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Ribonucleotide reductase subunit 2 (RRM2) predicts shorter survival in resected stage I–III non-small cell lung cancer (NSCLC) patients
Abstracts / Lung Cancer 77 (2012) S21–S27
Ribonucleotide reductase subunit 2 (RRM2) predicts shorter survival in resected stage I–III non-small cell lung cancer (NSCLC) patients F. Grossi 1 , E. Rijavec 1 , M.G. Dal Bello 1 , G. Savarino 2 , C. Sini 1 , G. Barletta 1 , C. Genova 1 , M. Truini 3 , U. Pfeffer 2 , D.F. Merlo 4 . 1 Lung Cancer Unit, National Institute for Cancer Research, Genova, Italy, 2 Integrated Molecular Pathology, National Institute for Cancer Research, Genova, Italy, 3 Department of Pathology, National Institute for Cancer Research, Genova, Italy, 4 Unit of Epidemiology and Biostatistics, National Institute for Cancer Research, Genova, Italy Background: Biomarkers can help in identifying patients (pts) with early-stage NSCLC with high risk of relapse and poor prognosis. The aim of this study was to investigate the relationship between the levels of expression of 7 biomarkers, various clinicopathological characteristics and their prognostic significance. Material and methods: Tumor tissue from 82 radically resected stage I–III NSCLC pts were consecutively collected to investigate the mRNA expression and protein levels of the following biomarkers using quantitative reverse transcriptase real-time PCR (qRT-PCR) and immunohistochemistry (IHC) with a tissue microarray technique: excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase subunit 1 (RRM1), RRM2, p53R2, thymidylate synthase (TS) and class III beta-tubulin (-Tub-III). Results: On a univariate analysis, p53R2 expression was significantly higher in adenocarcinoma (ADK) compared to squamous cell carcinoma (SSC) samples (p = 0.002) and in stage I compared to stage II–III (p ≤ 0.001). ERCC1 expression was significantly higher in females compared to males (p = 0.03), and -Tub-III expression was significantly higher in ADK than in SSC (p = 0.03). Pts with lower RRM2 expression survived longer than pts with higher RRM2 expression (p = 0.069). The multivariate analysis confirmed RRM2 as an independent prognostic marker of shorter survival (p = 0.031). The comparison between survival curves with qRT-PCR and IHC showed similar results with a trend towards longer survival among ERCC1 negative pts, BRCA1 negative pts, p53R2 positive pts and among pts with low levels of RRM1 and RRM2, although the difference was not statistically significant with both methods. qRT-PCR and IHC have shown that -Tub-III and TS had no significant impact on survival. Conclusions: This is the first study that identifies RRM2 expression as a negative prognostic factor in resected stage I–III NSCLC. Moreover, we have demonstrated the differential expression of p53R2 and -Tub-III in ADK compared to SSC and higher expression of p53R2 in pts with stage I compared to stage II–III NSCLC. http://dx.doi.org/10.1016/j.lungcan.2012.05.038 In vitro culturing of circulating tumor cells in lung cancer management Katarína Koloˇstová 1 , Marián Liberko 1 , Eva Hroncová 1 , Robert M. Hoffman 2 , Vladimír Bobek 1,3 . 1 Department of Tumor Biology, Third Faculty of Medicine, Charles University Prague, Czech Republic, 2 Department of Surgery, UCSD San Diego and Anticancer Ltd., San Diego, CA, USA, 3 Department of Surgery, Third Faculty of Medicine and FacultyHospital Kralovske Vinohrady, Prague, Czech Republic Introduction: In the present study circulating tumor cells (CTCs) have been isolated from H460-RFP orthotopic human lung cancer model in mice. In vitro culture of CTCs has enabled us to introduce additional testing describing the CTC-character. The main hypothesis was to compare the chemosensitivity of tumor cells from primary H460 culture and of CTCs solated from blood grown in vitro. Character of CTCs in 2D-culture has been
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described beside other means by confocal microscopy – in vitro life imaging. Material and methods: Cell line H460-RFP of human lung cancer used in this study was grown in growing media to 70–80% confluence. First, the primary tumor has been grown after subcutaneous injection of primary cell culture in nude mice. Following, pieces of tumor tissue (1 mm × 1 mm) were implanted into the lungs of nude mice. The dissemination process of the tumor has been further monitored by UV-transiluminator. As soon as dissemination has been documented (14 days) the mice have been sacrificied and blood was taken by cardiac puncture for CTC-analysis. The blood (0.5 ml), obtained by cardiac puncture, was put into an EDTA tube (BD) and the CTC cells were enriched by immunomagnetic separation targeted to eptihelial antigens. The enriched fraction or whole blood was put into the cultivation flask with growing medium. To monitor the growth Leica TCS SP5 AOBS, DFC350 FX Digital Camera (confocal) and Olympus OV 100 were used. The growth