DNA Panel Research Articles

Integrative genomic and transcriptomic profiling of pulmonary sarcomatoid carcinoma identifies molecular subtypes associated with distinct immune features and clinical outcomes

AbstractBackgroundPulmonary sarcomatoid carcinoma (PSC) is a rare and aggressive subtype of non‐small cell lung cancer (NSCLC), characterized by the presence of epithelial and sarcoma‐like components. The molecular and immune landscape of PSC has not been well defined.MethodsMultiomics profiling of 21 pairs of PSCs with matched normal lung tissues was performed through targeted high‐depth DNA panel, whole‐exome, and RNA sequencing. We describe molecular and immune features that define subgroups of PSC with disparate genomic and immunogenic features as well as distinct clinical outcomes.ResultsIn total, 27 canonical cancer gene mutations were identified, with TP53 the most frequently mutated gene, followed by KRAS. Interestingly, most TP53 and KRAS mutations were earlier genomic events mapped to the trunks of the tumors, suggesting branching evolution in most PSC tumors. We identified two distinct molecular subtypes of PSC, driven primarily by immune infiltration and signaling. The Immune High (IM‐H) subtype was associated with superior survival, highlighting the impact of immune infiltration on the biological and clinical features of localized PSCs.ConclusionsWe provided detailed insight into the mutational landscape of PSC and identified two molecular subtypes associated with prognosis. IM‐H tumors were associated with favorable recurrence‐free survival and overall survival, highlighting the importance of tumor immune infiltration in the biological and clinical features of PSCs.

Abstract 3659: Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract cancer

Abstract Biliary tract cancer (BTC) is a rare type of abdominal cancer and is more frequent in Asian countries than in Western. The 5-year survival rate for BTC is very low 7 to 20% and only 20% of BTC patients could undergo curative-intent surgery. BTC diagnosis based on biopsy and cytology are low in sensitivity that prompted the necessity to identify alternative biopsy. Liquid biopsy, specifically circulating tumor DNA (ctDNA) is known for the accessibility and low invasiveness received great interest for their potential uses in various clinical applications for patients with insufficient tumor tissue, including mutation profiling, treatment monitoring, and cancer detection. However, the panel available in the market up-to-date focused mainly targeted, actionable alterations and thus not suitable for minimal residual disease (MRD) monitoring. In this study, we aimed to develop a customized ctDNA panel for MRD monitoring in BTC. In this study, 124 BTC patients undergone curative resections were enrolled at Hokkaido University and Sapporo Medical University from May 2019 to April 22. All enrolled patients have BTC diagnosis confirmed by pathological diagnosis after surgery, and without other malignant tumors. Genomic profiles of FFPE tissue were conducted using Ampliseq CCP. 13 patients were excluded for high fragmentation. Referring to the genomic profiles of 111 patients in our study and 1924 patients in TCGA established a customized panel using Ion Ampliseq HD technology. The Ampliseq HD panel claims to detect variants at 0.1% LOD for ctDNA detection. It also integrates molecular barcodes to reduce NGS-related errors. The designed panel was 258 Amplicons and 20 genes including eleven therapeutically targetable genes. Clinical characteristics of ctDNA detection were also assessed. Results:The panel validation: concordance of the mutation allele frequency (MAF) between the NGS standard control and Ampliseq HD panel showed a coefficient of determination (R2) of 0.96. Also, concordance of MAF Ampliseq CCP and Ampliseq HD panel on FFPE tumor profiling showed R2 of 0.78. When assessing 70% of BTC patients were covered to have at least one tumor-derived Ampliseq HD panel. When assessing using plasma cell-free DNA, 55% of BTC patients were detected to have at least one tumor-derived ctDNA. Distribution of detected altered top4 genes using Ampliseq HD panels shows TP53 35%, KRAS 10%, SMAD4 5%, ARID1A 2.5%. Clinical characteristics may affected the concordance of plasma and FFPE tumor tissue; stage (P=0.035), size of tumors (P=0.0088), subtype (P=0.013), LN metastasis (P=0.11). Conclusions: A highly sensitive ctDNA customized panel for BTC was established. We also found that clinical characteristics of the BTC affects the detection of ctDNA from blood liquid biopsy. Future study: MRD evaluation using this customized panel. Citation Format: Yoshihito Shinohara, Siew-Kee Low, Toru Nakamura, Yasutoshi Kimura, Takeshi Murakami, Kazuma Kiyotani, Satoshi Hirano. Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract 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 3659.

Abstract 6460: Incidence of pathogenic germline variants and presumed germline pathogenic variants in Japanese lung cancer patients using comprehensive genomic profiling tests

