Pilot study in patients with congenital low-flow vascular malformation treated with low dose sirolimus

Pilot study in patients with congenital low-flow vascular malformation treated with low dose sirolimus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Pilot study in patients with congenital low-flow vascular malformation treated with low dose sirolimus Veroniek Harbers, Gerard Rongen, Carine Vleuten, van der, Bas Verhoeven, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-132412/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Patients with congenital low-flow vascular malformations (capillary (CM), lymphatic (LM), venous (VM) or combined) may have an impaired quality of life (QoL), due to their symptoms, which include pain, swelling, bleeding, thrombosis, and functional impairment. Unfortunately, current treatment methods are challenging and not always successful. Previous studies have shown that the mTOR-inhibitor sirolimus is an effective treatment for these patients. Target levels of 10–15 ng/ml were well tolerated; however, grade three adverse events were observed (ranged 20–40%). Methods A pilot study was performed using a Challenge–Dechallenge–Rechallenge (CDR) design to determine the pharmacodynamics of low target levels of sirolimus (target levels 4–10 ng/ml) in respect of efficacy and adverse events in patients with disabling low-flow vascular malformations without treatment alternatives. The patients received sirolimus over a three-to-six-month period (Challenge), followed by the withdrawal of sirolimus (Dechallenge). If the complaints returned, sirolimus was reintroduced during a twelve month period (Rechallenge). Efficacy was determined on pain (end point of the pilot study) and other symptoms related to the vascular malformation; and adverse events were determined in all phases of the study. Results An improvement in symptoms was seen in 92% (n = 11/12) of patients during the Challenge phase. In the Rechallenge phase, a positive response rate of 78% was found (n = 7/9). These response rates are comparable to those found in the literature despite low target levels of sirolimus. However, less serious adverse events were observed with low dose sirolimus, especially bone marrow toxicity and grade III liver toxicity. Conclusions This pilot using low dose sirolimus showed high efficacy in patients with therapy resistant and disabling low-flow malformation, with a lower incidence of serious adverse events (especially bone marrow toxicity and grade III liver toxicity). This is extremely relevant to patients with low-flow vascular malformation, as current clinical protocols tend to advise lifelong treatment. Trial registration The pilot study was part of a phase III study. Trial registration: EudraCT number: 2016-002157-38 and ClinicalTrials.gov Identifier: NCT03987152, registered 06/14/2019 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03987152?term=sirolimus&cond=Vascular+Malformations&cntry=NL&draw=2&rank=1 Clinical Pharmacology Cardiac & Cardiovascular Systems Vascular malformation lymphatic malformation venous malformation sirolimus low-dose sirolimus mTor inhibitor pain Figures Figure 1 Figure 2 Background Vascular malformations include a heterogenous group of developmental anomalies of the vascular system, capillaries, veins, arteries, lymphatics or any combination of these vessels may be involved. The International Society for the Study of Vascular Anomalies (ISSVA) classified vascular malformations into the following categories( 1 ): simple (low-flow vascular malformation: capillary (CM), lymphatic (LM), venous (VM) arteriovenous (AVM), combined, vascular malformation of major named vessels, and vascular malformation associated with other anomalies. Vascular malformations are congenital, however, they can be discovered at any life stage, depending on their size and associated symptoms. Current treatment options for low-flow vascular malformation may be conservative, with compression bandages, analgesics, anti-inflammatory or anti-coagulation drugs, or more invasive with intralesional sclerotherapy or embolization, and surgery( 2 ). Unfortunately, treatment is challenging and not always successful, and can leave patients with a high clinical burden and subsequently a reduced Quality of Life (QoL)( 3 ). Clinical symptoms that reduce the QoL in patients with low-flow vascular malformations include pain, functional impairment, bleeding, thrombophlebitis, ulceration, infections, and leakage (in LM)( 4 ). Sirolimus The PI3/AKT/mTOR pathway plays a pivotal role in low-flow vascular malformation( 5 ). The activation of mammalian Target of Rapamycin (mTOR), stimulates angiogenesis, cell proliferation, and glucose metabolism. Some activating somatic mutations in genes in a target of the mTOR pathway, such as PIK3CA, AKT-1, TEK/TIE-2 and PTEN, in patients with low-flow vascular malformation resulted in the increased activation of mTOR( 6 – 10 ). Therefore, the inhibition of this pathway in these patients seems a logical approach to treatment. In cells, sirolimus binds to the immunophilin FK Binding Protein-12 (FKBP-12), which in turn binds to and inhibits the activation of mTOR. This inhibition results in the obstruction of several signal transduction pathways, thereby inhibiting downstream protein biosynthesis, cell proliferation, and angiogenesis( 11 , 12 ). In theory, this should decrease the size of the low-flow vascular malformation or at least inhibit activity and stop further growth. Unfortunaly, the inhibition of lymphocyte activation also results in immunosuppression and might therefore be associated with the susceptibility to infections( 13 ). Several studies have been performed to explore the use of sirolimus as a treatment option in low-flow vascular malformations( 14 – 17 ). These prospective open-label trials used - high target sirolimus levels of 10–15 ng/ml leading to (partial) response in 85–100% patients( 14 – 17 ). As these trials were open label trials, the question remains what is the true efficacy of sirolimus and what is natural behavior of the vascular malformation. Ideally, a placebo controlled randomized trial should be performed, however, in respect to the severe clinical burden of the patients and the rarity of the disease, it is difficult to execute. Recently, it has been postulated that a different design can be used in rare diseases to identify true efficacy of a drug( 18 ). This design is based on the concept of Challenge, Dechallenge and Rechallenge (CDR) to proof the efficacy (or adverse events) caused by a single drug. Ideally, a future study should use this design to investigate true efficacy more in detail. Furthermore, more insight in the adverse events of sirolimus used as a single drug can be gained, as most information available so far, is based on adverse events observed in patients using a combination of drugs (e.g. renal transplant patients)( 13 , 19 – 23 ). The side effects of sirolimus described include oral ulceration, mucositis and stomatitis, interstitial lung disease, diabetes mellitus, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, gastro-intestinal side effects, angioedema, thrombo-embolic disease, anemia, leucopenia, thrombocytopenia, proteinuria, glomerulonephritis, and lymphedema( 13 ). Our knowledge regarding long term toxicities of sirolimus is still expanding and makes it necessary to minimalize the risk for these long term toxicities. For example, it has been observed that in patients with long-term sirolimus impaired insulin receptor substrate signaling and Akt activation can be found indicating a deterioration of glucose metabolism leading to an increase of development of diabetes( 24 ). As in most drugs observed, one can imagine that higher levels of a drug increase the risk of developing adverse events of which the most serious and sometimes even fatal complication is sirolimus-associated interstitial pneumonitis( 19 , 23 , 25 , 26 ). Bee et al. showed that low sirolimus serum levels (< 3 and 6.9 ng/ml) are related to less side effects without compromising efficacy of treatment in patients with diffuse lymphangioleiomyomatosis( 27 ). Additionally, Kahan et al. showed a significant relation between the occurrence of adverse events (hypertriglyceridemia, hypercholesterolemia, leukopenia and thrombocytopenia) and the steady state concentration value of sirolimus( 28 ). A C ss below 10 ng/L showed no toxic values. In the pilot study described here, we hypothesized that sirolimus used in low dosages with lower target levels (4–10 ng/ml) than previously described, is equally effective in the treatment of low flow vascular malformations however will lead to less serious adverse events as observed in those treated with high target levels. Treatment with low target levels (4–10 ng/ml) may require longer treatment than with high target levels (> 10 ng/ml), however low dose sirolimus will be more tolerable if less (serious) adverse events occur. This is especially important, as our knowledge regarding long term toxicities of sirolimus is still growing and makes it necessary to reduce the risk for these long term toxicities. Methods The pilot study was performed between 2015 and 2017. Patients included in the pilot study had a severe form of congenital vascular malformation, suffering severe pain and/or impairments. No predefined response criteria were used. All patients included underwent a Challenge-Dechallenge-Rechallenge to determine the efficacy of low dose sirolimus. Primary objectives of the pilot study were to determine the pharmacodynamics of low dose Sirolimus in respect of efficacy and adverse events. Patients received sirolimus for three to six months in the Challenge phase, after which sirolimus treatment stopped (Dechallenge phase). If complaints (e.g., pain) did not reoccur, the follow-up phase continued for at least one year, during which time the duration of the pain/symptom-free period and any serious adverse events was measured. If pain or other symptoms returned during the Dechallenge phase, a Rechallenge phase of one year of sirolimus treatment was initiated. Sirolimus was administered orally using a start dose of 0.8 mg/m 2 twice a day for children, and 1 mg twice a day for adults. This starting dose is based on the pharmacokinetic properties of sirolimus. During the treatment period, therapeutic drug monitoring was performed. Target