OS1.4 Liquid biopsy of the CSF in a series of GBM patients: preliminary results

Abstract BACKGROUND Liquid biopsy (LB) by cerebrospinal fluid (CSF) can be useful to identify circulating tumour DNA (ctDNA), thus offering information about the heterogeneity of the neoplastic genome. The aim of our study is to assess the effectiveness of LB of the CSF in detecting ctDNA which mirrors the genetic profile of the tumoural tissue, and to investigate the clinical and radiological aspects influencing the availability of ctDNA. MATERIAL AND METHODS Tumoral tissue and CSF samples of 13 GBM patients undergoing surgery was collected. CSF was withdrawn from the very proximity of the tumoural surface before the excision. DNA extracted from tissue samples was analysed by qPCR to identify typical genetic alterations such as copy number variations (EGFR, PDGFRA, CDK4, MDM2, CDKN2A), and point mutations (TP53, PTEN, IDH, NRAS, PI3K1, pTERT). CtDNA extracted from CSF was analysed by droplet digital PCR to assess the presence of the alterations found in the matching tissue. Both contrast-enhanced (CE) and FLAIR volumes of the lesions were measured in the pre-surgical MRI. Linear and logarithmic regressions were employed for the statistical analysis. RESULTS From June 2016 to February 2017 we prospectively collected 13 GBM patients. Median age was 73 years. All lesions showed CE at the MRI; other radiological findings included necrosis (84.6%), oedema (76.9%), cortical, ventricular or meningeal involvement (76.9%, 30.8%, and 15.4%). Median volumes of CE and FLAIR lesions were 28.6 and 25.5 cm3, with a median FLAIR/CE ratio of 72.9. Surgery was subtotal (<95%) in all patients. All GBM tissues were tested for the following alterations: EGFR, PDGFRA, CDK4, MDM2, CDKN2A; 76.9% were tested for TP53, PTEN, and IDH mutations; 38.5% for NRAS and pTERT mutations; 30.8% for PI3KR1 mutation. MGMT methylation was assessed in 12 cases (92.3%) and found in 7 (58.3%). Median CSF volume, ctDNA quantity and concentration were 0.45 mL, 59.64 ng, and 0.42 ng/μL. Processable DNA was found in 11 CSF specimens (84.6%), in 8 of which (61.5%) it carried the same alteration expressed by the tumoural cells of the matched tissue, while in 3 cases (23.1%) it seemed to have a different genetic profile; finally, in 2 cases it was not possible to detect any circulating DNA in the CSF. Preliminary data on 13 patients suggest that the ctDNA concentration in the CSF could be related to the FLAIR/CE ratio as measured in the MRI before surgery (p = 0.02). Other correlations between the molecular and the radiological features are still being exploring. CONCLUSION Our study confirms that LB of CSF can detect ctDNA carrying the same molecular profile harboured in the tumour. Therefore, it seems to be an accurate method to identify markers useful for the diagnosis and the monitoring of the disease. Additionally, our ongoing study is trying to demonstrate a potential correlation between radiological features of the tumour and availability of ctDNA in CSF.

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Acoustic array biochip combined with allele-specific PCR for multiple cancer mutation analysis in tissue and liquid biopsy

10.1101/2021.09.16.460590 ◽

2021 ◽

Author(s):

Nikoletta Naoumi ◽

Kleita Michaelidou ◽

George Papadakis ◽

Agapi E. Simaiaki ◽

Roman Fernandez ◽

...

