Normal DNA Samples Research Articles

Exploring the denaturations in cancer and non-cancer DNA molecules by optical absorption, thermal, and electric measurements: A case study

In this article, we carried out the temperature dependent UV-Visible (UV-Vis) spectroscopy, differential scanning calorimetry (DSC), and electrical studies for normal and cancerous (glioma) DNA samples of single patient. Based on this method, we were able to monitor the denaturation process and thermal stability in these molecules. From the temperature dependent optical absorption data, we calculated various optical parameters for these two types of samples. The optical band gap of these samples were also estimated and discussed as per the experimental conditions. The various optical parameters calculated indicate that mutated (tumor) DNA is less stable than the normal one. From the DSC data, clear melting peaks were observed for the tumor and normal samples. Also various thermodynamic parameters like change in enthalpy (ΔH), entropy (ΔS), and specific heat (Cp) were estimated. From the thermal study, it seems that the tumor DNA is less stable. Further from the electrical or current-voltage (I-V) characteristics data, the resistance for normal DNA decreases with temperature. But for tumor sample, it show anomalous behavior (like decreasing and then increasing trend) with temperature. For electrical transport, small polaron hopping could be the possible transport mechanism in the current sample. Here from these studies, the tumor sample seems more disordered, and structural fluctuations due to the speculated structure could be the best reason for this behavior. If such kind of molecular (at nano scale range) studied are done more vividly, then these calculated parameters of the molecule could be explored for further confirmation/diagnostics of the diseases in addition to clinical investigations.

Application of the TaqMan ARMS-PCR Approach for Genotyping Drug-Induced Hearing Loss Using Dried Blood Samples.

A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G mutant-type plasmids were constructed, respectively. In this study, a PCR method based on the TaqMan amplification refractory mutation system was proposed to detect mtDNA 1555A>G. A common upstream primer, a common TaqMan probe, and two downstream allele-specific primers with mismatched bases were designed. One-step amplification and detection of the wild-type and mutant type at the 1555 site were realized for the deafness-related gene through two reactions. Based on this detection method, the minimum detection limit of the wild-type and mutant type detection systems for plasmids was 50 copies/μL. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/μL. In the normal DBS DNA sample, the detection limit of the mutation abundance reached 0.78%. The specificity of the detection method was 100%, and the coefficient of variation was less than 3.36%. This approach was validated using clinical DNA extracted from 113 DBS samples of newborns. Additionally, it showed 100% agreement with bi-directional Sanger sequencing. It can be used as an optional method for the clinical detection of deafness-related genes.

Vibrational, Optical, Electrochemical, and Electrical Analysis of Normal and Cancer DNA

In the current article, we did characterizations like Fourier Transform Infrared (FT-IR) Spectroscopy, UV-Visible (UV–vis) spectroscopy, Photoluminescence (PL) spectroscopy, Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS), Current-Voltage (I-V) characteristics, dielectric spectroscopy, and transient time spectroscopy on normal and cancerous (esophagus) DNA samples. FT-IR confirms the associated functional groups of DNA. Also a significant change in these groups with mutations is observed. From the analysis of UV data, the various optical parameters like optical band gap, disorder energy were estimated and discussed. PL data demonstrate the various emissions and are described as per the existing structure of the molecule. From the CV plots, the energy levels, like highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) were also calculated. The EIS data interpretations show well developed changes in various parameters related with nature of the present molecules. Also from I-V characteristics, visible variations were observed and discussed. From the dielectric spectroscopy, a drastic change in the data were seen and described. Dynamic measurements like transient time demonstrates a vital impact on charge storage and hence on the rise and fall time of the molecules. The various calculated parameters related with these methods show changes with normal and mutated DNA. These observed properties shown by these techniques could be explored for further confirmation of the diagnostic of the disease.

Melt-Encoded-Tags for Expanded Optical Readout in Digital PCR (METEOR-dPCR) Enables Highly Multiplexed Quantitative Gene Panel Profiling.

Digital PCR (dPCR) is an important tool for precise nucleic acid quantification in clinical setting, but the limited multiplexing capability restricts its applications for quantitative gene panel profiling. Here, this work describes melt-encoded-tags for expanded optical readout in digital PCR (METEOR-dPCR), a simple two-step assay that enables simultaneous quantification of a large panel of arbitrary genes in a dPCR platform. Target genes are quantitatively converted into DNA tags with unique melting temperatures through a ligation approach. These tags are then counted and distinguished by their melt-curve profiles on a dPCR platform. A multiplexing capacity of M^N, where M is the number of resolvable melting temperature and N is the number of fluorescence channel, can be achieved. This work validates METEOR-dPCR with simultaneous DNA copy number profiling of 60 targets using dPCR in cancer cells, and demonstrates its sensitivity for estimating tumor fraction in mixed tumor and normal DNA samples. The rapid, quantitative, and highly multiplexed METEOR-dPCR assay will have wide appeal for many clinical applications.

