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Alignment views of four variants detected by NGS. Each panel depicts a representative set of variant reads for single nucleotide
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Next-generation sequencing (NGS) has been rapidly integrated into molecular pathology, dramatically increasing the breadthgenomic of information available to oncologists and their patients. This review will explore the ways in which this new technologyis currently applied to bolster care for patients with solid tumors and hematological malignancies...
Citations
... This also supports our previous conjecture to properly explain the age characteristics of osteosarcoma patients. A significant role of NGS in cancer precision medicine is to detect potential available targets (47). PDGFRA and KIT mutations are common in gastrointestinal stromal tumors (48). ...
Background
Osteosarcoma is a leading subtype of bone tumor affecting adolescents and adults. Comparative molecular characterization among different age groups, especially in pediatric, adolescents and adults, is scarce.
Methods
We collected samples from 194 osteosarcoma patients, encompassing pediatric, adolescent, and adult cohorts. Genomic analyses were conducted to reveal prevalent mutations and compare molecular features in pediatric, adolescent, and adult patients.
Results
Samples from 194 osteosarcoma patients across pediatric to adult ages were analyzed, revealing key mutations such as TP53, FLCN, NCOR1, and others. Children and adolescents showed more gene amplifications and HRD mutations, while adults had a greater Tumor Mutational Burden (TMB). Mutations in those over 15 were mainly in cell cycle and PI3K/mTOR pathways, while under 15s had more in cell cycle and angiogenesis with higher VEGFA, CCND3, TFEB mutations. CNV patterns varied with age: VEGFA and XPO5 amplifications more in under 25s, and CDKN2A/B deletions in over 25s. Genetic alterations in genes like MCL1 and MYC were associated with poor prognosis, with VEGFA mutations also indicating worse outcomes. 58% of patients had actionable mutations, suggesting opportunities for targeted therapies. Age-specific patterns were observed, with Multi-TKI mutations more common in younger patients and CDK4/6 inhibitor mutations in adults, highlighting the need for personalized treatment approaches in osteosarcoma. In a small group of patients with VEGFR amplification, postoperative treatment with multi-kinase inhibitors resulted in a PR in 3 of 13 cases, especially in patients under 15. A significant case involved a 13-year-old with a notable tumor size reduction achieving PR, even with other genetic alterations present in some patients with PD.
Conclusion
This study delineates the molecular differences among pediatric, adolescent, and adult osteosarcoma patients at the genomic level, emphasizing the necessity for precision diagnostics and treatment strategies, and may offer novel prognostic biomarkers for patients with osteosarcoma. These findings provide a significant scientific foundation for the development of individualized treatment approaches tailored to patients of different age groups.
... Since their introduction in 2005 [26] next generation sequencing (NGS) technologies have revolutionised genomics and have found many uses in fields such as clinical microbiology [27], oncology [28], and clinical genetics [29]. NGS allows sequencing of entire genomes cheaper and more rapidly than Sanger sequencing methods. ...
Celiac disease is an autoimmune disorder induced by consumption of gluten protein present in foods such as wheat and rye. In recent years there has been increasing evidence that changes in composition of gut microbiota may play a significant role in the pathogenesis of celiac disease. Multiple methods of bacterial identification may be used to find microbiota changes characteristic for celiac disease, and the latest methods such as next generation sequencing offer new possibilities of detecting previously unknown bacterial groups that may play a role in the occurrence of celiac disease. This review focuses on multiple methods of identifying bacterial gut microbiome and presents results of recent studies exploring the link between gut microbiota composition and celiac disease.
... The variants passed filters were annotated with the Genetic variant annotation toolbox SnpEff 46 . Variant allele frequency (VAF) was calculated as the ratio of observed variant depth divided by the overall depth at the variant locus 47 . Density of the variants was counted in 1mb interval across the genome using the bedtools 2.29.2 ...
