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Some patients experience mixed response to immunotherapy, whose biological mechanisms and clinical impact have been obscure. We obtained two tumor samples from lymph node (LN) metastatic lesions in a same patient. Whole exome sequencing for the both tumors and single-cell sequencing for the both tumor-infiltrating lymphocytes (TIL) demonstrated a significant difference in tumor clonality and TILs’ characteristics, especially exhausted T-cell clonotypes, although a close relationship between the tumor cell and T-cell clones were observed as a response of an overlapped exhausted T-cell clone to an overlapped neoantigen. To mimic the clinical setting, we generated a mouse model of several clones from a same tumor cell line. Similarly, differential tumor clones harbored distinct TILs, and one responded to programmed cell death protein 1 (PD-1) blockade but the other did not in this model. We further conducted cohort study (n = 503) treated with PD-1 blockade monotherapies to investigate the outcome of mixed response. Patients with mixed responses to PD-1 blockade had a poor prognosis in our cohort. Particularly, there were significant differences in both tumor and T-cell clones between the primary and LN lesions in a patient who experienced tumor response to anti–PD-1 mAb followed by disease progression in only LN metastasis. Our results underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome. Significance: Several patients experience mixed responses to immunotherapies, but the biological mechanisms and clinical significance remain unclear. Our results from clinical and mouse studies underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome.

Additional in vivo data using MC-38 cells.

Predicted neoantigen candidates with strong binding (%Rank < 0.5).

Representative photos of mice injected at different sites.

Additional in vitro data using the MC-38 clones.

Clinical course of a gastric cancer patient, IHC, and sequencing data.

Some patients experience mixed response to immunotherapy, whose biological mechanisms and clinical impact have been obscure. We obtained two tumor samples from lymph node (LN) metastatic lesions in a same patient. Whole exome sequencing for the both tumors and single-cell sequencing for the both tumor-infiltrating lymphocytes (TIL) demonstrated a significant difference in tumor clonality and TILs’ characteristics, especially exhausted T-cell clonotypes, although a close relationship between the tumor cell and T-cell clones were observed as a response of an overlapped exhausted T-cell clone to an overlapped neoantigen. To mimic the clinical setting, we generated a mouse model of several clones from a same tumor cell line. Similarly, differential tumor clones harbored distinct TILs, and one responded to programmed cell death protein 1 (PD-1) blockade but the other did not in this model. We further conducted cohort study (n = 503) treated with PD-1 blockade monotherapies to investigate the outcome of mixed response. Patients with mixed responses to PD-1 blockade had a poor prognosis in our cohort. Particularly, there were significant differences in both tumor and T-cell clones between the primary and LN lesions in a patient who experienced tumor response to anti–PD-1 mAb followed by disease progression in only LN metastasis. Our results underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome.Significance:Several patients experience mixed responses to immunotherapies, but the biological mechanisms and clinical significance remain unclear. Our results from clinical and mouse studies underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome.

Gastric cancer patient characteristics.

Additional analyses of scRNA-seq.

NSCLC patient characteristics.

Comparison of RNA-seq.

Peptide assay.

Sequencing summary.

Melanoma patient characteristics.

PDF file - 785K, Supplementary Figure 1. Expression of total BIM and ratio of BIM isoforms in NSCLC cell lines. Supplementary Figure 2. Flow cytometric analysis showing that EGFR mutated cell lines carrying the BIM polymorphism have low susceptibility to gefitinib-induced apoptosis. Supplementary Figure 3. Vorinostat-induced up-regulation of BIM enhances apoptosis in the HCC2279 cell line with the BIM polymorphism.Supplementary Figure 4. Knockdown or overexpression of BIM affects activation of caspase-3/7 induced by gefitinib and vorinostat in EGFR mutant cells with BIM polymorphism. Supplementary Figure 5. Detection of BIM polymorphism in human peripheral blood mononuclear cells (PBMC) and serum. Supplementary Table 1. Status of EGFR mutation and BIM polymorphism in NSCLC cell lines.

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Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets. [Cancer Res 2009;69(23):9073–82]

BIM (BCL2L11) is a BH3-only proapoptotic member of the Bcl-2 protein family. BIM upregulation is required for apoptosis induction by EGF receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) in EGFR-mutant forms of non–small cell lung cancer (NSCLC). Notably, a BIM deletion polymorphism occurs naturally in 12.9% of East Asian individuals, impairing the generation of the proapoptotic isoform required for the EGFR-TKIs gefitinib and erlotinib and therefore conferring an inherent drug-resistant phenotype. Indeed, patients with NSCLC, who harbored this host BIM polymorphism, exhibited significantly inferior responses to EGFR-TKI treatment than individuals lacking this polymorphism. In an attempt to correct this response defect in the resistant group, we investigated whether the histone deacetylase (HDAC) inhibitor vorinostat could circumvent EGFR-TKI resistance in EGFR-mutant NSCLC cell lines that also harbored the BIM polymorphism. Consistent with our clinical observations, we found that such cells were much less sensitive to gefitinib-induced apoptosis than EGFR-mutant cells, which did not harbor the polymorphism. Notably, vorinostat increased expression in a dose-dependent manner of the proapoptotic BH3 domain-containing isoform of BIM, which was sufficient to restore gefitinib death sensitivity in the EGFR mutant, EGFR-TKI–resistant cells. In xenograft models, while gefitinib induced marked regression via apoptosis of tumors without the BIM polymorphism, its combination with vorinostat was needed to induce marked regression of tumors with the BIM polymorphism in the same manner. Together, our results show how HDAC inhibition can epigenetically restore BIM function and death sensitivity of EGFR-TKI in cases of EGFR-mutant NSCLC where resistance to EGFR-TKI is associated with a common BIM polymorphism. Cancer Res; 73(8); 2428–34. ©2013 AACR.

