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USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION
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Journal Code
Manuscript No.
Dispatch: 23.4.14
CE: Sumithra
No. of pages: 10
PE: Suganya Raju © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Transplant Infectious Disease, ISSN 1398-2273
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Human herpesvirus 6 reactivation on the 30th day after allogeneic hematopoietic stem cell transplantation can predict grade 2–4 acute graft-versus-host disease M. Gotoh, S. Yoshizawa, S. Katagiri, T. Suguro, M. Asano, T. Kitahara, D. Akahane, S. Okabe, T. Tauchi, Y. Ito, K. Ohyashiki. Human herpesvirus 6 reactivation on the 30th day after allogeneic hematopoietic stem cell transplantation can predict grade 2–4 acute graft-versus-host disease. Transpl Infect Dis 2014. All rights reserved Abstract: Background. Viral infections and their occult reactivation occasionally cause not only organ damage, but also exacerbation of acute graft-versus-host disease (aGVHD), which may increase transplantation-related mortality synergistically. To determine correlations between viral reactivation and transplantation-related complications, we performed various viral screening tests on the 30th day after allogeneic hematopoietic stem cell transplantation (HSCT), and assessed the clinical implications. Patients and methods. Between August 2007 and January 2013, 49 patients (37 men, 12 women) underwent HSCT in our hospital. The stem cell sources were bone marrow (n = 21), peripheral blood (n = 13), and cord blood (n = 15). The presence of cytomegalovirus (CMV), Epstein– Barr virus (EBV), herpesvirus (HHV) 6, and HHV7 in plasma samples prospectively collected from HSCT recipients on day 30 after HSCT was assayed by quantitative polymerase chain reaction, and the correlations with transplantation-related complications were evaluated. Results. The positivities of CMV, EBV, HHV6, and HHV7 were 44.9%, 22.4%, 53.1%, and 18.3%, respectively. We analyzed transplantation-related complications, and a significant correlation was found only between HHV6 and grade 2–4 aGVHD from day 30 to day 100 (P < 0.001). Using a receiver operating characteristic curve, the area under the curve was calculated as 0.86 (95% confidence interval [CI], 0.74–0.98) between the viral load (VL) of HHV6 and grade 2–4 aGVHD. The sensitivity and specificity were 79% and 93%, respectively, when a cutoff value of 87 copies/mL was used. In multivariate analysis using the Fine and Gray proportional hazards model, the clinically determined high-risk patients (P = 0.004; hazard ratio [HR], 3.69; 95% CI, 1.52–9.00) and the positivity of HHV6 (P < 0.001; HR, 9.957; 95% CI, 2.68–37.06) were extracted as independent risk factors for the cumulative incidence of grade 2–4 aGVHD on or after post-HSCT day 30. The only risk factor extracted for the elevation of HHV6 VL >87 copies/mL was cord blood transplantation (P = 0.0032; odds ratio, 7.10; 95% CI, 1.98–30.00). Conclusion. All of the risk factors previously reported to predict severe aGVHD were obtained only during, but not after, HSCT. Our study suggests that the reactivation of HHV6 (≥87 copies/mL) at 30 days after HSCT is a possible predictive marker for grade 2–4 aGVHD on or after post-HSCT day 30.
M. Gotoh, S. Yoshizawa, S. Katagiri, T. Suguro, M. Asano, T. Kitahara, D. Akahane, S. Okabe, T. Tauchi, Y. Ito, K. Ohyashiki First Department of Internal Medicine, Tokyo Medical University, Tokyo, Japan
Key words: human herpesvirus 6; allogeneic hematopoietic stem cell transplantation; acute graft-versus-host disease Correspondence to: Moritaka Gotoh, MD, PhD, First Department of Internal Medicine (Division of Hematology), Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Tel: +81 3 3342 6111 (ext. 5895) Fax: +81 3 5381 6651 E-mail:
[email protected]
Received 28 July 2013, revised 4 December 2013, accepted for publication 30 January 2014 DOI: 10.1111/tid.12229 Transpl Infect Dis 2014: 0: 1–10
1
Gotoh et al: HHV6 reactivation can predict 2–4 acute GVHD
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A high incidence of viral infections and occult reactivation occurs in allogeneic stem cell transplantation (HSCT), because of the use of immunosuppressive agents during the immune reconstitution phase; this leads to organ damage and exacerbation of acute graftversus-host disease (aGVHD). As a result, transplantation-related mortality may be increased synergistically (1–4). There is strong evidence for the frequent reactivation of cytomegalovirus (CMV) and human herpesvirus 6A and B (HHV6) in HSCT (1–6). HHV6 is a member of the Roseolovirus genus of the b-herpesvirus subfamily. This virus reactivates in an immunosuppressed host, as do other human herpesviruses that reactivate about a month after HSCT, followed in some cases by spontaneous conversion into negativity (7–10). After HSCT, HHV6 may cause interstitial pneumonia (11, 12), encephalitis (7, 11), or bone marrow suppression (3, 7, 12–16). It has been suggested that HHV6 can cause transplantationassociated complications, such as aGVHD, and increase transplantation-related mortality (3, 9, 10, 14–16). CMV and HHV6 reactivate approximately 30 days after HSCT. Day 30 is also a critical stage when many recipients attain engraftment and active infections often improve. Complications, such as graft failure, residual severe infections, sinusoidal obstruction syndrome (SOS), and transplantation-associated microangiopathy (TAM), indicate a poor prognosis (17–20). We therefore make it a rule to confirm engraftment, evaluate the patient’s general condition and disease risk, and determine whether transplantation-related complications or viral reactivation has occurred at this stage. This study was conducted to determine whether reactivations of viruses are related to subsequent transplantation-associated complications, and whether they can predict the subsequent complications. We evaluated blood samples from patients on day 30 post-HSCT, to investigate whether or not the reactivation of viruses affects post transplantational complications on or after post-HSCT day 30 (7–10).
