Pentaplex PCR Research Articles

Assessment of STR Typing Success Rate in Soft Tissues from Putrefied Bodies Based on a Quantitative Grading System for Putrefaction.

To date, there is no systematic investigation of the association of short tandem repeat (STR) typing success rate in soft tissues with different signs of putrefaction. Herein, putrefaction was rated using a newly developed 19-parameter system in soft tissues from a collective of 68 decaying bodies, and DNA yield was determined in 408 samples. DNA integrity was rated using a self-devised pentaplex PCR generating an "integrity score" (Si). STR typing success rate was then assessed for selected cases. DNA yield and Si differed significantly between tissues with kidney on average exhibiting the highest Si values. Statistical analysis revealed that nine parameters were significantly and positively correlated with Si . The observed values for each of these nine parameters were summed up to generate a putrefaction score (Sp) for each sample. Our results show that STR typing success rate can be predicted based on Sp before expensive multiplex STR profiling is performed.

Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers

Staphylococcus aureus strains harbouring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbour mecA, the gene encoding staphylococcal methicillin-resistance. There have been previous attempts at distinguishing MRSA from MRCoNS, most of which were based on the detection of one of the pathognomonic markers of S. aureus, such as coa, nuc or spa. That approach might suffice for discrete colonies and mono-microbial samples; it is inadequate for identification of clinical specimens containing mixtures of S. aureus and CoNS. In the present study, a real-time pentaplex PCR assay has been developed which simultaneously detects markers for bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl) and methicillin resistance (mecA). Staphylococcal and non-staphylococcal bacterial strains (n = 283) were used to validate the new assay. The applicability of this test to clinical samples was evaluated using spiked blood cultures (n = 43) containing S. aureus and CoNS in mono-microbial and poly-microbial models, which showed that the 5 markers were all detected as expected. Cycling completes within 1 h, delivering 100% specificity, NPV and PPV with a detection limit of 1.0 × 101 to 3.0 × 101 colony forming units (CFU)/ml, suggesting direct applicability in routine diagnostic microbiology. This is the most multiplexed real-time PCR-based PVL-MRSA assay and the first detection of a unique marker for CoNS without recourse to the conventional elimination approach. There was no evidence that this new assay produced invalid/indeterminate test results.

Absence of Microsatellite Instability In Soft Tissue Sarcomas

Objective: Here, we analyze a series of soft tissue sarcomas (STS), which are a heterogeneous group of mesenchymal neoplasms, for the presence and frequency of microsatellite instability (MSI). MSI has been proposed to be clinically relevant for colorectal cancer, yet on STS its role is not consensual, partly due to the limited number of cases analyzed and methodology issues. Methods: The detailed evaluation of MSI in tumor samples from 71 STS patients was performed by pentaplex PCR of the MSI markers NR-27, NR-21, NR-24, BAT-25, and BAT-26, followed by capillary electrophoresis. The expression of DNA mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, and PMS2) was also evaluated in suspected MSI-positive cases by immunohistochemistry. Results: The MSI analysis showed instability of one MSI marker in a total of 3 cases (4.2%). However, MMR protein expression was not affected, demonstrating that all cases were microsatellite stable. Conclusion: Our results suggest that MSI does not play a role in STS tumorigenesis.

