Role of MEK1 and DIAPH3 expression in colorectal adenoma-carcinoma sequence.

BACKGROUND: It is well established that most colorectal carcinomas arise from conventional adenomas through the adenoma-carcinoma sequence (ACS) model. mitogen-activated protein kinases (MAPKs) pathway has been reported as a crucial player in tumorigenesis. The MAPK signaling pathway is activated by different extracellular signals involving the "mitogen-activated/extracellular signal-regulated kinase 1 (MEK1)", and this induces the expression of genes involved in proliferation and cellular transformation. Diaphanous-related formin-3 (DIAPH3) acts as a potential metastasis regulator through inhibiting the cellular transition to amoeboid behavior in different cancer types. OBJECTIVE: The aim of the study was to investigate the pattern of immunohistochemical expression of MEK1 and DIAPH3 in colorectal adenoma (CRA) and corresponding colorectal carcinoma (CRC) specimens. METHODS: The immunohistochemical expression of DIAPH3 and MEK1 was examined in 43 cases of CRC and their associated adenomas using tissue microarray technique. RESULTS: MEK1 was overexpressed in 23 CRC cases (53.5%) and in 20 CRA cases (46.5%). DIAPH3 was overexpressed in 11 CRA cases (about 29%) which were significantly lower than CRC (22 cases; 58%) (P = 0.011). Both MEK1 and DIAPH3 overexpression were significantly correlated in CRC (P = 0.009) and CRA cases (P = 0.002). Tumors with MEK1 overexpression had a significantly higher tumor grade (P = 0.050) and perineural invasion (P = 0.017). CONCLUSIONS: Both MEK1 and DIAPH3 are overexpressed across colorectal ACS with strong correlation between them. This co- expression suggests a possible synergistic effect of MEK1 and DIAPH-3 in colorectal ACS. Further large-scale studies are required to investigate the potential functional aspects of MEK1 and DIAPH3 in ACS and their involvement in tumor initiation and the metastatic process.

TGFß-Responsive Stromal Activation Occurs Early in Serrated Colorectal Carcinogenesis.

Activated TGFß signaling in the tumor microenvironment, which occurs independently of epithelial cancer cells, has emerged as a key driver of tumor progression in late-stage colorectal cancer (CRC). This study aimed to elucidate the contribution of TGFß-activated stroma to serrated carcinogenesis, representing approximately 25% of CRCs and often characterized by oncogenic BRAF mutations. We used a transcriptional signature developed based on TGFß-responsive, stroma-specific genes to infer TGFß-dependent stromal activation and conducted in silico analyses in 3 single-cell RNA-seq datasets from a total of 39 CRC samples and 12 bulk transcriptomic datasets consisting of 2014 CRC and 416 precursor samples, of which 33 were serrated lesions. Single-cell analyses validated that the signature was expressed specifically by stromal cells, effectively excluding transcriptional signals derived from epithelial cells. We found that the signature was upregulated during malignant transformation and cancer progression, and it was particularly enriched in CRCs with mutant BRAF compared to wild-type counterparts. Furthermore, across four independent precursor datasets, serrated lesions exhibited significantly higher levels of TGFß-responsive stromal activation compared to conventional adenomas. This large-scale analysis suggests that TGFß-dependent stromal activation occurs early in serrated carcinogenesis. Our study provides novel insights into the molecular mechanisms underlying CRC development via the serrated pathway.

Parathyroid tumors in the 5th edition of the WHO Classification of Tumors of the Endocrine Organs.

The diagnosis of pathological conditions of the parathyroid glands is the answer to clinically more frequently detected hypercalcemic conditions, including MEN syndromes. In routine biopsy practice, enlarged bodies are also a differential diagnosis for the diagnosis of thyroid nodules. In the chapter of parathyroid tumors, the 5th edition of the WHO classification brings changes influenced similarly to other endocrine organs by the increase in genetic information. At the terminological level, the concept of hyperplasia has been narrowed down to secondary hyperplasia, most of the previously primary hyperplasias are referred to as multiglandular parathyroid disease due to evidence of multiglandular clonal proliferations. The term atypical parathyroid tumor replacing atypical adenoma is newly introduced - the uncertain biological behaviour is emphasized. The basic examination includes parafibromin immunohis- tochemistry, the deficiency of parafibromin being an indicator of an inactivating CDC73 mutation and an increased risk of familial forms, or MEN. Methodologically, refinements are introduced in the quantification of mitotic activity per 10 mm2. Oncocytic subtypes have an arbitrarily declared threshold of more than 75% oncocytes. The definition of lipoadenoma (multiplication of both components, more than 50% of adipose tissue in the tumor) is similarly specified. The diagnosis of cancer remains histopathological with unequivocal evidence of invasion, or microscopically verified metastasis.

