Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality. Microsatellite instability-high (MSI-H) tumors, resulting from defective DNA mismatch repair (MMR), represent a well-defined subtype with distinctive biological behavior and immunogenicity. In contrast, tumors with elevated microsatellite alterations at tetranucleotide repeats (EMAST) are less well characterized. EMAST can manifest with MSI or arise as an isolated form of instability, delineating discrete phenotypes underpinned by distinct mechanisms. This study aimed to characterize MSI and EMAST status in CRCs. By integrating instability profiles with clinicopathological features and mutational profiles of key driver genes, we aimed to refine molecular classification and advance understanding of CRC tumorigenesis. A total of 332 CRCs were analyzed for MSI and EMAST using established panels. Clinicopathological characteristics were recorded, and mutational profiling of KRAS, BRAF, CTNNB1, PIK3CA, and TP53 was performed. MLH1 expression was assessed using immunohistochemistry. MSS/EMAST-S tumors displayed profiles typical of chromosomally stable CRC, dominated by KRAS and followed by TP53 and PIK3CA mutations. MSI-H/EMAST-H tumors were characterized by frequent BRAF mutations, right-sided location, female predominance, and lower TP53 mutation rate, consistent with the classical hypermutated, immunogenic subtype. In contrast, MSS/EMAST-H tumors exhibited unique features, including enrichment for PIK3CA and CTNNB1 mutations, larger tumor size, and poorer differentiation, suggesting an intermediate phenotype between MSS and MSI-H. MSS/EMAST-L tumors aligned with chromosomally stable, KRAS/Wnt-driven CRC. In conclusion, MSS/EMAST-H tumors represent an underrecognized CRC subtype with intermediate genomic instability and a distinctive molecular profile, with potential implications for prognostic assessment and personalized therapeutic strategies.

Microsatellite instability (MSI) has been recognized as an important factor in colorectal cancer (CRC). It arises due to deficient mismatch repair (MMR), mostly attributed to MLH1 and MSH2 loss of function leading to a global MMR defect affecting mononucleotide and longer microsatellite loci. Recently, microsatellite instability at tetranucleotide loci, independent of the global MMR defect context, has been suggested to represent a distinct entity with possibly different consequences for tumorigenesis. It arises as a result of an isolated MSH3 loss of function due to its translocation from the nucleus to the cytoplasm under the influence of interleukin-6 (IL-6). In this study the influence of MSH3 and IL-6 signaling pathway polymorphisms (MSH3 exon 1, MSH3+3133A/G, IL-6-174G/C, IL-6R+48892A/C, and gp130+148G/C) on the occurrence of different types of microsatellite instability in sporadic CRC was examined by PCR–RFLP and real-time PCR SNP analyses. A significant difference in distribution of gp130+148G/C genotypes (p = 0.037) and alleles (p = 0.031) was observed in CRC patients with the C allele being less common in tumors with di- and tetranucleotide instability (isolated MSH3 loss of function) compared to tumors without microsatellite instability. A functional polymorphism in gp130 might modulate the IL-6 signaling pathway, directing it toward the occurrence of microsatellite instability corresponding to the IL-6-mediated MSH3 loss of function.

Microsatellite instability (MSI) represents an accumulation of frameshifts in short tandem repeats, microsatellites, across the genome due to defective DNA mismatch repair (dMMR). MSI has been associated with distinct clinical, histological, and molecular features of tumors and has proven its prognostic and therapeutic value in different types of cancer. Recently, another type of microsatellite instability named elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) has been reported across many different tumors. EMAST tumors have been associated with chronic inflammation, higher tumor stage, and poor prognosis. Nevertheless, the clinical significance of EMAST and its relation to MSI remains unclear. It has been proposed that EMAST arises as a result of isolated MSH3 dysfunction or as a secondary event in MSI tumors. Even though previous studies have associated EMAST with MSI-low phenotype in tumors, recent studies show a certain degree of overlap between EMAST and MSI-high tumors. However, even in stable tumors, (MSS) frameshifts in microsatellites can be detected as a purely stochastic event, raising the question of whether EMAST truly represents a distinct type of microsatellite instability. Moreover, a significant fraction of patients with MSI tumors do not respond to immunotherapy and it can be speculated that in these tumors, EMAST might act as a modifying factor.

