Clinical significance of clonality and Epstein‐Barr virus infection in adult patients with hemophagocytic lymphohistiocytosis

We assessed the clinical significance of T or B cell clonality and Epstein-Barr virus (EBV) infection in adult patients with hemophagocytic lymphohistiocytosis (HLH) to identify factors related to prognosis. A total of 30 adult patients with diagnosed HLH were included in the study. In all patients, EBV-DNA in peripheral blood was examined by quantitative real-time polymerase chain reaction and bone marrow cells were examined for clonal rearrangement of T cell receptor gamma (TCRG) and immunoglobulin heavy chain (IGH) genes. TCRG clones were detected in 10 patients (33.3%) and IGH clones were detected in 8 patients (26.7%). We found no correlation between clonality and patient outcome. The patients less than 1,000 copies (mL)−1 of EBV-DNA showed a significantly higher clinical response (P = 0.008) and longer overall survival (P = 0.01) than those with high viral load of EBV-DNA. Our results suggest that TCRG and IGH rearrangement do not have any clinical significance in adult patients with HLH, but that high viral load of EBV-DNA may be a risk factor for poor outcomes. In HLH, high viral load of EBV-DNA should thus suggest a prompt approach with aggressive therapeutic interventions. In adults, HLH is often associated with a variety of infections, malignant neoplasms, drugs, autoimmune diseases, and various immunodeficiencies [1-4]. Lymphomas have been reported in secondary HLH associated with malignant disease, mostly in adults. Another common cause of secondary HLH is EBV associated HLH. The EBV genome can be detected in more than 80% of patients with T/NK cell lymphoma and may play a major role in the development of lymphoma-associated HLH. In fact, a substantial percentage of HLH may relapse or progress to T cell lymphoma in months to years [5]. Unfortunately, it is difficult to make a diagnosis of lymphoma-associated HLH, because fatal conditions delayed to perform the tissue biopsy and it took a long time to confirm diagnosis. A lack of histological proof of lymphoma can delay the choice of appropriate treatment for lymphoma-associated HLH at the initial stage. So, we hypothesized that early diagnosis may be supported by molecular study of the EBV genome and the clonal rearrangement of immunoglobulin (IG) and T cell receptor (TCR) genes. In this study, we assessed the clinical significance of T or B cell clonality and EBV infection in adult patients with HLH to identify factors related to prognosis. Thirty patients were enrolled in this study. There were 13 men and 17 women. The median age of the patients was 43 years (range, 17–75 years). Eighteen patients had viral infections (16 with EBV, 1 with cytomegalovirus, and 1 with Hantaan virus). Four patients had a malignant lymphoma (2 with a diffuse large B cell lymphoma, 1 with an extranodal NK/T cell lymphoma, and 1 with a peripheral T cell lymphoma). One patient also had Adult-onset Still's disease (AOSD). The remaining 7 patients (idiopathic HLH) had neither an underlying infection nor neoplasm. Thirteen (43.3%) of 30 patients were showed high viral copies for EBV-DNA [≥1000 (mL)−1], including one patient with lymphoma-associated HLH and one AOSD-associated HLH. There were no significant differences in laboratory and clinical findings between high-and low-viral load groups. The results for clonal detection by use of the specific primer set are summarized in Table I. TCRG clones were detected in 10 (33.3%) of 30 cases. In four cases, clonality was demonstrated by more than one primer set. IGH clones were detected in 8 (26.7%) of 30 cases. In one case, clonality was demonstrated by two primer sets. Among the cases of TCRG clonality, three cases were simultaneously detected by IGH rearrangement. One of eight cases with IGH clones and three of 10 cases with TCRG clones showed exceeded 1000 (mL)−1 of EBV-DNA. Of the 28 patients treated, the overall clinical response rate was 50% (14 patients) and the rate of inactive disease was 39.3% (11 patients). We analyzed, the response rate according to whether exceeded 1000 (mL)−1 of EBV-DNA. As shown in Table II, the patients with < 1000 copies (mL)−1 of EBV-DNA had