Development of a miRNA-based classifier for detection of colorectal cancer molecular subtypes.

Previously, colorectal cancer (CRC) has been classified into four distinct molecular subtypes based on transcriptome data. These consensus molecular subtypes (CMSs) have implications for our understanding of tumor heterogeneity and the prognosis of patients. So far, this classification has been based on the use of messenger RNAs (mRNAs), although microRNAs (miRNAs) have also been shown to play a role in tumor heterogeneity and biological differences between CMSs. In contrast to mRNAs, miRNAs have a smaller size and increased stability, facilitating their detection. Therefore, we built a miRNA-based CMS classifier by converting the existing mRNA-based CMS classification using machine learning (training dataset of n = 271). The performance of this miRNA-assigned CMS classifier (CMS-miRaCl) was evaluated in several datasets, achieving an overall accuracy of ~ 0.72 (0.6329-0.7987) in the largest dataset (n = 158). To gain insight into the biological relevance of CMS-miRaCl, we evaluated the most important features in the classifier. We found that miRNAs previously reported to be relevant in microsatellite-instable CRCs or Wnt signaling were important features for CMS-miRaCl. Following further studies to validate its robustness, this miRNA-based alternative might simplify the implementation of CMS classification in clinical workflows.

Variant profiling of colorectal adenomas from three patients of two families with MSH3-related adenomatous polyposis.

The spectrum of somatic genetic variation in colorectal adenomas caused by biallelic pathogenic germline variants in the MSH3 gene, was comprehensively analysed to characterise mutational signatures and identify potential driver genes and pathways of MSH3-related tumourigenesis. Three patients from two families with MSH3-associated polyposis were included. Whole exome sequencing of nine adenomas and matched normal tissue was performed. The amount of somatic variants in the MSH3-deficient adenomas and the pattern of single nucleotide variants (SNVs) was similar to sporadic adenomas, whereas the fraction of small insertions/deletions (indels) (21-42% of all small variants) was significantly higher. Interestingly, pathogenic somatic APC variants were found in all but one adenoma. The vast majority (12/13) of these were di-, tetra-, or penta-base pair (bp) deletions. The fraction of APC indels was significantly higher than that reported in patients with familial adenomatous polyposis (FAP) (p < 0.01) or in sporadic adenomas (p < 0.0001). In MSH3-deficient adenomas, the occurrence of APC indels in a repetitive sequence context was significantly higher than in FAP patients (p < 0.01). In addition, the MSH3-deficient adenomas harboured one to five (recurrent) somatic variants in 13 established or candidate driver genes for early colorectal carcinogenesis, including ACVR2A and ARID genes. Our data suggest that MSH3-related colorectal carcinogenesis seems to follow the classical APC-driven pathway. In line with the specific function of MSH3 in the mismatch repair (MMR) system, we identified a characteristic APC mutational pattern in MSH3-deficient adenomas, and confirmed further driver genes for colorectal tumourigenesis.

The recurring features of molecular subtypes in distinct gastrointestinal malignancies-A systematic review.

In colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC) and gastric cancer (GC) multiple studies of inter-tumor heterogeneity have identified molecular subtypes, which correlate with clinical features. Our aim was to investigate the attributes of molecular subtypes across three different gastrointestinal cancer types. We performed a systematic search for publications on molecular subtypes or classifications in PDAC and GC and compared the described subtypes with the established consensus molecular subtypes of CRC. Examining the characteristics of subtypes across CRC, PDAC and GC resulted in four categories of subtypes. We describe uniting and distinguishing features within a mesenchymal, an epithelial, an immunogenic and a metabolic and digestive subtype category. We conclude that molecular subtypes of CRC, PDAC and GC display relevant overlap in molecular features and clinical outcomes. This finding encourages quantitative studies on subtypes across different cancer types and could lead to a paradigm shift in future treatment strategies.

Intestinal region-specific Wnt signalling profiles reveal interrelation between cell identity and oncogenic pathway activity in cancer development.

