Genetic Variations and Frequencies of the Two Functional Single Nucleotide Polymorphisms of SLCO1B1 in the Thai Population.

AIM: To investigate the variations and the frequencies of the SLCO1B1 gene in the Thai population. METHODS: Collected samples were categorized into five regions of Thailand. DNA samples were genotyped for two variants, c.388A>G and c.521T>C of the SLCO1B1, using TaqMan® real-time PCR. RESULTS: The minor allele frequencies (MAFs) of two single nucleotide polymorphisms (SNPs) were not significantly different among the five regions. The most frequent haplotype was SLCO1B1*1b (frequency: 0.654), followed by *1a (frequency: 0.217), *15 (frequency: 0.128), and *5 (frequency: 0.001). We observed a similar frequency of OATP1B1 transporter phenotypes compared to other populations. 75.85% of the Thai subjects showed normal OATP1B1 activity, 22.5% showed intermediate OATP1B1 activity, and 1.58% showed low OATP1B1 activity. CONCLUSION: This study reported the frequencies of the SLCO1B1 variants and the subsequent OATP1B1 activity in a large cohort of Thais that can provide important information for the guidance of personalized drug therapy.

Droplet digital PCR using HER2/EIF2C1 ratio for detection of HER2 amplification in breast cancer tissues.

Breast cancers with amplification and overexpression of human epithelial growth factor receptor 2 (HER2) are associated with poor prognosis, and targeted for anti-HER2 therapy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently the recommended methods to asses HER2 overexpression/amplification. Droplet digital PCR (ddPCR), a highly accurate method to quantify DNA copy number, is potentially a robust alternative for HER2 diagnostics. In the FISH assay and most of previous ddPCR reports, chromosome 17 centromere (CEP17) has been used as the reference control to determine HER2/CEP17 ratio. Nevertheless, miss-classification could occur when HER2 is co-amplified with CEP17. To avoid this inherent defect, in the present study, we employed ddPCR assay using the human eukaryotic translation initiation factor 2C1 (EIF2C1) gene located at chromosome 1p34.3 as the reference control to quantify HER2 copy number in 31 frozen breast cancer tissues. HER2 status of these samples had been determined by FISH and classified as HER2-amplified and HER2-non-amplified breast cancers. The results showed that HER2 determined by ddPCR using HER2/EIF2C1 ratio was in good concordance with HER2 determined by FISH using HER2/CEP17 ratio, the concordance rate 87.1% (27/31), Kappa = 0.719. The sensitivity and specificity of ddPCR assay was 90% (9/10) and 85.7% (18/21), respectively. The median HER2/EIF2C1 copy number ratio in HER2-amplified cancers (6.55, range 1.3-17.3) was significantly higher than in HER2-non-amplified cancers (1.05, range 0.6-3.6, p < 0.001). This study demonstrated that ddPCR using HER2/EIF2C1 ratio could accurately assess HER2 status in frozen breast cancer tissues. Thus, our findings warrant further studies into breast cancer with HER2-equivocal by IHC/FISH.

Determination of irinotecan, SN-38 and SN-38 glucuronide using HPLC/MS/MS: Application in a clinical pharmacokinetic and personalized medicine in colorectal cancer patients.

BACKGROUND: Irinotecan (CPT-11) is chemotherapy used mainly in the metastatic colorectal cancer. The purpose of this study was to develop and validate the LC-MS/MS for the simultaneous determination of CPT-11, SN-38, and SN-38G. METHODS: A 100 µL of plasma was prepared after protein precipitation and analyzed on a C18 column using 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile as mobile phases. The mass spectrometer worked with multiple reaction monitoring (MRM) in positive scan mode. The standard curves were linear on a concentration range of 5-10 000 ng/mL for CPT-11, 5-1000 ng/mL for SN-38, and 8-1000 ng/mL for SN-38G. RESULTS: In this assay, the intra and interday precision consisted of ≤9.11% and ≤11.29% for CPT-11, ≤8.70% and 8.31% for SN-38, and ≤9.90 and 9.64% for SN-38G. CONCLUSION: This method was successfully used to quantify CPT-11, SN-38, and SN-38G and applied to a pharmacokinetic study.

ABCB1 and ABCC2 and the risk of distant metastasis in Thai breast cancer patients treated with tamoxifen.

