MutL homolog 1 methylation and microsatellite instability in sporadic colorectal tumors among Filipinos

BACKGROUND Colorectal cancer(CRC)ranks third in terms of incidence and second in mortality worldwide.In CRC,the silencing of mismatch repair genes,including the mutL homolog 1(hMLH1)has been linked to microsatellite instability(MSI),the lengthening or shortening of microsatellite repeats.Very limited data have been presented so far on the link of hMLH1 methylation and MSI in Southeast Asia populations with sporadic CRC,and on its clinical significance.AIM To investigate the significance of the MSI status and hMLH1 methylation in CRC Filipino patients.METHODS Fifty-four sporadic CRC patients with complete clinical data were included in this study.Genomic DNA from CRC tumor biopsies and their normal tissue counterparts were profiled for MSI by high resolution melting(HRM)analysis using the Bethesda Panel of Markers(BAT25,BAT26,D2S123,D5S346,and D17S250).hMLH1 methylation screening was performed using bisulfite conversion and methylation specific polymerase chain reaction.Statistical analysis was conducted to calculate their associations to clinicopathological characteristics and survival relevance(Kaplan-Meier curves and the log-rank test).RESULTS hMLH1 methylation was observed in 9%and 35%of CRC and normal samples,respectively.Higher incidence of consistently methylated hMLH1 found in both normal and CRC was noticed for relation to location of tumor(P<0.05).As for MSI status,D2S123 the most common unstable microsatellite and MSI-high(MSIH)was the most common MSI profile,counted for 46%and 50%of normal and CRC tissues,respectively.The presence of MSI-low(MSI-L)and microsatellite stable(MSS)was 43%and 11%for normal,and 31%and 19%for CRC samples.The mean month of patients’survival was shorter in patients whose normal and tumor tissues had methylated compared to those with unmethylated hMLH1 and with MSI-H compared to those with MSI-L/MSS(P<0.05).This was supported by significant difference in Kaplan-Meier with log-rank analysis.This data indicated that hMLH1 methylation and high MSI status have prognostic value.CONCLUSION This study showed the clinical significance of hMLH1 methylation and MSI status in sporadic CRC Filipino patients,especially in the normal part of the tumor.

Checkpoint with forkhead-associated and ring finger promoter hypermethylation correlates with microsatellite instability in gastric cancer

AIM:To examine the methylation status of the promoter region of the checkpoint with forkhead-associated and ring fi nger(CHFR) and microsatellite mutator status in 59 primary gastric cancers.METHODS:We investigated the promoter methylation of CHFR in 59 cases of gastric cancer using methylation-specifi c PCR.Five microsatellite loci were analyzed using high-intensity microsatellite analysis reported previously, and p53 gene mutations were investigated by direct sequencing.RESULTS:Twenty cases(33.9%) showed promoter methylation and no relation was observed with the clinicopathological factors.We found that the promoter methylation of CHFR was frequently accompanied with microsatellite instability(MIN).Seven of 20(35.0%) cases showed MIN in hypermethylation of the CHFR tumor, while three of 39(7.7%) cases showed MIN in the non-methylated CHFR tumor(P < 0.01).However, we failed to fi nd any relationship between CHFR methylation and p53 mutation status.CONCLUSION:The coordinated loss of both the mitotic check point function and mismatch repair system suggests the potential to overcome the cell cycle check point, which may lead to an accumulation of mutations.However, the p53 mutation was not related to hypermethylation of the CHFR promoter and MIN, which indicates that an abnormality in p53 occurs as an independent process from the mismatch repair deficiency in carcinogenesis.

Novel mutations and sequence variants in exons 3-9 of human T Cell Factor-4 gene in sporadic rectal cancer patients stratified by microsatellite instability

AIM: To establish the role of human T Cell Factor-4 (hTCF-4) gene exons 3-9 mutation status in association with sporadic rectal cancer with microsatellite instability (MSI). METHODS: Microsatellite markers were genotyped in 93 sporadic rectal cancer patients. Eleven cases were found to be high-frequency MSI (MSI-H). Sequence analysis of the coding region of the exons 3-9 of hTCF-4 gene was carried out for the 11 MSI-H cases and 10 controls (5 microsatellite stability (MSS) cases and 5 cases with normal mucosa). The sequencing and MSI identification were used. RESULTS: Several novel mutations and variants were revealed. In exon 4, one is a 4-position continuous alteration which caused amino acid change from Q131T and S132I (391insA, 392 G > A, 393 A > G and 395delC) and another nucleotide deletion (395delC) is present in MSI-H cases (5/10 and 4/10, respectively) but completely absent in the controls.CONCLUSION: Novel mutations in exon 4 of hTCF-4 gene were revealed in this study, which might be o importance in the pathogenesis of sporadic rectal cance patients with MSI-H.

