Novel Discovery of ROS1:PPFIBP1 fusion protein in General Lymphatic Anomaly: A Case Report and Review of the Literature : Journal of Vascular Anomalies

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Case Report

Novel Discovery of ROS1:PPFIBP1 fusion protein in General Lymphatic Anomaly

A Case Report and Review of the Literature

Kadenhe-Chiweshe, Angelaa; Baad, Michaelb; Kaicker, Shiprac; Mathew, Susand; Pua, Bradleye; Steigman, Shauna; McGuinn, Catherinec

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Journal of Vascular Anomalies 4(1):p e061, March 2023. | DOI: 10.1097/JOVA.0000000000000061
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Generalized lymphatic anomaly (GLA), kaposiform lymphangiomatosis (KLA), Gorham-Stoudt disease (GSD), and central conducting lymphatic anomaly (CCLA) are rare presentations of complex lymphatic malformations (CLMs) that originate from anomalies in embryogenesis but remain poorly understood.1–3 Usually extensive and multifocal in distribution, they are a source of significant morbidity, poor quality of life, and diminished life expectancy. Recently published consensus guidelines for the initial evaluation of patients with CLM provide an orderly, comprehensive, and systematic approach to the workup of these complicated disorders by a multidisciplinary group of specialists.1,4 Current interventions are not curative, and thus, treatment is aimed at symptom relief, improving function, and stemming disease progression.5 Despite a recent explosion of knowledge identifying the underlying pathogenic pathways involved in CLM, the genetic and biologic pathways underlying and driving these disorders have not been well elucidated. Next-generation sequencing (NGS) provides a unique tool that can unveil alterations in genes that are overactive or mutated in these disorders, as well as novel mutations which may be driving the disease process.


A 17-year-old female presented to the emergency room with 2 months of chest pain and dyspnea. She was a well-nourished Asian female in moderate respiratory distress but hemodynamically stable. She had an area of tenderness in her right mid back which was devoid of underlying bone. There were no overlying skin changes nor an accompanying palpable mass. A Computed Tomography scan of the chest revealed a large right pleural effusion, extensive lytic lesions of the T7, T8, and T9 vertebral bodies, and destruction of adjoining posterior elements and ribs at the same levels (Figure 1A1, A2). The cortex was predominantly thinned and intact; however, the cortex in portions of the eighth rib was imperceptible. Concomitant nondisplaced rib and pedicle fractures were also noted. The mediastinum was shifted to the left, but otherwise normal. Lung fields were normal. An extensive panel of laboratory results was unremarkable. She was admitted to the pediatric intensive care unit and underwent tube thoracostomy, with return of serosanguinous fluid. Approximately 6 L of pleural fluid drained within the first 2 days of admission and continued to drain 1–1.5 L daily, requiring closely monitored fluid and electrolyte replacement and nutritional repletion.

Figure 1.:
Clinical diagnosis and progression. Axial (A1) and sagittal (A2) images from contrast enhanced Computed Tomography: lytic lesion involving the right posterior 8th rib, transverse process, pedicle and vertebral body (arrows). A large right effusion (*) with leftward mediastinal shift is noted. B1, Coronal MRL 25 minutes following inguinal node: extensive subcutaneous collateral lymphatic flow (arrow) to the confluence of the right subclavian and internal jugular veins. Drainage through the cisterna chyli and a small-caliber thoracic duct (arrowheads) is also present. B2, Axial MRL delayed image: patchy enhancement of the lesion (arrow) and hyperintense contrast within the pleural space (arrowheads), consistent with chylous effusion. C, Representative intraoperative picture of appearance of soft-tissue disease—white, porous plaque appearance to pleura, with active fluid drainage, lymphorrhea, absence of ribs. D, Graphic representation of right chest tube output over time, with stated medical and procedural interventions.

