Targeted Sequencing Panel Research Articles

Switching anti-EGFR antibody re-sensitizes head and neck cancer patient following acquired resistance to cetuximab.

Cetuximab induces responses in about 13% of head and neck squamous cell carcinomas (HNSCC). We describe the molecular mechanism of acquired resistance to cetuximab, which could be overcome by switching to a different anti-EGFR antibody. Biopsies were collected at three different time points: before the start of cetuximab (PRE-cetux), at acquired resistance to cetuximab (AR-cetux), and at acquired resistance to duligotuzumab (AR-duligo). Biopsies were analyzed using tumor and normal whole-exome sequencing, RNASeq, and targeted panel sequencing with ultra-deep coverage to generate differential mutation and expression profiles. WES and targeted sequencing analysis identified an EGFR p.G465R extracellular domain mutation in AR-cetux biopsy. Furthermore, RNASeq confirmed the expression of this mutation in the tumor tissue. This mutation prevented the binding of cetuximab to EGFR and was not present in PRE-cetux and AR-duligo biopsies, suggesting a potential mechanism of acquired resistance to cetuximab. Molecular dynamic simulations confirmed that duligotuzumab effectively binds EGFR with a p.G465R mutation. Interestingly, the p.G465R mutation improved the stability of the duligotuzumab-EGFR complex as compared to the wild-type EGFR. This is the first report of an EGFR ECD mutation associated with acquired resistance to cetuximab, posing a need for further validation. We suggest appropriate serial mutational profiling to identify ECD mutations should be considered for select patients with initial cetuximab benefit.

Chromosome Segregation-1-like Gene Participates in Ferroptosis in Human Ovarian Granulosa Cells via Nucleocytoplasmic Transport.

Premature ovarian insufficiency (POI) is defined as the depletion of ovarian function before the age of 40 years. The global prevalence of POI is 3.5%. To date, genetic factors account for 23.5% of the etiology of POI. Herein, a previously uncharacterized pathogenic homozygous variant of the chromosome segregation-1-like gene (CSE1L) was identified in POI patients via targeted panel sequencing. It is reported that dysregulated iron metabolism is involved in many reproductive endocrine disorders; however, its precise role in POI remains obscure. In this study, we identified CSE1L as a potential candidate gene that plays an important role in maintaining iron homeostasis. Deficiency of CSE1L led to ferroptosis in human granulosa cells, which was confirmed by transmission electron microscopy. Mechanistically, coimmunoprecipitation identified the direct interaction between CSE1L and FoxO1. Inhibition of CSE1L led to the excessive accumulation of FoxO1 in the nucleus via nucleocytoplasmic transport. Then, FoxO1 bound to the promoter region of NCOA4 and promoted its transcription, which was verified by a chromatin immunoprecipitation assay. Moreover, inhibition of CSE1L in cumulus cell monolayer could impede oocyte maturation, which might be associated with oxidative stress. Consequently, our study first revealed that CSE1L participated in ferroptosis in human ovarian granulosa cells via nucleocytoplasmic transportation, which might be helpful in revealing the molecular mechanism of CSE1L in the development of POI. Importantly, these findings might provide new insights into the application of ferroptosis inhibitors in the treatment of POI.

Genomic Landscape of Branchio-Oto-Renal Syndrome through Whole-Genome Sequencing: A Single Rare Disease Center Experience in South Korea.

Branchio-oto-renal (BOR) and branchio-otic (BO) syndromes are characterized by anomalies affecting the ears, often accompanied by hearing loss, as well as abnormalities in the branchial arches and renal system. These syndromes exhibit a broad spectrum of phenotypes and a complex genomic landscape, with significant contributions from the EYA1 gene and the SIX gene family, including SIX1 and SIX5. Due to their diverse phenotypic presentations, which can overlap with other genetic syndromes, molecular genetic confirmation is essential. As sequencing technologies advance, whole-genome sequencing (WGS) is increasingly used in rare disease diagnostics. We explored the genomic landscape of 23 unrelated Korean families with typical or atypical BOR/BO syndrome using a stepwise approach: targeted panel sequencing and exome sequencing (Step 1), multiplex ligation-dependent probe amplification (MLPA) with copy number variation screening (Step 2), and WGS (Step 3). Integrating WGS into our diagnostic pipeline detected structure variations, including cryptic inversion and complex genomic rearrangement, eventually enhancing the diagnostic yield to 91%. Our findings expand the genomic architecture of BOR/BO syndrome and highlight the need for WGS to address the genetic diagnosis of clinically heterogeneous rare diseases.

