p53 alterations in chemically induced hamster cheek-pouch lesions

To confirm that the hamster cheek-pouch carcinogenesis model reflects development of human squamous cell carcinoma (SCC), we determined if and when p53 mutations occur in the development of SCC in this model by using immunohistochemical staining and polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis plus direct DNA sequencing. Twenty-four hamster cheek-pouches were treated with a solution of 0.5% 7,12-dimethylbenz[a]anthracene in mineral oil three times a week for 16 wk. The malignant endophytic and exophytic tumors induced with this protocol are preceded by a sequence of premalignant lesions such as hyperplasia with or without dysplasia and carcinoma in situ, similar to the development of this cancer in humans. For this study, p53 protein accumulation was evaluated by immunostaining of various hamster cheek-pouch exophytic and endophytic SCCs as well as flat dysplastic hyperplasia and carcinomas in situ. A moderate percentage (33.3%) of exophytic lesions and most endophytic carcinomas (90%) showed positive p53 staining. In addition we also found p53-positive staining in a number of preneoplastic lesions, including areas of focal hyperplasia, dysplastic hyperplasia, and carcinomas in situ. To determine whether the alterations in p53 staining were due to p53 gene mutation, we used PCR-SSCP analysis and direct sequencing. PCR products corresponding to exons 5a, 6, 7, and 8 from 40 tumors with the highest percentage of p53-stained cells were analyzed. We detected shifted bands in 17 lesions. Direct sequencing of eight selected shifted bands revealed four mutations, including two G→T transversions in codons 216 (tumor #1) and 252 (tumor #2) and one G→C transversion in codon 282 (tumor #3). Tumor #4 contained a frameshift mutation in codon 251. These mutations are consistent with those reported in many human cancers. Therefore, we concluded that in the hamster cheek-pouch model, p53 protein accumulation occurs frequently and early in carcinogenesis, as it does in human SCCs, and some of these p53 alterations are due to p53 gene mutations. These findings may help us better define the mechanisms of carcinogenesis in the hamster cheek-pouch model, and p53 alterations may be an early biomarker of progression for chemoprevention studies. © 1996 Wiley-Liss, Inc.     

Sequential p53 mutation analysis of pre-invasive and invasive head and neck squamous carcinoma

Single-stranded conformation polymorphism (SSCP) and direct sequencing were performed on uninvolved mucosa, severe dysplasia and invasive carcinoma samples from 20 patients with head and neck squamous carcinoma. Seven (35%) of the non-invasive lesions and 15 (75%) of the invasive carcinomas manifested p53 mutations. Although the majority of mutations were mis-sense, resulting in single amino acid substitution, a silent mutation encoding for the same amino acid and 2 non-sense mutations encoding a stop codon were also observed. Mutations in invasive carcinoma were mostly in exon 8 and involved codons 296, 288 and 298; non-invasive lesions showed more mutations at exons 5 to 7. Five lesions showed simultaneous mutations in 2 different exons; in 3 both non-invasive and invasive carcinomas showed primary mutation at exons 5 to 7, and invasive carcinoma showed a secondary mutation at exon 8. Different codon mutations in the same exon between dysplastic and the corresponding carcinoma samples were found in 2 cases. p53 alterations were not observed in any of the normal mucosa samples. No apparent association between p53 mutations and conventional clinicopathologic parameters, including DNA content, was found in this cohort. Our study indicates that (i) p53 alteration is an early event in the genesis of a subset of head and neck squamous carcinomas, (ii) normal mucosa within the resected specimens lacked p53 mutation, (iii) sequential mutations of different exons of the p53 gene suggests accumulation of genetic alterations during the neoplastic transformation of these lesions and (iv) the difference in codon mutation of the same exon between dysplastic and corresponding carcinoma suggests an independent clonal development. © 1995 Wiley-Liss, Inc.     

The p53 status of cultured human premalignant oral keratinocytes.