Supplementary Figures 1-3, Tables 1-5 from Epigenetic Profiles Distinguish Malignant Pleural Mesothelioma from Lung Adenocarcinoma

Background: Early-stage lung adenocarcinoma (LUAD) patients have substantial risk for recurrence and disease-related death. Cisplatin-based adjuvant chemotherapy remains the standard for care of LUAD patients who have undergone surgical resection with a high risk of recurrence. However, adjuvant chemotherapy is associated with increased risk of toxicity including chemotherapy-related death, with only a modest survival benefit. Therefore, there is an unmet need of biomarkers for assessment and identification of those in an early stage who would likely benefit from adjuvant chemotherapy. Circulating miRNAs are stably present in blood and potentially reflect different expressions in cancerous and non-cancerous tissues, making them attractive biomarkers. The purpose of this study was to identify circulating miRNAs useful for predicting recurrence in early-stage LUAD. Materials and Methods: miRNA microarray analysis was performed with pooled pretreatment plasma samples from stage I LUAD patients who developed recurrence within two years after curative surgery or remained recurrence free over a six-year follow-up period, as well as from healthy controls. miRNA biomarker candidates were assayed in two independent plasma sample sets from 85 stage I LUAD (validation set) and from 57 stage I and II LUAD (test set) patients. Results: Based on miRNA microarray data and previous reports, predictive performance of miR-23a-3p, miR-23b-3p, miR-191-5p, miR-185-5p, miR-151a-3p, miR-320c, miR-21-5p, miR-125b-5p, miR-30d-5p, and miR-197-3p was evaluated in the validation set. Plasma levels of miR-23a-3p, miR-185-5p, miR-320c, miR-21-5p, miR-125b-5p, miR-30d-5p, and miR-197-3p were significantly higher in those with recurrence as compared to those without. A miRNA panel comprised of miR-23a-3p, miR-320c, and miR-125b-5p was developed based on a logistic regression, with yielding an AUC of 0.776 (95% confidence interval [CI] = 0.660 to 0.893). The three-miRNA panel with fixed coefficients yielded an AUC of 0.804 (95% CI = 0.688 to 0.920) with a sensitivity of 45.8% at 95% specificity in the test set. The miRNA panel score was a significant and independent factor for predicting disease-free survival (DFS; P < 0.001, HR = 1.64, 95% CI = 1.51-4.22) and overall survival (OS; P = 0.001, HR = 1.51, 95% CI = 1.17-1.94). Conclusion: This circulating miRNA panel may serve as a noninvasive blood test for predicting DFS and OS in early-stage LUAD patients. Our findings provide rationale for further investigation to stratify early-stage LUAD patients using blood-based biomarkers to increase the ability to provide more personalized care. Citation Format: Mei-Chee Tai, Leonidas E. Bantis, Gargy Parhy, Taketo Kato, Ichidai Tanaka, Chi-Wan Chow, Junya Fujimoto, Carmen Behrens, Tetsunari Hase, Koji Kawaguchi, Johannes F. Fahrmann, Edwin Ostrin, Kohei Yokoi, Toyofumi F. Chen-Yoshikawa, Yoshinori Hasegawa, Samir M. Hanash, Ignacio I. Wistuba, Ayumu Taguchi. A circulating microRNA panel predicts recurrence and survival in early-stage lung adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3744.

Background: It is well known that the prognosis of non pCR TNBC patients was poor after anthracycline and taxan treatment. For such patients, capecitabine seems to be effective to reduce recurrence based on the HR 0.58 of the CREATE X trial (Masuda, N. et al. Adjuvant Capecitabine for Breast Cancer after Preoperative Chemotherapy. N Engl J Med. 376, 2147. 2017) . However, the target of capecitabine is still unclear for TNBC. We classified non pCR tumors as BRCAness and Sporadic using BRCAness test(MRC-Holland, Amsterdam, the Netherlands). The recurrence rate of the BRCAness group was about 70%. Carboplatine is expected to be effective against BRCAness tumors, as it is a DNA damaging agent. In this study BRCAness can be checked just before carboplatin treatment using surgical specimens. Then the efficacy of carboplatin will be directly known to make comparison between DFS in the carboplatin group and that of the observation group. Trial design: This is anopen label, randomized phase III study that will enroll TNBC with residual invasive cancer after surgery with preoperative chemotherapy including both anthracycrine and taxan. Patients are randomly assigned to either the carboplatin group or observation group. The patients in the carboplatin group are treated with carboplatin at AUC 6 and those in the observation group are observed at only 3 years. Eligibility criteria: 1) ER and PgR 2) Preoperative chemotherapy including both anthracycrine and taxan. 3) Residual invasive cancer on breast tumors or lymph node metastasis in surgical specimens. 4) 20-79 year old women. 5) No chemotherapy within 5 years. 6) Not bilateral breast cancer, without metastasis, no prior breast cancer. 7) No severe bone marrow suppression. Specific aims:Primary objective is DFS (Disease Free Survival). Secondary objectives are overall survival and safety. STATISTICAL METHODS: The 3 years recurrence rate of the observation group was estimated as 40% and hazard ratio at 0.58 based on the CREATE X trial. For both groups, 135 patients are necessary. This study is powered to approximately 80% to test the superiority of carboplatin group at a 2-sided α=0.05 using a stratified log-rank test. Activation Date:22ndMarch 2018. No patients had been enrolled till 3rd July. Citation Format: Tanino H, Suzuki M, Kaise H, Miyashita M, Chishima T, Hayashi M, Miyoshi Y, Futamura M, Ohtani S, Nagahashi M, Ohta T, Kosaka Y, Ishikawa T, Hasegawa Y, Kubota T, Sangai T, Iwatani T, Yamada A, Akazawa K, Kohno N. Phase 3 trial of carboplatin in triple negative breast cancer (TNBC) patients with residual invasive carcinoma after neoadjuvant chemotherapy (JONIE4:J-CAT trial) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-05-04.

