American Journal of Transplantation 2014; 14: 2838–2845 Wiley Periodicals Inc.
C
Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.12990
Donor or Recipient Origin of Posttransplant Lymphoproliferative Disorders Following Solid Organ Transplantation A. Kinch1,*, L. Cavelier2, M. Bengtsson2, E. Baecklund3, G. Enblad4, C. Backlin3, U. Thunberg4, C. Sundstro¨m2 and K. Pauksens1
priming; PTLD, posttransplant lymphoproliferative disorder; SOT, solid organ transplantation; WHO, World Health Organization
1
Received 02 July 2014, revised publication 25 August 2014
Department of Medical Sciences, Section of Infectious Diseases, Uppsala University, Uppsala, Sweden 2 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden 3 Department of Medical Sciences, Section of Rheumatology, Uppsala University, Uppsala, Sweden 4 Department of Radiology, Oncology and Radiation Sciences, Section of Oncology, Uppsala University, Uppsala, Sweden Corresponding author: Amelie Kinch, [email protected]
Previous studies of donor or recipient origin of posttransplant lymphoproliferative disorders (PTLDs) following solid organ transplantation (SOT) have either been small or with selected patient groups. We studied tumor origin in a population-based cohort of 93 patients with PTLD following SOT. Tumor origin of PTLD tissue was analyzed by fluorescence in situ hybridization of the sex chromosomes in cases of sex mismatch between donor and recipient (n ¼ 41), or HLA genotyping in cases of identical sex but different HLA type (n ¼ 52). Tumor origin of PTLD could be determined in 67 of the 93 cases. All 67 PTLDs were of recipient origin. They were found in recipients of kidney (n ¼ 38), liver (n ¼ 12), heart (n ¼ 10) and lung (n ¼ 7). The most common recipient-derived lymphomas were monomorphic B-cell PTLDs (n ¼ 45), monomorphic T cell PTLDs (n ¼ 9), indolent lymphomas (n ¼ 6), and polymorphic PTLD (n ¼ 4). Half of the recipient-derived PTLDs were Epstein–Barr virus-positive. Twelve of the recipient-derived PTLDs were located in the grafts: in four cases exclusively and in eight cases in combination with disseminated disease outside the graft. Tumor origin was indeterminable in 26 cases, probably due to low DNA quality. We conclude that the vast majority of PTLDs after SOT was of recipient origin. Abbreviations: DLBC, diffuse large B cell lymphoma; EBER, EBV-encoded RNA; EBV, Epstein–Barr virus; FISH, fluorescence in situ hybridization; ICD-7, Seventh edition of International Classification of Diseases; PCRSSP, polymerase chain reaction sequence-specific 2838
and accepted for
Introduction Posttransplant lymphoproliferative disorder (PTLD) is a rare but serious complication after transplantation (1,2). The majority of PTLDs following solid organ transplantation (SOT) is reported to arise from the recipient’s own lymphocytes, as opposed to PTLD after allogeneic hematopoietic stem cell transplantation, which is almost always of donor origin (3–5). Knowledge about the cell of origin in PTLD after SOT is important because it may influence the choice of PTLD therapy (4). Furthermore, donor-derived PTLDs could be more sensitive to reduction of immunosuppression since the restored immune system will recognize foreign antigens presented by the tumor cells (5). The frequency of donor-derived PTLD following SOT has not been investigated in any large study of randomly selected cases. There are several case reports and small studies indicating that donor-derived PTLDs are almost exclusively located in the graft (5–8). The largest study of tumor origin to date comprises 43 PTLDs in kidney recipients (5), in which notably half of the cases were selected because they were involving the graft. This proportion of graft PTLDs is clearly not representative of the distribution of PTLDs after kidney transplantation. Of importance is also the finding that the histopathology of donor-derived PTLDs may more frequently be polymorphic as opposed to monomorphic (5,6). Donor-derived PTLDs are reported to develop earlier after transplantation compared with recipient-derived PTLDs (5–7). It has been suggested that donor-derived PTLDs have a better outcome compared to recipient-derived PTLDs, but the studies so far have not been large enough to confirm this (5,7). We investigated the cell of origin in a large population-based cohort of PTLDs because the frequency of donor-derived PTLDs following SOT is not known, due to the small studies
Tumor Origin of PTLD
to date. Furthermore, many aspects of the differences between donor- and recipient-derived PTLDs are still unclear. We hypothesized that donor- and recipient-derived PTLDs would differ at the group level in PTLD subtype, presence of intratumoral Epstein–Barr virus (EBV), clinical characteristics and survival. The study population of 93 PTLDs was retrieved from a cohort of PTLDs following SOT in Sweden during almost 3 decades (2). In order to determine tumor origin, the PTLD specimens were either analyzed by fluorescence in situ hybridization (FISH) for the X and Y chromosomes, in the case of donor and recipient having different sex, or by HLA genotyping, in the case of sex match but HLA mismatch between donor and recipient.