Around 60% of oral squamous cell carcinomas (SCCs) have been shown to harbour p53 mutations, and other studies have demonstrated mutant p53 genes in normal and dysplastic squamous epithelium adjacent to these SCCs. In line with these earlier studies we show here that DOK, a keratinocyte cell line derived from a dysplasia, displays elevated levels of p53 protein and harbours a 12 bp in-frame deletion of the p53 gene spanning codons 188-191. In contrast, the coding region of the p53 gene was normal in a series of six benign recurrent laryngeal papillomas and a series of four premalignant oral erythroplakia biopsies and their cell cultures. All but one of these lesions were free of malignancy at the time of biopsy, in contrast to the premalignant lesions studied by previous investigators, but keratinocytes cultured from these lesions all displayed a partially transformed phenotype that was less pronounced than that of DOK. Since three out of four of the erythroplakia patients developed SCC within 1 year of biopsy, these lesions were by definition premalignant. The availability of strains of partially transformed keratinocytes from premalignant erythroplakias which possess normal p53 genes should enable us to test the role of mutant p53 in the progression of erythroplakia to SCC. The premalignant tissues and cultures were also tested for the presence of human papillomavirus (HPV), which is known to inactivate p53 function in some cases. Only the benign papillomas were shown to contain high levels of either HPV 6 or HPV 11 E6 DNA, but not both, and none of the samples contained detectable levels of HPV 16, HPV 18 or HPV 33 E6 DNA or L1 DNA of several other HPV types. There was therefore no evidence to suggest that p53 was being inactivated by a highly oncogenic HPV in these samples.     

Premalignant lesions of the oral mucosa. A discussion about the place of oral intraepithelial neoplasia (OIN)

Oral precancerous lesions are traditionally classified as leukoplakia, erythroplakia, erythroleukoplakia, and distinguished from precancerous conditions. Major attention is focused on leukoplakia, and no distinction made whether dysplasia is or not present. Malignant transformation is a multistep process that should be approached also from the histological, and not merely from the clinical standpoint. Intraepithelial neoplasia, a notion created for the uterine cervix and already extended to other mucosae, should be adapted to the oral mucosa and used as diagnostic term. OIN (oral intraepithelial neoplasia) is not only a change in terminology, but also a progress in the unifying concept of precursors of squamous cell carcinoma, suppressing the useless discussion between severe dysplasia and carcinoma in situ. Furthermore, grading lesions as low or high grade OIN increases diagnostic consistency. OIN is suspected on three clinical patterns reflecting histological changes: mosaic, irregular keratosis, erythroplakia (or intermediate aspects), but dysplastic mucosa may also appear normal clinically.     

p53 expression in oral cancer: observations of a South Indian study

Oral cancers constitute a significant proportion of hospital admissions among cancer patients in India. The aim of this study was to elucidate the role of tumour suppressor gene p53 in the pathogenesis of oral squamous cell carcinoma by immunohistochemistry. Though extensive studies on p53 alterations in oral cancers have been done in Western countries and some Asian countries, only a few studies have emerged from India, especially the Southern states. This study would therefore be helpful in providing an Indian perspective, with particular reference to South Indian states. A total of 110 cases of oral squamous cell carcinoma, 35 dysplastic lesions, 15 hyperplastic lesions, and 50 samples of normal mucosa were assessed for p53 expression. Out of 110 cases of oral carcinoma, 40 (36%) were p53 positive. Among 35 cases of dysplastic lesions studied, 6 (17%) showed p53 positive staining. None of the hyperplastic lesions and normal oral lesions showed any evidence of p53 positivity. However, in 9 out of 40 (23%) cases of positive infiltrating squamous cell carcinomas, the adjacent or overlying non-tumourous epithelium demonstrated focal areas of p53 positive staining in the basal and parabasal layers of the epithelium. In addition, in 7 out of 110 (6%) cases, cytoplasmic staining was observed. In these samples, nuclei were not stained. Our results indicate that p53 over-expression may be involved in only a certain proportion of oral carcinomas. The fact that 6% of the dysplastic lesions were p53 positive, and adjacent non-tumourous epithelium of 23% infiltrating squamous cell carcinomas showed positive staining for p53 in the progenitor compartment of the epithelium indicates that p53 immunoreactivity could be used to detect early tumours as well.     

p53 mutations in cutaneous lesions induced in the hairless mouse by a solar ultraviolet light simulator

