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Purpose NUT carcinoma (NC) is a solid tumor caused by the rearrangement of NUTM1 that usually develops in midline structures, such as the thorax. No standard treatment has been established despite high lethality. Thus, we investigated whether targeting the junction region of NUTM1 fusion breakpoints could serve as a potential treatment option for NC. Materials and Methods We designed and evaluated a series of small interfering RNAs (siRNAs) targeting the junction region of BRD4-NUTM1 fusion (B4N), the most common form of NUTM1 fusion. Droplet digital polymerase chain reaction using the blood of patients was also tested to evaluate the treatment responses by the junction sequence of the B4N fusion transcripts.Results As expected, the majority of NC fusion types were B4N (12 of 18, 67%). B4N fusion-specific siRNA treatment on NC cells showed specific inhibitory effects on the B4N fusion transcript and fusion protein without affecting the endogenous expression of the parent genes, resulting in decreased relative cell growth and attenuation of tumor size. In addition, the fusion transcript levels in platelet-rich-plasma samples of the NC patients with systemic metastasis showed a negative correlation with therapeutic effect, suggesting its potential as a measure of treatment responsiveness.Conclusion This study suggests that tumor-specific sequences could be used to treat patients with fusion genes as part of precision medicine for a rare but deadly disease.

Purpose The objective is to develop a real-time Mitochondrial ATP contents and Response Against Inhibiting and Stimulating Substrates (MitoRAISE) assay and assess its potential to evaluate the mitochondrial oxidative phosphorylation function. Methods MitoRAISE measures the total ATP contents, ATP synthesis capacity, and the response to inhibitory substrates. The measurements were all quantified with an ATP standard curve and validated with in vitro testing. MitoRAISE was then applied using peripheral blood mononuclear cells (PBMCs) from 35 healthy volunteers and 20 breast cancer patients. Result Results from isolated mitochondria, cells with different permeabilization percentages, and cells with damaged mitochondria demonstrated the ability of capturing ATP signals specifically from the mitochondria. Breast cancer PBMCs exhibited a significant increase in glutamic acid- and malic acid-induced mitochondrial ATP synthesis capacity yet a significant decrease in mitochondrial DNA copy number (mtDNA CN) compared to healthy PBMCs. Furthermore, breast cancer PBMCs mostly showed negative correlation between mtDNA CN and parameters from MitoRAISE but healthy individuals showed positive correlation. Conclusion We developed a quick and easy method to detect real-time mitochondrial activity in live cells. Monitoring mitochondrial ATP synthesis capacity and sensitivity to inhibitory substrates could aid in assessing the functional status of mitochondrial oxidative phosphorylation.

Supplementary Information

Combination therapy of ALK and β3 inhibitors shows promising results in vivo and in ALK-rearranged NSCLC

Treatment information of patients who received crizotinib

Purpose: Anaplastic lymphoma kinase (ALK)-positive cancers are sensitive to small-molecule ALK kinase inhibitors, but most cases experience failure following treatment. Hence, additional drug targets and combination therapeutic treatments are needed. We investigated gene expression that is regulated by the expression of ALK and explored its roles in cancer progression and therapeutic implication.Experimental Design: We screened ALK-rearranged non–small cell lung cancer (NSCLC) cases using immunohistochemistry and fluorescence in situ hybridization and then conducted multiplex gene expression analysis. We also performed a clinicopathologic analysis to validate the findings. Additional cellular experiments, including inhibition and migration assays, and in vivo lung cancer model studies were performed.Results: Among patients with ALK-rearranged NSCLC, integrin β3 (ITGB3) was one of the overexpressed genes in comparison with that in ALK-negative NSCLC (P = 0.0003). ALK and integrin β3 expression were positively correlated, and we discovered that high integrin β3 mRNA expression was associated with metastasis and more advanced tumor stages (P < 0.005; P < 0.05). Furthermore, we found that inhibition of both ALK and integrin β3 led to increased drug sensitivity in vitro and in vivo (both P < 0.05).Conclusions: We discovered a positive correlation between ALK and integrin β3 expression levels in ALK-rearranged NSCLC. Our findings suggest that high integrin β3 expression in ALK-rearranged NSCLC is associated with tumor progression and a worse prognosis. This finding demonstrates the prognostic value of integrin β3 and provides a rationale for combination treatment with ALK and integrin β3 inhibitors in patients with ALK-rearranged NSCLC. Clin Cancer Res; 24(17); 4162–74. ©2018 AACR.

