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Melanoma risk and survival among organ transplant recipients

Solid organ transplant recipients, who are medically immunosuppressed to prevent graft rejection, have increased melanoma risk, but risk factors and outcomes are incompletely documented. We evaluated melanoma incidence among 139,991 non-Hispanic white transplants using linked U.S. transplant-cancer registry data (1987–2010). We used standardized incidence ratios (SIRs) to compare incidence to the general population, and incidence rate ratios (IRRs) from multivariable Poisson models to assess risk factors. Separately, we compared post-melanoma survival among transplant recipients (N=182) and non-recipients (N=131,358) using multivariable Cox models. Among transplant recipients, risk of invasive melanoma (N=519) was elevated (SIR=2.20, 95%CI 2.01-2.39), especially for regional stage tumors (SIR=4.11, 95%CI 3.27–5.09). Risk of localized tumors was stable over time after transplantation, but higher with azathioprine maintenance therapy (IRR=1.35, 95%CI 1.03–1.77). Risk of regional/distant stage tumors peaked within 4 years following transplantation and increased with polyclonal antibody induction therapy (IRR=1.65, 95%CI 1.02–2.67). Melanoma-specific mortality was higher among transplant recipients than non-recipients (HR 2.98, 95%CI 2.26–3.93). Melanoma exhibits increased incidence and aggressive behavior under transplant-related immunosuppression. Some localized melanomas may result from azathioprine, which acts synergistically with ultraviolet radiation, while T-cell depleting induction therapies may promote late stage tumors. Our findings support sun safety practices and skin screening for transplant recipients.


INTRODUCTION
Melanoma is an aggressive form of skin cancer whose rapidly increasing incidence represents a major public health concern in the US (Siegel et al., 2014;United States Department of Health and Human Services, 2014). Risk factors include older age, family history, fair complexion, exposure to UV radiation (UVR; Rhodes et al., 1987), and higher counts of nevi (Olsen et al., 2010). Accordingly, incidence is concentrated in non-Hispanic white persons (Cormier et al., 2006) and among this group increases with decreasing latitude of residence (Eide and Weinstock, 2005).
Melanoma risk has been reported to be increased two to fivefold among solid organ transplant recipients, who are prescribed immunosuppressive medications to prevent graft rejection (Jensen et al., 1999;Kasiske et al., 2004;Hollenbeak et al., 2005;Grulich et al., 2007;Engels et al., 2011;Chatrath et al., 2013;Dahlke et al., 2014). This elevation could result from immune dysfunction or from direct carcinogenic effects of some medications (Hojo et al., 1999;O'Donovan et al., 2005;Han et al., 2012). In addition, transplant recipients are screened intensively for skin cancer due to a very high risk of cutaneous squamous-cell carcinoma (Ulrich et al., 2008). Thus, the melanoma excess could partly represent overdiagnosis, in which case it should be most pronounced for early-stage tumors (Welch et al., 2005).

See related commentary on pg 2560
ORIGINAL ARTICLE 1 Furthermore, immune response may be important in controlling melanoma after diagnosis. This possibility is supported by the recent success of immunomodulatory therapies in treating patients with metastatic melanoma (Hamid et al., 2013;Wolchok et al., 2013). If immune response is important, one would predict that melanomas would behave more aggressively when it is impaired, and there is some evidence for increased melanoma mortality among transplant recipients (Brewer et al., 2011;Vajdic et al., 2014).
Clinical management of transplant recipients would be informed by understanding of risk factors and outcomes for melanoma in this population. Prior studies have been small, and transplant-related risk factors are poorly documented. In the present study, we evaluated the epidemiology of melanoma in a large population of US transplant recipients and assessed the impact of transplantation on melanoma survival.

Incidence analysis
A total of 139,991 transplants in non-Hispanic white patients contributed 701,358 person-years of follow-up for incident melanoma (Table 1). Median follow-up time among all transplants was 4.0 years (interquartile range 1.4-7.6 years); this was similar across organ types with the exception of lung recipients who had a median follow-up of 2.6 years. Most transplants occurred in patients who were male (62.9%), aged 35-64 years (69.2%), and received a kidney (50.5%). Transplants of the liver (24.6%), heart (11.7%), lung (5.9%), and other or multiple organs (7.3%) were less common.
