The presence of busulfan metabolites and pharmacometabolomics in plasma drawn immediately before allograft infusion in hematopoietic cell transplant recipients.

Busulfan is hepatically metabolized through glutathione (GSH) conjugation; in vitro, this process depletes hepatocyte GSH stores and generates the cytotoxic metabolite γ-glutamyldehydroalanylglycine, which is too unstable to be quantitated in vivo. We sought to evaluate if pre-graft (i.e., immediately before allograft infusion) concentrations of busulfan metabolites' and of endogenous metabolomic compounds (EMCs) representing the glutathione pathway were associated with clinical outcomes in hematopoietic cell transplant (HCT) recipients receiving busulfan. The clinical outcomes evaluated were relapse, acute graft versus host disease (GVHD), chronic GVHD, non-relapse mortality, and neutrophil nadir. In pre-graft samples obtained from patients immediately before allograft infusion, our objectives were to evaluate for: (1) the presence of busulfan and its metabolites tetrahydrothiophenium ion (THT+), tetrahydrothiophene 1-oxide, sulfolane, and 3-hydroxysulfolane (N = 124); (2) EMCs using a global metabolomics assay (N = 77); and (3) the association of the busulfan metabolites and the EMCs with clinical outcomes. In the pre-graft samples, busulfan and THT+ could not be detected. THT 1-oxide, sulfolane, and 3-hydroxysulfolane were quantitated in 9.6%, 26%, and 58% of pre-graft samples; their concentrations were not associated with clinical outcomes. Four pre-graft EMCs were statistically significantly associated with the neutrophil nadir. The pre-graft EMCs were not associated with the other clinical outcomes. In conclusion, busulfan's metabolites are present in patients' plasma immediately before allograft infusion; the neutrophil nadir is associated with pre-graft EMCs. Future research should investigate the association of clinical outcomes with the concentrations of busulfan's metabolites and EMCs in the pre-graft plasma from allogeneic HCT recipients.

Prediction of Busulfan Clearance by Predose Plasma Metabolomic Profiling.

Intravenous busulfan doses are often personalized to a target plasma exposure (targeted busulfan) using an individual's busulfan clearance (BuCL). We evaluated whether BuCL could be predicted by a predose plasma panel of 841 endogenous metabolomic compounds (EMCs). In this prospective cohort of 132 hematopoietic cell transplantation (HCT) patients, all had samples collected immediately before busulfan administration (preBU) and 96 had samples collected 2 weeks before busulfan (2-week-preBU). BuCL was significantly associated with 37 EMCs after univariate linear regression analysis and controlling for false discovery (< 0.05) in the 132 preBU samples. In parallel, with preBU samples, we included all 841 EMCs in a least absolute shrinkage and selection operator-penalized regression which selected 13 EMCs as predominantly associated with BuCL. Then, we constructed a prediction model by estimating coefficients for these 13 EMCs, along with sex, using ordinary least-squares. When the resulting linear prediction model was applied to the 2-week-preBU samples, it explained 40% of the variation in BuCL (adjusted R2  = 0.40). Pathway enrichment analysis revealed 18 pathways associated with BuCL. Lysine degradation followed by steroid biosynthesis, which aligned with the univariate analysis, were the top two pathways. BuCL can be predicted before busulfan administration with a linear regression model of 13 EMCs. This pharmacometabolomics method should be prioritized over use of a busulfan test dose or pharmacogenomics to guide busulfan dosing. These results highlight the potential of pharmacometabolomics as a precision medicine tool to improve or replace pharmacokinetics to personalize busulfan doses.

Predictive Value of Clinical Findings and Plasma Biomarkers after Fourteen Days of Prednisone Treatment for Acute Graft-versus-host Disease.

We examined the hypothesis that plasma biomarkers and concomitant clinical findings after initial glucocorticoid therapy can accurately predict failure of graft-versus-host-disease (GVHD) treatment and mortality. We analyzed plasma samples and clinical data in 165 patients after 14 days of glucocorticoid therapy and used logistic regression and areas under receiver-operating characteristic curves (AUC) to evaluate associations with treatment failure and nonrelapse mortality (NRM). Initial treatment of GVHD was unsuccessful in 49 patients (30%). For predicting GVHD treatment failure, the best clinical combination (total serum bilirubin and skin GVHD stage: AUC, .70) was competitive with the best biomarker combination (T cell immunoglobulin and mucin domain 3 [TIM3] and [interleukin 1 receptor family encoded by the IL1RL1 gene, ST2]: AUC, .73). The combination of clinical features and biomarker results offered only a slight improvement (AUC, .75). For predicting NRM at 1 year, the best clinical predictor (total serum bilirubin: AUC, .81) was competitive with the best biomarker combination (TIM3 and soluble tumor necrosis factor receptor-1 [sTNFR1]: AUC, .85). The combination offered no improvement (AUC, .85). Infection was the proximate cause of death in virtually all patients. We conclude that after 14 days of glucocorticoid therapy, clinical findings (serum bilirubin, skin GVHD) and plasma biomarkers (TIM3, ST2, sTNFR1) can predict failure of GVHD treatment and NRM. These biomarkers reflect counter-regulatory mechanisms and provide insight into the pathophysiology of GVHD reactions after glucocorticoid treatment. The best predictive models, however, exhibit inadequate positive predictive values for identifying high-risk GVHD cohorts for investigational trials, as only a minority of patients with high-risk GVHD would be identified and most patients would be falsely predicted to have adverse outcomes.

