Identification of patients

Between June 1969 and December 2014, 2835 renal or simultaneous renal and pancreas transplantations were performed in 2437 patients at Uppsala University Hospital, Sweden. Ninety-two patients were lost to follow up, the remaining number of patients were 2345. All recipients received a kidney transplant or kidney and pancreas transplants according to the allocation rules of Scandiatransplant and according to Swedish legislation. None of the transplant donors was from a vulnerable population and all donors and the next of kin provided written informed consent that was freely given. Informed consent was obtained according to the Swedish law (National Board of Health and Welfare, SOSFS 2012:14) and according to Scandiatransplant allocation rules. The informed consent can, according to Swedish law, be found in a nationwide donor registry, be oral or written, or according to the interpretation of relatives to the donor. This is then signed by the treating physician.

Since September 2009 information on post-transplant malignancies have been retrieved by linkage between the Hospital Transplant Database and the Regional Tumour Register in the Uppsala-Örebro region (RTR). This allows for more than 95% detection of post-transplant malignancies [14]. In total 531 patients were diagnosed with post-transplant malignancies until August 2015. Forty-three patients were diagnosed with urologic malignancies, 13 patients with malignancy of the urinary tract and 30 patients with malignancy of the kidney. Of the 30 patients with malignancy in the kidney, 26 patients were found with tumour in the native kidney(s) and 4 patients with tumour in the kidney transplant (Fig 1).

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Fig 1. Flow diagram of donor-derived urologic cancers in renal transplanted patients.

https://doi.org/10.1371/journal.pone.0271293.g001

Seventeen patients were included in this partly retrospective study; all patients with malignancy of the urinary tract (n = 13) as well as all patients with malignancy in the renal transplant (n = 4). Clinical data, including patient survival, were collected from medical records. Information on sex, age and HLA genotype of donor and recipient were retrieved from the hospital transplant database at the Department of Transplantation. The primary endpoint was to analyse the donor or recipient origin of the tumour.

Archival paraffin blocks of urologic tumour tissue were obtained from the respective regional hospitals of the patients. There was insufficient tumour tissue for further analysis in one patient, and accidently tumour tissue from another patient was not correctly coded; hence these tumours of the urinary tract (n = 2) were excluded from the analyses. Thus, the final number of included patients in the study was 15 (ClinicalTrials.gov identifier NCT02241564).

This study was approved by the Regional Ethical Review Board in Uppsala, Sweden (Dnr 2007/032) and approved by the Uppsala Biobank, Sweden (ID: BbA-827-2015-081). Written informed consent was obtained from all living patients prior to inclusion. The use of data and cancer tissue for analysis from deceased patients was approved by the Regional Ethical Review Board.

Histology

An experienced pathologist re-evaluated the histology of the tumour tissue in the obtained archival paraffin blocks. Representative tumour material from transplantectomy specimens and biopsies from renal transplants and the urinary tract was fixed in 4% phosphate buffered formaldehyde and embedded in paraffin. Tissue sections (4 to 5 μm thick) were processed for routine histology and stained with haematoxylin-eosin, and Sirius red for collagen.

Analysis of donor or recipient origin of tumours by HLA genotyping

HLA genotyping of the tumour was performed when the donor and recipient had different HLA-A, HLA–B or HLA-DRB1. Genomic DNA was extracted from manually dissected tumour tissue by using Recover All Total Nucleic Acid Isolation kit (Ambion, Austin, TX, USA) following the protocol of the manufacturer. The HLA region was amplified by polymerase chain reaction (PCR) using sequence-specific primers (SSP; Olerup, Stockholm, Sweden) according to a standard protocol [15]. HLA-A, HLA-B and HLA-DRB1 were routinely tested [16]. The HLA type in the tumour was compared to the known HLA profile of the recipient and the donor.

