Abstract
Introduction
Autologous hematopoietic stem cell transplantation (ASCT) remains a cornerstone therapy for eligible patients with multiple myeloma. Cryopreservation of hematopoietic grafts provides logistical flexibility but may increase procedure-related toxicity and resource utilization.
Methods
We conducted a two-phase, non-randomized binational study at tertiary centers in Santiago, Chile, and Seville, Spain, comparing cryopreserved and non-cryopreserved grafts in patients undergoing ASCT. In the retrospective phase (2018–2023), outcomes were analyzed in 284 recipients of cryopreserved grafts and 148 recipients of non-cryopreserved grafts. In the prospective phase (2023–2025), 16 patients receiving cryopreserved grafts and 25 receiving non-cryopreserved grafts were evaluated for clinical endpoints and inflammatory biomarkers.
Results
Recipients of non-cryopreserved grafts achieved faster neutrophil engraftment (median 11 vs 14–15 days) and shorter hospital stays (median 13 vs 18 days), with reduced rates of febrile neutropenia (19% vs 50%) and grade ≥3 mucositis (19% vs 60%). No early mortality occurred in either group. Cryopreserved grafts were associated with higher levels of selected inflammatory and endothelial-associated biomarkers, including IL-8 and VEGF-D, whereas non-cryopreserved grafts showed a distinct inflammatory profile, including a non-statistically significant trend toward higher IFN-γ levels at the time of transplantation.
Discussion
Overall, the use of non-cryopreserved grafts was associated with reduced early transplant-related toxicity and comparable engraftment outcomes. Given the non-randomized design and center-specific supportive care practices, these findings should be interpreted as real-world associations and support further prospective evaluation of graft preservation strategies in autologous transplantation for multiple myeloma.
Keywords: autologous hematopoietic stem cell transplantation, cryopreservation, fresh grafts, engraftment kinetics, inflammatory biomarkers, multiple myeloma
Introduction
Autologous hematopoietic stem cell transplantation (ASCT) remains a key component of treatment for eligible patients with multiple myeloma, contributing to deeper responses and prolonged disease control when integrated into contemporary induction, consolidation, and maintenance strategies.1 Although therapeutic advances have substantially improved outcomes in multiple myeloma, ASCT continues to represent an important treatment platform in many healthcare systems, particularly for patients who are fit for high-dose melphalan-based therapy.
In standard clinical practice, peripheral blood stem cell grafts are commonly cryopreserved before infusion. Cryopreservation provides important logistical advantages, including flexibility in scheduling conditioning therapy, centralized processing, transport between institutions, and long-term graft storage. These advantages are particularly relevant in transplant programs with high procedural volume, complex referral pathways, or the need to coordinate collection and infusion across different clinical units. However, cryopreservation also introduces additional procedural complexity, costs, and exposure to cryoprotective agents, most commonly dimethyl sulfoxide (DMSO).2
The biological and clinical consequences of graft cryopreservation remain incompletely understood. DMSO exposure has been associated with infusion-related adverse events, while the freeze–thaw process may affect graft cellular integrity, viability, immune-cell composition, and the release of intracellular or damage-associated molecular signals. These events may theoretically contribute to early systemic inflammation, endothelial activation, and tissue injury after transplantation. In this context, inflammatory mediators such as IL-6, IL-8, IL-18, IFN-γ, MMP-1, and VEGF-D may provide exploratory insight into the biological effects associated with graft preservation strategies. Whether these biological alterations translate into clinically meaningful differences in transplant-related outcomes remains uncertain.
Early post-transplant toxicity remains clinically relevant in patients undergoing ASCT for multiple myeloma. Febrile neutropenia, severe mucositis, need for parenteral nutrition, opioid requirement, delayed hematopoietic recovery, and prolonged hospitalization all contribute to patient morbidity and healthcare resource utilization. These outcomes may be influenced not only by conditioning intensity and patient-related factors, but also by graft characteristics, preservation strategy, supportive care practices, and center-specific protocols.
