Although late-onset hemorrhagic cystitis (LOHC) is a common complication following allogeneic hematopoietic stem cell transplantation (HSCT), its pathogenesis remains to be defined.1-3 Most studies have reported that viral infection is significantly related to the pathogenesis of LOHC;4 however, in clinical practice, we found that the clearance of viremia or viruria did not necessarily lead to symptom relief of LOHC, and in some patients no evidence of a specific pathogen were found. As LOHC primarily occurs after allogeneic HSCT, researchers suspected that alloimmune injury might also be involved in the pathogenesis of LOHC. However, as a biopsy is not feasible in most situations and the pathological manifestations were non-specific, it is difficult to obtain objective evidence of alloimmune- related HC. To our knowledge, essentially no study has evaluated the role of immunosuppressants in the treatment of HC. In the present study, we retrospectively analyzed 11 patients with LOHC who responded to immune suppression treatment rather than anti-viral therapy.
A total of 250 allogeneic HSCTs were carried out at Peking University Institute of Hematology from April 2003 to November 2005. All patients had hematologic disorders. All patients provided written informed consent in accordance with the requirements of the institutional review board. Hemorrhagic cystitis (HC) occurred in 72 of the 250 patients within 180 days after transplantation. A total of 11 patients who were diagnosed with immune-related LOHC post allo-HSCT by retrospective analysis were included; among them, 7 patients with chronic myelogenous leukemia (CML) and 4 patients with acute lymphoblastic leukemia (ALL) were included in this study. Two patients underwent unrelated HLA-matched HSCT, while the other 9 underwent related HLA-mismatched HSCT. There were 7 male and 4 female patients with a median age of 32.5 years. Urine routine and renal function were normal before HSCT.
Conditioning regimen and graft-versus-host disease prophylaxis
Conditioning therapy was all BUCY-based (busulfan 4 mg∙kg-1∙d-1 administered orally for 3 days and 1.8 g∙m-2∙d-1 cyclophosphamide intravenously for 2 days); additional therapies were one simustine (Me-CCNU) 250 mg/m2 oral dose for all patients and cytarabine 2 g∙m-2∙d-1 intravenously for one day for patients with a matched sibling. For HLA-mismatched patients and unrelated- matched HCT patients, cytarabine was given at 4 g∙m-2∙d-1 for two days and ATG (thymoglobuline; SangStat, Lyon, France) 2.5 mg∙m-2∙d-1 intravenously for 4 consecutive days. Prophylaxis against graft-versus-host disease (GVHD) consisted of cyclosporine (CSA), a short course of methotrexate (MTX), and mycophenolate mofetil (MMF).5,6 Donors were primed with granulocyte-colony stimulating factor (G-CSF, Filgrastim, Kirin, Japan; 5 µg∙kg-1∙d-1) injected subcutaneously for 5–6 consecutive days. Bone marrow cells were harvested on the fourth day. The target mononuclear cell count (MNC) was 3×108–4×108 cells/kg recipient weight. On the fifth and sixth days, peripheral blood stem cells (PBSCs) were collected with a COBE Blood Cell Separator (Spectra LRS, COBE BCT, Inc., Lakewood, Colorado, USA) at a rate of 80 ml/min from a total blood volume of 10 L. In instances of ABO major blood group incompatibility, red cells were removed from bone marrow cells by density gradient sedimentation with Hespan according to the manufacturer's instructions (B. Braun Medical Inc, Irvine, CA, USA).
Evaluation of engraftment and chimerism
Follow-up assessment of engraftment and chimerism was done according to the methods as described previously.5,6
HC was defined as the presence of sustained microscopic or macroscopic hematuria from the day of transplantation in the absence of other conditions such as menstruation, gynecologic-related bleeding, and disseminated intravascular coagulation. 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.7 HC occuring within 48–72 hours of a conditioning regimen was defined as early onset HC (EOHC); LOHC occurred beyond 72 hours after the preparative regimen.8
Definition of immune-related LOHC
(1) Clinical manifestations and laboratory examinations met the criteria of LOHC. (2) No definite pathogen was found in urine samples, or symptoms were not relieved after the pathogen had been cleared by antibiotic therapy. (3) Good response to the immune suppression treatment, and remission of symptoms of HC continued without relapse.
