Insertion/deletion polymorphism of the angiotensin converting enzyme gene and loss of the insertion allele in aldosterone-producing adenoma

The genetic mechanisms responsible for the formation of adrenocortical adenomas which autonomously produce aldosterone are largely unknown, The adrenal renin-angiotensin system has been implicated in the pathophysiology of these tumours, Angiotensin-converting enzyme (ACE) catalyses the generation of angiotensin II, and the insertion/deletion (I/D) polymorphism of the ACE gene regulates up to 50% of plasma and cellular ACE variability in humans. We therefore examined the genotypic and allelic frequency distributions of the ACE gene I/D polymorphism in 55 patients with aldosterone-producing adenoma, APA, (angiotensin-unresponsive APA n = 28, angiotensin-responsive APA n = 27), and 80 control subjects with no family history of hypertension, We also compared the ACE gene I/D polymorphism allelic pattern in matched tumour and peripheral blood DNA in the 55 patients with APA, The frequency of the D allele was 0.518 and 0.512 and the I allele was 0.482 and 0.488 in the APA and control subjects respectively, Genotypic and allelic frequency analysis found no significant differences between the groups, Examination of the matched tumour and peripheral blood DNA samples revealed the loss of the insertion allele in four of the 25 patients who were heterozygous for the ACE I/D genotype. The I/D polymorphism of the ACE gene does not appear to contribute to the biochemical and phenotypic characteristics of APA, however, the deletion of the insertion allele of the ACE gene I/D polymorphism in 16% of aldosterone-producing adenomas may represent the loss of a tumour suppressor gene/s or other genes on chromosome 17q which may contribute to tumorigenesis in APA.

Presentation of the HPV16E7 protein by skin grafts is insufficient to allow graft rejection in an E7-primed animal

The E7 transforming protein of Human Papillomavirus type 16 (HPV16) is expressed in the skin of a line of RIB mice transgenic for the E6 and E7 open reading frames of HPV16 driven from the alpha A crystallin promoter (FVB alpha AcryHPV16E6E7). We have transferred skin from FVB alpha AcryHPV16E6E7 mice to naive or E7-primed syngeneic NE recipients to assess whether the E7 protein of HPV16 can function as a minor transplantation antigen (MTA) and promote skin graft rejection. FVB mice did not reject E7 expressing tail or flank skin grafts. E7 immunized FVB x C57BL/6J mice recipients of FVB alpha AcryHPV16E6E7 x C57BL/6J skin generated humoral and DTH responses to E7 in vivo and E7-specific CTL precursors in the spleen, but failed to reject 57 expressing tail skin grafts by 100 days posttransfer. Thus although HPV16 E7 + ve mesenchymal and endodermal tumors can be eliminated by an E7-specific immune response, the same protein is unable to act as a MTA and promote graft rejection when expressed in skin cells. Lack of rejection of grafts expressing MTAs such as E7 may be relevant to the immunology of epithelial tumors expressing tumor-specific antigens and to our understanding of the immunology of diseases of the skin. (C) 1997 Academic Press.

Hyperplastic polyps of the colorectum - Innocent or guilty?

Hyperplastic polyps have traditionally been regarded as nonneoplastic polyps lacking malignant potential. The demonstration of genetic alterations within these lesions indicates an underlying neoplastic cause. There is evidence that hyperplastic polyps are heterogeneous. Most are innocuous, but subsets may have malignant potential. Risk factors for neoplastic progression include multiple, large, and proximally located polyps. Aberrant methylation resulting in the silencing of cancer genes may be an important underlying mechanism, particularly in pathways progressing to tumors with DNA microsatellite instability. Lesions intermediate between hyperplastic polyp and cancer include admired polyps and serrated adenomas. Currently, pathologists have different thresholds for diagnosing serrated adenomas, including the distinction from large hyperplastic polyps. Reasons for over looking this pathway in the past may include rapid tumor progression and the fact that proximally located hyperplastic polyps may be flat and not especially numerous. Management of the serrated pathway of colorectal neoplasia may require novel approaches to screening, early detection, and prevention.

