Changes in screening behaviors and attitudes toward screening from pre-test genetic counseling to post-disclosure in Lynch syndrome families.

The purpose of this study was to examine colonoscopy adherence and attitudes toward colorectal cancer (CRC) screening in individuals who underwent Lynch syndrome genetic counseling and testing. We evaluated changes in colonoscopy adherence and CRC screening attitudes in 78 cancer-unaffected relatives of Lynch syndrome mutation carriers before pre-test genetic counseling (baseline) and at 6 and 12 months post-disclosure of test results (52 mutation negative and 26 mutation positive). While both groups were similar at baseline, at 12 months post-disclosure, a greater number of mutation-positive individuals had had a colonoscopy compared with mutation-negative individuals. From baseline to 12 months post-disclosure, the mutation-positive group demonstrated an increase in mean scores on measures of colonoscopy commitment, self-efficacy, and perceived benefits of CRC screening, and a decrease in mean scores for perceived barriers to CRC screening. Mean scores on colonoscopy commitment decreased from baseline to 6 months in the mutation-negative group. To conclude, adherence to risk-appropriate guidelines for CRC surveillance improved after genetic counseling and testing for Lynch syndrome. Mutation-positive individuals reported increasingly positive attitudes toward CRC screening after receiving genetic test results, potentially reinforcing longer term colonoscopy adherence.

Evolutionary evidence of the effect of rare variants on disease etiology.

The common disease/common variant hypothesis has been popular for describing the genetic architecture of common human diseases for several years. According to the originally stated hypothesis, one or a few common genetic variants with a large effect size control the risk of common diseases. A growing body of evidence, however, suggests that rare single-nucleotide polymorphisms (SNPs), i.e. those with a minor allele frequency of less than 5%, are also an important component of the genetic architecture of common human diseases. In this study, we analyzed the relevance of rare SNPs to the risk of common diseases from an evolutionary perspective and found that rare SNPs are more likely than common SNPs to be functional and tend to have a stronger effect size than do common SNPs. This observation, and the fact that most of the SNPs in the human genome are rare, suggests that rare SNPs are a crucial element of the genetic architecture of common human diseases. We propose that the next generation of genomic studies should focus on analyzing rare SNPs. Further, targeting patients with a family history of the disease, an extreme phenotype, or early disease onset may facilitate the detection of risk-associated rare SNPs.

Phase II trial of imatinib mesylate in patients with metastatic melanoma.

Metastatic melanoma cells express a number of protein tyrosine kinases (PTKs) that are considered to be targets for imatinib. We conducted a phase II trial of imatinib in patients with metastatic melanoma expressing at least one of these PTKs. Twenty-one patients whose tumours expressed at least one PTK (c-kit, platelet-derived growth factor receptors, c-abl, or abl-related gene) were treated with 400 mg of imatinib twice daily. One patient with metastatic acral lentiginous melanoma, containing the highest c-kit expression among all patients, had dramatic improvement on positron emission tomographic scan at 6 weeks and had a partial response lasting 12.8 months. The responder had a substantial increase in tumour and endothelial cell apoptosis at 2 weeks of treatment. Imatinib was fairly well tolerated: no patient required treatment discontinuation because of toxicity. Fatigue and oedema were the only grade 3 or 4 toxicities that occurred in more than 10% of the patients. Imatinib at the studied dose had minimal clinical efficacy as a single-agent therapy for metastatic melanoma. However, based on the characteristics of the responding tumour in our study, clinical activity of imatinib, specifically in patients with melanoma with certain c-kit aberrations, should be examined.

Genotype-phenotype correlations in Peutz-Jeghers syndrome.