BACKGROUND: Cancer results from the accumulation of mutations leading to the acquisition of cancer promoting characteristics such as increased proliferation and resistance to cell death. In colorectal cancer, an early mutation leading to such features usually occurs in the APC or CTNNB1 genes, thereby activating Wnt signalling. However, substantial phenotypic differences between cancers originating within the same organ, such as molecular subtypes, are not fully reflected by differences in mutations. Indeed, the phenotype seems to result from a complex interplay between the cell-intrinsic features and the acquired mutations, which is difficult to disentangle when established tumours are studied. METHODS: We use a 3D in vitro organoid model to study the early phase of colorectal cancer development. From three different murine intestinal locations we grow organoids. These are transformed to resemble adenomas after Wnt activation through lentiviral transduction with a stable form of ß-Catenin. The gene expression before and after Wnt activation is compared within each intestinal origin and across the three locations using RNA sequencing. To validate and generalize our findings, we use gene expression data from patients. RESULTS: In reaction to Wnt activation we observe downregulation of location specific genes and differentiation markers. A similar effect is seen in patient data, where genes with significant differential expression between the normal left and right colon are downregulated in the cancer samples. Furthermore, the signature of Wnt target genes differs between the three intestinal locations in the organoids. The location specific Wnt signatures are dominated by genes which have been lowly expressed in the tissue of origin, and are the targets of transcription factors that are activated following enhanced Wnt signalling. CONCLUSION: We observed that the region-specific cell identity has a substantial effect on the reaction to Wnt activation in a simple intestinal adenoma model. These findings provide a way forward in resolving the distinct biology between left- and right-sided human colon cancers with potential clinical relevance.

Diagnostic yield and clinical utility of a comprehensive gene panel for hereditary tumor syndromes.

BACKGROUND: In a considerable number of patients with a suspected hereditary tumor syndrome (HTS), no underlying germline mutation is detected in the most likely affected genes. The present study aimed to establish and validate a large gene panel for HTS, and determine its diagnostic yield and clinical utility. METHODS: The study cohort comprised 173 patients with suspected, but unexplained, HTS (group U) and 64 HTS patients with a broad spectrum of known germline mutations (group K). All patients in group U presented with early age at onset, multiple tumors, and/or a familial clustering of various tumor types; no germline mutation had been identified during routine diagnostics. Sequencing of leukocyte DNA was performed for the 94 HTS genes of the Illumina TruSight™Cancer Panel and 54 additional HTS genes. RESULTS: The sensitivity of the panel to identify known germline variants was 99.6%. In addition to known mutations, a total of 192 rare, potentially pathogenic germline variants in 86 genes were identified. Neither the proportion of rare variants per patient (group K: 0.9 variants; group U: 0.8 variants) nor the proportion of variants in the most frequently mutated, moderately penetrant genes CHEK2 and ATM showed significant inter-group difference. Four of the five patients from group U with a truncating CHEK2 mutation had thyroid cancer, pointing to a broader tumor spectrum in patients with pathogenic CHEK2 variants. In 22% of patients from group K, a further potential causative variant was identified. Here, the most interesting finding was an NF1 nonsense mutation in a child with a known TP53 frameshift mutation. In 17% of patients from group U, potential causative variants were identified. In three of these patients (2%), mutations in PMS2, PTEN, or POLD1 were considered to be causative. In both groups, incidental findings with presumptive predictive value were generated. CONCLUSIONS: The gene panel identified the genetic cause in some prescreened, unexplained HTS patients and generated incidental findings. Some patients harbored predicted pathogenic mutations in more than one established HTS gene, rendering interpretation of the respective alterations challenging. Established moderate risk genes showed an almost equal distribution among patients with known and unexplained disease.

Stem cell functionality is microenvironmentally defined during tumour expansion and therapy response in colon cancer.

Solid malignancies have been speculated to depend on cancer stem cells (CSCs) for expansion and relapse after therapy. Here we report on quantitative analyses of lineage tracing data from primary colon cancer xenograft tissue to assess CSC functionality in a human solid malignancy. The temporally obtained clone size distribution data support a model in which stem cell function in established cancers is not intrinsically, but is entirely spatiotemporally orchestrated. Functional stem cells that drive tumour expansion predominantly reside at the tumour edge, close to cancer-associated fibroblasts. Hence, stem cell properties change in time depending on the cell location. Furthermore, although chemotherapy enriches for cells with a CSC phenotype, in this context functional stem cell properties are also fully defined by the microenvironment. To conclude, we identified osteopontin as a key cancer-associated fibroblast-produced factor that drives in situ clonogenicity in colon cancer.

Chromoendoscopy in combination with random biopsies does not improve detection of gastric cancer foci in CDH1 mutation positive patients.