trough levels of sirolimus between 4–10 ng/ml were used, based on laboratory and clinical studies( 28 – 30 ). Since sirolimus has immunosuppressive properties, cotrimoxazole as a Pneumocystis Jiroveci Pneumonia prophylaxis was prescribed. Recent guidelines suggest that this prophylaxis is not necessary, citing no clear evidence of increased infection, however, at the time this study was performed these guidelines were not available( 31 ). Adverse events were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE). An MRI is only made in a subset of patients before and after each phase as this outcome measure was not prospectively defined for this pilot study. Therefore the evaluation of the effect of sirolimus on the size of the vascular malformation will be only possible in these patients. In case there was secondary material that was obtained after surgery, stored in the HECOVAN biobank it will be used for analyses. Comprehensive targeted Next Generation Sequencing screen using Unique Molecular Identifiers with a technical sensitivity of 1% mutant alleles was performed for frequently mutated positions using Single Molecule Molecular Inversion Probes( 6 ). The end point of the pilot study was pain reduction, therefore response was assigned to sirolimus based on pain reduction during the first period (Challenge phase), after which the patients stopped taking sirolimus (Dechallenge). When pain or other complaints returned, sirolimus was re-administered (Rechallenge). Results Twelve patients (aged between 1 and 50; 8 M, 4 F) were included in the pilot study. The clinical characteristics of the patients are shown in Supplemental Table S1. All patients had an otherwise untreatable form of a low-flow vascular malformation. All patients had a multiple sclerotherapy treatments and some had a partial resections in the past. These treatments were not possible or successful anymore. The initial treatment duration was at least three months (Challenge phase). At the end of the Challenge phase, eleven patients experienced pain reduction; of whom five became entirely pain free (pretreatment pain scores: 6–10); only one patient experienced no change in complaints. Pain reduction was achieved between one week and five months after start of sirolimus (mean: seven weeks). Table 1 summarizes the results per vascular malformation type. Table 1 Summary of clinical and MRI responses in the Challenge and Rechallenge periods. Challenge (n = 12) Rechallenge (n = 9) Vascular malformation type Response after six months of sirolimus Vascular malformation size as assessed using MRI Response after 12 months of sirolimus Vascular malformation size as assessed using MRI VM (n = 2) Improvement of symptoms (pain, QoL) (2) - Decrease (1) - No MRI (1) Pain free (1) Not restarted (1) - Decrease (1) - Not restarted (1) LM (n = 5) Improvement of symptoms (pain, QoL, leakage, infections, decrease of clinical size of LM, etc.) (5) - No change (2) - Decrease (2) - No MRI (1) - Improvement of symptoms (pain, QoL, leakage, decrease of clinical size of LM, etc.) (2) - No change (1) - Not restarted (1) - Restarted with mTOR inhibitor (1) - No change (1) - Decrease (1) - Not restarted (1) - Restarted with mTOR inhibitor (1) - Unknown (1) Lymphangiomatosis (n = 1) No change (1) Decrease (1) No change in symptoms (1) Increase (1) LVM (n = 2) Improvement of symptoms (pain, clinical size of LVM) (2) - No change (1) - No MRI (1) - Pain reduction (2) - Clinical size reduction LVM (1) - No change (1) - Unknown (1) KTS (n = 2) Improvement of symptoms (pain, QoL) (2) - No change (1) - No MRI (1) Improvement of symptoms (pain, clinical size reduction) (2) - No change (1) - Unknown (1) Total Improvement of symptoms (11; 92%) No change (1; 8%) - No change (4; 33%) - Decrease (4; 33%) - No MRI (4; 33%) - Improvement of symptoms (pain, QoL, leakage, decrease of clinical size of LM, etc.) (7; 78%) - No change (2; 122%) - Not restarted (2; 22%) - Restarted with mTOR inhibitor (1; 11%) - No change (3; 33%) - Decrease (2; 22%) - Increase (1; 11%) - Not restarted (2; 22%) - Restarted with mTOR inhibitor (1; 11%) - Unknown (3; 33%) VM: venous malformation, LM: lymphatic malformation, LVM: lymphatico-venous malformation, KTS: Klippel Trenaunay Weber syndrome. During the Dechallenge phase, ten patients had a return of pain/symptoms with a duration range of 10 days to 4 months. Nine patients restarted the sirolimus treatment shortly. In all patients, reasons for the re-administration of sirolimus were return of severe pain or other symptoms, such as leakage in the case of a lymphatic malformation or clear evidence of growth of the vascular malformation in the time period sirolimus was stopped. (see Supplemental Table S2). Three patients did not restart with sirolimus in the Rechallenge phase despite the positive response primarily observed. Reasons were a loss of energy (patient 6), logistic reasons (patient 12), or the initiation of a different mTOR inhibitor (everolimus) (patient 7) at a nearby hospital. The patient that switched to a different mTOR inhibitor showed a significant reduction in complaints again. A substantial relationship between sirolimus and the amelioration of symptoms was confirmed during the Rechallenge phase in seven patients by a substantial reduction of complaints after they resumed taking sirolimus. This indicates the efficacy of sirolimus at a low dose. All patients experienced adverse events when taking sirolimus. The most frequent adverse events that were likely related to sirolimus treatment were: aphthous stomatitis grade I (50% of patients) and menstrual disorder grades I-II (female patients only; 75%). No patients experienced grade III bone marrow toxicity in the pilot study (Table 2 ); however, two patients (16.7%) experienced grades I-II bone marrow toxicity. One adult patient developed hypophosphatemia that rapidly recovered after stopping sirolimus. One patient (patient 4) experienced a grade III sepsis due to an infected lymphatic cyst. Reasons for a temporary stop (several days) of sirolimus were: due to vaccination (n = 1), interventional radiology (bleomycin sclerotherapy n = 2), decannulation (n = 1), and adverse events: infections (n = 13) of which n = 1 sepsis, menstrual disorder (n = 2), aphthous stomatitis (n = 1), general malaise (n = 1), elevated liver enzymes (n = 1). Therapy-limiting adverse events were seen in two patients: one patient had due to a grade II increase of liver enzymes after five months sirolimus treatment (Challenge phase), and one patient had a grade II menorrhagia after 36 months sirolimus treatment (Rechallenge phase). All adverse events were resolved by interrupting treatment, and there have so far been no reported long-term adverse events (median follow-up three years; range 1–5 years). Table 2 Adverse events observed in our pilot study compared to other studies. Adverse events attributable to sirolimus Grade of toxicity Pilot study n = 12 Adams et al. n = 57 Hammer et al. n = 19 Nguyen et al. Review Blood/bone marrow toxicity Grade I and II 2 (17%) Grade II or higher: 30 (49%) 1(5%) 11–76% Grade ≥ III 0 (0%) 16 (27%) 1 (5%) Gastro-intestinal toxicity (e.g. mucositis) Grade I and II 9 (75%) Grade II or higher: 33 (55%) 19 (100%) 3–19% Grade ≥ III 0 (0%) 2 (3%) 2 (11%) Metabolic/laboratory toxicity Grade I and II 4 (33%) Grade II or higher: 12 (20%) 0 (0%) 20–64% Grade ≥ III 0 (0%) 2 (3%) 0 (0%) Infection Grade I and II 6 (50%) Grade II or higher: 9 (15%) 6 (32%) Unknown Grade ≥ III 0 (0%) 1 (2%) 0 (0%) Endocrine toxicity Grade I and II 3 (25%) Unknown Unknown 20–27% (Diabetes mellitus) Grade ≥ III 0 (0%) Unknown Unknown Dermatology toxicity Grade I and II 3 (25%) 5 (8%) 7 (36.9%) Unknown Grade ≥ III 0 (0%) Unknown 1 (5.3%) Neurologic toxicity Grade I and II 4(33%) Unknown 12 (63%) Unknown Grade ≥ III 0 (0%) Unknown 0 (0%) Pulmonary/upper respiratory toxicity Grade I and II 7 (58%) Grade II or higher: 1 (2%) 1 (5%) Unknown Grade ≥ III 0 (0%) 1 (2%) 0 (0%) Interstitial lung disease Grade I and II 0 (0%) Unknown Unknown 4–17% Grade ≥ III 0 (0%) Unknown Unknown Musculoskeletal/soft tissue Grade I and II 1 (8%) 0 (0%) 0 (0%) Unknown Grade ≥ III 0 (0%) 0 (0%) 0 (0%) General symptoms (for example hypertension/wound healing) Grade I and II 1 (8%) Cardiac general: 0 (0%) 20 (105%) Angioedema (2.2–15%), urologic (12%) Grade ≥ III 0 (0%) Cardiac general: 0 (0%) Constitutional symptoms: 0 (0%) 0 (0%) Lymphedema Grade I and II 0 (0%) Grade II or higher: 4 (7%) Unknown 6.4–12% Grade ≥ III 0 (0%) 1 (2%) Unknown Adverse events observed in our pilot study and those reported in the studies performed by Adams et al. and Hammer et al. and Nguyen et al.’s review. Adams et al.: patients with various complex vascular anomalies (including vascular tumors), target levels: 10–15 ng/ml ( 15 ). Hammer et al: patients with a vascular malformation using target levels: 10–15 ng/ml ( 17 ). The table showed a 105% percentage due to categorizing and summarizing patients with general symptoms. Nguyen et al.’s review of sirolimus in solid organ transplantation identified a wide array of adverse effects ( 13 ). After the Challenge phase, an MRI was performed in eight out of twelve patients. In four of the eight patients in which an MRI was performed, a visual size reduction of the low-flow vascular malformation was found. Stable disease was observed in the remaining four patients. MRIs were also performed in six of the nine patients who underwent the Rechallenge phase. Two patients showed a decrease in vascular malformations size compared to baseline as assessed before each treatment phase. Genetic testing of the vascular malformation was no performed routinely, however, in three out of four patients, DNA diagnostics on tissue revealed a PIK3CA mutation. In the other patient, no genetic aberrations could be found with our vascular anomalies panel ( 6 ) (Supplemental Table S1). Case Presentation To illustrate the efficacy of lower target levels of Sirolimus, patient 10 is described in more detail. This patient, was a two years old female at the start of treatment and suffered of a severe macroglossia due to a lymphatic malformation (Fig. 1 ). A life-threatening situation in the first months after birth led to the need for a tracheal cannula to guarantee an open airway for a longer time period. Before start of sirolimus, patient had a substantial reduced QoL. In the