BACKGROUND AND AIMS: Peutz-Jeghers syndrome (PJS) is a dominantly inherited disorder often caused by mutations in STK11. Time to onset of symptoms was characterised for a large collection of individuals with PJS who had been tested for STK11 mutations and genotype-phenotype correlations were evaluated. METHODS: We characterised mutations in 42 independent probands and also used a historical cohort design to study 51 individuals with Peutz-Jeghers syndrome who had completed self-administered questionnaires. RESULTS: Mutations were detected in 22/32 (69%) probands with PJS and 0/10 probands referred to rule out PJS. Real-time PCR analysis to quantitate DNA failed to detect any large deletions in PJS participants without STK11 mutations. The median time to onset for gastrointestinal symptoms or polypectomy was 13 years of age but showed a wide variability. Gastric polyps were frequent in PJS participants, with a median age at onset of 16 years. Individuals with missense mutations had a significantly later time to onset of first polypectomy (p = 0.04) and of other symptoms compared with those participants either with truncating mutations or no detectable mutation. CONCLUSION: STK11 mutation analysis should be restricted to individuals who meet PJS criteria or their close relatives. Direct sequencing of STK11 yields a high rate of point mutations in individuals who meet phenotypic PJS criteria. Individuals with missense mutations of STK11 typically had a later time to onset for PJS symptoms. The common occurrence of gastric polyps may facilitate chemopreventive studies for this disorder.

Cyclin D1 polymorphism and risk for squamous cell carcinoma of the head and neck: a case-control study.

A G-->A polymorphism (G870A) in exon 4 of the cyclin D1 (CCND1) gene creates an alternative splice site in its mRNA, encoding a protein with an altered C-terminal domain. It has been suggested that DNA damage in cells with the A allele bypasses the G(1)/S checkpoint of the cell cycle more easily than damage in cells without the A allele. Because CCND1 plays a critical role in cell cycle control and reduced DNA repair capacity is associated with an increased risk for squamous cell carcinoma of the head and neck (SCCHN), we hypothesize that this CCND1 polymorphism modulates individual susceptibility to SCCHN. To test this hypothesis we evaluated the frequency of the polymorphism in a hospital-based case-control study of 233 newly diagnosed SCCHN patients and 248 non-cancer controls. The cases and controls were frequency matched by age (+/-5 years), sex and tobacco use. All subjects were non-Hispanic whites. We found that the A allele frequency was slightly higher in the cases (0.485) than in the controls (0.425), but the difference was borderline statistically significant (P = 0.064). The frequencies of the CCND1 AA, GA and GG genotypes were 23.6, 49.8 and 26.6%, respectively, in cases and 16.5, 52.5 and 31.5%, respectively, in controls. Multivariate logistic regression analysis adjusting for age (in years), sex, smoking and alcohol use was performed to calculate odds ratios (OR) and 95% confidence intervals (CI). Compared with the wild-type CCND1 GG, the CCND1 A G genotype was associated with a non-significantly increased risk (adjusted OR 1.15, 95% CI 0.75-1.76), but the CCND1 AA genotype was associated with a significantly increased risk (adjusted OR 1.77, 95% CI 1.04-3.02) for SCCHN. Results from a trend test using a logistic regression model were statistically significant (P = 0.044). Among the cases the mean age of onset was 59.0, 56.8 and 55.5 years for the GG, GA and AA genotypes, respectively. In the stratification analysis the CCND1 AA variant genotype was associated with a >3-fold increased risk in individuals who were

Age-associated risk of cancer among individuals with N-acetyltransferase 2 (NAT2) mutations and mutations in DNA mismatch repair genes.

Mutations in N-acetyltransferase 2 (NAT2), a highly polymorphic enzyme involved in the metabolism of xenobiotics and carcinogens, may affect risk for colorectal cancer (CRC), especially among individuals with germ-line mutations in DNA mismatch repair genes. We determined the NAT2 genotypes and allele frequencies for 86 individuals with CRC who had mutations in hMLH1, hMSH2, or hPMS1. No significant difference in time to onset was observed between rapid (NAT2*4) and slow (NAT2*5, NAT2*6, and NAT2*7) acetylators. However, when individuals were stratified separately by NAT2 polymorphism (NAT2*5, NAT2*6, and NAT2*7), those who were heterozygous at the mutant locus NAT2*7 after adjustment for the NAT2 mutant loci NAT2*5 and NAT2*6 had a significantly higher risk of CRC (hazard ratio, 2.96; P = 0.012) and all of the cancers (hazard ratio, 3.37; P = 0.00004) than individuals homozygous for wild type at the NAT2*7 allele. These findings suggest that NAT2 genotype may be an important factor in tumorigenesis of CRC and cancers related to hereditary nonpolyposis CRC among individuals with mismatch repair defects.