BACKGROUND: Anthracycline-containing regimens are standard treatment options in adjuvant and neoadjuvant chemotherapy in breast cancer. Chemotherapy-induced nausea and vomiting (CINV) is experienced frequently in patients receiving these regimens, but the risk factors for CINV are unknown. OBJECTIVE: The aim of this study was to investigate risk factors for CINV in anthracycline-containing regimens retrospectively. METHODS: Data were collected from the JONIE study, which was conducted in order to estimate the efficacy of zoledronic acid in a neoadjuvant setting from March 2010 to June 2012 (UMIN000003261). A total of 180 patients were recruited, and we used CINV data from the first cycle of FEC100 treatment and patient backgrounds. As the protocol regulation allowed the use of antiemetic drugs,in the first cycle of the FEC100 regimen, patients received various types of antiemetic agents, which we classified into four groups: Dexamethasone (DEX)+5-HT3 receptor antagonist (5-HT3)+neurokinin-1 receptor antagonist (NK1) (DEX+5-HT3+NK1) group; Dexamethasone (DEX)+5-HT3 receptor antagonist (5-HT3) (DEX+5HT3) group; Dexamethasone (DEX)+5-HT3 receptor antagonist (5-HT3)+dopamine receptor antagonist (DRA) (DEX+5HT3+DRA) group; and Dexamethasone (DEX)+5-HT3 receptor antagonist (5-HT3)+neurokinin-1 receptor antagonist (NK1)+ dopamine receptor antagonist (DRA) (DEX+5-HT3+NK1+DRA) group. Risk factors were selected from patient backgrounds and the combinations of antiemetic drugs. In patient backgrounds, the body mass index (BMI) was stratified into 3 categories: Less than 18.5 (underweight group); equal to or more than 18.5 but less than 25 (standard BMI group); and equal to or more than 25 (overweight group). The risks for CINV were analyzed by univariate and multivariate analyses. P values of less than 0.05 were defined as significant. RESULTS: In a univariate analysis of nausea, the body mass index (BMI) was the only significant factor (P CONCLUSIONS: This study revealed that BMI was the most important risk factor for nausea, and that BMI and the combination of antiemetic drugs were risk factors for vomiting. Underweight-patients tend to have CINV in anthracycline-containing regimen. The DEX+5-HT3+NK1 group was the best antiemetic drug combination. These result show that following the CINV guideline treatment is mandatory in order to prevent CINV. Citation Format: Hayashi M, Nakazawa K, Hasegawa Y, Horiguchi J, Miura D, Ishikawa T, Takao S, Kim SJ, Yamagami K, Miyashita M, Konishi M, Shigeoka Y, Suzuki M, Taguchi T, Kubota T, Tanino Y, Yamada K, Kimura K, Akazawa K, Kohno N. Risk analysis for chemotherapy induced nausea and vomiting (CINV) in patients receiving FEC100 treatment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-11-07.

Background: Neoadjuvant chemotherapy (NAC) for breast cancer has been improved and pathological complete response (pCR) achieved in about 50% of Her2 positive tumor and over 30% of triple negative tumor. It is possible that not to perform surgery if pCR is diagnosed accurately. This study was performed to examine diagnostic ability of core needle biopsy (CNB) to detect pCR by NAC. (Study registry number: UMIN000012035) Methods: In this multicenter study, we registered histologically proved breast cancer patients who were diagnosed as clinical complete response (cCR) after NAC. The cCR was diagnosed by contrast-enhanced magnetic resonance imaging (MRI) after NAC by radiologists in each facility. Regimens of NAC were not specified by study protocol. A clip marker was not placed in the tumor before NAC. At the operating table, ultrasound-guided CNB is performed before starting the surgery. The CNB was performed with 14 gauge needle without vacuum assistance and requiring three biopsy specimens. The concordance of pathological results between a core needle biopsy and a surgical specimen is examined by pathologists at each facility. The pathological diagnosis was categorized as i) no carcinoma (pCR), ii) carcinoma in situ (CIS) and iii) invasive carcinoma. Results: The study included 86 women from 10 facilities, accrued from December 2013 to March 2017. Median age was 53.5 (31-75) years and median tumor size before NAC was 2.3 (0.9-7.3) cm. Estrogen receptor was positive in 32 (37%). HER2 was positive in 40 (47%). The clinical stage before NAC was stage I 19 (22%), II 48 (56%), III 16 (19%) and IV 3 (3%). All cases were administered cytotoxic agents. Both anthracycline and taxane were given in 70 cases (81%), and trastuzumab was administered in 35 (41%) cases. As for breast surgery, partial resection was performed in 53 cases (62%), whereas 33 cases (38%) underwent mastectomy. Pathological examination on surgical specimen revealed pCR (i) in 41 cases (48%), CIS (ii) in 17 cases (20%) and invasive carcinoma (iii) in 28 cases (32%). Results of pathology of CNB and surgical specimens are shown in table 1. Table 1. Pathological diagnosis of CNB and surgical specimens Surgical specimen No carcinoma (pCR)Residual carcinomaTotalCNBNo carcinoma (pCR)41 (NPV=63%, spec.=100%)24 (FNR=53%, 14 were CIS)65 (76%) Residual carcinoma021 (PPV=100%, sens.=47%)21 (24%)Total41 (48%)45 (52%)86 (100%)CNB: core needle biopsy, FNR: false-negative rate, CIS: carcinoma in situ, NPV: negative predictive value, PPV: positive predictive value, sens.: sensitivity, spec.: specificity. The kappa value was 0.455 which was not enough for accurate diagnosis. In 24 discordant cases, residual tumor was found in surgical specimen but not in CNB, residual disease was CIS in 14 cases. Conclusions: Up to 10% false negative rate of pCR may allow us to proceed to an observational study without performing surgery.This study revealed that the ultrasound-guided CNB for cCR cases by MRI was not accurate enough to omit surgery. Thus, imaging diagnosis and biopsy methods need to be improved. However, it is still possible to consider because the post-operative radiotherapy may compensate for surgery in cases with CIS after NAC. Citation Format: Narui K, Ishikawa T, Hasegawa Y, Kaise H, Kawate T, Yamada K, Suzuki Y, Niikura N, Kohno N, Kimoto T, Sugae S, Yamada A, Kosaka Y, Miyashita M, Okamura T, Shimizu D, Tanino H, Sakamaki K, Tokuda Y. Feasibility study to diagnose pathological complete response by neoadjuvant chemotherapy in breast cancer adding core needle biopsy (KBOG1301 supported by JONIE) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-22-09.

