Additional sex combs-like (ASXL) genes consist of three family members which are involved in epigenetic regulation and the three genes are: ASXL1, ASXL2, and ASXL3. Mutations in the family of ASXL have been identified in increased frequencies in myeloid neoplasms (MNs).¹ While ASXL1 acts as a haploinsufficient tumor suppressor, mice models have shown that ASXL2 gene plays an essential role in normal hematopoiesis and its deletion in hematopoietic stem cell leads to myeloid malignancies. Compared with the incidence of ASXL1 mutations, ASXL2 mutations are less frequent in myeloid malignancies.² Little is known about its clinical implications in patients with MNs.

In this analysis, we identified the frequency, clinical characteristics, and mutational co-occurrences of ASXL2 across MNs, including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN).

Among 5746 patients with MNs (AML (N = 2473), MDS (N = 1477), MPN (N = 1299), MDS/MPN overlap neoplasms (N = 497)) presented to our institution between 2017 through 2022, we identified 50 (0.9%) with ASXL2 mutations (Figure S1, Consort Diagram; Table S1, Next generation sequencing 81-gene Panel, Table S2, ASXL2 mutations). Baseline characteristics of the study patients are shown in Table 1.

Of the 50 patients, eight (6%), 20 (0.8%), 11 (0.7%), seven (0.5%), and four (0.8%) had CBF AML, non-CBF AML, MDS, MPN, and MDS/MPN, respectively. In the entire cohort, RAS (22%), SRSF2 (20%), TET2 (14%), and DNMT3A (14%), SF3B1 (12%) and RUNX1 mutation (12%) were the most commonly co-mutated genes (Figure S2, Bar plot of co-mutated genes). Six patients had coexisting ASXL1 mutations: three in AML, two in MPN, and one in MDS.

Out of 2473 AML patients, 28 (1%) had ASXL2 mutation. The incidence of ASXL2 mutation was the most common in patients with CBF AML, 6% (8 of 142) versus 0.8% (20 of 2331) in non-CBF AML. In addition, among CBF AML patients, ASXL2 mutations were detected only in patients with t(8;21) RUNX1/RUNX1T1 (AML1/ETO), 15% (8/53). Consistent with previously published literature, none of the patients with inv(16)/t(16;16) CBFB/MYH11 had ASXL2 mutations (0 of 89). The most common co-mutations were SRSF2 (18%), IDH2 (18%), RUNX1 (14%), SF3B1 (14%), KIT (14%), and DNMT3A (14%).

Of the 28 patients with AML, 16 were newly diagnosed (ND) and 12 had relapsed/refractory (R/R) disease. Among ND patients, all eight patients with CBF AML were treated with FLAG (fludarabine, cytarabine, and granulocyte colony-stimulating factor) based regimens and achieved complete remission with overall survival (OS) not reached. Because ASXL2 mutations were confined to the t(8;21) patients, we analyzed OS comparing t(8;21) with ASXL2 mutation versus t(8;21) with no ASXL2 mutations. The 3-year OS rates were not statistically different in t(8;21) patients with or without ASXL2 mutations, 87% versus 57%, respectively (p = .38) and median OS was not reached (Figure S3). Of the eight ND patients with non-CBF AML (two patients received intensive Rx, six patients received low-intensity Rx) had a median OS of 12.5 months.

In order to compare the clinical impact of ASXL2 with ASXL1 mutations, we identified ASXL1 mutated patients from our institution database between 2017 and 2022. In ND patients with t(8;21), patients with ASXL2 or ASXL1 mutated AML had similarly favorable OS, (median not reached for both, p = .65) (Figure S4A). In non-CBF AML patients, OS rates were similarly poor with ASXL2 or ASXL1 (median 12.5 vs. 14 months, p = .85) (Figure S4B).

Of the 12 R/R AML patients, all had non-CBF AML, the response rates after salvage chemotherapy and median OS rates were 67% and 6.2 months, respectively. There was no prior NGS data to evaluate whether ASXL2 emerged at relapse versus present at the time of first diagnosis.

