Detection of K-ras gene mutations at codon 12 in the pancreatic juice of patients with intraductal papillary mucinous tumors of the pancreas

BACKGROUND: The authors previously found specific mutations of the K-ras gene at codon 12 in the pancreatic juice of 67% of patients (6 of 9) with pancreatic ductal carcinoma, and the detection of these mutations was useful for diagnosis. This study was performed to detect and evaluate K-ras mutations in pancreatic juice from patients with intraductal papillary mucinous tumor of the pancreas, which is considered a low grade malignancy. The results were interpreted from the viewpoint of clinical significance. METHODS: K-ras mutations were examined using seminested polymerase chain reaction analysis combined with restriction enzyme digestion, followed by nonradioisotopic single strand DNA conformation polymorphism. RESULTS: Twelve of thirteen cases (92%) of intraductal papillary mucinous tumor of the pancreas, confirmed histologically (9 adenomas and 4 carcinomas), and 26 of 43 cases (60%) of ductal carcinoma showed specific K-ras gene mutations in the pancreatic juice. Furthermore, 4 of 22 patients (18%) with chronic pancreatitis, followed for more than 1 year without a sign of pancreatic tumor, showed K-ras mutations. In contrast, no mutations of the K-ras gene were detected in the pancreatic juice from 28 normal controls. CONCLUSIONS: K-ras mutations were found in the pancreatic juice of all but one patient with intraductal papillary mucinous tumor of the pancreas, but they were not useful for distinguishing carcinoma from adenoma. The authors concluded that K-ras mutations are not a specific marker for pancreatic neoplasms because similar mutations were detected in the pancreatic juice from patients with chronic pancreatitis. At the present time, the detection of K-ras mutations in pancreatic juice should be used clinically as an adjunct diagnostic modality for pancreatic diseases.     

Detection and clinical correlations of ras gene mutations in human ovarian tumors

In epithelial ovarian neoplasms K-ras codon 12 gene mutations show a wide variation fluctuating between 4-39% in invasive carcinomas and 20-48% in borderline malignant tumors. In this study, we showed the pattern of point mutations in codon 12 of the K-ras, H-ras and N-ras genes, using polymerase chain reaction restriction fragment length polymorphism analysis in 74 tissue specimens of Greek patients with epithelial ovarian tumors. K-ras and H-ras gene mutations were detected in 11/48 (23%) and 3/48 (6%) cases with primary invasive ovarian carcinomas, respectively, while N-ras gene mutations were not found. No mutation of K-, H- and N-ras genes was detected in 23 ovarian cystadenomas. In 1 out of 3 borderline ovarian tumors (33%) we found an H-ras gene mutation. The prevalence of mutations in K-ras gene was 1/8 (13%) in mucinous, 7/29 (24%) in serous, 1/3 (33%) in endometrioid and 2/8 (25%) in clear-cell adenocarcinomas and in H-ras gene 1/8 (13%) in mucinous and 2/29 (7%) in serous adenocarcinomas. Analysis of the results revealed no significant correlation between ras gene mutations and clinicopathological parameters or clinical outcome of this primary invasive ovarian carcinoma population. Our present data suggest that ras gene mutations in invasive ovarian carcinomas occur in 29% of Greek patients and are not associated with the differentiation of the epithelial cells or the response of patients to adjuvant platinum-based chemotherapy.     

Prevalence and clinical significance of combined K-ras mutation and p53 aberration in pancreatic adenocarcinoma