Abstract 3039: Evaluation of intra-tumoral pks+ E. coli, enterotoxigenic B. fragilis and Fusobacterium nucleatum, overall and in early onset disease, in colorectal cancer cases

Abstract Background: Colorectal cancer (CRC) incidence has increased in those 50 years or younger compared to older individuals in recent decades. While the intra-tumoral microbiome is a potent facilitator of tumorigenesis, the role of the microbiome in early onset (eo)CRC is unclear. Identification of the intra-tumoral microbiome may enhance prediction of adverse outcomes to improve survival of CRC overall age groups and eoCRC. Methods: We performed targeted sequencing of tumor and matched normal DNA samples from 3695 colorectal cancer cases within the Genetics and Colorectal Cancer Consortium (GECCO) and the Colon Cancer Family Registry (CCFR). Tumor DNA was extracted from formalin fixed paraffin embedded (FFPE) or fresh frozen tissue. Normal tissue DNA was extracted from blood or normal surrounding colorectal tissue. We designed probes for the bacterial pathogens enterotoxigenic Bacteroides fragilis (ETBF), polyketide synthase positive E. coli (pks+ EC), and Fusobacterium nucleatum (Fn). We used logistic regression to assess tumor-associated bacteria and clinical parameters, tumor molecular characteristics, and tumor stage adjusted for age, sex, and study and a Cox promotional hazard to assess association with CRC-specific survival, overall and in eoCRC, adjusted for age at diagnosis, sex, mutational burden, and microsatellite instability (MSI) and stratified baseline hazards by study. Survival data were available for 2357 and 308 patients, overall and in eoCRC, respectively. Results: The prevalence of different bacteria in tumors differed slightly overall vs. in eoCRC tumors: ETBF (5.2%, 6.3%), pks+ EC (12.9%, 14.8%), and Fn (10.4%, 8.2%). Overall, men had a lower probability of tumors with Fn (odds ratio (OR)=0.8, 95% confidence interval (CI)=0.64-0.99, p=0.04) but higher odds of tumors with pks+ EC (OR=1.38, CI=1.14-1.68, p=0.001), compared to women. The odds of prevalent Fn were higher for proximal vs distal tumors (OR=1.76, CI=1.38-2.26, p<0.001), but there was no association between tumor site and pks+ EC or ETBF. MSI-high tumors were more likely to carry Fn (OR=2.79, CI=2.08-3.75, p<0.001) and less likely to carry pks+ EC (OR=0.63, CI=0.44-0.88, p=0.008) when compared to patients with MSS tumors. In patients with non-hypermutated tumors, we observed that pks+ EC positive tumors were associated with better CRC-specific survival (HR=0.73, CI=0.57-0.93, p=0.01), whereas ETBF positive tumors (HR=1.99, CI=1,18-3.36,p=0.009) and Fn positive tumors (HR=1.33, CI=1.04-1.72, p=0.03) were associated with poorer survival. In eo-CRC patients, Fn positive status was associated with poorer survival (HR=2.4, CI= 1.056 - 5.492, p=0.026). Conclusions: We showed that the intra-tumoral microbiome differs in distribution across patient populations and patient survival over all age groups and, specifically, in eoCRC. Citation Format: Meredith A. Hullar, Keith R. Curtis, Tabitha Harrison, Yi Lin, Robert Steinfelder, Sonja I. Berndt, Daniel D. Buchanan, Andrew T. Chan, David A. Drew, Jane Figueiredo, Amy J. French, Tabitha A. Harrison, Mark A. Jenkins, Johannes Melaku, Victor Moreno, Tomotaka Ugai, Shuji Ogino, Conghui Qu, Chenxu Qu, Steven Thibodeau, Syed Zaidi, Amanda I. Phipps, Ulrike Peters, on behalf of GECCO and CCFR investigators. Evaluation of intra-tumoral pks+ E. coli, enterotoxigenic B. fragilis and Fusobacterium nucleatum, overall and in early onset disease, in colorectal cancer cases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3039.

Nanoscale Investigation of DNA Demethylation in Leukemia Cells by Means of Ultrasensitive Vibrational Spectroscopy.

