Advantages of non cryopreserved autologous hematopoietic stem cell transplantation against a cryopreserved strategy
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(2) Advantages of non-cryopreserved autologous hematopoietic stem…. 961. developing countries have reported their experience mainly in MM patients with a non-cryopreserved (non-CRYO) modality that avoids stem cell freezing on the basis of the stem cell ability to survive several days in non-freezing refrigeration [8–10]. Our transplantation program started in January 2015 a non-CRYO model in patients with MM and LYMPH in order to avoid a possible DMSO toxicity and cryopreservation costs; the preliminary information from this strategy indicated that it did not have any impact on the stem cell viability nor the engraftment capacity or immediate toxicities in our autografted population of patients. The present study was designed to compare our strategy (non-CRYO group) in terms of short-term transplant related toxicities and hematological recovery after auto-HSCT with the “standard of care” approach (CRYO group) of a Bone Marrow Transplantation Program from a Spanish University Hospital in Barcelona (Institut Català d’Oncologia (ICO)—Hospitalet). Herein, we are presenting the results of this study.. Transplantation modalities and peripheral blood stem cells (PBSC) storage. Patients and methods. Cryopreserved modality (CRYO). Characteristics of the transplantation programs. DMSO at 7.5% was the standard strategy for cryopreservation after the apheresis were completed and then stored until the infusion day. CryoStore freezing bags (Origen Biomedical) were used to freeze the apheresis product. DMSO was dissolved with the plasma obtained, after a 2400 rpm centrifugation, Bags were placed into an aluminum canister and, in a horizontal position, introduced into compartment of a −80 °C mechanical freezer (Thermo, Saint-Herblain, France). On day 0, bags were removed from liquid nitrogen storage and rapidly thawed in a 37–40 °C water bath and promptly reinfused into the patient, as previosuly reported [12, 13].. Both transplant centers are considered to be referral hospitals for HSCT in both countries. In total 20 allogeneic and 40 auto-HSCT are performed on average each year at the Universidad Católica Pontificia in Chile; 30 allogeneic and 45 auto-HSCT are correspondingly performed at ICO-Hospitalet in Barcelona, Spain. The Chilean program uses high-dose melphalan for patients with MM (200 or 140 mg/m2 according to kidney function) and BEC (BCNU 300 mg/m2 iv on day −5, etoposide 150 mg/m2 iv and cyclophosphamide 1500 mg/m2 iv from days −5 to −2) as a conditioning regimen for patients with LYMPH. This scheme was adopted in 2009 because of its lower cost [10]. The Catalan program also uses the same high-dose melphalan protocol for patients with MM but BEAM (BCNU 300 mg/m2 iv on day −6, cytarabine 200 mg/m2 iv and etoposide 150 mg/m2 iv from day −6 to −3 and melphalan 140 mg/m2 iv on day −2) in patients with LYMPH.. Mobilization and collection of PBSC All patients received filgrastim at a dose of 10 mcg/kg sc bid, 4–5 days before apheresis. Plerixafor at a dose of 0.24 mg/kg was used for bad mobilizers with CD34+ cell counts below 30,000 per uL on the first day of the apheresis procedure, as described previously [11]. All MM and LYMPH patients included in the Chilean program were harvested with GCSF±plerixafor. In the Catalan program, MM patients were harvested with GCSF±plerixafor and LYMPH patients were collected either after GCSF±plerixafor or chemotherapy and GCSF (n = 10). Apheresis procedures were performed with a Spectra Optia Apheresis System (Terumo Corporation, Tokyo Japan) in both institutions in the outpatient department. A successful apheresis procedure was established when the number of CD34+ cells collected/kg body weight was higher than 2.5 × 10E6/kg.. Non-cryopreserved modality (non-CRYO) Once patients achieved the best response, they received the same filgrastim±plerixafor treatment followed by CD34apheresis without DMSO freezing. The apheresis solution was acid citrate dextrose and the final product was stored at 4 °C during conditioning and then re-infused, as previously reported [14, 15].. Patient eligibility. Time of apheresis storage and viability assays. Eligible patients for the study were those aged 18 years or older that had undergone auto-HSCT for MM or LYMPH in both institutions between January/2015 to October/2016. Peripheral blood was the source of stem cells in 100% of the procedures. The study was approved by the Ethical Committees of both centers.. In non-CRYO, the apheresis product was kept at 4 °C for 2 days in MM patients and for 6 days in LYMPH patients. In CRYO, the cellular product was frozen at −80 °C, as described previously [16] and the median freezing time was 3 months (range 1–15 months). Analysis of CD34 cell viability using 7-amino actinomycyn D (7-AAD) flow.