A case study of a liver transplant-treated patient with glycogen storage disease type Ia presenting with multiple inflammatory hepatic adenomas: an analysis of clinicopathologic and genetic data.

BACKGROUND: Glycogen storage disease (GSD) is a disease caused by excessive deposition of glycogen in tissues due to genetic disorders in glycogen metabolism. Glycogen storage disease type I (GSD-I) is also known as VonGeirk disease and glucose-6-phosphatase deficiency. This disease is inherited in an autosomal recessive manner, and both sexes can be affected. The main symptoms include hypoglycaemia, hepatomegaly, acidosis, hyperlipidaemia, hyperuricaemia, hyperlactataemia, coagulopathy and developmental delay. CASE PRESENTATION: Here, we present the case of a 13-year-old female patient with GSD Ia complicated with multiple inflammatory hepatic adenomas. She presented to the hospital with hepatomegaly, hypoglycaemia, and epistaxis. By clinical manifestations and imaging and laboratory examinations, we suspected that the patient suffered from GSD I. Finally, the diagnosis was confirmed by liver pathology and whole-exome sequencing (WES). WES revealed a synonymous mutation, c.648 G > T (p.L216 = , NM_000151.4), in exon 5 and a frameshift mutation, c.262delG (p.Val88Phefs*14, NM_000151.4), in exon 2 of the G6PC gene. According to the pedigree analysis results of first-generation sequencing, heterozygous mutations of c.648 G > T and c.262delG were obtained from the patient's father and mother. Liver pathology revealed that the solid nodules were hepatocellular hyperplastic lesions, and immunohistochemical (IHC) results revealed positive expression of CD34 (incomplete vascularization), liver fatty acid binding protein (L-FABP) and C-reactive protein (CRP) in nodule hepatocytes and negative expression of ß-catenin and glutamine synthetase (GS). These findings suggest multiple inflammatory hepatocellular adenomas. PAS-stained peripheral hepatocytes that were mostly digested by PAS-D were strongly positive. This patient was finally diagnosed with GSD-Ia complicated with multiple inflammatory hepatic adenomas, briefly treated with nutritional therapy after diagnosis and then underwent living-donor liver allotransplantation. After 14 months of follow-up, the patient recovered well, liver function and blood glucose levels remained normal, and no complications occurred. CONCLUSION: The patient was diagnosed with GSD-Ia combined with multiple inflammatory hepatic adenomas and received liver transplant treatment. For childhood patients who present with hepatomegaly, growth retardation, and laboratory test abnormalities, including hypoglycaemia, hyperuricaemia, and hyperlipidaemia, a diagnosis of GSD should be considered. Gene sequencing and liver pathology play important roles in the diagnosis and typing of GSD.

Elucidating immunological characteristics of the adenoma-carcinoma sequence in colorectal cancer patients in South Korea using a bioinformatics approach.

Colorectal cancer (CRC) is one of the top five most common and life-threatening malignancies worldwide. Most CRC develops from advanced colorectal adenoma (ACA), a precancerous stage, through the adenoma-carcinoma sequence. However, its underlying mechanisms, including how the tumor microenvironment changes, remain elusive. Therefore, we conducted an integrative analysis comparing RNA-seq data collected from 40 ACA patients who visited Dongguk University Ilsan Hospital with normal adjacent colons and tumor samples from 18 CRC patients collected from a public database. Differential expression analysis identified 21 and 79 sequentially up- or down-regulated genes across the continuum, respectively. The functional centrality of the continuum genes was assessed through network analysis, identifying 11 up- and 13 down-regulated hub-genes. Subsequently, we validated the prognostic effects of hub-genes using the Kaplan-Meier survival analysis. To estimate the immunological transition of the adenoma-carcinoma sequence, single-cell deconvolution and immune repertoire analyses were conducted. Significant composition changes for innate immunity cells and decreased plasma B-cells with immunoglobulin diversity were observed, along with distinctive immunoglobulin recombination patterns. Taken together, we believe our findings suggest underlying transcriptional and immunological changes during the adenoma-carcinoma sequence, contributing to the further development of pre-diagnostic markers for CRC.

miR-200b-3p accelerates progression of pituitary adenomas by negatively regulating expression of RECK.

