Establishing Patient-Derived Cancer Cell Cultures and Xenografts in Biliary Tract Cancer / Journal of the Korean Cancer Association, 대한암학회지

Purpose@#Biliary tract cancers (BTCs) are rare and show a dismal prognosis with limited treatment options. To improve our understanding of these heterogeneous tumors and develop effective therapeutic agents, suitable preclinical models reflecting diverse tumor characteristics are needed. We established and characterized new patient-derived cancer cell cultures and patient-derived xenograft (PDX) models using malignant ascites from five patients with BTC. @*Materials and Methods@#Five patient-derived cancer cell cultures and three PDX models derived from malignant ascites of five patients with BTC, AMCBTC-01, -02, -03, -04, and -05, were established. To characterize the models histogenetically and confirm whether characteristics of the primary tumor were maintained, targeted sequencing and histopathological comparison between primary tissue and xenograft tumors were performed. @*Results@#From malignant ascites of five BTC patients, five patient-derived cancer cell cultures (100% success rate), and three PDXs (60% success rate) were established. The morphological characteristics of three primary xenograft tumors were compared with those of matched primary tumors, and they displayed a similar morphology. The mutated genes in samples (models, primary tumor tissue, or both) from more than one patient were TP53 (n=2), KRAS (n=2), and STK11 (n=2). Overall, the pattern of commonly mutated genes in BTC cell cultures was different from that in commercially available BTC cell lines. @*Conclusion@#We successfully established the patient-derived cancer cell cultures and xenograft models derived from malignant ascites in BTC patients. These models accompanied by different genetic characteristics from commercially available models will help better understand BTC biology.

BRCA-mutated gastric adenocarcinomas are associated with chromosomal instability and responsiveness to platinum-based chemotherapy

Background@#Homologous recombination defect is an important biomarker of chemotherapy in certain tumor types, and the presence of pathogenic or likely pathogenic mutations involving BRCA1 or BRCA2 (p-BRCA) mutations is the most well-established marker for the homologous recombination defect. Gastric cancer, one of the most prevalent tumor types in Asia, also harbors p-BRCA mutations. @*Methods@#To investigate the clinical significance of p-BRCA mutations, we analyzed 366 gastric cancer cases through next-generation sequencing. We determined the zygosity of p-BRCA mutations based on the calculated tumor purity through variant allelic fraction patterns and investigated whether the presence of p-BRCA mutations is associated with platinum-based chemotherapy and a certain molecular subtype. @*Results@#Biallelic p-BRCA mutation was associated with better response to platinum-based chemotherapy than heterozygous p-BRCA mutation or wild type BRCA genes. The biallelic p-BRCA mutations was observed only in the chromosomal instability subtype, while all p-BRCA mutations were heterozygous in microsatellite instability subtype. @*Conclusions@#In conclusion, patients with gastric cancer harboring biallelic p-BRCA mutations were associated with a good initial response to platinum-based chemotherapy and those tumors were exclusively chromosomal instability subtype. Further investigation for potential association with homologous recombination defect is warranted.

Clinical Utility of a Fully Automated Microsatellite Instability Test with Minimal Hands-on Time

BACKGROUND: Microsatellite instability (MSI) analysis is becoming increasingly important in many types of tumor including colorectal cancer (CRC). The commonly used MSI tests are either time-consuming or labor-intensive. A fully automated MSI test, the Idylla MSI assay, has recently been introduced. However, its diagnostic performance has not been extensively validated in clinical CRC samples.METHODS: We evaluated 133 samples whose MSI status had been rigorously validated by standard polymerase chain reaction (PCR), clinical next-generation sequencing (NGS) cancer panel test, or both. We evaluated the diagnostic performance of the Idylla MSI assay in terms of sensitivity, specificity, and positive and negative predictive values, as well as various sample requirements, such as minimum tumor purity and the quality of paraffin blocks.RESULTS: Compared with the gold standard results confirmed through both PCR MSI test and NGS, the Idylla MSI assay showed 99.05% accuracy (104/105), 100% sensitivity (11/11), 98.94% specificity (93/94), 91.67% positive predictive value (11/12), and 100% negative predictive value (93/93). In addition, the Idylla MSI assay did not require macro-dissection in most samples and reliably detected MSI-high in samples with approximately 10% tumor purity. The total turnaround time was about 150 minutes and the hands-on time was less than 2 minutes.CONCLUSIONS: The Idylla MSI assay shows good diagnostic performance that is sufficient for its implementation in the clinic to determine the MSI status of at least the CRC samples. In addition, the fully automated procedure requires only a few slices of formalin-fixed paraffin-embedded tissue and might greatly save time and labor.

