Abstract 2759: Technical limitations of capture-based, targeted deep sequencing for the detection of somatic variations in cell-free DNA

PURPOSE Novel sensitive methods for early detection of relapse and for monitoring therapeutic efficacy may have a huge impact on risk stratification, treatment, and ultimately outcome for patients with bladder cancer. We addressed the prognostic and predictive impact of ultra-deep sequencing of cell-free DNA in patients before and after cystectomy and during chemotherapy. PATIENTS AND METHODS We included 68 patients with localized advanced bladder cancer. Patient-specific somatic mutations, identified by whole-exome sequencing, were used to assess circulating tumor DNA (ctDNA) by ultra-deep sequencing (median, 105,000×) of plasma DNA. Plasma samples (n = 656) were procured at diagnosis, during chemotherapy, before cystectomy, and during surveillance. Expression profiling was performed for tumor subtype and immune signature analyses. RESULTS Presence of ctDNA was highly prognostic at diagnosis before chemotherapy (hazard ratio, 29.1; P = .001). After cystectomy, ctDNA analysis correctly identified all patients with metastatic relapse during disease monitoring (100% sensitivity, 98% specificity). A median lead time over radiographic imaging of 96 days was observed. In addition, for high-risk patients (ctDNA positive before or during treatment), the dynamics of ctDNA during chemotherapy was associated with disease recurrence ( P = .023), whereas pathologic downstaging was not. Analysis of tumor-centric biomarkers showed that mutational processes (signature 5) were associated with pathologic downstaging ( P = .024); however, no significant correlation for tumor subtypes, DNA damage response mutations, and other biomarkers was observed. Our results suggest that ctDNA analysis is better associated with treatment efficacy compared with other available methods. CONCLUSION ctDNA assessment for early risk stratification, therapy monitoring, and early relapse detection in bladder cancer is feasible and provides a basis for clinical studies that evaluate early therapeutic interventions.

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Targeted deep sequencing revealed variants in cell-free DNA of hormone receptor-positive metastatic breast cancer patients

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03189-z ◽

2019 ◽

Vol 77 (3) ◽

pp. 497-509 ◽

Cited By ~ 6

Author(s):

Corinna Keup ◽

Karim Benyaa ◽

Siegfried Hauch ◽

Markus Sprenger-Haussels ◽

Mitra Tewes ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Cancer Patients ◽

Deep Sequencing ◽

Hormone Receptor ◽

Metastatic Breast ◽

Breast Cancer Patients ◽

Hormone Receptor Positive ◽

Free Dna ◽

Targeted Deep Sequencing

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Monitoring of therapeutic efficacy to CDK4/6 inhibitors and early detection of metastatic relapse in breast cancer by ultra-deep sequencing of plasma cell-free DNA.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e15544 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e15544-e15544

Author(s):

Yoon Ming Chin ◽

Tomoko Shibayama ◽

Masumi Otaki ◽

Hiu Ting Chan ◽

Makiko Ono ◽

...

Keyword(s):

Breast Cancer ◽

Disease Progression ◽

Tumor Markers ◽

Plasma Cell ◽

Deep Sequencing ◽

Liquid Biopsy ◽

Therapeutic Efficacy ◽

Cell Free Dna ◽

Free Dna ◽

Metastatic Relapse

e15544 Background: CDK4/6 inhibition substantially improves progression-free survival for women with advanced estrogen receptor-positive breast cancer. Despite this, most patients experience acquired resistance. The analyses of cancer specific genomic aberrations in plasma cell free DNA, or commonly referred to as ‘liquid biopsy’, has generated immense interest. The role of liquid biopsy enables monitoring of therapeutic efficacy to detect relapse in a minimally invasive manner. It would drastically impact risk stratification, treatment selection and patient clinical outcome in advanced stage breast cancer. Methods: We have recruited consecutive patients with metastatic breast cancer who received CDK4/6 inhibitor treatment in combination with endocrine therapy between April 2018 and December 2019. Somatic mutations identified by the Oncomine Pan-Cancer cell free assay were used to assess the mutation profiles of circulating tumor DNA (ctDNA) by ultra-deep sequencing (~56,000 depth) of the plasma DNA. Plasma samples (N = 146) were collected before and during treatment (2 years) or until disease progression. Paired white blood cells (WBC) were also sequenced to exclude clonal hematopoiesis-derived mutations. Results: Twenty-eight patients were enrolled. Based on RECIST criteria, 1 patient showed complete response (CR) within 18 months of treatment, 5 patients showed partial response (PR) 6-18 months from start of treatment, 9 patients showed stable disease (SD) condition within 3-18 months and 13 patients developed progressive disease (PD) within 1-12 months. In 22 of 28 patients (79%), ctDNA was detected prior to start of treatment (12 responders; 10 PD) and used as a monitoring marker. Majority of mutations detected in pre-treatment samples were TP53, PIK3CA and ESR1. Among the 12 ctDNA positive responders, ctDNA levels of 9 patients were undetectable within 2 weeks to 6 months from start of treatment (median 3 months). ctDNA profiles were more reflective of clinical response compared to the tumor markers CEA and CA15-3. In 7 of the 10 PD patients with ctDNA monitoring markers, ctDNA showed higher sensitivity to predict disease progression compared to tumor markers that remained stagnant or below normal threshold. ctDNA also predicted PD earlier than radiological images with a median lead time of 3 months. Conclusions: ctDNA assessment for therapy monitoring and early relapse detection in advanced breast cancer is feasible and provides a basis to evaluate early therapeutic interventions.

