Pathogenic variants in PIK3CA are associated with clinical phenotypes of kaposiform lymphangiomatosis, generalized lymphatic anomaly, and central conducting lymphatic anomaly

Abstract

Complex lymphatic anomalies are debilitating conditions characterized by aberrant development of the lymphatic vasculature (lymphangiogenesis). Diagnosis is typically made by history, examination, radiology, and histologic findings. However, there is significant overlap between conditions, making accurate diagnosis difficult. Recently, genetic analysis has been offered as an additional diagnostic modality. Here, we describe four cases of complex lymphatic anomalies, all with PIK3CA variants but with varying clinical phenotypes. Identification of PIK3CA resulted in transition to a targeted inhibitor, alpelisib. These cases highlight the genetic overlap between phenotypically diverse lymphatic anomalies.

1 │. INTRODUCTION

Complex lymphatic anomalies, including generalized lymphatic anomaly (GLA), kaposiform lymphangiomatosis (KLA), and central conducting lymphatic anomaly (CCLA), are debilitating conditions characterized by aberrant lymphatic vessel development, resulting in diffuse lymphatic malformations, lymph leakage, and/or coagulopathy. Currently, diagnosis of specific anomalies relies on history, physical exam, characteristic imaging, and histology. However, recent studies have identified genetic variants associated with specific anomalies, providing hope for more accurate classification and treatment of these disorders.

KLA is a progressive condition consisting of multifocal infiltrative lymphatic malformations involving bones, thorax, and viscera, often with coagulopathy and effusions. Affected tissues contain focal areas of spindle-shaped D2–40⁺ PROX-1⁺ cells (markers of lymphatic endothelium); however, these foci are often sparse, making histologic diagnosis difficult.^1,2 Recently, several studies have discovered somatic NRAS, HRAS, and CBL pathogenic variants in KLA lesions.^3–6 Like KLA, GLA is marked by diffuse lymphatic malformations involving bones, thorax, and abdominal viscera, but spindle cells and coagulopathy are not typically observed. GLA has been associated with PIK3CA variants, which were suggested as an additional distinguishing feature between these two overlapping conditions.⁷ CCLA is characterized by improper drainage into the thoracic duct and retrograde reflux of lymphatic fluid, resulting in pleural and pericardial effusions, ascites, and protein-losing enteropathy. Diagnosis is often made by imaging.^1,2 JAG1, EPHB4, ARAF, and MDFIC pathogenic variants have been described in CCLA, and a recent cohort study has demonstrated an association between RASopathies, Trisomy 21, 22q11.2 deletion syndrome, and PIEZO1 generalized lymphatic dysplasia in CCLA.^8–12

Treatment of KLA and GLA has thus far involved sirolimus and interventional procedures such as sclerotherapy and lymphatic embolization. Treatment of CCLA consisted of surgical or interventional procedures to redirect or obliterate aberrant lymph flow, but novel therapies are still needed given the diffuse nature of the disease. More recently, targeted therapies such as PI and MEK inhibition have also been trialed.^1,2

In this article, we describe four cases of phenotypic KLA/GLA and CCLA associated with PIK3CA pathogenic variants. These results challenge the notion of a strong genotype–phenotype correlation in lymphatic anomalies and support an individualized approach in these patients.

2 │. CASE DESCRIPTIONS

A 7-year-old male was diagnosed with a lymphatic malformation shortly after birth, with subsequent evaluation revealing an extensive lymphatic malformation involving his left hand, wrist, forearm, chest, and spleen initially diagnosed as GLA (Figure 1A–C). As he aged, his disease became complicated by “flares” of extremity swelling, purpura, fevers, pain, elevated D-dimers, thrombocytopenia, and hypofibrinogenemia for which he received intermittent steroids. At 4 years old, he developed purpura, mild coagulopathy, and disease involving his soft tissue, spleen, and bones. Based on his diffuse disease and coagulopathy, he was reclassified as KLA, with subsequent biopsy showing D2–40⁺ lymphatic vessels without obvious spindle cells. Sequencing of affected tissue revealed a pathogenic variant NM_006218.4(PIK3CA):c.1093G>A with variant allele frequency (VAF) of 4%. He was transitioned from sirolimus to the PIK3CA inhibitor alpelisib after discovery of the variant. Two years later, he has improvement in pain, modest decrease in extent of the malformation, and decreased frequency of “flares.” He remains without appreciable coagulopathy, but continues with elevated D-dimers.

