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Endothelial nitric oxide synthase in colorectal cancer: A potential new therapeutic target in stem cell-like poor-prognosis subtype
S46
Poster abstracts
expression of EPCAM, MUC1 and ERBB2 was studied using multiplexPCR. CTC positivity and cutoffs, obtained by ROC curve analysis in healthy donors, were: 1 positive marker among EPCAM (0.40 ng/ml), MUC1 (0.10 ng/ml) and ERBB2 (0.20 ng/ml). CTC variation (t0/t2) was split in favorable (+/−, −/−, −/+) and unfavorable group (+/+) due to small numbers. Univariable analyses were undertaken for progression-free (PFS) and overall survival (OS). Multivariable analyses with bivariable associations with clinical factors were also done to improve understanding of effects. Results: Among the 31 analyzed pts, 17 (54.8%) were CTC+ at t0 and no association was found with any baseline pt and tumor characteristic, as well as with CTC status and objective response to MVAC. Unfavorable CTC trend was observed in 10/26 (38.5%) cases. CTC dynamic changes better predicted for 3-year (3y) PFS and OS probability compared to CTC status assessed at single time points. Unfavorable trend was univariably detrimental on both 3y PFS probability (10% vs 49.2%, p = 0.006) and 3y OS probability (20% vs 63.5%, p = 0.017). Significance was maintained after adjusting for liver metastases (p = 0.031 and p = 0.025 for PFS and OS) and MSKCC risk score (p = 0.014 and 0.025). Conclusions: We proposed a novel technique to early assess CTC status in metastatic UC receiving MVAC CT. Early CTC changes may be useful to improve our prognostic ability. Pending validation, these results may lead to improved trial designs and to refine the sequence of conventional CT options in the clinical setting. No conflict of interest. 122 Poster (Board P093) An evaluation of the association between molecular signature and postoperative recurrence in patients with non-small cell lung cancer A. Ono1 , M. Serizawa2 , M. Isaka3 , H. Kojima3 , S. Takahashi3 , K. Nakashima1 , S. Omori1 , K. Wakuda1 , H. Kenmotsu1 , T. Naito1 , H. Murakami1 , K. Urakami2 , Y. Ohde3 , T. Nakajima4 , M. Kusuhara2 , T. Takahashi1 , K. Yamaguchi5 . 1 Shizuoka Cancer Center, Division of Thoracic Oncology, Nagaizumi-cho, Shizuoka, Japan; 2 Shizuoka Cancer Center, Research Institute, Nagaizumi-cho, Shizuoka, Japan; 3 Shizuoka Cancer Center, Division of Thoracic Surgery, Nagaizumi-cho, Shizuoka, Japan; 4 Shizuoka Cancer Center, Division of Diagnostic Pathology, Nagaizumi-cho, Shizuoka, Japan; 5 Shizuoka Cancer Center, Hospital and Research Institute, Nagaizumi-cho, Shizuoka, Japan Background: Shizuoka Cancer Center launched the first prospective pancancer molecular profiling study in Japan in January 2014, identifying patient-specific molecular signatures via multi-omics analysis, with a view to developing cancer precision medicine. By May 2016, 2,683 patients (pts) were enrolled, and whole-exome sequencing (WES) for over 2,100 pts had been completed. This study aims to assess the association between molecular signature and clinical information in pts with postoperative recurrence (p-rec) of non-small cell lung cancer (NSCLC) for identification of novel prognostic factors, focusing on the association with somatic mutational burden. Material and Methods: Between September 2014 and September 2015, 247 pts with NSCLC, including 192 pts with adenocarcinoma (Ad) and 55 pts with squamous cell carcinoma (Sq), underwent surgery and were enrolled in this study. Surgically resected tissue was subjected to WES using an ion torrent proton platform (Thermo Fisher scientific). Mutations (mt) detected in 138 cancer-related genes listed in Vogelstein et al. [1] were evaluated as driver mt. Results: P-rec was observed in 26 (13.5%) and 13 (23.6%) pts with Ad and Sq, respectively. Median