Japanese Journal of Clinical Oncology 33:408-412 (2003)
© 2003 Foundation for Promotion of Cancer Research
Multiple Pulmonary Nodules Caused by B-Cell Post-transplant Lymphoproliferative Disorder after Bone Marrow Transplantation: Monitoring Epstein–Barr Virus Viral Load
1 First Department of Internal Medicine, 2 Intractable Disease Therapeutic Center and 3 First Department of Pathology, Tokyo Medical University, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONCASE REPORTDISCUSSIONAcknowledgmentsREFERENCES |
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We report a patient with myelodysplastic syndrome who underwent an allogeneic bone marrow transplantation during the first remission. On day 110 he had a low-grade fever and pulmonary nodules, without superficial lymphadenopathy, were observed. The pulmonary nodules gradually increased in size and in number despite administration of anti-fungal and anti-tuberculosis agents. Transbronchial lung biopsy was performed on day 204, yielding a diagnosis of polyclonal post-transplant lymphoproliferative disease (PTLD) positive for Epstein–Barr virus (EBV)-encoded RNA (EBER) and CD20. Subsequent measurement of herpesvirus viral load demonstrated a consistent elevation of EBV viral load from day 96 to day 221. After treatment with anti-CD20 monoclonal antibody (rituximab), regression of pulmonary nodules was confirmed and the number of EBV genome copies decreased to a normal range. This case suggests that monitoring the quantitative EBV viral load may be necessary in cases of EBV-associated PTLD, even in cases presenting pulmonary nodules. Solitary pulmonary nodules may be a rare symptom of PTLD, but in such cases, an aggressive approach may be necessary to obtain a correct diagnosis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCASE REPORTDISCUSSIONAcknowledgmentsREFERENCES |
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Pulmonary infiltrates are occasionally seen in immunocompromised hosts, especially solid-organ transplant recipients (1). Pulmonary nodules are one of the major complications in recipients with lung or cardiac transplantation. The causes for this are heterogeneous, such as infectious pathogens, Cytomegalovirus (CMV), Aspergillus or Nocardia organisms, bronchiolitis obliterans organizing pneumonia, or post-transplant lymphoproliferative disorders (PTLDs) (1,2). Improvement in survival after allogeneic stem cell transplantation (SCT) has resulted in the need to assess issues related to long-term complications, including PTLD (3,4). PTLDs are recognized as a severe complication arising in solid-organ transplant patients with an incidence of 1 to 20% (5). In most solid-organ transplant cases, Epstein–Barr virus (EBV) proteins (e.g., LMP, EBNA) or EBV-encoded RNA (EBER) can be detected in the affected tissues, and these cases are referred to as EBV-positive PTLDs (5). These PTLDs are considered to develop in chronic immunosuppressed patients from a multistep oncogenetic process triggered by EBV.
Although EBV-associated PTLDs presenting as pulmonary nodules in solid-organ recipients are well-known pulmonary complications (1,2,6), such lesions have rarely been reported in SCT recipients (7–11). The real-time quantification PCR assay of the EBV genome (EBV Q-PCR) in blood, which has recently become available, is a gold standard for B-cell PTLD (12–14). However, there is uncertainty regarding its diagnostic value in PTLD with pulmonary nodules in SCT recipients, since most of the reported PTLD patients have been assessed as solid-organ recipients with superficial lymphadenopathy. We report here a patient with myelodysplastic syndrome (MDS) who underwent allogeneic bone marrow transplantation, developed EBV-associated B-cell PTLDs, and presented pulmonary nodules without superficial lymphadenopathy. We found EBV Q-PCR to be useful to predict the onset of PTLD.
