The multitarget faecal immunochemical test for improving stool-based colorectal cancer screening programmes: a Dutch population-based, paired-design, intervention study.

BACKGROUND: The faecal immunochemical test (FIT) is widely employed for colorectal cancer screening. However, its sensitivity for advanced precursor lesions remains suboptimal. The multitarget FIT (mtFIT), measuring haemoglobin, calprotectin, and serpin family F member 2, has demonstrated enhanced sensitivity for advanced neoplasia, especially advanced adenomas, at equal specificity to FIT. This study aimed to prospectively validate and investigate the clinical utlitity of mtFIT versus FIT in a setting of population-based colorectal cancer screening. METHODS: Individuals aged 55-75 years and who were eligible for the Dutch national FIT-based colorectal cancer screening programme were invited to submit both a FIT and mtFIT sample collected from the same bowel movement. Positive FIT (47 µg/g haemoglobin cutoff) or mtFIT (based on decision-tree algorithm) led to a colonoscopy referral. The primary outcome was the relative detection rate of mtFIT versus FIT for all advanced neoplasia. Secondary outcomes were the relative detection rates of colorectal cancer, advanced adenoma, and advanced serrated polyps individually and the long-term effect of mtFIT-based versus FIT-based programmatic screening on colorectal cancer incidence, mortality, and cost, determined with microsimulation modelling. The study has been registered in ClinicalTrials.gov, NCT05314309, and is complete. FINDINGS: Between March 25 and Dec 7, 2022, 35 786 individuals were invited to participate in the study, of whom 15 283 (42·7%) consented, and 13 187 (86·3%) of 15 283 provided both mtFIT and FIT samples with valid results. Of the 13 187 participants, 6637 (50·3%) were male and 6550 (49·7%) were female. mtFIT showed a 9·11% (95% CI 8·62-9·61) positivity rate and 2·27% (95% CI 2·02-2·54) detection rate for advanced neoplasia, compared with a positivity rate of 4·08% (3·75-4·43) and a detection rate of 1·21% (1·03-1·41) for FIT. Detection rates of mtFIT versus FIT were 0·20% (95% CI 0·13-0·29) versus 0·17% (0·11-0·27) for colorectal cancer; 1·64% (1·43-1·87) versus 0·86% (0·72-1·04) for advanced adenoma, and 0·43% (0·33-0·56) versus 0·17% (0·11-0·26) for advanced serrated polyps. Modelling demonstrated that mtFIT-based screening could reduce colorectal cancer incidence by 21% and associated mortality by 18% compared with the current Dutch colorectal cancer screening programme, at feasible costs. Furthermore, at equal positivity rates, mtFIT outperformed FIT in terms of diagnostic yield. At an equally low positivity rate, mtFIT-based screening was predicted to further decrease colorectal cancer incidence by 5% and associated mortality by 4% compared with FIT-based screening. INTERPRETATION: The higher detection rate of mtFIT for advanced adenoma compared with FIT holds the potential to translate into additional and clinically meaningful long-term colorectal cancer incidence and associated mortality reductions in programmatic colorectal cancer screening. FUNDING: Stand Up to Cancer, Dutch Cancer Society, Dutch Digestive Foundation, and Health~Holland.

Systematically linking tranSMART, Galaxy and EGA for reusing human translational research data.

