Somatic Single Nucleotide Variants Research Articles

Comparative analysis of the targetable landscape in KRAS-mutant and wild-type pancreatic adenocarcinoma.

4155 Background: Pancreatic adenocarcinoma (PC) is the fourth leading cause of cancer deaths, with increased incidence among patients (pts) younger than 50 years old (yo). Small cohort studies suggest early-onset PC (EOPC, diagnosis at < 50 yo) tumors may have a unique biology, harboring a higher proportion of KRAS wild-type status ( KRASWT) and enrichment of targetable mutations/fusions compared to non-EOPC. In addition, therapy targeting oncogenic fusions in KRASWT tumors has shown meaningful responses in PC pts. Here, we investigate the prevalence of fusions, mutations, and homologous recombination deficiency (HRD) in KRASWT PC to identify potential targets for therapeutic intervention, and compare EOPC to non-EOPC to better characterize EOPC differences. Methods: De-identified records from 4,956 PC pts with tumor biopsies sequenced using the Tempus xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage; full transcriptome RNA-seq) were retrospectively reviewed. Fusions were detected from RNA-seq data via the Tempus bioinformatics pipeline. Mutations identified included germline and/or somatic single-nucleotide variants and insertions/deletions. HRD status was determined from RNA-seq data by the Tempus HRD test. Significance was determined as FDR-corrected p-values < 0.05. Results: Across the entire cohort, 21% tumors were KRASWT. HRD was more frequent in KRASWT (7.9%) compared to KRASMUT (3.1%) tumors ( P< 0.001). Significant somatic mutational differences between the KRASWT and KRASMUT cohorts included BRAF (5.0% vs 0.2%) , CDKN2A (5.4% vs 25%), ARID1A (3.6% vs 8.6%), CTNNB1 (1.3% vs 0.3%), and TSC2 (1.2% vs 0.1%) (all P< 0.001). Actionable rearrangements were enriched in the KRASWTcohort (10% vs 2.1%, P< 0.001); the most common fusion partners include NRG1, MET, RAF1, BRAF, NTRK1-3, FGFR 1-4, and RET. TMB-high (10 muts/Mb) and MSI-high were more common in KRASWT (2.9% and 1.0%) compared to KRASMUT (1.7% and 0.4%) tumors ( P= 0.015). Additionally, 382 pts were classified as EOPC. KRASWT tumors comprised 30% of EOPCs, 22% of the 50-70 yo cohort, and 17% of the > 70 yo cohort. Germline (pathogenic/likely pathogenic) alterations in BRCA1 and BRCA2 were more frequent in EOPCs (2.0% and 4.5%) compared to the 50-70 yo (0.4% and 1.7%) and > 70 yo (0.4% and 0.8%) cohorts ( P= 0.015 and < 0.001, for BRCA1 and BRCA2 respectively). There were no statistical differences in actionable genes detected in EOPC vs non-EOPC. HRD was found at higher frequency in EOPC (5.4%) and decreased in age cohorts 50-70 yo (4.7%) and > 70 yo (2.5%) ( P = 0.047). Rearrangements were more common in EOPC compared to non-EOPC (10% vs 1.1%, P< 0.001). Conclusions: HRD, TMB-H/MSI-H and oncogenic rearrangements are more prevalent in KRASWT PC when compared with KRASMUT. These molecular analyses may provide additional therapeutic options for PC pts, warranting comprehensive genomic and transcriptomic profiling for this population.

Serial postoperative ctDNA monitoring of breast cancer recurrence.

562 Background: Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive or reliable tests to monitor these patients and detect distant metastases before overt recurrence. Here, we demonstrate the use of personalized circulating tumour DNA (ctDNA) profiling performed postoperatively, postadjuvantly, and serially for detection of recurrence in breast cancer. Methods: Patients with primary breast cancer (n=188) were recruited following surgery and adjuvant therapy and were followed-up for up to 10 years with semi-annual blood sampling for ctDNA analysis. Patients (n=29) with insufficient residual tumour for whole exome sequencing (WES) were excluded from the analysis. Tumour WES profiles were generated for 159 patients; samples from 2 patients failed WES QC requirements and a personalised ctDNA panel could not be generated for 1 patient. In 156 patients, plasma samples (n=1141) were retrospectively tested for the presence of ctDNA using personalized Signatera assays (mPCR-NGS) targeting up to 16 somatic single nucleotide variants selected from primary tumour WES. Results: Plasma ctDNA was detected ahead of clinical or radiologic relapse in 30 of the 34 relapsed patients (sensitivity of 88%). Metastatic relapse was predicted with a lead interval of up to 2 years (median: 10 months, range: 0-39 months); median lead intervals for HR+/HER2- were 15 (2 - 39); for HR-/HER2+ 6 (0.5 – 12) for HR+/HER2+ 8 (5 – 14) and 9 (0-20) for TNBC. Patients with a positive ctDNA test had poorer relapse-free-survival (RFS) (HR=47.5; 95% CI 18.5-161.4; p <0.001) from surgery and all four breast cancer subgroups showed a similarly reduced RFS. Overall survival was also significantly reduced for patients who were ctDNA positive (HR=84.15; 95%CI 16.43-1538; p <0.001). The number of variants, mean VAF and MTM/mL varied between patients, with significantly higher values at the time closest to relapse than in the first ctDNA positive sample (p =0.0002). Among the 4 relapsed patients not detected in the study all were HR+/HER2-, 1 had a local recurrence, 2 had bone recurrence (1 with axillary LN involvement) and 1 had cancer cells in pleural fluid. Of the remaining 122 patients, only 5 developed ctDNA-positivity, all with low VAF, none of them have relapsed by the follow-up census date (31 December 2021). However, follow-up for some of these patients limits definitive assessment. Lastly, 4 patients developed a second primary cancer (2 breast, 2 lung) all of whom were ctDNA-negative. Conclusions: This study demonstrates that serial post-operative ctDNA analysis has strong prognostic value. More importantly, earlier detection of metastatic disease provides a possible window for therapeutic intervention, while repeated negative ctDNA tests can provide reassurance to patients. Future interventional studies may assess the clinical utility of ctDNA-based risk-stratification.

