Non-synonymous Somatic Mutations Research Articles

Tumour mutational burden: clinical utility, challenges and emerging improvements.

Tumour mutational burden (TMB), defined as the total number of somatic non-synonymous mutations present within the cancer genome, varies across and within cancer types. A first wave of retrospective and prospective research identified TMB as a predictive biomarker of response to immune-checkpoint inhibitors and culminated in the disease-agnostic approval of pembrolizumab for patients with TMB-high tumours based on data from the Keynote-158 trial. Although the applicability of outcomes from this trial to all cancer types and the optimal thresholds for TMB are yet to be ascertained, research into TMB is advancing along three principal avenues: enhancement of TMB assessments through rigorous quality control measures within the laboratory process, including the mitigation of confounding factors such as limited panel scope and low tumour purity; refinement of the traditional TMB framework through the incorporation of innovative concepts such as clonal, persistentor HLA-corrected TMB, tumour neoantigen load and mutational signatures; and integration of TMB with established and emerging biomarkers such as PD-L1 expression, microsatellite instability, immune gene expression profiles and the tumour immune contexture. Given its pivotal functions in both the pathogenesis of cancer and the ability of the immune system to recognize tumours, a profound comprehension of the foundational principles and the continued evolution of TMB are of paramount relevance for the field of oncology.

Subset of retinoblastoma tumours is associated with BRCA1/2 mutations

BackgroundWe investigated the potential association between pathogenic BRCA1/2 variants and retinoblastoma pathogenicity.MethodsIn this single-centre, retrospective case series, we performed hereditary cancer panel tests using blood samples for patients with retinoblastoma...

Genomic progression for local invasion of cutaneous squamous cell carcinoma from the superficial to the deep portion.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. While many treatments exist, our understanding of its genomic progression, especially from the epidermis to the deep dermis, remains limited. This study aims to identify genetic mutations associated with the progression of cSCC into the deep dermis, providing insights into its aggressive behavior and high-risk features. We performed high-depth whole-exome sequencing on 12 cSCC tissues, along with paired normal tissues from six patients, using microdissection techniques. The mutational analysis focused on identifying alterations enriched during cSCC progression. Gene Ontology enrichment analysis, immunohistochemical assays, and external single-cell RNA data were utilized for validation. A total of 8863 non-synonymous somatic mutations were identified in 4092 genes across the superficial and deep portions of cSCCs. Analysis of deep portion mutations revealed a significant correlation with gene ontology biological processes, particularly cell junction organization, and cell-cell adhesion. Clonal mutations in these processes were more prevalent in the deep portions, indicating their impact on the cSCC mutation landscape. Genetic evolution analysis identified 29 causal genes associated with dermal invasion in cSCC. We highlight somatic mutations in cSCC, revealing heterogeneity between superficial and deep regions. Altered genes in cell junction organization and cell-cell adhesion emerged as pivotal in dermal invasion. We identified 29 causal genes primarily in deep tumor regions. Our findings emphasize analyzing multiple tumor regions to capture varied mutational landscapes. These insights advance our understanding of cSCC progression, emphasizing genetic and cellular changes during tumor evolution.

Genomic analysis of oesophageal carcinoma (EC) to identify recurrent mutations in histone methyltransferases as a distinctive subset.

379 Background: Histone-lysine N-methyltransferase 2 (KMT2) family proteins methylate lysine 4 on the histone H3 tail at important regulatory regions in the genome and thereby impart crucial functions through modulating chromatin structures and DNA accessibility, which is associated with tumorigenesis and immune tolerance, indicating its possible correlation with the efficacy of immunotherapy. Recurrent mutations of KMT2 have been identified in EC, but data addressing the molecular features of KMT2 mutated (MT) EC are lacking. We aimed to understand the molecular profile of KMT2 -MT EC. Methods: A total of 787 oesophageal carcinoma [adenocarcinoma (EAC), N=604; squamous cell carcinoma (ESCC), N=183] were analyzed using next-generation sequencing (NGS) and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous mutations, and mismatch repair deficiency (dMMR)/microsatellite instability-high (MSI-H) status was evaluated by a combination of IHC, Fragment analysis and NGS. Results: The mutation spectrum of KMT2A/C/D was significantly different between ESCC and EAC ( KMT2A, 2.7% vs. 1.2%; KMT2C, 3.8% vs. 1.2%; KMT2D, 14.2% vs. 2.0%, P <0.05). KMT2-MT EAC tumors showed significantly higher mutation rates in CIC (13% vs 1.1%), NF1 (12.5% vs 2.2%), FBXW7 (12% vs 3.4%), ATM (11.5% vs 2.6%) and BRCA1 (11.5% vs 1%, all q<0.05), compared to KMT2-WT tumors. However, in ESCC there were no significant differences in gene mutations between the KMT2-MT and WT groups. Significantly higher rates of dMMR/MSI-H (19.2% vs 1.0% in EAC, 5.4% vs. 0% in ESCC, both p<0.01) were seen in both KMT2-MT ESCC and EAC, compared to KMT2-WT cohorts, respectively. TMB was significantly higher in both KMT2-MT ESCC (median: 10 vs 8.6 mut/Mb, P=0.042) and EAC (23.9 vs 8.0 mut/Mb, P<0.0001), compared to KMT2-WT cohorts, respectively. A numerically longer immune-related overall survival was observed in KMT2 -MT EC patients, compared to KMT2 -WT EC patients (9.05 vs 6.98 months, HR=0.744, 95%CI: 0.52-1.07, P =0.11). Conclusions: This is the largest study to investigate the distinct genomic landscapes between KMT2 -MT and WT EC to date. Our data showed the KMT2 -MT EC has a distinctive genetic profile, indicated by higher TMB, and higher frequency of dMMR/MSI-H and gene mutations involved in DDR and epigenetic regulation. Understanding these molecular characteristics may be informative in the development of effective treatment strategies in KMT2 -MT EC.