A wide differential was considered including leukemia, bony malignancy, infection, and/or Cerebral Spinal Fluid leak. Gram stain and culture of the pleural fluid did not show any signs of infection; cytopathology did not show any evidence of malignancy. Fluid analysis showed 80% lymphocytes consistent with lymphatic fluid. Beta-transferrin, specific for cerebrospinal fluid, was negative. An Magnetic Resonance Imaging of the cervical, thoracic, and lumbar spine echoed Computed Tomography findings with additional foci of disease noted at T10, but with preservation of the spinal canal and cord. Lesions were also noted in the bony pelvis. An MR lymphangiogram (Figure 1B1, B2) showed abnormal subcutaneous lymphatic collaterals with a diminutive thoracic duct as well as accumulation of contrast in the right pleural space, consistent with chylous effusion. An image-guided percutaneous biopsy of the T7 vertebral body, performed by Interventional Radiology, was inconclusive. One of these small biopsy fragments showed well-formed vessels with muscular walls surrounded by epithelioid appearing cells. These cells were positive for the endothelial marker ETS (erythroblast transformation-specific) Related Gene and negative for the histiocytic marker CD163 and the Langerhans cell marker CD1a. These findings were felt to be suggestive of a vascular tumor. Subsequently, the patient underwent right video-assisted thoracoscopic surgery (VATS) for better tissue sampling and optimal chest tube placement. At thoracoscopy (Figure 1C), the appearance of the pleura and soft tissue of T7-T8 were abnormal and clear fluid was visualized streaming into the chest. The destructive bony process involving the ribs at this level had left them comminuted and weak. Multiple biopsy specimens of bone and a pleura were obtained.

H&E immunostaining (Figure 2A) of the VATS biopsy samples demonstrated an abnormal vascular proliferation of bland cells with collapsed small spaces but without marked atypia or spindle cell morphology. This proliferation was seen throughout connective tissue, skeletal muscle and bone sampled from the area of the chest wall near the eighth and ninth rib and T8/T9 vertebra. Immunohistochemistry was positive for ETS (erythroblast transformation-specific) Related Gene and D2-40 (Figure 2B, C), while negative for macrophage markers (CD163) and Langerhans cell markers (CD1a). While the morphology was in keeping with GLA, aspects of the exuberant proliferation and the corresponding imaging made it difficult to rule out KLA. The clinical, radiographic, and pathology data were reviewed at our institution’s multi-disciplinary Comprehensive Vascular Anomalies Center meeting, as well as Tumor Board, and a consensus diagnosis of GLA was reached. The case was then sent to an outside Vascular Anomalies Group for second review with specific consideration of KLA. However, given a relative lack of pulmonary or mediastinal involvement, infiltrating soft tissue on imaging, as well as the absence of cutaneous involvement, consumptive coagulopathy, elevated serum angiopoietins, or NRAS mutation, KLA was less favored.

Figure 2.:
Analysis of tissue biopsy. A, H&E stains demonstrated an exuberant relatively bland proliferation of blood vessels is seen infiltrating in connective tissue (H&E, ×100). B, ERG, a marker of vascular endothelium, supports the proliferation as endothelial cells (DAB, ×100). C, D2-40, a marker of lymphatic endothelium, is strongly positive (DAB, ×150). Immunohistochemistry for ROS1 (D) shows positivity in biopsy specimens. E, FISH showing ROS1 gene rearrangement. The large yellow arrows indicate normal ROS1 allele (fusion signals), whereas the small red arrows indicate rearranged ROS1 allele (one red and one green signal). F, Next-generation sequencing with Oncomine reported single TIER 2 abnormality: presence of ROS1 fusion protein. Abbreviations: DAB, Diaminobenzidine; ERG, ETS (erythroblast transformation-specific) Related Gene; FISH, Fluorescence in situ hybridization.