Background and objectives: This study aimed to evaluate prognostic factors for post-recurrence survival in local and locally advanced colorectal cancer patients. Materials and Methods: A total of 273 patients with stage III and high-risk stage II colorectal cancer were prospectively enrolled. All patients underwent operative treatment of the primary tumor and adjuvant fluorouracil-based chemotherapy. Results: Over the three-year period (2008–2010), a cohort of 273 patients with stage III and high-risk stage II colorectal cancer had been screened. During follow up, 105 (38.5%) patients had disease recurrence. Survival rates 1-, 3- and 5-year after recurrence were 53.9, 18.2 and 6.5%, respectively, and the median post-recurrence survival time was 13 months. Survival analysis showed that age at diagnosis (p < 0.01), gender (p < 0.05), elevated postoperative Ca19-9 (p < 0.01), tumor histology (adenocarcinoma vs. mucinous vs. signet ring tumors, p < 0.01) and tumor stage (II vs. III, p < 0.05) had a significant influence on post-recurrence survival. Recurrence interval and metastatic site were not related to survival following recurrence. Multivariate analysis showed that older age (HR 2.43), mucinous tumors (HR 1.51) and tumors expressing Ca19-9 at baseline (HR 3.51) were independently associated with survival following recurrence. Conclusions: Baseline patient and tumor characteristics largely predicted patient outcomes after disease recurrence. Recurrence intervals in local and locally advanced colorectal cancer were not found to be prognostic factors for post-recurrence survival. Older age, male gender, stage III and mucinous histology were poor prognostic factors after the disease had recurred. Stage II patients had remarkable post-recurrence survival compared to stage III patients.

Proinflammatory counterworks are important at different stages of tumor development, particularly during invasion and metastasis. Immune cells and their signal molecules can influence all stages of tumor progression, as well as therapeutic intervention. Proinflammatory cytokines are known triggers of growth in gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). In this study, we explored the immunohistochemical expression of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), IL-2, and IL-6 in tissues from 43 GEP-NEN patients with tumors of gastric, duodenal, ileal, appendical, and colonic origin. The immunohistochemical expression of TNF-α was increased in tumor groups with high proliferation rates (Ki67; p = 0.034), as well as in those with higher tumor grades (p = 0.05). Moreover, the immunohistochemical expression of TNF-α positively correlated with death outcomes (p = 0.016). Expression of IL-6, IL-1β, and IL-2 displayed similar immunohistochemical expression patterns regardless of Ki67, although the expression between the ILs differed. Most GEP-NENs had high levels of IL-6 and lower levels of IL-1β and IL-2. Although further comprehensive studies are required for a complete understanding of activated mechanisms in proinflammatory protumoral microenvironment of GEP-NENs, TNF-α is a potential marker in the prognosis of those tumors.

Background/ Aim: Thrombin plays significant roles in various types of cancer. However, the expression levels of prothrombin, the thrombin precursor, in cancer remain unclear. Variants of the 3'end of the prothrombin gene lead to increased prothrombin expression. This study aimed to analyze prothrombin 3'end gene variants in colon tumor and adjacent normal tissue samples. Materials and Methods: The study group consisted of 93 patients suffering from colon adenocarcinoma. The 3'end of the prothrombin gene was analyzed by DNA sequencing. Results: Three variants, all previously associated with increased prothrombin expression were detected. Frequency of the FII 19911G allele was 46.77% and 47.85% in tumor and normal tissue, respectively. For the FII 20210A allele, the detected frequencies were 2.15% and 1.61%, respectively. The frequency of the FII c.1824T allele was 0.54% in both tissues. Four patients showed different genotypes in tumor and normal tissue. Conclusion: Prothrombin 3' end gene variants may play a role in colorectal cancer.

BACKGROUND mutS homolog 2 (MSH2) deficiency may be involved in the development of microsatellite instability found in certain sporadic colorectal tumors. In addition to mutations or loss of heterozygosity resulting in complete loss of MSH2 function, polymorphisms affecting MSH2 expression have been also identified. Therefore, the aim of this study was to examine MSH2 status in sporadic colon cancer. MATERIALS AND METHODS MSH2 status was examined at the DNA, RNA and protein levels through loss of heterozygosity (LOH) analysis, quantitative real-time PCR and immunohistochemistry. MSH2 IVS10+12A>G polymorphism was examined by real-time single nucleotide polymorphism genotyping. RESULTS MSH2 LOH was more frequent in tumors larger than 5 cm (p=0.032), mRNA expression was also significantly lower and the same expression pattern was present in the corresponding normal mucosa of the same patient (p=0.013 and p=0.008, respectively). No association was found between IVS10+12A>G polymorphism and susceptibility to sporadic colon cancer. CONCLUSION Altered MSH2 expression detected in sporadic colon tumors pointing to its role in colorectal tumorigenesis without a hereditary component.