Use of a High-Throughput Genotyping Platform (OncoMap) for RAS Mutational Analysis to Predict Cetuximab Efficacy in Patients with Metastatic Colorectal Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Cetuximab demonstrates improved efficacy outcomes in patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS exon 2. Resistance to cetuximab is mediated by activating less frequent mutations in the RAS genes beyond KRAS exon 2. We performed extended RAS Mutational analysis using a high-throughput genotyping platform (OncoMap) and evaluated extended RAS analysis for predicting cetuximab efficacy in patients harboring wild-type KRAS exon 2 tumors following Sanger sequencing. MATERIALS AND METHODS: Extended RAS analysis was performed on 227 wild-type KRAS exon 2 mCRC patients who received cetuximab as salvage treatment using OncoMap ver. 4.0. Targeted genes included exon 2, exon 3, and exon 4, both in KRAS and NRAS, and included BRAF exon 15. We assessed efficacy by the new RAS mutation status. RESULTS: The OncoMap detected 57 additional mutations (25.1%): 25 (11%) in KRAS exon 2 and 32 (14.1%) beyond KRAS exon 2. Survival differences were observed after dividing patients into the wild-type RAS group (n=170) and mutant RAS group (n=57) using OncoMap. Progression-free survival was 4.8 months versus 1.8 months (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.32 to 0.61), and overall survival was 11.9 months versus 8.4 months (HR, 0.65; 95% CI, 0.47 to 0.88). CONCLUSION: Sanger sequencing is not sufficient for selecting candidates for cetuximab treatment. High-throughput extended RAS genotyping is a feasible approach for this purpose and identifies patients who might benefit from cetuximab treatment.

Paired Primary and Metastatic Tumor Analysis of Somatic Mutations in Synchronous and Metachronous Colorectal Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Although the mutation status of KRAS is highly concordant in primary and metastatic lesions, it has not been generalized to other major pathway genes. MATERIALS AND METHODS: In this study, 41 genes were evaluated and the mutational profiles were compared in 46 colorectal cancer patients with paired surgical specimens of primary and metastatic lesions: synchronous (n=27) and metachronous (n=19) lesions. A high-throughput mass spectrometry-based genotyping platform validated by orthogonal chemistry, OncoMap v.4.4, was used to evaluate the formalin-fixed, paraffin-embedded surgical specimens. The patients’ demographics, tumor characteristics, and microsatellite instability status were analyzed by a retrospective chart review. RESULTS: In this study,with OncoMap, mutationswere identified in 80.4% of patientswith the following frequency: KRAS (39.1%), TP53 (28.3%), APC (28.3%), PIK3CA (6.5%), BRAF (6.5%), and NRAS (4.3%). Although 19.6% (9/46) of the patients showed no gene mutations, 43.5% (20/46) and 37.0% (17/46) had mutations in one and two or more genes, respectively. The synchronous and metachronous lesions showed similar mutational profiles. Paired samples between primary and metastatic tumors differed in 7.4% (2/27) and 10.5% (2/19) for synchronous and metachronous according to OncoMap. CONCLUSION: These findings indicate the major pathway genes, including KRAS, TP53, APC, PIK3CA, BRAF, and NRAS, are often concordant between the primary and metastatic lesions regardless of the temporal relationship of metastasis.

Good Laboratory Standards for Clinical Next-Generation Sequencing Cancer Panel Tests

Next-generation sequencing (NGS) has recently emerged as an essential component of personalized cancer medicine due to its high throughput and low per-base cost. However, no sufficient guidelines for implementing NGS as a clinical molecular pathology test are established in Korea. To ensure clinical grade quality without inhibiting adoption of NGS, a taskforce team assembled by the Korean Society of Pathologists developed laboratory guidelines for NGS cancer panel testing procedures and requirements for clinical implementation of NGS. This consensus standard proposal consists of two parts: laboratory guidelines and requirements for clinical NGS laboratories. The laboratory guidelines part addressed several important issues across multistep NGS cancer panel tests including choice of gene panel and platform, sample handling, nucleic acid management, sample identity tracking, library preparation, sequencing, analysis and reporting. Requirements for clinical NGS tests were summarized in terms of documentation, validation, quality management, and other required written policies. Together with appropriate pathologist training and international laboratory standards, these laboratory standards would help molecular pathology laboratories to successfully implement NGS cancer panel tests in clinic. In this way, the oncology community would be able to help patients to benefit more from personalized cancer medicine.