Targeted panel sequencing of pharmacogenes and oncodrivers in colorectal cancer patients reveals genes with prognostic significance

BackgroundColorectal cancer is still the second leading cause of cancer-related deaths and thus biomarkers allowing prediction of the resistance of patients to therapy and estimating their prognosis are needed. We designed a panel of 558 genes with pharmacogenomics records related to 5-fluorouracil resistance, genes important for sensitivity to other frequently used drugs, major oncodrivers, and actionable genes. We performed a target enrichment sequencing of DNA from tumors and matched blood samples of patients, and compared the results with patient prognosis stratified by systemic adjuvant chemotherapy.ResultsThe median number of detected variants per tumor sample was 18.5 with 4 classified as having a high predicted functional effect and 14.5 moderate effect. APC, TP53, and KRAS were the most frequent mutated genes (64%, 59%, and 42% of mutated samples, respectively) followed by FAT4 (23%), FBXW7, and PIK3CA (16% for both). Patients with advanced stage III had more frequently APC, TP53, or KRAS mutations than those in stages I or II. KRAS mutation counts followed an increasing trend with grade (G1 < G2 < G3). The response to adjuvant therapy was worse in carriers of frameshift mutations in APC or 12D variant in KRAS, but none of these oncodrivers had prognostic value. Carriage of somatic mutations in any of the genes ABCA13, ANK2, COL7A1, NAV3, or UNC80 had prognostic relevance for worse overall survival (OS) of all patients. In contrast, mutations in FLG, GLI3, or UNC80 were prognostic in the same direction for patients untreated, and mutations in COL6A3, LRP1B, NAV3, RYR1, RYR3, TCHH, or TENM4 for patients treated with adjuvant therapy. The first association was externally validated. From all germline variants with high or moderate predicted functional effects (median 326 per patient), > 5% frequency and positive Manhattan plot based on 3-year RFS, rs72753407 in NFACS, rs34621071 in ERBB4, and rs2444274 in RIF1 were significantly associated with RFS, OS or both.ConclusionsThe present study identified several putative somatic and germline genetic events with prognostic potential for colorectal cancer that should undergo functional characterization.

The Role of NOTCH Pathway Genes in the Inherited Susceptibility to Aortic Stenosis.

The NOTCH-signaling pathway is responsible for intercellular interactions and cell fate commitment. Recently, NOTCH pathway genes were demonstrated to play an important role in aortic valve development, leading to an increased calcified aortic valve disease (CAVD) later in life. Here, we further investigate the association between genetic variants in the NOTCH pathway genes and aortic stenosis in a case-control study of 90 CAVD cases and 4723 controls using target panel sequencing of full-length 20 genes from a NOTCH-related pathway (DVL2, DTX2, MFNG, NUMBL, LFNG, DVL1, DTX4, APH1A, DTX1, APH1B, NOTCH1, ADAM17, DVL3, NCSTN, DTX3L, ILK, RFNG, DTX3, NOTCH4, PSENEN). We identified a common intronic variant in NOTCH1, protecting against CAVD development (rs3812603), as well as several rare and unique new variants in NOTCH-pathway genes (DTX4, NOTCH1, DTX1, DVL2, NOTCH1, DTX3L, DVL3), with a prominent effect of the protein structure and function.

Similar genetic profile in early and late stage urothelial tract cancer

IntroductionUrothelial tract cancer (UTC) ranks as the tenth most prevalent cancer and holds the seventh position in terms of mortality worldwide. Despite its prevalence and mortality ranking, there are still gaps in the knowledge of the mutational landscape in patients with advanced disease who have limited therapeutic options after multiple lines of prior treatment. This study compares the genomic and transcriptomic landscape, and targeted treatment options between metastatic UTC (mUTC) patients treated with multiple lines of therapy compared to newly diagnosed, untreated Muscle Invasive Bladder Cancer (MIBC).MethodsWe compared genomic and clinical data from two cohorts: mUTC patients who received multiple lines of therapy and were referred to the Copenhagen Prospective Personalized Oncology (CoPPO) project at Rigshospitalet, University of Copenhagen. Data for MIBC UTC patients were acquired from the Cancer Genome Atlas Bladder Cancer (TCGA BLCA) cohort. Biopsies in CoPPO were performed at the time of enrollment. 523 highly important cancer-related genes (TrueSight Oncology-500 targeted sequencing panel) were used from both cohorts for comparative analysis. Analyses included RNA count data to compare predicted molecular subtypes in each cohort separately.ResultsPatients from the CoPPO cohort had a lower median age at first-line treatment than the TCGA BLCA cohort, with no significant gender disparity. The predominant histology was urothelial cell carcinoma in both cohorts. Genomic analysis revealed no significant difference between the top mutated genes in the two cohorts, specifically looking into DNA damage repair genes. Molecular subtyping indicated a higher frequency of neuroendocrine differentiation in the CoPPO cohort. 13% of patients in the CoPPO cohort received targeted therapy based on genomic findings, and 16% received non-targeted treatment, totaling 29% receiving CoPPO treatment (9 patients). The remaining 71% received best supportive care. Kaplan-Meier analysis showed a non-significant survival benefit for the intervention group in the CoPPO cohort.ConclusionWhen focusing on 523 highly relevant cancer genes, the mutational profile of mUTC patients who have undergone numerous treatment lines resembles that of newly diagnosed MIBC. These alterations can be targeted, indicating the potential advantage of early genomic testing for personalized treatment within clinical trials.

