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Genome-scale screening experiments in cancer produce long lists of candidate genes that require extensive interpretation for biological insight and prioritization for follow-up studies. Interrogation of gene lists frequently represents a significant and time-consuming undertaking, in which experimental biologists typically combine results from a variety of bioinformatics resources in an attempt to portray and understand cancer relevance. As a means to simplify and strengthen the support for this endeavor, we have developed oncoEnrichR, a flexible bioinformatics tool that allows cancer researchers to comprehensively interrogate a given gene list along multiple facets of cancer relevance. oncoEnrichR differs from general gene set analysis frameworks through the integration of an extensive set of prior knowledge specifically relevant for cancer, including ranked gene-tumor type associations, literature-supported proto-oncogene and tumor suppressor gene annotations, target druggability data, regulatory interactions, synthetic lethality predictions, as well as prognostic associations, gene aberrations and co-expression patterns across tumor types. The software produces a structured and user-friendly analysis report as its main output, where versions of all underlying data resources are explicitly logged, the latter being a critical component for reproducible science. We demonstrate the usefulness of oncoEnrichR through interrogation of two candidate lists from proteomic and CRISPR screens. oncoEnrichR is freely available as a web-based service hosted by the Galaxy platform (https://oncotools.elixir.no), and can also be accessed as a stand-alone R package (https://github.com/sigven/oncoEnrichR)., (© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

The value of high-throughput germline genetic testing is increasingly recognized in clinical cancer care. Disease-associated germline variants in cancer patients are important for risk management and surveillance, surgical decisions and can also have major implications for treatment strategies since many are in DNA repair genes. With the increasing availability of high-throughput DNA sequencing in cancer clinics and research, there is thus a need to provide clinically oriented sequencing reports for germline variants and their potential therapeutic relevance on a per-patient basis. To meet this need, we have developed the Cancer Predisposition Sequencing Reporter (CPSR), an open-source computational workflow that generates a structured report of germline variants identified in known cancer predisposition genes, highlighting markers of therapeutic, prognostic and diagnostic relevance. A fully automated variant classification procedure based on more than 30 refined American College of Medical Genetics and Genomics (ACMG) criteria represents an integral part of the workflow. Importantly, the set of cancer predisposition genes profiled in the report can be flexibly chosen from more than 40 virtual gene panels established by scientific experts, enabling customization of the report for different screening purposes and clinical contexts. The report can be configured to also list actionable secondary variant findings, as recommended by ACMG. CPSR demonstrates comparable sensitivity and specificity for the detection of pathogenic variants when compared to other algorithms in the field. Technically, the tool is implemented in Python/R, and is freely available through Docker technology. Source code, documentation, example reports and installation instructions are accessible via the project GitHub page: https://github.com/sigven/cpsr., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Colorectal cancer (CRC) is one of the most common cancers in Latin America and the Caribbean, with the highest rates reported for Uruguay, Brazil and Argentina. We provide a global snapshot of the CRC patterns, how screening is performed, and compared/contrasted to the genetic profile of Lynch syndrome (LS) in the region. From the literature, we find that only nine (20%) of the Latin America and the Caribbean countries have developed guidelines for early detection of CRC, and also with a low adherence. We describe a genetic profile of LS, including a total of 2,685 suspected families, where confirmed LS ranged from 8% in Uruguay and Argentina to 60% in Peru. Among confirmed LS, path_MLH1 variants were most commonly identified in Peru (82%), Mexico (80%), Chile (60%), and path_MSH2/EPCAM variants were most frequently identified in Colombia (80%) and Argentina (47%). Path_MSH6 and path_PMS2 variants were less common, but they showed important presence in Brazil (15%) and Chile (10%), respectively. Important differences exist at identifying LS families in Latin American countries, where the spectrum of path_MLH1 and path_MSH2 variants are those most frequently identified. Our findings have an impact on the evaluation of the patients and their relatives at risk for LS, derived from the gene affected. Although the awareness of hereditary cancer and genetic testing has improved in the last decade, it is remains deficient, with 39%-80% of the families not being identified for LS among those who actually met both the clinical criteria for LS and showed MMR deficiency., (© 2018 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Lung cancer is the leading cause of cancer related death, and the past years' improved insight into underlying molecular events has significantly improved outcome for specific subsets of patients. In particular, several new therapies that target protein kinases have been implemented, and many more are becoming available. We have investigated lung cancer specimens for somatic mutations in a targeted panel of 612 human genes, the majority being protein kinases. The somatic mutation profiles were correlated to profiles of immune cell infiltration as well as relapse-free survival. Targeted deep sequencing was performed on 117 tumour/normal pairs using the SureSelect Human Kinome kit (Agilent Technologies), with capture probes targeting 3.2 Mb of the human genome, including exons and untranslated regions of all known kinases, kinase receptors and selected cancer-related genes (612 genes in total). CD8 staining was determined using Ventana Benchmark. Survival analyses were performed using SPSS. The number of mutations per sample ranged from 0 to 50 (within the 612 genes tested), with a median of nine. The prognosis was worse for patients with more than the median number of mutations. A significant correlation was found between mutations in one of selected DNA-repair genes and the total number of mutations in that tumour (p < 0.001). There was a significant inverse correlation between the number of infiltrating stromal CD8+ lymphocytes and the presence of EGFR mutations., (© 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Robust markers of invasiveness may help reduce the overtreatment of in situ carcinomas. Breast cancer is a heterogeneous disease and biological mechanisms for carcinogenesis vary between subtypes. Stratification by subtype is therefore necessary to identify relevant and robust signatures of invasive disease. We have identified microRNA (miRNA) alterations during breast cancer progression in two separate datasets and used stratification and external validation to strengthen the findings. We analyzed two separate datasets (METABRIC and AHUS) consisting of a total of 186 normal breast tissue samples, 18 ductal carcinoma in situ (DCIS) and 1,338 invasive breast carcinomas. Validation in a separate dataset and stratification by molecular subtypes based on immunohistochemistry, PAM50 and integrated cluster classifications were performed. We propose subtype-specific miRNA signatures of invasive carcinoma and a validated signature of DCIS. miRNAs included in the invasive signatures include downregulation of miR-139-5p in aggressive subtypes and upregulation of miR-29c-5p expression in the luminal subtypes. No miRNAs were differentially expressed in the transition from DCIS to invasive carcinomas on the whole, indicating the need for subtype stratification. A total of 27 miRNAs were included in our proposed DCIS signature. Significant alterations of expression included upregulation of miR-21-5p and the miR-200 family and downregulation of let-7 family members in DCIS samples. The signatures proposed here can form the basis for studies exploring DCIS samples with increased invasive potential and serum biomarkers for in situ and invasive breast cancer., (© 2016 UICC.)