MicroRNA (miR)-200b-3p has been associated with many tumors, but its involvement in pituitary adenoma is unclear. This study investigated the molecular mechanism underlying miR-200b-3p regulation in pituitary adenomas to provide a theoretical basis for treatment. Bioinformatics was used to analyze pituitary adenoma-related genes and screen new targets related to RECK and miRNA. As well, the relationship between miR-200b-3p and RECK protein was verified using a double-luciferase reporter gene assay. The expression of miR-200b-3p in clinical samples was analyzed by in situ hybridization. Transfection of the miR-200b-3p inhibitor and small interfering-RECK (si-RECK) was verified by qPCR. GH3 cell viability and proliferation were detected using CCK8 and EdU assays. Apoptosis was detected by flow cytometry and western blotting. Wound healing and Transwell assays were used to detect cell migration and invasion. The effects of miR-200b-3p and RECK on GH3 cells were verified using salvage experiments. miR-200b-3p was highly expressed in pituitary tumor tissue. Inhibitors of miR-200b-3p inhibited cell proliferation promoted cell apoptosis, inhibited invasion and migration, and inhibited the expression of matrix metalloproteinases. Interestingly, miR-200b-3p negatively regulated RECK. The expression of RECK in pituitary adenoma tissues was lower than that in neighboring tissues. Si-RECK rescued the function of miR-200b-3p inhibitors in the above cellular behaviors, and miR-200b-3p accelerated the development of pituitary adenoma by negatively regulating RECK expression. In summary, this study investigated the molecular mechanism by which miR-200b-3p regulates the progression of pituitary adenoma through the negative regulation of RECK. The findings provide a new target for the treatment of pituitary adenoma.

Histopathology of growth hormone-secreting pituitary tumors: State of the art and new perspectives.

Somatotroph (GH) adenomas/PitNETs typically arise from adenohypophysis and are biochemically active, leading to acromegaly and gigantism. More rarely, they present with ectopic origin and do not present overt biochemical or clinical features (silent variants). Histopathological examination should consider the clinical and radiological background, and include multiple steps assessing tumor morphology, pituitary transcription factors (PTFs), hormone secretion, proliferation markers, granulation, and somatostatin receptors (STRs), aimed at depicting as better as possible tumor origin (in case of non-functioning and/or metastatic tumor), and clinical behavior, including response to treatment. GH-secreting tumors are part of the Pit-1 family tumors and can secrete GH only (pure somatotrophs) or co-secrete prolactin (mixed tumors; in this case, various histological subtypes have been identified). Each subtype presents unique radiological, biochemical, and clinical characteristic. Therefore, the integration of biochemical, clinical, radiological, and histopathological elements is fundamental for proper diagnosis and management of pituitary adenomas/PitNETs, to be performed in referral Centers. In more recent times, the importance of genetic and epigenetic evaluation in the characterization of pituitary tumors (i.e., early identification of aggressive variants) has been outlined by some large studies, with the intention of improving targeted treatments.

Colorectal cancer initiation: Understanding early-stage disease for intervention.

How tumors arise or the cause of precancerous lesions is a fundamental question in cancer biology. It is generally accepted that tumors originate from normal cells that undergo uncontrolled proliferation owing to genetic alterations. At the onset of adenoma formation, cancer driver mutations confer clonal growth advantage, enabling mutant cells to outcompete and eliminate the surrounding healthy cells. Hence, the development of precancerous lesions is not only attributed to the expansion of pre-malignant clones, but also relies on the relative fitness of mutated cells compared to the neighboring cells. Colorectal cancer (CRC) is an excellent model to investigate cancer origin as it follows a stereotypical process from mutant cell hyperplasia to adenoma formation and progression. Here, we review the evolving understanding of colonic tumor development, focusing on how cell intrinsic and extrinsic factors impact cell competition and the "clone war" between cancer-initiating cells and normal stem cells. We also discuss the promises and limitations of targeting cell competitiveness in cancer prevention and early intervention. The field of tumor initiation is currently in its infancy, elucidating the adenoma origin is crucial for designing effective prevention strategies and early treatments before cancer becomes incurable.