Neoantigen cancer vaccines: a new star on the horizon.

Immunotherapy represents a promising strategy for cancer treatment that utilizes immune cells or drugs to activate the patient's own immune system and eliminate cancer cells. One of the most exciting advances within this field is the targeting of neoantigens, which are peptides derived from non-synonymous somatic mutations that are found exclusively within cancer cells and absent in normal cells. Although neoantigen-based therapeutic vaccines have not received approval for standard cancer treatment, early clinical trials have yielded encouraging outcomes as standalone monotherapy or when combined with checkpoint inhibitors. Progress made in high-throughput sequencing and bioinformatics have greatly facilitated the precise and efficient identification of neoantigens. Consequently, personalized neoantigen-based vaccines tailored to each patient have been developed that are capable of eliciting a robust and long-lasting immune response which effectively eliminates tumors and prevents recurrences. This review provides a concise overview consolidating the latest clinical advances in neoantigen-based therapeutic vaccines, and also discusses challenges and future perspectives for this innovative approach, particularly emphasizing the potential of neoantigen-based therapeutic vaccines to enhance clinical efficacy against advanced solid tumors.

Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation

Loss-of-function of DDX3X is a leading cause of neurodevelopmental disorders (NDD) in females. DDX3X is also a somatically mutated cancer driver gene proposed to have tumour promoting and suppressing effects. We perform saturation genome editing of DDX3X, testing in vitro the functional impact of 12,776 nucleotide variants. We identify 3432 functionally abnormal variants, in three distinct classes. We train a machine learning classifier to identify functionally abnormal variants of NDD-relevance. This classifier has at least 97% sensitivity and 99% specificity to detect variants pathogenic for NDD, substantially out-performing in silico predictors, and resolving up to 93% of variants of uncertain significance. Moreover, functionally-abnormal variants can account for almost all of the excess nonsynonymous DDX3X somatic mutations seen in DDX3X-driven cancers. Systematic maps of variant effects generated in experimentally tractable cell types have the potential to transform clinical interpretation of both germline and somatic disease-associated variation.

Genomic landscape in Saudi patients with hepatocellular carcinoma using whole-genome sequencing: a pilot study

Background and aimsHepatocellular carcinoma (HCC) is the third most prevalent cancer in Saudi Arabia. HCC poses a significant clinical challenge due to the presence of resistance among certain patients to the standard therapeutic agent sorafenib. This study aims to unravel the genomic characteristics of HCC patients in Saudi Arabia, investigate the genetic makeup of tumors in both sorafenib-sensitive and sorafenib-resistant patients, and analyze the functional implications of genomic abnormalities observed in these individuals. The resistance displayed by some HCC patients toward sorafenib underscores the need for alternative treatment approaches to effectively combat this formidable disease burden.MethodsWhole-genome sequencing (WGS) was performed on 16 HCC samples and targeted sequencing was performed on seven additional tumors. We identified and validated somatic and germline genetic aberrations. Employing a prize-collecting Steiner tree algorithm, we identified important altered genetic modules and potential biomarkers for each patient. Furthermore, we analyzed non-synonymous germline and somatic mutations, specifically in patients who underwent sorafenib treatment.ResultsOut of the 13 patients who received sorafenib, three exhibited sorafenib sensitivity, while the others showed resistance to the drug. Notably, 3 out of 16 individuals carried cancer-predisposing mutations. Additionally, 8 out of 16 patients displayed non-synonymous somatic alterations in genes associated with cancer. In the targeted-sequencing samples, rare non-synonymous variants were observed across all seven cases. The study also revealed the presence of specific somatic aberrations, including TP53, PIK3CA, APOB, CTNNB1, DPYD, LRP1B, MYC, and NFE2L2, which were identified in two patients. Among the 42 genes linked to sorafenib treatment, 4 out of 10 resistant patients carried somatic non-synonymous variants. Furthermore, when analyzing the 5,000 genes most relevant to the 42 genes, 7 out of 10 resistant individuals exhibited rare non-synonymous germline variants. Interestingly, none of the three sorafenib-sensitive patients displayed any concerning variants in those genes.ConclusionOur findings indicate that most of the HCC patients possess cancer-related genetic variants, and the altered pathways in these patients exhibit similarities. Notably, resistant patients exhibit a higher frequency of aberrations in sorafenib-related genes than do sensitive patients. Specifically, 4 out of 10 resistant individuals demonstrated 13 somatic mutations, whereas none of the three sensitive patients exhibited any. Similarly, 7 out of 10 resistant patients possessed 30 germline mutations, while none were observed in the sensitive group (two-sided Fisher’s exact test; somatic: p=0.50, germline: 0.07). These results contribute to our understanding of the genetic landscape of HCC and highlight potential therapeutic targets that could aid in overcoming treatment resistance.

