Hee Joung Kim

Detection and comparison of peptide nucleic acid-mediated real-time polymerase chain reaction clamping and direct gene sequencing for epidermal growth factor receptor mutations in patients with non-small cell lung cancer

EGFR tyrosine kinase inhibitors (EGFR-TKIs) are recommended as first-line therapy in patients with advanced, recurrent, or metastatic non-squamous non-small cell lung cancer (NSCLC) that have active EGFR mutations. The importance of rapid and sensitive methods for the detection of EGFR mutations is emphasized. The aim of this study is to examine the EGFR mutational status by both direct DNA sequencing and peptide nucleic acid (PNA)-mediated real-time PCR clamping and to evaluate the correlation between the EGFR mutational status and the clinical response to EGFR-tyrosine kinase inhibitors. Clinical specimens from 240 NSCLC patients were analyzed for EGFR mutations in exons 18, 19, 20 and 21. All clinical data and tumor specimens were obtained from 8 centers of the Korean Molecular Lung Cancer Group (KMLCG). After genomic DNA was extracted from paraffin-embedded tissue specimens, we performed PNA-mediated real-time PCR clamping and direct DNA sequencing for the detection of EGFR mutations. Of 240 tumor samples, PNA-mediated PCR clamping was used to detect genomic alterations in 83 (34.6%) samples, including 61 identified by sequencing and 22 additional samples (10 in exon 19, 9 in exon 21, and 3 in both exons); direct DNA sequencing was used to identify a total of 63 (26.3%) mutations that contained 40 deletion mutations in exon 19 (63.5%) and 18 substitution mutations (28.6%) in exon 21. PNA-mediated PCR clamping was used to identify more mutations than clinical direct sequencing, whereas clinical outcomes were not significantly different between the groups harboring activating mutations detected by each method. These data suggest that PNA-mediated real-time PCR clamping exhibits high sensitivity and is a simple procedure relative to direct DNA sequencing that is a useful screening tool for the detection of EGFR mutations in clinical settings.

Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano‐LC–MS/MS following 1D SDS‐PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung‐enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.

The performance of a novel amino acid multivariate index for detecting lung cancer: A case control study in Korea

INTRODUCTION Previous studies have shown that plasma free amino acid (PFAA) profiles are altered in cancer patients compared with healthy controls. A multivariate index based on PFAAs was generated from a Japanese dataset and has been previously demonstrated to be clinically valuable for discriminating patients in the early stages of lung cancer. However, it remains unclear whether similar PFAA profile changes occur in cancer patients from other populations. Therefore, this study aimed to validate the performance of this index in discriminating lung cancer patients from controls in the Korean population. METHODS Samples were collected from a total of 142 Korean subjects (72 lung cancer/70 controls) for this study. PFAAs were quantified by high-performance liquid chromatography-electrospray ionization-mass spectrometry, and the clinical performance characteristics of the amino acid multivariate index were evaluated across cancer stages and histological types. RESULTS The concentrations of several PFAAs were significantly decreased in the Korean lung cancer patients compared with the controls. Significant decreases in threonine, citrulline, histidine and tryptophan and increases in proline, isoleucine, phenylalanine and ornithine were observed, which are similar to the PFAA changes reported by a previous Japanese study. The area under the receiver-operator characteristic curve (AUC of the ROC) for the index was 0.80, and similar performances were demonstrated for the different histological types. CONCLUSIONS These results suggest that the amino acid multivariate index previously developed from a Japanese dataset has the potential to aid in the early detection of lung cancers of different histological types in Korean patients.

P-glycoprotein confers acquired resistance to 17-DMAG in lung cancers with an ALK rearrangement

