A single nucleotide polymorphism in the matrix metalloproteinase 2 promoter is closely associated with high risk of nasopharyngeal carcinoma in Cantonese from southern China.

Matrix metalloproteinase 2 (MMP2) has been shown to play an important role in several steps of cancer development. The -1306C/T polymorphism of the MMP2 gene displays a strikingly lower promoter activity than the T allele, and the CC genotype in the MMP2 promoter has been reported to associate with the development of several cancers. To assess the contribution of the MMP2 -1306C/T polymorphism to the risk of nasopharyngeal carcinoma (NPC), we conducted a case-control study and analyzed MMP2 genotypes in 370 patients with NPC and 390 frequency-matched controls using real-time PCR-based TaqMan allele analysis. We found that subjects with the CC genotype had an increased risk (OR = 1.55, 95% CI = 1.05-2.27) of developing NPC compared to those with the CT or TT genotypes. Furthermore, we found that the risk of NPC was markedly increased in subjects who were smokers (OR = 15.04, 95% CI = 6.65-33.99), heavy smokers who smoked ≥ 20 pack-years (OR = 18.66, 95% CI = 7.67-45.38), or young (<60 years) at diagnosis (OR = 1.52, 95% CI = 1.01-2.29). Our results provide molecular epidemiological evidence that the MMP2 -1306C/T promoter polymorphism is associated with NPC risk, and this association is especially noteworthy in heavy smokers.

Polymorphisms of methylenetetrahydrofolate reductase are associated with a high risk of nasopharyngeal carcinoma in a smoking population from Southern China.

Methylenetetrahydrofolate reductase (MTHFR) plays a central role in converting folate to a compound which serves as a methyl donor for DNA methylation, an epigenetic modification known to be dysregulated in carcinogenesis. This case-control study assessed the contribution of MTHFR polymorphisms to the risk of nasopharyngeal carcinoma (NPC). MTHFR genotypes C677T and A1298C in 529 NPC patients and 577 frequency-matched controls were determined by PCR-based restriction fragment length polymorphism. We found a 1.57-fold increased risk of NPC in subjects with the MTHFR 1298AC genotype compared to subjects with the MTHFR 1298AA genotype. In addition, an elevated NPC risk was also found in subjects with both the MTHFR 677CT and 1298AC genotypes [odds ratio (OR) = 2.15, 95% confidence interval (CI) = 1.37-3.39] compared to subjects with the 677CC/1298AA genotypes. Furthermore, we observed an additive interaction between MTHFR polymorphisms and smoking status on the increased risk of NPC. The OR was 6.72 (95% CI = 1.85-24.48) among heavy smokers (pack-years ≥15) carrying 677TT compared with nonsmokers carrying the 677CC genotype. The OR was 7.23 (95% CI = 4.22-12.38) or 12.75 (95% CI = 2.74-59.3) among subjects carrying the 1298AC or 1298CC genotype in heavy smokers (pack-years ≥15) compared with 1298AA in nonsmokers. Our results provide the first molecular epidemiological evidence that MTHFR polymorphisms associate with the risk of NPC and this association is especially noteworthy in heavy smokers.

Primary small cell carcinoma of the esophagus: clinicopathological and immunohistochemical features of 21 cases.

BACKGROUND: Primary small cell carcinoma (SCC) of the esophagus is a rare and aggressive tumor with poor prognosis. In this study, we report the clinicopathological characteristics of 21 cases of small cell carcinoma of the esophagus treated at the Cancer Center of Sun Yat-Sen University, with particular focus on the histologic and immunohistochemical findings. METHODS: Twenty-one patient records were reviewed including presenting symptoms, demographics, disease stage, treatment, and follow-up. Histologic features were observed and immunohistochemical detection of cytokeratin (CK), epithelial membrane antigen (EMA), neuron specific enolase (NSE), synaptophysin (Syn), chromogranin A (CgA), neuronal cell adhesion molecules (CD56), thyroid transcriptional factor-1 (TTF-1) and S100 protein (S100) was performed. RESULTS: The median age of patients in the study was 56 years, with a male-to-female ratio of 3.2:1. Histologically, there were 19 "homogenous" SCC esophageal samples and 2 samples comprised of SCC and well-differentiated squamous cell carcinoma. The percentages of SCC samples with positive immunoreactivity were Syn 95.2%, CD56 76.2%, TTF-1 71.4%, NSE 61.9%, CgA 61.9%, CK 57.1%, EMA 61.9%, and S100 19.0%, respectively. The median patient survival time was 18.3 months after diagnosis. The 2-year survival rate was 28.6%. CONCLUSION: Our study suggests that esophageal SCC has similar histology to SCC that arises in the lung compartment, and Chinese patients have a poor prognosis. Higher proportion of positive labeling of Syn, CD56, CgA, NSE, and TTF-1 in esophageal SCC implicate that they are valuably applied in differential diagnosis of the malignancy.

[Surgical treatment for gastrointestinal stromal tumor].

OBJECTIVE: To analyze the effects of surgical treatment for gastrointestinal stromal tumors (GISTs) and influential factors of survival. METHODS: The clinical data and the tissue slices including immunohistochemical staining of 153 cases of GISTs from January 1990 to March 2006 were rechecked retrospectively. All patients were followed up carefully. More attention was paid to the surgical effects and the influential factors of survival. RESULTS: The overall survival rates at 1-, 2-, 3-, 4- and 5-year were 94.9%, 83.3%, 73.3%, 70.5% and 64.3%, respectively. The median survival time for patients with tumor resected completely was 66.0 months, and the 2- and 5-year survival rate were 89.4% and 70.9% respectively. The median survival time was 23.8 months for the patients with tumor resected partly, and only two of these patients survived over 2 years. Gender, tumor sites, preoperative metastasis, tumor size, pathological type, karyokinesis and recurrence and metastasis were related with survival rates for the patients with tumor resected completely on univariate analysis, but tumor size, pathology type, recurrence and metastasis were related with survival rates on Cox regression multivariate analysis (P < 0.05). CONCLUSIONS: Surgery should still be the main therapy for GISTs. Local complete resection is the principal treatment. The survival cannot be improved by extensive resection and lymph nodes clearance.

[Comparision of HER2/neu oncogene detected by chromogenic in-situ hybridization and immunohistochemistry in breast cancer].

OBJECTIVE: Through comparison of HER2/neu oncogene detected by chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) in breast cancer, to explore the effect of CISH on detecting gene amplification of HER2. METHODS: Selected formalin-fixed paraffin-embedded breast samples whose pathological types were infiltrating ductal carcinomas (255 retrospective samples, 271 prospective samples), and these samples were detected by IHC and CISH. RESULTS: (1) In the retrospective study, CISH identified gene amplification in 91.6% of IHC score 3+ tumors (120/131) and in 56.5% of IHC score 2+ tumors (39/69), thus the concordant ratio between IHC and CISH was 81.2% (207/255). The two results showed significant correlation (P<0.01). (2) In the prospective study, the ratio of HER2 protein over expression detected by IHC was 31.7%, the ratio of HER2 gene amplification detected by CISH was 27.3%. CISH identified gene amplification in 91.4% of IHC score 3+ tumors (53/58) and in 46.4% of IHC score 2+ tumors (13/28), Concordant ratio between IHC and CISH was 89.7% (243/271). Two results showed significant correlation (P<0.01). (3) Paired CISH/FISH results were concordant in 14 of 15 cases. The remaining case was detected by FISH, but showed no HER2 gene amplification by CISH. (4) The gene amplification by CISH had a significantly reverse correlation with ER and PR expression (P<0.01). CONCLUSIONS: The results of HER2 gene amplification detected by CISH have high concordance with the results detectd by IHC and FISH. CISH is a novel technique for detecting HER2 gene amplification.

