Alpelisib for the treatment of PIK3CA-related head and neck lymphatic malformations and overgrowth.

PURPOSE: PIK3CA-related overgrowth spectrum (PROS) conditions of the head and neck are treatment challenges. Traditionally, these conditions require multiple invasive interventions, with incomplete malformation removal, disfigurement, and possible dysfunction. Use of the PI3K inhibitor alpelisib, previously shown to be effective in PROS, has not been reported in PIK3CA-associated head and neck lymphatic malformations (HNLMs) or facial infiltrating lipomatosis (FIL). We describe prospective treatment of 5 children with PIK3CA-associated HNLMs or head and neck FIL with alpelisib monotherapy. METHODS: A total of 5 children with PIK3CA-associated HNLMs (n = 4) or FIL (n = 1) received alpelisib monotherapy (aged 2-12 years). Treatment response was determined by parental report, clinical evaluation, diary/questionnaire, and standardized clinical photography, measuring facial volume through 3-dimensional photos and magnetic resonance imaging. RESULTS: All participants had reduction in the size of lesion, and all had improvement or resolution of malformation inflammation/pain/bleeding. Common invasive therapy was avoided (ie, tracheotomy). After 6 or more months of alpelisib therapy, facial volume was reduced (range 1%-20%) and magnetic resonance imaging anomaly volume (range 0%-23%) were reduced, and there was improvement in swallowing, upper airway patency, and speech clarity. CONCLUSION: Individuals with head and neck PROS treated with alpelisib had decreased malformation size and locoregional overgrowth, improved function and symptoms, and fewer invasive procedures.

Cell-free DNA as a diagnostic analyte for molecular diagnosis of vascular malformations.

PURPOSE: Vascular malformations (VM) are primarily caused by somatic activating pathogenic variants in oncogenes. Targeted pharmacotherapies are emerging but require molecular diagnosis. Since variants are currently only detected in malformation tissue, patients may be ineligible for clinical trials prior to surgery. We hypothesized that cell-free DNA (cfDNA) could provide molecular diagnoses for patients with isolated VM. METHODS: cfDNA was isolated from plasma or cyst fluid from patients with arteriovenous malformations (AVM), venous malformations (VeM), or lymphatic malformations (LM), and assayed for known pathogenic variants using droplet digital polymerase chain reaction (ddPCR). Cyst fluid cfDNA from an independent cohort of LM patients was prospectively screened for variants using a multiplex ddPCR assay. RESULTS: Variants were detected in plasma cfDNA in patients with AVM (2/8) and VeM (1/3). Variants were detected in cyst fluid cfDNA (7/7) but not plasma (0/26) in LM patients. Prospective testing of cyst fluid cfDNA with multiplex ddPCR identified variants in LM patients who had never undergone surgery (4/5). CONCLUSION: Variants were detected in plasma from AVM and VeM patients, and in cyst fluid from patients with LM. These data support investigation of cfDNA-based molecular diagnostics for VM patients, which may provide opportunities to initiate targeted pharmacotherapies without prior surgery.

Primary targeted medical therapy for management of bilateral head and neck lymphatic malformations in infants.

OBJECTIVES: Patients born with bilateral head and neck lymphatic malformations (BHNLMs) often require multiple invasive treatments, including tracheostomy. We hypothesized that primary targeted medical therapy (pTMT) with diagnostic needle aspiration reduces the need for invasive therapy such as surgical resection and/or sclerotherapy. METHODS: Retrospective case review was performed of infants with BHNLMs (Grade 2 or De Serres stage IV and V) treated only at our institution from 2000 to 2021. Patients were divided into two cohorts: those managed with pTMT and those managed with observation, sclerotherapy, or surgical intervention (non-pTMT). Data regarding interventions, clinical outcomes, morbidity, and mortality were analyzed with descriptive statistics. RESULTS: Nine children with BHNLMs met inclusion criteria. Three (33%) were in the pTMT cohort and six (66%) were non-pTMT. Eight (89%) malformations were genotyped, and all demonstrated hotspot PIK3CA variants. All pTMT patients had sirolimus initiated in the first month of life and underwent needle aspiration of malformation cyst fluid for cell-free DNA samples. All pTMT patients tolerated medical therapy. For the non-pTMT cohort, primary treatment included none (deceased, n = 1, 17%), observation with needle aspiration (n = 1, 17%), surgical resection (n = 2, 33%), or combination surgery and sclerotherapy (n = 2, 33%). Intubation duration, intensive care and initial hospital length of stay were not different between cohorts. Four non-pTMT patients (67%) required tracheostomy, and two (33%) died prior to discharge. All pTMT patients survived and none required tracheostomy. Non-pTMT patients required a median of two invasive therapies prior to discharge (IQR 1-4) and a mean total of 13 over the course of their lifetime (IQR 1-16), compared to the pTMT group who did not require any lifetime invasive therapy, even after initial pTMT and discharge home. CONCLUSION: This study compares patients with BHNLMs (Grade 2) treated with pTMT versus those treated with observation or invasive therapy. Patients treated with pTMT required no surgical or invasive procedural treatment of their malformations, no tracheostomy placement, no unplanned readmissions after discharge, and had no mortalities. Needle aspiration was useful as a therapeutic adjunct for cell-free DNA diagnosis of PIK3CA variants, which guided TMT.