DNA methylation is a crucial epigenetic hallmark of cancer development but the experimental methods able to prove nanoscale modifications are very scarce. Over time, Raman and its counterpart, surface-enhanced Raman scattering (SERS), became one of the most promising techniques capable to investigate nanoscale modifications of DNA bases. In our study, we employed Raman/SERS to highlight the differences between normal and leukemia DNA samples and to evaluate the effects of a 5-azacytidine treatment on leukemia cells. To obtain spectral information related to DNA base modifications, a DNA incubation step of 4 min at 94 °C, similar to the one performed in the case of RT-PCR experiments, was conducted prior to any measurements. In this way, reproducible Raman/SERS spectra were collected for all genomic DNA samples. Our Raman results allowed discrimination between normal and cancer DNAs based on their different aggregation behavior induced by the distinct methylation landscape present in the DNA samples. On the other hand, the SERS spectra collected on the same DNA samples show a very intense vibrational band located at 1008 cm-1 assigned to a rocking vibration of 5-methyl-cytosine. The intensity of this band strongly decreases in cancer DNA due to the modification of the methylation landscape occurring in cancers. We believe that under controlled experimental conditions, this vibrational band could be used as a powerful marker for demonstrating epigenetic reprogramming in cancer by means of SERS.

Abstract PR008: Evaluation of intra-tumoral pathogenic bacteria pks+ E. coli, enterotoxigenic B. fragilis and fusobacterium nucleatum in 3695 colorectal cancer cases

Abstract Background: The microbiome in colorectal cancer (CRC) tumors is now recognized as a potent facilitator of tumorigenesis. Identification of the intra-tumoral microbiome may provide an avenue to enhance prediction of adverse outcomes and ultimately lead to improved survival of CRC, the second leading cause of cancer death. Methods: We performed targeted sequencing of tumor and matched normal DNA samples for 3695 colorectal cancer cases within the Genetics and Colorectal Cancer Consortium (GECCO) and the Colon Cancer Family Registry (CCFR). Tumor DNA was extracted primarily from formalin fixed paraffin embedded (FFPE) or from fresh frozen tissue. FFPE slides were dissected to ensure >70% tumor content. Normal DNA was primarily extracted from blood and otherwise normal surrounding colorectal tissue. We designed probes for bacterial pathogens including Bacteroides fragilis (ETBF), polyketide synthase positive E. coli (pks+ EC), enterotoxigenic and Fusobacterium nucleatum (Fn). Logistic regression was used to assess tumor-associated bacteria and various clinical parameters, tumor molecular characteristics, and tumor stage adjusted for age, sex, and study and a Cox promotional hazard to assess association with CRC-specific survival adjusted for age at diagnosis, sex, mutational burden and microsatellite instability (MSI) and stratified baseline hazards by study. Survival data were available for 2357 patients. Results: The prevalence of patients with bacteria in tumors varied for ETBF (5.2%), pks+ EC (12.9%), and Fn (10.4%). Men had a lower probability of tumors with Fn (odds ratio (OR)=0.8, 95% confidence interval (CI)=0.64-0.99,p=0.04) but higher odds of tumors with pks+ EC (OR=1.38, CI=1.14-1.68,p=0.001). Patients with proximal tumors had higher odds of carrying Fn (OR=1.76, CI=1.38-2.26, p<0.001) as did patients with MSI high tumors (OR 2.79, CI=2.08-3.74) p<0.001). Patients with MSI-high tumors had lower odds of carrying pks+ EC (OR=0.63, CI=0.44-0.88, p=0.008) when compared to patients with microsatellite stable tumors. Among non-hypermutated tumors, we observed that tumors positive for pks+ EC were associated with better CRC-specific survival (OR=0.73, CI=0.57-0.93,p=0.01 whereas in patients with ETBF positive tumors had shorter survival (OR=1.99,CI=1,18-3.36,p=0.009) as did patients with tumors positive for Fn (OR=1.33, CI=1.04-1.72, p=0.03). Conclusions: In this largest investigation of intra-tumoral bacteria in CRC, we demonstrate that inclusion of pathogenic intra-tumoral microbiome may enhance prediction of survival and can identify a subset of patients with CRC that may benefit from targeted therapies that modulate the tumor microbiome. Citation Format: Meredith A. J. Hullar, Keith R. Curtis, Tabitha A. Harrison, Yi Lin, Robert S. Steinfelder, Sonja I. Berndt, Daniel D. Buchanan, Andrew T. Chan, David Drew, Jane C. Figueiredo, Amy J. French, Ellen L. Goode, Mark A. Jenkins, Yohannes A. Melaku, Victor Moreno, Shuji Ogino, Stephen N. Thibideau, Amanda I. Phipps, Ulrike Peters. Evaluation of intra-tumoral pathogenic bacteria pks+ E. coli, enterotoxigenic B. fragilis and fusobacterium nucleatum in 3695 colorectal cancer cases [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR008.