Fluorescence in situ hybridization (FISH) analysis of different genders in tumours

FISH was used to determine donor or recipient origin of tumours in cases where the donor and recipient were HLA identical but had different gender. Representative tumour specimens, 4 μm thick, formalin-fixed and paraffin-embedded were analysed. First the tumour specimen was deparaffinised and incubated in hydrochloric acid (0.2M). After 20 min at room temperature the tumour specimen was rinsed and kept in VP2000 Pretreatment Reagent (Abbott Molecular Inc, Abbott Park, IL, USA). In order to separate the DNA strands a protease was added. DNA probes with fluorescent label, AneuVysion probe kit (Vysis CEP 18, X, Y-alpha satellite probe Abbott Molecular Inc), caused hybridization to the centromeres of the X- and Y-chromosomes. Vectashield mounting medium with 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc, Burlingame, CA, USA) was used to mount the chromosomes. To identify the XX or XY karyotype a fluorescence microscope (Zeiss, Oberkochen, Germany) was used. The analyses of the images were performed with the ISIS software (MetaSystems, Altlussheim, Germany).

Statistics

All data were presented as medians with ranges. Occurrence of donor-derived tumours were presented as ratios.

Patient characteristics

The characteristics of the patients are summarized in Table 1 and S1–S3 Tables. All study patients received a kidney transplant between 1981 and 2008. Eight patients received one renal transplant, five patients received two renal transplants, one of them had a simultaneous kidney and pancreas transplantation. Two patients received three renal transplants. Of all 24 renal allografts in these 15 patients 10 renal allografts originated from living donors and 14 renal allografts originated from deceased donors. The median age at transplantation was 43 years (range 22 to 64 years). Median duration from first transplantation to cancer diagnosis was 14 years (ranged 2 to 23 years). The gender distribution women: men was 1:2.

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Table 1. Patient characteristics.

https://doi.org/10.1371/journal.pone.0271293.t001

The immunosuppressive protocol was in concordance with the maintenance immunosuppression used at the Department of Surgery, Section of Transplantation Surgery in Uppsala at the time the patients were transplanted (intention to treat). All patients who were transplanted before the year 2000 (n = 11) received a T-cell depleting agent as induction theraphy while all patinets transplanted from the year 2000 and forth (n = 7) received an anti-IL2 receptor antagonist as induction therapy. Maintenance immunosuppression is mentioned in S4–S6 Tables for each patient. Acute rejections were treated with corticosteroids alone (n = 3), corticosteroids and anti-thymocyte globulin (n = 1) or corticosteroids and plasmapheresis (n = 1) (S4–S6 Tables).

Tumour characteristics

Of the 11 patients with cancer of the urinary tract, 9 patients suffered from cancer of the bladder and 2 patients experienced cancer of the ureter. Four patients were diagnosed with cancer of the renal allografts. The morphological diagnoses of the cancers in the bladder were distributed between urothelial cancer (n = 8) and adenocarcinoma (n = 1), while the cancers of the transplant ureter comprised urothelial cancer (n = 2). The morphological diagnoses of the renal cancers were clear cell carcinoma (n = 1), adenocarcinoma (n = 1) and papillary cancer (n = 2) (S4–S6 Tables). The patient with adenocarcinoma in the renal transplant died a few years later from advanced rectal cancer.

Origin of the tumour

The results of the HLA genotyping and FISH analyses showed that 6 of 14 (43%) examined tumours were of donor origin, 3 of 10 tumours (30%) in the urinary tract and 3 of 4 tumours (75%) in the renal allografts (S1–S3 Tables). One of the cancers in the renal allografts was identified to have tissue of recipient origin and another tumour was identified to have cells/tissue of two tissue types, i.e. cells/tissue of donor and recipient origin. In both cases the tumour tissue was extracted from renal allografts after transplantectomy. One male patient received renal allografts from live donors twice, both times from HLA identical brothers, and therefore the origin of tumour could not be analysed. The cancers developed 14 years (range 2 to 23 years) after the corresponding transplantation.

Treatment and outcome of the cancers

Treatment and outcomes are presented in data in S1 Text.