Circulating tumour DNA (ctDNA) has been established as a promising (prognostic) biomarker with the potential to personalise treatment in cancer patients. The objective of this systematic review is to ...provide an overview of the current literature and the future perspectives of ctDNA in non-metastatic rectal cancer.
A comprehensive search for studies published prior to the 4
of October 2022 was conducted in Embase, Medline, Cochrane, Google scholar, and Web of Science. Only peer-reviewed original articles and ongoing clinical trials investigating the association between ctDNA and oncological outcomes in non-metastatic rectal cancer patients were included. Meta-analyses were performed to pool hazard ratios (HR) for recurrence-free survival (RFS).
A total of 291 unique records were screened, of which 261 were original publications and 30 ongoing trials. Nineteen original publications were reviewed and discussed, of which seven provided sufficient data for meta-analyses on the association between the presence of post-treatment ctDNA and RFS. Results of the meta-analyses demonstrated that ctDNA analysis can be used to stratify patients into very high and low risk groups for recurrence, especially when detected after neoadjuvant treatment (HR for RFS: 9.3 4.6 - 18.8) and after surgery (HR for RFS: 15.5 8.2 - 29.3). Studies investigated different types of assays and used various techniques for the detection and quantification of ctDNA.
This literature overview and meta-analyses provide evidence for the strong association between ctDNA and recurrent disease. Future research should focus on the feasibility of ctDNA-guided treatment and follow-up strategies in rectal cancer. A blueprint for agreed-upon timing, preprocessing, and assay techniques is needed to empower adaptation of ctDNA into daily practice.
Epigenetic host cell changes involved in cervical cancer development following a persistent high-risk human papillomavirus (hrHPV) infection, provide promising markers for the management of ...hrHPV-positive women. In particular, markers based on DNA methylation of tumor suppressor gene promoters are valuable. These markers ideally identify hrHPV-positive women with precancer (CIN2/3) in need of treatment. Here, we set out to identify biologically relevant methylation markers by genome-wide methylation analysis of both hrHPV-transformed cell lines and cervical tissue specimens.
Genome-wide discovery by next-generation sequencing (NGS) of methyl-binding domain-enriched DNA (MBD-Seq) yielded 20 candidate methylation target genes. Further verification and validation by multiplex-targeted bisulfite NGS and (quantitative) methylation-specific PCR (MSP) resulted in 3 genes (
, and
) that showed a significant increase in methylation with severity of disease in both tissue specimens and cervical scrapes (
< 0.005). The area under the ROC curve for CIN3 or worse varied between 0.86 and 0.89. Within the group of CIN2/3, methylation levels of all 3 genes increased with duration of lesion existence (
< 0.0005), characterized by duration of preceding hrHPV infection, and were significantly higher in the presence of a 3q gain (
< 0.05) in the corresponding tissue biopsy.
By unbiased genome-wide DNA methylation profiling and comprehensive stepwise verification and validation studies using
and patient-derived samples, we identified 3 promising methylation markers (
, and
associated with a 3q gain for the detection of cervical (pre)cancer.
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Multiple prognostic biomarkers, including circulating tumor cell (CTC) counts, exist in metastatic castration-resistant prostate cancer (mCRPC) patients, but none of them have been implemented into ...daily clinical care. The modified fast aneuploidy screening test-sequencing system (mFast-SeqS), which yields a genome-wide aneuploidy score, is able to reflect the fraction of cell-free tumor DNA (ctDNA) within cell-free DNA (cfDNA) and may be a promising biomarker in mCRPC. In this study, we investigated the prognostic value of dichotomized aneuploidy scores (<5 vs ≥5) as well as CTC counts (<5 vs ≥5) in 131 mCRPC patients prior to treatment with cabazitaxel. We validated our findings in an independent cohort of 50 similarly treated mCRPC patients. We observed that, similar to the dichotomized CTC count HR: 2.92; 95% confidence interval (CI);1.84-4.62, dichotomized aneuploidy scores (HR: 3.24; CI: 2.12-4.94) significantly correlated with overall survival in mCRPC patients. We conclude that a dichotomized aneuploidy score from cfDNA is a prognostic marker for survival in mCRPC patients within our discovery cohort and in an independent mCRPC validation cohort. Therefore, this easy and robust minimally-invasive assay can be readily implemented as a prognostic marker in mCRPC. Dichotomized aneuploidy score might also be used as a stratification factor in clinical studies to account for tumor load.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating ...tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike‐in experiments using MCF‐7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI‐g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient‐derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient‐derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.
Here, we present a technical study on the development of a single circulating tumour cell (CTC) analysis pipeline to assess intrapatient heterogeneity by copy number profiling. We compared two whole genome amplification methods, two library preparation methods and three quality control assays for single‐cell analysis after CellSearch enrichment and VyCAP punching. The established modular pipeline was applied to blood‐derived single cells from patients with metastatic breast or prostate cancer.
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Detection of leptomeningeal metastasis is hampered by limited sensitivities of currently used techniques: MRI and cytology of cerebrospinal fluid (CSF). Detection of cell-free tumor DNA in CSF has ...been proposed as a tumor-specific candidate to detect leptomeningeal metastasis at an earlier stage. The aim of this study was to investigate mutation and aneuploidy status in CSF-derived cell-free DNA (cfDNA) of patients with breast cancer with a clinical suspicion of leptomeningeal metastasis.
cfDNA was isolated from stored remnant CSF and analyzed by targeted next-generation sequencing (NGS;
= 30) and the modified fast aneuploidy screening test-sequencing system (mFAST-SeqS;
= 121). The latter method employs selective amplification of long interspaced nuclear elements sequences that are present throughout the genome and allow for fast and cheap detection of aneuploidy. We compared these results with the gold standard to diagnose leptomeningeal metastasis: cytology.