Background and study aims: Hereditary diffuse gastric cancer (HGGC), an autosomal dominant tumor-syndrome, accounts for 1 % to 3 % of gastric cancers worldwide. Presumably 30 % to 40 % of all patients fulfilling the clinical guidelines for HDGC are carriers of a pathogenic mutation in the CDH1 gene. Patients often show multiple foci of signet ring cell carcinoma at early age and are advised to undergo prophylactic total gastrectomy (PTG). Our aim was to improve the endoscopic detection of HDGC by using an enhanced endoscopic protocol. Patient and methods: Patients with a proven CDH1 germline mutation identified in our institute were prospectively included. Patients were advised to undergo PTG and offered a baseline endoscopic examination prior surgery. Examination was performed by using high-resolution white-light endoscopy and pan-gastric chromoendoscopy with indigo carmine as dye combined with targeted and multiple random biopsies assessed by an expert histopathologist. Postoperative histopathology was compared with results from endoscopic biopsies. Results: Between September 2012 and November 2014 8 patients with a proven CDH1 germline mutation were included. We conducted 44 targeted (6.3/patient) and 225 random (32.1/patient) biopsies in 7 patients. We detected 1 gastric cancer by random biopsy (14 %). All other examinations showed no signs of cancer. Histopathology of gastrectomy specimen revealed multiple foci of gastric carcinoma in 6 patients (86 %) with a total number of 27 cancer foci. Conclusions: Examination with targeted and random biopsies combined with chromoendoscopy is not able to detect small foci of gastric cancer in CDH1 mutation carriers. Therefore PTG is advocated in these patients.

Prophylactic total gastrectomy in the management of hereditary tumor syndromes.

PURPOSE: Germline mutations in several genes confer a relevant lifetime risk of gastric cancer. In this context, an increasing involvement of a surgeon can be seen, mainly with the question of performing a prophylactic operation. METHODS: Patients with hereditary tumor syndromes predisposing for gastric cancer who received care leading to prophylactic total gastrectomy in our Center for Hereditary Tumor Syndromes were analyzed. For each patient, the multidisciplinary decision-making process, the perioperative course, and the histopathologic findings were assessed. Short-term morbidity was evaluated based on the medical reports. RESULTS: The analysis includes nine patients (six female, three male) with a median age of 41.6 (range 23-60) years. Indication for prophylactic total gastrectomy was based on family history and genetic analysis (eight patients with a germline mutation of the CDH1 gene and one patient with a SMAD4 mutation). Removal of the entire gastric mucosa was documented intraoperatively by fresh frozen section examination. Extended (DII) lymphadenectomy was performed in four patients. Histopathologic examination of gastrectomy specimens revealed six patients (6/9, 67 %) with multifocal signet ring cell carcinomas. In our series, prophylactic total gastrectomy was a safe procedure without mortality and low morbidity. CONCLUSIONS: Patients with hereditary syndromes predisposing for gastric cancer should be evaluated for this curative procedure in a specialized center. Further research is necessary, and the implementation of nationwide registers including patients with prophylactic gastrointestinal operations due to hereditary tumor syndrome is advisable.

Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis.

In ∼30% of families affected by colorectal adenomatous polyposis, no germline mutations have been identified in the previously implicated genes APC, MUTYH, POLE, POLD1, and NTHL1, although a hereditary etiology is likely. To uncover further genes with high-penetrance causative mutations, we performed exome sequencing of leukocyte DNA from 102 unrelated individuals with unexplained adenomatous polyposis. We identified two unrelated individuals with differing compound-heterozygous loss-of-function (LoF) germline mutations in the mismatch-repair gene MSH3. The impact of the MSH3 mutations (c.1148delA, c.2319-1G>A, c.2760delC, and c.3001-2A>C) was indicated at the RNA and protein levels. Analysis of the diseased individuals' tumor tissue demonstrated high microsatellite instability of di- and tetranucleotides (EMAST), and immunohistochemical staining illustrated a complete loss of nuclear MSH3 in normal and tumor tissue, confirming the LoF effect and causal relevance of the mutations. The pedigrees, genotypes, and frequency of MSH3 mutations in the general population are consistent with an autosomal-recessive mode of inheritance. Both index persons have an affected sibling carrying the same mutations. The tumor spectrum in these four persons comprised colorectal and duodenal adenomas, colorectal cancer, gastric cancer, and an early-onset astrocytoma. Additionally, we detected one unrelated individual with biallelic PMS2 germline mutations, representing constitutional mismatch-repair deficiency. Potentially causative variants in 14 more candidate genes identified in 26 other individuals require further workup. In the present study, we identified biallelic germline MSH3 mutations in individuals with a suspected hereditary tumor syndrome. Our data suggest that MSH3 mutations represent an additional recessive subtype of colorectal adenomatous polyposis.

Exome sequencing identifies potential novel candidate genes in patients with unexplained colorectal adenomatous polyposis.