The Significance of MicroRNA Let-7b, miR-30c, and miR-200c Expression in Breast Cancers

BACKGROUND: MicroRNA (miRNA) is a class of noncoding protein RNA as a promising biomarker for various diseases. In this study, the expression of let-7b, miR-30c, and miR-200c was studied in breast cancer tissues to evaluate the potential relationship with known clinicopathological parameters. METHODS: Quantitative real-time polymerase chain reaction was performed to determine the expression level of three miRNAs in 37 pairs of noncancerous normal and cancer tissues and an additional 38 cancer tissues from patients with invasive ductal carcinoma. RESULTS: miR-200c expression was higher in cancer tissues compared to noncancerous normal tissues, and its ratio was correlated with patient age at surgery, type of surgery, and Ki-67 expression. The expression level of let-7b in cancer tissues was inversely correlated with lymph node metastasis, histological grade, and Ki-67 expression but positively correlated with estrogen and progesterone receptor expression. miR-200c expression level was positively correlated with Her-2 expression. The miR-30c expression level in breast cancer was not correlated with any parameters. CONCLUSIONS: miR-200c and let-7b could be used as biomarkers in patients with breast cancer, but its pathological mechanism should be determined.

The Histone Acetyltransferase hMOF is Overexpressed in Non-small Cell Lung Carcinoma

BACKGROUND: One of the histone acetyltransferases (HATs) family of proteins, human MOF (hMOF, MYST1), is involved in histone H4 acetylation, particularly at lysine 16 (H4K16Ac), an epigenetic mark of active genes. Dysregulation of the epigenetic mark influences cellular biology and possibly leads to oncogenesis. We examined the involvement of hMOF and H4K16Ac in primary non-small cell lung cancer (NSCLC). METHODS: Reverse transcription polymerase chain reaction using fresh-frozen lung cancer tissues and lung cancer cell lines and immunohistochemistry for hMOF and H4K16Ac via tissue microarray of 551 formalin-fixed paraffin-embedded NSCLC tissue blocks were conducted. RESULTS: hMOF mRNA was frequently overexpressed in lung cancer tissues, compared with normal lung tissues (10/20, 50%). NSCLC tissues were positive for hMOF in 37.6% (184/489) and H4K16Ac in 24.7% (122/493) of cases. hMOF protein expression was tightly correlated with the H4K16Ac level in tumors (p<0.001). Knockdown of hMOF mRNA with siRNA led to a significant inhibition of growth in the Calu-6 cell line. CONCLUSIONS: hMOF was frequently expressed in NSCLC and was correlated with H4K16Ac. To our knowledge, this is the first study that has focused on the expression status of HATs and hMOF in NSCLC. Our results clearly suggest a potential oncogenic role of the gene and support its utility as a potential therapeutic target.

Unmet Healthcare Needs in People with Disabilities: Comparison with the General Population in Korea

OBJECTIVE: To determine the degree of disparity in unmet healthcare needs between people with disabilities and the general population in South Korea, and to analyze their perspective reasons. METHOD: Survey results of 9,744 subjects that participated in the 4th Korea National Health and Nutrition Examination Survey and 7,000 people that participated in the 2008 Survey of Disabled people were used in this population-based cross-sectional study. Unmet healthcare needs were identified as reporting experience during the last 12 months when there was a need to see a doctor, but were unable to get it. We assessed unmet healthcare needs by demographic variables, social variables and characteristics of disability. Chi-square test and logistic regression were used to determine which variables were related to unmet healthcare needs. RESULTS: A total of 22.1% of people with disabilities and 22.8% of the general population had unmet healthcare needs. However, brain impairment (25.3%) and physical impairment (25.2%) had more unmet healthcare needs than the general population and other disabilities. Unmet healthcare needs of people with disabilities was related to sex, age, self-perceived health, marital status, income, occupation and category of disability. Whereas the reasons for unmet healthcare needs in people with disabilities were lack of money (57.3%) and inaccessible transportation (12.8%), those for the general population were no available time (30.2%) and mild symptoms (23.8%). CONCLUSION: Brain and physical impairment cases with limitations to accessible transportation had the higher unmet healthcare needs. In addition, the reasons for people with these disabilities were more environmental than those of the general population.