first weeks after the start of the sirolimus treatment, a rapid decrease in the size of the low-flow vascular malformation was observed. The sirolimus target levels ranged between 4.4 and 6.0 ng/ml during this period. After six months of treatment, an MRI was performed to quantify the response to sirolimus, and a clear reduction in the lymphatic malformation was observed. During a six weeks Dechallenge period, the decision was made to restart sirolimus treatment, because of the increase of tongue volume, noduli and nodus tongue. After restarting, the vascular malformation further reduced in size and, at the age of three years, after using sirolimus for 21 months, bleomycin sclerotherapy, and a tongue resection, the cannula was removed. Photographs of the patient (Fig. 1 ) show a clinical reduction in the volume of the low-flow vascular malformation in the submandibular and neck regions after only four weeks of sirolimus treatment. A further reduction is seen after eleven months of treatment with sirolimus. MRI images also show a reduction in volume at six and twelve months post treatment initiation (Fig. 2 ). The possible related adverse events this patients experienced were intermittent aphthous stomatitis, upper airway infections, tonsillitis, and elevation of triglycerides. The results of patient 10 (Supplemental Table S1 and S2) in the pilot study are in line with our hypothesis that low target levels of sirolimus are sufficiently effective and lead to fewer and less severe adverse events. Discussion We present the results of a pilot study to determine the efficacy and safety of low dose sirolimus on low-flow vascular malformations. A positive response of 92% (n = 11/12) in the Challenge phase was achieved, and in the Rechallenge phase a response of 78% (n = 7/9) was achieved. Two patients did not experience a reduction in symptoms after restarting the treatment. In one patient, a positive response was seen in the Challenge phase but not during the Rechallenge phase. This could be attributed to resistance in the Rechallenge phase, or to a placebo effect during the Challenge phase; however, DNA diagnostics were not performed in this patient, so the genetic basis and resulting sensitivity of the vascular malformation could not be explored. In the other patient lacking a positive response in the Rechallenge phase; DNA diagnostics revealed no mutations. A hypothesis of these non-responders may be a mutation in a different pathway; for example the RAS/BRAF/MAPK/ERK pathway which stimulates angiogenesis also( 32 ). However, clinical appearance did not correspond with a mutation in this pathway. Another possible theory is that the duration of six or twelve months is not long enough for those patients who are potential late responders. Our data show a comparable response rate to that of the literature despite our lower target level (85% Adams, 20–80% Nadal et al. versus 78–83% in the pilot study( 15 , 33 )). Less serious adverse events in bone marrow toxicity were seen in this pilot study (0%) compared to other high dose clinical studies: 0% versus Adams et al. (27%), Schena et al (13.4–36.3%) in renal allograft recipients using target trough levels of 8–20 ng/mL( 21 ) and Nguyen et al: (review, 11%-76%)( 13 ) (see Table 2 ). A significant relationship was seen between steady state concentration (C ss ) values of sirolimus and the occurrence of thrombocytopenia and leukopenia by Kahan et al( 28 ). Taken together, these observations support our hypothesis that reduction of the target trough concentration range from high target level 10–15 ng/ml to low target level 4–10 ng/ml does not decrease efficacy but improves tolerance. Challenge-Dechallenge-Rechallenge-designs are typically used for single-subject clinical trials to investigate efficacy or verify causality when an adverse drug reaction is suspected( 18 ). As vascular malformations are rare and heterogenous, the use of a CDR design offers the opportunity to generate interpretable data on efficacy and safety by analyzing each patient as their own control. We consider this design to be suited to investigate the efficacy of new treatments in those rare diseases, for which randomized clinical trials are less feasible due to the low numbers of patients affected and double blind randomized repeated rechallenges (as implemented in ‘n = 1’ trials) are not possible due to carry-over effects( 34 ). During this pilot study a low target level of sirolimus (4–10 ng/ml) is being used in comparison with previous high target level studies (10–15 ng/ml). Parker et al. used a very low target level sirolimus of 2–6 ng/ml in 39 PIK3CA-Related Overgrowth Spectrum (PROS) patients. This study suggests that even very low target level sirolimus can modestly reduce overgrowth; a significant reduction of -7.2% was seen in the volume change of affected tissues( 35 ). We observed a clear clinical response during the Challenge phase using the CDR concept. After stopping the sirolimus treatment, all patients showed a relapse of complaints and regrowth of the vascular malformation; however, the Rechallenge with sirolimus again reduced the complaints, indicating the effectiveness of sirolimus treatment. The results of the pilot study have led to the development of a nationwide clinical trial, which is currently enrolling patients. This trial investigates whether low-dose sirolimus is effective for alleviating symptoms including pain, QoL, and lesion volume with an expected lower incidence of serious adverse events compared to high target levels (especially bone marrow toxicity and grade III liver toxicity, which at least have not been observed in the pilot study). The methods of the ongoing clinical trial are based on the data obtained in this pilot study. Conclusion This pilot using low dose Sirolimus showed high efficacy in patients with therapy resistant and disabling low-flow malformation, with a lower incidence of serious adverse events (especially bone marrow toxicity and grade III liver toxicity). This is extremely relevant to patients with low-flow vascular malformation, who are likely to require lifelong treatment for their condition. Declarations Ethics approval and consent to participate The pilot study was approved as non – Medical Research Involving Human Subjects Act (nWMO study) the Research Ethics Committee (CMO Regio Arnhem-Nijmegen – Institutional Review Board) in the Netherlands. The study was performed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Consent for publication Informed consent was obtained in all patients before start. Patient and her parents gave their consent for publication and image. Availability of data and materials The datasets supporting the conclusions of this article is(are) included within the article (and its additional files. Competing interests The authors declare that they have no competing interests. Funding Pfizer supports this clinical trial by offering sirolimus (Rapamune®) Authors' contributions All authors read and approved the final manuscript. VH writing the manuscript and analysed and interpreted the patient data, GR co-conducted the pilot study, substantively revised the manuscript, CvdV substantively revised the manuscript, PdL substantively revised the manuscript, CvdH substantively revised the manuscript, WK substantively revised the manuscript, LSK substantively revised the manuscript, MtL: conducted the pilot study, analysed and interpreted the patient data, contributor in writing the manuscript and responsible as principal investigator for the execution of the pilot study. All authors read and approved the final manuscript. Acknowledgements We would like to thank Ingrid van Rijnsoever for her excellent contribution of coordinating of this pilot. References Wassef M, Blei F, Adams D, Alomari A, Baselga E, Berenstein A, et al. Vascular Anomalies Classification: Recommendations From the International Society for the Study of Vascular Anomalies. Pediatrics. 2015;136(1):e203-14. Akita S, Houbara S, Hirano A. 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Lillie EO, Patay B, Diamant J, Issell B, Topol EJ, Schork NJ. The n-of-1 clinical trial: the ultimate strategy for individualizing medicine? Per Med. 2011;8(2):161-73. Wang WL, Yu LX. Acute respiratory distress attributed to sirolimus in solid organ transplant recipients. Am J Emerg Med. 2015;33(1):124 e1-4. Champion L, Stern M, Israel-Biet D, Mamzer-Bruneel MF, Peraldi MN, Kreis H, et al. Brief communication: sirolimus-associated pneumonitis: 24 cases in renal transplant recipients. Ann Intern Med. 2006;144(7):505-9. Schena FP, Pascoe MD, Alberu J, del Carmen Rial M, Oberbauer R, Brennan DC, et al. Conversion from calcineurin inhibitors to sirolimus maintenance therapy in renal allograft recipients: 24-month efficacy and safety results from the CONVERT trial. Transplantation. 2009;87(2):233-42. Rodriguez-Moreno A, Ridao N, Garcia-Ledesma P, Calvo N, Perez-Flores I, Marques M, et al. Sirolimus and everolimus induced pneumonitis in adult renal allograft recipients: experience in a center. Transplant Proc. 2009;41(6):2163-5. Garrean S, Massad MG, Tshibaka M, Hanhan Z, Caines AE, Benedetti E. Sirolimus-associated interstitial pneumonitis in solid organ transplant recipients. Clin Transplant. 2005;19(5):698-703. Altomare DA, Khaled AR. Homeostasis and the importance for a balance between AKT/mTOR activity and intracellular signaling. Curr Med Chem. 2012;19(22):3748-62. Morath C, Schwenger V, Ksoll-Rudek D, Sommerer C, Beimler J, Schmidt J, et al. Four cases of sirolimus-associated interstitial pneumonitis: identification of risk factors. Transplant Proc. 2007;39(1):99-102. Singer SJ, Tiernan R, Sullivan EJ. Interstitial pneumonitis associated with sirolimus therapy in renal-transplant recipients. N Engl J Med. 2000;343(24):1815-6. Bee J, Fuller S, Miller S, Johnson SR. Lung function response and side effects to rapamycin for lymphangioleiomyomatosis: a prospective national cohort study. Thorax. 2018;73(4):369-75. Kahan BD, Napoli KL, Kelly PA, Podbielski J, Hussein I, Urbauer DL, et al. Therapeutic drug monitoring of sirolimus: correlations with efficacy and toxicity. Clin Transplant. 2000;14(2):97-109. Mizuno T, Fukuda T, Emoto C, Mobberley-Schuman PS, Hammill AM, Adams DM, et al. Developmental pharmacokinetics of sirolimus: Implications for precision dosing in neonates and infants with complicated vascular anomalies. Pediatr Blood Cancer. 