BACKGROUND: Genetic polymorphisms of drug-metabolizing enzymes and transporters have been extensively studied with regard to tamoxifen treatment outcomes. However, the results are inconclusive. Analysis of organ-specific metastasis may reveal the association of these pharmacogenetic factors. The aim of this study is to investigate the impact of CYP3A5, CYP2D6, ABCB1, and ABCC2 polymorphisms on the risk of all distant and organ-specific metastases in Thai patients who received tamoxifen adjuvant therapy. METHODS: Genomic DNA was extracted from blood samples of 73 patients with breast cancer who received tamoxifen adjuvant therapy. CYP3A5 (6986A>G), CYP2D6 (100C>T), ABCB1 (3435C>T), and ABCC2 (-24C>T) were genotyped using allelic discrimination real-time polymerase chain reaction assays. The impacts of prognostic clinical factors and genetic variants on disease-free survival were analyzed using the Kaplan-Meier method and Cox regression analysis. RESULTS: In the univariate analysis, primary tumor size >5 cm was significantly associated with increased risk of distant metastasis (P=0.004; hazard ratio [HR] =3.05; 95% confidence interval [CI], 1.44-6.47). In the multivariate analysis, tumor size >5 cm remained predictive of distant metastasis (P<0.001; HR=5.49; 95% CI, 2.30-13.10). ABCC2 -24CC were shown to be associated with increased risk of distant metastasis (P=0.040; adjusted HR=2.34; 95% CI, 1.04-5.27). The combined genotype of ABCC2 -24CC - ABCB1 3435 CT+TT was associated with increased risk of distant and bone metastasis (P=0.020; adjusted HR=2.46; 95% CI, 1.15-5.26 and P=0.040; adjusted HR=3.70; 95% CI, 1.06-12.89, respectively). CONCLUSION: This study indicates that polymorphisms of ABCC2 and ABCB1 are independently associated with bone metastasis. Further prospective studies with larger sample sizes are needed to verify this finding.

Correlation of UGT1A1(*)28 and (*)6 polymorphisms with irinotecan-induced neutropenia in Thai colorectal cancer patients.

UDP-glucuronosyltransferase1A1 (UGT1A1) polymorphisms have been related with irinotecan toxicity. The purpose of this study was to determine the associations between UGT1A1(*)28 and (*)6 polymorphisms and irinotecan toxicity in Thai patients with metastatic colorectal cancer. 44 metastatic colorectal cancer patients received irinotecan-based chemotherapy. Hematologic toxicities were determined in the first and second cycles of treatment. The genotypes of UGT1A1(*)28 and (*)6 were analyzed by pyrosequencing technique. The frequencies of genetic testing for UGT1A1(*)28 and (*)6 polymorphisms were 22.8% (TA6/TA7; 20.5%, TA7/TA7; 2.3%) and 15.9% (GA), respectively. No patients had the homozygous UGT1A1(*)6 (AA). Neither UGT1A1(*)28 nor UGT1A1(*)6 polymorphisms were significantly associated with severe hematologic toxicities. However, analysis of UGT1A1(*)28 and (*)6 in combination revealed an association with severe neutropenia in the first and second cycles (P = 0.044, P = 0.017, respectively). Both UGT1A1(*)28 and (*)6 polymorphisms may have an increased risk of irinotecan-induced neutropenia in Thai colorectal cancer patients.

Hydroxyurea and colonic ulcers: a case report.

BACKGROUND: Hydroxyurea at a relatively low dose is frequently prescribed to induce hemoglobin F production in patients with sickle cell and ß-thalassemia diseases because of its good efficacy and safety profiles. However, a potentially fatal gastrointestinal ulceration was recently found and herein reported. CASE PRESENTATION: A thirty-seven-year-old man with transfusion dependent hemoglobin E/ß-thalassemia disease was treated with hydroxyurea to induce hemoglobin F production since 2007 without incident. From 2008 to April 2010, episodes of hematochezia, mucous diarrhea and epigastric pain intermittently manifested. Four colonoscopies done during the period repeatedly showed ulcerative lesions from the terminal ileum to the ascending colon with a non-specific histo-pathologic finding. Subsequently, ulcerative lesions also developed at the pharynx, histo-pathologic findings of which were not different from those in the colon. These ulcerative lesions resolved within a month after discontinuing hydroxyurea in April 2010 and have not recurred since. CONCLUSION: The findings suggested role of hydroxyurea in the pathogenesis of these ulcers, and that it must be immediately discontinued to prevent further damage to the digestive mucosa.