FIGURE 1.

Imaging for patients with KLA/GLA. (A–C) Five-year-old male with KLA. (A) Coronal fluid-sensitive (short tau inversion recovery) magnetic resonance imaging (MRI) showing infiltrative lymphatic malformation of the shoulder girdle and proximal humerus (arrows). (B) Axial T2-weighted MRI with fat saturation showing lymphatic malformation involving the forearm, chest wall, and spleen (short arrows). (C) Coronal fluid-sensitive (inversion recovery) sequence showing diffusely infiltrative lymphatic malformation of the forearm. (D–I) Twenty-year-old male with lymphatic anomaly (GLA vs. KLA). (D and E) Axial computed tomography (CT) images demonstrating a mesenteric mass with calcification (arrowheads) and pulmonary cysts and nodules (arrows). (F) Coronal CT image demonstrating massive portal and mesenteric thrombosis (arrowheads). (G and H) Axial T2-weighted MRI with fat saturation showing confluent lymphatic malformation in the posterior mediastinum and mesentery, with splenic cyst (asterix). (I) Axial T2-weighted MRI with fat saturation showing tiny cysts in the ilia (thick arrows). (J–M) An 11-year-old male with lymphatic anomaly (GLA vs. KLA) and massive portal vein thrombosis. (J) Axial T1-weighted with fat saturation post-contrast gradient echo MRI showing lymphatic malformation involving the posterior mediastinum and hila (arrows). (K) T2-weighted with fat saturation MRI showing diffuse microcystic lymphatic malformation in the abdomen (arrows) and cysts in the spleen (asterix). (L) Axial non-contrast-enhanced CT image (obtained 2 years prior to image J) showing interstitial thickening of the lungs (arrowheads). (M) Acute portal vein thrombosis in this patient, marked by expansion of the portal and superior mesenteric veins with massive thrombus (arrows).

A 20-year-old male presented to an outside institution with abdominal pain. Initial imaging revealed massive portal and mesenteric vein thrombosis, a mesenteric mass with calcification, posterior mediastinal thickening, and pulmonary cysts and nodules (Figure 1D–I). He began anticoagulation therapy and was referred to our institution, where magnetic resonance imaging (MRI) showed a diffuse slow-flow malformation throughout the mediastinum, peritoneum, spleen, and bones (Figure 1D–I). Biopsy of the mass revealed dilated venous structures and D2–40⁺ PROX1⁺ lymphatic channels, with areas of poorly staining spindle-shaped cells that appeared more reactive compared to those typically seen in KLA. Based on tumor encasement of vessels, pulmonary nodules, and histology, he was diagnosed with KLA. Analysis of genomic DNA from affected tissue revealed a pathogenic variant NM_006218.4(PIK3CA):c.1633G>A with VAF 13.8%–14.4% that was absent from blood. He underwent thrombectomy via creation of a transjugular intrahepatic portosystemic shunt (TIPS) and a short course of sirolimus before transitioning to alpelisib. His pain has since improved, with partial disease response noted on repeat imaging. His D-dimer has since normalized, with no bleeding or clotting events noted.

An 11-year-old male with history of mediastinal “hemangioma” resected at 6 months of age was referred to a Comprehensive Vascular Anomalies Program for dyspnea. Computed tomography (CT) and MRI identified an infiltrative “slow-flow malformation” of the mediastinum extending into the right lung and abdomen (Figure 1J–L) with associated splenic and skull lesions. Affected tissue showed D2–40⁺ CD31⁺ cells within cystic spaces, consistent with a lymphatic malformation, but no clearly identified spindle cell component. Dynamic contrast-enhanced magnetic resonance lymphangiography (DCMRL) demonstrated a vascular anomaly within the mediastinum, mesentery, and stomach, suggestive of KLA versus GLA. Six months later, the patient developed fulminant thrombosis of splenic, portal, and superior mesenteric veins (Figure 1M), requiring thrombectomy via TIPS. Sequencing of the resected mass revealed a somatic pathogenic variant NM_006218.4(PIK3CA):c.3140A>G with frequency of 12.4%– 13.7%. He was started on alpelisib therapy with disease progression on most recent evaluation.

