Could epithelial ovarian cancer be associated with chlamydial infection?

PURPOSE OF INVESTIGATION: Epithelial ovarian cancer (EOC) is the leading cause of death from gynaecological malignancy in the UK. The pathogenesis of this disease is poorly understood. Our hypothesis was that chlamydial infection might play a role in the pathogenesis of EOC. METHODS: 122 serum samples of patients undergoing surgery for benign or malignant gynaecological conditions were analysed. There was a total of 41 patients with EOC (33.6%), 27 with benign cystadenomas (22.1%) and 54 with normal ovaries (44.3%). RESULTS: There was a higher incidence of IgA seropositivity and lower incidence of IgG seropositivity in the EOC group compared with the other groups; however, this was not statistically significant. There was no statistical difference in the serum IgM antibodies to chlamydia in the three different groups. CONCLUSION: Although chronic infection and persistent inflammation may contribute to the pathogenesis of EOC, and chlamydia is a common genital tract pathogen, our study did not find an association between chlamydia and EOC.

Differential expression of alpha 2-adrenoceptor subtypes in purified rat pancreatic islet A- and B-cells.

In the endocrine pancreas, alpha 2-adrenoceptor stimulation reduces glucose-induced insulin secretion from islet B-cells. There is, however, controversy with regard to the effects of alpha 2-agonists on islet A-cell function. In this paper, we have used RNA samples prepared from whole rat islets and from FACS-purified rat A- and B-cells to study alpha 2-adrenoceptor subtype expression. RNase protection assays detected transcripts encoding alpha 2a and alpha 2b subtypes in the RNA pool of rat islets. Reverse transcription-PCR revealed that transcripts for all three alpha 2-adrenoceptor subtypes are present in rat islet cells in purified A-cell RNA. In contrast, RT-PCR of islet B-cell RNA yielded products corresponding to alpha 2a and alpha 2c, with no evidence for the presence of alpha 2b. Thus, the results reveal that both islet cell types express more than one receptor subtype and suggest that the distribution of the subtypes may differ between rat islet A- and B-cells.

p53 Protein accumulates in Cushings adenomas and invasive non-functional adenomas.

The p53 protein, a negative regulator of cell growth, plays an important role in the pathogenesis of many human tumours following gene mutation and/or deletion. We screened a large number of sporadic pituitary tumours for p53 protein accumulation suggestive of gene mutation. Samples were divided into benign adenomas (n = 95) and invasive tumours with local or distant invasion (n = 26). All main tumour classes were represented. Putative p53 mutations were detected by immunohistochemistry on paraffin-embedded sections using polyclonal CM-1 and monoclonal DO-7 and PAb1801 antibodies. Results were compared to normal post-mortem pituitary tissue controls (n = 17). p53 protein accumulation was detected in invasive tumours (16%), but only in corticotrophinomas (2/4) and non-functional tumours (4/15). In non-invasive adenomas, protein accumulation was observed only in ACTH-secreting tumours where 50% were positive (16/32). No protein accumulation was identified in any control tissue. These results indicate that p53 protein accumulation may play a role in the development of Cushings adenomas and in the progression of non-functional tumours to the invasive state.

Molecular genetic studies of sporadic pituitary tumors.

Tumor formation may result from the activation of dominant oncogenes or by inactivation of recessive, tumor suppressor genes. The role of such mutations in the development of pituitary tumors has been studied. Tumors from 88 patients, representing the 4 major classes of adenoma, were investigated. In DNA extracted from matched leukocyte and tumor samples, allelic deletions were sought with 15 probes identifying restriction fragment length polymorphisms on chromosomes 1, 5, 10, 11, 13, 17, 20, and 22. Evidence of amplification or rearrangement of 10 recognized cellular oncogenes (N-ras, mycL1, mycN, myc, H-ras, bcl1, H-stf1, sea, kraS2, and fos) was sought in tumor DNA. Activating dominant mutations of Gs alpha were detected using the polymerase chain reaction to amplify exons 7-10 and hybridizing the product to normal and mutant allele-specific oligonucleotides. Allelic deletions on chromosome 11 were identified in 16 tumors (18%) representing all 4 major subtypes. Deletions on other autosomes were observed in less than 6% of tumors. Three adenomas had deletions on multiple autosomes, 2 of these were aggressive and recurrent. Mutations of Gs alpha were confirmed to be specific to somatotrophinomas, being identified in 36% of such tumors in this series. No evidence of amplification or rearrangement of other recognized cellular oncogenes was found. Inactivation of a recessive oncogene on chromosome 11 is an important and possibly early event in the development of the four major types of pituitary adenoma, whereas activating mutations of Gs alpha are confirmed to be specific to somatotropinomas. Two aggressive tumors were found to have multiple autosomal losses, suggesting a multistep progression in the development of tumors of this phenotype.

P53 protein accumulates in Cushings adenomas and invasive non-functional adenomas.

The p53 protein, a negative regulator of cell growth, plays an important role in the pathogenesis of many human tumours following gene mutation and/or deletion. We screened a large number of sporadic pituitary tumours for p53 protein accumulation suggestive of gene mutation. Samples were divided into benign adenomas (n = 95) and invasive tumours with local or distant invasion (n = 26). All main tumour classes were represented. Putative p53 mutations were detected by immunohistochemistry on paraffin-embedded sections using polyclonal CM-1 and monoclonal DO-7 and PAb1801 antibodies. Results were compared to normal post-mortem pituitary tissue controls (n = 17). p53 protein accumulation was detected in invasive tumours (16%), but only in corticotrophinomas (2/4) and non-functional tumours (4/15). In non-invasive adenomas, protein accumulation was observed only in ACTH-secreting tumours where 50% were positive (16/32). No protein accumulation was identified in any control tissue. These results indicate that p53 protein accumulation may play a role in the development of Cushings adenomas and in the progression of non-functional tumours to the invasive state.

Allelic deletion in pituitary adenomas reflects aggressive biological activity and has potential value as a prognostic marker.

Tumors of the pituitary gland are usually benign adenomas and account for 10% of all intracranial neoplasms. Five pituitary tumors have previously been reported to harbor multiple allelic deletions. Of these, three displayed particularly aggressive biological behavior, whereas there were no clinical details provided for the others. This study was designed to test the hypothesis that genetic deletions are a marker of invasive behavior and to identify the loci most commonly involved. Accordingly, we studied two cohorts of pituitary tumors, classified radiologically as invasive or noninvasive, for loss of heterozygosity (LOH). There is a significantly higher frequency of LOH in invasive tumors (10.8% of all loci examined) compared to noninvasive tumors (2.4%; P < 0.001). Of the 11 loci investigated, 75% of the allelic deletions identified in invasive tumors were found at 4 loci: 11q13, 13q12-14, 10q, and 1p. Twenty of 47 invasive tumors had evidence of at least 1 allelic deletion, whereas 14 of 20 had more than 1. Of the 6 tumors with only 1 deletion, 5 involved the 11q13 locus, suggesting that this is an early change in the transition from noninvasive to invasive adenoma. Comparison of invasive and noninvasive tumors demonstrates a significantly higher frequency of deletions affecting 11q13 (P < 0.001), 13q12-14 (P < 0.05), and 10q26 (P < 0.05) in invasive tumors. In addition, allelic deletion correlates with increasingly invasive behavior (modified Hardy classification), as 73% of grade 4 tumors compared to 33% of grade 3 and 9.5% of grade 1 and 2 tumors demonstrated LOH at any locus. Furthermore, in some tumors we identified a breakpoint between markers intragenic and extragenic to the retinoblastoma gene (Rb1) on chromosome 13q, suggesting that tumor suppressor genes other than or in addition to Rb1 may be involved in pituitary tumorigenesis. This was further supported by the presence of Rb protein in two of four tumors where the genetic loss extended to include the intragenic marker D13S153. Early identification of tumors with likely invasive potential by means of genetic analysis (LOH) may provide useful information on potential tumor behavior and aid tumor management in a manner that is not possible using routine histological methods. A large prospective study is required in patients without radiological evidence of invasion to assess the value of LOH in predicting outcome and for planning treatment.

Expression of alpha 2- and beta-adrenoceptor subtypes in human islets of Langerhans.

Sequences from cDNA molecules encoding alpha 2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human alpha 2C2-, alpha 2C4- and alpha 2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of alpha 2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic beta-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both alpha 2 C2 and alpha 2 C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding alpha 2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each alpha 2-adrenoceptor subtype in sections of human pancreas. All three subtypes of alpha 2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffin-embedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three alpha 2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple alpha 2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of alpha 2-adrenoceptor subtype mRNA species in pancreatic beta-cells was confirmed by Northern blotting of RNA extracted from the clonal beta-cell line, HIT-T15. Transcripts encoding each of the three cloned alpha 2-adrenoceptor subtypes were detected in HIT-T15 cells. Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with beta 2-adrenoceptor mRNA revealed expression of this species in islet beta-cells but not in the exocrine tissue of the pancreas.

Changes in relative levels of specific brain mRNA species associated with schizophrenia and depression.

Total cellular polyadenylated RNA (poly(A)+ RNA, mRNA) was prepared after guanidinium thiocyanate extraction of frozen brain tissue from age-matched controls and patients suffering from schizophrenia and unipolar depression. These mRNA populations were analysed by in vitro translation followed by two-dimensional gel analysis. Data were obtained from fluorograms derived from 10 different schizophrenic patients, 10 different controls and 5 different depressive patients. The relative concentrations of mRNA species coding for 4 translation products (33 kDa, pI 5.8; 26 kDa, pI 5.8; 35 kDa, pI 7.1; 23 kDa, pI 6.1) were significantly reduced in schizophrenia compared to controls when determined by computerised image analysis of the fluorograms. In the case of depression, the relative concentrations of mRNA species coding for 6 translation products were significantly altered, 4 being increased (38 kDa, pI 6.2, 17 kDa, pI 5.7, 35 kDa, pI 7.1; 23 kDa, pI 6.1) and two decreased (34 kDa, pI 6.2; 33 kDa, pI 5.8). Three translation products were altered in both schizophrenia and depression, one (33 kDa, pI 5.8) being altered according to the same trend, a decrease relative to controls, but two (35 kDa, pI 7.1; 23 kDa, pI 6.1) being altered differently in schizophrenia (reduced) and depression (increased). The effects of post mortem delay, mode of death and drug treatment on mRNA composition were also examined and found not to affect the levels of these translation products significantly. The significance of these changes will be discussed in relation to their relevance of biological mechanisms in the psychoses.

Changes in brain gene expression in schizophrenic and depressed patients.

Poly(A+) mRNA was extracted from the post-mortem brain of schizophrenics (9 subjects), unipolar depressives (5 subjects) and controls (10 subjects) and used to direct the in vitro translation of radiolabelled protein in a cell-free reticulocyte-lysate system. Protein species were analysed on two-dimensional gels. Over 200 products were detected and, from these, 74 well-resolved species were chosen for further analysis. The optical density of each product was quantified by image analysis and normalised with respect to overall gel intensity. It was found that 7 novel, uncharacterised protein species, ranging from molecular weights (Mr) 17 kDa to 38 kDa and apparent isoelectric points (pI) 5.7-7.1, changed significantly in intensity in the psychotic groups compared to controls. One species changed only in the schizophrenia group (Mr = 26 kDa, pI = 5.8, 18% of control intensity) and 3 changed only in the depressive group (Mr = 38 kDa, pI = 6.2, 540% of control; Mr = 34 kDa, pI = 6.2, 6% of control; Mr = 17 kDa, pI = 5.7, 238% of control). Three further protein species were common to both psychotic groups (one species decreased in both schizophrenia and depression, Mr = 33 kDa, pI = 5.8; two species showed opposing intensity changes, decreasing in schizophrenia and increasing in depression, Mr = 35 kDa, pI = 7.1; Mr = 23 kDa, pI = 6.1). None of these changes was a function of post-mortem delay or mode of death. It is quite likely that such protein species reflect the abundance of specific mRNAs and target gene systems associated with the disease state.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiogenesis in benign, pre-malignant and malignant vulvar lesions.

