Jae-Kyung Won

A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing

The identification of the molecular events that drive cancer transformation is essential to the development of targeted agents that improve the clinical outcome of lung cancer. Many studies have reported genomic driver mutations in non-small-cell lung cancers (NSCLCs) over the past decade; however, the molecular pathogenesis of >40% of NSCLCs is still unknown. To identify new molecular targets in NSCLCs, we performed the combined analysis of massively parallel whole-genome and transcriptome sequencing for cancer and paired normal tissue of a 33-yr-old lung adenocarcinoma patient, who is a never-smoker and has no familial cancer history. The cancer showed no known driver mutation in EGFR or KRAS and no EML4-ALK fusion. Here we report a novel fusion gene between KIF5B and the RET proto-oncogene caused by a pericentric inversion of 10p11.22-q11.21. This fusion gene overexpresses chimeric RET receptor tyrosine kinase, which could spontaneously induce cellular transformation. We identified the KIF5B-RET fusion in two more cases out of 20 primary lung adenocarcinomas in the replication study. Our data demonstrate that a subset of NSCLCs could be caused by a fusion of KIF5B and RET, and suggest the chimeric oncogene as a promising molecular target for the personalized diagnosis and treatment of lung cancer.

Comparison of Shear-Wave and Strain Ultrasound Elastography in the Differentiation of Benign and Malignant Breast Lesions

OBJECTIVE The purpose of this article is to compare the diagnostic performances of shearwave and strain elastography for the differentiation of benign and malignant breast lesions. MATERIALS AND METHODS B-mode ultrasound and shear-wave and strain elastography were performed in 150 breast lesions; 71 were malignant. BI-RADS final assessment, elasticity values in kilopascals, and elasticity scores on a 5-point scale were assessed before biopsy. The results were compared using the area under the receiver operating characteristic curve (AUC). RESULTS The AUC for shear-wave elastography was similar to that of strain elastography (0.928 vs 0.943). The combined use of B-mode ultrasound and either elastography technique improved diagnostic performance in the differentiation of benign and malignant breast lesions compared with the use of B-mode ultrasound alone (B-mode alone, AUC = 0.851; B-mode plus shear-wave elastography, AUC = 0.964; B-mode plus strain elastography, AUC = 0.965; p < 0.001). With the best cutoff points of 80 kPa on shear-wave elastography and a score between 3 and 4 on strain elastography, the sensitivity was higher in shear-wave elastography, and specificity was higher in strain elastography (95.8% vs 81.7%, p = 0.002; 93.7% vs 84.8%, p = 0.016). In cases of infiltrating ductal carcinoma, mean elasticity scores were lower in grade 3 than in grade 1 and 2 cancers (p = 0.017) with strain elastography causing false-negative findings. CONCLUSION The diagnostic performance of shear-wave and strain elastography was similar. Either elastography technique can improve overall diagnostic performance in the differentiation of benign and malignant lesions when combined with B-mode ultrasound. However, the sensitivity and specificity of shear-wave and strain elastography were different according to lesion histologic profile, tumor grade, and breast thickness.

A Dramatic Response to Crizotinib in a Non–Small-Cell Lung Cancer Patient with IHC-Positive and FISH-Negative ALK

Journal of Thoracic Oncology • Volume 7, Number 12, December 2012 A 54-year-old woman presented with locally advanced pulmonary adenocarcinoma in December 2011. During the workup, lung mass increased rapidly and a large amount of pleural effusion was newly developed. The analysis of pleural effusion indicated malignant hemothorax, and more than 1 liter of bloody pleural effusion was drained daily through a chest tube. In the biomarker analysis, immunohistochemistry (IHC) with an antibody (clone 5A4, Novocastra, Newcastle, United Kingdom) for anaplastic lymphoma kinase (ALK) protein showed moderately positive staining (Fig. 1A). However, fluorescent in situ hybridization (FISH) using a probe specific to the ALK locus (Vysis LSI ALK dual-color, break-apart rearrangement probe; Abbott Molecular, Abbott Park, IL) did not show ALK translocation (Fig. 1B). There was neither ROS1 rearrangement nor MET amplification. We started chemotherapy with gemcitabine plus cisplatin. The follow-up chest radiograph and computed tomography, performed on the 17th day after starting chemotherapy, showed an unresolved primary lung mass and a mediastinal lymph node compressing the right main bronchus (Fig. 2A). We prescribed crizotinib (250 mg twice a day), starting February 16, 2012. The pleural effusion decreased rapidly, and the chest tube was removed 6 days after the initiation of crizotinib therapy. At day 32, chest images showed a great decrease in the cancerous lesions (Fig. 2B). The patient did not experience any noticeable hematologic or nonhematologic adverse events. At day 83, the lesions decreased further; this partial response was confirmed with chest images (Fig. 2C). At 27 weeks, the patient is still on crizotinib and is in good general condition.

