Human Papilloma Virus Genotyping Assay using Restriction Fragment Mass Polymorphism Analysis, and Its Comparison with Sequencing and Hybrid Capture Assays / 대한진단검사의학회지

BACKGROUND: Infection with human papilloma virus (HPV) is the main cause of cervical cancer, and HPV genotyping is of increasing importance for determining clinical course and management of the disease based on the HPV genotypes. Here, we established a novel matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) assay, termed restriction fragment mass polymorphism (RFMP) that is suitable for genotyping multiple HPV in an accurate and high-throughput manner. We evaluated the performance of the RFMP assay in HPV genotyping by comparing the results with those of direct or clonal sequencing and hybrid capture (HC) assays. METHODS: The study population consisted of 50 patients with histologically confirmed cervical lesions and a positive test for HPV DNA. HPV genotyping was performed with RFMP, sequencing, and HC assays. The assigned genotypes and risk groups were compared among the methods. RESULTS: Concordance rates in the genotype level between RFMP vs sequencing, sequencing vs HC, and HC vs RFMP were 98% (49/50), 88% (44/50), and 88% (44/50), respectivley. Especially, RFMP and sequencing were 100% concordant when assigned high-risk group was considered identical in 1 case of mixed genotypes identified only in RFMP. The observed discrepancy between HC and the other two methods is due to the assignment of six cases of low, intermediate, or unassigned risk genotypes as high-risk group in HC method. CONCLUSIONS: RFMP, sequencing, and HC assays were highly concordant with each other in HPV genotyping. Compared to sequencing assay, RFMP assay is found to be advantageous in detecting mixed genotype infections. The accuracy and amenability to high-throughput analysis should make the RFMP assay suitable for reliable screening of HPV genotypes in clinical laboratories.

Comparison of Efficacy of Human Papilloma Virus Genotyping Assays using Restriction Fragment Mass Polymorphism and DNA Chip Analysis in Patients with Abnormal Pap Smear and Uterine Cervical Cancer

BACKGROUND: High-risk human papilloma virus (HPV) infection is the primary cause of cervical cancer; there is a need for more sensitive and reliable methods for HPV genotyping to use as screening tools for early detection and intervention. METHODS: A novel MALDI-TOF MSbased assay, termed Restriction Fragment Mass Polymorphism (RFMP) was developed for multiple HPV genotyping. Its performance was compared with DNA chip technology. The study was based on 164 cases classified as normal (n=40), ASCUS (n=53) and invasive squamous cell carcinoma (SCC, n=71) by a PAP smear and/or cervical colposcopic biopsy. RESULTS: High-risk genotypes were detected in 7.5%, 47.2% and 97.2% in normal, ASCUS and SCC groups by RFMP, and in 20.0%, 41.5% and 90.1% using DNA chip technology, respectively. The results showed substantial concordance, with a kappa coefficient of 0.688, between the methods. Diagnostic sensitivity and specificity for cervical cancer were found to be 97.2% and 92.2% with RFMP and 90.1% and 80.0% using DNA chip microarrays. CONCLUSIONS: RFMP and DNA chip technologies were shown to be reliable methods for HPV genotyping with a high concordance. The improved sensitivity and specificity should make RFMP a viable option for the management of women with cervical neoplastic lesions.

Clinicopathologic Characteristics of Replication Error-Positive Gastric Adenocarcinoma in Korean

The purpose of this study is to obtain the clinicopathological characteristics of replication error-positive (RER ) gastric adenocarcinoma in Korean, and to identify the significance of RER in adenoma stage of gastric carcinogenesis. Microsatellite instability was examined at D2S71, D2S119, D3S1067, D6S87, D11S905, DM, AR, VWF, HPRT, and BAT-26 loci. Frameshift mutation of BAX gene was analyzed in RER tumors. Normal and tumor DNA of 76 cases of gastric carcinoma and 25 cases of adenoma were examined. RER was found in 8 of 76 cases (10.5%), and it was more frequently found in adenocarcinoma of female (17.7%) than those of male (4.8%). The frequency of RER was not different between the histologic types, age of the patient, anatomical location of the carcinoma, and the stage. The RER found in adenoma suggests that RER contributes to the malignant transformation early in the adenoma stage of the gastric carcinogenesis. None of the RER tumors revealed frameshift mutation of the BAX gene.