In up to 30% of patients with colorectal adenomatous polyposis, no germline mutation in the known genes APC, causing familial adenomatous polyposis, MUTYH, causing MUTYH-associated polyposis, and POLE or POLD1, causing Polymerase-Proofreading-associated polyposis can be identified, although a hereditary etiology is likely. To uncover new causative genes, exome sequencing was performed using DNA from leukocytes and a total of 12 colorectal adenomas from seven unrelated patients with unexplained sporadic adenomatous polyposis. For data analysis and variant filtering, an established bioinformatics pipeline including in-house tools was applied. Variants were filtered for rare truncating point mutations and copy-number variants assuming a dominant, recessive, or tumor suppressor model of inheritance. Subsequently, targeted sequence analysis of the most promising candidate genes was performed in a validation cohort of 191 unrelated patients. All relevant variants were validated by Sanger sequencing. The analysis of exome sequencing data resulted in the identification of rare loss-of-function germline mutations in three promising candidate genes (DSC2, PIEZO1, ZSWIM7). In the validation cohort, further variants predicted to be pathogenic were identified in DSC2 and PIEZO1. According to the somatic mutation spectra, the adenomas in this patient cohort follow the classical pathways of colorectal tumorigenesis. The present study identified three candidate genes which might represent rare causes for a predisposition to colorectal adenoma formation. Especially PIEZO1 (FAM38A) and ZSWIM7 (SWS1) warrant further exploration. To evaluate the clinical relevance of these genes, investigation of larger patient cohorts and functional studies are required.

Low-level APC mutational mosaicism is the underlying cause in a substantial fraction of unexplained colorectal adenomatous polyposis cases.

BACKGROUND: In 30-50% of patients with colorectal adenomatous polyposis, no germline mutation in the known genes APC, causing familial adenomatous polyposis, MUTYH, causing MUTYH-associated polyposis, or POLE or POLD1, causing polymerase-proofreading-associated polyposis can be identified, although a hereditary aetiology is likely. This study aimed to explore the impact of APC mutational mosaicism in unexplained polyposis. METHODS: To comprehensively screen for somatic low-level APC mosaicism, high-coverage next-generation sequencing of the APC gene was performed using DNA from leucocytes and a total of 53 colorectal tumours from 20 unrelated patients with unexplained sporadic adenomatous polyposis. APC mosaicism was assumed if the same loss-of-function APC mutation was present in ≥ 2 anatomically separated colorectal adenomas/carcinomas per patient. All mutations were validated using diverse methods. RESULTS: In 25% (5/20) of patients, somatic mosaicism of a pathogenic APC mutation was identified as underlying cause of the disease. In 2/5 cases, the mosaic level in leucocyte DNA was slightly below the sensitivity threshold of Sanger sequencing; while in 3/5 cases, the allelic fraction was either very low (0.1-1%) or no mutations were detectable. The majority of mosaic mutations were located outside the somatic mutation cluster region of the gene. CONCLUSIONS: The present data indicate a high prevalence of pathogenic mosaic APC mutations below the detection thresholds of routine diagnostics in adenomatous polyposis, even if high-coverage sequencing of leucocyte DNA alone is taken into account. This has important implications for both routine work-up and strategies to identify new causative genes in this patient group.

Genome-wide CNV analysis in 221 unrelated patients and targeted high-throughput sequencing reveal novel causative candidate genes for colorectal adenomatous polyposis.

To uncover novel causative genes in patients with unexplained adenomatous polyposis, a model disease for colorectal cancer, we performed a genome-wide analysis of germline copy number variants (CNV) in a large, well characterized APC and MUTYH mutation negative patient cohort followed by a targeted next generation sequencing (NGS) approach. Genomic DNA from 221 unrelated German patients was genotyped on high-resolution SNP arrays. Putative CNVs were filtered according to stringent criteria, compared with those of 531 population-based German controls, and validated by qPCR. Candidate genes were prioritized using in silico, expression, and segregation analyses, data mining and enrichment analyses of genes and pathways. In 27% of the 221 unrelated patients, a total of 77 protein coding genes displayed rare, nonrecurrent, germline CNVs. The set included 26 candidates with molecular and cellular functions related to tumorigenesis. Targeted high-throughput sequencing found truncating point mutations in 12% (10/77) of the prioritized genes. No clear evidence was found for autosomal recessive subtypes. Six patients had potentially causative mutations in more than one of the 26 genes. Combined with data from recent studies of early-onset colorectal and breast cancer, recurrent potential loss-of-function alterations were detected in CNTN6, FOCAD (KIAA1797), HSPH1, KIF26B, MCM3AP, YBEY and in three genes from the ARHGAP family. In the canonical Wnt pathway oncogene CTNNB1 (ß-catenin), two potential gain-of-function mutations were found. In conclusion, the present study identified a group of rarely affected genes which are likely to predispose to colorectal adenoma formation and confirmed previously published candidates for tumor predisposition as etiologically relevant.