Helicobacter pylori and EBV in gastric carcinomas:Methylation status and microsatellite instability

AIM:To verify the methylation status of CDH1, DAPK, COX2, hMLH1 and CDKN2A genes and to evaluate their association with Helicobacter pylori (H. pylori)-cagA+ and Epstein Barr virus (EBV) infections in gastric adenocarcinomas.METHODS: Methylation-specific PCR (MSP) assay was performed in 89 primary gastric carcinomas (intestinal and diffuse types). Microsatellite instability (MSI) analysis was performed using the BAT26 primer set and PCR products were analyzed with the ABI PRISM 3100 Genetic Analyzer using Genescan 3.7 software (Applied Biosystems). Detection of H. pylori and genotyping were performed by PCR, using specifi c primers for ureaseC and cagA genes. The presence of EBV was assessed by in situ hybridization. Statistical analyses were performed using the χ2 or Fisher's exact test.RESULTS: The most frequent hypermethylated gene was COX-2 (63.5%) followed by DAPK (55.7%), CDH1 (51%), CDKN2A (36%) and hMLH1 (30.3%). Intestinal and diffuse adenocarcinomas showed different methylation profiles and there was an association between methylation of E-CDH1 and H. pylori-cagA+ in the intestinal adenocarcinoma type. MSI was correlated with hMLH1 methylation. There was an inverse correlation between DAPK hypermethylation and MSI.CONCLUSION: We found a strong association between CDH1 methylation and H. pylori-cagA+ in intestinal-type gastric cancer, association of MSI and better prognosis and an heterogeneous COX-2 overexpression.

No Change in Frequency of Microsatellite Instability in Colorectal Cancers over a Period of 15 or More Years

Microsatellite instability (MSI) is a molecular change resulting from inactivation of DNA mismatch repair systems, occurring with a reported incidence between 15% - 20% of all sporadic colorectal cancers. Our aim was to determine whether a change in the incidence of MSI in colorectal cancer had occurred at our institution over time. We assayed 106 cases from the mid-1990s and 69 cases from 15 or more years later for MSI. Those tumors with MSI were assayed for BRAF mutation and methylation. MSI was detected in 15 (14.2%) of the early cases and 11 (15.9%) of the later cases. For the two groups with MSI, a similar percentage was methylated and had a BRAF mutation. One tumor in each group was MSI, unmethylated, and BRAF wild type. Our data indicate consistency in the frequency of microsatellite unstable colorectal cancer across a time span of 15 or more years.

MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults

AIM:To evaluate the association between Helicobacter pylori(H.pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability(MSI).METHODS:The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction(MSPPCR) in gastric biopsy samples from uninfected or H.pylori-infected children(n = 50),from adults with chronic gastritis(n = 97) and from adults with gastric cancer(n = 92).MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene(β actin).MSI analysis was performed by screening MSI markers at 4 loci(Bat-25,Bat-26,D17S250 and D2S123) with PCR;PCR products were analysed by single strand conformation polymorphism followed by silver staining.Statistical analyses were performed with either the χ 2 test with Yates continuity correction or Fisher's exact test,and statistical significance for expression analysis was assessed using an unpaired Student's t-test.RESULTS:Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children,regardless of H.pylori infection status.The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H.pylori infection(P < 0.05);this region was methylated in 66% of gastric cancer patients,and the difference in the percentage of methylated samples between these patients and those from H.pylori-infected chronic gastritis patients was statistically significant(P < 0.05).MLH1 methylation frequencies among H.pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%,respectively.We observed methylation of the MLH1 promoter(39%) and increased MSI levels(68%) in samples from gastric cancer patients in comparison to samples from H.pylori-infected adult chronic gastritis patients(P < 0.001 and P < 0.01,respectively).The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H.pylori-positive chronic gastritis samples(P < 0.05).The levels of MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated(P < 0.01).CONCLUSION:In summary,MGMT and MLH1 methylation did not occur in earlier-stage H.pylori infections and thus might depend on the duration of infection.

