Mutational Analysis Of Renal Angiomyolipoma Associated With Tuberous Sclerosis Complex And The Outcome Of Short-term Everolimus Therapy

BACKGROUNDTuberous sclerosis complex?TSC?is a systemic autosomal dominant genetic disorder that affects approximately 1-2 million individuals worldwide.It is usually caused by mutations in either the hamartin gene?TSC1?or tuberin gene?TSC2?,leading to the growth of nonmalignant hamartomas in various organs throughout the body,including the kidney,brain,lung,skin,and heart.Statistical studies show that approximately 80%of patients with TSC develop renal angiomyolipoma?AML?,which is rich in fat,muscle,and blood vessels.Unlike sporadic renal AML,most TSC-related renal AML?TSC-AML?patients often develop multiple bilateral lesions and experience a significant tumor burden on the kidneys.Furthermore,TSC-AML tend to be larger,grow faster and at higher risk of bleeding.In addition,AML can increase in size over time and may cause hypertension,renal failure or life-threatening hemorrhage,which causes the largest proportion of adult deaths from the disease.The TSC1 or TSC2 gene mutation occurs in approximately 80%of patients with TSC.The TSC1 gene,located on chromosome 9q34,consists of 23 exons and codes for hamartin,a novel 130-kDa protein.The TSC2 gene,located on chromosome 16p13.3,contains 41 exons and codes for tuberin,a 200-kDa protein.Hamartin and tuberin form a tumor suppressor complex that regulates the activity of rapamycin complex 1?mTORC1?,a critical regulator of cell growth and proliferation.Mutations in TSC1 or TSC2 result in the loss of the hamartin/tuberin complex,which leads to constitutive activation of mTORC1.Increased mTORC1 signaling results in the production of hamartomatous lesions of TSC.Growing knowledge about the molecular relationship between TSC and mTOR has led to the investigation of mTORC1 inhibition as a treatment approach in TSC.Moreover,the International Tuberous Sclerosis Complex Consensus Conference?ITSCCC?held in2012 has recommended mTOR inhibitors as the first-line treatment for TSC-AML when enlarged to 3 cm or more.Everolimus is a derivative of rapamycin that suppresses the enlargement of tumors and promotes their regression by inhibiting mTORC1.Clinically,a randomized,double-blind,placebo-controlled EXIST-2 trial and extension studies have demonstrated the efficacy and manageable safety of everolimus for TSC-AML.PURPOSEIn China,everolimus was included in national basic medical insurance,work injury insurance and the maternity insurance drug list in 2017.However,clinical information of TSC-AML patients in China and the effects of short-term everolimus therapy on TSC-AML are still limited.Thus,this study aimed to analyze the clinical characteristics and mutation spectra of patients with TSC-AML in Northwest China and to investigate the efficacy and safety of short-term everolimus therapy in patients with TSC-AML.Methods1.Patient SelectionFrom September 2015 to August 2018,a total of 167 patients with renal AML were screened at Xi Jing Hospital,82 of whom were enrolled in this study.All 82 patients had been clinically?definitively or potentially?diagnosed with TSC per ITSCCC diagnostic criteria¹⁵.All patients underwent a systematic evaluation that covered 11 major criteria and 6 minor criteria of TSC before any interventions.2.Mutational AnalysisPeripheral blood?10 mL?samples were collected from each patient and isolated from peripheral white blood cells.Then,DNA was extracted from peripheral blood leukocytes using standard methods.The mutational analysis of TSC genes was performed by the first generation sequencing and the next-generation sequencing?NGS?,including the preparation of DNA libraries,the hybridization capture of target area and single-ended sequencing.Then,the data were analyzed,and all variation results were validated by the PCR-SSCP method.Finally,all of the mutations were compared with the Tuberous Sclerosis Database?www.lovd.nl/TSC1;www.lovd.nl/TSC2?.3.Everolimus TreatmentEverolimus was administered orally at a dose of 10 mg/day,and the levels of everolimus in blood were measured at 2 weeks to ensure blood everolimus concentrations were between 5 and 15 ng/ml.All patients received everolimus for 12 weeks,while 25patients were followed for an additional 12 weeks after the therapy was stopped.The primary endpoint of this study was the efficacy of short-term everolimus therapy,which was assessed according to the reduction in the volume of renal AML.The volume of renal AML was evaluated using kidney magnetic resonance imaging or CT scanning,and all scans were assessed by central radiological review.The volume of renal AML before initiation of everolimus treatment was defined as“baseline”.Follow-up visits were performed at week 4 and 12 after the start of everolimus treatment and at week 12 after the treatments stopped.Treatment efficacy was defined as the volume reduction rate compared to the baseline,which was calculated with the formula:(V_Pre-Therapy-V_Post-Therapy)/V_Pre-Therapy×100%.