Gigaxonin is an E3 ubiquitin ligase that plays a role in cytoskeletal stability. Its role in cancer is not yet clearly understood. Our previous studies of head and neck cancer had identified gigaxonin interacting with p16 for NFκB ubiquitination. To explore its role in cancer cell growth suppression, we analyzed normal and tumor DNA from cervical and head and neck cancers. There was a higher frequency of exon 8 SNP (c.1293 C>T, rs2608555) in the tumor (46% vs. 25% normal, P = 0.011) pointing to a relationship to cancer. Comparison of primary tumor with recurrence and metastasis did not reveal a statistical significance. Two cervical cancer cell lines, ME180 and HT3 harboring exon 8 SNP and showing T allele expression correlated with higher gigaxonin expression, reduced in vitro cell growth and enhanced cisplatin sensitivity in comparison with C allele expressing cancer cell lines. Loss of gigaxonin expression in ME180 cells through CRISPR-Cas9 or siRNA led to aggressive cancer cell growth including increased migration and Matrigel invasion. The in vitro cell growth phenotypes were reversed with re-expression of gigaxonin. Suppression of cell growth correlated with reduced Snail and increased e-cadherin expression. Mouse tail vein injection studies showed increased lung metastasis of cells with low gigaxonin expression and reduced metastasis with reexpression of gigaxonin. We have found an association between C allele expression and RNA instability and absence of multimeric protein formation. From our results, we conclude that gigaxonin expression is associated with suppression of epithelial–mesenchymal transition through inhibition of Snail.
Significance
Our results suggest that GAN gene exon 8 SNP T allele expression correlates with higher gigaxonin expression and suppression of aggressive cancer cell growth. There is downregulation of Snail and upregulation of e-cadherin through NFκB ubiquitination. We hypothesize that exon 8 T allele and gigaxonin expression could serve as diagnostic markers of suppression of aggressive growth of head and neck cancer.
... De novo variants were subsetted based on standard somatic and germline variant allele frequencies (VAF). VAF, representing the percentage of sequencing reads matching a specific DNA variant 45 , was used as a surrogate measure of allele proportion. A VAF<0.3 indicated acquired somatic variants, while VAF≥0.3 ...
Bloom Syndrome (BSyn) is an autosomal recessive disorder caused by biallelic germline variants in BLM, which functions to maintain genomic stability. BSyn patients have poor growth, immune defects, insulin resistance, and a significantly increased risk of malignancies, most commonly hematologic. The malignancy risk in carriers of pathogenic variants in BLM (BLM variant carriers) remains understudied. Clonal hematopoiesis of indeterminate potential (CHIP) is defined by presence of somatic mutations in leukemia-related genes in blood of individuals without leukemia and is associated with increased risk of leukemia. We hypothesize that somatic mutations driving clonal expansion may be an underlying mechanism leading to increased cancer risk in BSyn patients and BLM variant carriers.
To determine whether de novo or somatic variation is increased in BSyn patients or carriers, we performed and analyzed exome sequencing on BSyn and control trios.
We discovered that both BSyn patients and carriers had increased numbers of low-frequency, putative somatic variants in CHIP genes compared to controls. Furthermore, BLM variant carriers had increased numbers of somatic variants in DNA methylation genes compared to controls. There was no statistical difference in the numbers of de novo variants in BSyn probands compared to control probands.
Our findings of increased CHIP in BSyn probands and carriers suggest that one or two germline pathogenic variants in BLM could be sufficient to increase the risk of clonal hematopoiesis. These findings warrant further studies in larger cohorts to determine the significance of CHIP as a potential biomarker of aging, cancer, cardiovascular disease, morbidity and mortality.
... But, if not, one way to solve this issue is to analyze the number of reads in each sense; if the reads are approximately the same, this provides an idea that it is a real variant, and not an artifact [47]. It is also important to confirm that the identified variant has a quality score over Q20 and that the quality of the reads containing it is good [48]. Moreover, the fact that the mutation identified is the one specific to that pathology also provides confidence, as seen in all of our samples. ...