Keyword(s):

Cost Effectiveness ◽

Liquid Biopsy ◽

Point Mutations ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Braf V600e ◽

Analytical Sensitivity ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

Regular screening of cancerous point mutations is of importance to cancer management and treatment selection. Although excellent techniques like next-generation sequencing and droplet digital PCR are available, these are still lacking in speed, simplicity and cost-effectiveness. Here a new approach is presented where allele-specific PCR (AS-PCR) is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) array biosensor for the amplification and detection, respectively, of cancer point mutations. For the proof-of-concept, the method was applied to the screening of the BRAF V600E and KRAS G12D mutations in spiked-in and clinical samples. Regarding the BRAF target, an analytical sensitivity of 0.01%, i.e., detection of 1 mutant copy of genomic DNA in an excess of 104 wild type molecules, was demonstrated; moreover, quantitative results during KRAS detection were obtained when an optimized assay was employed with a sensitivity of 0.05%. The assays were validated using tissue and plasma samples obtained from melanoma, colorectal and lung cancer patients. Results are in full agreement with Sanger sequencing and droplet digital PCR, demonstrating efficient detection of BRAF and KRAS mutations in samples having an allele frequency below 1%. The high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness and compatibility with routine work-flow, hold promise for the implementation of this AS-PCR/acoustic methodology in clinical oncology as a tool for tissue and liquid biopsy.

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Comprehensive Custom NGS Panel Validation for the Improvement of the Stratification of B-Acute Lymphoblastic Leukemia Patients

Journal of Personalized Medicine ◽

10.3390/jpm10030137 ◽

2020 ◽

Vol 10 (3) ◽

pp. 137

Author(s):

Adrián Montaño ◽

Jesús Hernández-Sánchez ◽

Maribel Forero-Castro ◽

María Matorra-Miguel ◽

Eva Lumbreras ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Wide Spectrum ◽

Lymphoblastic Leukemia ◽

Point Mutations ◽

Genetic Alterations ◽

Copy Number Variations ◽

Single Step ◽

Fusion Genes ◽

Single Nucleotide Variants ◽

Single Nucleotide

Background: B-acute lymphoblastic leukemia (B-ALL) is a hematological neoplasm of the stem lymphoid cell of the B lineage, characterized by the presence of genetic alterations closely related to the course of the disease. The number of alterations identified in these patients grows as studies of the disease progress, but in clinical practice, the conventional techniques frequently used are only capable of detecting the most common alterations. However, techniques, such as next-generation sequencing (NGS), are being implemented to detect a wide spectrum of new alterations that also include point mutations. Methods: In this study, we designed and validated a comprehensive custom NGS panel to detect the main genetic alterations present in the disease in a single step. For this purpose, 75 B-ALL diagnosis samples from patients previously characterized by standard-of-care diagnostic techniques were sequenced. Results: The use of the custom NGS panel allowed the correct detection of the main genetic alterations present in B-ALL patients, including the presence of an aneuploid clone in 14 of the samples and some of the recurrent fusion genes in 35 of the samples. The panel was also able to successfully detect a number of secondary alterations, such as single nucleotide variants (SNVs) and copy number variations (CNVs) in 66 and 46 of the samples analyzed, respectively, allowing for further refinement of the stratification of patients. The custom NGS panel could also detect alterations with a high level of sensitivity and reproducibility when the findings obtained by NGS were compared with those obtained from other conventional techniques. Conclusions: The use of this custom NGS panel allows us to quickly and efficiently detect the main genetic alterations present in B-ALL patients in a single assay (SNVs and insertions/deletions (INDELs), recurrent fusion genes, CNVs, aneuploidies, and single nucleotide polymorphisms (SNPs) associated with pharmacogenetics). The application of this panel would thus allow us to speed up and simplify the molecular diagnosis of patients, helping patient stratification and management.

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Comprehensive Genomic Analysis Reveals the Prognostic Role of LRRK2 Copy-Number Variations in Human Malignancies

Genes ◽

10.3390/genes11080846 ◽

2020 ◽

Vol 11 (8) ◽

pp. 846

Author(s):

Gianluca Lopez ◽

Giulia Lazzeri ◽

Alessandra Rappa ◽

Giuseppe Isimbaldi ◽

Fulvia Milena Cribiù ◽

...