(V_Pre-Therapyre-Therapy represents pre-everolimus therapy volume;V_Post-Therapyost-Therapy represents post-everolimus therapy volume).4.Statistical AnalysisClinical characteristics were collected from all patients screened for TSC-AML during the experiment period,and the results were presented as a frequency distribution?absolute frequencies and valid percentages;n,%?.The mutation spectra were the statistical result of the 47 patients who agreed to participate in the genetic testing,and the results were expressed in terms of frequency distribution.Efficacy and safety analyses were performed on all patients who received at least 12 weeks of everolimus treatment and had at least one follow-up assessment;missing data were not considered in the analyses.Descriptive analyses of patient characteristics were conducted,including central tendency and dispersion?mean±standard deviation[SD]?,or median and frequency distribution.The outcomes were analyzed using a t-test or?² test,and a P-value<0.05 was considered statistically significant.All statistical analyses were performed with the Statistical Package for the Social Sciences?SPSS,SPSS Inc,Chicago,USA?version 17.0.RESULT1.Clinical characteristics of patients with TSC-AML:A total of 82 Chinese patients with TSC-AML,including 65 definitively diagnosed patients and 17 patients with possible TSC-AML were enrolled in this study since 2015.The patients were 35±12 years old?range 18-68 years?,with a median age of 36 years.Among all patients,28 were males?34.15%?,and 54 were females?65.85%?.2.Mutation spectra and novel sequence variants:Out of 47 Chinese TSC-AML cases,TSC gene mutations were found in 22 patients?46.81%?.Among 22 TSC gene mutations,7?14.89%?were TSC1 gene mutations,13?27.66%?were TSC2 gene mutations and 2?4.26%?were both TSC1 and TSC2 gene mutations.Of these 22 patients,a total of 26 mutations were detected,and 3 patients had more than 1 type of mutation.Of all mutations,a total of 21 different kinds of mutations were identified,5 in TSC1 and 16 in TSC2.3.Comparison between TSC2 and non-TSC2 mutation populations:Patients with TSC2 mutations had a significantly larger average RAML largest diameter compared with patient populations with non-TSC2 mutations?16.37±9.61 cm vs9.146.84 cm,P=0.042?.Ten patients?66.67%?with TSC2 mutations had a RAML largest diameter of?10 cm,compared to only 11 patients?34.38%?in the non-TSC2mutation group?P=0.038?.In addition,hypomelanotic maculesoccurred more frequently in the TSC2 mutation group?P=0.007?.4.Treatment efficacy:All of the patients were given everolimus orally at doses of 10 mg per day.The duration of administration was 12 weeks.After 4 and 12 weeks of therapy,the mean renal AML volume decreased 36.51±23.94%?range:4.23%-84.47%;P<0.05 for the change from the baseline value?and 56.47±23.32%?range:4.88%-97.36%;P<0.05 for the change from the baseline value?,respectively.In most patients,the AML volume decreased rapidly within 4 weeks after treatment initiation,but the rate of decrease significantly slowed down for the next 8 weeks.5.Adverse events:The AEs were monitored throughout the treatment period.A wide variety of everolimus treatment-related AEs occurred,and they were consistent with the known safety profile of everolimus,which most commonly included stomatitis,headache,acne,cough and menstrual disorders.During the treatment period,everolimus treatment-related laboratory tests were also monitored,including blood,urine,liver and kidney functions.No other abnormalities were observed,except for 4 cases?13.33%?of hyperlipidemia?grade 1?and cytopenia?3 cases of grade 1 and 1 case of grade 2?.CONCLUSIONThis open single-center clinical study explored the clinical features and gene mutation information of TSC-AML patients in Northwest China and observed the efficacy and safety of short-term use of everolimus in patients with TSC-AML.Overall,the TSC gene mutation rate of TSC-AML patients in Northwest China is much lower than in other countries.The short-term everolimus treatment for TSC-AML is effective and safe,but the stability is much lower than long-term therapy.