In selectively neutral regions of the human genome, nucleotide substitutions do not occur at random with respect to the local DNA sequence neighborhood. However, apart from the hypermutability of methylated CpG dinucleotides, which can explain the overrepresentation of nucleotide transitions in this context, the sequence-specific factors underlying point mutation bias remain largely to be determined, both in nature and in quantitative impact. One hypothesis suggests that the physical characteristics of a DNA context could have a modulating effect on its mutability, adjusting the impact of damage or the efficiency of repair. Here, we report a genome-wide computational test of this hypothesis, in which we utilize a constrained set of human non-CpG SNPs as the source of selectively neutral germline mutations. Interestingly, we observe that the quantitative context-dependencies of some substitution types display significant associations to measures of local structural topography and helix stability in DNA. Most prominently, we find that the local sequence bias of transition mutations is significantly associated with the sequence-dependent level of helix instability imposed by the potentially underlying DNA mismatches. The results of our work indicate the extent to which DNA physical properties could have shaped the recent point mutational spectrum in the human genome., (© 2010 Wiley-Liss, Inc.)

The metastasis-promoting protein S100A4 stimulates metastatic progression through both intracellular and extracellular functions. Extracellular activities of S100A4 include stimulation of angiogenesis, regulation of cell death and increased cell motility and invasion, but the exact molecular mechanisms by which extracellular S100A4 exerts these effects are incompletely elucidated. The aim of the present study was to characterize S100A4-induced signal transduction mechanisms and to identify S100A4 target genes. We demonstrate that extracellular S100A4 activates the transcription factor NF-kappaB in a subset of human cancer cell lines through induction of phosphorylation and subsequent degradation of the NF-kappaB inhibitor IkappaBalpha. Concomitantly, S100A4 induced a sustained activation of the MAP kinase JNK, whereas no increased activity of the MAP kinases p38 or ERK was observed. Microarray analyses identified 136 genes as being significantly regulated by S100A4 treatment, and potentially interesting S100A4-induced gene products include IkappaBalpha, p53, ephrin-A1 and optineurin. Increased expression of ephrin-A1 and optineurin was validated using RT-PCR, Western blotting and functional assays. Furthermore, S100A4-stimulated transcription of these target genes was dependent on activation of the NF-kappaB pathway. In conclusion, these findings contribute to the understanding of the complex molecular mechanisms responsible for the diverse biological functions of extracellular S100A4, and provide further evidence of how S100A4 may stimulate metastatic progression., (Copyright 2008 Wiley-Liss, Inc.)

