第一部分 MYD88基因L265P和非L265P在弥漫大B细胞淋巴瘤中的临床特点和预后意义

目的：本研究旨在探讨MYD88基因L265P（MYD88^L265P）和非L265P（MYD88^other）在弥漫大B细胞淋巴瘤（diffuse large B-cell lymphoma，DLBCL）中的临床特点和预后意义。

方法：本研究收集了中国医学科学院肿瘤医院2007年2月6日至2022年5月20日期间的MYD88变异的DLBCL患者。对MYD88^L265P和MYD88^other患者的临床病理特征和治疗结局进行比较分析。

结果：本研究筛选了475例DLBCL患者，最终纳入132例MYD88变异患者，其中78例为MYD88^L265P，54例为MYD88^other。MYD88^L265P在非生发中心（non-germinal center B-cell，non-GCB）亚型中较MYD88^other更常见（83%对比60%，P=0.004）。此外，MYD88^L265P较MYD88^other有更高的睾丸/中枢神经系统受累比例（31% 对比6%，P<0.001）、PIM1变异率（71%对比39%，P＜0.001）和PIM1超变异率（28%对比11%，P=0.018）。与MYD88^L265P相比，MYD88^other更多见于晚期（60% 对比42%，P=0.044）、结外受累病灶数目≥2个（45% 对比28%，P=0.044）、乳酸脱氢酶升高（55%对比35%，P=0.033）、CD10表达阳性（36%对比16%，P=0.009）、BCL6易位（20%对比8%，P=0.033）和NOTCH通路基因变异（24% 对比13%，P=0.040）患者。在non-GCB亚型中，MYD88^other患者的无进展生存（progression free survival，PFS）显著劣于MYD88^L265P患者（P=0.010）。

结论：MYD88^L265P和MYD88^other变异的DLBCL患者可能是具有不同临床和分子特征的两个亚组。MYD88^other患者的预后并不优于MYD88^L265P患者，甚至在non-GCB亚型中，MYD88^other患者的PFS比MYD88^L265P患者更差。

第二部分 MYD88和CD79B基因变异在弥漫大B细胞淋巴瘤中的临床意义

目的：本研究旨在探讨MYD88-CD79B共变异（MYD88-CD79B^co-mut）、MYD88/CD79B单独变异（MYD88/CD79B^single-mut）和MYD88-CD79B共野生型（MYD88-CD79B^co-wt）弥漫大B细胞淋巴瘤（diffuse large B-cell lymphoma，DLBCL）患者的临床、基因特征以及治疗结局差异。

方法：本研究筛选了中国医学科学院肿瘤医院和六个公共数据库队列中接受R-CHOP或R-CHOP样方案治疗的DLBCL患者，对不同MYD88和CD79B变异状态患者的临床、基因特征以及预后进行分析，并通过基因集富集分析（gene set enrichment analysis，GSEA）和xCell方法探讨潜在的分子机制。

结果：本研究共纳入 2696 例 DLBCL 患者，包括来自中国医学科学院肿瘤医院队列的 295 例和来自公共数据库队列的 2401 例。MYD88变异和野生型患者的无进展生存（progression free survival，PFS）和总生存（overall survival，OS）并无显著差异。MYD88-CD79B^co-mut患者的OS显著劣于MYD88/CD79B^single-mut和MYD88-CD79B^co-wt患者。对于MCD亚型患者，MYD88-CD79B^co-mut患者的PFS和OS与MYD88/CD79B^single-mut或MYD88-CD79B^co-wt患者无显著差异。然而，在非MCD亚型中，MYD88-CD79B^co-mut患者的OS显著劣于MYD88/CD79B^single-mut或MYD88-CD79B^co-wt患者，而MYD88/CD79B^single-mut与MYD88-CD79B^co-wt患者的OS无显著差异[中位OS：68.8 （95% CI：22.0～无法评估）个月 对比 无法评估 （95% CI：112.0～无法评估）对比 177.7 （95% CI：159.0～无法评估）个月；MYD88-CD79B^co-mut 对比 MYD88/CD79B^single-mut：P=0.020；MYD88-CD79B^co-mut 对比MYD88-CD79B^co-wt：P=0.030；MYD88/CD79B^single-mut 对比 MYD88-CD79B^co-wt：P=0.330]。对于MYD88-CD79B^co-mut患者，MCD和非MCD亚型患者的PFS和OS无显著差异。对于MYD88-CD79B^co-mut患者，PIM1变异患者的PFS显著优于PIM1野生型患者[中位PFS：8.34 （95% CI：5.56～无法评估）个月对比 43.8 （95% CI：26.4～无法评估）个月，P=0.022]。GSEA结果显示，PIM1变异患者淋巴细胞介导的免疫和干扰素反应被激活；血管生成和上皮间质转化被抑制。xCell结果显示，PIM1变异患者的自然杀伤T细胞和浆细胞样树突状细胞比例更高；成纤维细胞比例和基质评分更低。

