Next generation sequencing of cell-free DNA (cfDNA) is a promising method for treatment monitoring and therapy selection in metastatic breast cancer (MBC). However, distinguishing tumor-specific ...variants from sequencing artefacts and germline variation with low false discovery rate is challenging when using large targeted sequencing panels covering many tumor suppressor genes. To address this, we built a machine learning model to remove false positive variant calls and augmented it with additional filters to ensure selection of tumor-derived variants. We used cfDNA of 70 MBC patients profiled with both the small targeted Oncomine breast panel (Thermofisher) and the much larger Qiaseq Human Breast Cancer Panel (Qiagen). The model was trained on the panels' common regions using Oncomine hotspot mutations as ground truth. Applied to Qiaseq data, it achieved 35% sensitivity and 36% precision, outperforming basic filtering. For 20 patients we used germline DNA to filter for somatic variants and obtained 245 variants in total, while our model found seven variants, of which six were also detected using the germline strategy. In ten tumor-free individuals, our method detected in total one (potentially germline) variant, in contrast to 521 variants detected without our model. These results indicate that our model largely detects somatic variants.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Offering self‐sampling for HPV testing improves the effectiveness of current cervical screening programs by increasing population coverage. Molecular markers directly applicable on self‐samples are ...needed to stratify HPV‐positive women at risk of cervical cancer (so‐called triage) and to avoid over‐referral and overtreatment. Deregulated microRNAs (miRNAs) have been implicated in the development of cervical cancer, and represent potential triage markers. However, it is unknown whether deregulated miRNA expression is reflected in self‐samples. Our study is the first to establish genome‐wide miRNA profiles in HPV‐positive self‐samples to identify miRNAs that can predict the presence of CIN3 and cervical cancer in self‐samples. Small RNA sequencing (sRNA‐Seq) was conducted to determine genome‐wide miRNA expression profiles in 74 HPV‐positive self‐samples of women with and without cervical precancer (CIN3). The optimal miRNA marker panel for CIN3 detection was determined by GRridge, a penalized method on logistic regression. Six miRNAs were validated by qPCR in 191 independent HPV‐positive self‐samples. Classification of sRNA‐Seq data yielded a 9‐miRNA marker panel with a combined area under the curve (AUC) of 0.89 for CIN3 detection. Validation by qPCR resulted in a combined AUC of 0.78 for CIN3+ detection. Our study shows that deregulated miRNA expression associated with CIN3 and cervical cancer development can be detected by sRNA‐Seq in HPV‐positive self‐samples. Validation by qPCR indicates that miRNA expression analysis offers a promising novel molecular triage strategy for CIN3 and cervical cancer detection applicable to self‐samples.
What's new?
MicroRNAs (miRNAs) are suspected of playing a role in cervical cancer development. They are also potential markers for the identification of human papillomavirus (HPV)‐infected women who are at risk of cervical cancer. Here, using small RNA sequencing of HPV‐positive self‐samples from women with and without cervical precancer (CIN3), the authors identify a miRNA signature consisting of multiple miRNAs that is strongly predictive of CIN3. Validation of this signature by qPCR revealed a good clinical performance for CIN3+ detection. The findings suggest that miRNA analysis is an effective means of CIN3+ prediction in HPV‐positive self‐samples obtained for cervical cancer screening.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Next to a persistent infection with high-risk human papillomavirus (HPV), molecular changes are required for the development of cervical cancer. To identify which molecular alterations drive ...carcinogenesis, we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines. Comparative genomic hybridization, mRNA, and miRNA expression analysis of four HPV-containing keratinocyte cell lines at eight different time points was performed. Data was analyzed using unsupervised hierarchical clustering, integrated longitudinal expression analysis, and pathway enrichment analysis. Biological relevance of identified key regulatory genes was evaluated in vitro and dual-luciferase assays were used to confirm predicted miRNA-mRNA interactions. We show that the acquisition of anchorage independence of HPV-containing keratinocyte cell lines is particularly associated with copy number alterations. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to copy number alterations. Focal adhesion, TGF-beta signaling, and mTOR signaling pathways were enriched among these genes. PITX2 was identified as key regulator of TGF-beta signaling and inhibited cell growth in vitro, most likely by inducing cell cycle arrest and apoptosis. Predicted miRNA-mRNA interactions miR-221-3p_BRWD3, miR-221-3p_FOS, and miR-138-5p_PLXNB2 were confirmed in vitro. Integrated longitudinal analysis of our HPV-induced carcinogenesis model pinpointed relevant interconnected molecular changes and crucial signaling pathways in HPV-mediated transformation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
CGHcall achieves high calling accuracy for array CGH data by effective use of breakpoint information from segmentation and by inclusion of several biological concepts that are ignored by existing ...algorithms. The algorithm is validated for simulated and verified real array CGH data. By incorporating more than three classes, CGHcall improves detection of single copy gains and amplifications. Moreover, it allows effective inclusion of chromosome arm information.
