BCR::ABL1-negative myeloproliferative neoplasms (MPNs) can evolve to secondary acute myeloid leukemia (sAML) or blast-phase (BP) MPN, a very severe condition with lack of effective therapy.¹ Leukemic transformation (LT) of MPNs displays a variable incidence according to MPN phenotype: 9%–13% in primary myelofibrosis (PMF), 3%–7% in polycythemia vera (PV), and 1%–4% in essential thrombocythemia (ET).¹ Here, we investigated the mutational landscape, copy number variations (CNVs), and uniparental disomy (UPD) events in BP-MPN cases that were diagnosed over a 6-year period of monitoring in three different hematology units (Fundeni Clinical Institute, Coltea Hospital and Emergency University Hospitals, Bucharest, Romania) and the patterns of clonal evolution in a subset of patients with available paired chronic phase (CP)-BP DNA samples.

The study was approved by the local ethics committee (No. 136/06.02.2017 rev. no.131/18.01.2019) and was performed in conformity with the Declaration of Helsinki. A written informed consent was provided by each patient at collection of samples that were referred to Stefan S Nicolau Institute of Virology, Romania, for molecular analysis. Clinical, morphological, and immunophenotypic data were provided from the medical records for all recruited patients. Peripheral blood or bone marrow (BM) mononuclear cells were isolated and processed to obtain various cell fractions. CD3+ T cells were used as reference for germline mutations. Molecular testing for MPN-driver mutations, targeted next-generation sequencing (NGS), whole-exome sequencing (WES), single nucleotide polymorphism (SNP) microarray analysis, and multiplex ligation-dependent probe amplification (MLPA) were performed as described by manufacturers (see Supplemental file; Data S1 for a complete description of methods).

A total of 33 patients (median age, 63 years; 57.6% males) were diagnosed with BP-MPN between 2017 and 2023, in the above-mentioned centers, including 20 post-PMF (60.4%), and 13 post-ET/PV (39.4%) cases (Table S1). A prior stage of secondary myelofibrosis was confirmed by BM biopsy in 8 out of 13 post-ET/PV AML (61.5%) patients. According to morphologic and immunophenotypic data, sAML cases were classified as AML with myelodysplasia-related changes (n = 4, 12.1%) and AML, not otherwise specified (n = 29, 87.9%), as follows: AML with minimal differentiation (n = 6, 18.2%), AML without maturation (n = 12, 36.4%), acute myelomonocytic leukemia (n = 6, 18.2%), acute monocytic leukemia (n = 1, 3%), pure erythroid leukemia (n = 1, 3%), and acute megakaryoblastic leukemia (n = 3, 9.1%). Concerning the MPN drivers detected at CP, 60.6% of patients carried JAK2 V617F mutation, 21.2% harbored calreticulin (CALR) mutations (5 type1/type 1-like, 2 type2/type-2 like), and 18.2% were classified as triple-negative (TN-MPNs). We report a median age at BP diagnosis significantly lower in CALR-mutated and TN-MPNs compared with JAK2-mutated ones (54, 56, vs. 67.5 years, p = .014), and a median time from MPN diagnosis to BP conversion significantly shorter in TN-MPNs compared with CALR and JAK2-mutated ones (1, 3 vs. 7 years, p = .0476) (Table S1). Overall, the median survival was 3 months (range, 1–54 months) without significant differences between the three groups of patients (Table S1).

By targeted NGS/WES testing of matched blast and CD3+ DNA samples, a total number of 62 somatic mutations apart from MPN-driver mutations were detected. The most frequent anomalies were represented by epigenetic mutations (72.8%), followed by TP53 mutations (33.3%), mutations of signaling molecules (24.2%, significantly more frequent in JAK2 V617F-negative groups, p = .005), transcriptional regulators (21.2%), and mRNA splicing factors (15.2%) (Table S1). The frequencies of individual mutations are shown in Figure 1A. Prevalence of prior exposure to ruxolitinib versus hydroxyurea was similar among the ASXL1, EZH2, and/or NRAS-mutated patients (Figure 1B). Importantly, a high occurrence of multiple CNVs (≥3) (42.4%) was detected by SNP array and MLPA. Del(17p) including TP53 gene was the most frequent anomaly, followed by del(5q), del(7q) including EZH2 gene, del(20q), gain of chromosome 21, and other less frequent anomalies. Also, UPD events involving JAK2 (9p), TP53 (17p), CBL (11q), and NRAS (1p) were observed in the analyzed cohort (Figure 1C). TP53 mutations co-occurred with multiple CNVs in 10 cases (30.3%) and with del(17p) in 9 cases (27.3%), being absent in CALR-mutated patients. All 5 detected RUNX1 mutations were identified as comutations to JAK2 V617F. The co-occurrence of JAK2-TP53 was found in 6 patients at BP (18.2%) (Figure 1D). Detailed information about demographics, genomic aberrations, therapy in CP and BP, clinical outcome, and survival for each BP-MPN case is displayed in Table S2.