The use of non-cryopreserved (fresh) grafts represents an alternative approach that may simplify transplant logistics and reduce procedure-related toxicity. Several studies have reported that infusion of fresh grafts is associated with comparable or faster hematopoietic recovery and reduced early transplant-related morbidity, including mucositis and supportive care requirements.2–6 In a prior multicenter analysis, our group reported faster engraftment and fewer early complications among recipients of non-cryopreserved grafts.4 Collectively, these observations suggest that avoiding cryopreservation may be feasible in selected settings while maintaining adequate transplant efficacy. However, most available reports have focused primarily on clinical outcomes, and few have incorporated systematic inflammatory or endothelial biomarker profiling. To our knowledge, prospective evaluation of inflammatory biomarkers according to graft preservation strategy in autologous transplantation for multiple myeloma remains scarce. Moreover, contemporary evidence remains limited in patients treated with modern induction regimens and current supportive care strategies, where the impact of graft preservation on early toxicity has not been fully characterized.4
Our group previously reported outcomes associated with non-cryopreserved autologous transplantation in a cohort that included patients with multiple myeloma and lymphoma transplanted up to 2016.4 Building upon this previous experience, the present study evaluates a larger and more contemporary cohort restricted to patients with multiple myeloma transplanted from 2018 onwards, with no patient overlap, while incorporating a prospective biomarker component designed to explore inflammatory and endothelial-associated profiles according to graft preservation strategy.
Despite accumulating evidence supporting the feasibility of non-cryopreserved transplantation, the clinical impact of avoiding cryopreservation in contemporary multiple myeloma practice remains incompletely defined, particularly with respect to early transplant-related toxicity, healthcare resource utilization, and the biological mechanisms that may underlie these differences. Furthermore, the biological mechanisms potentially linking graft preservation strategies to differences in early transplant-related toxicity remain poorly characterized.
In the present binational retrospective–prospective study, we compared cryopreserved and non-cryopreserved autologous grafts in patients undergoing ASCT for multiple myeloma. The primary objective was to evaluate early transplant-related toxicity, engraftment kinetics, supportive care requirements, and hospital length of stay. As an exploratory objective, we assessed plasma inflammatory and endothelial-associated biomarkers at predefined peri-transplant time points to generate hypotheses regarding potential biological differences associated with graft cryopreservation. Since all patients received the same high-dose melphalan conditioning strategy, the rationale for comparing graft preservation approaches was not to improve anti-myeloma efficacy per se, but rather to determine whether avoiding cryopreservation and DMSO exposure may reduce transplant-related toxicity, inflammatory activation, and supportive care requirements while maintaining effective hematopoietic recovery. Given the pragmatic, non-randomized nature of the study, the findings should be interpreted within the context of real-world transplant practice and center-specific supportive care strategies.
Materials and Methods
Patients
We conducted a combined retrospective–prospective study at Hospital Clínico Red Salud UC CHRISTUS/Pontificia Universidad Católica de Chile (Santiago, Chile) and Hospital Universitario Virgen del Rocío (Seville, Spain) between 2018 and 2025. The retrospective cohort included consecutive adult patients undergoing first ASCT for multiple myeloma transplanted from 2018 to 2023, while prospective sampling and clinical follow-up were carried out from 2023 to 2025.
Allocation to cryopreserved (CRYO) versus non-cryopreserved (Non-CRYO) grafts was not randomized, but determined by institutional practice and logistical considerations. In Chile, non-cryopreserved grafts represent the standard approach in routine clinical practice, with cryopreservation reserved for selected situations as previously described.4 In contrast, in Seville, Spain, cryopreservation is the predominant strategy. These complementary practice patterns between centers provided a pragmatic framework for comparative analysis of graft preservation strategies in real-world settings. As a result, comparisons between groups inherently reflect center-specific practices and should be interpreted as pragmatic, real-world comparisons rather than controlled experimental contrasts.
Additional differences in supportive care practices were noted between centers. Notably, granulocyte colony–stimulating factor (G-CSF) was routinely administered starting on day +5 post-transplant in the Chilean cohort, whereas this strategy was not systematically applied in the Spanish cohort. This represents one of the most relevant differences in post-transplant management between groups. In contrast, other key variables including pre-transplant treatment regimens, conditioning intensity (melphalan dosing), and general supportive care measures, were comparable between cohorts. Response assessments and maintenance strategies followed institutional standards and were not predefined study endpoints because the primary objective was evaluation of early transplant-related toxicity.
Exclusion criteria included active infection at the time of transplantation, uncontrolled comorbidities, or prior transplantation. The study was conducted in accordance with the Declaration of Helsinki and was approved by the local institutional review board at each center. Written informed consent was obtained from all prospectively enrolled participants, while retrospective data collection was approved with a waiver of consent under local regulations (Approvals: Junta de Andalucía, ID S2300220, Nov 29, 2023; Comité Ético Científico Salud UC, Pontificia Universidad Católica de Chile, ID 230302004, Apr 13, 2023).