2017;64(8). Czechowicz JA, Long-Boyle JR, Rosbe KW, Mathes EF, Frieden IJ, Shimano KA. Sirolimus for management of complex vascular anomalies - A proposed dosing regimen for very young infants. Int J Pediatr Otorhinolaryngol. 2018;105:48-51. Maschmeyer G, De Greef J, Mellinghoff SC, Nosari A, Thiebaut-Bertrand A, Bergeron A, et al. Infections associated with immunotherapeutic and molecular targeted agents in hematology and oncology. A position paper by the European Conference on Infections in Leukemia (ECIL). Leukemia. 2019;33(4):844-62. Queisser A, Boon LM, Vikkula M. Etiology and Genetics of Congenital Vascular Lesions. Otolaryngol Clin North Am. 2018;51(1):41-53. Nadal M, Giraudeau B, Tavernier E, Jonville-Bera AP, Lorette G, Maruani A. Efficacy and Safety of Mammalian Target of Rapamycin Inhibitors in Vascular Anomalies: A Systematic Review. Acta Derm Venereol. 2016;96(4):448-52. Wang Y, Schork NJ. Power and Design Issues in Crossover-Based N-Of-1 Clinical Trials with Fixed Data Collection Periods. Healthcare (Basel). 2019;7(3). Parker VER, Keppler-Noreuil KM, Faivre L, Luu M, Oden NL, De Silva L, et al. Safety and efficacy of low-dose sirolimus in the PIK3CA-related overgrowth spectrum. Genet Med. 2019;21(5):1189-98. Supplementary Files CONSORTextensionforPilotandFeasibilityTrialsChecklist.doc SupplementalTableS1.pdf SupplementalTableS2CV.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-132412","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":6788814,"identity":"df873845-e27b-442d-a049-684afb15e803","order_by":0,"name":"Veroniek Harbers","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0428-3242","institution":"Radboud University Nijmegen Medical Centre: , NL","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Veroniek","middleName":"","lastName":"Harbers","suffix":""},{"id":6788815,"identity":"7967d3b8-3892-48a4-93dc-9601886ace77","order_by":1,"name":"Gerard Rongen","email":"","orcid":"","institution":"Radboudumc","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gerard","middleName":"","lastName":"Rongen","suffix":""},{"id":6788816,"identity":"946a732b-4d06-447b-9c5f-4c763de623fe","order_by":2,"name":"Carine Vleuten, van der","email":"","orcid":"","institution":"Radboudumc","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"van","middleName":"der Carine","lastName":"Vleuten","suffix":""},{"id":6788817,"identity":"73db8b61-9aa1-41b6-9918-2ddcaf61fdea","order_by":3,"name":"Bas Verhoeven","email":"","orcid":"","institution":"Radboudumc","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bas","middleName":"","lastName":"Verhoeven","suffix":""},{"id":6788818,"identity":"97df7488-f3d2-460c-921f-19adf4e465a0","order_by":4,"name":"Peter Laat, de","email":"","orcid":"","institution":"Erasmusmc","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"de","middleName":"Peter","lastName":"Laat","suffix":""},{"id":6788819,"identity":"c3c83646-cf11-4b90-8a5f-93f0f35a7fd3","order_by":5,"name":"Chantal Horst, van der","email":"","orcid":"","institution":"Amsterdamumc","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"van","middleName":"der Chantal","lastName":"Horst","suffix":""},{"id":6788820,"identity":"e60f6307-fb6c-4be0-9aa8-bb189a4f9cc6","order_by":6,"name":"Willemijn Klein","email":"","orcid":"","institution":"Radboudumc","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Willemijn","middleName":"","lastName":"Klein","suffix":""},{"id":6788821,"identity":"457dd850-05fc-4947-9f90-bbd69ff5e85b","order_by":7,"name":"Leo Schultze Kool","email":"","orcid":"","institution":"Radboudumc","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Leo","middleName":"Schultze","lastName":"Kool","suffix":""},{"id":6788822,"identity":"21924680-f25f-4e3b-ba72-b1de3dcb5889","order_by":8,"name":"Maroeska Loo, te","email":"","orcid":"","institution":"Radboudumc","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"te","middleName":"Maroeska","lastName":"Loo","suffix":""}],"badges":[],"createdAt":"2020-12-19 19:06:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-132412/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-132412/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4486272,"identity":"e92ab908-f912-4ec0-97bb-25b2be193ea2","added_by":"auto","created_at":"2020-12-23 20:54:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11359466,"visible":true,"origin":"","legend":"Photographs obtained from patient 10 throughout the treatment period during the pilot study, showing the changes in the submandibular- and neck-localized lymphatic malformation. \n1a Five months before start of sirolimus\n1b After five months of sirolimus\n1c After eleven months of sirolimus\n1d Current clinical situation: after partial tongue resection and bleomycin sclerotherapy\n","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-132412/v1/e2416f5e268f9a37faf48550.png"},{"id":4486270,"identity":"637fd0d2-826c-45f2-bf6b-71ec5fea454c","added_by":"auto","created_at":"2020-12-23 20:54:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8457268,"visible":true,"origin":"","legend":"T2-weighted MRI images obtained from patient 10 throughout the treatment period during the pilot study, showing the changes in the submandibular- and neck-localized lymphatic malformation. The white boxes indicate the location of the lymphatic malformation.\n2a Before start of sirolimus treatment\n2b After six months of sirolimus treatment\n2c Before restarting sirolimus treatment after a partial tongue resection and bleomycin sclerotherapy\n2d After twelve months of sirolimus during the Rechallenge phase\n","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-132412/v1/19ca9886df6ddef0be1bbe7f.png"},{"id":13639084,"identity":"d98f9ec7-9564-4fc4-8792-50982a30f413","added_by":"auto","created_at":"2021-09-17 08:54:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2631243,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-132412/v1/bfac9052-8e69-4309-808b-4a39cd858a33.pdf"},{"id":4486271,"identity":"99c03420-9892-472f-bc1b-8903a363e9d0","added_by":"auto","created_at":"2020-12-23 20:54:40","extension":"doc","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":233984,"visible":true,"origin":"","legend":"","description":"","filename":"CONSORTextensionforPilotandFeasibilityTrialsChecklist.doc","url":"https://assets-eu.researchsquare.com/files/rs-132412/v1/458d9d724d285c1bb3effb46.doc"},{"id":4486186,"identity":"3240afd9-9d9c-4794-b1f3-9aca07170ec7","added_by":"auto","created_at":"2020-12-23 20:51:40","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":92180,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-132412/v1/686a0ba569e0d24d73940cd2.pdf"},{"id":4486188,"identity":"18d2770b-be53-46eb-9cc4-5ee56ba20b67","added_by":"auto","created_at":"2020-12-23 20:51:40","extension":"docx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":27786,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTableS2CV.docx","url":"https://assets-eu.researchsquare.com/files/rs-132412/v1/775d0e5795aaf3b7cc71ac04.docx"}],"financialInterests":"","formattedTitle":"Pilot study in patients with congenital low-flow vascular malformation treated with low dose sirolimus","fulltext":[{"header":"Background","content":" \u003cp\u003eVascular malformations include a heterogenous group of developmental anomalies of the vascular system, capillaries, veins, arteries, lymphatics or any combination of these vessels may be involved. The International Society for the Study of Vascular Anomalies (ISSVA) classified vascular malformations into the following categories(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e): simple (low-flow vascular malformation: capillary (CM), lymphatic (LM), venous (VM) arteriovenous (AVM), combined, vascular malformation of major named vessels, and vascular malformation associated with other anomalies.\u003c/p\u003e \u003cp\u003eVascular malformations are congenital, however, they can be discovered at any life stage, depending on their size and associated symptoms.\u003c/p\u003e \u003cp\u003eCurrent treatment options for low-flow vascular malformation may be conservative, with compression bandages, analgesics, anti-inflammatory or anti-coagulation drugs, or more invasive with intralesional sclerotherapy or embolization, and surgery(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Unfortunately, treatment is challenging and not always successful, and can leave patients with a high clinical burden and subsequently a reduced Quality of Life (QoL)(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Clinical symptoms that reduce the QoL in patients with low-flow vascular malformations include pain, functional impairment, bleeding, thrombophlebitis, ulceration, infections, and leakage (in LM)(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eSirolimus\u003c/h2\u003e \u003cp\u003eThe PI3/AKT/mTOR pathway plays a pivotal role in low-flow vascular malformation(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The activation of mammalian Target of Rapamycin (mTOR), stimulates angiogenesis, cell proliferation, and glucose metabolism. Some activating somatic mutations in genes in a target of the mTOR pathway, such as PIK3CA, AKT-1, TEK/TIE-2 and PTEN, in patients with low-flow vascular malformation resulted in the increased activation of mTOR(\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Therefore, the inhibition of this pathway in these patients seems a logical approach to treatment.