Abstract Introduction: Pathogenic germline variants (PGVs) and presumed germline pathogenic variants (PGPVs) are often detected with comprehensive genomic profiling (CGP) tests. Using the Center for Advanced Cancer Genomics and Therapeutics (C-CAT) database, we investigated the incidence of PGV and PGPV detected by CGP tests in Japanese lung cancer patients. Methods: Among 3,240 lung cancer cases registered in C-CAT between June 2019 and August 2023, 321 were tumor/normal paired NCC Oncopanel (NOP), 773 were FoundationOne Liquid CDx (F1L), and 2,145 were tumor-only FoundationOne CDx (F1CDx). We analyzed cancer susceptibility genes in accordance with the national guideline. In F1L and F1CDx, PGPV was determined based on variant allele frequency (VAF) and medical history. Differences between groups in the detection of PGV and PGPV were tested using chi-squared test, and differences in age were tested using t-test. Results: The incidence of PGV in NOP and PGPV in F1L and F1CDx were 4/321 (1.2%), 36/773 (4.7%), and 141/2145 (6.6%), respectively (NOP vs F1L vs F1CDx; p = 0.001 NOP vs F1L; p = 0.006, NOP vs F1CDx; p < 0.001, F1L vs F1CDx;p=0.190). The 4 PGVs detected by NOP were in the ATM, BRCA2, MSH6, and TP53 genes. Notably, two out of four patients were in their 30s while the other two were in their 70s. In the F1L dataset, 36 patients (4.7%) had PGPV: 8 patients (1.0%) had PALB2, 6 patients (0.8%) had BRCA2, and 4 patients (0.5%) had CHEK2 variants. In the F1CDx dataset, 141 had PGPV: 19 patients (0.9%) each had BRCA1 or BRCA2 variants and 18 patients (0.8%) had PALB2 variant. There was no significant age difference between PGPV(-) and PGPV(+) patients (F1L:63.3 vs. 62.4 years; p = 0.938, F1CDx: 65.5 vs 65.0 years; p = 0.749). Most cases with PGV or PGPV were non-small cell lung cancer: 4/4 in NOP, 34/36 in F1L, and 110/141 in F1CDx. Discussion: The incidence of PGV in lung cancer patients was at least 1% and found in both younger and older patients. A circulating tumor DNA panel and a tumor-only tissue panel detected PGPVs at a significantly higher rate than detection of true PGVs by tumor/normal paired tissue panel, suggesting the use of tumor/normal paired panels may mitigate the burden of both patients and clinicians with concerns from false positive PGPV. The majority of detected PGV/PGPV were in homologous recombination repair genes. Citation Format: Michiko Ueki, Kousuke Watanabe, Momoko Morishita, Koki Fujii, Hiroki Ikushima, Hideaki Isago, Katsutoshi Oda, Hidenori Kage. Incidence of pathogenic germline variants and presumed germline pathogenic variants in Japanese lung cancer patients using comprehensive genomic profiling tests [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 6460.

Identification of gene variation feature for targeted therapy of non-small cell lung cancer through combined method of DNA and RNA sequencing

Next generation sequencing (NGS) is typically used to reveal tumor gene variation feature for targeted therapy of various types of human cancers, including non-small cell lung cancer (NSCLC). Here, we report the role and potential applicable value of combining DNA and RNA sequencing in gene variation detection in NSCLC. 386 NSCLC patients with stage II-IV were enrolled and detected using NGS sequencing of DNA and RNA panels that covered all well-documented target driver genes from the Chinese Society of Clinical Oncology (CSCO). The rate of epidermal growth factor receptor (EGFR) single nucleotide variation (SNV)/indel, mesenchymal-epithelial transition factor (MET) copy number variation (CNV) and anaplastic lymphoma kinase (ALK) fusion were 52.1%, 4.1% and 6.0% in the NSCLC cohort. The landscapes of SNV/indel, CNV and gene fusion in the cohort were depicted as well. Meanwhile, we assessed detection efficacy of DNA and RNA sequencing in gene fusion. Detected number and types of gene fusion using the RNA sequencing were better than those using the DNA sequencing. Gene fusion with intergenic region was only detected by DNA sequencing and MET exon 14 skipping (METΔex14) was more easily identified by RNA sequencing. Finally, we investigated clinical correlations of SNV/indel/CNV/fusion with clinicopathologic features in the NSCLC cohort. Taken together, RNA sequencing significantly complements deficiency of DNA sequencing for gene fusion, which cooperatively presents comprehensive and reliable gene variation features and facilitate the identification of potential drug targets for NSCLC patients.

Abstract A107: Characterizing the genomic and immunologic landscape of serous borderline tumors and low-grade serous ovarian cancer

Abstract A107: Characterizing the genomic and immunologic landscape of serous borderline tumors and low-grade serous ovarian cancer

Genetic mutation profiling reveals biomarkers for targeted therapy efficacy and prognosis in non-small cell lung cancer

IntroductionThe genetic heterogeneity of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations may affect clinical responses and outcomes to EGFR tyrosine kinase inhibitors (EGFR-TKIs). This study aims to investigate the genomic factors that influence the efficacy and clinical outcomes of first-line, second-line and third-line treatments in NSCLC and explore the heterogeneity of resistance mechanisms. Materials and methodsThis real-world study comprised 65 patients with EGFR mutant NSCLC. Molecular alterations were detected using a customized DNA panel before and after administering targeted therapy. The efficacy and prognosis of each treatment line were evaluated. ResultsIn first-generation EGFR-TKIs treatment, gefitinib showed favorable efficacy compared to icotinib and erlotinib, particularly in patients with EGFR L858R mutations. The resistance mechanisms to first-generation EGFR-TKIs varied among different EGFR mutation cohorts and different first-generation EGFR-TKIs. In second-line EGFR-TKIs treatment, EPH receptor A3 (EPHA3), IKAROS family zinc finger 1 (IKZF1), p21 (RAC1) activated kinase 5 (PAK5), DNA polymerase epsilon, catalytic subunit (POLE), RAD21 cohesin complex component (RAD21) and RNA binding motif protein 10 (RBM10) mutations were markedly associated with poorer progression-free survival (PFS). Notably, EPHA3, IKZF1 and RBM10 were identified as independent predictors of PFS. The mechanisms of osimertinib resistance exhibited heterogeneity, with a higher proportion of non-EGFR-dependent resistant mutations. In third-line treatments, the combination of osimertinib and anlotinib demonstrated superior efficacy compared to other regimens. Glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A) mutation was an independent risk indicator of shorter OS following third-line treatments. ConclusionsComprehending the tumor evolution in NSCLC is advantageous for assessing the efficacy and prognosis at each stage of treatment, providing valuable insights to guide personalized treatment decisions for patients.

Long-read sequencing genome assembly of Ceratocystis fimbriata enables development of molecular diagnostics for sweetpotato black rot.