Effects of different immune microenvironment characteristics on predictive efficacy of PTCH1 mutations in immune checkpoint inhibitor therapy of solid tumors.

e21026 Background: The PTCH1 gene encoding the protein corticosteroid receptor, which regulates the activity of the Sonic Hedgehog (SHH) signaling pathway. Mutations in PTCH1 result in disruption of the SHH signaling pathway and are closely related to the development of various solid tumors. Previous research has shown that mutations of the PTCH1 gene may result in decreased of antigen expression on the surface of tumor cells. However, relevant correlation with the efficacy of ICI therapy and immune microenvironment are still lacking. Methods: NGS data from an independent cohort (the MSKCC study cohort) of 1661 patients and Chinese clinical dataset (panel on 381/733-gene) of 35337 patients with pan-cancer to explore the association between PTCH1 mutations and immune biomarkers. TMB was defined as total number of somatic non-synonymous mutations in coding region. MSI was evaluated by NGS of 500 known MSI loci. PD-L1 expression was evaluated using immunohistochemistry (Dako 22C3). Patients from three independent cohorts (OAK, POPLAR and MSKCC study cohort) and were used to analyze the correlation between PTCH1 mutations and the efficacy of immune checkpoint blockade immunotherapies (ICIs). Estimation of immune filtration cells scores were conducted via TIMER2.0 (http://timer.cistrome.org/). Results: In Chinese cohort, there were 1384 (3.91%) patients harboured PTCH1 mutation ( PTCH1mut), slightly more than the MSKCC cohort (2.52%, 42/1661). For the TMB, both Chinese (median, 24.14 Muts/Mb vs 8.52 Muts/Mb, P ＜0.0001) and MSKCC (median, 22.30 Muts/Mb vs 5.90 Muts/Mb, P = 0.0035) cohort were associated with higher TMB than the wild-type. Supplemental analysis of MSI in the Chinese cohort showed significant differences between the PTCH1mut and PTCH1wt groups (P ＜0.0001), but not on PD-L1 levels (P = 0.64). In terms of prognostic effect with ICIs therapy, PTCH1mut were significantly associated with the overall survival (OS) of NSCLC (median, 5 months vs 11 months; HR = 1.61; 95% CI, 1.00-2.60; P = 0.049) and colorectal (median, 11 months vs 8 months; HR = 0.27; 95% CI, 0.08–0.90; P = 0.023). Interestingly, PTCH1mut in NSCLC was inversely associated with ICIs efficacy, as opposed to other solid tumors. Exploring the correlation between PTCH1 and immune microenvironment, it was found that CD4+ T cell (P＜0.001), macrophages (P＜0.001), NK cells (P＜0.001), and CD8+ T cells (P = 0.015) infiltration was significantly positively correlated in colorectal. But the infiltration of most immune cells was negatively correlated with PTCH1mut in NSCLC, that the B cells (P = 0.005), macrophages (P = 0.049), and NK cells (P = 0.022) had a significant difference. Conclusions: The results showed that the PTCH1 had a high correlation in solid tumors with TMB and MSI. The PTCH1 might a potential biomarker for ICIs therapy, and the immune microenvironment may be a factor affecting of it.

Effects of different immune microenvironment characteristics on effect of immune checkpoint inhibitors in solid tumors with TGFBR2 mutation.

e21027 Background: TGFBR2 is a member of the TGF-β receptor family, the protein encoded by it was involved in the regulation of cell proliferation, apoptosis, differentiation and other biological processes. TGFBR2 mutation can affect the occurrence of tumors. previous research showed that the TGF-β signaling pathway suppresses the tumor immune response, so TGFBR2 mutations may lead to tumor resistance to immunotherapy. However, effect of TGFBR2 mutations on ICIs and its correlation with immune microenvironment are rarely explored. Methods: NGS data from an independent cohort (the MSKCC study cohort) of 1661 patients and Chinese clinical dataset (panel on 733-gene) of 35337 patients with pan-cancer to explore the association between PTCH1 mutations and immune biomarkers. TMB was defined as total number of somatic non-synonymous mutations in coding region. MSI was evaluated by NGS of 500 known MSI loci. PD-L1 expression was evaluated using immunohistochemistry (Dako 22C3). Patients from three independent cohorts (OAK, POPLAR and MSKCC study cohort) and were used to analyze the correlation between PTCH1 mutations and the efficacy of immune checkpoint blockade immunotherapies (ICIs). Estimation of immune filtration cells scores were conducted via TIMER2.0 (http://timer.cistrome.org/). Results: In Chinese cohort, there were 831 (4.32%) patients harboured TGFBR2 mutation ( TGFBR2mut), in which the most number patients with TGFBR2mut were gastric carcinoma (10.53%), followed by colorectal (8.63%) and NSCLC (2.98%). Same as the MSKCC cohort (2.77%). For the TMB, both Chinese (median, 28.64 Muts/Mb vs 8.39 Muts/Mb, P ＜0.001) and MSKCC (median, 28.53 Muts/Mb vs 6.89 Muts/Mb, P = 0.030) cohort were associated with higher TMB than the wild-type. Supplemental analysis of MSI in the Chinese cohort showed significant differences between the TGFBR2mut and TGFBR2wt groups (P ＜0.001), but not on PD-L1 levels (P = 0.50). In terms of prognostic effect with ICIs therapy, TGFBR2mut were significantly associated with the overall survival (OS) of NSCLC (median, 2 months vs 11 months; HR = 3.46; 95% CI, 1.63-7.35; P ＜0.001) in OAK and POPLAR cohort, same as MSKCC cohort (median, 3 months vs 10 months; HR = 2.21; 95% CI, 1.04-4.71; P = 0.034). Interestingly, In the MSKCC cohort, opposite with NSCLC, majority tumors with TGFBR2mut had a longer survival value, showing a trend toward longer survival. Exploring the correlation between TGFBR2 and immune microenvironment. The results showed NK cell (P = 0.042) and CD4+ T cell (P = 0.036) infiltration were significantly negatively correlated in NSCLC. However, other solid tumors such as colorectal and melanoma are opposite. Conclusions: The results showed that the TGFBR2 had a high correlation in solid tumors with TMB and MSI. The TGFBR2 might a potential biomarker for ICIs therapy in NSCLC, and the immune microenvironment may be a factor affecting of it.

