Early detection and stratification of lung cancer aided by a cost-effective assay targeting circulating tumor DNA (ctDNA) methylation.

BACKGROUND: Detection of lung cancer at earlier stage can greatly improve patient survival. We aim to develop, validate, and implement a cost-effective ctDNA-methylation-based plasma test to aid lung cancer early detection. METHODS: Case-control studies were designed to select the most relevant markers to lung cancer. Patients with lung cancer or benign lung disease and healthy individuals were recruited from different clinical centers. A multi-locus qPCR assay, LunaCAM, was developed for lung cancer alertness by ctDNA methylation. Two LunaCAM models were built for screening (-S) or diagnostic aid (-D) to favor sensitivity or specificity, respectively. The performance of the models was validated for different intended uses in clinics. RESULTS: Profiling DNA methylation on 429 plasma samples including 209 lung cancer, 123 benign diseases and 97 healthy participants identified the top markers that detected lung cancer from benign diseases and healthy with an AUC of 0.85 and 0.95, respectively. The most effective methylation markers were verified individually in 40 tissues and 169 plasma samples to develop LunaCAM assay. Two models corresponding to different intended uses were trained with 513 plasma samples, and validated with an independent collection of 172 plasma samples. In validation, LunaCAM-S model achieved an AUC of 0.90 (95% CI: 0.88-0.94) between lung cancer and healthy individuals, whereas LunaCAM-D model stratified lung cancer from benign pulmonary diseases with an AUC of 0.81 (95% CI: 0.78-0.86). When implemented sequentially in the validation set, LunaCAM-S enables to identify 58 patients of lung cancer (90.6% sensitivity), followed by LunaCAM-D to remove 20 patients with no evidence of cancer (83.3% specificity). LunaCAM-D significantly outperformed the blood test of carcinoembryonic antigen (CEA), and the combined model can further improve the predictive power for lung cancer to an overall AUC of 0.86. CONCLUSIONS: We developed two different models by ctDNA methylation assay to sensitively detect early-stage lung cancer or specifically classify lung benign diseases. Implemented at different clinical settings, LunaCAM models has a potential to provide a facile and inexpensive avenue for early screening and diagnostic aids for lung cancer.

Molecular analysis of cyst fluids improves the diagnostic accuracy of pre-operative assessment of pancreatic cystic lesions.

Pancreatic cystic lesions (PCL) are increasingly diagnosed. Endoscopic ultrasound fine-needle aspiration (EUS-FNA) cytology is often used for diagnostic confirmation but can be inconclusive. In this study, the role of molecular analyses in the pre-operative diagnostics of PCL is evaluated. Targeted Next Generation Sequencing (NGS) applied on cytology smears was retrospectively evaluated in a cohort of 37 resected PCL. Usefulness of NGS on fresh cyst fluids was tested in a prospective cohort of patients with newly diagnosed PCL (n = 71). In the retrospective cohort, cytology plus NGS displayed higher sensitivity (94.1% vs. 87.1%) and specificity (100% vs. 50%) than cytology alone for the detection of mucinous neoplasms. In the prospective cohort, sensitivity and specificity of conventional cytology alone were 54.2% and 100% for the detection of mucinous neoplasia and 50.0% and 100% for the detection of high-grade dysplasia, respectively. Adding NGS, all lesions which underwent histopathologic verification (12/71, 17%) could be classified without false positive or false negative results regarding the detection of mucinous neoplasm so far. NGS analysis of cfDNA in PCL fluids is feasible and can increase diagnostic accuracy in the detection of mucinous neoplasms compared to cytology alone. However, algorithms for the detection of high-risk lesions need further improvement.

Developing a Low-Cost, Simple-to-Use Electrochemical Sensor for the Detection of Circulating Tumour DNA in Human Fluids.