Retraction Notice

Journal of Food ScienceVolume 79, Issue 1 p. iv-iv Retraction NoticeFree Access Retraction Notice This article retracts the following: A Multiplex PCR Assay for the Rapid and Sensitive Detection of Methicillin-Resistant Staphylococcus aureus and Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci Benjin Xu, Ling Liu, Li Liu, Xinping Li, Xiaofang Li, Xin Wang, Volume 77Issue 11Journal of Food Science pages: M638-M642 First Published online: October 26, 2012 First published: 24 January 2014 https://doi.org/10.1111/1750-3841.12351AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Retraction: “A Multiplex PCR Assay for the Rapid and Sensitive detection of Methicillin-Resistant Staphylococcus aureus and Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci” by Benjin Xu, Ling Liu, Li Liu, Xinping Li, Xiaofang Li, and Xin Wang The above article, published online on 26 October 2012 in Wiley Online Library (wileyonlinelibrary.com), and in Volume 77, No. 11, pp. M638-642, November 2012, has been retracted by agreement between the authors, the journal Editor in Chief, E. Allen Foegeding, and Wiley Periodicals, Inc. The retraction has been agreed due to data fabrication and unattributed overlap with “A Pentaplex PCR Assay for the Rapid Detection of Methicillin-Resistant Staphylococcus aureus and Panton-Valtentine Leucocidin” by Hassanain Al-Talib, Chan Yean, Yean, Alyaa Al-Khateeb, Habsan Hassan, Kirnpal-Kaur Banga Singh, Karim Al-Jashamy, and Manickam Ravichandran (BMC Microbiology 9:113, May 2009, doi: 10.1186/1471-2180-9-113). Author Xu has confirmed that he alone committed this misconduct, submitting the article without the knowledge or agreement of his co-authors, and he accepts full responsibility for its publication. The Journal of Food Science holds its authors to a high ethical standard and will not tolerate such misconduct. From mid-2010 through 2012, we randomly checked submitted papers for similarity to other sources via CrossCheck powered by iThenticate. In late 2012, just after Xu's article was accepted, we began to require that all accepted papers had been run through a plagiarism check, and since May 2013, all papers are checked through iThenticate upon submission to ScholarOne Manuscripts. Volume79, Issue1January 2014Pages iv-iv RelatedInformation

Optimization of a pentaplex panel for MSI analysis without control DNA in a Brazilian population: correlation with ancestry markers

Microsatellite instability (MSI) testing has been advocated for all newly diagnosed colorectal cancer patients. One of the most common tests is composed by a pentaplex panel of mononucleotides markers (NR-27, NR-21, NR-24, BAT-25, and BAT-26), which allows the analysis of MSI in tumors without the need of reference DNA. For that, it is fundamental to establish a quasi-monomorphic variation range (QMVR) for each marker. Herein, we aimed to establish the QMVR in a Brazilian healthy population, to evaluate the feasibility of MSI determination of tumors, without the matching normal DNA. Furthermore, we intend to assess their ancestry using specific ancestry-informative markers (AIMs) and correlate with QMVR. The QMVR was assessed in 214 individuals, through a pentaplex PCR followed by fragment analysis. The ancestry analysis was done by 46 AIMs in a single multiplex PCR followed by capillary electrophoresis. Following QMVR establishment, we observed 23 individuals with alleles outside the QMVR. Importantly, none of them exhibited more than one marker outside the range. Therefore, individuals with instability at ≥2 markers would be accurately classified as MSI. The European ancestry proportion was the most frequent (67.5%), followed by the African (19.6%). The comparison of the individuals with alleles within (n=191) and outside (n=23) the QMVR showed statistical difference in the proportions of European and African alleles, confirming the higher polymorphic nature of African ancestry. In conclusion, the present study reports an accurate methodology to assess MSI status without matched-normal DNA and independently of the ethnicity, even in the highly admixed population of Brazil.

Detection of sixteen genetically modified maize events in processed foods using four event-specific pentaplex PCR systems

To efficiently identify genetically modified (GM) maize events in foods and feeds, we report here the development of four individual pentaplex PCR analysis systems for event-specific identification of sixteen GM maize events approved in South Korea. In addition to the maize endogenous reference gene, zSSIIb, the four pentaplex PCR assays target group 1 containing TC6275, MON810, T25, and NK603; group 2 with TC1507, MON863, GA21, and DAS-59122-7; group 3 with MIR604, Bt11, Bt176, and MON89034; group 4 with event 3272, LY038, MIR162, and MON88017. These amplicons were designed to be smaller than 200 bp to make the testing system suitable for analyzing processed foods/feeds derived from these 16 GM maize events. After optimizing the reaction condition, the limits of detection of these four assays were approximately 0.25% for all of the 16 GM maize events. This multiplex PCR method for sixteen GM maize events was validated by three operators, and the data confirmed the reliability of the developed assays. Furthermore, 74 food samples containing maize ingredients from the USA, China, Japan, and South Korea were analyzed, and we observed 18 food products containing one or more GM maize events. These results suggest that the developed multiplex system is applicable for use in specific testing of sixteen GM maize events in foods and feeds in South Korean market.

A Pentaplex PCR Assay for the Detection and Differentiation ofShigellaSpecies

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.