Effect of KDR mutations on efficacy of immune checkpoint inhibitors in patients with bladder cancer.

4521 Background: KDR (kinase insert domain receptor) is the most important VEGF (vascular endothelial growth factor) receptor that mediates the signaling transduction initiated by the VEGF ligands. VEGF signaling can activate angiogenesis which plays a crucial role in tumor growth, metastasis, and treatment response. Here we explore the relationship between KDR mutation and its efficacy of immune checkpoint inhibitors (ICIs) in patients with bladder cancer. Methods: To explore the association between KDR mutations and the efficacy of ICIs in bladder cancer patients, we collected clinical and mutation data of 215 patients who received ICIs (PD-(L)1 and/or CTLA-4 inhibitors) from MSKCC cohort. Gene expression data and WES mutation data of 413 samples in the TCGA database were obtained from the UCSC Xena for analysis of the differences of potential biological mechanisms between KDR-mutant and KDR-wildtype tumors. TMB was calculated as the total number of somatic non-synonymous mutations per megabase. CIBERSORT was applied to infer 22 immune cell type proportions in TCGA advanced bladder cancers. Results: In MSKCC cohort, we observed 5.6% (12/215) samples had KDR mutation. Survival analysis showed that KDR-mutant patients had longer overall survival (OS) than that of wildtype patients (hazard ratio (HR) = 0.27, 95% CI: 0.07-1.09, logrank test P = 0.048); median OS (not reached versus 15 months). Then we compared the TMB between KDR-mutant and KDR-wildtype tumors, the results showed that KDR-mutant group had higher TMB in MSKCC cohort (median [IQR]: 17.58 [11.64–47.81] versus 7.90 [4.92–14.76], P=0.002) and TCGA cohort (median [IQR]: 10.69 [5.87–19.85] versus 4.60 [2.52–8.08], P=0.001). Analysis of 22 immune cell subsets in the TCGA advanced bladder cancers, B cells naive, plasma cells and macrophages M1 were significantly abundant in KDR-mutant tumors (all P <0.05). Conclusions: In ICI-treated bladder cancers, KDR mutation is associated with prolonged OS, higher TMB and activated antitumor immunity, which suggests that KDR mutation may serve as a potential biomarker to guide ICI therapy in bladder cancer.

Abstract 4982: The somatic mutation signature detected in gynecological mucosal melanoma

Abstract Background: Gynecological Mucosal Melanoma(GMM) is aggressive disease with a poor prognosis, where few studies focusing on its target drug and mutation signatures. Cancer as outcome of DNA mutagenesis process and can be inferred from somatic mutations signature by analyzing the genome sequence. Due to the rapid development of next-generation sequencing (NGS) and molecularly driven targeted therapy, discovering different mutation signature in cancer plays an important role in precision medicine. Methods: The study was conducted at a high-volume melanoma center, the Peking University Cancer Hospital. We enrolled female patients with melanoma (n = 22) from 2017 to 2021. Subsequently, whole-exome sequencing performed on the Formalin-fixed paraffin-embedded (FFPE) specimens from 22 GMM patients and match normal tissues of patients. Tumor mutational burden (TMB) was measured as the number of nonsynonymous somatic mutations of whole-exome sequence coding region. Mutation signature detection base pair of six substitution subtypes: C>A, C>G, C>T, T>A, T>C, and T>G. NMF can be used to decompose matrix A into two nonnegative matrices. Results: In all mutations of 22 GMM patients, we screen two mutation signatures. Signature 1 showed a strong correlation [0.77;cosine similarity value (CSV)] to the signature 3 from the COSMIC database, which represents failure of DNA double-strand break-repair by homologous recombination, which predominated in 55%(12/22) GMM patients. Signature 2 showed a strong correlation to Cosmic signature 1 (CSV = 0.791), which is has been found in all cancer types correlated with age cancer diagnosis. Patient-18 did not any treatment in our hospital, follow-up was lost. Kaplan-Meier revealed that Signature 2 was associated with longer overall survival in GMM patients and later recurrence or progression. Conclusions: This study aimed to identify prognostic markers and the development of targeted therapy for the treatment of GMM. Citation Format: Zhongwu Li, Lianyi Shen, Siyao Liu. The somatic mutation signature detected in gynecological mucosal melanoma. [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 4982.