Identification of distinct genomic features reveals frequent somatic AHNAK and PTEN mutations predominantly in primary malignant melanoma presenting in the ureter.

Primary malignant melanoma of the ureter is extremely rare. Genetic variants to the increased risk of developing the disease have not yet been investigated. Tumour mutation profiling for primary malignant melanoma of the ureter was performed by whole-exome sequencing. Immunohistochemistry was performed to verify histopathological features and the variants of predisposing genes and driver mutation genes. Furthermore, we conducted a literature review and Surveillance, Epidemiology and End Result-based study by searching public databases. We identified 38 somatic single nucleotide variants and 9 somatic insertions and deletions (INDELs) in tumour specimens. After filtering with the Cancer Gene Census database, seven predisposing genes and two driver mutation genes were identified. Moreover, the immunohistochemical profile showed that tumour cells were positive for Melan-A, melanoma gp100 human melanoma black 45 (HMB45), S100 beta and P53. The expression levels of two driver mutation genes (phosphatase and tensin homolog (PTEN) and desmoyokin (AHNAK) and five predisposing genes (AT-rich interaction domain 1B (ARID1B), catalase, eukaryotic translation initiation factor 4 gamma 3 (EIF4G3), ANK3 and collagen type I) were significantly downregulated in tumour tissues compared to paracancerous tissues. In the literature review and Surveillance, Epidemiology and End Results-based study, patients with primary malignant melanoma of the urinary tract had worse clinical outcomes than patients with primary urothelial carcinoma after 1:2 propensity score matching (P=0.010). Additionally, Cox multivariate analysis for patients with primary malignant melanoma of the urinary tract indicated that distant metastasis (hazard ratio=1.185; P=0.044) was an independent predictor for overall survival, and tumour focality (hazard ratio=0.602; P=0.017) and non-surgery (hazard ratio=0.434; P=0.003) were independent factors for tumour progression. Our study is the first to provide evidence that the distinct phenotypes of primary malignant melanoma of the ureter may be due to different genetic variations. The prognosis of primary malignant melanoma of the urinary tract was poorer than that of primary urothelial carcinoma of the urinary tract.

Genetic Alterations in Papillary Thyroid Carcinoma With Hashimoto's Thyroiditis： ANK3, an Indolent Maintainer of Papillary Thyroid Carcinoma.

Hashimoto’s thyroiditis (TH) is a risk factor for the occurrence of papillary thyroid carcinoma (PTC), which is considered to be the most common type of thyroid cancer. In recent years, the prevalence of PTC with TH has been increasing, but little is known about the genetic alteration in PTC with TH. This study analyzed the mutation spectrum and mutation signature of somatic single nucleotide variants (SNV) for 10 non-tumor and tumor pair tissues of PTC with TH using whole-exome sequencing. The ANK3 protein expression was evaluated by immunohistochemistry in PTC with TH and PTC samples. Moreover, the functional role of ANK3 in PTC cells was determined by CCK-8 proliferation assay, colony formation assays, cell cycle analysis, cell invasion and migration and in vivo study through overexpression assay. Our results showed three distinct mutational signatures and the C>T/G>A substitution was the most common type of SNV. Gene-set enrichment analysis showed that most of the significantly mutated genes were enriched in the regulation of actin cytoskeleton signaling. Moreover, NCOR2, BPTF, ANK3, and PCSK5 were identified as the significantly mutated genes in PTC with TH, most of which have not been previously characterized. Unexpectedly, it was found that ANK3 was overexpressed in cytoplasm close to the membrane of PTC cells with TH and in almost all PTC cases, suggesting its role as a diagnostic marker of PTC. Ectopic expression of ANK3 suppressed invasion and migration, increased apoptosis of B-CPAP and TPC-1 cells. Moreover, our findings revealed that enhanced ANK3 expression inhibits growth of PTC cells both in vitro and in vivo. Ectopic expression of ANK3 significantly enhanced E-cadherin protein expression and inhibited PTC progression, at least in part, by suppression of epithelial-mesenchymal transition (EMT). Our study shows that ANK3 exerts an anti-oncogenic role in the development of PTC and might be an indolent maintainer of PTC.

Comprehensive molecular characterization of hypertension-related genes in cancer.

BackgroundDuring cancer treatment, patients have a significantly higher risk of developing cardiovascular complications such as hypertension. In this study, we investigated the internal relationships between hypertension and different types of cancer.MethodsFirst, we comprehensively characterized the involvement of 10 hypertension-related genes across 33 types of cancer. The somatic copy number alteration (CNA) and single nucleotide variant (SNV) of each gene were identified for each type of cancer. Then, the expression patterns of hypertension-related genes were analyzed across 14 types of cancer. The hypertension-related genes were aberrantly expressed in different types of cancer, and some were associated with the overall survival of patients or the cancer stage. Subsequently, the interactions between hypertension-related genes and clinically actionable genes (CAGs) were identified by analyzing the co-expressions and protein–protein interactions.ResultsWe found that certain hypertension-related genes were correlated with CAGs. Next, the pathways associated with hypertension-related genes were identified. The positively correlated pathways included epithelial to mesenchymal transition, hormone androgen receptor, and receptor tyrosine kinase, and the negatively correlated pathways included apoptosis, cell cycle, and DNA damage response. Finally, the correlations between hypertension-related genes and drug sensitivity were evaluated for different drugs and different types of cancer. The hypertension-related genes were all positively or negatively correlated with the resistance of cancer to the majority of anti-cancer drugs. These results highlight the importance of hypertension-related genes in cancer.ConclusionsThis study provides an approach to characterize the relationship between hypertension-related genes and cancers in the post-genomic era.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40959-022-00136-z.

