We retrospectively analyzed a cohort of 930 kidney transplant recipients at the Fondazione IRCCS Ca’ Granda Ospedale Policlinico Maggiore, Milan, Italy, who underwent transplantation between 2004 and 2021. Patients were followed until September 2023 with a median observation period of 7 [1–19] years. Data were recorded from both paper and electronic hospital records.

According to our protocols, blood and urine analyses were performed after a 12-h fast by the nursing staff of the Transplantation Outpatient Clinic.

For each of the 930 KTRs, the following data were recorded:

- at the time of KTx, anthropometric and clinical characteristics: age, sex, type of transplant, dialysis vintage and type, basic nephropathy, presence of diabetes mellitus, body mass index (BMI), steroid use before KTx, and HCV serology.

Biochemical parameters were measured according to the routine methodology used by our central laboratory. Serum creatinine was assessed by Jaffe’s reaction, whereas urinary protein excretion was assessed by measuring 24-h urine collection protein using the immunoturbidimetric method.

Data on the IS therapy regimen were collected during the first year after KTx; specifically, both the induction IS therapy with basiliximab or ATG and the subsequent maintenance regimen at T1 and T12 with steroids, calcineurin inhibitors (CNI), mycophenolate/mycophenolic acid (MMF), and/or mTORi were fully evaluated. For ATG use, the cumulative dose was expressed in mg/kg; for steroid use, the overall exposure of patients at T1 and T12 was calculated in mg. According to our internal protocol, basiliximab was used in cases of first KTx and low immunologic risk based on donor type and recipient immunologic status. ATG was administered to patients with high immunologic risk or in cases of re-transplantation.

At our center, KTRs undergo regular cancer screening, including an annual abdominal ultrasound and graft examination, along with skin, urological, and gynecological exams. Colorectal cancer screening follows general guidelines. We recorded the time of cancer onset, tumor details, recurrence, staging, management, and therapy. The cohort was divided into CA+ (KTRs who developed cancer) and CA- (KTRs who did not develop cancer). Cancer types included NMSC (graded according to Broder’s system [12]), solid tumors, PTLD, and Kaposi’s sarcoma. We analyzed survival from cancer and KTx, along with patient and graft outcomes post-cancer.

Patients were followed up for a median time of 7 [1–19] years. At the end of the follow-up, we evaluated as principal outcomes graft loss and death with functioning graft both globally and related to IS modifications after cancer diagnosis.

Continuous variables were presented as mean and standard deviation or median [25°–75° percentile] for non-normally distributed data. Qualitative data were presented as frequencies and percentages. Group differences were assessed using the Student’s t-test and the Wilcoxon-Mann-Whitney test for normally and non-normally distributed continuous variables, respectively. The nominal variable analysis employed the χ² and Fisher tests. Logistic regression models were used for multivariate analysis of cancer risk factors. Survival analysis utilized Kaplan-Meier curves with the Log-Rank test, a multivariable time-dependent Cox model, and competing risk analysis to assess the mortality risk associated with IS induction therapy. The multivariate analysis variables were determined based on univariate testing. ROC curve analyses with the evaluation of the Youden Index were used for discriminatory analyses. Statistical significance was set at p < 0.05. All the statistical analyses in this manuscript were conducted using SPSS 27^® software and JMP Pro 15^®. Treatments and procedures reported herein were in accordance with the ethical standards of the institutional committee where the study was conducted (Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Ethical Committee, Protocol ID 4759-1837/19), and with the tenets of the Helsinki declaration of 1964 and its later amendments, or comparable ethical standards.

The cohort consisted of 930 KTRs, 537 of them men, with a mean age at KTx of 49 ± 13 years. The main clinical characteristics of KTRs are summarized in Table 1.

The majority of patients were on dialysis prior to KTx, with a higher prevalence of hemodialysis and a mean dialysis vintage of 52 ± 51 months. The majority of patients received KTx from a deceased donor. The most common cause of kidney disease in the overall cohort was glomerulonephritis (20.5%) (Figure 1).

Data regarding induction and maintenance IS are shown in Supplementary Table S1. The majority of patients who received induction therapy had been treated with basiliximab (74%) while ATG was administered in 26% of cases, with the latter having a mean individual dose of 4.8 ± 1.1 mg. Of note, between 2004 and December 2015 (KTRs, n = 576), only 10.4% (KTRs, n = 60) of patients received ATG, whereas between 2016 and 2021 (KTRs, n = 355), ATG and basiliximab were used in 53.8% (KTRs, n = 191) and 46.2% of cases, respectively. Maintenance therapy included, in the majority of cases, the use of steroids with a cumulative dose of 1094 ± 438 mg at T1 and 2915 ± 1004 mg at T12; CNI (tacrolimus 91.9%, ciclosporin 7.7%) and mycophenolate/mycophenolic acid (94.6%) were the most represented categories. Only 2.6% of patients were prescribed mTORi. No significant differences were observed in the distribution of IS regimens from T1 to T12.

