Mutant Allele Frequency Research Articles

Analysis of mutation frequencies of the BRCA1 5382insC and BRCA2 6174delT genes in the population of Ufa city

BACKGROUND: Currently, there is an acute problem of assessing the real genetic danger and the impact of environmental pollution on human health. One of the cities that is affected by the whole complex of anthropogenic mutagenic factors is Ufa (Republic of Bashkortostan).
 AIM: The aim of the study was to analyze the frequency of pathogenic mutations in the BRCA1 5382insC and BRCA2 6174delT genes in healthy residents of an industrial city.
 MATERIALS AND METHODS: The mutations were determined by real-time PCR using the TaqMan probe technology on the Bio-Rad CFX96 device (Bio-Rad, USA) in accordance with protocol in 379 DNA samples of healthy residents of average age (18.32 0.45 years).
 RESULTS: The total mutations frequency in the BRCA1 and BRCA2 genes reached 4.22% (n = 16). The highest frequency of mutant alleles in the BRCA1 and BRCA2 genes of 6.18% (n = 11) was present in residents of the 1 observation area, where petrochemical production enterprises are concentrated. The mutant homozygous genotype of the BRCA2 gene was detected in 3.37% of cases only among residents of the 1 observation area (n = 6, p = 0.0002). The risk of formation of mutations in the BRCA2 gene is 6 times higher in residents of the 1 observation area (2 = 7.75; df 1; p = 0.006; OR 6.31; 95% CI 1.4427.67).
 CONCLUSIONS: The frequency of the BRCA family genes varies depending on the studied region, the degree of environmental pollution and the studied group.

Use of the Biocartis Idylla™ Platform for the Detection of Epidermal Growth Factor Receptor, BRAF and KRAS Proto-Oncogene Mutations in Liquid-Based Cytology Specimens from Patients with Non-Small Cell Lung Carcinoma and Pancreatic Adenocarcinoma

The study aimed to demonstrate rapid and effective molecular testing on liquid-based cytology (LBC) samples for EGFR, KRAS and BRAF mutations using the Biocartis Idylla™. Rapid on-site evaluation (ROSE) LBC samples for patients with non-small cell lung carcinoma (NSCLC) or pancreatic ductal adenocarcinoma (PDAC) were tested for EGFR, KRAS and BRAF mutations based on the relevance to tumour subtype. The quantification values (Cq values) and mutation detection status were compared between LBC samples and routine formalin-fixed paraffin-embedded (FFPE) clot samples. ROSE LBC samples (n = 54) showed a higher yield of well-preserved tumour and wild type (WT) DNA, demonstrated by lower quantification cycles, no false positives or false negatives, and a higher sensitivity for low allele frequency mutations when compared with FFPE clot samples. The Biocartis Idylla™ provides highly sensitive, reliable and rapid testing for LBC samples for the detection of EFGR and KRAS mutations. BRAF mutations were not detected in the participant cohort; however, all LBC WT BRAF results correlated with the results from the FFPE clot samples. Access to rapid molecular testing using LBC samples can detect the most frequent driver mutations closer to the time of diagnosis, enabling the selection of the most effective first-line targeted therapy sooner, reducing delays or side effects from suboptimal treatments, patient anxiety and costs to healthcare systems, whilst improving patient outcomes.

Carrier Rate and Mutant Allele Frequency of GM1 Gangliosidosis in Miniature Shiba Inus (Mame Shiba): Population Screening of Breeding Dogs in Japan.

