Jinseon Lee

Discovery of ALK‐PTPN3 gene fusion from human non‐small cell lung carcinoma cell line using next generation RNA sequencing

An increasing number of chromosomal aberrations is being identified in solid tumors providing novel biomarkers for various types of cancer and new insights into the mechanisms of carcinogenesis. We applied next generation sequencing technique to analyze the transcriptome of the non‐small cell lung carcinoma (NSCLC) cell line H2228 and discovered a fusion transcript composed of multiple exons of ALK (anaplastic lymphoma receptor tyrosine kinase) and PTPN3 (protein tyrosine phosphatase, nonreceptor Type 3). Detailed analysis of the genomic structure revealed that a portion of genomic region encompassing Exons 10 and 11 of ALK has been translocated into the intronic region between Exons 2 and 3 of PTPN3. The key net result appears to be the null mutation of one allele of PTPN3, a gene with tumor suppressor activity. Consistently, ectopic expression of PTPN3 in NSCLC cell lines led to inhibition of colony formation. Our study confirms the utility of next generation sequencing as a tool for the discovery of somatic mutations and has led to the identification of a novel mutation in NSCLC that may be of diagnostic, prognostic, and therapeutic importance.

A High-Dimensional, Deep-Sequencing Study of Lung Adenocarcinoma in Female Never-Smokers

Background Deep sequencing techniques provide a remarkable opportunity for comprehensive understanding of tumorigenesis at the molecular level. As omics studies become popular, integrative approaches need to be developed to move from a simple cataloguing of mutations and changes in gene expression to dissecting the molecular nature of carcinogenesis at the systemic level and understanding the complex networks that lead to cancer development. Results Here, we describe a high-throughput, multi-dimensional sequencing study of primary lung adenocarcinoma tumors and adjacent normal tissues of six Korean female never-smoker patients. Our data encompass results from exome-seq, RNA-seq, small RNA-seq, and MeDIP-seq. We identified and validated novel genetic aberrations, including 47 somatic mutations and 19 fusion transcripts. One of the fusions involves the c-RET gene, which was recently reported to form fusion genes that may function as drivers of carcinogenesis in lung cancer patients. We also characterized gene expression profiles, which we integrated with genomic aberrations and gene regulations into functional networks. The most prominent gene network module that emerged indicates that disturbances in G2/M transition and mitotic progression are causally linked to tumorigenesis in these patients. Also, results from the analysis strongly suggest that several novel microRNA-target interactions represent key regulatory elements of the gene network. Conclusions Our study not only provides an overview of the alterations occurring in lung adenocarcinoma at multiple levels from genome to transcriptome and epigenome, but also offers a model for integrative genomics analysis and proposes potential target pathways for the control of lung adenocarcinoma.

A randomized phase III trial of stereotactic radiosurgery (SRS) versus observation for patients with asymptomatic cerebral oligo-metastases in non-small cell lung cancer

BACKGROUNDnIt is unclear whether treating brain metastasis before starting systemic chemotherapy can improve survival compared with upfront chemotherapy in non-small-cell lung cancer (NSCLC) with asymptomatic cerebral oligo-metastases.nnnPATIENTS AND METHODSnWe undertook a randomized, controlled trial of 105 patients with one to four brain metastases, admitted to Samsung Medical Center between 2008 and 2013. Patients were randomly assigned to receive stereotactic radiosurgery (SRS) (49 patients) followed by chemotherapy or upfront chemotherapy (49 patients). The primary end point was overall survival (OS) and secondary end points included central nervous system (CNS) progression-free survival, progression to symptomatic brain metastasis and brain functional outcome.nnnRESULTSnThe median age was 58 years (range, 29-85) with ECOG 0-1 performance status, and 40% of patients were never smokers. Most patients had adenocarcinoma, and about half of patients had only one brain metastasis, while the rest had multiple cerebral metastases. The median OS time was 14.6 months [95% confidence interval (CI), 9.2-20.0] in the SRS group and 15.3 months (95% CI, 7.2-23.4) for the upfront chemotherapy group (P = 0.418). There was no significant difference in time to CNS disease progression [median, 9.4 months (SRS) versus 6.6 months (upfront chemotherapy), P = 0.248]. Symptomatic progression of brain metastases was observed more frequently in the upfront chemotherapy group (26.5%) than the SRS group (18.4%) but without statistical significance.nnnCONCLUSIONSnAlthough this study included smaller sample size than initially anticipated due to early termination, SRS followed by chemotherapy did not improve OS in oligo-brain metastases NSCLC patients compared with upfront chemotherapy. Further study with large number of patients should be needed to confirm the use of upfront chemotherapy alone in this subgroup of patients.nnnCLINICAL TRIALS NUMBERnNCT01301560.