Stem Cell Collection and Processing
Peripheral blood stem cells (PBSCs) were mobilized with granulocyte colony–stimulating factor (G-CSF), and on-demand plerixafor was administered according to peripheral blood CD34+ counts measured the day before apheresis, based on institutional algorithms and predefined thresholds. Cells were collected by leukapheresis under aseptic, GMP-compliant conditions. Processing procedures were standardized according to institutional protocols at each center.
For the cryopreserved (CRYO) cohort, products were formulated with 10% dimethyl sulfoxide (DMSO) as a cryoprotectant and stored in liquid nitrogen (≤−150 °C) until infusion. For the non-cryopreserved (Non-CRYO) cohort, products were stored at 2–8 °C and infused within 72 hours of collection without freezing.
Before release, routine product quality checks included volume, labeling, visual inspection, and continuous temperature monitoring during storage and transport. Viability assessment followed cohort-specific institutional practice: in the Non-CRYO group, viability was measured by flow cytometry using eBioscience™ 7-AAD Viability Staining Solution (Thermo Fisher Scientific) according to the manufacturer’s instructions; in the CRYO group, total nucleated cell counts were obtained with a Sysmex automated analyzer, and viability was determined by the trypan blue exclusion assay. Given differences in viability assessment methods between cohorts, these results should be interpreted primarily as descriptive.
Conditioning Regimen and Transplantation
Patients received high-dose melphalan intravenously as a single-day conditioning regimen before stem-cell infusion. Dose selection followed institutional algorithms incorporating age, renal function, and comorbidities: 200 mg/m2 for standard-fit candidates, 140 mg/m2 for older or frail patients, and 160 mg/m2 for selected intermediate-risk scenarios.1,7,8
Infusions were prepared in accordance with pharmacy compounding standards and administered via central venous access with standard antiemetic prophylaxis. Dose adjustments were applied for renal impairment or other predefined clinical criteria.
Post-conditioning supportive measures (hydration and oral cryotherapy, per local practice) and the timing of stem-cell infusion were standardized across sites. Regimen selection and dosing thresholds were harmonized between centers and prespecified in the study protocol.3,4 Post-transplant growth factor use differed between centers, as described above.
Clinical and Laboratory Assessments
Engraftment was defined as the first of three consecutive days with an absolute neutrophil count (ANC) ≥ 0.5 × 109/L and a platelet count ≥ 20 × 109/L without transfusion support.
Complete blood counts were performed daily until engraftment, then at least three times per week until hospital discharge, and weekly during outpatient follow-up for the first 100 days post-transplant.
Early post-transplant morbidity was systematically assessed, including duration of hospitalization (from transplant day 0 to discharge), incidence of febrile neutropenia (oral temperature ≥ 38.3 °C or ≥ 38.0 °C sustained for ≥ 1 hour with ANC < 0.5 × 109/L), and severity of mucositis, graded using the World Health Organization (WHO) oral toxicity scale.
The need for opioid analgesia was defined as any systemic administration for mucositis- or regimen-related pain, while parenteral nutrition was documented when required for ≥ 48 hours due to gastrointestinal toxicity.
Adverse events were categorized and graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. Supportive care measures, including antimicrobial prophylaxis, growth factor use, transfusion thresholds, and infection surveillance, were applied in line with harmonized institutional guidelines at both centers.
Inflammatory Marker Analysis
Biomarker analyses were exploratory and hypothesis-generating. No formal sample size calculation was performed for biomarker endpoints. Consecutive eligible patients undergoing ASCT during the prospective study period were invited to participate in biomarker sampling, and no selection was made based on disease characteristics, clinical status, or transplant outcomes. Peripheral blood samples were collected into EDTA-coated tubes at baseline (pre-transplant), on the day of transplantation (day 0) after graft infusion, and on day +15 post-transplant. Day 0 samples were collected before initiation of routine post-transplant G-CSF administration in the Chilean cohort. Samples were centrifuged at 600 × g for 15 minutes at 4°C to separate plasma. The plasma supernatant was carefully aspirated without disturbing the buffy coat and transferred to a fresh tube, followed by a second centrifugation at 600 × g for 15 minutes at 4°C. The plasma was then transferred to a fresh tube for a final centrifugation at 2,500 × g for 5 minutes at 4°C to remove residual cellular debris. The resulting cell-free plasma was aliquoted and stored at −80°C. Plasma samples were analyzed to quantify the expression of six inflammatory markers (IL-6, IL-8, IL-18, IFN-γ, MMP-1, and VEGF-D) using a customized ProcartaPlex Human Inflammation Panel (Thermo Fisher Scientific, USA) on the Luminex xMAP® platform. Measurements were performed on a MAGPIX® instrument, and data were analyzed with Xponent® 4.2 software. Biomarker analyses were exploratory and hypothesis-generating; no formal sample size calculation was performed, and the study was not powered for definitive mechanistic conclusions.
