Hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation: donor type matters

Hemorrhagic cystitis (HC) is a regularly observed problem after blood and marrow transplantation, frequently causing prolongation of hospitalization and occasionally death.¹  HC has a spectrum of manifestations that range from microscopic hematuria to severe hemorrhage with obstructive renal failure. Discrepancies in definition criteria are partially responsible for the wide range of reported incidence of this complication, from less than 10% to more than 60%.^2,3  HC is graded as mild, moderate, or severe according to degree of pain and amount of hematuria. Although mild forms usually resolve with supportive treatment, severe HC may require antiviral therapy such as vidarabine, hyperbaric oxygen treatment, amifostine, factor XIII, bladder irrigation with intravesicular instillation of E-aminocaproic acid, methyl prednisolone or formalin, cystoscopy and cauterization, and even cystectomy.^4-12 

In general, alkylating agents such as cyclophosphamide (CTX), radiation therapy, or adenovirus and BK polyoma virus infection have been implicated in the etiology of HC.^13-24  However, previous studies evaluating risk factors for HC have been carried out on either small numbers of patients or in heterogeneous populations including different preparative regimens.^1,16,20,21  It is largely unknown if hematopoietic stem cell grafts usually associated with delayed immune recovery and increased need of immunosuppressants have a higher incidence of HC.

We postulated that recipients of matched unrelated donor (MUD) or umbilical cord blood grafts have a higher incidence of HC and that there is a relationship between degree of immunosuppression and development of HC. To investigate this hypothesis, we limited our analysis to acute lymphocytic leukemia (ALL) patients who had 12 Gy total body irradiation (TBI)-based regimens followed by MUD, matched or one-antigen mismatched related donor (MRD or MM rel), or unrelated cord blood (UCB) transplantation.

Subjects were eligible for this study if they were diagnosed with ALL and had a hematopoietic stem cell transplant (HSCT) with a 12 Gy TBI-based regimen in our institution from 1990 to 2001. Patients were not to have a prior transplant.

Inclusion criteria was fulfilled by 105 patients treated with Institutional Review Board (IRB)-approved protocols. IRB approval for this retrospective chart review was obtained according to institutional guidelines. Data on patient, donor, and disease characteristics; conditioning regimen; graft-versus-host disease (GVHD) prophylaxis; and outcomes of transplantation were collected.

Our sources of information included patients' charts and our Department of Blood and Marrow Transplantation prospectively updated electronic database. HC that occurred within 12 months after transplantation was included in this study. All cultures and urinalysis were reviewed.

Detailed patient characteristics are listed in Table 1. From 1990 to 2001, a total of 105 patients (74 males and 31 females) with ALL underwent allogeneic HSCT at M D Anderson Cancer Center. Median age was 25 years (range, 4-56 years). Stem cell donors were siblings in 52 cases, of whom 20 were human leukocyte antigen (HLA) mismatched, unrelated volunteers in 38 cases, and unrelated umbilical cord in 15 cases. Stem cell grafts were bone marrow in 70 patients, granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood in 20, and cord blood in 15 patients. Twenty-nine patients were in first remission with high-risk cytogenetics, 28 patients were in second complete remission (CR), while 15 patients were in third CR. Six patients were primary induction failures, 12 were in first relapse, and 15 in second or subsequent relapses.

Conditioning therapies used were all TBI-based (4 daily fractions of 3 Gy for a total of 12 Gy). Eighty-six patients (82%) received cyclophosphamide 120 mg/kg either with thiotepa (n = 53) 5 to 10 mg/kg or etoposide (VP16) (n = 19) 750 to 1500 mg/m². Nineteen patients (18%) received noncyclophosphamide regimens, consisting of fludarabine 90 mg/m² with (n = 16) or without (n = 3) melphalan 140 mg/m². Anti-thymocyte globulin was incorporated in the regimen in 28 cases.

GVHD prophylaxis consisted of cyclosporine (CsA) and methotrexate, in 9 cases. Tacrolimus was substituted for CsA, in combination with methotrexate, in 60 patients. GVHD prophylaxis regimens are presented in Table 1.