Materials and Methods
‘‘non-Hodgkin lymphomas’’ after transplantation. We excluded cases where the lymphoma occurred pretransplant (n ¼ 2), PTLD was diagnosed before 1980 (n ¼ 2), patients had not undergone SOT according to medical records (n ¼ 2) and cases where the lymphoma diagnosis could not be confirmed (n ¼ 6). The tumor biopsies were re-evaluated by an experienced hematopathologist (CS) according to the 2008 WHO classification of lymphomas and analyzed for EBV by EBV-encoded RNA (EBER) in situ hybridization (3). Detailed clinical data were collected from the medical records from the transplantation centers as well as from the hospitals where the patients were followed posttransplant. These data included baseline clinical characteristics, complications posttransplant, PTLD therapy and survival for the organ recipients. Further, sex and results of HLA genotyping of the donor and recipient were registered. Six cases were excluded because the donor and the recipient had the same sex and were HLA-identical or because these data were not available. All other cases with sufficient material for further analysis were included (n ¼ 93). The study was approved by the Regional Ethical Review Board in Uppsala, Sweden (No. 2007/150).
Identification of cases
FISH analysis
The PTLD cases were retrieved from a previously reported populationbased cohort of lymphomas/PTLDs after SOT between 1980 and 2006 in Sweden (2). All patients (n ¼ 10 010) that had undergone SOT between 1970 and 2006 were identified from the Swedish National Inpatient Register (Figure 1). Through linkage to the Swedish Cancer Register, using the Seventh edition of International Classification of Diseases (ICD-7) codes 200, 201, 202 and 204.1, 147 cases of lymphoma/PTLD were identified. Plasma cell tumors were not included. Indolent B cell lymphomas were included although they are not defined as PTLDs according to the 2008 World Health Organization (WHO) classification of lymphomas, since these lymphomas commonly are included in studies of
FISH analysis was performed on PTLD tissue to determine the tumor cell origin in cases of sex mismatch between the recipient and the donor (n ¼ 41, Figure 1). Formalin-fixed, paraffin-embedded diagnostic specimens of PTLD were cut into 4 mm thick sections and mounted on slides. After deparaffinization, the slides were treated in hydrochloric acid (0.2 M) at room temperature for 20 min, rinsed and incubated in VP 2000 Pretreatment Reagent followed by protease treatment according to manufacturer’s instructions (Abbott Molecular, Inc., Abbott Park, IL). FISH was performed using the AneuVysion probe kit, that hybridize to the centromeres of the X and Y chromosomes, following the manufacturer’s instructions (Vysis CEP 18, X, Y-alpha satellite probe; Abbott Molecular, Inc.), and mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA). The area with tumor cells was localized by morphology and evaluated for the presence of XX or XY karyotype with the use of a fluorescence microscope (Zeiss, Germany) (Figure 2). When needed, a comparison was done with the hematoxylin and eosin stainings. Images were obtained using the ISIS software (MetaSystems, Altlussheim, Germany).
Swedish Naonal Inpaent Register: 10,010 SOT recipients 1970-2006 Linkage to the Swedish Cancer Register 147 lymphomas 1970-2006 Exclusion of cases before SOT, before 1980, no SOT performed, lymphoma not confirmed (n=12) 135 confirmed PTLDs 1980-2006
93 PTLDs were analyzed for tumor origin
Exclusion of cases where PTLD ssue was not available (n= 36), cases that were sex matched and HLA idencal (n=2) or HLA typing was missing (n=4).
