Introduction: Medulloblastoma is the most common malignant CNS tumor of childhood. High expression of folate receptor 1 (Folr1) was observed in some malignant epithelial tumors. However its expression and the role for clinicopathological significance and targeted therapeutic potential in MB still remain unclear.

Methods: In current study we have detected the differential expression of Folr1 in MB specimens and cells and identified its clinical, pathological and radiological values to be considered as a biomarker for diagnosis of MB. Then we have studied the targeted treatment of MB with Folr 1 targeted cytarabine (Folr1-Ara-C) both in vitro and in vivo.

Results: Folr1 protein and mRNA were overexpressed in MB specimens, while the expression level correlated with pathological subtypes. Folr1 expression was positively correlated with CSF spreading, Ki-67, MMP9, pathological subtypes and serum Folr1. Factors of age, CSF spreading, Ki-67, MMP9, strong Folr1 expression and pathological subtypes were found to be the independent prognostic values for patients with MB. Serum Folr1 presented increasing trends in turn with the different subgroups of MB, indicating that serum Folr1 showed rational sensitivity and specificity in demonstrating histological types. Folr1-Ara-C led to changes in cellular proliferation and invasion with down-regulation of MMPs proteins and activation of apoptosis in vitro. Using mouse xenograft models, Folr1-Ara-C suppressed tumor growth and improved survival of mouse by MRI and PET/CT. Immunohistochemical analysis of xenograft specimens showed decreased Ki-67 and MMP9 labelling index suggesting the effects on proliferation and invasion in vivo.

Conclusions: Folr1 may be considered as a molecular predictive candidate for histological types and serum Folr1 may be a novel noninvasive biomarker for diagnosis of MB. The application of Folr1-Ara-C contributed to be one kind of targeted therapies for MB.

Patient Care: Our research elucidated an important role for Folr1 expression in MB and unraveled a forward targeting Folr1 therapy for MB or other high-expressed cancers.

Learning Objectives: To discover the differential expression of Folr1 in MB and its relationship to clinical, pathological and radiological features as well as targeted therapy for MB indicating that Folr1 could be used as a potential candidate to diagnose and treat.

