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Expression profile of EPHB3 and its prognostic significance in colorectal cancer progression (Running head: Prognostic value of EPHB3 in colorectal cancers)
abstracts
Annals of Oncology
grant / Funding (institution): Merck Serono; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Beigene; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Servier; Advisory / Consultancy, Research grant / Funding (institution): Lilly; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): Takeda; Advisory / Consultancy, Research grant / Funding (institution): Astellas; Advisory / Consultancy: Pierre Fabre; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Fibrogen; Research grant / Funding (institution): Amcure; Research grant / Funding (institution): Sierra Oncology; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Medimmune; Research grant / Funding (institution): BMS; Research grant / Funding (institution): MSD; Speaker Bureau / Expert testimony: Amgen; Speaker Bureau / Expert testimony: Foundation Medicine. All other authors have declared no conflicts of interest.
639P
Microsatellite instability detection in colorectal cancer: 44-Center comparison between the Idylla MSI assay and routine molecular and immunohistochemistry tests on formalin-fixed paraffin-embedded tissue
X. Matias-Guiu Pathology, Hospital Universitario Arnau de Vilanova, Lleida, Spain Background: Microsatellite instability (MSI) is observed in 15% of all colorectal cancers (CRC), including Lynch syndrome caused by germline mutation of an MMR gene. Guidelines recommend assessing the MSI status of all CRC patients for screening for Lynch syndrome as well as for prognostic stratification. MSI-high tumors are more vulnerable to the immune system and therefore generally have a better prognosis. Methods: The performance of the IdyllaTM MSI Assay was compared to routine immunohistochemistry (IHC) and molecular tests (including Bethesda panel) on 1,301 archival CRC formalin-fixed, paraffin-embedded (FFPE) tissue sections, at 44 centers worldwide. Results: The failure rate of the IdyllaTM MSI Assay was 0.23%, while the molecular methods had a higher failure rate overall of 0.86% probably due to their use of a longer amplicon. IHC had a much higher failure rate of 4.37%, possibly related to interpretation difficulties of the results. Routine method failure rates might be an underestimation as the current analysis was done retrospectively on samples with known routine results. Compared to IHC, the results of 37 of the 1,075 samples tested were discordant, resulting in an overall concordance agreement of 96.6%. When taking molecular results and further analysis of discordant samples into account, the IdyllaTM MSI result was confirmed in 16 samples. Compared to molecular methods, 16 of the 812 samples tested were discordant, resulting in an overall concordance agreement of 98.0%. Taking IHC results and further analysis of discordant samples into account, the IdyllaTM MSI result was confirmed in 8 samples. Conclusions: Results of the IdyllaTM MSI Assay were highly concordant with results of routine tests and lower failure rates were observed. Further advantages are the lack of need for matched normal tissue, the low dependence on pre-analytical conditions, the simplified workflow, the short turnaround times, the automated result interpretation, and the very limited hands-on work. Editorial acknowledgement: Teams involved in the MSI, Idylla MSI Assay, Colon Rectal Cancer, Multicenter Study. Legal entity responsible for the study: Irblleida. Funding: Biocartis. Disclosure: X. Matias-guiu: Non-remunerated activity/ies: Biocartis.
Volume 30 | Supplement 5 | October 2019
640P
Expression profile of EPHB3 and its prognostic significance in colorectal cancer progression (Running head: Prognostic value of EPHB3 in colorectal cancers)
B. Jang, H. Kim Pathology, Jeju National University School of Medicine, Jeju-si, Republic of Korea Background: A tumour suppressive role for EPHB receptors in various malignancies has been described, however their expression profile and prognostic significance in human colorectal cancers (CRCs) remain unclear. In this study, we aimed to investigate the expression profile of EPHB3 during CRC progression and determined its prognostic impact in a large cohort of CRC samples. Methods: We examined the EPHB3 mRNA levels by real time PCR with fresh frozen CRC samples and evaluated protein expression by immunohistochemistry with tissue microarrays containing various benign tumours and 610 CRC samples. AOM-DSS induced colitis model was used to investigate the EPHB3 expression profile during the colitis-associated carcinogenesis. Results: EPHB3 expression was upregulated in CRCs than in normal colonic mucosa, and showed positive correlations with intestinal stem cell (ISC) markers (EPHB2, OLFM4, LRIG1) and CD44, a candidate cancer stem cell marker. EPHB3 positivity was observed in 24% of 610 CRCs and showed negative correlations with differentiation, lympho-vascular invasions and TNM stages. EPHB3 expression significantly declined during adenoma-carcinoma transition and invasion into deeper layers. In particular, a substantial reduction of EPHB3 was observed in the budding cancer cells at the invasive fronts. In AOM-DSS induced colitis-associated colon cancer model, EPHB3 expression increased along with tumor development. Notably, EPHB3 was positively associated with microsatellite instability (MSI) phenotype but was not associated with CpG island methylator phenotype, KRAS and BRAF mutations. Furthermore, EPHB3 positivity was a prognostic marker for better clinical outcomes in CRC patients. Conclusions: EPHB3 was upregulated in CRCs and showed positive correlations with candidate cancer stem cell marker CD44. EPHB3 expression decreased during adenoma-carcinoma transition and particularly in the budding cancer cells at the invasive fronts. EPHB3 positivity was associated with MSI phenotype and demonstrated to be a good prognostic marker in CRCs. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
