Targeted therapy in patients with PIK3CA-related overgrowth syndrome

Abstract

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.

Extended Data Fig. 1. p110* construction and mouse model characterization.

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 linker (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*). c, d, EGFP expression from flow cytometry experiments in the spleen of PIK3CA^WT mice (n = 12) and PIK3CA^CAGG-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 PIK3CA^CAGG-CreER mice before BYL719 treatment. This phenotype was reversed by BYL719 administration. Middle, arrows show scoliosis in PIK3CA^CAGG-CreER mice before BYL719 treatment, which was rescued by BYL719 administration. Bottom, arrows show arterial dilation in PIK3CA^CAGG-CreER mice before BYL719 treatment, which was reversed by BYL719 administration (n = 6 mice per group).

Extended Data Fig. 2. Quantification and vessel malformation.

a, Percentage of PIK3CA^WT and PIK3CA^CAGG-CreER mice with or without BYL719 treatment presenting organ abnormalities. b, Oil Red O staining of the livers of PIK3CA^WT and PIK3CA^CAGG-CreER mice demonstrating steatosis (n = 8 mice per group). Scale bars, 10 μm. c, CD31 (top) and CD34 (bottom) immunostaining in the liver of PIK3CA^WT and PIK3CA^CAGG-CreER mice with or without BYL719 (n = 8 mice per group). PIK3CA^CAGG-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 PIK3CA^WT and PIK3CA^CAGG-CreER mice (n = 8 mice per group). Scale bars, 10 μm. e, Representative western blot of LYVE-1 in the liver of PIK3CA^WT and PIK3CA^CAGG-CreER mice demonstrating lymphatic increased in the PIK3CA^CAGG-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). PIK3CA^CAGG-CreER versus PIK3CA^WT mice, ***P < 0.001.

Extended Data Fig. 3. BYL719 affects proliferation.

a, Ki67 immunostaining and quantification in liver, spleen and heart of PIK3CA^WT and PIK3CA^CAGG-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). PIK3CA^CAGG-CreER versus PIK3CA^WT mice, ***P < 0.001. PIK3CA^CAGG-CreER mice treated with vehicle versus PIK3CA^CAGG-CreER mice treated with preventive BYL719, ###P < 0.001. PIK3CA^CAGG-CreER mice treated with vehicle versus PIK3CA^CAGG-CreER mice treated with therapeutic BYL719, ⁺⁺⁺P < 0.001.

Extended Data Fig. 4. Senescence and BYL719.

a, β-galactosidase staining in the liver, heart, spleen and kidney of PIK3CA^WT and PIK3CA^CAGG-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 PIK3CA^WT and PIK3CA^CAGG-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).

Extended Data Fig. 5. p110* expression in affected tissues.

a, Western blot showing the expression of p110* in PIK3CA^CAGG-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 PIK3CA^WT and PIK3CA^CAGG-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).PIK3CA^CAGG-CreER versus PIK3CA^WT mice, ***P < 0.001. PIK3CA^CAGG-CreER mice treated with vehicle versus PIK3CA^CAGG-CreER mice treated with preventive BYL719, ^### P < 0.001. PIK3CA^CAGG-CreER mice treated with vehicle versusd PIK3CA^CAGG-CreER mice treated with therapeutic BYL719, ⁺⁺⁺ P < 0.001.

Extended Data Fig. 6. Ability of BYL719 to inhibit PIK3CA activation in different tissues.

Immunofluorescence staining of P-AKT (Ser⁴⁷³) and P-S6RP in the liver (a), heart (b), spleen (c) and muscles (d) of PIK3CA^WT and PIK3CA^CAGG-CreER mice treated with or without BYL719 (n = 8 mice per group). Scale bars, 10 μm.

Extended Data Fig. 7. Recruitment of the AKT/mTORC pathway by the different forms of mutant p110.

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 PIK3CA^CAGG-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. PIK3CA^CAGG-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 PIK3CA^WT and PIK3CA^CAGG-CreER mice treated with vehicle or BYL719 (n = 3 mice per group). b, CT scan evaluation and quantification of the fat tissue content in PIK3CA^WT and PIK3CA^CAGG-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 PIK3CA^CAGG-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.