Patients who received additional immunosuppression besides cyclosporine or tacrolimus-based regimens were considered to have received “intense” immunosuppression (n = 35). Thirteen patients received CsA, methotrexate, methylprednisolone, and xomazyme, an anti-CD5 antibody; 4 patients received T-cell-purged bone marrow with methyl prednisolone; and 2 patients received cyclosporine, methotrexate, and methylprednisolone. Patients receiving horse-derived anti-thymocyte globulin (ATG) with the conditioning regimen were included in this group (n = 28). ATG was given in a dose of 15 to 30 mg/kg for 2 or 3 days. Overall, in the “intense” immunosuppression group, 27 patients received CsA-based regimens, 6 received tacrolimus-based combinations, and 2 received other immunosuppressants.

Patients received intravenous fluids at approximately 1.5 to 2 L/m²/24 hours during the preparative regimen. In addition, subjects treated with cyclophosphamide were given sodium 2-mercaptoethane sulfonate (MESNA) 10 mg/kg intravenously 1 hour prior to cyclophosphamide administration, with repeated dosing every 4 hours for 12 total doses. Anti-emetics and other supportive measures were used according to institutional practice. All subjects received filgrastim (G-CSF, Neupogen; Amgen, Thousand Oaks, CA) subcutaneously daily from day 0 or day +7 until achievement of an absolute neutrophil count (ANC) of more than 1.5 × 10⁹/L for 3 days. Packed red blood cells were administered to maintain hemoglobin levels greater or equal to 8 gm/dL. Platelet transfusions were administered to keep the platelet count at a level greater or equal to 10-20 × 10⁹/L. All blood products were filtered and irradiated. Patients who developed grade 2 or greater acute GVHD received methylprednisolone 2 mg/kg/d.

Donor hematopoietic stem cells (HSCs) were procured using standard mobilization or harvesting protocols and pheresis techniques. HSCs were processed according to current institutional guidelines and protocols. All healthy donors signed written informed consent for the procedure. Bone marrow or peripheral blood stem cells procured from unrelated donors were obtained through the National Marrow Donor Program (NMDP).

HLA typing for class I antigens was performed using standard serological techniques. Low-resolution molecular typing using hybridization techniques of amplified DNA with sequence-specific oligonucleotide (SSO) probes, followed by high-resolution molecular typing using polymerase chain reaction (PCR) in the sampled DNA with sequence specific primers (SSPs) was performed for class II alleles (HLA-DRB1,-DQB1) in all patients, except those who received transplants before October 1992. Serological methods were used for both class I and class II antigens for donor-recipient pairs if the transplantation occurred before October 1992. Due to the heterogeneity of methods used for HLA matching during the study period, for the purpose of this analysis we considered complete HLA matches, donor-recipient pairs who matched for HLA-A,-B by serology, and for -DRB1 by molecular typing (6 of 6 matches).

HC was defined as the presence of sustained macroscopic hematuria from the day of transplantation in the absence of other conditions such as gynecologic-related bleeding, disseminated intravascular coagulation, multiple organ dysfunction, or sepsis.

HC was graded according to the following criteria: grade 0 = no HC, grade I = microscopic hematuria, grade II = macroscopic hematuria, grade III = macroscopic hematuria with clots, and grade IV = macroscopic hematuria requiring instrumentation for clot evacuation and/or causing urinary retention. In patients with intermittent or recurrent symptoms, the date of onset of HC was defined as the first day of symptoms or laboratory evidence appearing after transplant. The worst clinical presentation was considered as the maximum grading.

Day 0 was the stem cell infusion day. Engraftment was defined as the first of 3 consecutive days that the ANC exceeded 0.5 × 10⁹/L. Platelet engraftment was defined as the first of 7 consecutive days that the platelet count exceeded 20 × 10⁹/L without transfusion support. Failure to reach an ANC of 0.5 × 10⁹/L by HSCT day +30 characterized primary engraftment failure. Diagnosis of acute and chronic GVHD was based on standard clinical criteria with histopathological confirmation where possible.

The incidence of grade II or greater HC within the first year after transplantation was estimated using the cumulative incidence method.²⁵  This method accounts for death or stem cell re-infusion following graft failure as competing risks. Cox proportional hazards model was used to evaluate risk factors for the development of HC on univariate and multivariate analysis. It also was used to assess if the occurrence of HC increases the mortality rate. For this purpose HC was evaluated as a time-dependent variable. Risk factors considered include patient's age (as quartiles), donor's age (as quartiles), graft type (MRD, MM rel, MUD, and UCB), disease status at transplant (CR versus other), use of cyclophosphamide in the conditioning regimen, inclusion of VP16 in the preparative regimen, cell type (bone marrow versus peripheral blood stem cell [PBSC]), intense GVHD prophylaxis (defined as the addition of any immunosuppressive agent to the standard CsA), or tacrolimus-based GVHD prophylaxis and/or the use of ATG in the preparative regimen, platelet recovery, occurrence of grades II-IV acute GVHD, and the use of steroids after transplantation. The last 3 factors were evaluated as time-dependent variables. Patients who died within 30 days after transplantation without documentation of platelet recovery were excluded for the evaluation of platelet recovery on the occurrence of HC. Patients who received a second infusion of donor cells for the treatment of engraftment failure had the follow-up time censored at the time of the second infusion.

