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We evaluated the prognostic implications of the circulating tumor cell (CTC) count in non-metastatic, HER2-negative breast cancer patients who failed to achieve pathologic complete response (pCR) after neoadjuvant chemotherapy (NCT). A total of 173, non-metastatic breast cancer patients treated with NCT were prospectively enrolled. CTCs were obtained from blood drawn pre-NCT and post-NCT using a SMART BIOPSY SYSTEM isolation kit (Cytogen Inc., Seoul, Korea) with immunofluorescence staining. Excluding 26 HER2-positive patients, Relapse-free survival (RFS) and overall survival (OS) related to the CTC count and the association of the CTC count with the treatment response to given therapy were analyzed in 147 HER2-negative patients. Among 147 HER2-negative patients, 28 relapses (19.0%) and 13 deaths (8.8%, all breast cancer-specific) were observed during a median follow-up of 37.3 months. One hundred and seven patients (72.8%) were hormone receptor-positive, and 40 patients (27.2%) had triple-negative breast cancer (TNBC). One or more CTCs were identified in 88 of the 147 patients (59.9%) before NCT and 77 of the 134 patients (52.4%) after NCT. In the entire HER2-negative patient cohort, the initial nodal status was the most significant factor influencing RFS and OS. In TNBC, 11 patients (27.5%) achieved pCR and patients that failed to achieve pCR with ≥ 5 CTCs after NCT, showed worse RFS (HR, 10.66; 95% CI, 1.80–63.07; p = 0.009) and OS (HR, 14.00; 95% CI, 1.26–155.53; p = 0.032). The patients with residual tumor and a high number of the CTCs after NCT displayed the worse outcome. These findings could provide justification to launch a future, well designed trial with longer follow-up data to obtain regulatory approval for clinical use of the assay, especially for the ER-positive, HER2-negative breast cancer subset.

BACKGROUND/AIM Although it has been suggested that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) might be used in a complementary manner in lung cancer diagnosis, limited confirmatory data are available. In this prospective study, we evaluated the diagnostic performance of each assay separately and in combination. PATIENTS AND METHODS From March 2018 to January 2019, patients with suspected primary lung cancer, who underwent routine lung cancer work-up and peripheral blood sampling, were prospectively enrolled in the study. Epithelial cell adhesion molecule and cytokeratin served as markers of CTCs. In terms of ctDNA analysis, single-nucleotide variants were evaluated via next-generation sequencing. RESULTS We analyzed 111 patients, including 99 with primary lung cancer and 12 with benign pulmonary disease. The median number of CTCs in 10 ml of blood was 3. The most frequently detected single nucleotide variants of ctDNA were TP53, CDKN2A, and EGFR. The diagnostic sensitivity of conventional tumor marker (combination of carcinoembryonic antigen/CYFRA 21-1/neuron-specific enolase) was 66.7%, while those of the ctDNA and CTC assays were 72.7% and 65.7%, respectively. The sensitivity of the CTC/ctDNA combination (95.0%) was significantly greater than those of the CTC (p

Background: Circulating tumor cells (CTCs) and circulating free DNA (cfDNA) have a promising role for detecting early response and progression in breast cancer. Furthermore, change of immune-related gene signature during systemic treatment influence the treatment response of breast cancer. Herein we report the outcome of serial genomic analysis of CTC, cfDNA and change of immune-related gene signature in advanced, metastatic triple negative breast cancer (TNBC) and HER2 positive breast cancer (BC). Methods: Serial whole blood were prospectively collected in 18 early or advanced, and 10 metastatic BC patients periodically during systemic chemotherapy. CTC was isolated from whole blood through EpCAM positive bead selection, and ctDNA was isolated from plasma. For genomic profiling of CTC and ctDNA, Oncomine™ Comprehensive Assay Plus and Oncomine™ Pan-Cancer cfDNA assay (included BRCA1, BRCA2 and MYCN customized panel) was performed and analyzed, respectively. Total RNA was extracted using whole blood and analyzed using Nanostring Pancancer Immunology Panel. Results: Total 61 serial samples were obtained from 28 patients during the study. At baseline, FGFR4 mutation was the most commonly detected (10 patients, 76.9%) with median variant of allele frequency (VAF) of 54.5% (range 20.87~99.8%) in advanced and metastatic TNBC based on CTC analysis. In case of cfDNA, 11 patients (84.6%) showed TP53 mutation with low VAF (median 1.8%, range 0.1~12.3%). In HER2 positive BC, FGFR4 was also the most common mutation (5 patients [62.5%]; median VAF 57.9%) in CTC analysis and TP53 was most frequently detected (5 patients, [62.5%]; median VAF 3.0%) in cfDNA analysis. In three pathogenic gBRCA1 mutated patients, BRCA1 was identically detected in 2 patients based on CTC analysis and in 3 patients based on cfDNA analysis with VAF of approximately 50%. There were no significant changes of VAF in target mutations of CTC and cfDNA during systemic treatment, irrespective of tumor response and subtype. However, in one patients who harbored 13 mutations detected based on baseline CTC analysis, showed disappearance of 12 mutations in final CTC analysis with partial response based on radiologic findings. In contrary, there was another patient who gained multiple mutations during CTC analysis during neoadjuvant chemotherapy (NAC) with gain of tumor mutational burden (TMB). She did not achieve pathologic complete response and RCB score was 3 after completion of NAC. Most TNBC patients who received NAC and showed partial response, TMB showed gradual decrease during treatment. Baseline immune-related gene signatures were compared between HER2(+) BC and TNBC, and type 1 interferon signaling pathway was upregulated in HER2 (+) BC compared to TNBC. Conclusions: Our study suggest that serial follow up CTC and ctDNA, immune-related gene signature is feasible and reflect the general characteristics of baseline characteristics and dynamic molecular alteration of breast cancer. Further analysis with larger patient sample and correlation with tumor tissue is warranted in the future. Citation Format: Jieun Lee, Kabsoo Shin, Seung Yeon Joe, Hayoon Lee, Byung Hee Jeon, Ahwon Lee. Serial genomic analysis of circulating free DNA and change of immune-related gene signature in triple negative and HER2 positive advanced, metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-29.