Keyword(s):

Copy Number ◽

Prognostic Significance ◽

Point Mutations ◽

Genomic Analysis ◽

Genetic Alterations ◽

Copy Number Variations ◽

Cancer Development ◽

Future Research ◽

Gene Deletions ◽

Significant Difference

Genetic alterations of leucine-rich repeat kinase 2 (LRRK2), one of the most important contributors to familial Parkinson’s disease (PD), have been hypothesized to play a role in cancer development due to demographical and preclinical data. Here, we sought to define the prevalence and prognostic significance of LRRK2 somatic mutations across all types of human malignancies by querying the publicly available online genomic database cBioPortal. Ninety-six different studies with 14,041 cases were included in the analysis, and 761/14,041 (5.4%) showed genetic alterations in LRRK2. Among these, 585 (76.9%) were point mutations, indels or fusions, 168 (22.1%) were copy number variations (CNVs), and 8 (1.0%) showed both types of alterations. One case showed the somatic mutation R1441C. A significant difference in terms of overall survival (OS) was noted between cases harboring somatic LRRK2 whole deletions, amplifications, and CNV-unaltered cases (median OS: 20.09, 57.40, and 106.57 months, respectively; p = 0.0008). These results suggest that both LRRK2 amplifications and whole gene deletions could play a role in cancer development, paving the way for future research in terms of potential treatment with LRRK2 small molecule inhibitors for LRRK2-amplified cases.

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IDH-wild type glioblastomas featuring at least 30% giant cells are characterized by frequent RB1 and NF1 alterations and hypermutation

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01304-5 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Valeria Barresi ◽

Michele Simbolo ◽

Andrea Mafficini ◽

Maurizio Martini ◽

Martina Calicchia ◽

...

Keyword(s):

Giant Cells ◽

Genetic Alterations ◽

Copy Number Variations ◽

Molecular Profile ◽

Wild Type ◽

Mutational Burden ◽

Check Point Inhibitors ◽

Tumor Mutational Burden ◽

Molecular Landscape ◽

Promoter Mutations

AbstractGiant cell glioblastoma (GC-GBM) is a rare variant of IDH-wt GBM histologically characterized by the presence of numerous multinucleated giant cells and molecularly considered a hybrid between IDH-wt and IDH-mutant GBM. The lack of an objective definition, specifying the percentage of giant cells required for this diagnosis, may account for the absence of a definite molecular profile of this variant. This study aimed to clarify the molecular landscape of GC-GBM, exploring the mutations and copy number variations of 458 cancer-related genes, tumor mutational burden (TMB), and microsatellite instability (MSI) in 39 GBMs dichotomized into having 30–49% (15 cases) or ≥ 50% (24 cases) GCs. The type and prevalence of the genetic alterations in this series was not associated with the GCs content (< 50% or ≥ 50%). Most cases (82% and 51.2%) had impairment in TP53/MDM2 and PTEN/PI3K pathways, but a high proportion also featured TERT promoter mutations (61.5%) and RB1 (25.6%) or NF1 (25.6%) alterations. EGFR amplification was detected in 18% cases in association with a shorter overall survival (P = 0.004). Sixteen (41%) cases had a TMB > 10 mut/Mb, including two (5%) that harbored MSI and one with a POLE mutation. The frequency of RB1 and NF1 alterations and TMB counts were significantly higher compared to 567 IDH wild type (P < 0.0001; P = 0.0003; P < 0.0001) and 26 IDH-mutant (P < 0.0001; P = 0.0227; P < 0.0001) GBMs in the TCGA PanCancer Atlas cohort. These findings demonstrate that the molecular landscape of GBMs with at least 30% giant cells is dominated by the impairment of TP53/MDM2 and PTEN/PI3K pathways, and additionally characterized by frequent RB1 alterations and hypermutation and by EGFR amplification in more aggressive cases. The high frequency of hypermutated cases suggests that GC-GBMs might be candidates for immune check-point inhibitors clinical trials.