CONCLUSION: This study could not attribute survival differences to the coincident acquisition of two common genetic alterations, K-ras mutation and p53 overexpression in pancreatic adenocarcinoma patients. Additionally, our data indicate the converse to be true: Those patients lacking both K-ras mutation and aberrant p53 expression showed the shortest survival when compared with cases showing either alteration or both. This study also showed the negative effect of K-ras mutation and p53 expression on pancreas cancer patients' survival after treatment with either radiation therapy or chemotherapy. BACKGROUND: Mutations of the oncogene K-ras at codon 12 are reported to be the most common genetic alteration in pancreatic carcinoma, whereas either overexpression or mutation of the tumor suppressor p53 gene is considered the most common genetic alteration in neoplasia of all types. p53 overexpression has been attributed to survival differences in pancreatic carcinoma, but such association is still controversial. No studies have fully documented the combined incidence of K-ras and p53 alterations in pancreatic adenocarcinoma, or their combined effect on patient survival in a large case series. The influence of radiation or chemotherapy in groups showing both, either, or neither mutation is also undocumented. METHODS: Paraffin-embedded tissue sections from 76 cases of pancreatic adenocarcinoma were cut for DNA extraction for K-ras analysis and immunohistochemical staining for aberrant p53 expression. K-ras mutation was determined by single-strand conformation polymorphism (SSCP) and slot-blot allele-specific oligonucleotide (ASO) hybridization of PCR-amplified DNA product p53 expression was scored on the basis of percent nuclear staining with the MAb DO7. RESULTS: Sixty-four of 76 cases (84%) showed K-ras mutation, p53 expression, or both, K-ras was mutated in 55 of 76 cases (72%). p53 was expressed in 33 of 76 cases (43%). Twenty-four of 76 cases (31%) showed both K-ras mutation and p53 expression. The presence of both alterations was not related to significant differences in tumor grade, stage, or survival compared to either alteration alone. A sizable subset (16% of cases) lacked either alteration, and surprisingly, this group showed the shortest median survival compared to those with K-ras mutation, p53 expression, or both (p = 0.024). Patients whose tumors were K-ras-negative showed the greatest difference in median survival with radiation therapy (median survival 30.8 mo vs 7.8 mo with no radiation, p = 0.005).     

Prognostic impact of mutated K-ras gene in surgically resected non-small cell lung cancer patients

Mutated K-ras oncogenes have been detected in a third of lung adenocarcinomas, located usually in codon 12, its presence correlating negatively with survival. To further define the role of K-ras point mutations in non-small cell lung cancer, we studied the presence of mutated K-ras genes in surgical specimens from 66 patients. Polymerase chain reaction was performed from sections of formalin-fixed paraffin-embedded tissue. We screened for point mutations in codons 12, 13 and 61 of the K-ras gene by dot blot hybridization analysis with mutation-specific oligonucleotide probes. Ras gene mutations were present in 13 of 66 carcinomas (20%), nine in codon 12 and four in codon 61. Three squamous cell carcinomas harbored two different point mutations in K-ras codon 12. Mutated K-ras genes were found more frequently in squamous cell carcinomas (eight of 38) than in adenocarcinoma (three of 22). Analysis of nucleotide sequence disclosed a multifarious mutation pattern of K-ras codon 12, where the most common conversion was from glycine (GGT) to valine (GTT). K-ras point mutation positive subset had poorer survival, nine of the 13 patients died during the follow-up period as compared with 22 of 53 patients with no mutation in the K-ras gene (P = 0.01). The difference was also strikingly significant when stratified according to node status.     

Detection of K-ras mutations in lung carcinomas: relationship to prognosis

The K-ras mutation is one of the most common genetic alterations found in human lung cancer. To evaluate the prognostic value of ras gene alterations in lung cancer in a U.S. population, we have screened 173 human lung tumors, which included 127 adenocarcinomas, 37 squamous carcinomas, and 9 adenosquamous carcinomas, for mutations in the K-ras gene using the combination of the PCR and denaturing gradient gel electrophoresis. Forty-three tumors contained K-ras mutations. Of these, 41 were identified among the adenocarcinomas (32%), 1 among the squamous carcinomas (2.7%), and 1 among the adenosquamous carcinomas (11%). Forty of these mutations were found in codon 12 and consisted of 24 G to T transversions, 12 G to A transitions, 2 G to C transversions, and 1 double GG to TT mutation. Two other G to T transversions were found in codon 13, and 1 A to C transversion was found in codon 61. The data showed that gender did not seem to affect the incidence and the types of the K-ras mutations or amino acid changes. Examination of the mutations in adenocarcinomas in relation to overall survival showed no difference in adenocarcinomas with K-ras mutations compared with K-ras-negative adenocarcinomas. However, the substitution of the wild-type GGT (glycine) at codon 12 with a GTT (valine) or a CGT (arginine) showed a strong trend (P = 0.07) toward a poorer prognosis compared with wild-type or other amino acid substitutions. Substitution of the wild-type glycine for aspartate (GAT) showed a strong trend (P = 0.06) for a better outcome than the valine or arginine substitution. Although these trends will require larger patient populations for verification, these data suggest that the prognostic significance of K-ras mutations may depend on the amino acid substitution in the p21(ras) protein.     