Microsatellite instability and MLH1 promoter hypermethylation in colorectal cancer

Colorectal cancer (CRC) is caused by a series of genetic or epigenetic changes, and in the last decade there has been an increased awareness that there are multiple forms of colorectal cancer that develop through different pathways. Microsatellite instability is involved in the genesis of about 15% of sporadic colorectal cancers and most of hereditary nonpolyposis cancers. Tumors with a high frequency of microsatellite instability tend to be diploid, to possess a mucinous histology, and to have a surrounding lymphoid reaction. They are more prevalent in the proximal colon and have a fast pass from polyp to cancer. Nevertheless, they are associated with longer survival than stage-matched tumors with microsatellite stability. Resistance of colorectal cancers with a high frequency of microsatellite instability to 5-fluorouracil-based chemotherapy is well established. Silencing the MLH1 gene expression by its promoter methylation stops the formation of MLH1 protein, and prevents the normal activation of the DNA repair gene. This is an important cause for genomic instability and cell proliferation to the point of colorectal cancer formation. Better knowledge of this process will have a huge impact on colorectal cancer management, prevention, treatment and prognosis.

Comparative features of colorectal and gastric cancers with microsatellite instability in Chinese patients

Objective:The purpose of this study was to determine the unique and universal features of microsatellite instability-high(MSI-H) colorectal cancer(CRC) and MSI-H gastric cancer(GC) in the Chinese population.Methods:A new panel of mononucleotide MSI markers,BAT25,BAT26,NR21,NR24,and MONO-27,was used to define MSI status in 303 CRC and 288 GC subjects.Clinicopathological features of both types of MSI-H tumors were analyzed.Methylation analysis in the hMLH1 promoter region by methylation specific polymerase chain reaction(PCR) and mutation detection of hMSH2/hMLH1 genes by denaturing high-performance liquid chromatography(DHPLC) were carried out simultaneously.Results:MSI-H CRCs and MSI-H GCs account for 11.9% and 8.0% of unselected sporadic CRCs and GCs,respectively.MSI-H CRCs are strongly characterized by early onset,right-side location,low differentiation,mucinous tumor,less infiltration,less lymphatic metastasis,and more often familial tumor.MSI-H GCs only showed site preference for the antrum and less lymphatic metastasis.Genetic and epigenetic analyses were positive in 6/36 MSI-H CRCs and 0/23 MSI-H GCs with pathological mutation in major mismatch repair genes,and in 7/36 MSI-H CRCs and 18/23 MSI-H GCs with methylated hMLH1 promoter(P<0.01),respectively.Conclusions:Although there are many differences in the genetic basis and clinicopathological features between MSI-H CRC and MSI-H GC,when compared with their microsatellite stable(MSS) counterparts,site preference and lymphatic metastasis are features common to both types of MSI-H tumors.

Influence of methyl donor nutrients as epigenetic regulators in colorectal cancer:A systematic review of observational studies

BACKGROUND Dietary methyl donors might influence DNA methylation during carcinogenesis of colorectal cancer(CRC).However,whether the influence of methyl donor intake is modified by polymorphisms in such epigenetic regulators is still unclear.AIM To improve the current understanding of the molecular basis of CRC.METHODS A literature search in the Medline database,Reference Citation Analysis(https://www.referencecitationanalysis.com/),and manual reference screening were performed to identify observational studies published from inception to May 2022.RESULTS A total of fourteen case-control studies and five cohort studies were identified.These studies included information on dietary methyl donors,dietary components that potentially modulate the bioavailability of methyl groups,genetic variants of methyl metabolizing enzymes,and/or markers of CpG island methylator phenotype and/or microsatellite instability,and their possible interactions on CRC risk.CONCLUSION Several studies have suggested interactions between methylenetetrahydrofolate reductase polymorphisms,methyl donor nutrients(such as folate)and alcohol on CRC risk.Moreover,vitamin B6,niacin,and alcohol may affect CRC risk through not only genetic but also epigenetic regulation.Identification of specific mechanisms in these interactions associated with CRC may assist in developing targeted prevention strategies for individuals at the highest risk of developing CRC.

