Phenotypes of invasion in sporadic colorectal carcinomas related to aberrations of the adenomatous polyposis coli (APC ) gene.

AIMS: To determine whether the dissociation of tumour cells from neoplastic glands in colorectal carcinomas is caused by disruption of the wnt-signalling pathway and whether the adenomatous polyposis coli (APC) protein is implicated in this. METHODS AND RESULTS: In a series of 99 clinically sporadic colorectal carcinomas, APC exon 15 mutations, loss of heterozygosity (LOH) and promoter methylation were found in 49, 20 and 23 cases, respectively. Singly, these APC aberrations were not associated with the degree of tumour cell dissociation, but dissociation was higher for the cases with combined APC mutation and LOH. Immunohistochemical beta-catenin translocation to the nucleus correlated with APC aberrations. Tumour growth pattern (expansive/infiltrative/diffuse) and tumour stroma (desmoplastic common-type versus keloid-like) showed a statistically significant association with tumour cell dissociation and with beta-catenin translocation. Of other molecular alterations tested (p53 mutation; LOH at 17p13, 18q, 9p21; CpG island methylator phenotype), only the highly microsatellite unstable status (n = 11) was negatively associated. CONCLUSIONS: In colorectal carcinomas, wnt dysregulation relates to APC aberrations, but wnt dysregulation and APC aberrations are not strictly required for tumour cell dissociation, and additional and/or alternative factors must play a role. Of these, outside-in signalling by cancer cell-matrix interactions, as partially mirrored in histomorphological features, could be important.

p53 and K-ras mutations in pancreatic juice samples from patients with chronic pancreatitis.

BACKGROUND: Mutations in p53 and ras genes are frequent in pancreatic carcinoma. Several ras mutations are consistently detected in the pancreatic juice from patients with chronic pancreatitis. The p53 gene mutations have been detected occasionally in chronic pancreatitis tissue. It was the aim of this study to evaluate the presence and clinical significance of p53 and ras mutations in clinical pancreatic juice samples from patients with chronic pancreatitis. METHODS: Pancreatic juice was obtained from 66 patients with chronic pancreatitis and no evidence of pancreatic carcinoma (51 men, 15 women; age 17-86 years [mean 49.6 +/- 12.9]). Patients were followed prospectively for 26 +/- 3 (4-54) months. Detection of p53 gene mutations was by temperature gradient gel electrophoresis (TGGE) and single strand conformation polymorphism (SSCP) for exons 5-8. Analysis of ras mutations was performed by SSCP/polymerase chain reaction, restriction fragment length polymorphism/polymerase chain reaction. All mutations were confirmed by sequencing. RESULTS: Five of 66 (7.5%) pancreatic juice samples contained p53 mutations, and ras mutations were detected in 6 cases (9%). Cytology was negative in all cases. No pancreatic carcinoma developed during follow-up and neither cancer cells nor preneoplastic lesions could be detected histologically in resected specimens. Although no correlation between p53 mutations and duration of pancreatitis or drinking habits was found, K-ras mutations correlated with both heavy smoking and severity of the disease. CONCLUSION: p53 and ras mutations can be detected in a minority of pancreatic juice samples from patients with chronic pancreatitis in the absence of malignancy.

Low frequency of p53 and ras mutations in bile of patients with hepato-biliary disease: a prospective study in more than 100 patients.

