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Abstract Background Esophageal squamous cell carcinoma (ESCC) shows geographic variations in incidence, with high incidences (>50/105 person-years) in central Asia, including North Eastern Iran (Golestan) and Northern India (Kashmir). In contrast to Western countries, smoking does not appear to be a significant risk factor for ESCC in central Asia. In lung adenocarcinoma, activating mutations in the gene encoding epidermal growth factor receptor (EGFR) are frequent in tumors of never smokers of Asian origin, predicting therapeutic sensitivity to Egfr-targeting drugs. Methods In this study 152 cases of histologically confirmed ESCC from Iran (Tehran and Golestan Province) and North India (Kashmir Valley) have been analyzed for EGFR mutation by direct sequencing of exons 18–21. Egfr protein expression was evaluated by immunohistochemistry in 34 samples from Tehran and HER2 mutations were analyzed in 54 cases from Kashmir. Results A total of 14 (9.2%) EGFR variations were detected, including seven variations in exons. Among those, four (2.6%) were already documented in lung cancers, two were reported as polymorphisms and one was a potentially new activating mutation. All but one variation in introns were previously identified as polymorphisms. Over-expression of Egfr was detected in 22/34 (65%) of tested cases whereas no HER2 mutation was found in 54 cases from Kashmir. Conclusion Overall, EGFR mutations appear to be a rare event in ESCC in high incidence areas of central Asia, although a very small proportion of cases may harbor mutations predicting sensitivity to anti-Egfr drugs.

Abstract Background Squamous Cell Carcinoma of Esophagus is one of the most common malignancies in both men and women in eastern and south-eastern Africa. In Kenya, clinical observations suggest that this cancer is frequent in the Rift Valley area. However, so far, there has been no report on the molecular characteristics of esophageal squamous cell carcinoma (ESCC) in this area. Results We have analyzed TP53 mutations, the presence of human papilloma virus (HPV) DNA and expression of inflammation markers Cyclooxygenase 2 (Cox-2) and Nitrotyrosine (NTyR) in 28 cases (13 males and 15 females) of archived ESCC tissues collected at the Moi Teaching and Referral Hospital in Eldoret, Kenya. Eleven mutations were detected in TP53 exons 5 to 8 (39%). All ESCC samples were negative for HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82. Immunohistochemical analysis of Cox-2 and NTyR showed a low proportion of positive cases (17.4% and 39.1%, respectively). No association between the above markers and suspected risk factors (alcohol or tobacco use, hot tea drinking, use of charcoal for cooking) was found. Conclusion Our findings suggest that mechanisms of esophageal carcinogenesis in eastern Africa might be different from other parts of the world. Low prevalence of TP53 mutation compared with other intermediate or high incidence areas of the world highlights this hypothesis. Our data did not support a possible ole of HPV in this series of cases. Further studies are needed to assess and compare the molecular patterns of ESCC from Kenya with those of high-incidence areas such as China or Central Asia.

Abstract Background Gastric adenocarcinoma is rare in children and adolescents, with about 17 cases under age 21 in the world's literature. We report a case of invasive well-differentiated metastatic gastric cancer in a Brazilian 12-year-old boy without documented familial history of cancer. Case presentation The patient, diagnosed with metastatic disease, died seven months after surgery. DNA from intra-surgical specimens revealed a TP53 mutation at codon 337 (p.R337H) in samples with neoplastic cells (dysplasia, tumor and metastasis) but not in non-transformed cells (incomplete intestinal metaplasia and non-involved celiac lymph node). In all mutation-positive tissues, p.R337H occurred on the same background, a founder allele identified by a specific haplotype previously described in Brazilian Li-Fraumeni syndrome patients. The same mutant haplotype, corresponding to a founder mutation present in 0.3% of the general population in Southern Brazil, was found in the genome of the father. Presence of this inherited haplotype in the tumor as well as in the father's germline, suggests a rare case of microchimerism in this patient, who may have harbored a small number of mutant cells originating in another individual, perhaps a dizygotic twin that died early in gestation. Conclusion This case represents one of the earliest ages at diagnosis of gastric cancer ever reported. It shows that cancer inheritance can occur in the absence of an obvious germline mutation, calling for caution in assessing early cancers in populations with common founder mutations such as p.R337H in Southern Brazil.

