Somatic Single Nucleotide Variants Research Articles

Molecular Characterization and Therapeutic Opportunities in KRAS Wildtype Pancreatic Ductal Adenocarcinoma

Purpose: To investigate the molecular characteristics of and potential for precision medicine in KRAS wildtype pancreatic ductal adenocarcinoma (PDAC). Patients and Methods: We investigated 27 patients with KRASWT PDAC at our institution. Clinical data were obtained via chart review. Tumor specimens for each subject were interrogated for somatic single nucleotide variants, insertion and deletions, and copy number variants by DNA sequencing. Gene fusions were detected from RNA-seq. A patient-derived organoid (PDO) was developed from a patient with a MET translocation and expanded ex vivo to predict therapeutic sensitivity prior to enrollment in a phase 2 clinical trial. Results: Transcriptomic analysis showed our cohort may be stratified by the relative gene expression of the KRAS signaling cascade. The PDO derived from our patient harboring a TFG-MET rearrangement was found to have in vitro sensitivity to the multi-tyrosine kinase inhibitor crizotinib. The patient was enrolled in the phase 2 SPARTA clinical trial and received monotherapy with vebrelitinib, a c-MET inhibitor, and achieved a partial and durable response. Conclusions: KRASWT PDAC is molecularly distinct from KRASMUT and enriched with potentially actionable genetic variants. In our study, transcriptomic profiling revealed that the KRAS signaling cascade may play a key role in KRASWT PDAC. Our report of a KRASWT PDAC patient with TFG-MET rearrangement who responded to a cMET inhibitor further supports the pursuit of precision oncology in this sub-population. Identification of targetable mutations, perhaps through approaches like RNA-seq, can help enable precision-driven approaches to select optimal treatment based on tumor characteristics.

Abstract PO3-06-05: Association of tumor-derived extracellular vesicles with circulating tumor DNA alterations in metastatic breast cancer patients: exploring differences in inflammatory breast cancer

Abstract Background: Liquid biopsy provides a real-time assessment of metastatic breast cancer (MBC). Recently, the complementary prognostic value of tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) has been reported. We have previously confirmed the strong prognostic significance of CTCs and tdEVs in inflammatory breast cancer (IBC). While previous studies have reported the association of CTCs with circulating tumor DNA (ctDNA) alterations in MBC, no evidence is available for tdEVs. This study aimed to analyze the association of tdEVs with ctDNA alterations, to investigate the molecular pathways underlying their presence. Moreover, we explored potential differences of this association in patients (pts) with IBC to provide a comprehensive liquid biopsy-based portrait of this aggressive subtype of BC. Methods: Blood samples were collected from 355 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). For CTCs and tdEVs analysis, 7.5 mL of blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Positivity cutoff was ≥5 for CTCs, while tdEV cutoff points were < 20 (low), 20-79 (intermediate), and ≥80 (high). For ctDNA analysis, matched plasma samples (± 1 month) were analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs) and copy number variations (CNVs), which were classified into oncogenic pathways based on defined profiles generated on the Cancer Genome Atlas database (p53, PI3K, ER, RTK, RAS, RAF, WNT, MYC, cell cycle, notch). Associations between ctDNA pathways alterations and tdEVs were tested in the overall population by multinomial logistic regression and corrected for significant clinical characteristics. Differences in liquid biopsy features between IBC and non-IBC subgroups were analyzed through Fisher's exact test. Results: Of the 355 MBC pts, 210 (62%) had HR+ BC, 61 (17%) had HER2+ BC, and 68 (20%) had triple-negative BC. Eighty-three (23%) had a diagnosis of IBC and had a numerically lower tdEV count. Also, lower tdEVs were detected in HER2+ BC as compared with HR+. Significantly higher tdEVs were observed among pts with lobular histology, liver, and bone metastases. CTC count was significantly associated with tdEV number. A matched plasma sample for ctDNA analysis was available for 175 pts (62 IBC and 113 non-IBC). In the overall population, SNVs in ER pathway were associated with intermediated/high levels of tdEVs (p=0.004 and 0.008). A similar association was observed for CNVs in the cell cycle pathway (p=0.008 and 0.007). Moreover, associations with ≥80 tdEVs were observed for PI3K SNVs and CNVs (p=0.039 and 0.004, respectively), RTK CNVs (p=0.023), and MYC CNVs (p=0.001). In multivariable analysis, clinical characteristics associated with higher tdEVs were lobular histology (p=0.014 for 20-79 and < 0.001 for ≥80) and bone metastases (p=0.019 for 20-79 and 0.002 for ≥80). When considering clinical variables of interest, only ER SNVs and MYC CNVs were significantly associated with intermediate (p=0.031) and high (p=0.022) tdEV counts, respectively. While the association with ER SNVs and cell cycle CNVs was significant both in the IBC and non-IBC subgroups, others were specific for a certain subgroup: higher tdEVs were significantly associated with PI3K SNVs only in non-IBC pts whereas the significant association with CNVs in PI3K and MYC pathways was observed only in pts with IBC. Conclusion: Detection of tdEVs was associated with particular genomic profiles. These alterations seem to be different in IBC further underlying a different biology of this BC subtype. Additional studies are needed to explore how to integrate different liquid-biopsy based biomarkers in the management of pts with MBC. Citation Format: Eleonora Nicolò, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz Arcos, Mara Serena Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda Kaylan Strickland, Youbin Zhang, Paolo D’Amico, Andrew Davis, Jeannine Donahue, Huiping liu, William Gradishar, Ami Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Association of tumor-derived extracellular vesicles with circulating tumor DNA alterations in metastatic breast cancer patients: exploring differences in inflammatory breast cancer [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 PO3-06-05.