Availability: An R-package (GUI), a manual and an example data set are available at http://www.few.vu.nl/~mavdwiel/CGHcall.html.
Contact:
mark.vdwiel@vumc.nl
Supplementary information: Supplementary data are available at Bioinformatics online.
The progression of anchorage-dependent epithelial cells to anchorage-independent growth represents a critical hallmark of malignant transformation. Using an in vitro model of human papillomavirus ...(HPV)-induced transformation, we previously showed that acquisition of anchorage-independent growth is associated with marked (epi)genetic changes, including altered expression of microRNAs. However, the laborious nature of the conventional growth method in soft agar to measure this phenotype hampers a high-throughput analysis. We developed alternative functional screening methods using 96- and 384-well ultra-low attachment plates to systematically investigate microRNAs regulating anchorage-independent growth. SiHa cervical cancer cells were transfected with a microRNA mimic library (n = 2019) and evaluated for cell viability. We identified 84 microRNAs that consistently suppressed growth in three independent experiments. Further validation in three cell lines and comparison of growth in adherent and ultra-low attachment plates yielded 40 microRNAs that specifically reduced anchorage-independent growth. In conclusion, ultra-low attachment plates are a promising alternative for soft-agar assays to study anchorage-independent growth and are suitable for high-throughput functional screening. Anchorage independence suppressing microRNAs identified through our screen were successfully validated in three cell lines. These microRNAs may provide specific biomarkers for detecting and treating HPV-induced precancerous lesions progressing to invasive cancer, the most critical stage during cervical cancer development.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Prediction in high dimensional settings is difficult due to the large number of variables relative to the sample size. We demonstrate how auxiliary 'co-data' can be used to improve the performance of ...a Random Forest in such a setting.
Co-data are incorporated in the Random Forest by replacing the uniform sampling probabilities that are used to draw candidate variables by co-data moderated sampling probabilities. Co-data here are defined as any type information that is available on the variables of the primary data, but does not use its response labels. These moderated sampling probabilities are, inspired by empirical Bayes, learned from the data at hand. We demonstrate the co-data moderated Random Forest (CoRF) with two examples. In the first example we aim to predict the presence of a lymph node metastasis with gene expression data. We demonstrate how a set of external p-values, a gene signature, and the correlation between gene expression and DNA copy number can improve the predictive performance. In the second example we demonstrate how the prediction of cervical (pre-)cancer with methylation data can be improved by including the location of the probe relative to the known CpG islands, the number of CpG sites targeted by a probe, and a set of p-values from a related study.
