Effects of intensified conditioning on Epstein-Barr virus and cytomegalovirus infections in allogeneic hematopoietic stem cell transplantation for hematological malignancies

Background Intensified conditioning regimens (increasing the intensity of standard myeloablative conditioning) for hematological malignancies in allogeneic hematopoietic stem cell transplantation (allo-HSCT) could reduce the relapse rate of the underlying disease, but it might simultaneously increase the transplant-related mortality including the mortality of infections. To explore whether intensified conditioning affected Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections, 185 patients undergoing allo-HSCT were enrolled. Methods A total of 104 cases received standard and 81 intensified conditioning. Cyclosporine A (CsA) withdrawal and/or donor lymphocyte infusion (DLI) were conducted in high-risk patients. The EBV-DNA and CMV-DNA levels of blood were monitored regularly by quantitative real-time polymerase chain reaction (RQ-PCR) and immune reconstitution of recipients were analyzed by flow cytometry. Results The 3-year cumulative incidence of EBV viremia, EBV-associated diseases and mortality of EBV-associated diseases were 25.3% ± 4.6%, 10.5% ± 3.4% and 0.0% ± 0.0% in the standard group, compared with 45.6% ± 6.5%, 26.0% ±5.3% and 7.3% ± 3.1% in the intensified group (P = 0.002, P = 0.002, P = 0.008). The 3-year cumulative incidence of CMV viremia and CMV-associated diseases, mortality of CMV-associated diseases and incidence of bacterial and fungal infections were similar between the two groups (P = 0.855, P = 0.581, P = 0.933, P = 0.142, P = 0.182, respectively). Multivariate analysis showed that intensified conditioning was one of the risk factors for EBV viremia and EBV-associated diseases (P = 0.037, P = 0.037), but it had no effects on CMV infections. The percentage of CD4+ T cells and CD4+/CD8+ ratio at 3 months post-transplantation were lower in the intensified group (P = 0.032, P = 0.022). The 3-year OS and DFS in the standard group were 62.2% ± 5.8% and 60.6% ± 5.6%, compared with 51.6% ± 6.2% and 51.1% ± 5.9% in the intensified group (P = 0.029, P = 0.063). Conclusions Intensified conditioning represents a promising approach for high-risk hematological malignancies, although it affects early immune reconstitution of recipients and increases the incidence and mortality of EBV infections.


Introduction
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative approach for hematological malignancies [1,2]. Besides graft-versus-host disease (GVHD), the two main causes of death after allo-HSCT remain relapse of malignancy and infections [3,4]. The relapse rate exceeds 50% in patients with refractory hematological malignancies with a standard myeloablative (MA) regimen consisting of total body irradiation (TBI)/busulfan (Bu) combined with cyclophosphamide (CY) [5]. Reduced-intensity conditioning (RIC) regimens have been advocated to reduce transplantation-associated toxicity in elderly or medically unfit patients [6,7]; however, disappointing results have been reported with RIC transplantation in patients with refractory hematological malignancies [8,9]. Some studies suggested that intensified conditioning regimens, which increased the intensity of standard myeloablative conditioning, could reduce tumor relapse, but it might simutaneously increase the transplant-related mortality (TRM) including the mortality of infections [10][11][12]. Therefore, overall survival (OS) did not improve significantly. To improve the outcomes of allo-HSCT for refractory hematological malignancies, we introduced a strategy of sequential intensified conditioning and early rapid tapering of prophylactic immunosupressants therapy for GVHD during the early stage after transplantation [13]. The results of this trial suggested that this strategy might reduce tumor relapse. Moreover, the nonrandomized data suggested that this strategy did not increase the incidence and mortality of bacterial and fungal infections [13], but the effect of this strategy on opportunistic viral infections needed further investigation.
Opportunistic viral infections, especially Epstein Barr virus (EBV) and cytomegalovirus (CMV) infections are one of the common complications after allo-HSCT. Both primary infections and reactivations of EBV and CMV may result in life-threatening diseases in recipients of allo-HSCT [14][15][16][17]. The occurrence of EBV and CMV infections and reactivations is influenced by several factors and closely related to the immune function [18][19][20][21]. Some studies showed that the intensity of conditioning might affect immune reconstitution of recipients after allo-HSCT [22,23]. To explore whether intensified conditioning affected EBV and CMV infections, we prospectively studied the incidence and mortality of EBV and CMV infections in the recipients of allo-HSCT following intensified or standard conditioning.