inhibition has been tested by means of standard MTT-test and ×CELLigence system (RTDC, Roche) for different concetrations of cis-platinum, gemcitabine (incubation time 24–72 h). The isolated CTCs were profiled by means of gene expression for 24 markers. The samples were analyzed on microfluidic BioMark HD System using 48 × 48 Dynamic Array integrated fluidic circuits (Fluidigm, USA). Results: Based on the obtained data we may conclude that testing of the stem cell like character and drug resistance in the newly introduced in vitro cultures of CTCs could help to predict the therapy succes also in clinics. Conclusions: We assume that developing cultivation strategies for CTCs could bring us closer to the definition of the CTC-function in the whole dissemination process. http://dx.doi.org/10.1016/j.lungcan.2012.05.039 Targeted therapy of metastatic NSCLC in clinical setting at US Zdenko Krizan (M.D.). Community Cancer Center, Coldwater, Mi. Michigan State University, Lansing, MI, United States Exciting discoveries in basic science, especially in molecular biology and cytogenetics have shown great influence in the management of non small cell lung carcinoma (NSCLC). Histological classifications have been used to make therapeutic decisions regarding chemotherapeutic regimens for patients with metastatic disease. At present time over half of all lung adenocarcinomas can be also classified based on the presence of known genetic abnormalities. Some of these mutations have important therapeutic implications, with specific targeted therapeutic drugs that can be more effective and less toxic than traditional chemotherapies. Currently, the most important mutations in NSCLC are epidermal growth factor receptor (EGFR) mutations and fusions involving the anaplastic lymphoma kinase (ALK) gene. Others such as Kras gene mutation are associated with intrinsic tyrosine kinase inhibitor (TKI) resistance and could be useful for selection of patients for tyrosine kinase inhibitors (TKIs) therapy. Stimulation of the EGFR pathway leads to increased autophosporylation of tyrosinekinase pathway associated with EGFR. This leads to a series of intracellular events, with increased mitotic and growth potential, increased ability to metastasize, and increased angiogenesis. Gefitinib represents a class of EGFR TKIs that acts intracellularly to block activation of the EGFR pathway. Mok at al. presented a large study comparing gefitinib to carboplatin–paclitaxel as first line therapy. Asian patients with lung adenocarcinoma who never smoked, or were former (<15 years) light smokers, had higher response rates to gefitinib (43%)
Ribonucleotide reductase subunit 2 (RRM2) predicts shorter survival in resected stage I–III non-small cell lung cancer (NSCLC) patients F. Grossi 1 , E. Rijavec 1 , M.G. Dal Bello 1 , G. Savarino 2 , C. Sini 1 , G. Barletta 1 , C. Genova 1 , M. Truini 3 , U. Pfeffer 2 , D.F. Merlo 4 . 1 Lung Cancer Unit, National Institute for Cancer Research, Genova, Italy, 2 Integrated Molecular Pathology, National Institute for Cancer Research, Genova, Italy, 3 Department of Pathology, National Institute for Cancer Research, Genova, Italy, 4 Unit of Epidemiology and Biostatistics, National Institute for Cancer Research, Genova, Italy Background: Biomarkers can help in identifying patients (pts) with early-stage NSCLC with high risk of relapse and poor prognosis. The aim of this study was to investigate the relationship between the levels of expression of 7 biomarkers, various clinicopathological characteristics and their prognostic significance. Material and methods: Tumor tissue from 82 radically resected stage I–III NSCLC pts were consecutively collected to investigate the mRNA expression and protein levels of the following biomarkers using quantitative reverse transcriptase real-time PCR (qRT-PCR) and immunohistochemistry (IHC) with a tissue microarray technique: excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase subunit 1 (RRM1), RRM2, p53R2, thymidylate synthase (TS) and class III beta-tubulin (-Tub-III). Results: On a univariate analysis, p53R2 expression was significantly higher in adenocarcinoma (ADK) compared to squamous cell carcinoma (SSC) samples (p = 0.002) and in stage I compared to stage II–III (p ≤ 0.001). ERCC1 expression was significantly higher in females compared to males (p = 0.03), and -Tub-III expression was significantly higher in ADK than in SSC (p = 0.03). Pts with lower RRM2 expression survived longer than pts with higher RRM2 expression (p = 0.069). The multivariate analysis confirmed RRM2 as an independent prognostic marker of shorter survival (p = 0.031). The comparison between survival curves with qRT-PCR and IHC showed similar results with a trend towards longer survival among ERCC1 negative pts, BRCA1 negative pts, p53R2 positive pts and among pts with low levels of RRM1 and RRM2, although the difference was not statistically significant with both methods. qRT-PCR and IHC have shown that -Tub-III and TS had no significant impact on survival. Conclusions: This is the first study that identifies RRM2 expression as a negative prognostic factor in resected stage I–III NSCLC. Moreover, we have demonstrated the differential expression of p53R2 and -Tub-III in ADK compared to SSC and higher expression of p53R2 in pts with stage I compared to stage II–III NSCLC. http://dx.doi.org/10.1016/j.lungcan.2012.05.038 In vitro culturing of circulating tumor cells in lung cancer management Katarína Koloˇstová 1 , Marián Liberko 1 , Eva Hroncová 1 , Robert M. Hoffman 2 , Vladimír Bobek 1,3 . 