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Simple Summary: Colonoscopies are effective in the prevention of colorectal cancer (CRC) but considered burdensome. An alternative for or addition to this procedure might be found in circulating tumor DNA (ctDNA) analysis. However, to date, it is not clear which analysis is most suitable for such a ctDNA-based blood test for CRC. Therefore, we assessed this in ten patients with colonoscopies for Lynch syndrome or in the context of the Dutch national screening program who were diagnosed with CRC or its precursor lesion (advanced adenoma). The results of this proof-of-principle study could form the foundation for subsequent studies on ctDNA-based blood test development for CRC screening and management, specifically in carriers of Lynch syndrome. Colorectal cancer (CRC) colonoscopic surveillance is effective but burdensome. Circulating tumor DNA (ctDNA) analysis has emerged as a promising, minimally invasive tool for disease detection and management. Here, we assessed which ctDNA assay might be most suitable for a ctDNA-based CRC screening/surveillance blood test. In this prospective, proof-of-concept study, patients with colonoscopies for Lynch surveillance or the National Colorectal Cancer screening program were included between 7 July 2019 and 3 June 2022. Blood was drawn, and if advanced neoplasia (adenoma with villous component, high-grade dysplasia, ≥10 mm, or CRC) was detected, it was analyzed for chromosomal copy number variations, single nucleotide variants, and genome-wide methylation (MeD-seq). Outcomes were compared with corresponding patients' tissues and the MeD-seq results of healthy blood donors. Two Lynch carriers and eight screening program patients were included: five with CRC and five with advanced adenomas. cfDNA showed copy number variations and single nucleotide variants in one patient with CRC and liver metastases. Eight patients analyzed with MeD-seq showed clustering of Lynch-associated and sporadic microsatellite instable lesions separate from microsatellite stable lesions, as did healthy blood donors. In conclusion, whereas copy number changes and single nucleotide variants were only detected in one patient, cfDNA methylation profiles could discriminate all microsatellite instable advanced neoplasia, rendering this tool particularly promising for LS surveillance. Larger studies are warranted to validate these findings. [ABSTRACT FROM AUTHOR]

Among the structural variants observed in metastatic colorectal cancer (mCRC), deletions (DELs) show a size preference of ~10 kb–1 Mb and are often found in common fragile sites (CFSs). To gain more insight into the biology behind the occurrence of these specific DELs in mCRC, and their possible association with outcome, we here studied them in detail in metastatic lesions of 429 CRC patients using available whole-genome sequencing and corresponding RNA-seq data. Breakpoints of DELs within CFSs are significantly more often located between two consecutive replication origins compared to DELs outside CFSs. DELs are more frequently located at the midpoint of genes inside CFSs with duplications (DUPs) at the flanks of the genes. The median expression of genes inside CFSs was significantly higher than those of similarly-sized genes outside CFSs. Patients with high numbers of these specific DELs showed a shorter progression-free survival time on platinum-containing therapy. Taken together, we propose that the observed DEL/DUP patterns in expressed genes located in CFSs are consistent with a model of transcription-dependent double-fork failure, and, importantly, that the ability to overcome the resulting stalled replication forks decreases sensitivity to platinum-containing treatment, known to induce stalled replication forks as well. Therefore, we propose that our DEL score can be used as predictive biomarker for decreased sensitivity to platinum-containing treatment, which, upon validation, may augment future therapeutic choices. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer patients often receive anti-hormonal treatment if their tumor is positive for the Estrogen Receptor (ER), but tumors may become resistant to this therapy and still metastasize. We studied 101 of such metastatic lesions and investigated these lesions for mutated genes and mutation patterns, in combination with the level of expression of relevant genes. Our aim was to better understand the mechanisms that are involved in the resistance to anti-hormonal treatment. The analyses showed two distinct groups of patients, each with specific mutations. One group clearly showed an ongoing, active ER and its associated signal route; these patients probably still would benefit from ER-targeting agents. We advocate for combining mutation and expression analyses on metastatic lesions, to maximize the group of patients that still may benefit from existing or new anti-hormonal treatments targeting ER or its signaling network. Mutations in the estrogen receptor gene (ESR1), its transcriptional regulators, and the mitogen-activated protein kinase (MAPK) pathway are enriched in patients with endocrine-resistant metastatic breast cancer (MBC). Here, we integrated whole genome sequencing with RNA sequencing data from the same samples of 101 ER-positive/HER2-negative MBC patients who underwent a tumor biopsy prior to the start of a new line of treatment for MBC (CPCT-02 study, NCT01855477) to analyze the downstream effects of DNA alterations previously linked to endocrine resistance, thereby gaining a better understanding of the associated mechanisms. Hierarchical clustering was performed using expression of ESR1 target genes. Genomic alterations at the DNA level, gene expression levels, and last administered therapy were compared between the identified clusters. Hierarchical clustering revealed two distinct clusters, one of which was characterized by increased expression of ESR1 and its target genes. Samples in this cluster were significantly enriched for mutations in ESR1 and amplifications in FGFR1 and TSPYL. Patients in the other cluster showed relatively lower expression levels of ESR1 and its target genes, comparable to ER-negative samples, and more often received endocrine therapy as their last treatment before biopsy. Genes in the MAPK-pathway, including NF1, and ESR1 transcriptional regulators were evenly distributed. In conclusion, RNA sequencing identified a subgroup of patients with clear expression of ESR1 and its downstream targets, probably still benefiting from ER-targeting agents. The lower ER expression in the other subgroup might be partially explained by ER activity still being blocked by recently administered endocrine treatment, indicating that biopsy timing relative to endocrine treatment needs to be considered when interpreting transcriptomic data. [ABSTRACT FROM AUTHOR]