Abstract 6201: Cross-platform comparisons for DNA methylation sequencing

Abstract Introduction: Accurate, reproducible, and cost-effective NGS platform is essential to identify malignancy-related abnormal DNA methylation changes and translate them into clinical applications including cancer detection, prognosis, and surveillance. In this study we evaluated the performances by the MGISEQ-2000 sequencer in DNA methylation sequencing, using Illumina’s NovaSeq6000 as a benchmark. Materials and Methods: We performed both genome-wide methylation profiling and targeted methylation sequencing on reference DNA and clinical DNA samples. To minimize non-platform discrepancies, we prepared 2 libraries, one for NovaSeq600 and the other for MGISEQ-2000, from each DNA sample using the same experimental protocol except the last step, when we used indexed primers whose sequences and chemical modification are compatible with either NovaSeq600 or MGISEQ-2000 to amplify the corresponding library. We conducted standardized analyses on MGISEQ-2000’s reads quality, sequence bias, genome alignment and coverage, regional depth, limits of detection (LOD), etc. We compared methylation levels at CpG sites, and measurements of methylation haplotypes to assess the consistency in mapping and quantifying genome-wide and regional methylation by MGISEQ-2000 and between the 2 platforms. Furthermore, we analyzed inter-platform variations in classifying normal and malignant plasma DNA samples using targeted methylation sequencing data from either platform exclusively. Results: We prepared and sequenced libraries from over 100 DNA samples of normal or malignant tissue or blood samples for comparison, in addition to reference DNA. Overall, MGISEQ-2000 displayed high level of consistency, and little-to-no sequence bias in methylation sequencing. At genome-wide, regional, and individual-CpG levels, MGISEQ-2000 demonstrated high degree of concordance with NovaSeq6000 in quantifying DNA methylation, as the inter-platform Pearson correlation coefficients were consistently above 0.90 or higher for both targeted sequencing and whole-genome bisulfite sequencing, regardless of whether average methylation level (AMF) or methylation haplotype-specific metrics such as Methylation Haplotype Load (MHL) were used to quantify methylation. Additionally, MGISEQ-2000 is as sensitive as NovaSeq6000 at detecting spik-in methylation controls, as both reached an LOD of 0.1%. Lastly, MGISEQ-2000 produced similar results in classifying normal and malignant samples as NovaSeq6000, suggesting it had minimal distortion on the NovaSeq6000-based classifier. Conclusions: MGISEQ-2000 demonstrated comparable data quality, methylation-calling accuracy, and consistency as NovaSeq6000 in DNA methylation sequencing, supporting its application in future investigation on DNA methylation as cancer biomarkers. Citation Format: Mingyang Su, Jin Sun, Jianhua Ma, Jun Xia, Chengcheng Ma, Wei Li, Zhixi Su, Qiye He, Rui Liu. Cross-platform comparisons for DNA methylation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6201.

Consistency and reproducibility of large panel next-generation sequencing: Multi-laboratory assessment of somatic mutation detection on reference materials with mismatch repair and proofreading deficiency

IntroductionClinical precision oncology increasingly relies on accurate genome-wide profiling using large panel next generation sequencing; however, difficulties in accurate and consistent detection of somatic mutation from individual platforms and pipelines remain an open question. ObjectivesTo obtain paired tumor–normal reference materials that can be effectively constructed and interchangeable with clinical samples, and evaluate the performance of 56 panels under routine testing conditions based on the reference samples. MethodsGenes involved in mismatch repair and DNA proofreading were knocked down using the CRISPR-Cas9 technology to accumulate somatic mutations in a defined GM12878 cell line. They were used as reference materials to comprehensively evaluate the reproducibility and accuracy of detection results of oncopanels and explore the potential influencing factors. ResultsIn total, 14 paired tumor–normal reference DNA samples from engineered cell lines were prepared, and a reference dataset comprising 168 somatic mutations in a high-confidence region of 1.8 Mb were generated. For mutations with an allele frequency (AF) of more than 5% in reference samples, 56 panels collectively reported 1306 errors, including 729 false negatives (FNs), 179 false positives (FPs) and 398 reproducibility errors. The performance metric varied among panels with precision and recall ranging from 0.773 to 1 and 0.683 to 1, respectively. Incorrect and inadequate filtering accounted for a large proportion of false discovery (including FNs and FPs), while low-quality detection, cross-contamination and other sequencing errors during the wet bench process were other sources of FNs and FPs. In addition, low AF (<5%) considerably influenced the reproducibility and comparability among panels. ConclusionsThis study provided an integrated practice for developing reference standard to assess oncopanels in detecting somatic mutations and quantitatively revealed the source of detection errors. It will promote optimization, validation, and quality control among laboratories with potential applicability in clinical use.

