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P3.03-028 WINTHER – a Study of Cancer Therapy Based on Tumor and Normal-Matched Biopsies – the Sheba Medical Center Lung Cancer Experience
November 2017
Abstracts
P3.03-026 Cell-CT® Differential Detection of Dysplastic Bronchial Epithelial Cells from Patient Explants D. Sussman,1 M. Meyer,1 R. Katdare,1 C. Presley,1 T. Bell,1 J. Reyna,1 F. Lakers,1 C. Hamilton,1 J. Zulueta,1 Y. Miller,2 M. Ghosh,3 A. Nelson1 1Visiongate, Phoenix, AZ/US, 2Pulmonary and Critical Care Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO/US, 3Pediatrics, National Jewish Health, Denver/US Background: Chemoprevention could have a great impact on lung cancer prevention. While Iloprost treatment has shown a significant reduction of dysplasia in former smokers, the identification of patients who would benefit from the drug is seriously hampered due to the need to use invasive diagnostic procedures in patients who are typically asymptomatic. Published clinical data shows that non-invasive sputum analysis using the Cell-CT platform detects early stage lung cancer with high sensitivity (92%) and specificity (95%). This abstract reports the development of cell classifiers that distinguish cultured human lung dysplastic explants from malignant and normal sputum cells. This study represents a first important step toward developing a non-invasive diagnostic test for detecting patients with moderate to severe bronchial dysplasia who may then be treated with chemopreventive drugs such as Iloprost. Method: To achieve diagnostic classifications, sputum from patients without lung cancer (“normal cells”), small cell lung cancer and five adenocarcinoma cell lines, and cultured bronchial explants from three patients with moderate to severe dysplasia were analyzed using the Cell-CT. Result: 15,000 normal cells from sputum, 500 malignant cells from each of the five lung cancer cell lines and 264 cells from patient dysplastic explants were analyzed using Cell-CT platform, measuring 704 structural biomarkers to sub-classify the cancer cells by abnormality and dysplastic status. Cell classifiers were operated to drive the highest specificity (avoidance of false positives). The area under ROC (aROC), sensitivity and specificity for each classifier were (see Table). Conclusion: These results show strong discrimination by the Cell-CT in classifying normal cells from sputum versus cells from lung dysplastic explants and lung cancer cell lines grown in culture. These data suggest that a non-invasive test using sputum liquid biopsy analyzed on the Cell-CT platform could enable the detection of dysplasia in patients who would benefit from chemoprevention drug therapy. Keywords: chemoprevention, Cell-CT Platform, Dysplasia
Cell Classifiers
aROC
Sensitivity%
Specificity%
Lung Cancer cell lines Cells from patient dysplastic explants
0.999 0.995
93% 86%
99.99 99.99
P3.03-027 LKB1 Loss Is Associated with Resistance to AntiAngiogenic Therapy in Non-Small Cell Lung Cancer Mouse Models I. Guijarro,1 A. Poteete,1 Y. Fan,1 S. Cho,1 P. Tong,1 E. Roarty,1 M. Nilsson,1 J. Rodriguez-Canales,2 B. Mino,2 E. Parra Cuentas,2 I. Wistuba,3 J. Wang,4 J. Heymach5 1Thoracic, Head & Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX/US, 2Translational Molecular Pathology, MD Anderson, Houston, TX/ US, 3Translational Molecular Pathology, MD Anderson, Houston, AL/US, 4 Dept. Of Bioinformatics and Computational Biology, MD Anderson Cancer Center, Houston/US, 5Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX/US Background: LKB1 is a protein kinase that is mutated and downregulated in 20-30% of non-small cell lung cancer (NSCLC). LKB1 mutations co-occur with KRAS alterations in 7%-10% of NSCLC, resulting in an aggressive phenotype with short survival. Because LKB1
S2283