Human pancreatic ribonuclease 1: expression and distribution in pancreatic adenocarcinoma.

BACKGROUND: Human pancreatic ribonuclease (RNase 1) is a pancreatic enzyme that is present at high levels in the serum of most patients with pancreatic adenocarcinoma. For this reason, the authors studied its patterns of expression at the single-cell level in pancreatic adenocarcinoma tissues by immunohistochemical analysis and in situ hybridization (ISH). METHODS: Immunohistochemical analysis with polyclonal antibodies against RNase 1 and by ISH with digoxigenin-labeled RNase 1 probe were used to detect RNase 1 in the neoplastic cells of ductal type pancreatic adenocarcinomas. RESULTS: Fifteen of 18 carcinoma samples were positive for RNase 1, demonstrating that the expression of ribonuclease that the authors observed previously in human pancreatic adenocarcinoma cell lines was not an artifact of cell culture. The authors also found RNase 1 in some of the metaplastic ducts and atrophic islets in 4 of 6 chronic pancreatitis samples, and they observed RNase 1 immunostaining in hyperplastic ducts adjacent to one of the well-differentiated adenocarcinomas. CONCLUSIONS: The expression levels of RNase 1 by tumor cells from pancreatic adenocarcinomas are consistent with the high RNase 1 levels found in the serum of most patients with pancreatic adenocarcinoma. This expression of RNase 1, which is an acinar protein, demonstrates that the patterns of gene expression in pancreatic adenocarcinoma are distinct from those of normal pancreatic duct cells. Conversely, RNase 1 expression levels in altered ductal cells from some chronic pancreatitis tissues and hyperplastic ducts from carcinoma tissues suggest that abnormal expression levels may be an early event in pancreatic tumorigenesis.

ARHI is the center of allelic deletion on chromosome 1p31 in ovarian and breast cancers.

In our previous work, we had characterized ARHI as an imprinted putative tumor-suppressor gene in ovarian and breast cancers. ARHI is expressed in primary breast and ovarian cell lines but largely absent from the corresponding malignant tumors. Moreover, the non-imprinted functional allele is typically deleted in malignant cells. Since ARHI had been mapped to 1p31, a common deletion site in breast and ovarian cancer and male germ-cell tumors, in this study, we set out to define precisely the physical location of ARHI at 1p31 and to determine if this location lies within the smallest common region of deletion in breast and ovarian cancers. To this end, we first carried out radiation hybrid mapping of ARHI and surrounding markers, followed by a high-resolution study of loss of heterozygosity at 1p31 in 49 ovarian and breast cancers. Combining a radiation hybrid map and a physical map of the region encompassing ARHI, 3 discrete regions of minimal deletion were found at 1p31 in breast and ovarian cancers. ARHI is the most common deletion region at 1p31. Two other less common regions of deletion were found centromeric to this gene. One of them centered on D1S207 and the other one included and was proximal to D1S488. We also confirmed the preferential loss of non-imprinted functional allele in 7 of 9 tumor specimens. These data support the possibility that ARHI is a tumor-suppressor gene and suggest that additional tumor-suppressor genes may lie proximal to ARHI at 1p31. The data obtained from our study should aid in the identification and characterization of genes in this novel imprinted region.

DNA pooling in mutation detection with reference to sequence analysis.