Background: Although treatment of eribulin mesylate (E) improved overall survival in metastatic breast cancer (BC) patients, little is known about the efficacy in early BC. The hypothesis of this study is that sequential administration of E followed by FEC would have less toxic, particularly peripheral neuropathy, and also have similar effect compared to paclitaxel (P) followed by FEC as primary systemic therapy (PST) for woman with operable BC. Methods: This is a phase II multicenter open label study (UMIN000012817). Patients (pts) were randomly assigned to either E (1.4mg/m2, d1 and d8, q21 days, 4 cycles) + FEC (fluorouracil 500 mg/m2, epirubicin 100 mg/m2, and cyclophosphamide 500 mg/m2) or P (80mg/m2, weekly, 12 cycles) + FEC as PST. HER2+ patients were allowed to receive trastuzumab. Stratification factors were ER, HER2, and menopausal status. Primary endpoint was the incidence of peripheral sensory and motor neuropathy (PSN and PMN) with Grade 1 or higher according to CTCAE ver.4.0. Secondary endpoints were pathological complete response (pCR) rates (ypT0/is/ypN0), clinical response rates (CR+PR), and adverse events. Safety was assessed in all pts who received at least one dose of the study drug. Results: Between 12/2013 to 3/2016, 121 pts were randomly assigned equally to E + FEC and P + FEC. Excluding 5 pts from the primary assessment, 116 pts (58 in each group) were included in the full analysis set. The characteristics of the pts were similar in the two arms. At the end of E or P administration, the incidences of PSN were 55.4% and 92.9% in E and P arm, respectively (p Conclusion: This randomized phase II study revealed that eribulin had favorable peripheral neuropathy profile with modest efficacy in the neoadjuvant setting, compared with paclitaxel. Citation Format: Miura D, Hasegawa Y, Ishikawa T, Tachibana A, Horiguchi J, Hayashi M, Miyashita M, Kubota T, Narui K, Suzuki M, Akazawa K, Kohno N. Randomized controlled trial of neoadjuvant eribulin mesylate versus paclitaxel in women with operable breast cancer (JONIE-3 study) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-15-05.

Background: Pertuzumab provided overall and progression-free survival (PFS) benefits in HER2-positive metastatic breast cancer patients (pts) in the CLEOPATRA (Clinical evaluation of docetaxel, pertuzumab and trastuzumab) study. However, few studies have described the efficacy of other drugs in combination with pertuzumab plus trastuzumab. Here, we present a pre-specified analysis of eribulin in combination with pertuzumab plus trastuzumab as first- and second-line therapy for advanced or metastatic breast cancer (AMBC) in a multicenter, open-label phase II study (UMIN000012232, JBCRG-M03). Methods: HER2-positive AMBC with no or single prior chemotherapy for AMBC were enrolled. All pts were administered trastuzumab and taxane as adjuvant or first-line chemotherapy. Treatment consisted of eribulin 1.4 mg/m2 on days 1 and 8 of a 21-day cycle and trastuzumab (8 mg/kg loading dose, then 6 mg/kg) plus pertuzumab (840 mg/body loading dose, then 420 mg/ body) once every 3 weeks, all administered intravenously. The primary endpoint was PFS, and secondary endpoints included overall response rate (ORR) and safety. PFS was determined using Kaplan–Meier analysis. Tumor response was assessed according to RECIST ver. 1.1. Results: Fifty pts were enrolled from November 2013 to April 2016. Forty-nine pts were eligible for safety analysis and the full analysis set (FAS) included 46 pts. The median age was 56 years (23–70), and 8 (16%) and 41 (84%) pts were treated in first- and second-line settings, respectively. Eleven pts (23.9%) were de-novo Stage 4, and 35 pts (76.1%) had progressed in metastatic disease after completion of local therapy. Median PFS was 9.3 months (M) (95% confidence interval [CI]: 6.4–12.3). Table 1 shows the efficacy data for each treatment line and includes ORR, complete response rate (CR), partial response rate (PR), stable disease rate (SD), progressive disease rate (PD), not evaluable rate (NE) and PFS in the FAS. The median relative dose intensities of eribulin, trastuzumab, and pertuzumab were 93.3% (77.0%–100%), 100% (96.0%–100%), and 100% (89.7%–100%), respectively, in the FAS. The grade 3/4 adverse events (AE) were neutropenia in 5 pts (10.2%), including 2 pts (4.1%) with febrile neutropenia; hypertension in 3 pts (6.1%), and other AEs in only one patient. The average of the ejection fraction did not decrease significantly. Symptomatic left ventricular systolic dysfunction was not observed. Conclusion: In pts with HER2-positive AMBC, first- and second-line therapy of eribulin in combination with pertuzumab plus trastuzumab demonstrated substantial antitumor activity with an acceptable safety profile. We are planning a phase III study comparing eribulin with taxanes in combination with pertuzumab plus trastuzumab for the treatment of HER2-positive AMBC. Efficacy data for each treatment lineTreatment LineTotal (n=46)First line (n=8)Second line (n=38)PFS (95% CI), months9.3 (6.4-12.3)20.8 (2.8-38.7)8.7 (7.2-10.2)ORR (%)28 (60.9)7 (87.5)21 (55.3)CR (%)8 (17.4)3 (37.5)5 (13.2)PR (%)20 (43.5)4 (50.0)16 (42.1)SD (%)11 (23.9)1 (12.5)10 (26.3)PD (%)5 (10.9)05 (13.2)NE (%)2 (4.3)02 (5.3) Citation Format: Kawaguchi H, Yamashita T, Masuda N, Kitada M, Narui K, Hattori M, Yoshinami T, Matsunami N, Yanagihara K, Kawasoe T, Nagashima T, Bando H, Yano H, Hasegawa Y, Nakamura R, Kashiwaba M, Morita S, Ohno S, Toi M. Phase II study of eribulin in combination with pertuzumab plus trastuzumab for human epidermal growth factor receptor 2 (HER2)-positive advanced or metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-07.

