Source of funding: Project funded by UK Government Engineering and PhysicalSciences Research Council (EPSRC) and Bioprocessing Research Industry Club (BRIC)part of the Biotechnology and Biological Sciences Research Council (BBSRC).

Conflict of interest: None declared.Acknowledgements: Prof Nigel Slater, Dr Duncan Sharp, Dr Andy Picken, Karina

Brosnan, Kirsty-Louise Marrow, Nathalie Robinson, Alex Chan.Email address: T.J.Morris@lboro.ac.uk

http://dx.doi.org/10.1016/j.cryobiol.2013.09.121

116Applications of the DtBIG method of cooling rate quantification to cell therapycryopreservation under GMP compliance. David Frey, Jung Lee, Cellerant Thera-peutics, Inc., San Carlos, CA, USA

Cryopreservation is a commonly adopted method for preserving allogeneic celltherapies through long-term storage and distribution to end-users. For clinical andeventually commercial manufacturing of these products, the cooling leg of the cryo-preservation cycle is performed within a manufacturing environment that is regu-lated for Good Manufacturing Practices (GMP). To comply with GMP, the coolingprocess is held to high standards for process characterization and control, includingdefining critical process parameters, specifying acceptable ranges for those parame-ters, and verifying that each parameter is consistently controlled to within its speci-fied range during normal operation. For cell products of defined composition, coolingrate is the primary critical process parameter that drives the performance of the cool-ing process. To successfully describe product cooling, cooling rate should be quanti-fied in a specific and relevant manner that is independent of scale and freezer model,and enables measurement of multiple unit containers across a freezer load of interest.

To support process development, engineering, scale-up and validation of the con-trolled-rate freezing step for CLT-008, an ‘‘off-the-shelf” cryopreserved myeloid pro-genitor cell therapy for neutropenia, we have defined a term which quantifiesproduct cooling rate and serves as the primary critical process parameter linkingproduct cooling to recovery of function. This cooling rate term, DtBIG, represents thetime required for bulk ice growth and is defined as the time difference between prod-uct nucleation and achieving �20 �C: DtBIG ¼ t�20 � tn0 . DtBIG has been successfullyapplied to internal and external monitoring of cryobags and vials to determine cool-ing rates of multiple individual product containers across controlled-rate freezerloads. DtBIG distributions have served as the basis for load configuration and coolingprogram engineering as well as a means of setting acceptable ranges for performancequalification of GMP manufacturing freezes. The application of this cooling rate quan-tification method to cell cryopreservation development and GMP manufacturing isdescribed using CLT-008 as a case study.

Source of funding: Biomedical Advanced Research and Development Authority(BARDA).

Conflict of interest: Cellerant is developing CLT-008 as a commercial therapy forneutropenia.

Email address: dfrey@cellerant.com

http://dx.doi.org/10.1016/j.cryobiol.2013.09.122

117Cryosurgery can be effective therapeutic method at selected patients with Paget’sdisease of the nipple: A series of cases report. Marek Rzaca 1, Radoslaw Tarkowski 2,1 Regional Specialist Hospital, Department of Surgical Oncology, Wroclaw, Poland,2 Department of Oncology, Wroclaw Medical University, Poland

Introduction: Paget’s disease of the nipple is a rare form of breast malignancy.Underlying breast cancer, either in situ or invasive accompanies lesions of the nip-ple in most of the cases. The connection between both entities and their exact originremains unclear. Nevertheless, underlying ductal breast cancer is often confined tothe central, subareolar part of the breast. Radical mastectomy, although successful,seems to be too mutilating a treatment in the era of breast sparing surgery. Studiesdescribing breast conserving surgery performed without adjuvant treatment arerare and the patients not numerous. Due to low incidence of the disease, thereare no randomised trials conducted which could show the optimal method of treat-ment. We have not found any publications describing cryotherapy in Paget’s diseaseof the nipple. Due to our broad experience in cryodestruction of skin and mucosalcancers we find it very efficient therapeuthic method in mentioned indications.Despite of this we were encouraged with good effects obtained with local cryother-apy of extramammary Paget’s disease. We performed the cryodestruction of thenipple areola complex (NAC) in the group of patients who could not undergo mas-tectomy and axillary clearance as a standard form of treatment performed in thetime when the study was conducted. We followed the results during long periodof surveillance. Material and method: We performed cryosurgery of the affectednipple-areola complex (NAC) in treatment of 6 patients with Paget’s disease of