We investigated skin lesions induced in hairless SKH:HR1 mice by chronic exposure to a solar ultraviolet light (UV) simulator for alterations of the p53 gene in conserved domains. Mutations of exons 5–8 of the p53 gene in skin lesions were screened in 31 benign skin lesions (hyperplasias), 25 precancerous skin lesions (keratoacanthomas), and 25 malignant skin lesions (squamous cell carcinomas; SCC) by polymerase chain reaction–single-strand conformation polymorphism analysis. Most of the mutations occurred at dipyrimidine sequences located on the nontranscribed strand; the most frequent modifications were C→T transitions (77%) and CC→TT tandem mutations (5%); the latter are considered the UV fingerprint. p53 mutations were detected in 3% of the hyperplasias, 12% of the keratoacanthomas, and 52% of the SCCs. Hence, the high frequency of p53 mutations in SCCs compared with keratoacanthomas induced by a solar UV simulator suggested that, in our study, p53 mutations probably occurred as a late event in the skin carcinogenesis progression of SCC. Interestingly, the level of CC→TT tandem mutations in the SCCs (5%) was similar to that found in SCCs induced in hairless mice by UVB alone. p53 protein was also detected in the different types of skin lesions by immunohistochemical analysis. Thus, our data from hairless mouse skin tumors induced by a solar UV simulator confirmed the major role of UVB-induced DNA damage in skin carcinogenesis and suggested that UVA plays a minor role in bringing about p53 alterations. Mol. Carcinog. 22:167–174, 1998. © 1998 Wiley-Liss, Inc.     

Human papillomavirus and p53 mutations in head and neck squamous cell carcinoma among Japanese population

We aimed to reveal the prevalence and pattern of human papillomavirus (HPV) infection and p53 mutations among Japanese head and neck squamous cell carcinoma (HNSCC) patients in relation to clinicopathological parameters. Human papillomavirus DNA and p53 mutations were examined in 493 HNSCCs and its subset of 283 HNSCCs. Oropharyngeal carcinoma was more frequently HPV‐positive than non‐oropharyngeal carcinoma (34.4% vs 3.6%, P < 0.001), and HPV16 accounted for 91.1% of HPV‐positive tumors. In oropharyngeal carcinoma, which showed an increasing trend of HPV prevalence over time (P < 0.001), HPV infection was inversely correlated with tobacco smoking, alcohol drinking, p53 mutations, and a disruptive mutation (P = 0.003, <0.001, <0.001, and <0.001, respectively). The prevalence of p53 mutations differed significantly between virus‐unrelated HNSCC and virus‐related HNSCC consisting of nasopharyngeal and HPV‐positive oropharyngeal carcinomas (48.3% vs 7.1%, P < 0.001). Although p53 mutations were associated with tobacco smoking and alcohol drinking, this association disappeared in virus‐unrelated HNSCC. A disruptive mutation was never found in virus‐related HNSCC, whereas it was independently associated with primary site, such as the oropharynx and hypopharynx (P = 0.01 and 0.03, respectively), in virus‐unrelated HNSCC. Moreover, in virus‐unrelated HNSCC, G:C to T:A transversions were more frequent in ever‐smokers than in never‐smokers (P = 0.04), whereas G:C to A:T transitions at CpG sites were less frequent in ever‐smokers than in never‐smokers (P = 0.04). In conclusion, HNSCC is etiologically classified into virus‐related and virus‐unrelated subgroups. In virus‐related HNSCC, p53 mutations are uncommon with the absence of a disruptive mutation, whereas in virus‐unrelated HNSCC, p53 mutations are common, and disruptive mutagenesis of p53 is related with oropharyngeal and hypopharyngeal carcinoma.     

KRAS and GNAS mutations and p53 overexpression in biliary intraepithelial neoplasia and intrahepatic cholangiocarcinomas

BACKGROUND:

Similar to the pancreatic intraepithelial neoplasia (PanIN)‐pancreatic carcinoma sequence model, intrahepatic cholangiocarcinoma (ICC) also reportedly follows a stepwise carcinogenesis process through the a precursor lesion: biliary intraepithelial neoplasia (BilIN). For this study, the authors investigated the status of v‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and GNAS complex locus (GNAS) mutations and tumor protein 53 (p53) overexpression in the stepwise process of cholangiocarcinogenesis.

