scholarly journals Cell-free DNA captures tumor heterogeneity and driver alterations in rapid autopsies with pre-treated metastatic cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bernard Pereira ◽  
Christopher T. Chen ◽  
Lipika Goyal ◽  
Charlotte Walmsley ◽  
Christopher J. Pinto ◽  
...  
Keyword(s):  
Metastatic Cancer ◽  
Point Mutations ◽  
Tumor Biopsy ◽  
Cell Free Dna ◽  
Tissue Samples ◽  
Metastatic Lesions ◽  
Free Dna ◽  
Somatic Alterations ◽  
Rapid Autopsy ◽  
Single Tumor

AbstractIn patients with metastatic cancer, spatial heterogeneity of somatic alterations may lead to incomplete assessment of a cancer’s mutational profile when analyzing a single tumor biopsy. In this study, we perform sequencing of cell-free DNA (cfDNA) and distinct metastatic tissue samples from ten rapid autopsy cases with pre-treated metastatic cancer. We show that levels of heterogeneity in genetic biomarkers vary between patients but that gene expression signatures representative of the tumor microenvironment are more consistent. Across nine patients with plasma samples available, we are able to detect 62/62 truncal and 47/121 non-truncal point mutations in cfDNA. We observe that mutation clonality in cfDNA is correlated with the number of metastatic lesions in which the mutation is detected and use this result to derive a clonality threshold to classify truncal and non-truncal driver alterations with reasonable specificity. In contrast, mutation truncality is more often incorrectly assigned when studying single tissue samples. Our results demonstrate the utility of a single cfDNA sample relative to that of single tissue samples when treating patients with metastatic cancer.

scholarly journals Evaluation of Merkel Cell Polyomavirus DNA in Tissue Samples from Italian Patients with Diagnosis of MCC

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 61
Author(s):  
Carla Prezioso ◽  
Raffaella Carletti ◽  
Francisco Obregon ◽  
Francesca Piacentini ◽  
Anna Maria Manicone ◽  
...  
Keyword(s):  
Point Mutations ◽  
Merkel Cell Polyomavirus ◽  
Merkel Cell ◽  
Tissue Samples ◽  
Metastatic Lesions ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Metastatic Sites ◽  
Archived Specimens ◽  
Coding Control

Because the incidence of Merkel cell carcinoma (MCC) has increased significantly during the last 10 years and it is recognized that Merkel cell polyomavirus (MCPyV) and ultraviolet (UV) radiation represent two different etiological inputs sharing clinical, histopathological, and prognostic similar features, although with different prognosis, this study investigated the detection of MCPyV in skin and lymph nodes with histological diagnosis of MCC. Formalin-fixed paraffin-embedded tissue (FFPE) were retrieved from archived specimens and MCPyV non-coding control region (NCCR) and viral capsid protein 1 (VP1) sequences were amplified and sequenced. Results provide an interesting observation concerning the discrepancy between the MCPyV DNA status in primary and metastatic sites: in fact, in all cases in which primary and metastatic lesions were investigated, MCPyV DNA was detected only in the primary lesions. Our data further support the “hit-and-run” theory, also proposed by other authors, and may lead to speculation that in some MCCs the virus is only necessary for the process of tumor initiation and that further mutations may render the tumor independent from the virus. Few point mutations were detected in the NCCR and only silent mutations were observed in the VP1 sequence compared to the MCPyV MCC350 isolate. To unequivocally establish a role of MCPyV in malignancies, additional well-controlled investigations are required, and larger cohorts should be examined.

scholarly journals Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
A. Rose Brannon ◽  
Gowtham Jayakumaran ◽  
Monica Diosdado ◽  
Juber Patel ◽  
Anna Razumova ◽  
...  
Keyword(s):  
Cancer Patients ◽  
De Novo ◽  
Low Frequency ◽  
High Sensitivity ◽  
Clinical Analysis ◽  
De Novo Mutation ◽  
Cell Free Dna ◽  
Free Dna ◽  
Somatic Alterations ◽  
Mutation Profiling

AbstractCirculating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

scholarly journals Rapid response of stage IV colorectal cancer with APC/TP53/KRAS mutations to FOLFIRI and Bevacizumab combination chemotherapy: a case report of use of liquid biopsy