结论：在MCD亚型中，MYD88-CD79B^co-mut患者与MYD88/CD79B^single-mut或MYD88-CD79B^co-wt患者的PFS和OS无显著差异；而在非MCD亚型中，MYD88-CD79B^co-mut患者的OS显著劣于MYD88/CD79B^single-mut或MYD88-CD79B^co-wt患者。无论是在MCD亚型还是非MCD亚型，MYD88-CD79B^co-mut患者的PFS和OS无显著差异。在MYD88-CD79B^co-mut患者中，PIM1^mut患者显示出更好的PFS，这可能与复杂的免疫调控和肿瘤微环境变化有关。

第三部分 数字空间组学探讨弥漫大B细胞淋巴瘤浆细胞样表型异质性和治疗策略

目的：本研究旨在探讨弥漫大B细胞淋巴瘤（diffuse large B-cell lymphoma，DLBCL）中浆细胞样表型相关的异质性和治疗策略。

方法：本研究收集了2018年4月1日至2023年9月30日在中国医学科学院肿瘤医院诊治的DLBCL患者，基于CD20、HLA-DRA和PRDM1标记物，利用数字空间组学探讨了DLBCL中浆细胞样表型相关的异质性和预后价值。通过基因富集分析（gene set enrichment analysis，GSEA）探讨了不同DLBCL亚群细胞和浆母细胞淋巴瘤（plasmablastic lymphoma，PBL）转录组的相似性。同时，构建了随机森林预后模型用于预测伴浆细胞样表型的DLBCL，并探究其是否可以从硼替佐米治疗中获益。

结果：本研究纳入了81例DLBCL患者，识别出了4种DLBCL细胞亚群：CD20（+）HLA-DRA（+）PRDM1（-）、CD20（+）HLA-DRA（+）PRDM1（+）、CD20（+）HLA-DRA（-）PRDM1（-）和CD20（+）HLA-DRA（-）PRDM1（+）。CD20（+）HLA-DRA（+）PRDM1（-）亚群缺乏浆细胞样表型特征，与较好的预后相关，其余亚群则代表不同的浆细胞样表型细胞，与较差的预后相关。具有浆细胞样表型特征的DLBCL表现为增殖能力降低、免疫功能受损和肿瘤微环境重塑能力增强。与浆细胞样表型细胞缺乏型DLBCL（DLBCL plasma cell-like phenotype deficient，DLBCL^PCPD）患者相比，浆细胞样表型细胞为主型DLBCL（DLBCL plasma cell-like phenotype predominant，DLBCL^PCPP）患者的Ki-67≥85%的比例较低（29.6%对比53.9%，P=0.020）。两组患者对一线治疗的客观缓解率无区别（92.6%对比92.6%，P=1.000），但对于一线治疗获得缓解的患者，DLBCL^PCPP患者12个月内出现疾病进展比例显著高于DLBCL^PCPD患者（25.9% 对比3.7%，P=0.024），DLBCL^PCPP患者B2M基因变异的发生率较DLBCL^PCPD患者高（22.2% 对比3.7%，P=0.051）。GSEA分析揭示了伴有浆细胞样表型特征的DLBCL和PBL具有相似性。由13个浆细胞样表型相关基因构建的随机森林预后模型预测的高浆细胞样表型指数（plasma cell-like phenotype signature，PCPS）的患者的无进展生存（progression free survival，PFS）显著劣于低PCPS的患者，高PCPS的患者接受R-CHOP联合硼替佐米（RB-CHOP）方案治疗的PFS显著优于接受R-CHOP方案治疗的患者。相反，低PCPS的患者接受R-CHOP方案治疗的PFS显著优于接受RB-CHOP方案治疗的患者。

结论：本研究揭示了DLBCL中浆细胞样表型相关的异质性，伴有浆细胞样表型特征的DLBCL预后不佳。由浆细胞样表型相关基因构建的随机森林预后模型可以预测伴有浆细胞样表型特征的DLBCL，这部分患者可以从R-CHOP联合硼替佐米方案治疗中获益。

Part 1: Clinical Features and Prognostic Significance of MYD88 L265P and Non-L265P Variations in Diffuse Large B-Cell Lymphoma

Purpose: This study aims to investigate the clinical and prognosis differences between diffuse large B-cell lymphoma (DLBCL) patients with MYD88 L265P (MYD88^L265P) and non-L265P (MYD88^other) variations.