GNAS-activating somatic mutations give rise to Fibrous Dysplasia/McCune–Albright syndrome (FD/MAS). The low specificity of extra-skeletal signs of MAS and the mosaic status of the mutations generate some difficulties for a proper diagnosis. We studied the clinical and molecular statuses of 40 patients referred with a clinical suspicion of FD/MAS to provide some clues. GNAS was sequenced using both Sanger and Next-Generation Sequencing (NGS). We were able to identify the pathogenic variants in 25% of the patients. Most of them were identified in the affected tissue, but not in blood. Additionally, NGS demonstrated the ability to detect more patients with mosaicism (8/34) than Sanger sequencing (4/39). Even if in some cases, the clinical information was not complete, we confirmed that, as in previous works, when the patients were young children with a single manifestation, such as hyperpigmented skin macules or precocious puberty, the molecular diagnosis was usually negative. In conclusion, as FD/MAS is caused by mosaic variants, it is essential to use sensitive techniques that allow for the detection of low percentages and to choose the right tissue to study. When not possible, and due to the low positive genetic rate, patients with FD/MAS should only be genetically tested when the clinical diagnosis is really uncertain.
... Tumors exhibit clonal architecture with multiple mutations present in a subset of the tumor cell population (Yates and Campbell 2012). Subclonal mutations provide key insights into tumor evolution and subclonal variants can be detected (Xu 2018) using the deep coverage (over 10,000x) of next-generation sequencing (NGS) data (Strom 2016), but their distinction from sequencing errors, library preparation and alignment artifacts depend on the noise level (Schmitt, Kennedy et al. 2012). We perform variant calling via a package available at Bioconductor (Gentleman, Carey et al. 2004), which provides quantitative detection of subclonal mutations in ultra-deep sequencing data. ...
(See also the 5 PDF files appended below) I developed a tumor-agnostic approach for the detection of colorectal neoplasm based on the DNA fragment length distribution on- and off-panel. Tumors exhibit clonal architecture with multiple mutations present in a subset of the tumor cell population. Sub-clonal mutations provide key insights into tumor evolution and sub-clonal variants can be detected using the deep coverage (over 10,000x) of next-generation sequencing (NGS) data, but their distinction from sequencing errors, library preparation and alignment artifacts depend on the noise level. We perform variant calling via a package available at Bioconductor, which provides quantitative detection of sub-clonal mutations in ultra-deep sequencing data. The deepSNV algorithm is used for a comparative setup with a control experiment of the same loci and uses a beta-binomial model as well as a likelihood ratio test to discriminate sequencing errors and sub-clonal SNVs. The shearwater (SW) algorithm computes a Bayes classifier based on a beta-binomial model for variant calling with multiple samples for precisely estimating the detection error rates and the variance dispersion using prior knowledge, such as variation data from the COSMIC database. It computes a model based on the coverage, error rate, and a dispersion factor. The determination of statistical significance is made by comparing the expected number of nucleotide counts per read with the stringency level of the selected cut-off value based on computing a posterior probability as a function of a Bayesian factor. Analysis strategy: We perform both tumor-informed and non-tumor informed analysis. We use a prior in the SW call to improve specificity by making it harder to call mutations not seen in COSMIC. In the tumor-informed analysis of the Dilution Series (DS) of mixed tumor DNA set, there is, in principle, no need for using COSMIC as a prior, as we know that the mutations are in the tumor, and hence in the plasma too. We assess how different posterior cut off values impact specificity by analyzing the Cance Research UK (CRUK) and Qiagen healthy samples using the DS mutation profile of 90 mutations with the objective to identity a tradeoff between sensitivity and specificity, and to make sure none of the control samples is called positive. We also assess the specificity by switching the mutation profiles of patients and access the rate with which we call plasma samples positive when using the mutation profiles of other patient samples from pre-operative (preOP) clinical trials samples. In non-tumor informed analysis, our requirements for specificity are much higher. Here, we search all 15,465 positions (the number will be lowered to around 15,000 based on the exact regions sequenced) in the panel and therefore it becomes critical not to make false positive calls. Using COSMIC as a prior is one strategy we use to improve specificity. Another option we have is to require a mutation to be observed on both strands (the AND algorithm). Limit of detection: In every sample, there are a certain number of cfDNA molecules, which, after library preparation, are sequenced with a certain depth. In plasma, the tumors shed