We evaluated the prognostic implications of the circulating tumor cell (CTC) count in non-metastatic, HER2-negative breast cancer patients who failed to achieve pathologic complete response (pCR) after neoadjuvant chemotherapy (NCT). A total of 173, non-metastatic breast cancer patients treated with NCT were prospectively enrolled. CTCs were obtained from blood drawn pre-NCT and post-NCT using a SMART BIOPSY SYSTEM isolation kit (Cytogen Inc., Seoul, Korea) with immunofluorescence staining. Excluding 26 HER2-positive patients, Relapse-free survival (RFS) and overall survival (OS) related to the CTC count and the association of the CTC count with the treatment response to given therapy were analyzed in 147 HER2-negative patients. Among 147 HER2-negative patients, 28 relapses (19.0%) and 13 deaths (8.8%, all breast cancer-specific) were observed during a median follow-up of 37.3 months. One hundred and seven patients (72.8%) were hormone receptor-positive, and 40 patients (27.2%) had triple-negative breast cancer (TNBC). One or more CTCs were identified in 88 of the 147 patients (59.9%) before NCT and 77 of the 134 patients (52.4%) after NCT. In the entire HER2-negative patient cohort, the initial nodal status was the most significant factor influencing RFS and OS. In TNBC, 11 patients (27.5%) achieved pCR and patients that failed to achieve pCR with ≥ 5 CTCs after NCT, showed worse RFS (HR, 10.66; 95% CI, 1.80-63.07; p = 0.009) and OS (HR, 14.00; 95% CI, 1.26-155.53; p = 0.032). The patients with residual tumor and a high number of the CTCs after NCT displayed the worse outcome. These findings could provide justification to launch a future, well designed trial with longer follow-up data to obtain regulatory approval for clinical use of the assay, especially for the ER-positive, HER2-negative breast cancer subset.

e12561 Background: Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have a promising role for detecting early response and progression in breast cancer. We performed serial genomic analysis of CTC and ctDNA in advanced, metastatic triple negative breast cancer (TNBC) and HER2 positive breast cancer (BC). Methods: Serial CTCs, ctDNA were prospectively collected in 9 early or advanced, and 10 metastatic BC patients periodically during systemic chemotherapy. CTC was isolated from whole blood through EpCAM positive bead selection, and ctDNA was isolated from plasma. For genomic profiling of CTC and ctDNA, Oncomine™ Comprehensive Assay Plus and Oncomine™ Pan-Cancer cfDNA assay (included BRCA1, BRCA2 and MYCN customized panel) was performed and analyzed, respectively. Results: TP63 was most commonly detected mutation (13 patients, 68.4%) at serial CTC analysis with median variant of allele frequency (VAF) of 18.6% (range 11.5̃25.2%), irrespective of tumor subtype and stage. In ctDNA analysis, 11 patients showed TP53 mutation with low VAF (median 1.4%, range 0.3̃12.3%). FGFR4 was equally detected, with median variant of allele frequency (VAF) of 57.2% (range 46.6-99.8) and there was no change of FGFR4 VAF irrespective of tumor response in CTC analysis. In three gBRCA1 mutated patients, BRCA1 was identically detected in 3 patients based on CTC analysis and in 2 patients based on ctDNA analysis with VAF of approximately 50%. There were no significant changes of VAF in target mutations of CTC irrespective of tumor response and subtype. In TNBC patients who received neoadjuvant chemotherapy, tumor mutation burden (TMB) showed gradual decrease during treatment. However, there were initial surge of TMB in HER2 positive BC patients who received neoadjuvant chemotherapy, irrespective of presence of pathologic complete response. Conclusions: Our study suggest that serial follow up CTC and ctDNA is feasible and reflect the general characteristics of baseline characteristics and dynamic molecular alteration of breast cancer. Further analysis with larger patient sample and correlation with tumor tissue is warranted in the future.