Early screening of colorectal cancer using feature engineering with artificial intelligence-enhanced analysis of nanoscale chromatin modifications.

Colonoscopy is accurate but inefficient for colorectal cancer (CRC) prevention due to the low (~ 7 to 8%) prevalence of target lesions, advanced adenomas. We leveraged rectal mucosa to identify patients who harbor CRC field carcinogenesis by evaluating chromatin 3D architecture. Supranucleosomal disordered chromatin chains (~ 5 to 20 nm, ~1 kbp) fold into chromatin packing domains (~ 100 to 200 nm, ~ 100 to 1000 kbp). In turn, the fractal-like conformation of DNA within chromatin domains and the folding of the genome into packing domains has been shown to influence multiple facets of gene transcription, including the transcriptional plasticity of cancer cells. We deployed an optical spectroscopic nanosensing technique, chromatin-sensitive partial wave spectroscopic microscopy (csPWS), to evaluate the packing density scaling D of the chromatin chain conformation within packing domains from rectal mucosa in 256 patients with varying degrees of progression to colorectal cancer. We found average packing scaling D of chromatin domains was elevated in tumor cells, histologically normal-appearing cells 4 cm proximal to the tumor, and histologically normal-appearing rectal mucosa compared to cells from control patients (p < 0.001). Nuclear D had a robust correlation with the model of 5-year risk of CRC with r2 = 0.94. Furthermore, rectal D was evaluated as a screening biomarker for patients with advanced adenomas presenting an AUC of 0.85 and 85% sensitivity and specificity. artificial intelligence-enhanced csPWS improved diagnostic performance with AUC = 0.90. Considering the low sensitivity of existing CRC tests, including liquid biopsies, to early-stage cancers our work highlights the potential of chromatin biomarkers of field carcinogenesis in detecting early, significant precancerous colon lesions.

Persistent hypercalcaemia associated with two pathogenic variants in the CYP24A1 gene and a parathyroid adenoma-a case report and review.

Introduction: 24-Hydroxylase, encoded by the CYP24A1 gene, is a crucial enzyme involved in the catabolism of vitamin D. Loss-of-function mutations in CYP24A1 result in PTH-independent hypercalcaemia with high levels of 1,25(OH)2D3. The variety of clinical manifestations depends on age, and underlying genetic predisposition mutations can lead to fatal infantile hypercalcaemia among neonates, whereas adult symptoms are usually mild. Aim of the study: We report a rare case of an adult with primary hyperparathyroidism and loss-of-function mutations in the CYP24A1 gene and a review of similar cases. Case presentation: We report the case of a 58-year-old woman diagnosed initially with primary hyperparathyroidism. Preoperatively, the suspected mass adjoining the upper pole of the left lobe of the thyroid gland was found via ultrasonography and confirmed by 99mTc scintigraphy and biopsy as the parathyroid gland. The patient underwent parathyroidectomy (a histopathology report revealed parathyroid adenoma), which led to normocalcaemia. After 10 months, vitamin D supplementation was introduced due to deficiency, and the calcium level remained within the reference range. Two years later, biochemical tests showed recurrence of hypercalcaemia with suppressed parathyroid hormone levels and elevated 1,25(OH)2D3 concentrations. Further investigation excluded the most common causes of PTH-independent hypercalcaemia, such as granulomatous disease, malignancy, and vitamin D intoxication. Subsequently, vitamin D metabolites were measured using LC-MS/MS, which revealed high levels of 25(OH)D3, low levels of 24,25(OH)2D3 and elevated 25(OH)2D3/24,25(OH)2D3 ratios, suggesting a defect in vitamin D catabolism. Molecular analysis of the CYP24A1 gene using the NGS technique revealed two pathogenic variants: p.(Arg396Trp) and p.(Glu143del) (rs114368325 and rs777676129, respectively). Conclusions: The diagnostic process for hypercalcaemia becomes complicated when multiple causes of hypercalcaemia coexist. The measurement of vitamin D metabolites using LC-MS/MS may help to identify carriers of CYP24A1 mutations. Subsequent molecular testing may contribute to establishing the exact frequency of pathogenic variants of the CYP24A1 gene and introducing personalized treatment.