The diagnosis of biliary disease, namely malignant disorders, is frequently hampered by the inconclusive cytology. We investigated prospectively the frequency of molecular changes in p53 and ras compared with cytology in patients with primary or secondary hepato-biliary disease. We investigated 118 consecutive patients, aged 24-89 with the following clinical diagnoses: choledocho/cholecystolithiasis (28), cholangiocellular carcinoma (21), gall bladder tumor (8), liver metastasis (3), autoimmune disease (8), chronic pancreatitis (16), pancreatic carcinoma (11), papillary disease (4), hepatic cirrhosis (6), cholangitis (2), anomalies (2), and normal (9). Bile was aspirated during routine endoscopic retrograde cholangio pancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC). DNA was prepared freshly from a native aliquot. p53 mutations were detected by polymerase chain reaction (PCR) for exons 5 through 8 followed by TGGE. PCR for ras mutations was performed as RFLP-PCR with sequencing. In four cases, mutations in p53 could be found in exons 6 and 7. Twenty-two samples showed ras mutations; ras mutations were found in choledocholithiasis (4/28), bile duct (5/21), gall bladder (3/8) and pancreatic (1/11) carcinoma, liver metastasis (3/3), ulcerative colitis (2/3), PSC (1/2), and chronic pancreatitis (1/16). Cytology was clearly positive in seven cases, suspicious in three other, inconclusive in six, and negative in the rest. The molecular analysis resulted in a sensitivity of 33% and specificity of 87%, respectively, for the diagnosis of a malignant condition. PCR for p53 and ras mutations may aid the diagnosis of primary and secondary (metastatic) hepatobiliary disease if a malignant condition of the bile ducts and the liver is suspected and cytology is inconclusive or negative. However, the incidence of p53 and ras mutations in bile seems less frequent than in other malignant conditions of the gastrointestinal tract and the pancreas and lower than in tissue, leaving a poor sensitivity and specificity. Nevertheless, the presence of a p53 and/or ras mutation per se supports a clinical suspicion of malignancy, even when the conventional cytology is negative or inconclusive.

p53 mutational spectra are different between squamous-cell carcinomas of the lip and the oral cavity.

We studied the p53 mutational spectra of 34 lip and 60 intra-oral squamous-cell carcinomas and examined possible etiological and prognostic correlations for these tumor sites. For the p53 analysis of exons 5-8, we used PCR/TGGE screening followed by DNA sequencing. Mutations were found in 18/34 (53%) lip and 22/60 (38%) intra-oral carcinomas. The p53 mutational spectrum of the intra-oral carcinomas comprised transitions and transversions in nearly equal frequency (11 to 10). In comparison, transitions were 3.5 times more frequent than transversions (14 to 4) in carcinomas of the lip. The predominant types of base change found in intra-oral tumors were G:C-to-T:A transversions and G:C-to-A:T transitions (32% each), while in lip tumors G:C-to-A:T transitions (70%) were the most frequent. The rate of lip tumors with mutations was higher in non-smokers (8/13) than in smokers (9/20). In contrast, p53 mutations in intra-oral tumors clustered in smokers (18/47 vs. 2/10). G:C-to-T:A transversions, regarded as tobacco smoke-associated in lung cancer, were found in 2 moderate and 4 heavy smokers with intra-oral cancer. This base substitution was found in none of our lip cancers. In lip tumors, a high rate of mutations occurred at dipyridine sites (13/18); among these were 8 C-to-T transitions and 1 CC-to-TT tandem base transition. These changes are characteristic of DNA damage caused by UV light. The presence of mutational events at the DNA-binding surface of the p53 protein may correlate with poor clinical outcome. However, we could not find any statistically significant correlations between p53 status and survival. Only the recurrence-free interval was significantly shortened in cases with mutations affecting residues of the DNA-binding surface of the p53 protein.

Anal verrucous carcinoma and penile condylomata acuminata.

A 50-year-old man presented with recurrent tumorous lesions on the penis and the anal region. The anal lesion was histologically diagnosed as verrucous carcinoma (VC) and the penile lesions were in line with condylomata acuminata. Samples taken from tumors of both sites were human papillomavirus (HPV) DNA positive. Two of them taken from the penis and the perianal region scored HPV DNA 6 positive by using polymerase chain reaction and the Southern blot method. Treatment of both VC and condylomata acuminata consisted in surgery and adjuvant immune therapy. Neither tumor recurrence nor metastases occurred up until 6 months after therapy.

[p53 mutations and HPV infections in squamous epithelial carcinomas of the head-neck region. Long-term follow-up]. / p53-Mutationen und HPV-Infektionen in Plattenepithelkarzinomen der Kopf-Hals-Region. Langzeitverlaufsstudie.