Leptomeningeal metastasis was cytology proven in 13 of 121 patients. Low DNA yields resulted in insufficient molecular coverage of NGS for the majority of samples (success rate, 8/30). The mFAST-SeqS method, successful in 112 of 121 (93%) samples, detected genome-wide aneuploidy in 24 patients. Ten of these patients had cytology-proven leptomeningeal metastasis; 8 additional patients were either concurrently diagnosed with central nervous system metastases by radiological means or developed these soon after the lumbar puncture. The remaining six cases were suspected of leptomeningeal metastasis, but could not be confirmed by cytology or imaging. Aneuploidy was associated with development of leptomeningeal metastasis and significantly worse overall survival.
Aneuploidy in CSF-derived cfDNA may provide a promising biomarker to improve timely detection of leptomeningeal metastasis.
Monitoring treatment response in metastatic breast cancer currently consists mainly of radiological and clinical assessments. These methods have high inter-observer variation, suboptimal sensitivity ...to determine response to treatment and give little insight into the biological characteristics of the tumor. Assessing circulating tumor DNA (ctDNA) over time could be employed to address these limitations. Several ways to quantify and characterize ctDNA exist, based on somatic mutations, copy number variations, methylation, and global circulating cell-free DNA (cfDNA) fragment sizes and concentrations. These methods are being explored and technically validated, but to date none of these methods are applied clinically. We systematically reviewed the literature on the use of quantitative ctDNA measurements over time to monitor response to systemic therapy in patients with metastatic breast cancer. Cochrane, Embase, PubMed and Google Scholar databases were searched to find studies focusing on the use of cfDNA to longitudinally monitor treatment response in advanced breast cancer patients until October 2020. This resulted in a total of 33 studies which met the inclusion criteria. These studies were heterogeneous in (pre-)processing procedures, applied techniques and design. An association between ctDNA and treatment response was found in most of the included studies, independent of the applied assay. To implement ctDNA-based response monitoring into daily clinical practice for metastatic breast cancer patients, sample (pre-) processing procedures need to be standardized and large prospectively collected sample cohorts with well annotated clinical follow-up are required to establish its clinical validity.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Offering self-sampling of cervico-vaginal material for high-risk human papillomavirus (hrHPV) testing is an effective method to increase the coverage in cervical screening programs. Molecular triage ...directly on hrHPV-positive self-samples for colposcopy referral opens the way to full molecular cervical screening. Here, we set out to identify a DNA methylation classifier for detection of cervical precancer (CIN3) and cancer, applicable to lavage and brush self-samples.
We determined genome-wide DNA methylation profiles of 72 hrHPV-positive self-samples, using the Infinium Methylation 450K Array. The selected DNA methylation markers were evaluated by multiplex quantitative methylation-specific PCR (qMSP) in both hrHPV-positive lavage (
= 245) and brush (
= 246) self-samples from screening cohorts. Subsequently, logistic regression analysis was performed to build a DNA methylation classifier for CIN3 detection applicable to self-samples of both devices. For validation, an independent set of hrHPV-positive lavage (
= 199) and brush (
= 287) self-samples was analyzed.
Genome-wide DNA methylation profiling revealed 12 DNA methylation markers for CIN3 detection. Multiplex qMSP analysis of these markers in large series of lavage and brush self-samples yielded a 3-gene methylation classifier (
and
). This classifier showed a very good clinical performance for CIN3 detection in both lavage (AUC = 0.88; sensitivity = 74%; specificity = 79%) and brush (AUC = 0.90; sensitivity = 88%; specificity = 81%) self-samples in the validation set. Importantly, all self-samples from women with cervical cancer scored DNA methylation-positive.
By genome-wide DNA methylation profiling on self-samples, we identified a highly effective 3-gene methylation classifier for direct triage on hrHPV-positive self-samples, which is superior to currently available methods.
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Second‐line treatment with immune checkpoint inhibition in patients with metastatic urothelial cancer (mUC) has a low success rate (~ 20%). Circulating tumour‐derived DNA (ctDNA) levels may guide ...patient stratification, provided that an affordable and robust assay is available. Here, we investigate whether the modified fast aneuploidy screening test‐sequencing system (mFast‐SeqS) may provide such an assay. To this end, mFast‐SeqS was performed on cell‐free DNA (cfDNA) from 74 patients with mUC prior to treatment with pembrolizumab. Results were associated with corresponding tissue‐based profiles, plasma‐based variant allele frequencies (VAFs) and clinical response. We found that plasma‐derived mFast‐SeqS‐based aneuploidy scores significantly correlated with those observed in the corresponding tumour tissue as well as with the ctDNA level in the plasma. In multivariate logistic regression analysis, a high aneuploidy score was independently associated with lack of clinical benefit from treatment with pembrolizumab. In conclusion, mFast‐SeqS provides a patient‐friendly, high‐throughput and affordable method to estimate ctDNA level. Following independent validation, this test could be used to stratify mUC patients for response prior to the initiation of treatment with pembrolizumab.
Second‐line immune checkpoint inhibition in patients with metastatic urothelial cancer (mUC) has a low success rate. ctDNA levels may guide patient stratification; however, a high‐throughput and affordable method is lacking. Here, we report that a high aneuploidy score detected by the mFast‐SeqS method is independently associated to a lack of clinical benefit from pembrolizumab treatment. Following independent validation, mFast‐SeqS could stratify patients with mUC for response to therapy.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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