The contribution of rare copy number variants in FAS toward pathogenesis of autoimmune lymphoproliferative syndrome

AbstractAutoimmune lymphoproliferative syndrome (ALPS) is characterized by chronic nonmalignant lymphadenopathy, splenomegaly, cytopenias, and other autoimmune manifestations. ALPS is caused by lymphocyte accumulation from defects in FAS-mediated apoptosis. Heterozygous germline or somatic pathogenic single nucleotide variants in FAS are the most common molecular etiology of ALPS. Through the Centralized Sequencing Program at the National Institute of Allergy and Infectious Diseases, we performed exome sequencing on subjects with a clinical diagnosis of ALPS, with a subset receiving copy number variant (CNV) analysis. In this cohort, we identified 3 subjects from unrelated families with CNVs at the FAS locus. One subject had a de novo ∼0.828 Mb copy number loss encompassing all of FAS. The second subject had a maternally inherited ∼1.004 Mb copy number loss encompassing all of FAS. The third subject had a paternally inherited ∼0.044 Mb copy number loss encompassing exons 7 through 9 of FAS. Subjects with deletions in FAS had clinical presentations and biomarker profiles similar to those with ALPS and with germline and somatic FAS variants. We demonstrate that CNV analysis should be pursued if there is clinical and biomarker evidence of ALPS because it can lead to a molecular diagnosis and appropriate treatment when FAS sequencing is inconclusive.

Somatic Mutations Detected in Parkinson Disease Could Affect Genes With a Role in Synaptic and Neuronal Processes.

The role of somatic mutations in complex diseases, including neurodevelopmental and neurodegenerative disorders, is becoming increasingly clear. However, to date, no study has shown their relation to Parkinson disease’s phenotype. To explore the relevance of embryonic somatic mutations in sporadic Parkinson disease, we performed whole-exome sequencing in blood and four brain regions of ten patients. We identified 59 candidate somatic single nucleotide variants (sSNVs) through sensitive calling and a careful filtering strategy (COSMOS). We validated 27 of them with amplicon-based ultra-deep sequencing, with a 70% validation rate for the highest-confidence variants. The identified sSNVs are in genes with synaptic functions that are co-expressed with genes previously associated with Parkinson disease. Most of the sSNVs were only called in blood but were also found in the brain tissues with ultra-deep amplicon sequencing, demonstrating the strength of multi-tissue sampling designs.

Somatic variants in diverse genes leads to a spectrum of focal cortical malformations.

Post-zygotically acquired genetic variants, or somatic variants, that arise during cortical development have emerged as important causes of focal epilepsies, particularly those due to malformations of cortical development. Pathogenic somatic variants have been identified in many genes within the PI3K-AKT-mTOR-signalling pathway in individuals with hemimegalencephaly and focal cortical dysplasia (type II), and more recently in SLC35A2 in individuals with focal cortical dysplasia (type I) or non-dysplastic epileptic cortex. Given the expanding role of somatic variants across different brain malformations, we sought to delineate the landscape of somatic variants in a large cohort of patients who underwent epilepsy surgery with hemimegalencephaly or focal cortical dysplasia. We evaluated samples from 123 children with hemimegalencephaly (n = 16), focal cortical dysplasia type I and related phenotypes (n = 48), focal cortical dysplasia type II (n = 44), or focal cortical dysplasia type III (n = 15). We performed high-depth exome sequencing in brain tissue-derived DNA from each case and identified somatic single nucleotide, indel and large copy number variants. In 75% of individuals with hemimegalencephaly and 29% with focal cortical dysplasia type II, we identified pathogenic variants in PI3K-AKT-mTOR pathway genes. Four of 48 cases with focal cortical dysplasia type I (8%) had a likely pathogenic variant in SLC35A2. While no other gene had multiple disease-causing somatic variants across the focal cortical dysplasia type I cohort, four individuals in this group had a single pathogenic or likely pathogenic somatic variant in CASK, KRAS, NF1 and NIPBL, genes previously associated with neurodevelopmental disorders. No rare pathogenic or likely pathogenic somatic variants in any neurological disease genes like those identified in the focal cortical dysplasia type I cohort were found in 63 neurologically normal controls (P = 0.017), suggesting a role for these novel variants. We also identified a somatic loss-of-function variant in the known epilepsy gene, PCDH19, present in a small number of alleles in the dysplastic tissue from a female patient with focal cortical dysplasia IIIa with hippocampal sclerosis. In contrast to focal cortical dysplasia type II, neither focal cortical dysplasia type I nor III had somatic variants in genes that converge on a unifying biological pathway, suggesting greater genetic heterogeneity compared to type II. Importantly, we demonstrate that focal cortical dysplasia types I, II and III are associated with somatic gene variants across a broad range of genes, many associated with epilepsy in clinical syndromes caused by germline variants, as well as including some not previously associated with radiographically evident cortical brain malformations.

Panel Sequencing for Clinically Oriented Variant Screening and Copy Number Detection in Chronic Lymphocytic Leukemia Patients.

According to current guidelines, in chronic lymphocytic leukemia (CLL), only the TP53 molecular status must be evaluated prior to every treatment’s initiation. However, additional heterogeneous genetic events are known to confer a proliferative advantage to the tumor clone and are associated with progression and treatment failure in CLL patients. Here, we describe the implementation of a comprehensive targeted sequencing solution that is suitable for routine clinical practice and allows for the detection of the most common somatic single-nucleotide and copy number variants in genes relevant to CLL. We demonstrate that this cost-effective strategy achieves variant detection with high accuracy, specificity, and sensitivity. Furthermore, we identify somatic variants and copy number variations in genes with prognostic and/or predictive value, according to the most recent literature, and the tool provides evidence about subclonal events. This next-generation sequencing (NGS) capture-based target assay is an improvement on current approaches in defining molecular prognostic and/or predictive variables in CLL patients.