Concomitant ALK translocation and EGFR mutation in lung cancer: a comparison of direct sequencing and sensitive assays and the impact on responsiveness to tyrosine kinase inhibitor

BACKGROUND Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) translocation are considered mutually exclusive in nonsmall-cell lung cancer (NSCLC). However, sporadic cases having concomitant EGFR and ALK alterations have been reported. The present study aimed to assess the prevalence of NSCLCs with concomitant EGFR and ALK alterations using mutation detection methods with different sensitivity and to propose an effective diagnostic and therapeutic strategy. PATIENTS AND METHODS A total of 1458 cases of lung cancer were screened for EGFR and ALK alterations by direct sequencing and flourescence in situ hybridization (FISH), respectively. For the 91 patients identified as having an ALK translocation, peptide nucleic acid (PNA)-clamping real-time PCR, targeted next-generation sequencing (NGS), and mutant-enriched NGS assays were carried out to detect EGFR mutation. RESULTS EGFR mutations and ALK translocations were observed in 42.4% (612/1445) and 6.3% (91/1445) of NSCLCs by direct sequencing and FISH, respectively. Concomitant EGFR and ALK alterations were detected in four cases, which accounted for 4.4% (4/91) of ALK-translocated NSCLCs. Additional analyses for EGFR using PNA real-time PCR and ultra-deep sequencing by NGS, mutant-enriched NGS increased the detection rate of concomitant EGFR and ALK alterations to 8.8% (8/91), 12.1% (11/91), and 15.4% (14/91) of ALK-translocated NSCLCs, respectively. Of the 14 patients, 3 who were treated with gefitinib showed poor response to gefitinib with stable disease in one and progressive disease in two patients. However, eight patients who received ALK inhibitor (crizotinib or ceritinib) showed good response, with response rate of 87.5% (7/8 with partial response) and durable progression-free survival. CONCLUSIONS A portion of NSCLC patients have concomitant EGFR and ALK alterations and the frequency of co-alteration detection increases when sensitive detection methods for EGFR mutation are applied. ALK inhibitors appear to be effective for patients with co-alterations.

Targeted sequencing of cancer-related genes in colorectal cancer using next-generation sequencing.

Recent advance in sequencing technology has enabled comprehensive profiling of genetic alterations in cancer. We have established a targeted sequencing platform using next-generation sequencing (NGS) technology for clinical use, which can provide mutation and copy number variation data. NGS was performed with paired-end library enriched with exons of 183 cancer-related genes. Normal and tumor tissue pairs of 60 colorectal adenocarcinomas were used to test feasibility. Somatic mutation and copy number alteration were analyzed. A total of 526 somatic non-synonymous sequence variations were found in 113 genes. Among these, 278 single nucleotide variations were 232 different somatic point mutations. 216 SNV were 79 known single nucleotide polymorphisms in the dbSNP. 32 indels were 28 different indel mutations. Median number of mutated gene per tumor was 4 (range 0–23). Copy number gain (>X2 fold) was found in 65 genes in 40 patients, whereas copy number loss (<X0.5 fold) was found in 103 genes in 39 patients. The most frequently altered genes (mutation and/or copy number alteration) were APC in 35 patients (58%), TP53 in 34 (57%), and KRAS in 24 (40%). Altered gene list revealed ErbB signaling pathway as the most commonly involved pathway (25 patients, 42%). Targeted sequencing platform using NGS technology is feasible for clinical use and provides comprehensive genetic alteration data.

Comprehensive Analysis of the Transcriptional and Mutational Landscape of Follicular and Papillary Thyroid Cancers.

