The Effects of Late Toxicities on Outcomes in Long-Term Survivors of Ex-Vivo CD34+− Selected Allogeneic Hematopoietic Cell Transplantation

Abstract

The late adverse events in long-term survivors after myeloablative conditioned allogeneic hematopoietic cell transplantation with ex-vivo CD34+ selection are not well characterized. Using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0, we assessed all grade ≥ 3 toxicities from the start conditioning to date of death, relapse, or last contact in 131 patients that survived > 1-year post-transplantation, identifying 285 individual toxicities among 17 organ-based toxicity groups. Pre-transplantation absolute lymphocyte count > 0.5 K/mcL and albumin > 4.0 g/dL were associated with a reduced risk of toxicities, death and non-relapse mortality (NRM), while ferritin > 1000 ng/mL was associated with an increased risk of toxicities and NRM after 1-year. HCT-CI ≥ 3 was associated with an increased risk of all-cause death and NRM, but was not associated with a specific increased toxicity risk after 1-year. Those that incurred more than the median number of toxicities (n=7) among all patients within the first year subsequently had an increased risk of hematologic, infectious, and metabolic toxicities, and had an increased risk of NRM and inferior 4-year OS, 67% versus 86% (p=0.003) after the 1-year landmark. Developing grade 2-4 GVHD within the first year was associated with incurring > 7 toxicities within the first year (p=0.016), and was associated with an increased risk of all-cause death and NRM after 1-year. In multivariate models, cardiovascular, hematologic, hepatic, infectious, metabolic, neurologic, and pulmonary toxicities incurred after 1-year all independently increased the risk of death and NRM when adjusting for both HCT-CI and grade 2-4 GVHD within the first year. One-year survivors of ex-vivo CD34+ selection had a favorable 4-year OS of 77%, though the development of grade ≥ 3 toxicities after the first year was associated with poorer outcomes, emphasizing the fundamental importance of improving survivorship efforts that may improve long-term toxicity burden and outcome.

Introduction:

The advent of innovative supportive care and transplantation techniques has improved long-term survival following allogeneic hematopoietic cell transplantation (allo-HCT).¹ In particular, CD34+ stem cell enrichment and T cell depletion modalities have contributed to favorable outcomes free from graft-versus-host disease (GVHD). Multiple prior studies of patients with hematologic malignancies treated with myeloablative conditioning regimens and ex-vivo CD34+ stem cell selected donor allografts demonstrated durable disease control and low rates of acute and chronic GVHD without the need for prolonged pharmacologic immunosuppression.^2-13 The ongoing Bone Marrow Transplant Clinical Trials Network Protocol 1301 () [PROGRESS II] is prospectively comparing 3 GVHD prophylaxis strategies including: ex-vivo CD34+ selection with the CliniMACS® Reagent System (Miltenyi), post-transplantation cyclophosphamide and standard tacrolimus and methotrexate.^14-19 These transplantation platforms have unique features and are therefore differentiated by intrinsically distinctive toxicities.^20-23

Much of the available literature regarding long-term outcomes after allo-HCT has focused on disease-related mortality and broad causes of non-relapse mortality (NRM).^24,25 Moreover, there is a paucity of literature detailing the characteristic adverse events occurring in adults with hematologic malignancies undergoing ex-vivo CD34+ selected allo-HCT, particularly late adverse events. We aimed to elaborate on our experience by compiling a comprehensive database of high-grade toxicities incurred by patients undergoing allo-HCT using ex-vivo CD34+ selection as GVHD prophylaxis at Memorial Sloan Kettering Cancer Center (MSKCC). In this study, our specific focus was the assessment of toxicities occurring after the 1-year post-transplant landmark in an effort to identify factors that predicted for specific individual toxicities, non-relapse mortality (NRM) and overall survival (OS), and to identify patients at risk for late toxicity and unfavorable long-term outcomes.

Methods:

Patients, Graft Sources and Conditioning Regimens:

Eligible patients were ≥ 18 years old with adequate pre-transplant organ function who underwent matched-related donor (MRD), mismatched-related donor (MMRD), matched-unrelated donor (MUD) or mismatched unrelated donor (MMUD) granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cell (PBSC) allo-HCT for any hematologic malignancy using ex-vivo CD34+ selection (CliniMACS® CD34 Reagent System, Miltenyi, Gladbach, Germany) as GVHD prophylaxis between 2006 and 2012. Data was extracted from the electronic medical record with a data-cutoff of December 31, 2015. No further pharmacologic GVHD prophylaxis was given post-transplant. Human leukocyte antigen (HLA) typing was performed using high-resolution DNA sequence-specific oligonucleotide typing for the HLA−A, −B, −C, −DRB1 and −DQB1 loci. Patients who had been previously typed using intermediate resolution methods were re-typed with high-resolution methods that were used to define the level of mismatch. All patients were treated with one of four of the following conditioning regimens at the discretion of the treating physician based on age, comorbidities, and disease type: busulfan, melphalan, fludarabine (bu/mel/flu); clofarabine, melphalan, thiotepa (clo/mel/thio); total body irradiation, thiotepa, cyclophosphamide (TBI/thio/cy); or TBI, thiotepa, and fludarabine (TBI/thio/flu) as previously described.^4,6,7 All patients received rabbit anti-thymocyte globulin (ATG) 2.5 to 5 mg/kg/day on days −3, −2.^5,21 Patients who received a TBI-based preparative regimen received keratinocyte growth factor (KGF) 60 mcg/kg IV on days −13, −12, −11 and 0, +1, +2.²⁶ G-CSF was initiated on day +7 until absolute neutrophil count (ANC) recovery. For patients receiving PK-targeted Bu-based MAC regimens, first dose Bu pharmacokinetic was performed to a target area-under-the-curve (AUC) range of 4100 – 5300 micromol*min/L. All patients received standard supportive care for prevention of sinusoidal obstruction syndrome (SOS) and opportunistic anti-microbial prophylaxis according to standard MSKCC institutional guidelines.