activates AMPK, the master sensor of cellular energy, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations in the cells. However LKB1 also plays an important role in regulating angiogenesis, likely as a strategy to overcome energetic depletion of tumor microenvironment. Bevacizumab, the human anti-VEGF antibody, improves the PFS and OS of NSCLC patients combined with chemotherapy, but often the benefit is transient and therapeutic resistance occurs. Our laboratory has previously identified alterations in cell metabolism and in vasculature of LKB1-deficient tumors when compared to LKB1 wild type in NSCLC. Method: LKB1 KO murine NSCLC cell lines were generated using CRISPR/Cas9 system in a KRASG12D mutant background (LKR10 & LKR13). Syngeneic NSCLC models were established via s.c. injection of LKB1 intact and KO murine cells in immunocompetent mice. After tumors reached 150 mm3 mice were randomly assigned to treatment groups consisting of vehicle, mouse anti-VEGF and nintedanib. Tumor volumes were measured and compared using student’s t test and samples were collected for vasculature analysis. Survival curves will be calculated using log rank test. Hypoxia experiments were preformed and apoptosis was measured using annexin V and 7ADD staining. Result: Treatment with anti-VEGF or nintedanib significantly inhibited tumor progression in LKB1 wt KRASG12D mutant mouse model (p<0.001) but did not show any therapeutic effect in the LKB1 KO KRASG12D group. Furthermore in the LKB1 wt group, the median survival of anti-VEGF and nintedanib treated mice was 111 days and 84 days respectively and 37 days in the vehicle group. No improvement in survival was detected in the LKB1 KO group after treatment with anti-VEGF. In vitro studies showed that LKB1 loss is associated with a decrease in oxygen consumption and enhanced glycolysis. Furthermore LKB1 KO NSCLC cells showed a decrease in apoptosis under hypoxic and low nutrient conditions compared to LKR13 LKB1 wt cells. Conclusion: NSCLC LKB1-deficient tumors showed resistance to anti-angiogenic therapy and this effect is driven by the regulation of metabolic adaptations that allow cells to survive under hypoxic and low nutrient conditions. Keywords: LKB1, Anti-angiogenic therapy, Resistance
P3.03-028 WINTHER e a Study of Cancer Therapy Based on Tumor and Normal-Matched Biopsies e the Sheba Medical Center Lung Cancer Experience T. Sella,1 A. Ackerstein,2 S. Halperin,2 G. Hout-Siloni,2 S. Lieberman,2 I. Barshack,2 H. Nechushatan,3 R. Berger,2 J. Bar,2 A. Onn2 1Pulmonary Medicine, Sheba Medical Center, Ramat Gan/IL, 2 Sheba Medical Center, Ramat Gan/IL, 3Medical Oncology, Hadassah Medical Center, Jerusalem/IL Background: Patient-tailored therapy based on tumor genomics is limited to 30-40% of the patients whose tumor harbor actionable DNA mutations or amplifications. Method: WINTHER is an international open label non-randomized clinical trial developed by the WIN consortium. Matched tumor and normal tissue biopsies are collected and analyzed by NGS (Foundation Medicine) or by functional genomics utilizing a prediction model of efficacy developed at Ben Gurion University. Result: 56 patients were biopsied. 29 (52%) had lung cancer. Successful biopsies yielding sufficient material for full genomic analyzes were achieved in 29 subjects (53%). Lung biopsy success rates were 71% and 61% respectively for normal and tumor specimens. To date, 11 lung cancer patients were treated with chemotherapy (1) or biologic agents (11). Targeted genomic alterations included EGFR (3), RET (2), KRAS (2), ALK (1), ErbB2 (1), ErbB3 (1), BRAF (1). Clinical benefit rate (CBR) was 55% (6/11) with 1 subject achieving compete response, 2 partial responses and 3 stable disease. Response durations were 7, 14 and 18 months. Conclusion: Tumor genomic analysis based on the comparison of matched tumor and normal biopsies is acceptable and feasible. The experience of the multidisciplinary team is an important contributor to the program’s success. Keywords: clinical trial, genetic testing, biopsy