BACKGROUND and AIM: Findings from a randomized phase 2 JONIE1 trial in women with HER2-negative early breast cancer have shown that the addition of zoledronic acid (ZOL) to neoadjuvant chemotherapy (CT) has potential anticancer benefits in postmenopausal and triple-negative breast cancer patients. We report the data for the prespecified secondary endpoint of disease-free survival (DFS). METHODS: We enrolled women with HER2-negative early breast cancer and randomly assigned them to receive CT or CT+ZOL (CTZ). All patients received 4 cycles of FEC100 (fluorouracil 500 mg/m2, epirubicin 100 mg/m2, and cyclophosphamide 500 mg/m2), followed by 12 cycles of paclitaxel at 80 mg/m2 weekly. ZOL (4 mg) was administered 3-4 times weekly for 7 weeks to the CTZ group patients. Definitive surgery was performed 3-4 weeks after the last paclitaxel dose. The primary endpoint was pathological complete response (pCR). The secondary endpoints were the clinical response rates, rate of breast-conserving surgery, safety, and DFS (defined as the time from randomization to disease occurrence or death). The trial is registered as UMIN000003261 (www.umin.ac.jp/english/) with ongoing follow-up. FINDINGS: Of the 188 patients enrolled, 95 were assigned to the CT group and 93 to the CTZ group. The mean (95% CI) DFS time of the CT group was 5.15 years (4.83-5.47) and that of the CTZ group was 5.38 years (5.11-5.66). The 3-year DFS rate was 84.6% (95% CI 77.2-92.0) in the CT group and 90.7% (84.6-96.8) in the CTZ group with no significant difference (p = 0.120). The particular benefit from ZOL for the neoadjuvant CT seen as improvement of the pCR rate was indicated in the 3-year DFS period for triple-negative cancer cases (CT vs CTZ: 70.6% vs 94.1%), but not for postmenopausal cases. CONCLUSIONS: ZOL slightly improved DFS when combined with CT. Although a significant difference was not found in this study, plans are underway for conducting a combined analysis of 3 neoadjuvant CT trials together with ZOL. The improvement of the pCR rate may be associated with DFS in triple-negative cases. Previous studies have shown that ZOL was more efficacious in an estrogen-suppressed condition. However, the short-term application of ZOL in this study may not be sufficient to improve the outcome in postmenopausal patients. Citation Format: Ishikawa T, Akazawa K, Hasegawa Y, Tanino H, Horiguchi J, Miura D, Hayashi M, Takao S, Kim SJ, Yamagami K, Miyashita M, Konishi M, Shigeoka Y, Suzuki M, Taguchi T, Kubota T, Kohno N. Zoledronic acid combined with neoadjuvant chemotherapy for HER2-negative early breast cancer (JONIE 1 trial): Survival outcomes of a randomized multicenter phase 2 trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-16-10.

Background: There is a high risk of under-reporting subjective toxicities by physicians, even when collected prospectively in clinical trials. It has been recommended to include patient reported measures regarding symptoms in prospective clinical comparative effectiveness trials. However, there have been few reports of agreement in endocrine related symptoms between patient and physician reporting. Patients and Method: The National Surgical Adjuvant Study of Breast Cancer 06 (N-SAS BC 06) is a multicenter, randomized clinical trial of postmenopausal, hormone receptor-positive breast cancer patients, with a two-stage (preoperative and postoperative) enrollment, and intervention. The primary aim was to evaluate the need for adjuvant chemotherapy in the treatment of postmenopausal breast cancer patients who responded to neoadjuvant treatment with Letrozole (LET) for 24-28 weeks. After surgery, responders were randomized into two arms receiving either chemotherapy plus LET, or LET alone. The primary endpoint was disease-free survival, and the secondary endpoints included adverse events, quality of life and health economic evaluation. This study enrolled 497 subjects from the N-SAS BC 06 who were evaluated by Patient Reported Outcomes (PROs). The concordance rate between Clinician Reported Outcomes (CROs) and PROs in their endocrine symptoms during neoadjuvant endocrine therapy was examined. Symptoms were collected prospectively by physicians using the Common Toxicity Criteria for Adverse Events at enrollment, i.e., baseline, and 4 and 16 weeks after starting neoadjuvant LET. Patients also completed the FACT-G (General), B (Breast), ES (Endocrine Symptoms), and HADS. The endocrine symptoms according to the PROs, included nausea, hot flushes, cold sweats, headaches, and HADS-Depression score. In FACT, "Not at all" was used to express the absence of the symptoms, and "A little bit", "Some-what", "Quite a bit", and "Very much" were used to express the presence of symptoms. The HADS-Depression score threshold was 10/11. According to the CROs, grade 0 was defined as the absence of symptoms and grade 1 or more was defined as the presence of symptoms. Cohen's kappa was used to determine the concordance between CROs and PROs. The sensitivity of CROs was also calculated. Results: The calculated point estimates of Cohen's kappa at Weeks 4 and 16 after starting neoadjuvant LET were 0.12 and 0.01 for nausea, 0.16 and 0.18 for hot flushes, 0.12 and 0.09 for cold sweats, 0.03 and 0.02 for headaches, and 0.11 and 0.11 for dysthymia/depression, respectively; the concordance was quite low. The sensitivity of CROs at Weeks 4 and 16 after starting neoadjuvant LET was 0.07 and 0.03 for nausea, 0.16 and 0.17 for hot flushes, 0.1 and 0.08 for cold sweats, 0.03 and 0.03 for headaches, and 0.11 and 0.1 for dysthymia/depression, respectively; the sensitivity was quite low. Conclusion: This study showed that there were big differences between CROs and PROs in endocrine symptoms associated with endocrine therapy for breast cancer and that physicians could not obtain sufficient information on the endocrine symptoms. It is recommended that PROs be used to evaluate adverse events caused by endocrine therapy. Citation Format: Fujisawa T, Iwata H, Sakai T, Nakamura R, Hasegawa Y, Ohtani S, Kashiwaba M, Taira N, Toyama T, Masuda N, Yamamoto Y, Kihara K, Shimozuma K, Ohashi Y, Mukai H. Endocrine-related symptoms during neoadjuvant endocrine therapy for breast cancer: Agreement between patient and physician reporting in a prospective clinical trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-11-02.