Utility of Targeted Sequencing Compared to FISH for Detection of Chronic Lymphocytic Leukemia Copy Number Alterations.

Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNAs) and somatic mutations that are central to disease prognosis, risk stratification, and mechanisms of therapy resistance. Fluorescence in situ hybridization (FISH) panels are widely used in clinical applications as the gold standard for screening prognostic chromosomal abnormalities in CLL. DNA sequencing is an alternative approach to identifying CNAs but is not an established method for clinical CNA screening. We sequenced DNA from 509 individuals with CLL or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, using a targeted sequencing panel of 59 recurrently mutated genes in CLL and additional amplicons across regions affected by clinically relevant CNAs [i.e., del(17p), del(11q), del(13q), and trisomy 12]. We used the PatternCNV algorithm to call CNA and compared the concordance of calling clinically relevant CNAs by targeted sequencing to that of FISH. We found a high accuracy of calling CNAs via sequencing compared to FISH. With FISH as the gold standard, the specificity of targeted sequencing was >95%, sensitivity was >86%, positive predictive value was >90%, and negative predictive value was >84% across the clinically relevant CNAs. Using targeted sequencing, we were also able to identify other common CLL-associated CNAs, including del(6q), del(14q), and gain 8q, as well as complex karyotype, defined as the presence of 3 or more chromosomal abnormalities, in 26 patients. In a single and cost-effective assay that can be performed on stored DNA samples, targeted sequencing can simultaneously detect CNAs, somatic mutations, and complex karyotypes, which are all important prognostic features in CLL.

The Utility of Long-Read Sequencing in Diagnosing Genetic Autosomal Recessive Parkinson's Disease: a genetic screening study.

Mutations within the genes PRKN and PINK1 are the leading cause of early onset autosomal recessive Parkinson's disease (PD). However, the genetic cause of most early-onset PD (EOPD) cases still remains unresolved. Long-read sequencing has successfully identified many pathogenic structural variants that cause disease, but this technology has not been widely applied to PD. We recently identified the genetic cause of EOPD in a pair of monozygotic twins by uncovering a complex structural variant that spans over 7 Mb, utilizing Oxford Nanopore Technologies (ONT) long-read sequencing. In this study, we aimed to expand on this and assess whether a second variant could be detected with ONT long-read sequencing in other unresolved EOPD cases reported to carry one heterozygous variant in PRKN or PINK1. ONT long-read sequencing was performed on patients with one reported PRKN/PINK1 pathogenic variant. EOPD patients with an age at onset younger than 50 were included in this study. As a positive control, we also included EOPD patients who had already been identified to carry two known PRKN pathogenic variants. Initial genetic testing was performed using either short-read targeted panel sequencing for single nucleotide variants and multiplex ligation-dependent probe amplification (MLPA) for copy number variants. 48 patients were included in this study (PRKN "one-variant" n = 24, PINK1 "one-variant" n = 12, PRKN "two-variants" n = 12). Using ONT long-read sequencing, we detected a second pathogenic variant in six PRKN "one-variant" patients (26%, 6/23) but none in the PINK1 "one-variant" patients (0%, 0/12). Long-read sequencing identified one case with a complex inversion, two instances of structural variant overlap, and three cases of duplication. In addition, in the positive control PRKN "two-variants" group, we were able to identify both pathogenic variants in PRKN in all the patients (100%, 12/12). This data highlights that ONT long-read sequencing is a powerful tool to identify a pathogenic structural variant at the PRKN locus that is often missed by conventional methods. Therefore, for cases where conventional methods fail to detect a second variant for EOPD, long-read sequencing should be considered as an alternative and complementary approach.

Longitudinal circulating tumor DNA monitoring in predicting response to short-course neoadjuvant radiotherapy in locally advanced rectal cancer: Data from a phase III clinical trial (UNION).

