Incidence of Merkel cell carcinoma in renal transplant recipients.

BACKGROUND
The risk factors for Merkel cell carcinoma (MCC), a rare type of skin cancer, are poorly understood. Some evidence suggests that MCC is more common in individuals with abnormal immune function resulting from viral infection, autoimmune disease or organ trans- plantation.


METHODS
The national Renal Transplant Registry and the Finnish Cancer Registry data were searched for recipients of a renal transplant who were diagnosed with MCC. The MCC diagnoses were confirmed using immunohistochemistry.


RESULTS
Three cases of MCC were detected among 4200 individuals who underwent renal transplantation from 1967 to 2005 [expected number 0.05, standardized incidence ratio (SIR) 66, 95% CI 14-194, P <0.001]. The latency period between the transplant and detection of MCC ranged from 6 to 19 years. In all three cases, the cause of transplantation was an autoimmune disease. All three died from aggressive MCC with a survival time ranging from 0.5 to 2.1 years.


CONCLUSIONS
The results indicate that the risk of MCC is greatly increased among subjects who have undergone renal transplantation. The course of the disease appears aggressive in this patient population. The physicians who treat recipients of a kidney transplant should be aware of the substantially increased risk of MCC.

. Early MCC can be cured by surgery with or without postoperative radiation therapy, whereas advanced MCC is currently considered incurable. MCC often gives rise to metastases in the regional lymph nodes, which decreases disease-specific survival to 52% [2], and when distant metastases are present, expected survival is short with fewer than 10% surviving for 3 years [3]. The age-specific incidence of MCC is highest among the elderly in the Caucasian population [1]. Only few MCCs are diagnosed in individuals younger than 50. In such cases, the disease is usually associated with immunosuppression [4]. The causative factors remain largely unknown [5], but several articles have linked immunosuppression to MCC by case reports and cancer registry or transplant registry linkage studies [4,6,7]. Recently, a new virus named as Merkel cell polyoma virus (MCPyV) was detected in ∼80% of MCCs [8]. Patients whose MCC contains the MCPyV genome generally have a more favourable disease course than those whose cancer does not contain the viral DNA [9].
The purpose of this study was to investigate the incidence of MCC among patients who have undergone renal transplantation. To our knowledge, none of the prior studies addressing the incidence of MCC following organ transplantation is based on a nationwide database. In Finland, all renal transplants are performed at a single centre (Helsinki University Central Hospital), which allows centralized monitoring of survival and adverse effects associated with the procedure. Subjects diagnosed with cancer are registered in Finland in a nationwide cancer registry (the Finnish Cancer Registry) with a coverage approaching to 100%, which allows identification of subjects diagnosed with MCC. In the present study, we identified subjects diagnosed with MCC from the files of the Finnish Cancer Registry and the national Transplant Registry, and reviewed the histological diagnoses. These two nationwide registries allow reliable assessment of the incidence of MCC among renal transplant recipients.

Patients and methods
The nationwide population-based Finnish Cancer Registry (FCR) was founded in 1952. It covers the entire Finnish population [10], which was 5 276 955 on 31 December 2006. Hospitals, practicing physicians and pathological and haematological laboratories are requested to report all cases of cancer to the FCR. In addition, each year, data of all death certificates mentioning cancer are transferred to the FCR. All diagnoses of MCC in the FCR file are based on histological examination. Deaths and emigrations are registered in the Central Population Register and regularly linked to the FCR. The files of the Renal Transplant Registry of Finland, located at the Helsinki University Central Hospital, were reviewed to identify subjects diagnosed with MCC following transplantation of the kidney.
The control population is the entire Finnish population, which includes the patients with renal transplants. The proportion of population with renal transplants is, however, so small that it does not have effect on the reference rates.
The files of the Renal Transplant Registry of Finland, located at the Helsinki University Central Hospital, were reviewed to identify subjects diagnosed with MCC following transplantation of the kidney. All patients who have undergone transplantation of the kidney in Finland were included in this registry.
The hospital records of the MCC cases identified, and data on the reasons for performing the transplant, medication, MCC diagnosis, treatment and outcome were extracted. Subject follow-up data were obtained from the hospital records and the files of the Renal Transplant Registry and FCR.

Review of tumour histology
The formalin-fixed, paraffin-embedded tissue blocks of patients recorded as having been diagnosed with MCC were collected from the pathology archives. The tissue diagnoses were reviewed in a blinded fashion by two researchers with special expertise in MCC pathology (TB and HK). The samples were stained with haematoxylin & eosin, and we performed immunohistochemistry with antibodies for cytokeratin-20 (CK-20, DakoCytomation, Glostrup, Denmark) and thyroid transcription factor-1 (TTF-1; Novocastra, Balliol Business Park West, Benton Lane, Newcastle Upon Tyne, UK). For histological diagnosis of MCC, we required morphology compatible with MCC in light microscopy, positive staining for CK-20 and negative staining for TTF-1. The longest diameter of the tumour was measured from haematoxylin and eosin-stained slides.