BackgroundBecause anaplastic lymphoma kinase (ALK) is dependent on Hsp90 for protein stability, Hsp90 inhibitors are effective in controlling growth of lung cancer cells with ALK rearrangement. We investigated the mechanism of acquired resistance to 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin analogue Hsp90 inhibitor, in H3122 and H2228 non-small cell lung cancer cell lines with ALK rearrangement.MethodsResistant cell lines (H3122/DR-1, H3122/DR-2 and H2228/DR) were established by repeated exposure to increasing concentrations of 17-DMAG. Mechanisms for resistance by either NAD(P)H/quinone oxidoreductase 1 (NQO1), previously known as a factor related to 17-DMAG resistance, or P-glycoprotein (P-gp; ABCB1/MDR1) were queried using RT-PCR, western blot analysis, chemical inhibitors, the MTT cell proliferation/survival assay, and cellular efflux of rhodamine 123.ResultsThe resistant cells showed no cross-resistance to AUY922 or ALK inhibitors, suggesting that ALK dependency persists in cells with acquired resistance to 17-DMAG. Although expression of NQO1 was decreased in H3122/DR-1 and H3122/DR-2, NQO1 inhibition by dicumarol did not affect the response of parental cells (H2228 and H3122) to 17-DMAG. Interestingly, all resistant cells showed the induction of P-gp at the protein and RNA levels, which was associated with an increased efflux of the P-gp substrate rhodamine 123 (Rho123). Transfection with siRNA directed against P-gp or treatment with verapamil, an inhibitor of P-gp, restored the sensitivity to the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we also observed that the growth-inhibitory effect of 17-DMAG was decreased in A549/PR and H460/PR cells generated to over-express P-gp by long-term exposure to paclitaxel, and these cells recovered their sensitivity to 17-DMAG through the inhibition of P-gp.ConclusionP-gp over-expression is a possible mechanism of acquired resistance to 17-DMAG in cells with ALK rearrangement.

Paclitaxel-Loaded Polymeric Micelle (230 mg/m2) and Cisplatin (60 mg/m2) vs. Paclitaxel (175 mg/m2) and Cisplatin (60 mg/m2) in Advanced Non–Small-Cell Lung Cancer: A Multicenter Randomized Phase IIB Trial

INTRODUCTION The development of paclitaxel-loaded polymeric micelle (PPM) has circumvented many of the infusion-related difficulties associated with standard solvent-based paclitaxel. PPM plus cisplatin combination chemotherapy showed significant antitumor activity in phase I and II studies. This prospective randomized controlled phase IIB study assessed the noninferiority of the efficacy and tolerability of high-dose PPM plus cisplatin to a standard dose of paclitaxel plus cisplatin. PATIENTS AND METHODS Patients with stage IIIB/IV or recurrent non-small-cell lung cancer (NSCLC) who were chemonaive were eligible for participation. The patients were randomly assigned to receive PPM 230 mg/m(2) plus cisplatin 60 mg/m(2) or paclitaxel 175 mg/m(2) plus cisplatin 60 mg/m(2) once every 3-week cycle. The primary endpoint was to compare the response rate (RR) between the groups with coprimary analyses to assess noninferiority. Secondary endpoints included progression-free survival, overall survival, and safety. RESULTS A total of 276 patients were randomized to PPM plus cisplatin (n = 140) or paclitaxel plus cisplatin (n = 136). RR was 43.6% in the PPM plus cisplatin group and 41.9% in the paclitaxel plus cisplatin group. Noninferiority of PPM plus cisplatin compared with paclitaxel plus cisplatin was confirmed for RR. There were no differences in progression-free survival and overall survival between the groups. Although there was a higher rate of grade 3 neutropenia in the PPM plus cisplatin group, the overall rate of adverse events was comparable between the 2 groups. CONCLUSION PPM in combination with cisplatin was well tolerated, and its response rate was noninferior to that of paclitaxel plus cisplatin in patients with advanced NSCLC and who were chemonaive.

Clinical characteristics of non-small cell lung cancer patients who experienced acquired resistance during gefitinib treatment

BACKGROUND The NSCLC patients who experienced good clinical responses to an EGFR-TKI will inevitably develop acquired resistance. A great deal of research is being carried out to discover the molecular mechanisms underlying this resistance. In comparison, few studies have been conducted to find out about the clinical characteristics of acquired resistance in the patients who had responded to an EGFR-TKI. Herein we investigated clinical characteristics of NSCLC patients who experienced acquired resistance during gefitinib therapy. PATIENTS AND METHODS We reviewed NSCLC patients who showed a clinical benefit from initial gefitinib therapy. All clinical data were obtained from 11 centers of Korean Molecular Lung Cancer Group (KMLCG). The clinical manifestations of acquired resistance, time to progression (TTP), and post-progression survival (PPS) after gefitinib failure were analyzed retrospectively. RESULTS A total of 417 patients were recruited. Median TTP was 10.2 months (95% CI, 9.5-10.9). TTP showed a significant longer duration in female, non-smoker, and patients with adenocarcinoma. At the time of acquired resistance, 63.3% of the patients showed symptomatic deterioration. Sites of disease progression were as follows: primary lung lesion in 58.4%, previous metastasis in 38.3%, and new metastasis in 54.2%. Patients with EGFR wild type showed a tendency of higher frequency in symptomatic deterioration and newly development of CNS metastasis compared with patients with EGFR mutation. There was a significant difference in newly development of lung metastasis between patients with exon 19 deletion and those with L858R mutation (41.4% vs. 6.3%, p=0.02). PPS was 8.9 months (95% CI, 7.4-10.4). Smoking history, PS, new CNS lesion and subsequent chemotherapy were independent factors for PPS. CONCLUSION This study suggests that clinical manifestations of acquired resistance may be different according to EGFR mutation status and EGFR mutation genotype. In addition, subsequent chemotherapy confers clinical benefit in terms of PPS in NSCLC patients who experienced acquired resistance after gefitinib therapy.