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CASE REPORT |
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TOPABSTRACTINTRODUCTIONCASE REPORTDISCUSSIONAcknowledgmentsREFERENCES |
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Clinical Course
The patient was a 47-year-old male, who was diagnosed as MDS (RAEB-2; IPSS INT-2 score) with chronic hepatitis C in September 1999. He was administered intensive chemotherapy, according to JALSG (Japan Adult Leukemia Study Group)-MDS 200, and achieved complete remission (15). In October 2001, he received an HLA-matched ABO-major mismatch (AB-type donor to O-type recipient) related donor bone marrow transplantation (BMT) during his first complete remission. He received 120 mg/kg cyclophosphamide i.v., 1200 cGy total body irradiation, and graft versus host disease (GVHD) prophylaxis consisting of 10 mg/m2 methotrexate i.v. on day 1, and 7 mg/m2 i.v. on days +3 and +6, and cyclosporin A. Both the recipient and the donor were CMV-positive. The patient had acute GVHD (skin, Grade 2) on day 21 and received 50 mg/day prednisolone (then tapered) and 10 mg Taclorimus instead of cyclosporin A.
On day 110, he had a low grade fever, and computed tomography (CT) of the chest on day 165 showed a small pulmonary nodule in left S2 (1 cm in diameter) (Fig. 1A). After multiple pulmonary nodules gradually became evident, he was treated with amphotericin-B and anti-tuberculosis agents, based on the transient elevation of ß-D-glucan, but there was no noticeable change in the pulmonary nodules. The polymerase chain reaction (PCR) showed no positive results for tuberculosis. Similarly, serological studies for fungi showed no positive results.
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On day 219, multiple pulmonary nodules became prominent (the largest nodule was 4 cm in diameter) (Fig. 1B). Transbronchial lung biopsy (TBLB) was performed on day 204, and a pathological diagnosis of EBV-associated PTLD (CD20-positive, EBER-positive) was made. The patient also had chronic GVHD (skin) on day 235 though the FK506 dose had not been changed. On day 239, his pulmonary nodules decreased in diameter (Fig. 1D) but persisted, therefore, rituximab (375 mg/m2, for 4 times) was started on day 243 after obtaining informed consent. After the administration of rituximab, the pulmonary nodules decreased remarkably in size and number until November 2002 (Fig. 1C). There were no prominent changes in EBV-VCA IgG (x 40), EBV-VCA IgM (<x 10), and EBNA (x 40) titers before and after transplantation.
Morphologic and Immunologic Assessment
A TBLB on day 204 demonstrated infiltration of atypical lymphocytes, consisting of a mixture of intermediate-sized cells with irregularly shaped nuclei and large or small lymphocytes, around the sub-epithelial interstitium of the bronchioles and bronchial glands. Although the composition of the infiltrate was rather uniform, the variable sizes of the infiltrating lymphocytes led us to a diagnosis of polymorphic PTLD (Fig. 2A). An immunohistochemical study showed that some of them were positive for CD20 (Fig. 2B), and scattered CD3-positive cells were also present. Most of these cells were also CD8-positive (not shown). In situ hybridization of EBV-encoded RNA (EBER) demonstrated the presence of EBV (Fig. 2C). Thus, a diagnosis of EBV-associated PTLD was confirmed. The TBLB specimen was hybridized with X- and Y-chromosome-specific probes without positive information, since the TBLB specimen was too small to analyze. We also tried to perform a short tandem repeat (STR) analysis to ascertain whether the PTLD was derived from the recipient, but unfortunately, we could not obtain available data. PCR using the joining region probe did not detect any rearrangement in the immunoglobulin heavy-chain gene in the TBLB specimen, indicating that PTLD was polyclonal. Therefore, this case is considered to be Stage I according to Knowles’ PTLD classification (16), although we did not have a chance to analyze EBV clonality because DNA samples were not available.
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Herpesvirus Viral Load Measurement
Subsequential quantification of EBV, CMV, and human herpesvirus-6 (HHV-6) genomes of DNAs obtained from peripheral blood (1 ml, EDTA-N2 M treated) was conducted before and after transplantation using a LightCycler (11,12). For EBV Q-PCR, DNA was amplified by primers (5'-CGC ATA ATG GCG GAC CTA-3' and 5'-CAA ACA AGC CCA CTC CCC-3') and hybridized with probes (LCRed640-AAC CAT AGA CCC GCT TCC TG-P and 5'-AAA GAT AGA GCA GCG CAG C-F) (17). For CMV Q-PCR, DNA was amplified by primers (5'-GGC CTC TGA TAA CCA AGC C-3' and 5'-GCA CCA TCC TCC TCT TCC T-3'') and hybridized with probes (LCRed640-CCC TCC TCC TCT TCC TCA TCA CTC T-P and 5'-CCT CCC GCT CCT GAG CT-F) (17). For HHV-6 Q-PCR, DNA was amplified by primers (5'-ACC CGA GAG ATG ATT TTG CG-3' and 5'-GCA GAA GAC AGC AGC GCGAT-3') and hybridized with probes (LCRed640-GGG TCA TTT ATG TTA TAG ACG GT-P and 5'-TAA GTA ACC GTT TTC GTC CCA-F) (18).