The availability of high-throughput molecular profiling techniques has provided more accurate and informative data for regular clinical studies. Nevertheless, complex computational workflows are required to interpret these data. Over the past years, the data volume has been growing explosively, requiring robust human data management to organise and integrate the data efficiently. For this reason, we set up an ELIXIR implementation study, together with the Translational research IT (TraIT) programme, to design a data ecosystem that is able to link raw and interpreted data. In this project, the data from the TraIT Cell Line Use Case (TraIT-CLUC) are used as a test case for this system. Within this ecosystem, we use the European Genome-phenome Archive (EGA) to store raw molecular profiling data; tranSMART to collect interpreted molecular profiling data and clinical data for corresponding samples; and Galaxy to store, run and manage the computational workflows. We can integrate these data by linking their repositories systematically. To showcase our design, we have structured the TraIT-CLUC data, which contain a variety of molecular profiling data types, for storage in both tranSMART and EGA. The metadata provided allows referencing between tranSMART and EGA, fulfilling the cycle of data submission and discovery; we have also designed a data flow from EGA to Galaxy, enabling reanalysis of the raw data in Galaxy. In this way, users can select patient cohorts in tranSMART, trace them back to the raw data and perform (re)analysis in Galaxy. Our conclusion is that the majority of metadata does not necessarily need to be stored (redundantly) in both databases, but that instead FAIR persistent identifiers should be available for well-defined data ontology levels: study, data access committee, physical sample, data sample and raw data file. This approach will pave the way for the stable linkage and reuse of data.

Chromosomal gains and losses in human papillomavirus-associated neoplasia of the lower genital tract - a systematic review and meta-analysis.

BACKGROUND: Overexpression of the human papillomavirus (HPV) oncogenes E6 and E7 is necessary for the development of distinct lower genital tract cancers. However, secondary cellular genomic alterations are mandatory to promote progression of HPV-induced premalignant stages. We aimed at identifying the chromosomal regions most frequently gained and lost and the disease stage at which the latter occurs. These regions might be relevant for carcinogenesis and could serve as diagnostic markers to identify premalignant lesions with high progression risk towards invasive cancer. METHODS: We performed a systematic literature review and meta-analysis of studies listed in PubMed that analysed chromosomal copy number alterations by comparative genomic hybridisation (CGH) in HPV-positive and -negative cancers or premalignant lesions of the anogenital tract (cervix, anus, vagina, penis and vulva). FINDINGS: Data were extracted and analysed from 32 studies. The most common alterations in cervical squamous cell carcinoma (SCC) (12 studies, 293 samples) were gains at 3q with a rate of 0.55 (95% confidence interval (CI) 0.43-0.70), losses at 3p (0.36, 95%CI 0.27-0.48) and losses at 11q (0.33, 95%CI 0.26-0.43). Gains at 3q were particularly frequent in HPV16-positive cervical SCC (0.84, 95%CI 0.78-0.90). Also more than one quarter of high grade cervical intraepithelial neoplasia (CIN) harboured gains of 3q (0.27, 95%CI 0.20-0.36), but the rate in low grade CIN was low (0.02, 95%CI 0.00-0.09). For HPV-associated vulvar SCC (four studies, 30 samples) the same common alterations as in cervical SCC were reported. Studies on non-cervical and non-vulvar SCC and premalignant lesions of the lower genital tract are scarce. INTERPRETATION: 3q gains were most frequently found in HPV16-positive cervical SCC. The results suggest the selection of HPV-transformed cell clones harbouring 3q gains in high grade premalignant lesions, while alterations in low grade lesions are rare.

CADM1 and MAL promoter methylation levels in hrHPV-positive cervical scrapes increase proportional to degree and duration of underlying cervical disease.

Combined detection of cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation-associated protein (MAL) promoter methylation in cervical scrapes is a promising triage strategy for high-risk human papillomavirus (hrHPV)-positive women. Here, CADM1 and MAL DNA methylation levels were analysed in cervical scrapes of hrHPV-positive women with no underlying high-grade disease, high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. CADM1 and MAL methylation levels in scrapes were first related to CIN-grade of the corresponding biopsy and second to CIN-grade stratified by the presence of 'normal' or 'abnormal' cytology as present in the accompanying scrape preceding the cervical biopsy. The scrapes included 167 women with ≤ CIN1, 54 with CIN2/3 and 44 with carcinoma. In a separate series of hrHPV-positive scrapes of women with CIN2/3 (n = 48), methylation levels were related to duration of preceding hrHPV infection (PHI; <5 and ≥ 5 years). Methylation levels were determined by quantitative methylation-specific PCR and normal cytology scrapes of hrHPV-positive women with histologically ≤ CIN1 served as reference. CADM1 and MAL methylation levels increased proportional to severity of the underlying lesion, showing an increase of 5.3- and 6.2-fold in CIN2/3, respectively, and 143.5- and 454.9-fold in carcinomas, respectively, compared to the reference. Methylation levels were also elevated in CIN2/3 with a longer duration of PHI (i.e. 11.5- and 13.6-fold, respectively). Moreover, per histological category, methylation levels were higher in accompanying scrapes with abnormal cytology than in scrapes with normal cytology. Concluding, CADM1 and MAL promoter methylation levels in hrHPV-positive cervical scrapes are related to the degree and duration of underlying cervical disease and markedly increased in cervical cancer.