Aim: To evaluate the clinical efficacy of a circulating tumor cell (CTC) test by comparison between healthy volunteers and patients with localized prostate cancer including those under active surveillance. Materials and Methods: CTC counts in peripheral blood were compared between patients with prostate cancer (n=45) and healthy volunteers (n=17). CTCs were identified based on the expression of epithelial cell adhesion molecule (EpCAM) and counted using a SMART BIOPSY™ SYSTEM. Results: The number of EpCAM+ cells was significantly higher in patients with cancer than in healthy volunteers. Among the low-risk patients (n=9), two had up-staging and six had up-grading. Among those up-staged, there was one case which was EpCAM(+). Among those cases up-graded, three were EpCAM(+). In those with stage T2 tumors, the presence of Gleason pattern 5 was positively correlated with EpCAM positivity (rho=0.59, p< 0.001). Conclusion: CTC counts in localized prostate cancer were associated with Gleason pattern 5. Active treatment should be considered for patients with low-risk disease during active surveillance who are found to have EpCAM+ CTCs because of a risk of up-staging and up-grading.

Liquid biopsy isolation of circulating tumor cells (CTCs) allows the genomic analysis of CTCs, which is useful in the determination of personalized cancer therapy. In the present study, CTCs from patients with breast cancer were enriched and successfully analyzed using cancer gene panel analysis. Blood samples from 11 patients with breast cancer were collected and CTCs enriched for using size-based filtration. The enriched CTCs were analyzed using immunofluorescence staining with antibodies directed against epithelial cell adhesion molecule (EpCAM) and cluster of differentiation 45. The genomic DNA of CTCs was extracted, amplified and 50 genes screened for mutations using the Ion AmpliSeq™ Cancer Hotspot Panel v2. EpCAM staining detected CTCs in 10/11 patients and the average CTC count was 3.9 in 5 ml blood. The average purity of enriched CTCs was 14.2±29.4% and the average amount of amplified DNA was 28.6±11.9 µg. Catalogue Of Somatic Mutations In Cancer mutations were detected in the CTCs and included IDH2, TP53, NRAS, IDH1, PDGFRA, HRAS, STK11, EGFR, PTEN, MLH1, PIK3CA, CDKN2A, KIT and SMARCB1. In conclusion, a novel size-based filtration approach for the isolation of CTCs was evaluated and successfully applied for the genomic analysis of CTCs from patients with breast cancer.

Gefitinib for EGFR mutated non-small cell lung cancer (NSCLC) is showing acquired resistance after 8-11 months, and about 60% of its mechanisms have been reported due to T790M mutation. In about a third of NSCLC, AXL expression can be observed at baseline status. And because AXL expression could be a mechanism of drug resistance, we decided to monitor AXL expression before and after Gefitinib treatment in EGFR mutated NSCLC patients through circulating tumor cells (CTCs). Herein, Thirty-seven out of 55 participants who showed acquired resistance to EGFR-TKI were selected in this prospective analysis. To evaluate the AXL expression on the CTCs, serial blood was obtained (tracking for 4, 12, 24 and 36 weeks after EGFR-TKIs). Baseline tumor tissues were also obtained for genetic analysis. Progression-free survival (PFS) and overall survival (OS) were explored according to the baseline AXL expression. AXL expression was analyzed by observation and quantitative intensity-dependent fold change analysis using immunofluorescence analysis. CTCs were isolated and analyzed by CytoGen's Smart Liquid Biopsy platform that uses the size-based gravity filtration for CTC isolation. We found out that there were ten cases (27%) of the increasing pattern of AXL expression in CTCs during longitudinal monitoring in patients with progressive disease (PD). The most remarkable aspect is that AXL up-regulation in CTCs preceded the time point of clinical PD. These results imply that the expectation of clinical PD by blood CTC (liquid biopsy-based examination) can be combined with the T790M. Moreover, the PFS (p= 0.002) and OS (p= 0.007) were both worse in patients with AXL expression at the baseline status compared with patients without AXL expression (N=55). Conclusively, this study implicates that AXL in CTCs could be deployed for prognostic evaluation. And the clinical monitoring of specific target marker in CTC could help for further treatment strategy. Citation Format: Mi Young Choi, Da Hye Moon, Hae Ung Lee, Jong Min Jo, Young Woong Sohn, Byung Hee Jeon, Sang-We Kim. Longitudinal AXL assessment of circulating tumor cells (CTCs) and its clinical implication in the patients with EGFR mutated non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 663.