Statistical Analysis
Continuous variables were summarized as median (interquartile range) or mean ± standard deviation, as appropriate based on data distribution. Categorical variables were expressed as frequencies and percentages.
Comparisons between groups were performed using the Mann–Whitney U-test or Student’s t-test for continuous variables, depending on normality assumptions, and χ2 or Fisher’s exact test for categorical variables. Engraftment times and other continuous clinical outcomes were primarily analyzed using non-parametric methods.
For graphical representation, continuous variables were displayed using box-and-whisker plots with individual data points to illustrate data distribution.
Correlations between inflammatory markers and clinical outcomes were assessed using Spearman’s rank correlation coefficient. No adjustment for multiple comparisons was performed, given the exploratory nature of the biomarker analyses.
All statistical tests were two-sided, and a p-value < 0.05 was considered statistically significant. Statistical analyses were performed using IBM SPSS Statistics version 28.1 (IBM Corp., Armonk, NY) and GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA).
Given the observational design and the exploratory nature of the study objectives, no prespecified multivariable adjustment was performed.
Results
Cohort Characteristics
A total of 473 patients with multiple myeloma undergoing autologous hematopoietic stem cell transplantation (ASCT) were included, comprising 432 patients in the retrospective cohort (2018–2023) and 41 in the prospective cohort (2023–2025). The median age was 62 years (range, 41–79), and 49% of patients were female.
Baseline characteristics were comparable between patients receiving cryopreserved (CRYO) and non-cryopreserved (Non-CRYO) grafts in both cohorts, including disease status at transplantation, conditioning intensity, CD34+ cell dose, and graft viability (Tables 1 and 2).
Table 1.
Patient Characteristics (Retrospective Cohort)
| Variable | CRYO (n=284) | Non-CRYO (n=148) | p-value |
|---|---|---|---|
| Age, years | 62 (41–72) | 63 (40–79) | 0.9 |
| Pre-transplant treatment | 0.56 | ||
| – VRD | 35% | 45% | |
| – VTD | 33% | 25% | |
| – Daratumumab-based | 23% | 30% | |
| – RD | 2% | 0% | |
| – Isatuximab-based | 5% | 0% | |
| – Melphalan-based | 3% | 0% | |
| Disease status at transplant | |||
| – sCR | 35% | 40% | 0.7 |
| – CR | 32% | 35% | 0.8 |
| – VGPR | 23% | 20% | 0.6 |
| – PR | 9% | 5% | 0.07 |
| – Active disease | 1% | 0% | 0.55 |
| Conditioning | 0.27 | ||
| – Melphalan 200 mg/m2 | 195 | 110 | |
| – Reduced dose | 89 | 38 | |
| CD34+ cells (×106/kg) | 6.24 (2.5–9.7) | 6.9 (2.8–11) | 0.8 |
| Viability (%) | 95 (94–98) | 97 (92–98) | 0.9 |
| Platelet engraftment, days | 12 (11–25) | 13 (11–19) | 0.005 |
| Neutrophil engraftment, days | 14 (13–21) | 11 (9–15) | 0.006 |
| Grade ≥3 mucositis (%) | 66% | 20% | 0.001 |
| Parenteral nutrition (%) | 20% | 8% | 0.001 |
| Febrile neutropenia (%) | 45% | 22% | 0.005 |
| Length of stay, days | 19 | 14 | 0.005 |
Notes: CD34+ cell dose is expressed as ×106 cells/kg. Viability refers to the percentage of viable nucleated cells assessed prior to infusion. Platelet engraftment was defined as the first of three consecutive days with a platelet count ≥ 20 × 109/L without transfusion support. Neutrophil engraftment was defined as the first of three consecutive days with an absolute neutrophil count ≥ 0.5 × 109/L. Febrile neutropenia was defined as an oral temperature ≥ 38.3 °C or ≥ 38.0 °C sustained for ≥ 1 hour in the setting of neutropenia. Length of stay was defined as the number of days from transplantation (day 0) to hospital discharge.