HC prophylaxis and management
As prophylaxis for HC, all patients were given intravenous fluid at 3 L∙m-2∙d-1 from 4 hours before to 24 hours after the administration of cyclophosphamide. Sodium 2-mercaptoethanesulphonate was given at a dose of 15 mg/kg prior to cyclophosphamide administration and intravenously once every 8 hours thereafter over 24 hours until the last dose of cyclophosphamide. HC management was standardized at our institution. Surveillance urine cultures for bacteria and fungi were performed on a weekly basis when hematuria occurred. The initial treatment for HC consisted of intravenous fluid hydration, alkalization, and forced diuresis. Platelet transfusions were given to patients when clinically needed; for patients with grade III HC, bladder irrigation was administered to ameliorate symptoms and to prevent obstruction by blood clots. If the HC was not resolved, an empirical anti-viral therapy with ganciclovir or foscarnet was given later if clinically permitted, and an immunosuppressant taper was given to patients with partial HC and no GVHD but who had concomitant viremia or viruria. Patients with persistent bleeding despite these measures were defined as refractory HC. In order to consider alloimmune injury in the pathogenesis of HC, 11 patients with refractory HC were selected to receive tentative treatment with a short impact therapy with methylprednisolone (20–40 mg/d, intravenous injection, 2 to 3 days) from 2004 to 2005.
Cytomegalovirus and adenovirus surveillance
As cytomegalovirus (CMV) and adenovirus (ADV) infection is highly prevalent in the Chinese population, only CMV and ADV were surveyed in our protocol. All of the patients were screened for CMV serostatus pre-transplantation. Weekly polymerase chain reaction (PCR) (Roche, Amplicor, USA) was employed to survey the CMV reactivation in the blood, from the time of transplantation to 100 days afterward. Urine samples were sent for CMV-PCR assay when CMV-viremia was diagnosed. Once HC developed in the patients, blood and urine samples were sent for Adenovirus-specific quality-PCR assay (Qiagen, China) weekly. As a biopsy is unfeasible in most situations, we cannot confirm pathologically viral cystitis for each instance of HC. In this study, CMV-viruria (PCR) in cases of LOHC was thought to be CMV-associated viral cystitis.4
Criteria of therapeutic efficiency of immune suppression treatment
The criteria of therapeutic efficiency were as follows. (1) Complete remission: HC symptoms rapidly relieved within 3 days and gross hematuria disappeared within one week. (2) Partial remission: HC symptoms and gross hematuria significantly improved.
For nonnormal distributed values, data were summarized by means of median and ranges; otherwise, the arithmetic mean and standard deviation were used. The Cox regression model was used to evaluate risk factors for the development of HC with univariate and multivariate analysis. Actuarial survival from day of transplantation was estimated by the method of Kaplan-Meier. Statistical analysis was performed using SPSS 13.3. A P value less than 0.05 was considered statistically significant.
Onset of LOHC and immune-related LOHC
HC occurred in 72 of the 250 patients within 180 days after transplantation and with a cumulative incidence of 28.8%, 12.6% in HLA-matched related donor (MRD), 34.4% in matched unrelated donor (MUD) and 49.5% in mismatched related donor (MMRD). No early-onset HC developed in our cohort. The median time of onset was 33 days after HSCT (range 14–170 days), lasting from 3 to 186 days, 35 days being the median duration. HC was scored as grade I in 2/72 cases (2.8%), grade II in 49/72 (68.1%), grade III in 20/72 (27.8%) and grade IV in 1/72 (1.4%). In the Cox regression model analysis, predictive factors associated with the development of HC were CMV-viremia, graft received from MUD or MMRD and grades II–IV GVHD. A Kaplan-Meier survival analysis indicated that LOHC did not increase mortality.
A total of 11 patients were diagnosed as immune-related LOHC post allo-HSCT according to our criteria. There were 7 patients with grade III HC and 4 patients with grade II HC. The median time of onset was 42 days after HSCT (range 16–150 days) with a median duration of 43 days (range 29–47 days). All patients' HC occurred without any clinical signs of GVHD.
Detection of etiology in immune-related LOHC patients
No defined pathogen was detected in 2 patients. CMV viremia was detected before and after the onset of HC in 9 patients, all of whom were positive for the presence of CMV in urine samples. Although CMV infection was ablated after Ganciclovir treatment, symptoms were not relieved and 5 patients deteriorated. Bacteria were detected in the urine of 4 patients (2 Staphylococcal infections and 2 Escherichia coli infections) during later periods of HC. Symptoms did not improve after sufficient anti-infective therapy, but showed good response to the corticosteroid therapy.
Routine urine test and uric Addis count test in immune-related patients
Erythrocyturia was commonly found in the initial stage of HC. However, casts were rarely seen. A prolonged excessive proteinuria and protein casts occurred in 3 patients two weeks after the onset of HC. Protein casts and epithelial cell casts occurred in the 5 other patients, all of whom recovered after a short course of corticosteroid administration.
