Genomic landscape of lymphatic malformations: a case series and response to the PI3Kα inhibitor alpelisib in an N-of-1 clinical trial

Abstract

Background: Lymphatic malformations (LMs) often pose treatment challenges due to a large size or a critical location that could lead to disfigurement, and there are no standardized treatment approaches for either refractory or unresectable cases.

Methods: We examined the genomic landscape of a patient cohort of LMs (n = 30 cases) that underwent comprehensive genomic profiling using a large-panel next-generation sequencing assay. Immunohistochemical analyses were completed in parallel.

Results: These LMs had low mutational burden with hotspot PIK3CA mutations (n = 20) and NRAS (n = 5) mutations being most frequent, and mutually exclusive. All LM cases with Kaposi sarcoma-like (kaposiform) histology had NRAS mutations. One index patient presented with subacute abdominal pain and was diagnosed with a large retroperitoneal LM harboring a somatic PIK3CA gain-of-function mutation (H1047R). The patient achieved a rapid and durable radiologic complete response, as defined in RECIST1.1, to the PI3Kα inhibitor alpelisib within the context of a personalized N-of-1 clinical trial (NCT03941782). In translational correlative studies, canonical PI3Kα pathway activation was confirmed by immunohistochemistry and human LM-derived lymphatic endothelial cells carrying an allele with an activating mutation at the same locus were sensitive to alpelisib treatment in vitro, which was demonstrated by a concentration-dependent drop in measurable impedance, an assessment of cell status.

Conclusions: Our findings establish that LM patients with conventional or kaposiform histology have distinct, yet targetable, driver mutations.

Funding: R.P. and W.A. are supported by awards from the Levy-Longenbaugh Fund. S.G. is supported by awards from the Hugs for Brady Foundation. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of Arizona Cancer Center (CA023074), the University of New Mexico Comprehensive Cancer Center (CA118100), and the Rutgers Cancer Institute of New Jersey (CA072720). B.K.M. was supported by National Science Foundation via Graduate Research Fellowship DGE-1143953.

Clinical trial number: NCT03941782.

Keywords: NGS; PI3Kα; genomics; human; lymphatic malformations; medicine.

Conflict of interest statement

MS reports personal fees from Illumina, BMS, and Qiagen (outside of the submitted work). The author has no other competing interests to declare, JT, ES is an employee of Foundation Medicine, Inc, a wholly owned subsidiary of Roche, and owns equity in Roche. The author has no other competing interests to declare, MM, PB, IR, CT, AD, TS, TB, RG, JB, SN, GR No competing interests declared, BM is an employee of Foundation Medicine, Inc, a wholly owned subsidiary of Roche, and owns equity in Roche, RG reports research funding/grant support for clinical trials (to his institution) from Regeneron, BMS, Merck/EMD Serano, Amgen, Roche/Genentech, Philogen; consulting/advisory board fees from Regeneron; and speaker fees for Deciphera (all outside of the submitted work). These arrangements are managed in accordance with the established institutional conflict of interest policies of Rutgers, The State University of New Jersey. The author has no other competing interests to declare, RP, WA Reviewing editor, eLife, SG has consulting agreements with Merck, Roche, Novartis, Foundation Medicine, EQRX, Foghorn Therapeutics, Silagene, and KayoThera and owns equity in Silagene; his spouse is an employee of Merck and owns equity in Merck (all outside of the submitted work). These arrangements are managed in accordance with the established institutional conflict of interest policies of Rutgers, The State University of New Jersey. The author has no other competing interests to declare

© 2022, Shaheen, Tse et al.

Figures

Figure 1.. Mutational landscape and histopathology of lymphatic malformations (LMs).

(A) Oncoprint showing mutational landscape of 30 LM samples sequenced. (B) Lollipop plot showing spectrum of PIK3CA and NRAS mutations in this cohort. (C) Schema showing details of GOPC–ROS1 fusion identified in an NRAS and PIK3CA wild-type LM. (D) Representative histologic images for LMs with conventional and kaposiform histology. The relative frequencies of PIK3CA and NRAS mutations in the two histologic variants are plotted.

Figure 2.. Imaging and histological analysis of lymphatic malformation (LM) patient.

(A) Baseline CT abdomen scan at the time of presentation demonstrating a large retroperitoneal/pancreatic LM. (B) CT abdomen scan 6 weeks after the initiation of alpelisib. (C) CT abdomen scan 1 year into the trial. (D, E) Hematoxylin and eosin (H&E)-stained photomicrographs of the LM showing dilated lymphatic channels percolating through visceral fat and associated patchy lymphocytic inflammation (×4 and ×20, respectively). (F) Immunohistochemistry utilizing an anti-P-6S antibody demonstrates PI3Ka pathway activation within the channels’ lining cells. (G) Anti-P-AKT positivity in the lining endothelium of lymphatic channels as well.

Figure 3.. Alpelisib reduces lymphatic malformation-lymphatic endothelial cell (LM-LEC) viability.

(A) Logarithmic dose–response curve of alpelisib was performed using the xCELLigence RTCA system. 1, 3, 10, 30, and 100 nM (n = 5 replicates) of alpelisib were used to determine the concentration–response curve. The alpelisib half maximal inhibitory concentration (IC50) was calculated for LM-LEC at 24 hr after treatment as 4.72 × 10−9 M. Error bars are shown as mean +/- standard deviation (SD), which was automatically calculated for each data point by the xCELLigence RTCA system software (Version 2.0) based on five replicates per drug concentration. (B) Illustrative picture of LM-LEC clonogenic plaques at 24 hr after alpelisib treatment (4.72 × 10−9 M). Negative, no treatment; dimethyl sulfoxide (DMSO), vehicle control. Experiments were performed two times with similar results. LM-LEC colonies were stained with crystal violet (0.3%). (C) Colony count 24 hr after alpelisib treatment (4.72 × 10−9 M; n = 2 wells/condition). Error bars are shown as mean +/- SD calculated by GraphPad Prism by determining the square root of variance for each data point deviation relative to the mean.

Figure 4.. RNA-seq analysis of lymphatic malformation (LM) samples from index patient (#9).

(A) The heatmap summarizes the results of the differential gene expression analysis. Up- and downregulated genes are shaded red and blue, respectively. (B) The volcano plot summarizes the distribution of genes that were differentially expressed. The vertical axis shows the p value and the horizontal shows the fold-change. The genes that were more than twofold changed and had an adjusted p value less than 0.05 are shaded red. Similar numbers of genes were up- or downregulated.

Figure 5.. Graphical summary of the mutations found in genomic analysis of lymphatic malformation (LM) patient cohort (created with BioRender.com).

(A) The majority of LMs have driver mutations that are potentially targetable. (B) LMs with NRAS mutations had kaposiform histopathology. (C) An N-of-1 clinical trial is reported in a patient with a targetable PIK3CA mutation. (D) Comprehensive genomic analyses may reveal further actionable molecular insights.