We discuss pooling methods of mutation detection for identifying rare mutations. We provide mathematical formulae for obtaining the optimal pool size as a function of the mutation frequency in the study population and the specificity of the test. The optimal pool size depends strongly on the specificity of the test. With a test that has 99% specificity, pooling can reduce the number of tests that need to be performed by 80%, whereas, with a test with 95% specificity, pooling reduces the number of samples that must be tested by only 50%. We used the software PHRED to call mutations after sequencing of pooled samples with known STK11 mutations. We found that, when the area under the curve for the less prominent peak was used to call mutations, we were able to pool pairs of samples and correctly identify mutations. Pooling of three samples did not lead to an adequately specific test for the basic automated allele-calling procedures that we used. We discuss methods by which the specificity may be improved to permit pooling of three or more samples when testing for mutations by sequencing.

Effects of cyclin D1 polymorphism on age of onset of hereditary nonpolyposis colorectal cancer.

A common polymorphism in the cyclin D1 gene enhances the gene's alternate splicing. The alternatively spliced product encodes an altered protein that does not contain sequences involved in the turnover of the protein. We found that hereditary nonpolyposis colorectal carcinoma patients who were homozygous or heterozygous for the mutant allele developed colorectal cancer an average of 11 years earlier than patients who were homozygous for the normal alleles. This is the first report indicating that the cyclin D1 polymorphism influences age of onset of cancer. Because cyclin D1 plays an important role in the G1 to S phase transition of the cell cycle, our findings suggest that cells with the mutant allele accumulate mutations as a result of defective mismatch repair and may also bypass the G1-S checkpoint of the cell cycle more easily than in cells not carrying the polymorphism. The polymorphism has a dominant phenotype.

Ribonucleases expressed by human pancreatic adenocarcinoma cell lines.

Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N-glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N-glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.

Increased cyclooxygenase-2 expression in human pancreatic carcinomas and cell lines: growth inhibition by nonsteroidal anti-inflammatory drugs.

Cyclooxygenase (COX)-2 mRNA and protein expression were found to be frequently elevated in human pancreatic adenocarcinomas and cell lines derived from such tumors. Immunohistochemistry demonstrated cytoplasmic COX-2 expression in 14 of 21 (67%) pancreatic carcinomas. The level of COX-2 mRNA was found to be elevated in carcinomas, relative to histologically normal pancreas from a healthy individual, as assessed by reverse transcription-PCR. COX-2 protein expression was detected by the Western blot assay in three of five pancreatic carcinoma cell lines (BxPC-3, Capan-1, and MDAPanc-3), whereas COX-1 protein was detected in two of the five cell lines (BxPC-3 and Capan-1). Increased levels of COX-2 mRNA were found in four of five cell lines, and only in PANC-1 cells was the low level of transcript comparable to that in the normal pancreas. The level of COX-2 mRNA was positively correlated with the differentiation status of the tumor of origin for each cell line, COX-2 protein expression was up-regulated by epidermal growth factor when the cells were grown in absence of serum. Finally, two nonsteroidal anti-inflammatory drugs, sulindac sulfide and NS398, produced a dose-dependent inhibition of cell proliferation in all pancreatic cell lines tested. No correlation was found between the level of COX-2 or COX-1 expression and the extent of growth inhibition. Treatment of BxPC-3 cells with sulindac sulfide and NS398 resulted in an induction of COX-2 expression. Our findings indicate that COX-2 up-regulation is a frequent event in pancreatic cancers and suggest that nonsteroidal anti-inflammatory drugs may be useful in the chemoprevention and therapy of pancreatic carcinoma.

Gene expression in pancreatic adenocarcinoma.

Both acinar and duct cell-specific gene products are expressed by pancreatic adenocarcinoma. In order to begin to understand the mechanisms by which genes of both cell types are expressed in pancreatic adenocarcinoma, an understanding of the underlying transcription factors is important. PDX1 plays an important role in the development of the pancreas and is also expressed in the adult pancreas; it is known to be involved in the regulation of expression of both acinar and islet cell-specific gene products. We have examined pancreatic adenocarcinoma cell lines and have determined that they also express PDX1, making it a candidate transcription factor for the abnormal regulation of these acinar and duc cell gene products.