Simple SummaryGM1 gangliosidosis, a progressive neurodegenerative lysosomal storage disease, occurs in the Shiba Inu breed due to the GLB1:c.1649delC (p.P550Rfs*50) mutation. Previous surveys performed of the Shiba Inu population in Japan showed that the carrier rate was 1.02–2.94%. Currently, a miniature type of the Shiba Inu called “Mame Shiba”, bred via artificial selection to yield smaller individuals, is becoming more popular than the standard type. A GM1 gangliosidosis mutation survey has yet to be carried out in the Mame Shiba population. Therefore, this study aimed to determine the frequency of the mutant allele and carrier rate of GM1 gangliosidosis in the Mame Shiba breed. We surveyed 1832 adult Mame Shiba Inus used for breeding across 143 Japanese kennels using a genotyping assay. As a result, nine Mame Shiba Inus were found to be carriers, indicating that the carrier rate was 0.49% (corresponding mutant allele frequency = 0.00246). This study demonstrates that there are certain Mame Shiba Inus carrying the mutation for GM1 gangliosidosis and that their breeding should be prevented or controlled.GM1 gangliosidosis is a progressive, recessive, autosomal, neurodegenerative, lysosomal storage disorder that affects the brain and multiple systemic organs due to an acid β-galactosidase deficiency encoded by the GLB1 gene. This disease occurs in the Shiba Inu breed, which is one of the most popular traditional breeds in Japan, due to the GLB1:c.1649delC (p.P550Rfs*50) mutation. Previous surveys performed of the Shiba Inu population in Japan found a carrier rate of 1.02–2.94%. Currently, a miniature type of the Shiba Inu called “Mame Shiba”, bred via artificial selection to yield smaller individuals, is becoming more popular than the standard Shiba Inu and it is now one of the most popular breeds in Japan and China. The GM1 gangliosidosis mutation has yet to be surveyed in the Mame Shiba population. This study aimed to determine the frequency of the mutant allele and carrier rate of GM1 gangliosidosis in the Mame Shiba breed. Blood samples were collected from 1832 clinically healthy adult Mame Shiba Inus used for breeding across 143 Japanese kennels. The genotyping was performed using a real-time PCR assay. The survey found nine carriers among the Mame Shibas, indicating that the carrier rate and mutant allele frequency were 0.49% and 0.00246, respectively. This study demonstrated that the mutant allele has already been inherited by the Mame Shiba population. There is a risk of GM1 gangliosidosis occurrence in the Mame Shiba breed if breeders use carriers for mating. Further genotyping surveys are necessary for breeding Mame Shibas to prevent the inheritance of this disease.

Genetic changes during leukemic transformation to secondary acute myeloid leukemia from myeloproliferative neoplasms

Leukemic transformation (LT) is the main cause of death for patients with myeloproliferative neoplasms (MPNs). To study genetic changes associated with the LT, we performed targeted sequencing in 26 MPN patients including 21 with paired samples. We observed that, besides three driver genes, IDH2 (19%) and ASXL1 (14%) were also frequently mutated at MPN diagnosis. Although variant allele frequencies (VAFs) of mutations in DNA methylation and spliceosome did not expand during LT, they were enriched in patients with LT (the LT group). At follow-up, we also observed acquisition of mutations, mostly in the LT group. When considering dynamics of VAF from diagnosis to follow-up, VAFs in the LT group expanded (median VAF, 36.7–43.7%, p = 0.045). In contrast, mutations in patients with no clinical progression was stable (median VAF, 36.3–35.7%, p = 0.739). Overall, the present study demonstrates genetic changes during LT and provides the potential for prognostic application.

Screening and Carrier Rate of Neuronal Ceroid Lipofuscinosis in Chihuahua Dogs in Japan.

