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Application of sequencing, liquid biopsies and patient-derived xenografts for personalized medicine in melanoma
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 836 Application of sequencing, liquid biopsies and patient-derived xenografts for personalized medicine in melanoma M.R. Girotti1 , G. Gremel1 , R. Lee1 , E. Galvani1 , D. Rothwell2 , A.K. Mandal1 , K.H.J. Lim1 , G. Saturno1 , S.J. Furney1 , F. Baenke1 , M. Pedersen3 , M. Smith1 , A. Fusi4 , N. Dhomen1 , G. Brady2 , P. Lorigan4 , C. Dive2 , R. Marais1 . 1 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 Cancer Research UK Manchester Institute, Clinical and Experimental Pharmacology Team, Manchester, United Kingdom, 3 The Institute of Cancer Research, Targeted Therapy Team, London, United Kingdom, 4 The University of Manchester, The Christie NHS Foundation Trust, Manchester, United Kingdom Background: Targeted and immunotherapies have transformed melanoma care, extending median survival from ~9 to over 25 months but nevertheless, most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end we aimed to develop protocols to facilitate individualized treatment decisions for advanced melanoma patients. Material and Methods: We have analyzed 364 samples from 214 patients by Whole exome sequencing (WES), circulating tumor DNA (ctDNA), establishment of patient-derived xenografts (PDXs) and circulating tumor cellderived xenografts (CDX). Results: WES and targeted sequencing of ctDNA allowed us to monitor responses to therapy and to identify and then monitor mechanisms of resistance and we propose that longitudinal monitoring of BRAF and NRAS driver mutations should be used to prospectively follow patient responses to targeted therapy and immunotherapy. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and BRAFinhibitor-resistant BRAF mutant tumors, which were then validated in PDXs. For one of our patients, WES revealed that trametinib plus paclitaxel may have been an effective therapeutic option and we validated this combination in the patient’s PDX. We therefore propose that integration of WES and ctDNA sequencing with functional studies in PDXs provides a powerful combination that can change clinical practice and improve patient outcomes. We also developed CDXs as an alternative to PDXs when tumors are inaccessible or difficult to biopsy. We establish a proof-of-principle for this approach in melanoma and show that the CDXs resemble patient tumors and their response to therapies. Our data show that melanoma CTCs are tumorigenic and that in mice they have similar tropism to the tumors in the patients. Conclusions: Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses, and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments. No conflict of interest.
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microenvironment in larger cohorts is needed to identify robust determinants of response to immune checkpoint inhibitors. No conflict of interest. 839 Clinical significance of non-coding RNAs expression in human hepatocellular carcinoma K. Hur1 , S.Y. Jang2 , S.Y. Park2 , G. Kim1 , Y.H. Choi1 , Y.R. Lee2 , S.H. Lee2 , S.K. Jang2 , W.Y. Tak2 , Y.O. Kweon2 . 1 Kyungpook National University School of Medicine, Department of Biochemistry and Cell Biology, Daegu, South Korea, 2 Kyungpook National University Hospital, Department of Internal Medicine, Daegu, Korea Background: Hepatocellular carcinoma (HCC) is one of the most leading causes of cancer-related death worldwide. It is therefore important to understand the mechanistic roles of biomolecules involved in HCC development. Recently, non-coding RNAs (ncRNAs) including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have emerged as novel gene-regulators. Many studies have indicated that miRNAs and lncRNAs are frequently aberrantly expressed in various human cancers, which have potential roles in both oncogenes and tumor-suppressors. The aim of this study was to determine the clinical relevancies of expression of 2 lncRNAs (lncRNA-ATB and MEG3) and 2 miRNAs (miR-21 and miR-31) in HCC. Material and Methods: We analyzed clinical specimens from 100 pairs of HCC tissues and matched normal liver (NL) tissues. MiRNAs and lncRNAs expression levels were evaluated by quantitative real-time PCR (qRT-PCR) and their expression was normalized relative to U6 and GAPDH expression, respectively. In addition, we determined the clinical significance of the miR-21 and lncRNA-ATB expression in matching tissue and serum samples from HCC patients. Results: MiRNA-21 significantly increased in HCC (P < 0.01), whereas miRNA-31 decreased in HCC (P < 0.001) compared to corresponding NL tissues. LncRNA-ATB was significantly up-regulated in HCC (P < 0.05); however, lncRNA-MEG3 was down-regulated in HCC (P < 0.05) compared to corresponding NL tissues. In addition, expression of microRNA-21 and lncRNA-ATB was significantly associated with the tumor size (T stage; P = 0.0092) and the tumor size (T stage; P = 0.0013) and poor prognosis (BCLC stage; P = 0.0039) of HCC patients, respectively. More significantly, high expression of miR-21 and lncRNA-ATB was significantly associated with poor survival of HCC patients. Conclusions: We conclude that non-coding RNAs (lncRNA-ATB and miR-21) expression has the potential to serve as biomarkers for prognosis and targeted therapy in HCC patients. No conflict of interest. 840 Dickkopf-1 (DKK-1) expression is a novel lymph node metastasis predictor in early gastric cancer
