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Non-coding NOTCH1 mutations in chronic lymphocytic leukemia negatively impact prognosis
American Journal of Hematology ( IF 10.1 ) Pub Date : 2022-01-06 , DOI: 10.1002/ajh.26457
Fatima Zahra Jelloul 1 , Richard K Yang 1 , Peng Wang 2 , Sofia Garces 1 , Rashmi Kanagal-Shamanna 1 , Chi Y Ok 1 , Sanam Loghavi 1 , Mark J Routbort 1 , Zhuang Zuo 1 , Cheng Cameron Yin 1 , Kristen Floyd 1 , Roland L Bassett 3 , William G Wierda 4 , Nitin Jain 4 , Philip A Thompson 4 , Rajyalakshmi Luthra 1 , Leonard Jeffrey Medeiros 1 , Keyur P Patel 1
Affiliation  

NOTCH1 mutations occur in 6–10% of cases of chronic lymphocytic leukemia (CLL) at diagnosis. NOTCH1 mutations in CLL occur with increasing prevalence in patients with disease progression and transformation to diffuse large B-cell lymphoma (i.e., Richter transformation). NOTCH1 mutations in CLL have been associated with poorer patient response to therapies and outcomes.1-3

NOTCH1 mutations were detected initially in exon 34 and were shown to result in truncation of the C-PEST regulatory protein sequence. These mutations lead to impaired degradation of the NOTCH1 intracellular domain (NCID), constitutive activation of NOTCH1 signaling, and promotion of cell survival and resistance to apoptosis. Subsequently, recurrent non-coding NOTCH1 mutations involving the 3′-UTR of NOTCH1 were described in a limited number of untreated CLL patients.4, 5 The presence of these 3’-UTR mutations causes a novel splicing event, preferentially between a cryptic donor site located in the last exon and a newly created acceptor site in the 3′-UTR of exon 34, resulting in the removal of the PEST sequence and constitutive activation of downstream NOTCH1 signaling.4

Others have reported that non-coding NOTCH1 mutations are mutually exclusive with coding mutations, and result in constitutive activation of NOTCH1 signaling attributable to aberrant NOTCH1 splicing. In addition, non-coding NOTCH1 mutations in CLL patients have been associated with shorter time-to-first treatment and shorter overall survival, similar to coding mutations, when compared to CLL patients with wild type NOTCH1.4, 5 To further address these issues, we assessed in this study a cohort of CLL patients with non-coding NOTCH1 mutations.

Peripheral blood or bone marrow samples from 1574 CLL patients who had at least 10% monotypic CD5+/CD19+ B-cells as evaluated by flow cytometric immunophenotyping analysis were subjected to next-generation sequencing using a targeted 29-gene panel. Clinical, demographic and laboratory data were extracted from the medical records using an Institutional Review Board—approved chart review protocol. Fisher's exact test was used to assess the association between NOTCH1 status and categorical variables. The Wilcoxon rank-sum test was used to assess the association between NOTCH1 mutation status and continuous variables. The Kaplan and Meier method was used to estimate the time to start first treatment from the diagnosis date.

In this cohort, we detected coding NOTCH1 mutations in 220 (14%) patients. In addition, 24 (1.5%) patients had non-coding NOTCH1 mutations and 8 (0.5%) patients had mixed coding and non-coding NOTCH1 mutations. The non-coding NOTCH1 mutations involved the 3′ untranslated region (3′-UTR) of exon 34 and were represented by c.*370A > G 21 (7.7%) and c.*377A > G 12 (4.4%).