Fibroblasts from Patients with Melorheostosis Promote Angiogenesis in Healthy Endothelial Cells through Secreted Factors

Melorheostosis is a rare sclerosing bone disease with associated vascular abnormalities in skin and bone, which is caused by somatic mosaic single nucleotide variations in the MAP2K1 gene, which encodes MAPK/extracellular signal‒regulated kinase (ERK) kinase 1. However, disease pathogenesis is poorly understood. Using patient-derived cells, we found that affected skin fibroblasts carrying the single nucleotide variations have increased activation of ERK1/2, which results in increased expression and secretion of proangiogenic factors, including VEGF. VEGF secretion was strongly reduced in affected cells after treatment with MAPK/ERK kinase 1 inhibitor trametinib. Treatment of healthy endothelial cells on matrigel with conditioned medium from affected fibroblasts induces the adoption of a proangiogenic phenotype. Direct coculture of fibroblasts and endothelial cells further shows that both secreted factors and extracellular matrix are capable of inducing a proangiogenic phenotype in healthy endothelial cells. Blocking VEGF with bevacizumab reduces the proangiogenic effect of affected fibroblasts in both the matrigel and direct coculture angiogenesis models, indicating that elevated VEGF secretion is a key mediator of increased angiogenesis in melorheostosis tissue. In conclusion, this work identifies the role of several important molecular mediators in the pathogenesis of melorheostosis, including MAPK/ERK kinase 1, phosphorylated ERK1/2, and VEGF, all of which have clinically available pharmacologic inhibitors, which could be further explored as therapeutic targets.

Integrative Genomic and Transcriptomic Profiling Reveals a Differential Molecular Signature in Uterine Leiomyoma versus Leiomyosarcoma.

The absence of standardized molecular profiling to differentiate uterine leiomyosarcomas versus leiomyomas represents a current diagnostic challenge. In this study, we aimed to search for a differential molecular signature for these myometrial tumors based on artificial intelligence. For this purpose, differential exome and transcriptome-wide research was performed on histologically confirmed leiomyomas (n = 52) and leiomyosarcomas (n = 44) to elucidate differences between and within these two entities. We identified a significantly higher tumor mutation burden in leiomyosarcomas vs. leiomyomas in terms of somatic single-nucleotide variants (171,863 vs. 81,152), indels (9491 vs. 4098), and copy number variants (8390 vs. 5376). Further, we discovered alterations in specific copy number variant regions that affect the expression of some tumor suppressor genes. A transcriptomic analysis revealed 489 differentially expressed genes between these two conditions, as well as structural rearrangements targeting ATRX and RAD51B. These results allowed us to develop a machine learning approach based on 19 differentially expressed genes that differentiate both tumor types with high sensitivity and specificity. Our findings provide a novel molecular signature for the diagnosis of leiomyoma and leiomyosarcoma, which could be helpful to complement the current morphological and immunohistochemical diagnosis and may lay the foundation for the future evaluation of malignancy risk.

Abstract P2-07-01: Blood tumor mutational burden (bTMB) and blood copy number burden (bCNB) by genome-wide circulating tumor DNA (ctDNA) assessment predict outcome and resistance in hormone-receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients (pts) treated with CDK4/6 inhibitor (CDK4/6i)

Abstract Background: CDK4/6i combined with ET improves survival for pts with HR+, HER2- MBC. However, biomarkers to predict efficacy and resistance are needed. We hypothesized that a comprehensive next-generation sequencing (NGS)-based liquid biopsy assessment of ctDNA mutation and copy number analysis may identify novel prognostic and predictive biomarkers. Methods: We collected serial blood for ctDNA testing from the 51 pts with HR+, HER2- MBC enrolled in the Alt Dose Palbo trial (NCT03007979), a single-arm phase II study of palbociclib plus letrozole or fulvestrant at 5 days on/2 days off weekly schedule as the first- or second-line ET. The median follow up was 38.2 months at data cutoff for this analysis. Plasma collected at baseline (BL) from all 51 pts, at progression (PD) from all 20 pts who have progressed, and at additional interim timepoints from 2 pts to explore longitutinal changes, with paired germline DNA, were subjected to the PredicineWES+ assay and low-pass whole-genome sequencing (lpWGS). The PredicineWES+ assay provides deep sequencing of 600 genes in the PredicineATLAS panel combined with whole-exome sequencing (WES) of all exonic regions of 20,000 genes enabling genome-wide detection of somatic single nucleotide variation (SNV), indels, copy number variation (CNV) and determination of bTMB. LpWGS offers an unbiased and high-throughput assessment of CNVs and tumor fraction in cell free DNA to derive bCNB. Statistical associations of bTMB, bCNB, and individual alterations with clinical benefit (CB), defined as no PD at 24 weeks by RECIST 1.1, and progression-free survival (PFS) were examined by Fisher’s exact test, Kaplan-Meier analysis, and Cox model. P values were adjusted to control false discovery rate (FDR). Results: The BL median bTMB was 1.6 mutations per megabase pair [IQR 0.6-3.5]. Pts with CB (N=41, 80%) had significantly lower bTMB scores (median [IQR] 1.2 [0.6-2.6] vs. 8.3 [2.4-21.4], (P<0.01) and significantly lower bCNB (P=0.03). bTMB and bCNB were highly correlated (Spearman correlation ρ =0.77, P=6.2E-11). bTMB above the predefined cutoff of 16 identified pts with significantly shorter PFS (4.7 vs 18.2 months, HR 3.73, CI 1.24-11.27, P=0.01). In contrast, BL clinical features including ET sensitivity, bone only vs. visceral, or de novo vs. recurrent MBC, were not associated with CB. BL alterations (SNV or CNV) in 91 genes, occurring in at least 3 pts each, were significantly associated with worse PFS (adjusted P<0.05), 61 of which were outside of the 600-gene panel. The 91 genes included both novel and previously reported alterations implicated in CDK4/6i and ET resistance. Examples include AR, ATM, AURKA, BRCA2, CCND1, DDR2, ESR1, FAT1, FGFR4, FOXP1, MYC, RB1, and RUNX1T1, some of which were also enriched at PD. Additional novel variants were enriched at PD, including ABCC12, ABCA13, SHANK1, and TET1. Serial analysis at BL, cycle 1 day 15 (C1D15), C2D1, the last Q3-month staging scan without PD, and at imaging detection of PD from 2 pts revealed a decline in bCNB at C1D15, then plateau, followed by an increase in bCNB that preceded imaging detection of PD. Conclusion: The comprehensive ctDNA profiling approach with PredicineWES+ and lpWGS identified high BL bTMB and bCNB as poor prognostic biomarkers in pts on CDK4/6i and ET. Our study also discovered novel candidate genes involved in cell cycle and DNA damage repair pathways in association with poor outcome. Results from this study highlight the genomic heterogeneity of HR+, HER2- MBC and provide important insights in the understanding of CDK4/6i and ET resistance mechanisms to guide therapeutic development in pts with resistant disease. Citation Format: Andrew Davis, Jingqin Luo, Tiantian Zheng, Lu Tan, Amy Wang, Rama Suresh, Foluso Ademuyiwa, Caron Rigden, Timothy Rearden, Katherine Clifton, Katherine Weilbaecher, Ashley Frith, Pavan K Tandra, Tracy Summa, Brittney Haas, Shana Thomas, Leonel Hernandez-Aya, Lindsay Peterson, Shujun Luo, Bonnie L King, Shidong Jia, Jianjun Yu, Pan Du, Jairam Krishnamurthy, Cynthia X Ma, C Dai. Blood tumor mutational burden (bTMB) and blood copy number burden (bCNB) by genome-wide circulating tumor DNA (ctDNA) assessment predict outcome and resistance in hormone-receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients (pts) treated with CDK4/6 inhibitor (CDK4/6i) [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 P2-07-01.