Patients
From February 2009 to December 2011, 189 consecutive patients with hematological malignancies received allo-HSCT in our single institution. A total of 185 cases were enrolled in this prospective study, and 4 cases who died from regimen-related toxicity (RRT) or bacterial infection before hematopoietic reconstitution were not included. The median age was 28.0 years (range 12-63 years). Seventy-two patients were female, and 113 were male. The primary diseases included acute leukemia (AL, n = 149), chronic myeloid leukemia (CML, n = 28), lymphoma (n = 5), myelodysplastic syndrome (MDS, n = 2) and blastic plasmacytoid dendritic cell neoplasm (n = 1). One hundred and twenty-five patients were in the status of complete remission (CR) (including patients with CML-chronic phase [CP]), and 60 were not in CR (NR) at the time of transplantation. All recipients were EBV-DNA negative in blood and 99 were EBV-VCA (viral capsid antigen, IgG) seropositive before transplantation. Two donors were EBV-DNA positive and became EBV-DNA negative with antiviral agents before collection of stem cells; 111 donors were EBV-seropositive. Seven recipients and nine donors were CMV-DNA positive in blood before transplantation. After antiviral treatment, they were all CMV-DNA negative at the time of transplantation. One hundred and sixty-nine recipients and 171 donors were CMV-IgG positive ( Table 1). The study was performed in accordance with the modified Helsinki Declaration, and the protocol was approved by our ethical review boards before study initiation. All recipients, donors and/or guardians provided written informed consent.

Conditioning regimens
Five conditioning regimens, including three standard MA and two intensified MA conditioning regimens were administrated. The standard conditioning was as follows: ① TBI (4.5 Gy/day, -5, -4 days) + CY (60 mg/ kg/day, -3, -2 days) in 36 recipients; ② Bu (3.2 mg/kg/ day, -7 to −4 days) + CY (60 mg/kg/day, -3, -2 days) in 40 recipients; ③ Bu (3.2 mg/kg/day, -6 to −3 days) + Flu (fludarabine, 30 mg/m 2 , -6 to −2 days) in 28 recipients. The intensified conditioning included the following: ① TBI (4.5 Gy/day, -5, -4 days) + CY + VP-16 (etoposide, 10-15 mg/kg/day, -3, -2 days) in 32 recipients; ② Flu (30 mg/m 2 /day, -10 to −6 days) + Ara-C (cytarabine, 2.0 g/m 2 /day, -10 to −6 days) plus TBI (4.5 Gy/day, -5, -4 days) + CY in 49 recipients [13]. The alternative rules of conditioning depended on the high risk factors for primary diseases and comorbidities at the time of transplantation. Generally, except those with severe comorbidities, patients with high-risk genetics and/or in NR at the time of transplantation all received intensified conditioning; patients with intermediate/low-risk genetics and in CR at the time of transplantation all received standard conditioning (Table 1). According to the following criteria, CsA withdrawal and/or DLI were conducted in all patients with acute lymphoblastic leukemia (ALL) and high-risk factors (high-risk genetics or NR/>CR2 [second complete remission] at the time of transplantation). Depending on whether donor lymphocytes were available, CsA was withdrawn in two ways in patients who did not experience acute GVHD (aGVHD) by day +30 post-transplantation: if donor lymphocytes were unavailable, CsA was withdrawn rapidly in a stepwise fashion (ie, total dose reduced by 20%/week); if they were available, CsA was withdrawn in a stepwise fashion (ie, total dose reduced by 10%/week) and G-CSF mobilized donor lymphocytes (1.0 × 10 8 /kg, once a month, 4 doses totally) would be infused in patients without II°or more than II°aGVHD by day + 60 post-transplantation. Once patients developed GVHD after DLI, DLI would stop and methylprednisolone was added to the regimen.