Ceratocystis fimbriata is a destructive fungal pathogen of sweetpotato (Ipomoea batatas (L.) Lam.) that leads to losses at all stages of sweetpotato production. Accurate detection of C. fimbriata would allow for more efficient deployment of management tactics in sweetpotato production. To develop a diagnostic assay, a hybrid genome assembly of C. fimbriata isolate AS236 was generated. The resulting 31.7 MB assembly was near-chromosome level, with 18 contigs, 6,481 predicted genes, and a BUSCO completion score of 98.4% when compared to the fungi-specific lineage database. Additional Illumina DNA reads from C. manginecans, C. platani, and a second C. fimbriata isolate (C1421) were then mapped to the assembled genome using BOWTIE2 and counted using HTSeq, which identified 148 genes present only within C. fimbriata as molecular diagnostic candidates; 6 single-copy and 35 highly multi-copy (>40 BLAST hits), as determined through a self-BLAST-P alignment. Primers for PCR were designed in the 200 bp flanking region of the first exon for each candidate, and the candidates were validated against a diverse DNA panel containing Ceratocystis species, sweetpotato pathogens, and plants. After validation, two diagnostic candidates amplified only C. fimbriata DNA and were considered to be highly specific to the species. These genetic markers will serve as valuable diagnostic tools with multiple applications including the detection of C. fimbriata in seed, soil, and wash water in sweetpotato production.

Genomic and immune landscape of biliary tract cancers with ARID1A, PBRM1, and BAP1 alterations.

557 Background: Alterations in ARID1A, PBRM1 and BAP1 subunits of the SWI/SNF complex are common in biliary tract cancers (BTCs) and have been implicated in tumor microenvironment (TME) immunomodulation. However, there is heterogeneity in outcomes, with PBRM1 and BAP1 conferring improved survival vs worse survival with ARID1A. We explored relationships between ARID1A, PBRM1 and BAP1 alterations with putative immunotherapy biomarkers and genomic co-alterations. Methods: We examined BTC patients in the Tempus database with ARID1A, PBRM1 and BAP1 alterations. Co-alterations were identified through next-generation sequencing (NGS) using the Tempus xT assay, a targeted, tumor-normal-matched DNA panel that detects SNVs, indels, and CNVs in 648 genes, as well as chromosomal rearrangements in 22 genes with high sensitivity and specificity. Samples additionally underwent whole transcriptome RNA-seq via the Tempus xR assay (Tempus Labs, Chicago, IL). Immune biomarkers explored include MSI-H, PD-L1, Tumor Mutation Burden (TMB), and immune cell infiltration by RNA sequencing. Parameters were analyzed between two groups: BTC with ARID1A, vs BTC with PBRM1 and/or BAP1 alterations. Results: 696 BTC patients with ARID1A, PBRM1 and BAP1 alterations were included. Overall, there was low frequency of MSI-H (3.2%), PD-L1-positivity (5.7%) and TMB >10 mt/Mb (4.7%). Among the two groups, there was a higher proportion of MSI-H patients in ARID1A BTC vs PBRM1 and/or BAP1 BTC (4.9% vs 1.4%, p=0.01). Macrophages were the predominant immune cell (46%), followed by CD4+ T-cells (23%) and NK cells (12%). CD8+ T cells were the least encountered (5%). B-Cells were seen more in ARID1A vs PBRM1 and/or BAP1 (9% vs 6%, p <0.001) while macrophages were more frequent in PBRM1 and/or BAP1 vs ARID1A (49% vs 44%, P <0.001). Co-alterations in SMAD4, TP53, ARID2 and KRAS were more frequent in ARID1A BTC while IDH2 and NRAS were more common in PBRM1 and/or BAP1. Within the PBRM1 subgroup, TP53 was most frequently encountered (33%), while CDKN2A alterations were most frequently encountered in BAP1 BTC (35%) (Table 1). Conclusions: This is the largest known data set exploring the genomic and immune landscape of BTC with ARID1A, PBRM1 and BAP1 alterations. Macrophages were the dominant immune cell TME and may be a target of interest. Co-alteration profile is distinct between ARID1A- vs PBRM1- and/or BAP1-altered BTC.[Table: see text]

Next-generation Multi-target Stool DNA Panel Accurately Detects Colorectal Cancer and Advanced Precancerous Lesions.

This study highlights performance of the next-generation mt-sDNA test, which exhibits high sensitivity and specificity for detecting colorectal cancer and APLs. This noninvasive option has potential to increase screening participation and clinical outcomes. A multi-center, clinical validation trial is underway. See related commentary by Bresalier, p. 93.

Synthetic Nucleic Acid Antigens in Localized Scleroderma.

We investigated the impact of synthetic nucleic acid antigens on the autoantibody profiles in patients with localized scleroderma, an autoimmune skin disease. Anti-DNA antibodies, including double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), are common among autoimmune diseases, such as systemic lupus erythematosus and localized scleroderma. Based on recent studies, we hypothesized that the sequence of nucleic acid antigens has an impact on the autoimmune reactions in localized scleroderma. To test our hypothesis, we synthesized a panel of DNA and RNA antigens and used them for autoantibody profiling of 70 children with localized scleroderma compared with the healthy controls and patients with pediatric systemic lupus erythematosus (as a disease control). Among the tested antigens, dsD4, which contains the sequence of the human oncogene BRAF, showed a particularly strong presence in localized scleroderma but not systemic lupus erythematosus. Disease activity in patients was significantly associated with dsD4 autoantibody levels. We confirmed this result in vivo by using a bleomycin-induced mouse model of localized scleroderma. When administered intraperitoneally, dsD4 promoted an active polyclonal response in the mouse model. Our study highlights sequence specificity for nucleic acid antigens in localized scleroderma that could potentially lead to developing novel early-stage diagnostic tools.