[Background] As the development of endocrine resistance and late recurrences are the major clinical concerns in hormone receptor (HR)-positive/HER2-negative metastatic breast cancer (MBC) patients, biomarkers to predict the occurrence of endocrine resistance or disease progression are crucial for improving patient outcomes. Aberrant HGF/c-MET signaling pathway has been reported to play a role in various cellular processes in cancer. Estrogen Receptor 1 (ESR1) mutations, encoding estrogen receptor α, are associated with endocrine resistance in HR+ breast cancer. PIK3CA hotspot mutations that induce hyperactivation of the PI3K are found in 30-40% of HR+ advanced breast cancers. In this context, we evaluated the prognostic values of c-MET-enriched CTC, ESR1 mutations, PIK3CA mutations, and cfDNA concentrations detected in the blood of HR+HER2- MBC patients. [Methods] MBC patients were prospectively enrolled during standard treatments at Samsung Medical Center (IRB No.2019-08-119). Circulating tumor cells (CTCs) were isolated using the GenoCTC® with c-MET-enriched or EpCAM-enriched CTC isolation kits (Genobio Corp., South Korea) from 4mL of blood each. PIK3CA and ESR1 hotspot mutations were analyzed by droplet digital PCR kits (Gencurix Inc., South Korea). cfDNA concentrations were calculated using ESR1 gene copy numbers from plasma. To compare the proportion of c-MET overexpression between primary breast tumors and metastatic sites in HR+HER2- breast cancer patients, primary breast (n=358) and metastatic sites (n=27) were independently collected. c-MET expression was evaluated by an immunohistochemistry assay using an anti-total c-MET (SP44) antibody with a Ventana Discovery XY automated system according to the manufacturer’s instruction. c-MET overexpression was defined if the staining was scored as 2+ or 3+. Progression-free survival (PFS) was defined as the time from blood draw to the first of either disease progression or death during standard therapy. [Results] Out of 93 patients with HR+ MBC, analysis was performed in 63 HR+HER2- MBC patients. Seventeen patients (27%) had one or more EpCAM-enriched CTCs, and fourteen patients (22%) had one or more c-MET-enriched CTCs detected in their blood. The median follow-up time and median time to censoring were 8.4 months and 18.7 months, respectively. According to the Kaplan-Meier survival analysis by log-rank test, c-MET-enriched CTCs, cfDNA concentrations, and ESR1 mutations were significantly associated with PFS (p=0.0026, 0.0064, and 0.011, respectively). However, PIK3CA mutations and EpCAM-enriched CTCs were not statistically significant with PFS (p=0.38 and 0.86, respectively). Multivariate analysis showed that both c-MET-enriched CTCs (HR=3.5, p=0.014) and cfDNA concentrations (HR=2.2, p=0.031) were independent predictors for PFS in HR+HER2- MBC. The proportion of c-MET overexpression was significantly higher in metastatic sites (22.2%) than in primary breast tumors (4.7%) in HR+HER2- breast cancer patients (p=0.00002). As c-MET-enriched CTCs and cfDNA concentrations were independent predictors of disease progression, patients were divided into two groups depending on the result of c-MET-enriched CTCs and cfDNA concentration. When patients with low c-MET-enriched CTC and cfDNA concentrations were classified as a low-risk group and other patients into a high-risk group, the high-risk group had a shorter PFS than the low-risk group (p=0.003). [Conclusion] This study provided c-MET-enriched CTCs and cfDNA concentrations calculated by ESR1 copy numbers in patient blood were significant independent predictors of disease progression in HR+HER2- MBC. The poor prognosis in the c-MET-enriched CTC-high group and the difference in the c-MET overexpression rate between the primary breast and metastatic sites suggested the importance of monitoring c-MET-enriched CTCs in the blood of HR+HER2- MBC patients. Citation Format: Jieun Park, Eun Sol Chang, Ji-Yeon Kim, Chaithanya Chelakkot, Minjung Sung, Ji-Young Song, Kyungsoo Jung, Na Young Kim, Hyegyeong Lee, Mi-Ran Kang, Yeon Hee Park, Young Kee Shin, Yoon-La Choi. The prognostic value of c-MET monitoring by using c-MET-enriched circulating tumor cells in HR-positive HER2-negative 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 P5-06-04.

Small cell lung cancer (SCLC) is a fast-growing and malignant cancer that responds well to chemotherapy; however, the survival rate is less than 15% after 2 years of diagnosis. Therefore, novel therapeutic agents for treating SCLC patients need to be evaluated. This study aims to identify the therapeutic targets based on the comprehensive genomic profiling of SCLC patients. Among the molecular-profiled SCLC samples obtained using targeted sequencing, the array-based comparative genomic hybridization (array CGH) identified focal insulin receptor substrate 2 (IRS2) amplification in the SCLC patients. IRS2 amplification was confirmed in 5% of 73 SCLC patients. To determine whether IRS2 amplification could act as a therapeutic target, we generated a patient-derived xenograft (PDX) model and subsequently screened 43 targeted agents using the PDX-derived cells (PDCs). Ceritinib significantly inhibited the cell growth and impaired the tumor sphere formation in IRS2-expressing PDCs. Its effects were confirmed in various in vitro assays and were further validated in the mouse xenograft models. In this study, we present that IRS2 amplification and/or expression serve as preclinical implications for a novel therapeutic target in SCLC progression. Furthermore, we suggest that insulin-like growth factor-1 (IGF-1) receptor inhibitor-based therapy could be used for treating SCLC with IRS2 amplification. Keywords: IRS2, small cell lung cancer, SCLC, patient-derived xenograft, PDX, targeted therapy, Ceritinib