both mutated and non-mutated fragments, which is further mixed with normal cells fragments. We analyze the distributions of the error counts per transition to identify those resulting from clonal hematopoiesis vs the true noise on the panel. We further utilize background noise models based on using patient samples, patient-control samples, samples from healthy individuals, and dilution series to perform error suppression, which improves the accuracy of our analysis. For samples for which we have whole exome sequencing (WES) tumor tissue sequencing to guide the analysis, we require at least one patient mutation to be detected in the plasma to call it positive. For every patient tumor, we ask the question: What is the lowest ratio of tumor fragments over the total molecules for which we detect >2 tumor fragments? We investigate how the pattens of lengths’ characteristics of the tumor fragments differ those originating from normal cells for particular fragments lengths and genomic regions. We are interested in computing a comprehensive score and in classifying samples according to the likelihood of detecting cancer. Tumor informed strategy: To quantitatively evaluate the detection limits of our assay, we have generated samples consisting of DNA from 36 tumors with known mutations, which have been pooled together. For these 36 tumors, we have an a-priori knowledge that 12 samples carry a KRAS mutation, 12 samples carry a BRAF mutation, and 12 samples carry a PIK3CA mutation, among other 90 mutations representative of colorectal cancer (CRC). In addition, we have gradually lowered the tumor concentration by mixing the tumor with normal DNA and keeping a constant amount of DNA corresponding to 25,000 GE. This way, we have created new titrated samples and have gradually reduced the frequency of the mutations with the objective to benchmark the performance of our algorithm. We have evaluated the mutation detection error rates for (i) different read coverage levels, (ii) mutation detection stringency cut-off levels, and (iii) ranges of detectability in terms of variant allele frequency (VAF). We combined the 8 samples and considered six different ranges ([0.1-0.5] with 2 values, [0.01-0.1) with 49 values, [0.001-0.01) with 163 values, [0.0001-0.001) with 255values, (0-0.0001) with 105 values, and 146 entries have VAF=0) in terms of variant allele frequency (VAFs) and evaluated the success rate of detecting the 720 (90x8) mutations from our panel; see the table below. To evaluate the predictive power of our analysis, we compute specificity as True Negative / (True Negative + False Positive) and sensitivity as True Positive/ (True Positive + False Negative). In the long run, it will be our objective to perform liquid biopsies only. Specifically, the current tumor-informed procedure includes a WES step of resected tissue to identify the mutations foreach patient, which could be omitted and instead the entire unique molecular identifiers sequencing (UMI-seq) panel could be analyzed for significant mutations. Aggregating evidence from multiple mutations (non-tumor informed mutation score): For classification purposes, we propose to compute a score for each patient sample based on aggregating evidence from multiple mutations present in the plasma. We will assign different weights for each mutation based on a metric (Matov et al. 2011), such as the Mahalanobis distance, which accounts for the heterogeneity of each mutation in samples from healthy individuals. In addition, we assign another weighting factor based on the likelihood to have a mutation at the concrete position with the gene and the probability the gene to be mutated based on CRC statistics and will add a weight for short/long vs middle-length reads to further distinguish reads originating from tumor cells. We formulate our score as a function of multiple mutations, between five and eight, which collectively determine the presence of colorectal neoplasm. Each mutation is presented as its MAF weighted by the dispersion of the read counts for the same position in a cohort of normal samples. For positions with zero variance across the panel of normal (PON) samples, the zero is replaced with a very small value calculated, as the minimal value of the variance matrix divided by 1e+07. Additionally, we utilize a second weight term based on the probability of each of the mutated genes to carry a mutation in that position. For the analysis of patient samples, the Poisson distributions of the score values for a cohort of healthy individuals (normal) and CRC patients will be compared to determine a cut-off threshold score value. Further, we will add weights based on the DNA fragment length distribution for particular on-panel and off-panel genomic positions. See: github.com/amatov/TumorAgnosticUMI
... VAF is a metric of the fraction of alleles carrying a specific genomic alteration. In detail, VAF refers to the proportion of sequencing reads that support a specific variant allele relative to the total number of reads within a genomic locus [9,10]. By calculating this genomic biomarker, VAF may represent a surrogate for mutation clonality and can act as a tool to evaluate the genomic heterogeneity of tumors [10]. ...