Abstract 1923: Ancestry-associated differences in somatic mutation rates from tumor profiling data of a pan-cancer cohort of 100,000 patients

Abstract Racial and ethnic disparities span the continuum of cancer care and adversely impact screening, detection, diagnosis, treatment, and outcomes of cancer. These disparities are driven by a complex interplay among social, psychosocial, lifestyle, environmental, healthcare, and biological determinants of health. A challenge is that the social constructs of race and ethnicity may not fully capture shared genetic background. Genomics can capture ancestry in a more precise way, allowing genetic influences to be teased apart from the impact of social and environmental factors. Here, we searched for associations between continental genetic ancestry and somatic mutations in cancer genes in a pan-cancer cohort of 100,000 patients. A sample of de-identified records from patients who underwent tumor profiling with the Tempus xT 648-gene assay was selected from the Tempus Database. We inferred global African (AFR), Amerindian (AMR), East Asian (EAS), European (EUR), and South Asian (SAS) continental ancestry proportions with a custom set of 658 ancestry informative markers. To properly account for the collinearity of ancestry proportions, we performed logistic compositional data analysis between mutation counts and ancestry proportions from data of cancer types with at least 300 patients and for genes for which a minimum of 10 and at least 1% of patients harbored variants. Odds ratios (ORs) represent the effect of a doubling in ancestry proportion, adjusted for age, gender, assay version, and all other ancestries. We examined associations for the presence of nonsynonymous somatic mutations and predicted driver mutations with the boostDM algorithm. We adjusted for multiple comparisons at a 5% false discovery rate (FDR) with the Benjamini-Hochberg method. We identified previously reported significant (p<0.001) associations between EAS and predicted driver mutations in EGFR (OR=1.08) in lung cancer, and between AFR and APC (OR=1.03) and KRAS (OR=1.04) in colorectal cancer. Moreover, we found several novel associations, including AFR with TP53 (OR=1.06) and PTEN (OR=0.90) in endometrial cancer and BRAF (OR=0.93) in colorectal cancer, and EAS with CTNNB1 (OR=1.17) in lung cancer. The latter is noteworthy as CTNNB1, which has been proposed as a TTK inhibitor response biomarker, is overall infrequently mutated in lung cancer but activating mutations have been reported in East Asian patients. Indeed, we found that 7% of East Asian lung cancer patients in our cohort harbor a driver mutation in this gene vs. 1.6% in the other groups. Additionally, an association with nonsynonymous mutations was found between AMR with PTPN22 (OR=1.26) in colorectal cancer. While these associations can be the result of a combination of environmental, social, and biological factors, our results suggest that genetic ancestry can be useful to understand disparities in cancer incidence, progression, and outcomes. Citation Format: Brooke Rhead, Yannick Pouliot, Justin Guinney, Francisco M. De La Vega. Ancestry-associated differences in somatic mutation rates from tumor profiling data of a pan-cancer cohort of 100,000 patients [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 1923.

Abstract P2-23-15: Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas

Abstract Background: Invasive lobular carcinomas (ILCs) are the most frequent special histologic subtype of breast cancer, accounting for up to 15% of all breast cancer cases. ILCs are characterized by a distinctive discohesive growth pattern, with cells arranged in single cell infiltrative file and dispersed throughout the stroma, which stems from the loss of E-cadherin expression due to bi-allelic inactivation of the CDH1 gene. A subset of breast cancers display a similar single cell infiltrative growth pattern but, in contrast to classic ILC, display diffuse strong membranous E-cadherin reactivity and membranous p120 expression. We refer to such cases as “lobular-like invasive ductal carcinoma” (LLIDC), but it is unclear if this terminology is appropriate and if such cases show biallelic inactivation of CDH1, similarly to ILCs. Here, we sought to define whether LLIDCs would harbor bi-allelic alterations of CDH1 and to perform an exploratory, hypothesis generating analysis of the repertoire of somatic genetic alterations of LLIDCs and classic ILCs. Materials and methods: Representative H&Es, as well as sections subjected to E-cadherin and p120 immunohistochemistry from seven classic ILCs and seven bona fide “lobular-like invasive ductal carcinomas” were retrieved and independently reviewed by two pathologists with experience and expertise in breast pathology. DNA samples were extracted from representative sections from tumor and normal breast tissue from each patient and subjected to an FDA-approved targeted sequencing assay comprising the coding regions and selected regulatory elements of 515 genes. Somatic single nucleotide variants (SNVs) were detected with MuTect, indels with Strelka, Varscan2, Scalpel and Lancet. All mutations were manually inspected using the Integrative Genomics Viewer (IGV). The cancer cell fraction (CCF) of each mutation was inferred, as well as clonal probability, using ABSOLUTE. Copy number alterations and loss of heterozygosity were determined using FACETS. Mutational signatures were inferred using SigMA based on all synonymous and nonsynonymous somatic mutations. Results: Based on the histopathologic evaluation, of the 14 cases analyzed, seven were classified as ILC, and the other seven were classified as LLIDC. Sequencing analysis revealed that the classic ILCs harbored 16q LOH and CDH1 mutations (7/7), of which five were frameshift indel and two were splice site mutations consistently coupled with loss-of-heterozygosity (LOH) of the wild-type allele. Conversely, five of the seven LLIDCs did not harbor CDH1 mutations or genomic rearrangements. CDH1 mutations were identified in 2 LLIDCs: one harbored a subclonal CDH1 in-frame indel mutation coupled with LOH. This case displayed membranous E-cadherin and p120 expression with areas of aberrant expression. The other CDH1-mutated LLIDC harbored a complex in-frame indel with subclonal LOH. This case displayed membranous E-cadherin and p120 expression. The comparative analysis of the repertoire of somatic genetic alterations and mutational signatures present in LLIDCs and classic ILCs did not reveal any significant differences. Conclusion: Despite the histologic similarities, LLIDCs differ from classic lobular carcinomas based on the lack of CDH1 bi-allelic inactivation and the patterns of expression of E-cadherin and p120 catenin. Further whole-genome sequencing analyses are warranted to define the molecular basis of the discohesive cancer cells of LLIDC display. Citation Format: Edaise M. da Silva, Thais Basili, Jing Yu, Juan Blanco-Heredia, Pier Selenica, Qiqi Ye, Arnaud da Cruz Paula, Higinio Dopeso, Antonio Marra, Steffi Oesterreich, Jorge Reis-Filho, Rohit Bhargava. Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-15.