Methods: DLBCL patients with MYD88 variations were collected from the Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College from February 6, 2007 to May 20, 2022. Clinicopathological parameters and treatment outcomes between MYD88^L265P and MYD88^other were investigated.

Results: A total of 132 patients with MYD88 variations from a cohort of 475 DLBCL patients were included, among which, 78 were MYD88^L265P, while 54 were MYD88^other. MYD88^L265P was more common in non-germinal center B-cell (non-GCB) subtype than MYD88^other (83% vs. 60%, P=0.004). Besides, MYD88^L265P was significantly related to higher proportion of testicle/central nervous system involvement (31% vs. 6%, P<0.001), PIM1 variation (71% vs. 39%, P<0.001), and PIM1 hypervariability (28% vs. 11%, P=0.018), compared with MYD88^other. Compared with MYD88^L265P, MYD88^other were more likely to have higher percentage of advanced stage (60% vs. 42%, P=0.044), extranodal site number≥2 (45% vs. 28%, P=0.044), elevated lactate dehydrogenase (55% vs. 35%, P=0.033), positive CD10 expression (36% vs. 16%, P=0.009), BCL6 translocation (20% vs. 8%, P=0.033), and NOTCH pathway gene alteration (24% vs. 13%, P=0.040). In non-GCB DLBCL subtype, patients with MYD88^other were significantly associated with worse progression free survival (PFS) than those with MYD88^L265P (P=0.010).

Conclusions: DLBCL patients with MYD88^L265P and MYD88^other are likely to be two subgroups with different clinical and molecular characteristics. The survival of patients with MYD88^other is not superior than those with MYD88^L265P, even poorer when focusing on the non-GCB subtype.

Part 2: Clinical Significance of MYD88 and CD79B Variations in Diffuse Large B-Cell Lymphoma

Purpose: This study aims to investigate the distinctions in clinical and genetic characteristics, as well as treatment outcomes among diffuse large B-cell lymphoma (DLBCL) patients with MYD88-CD79B^co-mut, MYD88/CD79B^single-mut, and MYD88-CD79B^co-wt.

Methods: This study screened DLBCL patients who were treated with R-CHOP or R-CHOP-like regimens at Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CHCAMS) and six publicly available database cohorts. The clinical and genetic characteristics, as well as the prognosis of patients with different MYD88 and CD79B variations were analyzed. Additionally, potential molecular mechanisms were explored through gene set enrichment analysis (GSEA) and the xCell method.

Results: This study enrolled a total of 2,696 DLBCL patients, including 295 from the cohort of CHCAMS and 2,401 from publicly available database cohorts. No significant differences in progression free survival (PFS) or overall survival (OS) were observed between MYD88^mut and MYD88^wt patients. However, MYD88-CD79B^co-mut patients exhibited significantly inferior OS compared to those with MYD88/CD79B^single-mut or MYD88-CD79B^co-wt patients. Among the MCD subtype, no significant differences in PFS or OS were observed between MYD88-CD79B^co-mut patients and those with MYD88/CD79B^single-mut or MYD88-CD79B^co-wt. In contrast, within the non-MCD subtype, MYD88-CD79B^co-mut patients demonstrated significantly worse OS compared to those with MYD88/CD79B^single-mut or MYD88-CD79B^co-wt, while no significant OS difference was observed between MYD88/CD79B^single-mut and MYD88-CD79B^co-wt patients (median OS: 68.8 [95% CI: 22.0-NE] vs. NE [95% CI: 112.0-NE] months vs. 177.7 [95% CI: 159.0-NE]; MYD88-CD79B^co-mut vs. MYD88/CD79B^single-mut: P=0.020; MYD88-CD79B^co-mut vs. MYD88-CD79B^co-wt: P=0.030; MYD88/CD79B^single-mut vs. MYD88-CD79B^co-wt: P=0.330). For MYD88-CD79B^co-mut patients, no significant differences in PFS or OS were observed between the MCD and non-MCD subtypes. Within the MYD88-CD79B^co-mut group, patients with PIM1^mut had better PFS than PIM1^wt (median PFS: 8.34 [95% CI: 5.56-NE] vs. 43.8 [95% CI: 26.4-NE] months, P=0.022). GSEA analysis demonstrated activated lymphocyte mediated immunity and interferon response, alongside suppressed angiogenesis and epithelial-mesenchymal transition in PIM1^mut patients. xCell analysis demonstrated a higher proportion of natural killer T cells and plasmacytoid dendritic cells, coupled with lower fibroblast and stromal score in PIM1^mut patients.