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Molecular characterisation of longitudinally collected circulating cell-free DNA in HPV+ve and HPV-ve oropharyngeal cancer

10.1101/2020.09.07.20189704 ◽

2020 ◽

Author(s):

John P Thomson ◽

Sophie J Warlow ◽

Martyna Adamowicz ◽

Helen Thain ◽

Kate Cuschieri ◽

...

Keyword(s):

Deep Sequencing ◽

Clinical Decision Making ◽

Fragment Size ◽

Clinical Care ◽

Significant Risk ◽

Post Treatment ◽

Response To Therapy ◽

Cell Free Dna ◽

Tissue Biopsy ◽

Free Dna

Oropharyngeal squamous cell carcinoma (OPSCC) is an increasing global health problem and is divided into two types dependent on association with human papillomavirus (HPV), with a more favourable prognosis in virus-associated tumours. Current methods of establishing viral aetiology, assessing response to therapy and clinical monitoring rest on tissue biopsy, clinical examination and post-treatment imaging. However, tissue biopsy is invasive and carries significant risk of morbidity, and post-treatment scans are frequently indeterminate. Analysis of cell-free DNA (cfDNA) from the circulation provides a minimally invasive method for detecting and monitoring cancer-derived DNA fragments, with the potential for enhancing clinical care. Through the longitudinal collection of 166 blood samples in 67 OPSCC patients we evaluate the utility of three cfDNA analysis methods: droplet digital PCR (ddPCR) and fragment size analysis in both HPV+ve and HPV-ve disease, and ultra-deep sequencing in patients with HPV-ve disease. We show that ddPCR analysis of cfDNA for five HPV types (16, 18, 31, 33 & 35) is strongly concordant with existing clinical assays (p16 immunohistochemistry (IHC) and quantitative PCR analysis of solid tumour tissue) and that cfDNA fragment size was reduced in OPSCC patients compared to healthy controls. Sequential ddPCR measurements of cfDNA HPV copy number showed a decrease to undetectable levels in all 30 HPV+ve patients in at least one of their post-treatment samples and a corresponding increase in cfDNA fragment size in patients who had a complete response to chemoradiotherapy. In two HPV+ve patients, clinical decision-making based on HPV ddPCR of cfDNA may have led to earlier detection of relapse in one patient or avoided surgical exploration in a second patient, which led to resection of tissue that did not harbour malignancy. In HPV-ve disease, ultra-deep sequencing identified tumour-derived somatic mutations of circulating cfDNA in genes such as TP53 and members of the ERBB family that are potential markers of therapeutic responsiveness and patient prognosis. Together our data suggest that analysis of circulating cfDNA can enhance current clinical strategies for assessing therapeutic response and disease monitoring in both HPV+ve and HPV-ve OPSCC.

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Characterization of DNA lesions associated with cell-free DNA by targeted deep sequencing

BMC Medical Genomics ◽

10.1186/s12920-021-01040-8 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Seung-Ho Shin ◽

Woong-Yang Park ◽

Donghyun Park

Keyword(s):

Dna Damage ◽

Deep Sequencing ◽

Error Rates ◽

Dna Lesions ◽

Sequencing Data ◽

Substitution Error ◽

Cytosine Deamination ◽

Free Dna ◽

Targeted Deep Sequencing ◽

Dna Fragment

Abstract Background Recently, a next-generation sequencing (NGS)-based method has been used for the successful detection of circulating tumor DNA (ctDNA) in various cancer types. Thus, the use of NGS on liquid biopsies will improve cancer diagnosis and prognosis. However, the low-allelic fraction of ctDNA poses a challenge for the sensitive and specific detection of tumor variants in cell-free DNA (cfDNA). To distinguish true variants from false positives, the characteristics of errors that occur during sample preparation and sequencing need to be elucidated. Methods We generated capture-based targeted deep sequencing data from plasma cfDNA and peripheral blood leucocyte (PBL) gDNA to profile background errors. To reveal cfDNA-associated DNA lesions, background error profiles from two sample types were compared in each nucleotide substitution class. Results In this study, we determined the prevalence of single nucleotide substitutions in cfDNA sequencing data to identify DNA damage preferentially associated with cfDNA. On comparing sequencing errors between cfDNA and cellular genomic DNA (gDNA), we observed that the total substitution error rates in cfDNA were significantly higher than those in gDNA. When the substitution errors were divided into 12 substitution error classes, C:G>T:A substitution errors constituted the largest difference between cfDNA and gDNA samples. When the substitution error rates were estimated based on the location of DNA-fragment substitutions, the differences in error rates of most substitution classes between cfDNA and gDNA samples were observed only at the ends of the DNA fragments. In contrast, C:G>T:A substitution errors in the cfDNA samples were not particularly associated with DNA-fragment ends. All observations were verified in an independent dataset. Conclusions Our data suggested that cytosine deamination increased in cfDNA compared to that in cellular gDNA. Such an observation might be due to the attenuation of DNA damage repair before the release of cfDNA and/or the accumulation of cytosine deamination after it. These findings can contribute to a better understanding of cfDNA-associated DNA damage, which will enable the accurate analysis of somatic variants present in cfDNA at an extremely low frequency.

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