Department of Hematology, Ghent University Hospital, 9000 Ghent, Belgium.
OncoRNALab, Cancer Research Institute Ghent (CRIG), Ghent University, 9000 Ghent, Belgium.
Int J Mol Sci. 2024 Sep 16;25(18):9982. doi: 10.3390/ijms25189982.
The potential of RNA-based liquid biopsy is increasingly being recognized in diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma. This study explores the cell-free transcriptome in a humanized DLBCL patient-derived tumor xenograft (PDTX) model. Blood plasma samples (n = 171) derived from a DLBCL PDTX model, including 27 humanized (HIS) PDTX, 8 HIS non-PDTX, and 21 non-HIS PDTX non-obese diabetic (NOD)-scid IL2Rgnull (NSG) mice were collected during humanization, xenografting, treatment, and sacrifice. The mice were treated with either rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), CD20-targeted human IFNα2-based AcTaferon combined with CHOP (huCD20-Fc-AFN-CHOP), or phosphate-buffered saline (PBS). RNA was extracted using the miRNeasy serum/plasma kit and sequenced on the NovaSeq 6000 platform. RNA sequencing data of the formalin-fixed paraffin-embedded (FFPE) tissue and blood plasma samples of the original patient were included. Flow cytometry was performed on immune cells isolated from whole blood, spleen, and bone marrow. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Both R-CHOP and huCD20-Fc-AFN-CHOP were able to control tumor growth in most mice. Xenograft tumor volume was strongly associated with circulating tumor RNA (ctRNA) concentration ( < 0.001, R = 0.89), as well as with the number of detected human genes ( < 0.001, R = 0.79). Abundance analysis identified tumor-specific biomarkers that were dynamically tracked during tumor growth or treatment. An 8-gene signature demonstrated high accuracy for assessing therapy response (AUC 0.92). The tumoral gene detectability in the ctRNA of the PDTX-derived plasma was associated with RNA abundance levels in the patient's tumor tissue and blood plasma ( < 0.001), confirming that tumoral gene abundance contributes to the cell-free RNA (cfRNA) profile. Decomposing the transcriptome, however, revealed high inter- and intra-mouse variability, which was lower in the HIS PDTX mice, indicating an impact of human engraftment on the stability and profile of cfRNA. Immunochemotherapy resulted in B cell depletion, and tumor clearance was reflected by a decrease in the fraction of human CD45+ cells. Lastly, bulk deconvolution provided complementary biological insights into the composition of the tumor and circulating immune system. In conclusion, the blood plasma-derived transcriptome serves as a biomarker source in a preclinical PDTX model, enables the assessment of biological pathways, and enhances the understanding of cfRNA dynamics.
在弥漫性大 B 细胞淋巴瘤（DLBCL）中，RNA 为基础的液体活检的潜力正日益得到认可，DLBCL 是最常见的非霍奇金淋巴瘤亚型。本研究探讨了在人源化 DLBCL 患者来源肿瘤异种移植（PDTX）模型中的无细胞转录组。从 DLBCL PDTX 模型中收集了血浆样本（n = 171），包括 27 个人源化（HIS）PDTX、8 个人源化非 PDTX、和 21 个非人类源化非肥胖糖尿病（NOD）-scid IL2Rgnull（NSG）小鼠，这些样本在人源化、异种移植、治疗和牺牲期间采集。用利妥昔单抗、环磷酰胺、多柔比星、长春新碱和泼尼松（R-CHOP）、CD20 靶向的人 IFNα2 为基础的 AcTaferon 联合 CHOP（huCD20-Fc-AFN-CHOP）或磷酸盐缓冲盐水（PBS）对小鼠进行治疗。使用 miRNeasy 血清/血浆试剂盒提取 RNA，并在 NovaSeq 6000 平台上进行测序。包括原始患者的福尔马林固定石蜡包埋（FFPE）组织和血浆样本的 RNA 测序数据。对全血、脾脏和骨髓中分离的免疫细胞进行流式细胞术分析。使用 Tabula Sapiens v1 基础矩阵进行批量去卷积。R-CHOP 和 huCD20-Fc-AFN-CHOP 都能够控制大多数小鼠的肿瘤生长。异种移植肿瘤体积与循环肿瘤 RNA（ctRNA）浓度密切相关（<0.001，R = 0.89），与检测到的人类基因数量也密切相关（<0.001，R = 0.79）。丰度分析确定了在肿瘤生长或治疗过程中可动态跟踪的肿瘤特异性生物标志物。一个 8 基因特征显示出对评估治疗反应的高准确性（AUC 0.92）。PDTX 衍生血浆中 ctRNA 中的肿瘤基因检测能力与患者肿瘤组织和血浆中的 RNA 丰度水平相关（<0.001），证实肿瘤基因丰度有助于细胞游离 RNA（cfRNA）谱。然而，对转录组的分解显示出高度的内和间鼠变异性，在 HIS PDTX 小鼠中变异性较低，表明人源化对 cfRNA 的稳定性和谱的影响。免疫化学疗法导致 B 细胞耗竭，人类 CD45+细胞比例的下降反映了肿瘤清除。最后，批量去卷积为肿瘤和循环免疫系统的组成提供了补充的生物学见解。总之，在临床前 PDTX 模型中，血浆衍生的转录组可作为生物标志物来源，能够评估生物途径，并增强对 cfRNA 动力学的理解。