e15031 Background: Stage IV lung cancer is the most advanced lung cancer state accompanied by metastasized to the area around the lungs or distant major organs. The most common type of lung cancer is non-small cell lung cancer, which is more aggressive and may spread quickly due to organ-specific complex networks such as lymph and major blood vessels. Thus, only precise diagnostic strategy approaches will determine the effectiveness of the actual and successful clinical treatment. Until a recent date, Immunohistochemistry (IHC) for programmed death-ligand 1(PD-L1) test is the only available biomarker test that purpose diagnostics (CDx) and guide the treatment with immune checkpoint inhibitors in NSCLC. Methods: Given that CDx strategy, tissue biopsy has inevitable limitations, including patient risk, repetitive examination, sample preparation, sensitivity, and accuracy. For this reason, our research team contrived the best strategy for biomarker, PD-L1-specific CTCs in stage IV NSCLC group (N = 30) compared to pulmonary inflammatory patient groups (N = 30) CytoGen Smart biopsy platform. Herein, we removed false-positive cells for the first strategy of distinguishing between lymphoid/myeloid cells and the enriched-CTCs. And the second strategic approach is to calculate the pure CTCs (without false-positive cells) and then CTPS) as measured by the PD-L1 expression among pure-CTCs. That application is the percentage of viable CTCs showing partial or complete stained cells at the deducted cut-off value in each fluorescence, respectively. Results: Consequently, we demonstrated over 80% of the concordance rate between VENTANA PD-L1(SP263) and DAKO PD-L1(SP263) assay tested by the PD-L1 expression on stage IV NSCLC in tissue and pure-CTCs based CTPS from the blood. In contrast, pure-CTCs based CTPS in the pulmonary inflammatory group were all negative (recorded as zero). Conclusions: Conclusively, this study implicates that pure-CTCs based CTPS could be deployed for innovative diagnosis strategies as alternatives for tissue biopsy. Our clinical study's data suggested that the possibility for prompt decision for diagnosis and gain powerful insights to guide the personalized treatment in NSCLCs. Clinical trial information: 2020-0553.

e15557 Background: CTC (Circulating tumor cells) has the advantage of being able to effectively investigate non-invasive diagnosis, prediction, and prognosis for non-surgical carcinoma or tissue sample collection. We have developed WM-S1-030 (WMBIO) as a novel inhibitor for mtRTK (mutant receptor tyrosine kinase) identified important targets for cancer progression and metastasis in various cancers, including colorectal cancer, and have tried to prove the possibility of CTC diagnosis, prediction and prognosis of WM-S1-030 using tumor animal models. Methods: Six of colon cancer cell lines expressing mtRTK (pTyr-mtRTK) were transplanted into Balb.c nude mice and NSG mice to collect peripheral blood when the tumor size reached 1000 mm3. CTCs were quantified in peripheral blood using the Smart Biopsy cell isolator (Cytogen). The expression of pTyr-mtRTK in CTCs was analyzed via the Smart Biopsy Image Analyzer (Cytogen). Colon cancer cell lines expressing pTyr-mtRTK were transplanted into NSG mice and we examined the tumor suppression, the enumeration of CTCs and pTyr-mtRTK expression in CTCs after administration of WM-S1-030. We also identified CTCs in peripheral blood of colon cancer patients and analyzed expression of pTyr-mtRTK in CTCs through Smart Biopsy system. Results: As results of the enumeration of CTCs after tumor transplantation into Balb.c nude mice and NSG mice, the number of CTC was 8 ~ 20 times higher peripheral blood in the NSG model, which indicates innate immune responses are critical for the survival of CTCs. Half of CTCs isolated from the blood expressed pTyr-mtRTK. The tumor size, the number of CTCs, and the expression of pTyr-mtRTK in CTCs were examined after the administration of WM-S1-030. The volume of tumor decreased by 67% in the WM-S1-030 group compared to the vehicle. The number of CTCs was reduced by approximately three times after the treatment of WM-S1-030 and also the number of CTCs expressing pTyr-mtRTK was significantly decreased. CTC detection and pTyr-mtRTK expression analysis in the peripheral blood of colon cancer patients detected 9 CTCs per 10 ml of peripheral blood, of which 11 % of CTCs representing pTyr-mtRTK were confirmed. Conclusions: WM-S1-030 was developed and confirmed in the CTC-based animal model, which implies that the analysis of CTCs is the powerful tool for predicting/ prognosing WM-S1-030 in the pre-clinical analysis. Currently, we are conducting clinical diagnostic validation using CTC in the blood of colorectal cancer patients and preparing for clinical trial of WM-S1-030.

