1. Abbreviations: RECIST, Response Evaluation Criteria In Solid Tumors; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; mos, months; HR, hazard ratio; CI, confidence interval. * P value was based on Cochran-Armitage test for response, log-rank test for PFS and OS in the univariate analysis (†) and Wald test for PFS and OS in the multivariable Cox regression model (‡). EGFL7 Polymorphism Predicts Tumor Response in Metastatic Colorectal Cancer Patients Treated with FOLFIRI and Bevacizumab Joseph E. Li 1, Wu Zhang 2, Fotios Loupakis 3, Dongyun Yang 2, Takeru Wakatsuki 2, Yan Ning 2, Sebastian Stintzing 2, Rita E. El-Khoueiry 2, Nico B. Volz 2, Federica Marmorino 3, Marta Schirripa 3, Lisa Salvatore 3, Carlotta Antoniotti 3, Chiara Cremolini 3, Heinz-Josef Lenz 2 1 Keck School of Medicine of USC, Los Angeles, CA; 2 USC/Norris Comprehensive Cancer Center, Los Angeles, CA; 3 U.O. Oncologia Medica 2, Azienda Ospedaliero-Universitaria Pisana, Istituto Toscano Tumori, Pisa, Italy Introduction Methods Conclusion This study tested whether single nucleotide polymorphisms (SNPs) in EGFL7 (rs1051851, rs2297538) and ALK1 (rs2293094, rs11169953, rs706819) could predict efficacy of BV-based therapy in metastatic colorectal cancer patients. References Angiogenesis is the formation of new vessels from preexisting vascular plexuses. This process involves endothelial cells (ECs) loosening intercellular adhesions, proliferating, and sprouting, while the extracellular matrix (ECM) undergoes remodeling. Nascent vessels are stabilized by the recruitment of mural cells, such as pericytes and vascular smooth muscle cells (VSMCs), which provide structural and functional support. 1 This system is critical for tumor cell survival. The proteins Epidermal Growth Factor Domain- Like 7 (EGFL7) and Activin Receptor-like Kinase 1 (ALK1) play critical roles in this process. EGFL7 is predominantly expressed by sprouting ECs and associates with the ECM after secretion. 2,3 The protein modulates Notch signaling to promote EC proliferation, sprouting, and migration. 4 It also regulates ECM deposition and the recruitment of VSMCs. 3,5 ALK1 is an EC-specific TGF-β Type 1 receptor with downstream repression of Vascular Endothelial Growth Factor (VEGF) response. ALK1 is also required for mural cell recruitment. 6 ALK1 and EGFL7 are overexpressed in many tumors, including colon cancer. 7,8 Importantly, the inhibition of either protein enhances anti-tumor efficacy when combined with bevacizumab (BV) in murine models. 9,10 EGFL7 and ALK1 may be critical for the efficacy of anti-VEGF therapy. BV has been shown to yield advantages in terms of survival for metastatic colorectal cancer patients. 11 However, from a clinical perspective, the relatively small absolute benefit provided by this agent highlights the need for predictive biomarkers. There are currently no biomarkers available for BV-based therapies. Colorectal cancer was the third-leading cause of cancer-related incidence and death with 143,460 new cases and 51,690 deaths in 2012. 12 1.Carmeliet, P (2003) Angiogenesis in health and disease. Nature Med. 9: 653-660 2.Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H (2004) EGFL7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn. 230(2):316-324. 3.Soncin F, Mattot V, Lionneton F, et al. (2003). VE-statin, an endothelial repressor of smooth muscle cell migration. EMBO J. 22(21):5700-5711 4.Nichol D, Shawber C, Fitch MJ, et al. (2010) Impaired angiogenesis and altered Notch signaling in mice overexpressing endothelial Egfl7. Blood (115(26):6133-6143 5.Lelievre E, Hinek A, Lupu F, Buquet C, Soncin F, Mattot V (2008) VE-statin/egfl7 regulates vascular elastogenesis by interacting with lysyl oxidases. EMBO J. 27(12):1658-1670. 6. Larrivée B, Prahst C, Gordon E, et al. (2012) ALK1 signaling inhibits angiogenesis by cooperating with the Notch pathway. Dev Cell. 22(3):489-500. 7.Nichol D, Stuhlmann H (2012) EGFL7: a unique angiogenic signaling factor in vascular development and disease. Blood 199:1345-1352 8.Olivier Nolan-Stevaux and H. Toni Jun (2012). Beyond VEGF: The NOTCH and ALK1 Signaling Pathways as Tumor Angiogenesis Targets, Tumor Angiogenesis, Dr. Sophia Ran (Ed.), ISBN: 978-953-51-0009-6, InTech, DOI: 10.5772/26834. 