Relevance of mutations in protein deubiquitinases genes and TP53 in corticotroph pituitary tumors.

Introduction: Corticotroph pituitary neuroendocrine tumors (PitNETs) develop from ACTH-producing cells. They commonly cause Cushing's disease (CD), however, some remain clinically silent. Recurrent USP8, USP48, BRAF and TP53 mutations occur in corticotroph PitNETs. The aim of our study was to determine frequency and relevance of these mutations in a possibly large series of corticotroph PitNETs. Methods: Study included 147 patients (100 CD and 47 silent tumors) that were screened for hot-spot mutations in USP8, USP48 and BRAF with Sanger sequencing, while 128 of these patients were screened for TP53 mutations with next generation sequencing and immunohistochemistry. Results: USP8 mutations were found in 41% CD and 8,5% silent tumors, while USP48 mutations were found in 6% CD patients only. Both were more prevalent in women. They were related to higher rate of biochemical remission, non-invasive tumor growth, its smaller size and densely granulated histology, suggesting that these mutation may be favorable clinical features. Multivariate survival analyses did not confirm possible prognostic value of mutation in protein deubiquitinases. No BRAF mutations were found. Four TP53 mutations were identified (2 in CD, 2 in silent tumors) in tumors with size >10mm including 3 invasive ones. They were found in Crooke's cell and sparsely granulated tumors. Tumors with missense TP53 mutations had higher TP53 immunoreactivity score than wild-type tumors. Tumor with frameshift TP53 variant had low protein expression. TP53 mutation was a poor prognostic factor in CD according to uni- and multivariate survival analyses in spite of low mutations frequency. Conclusions: We confirmed high prevalence of USP8 mutations and low incidence of USP48 and TP53 mutations. Changes in protein deubiquitinases genes appear to be favorable prognostic factors in CD. TP53 mutations are rare, occur in both functioning and silent tumors and are related to poor clinical outcome in CD.

Double somatic mutations in CTNNB1 and GNA11 in an aldosterone-producing adenoma.

Double somatic mutations in CTNNB1 and GNA11/Q have recently been identified in a small subset of aldosterone-producing adenomas (APAs). As a possible pathogenesis of APA due to these mutations, an association with pregnancy, menopause, or puberty has been proposed. However, because of its rarity, characteristics of APA with these mutations have not been well characterized. A 46-year-old Japanese woman presented with hypertension and hypokalemia. She had two pregnancies in the past but had no history of pregnancy-induced hypertension. She had regular menstrual cycle at presentation and was diagnosed as having primary aldosteronism after endocrinologic examinations. Computed tomography revealed a 2 cm right adrenal mass. Adrenal venous sampling demonstrated excess aldosterone production from the right adrenal gland. She underwent right laparoscopic adrenalectomy. The resected right adrenal tumor was histologically diagnosed as adrenocortical adenoma and subsequent immunohistochemistry (IHC) revealed diffuse immunoreactivity of aldosterone synthase (CYP11B2) and visinin like 1, a marker of the zona glomerulosa (ZG), whereas 11ß-hydroxylase, a steroidogenic enzyme for cortisol biosynthesis, was mostly negative. CYP11B2 IHC-guided targeted next-generation sequencing identified somatic CTNNB1 (p.D32Y) and GNA11 (p.Q209H) mutations. Immunofluorescence staining of the tumor also revealed the presence of activated ß-catenin, consistent with features of the normal ZG. The expression patterns of steroidogenic enzymes and related proteins indicated ZG features of the tumor cells. PA was clinically and biochemically cured after surgery. In conclusion, our study indicated that CTNNB1 and GNA11-mutated APA has characteristics of the ZG. The disease could occur in adults with no clear association with pregnancy or menopause.

Downregulation of SMOC1 is associated with progression of colorectal traditional serrated adenomas.

