Perineural Invasion is a Better Prognostic Indicator than Lymphovascular Invasion and a Potential Adjuvant Therapy Indicator for pN0M0 Esophageal Squamous Cell Carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) at pN0M0 can be more locally aggressive and disseminated than those with lymph node and distant metastasis. Perineural invasion (PNI) is reported as a poor prognostic factor in cancer and is thought to be related to regional tumor spread and metastasis. However, its clinicopathological role and meaning for treatment in pN0M0 ESCC are unknown. PATIENTS AND METHODS: We applied scoring methods of PNI and lymphatic and vascular invasion (LI, VI) based on immunohistochemistry staining on tumor tissues of pN0M0 ESCC patients. ROC analyses, Kaplan-Meier analyses, Cox regression, and χ2 test were performed for survival analysis, comparison of PNI with LI and VI, and exploration of the relevance between PNI and other clinicopathological features. RESULTS: Presence of PNI was significantly associated with poor survival in pN0M0 patients, whereas LI and VI were not predictive of outcome (P > 0.05). Neural invasion index (NII), defined as the ratio of the number of tumor-invaded nerves to the total number of nerves per tumor microsection, was the most consistent measure of PNI (P = 0.006, HR = 6.892, 1.731-27.428). Postoperative radiotherapy significantly improved survival in high-NII patients (P = 0.035, HR = 0.390, 0.163-0.936). CONCLUSIONS: PNI is an important risk factor for the outcome of pN0M0 ESCC patients. NII can be used for risk assessment and to tailor adjuvant radiotherapy in this population.

Esophageal intraepithelial invasion of Helicobacter pylori correlates with atypical hyperplasia.

Helicobacter pylori (H. pylori), a common pathogen residing in the gastrointestinal tract, has been well characterized in stomach cancer,while its correlation with esophageal cancer remains poorly understood. In this study, we aim to assess the relationship between esophageal intraepithelial H. pylori invasion and inflammation as well as atypical hyperplasia in esophageal squamous epithelial tissues. Esophageal squamous cell carcinoma (ESCC) tissue samples from 196 individuals from both southern and northern esophageal carcinoma high-risk areas in China were examined (125 from northern high-risk areas, 71 from southern high-risk area), while additional 30 samples were collected adjacent to the esophageal squamous cell carcinoma (A-ESCC). H. pylori infection was identified by Giemsa staining, immuno-histochemical staining, and H. pylori 16S rRNA-based PCR. A significant increase of H. pylori infection was found in tumor tissues (including ESCC and A-ESCC samples) compared to that of non-tumor tissues (p < 0.05). The positive rate of H. pylori 16S rRNA in ESCC, A-ESCC, and normal groups were 62.5, 74.1, and 26.7%, respectively. The PCR results showed that the positive incidence of the H. pylori virulence factor CagA gene in tumor (ESCC and A-ESCC) and normal groups was 54.9 and 20%, respectively (p < 0.05). To explore the possible causes of CagA+ H. pylori infection leading to carcinogenesis, we found that CagA+ H. pylori filtrate induced DNA strand breaks in esophageal epithelial NE3 cells, suggesting that H. pylori infection may be an original cause leading to atypical hyperplasia of esophageal squamous epithelial tissues and contributed to pathological carcinogenesis of ESCC.

Identification and Prognostic Effect of Extramural Venous Invasion in Locally Advanced Esophageal Squamous Cell Carcinoma.

The role of extramural venous invasion (EMVI) in esophageal cancer is still unclear. This study aimed to identify EMVI and assess its impact on survival and recurrences in esophageal squamous cell carcinoma (ESCC). Retrospectively, we reviewed resection specimens of 147 locally advanced ESCC (pT3-T4aN0-3M0) patients who had a curative intended surgery alone at the Cancer Hospital of Shantou University from March 2009 to December 2013. After confirming pT≥3 in hematoxylin-eosin tumor slides, EMVI was evaluated by Verhoeff and Caldesmon staining. The impact of EMVI with other clinicopathological characteristics and survival were analyzed using the χ 2 test, Cox regression, and Kaplan-Meier method. EMVI was present in 30.6% (45/147) of the P ≥T3 ESCCs and associated with lymph-vascular invasion and poor differentiation grade ( P <0.05). Disease-free survival and overall survival in patients with EMVI-absent tumors were about 2.0 times longer than in those with EMVI-present tumors. In pN0 patients, EMVI-presence was associated with poor overall survival (HR 4.829, 95% CI 1.434-16.26, P =0.003) and Disease-free Survival (HR 4.026, 95% CI 0.685-23.32, P =0.018). In pN1-3 patients, EMVI had no additional effect on survival. Conclusions EMVI has an independent adverse prognostic effect on survival in ESCC patients after surgery alone. EMVI should be included in pathology reports as it might contribute to identify high-risk patients for potential additional treatment.

Analysis of basement membrane structure and inflammation during the development of esophageal squamous cell carcinoma in the Chinese Chaoshan high risk region.

BACKGROUND & AIMS: To investigate relationships between basement membrane structure, inflammation, beta1 integrin expression, activation of ERK/MAPK signaling pathways, and cell proliferation in esophageal mucosa at various stages during the evolution of esophageal squamous cell carcinoma. METHODS: Three tissue arrays were made of 228 tissue cores from 428 surgically-resected specimens. The arrays included 26 samples of normal epithelium, 28 with hyperplasia, 18 with dysplasia, 27 with carcinoma in situ and 129 with invasive carcinoma. In addition, 21 cases of hyperplasia, 13 cases of dysplasia and 13 case of carcinoma in situ were obtained by manual microdissection of unfixed frozen tissue. Hematoxylin and eosin stained sections were used to evaluate the epithelium and inflammation. The periodic acid-Schiff stain and an immunohistochemical stain for laminin were used to examine the structure of basement membranes. The expression of beta1 integrin, p-ERK, and Ki67 were evaluated by quantitative immunohistochemistry. RT-PCR and Western blots were also used to detect expression of beta1 integrin. RESULTS: Quantitative scales were developed to classify basement membrane structure and inflammation. Basement membrane alterations correlated with the degree of epithelial change (chi2 = 501.9, p < 0.01) and with the degree of lymphocytic infiltration in the lamina propria and epithelium (chi2 = 273.4, p < 0.01). There was a significant relationship between the extent of basement membrane alteration and the expression of beta1 integrin, p-ERK, and Ki67. CONCLUSIONS: The correlations suggest that there is a direct relationship between basement membrane structure and the development of esophageal squamous cell carcinoma.

Cognitive impairment in rats after long-term exposure to GSM-900 mobile phone radiation.

Considering the frequent use of mobile phones, we have directed attention to possible implications on cognitive functions. In this study we investigated in a rat model the long-term effects of protracted exposure to Global System for Mobile Communication-900 MHz (GSM-900) radiation. Out of a total of 56 rats, 32 were exposed for 2 h each week for 55 weeks to radio-frequency electromagnetic radiation at different SAR levels (0.6 and 60 mW/kg at the initiation of the experimental period) emitted by a (GSM-900) test phone. Sixteen animals were sham exposed and eight animals were cage controls, which never left the animal house. After this protracted exposure, GSM-900 exposed rats were compared to sham exposed controls. Effects on exploratory behaviour were evaluated in the open-field test, in which no difference was seen. Effects on cognitive functions were evaluated in the episodic-like memory test. In our study, GSM exposed rats had impaired memory for objects and their temporal order of presentation, compared to sham exposed controls (P = 0.02). Detecting the place in which an object was presented was not affected by GSM exposure. Our results suggest significantly reduced memory functions in rats after GSM microwave exposure (P = 0.02).

[Effect of selenium deficiency on the F344 inbred line offspring rats' neuro-behavior, ability of learning and memory].

OBJECTIVE: To establish the selenium (Se) deficient animal model on F344 inbred line rats and observe the effects of a long-term Se-deficiency on the offspring's neuro-behavior, abilities of learning and memory. METHODS: Feeding F344 inbred line rats on Se-deficient diet to establish Se-deficient animal model. For the offspring, the body weight, physiological indexes nervous reflections for growth and development were monitored during the early postnatal period. RESULTS: The Se-deficient diet contained less than 0.01 mg/kg and the glutathione peroxidase (GSH-Px) activity in blood of the Se-deficient group rats is lower than the Se-normal group after feeding on Se-deficient diet for 4 weeks. For the offspring, the birth weight and the body weight of Se-deficient group were obviously lower than the Se-normal group before weaning. Se-deficient offspring rats differed from Se-normal controls in lower scores in surface righting reflex (RR) test at postnatal 4th day after delivery, cliff avoidance test at postnatal 7th day and auditory acuity trial at postnatal 10th day respectively. But these differences disappear after a few days in the same tests. In addition, no significant differences between two groups in suspending test and walking ability test at postnatal 12th and 14th day. In open field test, Se-deficient male offspring stayed less time in the middle grid and moved less. In Morris water maze test, the Se-deficient offspring spent more time to find the hidden platform at the 6th and 9th training tests in the place navigation trial. Furthermore, the Se-deficient group spent less time in target quadrant when giving the spatial probe trial. CONCLUSION: A Se-deficient animal model have been established on F344 inbred line rats successfully. A long-term Se deficiency could retard the development of the offspring in uterus and after delivery. Se deficiency also decreased the offspring's abilities of spatial learning and memory in Morris water maze test and resulted in the male offspring's nervousness to new stimulant.

[Effects of selenium and B-27 supplements on viability and differentiation of neural stem cell in newborn rat].

OBJECTIVE: To assess how trace element selenium and B27 supplements affect the neural stem cell (NSc) differentiation in vitro. METHODS: The development and differentiation of NSc from the newborn rat were observed with primary culture and subculture during treating by sodium-selenite, and selenium-methyl-cysteine (SMC). The immunocytochemistry techniques were used to identify the NSc and mature protein expression with neuron marker beta-tubulin, astrocyte marker GFAP, and oligodendrocyte marker CNPase. The neurosphere morphology and neurite outgrowth were observed. RESULTS: Adding the complete B-27 serum-free supplement, Selenium could promote the neurosphere viability, development and differentiation. Without selenium and B-27, neurosphere could not survive and differentiate. Without B-27 in the medium but there containing selenium, the neurosphere could promote the viability and development into neuron, astrocyte and oligodendrocyte, as compared with the no-containing B-27 and selenium groups, these differentiated cells might have more quantity, more branches and better morphological nerve net. The count of the neuron, astrocyte and oligodendrocyte was 11.2/Hp, 16.1/Hp and 9.3/Hp. CONCLUSIONS: The selenium should be very important for neural stem cells' survival. Selenium could promote the neurosphere cells differentiation and development.

[Effects of prolonged selenium deficiency on synaptic structures in CA3 area of hippocampus in the third generation rats].

OBJECTIVE: The relationship between selenium deficiency and the changes of synaptic structure in the CA3 area of hippocampus were studied in the third generation rats. METHODS: A selenium deficiency model was established by feeding rats with selenium-deficient food. The rats were divided into 4 groups: control (Se+I+), selenium deficiency (Se-I+), iodine deficiency (Se+I-), and both deficient group (Se-I-). The hippocampuses were dissected from the third generation rats on the 21st gestational day and the ultrastructural features of hippocampal synapses were observed with electron microscope. The length of active zone, synaptic curvatures, post-synaptic density (PSD) and synaptic cleft were quantitatively described. RESULTS: Compared with the control, the length of active zone and the thickness of PSD were significantly decreased in Se-I+, Se+I- and Se-I- groups [(261.7 +/- 50.1) nm, (286.7 +/- 41.6) nm and (220.8 +/- 61.6) nm contrast to (312.4 +/- 47.7) nm, P < 0.01], so were the synaptic curvatures in Se-I+, Se+I- and Se-I- groups [(22.9 +/- 6.3) nm, (27.5 +/- 8.6) nm and (25.2 +/- 6.5) nm contrast to (48.1 +/- 12.3) nm, P < 0.01]; the width of synaptic cleft were also decreased significantly in Se-I- [(11.1 +/- 3.3) nm contrast to (16.1 +/- 4.0) nm, P < 0.01]. CONCLUSION: Selenium deficiency might cause changes of neuronal functions at the synaptic level, and furthermore, affect learning and memory.