Follicular thyroid carcinoma (FTC) and benign follicular adenoma (FA) are indistinguishable by preoperative diagnosis due to their similar histological features. Here we report the first RNA sequencing study of these tumors, with data for 30 minimally invasive FTCs (miFTCs) and 25 FAs. We also compared 77 classical papillary thyroid carcinomas (cPTCs) and 48 follicular variant of PTCs (FVPTCs) to observe the differences in their molecular properties. Mutations in H/K/NRAS, DICER1, EIF1AX, IDH1, PTEN, SOS1, and SPOP were identified in miFTC or FA. We identified a low frequency of fusion genes in miFTC (only one, PAX8–PPARG), but a high frequency of that in PTC (17.60%). The frequencies of BRAFV600E and H/K/NRAS mutations were substantially different in miFTC and cPTC, and those of FVPTC were intermediate between miFTC and cPTC. Gene expression analysis demonstrated three molecular subtypes regardless of their histological features, including Non–BRAF–Non–RAS (NBNR), as well as BRAF–like and RAS–like. The novel molecular subtype, NBNR, was associated with DICER1, EIF1AX, IDH1, PTEN, SOS1, SPOP, and PAX8–PPARG. The transcriptome of miFTC or encapsulated FVPTC was indistinguishable from that of FA, providing a molecular explanation for the similarly indolent behavior of these tumors. We identified upregulation of genes that are related to mitochondrial biogenesis including ESRRA and PPARGC1A in oncocytic follicular thyroid neoplasm. Arm-level copy number variations were correlated to histological and molecular characteristics. These results expanded the current molecular understanding of thyroid cancer and may lead to new diagnostic and therapeutic approaches to the disease.

Prognostic effects of TERT promoter mutations are enhanced by coexistence with BRAF or RAS mutations and strengthen the risk prediction by the ATA or TNM staging system in differentiated thyroid cancer patients.

Recent reports suggest that mutations in the promoter of the gene encoding telomerase reverse transcriptase (TERT) affect thyroid cancer outcomes.

Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

Expression of Ki-67 and COX-2 in Patients With Upper Urinary Tract Urothelial Carcinoma

OBJECTIVES To investigate the prognostic value of Ki-67, cyclooxygenase-2 (COX-2), E-cadherin, and retinoblastoma protein (pRB) in patients with urothelial carcinoma of the upper urinary tract. METHODS From January 1998 to December 2005, the specimens from 107 patients with urothelial carcinoma of the upper urinary tract who had undergone nephroureterectomy were analyzed. The expression of Ki-67, COX-2, E-cadherin, and pRB was examined by immunochemistry on tissue microarray sections. The correlation of the immunoreactivity with the pathologic parameters and progression-free and cancer-specific survival were examined. RESULTS Ki-67 and COX-2 were overexpressed in 26 (24%) and 38 patients (36%), respectively. The loss of E-cadherin expression was observed in 66 patients (62%). Altered pRB expression was found in 37 patients (34%). Overexpression of Ki-67 (P = .041 and P = .006, respectively) and COX-2 (P = .002 and P = .001, respectively) was associated with the pathologic stage and grade. Multivariate analysis showed that Ki-67 overexpression (P = .002), T stage (P = .009), and lymph node metastases (P = .009) were independent predictors of progression-free survival. In addition, Ki-67 overexpression (P = .007) and pathologic T stage (P = .003) were independent predictors of cancer-specific survival. No association was found between the pathologic findings and prognosis and the other markers (E-cadherin and pRB). CONCLUSIONS Our results suggest that Ki-67 overexpression is an independent predictor of the progression of urothelial carcinoma of the upper urinary tract. Patients with Ki-67 overexpression should be followed up more closely. In addition, they might be candidates for future prospective therapy trials.

The crossregulation between ERK and PI3K signaling pathways determines the tumoricidal efficacy of MEK inhibitor

MEK inhibitor has been highlighted as a promising anti-tumor drug but its effect has been reported as varying over a wide range depending on patho-physiological conditions. In this study, we employed a systems approach by combining biochemical experimentation with in silico simulations to investigate the resistance mechanism and functional consequences of MEK inhibitor. To this end, we have developed an extended integrative model of ERK and PI3K signaling pathways by considering the crosstalk between Ras and PI3K, and analyzed the resistance mechanism to the MEK inhibitor under various mutational conditions. We found that the phospho-Akt level under the Raf mutation was remarkably augmented by MEK inhibitor, while the phospho-ERK level was almost completely repressed. These results suggest that bypassing of the ERK signal to the PI3K signal causes the resistance to the MEK inhibitor in a complex oncogenic signaling network. We further investigated the underlying mechanism of the drug resistance and revealed that the MEK inhibitor disrupts the negative feedback loops from ERK to SOS and GAB1, but activates the positive feedback loop composed of GAB1, Ras, and PI3K, which induces the bypass of the ERK signal to the PI3K signal. Based on these core feedback circuits, we suggested promising candidates for combination therapy and examined the improved inhibitory effects.