CDKN2A is not the principal target of deletions on the short arm of chromosome 9 in neuroendocrine (Merkel cell) carcinoma of the skin

The majority of small-cell lung cancers (SCLCs) express p16 but not pRb, Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC acid MCC, we wished to determine if this was also the case in MCC, Twenty-nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and I on 9p. No loss of heterozygosity (LO H) was peen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p, Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined, Half of all informative cases had LOH at D95168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168, A second region (InFNA-D9S126) showed L0H in 10(44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti-p16 antibody was seen in all Il tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti-p 14' antibody, These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour-suppressor genes mapping both centromeric and telomeric of this locus. (C) 2001 Wiley-Liss, Inc.

Timely topic: Premalignant lesions associated with adenocarcinoma of the upper gastrointestinal tract

The changing incidence of adenocarcinomas, particularly in the oesophagus and gastric cardia, has led to the rapid expansion of screening programmes aimed at detecting the precursor lesion of dysplasia before adenocarcinoma develops. The pathologist now has an important role in first diagnosing patients at risk for developing dysplasia, and then correctly classifying dysplasia when it occurs. Barrett's oesophagus has had different diagnostic criteria in previous years but is currently diagnosed by the presence of intestinal metaplasia of any length in the true oesophagus. Intestinal metaplasia confined only to the gastro-oesophageal junction or cardia is of uncertain significance but is probably common, with less risk of progressing to dysplasia or malignancy. In the stomach, patients with autoimmune atrophic gastritis and Helicobacter-associated multifocal atrophic gastritis have an increased risk of adenocarcinoma, but screening protocols are not well-developed compared with those used for Barrett's oesophagus. Dysplasia of glandular epithelium can be classified using well-described criteria. Low grade dysplasia is the most common type and regresses or remains stable in the majority of patients. High grade dysplasia is more ominous clinically, with a propensity to coexist with or progress to adenocarcinoma.

Mutation analysis of the CDKN2A promoter in Australian melanoma families

Approximately 50% of all melanoma families worldwide show linkage to 9p21-22, but only about half of these have been shown to contain germ line CDKN2A mutations. It has been hypothesized that a proportion of these families carry mutations in the noncoding regions of CDKN2A. Several Canadian families have been reported to carry a mutation in the 5' UTR, at position -34 relative to the start site, which gives rise to a novel AUG translation initiation codon that markedly decreases translation from the wild-type AUG (Liu et al., 1999). Haplotype sharing in these Canadian families suggested that this mutation is of British origin. We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region. In addition, 431 bp upstream of the start codon was sequenced in an additional 253 affected probands from two-case melanoma families for which the CDKN2A mutation status was unknown. Several known polymorphisms at positions -33, -191, -493, and -735 were detected, in addition to four novel variants at positions 120, -252, -347, and -981 relative to the start codon. One of the probands from a two-case family was found to have the previously reported Q50R mutation. No family member was found to carry the mutation at position -34 or any other disease-associated mutation. For further investigation of noncoding CDKN2A mutations that may affect transcription, allele-specific expression analysis was carried out in 31 of the families with at least three affected members who showed either complete or indeterminate 9p haplotype sharing without CDKN2A exonic mutations. Reverse transcription polymerase chain reaction and automated sequencing showed expression of both CDKN2A alleles in all family members tested. The lack of CDKN2A promoter mutations and the absence of transcriptional silencing in the germ line of this cohort of families suggest that mutations in the promoter and 5' UTR play a very limited role in melanoma predisposition. (C) 2001 Wiley-Liss, Inc.