Abstract PS12-02: Exploration of ctDNA Dynamics in the PACE Trial: A Randomized Phase II Study of Fulvestrant, Palbociclib, and Avelumab for HR+/HER2- Metastatic Breast Cancer

Abstract Background The PACE trial for HR+/HER2- metastatic breast cancer (MBC) prospectively evaluated whether continuation of the CKD4/6 inhibitor (CDK4/6i) palbociclib (P) beyond progression on prior CDK4/6i and aromatase inhibitor (AI), with a change in endocrine therapy (ET) to fulvestrant (F), improved outcomes beyond change to F alone, as well as explored the activity of a palbociclib, fulvestrant, and avelumab (P+F+A) triplet. The primary analysis did not show improvement in progression-free survival (PFS) with F+P versus F alone. In this analysis, an evaluation of serial translational PACE samples was performed to gain insights into the mutational landscape of HR+/HER2- MBC after prolonged exposure to CDK4/6i, evaluate early mutational dynamics with ongoing therapy, and identify potential genetic markers predictive of treatment response. Additionally, the identification of acquired mutations at the time of tumor progression could provide valuable information about mechanisms of treatment resistance. Methods PACE is a multicenter randomized open-label investigator-initiated phase II trial. Eligible patients (pts) had HR+/HER2- evaluable MBC with prior progression on AI and any CDK4/6i after > 6 months (mo) of therapy in the MBC setting, or during/within 12 mo in the adjuvant setting, with no more than 1 prior line of chemotherapy for MBC. Pts were randomized 1:2:1 to F alone, F+P, or F+P+A, with tumor assessments every 8 weeks. Correlative samples including circulating tumor DNA (ctDNA) were collected at baseline (BL), times of tumor assessments (C3D1), and end of study/progression (EOT). Targeted NGS (Guardant360) to evaluate mutations (somatic single nucleotide variants and indels) was performed. Kaplan-Meier survival curves were used for assessing PFS and the log-rank test for comparisons. McNemar’s test with continuity correction was used to test the changes in the frequency of mutations. Results Of the 220 randomized pts, 211 contributed ctDNA samples (200 at BL, 129 at C3D1, 146 at EOT). Characteristics of the correlative science population were similar to the intention to treat population. At BL, the most common mutations observed were ESR1 (57%), TP53 (38%), PIK3CA (36%), GATA3 (20%) ATM (12%) and Rb1 (12%). The presence of a PIK3CA mutation vs WT-PIK3CA at BL was associated with shorter PFS in the F alone arm (p=0.001) but not in the F+P or F+P+A arms. ESR1 mutations present at baseline were D538G (34%), Y537S (21%), Y537N (15%), E380Q (10%) and L536H (4%). At C3D1, the most common mutations observed were ESR1 (47%), TP53 (33%), PIK3CA (26.1), GATA3 (18.1), ATM (13%) and PTEN (8.6%). Including only BL and C3D1 matched samples (N=124), the prevalence of ESR1 mutations decreased after 2 cycles of treatment (57% at BL vs 44% at C3D1), predominantly observed in the F+P+A arm. For those in any arm with at least 6 months of disease stability on study (N=53), the presence of an ESR1 D538G mutation decreased during therapy, with 38% having a mutation at BL, 24% at C3D1, and no pts gaining ESR1 D538G at C3D1. The prevalence of PIK3CA mutations also decreased on treatment (32% at BL to 24% at C3D1), with the greatest decrease in the F+P arm. A predictive model correlating early genomic dynamics and associations with outcome will be presented. Conclusions The PACE trial offers one of the largest clinical trial cohorts describing the genomic landscape of HR+/HER2- MBC post-CDK4/6 exposure. Results from this correlative study provide a comprehensive mutational landscape post-CDK4/6i, demonstrate associations between mutations and clinical outcomes, and suggest the potential value of early ctDNA dynamics in this setting. Citation Format: Rinath Jeselsohn, Jingxin Fu, Yue Ren, Reshma Mahtani, Cynthia Ma, Angela DeMichele, Massimo Cristofanilli, Jane Meisel, Kathy Miller, Yara Abdou, Elizabeth Riley, Rubina Qamar, Priyanka Sharma, Sonya Reid, Naomi Ko, Yuan Liu, Harold Burstein, Michelle DeMeo, Sara Tolaney, Erica Mayer. Exploration of ctDNA Dynamics in the PACE Trial: A Randomized Phase II Study of Fulvestrant, Palbociclib, and Avelumab for HR+/HER2- Metastatic Breast Cancer [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 PS12-02.

Abstract PO4-01-02: High throughput analysis in HER2 positive locally advanced breast cancer: pCR and mutational status

Abstract Background Pathologic complete response (pCR) after neoadjuvant treatment (NAD) is a strong prognostic biomarker associated with improved survival in breast cancer patients, especially with HER2-positive and Triple negative subtypes. PIK3CA is mutated in up to 20% of HER2 positive breast cancers, contributes to anti-HER-2 resistance and may be predictive of the lack of response to anti HER2 NAD. PIK3CA mutations in breast cancer occur primarily at hotspots E545K at exon 9 and H1047R at exon 20. Next generation sequencing has improved our knowledge regarding the biology of the mechanisms behind treatment resistance. The aim of this study is to evaluate the genomic landscape of tissue samples obtained from locally advanced breast cancer patients treated in an neoadjuvant setting, in order to correlate the genomic analysis to response and clinical outcome. Methods DNA extracted from the archival 84 formalin-fixed paraffin-embedded (FFPE) samples from core needle biopsies was subjected to deep sequencing using the TruSight Oncology (TSO) 500 panel (Illumina, San Diego, USA; 523 genes, size: 1.94 Mb), following the manufacturer’s protocol, which assesses microsatellite instability (MSI) status, tumor mutation burden (TMB), recurrent somatic copy number variations (sCNV), somatic single nucleotide variants (SNVs). Libraries were sequenced on a NovaSeq 6000 instrument (Illumina) to reach a minimum of 500 × read depth. All samples were evaluated for RNA single cell transcriptional profile. 73 of 84 patients were treated with traditional neoadjuvant chemotherapy plus trastuzumab meanwhile 13 of 84 received neoadjuvant chemotherapy plus trastuzumab and pertuzumab. Statistical analysis was performed to detect the genes whose mutation involved in the progression and/or non-response of the disease using the Statistical Package for Social Science (SPSS), release 23.0. Continuous variables were expressed as mean ± SD or median [range], categorical variables were displayed as frequencies and the appropriate parametric (Student‘s t-test) non-parametric test (X2-test or Mann-Whitney test) was used to assess significance of the differences between subgroups (patients with or without pCR). Results In total we analyzed samples of 84 patients. The median age was 48 years. 64 of 84 (76.2%) were triple positive, 66 (78.6%) with G3 and 29 (34.5%) with stage III. The most common frequently mutated genes were PIK3CA (16/84, 19%) and BRCA1 (7/84, 8.3%). The pathogenic variant of BRCA1 (sBRCA1) had a significant association with resistance to treatment: we found a pCR in only 14.3% of sBRCA1 versus 62.3% in sBRCA1 wild type (wt) patients (p=0.014). We also identified 11 somatic mutations in hereditary genes (ATM, BRCA1, BRCA2, PALB2, CHEK2, RAD51C, RAD51D). From a germline standpoint we detected 7 pathogenic variants (two affecting BRCA1, two affecting CHEK2, one affecting PALB2, 7/84, 8.3%), which will be clinically confirmed. Among patients with a mutated PI3KCA, 43.8% obtained a pCR versus 61.8% of patients with PI3KCA wt (p=0.19). This data probably presents an imbalance due to the sample size A PTEN mutation was identified in 2% of pts (2/84) which obtained no benefit in terms of pCR after NAD versus 59.8% in wt patients (p=0.09). Conclusions In this preliminary report, genetic drivers such as BRCA1 status may have a clinical implication for prognosis and treatment response after NAD in locally advanced HER2 positive breast cancer. More samples are needed to evaluate the significance of PI3K and PTEN mutations in this patient setting. We are waiting for the results of the RNA single cell transcriptional profile of these samples. Event free survival data is not yet mature at this moment. We would like to acknowledge the contribution of Multi-specialistic Biobank Research Core Facility G-STeP, Fondazione Policlinico Universitario “A. Gemelli” IRCCS (Biobank-FPG) who provided the bio-resources. Citation Format: Ida Paris, Angelo Minucci, Daniele Generali, simona Nanni, Tatiana D'angelo, Francesco Pavese, Alessandra Fabi, Maria De Bonis, Katia Cannita, Alessia Piermattei, Giordana Tiberi, Paola Fuso, Luisa Carbognin, Silvia Rotondaro, Camilla Nero, Flavia Giacomini, Tina Pasciuto, Maria Marrazzo, Luciano Giaco', Antonino Mulè, Riccardo Masetti, Gianluca Franceschini, Giorgia Garganese, Diana Giannarelli, Giovanni Scambia. High throughput analysis in HER2 positive locally advanced breast cancer: pCR and mutational status [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 PO4-01-02.