Eleven of 1477 (0.7%) MDS patients were identified to have ASXL2 mutation. Of the 11 patients, eight had ND disease and three had R/R disease. For the eight ND patients, most of the patients (63%) had lower risk disease based on the International Prognosis Scoring System (two low, five intermediate, one high). Most common co-mutations were SRSF2 (27%),TP53 (27%), one patient had co-occurring ASXL1 mutation. Of these eight patients, six received HMA based therapy (four had response −2 CR and 2 mCR- and two no response), one lenalidomide (achieved response). The OS was poor in patients with ND MDS with a median of 12.9 months only. There was no statistically significant OS difference between ASXL2 mutated ND MDS with historical ASXL1 mutated MDS (median OS 13 vs. 23 months, p = .29) (Figure S5).

Out of seven patients with MPN and ASXL2 mutation, five patients had primary MF, one patient had post-essential thrombocythemia MF, and one patient had post-Polycythemia-Vera MF. Five patients (71%) harbored JAK2 V617F mutation, two (29%) MPL mutation, and three (43%) ASXL1 mutation. Six out of seven patients had intermediate risk based on Dynamic International Prognostic Scoring System score. The 3-year OS rate was 100%.

Out of 497 patients with MDS/MPN neoplasm, four (0.8%) had ASXL2 mutation. Two patients had ND chronic myelomonocytic leukemia, one patient with ND MDS/MPN unclassified, and one patient with R/R MDS/MPN with fibrosis.

Our study reveals a distinct pattern regarding the occurrence of ASXL2 mutations in MNs, distinguishing them from the more prevalent ASXL1 mutations. ASXL1 mutations range from 5% to 40% in MNs, whereas ASXL2 mutations are notably less common in MNs, ranging from 0.5% to 1%.² Specifically, our data confirms the rarity of ASXL2 mutations, with the exception of cases involving CBF AML. Notably, these mutations were exclusively observed in patients harboring the t(8;21)/RUNX1-RUNX1T1 (AML1-ETO) fusion, constituting approximately 15% of the analyzed cohort, while they were absent in patients with inv(16)/t(16;16) CBFB/MYH11. Importantly, our findings demonstrate that ASXL2 mutations did not exhibit an association with a poor prognosis in CBF AML. However, in the context of non-CBF AML and MDS, like ASXL1 mutations, ASXL2 mutations emerged as significant indicators of an unfavorable prognosis.

Previous studies have reported a higher prevalence of ASXL2 mutations in CBF AML (RUNX1/RUNX1T1).³ Our study supports these findings and finds no evidence linking ASXL2 mutations to an increased risk of relapse or decreased survival in CBF AML. Notably, a study by Abdul-Wahab et al. delves further into the pathogenesis of ASXL2 mutations in CBF AML, unveiling ASXL2's crucial role in hematopoietic stem cell self-renewal and its contribution to RUNX1/RUNX1T1-induced leukemogenesis.¹ Furthermore, the target genes of ASXL2 exhibit overlap with those of RUNX1 and RUNX1/RUNX1T1, underscoring ASXL2's pivotal role as a regulatory factor in hematopoiesis, mediating transcriptional effects that promote leukemogenesis driven by RUNX1/RUNXT1.

Our study revealed a negative prognostic impact on non-CBF AML or MDS patients with ASXL2, similar to the poor prognosis associated with ASXL1 mutations. When comparing OS among patients with ASXL1 and ASXL2 mutated MDS or AML, ASXL2 mutations displayed a similarly unfavorable prognosis. The European Leukemia Network's AML risk criteria already designates ASXL1 mutations as adverse risk,⁴ and our research supports extending this classification to include ASXL2 mutations in MDS and non-CBF AML. However, further studies are necessary to provide additional evidence and validate these observations.

