CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal naevi, scoliosis/skeletal and spinal syndrome) is a genetic disorder that results from somatic, mosaic gain-of-function mutations of the PIK3CA gene, and belongs to the spectrum of PIK3CA-related overgrowth syndromes (PROS). This rare condition has no specific treatment and a poor survival rate. Here, we describe a postnatal mouse model of PROS/CLOVES that partially recapitulates the human disease, and demonstrate the efficacy of BYL719, an inhibitor of PIK3CA, in preventing and improving organ dysfunction. On the basis of these results, we used BYL719 to treat nineteen patients with PROS. The drug improved the disease symptoms in all patients. Previously intractable vascular tumours became smaller, congestive heart failure was improved, hemihypertrophy was reduced, and scoliosis was attenuated. The treatment was not associated with any substantial side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic strategy in patients with PROS.
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This project received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program grants, (STG-2015) 679254 and (PoC-2016) 737546 (both awarded to G.C.). This work was also supported by the Emmanuel BOUSSARD Foundation, the DAY SOLVAY Foundation, Fondation TOURRE, Fondation Simone et Cino Del Duca, INSERM, Assistance Publique—Hôpitaux de Paris and the University of Paris, Descartes. We would like to thank the two patients and their families. We also thank C. Semaille from the ANSM for help and advice; S. Bisot-Locard (Novartis) for the BYL719; G. Autret for performing the mouse MRIs; S. Berissi and colleagues at the Plateforme d’histologie et morphologie du petit animal, INEM, Paris; S. Principe for administrative management; A. Klippel for advice and for providing plasmids encoding Myr-p110*-myc and Myr-p110*KR-myc constructs; K. Rajewsky for advice; and the radiological team from the Centre d’Imagerie de Franconville and in particular M. Canaud and A. Scemama for their help.
Nature thanks E. Baselga, W. Dobyns, R. Semple and B. Vanhaesebroeck for their contribution to the peer review of this work.
A patent application (WO2017140828A1) has been filed by INSERM (Institut National de la Santé et de la Recherche Médicale), Centre National De La Recherche Scientifique (CNRS), Université Paris Descartes, and Assistance Publique-Hôpitaux De Paris (AP-HP) for the use of BYL719 (alpelisib) in the treatment of PIK3CA-related overgrowth spectrum (PROS/CLOVES syndrome). G.C. is the inventor.
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Extended data figures and tables
a, Left, Representation of p110 and iSH2 domain of the p85 subunit (striped bar). The iSH2 domain is important to stabilize the p110α protein. The p110* protein is a constitutively active chimaera that contains the iSH2 domain of p85 fused to the N terminus of p110 via a flexible glycine linker14 (right). b, To generate tissue-specific p110*-transgenic mice, a cloned loxP-flanked neoR-stop cassette was inserted into a modified version of pROSA26-1 followed by the cDNA encoding p110* and then a frt-flanked IRES–EGFP cassette and a bovine polyadenylation sequence (R26StopFLP110*)13. c, d, EGFP expression from flow cytometry experiments in the spleen of PIK3CAWT mice (n = 12) and PIK3CACAGG-CreER mice injected with either a single 40 mg kg−1 dose (c; n = 6 mice) or a single 4 mg kg−1 dose (d; n = 6 mice) of tamoxifen. Each curve is a different mouse. e, MRI examination of the PROS mouse model and efficacy of BYL719 treatment. Top, arrows show muscle hypertrophy in PIK3CACAGG-CreER mice before BYL719 treatment. This phenotype was reversed by BYL719 administration. Middle, arrows show scoliosis in PIK3CACAGG-CreER mice before BYL719 treatment, which was rescued by BYL719 administration. Bottom, arrows show arterial dilation in PIK3CACAGG-CreER mice before BYL719 treatment, which was reversed by BYL719 administration (n = 6 mice per group).