A 9-year-old female presented to an outside hospital with chylous leakage and casts from her rectum and vagina. CT and MRI confirmed a complex confluent lymphatic mass connecting to her sigmoid colon and vaginal canal. She was evaluated at two vascular anomalies centers and diagnosed with CCLA. She was treated with sirolimus, then everolimus, with initial improvement but notable drug toxicity and persistence of chylous leak, requiring frequent albumin and immunoglobulin replacement. She presented to our institution shortly thereafter. DCMRL showed multiple pelvic cystic lymphatic malformations with drainage into the peritoneum, descending and rectosigmoid colon, and vaginal cavity (Figure 2A–C). She underwent embolization of peri-colonic and peri-vaginal lymphatics and lymphovenous anastomosis to right gonadal vein. Her symptoms improved for several weeks, but ascites returned. Sequencing of affected tissue revealed a pathogenic variant NM_006218.4(PIK3CA):c.3129G>A with VAF of 5.6%. She started alpelisib therapy, with improvement in symptoms but continued accumulation of ascites requiring lymphatic embolization procedures and lymphovenous anastomoses.

FIGURE 2.

Imaging for 9-year-old female with central conducting lymphatic anomaly (CCLA) presenting as rectal and vaginal leak. (A) Axial T2-weighted turbo spin echo image showing diffuse microcystic lymphatic malformation in the pelvis (thick arrows). (B) Magnecitc resonance (MR) lymphangiogram (maximum-intensity projection image from T1-weighted spoiled gradient echo sequence) after injection of mesenteric lymphatics showing perfusion of the confluent abdominopelvic lymphatic malformation (long arrows) and leak of contrast into the sigmoid colon, peritoneum (arrowhead), and vagina (asterix). The thoracic duct was not seen during this lymphangiogram. (C) Inguinal nodal injection MR lymphangiogram obtained 3 months later, after initiation of sirolimus and pelvic lympho-venous anastomosis showing reduced perfusion of pelvic lymphatics, resolved leak, and now visualization of a dilated and tortuous thoracic duct (arrowheads).

3 │. DISCUSSION

Complex lymphatic anomalies comprise a collection of diseases of lymphatic development and dysfunction. Unfortunately, these diseases are often progressive, morbid, and difficult to treat.^1,2 Clinicians have used history, clinical exam, imaging, and histopathology to diagnose GLA, CCLA, and KLA; however, there is often significant overlap in anatomic and histologic phenotypes in these conditions. Because of this, diagnosis is often difficult.

Recent studies have begun to elucidate the molecular underpinnings of these rare disorders. Cells isolated from patients with KLA have shown increased signaling in the PI3K–AKT–mTOR and MAPK pathways; however, NRAS variants have been the most frequently identified pathogenic variants thus far.^3,4 In contrast, GLA has been associated with PIK3CA variants, suggesting that genetic analysis might help in diagnosis of these overlapping conditions.⁷ Unfortunately, these cases suggest this dichotomy is not absolute.

This report describes four cases of KLA/GLA and CCLA associated with somatic PIK3CA variants in absence of other known pathogenic variants. These cases highlight the genotypic overlap between clinically diverse lymphatic anomalies (Table 1). Future studies should assess whether variants not sequenced in these cases correlate to specific lymphatic anomalies. While our findings suggest that sequencing of a panel of genes may not help with diagnosis, it may allow for personalized directed therapy. Our experience suggests modest responses to alpelisib; however, future studies are needed to evaluate the efficacy of targeted therapies in patients with complex lymphatic anomalies.

TABLE 1.

Clinical features in patients.