Contrary to the prevailing literature indicating mutual exclusivity between ASXL1 and ASXL2 mutations,^{1, 5} our study revealed instances of co-occurrence between ASXL1 and ASXL2 mutations. Specifically, we identified six patients with coexisting ASXL1 mutations: three in AML, two in MPN, and one in MDS. However, it is important to note that the incidence of this co-occurrence was exceptionally rare within our large study cohort, comprising only six out of 5746 patients with myeloid malignancies.

Within our cohort of patients with MPNs, we observed that all seven individuals with ASXL2 mutations were diagnosed with MF, while none had ET or PV. Surprisingly, the 3-year OS rate for patients harboring ASXL2 mutations was 100%. This suggests that ASXL2 mutations may not have a negative prognostic impact in MF. When comparing our findings to a study conducted by Wang et al., which focused on ASXL1-mutated myelofibrosis patients with a 3-year OS rate of 70%, it becomes evident that ASXL2 mutations might not have the same adverse effects as ASXL1 mutations in myelofibrosis patients.⁶ However, it is important to note that our study had a small sample size, and therefore, it is not possible to draw a definitive conclusion based on these results.

In summary, our research findings indicate that ASXL2 mutations, although rare, can be detected across various myeloid malignancies, including AML, MDS, and MPNs. However, the frequency of ASXL2 mutations is generally low in myeloid malignancies, except in CBF AML, where they are limited to patients with the t(8;21)/RUNX1/RUNX1T1 (AML1-ETO) fusion, without significant prognostic implications. Conversely, in non-CBF AML or MDS, the presence of ASXL2 mutations was associated with an unfavorable prognosis. These results highlight the diverse impact of ASXL2 mutations in different subtypes of myeloid malignancies, emphasizing the need for further investigation into their underlying mechanisms and clinical implications.

额外的性梳样 ( ASXL ) 基因由参与表观遗传调控的三个家族成员组成，这三个基因是：ASXL1、ASXL2和ASXL3。已发现 ASXL 家族的突变在髓系肿瘤 (MN) 中的频率增加。¹虽然ASXL1是一种单倍体不足的肿瘤抑制因子，但小鼠模型表明ASXL2基因在正常造血中发挥着重要作用，其在造血干细胞中的缺失会导致骨髓恶性肿瘤。与ASXL1突变的发生率相比，ASXL2突变在骨髓恶性肿瘤中的发生率较低。²关于其对 MN 患者的临床影响知之甚少。

在这项分析中，我们确定了ASXL2在 MN 中的频率、临床特征和突变共现情况，包括急性髓系白血病 (AML)、骨髓增生异常综合征 (MDS) 和骨髓增殖性肿瘤 (MPN)。

在 2017 年至 2022 年间向我们机构就诊的5746 名 MN 患者（AML（N  = 2473）、MDS（N  = 1477）、MPN（N  = 1299）、MDS/MPN 重叠肿瘤（N  = 497））中，我们确定了 50 (0.9%) 具有ASXL2突变（图 S1，配对图；表 S1，下一代测序 81 基因组，表 S2，ASXL2突变）。研究患者的基线特征如表 1 所示。

在 50 名患者中，8 名 (6%)、20 名 (0.8%)、11 名 (0.7%)、7 名 (0.5%) 和 4 名 (0.8%) 患有 CBF AML、非 CBF AML、MDS、MPN 和 MDS /MPN，分别。在整个队列中，RAS（22%）、SRSF2（20%）、TET2（14%）、DNMT3A（14%）、SF3B1（12%）和RUNX1突变（12%）是最常见的共突变基因（图S2，共突变基因的条形图）。 6 名患者同时存在ASXL1突变：3 名患者患有 AML，2 名患者患有 MPN，1 名患者患有 MDS。