\u003c/p\u003e \u003cp\u003eIn cells, sirolimus binds to the immunophilin FK Binding Protein-12 (FKBP-12), which in turn binds to and inhibits the activation of mTOR. This inhibition results in the obstruction of several signal transduction pathways, thereby inhibiting downstream protein biosynthesis, cell proliferation, and angiogenesis(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In theory, this should decrease the size of the low-flow vascular malformation or at least inhibit activity and stop further growth. Unfortunaly, the inhibition of lymphocyte activation also results in immunosuppression and might therefore be associated with the susceptibility to infections(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have been performed to explore the use of sirolimus as a treatment option in low-flow vascular malformations(\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These prospective open-label trials used - high target sirolimus levels of 10\u0026ndash;15\u0026nbsp;ng/ml leading to (partial) response in 85\u0026ndash;100% patients(\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). As these trials were open label trials, the question remains what is the true efficacy of sirolimus and what is natural behavior of the vascular malformation. Ideally, a placebo controlled randomized trial should be performed, however, in respect to the severe clinical burden of the patients and the rarity of the disease, it is difficult to execute. Recently, it has been postulated that a different design can be used in rare diseases to identify true efficacy of a drug(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). This design is based on the concept of Challenge, Dechallenge and Rechallenge (CDR) to proof the efficacy (or adverse events) caused by a single drug. Ideally, a future study should use this design to investigate true efficacy more in detail. Furthermore, more insight in the adverse events of sirolimus used as a single drug can be gained, as most information available so far, is based on adverse events observed in patients using a combination of drugs (e.g. renal transplant patients)(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe side effects of sirolimus described include oral ulceration, mucositis and stomatitis, interstitial lung disease, diabetes mellitus, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, gastro-intestinal side effects, angioedema, thrombo-embolic disease, anemia, leucopenia, thrombocytopenia, proteinuria, glomerulonephritis, and lymphedema(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur knowledge regarding long term toxicities of sirolimus is still expanding and makes it necessary to minimalize the risk for these long term toxicities. For example, it has been observed that in patients with long-term sirolimus impaired insulin receptor substrate signaling and Akt activation can be found indicating a deterioration of glucose metabolism leading to an increase of development of diabetes(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs in most drugs observed, one can imagine that higher levels of a drug increase the risk of developing adverse events of which the most serious and sometimes even fatal complication is sirolimus-associated interstitial pneumonitis(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBee et al. showed that low sirolimus serum levels (\u0026lt;\u0026thinsp;3 and 6.9\u0026nbsp;ng/ml) are related to less side effects without compromising efficacy of treatment in patients with diffuse lymphangioleiomyomatosis(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Additionally, Kahan et al. showed a significant relation between the occurrence of adverse events (hypertriglyceridemia, hypercholesterolemia, leukopenia and thrombocytopenia) and the steady state concentration value of sirolimus(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). A C\u003csub\u003ess\u003c/sub\u003e below 10\u0026nbsp;ng/L showed no toxic values.\u003c/p\u003e \u003cp\u003eIn the pilot study described here, we hypothesized that sirolimus used in low dosages with lower target levels (4\u0026ndash;10\u0026nbsp;ng/ml) than previously described, is equally effective in the treatment of low flow vascular malformations however will lead to less serious adverse events as observed in those treated with high target levels. Treatment with low target levels (4\u0026ndash;10\u0026nbsp;ng/ml) may require longer treatment than with high target levels (\u0026gt;\u0026thinsp;10\u0026nbsp;ng/ml), however low dose sirolimus will be more tolerable if less (serious) adverse events occur. This is especially important, as our knowledge regarding long term toxicities of sirolimus is still growing and makes it necessary to reduce the risk for these long term toxicities.\u003c/p\u003e"},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section3\"\u003e \u003cp\u003eThe pilot study was performed between 2015 and 2017. Patients included in the pilot study had a severe form of congenital vascular malformation, suffering severe pain and/or impairments. No predefined response criteria were used.\u003c/p\u003e \u003cp\u003eAll patients included underwent a Challenge-Dechallenge-Rechallenge to determine the efficacy of low dose sirolimus. Primary objectives of the pilot study were to determine the pharmacodynamics of low dose Sirolimus in respect of efficacy and adverse events.\u003c/p\u003e \u003cp\u003ePatients received sirolimus for three to six months in the Challenge phase, after which sirolimus treatment stopped (Dechallenge phase). If complaints (e.g., pain) did not reoccur, the follow-up phase continued for at least one year, during which time the duration of the pain/symptom-free period and any serious adverse events was measured. If pain or other symptoms returned during the Dechallenge phase, a Rechallenge phase of one year of sirolimus treatment was initiated.\u003c/p\u003e \u003cp\u003eSirolimus was administered orally using a start dose of 0.8\u0026nbsp;mg/m\u003csup\u003e2\u003c/sup\u003e twice a day for children, and 1\u0026nbsp;mg twice a day for adults. This starting dose is based on the pharmacokinetic properties of sirolimus. During the treatment period, therapeutic drug monitoring was performed. Target trough levels of sirolimus between 4\u0026ndash;10\u0026nbsp;ng/ml were used, based on laboratory and clinical studies(\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince sirolimus has immunosuppressive properties, cotrimoxazole as a Pneumocystis Jiroveci Pneumonia prophylaxis was prescribed. Recent guidelines suggest that this prophylaxis is not necessary, citing no clear evidence of increased infection, however, at the time this study was performed these guidelines were not available(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Adverse events were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE).\u003c/p\u003e \u003cp\u003eAn MRI is only made in a subset of patients before and after each phase as this outcome measure was not prospectively defined for this pilot study. Therefore the evaluation of the effect of sirolimus on the size of the vascular malformation will be only possible in these patients. In case there was secondary material that was obtained after surgery, stored in the HECOVAN biobank it will be used for analyses. Comprehensive targeted Next Generation Sequencing screen using Unique Molecular Identifiers with a technical sensitivity of 1% mutant alleles was performed for frequently mutated positions using Single Molecule Molecular Inversion Probes(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe end point of the pilot study was pain reduction, therefore response was assigned to sirolimus based on pain reduction during the first period (Challenge phase), after which the patients stopped taking sirolimus (Dechallenge). When pain or other complaints returned, sirolimus was re-administered (Rechallenge).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cp\u003eTwelve patients (aged between 1 and 50; 8\u0026nbsp;M, 4\u0026nbsp;F) were included in the pilot study. The clinical characteristics of the patients are shown in Supplemental Table S1. All patients had an otherwise untreatable form of a low-flow vascular malformation. All patients had a multiple sclerotherapy treatments and some had a partial resections in the past. These treatments were not possible or successful anymore. The initial treatment duration was at least three months (Challenge phase). At the end of the Challenge phase, eleven patients experienced pain reduction; of whom five became entirely pain free (pretreatment pain scores: 6\u0026ndash;10); only one patient experienced no change in complaints. Pain reduction was achieved between one week and five months after start of sirolimus (mean: seven weeks). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the results per vascular malformation type.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of clinical and MRI responses in the Challenge and Rechallenge periods.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eChallenge (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eRechallenge (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVascular malformation type\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eResponse after six months of sirolimus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eVascular malformation size as assessed using MRI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eResponse after 12 months of sirolimus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eVascular malformation size as assessed using MRI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVM (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImprovement of symptoms (pain, QoL) (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- Decrease (1)\u003c/p\u003e \u003cp\u003e- No MRI (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePain free (1)\u003c/p\u003e \u003cp\u003eNot restarted (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e- Decrease (1)\u003c/p\u003e \u003cp\u003e- Not restarted (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImprovement of symptoms (pain, QoL, leakage, infections, decrease of clinical size of LM, etc.) (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- No change (2)\u003c/p\u003e \u003cp\u003e- Decrease (2)\u003c/p\u003e \u003cp\u003e- No MRI (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e- Improvement of symptoms (pain, QoL, leakage, decrease of clinical size of LM, etc.) (2)\u003c/p\u003e \u003cp\u003e- No change (1)\u003c/p\u003e \u003cp\u003e- Not restarted (1)\u003c/p\u003e \u003cp\u003e- Restarted with mTOR inhibitor (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e- No change (1)\u003c/p\u003e \u003cp\u003e- Decrease (1)\u003c/p\u003e \u003cp\u003e- Not restarted (1)\u003c/p\u003e \u003cp\u003e- Restarted with mTOR inhibitor (1)\u003c/p\u003e \u003cp\u003e- Unknown (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphangiomatosis (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo change (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDecrease (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo change in symptoms (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncrease (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLVM (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImprovement of symptoms (pain, clinical size of LVM) (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- No change (1)\u003c/p\u003e \u003cp\u003e- No MRI (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e- Pain reduction (2)\u003c/p\u003e \u003cp\u003e- Clinical size reduction LVM (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e- No change (1)\u003c/p\u003e \u003cp\u003e- Unknown (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKTS (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImprovement of symptoms (pain, QoL) (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- No change (1)\u003c/p\u003e \u003cp\u003e- No MRI (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eImprovement of symptoms (pain, clinical size reduction) (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e- No change (1)\u003c/p\u003e \u003cp\u003e- Unknown (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImprovement of symptoms (11; 92%)\u003c/p\u003e \u003cp\u003eNo change (1; 8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- No change (4; 33%)\u003c/p\u003e \u003cp\u003e- Decrease (4; 33%)\u003c/p\u003e \u003cp\u003e- No MRI (4; 33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e- Improvement of symptoms (pain, QoL, leakage, decrease of clinical size of LM, etc.) (7; 78%)\u003c/p\u003e \u003cp\u003e- No change (2; 122%)\u003c/p\u003e \u003cp\u003e- Not restarted (2; 22%)\u003c/p\u003e \u003cp\u003e- Restarted with mTOR inhibitor (1; 11%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e- No change (3; 33%)\u003c/p\u003e \u003cp\u003e- Decrease (2; 22%)\u003c/p\u003e \u003cp\u003e- Increase (1; 11%)\u003c/p\u003e \u003cp\u003e- Not restarted (2; 22%)\u003c/p\u003e \u003cp\u003e- Restarted with mTOR inhibitor (1; 11%)\u003c/p\u003e \u003cp\u003e- Unknown (3; 33%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eVM: venous malformation, LM: lymphatic malformation, LVM: lymphatico-venous malformation, KTS: Klippel Trenaunay Weber syndrome.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eDuring the Dechallenge phase, ten patients had a return of pain/symptoms with a duration range of 10 days to 4 months. Nine patients restarted the sirolimus treatment shortly. In all patients, reasons for the re-administration of sirolimus were return of severe pain or other symptoms, such as leakage in the case of a lymphatic malformation or clear evidence of growth of the vascular malformation in the time period sirolimus was stopped. (see Supplemental Table S2).\u003c/p\u003e \u003cp\u003eThree patients did not restart with sirolimus in the Rechallenge phase despite the positive response primarily observed. Reasons were a loss of energy (patient 6), logistic reasons (patient 12), or the initiation of a different mTOR inhibitor (everolimus) (patient 7) at a nearby hospital. The patient that switched to a different mTOR inhibitor showed a significant reduction in complaints again. A substantial relationship between sirolimus and the amelioration of symptoms was confirmed during the Rechallenge phase in seven patients by a substantial reduction of complaints after they resumed taking sirolimus. This indicates the efficacy of sirolimus at a low dose.\u003c/p\u003e \u003cp\u003eAll patients experienced adverse events when taking sirolimus. The most frequent adverse events that were likely related to sirolimus treatment were: aphthous stomatitis grade I (50% of patients) and menstrual disorder grades I-II (female patients only; 75%). No patients experienced grade III bone marrow toxicity in the pilot study (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e); however, two patients (16.7%) experienced grades I-II bone marrow toxicity. One adult patient developed hypophosphatemia that rapidly recovered after stopping sirolimus. One patient (patient 4) experienced a grade III sepsis due to an infected lymphatic cyst. Reasons for a temporary stop (several days) of sirolimus were: due to vaccination (n\u0026thinsp;=\u0026thinsp;1), interventional radiology (bleomycin sclerotherapy n\u0026thinsp;=\u0026thinsp;2), decannulation (n\u0026thinsp;=\u0026thinsp;1), and adverse events: infections (n\u0026thinsp;=\u0026thinsp;13) of which n\u0026thinsp;=\u0026thinsp;1 sepsis, menstrual disorder (n\u0026thinsp;=\u0026thinsp;2), aphthous stomatitis (n\u0026thinsp;=\u0026thinsp;1), general malaise (n\u0026thinsp;=\u0026thinsp;1), elevated liver enzymes (n\u0026thinsp;=\u0026thinsp;1). Therapy-limiting adverse events were seen in two patients: one patient had due to a grade II increase of liver enzymes after five months sirolimus treatment (Challenge phase), and one patient had a grade II menorrhagia after 36\u0026nbsp;months sirolimus treatment (Rechallenge phase). All adverse events were resolved by interrupting treatment, and there have so far been no reported long-term adverse events (median follow-up three years; range 1\u0026ndash;5\u0026nbsp;years).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAdverse events observed in our pilot study compared to other studies.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdverse events attributable to sirolimus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade of toxicity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePilot study n\u0026thinsp;=\u0026thinsp;12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdams et al.\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;57\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHammer et al.\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;19\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNguyen et al. Review\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBlood/bone marrow toxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGrade II or higher: 30 (49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1(5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e11\u0026ndash;76%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (27%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGastro-intestinal toxicity (e.g. mucositis)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGrade II or higher: 33 (55%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e3\u0026ndash;19%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (11%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMetabolic/laboratory toxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGrade II or higher: 12 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e20\u0026ndash;64%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInfection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGrade II or higher: 9 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEndocrine toxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e20\u0026ndash;27% (Diabetes mellitus)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDermatology toxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (36.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (5.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNeurologic toxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4(33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12 (63%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePulmonary/upper respiratory toxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (58%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGrade II or higher: 1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInterstitial lung\u003c/p\u003e \u003cp\u003edisease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e4\u0026ndash;17%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMusculoskeletal/soft tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGeneral symptoms (for example hypertension/wound healing)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCardiac general: 0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 (105%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAngioedema (2.2\u0026ndash;15%), urologic (12%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCardiac general: 0 (0%)\u003c/p\u003e \u003cp\u003eConstitutional symptoms: 0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLymphedema\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade I and II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGrade II or higher: 4 (7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e6.4\u0026ndash;12%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAdverse events observed in our pilot study and those reported in the studies performed by Adams et al. and Hammer et al. and Nguyen et al.\u0026rsquo;s review. Adams et al.: patients with various complex vascular anomalies (including vascular tumors), target levels: 10\u0026ndash;15\u0026nbsp;ng/ml\u003c/em\u003e (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). \u003cem\u003eHammer et al: patients with a vascular malformation using target levels: 10\u0026ndash;15\u0026nbsp;ng/ml\u003c/em\u003e (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). \u003cem\u003eThe table showed a 105% percentage due to categorizing and summarizing patients with general symptoms. Nguyen et al.\u0026rsquo;s review of sirolimus in solid organ transplantation identified a wide array of adverse effects\u003c/em\u003e (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter the Challenge phase, an MRI was performed in eight out of twelve patients. In four of the eight patients in which an MRI was performed, a visual size reduction of the low-flow vascular malformation was found. Stable disease was observed in the remaining four patients.\u003c/p\u003e \u003cp\u003eMRIs were also performed in six of the nine patients who underwent the Rechallenge phase. Two patients showed a decrease in vascular malformations size compared to baseline as assessed before each treatment phase.\u003c/p\u003e \u003cp\u003eGenetic testing of the vascular malformation was no performed routinely, however, in three out of four patients, DNA diagnostics on tissue revealed a PIK3CA mutation. In the other patient, no genetic aberrations could be found with our vascular anomalies panel (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) (Supplemental Table S1).