Abstract P3-09-04: Genomic landscape of HER2-negative advanced or metastatic breast cancer with PIK3CA gain-of-function mutations

Abstract Background: Alpelisib and fulvestrant are used as a combination treatment option for postmenopausal PIK3CA-mutated, hormone receptor positive (HR+), human epidermal growth factor receptor 2-negative (HER2-), advanced or metastatic breast cancer (a/mBC) patients. However, despite the presence of activating mutations in PIK3CA, the majority of patients do not derive benefit, or ultimately progress while on alpelisib therapy. Here, we investigate the genomic landscape of PIK3CA-mutated, HER2- a/mBC using next-generation sequencing (NGS) to provide insight into possible mechanisms of therapeutic resistance to alpelisib/fulvestrant and to identify potential targetable pathways. Methods: We utilized the Tempus LENS platform to retrospectively analyze de-identified NGS data from 2,918 a/mBC patients with formalin-fixed, paraffin-embedded tumor biopsies sequenced using the Tempus|xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage; full transcriptome RNA-seq). Mutations identified included germline and/or somatic single nucleotide variants, insertions/deletions and copy number variations (gains defined as ≥8 copies). We used curated clinical data to determine HER2 and hormone receptor (ER/PR) status. Results: Among 2,918 a/mBC patients, we identified somatic mutations in PIK3CA in 782 (26.8%). Within these tumors, 629 (80.4%) had one of the 11 mutations currently included in the alpelisib companion diagnostic, and we focused on this population (here defined as mut-PIK3CA). Of these 629, 546 (86.8%) were HER2-, with 176 (32.3%) and 370 (67.7%) derived from primary and metastatic tumors, respectively. Cases were further classified as HR+ (defined as ER+ or PR+) or triple negative (TNBC). While the majority of mutPIK3CA samples were identified in HR+ disease, 10% of the cases occurred in TNBC. Within the mutPIK3CA cohort, tumor mutational burden high (TMB-H; defined as ≥10 mutations/MB) was detected in 11.5% of samples, while microsatellite instability high (MSI-H) was detected in 0.5%. MSI-H was detected at a higher frequency in TNBC compared to HR+. Overall, the most commonly co-mutated genes among mutPIK3CA, HER2- samples were TP53 (34.6%), CDH1 (21.6%), ESR1 (12.3%), KMT2C (11%), MAP3K1 (9.5%), ARID1A (8.1%), PTEN (6.8%), GATA3 (6.6%), NF1 (5.9%), and TBX3 (5.9%) among others (Table 1); some of these genes have previously been implicated in resistance to endocrine therapy or PI3K inhibitor. In addition, in HR+ disease, metastatic samples had a higher frequency of mutations in genes implicated in endocrine resistance, such as ESR1 (18.7% vs 1.9%), ERBB2 (3.3% vs 2.6%), NF1 (6.8% vs 2.6%), compared to primary tumors. We also identified copy number gains (CNG) in several cell cycle genes, including: CCND1 (15.2%), CDK4 (2.7%), and AURKA (2.6%) (Table 1). Finally, further analyses at the transcript-level are the subject of on-going research. Conclusions: Our study highlights that there is substantial genomic heterogeneity among mutPIK3CA, HER2- a/mBCs. Across a series of comparisons between primary and metastatic samples, as well as HR+ and TNBC subtypes, we identified a number of co-mutations that occur alongside mutPIK3CA and which could be potentially exploited by targeted therapies. Future studies are needed to assess the prognostic/predictive role of these and other candidate gene alterations. Table 1. Genomic features of mutPIK3CA, HER2– a/mBCPrimaryMetastaticTotalAny PIK3CA Mutation1255527782mutPIK3CA2204425629HER2– (n=176)HER2– (n=370)546HR+ HER2– 154 (88%)TNBC 22 (12%)HR+ HER2– 337 (91%)TNBC 33 (9%)TMB-H16 (10.3%)2 (9.1%)41 (12%)4 (12%)63 (11.5%)MSI-H1 (0.6%)1 (4.5%)0 (0%)1 (3.0%)3 (0.5%)Co-mutations (mutPIK3CA): n (%)TP5347 (30.5%)14 (63.6%)101 (30%)27 (81.8%)189 (34.6%)CDH137 (24%)1 (4.5%)75 (22.3%)5 (15.2%)118 (21.6%)KMT2C17 (11%)1 (4.5%)40 (11.9%)2 (6.1%)60 (11%)MAP3K117 (11%)1 (4.5%)31 (9.2%)1 (3%)50 (9.2%)ARID1A15 (9.7%)0 (0%)26 (7.7%)2 (6.1%)43(7.9%)PTEN12 (7.8%)1 (4.5%)21 (6.2%)3 (9.1%)37 (6.8%)GATA311 (7.1%)0 (0%)23 (6.8%)2 (6.1%)35 (6.6%)TBX311 (7.1%)1 (4.5%)19 (5.6%)1 (3%)32 (5.9%)NCOR12 (1.3%)1 (4.5%)18 (5.3%)0 (0%)21 (3.8%)FOXA17 (4.5%)2 (9.1%)10 (3%)1 (3%)20 (3.7%)MAP2K41 (0.6%)1 (4.5%)12 (3.6%)1 (3%)15 (2.7%)ESR13 (1.9%)0 (0%)63 (18.7%)1 (3%)67 (12.3%)PIK3R13 (1.9%)0 (0%)4 (1.2%)0 (0%)7 (1.3%)AKT11 (0.6%)0 (0%)1 (0.6%)0 (0%)2 (0.4%)RB15 (3.2%)1 (4.5%)8 (2.4%)1 (3%)15 (2.7%)NF14 (2.6%)3 (13.6%)23 (6.8%)2 (6.1%)32 (5.9%)ERBB24 (2.6%)4 (18.2%)11 (3.3%)2 (6.1%)21 (3.8%)CCND1 CNG22 (14%)0 (0%)61 (18%)0 (0%)83 (15.2%)AURKA CNG3 (1.9%)0 (0%)11 (3.3%)0 (0%)14 (2.6%)CDK4 CNG5 (3.2%)1 (4.5%)7 (2.1%)1 (3.0%)14 (2.6%)1any somatic variant detected in PIK3CA2somatic PIK3CA mutations among the 11 currently included in the alpelisib companion diagnostic (C420R, E542K, E545A, E545D, E545G, E545K, Q546E, Q546R, H1047L, H1047R, and H1047Y) Citation Format: Jing Xi, Kathleen Harnden, Jingqin Luo, Greg S. Call, Elizabeth Mauer, Karyn Ronski, Cynthia X. Ma, Neil Vasan. Genomic landscape of HER2-negative advanced or metastatic breast cancer with PIK3CA gain-of-function mutations [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 P3-09-04.