Background: To determine which changes in the host cell genome are crucial for cervical carcinogenesis, a longitudinal in vitro model system of HPV-transformed keratinocytes was profiled in a genome-wide manner. Four cell lines affected with either HPV16 or HPV18 were assayed at 8 sequential time points for gene expression (mRNA) and gene copy number (DNA) using high-resolution microarrays. Available methods for temporal differential expression analysis are not designed for integrative genomic studies. Results: Here, we present a method that allows for the identification of differential gene expression associated with DNA copy number changes over time. The temporal variation in gene expression is described by a generalized linear mixed model employing low-rank thin-plate splines. Model parameters are estimated with an empirical Bayes procedure, which exploits integrated nested Laplace approximation for fast computation. Iteratively, posteriors of hyperparameters and model parameters are estimated. The empirical Bayes procedure shrinks multiple dispersion-related parameters. Shrinkage leads to more stable estimates of the model parameters, better control of false positives and improvement of reproducibility. In addition, to make estimates of the DNA copy number more stable, model parameters are also estimated in a multivariate way using triplets of features, imposing a spatial prior for the copy number effect. Conclusion: With the proposed method for analysis of time-course multilevel molecular data, more profound insight may be gained through the identification of temporal differential expression induced by DNA copy number abnormalities. In particular, in the analysis of an integrative oncogenomics study with a time-course set-up our method finds genes previously reported to be involved in cervical carcinogenesis. Furthermore, the proposed method yields improvements in sensitivity, specificity and reproducibility compared to existing methods. Finally, the proposed method is able to handle count (RNAseq) data from time course experiments as is shown on a real data set. [ABSTRACT FROM AUTHOR]

Background: A substantial number of microRNAs (miRNAs) is subject to epigenetic silencing in cancer. Although epigenetic silencing of tumour suppressor genes is an important feature of cervical cancer, little is known about epigenetic silencing of miRNAs. Since DNA methylation-based silencing of hsa-miR-124 occurs in various human cancers, we studied the frequency and functional effects of hsa-miR-124 methylation in cervical carcinogenesis. Results: Quantitative MSP analysis of all 3 loci encoding the mature hsa-miR-124 (hsa-miR-124-1/-2/-3) showed methylation in cervical cancer cell lines SiHa, CaSki and HeLa as well as in late passages of human papillomavirus (HPV) type 16 or 18 immortalised keratinocytes. Treatment of SiHa cells with a demethylating agent reduced hsa-miR-124 methylation levels and induced hsa-miR-124 expression. In HPV-immortalised keratinocytes increased methylation levels were related to reduced hsa-miR-124 expression and higher mRNA expression of IGFBP7, a potential hsa-miR-124 target gene. Ectopic hsa-miR-124 expression in SiHa and CaSki cells decreased proliferation rates and migratory capacity. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 139 cervical tissue specimens showed an increasing methylation frequency from 0% in normal tissues up to 93% in cervical carcinomas. Increased methylation levels of hsa-miR-124-1 and hsa-miR-124-2 were significantly correlated with reduced hsa-miR-124 expression in cervical tissue specimens. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 43 cervical scrapes of high-risk HPV positive women was predictive of underlying high-grade lesions. Conclusions: DNA methylation-based silencing of hsa-miR-124 is functionally involved in cervical carcinogenesis and may provide a valuable marker for improved detection of cervical cancer and its high-grade precursor lesions. [ABSTRACT FROM AUTHOR]