METHODS:

Thirty patients with hepatolithiasis were surveyed, and their lesions were categorized as follows: non‐neoplastic large bile duct (LBD) (n = 12), peribiliary gland (PBG) (n = 9), BilIN‐1 (low‐grade dysplasia; n = 12), BilIN‐2 (high‐grade dysplasia; n = 16), and BilIN‐3 (noninvasive or in situ carcinoma; n = 10). KRAS mutation at codons 12 and 13 and GNAS mutations at codons 601 and 602 were analyzed using genomic DNA extracted from isolated lesions by laser capture microdissection. Immunohistochemical expression of p53 also was evaluated in BilIN lesions, ICCs, and extrahepatic cholangiocarcinomas (ExCCs).

RESULTS:

A prevalence of KRAS mutations was identified in patients with ICC (31.5%), BilIN‐3 (30%), and BilIN‐2 (43.8%) compared with BilIN‐1 (25%). Furthermore, KRAS mutations were detected in LBD lesions (41.7%) and PBG lesions (44.4%), and these mutations were observed with greater frequency in patients who had BilIN with KRAS mutations. GNAS mutations were not identified in any of the ICCs or other lesions examined. The overexpression of p53 was not identified in BilIN lesions and was less frequent in ICCs (18.2%) compared with ExCCs (38.1%) and gallbladder carcinomas (61.5%).

CONCLUSIONS:

KRAS mutations, which were present in approximately 33% of BilIN lesions, occurred as an early molecular event during the progression of BilIN to ICC, whereas p53 overexpression was identified as a late molecular event. Furthermore, the current results indicted that BilIN also may arise from LBD and PBG lesions in patients who have hepatolithiasis with KRAS mutations. Cancer 2013. © 2013 American Cancer Society.     

Human papilloma viruses and p53 mutations in normal,pre-malignant and malignant oral epithelia

HPV infections have been previously observed in oral cancers, and inactivation of the p53 gene has been shown to be one of the most common genetic alterations in human tumors. We examined 179 oral specimens from 70 individuals with histologic findings of either normal mucosa (n = 6) or oral disease that ranged from mild dysplasia to invasive squamous-cell carcinoma (n = 64) to determine the occurrence of both HPV infection and p53 mutations and their relationship with several clinical factors. HPV infection was detected by PCR amplification of viral DNA, and the presence of p53 mutations was assayed using the single-strand conformation polymorphism (SSCP)-PCR technique. HPV infection was found in 31% of individuals with oral disease and was not seen in healthy individuals. Mutations in exons 5, 6, 7 or 8 of the p53 gene were detected in 37.5% of patients with oral lesions and in a biopsy from 1 healthy individual who was a heavy smoker. Approximately one-third of lesions classified as pre-malignant (dysplasia and carcinoma in situ) and 42% of invasive carcinomas contained p53 mutations. The majority of these mutations were G:T transversions located within exons 7 and 8. Tumor tissues from 6 patients with oral lesions were found both to be HPV-16-positive and to contain p53 mutations; of these, 4 were poorly differentiated carcinomas that were diagnosed as late-stage disease. In this study, p53 mutations were detected in the early stages of cancer development. © 1996 Wiley-Liss, Inc.     

Correlation between p53 gene mutations and circulating antibodies in betel- and tobacco-consuming North Indian population

Alterations in p53 tumour suppressor gene and its expression may be implicated in the pathogenesis of betel- and tobacco-related oral cancer. There is wide regional variation in betel- and tobacco-consuming habits in different parts of the Indian subcontinent. The purpose of this study was to determine the correlations between p53 gene mutations, protein accumulation and serum antibodies in oral precancer and cancer. We analysed 30 potentially malignant oral lesions (leukoplakia) and 30 oral squamous cell carcinomas (SCCs) from northern India because the betel quid-consuming habits are different from those prevalent in other regions of India. p53 mutations were analysed by polymerase chain reaction amplification of genomic DNA and direct sequencing, p53 protein accumulation by immunohistochemical analysis and circulating p53 antibodies by ELISA. p53 gene mutations, analysed within exons 5-9, were observed in five out of 30 (17%) potentially malignant oral lesions and seven out of 30 (23%) oral SCCs. All the mutations were base substitution mutations. Three missense and two nonsense mutations were observed in potentially malignant oral lesions, while six missense and one nonsense mutations were identified in oral SCCs. The probable hot spots for the mutations were identified at codons 126, 136 and 174, which have not been observed thus far. A good correlation was observed between p53 missense mutation, p53 antibodies and p53 protein accumulation in matched potentially malignant and malignant oral lesions. All the potentially malignant and cancerous lesions harbouring missense mutations showed accumulation of p53 protein and the majority of these patients showed circulating p53 antibodies suggesting that serological detection of p53 antibodies may serve as a surrogate marker for p53 alterations in oral lesions.     