Purpose The purpose of this study was to investigate the concordance rate of PIK3CA mutations between primary and matched distant metastatic sites in patients with breast cancer and to verify whether there are differences in the frequency of PIK3CA hotspot mutations depending on the metastatic sites involved.Materials and Methods Archived formalin-fixed paraffin-embedded (FFPE) specimens of primary breast and matched distant metastatic tumors were retrospectively obtained for 49 patients. Additionally, 40 archived FFPE specimens were independently collected from different breast cancer metastatic sites, which were limited to three common sites: the liver, brain, and lung. PIK3CA mutations were analyzed using droplet digital PCR, including hotspots involving exons 9 and 20. Results After analysis of 49 breast tumors with matched metastasis sites, 87.8% showed concordance in PIK3CA mutation status. According to PIK3CA hotspot mutation testing in 89 cases of breast cancer metastatic sites, the proportion of PIK3CA mutations at sites of metastasis involving the liver, brain, and lung was 37.5%, 28.6%, and 42.9%, respectively, which did not result in statistical significance. Conclusion The high concordance of PIK3CA mutation status between primary and matched metastasis sites suggests that metastatic sites, regardless of the metastatic organ, could be considered sample sources for PIK3CA mutation testing for improved therapeutic strategies in patients with metastatic breast cancer.

Amplification and overexpression of MDM2 and CDK4 are well-known diagnostic criteria for well-differentiated liposarcoma (WDLPS)/dedifferentiated liposarcoma (DDLPS). Although it was reported that the depletion of MDM2 or CDK4 decreased proliferation in DDLPS cell lines, whether MDM2 and CDK4 induce WDLPS/DDLPS tumorigenesis remains unclear. We examined whether MDM2 and/or CDK4 cause WDLPS/DDLPS, using two types of transformed human bone marrow stem cells (BMSCs), 2H and 5H, with five oncogenic hits (overexpression of hTERT, TP53 degradation, RB inactivation, c-MYC stabilization, and overexpression of HRASv12). In vitro functional experiments revealed that the co-overexpression of MDM2 and CDK4 plays a key role in tumorigenesis by increasing cell growth and migration and inhibiting adipogenic differentiation potency when compared with the sole expression of MDM2 or CDK4. Using mouse xenograft models, we found that the co-overexpression of MDM2 and CDK4 in 5H cells with five additional oncogenic mutations can cause proliferative sarcoma with a DDLPS-like morphology in vivo. Our results suggest that the co-overexpression of MDM2 and CDK4, along with multiple genetic factors, increases the tendency for high-grade sarcoma with a DDLPS-like morphology in transformed human BMSCs by accelerating their growth and migration and blocking their adipogenic potential., This paper examines whether the nuclear E3 ubiquitin ligase MDM2 and/or the cyclin-dependent kinase CDK4 produce well-differentiated liposarcoma (WDLPS)/dedifferentiated liposarcoma (DDLPS) using transformed human bone marrow stem cells through in vitro and in vivo functional experiments. The authors found that co-overexpression of MDM2 and CDK4 causes high-grade sarcoma with a dedifferentiated liposarcoma-like morphology.

PurposeTo find biomarkers for disease, there have been constant attempts to investigate the genes that differ from those in the disease groups. However, the values that lie outside the overall pattern of a distribution, the outliers, are frequently excluded in traditional analytical methods as they are considered to be ‘some sort of problem.’ Such outliers may have a biologic role in the disease group. Thus, this study explored new biomarker using outlier analysis, and verified the suitability of therapeutic potential of two genes (TM4SF4 and LRRK2).Materials and MethodsModified Tukey’s fences outlier analysis was carried out to identify new biomarkers using the public gene expression datasets. And we verified the presence of the selected biomarkers in other clinical samples via customized gene expression panels and tissue microarrays. Moreover, a siRNA-based knockdown test was performed to evaluate the impact of the biomarkers on oncogenic phenotypes.ResultsTM4SF4 in lung cancer and LRRK2 in breast cancer were chosen as candidates among the genes derived from the analysis. TM4SF4 and LRRK2 were overexpressed in the small number of samples with lung cancer (4.20%) and breast cancer (2.42%), respectively. Knockdown of TM4SF4 and LRRK2 suppressed the growth of lung and breast cancer cell lines. The LRRK2 overexpressing cell lines were more sensitive to LRRK2-IN-1 than the LRRK2 under-expressing cell linesConclusionOur modified outlier-based analysis method has proved to rescue biomarkers previously missed or unnoticed by traditional analysis showing TM4SF4 and LRRK2 are novel target candidates for lung and breast cancer, respectively.

