Journal of Pathology and Translational Medicine

Original Articles

KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay: Sung Hak Lee, Arthur Minwoo Chung, Ahwon Lee, Woo Jin Oh, Yeong Jin Choi, Youn-Soo Lee, Eun Sun Jung; J Pathol Transl Med. 2017;51(1):24-31. Published online December 25, 2016; DOI: https://doi.org/10.4132/jptm.2016.10.03

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Background
Mutations in the KRAS gene have been identified in approximately 50% of colorectal cancers (CRCs). KRAS mutations are well established biomarkers in anti–epidermal growth factor receptor therapy. Therefore, assessment of KRAS mutations is needed in CRC patients to ensure appropriate treatment.
Methods
We compared the analytical performance of the cobas test to Sanger sequencing in 264 CRC cases. In addition, discordant specimens were evaluated by 454 pyrosequencing.
Results
KRAS mutations for codons 12/13 were detected in 43.2% of cases (114/264) by Sanger sequencing. Of 257 evaluable specimens for comparison, KRAS mutations were detected in 112 cases (43.6%) by Sanger sequencing and 118 cases (45.9%) by the cobas test. Concordance between the cobas test and Sanger sequencing for each lot was 93.8% positive percent agreement (PPA) and 91.0% negative percent agreement (NPA) for codons 12/13. Results from the cobas test and Sanger sequencing were discordant for 20 cases (7.8%). Twenty discrepant cases were subsequently subjected to 454 pyrosequencing. After comprehensive analysis of the results from combined Sanger sequencing–454 pyrosequencing and the cobas test, PPA was 97.5% and NPA was 100%.
Conclusions
The cobas test is an accurate and sensitive test for detecting KRAS-activating mutations and has analytical power equivalent to Sanger sequencing. Prescreening using the cobas test with subsequent application of Sanger sequencing is the best strategy for routine detection of KRAS mutations in CRC.

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Single-center study on clinicopathological and typical molecular pathologic features of metastatic brain tumor
Su Hwa Kim, Young Suk Lee, Sung Hak Lee, Yeoun Eun Sung, Ahwon Lee, Jun Kang, Jae-Sung Park, Sin Soo Jeun, Youn Soo Lee
Journal of Pathology and Translational Medicine.2023; 57(4): 217. CrossRef
Assessment of KRAS and NRAS status in metastatic colorectal cancer: Experience of the National Institute of Oncology in Rabat Morocco
Chaimaa Mounjid, Hajar El Agouri, Youssef Mahdi, Abdelilah Laraqui, En-nacer Chtati, Soumaya Ech-charif, Mouna Khmou, Youssef Bakri, Amine Souadka, Basma El Khannoussi
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The current understanding on the impact of KRAS on colorectal cancer
Mingjing Meng, Keying Zhong, Ting Jiang, Zhongqiu Liu, Hiu Yee Kwan, Tao Su
Biomedicine & Pharmacotherapy.2021; 140: 111717. CrossRef
Droplet digital PCR revealed high concordance between primary tumors and lymph node metastases in multiplex screening of KRAS mutations in colorectal cancer
Barbora Vanova, Michal Kalman, Karin Jasek, Ivana Kasubova, Tatiana Burjanivova, Anna Farkasova, Peter Kruzliak, Dietrich Busselberg, Lukas Plank, Zora Lasabova
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Rachel L. Mintz, Madeleine A. Gao, Kahmun Lo, Yeh‐Hsing Lao, Mingqiang Li, Kam W. Leong
Advanced Biosystems.2018;[Epub] CrossRef

Comparison of Direct Sequencing, PNA Clamping-Real Time Polymerase Chain Reaction, and Pyrosequencing Methods for the Detection of EGFR Mutations in Non-small Cell Lung Carcinoma and the Correlation with Clinical Responses to EGFR Tyrosin: Hyun Ju Lee, Xianhua Xu, Hyojin Kim, Yan Jin, Pingli Sun, Ji Eun Kim, Jin-Haeng Chung; Korean J Pathol. 2013;47(1):52-60. Published online February 25, 2013; DOI: https://doi.org/10.4132/KoreanJPathol.2013.47.1.52

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Abstract PDF

Background

The aims of this study were to evaluate the abilities of direct sequencing (DS), peptide nucleic acid (PNA) clamping, and pyrosequencing methods to detect epidermal growth factor receptor (EGFR) mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung carcinoma (NSCLC) samples and to correlate EGFR mutational status as determined by each method with the clinical response to EGFR tyrosine kinase inhibitors (TKIs).

Methods

Sixty-one NSCLC patients treated with EGFR TKIs were identified to investigate somatic mutations in the EGFR gene (exons 18-21).

Results

Mutations in the EGFR gene were detected in 38 of the 61 patients (62%) by DS, 35 (57%) by PNA clamping and 37 (61%) by pyrosequencing. A total of 44 mutations (72%) were found by at least one of the three methods, and the concordances among the results were relatively high (82-85%; kappa coefficient, 0.713 to 0.736). There were 15 discordant cases (25%) among the three different methods.