time of p-rec (range) was 274 days (102–749). Patient background [recurrence (rec); non-recurrence (non-rec)]: median age (range) 72 (52−87); 69 (39−87), male 64%; 63%, smoker 77%; 70%, pathological stage (p-stage) (I/II/III) 41/33/26%; 72/19/9%, histological type (Ad/Sq) 67%/33%; 80%/20%, driver mutation (presence/absence) 95%/5%; 82%/18%, median somatic mutational burden (range) 2.7 mt/Mb (0.2–17.4); 1.7 mt/Mb (0.1–61.4). In the rec group, the most common driver mutation was TP53 mutation (54%, 21/39 pts). In Fisher’s exact test, the presence or absence of p-rec showed trend of association with presence or absence of driver mt (ie. driver mutation status) (p = 0.055) and significantly association with p-stage (I/II, III) (p = 0.0003), but not with histology type (p = 0.09), smoking status (p = 0.44), gender (p = 0.99) or age (<70/70) (p = 0.22). Multiple logistic regression analysis also revealed trend of association between driver mutation status and p-rec (p = 0.052, odds ratio [OR]: 3.56, 95% CI 0.98–22.9), when adjusted by histological type (p = 0.40), smoking status (p = 0.26), gender (p = 0.25), age (p = 0.15) and p-stage (p = 0.0002, OR: 4.12, 95% CI 1.97–8.93). The somatic mutational burden did not differ significantly between the rec and non-rec groups (p = 0.35). Conclusions: Driver mutation status may be associated with p-rec of NSCLC. References [1] Science. 2013; 339: 1546–1558. No conflict of interest.
Poster Session – Other, Wednesday 29 November 2016 123 Poster (Board P094) Endothelial nitric oxide synthase in colorectal cancer: A potential new therapeutic target in stem cell-like poor-prognosis subtype 1 1 J. Penarando ˜ , R. Mena1 , S. Guil1 , L.M. Lopez-S ´ anchez ´ , C. Villar2 , 2 , M. Centeno2 , A. Jimenez ´ Arranz1 , J. Gomez ´ Barbadillo3 , R. Sanchez ´ C. D´ıaz3 , J. De la Haba-Rodr´ıguez1 , E. Aranda1 , K. Myant4 , A. Rodr´ıguez ´ Institute of Biomedical Research IMIBIC, Reina Sof´ıa Ariza1 . 1 Maimonides ´ Hospital, University of Cordoba, Oncology, Cordoba, Spain; 2 Pathology ´ Department, Reina Sof´ıa Hospital, Pathology, Cordoba, Spain; 3 Surgery ´ department, Reina Sof´ıa Hospital, Surgery, Cordoba, Spain; 4 Edinburgh Cancer Research Centre, Oncology, Edinburgh, United Kingdom
Background: Nitric oxide (NO) has been highlighted as an important factor in tumor processes. Although the inducible nitric oxide synthase (iNOS) form has received most of the attention, recent studies indicate that endothelial form (eNOS) can also modulate different tumor processes including angiogenesis, invasion, and metastasis. However, the role of eNOS in cancer stem cell (CSC) biology is almost unknown, so it is essential to elucidate its potential implication in tumorigenesis. Material and Methods: The mouse model VIL-CRE-ERT2-APCfl/fl was used to obtain the histological sections and the 3D in vitro organoid culture. Carboxy-PTIO (c-PTIO) was used to selectively remove NO in organoid cultures and proliferation was determined by analyzing the organoid size with ImageJ software. Stem cell markers and nitric oxide synthases expression in organoids was analyzed by RT-qPCR. eNOS levels in mice and human tumors were determined by immunohistochemistry of histological sections. NO production was evaluated by using the fluorescent probe 1,2 diaminoanthraquinone (DAQ) in mice cryosections. We classified 40 human colorectal tumors from patients over 18 years with resectable colorectal cancer (CRC) and the expression analysis was performed using the nCounter system by NanoString. Chemoresistant cell lines were generated in our lab through progressive exposures to chemotherapy (5-FU+Oxaliplatin) Results: Here, we show that NO removal with the NO scavenger c-PTIO decreased the proliferation of intestinal Apcfl/fl organoids from VIL-CRE-ERT2-APCfl/fl mouse model and this effect