Prognostic values of chromosome 18q microsatellite alterations in stage Ⅱ colonic carcinoma

AIM: To investigate the prognostic value of chromosome 18q microsatellite alterations (MA) in stage Ⅱ colon cancer. METHODS: One hundred and six patients with sporadic stage Ⅱ colon cancer were enrolled in this study. DNA was extracted from formalin-fixed, paraffin-embedded tumor and adjacent normal mucosal tissue samples. MA, including loss of heterozygosity (LOH) and microsatellite instability (MSI), was analyzed by polymerase chain reaction, polyacrylamide gel-electrophoresis and DNA sequencing at 5 microsatellite loci on chromosome 18q (D18S474, D18S55, D18S58, D18S61 and D18S64).RESULTS: Among the 102 patients eligible for MA information, the overall frequencies of LOH, high and low frequency MSI/microsatellite stable were 49.0%, 17.6% and 82.4%, respectively. The high frequency of 18q-LOH was signif icantly associated with the poor 5-year overall survival (OS) (P=0.008) and disease free survival (P=0.006). High levels of MSI were significantly associated with a longer 5-year OS (P=0.045) while the higher frequency of 18q-LOH at the loci of D18S474 and D18S61 was significantly associated with a poorer 5-year OS (P=0.010 and 0.005, respectively). But multivariate analysis showed that only the frequency of 18q-LOH was significantly associated with the prognosis of the disease. CONCLUSION: High frequency of 18q-LOH is an independent prognostic factor indicating poor prognosis of the patients with stage Ⅱ colon cancer.

Molecular and prognostic heterogeneity of microsatellite-unstable colorectal cancer

Colorectal cancers(CRCs)with a high level of microsatellite instability(MSI-H)are clinicopathologically distinct tumors characterized by predominance in females,proximal colonic localization,poor differentiation,mucinous histology,tumor-infiltrating lymphocytes,a Crohn’s-like lymphoid reaction and a favorable prognosis.In terms of their molecular features,MSI-H CRCs are heterogeneous tumors associated with various genetic and epigenetic alterations,including DNA mismatch repair deficiency,target microsatellite mutations,BRAF mutations,a CpG island methylator phenotype-high(CIMP-H)status,and a low level of genomic hypomethylation.The molecular heterogeneity of MSI-H CRCs also depends on ethnic differences;for example,in Eastern Asian countries,relatively low frequencies of CIMP-H and BRAF mutations have been observed in MSI-H CRCs compared to Western countries.Although the prognostic features of MSI-H CRCs include a favorable survival of patients and low benefit of adjuvant chemotherapy,there may be prognostic differences based on the molecular heterogeneity of MSI-H CRCs.Here,we have reviewed and discussed the molecular and prognostic features of MSI-H CRCs,as well as several putative prognostic or predictive molecular markers,including HSP110 expression,beta2-microglobulin mutations,myosin 1a expression,CDX2/CK20 expression,SMAD4expression,CIMP status and LINE-1 methylation levels.

Prognostic and predictive significance of MSI in stages Ⅱ/Ⅲ colon cancer

In colon cancer,classic disease staging remains the key prognosis and treatment determinant.Although adjuvant chemotherapy has an established role in stageⅢcolon cancer patients,in stageⅡit is still a subject of controversy due to its restriction to a small subgroup of patients with high-risk histopathologic features.Patients with stageⅡtumors form a highly heterogeneous group,with five-year relative overall survival rates ranging from 87.5%(ⅡA)to 58.4%(ⅡC).Identifying those for whom adjuvant chemotherapy would be appropriate and necessary has been challenging,and prognostic markers which could serve in the selection of patients more likely to recur or benefit from adjuvant chemotherapy are eagerly needed.The stronger candidate in this category seems to be microsatellite instability(MSI).The recently reported European Society for Medical Oncology guidelines suggest that MSI should be evaluated in stageⅡcolorectal cancer patients in order to contribute in treatment decisionmaking regarding chemotherapy administration.Thehypothetical predictive role of MSI regarding its response to 5-fluorouracil-based adjuvant chemotherapy has proven a much more difficult issue to address.Almost every possible relation between MSI and chemotherapy outcome has been described in the adjuvant colon cancer setting in the international literature,and the matter is far from being settled.In this current report we critically evaluate the prognostic and predictive impact of MSI status in patients with stageⅡand stageⅢcolon cancer patients.

The path toward prognostication and prediction in stage Ⅱ colon cancer

Currently,there are several newer biomarkers that may be clinically useful in colon cancer. This paper focuses on a few of these biomarkers,namely microsatellite instability,loss of heterozygosity at chromosome 18q(LOH18q) and multi-gene assays,and discusses the clinical evidence behind their predictive or prognostic abilities. The results show that although there have been several newer prognostic factors identified,such as LOH18 q and multi-gene assays,none of these factors can predict benefit from treatment. Therefore,ongoing prospective clinical trials are still needed to further assess the role and optimal use of these tests.