K-ras point mutations are rare events in premalignant forms of Barrett's oesophagus

OBJECTIVE: In Barrett's adenocarcinomas, in contrast to squamous oesophageal carcinomas, K-ras point mutations are thought to be a frequent event. The frequency of K-ras point mutations in premalignant forms of Barrett's oesophagus (metaplasia, dysplasia) leading to adenocarcinoma with increased risk is currently not known. To establish the frequency of K-ras mutations in premalignant forms of Barrett's oesophagus, we investigated oesophageal biopsy specimens with Barrett's metaplastic and dysplastic epithelium for point mutations in the K-ras gene/codons 12, 13. DESIGN: A total of 412 biopsies from patients with Barrett's oesophagus were histologically classified into biopsies with metaplasia (n = 252), dysplasia (n = 105) and adenocarcinoma (n = 11), as well as biopsies distant from disease (normal, n = 37 and hyperplastic squamous epithelium, n = 7). METHODS: DNA from biopsy specimens was amplified by polymerase chain reaction (PCR) with a modified primer for generating a restriction site in the case of wild type in codon 12. Wild-type or point mutations in the K-ras gene/codons 12, 13 were detected by restriction fragment length analysis of the PCR products. RESULTS: Point mutations in K-ras/codon 12 were found in 9 biopsies (n = 1 in metaplasia, n = 4 in dysplasias, n = 4 in adenocarcinomas). All the other biopsies showed the wild type of K-ras/codon 12. No K-ras/codon 13 mutation (GGCgly-->GACasp) was observed. CONCLUSION: Mutations in K-ras/codon 12 were rarely found in premalignant forms of Barrett's oesophagus. Whereas the screening for K-ras point mutations in metaplastic sites of Barrett's epithelium seems not to be of practical value, the screening for mutations in dysplastic lesions might be helpful to estimate the individual risk for progression of Barrett's epithelium to adenocarcinoma. A further evaluation in larger numbers of patients is needed.     

A comparison of morphine, pethidine and fentanyl in the postsurgical patient-controlled analgesia environment

Oncogene and tumor suppressor gene mutations are candidate biomarkers for cancer risk assessment and lesion detection. The K-ras oncogene has previously been associated with non-small cell lung cancer (NSCLC), particularly adenocarcinomas in which reported rates of mutation have approached 30-40%. We have analyzed non-malignant lung tissue from patients with lung cancer and primary lung cancers for K-ras gene mutations. Mutations were detected in 32% cancers and 29% non-malignant lung tissue from patients with cancer. The majority of tumors testing positive were adenocarcinoma of the lung. Normal DNA controls, including peripheral blood lymphocytes and normal lung from non-smokers, were negative. The ability to detect genetic alterations in non-malignant lung tissues is consistent with the concept that genetic alterations are involved in field cancerization of the aerodigestive tract.     

Adenocarcinoma of the pancreas: detection of occult metastases in regional lymph nodes by a polymerase chain reaction-based assay