Background: Many anti-tumor agents are used in chemotherapy for cancers. However, they also have serious side effects because they express similar growth inhibitory activity in normal cells. Meanwhile, the development of molecular target drugs inhibiting biomolecules relating to tumor growth, invasion, and metastasis has been proceeding with the goal of selectively attacking cancer cells. It has been revealed that the proliferation of cancer stem cells involved in redevelopment, invasion, and metastasis is promoted by sphingosine-1-phosphate (S1P). We focused on the transmembrane domains of sphingosine 1 phosphate receptor 4 (S1PR4), and hypothesized that peptides which utilized these domains would exhibit a selective anti-tumor effect in cancer cells. We designed peptides based on the transmembrane domains of S1PR4 and evaluated whether the peptides showed tumor cell growth inhibitory activity against various kinds of human tumor cells. Material and methods: We chemically synthesized conjugation peptides of the human S1PR4 transmembrane domain sequences and a cell-penetrating peptide HIV TAT. The peptides were evaluated for growth inhibitory activity against human melanoma, non-small cell lung cancer (NSCLC),kidney cancer by WST assay. Results: The peptide based on the S1PR4 transmembrane domain 2 exhibited the strongest anti-tumor activity in the synthesized peptides based on the domain from 1 to 7. The result was that more than 95% of the tumor cells were inhibited in cells treated with a concentration of 25μM for 48 hours. Conclusions: We demonstrated a novel strategy for discovering an anti-tumor molecule by focusing on transmembrane domains of proteins involved in the proliferation of cancer cells. In particular, the peptide based on the transmembrane domain 2 of S1PR4 exhibits remarkable anti-tumor activity against human melanoma, NSCLC, kidney cancer which suggests that the peptide could be a candidate for a new anti-tumor agent. COI: I/we (in case of co-authors) have no potential conflict of interest to disclose Citation Format: Tetsuhiko Yoshida, Kenichi Tanaka, Shuichi Asakawa, Makoto Sawada, Yoshinori Hasegawa, Ikuhiko Nakase, Nahoko Bailey Kobayashi. A peptide based on transmembrane domain of S1PR4 as a tumor cell growth inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A106. doi:10.1158/1535-7163.TARG-19-A106

Background: Treatment with an aromatase inhibitor for 5 years as up-front monotherapy or after tamoxifen therapy for 2-3 years is the treatment of choice for hormone-receptor-positive breast cancer in postmenopausal women. Extending treatment with an aromatase inhibitor to 10 years may reduce the risk of breast cancer recurrence. Methods: We conducted a prospective randomized multi-center open-label phase III trial to assess the effect of the extended use of anastrozole for an additional 5 years. Postmenopausal patients with stageI-III, hormone-receptor-positive breast cancer, disease-free after 5 years of either anastrozole alone or tamoxifen 2-3 years followed by anastrozole 3-2 years were randomized to continual group with anastrozole for an additional 5 years or stop group without an additional anastrozole. Our primary end point was disease-free survival. Results: We enrolled 1697 women. After a median follow up of 4.9 years, there were 149 events involving disease recurrence or the occurrence of contralateral breast cancer (51 in continual group and 98 in stop group) and 7 deaths (3 in continual group and 4 in stop group). The 5-year disease-free survival rate was 91.9% (95% confidence interval [CI], 89.4 to 93.8) in continual group and 84.4% (95% CI: 80.0 to 88.0) in stop group (hazard ratio for disease-free survival,0.548 ;P=0.0004. by a two-sided log-rank test stratified according to nodal status, prior adjuvant chemotherapy, institution, and choice of anastrozole or tamoxifen). The rate of 5-year overall survival was 99.5% in continual group and 99.6% in stop group. (hazard ratio,1.389 ;P=0.665). The rate of 5-year distant disease-free survival was 97.2% in continual group and 94.3% in stop group (hazard ratio,0.514 ;P=0.0077). Bone-related adverse events were observed more frequently among patients in continual group than among patients in stop group, including a higher incidence of bone pain, stiff joints, bone fractures, and new-onset osteoporosis. Conclusion: The extension of treatment with an adjuvant aromatase inhibitor (anastrozole) to 10 years resulted in significantly higher rates of disease-free survival and distant disease-free survival than those with no additional anastrozole, but the rate of overall survival was not different between two groups. Our study shows that it is safe and beneficial for postmenopausal patients with hormone-receptor-positive breast cancer to take an anastrozole as adjuvant therapy for an additional 5 years after initial treatment. (UMIN:000000818) Citation Format: Ohtani S, Iijima K, Higaki K, Sato Y, Hozumi Y, Hasegawa Y, Takei H, Tanaka M, Yagata H, Masuoka H, Tanabe M, Egawa C, Komoike Y, Saji S, Nakamura T, Yanagita Y, Ohtsu H, Mukai H, Iwase T. A prospective randomized multi-center open-label phase III trial of extending aromatase-inhibitor adjuvant therapy to 10 years - Results from 1697 postmenopausal women in the N-SAS BC 05 trial: Arimidex extended adjuvant randomized study (AERAS) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr GS3-04.

Background: Neoadjuvant therapy for locally advanced breast cancer has the potential to improve surgical therapeutic outcomes without sacrificing the survival advantages of adjuvant therapy. However, determining whether ER+ patients (pts) will respond to neoadjuvant (NA) chemotherapy (CT) or hormone therapy (HT) can be difficult. Not all ER+ pts respond to NACT, while response to NAHT can vary across ER+ pts. Thus, the ability to select pts more likely to benefit from NAHT would represent progress in clinical management of breast cancer. NEOS is a randomized phase III study assessinglong-term prognosis of ER+ primary breast cancer with/without adjuvant CT following NAHT (UMIN 000001090, http://www.umin.ac.jp/). We used archived core biopsy tumor samples from the NEOS study to validate the RS result as a predictor of clinical response and its association with successful breast conserving surgery (BCS) in pts treated with 6 months of NAHT. Methods: NEOS enrolled 904 postmenopausal pts with ER+, HER2-, clinically node negative (cN0) breast cancer to evaluate whether adjuvant CT was necessary for pts who responded to NAHT. In this current study, we enrolled pts with tumors ≥2cm from the NEOS study. Biopsy samples of 333 pts were assessed for the Oncotype DX assay. Response to NAHT was recorded as complete/partial response (CR/PR), or stable/progressive disease (SD/PD). Primary endpoint of this study was to evaluate clinical response (CR/PR) to NA letrozole between pts with low ( Results: The analysis included 294 pts with median age of 63 yrs, median tumor size of 25mm, and 66% were nuclear grade 1. 156 (53.0%), 83 (28.6%) and 54(18.4%) cases were low, intermediate, and high RS groups by Oncotype DX, respectively. Six (2%), 126 (42.8%), 149 (50.3%), 13 (4.4%) cases experienced CR, PR, SD, PD as clinical response, respectively, similar to that of all NEOS pts. Clinical response rate was 54%, 42% and 22% in low, intermediate, and high RS groups, respectively. The proportion of pts with clinical response was significantly higher in the low RS group vs the high RS group (p Conclusion: The Oncotype DX RS test in core biopsy samples is validated as a predictive assay for clinical response of NAHT in postmenopausal, ER+/HER2-, cN0, primary early breast cancer pts. Further results on the association of RS results with BCS outcomes following NAHT will be presented. These results when combined with previously published data on RS in NACT studies help guide pts with ER+, HER2- breast cancer with NAHT vs NACT treatment options to maximize clinical response. Citation Format: Yamamoto Y, Iwata H, Masuda N, Fujisawa T, Toyama T, Kashiwaba M, Ohtani S, Taira N, Sakai T, Hasegawa Y, Nakamura R, Akabane H, Shibahara Y, Sasano H, Yamaguchi T, Sakamaki K, Chao C, McCullough D, Sugiyama N, Ohashi Y. TransNEOS: Validation of the oncotype DX recurrence score (RS) testing core needle biopsy samples from NEOS as predictor of clinical response to neoadjuvant endocrine therapy for postmenopausal estrogen receptor positive (ER+), HER2 negative (HER2-) breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD5-03.

Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets. [Cancer Res 2009;69(23):9073–82]

Krüppel-like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcriptional factor, which has been suggested to be a candidate tumor suppressor gene in prostate cancer and astrocytic glioma. Because KLF6 is located at chromosome 10p15, where non-small cell lung cancers (NSCLCs) also exhibit frequent allelic loss, we hypothesized that the inactivation of KLF6 is also involved in the development of NSCLC. To determine this, we performed mutational analysis for 105 NSCLCs, including 9 cell lines and 96 primary tumors, and Northern blot analysis for 74 NSCLCs, including the 9 cell lines and 65 primary tumors. Although somatic mutations were not detected in the coding sequence of KLF6, expression of KLF6 mRNA was down-regulated in the 9 cell lines and in 55 (85%) of the 65 primary tumors compared with normal lung tissue. Treatment of two cell lines expressing KLF6 at low levels with 5-azacytidine did not induce KLF6 expression, suggesting that KLF6 down-regulation is not due to promoter hypermethylation. We also performed loss of heterozygosity (LOH) analysis using the laser capture microdissection technique, and found that 21 of 62 (34%) informative samples had LOH in the KLF6 gene locus. Comparing the LOH status with mRNA expression of KLF6, we found that 14 of the 14 (100%) samples with LOH showed KLF6 down-regulation, and that even 23 of 31 (74%) samples without LOH also showed this down-regulation. We also studied the expression of the WAF1 gene, a possible downstream gene of KLF6, and detected simultaneous down-regulation of WAF1 and KLF6 mRNA in 6 of 9 (67%) cell lines and 48 of the 55 (87%) primary tumors, although there was not a significant association between loss of KLF6 and WAF1 expression. Furthermore, colony formation assay of two NSCLC cell lines (NCI-H1299 and NCI-H2009) induced a markedly reduced colony formation by KLF6 transfection, and Annexin V staining and terminal deoxynucleotidyl transferase-mediated nick end labeling assays revealed that KLF6 induced apoptosis. Our present studies demonstrated that KLF6 is frequently down-regulated in NSCLC and suppresses tumor growth via induction of apoptosis in NSCLC, which may suggest that KLF6 is a tumor suppressor for NSCLC.

Background: TIMELSS (TIM) is a circadian clock gene in Drosophila, but its role in mammals in circadian clock systems is not well-understood. Mammalian TIM is involved in Chk1 activation and intra-S checkpoint through genotoxic stress (Unsal-Kaçmaz et.al, Molecular and Cellular Biology, 27(8), 3131-3142(2007)). TIM is also associated with doxorubicin-induced G2/M cell cycle arrest and TIM depletion sensitizes HCT116 colon cancer cells to doxorubicin-induced cytotoxicity (Yang et.al, Journal of Biological Chemistry, 285(5), 3030-3034(2010)). Mutations in TIM in breast cancer were reported (Sjöblom et.al, Science, 314(5797), 268-274(2006)). Methods and Findings: The mRNA expression levels of TIM in 17 non-small cell lung cancer (NSCLC) cell lines and 4 small cell lung cancer (SCLC) cell lines were analyzed by reverse transcriptase PCR. HCC44, H460, H157 (NSCLC), H146 and H740 (SCLC) expressed higher levels of TIM than cdk4/hTERT-immortalized normal human bronchial epithelial cell line HBEC4. Sequencing analysis of TIM revealed no mutation in 21 lung cancer cell lines. RNAi-mediated knockdown of TIM in H157 suppressed proliferation in WST-1 assay and clonogenic growth in liquid colony formation assay. Western blot analysis revealed increased level of cleaved caspase-3 after TIM-knockdown, suggesting that apoptosis was involved in growth inhibition. TIM-knockdown induced increased sensitivity to cisplatin in drug sensitivity assay using WST-1 (IC50 were 1.70μM and 0.91μM in TIM-knocked down H157 compared to 4.87μM in control). Conclusions: TIM inhibition causes growth suppression, apoptosis and enhanced cytotoxicity of cisplatin. Our results suggest that TIM inhibition has a potential utility in the treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4678. doi:1538-7445.AM2012-4678

Introduction: Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers, including lung cancer, and its contribution to increased proliferation through up-regulating cell cycle accelerators such as cyclin A/E is well demonstrated in breast and gastric cancers. Nevertheless, very little is known about its role in survival of cancer cells. In addition, functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. Matrials and methods: 18 human non-small cell lung cancer cell lines and an immortalized normal human bronchial epithelial cell (HBEC4) lines were used. EpCAM expression was measured by quantitative real-time PCR (qRT-PCR) and FACS. RNA interference (RNAi)-mediated gene silencing for EpCAM was done in three non-small cell lung cancer cell and HBEC4 lines. Cell proliferation was measured by WST-1 and clonogenic growth was measured by liquid and soft agar colony formation assays. Apoptosis was evaluated by annexin V/7-AAD staining. FACS with PI staining was done to examine apoptosis and cell cycle. p27kip1 expression was evaluated by western blot analysis. In vitro invasion assay was done by using transwell chambers layered with matrigel and invading cells were stained and counted. Results: We analyzed the expression of EpCAM in a panel of lung cancer cell and HBEC4 lines and found that most of lung cancer cell lines expressed EpCAM. EpCAM knockdown suppressed proliferation and clonogenic growth of two EpCAM-expressing lung cancer cell lines in anchorage dependent and independent conditions. EpCAM knockdown suppressed invasiveness in a highly invasive line but not in a lowly invasive cell line. In addition, EpCAM knockdown induced massive apoptosis in both cell lines as well as another EpCAM-expressing lung cancer cell line but to a much lesser extent in HBEC4 line. EpCAM knockdown caused lung cancer cells to have increased responsiveness to contact inhibition in part through up-regulating p27kip1 cyclin dependent kinase inhibitor. Conclusion: These results demonstrate that EpCAM substantially contributes to the pathogenesis of lung cancer, especially in its survival, and the development of EpCAM-targeted therapy for lung cancer may have promise. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1657. doi:10.1158/1538-7445.AM2011-1657

