Identification of candidate genes for prostate cancer-risk SNPs utilizing a normal prostate tissue eQTL data set.

Multiple studies have identified loci associated with the risk of developing prostate cancer but the associated genes are not well studied. Here we create a normal prostate tissue-specific eQTL data set and apply this data set to previously identified prostate cancer (PrCa)-risk SNPs in an effort to identify candidate target genes. The eQTL data set is constructed by the genotyping and RNA sequencing of 471 samples. We focus on 146 PrCa-risk SNPs, including all SNPs in linkage disequilibrium with each risk SNP, resulting in 100 unique risk intervals. We analyse cis-acting associations where the transcript is located within 2 Mb (±1 Mb) of the risk SNP interval. Of all SNP-gene combinations tested, 41.7% of SNPs demonstrate a significant eQTL signal after adjustment for sample histology and 14 expression principal component covariates. Of the 100 PrCa-risk intervals, 51 have a significant eQTL signal and these are associated with 88 genes. This study provides a rich resource to study biological mechanisms underlying genetic risk to PrCa.

Oestrous synchronization, ovarian superovulation and intraspecific transfers from a closed breeding colony of inbred SLA miniature pigs.

The inbred SLA miniature pig is a unique animal model developed for organ transplantation studies and pre-clinical experimental purposes. Reported oestrous synchronization and superovulation treatments were examined in two SLA haplotypes (AA and DD) to allow collection of embryos for both practical embryo transfer and experimental technologies from a closed breeding colony. Pre-puberal miniature pigs were poor responders to oestrous synchronization treatments, while post-puberal sows were equivalent to commercial sows. Following superovulation, the ovulation number (corpora .hemorrhagica) was higher (p < 0.05) in the cycling sows when compared with non-cycling sows. Ovulations were equivalent to commercial pre-puberal gilts and non-cycling sows (p > 0.05). No difference in ovulation number between haplotypes was observed, which differs from the previous report (DD>AA). Collection of zygotes for pronuclear injection was the highest in the non-cycling post-puberal miniature pig group (p < 0.05), although significantly lower when compared with the commercial pig treatment groups (p < 0.05). The incidence of cystic endometrial hyperplasia in our colony was equivalent to rates observed in commercial pigs. Pronuclear visualization following centrifugation was the highest in the non-cycling miniature sow group and approximates to about 25% of ovulations and about half the rate observed in the commercial pigs (50%). Miniature pig embryos transferred between SLA haplotypes and transfer of DD embryos to commercial pigs resulted in live births at a higher efficiency than previously reported. This study demonstrates the feasibility of undertaking assisted reproductive technologies in a closed breeding colony of inbred SLA miniature pigs without compromise to the breeding programmes.

Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos.

The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos. The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest-1 showed differential expression in Day 21 parthenogenetic embryos compared to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation.

Gene expression analysis of single preimplantation bovine embryos and the consequence for developmental potential.

Preimplantation embryo development typically involves sequential morphological events connecting embryonic cleavage, morula compaction and blastocyst formation, and occurs in parallel with transcriptional regulation, specifically, the maternal to embryonic transition. The underlying homeostatic and metabolic mechanisms governing embryo development are influenced by both genetic and epigenetic factors that respond to environmental stimuli and may impact development during later gestational and fetal growth. There is a renewed interest in the identification and characterization of developmentally important genes during embryonic and fetal development. Perturbations in gene expression, resulting from environmental conditions, can have serious consequences on further embryonic development, homeostasis and disease pathogenesis. The bovine embryo is, however, capable of tolerating and adapting to a wide range of conditions, although little is known of the molecular fingerprint required for oocyte maturation, fertilization and development to term. The genomic revolution united with promising new technologies offer greater opportunity to elucidate the mechanisms behind this well-orchestrated biological process. This paper reviews the current literature on gene expression in the bovine embryo with reference to environmental interference and the development of new technologies to observe this biological process. Defining the difference in molecular signalling between in vivo and in vitro systems will undoubtedly improve the safety and efficiency of assisted reproductive technologies. The future challenge is to devise culture conditions that mimic the changing environment required by developing embryos to allow the correct temporal and spatial expression of a cohort of developmental genes in a manner similar to that seen in

BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing.