在 2473 名 AML 患者中，28 名 (1%) 患有ASXL2突变。ASXL2突变的发生率在 CBF AML 患者中最常见，为 6%（142 例中的 8 例），而非 CBF AML 患者中 ASXL2 突变的发生率为 0.8%（2331 例中的 20 例）。此外，在 CBF AML 患者中，仅在 t(8;21) RUNX1/RUNX1T1 ( AML1/ETO )患者中检测到ASXL2突变，占 15% (8/53)。与之前发表的文献一致，inv(16)/t(16;16) CBFB/MYH11患者均未出现 ASXL2 突变（89 例中有 0 例）。最常见的共突变是SRSF2 (18%)、IDH2 (18%)、RUNX1 (14%)、SF3B1 (14%)、KIT (14%) 和DNMT3A (14%)。

在 28 名 AML 患者中，16 名是新诊断的 (ND)，12 名是复发/难治性 (R/R) 疾病。在 ND 患者中，所有 8 名 CBF AML 患者均接受基于 FLAG（氟达拉滨、阿糖胞苷和粒细胞集落刺激因子）的治疗方案，并获得完全缓解，但未达到总生存期 (OS)。由于ASXL2突变仅限于 t(8;21) 患者，因此我们分析了 OS，将具有ASXL2突变的 t(8;21) 与无ASXL2突变的 t(8;21) 进行比较。在有或没有ASXL2突变的t(8;21) 患者中，3 年 OS 率没有统计学差异，分别为 87% 和 57% ( p  = .38)，并且未达到中位 OS（图 S3）。在 8 名患有非 CBF AML 的 ND 患者（两名患者接受强化 Rx，六名患者接受低强度 Rx）中，中位 OS 为 12.5 个月。

为了比较ASXL2与ASXL 1 突变的临床影响，我们从我们的机构数据库中确定了2017 年至 2022 年间ASXL1突变的患者。在 t(8;21) 的 ND 患者中，ASXL2或ASXL 1 突变的 AML 患者也有类似的有利结果OS（两者均未达到中位数，p  = .65）（图 S4A）。在非 CBF AML 患者中， ASXL2或ASXL1 的OS 率同样较差（中位 12.5 个月与 14 个月，p  = 0.85）（图 S4B）。

12 例 R/R AML 患者均为非 CBF AML，挽救性化疗后的缓解率和中位 OS 率分别为 67% 和 6.2 个月。之前没有 NGS 数据来评估ASXL2是否在复发时出现与首次诊断时出现。

1477 名 MDS 患者中有 11 名 (0.7%) 被确定患有ASXL2突变。在 11 名患者中，8 名患有 ND 疾病，3 名患有 R/R 疾病。根据国际预后评分系统，对于 8 名 ND 患者，大多数患者 (63%) 的疾病风险较低（两名低，五名中，一名高）。最常见的共突变是SRSF2 (27%)、TP53 (27%)，一名患者同时发生ASXL 1 突变。在这 8 名患者中，6 名接受基于 HMA 的治疗（4 名有反应 -2 CR 和 2 mCR-，2 名无反应），1 名来那度胺（已达到反应）。 ND MDS 患者的 OS 较差，中位生存期仅为 12.9 个月。ASXL2突变 ND MDS 与历史ASXL 1 突变 MDS之间没有统计学上显着的 OS 差异（中位 OS 13 个月与 23 个月， p  = 0.29）（图 S5）。

在七名患有 MPN 和ASXL2突变的患者中，五名患者患有原发性 MF，一名患者患有原发性血小板增多症后 MF，一名患者患有红细胞增多症后 MF。 5 名患者 (71%) 携带JAK2 V617F 突变、2 名 (29%) 名MPL突变和 3 名 (43%)名ASXL1突变。根据动态国际预后评分系统评分，七名患者中有六名具有中等风险。 3年总生存率为100%。

在 497 名 MDS/MPN 肿瘤患者中，4 名 (0.8%) 患有ASXL2突变。两名患者患有 ND 慢性粒单核细胞白血病，一名患者患有未分类的 ND MDS/MPN，一名患者患有 R/R MDS/MPN 并伴有纤维化。

