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ORIGINAL ARTICLE
Year : 2023 | Volume
: 34
| Issue : 4 | Page : 204-208
Genetic investigation of the origin of an allograft ureteral carcinoma with short tandem repeats: From recipient or donor?
Jia-Yuh Sheu1, Jeff Shih-Chieh Chueh2, Chao-Yuan Huang2, Hsiao-Lin Hwa3, Shuo-Meng Wang2
1 Department of Medical Education, National Taiwan University Hospital, Taipei, Taiwan
2 Department of Urology, College of Medicine, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan
3 Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University; Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan
Correspondence Address:
Shuo-Meng Wang
Room 11-06, Clinical Research Building, Department of Urology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei 100
Taiwan
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/UROS.UROS_124_22
Purpose: The purpose of this study was to demonstrate the usefulness of genetic analysis with short tandem repeats (STRs) to identify the cellular origin of an unusual allograft urothelial carcinoma (UC). Materials and Methods: A 30-year-old Taiwanese woman received a kidney transplant from her aunt in 2000. She was diagnosed with high-grade UC of her native upper urinary tract (urinary tract UC [UTUC]) in 2012. During a follow-up, tumors were discovered in both her native right ureter and graft ureter. The final pathology report identified this as a high-grade invasive UC. To investigate the origin of her allograft ureteral cancer to determine whether it originated from her own or the donor cells, we employed STR analysis because the recipient and donor were of the same gender. Results: We compared 23 autosomal STR loci and one amelogenin. Overall, the STR expression from the native right UTUC was identical to that of the recipient's buccal cells. The STR expression of the graft UTUC was similar to that of recipient cells, but importantly, some STR loci showed gene expressions that were only present in the donor's buccal cells. Conclusion: We concluded that the native right UTUC was of recipient origin and not metastatic from the donor. While we cannot be entirely sure of the tumor origin of the graft ureteral UC, we conjectured that it was not wholly from the donor source alone; either because of the intermixing with the donor stroma or due to microchimerism that developed after transplantation.
Keywords: Kidney transplant, short tandem repeats, ureteral cancer
Kidney transplantation could potentially achieve the best outcome as renal replacement therapy for patients with stage 4 or 5 chronic kidney disease (end-stage renal disease [ESRD]).[1],[2] However, the risk of developing cancer (posttransplant malignancy) in the organ recipients is higher than that of the general population,[3],[4],[5],[6] which might affect the recipients' subsequent survival. Moreover, when cancer of the transplant or native kidney or ureter occurs, whether the cancer cells originated from the recipient's native organ or they are a direct invasion or metastasis from the donor organ may affect cancer staging and lead to differences in treatment. Therefore, identifying the origin of the cancer cells becomes essential and is worth exploring. Here, we demonstrate the usefulness of genetic analysis using short tandem repeats (STRs) to solve this problem in a case scenario of a female who had received a kidney transplant and was later diagnosed with allograft ureteral urothelial carcinoma (UC). Moreover, since her transplanted kidney was from her aunt, it was impossible to compare the sex chromosome to determine the origin of the cancer cells.[7]
Materials and MethodsA 30-year-old Taiwanese woman received an allograft kidney from her aunt, the patient's father's sister, in 2000 for ESRD secondary to unknown etiology. There was no past history of cancer or posttransplant urinary tract diseases of the donor during follow-up. After the transplant, the patient was initially administered calcineurin inhibitor, mycophenolate mofetil, and steroid as base immunosuppressant agents. However, she was diagnosed with UC of her native left upper urinary tract (urinary tract UC [UTUC]) and underwent a left nephroureterectomy with bladder cuff resection in 2010. Subsequent follow-up identified a urinary bladder UC, and the patient has had multiple transurethral resections of her recurrent bladder tumors since 2017. Eventually, the urinary bladder showed no evidence of UC recurrence with repeated cystoscopic biopsies.
The patient suffered from graft kidney hydronephrosis with acute renal allograft dysfunction in September 2019 and underwent percutaneous nephrostomy (PCN). After PCN drainage, the patient's renal function gradually improved, and percutaneous ureteroplasty with antegrade double J catheter insertion was subsequently performed. Magnetic resonance imaging (MRI) showed an increased soft tissue mass at the lower segment of the allograft ureter, which was suspicious of malignancy. Ureteroscopic biopsy and laser ablation in November 2019 confirmed a tumor in the distal graft ureter near the ureteral orifice and the biopsy revealed a noninvasive high-grade papillary UC.
