Michio Sugita

Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines.

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, produce 9% to 27% response rates in NSCLC patients. E-Cadherin, a calcium-dependent adhesion molecule, plays an important role in NSCLC prognosis and progression, and interacts with EGFR. The zinc finger transcriptional repressor, ZEB1, inhibits E-cadherin expression by recruiting histone deacetylases (HDAC). We identified a significant correlation between sensitivity to gefitinib and expression of E-cadherin, and ZEB1, suggesting their predictive value for responsiveness to EGFR-tyrosine kinase inhibitors. E-Cadherin transfection into a gefitinib-resistant line increased its sensitivity to gefitinib. Pretreating resistant cell lines with the HDAC inhibitor, MS-275, induced E-cadherin along with EGFR and led to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive NSCLC cell lines including those harboring EGFR mutations. Thus, combined HDAC inhibitor and gefitinib treatment represents a novel pharmacologic strategy for overcoming resistance to EGFR inhibitors in patients with lung cancer.

EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to gefitinib and EGFR expression in cell lines

BACKGROUND Allelic loss in chromosome 3p is one of the most frequent and earliest genetic events in lung carcinogenesis. We investigated if the loss of microRNA-128b, a microRNA located on chromosome 3p and a putative regulator of epidermal growth factor receptor (EGFR), correlated with response to targeted EGFR inhibition. Loss of microRNA-128b would be equivalent to losing a tumor suppressor gene because it would allow increased expression of EGFR. PATIENTS AND METHODS We initially showed that microRNA-128b is a regulator of EGFR in non-small-cell lung cancer (NSCLC) cell lines. We tested microRNA-128b expression levels by quantitative RT-PCR, genomic copy number by quantitative PCR, and mutations in the mature microRNA-128b by sequencing. We determined whether microRNA-128b loss of heterozygosity (LOH) in 58 NSCLC patient samples correlated with response to gefitinib and evaluated EGFR expression and mutation status. RESULTS We determined that microRNA-128b directly regulates EGFR. MicroRNA-128b LOH was frequent in tumor samples and correlated significantly with clinical response and survival following gefitinib. EGFR expression and mutation status did not correlate with survival outcome. CONCLUSION Identifying microRNA regulators of oncogenes could have far-reaching implications for lung cancer patients including improving patient selection for targeted agents, development of novel therapeutics, or development as early biomarkers of disease.

Mutations of the β - and γ -catenin genes are uncommon in human lung, breast, kidney, cervical and ovarian carcinomas

β-catenin forms complexes with Tcf and Lef-1 and functions as a transcriptional activator in the Wnt signalling pathway. Although recent investigations have been focused on the role of the adenomatous polyposis coli (APC)/ β-catenin/Tcf pathway in human tumorigenesis, there have been very few reports on mutations of the β-catenin gene in a variety of tumour types. Using PCR and single-strand conformational polymorphism analysis, we examined 93 lung, 9 breast, 6 kidney, 19 cervical and 7 ovarian carcinoma cell lines for mutations in exon 3 of the β-catenin gene. In addition, we tested these same samples for mutations in the NH2-terminal regulatory region of the γ-catenin gene. Mutational analysis for the entire coding region of β-catenin cDNA was also undertaken in 20 lung, 9 breast, 5 kidney and 6 cervical carcinoma cell lines. Deletion of most β-catenin coding exons was confirmed in line NCI-H28 (lung mesothelioma) and a silent mutation at codon 214 in exon 5 was found in HeLa (cervical adenocarcinoma). A missense mutation at codon 19 and a silent mutation at codon 28 in the NH2-terminal regulatory region of the γ-catenin gene were found in H1726 (squamous cell lung carcinoma) and H1048 (small cell lung carcinoma), respectively. Neither deletions nor mutations of these genes were detected in the other cell lines examined. These results suggest that β- and γ-catenins are infrequent mutational targets during development of human lung, breast, kidney, cervical and ovarian carcinomas.

Baseline Gene Expression Predicts Sensitivity to Gefitinib in Non–Small Cell Lung Cancer Cell Lines

Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non–small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance. (Mol Cancer Res 2006;4(8):521–8)

KRAS Mutation: Comparison of Testing Methods and Tissue Sampling Techniques in Colon Cancer