Toxicity Collection and Biostatistical Methods

“Late survivor” was defined as being alive and without disease relapse or disease progression at 1-year post-HCT. For these patients, we retrospectively collected all individual grade ≥ 3 toxicities using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 from the start of the conditioning regimen to the date of death, relapse or progression of disease, or last contact. For the purposes of statistical analyses, diseases were separated into the following 3 groups: acute leukemia/myelodysplastic syndrome (MDS), multiple myeloma, and other malignancies. Also, conditioning regimen types were classified as chemotherapy-based or TBI-based. The post-transplant time-periods analyzed were: +1 to 2 years, +2 to 3 years, +3 to 4 years, and beyond 4 years. We employed a standard toxicity review structure with the guidance of CTCAE v. 4.0, and we performed audits wherein different reviewers performed toxicity assessments on several of the same patients in order to minimize inter-reviewer variability. Individual toxicities were organized into 17 organ- and laboratory parameter-based groups and further classified by specific toxicity as shown in Appendix 1. In order to avoid multiple counting, infectious toxicities were separated into: febrile neutropenia without a source, febrile neutropenia with an identified source, neutropenic sepsis, non-neutropenic sepsis, and non-neutropenic bacteremia. Viral reactivations without organ disease were excluded and recurrence intervals for all infections were based on the CIBMTR classification.²⁷ The highest individually graded toxicity per specified post-HCT time period was recorded and used for the purposes of statistical analyses.

The cumulative incidences (CI) of all individual toxicity groups occurring after 1-year were assessed; competing risk for this analysis included death, relapse or progression of disease, or second transplant. Cause-specific Cox regression assessed the risk of individual toxicities based on patient and transplantation characteristics including the hematopoietic cell transplantation co-morbidity index (HCT-CI), development of GVHD and the total toxicities observed in the initial year after transplantation.²⁸ Cox regression also assessed the univariate and multivariate associations for the endpoints of non-relapse mortality (NRM) and any-cause mortality. Toxicities were included as potential predictors in these models by treating each toxicity as a time-dependent covariate. Separate multivariate models adjusted for 1-year GVHD and HCT-CI; these were selected based on the number of events and the univariate associations of the two factors. Kaplan-Meier methods were used to estimate overall survival and progression-free survival after the 1-year post-transplantation landmark.²⁹ All analyses were conducted using the R v3.3.2.

Results:

There were 131 late survivors included in this analysis with a median follow-up among survivors of 36 months. Baseline patient and transplantation characteristics are shown in Table 1. Median age was 55 (range 19-72). Patients had the following diseases: 50 (38%) with acute myeloid leukemia, 12 (9%) with acute lymphoid leukemia, 3 (2%) with acute leukemia not otherwise specified, 32 (25%) with MDS, 15 (11%) with multiple myeloma, and 19 (15%) with other malignancies which included 8 with myeloproliferative neoplasms, 7 with chronic myeloid leukemia, 3 with non-Hodgkin lymphoma, and 1 patient with T-cell prolymphocytic leukemia. The most common toxicities in patients after 1-year post-transplantation were hematologic with a cumulative incidence at 4-years after the landmark of 33% [95% confidence interval (CI) 25-42%], infectious 27% (95% CI 20-35%), metabolic 22% (95% CI 15-29%), hepatic 17% (95% CI 11-23%), and cardiovascular 10% (95% CI 6-16%). Appendix 2 demonstrates the cumulative incidences of all the toxicity groups. Among all late survivors, there were 285 individual toxicities recorded during the study period, 215 (75%) of which occurred within the +1 to 2-year post-transplantation period, 49 (17%) occurred in the +2 to 3 year post-transplantation period, and 21 (7%) of which occurred > 3 years post-transplantation. The median number of toxicities per patient in the +1 to 2-year post-transplantation period was 2. Appendix 3 details all of the individual toxicities incurred among all patients during the study period categorized based on the respective toxicity group and subgroup.

Table 1.