Comprehensive characterization of somatic mutations associated with chimeric RNAs in human cancers.

Chimeric RNAs, which can arise from gene recombination at the DNA level or non-canonical splicing events at the RNA level, have been identified as important roles in human tumors. Dysregulated gene expression caused by somatic mutations and altered splicing patterns of oncogenes or tumor suppressor genes can contribute to the development of tumors. Therefore, investigating the formation mechanism of chimeric RNAs via somatic mutations is critical for understanding tumor pathogenesis. This project is the first to propose studying the association between somatic single nucleotide variants and chimeric RNAs, identifying around 2900 somatic SNVs affecting chimeric RNAs in pan-cancer level. The somatic SNVs on chimeric RNAs were commonly observed in various types of tumor tissues, providing a valuable resource for future study. Additionally, these SNVs show distinct tumor specificity, and those with high frequency had a significant impact on the survival time of patients with tumors. Further research revealed that somatic SNVs associated with chimeric RNA (chiR-SNVs) were typically found within 10 nt of the junction site of chimeric RNAs and had a particularly significant effect on chimeric RNAs from different chromosomes. The enrichment analysis revealed that chiR-SNVs were significantly overrepresented in oncogenes and genes related to RNA binding proteins involved in RNA splicing, which could imply that chiR-SNVs may disrupt the process of RNA splicing and induce the occurrence of chimeric RNAs. This study sheds light on the potential molecular interaction mechanism between somatic SNVs and chimeric RNAs, which opens up a new avenue for researching disease pathway and tumorigenesis development.

Abstract 2850: Noncoding regulatory mutations as driving event for the oncogenic core regulatory circuitries of neuroblastoma

Abstract Background and Aim: Neuroblastoma (NB) is the most aggressive solid tumor of infancy and arises from neural-crest-derived progenitor cells. It is composed by cell types reflecting developmental stages of the adrenergic lineage: mesenchymal (MES) and adrenergic (ADRN) cell types. These cell identities result from the activity of NB specific Core Regulatory Circuitries (CRCs), transcription factor (TF) sets that co-occupy regulatory regions together impacting the expression of their own genes and those of lineage-specific ones governing cell state transitions and cellular identities. Our hypothesis is that noncoding somatic single nucleotide variants (SNVs) located in NB CRCs TF binding sites (TFBSs) underlie NB CRCs activity, affecting cellular differentiation and promoting tumor onset. We aim to investigate such pattern of regulatory elements and identify putative driver SNVs, exploring their role and characterizing their action mechanism. Methods: Transcriptionally active TFBSs (aTFBSs) bound by ADRN CRC TFs (GATA3, HAND2, ISL1, TBX2, MYCN, PHOX2B, ASCL1 and LMO1) were identified integrating 27 ChIP-seq and 11 ATAC-seq experiments in 6 NB ADRN cell lines (SKNBE2C, Kelly, NGP, COGN415, LAN5 and NB1643). In particular, aTFBSs were defined as ChIP-seq peaks overlapping ATAC-seq signals in the same cell line. SNVs from 317 NB whole genomes were mapped on aTFBSs and the enrichment of mutations in core regions respect to flanking ones was tested using Fisher test. Observed mutation rate in aTFBSs was compared with background mutation rate due to local sequence context. SNVs in mutated aTFBSs were selected and their impact on TF binding affinity was evaluated with FABIAN-variant and motifbreakR tools. aTFBSs target-genes were identified through promoter capture HiC (CHiC) in ADRN NB cells (SKNBE2C and SHSY5Y). Results: We found a significant (FDR < 0.05) mutation enrichment in aTFBSs of ISL1, PHOX2B, TBX2, GATA3 and MYCN, suggesting a common somatic mutation burden for a subset of ADRN CRC TFBSs in ADRN NB cells. Starting from 839 SNVs falling in significantly mutated aTFBSs, we selected 269 mutations impacting the binding of ADRN CRC TFs. This mutations subset mapped in aTFBSs interacting with genes that significantly enriched pathways involved in neuronal development and migration (FDR < 0.05), thus suggesting their possible role in affecting cell identity definition. As an example, chr6:137753475:G>T mapped in aTFBSs interacting with the promoter of OLIG3, which takes part in neuronal differentiation. Interestingly, chr6:137753475:G>T was predicted to break the binding to ISL1 (FABIAN score: -0.24), a key regulator of ADRN CRC. Conclusions: These results demonstrate that somatic noncoding SNVs can act synergistically to promote NB onset, affecting CRCs activity and, in turn, the normal cell identity definition. Citation Format: Mario Capasso, Vincenzo Aievola, Vito Alessandro Lasorsa, Annalaura Montella, Ferdinando Bonfiglio, Marianna Avitabile, Teresa Maiorino, Matilde Tirelli, Giuseppe D'Alterio, Matthias Fischer, Frank Westermann, Achille Iolascon. Noncoding regulatory mutations as driving event for the oncogenic core regulatory circuitries of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2850.