Thirteen paired CP-BP DNA samples were available for combined genomic analysis. In CP-MPN patients, the median number of aberrations was 3 (maximum 8). Eleven patients carried MPN-driver mutations: seven JAK2 V617F, three CALR type1/type 1-like, and one with both CALR type 2 and low-burden JAK2 V617F. All of them had at least one additional genetic lesion, the most frequent being TP53 heterozygous mutations, del(5q), and ASXL1 mutations. In BP-MPN, the median number of aberrations increased to 6 (maximum 9). JAK2 V617F was lost in three BP-MPNs, while CALR mutations were present at both MPN and LT in all cases. Loss of heterozygosity (LOH) events affecting TP53, NF1 and SUZ12 (17q), and CBL, as well as RUNX1 mutations, EZH2 mutations, del(7q), and 21q gain were detected exclusively in BP (Figure 1E).

Based on the VAFs of the somatic mutations identified by targeted NGS/WES and also on the information provided by SNP array/MLPA in paired CP-BP DNA samples, four patterns of clonal evolution were inferred of which three involved the MPN phenotypic driver mutation clone(s), while one involved the non-MPN-mutated clone (Figure 1F): (i) a linear pattern, in which the leukemic clone developed from the driver mutation clone through acquisition of one or more genomic alterations; (ii) acquisition of a MPN-driver mutation in HSC carrying a pre-existing mutation that was also present in the leukemic clone; (iii) a branched subclonal evolution in which several subclones derived from the driver mutation clone coexisted and one of them, by acquiring novel genomic aberrations, gained growth advantage and promoted leukemogenesis; and (iv) acquisition of genomic aberrations leading to proliferation advantage and transformation in a non-MPN clone in patients where two independent clones or subclones existed at CP, one of them carrying the MPN-driver mutation. Additionally, a linear pattern was observed in TN-MPNs that progressed to BP, in which the leukemic clone developed from a TP53 clone or subclone present at CP that predominantly gained del(17p) among multiple CNVs. While these patterns have been described before, the precise acquired mutations in the patterns are of interest and show a diversity of pathways leading to LT. For example, signaling mutations in NRAS, CBL, NF1, and PTPN11 occurred at BP. It is also interesting that competition between mutated JAK2 and CALR clones has been detected, with one leading to LT, as well as competition between different TP53-mutated clones. Individual profiles of CP-BP genomic aberrations in 12 patients are described in Figure S1. Clonal evolution of patient 13# was not presented as it was previously published.²

Our results point to a dominance of epigenetic mutations in BP-MPN patients (72.8% of cases). When considering also CNVs that affect chromatin modifiers and DNA methylation, 84.8% of patients exhibited epigenetics alterations. Particularly, anomalies of EZH2 gene were relatively frequent in JAK2 V617F-mutated and TN-MPN patients. In serial DNA samples, EZH2 anomalies were detected exclusively in BP. In agreement, in a single-cell analysis of clonal evolution in paired CP-BP samples,³ EZH2 was recurrently mutated or affected by CNVs in transition to BP.

In BP-MPN patients carrying TP53 alterations, epigenetic modifications were observed in 75% of cases. In paired-sample analysis, the same TP53 mutations found in BP were already present in CP-MPNs in a heterozygous state, as previously reported.⁴ Chronic inflammation was recently demonstrated to play an important role in clonal evolution of TP53-mutant MPNs by suppressing unmutated HSCs, and therefore conferring a fitness advantage to TP53-positive HSCs and progenitors.⁵ As expected, in our BP-MPN patients, multi-hit TP53 were dominant, mostly represented by a single TP53 missense mutations accompanied by del(17p) (Supplemental file, Figure S2). Among TP53 missense mutations, structural mutants were more frequent than contact ones (Table S3).