(3) 962. M. Sarmiento et al.. Table 1 Clinical characteristics of the whole series CRYO (n = 74). Non-CRYO (n = 42). p value. 55 (22–69). 52 (22–68). NS. Male. 41 (55%). 23 (55%). NS. Female. 33 (45%). 19 (45%). 40 (54%). 29 (69%). Age (mean (range)) in years Sex. Diagnosis Multiple myeloma Amyloidosis AL Non-Hodgkin’s lymphoma DLBCL B cell Indolent lymphomas T lymphomas Hodgkin’s lymphoma. 2 (3%). 2 (5%). 26 (35%). 9 (21%). 22 (85%). 7 (90%). 3 (10%). 1 (19%). 1 (5%). 1 (1%). 6 (8%). 2 (5%). NS. CR1. 24 (32%). 24 (57%). CR2. 27 (36%). 8 (19%). PR. 23 (32%). 10 (24%). Time from diagnosis to auto-HSCT. 36 m (range 12–48). 33 m (range 12–40). NS. Treatment schemas previous to auto-HCST. 3 (range 1–4). 2 (range 1.3). NS. NS. BEC. −(40%). 11 (26%). 0.001. Melphalan. 39 (53%). 30 (71%). Conditioning regimen. 26 (35%). 1 (3%). Others. 9 (12%). 0. CR1 Complete remission 1, CR2 Complete remission 2, PR Partial remission, NS Non significant Melphalan: 200 mg/m2 iv in patients with a normal renal function and 140 mg/m2 iv in those with creatinine clearance below 30 mg/ml/min, as single dose at day −2 BEAM: carmustine 300 mg/m at day −6, cytarabine 200 mg/m and etoposide 150 mg/m2 from day −6 to −3 and melphalan 140 mg/m2 at day −2 2. Day 0 At day 0, all patients were infused with their apheresis product according to transplant modality. A protocol of infusion vigilance was performed in all CRYO patients [17]. In non-CRYO patients an expedited infusion was made.. Supportive measures. Diseases status at transplantation. BEAM. Barcelona (carmustine 300 mg/m2 iv on day −6, cytarabine 200 mg/m2 iv and etoposide 150 mg/m2 iv from day −6 to −3 and melphalan 140 mg/m2 iv on day −2) and BEC was used in Chile for the same subgroup of patients (carmustine 300 mg/m2 iv on day −5, etoposide 150 mg/m2 iv and cyclophosphamide 1500 mg/m2 iv from days −5 to −2).. All patients were hospitalized from the beginning of the conditioning regimen and during the period of pancytopenia and received transfusions, prophylactic antiviral, antifungal and antibiotics agents as per institutional guidelines. High doses of morphine were defined as the need of >10 mg/day of morphine or opioid equivalent in the context of severe oropharyngeal or gastrointestinal mucositis, defined by grades 3–4 of the WHO scale [16]. Total parenteral nutrition (TPN) was initiated when caloric requirements were under 1000 calories/day by oral nutrition or in those patients with severe mucositis and pain-induced ingestion impairment. Febrile neutropenia was defined as the combination of granulocyte counts below 500 cell/μL and temperature over 38 oC. Patients were discharged when they reached >500 neutrophils/μL for three consecutive days and 25,000 platelets/μL without the need of transfusions, with satisfactory oral intake and no active infections.. Objectives of the study. 2. BEC: carmustine 300 mg/m2 at day −5, etoposide 150 mg/m2 and cyclophosphamide 1500 mg/m2 from days −5 to −2. Others: melphalan+busulphan, Melphalan+bortezomib. cytometry test was performed in all apheresis products at day of infusion.. Conditioning regimen Conditioning regimen for MM patients was high-dose melphalan (200 mg/m2 iv in patients with a normal renal function and 140 mg/m2 iv in those with creatinine clearance below 30 mg/ml/min, as a single dose at day −2). The BEAM protocol was used for LYMPH patients in. The main objective of the study was to compare early hematological and non-hematological complications after auto-HSCT between both groups of patients, measured by: infusion reactions on day 0, incidence and grade of oropharyngeal and gastrointestinal mucositis and the use of highdose morphine, need for TPN, incidence of febrile neutropenia, hematopoietic recovery after auto-HSCT and length of hospital stay between non-CRYO group and CRYO group. We also wanted to compare the number of CD34+ cells collected, as well as cell viability between both groups of patients. The International Myeloma Working group (IMWG) Uniform Response criteria were applied to evaluate disease status and response to therapy in patients with MM. Briefly, patients were classified as being in complete response (stringent or not) and partial response (very good partial.