Analysis of tumour Merkel cell polyomavirus DNA
The presence of MCPyV DNA was analysed from DNA extracted from representative deparaffinized tumour sections using quantitative polymerase chain reaction (qPCR) as described in detail elsewhere [9]. In brief, quantitation of MCPyV DNA was done using real-time PCR with hydrolysis probes and primers specific for the viral LT3 coding region in a LightCycler 480 instrument (Roche Diagnostics GmbH, Mannheim, Germany). The relative DNA sequence copy number for each tissue sample was expressed as a ratio of MCPyV DNA-to-protein tyrosine phosphatase gamma receptor gene (PTPRG) DNA. Whenever MCPyV DNA was detected (i.e. when the MCPyV DNA-to-PTPRG DNA-ratio was >0), the sample was considered positive. Each PCR product generated was treated with an ExoSAP-IT enzyme mix (product number 78201, USB Corporation, Cleveland, OH, USA) according to the manufacturer's protocol and then sequenced by using BigDye3 termination chemistry and an ABI 3100 Genetic Analyzer (both from Applied Biosystems, Foster City, CA, USA). The sequences were compared with the reference sequences of Merkel cell polyomavirus isolates obtained from the National Center for Biotechnology Information (NCBI) Entrez Nucleotide database by using LaserGene 7.2 software (DNASTAR Inc, Madison, WI, USA).

Statistical analysis
The observed number of MCC cases was compared with the expected number of cases based on cancer incidence in the national population, stratified by age, gender, and calendar time. Standardized incidence ratios (SIRs) were calculated as observed to expected ratios. Exact 95% confidence intervals (CIs) were defined under the assumption that the observed number of cases followed a Poisson distribution.

Permissions
The study was approved by an Ethics Committee of Helsinki University Central Hospital. Permission to collect and analyse paraffin-embedded tumour tissue was obtained from the National Authority of Medicolegal Affairs, Finland.

Results
A total of 4200 individuals received a renal transplant during the study period, 1967-2005; this yielded a total of 36 811 person years at risk by the end of 2005. 60% were men, with the largest number of patients identified in the 45-59 age group (1516, 36%). Based on data from Finnish Cancer Registry and histopathological review of the tumour tissue samples, 172 patients (119 women and 53 men) were reported to FCR of being diagnosed with MCC within the time period 1979-2006. All the patients were Caucasian and mostly elderly. Only four patients (2.3%) were <45 years at diagnosis, while 60.5% were ≥75 years. Before 1 January 2007, 108 patients died.
Linkage of the national Renal Transplant Registry and the Finnish Cancer Registry data identified three recipients of a renal transplant who were diagnosed with MCC. The expected number of MCC based on the national MCC incidence rates is only 0.05; the standardized incidence ratio turned out to be highly elevated (SIR 66, 95% CI, 14-194; P < 0.001). All patients identified were male (P > 0.1). Their age ranged from 44 to 68 at the time of the diagnosis. The latency period between a renal transplant and the diagnosis of MCC ranged from 6 to 19 years.
The reason for renal transplant in all three cases was considered an autoimmune disease; two patients had chronic glomerulonephritis and one rheumatoid arthritis. Their mean age at the time of the MCC diagnosis was 59 years. All the patients were treated with methylprednisolone following the transplant; two also received azathioprine and two cyclosporin A.
All MCCs occurred in the skin of the head, two in a cheek and one in an earlobe. The longest tumour diameters at diagnosis were 15 mm, 16 mm and 20 mm; two patients had local (stage I) disease (patients 1 and 3 in Table 1), whereas one patient (patient 2, Table 1) presented with regional and systemic metastases (stage III). Patient 1 was also diagnosed with several basal cell carcinomas and squamous cell carcinomas in the skin of the head and neck region. The MCCs were treated with surgery and postoperative radiation therapy. All three patients died from progressive MCC with a survival time after the diagnosis ranging only from 0.5 to 2.1 years. In a quantitative PCR analysis, Merkel cell polyoma virus was present in only one of the three MCCs.

