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This study was a prospective single arm trial conducted in Zhejiang Jinhua Guangfu hospital from February 2018 to June 2020. A total of 39 patients (32 males and 7 females) with esophageal cancer, aged from 44 to 82 (69±9) years were enrolled. Diffusion weighted magnetic resonance imaging(MR-DWI) was implemented to evaluate the changes of apparent diffusion coefficient(ADC) value before and after chemoradiotherapy. The results showed that the ADC value after chemoradiotherapy was higher than that before treatment[(2.03±0.42)×10⁻³ mm 2 /s vs (1.60±0.28)×10⁻³ mm 2 /s], and there was a positive correlation between the increase of ADC value and the prognosis of patients.

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.

We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. We also examined 36 of these 50 carcinomas for expression of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene.

Amplification of the hst-1 and int-2 genes on chromosome 11q13 has previously been found in over 20% of human primary esophageal cancers. However, these two genes do not appear to be transcribed in appreciable amounts. Recently, the human cyclin D gene (also referred to as prad1) has been mapped to the 11q13 locus. Here, we report coamplification of the cyclin D and hst-1 genes in 5 of 20 (25%) human squamous esophageal tumors. We also detected significant levels of cyclin D transcription in two esophageal carcinoma cell lines, even though they did not express detectable amounts of hst-1 transcription. These findings provide the first evidence for the amplification of a cyclin gene in human esophageal cancer and suggest that an increase in cyclin D gene dosage could be an important factor in the pathogenesis of esophageal cancer. Additionally, because the 11q13 locus is found to be amplified in many types of human tumors, cyclin gene amplification could also play an important role in the development of other forms of human cancer.

Oesophageal cancer occurs at a very high frequency in certain areas of China, especially in Linxian county, Henan province. Previous studies suggested that N-nitroso compounds play a causative role. In order to study further the exposure of Linxian inhabitants, the intake of N-nitrosamines in the diet was determined. The total daily intake of volatile nitrosamines and of N-nitroso-N-methylbenzylamine (NMBzA) was higher in Linxian than in two other counties. NMBzA can induce cancer in animal and human oesophageal epithelium. Human fetal oesophageal epithelia were cultured with NMBzA for 4 h, and eight monkeys were treated with NMBzA. High-molecular-weight DNA extracted from explants and from the oesophageal epithelia of monkeys induced malignant transformation of NIH 3T3 cells. Alu and monkey-specific repeat sequences were present in transformed cells, and H-ras was found in the transforming DNA. Human fetal oesophageal epithelium cultured with NMBzA was transplanted into the mesentery of BALB/c nude mice. Tumours were found after eight months; and the Alu sequence was present in DNA extracted from tumours induced by NMBzA, showing that the tumours were of human origin. The results provide direct evidence that NMBzA is carcinogenic in human oesophageal epithelium and that N-nitrosamines are one of the causative factors of oesophageal cancer in Linxian county.

Studies on the relevance of the N-nitrosamines to esophageal cancer in China are reviewed. Esophageal cancer is a complex and multifactorial problem. Although a causal association between nitrosamines exposure and esophageal cancer in China has not yet been rigorously established, exposure of Lin-Xian subjects to nitrosamines either directly or as a result of their in vivo formation has been detected in our study. Several N-nitrosamines (NDMA, NDEA, NMBzA, NPyr, NPip, and NSAR) in gastric juice collected from Lin-Xian inhabitants have been detected. A correlation was found between the lesions of esophageal epithelium and the amount of nitrosamines present. In addition, the amounts of N-nitrosamino acids (N-nitrosoproline, N-nitrosothiazolidine 4-carboxylic acid, NSAR, and nitrates) excreted in 24-hr urine of subjects in Lin-Xian were significantly higher than those in Fan-Xian, indicating a higher exposure to N-nitroso compound and their precursors of the inhabitants in the high-risk area. The effect of nitrosamines on human esophagus has been investigated at the cellular levels. The amounts of O6-MedG in DNA of esophageal or stomach mucosa of patients from Lin-Xian were higher than that from Europe (Lyon and Essen). The presence of O6-MedG in the human fetal esophagus cultured with NMBzA was also detected. These findings indicate that the elevated levels of O6-MedG in esophageal DNA could be the result of a recent exposure to N-nitroso compounds or a genetically determined reduced cellular capacity for repair of O6-MedG from DNA. The hyperplasia was induced in the esophagus of human fetus that cultured with NMBzA for 2 weeks to 2 months. The intervention studies of esophageal cancer in Lin-Xian have been pursued. Intake of moderate doses of ascorbic acids by Lin-Xian subjects effectively reduced the urinary levels of N-nitrosamino acids to those found in un-dosed subjects in the low-risk area. If N-nitroso compounds are formed in vivo and are among the causative factors of esophageal cancer in Lin-Xian, ascorbic acid appears to be effective in lowering the body burden of these carcinogenic compounds. Thus, the plan of chemoprevention is carried out in Lin-Xian.

We have developed a rapid, nonradioactive large scale method for the detection of ras oncogenes in human tumors. DNA is amplified by the polymerase chain reaction (PCR), and then digested with specific restriction enzymes to detect either endogenous or primer-mediated Restriction Fragment Length Polymorphisms (RFLPs). We report here that three of 15 colon tumors tested contain K-ras codon 12 aspartic acid mutations and one, along with the HCT 116 colon carcinoma cell line, contains a K-ras codon 13 aspartic acid mutation. On the other hand, we did not detect H- or K-ras codon 12 mutations or the K-ras codon 13 aspartic acid mutation in 25 esophageal and 27 gastric cardia tumors isolated from patients in Lin-xing County, China. By incorporating nucleotide substitutions in PCR primers, this method can be applied towards the rapid, non-radioactive screening of virtually any genetic disease caused by known point mutations.

Possible etiological factors involved in oesophageal cancer in various parts of the world and in certain provinces in Northern China are summarized. Evidence is accumulating that N-nitroso compounds and their precursors are involved in the disease in Northern China, as shown in a recent study: excretion of urinary N-nitrosamino acids by inhabitants living in a high- (Linxian) and in a low-risk area (Fanxian) for oesophageal cancer was compared. Linxian subjects excreted significantly more nitrate and nitrosamino acids (N-nitrosoproline, N-nitrosothiazolidine-4-carboxylic acid, N-nitrososarcosine) than those in Fanxian. When Linxian subjects were given 100 mg vitamin C three times a day (after each meal) together with proline, the level of urinary N-nitrosamino acids was reduced to that found in Fanxian. Thus, vitamin C, an efficient inhibitor of endogenous nitrosation, should now be examined in intervention trials in subjects in whom endogenous formation of N-nitroso compounds is elevated.

Extracts of pickled vegetables commonly consumed in Linhsien County, a high-incidence area for esophageal cancer in Northern China, were studied for mutagenicity. The liquid residue from ethereal extracts produced a dose-dependent increase of mutants in Salmonella typhimurium TA98 and TA100 strains; mutagenicity required the presence of a fortified liver microsomal activation system induced by Aroclor 1254 in adult male BD VI inbred rats. An amount of extract equivalent to 2.8 g fresh pickled vegetables produced sixfold (75 revertants/g) and twofold (45 revertants/g) increases in revertant frequencies in strains TA98 and TA100, respectively. Roussin's red methyl ester, a tetranitroso compound, [(NO)2Fe(CH3S)]2, not previously reported to occur in nature, was isolated and identified from the ethereal extracts. The synthetic compound was mutagenic in strain TA100 in the presence of a liver activation system, producing 25 revertants/mumol. Findings on the presence of mutagenic compounds in pickled vegetables were discussed in relation to their possible etiologic role in cancer of the esophagus in Linhsien County.

A radioimmunoassay using monoclonal antibodies (MAb) against 0(6)-medGuo and 0(6)-etdGuo enabled the presence of these DNA adducts to be detected in human tissues. With this methodology and with 1 mg of hydrolysed DNA it is possible to detect approximately 25 fmol 0(6)-medGuo or 12.5 fmol 0(6)-etdGuo per mg DNA. The analysis comprised a total of 37 human tissue specimens derived from patients who underwent surgery for cancer of the oesophagus in Linxian County (People's Republic of China); for comparison 12 tissue samples obtained from hospitals in Europe were similarly analysed. Seventeen samples from Linxian County showed a level of 0(6)-medGuo ranging from 15 to 50 fmol/mg DNA, 10 showed higher levels up to 160 fmol/mg DNA, and the remaining 10 samples were below the limit of detection. All the tissue samples from Europe showed levels below 45 fmol 0(6)-medGuo/mg DNA, 7 being below the limit of detection. No 0(6)-etdGuo was detected in any of the samples. 0(6)-alkylguanine DNA alkyltransferase activity observed in tissue extracts from Linxian County ranged from 190 to 326 fmol (mean values) of 0(6)-medGuo removed per mg protein and lower values were obtained for the removal of 0(6)-etdGuo. The findings obtained using this approach appear promising in assessing the role of alkylating agents, e.g. nitrosamines, in the aetiology of human cancer.