Simple SummaryNeuronal ceroid lipofuscinosis (NCL) is a rare group of lethal neurodegenerative lysosomal storage diseases, in which a homozygous single base-pair deletion (c.846delT) in the canine CLN7/MFSD8 gene has been identified as a causative mutation in Chihuahuas. In this study, we aimed to determine the carrier rate of NCL in Chihuahuas in Japan based on the screening of puppies using a newly designed real-time PCR assay. A survey conducted among 1007 Chihuahua puppies indicated that the carrier rate was 1.29%. Given the lethality of this disease, the mutant allele frequency (0.00645) is sufficiently high to warrant measures for disease control and prevention using the genotyping assay.Neuronal ceroid lipofuscinosis (NCL) is a group of rare lethal neurodegenerative lysosomal storage diseases that occur in a range of dog breeds, including Chihuahuas. Recently, a homozygous single base-pair deletion (c.846delT), which causes a frame shift generating a premature stop codon (p.Phe282Leufs13*) in the canine CLN7/MFSD8 gene, has been identified as a causative mutation for NCL in Chihuahuas. The objective of this study was to determine the frequency of the mutant allele and/or carrier rate of NCL in Chihuahuas in Japan using a newly designed real-time PCR assay. Samples of saliva were randomly collected from 1007 Chihuahua puppies during physical examinations prior to the transportation to pet shops. Screening results revealed a carrier rate of 1.29%, indicating a mutant allele frequency (0.00645) that is considered sufficiently high to warrant measures for the control and prevention of this lethal disease. The genotyping assay designed in this study could make a valuable contribution to the control and prevention of NCL.

Spatial heterogeneity of knockdown resistance mutations in the dengue vector Aedesalbopictus in Guangzhou, China

BackgroundThe city of Guangzhou has been the epicenter of dengue fever in China since the 1990s, with Aedesalbopictus being the primary vector. The main method used to control vectors and prevent dengue fever has been the application of chemical insecticides; however, this control strategy has resulted in the development of resistance to these insecticides in mosquitoes. Here we report our investigation of the patterns of knockdown resistance (kdr) mutations in 15 field populations of Ae.albopictus collected from 11 districts in Guangzhou.ResultsFour mutant alleles (V1016G, F1534S, F1534C, F1534L) were detected in domain II and III of the voltage-gated sodium channel (VGSC) gene. Various allele frequencies of kdr mutations were observed (3.1–25.9% for V1016G, 22.6–85.5% for F1534S, 0–29.0% for F1534L, 0.6–54.2% for F1534C). Seven kdr haplotypes (VF, VS, VL, VC, GF, GC, GS) were identified; the highest frequency of haplotypes was found for the single mutant haplotype VS (50.8%), followed by the wild-type VF haplotype (21.7%) and the single mutant haplotype VC (11.9%). Of the three double mutant haplotypes, GF was the most frequent (8.8%), followed by GC (1.2%) and GS (0.8%). Aedesalbopictus showed spatial heterogeneity in deltamethrin resistance in populations collected in Guangzhou. We also observed significant differences in haplotype frequency. The frequency of the VC haplotype was significantly higher in high-risk dengue areas than in low-risk ones.ConclusionsThe kdr allele V1016G was discovered for the first time in Guangzhou. Genetic isolation in mosquito populations and long-term insecticide selection seem to be responsible for the persistent, patchy distribution of kdr mutant alleles. The small-scale spatial heterogeneity in the distribution and frequency of kdr mutations may have important implications for vector control operations and insecticide resistance management strategies.Graphical

A Common Missense Variant Causing Factor XI Deficiency and Increased Bleeding Tendency in Maine Coon Cats.

Hereditary factor XI (FXI) deficiency is characterized as an autosomal mild to moderate coagulopathy in humans and domestic animals. Coagulation testing revealed FXI deficiency in a core family of Maine Coon cats (MCCs) in the United States. Factor XI-deficient MCCs were homozygous for a guanine to adenine transition resulting in a methionine substitution for the highly conserved valine-516 in the FXI catalytic domain. Immunoblots detected FXI of normal size and quantity in plasmas of MCCs homozygous for V516M. Some FXI-deficient MCCs experienced excessive post-operative/traumatic bleeding. Screening of 263 MCCs in Europe revealed a mutant allele frequency of 0.232 (23.2%). However, V516M was not found among 100 cats of other breeds. Recombinant feline FXI-M516 (fFXI-M516) expressed ~4% of the activity of wild-type fFXI-V516 in plasma clotting assays. Furthermore, fFXIa-M516 cleaved the chromogenic substrate S-2366 with ~4.3-fold lower catalytic efficacy (kcat/Km) than fFXIa-V516, supporting a conformational alteration of the protease active site. The rate of FIX activation by fFXIa-M516 was reduced >3-fold compared with fFXIa-V516. The common missense variant FXI-V516M causes a cross-reactive material positive FXI deficiency in MCCs that is associated with mild-moderate bleeding tendencies. Given the prevalence of the variant in MCCs, genotyping is recommended prior to invasive procedures or breeding.