641P
A pan-ErbB family inhibitor, AF8c, promotes apoptosis by DR5/Nrf2 activation via ROS in colorectal cancer cells
S. Jeong1, H.K. Yun2, Y.A. Jeong2, D.Y. Kim2, M.J. Jo3, S.H. Park3, B.R. Kim1, J.L. Kim1, Y.J. Na3, D-H. Lee1, S.C. Oh4 1 Oncology/Hematology, Korea University Guro Hospital, Seoul, Republic of Korea, 2 Graduate School of Medicine, College of Medicine, Korea University, Seoul, Republic of Korea, 3Oncology, Korea University Guro Hospital, Seoul, Republic of Korea, 4 Department of Internal Medicine, Korea University Guro Hospital, Seoul, Republic of Korea Background: Multifunctional, antiproliferative small molecules are deemed to offer pharmacodynamic and pharmacokinetic benefits over combination therapy, in addition to reducing toxicity, cancer resistance, and therapy costs. In this study, we conducted an in vitro cellular screen of our recently reported series of EGFR/HER2 inhibitors. Methods: Cytotoxicity induced by AF8c was confirmed using WST-1 assay. An Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit, TUNEL assay, and Western blotting were carried out to invastigate apoptosis. A human phosphorylated kinase array was used to identify proteins differentially expressed between control cells and AF8c-treated cell lines. ROS generation was monitored by flow cytometry, confocal microscopy using dihydroethidium (DHE) and MitoSOX. For in vivo tumor xenograft study, four-week-old female BALB/c nude mice were injected subcutaneously with either HT29 Lucþ or HCT116 Lucþ cells (1 107 cells in 100 lL PBS). When the tumor had reached approximately 100 mm3 in size, the mice were randomly divided into three groups (n ¼ 8): DMSO-treated, treated with 10 mg/kg AF8c, and treated with 20 mg/kg AF8c. Results: Analysis of the AF8c mode of action in CRC cells revealed that it mediates apoptosis via the generation of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS), as well as selective activation of nuclear respiratory factor 2 alpha subunit (Nrf2) and death receptor 5 (DR5), but not DR4. Silencing of DR5 attenuated the expression levels of Nrf2 and partially inhibited AF8c-induced apoptosis. Additionally, upregulation of Nrf2 by AF8c evoked apoptosis through a decrease in antioxidant levels. Treatment of mice with AF8c also resulted in the upregulation of DR5, Nrf2, and CHOP proteins, subsequently leading to a significant decrease in tumor burden.
doi:10.1093/annonc/mdz246 | v241
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz246.117/5577989 by guest on 26 October 2019
sequenced with an internal customized panel. A comparison statistical test with matched tissue has been performed. Results: The success rate in CRC-PDOs establishment is around 80%. PDOs staining with H&E shows a morphology comparable to the original tissue. IHC shows ki67 staining, nuclear positivity for CDX2, cytoplasmic CK20 and MUC2/5. IF evidences the positivity for the intestinal stem cells marker Lgr5 and CDH1 thus confirms that PDOs faithfully recapitulate original tissue morphology and cell composition.Between 80 and 120 ng of genomic DNA has been sequenced. Spectrum of PDOs mutations and polimorphisms displays a good concordance with that of matched patients with an R of linear concordance of 0.9. Conclusions: We have developed a robust protocol for efficient development of CRC PDOs. Organoids recapitulate morphological and genomic features of the original patient. They work as a solid in vitromodel, suitable for functional studies and drug screening, helping in promoting precision medicine. Legal entity responsible for the study: The authors. Funding: Grants from the Instituto de Salud Carlos III (PI15/02180 to AC and PI18/ 01508 to TF). FP is supported by the ESMO 2018 fellowship programme. VG was supported by the ESMO 2014 fellowship programme and by Rio Ortega contract CM18/ 00241 from the Carlos III Health Institute; TF is supported by Joan Rodes contract 17/ 00026 from the Carlos III Health Institute. NT was supported by Rio Ortega contract CM15/00246 from the Carlos III Health Institute; DR was supported by Joan Rodes contract 16/00040 from the Carlos III Health Institute. MFGB is supported by a Santiago Grisolia fellowship. Disclosure: A. Cervantes: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research
Annals of Oncology
grant / Funding (institution): Merck Serono; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Beigene; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Servier; Advisory / Consultancy, Research grant / Funding (institution): Lilly; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): Takeda; Advisory / Consultancy, Research grant / Funding (institution): Astellas; Advisory / Consultancy: Pierre Fabre; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Fibrogen; Research grant / Funding (institution): Amcure; Research grant / Funding (institution): Sierra Oncology; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Medimmune; Research grant / Funding (institution): BMS; Research grant / Funding (institution): MSD; Speaker Bureau / Expert testimony: Amgen; Speaker Bureau / Expert testimony: Foundation Medicine. All other authors have declared no conflicts of interest.