\u003c/p\u003e "},{"header":"Case Presentation","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003cp\u003eTo illustrate the efficacy of lower target levels of Sirolimus, patient 10 is described in more detail. This patient, was a two years old female at the start of treatment and suffered of a severe macroglossia due to a lymphatic malformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A life-threatening situation in the first months after birth led to the need for a tracheal cannula to guarantee an open airway for a longer time period. Before start of sirolimus, patient had a substantial reduced QoL. In the first weeks after the start of the sirolimus treatment, a rapid decrease in the size of the low-flow vascular malformation was observed. The sirolimus target levels ranged between 4.4 and 6.0\u0026nbsp;ng/ml during this period. After six months of treatment, an MRI was performed to quantify the response to sirolimus, and a clear reduction in the lymphatic malformation was observed. During a six weeks Dechallenge period, the decision was made to restart sirolimus treatment, because of the increase of tongue volume, noduli and nodus tongue. After restarting, the vascular malformation further reduced in size and, at the age of three years, after using sirolimus for 21 months, bleomycin sclerotherapy, and a tongue resection, the cannula was removed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhotographs of the patient (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) show a clinical reduction in the volume of the low-flow vascular malformation in the submandibular and neck regions after only four weeks of sirolimus treatment. A further reduction is seen after eleven months of treatment with sirolimus. MRI images also show a reduction in volume at six and twelve months post treatment initiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The possible related adverse events this patients experienced were intermittent aphthous stomatitis, upper airway infections, tonsillitis, and elevation of triglycerides.\u003c/p\u003e \u003cp\u003eThe results of patient 10 (Supplemental Table S1 and S2) in the pilot study are in line with our hypothesis that low target levels of sirolimus are sufficiently effective and lead to fewer and less severe adverse events.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eWe present the results of a pilot study to determine the efficacy and safety of low dose sirolimus on low-flow vascular malformations. A positive response of 92% (n\u0026thinsp;=\u0026thinsp;11/12) in the Challenge phase was achieved, and in the Rechallenge phase a response of 78% (n\u0026thinsp;=\u0026thinsp;7/9) was achieved. Two patients did not experience a reduction in symptoms after restarting the treatment. In one patient, a positive response was seen in the Challenge phase but not during the Rechallenge phase. This could be attributed to resistance in the Rechallenge phase, or to a placebo effect during the Challenge phase; however, DNA diagnostics were not performed in this patient, so the genetic basis and resulting sensitivity of the vascular malformation could not be explored. In the other patient lacking a positive response in the Rechallenge phase; DNA diagnostics revealed no mutations. A hypothesis of these non-responders may be a mutation in a different pathway; for example the RAS/BRAF/MAPK/ERK pathway which stimulates angiogenesis also(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). However, clinical appearance did not correspond with a mutation in this pathway. Another possible theory is that the duration of six or twelve months is not long enough for those patients who are potential late responders.\u003c/p\u003e \u003cp\u003eOur data show a comparable response rate to that of the literature despite our lower target level (85% Adams, 20\u0026ndash;80% Nadal et al. versus 78\u0026ndash;83% in the pilot study(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)).\u003c/p\u003e \u003cp\u003eLess serious adverse events in bone marrow toxicity were seen in this pilot study (0%) compared to other high dose clinical studies: 0% versus Adams et al. (27%), Schena et al (13.4\u0026ndash;36.3%) in renal allograft recipients using target trough levels of 8\u0026ndash;20\u0026nbsp;ng/mL(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and Nguyen et al: (review, 11%-76%)(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) (see Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A significant relationship was seen between steady state concentration (C\u003csub\u003ess\u003c/sub\u003e) values of sirolimus and the occurrence of thrombocytopenia and leukopenia by Kahan et al(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Taken together, these observations support our hypothesis that reduction of the target trough concentration range from high target level 10\u0026ndash;15\u0026nbsp;ng/ml to low target level 4\u0026ndash;10\u0026nbsp;ng/ml does not decrease efficacy but improves tolerance.\u003c/p\u003e \u003cp\u003eChallenge-Dechallenge-Rechallenge-designs are typically used for single-subject clinical trials to investigate efficacy or verify causality when an adverse drug reaction is suspected(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). As vascular malformations are rare and heterogenous, the use of a CDR design offers the opportunity to generate interpretable data on efficacy and safety by analyzing each patient as their own control. We consider this design to be suited to investigate the efficacy of new treatments in those rare diseases, for which randomized clinical trials are less feasible due to the low numbers of patients affected and double blind randomized repeated rechallenges (as implemented in \u0026lsquo;n\u0026thinsp;=\u0026thinsp;1\u0026rsquo; trials) are not possible due to carry-over effects(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring this pilot study a low target level of sirolimus (4\u0026ndash;10\u0026nbsp;ng/ml) is being used in comparison with previous high target level studies (10\u0026ndash;15\u0026nbsp;ng/ml). Parker et al. used a very low target level sirolimus of 2\u0026ndash;6\u0026nbsp;ng/ml in 39 PIK3CA-Related Overgrowth Spectrum (PROS) patients. This study suggests that even very low target level sirolimus can modestly reduce overgrowth; a significant reduction of -7.2% was seen in the volume change of affected tissues(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe observed a clear clinical response during the Challenge phase using the CDR concept. After stopping the sirolimus treatment, all patients showed a relapse of complaints and regrowth of the vascular malformation; however, the Rechallenge with sirolimus again reduced the complaints, indicating the effectiveness of sirolimus treatment.\u003c/p\u003e \u003cp\u003eThe results of the pilot study have led to the development of a nationwide clinical trial, which is currently enrolling patients. This trial investigates whether low-dose sirolimus is effective for alleviating symptoms including pain, QoL, and lesion volume with an expected lower incidence of serious adverse events compared to high target levels (especially bone marrow toxicity and grade III liver toxicity, which at least have not been observed in the pilot study). The methods of the ongoing clinical trial are based on the data obtained in this pilot study.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eThis pilot using low dose Sirolimus showed high efficacy in patients with therapy resistant and disabling low-flow malformation, with a lower incidence of serious adverse events (especially bone marrow toxicity and grade III liver toxicity). This is extremely relevant to patients with low-flow vascular malformation, who are likely to require lifelong treatment for their condition.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pilot study was approved as non \u0026ndash; Medical Research Involving Human Subjects Act (nWMO study) the Research Ethics Committee (CMO Regio Arnhem-Nijmegen \u0026ndash; Institutional Review Board) in the Netherlands. The study was performed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained in all patients before start. Patient and her parents gave their consent for publication and image.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article is(are) included within the article (and its additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePfizer supports this clinical trial by offering sirolimus (Rapamune\u0026reg;)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors' contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eVH writing the manuscript and analysed and interpreted the patient data, GR co-conducted the pilot study, substantively revised the manuscript, CvdV substantively revised the manuscript, PdL substantively revised the manuscript, CvdH substantively revised the manuscript, WK substantively revised the manuscript, LSK substantively revised the manuscript, MtL: conducted the pilot study, analysed and interpreted the patient data, contributor in writing the manuscript and responsible as principal investigator for the execution of the pilot study. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Ingrid van Rijnsoever for her excellent contribution of coordinating of this pilot.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWassef M, Blei F, Adams D, Alomari A, Baselga E, Berenstein A, et al. Vascular Anomalies Classification: Recommendations From the International Society for the Study of Vascular Anomalies. Pediatrics. 2015;136(1):e203-14.\u003c/li\u003e\n\u003cli\u003eAkita S, Houbara S, Hirano A. Management of vascular malformations. Plast Reconstr Surg Glob Open. 2014;2(3):e128.\u003c/li\u003e\n\u003cli\u003eMulligan PR, Prajapati HJ, Martin LG, Patel TH. Vascular anomalies: classification, imaging characteristics and implications for interventional radiology treatment approaches. Br J Radiol. 2014;87(1035):20130392.\u003c/li\u003e\n\u003cli\u003eCox JA, Bartlett E, Lee EI. Vascular malformations: a review. Semin Plast Surg. 2014;28(2):58-63.\u003c/li\u003e\n\u003cli\u003eLindhurst MJ, Sapp JC, Teer JK, Johnston JJ, Finn EM, Peters K, et al. A mosaic activating mutation in AKT1 associated with the Proteus syndrome. N Engl J Med. 2011;365(7):611-9.\u003c/li\u003e\n\u003cli\u003eTen Broek RW, Eijkelenboom A, van der Vleuten CJM, Kamping EJ, Kets M, Verhoeven BH, et al. Comprehensive molecular and clinicopathological analysis of vascular malformations: A study of 319 cases. Genes Chromosomes Cancer. 2019;58(8):541-50.