Factors that were significant at the 0.1 level on univariate analysis or were considered to be clinically significant were evaluated in a multivariate model with allotype as a constant variable. Statistical significance was determined at the .05 level. All P values were 2 sided. Actuarial survival from day of transplantation was estimated by the method of Kaplan-Meier. Statistical analysis was performed using STATA 7.0 (Stata, College Station, TX).

Ninety-two percent (n = 97) of patients engrafted with a median time to ANC exceeding 0.5 × 10⁹/L of 15 days (range, 9-40 days). Three patients had primary graft failure; one died at day 48 after transplantation and 2 received autologous stem cells infusions at days 31 and 37 after transplantation, respectively. Five patients died within 30 days after transplantation before ANC recovery. Seventy-eight percent (n = 82) of patients achieved a platelet count higher than 20 × 10⁹/L at a median of 22 days (range, 4-163 days) (Table 2). Eleven patients died within 30 days after transplantation before achievement of platelet transfusion independence, and 2 patients had secondary graft failure and received a second allogeneic infusion at 35 and 38 days after transplantation, respectively. Median time to a platelet count higher than 100 × 10⁹/L was 32 days (range, 13-304 days, n = 47).

With a median follow-up of 17 months, 28 patients are alive (27%). Twenty-nine patients died within the first 100 days after transplantation and 48 patients within the first year, without a diagnosis of HC. The most common causes of death were disease progression (n = 24), GVHD (n = 17), and infection (n = 14). Other less frequent causes were bronchiolitis obliterans, multiorgan failure, vascular occlusive disease, and adult respiratory distress syndrome. One-year Kaplan Meier estimate of survival was 33% (95% CI 24-42). For patients older than 25 years it was 29% (95% CI 16-43), and for those subjects younger than 26 years it was 30% (95% CI 17-45), log rank P value = .9. Similarly, there was no significant difference in 100-day mortality between the 2 subgroups (age < 26 years: 41% [95% CI 28-57]; age > 25 years: 38% [95% CI 25-53]).

Twenty-two of 32 patients who were diagnosed with HC died at a median of 78 days (range, 21-574 days) from the date of onset. Causes of death included infection (n = 7), GVHD (n = 7), progressive disease (n = 5), engraftment failure (n = 1), acute renal failure (n = 1), and adult respiratory distress syndrome (n = 1). However, development of HC, evaluated as a time-dependent variable, was not associated with an increased mortality rate in our population, even when cases with intensity grade III-IV were evaluated.

Thirty-two patients developed HC within a year after transplantation with a cumulative incidence of 30% (SE 5%). The median time to onset of HC was 23 days (range, 1-165 days) with only 1 case presenting after day 100. In 20 patients, the onset of HC was within 28 days after transplantation and in 12 cases, it was later than day 28. The median duration of HC was 30 days (range, 3-167 days). Grade II HC occurred in 18 patients, and grades III and IV in 10 and 4 cases, respectively; 3 of the grade IV cases occurred among recipients of UCB.

Generally, patients were not tested for polyoma or adenovirus in the urine except after developing symptoms of HC; thus, we can only report the prevalence of these viruses in that population. Presumptive diagnosis of polyoma virus infection was made using urinary cytologic changes, since we did not perform PCR tests. Fifty-nine percent (n = 13) of tested patients with HC had urinary cytologic changes compatible with polyoma virus infection (test not done in 10 cases), while 17% (n = 4) were positive for adenovirus by urine culture (test not done in 8 cases). Cytomegalo-virus (CMV) reactivation detected by blood culture or antigenemia occurred in 63% (n = 20) of patients developing HC. In the whole cohort, 67% (n = 66) had evidence of CMV reactivation, 5% (n = 5) had positive adenovirus urine culture, and 15% (n = 16) had cytologic changes compatible with polyoma virus urinary tract infection. All cases of documented adenovirus infection occurred among patients younger than 25, while presumed infection with polyomavirus occurred in similar proportion among older and younger patients.