The impact of the Human Genome Project on medical genetics

The near completion of the Human Genome Project stands as a remarkable achievement, with enormous implications for both science and society. For scientists, it is the first step in a complex process that will lead to important advances in the diagnosis and treatment of many diseases. Society, meanwhile, must prevent genetic discrimination, and protect genetic privacy through appropriate legislation.

Papillomavirus research update: highlights of the Barcelona HPV 2000 international papillomavirus conference

The 18th international papillomavirus conference took place in Barcelona, Spain in July 2000. The HPV clinical workshop was jointly organised with the annual meeting of the Spanish Association of Cervical Pathology and Colposcopy. The conference included 615 abstracts describing ongoing research in epidemiology, diagnosis/screening, treatment/prognosis, immunology/human immunodeficiency virus, vaccine development/trials, transformation/progression, replication, transcription/translation, viral protein functions, and viral and host interactions. This leader summarises the highlights presented at the conference (the full text of the abstracts and lectures can be found at www.hpv2000.com). Relevant material in Spanish can be found at www.aepcc. org.

Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma

Epithelial ovarian carcinoma is often diagnosed at an advanced stage of disease and is the leading cause of death from gynaecological neoplasia. The genetic changes that occur during the development of this carcinoma are poorly understood. It has been proposed that IGFIIR, TGF beta1 and TGF beta RII act as a functional unit in the TGF beta growth inhibitory pathway, and that somatic loss-of-function mutations in any one of these genes could lead to disruption of the pathway and subsequent loss of cell cycle control. We have examined these 3 genes in 25 epithelial ovarian carcinomas using single-stranded conformational polymorphism analysis and DNA sequence analysis. A total of 3 somatic missense mutations were found in the TGF beta RII gene, but none in IGFRII or TGF beta1. An association was found between TGF beta RII mutations and histology, with 2 out of 3 clear cell carcinomas having TGF beta RII mutations. This data supports other evidence from mutational analysis of the PTEN and beta -catenin genes that there are distinct developmental pathways responsible for the progression of different epithelial ovarian cancer histologic subtypes. (C) 2001 Cancer Research Campaign.

Rearrangement of diflunisal acyl glucuronide into its beta-glucuronidase-resistant isomers facilitates transport through the small intestine to the colon of the rat

Many non-steroidal anti-inflammatory drugs (NSAIDs) which form acyl glucuronide conjugates as major metabolites have shown an antiproliferative effect on colorectal tumors. This study assesses the extent to which rearrangement of an acyl glucuronide metabolite of a model NSAID into beta -glucuronidase-resistant isomers facilitates its passage through the small intestine to reach the colon. Rats were dosed orally with diflunisal (DF), its acyl glucuronide (DAG) and a mixture of rearrangement isomers (iso-DAG) at 10 mg DF equivalents/kg. The parent drug DF appeared in plasma after all doses, with maximum concentrations of 20.5 +/- 2.5, 28.8 +/- 8.3 and 11.0 +/- 1.6 mug DF/ml respectively, obtained at 3.8 +/- 0.3, 3.6 +/- 1.8 and 7.5 +/- 0.9 hr after the DF, DAG and iso-DAG doses respectively. At 48 hr, 16.2 +/- 3.3, 19.8 +/- 0.8 and 42.9 +/- 10.1% of the doses respectively were recovered in feces, with less than or equal to 1% remaining in the intestine. About half of each dose was recovered as DF and metabolites in 48 hr urine: for DF and DAG doses, the majority was in the first 24 hr urine. whereas for iso-DAG doses, recoveries in the first and second 24 hr periods were similar. The results show that hydrolysis of both DAG and iso-DAG, and absorption of liberated DF, occur during passage through the gut, but that these processes occur more slowly and to a lesser degree for iso-DAG. The intrinsic hydrolytic capacities of various intestinal segments (including contents) towards DAG and iso-DAG were obtained by incubating homogenates under saturating concentrations of DAG/iso-DAG at 37 degreesC. Upper small intestine, lower small intestine, caecum and colon released 2400, 3200, 9200 and 22800 mug DF/hr/g tissue plus contents respectively from DAG substrate, and 18, 10, 140 and 120 mug DF/hr/g tissue plus contents respectively from iso-DAG substrate. The much greater resistance of iso-DAG to hydrolysis appears attributable to its resistance to beta -glucuronidases. The data suggest that in rats dosed with DF, DAG excreted in bile would be substantially hydrolysed in the small intestine and liberated DF reabsorbed, but that portion which rearranges to iso-DAG would likely reach the colon. (C) 2001 Elsevier Science Inc. All rights reserved.