Age/sex	Clinical presentation	Anatomic features	Histopathology	Diagnosis	Prior treatment	Genetic testing
7 years, M	Vascular malformation of left arm identified at birth. Recurrent episodes of fever, arm swelling, pain, thrombocytopenia, and hypofibrinogenemia	Extensive lymphatic malformation of left arm with bony erosion, chest, and spleen. Associated with overlying purpura on arm	D2–40+ endothelium without apparent spindle-shaped cells	KLA	Sirolimus, compression wraps, intermittent steroids, zolendronic acid for osteopenia	Brigham and Women’s Hospital OncoPanel NM_006218.4(P/K3CA):c.l093G>A (p.Glu365Lys) VAF 4% from lesion Known pathogenic varianta No NRAS variants detected
20 years, M	Sudden onset abdominal pain with imaging showing portal and mesenteric vein thrombus with associated abdominal mass Angiopoietin-2 4519 pg/mL	Mesenteric vascular mass, extending through peritoneum, spleen, bones, and encasing vessels. Associated portal and mesenteric vein thrombus	D2–40+ PROX1+ channels with nonspecific spindle-shaped cells	KLA	Thrombectomy and transjugular intrahepatic portosystemic shunt procedure, sirolimus	University of Pennsylvania Somatic Overgrowth & Vascular Malformations Panel (NGS) NM 006218.4(P/K3CA):c.l633G>A (p.Glu545Lys) VAF 13.8%−14.4% from lesion Known pathogenic variantb Concurrent leukocyte-derived DNA was negative for this variant No NRAS variants detected
11 years, M	Mediastinal “hemangioma” diagnosed around birth and resected at 6 months old. At 10 years old, presented with coughing up bronchial casts with subsequent development of portal, splenic, and superior mesenteric vein thrombus following lymphangiogram Angiopoietin-2 3834 pg/mL	Infiltrative vascular malformation of mediastinum extending to right lung and abdomen with associated splenic and skull lesions. Associated with splenic, portal, and superior mesenteric vein thrombus	Cystic tissue lined with D2–40+ CD31+ cells without spindle cell component	KLA/GLA spectrum	Resection at 6 months of age, thrombectomy and transjugular intrahepatic portosystemic shunt procedure, sirolimus after recurrence	University of Pennsylvania Lymphedema Panel (NGS) NM_006218.4(P/K3CA):c.3140A>G (p.Hisl047Arg) VAF 12.4%−13.7% from lesion. Known pathogenic variantc cfDNA from plasma demonstrated same variant with VAF 0.41% No NRAS variant detected
9 years, F	Chylous drainage from vagina and rectum, protein-losing enteropathy, and abdominal pain Angiopoietin-2 1613 pg/mL	Multiple pelvic lymphatic malformations draining into peritoneum, colon, and vaginal cavity	None obtained	CCLA	Sirolimus then everolimus, multiple lymphangiograms with embolization of aberrant lymphatic vessels and lymphovenous anastomoses	CHOP Center for Applied Genomics high-coverage exome sequencing NM 006218.4(P/K3CA):c.3129G>A (p.Metl043lle) VAF 5.6% from lesion Pathogenic variantd Concurrent leukocyte-derived DNA was negative for this variant No known CLA-causing variants detected

Note: Unless noted in the table, all testing was performed from genomic DNA.

Abbreviations: CCLA, central conducting lymphatic anomaly; cfDNA, cell-free DNA; GLA, generalized lymphatic anomaly; KLA, kaposiform lymphangiomatosis; VAF, variant allele frequency.

^aNational Center for Biotechnology Information. ClinVar; [VCV000419222.13], https://www.ncbi.nlm.nih.gov/clinvar/variation/VCV000419222.13 (accessed February 15, 2023).

^bNational Center for Biotechnology Information. ClinVar; [VCV000013655.34], https://www.ncbi.nlm.nih.gov/clinvar/variation/VCV000013655.34 (accessed February 15, 2023).

^cNational Center for Biotechnology Information. ClinVar; [VCV000013652.46], https://www.ncbi.nlm.nih.gov/clinvar/variation/VCV000013652.46 (accessed February 15, 2023).

^dNational Center for Biotechnology Information. ClinVar; [VCV000179173.il], https://www.ncbi.nlm.nih.gov/clinvar/variation/VCV000179173.ll (accessed February 15, 2023).

Abbreviations:

CCLA

central conducting lymphatic anomaly

GLA

generalized lymphatic anomaly

KLA

kaposiform lymphangiomatosis

VAF

variant allele frequency

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.