Real-Time PCR was performed using a LightCycler DNA Master Hybridization Probe kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. The estimation of viral genomes in each sample was arbitrarily expressed as the number of viral genomes per one µg of purified DNA using reference DNA with known amounts of viral DNA (17,18). Based on the results obtained from healthy volunteers, we assessed the cut-off level of CMV and HHV-6 as 102 copies/µg and that of EBV as 103 copies/µg, as reported previously (17,18). Experiments were carried out at least twice to ensure the reliability of the results.
After transplantation, a transient but slight elevation of CMV levels (1.5 x 102 copies/µg) was detected on October 26, 2001 (day 21). However, no particular elevation was detected after day 50 (depicted as squares in Fig. 1E). Elevated levels of HHV-6 were detected on day 15 (5 x 102 copies/µg) and day 21 (7 x 102 copies/µg) after transplantation, which then decreased from day 41 to day 96. The HHV-6 genome increased to 8.3 x 101 copies/µg, but the elevation was within the normal range (depicted as triangles in Fig. 1E). In contrast, EBV genome copies fluctuated until day 96 (January 10, 2002) from 1.1 x 102 to 6.7 x 103. However, a consistent elevation was detected from day 96 to day 221 (April 25, 2002) (9 x 102 to 1.6 x 103 copies/µg). After rituximab treatment (day 251), the elevated EBV genome copies decreased and then stayed within the normal range (depicted as diamonds in Fig. 1E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONCASE REPORTDISCUSSIONAcknowledgmentsREFERENCES |
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Pulmonary nodules as solitary lesions of PTLD are severe complications arising in solid-organ transplant recipients (1,2). In lung transplantation, six out of 64 developed multiple pulmonary nodules, and three had PTLD; two of the three PTLDs were polyclonal, and immunosuppressive therapy was effective in such cases (1). Although pulmonary complications, including pulmonary PTLDs, have been reported, recipients of allogeneic hematopoietic SCT rarely present pulmonary PTLDs. Tolar et al. described three childhood SCT recipients who developed PTLDs, presenting pulmonary nodules; two were treated with donor leukocyte infusion (DLI) and one received rituximab and ifofamide + etoposide, and all three survived (7). Although they did not determine the EBV viral load, they emphasized the need for early aggressive strategies to diagnose pulmonary nodules of unclear etiology (7).
EBV, which infects B-cells, induces immortalization of the infected B-cells and the virus remains in the cell in a latent state. In healthy people, EBV proteins such as EBNA1 and LMP1 act as antigens, therefore, immortalized B-cells are killed by cytotoxic T-cells (CTL) (19,20). Under administration of FK506 or cyclosporin A, the suppression of interleukin-2 production immobilizes CTLs. Therefore, infected B-cells proliferate from polyclonal or oligoclonal lymphoma and progress into monoclonal non-Hodgkin’s lymphoma. For this reason, it is important to diagnose EBV-associated disorders in the early stage and establish an EBV monitoring system as a diagnostic parameter. Weekly EBV DNA load monitoring using unfractionated whole blood showed that 78% of PTLD patients had quantitative competitive PCR values above the cut-off level (14), suggesting rapid increases in peripheral blood EBV DNA viral load. This enabled the prediction and diagnosis of PTLD. In contrast, several investigators have reported failure to detect EBV by real-time PCR in cases of PTLD with solitary extra-nodal regions (21,22). In the present case, EBV viral load was elevated to 6 x 103 copies/µg but the level of EBV viral load was somewhat lower than that of typical nodal PTLD (14). Recently, Gartner et al. reported the use of EBV viral load in patients after allogeneic SCT to diagnose and monitor PTLD, employing the quantitative competitive PCR method (23), which is different from the current study by real-time PCR. Due to the quantification system, the cut-off level of EBV load in their study is different from that analyzed in the present study. In the present case, pulmonary nodules were only manifesting lesions as PTLD; in such cases, EBV viral load could be a valuable parameter to guide diagnosis and therapy. Therefore, we should pay more attention to the persistent elevation of EBV viral load in order to predict extra-nodal PTLD lesions.