Frequency and phenotypic spectrum of germline mutations in POLE and seven other polymerase genes in 266 patients with colorectal adenomas and carcinomas.

In a number of families with colorectal adenomatous polyposis or suspected Lynch syndrome/HNPCC, no germline alteration in the APC, MUTYH, or mismatch repair (MMR) genes are found. Missense mutations in the polymerase genes POLE and POLD1 have recently been identified as rare cause of multiple colorectal adenomas and carcinomas, a condition termed polymerase proofreading-associated polyposis (PPAP). The aim of the present study was to evaluate the clinical relevance and phenotypic spectrum of polymerase germline mutations. Therefore, targeted sequencing of the polymerase genes POLD1, POLD2, POLD3, POLD4, POLE, POLE2, POLE3 and POLE4 was performed in 266 unrelated patients with polyposis or fulfilled Amsterdam criteria. The POLE mutation c.1270C>G;p.Leu424Val was detected in four unrelated patients. The mutation was present in 1.5% (4/266) of all patients, 4% (3/77) of all familial cases and 7% (2/30) of familial polyposis cases. The colorectal phenotype in 14 affected individuals ranged from typical adenomatous polyposis to a HNPCC phenotype, with high intrafamilial variability. Multiple colorectal carcinomas and duodenal adenomas were common, and one case of duodenal carcinoma was reported. Additionally, various extraintestinal lesions were evident. Nine further putative pathogenic variants were identified. The most promising was c.1306C>T;p.Pro436Ser in POLE. In conclusion, a PPAP was identified in a substantial number of polyposis and familial colorectal cancer patients. Screening for polymerase proofreading mutations should therefore be considered, particularly in unexplained familial cases. The present study broadens the phenotypic spectrum of PPAP to duodenal adenomas and carcinomas, and identified novel, potentially pathogenic variants in four polymerase genes.

De novo duplication of chromosome 16p in a female infant with signs of neonatal hemochromatosis.

Reported cases of "pure" duplication of the entire short arm of chromosome 16 (16p) are rare, with only 7 patients described in the literature. We report on a female infant with de novo 16p duplication localized to the short arm of chromosome 6, detected by chromosomal analysis and characterized by array CGH and fluorescence in situ hybridization. This baby girl presented with clinical symptoms characteristic of patients with duplications of the short arm of chromosome 16: psychomotor retardation, constitutional growth delay and specific dysmorphic features, including proximally placed hypoplastic thumbs. In addition, she exhibited evidence of neonatal hemochromatosis as shown by direct hyperbilirubinemia, iron overload and elevated liver enzyme levels. To our knowledge, this is the first report of signs of neonatal hemochromatosis in a patient with 16p duplication.

C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

A CRM1-dependent nuclear export pathway is involved in the regulation of MutLα subcellular localization.

MutLα plays an essential role in DNA mismatch repair (MMR) and is additionally involved in other cellular mechanisms such as the regulation of cell cycle checkpoints and apoptosis. Therefore, not only germline MMR gene defects but also the subcellular localization of MutLα might be of importance for the development of Lynch syndrome. Recently, we showed that MutLα contains functional nuclear import sequences and is most frequently localized in the nucleus. Here, we demonstrate that MutLα can move bidirectionally towards the nuclear membrane. Using MutLα transfected HEK293T cells we observed a significant shift of MLH1 and PMS2 from the nucleus to the cytoplasm after irradiation or cisplatin treatment. We analyzed both proteins for potential nuclear export sequences (NES) and identified one functional Rev-type NES (578LFDLAMLAL) in the C-terminal part of MLH1 that facilitates export via the CRM1/exportin pathway. Moreover, an MLH1-NES mutation detected in a patient with Lynch syndrome showed normal MMR activity but led to significantly impaired cytoplasmic transport after actinomycin D treatment. These results indicate that MutLα is able to shuttle from the nucleus to the cytoplasm, probably signaling DNA damages to downstream pathways. In conclusion, not only a defective MMR but also impaired nucleo-cytoplasmic shuttling might result in the onset of Lynch syndrome.