Prophylaxis and treatment for GVHD
CsA alone or CsA plus MTX (methotrexate) (on days +1 and +3) were administered in patients with NR undergoing HLA-matched sibling donor transplantation, and CsA plus MTX (on days +1, +3 and +6) were administered in patients with CR undergoing HLA matched sibling donor transplants for GVHD prophylaxis. CsA + MTX + ATG (antithymocyte globulin, for total doses of 6-10 mg/kg, on days −3 to −1 or −4 to 0) and/ or MMF (mycophenolate) were used in patients undergoing HLA-mismatched related and unrelated donor transplants. Methylprednisolone (1-2 mg/kg/day) was used to treat aGVHD. ATG or ATG combined with CD25 monoclonal antibody and other immunodepressants were used to treat glucocorticosteroid-resistant aGVHD. Corticosteroids and CsA were used initially to treat chronic GVHD (cGVHD) and were used in combination with various immunosuppressive agents to treat cGVHD that was unresponsive to initial therapy.

Infection prophylaxis
Oral sulfamethoxazole and norfloxacin were given to all patients. Acyclovir was given daily from the beginning of conditioning therapy to engraftment, and it was then administered daily for 7 days every 2 weeks until 1 year after transplantation. Ganciclovir was given for 2 weeks before transplantation for prophylaxis of CMV infections, and was administered once again when CMV viremia occurred. Antifungal agents were administered 5 days before transplantation. Fluconazole (0.3 g/day) or itraconazole (0.4 g/kg.d) was used for up to +60 days post-transplantation in patients with no history of invasive fungal infection (IFI); those with a history of IFI received itraconazole (0.4 g/day), voriconazole (0.4 g/day), caspofungin (50 mg/day) or Am-Bisome (2 mg/kg.day) intravenously. Oral itraconazole or voriconazole was started when the peripheral white blood cell count exceeded 2.0 × 10 9 /L and was discontinued after 90 days post-transplantation.

Monitoring of EBV-DNA and CMV-DNA levels in blood
Generally, the EBV-DNA and CMV-DNA levels of blood were monitored weekly for three months after transplantation. During the 4th to 9th month post-transplantation, the monitoring frequency was once every two weeks; the 10th to the 24th month, once a month; the 25th to 36th month, once every three months. If EBV-DNA or CMV-DNA was positive, it was monitored twice a week. The DNA levels of EBV and CMV in blood were detected by quantitative real-time polymerase chain reaction (RQ-PCR) [24,25]. The plasma (50 μl) was mixed with 50 μl of nucleic acid extract, and the mixture was  heated at 99°C for 10 minutes and then centrifuged at 13000 rpm for 10 minutes. The supernatant was collected for the next step. The PCR conditions for EBV were as follows: 37°C for 2 mins and 94°C for 2 mins followed by 40 cycles at 93°C for 15 s and 60°C for 1 min. The sequences of the TaqMan probes and primers for EBV were as follows: EBV TaqMan probe.

Intervention for EBV and CMV viremia
Once EBV-DNA or CMV-DNA in the blood was positive, the viral loads would be detected once again the next day. When EBV-DNA in the blood was positive twice consecutively, several measures of control were taken, including administration of antiviral agents (ganciclovir, acyclovir or foscarnet), immunoglobulin (0.4 g/kg/d × 3d) and reduction of immunosuppression if the condition of the patient was acceptable. If EBV-DNA in the blood was continuously positive four times with a rising trend, anti-CD20 antibody (rituximab, 375 mg/m 2 ) was administered weekly until EBV-DNA was negative or for a total of 4 weeks.
When CMV-DNA in the blood was positive twice consecutively, ganciclovir or foscarnet was administrated. If CMV-DNA in the blood was continuously positive four times with a rising trend, several measures of control were taken, including immunoglobulin (0.4 g/kg/d × 3d), reduction of immunosuppression and the combination of antiviral agents (ganciclovir and foscarnet).

Diagnosis of EBV-and CMV-associated diseases
EBV-associated diseases were classified into EBVassociated post-transplant lymphoproliferative diseases (PTLD) and EBV-associated other diseases. The diagnosis of EBV-associated PTLD was according to the criteria of World Health Organization (WHO) [26,27]. The diagnosis of EBV-associated other diseases was based on the criteria of the European Conference on Infections in Leukemia and literatures [17,28,29], which included EBV-associated fever without tissue involvement, EBV-associated diseases with tissue other than lymphatic tissue involvement.
CMV-associated diseases were defined according to published recommendations [15]. Briefly, CMV-associated disease was defined by the presence of clinical symptoms or signs of end organ disease, combined with the evidence of CMV infection in a tissue biopsy specimen. CMV pneumonia was diagnosed on the basis of signs and symptoms compatible with a diagnosis of pneumonia (hypoxemia, x-ray) and a bronchoalveolar lavage (BAL) fluid or lung biopsy specimen positive for CMV by immunohistology. CMV gastrointestinal (GI) disease was diagnosed when GI signs or symptoms occurred, and evidence of CMV in the GI tract was diagnosed by immunohistochemistry or in situ hybridization from biopsy specimens. CMV encephalitis was defined by the identification of central nervous system symptoms together with the detection of CMV-DNA in cerebrospinal fluid samples.