Samples of gastric juice were collected from a high-risk area for oesophageal cancer in China and analysed for N-nitroso compounds. N-Nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosomethylbenzylamine (NMBzA), N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP) were identified in descending order of concentration. Several unknown compounds were also detected in the fasting gastric juice. A positive correlation was found between the amount of nitrosamines in gastric juice and the degree of severity of lesions of the oesophageal epithelium: the amounts of nitrosamines in gastric juice from subjects with a normal oesophageal epithelium were lower than those in subjects with marked dysplasia or carcinoma of the oesophagus. In addition, 1500 samples of 24-h urine were collected from various communes in six high-risk areas and two low-risk areas for this cancer in China and analysed for N-nitrosamino acids. Subjects in high-risk areas excreted higher levels than those in low-risk areas. Intake of L-proline resulted in marked increases in levels of urinary N-nitrosoproline (NPRO) in inhabitants from both high- and low-risk areas. Intake of moderate doses of vitamin C, alpha-tocopherol and zinc by high-risk subjects reduced the urinary levels of N-nitrosamino acids to those found in undosed subjects in low-risk areas, suggesting a rational basis for prevention in high-risk areas.

Studies on the pattern of proliferation of epithelial cells from the oesophagus of 33 individuals from Linxian, a high-risk area for oesophageal cancer in China, and of 22 subjects from Jiaoxian, a low-risk area for the same cancer in China, were performed using thymidine labelling. Oesophageal biopsies were obtained during endoscopic surveys carried out in these 2 populations. A clear difference was observed between the 2 groups; the high-risk group showed cell proliferation in the upper layers of the epithelium more often than the low-risk group. No difference was found in the cell proliferation patterns of subjects with oesophagitis and those without oesophagitis in the high-risk area. This finding could suggest that the tritiated thymidine assay may be a more sensitive method to characterize the precancerous lesions of the oesophagus in high- and low-risk populations than simple histological evaluation.

N-Nitrosamines are alkylating agents capable of forming various covalent adducts with DNA in vivo. Since formation of promutagenic DNA adducts, particularly O6-methyldeoxyguanosine (O6-medGuo), has been implicated as an initiating event in nitrosamine-induced carcinogenesis, we have used radioimmunoassay (RIA) to probe for such damage in surgical tissue samples from cancer patients in populations likely to be exposed environmentally to nitrosamines.

China could be expected to offer good opportunities for the investigation of esophageal cancer since it has both high and low incidence areas. However, it seems impossible to attribute the induction of esophageal cancer to one particular substance--it rather seems to be caused by a variety of factors. To the authors' opinion, esophageal cancer may originate from exposure to N-nitroso compounds or mycotoxins. Lack of certain trace elements or essential vitamins may also play a role. Contrary to France, which also has high incidence areas of esophageal cancer, excessive alcohol consumption is of no importance in China. It is not clear whether genetic factors can be discussed.

The incidence of esophageal cancer is extremely high in Linxian County and certain other regions of the People's Republic of China. Epidemiologic and laboratory studies suggest that N-nitroso carcinogens and other environmental factors play a causative role. In the present study, employing over 100 DNA samples obtained from Lin-xian patients who underwent surgery for esophageal cancer, we have found a significant frequency of amplification of either the human epidermal growth factor receptor (HER-I) gene or the c-myc oncogene. These changes were found not only in tumor specimens, but also in adjacent non-tumor (grossly normal) tissue specimens obtained from patients with esophageal cancer. RNA samples were also obtained from over 30 tissue samples. These revealed considerable variation in the abundance of HER-I and c-myc transcripts in both the tumor and adjacent non-tumor specimens. A few samples revealed extremely high levels of these transcripts. Thus, changes in gene copy number or level of expression of HER-I or c-myc DNA sequences may play an important role in the pathogenesis of esophageal cancer in this high-risk region.

A total of 238 samples of 24-h urine were collected from inhabitants of high-risk (Lin-xian) and low-risk (Fan-xian) areas for esophageal cancer in northern China, according to three protocols: (a) from undosed subjects; (b) from subjects who had ingested 100 mg L-proline three times a day 1 h after each meal; and (c) from subjects in Lin-xian who had ingested 100 mg ascorbic acid together with 100 mg L-proline three times a day 1 h after each meal. As an index of individual exposure to N-nitroso compounds or their precursors, ingested in food and/or formed endogenously, the levels of four urinary N-nitrosamino acids and nitrate were determined. The amounts of N-nitrosoproline, N-nitrosothiazolidine 4-carboxylic acid, N-nitrososarcosine, and nitrate excreted in the 24-h urine of undosed subjects in Lin-xian were significantly higher than those in Fan-xian, indicating a higher exposure of the inhabitants in the high-risk area to N-nitroso compounds and their precursors. Ingestion of L-proline resulted in a marked increase in urinary N-nitrosoproline levels in inhabitants from both areas, suggesting that endogenous nitrosation may occur to a larger extent when appropriate amine precursors are ingested in foods. Intake of moderate doses of ascorbic acid by high-risk subjects effectively reduced the urinary levels of N-nitrosamino acids to those found in undosed subjects in the low-risk area. If N-nitroso compounds formed in vivo are among the causative factors for esophageal cancer in this area of northern China, ascorbic acid appears to be effective in lowering the body burden of these carcinogenic compounds, thus offering a rational basis for long-term intervention studies in this area.