Absence of p53 mutations in benign and pre-malignant male genital lesions with over-expressed p53 protein

Mutations of the tumor-suppressor gene p53 are common in epithelial tumors. Clonal mutations of p53 have been found in cervical and vulvar carcinomas negative for human papillomavirus (HPV), though at least in cervical cancer HPV infection and p53 mutations are not mutually exclusive. We have previously shown that about 40% of male genital warts and bowenoid papulosis lesions exhibit immunohistochemically detectable aberrant p53 protein, irrespective of the presence of HPV DNA. We studied p53 mutations in exons 4–8 with SSCP and sequencing in 13 male patients with 1 to 3 therapy-resistant genital warts or intra-epithelial neoplasias each and in 4 patients with penile squamous cell carcinoma. Thus, 13 genital warts, 6 bowenoid papulosis, 1 Queyrat's erythroplasia and 1 carcinoma in situ were studied. p53 protein was detected immunohistochemically, and HPV status was analyzed with DNA in situ hybridization and amplification of HPV-specific DNA. There was no correlation between p53 protein expression and HPV status. No mutations in exons 5–8 of the p53 gene were found in any of the lesions, and furthermore, no exon 4 mutations were found in lesions positive in p53 immunohistochemistry. In conclusion, over-expression of p53 does not indicate a p53 mutation in male genital warts, pre-malignant lesions or malignant squamous cell carcinomas. Our study thus suggests that p53 mutations are not important, or at least not early, events in male genital carcinogenesis. Int. J. Cancer 77:674–678, 1998. © 1998 Wiley-Liss, Inc.     

Infrequent Ha-ras mutations and absence of Ki-ras,N-ras,and p53 mutations in 4-nitroquinoline 1-oxide-induced rat oral lesions

The alkylating agent 4-nitroquinoline 1-oxide (4-NQO) is a powerful carcinogen and induces squamous cell hyperplasia, squamous cell dysplasia, papilloma, and squamous cell carcinoma (a) in rat oral epithelia. Oral cancers induced by a single application of 4-NQO develop through a multistage process in a way similar to the development of this cancer in humans. In this study, mutations in exons 1 and 2 of Ki-ras, N-ras, and Ha-ras and exons 4–7 of p53 were examined by polymerase chain reaction (a) -single strand conformation polymorphism (a) analysis, followed by PCR-direct sequencing for the confirmation of mutations. Samples for the mutation analysis were obtained from dysplasias, papillomas, and SCCs on the tongue epithelia induced in F344 rats by adding 4-NQO (20 ppm) to their drinking water for 8 wk. The Ha-ras mutations (⁶¹A→T transversions in the second position) were found in five of 29 (17%) samples (one dysplasia and four SCCs). However, no mutations were detected in either Ki-ras, N-ras, or p53 under two different conditions of PCR-SSCP analysis. We suggest that some neoplasms in oral carcinogenesis induced by 4-NQO may involve Ha-ras mutations but not mutations in Ki-ras, N-ras, or p53. The 4-NQO-induced rat oral carcinogenesis model may provide a system for evaluation of the mechanisms of multistage oral carcinogenesis associated with Ha-ras mutation without Ki-ras, N-ras, or p53 mutation. © 1995 Wiley- Liss, Inc.     