Invasive melanoma was diagnosed in 519 transplants and in situ melanoma in 190 transplants (incidence rates 74.0 and 27.1 per 100,000 person-years, respectively). Risk of invasive melanoma was elevated more than twofold above the general population (standardized incidence ratio (SIR) 2.20, 95% confidence interval (CI) 2.01-2.39; Table 2). Although risk was elevated across tumor stages, the greatest increase was for regional stage melanoma (SIR = 4.11, 95% CI 3.27-5.09). By tumor site, risk was strongly elevated for melanomas on the head and neck (SIR = 3.34, 95% CI 2.85-3.90), with more modest increases for other sites. Risk was also elevated for in situ melanoma (SIR = 1.47, 95% CI 1.27-1.69, Table 2).
Examination of melanoma risk by stage and time since transplantation revealed two distinct patterns. Risk of regional and distant stage melanoma increased markedly within 4 years after transplantation (up to sixfold for regional stage tumors) and then declined, whereas risk of in situ and localized melanoma was elevated~1.5-to 2-fold consistently over time ( Figure 1).
To investigate these patterns, we separately evaluated risk factors for localized and regional/distant stage melanoma. Supplementary Table 1 (online) shows adjusted associations for UVR, medications, and transplant-related characteristics, separately according to stage. We did not observe any statistically significant associations with our ecological measures of UVR exposure, thus preventing their inclusion in the final models. However, we note that the trends for localized stage tumors were suggestive (P-values for trend across quintiles of 0.052 for average daily global solar radiation (AVGLO) and 0.079 for latitude, Supplementary Table 1 online). In our final multivariable model for localized melanoma (Table 3), higher risk was associated with male sex, increasing age, and azathioprine maintenance therapy (incidence rate ratio (IRR) = 1.35, 95% CI 1.03-1.77). Compared with kidney recipients, risk was lower in liver recipients (IRR = 0.60, 95% CI 0.45-0.80) and lung recipients (IRR = 0.50, 95% CI 0.26-0.95). Incidence did not vary significantly by time since transplantation, although it was suggestively higher in some later intervals.
In the multivariable model for regional/distant stage melanoma (Table 3), risk increased with male sex, increasing age, and polyclonal antibody induction therapy (IRR = 1.65, 95% CI 1.02-2.67). Incidence increased sharply in the first 4 years after transplantation before steadily declining.

Survival analysis
For survival, we evaluated 131,540 patients diagnosed with invasive melanoma, of whom 96% were of white race. Melanomas were largely local stage (76%), with smaller proportions of regional (8%), distant (4%), or unknown stage (13%). On the basis of linkage to the Scientific Registry of Transplant Recipients (SRTR), 182 melanomas (0.14%) occurred in transplant recipients.

DISCUSSION
In this large, representative series of solid organ transplant recipients, invasive melanoma incidence was increased over twofold above rates seen in the general population.
We observed notable differences by tumor stage in the timing of onset and melanoma risk factors. Also, melanoma-specific mortality was elevated threefold compared with non-recipients, suggesting that melanoma behaves aggressively under transplant-related immunosuppression.
One possible interpretation of our results for localized melanoma is that medications that increase UVR-induced DNA damage, coupled with continued UVR exposure, contribute to the development of early melanomas. Incidence of localized tumors was increased in recipients prescribed azathioprine, which may accelerate UVR-induced DNA damage (O'Donovan et al., 2005;Clarke et al., 2015). In addition, we observed suggestive increases with two measures of UVR exposure, AVGLO (P trend across quintiles = 0.052) and latitude (P trend across quintiles = 0.079; Supplementary Table 1 online); we note that these measures are ecological and may not be good proxies for individual-level exposure. Although UVR exposure in early life may be most relevant for melanoma (Holman et al., 1986;Nelemans et al., 1993), the association with azathioprine suggests that UVR exposure occurring after transplantation could also affect risk. One caveat is that we only examined immunosuppressive medications indicated at baseline. Also, although we adjusted for calendar year of transplantation, there have been strong time trends in medication use, which may have led to residual confounding and impacted these results. Compared with kidney recipients, we observed lower risk for localized melanoma among liver and lung recipients, but we do not have an explanation for this particular result.