Reduced expression of cyclooxygenase 2 proteins in hereditary nonpolyposis colorectal cancers relative to sporadic cancers.

BACKGROUND & AIMS: Cyclooxygenase (COX) enzymes catalyze the conversion of arachidonic acid to prostaglandins. Evidence suggests that nonsteroidal anti-inflammatory drugs reduce the risk of colorectal cancer (CRC) and that this effect is mediated through COX inhibition. We analyzed and compared expression of the inducible COX-2 isoform in colorectal neoplasms from patients with hereditary nonpolyposis colorectal cancer (HNPCC), familial adenomatous polyposis (FAP), and sporadic CRC. Given that COX-2 is induced by transforming growth factor (TGF)-beta and that TGF-beta type II receptor (RII) mutations are found in HNPCCs, we determined the relationship between RII status and COX-2 expression. METHODS: COX-2 protein expression was determined in colorectal epithelia using immunohistochemistry and Western blotting. Patients with HNPCC had known mutations in hMLH1 or hMSH2 genes and/or met the Amsterdam criteria. In CRCs from HNPCC cases, mutations were sought in the coding region of the RII gene using the polymerase chain reaction. RESULTS: COX-2 was detected in adenomas from 2 of 3 HNPCC, 6 of 7 FAP, and 5 of 8 sporadic cases. In CRCs, COX-2 staining was found in 16 of 24 (67%) HNPCC vs. 24 of 26 (92%) sporadic cases (P = 0.035) and in 2 of 2 FAP cases. Staining intensity was reduced in HNPCCs compared with sporadic CRCs (P = 0.035). Staining localized to the cytoplasm of neoplastic cells; normal epithelial cells were negative for COX-2. Overexpression of COX-2 in CRCs relative to normal mucosa was confirmed by Western blotting. TGF-beta RII mutations were detected in 12 of 14 HNPCCs examined, including 3 of 4 COX-2-negative and 9 of 10 COX-2-positive cancers. CONCLUSIONS: The frequency and intensity of COX-2 expression was significantly reduced in HNPCCs relative to sporadic CRCs, and was not a consequence of RII mutations. Given that many HNPCCs express COX-2, inhibition of this enzyme may be an important strategy to prevent CRC in these patients.

Loci for efficient detection of microsatellite instability in hereditary non-polyposis colorectal cancer.

Most hereditary non-polyposis colorectal cancer (HNPCC) is due to germline mutations in DNA mismatch repair genes. Tumors arising as a result of these mutations display instability in microsatellites, which are short tandem repeats of DNA that are distributed throughout the genome. Although a subset of sporadic colorectal carcinomas also have microsatellite instability (MSI), the phenotype is a useful screening test in identifying patients with HNPCC caused by mutations in mismatch repair (MMR) genes. Studies have shown that some microsatellite markers are more efficient than others in identifying tumors with MSI. Furthermore, the frequency of instability can be assessed by categorizing patients into high (MSI-H, >/= 30-40% positive markers), low (MSI-L), and microsatellite stable (MSS) groups. Using a panel of 28 microsatellite markers, tumor and normal DNA from 10 HNPCC patients was used to identify the five most efficient markers for detecting MSI (BAT26, D2S123, FGA, D18S35, and TP53-DI). Each of the five markers detected MSI in 80-100% of the cases examined. We then expanded the sample size to 17 tumors from HNPCC patients. Each case had evidence for a mutation in either hMSH2 or hMLH1. We compared the efficiency of our panel of five best markers with another panel of five markers (BAT25, BAT26, D2S123, D17S250, and D5S346) identified as being efficient markers for detection of MSI at a recent NCI workshop. Our five selected markers were more efficient (85% vs. 79%) in detecting MSI. However, using either panel, 100% of the cases fell into the MSI-H category and the probability of misclassifying an MSI-H case as MSI-L is very low (0.002-0.008). We also examined four cases meeting the Amsterdam criteria for HNPCC, but with no evidence for mutation in either the hMSH2 or hMLH1 gene. With our panel, three were classified as MSI-H, while only two were classified as such with the NCI reference panel. The probability of misclassifying an MSI-L case as an MSI-H, using a panel of five markers is high (0.263).