Analysis of anaplastic lymphoma kinase (ALK) rearrangement in non-small cell lung cancer (NSCLC) is considered to be a useful tool when considering predictive biomarker detection for evaluating eligibility for targeted therapy. It is not always possible to perform a tumor biopsy in patients. Isolation and culturing of circulating tumor cells (CTCs) may be an alternative to tumor biopsies for the diagnosis of ALK rearrangement. Blood was collected from 22 patients with NSCLC harboring ALK rearrangement and was divided into two groups: One for immunofluorescence staining and the other for culture. Samples were filtered by size and cultured CTCs were analyzed for echinoderm microtubule-associated protein-like 4-ALK translocation using fluorescence in situ hybridization. CTCs positive for epithelial cell adhesion molecule and CTCs exhibiting ALK rearrangement were detected. Therefore, CTCs may be used as a potential alternative method to tissue biopsy for diagnosing ALK rearrangement. Additionally, this method may have clinical applications including serial blood sampling for the development of personalized cancer therapy based on individual genomic information.

Background: The EGFR (epidermal growth factor receptor) TKI (tyrosine kinase inhibitor) has become the standard treatment in lung cancer patients with EGFR mutations. When these patients are treated with EGFR TKI, 80~90% of the patients show responses to the drug. However the tumor begins to progress again following the development of resistance about 1 year later on average. About a half of resistance mechanisms are caused by the additional mutation of the EGFR gene (T790M), and the other half of the resistance mechanisms are caused by various mechanisms, with one of them being the overexpression of the AXL protein. Confirming AXL overexpression in circulating tumor cells (CTCs) can be an alternative method for tissue biopsy, but almost no research has been conducted on this so far. Here, we evaluate the AXL expression in CTC in case resistance occurs after the EGFR inhibitor treatment. Materials and Methods: The blood samples (10 ml) were collected from 10 EGFR TKI treated and relapsed lung cancer patients (TKI group) and 10 non-treated patients (control group). The blood samples were processed through Cytogen protocol to enrich CTCs. The enriched cells were immunofluorescent stained for CTC markers (EpCAM or Vimentin), AXL and WBC marker (CD45). The immunofluorescent stained cells were analyzed for each markers using Image Analysis program. Result: CTCs were detected by CTC markers in all patients from TKI group (range 0-210) and control group (range 1-42). AXL positive CTCs were detected in 9 out of 10 cases from TKI group and in all cases from control group. Interestingly, the quantification of AXL staining intensity in CTCs showed that AXL overexpression (3 fold or greater than baseline) was in 50% of TKI group and 20 % of control group. Conclusion: We have tested a new approach to evaluate the AXL overexpression in tumor cells of EGFR TKI resistant patients, and showed the feasibility of the this method. Most of lung cancer patients who undergo biopsy require hospitalization and some degree of complication due to biopsy is inevitable. Here we suggested that the blood-based method as a good alternative to the tissue biopsy in patients with tolerance. Citation Format: Young Hun Kim, Myoung Shin Kim, Jun Sup Lee, Hyun Kyung Lee, Jae Hyuk Lee, Young Woong Sohn, Koichi Tazaki, Kenji Nakamaru, Kenichi Wakita, Byung Hee Jeon, Seokhyung Kim, Se-Hoon Lee. Evaluation of AXL expression on circulating tumor cells from EGFR mutated lung cancer patients who have relapsed after the EGFR TKI treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1586.

The brown algal species Sargassum thunbergii (Fucales, Phaeophyceae) is widely distributed in coastal area of East Asia. This alga is ecologically and economically important seaweeds; however, no genome data was reported thus far. To get a better understanding of organellar genome of S. thunbergii, we sequenced and annotated its complete plastid genome. The circular plastid genome is 124,592 bp in size with 30.4% GC content, which is composed of a large single-copy (LSC) region of 73,668 bp, a small single-copy (SSC) region of 40,032 bp, and two inverted repeat (IR) of 5446 bp each. The plastid genome of S. thunbergii includes 139 protein-coding genes, six ribosomal RNA (rRNA) operons, 28 transfer RNA (tRNA) sequences, and one intron (214 bp) in tRNA-Leu (trnL) gene. Five overlapping genes were identified in the compact plastid genome. Base on the comparison with previously published five brown algae plastid genomes, we found that the gene content and gene order of S. thunbergii are identical to that of other Fucales species Fucus vesiculosus.