It is well-known that two major issues, preventing improved outcomes from cancer are late diagnosis and the evolution of drug resistance during chemotherapy, therefore technologies that address these issues can have a transformative effect on healthcare workflows. In this work we present a simple, low-cost DNA biosensor that was developed specifically to detect mutations in a key oncogene (KRAS). The sensor employed was a screen-printed array of carbon electrodes, used to perform parallel measurements of DNA hybridisation. A DNA amplification reaction was developed with primers for mutant and wild type KRAS sequences which amplified target sequences from representative clinical samples to detectable levels in as few as twenty cycles. High levels of sensitivity were demonstrated alongside a clear exemplar of assay specificity by showing the mutant KRAS sequence was detectable against a significant background of wild type DNA following amplification and hybridisation on the sensor surface. The time to result was found to be 3.5 h with considerable potential for optimisation through assay integration. This quick and versatile biosensor has the potential to be deployed in a low-cost, point-of-care test where patients can be screened either for early diagnosis purposes or monitoring of response to therapy.

Early Assessment of Molecular Progression and Response by Whole-genome Circulating Tumor DNA in Advanced Solid Tumors.

Treatment response assessment for patients with advanced solid tumors is complex and existing methods require greater precision. Current guidelines rely on imaging, which has known limitations, including the time required to show a deterministic change in target lesions. Serial changes in whole-genome (WG) circulating tumor DNA (ctDNA) were used to assess response or resistance to treatment early in the treatment course. Ninety-six patients with advanced cancer were prospectively enrolled (91 analyzed and 5 excluded), and blood was collected before and after initiation of a new, systemic treatment. Plasma cell-free DNA libraries were prepared for either WG or WG bisulfite sequencing. Longitudinal changes in the fraction of ctDNA were quantified to retrospectively identify molecular progression (MP) or major molecular response (MMR). Study endpoints were concordance with first follow-up imaging (FFUI) and stratification of progression-free survival (PFS) and overall survival (OS). Patients with MP (n = 13) had significantly shorter PFS (median 62 days vs. 310 days) and OS (255 days vs. not reached). Sensitivity for MP to identify clinical progression was 54% and specificity was 100%. MP calls were from samples taken a median of 28 days into treatment and 39 days before FFUI. Patients with MMR (n = 27) had significantly longer PFS and OS compared with those with neither call (n = 51). These results demonstrated that ctDNA changes early after treatment initiation inform response to treatment and correlate with long-term clinical outcomes. Once validated, molecular response assessment can enable early treatment change minimizing side effects and costs associated with additional cycles of ineffective treatment.

Monitoring tumour burden and therapeutic response through analysis of circulating tumour DNA and extracellular RNA in multiple myeloma patients.

Monitoring tumour burden and therapeutic response through analyses of circulating cell-free tumour DNA (ctDNA) and extracellular RNA (exRNA) in multiple myeloma (MM) patients were performed in a Phase Ib trial of 24 relapsed/refractory patients receiving oral azacitidine in combination with lenalidomide and dexamethasone. Mutational characterisation of paired BM and PL samples at study entry identified that patients with a higher number of mutations or a higher mutational fractional abundance in PL had significantly shorter overall survival (OS) (p = 0.005 and p = 0.018, respectively). A decrease in ctDNA levels at day 5 of cycle 1 of treatment (C1D5) correlated with superior progression-free survival (PFS) (p = 0.017). Evaluation of exRNA transcripts of candidate biomarkers indicated that high CRBN levels coupled with low levels of SPARC at baseline were associated with shorter OS (p = 0.000003). IKZF1 fold-change <0.05 at C1D5 was associated with shorter PFS (p = 0.0051) and OS (p = 0.0001). Furthermore, patients with high baseline CRBN coupled with low fold-change at C1D5 were at the highest risk of progression (p = 0.0001). In conclusion, this exploratory analysis has provided the first demonstration in MM of ctDNA for predicting disease outcome and of the utility of exRNA as a biomarker of therapeutic response.

Resectable lung lesions malignancy assessment and cancer detection by ultra-deep sequencing of targeted gene mutations in plasma cell-free DNA.