837 Modelling anti-PD-1 treatment in a transgenic murine model of BRAFV600E-driven melanoma E. Galvani1 , K. Hogan1 , L. Boon2 , G. Gremel1 , A. Viros1 , A. Mandal1 , M. Smith1 , J. Swan3 , A. Banyard3 , G. Ashton3 , N. Dhomen1 , R. Marais1 . 1 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 EPIRUS Biopharmaceuticals Netherlands, CLG- Upstream PD & Bioassay Development, Utrecht, Netherlands, 3 Cancer Research UK Manchester Institute, Research Services, Manchester, United Kingdom Introduction: Immune checkpoint inhibitors have recently revolutionised the field of cancer treatment providing for the first time durable responses in metastatic melanoma and non-small cell lung cancer patients. It is imperative, moving this area of treatment forward, to understand the basis of such responses and be able to identify the patients most likely to benefit from these therapies. Material and Methods: Melanomas derived from a BRAFV600E transgenic mouse model in which chronic exposure to ultraviolet radiation (UVR) was used to simulate the effect of sunburn, were compared for their response to programmed cell death-1 (PD-1) blockade. Tumours were subsequently subjected to comprehensive molecular and immune profiling using whole exome sequencing, immunohistochemistry, and flow cytometry. Results and Discussion: BRAFV600E-driven melanomas developed in mice exposed to UVR presented with significant increase in single nucleotide variants (SNVs), and thus higher mutational load, as compared to non-UVR treated tumours. Responses to anti-PD-1 treatment were compared between tumours having low (~10 total missense SNVs) and high (>100 total missense SNVs) non-synonymous mutational load. No significant association between tumour mutation burden and response to PD-1 blockade was observed in our BRAFV600E-driven melanoma model. Detailed analysis of low- and highmutation burden tumours in light of their response to treatment will be presented. Conclusions: Consistent with previous studies, our data highlight that integrated molecular characterisation of tumour tissue and associated
Y.J. Huh1 , H.M. Lee1 , M.S. Cho2 , J.H. Lee1 . 1 Ewha Womans University Mokdong hospital, Surgery, Seoul, Korea, 2 Ewha Womans University Mokdong hospital, Pathology, Seoul, Korea Background: In early gastric cancer (EGC), predicting the lymph node metastasis (LNM) is important to increase the efficacy of surgical treatment and to select the minimally invasive endoscopic treatment. Therefore, novel method for predicting LNM is required. Although radiologic modalities such as CT and MRI are widely used for LNM diagnosis, it has several limitations including insufficient resolution and sensitivity. Dickkopf-1 (DKK-1), an inhibitor of Wnt signaling pathway, has been reported to promote cancer cell invasion and migration. However, the clinical significance of DKK-1 for predicting LNM has not been previously investigated in GC patients. The aim of this study was to determine the potential ability of DKK-1 expression to predict LNM in EGC. Material and Methods: We analyzed expression of DKK-1 in 126 EGC tissue specimens, which included LNM-positive (LN(+), n = 42) and LNMnegative (LN(-), n = 84). Expression status of DKK-1 protein was determined by immunohistochemical staining using formalin-fixed, paraffin-embedded tissue blocks from the patients underwent surgery due to the EGC from 2002 to 2013 at Ewha Womans University Mokdong Hospital. The area of staining was quantified according to the proportion of stained cells and categorized as low; 0−30% and high; >30%. In clinicopathological analyses, the tumor size and depth of invasion were matched between two groups (LN(+) and LN (-)) to exclude the effect to LNM of those factors. Results: We observed significant up-regulation of DKK-1 in EGC tissues compared to corresponding normal gastric mucosa tissues. In addition, DKK-1 was more frequently elevated in LN(+) EGC patient group than in LN(-) EGC patient group (40.7% vs. 26.9%, P = 0.073). In further subgroup analyses, male gender (P = 0.036) and undifferentiated histologic type (P = 0.038) were significantly correlated with LN metastasis in EGC. More importantly, high DKK-1 protein expression was an independent predictor for lymph node metastasis (odds ratio: 2.55, 95% confidence interval: 1.12–5.83, P = 0.027).
