Transplantation is the treatment of choice for patients with end-stage organ failure. Progressive advances in clinical management and immunosuppressive regimens have resulted in a significant reduction in the number of acute rejection episodes and increased 1-year graft survival rates in transplant recipients (1). However, despite these advances in short-term outcomes after transplantation, there has been minimal change in long-term allograft survival (1–3). No significant changes were observed in a recent study investigating long-term graft failure rates of solid-organ transplants over the past 20 years, with annual graft failure rates 5 to 10 years after transplantation only improving from 4.7% to 4.3% in liver and from 6.4% to 5.1% in heart (1). A key factor in this lack of correlation between short-term and long-term outcomes is the inability for current immunosuppressants to induce a state of specific immunologic unresponsiveness or operational tolerance to donor alloantigens. This results in the patient requiring maintenance immunosuppression for the duration of the graft survival to avoid rejection.
Chronic exposure to these immunosuppressive drugs results in a substantially increased risk of malignancy, which is becoming the most important cause of morbidity and death with a functioning transplant (4–7). U.S. Registry data of 175,732 solid-organ transplant recipients (58.4% kidney, 21.6% liver, 10.0% heart, and 4.0% lung) between 1987 and 2008 detected an overall twofold increased cancer risk in comparison with the general population (standardized incidence ratio [SIR], 2.1; 95% confidence interval [CI], 2.06–2.14) (8). Although there is a general increase in overall cancer risk, there is specifically marked increase cancer risk of all types of skin cancer, particularly nonmelanoma skin cancer (NMSC), as well as virally driven tumors (Table 1) (8–12).
There are multiple mechanisms through which immunosuppression is believed to increase the risk of developing cancer in transplant patients (Fig. 1). The first mechanism is that long-term immunodeficiency may increase the risk of oncoviral-driven malignancy (13). Second, impaired immunosurveillance of neoplastic cells due to the nonspecific mode of action of the majority of immunosuppressive drugs is thought to play a role. Finally, some immunosuppressive drugs may have pro-oncogenic properties. For example, the calcineurin inhibitor (CNI), cyclosporine A (CsA), has been demonstrated to cause a significant reduction in DNA repair mechanisms, as well as azathioprine and prednisolone to a smaller extent, which may in part contribute to the elevated cancer risk observed in these patients (14). In addition to cyclosporine’s effect on DNA repair mechanisms, it has been shown to promote angiogenesis and invasiveness of nontransformed cells in vitro, considered to be a result of cyclosporine-induced transforming growth factor-β production (15, 16). Cancer incidence among transplant recipients is consequently greater than in the general population (17).
As well as being at an increased risk of developing cancer, it has been demonstrated that transplant patients experience worse outcomes with increased tumor development and metastases compared with that of nonimmunosuppressed patients. Several common cancers, including prostate, breast, and colon, have been identified to have a worse disease-specific survival rate in transplant patients compared with the general population. A multivariate analysis of cancer patient survival identified transplantation as a negative risk factor for each of the common cancers tested. Furthermore, transplantation was found to have a greater negative effect on patient survival than the stage of the cancer at diagnosis for both prostate and bladder cancers (18).
Using clinical and laboratory biomarkers may aid identification of transplant patients who are at an increased risk of developing cancer. The resultant risk stratification is important for developing appropriate prevention and early intervention programs for transplant recipients requiring long-term immunosuppression. This review will describe clinical markers of increased cancer risk after transplantation that allow stratification of those transplant recipients at low, medium, and high risks of malignancy. An increasing number of genetic and immunologic biomarkers have been identified that are complementary to existing clinical markers and allow enhanced detection of individual transplant recipients at increased risk of cancer. We will systematically describe these emerging translational genetic and immunologic markers relevant to clinical practice.
