bluuebunny/crossref_metadata_2025_split huggingface dataset:
Early detection of cancer using cell-free DNA (cfDNA) size analysis on a multiplexed amplicon-based next-generation sequencing (NGS) platform.
Authors: Choudhury Yukti , Pinweha Pannapa , Poh Jonathan , Lim Jing Shan , Tan Min-Han 
Published: June 2022

3041 Background: Tumor-derived cfDNA fragments are observed to be shorter in length than normal cfDNA. This size (length) difference can be analyzed as a tumor-specific signal. Whole genome or probe hybridization-based NGS methods can capture cfDNA fragments of native sizes. However, amplicon-based NGS assays are not directly amenable to cfDNA size analysis due to predetermination of amplicon sizes by design. Here we present a method to extract relative distribution of cfDNA fragment lengths from a targeted amplicon-based assay and show its utility in cancer detection. Methods: The LiquidHALLMARK cfDNA assay is an amplicon-based NGS test for the sensitive detection of genomic alterations in 80 genes. Although panel design is optimized for cfDNA with average amplicon length of ̃150 bp, the consecutive tiling design of amplicons for genes with contiguously targeted regions, e.g. BRCA permits the formation of longer amplicons (> 150 bp) from physically subsequent primer pairs, provided longer template cfDNA molecules are present. Cancer samples (n = 281), clinically tested by LiquidHALLMARK during Sep 2020-Sep 2021, and healthy samples (n = 28) were included for analysis. For each sample, fragment lengths were inferred from sequencing alignment files, and binned into “short” (0-150 bp) and “long” (151-500 bp) groups. A relative “size ratio” of the total number of short vs. long fragments per sample was calculated, and examined with clinical features, plasma cfDNA concentration (cfDNA/ml) and highest mutation allele frequency (AF%), in a model to predict cancer. Results: Calculated size ratios (relative abundance of short fragments) were higher in cancer than normal samples (median 47.6 vs. 31.2, p < 0.001). Cholangiocarcinoma and colorectal cancer samples had the highest size ratios (medians: 62.9 and 60.9, respectively) in agreement with a genome-wide NGS study that profiled cfDNA sizes. Size ratios were higher in metastatic (n = 143) compared to early stage (n = 30) lung cancers (p = 0.0039), indicating a stage-dependent accumulation of shorter cfDNA fragments. Size ratio was correlated with cfDNA/ml ( r= 0.63, p < 0.01) and AF% ( r= 0.42, p < 0.01). In 20-fold cross-validation of a logistic regression model trained to predict cancer, average area under curve (AUC) was 0.82 using size ratio, 0.86 using cfDNA/ml, and increased to 0.95 with the two features combined. Conclusions: Our analysis shows that it is feasible to derive meaningful cfDNA fragment size information from amplicon-based NGS data. Importantly, relative fragment size distributions observed in cancer and healthy plasma samples by this method are concordant with alternate target capture methods. Fragment size ratios derived from relatively small, targeted amplicon panels are a novel feature that, combined with other molecular and clinical features, can enhance non-invasive methods of cancer detection.
PD-L1 is overexpressed on tumorspheres cultured from circulating cancer stem cells in patients with breast cancer.
Authors: Pizon Monika , Schott Dorothea , Pachmann Ulrich A. , Pizon Marek , Pachmann Katharina 
Published: June 2022

3052 Background: Circulating cancer cells, and in particular their very rare subpopulation, circulating cancer stem cells (cCSCs), are responsible for recurrence and metastasis. The exact role of cCSCs in escape of cancer from immunosurveillance is still unknown, but recent studies revealed that enhanced PDL-1 expression in cancer stem cells is linked to immune evasion and could crucially contribute to the maintenance of CSC self-renewal. Understanding the mechanisms behind this PDL-1 overexpression in cancer stem cells is critical for developing more effective anti-PD-1/PD-L1 therapy. Therefore the aim of the study was to determine the number of tumorspheres and expression of PD-L1 on tumorspheres cultured from cCSC in breast cancer patients. Methods: 110 patients with breast cancer in different stages of disease were included in this study. The determination of circulating cancer stem cells was performed using the sphere-forming assay. Additionally anti-PDL-1 antibody staining was applied to examine PDL-1 expression on breast tumorspheres. Results: We have developed an innovative in vitro platform for detection of cCSCs from peripheral blood of cancer patients. The number of tumorspheres increased significantly with tumor progression and aggressiveness of primary tumor. Patients with metastatic disease had statistically more tumorspheres as compared to patients without metastasis (30 vs 10/100µl blood, p < 0.05). Patients with multiple metastasis had more tumorspheres compared to patients with single metastases (60 vs 30/100µl blood, p < 0.05). The number of tumorspheres was positively correlated with Ki-67, Her2 status and grade score in primary breast tumors. We observed high PDL-1 expression and their considerable heterogeneity in enriched tumorspheres. Conclusions: The number of tumorspheres cultured from peripheral blood directly reflects aggressiveness and proliferation capacity of primary tumor. The presence of tumorspheres with expression of PDL-1 might suggest their immunregulatory potential. Better understanding of the interaction between cCSCs and tumor immunology may help to identify strategies to eradicate the minor subpopulation that escapes conventional therapy attack, thus providing a solution to the problem of drug resistance and metastasis.