Abstract 4336: Homologous recombination repair deficiency, and not recurrence, determines mutational burden and clonal dynamics in high grade serous ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy, killing more than 9,000 women each year in the United States. Nearly 80% of patients with HGSOC will experience recurrence within 5 years, but little is known about the mechanisms that drive this process. Up to 60% of the genome in an HGSOC tumor is impacted by structural variant mutations and pronounced intratumoral heterogeneity. Intratumor heterogeneity is believed to be a key feature of recurrence and therapy resistance with some evidence indicating diverse and complex mechanisms of the seeding of metastatic sites and recurrent tumors. We included 35 HGSOC patients for whom paired chemo-naive and chemoresistant tumors as well as germline DNA was available. Whole genome sequencing was used to identify somatic single nucleotide variants (SNV) insertion/deletion variants (indels), and structural variants (SVs) in each tumor. The number of somatic SVs and indels was higher in tumors with homologous recombination repair deficiency (HRD; P=0.000252 SVs, P=0.00385 Indel) but was not affected by recurrent status (P=0.826 SV, P=0.145 SNV/indel). Clonal composition and dynamics were measured using SNVs/indels as tumors progressed from chemo-naïve primary samples to recurrent chemo-resistant tumors. Surprisingly, few changes were observed in clonal abundance and complexity through progression. When this analysis was repeated with SVs homologous recombination repair proficient tumors tend to be polyclonal while HRD tumors tend to be monoclonal. Five denovo structural variant signatures were identified in our cohort and led to three distinct structural variant classes tumors defined by deletion, tumors defined by duplication, and tumors defined by copy number neutral changes (inversions, translocations and balanced complex events). Patients with tumors defined by deletions and copy number neutral changes carry pathogenic SVs correlating with reduced survival (P=0.00023), while DNA gains are less subject to pathogenic alterations. Each class displayed distinct regions of the chromosome that was frequently affected by large scale SV events (>5Mb), which was also observed in the HRP/HRD status. Although no regions were notably altered across recurrence, GO analysis revealed that recurrent tumors have a significantly reduced immune response, which was not seen in the primary tumors. Within a subset of tumors, we are plan to utilize Oxford Nanopore Technologies ultra long read sequencing which will allow for confirmation of SVs observed in the WGS as well as identification of more complex events that cannot be deciphered using WGS techniques. Citation Format: Michael A. Diaz, Nicole Gull, Pei-Chen Peng, Kate Lawrenson, Bobbie J. Rimel, Jenny Lester, Beth Karlan, Simon A. Gayther, Michelle R. Jones. Homologous recombination repair deficiency, and not recurrence, determines mutational burden and clonal dynamics in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4336.

Abstract 7369: Passenger mutations link cellular origins and transcriptional identity in human lung adenocarcinoma

Abstract Lineage plasticity hinders identification of cellular origin of cancers, as malignant cells cease to resemble benign cells from which they presumably evolved. Lung adenocarcinoma (LUAD), the most common primary lung cancer in United States is thought to arise from alveolar type II (AT2) cells. However recent mouse models have shown that LUADs may also arise from other cell types such as basal and club. Although such mouse models are considered to be gold standard for identifying cell of origin for cancers, they are generally limited to a specific genetic context and lack all cell types that are present in human lung. Thus, to better understand the cellular origins of LUAD, we utilized a publicly available lung scRNA-seq atlas from Human Cell Atlas, spanning over 584,000 cells from 107 individuals to investigate cell type specific passenger mutational footprints of transcriptional coupled repair process (a sub-pathway of nucleotide excision repair that preferentially corrects mutations in highly expressed genes) across 295 lung cancer WGS profiles. As most mutations in self-renewing tissues are thought to occur prior to the onset of tumorigenesis, genes that are most highly transcribed in the cell of origin would have fewer somatic single nucleotide variants (SNV). To test this hypothesis, our study correlated somatic mutational density with gene expression profiles of cell types to identify Alveolar Type 0 (AT0, newly identified cell type) cells to be most strongly associated with LUAD mutation density, suggesting that the ancestor of LUAD cells in tumor spent most of their non-cancerous time in AT0 state. Interestingly studies have shown that following DNA damage in AT2 cells, transition to AT1 occurs to repair the damage. However, if the damage is detrimental, the transition of AT2 to AT1 is stalled and the cells remain in AT0 (i.e., a transitionary) state. We propose that such stalling for a long period of time can give rise to LUAD. Further our study identified a subset of LUAD patients with non-AT2 (i.e., proximal) origin. In particular, we observed significantly higher KRAS mutations in distal origin LUAD patients compared to those in proximal origin LUADs. Finally, a subset of LUAD patients with distal cell of origin adopted a more proximal transcriptional identity. Extending our analysis to lung squamous cell carcinoma (LUSC) patients, we observed basal resting cells to be the Cell of Origin (COO) for LUSC. Our study provides a novel approach for identifying cell of origin for cancers and points to a complex interplay between origin and identify in lung adenocarcinoma evolution. Citation Format: Sukanya Panja, Padmaja Mantri, Juan Andrade Martinez, Marcin Imielinski. Passenger mutations link cellular origins and transcriptional identity in human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7369.