The biochemical and anthropometric parameters of the entire cohort at T1 and T12 are summarized in Table 2. Among the anthropometric characteristics, BMI at T1 showed a substantial increase during the first year post-KTx. No differences were found in blood pressure control. Kidney function did not differ significantly between T1 and T12. Regarding the biochemical data, the levels of hemoglobin, albumin, uric acid, and 25-OH-vitamin D increased during the 12 months; lipid control remained stable during the first year after KTx.

During the follow-up, 177 KTRs (19%) were diagnosed with at least one cancer, with a mean time to development from KTx of 83 ± 48 months.

Available data showed the occurrence of de novo cancer in 113 patients and the recurrence of a pre-KTx tumor in 9 cases; in 55 subjects, cancer could not be defined as de novo nor recurrent. NMSC was the most common cancer type (55.1%), while solid tumors were observed in 69 patients (38.6%); PTLD and Kaposi’s sarcoma were diagnosed in 4% and 2.3% of cases, respectively (Supplementary Table S2).

In one-third of the KTRs (n = 58), a second cancer further developed (specifically 36 NMSC, 19 solid tumors, 2 PTLD and 1 Kaposi’s sarcoma), with recurrence in half of the cases (50% de novo cancer). The mean time to the onset of the second neoplasia after KTx was 122 ± 53 months. For both first and second tumors, the most prevalent grade of NMSC was G2; among solid cancers, the most frequent primary cancer sites were breast (13.2%), prostate (11.7%), cervix (10.3%), lung (10.3%), and urothelial carcinoma (8.8%). Considering first malignancies, 6% of cases were diagnosed with lymph node involvement and 4% with metastatic disease at diagnosis.

Table 3 and Supplementary Table S3 summarize the clinical, biochemical, and anthropometric differences observed between CA+ and CA- during follow-up.

CA+ exhibited a statistically significant difference in age, being older than CA- (p < 0.001). The incidence of malignancy was higher in men, with a mean age at diagnosis of 54 ± 10 years. Although not of causal meaning, CA+ correlated significantly with KTx from a deceased donor (p = 0.04) and dialysis treatment by any method (p = 0.06) with a mean dialysis vintage of 57.8 ± 57.1 months. There was no statistically significant difference between the two groups regarding diabetes, steroids pre-KTx or HCV positivity before KTx.

CA+ showed a higher prevalence of vasculitis and ADPKD, as well as basal nephropathy.

Moreover, CA+ correlated with significantly higher BMI values at T1 and T12 (p = 0.018 and p = 0.08, respectively). No other anthropometric differences were found.

When comparing biochemical variables, no significant differences were found between CA+ and CA- for renal function values such as serum creatinine and proteinuria at T1 and T12. In addition, no significant difference was found for the values of hemoglobin, albumin, blood glucose and uric acid at T1 and T12. However, during the regular management of KTRs, higher levels of total cholesterol at T12 (p = 0.034) and lower levels of 25-OH-vitamin D at T1 and T12 (p = 0.08 and p = 0.033) were reported in CA+ compared to CA-.

In the first year after KTx (Table 4), a significant correlation with higher cumulative therapy with steroids at T1 was found in CA- (p = 0.03), a result that was not confirmed at T12. Furthermore, the significance of the results of the IS regimen containing CNI at T1 and T12 remains difficult to interpret considering the almost ubiquitous use of tacrolimus in the maintenance regimens of the entire cohort.

Regarding induction IS therapy, in the entire follow-up cohort, basiliximab showed a significant correlation with cancer development, a result that was not confirmed when the induction therapy was evaluated between 2016 and 2021. In this specific period, characterized by a homogeneous and comparable administration of two different drugs in induction, the use of ATG proved to be significantly associated with cancer development after KTx.

We did not detect a significant difference in the average individual dose of ATG between the groups, which, according to the Center’s strategy, we attempted to maintain as a target of <5 mg/kg.

In the multivariate analysis, BMI at T1, cumulative exposure to steroid therapy at T12, and use of basiliximab or ATG were included as dependent variables, with post-KTx cancer as the independent variable.

