The impact of everolimus on hematologic parameters in patients with renal angiomyolipoma associated with tuberous sclerosis complex

Abstract

Background

Everolimus is an effective treatment for renal angiomyolipoma associated with TSC (TSC-RAML). However, its impact on hematologic parameters in TSC-RAML patients remains unclear.

Methods

Hematologic data were collected from TSC-RAML patients undergoing everolimus treatment in two registered clinical trials. Dynamic changes in hematologic parameters during treatment were analyzed. Additionally, we also explored variations in hematologic impact based on gender and age within the patient population.

Result

A total of 55 patients from the two clinical trials are included in this analysis. Hemoglobin, white blood cells (WBC), lymphocytes, neutrophils, and platelet showed significant decreases during everolimus treatment (P < 0.05). However, the decline in hemoglobin, WBC, and neutrophils attenuated by the 12th month (P ≥ 0.05). Aspartate transaminase (AST), Alanine transferase (ALT), total cholesterol (TC), and triglyceride (TG) increased significantly during everolimus treatment (P < 0.05), and these increases persisted throughout the year-long treatment. Hemoglobin decreased significantly more in male patients (− 15 vs − 6, P = 0.010), and AST showed a more significant increase in males (7.0 vs 3.0, P = 0.041). Platelet counts decreased significantly more in younger patients (≤ 30 years old) compared to older patients (− 50 vs − 14, P = 0.020).

Conclusion

Everolimus administration in TSC-RAML patients may increase hematologic risks, with male and younger patients potentially exhibiting greater susceptibility to these effects.

Introduction

Tuberous sclerosis complex (TSC) is a rare genetic disorder characterized by multiple organ lesions resulting from mutations in either TSC1 or TSC2 genes. The protein products of these genes negatively regulate the mammalian target of rapamycin (mTOR) signaling pathway [1, 2]. TSC-related renal lesion is prevalent among TSC patients, with up to 80% exhibiting renal abnormality, among which angiomyolipoma is the most common, affecting 56% of boys and 66% of girls [3]. Renal disease also is reported as a leading cause of mortality in these TSC patient [4]. Renal angiomyolipoma associated with TSC (TSC-RAML) predominantly affects younger individuals and is characterized by larger, faster-growing, multiple, and bilateral lesions, posing a higher risk of renal rupture and hemorrhage compared to sporadic renal angiomyolipoma (S-RAML) [5–7]. Treatment options were historically limited until the emergence of everolimus as a promising therapy in recent years [8]. Several clinical studies have demonstrated the efficacy of everolimus, an mTOR inhibitors, in reducing TSC-RAML volume, preventing tumor rupture and bleeding, and preserving renal function. Discontinuation of everolimus results in tumor rebound, necessitating its continuous administration to suppress tumor growth [9–11]. However, the use of everolimus is associated with significant side effects that can diminish a patient's quality of life, necessitating dose adjustments or treatment discontinuation [12]. Studies indicate that everolimus administration in TSC-RAML patients may increase hematologic risks, but understanding of its specific impact on hematologic parameters remains limited [10, 11].

In this study, we aim to elucidate the dynamic changes in hematologic parameters during one year of everolimus treatment among TSC-RAML patients. Furthermore, we investigate how these changes vary based on gender and age within the patient population. We anticipate that our research will provide valuable insights and guidance for optimizing everolimus management in TSC-RAML patients, thereby minimizing potential risks associated with treatment.

Materials and methods

Study design

Our research team conducted two previous clinical trials aimed at evaluating the effects of everolimus on TSC-RAML in the Chinese population. One trial, referred to as clinical trial 1, was a 2-year, nonrandomized, open-label trial, phase 2 study (ChiCTR-OPC-14005488, registered on the Chinese Clinical Trial Registry, https://www.chictr.org) [10]. The other trial, referred to as clinical trial 2, was a 1-year open-label, single-arm, multi-center Phase IV study (NCT03525834, registered on ClinicalTrials.gov, https://clinicaltrials.gov/study/NCT03525834) [11]. Retrospectively, we analyzed the prospectively collected hematologic data from TSC-RAML patients undergoing everolimus treatment in these two clinical trials to investigate the potential impact of everolimus on hematologic parameters.