Extended Data Fig. 9. Effect of rapamycin treatment on the different PIK3CA^CAGG-CreER mouse models.

a, Kaplan–Meier survival curves of PIK3CA^CAGG-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 PIK3CA^CAGG-CreER mice treated with rapamycin 40 days after Cre induction. c, Morphology of livers and spleens from PIK3CA^WT and PIK3CA^CAGG-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 PIK3CA^WT and PIK3CA^CAGG-CreER mice treated with vehicle or rapamycin directly after Cre induction.e, PIK3CA^CAGG-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). PIK3CA^CAGG-CreER versus PIK3CA^WT mice, ***P < 0.001. PIK3CA^CAGG-CreER mice treated with rapamycin compared with PIK3CA^CAGG-CreER mice treated with vehicle, ###P < 0.001.

Extended Data Fig. 10. In vitro effect of BYL719 and rapamycin on fibroblasts from PIK3CA^CAGG-CreER mice.

a, Skin fibroblasts from PIK3CA^WT and PIK3CA^CAGG-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.

Extended Data Fig. 11. Effect of BYL719 in patients with PROS.

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.

Fig. 1. Characterization of the PROS mouse model and efficacy of BYL719.

a, Kaplan-Meier survival curves of PIK3CA^WT and PIK3CA^CAGG-CreER mice (n = 16 mice per group) after tamoxifen administration (log-rank test, P < 0.0001). b, Representative necropsy examination pictures of PIK3CA^CAGG-CreER mice. The mice displayed sudden intra-abdominal and spontaneous hepatic haemorrhage (n = 16 mice). c, Morphology of livers (top), spleens (middle) and kidneys (bottom two rows) from PIK3CA^WT and PIK3CA^CAGG-CreER mice that were treated with or without BYL719 directly after Cre induction (preventive) or seven days later (therapeutic) (n = 8 mice per group). d, Western blot of P-AKT (Ser⁴⁷³), P-AKT (Thr³⁰⁸) and P-S6RP in liver, heart and muscles, respectively, from PIK3CA^WT and PIK3CA^CAGG-CreER mice treated with or without BYL719 directly after Cre induction (preventive) or seven days later (therapeutic) (n = 8 mice per group). e, Experimental design. f, Kaplan–Meier survival curves of PIK3CA^CAGG-CreER mice treated with or without BYL719 after tamoxifen administration (n = 16 mice per group). After 40 days, BYL719 treatment was withdrawn (log-rank test, P < 0.0001). g, Kaplan–Meier survival curves of PIK3CA^CAGG-CreER mice treated with or without BYL719 (n = 12 mice per group) seven days after tamoxifen administration (log-rank test, P < 0.0001). Scale bars, 20 μm.

Fig. 2. Characterization of the second mouse model of PROS and efficacy of BYL719.

a, Morphology of PIK3CA^CAGG-CreER mice one month after Cre induction with a single dose of 4 mg kg⁻¹ tamoxifen. The mice displayed multiple visible deforming tumours across the whole body (red arrows). Some of these tumours had huge vascular irregularities. The mice progressively developed hypertrophic extremities, enlarged liver with aberrant vessels, and multiple intraabdominal vascular malformations (pictures representative of nine mice). b–d, Histopathological examination of the tumours revealed the presence of lipomatous tumours (b; n = 10 mice examined) and multiple venous malformations composed of ecstatic venous channels with a thin endothelial cell lining, surrounded by sparse, erratically distributed vascular smooth muscle cells and a disorganized extracellular matrix (c, d; n = 10 mice examined). Scale bars, 50 μm. e, Coimmunostaining of CD34 and α-smooth muscle cells in venous malformation (n = 10 mice examined). f, Immunofluorescence staining for P-AKT (Ser⁴⁷³) and P-S6RP in the liver, subcutaneous tumour, ear tumour and kidneys of PIK3CA^WT and PIK3CA^CAGG-CreER mice (n = 10 mice examined per group). Scale bars, 10 μm. g, PIK3CA^CAGG-CreER mice were injected with a single dose of 4 mg kg⁻¹ tamoxifen and followed for one month after Cre induction. Once the tumours reached a certain volume, the mice were treated with BYL719 for two weeks and this led to the disappearance of all tumours (n = 18 mice). h, PIK3CA^CAGG-CreER mice were injected with a single dose of 4 mg kg⁻¹ tamoxifen and followed for one month after Cre induction. Once the tumours reached a certain volume, the mice were treated with BYL719 for two weeks and this led to the disappearance of all tumours (n = 7 mice). Then, BYL719 treatment was withdrawal and we observed the recurrence of tumours and vascular malformations in PIK3CA^CAGG-CreER mice within the next four weeks.