a, Percentage of PIK3CAWT and PIK3CACAGG-CreER mice with or without BYL719 treatment presenting organ abnormalities. b, Oil Red O staining of the livers of PIK3CAWT and PIK3CACAGG-CreER mice demonstrating steatosis (n = 8 mice per group). Scale bars, 10 μm. c, CD31 (top) and CD34 (bottom) immunostaining in the liver of PIK3CAWT and PIK3CACAGG-CreER mice with or without BYL719 (n = 8 mice per group). PIK3CACAGG-CreER mice treated with vehicle showed vessel dilation that was prevented or reversed by BYL719. Scale bars, 10 μm. d, Representative picture of lymphatic malformation as assessed by podoplanin immunostaining in the liver of PIK3CAWT and PIK3CACAGG-CreER mice (n = 8 mice per group). Scale bars, 10 μm. e, Representative western blot of LYVE-1 in the liver of PIK3CAWT and PIK3CACAGG-CreER mice demonstrating lymphatic increased in the PIK3CACAGG-CreER mice (n = 8 mice per group). All data are shown as the means ± s.e.m. Mann–Whitney test (two-tailed, P = 0.001). PIK3CACAGG-CreER versus PIK3CAWT mice, ***P < 0.001.
a, Ki67 immunostaining and quantification in liver, spleen and heart of PIK3CAWT and PIK3CACAGG-CreER mice with or without BYL719 treatment (n = 8 mice per group, 10 randomly selected fields per mice, ×400). b, TUNEL assay. The graphs show the quantification of TUNEL-positive cells per field (n = 8 mice per group, 10 randomly selected fields per mice, ×400). Scale bars, 10 μm. All data are shown as mean ± s.e.m. ANOVA followed by Tukey–Kramer test (two-tailed). PIK3CACAGG-CreER versus PIK3CAWT mice, ***P < 0.001. PIK3CACAGG-CreER mice treated with vehicle versus PIK3CACAGG-CreER mice treated with preventive BYL719, ###P < 0.001. PIK3CACAGG-CreER mice treated with vehicle versus PIK3CACAGG-CreER mice treated with therapeutic BYL719, +++P < 0.001.
a, β-galactosidase staining in the liver, heart, spleen and kidney of PIK3CAWT and PIK3CACAGG-CreER mice with or without BYL719 and quantification of β-galactosidase-positive cells per field (n = 8 mice per group, 10 randomly selected fields, ×400). C+: positive control. Scale bars, 10 μm. b, p16 mRNA expression in liver, heart and spleen of PIK3CAWT and PIK3CACAGG-CreER mice treated with or without BYL719 (n = 8 mice per group). A.U., arbitrary unit. All data are shown as mean ± s.e.m. ANOVA followed by Tukey–Kramer test (two-tailed).
a, Western blot showing the expression of p110* in PIK3CACAGG-CreER mice (n = 8 mice per group). b, p110* is not expressed in the brain or lungs (n = 8 mice per group). c–e, Western blot quantification of Fig. 1d, in the liver (c), heart (d) and muscle (e) of PIK3CAWT and PIK3CACAGG-CreER mice treated with or without BYL719 (n = 8 mice per group). All data are shown as mean ± s.e.m. ANOVA followed by Tukey–Kramer test (two-tailed). PIK3CACAGG-CreER versus PIK3CAWT mice, ***P < 0.001. PIK3CACAGG-CreER mice treated with vehicle versus PIK3CACAGG-CreER mice treated with preventive BYL719, ###P < 0.001. PIK3CACAGG-CreER mice treated with vehicle versusd PIK3CACAGG-CreER mice treated with therapeutic BYL719, +++P < 0.001.