The potential of miR-153 as aggressive prostate cancer biomarker.

Prostate cancer (PC) is one of the most frequently diagnosed cancers in males. MiR-153, as a member of the microRNA (miRNA) family, plays an important role in PC. This study aims to explore the expression and possible molecular mechanisms of the miR-153 action. Formalin-fixed paraffin-embedded (FFPE) tissues were collected from prostatectomy specimens of 29 metastatic and 32 initial stage PC patients. Expression levels of miR-153 were measured using real-time reverse transcription polymerase chain reaction (qRT-PCR). 2-ΔΔCT method was used for quantitative gene expression assessment. The candidate target genes for miR-153 were predicted by TargetScan. Mutations in target genes of miR-153 were identified using exome sequencing. Protein-protein interaction (PPI) networks, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to investigate the potential molecular mechanisms of miR-153 in PC. MiR-153 was significantly up-regulated in PC tissues compared to non-cancerous tissues. The analysis of correlation between the expression level of miR-153 and clinicopathological factors revealed a statistically significant correlation with the stage of the tumor process according to tumor, node, metastasis (TNM) staging system (p=0.0256). ROC curve analysis was used to evaluate the predictive ability of miR-153 for metastasis development and it revealed miR-153 as a potential prognostic marker (AUC=0.85; 95%CI 0.75-0.95; sensitivity=0.72, specificity=0.86)). According to logistic regression model the high expression of miR-153 increased the risk of metastasis development (odds ratios=3.14, 95% CI 1.62-8.49; p-value=0.006). Whole exome sequencing revealed nonsynonymous somatic mutations in collagen type IV alpha 1 (COL4A1), collagen type IV alpha 3 (COL4A3), forkhead box protein O1 (FOXO1), 2-hydroxyacyl-CoA lyase 1 (HACL1), hypoxia-inducible factor 1-alpha (HIF-1A), and nidogen 2 (NID2) genes. Moreover, KEGG analysis revealed that the extracellular matrix-receptor (ECM-receptor) interaction pathway is mainly involved in PC. MiR-153 is up-regulated in PC tissues and may play an important role in aggressive PC by targeting potential target genes.

Proteomic and single-cell landscape reveals novel pathogenic mechanisms of HBV-infected intrahepatic cholangiocarcinoma

Despite the epidemiological association between intrahepatic cholangiocarcinoma (ICC) and hepatitis B virus (HBV) infection, little is known about the relevant oncogenic effects. A cohort of 32 HBV-infected ICC and 89 non-HBV-ICC patients were characterized using whole-exome sequencing, proteomic analysis, and single-cell RNA sequencing. Proteomic analysis revealed decreased cell-cell junction levels in HBV-ICC patients. The cell-cell junction level had an inverse relationship with the epithelial-mesenchymal transition (EMT) program in ICC patients. Analysis of the immune landscape found that more CD8 Tcells and Th2 cells were present in HBV-ICC patients. Single-cell analysis indicated that transforming growth factor beta signaling-related EMT program changes increased in tumor cells of HBV-ICC patients. Moreover, ICAM1+ tumor-associated macrophages are correlated with a poor prognosis and contributed to the EMT in HBV-ICC patients. Our findings provide new insights into the behavior of HBV-infected ICC driven by various pathogenic mechanisms involving decreased cell junction levels and increased progression of the EMT program.