Mutations of the p53 gene are the most commonly observed genetic alterations in malignant tumors and are often associated with a loss of the tumor suppressor function of the p53 protein. We have analyzed specimens of head and neck squamous cell carcinomas (HNSCC) from 110 patients for p53 gene mutations and 92 of them additionally for human papillomavirus (HPV) infection in order to evaluate the prognostic significance of these factors by comparison with clinical follow-up data. Using the method of polymerase chain reaction (PCR)/temperature gradient gel electrophoresis (TGGE), mutations within the exons 5 to 8 of the p53 gene were found in 48 tumors (44%). Sequencing revealed missense mutations in most cases (15/20). Frequency of p53 gene mutations was not related to the tumor stage, the grade of differentiation, the presence of lymph node metastases, or the smoking history of the patients. With the help of a highly sensitive PCR/hybridization assay, an infection with the high-risk HPV types 16 and 18 could be detected in 39/92 tumor specimens (42%). Follow-up data were obtained from 99 patients with a range of 2-112 months. No correlation of overall survival on the presence of p53 gene mutations or HPV infection could be observed. The absence of statistically significant correlations between p53 gene mutations and progressive disease, however, does not exclude its putative relevance in early phases of tumor development.

p53 gene mutations and HPV infection in primary head and neck squamous cell carcinomas do not correlate with overall survival: a long-term follow-up study.

We analyzed specimens of head and neck squamous cell carcinomas (HNSCC) from 110 patients for p53 gene mutations, and 92 of them for human papillomavirus (HPV) infection, in order to evaluate the prognostic significance of these factors by comparison with clinical follow-up data. Mutations within the exons 5 to 8 of the p53 gene were found in 48 tumors (44%). Sequencing revealed in most cases mis-sense mutations (16/21). Frequency of p53 gene mutations was not related to the tumor stage or the presence of lymph node metastases. Of the 46 tumors that were analyzed by immunohistochemistry, 26 stained positively (56%). The number of positively stained nuclei increased slightly with decreasing differentiation of the tumors, whereas no correlation was found between tumor stage and immunoreactivity. An infection with the high-risk HPV types 16 and 18 could be detected in 39/92 tumor specimens (42%). Follow-up data were obtained from 99 patients within a range of 2 to 112 months. No dependence of overall survival on the presence of p53 gene mutations or HPV infection could be observed. The absence of statistically significant correlations between p53 gene mutation and progressive disease, however, does not deny its putative relevance in early phases of tumor development.

HPV DNA and p53 alterations in oropharyngeal carcinomas.

We have examined a series of 37 oropharyngeal squamous cell carcinomas for the presence of HPV 6/11, 16, and 18 DNA by polymerase chain reaction (PCR)/Southern blotting and for p53 alterations by immunohistochemistry and mutation screening with temperature gradient gel electrophoresis (TGGE). HPV sequences were found in a total of 26 of 37 cancers (70.3%), most frequently HPV 16 (20/37) followed by HPV 18 (11/37). Double infections with HPV 16 and 18 were present in 5 tumours. p53 accumulation was detectable immunohistochemically in 21 of 37 carcinomas (56.8%). There were remarkable differences in the distribution of immunoreactive tumour cells in relation to the tumour grade. A mutation screening for p53 by TGGE, directed to the amplified exons 5-8, revealed p53 mutations in 14 of 37 carcinomas (37.8%). Mutations in two different exons were present in 3 tumours, 11 tumours being hit once. Exon 7 was mutated in 6 carcinomas, exons 5 and 8 in 4 cases, and exon 6 in 3 cases. When grouping the tumours with p53 mutation according to their HPV state, HPV-positive cases showed slightly more mutations (11/26) than HPV-negative cases (3/11). Only 5 of 37 carcinomas (13.5%) contained neither HPV DNA nor p53 alterations. Our results indicate that high-risk HPV and p53 mutations frequently coexist in oropharyngeal carcinomas, in contrast to genital tumours, notably carcinomas of the cervix uteri. This may reflect different pathways in carcinogenesis in squamous cell epithelium from different sites.

Human papillomavirus DNA in oral squamous cell carcinomas and normal mucosa.