[Primary adenoid cystic carcinoma of the nasopharynx and its relation to Epstein-Barr virus infection].

OBJECTIVE: To investigate the clinicopathologic characteristics of primary nasopharyngeal adenoid cystic carcinoma (NPACC) and its relation to Epstein-Barr virus (EBV) infection in Guangzhou where is a high-incidence area of EBV-associated nasopharyngeal carcinoma (NPC). METHODS: 17 cases of NPACC with clinical record and biopsy samples were collected in Guangzhou and their clinical manifestations were reviewed. Besides HE, Alcian blue and PAS, LSAB immunohistochemistry was performed for detecting the expression of a variety of epithelial markers, CD21 and EBV encoded LMP1. EBV encoded early RNAs (EBER) was detected by using in-situ hybridization. Nested PCR was applied for studying the presence of EBV W-fragment in tissues. RESULTS: The ratio of male to female was 7:10. The patients' age ranged from 30 to 63 years, and the median age was 46 years. 14 out of 17 tumors showed markedly local aggressive growth, presenting as T3 or T4. However, only 1 patient had metastasis of an ipsilateral cervical lymph node. The majority of neoplastic cells were basal-cell like in shape and with scanty cytoplasm and a deeply stained nucleus. Intercellular hyaline or mucinous substance was always present in between the carcinoma cells. Cribriform structure formed by the neoplastic cells could be found in 16 out of these 17 biopsies. The NPACC always express the wide-spectrum cytokeratin and the epithelium membrane antigen. Only a few or a small number of carcinoma cells showed nuclear EBER-signals in 9 cases (9/17). Concurrently, these 9 NPACCs showed a 192 bp W-fragment positive band on electrophoresis gel by nested PCR. LMP1 expression had been found in 5 out of the 9 NPACCs (55.6%) accompanying with EBER-positive carcinoma cells. The EBER-positive infiltrating lymphocytes could also be found in the stroma of 3 out of the 9 EBER-stained NPACC slides. All the tumor cells, including the EBER-positive cell of the 17 NPACCs showed no CD21 expression. CONCLUSIONS: The female is predominant over the male in development of NPACC, which often accompanied with a markedly invasive capacity at the nasopharynx and its neighboring sites. Only a small number of tumor cells, nearly a half of the studied cases were infected with EBV. Therefore, it's postulated that there seems no close relation present between NPACC and EBV infection.

Mitochondrial DNA somatic mutations are frequent in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China, and is highly sensitive to radiotherapy. The control region (D-loop) of mtDNA is a polymorphic region in which point mutations occur frequently. In this study, point mutation and common deletion (CD) mutations were investigated in 23 samples of NPC tumor tissue and in the radiation-treated NPC cell line CNE2. Polymorphisms at 72 (7.28%, 72/988) nucleotide positions in D-loop region and 6 (0.75%, 6/795) nucleotide positions in part of the functional gene encoding regions were detected in all NPC patients. Of the detected polymorphisms, 8 are novel. These variants are nonencoding transitions, including np292T-->C , np517G-del, np16038A-->G, np513G-del, np16242C-->A, np513G-del, np16242C-->A and np15787T-->C transition. A total of 39 point mutations in the D-loop region of mtDNA were detected in 43.5% (10/23) of the NPC patients. Three point mutations in the functional gene encoding regions of mtDNA were detected in only 8.7% (2/23) of NPC patients. The effect of he mutation at np709G-->A in the 12sRNA gene is unclear, and the A-->G substitution at np15769 in the cytochrome B gene is a synonymous mutation. The C-->T substitution at np15970 in the T Psi C loop of the tRNA(pro) gene could alter the position of the proline residue. After irradiation, the survival fraction of CNE2 cells decreased as X-ray dose increased. Moreover, X-ray radiation could induce apoptosis and the CD mutation in a time- and dose-dependent manner, but did not induce mtDNA point mutations. A positive correlation between the apoptosis index and the ratio of CD/WT mtDNA was observed in irradiated CNE2 cells. Our results suggest that CD mutation induced by irradiation is one of the late events after apoptosis of the cancer cells, and the mtDNA CD mutation may associated with the susceptibility of NPC cells to IR-induced apoptosis.

[Detection of sentinel lymph node in patients with early stage breast cancer].

BACKGROUND & OBJECTIVE: Axillary lymph node dissection (ALND) is routinely performed during surgery for breast cancer, but whether ALND could increase survival rate of early stage breast cancer patients remains controversial. Recently, replacing ALND with sentinel lymph node (SLN) biopsy has became a hotspot in breast cancer research. This study aimed to evaluate the reliability and accuracy of SLN biopsy for early stage breast cancer, and to discover the significance of multiple step section level cytokeratin immunohistochemistry in identifying micrometastatic disease. METHODS: SLN biopsy was performed on 121 patients with T1 or T2 breast cancer: methylene blue-labeling was used in 38 patients (methylene blue group), double-labeling of (99m)Tc sulfur colloid and methylene blue was used in 83 patients (combination group). Lymphoscintigraphy and hand-hold gamma detector were used to localize SLNs before operation. All SLNs and ALND lymph nodes were pathologically examined. The tumor-negative SLNs were cut at three levels, and detected by immunohistochemistry. RESULTS: Success rates were 81.6% in methylene blue group, and 95.2% in combination group; accurate detection rates of axillary lymph nodes were 93.5% in methylene blue group, and 97.5% in combination group. SLNs were found in 19 patients (23%) by lymphoscintigraphy, and 76 patients (92%) by hand-hold gamma detector, respectively (P=0.04). A total of 194 negative SLNs, detected by routine pathologic examination, were re-examined by multiple step section level cytokeratin immunohistochemistry; micrometastatic diseases were identified in 21 SLNs of 13 patients. The accuracy rate of combined examinations was 98.7%, and the false-negative rate was 3.2%. CONCLUSIONS: The axillary node status can be predicted by SLN biopsy; double-staining is better than methylene blue-labeling. The role of lymphoscintigraphy in SLN biopsy needs further explore. Multiple step section level cytokeratin immunohistochemistry can improve detection rate of micrometastatic diseases.

Micrometastases of solitary hepatocellular carcinoma and appropriate resection margin.

We studied the pattern of intrahepatic micrometastases using large pathologic sections on liver resection specimens with ample resection margins from 113 patients with a solitary hepatocellular carcinoma (HCC). The liver tissues around the HCC were divided into proximal and distal areas according to the direction of the portal vein flow. These areas were further divided into zones based on fixed criteria. Altogether, 273 micrometastases were identified, including 254 (93.0%) intravascular micrometastases and 19 (7.0%) tumor satellite micronodules. The distance of spread of these micrometastases ranged from 0.05 to 6.10 cm. The number of micrometastases was less in the proximal area than in the distal area. In addition, the farther the distance away from the primary tumor, the fewer micrometastases there were. Micrometastases extended beyond the 2 cm margin in only nine (8.0%) patients. In conclusion, micrometastases could spread via invasion of portal vein branches at an early stage even when the tumor was solitary and small. Anatomic segment resection is preferred for patients with a solitary HCC. Nonanatomic resection may be used as an alternative in patients with impaired liver function, but adequate resection margins should be achieved. For HCCs < or = 3 cm, a proximal resection margin and a distal margin of 1.0 cm are recommended. For HCCs > 3 cm, a 1.0 proximal resection margin and a 2.0 cm distal margin are recommended.

[Correlation analysis among expression of ERCC-1, metallothionein, p53 and platinum resistance and prognosis in advanced non-small cell lung cancer].

BACKGROUND & OBJECTIVE: The molecular predictor for cisplatin sensitivity in advanced non-small cell lung cancer (NSCLC) is still an open question at present. The purpose of this study was to evaluate the relationship of the cisplatin sensitivity/prognosis with the expression of excision repair cross complement-1 (ERCC-1), metallothionein (MT), and p53 in the paraffin-embedded tissue of advanced non-small cell lung cancer (NSCLC). METHODS: From January 1994 to December 2001, 51 pathologically confirmed advanced NSCLC patients were included. All patients received cisplatin contained regimen chemotherapy (Gemzar + DDP or NVB + DDP) as the first line treatment. At the same time, the tumor samples were collected and the expression of ERCC-1, MT, and p53 in the tumor samples were examined by immunohistochemical method. The response rates and survival data were analyzed according to the over-expression or not of these three parameters (negative group/over-expression group). The response rates were compared by Chi(2) test. Kaplan-Meier method and Cox proportional hazards model were used for survival analysis. RESULTS: In these 51 patients, the expression rates of ERCC-1, MT, and p53 were 42.8%, 57.5%, and 37.8%, respectively. The response rates in negative group/over-expression group of ERCC-1, MT, and p53 were 33.3%/16.7%, 35.3%/27.3%, and 21.4%/35.3%, respectively (1.99, 1.29, and 0.61 times, respectively, P >0.05). The response rate in both ERCC-1 and MT negative group was 37.5%. In both ERCC-1 and MT over-expression (co-expression) group, the response rate was 14.3%. The former was 2.6 times of the latter (P >0.05). The average survival time in negative group/over-expression groups were 621/523 days (for ERCC-1), 556/479 days (for MT), 599/416 days (for p53), respectively. ERCC-1, MT, or p53 over-expression group showed poorer prognosis than those of negative group by Kaplan-Meier analysis. But in multivariate analysis, only p53 over-expression was an independent poor prognostic factor of survival time (Cox regression, P=0.009). CONCLUSION: Whether over-expression of ERCC-1 and MT can be used as a molecular marker of cisplatin resistance in advanced NSCLC patients need further study. Over-expression of p53 predicates poor prognosis for patients with advanced NSCLC.

[Clinicopathological characteristics of primary nasopharyngeal adenocarcinoma].

BACKGROUND & OBJECTIVE: The primary nasopharyngeal adenocarcinoma (NPAC) is rare. This study was designed to investigate the clinicopathological characteristics of primary NPAC. METHODS: A series of carcinoma biopsies taken from the nasopharynx of the patients followed at Cancer Center, Sun Yat-sen University were reviewed during 24 years. The clinical data and paraffin blocks of 39 NPAC patients were used for this study. In addition, the clinical manifestations of 177 nasopharyngeal carcinoma (NPC) patients were adopted as control. HE, Alcian blue, and PAS histochemical staining were performed and then the morphology was observed under microscope. RESULTS: (1) There were 153 NPACs found in 31791 carcinomas of the nasopharynx; thus the hospital-based frequency of NPAC was 0.48% (153/31791). (2) The median age of 39 NPAC patients was 46.0 years old and the age peak was 40-49 age group. (3) Seventeen out of 39 NPAC patients (43.5%) were female. (4) Most of NPAC tumors (23/38) took growth beyond the nasopharyngeal cavity (T3) and/or invasion to the cranial bone and/or cranial nerves (T4). Furthermore, only 7 NPAC patients (7/39, 17.95%) had lymph node metastasis; and on the contrary, there were 136 out of the 177 patients (136/177, 76.84%) with NPC had lymph node(s) metastasis. There was significant difference between these two percentages (17.95% vs. 76.84%, P< 0.01). (5) The 39 NPACs could be classified into 2 categories, namely conventional type (16 cases) and salivary gland type (23 cases). The conventional type could further be graded as low-grade (9 cases) and high-grade (7 cases). The 23 salivary-gland type NPACs consisted of 17 adenoid cystic carcinomas and 6 mucoepidermoid carcinomas. (6)The continuation of neoplastic cells to surface lining epithelium was presented in 7 conventional type adenocarcinomas (including all 5 papillary adenocarcinomas) and 4 mucoepidermoid carcinomas. The transition from the adenoid cystic carcinoma cells to the atypical hyperplastic lesion of the ductal epithelium of minor salivary gland was observed in 2 cases. CONCLUSION: The primary NPAC is uncommon. The age distribution of NPACs is not different from that of NPCs. Most patients show significant local invasion, but scarce cervical lymph node metastasis, especially adenoid cystic carcinomas.