Background: Epithelial-to-mesenchymal transition (EMT), an essential process during embryonic development and in wound healing, has been shown to be a key event in tumor migration, invasion and metastasis. EMT program is controlled by several master regulators including Twist, ZEB1, SIP1, Snail, Slug, and Goosecoid which are called master EMT genes1),2). ZEB1 protein binds E-boxes within the promoter region of E-cadherin gene, leading to its transcriptional repression. Also, it directly represses many other genes encoding components of the epithelial junctional complex and cell polarity factors. ZEB1 was shown to promote metastasis in colorectal cancer and breast cancer3), and the association between ZEB1 and tumor progression has been studied in several human cancers. Purpose: To evaluate the association between ZEB1 expression and mesenchymal phenotype in lung cancer, and to test the effect of ZEB1 knockdown with RNA interference on the growth of lung cancer cells. Methods: We analyzed the expression of E-cadherin (epithelial marker), Vimentin (mesenchymal marker), and four master EMT genes (Snail, Slug, ZEB1, SIP1) in 19 NSCLC cell lines and 32 NSCLC tumor tissues. Transient knockdown of ZEB1 with RNA interference was done in three NSCLC cell lines with high expression of ZEB1: H157, H1299, and H460. Quantitative real-time RT-PCR and western blot of ZEB1, E-cadherin and Vimentin were done. Cellular proliferation was measured by WST-1 assay and clonogenic growth was measured by liquid (anchorage-dependent) and soft agar (anchorage-independent) colony formation assays. Apoptosis was evaluated by FACS and western blot of cleaved caspase-3. Results: In cell lines, of four master EMT genes, only ZEB1 expression significantly correlated with both E-cadherin and Vimentin expression. Most EGFR mutant lines showed epithelial phenotype (ratio of Vimentin to E-cadherin (RVE) < 1.0). ZEB1 expression was also inversely correlated with miR-200c and miR-205. In tumor tissues, ZEB1 and Snail expression correlated with the ratio of Vimentin to E-cadherin, and ZEB1 expression highly significantly correlated with Vimentin expression. After ZEB1 knockdown E-cadherin protein was reexpressed in H460 but not in H1299 and H157 cells. ZEB1 knockdown suppressed cell proliferation and liquid colony formation significantly in H1299 and modestly in H157 and H460. Notably, ZEB1 knockdown dramatically suppressed growth of soft agar in the all three cell lines studied. FACS and western blot of cleaved caspase-3 showed apoptosis induction by ZEB1 knockdown in H460, suggesting that growth inhibitory effect of ZEB1 knockdown in lung cancer was caused in part by apoptosis. Conclusions: ZEB1 has the dominant role in maintaining mesencymal phenotype in NSCLC and removal of ZEB1 in lung cancer cell lines induces inhibition of soft agar growth. These results suggest that ZEB1 could be a therapeutic target for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2295.

Background and Objective: Malignant pleural mesothelioma (MPM) is a highly aggressive disease, usually caused by asbestos exposure. The underlying molecular mechanism contributing to aggressive phenotype of MPM remains to be fully elucidated. Epithelial-to-mesenchymal transition (EMT), an early developmental program, causes a wide variety of human epithelial cancers to have the ability to invade surrounding tissues and metastasize to distant sites. MPM cells usually exhibit mesenchymal characteristics probably because of their mesodermal origin. Such characteristics include fibroblast-like morphology and expression of master EMT-inducing genes. Thus, EMT may contribute to aggressive phenotype of MPM. Among EMT-inducing genes, ZEB1, a repressor of E-cadherin, has emerged as a key player in the progression of several epithelial cancers. To explore the potential of ZEB1 as a therapeutic target for MPM, we examined the effect of ZEB1 knockdown on growth of MPM cell lines. In addition, we evaluated the effect of ZEB1 knockdown on the levels of IL-6, which is known to induce growth of mesothelioma cells. Materials and Methods: 18 human MPM cell lines and one non-tumorigenic mesothelial cell line were used. Transient and stable knockdown of ZEB1 were done in two MPM cell lines. Quantitative real-time RT-PCR and western blot of ZEB1, E-cadherin, and Vimentin were done. Soluble IL-6 in cellular supernatants was measured by enzyme-linked immunosorbent assay. Cell proliferation was measured by WST-1 and clonogenic growth was measured by liquid and soft agar colony formation assays. FACS with PI staining was done to examine apoptosis and cell cycle. Apoptosis was also evaluated by western blot of cleaved caspase-3. Results: The majority of MPM cells expressed higher levels of ZEB1 than a non-tumorigenic mesothelial cell. We performed ZEB1 knockdown experiments in two MPM cells, ACC-MESO-1(MESO-1) and H2052, which express high and moderate levels of ZEB1. Transient knockdown of ZEB1 caused MESO-1 but not H2052 cells to reexpress E-cadherin. Transient ZEB1 knockdown suppressed cell proliferation and liquid colony formation in the two lines. Most importantly, the ZEB1 knockdown dramatically suppressed soft agar colony formation in the two lines. We did not see apoptosis or cell cycle arrest in either of the two lines. Stable knockdown of ZEB1 induced morphologic changes suggestive of mesenchymal-to-epithelial transition (MET) in MESO-1 cells but not in H2052 cells. ZEB1 stable knockdown resulted in a decreased IL-6 production in MESO-1 cells, suggesting that growth inhibition by ZEB1 knockdown may in part due to decreased IL-6 production. Conclusion: These results suggest that ZEB1 serves as an attractive therapeutic target for MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2302.