Baseline Patient and HCT Characteristics for Patients Alive and Relapse-Free at 1-Year Post-Transplantation

Characteristic	N=131 (%)
Age, Median (Range)	55 (19-72)
< 60	85 (65)
≥ 60	46 (35)
Gender
Female	69 (53)
Male	62 (47)
Disease
AML/ALL/MDS	97 (74)
MM	15 (11)
Other	19 (15)
Regimen
Chemo-based	83 (71)
TBI-based	38 (29)
HLA Matching
MRD	51 (39)
MMRD	1 (<1)
MURD	53 (40)
MMURD	26 (20)

AML indicates acute myeloid leuekemia, ALL, acute lymphocytic leukemia; MDS, myelodysplastic syndrome; MM, multiple myeloma; TBI, total-body irradiation; HLA, human-leukocyte antigen; MRD, matched related donor; MMRD, mismatched-related donor; MURD, matched-unrelated donor; MMURD, mismatched unrelated donor.

Notably, there were 69 individual infectious toxicities among all patients over the study period, the majority of which included: lung infections (32%), bacteremias (22%), upper respiratory infections (7%), sepsis (6%), urinary tract infections (4%), Epstein-Barr virus post-transplant lymphoproliferative disorders (EBV-PTLD, 3%), cytomegalovirus (CMV) reactivations (3%) and human herpesvirus-6 reactivations (3%). The 56 individual hematologic toxicities among all patients were comprised mostly of cytopenias (80%). The 23 individual hepatic toxicities largely included lab abnormalities (96%) such as transaminitis, alkaline phosphatase elevations and hyperbilirubinemia; there were no cases of late sinusoidal obstruction syndrome. A majority of individual cardiovascular toxicities were related to hypertension (32%), and of all 12 late pulmonary toxicities, 25% were cases of acute respiratory distress syndrome. There were few late neurologic events, and of the 11 total toxicities, 27% were cognitive disturbances. The median number of individual toxicities incurred per patient within the first year post-transplantation was 7. Figure 1 shows the distribution of toxicities by number of months post-transplantation.

Figure 1.

Distribution of Late Toxicities After Transplantation

Among all late survivors, there were 285 individual toxicities recorded during the study period: 215 (75%) in the +1-2 year post-transplantation period, 49 (17%) in the +2 to 3 year post-transplantation period, and 21 (7%) occurred > 3 years post-transplantation.

Risk Factors for Individual Toxicities, Any-Cause Death and NRM

When assessing the associations of patient and transplantation characteristics with the development of the most common toxicities after 1-year post-transplantation, we found that a pre-transplantation patient absolute lymphocyte count (ALC) > 0.5 K/mcL was associated with a reduced risk of developing hematologic [hazard ratio (HR) 0.3 (0.14-0.6), p=0.001], infectious [HR 0.36 (0.15-0.9), p=0.023], metabolic [HR 0.36 (0.14-0.96), p=0.04], and pulmonary [HR 0.23 (0.06-0.9), p=0.03] toxicities. A pre-HCT albumin > 4.0 g/dL was associated with a reduced risk of developing hepatic [HR 0.25 (0.1-0.7), p=0.012] and infectious [HR 0.47 (0.2-0.98), p=0.05] toxicities. In contrast, a pre-HCT ferritin > 1000 ng/mL was associated with an increased risk of metabolic [HR 2.77 (1.2-6.3), p=0.05] toxicities. HCT-CI was not associated with an increased risk of specific toxicities. Developing grade 2-4 GVHD within the first-year post-transplantation correlated increased the risk of hematologic [HR 2.7 (1.44-5.1), p=0.002], infectious [HR 2.8 (1.4-5.6), p=0.004], metabolic [HR 3.65 (1.7-7.8), p=0.001] toxicities. Recipients of cytomegalovirus (CMV) positive donor allografts had an increased risk of metabolic toxicities [HR 2.6 (1.2-5.6), p=0.02], and host CMV status did not predict for toxicities. Patients with > 7 toxicities within the first year had associated risk of hepatic [HR 2.77 (1.5-5.1), p=0.001], metabolic [HR 3.55 (1.7-7.4), p=0.001], and oral/gastrointestinal [HR 3.36 (1.5-7.6), p=0.004] toxicities.

Appendix 4 details the characteristics and toxicities whose development after the first year post-transplantation were associated with an increased risk of all-cause death and NRM by univariate Cox regression. Incurring a cardiovascular, hematologic, infectious, metabolic, neurologic, pulmonary or hepatic toxicity after the first year was associated with an increased risk of all-cause death and NRM. Development of grades 2-4 GVHD in the first year post-transplantation and HCT-CI ≥ 3 were both associated with an increased risk of all-cause death and NRM, and ferritin > 1000 ng/ml was associated with an increased risk of NRM. In contrast, absolute lymphocyte count (ALC) >0.5 K/mcl and albumin >4.0 g/l were associated with a decreased risk of all-cause death, and ALC >0.5 K/mcl was associated with a decreased risk of NRM. Age, gender, disease, HLA match, conditioning regimen type, CMV status, busulfan AUC level, and use of palifermin were not associated with an increased risk of specific toxicities, all-cause mortality or NRM.