Clinical Markers of Posttransplantation Malignancy Risk
The identification of biomarkers that can accurately distinguish patients who are at a higher risk of developing cancer would undoubtedly prove to be clinically useful and potentially guide tailoring an individualized immunosuppression regimen. A prospective UK study of renal transplant recipients used risk factors, including age, Fitzpatrick skin type, outdoor occupation, male gender, duration of immunosuppression, and smoking history, to develop a predictive index of skin cancer risk. A predictive index based on sex, age at transplantation, and eye color was able to identify individuals who went on to develop NMSC with 80% sensitivity and 75% specificity (19). In a follow-up study, in a cohort of Australian renal transplant patients, a simplified clinical predictive index tailored toward the Australian population was developed. This index was able to identify patients who then went on to develop posttransplantation NMSC with a sensitivity of 78.7% and specificity of 89.3% (positive predictive value 87.1%, negative predictive value 82.1%, and accuracy 84.3%; P<0.0001) (20). Importantly, the development and use of a clinical phenotypic predictive index needs to be tailored to the population to which it is being applied.
Suppression of the immune system by immunosuppressive drugs has been shown to allow increased viral loads of oncoviral infections, which is thought to be responsible for the increased prevalence of particular posttransplantation malignancies (21–23). Studies have revealed that monitoring of viral-DNA load in patients with chronic viral infections such as cytomegalovirus, hepatitis B virus, hepatitis C virus, and Epstein–Barr virus (EBV) is important in identifying patients at a higher risk of developing virus-related malignancy (24–27). A specific clinically important example is EBV infection, which is associated with life-threatening cancers, including posttransplantation lympho-proliferative disorder (PTLD), Hodgkin’s and non-Hodgkin’s lymphomas, and nasopharyngeal carcinoma (28). EBV viral load can be used in transplant patients at higher risk of developing lymphoma, although it is not routinely used in all transplant patients despite the increased risk of PTLD (29). There remains the problem that there is no cutoff point of EBV-viral load that identifies patients at high risk of developing an EBV-related malignancy (30). Additionally, solely monitoring viral load has limited use in reducing the numbers of transplant patients who will go on to develop oncovirus-induced malignancy without preemptive measures in place. Studies have shown that the use of antiviral prophylactic treatment when appropriate, in addition to monitoring EBV viral load, has been shown to significantly reduce PTLD-related mortality (31, 32).
After transplantation, active long-term screening protocols are recommended to allow for early identification of certain transplant-related malignancies, allowing intervention before tumor progression. In patients having a liver transplant for a primary cancer, those undergoing intensive posttransplantation screening had significantly improved survival compared with those under standard care (33, 34). In addition, a set of recommendations has recently been published regarding posttransplantation monitoring for cancer detection by the Kidney Disease: Improving Global Outcomes group, with particular focus on patient education in identifying skin and lip cancers (35).
Although these strategies currently used in the clinic have potentially reduced the number of posttransplantation malignancies, new markers need to be developed and validated to accurately identify recipients at particular susceptibility of developing a malignancy after transplantation. Currently, few investigations have shown the capability to identify patients at particular risk of developing malignancy after transplantation. An informed algorithm using translational markers for the classification of patient cancer risk would allow for better minimization of immunosuppressants without the risk of rejection (Fig. 2).
Genetic Markers of Posttransplantation Cancer Risk
Several genetic polymorphisms have been implicated in influencing the risk of developing a de novo posttransplantation malignancy. In a case-control study of 1765 transplant patients, polymorphisms in interferon (IFN)-γ, tumor necrosis factor-α, transforming growth factor-β, and interleukin (IL)-10 were analyzed, which had been previously been linked with EBV-associated diseases in nonimmunocompromised patients (36–38). This study identified that specific polymorphisms in two of the anti-inflammatory cytokines, IL-10 and transforming growth factor-β, were associated with the development of EBV associated PTLD in transplant recipients.