this study, and the study was approved by the institutional review board of Tokyo Medical University. Table 1 summarizes their age, gender, performance status (PS), underlying disease and disease risk, previous treatment, time from diagnosis to HSCT, type of preconditioning regimen (myeloablative conditioning [MAC] or reduced-intensity conditioning [RIC]), GVHD prophylaxis (cyclosporine or tacrolimus), stem cell source (umbilical cord blood or other), and human leukocyte antigen (HLA) disparity. Acute leukemia in the first or second remission was defined as standardrisk diseases, and others were defined as high-risk Patient characteristics Gender Male/Female Median age at HSCT (range)
37/12 45 (17–69)
Performance status 0–1/2–4
31/18
Diagnosis AML/ALL/MPAL/CML/CLL/MDS CMPN/ML
23/15/2/1/1/1/6
Disease risk Standard1/High2
20/29
Previous chemotherapy Median number of times (range)
4 (0–17)
Duration from diagnosis to HSCT Median number of months (range)
8 (1–120)
Type of conditioning Myeloablative/Reduced intensity
20/29
GVHD prophylaxis CsA base/TAC base
27/22
Source of stem cells BM/PB/CB
21/13/15
Donor type Related/Unrelated
15/34
HLA disparity Matched/Mismatched
Materials and methods 1
In total 49 patients (17–69 years old), who received an HSCT at Tokyo Medical University for different indications between August 2007 and January 2013, were included in this study. They had serial polymerase chain reaction (PCR) DNA tests for CMV, Epstein–Barr virus (EBV), HHV6, and HHV7 at the First Department of Internal Medicine at Tokyo Medical University. All patients gave written informed consent to participate in
2
Standard risk: first or second remission of acute leukemia. High risk: over third remission of acute leukemia, or disease leukemia and malignant lymphoma. HSCT, hematopoietic stem cell transplantation; AML, acute myeloid leukemia; ALL, acute lymphoid leukemia; MPAL, mixed phenotype acute leukemia; CML, chronic myelogenous leukemia; CLL, chronic lymphocytic leukemia; MDS, myelodysplastic syndromes; MPN, myeloproliferative neoplasms; ML, malignant lymphoma; GVHD, graft-versus-host disease; CsA, cyclosporine; TAC, tacrolimus; BM, bone marrow; PB, peripheral blood; CB, cord blood; HLA, human leukocyte antigen. 2
Patients
Transplant Infectious Disease 2014: 0: 1–10
27/22
Table 1
Gotoh et al: HHV6 reactivation can predict 2–4 acute GVHD
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diseases based on the previous Japanese studies (21, 22). We set post-HSCT day 30 as the landmark point for analyzing various transplantation-related complications and viral load (VL). The factors considered at the landmark point were leukocyte count, neutrophil count, lymphocyte count, aGVHD features within 0–30 days after HSCT, administration of ≥0.5 mg/kg prednisolone, TAM, SOS, active documented infections, use of antiviral agents, viral reactivations, and transplantationassociated morbidity (Table 2).
