In addition to its adverse effect on survival and leukemic transformation, in both polycythemia vera (PV) and essential thrombocythemia (ET), leukocytosis has been implicated as a risk factor for thrombosis, in both of these myeloproliferative neoplasms (MPNs).¹ Recent studies have underscored anatomical and biologic differences as well as common traits between arterial (AT) and venous (VTE) thrombosis. Consequently, different sets of risk factors for AT versus VTE in PV versus ET, have been promoted; in PV, these include prior AT event, hypertension, and hyperlipidemia, for AT, and prior venous event, older age, major hemorrhage, and leukocytosis, for VTE^{2, 3}; in ET, the corresponding variables for AT include prior AT event, age > 60 years, cardiovascular risk factors, presence of JAK2V617F, and leukocytosis and, for VTE, male gender.^4-6 In the current study, we sought to clarify the individual prognostic contribution of absolute neutrophil (ANC) and monocyte (AMC) counts for the association between leukocytosis and thrombosis in ET and PV.

The current study included 487 patients with PV (n = 138) or ET (n = 349), recruited from the Mayo Clinic MPNs database, based on the availability of information on ANC and AMC, within 1 year of diagnosis. An external cohort of 424 patients with PV, from the University of Florence, Italy, was used to validate the observations from the Mayo Clinic PV cohort. Conventional criteria were used for diagnosis⁷ and definitions of major vascular events.⁶ Most patients were managed according to standard practice that included phlebotomy and aspirin therapy in low-risk patients and the addition of cytoreductive therapy, often in the form of hydroxyurea, in high-risk patients. Statistical analyses were performed using JMP Pro 14.0.0 software package, SAS Institute, Cary, NC. Multivariable analyses included previously established risk factors for AT or venous thrombosis.

The Mayo Clinic PV patient cohort included 138 patients (median age 62 years, range 20–94; females 50%); presenting median (range) values were 17.9 g/dL (16.1–24) for hemoglobin, 11.8 × 10 (9)/L (2.7–65.8) for leukocyte count, and 434 × 10 (9)/L (44–1679) for platelet count; 57% of patients presented with leukocytosis >11 × 10 (9)/L; palpable splenomegaly was present in 37 (27%) patients; abnormal karyotype was documented in 17% of patients. Conventional risk category was high in 86 (62%) patients and low in 52 (38%). Median follow-up was 11 years (range 0.03–36.7). There were 22 (16%) documented venous events at diagnosis and 20 (15%) after diagnosis and 28 (20%) AT events at diagnosis and 15 (11%) after diagnosis.

Neither ANC (p = .85) nor AMC (p = .14) correlated with AT at or prior to diagnosis, advanced age was the only other parameter with significant association in this regard (p = .01). Similarly, AT-free survival was not affected by either ANC (0.31) or AMC (0.27); advanced age was again the only other parameter tested that showed significance (p < .001) while prior AT events showed borderline significance (p = .05) that was no longer apparent during multivariable analysis. By contrast, VTE at or prior to diagnosis correlated with both ANC (p = .048) and AMC (0.06; borderline significance) while no other parameter tested, including age (0.7) or sex (p = .16), showed significant association. In univariate analysis, VTE-free survival was negatively affected by higher ANC (p = .001) and higher AMC (p = .036); ANC (p = .02) but not AMC (p = .71) retained significance during multivariable analysis that included age (p = .68), sex (p = .18) and prior venous events (p = .68). The adverse effect of higher ANC on VTE-free survival was most apparent in high (p = .004) versus low (p = .14) risk disease.

The external validation cohort of PV patients was recruited from the University of Florence, Italy: median age 62 years (range 18–92); females 40%; presenting median (range) values of 17.8 g/dL (16.1–24) for hemoglobin, 9.8 × 10 (9)/L (4.5–26.9) for leukocyte count, and 461 × 10 (9)/L (154–1352) for platelet count. Median follow-up was 8 years (range 0.5–30.8); 46 (10.8%) venous events were documented at diagnosis and 33 (7.8%) after diagnosis; 58 (13.7%) AT events were documented at diagnosis and 39 (9.2%) after diagnosis. As was the case with the Mayo Clinic cohort, neither ANC nor AMC correlated with AT events at/prior to or after diagnosis. By contrast, VTE-free survival was negatively affected by higher ANC (p = .003) and prior venous events (p = .01), but not by AMC (p = .7), age (p = .4) or gender (p = .3); ANC (p = .01) and prior venous events (p = .04) retained significance during multivariable analysis. In the Florence cohort, the adverse effect of higher ANC on VTE-free survival was more prominent in low-risk diseases (p = .02), although a trend was also apparent in high-risk diseases (p = .07). On the other hand, unlike the case with the Mayo Clinic cohort, VTE at or prior to PV diagnosis did not correlate with either ANC (p = .4) or AMC (p = .2). The optimal ANC cutoff level for VTE-free survival risk stratification, determined by receiver operating characteristic (ROC) plot analysis, was 22 × 10 (9)/L for the Mayo Clinic cohort (Figure 1A) and 10 × 10 (9)/L for the Florence cohort (Figure 1B).