BACKGROUND: Aberrant DNA methylation is prevalent in colorectal serrated lesions. We previously reported that the CpG island of SMOC1 is frequently methylated in traditional serrated adenomas (TSAs) and colorectal cancers (CRCs) but is rarely methylated in sessile serrated lesions (SSLs). In the present study, we aimed to further characterize the expression of SMOC1 in early colorectal lesions. METHODS: SMOC1 expression was analyzed immunohistochemically in a series of colorectal tumors (n = 199) and adjacent normal colonic tissues (n = 112). RESULTS: SMOC1 was abundantly expressed in normal colon and SSLs while it was significantly downregulated in TSAs, advanced adenomas and cancers. Mean immunohistochemistry scores were as follows: normal colon, 24.2; hyperplastic polyp (HP), 18.9; SSL, 23.8; SSL with dysplasia (SSLD)/SSL with early invasive cancer (EIC), 15.8; TSA, 5.4; TSA with high grade dysplasia (HGD)/EIC, 4.7; non-advanced adenoma, 21.4; advanced adenoma, 11.9; EIC, 10.9. Higher levels SMOC1 expression correlated positively with proximal colon locations and flat tumoral morphology, reflecting its abundant expression in SSLs. Among TSAs that contained both flat and protruding components, levels of SMOC1 expression were significantly lower in the protruding components. CONCLUSION: Our results suggest that reduced expression of SMOC1 is associated with progression of TSAs and conventional adenomas and that SMOC1 expression may be a biomarker for diagnosis of serrated lesions and risk prediction in colorectal tumors.

Dickkopf 1 is expressed in normal fibroblasts during early stages of colorectal tumorigenesis.

BACKGROUND AND PURPOSE: Colorectal cancer progression from adenoma to cancer is a time-intensive process; however, the interaction between normal fibroblasts (NFs) with early colorectal tumors, such as adenomas, remains unclear. Here, we analyzed the response of the microenvironment during early tumorigenesis using co-cultures of organoids and NFs. MATERIALS AND METHODS: Colon normal epithelium, adenoma, cancer organoid, and NFs were established and co-cultured using Transwell inserts. Microarray analysis of NFs was performed to identify factors expressed early in tumor growth. Immunostaining of clinical specimens was performed to localize the identified factor. Functional analysis was performed using HCT116 cells. Serum DKK1 levels were measured in patients with colorectal cancer and adenoma. RESULTS: Colorectal organoid-NF co-culture resulted in increased organoid diameter and cell viability in normal epithelial and adenomatous organoids but not in cancer organoids. Microarray analysis of NFs revealed 18 genes with increased expression when co-cultured with adenoma and cancer organoids. Immunohistochemical staining revealed DKK1 expression in the tumor stroma from early tumor growth. DKK1 stimulation reduced HCT116 cell proliferation, while DKK1 silencing by siRNA transfection increased cell proliferation. Serum DKK1 level was significantly higher in patients with advanced cancer and adenoma than in controls. Serum DKK1 level revealed area-under-the-curve values of 0.78 and 0.64 for cancer and adenoma, respectively. CONCLUSION: These findings contribute valuable insights into the early stages of colorectal tumorigenesis and suggest DKK1 as a tumor suppressor. Additionally, serum DKK1 levels could serve as a biomarker to identify both cancer and adenoma, offering diagnostic possibilities for early-stage colon tumors. The present study has a few limitations. We considered using DKK1 as a candidate gene for gene transfer to organoids and NFs; however, it was difficult due to technical problems and the slow growth rate of NFs. Therefore, we used cancer cell lines instead. In addition, immunostaining and ELISA were based on the short-term collection at a single institution, and further accumulation of such data is desirable. As described above, most previous reports were related to advanced cancers, but in this study, new findings were obtained by conducting experiments on endoscopically curable early-stage tumors, such as adenomas.

In persons aged ≥45 y, the mt-sRNA test had 94% sensitivity for colorectal cancer and 46% for advanced adenomas.

SOURCE CITATION: Barnell EK, Wurtzler EM, La Rocca J, et al. Multitarget stool RNA test for colorectal cancer screening. JAMA. 2023;330:1760-1768. 37870871.

Deficient mismatch repair screening of advanced adenomas in the population screening program for colorectal cancer is not effective.