Medicinska genetika obilježena je dinamicnom izmjenom informacija i stalnom usložavanju problema zahvaljujuci velikom znanstveno- tehnoloskom napredku medicine. Zato je prepoznavanje i dijagnosticiranje bolesti uzrokovanih poremecajima kromosoma/gena danas znatno obimnije nego prijasnjih desetljeca pa su tako postale dio svakodnevnog razmisljanja strucnjaka razlicitih profila koji dolaze u dodir s bolesnicima i njihovim obiteljima. Namjera nam je bila pokazati raznolikost medicinske/klinicke genetike unutar istaknutih specijalnosti koje su najtjesnje povezane s procesom genetickog informiranja. Kako ce proci jos podosta vremena prije nego li ce se u Republici Hrvatskoj doskolovati prvi specijalisti samostalne specijalizacije klinicke/medicinske genetike, smatrale smo da ce studentima i lijecnicima, specijalistima raznih podrucja medicine, „dobro doci“ knjiga u kojoj se na razlicite nacine razmatra geneticko informiranje bolesnika i clanova njihovih obitelji. Ova je knjiga osmisljena na takav nacin. Citateljima je izložena sva složenost genetickog informiranja, a pravo bogatstvo rjesenja nudi timski pristup, ne bolesti, vec bolesnicima. Suocavanje s razlicitim pogledima, profesionalnim usmjerenjima i senzibilitetom nudi dijalog, razmjenu teorijskih i prakticnih misljenja, gledista i stajalista cime umnaža vrijednost napora autora.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are highly vascularized neoplasms, capable of synthethisizing VEGF-A, a key mediator of angiogenesis. In pancreatic neuroendocrine tumors (pNETs) VEGF expression is higher in benign and low-grade tumors and associated with good prognosis (neuroendocrine paradox) while the VEGF role in gastrointestinal NETs (GI-NETs) is still unclear. In this study, we examined the VEGF-1154A/G polymorphism in 145 GEP-NET patients and 150 controls. Next, we measured VEGF serum levels and VEGF tumor protein expression, comparing it with Ki67 and tumor grade. Patients' VEGF serum levels were compared with VEGF -1145A/G genotypes and metastatic status as well as with chromogranin A (CgA) and 5-hydroxyindolacetic acid (5-HIAA) in case of GI-NET patients. In this study GEP-NET patients had elevated VEGF serum values when compared to healthy controls (p = 0.0013). VEGF-1145G allele correlated with higher VEGF serum levels (p = 0.002). Patients with metastatic tumors had higher VEGF serum values when compared to patients without metastases (p = 0.033), and highest levels were observed in case of lymph node metastases (p = 0.008). VEGF-1145G allele was more frequent in non-functional GI-NET patients than in healthy controls (p = 0.041). CgA was superior to VEGF in tumor detection, while VEGF was superior to 5-HIAA. A correlation was observed between VEGF immunohistochemical staining and Ki-67 (p = 0.028). Tumours with weaker VEGF protein expression were more aggressive than tumours with stronger VEGF expression, confirming a "neuroendocrine paradox" in GI-NETs. Our results suggest the role of VEGF in GI-NETs locoregional spread.

Many scenarios occurring in genomics and proteomics involve small number of labeled data and large number of variables. To create prediction models robust to overfitting variable selection is necessary. We propose variable selection method using nonlinear sparse component analysis with a reference representing either negative healthy or positive cancer class. Thereby, component comprised of cancer related variables is automatically inferred from the geometry of nonlinear mixture model with a reference. Proposed method is compared with 3 supervised and 2 unsupervised variable selection methods on two-class problems using 2 genomic and 2 proteomic datasets. Obtained results, which include analysis of biological relevance of selected genes, are comparable with those achieved by supervised methods. Thus, proposed method can possibly perform better on unseen data of the same cancer type.