the nipple who presented general contraindications or lack of consent for surgery.All the patients underwent cryoablation of the changed NAC performed in localanesthesia of 1% lignocaine solution injected subcutaneously. The device used dur-ing the procedure was Kriopol K26-M1, produced by Kriomedpol (liquid nitrogenused as a cryogen, tank capacity 26 dm3, surface temperature of the tip cryoappli-cator: �190 �C as described by procucer , pressure 50 kPa (0.5 bar), time to achieve-ment 50 kPa pressure in the tank: 20 min, power: V 230 ± 10%, frequency 50 Hz).Median diameter of cryoprobe used separately was 25 mm, less than the averagediameter of the NAC. At least three successive freeze-thaw cycles were performedsuccessfully in each case. Results: With a follow up ranging from 60 to 121 months(average 94) we obtained very good results in terms of disease specific survival:there were 2 cases of recurrent disease, confined to the scar and treated successfullywith cryosurgery again; 4 patients are alive without disease. Death was not relatedto cancer in the remained two cases. Conclusions: Cryotherapy is successful form oftreatment of localised Paget‘s disease of the nipple – especially in face of contrain-dications for surgery or lack of patient’s agreement for operational treatment.

Source of funding: Own sources.Conflict of interest: None declared.Email address: rt@tarkowski.eu

http://dx.doi.org/10.1016/j.cryobiol.2013.09.123

118Cryodestruction of regionally metastatic cutaneous melanoma (in-transitmetastases) is effective therapeutic method in terms of survival: A series of casesreport. Radoslaw Tarkowski 1, Marek Rzaca 2, Roman Czarnecki 2, Marek Bebenek 3,1 Department of Oncology, Wroclaw Medical University, Poland, 2 Regional SpecialistHospital, Department of Surgical Oncology, Wrocław, Poland, 3 Lower Silesian CancerCenter, Wrocław, Poland

Introduction: Cutaneous melanoma of the limb, treated primarily with surgicalresection and sentinel node biopsy followed by lymphadenctomy if necessary canrecur as multiple in-transit metastases localised within the skin of the extremity.This feature means poor prognosis, although patients with such spread of diseasestill have a chance for a long-therm survival, so they should be treated further withcurative intent. There are several options of treatment in this setting: surgical resec-tion of small number of lesions or isolated limb perfusion in hiperthermia or evenlimb amputation in very select cases. There is some morbididity associated witheach of the methods and melanoma usually continues to progress despite of ther-apy applied. Cryodestruction is a method which obtains good and sometimes excel-lent local control in this setting of recurrent melanoma. According to Neel (Vet ClinNorth Am 1980) cryoablation can stimulate immunological response against theantigens of tumor treated with this kind of therapy. Materials and methods: 3patients treated previously with surgery and selective groin lymphadenectomywith in-transit metastases as recurent disease which occurred 6 and 12 monthsafter first surgery within skin of lower extremities. The number of tumors wasuncountable. There were no distant metastases found in imaging studies in any casetumors were treated with cryodestruction performed with Kriomed device (liquidnitrogen used as a cryogen). There were three freeze-thaw cycles performed forevery tumor. Each freeze cycle lasted 60 seconds from the point when cryoprobereached the temperature �180 �C to the point of 0 �C, the depth of freezing wasnot measured; thawing lasted until the temperature of cryoprobe reached the tem-perature of 36 �C. The probe was applied at room temperature in the first cycle, then36 �C within the next two of three cycles. One of the patients was treated with ourown method: hypothermic syringe cut and filled with 5–10 cm2 of liquid nitrogenwas placed with the opened end directly over the tumor allowing direct contact ofthe cryogen with tumors surface. Results: All of the metastases treated with cryo-surgery were destroyed. Usually there were consecutive phases of edema and ser-ous blister formation replaced by crust. In one patients we noticed disappearence ofthe tumors that were not treated with cryodestruction, as a symptom of systemicresponse to cryotherapy – this patient has stayed alive free of disease for 48months. Overall survivall is 12–98 months. There were other recurrent tumorsnoticed during follow up control at two other patients, which were treated againwith cryosurgery. None of the patients obtained systemic therapy (i.e. chemother-apy). There were no distant metastases in any case observed. There were no majorcomorbidities noted. Conclusions: Cryodestruction performed on patients with intransit metastases of skin melanoma can be an effective, cost-effective method ofpaliative treatment and allows good local control. Besides the direct ablative effecton frozen tissues there is perhaps another mechanism enhancing effectiveness ofthe method: stimulation of the immunological response due to presentation ofthe antigens of the cryodestroyed metastatic tumors.

Source of funding: Own sources.Conflict of interest: None declared.Email address: rt@tarkowski.eu

http://dx.doi.org/10.1016/j.cryobiol.2013.09.124

Abstracts / Cryobiology 67 (2013) 398–442 431