Identification of eight candidate target genes of the recurrent 3p12-p14 loss in cervical cancer by integrative genomic profiling.

The pathogenetic role, including its target genes, of the recurrent 3p12-p14 loss in cervical cancer has remained unclear. To determine the onset of the event during carcinogenesis, we used microarray techniques and found that the loss was the most frequent 3p event, occurring in 61% of 92 invasive carcinomas, in only 2% of 43 high-grade intraepithelial lesions (CIN2/3), and in 33% of 6 CIN3 lesions adjacent to invasive carcinomas, suggesting a role in acquisition of invasiveness or early during the invasive phase. We performed an integrative DNA copy number and expression analysis of 77 invasive carcinomas, where all genes within the recurrent region were included. We selected eight genes, THOC7, PSMD6, SLC25A26, TMF1, RYBP, SHQ1, EBLN2, and GBE1, which were highly down-regulated in cases with loss, as confirmed at the protein level for RYBP and TMF1 by immunohistochemistry. The eight genes were subjected to network analysis based on the expression profiles, revealing interaction partners of proteins encoded by the genes that were coordinately regulated in tumours with loss. Several partners were shared among the eight genes, indicating crosstalk in their signalling. Gene ontology analysis showed enrichment of biological processes such as apoptosis, proliferation, and stress response in the network and suggested a relationship between down-regulation of the eight genes and activation of tumourigenic pathways. Survival analysis showed prognostic impact of the eight-gene signature that was confirmed in a validation cohort of 74 patients and was independent of clinical parameters. These results support the role of the eight candidate genes as targets of the 3p12-p14 loss in cervical cancer and suggest that the strong selection advantage of the loss during carcinogenesis might be caused by a synergetic effect of several tumourigenic processes controlled by these targets.

Focal aberrations indicate EYA2 and hsa-miR-375 as oncogene and tumor suppressor in cervical carcinogenesis.

Cervical cancer results from persistent infection with high-risk human papillomavirus (hrHPV). Common genetic aberrations in cervical (pre)cancers encompass large genomic regions with numerous genes, hampering identification of driver genes. This study aimed to identify genes functionally involved in HPV-mediated transformation by analysis of focal aberrations (<3 Mb) in high-grade cervical intraepithelial neoplasia (hgCIN). Focal chromosomal aberrations were determined in high-resolution array comparative genomic hybridization data of 60 hgCIN. Genes located within focal aberrations were validated using 2 external gene expression datasets or qRT-PCR. Functional roles of candidate genes EYA2 (20q13) and hsa-miR-375 (2q35) were studied by siRNA-mediated knock-down and overexpression, respectively, in hrHPV-containing cell lines. We identified 74 focal aberrations encoding 305 genes. Concurrent altered expression in hgCIN and/or cervical carcinomas compared with normal cervical samples was shown for ATP13A3, HES1, OPA1, HRASLS, EYA2, ZMYND8, APOBEC2, and NCR2. Gene silencing of EYA2 significantly reduced viability, migratory capacity, and anchorage-independent growth of HPV16-transformed keratinocytes. For hsa-miR-375, a direct correlation between a (focal) loss and significantly reduced expression was found. Downregulation of hsa-miR-375 expression was confirmed in an independent series of cervical tissues. Ectopic expression of hsa-miR-375 in 2 cervical carcinoma cell lines reduced cellular viability. Our data provide a proof of concept that chromosomal aberrations are actively contributing to HPV-induced carcinogenesis and identify EYA2 and hsa-miR-375 as oncogene and tumor suppressor gene, respectively.

