Allogeneic stem cell transplant recipients surviving at least 2 years without relapse: outcome and risk factors

Abstract Outcomes of 2‐year survivours undergoing allo‐haematopoietic stem cell transplantation at Oslo University Hospital were retrospectively assessed with the objectives of identification of risk factors for late death as possible means for precautionary measures and interventions to improve long‐term survival. 421 patients with haematological malignancy, transplanted between 2005 and 2019, alive and free of disease after 2 years were included with data reported from The OUS‐HSCT registry. Median follow‐up was 6.2 years (2.016.1), and 232 patients (55%) were observed for minimum 5 years. The probability of being alive 5 and 10 years after HSCT was 86% and 76%. Primary risk factors for late death included initial diagnosis of age ≥ 60 years, chronic lymphocytic leukaemia (CLL), previous blood stream‐ or invasive fungal infection (BSI, IFI), and chronic graft‐versus‐host disease (cGVHD). Transplant‐related mortality (TRM) and relapse at 5 years were 9.0% and 7.7%, respectively. Two factors were associated with the latter: cytomegalovirus (CMV) seronegative donor and CLL. Compared with the age‐ and gender‐matched Norwegian general population, life expectancy was lower for each disease, except for CML. The prospect for the long‐term survival is good for 2‐year survivors of the allogeneic hematopoietic stem cell transplantation. However, life expectancy remains inferior to the age‐ and gender‐matched general population. Optimising prophylaxis and treatment for chronic GVHD, BSI and IFI are needed along with the improved adherence to guidelines for early detection of secondary malignancies. Measures to improve immune reconstitution, possibly the microbiota, and the use of CMV seropositive donors regardless of recipient sero‐status may be warranted and should be addressed in further studies.


K E Y W O R D S
aGvHD, Allo-HSCT, cGvHD, GvHD-prophylaxis, long-term follow-up

BACKGROUND
Outcome after allogeneic haematopoietic stem cell transplantation (HSCT) has improved in recent years [1] even though patients are older and alternative donors are used more frequently [2].Reasons for this are various, including use of less toxic conditioning protocols, advancement in human leukocyte antigen (HLA)-typing and extended donor availability, increased knowledge on prophylaxis and treatment of graft versus host disease (GVHD), better supportive care, testing for minimal residual disease (MRD), use of new prognostic models, and measures to prevent relapse [3,4].
Most deaths' post transplantation occurs during the first 2 years [5,6].In patients who survive longer, the leading causes of longterm mortality are chronic GVHD, relapse of malignant disease, infections and secondary malignancies [7].Compared to the general population, survivors beyond 2-and even 5 years still have lower life expectancy and two-to nine-fold increased mortality ranging in studies [8].
In the largest retrospective study to date, the estimated 10-year survival was 85% in 2-year survivors, based on records from the Centre of International Blood and Marrow Transplant Research (CIBMTR) [9].
In this study, we evaluated the outcome of patients who were alive without relapse 2 years after HSCT, and compared survival of these patients with an age-and gender-matched Norwegian population.Our main objective was identification of risk factors for late death as possible means for precautionary measures and interventions to improve long-term survival.

Patients
Adult (≥18 years) patients with malignant disorder who underwent allo-HSCT at OUS from 2005 to 2019 and were alive without relapse 2 years post-transplant were eligible for the study.OUS is a national referral centre conducting the vast majority of allo-HSCT in Norway.Patient characteristics are displayed in Table 1.The study was approved by the Regional Committee for Medical and Health Research Ethics of South-East Norway.The procedures were in accordance with the Helsinki Declaration.

HLA typing
All patients and donors were typed using polymerase chain reaction (PCR)-SSP high-resolution typing for both HLA class I and II alleles.

Conditioning regimen and GVHD prophylaxis
Reduced Seventeen patients transplanted with an HLA-haploid family donor received CsA+MMF and post-transplant cyclophosphamide.During the first month, blood CsA trough levels were kept at 200-300 ng/mL, depending on the transplant protocol.In the absence of GvHD, CsA was tapered with the aim of discontinuation after 4-6 months.

Stem cell source
Three hundred and twelve patients (74%) received peripheral blood stem cells (PBSC) and 110 received bone marrow (BM).