Tracking changes in circulating stromal cells and circulating tumor cells predicts responsiveness of new line induction in metastatic breast cancer after 1 cycle of therapy.
Authors: Adams Daniel L , Alpaugh R. Katherine , Lapidus Rena G. , Chumsri Saranya , Reduzzi Carolina , Tang Cha-Mei , Williams William , Cristofanilli Massimo 
Published: June 2022

3056 Background: In metastatic Breast Cancer (mBC), Circulating Tumor Cells (CTCs) are established prognostic indicators of patients (pts) not responding to new lines of therapy and who have poor clinical outcomes. However, CTCs are typically found in < 20% of mBC pts and many pts without CTCs will also progress. Recently an inflammatory pro-tumorigenic macrophage emanating from tumor stroma (i.e. Cancer associated macrophage-like cell [CAML]) was found in > 90% of mBC pts and were also indicative of poor clinical outcomes. As CTCs & CAMLs are isolated in conjunction from a single blood sample, and both are prognostic for therapy response, we evaluated CTCs & CAMLs before and after initiation of new therapies in mBC to determine their combined prognostic/predictive values. Methods: An observational prospective 2-year multi-institutional study was undertaken to evaluate CTCs & CAMLs before, and after, induction of any new line of therapy in pts with diagnosed mBC (n = 101). Anonymized and blinded blood samples were taken at baseline (BL), prior to starting a new systemic therapy, and a 2nd sample (T1) taken after therapy initiation (̃30 days). Blood was filtered by CellSieve filtration. The quantities and subtypes of CTCs & CAMLs were analyzed based on RECIST v1.1 for progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) by censored univariate & multivariate analysis at 2 years. Results: CTCs were identified in 35% (n = 35/101) of pts at BL & 24% (n = 24/101) at T1, with a single CTC at T1 being highly prognostic for worse PFS HR = 6.2 95%CI 3.0-13.2, p < 0.001 & OS (HR = 5.1 95%CI 2.0-13.4, p = 0.002. In parallel, CAMLs were found in 93% of BL and 86% of T1 samples, and whose decreases were significantly prognostic for improved PFS (HR = 2.7, 95%CI 1.4-5.1, p = 0.006) and OS (HR = 4.4 95%CI 1.5-13.2, p = 0.018) when CTCs were absent. Overall ≥1 CTC at T1 (n = 24) had median PFS = 2.4 & mOS = 4.8 months (mos), however, in pts without CTCs plus an increase in CAMLs (n = 36) had mPFS = 5.9 & mOS = 14.1 mos, while in pts without CTCs plus a decrease in CAMLs (n = 41) had mPFS = 14.8 & mOS = 18.8 mos. Conclusions: Our data confirms that pts with persistent CTCs have the worst clinical outcomes. Further, simultaneous CAML quantification provided a new dynamic predictive blood based biomarker in pts without detectable CTCs which may be useful to better individualize therapy and improve outcomes, though future studies are need to validate these findings.
Resolution ctDx FIRST plasma assay as a companion diagnostic for adagrasib and its application to longitudinal monitoring.