Patients and clinical data

The patients enrolled in both trials were diagnosed with definite TSC based on the consensus criteria published in 2013 [13]. All participants were Chinese adults aged 18 years or older. The two published studies provided comprehensive details regarding the inclusion and exclusion criteria [10, 11]. Initially, patients received an oral dose of 10 mg of everolimus per day, which was subsequently adjusted based on safety considerations during treatment.

Blood samples for hematological evaluation, including blood cell counts and biochemical analyses, were collected from patients between 8:00 and 10:00 a.m. at baseline, and at the 3rd, 6th, and 12th months of treatment. If potential factors, such as active infection or inflammation, were suspected to cause significant fluctuations in hematologic parameters, the patients would undergo re-evaluation in a timely manner.

The protocols of both studies were reviewed and approved by the respective local independent ethics committees at each center. The studies were conducted in compliance with the guidelines of Good Clinical Practice (GCP) and the ethical principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all patients before their enrollment.

Statistical methods

Numerical variables were presented as the mean ± standard deviation (SD) if they followed a normal distribution, or as the median (first quartile–third quartile) if they exhibited a non-normal distribution. Categorical variables were reported as frequencies and percentages.

Paired t-tests were conducted to analyze the differences in hematologic parameter values between the baseline and the treatment periods (3rd, 6th, or 12th month) for normally distributed data, while the Wilcoxon test was employed for non-normally distributed data. Additionally, the differences in value changes of hematological parameters among populations with varying genders or ages at the time point that showed the greatest increase or decrease (3rd, 6th, or 12th month), as determined from the analysis, were assessed using appropriate statistical tests. Specifically, unpaired t-tests were used for normally distributed data with equal SD, Welch’s t-test was employed for normally distributed data without equal SD, and the Mann–Whitney test was applied for non-normally distributed data. The analysis of categorical data was performed using the Chi-Squared test. Statistical analyses were conducted using GraphPad Prism 9.0 for Windows (GraphPad Software, Inc.). A significance level of P < 0.05 was adopted to determine statistical significance.

Result

A total of 55 patients were included in this analysis, comprising 15 patients from clinical trial 1 (out of the initially enrolled 18 patients, 3 patients discontinued the study within the first year) and 40 patients from clinical trial 2. The demographic and clinical characteristics of the patients are presented in Table 1. The mean age of the patients was 31, with 30 patients being above 30 years old. Of the patients, 36 were male, while 4 female patients presented with lymphangioleiomyomatosis (LAM). Four patients were diagnosed with subependymal giant cell astrocytoma (SEGA), and all 55 patients exhibited skin lesions.

Table 1.

Demographics and baseline characteristics

Characteristics	Clinical trial 1	Clinical trial 2	Total
Number of patients	15	40	55
Age in years, median (range)	30 (20–46)	33 (18–60)	31 (18–60)
≦ 30y	8 (53.3%)	17 (42.5%)	25 (45.5%)
> 30y	7 (46.7%)	23 (57.5%)	30 (54.5%)
Sex
Female	11 (73.3%)	26 (65.0%)	37 (67.3%)
Male	4 (26.6%)	14 (35.0%)	18 (32.7%)
Race (n, %)
Asian	15 (100%)	40 (100%)	55 (100%)
Presence of SEGA	0 (0%)	3 (7.5%)	3 (5.5%)
Diagnosis of LAM	5 (40.0%)	13 (32.5%)	18 (32.7%)
Skin lesion (≥ 1)	15 (100%)	40 (100%)	55 (100%)

SEGA subependymal giant cell astrocytoma, LAM lymphangioleiomyomatosis

Significant reductions were observed in hemoglobin, white blood cells (WBC), lymphocytes, neutrophils, and platelets during everolimus treatment (P < 0.05). However, the decreases in hemoglobin, WBC, and neutrophils attenuated in the 12th month (P > 0.05) (Fig. 1A, B, E, F). Importantly, lymphocytes exhibited a persistent and significant decrease throughout the entire year of treatment (Fig. 1C). Conversely, monocytes showed a consistent increasing trend, although this change was not statistically significant (Fig. 1D).

Fig. 1.