For regional and distant stage melanoma, we found a high risk soon after transplantation that may relate to short-term, intense immunosuppression. Risk of regional/distant stage tumors peaked within 4 years of transplantation and increased with T-cell-depleting polyclonal antibody induction therapy. Each of these patterns was also observed for melanoma overall in an Australian study (Vajdic et al., 2009). Consistent with a short-term effect of intense immunosuppression, melanoma incidence declines after graft failure in kidney recipients, when patients return to dialysis and immunosuppressive therapy is ceased or reduced (Vajdic et al., 2009;van Leeuwen et al., 2010).
Although the steady incidence of localized melanomas after transplantation may represent the occurrence of de novo tumors, a plausible explanation for the sharp increase in regional and distant melanoma is that melanocytic precursors or early-stage melanomas were already present but undiagnosed at the time of transplant, and they progressed rapidly with intense immune suppression. Consistent with this model, melanocytic nevus counts increase after transplantation (Smith et al., 1993;Grob et al., 1996), sometimes in an eruptive manner and/or with the presence of dysplasia (Barker and MacDonald, 1988;McGregor et al., 1991). Melanomas express a range of neoantigens that can serve as targets for T cells (Lennerz et al., 2005), and among melanoma patients lower host immune response to the tumor (as measured by tumor-infiltrating lymphocytes) is associated with a larger tumor size and a greater likelihood of sentinel lymph node positivity (Taylor et al., 2007;Azimi et al., 2012). The excess risk of regional and distant stage melanoma observed here is inconsistent with overdiagnosis, as frequent skin cancer screening in transplant recipients would shift the stage distribution downward (Welch et al., 2005). In passing, we note that melanoma can be transmitted from donors to recipients through the donated organ, but such a transmission is very rare and does not likely account for our findings (MacKie et al., 2003;Strauss and Thomas, 2010).
Our survival analysis supports a further role for immune response in controlling melanoma progression after clinical diagnosis. Transplant recipients had a threefold increased risk of dying from their melanoma compared with melanoma patients without a transplant; this is generally consistent with most (  The localized tumors-including for thin tumors with Breslow thickness o1 mm-implies that there may be subclinical spread of these tumors in transplant recipients and, in turn, that intact immune responses may normally prevent this spread. Importantly, although our data on surgical treatment were limited, our sensitivity analysis did not support that treatment differences explain the decreased survival of transplant recipients. In other contexts, survival following a melanoma diagnosis correlates with multiple measures of the cellular immune response including density, distribution, and activation of tumor-infiltrating lymphocytes (van Houdt et al, 2008;Azimi et al, 2012). Melanoma risk is increased among immunocompromised populations other than transplant recipients (Kubica and Brewer, 2012). People infected with HIV experience an excess (Grulich et al, 2007), but it is smaller, possibly due to differences in the mechanism or rapidity of onset of immunosuppression or to differences in population structure, which can confound SIR comparisons. Chronic lymphocytic leukemia (CLL) is a malignancy characterized by immunosuppression, which can be intensified by CLL treatment, and melanoma risk is approximately threefold increased among CLL patients (Travis et al, 1992;Adami et al, 1995;Hisada et al, 2001). Survival after melanoma may also be decreased in these populations (Rodrigues et al, 2002;Brewer et al, 2012).
Our complementary analyses of melanoma incidence and survival allowed us to assess the impact of immunosuppression along a continuum of outcomes. Linkage of transplant and cancer registries yielded a population-based sample of nearly half of the US transplant population, and cancer registries provided systematic ascertainment of melanomas, as well as information on stage, site, and melanoma-specific mortality. Our study is also subject to the typical limitations of analyses based on registry data. We were limited in our ability to assess some clinically relevant information (e.g., tumor Clark's level and Breslow thickness, sentinel lymph node biopsy, details on surgeries) because data were incomplete or unavailable. Our survival analyses were based on death certificate-coded cause of death, which could be inaccurate for transplant recipients who have multiple chronic medical issues. There is also the possibility of differential diagnosis, staging, or reporting of melanoma for transplant recipients compared with non-recipients. For example, transplant recipients may be more likely to be diagnosed with advanced melanomas in hospital settings, where reporting to cancer registries is more complete than for thinner tumors diagnosed in dermatology offices (Cockburn et al., 2008), and because of their poorer health they may receive a different diagnostic work-up compared with non-recipients. On the other hand, the identification of melanoma may be more difficult among transplant recipients if the frequent presence of other skin lesions makes some melanomas difficult to identify. Finally, although the associations that we observed with melanoma incidence (e.g., for azathioprine maintenance and polyclonal antibody induction) could indicate causal effects, we cannot rule out that they are due to bias, chance, unmeasured confounding factors, or other complexities related to the analysis of linked data sets.