DrOGA: An Artificial Intelligence Solution for Driver-Status Prediction of Genomics Mutations in Precision Cancer Medicine

Precision cancer medicine suggests that better cancer treatments would be possible guiding therapies by tumor’s genomics alterations. This hypothesis boosted exome sequencing studies, collection of cancer variants databases and developing of statistical and Machine Learning-driven methods for alterations’ analysis. In order to extract relevant information from huge exome sequencing data, accurate methods to distinguish driver and neutral or passengers mutations are vital. Nevertheless, traditional variant classification methods have often low precision in favour of higher recall. Here, we propose several traditional Machine Learning and new Deep Learning techniques to finely classify driver somatic non-synonymous mutations based on a 70-features annotation, derived from medical and statistical tools. We collected and annotated a complete database containing driver and neutral alterations from various public data sources. Our framework, called Driver-Oriented Genomics Analysis (DrOGA), presents the best performances compared to individual and other ensemble methods on our data. Explainable Artificial Intelligence is used to provide visual and clinical explanation of the results, with a particular focus on the most relevant annotations. This analysis and the proposed tool, along with the collected database and the feature engineering pipeline suggested, can help the study of genomics alterations in human cancers allowing precision oncology targeted therapies based on personal data from next-generation sequencing.

Integrated multi-omic analysis of low-grade ovarian serous carcinoma collected from short and long-term survivors

BackgroundLow-grade serous ovarian cancer (LGSOC) is a rare disease that occurs more frequently in younger women than those with high-grade disease. The current treatment is suboptimal and a better understanding of the molecular pathogenesis of this disease is required. In this study, we compared the proteogenomic analyses of LGSOCs from short- and long-term survivors (defined as < 40 and > 60 months, respectively). Our goal was to identify novel mutations, proteins, and mRNA transcripts that are dysregulated in LGSOC, particularly in short-term survivors.MethodsInitially, targeted sequencing of 409 cancer-related genes was performed on 22 LGSOC and 6 serous borderline ovarian tumor samples. Subsequently, whole-genome sequencing analysis was performed on 14 LGSOC samples (7 long-term survivors and 7 short-term survivors) with matched normal tissue samples. RNA sequencing (RNA-seq), quantitative proteomics, and phosphoproteomic analyses were also performed.ResultsWe identified single-nucleotide variants (SNVs) (range: 5688–14,833 per sample), insertion and deletion variants (indels) (range: 880–1065), and regions with copy number variants (CNVs) (range: 62–335) among the 14 LGSOC samples. Among all SNVs and indels, 2637 mutation sites were found in the exonic regions. The allele frequencies of the detected variants were low (median12%). The identified recurrent nonsynonymous missense mutations included KRAS, NRAS, EIF1AX, UBR5, and DNM3 mutations. Mutations in DNM3 and UBR5 have not previously been reported in LGSOC. For the two samples, somatic DNM3 nonsynonymous missense mutations in the exonic region were validated using Sanger sequencing. The third sample contained two missense mutations in the intronic region of DNM3, leading to a frameshift mutation detected in RNA transcripts in the RNA-seq data. Among the 14 LGSOC samples, 7754 proteins and 9733 phosphosites were detected by global proteomic analysis. Some of these proteins and signaling pathways, such as BST1, TBXAS1, MPEG1, HBA1, and phosphorylated ASAP1, are potential therapeutic targets.ConclusionsThis is the first study to use whole-genome sequencing to detect somatic mutations in LGSOCs with matched normal tissues. We detected and validated novel mutations in DNM3, which were present in 3 of the 14 samples analyzed. Additionally, we identified novel indels, regions with CNVs, dysregulated mRNA, dysregulated proteins, and phosphosites that are more prevalent in short-term survivors. This integrated proteogenomic analysis can guide research into the pathogenesis and treatment of LGSOC.

High prevalence of low-allele-fraction somatic mutations in STAT3 in peripheral blood CD8+ cells in multiple sclerosis patients and controls.

Somatic mutations have a central role in cancer, but there are also a few rare autoimmune diseases in which somatic mutations play a major role. We have recently shown that nonsynonymous somatic mutations with low allele fractions are preferentially detectable in CD8+ cells and that the STAT3 gene is a promising target for screening. Here, we analyzed somatic mutations in the STAT3 SH2 domain in peripheral blood CD8+ cells in a set of 94 multiple sclerosis (MS) patients and 99 matched controls. PCR amplicons targeting the exons 20 and 21 of STAT3 were prepared and sequenced using the Illumina MiSeq instrument with 2x300bp reads. We designed a novel variant calling method, optimized for large number of samples, high sequencing depth (>25,000x) and small target genomic area. Overall, we discovered 64 STAT3 somatic mutations in the 193 donors, of which 63 were non-synonymous and 77% have been previously reported in cancer or lymphoproliferative disease. The overall median variant allele fraction was 0.065% (range 0.007-1.2%), without significant difference between MS and controls (p = 0.82). There were 26 (28%) MS patients vs. 24 (24%) controls with mutations (p = 0.62). Two or more mutations were found in 9 MS patients vs. 2 controls (p = 0.03, pcorr = 0.12). Carriership of mutations associated with older age and lower neutrophil counts. These results demonstrate that STAT3 SH2 domain is a hotspot for somatic mutations in CD8+ cells with a prevalence of 26% among the participants. There were no significant differences in the mutation prevalences between MS patients and controls. Further research is needed to elucidate the role of antigenic stimuli in the expansion of the mutant clones. Furthermore, the high discovered prevalence of STAT3 somatic mutations makes it feasible to analyze these mutations directly in tissue-infiltrating CD8+ cells in autoimmune diseases.

Somatic mutations in tumor and plasma of locoregional recurrent and/or metastatic head and neck cancer using a next-generation sequencing panel: A preliminary study.