Patients' characteristics and their association with HC are presented in Table 3. Subjects who received cord blood grafts were not included in the evaluation of risk factors for HC because of the small sample size and different kinetics of engraftment. On univariate analysis, the most significant risk factors associated with HC were donor type and patient's age.

Patients who received a graft from a related donor had the lowest rate of HC, followed by patients with MM rel donor (HR 2.0, 95% CI 0.6-6.7), cord blood grafts (HR 2.6, 95% CI 0.8-8.5), and MUD (HR = 2.9, P = .04). Recipients of UCB transplants developed HC in 40% of the cases. All cases in the matched related group occurred within the first month after transplantation (Figure 1).

Fifty-three percent of the patients in the MM rel and MUD groups received “intense” GVHD prophylaxis. HC rate was higher among these patients compared to those receiving standard prophylaxis (HR = 1.7, 95% CI 0.7-4.1). This effect did not reach statistical significance, possibly due to a small sample size. However, when we restricted the comparison of HC rate according to donor type to the group of patients who received standard GVHD prophylaxis, we still observed a significantly higher rate among the MM rel and MUD groups compared with the MRD group (HR = 2.6 (95% CI 1.0-6.9).

There was a trend for lower rate of HC for patients with active disease at the time of transplant (HR 0.4 [95% CI 0.1-1.1]). This effect was seen mostly in the MUD group. Platelet recovery, patient's gender, donor's age, preparative regimen, and cell type (BM [bone marrow] vs PBSC) were not associated with the risk of HC in this study.

A patient's age was significantly associated with the rate of HC, with a higher rate among patients younger than 26 (Figure 2). Eighteen of 45 patients (40%) who were 25 years or younger developed HC, while only 8 of 45 patients older than 25 years developed HC (HR = 2.5 [95% CI 1.1-5.8], P = .03). This effect was consistent within graft type groups.

Cyclophosphamide was part of the conditioning regimen in 82% of the cases (n = 86). The incidence of HC in this subgroup was 27% (n = 24), while among 19 patients who did not receive the alkylator, the incidence of HC was 42% (n = 8). In the MRD group, HC occurred in 2 of 4 patients who did not receive cyclophosphamide and in 11% of the 28 patients receiving it. All MM rel patients received cyclophosphamide (HC incidence of 30%), while only one patient in the MUD group did not receive it. Among recipients of UCB transplants, only one patient received cyclophosphamide (HC incidence of 43% among 14 patients treated without the alkylating agent).

Among 70 patients who received MUD or MM rel grafts, use of cyclophosphamide in the preparative regimen was not significantly associated with the development of HC (HR 0.7 [95% CI 0.16-2.95]; P = .6). Furthermore, we evaluated the effect of donor type (MM rel and MUD versus MRD) among patients who received cyclophosphamide in the preparative regimen, and the donor type effect remained significant (HR 2.4 [95% CI 1.06-5.6]; P = .035).

Graft type (MUD) and patient's age were evaluated in a Cox proportional hazards model to adjust for potential confounding effects (Table 4). Results were similar to those obtained in the univariate analysis with the exception that the effect of graft type became marginally significant (HR 2 95% CI 0.9-4.5, P = .07), while younger age remained significant (HR 2.5, 95% CI 1.1-5.7, P = .03). Although not reaching statistical significance, these results are still consistent with a clinically significant higher risk of development of HC among recipients of MUD transplants.

Our study was designed to minimize the influence of 2 major biases common to the majority of studies that aimed to describe the incidence and risk factors involved in the development of HC: heterogeneity of diagnosis and preparative regimens. For that purpose, we limited our analysis to patients with ALL treated with 12 Gy TBI-based regimens. That enabled us to define the role of donor type in this context. We showed that recipients of unrelated donor HSCTs have a higher incidence of HC when compared to recipients of matched related transplants. The cumulative incidence was 16%, 30%, 40%, and 40% among matched related donor, mismatched related, unrelated donor HSCT, and UCB transplant recipients, respectively.