Molecular Epidemiology of Na+-Taurocholate Cotransporting Polypeptide Deficiency in Guangdong Province, China: A Pilot Study by Screening for Four Prevalent Variants of the Causative Gene SLC10A1.

Na+-taurocholate cotransporting polypeptide deficiency (NTCPD) is an autosomal recessive disorder arising from biallelic SLC10A1 mutations. As a newly-described inborn error of bile acid metabolism, the epidemiology of this condition remains largely unclear in Chinese population so far. In this study, a total of 2,828 peripheral blood samples were collected from 12 cities in Guangdong, a province with the largest population in China, and the four prevalent SLC10A1 variants c.800C > T (p.Ser267Phe), c.263T > C (p.Ile88Thr), c.595A > C (p.Ser199Arg) and c.665T > C (p.Leu222Ser) were screened for by using polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP). As a result, 663 mutated SLC10A1 alleles were detected, and the mutated allele frequency was calculated to be 11.72% (663/5,656), with a carrier frequency 20.69% (1/5) and a theoretical morbidity rate 1.37% (1/73) of NTCPD in Guangdong province. The variant c.800C > T (p.Ser267Phe) exhibited highest allele frequency among the four prevalent variants (χ2 = 1501.27, p < 0.0001) as well as higher allele frequency in the peripheral region than that within the Pearl River Delta (χ2 = 4.834, p < 0.05). The results suggested that NTCPD might be a disorder rather common in Guangdong province. The findings depicted the molecular epidemiologic features of NTCPD, providing preliminary but significant laboratory evidences for the subsequent NTCPD diagnosis and management in Guangdong population.

Anchored Multiplex PCR Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Detection of melanoma mutations using circulating tumor DNA (ctDNA) is a potential alternative to using genomic DNA from invasive tissue biopsies. To date, mutations in the GC-rich TERT promoter region, which is commonly mutated in melanoma, have been technically difficult to detect in ctDNA using next-generation sequencing (NGS) panels. In this study, we developed a custom melanoma NGS panel for detection of ctDNA, which encompasses the top 15 gene mutations in melanoma including the TERT promoter. We analyzed 21 stage III and IV melanoma patient samples who were treatment-naïve or on therapy. The overall detection rate of the custom panel, based on BRAF/NRAS/TERT promoter mutations, was 14/21 (67%) patient samples which included a TERT C250T mutation in one BRAF and NRAS mutation negative sample. A BRAF or NRAS mutation was detected in the ctDNA of 13/21 (62%) patients while TERT promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of TERT promoter mutations with BRAF or NRAS mutations was found in 9/10 (90%) patients. The custom ctDNA panel showed a concordance of 16/21 (76%) with tissue based-detection and included 12 BRAF/NRAS mutation positive and 4 BRAF/NRAS mutation negative patients. The ctDNA mutation detection rate for stage IV was 12/16 (75%) and for stage III was 1/5 (20%). Based on BRAF, NRAS and TERT promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency and showed significant correlation with droplet digital PCR. For one patient, a novel MAP2K1 H119Y mutation was detected in an NRAS/BRAF/TERT promoter mutation negative background. To increase the detection rate to >90% for stage IV melanoma patients, we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect TERT promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel.

Next-generation T cell-activating vaccination increases influenza virus mutation prevalence.