... Following library preparation, DNA fragments are sequenced using NGS platforms. Finally, to assess VAF, bioinformatics pipelines are employed to align the sequencing reads to the reference genome, call variants, and calculate the VAF for each variant [9]. ...
... If the VAF is approximately 50% or 100%, this variant may represent a germline mutation with heterozygous or homozygous loci, respectively [1]. VAF in somatic testing can vary depending on tumor heterogeneity and contamination from normal cells [2]. In this patient's case, subsequent germline testing was performed with an 84-gene panel using Invitae and was negative for a SDHB variant. ...
Cardiac paragangliomas are extremely rare tumors derived from chromaffin cells of the neural crest. Succinate dehydrogenase B (SDHB) mutations are associated with metastatic potential and potentially worse prognosis. Here we describe the case of a 64-year-old man who presented with chest pain, fatigue, and weight loss. Cardiac workup revealed a nearly 7-cm cardiac mass in the right lateral wall. Incisional biopsy demonstrated paraganglioma. Plasma free normetanephrine and chromogranin A were elevated. A DOTATATE positron emission tomography/computed tomography (PET/CT) revealed avidity of the mass with no evidence of distant metastases. Next-generation sequencing of the specimen demonstrated a variant of unknown significance of SDHB at H244D. Germline testing was negative. Surgical resection was aborted due to involvement of critical structures of the heart. Systemic treatment with the multi-tyrosine kinase inhibitor cabozantinib was initiated with subsequent improvements in biochemical markers as well as reductions in maximum standardized uptake value (SUVmax) on Ga-68 DOTATATE PET/CT. After 5 months of cabozantinib, he was unable to tolerate the side effects and external beam radiation therapy was completed. In this case, we report a novel somatic SDHB mutation at H244D in a sympathetic paraganglioma presenting as a cardiac mass.
... In addition, targeted therapy with a RET inhibitor such as prelsetinib resulted in a reduction of metastatic lesion volume and a concomitant decrease in VAF value. As NGS provides a near random sample of the DNA molecules, VAF is thus a surrogate measure of the proportion of DNA molecules in the original specimen carrying the variant (45). A retrospective cohort analysis of 561 patients with advanced solid cancers, including NSCLC, pancreatic, prostate, colon and breast cancer, reported that there was a positive association between maximal VAF levels with the diameter or volume of all lesions, and in patients with undetectable ctDNA, a lower ctDNA VAF was associated with improved survival (46). ...