重组人p53腺病毒注射液治疗口腔白斑诱导病变细胞凋亡的研究

 目的　观察重组人p53腺病毒注射液（rAd-p53）治疗上皮异常增生型口腔白斑引起的细胞凋亡改变。方法　对18例上皮异常增生型口腔白斑患者进行rAd-p53局部黏膜内注射治疗15 d。观察rAd-p53注射后病变组织的组织病理学改变,并通过免疫组化观察细胞中p53蛋白、bcl-2蛋白的表达水平,应用TUNEL染色观察细胞凋亡情况。结果　rAd-p53治疗后组织内p53蛋白和bcl-2 蛋白的阳性表达率分别为100 %（18／18）和17 %（3／18）,二者阳性表达呈负相关（r＝-0.837,P＜0.01）。TUNEL染色发现,在83 %（15／18）的组织中检测到凋亡细胞,尤其见于p53蛋白强阳性表达的组织（r＝0.684,P＜0.01）。结论　rAd-p53局部注射能诱导异常增生上皮细胞凋亡,对口腔白斑治疗有良好的临床应用前景。     

P‐glycoprotein is positively correlated with p53 in human oral pre‐malignant and malignant lesions and is associated with poor prognosis

P‐glycoprotein (Pgp) encoded by the MDR1 gene, a predictor of chemoresistance, may also serve as a prognosticator of clinical outcome in cancer patients. The mutant tumour‐suppressor p53 protein has been shown to activate the MDR1 promoter, whereas the wild‐type p53 represses this activity in cultured cells. We have described the differential expression of Pgp and p53 proteins in betel‐ and tobacco‐related oral tumorigenesis in the Indian population. Herein, Pgp expression was analysed in relation to p53 protein accumulation in pre‐malignant and malignant oral lesions by immuno‐histochemical and flow‐cytometric analyses. The relationship between Pgp and p53 protein accumulation and clinico‐pathological parameters as well as prognosis was determined. Expression of Pgp was observed in 81% of oral squamous cell carcinomas (SCCs) and 71% of pre‐malignant lesions. Sixty‐five of 75 p53‐positive oral SCCs and 21/24 p53‐positive pre‐malignant lesions showed expression of Pgp. Significant correlation between Pgp and p53 expression was found not only in oral SCCs but also in pre‐malignant lesions. Co‐expression of Pgp and p53 proteins was indicative of poor prognosis. Follow‐up studies of 35 patients showed that 7 of 10 oral SCCs with accumulation of Pgp and p53 proteins also exhibited shorter disease‐free survival (recurrence/metastases). Our findings provide clinical evidence for a significant association between Pgp and p53 protein expression in oral tumorigenesis and may account for the aggressive nature of the tumour and poor prognosis. Int. J. Cancer (Pred. Oncol.) 84:80–85, 1999. © 1999 Wiley‐Liss, Inc.     

Serum p53 antibodies in patients with oral lesions: Correlation with p53/HSP70 complexes

The functional significance of alterations in expression of tumour-suppressor gene p53 and 70-kDa heat-shock protein (HSP70) in eliciting p53-specific humoral response in oral-cancer patients is not yet known. In this study, p53 auto-antibodies were analyzed in sera from oral-cancer patients by immunoblotting and results were correlated with clinicopathological features of the patients as well as with the levels of HSP70, p53 and p53-HSP70 complexes in matched patients' tumour tissue, for determining their diagnostic/prognostic significance and relationship to survival. Circulating anti-p53 antibodies were observed in 7 of 30 cancer patients and 3 of 25 patients with pre-malignant lesions. Over-expression of p53 protein in matched oral lesions was observed in 22 of these 30 cancer patients and 14 of 25 patients with dysplastic lesions. No detectable levels of p53 protein or anti-p53 antibodies were observed in normal subjects (15 cases). Elevated levels of HSP70 were observed in 23 of these 30 oral tumours and 17 of 25 dysplastic lesions. All the anti-p53-antibody-seropositive cases showed elevated levels of p53 and HSP70 proteins, as well as formation of p53-HSP70 complexes, in matched dysplastic or malignant lesions, suggesting that these molecular alterations may be early events in oral tumorigenesis and are implicated in eliciting p53-specific humoral immune response in these patients. Anti-p53-antibody-seropositive cases showed poor prognosis and significantly decreased overall disease-free survival in comparison with the seronegative cases. Detection of circulating anti-p53 antibodies may serve as a useful non-invasive marker for identifying oral tumours having poor prognosis. Int. J. Cancer 74:609–613, 1997.© 1997 Wiley-Liss, Inc.     