Follow-up whole-body positron emission tomography (PET) scan revealed metastatic paracaval, left common iliac and left external iliac nodes. After a discussion with the family, the patient received chemotherapy with a regimen of gemcitabine plus carboplatin. She completed the third cycle of chemotherapy in November 2020. However, a follow-up MRI in March 2021 identified a suspicious right ureteral tumor. A whole-body PET scan showed fluorodeoxyglucose-avid malignancy within the graft ureter. Since she and her family were strongly urged to preserve her functioning allograft kidney, and after the risks, benefits, alternatives, personnel involved, and possible complications were fully explained and agreed upon, she underwent segmental resection of her distal graft ureter and reconstruction repair, in addition to a right native nephroureterectomy and right iliac lymph node dissection in October 2021. The final pathology report revealed a high-grade invasive UC in the graft ureter, right native UTUC, with no evidence of malignancy in the right urinary bladder cuff or metastasis in the right iliac, paracaval, perinephric, and para-ureteral lymph nodes.
To determine the origin of the patient's UC as either recipient- or donor-derived, we used STR analysis, like in forensic medicine, since the recipient and donor were of the same gender. We used the paraffin-embedded tumor specimens and collected buccal mucosa swabs from the patient and her aunt for STR analysis and genetic comparison. The paraffin-embedded tumor specimens included the pathological sections of the graft ureteral tumor and the right native ureteral tumor. Ethical approval (202110034RINC, approval date November 11, 2021, and 202204088RIND, approval date May 18, 2022) was obtained from the institutional review board of the National Taiwan University Hospital. All procedures were conducted in accordance with relevant guidelines and regulations of the committee, and informed consent was obtained at the time of STR analysis from each participant after the purpose and nature of the procedures/tests performed had been explained in detail.
A QIAamp DNA Mini kit (QIAGEN, Hilden, Germany) and QIAcube (QIAGEN) were used for fully automated DNA extraction from the paraffin-embedded ureter tumor tissues (tumor and nontumor portions) as well as buccal cells from the recipient and donor.
All samples were prepared for polymerase chain reaction (PCR) analysis using the VeriFiler™ Express PCR Amplification Kit (Life Technologies LTD, Warrington, UK) in accordance with manufacturer protocols. Briefly, PCR reactions were performed in a total volume of 5 μL containing 1 μL (0.5 ng/μL) of genomic DNA, 2 μL of VeriFiler™ Express Master Mix, and 2 μL of VeriFiler™ Express Primer Set. PCR was performed using a GeneAmp 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) in 9600 modes. The cycling programs consisted of predenaturation at 95°C for 1 min, followed by 28 cycles of denaturation at 94°C for 3 s, annealing at 59°C for 16 s, extension at 65°C for 29 s, and a final extension at 60°C for 5 min.[8] The loci and alleles of the VeriFiler™ Express PCR Amplification Kit (referenced from the manufacturer's user guide) are listed in [Supplementary Table 1] [Additional file 1].
The PCR product was detected on the ABI PRISM 3500 Genetic Analyzer (Applied Biosystems) electrophoresis system and analyzed using GeneMapper Software v5.1 (Applied Biosystems) by comparison with an allelic ladder and reference DNA control sample 007 (Life Technologies LTD, Warrington, UK). The reference DNA control sample 007 contained 2.0 ng/μL human male genomic DNA in 0.05% sodium azide and buffer, with an analytic threshold at 150 relative fluorescence units.[9]
ResultsAltogether 23 autosomal STR loci and one amelogenin were compared [Figure 1] and [Table 1]. Overall, the STR expression in the recipient's native right ureteral UC was identical to the STR expression in her own buccal cells [Figure 1]a, [Figure 1]b and [Figure 1]d, and there was no evidence of different STR expressions that belonged to the buccal cells of the donor. It can be deduced that the native right ureteral cancer (UTUC) was not donor-derived.