Because risk for multiple types of skin cancer is high, transplant recipients should be encouraged to minimize unnecessary UVR exposure and adopt sun-protective behaviors (Ulrich et al., 2009). Our results also highlight the importance of a thorough dermatologic evaluation for transplant candidates before transplantation, with the goal of detecting and removing both small melanomas and precursor lesions that could rapidly progress to invasive melanoma. Close monitoring within 4 years of transplantation is warranted, particularly for recipients with risk factors for late-stage melanoma such as male sex, older age, or receipt of T-cell-depleting induction therapy. For transplant recipients Abbreviations: CI, confidence interval; NOS, not otherwise specified; SIR, standardized incidence ratio. who do develop melanoma, physicians should perform an appropriate staging evaluation, including a clinical assessment of lymph node involvement and other distant spread (Fong and Tanabe, 2014). Along with surgery directed at the primary tumor, treatment should incorporate reduction or revision of immunosuppression, to the extent possible, to facilitate immunologic control of the tumor.
In conclusion, transplant recipients have an elevated risk of melanoma that may be related to immunosuppressive medications used for transplant induction and maintenance. Compared with melanomas in immunocompetent people, melanomas in transplant recipients occur at advanced stage and are associated with poor survival. Evaluation of risk in other immunocompromised populations, as well as molecular characterization of tumors in immunosuppressed patients, may yield further clues to the relationship between immune responses and melanoma.

Incidence analysis
The Transplant Cancer Match Study (www.transplantmatch.cancer. gov) links the SRTR, which captures data on all transplants occurring in the US, with 15 population-based cancer registries (Engels et al, 2011). Linkage between the SRTR and cancer registries was performed using a probabilistic matching algorithm based on name, sex, date of birth, and social security number, followed by clerical review of potential matches. The resulting cohort includes 46.5% of the US transplant population during 1987-2010-specifically, transplant recipients in California (years of follow-up: 1988), Colorado (1988), Connecticut (1987), Florida (1987), Georgia (1995), Hawaii (1987, Illinois , Iowa (1987), Michigan (1987), New Jersey (1987), New York (1987), North Carolina (1990), Seattle (1987), Texas (1995), and Utah (1987 see Table 1 footnote for numbers of transplants by registry). The Transplant Cancer Match Study was approved by human subjects research review committees at the National Cancer Institute and, as required, at participating cancer registries. The outcome for our incidence analysis was first diagnosis of cutaneous melanoma (invasive or in situ); subsequent melanoma diagnoses were not further considered. Transplant recipients were followed from the later of transplantation or beginning of cancer registry coverage and exited at the earliest of melanoma diagnosis, organ failure, a subsequent transplant, loss to follow-up, death, or end of cancer registry coverage. Transplants performed at different times on the same individual were considered separately. We restricted analysis to non-Hispanic whites, as only 26 invasive melanoma cases occurred outside of this group. We further excluded 320 transplants with melanoma diagnosed before transplantation and 128 transplants in people with HIV infection.
We compared melanoma risk in transplant recipients with the general population using SIRs. SIRs were calculated as the number of observed melanoma cases divided by the number expected, based on general population rates specific to registry, 5-year age group, sex, race/ethnicity, and calendar year. We estimated SIRs overall, by tumor stage and site, and in cross-classified categories by tumor stage and time since transplantation. For tumor stage, we used the summary-stage variable, which has three levels (local, regional, and distant) and is largely complete in cancer registries. Summary stage allows summarization of different and regularly updated clinical staging systems (e.g., American Joint Committee on Cancer editions), thus enabling reliable classification of stage for patients diagnosed over time.