Polymorphisms in the genes encoding vitamin D receptor and methylenetetrahydrofolate reductase (MTHFR) have also been shown to increase the risk of development of several cancers, including squamous cell carcinoma (SCC), in otherwise healthy individuals (39–41). However, polymorphisms in the vitamin D receptor were found to have no association with skin cancer in kidney transplant recipients (KTRs) once adjusted for other risk factors involved in the development of SCC. In contrast, KTRs with the MTHFR 677T polymorphism were found to have a significant increase in the risk of developing an SCC (odds ratio, 2.54; 95% CI, 1.41; P=0.002) (42). A subsequent study by the same group revealed that hypermethylation of the genome could be a potential mechanism by which the increase risk of developing cancer is conferred. They found higher levels of global methylation in both the tumors and the normal skin in patients with the MTHFR C677T polymorphism compared with those without (P<0.002) (43). Although there are several studies that present data on the use of genetic markers identifying transplant patients at particular risk of developing a malignancy, at present, genetic markers are of limited value. Large prospective independent studies are required to confirm the relationship between malignancy and the presence of specific genetic polymorphisms.
Immunologic Markers of Posttransplantation Cancer Risk
The immune system plays a significant role in the prevention of cancers that occur more commonly in transplant patients. Type, duration, and levels of immunosuppression are associated with the frequency and severity of posttransplantation malignancy. For this reason, many studies have strived to identify particular immune phenotypes, which may predispose individuals to developing a cancer after transplantation. These immune biomarkers usually relate to the functionality of the immune system allowing analysis of immunoregulatory or effector populations of cells. A recent study looked into T-regulatory cell populations and their ability to predict risk of cutaneous SCC (cSCC) after renal transplantation hypothesized that high numbers of this subpopulation of cells would result in increased development of cSCCs. The study revealed that high numbers of peripheral FOXP3+CD4+CD127low regulatory T cells (>35 cells/μL) and low numbers of natural killer cells (<100 cells/μL) in KTRs identified patients that were at a higher risk of developing a de novo cSCC. Furthermore, patients who had both high levels of regulatory T cells (>35 FOXP3+ cells/μL) along with low numbers of NK cells (<100 cells/μL) were identified as a high-risk phenotype and had a much higher risk of developing an SCC compared with those without this phenotype (hazard ratio, 3.77; 95% CI, 1.42–10.00; P=0.008 for high-risk phenotype) (44). Phenotyping of peripheral blood cells in another study to correlate findings with clinical outcomes observed a higher number of CD4+CD25+ cells in the CD4+ population in KTRs who had developed a posttransplantation malignancy (9.2%±5.7% vs. 7.0%±3.3%; P=0.04). However, no significant correlation between posttransplantation malignancy and the proportion of CD4+CD25+FOXP3+ (T-regulatory) cells of CD4+ cells was observed. Additional phenotypic markers for effector and regulatory cell populations are required for an accurate predictive value of peripheral blood analysis (45).
The use of T lymphocyte subset analysis, expressly CD4+ T-cell counts, is considered a well-founded predictor of opportunistic infections within immunocompromised hosts (46). Nevertheless, the correlation between CD4 lymphopenia and the risk of developing posttransplantation malignancies remains controversial. In a prospective study, CD3, CD4, and CD8 levels were measured annually over 10 years in a large cohort of renal transplant recipients. They found that patients in this study who developed multiple cancers displayed lower levels of CD4 cells compared with patients without cancer (P=0.003) and even to patients who developed only one cancer during the follow-up period (P=0.02) (47). Unfortunately, a specific threshold or sensitivity for identifying patients at an increased risk of malignancy could not be determined. Furthermore, another large retrospective study involving renal transplant recipients showed no association between CD4 lymphopenia and the risk of developing posttransplantation malignancy (48). In a study of liver transplant recipients, the mean frequency of CD28+CD8+ T cells was significantly lower in patients who went on to develop a de novo malignancy (39%±22% vs. 51%±21%; P=0.008). Moreover, a frequency of CD28+CD8+ cells greater than or equal to 40% in these patients resulted in a significantly reduced proportion of patients who developed a de novo malignancy compared with those with less than 40% (P=0.01) (49).
In summary, many studies have shown a correlation between subsets of immune cells and transplant patients that are at risk of developing malignancy after transplantation. However, much of the data needs to be validated in larger independent studies to confirm predictive value before translation to the clinic. In unpublished data expanding on previously collected data by Carroll et al., we have validated the previous immunophenotype identifying patients at greater risk of developing an SCC as well as including a much broader analysis of effector and regulatory cells of the immune system (44) (unpublished data). Studies investigating a broader analysis of multiple effector and regulatory populations in the blood may prove more useful in the future and provide a more accurate representation of a patient’s immune status.