Changes in hematologic parameters in TSC-RAML patients during everolimus treatment

Aspartate transaminase (AST), alanine transferase (ALT), and total cholesterol (TC) exhibited a sharp and significant increase during the initial 3 months of treatment (P < 0.05), and these levels remained significantly elevated thereafter (Fig. 1G, H, K). Triglyceride levels showed a significant increase starting from the 3rd month and continued to rise throughout the treatment period, despite a subsequent decrease after peaking in the 6th month (Fig. 1L). Although creatinine and glucose levels displayed a general increasing trend over the one-year treatment period, these changes were not statistically significant (Fig. 1I, J).

The differences in the changes in hematologic parameters between female and male patients are presented in Table 2. The age difference between females and males was not statistically significant (31 vs 33, P = 0.620). Male patients exhibited a significantly greater decrease in hemoglobin compared to female patients (− 15 vs − 6, P = 0.010). The increase in AST was significantly higher in male patients compared to female patients (7.0 vs 3.0, P = 0.041), and a similar trend was observed for ALT, indicating a higher increase in male patients. Furthermore, female patients exhibited a higher increase in creatine, glucose, total cholesterol (TC), and triglycerides, although these differences were not statistically significant. The gender distribution between younger and older patients were not statistically significant (female/male: 18/7 vs 18/12, P = 0.351). However, Table 3 demonstrates that the decrease in platelet count (− 50 vs − 14, P = 0.020) was significantly greater in younger patients (≦30 years old) compared to older patients (> 30 years old).

Table 2.

Differences in hematologic parameter changes between females and males

Parameter	Female (n = 29)	Male (n = 16)	P
Hemoglobin (g/L)	− 6 (− 12, − 4)	− 15 (− 20, − 5)	0.010
WBC (× 109/L)	− 0.64 ± 0.93	− 0.62 ± 1.98	0.965
Lymphocyte (× 109/L)	− 0.20 ± 0.41	− 0.20 ± 0.70	0.998
Monocyte (× 109/L)	0.052 ± 0.136	− 0.001 ± 0.143	0.191
Neutrophil (× 109/L)	− 0.38 (− 0.84, 0.12)	− 0.43 (− 2.06, 1.27)	0.614
Platelet (× 109/L)	− 32 ± 59	− 30 ± 51	0.899
AST (U/L)	3.0 (0.3, 7.0)	7.0 (3.0, 16.0)	0.041
ALT (U/L)	4.5 (0.3, 8.8)	13.0 (− 1.0, 30.0)	0.132
Creatine (μmol/L)	3.0 (− 2.0, 7.3)	1.5 (− 6.8, 5.0)	0.388
Glucose (nmol/L)	0.13 (− 0.20, 0.58)	− 0.06 (− 0.40, 0.30)	0.150
TC (nmol/L)	1.47 ± 1.03	1.02 ± 1.01	0.151
Triglyceride (nmol/L)	1.07 (0.52, 1.73)	0.75 (− 0.07, 1.16)	0.084

WBC white blood cell, TC Total cholesterol

Table 3.

Differences in hematologic parameter changes between the younger and older

Parameters	Younger (≦ 30 y, n = 25)	Elder (> 30 y, n = 30)	P
Hemoglobin (g/L)	− 6 ± 11	− 11 ± 16	0.265
WBC (× 109/L)	− 0.39 ± 1.13	− 0.85 ± 1.49	0.223
Lymphocyte (× 109/L)	− 0.15 (− 0.60, 0.09)	− 0.19 (− 0.44, 0.12)	0.703
Monocyte (× 109/L)	0.019 ± 0.117	0.039 ± 0.144	0.608
Neutrophil (× 109/L)	− 0.46 ± 1.27	− 0.60 ± 1.73	0.366
Platelet (× 109/L)	− 50 ± 54	− 14 ± 54	0.020
AST (U/L)	4.0 (0, 8.0)	4.0 (1, 12.5)	0.407
ALT (U/L)	7.0 (2.5, 20)	3.5 (− 3.0, 10.0)	0.095
Creatine (μmol/L)	2.32 ± 7.02	5.63 ± 16.80	0.355
Glucose (nmol/L)	0.07 ± 0.41	0.20 ± 0.67	0.420
TC (nmol/L)	1.35 ± 0.90	1.32 ± 1.16	0.913
Triglyceride (nmol/L)	0.94 (0.42, 1.49)	0.84 (0.42, 1.60)	0.933