Gain of chromosome 21 was observed in one type-2 CALR and two JAK2 V617F-positive patients (trisomy 21 in one patient and a duplication of 21q11.2-q22.3 in two cases, both associated with multiple CNVs and one with TP53 mutations). This is in agreement with a recent study⁶ that identified an amplified region of chromosome 21 as a recurrent event in BP-MPN patients, being accompanied by chromothripsis and displaying a very aggressive phenotype.⁶ This genetic alteration leads to an upregulation of DYRK1A gene (21q.22) that in functional studies promoted genomic instability and increased JAK/STAT signaling.⁶

Compared with JAK2-mutated MPNs that exhibited complex patterns of clonal evolution, giving rise to either JAK2-mutated AML or JAK2 wild-type AML, CALR-mutated PMF patients evolved to BP by acquiring novel genomic alterations on the top of MPN-driver mutation. Paired-sample analysis in two PMF patients carrying type 1 CALR mutation that developed sAML with hyperleukocytosis resembling BP chronic myeloid leukemia detected the presence of LOH events leading to a hyperactive RAS/tyrosine kinase receptor signaling, respectively. At time of LT, one patient displayed NF1 biallelic inactivation, while another carried a homozygous CBL mutation due to an UPD11q. Regarding mutated CALR proteins and granulocytosis, it has been shown that besides activating thrombopoietin receptor, which explains the ET and PMF phenotypes, these mutated proteins can also activate granulocyte colony-stimulating factor receptor (GCSFR).⁷ Further studies are required to assess if extreme granulocytosis was related to activation of GCSFR or to other cytokines associated with inflammation in PMF, for example, granulocyte-macrophage colony-stimulating factor.

The profile of genomic aberrations in AML post-TN-PMF was characterized by a high-incidence of biallelic TP53 alterations (66.6%) and multiple CNVs (66.6%). The spectrum of anomalies together with a shorter median time from diagnosis to BP revealed common features with fibrotic myelodysplastic neoplasms (f-MDS). The distinction between TN-PMF and f-MDS is often challenging, both entities being associated with a higher risk of LT at 3 years from diagnosis.⁸

As a limitation of our study, inherently related to the small number of patients included in the study, we could not assess the impact on survival of the detected genomic alterations in a multivariable analysis.

To conclude, we highlight the overwhelming prominence of epigenetic alterations and importance of serial evaluation of clonal evolution in MPNs for early prediction of disease transformation. We also describe a diversity of signaling mutations contributing to transformation.

BCR：：ABL1 阴性骨髓增生性肿瘤 （MPN） 可演变为继发性急性髓性白血病 （sAML） 或急变期 （BP） MPN，这是一种非常严重的疾病，缺乏有效的治疗。¹ MPN 的白血病转化 （LT） 根据 MPN 表型显示可变的发生率：原发性骨髓纤维化 （PMF） 为 9%-13%，真性红细胞增多症 （PV） 为 3%-7%，原发性血小板增多症 （ET） 为 1%-4%。¹ 在这里，我们调查了在三个不同的血液学单位（Fundeni 临床研究所、Coltea 医院和罗马尼亚布加勒斯特急诊大学医院）诊断的 BP-MPN 病例的突变景观、拷贝数变异 （CNV） 和单亲二倍体 （UPD） 事件，以及具有可用配对慢性期 （CP）-BP DNA 样本的患者子集的克隆进化模式。

该研究得到了当地伦理委员会的批准（第 136/06.02.2017 修订版第 131/18.01.2019），并按照赫尔辛基宣言进行。每位患者在采集样本时提供书面知情同意书，这些样本被转介到罗马尼亚 Stefan S Nicolau 病毒学研究所进行分子分析。从所有招募患者的病历中提供临床、形态学和免疫表型数据。分离外周血或骨髓 （BM） 单核细胞并加工以获得各种细胞组分。CD3 + T 细胞用作种系突变的参考。按照制造商的说明进行 MPN 驱动突变的分子检测、靶向下一代测序 （NGS）、全外显子组测序 （WES）、单核苷酸多态性 （SNP） 微阵列分析和多重连接依赖性探针扩增 （MLPA）（见补充文件;数据 S1 获取方法的完整说明）。

2017 年至 2023 年期间，上述中心共有 33 名患者 （中位年龄 63 岁;57.6% 为男性） 被诊断为 BP-MPN，包括 20 例 PMF 后 （60.4%） 和 13 例 ET/PV 后 （39.4%） 病例（表 S1）。13 例 ET/PV AML 后患者中有 8 例 （61.5%） 通过 BM 活检证实了继发性骨髓纤维化的既往阶段。根据形态学和免疫表型数据，sAML 病例分为骨髓增生异常相关变化的 AML （n = 4， 12.1%） 和未另行说明的 AML （n = 29， 87.9%），如下所示：最小分化的 AML （n = 6， 18.2%）、未成熟的 AML （n = 12， 36.4%）、急性粒单核细胞白血病 （n = 6， 18.2%）、急性单核细胞白血病 （n = 1， 3%）、纯红细胞白血病 （n= 1， 3%）和急性巨核细胞白血病 （n = 3， 9.1%）。关于 CP 检测到的 MPN 驱动因素，60.6% 的患者携带 JAK2 V617F 突变，21.2% 携带钙网蛋白 （CALR） 突变 （5 个 1 型/1 型样，2 个 2 型/2 型样），18.2% 被归类为三阴性 （TN-MPN）。我们报告说，与 JAK2 突变相比，CALR 突变和 TN-MPN 的 BP 诊断中位年龄显着降低（54、56 对 67.5 岁，p = .014），并且与 CALR 和 JAK2 突变相比，TN-MPN 从 MPN 诊断到 BP 转换的中位时间显著缩短（1、3 对 7 岁，p= .0476）（表 S1）。总体而言，中位生存期为 3 个月 （范围，1-54 个月），三组患者之间没有显着差异 （表 S1）。