Somatic Mutations Enriched in Cis-Regulatory Elements Affect Genes Involved in Embryonic Development and Immune System Response in Neuroblastoma.

Assessment of noncoding cis-regulatory variants and long-range interaction data highlight the combined effect of somatic mutations in regulatory elements in driving neuroblastoma.

Somatic loss of the remaining allele occurs approximately in half of CHEK2-driven breast cancers and is accompanied by a border-line increase of chromosomal instability.

Germline mutations in CHEK2 gene represent the second most frequent cause of hereditary breast cancer (BC) after BRCA1/2 lesions. This study aimed to identify the molecular characteristics of CHEK2-driven BCs. Loss of heterozygosity (LOH) for the remaining CHEK2 allele was examined in 50 CHEK2-driven BCs using allele-specific PCR assays for the germline mutations and analysis of surrounding single-nucleotide polymorphisms (SNPs). Paired tumor and normal DNA samples from 25 cases were subjected to next-generation sequencing analysis. CHEK2 LOH was detected in 28/50 (56%) BCs. LOH involved the wild-type allele in 24 BCs, mutant CHEK2 copy was deleted in 3 carcinomas, while in one case the origin of the deleted allele could not be identified. Somatic PIK3CA and TP53 mutations were present in 13/25 (52%) and 4/25 (16%) tumors, respectively. Genomic features of homologous recombination deficiency (HRD), including the HRD score ≥ 42, the predominance of BRCA-related mutational signature 3, and the high proportion of long (≥ 5bp) indels, were observed only in 1/20 (5%) BC analyzed for chromosomal instability. Tumors with the deleted wild-type CHEK2 allele differed from LOH-negative cases by elevated HRD scores (median 23 vs. 7, p = 0.010) and higher numbers of chromosomal segments affected by copy number aberrations (p = 0.008). Somatic loss of the wild-type CHEK2 allele is observed in approximately half of CHEK2-driven BCs. Tumors without CHEK2 LOH are chromosomally stable. BCs with LOH demonstrate some signs of chromosomal instability; however, its degree is significantly lower as compared to BRCA1/2-associated cancers.

Thymidylate synthase locus LOH in combination with genotype has prognostic and predictive significance in colorectal cancer.

The aim of the current study was to investigate the prognostic and predictive significance of polymorphisms in the thymidylate synthase (TS) gene, alongside the loss of heterozygocity (LOH) at this gene locus in patients with colorectal cancer. Genotyping was carried out for a variable number tandem repeat (VNTR) polymorphism in the TS 5'-untranslated region, a G/C single nucleotide polymorphism (SNP) located within this VNTR, and for TS LOH status in 246 colorectal cancer and paired normal DNA samples. The results were analyzed in relation to clinicopathological features, including the prognostic and predictive significance of TS genotype in patients who underwent curative surgery. Complete VNTR, SNP and LOH information for TS was obtained in 226 cases. No significant associations were observed between normal tissue TS genotype status and clinicopathological features. LOH of TS was observed in 58% of tumor samples and was associated with poor prognosis independently of clinical stage. Cases exhibiting TS LOH were classified into the three groups of 2R/loss, 3G/loss and 3C/loss. Patients with 3C/loss genotype status had poor outcomes when treated by surgery alone, but their survival was similar to patients with other genotypes following Fluorouracil (5-FU)-based adjuvant chemotherapy. The results suggested that LOH of the TS locus may be a significant prognostic factor in colorectal cancer, with the genotype of the residual allele also demonstrating an influence on prognosis. In conclusion, LOH status should be considered when TS genotype is explored as a potential prognostic and predictive marker for 5-FU-based adjuvant chemotherapy in colorectal cancer.

Assessment of Clonal Expansion Using CarcSeq Measurement of Lung Cancer Driver Mutations and Correlation With Mouse Strain- and Sex-Related Incidence of Spontaneous Lung Neoplasia.