LBA3606 Background: The data presented here is from a multicenter, randomized, open-label, controlled Phase III clinical study evaluating the feasibility of short-course radiotherapy (shortRT) sequentially combined with camrelizumab and chemotherapy as neoadjuvant therapy (NAT) for locally advanced rectal cancer (LARC)(UNION). Our aim is to explore the value of circulating tumor DNA (ctDNA)-based minimal residual disease (MRD) in assessing the comparative efficacy of short-course and long-course chemoradiotherapy (CRT). Methods: A total of 244 plasma samples from 79 LARC patients, who underwent NAT prior to curative surgery, were collected at baseline (C1), on-NAT (C2), post-NAT (C3), and post-surgery (C4). Deep targeted panel sequencing of 556 cancer-related genes was performed. The changes in genomic features and ctDNA-MRD status during treatment were monitored, and the relationship between these changes and treatment response were explored. Results: During NAT, the ctDNA-MRD positivity rate showed significant declining trends. Patients with high baseline TMB tend to show a significant inclination towards major pathological response and tumor regression grade 0/1 after NAT, while there is no significant correlation observed between baseline ctDNA-MRD status and treatment response. Interestingly, compared to long-course radiotherapy, microsatellite instability is more pronounced after shortRT ( P=0.042), and ctDNA negativity is significantly associated with pathological complete response (pCR) ( P=0.022). Furthermore, both ctDNA clearance ( P=0.049) and MRD clearance ( P=0.015) after shortRT are significantly correlated with pCR. A risk scoring predictive model based on ctDNA-MRD was established, with achieving the highest C-index at the C2 time point. This model, combining MRD clearance and CEA, outperforms models using only MRD clearance (AUC=0.917, 95% CI=0.753 to 1.000) or only CEA (AUC=0.733, 95% CI=0.449 to 1.000), demonstrating superior performance in predicting pCR/non-pCR (AUC=0.983, 95% CI=0.937 to 1.000). Conclusions: These findings offers valuable insights into the dynamic landscape of NAT for LARC management and emphasize the potential of ctDNA-based MRD assessment as a valuable tool for tailoring treatment strategies. The differences observed between shortCRT and longCRT regimens underscore the need for personalized treatment approaches. Overall, our study contributes valuable insights into optimizing treatment decision-making and predicting treatment response in LARC patients, ultimately advancing the field of rectal cancer management.

Molecular profiling using next generation sequencing with high-purity enrichment of circulating tumor cells.

e15054 Background: In the field of liquid biopsy, coupled with next generation sequencing (NGS), circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can serve as an alternative substrate to tumor tissue for mutation detection and companion diagnostic purpose. Emerging evidence suggests that complementary sequential CTCs and ctDNA profiling offers promising benefits for prognosis and treatment. In contrast to well-established ctDNA extraction methods, the process of isolation of intact CTCs for NGS analysis has not yet been fully elucidated. Here we show that isolation and harvesting of viable CTCs using CytoGen's Smart Biopsy™ System achieves high CTC purity for NGS analysis in breast cancer and non-small cell lung cancer (NSCLC). Methods: Targeted panel sequencing for CTC was developed to detect representative mutated genes using the Oncomine™ Comprehensive Assay Plus (501 genes) and the Oncomine™ Pan-Cancer Cell-Free Assay (52 genes). Analytical validation was performed with breast cancer cells MDA-MB-231 (50 to 5 cells) and NSCLC cells H358 (10 and 5 cells) which were spiked into 5 mL of healthy blood to mimic CTCs. CytoGen's Smart Biopsy™ System was applied to isolate the CTCs. Subsequently, CD45 depletion and whole genome amplification were performed. Sequencing was performed on Ion S5™ XL Systems using an Ion 550™ chip kit. The sequencing data were then analyzed using the Ion Torrent software with the default configurations. Results: As a result of targeted panel sequencing with Oncomine™ Comprehensive Assay Plus, representative breast cancer mutation genes (BRAF, KRAS, TP53, NF2 and NOTCH3) were detected in at least 20 MDA-MB-231 cells. In the results using Oncomine™ Pan-Cancer Cell-Free Assay, mutated genes (BRAF, KRAS and TP53) were successfully detected in as few as 5 MDA-MB-231 cells and, in addition, KRAS mutation was detected even with only 5 H358 cells. These results suggest that CytoGen's Smart Biopsy™ System could be applied to detect representative mutated genes in clinical breast cancer and NSCLC patient samples with detection technology that require high sensitivity and specificity. Conclusions: Molecular analysis of CTCs is a challenge since CTCs are very rare in the blood and there are technical limitations in isolating CTCs with sufficient purity for NGS analysis. In this study, we developed the process for NGS analysis on very small number of high-purity CTCs using CytoGen's Smart Biopsy™ System. Taken together with the results from our ongoing clinical trials, this process could provide information for the diagnosis and appropriate treatment of breast cancer and NSCLC patients.