FISH of X and Y chromosomes: 41 cases with sex mismatch
HLA genotyping: 52 cases with sex match but HLA mismatch
HLA genotyping HLA genotyping was performed in cases in which the donor and the recipient had the same sex, but differed in HLA-A, -B or -DRB1 locus (n ¼ 52, Figure 1). Genomic DNA was extracted from the dissected tissue using the Recover All Total Nucleic Acid Isolation kit by the standard protocol (Ambion, Austin, TX). Ten 4 mm thick sections of formalin-fixed, paraffin-embedded PTLD tissue were used for each case. The DNA yield ranged from 4 to 2925 ng/mL. We used polymerase chain reaction sequence-specific priming (PCR-SSP) for HLA typing using commercially available reagents (Olerup, Stockholm, Sweden) (9). HLA typing was performed on HLA-A, -B or -DRB1 since only those data were available on the donor-recipient pairs. The most informative loci were chosen so that both donor and recipient HLA type could be identified both with positive and negative signals.
Statistical analysis 29 recipient origin
12 indeterminable
38 recipient origin
14 indeterminable
Figure 1: Flowchart for identification of the study population and results of the analysis of tumor origin of posttransplant lymphoproliferative disorder (PTLD).
American Journal of Transplantation 2014; 14: 2838–2845
Univariate analyses were performed using the chi-square or the Fisher’s exact test for categorical variables and the Mann–Whitney U-test for continuous variables. End of follow-up for survival was October 25, 2012. Overall survival was defined as time from PTLD diagnosis to death. Cases diagnosed at autopsy were excluded from survival analysis. Survival curves were generated using the Kaplan–Meier method. p < 0.05 was considered statistically significant. Statistical analyses were performed using Statistica Software (version 12, Stat Soft, Inc., Tulsa, OK).
2839
Kinch et al Table 1: Clinical characteristics of the study population and distribution of PTLD subtypes n (%) or median years (range, min–max)
Number of patients Tumor origin Recipient Donor Indeterminable Follow-up time Type of transplant Kidney Kidney þ pancreas Liver Lung Heart Retransplantation before PTLD Immunosuppressive regimen Corticosteroids Calcineurin inhibitors Azathioprine Mycophenolic acid Anti-T cell globulin Gender, male Age at first transplantation Age
C
Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.12990
Donor or Recipient Origin of Posttransplant Lymphoproliferative Disorders Following Solid Organ Transplantation A. Kinch1,*, L. Cavelier2, M. Bengtsson2, E. Baecklund3, G. Enblad4, C. Backlin3, U. Thunberg4, C. Sundstro¨m2 and K. Pauksens1
priming; PTLD, posttransplant lymphoproliferative disorder; SOT, solid organ transplantation; WHO, World Health Organization
1
Received 02 July 2014, revised publication 25 August 2014
Department of Medical Sciences, Section of Infectious Diseases, Uppsala University, Uppsala, Sweden 2 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden 3 Department of Medical Sciences, Section of Rheumatology, Uppsala University, Uppsala, Sweden 4 Department of Radiology, Oncology and Radiation Sciences, Section of Oncology, Uppsala University, Uppsala, Sweden Corresponding author: Amelie Kinch, [email protected]
Previous studies of donor or recipient origin of posttransplant lymphoproliferative disorders (PTLDs) following solid organ transplantation (SOT) have either been small or with selected patient groups. We studied tumor origin in a population-based cohort of 93 patients with PTLD following SOT. Tumor origin of PTLD tissue was analyzed by fluorescence in situ hybridization of the sex chromosomes in cases of sex mismatch between donor and recipient (n ¼ 41), or HLA genotyping in cases of identical sex but different HLA type (n ¼ 52). Tumor origin of PTLD could be determined in 67 of the 93 cases. All 67 PTLDs were of recipient origin. They were found in recipients of kidney (n ¼ 38), liver (n ¼ 12), heart (n ¼ 10) and lung (n ¼ 7). The most common recipient-derived lymphomas were monomorphic B-cell PTLDs (n ¼ 45), monomorphic T cell PTLDs (n ¼ 9), indolent lymphomas (n ¼ 6), and polymorphic PTLD (n ¼ 4). Half of the recipient-derived PTLDs were Epstein–Barr virus-positive. Twelve of the recipient-derived PTLDs were located in the grafts: in four cases exclusively and in eight cases in combination with disseminated disease outside the graft. Tumor origin was indeterminable in 26 cases, probably due to low DNA quality. We conclude that the vast majority of PTLDs after SOT was of recipient origin. Abbreviations: DLBC, diffuse large B cell lymphoma; EBER, EBV-encoded RNA; EBV, Epstein–Barr virus; FISH, fluorescence in situ hybridization; ICD-7, Seventh edition of International Classification of Diseases; PCRSSP, polymerase chain reaction sequence-specific 2838
