Frequency Of Non-synonymous Mutations Research Articles

Abstract P5-01-03: Mouse-intraductal (MIND): An in vivo model for studying the underlying mechanisms of DCIS malignancy

Abstract Background: Due to widespread adoption of screening mammography, there has been a significant increase in new diagnoses of ductal carcinoma in situ (DCIS). However, DCIS prognosis remains unclear. Methods: To address this gap, we developed an in vivo model, Mouse-INtraDuctal (MIND), by which patient-derived DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. The source of DCIS samples reflected clinical practice as predominantly high grade (70%), but also included intermediate grade (27%) and low grade (3%). Thirty-seven patient samples were injected into 202 mouse mammary glands and evaluated for invasive progression at a median duration of 9 months. The expression of clinically relevant biomarkers (ER, PR, Ki-67, HER2 and p53) on patient DCIS FFPE sections and xenografts’ extent of in vivo growth were evaluated for their utility in predicting DCIS invasive progression in the xenografts. Targeted DNA sequencing using Tempus XT oncology assay was used on patient DCIS in order to find a unique pattern of cancer related gene mutations that predicted DCIS invasiveness in the xenografts. Results: Similar to human DCIS, the cancer cells formed in situ lesions inside the mouse mammary ducts and mimicked all histologic subtypes including micropapillary, papillary, cribriform, solid, and comedo. Among 37 patient samples injected into 202 xenografts, at median duration of 9 months, 20 samples (54%) injected into 95 xenografts showed in vivo invasive progression while 17 (46%) samples injected into 107 xenografts remained noninvasive. Among the 20 samples that showed invasive progression in the MIND model, 9 patient samples injected into 54 xenografts exhibited a mixed pattern in which some xenografts showed invasive progression while others remained noninvasive. The mean duration of follow-up was not significantly different among the progressed, non-progressed or mixed groups (ANOVA; p-value=0.44). Among the clinically relevant biomarkers, only elevated progesterone receptor expression in patient DCIS and extent of in vivo growth in xenografts predicted an invasive outcome in the xenografts. Tempus XT oncology assay was used on 16 patient DCIS FFPE sections including eight patient DCIS that showed invasive progression (P), five patient DCIS that remained non-invasive (NP) and three patient DCIS that showed a mixed pattern (M) in the xenografts. Variant severity was called using SnpSift which is a program for identifying phenotype-relevant variants and predicts the severity of SNPs based on their effect on gene expression and function. COSMIC database was also used to identify mutations with pathogenic scores >0.5. Analysis of the frequency of cancer related pathogenic mutations showed no significant differences (P=27, NP=79, M=43, Kruskal-Wallis: P value=>0.05). There were also no differences in the frequency of low, moderate or high severity mutations (P= 25 highly severe, 120 moderately severe and 50 low-severity; NP=9 highly severe, 58 moderately severe and 28 low severity; M=3 highly severe, 33 moderately severe and 14 low severity; Kruskal-Wallis; P value >0.05). Conclusions: Highly severe and pathogenic variants in the patient’s DCIS were not associated with whether the DCIS developed into invasive lesions or remained non-invasive in the MIND models. These results are in agreement with previous studies that showed no significant differences in frequency of non-synonymous mutations and CNAs when comparing pure DCIS with synchronous IDC-DCIS. The MIND models are in immunocompromised mice, so the contribution of the immune system to DCIS progression may not recapitulate cancer progression in an immunocompetent state. However, the MIND model suggests that genetic changes in the DCIS are not the primary driver for the development of invasive disease. Citation Format: Aditi Rastogi, Jerome Lin, Yan Hong, Darlene Limback, Hanan S. Elsarraj, Haleigh Harper, Haley Haines, Hayley Hansford, Michael Ricci, Carolyn Kaufman, Emily Wedlock, Mingchu Xu, Jianhua Zhang, Lisa May, Terri Cusick, Marc Inciardi, Mark Redick, Jason Gatewood, Onalisa Winblad, Allison Aripoli, Ashley Huppe, Christa Balanoff, Jamie Wagner, Amanda Amin, Kelsey E. Larson, Lawrence Ricci, Ossama Tawfik, Hana Razek, Ruby O Meierotto, Rashna Madan, Andrew K. Godwin, Jeffrey Thompson, Susan G. Hilsenbeck, Andy Futreal, Alastair Thompson, E. Shelley Hwang, Fang Fan, Nicholas Navin, Fariba Behbod, Grand Challenge PRECISION Consortium. Mouse-intraductal (MIND): An in vivo model for studying the underlying mechanisms of DCIS malignancy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-03.