HC is a major complication in patients undergoing HSCT. Early occurring HC is frequently ascribed to the preparative regimen (such as those including cyclophosphamide), while those that occur later after transplantation are more often credited to viral infections such as adenovirus.^20-24,26-32  The cumulative incidence of HC was comparable during the first month after transplant within all donor type groups. After the first 30 days, it remained stable for recipients of matched related donor transplants but increased significantly among the other groups up to transplantation day 100. There were no cases among HLA-identical sibling transplant recipients later than 30 days after treatment. Therefore, the statistically significant difference in the rates of HC is due to bleeding occurring after the first month, a period of time when the influence of preparative regimen-related factors is thought to be decreased.

Among 1402 transplants performed at Johns Hopkins, the incidence of bleeding episodes after unrelated donor transplantations was 62.5%, while allogeneic and autologous transplantations in general had incidences of 31% and 18.5%, respectively. In that series, severe HC occurred in 6.4% of the MUD recipients.³³  Delayed platelet engraftment is an obvious cause of increased risk of bleeding, usually associated with transplants from donors other than HLA-identical siblings. In our group of poor prognosis patients with ALL, platelet engraftment analyzed as a time-dependent variable had no correlation with development of HC. Subjects with HC were similarly distributed between patients who had achieved platelet transfusion independence or were dependent on transfusions at the time of bleeding.

A possible interaction between degree of pharmacologic immunosuppression, donor type, and development of HC may exist. There was a trend toward higher incidence of HC among MM rel and MUD patients receiving more intense immunosuppression compared to those receiving “standard” GVHD prophylaxis, but this effect did not reach statistical significance (HR = 1.7, 95% CI 0.7-4.1). When we restricted the comparison of HC incidence according to donor type to patients who received standard GVHD prophylaxis, we still observed a higher rate of HC among the MM rel and MUD groups compared with the MRD group (HR = 2.6 [95% CI 1.0-6.9]). This suggests that “intense” GVHD prophylaxis may contribute to the increased risk among the MM rel and MUD groups; however, it does not totally account for the graft type effect.

It has been suggested that a correlation between acute or chronic GVHD and HC might exist or that immunosuppressive therapies used to treat GVHD increase the chance of opportunistic infections that subsequently cause HC.^34-38  Nevertheless, Sencer et al²¹  did not find GVHD to be significantly associated with HC in their large series. Similarly, Bedi et al³⁸  failed to establish such a correlation.³⁸  Here, development of grades II-IV acute GVHD was not associated with HC.

Younger age was independently associated with HC in our series, even after adjusting for the donor type-confounding factor. Subjects younger than 26 had a cumulative incidence of HC of 40% versus 17% among those older than 25. This is a somewhat counterintuitive result, however. Young age has been inconsistently described as a risk factor for ifosfamide-induced nephrotoxicity, while studies showing a higher incidence of HC among older patients can be found in the literature.^{1,20,21,39-42}  Age-related prevalence of latent viral infections might provide a possible explanation. Dei et al⁴³  have reported that prevalence rates of BK polyomavirus infection increase from birth until the age of 12. Latent infection with adenovirus is lower in younger children, and Kondo et al⁴²  suggested that this could justify a lower incidence of late-onset HC in younger children. Conversely, Bogdanovic et al detected human polyomaviruses in urine and blood samples of allogeneic and autologous bone marrow transplantation patients with and without HC.⁴⁴  Recently, Leung et al³¹  conducted a study on 50 BMT patients and found that patients with HC had significantly higher peak BK viruria (6 × 10¹² versus 5.7 × 10⁷ genome copies/d) and larger total amounts of virus excreted during BMT. BK viruria was the only risk factor for HC. They found no correlation with age, conditioning regimen, type of BMT, and development of GVHD. Our study is limited by the lack of prospective, universal investigation of viral infection, and no conclusions can be drawn regarding the role of BK or adenovirus infection.

We confirmed our hypothesis that sources of hematopoietic stem cells other than HLA-identical siblings are associated with an excessive incidence of HC. Risk of early HC (< 28 days) was similar for all subgroups and possibly determined mainly by preparative regimen toxicity. Late-onset cases were responsible for the increased rate documented in this analysis. It is possible that intrinsic and pharmacologic-induced immunosuppression predis-posed to the development or reactivation of HC causing latent virus infection.

Prospective monitoring of BK and adenovirus viruria in parallel with evaluations of immune reconstitution after transplant are likely to further contribute to the understanding of this complication. Patients receiving unrelated bone marrow or cord blood transplants are at high risk of developing HC, and early monitoring should be implemented.