and accepted for
Introduction Posttransplant lymphoproliferative disorder (PTLD) is a rare but serious complication after transplantation (1,2). The majority of PTLDs following solid organ transplantation (SOT) is reported to arise from the recipient’s own lymphocytes, as opposed to PTLD after allogeneic hematopoietic stem cell transplantation, which is almost always of donor origin (3–5). Knowledge about the cell of origin in PTLD after SOT is important because it may influence the choice of PTLD therapy (4). Furthermore, donor-derived PTLDs could be more sensitive to reduction of immunosuppression since the restored immune system will recognize foreign antigens presented by the tumor cells (5). The frequency of donor-derived PTLD following SOT has not been investigated in any large study of randomly selected cases. There are several case reports and small studies indicating that donor-derived PTLDs are almost exclusively located in the graft (5–8). The largest study of tumor origin to date comprises 43 PTLDs in kidney recipients (5), in which notably half of the cases were selected because they were involving the graft. This proportion of graft PTLDs is clearly not representative of the distribution of PTLDs after kidney transplantation. Of importance is also the finding that the histopathology of donor-derived PTLDs may more frequently be polymorphic as opposed to monomorphic (5,6). Donor-derived PTLDs are reported to develop earlier after transplantation compared with recipient-derived PTLDs (5–7). It has been suggested that donor-derived PTLDs have a better outcome compared to recipient-derived PTLDs, but the studies so far have not been large enough to confirm this (5,7). We investigated the cell of origin in a large population-based cohort of PTLDs because the frequency of donor-derived PTLDs following SOT is not known, due to the small studies
Tumor Origin of PTLD
to date. Furthermore, many aspects of the differences between donor- and recipient-derived PTLDs are still unclear. We hypothesized that donor- and recipient-derived PTLDs would differ at the group level in PTLD subtype, presence of intratumoral Epstein–Barr virus (EBV), clinical characteristics and survival. The study population of 93 PTLDs was retrieved from a cohort of PTLDs following SOT in Sweden during almost 3 decades (2). In order to determine tumor origin, the PTLD specimens were either analyzed by fluorescence in situ hybridization (FISH) for the X and Y chromosomes, in the case of donor and recipient having different sex, or by HLA genotyping, in the case of sex match but HLA mismatch between donor and recipient.
Materials and Methods
‘‘non-Hodgkin lymphomas’’ after transplantation. We excluded cases where the lymphoma occurred pretransplant (n ¼ 2), PTLD was diagnosed before 1980 (n ¼ 2), patients had not undergone SOT according to medical records (n ¼ 2) and cases where the lymphoma diagnosis could not be confirmed (n ¼ 6). The tumor biopsies were re-evaluated by an experienced hematopathologist (CS) according to the 2008 WHO classification of lymphomas and analyzed for EBV by EBV-encoded RNA (EBER) in situ hybridization (3). Detailed clinical data were collected from the medical records from the transplantation centers as well as from the hospitals where the patients were followed posttransplant. These data included baseline clinical characteristics, complications posttransplant, PTLD therapy and survival for the organ recipients. Further, sex and results of HLA genotyping of the donor and recipient were registered. Six cases were excluded because the donor and the recipient had the same sex and were HLA-identical or because these data were not available. All other cases with sufficient material for further analysis were included (n ¼ 93). The study was approved by the Regional Ethical Review Board in Uppsala, Sweden (No. 2007/150).