Germline TP53 mutations predispose individuals to multiple cancers and are associated with Li-Fraumeni/Li-Fraumeni-Like Syndromes (LFS/LFL). The founder mutation TP53 p.R337H is detected in 0.3% of the general population in southern Brazil. This mutation is associated with an increased risk of childhood adrenal cortical carcinoma (ACC) but is also common in Brazilian LFS/LFL families. Breast Cancer (BC) is one of the most common cancers diagnosed in TP53 mutation carriers. We have assessed the prevalence of p.R337H in two groups: (1) 59 BC affected women with a familial history (FH) suggestive of hereditary cancer syndrome but no LFS/LFL features; (2) 815 BC affected women unselected for cancer FH, diagnosed with BC at or before age 45 or at age 55 or older. Among group 1 and group 2 patients, 2/59 (3.4%, CI95%: 0.4%-11.7%) and 70/815 (8.6%, CI95%: 6.8%-10.7%), respectively, were p.R337H carriers in the germline. The prevalence of p.R337H was higher in women diagnosed with BC at or before age 45 (12.1%, CI95%: 9.1%-15.8%) than at age 55 or older (5.1%, CI95%: 3.2%-7.7%), p<0.001). The Brazilian founder p.R337H haplotype was detected in all carriers analysed. These results suggest that inheritance of p.R337H may significantly contribute to the high incidence of BC in Brazil, in addition to its recently demonstrated impact on the risk of childhood ACC.

Germline TP53 mutations predispose to multiple cancers defining Li-Fraumeni/Li-Fraumeni-like syndrome (LFS/LFL), a disease with large individual disparities in cancer profiles and age of onset. G-quadruplexes (G4s) are secondary structural motifs occurring in guanine tracks, with regulatory effects on DNA and RNA. We analyzed 85 polymorphisms within or near five predicted G4s in TP53 in search of modifiers of penetrance of LFS/LFL in Brazilian cancer families with (n = 35) or without (n = 110) TP53 mutations. Statistical analyses stratified on family structure showed that cancer tended to occur ~15 years later in mutation carriers who also carried the variant alleles of two polymorphisms within predicted G4-forming regions, rs17878362 (TP53 PIN3, 16 bp duplication in intron 3; P = 0.082) and rs17880560 (6 bp duplication in 3' flanking region; P = 0.067). Haplotype analysis showed that this inverse association was driven by the polymorphic status of the remaining wild-type (WT) haplotype in mutation carriers: in carriers with a WT haplotype containing at least one variant allele of rs17878362 or rs17880560, cancer occurred ~15 years later than in carriers with other WT haplotypes (P = 0.019). No effect on age of cancer onset was observed in subjects without a TP53 mutation. The G4 in intron 3 has been shown to regulate alternative p53 messenger RNA splicing, whereas the biological roles of predicted G4s in the 3' flanking region remain to be elucidated. In conclusion, this study demonstrates that G4 polymorphisms in haplotypes of the WT TP53 allele have an impact on LFS/LFL penetrance in germline TP53 mutation carriers.

Background: In lung adenocarcinoma, inactivation of the tumor suppressor p53 may abrogate a safeguard mechanism preventing the development of tumors with activating mutations in EGFR or KRAS. To assess this hypothesis, we analyzed TP53 mutations and downregulation of p14(arf), a negative regulator of p53 activated by oncogenic signals, in a retrospective series of 96 patients with primary adenocarcinoma of the lung., Patients and Methods: Mutations in TP53 (exons 4-9), KRAS (exon 1), and EGFR (exons 18-21) were identified by direct sequencing of DNA from formalin-fixed, paraffin-embedded resected tumors. Expression of p14(arf) was semiquantitatively evaluated by immunohistochemical analysis., Results: TP53, KRAS, and EGFR mutations were detected in 42 of 93 (45.2%), 15 of 95 (15.8%), and 31 of 90 (34.4%) cases, respectively. Low p14(arf) expression was observed in 19 of 91 cases (20.9%). Disruption of the p53/p14(arf) pathway (defined as TP53 mutation or decreased p14(arf) expression, or both) was observed in 18 of 31 EGFR-mutated (58.1%) tumors and in 9 of 13 KRAS-mutated (69.2%) tumors., Conclusion: Inactivation of the p53/p14(arf) pathway is common but not systematic in EGFR- or KRAS-mutated lung adenocarcinomas. Our work highlights the need to better investigate the association between EGFR and KRAS mutations and alterations in tumor suppressor pathways., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Pediatric cancers are a feature in patients with Li-Fraumeni syndrome and its variant Li-Fraumeni-like syndrome (LFS/LFL). To the best of the authors' knowledge, TP53 germline mutations are currently the only molecular defect known to be associated with this disease. Recently, a specific germline mutation in this gene, p.R337H, has been reported at a high prevalence in Brazil., Methods: The prevalence of LFS/LFL was investigated in children with cancer who were diagnosed with tumors on the LFS/LFL spectrum and in a small consecutive series of controls without cancer. The prevalence of the germline p.R337H mutation and of other germline TP53 mutations was investigated in a general group of children with cancer and exclusively in children fulfilling the clinical criteria for LFS/LFL, respectively., Results: Among the 65 children without cancer, 1.5% had a family history of LFL whereas of the 292 children with cancer, 25.3% had a family history of LFL (P < .001). Screening for the p.R337H mutation identified 11 carriers (3.7%), 9 of whom were diagnosed with adrenocortical carcinomas (ACC) and 2 of whom were diagnosed with choroid plexus carcinomas. One of the ACC probands was homozygous mutant. The Brazilian founder haplotype and loss of heterozygosity at the p.R337H locus were present in all carriers. In addition, direct sequencing of the entire TP53 coding region and gene rearrangement analysis of probands fulfilling the criteria for LFL (Eeles 2 criteria, Birch and/or Chompret criteria) and who were negative for the p.R337H mutation revealed a DNA-binding domain pathogenic mutation, p.G245S, in 1 child., Conclusions: TP53 p.R337H testing should be offered to Brazilian children diagnosed with ACC and choroid plexus carcinoma. A significant percentage of children with cancer in southern Brazil fulfill the criteria for LFL and should be referred for genetic risk assessment., (© 2013 American Cancer Society.)

Infection by hepatitis B virus (HBV) and dietary exposure to aflatoxin B(1) (AFB(1)) are the main risk factors for the development of chronic liver disease and hepatocellular carcinoma (HCC). How these factors cooperate is still largely unknown. AFB(1) activation leads to DNA adduction and mutagenesis, with a specific mutation at codon 249 in TP53 (p.R249S). So far, only limited studies have addressed the effects of AFB(1) on HBV replication. We have analysed the effects of both risk factors on p53 induction during HBV infection in HepaRG, a cell line with hepatocyte-like morphology that metabolizes AFB(1) and supports HBV infection. Exposure to AFB(1) up to 5 µM induced a downregulation of HBV replication after 48 h, as measured by a decrease in viral antigens in the culture medium (HBsAg, HBeAg and large envelope protein) and in intracellular levels of HBV transcripts, DNA and HBsAg. Conversely, HBV infection did not significantly modify AFB(1)-DNA adduct formation or repair as assessed by immunodot-blot assay, and the induction of p53 in response to AFB(1) was similar in infected and non-infected HepaRG cells. Overall, our results suggest that AFB(1) exposure decreases HBV replication, whereas DNA damage by AFB(1) and subsequent p53 induction is not affected by the presence of the virus. Thus, in HepaRG cell line, AFB(1) and HBV do not cooperate to increase DNA damage by AFB(1). Further studies on the effects of both factors in a context of chronicity are needed to better understand synergistic effects.

Background: Squamous Cell Carcinoma of Esophagus is one of the most common malignancies in both men and women in eastern and south-eastern Africa. In Kenya, clinical observations suggest that this cancer is frequent in the Rift Valley area. However, so far, there has been no report on the molecular characteristics of esophageal squamous cell carcinoma (ESCC) in this area., Results: We have analyzed TP53 mutations, the presence of human papilloma virus (HPV) DNA and expression of inflammation markers Cyclooxygenase 2 (Cox-2) and Nitrotyrosine (NTyR) in 28 cases (13 males and 15 females) of archived ESCC tissues collected at the Moi Teaching and Referral Hospital in Eldoret, Kenya. Eleven mutations were detected in TP53 exons 5 to 8 (39%). All ESCC samples were negative for HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82. Immunohistochemical analysis of Cox-2 and NTyR showed a low proportion of positive cases (17.4% and 39.1%, respectively). No association between the above markers and suspected risk factors (alcohol or tobacco use, hot tea drinking, use of charcoal for cooking) was found., Conclusion: Our findings suggest that mechanisms of esophageal carcinogenesis in eastern Africa might be different from other parts of the world. Low prevalence of TP53 mutation compared with other intermediate or high incidence areas of the world highlights this hypothesis. Our data did not support a possible ole of HPV in this series of cases. Further studies are needed to assess and compare the molecular patterns of ESCC from Kenya with those of high-incidence areas such as China or Central Asia.

Background: Gastric adenocarcinoma is rare in children and adolescents, with about 17 cases under age 21 in the world's literature. We report a case of invasive well-differentiated metastatic gastric cancer in a Brazilian 12-year-old boy without documented familial history of cancer., Case Presentation: The patient, diagnosed with metastatic disease, died seven months after surgery. DNA from intra-surgical specimens revealed a TP53 mutation at codon 337 (p.R337H) in samples with neoplastic cells (dysplasia, tumor and metastasis) but not in non-transformed cells (incomplete intestinal metaplasia and non-involved celiac lymph node). In all mutation-positive tissues, p.R337H occurred on the same background, a founder allele identified by a specific haplotype previously described in Brazilian Li-Fraumeni syndrome patients. The same mutant haplotype, corresponding to a founder mutation present in 0.3% of the general population in Southern Brazil, was found in the genome of the father. Presence of this inherited haplotype in the tumor as well as in the father's germline, suggests a rare case of microchimerism in this patient, who may have harbored a small number of mutant cells originating in another individual, perhaps a dizygotic twin that died early in gestation., Conclusion: This case represents one of the earliest ages at diagnosis of gastric cancer ever reported. It shows that cancer inheritance can occur in the absence of an obvious germline mutation, calling for caution in assessing early cancers in populations with common founder mutations such as p.R337H in Southern Brazil.

The tumor suppressor gene TP53, encoding p53, is expressed as several transcripts. The fully spliced p53 (FSp53) transcript encodes the canonical p53 protein. The alternatively spliced p53I2 transcript retains intron 2 and encodes Δ40p53 (or ΔNp53), an isoform lacking first 39 N-terminal residues corresponding to the main transactivation domain. We demonstrate the formation of G-quadruplex structures (G4) in a GC-rich region of intron 3 that modulates the splicing of intron 2. First, we show the formation of G4 in synthetic RNAs encompassing intron 3 sequences by ultraviolet melting, thermal difference spectra and circular dichroism spectroscopy. These observations are confirmed by detection of G4-induced reverse transcriptase elongation stops in synthetic RNA of intron 3. In this region, p53 pre-messenger RNA (mRNA) contains a succession of short exons (exons 2 and 3) and introns (introns 2 and 4) covering a total of 333 bp. Site-directed mutagenesis of G-tracts putatively involved in G4 formation decreased by ~30% the excision of intron 2 in a green fluorescent protein-reporter splicing assay. Moreover, treatment of lymphoblastoid cells with 360A, a synthetic ligand that binds to single-strand G4 structures, increases the formation of FSp53 mRNA and decreases p53I2 mRNA expression. These results indicate that G4 structures in intron 3 regulate the splicing of intron 2, leading to differential expression of transcripts encoding distinct p53 isoforms.

Background: The objective of this study was to determine whether the mutation status of the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) differed between primary tumors and matched distant metastases in nonsmall cell lung cancer (NSCLC)., Methods: Patients who underwent resection for both primary NSCLC and matched distant metastases were included in the study. KRAS and EGFR mutation status were assessed by polymerase chain reaction (PCR) amplification and direct sequencing on both primary tumors and metastases. For KRAS analysis, mutant-enriched PCR (ME-PCR) was performed in case of discordance between a primary tumor and its matched metastasis., Results: Twenty-one patients were included. No EGFR mutations were detected. KRAS mutations were detected in 6 patients (28%). In all patients, the mutations identified by direct sequencing were discordant between the primary tumor and the matched metastasis. The use of ME-PCR allowed a resolution of the discordance in 3 of the 6 cases by demonstrating the presence of low levels of mutant KRAS in lesions that were negative by direct sequencing., Conclusions: Highly sensitive tools are required to identify biomarkers. The KRAS mutation status mostly was concordant between primary tumors and matched distant metastases. In a few patients, KRAS mutation status differed between different tumor sites., ((c) 2010 American Cancer Society.)

Due to patterns of migration, selection, and population expansion, founder effects are common among humans. In Southern Brazil, a recurrent TP53 mutation, p.R337H, is detected in families with cancer predisposition. We have used whole locus resequencing and high-density single nucleotide polymorphism (SNP) genotyping to refine TP53 locus haplotype definitions. Haplotyping of 12 unrelated p.R337H carriers using a set of 29 tag SNPs, revealed that all subjects carried the same haplotype, and presence of the mutation on this haplotype was confirmed by allele-specific PCR. The probability that this haplotype occurs independently in all index cases was of 3.1x10(-9), demonstrating a founder effect. Analysis of the patterns of 103 tumors diagnosed in 12 families showed that the presence of p.R337H is associated with multiple cancers of the Li-Fraumeni Syndrome (LFS) spectrum, with relatively low penetrance before the age of 30 but a lifetime risk comparable to classical LFS. The p.R337H families are mostly distributed along a road axis historically known as the main route used by merchants of Portuguese origin in the XVIII and XIX century. This historical circumstance and the relatively low penetrance before the age of 30 may have contributed to the maintenance of this pathogenic mutation in a large, open population.