BACKGROUND: According to the international criteria for hereditary non-polyposis colorectal cancer (HNPCC) diagnostics, cancer patients with a family history or early onset of colorectal tumours showing high microsatellite instability (MSI-H) should receive genetic counselling and be offered testing for germline mutations in DNA repair genes, mainly MLH1 and MSH2. Recently, an oncogenic V600E hotspot mutation within BRAF, a kinase encoding gene from the RAS/RAF/MAPK pathway, has been found to be associated with sporadic MSI-H colon cancer, but its association with HNPCC remains to be further clarified. METHODS: BRAF-V600E mutations were analysed by automatic sequencing in colorectal cancers from 206 sporadic cases with MSI-H and 111 HNPCC cases with known germline mutations in MLH1 and MSH2. In addition, 45 HNPCC cases showing abnormal immunostaining for MSH2 were also analysed. RESULTS: The BRAF-V600E hotspot mutation was found in 40% (82/206) of the sporadic MSI-H tumours analysed but in none of the 111 tested HNPCC tumours or in the 45 cases showing abnormal MSH2 immunostaining. CONCLUSIONS: Detection of the V600E mutation in a colorectal MSI-H tumour argues against the presence of a germline mutation in either the MLH1 or MSH2 gene. Therefore, screening of these mismatch repair (MMR) genes can be avoided in cases positive for V600E if no other significant evidence, such as fulfilment of the strict Amsterdam criteria, suggests MMR associated HNPCC. In this context, mutation analysis of the BRAF hotspot is a reliable, fast, and low cost strategy which simplifies genetic testing for HNPCC.

Investigation of sequential mitomycin C and photodynamic therapy in a mitomycin-resistant bladder cancer cell-line model.

OBJECTIVES: To investigate the hypothesis that sequential mitomycin C and 5-aminolaevulinic acid (ALA)-mediated photodynamic therapy (PDT) interact additively in both the J82 bladder cancer cell line and its mitomycin-C-resistant derivative, J82/MMC, and to assess the theoretical basis of this interaction by measuring the relative mitochondrial density of the respective cell lines, on the basis that the mitochondria are the intracellular site where ALA is metabolized to the active photosensitizer, protoporphyrin IX. MATERIALS AND METHODS: Cell survival was assayed in J82 cell line and the J82/MMC derivative after treating them with sequential ALA-mediated PDT and mitomycin C, and with the sequence of treatments reversed. Cell survival was estimated using the tetrazolium assay. The relative mitochondrial density of the two cell lines was estimated using flow cytometry to measure 123rhodamine fluorescence. RESULTS: The effect of sequential mitomycin C followed by ALA-mediated PDT enhanced the effect of PDT in both cell lines. In J82/MMC this effect was marginally supra-additive. When ALA-mediated PDT was administered before mitomycin C, the combined effect was 'sub-additive'. 123Rhodamine fluorescence was > 10 times greater in J82/MMC than J82, suggesting a significantly higher mitochondrial density in the former than the latter. CONCLUSION: Mitomycin C appears to enhance ALA-mediated PDT when administered first. This appears to be particularly so in J82/MMC. This phenomenon may have clinical significance in recurrent superficial bladder cancer.

Confirmation of linkage of prostate cancer aggressiveness with chromosome 19q.

Regions on chromosomes 7 and 19 were recently reported to contain susceptibility loci that regulate tumor aggressiveness of prostate cancer. To confirm these findings, we analyzed genome scan data from 161 pedigrees affected with prostate cancer. Using the Gleason score as a quantitative measure of tumor aggressiveness, we regressed the squared trait difference, as well as the mean-corrected cross product, on the estimated proportion of alleles shared identical-by-descent at each marker position. Our results confirm the previous linkage results for chromosome 19q (D19S902, P<.00001). In addition, we report suggestive evidence for linkage on chromosome 4 (D4S403, P=.00012). The results of previous findings, together with our results, provide strong evidence that chromosome 19 harbors a gene for tumor aggressiveness.

Birth of a cloned calf derived from a vitrified hand-made cloned embryo.

The hand-made cloning (HMC) technique describes a simplified nuclear transfer process without the need for micromanipulators. The technique involves manual bisection of zona-free oocytes, selection of cytoplasts by Hoechst staining and fusion of a single somatic cell and two cytoplasts. In this proof-of-principle experiment, the objective was to examine the developmental competence of HMC embryos following embryo transfer. Modifications to the original method include not selecting of matured oocytes and simultaneous fusion of cytoplasts and karyoplast. Blastocyst rates for embryos cultured in the glass oviduct system as singles (10.5%; 24/228) or in pairs (16.1%; 36/224) did not differ significantly. Fresh and vitrified-thawed blastocysts were transferred to 16 synchronised recipients (three to four embryos per recipient). Ultrasound examination on Days 35-45 showed an initial pregnancy rate of 43.8% (7/16) and a pregnancy rate >8 months of 12.5% (2/16). A male cloned calf (42 kg) derived from a vitrified HMC blastocyst was delivered by Caesarean section on Day 271. The birth and ongoing survival (15 months, 243 kg) of a healthy and apparently normal calf, combining both HMC and vitrification technologies, provides a 'proof of principle' of the technology and a promising alternative to traditional nuclear-transfer techniques.