Quantification of variation in levels of spontaneously occurring cancer driver mutations (CDMs) was developed to assess clonal expansion and predict future risk of neoplasm development. Specifically, an error-corrected next-generation sequencing method, CarcSeq, and a mouse CarcSeq panel (analogous to human and rat panels) were developed and used to quantify low-frequency mutations in a panel of amplicons enriched in hotspot CDMs. Mutations in a subset of panel amplicons, Braf, Egfr, Kras, Stk11, and Tp53, were related to incidence of lung neoplasms at 2 years. This was achieved by correlating median absolute deviation (MAD) from the overall median mutant fraction (MF) measured in the lung DNA of 16-week-old male and female, B6C3F1 and CD-1 mice (10 mice/sex/strain) with percentages of spontaneous alveolar/bronchioloalveolar adenomas and carcinomas reported in bioassay control groups. A total of 1586 mouse lung mutants with MFs >1 × 10-4 were recovered. The ratio of nonsynonymous to synonymous mutations was used to assess the proportion of recovered mutations conferring a positive selective advantage. The greatest ratio was observed in what is considered the most lung tumor-sensitive model examined, male B6C3F1 mice. Of the recurrent, nonsynonymous mouse mutations recovered, 55.5% have been reported in human tumors, with many located in or around the mouse equivalent of human cancer hotspot codons. MAD for the same subset of amplicons measured in normal human lung DNA samples showed a correlation of moderate strength and borderline significance with age (a cancer risk factor), as well as age-related cumulative lung cancer risk, suggesting MAD may inform species extrapolation.

Abstract LB090: Associations of somatically mutated genes and pathways with colorectal cancer specific survival in 4,500 colorectal cancer patients

Abstract Colorectal cancer (CRC) is a heterogenous disease that develops through somatic mutations in driver genes, leading to activation of diverse neoplastic pathways. To systematically examine if somatically mutated genes and pathways impact survival, we sequenced tumor and normal DNA samples for 4,512 CRC cases using a targeted panel. We performed Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CIs) for associations of disease-specific (DS)-survival with somatically mutated genes and pathways, adjusting for age and sex, and stratifying baseline hazards by study population. We assessed statistical significance using Bonferroni p-value thresholds to account for multiple testing of 214 genes (2.34x10-4) and 6 key CRC pathways (8.3x10-3). We limited analyses to non-silent mutations. We observed that DS-survival was significantly more favorable among individuals with hypermutated (HM) tumors, primarily consisting of microsatellite unstable and POLE-mutated tumors, as compared to those with non-hypermutated (NHM) tumors (HR=0.4, 95% CI: 0.3-0.5, p=1.2x10-17). BRAF V600E mutations were associated with poorer survival (HR=2.0, 95% CI: 1.6-2.5, p=1.6x10-10). This association was more pronounced among NHM tumors (HR=2.3, 95% CI: 1.8-2.9, p=4.3x10-12). We identified suggestive associations (p-values &amp;lt; 5.0x10-3 in overall or stratified analyses) between DS-survival and mutations in B2M, TP53, and SMAD4. Mutations in B2M may provide more favorable prognosis for survival (HR=0.5, 95% CI: 0.3-0.8, p=4.4x10-3), with similar effect sizes in HM and NHM tumors. Poorer survival may be associated with mutations in TP53 (HR=1.2, 95% CI:1.2-1.4, p=9.3x10-4) and SMAD4 (HR=1.3, 95% CI:1.1-1.6, p=3.2x10-3) in NHM tumors. We further observed statistically significant associations between survival and three mutated pathways: TP53/ATM (HR=1.2, 95% CI:1.1-1.4, p=9.0x10-4), receptor tyrosine kinases (RTK) and RAS (HR=1.3, 95% CI:1.1-1.5, p=4.5x10-5), and TGF-beta (HR=1.3, 95% CI:1.1-1.5, p=2.9x10-4). Findings for TP53/ATM and RTK/RAS were primarily due to mutations in one gene within each pathway (TP53 and BRAF, respectively). However, the TGF-beta pathway finding, which was more pronounced in NHM tumors, included two genes with p-values less than 0.05 (SMAD4 p=3.2x10-3 and TGFBR2 p=0.01). Despite our large sample size, relatively few somatically mutated genes were significantly associated with DS-survival. It may be that pathway-level testing affords more power for analyses and that larger sample sizes are needed. It is of interest that non-silent mutations in B2M, whose product is a component of the class I major histocompatibility complex, was associated with improved DS-survival, though our finding was not statistically significant after multiple testing correction so further exploration and replication is needed. Citation Format: Tabitha A. Harrison, Syed H. Zaidi, Conghui Qu, Amanda I. Phipps, Robert S. Steinfelder, Quang M. Trinh, Sonja I. Berndt, Daniel D. Buchanan, Peter T. Campbell, Andrew T. Chan, Kim F. Doheny, David A. Drew, Jane C. Figueiredo, Steven J. Gallinger, Marios Giannakis, Andrea Gsur, Marc J. Gunter, Michael Hoffmeister, Wen-Yi Huang, Paul J. Limburg, Victor Moreno, Polly A. Newcomb, Shuji Ogino, Ross L. Prentice, Tameka Shelford, Wei Sun, Stephen N. Thibodeau, Li Hsu, Ulrike Peters. Associations of somatically mutated genes and pathways with colorectal cancer specific survival in 4,500 colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB090.