(4) Advantages of non-cryopreserved autologous hematopoietic stem…. 963. Table 2 Transplantation outcomes CRYO (n = 74). Non-CRYO (n = 42). p value. Incidence of graft failure. 0. 0. NS. Total nucleated cells 108/Kg [median (range)] collected. 3.8 (3.5–4). 3.7 (3.6–4.1). NS. Apheresis volume infused [ml (range)]. 630 (520–750). 590 (500–790). NS. CD34+ cells x 106/kg (median (range)) collected. 4.9 (2.2–7.8). 5.1 (2.5–7.6). NS. CD34 cell viability before infusion (median (range)). 95% (91–97%). 96% (92–96%). NS. Neutrophil engraftment, days (median (range)). 13 (12–40). 9 (9–16). < 0.001. Platelet engraftment, days (median (range)). 14 (14–48). 11 [10–19]. 0.003. Incidence of febrile neutropenia. 69 (90%). 17 (14%). < 0.0001. Days of antibiotic use. 10 (range 4–14). 4 (range 2–9). > 0.0001. Mucositis grade 3–4. 48 (65%). 4 (11%). 0.003. Total TPN requirements. 16 (19%). 3 (10%). 0.003. Morphine use >10 mg/day. 47 (64%). 4 (11%). 0.004. Inpatient hospital stay (median (range)) in days. 20 (14–54). 15 (9–20). 0.001. 30-days NRM. 0. 1 (0.5%). 0.9 h 1.5 CI 95% 0.2–7.8. TPN Total parenteral nutrition, NRM Non-relapse mortality, RR Relapse rate, OS Overall survival. response) for the present analysis [17]. Lugano criteria [18] were used for patients with LYMPH and were classified as in complete response (CR) and partial response (PR) after salvage treatment strategies. Patients with refractory disease were not considered candidates to auto-HSCT.. Statistical analysis Patient characteristics were compared using the Kruskall–Wallis test for quantitative variables, and chisquare or Fisher’s exact test for categorical variables. Data are presented as frequency (percent) or median (range). The paired student's t test and Pearson correlation coefficient were used to compare CD34+cells count and viability. Fischer exact test and independent student's t test were used to compare engraftment after auto-HSCT, incidence of oropharyngeal mucositis, incidence of febrile neutropenia, use of TPN and morphine. To evaluate the impact of avoid DMSO cryopreservation and conditioning length, we made statistical analysis of each disease in both CRYO and nonCRYO groups. All tests were two-sided and p-values < 0.05 were considered as indicating significant associations. SPSS 21.0 (IBM, USA) and GraphPad Prism 6.0 (US) were used to perform the statistical analysis.. characteristics are depicted in Table 1. There were no significant differences between both groups of patients in terms of demography, underlying hematological disorder and disease status at auto-HSCT with the exception of the conditioning regimen used: BEAM was the conditioning protocol used for LYMPH in Barcelona while BEC was the one used in Chile.. Cellularity obtained and infused Median number of CD34+ cells/kg collected in the nonCRYO group was 5.1 (range 2.5–7.6) and in the CRYO one was 4.9 (range 2.2–7.8). There were no differences in volume infused nor total mononuclear cells/kg. Plerixafor was added in the mobilization process In 10 patients (25%) from the non-CRYO Group; 28 patients (35%) needed it in the CRYO group (p = 0.04). In CRYO group the average total amount of DMSO was 30 gr (range 50–80) with doses of 0.6–1 gr/kg of patient body weight. Viability assays 7-AAD flow cytometry CD34 viability average in the nonCRYO group was 96% (range 92–98%). In the CRYO group, CD34 viability was 95% (range 92–98%) without significant differences between both groups.. Results Infusion-related events and engraftment Baseline characteristics A total of 116 patients (42 non-CRYO and 74 CRYO) were treated with auto-HSCT because of a MM or LYMPH in both centers during the inclusion period. Clinical. No severe infusion-related events were reported in the whole series. All patients engrafted in both groups. As shown in Table 2, median neutrophil and platelet engraftment in the non-CRYO group was 9 days (range 9–16) and.