Liquid biopsy using circulating tumor cells (CTCs) is a noninvasive and repeatable procedure, and is therefore useful for molecular assays. However, the rarity of CTCs remains a challenge. To overcome this issue, our group developed a novel technology for the isolation of CTCs on the basis of cell size difference. The present study isolated CTCs from patients with breast cancer using this method, and then used these cells for cancer gene panel analysis. Blood samples from eight patients with breast cancer were collected, and CTCs were enriched using size-based filtration. Enriched CTCs were counted using immunofluorescent staining with an epithelial cell adhesion molecule (EpCAM) and CD45 antibodies. CTC genomic DNA was extracted, amplified, and screened for mutations in 400 genes using the Ion AmpliSeq Comprehensive Cancer Panel. White blood cells (WBCs) from the same patient served as a negative control, and mutations in CTCs and WBCs were compared. EpCAM+ cells were detected in seven out of eight patients, and the average number of EpCAM+ cells was 8.6. The average amount of amplified DNA was 32.7 µg, and the percentage of reads mapped to any targeted region relative to all reads mapped to the reference was 98.6%. The detection rate of CTC-specific mutations was 62.5%. The CTC-specific mutations were enhancer of zeste polycomb repressive complex 2 subunit, notch 1, AT-rich interaction domain 1A, serine/threonine kinase 11, fms related tyrosine kinase 3, MYCN proto-oncogene, bHLH transcription factor, APC, WNT signaling pathway regulator, and phosphatase and tensin homolog. The technique used by the present study was demonstrated to be effective at isolating CTCs at a sufficiently high purity for genomic analysis, and supported the use of comprehensive cancer panel analysis as a potential application for precision medicine.

Although numerous effective therapies have improved the survival rate of patients with breast cancer, a number of patients present with tumor recurrence and metastasis. A liquid biopsy of circulating tumor cells (CTC) is a non-invasive method to obtain tumor cells and may be used as substitute for a tumor tissue biopsy. The present study focuses on determining whether CTC culture is an optimal method of obtaining sufficient amounts of CTCs for molecular analysis. The current study demonstrates a method of isolating and culturing CTCs from patients with breast cancer and the construction of a molecular profile of cultured cells using the Ion AmpliSeq Cancer Gene Panel V2. Gene mutations that were observed in cultured CTCs were compared with those observed in primary tumor tissues. CTCs were isolated and cultured from the blood of six patients with breast cancer. Mutations from the Catalogue Of Somatic Mutation In Cancer (COSMIC) were detected in Platelet-Derived Growth Factor Receptor Alpha, MET (also known as Hepatocyte Growth Factor Receptor), Phosphatase and Tensin Homolog, Harvey Rat Sarcoma Viral Oncogene Homolog, SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin Subfamily B Member 1, Cyclin Dependent Kinase Inhibitor 2A and MutL Homolog 1 genes in 5/6 samples. A comparison between mutations detected in cultured CTCs and mutations detected in primary tumor tissues demonstrated that a large number of mutations that were identified in CTCs were also detected in primary tumor tissues. The results from the current study describe a novel cell culture approach that may be used to obtain an optimal number of CTCs for molecular analysis. This novel approach is able to be used as a tool for liquid biopsy during breast cancer treatment.

Hydroforming process recently draws attention of automotive industries due to its advantages like the increased strength, weight reduction, improved quality and reduced tooling cost. This paper summarizes some of our experiences of tube and sheet hydroforming process design by simulation technique through actual tryout. Parts included in this paper are tie bar, subframe and engine mount bracket. The simulations for the entire hydroforming processes were performed and their results were used to predict and fix the forming failures like the wrinkling and the fracture. Based on this experience, we also suggested necessary improvements in the design procedure.

Hydroforming is the technology that utilizes hydraulic pressure to form tube and sheet materials into desired shapes inside die cavities. It can be characterized as tube hydroforming and sheet hydroforming depending on the shape of used blank. Hydroforming offers several advantages as compared with conventional manufacturing via stamping and welding, such as part consolidation, weight reduction, improved structural strength and stiffness, lower tooling costs, fewer secondary operations, tight dimensional tolerances, low springback, and reduced scrap. In this paper, the welded blank hydroforming (WBH) technology is discussed in the aspects of formability, and the engine mount bracket and the subframe are analyzed and manufactured for the application of the technology, which have been manufactured by stamping and welding processes. Die geometry and process parameters are suggested by the computer simulation using the explicit finite element code, PAM-STAMP™. Various defects, especially wrinkling in the area of big difference of die depth and large height deviation of the die flange face, are investigated and analyzed. The numerical results are compared with prototype tryout results, and the wrinkling and the local thinning under different forming conditions are predicted.

