DOI: 10.5281/zenodo.17527214
title: Multiomic Study of Cutaneous T-Cell Lymphoma Reveals Single Cell Clonal Evolution in Progression and Therapy Resistance

description: Cutaneous T-cell lymphoma (CTCL) remains a challenging disease due to its significant heterogeneity, therapy resistance, and relentless progression. Multi-omics technologies offer the potential to provide uniquely precise views of disease progression and response to therapy. We present here a comprehensive multi-omics view of CTCL clonal evolution, incorporating exome, whole genome, epigenome, bulk-, single cell (sc) VDJ-, and scRNA- sequencing of 114 clinically annotated serial skin, peripheral blood, and lymph node samples from 35 CTCL patients. We leveraged this extensive dataset to define the molecular underpinnings of CTCL progression in individual patients at single cell resolution with the goal of identifying clinically useful biomarkers and therapeutic targets. Our studies identified a large number of recurrent progression-associated clonal genomic alterations; we highlight mutation of CCR4, PI3K signaling, and PD-1 checkpoint pathways as evasion tactics deployed by malignant T cells. We also identified a gain of function mutation in STAT3 (D661Y) and demonstrated by CUT&RUN-seq that it enhances binding to transcription start sites of genes in Rho GTPase pathways, which we previously reported to have activated chromatin and increased expression in HDACi-resistant CTCL. These data provide further support for a previously unrecognized role for Rho GTPase pathway dysregulation in CTCL progression. A striking number of progression-associated mutations occurred in chromatin methylation modifiers, including EZH2, suggesting that EZH1/2 inhibition may also benefit patients with CTCL. Knowledge of these molecular changes should be leveraged for improved disease monitoring, biomarker-informed clinical trial design, and new therapeutic strategies in this challenging and incurable cancer.
description: Single cell (sc)RNA sequencing were performed on Cutaneous T-cell Lymphoma (CTCL) samples, including skin and lymph node biopsies of CTCL, peripheral blood mononuclear cells, purified malignant and non-malignant T cells and matched normal monocytes or NK cells from peripheral blood, as well as peripheral blood T cells from healthy donors.

Treatment protocol: Peripheral blood mononuclear cells were subjected to CD4+ enrichment (EasySep Human CD4 Positive Selection Kit or CD4+ staining and FACS) and/or monocyte depletion (RosetteSep Human Monocyte (CD36) Depletion Cocktail) and viable cells sorted using a 7-AAD viability stain on a Sony iCyt Synergy SY3200.

Extraction protocol: For each of the PBMC samples, 10,000 to 20,000 viable cells were submitted for processing using the 10x Genomics Chromium Controller and the Chromium Single Cell 5′ Library & Gel Bead Kit v2 (PN-1000006) following the manufacturer’s protocols. cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 10-14 cycles before being cleaned up using SPRIselect beads. Samples were then run on a Bioanalyzer to determine the cDNA concentration. TCR target enrichments were performed on the full-length cDNA. GEX and Enriched TCR libraries were prepared as recommended by the 10x Genomics Chromium Single Cell V(D)J Reagent Kits (v1.0 Chemistry) user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium Single Cell 5’ Library and Gel Bead Kit v2 (PN-1000006), Chromium Single Cell A Chip Kit (PN-1000152), Chromium Single Cell V(D)J Enrichment Kit, Human, Tcell (96rxns)(PN-1000005), and Chromium Single Index Kit T (PN-1000213) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 151x10x10x151 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50,000 reads/cell was targeted for each sample.

Data processing: Alignment and gene counting were performed using the Cell Ranger pipeline (10x Genomics, v3.0, Pleasanton, CA) with genome assembly GRCh38/hg38. Genes found in fewer than 15 cells in each sample were removed. For each patient, gene counts and cells were pooled into a single Seurat (v5.1.0)(Butler et al., 2018; Hao et al., 2024; Stuart et al., 2019) object, and cells containing fewer than 200 or more than 3000-3750 expressed genes, more than 8-10% mitochondrial reads, fewer than 300 or more than 10000UMIs, or classification as a doublet by the R package scDblFinder(Germain et al., 2022) with parameters dims = 30, clust.method = “fast_greedy” were removed. In total, 118,608 cells were sequenced, with 92,496 passing initial QC filters after removing dead cells, empty droplets, and suspected multiplets.