Advances in Molecular Diagnostics

Advances in Molecular Diagnostics

Comparison of real-time PCR and MassTag PCR for the multiplex detection of highly pathogenic agents

Multiplex PCR assays are a cost- as well as labour-effective way to analyse one sample for several pathogens simultaneously. Besides the mutual competition of the individual PCR reactions included in a multiplex PCR assay, their specific read-out displays a limiting factor for the total number of PCR reactions that can be multiplexed.In this study, two PCR systems with different read-out approaches are compared, using a pentaplex PCR assay for the detection of highly pathogenic agents. A pentaplex assay was used since five represents the current limit of real-time PCR multiplexing capacity due to the low resolution of fluorescence emission peaks of the current equipment. In contrast, MassTag PCR as a quite new technique offers the possibility to detect up to 20–30 target sequences from one reaction.After extensive and separate optimisation of the PCR protocol for both platforms, a comparative probit analysis showed good sensitivities for MassTag and real-time PCR detection. Nevertheless, the detection limits of MassTag PCR have been undercut by the real-time PCR for each target.We therefore conclude that MassTag PCR is a useful diagnostic technique for the sensitive screening for pathogens by highly multiplexed PCR assays, but cannot reach the sensitivity of real-time PCR for lower multiplexed PCR assays.

Microsatellite Instability and DNA Mismatch Repair Protein Deficiency in Lynch Syndrome Colorectal Polyps

Colorectal cancers associated with Lynch syndrome are characterized by deficient DNA mismatch repair (MMR) function. Our aim was to evaluate the prevalence of microsatellite instability (MSI) and loss of MMR protein expression in Lynch syndrome-associated polyps. Sixty-two colorectal polyps--37 adenomatous polyps, 23 hyperplastic polyps, and 2 sessile serrated polyps (SSP)--from 34 subjects with germline MMR gene mutations were tested for MSI using a single pentaplex PCR for five mononucleotide repeat microsatellite markers, and also for expression of MLH1, MSH2, MSH6, and PMS2 proteins by immunohistochemistry. High-level MSI (MSI-H) was seen in 15 of 37 (41%) adenomatous polyps, one of 23 (4%) hyperplastic polyps, and one of two (50%) SSPs. Loss of MMR protein expression was seen in 18 of 36 (50%) adenomatous polyps, zero of 21 hyperplastic polyps, and zero of two SSPs. Adenomatous polyps 8 mm or larger in size were significantly more likely to show MSI-H [OR, 9.98; 95% confidence interval (CI), 1.52-65.65; P = 0.02] and deficient MMR protein expression (OR, 3.17; 95% CI, 1.20-8.37; P = 0.02) compared with those less than 8 mm in size. All (six of six) adenomatous polyps 10 mm or larger in size showed both MSI-H and loss of MMR protein expression by immunohistochemistry. Our finding that the prevalence of MMR deficiency increases with the size of adenomatous polyps suggests that loss of MMR function is a late event in Lynch syndrome-associated colorectal neoplasia. Although testing large adenomatous polyps may be of value in the diagnostic evaluation of patients with suspected Lynch syndrome, the absence of an MMR-deficient phenotype in an adenoma cannot be considered as a strong evidence against Lynch syndrome, as it is with colorectal carcinomas.

Combination of multiplex PCR and DHPLC-based strategy for CYP2D6 genotyping scheme in Thais

ObjectiveTo develop CYP2D6 genotyping scheme for accurate allele calling and reliable estimation of functional allele dosage in Thais. Design and methodsWe analyzed CYP2D6 copy numbers by pentaplex PCR coupled with semi-quantitative denaturing high performance liquid chromatography (DHPLC)-based technique. Ten common SNPs were genotyped from CYP2D6 gene product using single base extension (SBE) followed by DHPLC analysis. This detection scheme was compared with real-time PCR and conventional PCR-RFLP for cost-effectiveness. ResultsThe distribution of CYP2D6 gene copy numbers in our population ranged from zero (0.69%), one (7.99%), two (60.07%), three (28.13%) and four (3.13%). The most commonly detected SNPs were related to CYP2D6*10 haplotype. CYP2D6*36 in tandem with CYP2D6*10B is the major rearrangement type in Thais (18.75%). ConclusionsMultiplex PCR coupled with DHPLC-based strategy is convenient and reliable method for CYP2D6 genotyping offering sufficient allele coverage for Asians. Both cost and analytical time saving were shown and the method could potentially be modified to accommodate CYP2D6 genotyping in other ethnics.

Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese

A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10(3)CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species.

An in-house multiplex pcr method to detect of putative virulence factors in aeromonas species.

A pentaplex PCR was developed and optimised to detect the genes that encode the five most important putative virulence factors in Aeromonas isolates. It seems to be more efficient than previously reported techniques and promises to be a powerful tool for more accurate risk assessments and for monitoring pathogenic strains.

A Japanese-specific allele in the GALNT11 gene

In this study, five single nucleotide polymorphisms (SNPs) in the ABCC4, FBN1, CEP152, ZNF804B, and GALNT11 genes were investigated to assess allele frequencies in 14 different populations by a novel pentaplex PCR method. All SNPs were polymorphic in East Asians, whereas mutant alleles were absent or rare in non-East Asians. The frequencies of a mutant allele in FBN1 (rs140598) showed a north–south downward cline in East Asia, whereas those of a mutant allele in ZNF804B (rs1916830) were relatively uniform in East Asia. The highest frequencies of mutant alleles in ABCC4 (rs3765534), CEP152 (rs2289178), and GALNT11 (rs3778922) were observed in Okinawa. The mutant allele in GALNT11 was found only in Far-East Asian populations: the frequencies were about 0.153 in Okinawa, 0.076 in the main island of Japan, and 0.017–0.004 in Korea. These five East Asian- and Japanese-specific SNPs would be useful markers for forensic individualization, in particular, as ancestry-informative markers.

The number of lymph nodes is influenced by the MSI phenotype in resected colorectal cancers (CRC): A study in a large monocentric series.

3607 Background: The presence of lymph node (LN) metastasis has important prognostic implications in colorectal cancer (CRC). The number of LN assessed by pathologic examination is positively associated with longer survival. The presence of microsatellite instability (MSI) defines a group of CRCs with a favorable prognosis, which may be due to different immune reaction and LN status. Methods: The aim of the study was to compare the number of LN removed from surgical specimens between MSI and MSS (microsatellite stable) CRCs in a large monocentric series. 1,421 consecutive CRCs with curative surgery were included (364 right colon, 448 left colon-sigmoid, 609 rectum). MSI was assessed by MMR proteins immunohistochemistry in all patients, and a mononucleotidic pentaplex PCR in 796 patients. Results: There were 147 (10.3%) MSI CRCs (109 right colon; 21 left colon; 17 rectum/114 hMLH1 neg; 30 hMSH2 neg; 3 hMSH6 neg). The mean LN number was 32.99 ± 17.08 in MSI vs. 26.45 ± 15.31 in MSS CRC (p < 10-5). 97 (66%) MSI CRCs had no LN metastasis (N0) vs. 647 (50.8%) MSS. The mean LN number in N0 CRCs was 34.15 ± 19.3 in MSI vs. 26.43 ± 16.26 in MSS (p = 0.0003); in N1-N2 CRCs, it was 30.72 ± 11.46 in MSI (p = 0.18 vs N0 MSI) vs. 26.50 ± 14.29 in MSS (p = 0.0187 vs. N1-N2 MSI; p = 0.93 vs. N0 MSS). In MSI CRCs, the LN number was higher in hMLH1 neg (34 ± 17.99) vs. MSH2 neg (30 ± 13.58), but this difference was not significant (p = 0.187) and was only observed in N0 cancers (35.88 ± 20.18 in hMLH1 neg vs. 28.8 ± 15.46 in hMSH2 neg; p = 0.09). There was a relation between the LN number and the localization of the tumour, both in MSI and MSS CRCs. Conclusions: Our results show that MSI CRCs have a higher LN number found in their surgical specimen than MSS CRCs, especially in cases without LN metastasis, in right colon tumors, and in hMLH1 neg cases. This may participate to the favorable prognosis of MSI CRCs. No significant financial relationships to disclose.