Patients with > 7 toxicities within the first year had an increased risk of death [HR 3.2 (1.4-7.2), p=0.006] and NRM [HR 4.8 (1.6-14.4), p=0.005]; this translated into a 4-year OS after the 1-year landmark of 67% versus 86% (p=0.003) for those who did and did not have > 7 toxicities, respectively, as shown in Figure 2. Grades 2-4 acute GVHD occurred in 13 (10%) patients, and 22 (17%) patients had late acute GVHD. Chronic GVHD occurred in 9 (7%) of patients, 5 of moderate and 4 of mild grade, respectively. Of the patients that did not develop grade 2-4 GVHD in the first year, 43% had more than 7 toxicities in the first year whereas 70% of patients who developed grade 2-4 GVHD in the first year had more than 7 toxicities in the first year (p=0.016).

Figure 2.

Overall Survival by Median Number of Early Toxicities

We investigated two multivariate models: the first analyzed the association between individual toxicities and the risk of death and NRM while adjusting for HCT-CI, and the second analyzed similar associations but adjusted for the development of grade 2-4 GVHD within the first year. In the multivariate model adjusting for HCT-CI, we found that cardiovascular, hematologic, hepatic, infectious, metabolic, neurologic, and pulmonary toxicities incurred after 1-year all independently increased the risk of any-cause death and NRM. The same toxicities remained independently significant for both outcomes in the second model adjusting for grade 2-4 GVHD within the first year, as shown in Table 2.

Table 2.

Multivariate Models Assessing Association of Toxicity with Risk of Death and NRM Adjusting for HCT-CI and GVHD in the 1st Year

			Risk of death		Risk of NRM
Model			HR (95% CI)	P-value	HR (95% CI)	P-value
1a	Cardiovascular		7.1 (2.9-17.2)	<0.001	8.5 (3.1-22.8)	<0.001
	HCT-CI	0	(reference)		(reference)
		1-2	1.0 (0.3-3.3)	0.96	1.14 (0.2-6.9)	0.89
		≥3	2.2 (0.8-6.1)	0.12	4.8 (1.1-21.1)	0.04
1b	Cardiovascular		6.6 (2.8-16)	<0.001	7.8 (2.9-21.2)	<0.001
	Grade 2-4 GVHD		2.8 (1.3-6)	0.009	4.6 (1.8-11.4)	0.001
2a	Hematologic		6.9 (3.1-15.3)	<0.001	9.4 (3.4-26)	<0.001
	HCT-CI	0	(reference)		(reference)
		1-2	1.2 (0.4-3.7)	0.80	1.3 (0.2-8.0)	0.76
		≥3	2.9 (1.1-7.9)	0.04	6.4 (1.5-28)	0.01
2b	Hematologic		5.7 (2.5-12.9)	<0.001	6.7 (2.3-19.4)	<0.001
	Grade 2-4 GVHD		2.1 (0.9-4.4)	0.07	3.3 (1.3-8.3)	0.01
3a	Hepatic		3.3 (1.4-7.8)	0.008	4.4 (1.7-11.8)	0.003
	HCT-CI	0	(reference)		(reference)
		1-2	0.8 (0.3-2.6)	0.75	0.9 (0.2-5.4)	0.90
		≥3	2.2 (0.8-6.0)	0.12	4.6 (1.1-20.3)	0.04
3b	Hepatic		3.3 (1.4-7.8)	0.007	4.4 (1.7-11.8)	0.003
	Grade 2-4 GVHD		3.3 (1.6-6.9)	0.002	5.4 (2.2-13.1)	<0.001
4a	Infectious		5.8 (2.7-12.3)	<0.001	9.6 (3.8-24.3)	<0.001
	HCT-CI	0	(reference)		(reference)
		1-2	0.9 (0.3-2.9)	0.90	1.0 (0.2-6)	0.99
		≥3	2.2 (0.8-5.9)	0.13	4.3 (0.98-19)	0.053
4b	Infectious		5.7 (2.7-12.2)	<0.001	9.7 (3.8-24.6)	<0.001
	Grade2-4 GVHD		2.9 (1.4-6.2)	0.005	4.6 (1.9-11.3)	0.001
5a	Metabolic		6.1 (2.8-13.1)	<0.001	8.2 (3.3-20.5)	<0.001
	HCT-CI	0	(reference)		(reference)
		1-2	0.9 (0.3-2.7)	0.78	0.9 (0.2-5.6)	0.96
		≥3	1.7 (0.6-4.7)	0.31	3.4 (0.8-15.3)	0.11
5b	Metabolic		5.7 (2.6-12.4)	<0.001	7.6 (3-19.6)	<0.001
	Grade 2-4 GVHD		2.2 (1-4.9)	0.046	3.3 (1.3-8.6)	0.013
6a	Neurologic		8.8 (3.5-21.8)	<0.001	14.4 (5.4-38)	<0.001
	HCT-CI	0	(reference)		(reference)
		1-2	0.9 (0.3-2.9)	0.86	1.0 (0.2-6.3)	0.97
		>3	2.4 (0.9-6.5)	0.09	5.2 (1.2-22.6)	0.03
6b	Neurologic		6.6 (2.6-16.9)	<0.001	9.5 (3.4-26.4)	<0.001
	Grade 2-4 GVHD		2.7 (1.3-6)	0.012	4.1 (1.6-10.7)	0.003
7a	Pulmonary		5.9 (1.98-17.6)	<0.001	12.7 (3.9-41)	<0.001
	HCT-CI	0	(reference)		(reference)
		1-2	0.84 (0.3-2.6)	0.8	0.87 (0.14-5)	0.88
		≥3	2.8 (1.0-7.7)	0.05	6.8 (1.5-30.7)	0.01
7b	Pulmonary		3.1 (1-9.1)	0.04	4.4 (1.4-13.8)	0.011
	Grade 2-4 GVHD		3.1 (1.5-6.7)	0.003	5 (2-12.3)	0.001