References: 1. Northcott PA, Jones DT, Kool M, et al. Medulloblastomics: the end of the beginning. Nat Rev Cancer. 2012;12(12):818-34. 2. Paul Gibson, Yiai Tong, Giles Robinson, et al. Subtypes of medulloblastoma have distinct developmental origins. Nature. 2010;12(468):1095-99. 3. Vijay R, Marc Remke, Eric B, et al. Recurrence patterns across medulloblastoma subgroups: an integrated clinical and molecular analysis. Lancet Oncol. 2013;14(12):1200-07. 4. David N.Louis, Hiroko O, et al. The 2007 WHO classification of tumors of the central nervous system. Acta Neuropathol. 2007;114(7):97-109. 5. David N.Louis, Arie Perry, Guido Reifenberger, et al. The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol. 2016;113 (6):803-20. 6. Nicholas G, Gottardo, Jordan R, et al. Medulloblastoma down under 2013: a report from the third annual meeting of the international medulloblastoma working group. Acta Neuropathol. 2014;127(11):189-01. 7. Tarbell NJ, Friedman H, Polkinghorn WR, et al. High-risk medulloblastoma: a pediatric oncology group randomized trial of chemotherapy before or after radiation therapy. J Clin Oncol. 2013;31(23):2936-41. 8. Ellison DW, Kocak M, Dalton J, et al. Definition of disease-risk stratification groups in childhood medulloblastoma using clinical, pathologic, and molecular variables. J Clin Oncol. 2011;29(11):1400-07. 9. Ramaswamy V, Northcott PA, Taylor M, et al. FISH and chips: the recipe for improved prognostication and outcomes for children with medulloblastoma. Cancer Genet. 2011;204(11): 577-88. 10. Choi SW, Mason JB. Folate and carcinogenesis: an integrated scheme. J Nutr. 2000;130(2): 129-32. 11. N.Solanky, A.Requena Jimene, S.W. D’Souza, et al. Expression of folate transporters in human placenta and implications for homocysteine metabolism. Placenta. 2010;13(6):134-43. 12. Steven D, Weitman, Richard H.Lark, et al. Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues. Cancer Res. 1992;52(11):3396-01. 13. Daniel J, O’Shannessy, Gordon Yu, et al. Folate receptor Alpha expression in lung cancer: diagnostic and prognostic significance. Oncotarget. 2012;3(4):414-25. 14. Akira Kurosaki, Kosei Hasegawa, Tomomi Kato, et al. Serum folate receptor alpha as a biomarker for ovarian cancer: implications for diagnosis, prognosis and predicting its local tumor expression. Int. J. Cancer. 2016;1388(8):1994-02. 15. S.D.Weitman, K.M.Frazier, B.A.Kamen, et cl. The folate receptor in central nervous system malignancies of childhood. J Neurooncol, 1994,21:107-11. 16. Wu Ht, Zhan Yp Qu Ym, et al. Changes of folate receptors-a protein expression in human gliomas and its clinical relevance. Chin J Surg. 2014;3(12):202-07. 17. Xiaohai Liu, Sihai Ma, Yong Yao, et al. Differential expression of folate receptor in pituitary adenomas and its relationship to tumor behavior. Neurosurg. 2012;70(5):1274-80. 18. Jose Pablo, Rohit Bhargava, Brian K, et al. Expression of high affinity folate receptor in breast cancer brain metastasis. Oncotarget. 2015;6(30):30327-33. 19. Hailong Liu, Yanming Qu, Chunjiang Yu, et al. An experimental study in vitro on folate receptor targeted cytarabine therapy for medulloblastoma. Chin J Neurosurg. 2015;31(5):514-18. 20. Basal E, Eghbali-Fatourechi GZ, Kalli KR, et al. Functional folate receptor alpha is elevated in the blood of ovarian cancer patients. PLoS One. 2009;4(7):e6292. 21. Kelemen LE. The role of folate receptor alpha in cancer development, progression and treatment: Cause, consequence or innocent bystander. Int J Cancer. 2006;119(2):243–50. 22. Linda E, James D, Christine Parkinson, et al. Conditions associated with circulating tumor associated folate receptor 1 protein in healthy men and women. PloS One. 2014;5(9):e96542. 23. Jones SK, Lizzio Y, Merkel OM. Folate receptor targeted delivery of siRNA and paclitaxel to ovarian cancer cells via folate conjugated triblock copolymer to overcome TLR4 driven chemotherapy resistance. Biomac. 2016;17(1):76-87. 24. Boyuan Huang, Chunjiang Yu, Qanming Qu, et al. The PD-1/B7-H1 pathway modulates the natural killer cells versus mouse glioma stem cells. PloS One. 2015;10(8):1-14. 25. Tadashi O, Masahiko T, Masaya N, et al. Factors affecting peritumoral brain edema in meningioma: special histological subtypes with prominently extensive edema. J Neurocol. 2013;111(10):49-57. 26. Soutoo AA, Chimelli L, Takya CM, et al. Brain edema in meningiomas: radiological and histological factors. Arq Neuropsiquiatr. 2002;60(3-B):807-17. 27. Dong Chi, Zhou Junlin. Correlation between peritumorous edema on MRI and P73 protein expression in intracal haemangiopericytoma. Chin J Med Imaging Technol. 