Single-cell transcriptomic profiling reveals tumor-intrinsic heterogeneity and immune-microenvironment dynamics in the order trial for mantle-cell lymphoma Free

Background: We conducted an open-label, randomized, multicenter trial (NCT06496308) enrolling previously untreated patients with intermediate- or high-risk mantle-cell lymphoma (MCL) who were ineligible for autologous stem-cell transplantation (Order Trail).

Aims: Using single-cell RNA-seq, we will dissect the microenvironmental mechanisms of resistance in responders versus non-responders, identify biomarkers for efficacy, and characterize the tumor and microenvironmental features of blastoid, refractory patients.

Methods: Single-cell RNA sequencing (scRNA-seq) were performed on 33 enrolled patients with available samples (25 classical, 6 blastoid, and 2 pleomorphic), yielding 370,392 high-quality cells.

Results: UMAP clustering identified 21 distinct cell populations, which were further annotated into 13 B cell subclusters, 5 T cell subclusters, 1 myeloid cell population and 1 epithelial population, based on the canonical markers, BCR/TCR profiling, and functional signatures.

RNA velocity analysis revealed that MCL tumor cells originated from the MCL-Precursor populations and subsequently differentiated into heterogeneous subclusters, including two patient-unique subclusters (Patient1-unique B cells and Patient21-unique B cells). Cell cycle analysis showed elevated S-phase fractions in MCL-6, MCL-7, and MCL-8 subclusters. BCR repertoire analysis revealed that Memory B-like and Plasma cell-like subclusters were enriched in monoclonal B cells, with IgH heavy chains expressing IGHG/IGHA, indicating that these two subclusters have undergone class switching and were closer to normal B cells.

The immunue microenvironment characterization identified 10 T-cell subclusters, including three CD4+ subclusters, three CD8+ subclusters, 1 NKT population, and 3 TOX-high DNT populations. Concurrently, the cytotoxic score, exhausted score and regulatory score for each T cell subclusters were calculated. Myeloid compartments were predominantly composed of DCs, M1/M2 macrophages, neutrophils and mast cells.

Subsequently, we compared the characteristics of tumor B cell and immune microenvironment among classical, blastic, and pleomorphic MCL. For B cells, classical MCL showed predominant MCL-3 and MCL-4 B-cell populations, while blastoid cases exhibited MCL-7 expansion. Pleomorphic samples demonstrated exceptional heterogeneity with patient-specific clusters. Pathway analysis showed that cell cycle-related pathways were significantly activated in blastoid B-cells, while pleomorphic cases exhibited B cell differentiation and stemness maintenance signatures. Regarding the immune microenvironment, classical MCL presented higher proportion of CD4+ naïve T cells, while blastoid/pleomorphic subtypes displayed elevated proportions of CD8+ CTLs compared to the classical subtype. Gene Ontology analysis revealed that the activated genes of T cells in blastic MCL were mainly enriched in cytokine secretion pathways, contrasting with the enriched immune-regulation pathways in pleomorphic cases. Myeloid compartments in pleomorphic cases exhibited distinct proportional differences compared to classical and blastic subtypes, with significantly increased mast cells and neutrophils, and reduced macrophages and DCs.

To investigate the underlying molecular mechanisms between patients with different treatment responses, we compared the proportions of the cell populations between patients achieving CR/PR and those with PD/SD. Compared to CR/PR responders, PD/SD patients had higher MCL-Precursor, MCL-4, DNT-2/3 proportions, but lower MCL-1/5 frequncies, alongside increased DCs and decreased neutrophils and mast cells. Pathway enrichment analysis demonstrated that upregulated genes in B cells from the PD/SD group were significantly enriched in cell cycle-related pathways, compared to the CR/PR group. Additionally, MCL-Precursor of PD/SD responders showed activation of the NF-κB and JAK-STAT signaling pathways, suggesting stronger malignant proliferation and earlier malignant transformation of tumor cells in patients with poor prognosis.

Summary and conclusion: This is a preliminary micro-environment-based efficacy analysis derived from the ORDER study. We will further interrogate the RNA transcriptomes of responders and non-responders—integrated with single-cell data—to uncover resistance mechanisms and identify novel therapeutic targets for progressive disease.