Background: It is well known that a persistent infection with high-risk human papillomavirus (hrHPV) is causally involved in the development of squamous cell carcinomas of the uterine cervix (CxSCCs) and a subset of SCCs of the head and neck (HNSCCs). The latter differ from hrHPV-negative HNSCCs at the clinical and molecular level. Methods: To determine whether hrHPV-associated SCCs arising from different organs have specific chromosomal alterations in common, we compared genome-wide chromosomal profiles of 10 CxSCCs (all hrHPV-positive) with 12 hrHPV-positive HNSCCs and 30 hrHPV-negative HNSCCs. Potential organ-specific alterations and alterations shared by SCCs in general were investigated as well. Results: Unsupervised hierarchical clustering resulted in one mainly hrHPV-positive and one mainly hrHPV-negative cluster. Interestingly, loss at 13q and gain at 20q were frequent in HPV-positive carcinomas of both origins, but uncommon in hrHPV-negative HNSCCs, indicating that these alterations are associated with hrHPV-mediated carcinogenesis. Within the group of hrHPV-positive carcinomas, HNSCCs more frequently showed gains of multiple regions at 8q whereas CxSCCs more often showed loss at 17p. Finally, gains at 3q24-29 and losses at 11q22.3-25 were frequent (>50%) in all sample groups. Conclusion: In this study hrHPV-specific, organ-specific, and pan-SCC chromosomal alterations were identified. The existence of hrHPV-specific alterations in SCCs of different anatomical origin, suggests that these alterations are crucial for hrHPV-mediated carcinogenesis. [ABSTRACT FROM AUTHOR]

Advances in therapeutic approaches for melanoma urge the need for biomarkers that can identify patients at risk for recurrence and to guide treatment. The potential use of liquid biopsies in identifying biomarkers is increasingly being recognized. Here, we present a head‐to‐head comparison of several techniques to analyze circulating tumor cells (CTCs) and cell‐free DNA (cfDNA) in 20 patients with metastatic melanoma. In this study, we investigated whether diagnostic leukapheresis (DLA) combined with multimarker flow cytometry (FCM) increased the detection of CTCs in blood compared to the CellSearch platform. Additionally, we characterized cfDNA at the level of somatic mutations, extent of aneuploidy and genome‐wide DNA methylation. Both CTCs and cfDNA measures were compared to tumor markers and extracranial tumor burden on radiological imaging. Compared to the CellSearch method applied on peripheral blood, DLA combined with FCM increased the proportion of patients with detectable CTCs from 35% to 70% (P = 0.06). However, the median percentage of cells that could be recovered by the DLA procedure was 29%. Alternatively, cfDNA mutation and methylation analysis detected tumor load in the majority of patients (90% and 93% of samples successfully analyzed, respectively). The aneuploidy score was positive in 35% of all patients. From all tumor measurements in blood, lactate dehydrogenase (LDH) levels were significantly correlated to variant allele frequency (P = 0.004). Furthermore, the presence of CTCs in DLA was associated with tumor burden (P < 0.001), whereas the presence of CTCs in peripheral blood was associated with number of lesions on radiological imaging (P < 0.001). In conclusion, DLA tended to increase the proportion of patients with detectable CTCs but was also associated with low recovery. Both cfDNA and CTCs were correlated with clinical parameters such as LDH levels and extracranial tumor burden. [ABSTRACT FROM AUTHOR]

Circulating tumour DNA (ctDNA) is a potential biomarker that could contribute to more judicious patient selection for personalised treatment. This review and meta-analysis gives an overview of the current knowledge in the literature investigating the value of ctDNA in patients with colorectal liver metastases (CRLM). A systematic search was conducted in electronic databases for studies published prior to the 26th of May 2023. Studies investigating the association between ctDNA and oncological outcomes in patients undergoing curative-intent local therapy for CRLM were included. Meta-analyses were performed to pool hazard ratios (HR) for the recurrence-free survival (RFS) and overall survival (OS). A total of eleven studies were included and nine were eligible for meta-analyses. Patients with detectable ctDNA after surgery experienced a significantly higher chance of recurrence (HR 3.12, 95% CI 2.27–4.28, p < 0.000010) and shorter OS (HR 5.04, 95% CI 2.53–10.04, p < 0.00001) compared to patients without detectable ctDNA. A similar association for recurrence was found in patients with detectable ctDNA after the completion of adjuvant therapy (HR 6.39, 95% CI 2.13–19.17, p < 0.0009). The meta-analyses revealed no association between detectable ctDNA before surgery and the RFS and OS. These meta-analyses demonstrate the strong association between detectable ctDNA after treatment and oncological outcomes in CRLM patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Hepatocellular carcinoma (HCC) remains a significant cause of cancer-related deaths worldwide. Although most HCC cases have a background of cirrhosis, up to 20–30% of patients develop HCC in a non-cirrhotic liver. The prognosis of these unusual HCC cases is poor, since surveillance is not common. Therefore, investigating sensitive and specific biomarkers to detect non-cirrhotic HCC is crucial. Aberrant DNA methylation has been reported to play an important role in the development of cirrhotic HCC, while limited information can be found on non-cirrhotic HCC. This study is the first to determine the performance of reported promising DNA methylation markers in non-cirrhotic HCC. A total of 146 liver tissues were tested for 4 methylation markers using PCR- and sequencing-related techniques. We demonstrated significant DNA methylation changes in non-cirrhotic HCC compared to non-HCC benign lesions. These findings may be highly relevant to the future application of DNA methylation markers in non-cirrhotic HCC surveillance. 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. [ABSTRACT FROM AUTHOR]