HCT-CI indicates hematopoietic cell transplantation-comorbidity index; GVHD, graft-versus-host disease

Causes of Death

During the study period, 8 patients (6%) died of relapsed disease, 20 patients (15%) died of NRM, and 9 patients (7%) had relapse without death. Of the 20 patients who died of NRM: 9 died of infection, 8 died of GVHD, and 3 of other causes. At 4-years after the 1-year landmark, PFS and OS for the entire cohort were 70% and 77%, respectively (Figure 3).

Figure 3.

Progression-Free and Overall Survival

Discussion:

There has been limited understanding of the long-term toxicities of ex-vivo CD34+−selected allo-HCT. Our study is the most comprehensive assessment of adverse events in late survivors of ex-vivo CD34+−selected allo-HCT. It represents an essential initial step in identifying the incidences of specific organ-based toxicities and the risk factors that predict for these toxicities and unfavorable outcomes, with the goal of designing future targeted prospective clinical trials aimed at improving long-term survivorship and quality of life. We reaffirmed that 1-year survivors of ex-vivo CD34+ selected MAC allo-HCT have excellent long-term OS. Despite this, patients who develop GVHD within the first year remain at a higher risk of incurring multiple toxicities and a greater risk of NRM, which ultimately translates into inferior OS. There are no head-to-head adverse event comparisons of ex-vivo CD34-selected MAC allo-HCT and un-manipulated MAC allo-HCT; however, this type of analysis is highly relevant and warranted. Comprehensive toxicity data collected from several, previously published, prospective BMT CTN clinical trials could serve as an important comparative database to do so.³⁰

Our group has previously reported the causes of death in 1-year survivors of ex-vivo CD34+ selected MAC allo-HCT yielding results congruent with our current findings in that late deaths are caused either by relapse or NRM, with the latter driven largely by GVHD and infection.³¹ A HCT-CI ≥ 3 was associated with an increased risk of death and NRM in both studies. Complementary to this, our current study shows that specific toxicities, both infectious and non-infectious, are also independently associated with an increased risk of death and NRM when adjusting for both HCT-CI and the development of GVHD in the first year. Additionally, patients that incur more individual toxicities within the first year post-transplantation, regardless of etiology, have inferior OS. This underscores the detrimental effects of individual toxicities as well as the cumulative effects of accumulating numerous toxicities over time.

While T-cell depletion techniques typically delay immune reconstitution post-HCT compared to un-modified allografts, we found that among 1-year survivors of ex-vivo CD34+ selected allo-HCT, only 9 died of infection-related toxicities.^{21, 32, 33} Moreover, we noted low rates of high-grade viral reactivation, viral organ-disease and viral-related deaths among 1-year survivors, suggesting adequate T-cell immunity in the majority of patients who did not develop GVHD in the first year. Several previous studies have demonstrated the significance of a patient’s ALC at day +30 post-transplantation as a predictive biomarker of immune reconstitution and overall survival.^34-38 Similarly, our study demonstrated that a higher baseline pre-transplantation ALC portends a lower risk of hematologic, infectious, metabolic and pulmonary toxicities in patients undergoing ex-vivo CD34+ selection. Previously well-characterized and widely clinically available pre-transplantation biomarkers of outcome such as albumin and ferritin were also important predictive biomarkers in our patients, though our analysis emphasizes the unique association of these biomarkers with the development of specific toxicities.^39,40 Further, given that HCT-CI was not associated with an increased risk of specific toxicities, our data suggest that simple serum assays such as ferritin, ALC and albumin may complement and contribute to previously validated and widely used prognostic tools such as, HCT-CI, to predict for specific toxicities in this distinct allo-HCT patient population.²⁸

Our study comes with several limitations primarily related to the retrospective collection of adverse events which may over- or under-estimate the incidence and degree of adverse events occurring in complex patients with multiple, prolonged hospitalizations.⁴¹ Our solution was to perform inter-reviewer audits in order to enhance reviewer consistency and reduce variability. Additionally, given the limited number of certain individual toxicity events in late survivors, we were only able to analyze separate sequential multivariate models. We specifically chose to build separate models for two factors, HCT-CI and the development of GVHD within the first year, given that these were both important predictors in our univariate models and are known risk factors for NRM ^25,42

In summary, 1-year survivors of ex-vivo CD34+ selection-based allo-HCT achieve favorable and durable clinical outcomes, and their expected toxicities after 1-year are largely driven by specific pre-transplantation variables, the type and frequency of post-transplantation toxicities, and whether they develop GVHD within the first year. Our comprehensive analysis emphasizes that determining ways to limit the accumulation of toxicities within the first year may improve outcomes, and identification of patients at continued risk for late toxicity will enhance survivorship care in this population. In light of the PROGRESS II trial, our study may also serve as an essential reference guide for patients receiving ex-vivo CD34+ selection-based allo-HCT and for the healthcare providers caring for them.