Figure 1: Comparison of five selected STR loci (vWA, D1S1656, D12S391, D2S1338, Penta_D). Among 4 cell/tissue types – (a) The donor's buccal sample, (b) The recipient's buccal sample, (c) The graft ureter tumor, (d) The right native ureter tumor. Such comparisons allowed us to judge the origin of her various UC. UC: Urothelial carcinoma, STR: Short tandem repeatThe majority of the STR expression in the allograft ureteral UC were similar, but not totally identical, to those of the patients' own buccal cells. However, some STR loci from the allograft ureteral tumor specimen showed STR expression patterns that matched those which only appeared in the donor's buccal cells [Figure 1]a, [Figure 1]b, [Figure 1]c.
DiscussionIn previous studies, the most common and straightforward method to distinguish between donor-derived and recipient-derived cancers in transplant patients was fluorescence in situ hybridization analysis for the sex chromosomes.[10],[11] However, in this case, this method was limited because the premise of the method is that the donor and recipient must be of different genders. Therefore, we used the comparison of STR microsatellite markers to distinguish between the donor and recipient cells.
Genetic analysis using STRs is one of forensic medicine's most commonly used methods for paternity tests. Herein, we utilized this method to distinguish whether the cancer cells were donor- or recipient-derived; this result could benefit medical judgment and/or treatment planning. Our study confirmed that the recipient's native right ureteral UC was completely recipient-derived and was not a metastasis originating from the grafted kidney/ureter.
Although we found more than one STR pattern specifically from the donor as the tumor origin of the allograft ureter, we identified more STR similarity with the patient's own buccal cells in the allograft ureteral tumoral specimen. We are certain that the cancer cells were not solely of donor origin, but included donor DNA either because of the confounding factor of the donor's stromal involvement or the occurrence of the phenomenon of “microchimerism” had developed in the patient's allograft ureter after over almost 20 years' posttransplantation. Another possible explanation is the admix of the donor's normal stromal cells combined with the allograft UC cells, which were direct invasions and migrations from the recipient's urinary bladder UC. Nonetheless, we could not fully conclude whether the allograft ureteral tumor was completely recipient-derived. If the laser capture microdissection (LCM) technique was available to dissect only the cancerous allograft ureteral tissues, we could more confidently define the origin of the allograft ureteral UC to rule out or in the participation of the donor component. According to Espina et al.,[12] LCM is a method to obtain cells under direct microscopic visualization that allows the selection of nonmalignant and malignant cells. For tumor-stromal interactions, which serve as a communication interface during tumor development,[13],[14],[15],[16] the LCM technique can isolate tumor cells through the excision of unwanted stromal cells, and thereby provide a histologically pure population of cancer cells.
From another perspective, because the allograft ureter should theoretically only possess donor cells, it can be deduced that the allograft ureteral tumor is at least partially recipient derived. In this situation, the recipient's cells might directly invade from the bladder or metastasize to the transplanted organ via the blood or lymphatic vessels. To match the genetic analysis findings meaningfully with her medical history, the hydronephrosis of her transplant kidney, ureteral double J stent placement, and allograft ureteral pathology of UC may play a role in the final long-segment distal allograft ureteral cancer. However, it is challenging to identify which factor was the earliest or most critical contributor to the final picture. Therefore, we could only speculate that the patient's UC has a higher chance of recurrence if derived from herself than if it originated from the donor tissue, and we did not observe evidence of donor-originated UC in her native UTUC.
ConclusionWe demonstrated the usefulness of genetic analysis using STRs to identify the cellular origin of an unusual allograft UC.
Data Availability Statement
The datasets generated and analyzed during the present study are available from the corresponding authors on request.
Acknowledgments
We would like to thank the National Taiwan University Hospital, Taiwan's National Health Research Institutes, and Taiwan's Ministry of Science and Technology.
Financial support and sponsorship
The authors greatly appreciate the Laboratory of Paternity Test in the National Taiwan University Hospital for technical assistance. This study was supported by NTUH-TMU cooperative grant 111-TMU124 with the NTUH IRB approval number of 202110034RINC.
Conflicts of interest
Prof. Jeff Shih-Chieh Chueh, and Prof. Chao-Yuan Huang, the editorial board members at Urological Science, had no roles in the peer review process of or decision to publish this article. The other authors declared no conflicts of interest in writing this paper.
Supplementary Materials
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