通过对匹配的原始细胞和 CD3+ DNA 样本进行靶向 NGS/WES 检测，除 MPN 驱动突变外，共检测到 62 个体细胞突变。最常见的异常是表观遗传突变 （72.8%），其次是 TP53 突变 （33.3%）、信号分子突变 （24.2%，在 JAK2 V617F 阴性组中明显更频繁，p = .005）、转录调节因子 （21.2%） 和 mRNA 剪接因子 （15.2%） （表 S1）。单个突变的频率如图 1A 所示。ASXL1 、 EZH2 和/或 NRAS 突变患者先前暴露于 ruxolitinib 与羟基脲的患病率相似（图 1B）。重要的是，SNP 阵列和 MLPA 检测到多个 CNV （≥3） （42.4%） 的高发生率。包括 TP53 基因的 Del （17p） 是最常见的异常，其次是 del（5q）、包括 EZH2 基因的 del（7q）、del（20q）、21 号染色体的获得和其他不太常见的异常。此外，在分析的队列中观察到涉及 JAK2 （9p）、TP53 （17p）、CBL （11q） 和 NRAS （1p） 的 UPD 事件（图 1C）。TP53 突变在 10 例 （30.3%） 中与多个 CNV 同时发生，在 9 例 （27.3%） 中与 del（17p） 同时发生，在 CALR 突变患者中不存在。所有检测到的 5 个 RUNX1 突变均被鉴定为 JAK2 V617F 的共突变。在 6 例 BP 患者 （18.2%） 中发现 JAK2-TP53 的共存 （图 1D）。表 S2 显示了有关每个 BP-MPN 病例的人口统计学、基因组畸变、CP 和 BP 治疗、临床结果和生存率的详细信息。

13 个配对 CP-BP DNA 样品可用于联合基因组分析。在 CP-MPN 患者中，畸变的中位数为 3 （最多 8 个）。11 例患者携带 MPN 驱动基因突变： 7 例 JAK2 V617F，3 例 CALR 1 型/1 型样突变，1 例同时具有 CALR 2 型和低负荷 JAK2 V617F。他们都至少有 1 个额外的遗传病灶，最常见的是 TP53 杂合突变、 del（5q） 和 ASXL1 突变。在 BP-MPN 中，像差的中位数增加到 6 （最多 9 个）。JAK2V617F 在 3 个 BP-MPN 中丢失，而在所有病例中，MPN 和 LT 均存在 CALR 突变。影响 TP53 、 NF1 和 SUZ12 （17q） 和 CBL 的杂合性缺失 （LOH） 事件，以及 RUNX1 突变、EZH2 突变、 del（7q） 和 21q 增益仅在 BP 中检测到（图 1E）。

根据靶向 NGS/WES 鉴定的体细胞突变的 VAF 以及 SNP 阵列/MLPA 在配对 CP-BP DNA 样本中提供的信息，推断出四种克隆进化模式，其中三种涉及 MPN 表型驱动突变克隆，而一种涉及非 MPN 突变克隆（图 1F）：（i） 线性模式， 其中白血病克隆通过获得一个或多个基因组改变从驱动突变克隆发育而来;（ii） 在 HSC 中获得 MPN 驱动突变，携带也存在于白血病克隆中的预先存在的突变;（iii） 一种分支亚克隆进化，其中来自驱动突变克隆的几个亚克隆共存，其中一个通过获得新的基因组畸变，获得了生长优势并促进了白血病发生;（iv） 在 CP 处存在两个独立克隆或亚克隆，其中一个携带 MPN 驱动突变的患者中，获得基因组畸变，导致非 MPN 克隆的增殖优势和转化。此外，在进展为 BP 的 TN-MPN 中观察到线性模式，其中白血病克隆从 CP 中存在的 TP53 克隆或亚克隆发展而来，该克隆主要在多个 CNV 中获得 del（17p）。虽然这些模式之前已经描述过，但这些模式中精确的获得性突变很有趣，并显示了导致 LT 的多种途径。例如，NRAS 、 CBL 、 NF1 和 PTPN11 的信号突变发生在 BP 处。 同样有趣的是，已经检测到突变的 JAK2 和 CALR 克隆之间的竞争，其中一个导致 LT，以及不同 TP53 突变克隆之间的竞争。图 S1 描述了 12 名患者的 CP-BP 基因组畸变的个体概况。患者 13# 的克隆进化没有像以前发表的那样呈现。^{阿拉伯数字}

