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 [9,10,11]. 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 [16,17,18,19], 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.