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Alexander Hendricks ◽  
Philip Rosenstiel ◽  
Sebastian Hinz ◽  
Greta Burmeister ◽  
Christoph Röcken ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Treatment Response ◽  
Liquid Biopsy ◽  
Metastatic Cancer ◽  
Stage Iv ◽  
Liquid Biopsies ◽  
Kras Mutations ◽  
Cell Free Dna ◽  
Stage Iv Colorectal Cancer ◽  
Free Dna

Abstract Background Liquid biopsies of blood plasma cell free DNA can be used to monitor treatment response and potentially detect mutations that are present in resistant clones in metastatic cancer patients. Case presentation In our non-interventional liquid biopsy study, a male patient in his fifties diagnosed with stage IV colorectal cancer and polytope liver metastases rapidly progressed after completing chemotherapy and deceased 8 months after diagnosis. Retrospective cell free DNA testing showed that the APC/TP53/KRAS major clone responded quickly after 3 cycles of FOLFIRI + Bevacizumab. Retrospective exome sequencing of pre-chemotherapy and post-chemotherapy tissue samples including metastases confirmed that the APC/TP53/KRAS and other major clonal mutations (GPR50, SLC5A, ZIC3, SF3A1 and others) were present in all samples. After the last chemotherapy cycle, CT imaging, CEA and CA19–9 markers validated the cfDNA findings of treatment response. However, 5 weeks later, the tumour had rapidly progressed. Conclusion As FOLFIRI+Bevacizumab has recently also been associated with sustained complete remission in a APC/TP53/KRAS triple-mutated patient, these driver genes should be tested and monitored in a more in-depth manner in future patients. Patients with metastatic disease should be monitored more closely during and after chemotherapy, ideally using cfDNA.

scholarly journals Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA

2018 ◽  
Vol 64 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Amin Aalipour ◽  
Jonathan C Dudley ◽  
Seung-min Park ◽  
Surya Murty ◽  
Jacob J Chabon ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Nsclc Patient ◽  
Point Mutations ◽  
Growth Factor Receptor ◽  
Ease Of Use ◽  
Minor Allele ◽  
Detection Capability ◽  
New Paradigm ◽  
Cell Free Dna ◽  
Free Dna

Abstract BACKGROUND Cell-free DNA (cfDNA) diagnostics are emerging as a new paradigm of disease monitoring and therapy management. The clinical utility of these diagnostics is relatively limited by a low signal-to-noise ratio, such as with low allele frequency (AF) mutations in cancer. While enriching for rare alleles to increase their AF before sample analysis is one strategy that can greatly improve detection capability, current methods are limited in their generalizability, ease of use, and applicability to point mutations. METHODS Leveraging the robust single-base-pair specificity and generalizability of the CRISPR associated protein 9 (Cas9) system, we developed a deactivated Cas9 (dCas9)-based method of minor-allele enrichment capable of efficient single-target and multiplexed enrichment. The dCas9 protein was complexed with single guide RNAs targeted to mutations of interest and incubated with cfDNA samples containing mutant strands at low abundance. Mutation-bound dCas9 complexes were isolated, dissociated, and the captured DNA purified for downstream use. RESULTS Targeting the 3 most common epidermal growth factor receptor mutations (exon 19 deletion, T790M, L858R) found in non-small cell lung cancer (NSCLC), we achieved >20-fold increases in AF and detected mutations by use of qPCR at an AF of 0.1%. In a cohort of 18 NSCLC patient-derived cfDNA samples, our method enabled detection of 8 out of 13 mutations that were otherwise undetected by qPCR. CONCLUSIONS The dCas9 method provides an important application of the CRISPR/Cas9 system outside the realm of genome editing and can provide a step forward for the detection capability of cfDNA diagnostics.

scholarly journals Two‐point‐NGS analysis of cancer genes in cell‐free DNA of metastatic cancer patients