Somatic activating BRAF variants cause isolated lymphatic malformations.

Somatic activating variants in PIK3CA, the gene that encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), have been previously detected in ∼80% of lymphatic malformations (LMs).1 , 2 We report the presence of somatic activating variants in BRAF in individuals with LMs that do not possess pathogenic PIK3CA variants. The BRAF substitution p.Val600Glu (c.1799T>A), one of the most common driver mutations in cancer, was detected in multiple individuals with LMs. Histology revealed abnormal lymphatic channels with immunopositivity for BRAFV600E in endothelial cells that was otherwise indistinguishable from PIK3CA-positive LM. The finding that BRAF variants contribute to low-flow LMs increases the complexity of prior models associating low-flow vascular malformations (LM and venous malformations) with mutations in the PI3K-AKT-MTOR and high-flow vascular malformations (arteriovenous malformations) with mutations in the RAS-mitogen-activated protein kinase (MAPK) pathway.3 In addition, this work highlights the importance of genetic diagnosis prior to initiating medical therapy as more studies examine therapeutics for individuals with vascular malformations.

Cerebrofacial vascular metameric syndrome is caused by somatic pathogenic variants in PIK3CA.

Disorganized morphogenesis of arteries, veins, capillaries, and lymphatic vessels results in vascular malformations. Most individuals with isolated vascular malformations have postzygotic (mosaic), activating pathogenic variants in a handful of oncogenes within the PI3K-RAS-MAPK pathway (Padia et al., Laryngoscope Investig Otolaryngol 4: 170-173 [2019]). Activating pathogenic variants in the gene PIK3CA, which encodes for the catalytic subunit of phosphatidylinositol 3-kinase, are present in both lymphatic and venous malformations as well as arteriovenous malformations in other complex disorders such as CLOVES syndrome (congenital, lipomatous, overgrowth, vascular malformations, epidermal anevi, scoliosis) (Luks et al., Pediatr Dev Pathol 16: 51 [2013]; Luks et al., J Pediatr 166: 1048-1054.e1-5 [2015]; Al-Olabi et al., J Clin Invest 128: 1496-1508 [2018]). These vascular malformations are part of the PIK3CA-related overgrowth spectrum, a spectrum of entities that have regionalized disordered growth due to the presence of tissue-restricted postzygotic PIK3CA pathogenic variants (Keppler-Noreuil et al., Am J Med Genet A 167A: 287-295 [2015]). Cerebrofacial vascular metameric syndrome (CVMS; also described as cerebrofacial arteriovenous metameric syndrome, Bonnet-Dechaume-Blanc syndrome, and Wyburn-Mason syndrome) is the association of retinal, facial, and cerebral vascular malformations (Bhattacharya et al., Interv Neuroradiol 7: 5-17 [2001]; Krings et al., Neuroimaging Clin N Am 17: 245-258 [2007]). The segmental distribution, the presence of tissue overgrowth, and the absence of familial recurrence are all consistent with CVMS being caused by a postzygotic mutation, which has been hypothesized by previous authors (Brinjiki et al., Am J Neuroradiol 39: 2103-2107 [2018]). However, the genetic cause of CVMS has not yet been described. Here, we present three individuals with CVMS and mosaic activating pathogenic variants within the gene PIK3CA We propose that CVMS be recognized as part of the PIK3CA-related overgrowth spectrum, providing justification for future trials using pharmacologic PIK3CA inhibitors (e.g., alpelisib) for these difficult-to-treat patients.

Acetylsalicylic acid suppression of the PI3K pathway as a novel medical therapy for head and neck lymphatic malformations.

OBJECTIVES: Head and neck lymphatic malformations (HNLM) are caused by gain-of-function somatic mutations in PIK3CA. Acetylsalicylic acid (ASA/aspirin) is thought to limit growth in PIK3CA-mutated neoplasms through PI3K pathway suppression. We sought to determine if ASA could be beneficial for HNLM. METHODS: Retrospective case series of patients (0-18 years) offered ASA (3-5 mg/kg/day) for HNLM treatment (2010-2018). Clinical and treatment characteristics, patient-reported symptom improvement, medication tolerance, compliance, and complications were recorded. Treatment response was determined by change in patient/caregiver-reported symptoms, or HNLM size [complete (resolved), partial (decreased), or stable]. RESULTS: Fifty-three patients were offered ASA, 23 (43%) accepted (median age 10 years, IQR 6-14). Compared to patients who declined, patients receiving ASA were more likely to have extensive malformations: ex-utero intrapartum treatment procedure, bilateral malformations, oral cavity location, ≥2 invasive treatments, or tracheotomy (p < 0.05). All patients with tissue available had PIK3CA mutations (13/23). Treatment indications included oral pain/blebs (12, 52%), recurrent pain/swelling (6, 26%), or sudden/persistent swelling (5, 22%). Treatment plan was commonly one 81 mg tablet daily (19, 83%) for 3-12 months (8, 42%). Therapeutic adherence was reported by 18 patients (78%). Symptoms improved in 18 patients [78%; decreased pain (9, 39%) and swelling (8, 35%)]. Treatment resulted in partial (14, 61%) or complete response (4, 17%). Three patients developed oral bleb bleeding, which resolved with medication discontinuation. CONCLUSION: ASA seems to be a well-tolerated, low-risk medication for HNLM treatment. This pilot study suggests that it often improves symptoms and reduces HNLM size. Further prospective, randomized studies are warranted to comprehensively assess indications, safety, and efficacy. LEVEL OF EVIDENCE: Level 4.

