Background Analysis of circulating free DNA (cfDNA) is a promising tool for personalized management of colorectal cancer (CRC) patients. Untargeted cfDNA analysis using whole-genome sequencing (WGS) ...does not need a priori knowledge of the patient´s mutation profile. Methods Here we established LIquid biopsy Fragmentation, Epigenetic signature and Copy Number Alteration analysis (LIFE-CNA) using WGS with ~ 6x coverage for detection of circulating tumor DNA (ctDNA) in CRC patients as a marker for CRC detection and monitoring. Results We describe the analytical validity and a clinical proof-of-concept of LIFE-CNA using a total of 259 plasma samples collected from 50 patients with stage I-IV CRC and 61 healthy controls. To reliably distinguish CRC patients from healthy controls, we determined cutoffs for the detection of ctDNA based on global and regional cfDNA fragmentation patterns, transcriptionally active chromatin sites, and somatic copy number alterations. We further combined global and regional fragmentation pattern into a machine learning (ML) classifier to accurately predict ctDNA for cancer detection. By following individual patients throughout their course of disease, we show that LIFE-CNA enables the reliable prediction of response or resistance to treatment up to 3.5 months before commonly used CEA. Conclusion In summary, we developed and validated a sensitive and cost-effective method for untargeted ctDNA detection at diagnosis as well as for treatment monitoring of all CRC patients based on genetic as well as non-genetic tumor-specific cfDNA features. Thus, once sensitivity and specificity have been externally validated, LIFE-CNA has the potential to be implemented into clinical practice. To the best of our knowledge, this is the first study to consider multiple genetic and non-genetic cfDNA features in combination with ML classifiers and to evaluate their potential in both cancer detection and treatment monitoring. Trial registration DRKS00012890. Keywords: ctDNA, Colorectal cancer, Liquid biopsy, Whole-genome sequencing, Somatic copy number alterations, cfDNA fragmentation, Chromatin signatures
Liquid biopsy (LB) is a promising complement to tissue biopsy for detection of clinically relevant genetic variants in cancer and mosaic diseases. A combined workflow to enable parallel tissue and LB ...analysis is required to maximize diagnostic yield for patients.
We developed and validated a cost-efficient combined next-generation sequencing (NGS) workflow for both tissue and LB samples, and applied Duplex sequencing technology for highly accurate detection of low frequency variants in plasma. Clinically relevant cutoffs for variant reporting and quantification were established.
We investigated assay performance characteristics for very low amounts of clinically relevant variants. In plasma, the assay achieved 100% sensitivity and 92.3% positive predictive value (PPV) for single nucleotide variants (SNVs) and 91.7% sensitivity and 100% PPV for insertions and deletions (InDel) in clinically relevant hotspots with 0.5-5% variant allele frequencies (VAFs). We further established a cutoff for reporting variants (i.e. Limit of Blank, LOB) at 0.25% VAF and a cutoff for quantification (i.e. Limit of Quantification, LOQ) at 5% VAF in plasma for accurate clinical interpretation of analysis results. With our LB approach, we were able to identify the molecular cause of a clinically confirmed asymmetric overgrowth syndrome in a 10-year old child that would have remained undetected with tissue analysis as well as other molecular diagnostic approaches.
Our flexible and cost-efficient workflow allows analysis of both tissue and LB samples and provides clinically relevant cutoffs for variant reporting and precise quantification. Complementing tissue analysis by LB is likely to increase diagnostic yield for patients with molecular diseases.
Background: Liquid biopsy enables the non-invasive analysis of genetic tumor variants in circulating free DNA (cfDNA) in plasma. Accurate analytical validation of liquid biopsy NGS assays is required ...to detect variants with low variant allele frequencies (VAFs). Methods: Six types of commercial cfDNA reference materials and 42 patient samples were analyzed using a duplex-sequencing-based liquid biopsy NGS assay. Results: We comprehensively evaluated the similarity of commercial cfDNA reference materials to native cfDNA. We observed significant differences between the reference materials in terms of wet-lab and sequencing quality as well as background noise. No reference material resembled native cfDNA in all performance metrics investigated. Based on our results, we established guidelines for the selection of appropriate reference materials for the different steps in performance evaluation. The use of inappropriate materials and cutoffs could eventually lead to a lower sensitivity for variant detection. Conclusion: Careful consideration of commercial reference materials is required for performance evaluation of liquid biopsy NGS assays. While the similarity to native cfDNA aids in the development of experimental protocols, reference materials with well-defined variants are preferable for determining sensitivity and precision, which are essential for accurate clinical interpretation.