Abstract B078: Unraveling resistance and optimizing treatment through molecular profiling in patients with NSCLC and oncogenic fusions

Abstract Introduction: In non-small cell lung cancer (NSCLC) with oncogenic fusions, resistance development complicates optimal therapeutic sequencing. This study investigates the resistance landscape in NSCLC with oncogenic fusions and evaluates the efficacy of subsequent treatment tailoring based on the molecular profile at progression. Methods: This is a single center, retrospective study, including patients with advanced NSCLC and ALK, ROS1, RET or NTRK fusions, with a molecular profile at progression after targeted therapy (TKI), between January 2015 and February 2023. Molecular profile was performed on tissue or liquid samples, with next-generation sequencing (NGS) (targeted DNA and/or RNA panels, WES/RNA sequencing). In case of TKI sequencing, more than one NGS results were available for selected patients. Alterations at time of resistance were classified into on-target, bypass alterations, and polyclonal/complex resistance (with at least two types of resistance concurrently present in the same biopsy). The primary objective was to assess the clinical utility of NGS to guide subsequent treatment at progression after TKI. Results: A total of 95 patients (61 ALK, 21 ROS1, 12 RET, 1 NTRK) had 179 samples collected. Patients were female in 51 (54%), had a median age of 53 years (IQR 41-60), never smokers in 52 (55%) cases and adenocarcinoma histology in 88 (94%) of cases. Molecular profile was performed by liquid biopsy in 83 (66%) cases, other liquids in 3 (2%) and tissue in 40 (32%) cases. There were 126 contributive samples (70%) for 69 patients. Resistance was explained by 60/126 (48%) samples for 41 patients: on-target in 27 (47%), bypass in 22 (39%), and polyclonal resistance in 8 (14%) samples. Three out of 7 samples evaluable for mutational signatures via WES showed Apolipoprotein B mRNA Editing Catalytic Polypeptide-like (APOBEC) signatures. The time until the first occurrence of resistance was 34.2 months (95%CI 26-65.3) for on-target, 23.6 months (95%CI 18.8-97.4) for by-pass and 16.5 months (95%CI 7.2-NR) for polyclonal resistance. The early occurrence of by-pass or polyclonal resistance in the first 12 months was associated with a younger age (median 37 vs 52 years, p=0.044). Among the 60 NGS results explaining resistance for 41 patients, 56 (93%) NGS results informed treatment tailoring. Drug tailoring was conducted in 16 patients for 23 NGS reports, revealing a median overall survival of 18.6 months (95% CI 11.8 – NR) for patients receiving an effective tailored treatment, versus 7.3 months (95%CI 2.9 – NR) for those without tailoring (p=0.088). Conclusion: Our findings underscore the value of using NGS to inform treatment tailoring in NSCLC patients with oncogenic fusions. Special focus should be placed on younger patients who might be prone to early development of bypass or polyclonal resistance. Citation Format: Mihaela Aldea, Vlad Pop, Arianna Marinello, Andrei Ivashkin, Damien Vasseur, Sebastien Gendarme, Marco Tagliamento, Anas Gazzah, Jordi Remon, Imen Ben-Ammar, Maxime Frelaut, Pamela Abdayem, Pernelle Lavaud, Antoine Hollebecque, Etienne Rouleau, Yohann Loriot, Luc Friboulet, Sergey Nikolaev, Antoine Italiano, Fabrice Barlesi, David Planchard, Benjamin Besse. Unraveling resistance and optimizing treatment through molecular profiling in patients with NSCLC and oncogenic fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B078.

Abstract C045: Uncovering molecular differences in pancreatic ductal adenocarcinoma tissues from Black and White patients in the US

Abstract Background. Pancreatic cancer is the 3rd leading cause of cancer-related deaths in the US, with a 5-year survival rate of approximately 12%. The occurrence of new pancreatic cancer cases has continued to rise in the last two decades, with the highest incidence observed in the Black and African American (BAA) population. While socioeconomic and environmental factors may contribute to observed racial disparities in the occurrence of pancreatic cancer, it is unclear whether there are molecular differences in pancreatic tumors across racially and ethnically diverse populations. The goal of this study was to identify key molecular differences between BAA and White patients’ pancreatic tumors. Methods. We analyzed 4620 de-identified records with a primary diagnosis of pancreatic ductal adenocarcinoma (PDAC) who underwent somatic NGS testing via a commercially available 595-648 gene DNA panel as well as whole transcriptome RNA-seq. Race was extracted from clinical records (e.g., order forms, patient records, etc.). Somatic mutations, microsatellite instability (MSI), and tumor mutational burden (TMB) were compared between White (n=3797) and BAA (n=452) samples by Chi-squared or Fisher’s Exact tests. False discovery rate (FDR) corrections were applied to pairwise comparisons. Results. The combined cohort had a median age of 68 years and 88% of records had stage 3 or 4 disease at the time of diagnosis. We found that alterations in TP53 (70% vs 62%, q=0.014) and KMT2C (6.9% vs 2.3% q<0.001) were at significantly higher frequencies in BAA vs White samples. Weakly significant differences were observed in BAA vs White samples based on alterations in KRAS (79% vs 74%, p=0.02), RNF43 (6% vs 3.7% p=0.022), and GNAS (0.2% vs 1.6%, p=0.022), all of which had q-values of 0.14 after FDR correction. When stratifying by detailed alterations, KRAS G12R was significantly more frequent in BAA than White (14% vs 10%, q=0.037), and GNAS R201H was more frequent in White vs BAA (0.9% vs. 0%, q=0.056) samples. Finally, when assessing biomarkers associated with immunotherapy response in solid tumors, we found that neither the frequency of TMB-H (≥ 10 mut/MB) nor MSI-high status were significantly different between BAA and White samples. Interestingly, in the subset of 676 PDAC patients who had PD-L1 IHC performed previously, BAA samples had a higher frequency of PD-L1 positivity compared with White samples (20% vs 12%, p=0.039). Conclusions. We assessed molecular differences between BAA and White patients’ tumors in the largest cohort of PDAC records reported to date. Our findings suggest that there are key molecular tumor characteristics which occur more frequently in BAA PDAC tumors. In particular, alterations in genes such as KMT2C, which were more frequent in our cohort of BAA tumors, are known contributors to pancreatic tumorigenesis and represent potential therapeutic targets. These findings highlight the importance of studying racially and ethnically diverse populations to better understand key molecular alterations. Citation Format: Emily Teslow, Minxuan Huang, Melissa Stoppler, Albert Levin, Ling Huang. Uncovering molecular differences in pancreatic ductal adenocarcinoma tissues from Black and White patients in the US [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C045.