Discussion
We found a SIR as high as 66 (95% CI,  for a diagnosis of MCC among individuals who had undergone kidney transplantation. To the best of our knowledge, the current study is the first one to provide a SIR for the risk of MCC following renal transplantation. This SIR is based on linkage of two nationwide registries that both cover the entire population of Finland. The largest epidemiological study on the incidence of MCC among organ transplant patients performed thus far is based on the Cincinnati Transplant Tumor Registry (now called the Israel Penn International Transplant Tumor Registry), which is a web-based registry that collects tumour data from organ transplant recipients worldwide. A report from this registry included 41 cases of MCC diagnosed following an organ transplant suggesting that the incidence of MCC may be increased among organ recipients, but no SIR could be provided [4]. An approximate incidence of MCC in the Cincinnati Transplant Tumor Registry is three cases per 1000 patients in 1997 [11]. Since the demographic features and follow-up times may differ markedly between study cohorts, we suggest that the cohorts are best compared using absolute estimates such as the SIR or the absolute incidence of cancers per 100 000 person years.
None of the prior registry-based studies [4,6,7] included verification of the diagnosis of MCC using immunohistochemistry, which is a limitation, because the histopathological diagnosis of MCC may be demanding and MCC also lacks distinguishing clinical features [12]. The diagnosis of MCC should be confirmed by immunohistochemistry [13], since no tissue morphological features distinguish MCC with certainty from other small round blue cell tumours.
Organ transplantation and subsequent immunosuppression are risk factors for some epithelial and nonepithelial cancers [14,15]. The risk of MCC related to any single immunosuppressive drug is difficult to estimate from the available data. The reports addressing the role of cyclosporin A in the pathogenesis of skin malignancies in organ transplant patients are conflicting [16,17], and these risks may be related to the degree of global immunosuppression rather than to any one specific drug [18,19]. Friedlaender et al. reported a terminally ill patient where withdrawal of cyclosprin may have resulted in the temporary regression of MCC [20]. Ultraviolet radiation may contribute to the development in MCC [21,22], and the risk of MCC may rise along with an increased exposure to solar UV-B [7].
Recently, a small polyomavirus with double-stranded DNA, MCPyV, was found to be clonally integrated in the MCC tumour genomes, suggesting a role for this virus in the pathogenesis of this disease [8]. MCPyV has been found in ∼70-80% of the MCCs examined [8,23,24,25,26]. Another polyomavirus, the BK virus, can cause haemorrhagic cystitis, urethral stenosis and interstitial nephritis leading to irreversible chronic allograft dysfunction in kidney transplant recipients [27,28].
MCCs that arise following an organ transplant may not be similar to those that occur in other patient populations. Our patients were relatively young in this survey in keeping with previous studies [4,29], and the clinical behaviour of the tumours was aggressive. All tumours were located in the head, but ∼50% of MCCs occur in skin of the head and neck area [30]. In the present study, only one of the three MCC cases was MCPyV positive, suggesting that MCPyV is not associated with development of MCC in all organ transplant recipients.
Non-melanoma skin cancers are by far the most common type of malignancy among organ transplant recipients [31].
The proportions of skin cancers also differ among organ recipients from those found in the general population. Basal cell carcinomas are more frequent than squamous cell carcinomas in the general population, whereas the opposite may be the case among organ transplant recipients [32]. In the present study, MCCs were diagnosed 6-19 years after receiving a renal transplant, a latency time in line with case reports [11,33,34,35] and data from the Cincinnati Transplant Tumor Registry, where the mean time from renal transplant to the diagnosis of MCC was 7.5 years (range, from 5 months to 23 years) [4].
The rate of MCC increased from 0.15 cases per 100 000 in 1986 to 0.44 cases per 100 000 in 2001 [1], and agespecific incidence is highest in the most elderly, 4.28 per 100 000 in the 85+ age group [1]. In the literature, it was difficult to find standardized incidence ratios of MCC in organ recipients; this may be due to the rarity of the tumour and lack of proper registries. The rarity of the tumour makes it tempting to publish case reports and reciprocally makes it difficult to collect large series, producing a publication bias.
Eventually we found that Williams et al. referred to a personal communication by Dr I Penn [11]; the approximate incidence of MCC in Cincinnati Transplant Tumor Registry was three cases per 1000 patients in 1997. Further, many of the compared variables (e.g. ages, mean ages, types of concurrent cancers) are not comparable as such, rather depending on the follow-up time and age of the patient at the time of the transplant operation. Therefore, we suggest that instead of constantly changing variables, the comparison should be based on absolute estimates like the standardized incidence ratio (SIR) or the absolute incidence such as cancers per 100 000 person years. These absolute estimates are comparable among different cohorts and eventually provide accurate and equal information, translated to better patient care.
We conclude that incidence of MCC is very high among subjects who have received a renal transplant. MCCs are usually diagnosed only several years after transplant surgery. In this patient population, MCC appears often to have an aggressive clinical course, frequently resulting in a fatal outcome. Although MCCs are rare, the risk of MCC needs to be borne in mind when organ transplant has been carried out, since early detection and surgical removal might lead to cure. Other skin cancers than MCC are also common among renal transplant recipients, which places further emphasis on the importance of inspecting the skin as a part of the physical examination of these patients.
Conflict of interest statement. None declared.