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microenvironment in larger cohorts is needed to identify robust determinants of response to immune checkpoint inhibitors. No conflict of interest. 839 Clinical significance of non-coding RNAs expression in human hepatocellular carcinoma K. Hur1 , S.Y. Jang2 , S.Y. Park2 , G. Kim1 , Y.H. Choi1 , Y.R. Lee2 , S.H. Lee2 , S.K. Jang2 , W.Y. Tak2 , Y.O. Kweon2 . 1 Kyungpook National University School of Medicine, Department of Biochemistry and Cell Biology, Daegu, South Korea, 2 Kyungpook National University Hospital, Department of Internal Medicine, Daegu, Korea Background: Hepatocellular carcinoma (HCC) is one of the most leading causes of cancer-related death worldwide. It is therefore important to understand the mechanistic roles of biomolecules involved in HCC development. Recently, non-coding RNAs (ncRNAs) including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have emerged as novel gene-regulators. Many studies have indicated that miRNAs and lncRNAs are frequently aberrantly expressed in various human cancers, which have potential roles in both oncogenes and tumor-suppressors. The aim of this study was to determine the clinical relevancies of expression of 2 lncRNAs (lncRNA-ATB and MEG3) and 2 miRNAs (miR-21 and miR-31) in HCC. Material and Methods: We analyzed clinical specimens from 100 pairs of HCC tissues and matched normal liver (NL) tissues. MiRNAs and lncRNAs expression levels were evaluated by quantitative real-time PCR (qRT-PCR) and their expression was normalized relative to U6 and GAPDH expression, respectively. In addition, we determined the clinical significance of the miR-21 and lncRNA-ATB expression in matching tissue and serum samples from HCC patients. Results: MiRNA-21 significantly increased in HCC (P < 0.01), whereas miRNA-31 decreased in HCC (P < 0.001) compared to corresponding NL tissues. LncRNA-ATB was significantly up-regulated in HCC (P < 0.05); however, lncRNA-MEG3 was down-regulated in HCC (P < 0.05) compared to corresponding NL tissues. In addition, expression of microRNA-21 and lncRNA-ATB was significantly associated with the tumor size (T stage; P = 0.0092) and the tumor size (T stage; P = 0.0013) and poor prognosis (BCLC stage; P = 0.0039) of HCC patients, respectively. More significantly, high expression of miR-21 and lncRNA-ATB was significantly associated with poor survival of HCC patients. Conclusions: We conclude that non-coding RNAs (lncRNA-ATB and miR-21) expression has the potential to serve as biomarkers for prognosis and targeted therapy in HCC patients. No conflict of interest. 840 Dickkopf-1 (DKK-1) expression is a novel lymph node metastasis predictor in early gastric cancer
837 Modelling anti-PD-1 treatment in a transgenic murine model of BRAFV600E-driven melanoma E. Galvani1 , K. Hogan1 , L. Boon2 , G. Gremel1 , A. Viros1 , A. Mandal1 , M. Smith1 , J. Swan3 , A. Banyard3 , G. Ashton3 , N. Dhomen1 , R. Marais1 . 1 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 EPIRUS Biopharmaceuticals Netherlands, CLG- Upstream PD & Bioassay Development, Utrecht, Netherlands, 3 Cancer Research UK Manchester Institute, Research Services, Manchester, United Kingdom Introduction: Immune checkpoint inhibitors have recently revolutionised the field of cancer treatment providing for the first time durable responses in metastatic melanoma and non-small cell lung cancer patients. It is imperative, moving this area of treatment forward, to understand the basis of such responses and be