639P
Microsatellite instability detection in colorectal cancer: 44-Center comparison between the Idylla MSI assay and routine molecular and immunohistochemistry tests on formalin-fixed paraffin-embedded tissue
X. Matias-Guiu Pathology, Hospital Universitario Arnau de Vilanova, Lleida, Spain Background: Microsatellite instability (MSI) is observed in 15% of all colorectal cancers (CRC), including Lynch syndrome caused by germline mutation of an MMR gene. Guidelines recommend assessing the MSI status of all CRC patients for screening for Lynch syndrome as well as for prognostic stratification. MSI-high tumors are more vulnerable to the immune system and therefore generally have a better prognosis. Methods: The performance of the IdyllaTM MSI Assay was compared to routine immunohistochemistry (IHC) and molecular tests (including Bethesda panel) on 1,301 archival CRC formalin-fixed, paraffin-embedded (FFPE) tissue sections, at 44 centers worldwide. Results: The failure rate of the IdyllaTM MSI Assay was 0.23%, while the molecular methods had a higher failure rate overall of 0.86% probably due to their use of a longer amplicon. IHC had a much higher failure rate of 4.37%, possibly related to interpretation difficulties of the results. Routine method failure rates might be an underestimation as the current analysis was done retrospectively on samples with known routine results. Compared to IHC, the results of 37 of the 1,075 samples tested were discordant, resulting in an overall concordance agreement of 96.6%. When taking molecular results and further analysis of discordant samples into account, the IdyllaTM MSI result was confirmed in 16 samples. Compared to molecular methods, 16 of the 812 samples tested were discordant, resulting in an overall concordance agreement of 98.0%. Taking IHC results and further analysis of discordant samples into account, the IdyllaTM MSI result was confirmed in 8 samples. Conclusions: Results of the IdyllaTM MSI Assay were highly concordant with results of routine tests and lower failure rates were observed. Further advantages are the lack of need for matched normal tissue, the low dependence on pre-analytical conditions, the simplified workflow, the short turnaround times, the automated result interpretation, and the very limited hands-on work. Editorial acknowledgement: Teams involved in the MSI, Idylla MSI Assay, Colon Rectal Cancer, Multicenter Study. Legal entity responsible for the study: Irblleida. Funding: Biocartis. Disclosure: X. Matias-guiu: Non-remunerated activity/ies: Biocartis.
Volume 30 | Supplement 5 | October 2019
640P
Expression profile of EPHB3 and its prognostic significance in colorectal cancer progression (Running head: Prognostic value of EPHB3 in colorectal cancers)
B. Jang, H. Kim Pathology, Jeju National University School of Medicine, Jeju-si, Republic of Korea Background: A tumour suppressive role for EPHB receptors in various malignancies has been described, however their expression profile and prognostic significance in human colorectal cancers (CRCs) remain unclear. In this study, we aimed to investigate the expression profile of EPHB3 during CRC progression and determined its prognostic impact in a large cohort of CRC samples. Methods: We examined the EPHB3 mRNA levels by real time PCR with fresh frozen CRC samples and evaluated protein expression by immunohistochemistry with tissue microarrays containing various benign tumours and 610 CRC samples. AOM-DSS induced colitis model was used to investigate the EPHB3 expression profile during the colitis-associated carcinogenesis. Results: EPHB3 expression was upregulated in CRCs than in normal colonic mucosa, and showed positive correlations with intestinal stem cell (ISC) markers (EPHB2, OLFM4, LRIG1) and CD44, a candidate cancer stem cell marker. EPHB3 positivity was observed in 24% of 610 CRCs and showed negative correlations with differentiation, lympho-vascular invasions and TNM stages. EPHB3 expression significantly declined during adenoma-carcinoma transition and invasion into deeper layers. In particular, a substantial reduction of EPHB3 was observed in the budding cancer cells at the invasive fronts. In AOM-DSS induced colitis-associated colon cancer model, EPHB3 expression increased along with tumor development. Notably, EPHB3 was positively associated with microsatellite instability (MSI) phenotype but was not associated with CpG island methylator phenotype, KRAS and BRAF mutations. Furthermore, EPHB3 positivity was a prognostic marker for better clinical outcomes in CRC patients. Conclusions: EPHB3 was upregulated in CRCs and showed positive correlations with candidate cancer stem cell marker CD44. EPHB3 expression decreased during adenoma-carcinoma transition and particularly in the budding cancer cells at the invasive fronts. EPHB3 positivity was associated with MSI phenotype and demonstrated to be a good prognostic marker in CRCs. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
