Gene Expression Profiling of Mycosis Fungoides in Early and Tumor Stage—A Proof-of-Concept Study Using Laser Capture/Single Cell Microdissection and NanoString Analysis

Abstract

A subset of patients with mycosis fungoides (MF) progress to the tumor stage, which correlates with a worse clinical outcome. The molecular events driving this progression are not well-understood. To identify the key molecular drivers, we performed gene expression profiling (GEP) using NanoString. Ten formalin-fixed/paraffin-embedded skin biopsies from six patients (six non-tumor and four tumor MF) were included; non-tumor and tumor samples were available in three patients. Laser capture/single cell microdissection of epidermotropic MF cells was used for non-tumor cases. We found that the RNA extracted from 700–800 single cells was consistently sufficient for GEP, provided that multiplexed target enrichment amplification was used. An un-supervised/hierarchical analysis revealed clustering of non-tumor and tumor cases. Many of the most upregulated or downregulated genes are implicated in the PI3K, RAS, cell cycle/apoptosis and MAPK pathways. Two of the targets, HMGA1 and PTPN11 (encodes SHP2), were validated using immunohistochemistry. HMGA1 was positive in six out of six non-tumor MF samples and negative in five out of five tumor MF samples. An opposite pattern was seen with SHP2. Our study has provided a proof-of-concept that single-cell microdissection/GEP can be applied to archival tissues. Some of our identified gene targets might be key drivers of the disease progression of MF.

Keywords

• mycosis fungoides
• single cell microdissection
• NanoString
• gene expression profiling
• immunohistochemistry