Conclusions

All three EGFR mutation tests had good concordance rates (over 82%) for FFPE samples. These results suggest that if the DNA quality and enrichment of tumor cells are assured, then DS, PNA clamping, and pyrosequencing are appropriate methods for the detection of EGFR mutations.

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Aeri Kim, Min Hye Jang, Soo Jung Lee, Young Kyung Bae
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MGMT Gene Promoter Methylation Analysis by Pyrosequencing of Brain Tumour.: Young Zoon Kim, Young Jin Song, Ki Uk Kim, Dae Cheol Kim; Korean J Pathol. 2011;45(5):455-462.; DOI: https://doi.org/10.4132/KoreanJPathol.2011.45.5.455

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Abstract PDF

BACKGROUND
The aim of this study was to determine whether pyrosequencing (PSQ) might be useful to achieve O6-methyl guanine methyltransferase (MGMT) promoter methylation using 1- to 13-year-old archival tissues as a clinical biomarker in routine practice.
METHODS
The study included 141 formalin-fixed paraffin-embedded (FFPE) glial tumors from the archives of the Pathology Department from 1997-2010.
RESULTS
The average percentage of methylation (MP) of the 141 cases was 14.0+/-16.8%, and methylated cases were 32.3+/-14.9%. The average MP of each year did not show a linear increasing or decreasing pattern according to the age of the FFPE block (p=0.771). The average MP of methylated glioblastomas was 35.8+/-14.7%, 31.8+/-15.5% for anaplastic astrocytomas, and 22.4+/-15.1% for astrocytoma. A tendency was observed toward an increasing pattern of average MP with World Health Organization (WHO) grade (p=0.063) in astrocytic tumors. A correlation was observed between average MP and WHO grade (p=0.038) and a bimodal distribution was observed between the methylated and unmethylated cases, using a 9% cut-off value (p<0.001).
CONCLUSIONS
The results showed that a quantitative approach for MGMT promoter methylation yielded a 100% success rate for FFPE tissues from archives. PSQ can be used in a retrospective trial, but the cut-off value and calculation method should be further validated.

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Immunohistochemical Classification of Primary and Secondary Glioblastomas
Kyu Sang Lee, Gheeyoung Choe, Kyung Han Nam, An Na Seo, Sumi Yun, Kyung Ju Kim, Hwa Jin Cho, Sung Hye Park
Korean Journal of Pathology.2013; 47(6): 541. CrossRef

A Consideration of MGMT Gene Promotor Methylation Analysis for Glioblastoma Using Methylation-Specific Polymerase Chain Reaction and Pyrosequencing.: Sang Hwa Lee, Tae Sook Hwang, Young Cho Koh, Wook Youn Kim, Hye Seung Han, Wan Seop Kim, Young Sin Ko, So Dug Lim; Korean J Pathol. 2011;45(1):21-29.; DOI: https://doi.org/10.4132/KoreanJPathol.2011.45.1.21

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Abstract PDF

BACKGROUND
O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation is currently the most promising predictive marker for the outcome and benefit from temozolomide treatment in patients with glioblastoma, but there is no consensus on the analysis method for assessing the methylation status in the molecular diagnostic field. The objective of this study was to evaluate methylation-specific polymerase chain reaction (MSP) and pyrosequencing methods for assessing MGMT gene promotor methylation of glioblastoma as well as assessing the MGMT protein expression by immunohistochemistry.
METHODS
Twenty-seven cases of glioblastoma from the archives at the Department of Pathology Konkuk University Hospital were selected. MGMT promoter methylation was evaluated by MSP and the pyrosequencing methods. The MGMT expression was also measured at the protein level by immunohistochemistry.
RESULTS
Overall, MGMT hypermethylation was observed in 44.4% (12/27 cases) of the case of glioblastoma using either MSP or pyrosequencing. The concordant rate was 70.3% (19/27 cases) between MSP and pyrosequencing for MGMT methylation. There was no correlation between MGMT methylation and the protein expression. No significant differences in progression free survival and overall survival were seen between the methylated group and the unmethylated group by using either MSP or pyrosequencing. The status of the MGMT protein expression was correlated with progression free survival (p=0.026).
CONCLUSIONS
In this study the concordance rate between MSP and the pyrosequencing methods for assessing MGMT gene promotor methylation was relatively low for the cases of glioblastoma. This suggests that more reliable techniques for routine MGMT methylation study of glioblastoma remain to be developed because of quality control and assurance issues.

Citations

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Prognostic Role of Methylation Status of theMGMTPromoter Determined Quantitatively by Pyrosequencing in Glioblastoma Patients
Dae Cheol Kim, Ki Uk Kim, Young Zoon Kim
Journal of Korean Neurosurgical Society.2016; 59(1): 26. CrossRef
Distinct genetic alterations in pediatric glioblastomas
Sun-ju Byeon, Jae Kyung Myung, Se Hoon Kim, Seung-Ki Kim, Ji Hoon Phi, Sung-Hye Park
Child's Nervous System.2012; 28(7): 1025. CrossRef
MGMTGene Promoter Methylation Analysis by Pyrosequencing of Brain Tumour
Young Zoon Kim, Young Jin Song, Ki Uk Kim, Dae Cheol Kim
The Korean Journal of Pathology.2011; 45(5): 455. CrossRef

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