Highlights:

• We evaluated all high-grade toxicities in 1-year survivors of CD34+− selected allo-HCT.

• HCT-CI ≥ 3 and GVHD in the 1^st year resulted in more late toxicities, late NRM, and poorer OS.

• Limiting toxicities in the 1^st year and identifying those at risk for late toxicity may improve outcomes.

Appendix

Appendix 1.

Toxicity Groups and Sub-groups

Group Code	Toxicity Group
1	Cardiovascular
2	Endocrine
3	Hematologic
4	Hepatic
5	Immune System
6	Infection
7	Metabolic
8	Musculoskeletal and connective tissue
9	Neurologic
10	Ocular
11	Oral/Gastrointestinal
12	Other
13	Psychiatric
14	Pulmonary
15	Renal and Urinary
16	Reproductive system
17	Skin
Toxicity		Group Code	Toxicity Code
Cardiovascular - Atrial arrhythmia		1	1
Cardiovascular - Atrial fibrillation		1	2
Cardiovascular - Ejection fraction decrease		1	3
Cardiovascular - Heart failure		1	4
Cardiovascular - Hypertension		1	5
Cardiovascular - Hypotension		1	6
Cardiovascular - Myocardial infarction		1	7
Cardiovascular - Pericardial effusion		1	8
Cardiovascular - Pulmonary Hypertension		1	9
Cardiovascular - QTc prolongation		1	10
Cardiovascular - Syncope		1	11
Cardiovascular - Ventricular arrhythmia		1	12
Cardiovascular - Volume overload		1	13
Cardiovascular - Other		1	14
Endocrine - Endocrine disorders, other		2	15
Hematologic - DIC		3	16
Hematologic - Hemolysis		3	17
Hematologic - HUS/TTP		3	18
Hematologic - Coagulation labs		3	19
Hematologic - VTE		3	20
Hematologic - Cytopenia		3	21
Hematologic - Other		3	22
Hepatic - ALT/AST/ALK Phos/BILI		4	23
Hepatic - Cholecystitis		4	24
Hepatic - Hepatic failure		4	25
Hepatic - Portal hypertension		4	26
Hepatic - Hepatobiliary disorders, other		4	27
Immune System - Autoimmune disorder		5	28
Immune System - Immune System disorders, other		5	29
Infection - Adenovirus Organ Disease		6	30
Infection - Adenovirus Reactivation		6	31
Infection - Bacteremia		6	32
Infection - Bladder Infection (BK)		6	33
Infection - C. difficile		6	34
Infection - CMV Organ Disease		6	35
Infection - CMV Reactivation		6	36
Infection - EBV PTLD		6	37
Infection - EBV Reactivation		6	38
Infection - Febrile Neutropenia no source		6	39
Infection - Febrile Neutropenia w/ Source		6	40
Infection - HHV-6 Organ Disease		6	41
Infection - HHV-6 Reactivation		6	42
Infection - Lung Infection		6	43
Infection - Neutropenic Sepsis		6	44
Infection - Sepsis		6	45
Infection - Upper Respiratory Infection		6	46
Infection - Urinary Tract Infection		6	47
Infection - Infections and infestations, other		6	48
Metabolic - Acidosis		7	49
Metabolic - Anorexia		7	50
Metabolic - Dehydration		7	51
Metabolic - Electrolyte abnormality		7	52
Metabolic - Hyperglycemia (diabetes)		7	53
Metabolic - Other		7	54
Musculoskeletal and connective tissue - Avascular necrosis		8	55
Musculoskeletal and connective tissues - Disorder, other		8	56
Neurologic - Cognitive disturbance		9	57
Neurologic - Leukoencephalopathy		9	58
Neurologic - Peripheral motor neuropathy		9	59
Neurologic - Peripheral sensory neuropathy		9	60
Neurologic - Seizure		9	61
Neurologic - Stroke		9	62
Neurologic - Nervous system disorder, other		9	63
Ocular - Cataracts		10	64
Ocular - Other		10	65
Oral/Gastrointestinal - Ascites		11	66
Oral/Gastrointestinal - Colitis		11	67
Oral/Gastrointestinal - Diarrhea		11	68
Oral/Gastrointestinal - GI hemorrhage (Upper or Lower)		11	69
Oral/Gastrointestinal - Mucositis oral		11	70
Oral/Gastrointestinal - Nausea/Vomiting		11	71
Oral/Gastrointestinal - Other		11	72
Other - Fatigue		12	73
Other - Pain		12	74
Other - Treatment related secondary malignancy		12	75
Other - Weight Loss		12	76
Other - SPECIFY		12	77
Psychiatric		13	78
Pulmonary - Acute Respiratory distress syndrome		14	79
Pulmonary - Pneumonitis		14	80
Pulmonary - Pulmonary edema		14	81
Pulmonary - Respiratory, thoracic and mediastinal disorders, other		14	82
Renal and Urinary - Chronic Kidney Disease		15	83
Renal and Urinary - Creatinine increased		15	84
Renal and Urinary - Cystitis noninfective		15	85
Renal and Urinary - Hematuria		15	86
Renal and urinary - other		15	87
Reproductive system - Erectile dysfunction		16	88
Reproductive system - Other		16	89
Skin - Erythroderma		17	90
Skin - Rash maculo-papular		17	91