P-050: Whole Exome Sequencing provides novel insights in synonymous and non-synonymous mutational landscapes of Multiple Myeloma

<h3>Background</h3> Recent NGS based genomic studies have established the role of temporal precedence of mutational signatures that initiate and drive pathogenesis of Multiple Myeloma (MM). Almost 100 driver genes have been identified and investigated extensively for driver mutations in MM but their clinical impact remains underestimated. While coding nonsynonymous mutations have been well characterized, the synonymous mutations that can regulate and impact gene function have not been investigated in depth. In this study, we have co-analyzed both synonymous and nonsynonymous mutations in whole exomes of MM and compared with reported literature for ethnic differences, if any. <h3>Methods</h3> Whole exome sequencing was performed on malignant plasma cells (PCs) obtained from 71 newly diagnosed MM patients. <h3>Results</h3> Both synonymous (S) and nonsynonymous (NS) substitutions were analyzed. The C>T substitutions were most common. Other than age linked SBS5, SBS1, ID2, ID8; mutational signatures such as APOBEC related DBS11, defective DNA mismatch repair related SBS15 were also identified. The patients had an average tumor mutation burden of ~10. Nearly half of nonsynonymous mutations were clonal. The most common NS mutations were missense (~18000) followed by those in splice (~3000), 3' or 5'UTR regions (~1500) and nonsense or frameshift (~200). The most frequently mutated oncogenes with NS mutations included KRAS, BRAF, DIS3, TET2 and CREBBP while the commonly NS mutated tumor suppressor genes were KMT2C, ATM, KMT2B and TP53. Frequency of NS mutations in TP53 (~7%) in this study were significantly different from those reported for African American (1.6%) and more closer to Caucasian (6.3%). Mutations (NS) in PARP4 were found at a frequency of 3.9% which is similar to African American (3.9%) and different from Caucasian (1%). In addition, 6 different synonymous mutations were also observed in this gene at a frequency ~ 2% in MM patients in this study. Gene IRF4 also had both NS (3%) and S (~1.5%) mutations. The IRF4 NS mutation frequency (3%) is comparable with Caucasian (3.2%) while it doesnot exist among African Americans (0%). <h3>Conclusion</h3> In analogy, oncogenes (e.g., LMO2, ARHGEF28, NOTCH1, RET, NOTCH2, DDR2) and tumor suppressor genes (like KMT2C, EP400, RASA2, CYLD) were enriched in synonymous mutations. A consolidated analysis of co-occurrence of both synonymous and NS mutations may help revisit their mechanistic oncogenic and clinical significance in MM.

Genetic mutational analysis of β-catenin gene affecting GSK-3β phosphorylation plays a role in gallbladder carcinogenesis: Results from a case control study

This manuscript has reported different mutations of β-catenin gene in gallbladder cancer patients which affect GSK-3β phosphorylation site. PurposeGallbladder carcinoma (GBC) is a relatively rare and fatal cancer with poor prognosis. The molecular mechanism of gallbladder carcinogenesis is still not clear. Wnt signaling pathway is a highly conserved pathway that regulates proliferation, differentiation, migration, genetic stability, apoptosis, and stem cell renewal. β-catenin plays major role in Wnt signaling and aberrations in β-catenin has found to be involved in several cancers pathogenesis. This study was carried out to document the mutations of β-catenin gene in gallbladder cancer and to evaluate its possible role in gallbladder carcinogenesis. MethodsPCR-SSCP (Single Stranded Conformation Polymorphism) for ctnnb1 was performed in 50 patients each of gallbladder cancer, cholelithiasis and 50 healthy controls. Samples that showed variation in banding pattern were sequenced. ResultsVariation in banding pattern was observed in 9 (18%) samples of GBC, 4 (8%) of cholelithiasis and 2 (4%) of control. Sequencing analysis showed 9 novel mutations of ctnnb1 in exon 3 in 18% of gallbladder cancer (χ2 = 5.778; p < 0.05). Six point mutations, 1 deletion and 1 insertion mutation were found in 9 cases of gallbladder cancer. All point mutations were mis-sense mutation that affected highly conserved serine or threonine region that is important for GSK-3β phosphorylation. ConclusionFindings of the study suggests that high frequency of non synonymous mutations of β-catenin gene (ctnnb1) occurs in patients with gallbladder cancer. As these mutations mainly effect GSK 3β phosphorylation, it may be concluded that this might be an important step in gallbladder carcinogenesis. These β-catenin mutations lead to Wnt pathway activation and appear to have a role in progression from inflammation to cancer in gallbladder.