Investigations into overimmunosuppression in transplant patients have revealed translational markers that can help identify or predict patients at particular risk of developing cancer after transplantation. For example, analysis of the transcription factor nuclear factor of activated T cells (NFAT)–related gene expression has been used as a biological marker for the effect of the CNI, CsA (50). Traditionally, levels of immunosuppression have been tailored through obtaining trough blood levels of the CNI and adjusted to within a target range (51). However, this technique is highly inaccurate as there is huge intrapatient and interpatient variation on the degree of suppression of the immune system for any given immunosuppressive drug level (52, 53).
CsA works by inhibiting calcineurin and down-regulating downstream transcription factors, mainly NFAT (Fig. 3). NFAT is responsible for the transcription of numerous genes that promote lymphocyte activation and proliferation in response to an antigen. In this study, they found that IL-2, IFN-γ, and granulocyte-macrophage colony-stimulating factor were the optimum genes to analyze the effects of CsA on the suppression of NFAT. mRNA levels of IL-2, IFN-γ, and granulocyte-macrophage colony-stimulating factor were measured at trough levels of CsA and 2 hr postdose. Patients with a strong suppression of NFAT-regulated genes, defined as a residual level of transcription of less than 15% after drug dose, were found to develop increased numbers of malignancies (22% vs. 4%; P=0.002) (50). This was then repeated in another population of KTRs, and again, NFAT-regulated gene expression was found to be significantly lower in patients with skin cancer compared with patients without skin cancer (4.94% [0.91–13.4] vs. 11.6% [3.3–40.8]; P<0.001) (54). The same group then demonstrated that NFAT expression monitoring was also useful in both liver transplant recipients and patients on an alternative CNI, tacrolimus (FK506). Once again, they were able to show a strong negative correlation between the peak levels of CsA and FK506 and the residual gene expression of all NFAT-regulated genes (P<0.0001; r=−0.8026 and P<0.0001; r=−0.6982, respectively). These data show promising results and clinical reliability of this assay, which is a step forward to individualized pharmacodynamic drug monitoring. Further large prospective studies are needed to confirm the use of a more individualized assay with respect to clinical outcomes (55).
Malignancy is a major cause of morbidity and mortality after transplantation. Identifying translational markers for the detection of patients at particular risk remains an important clinical challenge. Individual clinical and immunologic markers alone have limited ability to predict risk of malignancy in transplant recipients. A combination of complementary clinical and laboratory-based markers are required to identify transplant patients who are at risk of developing cancer with high degrees of sensitivity and specificity.
Standard clinical practice is to reduce and modify immunosuppression once a transplant recipient has developed cancer (56–59). In the future, the clinical and laboratory-based markers outlined in this review may prove to be powerful tools in identifying patients at high risk of cancer to allow tailoring of immunosuppression to reduce future risk of cancer development. Reduction or discontinuation of immunosuppression is a major challenge to the transplant clinician because of the risk of irreversible damage to the graft (60). An alternative approach is to consider switching patients with a CNI-based immunosuppression protocol to a mammalian target of rapamycin inhibitor, such as sirolimus, which has been shown to have cancer-inhibiting effects in experimental models (16). Registry data outcomes have reported reduced cancer risk in patients on sirolimus-based immunosuppressive regimens (61, 62). In a recent randomized, multicenter trials, patients who were treated with sirolimus and steroids on a CNI-free immunosuppressive regimen 3 months after renal transplantation showed a reduced incidence of both skin and nonskin malignancies 5 years after transplantation compared with patients on a regimen of sirolimus, steroids, and cyclosporine (63). Two recent randomized controlled multicenter trials have shown a reduction in the recurrence rate of cutaneous SCC twelve months after conversion to rapamycin in long-term kidney transplant recipients (64, 65).
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