was higher than in WT organoids. Besides, NO scavenging decreased the expression of stem cell markers such as Lgr5, Troy, Vav3 and Slc14a1 in intestinal Apcfl/fl organoids. The expression of eNOS was elevated in intestinal Apcfl/fl organoids and tissue, where the immunostaining was found in both the epithelial and non-epithelial compartments of intestinal crypts. Furthermore, we found a higher production of NO in Apcfl/fl crypts. The classification into five molecular subtypes using different genetic signatures showed a higher eNOS expression at RNA and protein levels in the stem cell-like subtype, which is characterized by poor prognosis and survival. We also found a high expression of eNOS in advanced (T4) and poorly differentiated human tumors. Finally, chemoresistant (5-FU+oxaliplatin) human cancer cell lines (HCT116 and DLD1) generated in our laboratory showed higher eNOS expression compared with parental chemo-sensitive cells. Conclusion: Our data show that eNOS is a new and unexpected potential new target in poor-prognosis stem cell-like colorectal tumors. Conflict of interest: Funded by ISClll (PI13–00553 and RD12/0036/0038). 124 Poster (Board P095) Revisiting the roles of TP53, IDH1/IDH2, BRAF, H3F3A, HIST1H3B as diagnostic markers and their clinical significance in glioma tumorigenesis P.P. Sarma1 , K.K. Saikia1 , D. Dutta2 , P. Chandra3 . 1 Gauhati University, Bioengineering & Technology, Guwahati, India; 2 Gauhati Medical College and Hospital, Neurosurgery, Guwahati, India; 3 Hamad Medical Corporation, Medical Research Center, Doha, Qatar Background: With the advent of novel technology platforms molecular biomarkers have proved to be of immense importance in diagnosis and management of glioma. The objective of this study was to re-evaluate the potential of known glioma markers as potential diagnostic and prognostic factors in patients with glioma. Patients and Methods: Non duplicate 55 newly diagnosed glioma patients, who underwent surgery at the Neurosurgery Department, Gauhati Medical College and Hospital, were considered for this study. Written informed consent from all patients was collected post surgery and the study was approved by the institutional ethics committee of Gauhati University. Nucleic acids were extracted using commercially available kit and used to identify genetic alterations present in the studied biomarkers. Presence of SNPs in exon5−8 of TP53, exon 4 of IDH1 and IDH2, exon 11 and 15 of BRAF, and exon 1 of H3F3A and HIST1H3B genes were screened using PCR-sequencing assay. Expression pattern of EGFR gene was studied
Poster abstracts
expression of EPCAM, MUC1 and ERBB2 was studied using multiplexPCR. CTC positivity and cutoffs, obtained by ROC curve analysis in healthy donors, were: 1 positive marker among EPCAM (0.40 ng/ml), MUC1 (0.10 ng/ml) and ERBB2 (0.20 ng/ml). CTC variation (t0/t2) was split in favorable (+/−, −/−, −/+) and unfavorable group (+/+) due to small numbers. Univariable analyses were undertaken for progression-free (PFS) and overall survival (OS). Multivariable analyses with bivariable associations with clinical factors were also done to improve understanding of effects. Results: Among the 31 analyzed pts, 17 (54.8%) were CTC+ at t0 and no association was found with any baseline pt and tumor characteristic, as well as with CTC status and objective response to MVAC. Unfavorable CTC trend was observed in 10/26 (38.5%) cases. CTC dynamic changes better predicted for 3-year (3y) PFS and OS probability compared to CTC status assessed at single time points. Unfavorable trend was univariably detrimental on both 3y PFS probability (10% vs 49.2%, p = 0.006) and 3y OS probability (20% vs 63.5%, p = 0.017). Significance was maintained after adjusting for liver metastases (p = 0.031 and p = 0.025 for PFS and OS) and MSKCC risk score (p = 0.014 and 0.025). Conclusions: We proposed a novel technique to early assess CTC status in metastatic UC receiving MVAC CT. Early CTC changes may be useful to improve our prognostic ability. Pending validation, these results may lead to improved trial designs and to refine the sequence of conventional CT options in the clinical setting. No conflict of interest. 