The ET patient cohort included 349 patients (median age 57 years, range 18–89; females 61%): 46% JAK2, 34% CALR, 16% triple-negative, and 4% MPL mutated; international prognostic system for thrombosis in ET (IPSET) risk category was high 24%, intermediate 41%, and low 35%; presenting median (range) values were 13.8 g/dL (11.1–16.4) for hemoglobin, 8.2 × 10 (9)/L (3.2–29.8) for leukocyte count, and 859 × 10 (9)/L (451–3460) for platelet count; palpable splenomegaly was present in 48 (14%) patients and cardiovascular risk factors in 56%; median follow-up was 10 years (range 0–47). There were 38 (11%) documented venous events at diagnosis and 31 (9%) after diagnosis and 42 (12%) AT events at diagnosis and 64 (18%) after diagnosis.

Neither ANC (p = .8) nor AMC (p = .38) correlated with AT at or prior to diagnosis; instead, parameters of significance included advanced age (p = .003), JAK2 mutation (p = .01), male sex (p = .03) and, of borderline significance, cardiovascular risk factors (p = .07). Similarly, AT-free survival was not affected by either ANC (0.1) or AMC (0.1); in multivariable analysis, significant variables included advanced age (p = .03), prior AT event (p = .04), and cardiovascular risk factors (p = .04). As was the case with PV, VTE at or prior to diagnosis correlated with both AMC (0.003) and ANC (p = .06), the latter with borderline significance; other parameters with borderline significance in this regard included JAK2 mutation (p = .06), advanced age (p = .08), and female sex (p = .08). During multivariable analysis, VTE-free survival was affected by advanced age only (p = .001).

Observations from the current study flag neutrophils, and possibly monocytes, as potential contributors to venous thrombosis in PV, and possibly in ET as well. This would be consistent with emerging evidence on the role of neutrophils in venous thrombosis where the underlying mechanisms might also involve platelets, monocytes, the endothelium, procoagulant proteins, and neutrophil extracellular traps (NETs), with resulting interactive state of hypercoagulability and immune-mediate inflammation.^{8, 9} The persistent VTE risk in both high and low-risk PV patients presenting with increased ANC, as suggested by their inferior VTE-free survival, raises questions on the adequacy of current treatment in preventing venous thrombosis. A similar notation was made in a previously published controlled study of patients with ET where the risk of VTE was higher in patients receiving hydroxyurea versus anagrelide, both in combination with aspirin therapy.¹⁰ In two separate collaborative work involving patients with PV, we also showed adverse impact on VTE-free survival from higher JAK2V617F allele burden¹¹ and neutrophil-lymphocyte ratio,¹² which further supports the detrimental role of pro-thrombotic clonal burden associated with neutrophilia and associated inflammatory state, marked by lower lymphocyte count. Taken together, the three parallel studies,^{11, 12} including the current work, identify risk factors for VTE that are different than those for AT. Whether or not such information is actionable, in the context of current therapy, cannot be surmised at the present time, but signifies the need for venous thrombosis-focused analysis of future prospective and retrospective studies.

除了对生存和白血病转化的不利影响外，在真性红细胞增多症 (PV) 和原发性血小板增多症 (ET) 中，白细胞增多已被认为是这两种骨髓增生性肿瘤 (MPN) 中血栓形成的危险因素。¹最近的研究强调了动脉 (AT) 和静脉 (VTE) 血栓形成之间的解剖学和生物学差异以及共同特征。因此，促进了 PV 与 ET 中 AT 与 VTE 的不同风险因素；在 PV 中，这些包括先前的 AT 事件、高血压和高脂血症（对于 AT），以及先前的静脉事件、年龄较大、大出血和白细胞增多（对于 VTE ^2、3）；在 ET 中，AT 的相应变量包括先前的 AT 事件、年龄 > 60 岁、心血管危险因素、存在JAK2 V617F，白细胞增多，对于 VTE，男性。^4-6在目前的研究中，我们试图阐明绝对中性粒细胞 (ANC) 和单核细胞 (AMC) 计数对 ET 和 PV 中白细胞增多和血栓形成之间关联的个体预后贡献。