Reduction in hMSH2 mRNA levels by premature translation termination: implications for mutation screening in hereditary nonpolyposis colorectal cancer.

We have used single-strand conformational polymorphism (SSCP) and heteroduplex analysis to examine DNA from 50 colorectal carcinoma patients coming from families meeting the Amsterdam criteria for hereditary nonpolyposis colorectal cancer (HNPCC) or having developed colorectal carcinoma at age 45 or younger. We identified mutations in 12 of these patients, with seven of these being novel mutations. We examined four of the truncating mutations using in vitro transcription and translation (IVTT) assays and determined that the mutation causing an in-frame deletion of exon 5 could easily be detected by the IVTT assay, but the three mutations resulting in premature translation termination were not detected because the steady-state levels of the mutant allele transcripts are too low. Our findings suggest that some but not all mutant hMSH2 alleles have significantly lower steady-state mRNA levels than the normal allele. Under ideal circumstances, where lymphoblastoid cell lines are available for RNA extraction, IVTT may be useful for detecting truncating mutations. However, our data suggest that caution should be taken in using IVTT in routine screening of clinical samples for truncating HNPCC mutations, as many mutations may go undetected.

Loss of p21WAF1/Cip1 protein expression accompanies progression of sporadic colorectal neoplasms but not hereditary nonpolyposis colorectal cancers.

p21 (p21WAF1/Cip1), a cyclin-dependent kinase inhibitor, induces G1 arrest and can inhibit the activity of the proliferating cell nuclear antigen (PCNA). We analyzed p21 expression during colorectal tumorigenesis, its association with its transcriptional regulator p53, and its relationship to rates of cell proliferation and apoptosis. p21 and p53 protein expression were examined in sporadic tumors and hereditary nonpolyposis colorectal cancers (HNPCCs) by immunohistochemistry (IHC) and immunoblotting. Apoptosis was examined using a DNA nick end-labeling assay, and cell proliferation was examined by PCNA staining. In normal colorectal epithelia, nuclear p21 staining was uniformly detected in crypt cells of the superficial compartment (upper one-third) that stained negatively for PCNA. p21 and PCNA expression were, therefore, mutually exclusive. In sporadic cases, a decrease in the frequency of p21 expression accompanied adenoma development and progression to carcinoma. Specifically, p21 was detected in 12 of 16 (75%) adenomas and 10 of 32 (31%) carcinomas. In contrast to sporadic cases, HNPCCs with known mutations in DNA mismatch repair genes expressed p21 in 12 of 15 (80%) carcinomas. An inverse relationship between p21 and p53 was observed wherein mutant p53 proteins were detected in 4 of 15 (27%) HNPCCs versus 22 of 32 (69%) sporadic carcinomas. Although p21+ carcinoma cells were generally negative for p53, IHC revealed that some carcinoma cells expressed both p21 and p53 proteins. Furthermore, p53-mutated SW480 colon carcinoma cells were found to coexpress p21 and p53, suggesting that p21 can also be activated by a p53-independent mechanism. No association was found between p21 or PCNA and apoptotic labeling indices in adenomas or carcinomas. In conclusion, a decrease in p21 expression accompanies neoplastic progression in sporadic cases but not in HNPCCs. This finding appears related to p53 status in that the frequency of p53 expression was significantly reduced in HNPCCs compared to sporadic cases, suggesting a difference in their molecular pathways of tumorigenesis.