We compared the two groups of CLL patients with coding and non-coding NOTCH1 mutations to CLL patients with wild type NOTCH1. CLL patients with coding NOTCH1 mutations were significantly more prevalent in patients with unmutated IGHV (81.4% vs. 47.3%, p < .05), ZAP70 positivity (77.5% vs. 50.3%, p < .05), and CD38 positivity (70.6% vs. 44.7%, p < .05). Similarly, patients with non-coding NOTCH1 mutations were significantly more prevalent in patients with unmutated IGHV (80.9% vs. 47.3%, p < .05), ZAP70 positivity (70.8% vs. 50.3%, p < .05), and CD38 positivity (83.3% vs. 44.7%, p < .05). Patients with coding NOTCH1 mutations were also significantly more prevalent in patients with trisomy 12 compared to patients wild type NOTCH1 (26% vs. 14.5%, p < .05), whereas there was no association between non-coding NOTCH1 mutations and trisomy 12 in this cohort (12.5% vs. 14.5%, respectively, p = .7).

We evaluated whether coding and non-coding NOTCH1 mutations in CLL patients confer an adverse prognosis compared to CLL patients with wild type NOTCH1. Given the short followup time in this cohort that included treatment naïve and relapsed/refractory patients, and to avoid the confounding effects of different therapies in previously treated patients, the survival analysis was restricted to the assessment of time-to-first treatment in treatment naïve patients (coding NOTCH1 = 134, non-coding NOTCH1 = 14, mixed NOTCH1 = 7, wild type NOTCH1 = 446).

CLL patients with non-coding NOTCH1 mutations had a significantly shorter time-to-first treatment than patients with coding NOTCH1 mutations (2.3 vs. 5.3 years, p < .05). CLL patients with non-coding NOTCH1 mutations also had a shorter time-to-first treatment than patients with wild type NOTCH1 (2.3 vs. 10.0 years, p < .05). CLL patients with coding and mixed NOTCH1 mutations also had a significantly shorter time-to-first treatment than patients with wild type NOTCH1 (5.3 vs. 10.0 and 2.1 vs. 10.0 years, respectively, p < .05). There was no difference in time-to-first treatment between CLL patients with non-coding NOTCH1 mutations versus mixed NOTCH1 mutations (2.3 vs. 2.1 years, p = .82), and between those with coding NOTCH1 mutations versus mixed NOTCH1 mutations (5.3 vs. 2.1 years, p = .06) (Figure 1).

Details are in the caption following the image
FIGURE 1
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Kaplan–Meir plots showing time-to-first treatment in our CLL cohort. CLL patients with non-coding NOTCH1 mutations had a significantly shorter time-to-first treatment than patients with coding NOTCH1 mutations and those with wild type NOTCH1 mutations (2.3 vs. 5.3 years and 2.3 vs. 10.0 years, respectively, p < 0.05). CLL patients with coding and mixed NOTCH1 mutations also had a significantly shorter time-to-first treatment than patients with wild type NOTCH1 mutations (5.3 vs. 10.0 and 2.1 vs. 10.0 years respectively, p < 0.05). There was no statistically significant difference in time-to-first treatment between CLL patients with non-coding and mixed NOTCH1 mutations (2.3 vs. 2.1 years, p = 0.82), and between those with coding and mixed NOTCH1 mutations (5.3 vs. 2.1 years, p = 0.06)

Non-coding NOTCH1 mutations have been initially identified in 2% of previously untreated patients with CLL or monoclonal B-cell lymphocytosis.4 Others have studied the clinicopathological features and prognosis of a limited number of CLL patients with non-coding NOTCH1 mutations. The presence of non-coding NOTCH1 mutations has been associated with reduced overall survival and progression-free survival.4, 5

In the current study, we show that the most common non-coding 3′-UTR NOTCH1 mutations are c.*370A > G (63.6%) followed by c.*377A > G (36.4%). These data extend and support the findings reported previously. We also show that non-coding NOTCH1 mutations are significantly more common in CLL cases with unmutated IGHV genes, CD38 positivity, and ZAP70 positivity, consistent with earlier studies.4, 5 We showed no association between non-coding NOTCH1 mutations and trisomy 12.

In earlier studies, non-coding NOTCH1 mutations were reported to be mutually exclusive with coding NOTCH1 mutations, however, the results of this study show that non-coding NOTCH1 mutations can co-occur with coding NOTCH1 mutations, in less than 1% of patients in the cohort presented.