Impact of donor age on outcome after allogeneic hematopoietic cell transplantation.

As older patients are eligible for allogeneic hematopoietic cell transplantation (HCT), older siblings are increasingly proposed as donors. We studied the impact of donor age on the tempo of hematopoietic engraftment and donor chimerism, acute and chronic graft-versus-host disease (GVHD), and nonrelapse mortality (NRM) among 1174 consecutive patients undergoing myeloablative and 367 patients undergoing nonmyeloablative HCT from HLA-matched related or unrelated donors with granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell allografts. Sustained engraftment rates were 97% and 98% in patients undergoing myeloablative and nonmyeloablative conditioning, respectively, for grafts from donors < 60 years old (younger; n = 1416) and 98% and 100%, respectively, for those from donors ≥ 60 years old (older; n = 125). No significant differences were seen in the tempo of neutrophil and platelet recoveries and donor chimerism except for an average 1.3-day delay in neutrophil recovery among myeloablative patients with older donors (P = .04). CD34(+) cell dose had an independent effect on the tempo of engraftment. Aged stem cells did not convey an increased risk of donor-derived clonal disorders after HCT. Myeloablative and nonmyeloablative recipients with older sibling donors had significantly less grade II to IV acute GVHD than recipients with grafts from younger unrelated donors. Rates of grade III and IV acute GVHD, chronic GVHD, and NRM for recipients with older donors were not significantly different from recipients with younger donors. In conclusion, grafts from donors ≥ 60 years old do not adversely affect outcomes of allogeneic HCT compared with grafts from younger donors.

Cyclophosphamide following targeted oral busulfan as conditioning for hematopoietic cell transplantation: pharmacokinetics, liver toxicity, and mortality.

The pharmacokinetics of cyclophosphamide (CY) and its metabolites hydroxycyclophosphamide and carboxyethylphosphoramide mustard were determined in 75 patients receiving targeted oral busulfan followed by i.v. CY ((T)BU/CY) and in 147 patients receiving i.v. CY followed by total body irradiation (CY/TBI) in preparation for hematopoietic cell transplantation (HCT). In the (T)BU/CY patients only, the association of the pharmacokinetic data with liver toxicity, relapse, and survival was evaluated. CY was infused at 60 mg/kg/day over 1 or 2 hours on 2 consecutive days; the majority of patients had BU levels targeted to a steady state plasma concentration (Css) of 800-900 ng/mL. Systemic exposure (i.e., area under the concentration-time curve [AUC]) of CY, hydroxycyclophosphamide, and carboxyethylphosphoramide mustard was measured. Liver toxicity was assessed as the development of hepatic sinusoidal obstruction syndrome (SOS). CY metabolism was highly variable and age dependent. (T)BU/CY-treated patients had lower AUC(CY) (P < .0001), higher AUC(HCY) (P < .0001), and higher AUC(CEPM) (P = .15) than CY/TBI-conditioned patients. Among patients receiving (T)BU/CY, 17 (23%) developed SOS, and there were no statistically significant associations between the AUC of CY or its metabolites and SOS, nonrelapse mortality, relapse, or survival (all P >.15). In conclusion, CY exhibits conditioning-regimen dependent pharmacokinetics and pharmacodynamics, suggesting that lowering CY doses is unlikely to improve outcomes to (T)BU/CY. Alternative strategies, such as administering i.v. busulfan or CY before BU, should be explored.

Gastrointestinal graft-versus-host disease in recipients of autologous hematopoietic stem cells: incidence, risk factors, and outcome.

Graft-versus-host disease (GVHD) is seen in skin, intestinal mucosa, and liver after autologous stem cell transplantation. We reviewed 681 consecutive patients to estimate the probability of gastrointestinal (GI) GVHD, response to treatment, risk factors for development, and effect on survival. GI GVHD was defined by persistent symptoms, mucosal abnormalities at endoscopy, and histology showing apoptotic crypt cells with or without lymphoid infiltrates. The proportion of patients with GI GVHD was 90/681 (13%). Nausea and vomiting occurred in 90% and diarrhea in 40%. The mean time to developing symptoms was day +15, that to histologically proven diagnosis was day +42, and that to starting prednisone treatment was day +45 after stem cell infusion. Treatment with a short course of prednisone effected durable responses in 79% of patients, and an additional 18% responded to a second course of prednisone. A multivariable logistic regression model demonstrated that the combined factor of a diagnosis of breast cancer or hematologic malignancy and female sex was statistically significantly associated with the probability of GI GVHD (P = .003). Survival in patients with GI GVHD was not statistically different than that in those without GVHD. We conclude that women with breast cancer or hematologic malignancy are more likely to develop GI GVHD after autologous transplantation, and that treatment with prednisone was effective.

