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Monitoring metastatic melanoma treatment resistance using circulating tumour DNA
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EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
validated acceptance criteria. Amplification profile for EpCAM gene expression identified the presence of CTCs in specific wells in qualitative manner. Conclusions: Following capture of CTCs using the Parsortix system, gentle contactless cell transfer using the Echo liquid handler enabled standardisation of CTC enrichment allowing detection by qPCR. Miniaturisation of reagents volumes greatly reduced the cost of the full workflow. This work provided a proof of concept that used the Parsortix system and Echo liquid handler as an end-to-end platform to capture, enrich and profile CTCs. No conflict of interest. 851 Association of angiopoietin-2 and Ki-67 expression with vascular density and sunitinib response in metastatic renal cell carcinoma A. Lampinen1 , J. Rautiola2 , T. Mirtti3 , A. Ristimaki ¨ 4 , H. Joensuu2 , P. Bono2 , P. Saharinen5 . 1 University of Helsinki, Research Programs Unit- Translational Cancer Biology Program, Helsinki, Finland, 2 Helsinki University Central Hospital, Comprehensive Cancer Center, Helsinki, Finland, 3 University of Helsinki, Institute for Molecular Medicine Finland, Helsinki, Finland, 4 Helsinki University Central Hospital, Department of Pathology, Helsinki, Finland, 5 University of Helsinki, Translational Cancer Biology Program and Wihuri Research Institute, Helsinki, Finland Background: The angiopoietin-2 (Ang2, Angpt2) growth factor is a contextdependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase known to promote tumour angiogenesis and metastasis. In clinical cancer trials, angiopoietin antagonists are combined with VEGF-based antiangiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. The purpose of this study was to correlate the baseline expression of endothelial angiopoietin-2 (Ang2), CD31 and the cell proliferation marker Ki-67 to the outcome of first-line sunitinib therapy in mRCC. Materials and Methods: We evaluated using immunohistochemistry pretherapeutic Ang2, CD31 and Ki67 expression in formalin-fixed paraffinembedded tumor samples from 136 patients with metastatic RCC, who were subsequently treated with first-line sunitinib. Results: Ang2 was exclusively expressed in the endothelial cells of tumor blood vessels. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS 15.7 vs. 28.5 months, P = 0.015). Conclusions: In summary, high cancer Ang2 expression was associated with high CBR, but not with PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS in mRCC patients treated with first-line sunitinib. No conflict of interest. 852 Combined anti-MET/EGFR treatment results in complete tumor regression and prevents resistance onset in a MET-amplified gastroesophageal xenopatient cohort S. Giordano1 , M. Apicella1 , C. Migliore1 , T. Capeloa2 , S. Menegon2 , M. Cargnelutti1 , A. Sapino3 , P. Cassoni4 , S. Marsoni5 , S. Corso1 . 1 University of Torino- Candiolo Cancer Institute- FPO-IRCCS- Candiolo- Italy, Department of Oncology, Candiolo, Italy, 2 University of Torino- Candiolo Cancer InstituteFPO-IRCCS- Candiolo- Italy, Department of clinical and biological sciences, Candiolo, Italy, 3 University of Torino- Candiolo Cancer Institute- FPO-IRCCSCandiolo- Italy, Department of Medical Sciences, Candiolo, Italy, 4 University of Torino- Italy, Department of Medical Sciences, Candiolo, Italy, 5 Candiolo Cancer Institute- FPO-IRCCS- Candiolo- Italy, clinical trials, Candiolo, Italy Introduction: Gastric cancer is the world third leading cause of cancer mortality. In spite of the significant therapeutic advances, the overall clinical outcome for patients with advanced gastric cancer is poor, with 5−20% 5-year survival. The only targeted therapy approved so far are trastuzumab, and Ramucirumab which have given unsatisfactory results. Around 50% of gastric tumors bear genetic alterations affecting tyrosine kinase pathways (mainly HER2, EGFR, HER3, FGFR2 and MET pathways) but their clinical validation as tumor drivers is missing. The need for new therapeutic options and the possible presence of ‘druggable’ targets prompted us to investigate potential targeted therapies for this disease. Material and Methods: We generated a platform of gastroesophageal tumor patient-derived xenografts (PDXs), in which tumor surgical specimens are directly transferred into mice. Upon engraftment, the tumor is split and reimplanted in a cohort of mice, allowing the simultaneous testing of different drugs on the same tumor. Thanks to the establishment of a network of 15 Italian centers for samples collection, we generated around 80 gastric PDXs and successfully derived cell lines and organoids from engrafted tumors.
