Mutations in the let-7 binding site - a mechanism of RAS activation in juvenile myelomonocytic leukemia?

Cryopreservation is the technique of choice to store autologous peripheral blood stem cells (PBSC) and allogeneic umbilical cord blood stem cells. In contrast, donor stem cells for allogeneic hematopoietic stem cell transplantation (alloHSCT) are usually collected immediately prior to transplant and transfused “fresh” into the recipient. However, for medical reasons it may be necessary to delay conditioning and subsequent graft infusion when stem cell collection is already planned and cannot be rescheduled. In such situations allogeneic grafts may be cryopreserved and patients receive thawed products.

In 2009, 4 pediatric patients (male: female = 1:3; median age: 7 years) suffering from relapsed acute myeloid leukemia, Mucopolysaccharidosis I Hurler, relapsed severe aplastic anemia and metastatic osteosarcoma, respectively, were scheduled for allogeneic stem cell transplantation (patients’ characteristics are summarized in Table 1). Conditioning had to be postponed for acute infections in 3 patients and for persistence of blasts in one patient. Stem cell donors (3 unrelated, one related) gave their consent to short-term stem cell cryopreservation and graft manipulation.

Cell doses of around 10×10/kg recipient body weight were requested. PBSC products were CD3/19-depleted, in 2 patients half of the product was CD34+selected; the median number of CD34cells/kg recipient body weight was 14.1×10/kg (median CD3cells/kg: 1.1×10). The manipulated products were split into aliquots/bags that could be infused several hours apart or on subsequent days with regard to potential dose-dependent DMSO-neurotoxicity especially in children with low body weight (≤ 10 kg).

The time interval from the end of the donation to freezing was 29, 28 and 27 h, respectively, for externally harvested apheresis products (n=3, patients 1–3), and 10 and 5 h, respectively, for in-house harvested products (n=2, patient 4).

Figure 1.Donor chimerism of patients 1–4 as assessed by SNP-analysis on days +7,+14,+21,+28, + 60, +90, +120, +150, +180, +210 and +240 (upper panel). Absolute numbers of T-lymphocytes, B-lymphocytes, NK-cells, and monocytes at the same time points (lower panel).

Cryoprotectant solutions for unmanipulated PBSC consisted of 80% autologous plasma and 20% DMSO (CryoSure, WAK Chemie, Steinbach, Germany), for CD34 selected and CD3/19 depleted products of 70% MEM medium, 10% human albumin 20% and 20% DMSO-CryoSure. Apheresis products were added to equal volumes of cryoprotectant solution to a final DMSO-concentration of 10%. Cryopreservation was performed using a controlled phased freezing procedure within one hour to a temperature of −120°C (Ice-Cube 1810 Computer Freezer, Sylab). Cryo-bags were stored in liquid nitrogen at a temperature of −196°C. At the time of alloHSCT, frozen grafts were thawed rapidly in a warm water bath at 37ºC and infused via a central venous catheter. Stem cell products were cryopreserved for a median 12 days (range 6–102 days).

Sterility testing by bacterial cultures of aliquots yielded negative results in all patients. CD34 cells and CD3 cells were measured by flow cytometry in aliquots of PBSC products before cryopreservation and after thawing; correlation coefficient for CD34 cell numbers was 0.9557, for CD3 cell numbers 0.9932. Viability after thawing was assessed by trypan blue staining and was 91±4.08%.

Three patients received fludarabine-based conditioning, one patient a busulfan-based regimen.

Stem cell infusion was tolerated without side effects.

All patients had three-lineage hematopoietic engraftment. The posttransplant course of patients 2 to 4 was uneventful and without signs of GVHD. Patient 1 with a refractory high-risk AML, who received a partly unmanipulated PBSC-graft, reactivated the hemophagocytic syndrome, which she had manifested just before the start of conditioning, in the early posttransplant period, and developed acute graft-versus-host-disease (GVHD) of skin and gut up to grade IV. She had a poor graft function due to the cumulative toxicity of the intense multimodal immunosuppressive and virostatic treatment, and succumbed to aspergillus pneumonia on day +140 in remission from her refractory AML.

Transplant characteristics as well as engraftment data are summarized in Table 1. Chimerism and immune reconstitution data are shown in Figure 1.

Informed consent was obtained from the patients’ parents to stem cell transplantation including cryopreservation and to the study, which was approved by the Ethics Committee of the Medical University of Graz.

Table 1.Patients’ characteristics, details of transplant procedure and posttransplant course.

There are several advantages and disadvantages regarding cryopreservation of allogeneic stem cells as discussed in a recent review.1 Data on the use of cryopreserved allogeneic grafts are limited and almost exclusively restricted to adults. For cryopreserved compared to fresh bone marrow transplants from related and unrelated donors no differences were found with regard to time to myeloid or platelet engraftment, incidence or severity of acute and chronic GVHD, day 100 survival and long-term survival.2–4 A trend toward less acute GVHD in patients who received cryopreserved bone marrow reported by one group5 was not confirmed by others.2 Although the use of cryopreserved allogeneic PBSC grafts has increased over recent years,1 few reports have been published so far demonstrating conflicting results.6–8

In 2006, Frey et al. stated in their review that the “available literature does not sufficiently justify the dogmatic use of fresh over frozen allografts”.1 Since then, contradictory data on cryopreserved alloPBSC grafts were added. In modern and increasingly sophisticated transplant settings including alloHSCT from haploidentical family donors and posttransplant adoptive immunotherapy, graft manipulation is a prerequisite and temporary cryopreservation of certain grafts as well as donor lymphocytes is being performed.9–11

There is a concern that grafts might be cryopreserved in advance but not utilized and that donors might be subjected unnecessarily to the potentially harmful procedure of stem cell collection.12 This could be met by keeping the time interval between harvest and the definitive start of the transplant procedure as short as possible (e.g. around 30 days) as suggested by Frey based on reported median storage times ranging from 10.5 to 38 days.1

We conclude that short-term cryopreservation of unrelated PBSC allografts in pediatric patients is feasible for compelling medical reasons. The advantages of the use of cryopreserved grafts must be individually outweighed against the concerns raised but not definitely answered by the available data. Looking to the future, and in view of the increasing use of manipulated grafts, we suggest that short-term cryopreservation might be an option to ensure graft quality and to enhance procedure safety for the patient without increasing the risk for the donor. Therefore, further studies regarding cryopreservation of allogeneic PBSC, including manipulated grafts on a larger and more homogenous patient cohort, are required.

Acknowledgments

the authors would like to thank Andrea Raicht, B.Sc., and Barbara Egner, B.Sc., for performing SNP-and FACS analyses and for their technical assistance.

Footnotes

• The authors reported no potential conflicts of interest.