Clinical outcome of immune-related LOHC by corticosteroid therapy
The initial treatment for HC consisted of intravenous fluid hydration, alkalization and forced diuresis. If the HC could not be resolved, an empirical anti-viral therapy with cost-effectiveness of ganciclovir (DHPG) 5 mg∙kg-1∙d-1 or foscarnet 80–120 mg∙kg-1∙d-1 was used. After the treatment, absence of CMV in the urine was confirmed by PCR in 9 patients who had activated CMV infection; however, the symptoms remained. Broad-spectrum antibiotics were used to treat all the cases of bacterial infection until the bacteriological tests had become negative. Clinical symptoms and hematuria did not improve until corticosteroid therapy was conducted. All patients achieved complete remission with macroscopic hematuria and clots disappearing within 3 days and continued remission of symptoms without relapse.
With a median follow-up of 180 days after transplantation (range 90–330 days), 2 of 11 patients with immune-related LOHC died within 180 days after transplantation. The causes of death were multiple organ failure and relapse. The other 9 patients were alive with complete continued remission (CCR).
The pathogenesis leading to HC has not yet been elucidated. A number of retrospective studies have identified the risk factors for LOHC, which include viral infections due to immunosuppressants, chemo-irradiation, cytopenia, and GVHD; of these, reactivation of a virus during HSCT is the most common and most consistent risk factor for HC.1-4,8,9 However, no definitive and effective therapy for LOHC has been established to date,4 which suggests that the clinical course of this complication is heterogeneous. The observation that some patients present with an idiopathic etiology led us to examine the immune aetiology of LOHC.
Only a few studies have focused on the immune aetiology of LOHC. In 1985, Ruutu et al10 firstly reported an LOHC patient with chronic GVHD that recovered following anti-GVHD treatment. It was hypothesized that the bladder mucosa of the host might be one of the target organs of GVHD. Data from Ost et al11 (1987) demonstrated a correlation between GVHD and LOHC in incidence, severity and timing of onset. They postulated that LOHC might be one of the clinical manifestations of GVHD. However, as viral infection and GVHD usually coexist, it was difficult to determine whether GVHD per se or GVHD-related immunosuppressant therapy causing viral infection was responsible for the high incidence of HC. To date, no study has evaluated whether the immuno- suppressants used for the treatment of systemic GVHD improve HC.
In our study, HC occurred in 72 of the 250 patients within 180 days after transplantation. Eleven patients were diagnosed as having immune-related LOHC, since all these patients showed a particularly good response to steroid rather than anti-viral therapy. The favorable impact of short-term steroid prescription could be explained by decreased damage from post-HSCT immune reactions. Although several measures had been taken, such as treatment with ganciclovir, and supportive care with platelet transfusion can also produce symptomatic improvement, the therapeutic result is attributed to steroids since all of these patients failed to respond to our conservative therapy. HC was reversed quickly after steroid administration and symptoms continued in remission without relapse. This indicated that an immune mechanism may be involved in these occurrences of LOHC, and though our study was not randomized and the sample size was limited, no other studies have focused on the seemingly trivial issue of LOHC.
It is also intriguing to note that HC occurred without any clinical signs of GVHD in all of these patients, which is obviously distinct from previous studies concerning GVHD-related LOHC.10,11 This observation suggests that the immune mechanism associated with LOHC in these patients might be different from the mechanism of GVHD.
The accurate immune pathogenesis of LOHC in these patients is not clear. Though the therapeutic outcome supports the hypothesis that immune pathogenesis was the main cause of HC in these patients, it could not excluded that this immune pathogenesis might be associated with virus reactivation, since 9 patients developed CMV-reactivation at the onset of LOHC. There is some evidence that virus infection post-HSCT may provoke a general immunostimulatory response with concurrent alloantigen stimulation.12 Gluck et al13 reports that virus-related HC in patients with HIV often follows the increase in CD4 lymphocyte count after anti-retroviral therapy. These results rationally suggest that viral antigen and viral specific immunological reconstitution might be involved in the pathogenesis of LOHC. For these reasons, the clinical course of LOHC might not be determined as a direct cytopathic effect of virus infection, at least in some patients. Low doses of immune suppression therapy might quickly arrest the local inflammatory environment and shorten the course of HC.
Diagnosis of immune-related LOHC is never straightforward since there are no clinical or laboratory tests that are specific and signs of a preexisting infection should be carefully investigated because such presentations may be a result of inadequate antiviral therapy or development of other viral infection. In our group, the immune-related LOHC was characterized by the following characteristics. (1) Long-lasting bleeding with alternation of relief and exacerbation with no specific pathogen being detected. (2) An increase in casts and protein in urine as observed microscopically. (3) Good response to immune suppression treatment as opposed to anti-viral therapy, and symptoms of HC that continue to be in remission without relapse.
Although the present study provides novel information concerning LOHC post-HSCT, it contains certain limitations. This was a small retrospective study, and unrecognized bias caused by a heterogeneous patient background might have influenced the results. Furthermore, the diagnostic criteria for immune-related LOHC were only based on the therapeutic outcome and lack of pathologic evidence; however, our clinical observation suggested that an immune mechanism may be involved in at least some patients with LOHC.
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