Abstract P3-08-05: Multi-omic profiling of simultaneous ductal carcinoma in situ and invasive breast cancer

Abstract Background: Simultaneous ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC) is very common. However, it is not clear how often these histologies are clonally related or the mechanism by which DCIS evolves into IBC. In this study we evaluated tissue samples of DCIS and IBC by DNA and RNA sequencing from 39 patients with concurrent disease.Methods: Patients were identified from the Swedish Cancer Institute Breast Cancer Database starting sequentially with 2018 backwards to identify candidates with simultaneous DCIS and IBC. Individual pathologic review was performed from the cohort of patients who had had modified radical mastectomy to select 50 cases for analysis of normal breast, DCIS, and invasive disease. Sequencing was performed using the Tempus xT solid tumor assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). The mutations identified by this assay include germline and/or somatic single nucleotide variants, insertions/deletions, and copy number variations. Results: Thirty-nine cases had adequate tissue for both DCIS and IBC for DNA and 31 for RNA analysis. DCIS and IBC were in the same quadrant of the breast for all cases. All IBC cases were ductal carcinoma. 38/39 IBC were estrogen receptor (ER) positive, 33/39 were progesterone receptor (PR) positive. 5 were HER2/neu amplified (three ER+/PR-, 1 ER+/PR+, 1 ER/PR-). 32/39 had Ki67 of 20% or less (IQR 14(9,19.5)). 35/39 were T1 or T2. Three were T3 and one T4. 21/39 were N0, 4/39 N1mic, 9/39 N1a, 3/33 N2a 2/39 N3a.For DCIS 24/39 had both genomic variants and copy number changes (V+C+), 7/39 were V+C-, 5/39 were V-C+ and 3/39 V-C-. For IBC 25/39 were V+C+, 7/39 were V+C-, 6/39 were V-C+, and 1/39 V-C-. In total, there were 36 unique genomic variants seen in DCIS but not the corresponding invasive sample. In contrast, there were 32 total unique variants seen in invasive but not the corresponding DCIS and 38 variants were seen in both samples. The one BRCA variant seen in both samples of a single patient was BRCA1 p.Q284, a known pathogenic variant but gBRCA normal. There were 86 copy number changes seen in DCIS but not IBC, 75 copy number changes seen in IBC but not DCIS and 47 copy number changes seen in both DCIS and IBC. 24/39 patients shared at least one common variant and 22/39 shared at least one copy number change. 4/39 had no copy changes in either sample but all of these had genomic variants. 5/39 had no variants in either sample but all of these had copy number changes. PIK3CA was seen in 2/39 IBC, 2/39 DCIS and 2/39 in both. TMB IQR was 1.6(1.1, 3.4) for IBC and 2.1 (1.1,3.2) for DCIS. All samples tested for MSI were stable. Only one patient had identical genomic variants and copy changes in both DCIS and IBC. The most frequent changes were: RNA sequencing revealed 1178 genes with significant gene expression differences between paired DCIS and IBC (q<0.001). RNA-seq data was quantified using Kallisto and differential gene expression was assessed using DESeq2. Pathway enrichment was performed using rank GSEA against the MSigDB C2 collection (filtered to KEGG, Reactome and Biocarta pathways only). Upregulated pathways in DCIS involved more immunogenic pathways while IBC upregulated pathways involved proliferation and cell cycling. Conclusions: The transition from DCIS to IBC is a dynamic process. How much of these differences are the result of tumor heterogeneity and temporal factors as opposed to truly separate clones will require more study. This could have significant implications for the treatment of DCIS. VariantGain/Loss of FunctionIBC onlyDCIS onlySame in BothTotalTP53Loss53614MAP3K1Loss51511GATA3Gain3249KM2TCLoss0538PTENLoss2237Copy ChangeMCL1 (1q21.2)Gain68822CKS1B (1q21.3)Gain95619ERRB2 (17q12)Gain0459JAK1 (1p31.3)Loss2428IKZF1 (7p12.1)Loss2327 Citation Format: Henry G Kaplan, Anna B Berry, Alexa Dowdell, Brian Piening. Multi-omic profiling of simultaneous ductal carcinoma in situ and invasive breast cancer [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 P3-08-05.