Statistics
Overall survival (OS) and relapse-free survival (RFS) were calculated using the Kaplan-Meier method and survival curves were compared using the log-rank test.Survival was calculated from 2 years after transplantation until death or last follow-up.Expected survival for a comparable general population was estimated using the Ederer2-estimator in combination with mortality rates from a general lifetable stratified by sex, age (1-year groups) and calendar year (1-year groups) [10].The incidence of transplant-related mortality (TRM) and relapse were estimated using the competing risk analysis by Fine and Gray-considering relapse as a competing event for TRM and death without relapse as competing event for relapse.Patients were censored at the time of last follow-up, 31 Jan 2021.
Uni-and multivariate predictive analyses for relapse and TRM were performed with the proportional sub-distribution hazard regression model of Fine and Gray, while analysis of OS and RFS were performed using the Cox proportional hazards model.

RESULTS
Of 1,000 adult patients with a malignant disease transplanted during the study period, 421 were alive and disease free 2 years posttransplant.Median follow-up were 6.2 years (2.0-16.1).
By the end of follow-up, 69 (16%) patients had died.

DISCUSSION
In this study, we analysed outcome of patients alive without relapse one from 1999 [13] with probability of surviving 5 years at 89% and the other from 2011 with 85% at 10 years [9], our results are slightly inferior.However, cited studies included only transplants with MAC, recipients were young with a median age of 16-34 years, and nonmalignant diseases as aplastic anaemia were part of the cohort.Our population had a higher median age of 50 years, nearly half of them received RIC with PBSC as stem cell source, only malignant diagnoses were included, and in contrast to the 1999 study with CML making up nearly one-third of the patients only 4% were patients with CML in our cohort.These are factors known to be associated with increased risk of relapse or chronic GVHD, which is a likely explanation for the difference in OS.A more recent single centre analysis from 2018 with patient characteristics comparable to ours reported 5 years OS at 78% in a cohort of patients alive and free of relapse at 1 year after HSCT [14].
Factors associated with impaired late OS and NRM were mainly transplant related complications occurring during the two first years after HSCT: chronic GVHD, BSI and IFI.Age was of significance with inferior prospects for older patients.These are factors known to increase early mortality [15,16], and chronic GVHD and age being major contributors to late mortality are well established [14,17].
Regarding BSI, several studies have addressed this issue: the largest from 2019 showing an association between BSI by day 100 and impaired 1-year survival [18].Here, we show that BSI also has negative impact on long-term survival.Reasons may be numerous.BSI may be a surrogate marker for comorbidity and frailty.Further, early BSI is associated with acute GVHD [19] either manifest or subclinical, facilitating thymus injury either from allo-reactive T-cells and/or immunosuppressant drugs, corticosteroids in particular [20].Net result being impaired function for thymus epithelial cells with reduced thymopoiesis and negative selection, resulting in impaired regeneration of T-cells and reduced elimination of allo-reactive T-cells, negatively affecting adaptive immunity [21].This sets the scene for long-term complications such as immunodeficiency, reduced GVL with subsequent relapse, or release of allo-reactive T-cells resulting in chronic GVHD [22].
Another possible mechanism may be through loss of gut microbiota diversity after BSI treatment with broad-spectrum antibiotics.High microbiota diversity during engraftment is associated with superior survival compared to low diversity, with positive impact on NRM and less GVHD in the former group [23,24].Interestingly, recent data indicate that gut microbiota influence immune reconstitution, and that low microbiota diversity may impair neutrophil and lymphocyte recovery [25,26].
Regarding IFI, increased NRM for up to 2 years has been reported, and risk factors for IFI such as impaired immune reconstitution, acute GVHD, and subsequent prolonged immunosuppressive therapy may lead to impaired GVL with relapse or chronic GVHD in the long run [27,28].Thus, IFI may serve as a surrogate marker for factors associated with risk for long-term mortality.As for BSI, here we show that IFI is associated with increased long-term mortality.Taken together, early BSI and IFI are associated with increased long-term NRM where measures for optimising prophylaxis, treatment, immune reconstitution, and microbiota may be warranted, and should be addressed in future studies.
Relapse was the leading cause of late deaths (32% of all deaths) in line with comparable studies [9,29].Nearly all relapses (27/30) occurred between years 2 to 5 after HSCT, and relapse beyond that time point was very rare.In multivariate analysis, only CLL and CMV negative donor were of statistical significance.CMV latency or reactivation and its association with enhanced GVL effect and reduction in short-term relapse incidence has been shown in several publications with the proposed main mechanism being CMVs induction of NKG2C+/NKG2A− NK cells with NKG2C mediated cytotoxicity against HLA-E expressing malignant cells [30][31][32].There is evidence of a similar response by a subset of γδ T-cells to an undefined antigen, but the role of αβ T-cells in this setting is less clear [33].Our findings indicate that this antitumour activity also holds long-term.In our cohort, the protective effect of a CMV-positive donor was present regardless of manifest CMV-reactivation or not, and regardless of recipient serostatus (data not shown).Choosing a CMV+ donor for CMV+ recipient is supported, but in the era of improved CMV monitoring, CMV treatment, and reduction in risk factors for CMV-reactivation such as better GVHD-prophylaxis with ATG or PTCy, we hypothesise that the protective effect of a CMV-positive donor might also warrant a CMV+ donor for a CMV− recipient.However, this should be addressed in future studies.
Other major causes of death were chronic GVHD (28%), secondary malignancies (13%) and infections (12%).The importance of the ever prominent chronic GVHD is of no surprise and well documented [8,9].Thymus injury from radio-and/or chemotherapy as stated above [21] is a risk factor.This might explain the high incidence among HSCT CLL and lymphoma patients in our cohort, being a particularly heavily pre-treated group, many transplanted in third remission or even beyond.The incidence of GVHD is fortunately decreasing, in particular related to increased use of ATG or post-transplantation cyclophosphamide (PTCy) as is our experience [34] as well as that of others [35,36].Increased risk of death due to secondary malignancies for long-term survivors after HSCT, another known significant complication [37] may be even more apparent as HSCT survivors live longer.
However, changes in treatment protocols with less toxic conditioning regimens, for example, less use of TBI, measures for optimising immune reconstitution, reduction in chronic GVHD with subsequent less use of immunosuppressive drugs, and adequate guidelines for screening and long-term follow-up may reduce the incidence of secondary malignancies.
Our findings must be interpreted with caution.This is a single centre study with limited number of patients, and transplantation practices have changed over the 14 years spanning the study period.In particular, use of ATG and PTCy as GVHD prophylaxis, implementation of haploidentical donors and steadily increasing number of older patients with use of RIC protocols may affect future outcome.

CONCLUSION
The prospect for long-term survival is good for 2-year survivors of HSCT.However, life expectancy remains inferior to the general population.Optimising prophylaxis and treatment for chronic GVHD, BSI and IFI are needed, along with guidelines for follow-up regarding secondary malignancies.Measures to improve immune reconstitution, microbiota, and use of CMV seropositive donors regardless of donor serostatus may be warranted.

Factors
analysed: patient and donor age, gender and pre-HSCT cytomegalovirus (CMV) sero-status, diagnosis, gender match, HLAmatch, donor type, stem cell source, CD34+ cell dose, conditioning intensity, ATG, GVHD prophylaxis, previous acute and chronic GVHD, previous blood-stream infection, invasive fungal infection and CMV reactivation.Since acute GVHD, IFI and BSI antedated the 2 years analysis, these factors were treated as non-time-dependent factors.Most (95%) chronic GVHD already existed at 2 years, and only a few patients (n = 15, 8 limited and 7 extensive) developed chronic GVHD beyond 2 years.For this reason, we treated chronic GVHD as a time-dependent covariate in the Cox proportional hazards model.The proportional hazards assumptions were tested with scaled Schoenfeld residuals.Analyses were performed using the EZR freely available software, and Statistica 13 (Tulsa, OK, USA) and Stata version 16.1 software.

F U E 2
Overall survival in patients alive without relapse 2 years after HSCT compared with Norwegian age-and gender-matched population.HSCT, haematopoietic stem cell transplantation.F I G U R E 3 Transplant-related mortality (TRM) in patients alive without relapse 2 years after HSCT.HSCT, haematopoietic stem cell transplantation.Most relapses (27/30) occurred between 2 and 5 years after HSCT, while only three occurred beyond 5 years post HSCT.The proportional hazard assumption was not fulfilled in the analysis of CLL diagnosis due to crossing K-M curves.For this reason, we split the follow-up period into 2-year intervals.However, the number of patients with CLL was very limited (n = 12) and events (relapse) were few (n = 3) making the interpretation very doubtful.

2
years after HSCT.Acceptable long-term survival was achieved with 86% and 76% at 5 and 10 years, respectively.Results varied across different diagnosis.Only patients transplanted for CML were in accordance with survival in the Norwegian age-and gender-matched population.Superior long-term outcome for CML after HSCT has been reported previously[11].Dismal prospects for lymphoproliferative disorders after HSCT, CLL in particular, are in our material as well as others[12] mainly due to high NRM; 36% among our patients with chronic GVHD being the main culprit.Compared with two larger previous studies of patients alive and free of disease at 2 years post HSCT, F I G U R E 4 Relapse incidence in patients alive without relapse 2 years after HSCT.HSCT, haematopoietic stem cell transplantation.

TA B L E 1
Characteristics of patients alive without relapse 2 years after HSCT.
Results from multivariate analyses (MVA) of factors affecting outcome in patients alive without relapse 2 years after HSCT.