Treatment of EBV-and CMV-associated diseases
Once EBV-associated diseases were diagnosed, several measures would be taken promptly, including antiviral agents, reduction of immunosuppression, rituximab, combination chemotherapy, DLI and EBV-specific cytotoxic lymphocyte (EBV-CTL) treatment.
Once CMV-associated diseases were diagnosed, several measures would also be taken promptly, including administration of ganciclovir and foscarnet, immunoglobulin (0.4 g/kg/d × 3d) and reduction of immunosuppression.

Evaluation points and statistics
Our data was analyzed on May 31, 2012. The main evaluation points included EBV and CMV infections within 3 years post-transplantation as well as bacterial and fungal infections within 100 days posttransplantation. The secondary evaluation points included hematopoietic engraftment, primary disease response, aGVHD, cGVHD, immune reconstitution, recurrence and survival. Comparisons of categorical variables were made by means of chi-squared and Fisher exact tests for small numbers. Differences between numerical variables were calculated by means of the Mann-Whitney U-test. Incidence of timedependent variables was estimated by the method of Kaplan-Meier. Intervals were measured from the day of transplantation until first diagnosis of EBV or CMV infections or until the last day of followup, transplant-related death or relapse. Univariate and multivariate Cox regression models were used to analyze risk factors for EBV and CMV infections after transplantation as well as OS and DFS (disease-free survival). EBV and CMV infections as well as OS and DFS were entered as time-dependent covariates. Variables for the multivariate models were selected with backward stepwise elimination with significance exceeding 0.05 as the criterion for removal from the models. A variable indicating whether patients were in the intensified or standard group was included in the models regardless of its significance.

Patient, donor and transplants characteristics
The characteristics of patients, donors and transplants are summarized in Table 1. There were significant differences between standard and intensified group in the category of diseases (P<0.001), disease status at the time of transplantation (P<0.001) and donor type (P = 0.010). As could be seen from the comparison, more patients in the intensified group were cases with ALL and/or in NR, receiving more family or unrelated donor transplants compared with standard group.

Hematopoietic engraftment and primary disease response
Of the 189 consecutive patients undergoing transplantation, 4 cases (3 in the intensified group, 1 in the standard group) died from RRT or bacterial infection before hematopoietic reconstitution and were not included. Regeneration of neutrophil counts > 0.5 × 10 9 /L took a median of 11 days (range 9-22 days) and 12 days (range 9-31 days) in the standard and intensified group (P = 0.486), respectively. Platelet counts > 20 × 10 9 /L were reached after a median of 12 days (range 9-40 days) and 13 days (range 9-70 days) in the standard and intensified group (P = 0.029), respectively. The sixty patients in NR at the time of transplantation, including 50 cases in the intensified group and 10 in the standard group, all achieved CR by day +30 post-transplantation.

CsA withdrawal and DLI
CsA was withdrawn in 42 (40.4%) cases in the standard group and 50 (61.7%) in the intensified group according to the criteria aforementioned (P = 0.004). 16.3% (17/ 104) and 45.7% (37/81) cases received DLI in the standard and intensified group, respectively (P<0.001). Thirtyeight cases who met the criteria of DLI did not receive DLI because of the limitation of the donor lymphocytes source, including 25 cases in the standard group and 13 in the intensified group (P = 0.182). The infection rates and the incidence of bacterial and fungal infections within 100 days post-transplantation were 49.0%, 32.7% and 11.5% in the standard group, compared with 61.7%, 43.2% and 18.5% in the intensified group (P = 0.085, P = 0.142, P = 0.182, respectively). Eight cases died of infections within 100 days post-transplantation, including three who died of CMV-associated diseases and two who died of EBV-associated diseases.