Sarcomatoid carcinomas (SC) of the lung are uncommon malignant tumors composed of carcinomatous and sarcomatous cell components and characterized by a more aggressive outcome than other histological subtypes of nonsmall cell lung cancer (NSCLC). Although epidermal growth factor receptor (EGFR)-targeted therapies have emerged as a promising therapeutic approach in patients with advanced typical NSCLC such as adenocarcinoma, the potential clinical activity of these drugs in lung SC is still unknown. To investigate this point, we have analyzed the status of 4 EGFR pathways biomarkers in a series of lung SC. EGFR protein expression, EGFR gene copy number, EGFR mutational status and KRAS mutational status were assessed in a series of 22 consecutive cases of primary lung SC. EGFR protein overexpression was observed in all the cases. High level of polysomy (>or=4 copies of the gene in >40% of cells) was detected in 5 cases (23%). No EGFR mutation was detected. KRAS mutations were found in 8 patients (38%; Gly12Cys in 6 cases and Gly12Val in 2 cases). The consistent EGFR protein overexpression and the high rate of KRAS mutation may contribute to the poorer outcome of lung SC in comparison with typical NSCLC. The rare incidence of increased EGFR gene copy number, the lack of EGFR mutation and the high rate of KRAS mutation observed in our series also suggest that most patients with lung SC are not likely to benefit from anti-EGFR therapies.

We report on a Malaysian kindred with Li-Fraumeni syndrome. The proband was an 8-year-old girl who presented with embryonal rhabdomyosarcoma of the trunk at the age of 8 months and developed a brain recurrence at the age of 7 years, which was 5 years after remission. A younger sister later developed adrenocortical carcinoma at the age of 6 months. Their mother and maternal grandmother were diagnosed with breast cancer at the ages of 26 and 38 years, respectively. TP53 mutation detection in this family revealed a duplication of a GGCGTG motif starting at nucleotide 17579 in exon 10, resulting in an in-frame insertion of two amino acids between residues 334 and 336 in the tetramerization domain of the p53 protein. This mutation was found in the proband and her affected sister as well as her mother. In addition, the mutation was detected in two other siblings (a brother aged 3 years and a sister aged 18 months) who have not yet developed any malignancy. Sequencing of TP53 in the father and two other asymptomatic siblings revealed wild-type TP53. To our knowledge, this is a first report of a Li-Fraumeni syndrome family in Southeast Asia.

Germline TP53 mutations predispose to a rare familial cancer syndrome, the Li-Fraumeni Syndrome (LFS), characterized by the early onset of multiple cancers including childhood adrenocortical carcinomas, sarcomas and brain tumors, and breast and colon cancer in young adults. An identical germline mutation at codon 337 in TP53 (R337H) has been shown to be causally related to an increased risk of multiple cancers in unrelated subjects with familial cancer risk in Southern Brazil. Here we have assessed the prevalence of R337H in 750 healthy women participating in a community-based breast cancer screening program in the area of Porto Alegre. The mutant was detected in two participants (0.3%) who were fourth-degree relatives and reported a familial history of cancer at multiple sites that did not match classical criteria for LFS and its variants. Testing in additional family members detected the mutation in three subjects, one of whom developed breast cancer at the age of 36. These findings indicate that R337H may be a low penetrance mutant which predisposes to multiple cancers and occurs in the population of Southern Brazil at a frequency 10-20 times higher than other TP53 mutants commonly associated with LFS.

A TP53 germline mutation, R337H, has been previously described in children from southern Brazil with adrenocortical tumours but no documented familial history of other cancers. Here, we have screened for TP53 mutation 45 Brazilian unrelated individuals with family histories fulfilling the clinical definitions of Li-Fraumeni (LFS) or Li-Fraumeni-like (LFL) syndromes. Mutations were found in 13 patients (28.9%), including six (46.1%) R337H mutations, and four novel germline mutations (V173M, V197M, G244D and IVS6+1G>T). Families with the R337H mutation presented a wide spectrum of tumours, including breast cancers (30.4%), brain cancers (10.7%), soft tissue sarcomas (10.7%) and adrenocortical carcinomas (8.9%). Testing of 53 Brazilian subjects with no cancer history showed that R337H was not a common polymorphism in that population. Moreover, loss of heterozygocity with retention of the R337H allele was observed in a breast adenocarcinoma, supporting a role for this mutation in breast tumorigenesis. These results show that the TP53 R337H germline mutation predisposes to a larger spectrum of tumours, similar to the one reported for other TP53 mutations.

Extremely high rates of squamous cell carcinoma of the esophagus (SCCE) are observed in Iran, reflecting unknown, genetic and/or epidemiological risk factors. Among genetic alterations in SCCE, TP53 mutations are the most frequent, vary among populations, and may provide clues on etiological mechanisms. We have analysed mutations in TP53 (exons 5-8) in 98 SCCE from Iran by temporal temperature gel electrophoresis and direct sequencing. We found 58 mutations in 49 patients (50%), with a high prevalence of C to T transitions at CpG dinucleotides (29.3%). The TP53 mutation pattern in Iran was significantly different from that observed in SCCEs from high incidence areas of China and Western Europe (P=0.007). Moreover, the prevalence of mutations at A : T base pairs (transitions and transversions) was higher in men than in women (38.7% vs 11.1%, P=0.033). COX-2 overexpression was detected in 69% of the cases evaluated (24/35), without significant association with TP53 mutation. Accumulation of nitrotyrosine, a marker of protein damage by excess levels of nitric oxide, was observed in tumor cells in six of 18 [corrected] cases analysed. These results are consistent with the hypothesis that several factors are involved in TP53 mutagenesis in Iran. These factors include a baseline of chronic inflammatory stress, which may have a multiplicative impact on the sensitivity of esophageal cells to exogenous factors of risk.

Adenocarcinoma of the esophagus (ADCE) with Barrett's mucosa and adenocarcinoma of the cardia (ADCC) are often reported as a single pathological entity. In this study we have used strict anatomical-pathological criteria to distinguish between these two lesions and we have investigated their differences in TP53 mutations, MDM2 gene amplification, and cytokeratin expression. DNA was extracted from the tumor areas of formalin-fixed, paraffin-embedded sections in 26 ADCC and 28 ADCE patients. TP53 mutations were detected by temporal temperature gradient electrophoresis and identified by sequencing. MDM2 amplification was assessed by differential polymerase chain reaction. The expression of cytokeratins 4, 7, and 13 was examined by immunohistochemistry. In ADCC, the male to female ratio was 1.8:1, compared to 27:1 in ADCE. Five ADCC patients had a history of other neoplasms, compared to only one ADCE patient. The two types of tumor differed in the prevalence of TP53 mutations (31% in ADCC and 50% in ADCE) and of MDM2 gene amplification (19% in ADCC and 4% in ADCE), and in the pattern of expression of cytokeratin 7 (positive in 100% of ADCE and in 41% of ADCC) and cytokeratin 13 (positive in 81% of ADCE and in 36.5% of ADCC). ADCE and ADCC differ in their clinical characteristics, in the prevalence of TP53 mutations and MDM2 amplifications, and in the patterns of cytokeratin expression. These results support the notion that ADCC and ADCE are distinct pathological entities.

Squamous-cell carcinoma of the esophagus (SCCE) shows geographic variations in incidence that are thought to reflect the etiological involvement of environmental or dietary risk factors. Mutations of TP53 are frequent in SCCE, and there is evidence that both the frequency and type of these mutations may differ from one geographic area to the other. Although SCCE is relatively rare in most parts of Thailand, the province of Songkhla (south Thailand) has been described as a high-risk area for SCCE. We have analyzed 56 SCCE cases from this area for TP53 mutations by denaturing gradient gel electrophoresis (DGGE, exons 5-8) and direct DNA sequencing. The same tumors were also analyzed for MDM2 gene amplification by differential PCR. TP53 mutations were detected in 23 cases (41%). In contrast, clear amplification of MDM2 was detected in only 2 cases (4%), both of which contained wild-type TP53. Comparison with published results from other geographic areas of high SCCE incidence revealed that the spectrum of TP53 mutations in south Thailand is similar to that observed in central China (Henan Province) but clearly differs from that of SCCE from western Europe (Normandy, France; northern Italy), with more G:T transversions and fewer mutations affecting A and T base pairs. These results suggest that SCCE from south Thailand and from central China may involve similar risk factors., (Copyright 2000 Wiley-Liss, Inc.)

The ataxia telangiectasia gene (ATM) has been implicated as a risk factor in the development of sporadic breast carcinomas. ATM protein expression was analyzed by immunohistochemistry in 17 breast carcinomas with two monoclonal antibodies whose immunohistochemical use was first validated by comparing the immunoreactivity observed in spleen samples from ataxia telangiectasia and trauma patients. In normal breast ducts, ATM showed nuclear expression in the epithelial but not in the myoepithelial cells. In contrast, this nuclear expression was absent or low in the epithelial cancer cells in 10 of 17 (59%) of the tumors studied. Allelic imbalance in the ATM gene was found in three of seven tumors examined. Two of these showed reduced ATM protein expression, but this did not correlate with the presence of ATM mutations in the tumor DNA detected by restriction endonuclease fingerprinting screening. These results suggest that the reduced ATM protein expression could be attributable, in certain tumors, to deletions or rearrangements within or close to the ATM gene. Positive p53 immunostaining was found in 10 tumors, with TP53 mutations detected in 8. Three tumors had both low ATM expression and mutated TP53. Our results indicate that in the majority (15 of 17) of the sporadic breast carcinomas examined, not only is the functionality of the ATM-p53-mediated DNA damage response compromised, but also other signaling pathways activated by these two multifunctional proteins are likely to be impaired, which could be a contributing factor to tumor development and progression.

Background/aim: Fibrosis and/or cirrhosis are present in the precursor stages of most liver cancers. However, little is known about the reciprocal interactions of fibroblasts, mainly responsible for fibrosis, and the other liver cells. We report here the isolation of a new liver myofibroblast cell line from a human liver angiosarcoma and its characterization., Methods: The cells were isolated by the explant technique and characterization was performed, on one hand, using immunohistochemical and ultrastructural analysis and, in the other hand, by determining their karyotype, ras and p53 status and their tumorigenic properties., Results: To date, the cells have undergone approximately 170 population doublings and are still proliferating. Immunohistochemically, they were negative for desmin, smooth muscle myosin, cytokeratin 19 and von Willebrand factor, positive for vimentin and alpha-smooth muscle actin, with an important deposition of fibronectin around the cells. Ultrastructure showed particularly cytoplasmic microfilament bundles. Their chromosome number ranged from 38 to 168 with a bimodal population, near diploid and hypotetraploid. No mutations were found in codons 12, 13 or 61 of Ha-, Ki- and N-ras genes but a homozygous missense mutation in codon 179 (CAT-->CTT) was detected in the p53 gene. They were unable to form foci in soft agar or tumors in nude mice., Conclusions: Taken together, these results show that these cells, called BM 2.2.1, exhibited typical myofibroblast-like features. Although they contained a karyotype suggestive of tumoral cells and a homozygous mutated p53 gene, they were not tumorigenic. The nature of these cells and the abnormalities of the p53 gene and the karyotype, suggest that: i) they were a component of the tumor stroma, and ii) they could have been involved in angiosarcoma development. Thus, this cell line may be valuable for the study of cellular interactions in liver carcinogenesis.

Alteration of the p53 gene is thought to be important in the early stages of human esophageal cancers, but how this confers a selective advantage to esophageal cancer cells is unknown. In this report, we analyzed 9 cell lines derived from human esophageal cancers (TE-1, TE-3, TE-6, TE-7, TE-9, TE-10, TE-11, TE-13 and TE-15) for mutations in the p53 sequence, p53 protein expression and p53 protein DNA-binding activity. The cell lines could be grouped in 3 categories, including (1) cell lines with mis-sense mutations in the coding sequence and accumulation of mutant proteins (TE-1, TE-6, TE-10 and TE-11); (2) cell lines expressing truncated forms of p53 as a result of frameshift (TE-9) or splice-site (TE-15) mutations; and (3) cell lines with wild-type p53 sequences but with impaired expression of p53 mRNA and protein, suggesting that p53 is inactivated by transcriptional repression (TE-3, TE-7 and TE-13). With the exception of TE-1, none of the cell lines exhibited p53-DNA-binding activity. In TE-1, a mutation at codon 272 (methionine to valine) generated a protein that retains basal DNA-binding activity, but that was not activated in response to DNA damage, suggesting that this mutation prevented p53 induction by genotoxic stress. Thus, p53 activity was impaired in all esophageal cell lines, including those containing wild-type p53 sequences.

Mutational analysis of the p16/CDKN2 gene was conducted by direct sequencing of the whole coding sequence (exons 1-3 and flanking splicing sites) in 21 esophageal squamous-cell carcinomas and 3 adenocarcinomas from a high-incidence area of Italy. Two inactivating mutations were found in exon 1 of the gene (both in squamous-cell carcinoma), whereas no mutations were detected in exon 2, where most of the sequence changes reported so far have been located, or in exon 3. Southern blot analysis of exon 2 in this set of samples and in a complementary set of 12 tumor samples from France did not show homozygous deletions or detectable gene rearrangements. Thus, p16/CDKN2 gene alterations do not appear to play a major role in the group of patients examined.