Abbreviations: VRD, bortezomib, lenalidomide, and dexamethasone; sCR, stringent complete response; CR, complete response; VGPR, very good partial response.
Table 2.
Patient Characteristics (Prospective Cohort)
| Variable | CRYO (n=16) | Non-CRYO (n=25) | p-value |
|---|---|---|---|
| Age, years | 64 (41–72) | 63 (40–79) | 0.9 |
| Pre-transplant treatment | NS | ||
| – VRD | 50% | 48% | |
| – Daratumumab-based | 50% | 52% | |
| Disease status at transplant | |||
| – sCR | 50% | 52% | 0.9 |
| – CR | 40% | 35% | 0.8 |
| – VGPR | 10% | 13% | 0.7 |
| Conditioning | 0.8 | ||
| – Melphalan 200 mg/m2 | 12 | 18 | |
| – Reduced dose | 4 | 7 | |
| CD34+ cells (×106/kg) | 6.59 (2.5–9.7) | 7.1 (2.8–11) | 0.8 |
| Viability (%) | 95 (94–98) | 97 (92–98) | 0.9 |
| Platelet engraftment, days | 11 (11–24) | 13 (11–19) | 0.005 |
| Neutrophil engraftment, days | 15 (13–21) | 11 (9–15) | 0.006 |
| Grade ≥3 mucositis (%) | 60% | 19% | 0.009 |
| Parenteral nutrition (%) | 25% | 4% | 0.006 |
| Febrile neutropenia (%) | 50% | 19% | 0.005 |
| Length of stay, days | 18 | 13 | 0.005 |
Notes: CD34+ cell dose is expressed as ×106 cells/kg. Viability refers to the percentage of viable nucleated cells assessed prior to infusion. Platelet engraftment was defined as the first of three consecutive days with a platelet count ≥ 20 × 109/L without transfusion support. Neutrophil engraftment was defined as the first of three consecutive days with an absolute neutrophil count ≥ 0.5 × 109/L. Febrile neutropenia was defined as an oral temperature ≥ 38.3 °C or ≥ 38.0 °C sustained for ≥ 1 hour in the setting of neutropenia. Length of stay was defined as the number of days from transplantation (day 0) to hospital discharge.
Abbreviations: VRD, bortezomib, lenalidomide, and dexamethasone; sCR, stringent complete response; CR, complete response; VGPR, very good partial response, NS: non significative.
The prospective cohort included 16 CRYO and 25 Non-CRYO patients, with no relevant differences in baseline clinical or transplant-related variables (Table 2).
Early Transplant Outcomes
Platelet engraftment occurred slightly earlier in the CRYO group (retrospective: median 12 vs 13 days; prospective: 11 vs 13 days; p = 0.005 for both comparisons). In contrast, neutrophil engraftment was faster in the Non-CRYO group (retrospective: 11 vs 14 days; prospective: 11 vs 15 days; p = 0.006 for both comparisons).
Patients receiving cryopreserved grafts experienced a higher burden of early transplant-related toxicity. Febrile neutropenia was more frequent in the CRYO group (retrospective: 45% vs 22%; prospective: 50% vs 19%; p = 0.005), as was grade ≥3 mucositis (retrospective: 66% vs 20%; prospective: 60% vs 19%; p = 0.001).
These complications were associated with increased supportive care requirements, including higher use of total parenteral nutrition (retrospective: 20% vs 8%; prospective: 25% vs 4%; p = 0.001) and more frequent opioid analgesic use in patients with moderate-to-severe mucositis. In addition, hospital length of stay was longer in the CRYO group (retrospective: median 19 vs 14 days; prospective: 18 vs 13 days; p = 0.005).
No early transplant-related mortality was observed in either group.
Inflammatory Markers
Plasma samples were analyzed at three time points: baseline (PRE), transplant day (Tx), and day +15 post-transplant (POST).
Higher levels of interferon-γ (IFN-γ) were observed in the Non-CRYO group at the time of transplantation (Tx), although this difference did not reach statistical significance. In contrast, the CRYO group showed higher levels of selected pro-inflammatory and endothelial-associated markers, including IL-8 and VEGF-D, at both Tx and POST time points. Median IL-8 levels were 3.59 vs 1.25 at Tx (p = 0.007) and remained higher at POST (p = 0.002). Similarly, VEGF-D levels were higher in the CRYO group at Tx (4.96 vs 1.22, p = 0.001) and POST (5.63 vs 3.03, p = 0.044). These findings are summarized in Table 3 and illustrated in Figure 1.