Purpose: Anaplastic lymphoma kinase (ALK)-positive cancers are sensitive to small-molecule ALK kinase inhibitors, but most cases experience failure following treatment. Hence, additional drug targets and combination therapeutic treatments are needed. We investigated gene expression that is regulated by the expression of ALK and explored its roles in cancer progression and therapeutic implication. Experimental Design: We screened ALK-rearranged non–small cell lung cancer (NSCLC) cases using immunohistochemistry and fluorescence in situ hybridization and then conducted multiplex gene expression analysis. We also performed a clinicopathologic analysis to validate the findings. Additional cellular experiments, including inhibition and migration assays, and in vivo lung cancer model studies were performed. Results: Among patients with ALK-rearranged NSCLC, integrin β3 (ITGB3) was one of the overexpressed genes in comparison with that in ALK-negative NSCLC (P = 0.0003). ALK and integrin β3 expression were positively correlated, and we discovered that high integrin β3 mRNA expression was associated with metastasis and more advanced tumor stages (P < 0.005; P < 0.05). Furthermore, we found that inhibition of both ALK and integrin β3 led to increased drug sensitivity in vitro and in vivo (both P < 0.05). Conclusions: We discovered a positive correlation between ALK and integrin β3 expression levels in ALK-rearranged NSCLC. Our findings suggest that high integrin β3 expression in ALK-rearranged NSCLC is associated with tumor progression and a worse prognosis. This finding demonstrates the prognostic value of integrin β3 and provides a rationale for combination treatment with ALK and integrin β3 inhibitors in patients with ALK-rearranged NSCLC. Clin Cancer Res; 24(17); 4162–74. ©2018 AACR.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by poor patient prognosis and for which no targeted therapies are currently available. TNBC can be further categorized as either basal-like (BLBC) or quintuple-negative breast cancer (QNBC). In the present study, we aimed to identify novel molecular therapeutic targets for TNBC by analyzing the mRNA expression of TNBC-related genes in publicly available microarray data sets. We found that Engrailed 1 (EN1) was significantly overexpressed in TNBC. Using breast cancer cell lines, we found that EN1 was more highly expressed in TNBC than in other breast cancer subtypes. EN1 expression was analyzed in 199 TNBC paraffin-embedded tissue samples by immunohistochemistry. EN1 protein expression was positively associated with reduced overall survival (OS) rate in patients with QNBC, but not those with BLBC. The importance of EN1 expression in QNBC cell viability and tumorigenicity was evaluated using the QNBC cell lines, HCC38 and HCC1395. Based on our data, EN1 may promote the proliferation, migration, and multinucleation of QNBC cells, likely via the transcriptional activation of HDAC8, UTP11L, and ZIC3. We also demonstrated that actinomycin D effectively inhibits EN1 activity in QNBC cells. The results of the present study suggest that EN1 activity is highly clinically relevant to the survival prognosis of patients with QNBC and EN1 is a promising potential therapeutic target for future QNBC treatment.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancers (NSCLCs) show good clinical response to ALK inhibitors. However, some ALK-rearranged NSCLC patients show various primary responses with unknown reasons. Previous studies focused on the clinical aspects of ALK fusions in small cohorts, or were conducted in vitro and/or in vivo to investigate the function of ALK. One of the suggested theories describes how echinoderm microtubule-associated protein-like 4 (EML4)-ALK variants play a role towards different sensitivities in ALK inhibitors. Until now, there has been no integrated comprehensive study that dissects ALK at the molecular level in a large scale. Here, we report the largest extensive molecular analysis of 158 ALK-rearranged NSCLCs and have investigated these findings in a cell line construct experiment. We discovered that NSCLCs with EML4-ALK short forms (variant 3/others) had more advanced stage and frequent metastases than cases with the long forms (variant 1/others) (p = 0.057, p < 0.05). In vitro experiments revealed that EML4-ALK short forms show lower sensitivity to ALK inhibitors than do long forms. Clinical analysis also showed a trend for the short forms showing worse PFS. Interestingly, we found that breakpoints of ALK are evenly distributed mainly in intron 19 and almost all of them undergo a non-homologous end-joining repair to generate ALK fusions. We also discovered four novel somatic ALK mutations in NSCLC (T1151R, R1192P, A1280V, and L1535Q) that confer primary resistance; all of them showed strong resistance to ALK inhibitors, as G1202R does. Through targeted deep sequencing, we discovered three novel ALK fusion partners (GCC2, LMO7, and PHACTR1), and different ALK fusion partners showed different intracellular localization. With our findings that the EML4-ALK variants, new ALK somatic mutations, and novel ALK-fusion partners may affect sensitivity to ALK inhibitors, we stress the importance of targeted therapy to take the ALK molecular profiling into consideration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Solitary fibrous tumors (SFTs) are NAB2-STAT6 fusion-associated neoplasms. There are several subtypes of NAB2-STAT6 fusions, but their clinical significances are still unclear. Moreover, the mechanisms of malignant progression are also poorly understood. In this study, using 91 SFT cases, we examined whether fusion variants are associated with clinicopathological parameters and also investigated the molecular mechanism of malignant transformation using whole-exome sequencing. We detected variant 1b (NAB2ex4-STAT6ex2) in 51/91 (56%) cases and variants 2a/2b (NAB2ex6-STAT6ex16/17) in 17/91 (19%) cases. The NAB2-STAT6 fusion variant types were significantly associated with their primary site (P