The evaluation of ALK rearrangement in non-small-cell lung cancer (NSCLC) is a significant tool when considering chemotherapy. It is not always possible to perform a tumor biopsy in patients. We suggest isolation and culturing of circulating tumor cells (CTCs) as an alternative tool to a tumor biopsy for the diagnosis of ALK rearrangement. From 22 patients with NSCLC harboring ALK rearrangement, blood samples were collected and divided into two parts: one for immunofluorescence staining of CTC marker and the other for culturing of CTCs. Both samples were processed by size-based filtration, and Cultured CTCs were analyzed for EML4-ALK translocation by fluorescence in situ hybridization (FISH) using Vysis ALK break apart FISH probe kit. CTC culturing was successful in 18 of 22 cases (81.8%). Among 18 cases of successful CTC cultures, 13 cases showed ALK rearrangement positivity (72.2%). Therefore, we suggest that the CTCs can be used as an alternative method to tissue biopsy for diagnosing ALK rearrangement. In addition, this method may have clinical applications including serial blood sampling for the development of personalized cancer therapy based on individual genomic information. Citation Format: Min Kyung Jeon, Young Hun Kim, Eunjoo Hwang, Hye Seon Lee, Ji-hyun Uh, Myoung Shin Kim, JooKyung Park, Byung Hee Jeon, Se-Hoon Lee. ALK rearrangement analysis in circulating tumor cells of lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1737. doi:10.1158/1538-7445.AM2017-1737

Tears and wrinkles were generated during the forming of a B-pillar, a component of the automotive side-frame. Employing tailor-welded blanks in the B-pillar, a stretch flanging condition gave rise to such forming problems as tearing along the weld bead. The authors simplified B-pillar lower part to a T shaped section to investigate the forming behaviors of the tailor-welded blanks. The effects of the weld line locations and the section end shapes on tearing and wrinkling were studied by way of finite element simulations and several experiments. An appropriate weld bead modeling technique was selected producing a deformation pattern similar to those of the experiments. The concept of the counter-punch was introduced to eliminate body wrinkles.

Tube hydroforming has recently been drawing the attention of the automotive industries due to its several advantages over conventional methods. It can produce a wide range of products such as sub-frames, engine cradles, and exhaust manifolds with cheaper production cost by reducing the overall number of processes. The tube hydroforming process is based on the use of internal pressure combined with axial load. Successful tube hydroforming depends on the reasonable combination of the internal pressure and the axial load at the tube ends. This paper deals with the optimal process design (internal pressure and axial load) of the hydroforming process using numerical simulation by the explicit finite element code combined with an optimization tool. An optimization technique is used in order to minimize the tube thickness variation by determining the optimal loading path in the tube expansion forming and the sub-frame forming process. The optimization is performed by means of a gradient-based method including sensitivity analysis.

This paper presents the simulation results on prebending and hydroforming processes that are used to form an automotive part, a tie bar. Two prebending simulations, by a rotary draw bending machine and a bend die, are carried out to obtain the shape change of the cross-section and the thinning of the tube and the results are compared with each other. In prebending simulation with a rotary draw bending machine, to avoid wrinkling in compressive area of the tube, a wiper die is included. A parametric study is carried out to obtain the effect of the forming parameters such as bend radius and tube thickness. The hydroforming process is simulated considering the results of the prebending simulation.

Enrichment of circulating tumor cells (CTCs) from whole blood is very challenging due to its rarity. We have developed a new CTC enrichment method using a microfabricated filter. The filter was designed to fractionate tumor cells by cell size and optimized to have high porosity and proper pore distribution. When cancer cells were spiked in whole blood, the average recovery rate was 82.0 to 86.7% and the limit of detection by filtration process was approximately 2 cancer cells in a testing volume of blood. The results indicate that the microfabricated filter-based enrichment would be useful to retrieve and analyze CTCs in practice.

Multi-stage deep-drawing processes, including normal drawing, reverse drawing, and redrawing, to shape the front shell of a master VAC for an automobile have been analyzed sequentially by the use of the rigid-plastic finite-element method, computational results on the punch/die loads and thickness distributions being obtained. The thickness strains were compared with the results of experiments on current drawing processes, good agreement being found. Deep-drawing processes of the redesigned shell to improve the specific strength and stiffness were simulated with the numerical method developed. By varying several process parameters, such as the blank size, the corner radii of the tools, and the clearances, the simulation results showed improvements in reducing the forming loads. Also, forming defects were found during simulation and the appropriate blank size could be verified.

e20579Background: The evaluation of ALK rearrangement in non-small-cell lung cancer (NSCLC) is a useful and significant tool for the decision of crizotinib treatment. However, tumor biopsy in patie...