AIM: Currently, screening of colorectal cancers (CRC) by assessing mismatch repair deficiency (dMMR) or microsatellite instability (MSI) is used to identify Lynch syndrome (LS) patients. Advanced adenomas are considered immediate precursor lesions of CRC. In this study we investigate the relevance of screening of advanced adenomas for LS in population screening. METHODS AND RESULTS: Advanced adenomas (n = 1572) were selected from the Dutch colorectal cancer population screening programme, based on one or more of the criteria: tubulovillous (n = 848, 54%) or villous adenoma (n = 118, 7.5%), diameter ≥ 1 cm (n = 1286, 82%) and/or high-grade dysplasia (n = 176, 11%). In 86 cases (5%), all three criteria were fulfilled at the same time. MMR-IHC and/or MSI analyses were performed on all cases. Only five advanced adenomas (0.3%) showed dMMR and MSI, including two cases with hypermethylation. In at least two patients a germline event was suspected based on allelic frequencies. No pathogenic explanation was found in the last case. CONCLUSION: Timely testing of precursor lesions would be preferable to detect new LS patients before CRC development. However, standard assessment of dMMR of advanced adenomas from the population screening is not effective.

Characterization of sessile serrated adenomas with dysplasia including intramucosal adenocarcinoma and colorectal carcinoma with a microsatellite instability phenotype.

Recent studies have shown that sessile serrated lesions (SSLs) lead to the development of colorectal cancer (CRC) with a microsatellite instability (MSI) phenotype via a dysplasia-carcinoma sequence. However, the pathological and molecular mechanisms of SSL with dysplasia (SSLD) are unclear. Here, we aimed to examine the clinicopathological and molecular alterations in SSLD and to evaluate the significance of such alterations with regard to lesion progression. Fifty-four SSLDs (20 serrated dysplasia cases and 17 intestinal dysplasia cases, including 30 low-grade dysplasia [LGD] cases, 7 high-grade dysplasia [HGD] cases, and 17 intramucosal adenocarcinomas [IMAs]) were evaluated. Molecular alterations, including immunohistochemical expression of various markers, DNA methylation status, and multiple genetic mutations (using next-generation sequencing), were assessed. Additionally, such alterations were also investigated in 41 CRCs with an MSI phenotype (invasion beyond submucosa). The frequency of mismatch repair (MMR) deficiency in SSLD was 12 of 39 cases (32.4 %), whereas the MMR proficient type was observed in 17 of 39 SSLD cases. SSLD with serrated dysplasia showed a significantly higher frequency of loss of MMR protein expression and methylation status. Moreover, loss of MMR protein expression differed significantly between LGD and IMA. Furthermore, the frequency of TP53 mutation was significantly higher in IMA than in LGD. The current findings demonstrated that SSL with serrated dysplasia may be associated with an increased risk of malignant transformation compared with intestinal dysplasia. Loss of MMR proteins and mutation of TP53 may play important roles in tumor progression from dysplasia to carcinomatous lesions.

Enrichment of colibactin-associated mutational signatures in unexplained colorectal polyposis patients.

BACKGROUND: Colibactin, a genotoxin produced by polyketide synthase harboring (pks+) bacteria, induces double-strand breaks and chromosome aberrations. Consequently, enrichment of pks+Escherichia coli in colorectal cancer and polyposis suggests a possible carcinogenic effect in the large intestine. Additionally, specific colibactin-associated mutational signatures; SBS88 and ID18 in the Catalogue of Somatic Mutations in Cancer database, are detected in colorectal carcinomas. Previous research showed that a recurrent APC splice variant perfectly fits SBS88. METHODS: In this study, we explore the presence of colibactin-associated signatures and fecal pks in an unexplained polyposis cohort. Somatic targeted Next-Generation Sequencing (NGS) was performed for 379 patients. Additionally, for a subset of 29 patients, metagenomics was performed on feces and mutational signature analyses using Whole-Genome Sequencing (WGS) on Formalin-Fixed Paraffin Embedded (FFPE) colorectal tissue blocks. RESULTS: NGS showed somatic APC variants fitting SBS88 or ID18 in at least one colorectal adenoma or carcinoma in 29% of patients. Fecal metagenomic analyses revealed enriched presence of pks genes in patients with somatic variants fitting colibactin-associated signatures compared to patients without variants fitting colibactin-associated signatures. Also, mutational signature analyses showed enrichment of SBS88 and ID18 in patients with variants fitting these signatures in NGS compared to patients without. CONCLUSIONS: These findings further support colibactins ability to mutagenize colorectal mucosa and contribute to the development of colorectal adenomas and carcinomas explaining a relevant part of patients with unexplained polyposis.