纳武利尤单抗联合伊匹木单抗治疗MSI-H晚期结肠癌1例报道

本文报告1例晚期结肠癌患者,既往术后标本免疫组化结果提示错配修复完整(proficient mismatch repair, pMMR),予术后化疗等全身治疗后病情进展。随后行二代测序(next-generation sequencing, NGS)提示该患者微卫星高度不稳定(microsatellite instability-high, MSI-H)以及高水平的肿瘤突变负荷值(tumor mutation burden, TMB)。

hMLH1和miR-148a表达与散发性结直肠癌微卫星不稳定性的关系

目的检测散发性结直肠癌(sCRC)组织中人类错配修复基因1(hMLH1)蛋白和miR-148a表达及其与微卫星不稳定性(MSI)的关系。方法收集黄冈市中心医院病理科保存的sCRC组织标本90例及其对应癌旁正常组织。收集患者临床病历资料,采用多重荧光PCR法检测组织DNA微卫星不稳定性,免疫组化染色法检测组织hMLH1蛋白表达,实时荧光定量PCR(qPCR)检测组织miR-148a表达量,双荧光素酶报告实验分析miR-148a和hMLH1 mRNA靶向关系。结果90例sCRC组织中,检出MSI 29例(32.22%),hMLH1蛋白表达缺失24例(26.67%)。经荧光素酶报告基因分析,miR-148a mimics可抑制含有hMLH1 mRNA 3′UTR-WT序列的报告基因质粒的荧光素酶活性;而且hMLH1蛋白表达缺失组织中miR-148a相对表达量高于未缺失组织(P<0.05);此外MSI组织中hMLH1蛋白表达缺失率和miR-148a相对表达量均高于微卫星稳定性(MSS)组织。右半结肠和直肠部位、pN_(0/1)期、pM _(0)分期hMLH1蛋白表达缺失率和MSI比例高于左半结肠、pN_(2)期、pM_(0)期(P<0.05);另外肿瘤直径>5.0 cm、右半结肠和直肠、pT_( 3/4)期、pN_(2)期、pM_(1)分组织miR-148a相对表达量低于肿瘤直径≤5.0 cm、左半结肠、pT_(1/2)期、pN_(0/1)期、pM_(0)期组织(P<0.05)。结论约30%的sCRC患者存在hMLH1蛋白表达缺失和MSI状态;sCRC肿瘤组织miR-148a相对表达量降低,导致对hMLH1基因表达的抑制作用减弱,可能是影响sCRC肿瘤细胞MSI状态的重要原因之一。

Clinical and molecular features of young-onset colorectal cancer

Colorectal cancer(CRC) is one of the leading causes of cancer related mortality worldwide. Although young-onset CRC raises the possibility of a hereditary component, hereditary CRC syndromes only explain a minority of young-onset CRC cases. There is evidence to suggest that young-onset CRC have a different molecular profile than late-onset CRC. While the pathogenesis of young-onset CRC is well characterized in individuals with an inherited CRC syndrome, knowledge regarding the molecular features of sporadic young-onset CRC is limited. Understanding the molecular mechanisms of young-onset CRC can help us tailor specific screening and management strategies. While the incidence of late-onset CRC has been decreasing, mainly attributed to an increase in CRC screening, the incidence of young-onset CRC is increasing. Differences in the molecular biology of these tumors and low suspicion of CRC in young symptomatic individuals, may be possible explanations. Currently there is no evidence that supports that screening of average risk individuals less than 50 years of age will translate into early detection or increased survival. However, increasing understanding of the underlying molecular mechanisms of young-onset CRC could help us tailor specific screening and management strategies. The purpose of this review is to evaluate the current knowledge about young-onset CRC, its clinicopathologic features, and the newly recognized molecular alterations involved in tumor progression.

Use of blood-based biomarkers for early diagnosis and surveillance of colorectal cancer

Early screening for colorectal cancer(CRC) holds the key to combat and control the increasing global burden of CRC morbidity and mortality. However, the current available screening modalities are severely inadequate because of their high cost and cumbersome preparatory procedures that ultimately lead to a low participation rate. People simply do not like to have colonoscopies. It would be ideal, therefore, to develop an alternative modality based on blood biomarkers as the first line screening test. This will allow for the differentiation of the general population from high risk individuals. Colonoscopy would then become the secondary test, to further screen the high risk segment of the population. This will encourage participation and therefore help to reach the goal of early detection and thereby reduce the anticipated increasing global CRC incidence rate. A blood-based screening test is anappealing alternative as it is non-invasive and poses minimal risk to patients. It is easy to perform, can be repeated at shorter intervals, and therefore would likely lead to a much higher participation rate. This review surveys various blood-based test strategies currently under investigation, discusses the potency of what is available, and assesses how new technology may contribute to future test design.