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Liquid Biopsy Detects Relapse Five Months Earlier than Regular Clinical Follow-Up and Guides Targeted Treatment in Breast Cancer

Case Reports in Oncological Medicine ◽

10.1155/2019/6545298 ◽

2019 ◽

Vol 2019 ◽

pp. 1-4

Author(s):

Fiona Tsui-Fen Cheng ◽

Nina Lapke ◽

Chin-Chu Wu ◽

Yen-Jung Lu ◽

Shu-Jen Chen ◽

...

Keyword(s):

Breast Cancer ◽

Early Detection ◽

Liquid Biopsy ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Genetic Alterations ◽

Targeted Treatment ◽

Breast Cancer Patients ◽

Ctdna Analysis

Genetic alterations in circulating tumor DNA (ctDNA) are an emerging biomarker for the early detection of relapse and have the potential to guide targeted treatment. ctDNA analysis is often performed by droplet digital PCR; however, next-generation sequencing (NGS) allows multigene testing without having to access a tumor sample to identify target alterations. Here, we report the case of a stage III hormone receptor-positive breast cancer patient who remained symptomless after receiving surgery and adjuvant chemotherapy. Liquid biopsy analysis by NGS revealed the presence of a ctDNA PIK3CA N345K mutation five months before the detection of relapse with multiple liver metastases by regular clinical follow-up. To date, clinical implications of the PIK3CA N345K variant remain insufficiently investigated; however, everolimus treatment resulted in the shrinkage of tumor lesions and decreased the levels of tumor markers. Four months after treatment initiation, a second ctDNA analysis suggested a relapse, and the patient clinically progressed after five months of everolimus therapy. This case report demonstrates the value of ctDNA analysis by NGS for the early detection of relapse in breast cancer patients. The study further indicates its usefulness for the choice of targeted treatments, suggesting that the variant PIK3CA N345K might be associated with everolimus sensitivity.

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Genetic Characterization of Cancer of Unknown Primary Using Liquid Biopsy Approaches

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.666156 ◽

2021 ◽

Vol 9 ◽

Author(s):

Noemi Laprovitera ◽

Irene Salamon ◽

Francesco Gelsomino ◽

Elisa Porcellini ◽

Mattia Riefolo ◽

...

Keyword(s):

Liquid Biopsy ◽

Tumor Tissue ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Genetic Alterations ◽

Unknown Primary ◽

Tumor Evolution ◽

Dismal Prognosis ◽

Ffpe Tumor ◽

Custom Panel

Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical–pathological investigations. CUP patients are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH® and DEPArrayTM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with Ampli1TM kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in APC gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. FGFR2 and CCNE1 gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in ARID1A gene (p.R1276∗) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in ASPM and SEPT9 genes and loss of FANCC. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs∗19) and a somatic mutation (p. A1487V) in ARID1A gene and a point mutation in FGFR2 gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes.

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Circulating tumor DNA is detectable in canine histiocytic sarcoma, oral malignant melanoma, and multicentric lymphoma

Scientific Reports ◽

10.1038/s41598-020-80332-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anaïs Prouteau ◽

Jérôme Alexandre Denis ◽

Pauline De Fornel ◽

Edouard Cadieu ◽

Thomas Derrien ◽

...

Keyword(s):

Residual Disease ◽

Specific Antigen ◽

Point Mutations ◽

Antigen Receptor ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Histiocytic Sarcoma ◽