Comparison of gene transcription in cloned bovine embryos produced by different nuclear transfer techniques.

The efficiency of animal production using cloning technology is still relatively low and research to determine a more efficient nuclear transfer procedure is ongoing. One approach which may be informative in assessing the viability of nuclear transfer embryos is the analysis of embryonic gene expression. Using RT-PCR techniques we have previously detected the aberrant expression of FGF4, FGFr2 and IL6 in a significant proportion of bovine granulosa cell-derived nuclear transfer embryos, which correlated with a limited developmental potential in vivo. In order to analyse the effect of different donor cell nuclei on embryonic gene expression we have now analysed the expression of these genes in nuclear transfer embryos reconstructed with fetal epithelial cell nuclei. In addition, we have compared the expression of these genes in bovine nuclear transfer embryos produced by cell fusion or direct injection with variations in the timing of oocyte activation. In all nuclear transfer embryos analysed, FGFr2 and IL6 transcripts were detected at a similar rate to that in IVF embryos. However, the absence of FGF4 transcripts was again evident in a large proportion of nuclear transfer embryos and most significantly in those embryos whose development was activated almost immediately following the transfer of the donor nucleus. The results demonstrate the effects that different donor cell lines and different nuclear transfer procedures may have on the expression of developmentally important genes in nuclear transfer embryos.

Analysis of the prostate cancer-susceptibility locus HPC20 in 172 families affected by prostate cancer.

A recent study of hereditary prostate cancer has provided evidence for a prostate cancer-susceptibility locus, HPC20, which maps to 20q13. To assess further the potential contribution of this locus to prostate cancer susceptibility, we studied 172 unrelated families affected by prostate cancer, using 17 polymorphic markers across a 98.5-cM segment of chromosome 20 that contains the candidate region. Parametric analysis, allowing for heterogeneity, resulted in an overall HLOD score of 0.09 (P=.39) at D20S171, under the assumption of linkage in 6% of families. Mode-of-inheritance-free analysis of the entire data set resulted in a maximal Zlr score of 0.76 (LOD 0.13; P=.22) at the same location. The strongest evidence for linkage was seen in the subset of 16 black families (LOD 0.86; Zlr=1.99; P=.023) between markers D20S893 and D20S120, near the putative location of HPC20. Although some positive results were observed, our linkage study does not provide statistically significant support for the existence of a prostate cancer-susceptibility locus HPC20 at 20q13.

Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts.

Histological staining and counting of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells differentially with chromatin-specific dyes is a more accurate indicator of cultured blastocyst quality and normality than total cell number assessment. The aim of this study was to test the effectiveness of a simplified method of chemically-defined differential blastocyst staining. The TE of cultured mouse and bovine blastocysts of different developmental stages was stained when blastocysts were treated with a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide. Blastocysts were then incubated in a second solution containing 100% ethanol (for fixation) and the secondary fluorochrome bisbenzimide. Fixed and stained whole blastocysts were mounted and assessed for cell number using ultraviolet fluorescent microscopy. Using this method, in-vitro cultured mouse blastocysts (day 4.5) were shown to have an ICM:TE ratio of 1:2.63 with an average total cell count of 75.3 +/- 3. While day 7 and 8 in-vitro produced bovine blastocysts were shown to have an ICM:TE ratio of 1:3.42 and 1:3.36 with an average total cell count of 151.3 +/- 5.48 and 217.8 +/- 8.75 respectively. Blastocyst staining patterns indicate that this modified technique represents a simple and reliable alternative to current bichromatic blastocyst staining techniques for the differential assessment of cell numbers and may be useful for the assessment of blastocysts derived from in-vitro maturation, novel culture systems and advanced reproductive technologies such as cloning.

Evidence for a prostate cancer-susceptibility locus on chromosome 20.