Mutational hotspots in the mitochondrial genome of lung cancer.

We determined the somatic mutations in the mitochondrial genomes of 70 lung cancer patients by pair-wise comparative analyses of the normal- and tumor-genome sequences acquired using Affymetrix Mitochondrial Resequencing Array 2.0. The overall mutation rates in lung cancers were Approximately 100 fold higher than those in normal cells, with significant statistical correlation with smoking (p=0.00088). Total of 532 somatic mutations were evenly distributed in 499 positions with very low overall frequency (1.07/bp), but the non-synonymous mutations causing amino acid substitution occurred more frequently (1.83/bp), particularly at two positions, 8701 and 10398 (10.5/bp) that code for ATPase6 and NADH dehydrogenase 3, respectively. Despite the randomness or even distribution of the mutations, these two mutations occurred together in 86% of the cases. The linkage between the two most frequent mutations suggests that they were selected together, possibly due to their cooperative role during cancer development. Indeed, the mutation at 10398 was shown by Canter, Pezzotti, and their colleagues in 2009, as a risk factor for breast cancer. In this study, we identified two potential biomarkers that might be functionally linked together during the development of cancer.

Prediction of lymph node metastasis using the combined criteria of helical CT and mRNA expression profiling for non-small cell lung cancer

To improve the diagnostic accuracy of nodal metastasis, we suggest new criteria for the prediction of nodal metastasis with combining CT and mRNA expression profiling. Gene signatures related to nodal metastasis were selected from a microarray using extracted mRNA of 112 patients who underwent surgical resection for non-small cell lung cancer. Included patients were randomized into two groups; the training set (n=79) and the test set (n=33). On the basis of the gene signatures, the chest CTs of the training set of patients were re-analyzed and we set up hypothetical criteria for nodal diagnosis. Thirty-one genes were selected from the mRNA expression profiling to separate the LN-metastasis prediction (+) and LN-metastasis prediction (-) groups. On the basis of these signatures, the criteria of lymph node was adjusted (1) in cases of LN-metastasis prediction (+), mediastinal nodes greater than a 5mm in short axis diameter and detectable hilar nodes were considered as metastatic, and (2) in cases of LN-metastasis prediction (-), the conventional size criterion was applied for both mediastinal and hilar lymphadenopathies, except for enlarged nodes along with obstructive pneumonia. The sensitivity and accuracy for the nodal diagnosis were improved from 31% to 85% and 58% to 86%, respectively (p<0.05) by using the combined criteria of CT and the microarray results in the test set as compared to those of CT alone. Prediction of lymph node metastasis using combination of gene signatures and chest CT is superior to the CT-only diagnosis.

Systematic identification of cancer-related long noncoding RNAs and aberrant alternative splicing of quintuple-negative lung adenocarcinoma through RNA-Seq