Our data confirm the adverse prognostic impact of non-coding NOTCH1 mutations. Similar to CLL patients with coding NOTCH1 mutations, CLL patients with non-coding NOTCH1 mutations have a shorter time-to-first treatment and shorter overall survival compared to patients with wild type NOTCH1.4, 5 We also show that CLL patients with coding and non-coding NOTCH1 mutations have shorter time-to-first treatment than patients with CLL and wild type NOTCH1. In addition, our results show that patients with non-coding NOTCH1 mutations had a significantly shorter time-to-first treatment than patients with coding NOTCH1 mutations. Similarly, patients with mixed NOTCH1 mutations had a significantly shorter time-to-first treatment than patients with wild type NOTCH1 mutations. There was no statistically significant difference in time-to-first treatment between CLL patients with non-coding and mixed NOTCH1 mutations, and between those with coding and mixed NOTCH1 mutations.

Fabbri et al. were the first to identify an association between NOTCH1 mutation and refractoriness to chemotherapy as well as disease progression to large cell lymphoma (Richter transformation).1 Others have confirmed that NOTCH1 mutations are associated with a poorer clinical outcome, and they showed an association with other genetic markers, including unmutated IGHV and trisomy 12. Based on these observations, NOTCH1 mutations have been integrated into a hierarchical prognostic stratification model6 that independently predicts survival and improves CLL prognostication compared with fluorescence in situ hybridization and karyotype data.

Despite the increase in knowledge of CLL biology that has led to significant progress and transformative treatments, CLL remains an incurable disease. B-cell receptor and BCL-2 inhibitors are revolutionizing the treatment landscape of this disease, indicating that a molecularly targeted therapy can lead to improved outcomes for CLL patients. Preclinical studies targeting the NOTCH1 receptor have shown promise in T-ALL and are emerging as a promising therapeutic strategy for CLL patients. Notably, a broad number of patients may benefit from anti-NOTCH1 therapies given the importance of NOTCH1-deregulated signaling in CLL; even patients with wild type NOTCH1 mutation may benefit.

In summary, we have assessed a large cohort of CLL patients for NOTCH1 mutations. Our data show that non-coding NOTCH1 mutations carry adverse prognostic significance and can co-occur with coding NOTCH1 mutations. These findings emphasize the importance of screening for non-coding NOTCH1 mutations by analysis of the NOTCH1 3’-UTR region, to facilitate identification of CLL patients with an adverse prognosis who may benefit from targeted therapies.



中文翻译:

慢性淋巴细胞白血病的非编码 NOTCH1 突变对预后有负面影响

NOTCH1突变发生在 6-10% 的慢性淋巴细胞白血病 (CLL) 病例中。CLL 中的NOTCH1突变随着疾病进展和转化为弥漫性大 B 细胞淋巴瘤(即里氏转化)患者的患病率增加而发生。CLL 中的NOTCH1突变与患者对治疗和结果的反应较差有关。1-3

NOTCH1突变最初在外显子 34 中检测到,并显示会导致 C-PEST 调节蛋白序列的截断。这些突变导致NOTCH1细胞内结构域 (NCID) 的降解受损、NOTCH1 信号的组成型激活以及细胞存活的促进和细胞凋亡的抵抗。随后,在有限数量的未经治疗的 CLL 患者中描述了涉及NOTCH1的 3'-UTR 的复发性非编码NOTCH1突变。4, 5这些 3'-UTR 突变的存在导致新的剪接事件,优先发生在位于最后一个外显子的隐蔽供体位点和外显子 34 的 3'-UTR 中新创建的受体位点之间,导致 PEST 序列的去除和下游 NOTCH1 信号的组成性激活。4个

其他人报告说,非编码NOTCH1突变与编码突变相互排斥,并导致 NOTCH1 信号的组成性激活,这归因于异常的NOTCH1剪接。此外,与具有野生型NOTCH1的 CLL 患者相比,CLL 患者的非编码NOTCH1突变与更短的首次治疗时间和更短的总生存期相关,类似于编码突变。4, 5为了进一步解决这些问题,我们在本研究中评估了一组具有非编码NOTCH1突变的 CLL 患者。