Immunofluorescence staining of P-AKT (Ser473) and P-S6RP in the liver (a), heart (b), spleen (c) and muscles (d) of PIK3CAWT and PIK3CACAGG-CreER mice treated with or without BYL719 (n = 8 mice per group). Scale bars, 10 μm.
a, Western blot and quantification of p110, P-AKT (Ser473), P-S6RP and GFP in HeLa cells transfected with plasmids containing cDNA encoding p110*, p110* kinase-dead mutant (p110* KD) as a control, H1047R mutation or E545K mutation. Cells transfected with the p110* mutant showed a more powerful effect on the activation of the AKT/mTORC pathway than the others (n = 4 independent experiments). All data are shown as mean ± s.e.m. ANOVA followed by Tukey–Kramer test (two-tailed). p110* versus H1047R mutation, ***P < 0.001. p110* versus E545K mutation, ###P < 0.001. p110* versus wild-type p110, +++P < 0.001. p110* versus p110* KD, $$$P < 0.001. Negative control is a vector that contains cDNA encoding GFP. b, Histological examination of different tissues from PIK3CACAGG-CreER mice. Left column, from top to bottom, liver, abdominal tumour, leg and ear abnormalities. Middle column, PAS or HE staining of the tissue. Right column, Ki67 staining of the same tissue (n = 8 mice). Scale bars, 20 μm. c, Design of the experiment shown in Fig. 2h. PIK3CACAGG-CreER mice received a single dose of 4 mg kg−1 tamoxifen and were followed for one month. Once the tumours became visible, BYL719 was started for two weeks and then withdrawn.
Extended Data Fig. 8 CT scan evaluation of the tumours and adipose tissue before and after BYL719 introduction.
a, Body weight evolution of PIK3CAWT and PIK3CACAGG-CreER mice treated with vehicle or BYL719 (n = 3 mice per group). b, CT scan evaluation and quantification of the fat tissue content in PIK3CAWT and PIK3CACAGG-CreER mice treated with vehicle or BYL719. Subcutaneous and visceral fat content were measured before treatment and 7 and 14 days after onset of treatment with vehicle or BYL719 (n = 3 mice per group). c, CT scan evaluation and quantification of tumour volume in PIK3CACAGG-CreER mice before and after two weeks of BYL719 treatment (arrows) (n = 3 mice per group). All data are shown as mean ± s.e.m.
a, Kaplan–Meier survival curves of PIK3CACAGG-CreER mice that received a single dose of 40 mg kg−1 tamoxifen and were treated with or without rapamycin after tamoxifen administration. b, Representative pictures of the liver of PIK3CACAGG-CreER mice treated with rapamycin 40 days after Cre induction. c, Morphology of livers and spleens from PIK3CAWT and PIK3CACAGG-CreER mice that were treated with or without rapamycin after Cre induction. Scale bars, 10 μm. d, Western blot and quantification of P-AKT (Ser473) and P-S6RP in the liver, heart and muscle, respectively, of PIK3CAWT and PIK3CACAGG-CreER mice treated with vehicle or rapamycin directly after Cre induction. e, PIK3CACAGG-CreER mice were treated with rapamycin one month after Cre induction with a single dose of 4 mg kg−1 tamoxifen and followed for one month. All data are shown as mean ± s.e.m. ANOVA followed by Tukey–Kramer test (two-tailed). PIK3CACAGG-CreER versus PIK3CAWT mice, ***P < 0.001. PIK3CACAGG-CreER mice treated with rapamycin compared with PIK3CACAGG-CreER mice treated with vehicle, ###P < 0.001.
Extended Data Fig. 10 In vitro effect of BYL719 and rapamycin on fibroblasts from PIK3CACAGG-CreER mice.
a, Skin fibroblasts from PIK3CAWT and PIK3CACAGG-CreER mice were isolated and exposed to vehicle or increasing concentrations of BYL719 or rapamycin for 24 h. b, Quantification. White column, without 4-OHT; black column, with 4-OHT. All data are shown as mean ± s.e.m. ANOVA followed by Tukey–Kramer test (two-tailed). Before versus after Cre induction with 4-OHT, ***P < 0.001. BYL719 or rapamycin exposure compared with cells treated with vehicle, ##P < 0.01 and ###P < 0.001.