Development and validation of a breast cancer risk prediction model for Thai women: a cross-sectional study.

BACKGROUND: Breast cancer risk prediction models are widely used in clinical practice. They should be useful in identifying high risk women for screening in limited-resource countries. However, previous models showed poor performance in derived and validated settings. Therefore, we aimed to develop and validate a breast cancer risk prediction model for Thai women. MATERIALS AND METHODS: This cross-sectional study consisted of derived and validation phases. Data collected at Ramathibodi and other two hospitals were used for deriving and externally validating models, respectively. Multiple logistic regression was applied to construct the model. Calibration and discrimination performances were assessed using the observed/expected ratio and concordance statistic (C-statistic), respectively. A bootstrap with 200 repetitions was applied for internal validation. RESULTS: Age, menopausal status, body mass index, and use of oral contraceptives were significantly associated with breast cancer and were included in the model. Observed/expected ratio and C-statistic were 1.00 (95% CI: 0.82, 1.21) and 0.651 (95% CI: 0.595, 0.707), respectively. Internal validation showed good performance with a bias of 0.010 (95% CI: 0.002, 0.018) and C-statistic of 0.646(95% CI: 0.642, 0.650). The observed/expected ratio and C-statistic from external validation were 0.97 (95% CI: 0.68, 1.35) and 0.609 (95% CI: 0.511, 0.706), respectively. Risk scores were created and was stratified as low (0-0.86), low-intermediate (0.87-1.14), intermediate-high (1.15-1.52), and high-risk (1.53-3.40) groups. CONCLUSIONS: A Thai breast cancer risk prediction model was created with good calibration and fair discrimination performance. Risk stratification should aid to prioritize high risk women to receive an organized breast cancer screening program in Thailand and other limited-resource countries.

Risk factors of breast cancer: a systematic review and meta-analysis.

The etiology of breast cancer might be explained by 2 mechanisms, namely, differentiation and proliferation of breast epithelial cells mediated by hormonal factors. We performed a systematic review and meta-analysis to update effects of risk factors for both mechanisms. MEDLINE and EMBASE were searched up to January 2011. Studies that assessed association between oral contraceptives (OC), hormonal replacement therapy (HRT), diabetes mellitus (DM), or breastfeeding and breast cancer were eligible. Relative risks with their confidence intervals (CIs) were extracted. A random-effects method was applied for pooling the effect size. The pooled odds ratios of OC, HRT, and DM were 1.10 (95% CI = 1.03-1.18), 1.23 (95% CI = 1.21-1.25), and 1.14 (95% CI = 1.09-1.19), respectively, whereas the pooled odds ratio of ever-breastfeeding was 0.72 (95% CI = 0.58-0.89). Our study suggests that OC, HRT, and DM might increase risks, whereas breastfeeding might lower risks of breast cancer.

Androgenic pathway in triple negative invasive ductal tumors: its correlation with tumor cell proliferation.

Triple negative breast cancer (TNBC) is defined by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 negativity. Patients with TNBC frequently undergo an aggressive clinical course due to the unavailability of specific targeted therapies. Androgen receptor (AR) was reported to be expressed in up to 60% of TNBC cases but there have been controversies as to the roles of androgen signaling through AR in TNBC. Therefore, in this study, we analyzed the status of AR in combination with androgen synthesizing enzymes (5α-reductase type 1 (5αR1) and 17ß-hydroxysteroid dehydrogenase type 5 (17ßHSD5)] in order to further understand androgenic actions in TNBC. Androgen receptor, 5αR1, and 17ßHSD5 were immunolocalized in a cohort of 203 TNBC patients from Thailand and Japan. We then correlated the findings with clinicopathological characteristics (age, stage, tumor diameter, lymph node invasion, metastatic spread, Ki-67 labeling index, disease-free survival, and overall survival) of the patients. Univariate analysis revealed that AR+/enzyme+ cases were associated with a significantly lower Ki-67 labeling index than AR-/enzyme- samples. Multivariate analysis indicated the presence of significant positive correlations between AR and enzyme status in tumor cells, and between tumor diameter, lymph node invasion, and distant metastasis. Significant negative correlations were also detected between Ki-67 labeling index and AR status (P = 0.04) or 5αR1 (P < 0.001). Cox proportional hazards analysis showed that Ki-67 labeling index and stage were the only factors predicting disease-free and overall survival of the patients, although univariate Kaplan-Meier analysis revealed AR/5αR1 negativity suggested a more adverse clinical course up to 80 months after surgery. These results suggest that the presence of androgen synthesizing pathways in addition to AR expression in tumor cells could confer a better clinical outcome through suppression of cell proliferation.