Sirolimus (Rapamune, Pfizer), a known treatment for GLA, was promptly started and titrated to a therapeutic range of 10–15 ng/mL over the ensuing 4 weeks.6–8 Over this time, fluid production gradually decreased to an average of 500 mL/d, but the patient remained chest tube-dependent and demonstrated persistent deficiencies in immunoglobulins and nutrition. After multidisciplinary reviews, both internally and with national experts, and extensive discussion with patient and family, we performed right VATS with talc pleurodesis. We initiated zoledronic acid (Zometa, Novartis), a bisphosphonate, used in lymphatic malformations with osteolytic bone lesions and metastatic malignancies. Zoledronic acid is postulated to work synergistically with sirolimus, when used in complex lymphatic malformations with bony involvement.4,9 Chest drainage decreased significantly over the ensuing weeks and the chest tubes were removed sequentially (Figure 1D). The patient was discharged to home after a 3-month hospitalization. She is currently doing well, on room air, and enrolled in college. She is on a maintenance dose of sirolimus that provides a therapeutic serum level of 10 ng/mL, as well as scheduled infusions of zoledronic acid.

To further delineate the lesion, NGS testing was performed using the Oncomine Comprehensive v2 (Thermo Fisher Scientific, Waltham, MA) platform on DNA extracted from formalin-fixed paraffin-embedded pleural biopsy specimens. This assay was run using the clinical pipeline, and it is validated to a variant allele frequency limit of detection of 3%. It has been clinically validated by the Weill Cornell Medicine Clinical Genomics Laboratory, which is a Clinical Laboratory Improvement Amendments-approved and Collge of American Pathologists-accredited laboratory. In this validation, it was demonstrated that the limit of detection for single nucleotide variants was 3% at a coverage depth of 1000×. The validation was reviewed and approved by New York State as part of the Clinical Laboratory Evaluation Program (

A tier-2 variant was identified, a ROS1 fusion protein. The annotation of this fusion is: PPFIBP1(9):ROS1(35). Breakpoints are chr12:27809663 -chr6:117642557 (Figure 2F). NGS did not detect any mutations in the PI3KCA pathway in the lesional tissue. These findings were confirmed by performing immunohistochemistry for ROS1 (Figure 2D) using Clone SP384 (Ventana Medical Systems) ready-to-use rabbit monoclonal primary antibody using OptiView Diaminobenzidine IHC Detection Kit on a Ventana Ultra automated instrument. Fluorescence in situ hybridization was also performed on formalin-fixed paraffin-embedded tissue slides, using multiplex ROS1(6q22.1) dual color break-apart rearrangement probe (Abbott Molecular Inc., Des Plaines, IL) to interrogate for rearrangement of the ROS1 gene (Figure 2E). Two hundred interphase nuclei were evaluated. Sixty-seven percent of cells were POSITIVE for ROS1 gene rearrangement. A literature search demonstrated that this particular fusion protein has not previously been reported in vascular anomalies, including complicated lymphatic anomalies.


Vascular malformations, including CLM, are poorly understood in terms of how and when in gestational development these malformations occur, and what biologic pathways play essential roles in their pathobiology, phenotype, and progression of disease. NGS has been an important tool for molecular characterization and diagnosis, which can translate to targeted treatment strategies.10,11 Approximately 80% of lymphatic anomalies and venous malformations demonstrate somatic mutations in the PI3KCA pathway.10,12 Several studies have shown that mutations in the PI3KCA pathway are integral to cell survival, proliferation, angiogenesis, and metabolism in lymphatic malformations although the mechanism by which these PI3KCA mutations contribute to the development and progression of these malformations remains unclear.10

Sirolimus (Rapamune) targets mTOR in the PI3KCA signaling pathway and has been used for patients with both extensive isolated, multifocal, and syndromic malformations.13 Additional insights from NGS have identified other therapeutic targets for complex lymphatic anomalies, including somatic activating NRAS mutations14 and an upstream, casitas B lineage lymphoma (CBL) proto-oncogene mutations15 in patients with KLA, allowing integration of MEK (mitogen-activated protein kinase) inhibitors, such as trametinib (Mekinist, Novartis), directed at the closely related RAS (rat sarcoma) pathway16 and MAPK (mitogen-activated protein kinase) pathways.3,17