To determine the potential for viral adaptation to T cell responses, we probed the full influenza virus genome by next-generation sequencing directly ex vivo from infected mice, in the context of an experimental T cell–based vaccine, an H5N1-based viral vectored vaccinia vaccine Wyeth/IL-15/5Flu, versus the current standard-of-care, seasonal inactivated influenza vaccine (IIV) and unvaccinated conditions. Wyeth/IL-15/5Flu vaccination was coincident with increased mutation incidence and frequency across the influenza genome; however, mutations were not enriched within T cell epitope regions, but high allele frequency mutations within conserved hemagglutinin stem regions and PB2 mammalian adaptive mutations arose. Depletion of CD4+ and CD8+ T cell subsets led to reduced frequency of mutants in vaccinated mice; therefore, vaccine-mediated T cell responses were important drivers of virus diversification. Our findings suggest that Wyeth/IL-15/5Flu does not generate T cell escape mutants but increases stochastic events for virus adaptation by stringent bottlenecks.

The role of clonal hematopoiesis as driver of therapy-related myeloid neoplasms after autologous stem cell transplantation.

Therapy-related myeloid neoplasm (t-MN) is a threatening complication of autologous stem cell transplantation (ASCT). Detecting clonal hematopoiesis (CH) mutations in cryopreserved cells before ASCT has been associated with a higher risk of t-MN, but the evolution of molecular abnormalities from pre-ASCT to t-MN, within the same patient, remains to be elucidated. We evaluated the mutational profile of 19 lymphoma/myeloma patients, at both pre-ASCT and t-MN diagnosis, using a targeted NGS approach; 26 non-developing t-MN control patients were also studied pre-ASCT. At ASCT, we found a higher frequency of CH in patients developing t-MN (58%) than in those who did not (23%) (P = 0.029); mutations in epigenetic (DNMT3A, TET2, and ASXL1) and DNA repair genes (PPM1D, RAD21, TP53, and STAG2) were the most represented. At t-MN, CH increased to 82% of patients. Cumulative mutational burden and variant allele frequency (VAF) also increased at t-MN. CH clones detected at ASCT were found at t-MN in eight out of 16 patients, mainly with stable VAF. Among the new driver mutations appeared at t-MN, TP53 increased from one to 13 mutations, in nine patients; being associated with complex karyotype. Mutations in transcription factor (RUNX1, CEBPA) and intracellular signaling genes (FLT3, RAS genes) also increased from three to 17 mutations in eight patients, presenting with a normal karyotype. Overall, we found that preexisting CH at ASCT rarely causes t-MN directly, but may rather facilitate the appearance of new mutations, especially those involving TP53, RUNX1, and RAS, that can drive the evolution to t-MN of at least two distinct types.

Droplet digital PCR-based analyses for robust, rapid, and sensitive molecular diagnostics of gliomas

Classification of gliomas involves the combination of histological features with molecular biomarkers to establish an integrated histomolecular diagnosis. Here, we report on the application and validation of a set of molecular assays for glioma diagnostics based on digital PCR technology using the QX200™ Droplet Digital™ PCR (ddPCR) system. The investigated ddPCR-based assays enable the detection of diagnostically relevant glioma-associated mutations in the IDH1, IDH2, H3-3A, BRAF, and PRKCA genes, as well as in the TERT promoter. In addition, ddPCR-based assays assessing diagnostically relevant copy number alterations were studied, including 1p/19q codeletion, gain of chromosome 7 and loss of chromosome 10 (+ 7/-10), EGFR amplification, duplication of the BRAF locus, and CDKN2A homozygous deletion. Results obtained by ddPCR were validated by other methods, including immunohistochemistry, Sanger sequencing, pyrosequencing, microsatellite analyses for loss of heterozygosity, as well as real-time PCR- or microarray-based copy number assays. Particular strengths of the ddPCR approach are (1) its high analytical sensitivity allowing for reliable detection of mutations even with low mutant allele frequencies, (2) its quantitative determination of mutant allele frequencies and copy number changes, and (3) its rapid generation of results within a single day. Thus, in line with other recent studies our findings support ddPCR analysis as a valuable approach for molecular glioma diagnostics in a fast, quantitative and highly sensitive manner.