Primary Renal Well Differentiated Neuroendocrine Neoplasm with Nodal Metastasis Possibly Originating from A Teratoma

Abstract Introduction/Objective Well differentiated neuroendocrine tumors (WDNETs) of the kidney are extremely rare and are aggressive tumors with higher rate of metastasis, even those with low proliferative rates. Primary WDNET arising in a renal teratoma is an exceedingly rare entity. Methods/Case Report Herein, we describe a case with a primary WDNET and possible mature cystic teratoma of the right kidney of a 22-year old male who presented with an epigastric/right upper quadrant abdominal pain. Abdominal MRI identified a 4.4cm isointense mass in the mid-lower pole of the right kidney within renal sinus. Robotic assisted partial nephrectomy was performed. Grossly, the 56gm excised mass is well circumscribed with tan- brown friable homogeneous cut surface. Histologically, tumor cells show round to oval nuclei with speckled chromatin and calcifications. The tumor metastasized to two hilar lymph nodes. The tumor is positive for AE1/AE3, vimentin, synaptophysin, CD99, INI-1 (SMARCB1), FLI-1, and chromogranin. While negative for CK7, CK20, PAX-8, CD10, WT-1, CEA, and CDX-2. Ki-67 is 2% and the mitotic count is < 1 per 10 HPF. The histology and immunoprofile are consistent with WDNET. No pathogenic fusions detected on Next Generation Sequencing (NGS) targeting RNA and DNA panels. Adjacent to the tumor, there is a cystic structure lined by intestinal mucosa positive for CDX2. Results (if a Case Study enter NA) N/A Conclusion Neuroendocrine cells are not identified in the normal parenchyma of the kidney, while noted in adjacent intestinal mucosa raising the possibility that renal WDNET is arising from mature renal teratoma.

Clinical Practice Recommendations for the Use of Next-Generation Sequencing in Patients with Solid Cancer: A Joint Report from KSMO and KSP.

In recent years, next-generation sequencing (NGS)-based genetic testing has become crucial in cancer care. While its primary objective is to identify actionable genetic alterations to guide treatment decisions, its scope has broadened to encompass aiding in pathological diagnosis and exploring resistance mechanisms. With the ongoing expansion in NGS application and reliance, a compelling necessity arises for expert consensus on its application in solid cancers. To address this demand, the forthcoming recommendations not only provide pragmatic guidance for the clinical use of NGS but also systematically classify actionable genes based on specific cancer types. Additionally, these recommendations will incorporate expert perspectives on crucial biomarkers, ensuring informed decisions regarding circulating tumor DNA panel testing.

A Multiomic, Single-Cell Measurable Residual Disease (scMRD) Assay for Phasing DNA Mutations and Surface Immunophenotypes

The small population of cancerous cells that remain following treatment, known as measurable residual disease (MRD), is the major cause of relapse in acute myeloid leukemia (AML). Usually, these refractory cells have gained additional resistance mutations or changed their surface immunophenotypes in ways that preclude detection and phasing by current gold standard flow cytometry or bulk next-generation sequencing assays. For this reason, a multiomic single-cell MRD (scMRD) assay could offer a more comprehensive indicator of relapse and the potential for faster response. Here, we present a new scMRD assay with a 0.01% limit of detection that provides single-cell clonal architecture and immunophenotyping to not only identify residual leukemia cells, but also identify putative DNA or protein targets for salvage therapy. The assay enables rare-cell detection on a standard Mission Bio Tapestri run by adding (i) an upfront bead-based protocol to enrich for blast cells, (ii) a DNA and protein panel specifically designed for AML MRD diagnosis and treatment, and (iii) a new, automated analysis pipeline to evaluate single-cell multiomics output. By utilizing Mission Bio's single-molecule DNA sensitivity for single cells, this pipeline can identify and correlate co-occurring de novo variants, thereby reducing false positive rates over bulk assays that do not correlate variants. It furthermore can create phylogenetic trees of the detected MRD cells and present their surface protein signature and arm-level copy number. In addition, the multiplexing of up to three patient samples combined in one run via germline identification further reduces per sample costs and increases throughput. To demonstrate these features on 0.01% MRD, samples were constructed by titrating diseased cells into healthy bone marrow cells before processing them with the scMRD assay. We detected 0.01% spike-in (CD34+) and 0.1% spike-in (CD117+) in 6 of 6 samples, with an average enrichment of 41x and 13x, respectively. Further testing detected CD34+ 0.1% spike-ins in 10 of 10 samples (32x average enrichment). We applied the scMRD assay to banked bone marrow aspirate samples from 3 AML patients. The scMRD assay resolved the clonal architecture identifying multiple leukemic clones with co-occurring mutations. The assay readily distinguished pre-leukemic from leukemic clones thereby increasing the specificity of MRD results. The integration of genotype and immunophenotypic further enhanced MRD detection by identifying genotype-specific protein expression patterns. By combining high sensitivity with multiomics, this assay offers a potential scalable solution for comprehensive MRD detection that guides therapeutic decision-making.