The human malignant B-lymphocyte cell lines Reh and U698 show arrest in G2 phase after ionizing radiation (IR), but only Reh cells arrest in G1 phase and die by apoptosis. We have used cDNA microarrays to measure changes in gene expression at 2, 4 and 6 hr after irradiation of Reh and U698 cells with 0.5 and 4 Gy in order to begin exploring the molecular mechanisms underlying the phenotypic changes. We also investigated whether gene expression changes could be caused by possible aberrations of genes, as measured by comparative genomic hybridization. Reh cells showed upregulation of CDKN1A that likely mediated the G1 arrest. In contrast, U698 cells have impaired function of TP53 protein and no activation of CDKN1A, suppressing the arrest in G1. The G2 arrest in both cell lines was likely due to repression of PLK1 and/or CCNF. IR-induced apoptosis in Reh cells was probably mediated by TP53 and CDKN1A, whereas a high expression level of MCL1, caused by gene amplification, and activation of the NFKB pathway may have suppressed the apoptotic response in U698 cells. Genes suggested to be involved in apoptosis were activated long before this phenotype was detectable and showed the same temporal expression profiles as genes involved in cell cycle arrest. Our results suggest that differences in functionality and/or copy number of several genes involved in IR-regulated pathways contributed to the phenotypic differences between Reh and U698 cells after IR, and that multiple molecular factors control the radiation response of malignant B lymphocytes., (Copyright 2005 Wiley-Liss, Inc.)

The red bone marrow (BM) is an important indicator organ of hematogenous micrometastatic spread of carcinomas. Characterization of biological properties specific for BM micrometastatic cells, however, is technically challenging due to the limited number of target cells usually available for the purpose. This report provides referrals to qualitative gene expression profiling of BM micrometastatic cells enriched by immunomagnetic selection. First, an experimental strategy was used to study regulatory mechanisms involved when BM micrometastatic cells colonize distant organs. The MA-11 cells, originating from BM micrometastases in a breast cancer patient clinically devoid of overt metastatic disease, were injected into immunodeficient rats. Metastatic MA-11 cells were subsequently immunoselected from the resulting in vivo lesions. The selected cell populations were compared to the injected cells by differential display analysis, and several genes possibly involved in tumor cell invasion and proliferation were confirmed as differentially expressed among the various MA-11 cell populations. A direct approach to qualitative gene expression profiling of BM micrometastatic cells was also explored. Carcinoma cells were immunoselected from BM and axillary lymph nodes obtained from breast cancer patients, and the isolated cell populations were compared by differential display analysis. Two candidate genes, identified as factors involved in cellular growth control, appeared as differentially expressed by the target cells from BM. Our study provides detailed information on how to combine an immunomagnetic selection procedure and differential display analysis to reveal gene expression profiles that may characterize BM micrometastatic cells., (Copyright 2002 Wiley-Liss, Inc.)

In many recent publications, it has been claimed that reverse transcriptase-polymerase chain reaction (RT-PCR) assays involving genes with tissue-restricted expression can be used for specific and sensitive detection of cancer cells in blood, bone marrow and lymph nodes. Many different target mRNAs have been evaluated for such purposes. One of the most extensively studied genes, CK19, is predominantly expressed in cells of epithelial origin and normally not at detectable levels in hematopoietic or lymphatic tissues. Based on previous reports on CK19 we wanted to establish a useful assay for detection of micrometastatic cells. RNA and DNA specimens extracted from peripheral blood nucleated cells of healthy volunteers, as well as cell lines positive and negative for CK19 expression, were used in nested RT-PCR assays. Using previously published primers, we found a novel pseudogene that shows a high degree of identity with the CK19 gene sequence, except for differences caused by 3 small deletions and a number of point mutations, resulting in termination codons and frameshifts. The gene has therefore no coding potential. Importantly, published primer sequences and reaction conditions used by several other groups to detect CK19 mRNA may have led to the amplification of this pseudogene. The data illustrate one of the problems that must be addressed in validating RT-PCR assays for micrometastasis detection, and it is suggested that previous work using CK19 as a marker should be reassessed in view of the present finding.