Background: 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. Results: 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. Conclusion: The proposed method is able to utilize auxiliary co-data to improve the performance of a Random Forest. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: A subset of patients with an intermediate risk for renal cell carcinoma have an indolent disease course. For these patients, the initiation of treatment for metastatic disease can be safely delayed by entering a watchful waiting period. At present, we are not able to identify those patients with indolent disease and the minor group of patients with rapidly progressive disease requiring earlier initiation of systemic treatment. In this study, we investigate whether cell-free DNA (cfDNA) can be used as a blood-based biomarker to identify those patients with rapid progression. Methylated cfDNA profiles were used as a proxy for tumor load in blood. cfDNA methylation patterns were associated with the time to radiological progression, but not with the watchful waiting time. The results of this study do not provide definite proof that cfDNA methylation patterns are associated with WW time. According to the current guidelines, watchful waiting (WW) is a feasible option for patients with good or intermediate prognosis renal-cell carcinoma (RCC). However, some patients rapidly progress during WW, requiring the initiation of treatment. Here, we explore whether we can identify those patients using circulating cell-free DNA (cfDNA) methylation. We first defined a panel of RCC-specific circulating methylation markers by intersecting differentially methylated regions from a publicly available dataset with known RCC methylation markers from the literature. The resulting RCC-specific methylation marker panel of 22 markers was subsequently evaluated for an association with rapid progression by methylated DNA sequencing (MeD-seq) in serum from 10 HBDs and 34 RCC patients with a good or intermediate prognosis starting WW in the IMPACT-RCC study. Patients with an elevated RCC-specific methylation score compared to HBDs had a shorter progression-free survival (PFS, p = 0.018), but not a shorter WW-time (p = 0.15). Cox proportional hazards regression showed that only the International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) criteria were significantly associated with WW time (HR 2.01, p = 0.01), whereas only our RCC-specific methylation score (HR 4.45, p = 0.02) was significantly associated with PFS. The results of this study suggest that cfDNA methylation is predictive of PFS but not WW. [ABSTRACT FROM AUTHOR]

Background: The development of cervical cancer and its high-grade precursor lesions (Cervical Intraepithelial Neoplasia grade 2/3 [CIN2/3]) result from a persistent infection with high-risk human papillomavirus (hrHPV) types and the accumulation of (epi)genetic host cell aberrations. Epidemiological studies have demonstrated variable CIN2/3 and cancer risks between different hrHPV types. Recent genomic profiling studies revealed substantial heterogeneity in the chromosomal aberrations detected in morphologically indistinguishable CIN2/3 suggestive of varying cancer risk. The current study aimed to investigate whether CIN2/3 with different hrHPV types vary with respect to their chromosomal profiles, both in terms of the number of aberrations and chromosomal loci affected.Methods: Chromosomal profiles were determined of 43 p16INK4a-immunopositive CIN2/3 of women with long-term hrHPV infection (≥ 5 years). Sixteen lesions harboured HPV16, 3 HPV18, 14 HPV31, 1 HPV33, 4 HPV45, 1 HPV51, 2 HPV52 and 2 HPV58.Results: Unsupervised hierarchical clustering analysis of the chromosomal profiles revealed two major clusters, characterised by either few or multiple chromosomal aberrations, respectively. A majority of 87.5% of lesions with HPV16 were in the cluster with relatively few aberrations, whereas no such unbalanced distribution was seen for lesions harbouring other hrHPV types. Analysis of the two most prevalent types (HPV16 and HPV31) in this data set revealed a three-fold increase in the number of losses in lesions with HPV31 compared to HPV16-positive lesions. In particular, losses at chromosomes 2q, 4p, 4q, 6p, 6q, 8q & 17p and gain at 1p & 1q were significantly more frequent in HPV31-positive lesions (FDR < 0.2).Conclusions: Chromosomal aberrations in CIN2/3 are at least in part related to the hrHPV type present. The relatively low number of chromosomal aberrations observed in HPV16-positive CIN2/3 suggests that the development of these lesions is less dependent on genetic insult than those caused by other types like HPV31. [ABSTRACT FROM AUTHOR]