The proposed method is able to utilize auxiliary co-data to improve the performance of a Random Forest.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aberrant DNA methylation changes have been reported to be associated with carcinogenesis in cirrhotic HCC, but DNA methylation patterns for these non-cirrhotic HCC cases were not examined. Therefore, ...we sought to investigate DNA methylation changes on non-cirrhotic HCC using reported promising DNA methylation markers (DMMs), including HOXA1, CLEC11A, AK055957, and TSPYL5, on 146 liver tissues using quantitative methylation-specific PCR and methylated DNA sequencing. We observed a high frequency of aberrant methylation changes in the four DMMs through both techniques in non-cirrhotic HCC compared to cirrhosis, hepatitis, and benign lesions (p < 0.05), suggesting that hypermethylation of these DMMs is specific to non-cirrhotic HCC development. Also, the combination of the four DMMs exhibited 78% sensitivity at 80% specificity with an AUC of 0.85 in discriminating non-cirrhotic HCC from hepatitis and benign lesions. In addition, HOXA1 showed a higher aberrant methylation percentage in non-cirrhotic HCC compared to cirrhotic HCC (43.3% versus 13.3%, p = 0.039), which was confirmed using multivariate linear regression (p < 0.05). In summary, we identified aberrant hypermethylation changes in HOXA1, CLEC11A, AK055957, and TSPYL5 in non-cirrhotic HCC tissues compared to cirrhosis, hepatitis, and benign lesions, providing information that could be used as potentially detectable biomarkers for these unusual HCC cases in clinical practice.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Purpose
HER2 overexpressing circulating tumor cells (CTCs) are observed in up to 25% of HER2-negative metastatic breast cancer patients. Since targeted anti-HER2 therapy has drastically improved ...clinical outcomes of patients with HER2-positive breast cancer, we hypothesized that patients with HER2 overexpressing CTCs might benefit from the addition of trastuzumab to chemotherapy.
Methods
In this single-arm, phase II trial, patients with HER2-positive CTCs received trastuzumab as addition to first-line treatment with taxane chemotherapy. Patients with detectable CTCs but without HER2 overexpression that received taxane chemotherapy only, were used as control group. The primary outcome measure was progression-free rate at 6 months (PFR6), with a target of 80%. In November 2022, the study was terminated early due to slow patient accrual.
Results
63 patients were screened, of which eight patients had HER2-positive CTCs and were treated with trastuzumab. The median number of CTCs was 15 per 7.5 ml of blood (range 1–131) in patients with HER2-positive CTCs, compared to median 5 (range 1–1047) in the control group. PFR6 was 50% in the trastuzumab group and 54% in the taxane monotherapy group, with no significant difference in median PFS (8 versus 9 months, p = 0.51).
Conclusion
No clinical benefit of trastuzumab was observed, although this study was performed in a limited number of patients. Additionally, we observed a strong correlation between the number of evaluable CTCs and the presence of HER2-positive CTCs. We argue that randomized studies investigating agents that are proven to be solely effective in the HER2-positive patient group in patients with HER2-positive CTCs and HER2-negative tissue are currently infeasible. Several factors contribute to this impracticality, including the need for more stringent thresholds, and the rapidly evolving landscape of cancer treatments.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high‐throughput method for fast, reproducible, and efficient isolation of ...ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study was to compare two competing automated DNA isolation platforms Maxwell (MX) and QIAsymphony (QS) to the current ‘gold standard’ QA to facilitate high‐throughput processing of samples in prospective trials. We obtained blood samples from healthy blood donors and metastatic cancer patients for plasma isolation. Total cell‐free DNA (cfDNA) quantity was assessed by TERT quantitative PCR. Recovery efficiency was investigated by quantitative PCR analysis of spiked‐in synthetic plant DNA. In addition, a β‐actin fragmentation assay was performed to determine the amount of contamination by genomic DNA from lysed leukocytes. ctDNA quality was assessed by digital PCR for somatic variant detection. cfDNA quantity and recovery efficiency were lowest using the MX platform, whereas QA and QS showed a comparable performance. All platforms preferentially isolated small (136 bp) DNA fragments over large (420 and 2000 bp) DNA fragments. Detection of the number variant and wild‐type molecules was most comparable between QA and QS. However, there was no significant difference in variant allele frequency comparing QS and MX to QA. In summary, we show that the QS platform has comparable performance to QA, the ‘gold standard’, and outperformed the MX platform depending on the readout used. We conclude that the QS can replace the more laborious QA platform, especially when high‐throughput cfDNA isolation is needed.
To facilitate high‐throughput isolation of cell‐free DNA (cfDNA) in prospective trials, we compared two competing automated DNA isolation platforms (Maxwell and QIAsymphony) to the current manual ‘gold standard’ QIAamp. We evaluated cfDNA and circulating tumor DNA quantity and quality. We observed that the QIAsymphony has comparable performance to QIAamp and outperformed the Maxwell (dependent on the read‐out used).
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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.
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