Results and Discussion: Among the tumors collected so far, we found HER2, EGFR, FGFR2, MET and KRAS amplifications. We exploited this gastric PDX platform for: (1) Validation of candidate oncogenes as relevant targets and identification of efficient therapeutic strategies; (2) identification of novel molecular targets; (3) identification of genetic predictors of response/resistance. In this PDX platform we identified one tumor bearing high level of MET gene amplification (26 copies). We found report that despite the high MET amplification level, in the absence of qualitative or quantitative alterations of EGFR, MET inhibitors induced only tumor growth inhibition, while dual MET/EGFR inhibition led to complete tumor regression. Importantly, the combo treatment completely prevented the onset of resistance, which quite rapidly appeared in tumors treated with anti-MET monotherapy. We found that this secondary resistance was due to EGFR activation and could be overcome by dual MET/EGFR inhibition. In vitro experiments performed on tumor-derived primary cells confirmed that MET inhibitors were not able to abrogate the activation of downstream transducers and that only the combined anti-MET/EGFR treatment completely shut off the signaling. Conclusion: Previously reported cases, as well as the one described here, showed only partial and transient sensitivity to anti-MET therapy. The finding that combined anti-MET/EGFR therapy − even in the absence of EGFR genetic alterations − induced complete and durable response, represents a proof of concept and guarantees further investigations, opening a new perspective of treatment for these patients. No conflict of interest. 853 Monitoring metastatic melanoma treatment resistance using circulating tumour DNA S. Murphy1 , J. Wan1 , D. Gale1 , J. Morris1 , F. Mouliere1 , G. Bignell2 , C. Alifrangis2 , C. Parkinson3 , A. Durrani4 , U. McDermott2 , C. Massie1 , P. Corrie3 , N. Rosenfeld1 . 1 Cancer Research UK Cambridge Research Institute, Rosenfeld Group, Cambridge, United Kingdom, 2 Wellcome Trust Sanger Institute, Hinxton, United Kingdom, 3 Addenbrooke’s Hospital, Department of Oncology, Cambridge, United Kingdom, 4 Addenbrooke’s Hospital, Department of Plastic Surgery, Cambridge, United Kingdom Background: Metastatic melanoma is an advanced skin cancer with a life expectancy of 2−7 months. Although patients may initially respond to novel molecular therapies, patients inevitably relapse due to the emergence of drug resistance. Non-invasive methods to monitor treatment response and resistance are needed, which may facilitate personalised treatment decisions. Short DNA fragments are continuously released by cancer cells into the bloodstream, termed as circulating tumour DNA (ctDNA). ctDNA levels have been shown to reflect tumour burden at the time of sampling. Here, ctDNA analysis was carried out to monitor for treatment resistance on a molecular level via serial blood tests. Materials and Methods: Tumour and plasma samples were collected from AJCC Stage III or IV melanoma patients enroled on MelResist, a translational, multi-centre research study. Exome sequencing was carried out on melanoma tumour samples at baseline and disease progression. Variants identified by exome sequencing were used to design patient-specific panels for TaggedAmplicon sequencing (TAm-seq). TAm-seq was carried out on serial plasma DNA samples, enabling the longitudinal monitoring of multiple mutations. Results: To date, 72 patients have been recruited. Individualised Tam-Seq panels were run on plasma from 8 patients, tracking ~50 mutations per patient. Using this approach, tumour dynamics across multiple mutations were monitored, providing insight into the processes underlying the emergence of treatment resistance. ctDNA dynamics correlate with clinical progression data, and precede LDH increases in a number of cases. Conclusion: Monitoring multiple mutations per patient in ctDNA may provide insight into clonal evolution during treatment, and may have utility for identifying treatment resistance earlier than conventional markers. Conflict of interest: Board of Directors: Nitzan Rosenfeld is the CSO of Inivata. Other Substantive Relationships: Nitzan Rosenfeld and Davina Gale are cofounders of Inivata. Nitzan Rosenfeld is a coinventor of patent applications describing methods for analysis of rare DNA fragments. 854 Development of a tool inhibitor of GCN5 towards the treatment of ccRCC S. Walker1 , K. Duffell1 , J. Downs2 , S. Ward1 . 1 University of Sussex, Sussex Drug Discovery Centre, Brighton- East Sussex, United Kingdom, 2 University of Sussex, Genome Damage and Stability Centre, Brighton- East Sussex, United Kingdom BAF180 (BRG1-associated factor 180) is a subunit of PBAF (Polybromo BRG1-Associated Factor), a chromatin remodelling complex. Recently it was identified as a major ccRCC (clear cell Renal Cell Carcinoma) cancer gene, exhibiting inactivating mutations in 41% of samples of primary ccRCC, and thus represents an opportunity to target ccRCC via a synthetic lethality approach. In unpublished work (Hopkins, S. and Downs, J.), GCN5 (general control of amino acid synthesis protein 5) has been identified as synthetically lethal with