We used zip codes of residence provided by the SRTR to link transplants to two ecological measures of UVR exposure, which we divided into quintiles of equal range (after excluding extreme outliers). The first was latitude, which we assigned using a public database (CivicSpace Labs, 2004). The second was a measure of predicted 30-year AVGLO that has been associated with melanoma risk (Tatalovich et al., 2006a,b). Some recipients could not be assigned these measures based on their zip code. For recipients in states where the range of latitude or county-level AVGLO fit completely or very nearly within a pre-defined quintile, we imputed quintiles with a maximum error of 0.5 degrees (latitude) or 41 watt-hours per square kilometer (AVGLO). For latitude, we combined the lower two quintiles (high/highest UVR) owing to sparse observation time.  -up: 1988number melanoma cases = 14,367), Connecticut (1987, n = 15,103), Georgia (1995, n = 22,331), Iowa (1987, n = 11,189), New Jersey (1987, n = 31,883), and Texas (1995. HRs are adjusted for age (in categories of 0-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, and 490 years), sex, race (white, non-white), year of melanoma diagnosis (1987-1994, 1995-1999, 2000-2005, and 2006-2010), and primary site (head and neck, upper limb and shoulder, trunk, lower limb and hip, and overlapping/not otherwise specified). The overall HR for all invasive melanomas is additionally adjusted for tumor stage (localized, regional, distant, and unknown).
We calculated IRRs to compare incidence between groups of transplant recipients. We adjusted, a priori, for age, sex, transplanted organ, time since transplantation, and year of transplantation (see Table 3 for details). We estimated adjusted IRRs for groups defined by receipt of individual induction and maintenance medications (as recorded at the time of transplant), UVR exposure (latitude and AVGLO) and for kidney recipients, by living/deceased donor status and history of acute rejection. On the basis of these results, we included variables with significant IRRs (Po0.05) in multivariable Poisson models. As described in the Results section, these models were fit separately for localized and regional/distant stage melanoma, because we observed different patterns of incidence over time by tumor stage suggestive of distinct biological processes.

Survival analysis
To compare survival after melanoma diagnosis between transplant recipients and non-recipients, we used data from a subset of the cancer registries in the Transplant Cancer Match Study. Of the eight registries providing vital status follow-up and cause of death information, we eliminated two that appeared to have incomplete follow-up for mortality. Our study population for survival analysis thus included data from Colorado (years of melanoma diagnosis and follow-up: 1988), Connecticut (1987), Georgia (1995), Iowa (1987), New Jersey (1987, and Texas (1995-2010; see Table 4 footnote for numbers of melanoma cases by registry). Among individuals in the general population who were diagnosed with melanoma (i.e., both transplant recipients and non-recipients), we restricted to melanoma cases occurring as an individual's first diagnosis of invasive cutaneous melanoma (n = 134,096). We then excluded cases with missing/unknown cause of death (n = 2,556). The survival analysis included patients of all races/ethnicities. Melanoma patients were classified as transplant recipients if they linked to a transplant in the SRTR that occurred before their melanoma diagnosis. Other individuals were classified as nonrecipients. Individuals who received transplants after melanoma diagnosis (n = 72) were classified as non-recipients and were censored at transplantation.
The primary survival outcome was death due to melanoma, which we assessed using underlying cause of death codes indicated on death certificates. Follow-up time began at melanoma diagnosis and ended at the first of death, loss to follow-up, or the end of cancer registry ascertainment of deaths. Individuals were censored if they   died of another cause or were still living at the end of cancer registry coverage. We calculated melanoma-specific survival estimates using the Kaplan-Meier method.
We fit a Cox proportional hazards model to assess the effect of transplant status, adjusting for age, sex, race, diagnosis year, tumor site, and tumor stage (see Table 4 footnote for details). We tested the proportional hazards assumption for transplant status by allowing different HRs for four intervals after melanoma diagnosis (o1, 1-1.9, 2-2.9, and ⩾ 3 years). Separately, we fit models stratified by tumor stage.
We performed three sensitivity analyses. First, we restricted to non-Hispanic whites diagnosed beginning in 1992 (when Hispanic ethnicity data became available). Second, we aimed to assess whether possible treatment differences between transplant recipients and non-recipients influenced survival differences. Localized melanomas comprised the majority of cases, and transplantation was most strongly associated with mortality in this group. Therefore, in the second sensitivity analysis, we restricted to localized cases who were reported by cancer registries to have received surgical treatment. Finally, Breslow thickness was unknown or missing for 42% of melanoma cases, precluding its inclusion in the primary analysis. As a sensitivity analysis, where Breslow thickness was known, we classified localized melanomas as thin (o1 mm) or thick (⩾1 mm) and evaluated the effect of transplantation in each category.