ESR1 methylation was proposed as mechanism for endocrine resistance in metastatic breast cancer patients. To evaluate its potential as a minimally invasive biomarker, we investigated the feasibility of measuring ESR1 methylation in cell-free DNA (cfDNA) and its association with endocrine resistance. First, we provided evidence that demethylation in vitro restores ER expression. Subsequently, we found that ESR1 methylation in cfDNA was not enriched in endocrine-resistant versus endocrine-sensitive patients. Interestingly, we found a correlation between ESR1 methylation and age. Publicly available data confirm an age-related increase in ESR1 methylation in leukocytes, confounding the determination of the ESR1 methylation status of tumors using cfDNA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Our aim is to improve omics based prediction and feature selection using multiple sources of auxiliary information: co-data. Adaptive group regularized ridge regression (GRridge) was proposed to achieve this by estimating additional group-based penalty parameters through an empirical Bayes method at a low computational cost. We illustrate the GRridge method and software on RNA sequencing datasets. The method boosts the performance of an ordinary ridge regression and outperforms other classifiers. Post-hoc feature selection maintains the predictive ability of the classifier with far fewer markers. [ABSTRACT FROM AUTHOR]

Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples. In the present study, we evaluated whether mFAST-SeqS is suitable to assess CNA in small formalin-fixed paraffin-embedded (FFPE) tissue specimens, using vulva and anal HPV-related lesions. Seventy-two FFPE samples, including 36 control samples (19 vulva;17 anal) for threshold setting and 36 samples (24 vulva; 12 anal) for clinical evaluation, were analyzed by mFAST-SeqS. CNA in vulva and anal lesions were determined by calculating genome-wide and chromosome arm-specific z-scores in comparison with the respective control samples. Sixteen samples were also analyzed with the conventional Shallow-seq approach. Genome-wide z-scores increased with the severity of disease, with highest values being found in cancers. In vulva samples median and inter quartile ranges [IQR] were 1[0–2] in normal tissues (n = 4), 3[1–7] in premalignant lesions (n = 9) and 21[13–48] in cancers (n = 10). In anal samples, median [IQR] were 0[0–1] in normal tissues (n = 4), 14[6–38] in premalignant lesions (n = 4) and 18[9–31] in cancers (n = 4). At threshold 4, all controls were CNA negative, while 8/13 premalignant lesions and 12/14 cancers were CNA positive. CNA captured by mFAST-SeqS were mostly also found by Shallow-seq. mFAST-SeqS is easy to perform, requires less DNA and less sequencing reads reducing costs, thereby providing a good alternative for Shallow-seq to determine CNA in small FFPE samples. • The targeted mFAST-SeqS approach is a robust method to detect copy number aberrations in small FFPE biopsies • Genome-wide z-scores determined with the mFAST-SeqS approach increased with the severity of HPV-induced disease • Copy number aberrations captured by mFAST-SeqS and untargeted Shallow-seq method show high concordance • mFAST-SeqS is a promising method requiring less DNA, less sequencing reads and costs than Shallow-seq to determine CNA [ABSTRACT FROM AUTHOR]

Cellular senescence is a state of stable growth arrest and a desired outcome of tumor suppressive interventions. Treatment with many anti‐cancer drugs can cause premature senescence of non‐malignant cells. These therapy‐induced senescent cells can have pro‐tumorigenic and pro‐disease functions via activation of an inflammatory secretory phenotype (SASP). Inhibitors of cyclin‐dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence‐like program in cancer cells. However, the physiological consequence of exposing the whole organism to pharmacological CDK4/6i remains poorly characterized. Here, we show that exposure to CDK4/6i induces non‐malignant cells to enter a premature state of senescence dependent on p53. We observe in mice and breast cancer patients that the CDK4/6i‐induced senescent program activates only a partial SASP enriched in p53 targets but lacking pro‐inflammatory and NF‐κB‐driven components. We find that CDK4/6i‐induced senescent cells do not acquire pro‐tumorigenic and detrimental properties but retain the ability to promote paracrine senescence and undergo clearance. Our results demonstrate that SASP composition is exquisitely stress‐dependent and a predictor for the biological functions of different senescence subsets. Synopsis: Pharmacological CDK4/6 inhibitors lead to premature senescence in culture and in vivo. CDK4/6 inhibitors‐induced senescence is p53‐dependent and not associated with the detrimental pro‐inflammatory secretory phenotype, SASP. Exposure to pharmacological CDK4/6 inhibitors (CDK4/6i) causes premature senescence.CDK4/6i‐induced senescence is dependent on p53.CDK4/6i‐induced senescence is well tolerated in vivo.CDK4/6i‐induced senescent cells exhibit a p53‐associated secretory phenotype (PASP) but lack the NF‐kB‐associated secretory phenotype (NASP). [ABSTRACT FROM AUTHOR]