Abstract 3692: Genomic profiling with the novel single-cell IMPACT assay reveals evidence of genetic heterogeneity among CTCs in late-stage breast cancer patients

Abstract Introduction: Liquid biopsy enables minimally invasive molecular profiling of systemic cancer for diagnosis, therapy selection and longitudinal monitoring of minimal residual disease. Cell-based liquid biopsy methods are particularly well suited to examine the extent of genetic heterogeneity and clonal diversity among metastatic cancer cells, which are believed to be the major determinants of therapeutic failure. However, isolation and molecular profiling of circulating tumor cells (CTCs) from body fluids is challenging due to methodological constrains. Methods: To overcome these challenges, we have developed a workflow for detection, isolation, and mutational profiling of CTCs. The workflow utilizes the FDA-approved CellSearch System® and a novel method for single-cell Integrated Mutation Profiling of Actionable Cancer Targets (scIMPACT). Our workflow was optimized for reliable detection of copy number variations (CNVs) and single nucleotide variations (SNVs) in single CTCs. The method was then applied to 31 CTCs alongside with 15 matched germline samples. Results: After implementation of a customized Bayesian neural network based algorithm, accounting for biases introduced during amplification of single-cell DNA and prioritizing the detected mutations, we achieved a sensitivity of 91% and a specificity of 96%, AUC = 0.98. The CTC analysis revealed presence of CNVs affecting known oncogenes and tumor suppressor genes (including MYC, CCND1, FGF4, TP53 and RB1) as well as SNVs classified as pathogenic (e.g., ARID1A Q775*). Notably, in the examined specimens most of the chromosomal aberrations (51-82%) were clonal across all cells of a given patient. In contrast, most of the detected somatic SNVs (67-96%) were sub-clonal and thus present only in a subset of CTCs. Conclusion: In summary, the novel scIMPACT workflow enabled reliable and accurate genomic profiling of patient-derived CTCs in late-stage breast cancer patients. Our proof-of-concept study revealed evidence of genetic diversity of SNVs among the examined CTCs, which might have been acquired late in progression but relevant for therapy escape in the studied cases. Further analyses are still needed for assessing the generalizability of our finding as well as for deeper understanding of the molecular basis orchestrating the establishment and maintenance of heterogeneous genotypes of CTCs in breast cancer. Citation Format: Zbigniew T. Czyż, Clara Chaiban, Adithi Ravikumar Varadarajan, Cäcilia Köstler, Vadim Dechand, Jonas Grote, Thomas Ragg, Bernhard Polzer, Jens Warfsmann, Christoph A. Klein. Genomic profiling with the novel single-cell IMPACT assay reveals evidence of genetic heterogeneity among CTCs in late-stage breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3692.

Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome

BackgroundHybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors.ResultsWe used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation “blacklist” in the BED region to reduce errors in downstream analyses.ConclusionsWe first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

Contrasting somatic mutation patterns in aging human neurons and oligodendrocytes

Characterizing somatic mutations in the brain is important for disentangling the complex mechanisms of aging, yet little is known about mutational patterns in different brain cell types. Here, we performed whole-genome sequencing (WGS) of 86 single oligodendrocytes, 20 mixed glia, and 56 single neurons from neurotypical individuals spanning 0.4-104 years of age and identified >92,000 somatic single-nucleotide variants (sSNVs) and small insertions/deletions (indels). Although both cell types accumulate somatic mutations linearly with age, oligodendrocytes accumulated sSNVs 81% faster than neurons and indels 28% slower than neurons. Correlation of mutations with single-nucleus RNA profiles and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed across the genome similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These stark differences suggest an assortment of active mutagenic processes in oligodendrocytes and neurons.

Analytical validation and clinical utilization of K-4CARE™: a comprehensive genomic profiling assay with personalized MRD detection.

Background: Biomarker testing has gradually become standard of care in precision oncology to help physicians select optimal treatment for patients. Compared to single-gene or small gene panel testing, comprehensive genomic profiling (CGP) has emerged as a more time- and tissue-efficient method. This study demonstrated in-depth analytical validation of K-4CARE, a CGP assay that integrates circulating tumor DNA (ctDNA) tracking for residual cancer surveillance. Methods: The assay utilized a panel of 473 cancer-relevant genes with a total length of 1.7Mb. Reference standards were used to evaluate limit of detection (LOD), concordance, sensitivity, specificity and precision of the assay to detect single nucleotide variants (SNVs), small insertion/deletions (Indels), gene amplification and fusion, microsatellite instability (MSI) and tumor mutational burden (TMB). The assay was then benchmarked against orthogonal methods using 155 clinical samples from 10 cancer types. In selected cancers, top tumor-derived somatic mutations, as ranked by our proprietary algorithm, were used to detect ctDNA in the plasma. Results: For detection of somatic SNVs and Indels, gene fusion and amplification, the assay had sensitivity of >99%, 94% and >99% respectively, and specificity of >99%. Detection of germline variants also achieved sensitivity and specificity of >99%. For TMB measurement, the correlation coefficient between whole-exome sequencing and our targeted panel was 97%. MSI analysis when benchmarked against polymerase chain reaction method showed sensitivity of 94% and specificity of >99%. The concordance between our assay and the TruSight Oncology 500 assay for detection of somatic variants, TMB and MSI measurement was 100%, 89%, and 98% respectively. When CGP-informed mutations were used to personalize ctDNA tracking, the detection rate of ctDNA in liquid biopsy was 79%, and clinical utility in cancer surveillance was demonstrated in 2 case studies. Conclusion: K-4CARE™ assay provides comprehensive and reliable genomic information that fulfills all guideline-based biomarker testing for both targeted therapy and immunotherapy. Integration of ctDNA tracking helps clinicians to further monitor treatment response and ultimately provide well-rounded care to cancer patients.

Feasibility of whole-exome sequencing in fine-needle aspiration specimens of papillary thyroid microcarcinoma for the identification of novel gene mutations.

Genetic profiling is important for assisting the management of papillary thyroid microcarcinoma (PTMC). Although whole-exome sequencing (WES) of surgically resected PTMC tissue has been performed and revealed potential prognostic biomarkers, its application in PTMC fine-needle aspiration (FNA) specimens has not been explored. This study aimed to evaluate the feasibility of WES using FNA specimens of PTMC. Five PTMC patients were enrolled with clinical characteristics gathered. Fine aspiration cytology needle (23 gauges) was used to collect FNA biopsy with ultrasound guidance. WES analysis of FNA specimens from five PTMC patients and matched blood samples was performed. The WES of FNA samples yielded an average sequencing depth of 281× and average coverage of 99.5%. We identified 534 somatic single-nucleotide variants and 13 indels in total, and per sample, we found a mean of 24 exonic mutations, which affected a total of 120 genes. In the PTMC FNA samples, the most frequently mutated genes were BRAF and ANKRD18B, and the four driver genes were BRAF, AFF3, SRCAP, and EGFR. We also identified several germline cancer predisposing gene mutations. The results suggest that WES of FNA specimens is feasible for PTMC and can identify novel genetic mutations.