KRAS mutation allele frequency threshold alters prognosis in right-sided resected pancreatic cancer.

Next-generation sequencing (NGS) provides information on genetic mutations and mutant allele frequency in tumor specimens. We investigated the prognostic significance of KRAS mutant allele frequency in patients with right-sided pancreatic ductal adenocarcinoma (PDAC) treated with surgical resection. A retrospective study reviewed patients who underwent surgical resection for PDAC and analyzed tumors with an in-house mutational panel. Microdissected samples were studied using an NGS-based assay to detect over 200 hotspot mutations in 42 genes (Pan42) commonly involved in PDAC. A total of 144 PDAC right-sided surgical patients with a Pan42 panel were evaluated between 2015 and 2020; 121 patients (84%) harbored a KRAS mutation. Detected mutant allele frequencies were categorized as less than 20% (low mKRAS, n = 92) or greater than or equal to 20% (high mKRAS, n = 29). High mKRAS (KRAS ≥ 20%) patients were noted to have shorter disease-free survival after surgery (11.5 ± 2.1 vs. 19.5 ± 3.5 months, p = 0.03), more advanced tumor stage (p = 0.02), larger tumors (3.6 vs. 2.7 cm, p = 0.001), greater tumor cellularity (26% vs. 18%, p = 0.001), and higher rate of distant recurrence (p = 0.03) than low mKRAS patients. This study demonstrates the importance of KRAS mutant allele frequency on pathological characteristics and prognosis in right-sided PDAC treated with surgery.

Comparison of clonal architecture between primary and immunodeficient mouse-engrafted acute myeloid leukemia cells

Patient-derived xenografts (PDX) are widely used as human cancer models. Previous studies demonstrated clonal discordance between PDX and primary cells. However, in acute myeloid leukemia (AML)-PDX models, the significance of the clonal dynamics occurring in PDX remains unclear. By evaluating changes in the variant allele frequencies (VAF) of somatic mutations in serial samples of paired primary AML and their PDX bone marrow cells, we identify the skewing engraftment of relapsed or refractory (R/R) AML clones in 57% of PDX models generated from multiclonal AML cells at diagnosis, even if R/R clones are minor at <5% of VAF in patients. The event-free survival rate of patients whose AML cells successfully engraft in PDX models is consistently lower than that of patients with engraftment failure. We herein demonstrate that primary AML cells including potentially chemotherapy-resistant clones dominantly engraft in AML-PDX models and they enrich pre-existing treatment-resistant subclones.

Urine- and Blood-Based Molecular Profiling of Human Prostate Cancer.

ObjectiveProstate cancer (PCa) is one of the most common malignant tumors, accounting for 20% of total tumors ranked first in males. PCa is usually asymptomatic at the early stage and the specificity of the current biomarkers for the detection of PCa is low. The present study evaluates circulating tumor DNA (ctDNA) in blood or urine, which can be used as biomarkers of PCa and the combination of these markers may increase the sensitivity and specificity of the detection of PCa.MethodsTissue, blood, and urine samples were collected from patients with PCa. All prostate tissue specimens underwent pathological examination. A hybrid-capture-based next-generation sequencing assay was used for plasma and urinary ctDNA profiling. Sequencing data were analyzed by an in-house pipeline for mutation calling. Mutational profiles of PCa and BPH were compared in both plasma and urine samples. Associations of detected mutations and clinical characteristics were statistically analyzed.ResultsA significant association of mutation allele frequencies (MAFs) in the blood samples with patients with metastatic PCa rather than patients with primary PCa, and MAFs are changed after treatment in patients with PCa. Further, the number of mutations in urine is not associated with clinical characteristics of PCa patients, but the frequencies of mutation alleles in the urine are associated with patient age. Comparison of cfDNA aberration profiles between urine and blood reveals more alterations in urine than in blood, including TP53, AR, ATM, MYC, and SPOP mutations.ConclusionThis work provides the potential clinical application of urine, in addition to blood, as a powerful and convenient non-invasive approach in personalized medicine for patients with PCa.