The process of occurrence of genomic aberrations over time in the genetic material of cancer cells reflects the progression of the cancer. Modern technologies like aCGH (array Comparative Genomic Hybridization) and MPS (Massive Parallel Sequencing) provide high-resolution measurements of DNA copy number aberrations, that reveal the full scale of genomic aberrations. A continuous time Markov chain model is proposed to describe the accumulation of aberrations over time. Time however is a latent variable (with the number of aberrations as a proxy). Integrating out time, yields the distribution of the observed DNA copy number data. The model parameters are estimated from high-dimensional DNA copy number data by means of penalized maximum pseudo- and likelihood and method of moments procedures. Having fitted the model, posterior time estimates of the advancement of each sample's cancer are obtained and the most likely locations of a sample's aberrations are predicted. The three estimation methods are compared in a simulation study. The paper closes with an application of the proposed methodology on cancer data. [ABSTRACT FROM AUTHOR]

Abstract: Infection with high-risk human papillomavirus (HR-HPV) has been associated with intraepithelial neoplasia and carcinomas at various sites of the anogenital tract, including the cervix, vulva, vagina, penis and anus. Although HR-HPV is a necessary cause for cervical cancer, the majority of anal cancers and a subset of cancers at other genital sites, additional (epi)genetic events are required for malignant transformation. HPV-mediated transformation of human epithelial cells has been recognized as a multi-step process resulting from deregulated transcription of the viral oncogenes E6 and E7 in the proliferating cells. Interference of E6 and E7 with cell cycle regulators induces genetic instability, which drives the continuous selection of oncogenic alterations providing cells with a malignant phenotype. Early genetic events during cervical carcinogenesis associated with immortalization, include deletions at chromosomes 3p, 6 and 10p, whereas amongst others gain of chromosome 3q, loss of chromosome 11 and epigenetic alterations such as inactivation of the TSLC1 tumor suppressor gene represent later events associated with tumor invasion. [Copyright &y& Elsevier]

Background: DNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here, we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery. Results: The MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell® RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QiaSymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (p < 0.001). Vacuum concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4 were validated by quantitative methylation specific PCR. Conclusions: The MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients. [ABSTRACT FROM AUTHOR]

The cyclin-dependent kinase (CDK) 4/6 inhibitors belong to a new class of drugs that interrupt proliferation of malignant cells by inhibiting progression through the cell cycle. Three such inhibitors, palbociclib, ribociclib, and abemaciclib were recently approved for breast cancer treatment in various settings and combination regimens. On the basis of their impressive efficacy, all three CDK4/6 inhibitors now play an important role in the treatment of patients with HR+, HER2− breast cancer; however, their optimal use still needs to be established. The three drugs have many similarities in both pharmacokinetics and pharmacodynamics. However, there are some differences on the basis of which the choice for a particular CDK4/6 inhibitor for an individual patient can be important. In this article, the clinical pharmacokinetic and pharmacodynamic profiles of the three CDK4/6 inhibitors are reviewed and important future directions of the clinical applicability of CDK4/6 inhibitors will be discussed. [ABSTRACT FROM AUTHOR]

Squamous cell carcinoma (SCC) and adenocarcinoma (AC) represent the major cervical cancer histotypes. Both histotypes are caused by infection with high-risk HPV (hrHPV) and are associated with deregulated microRNA expression. Histotype-dependent expression has been observed for miR-9-5p, showing increased expression in SCC and low expression in AC. Here, we studied the regulation and functionality of miR-9-5p in cervical SCCs and ACs using cervical tissue samples and hrHPV-containing cell lines. Expression and methylation analysis of cervical tissues revealed that low levels of miR-9-5p in ACs are linked to methylation of its precursor genes, particularly miR-9-1. Stratification of tissue samples and hrHPV-containing cell lines suggested that miR-9-5p depends on both histotype and hrHPV type, with higher expression in SCCs and HPV16-positive cells. MiR-9-5p promoted cell viability and anchorage independence in cervical cancer cell lines SiHa (SCC, HPV16) and CaSki (metastasized SCC, HPV16), while it played a tumor suppressive role in HeLa (AC, HPV18). TWIST1, a transcription factor involved in epithelial-to-mesenchymal transition (EMT), was established as a novel miR-9-5p target. Our results show that miR-9-5p plays a dual role in cervical cancer in a histotype- and hrHPV type-dependent manner. MiR-9-5p mediated silencing of TWIST1 suggests two distinct mechanisms towards EMT in cervical cancer. [ABSTRACT FROM AUTHOR]