This study was aimed at the analysis of mononucleotide repeats -462T(15) and -4T(12) in the SMAD4 gene promoter in sporadic colon adenocarcinoma tissue of Croatian patients. The analysis has included 60 pairs of samples of colon tumor and adjacent normal tissue. The number of thymidines in the tracts -462T(15) and -4T(12) of the SMAD4 gene promoter was determined by PCR with fluorescently labeled primers followed by the analysis of obtained DNA fragments by capillary electrophoresis. In the normal colon tissue two haplotypes were present: -462T(15)/-4T(12) in 51 patients (85%) and -462T(16)/-4T(12) in 9 patients (15%). Among the cases with haplotype -462T(15)/-4T(12) detected in normal colon tissue, in 5 cases (8%) malignant tissue displayed different haplotypes: 462T(10)/-4T(10), -462T(12)/-4T(12), 462T(13)/-4T(11), -462T(14)/-4T(10) and -462T(15)/-4T(11). Haplotype -462T(14)/-4T(10) was previously found to be associated with significantly decreased SMAD4 gene promoter activity in comparison to the wild type, while the other detected haplotypes remain to be functionally characterized. This study has shown that functionally relevant somatic alterations of the SMAD4 gene promoter are found in some colon cancer tumors. Although not as frequent in colon as in pancreatic cancer, they may be of significance for certain cases and their role in colon tumorigenesis should be investigated further.

Purpose NF2 /Merlin was first identified through its association with neurofibromatosis type 2 (NF2). However, accumulating evidence suggests a more general involvement in tumorigenesis and, in particular, a broader role in tumor suppression. The aim of this study was to examine NF2 /Merlin involvement in sporadic colorectal cancer. Methods This study is the first to examine the role of NF2 /Merlin in sporadic colorectal cancer through LOH analysis at the NF2 locus and mRNA expression analysis via quantitative RT-PCR of total NF2, NF2 isoform I and II. In addition, Merlin protein expression was assessed by immunohistochemistry and Western blotting. Results NF2 LOH was detected in 20.0 % of heterozygous cases and was found to be more frequent in tumors larger than 5 cm in diameter ( p = 0.041) and in tumors with a less differentiated phenotype ( p = 0.027). No differences were observed in total NF2 and NF2 isoform I/isoform II mRNA expression between the tumors and their corresponding normal mucous tissues. NF2 isoform II was the most predominant isoform in all samples analyzed. mRNA expression levels of total NF2 and isoforms I and II were significantly lower in poorly differentiated tumors ( p = 0.033, p = 0.036 and p = 0.044, respectively). Weak Merlin immunostaining was more frequent in poorly differentiated tumors ( p = 0.034) and tumors classified as Dukes’ C ( p = 0.023). A distinct pattern of Merin phosphorylation was observed in tumors compared to normal mucous tissues. Conclusion Our data indicate that NF2 /Merlin may serve as a potential target in the management of colorectal cancer.

Although previously considered rare, recent epidemiological studies have revealed that the incidence (3.6/100,000) and prevalence (35/100,000) of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has increased over the past few decades. Despite the progress in the understanding of GEP-NET molecular biology, there is still little advance in the early diagnosis due to lack of specific tumor markers. As the tumors are mostly detected in their late stage, they are not well controlled by either biotherapy or conventional chemotherapy, and thus represent a significant clinical issue. Chronic inflammation has been implicated in the development of GEP-NETs. This review presents recent findings that link pro-inflammatory cytokines to the molecular basis of GEP-NET tumorigenesis, leading to a more personalized approach to disease management and therapy.

Purpose: Inherited polymorphisms in immunomodulatory genes such as cytokines may contribute to variation in immunological response and genetic susceptibility for complex diseases, including cancer. TNF alpha can mediate tumor progression by inducing proliferation, invasion and metastasis of tumor cells. The aim of our study was to examine the allelic frequencies of TNF alpha promoter SNPs, -1031 T/C, -857 C/T, -308 G/A and -238 G/A, in patients with sporadic colon adenocarcinoma in order to investigate the possible role of these SNPs in susceptibility to sporadic colon cancer. Another aim of this study was to examine the influence of TNF alpha SNPs on TNF alpha mRNA and protein expression in colon tumors and their possible role in the development and progression of this type of tumor.Results: The distribution of all four TNF alpha SNP genotypes in patients showed no significant difference compared to controls. No statistically significant difference in TNF alpha mRNA expression in tumors and corresponding normal mucous tissue was found (p = 0.14). A statistically significant (p = 0.028) difference was found in TNF alpha mRNA expression between histological grade I and histological grade 2 and 3 tumors. Additionally, a statistically significant correlation (p = 0.03) was found between TNF alpha-857 C/T genotypes and TNF alpha mRNA expression in tumor tissue. TNF alpha mRNA expression was significantly higher in the tumor tissue of patients with -857 CT and -857 TT genotypes. Most of the tumors (78.26%) were positive for TNF alpha protein. No correlation was found between the TNF alpha protein expression and clinicopathological characteristics as well as TNF alpha genotypes. However, patients with TNF alpha protein negative tumors had longer survival but the result was not statistically significant (p = 0.365).Conclusion: Our results suggest the role of TNF alpha as one of the immunomodulatory genes in the progression of sporadic colon cancer. (C) 2014 Elsevier Inc. All rights reserved.