Abstract P1-18-05: Early changes in circulating tumor DNA and its effect on clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib: Genotyping results from POLARIS

Abstract Background: POLARIS is a prospective, real-world study of palbociclib in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC) in the United States and Canada. These analyses evaluated the difference in tumor mutation profiles of patients receiving palbociclib in the first line versus second line or greater and examined the applicability of circulating tumor DNA (ctDNA) monitoring in a real-world setting. Methods: The clinical database cut-off date was March 16, 2021. Patients in the biomarker analysis group provided consent for serial blood sample collections, received ≥1 dose of initial palbociclib combination treatment, and had ≥1 ctDNA measurement available. The Guardant360 platform with somatic single-nucleotide variants in complete or critical exons of 73 genes was used. The association between early changes in ctDNA mutation allele fractions at Cycle 2 Day 1 (C2D1) and disease progression at Week 24 was evaluated using univariate and multivariable logistic regression analysis adjusting for line of therapy. Results: Among patients with HR+/HER2– ABC (N=347), 93.9% (n=326) had ctDNA measured at baseline, of which 85.9% (n=280) had ≥1 ctDNA alteration detected; 66.6% of patients received palbociclib treatment in the first line and 33.4% as second line or greater ABC setting. The frequencies of gene mutations at baseline were generally higher in patients receiving palbociclib as a second-line or greater therapy compared to those who received it as first-line therapy, particularly for ESR1 mutations (30% vs 15%) and FGFR1 mutations (13% vs 9%). With a median (range) follow-up duration of 18.5 (0.1-46.3) months, patients with total ctDNA increase at C2D1 (log ratio change >0) were 3.74 times more likely to have disease progression at Week 24 (odds ratio, 3.74 [95% CI, 1.71-8.17]; P=0.001) compared with those without change or with a decrease in ctDNA at C2D1 (log ratio change ≤0). The observed significant association remained after adjusting for line of therapy in the multivariable regression analysis (odds ratio, 2.62 [95% CI, 1.14-6.04]; P=0.023). Conclusions: Among patients with HR+/HER2- ABC receiving palbociclib, early changes in ctDNA mutations were significantly associated with disease progression. Further studies are needed to confirm these findings and to evaluate the clinical utility of ctDNA-guided “adaptive” early therapeutic interventions to improve outcomes in patients with metastatic breast cancer. Pfizer; NCT03280303 Citation Format: Debu Tripathy, Zhe Zhang, Joanne L. Blum, Meghan S. Karuturi, Steven L. McCune, Bijoy Telivala, Shailendra Lakhanpal, Kamal Patel, Richard C. Frank, Kit Lu, Chetan Deshpande, Yao Wang, Yuan Liu, Aditya Bardia. Early changes in circulating tumor DNA and its effect on clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib: Genotyping results from POLARIS [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 P1-18-05.

Abstract PD14-01: Comprehensive molecular characterization of patients with metastatic invasive lobular carcinoma (ILC): Using real-world data to describe this unique clinical entity

Abstract Background: ILC is the second most common type of breast cancer and accounts for approximately 10% of all invasive breast cancers. A hallmark of ILC is the lack of E-cadherin (CDH1) expression, which is frequently used to discriminate between lesions with borderline ductal and lobular histologies. While the genomic landscape of primary ILCs has been well described, less is known about patients (pts) with metastatic-ILC (mILC). Better characterization of the genomic landscape associated with mILC is critical for identifying biomarkers that may provide new insight into ILC tumor biology and ultimately improve long-term outcomes in pts with mILC. Methods: We retrospectively analyzed de-identified next-generation sequencing (NGS) data from 150 advanced/metastatic pts with ILC and 51 with mixed lobular/ductal histology, defined using the histology of the sequenced biopsy. Diagnoses were abstracted from pathology reports submitted at the time of sequencing. We used the stage documented closest in time to biopsy collection, and samples were excluded if the staging date was unknown or exceeded 180 days after the biopsy date. Our dataset consisted of samples that were molecularly profiled using the Tempus xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage, full-transcriptome RNA-seq). The mutations identified for this study include somatic single-nucleotide variants and insertions/deletions. Furthermore, we examined the co-mutational landscape of CDH1-mutant disease and investigated transcript-level expression variation. Results: Mutations in CDH1 occurred in 65.3% of all mILC samples (98/150). CDH1 expression was similar between CDH1-mutant and WT mILC samples (Wilcoxon rank sum test, p=0.8). The median tumor mutational burden (TMB) score was significantly higher in CDH1-mutant samples (Wilcoxon rank-sum test, p=0.010). CDH1-mutant samples were more likely to have a high TMB (≥10 mutations/MB) when compared with the wild-type CDH1 cohort (10% vs. 6.2%), but this difference was not statistically significant (Fisher’s Exact test, p=0.5). Additionally, we observed that the ER+ subtype was more frequent in CDH1-mutant samples, although this difference was not statistically significant (97% vs 88%; Fisher’s exact test, p=0.063). PIK3CA mutations were enriched in CDH1-mutant mILC (Table 1). TBX3 and NCOR1 mutations were also mildly enriched in CDH1-mutant mILC, but these results were not significant when correcting for multiple testing (Table 1). CDH1-mutant mixed histology pts had lower CDH1 expression than WT pts (p<0.001, Wilcoxon rank sum exact test). PIK3CA mutations were enriched in CDH1-mutant mixed histology pts, but this difference was not statistically significant (50% vs. 31%; p=0.3, Fisher’s exact test). Log10 CDH1 expression across all mILC pts was lower than in mixed histology pts (3.01 vs 3.53; p<0.001, Wilcoxon rank-sum test). Conclusions: Our real-world dataset illustrates that the molecular landscape of CDH1-mutant mILC pts is distinct from CDH1-WT mILC pts. Additionally, mILC differs from mixed histology at a transcriptional level, with lower CDH1 expression regardless of CDH1 mutational status. Our findings suggest a use for CDH1 RNA expression levels in reclassifying mixed histology samples as mILC. Additionally, therapies targeting PIK3CA may be further investigated for their actionability in CDH1-mutant mILC cases. Table 1.Frequency of co-mutations in CDH1-mutant vs. WT mILC cohortsGenesCDH1-mutant (n=98)CDH1 WT (n=52)p-value1q-value2n (%)n (%)PIK3CA53 (54%)6 (12%)<0.001<0.001TBX313 (13%)0 (0%)0.0040.13NCOR111 (11%)0 (0%)0.0090.21Pearson’s Chi-squared test; Fisher’sexact test. 2Falsediscovery rate correction for multiple testing Citation Format: Andrew A Davis, Amir Behdad, Kayla Viets Layng, Firas Wehbe, Lorenzo Gerratana, Elizabeth Mauer, Alex Barrett, Ami N Shah, Paolo D’Amico, Lisa Flaum, William J Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Comprehensive molecular characterization of patients with metastatic invasive lobular carcinoma (ILC): Using real-world data to describe this unique clinical entity [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 PD14-01.