(5) 964. M. Sarmiento et al.. 11 days (range 10–19) respectively. In the CRYO group, neutrophil and platelet recovery were 12 days (range 13–40) and 14 days (range 14–48), respectively (p = 0.003).. Short-term complications and length of hospital stay As shown in Fig. 1, incidence of severe mucositis and highdose morphine use were 14% (n = 5 patients) and 11% (n = 4 patients) respectively in the non-CRYO group and 42% (n = 31) and 64% (n = 47), respectively in the CRYO group (p = 0.004). The use of TPN was 19% (n = 16) in CRYO group and 10% (n = 3) in non-CRYO group (p = 0.003). Incidence of febrile neutropenia was also higher in the CRYO group (40%, n = 17 vs 92%, n = 69, p = 0.0001). In 40% of the cases, neutropenic fever associated to enterobacteriae and gram positive bacteria detected in blood cultures (no differences between both groups). There were no fungal infections diagnosed during this time period in both hospitals. Mean hospital stay was 20 days (range 14–54) in the CRYO group vs 15 days (range 9–20) in the non-CRYO group (p = 0.0001) Table 3.. Short-term complications according to the underlying disease and conditioning regimen used 42 MM and AL amyloidosis patients were included in the CRYO and 31 in the non-CRYO group. In this subgroup of patients, the incidence of severe mucositis and high-dose 80. 60. 40 P <0.0001. morphine use was 50% (n = 21) in the CRYO group and 11% (n = 3) in the non-CRYO (p = 0.001). The use of TPN was 14% (n = 6 patients) in the CRYO group and 7% (n = 2) in the non-CRYO group (p = 0.004). Incidence of febrile neutropenia was also higher in the CRYO group (89%, n = 37 vs. 36%, n = 11, p = 0.0001). Conversely, length of hospitalization was 5 days shorter in the non-CRYO group (p = 0.001). Engraftment of neutrophils and platelets were 8 and 10 days (range 8–11) in the non-CRYO group and 12 and 14 days (range 12–18) in the CRYO one (p = 0.001). In total 43 patients with LYMPH were included in the study: 32 LYMPH in the CRYO group and 11 in the nonCRYO modality. The incidence of severe mucositis and high doses of morphine were 60% (n = 19) in the CRYO group and 20% (n = 3) in the non-CRYO (p = 0.005). TPN use was 18% (n = 5) in the CRYO group and 10% (n = 1) in the non-CRYO group (p = 0.0032). Both incidence of febrile neutropenia and length of hospital stay were lower in the non-CRYO group. Neutrophil and platelet engraftment were 9 and 11 days (range 8–12), respectively in the nonCRYO group and 11 and 16 days (range 12–18) in the CRYO one (p = 0.003). With a median follow-up of 14 months (range 6–28) for surviving patients in the Chilean Program and 13 months (range 8–32) in the Catalan one, 30-day NRM was 0% in the non-CRYO group and 0.5% in the CRYO group (Table 2). Five patients with a prior history of MM that were treated with auto-HSCT within the CRYO group before 2012, were treated with a second non-CRYO auto-HSCT upon relapse and reinduction to a second response with the appropriate therapy. Peripheral blood stem cell harvest was satisfactory in the five of them, two of them needing additional treatment with plerixafor. When comparing CD34+ collected cells and early complications of these five patients with those of MM patients undergoing a single CRYO transplant, there were no significant differences (p = 0.09).. 20 P =0.004. P =0.003. P =0.004. 0 CRYO. Non-CRYO. TPN. CRYO. Non-CRYO. Febrile neutropenia. CRYO. CRYO. Non-CRYO. Severe mucositis. Non-CRYO. Morphine use >10mg/d. Fig. 1 Short-term complications in CRYO vs non-CRYO patients. Discussion In recent years progress has been made in the process of HSCT to make it more accessible. Several groups have. Table 3 Summary data from some studies without cryopreservation for periods greater than 72 h Author (reference). n. Conditioning. Storage a 4 °C duration. Engraftment (%). Graft failure. Ager et al. [14]. 10. MEC. 96 h. 100. 0. Ceullar-Ambrosi et al. [22]. 47. CEB, Melphalan, Carboplatine. 144 h. 100. 0. Ahmed et al. [16]. 47. CEB, Cyclophosphamide+total body irradiation. 120 h. 100. 0. MEB melphalan etoposide carmustine, CEB Cyclophosphamide etoposide carmustine.