9.http://ip.com/patapp/US20110200602 10.Hu-Lowe DD, Chen E, Zhang L, et al. (2011) Targeting Activin Receptor-Like Kinase 1 inhibits angiogenesis and tumorigenesis through a mechanism of action complementary to anti-VEGF therapies. Cancer Res 71:1362-1373 11.Hurwitz H, Fehrenbacher L, Novotny W et al. (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 350(23):2335-42 12.American Cancer Society. Cancer Facts and Figures, 2012. www.cancer.org/ Results Objective Table 1. Single Nucleotide Polymorphisms Analyzed Table 2. Patient Characteristics and Clinical Outcome Table 3. rs1051851 and Clinical Outcomes in Metastatic Colorectal Cancer Patients Treated with FOLFIRI+BV Currently, there are no predictive biomarkers for BV-based therapies. This study demonstrates for the first time that the EGFL7 SNP rs1051851 predicts tumor RR in metastatic colorectal cancer patients treated with FOLFIRI+BV, which may be clinically important in patients with borderline resectable disease. The association between rs1051851 and RR but not PFS or OS may be explained by the fact that the depth of response influences the occurrence of RECIST defined “progression”. Importantly, this is the first study evaluating polymorphisms in genes responsible for blood vessel sprouting and maturation beyond VEGF and examining their relationship to cancer therapy. Prospective validation of this result and analysis of BV-free patient cohorts is warranted. This study analyzed patients combined from a phase III trial (TRIBE NCT00719797) and a phase II trial (PROVETTA NCT01363739). Trial participants had histologically confirmed metastatic colorectal adenocarcinoma with at least one measurable lesion according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.0. Patients received BV and FOLFIRI (folinic acid, fluorouracil, irinotecan). The primary endpoint of these studies was progression-free survival (PFS). The trials were approved by the local Ethics Committee, and written informed consent was collected from all patients. Genomic DNA was extracted from peripheral blood samples from all patients. SNPs were analyzed by PCR-based direct DNA sequencing. The Cochran-Armitage test was used to test for association with tumor response rate (RR). Logistic regression was used to adjust for baseline patient characteristics. Association between SNPs with overall survival (OS) and PFS was tested with Kaplan-Meier curves, log-rank test, and multivariable Cox regression. PFS was defined as the time from the first administration of study treatment until the first documentation of objective disease progression according to RECIST 1.0, or death due to any cause, whichever occurs first. Overall survival (OS) was defined as the time from the first administration of study treatment until the date of death due to any cause. Figure 1. EGFL7 rs1051851 and survival in patients with metastatic colorectal cancer treated with FOLIFIRI+BV. (A) rs1051851 and progression-free survival. (B) rs1051851 and overall survival. P-value based on multivariable Cox regression. Figure 2. EGFL7 rs1051851 and RR in patients with metastatic colorectal cancer treated with FOLFIRI+BV. P-value based on univariate Cochran-Armitage trend test. Patients carrying the EGFL7 rs1051851 G/G alleles had a 62% RR versus 55% in patients with A/G and 29% in patients with A/A (p=0.008, Cochran-Armitage trend test). The result remained significant after adjustment for baseline patient characteristics (p=0.035, Multivariable logistic regression test). There was no statistically significant association between the tested polymorphisms and OS or PFS. Geners-numberBase ExchangeRegionFunction EGFL7rs1051851G>A3’UTRUnknown rs2297538G>AnsCodingProtein coding, Splicing regulation ALK1rs2293094T>CIntronUnknown rs11169953C>TIntronTranscriptional regulation rs706819C>TIntronUnknown Abbreviations: UTR, untranslated region; nsCoding; nonsynonymous coding N% Age, years Median (range)62 (25-81) ≤6527464.6 > 6515035.4 Sex M25259.4 F17240.6 Caucasian 424100 Follow-Up, Median (range), mos45.1 (9.0-72.1) RR24458.5 Survival, mos OS, Median (95% CI)27.3 (24.3-30.9) PFS, Median (95% CI)10.4 (9.8-10.9) Abbreviations: mos, months; RR, response rate; OS, overall survival; CI, confidence interval; PFS, progression-free survival; ECOGPS, Eastern Cooperative Oncology Group Performance Status; LDH, lactate dehydrogenase Tumor Response, RECIST Progression-Free Survival (PFS) Overall Survival (OS) NCR+PRSD+PD Median, mos (95%CI) HR (95%CI)†HR (95%CI)‡ Median, mos (95%CI) HR (95%CI)†HR (95%CI)‡ rs1051851 G/G 279 171 (62%)103 (38%) 10.2 (9.6, 10.9)1 (reference) 29.1 (23.9, 33.0)1 (reference) G/A 121 65 (55%)54 (45%) 10.8 (9.7, 11.6)0.95 (0.75, 1.20)1.00 (0.78, 1.27) 26.1 (23.5, 31.6)1.02 (0.79, 1.32)1.17 (0.90, 1.52) A/A 17 5 (29%)12 (71%) 8.6 (2.8, 11.5)1.02 (0.59, 1.75)0.92 (0.52, 1.65) 21.8 (11.8, 43.9)1.05 (0.58, 1.90)0.91 (0.48, 1.71) P value*0.008 0.900.97 0.980.46 Progression-Free Survival: EGFL7 rs1051851 P-value = 0.90 A Overall Survival: EGFL7 rs1051851 B P-value = 0.98 N% ECOGPS 035784.2 1-26715.8 Primary tumor site Right colon10725.2 Left colon18042.5 Rectum12228.8 Colon, rectum10.2 Unknown143.3 High LDH Yes18844.3 No18844.3 NA4811.3 Response Rate: EGFL7 rs1051851 Abstract ID: 3565