LT and SOX9 expression are associated with gene sets that distinguish Merkel cell polyomavirus-positive and -negative Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive malignant neuroendocrine tumour. There are two subsets of MCC, one related to Merkel cell polyomavirus (MCPyV) and the other to ultraviolet (UV) radiation. MCPyV-positive and MCPyV-negative MCCs have been considered to be different tumours, since the former type harbours few DNA mutations and is not related to UV radiation, and the latter usually arises in sun-exposed areas and may be found in conjunction with other keratinocytic tumours, mostly squamous cell carcinomas. Two viral oncoproteins, large T antigen (LT, coded by MCPyV_gp3) and small T antigen (sT, coded by MCPyV_gp4), promote different carcinogenic pathways. We hypothesized that the biological behaviours of MCPyV-positive and MCPyV-negative MCCs are different. We aimed to determine which genes are differentially expressed in MCPyV-positive and MCPyV-negative MCC, to describe the mutational burden and the most frequently mutated genes in the two MCC types, and to identify the clinical and molecular factors that may be related to patient survival. Ninety-two cases with a diagnosis of MCC were identified from the medical databases of the participating centres.To study gene expression, a customized panel of 172 genes was developed. Gene expression profiling was performed with nCounter Technology (NanoString Technologies, Seattle, WA, USA).For mutational studies, a customized panel of 26 genes was designed. Somatic single nucleotide variants (SNVs) were identified following the best practices GATK workflow for somatic mutations. The expression of LT enabled the series to be divided into two groups, (LT-positive, n=55; LT-negative, n=37). Genes differentially expressed in LT-negative cases were related to epithelial differentiation, especially SOX9, or proliferation and cell cycle (MYC, CDK6), among others. Congruently, LT displayed lower expression in SOX9-positive cases, and differentially expressed genes in SOX9-positive cases were related to epithelial/squamous differentiation.In LT-positive cases, the mean SNV frequency was 4.3 per case, and 10 per case in LT-negative cases (p=0.03).The expression of SNAI1 (HR=1.046, 95% CI=1.007-1.086, p=0.021) and CDK6 (HR=1.049, 95% CI=1.020-1.080, p=0.001) were identified as risk factors in a multivariate survival analysis. Tumours with weak expression of LT tend to co-express genes related to squamous differentiation and cell cycle, and to have a higher mutational burden. These findings are congruent with those of earlier studies.

Comparison of colorectal cancer (CRC) characteristics across genetic ancestries: Implications for early cancer detection (ECD).

164 Background: Blood-based ECD has the potential to reduce mortality rates by improving strategies for, adoption of, and adherence to screening. A deeper understanding of the cancer genomic landscape across genetic ancestries will aid comparable detection performance across a broad population. We evaluated the mutational landscape in patients with CRC across genetic ancestries. Methods: 16,080 de-identified patients with stage I-IV CRC who had personalized, tumor-informed commercial ctDNA testing (Signatera) based on whole exome sequencing (WES) were included. Genetic ancestry determined via supervised local ancestry inference (pmid36042219) included African (AFR), East Asian (EAS), European (EUR), Latino (LAT), and South Asian (SAS). Patients assigned to a single ancestry were included (except SAS due to small cohort size). Somatic single nucleotide variants called from WES were used for mutational signature inference. EUR was the reference group for standard statistical tests with corrections for multiple hypothesis testing. Results: A single genomic ancestry was assigned for 16,257 (99.2%) patients (AFR n=1697, 10%; EAS n=2247, 14%; EUR n=10,526, 64%; LAT n=1291, 1%; SAS n=184, 1%). Across AFR, EAS, EUR, and LAT, approximately half of patients were male (range, 47-54%) and most were aged >50 years. Microsatellite instability (MSI) rates were significantly higher in EUR (14.5%) v AFR (11%, p<0.001), EAS (8%, p<0.001), and LAT (11%, p=0.002). Compared to EUR (range 0.027-875.1 mut/Mb), the distribution of TMB was significantly higher in EAS (0.304-906.2 mut/Mb, p<0.001) and lower in LAT (0.053-476.7 mut/Mb, p<0.001). The POLE hyper-mutator signature was seen in 1.2% of all patients and in 7.6% of all hyper-mutated cases. Patients with the POLE signature were less common in EUR (1.1%) v AFR (2%, p=0.001). There were significant differences in the frequency of driver mutations in APC, BRAF, KRAS, TP53, and PIK3CA between EUR and the other ancestry groups in both MSI and MSS tumors (Table). Median exposure levels to alkylation were higher in EAS v EUR. Conclusions: These data confirm previous results in AFR populations, and reveal novel differences in the EAS and LAT populations. Findings from this study may provide information for developing risk stratification and prevention strategies for the early detection of cancer and provide rationale for precision treatment based on ancestry. [Table: see text]

Distinct Circulating Genomic Features of Classical Hodgkin Lymphoma of Older Adults