Relationship between perioperative oncological evaluation and recurrence using circulating tumor DNA with KRAS mutation in patients with colorectal cancer.

BackgroundThe detection of circulating tumor DNA (ctDNA) in colorectal cancer (CRC) by liquid biopsy may have prognostic information. In this perioperative study, we evaluate if there is a relationship between mutant allele frequency (MAF) of Kirsten rat sarcoma viral oncogene homolog (KRAS) and tumor recurrence and how that could be useful in the early detection of recurrence.MethodsAmong 304 cases of colorectal cancer surgery, ctDNA was sampled from the perioperative blood of 84 patients with CRC with KRAS mutation (exon 4 p.A146T, exon 4 p.A146V, exon 2 p.G12A, exon 2 p.G12C, exon 2 p.G12D, exon 2 p.G12S, exon 2 p.G12V, exon 2 p.G13D, exon 3 p.Q61H) and analyzed using the digital polymerase chain reaction system. The median observation period was 26 months.ResultsAlthough the relationship between the perioperative MAF of KRAS and recurrence was not proved, tumor diameter, tumor depth, and stage were correlated with the preoperative MAF of KRAS (p = 0.034, p = 0.002, p = 0.008). However, tumor diameter, tumor depth, and stage did not correlate with MAF of KRAS at postoperative day 30.ConclusionsIn this study, pathological tumor size, tumor depth, and stage were correlated with preoperative MAF of KRAS, but it was unreliable to predict recurrence by detection of ctDNA with KRAS mutation in the perioperative period of colorectal surgery.

Mutations in the telomerase reverse transcriptase promoter and PIK3CA gene are common events in penile squamous cell carcinoma of Italian and Ugandan patients.

Penile carcinoma develops either through human papillomavirus (HPV) related or unrelated carcinogenic pathways. Genetic alterations and nucleotide changes in coding regions (ie, TP53, CDKN2A, PIK3CA and NOTCH1) are main cancer driver events either in HPV positive or in HPV negative tumours. We investigated the presence of hotspot nucleotide mutations in TERT promoter (TERTp) and PIK3CA exon 9 and their relationship with HPV status in 69 penile cancer cases from Italian and Ugandan patients. Genetic variations and viral sequences have been characterised by end‐point polymerase chain reaction (PCR) and Sanger sequencing. The mutant allele frequencies (MAFs) of TERTp −124A/−146A and PIK3CA E545K have been determined by droplet digital PCR (ddPCR) assays. The results showed that TERTp mutations are highly prevalent in penile carcinoma (53.6%) and significantly more frequent in HPV negative (67.6%) than HPV positive (32.4%) cases (P = .0482). PIK3CA mutations were similarly distributed in virus‐related and unrelated cases (25.9% and 26.7%, respectively) and coexisted with TERTp changes in 15.8% of penile carcinoma samples. Notably, MAFs of co‐occurring mutations were frequently discordant indicating that PIK3CA E545K nucleotide changes are subsequent genetic events occurring in subclones of TERTp mutated cells. The frequencies of TERTp and PIK3CA mutations were higher among Italian compared to Ugandan cases and inversely correlated with the HPV status. In conclusion, TERTp mutations are very common in penile carcinoma and their coexistence with PIK3CA in a substantial number of cases may represent a novel oncogenic synergy relevant for patient stratification and use of therapeutic strategies against new actionable targets.

Custom multi‑tumor next‑generation sequencing panel for routine molecular diagnosis of solid tumors: Validation and results from three‑year clinical use.