Appendix 2.

Cumulative Incidence of Toxicities

	1-2 Years	1-3 Years	1-4 Years
Cardiovascular	0.06 (0.03-0.11)	0.08 (0.040-0.14)	0.1 (0.06-0.16)
Endocrine	0.01 (0-0.04)	0.01 (0-0.04)	0.01 (0-0.04)
Hematologic	0.29 (0.22-0.37)	0.31 (0.24- 0.39)	0.33 (0.25-0.42)
Hepatic	0.13 (0.08-0.19)	0.16 (0.1- 0.22)	0.17 (0.11-0.23)
Immune System	0.02 (0.01- 0.06)	0.24 (0.17- 0.32)	0.27 (0.2- 0.35)
Infection	0.19 (0.13- 0.26)	0.24 (0.17-0.32)	0.27 (0.2-0.35)
Metabolic	0.14 (0.09- 0.21)	0.19 (0.13- 0.27)	0.22 (0.15- 0.29)
Musculoskeletal	0.03 (0.01- 0.07)	0.05 (0.02- 0.09)	0.05 (0.02- 0.09)
Neurologic	0.04 (0.01-0.08)	0.08 (0.04- 0.13)	0.08 (0.04- 0.13)
Ocular	0.02 (0.01-0.06)	0.02 (0.01- 0.06)	0.02 (0.01-0.06)
Oral/Gastrointestinal	0.04 (0.01-0.08)	0.04 (0.01-0.08)	0.04 (0.01-0.08)
Other	0.06 (0.03-0.11)	0.08 (0.04-0.14)	0.09 (0.05-0.15)
Psychiatric	0	0.01 (0- 0.04)	0.01 (0-0.04)
Pulmonary	0.06 (0.03-0.11)	0.07 (0.04-0.13)	0.09 (0.04-0.14)
Renal and Urinary	0.02 (0.01-0.06)	0.05 (0.02- 0.09)	0.05 (0.02-0.09)
Reproductive System	0.01 (0-0.04)	0.01 (0-0.04)	0.01 (0-0.04)
Skin	0.02 (0.01-0.06)	0.02 (0.01-0.06)	0.02 (0.01-0.06)

Appendix 3.

Total Individual Toxicities During the Study Period Among All Patients

Toxicity Subgroups	Number of Individual Toxicities
Cardiovascular - Atrial arrhythmia	2
Cardiovascular - Heart failure	2
Cardiovascular - Hypertension	6
Cardiovascular - Hypotension (Orthostatic)	2
Cardiovascular - Myocardial infarction	1
Cardiovascular - Pericardial effusion	1
Cardiovascular - QTc prolongation	1
Cardiovascular - Ventricular arrhythmia	1
Cardiovascular - Volume overload	1
Cardiovascular - Other	2
Endocrine - Endocrine disorders, other	1
Hematologic - Coagulation labs	8
Hematologic - VTE	3
Hematologic - Cytopenia	45
Hepatic - ALT/AST/ALK Phos/BILI	22
Hepatic - Hepatic failure	1
Immune System - Autoimmune disorder	3
Immune System - Immune System disorders, other	1
Infection - Adenovirus Organ Disease	1
Infection - Bacteremia	15
Infection - Bladder Infection (BK cystitis)	1
Infection - C. difficile	1
Infection - CMV Reactivation	2
Infection - EBV PTLD	2
Infection - Febrile Neutropenia w/ Source	2
Infection - HHV-6 Reactivation	2
Infection - Lung Infection	22
Infection - Sepsis	4
Infection - Upper Respiratory Infection	5
Infection - Urinary Tract Infection	3
Infection - Infections and infestations, other	9
Metabolic - Anorexia	2
Metabolic - Electrolyte abnormality	20
Metabolic - Hyperglycemia	21
Metabolic - Other	5
Musculoskeletal and connective tissue - Avascular necrosis	1
Musculoskeletal and connective tissues - Disorder, other	5
Neurologic - Cognitive disturbance	3
Neurologic - Leukoencephalopathy	1
Neurologic - Seizure	1
Neurologic - Stroke	1
Neurologic - Nervous system disorder, other	5
Ocular - Cataracts	4
Ocular - Other	1
Oral/Gastrointestinal - Colitis	2
Oral/Gastrointestinal - Diarrhea	1
Oral/Gastrointestinal - Mucositis oral	1
Oral/Gastrointestinal - Nausea/Vomiting	1
Other - Fatigue	1
Other - Pain	5
Other - Treatment related secondary malignancy	7
Other	1
Psychiatric	1
Pulmonary - Acute Respiratory distress syndrome	3
Pulmonary - Respiratory, thoracic and mediastinal disorders, other	9
Renal and Urinary - Creatinine increased	5
Renal and Urinary - Hematuria	2
Reproductive system - Erectile dysfunction	1
Skin - Rash maculo-papular	3