WBC white blood cell, TC Total cholesterol

Discussion

The TSC1-TSC2 complex, composed of hamartin and tuberin encoded by the TSC1 and TSC2 genes, respectively, is a crucial inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1). Mutations in either TSC1 or TSC2 can lead to the functional loss of the TSC1-TSC2 complex, resulting in unregulated and constitutive activation of mTORC1. This dysregulation causes uncontrolled protein synthesis, cellular growth, proliferation, and angiogenesis [14]. Everolimus, an mTOR inhibitor, has shown efficacy in reducing tumor size, preventing tumor rupture and bleeding, and preserving renal function, making it the recommended first-line therapy for TSC patients [9–11]. However, the potential risks associated with its treatment cannot be overlooked. Our study specifically examined the potential hematologic impact and risks of everolimus in patients with TSC-RAML.

Many hematologic parameters were significantly impacted by everolimus throughout the treatment period, with most blood cells, except for monocytes, as well as hemoglobin showing significant decreases. A published study on the effectiveness and safety of everolimus treatment in TSC patients also demonstrated that anemia and leukopenia were the most frequent laboratory abnormalities during the entire treatment period [12]. In our study, hemoglobin, WBC, neutrophil, and platelet exhibited significant decreases followed by rebound increases, which may be attributed to the homeostatic regulation of blood cells to maintain parameter fluctuations around a certain level [15]. Hemoglobin and lymphocyte exhibited significant decreases in a shorter duration (≤ 3 months) compared to WBC, neutrophils, and platelets, suggesting that hemoglobin and lymphocytes were more susceptible to the effects of everolimus. This vulnerability may be due to the dynamic turnover of these cell types. Notably, red blood cells and lymphocytes have longer lifespans than neutrophils and platelets [16–19], indicating lower turnover activity in red blood cells and lymphocytes, and thus resulting in significant decreases within a shorter timeframe under the influence of everolimus. The decrease may be associated with inhibition on the cell cycle and subsequent proliferation of normal cells when everolimus suppresses tumor progression. Additionally, mTOR inhibitors block the interleukin-2 receptor and CD28-dependent signaling pathways, which could further contribute to the observed myelosuppression and inhibition of blood cell proliferation [20]. Everolimus also is used as an immune system suppressor in transplantation recipients, inhibiting the ability of white blood cells (WBC) to reject the transplanted organ [21]. The significant decrease in WBC, lymphocyte, and neutrophil levels, indicating immune suppression and toxicity of everolimus, may weaken the anti-infection capacity of TSC-RAML patients undergoing everolimus treatment. It is important to note that lymphocyte levels consistently decrease during the treatment and may further decline with continued therapy. Hematological toxicity and bone marrow suppression are common side effects of mTOR inhibitor treatment. Thrombocytopenia and neutropenia rarely lead to clinically significant bleeding or infection, and thus usually do not require platelet transfusions or growth factor support. However, in cases of grade 3 toxicity, it is necessary to interrupt the treatment and reduce the dose when resuming. Additionally, everolimus should be discontinued immediately if any life-threatening toxicity occurs [22].

Everolimus undergoes extensive metabolism primarily through the cytochrome P450 system (CYP3A4) and P-glycoprotein in the liver [23]. Liver injury may result from the direct effects of everolimus or intermediate toxicity generated during its metabolism. Both ALT and AST, key markers of liver function in clinical practice, showed a significant increase after 3 months and remained elevated throughout the treatment period. Therefore, careful monitoring of liver function is essential in TSC-RAML patients during treatment, especially when everolimus is co-administered with inhibitors or inducers of cytochrome P450 drug-metabolizing enzymes, as potential drug interactions may occur. Many studies have also documented hyperlipidemia as another common adverse effect of everolimus [10, 12]. Lipid levels, including total cholesterol (TC) and triglycerides (TG), also exhibited significant increases in the short term, specifically within less than 3 months in our study. Research has shown that everolimus can significantly elevate serum lipid levels by reducing lipoprotein lipase activity [24]. On the other hand, the metabolism of apolipoprotein B (apoB) is mediated through the mTOR signaling pathway, and the catabolism of apoB-containing lipoproteins is diminished by mTOR inhibitors [25]. Both cholesterol and triglycerides are apoB-containing particles, and the increased levels of apoB lipoproteins may contribute to elevated lipid levels [26]. We recommend drug intervention for severe hyperlipidemia or grade 3 liver injury. However, elevated lipid levels, particularly in TSC-RAML patients with comorbidities such as hyperlipidemia, hyperglycemia, and hypertension, may significantly increase the risk of cardiovascular and cerebrovascular diseases [22].