Breast cancer cells in the afferent lymphatic tracts of sentinel lymph nodes.

BACKGROUND: To identify breast cancer cells in the afferent lymphatic tracts of axillary sentinel lymph nodes (SLNs). MATERIAL AND METHOD: The authors performed a prospective study of 1 00 breast cancer patients who underwent SLN biopsy between June 2009 and January 2010. The afferent lymphatic tracts of SLNs were identified by isosulfan blue or radiocolloid or both and were examined histologically. RESULTS: One hundred three SLNs and afferent lymphatic tracts were examined. The mean age of the patients was 53.2 years (range, 24 to 78 years). The median number of SLNs was 2 (range, 1 to 7). Twenty-four (24%) patients had positive SLNs. Most patients had stage I breast cancer (67%). Three patients with positive SLNs (13%) and stages IIB-IIIC breast cancers had tumor cells in the afferent tract tissue. There were no tumor cells in the afferent tracts of negative SLNs. CONCLUSION: Only a small proportion of operable breast cancer patients have tumor cells in the afferent lymphatic tract tissue of SLNs. There was a probable trend for more advanced stage breast cancer to harbor tumor cells in the afferent lymphatic tract tissue.

Coexisting vulvar Paget's disease and mucinous carcinoma of the breast: a case report and review of the literature.

Vulvar Paget 'disease is the most common site of extramammary Paget's disease (EMPD). The disease frequently associated with the underlying invasive skin adnexal carcinoma or representing the migration of underlying internal malignancy, especially anorectal and genitourinary cancer, but the coexisting with primary breast cancer is rare. Herein, the authors report a case of a 46-year-old Thai woman who had vulvar Paget's disease with subsequent development of mucinous carcinoma of the breast. Interestingly, the overexpression of HER-2/neu in vulvar Paget's disease raises the additional option of anti-HER-2/neu antibody therapy in highly aggressive or recurrent disease. In conclusion, primary breast cancer should be of concern in patients with vulva Paget's disease, even though it is an uncommon association.

HER2 expression in breast cancer with nonamplified HER2 and gains of chromosome 17 centromere.

Gains of chromosome 17 centromere (CEP17) may be accompanied by gains of chromosome 17q. To evaluate the effect of CEP17 gains (CEP17>3 copies per tumor nucleus) on the expression of the HER2 gene, which is located on chromosome 17q12-21.32, we analyzed HER2 amplification and expression in breast carcinomas with and without CEP17 gains. We isolated tumor nuclei from frozen tissues of 37 breast carcinomas for analysis of the HER2 gene and CEP17 by fluorescence in situ hybridization. HER2 expression was detected by immunohistochemistry (IHC) performed on formalin-fixed, paraffin-embedded sections of the corresponding tumors. Tumors with amplified HER2 as determined by both HER2 copy number and HER2/CEP17 ratio were detected in 29.7% (11/37). CEP17 gains were significantly associated with HER2 amplification (P=0.005) but not associated with estrogen receptor status, tumor grade, and lymph node status (P>0.05). In contrast, HER2 amplification was significantly associated with estrogen receptor negativity (P=0.020) but not with tumor grade and lymph node status (P>0.05). IHC analysis was performed in 7 HER2-amplified tumors and all of these were IHC 3+, which were used as positive controls. Among HER2-non-amplified tumors with CEP17 gains, only 1 tumor (1/8, 12.5%) was IHC 3+. However, none of the HER2-non-amplified tumors without CEP17 gains was IHC 3+. In HER2-non-amplified tumors, there was no significant association between HER2 protein expression as detected by IHC and CEP17 or HER2 copy number (P=0.999, P=0.785, respectively). These findings indicate that in the absence of HER2 amplification, CEP17 gains do not have a significant effect on HER2 protein expression.

