Mosaic overgrowth with fibroadipose hyperplasia is caused by somatic activating mutations in PIK3CA

Abstract

The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2 or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells from an individual with an unclassified syndrome of congenital progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated mutation encoding p.His1047Leu in PIK3CA, the gene that encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in ten additional individuals with overlapping syndromes identified either the p.His1047Leu alteration or a second cancer-associated alteration, p.His1047Arg, in nine cases. Affected dermal fibroblasts showed enhanced basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) generation and concomitant activation of downstream signaling relative to their unaffected counterparts. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.

Figure 1. Spectrum of Overgrowth in Patients with Activating PIK3CA Mutations

Patient C1 showing (A)&(B) Lower extremity overgrowth with a paucity of facial adipose tissue at ages 12 months and 7 years progressing to (C) Massive leg overgrowth with lack of upper body adipose tissue at 37 years old, by which time left above knee amputation had been undertaken. (D) Right foot overgrowth at 35 years old (E) Feet at 35 years showing overgrowth, cutaneous syndactyly of the left foot and rotational deformity of the right foot .(F) and (G). Patient N7 showing his leg status post debulking surgery and massive overgrowth of the left foot, which was partially amputated. (H) and (I) Patient N99 showing more limited overgrowth confined to several rays of the feet. Informed consent to publish photographs was obtained from the identifiable patient in this figure.

Figure 2. Segmental Skeletal and Fibroadipose Overgrowth in Patients with Activating PIK3CA Mutations

Patient C1 (A) Reformatted coronal CT of the legs, showing bony overgrowth, destructive arthropathy, adipose expansion and relative lack of muscle (B) Sagittal T2-weighted MRI images of lumbar spine depicting enlarged neural structures within the foramina (circled 12-13 mm diameter). Normal nerve roots in this region are up to 5-6 mm and up to 8-9 mm for the dorsal root ganglion. (C) & (D) transverse CT image and photograph of anterior abdomen showing right-sided adipose overgrowth (arrow) and left-sided lack of adipose tissue (arrowhead) to the level of insertion of erector spinae. (E) Hematoxylin- and eosin-stained left foot skin showing marked dermal thickening (scale bar 15 μm). Inset - myofibroblasts with plump nuclei are prominent in the dermis (arrow) in contrast to normal skin(F). (G) Muscle is replaced by fibrous and adipose tissue with occasional residual muscle fibers (arrow) (scale bar 80 μm). (H) Normal muscle. Imaging of patient N45 showing (I)&(K) fibroadipose overgrowth of the legs including subcutaneous and muscular tissue,(J) fibroadipose overgrowth of the pelvis, and (L) &(M) plain radiographs showing distorting overgrowth of the knees . Plain radiograph of the hand of patient N68 showing distorting overgrowth of the second and third ray of the right hand (N). Plain radiograph (O) and photograph (P) of the left foot of patient N110 show overgrowth limited to the first and second rays of the left foot.

Figure 3. Identification of PIK3CA mutations in affected cells and tissues

A. PIK3CA c.3140A>T (p.His1047Leu) identified in cultured dermal fibroblasts from the affected left leg but not the unaffected right arm of patient C1 (left) and present at varying levels in left leg tissues (right) B. Location of His1047 near the carboxy-terminus of the kinase domain of the p110α catalytic subunit of type 1A PI3K. ABD = adaptor binding domain; RBD = ras binding domain C. PIK3CA c.3140A>G (p.His1047Arg) identified in cells derived from a variety of tissues of patients N7, N99, and N45.

Figure 4. Hyperactivity of phosphatidylinositol-3-kinase in cells harboring PIK3CA mutations

A. PIP3 levels determined by mass spectroscopy are elevated in fibroblasts harboring an activating PIK3CA mutation both in the basal state and after stimulation with EGF compared to unaffected cells. Cells from a healthy control (Cntrl), Patient C1 (unaffected arm and affected leg) and from patients N7 and N99 (both affected) are shown. * = Significantly higher than unstimulated unaffected cells (p<0.01); ∞ = significantly higher than EGF-stimulated unaffected cells (p<0.01); # = not significantly different; error bars are + 2 SEM. (B) & (C). Representative immunoblot showing abnormal basal phosphorylation of AKT and p70 S6 kinase (p70S6K) in affected dermal fibroblasts (B) but no increase in basal ERK phosphorylation (C). (D). Infrared detection of phosphorylated and total AKT in patients with PIK3CA or AKT1 mutations. Fibroblasts were serum starved for 24 hours before solubilization. Patients N7 and N99 were positive for the p.His1047Arg mutation in PIK3CA, PS53 and PS75 were positive for the p.Glu17Lys mutation in AKT1 (ref. 3), Cntrl1 and Cntrl2 were from unaffected individuals and negative for both mutations. Antibodies used in these experiments are described in Supplementary Table 3).