HPV type-related chromosomal profiles in high-grade cervical intraepithelial neoplasia.

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.

Chromosomal profiles of high-grade cervical intraepithelial neoplasia relate to duration of preceding high-risk human papillomavirus infection.

High-grade cervical intraepithelial neoplasia (CIN2/3) represents a heterogeneous disease both with respect to clinical behavior and chromosomal aberrations detected. We hypothesized that the extent of chromosomal aberrations reflects the duration of their existence. Chromosomal profiles were determined of CIN3 of women with a known 5-year history of high-risk human papillomavirus virus (hrHPV) infection, in which duration of prior hrHPV infection was considered a proxy for duration of CIN3 existence. Eleven women had a <5 year preceding hrHPV infection (CIN3<5yrPHI) and 24 had a PHI lasting ≥5 years (CIN3≥5yrPHI). For comparison, six CIN3 adjacent to squamous cell carcinomas (CIN3-SCC), the corresponding SCCs, and six CIN1 were included. Unsupervised hierarchical clustering analysis of the chromosomal profiles revealed two clusters. One was characterized by a low number of chromosomal aberrations and included all CIN1, 81.8% of CIN3<5yrPHI and 33.3% of CIN3≥5yrPHI. Samples in the second cluster, displaying multiple aberrations, included 18.2% of CIN3<5yrPHI, 66.7% CIN3≥5yrPHI, all except one CIN3-SCC and all SCCs. The number of genomic aberrations increased according to lesion grade and also with longer duration of PHI. The increase in aberrations in CIN3≥5yrPHI compared to <5yrPHI was highly significant (p = 0.001), suggesting that CIN3≥5yrPHI represent more severe lesions. In conclusion, longer duration of preceding hrHPV infection is associated with an increased number of chromosomal aberrations. Hence, CIN3 with a longer duration of existence are likely more prone to have an increased short-term risk of cervical cancer.

Repression of MAL tumour suppressor activity by promoter methylation during cervical carcinogenesis.

We recently identified MAL (T-lymphocyte maturation associated protein) as the most down-regulated gene in cervical oncogenesis. Here, we examined the mechanism underlying MAL silencing, its functional role in cervical carcinogenesis, and the relevance of detecting MAL alterations for risk assessment of hrHPV-positive women. MAL mRNA expression and promoter methylation were analysed in primary keratinocytes, hrHPV-immortalized keratinocytes, cervical cancer cell lines, biopsies, and scrapings by quantitative (methylation-specific) PCR. SiHa cells were transfected with MAL cDNA and assayed for proliferation, migration, and anchorage-independent growth. MAL mRNA was (nearly) undetectable in all HPV-immortalized and cervical cancer cells, but could be up-regulated upon methylation inhibition. MAL promoter methylation at two promoter regions (M1 and M2) was detected in all HPV-immortalized cells and cancer cells. Ectopic expression of MAL in SiHa cells suppressed proliferation, migration, and anchorage-independent growth. None (0/22) of normal cervical biopsies, 9% (6/66) of CIN1 lesions, 53% (34/64) of CIN3 lesions, 90% (85/94) of cervical squamous cell carcinomas (SCCs), and 93% (26/28) of cervical adenocarcinomas (AdCAs) demonstrated MAL promoter methylation at both promoter regions. Moreover, detection of MAL promoter methylation in cervical scrapings was predictive for underlying high-grade lesions. Both in biopsies and in scrapings, MAL promoter methylation was significantly correlated with reduced mRNA expression. MAL gene silencing by promoter methylation is a frequent and biologically essential event in HPV-induced cervical carcinogenesis. Hence, MAL promoter methylation and/or mRNA expression analysis on cervical scrapings may provide a valuable diagnostic tool to improve the detection of CIN3, SCC, and AdCA.