BACKGROUND: Stage I (T1-2NOM0) adenocarcinoma of the pancreas is associated with a 5-year survival rate of 15-25%. Despite apparently curative resection and pathologic staging indicating localized disease, these cancers recur. The authors hypothesized that there exists microscopic regional disease that is not detected by surgical exploration or routine histopathology. METHODS: Because 90-95% of pancreatic cancers exhibit codon 12 K-ras mutations, the authors examined regional lymph nodes for mutated K-ras as a marker of metastasis. DNA was extracted from paraffin embedded archival specimens (primary tumors and histologically negative lymph nodes) of patients with Stage I pancreatic adenocarcinoma. The target region of K-ras was amplified by polymerase chain reaction (PCR) and tested for codon 12 mutation by BstN1 restriction digestion (restriction fragment length polymorphism [RFLP]) that recognized normal but not mutated sequences. Cell lines that harbored normal or mutated K-ras and resected jejunum or gallbladder were used as controls. The regional lymph nodes of 22 patients whose tumors harbored mutated K-ras were tested. RESULTS: Dilution experiments with normal and mutant control cell line DNA demonstrated an assay sensitivity for mutated K-ras of 0.1%. Mutated K-ras was found in at least 1 regional lymph node in 16 (73%) of 22 patients with pathologic Stage I pancreatic adenocarcinoma, which suggested metastases not detected by routine histopathology. DNA sequence analysis was performed in four patients and confirmed identical point mutations in the primary tumor and accompanying PCR/RFLP positive lymph nodes. CONCLUSIONS: Pathologic examination of regional lymph nodes in pancreatic adenocarcinoma specimens fails to detect metastases in many patients. Lymph node micrometastasis is one reason for the poor survival rates observed among patients with Stage I cancers. PCR/RFLP may have a role in staging early pancreatic cancers.     

Increased telomerase activities in human pancreatic duct adenocarcinomas

Telomerase is a key enzyme with regard to immortalization of cancer cells and increased activity has been demonstrated in various human malignant neoplasms. Since little is known of its role in pancreatic cancers, we investigated changes in telomerase activity in human pancreatic duct adenocarcinomas and compared the frequency of increased telomerase activity with the presence of K-ras gene mutations. The samples were obtained from 38 pancreatic duct adenocarcinomas and 7 tumor surrounding tissues at surgical resection. Telomerase activity was examined by telomeric repeat amplification protocol assay and terminal restriction fragment (TRF) length was examined by Southern analysis. K-ras mutation was examined by means of polymerase chain reaction-single strand conformation polymorphism analysis. Among 38 pancreatic carcinomas, 32 (84%) exhibited increased telomerase activities with no apparent relation to the histological type of tumor, tumor size, regional lymphnode involvement and distant metastasis or clinical stage. In tissue surrounding the tumor, telomerase activity was not detected. TRF length tended to be reduced in pancreatic carcinomas. Mutations of K-ras gene were found in 24 out of the 38 (63%) cases. Among the 38 cases, 14 showed increased telomerase activity without K-ras mutation and 4 cases showed K-ras mutation without telomerase activity. These results suggest that increased telomerase activity might be a sensitive genetic diagnostic marker and could be a target for future therapy of pancreatic duct carcinomas.     

Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma

Pancreatic adenocarcinomas are known to have a high incidence of K-ras gene mutations. Differential diagnosis of pancreatic cancer and chronic pancreatitis sometimes presents a clinical dilemma. We recently developed a highly sensitive and specific polymerase chain reaction capable of detecting 3-30 copies of mutant K-ras genes harboring codon 12 single base changes in the presence of 300,000 normal copies. Mutant ras genes were detected in DNA purified from pancreatic juice from all 6 cases of pancreatic adenocarcinoma and 1 case of intraductal papillary neoplasms of the pancreas. In 2 of 6 other cases with pancreatic adenocarcinoma, circulating metastatic cells were detected in DNA purified from peripheral blood. Activated ras genes were not found in pancreatic juice of three control cases (chronic pancreatitis and choledocholithiasis) or in the peripheral blood of two patients with insulinomas. Notable conclusions of this study are that there can be significant levels of shed tumor cells in peripheral blood and an even higher number in pancreatic juice. In addition, two different K-ras mutations were found in some patients.     

