c-MET-positive circulating tumor cells and cell-free DNA as independent prognostic factors in hormone receptor-positive/HER2-negative metastatic breast cancer.

BACKGROUND: Endocrine therapy resistance in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) is a significant clinical challenge that poses several unmet needs in the management of the disease. This study aimed to investigate the prognostic value of c-MET-positive circulating tumor cells (cMET+ CTCs), ESR1/PIK3CA mutations, and cell-free DNA (cfDNA) concentrations in patients with hormone receptor-positive (HR+) metastatic breast cancer (mBC). METHODS: Ninety-seven patients with HR+ mBC were prospectively enrolled during standard treatment at Samsung Medical Center. CTCs were isolated from blood using GenoCTC® and EpCAM or c-MET CTC isolation kits. PIK3CA and ESR1 hotspot mutations were analyzed using droplet digital PCR. CfDNA concentrations were calculated using internal control copies from the ESR1 mutation test. Immunocytochemistry was performed to compare c-MET overexpression between primary and metastatic sites. RESULTS: The proportion of c-MET overexpression was significantly higher in metastatic sites than in primary sites (p = 0.00002). Survival analysis showed that c-MET+ CTC, cfDNA concentration, and ESR1 mutations were significantly associated with poor prognosis (p = 0.0026, 0.0021, and 0.0064, respectively) in HR+/HER2- mBC. By contrast, EpCAM-positive CTC (EpCAM+ CTC) and PIK3CA mutations were not associated with progression-free survival (PFS) in HR+/HER2- mBC. Multivariate analyses revealed that c-MET+ CTCs and cfDNA concentration were independent predictors of PFS in HR+/HER2- mBC. CONCLUSIONS: Monitoring c-MET+ CTC, rather than assessing c-MET expression in the primary BC site, could provide valuable information for predicting disease progression, as c-MET expression can change during treatment. The c-MET+ CTC count and cfDNA concentration could provide complementary information on disease progression in HR+ /HER2- mBC, highlighting the importance of integrated liquid biopsy.

Cancer-Specific Sequences in the Diagnosis and Treatment of NUT Carcinoma.

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.

Analysis of PIK3CA Mutation Concordance and Frequency in Primary and Different Distant Metastatic Sites in Breast Cancer.

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.

TM4SF4 and LRRK2 Are Potential Therapeutic Targets in Lung and Breast Cancers through Outlier Analysis.

PURPOSE: To 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 METHODS: Modified 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. RESULTS: TM4SF4 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 lines. CONCLUSION: Our 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.

Therapeutic Efficacy of ABN401, a Highly Potent and Selective MET Inhibitor, Based on Diagnostic Biomarker Test in MET-Addicted Cancer.

The receptor tyrosine kinase c-MET regulates processes essential for tissue remodeling and mammalian development. The dysregulation of c-MET signaling plays a role in tumorigenesis. The aberrant activation of c-MET, such as that caused by gene amplification or mutations, is associated with many cancers. c-MET is therefore an attractive therapeutic target, and inhibitors are being tested in clinical trials. However, inappropriate patient selection criteria, such as low amplification or expression level cut-off values, have led to the failure of clinical trials. To include patients who respond to MET inhibitors, the selection criteria must include MET oncogenic addiction. Here, the efficacy of ABN401, a MET inhibitor, was investigated using histopathologic and genetic analyses in MET-addicted cancer cell lines and xenograft models. ABN401 was highly selective for 571 kinases, and it inhibited c-MET activity and its downstream signaling pathway. We performed pharmacokinetic profiling of ABN401 and defined the dose and treatment duration of ABN401 required to inhibit c-MET phosphorylation in xenograft models. The results show that the efficacy of ABN401 is associated with MET status and they highlight the importance of determining the cut-off values. The results suggest that clinical trials need to establish the characteristics of each sample and their correlations with the efficacy of MET inhibitors.

IRS2 Amplification as a Predictive Biomarker in Response to Ceritinib in Small Cell Lung Cancer.

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.

Molecular changes in solitary fibrous tumor progression.

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 < 0.001). In addition, a TERT promoter mutation was detected in 7/73 (10%) cases, and it showed a significant association with malignant SFTs (P = 0.003). To identify molecular changes during malignant progression, we selected an index patient to obtain parallel tissue samples from the primary and metastatic tumors. In the metastatic tissue, 10 unique molecular alterations, including those in TP53 and APAF1, were detected. In vitro functional experiments showed that APAF1 depletion increased the tumor potency of cells expressing NAB2-STAT6 fusion protein under treatment with staurosporine. We found that TP53 immunopositivity (P = 0.006) and loss of APAF1 immunoreactivity (P < 0.001) were significantly associated with malignant SFTs. Our study suggests that dysfunction of TP53 and APAF1 leads to impaired apoptotic function, and eventually contributes toward malignant SFT transformation. KEY MESSAGES: We firstly found that the TERT promoter mutation was strongly associated with malignant SFTs (P = 0.003) and the representative 1b (NAB2ex4-STAT6ex2) or 2a (NAB2ex6-STAT6ex16) fusion variants similarly contribute to tumorigenicity. We also found that TP53 immunopositivity (P = 0.006) and loss of APAF1 immunoreactivity (P < 0.001) were significantly associated with malignant SFTs. Our study suggests that dysfunction of TP53 and APAF1 leads to impaired apoptotic function, and eventually contributes toward malignant SFT transformation.