2020 ◽  
Vol 9 (6) ◽  
pp. 2052-2061
Author(s):  
Maria Palmieri ◽  
Margherita Baldassarri ◽  
Francesca Fava ◽  
Alessandra Fabbiani ◽  
Elisa Gelli ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Metastatic Cancer ◽  
Cancer Genes ◽  
Cell Free Dna ◽  
Free Dna

scholarly journals Bile-Based Cell-Free DNA Analysis Is a Reliable Diagnostic Tool in Pancreatobiliary Cancer

Cancers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 39
Author(s):  
Caroline Driescher ◽  
Katharina Fuchs ◽  
Lena Haeberle ◽  
Wolfgang Goering ◽  
Lisa Frohn ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Invasive Disease ◽  
Primary Diagnosis ◽  
Ductal Adenocarcinoma ◽  
Extrahepatic Cholangiocarcinoma ◽  
Cell Free Dna ◽  
Tissue Samples ◽  
Invasive Approach ◽  
Less Invasive ◽  
Free Dna

Currently available serum biomarkers for pancreatobiliary cancers lack sensitivity and specificity and ultimate diagnosis still requires invasive procedures for histological confirmation. The detection of tumor-specific genetic aberrations with utilization of cell free DNA (cfDNA) is a less invasive approach than traditional tissue biopsies; however, it has not been implemented into clinical routine. In this study, we investigated bile as a liquid biopsy source in pancreatobiliary cancers and compared its potential as cell-free DNA source to plasma. Blood (n = 37) and bile (n = 21) samples were collected from patients affected by pancreatic ductal adenocarcinoma (PDAC) and extrahepatic cholangiocarcinoma (CCA) or with non-malignant biliary obstructions (blood n = 16; bile n = 21). Panel-based next generation sequencing (NGS) and digital droplet PCR (ddPCR) were applied for tumor mutation profiling. NGS results from matched tumor tissues (n = 29) served as comparison. Sequencing of cfDNA from bile resulted in detection of 96.2% of the pathogenic tumor mutations found in matched tissue samples. On the other hand, only 31.6% of pathogenic tumor mutations found in tissue could be detected in plasma. In a direct comparison, only half of the mutations detected in bile cfDNA were concordantly detected in plasma from the same patients. Panel NGS and ddPCR displayed comparable sensitivity. In conclusion, bile is a suitable source of cfDNA for the diagnosis of pancreatobiliary cancer and performs more reliably than plasma. Although primary diagnosis still requires histologic confirmation, bile-derived cfDNA could offer an alternative if tissue sampling is not feasible and might allow less invasive disease monitoring.

scholarly journals Detection of CTNNB1 Hotspot Mutations in Cell-Free DNA from the Urine of Hepatocellular Carcinoma Patients

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1475
Author(s):  
Selena Y. Lin ◽  
Ting-Tsung Chang ◽  
Jamin D. Steffen ◽  
Sitong Chen ◽  
Surbhi Jain ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Mutational Analysis ◽  
Residual Disease ◽  
Tumor Resection ◽  
Beta Catenin ◽  
Cell Free Dna ◽  
Tissue Samples ◽  
Free Dna ◽  
Minimum Residual ◽  
Hcc Recurrence

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. The beta-catenin gene, CTNNB1, is among the most frequently mutated in HCC tissues. However, mutational analysis of HCC tumors is hampered by the difficulty of obtaining tissue samples using traditional biopsy. Here, we explored the feasibility of detecting tumor-derived CTNNB1 mutations in cell-free DNA (cfDNA) extracted from the urine of HCC patients. Using a short amplicon qPCR assay targeting HCC mutational hotspot CTNNB1 codons 32–37 (exon 3), we detected CTNNB1 mutations in 25% (18/73) of HCC tissues and 24% (15/62) of pre-operative HCC urine samples in two independent cohorts. Among the CTNNB1-mutation-positive patients with available matched pre- and post-operative urine (n = 13), nine showed apparent elimination (n = 7) or severalfold reduction (n = 2) of the mutation in urine following tumor resection. Four of the seven patients with no detectable mutations in postoperative urine remained recurrence-free within five years after surgery. In contrast, all six patients with mutation-positive in post-operative urine recurred, including the two with reduced mutation levels. This is the first report of association between the presence of CTNNB1 mutations in pre- and post-operative urine cfDNA and HCC recurrence with implications for minimum residual disease detection.

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