122 Poster (Board P093) An evaluation of the association between molecular signature and postoperative recurrence in patients with non-small cell lung cancer A. Ono1 , M. Serizawa2 , M. Isaka3 , H. Kojima3 , S. Takahashi3 , K. Nakashima1 , S. Omori1 , K. Wakuda1 , H. Kenmotsu1 , T. Naito1 , H. Murakami1 , K. Urakami2 , Y. Ohde3 , T. Nakajima4 , M. Kusuhara2 , T. Takahashi1 , K. Yamaguchi5 . 1 Shizuoka Cancer Center, Division of Thoracic Oncology, Nagaizumi-cho, Shizuoka, Japan; 2 Shizuoka Cancer Center, Research Institute, Nagaizumi-cho, Shizuoka, Japan; 3 Shizuoka Cancer Center, Division of Thoracic Surgery, Nagaizumi-cho, Shizuoka, Japan; 4 Shizuoka Cancer Center, Division of Diagnostic Pathology, Nagaizumi-cho, Shizuoka, Japan; 5 Shizuoka Cancer Center, Hospital and Research Institute, Nagaizumi-cho, Shizuoka, Japan Background: Shizuoka Cancer Center launched the first prospective pancancer molecular profiling study in Japan in January 2014, identifying patient-specific molecular signatures via multi-omics analysis, with a view to developing cancer precision medicine. By May 2016, 2,683 patients (pts) were enrolled, and whole-exome sequencing (WES) for over 2,100 pts had been completed. This study aims to assess the association between molecular signature and clinical information in pts with postoperative recurrence (p-rec) of non-small cell lung cancer (NSCLC) for identification of novel prognostic factors, focusing on the association with somatic mutational burden. Material and Methods: Between September 2014 and September 2015, 247 pts with NSCLC, including 192 pts with adenocarcinoma (Ad) and 55 pts with squamous cell carcinoma (Sq), underwent surgery and were enrolled in this study. Surgically resected tissue was subjected to WES using an ion torrent proton platform (Thermo Fisher scientific). Mutations (mt) detected in 138 cancer-related genes listed in Vogelstein et al. [1] were evaluated as driver mt. Results: P-rec was observed in 26 (13.5%) and 13 (23.6%) pts with Ad and Sq, respectively. Median time of p-rec (range) was 274 days (102–749). Patient background [recurrence (rec); non-recurrence (non-rec)]: median age (range) 72 (52−87); 69 (39−87), male 64%; 63%, smoker 77%; 70%, pathological stage (p-stage) (I/II/III) 41/33/26%; 72/19/9%, histological type (Ad/Sq) 67%/33%; 80%/20%, driver mutation (presence/absence) 95%/5%; 82%/18%, median somatic mutational burden (range) 2.7 mt/Mb (0.2–17.4); 1.7 mt/Mb (0.1–61.4). In the rec group, the most common driver mutation was TP53 mutation (54%, 21/39 pts). In Fisher’s exact test, the presence or absence of p-rec showed trend of association with presence or absence of driver mt (ie. driver mutation status) (p = 0.055) and significantly association with p-stage (I/II, III) (p = 0.0003), but not with histology type (p = 0.09), smoking status (p = 0.44), gender (p = 0.99) or age (<70/70) (p = 0.22). Multiple logistic regression analysis also revealed trend of association between driver mutation status and p-rec (p = 0.052, odds ratio [OR]: 3.56, 95% CI 0.98–22.9), when adjusted by histological type (p = 0.40), smoking status (p = 0.26), gender (p = 0.25), age (p = 0.15) and p-stage (p = 0.0002, OR: 4.12, 95% CI 1.97–8.93). The somatic mutational burden did not differ significantly between the rec and non-rec groups (p = 0.35). Conclusions: Driver mutation status may be associated with p-rec of NSCLC. References [1] Science. 2013; 339: 1546–1558. No conflict of interest.