Clinicopathologic analysis of esophageal and cardiac cancers and survey of molecular expression on tissue arrays in Chaoshan littoral of China.

AIM: To investigate clinical and pathologic data of esophageal carcinoma (EC) and cardiac carcinoma (CC) among residents in Chaoshan region of China. METHODS: Clinical and pathologic data of 9 650 patients with EC and 4 173 patients with CC in the Chaoshan population were collected and analyzed. Moreover, Chaoshan esophageal carcinoma tissue arrays were made for high-throughput study. RESULTS: Male to female ratio was 3:1 in patients with EC and 4.75:1 in CC. The average age of the occurrence of EC was 54.6 years, and of CC was 58.1 years. For both EC and CC, age at diagnosis was a little younger in Chaoshan region than in most other areas. The most commonly affected site of esophageal carcinoma was the middle third of esophagus (72.0%); the second was the lower third (15.3%). The main gross type of esophageal carcinoma was ulcerative type (41.50%); the medullary type was the second (39.6%). Squamous cell carcinoma accounted for the overwhelming majority of esophageal cancer (96.4%); adenocarcinoma accounted for the overwhelming majority of cardiac carcinoma (94.5%). Chaoshan esophageal carcinoma tissue arrays were easily for high-throughput study, and tissue cores with a diameter of 1.5 mm could better keep more structure for molecular expression study. CONCLUSION: Both EC and CC are common in males. The average occurrence age of EC and CC is younger in Chaoshan than in most other regions of China. The most commonly affected site of esophageal carcinoma was the middle third of esophagus (72.0%). Squamous cell carcinoma accounted for the overwhelming majority of esophageal cancer; adenocarcinoma accounted for the overwhelming majority of cardiac carcinoma. Tissue arrays technology is applicable for rapid molecular profiling of large numbers of cancers in a single experiment.

MCM2 expression levels predict diagnosis and prognosis in gastric cardiac cancer.

BACKGROUND: Gastric Cardiac Cancer (GCC) has high incidence and poor prognosis requiring early screening of high-risk populations. Minichromosome maintenance (MCM) proteins are used as diagnostic-biomarkers in many cancers but not validated for GCC. We evaluate MCM protein 2 (MCM2), comparing it with the validated markers Ki67 and PCNA. METHODS: GCC and corresponding cardiac precancerous samples were immunostained with Ki67, MCM2 and PCNA antibodies. RESULTS: 90% of dysplasia samples expressed MCM2, whereas Ki67 and PCNA were expressed in 67% and 80% respectively. The sensitivity and negative predictive values of MCM2 were also superior at 90% and 87%, respectively. Ki67 and PCNA expression was correlated with MCM2, but their expressions seldom reached surface layers, whereas MCM2 manifested mostly in easily accessible superficial layers. Labeling indices (LI) of Ki67 and PCNA were also lower. Significant associations between LI (MCM2), LI (PCNA), and TNM-stages, lymph node metastases and GCC grade were found (P<0.05). Increased protein expressions were associated with reduced overall and disease-free survival (P<0.05). Although Ki67 and PCNA were significant prognostic factors, there was no significant improvement in multivariate statistical analyses, in contrast to LI (MCM2) findings. CONCLUSIONS: MCM2 is a sensitive, specific and efficient biomarker of GCC having potential use in clinic.

Tetramine poisoning: A case report and review of the literature.

BACKGROUND: Tetramethylene disulfotetramine (TETS), a banned neurotoxic rodenticide, has accounted for numerous intentional and unintentional poisonings in mainland China. Since the first known case of human illness caused by tetramine occurred in NewYork, in May 2002, TETS has caused more than 50 human poisonings in Western countries. AIM: To analyze pathological changes of TETS poisoning and to provide evidence for forensic identification. METHODS: We report the case of a 28-year-old female who suffered from tetramine poisoning and died of multi-organ failure. We also performed a retrospective study of 40 cases of poisoning, from pathological autopsy reports, by analyzing and summarizing the related the literature from 1996 to 2010. Based on pathologic autopsies and the literature, we summarize the pathological changes related to tetramine poisoning. RESULTS: Signs of asphyxia were obvious upon pathological examination. Edema and congestion of organs, particularly in the brain, were seen in all cases. Subarachnoid and cerebral hemorrhaging were also common signs of tetramine poisoning. CONCLUSION: In forensic practice, tetramine poisoning should be considered when the patient has signs of abnormal excitation of the central nervous system, convulsions, hyperspasmia, and cerebral hemorrhage.

Association of mitochondrial haplogroup D and risk of esophageal cancer in Taihang Mountain and Chaoshan areas in China.

Both the Taihang Mountain area in north-central China and Chaoshan area in the southeastern littoral of China are areas with high risk of esophageal cancer (EC). Our previous study confirmed that populations from the two areas might share similar matrilineal backgrounds and found that mitochondrial DNA (mtDNA) haplogroup D, especially subhaplogroups D4a and D5a, might be genetic background markers of EC in Chaoshan area. Here, to further determine whether D4a, D5a, and D might be susceptibility markers for EC in the two high-risk areas, we performed a case-control study with larger samples and analyzed the distributions of these three haplogroups in subjects (controls [n = 898] and patients [n = 768]) from the two areas. D4a haplogroup was significantly associated with increased risk of EC in Taihang Mountain subjects, especially women. D5 haplogroup was associated with EC at the general population level in the Taihang Mountain area and in subjects ≤ 60 years, especially women ≤ 60 years, in the Chaoshan area. D haplogroup was associated with EC only in subjects ≤ 60 years, especially men ≤ 60 years, in the Chaoshan area. D4a and D5 showing positive association with EC in the Taihang Mountain area became the predominant subhaplogroups of D in Chaoshan controls. In conclusion, D, D4a, and D5 haplogroups might be susceptibility markers for EC in the two high-risk areas in China, particularly D4a and D5 for the Taihang Mountain area and D and D5 for the Chaoshan area.

Heredity, diet and lifestyle as determining risk factors for the esophageal cancer on Nanao Island in Southern China.

In this case-control study we evaluated contribution of environmental and genetic factors for risk of esophageal cancer (EC) by studying populations on Nanao Island (highest risk area for EC in China) and Shanwei (low risk region). Data on lifestyle, diet and family history were collected from the 166 newly diagnosed EC patients on Nanao between 2003 and 2004, from their 1450 first degree relatives and from controls on Nanao and Shanwei. Univariate and logistic regression analysis, family aggregation patterns, standardized incidence ratio (SIR), segregation ratio and heritability index were evaluated. The family cancer history was a significant risk factor for the two scenarios; Nanao cases versus Nanao controls, and Nanao controls versus Shanwei controls. Other risk factors included smoking, alcohol and fermented fish sauce. After adjusting for confounding variables, family history was independently associated with the occurrence of EC in Nanao cases versus Nanao controls. The incidence in the first degree relatives of Nanao cases was 0.86%, significantly higher than that of the public (0.12%) and SIR value was 1.44 in the first degree relatives of the 166 EC cases. The segregation ratio was 0.11 and the heritability index among first degree relatives was 40%. Our study indicates that there are steady pathogenic risk factors in the Nanao population's lifestyle but genetic factors also play an important role for EC onset.

mtDNA evidence: genetic background associated with related populations at high risk for esophageal cancer between Chaoshan and Taihang Mountain areas in China.

There are three major geographic regions in China known for their high incidences of esophageal cancer (EC): the Taihang Mountain range of north-central China, the Minnan area of Fujian province, and the Chaoshan plain of Guangdong province. Historically, waves of great population migrations from north-central China through coastal Fujian to the Chaoshan plain were recorded. To study the genetic relationship among the related EC high-risk populations, we analyzed mitochondrial DNA (mtDNA) haplogroups based on 30 EC patients from Chaoshan and used control samples from the high-risk populations, including 48, 73, and 89 subjects from the Taihang, Fujian, and Chaoshan areas, respectively. The principal component of all haplogroups, correlation analysis of haplogroup frequency distributions between populations, and haplogroup D network analysis showed that compared with other Chinese populations, populations in the three studied areas are genetically related. The highest haplogroup frequency shared by all studied populations was haplogroup D, with much higher frequency in the Chaoshan area EC patients. The majority of haplogroup D individuals among the Chaoshan area EC patients belonged to subhaplogroups D4a and D5a, with the total frequency of these two haplogroups significantly higher than that in the high-risk population in the same area (chi(2)=9.017, p<0.01). In conclusion, EC high-risk populations in these three areas share a similar matrilineal genetic background, and D4a and D5a might be candidate genetic markers for screening populations susceptible to EC in the Chaoshan area. Ours is the first report to show the association between mtDNA haplogroups (D4a and D5a) and esophageal cancer.

Temporal trends of esophageal cancer during 1995-2004 in Nanao Island, an extremely high-risk area in China.

The purpose of our study was to investigate the temporal malignant tumor incidence rates among the 70,000 residents at the relatively isolated Nanao Island in South China Sea. The data on all malignant tumor cases from Nanao Cancer Registry during 1995-2004 were coded, computerized, and analyzed using the software SPSS10.0. The tumor incident cases, crude incident rate, age-standardized incidence rate, their sex distribution and temporal trend were assessed. A total of 1450 new cancer cases (990 males and 460 females) were identified. The annual average age-standardized incidence rate (ASR) of malignant tumors was 208.18/100,000. The age-standardized incidence rate of the ten leading cancers in both sexes combined per 100,000 population were 74.47 for esophageal cancer (EC), 34.81 for cardiac cancer (CC), 25.66 for liver cancer, 26.01 for lung cancer, 18.52 for stomach cancer, 4.45 for nasopharyngeal cancer, 3.91 for breast cancer, 2.53 for colon/rectum cancer, 2.45 for bladder cancer and 1.92 for pancreatic cancer. These ten types of cancers make up to 93% of all cancer cases, with EC and CC being the most prevalent and making up 52% of the total cases. The incidence rates of esophagus, liver, lung, breast, nasopharyngeal, and colon/rectum cancers showed increasing trends during the period from 1995 to 2004 in Nanao Island. Astounding the EC ASR were 72-150/100,000 among male and 26-64/100,000 among female in Nanao Island during 1995-2004. The EC incidence rate in Nanao population is among the highest across the world, which suggests that there are potential genetic and/or environmental factors affecting this particular population.

[Effect of chronic inflammation-induced basement membrane changes on esophageal carcinogenesis].

BACKGROUND & OBJECTIVE: Basement membrane (BM) could regulate differentiation, proliferation, and polarity of esophageal epithelium, and its integrity is important for carcinogenesis. This study was to explore the effect of the BM changes induced by chronic inflammation on esophageal carcinogenesis and its possible mechanisms through investigating the relationship of BM changes and epithelial proliferation. METHODS: The morphologic changes of BM in 10 specimens of invasive esophageal carcinoma tissue and 200 specimens of carcinoma adjacent tissue were observed with periodic acide-chiff staining (PAS); the correlations of inflammation score and histological type of esophageal epithelia to BM changes were analyzed. Of preceding specimens, the expression of laminin (LN), proliferating cell nuclear antigen (PCNA), and Ki-67 in 51 cases with typical BM changes were detected by immunohistochemistry; the correlation of LN to epithelial proliferation was analyzed. RESULTS: Dotted loss, piece loss, zone loss, and fragment thickening were observed at BM of hyperplasia, dysplasia, in situ carcinoma, and invasive carcinoma of esophageal epithelia. BM changes were related with inflammation score in epithelia (rs=0.795, P<0.05). PCNA and Ki-67 were expressed highly in the epithelia with changed BM (P<0.01). CONCLUSION: BM changes induced by chronic inflammation are correlated with aberrant proliferation of esophageal epithelia.