1 Department of Tumor Biology, Third Faculty of Medicine, Charles University Prague, Czech Republic, 2 Department of Surgery, UCSD San Diego and Anticancer Ltd., San Diego, CA, USA, 3 Department of Surgery, Third Faculty of Medicine and FacultyHospital Kralovske Vinohrady, Prague, Czech Republic Introduction: In the present study circulating tumor cells (CTCs) have been isolated from H460-RFP orthotopic human lung cancer model in mice. In vitro culture of CTCs has enabled us to introduce additional testing describing the CTC-character. The main hypothesis was to compare the chemosensitivity of tumor cells from primary H460 culture and of CTCs solated from blood grown in vitro. Character of CTCs in 2D-culture has been
S23
described beside other means by confocal microscopy – in vitro life imaging. Material and methods: Cell line H460-RFP of human lung cancer used in this study was grown in growing media to 70–80% confluence. First, the primary tumor has been grown after subcutaneous injection of primary cell culture in nude mice. Following, pieces of tumor tissue (1 mm × 1 mm) were implanted into the lungs of nude mice. The dissemination process of the tumor has been further monitored by UV-transiluminator. As soon as dissemination has been documented (14 days) the mice have been sacrificied and blood was taken by cardiac puncture for CTC-analysis. The blood (0.5 ml), obtained by cardiac puncture, was put into an EDTA tube (BD) and the CTC cells were enriched by immunomagnetic separation targeted to eptihelial antigens. The enriched fraction or whole blood was put into the cultivation flask with growing medium. To monitor the growth Leica TCS SP5 AOBS, DFC350 FX Digital Camera (confocal) and Olympus OV 100 were used. The growth inhibition has been tested by means of standard MTT-test and ×CELLigence system (RTDC, Roche) for different concetrations of cis-platinum, gemcitabine (incubation time 24–72 h). The isolated CTCs were profiled by means of gene expression for 24 markers. The samples were analyzed on microfluidic BioMark HD System using 48 × 48 Dynamic Array integrated fluidic circuits (Fluidigm, USA). Results: Based on the obtained data we may conclude that testing of the stem cell like character and drug resistance in the newly introduced in vitro cultures of CTCs could help to predict the therapy succes also in clinics. Conclusions: We assume that developing cultivation strategies for CTCs could bring us closer to the definition of the CTC-function in the whole dissemination process. http://dx.doi.org/10.1016/j.lungcan.2012.05.039 Targeted therapy of metastatic NSCLC in clinical setting at US Zdenko Krizan (M.D.). Community Cancer Center, Coldwater, Mi. Michigan State University, Lansing, MI, United States Exciting discoveries in basic science, especially in molecular biology and cytogenetics have shown great influence in the management of non small cell lung carcinoma (NSCLC). Histological classifications have been used to make therapeutic decisions regarding chemotherapeutic regimens for patients with metastatic disease. At present time over half of all lung adenocarcinomas can be also classified based on the presence of known genetic abnormalities. Some of these mutations have important therapeutic implications, with specific targeted therapeutic drugs that can be more effective and less toxic than traditional chemotherapies. Currently, the most important mutations in NSCLC are epidermal growth factor receptor (EGFR) mutations and fusions involving the anaplastic lymphoma kinase (ALK) gene. Others such as Kras gene mutation are associated with intrinsic tyrosine kinase inhibitor (TKI) resistance and could be useful for selection of patients for tyrosine kinase inhibitors (TKIs) therapy. Stimulation of the EGFR pathway leads to increased autophosporylation of tyrosinekinase pathway associated with EGFR. This leads to a series of intracellular events, with increased mitotic and growth potential, increased ability to metastasize, and increased angiogenesis. Gefitinib represents a class of EGFR TKIs that acts intracellularly to block activation of the EGFR pathway. Mok at al. presented a large study comparing gefitinib to carboplatin–paclitaxel as first line therapy. Asian patients with lung adenocarcinoma who never smoked, or were former (<15 years) light smokers, had higher response rates to gefitinib (43%)