Ongoing investigations of targeted therapeutic agents and their increased clinical applications, together with research in genomics and proteomics, have explored a variety of novel approaches for treatment of lung cancer, and 'molecular subtypes' have been defined based on specific actionable genetic aberrations. Liquid biopsies, including circulating tumor DNA (ctDNA) testing, are of value for cancer diagnosis and comprehensive genomic profiling, such as the identification of cancer subtypes and major genetic alterations in cancer cells. The case of a 66-year-old male patient with newly-diagnosed driver mutation-negative advanced non-small cell lung cancer (NSCLC) who underwent conventional therapy is described in the present report. The patient underwent regular monitoring, including continuous ctDNA analysis, imaging and assessment of tumor marker levels such as carcinoembryonic antigen (CEA). The patient initially presented with deep vein thrombosis which affected both lower extremities and without any symptoms in the lung, with a positron emission tomography scan identifying irregular pulmonary nodules in the right lower lobe and enlarged right supraclavicular lymph nodes. Subsequent ultrasound-guided fine-needle aspiration with nodule biopsy indicated advanced unresectable disease at stage IIIB based on the Tumor-Node-Metastasis staging system by the American Joint Committee on Cancer. Next-generation sequencing of tumor tissue and peripheral blood confirmed driver mutation-negative genes, including epidermal growth factor receptor, rat sarcoma, ALK receptor tyrosine kinase, ROS1 proto-oncogene receptor tyrosine kinase and rearrangement during transfection (RET). After 5 years of chemoradiotherapy and surveillance of ctDNA and CEA levels, detectable kinesin family member 5B (KIF5B)-RET fusion in ctDNA and rising CEA levels prompted early scans, which identified disease progression. The patient subsequently received the oral RET inhibitor pralsetinib, with treatment being currently ongoing for ≥17 months without detectable KIF5B-RET ctDNA or elevated CEA levels, with an ongoing minor response and stable disease based on Response Evaluation Criteria in Solid Tumors v1.1 on imaging. The present case illustrates the potential role of on-therapy circulating tumor biomarker monitoring as a non-traumatic method to evaluate therapy response and detect early disease progression in patients with advanced NSCLC. Integration of circulating tumor biomarker testing into the management of patients with advanced NSCLC requires additional prospective studies to actively assess and elucidate optimal treatment strategies.
... Although the DNA quality of the degenerative component was low, the VAF and total passing filter (PF) reads of this component did not differ from those of samples with well-preserved morphology. VAF is expressed as the percentage of sequence reads observed by matching a specific mutation derived from the overall coverage of a given locus [21,22]. When used in conjunction with the percentage of cancer cells, VAF can be beneficial in interpreting the mutation type and distribution [7,21,22]. ...
... VAF is expressed as the percentage of sequence reads observed by matching a specific mutation derived from the overall coverage of a given locus [21,22]. When used in conjunction with the percentage of cancer cells, VAF can be beneficial in interpreting the mutation type and distribution [7,21,22]. In histological evaluations, non-tumor cells with degenerated features are not counted as tumor content because their DNA is considered incapable of amplification, and it does not enhance the total PF read number. ...
Degenerated tissues are frequently observed in malignant tumors, but are not analyzed. We investigated whether nuclear streaming and necrosis samples could be used for genetic analysis to expand the sample pool. A total of 81 samples were extracted from small cell carcinoma and lymphoma FFPE tissue blocks and classified into three histological cohorts: 33 materials with well-preserved tumor morphology, 31 nuclear streaming samples, and 17 necrosis samples. DNA and RNA integrity numbers, percentage of RNA fragments with >200 nucleotides, and next-generation sequencing quality metrics were compared among the cohorts. DNA quality did not significantly differ between nuclear streaming materials and materials with well-preserved morphology, whereas that of the necrosis samples was inferior. RNA quality decreased in the following order: materials with well-preserved morphology > nuclear streaming > necrosis. The sequencing metrics did not differ significantly between the nuclear streaming samples and materials with well-preserved morphology, and reliable variants were detected. The necrosis samples extracted from resections exhibited sequencing failure and showed significantly fewer on-target aligned reads and variants. However, variant allele frequency did not differ among the cohorts. We revelated that DNA in nuclear streaming samples, especially within biopsies, could be used for genetic analysis. Moreover, degenerated non-tumor cells should be counted when evaluating tumor content to avoid misinterpreting the variant allele frequency.