Co-expression of MDM2 and CDK4 in transformed human mesenchymal stem cells causes high-grade sarcoma with a dedifferentiated liposarcoma-like morphology.

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.

Integrin ß3 Inhibition Enhances the Antitumor Activity of ALK Inhibitor in ALK-Rearranged NSCLC.

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 ß3 (ITGB3) was one of the overexpressed genes in comparison with that in ALK-negative NSCLC (P = 0.0003). ALK and integrin ß3 expression were positively correlated, and we discovered that high integrin ß3 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 ß3 led to increased drug sensitivity in vitro and in vivo (both P < 0.05).Conclusions: We discovered a positive correlation between ALK and integrin ß3 expression levels in ALK-rearranged NSCLC. Our findings suggest that high integrin ß3 expression in ALK-rearranged NSCLC is associated with tumor progression and a worse prognosis. This finding demonstrates the prognostic value of integrin ß3 and provides a rationale for combination treatment with ALK and integrin ß3 inhibitors in patients with ALK-rearranged NSCLC. Clin Cancer Res; 24(17); 4162-74. ©2018 AACR.

Engrailed 1 overexpression as a potential prognostic marker in quintuple-negative breast cancer.

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.

Precision medicine approaches to lung adenocarcinoma with concomitant MET and HER2 amplification.

BACKGROUND: Patient-derived xenograft (PDX) models are important tools in precision medicine and for the development of targeted therapies to treat cancer patients. This study aimed to evaluate our precision medicine strategy that integrates genomic profiling and preclinical drug-screening platforms, in order to personalize cancer treatments using PDX models. METHODS: We performed array-comparative genomic hybridization, microarray, and targeted next-generation sequencing analyses, in order to determine the oncogenic driver mutations. PDX cells were obtained from PDXs and subsequently screened in vitro with 17 targeted agents. RESULTS: PDX tumors recapitulated the histopathologic and genetic features of the patient tumors. Among the samples from lung cancer patients that were molecularly-profiled, copy number analysis identified unique focal MET amplification in one sample, 033 T, without RTK/RAS/RAF oncogene mutations. Although HER2 amplification in 033 T was not detected in the cancer panel, the selection of HER2-amplified clones was found in PDXs and PDX cells. Additionally, MET and HER2 overexpression were found in patient tumors, PDXs, and PDX cells. Crizotinib or EGFR tyrosine kinase inhibitor treatments significantly inhibited cell growth and impaired tumor sphere formation in 033 T PDX cells. CONCLUSIONS: We established PDX cell models using surgical samples from lung cancer patients, and investigated their preclinical and clinical implications for personalized targeted therapy. Additionally, we suggest that MET and EGFR inhibitor-based therapy can be used to treat MET and HER2-overexpressing lung cancers, without receptor tyrosine kinase /RAS/RAF pathway alterations.

Molecular breakdown: a comprehensive view of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer.

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.

MET Exon 14 Skipping Mutations in Lung Adenocarcinoma: Clinicopathologic Implications and Prognostic Values.

INTRODUCTION: Response to mesenchymal-epithelial transition (MET) inhibitors in NSCLC with mesenchymal-epithelial transition gene (MET) exon 14 skipping (METex14) has fueled molecular screening efforts and the search for optimal therapies. However, further work is needed to refine the clinicopathologic and prognostic implications of METex14 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 METex14 skipping. We also investigated the effect of small interfering RNA (siRNA) targeting skipping junction in cells with METex14 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 METex14 skipping had a higher recurrence rate than those with ALK fusion (versus METex14 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 METex14 skipping was quite high in East Asian patients without other driver mutations in lung adenocarcinomas. METex14 skipping was associated with old age, the acinar or solid histologic subtype, and high MET immunohistochemical expression. The prognosis of patients with METex14 skipping was similar to that of patients with major driver mutations. siRNA targeting the junction of METex14 skipping could inhibit MET-driven signaling pathways in cells with METex14 skipping.

LYN expression predicts the response to dasatinib in a subpopulation of lung adenocarcinoma patients.

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.

Phenylethanolamine N-methyltransferase downregulation is associated with malignant pheochromocytoma/paraganglioma.

Malignant pheochromocytoma/paraganglioma (PCC/PGL) is defined by the presence of metastases at non-chromaffin sites, which makes it difficult to prospectively diagnose malignancy. Here, we performed array CGH (aCGH) and paired gene expression profiling of fresh, frozen PCC/PGL samples (n = 12), including three malignant tumors, to identify genes that distinguish benign from malignant tumors. Most PCC/PGL cases showed few copy number aberrations, regardless of malignancy status, but mRNA analysis revealed that 390 genes were differentially expressed in benign and malignant tumors. Expression of the enzyme, phenylethanolamine N-methyltransferase (PNMT), which catalyzes the methylation of norepinephrine to epinephrine, was significantly lower in malignant PCC/PGL as compared to benign samples. In 62 additional samples, we confirmed that PNMT mRNA and protein levels were decreased in malignant PCC/PGL using quantitative real-time polymerase chain reaction and immunohistochemistry. The present study demonstrates that PNMT downregulation correlates with malignancy in PCC/PGL and identifies PNMT as one of the most differentially expressed genes between malignant and benign tumors.

HER2 as a novel therapeutic target for cervical cancer.

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.

Lung cancer in never-smoker Asian females is driven by oncogenic mutations, most often involving EGFR.

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.

Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.

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 is a potential target for use in combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer.

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.