The variations of the structural vibration characteristics such as the natural frequency, damping, and mode shape can be observed under thermal loading conditions. Therefore, the free vibration characteristic of a thermally loaded cylindrical shell was analyzed in this study. ABAQUS was used for the analysis and a thermo-elastic experimental modal test was conducted. Configuration of the shell is 5 m long, 0.4 m diameter, 0.002 m thick and its material was combined by stainless-steel 304 and aluminum 6,061. The cylindrical shell was heated by the temperature of 100, 200 and 400°C and divided longitudinally by five zones. The theoretical analysis was verified by the experimental modal analysis of the cylindrical shell which has the free–free boundary condition and the elevated thermal conditions. After that, the free vibration analysis of the thermally loaded cylindrical shell was performed according to several thermally loaded conditions.

Circulating tumor cells (CTCs), known as the “leukemic phase” of solid tumor, have been associated with poor prognosis and clinical outcome in various cancer including breast and colorectal. However, fewer studies reported characteristic and usefulness of CTC as an indicator in gastric cancer (GC). In this study, we evaluated utility of CTC in advanced GC cancer. First, we confirmed detection rate of GC cancer cell lines as a spiking test, and then defined CTC as DAPI+, EpCAM+ and CD45- cells through immunofluorescence stating. To determine a cut-off for CTC of GC, we assessed the number of CTCs in blood of 22 healthy volunteers, and observed 1 (3/22) to 2 (1/22) of CTCs. Based on these results, we determined CTC positivity of GC as more than 3 CTCs. When we evaluated CTC positive rate in Stage IV GC patients, 28 patients were CTC positive (30.1%) with the median number of CTC 5 (range 3 to over 100). Interestingly, 3 out of 28 patients were showed more than 100 CTCs. While, in operable GC patients (Stage I to III), CTC positivity was 18.4% (7/38) and median number of CTC was 4 (range 3 to 9) in CTC positive patients. These results showed that Stage IV GC patients have high number of CTCs than operable GC patients. Tumor histology and Lauren classification did not affect to CTC positivity in Stage IV and operable GC patients. We also observed the potential use of CTC in serial follow-ups and one of the cases shows the decreasing CTCs followed by clinical response. In conclusion, this study demonstrates that detection of CTC may be useful prognostic indicator of GC cancer patients. Citation Format: Won Suk Lee, Jeong Min Kim, Woo Sun Kwon, So Jung Lim, Tae Soo Kim, Ga Yun Kim, Sung Ho Choi, Byung Hee Jeon, Min Kyu Jung, Joong Bae Ahn, Hyun Cheol Chung, Sun Young Rha. Utility of circulating tumor cell (CTC) in advanced gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 391. doi:10.1158/1538-7445.AM2015-391

e22033 Non-invasive and repeatable EGFR analysis using novel enrichment method for circulating tumor cells Background: The first-line treatment in confirmed NSCLC (non-small cell lung cancer) patie...

e22029 Background: Although many effective therapies improve the survival rate in breast cancer patients, a significant number of patients could not avoid recurrence and metastasis. Liquid biopsy u...

Molybdenum, a bcc refractory metal with a melting point of about 2600°C, has a high heat and electrical conductivity. In addition, it remains strong mechanically at high temperatures as well as at low temperatures. Therefore it is a technologically very important material for the applications operating at high temperatures. However, a multi‐stage process is required due to the low drawability for making a deep drawn part from the molybdenum sheet. In this study, a multi‐stage deep drawing process for a molybdenum circular cup was designed by combining the drawing with the ironing, which was effective for the low drawability materials. A parametric study by FE analysis for the multi‐stage deep drawing was conducted for evaluation of the design variables effect. Based on the FE analysis result, the multi‐stage deep drawing process was parameterized by the design variables, and an optimum process design was obtained by the process optimization based on the FE simulation at each stage.

Circulating tumor cells (CTCs) are rare cells that have shed into the bloodstream from primary tumor, and potentiallyprovidea tool for the better understanding of tumor metastasis and noninvasive monitoring of the disease progression. However their isolation and characterization has been a major technological challenge due to their rareness. Here, we suggest the CTC culture as an effective method to obtain CTCs sufficient in numberfor molecular analysis of original tumor characteristics. We isolated and successfully cultured the CTCs from four lung cancer patients, and then analyzed those cells for ALK (anaplastic lymphoma kinase) fusion using real-time PCR method, and confirmed that the cultured CTCs have retained thefusion the same as those found in primary tumors. These results suggest that the isolation and culture of CTCs can be a substitutive method for tumor tissue biopsy, and may provide practically useful clinical applications, such as personalized cancer therapy based on their genomic information through serial blood samplings from the cancer patients.