我们的结果表明，BP-MPN 患者 （72.8% 的病例） 表观遗传突变占主导地位。当还考虑影响染色质修饰剂和 DNA 甲基化的 CNV 时，84.8% 的患者表现出表观遗传学改变。特别是 EZH2 基因异常在 JAK2 V617F 突变和 TN-MPN 患者中相对常见。在连续 DNA 样本中，仅在 BP 中检测到 EZH2 异常。一致，在配对 CP-BP 样本克隆进化的单细胞分析中，³EZH2 在转化为 BP 的过程中反复突变或受 CNV 影响。

在携带 TP53 改变的 BP-MPN 患者中，在 75% 的病例中观察到表观遗传修饰。在配对样本分析中，正如之前报道的那样，在 BP 中发现的相同 TP53 突变已经存在于杂合状态的 CP-MPN 中。⁴ 最近证明，慢性炎症通过抑制未突变的 HSC 在 TP53 突变 MPN 的克隆进化中发挥重要作用，从而赋予 TP53 阳性 HSC 和祖细胞适应性优势。⁵ 正如预期的那样，在我们的 BP-MPN 患者中，多发 TP53 占主导地位，主要由单个 TP53 错义突变伴有 del（17p） 表示（补充文件，图 S2）。在 TP53 错义突变中，结构突变体比接触突变更频繁 （表 S3）。

在 1 例 2 型 CALR 和 2 例 JAK2 V617F 阳性患者中观察到 21 号染色体的增加 （1 例患者 21 三体，2 例 21q11.2-q22.3 重复，均与多个 CNV 相关，1 例与 TP53 突变相关）。这与最近的一项研究⁶ 一致，该研究将 21 号染色体的扩增区域确定为 BP-MPN 患者的复发事件，伴有染色体裂解并表现出非常具有侵袭性的表型。⁶ 这种遗传改变导致 DYRK1A 基因 （21q.22） 上调，在功能研究中促进基因组不稳定性并增加 JAK/STAT 信号传导。⁶

与表现出复杂克隆进化模式的 JAK2 突变 MPN 相比，导致 JAK2 突变的 AML 或 JAK2 野生型 AML，CALR 突变的 PMF 患者通过在 MPN 驱动突变的基础上获得新的基因组改变而进化为 BP。对两名携带 1 型 CALR 突变的 PMF 患者进行配对样本分析，这些患者发展为类似于 BP 慢性粒细胞白血病的高白细胞增多症 sAML，分别检测到导致 RAS/酪氨酸激酶受体信号过度活跃的 LOH 事件的存在。在 LT 时，一名患者表现出 NF1 双等位基因失活，而另一名患者由于 UPD11q 而携带纯合 CBL 突变。关于突变的 CALR 蛋白和粒细胞增多症，已经表明，除了激活解释 ET 和 PMF 表型的血小板生成素受体外，这些突变蛋白还可以激活粒细胞集落刺激因子受体 （GCSFR）。⁷ 需要进一步的研究来评估极度粒细胞增多症是否与 GCSFR 的激活或与 PMF 炎症相关的其他细胞因子有关，例如粒细胞-巨噬细胞集落刺激因子。

TN-PMF 后 AML 基因组畸变的特征是双等位基因 TP53 改变 （66.6%） 和多个 CNV （66.6%） 的高发生率。异常范围以及从诊断到 BP 的中位时间较短揭示了纤维化骨髓增生异常肿瘤 （f-MDS） 的共同特征。TN-PMF 和 f-MDS 之间的区别通常具有挑战性，这两种疾病在诊断后 3 年与 LT 风险较高相关。⁸

作为我们研究的局限性，本质上与研究中纳入的患者数量少有关，我们无法在多变量分析中评估检测到的基因组改变对生存的影响。

总而言之，我们强调了表观遗传改变的压倒性突出以及 MPN 中克隆进化的连续评估对于疾病转化的早期预测的重要性。我们还描述了多种有助于转化的信号转导突变。