OBJECTIVESnLung adenocarcinoma (LUAD) is a common subtype of non-small cell lung cancer prevalent in Asia. There is a dearth of understanding regarding the transcriptome landscape of LUAD without primary known driver mutations. In this study, LUAD samples without well-known driver mutations occurring in EGFR, KRAS, ALK, ROS1 or RET (quintuple-negative) were used for transcriptome study with a focus on long noncoding RNAs (lncRNAs), alternative splicing and gene fusions.nnnMATERIALS AND METHODSn24 pairs of LUAD and adjacent normal samples and 13 tumor-only samples derived from 37 quintuple-negative patients were used. Differentially expressed lncRNA transcripts were detected by paired t-test and were validated by qPCR. Functions of lncRNAs were predicted by co-expressed mRNAs. Aberrant splicing events in LUAD were identified using MISO. In addition, gene fusions were screened by SOAPfuse.nnnRESULTS AND CONCLUSIONnIn total, 90 and 153 up- or down-regulated lncRNA transcripts were detected in LUAD samples in comparison with the adjacent normal samples. The most significantly differentially expressed lncRNA transcript was ENST00000598996.1 (FENDRR) down-regulated in LUAD. By lncRNA-mRNA co-expression analysis, functions of 14 lncRNAs were predicted. The predicted functions included vasculature development, immune response, cell cycle and respiratory gaseous exchange. Furthermore, six co-expressed pairs of lncRNAs and their nearby protein coding genes were identified as associated with lung development. This study also identified two highly recurrent (22 in 24) differential exon skipping events occurring in MYH14 and ESYT2 with exon including isoforms of both genes up-regulated in isoform percentage in LUAD samples. On the other hand, two out of 24 LUAD samples possessed the driver mutation exon 14 skipping of MET. The transcriptional alterations of LUAD samples without well-known driver mutations identified in the study can be used as references for future research. The translational values of these transcriptional changes are also worthy of further investigation.

Detection of circulating tumor cells in patients with non-small cell lung cancer using a size-based platform

The detection of circulating tumor cells (CTCs) is limited by the rarity of these cells in the peripheral blood of patients with cancer. Understanding tumor biology may be useful in the development of novel therapeutic strategies for patients with lung cancer. The present study evaluated a novel size-based filtration platform for enriching CTCs from patients with lung cancer. Blood samples were obtained from 82 patients with lung cancer for CTC analysis. CTC enrichment by size-based filtration was performed on 5-ml blood samples. The collected cells were detected by immunofluorescence using monoclonal anti-human antibodies against protein tyrosine phosphatase, receptor type C (CD45) and epithelial cell adhesion molecule (EpCAM; an epithelial cell marker), as well as a DAPI nucleic acid stain. CTCs were detected in 57 patients (69.5%) using the size-based filtration platform. The mean CTC counts, defined as the number of cells with DAPI-positive, CD45-negative and EpCAM-positive staining, were 1.48±1.71 per 5 ml blood for the 66 stage I-III patients and 8.00±9.95 per 5 ml blood for the 16 stage IV patients. The presence of ≥1 CTCs per 5-ml blood sample was significantly associated with pathological stage (stage IV vs. stage I-III, P=0.009), but not with patient age or gender, tumor histology, tumor size or lymphovascular invasion. The mean CTC count of healthy donors was 0.25±0.55 per 5 ml blood. In summary, CTCs from the blood of patients with lung cancer were enriched using a size-based filtration platform and immunofluorescent staining with DAPI, CD45 and EpCAM. The CTC counts of patients with stage IV cancer were higher than those of patients with stages I-III cancer. These results suggest that this novel platform may be a useful tool for determining the prognosis of patients with lung cancer.

680PPHASE II STUDY OF A COMBINATION CHEMOTHERAPY WITH WEEKLY DOCETAXEL AND GEMCITABINE IN PREVIOUSLY TREATED METASTATIC ESOPHAGEAL SQUAMOUS CELL CANCER