Human papillomavirus (HPV) infections in oral carcinomas and normal oral mucosa were studied by consensus primer screening and typing for HPV types 6/11, 16 and 18 DNA. After polymerase chain reaction (PCR) the DNA species of interest were identified by Southern blot hybridization with digoxigenin-labeled oligonucleotide probes. Frozen tissue and scrapings were equally suitable for HPV testing and yielded high HPV detection rates in carcinomas. By comparison, HPV analysis of paraffin-embedded material was much less efficient. HPV were demonstrated in 61.5% (16/26) of oral squamous cell carcinomas, high risk HPV 16 and 18 being the preferential types. The frequency of HPV detection in non-neoplastic mucosa of tumor patients decreased clearly with increasing distance from the tumor (range 26.9-3.8%) suggesting focal HPV infections. In contrast, normal buccal mucosa of a group of healthy volunteers contained HPV DNA only in 1% (1/97).

[p53 alterations and HPV status in oral squamous cell carcinomas]. / p53 Alterationen und HPV Status in oralen Plattenepithelkarzinomen.

Immunohistochemical examination of p53 protein accumulation, analysis of the p53 gene using amplification of the exons 5-8 followed by TGGE, and PCR based HPV typing were carried out on 40 oral squamous cell carcinomas. 28 of 40 oral carcinomas (70%) contained DNA of the oncogenic HPV types 16 and 18. A p53 accumulation was present in 20 of 37 tumors (54%). Mutations of the p53 gene were detected in 15 of 39 carcinomas (38%). 15 of 20 oral carcinomas with abnormal p53 expression and 11/15 tumors with mutations of the p53 gene were HPV positive. Our results demonstrate a frequent occurrence of p53 abnormalities in HPV positive oral squamous cell carcinomas. This pattern is divergent from HPV related genital carcinomas and could reflect differences in the etiology of squamous cell carcinomas of different sites.

Binding studies of gold labelled lectins on carbohydrate compounds of the flask cells in claw-frog kidney.

The carbohydrate compounds of the mucus of flask cells in the kidney of claw-frogs (Xenopus laevis) were studied by gold marked lectins (WGA, RCA, L, LCA, HPA, PNA). We used a post-embedding technique. Seminthin or ultrathin sections of Lowieryl K H M-embedded kidney tissue were incubated. For light microscopy, a gold-silver technique was used. The mucus of the flask cells reacted strongly with WGA, RCA L and HPA, whereas LCA and PNA showed no binding. The Golgi apparatus and small cytoplasmatic vesicles reacted also positively with WGA, RCA L and HPA. The autoradiographically detected secretion routes of the glycosaminoglycan-rich secretion of flask cells are also demonstrable by lectins.

Light and electron microscopic studies of lectin binding on the glycocalyx of rat pancreatic cells. I. Normal tissue and isolated cells.

Lectin binding of the glycocalyx of pancreatic tissue sections as well as of isolated pancreatic acini and acinar cells was studied of healthy wistar rats by light and electron microscopy. For light microscopy, we used FITC (WGA, RCAI, LCA) or peroxidase marked (WGA, RCAI, PNA, PHA, LCA, UEAI, LPA) as well as unmarked lectins (Con A, VAA I). Gold marked lectins were used for electron microscopy (WGA, RCAI, LCA, HPA, PNA, VAAI-B). Intact acinar cells in pancreatic tissue sections and isolated acini showed a strong binding of WGA, RACI, and HPA on the apical cell surface, whereas VAAI, UEAI, LCA, and Con A reacted strongly with the basolateral glycocalyx, but not with the apical surface. The 2 main domains of the glycocalyx of pancreatic cells showed their specific lectin binding so long as the junctional complexes between the cells are intact. The polarity of the cell surface of pancreatic acinar cells is discussed in regard to the possible function of the 2 domains.

Lectin binding on carbohydrate compounds of the flask cells in the claw-frog kidney.

The carbohydrate compounds of the mucus of flask cells in the kidney of claw-frogs (Xenopus laevis) were analysed through lectin binding studies. After removing epoxy resin semithin sections were incubated with 7 lectins (WGA, RCA I, PNA, LCH, UEA, LPA) marked by horseradish peroxidase and 2 unmarked lectins (VAA, Con A). The glycosaminoglycans in the canalicular lumen of flask cells showed a strong reaction with WGA and RCA, whereas the binding of PHA, Con A, and LCH was weaker. No reaction was observed with PNA, UEA, LPA, and VAA. The mucus of the flask cells seems to be rich in N-acetyl-glycosamine and -galactosamine. It contains also mannose, glucose, and galactose, but seems to have no fucose or N-acetyl-sialic acid residues.