Clinical application of molecular genetics in lymphatic malformations.

OBJECTIVES: To describe the clinical presentation of lymphatic malformations (LM) and genotypically associated disorders and to summarize the recent literature regarding the genetic etiology of LM and provide a biologic correlation to medical and surgical management. RESULTS: LM are congenital lesions derived from a developmental abnormality of the lymphatic vessels. The severity of disease varies widely and complications can occur with higher staged disease and those associated with a known constellation of symptoms. Somatic mutations of the PIK3CA gene have been found to be an etiologic factor in the development of LM and associated overgrowth syndromes. Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor that inhibits the pathway downstream of PIK3CA. Preliminary studies in select groups of patients suggest that sirolimus has a role in the medical management of certain aspects of this disease. CONCLUSIONS: Discovery of LM molecular genetics has led to the possibility of targeted therapies and highlights the importance of precision medicine in rare diseases. Identifying genetic mutations in larger cohorts of patients with LM will lead to additional insights. Knowledge of the genetic basis for disease can then lead to discovery of directed medical therapy. A specific molecular diagnosis can also help families understand better why their child is different and provide accurate counseling for subsequent pregnancies. LEVEL OF EVIDENCE: 6.

Genotype correlates with clinical severity in PIK3CA-associated lymphatic malformations.

Lymphatic malformations (LMs) are congenital, nonneoplastic vascular malformations associated with postzygotic activating PIK3CA mutations. The mutation spectrum within LMs is narrow, with the majority having 1 of 3 hotspot mutations. Despite this relative genetic homogeneity, clinical presentations differ dramatically. We used molecular inversion probes and droplet digital polymerase chain reaction to perform deep, targeted sequencing of PIK3CA in 271 affected and unaffected tissue samples from 81 individuals with isolated LMs and retrospectively collected clinical data. Pathogenic PIK3CA mutations were identified in affected LM tissue in 64 individuals (79%) with isolated LMs, with variant allele fractions (VAFs) ranging from 0.1% to 13%. Initial analyses revealed no correlation between VAF and phenotype variables. Recognizing that different mutations activate PI3K to varying degrees, we developed a metric, the genotype-adjusted VAF (GVAF), to account for differences in mutation strength, and found significantly higher GVAFs in LMs with more severe clinical characteristics including orofacial location or microcystic structure. In addition to providing insight into LM pathogenesis, we believe GVAF may have broad applicability for genotype-phenotype analyses in mosaic disorders.

Involvement of epigenetics and EMT-related miRNA in arsenic-induced neoplastic transformation and their potential clinical use.

Exposure to toxicants leads to cumulative molecular changes that overtime increase a subject's risk of developing urothelial carcinoma. To assess the impact of arsenic exposure at a time progressive manner, we developed and characterized a cell culture model and tested a panel of miRNAs in urine samples from arsenic-exposed subjects, urothelial carcinoma patients, and controls. To prepare an in vitro model, we chronically exposed an immortalized normal human bladder cell line (HUC1) to arsenic. Growth of the HUC1 cells was increased in a time-dependent manner after arsenic treatment and cellular morphology was changed. In a soft agar assay, colonies were observed only in arsenic-treated cells, and the number of colonies gradually increased with longer periods of treatment. Similarly, invaded cells in an invasion assay were observed only in arsenic-treated cells. Withdrawal of arsenic treatment for 2.5 months did not reverse the tumorigenic properties of arsenic-treated cells. Western blot analysis demonstrated decreased PTEN and increased AKT and mTOR in arsenic-treated HUC1 cells. Levels of miR-200a, miR-200b, and miR-200c were downregulated in arsenic-exposed HUC1 cells by quantitative RT-PCR. Furthermore, in human urine, miR-200c and miR-205 were inversely associated with arsenic exposure (P = 0.005 and 0.009, respectively). Expression of miR-205 discriminated cancer cases from controls with high sensitivity and specificity (AUC = 0.845). Our study suggests that exposure to arsenic rapidly induces a multifaceted dedifferentiation program and miR-205 has potential to be used as a marker of arsenic exposure as well as a maker of early urothelial carcinoma detection.