Prenatal diagnosis and pathology of laryngeal atresia in congenital high airway obstruction syndrome.

Congenital high airway obstruction syndrome is a rare but life-threatening condition. Therefore, prenatal diagnosis is important. The obstruction can be due to laryngeal/tracheal atresia or external compression. While a differential diagnosis with congenital cystic adenomatoid malformation (CCAM) type III may be difficult, it is still possible with ultrasonography. In this study, we report a case of bilateral echogenic lungs with hydrops fetalis. After the prenatal diagnosis of laryngeal atresia, the couple opted to have an elective termination of pregnancy performed at 20 weeks of gestation. The diagnosis was confirmed by a complete pathological examination.

Glioblastoma multiforme at the corpus callosum with spinal leptomeningeal metastasis.

Glioblastoma multiforme (GBM) often occurs in the supratentorial white matter including corpus callosum. However, spinal leptomeningeal metastasis in cases of supratentorial GBM has been reported to be rare and there is usually a long interval between the cerebral lesion and the spinal seeding. We report here a case of GBM at the corpus callosum and other parts of the brain with simultaneous manifestation of spinal leptomeningeal seeding. The patient exhibited an abnormal motor behavior of the left hand as mirror movement when the right hand was performing a unimanual task (diagonistic dyspraxia) which is a sign of lesion of the posterior part and splenium of the corpus callosum. There were also signs of peripheral nerve or nerve root involvement suggestive of spinal metastasis without any sensory symptoms. He died 3 months after the onset of the symptoms confirming the poor prognosis and short survival time in cases with spinal leptomeningeal metastasis reported previously. The cerebral GBM with spinal seeding was disclosed at autopsy.

Double check up of malignancy biopsy specimens for patient safety.

BACKGROUND: The diagnostic of malignancy in biopsy specimens is very important because it guides to selected treatment option and prognostic prediction. However biopsy specimens usually have small pieces leading to variations of the interpretation by anatomical pathologists. OBJECTIVE: To detect and correct the errors or the significant discrepancies in the diagnosis of biopsy specimens before sign-out and to determine the frequency of anatomic pathology significant discrepancies. DESIGN: The application of the mutually agreed work instructions (record) for the detection of errors or the significant discrepancies and their process of sign-out. The record of biopsy specimen that received a secondary check (1959 cases, 2005-2007) was analyzed. RESULTS: After a secondary check, 53 cases of non-malignancy for any reason by a second pathologist were included. However when using our definition on significant discrepancies, only 37 cases were considered. Another seven cases with the opinions with malignancy that were of different cell types that do harm to the patients were added. Therefore, 44 cases (2.25%) had truly significant discrepancies. CONCLUSION: The truly significant discrepancy frequency was 2.25% during the process of pre-sign-out secondary check of malignancy of biopsy specimens. The project has been applied as a routine daily work. It can be an innovative safety program for patient in Thailand.

Prognostic significance of microvessel density in breast cancer of Thai women.

OBJECTIVE: Angiogenesis is important in the process of tumor growth and progression of breast cancer Microvessel density (MVD) is the most commonly used technique to quantify intratumoral angiogenesis in breast cancer. In the present study, the authors investigated the prognostic indicator of intratumoral MVD in predicting overall survival, disease recurrence, and distance metastasis of breast cancer. MATERIAL AND METHOD: Two hundred patients who were diagnosed as invasive breast cancer from January 2000 to December 2004 were included in the present study, but only 64 patients had complete pathological specimens and tumor receptor studies. Representative paraffin sections of the primary tumor including the tumor border were immunostained with a monoclonal anti-CD34 antibody. The area of highest vascular density ("hot spot") was identified and the average count of three hot spots in each tumor was used for analyses. The distribution of MVD was categorized into high and low MVD as more than 76 and less than 76 respectively. Overall survival probability was estimated by the Kaplan-Meier method. A multivariate Cox regression was employed to examine the relationship between MVD and disease outcomes while adjusting for other concomitant variables. RESULT: The tumor size and more advanced disease have correlated with poor outcomes of invasive breast cancer. Tumor size is a poor predictor for local recurrence [Harzard ratio 1.02 (95% CI 0.99-1.04)] and more disease staging have correlated with distance metastases [Harzard ratio 1.48 (95% CI 0.98-2.24)]. The cancer staging only predicted poor outcome in invasive breast cancer in overall recurrence [Harzard ratio 1.51 (95% CI 1.05-2.16)]. MVD is not correlated with both tumor recurrence and distance metastases [Hazard ratio for local recurrence 1.01 (95% CI 0.99-1.04) and Harzard ratio 1.00 (95% CI 0.97-1.02)]. CONCLUSION: The microvessel density (MVD) has not predicted poor outcomes of invasive breast cancer in Thai woman.