Recent studies suggest that hereditary prostate cancer is a complex disease involving multiple susceptibility genes and variable phenotypic expression. While conducting a genomewide search on 162 North American families with > or =3 members affected with prostate cancer (PRCA), we found evidence for linkage to chromosome 20q13 with two-point parametric LOD scores >1 at multiple sites, with the highest two-point LOD score of 2.69 for marker D20S196. The maximum multipoint NPL score for the entire data set was 3.02 (P=.002) at D20S887. On the basis of findings from previous reports, families were stratified by the presence (n=116) or absence (n=46) of male-to-male transmission, average age of diagnosis (<66 years, n=73; > or =66 years, n=89), and number of affected individuals (<5, n=101; > or =5, n=61) for further analysis. The strongest evidence of linkage was evident with the pedigrees having <5 family members affected with prostate cancer (multipoint NPL 3.22, P=.00079), a later average age of diagnosis (multipoint NPL 3.40, P=.0006), and no male-to-male transmission (multipoint NPL 3.94, P=.00007). The group of patients having all three of these characteristics (n=19) had a multipoint NPL score of 3.69 (P=.0001). These results demonstrate evidence for a PRCA susceptibility locus in a subset of families that is distinct from the groups more likely to be linked to previously identified loci.

HMSH6 alterations in patients with microsatellite instability-low colorectal cancer.

Two microsatellite instability (MSI) phenotypes have been described in colorectal cancer (CRC): MSI-H (instability at >30% of the loci examined) and MSI-L (MSI at 1-30% of the loci examined). The MSI-H phenotype, observed in both hereditary nonpolyposis colon cancer-associated CRC and approximately 15% of sporadic CRC, generally results from mutational or epigenetic inactivation of the DNA mismatch repair (MMR) genes hMSH2 or hMLH1. The genetic basis for the MSI-L phenotype, however, is unknown. Several other proteins, including hMSH3 and hMSH6, also participate in DNA MMR. Inactivating mutations of MSH6 in yeast and human tumor cell lines are associated with an impaired ability to repair single-base mispairs and small insertion-deletion loops but not large insertion-deletion loops. This suggests that hMSH6 mutations are more likely to be associated with a MSI-L phenotype than a MSI-H phenotype in CRC. To explore this possibility, we screened tumors from 41 patients with MSI-L CRC for hMSH6 mutations with conformation-sensitive gel electrophoresis (CSGE) and for hMSH6 protein expression by immunohistochemistry. Alterations found with CSGE were confirmed by DNA sequencing of normal and tumor tissue. One somatic (Asp389Asn) and 15 germ-line changes were found. Of the 15 germ-line changes, 9 were found in an intron (none involving splice junctions), and 6 were found in an exon (Gly39Glu, Leu395Val, and 4 silent alterations). Immunohistochemical staining for hMSH6 performed on 34 of the 41 tumors revealed strong nuclear hMSH6 expression in all of the cases. Overall, our results suggest that hMSH6 mutations do not play a major role in the development of sporadic CRC with a MSI-L phenotype.

Linkage analyses at the chromosome 1 loci 1q24-25 (HPC1), 1q42.2-43 (PCAP), and 1p36 (CAPB) in families with hereditary prostate cancer.

Recent studies suggest that hereditary prostate cancer (PRCA) is a complex disease, involving multiple susceptibility genes and variable phenotypic expression. Through linkage analysis, potential prostate cancer susceptibility loci have been mapped to 3 regions on chromosome 1. To investigate the reported linkage to these regions, we conducted linkage studies on 144 PRCA families by using microsatellite markers in regions 1q24-25 (HPC1) and 1q42.2-43 (PCAP). We also examined the 1p36 (CAPB) region in 13 PRCA families with at least one case of brain cancer. No significant evidence of linkage to the HPC1 or PCAP region was found when the entire data set was analyzed. However, weak evidence for linkage to HPC1 was observed in the subset of families with male-to-male transmission (n=102; maximum multipoint nonparametric linkage [NPL] 1.99, P=.03). Weak evidence for linkage with heterogeneity within this subset was also observed (HLOD 1.21, P=.02), with approximately 20% of families linked. Although not statistically significant, suggestive evidence for linkage to PCAP was observed for the families (n=21) that met the three criteria of male-to-male transmission, average age of diagnosis <66 years, and >/=5 affected individuals (maximum multipoint NPL 1.45, P=.08). There was no evidence for linkage to CAPB in the brain cancer-prostate cancer subset. These results strengthen the argument that prostate cancer is a heterogeneous disease and that multiple genetic and environmental factors may be important for its etiology.