We explore the utility of TruSight Tumor 170 panel (TST170) for detecting somatic mutations in tumor and cfDNA from locoregional recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC). Targeted NGS of tumor DNA and plasma cfDNA was performed using TST170 panel. In addition, a set of somatic mutations previously described in HNSCC were selected for validating in tumor, plasma, and saliva by digital droplet PCR. The TST170 panel identified 13 non-synonymous somatic mutations, of which five were detected in tumoral tissue, other five in plasma cfDNA, and three in both tissue and plasma cfDNA. Of the eight somatic mutations identified in tissue, three were also identified in plasma cfDNA, showing an overall concordance rate of 37.5%. This preliminary study shows the possibility to detect somatic mutations in tumor and plasma of HNSCC patients using a single assay that would facilitate the clinical implementation of personalized medicine in the clinic.

Molecular Clustering on Ctdna May Improve Prognostic Stratification of DLBCL Patients

Background. Diffuse large B-cell lymphoma (DLBCL) displays a high degree of molecular heterogeneity and may be classified into molecular clusters harboring specific lesions and therapeutic vulnerabilities. To date, DLBCL molecular clusters have been identified on the tissue biopsy and do not consider mutations deriving from different anatomical compartments. Currently, DLBCL prone to become long-term survivors after R-CHOP or, alternatively, destined to relapse early after treatment initiation cannot be unequivocally identified a priori. The quantification of circulating tumor DNA (ctDNA) on the liquid biopsy at the time of diagnosis allows to stratify the outcome of DLBCL patients but its integration with molecular clustering has so far not been evaluated. Purpose. The aims of this study were: i) to evaluate the potential contribution of liquid biopsy to the identification of DLBCL molecular clusters; and ii) to evaluate whether the assignment to a specific molecular cluster may improve patients’ stratification in combination with ctDNA load. Methods. A multicenter cohort of newly diagnosed DLBCL patients provided with ctDNA from the plasma and genomic DNA (gDNA) from the lymph node (LN) biopsy represented the basis of this study. gDNA extracted from granulocytes was analyzed for comparative purposes. The LyV4.0 CAPP-Seq assay comprising a panel of 59 genes relevant to B-cell malignancies was used. The LymphGen tool was utilized for cluster analysis. Results. The cohort included 77 newly diagnosed DLBCL patients treated prospectively with R-CHOP. The median age of the studied cohort was 67 years, 68.8% presented with stage III-IV and 22.1% had a high-risk IPI. After a median follow-up of 33.9 months, the 40-month progression-free survival (PFS) and overall survival (OS) were 66.5% and 80.2%, respectively. Mutational analysis identified at least one somatic non-synonymous mutation in 92.2% of patients (71/77) in the LN biopsy and in 87.0% (67/77) in the ctDNA. The most frequently mutated genes in both compartments and in more than 20% of patients were KMT2Dfollowed by PIM1 and HIST1H1E. To assign patients to molecular clusters, we applied the LymphGen tool to both ctDNA and LN. This tool allowed the cluster assignment in 40.3% (27/67) DLBCL on ctDNA and in 46.5% (33/71) on the LN biopsy. On ctDNA, 9 patients were classified as MCD, 5 as ST2, 7 as EZB, 5 as BN2 and 1 as molecular composite (BN2/ST2). By evaluating the clinical impact of molecular clusters, patients assigned to the ST2 and BN2 clusters in both ctDNA (p=0.032) and in LN biopsy (p=0.007), displayed a very good PFS compared to other patients. By recursive partitioning for PFS, patients with a baseline ctDNA load <2Log10hGE (N=17) had a 40-month PFS of 94.1% compared to 54.6% for patients (N=49) with higher ctDNA levels (p=0.003) and were not further stratified. Conversely, patients with ctDNA levels >2Log10hGE were further stratified by the assignment to the ST2 and/or BN2 clusters. Patients assigned to cluster ST2 or BN2 (N=10), despite having ctDNA levels >2Log10hGE, had a 40-month PFS of 100% compared to 44.4% for patients (N=39) classified as MCD or EZB or not classified (p=0.004). Therefore, by combining ctDNA levels and molecular clusters identified on the liquid biopsy, we identified 2 different groups of patients with a significantly different PFS and OS (both p<0.001). Patients with a <2Log10hGE and/or assigned to cluster BN2 and ST2 presented a 40-month PFS and OS of 96.2% and 100%, respectively. Conversely, patients with a ctDNA level >2Log10hGE and not assigned to cluster ST2 and BN2 presented a 40-month PFS and OS of 44.4% and 61.5%, respectively (Figure 1). To evaluate the ctDNA dynamics according to a specific molecular cluster, we analyzed the ctDNA of 48 patients after one cycle of R-CHOP. Ten were classified as ST2 or BN2 at the time of diagnosis of which, after 1 cycle of R-CHOP, eight (80%) reached an early molecular response (EMR). Interestingly, the 2 ST2/BN2 patients who did not achieve an EMR have remained in CR and have not progressed at the last follow-up. Conclusions. Molecular clustering on the liquid biopsy reflects in most cases the prognostic impact of clusters identified in the tissue biopsy. If validated, the combination of ctDNA levels <2 Log10hGE and ST2/BN2 molecular clusters on the liquid biopsy represents a powerful biomarker to identify patients who can benefit most from R-CHOP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal