Immunohistochemical overexpression of p16 protein associated with cervical cancer in Thailand.

Cervical cancer is caused by persistent infections through high risk (HR) types of human HPVs, particularly HPV 16 and 18. HR-HPV types encode two potent oncogenes, referred to as E6 and E7. Both are required to induce and maintain neoplastic growth of cervical cancer cells. Cyclin dependent kinase inhibitor genes as for example p16INK4A were shown to be negative regulated by active pRb. Inactivation of pRb by E7 thus releases the p16 gene from its negative transcriptional control and results in significant overexpression of p16 encoded protein in HPV transformed cells. It has been demonstrated that p16 protein can be detected in cervical preneoplasia all high grade SIL or invasive cancers, whereas no expression was detected in normal, metaplastic or inflammatory cervical lesions. Moreover, low grade cervical lesions induced by low risk HPV infection but histological indistinguishable from low grade lesions induced by HR-HPV-infections could be clearly differentiated by p16INK4A immunohistochemistry, showing negative staining for p16 protein. The objective of this study is to examine the expression of p16 protein in cervical carcinoma in Thailand. Immunohistochemical analysis of p16INK4A was performed on 53 formalin fixed and paraffin embedded samples of various stages of cervical neoplastic lesions. There are squamous cell carcinoma in situ 8 cases, squamous cell carcinoma in situ with glandular involvement 16 cases, microinvasive squamous cell carcinoma 13 cases and invasive squamous cell carcinoma 16 cases. The specimens were taken from cervical biopsy, cervical conization and hysterectomy in the year 2000 at National Cancer Institute. Strong immunoreactivity for the p16 protein was observed in only the nuclei and cytoplasm of all cervical neoplastic cells. This study supported the idea that immunohistochemical overexpression of the p16 protein may be a useful screening test for cervical cancer. In addition, p16 immunohistochemistry is useful for helping in the interpretation of cervical histology samples, facilitating more rapid diagnosis.

Interobserver reproducibility in determining p16 overexpression in cervical lesions : use of a combined scoring method.

OBJECTIVES: To evaluate interobserver reproducibility of a combined scoring method for immunohistochemical interpretation of p16 overexpression in cervical lesions. MATERIALS AND METHODS: p16 immunostaining was performed in cervical samples from 183 patients, including 69 normal, 42 low grade squamous intraepithelial lesions(LSIL), 36 high grade SIL (HSIL), and 36 squamous cell carcinomas(SCCAs). Each case was evaluated by a combined scoring method based on the percentage of positive cells (score 0-3), the intensity of staining (score 0-3), and the distribution pattern (score 0-2). Immunoexpression for p16 was considered as positive when the combined score was 4-8 and negative with a score of 0-3. Ten pathologists with varied experience in interpretating p16 immunostains evaluated each slide independently. RESULTS: All normal cervical squamous epithelia (69/69) were uniformly negative for p16. All HSILs (36/36), all SCCAs (100/100), and all but one of the LSILs (40/41, 97.6%) showed positive expression. In 172 of 183 cases (93.9%), p16 interpretation was concordant with all pathologists. Eleven cases with discordant results included 10 LSILs and 1 normal mucosa sample. Percentage of agreement of each pathologist pair ranged from 96.7-100% (mean 98.1%) with mean kappa value of 0.96 (range 0.93-1.000). CONCLUSION: The proposed combined scoring method shows good reproducibility among the participating pathologists and good correlation with the histologic diagnosis. This method may be a useful guide in the interpretation of p16 expression in cervical epithelial lesions.

Real-time PCR assay for rapid detection of GSTM1 polymorphism in nasopharyngeal carcinoma patients.

Nasopharyngeal carcinoma (NPC) is a common public health problem in Thailand. Glutathione S-transferase M1 gene deletion (GSTM1 null genotype) carriers have been reported to be at increased risk and therefore this parameter is a potential marker for screening of NPC high-risk individuals. However, the conventional polymerase chain reaction (C-PCR) assay commonly used for GSTM1 null genotype detection is not suitable for mass screening since it is inconvenient, time consuming and unsafe due to the use of a toxic chemical. Currently, real-time PCR (R-PCR) assay is recommended for quicker and safer detection of various genetic polymorphisms. The aim of this study was to develop a SYBR green I R-PCR assay combined with melting curve analysis for GSTM1 polymorphism detection in Thai NPC patients. The results were compared to those from the C-PCR assay using DNA samples from peripheral blood leukocytes of 120 Thai NPC patients. The frequencies of GSTM1 polymorphism detected by the R-PCR and the C-PCR were the same. Forty-eight individuals that were GSTM1+ in the R-PCR assay showed 2 peaks with melting points of 82.5 and 87.5 that correlated with the appearance of 2 DNA bands in the C-PCR assay (i.e., one for GSTM1 at 215 base pairs (bp) and one for ?-globin at 268 bp). By contrast, 72 individuals that were GSTM1?- in the R-PCR assay showed 1 peak with a melting point of 87.5C that correlated with the appearance of 1 DNA band for -globin at 268 bp in the C-PCR assay. The R-PCR assay using SYBR Green I and melting curve analysis for GSTM1 polymorphism detection was as reliable as C-PCR assay but was quicker and safer and more amenable to large scale screening in Thai NPC cases.

Detection of GSTM1 polymorphism in patient with nasopharyngeal carcinoma by real-time PCR.

Objective: To investigate whether the real-time polymerase chain reaction (R-PCR) assay with SYBR green I and melting curve analysis could be used for glutathione S-transferase M1 gene (GSTM1) polymorphism detection in Thai nasopharyngeal carcinoma (NPC) patients by comparing the results of this assay with the conventional PCR (C-PCR) assay. Methods: DNA samples from peripheral blood leukocytes of 60 Thai NPC patients were investigated in this study. GSTM1 polymorphism [GSTM1 normal genotype (GSTM1+) and GSTM1 null genotype (GSTM1-)] were examined by using the R-PCR assay with SYBR green I and melting curve analysis and the C-PCR assay. Results: The results of GSTM1 polymorphism detection by the R-PCR assay were in concordance with the C-PCR assay (k = 1.0). Twenty-six individuals with GSTM1+ in the R-PCR assay showed 2 peaks of melting point at 82.5oC and 87.5oC that correlated with the appearance of 2 DNA bands of GSTM1 [215 base pair (bp)] and b-globin (268 bp) in the C-PCR assay, respectively. In addition, thirty-four individuals with GSTM1- in the R-PCR assay showed only 1 peak of melting point at 87.5oC that correlated with the appearance of 1 DNA band of b-globin (268 bp) in the C-PCR assay. Moreover, we found that the R-PCR assay was a faster and safer method for detection of GSTM1 polymorphism than the C-PCR assay. Conclusion: The present study suggests that the R-PCR assay with SYBR Green I and melting curve analysis may be a useful screening tool for more convenient, rapid, reliable, and safer detection of GSTM1 polymorphism in Thai NPC as compared to the C-PCR assay.

Real-time quantitative PCR analysis of amplified DNA on chromosomes 4p15.2 and 6q23-24 from formalin-fixed, paraffin-embedded breast cancer tissues.

This study was performed to detect amplification of DNA sequences on chromosomes 4p15.2 and 6q23-24, obtained from formalin-fixed, paraffin-embedded, breast-cancer tissues. The prognostic relevance of the amplification was also demonstrated. DNA from formalin-fixed, paraffin-embedded tumor and corresponding normal tissues of 53 patients with breast cancer was extracted and amplified by real-time quantitative PCR technique. Amplification of the DNA sequences on chromosomes 4p15.2 and 6q23-24 was detected in 23 (43%) and 32 (60%) cases, respectively. Thirty-six (68%) cases showed amplification on both or one of the chromosomes. These frequencies are similar to that obtained from fresh samples in our previous study. In addition, amplification of the DNA on chromosomes 4p15.2 and / or 6q23-24 was predominantly observed in tumors with invasive ductal carcinoma. The findings in this study demonstrate that DNA extracted from formalin-fixed, paraffin-embedded breast tumors can be used to determine amplification of DNA sequences on selective chromosomal regions. We also suggest that the amplified DNA on chromosomal regions 4p15.2 and 6q23-24 might be involved in the development and progression of breast cancer.

Genetic instability in cervical cancer detected by arbitrarily primed polymerase chain reaction.

The genetic instability in 54 Thai cervical cancer tissues were analyzed by Arbitrarily Primed Polymerase Chain Reaction (AP-PCR). The band alterations produced from 54 arbitrary primers were compared between the DNA finger printing from the patients and their corresponding normal cervical tissues. Results revealed 7 arbitrary primers provided DNA alteration patterns. Of these, an allelic loss in tumor DNA was found in DNA fingerprinting obtained from primers F-2 (64.8%), F-11 (68.5%), U-8 (51.9%), AE-3 (75.9%), AE-11 (53.7%), respectively. Moreover, DNA amplification was exhibited in patterns with primers B-12 (42.6%), J-16 (24.1%) and U-8 (70.4%). When genetic instability was investigated for associations with clinicopathological features, only the DNA amplified fragment with primer U-8 was significantly associated with stage II (P=0.030). Likewise, allelic loss amplified from arbitrary primer AE-3 showed significantly associate with age lower than 50 years old (P=0.003). Our findings suggest that the DNA alteration fragments produced from arbitrary primers of U-8 and AE-11 might be relevant to the pathogenesis of cervical cancer in Thai patients.