Spatial Multiomics Analysis Reveals Only Minor Genetic and Epigenetic Changes in Human Liver Cancer Stem-Like Cells Compared With Other Tumor Parenchymal Cells.

Cancer stem cells (CSCs) usually account for a very small tumor cell population but play pivotal roles in human cancer development and recurrence. A fundamental question in cancer biology is what genetic and epigenetic changes occur in CSCs. Here we show that the in-situ global levels of DNA cytosine modifications, including 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC), are similar between liver cancer stem-like (LCSL) cells and paratumor liver cells of liver cancer patients. We then developed a robust method combining immunohistochemistry, laser capture microdissection and genome sequencing with ultra-low-input cells (CIL-seq) to study the detailed genetic and DNA methylation changes in human LCSL cells. We first used clinical samples of mixed hepatocellular carcinoma-cholangiocarcinoma (HCC-CCA) with stem cell features to investigate human LCSL cells. The CIL-seq analysis of HCC-CCA and HCC patients showed that LCSL cells had strong spatial genetic and epigenetic heterogeneity. More interestingly, although the LCSL cells had some potential key changes in their genome, they had substantially fewer somatic single nucleotide variants (SNVs), copy number alterations (CNAs) and differentially methylated regions than other tumor parenchymal cells. The cluster analysis of SNVs, CNAs, DNA methylation patterns and spatial transcriptomes all clearly showed that the LCSL cells were clustered with the paratumor liver cells. Thus, spatial multiomics analysis showed that LCSL cells had only minor genetic and epigenetic changes compared with other tumor parenchymal cells. Targeting key changes in CSCs, not just changes in bulk tumor cells, should be more effective for human cancer therapy.

Neoantigens as potential vaccines in hepatocellular carcinoma

BackgroundNeoantigens, new immunogenic sequences arising from tumor mutations, have been associated with response to immunotherapy and are considered potential targets for vaccination. Hepatocellular carcinoma (HCC) is a moderately mutated tumor,...

CellPhy: accurate and fast probabilistic inference of single-cell phylogenies from scDNA-seq data

We introduce CellPhy, a maximum likelihood framework for inferring phylogenetic trees from somatic single-cell single-nucleotide variants. CellPhy leverages a finite-site Markov genotype model with 16 diploid states and considers amplification error and allelic dropout. We implement CellPhy into RAxML-NG, a widely used phylogenetic inference package that provides statistical confidence measurements and scales well on large datasets with hundreds or thousands of cells. Comprehensive simulations suggest that CellPhy is more robust to single-cell genomics errors and outperforms state-of-the-art methods under realistic scenarios, both in accuracy and speed. CellPhy is freely available at https://github.com/amkozlov/cellphy.

ATM Germline-Mutated Gastroesophageal Junction Adenocarcinomas: Clinical Descriptors, Molecular Characteristics, and Potential Therapeutic Implications.

BackgroundGastroesophageal junction (GEJ) adenocarcinoma is a rare cancer associated with poor prognosis. The genetic factors conferring predisposition to GEJ adenocarcinoma have yet to be identified.MethodsWe analyzed germline testing results from 23 381 cancer patients undergoing tumor-normal sequencing, of which 312 individuals had GEJ adenocarcinoma. Genomic profiles and clinico-pathologic features were analyzed for the GEJ adenocarcinomas. Silencing of ATM and ATR was performed using validated short-interfering RNA species in GEJ, esophageal, and gastric adenocarcinoma cell lines. All statistical tests were 2-sided.ResultsPathogenic or likely pathogenic ATM variants were identified in 18 of 312 patients (5.8%), and bi-allelic inactivation of ATM through loss of heterozygosity of the wild-type allele was detected in all (16 of 16) samples with sufficient tumor content. Germline ATM-mutated GEJ adenocarcinomas largely lacked somatic mutations in TP53, were more likely to harbor MDM2 amplification, and harbored statistically significantly fewer somatic single nucleotide variants (2.0 mutations/Mb vs 7.9 mutations/Mb; P < .001). A statistically significantly higher proportion of germline ATM-mutated than ATM–wild-type GEJ adenocarcinoma patients underwent a curative resection (10 [100%] vs 92 [86.8%], P = .04; Fisher’s exact test.), A synthetic lethal interaction between short-interfering RNA silencing of ATM and ATR was observed in the models analyzed.ConclusionsOur results indicate that germline pathogenic variants in ATM drive oncogenesis in GEJ adenocarcinoma and might result in a distinct clinical phenotype. Given the high prevalence of germline ATM-mutated GEJ adenocarcinomas, genetic testing for individuals with GEJ adenocarcinomas may be considered to better inform prognostication, treatment decisions, and future cancer risk.