Conclusions: In the MCD subtype, patients with MYD88-CD79B^co-mut showed comparable PFS and OS compared to MYD88/CD79B^single-mut or MYD88-CD79B^co-wt, while in non-MCD subtype, they exhibited significantly inferior OS. There was no significant disparity in PFS and OS of MYD88-CD79B^co-mut between the MCD and non-MCD subtypes. The presence of PIM1^mut within the MYD88-CD79B^co-mut group correlated with better PFS, which may result from an intricate interplay of immune processes and tumor microenvironment alterations.

Part 3: Spatially-Resolved Transcriptomics Reveals Plasma Cell-like Phenotypic Heterogeneity and Therapeutic Implication in Diffuse Large B-cell Lymphoma

Purpose: This study aimed to investigate the heterogeneity and therapeutic implication associated with plasma cell-like phenotype in diffuse large B-cell lymphoma (DLBCL).

Methods: DLBCL patients diagnosed and treated at the Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College between April 1, 2018, and September 30, 2023, were collected. Digital spatial profiling based on CD20, HLA-DRA, and PRDM1 markers was utilized to explore plasma cell-like phenotype-related heterogeneity and its prognostic significance. Gene set enrichment analysis (GSEA) was performed to assess transcriptional similarities between distinct DLBCL subpopulations and plasmablastic lymphoma (PBL). Additionally, a random forest prognostic model was developed to predict DLBCL with plasma cell-like phenotype and to evaluate its potential therapeutic benefit from bortezomib treatment.

Results: A total of 81 DLBCL patients were included, and four DLBCL subclusters were identified: CD20(+)HLA-DRA(+)PRDM1(−), CD20(+)HLA-DRA(+)PRDM1(+), CD20(+)HLA-DRA(−)PRDM1(−), and CD20(+)HLA-DRA(−)PRDM1(+). The CD20(+)HLA-DRA(+)PRDM1(−) subcluster lacked plasma cell-like phenotype features and was associated with favorable outcomes, while the remaining subpopulations represented varying plasma cell-like phenotypes and correlated with inferior prognosis. DLBCL with plasma cell-like phenotype features exhibited reduced proliferative capacity, impaired immune function, and enhanced tumor microenvironment remodeling. Compared to DLBCL plasma cell-like phenotype deficient (DLBCL^PCPD), those with DLBCL plasma cell-like phenotype predominant (DLBCL^PCPP) showed a lower proportion of Ki-67≥85% (29.6% vs. 53.9%, P=0.020). Although objective response rates to first-line therapy were comparable between groups (92.6% vs. 92.6%, P=1.000), DLBCL^PCPP patients experienced higher rates of disease progression within 12 months after response (25.9% vs. 3.7%, P=0.024) and a higher incidence of B2M variation (22.2% vs. 3.7%, P=0.051) compared to DLBCL^PCPD patients. GSEA demonstrated that DLBCL with plasma cell-like phenotype features exhibits transcriptional similarities to PBL. The random forest model constructed using 13 plasma cell-like phenotype-associated genes predicted that patients with a high plasma cell-like phenotype signature (PCPS) had significantly worse progression free survival (PFS) compared to those with low PCPS. Notably, high PCPS patients treated with R-CHOP plus bortezomib (RB-CHOP) achieved superior PFS compared to those receiving R-CHOP alone, whereas low PCPS patients exhibited better PFS with R-CHOP alone versus RB-CHOP treatment.

Conclusions: This study revealed the heterogeneity associated to plasma cell-like phenotype in DLBCL, with such differentiation correlating with unfavorable prognosis. The random forest model, incorporating plasma cell-like phenotype-related genes, identified DLBCL patients with plasma cell-like phenotype features who may benefit from RB-CHOP treatment.