Identification of cases
FISH analysis
The PTLD cases were retrieved from a previously reported populationbased cohort of lymphomas/PTLDs after SOT between 1980 and 2006 in Sweden (2). All patients (n ¼ 10 010) that had undergone SOT between 1970 and 2006 were identified from the Swedish National Inpatient Register (Figure 1). Through linkage to the Swedish Cancer Register, using the Seventh edition of International Classification of Diseases (ICD-7) codes 200, 201, 202 and 204.1, 147 cases of lymphoma/PTLD were identified. Plasma cell tumors were not included. Indolent B cell lymphomas were included although they are not defined as PTLDs according to the 2008 World Health Organization (WHO) classification of lymphomas, since these lymphomas commonly are included in studies of
FISH analysis was performed on PTLD tissue to determine the tumor cell origin in cases of sex mismatch between the recipient and the donor (n ¼ 41, Figure 1). Formalin-fixed, paraffin-embedded diagnostic specimens of PTLD were cut into 4 mm thick sections and mounted on slides. After deparaffinization, the slides were treated in hydrochloric acid (0.2 M) at room temperature for 20 min, rinsed and incubated in VP 2000 Pretreatment Reagent followed by protease treatment according to manufacturer’s instructions (Abbott Molecular, Inc., Abbott Park, IL). FISH was performed using the AneuVysion probe kit, that hybridize to the centromeres of the X and Y chromosomes, following the manufacturer’s instructions (Vysis CEP 18, X, Y-alpha satellite probe; Abbott Molecular, Inc.), and mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA). The area with tumor cells was localized by morphology and evaluated for the presence of XX or XY karyotype with the use of a fluorescence microscope (Zeiss, Germany) (Figure 2). When needed, a comparison was done with the hematoxylin and eosin stainings. Images were obtained using the ISIS software (MetaSystems, Altlussheim, Germany).
Swedish Naonal Inpaent Register: 10,010 SOT recipients 1970-2006 Linkage to the Swedish Cancer Register 147 lymphomas 1970-2006 Exclusion of cases before SOT, before 1980, no SOT performed, lymphoma not confirmed (n=12) 135 confirmed PTLDs 1980-2006
93 PTLDs were analyzed for tumor origin
Exclusion of cases where PTLD ssue was not available (n= 36), cases that were sex matched and HLA idencal (n=2) or HLA typing was missing (n=4).
FISH of X and Y chromosomes: 41 cases with sex mismatch
HLA genotyping: 52 cases with sex match but HLA mismatch
HLA genotyping HLA genotyping was performed in cases in which the donor and the recipient had the same sex, but differed in HLA-A, -B or -DRB1 locus (n ¼ 52, Figure 1). Genomic DNA was extracted from the dissected tissue using the Recover All Total Nucleic Acid Isolation kit by the standard protocol (Ambion, Austin, TX). Ten 4 mm thick sections of formalin-fixed, paraffin-embedded PTLD tissue were used for each case. The DNA yield ranged from 4 to 2925 ng/mL. We used polymerase chain reaction sequence-specific priming (PCR-SSP) for HLA typing using commercially available reagents (Olerup, Stockholm, Sweden) (9). HLA typing was performed on HLA-A, -B or -DRB1 since only those data were available on the donor-recipient pairs. The most informative loci were chosen so that both donor and recipient HLA type could be identified both with positive and negative signals.
Statistical analysis 29 recipient origin
12 indeterminable
38 recipient origin
14 indeterminable
Figure 1: Flowchart for identification of the study population and results of the analysis of tumor origin of posttransplant lymphoproliferative disorder (PTLD).
American Journal of Transplantation 2014; 14: 2838–2845
Univariate analyses were performed using the chi-square or the Fisher’s exact test for categorical variables and the Mann–Whitney U-test for continuous variables. End of follow-up for survival was October 25, 2012. Overall survival was defined as time from PTLD diagnosis to death. Cases diagnosed at autopsy were excluded from survival analysis. Survival curves were generated using the Kaplan–Meier method. p < 0.05 was considered statistically significant. Statistical analyses were performed using Statistica Software (version 12, Stat Soft, Inc., Tulsa, OK).
2839
Kinch et al Table 1: Clinical characteristics of the study population and distribution of PTLD subtypes n (%) or median years (range, min–max)
Number of patients Tumor origin Recipient Donor Indeterminable Follow-up time Type of transplant Kidney Kidney þ pancreas Liver Lung Heart Retransplantation before PTLD Immunosuppressive regimen Corticosteroids Calcineurin inhibitors Azathioprine Mycophenolic acid Anti-T cell globulin Gender, male Age at first transplantation Age