// Yu Jin Kim 1 , Sungyoul Hong 2 , Minjung Sung 1 , Min Jeong Park 2 , Kyungsoo Jung 1, 3 , Ka-Won Noh 1, 3 , Doo-Yi Oh 1, 3 , Mi-Sook Lee 1, 3 , Ensel Oh 1, 3 , Young Kee Shin 2, 4 , Yoon-La Choi 1, 3, 5 1 Laboratory of Cancer Genomics and Molecular Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 2 Laboratory of Molecular Pathology and Cancer Genomics, Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Korea 3 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Korea 4 The Center for Anti-cancer Companion Diagnostics, Bio-MAX/N-Bio, Seoul National University, Seoul, Korea 5 Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Correspondence to: Yoon-La Choi, email: ylachoi@skku.edu Young Kee Shin, email: ykeeshin@snu.ac.kr Keywords: LYN, dasatinib, lung adenocarcinoma subgroup, SRC, YES Received: March 16, 2016 Accepted: October 01, 2016 Published: October 14, 2016 ABSTRACT Therapies targeting SRC family kinases (SFKs) have shown efficacy in treating non-small cell lung cancer (NSCLC). However, recent clinical trials have found that the SFK inhibitor dasatinib is ineffective in some patient cohorts. Regardless, dasatinib treatment may benefit some NSCLC patient subgroups. Here, we investigated whether expression of LYN, a member of the SFK family, is associated with patient survival, the efficacy of dasatinib, and/or NSCLC cell viability. LYN expression was associated with poor overall survival in a multivariate analysis, and this association was strongest in non-smoker female patients with adenocarcinoma (ADC). In lung ADC cells, LYN expression enhanced cell proliferation, migration, and invasion. Dasatinib inhibited LYN activity and decreased cell viability in LYN-positive ADC cell lines and xenografts. Additionally, we identified the SFKs SRC and YES as candidate dasatinib targets in LYN-negative ADC cell lines. Our findings suggest that LYN is a useful prognostic marker and a selective target of dasatinib therapy in the lung ADC subpopulation especially in female non-smokers with lung ADC.

Amplification and overexpression of MDM2 and CDK4 are well-known diagnostic criteria of well-differentiated liposarcoma (WDLPS)/dedifferentiated liposarcoma (DDLPS). Although it was reported that depletion of MDM2 or CDK4 decreased proliferation in DDLPS cell lines, it remains unclear whether MDM2 and CDK4 induce WDLPS/DDLPS tumorigenesis. We examined whether MDM2 and/or CDK4 produce WDLPS/DDLPS using transformed human bone marrow stem cells (BMSCs), 2H and 5H, with five oncogenic hits (overexpression of hTERT, TP53 degradation, RB inactivation, c-MYC stabilization, and overexpression of HRASv12). In vitro functional experiments revealed that co-overexpression of MDM2 and CDK4 plays key roles in tumorigenesis by increasing cell growth and migration and inhibiting adipogenic differentiation potency compared to sole expression of MDM2 or CDK4. Using mouse xenograft models, we found that co-overexpression of MDM2 and CDK4 in 5H cells with five additional oncogenic mutations can develop proliferative DDLPS in vivo. Our results suggest that co-overexpression of MDM2 and CDK4 induces DDLPS tumour potency in transformed human BMSCs by accelerating cell growth and migration and blocking adipogenetic potential incooperation with multiple genetic factors.

Surgery and radiation are the current standard treatments for cervical cancer. However, there is no effective therapy for metastatic or recurrent cases, necessitating the identification of therapeutic targets. In order to create preclinical models for screening potential therapeutic targets, we established 14 patient-derived xenograft (PDX) models of cervical cancers using subrenal implantation methods. Serially passaged PDX tumors retained the histopathologic and genomic features of the original tumors. Among the 9 molecularly profiled cervical cancer patient samples, a HER2-amplified tumor was detected by array comparative genomic hybridization and targeted next-generation sequencing. We confirmed HER2 overexpression in the tumor and serially passaged PDX. Co-administration of trastuzumab and lapatinib in the HER2-overexpressed PDX significantly inhibited tumor growth compared to the control. Thus, we established histopathologically and genomically homologous PDX models of cervical cancer using subrenal implantation. Furthermore, we propose HER2 inhibitor-based therapy for HER2-amplified cervical cancer refractory to conventional therapy.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancers (NSCLCs) show good clinical response to ALK inhibitors. However, some ALK-rearranged NSCLC patients show various primary responses with unknown reasons. Previous studies focused on the clinical aspects of ALK fusions in small cohorts, or were conducted in vitro and/or in vivo to investigate the function of ALK. One of the suggested theories describes how echinoderm microtubule-associated protein-like 4 (EML4)-ALK variants play a role towards different sensitivities in ALK inhibitors. Until now, there has been no integrated comprehensive study that dissects ALK at the molecular level in a large scale. Here, we report the largest extensive molecular analysis of 158 ALK-rearranged NSCLCs and have investigated these findings in a cell line construct experiment. We discovered that NSCLCs with EML4-ALK short forms (variant 3/others) had more advanced stage and frequent metastases than cases with the long forms (variant 1/others) (p = 0.057, p0.05). In vitro experiments revealed that EML4-ALK short forms show lower sensitivity to ALK inhibitors than do long forms. Clinical analysis also showed a trend for the short forms showing worse PFS. Interestingly, we found that breakpoints of ALK are evenly distributed mainly in intron 19 and almost all of them undergo a non-homologous end-joining repair to generate ALK fusions. We also discovered four novel somatic ALK mutations in NSCLC (T1151R, R1192P, A1280V, and L1535Q) that confer primary resistance; all of them showed strong resistance to ALK inhibitors, as G1202R does. Through targeted deep sequencing, we discovered three novel ALK fusion partners (GCC2, LMO7, and PHACTR1), and different ALK fusion partners showed different intracellular localization. With our findings that the EML4-ALK variants, new ALK somatic mutations, and novel ALK-fusion partners may affect sensitivity to ALK inhibitors, we stress the importance of targeted therapy to take the ALK molecular profiling into consideration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