Immunohistochemical detection of p53 in non-malignant and malignant oral lesions associated with snuff dipping in the Sudan and Sweden

Immunohistochemistry was used to examine the expression of p53 in pre-malignant oral lesions and oral squamous-cell carcinomas (SCCs) from Swedish and Sudanese snuff-dippers, as well as in pre-malignant oral lesions and oral SCCs from non-snuff-dippers from the Sudan, Sweden and Norway. Of the 14 SCCs from Sudanese snuff-dippers, 21% (3/14) expressed p53. Of the 14, 60 and 41 SCCs from non-snuff-dippers from the Sudan, Sweden and Norway, 64% (9/14), 65% (39/60) and 68% (28/41) expressed p53, respectively. A statistically significant difference in expression of p53 was found in SCCs from Sudanese snuff-dippers compared to those from non-snuff-dippers from all/or any of the 3 countries. None of the suspected pre-malignant oral lesions from Sudanese snuff dippers or non-snuff-dippers expressed p53. Only 2 out of the 15 oral fibro-epithelial hyperplastic lesions from Swedish snuff-dippers expressed p53. Some of the oral epithelial dysplastic lesions, as well as the carcinoma in situ lesions from Norwegian non-snuffdippers, expressed p53, while the oral fibro-epithelial hyperplastic lesions did not. The low relative frequency of p53 expression found in oral SCCs from snuff-dippers compared to those from non-snuff-dippers might suggest differences in mechanisms of oncogenic action induced by snuff. Alternatively, the pathogenesis of malignant oral lesions from snuff-dippers may follow a p53-independent pathway. In view of the unusually high levels of the tobacco-specific nitrosamines (TSNA) found in the type of snuff used in the Sudan, investigations of p53 mutations or oncogenes are needed. © 1996 Wiley-Liss, Inc.     

Frequent mutations of Ki-ras but no mutations of Ha-ras and p53 in lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine in rats

Point mutations of the Ki-ras and p53 genes in rat lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated by polymerase chain reaction-single-strand conformation polymorphism analysis followed by direct sequencing using paraffin-embedded tissues. Male Wistar rats 6 wk old were given 2000 ppm BHP in drinking water for 15 wk. Another group was given drinking water without BHP. The rats were killed 20–27 wk after the beginning of the experiment. Lung adenomatous and squamous lesions, including carcinomas, were induced. The frequencies of Ki-ras mutations were 40% (six of 15) in alveolar hyperplasias, 36% (five of 14) in adenomas, 72% (18 of 25) in adenocarcinomas, 20% (three of 15) in squamous metaplasias, 50% (three of six) in squamous cell carcinomas, and 50% (five of 10) in adenosquamous carcinomas. The mutations were all G → A transitions at the second position of codon 12; no other mutations were detected. However, Ha-ras mutations in exons 1 and 2 and p53 mutations in exons 5, 6, and 7 were not detected in adenocarcinomas and squamous cell carcinomas. These results indicate that Ki-ras mutation is an early genetic event in some adenomatous and squamous lung carcinogenesis and that Ki-ras mutations can cause benign lesions to convert to malignant lesions. The results also show that Ha-ras and p53 mutations are not involved in rat lung carcinogenesis induced by BHP. © 1996 Wiley-Liss, Inc.     

MDM2/p53 co-expression in oral premalignant and malignant lesions: potential prognostic implications.