Limited studies have investigated hematologic response differences among TSC-RAML patients of different genders and age groups to everolimus treatment. Our analysis revealed that male patients experienced a greater decrease in hemoglobin levels and a greater increase in AST levels due to everolimus treatment. Estrogen has the potential to stimulate hematopoiesis in females and enhance the proliferation of hematopoietic stem cells (HSCs) and various blood cell types, including erythrocytes [27]. The higher renewal capacity of erythrocytes in females may help mitigate the side effects on hemoglobin levels. Although females are generally considered to be at higher risk for drug-induced liver injury [28], gender disparities in drug-induced liver injury vary depending on the specific drugs, with a higher frequency of reported drug-induced liver events in males for certain medications [29]. Our findings indicated that male patients exhibited higher elevations in alanine aminotransferase (ALT) levels, suggesting a potentially greater risk of drug-induced liver injury associated with everolimus in males.

Our results also revealed a greater decrease in platelet levels among younger patients. The tortuous aneurysmal vessels within the angiomyolipoma, which are more susceptible to rupture and bleeding compared to normal vessels, constitute a high-risk factor for tumor hemorrhage [30]. Tumor rupture and hemorrhage pose a significant life-threatening risk for TSC-RAML patients. The decrease in platelet count may further enhance the risk of hemorrhage due to the potential platelet-related coagulation dysfunction. Everolimus is an immune inhibitor that can potentially result in immune system dysfunction. Besides inhibiting platelet proliferation, it remains uncertain whether the decrease in platelets is related to drug-induced immune thrombocytopenia, as everolimus may lead to possible immune dysfunction. Furthermore, significant differences exist in the immune systems of younger and older individuals, which may contribute to the varying platelet decreases observed between younger and older patients. Additional research is necessary to further elucidate this potential relationship.

There are several limitations in our study: (1) The sample size was relatively small due to the rarity of TSC-RAML. (2) The generalizability of our findings might be limited because all patients included were Asians. (3) Hematologic parameters can be affected by various factors, such as active infection or inflammation. These factors might have influenced the accuracy of our results, although measures were taken to minimize potential influences as much as possible.

Conclusion

Hemoglobin, WBC, neutrophil, platelet and lymphocytes underwent a significant decrease during everolimus treatment in TSC-RAML patients, whereas AST, ALT, TC, and TG demonstrated significant increases. Male patients exhibited higher vulnerability to everolimus-induced decreases in hemoglobin and platelet counts, while younger patients showed greater susceptibility to everolimus-induced platelet reduction.

Author contributions

Study concept and design: ZCL and YSZ. Acquisition of data: ZCL DXQ, YZ, ZW. Analysis and interpretation of data: WDW, ZCL, DXQ, YZ. Drafting of the manuscript: ZCL. Statistical analysis: ZCL, ZW, XW. Critical revision of the manuscript for important intellectual content: YSZ. Administrative, technical, or material support: YSZ, WDW.

Funding

This study was supported by National High-Level Hospital Clinical Research Funding (2022-PUMCH-B-010), National High Level Hospital Clinical Research Funding (2022-PUMCH-A-151), and Postdoctoral Fellowship Program of CPSF (Grant Number: GZC20230301).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was done in accordance with the Declaration of Helsinki and local regulations and was approved by the ethics committee of Peking Union Medical College Hospital, the ethics committee of Chinese PLA General Hospital, the ethics committee of Cancer Hospital Afliated to Fudan University, the ethics committee of West China Hospital, the ethics committee of Tongji Hospital.

Patient consent statement

Informed written consent was obtained from all participants included.

Competing interests

The authors declare no competing interests.