Cilj: Rizik obolijevanja od raka kao i napredovanje tumora u velikoj mjeri ovise o genetickoj podlozi pojedinca. Prisutnost polimorfnih mjesta u genomu covjeka omogucuje varijabilnost koja utjece na normalne fizioloske mehanizme kao i na patogenezu raka. Citokini djeluju kao geni modifikatori u procesu tumorigeneze s nizom funkcija cija regulacija nije do kraja razjasnjena te je ponekad cak i kontradiktorna. Koja ce od funkcija prevladati ovisi prvenstveno o biologiji samog tumora kao i njegovoj mikrookolini. Metode: U ovom istraživanju ispitana je uloga IL- 6, IL-8, IL-10, TNFα te VEGF u nastanku i napredovanju sporadicnih karcinoma debelog crijeva kroz analizu ucestalosti SNP polimorfizama ovih gena u oboljelih u odnosu na zdravu populaciju. Također je ispitana i njihova ekspresija na razini mRNA i proteina metodom real-time PCR te imunohistokemijom. Testovima migracije i invazije ispitan je utjecaj IL-8 i IL-10 u uvjetima in vitro. Rezultati: Opažena je statisticki znacajna razlika u raspodjeli pojedinih genotipova SNP polimorfizma u genu IL-10, -819C/T te -592C/A, u skupini oboljelih u odnosu na zdravu populaciju. Dokazana je povezanost ekspresije IL-10 mRNA s genotipom polimorfizama -1082A/G, -819 C/T te -592C/A. Ekspresija IL-10 mRNA je u tkivu tumora bila niža, dok je ekspresija IL-8 mRNA bila visa u odnosu na ekspresiju u pripadajucoj normalnoj sluznici. Ekspresija proteina IL-8 bila visa u umjereno i slabo diferenciranim tumorima. Ekspresija proteina IL-10 bila visa u dobro i umjereno diferenciranim tumorima, dok je u tumorima klasificiranim kao Dukes' C ekspresija IL-10 bila niža u odnosu na tumore klasificirane kao Dukes' A i B. U ispitivanju utjecaja na migraciju i invazivnost stanica trajne stanicne linije adenokarcinoma debelog crijeva HT-29 u uvjetima in vitro, IL-8 je stimulirao dok je IL-10 imao slab inhibitorni ucinak na invazivnost stanica. Zakljucak: Na temelju provedenih analiza može se zakljuciti kako IL-8 i IL-10 imaju znacajnu ulogu u nastanku i napredovanju sporadicnih karcinoma debelog crijeva.

Purpose: Tumor supressor gene FHIT was identified at chromosome 3p14.2 spanning the FRA3B fragile site and is very often inactivated in different types of cancer. The aim of this study was to examine the frequency of FHIT gene LOH as well as FHIT mRNA and protein expression in sporadic colon adenocarcinoma.Methods: The results of LOH, real-time qRT-PCR and imunohistochemical analyses were correlated with clinicopathological characteristics of patients and their tumors in order to evaluate the role of FHIT gene/protein in sporadic colon adenocarcinoma tumorigenesis.Results: One hundred and thirty one (96.3%) samples were informative for both markers and 33/131 (25.2%) demonstrated LOH. Expression of FHIT mRNA was significantly decreased in colon tumors relative to that in corresponding normal tissue (p = 7.2 x 10(-6)). Most of the samples (54.0%) were negative for FHIT protein, 26.4% adenocarcinomas showed a weak to moderate immunostaining and 19.6% adenocarcinomas showed strong FHIT immunostaining. No correlation was found between FHIT gene LOH status, mRNA expression or FHIT protein immunostaining and clinico-pathological characteristics. Expression of FHIT mRNA was significantly decreased in FH/TLOH positive tumors (p = 0.027). Patients with LOH negative tumors or FHIT protein positive tumors had longer survival but this findings were not statistically significant.Conclusions: Our overall results suggest that reduced expression of FHITgene may be associated with the progression of these malignant tumors. (C) 2013 Elsevier Inc. All rights reserved.