able to identify the patients most likely to benefit from these therapies. Material and Methods: Melanomas derived from a BRAFV600E transgenic mouse model in which chronic exposure to ultraviolet radiation (UVR) was used to simulate the effect of sunburn, were compared for their response to programmed cell death-1 (PD-1) blockade. Tumours were subsequently subjected to comprehensive molecular and immune profiling using whole exome sequencing, immunohistochemistry, and flow cytometry. Results and Discussion: BRAFV600E-driven melanomas developed in mice exposed to UVR presented with significant increase in single nucleotide variants (SNVs), and thus higher mutational load, as compared to non-UVR treated tumours. Responses to anti-PD-1 treatment were compared between tumours having low (~10 total missense SNVs) and high (>100 total missense SNVs) non-synonymous mutational load. No significant association between tumour mutation burden and response to PD-1 blockade was observed in our BRAFV600E-driven melanoma model. Detailed analysis of low- and highmutation burden tumours in light of their response to treatment will be presented. Conclusions: Consistent with previous studies, our data highlight that integrated molecular characterisation of tumour tissue and associated
Y.J. Huh1 , H.M. Lee1 , M.S. Cho2 , J.H. Lee1 . 1 Ewha Womans University Mokdong hospital, Surgery, Seoul, Korea, 2 Ewha Womans University Mokdong hospital, Pathology, Seoul, Korea Background: In early gastric cancer (EGC), predicting the lymph node metastasis (LNM) is important to increase the efficacy of surgical treatment and to select the minimally invasive endoscopic treatment. Therefore, novel method for predicting LNM is required. Although radiologic modalities such as CT and MRI are widely used for LNM diagnosis, it has several limitations including insufficient resolution and sensitivity. Dickkopf-1 (DKK-1), an inhibitor of Wnt signaling pathway, has been reported to promote cancer cell invasion and migration. However, the clinical significance of DKK-1 for predicting LNM has not been previously investigated in GC patients. The aim of this study was to determine the potential ability of DKK-1 expression to predict LNM in EGC. Material and Methods: We analyzed expression of DKK-1 in 126 EGC tissue specimens, which included LNM-positive (LN(+), n = 42) and LNMnegative (LN(-), n = 84). Expression status of DKK-1 protein was determined by immunohistochemical staining using formalin-fixed, paraffin-embedded tissue blocks from the patients underwent surgery due to the EGC from 2002 to 2013 at Ewha Womans University Mokdong Hospital. The area of staining was quantified according to the proportion of stained cells and categorized as low; 0−30% and high; >30%. In clinicopathological analyses, the tumor size and depth of invasion were matched between two groups (LN(+) and LN (-)) to exclude the effect to LNM of those factors. Results: We observed significant up-regulation of DKK-1 in EGC tissues compared to corresponding normal gastric mucosa tissues. In addition, DKK-1 was more frequently elevated in LN(+) EGC patient group than in LN(-) EGC patient group (40.7% vs. 26.9%, P = 0.073). In further subgroup analyses, male gender (P = 0.036) and undifferentiated histologic type (P = 0.038) were significantly correlated with LN metastasis in EGC. More importantly, high DKK-1 protein expression was an independent predictor for lymph node metastasis (odds ratio: 2.55, 95% confidence interval: 1.12–5.83, P = 0.027).