Microsatellite instability and 8p allelic imbalance in stage B2 and C colorectal cancers.

BACKGROUND: Microsatellite instability (MSI) and allelic imbalance involving chromosome arms 5q, 8p, 17p, and 18q are genetic alterations commonly found in colorectal cancer. We investigated whether the presence or absence of these genetic alterations would allow stratification of patients with Astler-Coller stage B2 or C colorectal cancer into favorable and unfavorable prognostic groups. METHODS: Tumors from 508 patients were evaluated for MSI and allelic imbalance by use of 11 microsatellite markers located on chromosome arms 5q, 8p, 15q, 17p, and 18q. Genetic alterations involving each of these markers were examined for associations with survival and disease recurrence. All P values are two-sided. RESULTS: In univariate analyses, high MSI (MSI-H), i.e., MSI at 30% or more of the loci examined, was associated with improved survival (P =.02) and time to recurrence (P =.01). The group of patients whose tumors exhibited allelic imbalance at chromosome 8p had decreased survival (P =.02) and time to recurrence (P =.004). No statistically significant associations with survival or time to recurrence were observed for markers on chromosome arms 5q, 15q, 17p, or 18q. In multivariate analyses, MSI-H was an independent predictor of improved survival (hazard ratio [HR] = 0.51; 95% confidence interval [CI] = 0.31-0.82; P =.006) and time to recurrence (HR = 0.42; 95% CI = 0.24-0.74; P =.003), and 8p allelic imbalance was an independent predictor of decreased survival (HR = 1.89; 95% CI = 1.25-2.83; P =. 002) and time to recurrence (HR = 2.07; 95% CI = 1.32-3.25; P =.002). CONCLUSIONS: Patients whose tumors exhibited MSI-H had a favorable prognosis, whereas those with 8p allelic imbalance had a poor prognosis; both alterations served as independent prognostic factors. To our knowledge, this is the first report of an association between 8p allelic imbalance and survival in patients with colorectal cancer.

Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1.

Recent studies have demonstrated the presence of microsatellite instability (MSI) in tumors from patients with hereditary nonpolyposis colorectal cancer and in a large number of sporadic tumors. To further characterize the type of alterations at these loci and their frequency of involvement in colon cancer, we studied DNA extracted from paraffin-embedded tissue from 508 patients using 11 microsatellites localized to chromosomes 5, 8, 15, 17, and 18. Overall, MSI at each locus varied in character and frequency and was observed with at least one marker in 191 cases (37.6%). Based on the number of markers displaying instability per tumor, three groups of patients were defined: those with <30% of the markers showing instability (MSI-L,, n = 109, 21.5%); those with > or = 30% (MSI-H, n = 82, 16.1%); and those showing no instability (MSS, n = 317, 62.4%). These groups were tested for correlations with a number of clinical and pathological parameters, including age, sex, stage, ploidy status, and site of tumor. Comparing across the three groups and verified by pair-wise comparisons, the MSI-H group was associated with tumor site (proximal colon, P = 0.001), sex (females, P = 0.005), stage (Dukes' B, P = 0.01), and ploidy status (diploid, P = 0.03). No significant differences were noted between the MSI-L and MSS group for any of the parameters tested. An additional 188 consecutive surgical colorectal cancer cases were examined for the presence of MSI and for the immunohistochemical expression of hMLH1 and hMSH2 proteins. Of this group, 129 (68.6%) were classified as MSS, 17 (9.0%) as MSI-L, and 42 (22.3%) as MSI-H. None of the MSS and none of the MSI-L tumors had altered expression of either hMLH1 or hMSH2. However, the majority of MSI-H (40 of 42, 95%) cases demonstrated absence of staining for these proteins. The most frequently altered protein was hMLH1, occurring in 95% of the tumors with altered expression. Cumulatively, these data suggest that the tumor phenotype MSI-H is distinct from tumor phenotypes MSI-L and MSS, with no apparent differences between MSI-L and MSS. Furthermore, altered hMLH1 protein expression appears to be responsible for the mutator phenotype in the vast majority of MSI-H tumors.

Current and future prospects for xenotransplantation.