Inactivation of the p16 (INK4A) tumor-suppressor gene in pancreatic duct lesions: loss of intranuclear expression

Pancreatic adenocarcinoma develops from histologically identifiable intraductal lesions that undergo a series of architectural, cytological, and genetic changes. Limited genetic evidence recently suggested that the p16 gene plays a role in the progression of these "duct lesions." Duct lesions were identified in pancreata from 33 pancreaticoduodenectomies performed for infiltrating adenocarcinoma. All of these infiltrating adenocarcinomas were previously shown to contain alterations in the p16 gene or its promoter. Monoclonal and polyclonal anti-p16 antibodies were used for histological immunodetection. One hundred twenty-six duct lesions were identified. Nine (30%) of 30 flat, 4 (27%) of 15 papillary, 37 (55%) of 67 papillary with atypia, and 10 (71%) of 14 carcinoma in situ duct lesions showed loss of p16 expression. These included 30% of the flat lesions versus 53% of the nonflat lesions and 29% of the nonatypical lesions versus 58% of the atypical lesions. For both comparisons, the differences were statistically significant (P = 0.036 and P = 0.003, respectively). Loss of p16 expression occurs more frequently, but not exclusively, in higher-grade duct lesions. These data support the hypothesis that pancreatic duct lesions are neoplastic and that they represent the precursors of infiltrating adenocarcinoma. Immunohistochemical detection of p16 provides a new technology to study the genetic alterations in and stages of progression of large numbers of morphologically defined pancreatic duct lesions.     

Quantitation of endopeptidase 24.11 and endopeptidase 24.15 in human blood leukocytes

In order to know the involvement of multiple gene alterations in the pathogenesis of human lung cancer, we examined the genes of K-, H-ras (codons 12, 13, 61), p53(exons 5-9) and the retinoblastoma susceptibility gene (RB)(exons 20-22) using the polymerase chain reaction/single-strand conformation polymorphism method in 32 human lung cancer cell lines (5 squamous-cell carcinomas, 10 adenocarcinomas, 3 large-cell carcinomas, 14 small-cell carcinomas). In 18 non-small-cell lung cancer lines, gene alterations were found in 4 for K-ras (22%), none for H-ras (0%), 4 for p53 (22%) and none for the RB (0%) gene. In 14 small-cell lung cancer (SCLC) lines, no gene alterations were found in K-ras (0%), or H-ras (0%), but 6 were found for p53 (43%) and 3 for the RB (21%) gene. Coincident abnormalities of K-ras and p53, or K-ras and RB genes were not found in any cell lines, and those of the p53 and RB genes were found in only 2 SCLC lines. No association was observed between these three gene alterations and N-myc amplification. Although the above three genes may be involved to some extent in the pathogenesis of lung cancer, more factors are required for its development.     

Validation of the Point-EXACCT method in non-small cell lung carcinomas

K-ras point mutations are often detected in part of the lung carcinomas. For the validation of a highly sensitive and rapid assay for known point mutations, Point-EXACCT (Biochim Biophys Acta 1998; 1379:42-52), we analyzed 89 non-small cell lung carcinomas and compared the results with two sequencing methods. No point mutations were found with double-stranded sequencing. Single-stranded sequencing detected six patients positive for K-ras codon 12. When Point-EXACCT was used, K-ras codon 12 mutations were detected in 8 of 52 patients with squamous cell carcinomas, 10 of 29 patients with adenocarcinomas, and 3 of 8 patients with large cell carcinomas. The finding of K-ras mutations in squamous cell carcinomas is explained by the high sensitivity of the method. Therefore, Point-EXACCT may be applicable to detection of those alterations occurring at a low frequency among an excess of cells with wild-type DNA.     

Papillomavirus, p53 alteration, and primary carcinoma of the vulva

Twenty-nine samples from 28 cases of vulvar squamous cell carcinoma, of which 13 fulfilled the criteria of the bowenoid subtype (mean age 45 years, range 31-68) and 16 of the usual subtype of invasive squamous cell carcinoma (ISCC) (mean age 67.5 years, range 34-83) were investigated for human papillomavirus (HPV) DNA, TP53 alterations, and mdm2 and bcl-2 gene product deregulation. Microscopically all the bowenoid subtype cases (group I) showed a high-grade intraepithelial (VIN 3, carcinoma in situ) lesion associated with early invasive carcinoma in six cases and overt invasive carcinoma in one. By contrast, no evidence of early carcinoma was present in the ISCCs (group II). By in situ hybridization and/or Southern blot hybridization or polymerase chain reaction (PCR), HPV DNA was detected in all cases of group I and in four of 16 cases (25%) of group II, two only by Southern blot after PCR. By single-strand conformation polymorphism and immunocytochemistry only wild-type TP53 and absence of detectable p53 product, respectively, were found in all cases of group I, i.e., in high-risk HPV-positive carcinomas, whereas mutations and/or p53 overexpression accounted for 75% in group II, i.e., in mainly HPV-negative carcinomas. The TP53 gene mutations observed in invasive carcinomas were significantly related to node-positive cases (p = 0.04). Taken together and in agreement with in vitro data, these results support the view that an alteration of TP53, gained either by interaction with viral oncoproteins or by somatic mutations, is a crucial event in the pathogenesis of vulvar carcinomas, but that TP53 mutations are mainly associated with disease progression. Finally, a preliminary immunocytochemical analysis seems to speak against the possible involvement of both MDM2 and BCL-2 gene products in the development of vulvar carcinoma.     

Haemodynamic changes in patients undergoing high-risk PTCA under protection of transfemoral heart- lung machine support with centrifugal pumps

Deletions, mutations and the functional inactivation of tumor suppressor gene p16 are involved in the genesis of different neoplasias. Little is known about the role of p16 gene alterations in the genesis of gastric carcinomas. This study aimed to detect genetic alterations of the p16 gene in gastric carcinomas. We analyzed p16 gene mutations and the frequency of loss of heterozygosity (LOH) at the p16 locus in 43 gastric carcinomas. PCR-SSCP analysis of exons 1 and 2 revealed only one gene mutation in a carcinoma of the diffuse type. Besides carcinomas of the diffuse, intestinal and the mixed type, we also investigated a small-cell primary gastric carcinoma, which was the only one to show a deletion in the p16 gene. LOH analysis was performed using two polymorphic markers located near the p16 gene (D9S171, D9S162) and a sequence-tagged-site marker (c5.1). Allelic loss was noted in two carcinomas of the diffuse type and in one carcinoma of the intestinal type. Allelic instabilities were found in one tumor of the intestinal type and diffuse type each. Although only five of 43 (11.6%) gastric carcinomas had p16 alterations, tumors of the diffuse type tend to show a higher number of genetic alterations near the p16 locus.     

Multiple early gastric stump carcinomas after gastrectomy for peptic ulcer

The remnant stomach after partial gastrectomy is considered to have a predilection for the development of primary gastric carcinoma. However, early gastric stump carcinomas are uncommon because the diagnosis of gastric stump carcinoma is more difficult than that of carcinoma in the intact stomach. Triple early gastric stump carcinomas, as in the present case, are exceedingly rare and may provide some clues for further investigation of carcinogenesis in the gastric stump. We studied about the histological appearance, genetic alterations (P-53 gene, c-erbB-2 gene and K-ras gene), and expression of tumor-associated antigens (carcinoembryonic antigen, carbohydrate antigen 19-9, and sialyl-Tn) in this rare case. The three carcinomas differed from each other histologically. With respect to genetic alterations, c-erbB-2 was amplified in one lesion, but no mutations of K-ras and P-53 gene were detected. The three carcinomas also differed from each other on the expression of tumor-associated antigens. In noncancerous mucosal epithelium at the anastomosis showing hyperplasia and cystic formation of glandular epithelial cells, no genetic alterations were detected, but sialyl-Tn and carbohydrate antigen 19-9 were expressed. These results suggest that there may be different processes of carcinogenesis of the three carcinomas even though they occurred under identical environmental conditions to those that have increased cancer risk.     