Abstracts
P3.03-026 Cell-CT® Differential Detection of Dysplastic Bronchial Epithelial Cells from Patient Explants D. Sussman,1 M. Meyer,1 R. Katdare,1 C. Presley,1 T. Bell,1 J. Reyna,1 F. Lakers,1 C. Hamilton,1 J. Zulueta,1 Y. Miller,2 M. Ghosh,3 A. Nelson1 1Visiongate, Phoenix, AZ/US, 2Pulmonary and Critical Care Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO/US, 3Pediatrics, National Jewish Health, Denver/US Background: Chemoprevention could have a great impact on lung cancer prevention. While Iloprost treatment has shown a significant reduction of dysplasia in former smokers, the identification of patients who would benefit from the drug is seriously hampered due to the need to use invasive diagnostic procedures in patients who are typically asymptomatic. Published clinical data shows that non-invasive sputum analysis using the Cell-CT platform detects early stage lung cancer with high sensitivity (92%) and specificity (95%). This abstract reports the development of cell classifiers that distinguish cultured human lung dysplastic explants from malignant and normal sputum cells. This study represents a first important step toward developing a non-invasive diagnostic test for detecting patients with moderate to severe bronchial dysplasia who may then be treated with chemopreventive drugs such as Iloprost. Method: To achieve diagnostic classifications, sputum from patients without lung cancer (“normal cells”), small cell lung cancer and five adenocarcinoma cell lines, and cultured bronchial explants from three patients with moderate to severe dysplasia were analyzed using the Cell-CT. Result: 15,000 normal cells from sputum, 500 malignant cells from each of the five lung cancer cell lines and 264 cells from patient dysplastic explants were analyzed using Cell-CT platform, measuring 704 structural biomarkers to sub-classify the cancer cells by abnormality and dysplastic status. Cell classifiers were operated to drive the highest specificity (avoidance of false positives). The area under ROC (aROC), sensitivity and specificity for each classifier were (see Table). Conclusion: These results show strong discrimination by the Cell-CT in classifying normal cells from sputum versus cells from lung dysplastic explants and lung cancer cell lines grown in culture. These data suggest that a non-invasive test using sputum liquid biopsy analyzed on the Cell-CT platform could enable the detection of dysplasia in patients who would benefit from chemoprevention drug therapy. Keywords: chemoprevention, Cell-CT Platform, Dysplasia
Cell Classifiers
aROC
Sensitivity%
Specificity%
Lung Cancer cell lines Cells from patient dysplastic explants
0.999 0.995
93% 86%
99.99 99.99
P3.03-027 LKB1 Loss Is Associated with Resistance to AntiAngiogenic Therapy in Non-Small Cell Lung Cancer Mouse Models I. Guijarro,1 A. Poteete,1 Y. Fan,1 S. Cho,1 P. Tong,1 E. Roarty,1 M. Nilsson,1 J. Rodriguez-Canales,2 B. Mino,2 E. Parra Cuentas,2 I. Wistuba,3 J. Wang,4 J. Heymach5 1Thoracic, Head & Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX/US, 2Translational Molecular Pathology, MD Anderson, Houston, TX/ US, 3Translational Molecular Pathology, MD Anderson, Houston, AL/US, 4 Dept. Of Bioinformatics and Computational Biology, MD Anderson Cancer Center, Houston/US, 5Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX/US Background: LKB1 is a protein kinase that is mutated and downregulated in 20-30% of non-small cell lung cancer (NSCLC). LKB1 mutations co-occur with KRAS alterations in 7%-10% of NSCLC, resulting in an aggressive phenotype with short survival. Because LKB1
S2283