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
validated acceptance criteria. Amplification profile for EpCAM gene expression identified the presence of CTCs in specific wells in qualitative manner. Conclusions: Following capture of CTCs using the Parsortix system, gentle contactless cell transfer using the Echo liquid handler enabled standardisation of CTC enrichment allowing detection by qPCR. Miniaturisation of reagents volumes greatly reduced the cost of the full workflow. This work provided a proof of concept that used the Parsortix system and Echo liquid handler as an end-to-end platform to capture, enrich and profile CTCs. No conflict of interest. 851 Association of angiopoietin-2 and Ki-67 expression with vascular density and sunitinib response in metastatic renal cell carcinoma A. Lampinen1 , J. Rautiola2 , T. Mirtti3 , A. Ristimaki ¨ 4 , H. Joensuu2 , P. Bono2 , P. Saharinen5 . 1 University of Helsinki, Research Programs Unit- Translational Cancer Biology Program, Helsinki, Finland, 2 Helsinki University Central Hospital, Comprehensive Cancer Center, Helsinki, Finland, 3 University of Helsinki, Institute for Molecular Medicine Finland, Helsinki, Finland, 4 Helsinki University Central Hospital, Department of Pathology, Helsinki, Finland, 5 University of Helsinki, Translational Cancer Biology Program and Wihuri Research Institute, Helsinki, Finland Background: The angiopoietin-2 (Ang2, Angpt2) growth factor is a contextdependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase known to promote tumour angiogenesis and metastasis. In clinical cancer trials, angiopoietin antagonists are combined with VEGF-based antiangiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. The purpose of this study was to correlate the baseline expression of endothelial angiopoietin-2 (Ang2), CD31 and the cell proliferation marker Ki-67 to the outcome of first-line sunitinib therapy in mRCC. Materials and Methods: We evaluated using immunohistochemistry pretherapeutic Ang2, CD31 and Ki67 expression in formalin-fixed paraffinembedded tumor samples from 136 patients with metastatic RCC, who were subsequently treated with first-line sunitinib. Results: Ang2 was exclusively expressed in the endothelial cells of tumor blood vessels. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS 15.7 vs. 28.5 months, P = 0.015). Conclusions: In summary, high cancer Ang2 expression was associated with high CBR, but not with PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS in mRCC patients treated with first-line sunitinib. No conflict of interest. 852 Combined anti-MET/EGFR treatment results in complete tumor regression and prevents resistance onset in a MET-amplified gastroesophageal xenopatient cohort S. Giordano1 , M. Apicella1 , C. Migliore1 , T. Capeloa2 , S. Menegon2 , M. Cargnelutti1 , A. Sapino3 , P. Cassoni4 , S. Marsoni5 , S. Corso1 . 1 University of Torino- Candiolo Cancer Institute- FPO-IRCCS- Candiolo- Italy, Department of Oncology, Candiolo, Italy, 2 University of Torino- Candiolo Cancer InstituteFPO-IRCCS- Candiolo- Italy, Department of clinical and biological sciences, Candiolo, Italy, 3 University of Torino- Candiolo Cancer Institute- FPO-IRCCSCandiolo- Italy, Department of Medical Sciences, Candiolo, Italy, 4 University of Torino- Italy, Department of Medical Sciences, Candiolo, Italy, 5 Candiolo Cancer Institute- FPO-IRCCS- Candiolo- Italy, clinical trials, Candiolo, Italy Introduction: Gastric cancer is the world third leading cause of cancer mortality. In spite of the significant therapeutic advances, the overall clinical outcome for patients with advanced gastric cancer is poor, with 5−20% 5-year survival. The only targeted therapy approved so far are trastuzumab, and Ramucirumab which have given unsatisfactory results. Around 50% of gastric tumors bear genetic alterations affecting tyrosine kinase pathways (mainly HER2, EGFR, HER3, FGFR2 and MET pathways) but their clinical validation as tumor drivers is missing. The need for new therapeutic options and the possible presence of ‘druggable’ targets prompted us to investigate potential targeted therapies for this disease. Material and Methods: We generated a platform of gastroesophageal tumor patient-derived xenografts (PDXs), in which tumor surgical specimens are directly transferred into mice. Upon engraftment, the tumor is split and reimplanted in a cohort of mice, allowing the simultaneous testing of different drugs on the same tumor. Thanks to the establishment of a network of 15 Italian centers for samples collection, we generated around 80 gastric PDXs and successfully derived cell lines and organoids from engrafted tumors.