1574 名 CLL 患者的外周血或骨髓样本通过流式细胞术免疫表型分析评估至少有 10% 的单型 CD5+/CD19+ B 细胞,并使用靶向 29 基因组进行下一代测序。使用机构审查委员会批准的图表审查协议从医疗记录中提取临床、人口统计和实验室数据。Fisher 精确检验用于评估NOTCH1状态与分类变量之间的关联。Wilcoxon 秩和检验用于评估NOTCH1突变状态与连续变量之间的关联。Kaplan 和 Meier 方法用于估计从诊断日期开始首次治疗的时间。

在该队列中,我们在 220 名 (14%) 患者中检测到编码NOTCH1突变。此外,24 名 (1.5%) 患者有非编码NOTCH1突变,8 名 (0.5%) 患者有混合编码和非编码NOTCH1突变。非编码NOTCH1突变涉及外显子 34 的 3' 非翻译区 (3'-UTR),表现为 c.*370A > G 21 (7.7%) 和 c.*377A > G 12 (4.4%)。

我们将两组具有编码和非编码NOTCH1突变的 CLL 患者与具有野生型NOTCH1的 CLL 患者进行了比较。编码NOTCH1突变的 CLL 患者在IGHV未突变(81.4% 对 47.3%,p  < .05)、ZAP70 阳性(77.5% 对 50.3%,p  < .05)和 CD38 阳性(70.6 % 对比 44.7%,p  < .05)。同样,具有非编码NOTCH1突变的患者在具有未突变IGHV(80.9% 对 47.3%,p  < .05)、ZAP70 阳性(70.8% 对 50.3%,p)的患者中更为普遍 < .05)和 CD38 阳性(83.3% 对 44.7%,p  < .05)。与野生型NOTCH1患者相比,编码NOTCH1突变的患者在 12 三体性患者中也更为普遍(26% 对 14.5%,p  < .05),而非编码NOTCH1突变与 12 三体性之间没有关联这个队列(分别为 12.5% 和 14.5%,p  = .7)。

我们评估了与具有野生型NOTCH1的 CLL 患者相比,CLL 患者的编码和非编码NOTCH1突变是否会带来不良预后。鉴于该队列中包括初治患者和复发/难治性患者的随访时间较短,并且为了避免不同疗法对既往治疗患者的混杂影响,生存分析仅限于评估初治患者的首次治疗时间患者(编码NOTCH1  = 134,非编码NOTCH1  = 14,混合型NOTCH1  = 7,野生型 NOTCH1 = 446)。

具有非编码NOTCH1突变的 CLL 患者的首次治疗时间明显短于具有编码NOTCH1突变的患者(2.3 年对 5.3 年,p  < .05)。具有非编码NOTCH1突变的 CLL 患者的首次治疗时间也比具有野生型NOTCH1的患者短(2.3 年对 10.0 年,p  < .05)。具有编码和混合NOTCH1突变的 CLL 患者的首次治疗时间也明显短于野生型NOTCH1患者(分别为 5.3 年与 10.0 年和 2.1 年与 10.0 年,p < .05)。具有非编码NOTCH1突变与混合NOTCH1突变的 CLL 患者(2.3 年与 2.1 年,p  = .82)以及具有编码NOTCH1突变与混合NOTCH1突变(5.3对比 2.1 年,p  = .06)(图 1)。

详细信息在图片后面的标题中
图1
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Kaplan-Meir 图显示了我们 CLL 队列中的首次治疗时间。具有非编码 NOTCH1 突变的 CLL 患者的首次治疗时间明显短于具有编码 NOTCH1 突变的患者和具有野生型 NOTCH1 突变的患者(分别为 2.3 年与 5.3 年和 2.3 年与 10.0 年,p < 0.05  ) . 具有编码和混合 NOTCH1 突变的 CLL 患者的首次治疗时间也明显短于具有野生型 NOTCH1 突变的患者(分别为 5.3 年与 10.0 年和 2.1 年与 10.0 年,p < 0.05  。具有非编码和混合 NOTCH1 突变的 CLL 患者的首次治疗时间无统计学显着差异(2.3 年与 2.1 年,p = 0.82),以及编码和混合 NOTCH1 突变之间(5.3 年与 2.1 年,p  = 0.06)