Poster Session – Other, Wednesday 29 November 2016 123 Poster (Board P094) Endothelial nitric oxide synthase in colorectal cancer: A potential new therapeutic target in stem cell-like poor-prognosis subtype 1 1 J. Penarando ˜ , R. Mena1 , S. Guil1 , L.M. Lopez-S ´ anchez ´ , C. Villar2 , 2 , M. Centeno2 , A. Jimenez ´ Arranz1 , J. Gomez ´ Barbadillo3 , R. Sanchez ´ C. D´ıaz3 , J. De la Haba-Rodr´ıguez1 , E. Aranda1 , K. Myant4 , A. Rodr´ıguez ´ Institute of Biomedical Research IMIBIC, Reina Sof´ıa Ariza1 . 1 Maimonides ´ Hospital, University of Cordoba, Oncology, Cordoba, Spain; 2 Pathology ´ Department, Reina Sof´ıa Hospital, Pathology, Cordoba, Spain; 3 Surgery ´ department, Reina Sof´ıa Hospital, Surgery, Cordoba, Spain; 4 Edinburgh Cancer Research Centre, Oncology, Edinburgh, United Kingdom
Background: Nitric oxide (NO) has been highlighted as an important factor in tumor processes. Although the inducible nitric oxide synthase (iNOS) form has received most of the attention, recent studies indicate that endothelial form (eNOS) can also modulate different tumor processes including angiogenesis, invasion, and metastasis. However, the role of eNOS in cancer stem cell (CSC) biology is almost unknown, so it is essential to elucidate its potential implication in tumorigenesis. Material and Methods: The mouse model VIL-CRE-ERT2-APCfl/fl was used to obtain the histological sections and the 3D in vitro organoid culture. Carboxy-PTIO (c-PTIO) was used to selectively remove NO in organoid cultures and proliferation was determined by analyzing the organoid size with ImageJ software. Stem cell markers and nitric oxide synthases expression in organoids was analyzed by RT-qPCR. eNOS levels in mice and human tumors were determined by immunohistochemistry of histological sections. NO production was evaluated by using the fluorescent probe 1,2 diaminoanthraquinone (DAQ) in mice cryosections. We classified 40 human colorectal tumors from patients over 18 years with resectable colorectal cancer (CRC) and the expression analysis was performed using the nCounter system by NanoString. Chemoresistant cell lines were generated in our lab through progressive exposures to chemotherapy (5-FU+Oxaliplatin) Results: Here, we show that NO removal with the NO scavenger c-PTIO decreased the proliferation of intestinal Apcfl/fl organoids from VIL-CRE-ERT2-APCfl/fl mouse model and this effect was higher than in WT organoids. Besides, NO scavenging decreased the expression of stem cell markers such as Lgr5, Troy, Vav3 and Slc14a1 in intestinal Apcfl/fl organoids. The expression of eNOS was elevated in intestinal Apcfl/fl organoids and tissue, where the immunostaining was found in both the epithelial and non-epithelial compartments of intestinal crypts. Furthermore, we found a higher production of NO in Apcfl/fl crypts. The classification into five molecular subtypes using different genetic signatures showed a higher eNOS expression at RNA and protein levels in the stem cell-like subtype, which is characterized by poor prognosis and survival. We also found a high expression of eNOS in advanced (T4) and poorly differentiated human tumors. Finally, chemoresistant (5-FU+oxaliplatin) human cancer cell lines (HCT116 and DLD1) generated in our laboratory showed higher eNOS expression compared with parental chemo-sensitive cells. Conclusion: Our data show that eNOS is a new and unexpected potential new target in poor-prognosis stem cell-like colorectal tumors. Conflict of interest: Funded by ISClll (PI13–00553 and RD12/0036/0038). 124 Poster (Board P095) Revisiting the roles of TP53, IDH1/IDH2, BRAF, H3F3A, HIST1H3B as diagnostic markers and their clinical significance in glioma tumorigenesis P.P. Sarma1 , K.K. Saikia1 , D. Dutta2 , P. Chandra3 . 1 Gauhati University, Bioengineering & Technology, Guwahati, India; 2 Gauhati Medical College and Hospital, Neurosurgery, Guwahati, India; 3 Hamad Medical Corporation, Medical Research Center, Doha, Qatar Background: With the advent of novel technology platforms molecular biomarkers have proved to be of immense importance in diagnosis and management of glioma. The objective of this study was to re-evaluate the potential of known glioma markers as potential diagnostic and prognostic factors in patients with glioma. Patients and Methods: Non duplicate 55 newly diagnosed glioma patients, who underwent surgery at the Neurosurgery Department, Gauhati Medical College and Hospital, were considered for this study. Written informed consent from all patients was collected post surgery and the study was approved by the institutional ethics committee of Gauhati University. Nucleic acids were extracted using commercially available kit and used to identify genetic alterations present in the studied biomarkers. Presence of SNPs in exon5−8 of TP53, exon 4 of IDH1 and IDH2, exon 11 and 15 of BRAF, and exon 1 of H3F3A and HIST1H3B genes were screened using PCR-sequencing assay. Expression pattern of EGFR gene was studied