2009;25(11):1980-83. 28. Zhang MS, Ou YW, Zhang HW, et al. Leptomeningeal metastasis from central nervous system tumors: a study of classification and stage in the spinal canal of 58 patients. Chin Sci Bull. 2012;57(8):2914-19. 29. Yunwei O, Lingyan L, Liyan X, et al. TRAP1 shows clinical significance and promotes cellular migration and invasion through STAT3/MMP2 pathway in human esophageal squamous cell cancer. J Gen Genom. 2014;41(9):529-37. 30. Jie Chen, Weimin Zhang, Lijia Dong, et al. Feed-forward reciprocal activation of PAFR and Stat3 regulates epithelial-mesenchymal transition in non-small cell lung cancer. Cancer Res. 2015;75(19):4198-10. 31. Eric M, Thomas Hielscher, Eric Bouffet, et al. Prognostic value of medulloblastoma extent of resection after accounting for molecular subgroup: a retrospective integrated clinical and molecular analysis. Lancet Oncol. 2016;17(4):484-95. 32. Ximbo Zhang, Frederick L. Cytosine arabinoside substitution decreases transcription factor-DNA binding element complex formation. Arch Pathol Lab Med. 2004;128(12):1364-71. 33. Wang CY, Cusack JC, Liu R, et al. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-?B. Nat Med. 1999;5(5):412-17. 34. Yanyan L, Lixiao D, Edwin Roger Parra Cuentas, et al. Epithelial-mesenchymal transition is associated with a distinct tumor microenviroment including elevation of inflammatory signals and multiple immune checkpoint in lung adenocarcinoma. Clin Cancer Res. 2016;22(14):3630-43. 35. Morrissy AS, Garzia L, Shih DJ, et al. Divergent clonal selection dominate medulloblastoma at recurrence. Nature. 2016;529(7586):351-57. 36. O’shannessy J, Grasso L, WAN S, et al. Folate receptor alpha as a diagnostic and prognostic marker for folate receptor alpha-expressing cancers. Patent Cooperation Treaty (PCT). 2012;6:1-178. 37. Vergote I, Armstrong D. Phase 3 double-blind placebo-controlled study of weekly farletuzumab with carboplatin/taxane in subjects with platinum sensitive ovarian cancer in first replace. Int J Gynecol Cancer. 2013;23(8):11. 38. Jasson, A, Katherine M, Bell-McGuinn, et al. Farletuzumab, a humanized monclonal antibody against Folate Receptor 1, in epithelial ovarian cancer: a Phase I study. Clin Cancer Res. 2010;16(21):5288-96. 39. Jeffrey M, Vincent Th, Ramaekers, et al. The diagnostic utility of folate receptor autoanbibodies in blood. Clin Chem Lab Med. 2013;51(3):545-54. 40. Molloy AM, Quadros EV, Sequeria JM, et al. Lack of association between folate-receptor autoantibodies and neural-tube defects. N Eng J Med. 2009;361(2):152-60. 41. Ramaekers VT, Quadros EV, Sequeira JM, et al. Role of folate receptor autoantbodies in infantile autism. Mol Psychiatry. 2013;18(3):270-71. 42. Parker N, Turk MJ, Westrick E, et al. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem. 2005;338(3): 284-93. 43. Trump DP, Mathias CJ, Yang Z, et al. Synthesis and evaluation of 99mTc(CO)(3)-DTPA-folate as a folate receptor targeted radiopharmaceutical. Nucl Med Biol. 2002;29(5):569-73. 44. Juan Q, Ping D, Xiaoyu M, et al. Folic acid-conjugated fluorescent polymer for up-regulation folate receptor expression study via targeted imaging of tumor cells. Biosen and Biolectron. 2016;78(1):147-53. 45. Ning C, Chen S, Shuai L, et al. Folic Acid-Conjugated MnO Nanoparticles as a T1 Contrast Agent for Magnetic Resonance Imaging of Tiny Brain Gliomas. ACS Appl Mater Interfaces. 2014;6(22):19850-57. 46. Xiaohai Liu, Sihai Ma, Congxin Dai, et al. Antiproliferative, antiinvasive, and proapoptotic activity of folate receptor a-targeted liposomal doxorubicin in nonfunctional pituitary adenoma cells. Endocrinology. 2013;154(4):1414-23. 47. Rao SI, Pugh M, Nelson M, et al. Development and validation of a UPLC-MS/MS method for the novel folate-targeted small molecule drug conjugate EC1456 and its metabolites in tumor homogenates from mice. J Pharm Biomed Anal. 2016;122(4):148-56. 48. Li L, Mengyao Z, Damiano Librizzi, et al. Efficient and tumor targeted siRNA delivery by polyethylenimiegraft-polycaprolactone-block-poly (ethylene glycol)-folate (PEI-PCL-PEG-Fol). Mol Pharmaceutics. 2016;13(1):134-43. 49. Hockenbery DM, Oltvai ZN, Yin XM, et al. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993;75(2):241-51. 50. Jin Y, Lu Z, Ding K, et al. Antineoplastic mechanism of niclosamide in acute myelogenous leukemia stem cells: inactivation of the NF-kappaB pathway and generation of reactive oxygen species. Cancer Res. 2010;70(6):2516-27. 51. Yao X, Zhu F, Zhao Z, et al. Arctigenin enhances chemosensitivity of cancer cells to cisplatin through inhibition of the Stat3 signaling pathway. J Cell Biochem. 2011;112(10):2837-49.