Treatment of colon carcinoma with the anti-epidermal growth factor receptor antibody Cetuximab is reported to be ineffective in KRAS-mutant tumors. Mutation testing techniques have therefore become an urgent concern. We have compared three methods for detecting KRAS mutations in 59 cases of colon carcinoma: 1) high resolution melting, 2) the amplification refractory mutation system using a bifunctional self-probing primer (ARMS/Scorpion, ARMS/S), and 3) direct sequencing. We also evaluated the effects of the methods of sectioning and coring of paraffin blocks to obtain tumor DNA on assay sensitivity and specificity. The most sensitive and specific combination of block sampling and mutational analysis was ARMS/S performed on DNA derived from 1-mm paraffin cores. This combination of tissue sampling and testing method detected KRAS mutations in 46% of colon tumors. Four samples were positive by ARMS/S, but initially negative by direct sequencing. Cloned DNA samples were retested by direct sequencing, and in all four cases KRAS mutations were identified in the DNA. In six cases, high resolution melting abnormalities could not be confirmed as specific mutations either by ARMS/S or direct sequencing. We conclude that coring of the paraffin blocks and testing by ARMS/S is a sensitive, specific, and efficient method for KRAS testing.

Molecular Definition of a Small Amplification Domain within 3q26 in Tumors of Cervix, Ovary, and Lung

A common amplification target encompassing chromosome region 3q25 to q27 has been identified by comparative genomic hybridization analyses in tumors of the cervix, ovary, endometrium, lung, and head and neck. Because this segment spans at least 30 megabases, we undertook a molecular analysis of copy number to more precisely define the amplification domain. Our Southern blot and fluorescence in situ hybridization results with the use of 17 markers confirmed the presence of low-level 3q amplification events in cervical, ovarian, and variant SCLC tumors. Most of the tumor types studied appeared to have similar, broad amplification domains centered within 3q26.2, suggesting that the same target is being affected in all. The ovarian carcinoma cell line NIH:OVCAR3 had a highly restricted amplification domain spanned by four overlapping YAC clones, suggesting a small target. The region of highest amplification included the gene for the RNA component of telomerase (hTR), supporting it as a potential target. Although the importance of low-level amplification is unknown, the consistent and reproducible nature of this event in a variety of carcinomas suggests that 3q26.2 harbors an oncogene whose low-level amplification has a significant influence on tumor biology.

Altered Cell-Cycle Control, Inflammation, and Adhesion in High-Risk Persistent Bronchial Dysplasia

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell-cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G2-M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention.Significance: Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell-cell adhesion that may underlie progression to invasive SCC. Cancer Res; 78(17); 4971-83. ©2018 AACR.

Assessing lung cancer risk in bronchial dysplasia by identification of prognostic gene expression profiles

Bronchial dysplasia (BD) is a premalignant lesion that develops frequently in smokers and is thought to be a direct precursor of squamous cell carcinoma of the lung. Identification of BD lesions that are associated with high risk for progression to cancer is important so that adequate follow-up and establishment of chemopreventive therapy can be provided. We have reviewed specimens from the Colorado SPORE in lung cancer tissue bank to determine if persistence of BD over time is related to the initial degree of histologic atypia and the development of lung cancer, and to select appropriate groups of BD to assess for the presence of potential prognostic markers by microarray gene expression analysis. Histologic diagnoses (1=normal, 2=hyperplasia, 3=metaplasia, 4-6=mild, moderate and severe dysplasia, 7=CIS and 8=cancer) and clinical data from 186 subjects enrolled in SPORE bronchoscopy protocols that underwent at least two bronchoscopies were obtained from the SPORE database. 2,847 biopsies from 1,119 sites were assigned to baseline diagnostic groups according to the first non-normal biopsy obtained at a site. Atypia in follow-up biopsies was assessed per annum. Aditionally, RNA was extracted from bronchoscopic biopsies snap frozen in liquid nitrogen at collection. Cryostat sections were produced to establish histology and RNA was then extracted from the remaining tissue for gene expression analysis. Gene expression profiles for persistent and non-persistent BDs are being assessed on the Affymetrix Human Gene 1.0 ST array. A direct correlation between the degree of atypia at baseline and degree of atypia at follow-up was found at all time intervals (.25-1, 1-2, 2-3, 3-4 and >4 years follow-up biopsies) except for the 3-4 year timeframe. Biopsy sites from subjects with squamous cell lung cancer diagnosed prior to, during or after participation in bronchoscopy studies showed one full point higher histologic diagnosis on follow-up than sites with equivalent baseline diagnoses in subjects without known cancer. An interim assessment of data quality in 20 RNA specimens from baseline biopsies processed for gene expression analysis has been completed. Quality measurements show that distribution of signal intensity is similar across the cohort indicating comparability of specimens from the different study groups as defined by their histologic diagnoses on follow-up biopsy. Expression data from 40 specimens is targeted for the final dataset. The degree of atypia in BD is higher at sites with higher levels of a baseline atypia and in subjects with a history of or subsequent development of squamous cell carcinoma of the lung. These features suggest that persistence of BD over time may be indicative of risk for the development of invasive lung cancer. Preliminary data from ongoing gene expression analyses of BD of differing outcomes indicates that consistent data quality can be produced across the specimen types to be studied. This suggests that the gene expression analysis will be adequate to detect differentially expressed genes with the potential to provide prognostic information in BD and reveal targets for the chemoprevention of lung cancer.