MET, a cell surface receptor for hepatocyte growth factor, is involved in the development of triple-negative/basal-like breast cancer (TNBC/BLBC). However, its utility as a therapeutic target in this subtype of breast cancer is poorly understood. To evaluate MET fully as a potential therapeutic target for TNBC/BLBC, we investigated the relationship between MET expression and clinical outcomes of patients with breast cancer and the functional effect of MET inhibition. Using automated immunohistochemistry (Ventana), we analyzed MET expression in 924 breast cancer patients with relevant clinicopathologic parameters. BLBC showed the strongest relationship with MET expression (57.5%, p

Introduction Response to mesenchymal-epithelial transition (MET) inhibitors in NSCLC with mesenchymal-epithelial transition gene ( MET ) exon 14 skipping ( MET ex14) has fueled molecular screening efforts and the search for optimal therapies. However, further work is needed to refine the clinicopathologic and prognostic implications of MET ex14 skipping. Methods Among 795 East Asian patients who underwent a surgical procedure for NSCLC, we screened 45 patients with quintuple-negative ( EGFR -negative /KRAS -negative / anaplastic lymphoma kinase gene [ ALK ]-negative /ROS1 -negative / ret proto-oncogene [ RET ]-negative) lung adenocarcinomas by using reverse-transcriptase polymerase chain reaction and found 17 patients (37.8%) with MET ex14 skipping. We also investigated the effect of small interfering RNA (siRNA) targeting skipping junction in cells with MET ex14 skipping. Results The median age of the 17 patients was 73 years. The acinar subtype was predominant (52.9%), followed by the solid subtype (35.3%). MET immunohistochemistry demonstrated 100% sensitivity and 70.4% specificity. Multivariate analyses showed that patients with MET ex14 skipping had a higher recurrence rate than those with ALK fusion (versus MET ex14 skipping) (hazard ratio = 0.283, 95% confidence interval: 0.119 – 0.670) in stage I to IIIA disease; however, the differences in overall survival were not significant after adjustment for pathologic stage ( p = 0.669). Meanwhile, siRNA decreased MET-driven signaling pathways in Hs746T cells, and combined treatment with siRNA and crizotinib inhibited cell proliferation in crizotinib-resistant H596 cells. Conclusions The prevalence of MET ex14 skipping was quite high in East Asian patients without other driver mutations in lung adenocarcinomas. MET ex14 skipping was associated with old age, the acinar or solid histologic subtype, and high MET immunohistochemical expression. The prognosis of patients with MET ex14 skipping was similar to that of patients with major driver mutations. siRNA targeting the junction of MET ex14 skipping could inhibit MET-driven signaling pathways in cells with MET ex14 skipping.

The receptor tyrosine kinase c-MET regulates essential cellular processes in tissue remodeling and homeostasis, and mammalian development. Although c-MET is an important in normal physiology, the dysregulation of c-MET signaling has been strongly implicated in tumorigenic transformation, tumor growth, angiogenesis, invasiveness, and metastasis. The aberrant activation of c-MET, particularly in tumors with gene amplification and mutations, reported in a wide variety of cancers. In approximately 10% of gastric cancer, MET is amplified and is activated in the ligand-independent pathway. MET-amplified cancer cell lines are dependent on its signaling for survival and proliferation. That being so, c-MET is attractive target and a number of clinical trials are ongoing. However, some clinical trials were failed due to inappropriate patient selection criteria such as low cut-off. To select patients who respond to c-MET inhibitor, the selection criteria by using both IHC and FISH is necessary. In this study, we determined the efficacy of ABN401 based on histopathologic and genetic analysis using cell lines and patient-derived xenograft (PDX) models for gastric cancer and other c-MET related cancers as well. The cytotoxic effect of ABN401 was evaluated in six gastric cancer cell lines and one normal fibroblast cell as control by using cell viability assay. c-MET and its downstream signaling were assessed by western blot assay. In vivo anti-tumor therapeutic efficacy of ABN401 was evaluated in two gastric cancer cell xenograft models and eight PDX models. MET copy number, c-MET protein expression and genetic aberration were estimated by diagnostic test such as IHC and FISH, NGS, and array CGH in cell lines and PDX models. Our results revealed that ABN401 showed over 90% of cytotoxicity in five gastric cancer cell lines (SNU5, SNU620, SNU638, Hs746T, MKN45) and inhibited auto-phosphorylation of the c-MET as well as its downstream signal. Except for SNU638, the other four cell lines were MET copy number > 10, MET/CEP7 > 5, and c-MET expression (IHC 3+). Moreover, ABN401 significantly suppressed tumor growth in three of eight PDX models with MET copy number >5. MET/CEP7 > 5 and c-MET expression level (IHC 3+). However, ABN401 had no effect in gastric PDX model with MET copy number 5, MET/CEP7 ratio > 5, c-MET protein level IHC 2+ and esophagus PDX model with MET copy number 6, MET/CEP7 ratio = 2.2, IHC 1+. Both in vivo and in vitro experiment suggest that MET copy number and c-MET expression level affects effectiveness of c-MET inhibitor ABN401, which alert us the importance of cut-off. Based on this study, we conclude that it would be possible to make a conjecture in future clinical trials by establishing the characteristics of each sample and find the correlation with the efficacy of c-MET inhibitor. Citation Format: Jooseok Kim, Kyungsoo Jung, Kyungeui Park, Minji Seo, Yoonla Choi, Junyoung Choi, Sungyoul Hong, Youngkee Shin. Therapeutic efficacy of ABN401, a highly potent and selective c-MET inhibitor, on the basis of diagnostic test for biomarkers in MET-amplified cancers [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 LB-194.