Clinical and analytical validation for HRD status using targeted panel sequencing.

e17501 Background: In cancer therapy, understanding homologous recombination deficiency (HRD) is paramount, particularly due to its positive association with sensitivity to PARP inhibitor (PARPi) therapy. We propose using a targeted sequencing approach as a cost-effective and efficient alternative to the current standard of whole genome sequencing (WGS). In this study, we determine the sensitivity and clinical applications of a panel of 437 genes and over 12000 heterozygous SNP loci for evaluating HRD. Methods: HRD positivity for this assay was set at an HRD score of ≥38. To determine the limit of detection (LOD), we diluted 19 samples (4 tiers, 20 repeats each) with known HRD status. Accuracy of the assay was determined by comparing HRD status calls of 40 samples with those obtained through whole genome sequencing. Reproducibility of the assay was tested by measuring variance of HRD status for 30 samples between experimental conditions (2 operators* 3 repeats). Samples were obtained from 85 ovarian cancer (OC) patients undergoing platinum-based (Pt) chemotherapy and were profiled using our targeted sequencing assay. Results: The LOD for BRAC1/2 copy number deletions was established at a tumor purity (TP) of 30%. On the level of individual mutations, LODs range between 1.74% to 5.85% AF with a median of 2.26% AF. Repeatability analysis yields high concordance (APA= 97.83%, 95%CI= 95.34% - 99.00%; ANA= 97.73%, 95%CI= 95.13% - 98.95%), which emphasizes the precision of the assay. Comparisons against WGS based HRD scores reveal strong correlations, with Pearson coefficients of 0.975 for single sample (tumor sample only) and 0.987 for paired sample (tumor + normal sample) HRD score analysis. Through tumor profiling, we identified novel gene mutations associated with resistance to PARPi therapy in the HRD-positive population of our cohort. In the clinical setting, HRD-positive OC patients were found to experience longer progression free survival than their HRD-negative counterparts (median PFS = 18.3 vs 11.4 months, p&lt;0.05). Upon further investigation, we observed that HRD-positive patients who did not carry pathogenic alterations on BRCA1/2 gene showed significantly longer PFS than HRD-negative patients without pathogenic BRCA1/2 alterations (median PFS = 18.5 vs 11.6 months, p&lt;0.05). Conclusions: In summary, this targeted panel offers enhanced sensitivity, reliability, and precision in the assessment of HRD status for informed cancer therapy decisions. In practice, we showed that the targeted sequencing approach is a viable solution for determining HRD status of OC patients and can potentially serve as a predictive biomarker for treatment response. We believe that out assay emerges as a cornerstone in the advancement of personalized cancer care, and offers a sensitive, reliable, and precise approach to HRD assessment for improving patient outcomes.

CCNE1 amplification as marker of poor prognosis and novel therapeutic target in advanced breast cancer.

1040 Background: Cyclin E1 (CCNE1) overexpression or gene amplification is linked to dismal outcomes in various tumors, including breast cancer (BC). Recent studies suggest that CCNE1 amplification is a potential target for new synthetic lethality-based treatments, including CDK2-selective and PKMYT1 inhibition. This study explores the clinical and genomic characteristics of CCNE1-amplified BCs. Methods: We analyzed genomic and clinical data from consecutive BCs that underwent clinical tumor-normal targeted panel sequencing (MSK-IMPACT) between April 2014 and December 2021. Allele-specific copy number, including CCNE1 amplification, and fraction genome altered (FGA) were calculated by FACETS. Mutual exclusivity and co-occurrence analyses were performed using CoMEt. Benjamini–Hochberg method for multiple testing correction (q) was applied. Real-world progression-free survival (rwPFS) was assessed by the Kaplan Meier method and Cox models, focusing on patients with available pre-treatment samples. Results: Out of 3,753 BCs, 125 (3.3%) had CCNE1 amplification. A higher occurrence of CCNE1 amplification was noted in post-treatment (n=2,368) compared to treatment-naïve tumors (n=1,385; 4% vs 2.4%, p=0.007). CCNE1 amplification was less common in hormone receptor (HR)+/HER2- BCs (2%) compared to HER2+ (7.6%) and triple-negative (7.2%) tumors (p&lt;0.001). BCs with CCNE1 amplification showed higher median FGA, indicating increased genomic instability. In primary BC, TP53 alterations were more frequent in CCNE1-amplified tumors (q&lt;0.001), while CDH1 alterations were mutually exclusive (q&lt;0.001) suggesting that lobular BCs rarely harbor CCNE1 amplification. Similar trends were seen in post-treatment samples. CCNE1 amplification detected at baseline was linked to shorter median rwPFS in HR+/HER2- metastatic BCs treated with CDK4/6 inhibitors plus endocrine therapy (8.8 vs 15.2 months in CCNE1-amplified [n=9] vs CCNE1-non amplified [n=402]; hazard ratio [HR] 2.82, 95% CI 1.38-5.75), and in HER2+ metastatic BCs treated with first-line THP regimen (7.3 vs 20.8 months in CCNE1-amplified [n=5] vs CCNE1-non amplified [n=106]; HR 3.1, 95% CI 1.24-7.87). In a CCNE1-amplified triple-negative cell model (HCC1569), treatment with the PKMYT1 inhibitor RP-6306 significantly reduced tumor growth. Conclusions: CCNE1 amplification in BCs is associated with greater genomic instability and characterizes a group of metastatic BCs with poor response to standard of care therapies. Novel therapies targeting CCNE1-amplified tumors are under evaluation in preclinical and clinical studies.