(6) Advantages of non-cryopreserved autologous hematopoietic stem…. shown the possibility of performing transplants in the outpatient setting and the use of plerixafor has allowed satisfactory harvests in the majority of patients [12, 13]. The possibility to carry out less expensive procedures and with less toxicity that may allow its use in elderly or fragile patients has the same underlying objective. Our analysis shows that performing auto-HSCT without cryopreservation is possible. Although toxicity and directly related side effects of DMSO infusion were negligible in our population of CRYO patients, non-cryopreserved products were associated with a lower incidence of severe mucositis, febrile neutropenia and TPN requirements and thus, a significantly shorter hospital stay without sacrificing overall response rate and survival (data not shown). We believe that these results are of most importance, because they favor better treatment tolerance and, by being less expensive, they clearly give relief to our increasingly overloaded public health care systems. Several groups have previously shown that transplantation without cryopreservation is feasible. Ruiz-Arguelles et al. [19] showed that non-cryopreserved transplantation was possible in an outpatient setting and that >50% of transplant costs were saved, avoiding hospitalizations. However, this study used a one-fit all of melphalan conditioning in myeloablative doses for patients with lymphoma, myeloma, and leukemias. They used melphalan in all patients to perform single-dose chemotherapy and keep the cells refrigerated without DMSO for a few days. Other groups from developing countries have also shown their experience in autologous non-cryopreserved transplantation in patients with myeloma, because an easier conditioning [19–25]. A systemic review of the available literature available until 2007 [26] including 60 patients reported in 16 non-randomized retrospective analysis concluded that graft failure was very low (0.36%) and that a non-CRYO approach was safe and feasible. Again, high-dose melphalan was the universally conditioning regimen in these studies. According to the information available, our study is possibly the first one to show that cells can be refrigerated for longer without compromising viability, which allows the administration of appropriate conditioning schemes for patients with lymphoma or leukemia and that comparatively, non-CRYO offers better early outcomes. One of the possible criticisms of our data is the short follow-up time. However, our main objective was to assess short-term complications and this analysis was robust. Another potential shortcoming of this analysis is that although highdose melphalan was the conditioning regimen used for patients with MM in both HSCT programs, BEAM was the protocol used for lymphoma patient in the Barcelona program while BEC was the most frequently used for lymphoma patients in Chile; it could be argued that BEC is less toxic than BEAM and this fact could account for the lower. 965. intensity of early side effects in this group of patients. However, comparative studies have shown that this scheme is more toxic than the BEAM [20, 27]. Increasing complexity of the HSCT is another issue to be taking into consideration. One might think that the use of a non-CRYO approach mandates a very precise planning of patients hospital stay, conditioning and reinfusion and this require an efficient program coordination. However in our experience this could be made without distress of the blood banking, nurse and medical staff and carries a significant benefit of avoiding the toxicities, costs, and facilities for cryopreservation. Traditionally there is the practice of not maintaining cells without cryopreservation for >72 h due to the loss of viability, but this information has not been studied in a clinical setting. In fact, the first papers reported on successful autologous transplantation were developed without cryopreservation [27–29]. The development of protocols with cryopreservation allowed more complex and extensive conditioning schemes and the possibility of keeping healthy hematopoietic stem cells without exposition to chemotherapy, making a widespread use of DMSO in regular practice [30]. However the possible toxic effect of DMSO is known and avoiding this process is beneficial, as it allows to use more prolonged schemes of conditioning without viability impairment, as we have demonstrated. In addition to acute toxicity in the transplant infusion process, Parody et al. [31] showed that cryopreserved cells with DMSO for allogeneic transplantation favored a higher incidence of GVHD. In summary, our analysis shows that stem cell cryopreservation in patients who are to be treated with autologous hematopoietic transplantation can be avoided and seems to be unnecessary. Non-CRYO transplantation is safer and equally effective. With a good organization and coordination of the transplant team this process can be used in any transplant unit, avoiding DMSO use and delivering shorter hospitalizations results in a more inexpensive setting. Our plans for future investigations include the measurement of pro inflammatory cytokines in both groups of patients to try to discern which is the mechanism of our findings.. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.. References 1. Naumann-Winter F, Greb A, Borchmann P, Bohlius J, Engert A, Schnell R. First-line tandem high-dose chemotherapy and autologous stem cell transplantation versus single high-dose chemotherapy and autologous stem cell transplantation in.
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