Deciphering the Structural Variants in Acute Myeloid Leukemia and Myelodysplastic Neoplasms By Optical Genome Mapping

Introduction Acute myeloid leukemia (AML) and myelodysplastic neoplasm (MDS) are common hematological malignancies, and the majority of patients carry genetic mutations, structural variants (SVs) and copy number variants (CNVs). Identifying these variants is important for diagnosis and prognosis, and in some cases can alter treatment management and outcomes. Methods for analyzing SVs and CNVs include karyotype analysis, fluorescence in situ hybridization, gene chips, and high-throughput gene sequencing technologies. Optical genome mapping (OGM) is a novel ultra-long single-molecule DNA detection based high throughput SVs analysis technology, which has the characteristics of whole genome coverage, high detection incidence and sensitivity. There were few publications presenting technical concordance of SVs detection between karyotyping and OGM, and there are no systematic studies comparing the concordance of OGM versus transcriptome sequencing (RNA-seq) in detecting gene fusion. This study aimed to compare the concordance of OGM and RNA-seq in the detection of fusion genes in AML and MDS cases, and the additional findings using OGM technology. Methods Bone marrow aspirates of 52 newly diagnosed or relapsed/refractory AML/MDS patients were recruited for OGM, RNA-seq, and leukemia-related gene DNA panel sequencing. Among these cases, 36 males and 16 were females, 15 pediatric (< 18 years) and 37 adults (≥18 years), with a median age of 31 years. Results Based on the RNA-seq results, 29/52 (55.8%) cases were detected with pathogenic gene fusions, including RUNX1::RUNX1T1, CBFB::MYH11, KMT2Aretc. (Figure 1). All those fusions were detected by OGM. For SVs with VAF>5%, the concordance was 98.1% (51/52 cases) when comparing OGM with karyotyping. OGM missed one case whose translocation happened close to the centromere. OGM reported more pathological variants with clinically significant in 5/52 cases (9.62%) and helped revise karyotype results in 1 case with clarifying marker chromosome. According to the ELN-2022 risk stratification mainly based on karyotype results, OGM's report results helped revise the risk stratification of 3 patients from intermediate to high risk. Fifty of 52 (96.2%) patients carried the gene mutation detected by the gene sequencing panel (Figure 1). In the samples of 4 patients who relapsed after receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) with donors and patients of different genders, we can infer the proportion of male and female DNA based on the copy number of chr Y detected by OGM, and then determine the proportion of donor cells. Conclusion This is the first AML cohort study to systematically compare the performance of OGM and RNA-seq, and the reported results of fusion genes are completely consistent. This is also the first report of applying the OGM technology in patients who relapsed after allo-HSCT and for chimerism analysis. Combined using OGM and targeted sequencing to fully characterize the CNV, SV, and mutation can be used for accurate stratification risk of AML/MDS. In conclusion, combining OGM and NGS can significantly improve the detection rate of genetic abnormalities with important pathological/diagnostic significance, and may bring new discoveries on SVs and pathogenic mechanisms.

Prognostic Value of Tumor Mutational Burden in Follicular Lymphoma Patients Treated with Immunochemotherapy

INTRODUCTION: Follicular lymphoma (FL) is the most prevalent indolent non-Hodgkin lymphoma subtype. It is characterized by frequent relapses, and patients who progress or relapse within 24 months (POD24) of front-line immunochemotherapy (ICT) have a poor outcome. Several prognostic indices have been proposed to identify high-risk patients, but none of them are currently used in clinical practice to guide treatment. Tumor mutational burden (TMB), defined as the number of somatic mutations/megabase (mut/Mb), has been proposed as a biomarker for predicting response to immune checkpoint inhibitors in several types of cancer. Therefore, this study aimed to explore the role of TMB in FL patients treated with frontline ICT. METHODS: The study included a total of 103 patients with FL grade 1-3a treated with frontline ICT. TMB was determined by Next Generation Sequencing (NGS) in 98 diagnostic lymph node biopsies with enough material available, with the Oncomine Tumor Mutational Load Assay (Thermo Fisher). This assay covers 1.65Mb across 409 genes frequently mutated in cancer, allowing an accurate estimation of TMB. Sequencing was performed on the Ion GeneStudio S5 system with >300X mean read depth. Only exonic non-synonymous variants remaining after manual variant refinement to exclude artefacts were considered. In addition, the mutational profile of 56 of the 98 patients was analyzed using a QIAgen custom DNA panel which covered 64 genes frequently mutated in FL. Sequencing was performed using NextSeq (Illumina) with >3000X mean read depth. Variants were analyzed and interpreted for pathogenicity using several databases, and only pathogenic or presumed pathogenic variants (P/PP) were considered. Statistical analysis was performed using SPSS and R. RESULTS: The final cohort included 94 patients, as in 4/98 patients (4%) TMB could not be accurately estimated due to a high frequency of artefacts caused by cytosine deamination. Median age at diagnosis of 61 years (range 24-86), 51% males and 49% females. Patients with advanced disease stage at diagnosis represented 87% of the cohort. Median TMB was 5.08mut/Mb (range 1.69-13.80 mut/Mb), and all cases genetically profiled presented at least one P/PP variant. The number of P/PP mutations correlated with TMB at diagnosis (p=0.004). TMB did not correlate with clinical parameters, such as age at diagnosis (p=0.891), histologic grade (p=0.243), Ann Arbor stage (p=0.200), ECOG (p=0.190), FLIPI (p=0.363), FLIPI2 (p=0.693), or LDH levels (p=0.483). However, lower TMB at diagnosis was associated with the absence of the t(14;18)(q32;q21) translocation (mean 3.93 vs 5.42 mut/Mb, p=0.006). The analysis of the mutational status of the 64 genes frequently mutated in FL revealed that mutations in genes involved in cellular migration ( GNA13, GNAI2) were associated with higher TMB values (4.50 vs 7.56 mut/Mb, p=0.001). An statistically significant association between low TMB and shorter progression-free survival (PFS) (p=0.011) was found when stratifying patients into two groups: high basal TMB (>2.55mut/Mb) and low basal TMB (≤2.55mut/Mb) (Figure 1). 2-year PFS was 54.5% (95%CI: 33.2-89.5%) in low-TMB patients and 84.9% (95%CI: 77.3-93.7%) in high-TMB patients. 5-year PFS was 39.0% (95%CI: 19.7-77.0%) in low-TMB patients and 70.5% (95%CI: 60.0-82.9%) in high-TMB patients. The proportion of POD24 cases was higher in the low-TMB group (46.1% vs 16.9%, p=0.039), none of them being refractory to treatment (7/70 in the high-TMB group). Genetically, the low-TMB group was enriched in patients with mutations affecting genes involved in the mTOR signaling pathway ( ATP6AP1, ATP6AP2, ATP6V1B2, RRAGC) (50.0% vs 8.7%, p=0.006). CONCLUSIONS: FL patients harboring t(14;18) or mutations in genes involved in migration have higher TMB values at diagnosis. Patient stratification based on TMB values allows the identification of a subgroup of FL patients with ≤2.55 mut/Mb with shorter PFS after treatment with frontline ICT. The prognostic usefulness of TMB should also be explored at relapse, in particular in patients receiving novel immunotherapies such as bispecific antibodies and CAR T-cell therapies, that are being approved for relapsed/refractory FL. ACKNOWLEDGMENTS: Work supported by Institut de Salud Carlos III-Federación Española de Enfermedades Raras (ISCIII-FEDER) grant PI19/00034, GLD18/00117, 2017SGR205, PT20/00023, and Xarxa de Banc de Tumors de Catalunya.

Evaluation of the Thermo Fisher Oncomine Myeloid MRD DNA Assay on the Ion Torrent Platform

Introduction Malignant myeloid diseases are a group of blood cancers that arise from genetic changes in hematopoietic cells in the bone marrow or peripheral blood. The presence of measurable residual disease (MRD), or detection of abnormal cells in the body following completion of therapy, is associated with a higher risk of disease relapse, and decreased overall survival (Schuurhuis 2018). Next-generation sequencing (NGS) can be leveraged as a high-sensitivity, minimally invasive method for detection of MRD. The Ion Torrent Oncomine Myeloid MRD Assay (Myeloid MRD) is a targeted NGS assay designed for the detection of single nucleotide variants (SNVs) and insertions/deletions in a curated selection of clinically relevant myeloid disease-related genes at low limits of detection. Assay performance was evaluated using normal and diseased clinical blood and bone marrow samples and NGS reference materials. Methods The Myeloid MRD DNA panel targets hotspot regions across 33 genes and uses Ion AmpliSeq HD technology with unique molecular tagging to suppress sequencing artifacts and improve analytical sensitivity. Briefly, DNA was extracted from whole blood or bone marrow samples and 120 ng of nucleic acid was used as input for the assay. The DNA was divided between 2 polymerase chain reactions (PCR), where it served as the template for target amplifications, before combining reaction products by sample. Library preparation followed the standard Ion AmpliSeq HD protocol, where amplicons were partially enzymatically digested, and a second round of amplification was performed using barcoded primers. Following bead purification, the final library was measured by quantitative PCR (qPCR) to inform final library loading onto the sequencer. Six libraries were pooled and sequenced on an Ion 550 chip using the Ion GeneStudio S5 System. Primary and secondary analyses were performed using the Torrent Suite software, and results were exported to the Ion Reporter software for variant calling. From the variant calling results, a probit model was utilized to determine the variant allele frequency (VAF) at which the assay achieved 90% power to call an expected variant, defining the limit of detection (LoD). Other parameters evaluated included analytical sensitivity, analytical specificity, positive predictive value (PPV), accuracy, and precision. Results Overall, consistent, high quality on-target reads were observed across 22 sequencing runs with a median Ion Sphere Particle (ISP) loading of 90%, usable sequence of 59%, and over 130 million total reads per chip. When evaluating individual samples according to the vendor-recommended number of mapped reads (> 10 million), 98.3% of samples tested passed quality control (QC). Additional sample QC metrics established during assay validation included thresholds of > 80% reads on-target, > 80% uniformity of base coverage, and a mean read length of at least 80 base pairs. The estimated LoD for the assay was 0.1% VAF for small variants (SNVs, insertions/deletions).Above the LoD, the assay demonstrated 96.03% sensitivity, 99.89% accuracy of base-calling, 100% specificity, and 98.83% PPV.Additionally,a comparison was performed usingmatched whole blood and bone marrow samples collected from patients with acute myeloid leukemia (AML) to evaluate the concordance of variant calling. It was determined that the same variants were identified across both sample types, although at higher allele frequency in bone marrow compared to whole blood. Conclusion This evaluation of the Myeloid MRD assay demonstrates that it is a precise and accurate assay with sensitivity that is acceptable for MRD analysis. Notably, variant detection was concordant between whole blood and bone marrow sample types collected from AML patients, with bone marrow providing improved sensitivity for low-frequency variant detection. The user-friendly workflow and integrated informatics pipeline offer opportunities for streamlined testing and interpretation of results. The Ion Torrent Myeloid MRD DNA assay is a powerful new tool for MRD detection across a growing myeloid disease target landscape.