Table 3.
Comparison of Inflammatory Biomarkers Between Cryopreserved and Non-Cryopreserved Graft Recipients
| Marker | Time Point | CRYO, Median (IQR) | Non-CRYO, Median (IQR) | p-value |
|---|---|---|---|---|
| IFN-γ | PRE | 7.72 (2.86–11.30) | 6.16 (3.46–10.78) | 0.805 |
| Tx | 5.78 (4.68–7.52) | 20.06 (5.41–29.59) | 0.114 | |
| POST | 11.83 (5.78–20.60) | 10.57 (1.89–21.17) | 0.591 | |
| IL-8 | PRE | 1.88 (1.20–3.08) | 0.81 (0.53–1.51) | 0.020 |
| Tx | 3.59 (2.94–4.54) | 1.25 (0.71–2.21) | 0.007 | |
| POST | 3.63 (2.79–4.69) | 1.25 (0.77–2.17) | 0.002 | |
| VEGF-D | PRE | 3.31 (2.17–5.37) | 1.82 (0.98–3.76) | 0.104 |
| Tx | 4.96 (3.81–6.85) | 1.26 (0.71–1.90) | <0.001 | |
| POST | 5.63 (3.51–6.94) | 3.03 (1.90–4.47) | 0.044 | |
| IL-6 | PRE | 1.39 (0.93–1.49) | 1.39 (1.39–2.00) | 0.293 |
| Tx | 2.21 (1.39–3.10) | 1.39 (0.93–1.39) | 0.030 | |
| POST | 2.64 (2.00–3.66) | 3.08 (1.39–6.07) | 0.666 | |
| IL-18 | PRE | 68.61 (40.26–131.28) | 84.08 (26.33–129.89) | 0.824 |
| Tx | 122.65 (79.30–157.36) | 71.47 (37.02–142.88) | 0.188 | |
| POST | 108.36 (70.69–165.45) | 54.95 (38.81–78.84) | 0.025 | |
| MMP-1 | PRE | 3.68 (1.42–8.53) | 4.88 (1.77–9.54) | 0.660 |
| Tx | 7.52 (6.10–11.82) | 4.88 (1.42–7.32) | 0.064 | |
| POST | 3.88 (2.27–5.28) | 2.89 (2.13–7.11) | 0.810 |
Notes: Data are presented as median (interquartile range). P-values were calculated using the Mann–Whitney U-test.
Abbreviations: IFN-γ, interferon gamma; IL-8, interleukin-8; VEGF-D, vascular endothelial growth factor D; IL-6, interleukin-6; IL-18, interleukin-18; MMP-1, matrix metalloproteinase-1. PRE, baseline (pre-transplant); Tx, day of transplantation (day 0); POST, day +15 post-transplant.
Figure 1.
Inflammatory marker profiles according to graft preservation strategy. Box-and-whisker plots showing plasma levels of (a) IFN-γ, (b) IL-18, (c) VEGF-D, (d) IL-8, (e) MMP-1 and (f) IL-6 at baseline (PRE), transplant day (Tx), and day +15 post-transplant (POST). Individual data points are displayed. CRYO indicates cryopreserved grafts; Non-CRYO, non-cryopreserved grafts. Asterisks indicate statistically significant differences between the CRYO and Non-CRYO groups at the indicated time point.
Differences in selected inflammatory markers, particularly IL-8 and VEGF-D, were already evident at the time of transplantation (Tx), prior to the onset of febrile neutropenia in most patients, suggesting that these variations may not be fully explained by infection-related inflammatory responses. For other markers (MMP-1, IL-18, and IL-6), no consistent patterns were observed across time points, despite isolated statistically significant differences, including IL-6 at Tx and IL-18 at POST.
Discussion
In this multicenter study, the use of non-cryopreserved grafts in autologous stem cell transplantation for multiple myeloma was associated with comparable engraftment and a consistently lower burden of early transplant-related toxicity. While platelet recovery occurred slightly earlier in the cryopreserved group, neutrophil engraftment was faster in recipients of non-cryopreserved grafts. More importantly, patients receiving cryopreserved grafts experienced higher rates of febrile neutropenia, severe mucositis, increased need for parenteral nutrition, and longer hospital stays, reflecting a less favorable early clinical course. These findings were observed across both the retrospective and prospective cohorts, supporting the overall consistency of the clinical signal.