Multiple primary colorectal cancer: Individual or familial predisposition?

Colorectal carcinoma(CRC) is one of the most frequent cancers. Along the surface of the large bowel, several foci of CRC may appear simultaneously or over the time. The development of at least two different tumours has been defined as multiple primary CRC(MPCRC):When more than one tumour is diagnosed at the same time, it is known as synchronous CRC(SCRC), while when a second neoplasm is diagnosed some time after the resection and/or diagnosis of the first lesion, it is called metachronous CRC(MCRC). Multiple issues can promote the development of MPCRC, ranging from different personal factors, such as environmental exposure, to familial predisposition due to hereditary factors. However, most studies do not distinguish this dichotomy. High- and low-pentrance genetic variants are involved in MPCRC. An increased risk for MPCRC has been described in Lynch syndrome, familial adenomatous polyposis, and serrated polyposis. Non-syndromic familial CRCs should also be considered as risk factors for MPCRC. Environmental factors can promote damage to colon mucosae that enable the concurrence of MPCRC. Epigenetics are thought to play a major role in the carcinogenesis of sporadic MPCRC. The methylation state of the DNA depends on multiple environmental factors(e.g., smoking and eating foods cooked at high temperatures), and this can contribute to increasing the MPCRC rate. Certain clinical features may also suggest individual predisposition for MPCRC. Different etiopathogenic factors are suspected to be involved in SCRC and MCRC, and different familial vs individual factors may be implicated. MCRC seems to follow a familial pattern, whereas individual factors are more important in SCRC. Further studies must be carried out to know the molecular basis of risks for MPCRC in order to modify, if necessary, its clinical management, especially from a preventive point of view.

Early-onset colorectal cancer:A separate subset of colorectal cancer

Colorectal cancer(CRC)has a great impact on the world population.With increasing frequency,CRC is described according to the presenting phenotype,based on its molecular characteristics.Classification of CRC tumors according to their genetic and/or epigenetic alterations is not only important for establishing the molecular bases of the disease,but also for predicting patient outcomes and developing more individualized treatments.Early-onset CRC is a heterogeneous disease,with a strong familial component,although the disease is sporadic in an important proportion of cases.Different molecular alterations appear to contribute to the apparent heterogeneity of the early-onset population and subgroups can be distinguished with distinct histopathologic and familial characteristics.Moreover,compared with late-onset CRC,there are characteristicsthat suggest that early-onset CRC may have a different molecular basis.The purpose of this review was to analyze the current state of knowledge about earlyonset CRC with respect to clinicopathologic,familial and molecular features.Together,these features make it increasingly clear that this subset of CRC may be a separate disease,although it has much in common with late-onset CRC.

Novel MLH1 frameshift mutation in an extended hereditary nonpolyposis colorectal cancer family

AIM: To present novel frameshift mutation c.31delC [p.L11X] in the MLH1 gene identified in an extended Bulgarian hereditary non-polyposis colorectal cancer (HNPCC) family and to analyze the molecular and clinical findings within the pedigree concerning the proposal of adequate individual prophylactic strategy for all mutation carriers. METHODS: The pedigree of the family consists of 42 members in four generations. Search for mutations in the MLH1 and hMSH2 genes was performed in the pro-band. After PCR amplification of all exons including flanking intronic regions, amplicons were directly sequenced. RESULTS: The mutation was found in nine from the thirteen pedigree members who signed informed consent to participate in the study. In three adenocarcinomas, microsatellite instability and lack of the MLH1 protein expression were detected. The only one tubulovillous adenoma analyzed was microsatellite stable and the MLH1 protein showed an intact staining. CONCLUSION: The newly described mutation c.31delC is HNPCC causative. Besides the typical clinical features of the syndrome, we found a specific pathologic manifestation such as moderate to high differentiated adenocarcinomas of the colon. One of the mutation carriers developed a benign giant cell soft tissue tumor. The primary tumor localizations were frequently extracolonic and detailed yearly gastrointestinal and gynecological examinations have been proposed to the mutation carriers. We emphasize the importance of including the HNPCC genetic counseling and testing as well in the following surveillance of all patients at risk in the services covered by the health insurance in Bulgaria.