A two-phase study model for the standardization of HER2 immunohistochemical assay on invasive ductal carcinoma of the breast.

OBJECTIVES: To develop and verify a standardized protocol for HER2 immunohistochemical assays on invasive ductal carcinoma of the breast in Thailand. MATERIAL AND METHOD: A two-phase study approach was employed. In the Phase One, after verifying the proposed protocol that adopted the HercepTest procedure using readily available primary antibodies, CB11 and A0485, Lab 1 performed the HER2 immunohistochemical staining for 137 cases of invasive ductal carcinoma twice with two types of the antibody. Nine pathologists from 8 centers independently examined and scored all the 2 x 137 stained slides that were blinded for antibody type. Interobserver reliability was calculated using pair-wise kappa. Following discussion of the results, the Phase Two study was planned. Lab 2 and Lab 3 independently performed the HER2 staining according to the protocol for 60 invasive breast carcinoma cases. The same group of pathologists scored 2 x 60 stained slides that were masked for laboratories. Interobserver reliability and interlaboratory agreement from each pathologist were calculated using kappa statistics. Three interpreted categories--namely negative, equivocal and positive tests were used in the analyses. RESULTS: Phase One study showed interobserver agreement between pairs varied from kappa 0.75 (95%CI, 0.68-0.82) to 0.06 (95%CI, 0-0.14) while Phase Two study obtained pair-wise kappa scores ranged from 0.84 (95%CI, 0. 80-0.89) to 0. 65 (95%CI, 0.59-0.71). Interlaboratory kappa for each pathologist was 0.67 (95%CI, 0.61-0.73). CONCLUSION: The standardization of HER2 immunohistochemical assay was achieved through this two-phase study model. It had added benefits of improving pathologists' expertise and verifying the HER2 testing protocol to be used in Thailand.

Glutathione S-transferase M1 gene polymorphism in Thai nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a serious health problem in Thailand. It is caused by the combined effects of Epstein-Barr virus (EBV), carcinogens and genetic susceptibility. The glutathione S-transferase M1 gene (GSTM1) encodes a phase II enzyme responsible for detoxifying carcinogenic electrophiles. Polymorphic null forms of the gene GSTM1 lack enzyme activity and have been associated with susceptibility to several cancers including NPC. To examine the association between GSTM1 polymorphism and NPC susceptibility in Thais, GSTM1 genotypes (normal and null genotypes) in 78 NPC patients and 145 age-matched healthy controls were determined using PCR assays. Overall, no statistically significant differences were observed in the frequency of GSTM1 genotypes between cases and controls, nor among NPC patients compared on the basis of sex and clinical stage of disease. Carriers with the GSTM1 null genotype had a 2.9-fold increased risk for NPC of WHO type III when compared to those with GSTM1 normal genotype (P < 0.05 with OR =2.9, 95% CI = 1.2-6.8). When cases and controls were categorized into 3 age groups (>40, (>45 and (>50 years), the frequencies of GSTM1 null genotype in cases the (>45 and (>50 age groups were significantly different from controls (P< 0.05). In addition, carriers of the GSTM1 null genotype in age groups (>45 and (>50 years had a 2-fold and 3-fold increased risk for NPC when compared to those with GSTM1 normal genotype (OR = 2.2, 95% CI = 1.1-4.7 and OR = 3.0, 95% CI = 1.2-7.5). We suggest that GSTM1 polymorphism may be associated with NPC susceptibility in Thais, especially for GSTM1 null genotype carriers of age higher than 45 years. The GSTM1 null genotype may be a useful genetic marker for predicting Thai NPC and for screening of early stages of Thai NPC.

Sentinel node localization in breast cancer using intradermal dye injection: results, influencing factors and learning curve.

To evaluate the identification rate, false negative rate, concordance, negative predictive value of sentinel node localization in breast cancer using intradermal isosulfan blue injection whether this is accurate enough for surgical approach in breast cancer surgery and whether there is a significant learning curve for this technique. Factors affecting the outcomes of the procedure are also determined. From August 2002 to September 2003, 66 cases of stage 0-IIIB operable breast cancer patients underwent sentinel lymph node biopsy before standard breast cancer operation. Overall, identification rate was 80.3%, false negative rate was 10.6%, concordance was 86.8%, negative predictive value was 83.3%, sentinel node was the only node that was positive in 45.5%, and mean operative time was 55.1 minutes. Factors found to lower sentinel node identification rate are neoadjuvant chemotherapy and large tumor (T3-4) while previous excision was not found to affect the identification rate. There is significant learning curve in this technique and this should be performed at least 40-45 cases in the learning phase to accomplish a high identification rate and lower false negative rate before implicating into clinical practice.