Squamous cell carcinoma of the vulva (SCC) accounts for about 4% of all gynaecological malignancies. It has an incidence of about 1.8 per 100,000. However, after the age of 75 the incidence of vulvar carcinoma peaks at approximately 20 per 100,000, making it as common as cervical carcinoma. Benign and pre-malignant vulvar lesions can be broadly divided into non-neoplastic epithelial disorders of the vulva (NNEDV), vulvar intraepithelial neoplasia (VIN) and Paget's disease of the vulva (PDV). NNEDV lesions include lichen sclerosus (LS) and squamous hyperplasia (SH). To date no solid prognostic tools are available to predict which pre-malignant vulvar lesions will progress to SCC. About 4.5% of patients develop SCC following a history of LS and ca. 4% of treated VIN lesions go on to become vulvar SCC. In PDV, 28% of patients have an underlying cancer. Angiogenesis, the development of new blood vessels from existing vasculature, is an essential component of solid tumour growth and metastasis. Several angiogenic factors are expressed by many tumours, suggesting that tumours promote their own vascularisation by activating the host endothelium. This review follows the progress of research in angiogenesis in vulvar disease as assessed by a variety of methods, such as in situ hybridisation (ISH), microvessel density measurements (MVD), immunohistochemically-detected angiogenic growth factor expression, including vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), and serum concentrations of VEGF. Thus, the potential of angiogenesis as a prognostic and predictive tool for identifying which pre-malignant lesions could progress to SCC is discussed. A relatively high MVD and strong VEGF expression may serve as prognostic indicators of survival in invasive SCC. MVD and VEGF expression are also predictive factors in identifying which VIN lesions are more likely to become invasive. However VEGF is not a predictive marker in cases of LS likely to progress to carcinoma. The expression of PD-ECGF/TP in all types of lesions tested prevents its use as a prognostic tool, unlike VEGF. However, PD-ECGF/TP is involved in PDV pathogenesis, but is not a marker of the malignant progression of PDV. In PDV, VEGF was not expressed even in those cases associated with invasive disease. Serum concentrations of VEGF play a functional role in vulvar carcinogenesis. Although associated with impaired disease-free and overall survival, pre-treatment serum concentrations of VEGF are not an independent predictor of outcome in patients with vulvar cancer. These studies suggest that further angiogenic research will be important in both the therapy and prognosis of malignant and pre-malignant vulvar disease.

Proliferation and p53 expression in anal cancer precursor lesions.

BACKGROUND: Anal squamous cell carcinoma (SCC) develops from dysplastic anal warts. This study quantifies the expression of p53 and Ki67 in pre-invasive and invasive anal lesions. MATERIALS AND METHODS: Samples of 70 patients with anal warts (n = 20), low grade anal intraepithelial neoplasia (LG AIN) (n = 12), high grade anal intraepithelial neoplasia (HG AIN) (n = 27) and anal SCC (n = 11) were stained using immunohistochemical techniques. Eight patients with normal anal skin were used as controls. RESULTS: Both the expression of p53 and Ki67 increased significantly (p < 0.001) and gradually as the lesions became dysplastic and invasive. The main increase in p53 expression was as the lesions progressed from anal warts (7.38 +/- 11.93-mean +/- SD) to low grade AIN (20.778 +/- 13.14). CONCLUSION: p53 is involved in the progression of anal cancer and its expression increases from early in the development of pre-invasive anal lesions. p53 and Ki67 may be useful markers of early dysplasia and should be considered in the screening of high risk patients.

Role of angiogenesis in benign, premalignant and malignant vulvar lesions.

OBJECTIVE: To investigate the presence of angiogenic factors in benign, premalignant and malignant vulvar lesions. STUDY DESIGN: Immunohistochemical demonstration of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) in normal vulvar skin, lichen sclerosus, vulvar intraepithelial neoplasia (VIN) and vulvar cancer. RESULTS: VEGF was found in the majority of vulvar cancers but only a minority of VIN lesions. PD-ECGF was found in the majority of lesions. CONCLUSION: Demonstration of angiogenesis may suggest which preinvasive lesions will progress to invasive cancer.