activates AMPK, the master sensor of cellular energy, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations in the cells. However LKB1 also plays an important role in regulating angiogenesis, likely as a strategy to overcome energetic depletion of tumor microenvironment. Bevacizumab, the human anti-VEGF antibody, improves the PFS and OS of NSCLC patients combined with chemotherapy, but often the benefit is transient and therapeutic resistance occurs. Our laboratory has previously identified alterations in cell metabolism and in vasculature of LKB1-deficient tumors when compared to LKB1 wild type in NSCLC. Method: LKB1 KO murine NSCLC cell lines were generated using CRISPR/Cas9 system in a KRASG12D mutant background (LKR10 & LKR13). Syngeneic NSCLC models were established via s.c. injection of LKB1 intact and KO murine cells in immunocompetent mice. After tumors reached 150 mm3 mice were randomly assigned to treatment groups consisting of vehicle, mouse anti-VEGF and nintedanib. Tumor volumes were measured and compared using student’s t test and samples were collected for vasculature analysis. Survival curves will be calculated using log rank test. Hypoxia experiments were preformed and apoptosis was measured using annexin V and 7ADD staining. Result: Treatment with anti-VEGF or nintedanib significantly inhibited tumor progression in LKB1 wt KRASG12D mutant mouse model (p<0.001) but did not show any therapeutic effect in the LKB1 KO KRASG12D group. Furthermore in the LKB1 wt group, the median survival of anti-VEGF and nintedanib treated mice was 111 days and 84 days respectively and 37 days in the vehicle group. No improvement in survival was detected in the LKB1 KO group after treatment with anti-VEGF. In vitro studies showed that LKB1 loss is associated with a decrease in oxygen consumption and enhanced glycolysis. Furthermore LKB1 KO NSCLC cells showed a decrease in apoptosis under hypoxic and low nutrient conditions compared to LKR13 LKB1 wt cells. Conclusion: NSCLC LKB1-deficient tumors showed resistance to anti-angiogenic therapy and this effect is driven by the regulation of metabolic adaptations that allow cells to survive under hypoxic and low nutrient conditions. Keywords: LKB1, Anti-angiogenic therapy, Resistance
P3.03-028 WINTHER e a Study of Cancer Therapy Based on Tumor and Normal-Matched Biopsies e the Sheba Medical Center Lung Cancer Experience T. Sella,1 A. Ackerstein,2 S. Halperin,2 G. Hout-Siloni,2 S. Lieberman,2 I. Barshack,2 H. Nechushatan,3 R. Berger,2 J. Bar,2 A. Onn2 1Pulmonary Medicine, Sheba Medical Center, Ramat Gan/IL, 2 Sheba Medical Center, Ramat Gan/IL, 3Medical Oncology, Hadassah Medical Center, Jerusalem/IL Background: Patient-tailored therapy based on tumor genomics is limited to 30-40% of the patients whose tumor harbor actionable DNA mutations or amplifications. Method: WINTHER is an international open label non-randomized clinical trial developed by the WIN consortium. Matched tumor and normal tissue biopsies are collected and analyzed by NGS (Foundation Medicine) or by functional genomics utilizing a prediction model of efficacy developed at Ben Gurion University. Result: 56 patients were biopsied. 29 (52%) had lung cancer. Successful biopsies yielding sufficient material for full genomic analyzes were achieved in 29 subjects (53%). Lung biopsy success rates were 71% and 61% respectively for normal and tumor specimens. To date, 11 lung cancer patients were treated with chemotherapy (1) or biologic agents (11). Targeted genomic alterations included EGFR (3), RET (2), KRAS (2), ALK (1), ErbB2 (1), ErbB3 (1), BRAF (1). Clinical benefit rate (CBR) was 55% (6/11) with 1 subject achieving compete response, 2 partial responses and 3 stable disease. Response durations were 7, 14 and 18 months. Conclusion: Tumor genomic analysis based on the comparison of matched tumor and normal biopsies is acceptable and feasible. The experience of the multidisciplinary team is an important contributor to the program’s success. Keywords: clinical trial, genetic testing, biopsy