Three members of the S100 gene family, S100A2, S100A4 and S100A6, have been suggested to be associated with cancer development and metastasis. To study their involvement in the tumorigenesis of human melanoma, we examined the mRNA expression levels of the 3 genes in 45 melanoma metastases and in 20 benign nevi. Interestingly, whereas none of the metastases expressed S100A2 mRNA, and the expression level was low in 6 cell lines established from primary melanomas, all nevi showed moderate to high expression levels. Our results suggest that loss of S100A2 gene expression may be an early event in melanoma development. A significant correlation was found between the expression of S100A6 in melanoma metastases and both the survival time of the patients and the thickness of the corresponding primary tumors. For the S100A4 gene, however, no relationship was found between gene expression and clinical parameters of melanoma malignancy. The observed differences in expression patterns of the 3 S100 genes suggest distinct roles of their products in melanoma tumorigenesis and/or metastasis, and the results encourage studies to evaluate the potential value of using S100A2 and S100A6 expression levels as markers in the clinical management of melanoma.

Our objective was to develop and study the feasibility of a quantitative, nested reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of micrometastatic, epithelial tumor cells using the epithelial glycoprotein EGP-2 gene as a target. Several carcinoma cell lines and peripheral blood samples of 10 healthy volunteers were screened for levels of EGP-2 mRNA. The assay included EGP-2 competitor molecules, carrying an internal deletion, that had been titrated by limiting dilution. Seven carcinoma cell lines showed a wide spectrum of EGP-2 mRNA expression levels, with the highest values (20-100 molecules/cell) seen in 3 breast-cancer cell lines. Unexpectedly, a consistent low level of EGP-2 mRNA expression (0.0004 molecules/cell) was observed in peripheral blood mononuclear cells, probably representing ectopic non-functional expression. Because of this background level, spiking experiments with T47D breast-carcinoma cells added to blood mononuclear cells exhibited a detection limit that was not better than approximately one tumor cell in 2 x 10(4) normal cells. Together with the considerable variation of EGP-2 transcript levels that is observed in different carcinoma cell lines, the extent of expression in normal blood cells would prevent a reliable estimation of low numbers of carcinoma cells in clinical samples. A similar situation might well apply for other target genes. This emphasizes the need for a critical evaluation of the different steps involved in the methods used for RT-PCR detection of micrometastatic tumor cells.

The cell cycle is composed of a series of steps that can be negatively or positively regulated by various factors. A group of low-molecular-weight proteins have recently been identified that specifically inhibit the function of cyclin-dependent kinases in mammalian cells. Inactivation of the CDKN2A gene (also known as p16INK4A and MTS1) attracted considerable interest after it was mapped to 9p21, a locus for familial melanoma. In an effort to standardize the information regarding human CDKN2A mutations detected in cancers, a database with information of 146 point mutations has been created. Cancer type, origin of cells, specific mutation, amino acid change, literature citation, and other data are provided for each mutation entry. Studies of biochemical and biological functions of both wild-type and mutant proteins are central to our understanding of the role of p16INK4a mutations in tumorigenesis, a summary of these studies is also included in the present update.

The spectrum of somatic TP53 single basepair substitutions detected in 955 cancers was compared with that of 2,224 different germline mutations in 279 different human genes (other than TP53), reported as the cause of inherited disease. This comparison reveals that, disregarding a relatively small subset (12%) of TP53 mutations that probably result from the action of exogenous mutagens, both the relative rates and the nearest-neighbor spectra of single basepair substitutions are similar in the two datasets. This spectral resemblance suggests that a substantial proportion of cancer-associated somatic TP53 mutations result from endogenous cellular mechanisms. The likelihood of clinical observation of a particular mutation type differs, however, between tumors and genetic diseases, when the chemical properties of the resulting amino acid substitutions are considered. Together with a sixfold higher observation likelihood for mutations at evolutionarily conserved residues, this finding argues that selection is a critical factor in determining which TP53 mutations are found to be associated with human cancer.

Studies of mutant genotypes of the retinoblastoma susceptibility gene (RB1) in different solid tumors have mainly been concentrated on the demonstration of loss of heterozygosity (LOH) at both internal and external polymorphic sites. One reason for this is the complex organization of the gene. The p105RB protein has been shown to interact with both DNA and regulatory cellular proteins and oncoproteins. The amino acids encoded by exon 21 are implicated in several of these interactions. Both point mutations and intragenic deletions involving exon 21 have previously been reported in human tumors. We have examined RB1 exon 21 from a number of human tumor types where significant LOH in or around the RB1 gene has been reported. DNA from 78 primary tumors was amplified using the polymerase chain reaction (PCR) with primers covering exon 21, followed by constant denaturant gel electrophoresis (CDGE). The 78 tumors included 11 breast carcinomas, 30 nonsmall cell lung carcinomas, 6 colon carcinomas, and 31 sarcomas. The small cell lung cancer cell line NCI-H209, previously shown to harbour a point mutation in codon 706: TGT- greater than TTT (Cys- greater than Phe), was detected using CDGE. Apart from this control mutant cell line, we did not detect any mutations in the examined region in any of the tumors.