非编码NOTCH1突变最初已在 2% 的先前未治疗的 CLL 或单克隆 B 细胞淋巴细胞增多症患者中被发现。4其他人研究了数量有限的具有非编码NOTCH1突变的 CLL 患者的临床病理学特征和预后。非编码NOTCH1突变的存在与总生存期和无进展生存期的缩短有关。4, 5

在当前的研究中,我们发现最常见的非编码 3'-UTR NOTCH1突变是 c.*370A > G (63.6%),其次是 c.*377A > G (36.4%)。这些数据扩展并支持了之前报告的发现。我们还表明,非编码NOTCH1突变在 IGHV 基因未突变、CD38 阳性和 ZAP70 阳性的 CLL 病例中更为常见,这与早期研究一致。4, 5我们发现非编码NOTCH1突变与 12 三体之间没有关联。

在早期的研究中,据报道非编码NOTCH1突变与编码NOTCH1突变相互排斥,然而,这项研究的结果表明,非编码NOTCH1突变可以与编码NOTCH1突变同时发生,发生在不到 1% 的患者中在提出的队列中。

我们的数据证实了非编码NOTCH1突变的不良预后影响。与具有编码NOTCH1突变的 CLL 患者类似,具有非编码NOTCH1突变的 CLL 患者与具有野生型NOTCH1的患者相比具有更短的首次治疗时间和更短的总生存期。4, 5我们还表明,与 CLL 和野生型NOTCH1患者相比,具有编码和非编码NOTCH1突变的 CLL 患者的首次治疗时间更短。此外,我们的结果表明,与编码NOTCH1的患者相比,具有非编码NOTCH1突变的患者的首次治疗时间明显更短突变。同样,具有混合NOTCH1突变的患者的首次治疗时间明显短于具有野生型NOTCH1突变的患者。具有非编码和混合NOTCH1突变的 CLL 患者以及具有编码和混合NOTCH1突变的 CLL 患者之间的首次治疗时间没有统计学显着差异。

法布里等。是第一个确定NOTCH1突变与化疗难治性以及疾病进展为大细胞淋巴瘤(里氏转化)之间关联的人。1其他人已证实NOTCH1突变与较差的临床结果相关,并且它们显示与其他遗传标记物相关,包括未突变的IGHV和三体 12。基于这些观察结果,NOTCH1突变已被整合到分级预后分层模型6中与荧光原位杂交和核型数据相比,独立预测生存并改善 CLL 预后。

尽管对 CLL 生物学知识的增加导致了重大进展和变革性治疗,但 CLL 仍然是一种无法治愈的疾病。B 细胞受体和 BCL-2 抑制剂正在彻底改变这种疾病的治疗前景,表明分子靶向治疗可以改善 CLL 患者的预后。针对 NOTCH1 受体的临床前研究在 T-ALL 中显示出前景,并正在成为 CLL 患者的一种有前途的治疗策略。值得注意的是,考虑到 CLL 中 NOTCH1 失调信号的重要性,大量患者可能会受益于抗 NOTCH1 疗法;即使是具有野生型NOTCH1突变的患者也可能受益。

总之,我们评估了一大群 CLL 患者的NOTCH1突变。我们的数据表明,非编码NOTCH1突变具有不良预后意义,并且可能与编码NOTCH1突变同时发生。这些发现强调了通过分析NOTCH1 3'-UTR 区域来筛查非编码NOTCH1突变的重要性,以促进识别可能受益于靶向治疗的预后不良的 CLL 患者。

更新日期:2022-02-10
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