Is there a link between vulval cancer and blood group?

Risk factors for squamous cell vulval cancer (SCC) remain unclear though there have been associations with lichen sclerosis, smoking, and vulval intraepithelial neoplasia (VIN). We studied 191 patients who had been referred to the vulval clinic at the Royal Free Hospital and who had both blood group and histopathology results available. Seventy-two percent of patients with SCC and non-neoplastic epithelial disorders of the vulva (NNEDV) were found to be in blood group A with only 17% in blood group O. Those with SCC associated with VIN had only 30% in blood group A with 50% in blood group O. The control population showed that 38% of the population were in blood group A and 43% were in blood group O. Our results suggest that blood group A is prevalent in patients with SCC associated with NNEDV but not in those women with squamous vulval cancer and associated VIN.

Familial prevalence of uterine fibroids is associated with distinct clinical and molecular features.

BACKGROUND: Although uterine fibroids are very common, their pathogenesis and clinical behaviour are poorly understood. Since they may be prevalent in some families, we investigated whether such a prevalence was associated with distinctive clinical and molecular features. METHODS: A case-control questionnaire study of 300 multi-ethnic women with uterine fibroids at a London university hospital was undertaken, with review of case notes and immunohistochemical determination of vascular endothelial growth factor (VEGF-A) in fibroids. RESULTS: When compared with families with sporadic fibroids, familial prevalence of fibroids was associated with a higher incidence of abdominal swelling (59.1% versus 41.6%; P=0.037), menorrhagia (84.4% versus 51.9%; P=0.042), dysmenorrhoea (64.4% versus 46.3%; P=0.004), dyspareunia (43.2% versus 27.9%; P=0.012) and family history of cancers (52.3% versus 32.4%; P<0.01). The fibroids were also more multiple (mean +/- SEM: 7 +/- 0.86 versus 3 +/- 0.42; P<0.011) and strong VEGF-A expression in fibroids was more common in the familial group (64% versus 28%). Racial distribution was the same in both groups (blacks 49%, whites 33.4%, others 18.6%). CONCLUSIONS: Familial prevalence of uterine fibroids is associated with distinct clinical and molecular features that differ from those found when fibroids occur sporadically in families.

Microvessel quantification in benign and malignant ovarian tumors.

Microvessel density (MVD) in 92 paraffin sections of ovarian samples of different histologic subtypes was correlated with microvessel counts from 58 corresponding frozen sections. Anti-human von Willebrand factor antibody was used as an endothelial marker. MVD was performed in neovascular hotspots using a Quantimet 500+ Image Analyzer. The highest vessel density (HVD) and average vessel density (AVD) of three fields at the x 200 and x 400 magnification were recorded. There was a strong correlation between the HVD and AVD at the x 200 and x 400 magnifications when comparing fixed with frozen sections (correlation coefficients at x 200 for the HVD was 0.37, P = 0.005 and AVD was 0.30, P = 0.02; correlation coefficients at x 400 for the HVD was 0.38, P = 0.003 and AVD was 0.37, P = 0.004). In the fixed tissue, the HVD and AVD at both these magnifications were significantly greater in the group containing functional cysts; this was also the case for the frozen sections. These findings are consistent with the development of a microcirculation necessary for the growth and maturation of such cysts, and this appears to be greater than that in tumors. The good correlation between MVD in fixed and frozen sections suggests that such observations represent a true reflection of ovarian angiogenesis in both physiologic and pathologic states.

An estimation of the sensitivity of in vitro translation using two-dimensional gel analysis.

Poly (A+ mRNA species, isolated from 100-day-old rat brain, were analysed by in vitro translation and two-dimensional gel electrophoresis. The synthesis of selected protein species was compared to actin on the basis of [35S]methionine incorporation. The estimated molar abundance of translation products varied from abundant species at 0.78% of the total to several are species, detectable below the 0.02% level. If these synthesis rates reflect the abundance of particular mRNAs in the mixture, this sensitivity limit compares well with accepted values using differential cDNA screening techniques. This analysis provides evidence that in vitro translation methodology is able to detect rarer mRNA species than is usually expected--these include similar abundance classes to library screening procedures.