641P
A pan-ErbB family inhibitor, AF8c, promotes apoptosis by DR5/Nrf2 activation via ROS in colorectal cancer cells
S. Jeong1, H.K. Yun2, Y.A. Jeong2, D.Y. Kim2, M.J. Jo3, S.H. Park3, B.R. Kim1, J.L. Kim1, Y.J. Na3, D-H. Lee1, S.C. Oh4 1 Oncology/Hematology, Korea University Guro Hospital, Seoul, Republic of Korea, 2 Graduate School of Medicine, College of Medicine, Korea University, Seoul, Republic of Korea, 3Oncology, Korea University Guro Hospital, Seoul, Republic of Korea, 4 Department of Internal Medicine, Korea University Guro Hospital, Seoul, Republic of Korea Background: Multifunctional, antiproliferative small molecules are deemed to offer pharmacodynamic and pharmacokinetic benefits over combination therapy, in addition to reducing toxicity, cancer resistance, and therapy costs. In this study, we conducted an in vitro cellular screen of our recently reported series of EGFR/HER2 inhibitors. Methods: Cytotoxicity induced by AF8c was confirmed using WST-1 assay. An Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit, TUNEL assay, and Western blotting were carried out to invastigate apoptosis. A human phosphorylated kinase array was used to identify proteins differentially expressed between control cells and AF8c-treated cell lines. ROS generation was monitored by flow cytometry, confocal microscopy using dihydroethidium (DHE) and MitoSOX. For in vivo tumor xenograft study, four-week-old female BALB/c nude mice were injected subcutaneously with either HT29 Lucþ or HCT116 Lucþ cells (1 107 cells in 100 lL PBS). When the tumor had reached approximately 100 mm3 in size, the mice were randomly divided into three groups (n ¼ 8): DMSO-treated, treated with 10 mg/kg AF8c, and treated with 20 mg/kg AF8c. Results: Analysis of the AF8c mode of action in CRC cells revealed that it mediates apoptosis via the generation of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS), as well as selective activation of nuclear respiratory factor 2 alpha subunit (Nrf2) and death receptor 5 (DR5), but not DR4. Silencing of DR5 attenuated the expression levels of Nrf2 and partially inhibited AF8c-induced apoptosis. Additionally, upregulation of Nrf2 by AF8c evoked apoptosis through a decrease in antioxidant levels. Treatment of mice with AF8c also resulted in the upregulation of DR5, Nrf2, and CHOP proteins, subsequently leading to a significant decrease in tumor burden.
doi:10.1093/annonc/mdz246 | v241
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz246.117/5577989 by guest on 26 October 2019
sequenced with an internal customized panel. A comparison statistical test with matched tissue has been performed. Results: The success rate in CRC-PDOs establishment is around 80%. PDOs staining with H&E shows a morphology comparable to the original tissue. IHC shows ki67 staining, nuclear positivity for CDX2, cytoplasmic CK20 and MUC2/5. IF evidences the positivity for the intestinal stem cells marker Lgr5 and CDH1 thus confirms that PDOs faithfully recapitulate original tissue morphology and cell composition.Between 80 and 120 ng of genomic DNA has been sequenced. Spectrum of PDOs mutations and polimorphisms displays a good concordance with that of matched patients with an R of linear concordance of 0.9. Conclusions: We have developed a robust protocol for efficient development of CRC PDOs. Organoids recapitulate morphological and genomic features of the original patient. They work as a solid in vitromodel, suitable for functional studies and drug screening, helping in promoting precision medicine. Legal entity responsible for the study: The authors. Funding: Grants from the Instituto de Salud Carlos III (PI15/02180 to AC and PI18/ 01508 to TF). FP is supported by the ESMO 2018 fellowship programme. VG was supported by the ESMO 2014 fellowship programme and by Rio Ortega contract CM18/ 00241 from the Carlos III Health Institute; TF is supported by Joan Rodes contract 17/ 00026 from the Carlos III Health Institute. NT was supported by Rio Ortega contract CM15/00246 from the Carlos III Health Institute; DR was supported by Joan Rodes contract 16/00040 from the Carlos III Health Institute. MFGB is supported by a Santiago Grisolia fellowship. Disclosure: A. Cervantes: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research