Serum bilirubin levels and mortality after myeloablative allogeneic hematopoietic cell transplantation.

Many patients who undergo hematopoietic cell transplantation experience liver injury. We examined the association of serum bilirubin levels with nonrelapse mortality by day +200, testing the hypothesis that the duration of jaundice up to a given point in time provides more prognostic information than either the maximum bilirubin value or the value at that point in time. We studied 1,419 consecutive patients transplanted from allogeneic donors. Total serum bilirubin values up to day +100, death, or relapse were retrieved-along with nonrelapse mortality by day +200 as an outcome measure--using Cox regression models with each bilirubin measure modeled as a time-dependent covariate. The bilirubin value at a particular point in time provided the best fit to the model for mortality. With bilirubin at a point in time modeled as an 8th-degree polynomial, an increase in bilirubin from 1 to 3 mg/dL is associated with a mortality hazard ratio of 6.42. An increase from 4 to 6 mg/dL yields a hazard ratio of 2.05, and an increase from 10 to 12 mg/dL yields a hazard ratio of 1.17. Among patients who were deeply jaundiced, survival was related to the absence of multiorgan failure and to higher platelet counts. In conclusion, the value of total serum bilirubin at a particular point in time after transplant carries more informative prognostic information than does the maximum or average value up to that point in time. The increase in mortality for a given increase in bilirubin value is larger when the starting value is lower.

Hepatic injury after nonmyeloablative conditioning followed by allogeneic hematopoietic cell transplantation: a study of 193 patients.

Liver injury is a frequent, serious complication of allogeneic hematopoietic cell transplantation (HCT) following myeloablative preparative regimens. We sought to determine the frequency and severity of hepatic injury after nonmyeloablative conditioning and its relationship to outcomes. One hundred ninety-three consecutive patients who received 2 Gy total body irradiation with or without fludarabine were evaluated for end points related to liver injury. Patients with diseases treatable by HCT who were ineligible for conventional myeloablative allogeneic HCT because of advanced age and/or comorbid conditions were included. Fifty-one patients (26%) developed hyperbilirubinemia of 68.4 microM (4 mg/dL) or greater, most commonly resulting from cholestasis due to graft-versus-host disease (GVHD) or sepsis. Pretransplantation factors associated with liver dysfunction were a diagnosis of aggressive malignancy (hazard ratio [HR] 1.9; P =.04) and the inclusion of fludarabine in the conditioning regimen (HR 1.8; P =.07). Overall survival at 1 year was superior for patients who had maximal serum bilirubin levels in the normal (78%) or minimally elevated (22.23-66.69 microM [1.3-3.9 mg/dL]) ranges (69%) compared with those in the 68.4 to 117.99 microM (4-6.9 mg/dL; 20%), 119.7 to 169.29 microM (7.0-9.9 mg/dL; 17%), and 171.0 microM (10 mg/dL; 19%) or greater groups. In summary, significant jaundice occurred in 26% of patients and was predominantly due to cholestasis resulting from GVHD and/or sepsis. Aggressive malignancies (mainly advanced disease) and later development of jaundice after transplantation predicted inferior survival.

Cyclophosphamide metabolism, liver toxicity, and mortality following hematopoietic stem cell transplantation.

Liver toxicity caused by high-dose myeloablative therapy leads to significant morbidity after hematopoietic cell transplantation. We examined the hypothesis that liver toxicity after cyclophosphamide and total body irradiation is related to cyclophosphamide through its metabolism to toxins. Cyclophosphamide was infused at 60 mg/kg over 1 to 2 hours on each of 2 consecutive days, followed by total body irradiation. Plasma was analyzed for cyclophosphamide and its major metabolites. Liver toxicity was scored by the development of sinusoidal obstruction syndrome (veno-occlusive disease) and by total serum bilirubin levels. The hazards of liver toxicity, nonrelapse mortality, tumor relapse, and survival were calculated using regression analysis that included exposure to cyclophosphamide metabolites (as the area under the curve). Of 147 patients, 23 (16%) developed moderate or severe sinusoidal obstruction syndrome. The median peak serum bilirubin level through day 20 was 2.6 mg/dL (range, 0.5-41.1 mg/dL). Metabolism of cyclophosphamide was highly variable, particularly for the metabolite o-carboxyethyl-phosphoramide mustard, whose area under the curve varied 16-fold. Exposure to this metabolite was statistically significantly related to sinusoidal obstruction syndrome, bilirubin elevation, nonrelapse mortality, and survival, after adjusting for age and irradiation dose. Patients in the highest quartile of o-carboxyethyl-phosphoramide mustard exposure had a 5.9-fold higher risk for nonrelapse mortality than did patients in the lowest quartile. Engraftment and tumor relapse were not statistically significantly related to cyclophosphamide metabolite exposure. Increased exposure to toxic metabolites of cyclophosphamide leads to increased liver toxicity and nonrelapse mortality and lower overall survival after hematopoietic cell transplantation.