Treatment of patients Details of conditioning regimens and GVHD prophylaxis are described below. In brief, patients with
Allogeneic stem cell transplantation outcomes on day 30 (Landmark point) Median leukocyte (range) 9 109/L
2.90 (2.0–21.1)
Median neutrophil (range) 9 109/L
1.89 (0–19.2)
9
Median lymphocyte (range) 9 10 /L
0.35 (0–4.15)
Median (range) IgG level (mg/dL)
864 (523–1374)
Acute GVHD Within 30 days grade 0/1/2/3/4
27/14/7/1/0
At 30 days grade 0/1/2/3/4
37/7/4/1/0
>0.5 mg/kg PSL administration Yes/No
20/29
Sinusoidal obstruction syndrome
6/49
Transplantation-associated microangiopathy
8/49
Active documented infection Bacterial
10/49 5/49
Fungal
1/49
Viral
5/49
Antiviral administration ACV/GCV/FN Viral positivity and titer (range)
31/10/8 Median copies/mL (range)
CMV positive
22/49
69 (20–5100)
EBV-positive
11/49
94 (21–2800)
HHV6 positive
26/49
92 (20–18,000)
HHV7 positive
9/49
28 (25–85)
IgG, immunogloublin G; GVHD, graft-versus-host disease; PSL, prednisolone; ACV, acyclovir; GCV, ganciclovir; FN, foscarnet; CMV, cytomegalovirus; EBV, Epstein–Barr virus; HHV, human herpesvirus.
Table 2
hematological malignancies underwent MAC or RIC. MAC consisted of total body irradiation (TBI), at 12 Gy with high-dose cyclophosphamide (120 mg/kg). RIC included fludarabine (150 mg/m2) plus melphalan (140 mg/m2) with or without 4 Gy TBI, fludarabine (180 mg/m2) plus intravenous busulfan (3.2 mg/kg or 6.4 mg/kg) with or without 4 Gy TBI, or fludarabine (150 mg/m2) plus melphalan (80 mg/m2) plus combined filgrastim and cytarabine (6 g/m2) with 4 Gy TBI. GVHD prophylaxis consisted of cyclosporine or tacrolimus with or without short-term methotrexate (10 mg/m2 on day 1, 7 mg/m2 on days 3 and 6, and an optional dose on day 11). In patients who underwent cord blood transplantation (CBT), GVHD prophylaxis consisted of tacrolimus only. GVHD was clinically diagnosed in combination with skin or gut biopsies after engraftment or attainment of 100% donor chimerism. AGVHD and chronic GVHD were graded according to the established criteria (23, 24). All patients received oral acyclovir, at a dosage of 1000 mg per day from day 7 to day 35, followed by long-term, low-dose administration of acyclovir for Varicella zoster reactivation (200 mg daily) (25). At the time of neutrophil engraftment, we started to monitor weekly CMV antigenemia using the peroxidase-conjugated monoclonal C7 antibody (26). When the assay showed >10 CMV-positive cells per 5.0 9 104 white blood cells (WBC) in the bone marrow and peripheral blood (27) or >1–2 CMV-positive cells per 5.0 9 104 WBC in cord blood, we started preemptive administration of ganciclovir. Before platelet engraftment, we used foscarnet instead of ganciclovir. We did not administer other antiherpesvirus drugs. Engraftment was defined as WBC counts ≥1.0 9 109/L or absolute neutrophil counts ≥0.5 9 109/L for 2 consecutive days. Recombinant granulocyte-colony stimulating factor (filgrastim) at 300 lg/m2/day was administered by continuous intravenous infusion from day 1 until neutrophil engraftment.
Sample preparation and real-time quantitative PCR assay EDTA-treated peripheral blood plasma samples were collected on post-HSCT day 30. Viral DNA was detected by the use of a real-time quantitative PCR assay. DNA was extracted from 200 lL of serum with a QIAamp Blood Kit (Qiagen, Hilden, Germany), eluted in 50 lL of distilled water, and stored at 20°C until use. For the real-time PCR assay 5 lL of the DNA solution was used. The primers and probe conditions for EBV, CMV, HHV6, and HHV7 were as described previously
Transplant Infectious Disease 2014: 0: 1–10
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Gotoh et al: HHV6 reactivation can predict 2–4 acute GVHD
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(28–31). The PCR reaction was performed using a TaqMan PCR kit (PE Applied Biosystems, Foster City, California, USA) as previously described (29). A standard graph was constructed from the threshold cycle values obtained from the serially diluted positive control plasmid. The threshold cycle values from clinical samples were plotted on the standard curve, and the copy number was automatically calculated by the Sequence Detector v1.6 (PE Applied Biosystems), which is a software package for data analysis. Samples were defined as negative when the threshold cycle values exceeded 50 cycles. The copy number of each virus DNA was expressed as copies/mL. The detection limit for each viral DNA was ≤20 copies/mL plasma; this was defined as reactivation.
level (