Breast cancer underestimation rate of atypical ductal hyperplasia diagnosed by core-needle biopsy under imaging guidance.

OBJECTIVE: To evaluate breast cancer underestimation rate of atypical ductal hyperplasia (ADH) diagnosed by core-needle biopsy (CNB) under imaging guidance in Ramathibodi Hospital and to determine the difference between the malignant and benign groups in terms of clinical and imaging characteristics. MATERIAL AND METHOD: The pathological records of 1521 patients who underwent CNB under imaging guidance were reviewed. Thirty-nine patients diagnosed with ADH were enrolled into the present study. Clinical data, imaging features, biopsy technique and result of excisional biopsy as well as follow-up data were retrospectively reviewed. RESULTS: Of 39 ADH cases, eight (20.5%) were found to have malignancy on subsequent excisional biopsy. The majority of these were ductal carcinoma in situ (DCIS) (62.5%). Lesion categorized as category 5 according to BI-RADS (Breast imaging reporting and data system) was the only feature which was statistically different between the benign and malignant groups. No statistically significant difference was found between the benign and malignant groups in terms of age, personal and family history of breast cancer, clinical finding, mammographic lesion type, size of lesion, image-guided technique and percentage of lesion removal. CONCLUSION: The underestimation rate of ADH in the present study was comparable to other studies. The finding of Bl-RADS category 5 in patients with ADH diagnosed from CNB is a strong indication for subsequent excisional biopsy.

Nondiagnostic core needle biopsy of the breast under imaging guidance: result of rebiopsy.

OBJECTIVE: To determine the rate and the clinical application of recommendation for repeat biopsy after core needle biopsy (CNB) under imaging guidance and to determine the result of rebiopsy. MATERIAL AND METHOD: A retrospective review was performed in 1,306 consecutive women who underwent core needle biopsy under imaging guidance at the breast diagnostic center, the Faculty of Medicine, Ramathibodi Hospital from October 1997 to March 2004. RESULTS: Among 1,306 patients, there were 44 patients (3.37%) who had undergone rebiopsy. The three most common reasons for recommendation of rebiopsy were discordant imaging and pathology, atypical ductal hyperplasia and inadequate specimen. The authors found 12 malignancies subsequently found in rebiopsy (27.3%). The most common reason for rebiopsy in this group was inadequate specimen. CONCLUSION: Core needle biopsy under imaging guidance is a minimally invasive diagnostic tool and promises high accuracy and reliability. However, some patients need rebiopsy to exclude hidden malignancy. The cooperation between the radiologists, surgeons and pathologists are prudent for giving the best care to the 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.

Ocular pythiosis: is it under-diagnosed?

PURPOSE: To increase awareness of ocular pythiosis by presenting a typical case and summarizing clinical data of 11 ocular pythiosis cases in Ramathibodi Hospital. DESIGN: Interventional case report. METHODS: A 48-year-old healthy woman with a history of 3-week painful corneal ulcer of left eye was treated with enucleation. RESULTS: The histopathology of enucleated eye revealed endophthalmitis and ulcerative keratitis with numerous hyphae in full-thickness of corneal stroma. The culture identification of the causative organism was Pythium insidiosum. The final diagnosis was ocular pythiosis. CONCLUSIONS: Pythium insidiosum is a causative agent of pythiosis and is distributed worldwide. Ocular pythiosis may not be uncommon, as it may be underdiagnosed due to unfamiliarity among clinicians and microbiologists. Diagnosis of pythiosis is difficult. The disease has high morbidity, as evidenced by nearly evisceration or enucleation among all patients at Ramathibodi Hospital. Early detection and effective treatment are needed for possible cure.