Authors: Pekker Ira , Pollak Julia , Potts Kristy , Chan PuiYee , Tsai Chen-Hsun , Liao Angela , Garrison Carly , Brown Taylor , Stull Paul , Bianchi-Frias Daniella , Li Zhen , Baker Christine , Garg Kavita 
Published: June 2022

3057 Background: KRAS is one of the most commonly mutated oncogenes in cancer. There is a significant need for new treatment options for patients with non–small cell lung cancer (NSCLC) harboring the KRAS G12C mutation. Adagrasib is an investigational, highly selective, oral small molecule inhibitor of KRAS G12C that has demonstrated clinical benefit in patients with KRAS G12C–mutant NSCLC and CRC. Detection of KRAS G12C in cfDNA is minimally invasive and is of benefit to NSCLC patients, many without lesions accessible by tissue biopsy testing. Methods: Resolution ctDx FIRST is a 113 gene comprehensive genomic profiling assay that identifies oncogenic alterations including substitutions, insertions, deletions, gene fusions, and homozygous deletions using targeted NGS sequencing of cfDNA. The Resolution ctDx FIRST assay is being developed as a companion diagnostic for adagrasib in NSCLC patients of the Mirati Study 849-001. Results: The LOD95 for SNVs and indels in KRAS and EGFR at a cfDNA input level of 15ng ranged from 0.34% to 0.82%. No false positives were detected in any samples from healthy donors (N = 60). A total of 230 NSCLC plasma samples were orthogonally tested using a ddPCR assay for KRAS G12C, using 76 plasma samples from Study 849-001 where KRAS G12C positive results had previously been obtained by a tissue assay, and 154 commercially procured NSCLC samples representative of the trial population. The PPA and NPA for Resolution ctDx FIRST plasma testing relative to ddPCR assay (95% CI) were 87.0% (75.1–94.6%) and 97.6% (94.1–99.4%) respectively. Of 112 Study 849-001 patients, 71 (63.4%) were tested for KRAS G12C mutations with Resolution ctDx FIRST in pretreatment plasma samples. Seventy-four commercially procured matched tissue and plasma samples were tested by Resolution ctDx FIRST and tissue assay. The PPA and NPA for Resolution ctDx FIRST plasma testing relative to tissue assay (95% CI) were 66.2% (54.0-77.0%) and 100% (94.7-100%) respectively. The detection of EGFR variants in the Resolution ctDx FIRST assay was compared to results from a ddPCR assay for each variant. A total of 165 NSCLC plasma samples generated a total of 317 comparative valid results. PPA was 100% for EGFR L858R, 88.9% for EGFR T790M, and 91.3% for EGFR Exon 19 Deletions. NPA was 97.8% for EGFR L858R, 100% for EGFR T790M, and 100% for EGFR Exon 19 Deletions. Application of the assay in longitudinally collected NSCLC and CRC patient samples will be presented highlighting changes of VAF over time including identification of oncogenes involved in emerging resistance. Conclusions: The Resolution ctDx FIRST assay offers highly sensitive, specific, and robust test results, and meets analytical requirements for clinical applications. [Table: see text]
T-cell receptor repertoire analysis based on RNA sequencing data from tumor cells and tumor-infiltrating lymphocytes.
Authors: Du Cheng , Cui Lili , Wang Meiling , Li Long , Zheng Zhendong , Liu Danni , Li Yingmei , Mu Tianhao 
Published: June 2022

3058 Background: T-cell receptor (TCR) repertoire has been thought to be indicative in cancer progression and treatment response. Previous methods mainly focused on peripheral blood or fresh tumor tissue, which were sometimes logistically limited in clinical settings. Tumor-infiltrating lymphocytes (TILs), which harbored TCR characteristics, were also mingled with tumor cells (TCs), which brought hurdles in extracting TCR signals from bulk RNA sequencing data. Here we employed a set of RNA sequencing data from paired FFPE tumor samples and their micro-dissected TILs, to analyze and compare the TCR features in tumor cells and TILs. Methods: RNA sequencing data of 14 tumor cell samples and matched TILs were downloaded from NCBI-SRA (accession: PRJEB36554). Raw data were cleaned up by Trim Galore (v0.6.2). TCR clonotypes were assembled and quantified from clean fastq files by MiXCR (v3.0.4). Diversity and clonality metrics were analyzed using VDJtools (v1.2.1) and in-house Pearl scripts. Results: The median mapping rates of sequencing reads to TCR regions were 0.29% and 1.80% for TCs and TILs respectively (p = 0.00051). TCR diversity of TCs and TILs was characterized by Shannon and Simpson index respectively. The median Shannon index was 1.889 and 2.694 in TCs and TILs (p = 0.00034, unpaired Wilcoxon rank-sum test); the median Simpson index was 0.8724 and 0.9420 in TCs and TILs (p = 0.0058). Conclusions: RNA isolated from clinical FFPE samples could be used for TCR analysis. Micro-dissection of TILs could enhance TCR signals of unprocessed tumor tissues.
Frequency of practice-changing findings identified by comprehensive genomic profiling in non-myeloid hematologic malignancies.
Authors: Zhou Katherine I. , Lin Chenyu , Green Michelle , Caughey Bennett Adam , Datto Michael , Strickler John H , McKinney Matthew 
Published: June 2022

3060 Background: Comprehensive genomic profiling (CGP) is increasingly used to guide management of myeloid and advanced solid malignancies, but its role in non-myeloid hematologic malignancies is less clear. Studies have found a high rate of potentially actionable variants by CGP in this population, but these do not always translate into clinical practice changes. We aimed to determine the rate at which variants found on CGP changed clinical practice. Methods: We retrospectively reviewed a cohort of 101 consecutive patients with non-myeloid hematologic malignancies at Duke, comprising a total of 105 samples that were sent for CGP by FoundationOne Heme (104) or HemeComplete (1) in 2014–2021. We identified variants of clinical significance and classified them by evidence level according to the AMP/ASCO/CAP 2017 guidelines (e.g., for therapies, level A: FDA-approved / in guidelines; B: expert consensus; C: clinical trial / FDA-approved in different tumor type; D: preclinical data). We further identified documented changes in clinical practice that occurred in direct response to CGP results. Results: Commonly cited reasons for CGP included guiding therapy selection (27), identifying resistance mutations (19), refining prognosis (14), and clarifying diagnosis (11). Of the 105 samples sent for sequencing, 92 (88%) yielded at least one pathogenic or likely pathogenic variant. CGP resulted after death in 12 patients and within 1 month of death in another 11 patients. Seventy-three out of 101 patients (72%) had at least one variant with therapy sensitizing, diagnostic, or prognostic significance (levels A–C) or associated with therapy resistance (levels A/D). While 61 patients (60%) had a therapy sensitizing variant, only 6 patients (10%) were offered a biomarker-directed therapy. In contrast, the presence of a resistance mutation led to discontinuation of current therapy or influenced future therapy selection in 9 of 13 patients (69%). The absence of a resistance mutation influenced choice of therapy in another 4 patients. Sequencing results also helped clarify a previously uncertain diagnosis in 4 patients and led to medical genetics referrals in 3 patients. Conclusions: Comprehensive genomic profiling of non-myeloid hematologic malignancies identified variants of clinical significance in 72% of patients and led to changes in practice in 22% of patients. CGP was often sent late in the clinical course. [Table: see text]
Use of clinical RNA-sequencing in the detection of actionable fusions compared to DNA-sequencing alone.
Authors: Michuda Jackson , Park Ben Ho , Cummings Amy Lauren , Devarakonda Siddhartha , O'Neil Bert , Islam Sumaiya , Parsons Jerod , Ben-Shachar Rotem , Breschi Alessandra , Blackwell Kimberly L. , Chen James Lin , Dudley Joel , Stumpe Martin , Guinney Justin , Cohen Ezra E.W. 
Published: June 2022

3077 Background: While targeted DNA-seq can detect clinically actionable fusions in tumor tissue samples, technical and analytical challenges may give rise to false negatives. RNA-based, whole-exome sequencing provides a complementary method for fusion detection, and may improve the identification of actionable variants. In this study, we quantify this benefit using a large, real-world clinical dataset to assess actionable fusions detected from RNA in conjunction with DNA profiling. Methods: Using the Tempus Research Database, we retrospectively analyzed a de-identified dataset of ̃80K samples (77.4K patients) profiled with the Tempus xT assay (both DNA-seq with fusion detection in 21 genes and whole exome capture RNA-seq). Only patients that had successful RNA- and DNA-seq were included. Fusions were detected using the Tempus bioinformatic and clinical workflow. Candidate fusions were filtered based on read support thresholds, fusion annotation ( i.e., breakpoints, reading frame, conserved domains), and manual review. OncoKB was used to select fusion alterations in levels 1 and 2 and to identify those indication-matched to targeted therapies. Results: We identified 2118 level 1 and 2 fusion events across 1945 patients across 20 different cancer types. Most fusions were observed in non-small cell lung cancer (NSCLC) (25%) and biliary cancer (9%) samples. Of the 2118 fusion events, 29.1% (616) were detected only through RNA-seq while 4.8% (101) of the events were identifiable only through DNA-seq. Notably, 69.4% of fusions in low-grade glioma and 58.2% in sarcomas were detected only by RNA-seq. When evaluating specific gene fusion events, RNA-seq consistently improved the detection of fusions compared to DNA-seq alone (Table) across all cancer types. A total of 1106 fusions were classified as targetable by OncoKB indication-matched therapies with 19% (214) of these identifiable through RNA-seq alone, 5% (54) by DNA-seq alone, and 76% (838) identifiable through RNA- and DNA-seq. Overall, fusions identified through RNA-seq alone led to a 24% increase in the number of patients who were eligible to receive matched therapies (214 / 892). This included imatinib for patients with CML/BLCL (69.8%), crizotinib for NSCLC (40.3%) and entrectinib for NTRK and ROS1 fusions (32.5%). Conclusions: The addition of RNA-seq to DNA-seq significantly increased the detection of fusion events and ability to match patients to targeted therapies. Results support consideration of combined RNA-DNA-seq for standard-of-care fusion calling. [Table: see text]
Comprehensive genomic profiling to identify gene alterations in DNA repair pathway across solid tumors.
Authors: McDonnell Kevin , Basu Gargi D. , Hoag Jess W. , Hall David W. , Alyaqoub Fadel S. , Dombrowski Susan M. , Noel Pawan , Szelinger Szabolcs , Udhane Sameer S. , Wang Min , LoBello Janine R. , Thakkar Snehal Govind , Baehner Frederick L. , Hong Christine , Park Wai , Idos Gregory , Gray Stacy W. , Gruber Stephen B. 
Published: June 2022

3124 Background: Deleterious events in DNA damage response (DDR) are hallmarks of cancer associated with sensitivity to PARP inhibitors (PARPi) and immune checkpoint inhibitors (CPI). This study investigated DDR pathway alterations across solid tumors. Methods: Samples were sequenced with the Oncomap ExTra assay using tumor-normal paired whole-exome DNA sequencing to detect single base substitutions, indels, and copy number alterations that were clinically actionable, defined as associated with FDA approved drugs or clinical trial enrollment. Here, we report the frequency of MSI-high and TMB-high (>10 mutations/Mb), and clinically-actionable alterations for the following 49 DDR genes: ARID1A, ATM, ATR, ATRX, BAP1, BARD1, BLM, BRCA1/2, BRIP1, CDK12, CHEK1/2, EPCAM, ERCC1/2/3/4/5, FANCA/C/D2/E/F/G/I/L/M, MLH1, MRE11A, MSH2/6, MUTYH, NBN, PALB2, PMS2, PPP2R2A, PTEN, RAD21/50/51/51B/51C/51D/52/54L, XRCC1/2/3. Results: Of the 6055 patient samples profiled, 1633 (27.0%) had clinically actionable alterations in DDR genes; DDR alterations varied from 0-64.3% across tumor types. For the 5657 samples with TMB/MSI data, 429 (7.6%) had high TMB and 193 (3.4%) were MSI-high; MSI-high samples were usually TMB-high (n = 180; 93.2%). The percent of cancers that were TMB-high or MSI-high varied from 0-64.8% and 0-20.8% respectively. Actionable BRCA1/2 gene alterations were present in 319 patients (5.3%, range 0-15.3%). Three cancers (biliary, brain and liver) had BRCA1/2 alterations in less than 2% of patients but across the 49 DDR genes alterations were present in more than 20% of patients, representing a greater than 10-fold difference. Across solid tumors, this analysis identifies a group of 1314 patients (21.7%) who harbor a DDR gene alteration other than BRCA1/2. Conclusions: Defective DNA repair as detected by deleterious alterations in DDR genes along with TMB/MSI status has the potential to guide clinicians to FDA-approved therapy or clinical trial enrollment in a large percentage of patients across solid tumor types.[Table: see text]