Single-Cell Lymphocyte Heterogeneity in Advanced Cutaneous T-cell Lymphoma Skin Tumors

Abstract

Purpose: The heterogeneity of tumor cells presents a major challenge to cancer diagnosis and therapy. Cutaneous T-cell lymphomas (CTCL) are a group of T lymphocyte malignancies that primarily affect skin. Lack of highly specific markers for malignant lymphocytes prevents early diagnosis, while only limited treatment options are available for patients with advanced stage CTCL. Droplet-based single-cell transcriptome analysis of CTCL skin biopsies opens avenues for dissecting patient-specific T lymphocyte heterogeneity, providing a basis for identifying specific markers for diagnosis and cure of CTCL. Experimental Design: Single-cell RNA-sequencing was performed by Droplet-based sequencing (10X Genomics), focusing on 14,056 CD3 þ lymphocytes (448 cells from normal and 13,608 cells from CTCL skin samples) from skin biopsies of 5 patients with advanced-stage CTCL and 4 healthy donors. Protein expression of identified genes was validated in advanced stage CTCL skin tumors by immunohistochemistry and confocal immunofluorescence microscopy. Results: Our analysis revealed a large inter-and intratumor gene expression heterogeneity in the T lymphocyte subset, as well as a common gene expression signature in highly proliferating lymphocytes that was validated in multiple advanced-stage skin tumors. In addition, we established the immunologic state of reactive lymphocytes and found heterogeneity in effector and exhaustion programs across patient samples. Conclusions: Single-cell analysis of CTCL skin tumor samples reveals patient-specific landscapes of malignant and reactive lymphocytes within the local microenvironment of each tumor, giving an unprecedented view of lymphocyte heterogeneity and identifying tumor-specific molecular signatures, with important implications for diagnosis and personalized disease treatment.

Branched evolution and genomic intratumor heterogeneity in the pathogenesis of cutaneous T-cell lymphoma

“…It has recently been discovered by us and other groups that CTCL is heterogenous at transcriptomics and protein level. [45][46][47][48] Single cell approach would allow for precise mapping of malignant T-cell clones identified by a unique pair of rearranged TCRA and TCRB sequences to the mutational pattern and gene expression profiles. However, our results identified one potential problem in studies on ITH in multifocal tumor such as MF.…”

An Update on Molecular Biology of Cutaneous T Cell Lymphoma

“…Although this technology is still relatively contained within the research space, clinical applications using this technology will likely advance patient care in the next decade. Single cell sequencing studies have shown evidence of tumor heterogeneity in CTCLs and there is also preliminary evidence that there may be a prognostic impact (45)(46)(47). Demonstration of specific gene expression profiles enabling examination of the tumor microenvironment along with the tumor cells in the same assay provides useful insights to tumor biology and immune response, which ultimately helps in tailoring personalized treatments, understanding prognosis, response to therapy, and reasons for drug resistance (48,49).…”

“…Many CTCL's had differential expression of genes that was unique to that tumor signifying its importance in personalizing patient specific therapy. A common gene expression profile of highly proliferating lymphocytes was identified which was present in all tumors indicating its potential diagnostic role (47).…”

A skin liquid biopsy method to assess the immune environment of cutaneous T-cell lymphoma

“…Polo-like kinase4 (PLK4) is a serine/threonine kinase crucial for mitosis and DNA integrity and has emerged as a therapeutic target for the treatment of multiple cancers 29,30 . The serum and glucocorticoidregulated kinase 1 (SGK1) and the receptor of the G-protein signaling 1 (RGS1) are reportedly upregulated in CTCL, as determined using genome wide and single cell analyses 31,32 . The principal component analysis of full transcriptomes in this study revealed a high degree of individual variation in gene expression ( Figure 5e, j).…”