\u003c/li\u003e\n\u003cli\u003eKeppler-Noreuil KM, Rios JJ, Parker VE, Semple RK, Lindhurst MJ, Sapp JC, et al. PIK3CA-related overgrowth spectrum (PROS): diagnostic and testing eligibility criteria, differential diagnosis, and evaluation. Am J Med Genet A. 2015;167A(2):287-95.\u003c/li\u003e\n\u003cli\u003eFreed D, Stevens EL, Pevsner J. Somatic mosaicism in the human genome. Genes (Basel). 2014;5(4):1064-94.\u003c/li\u003e\n\u003cli\u003eBoscolo E, Limaye N, Huang L, Kang KT, Soblet J, Uebelhoer M, et al. Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects. J Clin Invest. 2015;125(9):3491-504.\u003c/li\u003e\n\u003cli\u003eNathan N, Keppler-Noreuil KM, Biesecker LG, Moss J, Darling TN. Mosaic Disorders of the PI3K/PTEN/AKT/TSC/mTORC1 Signaling Pathway. Dermatol Clin. 2017;35(1):51-60.\u003c/li\u003e\n\u003cli\u003eGuertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell. 2007;12(1):9-22.\u003c/li\u003e\n\u003cli\u003eLaplante M, Sabatini DM. mTOR signaling at a glance. J Cell Sci. 2009;122(Pt 20):3589-94.\u003c/li\u003e\n\u003cli\u003eNguyen LS, Vautier M, Allenbach Y, Zahr N, Benveniste O, Funck-Brentano C, et al. Sirolimus and mTOR Inhibitors: A Review of Side Effects and Specific Management in Solid Organ Transplantation. Drug Saf. 2019;42(7):813-25.\u003c/li\u003e\n\u003cli\u003eHammill AM, Wentzel M, Gupta A, Nelson S, Lucky A, Elluru R, et al. Sirolimus for the treatment of complicated vascular anomalies in children. Pediatr Blood Cancer. 2011;57(6):1018-24.\u003c/li\u003e\n\u003cli\u003eAdams DM, Trenor CC, 3rd, Hammill AM, Vinks AA, Patel MN, Chaudry G, et al. Efficacy and Safety of Sirolimus in the Treatment of Complicated Vascular Anomalies. Pediatrics. 2016;137(2):e20153257.\u003c/li\u003e\n\u003cli\u003eLackner H, Karastaneva A, Schwinger W, Benesch M, Sovinz P, Seidel M, et al. Sirolimus for the treatment of children with various complicated vascular anomalies. Eur J Pediatr. 2015;174(12):1579-84.\u003c/li\u003e\n\u003cli\u003eHammer J, Seront E, Duez S, Dupont S, Van Damme A, Schmitz S, et al. Sirolimus is efficacious in treatment for extensive and/or complex slow-flow vascular malformations: a monocentric prospective phase II study. Orphanet J Rare Dis. 2018;13(1):191.\u003c/li\u003e\n\u003cli\u003eLillie EO, Patay B, Diamant J, Issell B, Topol EJ, Schork NJ. The n-of-1 clinical trial: the ultimate strategy for individualizing medicine? Per Med. 2011;8(2):161-73.\u003c/li\u003e\n\u003cli\u003eWang WL, Yu LX. Acute respiratory distress attributed to sirolimus in solid organ transplant recipients. Am J Emerg Med. 2015;33(1):124 e1-4.\u003c/li\u003e\n\u003cli\u003eChampion L, Stern M, Israel-Biet D, Mamzer-Bruneel MF, Peraldi MN, Kreis H, et al. Brief communication: sirolimus-associated pneumonitis: 24 cases in renal transplant recipients. Ann Intern Med. 2006;144(7):505-9.\u003c/li\u003e\n\u003cli\u003eSchena FP, Pascoe MD, Alberu J, del Carmen Rial M, Oberbauer R, Brennan DC, et al. Conversion from calcineurin inhibitors to sirolimus maintenance therapy in renal allograft recipients: 24-month efficacy and safety results from the CONVERT trial. Transplantation. 2009;87(2):233-42.\u003c/li\u003e\n\u003cli\u003eRodriguez-Moreno A, Ridao N, Garcia-Ledesma P, Calvo N, Perez-Flores I, Marques M, et al. Sirolimus and everolimus induced pneumonitis in adult renal allograft recipients: experience in a center. Transplant Proc. 2009;41(6):2163-5.\u003c/li\u003e\n\u003cli\u003eGarrean S, Massad MG, Tshibaka M, Hanhan Z, Caines AE, Benedetti E. Sirolimus-associated interstitial pneumonitis in solid organ transplant recipients. Clin Transplant. 2005;19(5):698-703.\u003c/li\u003e\n\u003cli\u003eAltomare DA, Khaled AR. Homeostasis and the importance for a balance between AKT/mTOR activity and intracellular signaling. Curr Med Chem. 2012;19(22):3748-62.\u003c/li\u003e\n\u003cli\u003eMorath C, Schwenger V, Ksoll-Rudek D, Sommerer C, Beimler J, Schmidt J, et al. Four cases of sirolimus-associated interstitial pneumonitis: identification of risk factors. Transplant Proc. 2007;39(1):99-102.\u003c/li\u003e\n\u003cli\u003eSinger SJ, Tiernan R, Sullivan EJ. Interstitial pneumonitis associated with sirolimus therapy in renal-transplant recipients. N Engl J Med. 2000;343(24):1815-6.\u003c/li\u003e\n\u003cli\u003eBee J, Fuller S, Miller S, Johnson SR. Lung function response and side effects to rapamycin for lymphangioleiomyomatosis: a prospective national cohort study. Thorax. 2018;73(4):369-75.\u003c/li\u003e\n\u003cli\u003eKahan BD, Napoli KL, Kelly PA, Podbielski J, Hussein I, Urbauer DL, et al. Therapeutic drug monitoring of sirolimus: correlations with efficacy and toxicity. Clin Transplant. 2000;14(2):97-109.\u003c/li\u003e\n\u003cli\u003eMizuno T, Fukuda T, Emoto C, Mobberley-Schuman PS, Hammill AM, Adams DM, et al. Developmental pharmacokinetics of sirolimus: Implications for precision dosing in neonates and infants with complicated vascular anomalies. Pediatr Blood Cancer. 2017;64(8).\u003c/li\u003e\n\u003cli\u003eCzechowicz JA, Long-Boyle JR, Rosbe KW, Mathes EF, Frieden IJ, Shimano KA. Sirolimus for management of complex vascular anomalies - A proposed dosing regimen for very young infants. Int J Pediatr Otorhinolaryngol. 2018;105:48-51.\u003c/li\u003e\n\u003cli\u003eMaschmeyer G, De Greef J, Mellinghoff SC, Nosari A, Thiebaut-Bertrand A, Bergeron A, et al. Infections associated with immunotherapeutic and molecular targeted agents in hematology and oncology. A position paper by the European Conference on Infections in Leukemia (ECIL). Leukemia. 2019;33(4):844-62.\u003c/li\u003e\n\u003cli\u003eQueisser A, Boon LM, Vikkula M. Etiology and Genetics of Congenital Vascular Lesions. Otolaryngol Clin North Am. 2018;51(1):41-53.\u003c/li\u003e\n\u003cli\u003eNadal M, Giraudeau B, Tavernier E, Jonville-Bera AP, Lorette G, Maruani A. Efficacy and Safety of Mammalian Target of Rapamycin Inhibitors in Vascular Anomalies: A Systematic Review. Acta Derm Venereol. 2016;96(4):448-52.\u003c/li\u003e\n\u003cli\u003eWang Y, Schork NJ. Power and Design Issues in Crossover-Based N-Of-1 Clinical Trials with Fixed Data Collection Periods. Healthcare (Basel). 2019;7(3).\u003c/li\u003e\n\u003cli\u003eParker VER, Keppler-Noreuil KM, Faivre L, Luu M, Oden NL, De Silva L, et al. Safety and efficacy of low-dose sirolimus in the PIK3CA-related overgrowth spectrum. Genet Med. 2019;21(5):1189-98.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vascular malformation, lymphatic malformation, venous malformation, sirolimus, low-dose sirolimus, mTor inhibitor, pain","lastPublishedDoi":"10.21203/rs.3.rs-132412/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-132412/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePatients with congenital low-flow vascular malformations (capillary (CM), lymphatic (LM), venous (VM) or combined) may have an impaired quality of life (QoL), due to their symptoms, which include pain, swelling, bleeding, thrombosis, and functional impairment. Unfortunately, current treatment methods are challenging and not always successful. Previous studies have shown that the mTOR-inhibitor sirolimus is an effective treatment for these patients. Target levels of 10\u0026ndash;15\u0026nbsp;ng/ml were well tolerated; however, grade three adverse events were observed (ranged 20\u0026ndash;40%).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA pilot study was performed using a Challenge\u0026ndash;Dechallenge\u0026ndash;Rechallenge (CDR) design to determine the pharmacodynamics of low target levels of sirolimus (target levels 4\u0026ndash;10\u0026nbsp;ng/ml) in respect of efficacy and adverse events in patients with disabling low-flow vascular malformations without treatment alternatives. The patients received sirolimus over a three-to-six-month period (Challenge), followed by the withdrawal of sirolimus (Dechallenge). If the complaints returned, sirolimus was reintroduced during a twelve month period (Rechallenge). Efficacy was determined on pain (end point of the pilot study) and other symptoms related to the vascular malformation; and adverse events were determined in all phases of the study.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAn improvement in symptoms was seen in 92% (n\u0026thinsp;=\u0026thinsp;11/12) of patients during the Challenge phase. In the Rechallenge phase, a positive response rate of 78% was found (n\u0026thinsp;=\u0026thinsp;7/9). These response rates are comparable to those found in the literature despite low target levels of sirolimus. However, less serious adverse events were observed with low dose sirolimus, especially bone marrow toxicity and grade III liver toxicity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis pilot using low dose sirolimus showed high efficacy in patients with therapy resistant and disabling low-flow malformation, with a lower incidence of serious adverse events (especially bone marrow toxicity and grade III liver toxicity). This is extremely relevant to patients with low-flow vascular malformation, as current clinical protocols tend to advise lifelong treatment.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e \u003cp\u003eThe pilot study was part of a phase III study. Trial registration: EudraCT number: 2016-002157-38 and ClinicalTrials.gov Identifier: NCT03987152, registered 06/14/2019 - Retrospectively registered, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://clinicaltrials.gov/ct2/show/NCT03987152?term=sirolimus\u0026amp;cond=Vascular+Malformations\u0026amp;cntry=NL\u0026amp;draw=2\u0026amp;rank=1\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e","manuscriptTitle":"Pilot study in patients with congenital low-flow vascular malformation treated with low dose sirolimus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-23 20:51:38","doi":"10.21203/rs.3.rs-132412/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c9e8f2f9-6522-4c9a-90c1-69492ee46843","owner":[],"postedDate":"December 23rd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1609431,"name":"Clinical Pharmacology"},{"id":1609432,"name":"Cardiac \u0026 Cardiovascular Systems"}],"tags":[],"updatedAt":"2021-01-06T23:41:00+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-23 20:51:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-132412","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-132412","identity":"rs-132412","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}