BACKGROUND: Early detection of lung cancer to allow curative treatment remains challenging. Cell-free circulating tumour (ct) DNA (ctDNA) analysis may aid in malignancy assessment and early cancer diagnosis of lung nodules found in screening imagery. METHODS: The multicentre clinical study enrolled 192 patients with operable occupying lung diseases. Plasma ctDNA, white cell count genomic DNA (gDNA) and tumour tissue gDNA of each patient were analysed by ultra-deep sequencing to an average of 35 000× of the coding regions of 65 lung cancer-related genes. RESULTS: The cohort consists of a quarter of benign lung diseases and three quarters of cancer patients with all histopathology subtypes. 64% of the cancer patients are at stage I. Gene mutations detection in tissue gDNA and plasma ctDNA results in a sensitivity of 91% and specificity of 88%. When ctDNA assay was used as the test, the sensitivity was 69% and specificity 96%. As for the lung cancer patients, the assay detected 63%, 83%, 94% and 100%, for stages I, II, III and IV, respectively. In a linear discriminant analysis, combination of ctDNA, patient age and a panel of serum biomarkers boosted the overall sensitivity to 80% at a specificity of 99%. 29 out of the 65 genes harboured mutations in the patients with lung cancer with the largest number found in TP53 (30% plasma and 62% tumour tissue samples) and EGFR (20% and 40%, respectively). CONCLUSION: Plasma ctDNA was analysed in lung nodule assessment and early cancer detection, while an algorithm combining clinical information enhanced the test performance. TRIAL REGISTRATION NUMBER: NCT03081741.

Clinical determinants for successful circulating tumor DNA analysis in prostate cancer.

BACKGROUND: Plasma-based cell-free DNA is an attractive biospecimen for assessing somatic mutations due to minimally-invasive real-time sampling. However, next generation sequencing (NGS) of cell-free DNA (cfDNA) may not be appropriate for all patients with advanced prostate cancer (PC). METHODS: Blood was obtained from advanced PC patients for plasma-based sequencing. UW-OncoPlex, a ∼2 Mb multi-gene NGS panel performed in the CLIA/CAP environment, was optimized for detecting cfDNA mutations. Tumor tissue and germline samples were sequenced for comparative analyses. Multivariate logistic regression was performed to determine the clinical characteristic associated with the successful detection of somatic cfDNA alterations (ie detection of at least one clearly somatic PC mutation). RESULTS: Plasma for cfDNA sequencing was obtained from 93 PC patients along with tumor tissue (N = 67) and germline (N = 93) controls. We included data from 76 patients (72 prostate adenocarcinoma; 4 variant histology PC) in the analysis. Somatic DNA aberrations were detected in 34 cfDNA samples from patients with prostate adenocarcinoma. High PSA level, high tumor volume, and castration-resistance were significantly associated with successful detection of somatic cfDNA alterations. Among samples with somatic mutations detected, the cfDNA assay detected 93/102 (91%) alterations found in tumor tissue, yielding a clustering-corrected sensitivity of 92% (95% confidence interval 88-97%). All germline pathogenic variants present in lymphocyte DNA were also detected in cfDNA (N = 12). Somatic mutations from cfDNA were detected in 30/33 (93%) instances when PSA was >10 ng/mL. CONCLUSIONS: Disease burden, including a PSA >10 ng/mL, is strongly associated with detecting somatic mutations from cfDNA specimens.

[Circulating tumor DNA assessment for gynaecological cancers management]. / Apports de l'ADN tumoral circulant dans la compréhension et la prise en charge des carcinomes d'origine gynécologique.