Circulating tumor DNA (ctDNA) is a promising liquid biopsy (LB) marker to support clinical decisions in precision medicine. For implementation into routine clinical practice, clinicians need precise ...ctDNA level cutoffs for reporting residual disease and monitoring tumor burden changes during therapy. We clinically validated the limit of blank (LOB) and the limit of quantification (LOQ) of assays for the clinically most relevant somatic variants
p.V600E and
p.G12/p.G13 in colorectal cancer (CRC) in a study cohort encompassing a total of 212 plasma samples. We prove that residual disease detection using the LOB as a clinically verified cutoff for ctDNA positivity is in concordance with clinical evidence of metastasis or recurrence. We further show that tumor burden changes during chemotherapy and the course of disease are correctly predicted using the LOQ as a cutoff for quantitative ctDNA changes. The high potential of LB using ctDNA for accurately predicting the course of disease was proven by direct comparison to the routinely used carcinoembryonic antigen (CEA) as well as the circulating free DNA (cfDNA) concentration. Our results show that LB using validated ctDNA assays outperforms CEA and cfDNA for residual disease detection and the prediction of tumor burden changes.
Depletion of tryptophan and the accumulation of tryptophan metabolites mediated by the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1), trigger immune cells to undergo apoptosis. ...However, cancer cells in the same microenvironment appear not to be affected. Mechanisms whereby cancer cells resist accelerated tryptophan degradation are not completely understood. We hypothesize that cancer cells co-opt IMPACT (the product of IMPrinted and AnCienT gene), to withstand periods of tryptophan deficiency.
A range of bioinformatic techniques including correlation and gene set variation analyses was applied to genomic datasets of cancer (The Cancer Genome Atlas) and normal (Genotype Tissue Expression Project) tissues to investigate IMPACT's role in cancer. Survival of IMPACT-overexpressing GL261 glioma cells and their wild type counterparts cultured in low tryptophan media was assessed using fluorescence microscopy and MTT bio-reduction assay. Expression of the Integrated Stress Response proteins was measured using Western blotting.
We found IMPACT to be upregulated and frequently amplified in a broad range of clinical cancers relative to their non-malignant tissue counterparts. In a subset of clinical cancers, high IMPACT expression associated with decreased activity of pathways and genes involved in stress response and with increased activity of translational regulation such as the mTOR pathway. Experimental studies using the GL261 glioma line showed that cells engineered to overexpress IMPACT, gained a survival advantage over wild-type lines when cultured under limiting tryptophan concentrations. No significant difference in the expression of proteins in the Integrated Stress Response pathway was detected in tryptophan-deprived GL261 IMPACT-overexpressors compared to that in wild-type cells. IMPACT-overexpressing GL261 cells but not their wild-type counterparts, showed marked enlargement of their nuclei and cytoplasmic area when stressed by tryptophan deprivation.
The bioinformatics data together with our laboratory studies, support the hypothesis that IMPACT mediates a protective mechanism allowing cancer cells to overcome microenvironmental stresses such as tryptophan deficiency.
Zusammenfassung
Patienten mit einem hereditären Tumorprädispositionssyndrom haben ein deutlich erhöhtes Tumorrisiko und erkranken oft schon im jungen Erwachsenenalter. Sie benötigen daher eine ...intensivierte Überwachung, um Tumoren bereits in einem frühen Stadium detektieren und behandeln zu können. Entsprechend werden bei Patienten mit einem Lynch-Syndrom, der häufigsten erblichen Darmkrebsprädisposition, regelmäßige Koloskopien zur Vorsorge empfohlen. Eine Liquid Biopsy ermöglicht die nicht- bzw. minimal-invasive Untersuchung von zirkulierenden Tumormarkern. Insbesondere die Analyse von zirkulierender Tumor-DNA (ctDNA) wird schon jetzt zur Therapiesteuerung von Tumorpatienten eingesetzt und ermöglicht darüber hinaus den Nachweis einer minimale Resterkrankung oder eines Rezidivs. Fortschritte im Bereich des hochsensitiven, nicht zielgerichteten Nachweises von ctDNA sind vielversprechend für die Früherkennung bei Patienten mit einer hereditären Tumorprädisposition. Hinsichtlich des Nachweises von Tumorerkrankungen im Frühstadium müssen diese Analyseverfahren jedoch noch verbessert und ihre Spezifität und Sensitivität in klinischen Studien bewertet werden. Die Herausforderung ist die sensitive Erkennung von Vorstufen, wie z. B. fortgeschrittenen Adenomen, um maligne Tumorerkrankungen vorzubeugen oder diese möglichst frühzeitig behandeln zu können.