a, Patients 10–17 before and after 180 days of BYL719 treatment. Patient 10 was a 14-year-old boy with severe asthenia, dyspnea and bilateral overgrowth of lower limbs. After 180 days of treatment asthenia resolved and we observed a marked reduction in hypertrophy of the limbs. Patient 11 was a 14-year-old boy with overgrowth of the right buttock and an intra-abdominal vascular tumour infiltrating the left kidney and spinal nerve. He had chronic haematuria and was permanently confined to bed owing to pain. After 180 days, haematuria resolved and the volume of the intraabdominal vascular malformation was reduced by up to 68%. He had no more pain and became capable of walking. Patient 12 was a 15-year-old boy with multiple large tumours of the trunk and the back. After 180 days of treatment the tumours had reduced in size. Patient 13 was a 16-year-old boy with megalencephaly-capillary malformation (MCAP) and left hemifacial hyperplasia. Treatment led to a reduction in hemifacial hypertrophy and cognitive improvement. Owing to the deformation, this patient was not able to open the left eye. After 180 days of BYL719 treatment, he was able to open the eye (not shown for confidentiality reasons). Patient 14 was a 16-year-old girl with MCAP and a chronic noninfectious palpebral cellulitis who was steroid-dependent. BYL719 treatment led to the healing of the cellulitis and steroids were stopped without a flare. We also observed enhancement of cognitive function and behaviour and improvement of scoliosis. Patient 15 was a 19-year-old man with overgrowth of the left foot and unstable and painful walking. Treatment led to an improvement in the overgrowth as well as an improvement in walking distance. Patient 16 was a 32-year-old man with overgrowth of the right foot and unstable and painful walking. Treatment led to an improvement in the overgrowth as well as an improvement in walking distance. Patient 17 was 50-year-old woman with generalized hypertrophy, and severe and diffuse pain with opioid dependency. She was permanently confined to bed. After six months of treatment we observed an improvement in tiredness, and resolution of pain, and we were able to stop opioids within two weeks. The patient became able to walk again. b, PIK3CA mutations identified in the 17 patients. c, For each patient we determined a target lesion (see Supplementary Table 2) that was clinically measured at each time point. The graph represents the changes (per cent) during the 180 days of treatment with BYL719. Each line is a single patient. d, Mean body weight changes (per cent) during the 180 days of treatment with BYL719 (n = 13 patients, patients 1–13), excluding the four obese patients (patients 14, 15, 16 and 17). e, Mean body weight loss in the four obese patients during the 180 days of treatment with BYL719. All data are shown as mean ± s.e.m.
Extended Data Fig. 12 Height changes in children during treatment period and radiological changes with BYL719 treatment.
a, Height changes in children during the 180 days of treatment with BYL719. b, MRI scans of patient 1 before and after 180 days of BYL719 treatment. Arrows show the target lesion. c, Three-dimensional MRI-based reconstruction of the chest tumour in patient 1 before and after 180 days of BYL719 treatment. d, Examples of MRI showing the evolution of the target lesions in patients 9 and 11. e, Volume evolution of the radiological target lesion after 180 days of BYL719 treatment. f, Diffusion MRI demonstrating the enhancement of brain perfusion in patient 14 after 180 days of BYL719. g, PET scan images of patients 6, 9, 15 and 17, before and after 90 days of BYL719 treatment. The arrows delineate hypermetabolic activity before and after 90 days of treatment.
Supplementary Table 1 contains the principal demographic characteristics of the patients, the clinical manifestation and the PIK3CA mutation, and Supplementary Table 2 shows the responses to treatment.
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Venot, Q., Blanc, T., Rabia, S.H. et al. Targeted therapy in patients with PIK3CA-related overgrowth syndrome. Nature 558, 540–546 (2018). https://doi.org/10.1038/s41586-018-0217-9
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