Gynaecological cancers are frequent, with more than 16,000 cases per year in France for 6500 deaths. Few improvements in diagnostic methods, prognostic tools, and therapeutic strategies have occurred in the last two decades. Tumour genomic analyses from, at least in part, the Cancer Genome Atlas have identified some of the molecular alterations involved in gynaecological tumours growth and spreading. However, these data remain incomplete and have not led to dramatic changes in the clinical management of our patients. Moreover, they require invasive samples that are not suitable to objectives like screening/early diagnosis, assessment of treatment efficacy, monitoring of residual disease or early diagnosis of relapse. In the last years, the analysis of circulating tumour biomarkers (also called "liquid biopsies") based on tumour cells (circulating tumour cells) or tumour nucleotides (circulating DNA or RNA) has been massively explored through various indications, platforms, objectives; data related to circulating tumour DNA being the most important in terms of number of publications and interest for clinical practice. This review aims to describe the methods of analysis as well as the observations from the analysis of circulating tumour DNA in gynaecological tumours, from screening/early diagnosis to the adaptation of treatment for advanced stages, through choice of treatments and monitoring of subclinical disease.

Assessment of circulating tumor DNA in pediatric solid tumors: The promise of liquid biopsies.

Circulating tumor DNA can be detected in the blood and body fluids of patients using ultrasensitive technologies, which have the potential to improve cancer diagnosis, risk stratification, noninvasive tumor profiling, and tracking of treatment response and disease recurrence. As we begin to apply "liquid biopsy" strategies in children with cancer, it is important to tailor our efforts to the unique genomic features of these tumors and address the technical and logistical challenges of integrating biomarker testing. This article reviews the literature demonstrating the feasibility of applying liquid biopsy to pediatric solid malignancies and suggests new directions for future studies.

Duplex Proximity Sequencing (Pro-Seq): A method to improve DNA sequencing accuracy without the cost of molecular barcoding redundancy.

A challenge in the clinical adoption of cell-free DNA (cfDNA) liquid biopsies for cancer care is their high cost compared to potential reimbursement. The most common approach used in liquid biopsies to achieve high specificity detection of circulating tumor DNA (ctDNA) among a large background of normal cfDNA is to attach molecular barcodes to each DNA template, amplify it, and then sequence it many times to reach a low-error consensus. In applications where the highest possible specificity is required, error rate can be lowered further by independently detecting the sequences of both strands of the starting cfDNA. While effective in error reduction, the additional sequencing redundancy required by such barcoding methods can increase the cost of sequencing up to 100-fold over standard next-generation sequencing (NGS) of equivalent depth. We present a novel library construction and analysis method for NGS that achieves comparable performance to the best barcoding methods, but without the increase in sequencing and subsequent sequencing cost. Named Proximity-Sequencing (Pro-Seq), the method merges multiple copies of each template into a single sequencing read by physically linking the molecular copies so they seed a single sequencing cluster. Since multiple DNA copies of the same template are compared for consensus within the same cluster, sequencing accuracy is improved without the use of redundant reads. Additionally, it is possible to represent both senses of the starting duplex in a single cluster. The resulting workflow is simple, and can be completed by a single technician in a work day with minimal hands on time. Using both cfDNA and cell line DNA, we report the average per-mutation detection threshold and per-base analytical specificity to be 0.003% and >99.9997% respectively, demonstrating that Pro-Seq is among the highest performing liquid biopsy technologies in terms of both sensitivity and specificity, but with greatly reduced sequencing costs compared to existing methods of comparable accuracy.

Seminal Cell-Free DNA Assessment as a Novel Prostate Cancer Biomarker.

Cell-free DNA (cfDNA) includes circulating DNA fragments, which can be obtained from different human biological samples. cfDNA originates either from apoptotic and/or necrotic cells or is actively secreted by cancer cells. As yet, a quantification and size distribution assessment of seminal plasma cfDNA from prostate cancer patients has never been assessed. To discover a novel, sensitive, non-invasive biomarker of prostate cancer, through the fluorometric quantification and the electrophoretic analysis of seminal cfDNA in prostate cancer patients compared to healthy individuals. The concentration of seminal plasma cfDNA in prostate cancer patients was 2243.67 ± 1758 ng/µl, compared to 57.7 ± 4.8 ng/µl in healthy individuals (p < 0.05). Electrophoresis sites distribution patterns were different; ladder fragmentation was associated with prostate cancer patients and apoptotic electrophoretic fragmentation with healthy individuals. Human seminal fluid can be a valuable source of cfDNA in the setting of liquid biopsy procedures for the identification of novel oncological biomarkers. Seminal plasma cfDNA in prostate cancer patients is significantly more concentrated than that of age-matched, healthy controls. Fluorometric measurement and electrophoretic assessment allow a reliable quantification and characterization of seminal plasma cfDNA, which can be used routinely in prostate cancer screening programs.