Subcellular localization of the steroid receptor coactivators (SRCs) and MEF2 in muscle and rhabdomyosarcoma cells

Skeletal muscle differentiation and the activation of muscle-specific gene expression are dependent on the concerted action of the MyoD family and the MADS protein, MEF2, which function in a cooperative manner. The steroid receptor coactivator SRC-2/GRIP-1/TIF-2, is necessary for skeletal muscle differentiation, and functions as a cofactor for the transcription factor, MEF2. SRC-P belongs to the SRC family of transcriptional coactivators/cofactors that also includes SRC-1 and SRC-3/RAC-3/ACTR/ AIB-1. In this study we demonstrate that SRC-P is essentially localized in the nucleus of proliferating myoblasts; however, weak (but notable) expression is observed in the cytoplasm. Differentiation induces a predominant localization of SRC-P to the nucleus; furthermore, the nuclear staining is progressively more localized to dot-like structures or nuclear bodies. MEF2 is primarily expressed in the nucleus, although we observed a mosaic or variegated expression pattern in myoblasts; however, in myotubes all nuclei express MEF2. GRIP-1 and MEF2 are coexpressed in the nucleus during skeletal muscle differentiation, consistent with the direct interaction of these proteins. Rhabdomyosarcoma (RMS) cells derived from malignant skeletal muscle tumors have been proposed to be deficient in cofactors. Alveolar RMS cells very weakly express the steroid receptor coactivator, SRC-P, in a diffuse nucleocytoplasmic staining pattern. MEF2 and the cofactors, SRC-1 and SRC-3 are abundantly expressed in alveolar and embryonal RMS cells; however, the staining is not localized to the nucleus. Furthermore, the subcellular localization and transcriptional activity of MEF2C and a MEF2-dependent reporter are compromised in alveolar RMS cells. In contrast, embryonal RMS cells express SRC-2 in the nucleus, and MEF2 shuttles from the cytoplasm to the nucleus after serum withdrawal. In conclusion, this study suggests that the steroid receptor coactivator SRC-P and MEF2 are localized to the nucleus during the differentiation process. In contrast, RMS cells display aberrant transcription factor SRC localization and expression, which may underlie certain features of the RMS phenotype.

A for stage III and IV squamous carcinoma of the head and neck: a Trans-Tasman Radiation Oncology Group Study