Clinical utility of AlphaMissense in predicting pathogenicity of DNA damage repair genes in cancer.

10581 Background: Deficiencies in DNA damage repair (DDR) impact cancer predisposition and various treatment responses.Functional implications of missense variants in DDR genes remain elusive.AlphaMissense, a new AI-based method, accurately predicts the pathogenicity of missense variants, but its clinical utility requires validation. We evaluated the accuracy of AlphaMissense predictions by analyzing known mutational signatures, genomic characteristics, and therapeutic vulnerabilities of DDR-deficient tumors. Methods: We analyzed data from TCGA (n=8,178) and MSK-IMPACT targeted panel sequencing (n=56,695) to evaluate the performance of AlphaMissense in predicting clinically actionable DDR genes, including core homologous recombination (HR) genes ( BRCA1/2, PALB2, RAD51C), POLE, and ATM. Missense mutations were classified as known pathogenic by OncoKB or previous literature, newly pathogenic identified with AlphaMisense, or benign. Mutational signatures were computed using SigMA algorithm, classifying each signature as positive at &gt;20%. The irradiated tumor control was evaluated in patients with ATM missense mutations who received radiotherapy. Results: Among 6,743 unique DDR missense mutations from MSK-IMPACT, 372 (3.5%) were known pathogenic, and 1,182 (17.5%) were new pathogenic mutations. In 205 tumors with bi-allelic core-HR missense mutations in breast, ovarian, pancreatic, and prostate cancers, 20 (9.8%) had new pathogenic mutations. Tumors with new pathogenic mutations more frequently exhibited HR-deficient signatures (65%) than wild-type (31.8%, p &lt; .001), similar to tumors with known pathogenic mutations (62.8%, p = 1.0). In TCGA, HR-deficient signatures tended to be more frequent in tumors with new pathogenic missense mutations than in wild-type tumors (41.7% vs. 21.0%, p = .16). Of 1,010 tumors with POLE missense mutations from MSK-IMPACT, 300 (30%) had new pathogenic mutations. The presence of POLE signatures in tumors with new pathogenic mutations (11.9%) was not significantly different from those with benign mutations (4.3%, p = .19) and less frequent than in tumors with known pathogenic mutations (91.0%, p &lt; .001). In MSK-IMPACT, tumors with new pathogenic ATM missense mutations were less likely to have TP53 mutations than benign missense mutations (32.7% vs. 56.6%, p &lt; .001), consistent with bona-fide pathogenicity. Further, patients with new pathogenic ATM missense mutations showed increased radiosensitivity, characteristic of ATM deficiency, and improved irradiated tumor control compared to those with benign missense mutations (Hazard ratio 0.58, 95% Cl 0.35-0.95, p = .03). Conclusions: AlphaMissense can identify previously unknown pathogenic DDR missense mutations, but its accuracy is gene-dependent. AlphaMissense pathogenicity predictions for DDR genes can utilized to help classify mutations, but may not be sufficient on their own.

Detection of tumor-derived cell-free DNA in cerebrospinal fluid using a clinically validated targeted sequencing panel for pediatric brain tumors.

Clinical sequencing of tumor DNA is necessary to render an integrated diagnosis and select therapy for children with primary central nervous system (CNS) tumors, but neurosurgical biopsy is not without risk. In this study, we describe cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) as sources for "liquid biopsy" in pediatric brain tumors. CSF samples were collected by lumbar puncture, ventriculostomy, or surgery from pediatric patients with CNS tumors. Following extraction, CSF-derived cfDNA was sequenced using UW-OncoPlex™, a clinically validated next-generation sequencing platform. CSF-derived cfDNA results and paired plasma and tumor samples concordance was also evaluated. Seventeen CSF samples were obtained from 15 pediatric patients with primary CNS tumors. Tumor types included medulloblastoma (n = 7), atypical teratoid/rhabdoid tumor (n = 2), diffuse midline glioma with H3 K27 alteration (n = 4), pilocytic astrocytoma (n = 1), and pleomorphic xanthoastrocytoma (n = 1). CSF-derived cfDNA was detected in 9/17 (53%) of samples, and sufficient for sequencing in 8/10 (80%) of extracted samples. All somatic mutations and copy-number variants were also detected in matched tumor tissue, and tumor-derived cfDNA was absent in plasma samples and controls. Tumor-derived cfDNA alterations were detected in the absence of cytological evidence of malignant cells in as little as 200µl of CSF. Several clinically relevant alterations, including a KIAA1549::BRAF fusion were detected. Clinically relevant genomic alterations are detectable using CSF-derived cfDNA across a range of pediatric brain tumors. Next-generation sequencing platforms are capable of producing a high yield of DNA alterations with 100% concordance rate with tissue analysis.

Genetic and epigenetic alterations in precursor lesions of endometrial endometrioid carcinoma.

The hyperplasia-carcinoma sequence is a stepwise tumourigenic programme towards endometrial cancer in which normal endometrial epithelium becomes neoplastic through non-atypical endometrial hyperplasia (NAEH) and atypical endometrial hyperplasia (AEH), under the influence of unopposed oestrogen. NAEH and AEH are known to exhibit polyclonal and monoclonal cell growth, respectively; yet, aside from focal PTEN protein loss, the genetic and epigenetic alterations that occur during the cellular transition remain largely unknown. We sought to explore the potential molecular mechanisms that promote the NAEH-AEH transition and identify molecular markers that could help to differentiate between these two states. We conducted target-panel sequencing on the coding exons of 596 genes, including 96 endometrial cancer driver genes, and DNA methylome microarrays for 48 NAEH and 44 AEH lesions that were separately collected via macro- or micro-dissection from the endometrial tissues of 30 cases. Sequencing analyses revealed acquisition of the PTEN mutation and the clonal expansion of tumour cells in AEH samples. Further, across the transition, alterations to the DNA methylome were characterised by hypermethylation of promoter/enhancer regions and CpG islands, as well as hypo- and hyper-methylation of DNA-binding regions for transcription factors relevant to endometrial cell differentiation and/or tumourigenesis, including FOXA2, SOX17, and HAND2. The identified DNA methylation signature distinguishing NAEH and AEH lesions was reproducible in a validation cohort with modest discriminative capability. These findings not only support the concept that the transition from NAEH to AEH is an essential step within neoplastic cell transformation of endometrial epithelium but also provide deep insight into the molecular mechanism of the tumourigenic programme. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract PS06-09: Fragmentomic analysis of a circulating tumor DNA targeted cancer gene panel discriminates ER status in metastatic breast cancer liquid biopsies

Abstract Background: Circulating tumor DNA (ctDNA) isolated from the peripheral blood of patients with cancer is now routinely used in the clinic to detect tumor somatic mutations, with multiple ctDNA-targeted sequencing panels commercially available for Food and Drug Administration (FDA)-approved biomarker indications to guide targeted therapy options. More recently, it has been appreciated that ctDNA fragmentation patterns are influenced by the chromatin structure of the cell of origin, with greater diversity in fragment size in areas with open chromatin and less diversity in fragment size in areas with closed chromatin. In consequence, multiple studies have found that ctDNA fragmentation patterns can be used to accurately infer epigenomic and transcriptomic phenotypes from their tumor cells of origin. However, these analyses have previously relied on whole-genome sequencing (WGS), and it is not feasible to identify FDA-approved biomarker indications in a cost-effective manner with WGS. Methods: Peripheral blood was collected from a cohort of 90 patients with metastatic HER2-negative breast cancer (ER-positive n=71, ER-negative n=19). Cell-free DNA was extracted from plasma and sequenced using a custom 822 gene pan-cancer targeted sequencing panel (IDT). Shannon entropy of first coding exon fragment sizes (E1SE) was used to quantify fragmentation patterns. Higher diversity of fragment sizes, leading to a higher E1SE, are expected in ctDNA derived from areas of the genome with more open chromatin. A machine learning model utilizing the E1SE metric across all genes in the targeted panel was developed to distinguish between ER-positive and ER-negative disease. Results: Training cross-validated accuracy of the machine learning model was 87.8% with AUC of 0.91 to distinguish between ER-positive and ER-negative disease, despite a median ctDNA fraction of only 0.02. E1SE metrics for ESR1 and CCND1 were significantly higher in ER-positive than ER-negative samples, while E1SE metrics for EGFR, c-Kit and members of the MAP kinase family were significantly higher in ER-negative than ER-positive samples, consistent with anticipated differences in gene expression between ER-positive and ER-negative triple negative breast cancers. In contrast, HER2 E1SE was similar between groups, as expected in this clinically HER2-negative cohort. Conclusions: We have shown for the first time that sequencing from standard targeted ctDNA panels can be utilized to infer phenotypic information through analysis of ctDNA fragmentation patterns in patients with metastatic breast cancer. This expands the depth of potential biologic data that can be extracted from targeted ctDNA sequencing panels in addition to DNA mutation status in a cost-effective manner. In the future, this approach can be exploited to expand ctDNA-based biomarker development beyond DNA mutation status. Citation Format: Kyle Helzer, Jamie Sperger, Yue Shi, Viridiana Carreno, Hannah Krause, Katherine Kaufmann, Leilani Mora-Rodriguez, Matthew Bootsma, Mark Burkard, Ruth O'Regan, Kari Wisinski, Joshua Lang, Shuang (George) Zhao, Malinda West, Marina Sharifi. Fragmentomic analysis of a circulating tumor DNA targeted cancer gene panel discriminates ER status in metastatic breast cancer liquid biopsies [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS06-09.

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater.

Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

Screening of premature ovarian insufficiency associated genes in Hungarian patients with next generation sequencing

BackgroundPremature ovarian insuffiency (POI) is one of the main cause behind infertility. The genetic analysis of POI should be part of the clinical diagnostics, as several genes have been implicated in the genetic background of it. The aim of our study was to analyse the genetic background of POI in a Hungarian cohort.MethodsThe age of onset was between 15 and 39 years. All patients had the 46,XX karyotype and they were prescreened for the most frequent POI associated FMR1 premutation. To identify genetic alterations next-generation sequencing (NGS) of 31 genes which were previously associated to POI were carried out in 48 unrelated patients from Hungary.ResultsMonogenic defect was identified in 16.7% (8 of 48) and a potential genetic risk factor was found in 29.2% (14 of 48) and susceptible oligogenic effect was described in 12.5% (6 of 48) of women with POI using the customized targeted panel sequencing. The genetic analysis identified 8 heterozygous damaging and 4 potentially damaging variants in POI-associated genes. Further 10 potential genetic risk factors were detected in seven genes, from which EIF2B and GALT were the most frequent. These variants were related to 15 genes: AIRE, ATM, DACH2, DAZL, EIF2B2, EIF2B4, FMR1, GALT, GDF9, HS6ST2, LHCGR, NOBOX, POLG, USP9X and XPNPEP2. In six cases, two or three coexisting damaging mutations and risk variants were identified.ConclusionsPOI is characterized by heterogenous phenotypic features with complex genetic background that contains increasing number of genes. Deleterious variants, which were detected in our cohort, related to gonadal development (oogenesis and folliculogenesis), meiosis and DNA repair, hormonal signaling, immune function, and metabolism which were previously associated with the POI phenotype. This is the first genetic epidemiology study targeting POI associated genes in Hungary. The frequency of variants in different POI associated genes were similar to the literature, except EIF2B and GALT. Both of these genes potential risk factor were detected which could influence the phenotype, although it is unlikely that they can be responsible for the development of the disease by themselves. Advances of sequencing technologies make it possible to aid diagnostics of POI Since individual patients show high phenotypic variance because of the complex network controlling human folliculogenesis. Comprehensive NGS screening by widening the scope to genes which were previously linked to infertility may facilitate more accurate, quicker and cheaper genetic diagnoses for POI. The investigation of patient’s genotype could support clinical decision-making process and pave the way for future clinical trials and therapies.

Abstract NG07: Association between somatic microsatellite instability, hypermutation status, and specific T cell subsets in colorectal cancer tumors

Abstract NG07: Association between somatic microsatellite instability, hypermutation status, and specific T cell subsets in colorectal cancer tumors

TMBcalc: a computational pipeline for identifying pan-cancer Tumor Mutational Burden gene signatures.

In the precision medicine era, identifying predictive factors to select patients most likely to benefit from treatment with immunological agents is a crucial and open challenge in oncology. This paper presents a pan-cancer analysis of Tumor Mutational Burden (TMB). We developed a novel computational pipeline, TMBcalc, to calculate the TMB. Our methodology can identify small and reliable gene signatures to estimate TMB from custom targeted-sequencing panels. For this purpose, our pipeline has been trained on top of 17 cancer types data obtained from TCGA. Our results show that TMB, computed through the identified signature, strongly correlates with TMB obtained from whole-exome sequencing (WES). We have rigorously analyzed the effectiveness of our methodology on top of several independent datasets. In particular we conducted a comprehensive testing on: (i) 126 samples sourced from the TCGA database; few independent whole-exome sequencing (WES) datasets linked to colon, breast, and liver cancers, all acquired from the EGA and the ICGC Data Portal. This rigorous evaluation clearly highlights the robustness and practicality of our approach, positioning it as a promising avenue for driving substantial progress within the realm of clinical practice.