The expression of MDM2 protein in betel and tobacco related oral malignancies in Indian population, its relationship to clinicopathological parameters and p53 protein expression was investigated. Sixty five oral squamous cell carcinomas (SCCs), 33 premalignant lesions (leukoplakia) and 30 normal oral tissues were assessed by immunohistochemical analysis. MDM2 protein was overexpressed in 51/65 (78%) oral SCCs and 17/33 (52%) premalignant lesions; 11/23 hyperplastic lesions and 6/10 dysplastic lesions. mdm2 gene amplification is an infrequent event in oral tumorigenesis. Elevation in the level of MDM2 protein not only in oral SCCs but also in premalignant lesions suggests that altered MDM2 expression is an early even in the pathogenesis of oral neoplasia. The hallmark of the study was the significant association of MDM2 expression with the p53 protein accumulation in 16/33 (49%) oral premalignant lesions (p = 0.001) and 39/65 (60%) malignant lesions (p = 0.021), suggesting an active role for MDM2 in binding and inactivating p53 in oral tumorigenesis. Further, significant association of MDM2/p53 co-expression was observed with advanced tumour stage (p = 0.0009), as well as lymph node metastasis (p = 0.0325) features associated with aggressive tumour behaviour and poor prognosis. Discordant MDM2+/p53-phenotype was observed in 12/65 (18%) oral SCCs suggesting a p53-independent role for MDM2 in the pathogenesis of a subset of oral carcinomas. In conclusion, alterations in MDM2 and p53 expression are early events likely to be involved in preinvasive stages in oral tumorigenesis and may be indicative of a 'gain of function' phenotype with more aggressive characteristics.     

Overexpression and mutations of p53 in metastatic malignant melanomas

Alterations of the p53 tumor suppressor gene are the most frequent genetic abnormalities in human malignancies, but the role of p53 in the etiology of malignant melanomas is unclear. Fifty unselected malignant melanomas were analyzed for p53 overexpression by immunohistochemistry using 3 monoclonal antibodies (MAbs). Fifteen tumors (29.4%) showed positive staining with at least 2 different antibodies. In the first 20 consecutive tumors exons 5–9 and adjacent splice sites of the p53 gene were analyzed by genomic sequencing. There were 4 mutations in 20 metastatic melanomas. Three of 4 mutations were C:G → T:A transitions. A search of our database of p53 mutations revealed that out of 8 p53 mutations reported by others, 4 are C:G → T:A transitions at dipyrimidine sites, and one is a tandem CC → TT mutation. This mutational pattern is comparable with the pattern of p53 mutations in squamous cell and basal cell carcinomas of the skin and is related to exposure to ultraviolet B (UV-B) wavelength radiation. Taken together with a predominance of UV-induced mutations in the CDKN2/p16 gene demonstrated in melanoma cell lines, our data support a role of sunlight exposure in the etiology of malignant melanoma. The low frequency of p53 mutants in melanomas compared with other types of skin cancers suggests that although mutations in this gene are likely to be involved in the development of some malignant melanomas, they do not play as large a role as in squamous and basal cell carcinomas of the skin. © 1996 Wiley-Liss, Inc.     

Receptor tyrosine kinase amplification is predictive of distant metastasis in patients with oral squamous cell carcinoma

This study aimed to clarify the genomic factors associated with the diagnosis and prognosis of oral squamous cell carcinoma via next‐generation sequencing. We evaluated data from 220 cases of oral squamous cell carcinoma. Genomic DNA was eluted using formalin‐fixed, paraffin‐embedded samples, and targeted resequencing of 50 cancer‐related genes was performed. In total, 311 somatic mutations were detected in 220 patients, consisting of 68 synonymous mutations and 243 non‐synonymous mutations. Genes carrying mutations included TP53, CDKN2A, and PIK3CA in 79 (35.9%), 35 (15.9%), and 19 patients (8.6%), respectively. Copy number analysis detected amplification of PIK3CA and AKT1 in 38 (17.3%) and 11 patients (5.0%), respectively. Amplification of receptor tyrosine kinases was found in 37 patients (16.8%). Distant metastasis was noted in nine of 37 patients (24%) with receptor tyrosine kinase amplification, accounting for 43% of the 21 cases of distant metastasis. The cumulative 5‐year survival rate was 64.6% in the receptor tyrosine kinase amplification group vs 85.2% in the no receptor tyrosine kinase amplification group. Moreover, we identified significantly poorer prognosis in the TP53 mutation/receptor tyrosine kinase amplification group, for which the cumulative 5‐year survival rate was 41.6%. In conclusion, the results of this study demonstrated that receptor tyrosine kinase amplification is a prognostic factor for distant metastasis of oral squamous cell carcinoma, indicating the necessity of using next‐generation sequencing in clinical sequencing.