Abstract: Vulvar squamous cell carcinoma (VSCC) and precancerous vulvar intraepithelial neoplasia (VIN) can develop through human papillomavirus (HPV)‐dependent and ‐independent pathways, indicating a heterogeneous disease. Only a minority of VIN progress, but current clinicopathological classifications are insufficient to predict the cancer risk. Here we analyzed copy number alterations (CNA) to assess the molecular heterogeneity of vulvar lesions in relation to HPV and cancer risk. HPV‐status and CNA by means of whole‐genome next‐generation shallow‐sequencing were assessed in VSCC and VIN. The latter included VIN of women with associated VSCC (VINVSCC) and women who did not develop VSCC during follow‐up (VINnoVSCC). HPV‐testing resulted in 41 HPV‐positive (16 VINVSCC, 14 VINnoVSCC, and 11 VSCC) and 24 HPV‐negative (11 VINVSCC and 13 VSCC) lesions. HPV‐positive and ‐negative VSCC showed a partially overlapping pattern of recurrent CNA, including frequent gains of 3q and 8q. In contrast, amplification of 11q13/cyclinD1 was exclusively found in HPV‐negative lesions. HPV‐negative VINVSCC had less CNA than HPV‐negative VSCC (P = .009), but shared chromosome 8 alterations. HPV‐positive VINnoVSCC had less CNA than VINVSCC (P = .022). Interestingly, 1pq gain was detected in 81% of HPV‐positive VINVSCC and only in 21% of VINnoVSCC (P = .001). In conclusion, HPV‐dependent and ‐independent vulvar carcinogenesis is characterized by distinct CNA patterns at the VIN stage, while more comparable patterns are present at the cancer stage. Cancer risk in VIN seems to be reflected by the extent of CNA, in particular chromosome 1 gain in HPV‐positive cases. [ABSTRACT FROM AUTHOR]

Background: Current normalization methods for RNA-sequencing data allow either for intersample comparison to identify differentially expressed (DE) genes or for intrasample comparison for the discovery and validation of gene signatures. Most studies on optimization of normalization methods typically use simulated data to validate methodologies. We describe a new method, GeTMM, which allows for both inter- and intrasample analyses with the same normalized data set. We used actual (i.e. not simulated) RNA-seq data from 263 colon cancers (no biological replicates) and used the same read count data to compare GeTMM with the most commonly used normalization methods (i.e. TMM (used by edgeR), RLE (used by DESeq2) and TPM) with respect to distributions, effect of RNA quality, subtype-classification, recurrence score, recall of DE genes and correlation to RT-qPCR data. Results: We observed a clear benefit for GeTMM and TPM with regard to intrasample comparison while GeTMM performed similar to TMM and RLE normalized data in intersample comparisons. Regarding DE genes, recall was found comparable among the normalization methods, while GeTMM showed the lowest number of false-positive DE genes. Remarkably, we observed limited detrimental effects in samples with low RNA quality. Conclusions: We show that GeTMM outperforms established methods with regard to intrasample comparison while performing equivalent with regard to intersample normalization using the same normalized data. These combined properties enhance the general usefulness of RNA-seq but also the comparability to the many array-based gene expression data in the public domain. [ABSTRACT FROM AUTHOR]

For many high-dimensional studies, additional information on the variables, like (genomic) annotation or external p-values, is available. In the context of binary and continuous prediction, we develop a method for adaptive group-regularized (logistic) ridge regression, which makes structural use of such 'co-data'. Here, 'groups' refer to a partition of the variables according to the co-data. We derive empirical Bayes estimates of group-specific penalties, which possess several nice properties: (i) They are analytical. (ii) They adapt to the informativeness of the co-data for the data at hand. (iii) Only one global penalty parameter requires tuning by cross-validation. In addition, the method allows use of multiple types of co-data at little extra computational effort. We show that the group-specific penalties may lead to a larger distinction between 'near-zero' and relatively large regression parameters, which facilitates post hoc variable selection. The method, termed GRridge, is implemented in an easy-to-use R-package. It is demonstrated on two cancer genomics studies, which both concern the discrimination of precancerous cervical lesions from normal cervix tissues using methylation microarray data. For both examples, GRridge clearly improves the predictive performances of ordinary logistic ridge regression and the group lasso. In addition, we show that for the second study, the relatively good predictive performance is maintained when selecting only 42 variables. [ABSTRACT FROM AUTHOR]