Identification of the distinct genomic features in gastroesophageal junction adenocarcinoma and its Siewert subtypes.

Rates of gastroesophageal junction adenocarcinomas (GEJAs) have shown an alarming increase; however, the genetic background of GEJA and its Siewert classification have yet to be uncovered. Here, 60 paired tumor and normal DNA samples from GEJA patients were analyzed by whole-exome sequencing. Among them, 13 were Siewert type I, 14 were type II, and 33 were type III. A predominance of C/G>T/A substitutions was discovered in GEJA, followed by C/G>A/T substitutions. Notably, Siewert type I and type II/III display distinct sets of driver genes, mutational spectrum, and recurrently disrupted pathways. Siewert type I showed similarity to esophageal adenocarcinomas (EACs) and the chromosomal instability subtype of stomach adenocarcinomas, while Siewert type II/III showed similarity to the genomic stable subtype of stomach adenocarcinoma. We also found that mutation of FBXW7, a driver gene of GEJA, was enriched in Siewert type I. Our data identify differences between GEJA and stomach/EACs at the genomic level and provide evidence for differential treatment based on Siewert classification of GEJA. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Assessment of tumor suppressor promoter methylation in healthy individuals

BackgroundThe number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes.ResultsIn this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters.ConclusionsMethylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.

Whole-exome analysis of metaplastic breast carcinomas with extensive osseous differentiation.

Metaplastic breast carcinoma (MBC) is a rare type of triple-negative breast cancer that shows vast histological and genetic heterogeneity. Osseous differentiation can be found in different subtypes of MBC. Whether MBCs with osseous differentiation are underpinned by specific genetic alterations has yet to be defined. The aim of this study was to investigate the repertoire of somatic mutations and copy number alterations (CNAs) in three MBCs with extensive osseous differentiation. Tumour and normal DNA samples from three MBCs with extensive osseous differentiation were subjected to whole-exome sequencing. Somatic mutations, CNAs and mutational signatures were determined by use of a validated bioinformatics pipeline. Our analyses revealed clonal TP53 hotspot mutations associated with loss of heterozygosity of the wild-type allele coupled with mutations affecting genes related to the Wnt and/or the phosphoinositide 3-kinase-AKT-mammalian target of rapamycin pathways in all cases analysed. All cases showed a dominant mutational signature 1, with two cases showing a secondary signature 3 in addition to other features of homologous recombination DNA repair defects. The oncostatin M receptor gene, which plays a role in mesenchymal differentiation and bone formation, was found to be mutated in two MBCs with extensive osseous differentiation and in none of 35 previously published 35 MBCs. Our findings suggest that MBCs with osseous differentiation have somatic mutations similar to those of other forms of MBC.

Somalier: rapid relatedness estimation for cancer and germline studies using efficient genome sketches

BackgroundWhen interpreting sequencing data from multiple spatial or longitudinal biopsies, detecting sample mix-ups is essential, yet more difficult than in studies of germline variation. In most genomic studies of tumors, genetic variation is detected through pairwise comparisons of the tumor and a matched normal tissue from the sample donor. In many cases, only somatic variants are reported, which hinders the use of existing tools that detect sample swaps solely based on genotypes of inherited variants. To address this problem, we have developed Somalier, a tool that operates directly on alignments and does not require jointly called germline variants. Instead, Somalier extracts a small sketch of informative genetic variation for each sample. Sketches from hundreds of germline or somatic samples can then be compared in under a second, making Somalier a useful tool for measuring relatedness in large cohorts. Somalier produces both text output and an interactive visual report that facilitates the detection and correction of sample swaps using multiple relatedness metrics.ResultsWe introduce the tool and demonstrate its utility on a cohort of five glioma samples each with a normal, tumor, and cell-free DNA sample. Applying Somalier to high-coverage sequence data from the 1000 Genomes Project also identifies several related samples. We also demonstrate that it can distinguish pairs of whole-genome and RNA-seq samples from the same individuals in the Genotype-Tissue Expression (GTEx) project.ConclusionsSomalier is a tool that can rapidly evaluate relatedness from sequencing data. It can be applied to diverse sequencing data types and genome builds and is available under an MIT license at github.com/brentp/somalier.