ABSTRACT Aim: This multicenter, phase II study was conducted to assess the efficacy and safety of weekly docetaxel plus fixed-dose rate (FDR) gemcitabine as second-line chemotherapy in patients with metastatic esophageal squamous cell carcinoma (SCC). Methods: Esophageal SCC patients with documented progression after fluoropyrimidine-based first-line chemotherapy were treated with docetaxel 35u2003mg/m2 and gemcitabine 1,000u2003mg/m2 iv at FDR (10u2003mg/m2/min) on days 1 and 8. Treatment was repeated every 21 days until disease progression, unacceptable toxicity, or consent withdrawal. Primary endpoint was response rate (RR), and secondary endpoints were safety, progression-free survival (PFS) and overall survival (OS). Results: A total of 33 patients were registered onto this prospective study. Combination or weekly docetaxel and FDR gemcitabine was well tolerated: the most common treatment-related adverse events were anemia (97%), fatigue (64%) and neutropenia (55%). Grade 3 or 4 neutropenia was developed in 13 patients (39%) and three episodes of febrile neutropenia were observed. One patient with lung and extensive lymph node metastases died of respiratory failure after receiving fourth cycles of chemotherapy, and the possibility of drug-induced pneumonitis could not be completely excluded. The overall RR was 30% with one complete and 9 partial responses. Stable disease was documented in 19 patients (58%). The median PFS and OS were 6 (95% CI 5-8) and 9 (95% CI 7-11) months, respectively. Conclusions: The weekly combination of docetaxel and FDR gemcitabine showed a promising antitumor activity and tolerability in previously treated, metastatic esophageal SCC. Disclosure: All authors have declared no conflicts of interest.

Early progression (within 12 months) after autologous stem-cell transplantation in patients with multiple myeloma

Background: Autologous stem cell transplantation (ASCT) has become the standard of care for eligible patients with newly diagnosed multiple myeloma (MM). Although, attaining best pre-transplant response is important for long-term disease control, early progressive disease after ASCT is not fully understood in a subset ofMMpatients. The aim of this study is was to evaluate risk factors and clinical features of patients with early progression of disease after ASCT. Methods: We retrospectively reviewed 330 patients diagnosed with MM based on the International Myeloma Working Group diagnostic criteria and underwent ASCT between October 1998 and February 2015. Patients who progressed within 12months after ASCT were classified as the study group.Univariate andmultivariate analyses were performed on the recognized baseline parameters, and treatment related factors were also evaluated.Results: Among 324 patients, 103 (32%) progressed within 12 months after stem cell transplantation. Multivariate logistic regression analyses revealed that azotemia (Cr 2.0mg/dL) (RR1⁄43.2) and non-hyperdiploidy (RR1⁄42.6) were independently associated with early progressive disease (P<0.05). During a median 2.8 years of follow-up, the median overall survival of patients who progressed within 12 months after ASCT was significant poor (29.1months, 95% CI, 22.3-35.9) compared to those who did not progress within 12 months (94.5months, 95% CI, 78.5-110.5, p < 0.001). Conclusions: Early progression of disease after ASCT is associated with a poor prognosis in MM patients. PreASCT response ( PR versus < PR) did not affect early disease progression after stem cell transplantation. Patients with nonhyperdiploidy and azotemia may experience a limited benefit from ASCT. Further tailored therapy is needed for these patients.

Autologous Stem Cell Transplantation for AL Amyloidosis

2 and 4% grade 3), gastrointestinal toxicity in 6 patients (26%) only a 4% grade 3, 7 patients had hematologic toxicity, 5 patients anemia (1 grade1 and 4 grade2), 4 patients developed neutropenia (2 patients with 3) and there were no cases of thrombocytopenia. As a result of this toxicity 44% of patients required dose adjustment of bortezomib and 52% of thalidomide. Three patients discontinued treatment, 2 for progression and one voluntarily. After a median follow-up of 19 months 11 patients have progressed, 3 of them (13%) before TAPH and 8 post-transplantation with a median time to progression of 9 months (4-18). The overall survival is 83%. Conclusions: The results of our study demonstrate that the use of bortezomib SC within the scheme VTD as induction treatment not affects its efficacy with high CR rate pre and post-transplantation. The SC administration of bortezomib may reduce the incidence of peripheral neuropathy and the intensity thereof. Characteristics of the patients Number of patients (%) Men/women 11(48)/12(52) Median of age 54 anos (44-68) IgG/IgA/light chain/others 12 (52)/7 (30)/3 (13)/1 (4) ISS I/II/III 5 (22)/9 (39)/9 (39) Intramedullary disease 9 (39) Genetic alterations (FISH) 7 (30) Post induction response (N1⁄421)