WES and 8 were selected for following TES to detect their association with distant metastasis. We found that four candidate mutations were related to distant metastasis and mutated TP53 and MADCAM1 were significantly associated with poor metastasis-free survival. It’s worth noting that though with low mutational rate, MADCAM1 mutations were only observed in patients with metastasis within three years after surgery or at diagnosis. We demonstrated that MADCAM1-mutated proteins act as GOF mutants and could not only directly promote cancer cells migration, but also could trigger tumor metastasis by establishing complicated immunosuppressive microenvironment, including promoting PD-L1-mediated immune escape and reprogramming tumor-associated macrophages (TAMs). Conclusions: Our results showed that the GCs with different metastatic potential are distinguishable at the genetic level. A number of potential metastatic driver mutations was revealed in this study including known cancer-associated genes TP53. We also discovered some novel driver mutations, such as MADCAM1. The results indicate driver mutations in early-onset metastatic GC could promote metastasis not only by directly empower cancer cells to disseminate but also by establishing an immunosuppressive microenvironment. This study increased the understanding of molecular landscape of GC patients and provided possibility for future target therapy.

Analysis of the Relationship Between the Quality of Small Biopsy Specimens of Non-small Cell Lung Cancer and the Mutation Rate of EGFR Gene

背景与目的表皮生长因子受体（epidermal growth factor receptor, EGFR）是非小细胞肺癌（non-small cell lung cancer, NSCLC）患者中突变率最高的基因，准确检测出其突变类型有助于指导患者接受靶向药物治疗从而延长患者生存期。为了得到准确的检查结果，基因检测平台对标本质量有一定要求。已有文献报道标本的肿瘤细胞数量及其比例等会影响EGFR基因突变检出率，本研究旨在分析NSCLC小活检标本质量与突变扩增系统（amplification refractory mutation system, ARMS）法检测EGFR基因突变检出率的关系。方法收集299例小活检肺腺癌病例的临床特征、在显微镜下评估切片的肿瘤细胞数量及比例、标本提取的DNA浓度、EGFR基因突变检测结果，分析标本质量与EGFR基因突变检出率的关系。结果肿瘤细胞数≤500组与 > 500组EGFR基因突变阳性率分别为40.7%（11/27）、43.8%（119/272），两者间无统计学差异（P=0.764）。DNA浓度≤20.4 ng/μL组及 > 20.4 ng/μL组的基因突变阳性率分别为42.7%（64/150）、44.3%（66/149），两者间无统计学差异（P=0.776）。肿瘤细胞比例≤30%及 > 30%组的阳性率分别为29.4%（20/68）、47.6%（110/231），两者间存在统计学差异（P=0.008）。多因素Logistic分析显示男性、甲状腺转录因子1（thyroid transcription factor-1, TTF-1）阴性、有吸烟史及肿瘤细胞比例 < 30%是影响EGFR基因突变低检出率的主要因素。结论在达到检测的最低要求后，肿瘤细胞比例仍可影响EGFR基因突变检出率。有必要在基因检测切片后再次评估最后一张切片的肿瘤细胞比例，对于肿瘤细胞比例过低的标本，建议通过显微切割等方法富集肿瘤细胞，提高其肿瘤细胞比例，得出更准确的检测结果。对于无法进行肿瘤细胞富集的标本，可行循环肿瘤DNA（circulating tumor DNA, ctDNA）检测作为补充，若结果仍为阴性才需考虑再次活检获取足够的肿瘤标本进行分子检测。

The mutational landscape of older patients with IDH wild-type glioblastoma (GBM).

e14033 Background: Advanced age is associated with poorer outcomes in GBM and current NCCN guidelines distinguish older GBM patients (≥70 years, oGBM) from their younger counterparts ( &lt; 70 years, yGBM). We aim to characterize age-related comprehensive mutational profiles with the long-term goal of improving treatment strategies, outcomes, and rational clinical trial design. Specifically, we focused on IDH-wildtype (WT) oGBM, investigated if oGBM are more likely to acquire temozolomide-induced hypermutations, and finally, compared the frequency of high-tumor mutational burden (TMB) between oGBM vs. yGBM. Methods: Comprehensive molecular profiles of 1,657 adult IDH-WT GBM tumors tested at Caris Life Sciences (Phoenix, AZ) were queried. Tests included NGS of DNA (NextSeq, 592 Genes and NovaSEQ, WES) and RNA (NovaSeq) sequencing. SBS11 gene signature (i.e temozolomide-induced hypermutational profile) was queried using SigProfiler (Alexandrov 2020, Nature). Significance was determined by X2 and Fisher-Exact and p adjusted for multiple comparisons ( q) was &lt; 0.05. Results: We identified 1,657 patients (range 21-89 years old, median 61 years) with IDH-wildtype GBM, 22% (360) of whom were ≥ 70 years. There was a slight male predominance (60%) for all ages. The most prevalent alterations in oGBM were TERT promoter mutation (105/131,80%), MGMT promoter methylation (pMe) (175/346, 51%), and PTEN mutation (129/349, 37%). EGFR amplification was seen in 35% (125/356) and EGFRvIII in 23% (81/360); Overall, fusions were seen in 12% (44 of 360) oGBM; events &gt; 1% included MET (3.6%), FGFR3 (3.1%), EGFR (2.6%) and ROS1 (1.4%). 17% (56/349) of oGBM had positive PD-L1 by IHC. High TMB ( &gt; 10mt/Mb) tumors were rare (3.1%) and MSI-high tumors even rarer (0.8%) in oGBM. When compared to yGBM, MGMT pMe was more prevalent (51% vs 38%, risk ratio (RR) 1.35 [1.19-1.52], q &lt; 0.05) and NF1 mutations were less frequent in oGBM (21% v 34%, RR 0.62 [0.50-0.77], q &lt; 0.05). No significant differences were seen in other key markers examined. The prevalence of SBS11 gene signature across all ages (data available for 1,141 patients) was 1.2% and was comparable across the age spectrum; no significant difference seen in the MGMT pMe group when oGBM was compared to yGBM (3.9% vs. 1.8 %, p= 0.3). Conclusions: This study represents the largest comprehensive molecular characterization of older IDH-WT GBM patients. We show that molecular profiles of IDH-WT GBM are remarkably similar across the age spectrum, including immunotherapy-associated markers, gene fusion landscape, EGFR amplification, and TMB. The significantly higher prevalence of MGMT pMe and lower NF-1 mutation rate in the older population bear significant prognostic and therapeutic implications.

Analysis of circulating tumor DNA identifies homologous recombination repair molecular features of Chinese breast cancer.

e13006 Background: Breast cancer (BC) is the most common cancer in women world-wide. Assessments of genomic variants in circulating tumor DNA (ctDNA) have generated great enthusiasm for their potential application as clinically actionable biomarkers in the management of BC. Alterations in homologous recombination repair (HRR), are a determinant of sensitivity to platinum chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPi) in BC. However, as far as we known, most of HRR genes, except BRCA1/2, have not yet been fully understood to date. In this study, we aimed to elucidate the comprehensive HRR genetic alteration profile of breast tumors among Chinese patients by ctDNA analysis. Methods: Plasma ctDNA from 259 patients with BC were deeply sequenced via next-generation sequencing (NGS) techniques using AcornMed Biotechnology for 808 genes panel. Molecular profiles were reviewed to identify somatic and germline pathogenic mutations in the 14 HRR genes ( ATM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, FANCA, PALB2, RAD51B, RAD51C, RAD51D, RAD54L, CDK12, and BARD1) . Results: At our institution, 259 patients underwent NGS analysis of BC specimens. The median age was 46 (range from 31 to 79). Based on the analysis of the genetic alteration profile from our cohort, at least one of the HRR genes was observed from 29.34% of the tumor samples, with BRCA1 (8.11%), BRCA2 (5.79%), RAD51C (5.02%), CDK12 (4.63%), CHEK2 (1.93%), ATM (1.16%), and BARD1 (1.16%) as the most commonly altered genes. As compared with MSKCC dataset (Table), genetic alterations detected from our cohort affected genes involved in HRR (29.34% vs. 14.03%, P＜0.0001) pathways, with statistically different genetic alteration rates. Moreover, BRCA1/2, RAD51C, CDK12 and CHEK2 mutations exhibited higher mutation rates, whereas ATM, BRIP1 and PALB2 mutations presented a lower mutation rate in Chinese patients with breast cancer (P＜0.05). Conclusions: CtDNA can characterize the mutational feature of HRR in BC. our study contributes to the understanding of the HRR pathways and specific genetic alterations harbored by Chinese patients with BC that could potentially be developed as markers of treatment response to targeted therapeutics. Ref: Razavi P, Chang MT, Xu GT, et al. The Genomic Landscape of Endocrine-Resistant Advanced Breast Cancers. Cancer Cell. 2018 Sep 10;34(3):427-438.e6. doi: 10.1016/j.ccell.2018.08.008.[Table: see text]

Distinct Genomic Landscape of Colorectal Mucinous Carcinoma Determined via Comprehensive Genomic Profiling: Steps to a New Treatment Strategy.

Colorectal mucinous carcinoma (MC) is associated with inferior prognosis and response to treatment compared to adenocarcinoma (AC). The molecular landscapes of MC and adenocarcinoma with mucous composition (AMC) are not well-defined. We aimed to describe the genomic landscape of MC and AMC in a large colorectal cancer cohort. Tumor samples from patients with MC, AMC, or AC were analyzed using next-generation sequencing. MC had a molecular signature distinct from that of AC; genomic features were similar between AMC and MC but not between AMC and AC. HER2 amplification and TP53 and APC mutation rates were lower, whereas SMAD4, PIK3CA, ACVR2A, KMT2D, LRP1, TGFBR2, GRIN2A, BRAF V600E, PTEN, and BRCA2 mutation rates were higher in MC than in AC. The mutation frequencies in MAPK, PI3K, and TGF- pathways were higher, whereas those of cell cycle proteins and Wnt were lower in MC and AMC than in AC. The proportion of hypermutated tumors was significantly higher in MC and AMC than in AC. As MC has a distinct molecular signature from AC, immunotherapy can be potentially applied in treating MC. Similar molecular profiles of AMC and MC suggest that treatment strategies for MC, but not AC, can be used for AMC treatment.