Oncology Research ◽

Tumor Dna

AbstractCirculating tumor DNA (ctDNA) has become an attractive biomarker in human oncology, and its use may be informative in canine cancer. Thus, we used droplet digital PCR or PCR for antigen receptor rearrangement, to explore tumor-specific point mutations, copy number alterations, and chromosomal rearrangements in the plasma of cancer-affected dogs. We detected ctDNA in 21/23 (91.3%) of histiocytic sarcoma (HS), 2/8 (25%) of oral melanoma, and 12/13 (92.3%) of lymphoma cases. The utility of ctDNA in diagnosing HS was explored in 133 dogs, including 49 with HS, and the screening of recurrent PTPN11 mutations in plasma had a specificity of 98.8% and a sensitivity between 42.8 and 77% according to the clinical presentation of HS. Sensitivity was greater in visceral forms and especially related to pulmonary location. Follow-up of four dogs by targeting lymphoma-specific antigen receptor rearrangement in plasma showed that minimal residual disease detection was concordant with clinical evaluation and treatment response. Thus, our study shows that ctDNA is detectable in the plasma of cancer-affected dogs and is a promising biomarker for diagnosis and clinical follow-up. ctDNA detection appears to be useful in comparative oncology research due to growing interest in the study of natural canine tumors and exploration of new therapies.

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Virtual Fluorescence Color Channels by Selective Photobleaching in Digital PCR Applied to the Quantification of KRAS Point Mutations

Analytical Chemistry ◽

10.1021/acs.analchem.1c01488 ◽

2021 ◽

Author(s):

Franziska Schlenker ◽

Elena Kipf ◽

Nadine Borst ◽

Tobias Hutzenlaub ◽

Roland Zengerle ◽

...

Keyword(s):

Point Mutations ◽

Digital Pcr ◽

Fluorescence Color ◽

Color Channels

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The Liquid Biopsy for Lung Cancer: State of the Art, Limitations and Future Developments

Cancers ◽

10.3390/cancers13163923 ◽

2021 ◽

Vol 13 (16) ◽

pp. 3923

Author(s):

Daniel Di Capua ◽

Dara Bracken-Clarke ◽

Karine Ronan ◽

Anne-Marie Baird ◽

Stephen Finn

Keyword(s):

Lung Cancer ◽

Lung Carcinoma ◽

Targeted Therapies ◽

Liquid Biopsy ◽

Genetic Alterations ◽

Rapid Progression ◽

Liquid Biopsies ◽

Cell Lung Carcinoma ◽

Genetic Sequencing ◽

Tissue Biopsy

Lung cancer is a leading cause of cancer-related deaths, contributing to 18.4% of cancer deaths globally. Treatment of non-small cell lung carcinoma has seen rapid progression with targeted therapies tailored to specific genetic drivers. However, identifying genetic alterations can be difficult due to lack of tissue, inaccessible tumors and the risk of complications for the patient with serial tissue sampling. The liquid biopsy provides a minimally invasive method which can obtain circulating biomarkers shed from the tumor and could be a safer alternative to tissue biopsy. While tissue biopsy remains the gold standard, liquid biopsies could be very beneficial where serial sampling is required, such as monitoring disease progression or development of resistance mutations to current targeted therapies. Liquid biopsies also have a potential role in identifying patients at risk of relapse post treatment and as a component of future lung cancer screening protocols. Rapid developments have led to multiple platforms for isolating circulating tumor cells (CTCs) and detecting circulating tumor DNA (ctDNA); however, standardization is lacking, especially in lung carcinoma. Additionally, clonal hematopoiesis of uncertain clinical significance must be taken into consideration in genetic sequencing, as it introduces the potential for false positives. Various biomarkers have been investigated in liquid biopsies; however, in this review, we will concentrate on the current use of ctDNA and CTCs, focusing on the clinical relevance, current and possible future applications and limitations of each.

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A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

Genome Biology ◽

10.1186/s13059-021-02316-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Wendell Jones ◽

Binsheng Gong ◽

Natalia Novoradovskaya ◽

Dan Li ◽

Rebecca Kusko ◽

...

Keyword(s):

Quality Control ◽

Allele Frequency ◽

Liquid Biopsy ◽

Limit Of Detection ◽

Reference Sample ◽

Digital Pcr ◽

Detection Sensitivity ◽

Analytical Accuracy ◽

And Performance ◽

Reference Samples

Abstract Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

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