我们的研究揭示了 MN 中ASXL2突变发生的独特模式，将其与更常见的ASXL1突变区分开来。ASXL1突变在 MN 中的范围为 5% 至 40%，而ASXL2突变在 MN 中明显不常见，范围为 0.5% 至 1%。²具体而言，我们的数据证实了ASXL2突变的罕见性，涉及 CBF AML 的病例除外。值得注意的是，这些突变仅在携带 t(8;21)/RUNX1-RUNX1T1 (AML1-ETO) 融合的患者中观察到，约占分析队列的 15%，而在 inv(16)/t 患者中则不存在这些突变。 (16;16) CBFB/MYH11。重要的是，我们的研究结果表明ASXL2突变与 CBF AML 的不良预后无关。然而，在非 CBF AML 和 MDS 的背景下，与ASXL1突变一样，ASXL2突变成为不良预后的重要指标。

先前的研究报道CBF AML (RUNX1/RUNX1T1) 中ASXL2突变的患病率较高。 ³我们的研究支持这些发现，并且没有发现任何证据表明ASXL2突变与 CBF AML 复发风险增加或生存率降低有关。值得注意的是，Abdul-Wahab 等人的一项研究。进一步深入研究了CBF AML 中ASXL2突变的发病机制，揭示了ASXL2在造血干细胞自我更新中的关键作用及其对 RUNX1/RUNX1T1 诱导的白血病发生的贡献。¹此外， ASXL2的靶基因与 RUNX1 和 RUNX1/RUNX1T1 的靶基因重叠，强调了ASXL2作为造血调节因子的关键作用，介导转录效应，促进 RUNX1/RUNXT1 驱动的白血病发生。

我们的研究揭示了ASXL2对非 CBF AML 或 MDS 患者的负面预后影响，类似于ASXL1突变相关的不良预后。当比较ASXL1和ASXL2突变 MDS 或 AML患者的 OS 时， ASXL2突变显示出类似的不利预后。欧洲白血病网络的 AML 风险标准已将ASXL1突变指定为不良风险⁴，我们的研究支持扩展此分类以包括MDS 和非 CBF AML 中的ASXL2突变。然而，需要进一步的研究来提供更多证据并验证这些观察结果。

与表明ASXL1和ASXL2突变之间相互排斥的主流文献相反，^{1, 5}我们的研究揭示了ASXL1和ASXL2突变同时发生的情况。具体来说，我们确定了六名同时存在ASXL1突变的患者：三名患有 AML，两名患有 MPN，一名患有 MDS。然而，值得注意的是，这种同时发生的情况在我们的大型研究队列中极为罕见，在 5746 名骨髓恶性肿瘤患者中仅占 6 名。

在我们的 MPN 患者队列中，我们观察到所有 7 名携带ASXL2突变的患者均被诊断为 MF，而没有人患有 ET 或 PV。令人惊讶的是，携带ASXL2突变的患者的 3 年 OS 率为100%。这表明ASXL2突变可能不会对 MF 产生负面预后影响。将我们的研究结果与 Wang 等人进行的一项研究进行比较时，该研究重点关注ASXL1突变的骨髓纤维化患者，其 3 年 OS 率为 70%，很明显，ASXL2突变可能不会产生与ASXL1突变相同的副作用。骨髓纤维化患者。⁶然而，值得注意的是，我们的研究样本量较小，因此不可能根据这些结果得出明确的结论。

总之，我们的研究结果表明，ASXL2突变虽然罕见，但可以在各种骨髓恶性肿瘤中检测到，包括 AML、MDS 和 MPN。然而， ASXL2突变在骨髓恶性肿瘤中的频率通常较低，但 CBF AML 除外，这种突变仅限于 t(8;21)/RUNX1/RUNX1T1 (AML1-ETO) 融合的患者，没有显着的预后影响。相反，在非 CBF AML 或 MDS 中，ASXL2突变的存在与不良预后相关。这些结果强调了ASXL2突变对不同亚型的骨髓恶性肿瘤的不同影响，强调需要进一步研究其潜在机制和临床意义。