This study was conducted to analyze the clinical significance of follow-up diagnostic methods of atypical squamous cells (ASC) (the 2001 Bethesda System) cases according to age. A computerized search of the cytology database was performed to retrieve all cases diagnosed as ASC from 2001 to 2003. The pathologic reports for all follow-up diagnoses were reviewed. We divided the patients into two groups according to their age, younger than 50 years of age and 50 years and older, and follow-up diagnoses were compared between the two groups. ASC was identified in 1035 (2.0%) of 49,882 women screened, and a total of 914 patients were eligible. In atypical squamous cells of undetermined significance (ASC-US) cases, colposcopically directed biopsy showed CIN I (CIN is cervical intraepithelial neoplasia) or higher grade lesions in 34.9% of cases younger than 50 years of age and in 17.4% of cases 50 years and older (P= 0.000). However, repeat Pap smears and human papillomavirus DNA testing showed no differences between the two groups. In contrast, the three methods did not exhibit significant difference between the two groups in patients with atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) (P= 0.743). Colposcopically directed biopsy for the ASC-US was more useful in patients younger than 50 years of age than in those who were 50 years and older. It is suggested that age should be considered in deciding follow-up diagnostic methods in patients with ASC-US.

// Sang Yun Ha 1, * , So-Jung Choi 2, * , Jong Ho Cho 3, * , Hye Joo Choi 2 , Jinseon Lee 2 , Kyungsoo Jung 4 , Darry Irwin 5 , Xiao Liu 6, 7 , Maruja E. Lira 8 , Mao Mao 9 , Hong Kwan Kim 3 , Yong Soo Choi 3 , Young Mog Shim 3 , Woong Yang Park 10 , Yoon-La Choi 1, 4, 10 , Jhingook Kim 2, 3 1 Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 2 Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 3 Department of Thoracic and Cardiovascular Surgery, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea 4 Samsung Advanced Institute for Health Sciences & Technology, Sungkyunkwan University School of Medicine, Seoul, Korea 5 Agena Bioscience, Sequenom, San Diego, CA, USA 6 BGI-Shenzhen, Shenzhen, China 7 Department of Biology, University of Copenhagen, Copenhagen, Denmark 8 Oncoloy Research Unit, Pfizer Worldwide Research and Development, San Diego, CA, USA 9 WuXi AppTec, Shanghai, China 10 Samsung Genome Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea * These authors contributed equally to this work Correspondence to: Jhingook Kim, e-mail: jhingookkim@gmail.com Yoon-La Choi, e-mail: ylachoi@skku.edu Keywords: non-small cell lung cancer, adenocarcinoma, never-smoker female, driver mutation, EGFR Received: August 07, 2014 Accepted: December 16, 2014 Published: March 09, 2015 ABSTRACT The aim of this study was to determine the distribution of known oncogenic driver mutations in female never-smoker Asian patients with lung adenocarcinoma. We analyzed 214 mutations across 26 lung cancer-associated genes and three fusion genes using the MassARRAY® LungCarta Panel and the ALK, ROS1 , and RET fusion assays in 198 consecutively resected lung adenocarcinomas from never-smoker females at a single institution. EGFR mutation, which was the most frequent driver gene mutation, was detected in 124 (63%) cases. Mutation of ALK, KRAS, PIK3CA, ERBB2, BRAF, ROS1, and RET genesoccurred in 7% , 4%, 2.5%, 1.5%, 1%, 1%, and 1% of cases, respectively. Thus, 79% of lung adenocarcinomas from never-smoker females harbored well-known oncogenic mutations. Mucinous adenocarcinomas tended to have a lower frequency of known driver gene mutations than other histologic subtypes. EGFR mutation was associated with older age and a predominantly acinar pattern, while ALK rearrangement was associated with younger age and a predominantly solid pattern. Lung cancer in never-smoker Asian females is a distinct entity, with the majority of these cancers developing from oncogenic mutations.

The success of crizotinib in ALK-positive patients has elicited efforts to find new oncogenic fusions in lung cancer. These efforts have led to the discovery of novel oncogenic fusion genes such as ROS1 and RET. However, the molecular and clinicopathologic characteristics associated with RET or ROS1 fusion, compared with ALK fusion-positive lung cancer, remain unclear. We accordingly analyzed the clinicopathologic characteristics of RET- and ROS1-fusion-positive lung adenocarcinomas. We further performed immunohistochemistry and fluorescence in situ hybridization analysis (FISH) in 15 cases of RET and 9 cases of ROS1 fusion tumors by identified NanoString's nCounter screening. RET fusion-positive patients were younger in age, never-smokers, and in early T stage; ROS1 fusion-positive patients had a higher number of never-smokers compared with patients with quintuple-negative (EGFR-/KRAS-/ALK-/ROS1-/RET-) lung adenocarcinoma. Histologically, RET and ROS1 fusion tumors share the solid signet-ring cell and mucinous cribriform pattern, as previously mentioned in the histology of ALK fusion tumors. Therefore, it can be presumed that fusion gene-associated lung adenocarcinomas share similar histologic features. In immunohistochemistry, the majority of 15 RET and 9 ROS1 fusion-positive cases showed positivity of more than moderate intensity and cytoplasmic staining for RET and ROS1 proteins, respectively. In FISH, the majority of RET and ROS1 rearrangement showed two signal patterns such as one fusion signal and two separated green and orange signals (1F1G1O) and an isolated 3' green signal pattern (1F1G). Our study has provided not only characteristics of fusion gene-associated histologic features but also a proposal for a future screening strategy that will enable clinicians to select cases needed to be checked for ROS1 and RET rearrangements based on clinicohistologic features.

