Introduction Due to the higher metabolic demand, malignant cells have an increased dependency on the nucleocytoplasmic trafficking of proteins, as compared to normal cells. Chromosome region maintenance protein 1 (CRM1), encoded by the XPO1 gene, is the main protein receptor which facilitates export of molecules, including tumor suppressor proteins, from the nucleus to the cytoplasm thereby making them inactive. Expression of CRM1 in tumor tissue has been shown to be an independent prognostic marker in several solid tumors and in acute myeloid leukemia; high CRM1 expression by immunohistochemistry (IHC) was associated with more aggressive disease and shorter survival. Importantly, selinexor, a first in class small molecule inhibitor of CRM1, was recently approved for the treatment of relapsed diffuse large B-cell lymphoma (DLBCL). The expression of CRM1 on tumor cells and the assessment of its prognostic impact have not been studied in patients (pts) with DLBCL or primary mediastinal B-cell lymphomas (PMBCL). Methods Paraffin embedded tumor tissue from pts with DLBCL or PMBCL treated with immunochemotherapy was assessed for CRM1 expression through IHC on tissue microarray (TMA). CRM1 anti-rabbit monoclonal antibody (Cell Signaling, catalog-no: 46249) was used at 1:100 dilution. Tumor cell grading was based on CRM1 staining in tumor cells compared to background non-malignant lymphocytes and non-malignant lymphocytes in spleen and tonsillar tissue controls. Renal cell carcinoma (RCC) was used as a positive control [known high levels of CRM1 staining (Inoue et al, J Urol, 2012)]. Two expert hematopathologists (RLK & AJW) independently scored CRM1 nuclear staining and assigned a grade of 0-3; 0 (no definitive nuclear staining, equal to background lymphocytes), 1 (dim nuclear staining), 2 (consistent nuclear staining, nuclear detail still visible behind the stain) and 3 (strong nuclear staining obscuring most nuclear detail, staining equivalent to RCC control). The average CRM1 score per case across all available cores on the TMA was calculated. Low CRM1 expression for a case was arbitrarily defined as a score of 0-2.0; high CRM1 expression was score 2.1-3.0. Scoring reliability between reviewers and between cores was assessed using intra-class correlation coefficient; score 0.75-0.90 was considered as a good scoring reliability. Event-free survival (EFS) was defined as time from diagnosis to progression, relapse, retreatment, or death. The association of CRM1 expression and risk of failing to achieve EFS at 24 months after diagnosis (EFS24) was estimated using odds ratios (OR) and 95% confidence intervals (CI) from logistic regression models, while the association of CRM1 expression with continuous EFS and overall survival (OS) was estimated using Kaplan-Meier curves and hazard ratios (HR) and 95% CI from Cox regression models. Results Tumor tissue from 282 pts was studied for CRM1 staining, including 275 pts with DLBCL and 7 pts with PMBCL. Median age of the study population was 61 years (range: 18-93) and 59% were male. The median follow-up for the entire cohort was 88.6 months. The first-line treatment regimens and baseline patient characteristics at diagnosis are outlined in Figure 1A. Of the 282 pts, 200 (71%) had high level of CRM1 expression and 82 (29%) had low CRM1 expression [only 4 (1.4%) had no or 0 staining]. Intra-class correlation coefficient to measure scoring reliability was 0.8. There was no difference in International Prognostic Index (IPI), ECOG performance score, lactate dehydrogenase or age at diagnosis among the groups with high CRM1 expression compared to low CRM1 expression (Figure 1A). The EFS24 failure was 29% for pts with low CRM1 expression while 26% in pts who had high CRM1 expression, OR=1.16, 95% CI 0.63-2.07; p=0.63. Null associations were also observed for EFS (HR=1.21, 95% CI 0.80-1.83; p=0.38) and OS (HR=1.02, 95% CI 0.61-1.69; p=0.95), (Figure 1B, C). Results were similar when adjusted for gender and IPI. Conclusion CRM1 expression by IHC on paraffin embedded tumor tissue is feasible in DLBCL and PMBCL. These data demonstrate that the CRM1 protein, the target for selinexor, is indeed expressed in the vast majority of these tumors; only 1.4% had no staining. However, CRM1 expression by IHC is not a prognostic marker for EFS24, EFS or OS. Whether CRM1 staining predicts selinexor response has not been studied but should be included in any new studies using CRM1 inhibitors. Disclosures Maurer: Nanostring: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Celgene / BMS: Research Funding. Cerhan:BMS/Celgene: Research Funding; NanoString: Research Funding. Witzig:Acerta: Research Funding; Immune Design: Research Funding; Incyte: Consultancy; MorphSys: Consultancy; Celgene: Consultancy, Research Funding; Spectrum: Consultancy; Karyopharm Therapeutics: Research Funding; AbbVie: Consultancy.