Abstract 1598: Deep targeted sequencing of colorectal cancer cases to study associations of molecular subtypes with clinical, genetic, and lifestyle risk factors

Abstract Colorectal cancer (CRC) is the 3rd most common cancer in men and women, excluding skin cancer, and the 2nd leading cause of cancer death in the United States. In this biologically heterogeneous disease, a comprehensive molecular characterization is valuable for understanding tumorigenesis and studying associations with clinical, lifestyle, environmental, and germline genetic factors. The Colon Cancer Family Registry (CCFR) and Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) collected clinical, germline genetic, and epidemiologic data from over 125,000 CRC cases. In a subset of 2,500 CRC cases, we conducted targeted deep sequencing of tumor and matching normal DNA. The custom AmpliSeq sequencing panel includes 205 genes, which were prioritized from literature and analyses of existing CRC tumor whole exome sequencing data. Next-generation sequencing was conducted on Illumina Hi-Seq 2500, and mean sequence coverages of 748x and 287x were attained for tumors and normal DNA samples, respectively. So far, 1,705 cases have been analyzed. Strelka and MuTect were used to identify somatic single nucleotide variants, and VarsScan2, Verdict, and Strelka were used to call indels. Orthogonal technologies were used for validation and improving mutation calling algorithms. Approximately 18% of tumors were hypermutated, among which 70% exhibited microsatellite instability (MSI). Hypermutated tumors with MSI occurred more frequently in proximal colon compared to distal colon and rectum (OR, 10.10; 95% CI, 6.98-14.63; P&amp;lt;0.0001). Hypermutated tumors carried non-silent mutations in DNA mismatch repair genes (MSH2, MSH6, MLH1, MLH3, and PMS2), POLE, and POLD1. In POLE, in microsatellite stable hypermutated tumors, non-silent mutations were frequent in the exonuclease domain. Approximately 96% of non-hypermutated and 99% of hypermutated tumors contained non-silent mutations in genes in the Wnt/beta-catenin, p53, receptor tyrosine kinases/RAS, transforming growth factor-beta, and phosphatidylinositide 3-kinases pathways. Most tumors carried non-silent mutations in genes in more than one of these signaling pathways. The APC gene was the most significantly mutated gene in non-hypermutated tumors followed by TP53, KRAS, and PIK3CA. In hypermutated tumors, RNF43 was the most significantly mutated gene followed by BMPR2, APC, and BRAF. CRC-specific survival was significantly more favorable among individuals with hypermutated tumors, regardless of POLE mutation status (HR=0.36, 95% CI: 0.23-0.57). This survival benefit was attenuated in analyses of overall survival (HR=0.82, 95% CI: 0.65-1.02). This large dataset is being used to study associations with clinical, lifestyle, and environmental factors. This study will provide valuable information to develop better strategies for the prevention, diagnosis, and treatment of CRC. Citation Format: Syed H. Zaidi, Amanda I. Phipps, Tabitha A. Harrison, Robert S. Steinfelder, Quang Trinh, Barbara L. Banbury, Adilya Rafikova, Megan Van Tassel, Emma Reid, Stefanie Brezina, Marios Giannakis, Charles S. Fuchs, Li Hsu, Andrea Gsur, Shuji Ogino, Steven Gallinger, Polly A. Newcomb, Peter T. Campbell, Wei Sun, Thomas J. Hudson, Ulrike Peters. Deep targeted sequencing of colorectal cancer cases to study associations of molecular subtypes with clinical, genetic, and lifestyle risk factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1598.

Within-sample co-methylation patterns in normal tissues

BackgroundDNA methylation is an epigenetic event that may regulate gene expression. Because of this regulation role, aberrant DNA methylation is often associated with many diseases. Within-sample DNA co-methylation is the similarity of methylation in nearby cytosine sites of a chromosome. It is important to study co-methylation patterns. However, it is not well studied yet, and it is unclear to us what co-methylation patterns normal DNA samples have. Are the co-methylation patterns of the same tissue across several samples different? Are the co-methylation patterns of various tissues of the same sample different? To answer these questions, we conduct analyses using two sets of data: 3-sample-1-tissue (3S1T) and 1-sample-8-tissue (1S8T).ResultsTo study the co-methylation patterns of the two datasets, 3S1T and 1S8T, we investigate the following questions: How often does one methylation state change to other methylation states and how is this change associated with chromosome distance? Based on the 3S1T data, we find there is not significant co-methylation difference among the same spleen tissues of three different samples. However, the analysis results of 1S8T data show that there were significant differences among eight tissues of one sample. For both 3S1T and 1S8T data, we find that the no/low methylation state A and high/full methylation state D tend to remain the same along a chromosome region. We also find that the low/partial methylation state B and partial/high methylation state C tend to change to higher methylation states along a chromosome. Finally, we find that lengths of most co-methylation regions are very short with only a few hundred base pairs. In fact, only a small proportion of methylated regions are longer than 1000 base pairs.ConclusionsIn this paper, we have addressed a few questions regarding within-sample co-methylation patterns in normal tissues. Our statistical analysis results and answers may help researchers to better understand the biological process of DNA methylation. This may pave the way to develop better analysis methods for future methylation research.