Purpose: The aims of this randomized controlled trial were to determine whether there were differences in the disease-free survival (DFS) and toxicity between conventional radiotherapy (CRT) and a continuous 3 week accelerated radiotherapy regimen (ART) in stage III and IV squamous cell carcinoma of the oral cavity, oropharynx, larynx and hypopharynx. Patients and methods: Patients from 14 centres throughout Australia and New Zealand were randomly assigned to either CRT, using a single 2 Gy/day to a dose of 70 Gy in 35 fractions in 49 days or to ART, using 1.8 Gy twice a day to a dose of 59.4 Gy in 33 fractions in 24 days. Treatment allocation was stratified for site and stage. The accrual began in 1991 and the trial was closed in 1998 when the target of 350 patients was reached. Results: The median potential follow-up time was 53 months (range, 14-101). The DFS at 5 years was 41% (95% CI, 33-50%) for ART and 35% (95% CI, 27-43%) for CRT (P = 0.323) and the hazard ratio was 0.87 in favour of ART (95% CI, 0.66-1.15). The 5-year disease-specific survival rates were 40% for CRT and 46% for ART (P = 0.398) and the loco-regional control was 47% for CRT vs. 52% for ART (P = 0.300). The respective hazard ratios were 0.88 (95% CI, 0.65-1.2) and 0.85 (0.62-1.16), favouring the accelerated arm. In the ART arm, confluent mucositis was more severe (94 vs. 71%; P < 0.001) and peaked about 3 weeks earlier than in the CRT arm, but healing appeared complete in all cases. There were statistically significant reductions in the probability of grade 2 or greater late soft tissue effects over time in the ART arm (P < 0.05), except for the mucous membrane where late effects were similar in both arms. Conclusions: Differences in DFS, disease-specific survival and loco-regional control have not been demonstrated. ART resulted in more acute mucosal toxicity, but this did not result in greater prolongation of the treatment time compared with the CRT arm. There were less late effects in the ART arm, with the exception of late mucosal effects. This trial has confirmed that tumour cell repopulation occurs during conventionally fractionated radiotherapy for head and neck cancer. However, it has also provided additional evidence that overall improvements in the therapeutic ratio using accelerated fractionation strategies are seriously constrained by the need to limit total doses to levels that do not exceed acute mucosal tolerance. The accelerated schedule tested has been shown in this trial to be an acceptable alternative to conventionally fractionated irradiation to 70 Gy. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Photosensitization of the sunscreen octyl p-dimethylaminobenzoate by UVA in human melanocytes but not in keratinocytes

Sunscreens penetrate human epidermis and modify the biology of proliferating cells. This study addressed the question whether the UV response of cultured human cells is affected by direct treatment with nontoxic levels of sunscreens. Cell survival following exposure to UVC or unfiltered UVB was not altered by preincubation with 25 μg/mL of octyl p-dimethylaminobenzoate (o-PABA), 2-ethylhexyl p-methoxycinnamate (EHMC) or oxybenzone. However, UVA or UVB filtered to reproduce the solar UV spectrum penetrating to the basal layer of the epidermis, highly sensitized cells to killing by o-PABA but not by its hydrolysis product, 4-dimethylaminobenzoic acid. Sensitization was found in all cell types tested, except normal keratinocytes, and could be prevented by certain antioxidants particularly pyruvate and the hydroxyl radical scavenger mannitol. o-PABA and EHMC applied without UV reduced the adherence of cells. The results indicate that sunscreens may increase cell mobility and the combination of o-PABA with solar UV may selectively damage melanocytes in the skin.

Phyllodes tumors of the breast: Correlation of nucleolar organizer regions with histopathological malignancy grading, flow cytometric DNA analysis and clinical outcome

To examine whether nucleolar organizer regions detected by argyrophilia (Ag-NOR counts) can be used as a prognostic indicator in phyllodes tumors of the breast, and to compare its usefulness with that of DNA flow cytometric analysis, 28 cases of breast phyllodes tumors (including 15 benign, two borderline and 11 malignant tumors) were subjected to Ag-NOR staining and counting as well as DNA flow cytometric analysis. S-phase fraction and DNA ploidy analysis showed useful trends for improving outcome predictions in malignant phyllodes tumors. However, high Ag-NOR counts were significant in predicting survival status (P = 0.013) and reached near statistical significance in predicting survival times (P = 0.07). In predicting survival status, results for Ag-NOR counts were significantly better than those for ploidy analysis (P = 0.02) and S-phase fraction (P < 0.01). Only S-phase fraction was significantly predictive of survival times (P = 0.025). It is concluded that Ag-NOR counts and DNA flow cytometric analysis, easily performed using paraffin sections, give information that can improve predictions made by histopathological classification. Ag-NOR counts are significant in predicting survival in the presence of histopathological features of malignancy.