The expression of UNC5D is abnormal in the early stage of colorectal tumors associated with its proliferation and migration.

OBJECTIVE: Colorectal adenomas are an important precancerous lesion of colorectal adenoma with a high incidence. This study aims to explore new prognostic targets for colorectal adenomas through bioinformatics techniques. MATERIALS AND METHODS: In this study, data from 29 colonic adenomas and 38 normal colonic mucosa in GSE37364 were analyzed to screen for differentially expressed genes (DEGs). Then, batch survival analysis, construction of risk model, mutation analysis, Cox regression analysis and expression analysis were performed on DEGs to determine the hub genes of this study. Finally, immune correlation analysis and cell experiments were carried out on the hub gene to explore its potential mechanism. RESULTS: In our study, a total of 431 up-regulated and 809 down-regulated differentially expressed genes (DEGs) were identified. Among these, Unc-5 Netrin Receptor D (UNC5D) emerged as a pivotal gene associated with colorectal adenoma. Notably, UNC5D expression levels were found to be significantly higher in normal tissues compared to colorectal adenoma tissues. Furthermore, our analysis demonstrated that UNC5D showed promising diagnostic potential for patients with colon adenocarcinoma. In vitro experiments revealed that the overexpression of UNC5D had a profound impact on the behavior of colorectal tumor cells. Specifically, it led to a substantial reduction in the proliferation, motility, and invasion of these tumor cells. Additionally, UNC5D was shown to exert control over STAT1/STAT3 phosphorylation, which in turn regulated the expression of PD-L1 in response to interferon (IFN) stimulation. These findings highlight the significant role of UNC5D in modulating immune responses and the development of colorectal adenoma. UNC5D emerges as a potential diagnostic biomarker and an attractive immunotherapeutic target in the context of colorectal malignancies. These results call for further exploration of UNC5D-based strategies for the diagnosis and treatment of colorectal adenoma and adenocarcinoma. CONCLUSIONS: In addition to having the potential to be used as a diagnostic biomarker and an immunotherapeutic target in colorectal malignancies, UNC5D is necessary for the growth of colorectal adenomas. Additionally, UNC5D controlled STAT1/STAT3 phosphorylation to suppress the growth of colorectal cancers by regulating IFN-induced PD-L1 expression.

Significance of SDC2 and NDRG4 methylation in stool for colorectal cancer diagnosis.

BACKGROUND: Recent studies have identified methylated SDC2 and NDRG4 in colorectal cancer (CRC), however, the diagnostic value of the combined two genes remains undefined. This study aims to investigate the methylation of SDC2 and NDRG4 in stool samples and their application in diagnosis of CRC. METHODS: Five groups were enrolled in our study which consisted of CRC (n = 138), advanced adenomas (n = 27), polyp (n = 35), intestinal disease control (n = 150), and healthy individuals (n = 28). Methylation status of SDC2 and NDRG4 in fecal samples were tested with appropriate commercial kits. Primary data were collected and statistical analyses were performed. RESULTS: The positive rates of both SDC2 and NDRG4 methylation in stool samples of CRC group were significantly higher (P < 0.001) than those of either group of advanced adenomas, or polyp, or intestinal disease or the healthy control. It was suggested that both methylated SDC2,NDRG4, SDC2/NDRG4 and age were independent risk factors for CRC. The sensitivity of SDC2 and NDRG4 for CRC diagnosis were 73.9 % and 63.0 %, respectively, while SDC2 combined with NDRG4 had a higher sensitivity of 85.5 %. The specificity of SDC2, NDRG4 and SDC2 combined with NDRG4 achieved 91.6 %, 88.3 % and 84.6 %, respectively. The AUC for methylated SDC2 and NDRG4 were 0.828 (95 % CI: 0.780-0.876) and 0.757 (95 % CI: 0.703-0.811), respectively. In contrast, SDC2 combined with NDRG4 improved the AUC to 0.850 (95 % CI: 0.807-0.893). CONCLUSIONS: This research confirmed the significance of detection of SDC2 and NDRG4 methylation by using noninvasive samples of stool. More importantly, attributing to their high level and frequency of methylation in stool, SDC2 and NDRG4 could be promising biomarkers for stool-based method for screening and early diagnosis of CRC, especially when combined.