Bilateral Ovarian Metastases from ALK Rearranged Non-Small Cell Lung Cancer

Anaplastic lymphoma kinase (ALK) rearrangement, is a kind of driver mutation, accounts for 3%-5% of non-small cell lung cancer (NSCLC). NSCLC patients harboring ALK fusion genes have distinct clinical features and good response to ALK inhibitors. Metastasis from lung cancer to the ovary has rarely been known. We report a case of a 54-year-old woman with bilateral ovarian metastases from ALK rearranged NSCLC. She underwent bilateral salpingo-oophorectomy for ovary masses, which were progressed after cytotoxic chemotherapy although primary lung mass was decreased. Histopathological examination of the ovary tumor showed characteristic adenocarcinoma patterns of the lung and ALK rearrangement.

Detection of EGFR and KRAS Mutation by Pyrosequencing Analysis in Cytologic Samples of Non-Small Cell Lung Cancer

EGFR and KRAS mutations are two of the most common mutations that are present in lung cancer. Screening and detecting these mutations are of issue these days, and many different methods and tissue samples are currently used to effectively detect these two mutations. In this study, we aimed to evaluate the testing for EGFR and KRAS mutations by pyrosequencing method, and compared the yield of cytology versus histology specimens in a consecutive series of patients with lung cancer. We retrospectively reviewed EGFR and KRAS mutation results of 399 (patients with EGFR mutation test) and 323 patients (patients with KRAS mutation test) diagnosed with lung cancer in Konkuk University Medical Center from 2008 to 2014. Among them, 60 patients had received both EGFR and KRAS mutation studies. We compared the detection rate of EGFR and KRAS tests in cytology, biopsy, and resection specimens. EGFR and KRAS mutations were detected in 29.8% and 8.7% of total patients, and the positive mutation results of EGFR and KRAS were mutually exclusive. The detection rate of EGFR mutation in cytology was higher than non-cytology (biopsy or resection) materials (cytology: 48.5%, non-cytology: 26.1%), and the detection rate of KRAS mutation in cytology specimens was comparable to non-cytology specimens (cytology: 8.3%, non-cytology: 8.7%). We suggest that cytology specimens are good alternatives that can readily substitute tissue samples for testing both EGFR and KRAS mutations. Moreover, pyrosequencing method is highly sensitive in detecting EGFR and KRAS mutations in lung cancer patients.

A Case of Congenital Cystic Adenomatoid Malformation Infected with Mycobacterium avium-intracellulare Complex

We present a case of congenital cystic adenomatoid malformation (CCAM) in a 25-year-old male who was presented with chronic cough. Chest radiography revealed an abnormal mass-like shadow in the right lower pulmonary zone. A contrast enhanced computed tomography showed an 11 cm solid, cystic mixed mass on the right lower lobe. A right lower lobectomy was performed by video-assisted thoracoscopic surgery without complications. The gross specimen showed a massive cavitation with multiloculated cysts of varying size, consistent with CCAM, along with noticeable granulomatous inflammation. Non-tuberculosis mycobacteria were isolated from a bronchial wash specimen, and the resected tissue homogenates were positive for Mycobacterium avium-intracellulare complex by polymerase chain reaction.

Clinical features of lung adenocarcinomas with epidermal growth factor receptor mutations and miliary disseminated carcinomatosis.

We have occasionally encountered advanced lung cancer patients with disseminated carcinomatosis throughout the body and/or within the lung. This study investigated the clinical characteristics and outcomes of advanced lung adenocarcinoma patients with miliary disseminated carcinomatosis.