Introduction: Despite significant progress in curing younger patients with classical Hodgkin lymphoma (cHL), older patients continue to have substantially inferior outcomes. The bimodal distribution of age at cHL diagnosis has suggested distinct underlying pathogenesis in older adults. For example, among older patients where EBV+ disease is more prevalent, EBV+ status is associated with worse outcomes, unlike younger patients where it confers more favorable prognosis (Keegan, JCO 2005). The reasons for this disparity remain enigmatic. To address these challenges, we used circulating tumor DNA (ctDNA) profiling, enabling noninvasive genotyping, risk stratification, and classification into distinct molecular subtypes (Spina, Blood 2018; Alig, ASH 2022). We leveraged several non-invasive techniques to systematically characterize the genomic peculiarities of elderly cHL patients distinguishing them from younger counterparts, with special attention to EBV status. Methods: We comprehensively profiled newly diagnosed cHL patients from 3 prospective clinical trials: LYSA-PVAB (NCT02414568) for subjects ≥60 years (Ghesquières, ASH 2019), AHL2011 (NCT01358747) for patients aged 16-59 years with advanced cHL, and BIO-LYMPH (NCT04417803) for patients ≥18 years. We used age ≥60 as a threshold for stratification of older versus younger patients, a common cutoff in clinical trials. We identified somatic single nucleotide variants using CAPP-Seq (Newman et al, Nature Biotech 2016); and analyzed copy number alterations with CANARy (Chabon, Nature 2020). Patients were categorized into high vs low pretreatment ctDNA levels using a predefined threshold (2.5 log hGE/mL; Kurtz, JCO 2018). We determined EBV status using EBER and/or LMP1 staining of FFPE tumor tissue or plasma EBV levels (>32 copies/mL) by VirCAPP-Seq (Garofalo, ASH 2022). Results: A total of 215 newly diagnosed cHL patients were evaluable, with a median age of 51 years, and 35% being older (≥60 years). In this cohort, 82% had advanced stage disease, 68% had high IPS (≥3), and 57% had extra nodal involvement. At 4-year follow-up, OS and PFS were 84% and 74%, respectively. At presentation, older patients (≥60y) had more advanced stage (93% vs 73%, p<0.001), higher IPS (78% vs 61%, p<0.001), and greater EBV+ prevalence (63% vs 36%, p<0.001). Older patients also experienced significantly more relapses (39% vs 17%, p<0.001). and mortality (29% vs 6%, p<0.001). The median pre-treatment ctDNA level was 982 hGE/mL, but not significantly different in older vs younger patients (p=0.28). Higher ctDNA levels were positively associated with risk-associated features including B symptoms (p=0.01), IPS (p<0.001), bulky disease (p=0.04), extra nodal disease (p=0.02) and correlated with TMTV (R=0.47, p<.001). Older patients had significantly fewer somatic mutations than younger counterparts (Median 107 vs 192 SNVs within targeted genomic regions, p<0.001; Fig. 1A top); among older patients, EBV+ cases had even fewer mutations than EBV- counterparts (Median 83 vs 142 SNVs, p=0.05). Older cHL cases harbored significantly more BCL2 mutations and recurrent copy number anomalies, but had significantly fewer mutations in STAT6, ITPKB, B2M, GNA13, PAX5 and XPO1 among others(all p<0.05 Fig. 1A bottom). The H2 genomic subtype prevalence, characterized by genomic instability as well as mutations in TP53, KMT2D, and BCL2 (Alig et al, ICML 2023), was higher among older patients (56%) than younger patients (21%) (p=0.01). Older H2 cHL showed no significant difference in EBV+ status compared to H1 counterparts (p=0.9). In older patients, multivariate Cox regression (including EBV status, ctDNA, and IPS) identified EBV+ status (HR=2.2, p=0.02) as an independent predictor of worse PFS and DFS outcomes (HR=2.5, p=0.02 and HR=2.7, p=0.04). When older cHL patients were stratified by baseline ctDNA level and EBV status, high-risk cases (EBV+ and ctDNA High) had particularly poor outcomes in terms of both PFS and DFS ( Fig. 1B). Conclusion: Older cHL patients exhibit unique genomic characteristics compared to younger counterparts, including more prevalent H2 tumor genetic subtype and EBV positivity. Identifying of a high-risk subgroup of older cHL underscores the importance of ctDNA levels and EBV status. We envision future trials of personalized biomarker driven treatment strategies could therapeutically target this risk, including by PD1 blockade.

A Personalized Whole Genome-Informed Assay Targeting Single Mutant in Circulating Tumor DNA Can Identify MRD and Predict Relapse in DLBCL

Introduction: Minimal residual disease (MRD) detected by circulating tumor DNA (ctDNA) has emerged as a promising biomarker in diffuse large B-cell lymphoma (DLBCL). MAESTRO (minor-allele-enriched sequencing through recognition oligonucleotides) was recently developed to aid the detection of low-frequency mutations by enriching for mutant alleles using probes preferentially capturing single-nucleotide variants. We have extended this application - termed MAESTRO-Pool - to analyze personalized MRD variant detection within a cohort-level single assay. We demonstrate high sensitivity to detect MRD using MAESTRO-Pool and the detection of emergent mutations using targeted sequencing of the same samples. Methods: Fifty-nine plasma specimens from 9 patients with relapsed/refractory (R/R) DLBCL treated on a phase II trial (NCT02362997) of post-autologous stem cell transplant (ASCT) pembrolizumab maintenance were tested. Cases were selected based on the availability of genomic DNA from tumor tissue, patient-matched germline DNA, and serial post-ASCT plasma samples (≥ 3 time points). MAESTRO probes were designed to target patient tumor-specific somatic variants using results of baseline tumor-normal whole genome sequencing. Probes were then pooled into an integrated, cohort-level assay (MAESTRO-Pool). Serial samples were compared with an orthogonal, whole-genome, tumor-informed MRD test which does not use mutation enrichment (MRD Tracker; Parsons, HA et al. Clin Cancer Res26, 2556-2564 (2020)) for sensitivity and specificity of variant detection. In addition, sensitivity to detect MRD and predict relapse was compared to that observed with immunoglobulin locus high-throughput sequencing (IgHTS). Additional baits were designed to capture single nucleotide variants (SNVs) previously reported in R/R DLBCL (63 loci in 12 genes), enabling detection of treatment-emergent mutations not identified in baseline tumor specimens. Results: Tumor-normal WGS revealed a median of 433 somatic SNVs per tumor (range 81-1653). The pooled assay comprised 6044 SNV-specific probes. MRD identification was similar for MAESTRO-Pool and MRD Tracker. Among 59 samples, a discrepant MRD call was observed for a single sample where ctDNA was detected at 4 ppm using MRD Tracker, but not detected using MAESTRO-Pool (limit of detection [LoD] 16 ppm). Estimated tumor fractions using MRD Tracker and MAESTRO-Pool were concordant. Even with reduced sequencing requirements of MAESTRO-Pool, we observed a similar median limit of detection (LoD) for MAESTRO-Pool (median 30 ppm, range 1-18,243) and MRD Tracker (median 40 ppm, range 1-4,727 ppm). MAESTRO-Pool identified ctDNA prior to recurrence for all 5 patients who relapsed, including at the earliest available post-ASCT timepoint for 4 of 5 relapsing patients. The time from ctDNA detection to clinical relapse (lead time) was the same or longer for each patient using MAESTRO-Pool (median 178 days, range 69-518) compared to IgHTS (median 44 days, range not detected to 518 days) (p=0.37) (Fig.1a). MAESTRO-POOL was associated with improved sensitivity compared to IgHTS (MAESTRO-Pool sensitivity of 90.5% for samples with matched IgHTS results versus IgHTS sensitivity of 61.9%, p=0.006). Superior sensitivity was primarily driven by MAESTRO-Pool's improved detection of low-frequency (<1000 ppm) mutant alleles. In addition to tracking molecular tumor burden, we identified several de novo mutations in relapsing patients using targeted sequencing of the same samples. Notably, plasma from a patient who progressed at 18.5 months post-ASCT (DL-015) manifested an emergent CREBBP R1446H mutation not detected in the baseline tumor whose allele frequency steadily increased from 0.048% one week after ASCT to 30.533% at relapse (Fig 1b). Conclusion: In this pilot study, MAESTRO-Pool enabled ultrasensitive detection and quantification of MRD with superior sensitivity compared to IgHTS. Complementary targeted sequencing also characterized genetic evolution, including detection of treatment-emergent mutations. Our results support the incorporation of ctDNA testing using MAESTRO-Pool in future prospective trials in DLBCL.

Personalized Residual Disease Detection in Acute Myeloid Leukemia with a Multiplexed Genomic Fingerprint Predicts Patient Outcomes

Background: Acute myeloid leukemia (AML) is the most common leukemia in adults. While therapeutic options for AML have continued to improve, nearly half of all patients who achieve clinical remission will relapse within several years after diagnosis. Risk factors for relapse include age, cytogenetics, specific gene mutations, and the detection of residual disease after treatment. Current molecular approaches for measuring residual disease are designed primarily around a single gene. This limits both the sensitivity and the applicability of the assay to all patients, as AML is a highly heterogenous genetic disease. In this study, we demonstrate the utility of a comprehensive, personalized, multiplexed genomic assay that exhibits high sensitivity, excellent quantitation, and promising performance across tissue types. This approach enables the serial, non-invasive monitoring of patients with AML and the prediction of patient outcome. Methods: Serial samples of bone marrow, peripheral blood, and urine were collected at multiple time points from 12 patients with AML who were treated with standard-of-care therapy. Time points range from presentation to relapse, with a median of 4 time points per patient (range of 2-10). A median of 7 samples were collected per patient (range of 2 to 16). DNA was extracted from the peripheral blood mononuclear cells (PBMCs), the cell-free fraction of peripheral blood, bone marrow mononuclear cells, and the cell-free fraction of urine. Deep whole genome sequencing of paired presentation and remission samples identified hundreds of candidate leukemia-associated somatic single-nucleotide variants comprising the personalized genomic fingerprint of each patient's disease. A representative set of variants (n=30-50) was selected for each patient, and a personalized set of primers was designed for the Multiplex Accurate Sensitive Quantification (MASQ) next-generation sequencing protocol. The sensitivity of this assay allows for single variants to be quantified down to at least 1 in 100,000, resulting in an overall sensitivity below 1 in 1 million for residual disease detection. Quantification of the individual variants from the fingerprint was evaluated in samples of all tissue types collected (bone marrow, blood cells, blood cell-free DNA, and urine cell-free DNA) and all time points collected (presentation, post-induction, post-consolidation, and follow-up visits). Results: Serial monitoring of the leukemia genomic fingerprint enables risk assessment after each stage of treatment. Using Multiplex Accurate Sensitive Quantification (MASQ), we detected leukemia-associated variants in the blood and bone marrow of all patients during remission, at levels ranging from less than 10^-6 to 10^-2. We find consistent low signal, less than 10^-4, in both bone marrow and blood in patients with good prognosis. In contrast, we find high signal in remission, greater than 10^-3, in patients with poor prognosis. In the analysis of serial samples, frequencies drop over time with additional treatment and increase preceding clinical identification of relapse. Figure 1A shows the trajectory of 50 variants over 5 time points in PBMCs, preceding relapse. The overall signal increases at 7 months prior to clinical relapse and again at 5 months prior to clinical relapse. Figure 1B shows a comparison of the aggregate estimates of the leukemia genomic fingerprint in the cellular and cell-free components of the blood. While total DNA yields from the cell-free fraction of blood are lower than from PBMCs, we find that the frequency of the leukemia genomic fingerprint in the two sources of DNA is highly similar. Bone marrow and peripheral blood also show highly similar measurements of residual disease, suggesting that non-invasive monitoring with only a blood draw is a promising approach. Signal of the leukemia fingerprint is also detectable in urine, but at reduced levels and much more variable across patients. Conclusion: Our study shows the capability and utility of a personalized genomic residual disease detection method using MASQ to monitor patients and predict patient outcome. The high sensitivity and accurate quantitation of MASQ are demonstrated in a case study where relapse could be detected molecularly 7 months prior to clinical identification. Collection and analysis of a larger case series is ongoing.

Genomic data resources of the Brain Somatic Mosaicism Network for neuropsychiatric diseases

Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.

Quantification of rare somatic single nucleotide variants by droplet digital PCR using SuperSelective primers

Somatic single-nucleotide variants (SNVs) occur every time a cell divides, appearing even in healthy tissues at low frequencies. These mutations may accumulate as neutral variants during aging, or eventually, promote the development of neoplasia. Here, we present the SP-ddPCR, a droplet digital PCR (ddPCR) based approach that utilizes customized SuperSelective primers aiming at quantifying the proportion of rare SNVs. For that purpose, we selected five potentially pathogenic variants identified by whole-exome sequencing (WES) occurring at low variant allele frequency (VAF) in at-risk colon healthy mucosa of patients diagnosed with colorectal cancer or advanced adenoma. Additionally, two APC SNVs detected in two cancer lesions were added to the study for WES-VAF validation. SuperSelective primers were designed to quantify SNVs at low VAFs both in silico and in clinical samples. In addition to the two APC SNVs in colonic lesions, SP-ddPCR confirmed the presence of three out of five selected SNVs in the normal colonic mucosa with allelic frequencies ≤ 5%. Moreover, SP-ddPCR showed the presence of two potentially pathogenic variants in the distal normal mucosa of patients with colorectal carcinoma. In summary, SP-ddPCR offers a rapid and feasible methodology to validate next-generation sequencing data and accurately quantify rare SNVs, thus providing a potential tool for diagnosis and stratification of at-risk patients based on their mutational profiling.