Results of the first external quality assessment scheme (EQA) for isolation and analysis of circulating tumour DNA (ctDNA).

BACKGROUND: Circulating tumour DNA (ctDNA) is considered to have a high potential for future management of malignancies. This pilot external quality assessment (EQA) scheme aimed to address issues of analytical quality in this new area of laboratory diagnostics. METHODS: The EQA scheme consisted of three 2-mL EDTA-plasma samples spiked with fragmented genomic DNA with a mutant allele frequency ranging from 0% to 10% dedicated to the analysis of nine known sequence variations in KRAS codon 12/13 and of BRAF V600E. Laboratories reported: (1) time elapsed for processing, (2) storage temperatures, (3) methods for extraction and quantification, (4) genotyping methodologies and (5) results. RESULTS: Specimens were sent to 42 laboratories from 10 European countries; 72.3% reported to isolate cell-free DNA (cfDNA) manually, 62.5% used the entire plasma volume for cfDNA isolation and 38.5% used >10% of cfDNA extracted for downstream genotyping. Of the methods used for quantification, PicoGreen demonstrated the lowest coefficient of variation (33.7%). For genotyping, 11 different methods were reported with the highest error rate observed for Sanger sequencing and the lowest for highly sensitive approaches like digital PCR. In total, 197 genotypes were determined with an overall error rate of 6.09%. CONCLUSIONS: This pilot EQA scheme illustrates the current variability in multiple phases of cfDNA processing and analysis of ctDNA resulting in an overall error rate of 6.09%. The areas with the greatest variance and clinical impact included specimen volume, cfDNA quantification method, and preference of genotyping platform. Regarding quality assurance, there is an urgent need for harmonisation of procedures and workflows.

Next-generation sequencing (NGS) of cell-free circulating tumor DNA and tumor tissue in patients with advanced urothelial cancer: a pilot assessment of concordance.

BACKGROUND: Advances in cancer genome sequencing have led to the development of various next-generation sequencing (NGS) platforms. There is paucity of data regarding concordance of different NGS tests carried out in the same patient. METHODS: Here, we report a pilot analysis of 22 patients with metastatic urinary tract cancer and available NGS data from paired tumor tissue [FoundationOne (F1)] and cell-free circulating tumor DNA (ctDNA) [Guardant360 (G360)]. RESULTS: The median time between the diagnosis of stage IV disease and the first genomic test was 23.5 days (0-767), after a median number of 0 (0-3) prior systemic lines of treatment of advanced disease. Most frequent genomic alterations (GA) were found in the genes TP53 (50.0%), TERT promoter (36.3%); ARID1 (29.5%); FGFR2/3 (20.5%), PIK3CA (20.5%) and ERBB2 (18.2%). While we identified GA in both tests, the overall concordance between the two platforms was only 16.4% (0%-50%), and 17.1% (0%-50%) for those patients (n = 6) with both tests conducted around the same time (median difference = 36 days). On the contrary, in the subgroup of patients (n = 5) with repeated NGS in ctDNA after a median of 1 systemic therapy between the two tests, average concordance was 55.5% (12.1%-100.0%). Tumor tissue mutational burden was significantly associated with number of GA in G360 report (P < 0.001), number of known GA (P = 0.009) and number of variants of unknown significance (VUS) in F1 report (P < 0.001), and with total number of GA (non-VUS and VUS) in F1 report (P < 0.001). CONCLUSIONS: This study suggests a significant discordance between clinically available NGS panels in advanced urothelial cancer, even when collected around the same time. There is a need for better understanding of these two possibly complementary NGS platforms for better integration into clinical practice.