Our findings demonstrate that autologous transplantation with non-cryopreserved grafts performs at least as well as cryopreserved grafts with respect to early post-transplant outcomes in patients with multiple myeloma receiving high-dose melphalan conditioning. Rather than altering the anti-myeloma efficacy of transplantation, the non-cryopreserved approach appears to facilitate efficient delivery of hematopoietic progenitor cells while reducing the burden of early transplant-related complications. The consistency observed across both retrospective and prospective cohorts strengthens the robustness of these observations and suggests that the clinical differences identified are unlikely to be attributable to random variation alone.
These findings are consistent with a growing body of evidence supporting the feasibility and safety of non-cryopreserved autologous transplantation. Previous studies have demonstrated adequate hematopoietic recovery, acceptable toxicity profiles, and comparable clinical outcomes when fresh grafts are infused after short-term refrigerated storage. Collectively, these reports suggest that avoidance of cryopreservation may represent a viable alternative in selected clinical settings without compromising the therapeutic intent of transplantation.
Several independent groups have reported comparable findings.9 Noiperm et al demonstrated that non-cryopreserved peripheral blood stem-cell grafts achieved timely engraftment and acceptable survival outcomes in patients with myeloma and lymphoma.10 Similarly, Pessoa et al directly compared cryopreserved and non-cryopreserved autografts in multiple myeloma and reported comparable transplant outcomes, with higher cellular viability observed in fresh grafts.11 Long-term experiences from Algeria confirmed the efficacy and safety of non-cryopreserved transplantation in both myeloma and lymphoma, while Jennane et al and Kulkarni et al demonstrated adequate engraftment and favorable toxicity profiles across different healthcare settings.12–14 Taken together, these studies suggest that the benefits of non-cryopreserved transplantation are reproducible across diverse transplant programs and resource environments.
At the same time, the longstanding success of cryopreserved autologous transplantation should not be overlooked. Cryopreservation provides important logistical advantages, including scheduling flexibility, long-term graft storage, centralized processing, and contingency planning. Gertz et al demonstrated successful use of autologous grafts stored for more than 15 years, highlighting the durability and clinical utility of cryopreserved products.15 Consequently, cryopreservation remains an essential component of modern transplant programs. However, implementation of cryopreservation entails additional infrastructure requirements, operating costs, quality-control procedures, and exposure to cryoprotective agents such as DMSO. These considerations become particularly relevant in high-volume transplant programs and resource-constrained healthcare systems. In this context, non-cryopreserved transplantation should be viewed as a complementary strategy that may simplify logistics and reduce resource utilization when clinically feasible.
The incorporation of inflammatory biomarker profiling provides additional biological context for the observed clinical differences. Notably, IL-8 and VEGF-D levels were consistently higher in recipients of cryopreserved grafts at the time of transplantation and during early post-transplant follow-up. IL-8 is a key mediator of neutrophil recruitment and amplification of inflammatory responses, whereas VEGF-D has been associated with endothelial activation, vascular remodeling, and tissue repair processes. Although the mechanistic implications of these findings remain speculative, the observed pattern is consistent with the presence of a more pronounced early inflammatory and endothelial-associated response in the cryopreserved cohort.
Interestingly, IFN-γ levels tended to be higher in recipients of non-cryopreserved grafts at the time of transplantation, although this difference did not reach statistical significance. While these findings should be interpreted cautiously, they may suggest qualitative differences in early immune activation between preservation strategies. The overall biomarker profile observed in the non-cryopreserved cohort was characterized by lower levels of endothelial-associated and pro-inflammatory mediators despite favorable clinical outcomes, raising the possibility that graft handling and preservation methods influence the early inflammatory milieu after transplantation.
These observations may plausibly relate to cellular stress induced by cryopreservation and thawing procedures, exposure to DMSO, endothelial perturbation, or the release of intracellular mediators from damaged cells. Experimental and clinical studies have suggested that cryopreservation may affect cellular integrity, membrane stability, and immune-cell function, potentially promoting inflammatory signaling after graft infusion. Although the precise mechanisms remain incompletely characterized, our findings are compatible with the hypothesis that graft preservation strategy may influence early biological responses beyond simple preservation of cell viability.