Fig. 3. Efficacy of BYL719 treatment in an adult patient with severe CLOVES syndrome.

a, Representative pictures of patient 1 before and 360 and 540 days after onset of BYL719 treatment. b, CT scans of patient 1 before and 360 and 540 days after onset of BYL719 treatment. The magnifications are the same. c, Clinical parameters (weight, chest girth and waist circumference) of patient 1 before and after onset of BYL719 treatment. d, Serum BNP and creatinine levels before and after onset of BYL719 treatment. e, Three-dimensional MRI-based reconstructions of the intra-abdominal vessels before and after onset of BYL719 treatment. The total volume of the venous malformation was reduced by up to 72% after 540 days of treatment.

Fig. 4. Efficacy of BYL719 treatment in a child patient with severe CLOVES syndrome.

a, Representative pictures of patient 2 before and 180 and 360 days after onset of BYL719 treatment. b, Clinical parameters (thigh and waist circumferences) of patient 2 before and after onset of BYL719 treatment. c, Three-dimensional CT scan reconstruction of the spine before and 180 and 360 days after onset of BYL719 treatment.d, PET scan images of patient 2 before and 120, 180 and 360 days after onset of BYL719 treatment. Arrows indicate the hypermetabolic tissues before and during treatment. e, Three-dimensional MRI-based reconstruction of the intra-abdominal tumour before and 120, 180 and 360 days after onset of BYL719 treatment. The tumour volume was reduced by up to 71% after 360 days of treatment. Scale bar, 1 cm.

Fig. 5. Efficacy of BYL719 treatment in patients with PROS.

Images of patients 1–9 before and after six months of BYL719 treatment. Patient 1 is a four-year-old girl suffering from severe vascular malformations involving the right arm and chest with permanent pain. After six months of treatment we saw a marked improvement in all vascular malformations as well as the scoliosis. Patient 2 is a 4-year-old girl with scoliosis and hypertrophic left buttock who had already undergone left foot partial amputation. After six months of treatment we saw an improvement in the scoliosis and reduction of the hypertrophic lesion. Patient 3 is a 5-year-old girl with CLOVES syndrome and chronic gastrointestinal bleeding. After six months of treatment, chronic bleeding stopped. Patient 4 is a 5-year-old girl with left hemifacial hyperplasia that was progressing despite multiple surgeries. After six months of treatment we saw for the first time an improvement in the hypertrophy (not shown for reasons of confidentiality). Patient 5 is a 6-year-old boy with CLOVES syndrome. The treatment led to a reduction in lipomatous tumours and scoliosis. Patient 6 is a 10-year-old girl with CLOVES syndrome and a severe lipomatous tumour on her back. BYL719 led to a marked improvement in the scoliosis and shrinkage of the tumour. Patient 7 is an 11-year-old boy with CLOVES syndrome, chronic gastrointestinal bleeding and severe dyspnea. Treatment improved all symptoms and the bleeding stopped. Patient 8 is an 11-year-old girl with CLOVES syndrome and severe dyspnea. Treatment improved the subcutaneous lipoma as well as dyspnea. Patient 9 is a 13-year-old girl with CLOVES syndrome, splenomegaly and severe vascular malformations involving the left kidney and limbs. Vascular malformations were markedly improved and the size of the spleen reduced after treatment.