Induction therapy with ATG emerged as an independent and modifiable risk factor with a statistically significant association with cancer development after KTx (p = 0.014, HR 3.43, CI 1.287–9.153) (Table 5).

The critical role of ATG in oncological risk was confirmed by survival analyses. This was first demonstrated in the Kaplan-Meier analysis (p < 0.0001) and subsequently confirmed by both the Cox (p < 0.01 HR 3.4) and competing risk (p < 0.0001, time to cancer onset: basiliximab 89 ± 4 vs. ATG 40 ± 4 months) analyses (Figure 2; Table 6).

Oncological management for the different types of cancer included a surgical approach (75.1%), radiotherapy (8.4%), and/or active oncological treatment (17.5%). In the majority of individuals who underwent active systemic treatment, cytotoxic chemotherapy (61.3%) was the leading indication, while some CA+ patients started targeted therapy, immunotherapy or a combination of different classes of drugs (Figure 3). In the subgroup with second post-KTx neoplasia, active oncological treatment was initiated in 7 cases. Of note, at least 10 patients did not receive the optimal oncological treatment because of the potential risk of toxicity for the transplanted graft (i.e., no treatment or choice of less nephrotoxic drugs).

At cancer diagnosis, the mean serum creatinine was 1.53 ± 0.74 mg/dL. There were no reports of nephrotoxicity or renal adverse events directly associated with oncological therapy (acute kidney injury, proteinuria, electrolyte imbalance, microangiopathy, etc.); in two cases, acute renal failure of pre-renal origin occurred.

In 113 cases (65.7%), no major changes in the IS maintenance therapy regimen were made after cancer diagnosis, except for a general reduction in trough levels (trough levels: tacrolimus 3–5 μg/L–cyclosporine 80 μg/L). In total, 39.3% of CA+ subjects were subjected to modifications of the IS therapy, 12.2% of them in combination with the start of an active oncological therapy. These modifications consisted of the introduction in 38 cases (64.4%) of the mTORi regimen, which allowed the serum levels of CNI to be consensually minimized. In 16 cases (27.1%) an antimetabolite drug was permanently discontinued to achieve a dual IS regimen; of the available data, five patients (8.4%) underwent both therapeutic indications.

A total of 125 patients (13.4%) experienced graft failure and returned to dialysis after a median time from KTx of 57.3 [8.4–103.1] months. Of these, 9.6% were CA+ and in four cases a cancer-related cause was associated with the return to dialysis (in most cases due to explantation for cancer involving the graft).

During follow-up, 124 patients (13.3%) died, of whom 31.6% were CA+ and 9% CA-. Mortality was mainly due to septic shock (24.1%), cancer-related causes (23.3%), or cardiovascular events (18.5%). Median survival from cancer diagnosis to death was 23 [7.9–59.4] months, with a higher prevalence of cancer-related causes of mortality (51.7%) than all other causes in CA+.

Comparing CA+ and CA-, the median survival from KTx to the end of the follow-up was 148 [88–191] months in CA+ and 82 [45–147] months in CA-, confirming that tumor development and cancer-related mortality are common complications in long-term transplantation. Using competing risk analyses, we investigated the possible relationship between induction therapy and mortality. Interestingly, we found a significantly lower survival in the ATG-treated group (148 ± 2 vs. 89 ± 3 months, p < 0.0001).

We investigated the correlation between IS therapy strategies following cancer diagnosis and survival outcomes in CA+. Although no significant differences were observed when comparing various modifications in IS therapy, with no change in median survival (60.3 ± 47 vs. 63.3 ± 43 months, p = 0.339), a significant prognostic impact was identified regarding changes in the type of IS therapy regimen. Specifically, switching to mTORi and reducing CNI were associated with prolonged overall survival post-cancer diagnosis compared to patients who definitively discontinued one drug, while maintaining a dual therapy approach (67.4 ± 40 vs. 34.4 ± 40 months, p = 0.004). This was also investigated, by non-parametric analyses taking into account the type of cancer, dividing our cohort into NMSC and other cancers. Both in the NMSC group and the other cancers group, switching to mTORi and reducing CNI were associated with prolonged post-cancer overall survival: NMSC: 61 ± 35 vs. 34 ± 48 months, p = 0.002; other cancers: 61 ± 35 vs. 34 ± 40 months, p = 0.01.

No differences in principal anamnestic and biochemical parameters were found between those patients who switched to mTORi and those who did not.