Development of a Targeted NGS Panel for Hematologic Malignancies According to Who/ICC 2022 Guidelines

Introduction: Since 2020, we have successfully developed a targeted blood cancer NGS panel and have been using it efficiently in clinical settings. However, it needed to be upgraded due to recent new blood cancer diagnosis and classification guidelines by the World Health Organization and the International Collaboration for Cancer Classification (ICC) in 2022. Therefore, in this study, an updated targeted NGS panel that covers the updated guidelines was evaluated using clinical samples. Methods: To adhere to the recent guidelines, the currently used targeted NGS panel (KBB DNA/RNAseq NGS Leukemia PHB; KBlueBio Inc., Hwasun, Korea) was reviewed. Its analytical performance was validated using standard NA12878 and clinical samples. The clinical samples consisted of 19 genomic DNA and 20 total RNA samples extracted from the bone marrow or peripheral blood of patients with hematologic malignancies, including acute myeloid leukemia, acute lymphocytic leukemia, myelodysplastic syndrome/myeloproliferative neoplasm, multiple myeloma, and lymphoma. Results: The updated DNA panel comprises 125 genes including 6 new genes ( BCORL1, BLM, GNB1, PRPF8, SAMD9 and SAMD9L). The updated RNA panel has 116 genes including 31 new genes ( FLT3, CBFA2T3, RPN1, BCL11B, ZNF362, HLF, NUTM1, UBRF, CDX2, ZEB2, CDK6, ZMYM2, AFDN, ELL, NSD1, HOXA9, KDM5A, ZBTB16, GLIS2, PRDM16, NPM1, KAT6A, MNX1, TAF15, MLLT1, P2RY8, HNRNPUL1, ECM1, ENAM, JCHAIN (IGJ) and MDFIC). The accuracy, repeatability, reproducibility, sensitivity, and detection limit of the updated NGS panels were evaluated using standard materials, and the results met the predefined criteria. A comparison of the updated DNA panel with the old panel using 19 clinical samples revealed an overall concordance rate of 100% between the two panels for all mutations (95% confidence interval (CI): 99.72-100.00%). Similarly, the updated RNA panel also met the predefined criteria. The overall concordance rate between the updated RNA panel and the old panel was 99.72% in 20 clinical samples (95% CI: 99.00-99.72%). Conclusions: The old targeted DNA/RNA NGS panel was successfully updated according to the 2022 WHO and ICC guidelines, and can be used to accurately and efficiently detect the genetic variants of blood cancers.

BAX Mutated Clonal Hematopoiesis Arises Following Treatment with the BCL2 Inhibitor Class of Therapeutics across a Range of Hematological and Non-Hematological Neoplasms

Clonal hematopoiesis (CH) is an age-related phenomenon characterized by the expansion of genomically mutated hematopoietic clones with a selective fitness advantage. Venetoclax, a selective BCL2 inhibitor, has established efficacy in hematological malignancies such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Our previous studies described the emergence of deleterious BAX variants within the myeloid compartment of patients (pts) receiving venetoclax for CLL (Blombery, Blood2022). Whether this phenomenon is specific to venetoclax and the CLL disease context is currently unknown. We therefore aimed to investigate the prevalence and pattern of BAX-mutatedCH across different hematological and non-hematological neoplasms in response to BCL2 inhibitor therapy (both venetoclax and BGB-11417). We first examined pts with AML in complete morphologic remission after venetoclax exposure in both the newly diagnosed (CAVEAT; Chua, JCO 2020) and early relapse (VALDAC; Tiong, ASH 2022) settings using unique molecular index-based error-corrected next generation sequencing with a sensitivity of 0.5% (QIAseq targeted DNA panel). BAX variants were detected in remission samples in 11/20 (55%) and 9/26 (35%) pts in these cohorts, respectively, after a median of 9- and 4-months post venetoclax. This represented a significant higher incidence compared to remission samples from pts with newly diagnosed AML post conventional cytotoxic chemotherapy without venetoclax (6/87 [7%]; adjusted p<0.01 for both pairwise comparisons). Having observed the increased frequency of BAX-mutated CH in remission in myeloid (AML) and lymphoid (CLL) neoplasms, we then investigated whether BAX variants may be acquired in response to BCL2 inhibitor therapy outside the context of hematological neoplasm. We assessed a cohort of pts with metastatic breast cancer who received venetoclax in combination with tamoxifen (mBEP; Lok, Cancer Discover 2019), and detected BAX variants from peripheral blood samples in 5/16 (31%) pts (median age 66) after a median duration of 20 (range 13-44) months on venetoclax. In contrast, no BAX variants were detected in a similarly aged control cohort of 50 pts (median age 62) with breast cancer without exposure to venetoclax (p=0.01). The rates of any CH-related mutation were 75% and 50% pts, respectively (p=0.09). No pt developed MDS or AML during follow up in the mBEP cohort. Finally, to explore whether emergence of BAX-mutated CH is specific to venetoclax as an agent, we studied a cohort of 9 pts with CLL who had received BGB-11417, a more potent and selective BCL2 inhibitor; median treatment duration 13 months. Notably, we observed the emergence of BAX variants in 2/9 (22%) pts in this cohort. The characteristics of the BAX variants (n=88) in 28 pts (AML, CLL or breast cancer) are summarized in Figure 1; 3 variants (in 3 pts) were detectable in pre-treatment samples. Over half of the missense (n=22) or in-frame variants (n=1) were clustered in the C-terminus of the BAX protein, while truncating variants (n=65) were observed throughout the gene. The median number of BAX variants per pt was 2, with 32% of pts having ≥3 variants (max=13). The median VAF was 1.4%, and only 16% of variants had a VAF >5%. The number and VAF of BAX variants did not significantly correlate with the duration of therapy. Among 12 pts with AML who had serial testing (median inter-test interval 10 months), BAX variants (n=24) did not significantly change in VAF (median Δ 0.7%) with time after initial acquisition (Pearson's coefficient 0.17). To clarify the compartments affected by BAX variants we performed single-cell combined DNA/protein sequencing (Tapestri) of a sample with multiple BAX variants and demonstrated their presence in multiple independent clones with myeloid-biased lineage but also within the natural killer cell compartment ( Figure 2). In summary, the emergence of BAX-mutated CH appears to be a generalized phenomenon of adaptive hematopoiesis that occurs across both hematological and non-hematological malignancy settings as well being observed with other BCL2 inhibitors consistent with a therapeutic class effect. Further research is needed to understand the contribution of this phenomenon to the subsequent development of clinically relevant hematological abnormalities and its implications for future therapeutic resistance.