Evidence for reduced selection pressure on the hepatitis B virus core gene in hepatitis B e antigen-negative chronic hepatitis B

The mechanisms underlying the high levels of hepatitis B virus (HBV) replication that cause hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (e-CHB) are unknown. Impaired anti-HBV immunity, which may be measurable as a relaxation of selection pressure on the virus, is possible. A group of Tongans (n = 345) with a chronic HBV infection, including seven with e-CHB, were genotyped at HLA class I. The repertoire of HBV core-gene codons under positive selection pressure was defined by phylogenetic analysis (by using the paml program) of 708 cloned sequences extracted from the 67 of these 345 subjects with the same repertoire of HLA class I alleles as the seven e-CHB individuals and matched controls (see below). The frequency of non-synonymous mutations at these codons was measured in longitudinal data from 15 subjects. Finally, the number of non-synonymous mutations at these codons was compared in seven groups comprised of one subject with e-CHB and 1-3 HLA class I-matched controls with an inactive, HBeAg-negative chronic HBV infection (e-InD). Nineteen codons in the core gene were under positive selection pressure. There was a high frequency of new non-synonymous mutations at these codons (P<0.0001) in longitudinal data. The mean number of these 19 codons with non-synonymous mutations was lower (P = 0.02) in HBV from subjects with e-CHB (4.4±0.5 codons per subject) versus those with e-InD (6.4±0.4 codons per subject). There is a subtle relaxation in selection pressure on the HBV core gene in e-CHB. This may be due to impaired antiviral immunity, and could contribute to the high levels of viral replication that cause liver inflammation in this disease.

CD8 + T-Cell Response Promotes Evolution of Hepatitis C Virus Nonstructural Proteins

Hepatitis C virus (HCV) acquires mutations that allow it to escape the CD8+ T-cell response, although the extent to which this process contributes to viral evolution at the population level is not clear. We studied viral adaptation using data from a large outbreak of HCV genotype 1b infection that occurred among women immunized with contaminated immunoglobulin from 1977 to 1978. The HCV nonstructural protein coding regions NS3-NS5B were sequenced from 78 patients, and mutations were mapped according to their location inside or outside previously described CD8+ T-cell epitopes. A statistical approach was developed to identify sites/regions under reproducible selection pressure associated with HLA class I. The frequency of nonsynonymous mutations was significantly higher inside previously described CD8+ T-cell epitopes than outside-particularly in NS3/4A and NS5B. We identified new regions that are under selection pressure, indicating that not all CD8+ T-cell epitopes have been identified; 6 new epitopes that interact with CD8+ T cells were identified and confirmed in vitro. In some CD8+ T-cell epitopes mutations were reproducibly identified in patients that shared the relevant HLA allele, indicating immune pressure at the population level. There was statistical support for selection of mutations in 18 individual epitopes. Interestingly, 14 of these were restricted by HLA-B allele. HLA class I-associated selection pressure on the nonstructural proteins and here predominantly on NS3/4A and NS5B promotes evolution of HCV. HLA-B alleles have a dominant effect in this selection process. Adaptation of HCV to the CD8+ T-cell response at the population level creates challenges for vaccine design.

Patterns of intra- and interhost nonsynonymous variation reveal strong purifying selection in dengue virus.

Considerable uncertainty surrounds the evolutionary rates of and selection pressures acting on arthropod-borne RNA viruses (arboviruses). In particular, it is unclear why arboviruses such as dengue virus show substantial genetic variation within individual humans and mosquitoes yet low long-term rates of amino acid substitution. To address this question, I compared patterns of nonsynonymous variation in populations of dengue virus sampled at different levels of evolutionary divergence. Although nonsynonymous variation was abundant in viral populations within individual humans, there was a marked reduction in the frequency of nonsynonymous mutations in interhost comparisons. Moreover, intrahost genetic variation corresponded to a random pattern of mutation, and most of the sites that exhibited nonsynonymous variation within hosts were invariant at deeper phylogenetic levels. This loss of long-term nonsynonymous variation is the signature of extensive purifying selection such that more than 90% of all nonsynonymous mutations are deleterious. Consequently, although arboviruses are able to successfully adapt to diverse cell types, they are characterized by a high rate of deleterious mutation.