Some investigators have suggested that the detection of cell-free DNA by PCR in plasma might be more useful for discriminating active infection from the latent state (22). However, EBV Q-PCR using cell fraction is more useful to predict PTLD, even in the pre-clinical stage, because PTLD originates in tumorous proliferation by division of EBV-infected B-cells. Yang et al. demonstrated that the EBV load in peripheral blood increased in patients with PTLD, in comparison with healthy seropositive controls (14). However, this evidence may be accounted for by an increased number of infected B-cells in the blood. There was no relationship between the disappearance of EBV viral load with rituximab therapy and the clinical response (14). This clearly indicates that monitoring of EBV DNA load may be important to predict and diagnose EBV-associated PTLD, but it cannot predict the effectiveness of rituximab therapy (14). Unfortunately, we did not have a chance to analyze EBV-specific CTL which might be useful to make treatment decisions.
In the present case, pulmonary nodules spontaneously decreased in size and number during the course, perhaps due to the presence of GVHD at the time of PTLD diagnosis. The patient experienced GVHD before rituximab administration, thus indicating that GVHD may suppress EBV and could be linked to partial spontaneous regression of PTLD. However, since the reduction in pulmonary nodules due to PTLD was limited and the EBV DNA load in the blood was maintained, we decided to administer rituximab, and achieved successful results. Rituximab is now available for EBV-associated PTLD, though it is reported that some patients with PTLD were actually EBV-negative forms (6). Faye et al. reported 12 children with B-cell PTLD, including four patients without tumoral involvement, all of whom responded to rituximab therapy, while only four of eight patients with tumoral involvement responded to the treatment with complete remission (24). This suggests that rituximab is effective for early onset B-PTLD, for example, polyclonal B-cell PTLD. They suggested that peripheral B-cells in PTLD patients following SCT are tumoral, compared to those following solid-organ transplants (24). Therefore, monitoring of EBV DNA load is important to diagnose early onset B-cell PTLD even in SCT. In the present case, no rearrangement in the immunoglobulin heavy-chain genes was detected. Thus, the PTLD our patient had might have been polyclonal in nature.
In the past, only one childhood EBV-associated PTLD has been treated with rituximab with successful results after SCT presenting pulmonary nodules (7). Micallef et al. demonstrated that eight out of 428 patients who underwent allogeneic BMT developed PTLDs and two had localized lesions of monoclonal nature (25). Our findings, together with other observations (13,14), suggest that monitoring of quantitative EBV DNA load may be necessary to approach EBV-associated PTLD. Furthermore, even in cases of SCT, aggressive approaches to identify pulmonary nodules of unknown etiology are necessary, since EBV-associated PTLD presents a challenge for treatment with rituximab (26).
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONCASE REPORTDISCUSSIONAcknowledgmentsREFERENCES |
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The authors are indebted to Prof. J. Patrick Barron of the International Medical Communications Center, Tokyo Medical University for his review of this manuscript.
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FOOTNOTES |
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+ For reprints and all correspondence: Kazuma Ohyashiki, First Department of Internal Medicine (Hematology/Oncology Division), Tokyo Medical University, 6–7–1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan. E-mail: ohyashik{at}rr.iij4u.or.jp
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REFERENCES |
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TOPABSTRACTINTRODUCTIONCASE REPORTDISCUSSIONAcknowledgmentsREFERENCES |
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Received March 16, 2003; accepted July 28, 2003
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