Abstract
Background
Analysis of circulating tumor DNA (ctDNA) in plasma is a powerful approach to guide decisions in personalized cancer treatment. Given the low concentration of ctDNA in plasma, ...highly sensitive methods are required to reliably identify clinically relevant variants.
Methods
We evaluated the suitability of 5 droplet digital PCR (ddPCR) assays targeting KRAS, BRAF, and EGFR variants for ctDNA analysis in clinical use.
Results
We investigated assay performance characteristics for very low amounts of variants, showing that the assays had very low limits of blank (0% to 0.11% variant allele frequency, VAF) and limits of quantification (0.41% to 0.7% VAF). Nevertheless, striking differences in detection and quantification of low mutant VAFs between the 5 tested assays were observed, highlighting the need for assay-specific analytical validation. Besides in-depth evaluation, a guide for clinical interpretation of obtained VAFs in plasma was developed, depending on the limits of blank and limits of quantification values.
Conclusion
It is possible to provide comprehensive clinical reports on actionable variants, allowing minimal residual disease detection and treatment monitoring in liquid biopsy.
Abstract
A broad range of human malignancies overexpress an enzyme called indoleamine 2,3-dioxygenase 1 (IDO1) to suppress antitumor immunity which associates with poor patient prognosis. One ...mechanism whereby IDO1 activity suppresses the host’s immune cells is mediated by deprivation of the essential amino acid tryptophan (Trp) which raises an intriguing question. How do the cancer cells overcome low levels of the essential amino acid when the immune T-cells are triggered to self-destruct? A protein called IMPACT (product of IMPrinted gene with AnCienT domain) was reported to confer skin cells and neuronal cells increased survival in Trp-deprived environments. Based on these reports, we hypothesise that cancer cells hijack IMPACT to gain survival advantage in Trp-deprived environments. To obtain evidence for this hypothesis, we analysed publicly available genomics datasets and performed experimental assays.
Murine GL261 glioblastoma cells overexpressing a full-length mouse IMPACT gene (GL261-IMPACT) were generated by lipofection. IMPACT overexpression was confirmed by Western blot. Metabolic activity and survival of the GL261 cells in Trp deprivation experiments were evaluated using MTT assay and staining with live/dead indicators fluorescein diacetate and propidium iodide, respectively.
Meta-analysis of the publicly available RNA-sequencing datasets, The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx), provided evidence that IMPACT was overexpressed in the majority of the 24 analysed human cancers compared to their non-malignant tissue counterparts. Consistent with the high IMPACT expression in prostate cancer patients, 12-25% prostate tumors show amplification of the IMPACT gene (source: cBioPortal for Cancer Genomics). In addition, we found an association of high IMPACT expression with poorer survival for certain leukemia and lung cancer patients in the Gene Expression Omnibus (GEO) portal. To obtain experimental evidence that IMPACT protects cancer cells during Trp deprivation, GL261-IMPACT cells were cultured in media containing 2.5 - 50 μM Trp. Metabolic activity of GL261-IMPACT cells decreased at a slower rate than that of GL261-wild-type cells after 3 to 5 days in media containing limiting Trp concentration (2.5 - 10 μM). On day 5, GL261-wild-type and GL261-IMPACT cells cultivated in 10 μM Trp showed 4.5% and 23% metabolic activity (p = 2.8x10-8), respectively, relative to the same cells grown in 50 μM Trp. Fluorescence microscopy utilising live/dead staining showed that after 5 days of incubation at limiting Trp concentrations (5 - 10 μM), the majority of GL261-wild-type cells were non-viable whereas GL261-IMPACT cells were predominantly viable.
Our initial results support the hypothesis that IMPACT aids cancer cells to overcome periods of tryptophan deprivation.
Citation Format: Petr Tomek, Ariane Hallermayr, Michael Kilian, Cristin Gregor Print, Lai-Ming Ching. The role of IMPACT in survival of cancer cells during tryptophan deprivation by immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO1) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4338. doi:10.1158/1538-7445.AM2017-4338
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