Molecular testing is extremely important in cancer care, starting as early as at diagnosis. In order to address the challenge of providing reliable results within the timeframe adapted to patient management and suitable to guide clinical decisions, a capture-based next-generation sequencing (NGS) panel focusing on ten genes known to harbor genetic variations which may be targeted by approved drugs in patients with cancer was designed and validated. Very favorable analytical performances were obtained for both solid and liquid biopsies. For solid biopsies, a low read depth (80X per nucleotide) led to the genotype detection accuracy of 100%. The read of raw data for liquid biopsies resulted in the 91.19% result concordance between paired solid and liquid samples. The present method met all the requirements for the ISO15189 certification. During our three-year experience of routinely using this panel, almost 2,300 samples from lung and colorectal cancers, melanomas and gastrointestinal stromal tumors have been analyzed. It was found that our panel detected slightly more gain-of-function variants than described in the literature. Surprisingly, loss-of-function variants were also detected in certain of the analyzed genes. Finally, liquid biopsy data revealed statistically different mutated allele frequencies between tumor types, but also between mutated genes and variants themselves. In conclusion, the use of our capture-based NGS panel is perfectly adapted to perform relevant molecular diagnosis in a time frame compatible with patient care.

Allele Frequency of APAF1 Mutation in Holstein Cattle in Brazil.

APAF1 is an autosomal recessive inherited mutation, associated with Holstein haplotype 1 (HH1) and characterized by a substitution of cytosine for a thymine (c.1741C>T) in chromosome 5. The mutation causes fetal and embryonic loss, between 60 and 200 days of gestation, and reduced conception rate. The ARMS-PCR is considered a simple and low-cost method to determine single nucleotide polymorphism (SNP) with no need for genetic sequencing of the animal genome. This study aimed to verify the allelic frequency of APAF1 mutation in Brazilian Holstein cattle. A total of 248 Holstein DNA samples (210 cows and 38 bulls) were analyzed, and synthetic genes were manufactured to validate the primers developed by the authors. All animals assessed in this study were classified as wild-type for APAF1 mutation. The primers and protocol developed for the ARMS-PCR technique work with 100% specificity and efficiency since the amplicon formations are as expected according to the genotypes. In conclusion, the mutation responsible for APAF1 was not detected in the Brazilian Holstein cattle population assessed in this prevalence study, although it is not possible to affirm that APAF1 does not occur in Brazilian Holstein animals. The tetra-primer ARMS-PCR protocol for APAF1 mutation that has been validated here may be a relatively simple and economical method to determine the animals' genotype.

KRAS gene mutation quantification in the resection or venous margins of pancreatic ductal adenocarcinoma is not predictive of disease recurrence

Pancreatic ductal adenocarcinoma (PDAC) patients eligible for curative surgery undergo unpredictable disease relapse. Even patients with a good pathological response after neoadjuvant treatment (NAT) remain susceptible to recurrent PDAC. Molecular analysis of R0 margins may identify patients with a worse prognosis. The molecular status of mutant KRAS (exon 2, codon 12/13) was analysed retrospectively by digital droplet PCR in tumour areas, venous and resection margins of resected tumours, either undergoing up-front surgery (UFS) or after NAT with a good pathological response. Expectedly, tumour tissues or remnants from patients who underwent NAT presented lower KRAS mutant allele frequencies (MAF) than patients eligible for UFS. Similarly, ypT1 tumour MAFs were greater than the ypT0 tumour remnant MAFs in the NAT group. Mutant KRAS status in margins did not distinguish NAT subgroups. It was not predictive of shorter recurrence-free or overall survival within or between groups. KRAS-double negativity in both venous and resection margins did not identify patients with a better prognosis, regardless of the groups. The cohorts ‘sizes were small due to limited numbers of patients meeting the inclusion criteria, but KRAS-positivity or MAFs in resection and venous margins did not carry prognostic value. Comparison of margins from good versus bad responders receiving NAT may provide better clinical value.