a significantly higher clinical response rate than ≥ 1000 copies (mL)−1 (P = 0.008). With a median follow-up of 4.6 months (range, 0.1–93.1 months), the median overall survival of the 28 patients was 6.9 months and the estimated overall survival rate at 1 year was 47.6 ± 9.7%. When we analyzed the factors affecting survival, none of the clinical features or laboratory findings was found to affect survival except for excess viral load of EBV-DNA. Patients with ≥ 1000 (mL)−1 copies of EBV-DNA showed poor overall survival compared with those with low EBV-DNA load (P = 0.010) (Fig. 1). However, the clonality of TCRG and IGH did not have any prognostic role in patients with HLH (Fig. 2). Overall survival of the 28 patients with HLH according to EBV-DNA viral load. One year survival rates of patients with EBV DNA copies < 1000 (mL)−1 was 73.3 ± 11.4%, those of patients with EBV DNA copies ≥ 1000 (mL)−1 was 16.9 ± 10.9%. Overall survival of the 28 patients with HLH according to clonality. (a) One year survival rates of patients with and without TCRG clonality was 56.3 ± 16.5% and 43.2 ± 11.9%, respectively (P = 0.539) and in (b) that of patients with and without IGH clonality was 71.4 ± 17.1% and 38.8 ± 11.2%, respectively (P = 0.182). (c) Patients with and without any clonality showed 58.7 ± 14.2% and 38.1 ± 12.9% in one year survival rate, respectively (P = 0.15). EBV-infected LMP-1 expressed T cells may escape from TNF-α-induced cytokine injuries and, therefore, EBV-infected T cells tend to survive and proliferate, explaining the relapsing or disease progression from HLH to T cell lymphoma [5]. The prognosis of lymphoma-associated HLH is fatal and the overall survival rate to be less than 10% [6]. It is difficult to make a diagnosis of lymphoma-associated HLH from other causes of HLH. Detection of lymphocyte clonality by analysis of rearranged TCR and IG plays a critical role in the diagnosis and differential diagnosis of lymphoma [7-9]. The molecular study of clonal rearrangement for TCR and IGH may support the early diagnosis of lymphoma and may even contribute to detecting hidden malignancies of lymphoid lineage. In EBV-associated HLH, clonality study by immunophenotyping and southern blot analysis has been studied in several reports [10, 11]. In our study, clonal rearrangements of TCRG and IGH did not show any clinical significance, especially in terms of therapeutic response and survival in HLH. Similarly, Imashuku et al. suggested that TCR clonality does not have significant poor prognostic role compare with chromosomal abnormality in HLH [12]. Lin et al. reported the monoclonal T cell proliferation with reactive, probably driven by a strong immune reaction against EBV infection instead of a malignant process [10]. Our study could be interpreted to be that it might be difficult to diagnosis the existence of underlying lymphoid malignancies in HLH patients having clonal rearrangements of TCRG and IGH and, clonality does not seem to be helpful in identifying patients with lymphoma associated HLH. Clonality of EBV in patients with chronic, active EBV infection has been investigated by sequence variation of latent membrane protein 1 gene, which has a high degree of sequence heterogeneity. It was suggested that EBV in patients with chronic, active EBV infection was mostly polyclonal expansion. Su et al. also reported that the polyclonal T cell proliferation of EBV associated HLH in children from Asia [13] This could explain the result of our study that high-viral load of EBV-DNA in HLH patients had no correlation with TCRG or IGH rearrangement. Although patients with EBV-associated HLH have been reported to have a better prognosis compared with familial HLH in children [14, 15]. Imashuku [16] reported a high mortality rate (41%) of EBV-associated HLH in children. Early treatment, such as etoposide-based immunochemotherapy or stem cell transplantation improved the clinical outcome, longitudinal follow-up study was showed the good survival rate (75.6%) in EBV-HLH [14, 15]. Conversely, our study showed the result of poor prognosis in the group of high-viral load EBV-HLH. However, there were several limitations in interpreting the clinical response according to viral load of EBV-DNA. First, treatment