Augmentation of the affinity of HLA class I-binding peptides lacking primary anchor residues by manipulation of the secondary anchor residues

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.     

p53 protein overexpression and K-ras codon 12 mutation in pancreatic ductal carcinoma: correlation with histologic factors

The correlation of p53 protein overexpression and the K-ras codon 12 mutation with histologic type, grade of cytologic atypia, depth of invasion and other histologic prognostic factors was studied in paraffin sections from 43 ductectatic- and 70 solid-type pancreatic ductal carcinomas. Overexpression of p53 was found in 23.3% (10/43) of ductectatic carcinomas (17.2% of intraductal and 35.7% of invasive carcinomas) and in 61.4% (43/70) of solid carcinomas. In ductectatic cancers, p53 overexpression was detected in 14.8% (4/27) of carcinomas with low-grade atypia (CAL), 50.0% (5/10) of carcinomas with high-grade atypia (CAH) and in 16.7% (1/6) of mixed low- and high-grade cancers. In the last group, expression was restricted to an area of CAH. In solid cancers, p53 overexpression did not differ by histologic type or grade. Overexpression of p53 and K-ras mutations did not correlate with histologic prognostic factors (lymphatic, venous and perineural invasion, and lymph node metastasis) in ductectatic and solid cancers or depth of invasion of solid carcinomas. Our data suggest that p53 alteration occurs at an early intraductal stage of solid carcinoma, irrespective of cellular atypia, but is low in ductectatic CAL and becomes higher in ductectatic CAH. K-ras mutation, present in a high percentage of tumors of all groups and not correlating with the factors above, showed no changes in frequency with tumor progression.     

Experimental milk formulae with reduced protein content and desialylated milk proteins: influence on the faecal flora and the growth of term newborn infants

Diagnosing and monitoring pancreatic cancer is an ongoing challenge. Conventional markers such as tumor-associated antigens might be supplemented by molecular markers such as gene mutations and growth factor/growth factor receptor alterations in the future. Tumor-associated antigens can easily be measured by different EIA/ELISA systems, but the analysis of gene mutations, growth factors and their receptors requires advanced molecular techniques. CA 19-9 is the most widely used conventional marker for pancreatic cancer and is a useful tool in the diagnosis and follow-up of patients after tumor resection. Nonetheless, its role in detecting early pancreatic cancer is limited. The detection of K-ras and p53 mutations, which occur in about 90 and 50% of pancreatic cancers, respectively, in blood, stool, or bile samples, seems to be a promising approach in the diagnosis. Growth factor and growth factor receptor alterations are often associated with increased tumor aggressiveness and shorter survival following tumor resection. To date the analysis of growth factors/growth factor receptors in pancreatic cancer has not entered clinical use, but further molecular characterization of pancreatic cancer is necessary for earlier and more accurate diagnosis and it may result in new treatment options.     

Can K-ras codon 12 mutations be used to distinguish benign bile duct proliferations from metastases in the liver? A molecular analysis of 101 liver lesions from 93 patients

It can be difficult to distinguish benign bile duct proliferations (BDPs) from well-differentiated metastatic peripancreatic adenocarcinomas on histological grounds alone. Most peripancreatic carcinomas harbor activating point mutations in codon 12 of the K-ras oncogene, suggesting that K-ras mutational status may provide a molecular basis for distinguishing BDPs from liver metastases. The ability of tests for mutations in codon 12 of K-ras to make this distinction was examined in a two-part study. In the first part we determined the K-ras mutational status of 56 liver lesions and 48 primary peripancreatic adenocarcinomas obtained from 48 patients. In the second part of this study an additional 45 liver lesions were studied. In the first 48 patients, activating point mutations in codon 12 of K-ras were detected in 28 (61%) of the 46 primary carcinomas, in 8 (100%) of 8 liver metastases, in 2 (6.5%) of 31 BDPs, and in none (0%) of 14 liver granulomas. Three BDPs and two primary carcinomas did not amplify. To further estimate the prevalence of K-ras mutations in BDPs we analyzed an additional series of 45 mostly incidental BDPs for K-ras mutations. Three (6.7%) of these 45 harbored K-ras mutations. These results suggest that K-ras mutations may be useful in distinguishing BDPs from metastases in the liver; however, there is some overlap in the mutational spectra of BDPs and pancreatic carcinomas.