We read with great interest the article by Fernyhough et al.1 about the recovery of hematopoietic stem cells (HSCs) after extended cryostorage. As successful engraftment of HSCs depends on several factors related to the HSC collection itself (CD34+ cell count, cryopreservation techniques and constant temperature monitoring) and to the receiving host (myeloablative chemotherapy, BM microenvironment and therapeutic history), we want to report our scientific and clinical experience in this field, adding some comments about the currently used management of cryosystems. Studies by other groups about the relationship between cryostorage duration and hematopoietic engraftment potential have been performed mostly with in vitro assays on cord blood cells2 or, in vivo, using only BM-derived HSCs cryostored for a limited amount of time.3, 4 In addition, in the latter reports, data are available only for patients engrafted once, taking as a reference for hematological recovery the accepted time frame for BM reconstitution;5 these studies, although reliable, lack an internal control to compare the engraftment potential of hematopoietic stem cells after a short and a long period of cryostorage.
We want to report data from two of our patients engrafted successfully twice, each time after myeloablative conditioning. These data come from a survey performed in our database on more than 400 transplanted, consecutive and unselected patients; in addition, the same search was not able to identify any graft failure. In both cases, HSCs harvested from peripheral blood were cryopreserved and reinfused for the first time after a few months and after a cryostorage lasting about 4 and 10 years the second time. As for clinical practice and European Group for Blood and Marrow Transplantation (EBMT) guidelines, hematopoietic growth factors (that is, G-CSF, 300 μg/day, subcutaneously) were administered after each reinfusion until WBC count reached 5.0 × 103/μL. Both patients gave written informed consent before myeloablative therapy.
A 67-year-old man was diagnosed with mantle cell non-Hodgkin’s lymphoma and referred to our institution in June 2008. Owing to clinical conditions (stage IV disease for multiple lymphadenopathies, involvement of spleen, BM and gastrointestinal tract) and age, he was treated, after a CHOP-like therapy induction phase, with a high-dose chemotherapy (CY, 5.6 g/sm; cytarabine, 2 g/sm every 12 h for 4 days), achieving CR. Hematopoietic progenitor cells (HPCs) were collected by leukapheresis after both high-dose chemotherapy courses and cryopreserved using the following conditions: cells were diluted with an equal volume of 20% DMSO and 20% human albumin and were frozen in a temperature-controlled cryostat (freezing temperature rate, 1 °C/min) and stored in the vapor phase of liquid nitrogen. The patient was then conditioned with high-dose yttrium-90-ibritumomab tiuxetan6 (1.2 mCi/kg of body weight (b.w.)) and grafted with 9.4 × 106 CD34+ cells per kg b.w.; engraftment was successful and the patient achieved a granulocyte count of 0.5 × 109/L and a platelet count of 20 × 109/L after 8 and 7 days, respectively. A first relapse occurred in March 2011, CR was then obtained after six cycles of immunochemotherapy with rituximab (375 mg/sm, day 1) and bendamustine (120 mg/sm, days 2 and 3).7 The patient experienced a second relapse in May 2012, he was treated with chemotherapy (R-EPOCH) in association with bortezomib8 obtaining VGPR and underwent a second autologous transplant. High-dose melphalan (120 mg/sm) was used as the conditioning regimen and the patient was grafted in December 2012 with 6.7 × 106 CD34+ cells per kg b.w. that had been collected in August 2008. Again, engraftment was successful and the patient achieved a granulocyte count of 0.5 × 109/L and a platelet count of 20 × 109/L after 9 and 11 days, respectively. The patient is now in stable disease (SD) since the engraftment.
A 54-year-old man was diagnosed with multiple myeloma (International Staging System stage I) and referred to our institution in October 2000. After the induction phase consisting of three cycles of VAD (vincristine, 0.4 mg daily, days 1–4; adryamicin, 9 mg/sm daily, days 1–4; dexamethasone, 40 mg daily, days 1–4) chemotherapy, he underwent high-dose CY (5.6 g/sm) and HPCs were collected by leukapheresis, frozen and cryopreserved as described above. The patient obtained a VGPR and was thus myeloablated with high-dose melphalan (180 mg/sm) and transplanted with 7.3 × 106 CD34+ cells per kg b.w.; engraftment was successful and full hematopoietic recovery was obtained (granulocyte count of 0.5 × 109/L and platelet count of 20 × 109/L after 10 and 11 days, respectively). Thalidomide maintenance was started and continued until July 2006 when progressive disease (PD) was documented on bone X-rays; in consideration of the limited extension of the disease, the patient was treated with PAD9 chemotherapy for six cycles obtaining SD. A second PD was documented in December 2009: induction chemotherapy consisted of four courses of VCD (bortezomib, 1,3 mg/sm days 1, 4, 8, 11; CY, 300 mg/sm, day 1; dexamethasone, 40 mg, days 1–4, 8–11, 17–20) followed by myeloablative conditioning with high-dose melphalan (200 mg/sm). HPCs collected at the time of the first treatment (January 2001; 4 × 106 CD34+ cells per kg b.w.) were used as the source of graft; engraftment was successful in this case also and the patient reached a granulocyte count of 0.5 × 109/L and platelet count of 20 × 109/L after 14 days. Unfortunately, a third relapse occurred after a short time and patient died due to interstitial pneumonia after allogeneic BMT.
To our knowledge, this report is the first to show that hematopoietic reconstitution is not only possible with HSCs cryostored in standard conditions for several years, but, thanks to the possibility of having data from the same BM microenvironment (that is the same patient), engrafted twice many years apart, it demonstrates that, under constant temperature control, HSCs can be used safely even after extended cryostorage. In particular, in our two patients, the slower hematological recovery after the second HSC reinfusion might be because of the independent contribution of several factors, that is (i) lower number of reinfused cells, (ii) therapy-induced changes in host marrow stromal cell function and (iii) a progressive loss of hematopoietic reconstituting potential during storage. In this respect, while the first two factors have to be taken seriously into account, for the third hypothesis we have records of temperature control over time (not showing any significant fluctuation) but, unfortunately, no viability data; however, for this latter issue, it must be considered that both trypan blue viability and CD34 Ag detection have been reported as potentially unreliable in detecting clonogenic cells in frozen/thawed cell populations.10 In addition, as colony forming units granulocyte-macrophage (CFU-GM) counts do not decrease over time,1 our in vivo data seem to provide support for the use of more functional methods (such as CFU-GM assay) to evaluate the real hematological clonogenic potential of cryopreserved stem cells. It is to be hoped that in the near future other cases similar to ours will help in clarifing this issue, performing biological assays (CD34 antigen detection, viability and CFU-GM assays) before and after HSC cryopreservaton and then correlating in vitro and clinical data.
One of the reasons for the engraftment or colony assay failure reported by others might be explained by variations or lack of accurate temperature control over time. As requested by the JACIE/FACT Program,11 to which our institution is accredited, each BMT center and each cryopreservation facility should not only have constant monitoring of the conditions of temperature and liquid nitrogen levels of the containers where HPCs are stored, but should also adopt and run a disaster plan to avoid the possibility of losing HPCs in case of power failure or other accidents.11 We do believe that adopting this quality program will improve the safety level and the accuracy that we all use in storing the HPCs of our patients, considering the increasing collection and storing needs of allogeneic and cord blood-derived HPCs.
This paper is in memory of Mrs Adele Piccinini.