Utilization of an NGS platform identified a ROS1 fusion protein, ROS1:PPFIBP1, in the tissue biopsy of our patient with GLA, confirmed by fluorescence in situ hybridization and immunostaining. This fusion protein has only previously been reported in a small case series of spitzoid nevus.18 Barclay et al, however, have reported the identification of a KHE occurring in the maxilla of a 10-year-old containing a GOPC:ROS1 fusion using Oncomine Childhood Cancer Research Assay (Thermo Fisher Scientific).19ROS1 is a proto-oncogene located on chromosome 6 that encodes a receptor tyrosine kinase expressed in several human tissues, whose function is not clearly elucidated.20PPFIBP1, also named Liprin-β1, is one of the ubiquitously expressed liprins, a family of leukocyte common antigen–related (LAR) protein-tyrosine phosphatase-interacting protein.21 It plays a key role in lymphatic vessel development and integrity.

ROS1 fusion proteins have been identified in adult and pediatric cancers, including inflammatory myofibroblastic tumors, glioblastoma, spitzoid neoplasms and non–small cell lung cancer (NSCLC).18,20 Studies have shown that ROS1 fusions exhibit ligand-independent constitutive activation. These fusions are sufficient to induce tumorigenesis in murine models independent of any other mutations.20ROS1 fusion proteins have not been previously reported in vascular anomalies, including CLM. Cell signaling that is induced by ROS1 upstream activation includes the MAPK and PI3KCA pathways.22 We speculate that ROS1 activation is an upstream driver to abnormal PI3KCA signaling in our patient despite the absence of PI3KCA mutations, evidenced by her clinical response to sirolimus. Inhibitors of ROS1 thus present an additional target for treatment. Fortuitously, therapeutic tyrosine kinase inhibitors directed at ROS1 have recently emerged, leading to recent Food and Drug Administration approval of entrectinib (Rozlytrek, Genentech), for the treatment of NSCLC. The drug also carries indications for adult and pediatric patients >12 years old with NTRK fusion+ solid tumors. This approval was based on single-arm clinical trials,23 with inclusion of pediatric and adolescent patients enrolled in STARTRK-NG.24 Having access to novel therapeutics with an approved pediatric indication based on a molecular classification unlocks real potential for access and extrapolation of dosing and safety data for our patient with a rare disease.

Currently, our patient is stable and has a good quality of life with minimal side effects to her treatment with sirolimus. After a multidisciplinary review of her case at Molecular Tumor Board, we elected to continue sirolimus given her excellent clinical response, and to reserve ROS1 targeted therapy such as entrectinib as alternate therapy to offer in the event of symptoms, disease progression, or intolerance to sirolimus. Our future endeavors will focus on analyzing additional patient samples and translational investigations into the role of aberrant ROS1 signaling in the pathobiology of complex lymphatic malformations. We have developed a construct of the fusion protein for in vitro investigation as to its potential causative role in complex lymphatic anomalies. In vitro studies will include transfection into lymphatic endothelial cells and examination of end points such as of proliferation, migration, as well as upregulation of PIK3CA pathway; and inhibition with ROS1 inhibitors. We also intend to utilize zebra fish for modeling of this vascular phenotype and attempting rescue/treatment with ROS1 inhibitors.


We present a case of generalized lymphatic anomaly where NGS identified a ROS1 fusion protein that may be a newly discovered driver of disease in these conditions. Additional translational studies are needed to understand its contribution to the pathobiology of these disease and potentially justify use of recently Food and Drug Administration approved ROS1 tyrosine kinase inhibitors in complex lymphatic malformations.


The authors would like to thank the patient and family who gave permission to publish this case and discovery.


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complex lymphatic malformations; generalized lymphatic anomaly; PIK3CA; ROS1 fusion protein

Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Society for the Study of Vascular Anomalies.