title: Freezing Nitrogen Ethanol Composite May be a Viable Approach for Cryotherapy of Human Giant Cell Tumor of Bone.
abstract: BACKGROUND
  Liquid nitrogen has been used as adjuvant cryotherapy for treating giant cell tumor (GCT) of bone. However, the liquid phase and ultrafreezing (-196° C) properties increase the risk of damage to the adjacent tissues and may lead to perioperative complications. A novel semisolid cryogen, freezing nitrogen ethanol composite, might mitigate these shortcomings because of less-extreme freezing. We therefore wished to evaluate freezing nitrogen ethanol composite as a coolant to determine its properties in tumor cryoablation.
  
  
  OBJECTIVE
  (1) Is freezing nitrogen ethanol composite-mediated freezing effective for tumor cryoablation in an ex vivo model, and if yes, is apoptosis involved in the tumor-killing mechanism? (2) Does freezing nitrogen ethanol composite treatment block neovascularization and neoplastic progression of the grafted GCTs and is it comparable to that of liquid nitrogen in an in vivo chicken model? (3) Can use of freezing nitrogen ethanol composite as an adjuvant to curettage result in successful short-term treatment, defined as absence of GCT recurrence at a minimum of 1 year in a small proof-of-concept clinical series?
  
  
  METHODS
  The cryogenic effect on bone tissue mediated by freezing nitrogen ethanol composite and liquid nitrogen was verified by thermal measurement in a time-course manner. Cryoablation on human GCT tissue was examined ex vivo for effect on morphologic features (cell shrinkage) and DNA fragmentation (apoptosis). The presumed mechanism was investigated by molecular analysis of apoptosis regulatory proteins including caspases 3, 8, and 9 and Bax/Bcl-2. Chicken chorioallantoic membrane was used as an in vivo model to evaluate the effects of freezing nitrogen ethanol composite and liquid nitrogen treatment on GCT-derived neovascularization and tumor neoplasm. A small group of patients with GCT of bone was treated by curettage and adjuvant freezing nitrogen ethanol composite cryotherapy in a proof-of-concept study. Tumor recurrence and perioperative complications were evaluated at a minimum of 19 months followup (mean, 24 months; range, 19-30 months).
  
  
  RESULTS
  Freshly prepared freezing nitrogen ethanol composite froze to -136° C and achieved -122° C isotherm across a piece of 10 ± 0.50-mm-thick bone with a freezing rate of -34° C per minute, a temperature expected to meet clinical tumor-killing requirements. Human GCT tissues revealed histologic changes including shrinkage in morphologic features of multinucleated giant cells in the liquid nitrogen (202 ± 45 μm; p = 0.006) and freezing nitrogen ethanol composite groups (169 ± 27.4 μm; p < 0.001), and a decreased nucleated area of neoplastic stromal cells for the 30-second treatment. Enhanced counts of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells verified the involvement of DNA fragmentation in cryoablated GCT tissues. Western blotting analysis on the expression of apoptosis regulatory proteins showed enhancement of proteocleavage-activated caspases 3, 8, and 9 and higher ratios of Bax/Bcl2 in the liquid nitrogen- and freezing nitrogen ethanol composite-treated samples. Numbers of blood vessels and human origin tumor cells also were decreased by freezing nitrogen ethanol composite and liquid nitrogen treatment in the GCT-grafted chicken chorioallantoic membrane model. Seven patients with GCT treated by curettage and adjuvant cryotherapy by use of freezing nitrogen ethanol composite preparation had no intra- or postoperative complications related to the freezing, and no recurrences during the study surveillance period.
  
  
  CONCLUSIONS
  These preliminary in vitro and clinical findings suggest that freezing nitrogen ethanol composite may be an effective cryogen showing ex vivo and in vivo tumor cryoablation comparable to liquid nitrogen. The semisolid phase and proper thermal conduction might avoid some of the disadvantages of liquid nitrogen in cryotherapy, but a larger clinical study is needed to confirm these findings.
  
  
  METHODS
  Level IV, therapeutic study.
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.|Department of Orthopaedics, Therapeutical and Research Center of Musculoskeletal Tumor, Taipei Veterans General Hospital, Taipei, Taiwan.|Department of Orthopaedics & Traumatology, Taipei Veterans General Hospital, 201, Sec 2, Shih-Pai Road, Taipei, 112, Taiwan.|Department of Orthopedics, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Po-Kuei
authors.initials: PK
authors.lastname: Wu
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.|Department of Orthopaedics, Therapeutical and Research Center of Musculoskeletal Tumor, Taipei Veterans General Hospital, Taipei, Taiwan.|Department of Orthopaedics & Traumatology, Taipei Veterans General Hospital, 201, Sec 2, Shih-Pai Road, Taipei, 112, Taiwan.|Department of Orthopedics, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Cheng-Fong
authors.initials: CF
authors.lastname: Chen
authors.affiliation: Department of Orthopaedics, Therapeutical and Research Center of Musculoskeletal Tumor, Taipei Veterans General Hospital, Taipei, Taiwan.|Department of Orthopaedics & Traumatology, Taipei Veterans General Hospital, 201, Sec 2, Shih-Pai Road, Taipei, 112, Taiwan.|Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Jir-You
authors.initials: JY
authors.lastname: Wang
authors.affiliation: Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.
authors.forename: Paul Chih-Hsueh
authors.initials: PC
authors.lastname: Chen
authors.affiliation: Department of Orthopaedics & Traumatology, Taipei Veterans General Hospital, 201, Sec 2, Shih-Pai Road, Taipei, 112, Taiwan.|Department of Orthopedics, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Ming-Chau
authors.initials: MC
authors.lastname: Chang
authors.affiliation: Integrative Stem Cell Center, China Medical University Hospital, Institute of Clinical Medicine, China Medical University, Taichung, Taiwan.
authors.forename: Shih-Chieh
authors.initials: SC
authors.lastname: Hung
authors.affiliation: Department of Orthopaedics, Therapeutical and Research Center of Musculoskeletal Tumor, Taipei Veterans General Hospital, Taipei, Taiwan. wmchen2499@gmail.com.|Department of Orthopaedics & Traumatology, Taipei Veterans General Hospital, 201, Sec 2, Shih-Pai Road, Taipei, 112, Taiwan. wmchen2499@gmail.com.|Department of Orthopedics, School of Medicine, National Yang-Ming University, Taipei, Taiwan. wmchen2499@gmail.com.
authors.forename: Wei-Ming
authors.initials: WM
authors.lastname: Chen
mesh_terms: D000328:Adult; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001859:Bone Neoplasms / Q000473:pathology / Q000601:surgery*; D001842:Bone and Bones / Q000473:pathology / Q000601:surgery; D002645:Chickens; D003452:Cryosurgery / Q000379:methods*; D053938:DNA Fragmentation; D000431:Ethanol / Q000627:therapeutic use*; D005260:Female; D005615:Freezing; D018212:Giant Cell Tumor of Bone / Q000473:pathology / Q000601:surgery*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009584:Nitrogen / Q000627:therapeutic use*; D016896:Treatment Outcome; D055815:Young Adult
chemical_list: D000970:Antineoplastic Agents; D000431:Ethanol; D009584:Nitrogen