varied from only supportive to combination chemotherapy and stem cell transplantation by patients' clinical features. Second, this study was analyzed retrospectively, so only serology for CMV and hepatitis viral marker was performed, infections with other viruses cannot be excluded. Third, we could not exclude familial HLH in young adult because they only have no familial history and this study was performed retrospectively. However, we could find a clinical significance in this study because this is a rare report in adult group and we assume that it could influence the difference of clinical response and survival compare to previous data. In conclusion, the results of our study indicate that adult with high-viral load of EBV-DNA should consider a prompt approach with aggressive therapeutic interventions. However, as we analyzed a small number of patients, examination of more cases is necessary to verify this conclusion. We retrospectively analyzed, between March 2000 and June 2009, patients with HLH diagnosed by the revised diagnostic guidelines. Patients for whom data were available on EBV-DNA by real-time polymerase chain reaction (PCR) and on the clonal rearrangement of IG and TCR genes were included in this study. Additionally, to detect viral-associated HLH, serologic tests for EBV and antibody titers for cytomegalovirus and hepatitis virus were performed at the time of diagnosis. Markers of autoimmune disease were studied in all patients, and imaging studies were performed. Twenty of 30 cases were treated with systemic immunotherapy or immunochemotherapy and four cases were treated with combination chemotherapies of lymphoma regimen initially. Four patients received conservative therapy only, and 2 patients were transferred to other hospitals. Peripheral blood and bone marrow samples were obtained from patients at the time of diagnosis after informed consent was obtained. Viral DNA was extracted from whole blood by using QIAamp DNA blood mini kits (Qiagen, Valencia, CA) according to the manufacturer's protocol. EBV load was quantified by using commercially available Real-Q EBV quantification kits (Biosewoom, Seoul, Korea). Mononuclear cells (MNCs) of bone marrow were first isolated by means of density gradient centrifugation with Ficoll-Hypaque (Lymphoprep, Nycomed, Oslo, Norway) and were then stored at −80°C until use. Genomic DNA was extracted from the MNCs by use of the AccuPrep Genomic DNA Extraction Kit (Bioneer, Daejon, Korea) according to the manufacturer's protocol. Clonality was assessed by PCR assay. Genomic DNA from the bone marrow samples was analyzed for TCRG chain genes and IGH gene by use of commercial kits (IdentiClone; InVivoScribe Technologies, San Diego, CA), which represent a recent European collaborative study (BIOMED-2 Concerted Action) [17]. All statistical analyses were performed with the program SPSS version 17.0 (SPSS, Chicago, IL). The Mann-Whitney U test was performed to analyze the statistical significance of nonparametric differences between patients with high viral copies for EBV-DNA [≥1000 (mL)−1] and patients with low-viral copies for EBV-DNA [<1000 (mL)−1], and the χ2 test was performed in the case of binary variables. Overall survival was measured from the time of diagnosis to the time of the last follow-up or death. Survival analyses were performed by using the Kaplan-Meier method. Significances of survival among subgroups were calculated by the log rank test. P < 0.05 was considered statistically significant. Additional supporting information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Jae-Sook Ahn*, Sung-Yoon Rew*, Myung-Geun Shin , Hye-Ran Kim , Deok-Hwan Yang*, Duck Cho , Soo-Hyun Kim , Soo Young Bae*, Se Ryeon Lee§, Yeo-Kyeoung Kim*, Hyeoung-Joon Kim*, Je-Jung Lee* , * Department of Hematology-Oncology, Chonnam National University Hwasun Hospital, Jeollanamdo, Republic of Korea, Department of Laboratory Medicine, Chonnam National University Hwasun Hospital, Jeollanamdo, Republic of Korea, The Brain Korea 21 Project, Center for Biomedical Human Resources at Chonnam National University Medical School, Gwangju, Republic of Korea, § Division of Oncology/Hematology, Korea University Medical Center, Seoul, Republic of Korea.