[Study of CD44v6 protein expression in intraductal papilloma and its malignant transformation of breast].

BACKGROUND & OBJECTIVE: CD44v6 plays an important role in the malignant transformation of breast epithelia and is differentially expressed in normal and neoplastic breast tissue. The diagnostic value of this marker in differentiating malignant from benign breast lesions has not been well examined. This study was designed to evaluate the diagnostic value of CD44v6 protein in distinguishing between benign and malignant papillary lesions of the breast, which is be difficult morphologically, by determining the level of CD44v6 expression in different lesions of the breast. METHODS: The level of CD44v6 protein was detected by immunohistochemistry in 111 paraffin-embeded blocks. RESULT: The positive expressive rates of CD44v6 protein in the basal epithelial cells of the normal tissue, intraductal papilloma, and its malignant transformation, intraductal carcinoma, invasive ductal carcinoma were 95.00%, 85.72%, 66.66%, 66.66%, and 0.00% respectively. The difference between the intraductal papilloma and its malignant transformation was not significant(P > 0.05). However, the positive expressive rates of CD44v6 protein in the luminal epithelial cells of the above lesions were 5.00%, 20.41%, 83.34%, 93.33%, and 100% respectively. There was difference between intraductal papilloma and its malignant transformation(P < 0.01). CONCLUSION: CD44v6 detection by immunohistochemistry is useful in distinguishing intraductal papilloma from its malignant transformation of the breast.

[Advancement of studying the biological characteristics of nasopharyngeal carcinogenesis].

This review is to summarize the main achievements of studying the biological characteristics of nasopharyngeal carcinogenesis performed by the authors' research team and the recent advancement in this field during the past 5 years as well as to explain the authors' viewpoints concerning the nasopharyngeal carcinogenesis. In order to study the nasopharyngeal carcinogenesis, more than 20,000 nasopharyngeal carcinoma biopsies and more than 600 nasopharyngeal biopsies of Epstein-Barr virus seropositive persons who had been got follow-up over 12 years, were collected. In addition, nude mice and cell lines were also to be utilized. Besides histopathological staining, methods of molecular biology, including in-situ hybridization, PCR etc. were applied. Up to date, 26 papers concerning this subject had been formally published in the medico-biological journals at home and abroad. The results and conclusions were as follows. (1) The squamous metaplasia, epithelial dysplasia, carcinoma in-situ and microinvasive carcinoma are the morphogenetic sequence found in nasopharyngeal carcinogenesis. (2) This morphogenetic sequence is frequently observed in a restricted area of nasopharyngeal mucosal epithelium, representing as an appearance of field carcinogenesis. (3) EB virus may play a critical role in nasopharyngeal carcinogenesis, since EB virus DNA and small RNAs could be detected in epithelial dysplasia first and several viral encoded products, especially LMP1, might be expressed in dysplasia, carcinoma in-situ and microinvasive carcinoma. (4) The multigenic mechanisms, including interactions between EB viral genes encoded products and the products abnormally expressed step by step from genes related to cell-cycle regulation, are the molecular events involved in nasopharyngeal carcinogenesis. (5) The cellular immunity of individuals should also be considered as an important factor influencing nasopharyngeal carcinogenesis, because EB virus specific cytotoxic T-lymphocytes could not only be observed in carcinoma nests but also detected in peripheral blood.

Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix.

It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix.