Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives.

Formalin fixation preserves tissue morphology at the expense of macromolecule integrity. Freshly frozen samples are the golden standard for DNA and RNA analyses but require laborious deep-freezing and frozen sectioning for morphological studies. Alternative tissue stabilisation methods are therefore needed. We analysed the preservation of nucleic acids, immunohistochemical staining properties and tissue morphology in paraffin-embedded clinical tissue samples fixed with Z7, RCL2, PAXgene, Allprotect and RNAlater. Formalin-fixed and deep-frozen samples were used as controls. Immunohistochemical analyses showed good preservation of antigenicity in all except Allprotect and RNAlater-fixed samples. RNA quality, based on RNA integrity number value by Bioanalyzer, was comparable with freshly frozen samples only in PAXgene-fixed samples. According to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, RNA from PAXgene samples yielded results similar to freshly frozen samples. No difference between fixatives was seen in DNA analyses (PCR and real-time PCR). In conclusion, PAXgene seems to be superior to other molecular fixatives and formaldehyde.

Hereditary prostate cancer in Finland: fine-mapping validates 3p26 as a major predisposition locus.

In a recent genome-wide linkage (GWL) analysis of Finnish families at high risk for prostate cancer, we found two novel putative susceptibility loci at 3p25-p26 and 11q14. Here, we report the fine-mapping of these two critical regions at high resolution with 39 microsatellite markers in 16 families, including multiplex families that were not used in the GWL scan. The maximum multipoint HLOD was 3.39 at 3p26 and 1.42 at 11q14. The highest LOD scores were seen around markers D3S1270 and D3S4559 (alpha=0.89), covering approximately two megabases. The two known genes in this region CHL1 (cell adhesion molecule with homology to L1CAM) and CNTN6 (contactin 6) were screened for exonic mutations in the families showing the strongest linkage, but no disease-segregating sequence variants were observed. The recombination map pointed to a region proximal to the area of best linkage, suggesting that more genes may need to be investigated as candidates. These results provide strong evidence for the existence of a prostate cancer susceptibility gene at 3p26 in Finnish prostate cancer families. This locus has not been strongly linked with hereditary prostate cancer in other populations. However, the mildly positive 3p LOD scores in a recent GWL analysis of patients from the United States suggest that the locus may also be important in other populations.

Combined genome-wide scan for prostate cancer susceptibility genes.

BACKGROUND: Prostate cancer represents a substantial public health burden worldwide. It is the second leading cause of cancer death among men in the United States. A family history of the disease is among the most well-established risk factors for prostate cancer. Efforts to localize prostate cancer susceptibility alleles by using genetic linkage analysis methods have been hindered by genetic heterogeneity, incomplete penetrance, disease phenocopies, and the lack of DNA samples from parents of individuals with late-onset prostate cancer. METHODS: We performed a combined genome-wide linkage analysis among 426 families from four existing hereditary prostate cancer (HPC) study populations to systematically search for prostate cancer susceptibility genes. To decrease the degree of locus heterogeneity, we analyzed subsets of families with similar clinical and demographic characteristics. Nonparametric multipoint linkage was the primary method of analysis. Results are presented as allele-sharing logarithm of the odds (LOD) scores, and all reported P values are two-sided. RESULTS: The strongest evidence for prostate cancer linkage was found at chromosome region 17q22 (nonparametric multipoint Kong and Cox allele-sharing LOD score = 3.16 at marker D17S787; P =.00007). Stratified analyses revealed several additional chromosomal regions that are likely to segregate prostate cancer susceptibility genes among specific subsets of HPC families, including 15q11 among families with late-onset disease (allele-sharing LOD = 5.57 at marker D15S128; P<.00001) and 4q35 among families with four or more affected family members (allele-sharing LOD = 3.10 at marker D4S1615; P =.00008). CONCLUSION: Fine mapping studies to facilitate identification of prostate cancer susceptibility genes in these linked regions are warranted.

Germ-line alterations in MSR1 gene and prostate cancer risk.

PURPOSE: The MSR1 gene maps to 8p22-23, a novel susceptibility locus for hereditary prostate cancer (HPC). Mutations in MSR1 have been reported to associate with prostate cancer (PRCA) risk. Here we report a follow-up study from Finland to evaluate the association between PRCA and MSR1 gene. EXPERIMENTAL DESIGN: The youngest affected patient from each of 120 HPC families was initially used for the screening of MSR1 mutations by single-strand conformational polymorphism analysis. Selected variants of MSR1 gene were then screened in 537 unselected PRCA cases and in 480 controls. RESULTS: Among 120 HPC families, five MSR1 sequence variants were identified. The carrier frequencies of the R293X, P275A, and -14743A>G variants were compared between the probands with HPC, unselected PRCA cases, and healthy male blood donors. No significant differences were observed. The odds ratios for R293X, P275A, and -14743A>G mutations were also calculated to estimate the PRCA risk. No significantly elevated or lowered risks for PRCA among these three variants were detected. However, the mean age at diagnosis of the R293X mutation carriers among HPC probands was significantly lower compared with noncarriers (55.4 versus 65.4 years; t test, P = 0.04). The same trend was observed among unselected PRCA cases (65.7 versus 68.7 years; t test, P = 0.37). CONCLUSIONS: Our results do not support a major role for the MSR1 gene in the causation of hereditary or unselected PRCAs but suggest a possible modifying role in cancer predisposition.

Androgen receptor CAG polymorphism and prostate cancer risk.

Recent studies have suggested that polymorphisms of the androgen receptor gene ( AR) may influence the risk of prostate cancer (PC) development and progression. Here, we analyzed the length of the CAG repeat of the AR gene in 1363 individuals, including patients with PC, benign prostate hyperplasia (BPH), and population controls. There was a tendency for short CAG repeats to be associated with PC. The Odds Ratio (OR) for PC was 1.47 ( P=0.05) when individuals with short CAG repeats (18). CAG repeat length was not significantly associated with family history, disease stage, grade, age at diagnosis, prostate-specific antigen (PSA) level at diagnosis, or prognosis of the patients. Unexpectedly, short CAG repeats were significantly less common in patients with BPH compared with controls (OR=0.47, P=0.03). Our results suggest that the CAG polymorphism of the AR gene is unlikely to have a major role in the development or progression of PC in the Finnish population. The association of CAG repeats with the risk of BPH warrants further study.

Germline alterations of the RNASEL gene, a candidate HPC1 gene at 1q25, in patients and families with prostate cancer.

The RNASEL gene (2',5'-oligoisoadenylate-synthetase dependent) encodes a ribonuclease that mediates the antiviral and apoptotic activities of interferons. The RNASEL gene maps to the hereditary-prostate-cancer (HPC)-predisposition locus at 1q24-q25 (HPC1) and was recently shown to harbor truncating mutations in two families with linkage to HPC1. Here, we screened for RNASEL germline mutations in 66 Finnish patients with HPC, and we determined the frequency of the changes in the index patients from 116 families with HPC, in 492 patients with unselected prostate cancer (PRCA), in 223 patients with benign prostatic hyperplasia (BPH), and in 566 controls. A truncating mutation, E265X, was found in 5 (4.3%) of the 116 patients from families with HPC. This was significantly higher (odds ratio [OR] =4.56; P=.04) than the frequency of E265X in controls (1.8%). The highest mutation frequency (9.5%) was found in patients from families with four or more affected members. Possible segregation was detected only in a single family. However, the median age at disease onset for E265X carriers was 11 years less than that for noncarriers in the same families. In addition, of the four missense variants found, R462Q showed an association with HPC (OR=1.96; P=.07). None of the variants showed any differences between controls and either patients with BPH or patients with PRCA. We conclude that, although RNASEL mutations do not explain disease segregation in Finnish families with HPC, the variants are enriched in families with HPC that include more than two affected members and may also be associated with the age at disease onset. This suggests a possible modifying role in cancer predisposition. The impact that the RNASEL sequence variants have on PRCA burden at the population level seems small but deserves further study.