An important potential confounder is the difference in post-transplant G-CSF administration between centers. Because G-CSF was routinely administered in the non-cryopreserved cohort but not systematically in the cryopreserved cohort, its influence on engraftment kinetics cannot be excluded. However, this factor is less likely to explain the observed biomarker profile. Notably, higher levels of IL-8 and VEGF-D were already evident at the time of transplantation (day 0), before initiation of post-transplant G-CSF and prior to the development of most infectious complications. Because routine G-CSF administration in the non-cryopreserved cohort was initiated on day +5 after transplantation, the differences observed in inflammatory biomarkers at earlier sampling time points are unlikely to be explained by exposure to this cytokine-stimulating agent. Theoretically, routine G-CSF administration would be expected to promote a more pronounced inflammatory profile in the non-cryopreserved cohort. Instead, the opposite pattern was observed, with higher levels of selected inflammatory and endothelial-associated markers in recipients of cryopreserved grafts. Although these findings do not establish causality, they are consistent with the hypothesis that graft preservation strategy may contribute to early biological differences observed after transplantation.
Importantly, our findings emphasize that the objective of avoiding cryopreservation is not to enhance the anti-myeloma activity of transplantation. All patients received comparable high-dose melphalan conditioning and underwent ASCT according to standard practice. Rather, the potential benefit of non-cryopreserved grafts lies in reducing procedure-related toxicity, inflammatory responses, and healthcare resource utilization while preserving the therapeutic efficacy of transplantation. From this perspective, non-cryopreserved transplantation may be viewed as a DMSO-sparing strategy aimed at improving transplant tolerability and simplifying logistical requirements without compromising the fundamental therapeutic goals of high-dose therapy and stem-cell rescue. The lower burden of early toxicity and the more favorable inflammatory profile observed in the non-cryopreserved cohort support further investigation of this approach in appropriately selected patients.
Our observations are also consistent with emerging evidence suggesting that cryopreservation may influence graft performance beyond the autologous setting. During the COVID-19 pandemic, De Mello Costa et al compared cryopreserved and fresh bone marrow grafts in allogeneic transplantation and reported delayed engraftment, higher transfusion requirements, and more infusion-related complications among recipients of cryopreserved products.16 Similarly, Parody et al reported slower hematologic recovery associated with cryopreserved grafts in matched-related donor transplantation.3 Although these studies were conducted in different transplant settings, they support the concept that preservation methods may influence both cellular integrity and early clinical outcomes.
This study has several limitations. First, the non-randomized design introduces the possibility of center-related confounding, particularly because graft preservation strategy was closely linked to institutional practice. Differences in supportive care, including post-transplant G-CSF administration, hospitalization practices, discharge criteria, and management of mucositis or febrile neutropenia, may have influenced some of the observed outcomes. Second, the prospective cohort was intentionally limited in size, reflecting the exploratory nature of the biomarker analyses and available resources. Recruitment in Spain was further affected by increasing availability of CAR-T cell therapies, which reduced the number of patients proceeding to autologous transplantation during the study period. Third, biomarker analyses were exploratory and not powered for definitive mechanistic conclusions. Finally, although the observed biomarker patterns are biologically plausible, the study design does not permit definitive causal inferences regarding the relationship between cryopreservation, endothelial activation, and transplant-related toxicity.
Despite these limitations, the consistency of the clinical findings across retrospective and prospective cohorts, together with the reproducible differences observed in inflammatory biomarkers, provides a meaningful foundation for future investigation. To our knowledge, this is among the first studies to integrate prospective inflammatory and endothelial-associated biomarker profiling with clinical outcomes in the comparison of cryopreserved and non-cryopreserved autologous grafts in multiple myeloma. By combining biological and clinical observations, the present work generates the hypothesis that graft preservation strategies may influence the early inflammatory environment after transplantation and thereby contribute to differences in transplant-related toxicity.
In conclusion, non-cryopreserved autologous transplantation represents a safe and pragmatic approach in multiple myeloma, associated with reduced early toxicity and comparable engraftment outcomes. These findings are supported by a more favorable inflammatory profile characterized by lower levels of selected inflammatory and endothelial-associated biomarkers. Although prospective controlled studies are needed to confirm these observations, the present results support continued evaluation of non-cryopreserved and DMSO-sparing strategies aimed at improving the tolerability and efficiency of autologous transplantation.
Acknowledgments
The authors used ChatGPT (OpenAI) for language editing and clarity. No involvement occurred in study design, data analysis, or interpretation. The authors take full responsibility for the content of the manuscript.
Disclosure
Patricia Alcalde-Mellado reports Grants or contracts from Junta de Andalucía, outside the submitted work.The authors declare no other conflicts of interest in this work.
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