Appendix 4.

Association of Toxicities and Transplantation Characteristics with Outcome Using Univariate Cox Regression

	Risk of Death		Risk of NRM
	HR (95% CI)	p-value	HR (95% CI)	p-value
Toxicity
Cardiovascular	8.6 (3.7-20.3)	<0.001	11.6 (4.4-30.5)	<0.001
Endocrine*	-	-	-	-
Hematologic	6.9 (3.1-15.1)	<0.001	9.5 (3.5-26.3)	<0.001
Hepatic	3.6 (1.5-8.6)	0.003	5.4 (2-14.3)	0.001
Immune System*	-	-	-	-
Infectious	6.4 (3-13.6)	<0.001	11.7 (4.6-29.6)	<0.001
Metabolic	7.2 (3.5-15.2)	<0.001	11.5 (4.7-28.1)	<0.001
Musculoskeletal	1.4 (0.2-10.8)	0.73	2.6 (0.33-20.4)	0.37
Neurologic	9.4 (3.8-23.7)	<0.001	17 (6.3-45.2)	<0.001
Ocular*	-	-	-	-
Oral/Gastrointestinal*	-	-	-	-
Other	1.4 (0.3-6.1)	0.64	2.4 (0.54-10.8)	0.25
Psychiatric*	-	-	-	-
Pulmonary	4.3 (1.5-12.4)	0.008	7.2 (2.4-21.9)	<0.001
Renal and Urinary*	-	-	-	-
Reproductive System*	-	-	-	-
Skin*	-	-	-	-
Transplantation Characteristics
Grade 2-4 GVHD in 1st Year	3.5 (1.7-7.3)	0.001	5.9 (2.4-14.3)	<0.001
Grade 2-4 GVHD in 1st 100 Days	1.2 (0.4-4.0)	0.77	1.8 (0.5-6.3)	0.33
HCT-CI
0	(reference)	0.04	(reference)	0.004
1-2	0.9 (0.3-2.8)		0.9 (0.2-5.6)
≥ 3	2.5 (0.9-6.6)		5.3 (1.2-23)
ALC > 0.5 K/mcl	0.3 (0.1-0.7)	0.004	0.3 (0.1-0.8)	0.15
Ferritin
<1000 ng/ml	(reference)	0.08	(reference)	0.03
1000-2500 ng/ml	2.3 (1-5.1)		3.6 (3.3-10.3)
>2500 ng/ml	2.7 (0.9-8.4)		4.0 (0.99-16)
Albumin > 4.0 g/l	0.4 (0.2-0.9)	0.03	0.4 (0.2-1.11)	0.08
Busulfan
No Change	(reference)	0.94	(reference)	0.96
Dose Decrease	0.7 (0.1-5.6)		1.4 (0.2-12.5)
Dose Increase	0.9 (0.3-2.5)		1.1 (0.3-4.2)
More Than 7 Toxicities in 1st Year	3.2 (1.4-7.2)	0.006	4.8 (1.6-14.4)	0.005
Use of Palifermin	0.7 (0.3-1.4)	0.25	0.6 (0.3-1.5)	0.29
Patient Age	1.0 (0.98-1.1)	0.31	1.01 (0.97-1.1)	0.66
Patient Gender – Male	0.8 (0.4-4.4)	0.56	1.1 (0.5-2.7)	0.78
Disease Group
Acute Leukemia/MDS	(reference)	0.88
Multiple Myeloma	0.9 (0.3-3.1)
Other	1.3 (0.5-3.4)
Matched unrelated donor	1.1 (0.5-2.4)	0.73	1.6 (0.6-4.2)	0.33
TBI-based Conditioning	0.6 (0.2-1.5)	0.25	0.6 (0.2-1.7)	0.32
CMV Positive Donor	1.5 (0.7-3.1)	0.28	1.4 (0.6-3.3)	0.5
CMV Positive Patient	0.8 (0.4-1.6)	0.5	0.8 (0.3-1.9)	0.6

HR indicates hazard ratio; GVHD, graft-versus-host disease; HCT-CI, hematopoietic cell transplantation-comorbidity index; ALC, absolute lymphocyte count; MDS, myelodysplastic syndrome; TBI, total-body irradiation; CMV, cytomegalovirus.

^*Could not be estimated based on the number who developed each toxicity