Simple Summary: Colon cancer (CC) is one of the most common types of cancer. Circular RNAs (circRNAs) appear to play an important role in tumor progression of CC. They are stably expressed in saliva, blood, and exosomes, potentially rendering them promising biomarkers for the diagnosis, prognosis, and treatment of CC. In this study we describe the identification of an extensive catalog of circRNAs in a large cohort of 181 chemonaive, stage I/II primary colon tumors and related circRNA expression to consensus molecular subtypes (CMS), microsatellite instability (MSI) status and clinical outcome. We observed that a high diversity in circRNAs was associated with favorable disease-free survival, and that several circRNAs were associated with MSI and CMS, demonstrating the potential clinical value of circRNAs in CC. Circular RNAs (circRNAs) appear important in tumor progression of colon cancer (CC). We identified an extensive catalog of circRNAs in 181 chemonaive stage I/II colon tumors, who underwent curative surgery between 2007 and 2014. We identified circRNAs from RNAseq data, investigated common biology related to circRNA expression, and studied the association between circRNAs and relapse status, tumor stage, consensus molecular subtypes (CMS), tumor localization and microsatellite instability (MSI). We identified 2606 unique circRNAs. 277 circRNAs (derived from 260 genes) were repeatedly occurring in at least 20 patients of which 153 showed a poor or even negative (R < 0.3) correlation with the expression level of their linear gene. The circular junctions for circSATB2, circFGD6, circKMT2C and circPLEKHM3 were validated by Sanger sequencing. Multiple correspondence analysis showed that circRNAs were often co-expressed and that high diversity in circRNAs was associated with favorable disease-free survival (DFS), which was confirmed by Cox regression analysis (Hazard Ratio (HR) 0.60, 95% CI 0.38–0.97, p = 0.036). Considering individual circRNAs, absence of circMGA was significantly associated with relapse, whereas circSATB2, circNAB1, and circCEP192 were associated with both MSI and CMS. This study represents a showcase of the potential clinical utility of circRNAs for prognostic stratification in patients with stage I–II colon cancer and demonstrated that high diversity in circRNAs is associated with favorable DFS. [ABSTRACT FROM AUTHOR]

Simple Summary: Currently, the most commonly used method to monitor response to treatment in metastatic breast cancer patients is by radiological imaging. However, these imaging techniques are relatively insensitive and give little to no insight into biological tumor characteristics that might be relevant for the choice of treatment. Circulating tumor DNA (ctDNA), released by tumor cells into the blood of cancer patients, can be used to overcome these shortcomings. Besides the fact that specific alterations are known to predict response to treatment and development of resistance, the total amount of ctDNA is believed to reflect the proliferation rate of the tumor, suggesting ctDNA levels can be used as a general tool to evaluate treatment response. Different methods are available to measure ctDNA primarily based on detection of cancer-specific somatic mutations, DNA methylation, and copy number variations. In this review we have critically analyzed recently published studies using blood-derived ctDNA of metastatic breast cancer patients on multiple time points to monitor disease response in respect to analytical validity and clinical utility. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Primary testing for high-risk HPV (hrHPV) is increasingly implemented in cervical cancer screening programs. Many hrHPV-positive women, however, harbor clinically irrelevant infections, demanding additional disease markers to prevent over-referral and over-treatment. Most promising biomarkers reflect molecular events relevant to the disease process that can be measured objectively in small amounts of clinical material, such as miRNAs. We previously identified eight miRNAs with altered expression in cervical precancer and cancer due to either methylation-mediated silencing or chromosomal alterations. In this study, we evaluated the clinical value of these eight miRNAs on cervical scrapes to triage hrHPV-positive women in cervical screening. Results: Expression levels of the eight candidate miRNAs in cervical tissue samples (n = 58) and hrHPV-positive cervical scrapes from a screening population (n = 187) and cancer patients (n = 38) were verified by quantitative RT-PCR. In tissue samples, all miRNAs were significantly differentially expressed (p < 0.05) between normal, high-grade precancerous lesions (CIN3), and/or cancer. Expression patterns detected in cervical tissue samples were reflected in cervical scrapes, with five miRNAs showing significantly differential expression between controls and women with CIN3 and cancer. Using logistic regression analysis, a miRNA classifier was built for optimal detection of CIN3 in hrHPV-positive cervical scrapes from the screening population and its performance was evaluated using leave-one-out cross-validation. This miRNA classifier consisted of miR-15b-5p and miR-375 and detected a major subset of CIN3 as well as all carcinomas at a specificity of 70%. The CIN3 detection rate was further improved by combining the two miRNAs with HPV16/18 genotyping. Interestingly, both miRNAs affected the viability of cervical cancer cells in vitro. Conclusions: This study shows that miRNA expression analysis in cervical scrapes is feasible and enables the early detection of cervical cancer, thus underlining the potential of miRNA expression analysis for triage of hrHPV-positive women in cervical cancer screening. [ABSTRACT FROM AUTHOR]