中文翻译：

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染色体区域维持蛋白 1 (CRM1) 在大细胞淋巴瘤中的表达

简介 由于更高的代谢需求，与正常细胞相比，恶性细胞对蛋白质核质运输的依赖性增加。由 XPO1 基因编码的染色体区域维持蛋白 1 (CRM1) 是主要的蛋白质受体，它促进包括肿瘤抑制蛋白在内的分子从细胞核输出到细胞质，从而使它们失活。CRM1 在肿瘤组织中的表达已被证明是几种实体瘤和急性髓性白血病的独立预后标志物。免疫组织化学 (IHC) 的高 CRM1 表达与更具侵袭性的疾病和更短的生存期相关。重要的是，selinexor 是 CRM1 的首个小分子抑制剂，最近被批准用于治疗复发性弥漫性大 B 细胞淋巴瘤 (DLBCL)。尚未在 DLBCL 或原发性纵隔 B 细胞淋巴瘤 (PMBCL) 患者 (pts) 中研究 CRM1 在肿瘤细胞上的表达及其对预后影响的评估。方法 通过组织微阵列 (TMA) 上的 IHC 评估来自经免疫化疗治疗的 DLBCL 或 PMBCL 患者的石蜡包埋肿瘤组织的 CRM1 表达。CRM1 抗兔单克隆抗体（Cell Signaling，目录号：46249）以 1:100 稀释度使用。肿瘤细胞分级基于肿瘤细胞中的 CRM1 染色与脾脏和扁桃体组织对照中的背景非恶性淋巴细胞和非恶性淋巴细胞的比较。肾细胞癌 (RCC) 用作阳性对照 [已知高水平的 CRM1 染色 (Inoue et al, J Urol, 2012)]。两位血液病理学专家 (RLK & AJW) 独立评分 CRM1 核染色并分配 0-3 级；0（无明确的核染色，与背景淋巴细胞相同）、1（核染色较暗）、2（核染色一致，染色后仍可见核细节）和 3（强核染色掩盖了大部分核细节，染色相当于 RCC 对照）。计算了 TMA 上所有可用内核的每个案例的平均 CRM1 分数。病例 CRM1 低表达任意定义为 0-2.0 分；CRM1 高表达得分为 2.1-3.0。使用类内相关系数评估审稿人之间和核心之间的评分可靠性；得分0.75-0.90被认为是良好的得分可靠性。无事件生存期（EFS）定义为从诊断到进展、复发、再治疗或死亡的时间。使用逻辑回归模型的优势比 (OR) 和 95% 置信区间 (CI) 估计 CRM1 表达与诊断后 24 个月未能达到 EFS 的风险 (EFS24) 的关联，而 CRM1 表达与连续 EFS 的关联使用 Kaplan-Meier 曲线和风险比 (HR) 以及 Cox 回归模型的 95% CI 估计总生存期 (OS)。结果 282 例肿瘤组织进行了 CRM1 染色研究，其中 275 例 DLBCL 和 7 例 PMBCL。研究人群的中位年龄为 61 岁（范围：18-93），59% 为男性。整个队列的中位随访时间为 88.6 个月。图 1A 概述了诊断时的一线治疗方案和基线患者特征。在 282 分中，200 (71%) 的 CRM1 表达水平高，82 (29%) 的 CRM1 表达水平低[只有 4 (1.4%) 没有染色或 0 染色]。衡量评分可靠性的类内相关系数为0.8。与 CRM1 低表达组相比，CRM1 高表达组与 CRM1 低表达组的国际预后指数 (IPI)、ECOG 性能评分、乳酸脱氢酶或诊断年龄没有差异（图 1A）。EFS24失败率在CRM1低表达的患者中为29%，而在CRM1高表达的患者中为26%，OR=1.16，95% CI 0.63-2.07；p=0.63。EFS (HR=1.21, 95% CI 0.80-1.83; p=0.38) 和 OS (HR=1.02, 95% CI 0.61-1.69; p=0.95) (图 1B, C) 也观察到了空关联。调整性别和 IPI 后，结果相似。结论 在DLBCL和PMBCL中IHC对石蜡包埋的肿瘤组织表达CRM1是可行的。这些数据表明，selinexor 的靶标 CRM1 蛋白确实在绝大多数这些肿瘤中表达。只有 1.4% 没有染色。然而，IHC 的 CRM1 表达不是 EFS24、EFS 或 OS 的预后标志物。尚未研究 CRM1 染色是否预测 selinexor 反应，但应包括在任何使用 CRM1 抑制剂的新研究中。Maurer：Nanostring：研究经费；Morphosys：实体董事会或咨询委员会的成员；辉瑞：实体董事会或咨询委员会的成员；风筝：实体董事会或咨询委员会的成员；Celgene / BMS：研究经费。Cerhan：BMS/Celgene：研究经费；NanoString：研究经费。Witzig：Acerta：研究经费；免疫设计：研究经费；Incyte：咨询；MorphSys：咨询；Celgene：咨询、研究经费；频谱：咨询；Karyopharm Therapeutics：研究经费；艾伯维：咨询。