Angiogenesis in epithelian ovarian cancer.

Angiogenesis, the development of new blood vessels from the existing vasculature, is an essential component of solid tumour growth and metastasis. Several angiogenic factors are expressed by many tumours, suggesting that tumours promote their own vascularisation by activating the host endothelium. This review will discuss various angiogenic stimulators and inhibitors in epithelian ovarian cancer (EOC), including vascular endothelial growth factor and platelet derived endothelial cell growth factor/thymidine phosphorylase. The analysis of tumour vascularisation by microvessel density will also be discussed and the relevance of these markers of angiogenesis in the prognosis of EOC will be assessed.

Characterisation of messenger RNA extracted post-mortem from the brains of schizophrenic, depressed and control subjects.

Messenger RNA, obtained from post-mortem brain of 10 schizophrenics, five depressed patients and 10 control subjects, was characterised with respect to a number of parameters. It was found that post-mortem delay was not the major factor in determining RNA yield, size (as determined by cDNA synthesis) and biological activity. Biological activity, as determined by in vitro translation in a reticulocyte-lysate system, could be observed using messenger RNA from periods of 0 to 84 hours post-mortem. Two-dimensional gel analysis of the newly-synthesised radiolabelled products obtained from this material revealed several hundred individual species but no consistent degradation of any particular species with post-mortem delay. It is suggested, therefore, that premortem changes are as important as post-mortem changes in determining RNA yield, size and biological activity. Although no consistent difference could be found between patients and controls using any of these parameters, this study confirms that, by isolating messenger RNA from post-mortem human brain, valuable information can be gained on gene expression in psychiatric disorders.

Analysis of relative mRNA levels and protein patterns in brains of rat strains bred for differing levels of emotionality.

mRNA and protein populations were studied in the brains of Maudsley reactive (MR) and Maudsley nonreactive (MNR) rat strains, which exhibit differing levels of emotionality. Translational analysis of forebrain mRNA indicated that the relative levels of two translation products (42 kDA, pI 5.0; 30 kDa, pI 5.8) were increased in the MR compared to the MNR strain. In addition, a charge-shift variant of a 36 kDa protein was present in the MR strain. Analysis of brain protein patterns indicated that a protein of 39 kDa, pI 5.0, was found to be more abundant in MR compared with MNR strains in both frontal cortex and hippocampus and the relative level of one protein (40 kDa, pI 5.8) was decreased in the frontal cortex.

TP53 mutations in vulval lichen sclerosus adjacent to squamous cell carcinoma of the vulva.

Non-neoplastic epithelial lesions of the vulva (NNEDV) lichen sclerosus (LS) and squamous hyperplasia (SH) have been implicated in the pathogenesis of squamous cell carcinoma of the vulva (SCC). To date, there have been no recognisable precursor lesions for SCC associated with NNEDV. TP53 is the most frequent genetic change in human cancers and can indicate both aetiology and molecular pathogenesis of tumours. A total of 27 SCC patients underwent immunohistochemistry (IHC) and TP53 mutational analysis using microdissection and direct sequencing. There were 19 patients with areas of adjacent epidermis: 17 had NNEDV (four SCCs had more than one adjacent lesion) and two had normal epidermis. In all, 70.4% of the SCCs, 40% LS and 22.2% SH demonstrated overexpression of p53. In total, 77.8% of SCCs, 46.7% of LS and 22.2% SH demonstrated mutations in TP53, with the majority of lesions having a mutation in codon 136. Eight cases were identified where the same mutation was identified in the SCC and in the adjacent area. These data suggest that TP53 mutations develop in NNEDV and are intrinsic to the clonal evolution that leads to SCC. The type of mutation detected is more likely to occur due to endogenous cellular changes rather than exogenous carcinogen exposure.