P1-200: Saliva mRNA expression profiling for early stage non-small cell lung cancer (NSCLC) screening

Saliva mRNA expression profiling for early stage non-small cell lung cancer (NSCLC) screening Weiss, Glen J.1 Hu, Zhanzhi2 Peake, Delee1 Kelly, Karen3 Bunn, Jr, Paul A.1 Zhou, Michaeal2 Sugita, Michio1 Keith, Robert4 Wong, David T.2 1 University of Colorado Health Sciences Center, Aurora, CO, USA 2 University of California-Los Angeles, Los Angeles, CA, USA 3 University of Kansas Medical Center, Kansas City, KS, USA 4 Denver VA Medical Center, Denver, CO, USA Background: Strategies to identify high risk individuals who may develop lung cancer are sorely needed. Radiographic screening has several drawbacks including cost, high false positive rate, and exclusion of never-smokers who account for up to 13% of new diagnoses. Sputum collection can be difficult in up to half of former smokers. Alternatively, saliva is readily available and easy to collect. We have previously shown high sensitivity and specificity in distinguishing newly diagnosed oral squamous cell carcinoma from matched controls (Li et al. Clin Cancer Res 2004). Others have shown a correlation of salivary and serum levels of soluble Her2/neu in women with breast cancer compared to benign tumor and healthy controls (Streckfus et al. Clin Cancer Res 2000). These studies led us to embark on a pilot study to validate mRNA expression on a gene exon array platform and to explore gene exon signatures discriminating between early-stage NSCLC and healthy controls. Methods: Saliva is currently being collected from healthy subjects ages 40-79, (current and former smokers [≥20 pack-year] and never-smokers [<100 cigarettes/lifetime] and histologically-confirmed, untreated stage I-II NSCLC patients along with a medical history questionnaire. Exclusion criteria included: active pulmonary infection within 6 months, steroid inhaler use ≥ 6 months, or history of other invasive cancer within past 5 years. A one-time saliva specimen is being obtained between 910 am to avoid diurnal variation, mixed with a RNA stabilization agent, and stored at -70 ̊C. RNA is then extracted, amplified, and hybridized to the Affymetrix Human Exon 1.0 arrays (which contain 1.4 million probe sets). Array data is analyzed after appropriate normalization. Results: As proof-of-principal of the value of the exon array discovery platform, saliva mRNA expression was performed on 18 healthy subjects [14 males (3 never-smokers), 11 females (8 never-smokers)]. All of whom were able to provide adequate sample for analysis. Our data demonstrate that we have successfully amplified salivary RNA, hybridized to the exon array and have developed the statistical and informatics tools to harness the diagnostic information. Using these developed tools and the healthy cohort of subjects, we have identified and validated gender-specific salivary exon signatures. Conclusions: Preliminary results with saliva mRNA exon-level expression profiling suggest that quality samples are easily obtainable and the technique is feasible. Updated results with NSCLC specimens will be presented. Supported by International Association for the Study of Lung Cancer Fellowship Award (GJW) and R01-DE15970-03 (DTW).

Management of pregnant women with abnormal cervical cytology.

過去3年間において当科を受診した妊婦2972例に対して綿棒または湿綿球にて子宮頸部擦過細胞診を実施した.結果はclass III aが16例 (0.54%), class III bが4例 (0.13%), class IVが2例 (0.07%) であった.当科ではこのような細胞診異常を呈した妊婦については極力円錐切除を回避するよう心がけており, 細胞診やコルポスコープで進行癌を示唆する所見がない場合は, 円錐切除を敢えて行っていない.初回細胞診にてclass IVを呈した2例は狙い組織診にて1例はcarcinoma in situ (CIS), 1例はsevere dysplasiaであった.2例とも円錐切除は実施せず, 保存的に経過観察を行ったところ, いずれも細胞診上はclass IVを持続した.この2例は産後の子宮全摘, 円錐切除により, 組織学的にCISであった.また, 今回の検討期間中には進行癌を示唆する症例は認められなかった.当科の管理方針は妊娠中の円錐切除に起因するさまざまな合併症を回避できる点において有用であるが, 浸潤癌におけるunder diagnosisの危険性については今後症例を重ね検討する必要があると思われた.