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.
Highlights
Mycosis fungoides (MF), the most common form of primary cutaneous lymphoid malignancy, is typically characterized by an infiltration of neoplastic CD4-positive T lymphocytes in the skin [1]
Since we identified two prior publications in which RNA extracted from 60–200 singly dissected neurons/astrocytes from frozen tissue sections was sufficient for NanoString [11,12], we chose to microdissect 700–800 single cells from FFPE tissue sections
Single-cell microdissection is well-suited for gene expression profiling (GEP) of MF cells, since these neoplastic cells are often surrounded by an abundance of benign epithelial cells, stromal cells and reactive immune cells
Summary
Mycosis fungoides (MF), the most common form of primary cutaneous lymphoid malignancy, is typically characterized by an infiltration of neoplastic CD4-positive T lymphocytes in the skin [1]. The histology of the early-stage skin lesions is variable, but one of the most consistent features is that of epidermotropism of cytologically atypical lymphoid cells with or without the formation of Pautrier microabscesses. With progression to the tumor stage (i.e., ≥1 cm in diameter), epidermotropism often diminishes and the dermal infiltrates of MF cells become more diffuse and confluent. Large cell transformation, defined by the presence of >25% large cells in the infiltrate, occurs mostly in the tumor stage [2]. While MF is generally an indolent disease, progression to the tumor stage elevates the clinical stage of the disease, which is the single most important prognostic factor [1,3,4]. There are no biomarkers that are predictive of progression to the tumor stage
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