MET, a cell surface receptor for hepatocyte growth factor, is involved in the development of triple-negative/basal-like breast cancer (TNBC/BLBC). However, its utility as a therapeutic target in this subtype of breast cancer is poorly understood. To evaluate MET fully as a potential therapeutic target for TNBC/BLBC, we investigated the relationship between MET expression and clinical outcomes of patients with breast cancer and the functional effect of MET inhibition. Using automated immunohistochemistry (Ventana), we analyzed MET expression in 924 breast cancer patients with relevant clinicopathologic parameters. BLBC showed the strongest relationship with MET expression (57.5%, p 0.001). High expression of MET in breast cancer resulted in poor overall survival (p = 0.001) and disease-free survival (DFS, p = 0.010). MET expression was relatively high in TNBC cell lines, and the silencing of MET via small interfering RNA reduced cell proliferation and migration. We observed reduced TNBC cell viability after treatment with the MET inhibitor PHA-665752. In the most drug-resistant cell line, MDA-MB-468, which showed elevated epidermal growth factor receptor (EGFR) expression, silencing of EGFR resulted in increased sensitivity to PHA-665752 treatment. We confirmed that PHA-665752 synergizes with the EGFR inhibitor erlotinib to decrease the viability of MDA-MB-468 cells. TNBC patients coexpressing MET and EGFR showed significantly worse DFS than that in patients expressing EGFR alone (p = 0.021). Our findings strongly suggest that MET may be a therapeutic target in TNBC and that the combined therapy targeting MET and EGFR may be beneficial for the treatment of TNBC/BLBC patients.

The paper presents the design of a wide-band active-RC filter with a fast tuning circuit for wireless communication receiver applications. The filter topology is the 5/sup th/-order Chebyshev-II lowpass filter type and the programmable bandwidth can be extended up to 10MHz while the stopband attenuation larger than 40dB is obtained. The successive approximation register (SAR) scheme is incorporated for a prompt on-chip tuning operation that should be needed for compensating RC variations. The filter is fabricated in a 0.18-/spl mu/m standard digital CMOS technology and dissipates 20.7mW for a supply voltage of 1.8V. The measured 3/sup rd/-order harmonic input intercept point (IIP3) is larger than 32dBm.

This paper presents a high efficiency CMOS DC-DC boost converter with current sensing feedback. The internal 500 kHz oscillator for switching operation is used to make set control signal in pulse-width modulation topology. To sense the amount of current through the inductor, the high accuracy current copy circuit using operational amplifier is included. The load regulation is 11.5%/A with compensation scheme that increases the feedback loop gain and accuracy of the output voltage. To guarantee the stability of the converter, the artificial ramp signal is used. The measurement results show that the efficiency is up to 90.7% with 2.2 V input and 220 mA load current for 3.3V output. This converter is fabricated in 0.18-mum CMOS standard technology with thick gate oxide option

This paper presents a fast tuning scheme for active-RC filters of monolithic implementation. Due to the significant variations of passive resistor and capacitor values, it should be necessary for the filter to be equipped with the on-chip tuning circuitry. In this paper, the successive approximation register (SAR) scheme is incorporated for a prompt compensation of RC variations. The tuning time can be proportional to the number of bits in the tuning resolution scheme. The propose tuning scheme is included in implementation of the 5th-order Chebyshev-II lowpass filter, where 5-bit is used to achieve less than 2.5% cutoff frequency error with respect to RC time constant variations of plusmn40%

Triple-negative breast cancer (TNBC)/basal-like breast cancer (BLBC) are known as poor outcome subtypes with deficiency of targeted molecular therapy. Previous studies showed that MET may play a critical role in the development of TNBC and may be a rational therapeutic target. In order to evaluate MET as a potential therapeutic target for TNBC, we investigated genomic aberration and expression status of MET and evaluated efficacy of MET inhibitor, PHA-665752 in TNBC cell lines. MET protein expression was relatively high in TNBC cell lines. The depletion of MET using small interfering RNA (siRNA) led decreased cell proliferation and migration. PHA-665752 treatment resulted in decreased cell viability, and most of these cell lines were found to be moderately sensitive in their response to PHA-665752. The most resistant cell line, MDA-MB-468, showed exclusively high expression of EGFR, and then EGFR depletion using siRNA induced increased sensitivity to PHA-665752. In our cohort, BLBC was the subtype showing the highest relationship with MET expression among 5 subtypes (77/134, 57.5%, P Citation Format: Yu Jin Kim, Jong-Sun Choi, Ji-Young Song, Kyungsoo Jung, Ensel Oh, Young Kee Shin, Yoon-La Choi. MET is a potential therapeutic target and a candidate for combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2013-4391