Results and Discussion: Among the tumors collected so far, we found HER2, EGFR, FGFR2, MET and KRAS amplifications. We exploited this gastric PDX platform for: (1) Validation of candidate oncogenes as relevant targets and identification of efficient therapeutic strategies; (2) identification of novel molecular targets; (3) identification of genetic predictors of response/resistance. In this PDX platform we identified one tumor bearing high level of MET gene amplification (26 copies). We found report that despite the high MET amplification level, in the absence of qualitative or quantitative alterations of EGFR, MET inhibitors induced only tumor growth inhibition, while dual MET/EGFR inhibition led to complete tumor regression. Importantly, the combo treatment completely prevented the onset of resistance, which quite rapidly appeared in tumors treated with anti-MET monotherapy. We found that this secondary resistance was due to EGFR activation and could be overcome by dual MET/EGFR inhibition. In vitro experiments performed on tumor-derived primary cells confirmed that MET inhibitors were not able to abrogate the activation of downstream transducers and that only the combined anti-MET/EGFR treatment completely shut off the signaling. Conclusion: Previously reported cases, as well as the one described here, showed only partial and transient sensitivity to anti-MET therapy. The finding that combined anti-MET/EGFR therapy − even in the absence of EGFR genetic alterations − induced complete and durable response, represents a proof of concept and guarantees further investigations, opening a new perspective of treatment for these patients. No conflict of interest. 853 Monitoring metastatic melanoma treatment resistance using circulating tumour DNA S. Murphy1 , J. Wan1 , D. Gale1 , J. Morris1 , F. Mouliere1 , G. Bignell2 , C. Alifrangis2 , C. Parkinson3 , A. Durrani4 , U. McDermott2 , C. Massie1 , P. Corrie3 , N. Rosenfeld1 . 1 Cancer Research UK Cambridge Research Institute, Rosenfeld Group, Cambridge, United Kingdom, 2 Wellcome Trust Sanger Institute, Hinxton, United Kingdom, 3 Addenbrooke’s Hospital, Department of Oncology, Cambridge, United Kingdom, 4 Addenbrooke’s Hospital, Department of Plastic Surgery, Cambridge, United Kingdom Background: Metastatic melanoma is an advanced skin cancer with a life expectancy of 2−7 months. Although patients may initially respond to novel molecular therapies, patients inevitably relapse due to the emergence of drug resistance. Non-invasive methods to monitor treatment response and resistance are needed, which may facilitate personalised treatment decisions. Short DNA fragments are continuously released by cancer cells into the bloodstream, termed as circulating tumour DNA (ctDNA). ctDNA levels have been shown to reflect tumour burden at the time of sampling. Here, ctDNA analysis was carried out to monitor for treatment resistance on a molecular level via serial blood tests. Materials and Methods: Tumour and plasma samples were collected from AJCC Stage III or IV melanoma patients enroled on MelResist, a translational, multi-centre research study. Exome sequencing was carried out on melanoma tumour samples at baseline and disease progression. Variants identified by exome sequencing were used to design patient-specific panels for TaggedAmplicon sequencing (TAm-seq). TAm-seq was carried out on serial plasma DNA samples, enabling the longitudinal monitoring of multiple mutations. Results: To date, 72 patients have been recruited. Individualised Tam-Seq panels were run on plasma from 8 patients, tracking ~50 mutations per patient. Using this approach, tumour dynamics across multiple mutations were monitored, providing insight into the processes underlying the emergence of treatment resistance. ctDNA dynamics correlate with clinical progression data, and precede LDH increases in a number of cases. Conclusion: Monitoring multiple mutations per patient in ctDNA may provide insight into clonal evolution during treatment, and may have utility for identifying treatment resistance earlier than conventional markers. Conflict of interest: Board of Directors: Nitzan Rosenfeld is the CSO of Inivata. Other Substantive Relationships: Nitzan Rosenfeld and Davina Gale are cofounders of Inivata. Nitzan Rosenfeld is a coinventor of patent applications describing methods for analysis of rare DNA fragments. 854 Development of a tool inhibitor of GCN5 towards the treatment of ccRCC S. Walker1 , K. Duffell1 , J. Downs2 , S. Ward1 . 1 University of Sussex, Sussex Drug Discovery Centre, Brighton- East Sussex, United Kingdom, 2 University of Sussex, Genome Damage and Stability Centre, Brighton- East Sussex, United Kingdom BAF180 (BRG1-associated factor 180) is a subunit of PBAF (Polybromo BRG1-Associated Factor), a chromatin remodelling complex. Recently it was identified as a major ccRCC (clear cell Renal Cell Carcinoma) cancer gene, exhibiting inactivating mutations in 41% of samples of primary ccRCC, and thus represents an opportunity to target ccRCC via a synthetic lethality approach. In unpublished work (Hopkins, S. and Downs, J.), GCN5 (general control of amino acid synthesis protein 5) has been identified as synthetically lethal with