Discussion
In allo-HSCT, the relapse of the underlying disease is the main factor that affects survival. The intensity of conditioning regimen has been shown to directly affect the relapse and survival [30,31]. Some studies suggested that intensified conditioning could reduce tumor relapse, but it might simutaneously increase TRM including infection-related mortality [10][11][12]. In addition to the anti-tumor effect of conditioning regimens, the therapeutic efficacy of allo-HSCT also relies on the graftversus-tumor (GVT) effect [1,32]. In this study, based on the results of our previous studies [13], we introduced the regimen of intensified conditioning, early tapering of prophylactic immunosuppressants followed by DLI for inducing GVT effect for patients with highrisk and refractory hematological malignancies, with 3year OS and DFS of 51.6% ±6.2% and 51.1% ± 5.9%. The results once again proven that intensified conditioning followed by inducing GVT effect was effective for patients with high-risk and refractory hematological malignancies.
Infections are another leading cause of death after allo-HSCT. Some studies reported that the incidence of infections and the infection-related mortality might reach up to 77% and 20% after allo-HSCT, respectively [3,33,34]. Recently, with wide applications of antibacterial and antifungal drugs in the prophylaxis and therapy of infections, the incidence and mortality of bacterial and fungal infections post-transplantation decrease markedly. However, due to the absence of effective preventive and therapeutic drugs for most viruses, the incidence and mortality of viral infections increase relatively, especially in the early period after transplantation. Some studies suggested that intensified conditioning was accompanied by an increasing incidence and mortality of early-stage infections, due to aggravated tissue and organ damage as well as the delay of immune reconstitution after HSCT [10][11][12]. In this study, we prospectively compared the effects of standard and intensified conditioning on infections, especially EBV and CMV infections. Our data further confirmed our previous results that intensified conditioning did not increase the incidence and mortality of bacterial and fungal infections early post-transplantation [13]. Meanwhile, our data showed that intensified conditioning might increase the incidence of EBV viremia and EBV-associated diseases as well as the mortality of EBV-associated diseases, but it did not affect the incidence of CMV viremia and CMV-associated diseases as well as the mortality of CMV-associated diseases. The differences might be associated with the fact that there was optimal strategy for prevention and treatment of CMV infections, but lack of effective methods to prevent and treat EBV infections.
Although EBV and CMV infections are the most common opportunistic viral infections and closely related to the immune function, the risk factors for both infections are different in recipients of allo-HSCT. Recognized main risk factors for EBV infections include T-cell depletion, use of ATG or anti-CD3 monoclonal antibody, HLA mismatch, unrelated donor and so on [35][36][37][38]. Important risk factors for CMV infections are associated with the serological status of donor and recipient, aGVHD, T-cell depletion and use of ATG [18,23]. In this study, we analyzed the risk factors for EBV and CMV infections. Univariate analysis revealed that HLA mismatch, unrelated donor, use of ATG, advanced disease status and aGVHDII-IV were associated with EBV infections; use of ATG, aGVHDII-IV, HLA mismatch, early CsA withdrawal and DLI were associated with CMV infections. Upon multivariate analysis, use of ATG was found to be the risk factor for EBV viremia and EBVassociated diseases; HLA mismatch and early CsA withdrawal were the risk factors for CMV viremia; aGVHDII-IV was the only risk factor for CMVassociated diseases. These results were consistent with current studies [18,23,36,38], except the finding that CMV infections was associated with early CsA withdrawal and DLI. The reasonable explanation for this finding was that early CsA withdrawal or DLI could increase the incidence of GVHD, and GVHD was the risk factor for CMV infections. Interestingly, univariate and multivariate analysis both revealed that intensified conditioning was the risk factor for EBV viremia and EBVassociated diseases. The mechanisms that intensified conditioning increased EBV infections might be associated with the effects of intensified conditioning on early immune reconstitution. Therefore, we analyzed the immune reconstitution of recipients early posttransplantation and found that the percentage of CD4 + T cells and ratio of CD4 + /CD8 + T cells at 3 months post-transplantation were significantly lower in the intensified group.

Conclusions
Intensified conditioning represents a promising approach for high-risk hematological malignancies, although it affects early immune reconstitution of recipients and increases the incidence and mortality of EBV infections.