The transplantation of organs and tissues between animal species, or xenotransplantation, is the focus of a growing field of research, owing primarily to the increasing shortage of allogeneic donor organs. The pig stands out as the most suitable donor animal for humans; however, xenografts (e.g. pig organs) used for human transplantation are normally destroyed by the host within minutes by hyperacute xenograft rejection. An improved understanding of the immune recognition and rejection of xenografts has resulted in new therapies that can partially overcome hyperacute rejection (HAR), delayed xenograft rejection (DXR) or acute vascular xenograft rejection. Strategies to diminish immunogenicity following xenotransplantation can be divided into two approaches: those directed at the recipient (e.g. antibodies or complement depletion or inhibition and tolerance induction) and those directed at the donor (e.g. transgenic modifications to express human complement-regulatory proteins or removal or displacement of alphaGal epitopes). DXR is likely to be controlled by transgenic inhibition of endothelial cell activation (e.g. inhibition of NF-kappaB). Transgenic pigs required for xenotransplantation will soon be generated at a greater efficiency and precision using nuclear transfer and cloning when compared to pronuclear injection. Of greater significance is that nuclear transfer offers the ability to target gene insertion selectively to specific gene loci and to delete specific genes in the pig. Experimental pig-to-primate organ xenotransplantation is currently under way, and results show increased transplant function from minutes to days and weeks. The final therapeutic regimen that allows survival of a discordant xenograft is likely to involve a combination of 'modified' functional genes in the donor organ, the development of immunological tolerance to pig antigens and administration of novel therapeutic agents, including immunosuppressants, that can control natural killer (NK) cell and monocyte mediated responses.

Allelic imbalance and microsatellite instability in resected Duke's D colorectal cancer.

Hepatic resection is the treatment of choice for selected patients with liver metastases from colorectal cancer (CRC). Although the 5-year survival rate among patients after liver resection is 25-45%, 55-75% of patients die from progressive disease. The purpose of this study was to characterize molecular genetic alterations, including microsatellite instability and allelic imbalance, in patients with potentially curative resected liver metastases from CRC and to correlate these molecular features with clinical and pathologic characteristics. We examined DNA from formalin-fixed, paraffin-embedded archival tumor specimens from 141 surgically resected hepatic metastases from CRC. We used microsatellite markers localized to chromosome arms 5q, 8p, 10q, 15q, 17p, 18p, and 18q in a polymerase chain reaction-based assay. Allelic imbalance at each locus and the presence of tumor microsatellite instability were correlated with clinicopathologic features of the tumor and clinical course of the patient. Microsatellite instability at multiple loci was seen in only 2.5% of resected liver metastases, a frequency significantly lower than that previously detected for primary CRC. Additionally, these findings had no significant correlation with disease-free survival or overall survival. Allelic imbalance at one or more loci was seen in 87% of informative tumors. Allelic imbalance on chromosome 17p was seen in 84% of informative tumors, and its presence was associated with a significantly poor disease-free survival (p = 0.015) and overall survival (p = 0.05). These data suggest that allelic imbalance on chromosome 17p is an independent prognostic parameter in patients with potentially curative resected liver metastases from CRC. Such alterations could provide a useful stratification criterion for adjuvant therapy for patients who have undergone curative resection of liver metastases from CRC.

Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes.

To date, at least four genes involved in DNA mismatch repair (MMR) have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colon cancer: hMSH2, hMLH1, hPMS1, and hPMS2. Additionally, loss of MMR function has been demonstrated to lead to the phenomenon of microsatellite instability (MIN) in tumors from these patients. In this study, we have examined the protein expression pattern of hMSH2 and hMLH1 by immunohistochemistry in paraffin-embedded tumors from 7 patients with MIN+ sporadic cancer, 13 patients with familial colorectal cancer, and 12 patients meeting the strict Amsterdam criteria for hereditary nonpolyposis colon cancer. The relationship between the expression of these two gene products, the presence of germline or somatic mutations, and the presence of tumor MIN was examined. Nineteen of the 28 tumors studied demonstrated MIN, whereas mutations in hMLH1 and hMSH2 were detected in 6 and 2 patients, respectively. Of the eight MIN+/mutation+ cases, the absence of protein expression was observed for the corresponding gene product in all but one case (missense mutation in hMLH1). However, seven MIN+/mutation- cases also showed no expression of either hMLH1 (n = 5), hMSH2 (n = 1), or both (n = 1), whereas four MIN+/mutation- cases demonstrated normal expression for both. None of the MIN-/mutation- cases (n = 9) demonstrated an altered expression pattern for either protein. These data suggest that examination of protein expression by immunohistochemistry may be a rapid method for prescreening tumors for mutations in the MMR genes.