Carcinomas of the upper digestive tract (squamous-cell carcinoma of the esophagus, adenocarcinoma of the cardia) from 24 patients residing in Linxian (China) and near-by high-incidence areas were analyzed for mutations in exons 5-8 of the p53 tumor-suppressor gene. Mutations were identified by polymerase chain reaction amplification and direct sequencing in 50% of the specimens. Eleven tumors harbored a single base-pair substitution leading to either an amino-acid substitution (8 tumors) or a chain-termination signal (3 tumors), and one tumor revealed a 15-bp deletion in exon 7 with a silent base substitution adjacent to the deletion site. Mutations occurred in all 4 exons examined, with a preponderance in exon 5. Of the 6 mutations identified among the 14 adenocarcinomas examined, 3 were G to T transversions, a mutation that has thus far been absent from reported mutations in Barrett's esophageal adenocarcinomas and dysplasias from patients residing in Europe and North America.

Point mutations in the p53 tumor suppressor gene are the most common genetic alterations in human cancers. The nature and location of these mutations can be informative in assessing the importance of putative carcinogenic agents. Potential associations between a given carcinogen and a specific mutation pattern can be substantiated when the exposure history of the patients is known. While the past exposure to environmental risk factors is often difficult to determine, documented occupational exposure to carcinogens presents a unique situation for evaluating this approach. Analysis usually involves working with paraffin-embedded tissues, fixed under conditions suboptimal for genetic analysis and stored for many years, since frozen tissues are not available in sufficient numbers. The particular methodological problems encountered with fixed samples are discussed here, using as illustration an ongoing study of oncogene and tumor suppressor gene mutations in archived bladder tumors of workers exposed to aromatic amines and nonexposed patients.

A procedure for measuring the level of O6-methylguanine (O6-meG) in DNA is described. Repair of 32P-oligodeoxynucleotides containing O6-meG adducts by O6-alkylguanine alkyltransferase (AGT) was performed in the presence of different quantities of DNA containing unknown concentrations of O6-meG. Each methylated DNA sample inhibited the repair of oligodeoxynucleotide substrate to an extent dependent upon O6-meG concentration. Each DNA sample tested at different concentrations in the assay therefore had a characteristic inhibition curve and could be compared to the curves generated using reference DNA samples of known O6-meG concentration. We report the method of calculation of the O6-meG level in a given DNA sample by comparison of its inhibition curve with that of reference DNAs. This method of calculation does not require a knowledge of the exact quantity of the labelled substrate or AGT used. The method requires only 0.1-10 micrograms of DNA, with a limit of detection of 0.8 fmol of O6-meG per microgram of DNA.

Distinct species differences exist between BDIV rats and Syrian Golden hamsters in the repair of methylated DNA lesions, after single exposures to dimethylnitrosamine (DMN). The promutagenic lesions O6-methylguanine (O6-MeG) and O4-methylthymidine were actively repaired in rat liver; in contrast, in hamster liver the levels of O6-MeG remained relatively stable while O4-methylthymidine levels were reduced. Species differences in the levels of two enzymes involved in the repair of DNA alkylation damage were also noted. An increase in the methylpurine-DNA glycosylase levels was seen in both species following DMN exposure; however, significant species differences in the inactivation and subsequent time course of recovery of the "suicide protein" O6-MeG-DNA methyltransferase were observed. In the rat a rapid recovery of activity began within 24 h of DMN exposure (20 mg/kg) and an approximately 3-fold induction in enzyme levels was observed at 96 h. In hamster liver, in which the constitutive level of expression of this enzyme is similar, no activity was detectable up to 96 h after treatment (25 mg/kg DMN). Only in animals in the lowest treatment group (2.5 mg/kg DMN) was a significant recovery seen, 264 h after treatment. The data presented suggest that the schedule of DMN treatment, in particular the time between doses of the carcinogen and the regeneration of the O6-MeG-DNA methyltransferase, would evoke different carcinogenic responses in hamster and rat liver following chronic exposure to alkylating agents.

Various methods for detecting DNA alkylation adducts are described briefly, with emphasis on immunoassays using antibodies against O6-methyldeoxyguanosine (O6-medGua), O4-methylthymidine (O4-meThy) and 7-methyldeoxyguanosine (7-medGua). The application of these methods to epidemiological studies is discussed, and results obtained so far on the presence of DNA alkylation adducts in human tissues are presented.

A sensitive and rapid procedure for measurement of alkyltransferase repair activity involving oligodeoxynucleotides followed by immunoprecipitation is described. Dodecadeoxynucleotides containing O6-methylguanine or O4-methylthymine were used as substrates for alkyltransferases and the reaction products of methylated or demethylated substrates were separated by precipitation with highly specific antibodies. This approach for O6-alkylguanine-DNA alkyltransferase measurement is far more rapid than when the reaction products are separated by chromatography. This technique makes the assay applicable to large-scale epidemiological or clinical studies and suggests a similar methodology could be applied for other DNA repair enzymes.