Correction: An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

The affiliations of the second author, Richard Hamelin, are incorrect. First, affiliation number 2 is incorrect. It should read: INSERM, UMRS 938, Paris, France. Second, an affiliation for this author was omitted. Richard Hamelin is also affiliated with Universite Pierre et Marie Curie-Paris 6, Paris, France.

An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

PurposeMicrosatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI.Experimental DesignWe investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA.ResultsAmplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI.ConclusionsAn optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.

Chromosome X centromere region—Haplotype frequencies for different populations

Searching for suitable and closely linked STRs on the X-chromosome (ChrX) we evaluated several polymorphic markers located within the human ChrX centromere region. Stable haplotypes can be expected in this region because of low recombination rates. The five markers investigated here show a tetranucleotide or pentanucleotide structure and exhibit high or medium polymorphic information content. We validated a pentaplex PCR for these STRs with a special 3 primer system for the amplification of the combined STR-INDEL polymorphism DXS10163. Additionally we present allele and haplotype frequency data for populations from Germany, Ethiopia, Egypt and Somalia.

Analysis of microsatellite instability in medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children. The presence of microsatellite instability (MSI) in brain tumors, particularly medulloblastomas, has not been properly addressed. The aim of the present study was to evaluate the role of MSI in medulloblastoma carcinogenesis. MSI status was determined in 36 patients using a pentaplex PCR of quasimonomorphic markers (NR27, NR21, NR24, BAT25, and BAT26). Methylation status of mismatch repair (MMR) genes was achieved by methylation-specific multiplex ligation-dependent probe amplification (MLPA). In addition, MutS homolog 6 (MSH6) expression was determined by immunohistochemistry. Mutations of 10 MSI target genes (TCF4, XRCC2, MBD4, MRE11, ATR, MSH3, TGFBR2, RAD50, MSH6, and BAX) were studied by pentaplex PCR followed by analysis with GeneScan 3.7 software. Mutation analysis of hotspot regions of beta-catenin (CTNNB1) and BRAF (v-raf murine sarcoma viral oncogene homolog B1) oncogenes was performed by PCR single-strand conformation polymorphism analysis followed by direct sequencing. Among the 36 tumors, we found four (11%) cases with instability, one with high MSI and three with low MSI. Methylation analysis of MMR genes in cases presenting shifts on the MSI markers revealed mild hypermethylation of MSH6 in 75% of cases, yet MSH6 was expressed in all the tumors. The MSI target genes MBD4 (methyl-CpG binding domain protein 4) and MRE11 (meiotic recombination 11 homolog A) were mutated in two different tumors. No CTNNB1 or BRAF mutations were found. This study is the most comprehensive analysis of MSI in medulloblastomas to date. We observed the presence of MSI together with mutations of MSI target genes in a small fraction of cases, suggesting a new genetic pathway for a role in medulloblastoma development.

Microsatellite instability in hepatocellular carcinoma in non‐cirrhotic liver in patients older than 60 years

Hepatocellular carcinoma (HCC) in otherwise normal liver is rare, its pathogenesis remains obscure and the literature on the subject is scarce. We investigated microsatellite instability (MSI) in eight elderly patients (median age 70.7, range 63-76 years) without a clinical history of liver disease and who underwent liver resection for HCC in otherwise normal background liver between 2001 and 2005 at King's College Hospital, London. Immunohistochemistry for mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), mutS homolog 6 (MSH6) and post-meiotic segregation increased 2 (PMS2) was carried out on formalin-fixed and paraffin-embedded sections of tumor and background liver. MSI analysis was performed using a panel of monomorphic microsatellites markers: BAT-25, BAT-26, NR21, NR24 and NR27 and pentaplex PCR. All HCC were solitary large tumors. Two also had satellite nodules. The background liver was usually unremarkable. There was nuclear expression of MLH1, MSH2, MSH6 and PMS2 in all tumors excluding a DNA mismatch repair defect. The same pattern of staining was noted in the hepatocytes of the background liver of all cases. No differences between microsatellite lengths in the background liver and in the tumor, as assessed in PCR products, were found for any of the five microsatellite markers in any patients. These findings provided no evidence for MSI. Our study showed that MSI is not implicated in the pathogenesis of a subset of HCC affecting elderly patients without chronic liver disease. Further studies are needed to clarify the pathogenesis of HCC in this particular setting.