目前的研究包括 487 名 PV ( n  = 138) 或 ET ( n  = 349) 患者，这些患者在诊断后 1 年内从 Mayo Clinic MPNs 数据库中招募，基于 ANC 和 AMC 信息的可用性。来自意大利佛罗伦萨大学的 424 名 PV 患者的外部队列用于验证 Mayo Clinic PV 队列的观察结果。常规标准用于诊断⁷和主要血管事件的定义。⁶大多数患者根据标准实践进行管理，包括低风险患者的放血和阿司匹林治疗，以及高风险患者的细胞减灭疗法，通常以羟基脲的形式。使用 JMP Pro 14.0.0 软件包（SAS Institute, Cary, NC）进行统计分析。多变量分析包括先前确定的 AT 或静脉血栓形成的危险因素。

Mayo Clinic PV 患者队列包括 138 名患者（中位年龄 62 岁，范围 20-94；女性 50%）；呈现中位数（范围）值为血红蛋白 17.9 g/dL (16.1-24)，白细胞计数 11.8 × 10 (9)/L (2.7-65.8) 和 434 × 10 (9)/L (44-1679)用于血小板计数；57% 的患者出现白细胞增多 >11 × 10 (9)/L；37 名 (27%) 患者出现可触及的脾肿大；在 17% 的患者中记录了异常的核型。86 名 (62%) 患者的常规风险类别为高，52 名 (38%) 为低。中位随访时间为 11 年（范围 0.03-36.7）。诊断时记录了 22 例 (16%) 的静脉事件，诊断后记录了 20 例 (15%)，诊断时记录了 28 例 (20%) 静脉事件，诊断后记录了 15 例 (11%)。

ANC ( p  = .85) 和 AMC ( p  = .14) 均与诊断时或诊断前的 AT 无关，高龄是唯一在这方面具有显着相关性的其他参数 ( p  = .01)。同样，无 AT 生存不受 ANC (0.31) 或 AMC (0.27) 的影响；高龄再次是唯一显示显着性的其他测试参数 ( p  < .001)，而先前的 AT 事件显示出临界显着性 ( p  = .05)，这在多变量分析中不再明显。相比之下，诊断时或诊断前的 VTE 与 ANC ( p  = .048) 和 AMC (0.06；临界显着性) 相关，而没有测试其他参数，包括年龄 (0.7) 或性别 ( p = .16)，显示出显着的关联。在单变量分析中，无 VTE 生存率受到较高 ANC ( p  = .001) 和较高 AMC ( p  = .036) 的负面影响；ANC ( p  = .02) 但 AMC ( p = .71) 在包括年龄 ( p  = .68)、性别 ( p  = .18) 和既往静脉事件 ( p = .68) 在内的多变量分析中保留了显着性 。较高的 ANC 对无 VTE 生存的不利影响在高风险 ( p  = .004) 与低风险 ( p  = .14) 疾病中最为明显。

PV 患者的外部验证队列来自意大利佛罗伦萨大学：中位年龄 62 岁（范围 18-92）；女性 40%；显示血红蛋白的中值（范围）为 17.8 g/dL (16.1-24)，白细胞计数为 9.8 × 10 (9)/L (4.5-26.9)，和 461 × 10 (9)/L (154-1352)用于血小板计数。中位随访时间为 8 年（范围 0.5-30.8）；诊断时记录了 46 例 (10.8%) 静脉事件，诊断后记录了 33 例 (7.8%) 静脉事件；诊断时记录了 58 例（13.7%）AT 事件，诊断后记录了 39 例（9.2%）。与 Mayo Clinic 队列的情况一样，ANC 和 AMC 均与诊断时/之前或之后的 AT 事件相关。相比之下，无 VTE 生存率受到较高 ANC ( p  = .003) 和既往静脉事件 ( p  = .01) 的负面影响，但不受 AMC (p  = .7)、年龄 ( p  = .4) 或性别 ( p  = .3)；ANC ( p  = .01) 和先前的静脉事件 ( p  = .04) 在多变量分析中保持显着性。在 Florence 队列中，较高 ANC 对无 VTE 生存的不利影响在低风险疾病中更为显着 ( p  = .02)，尽管在高风险疾病中也有明显的趋势 ( p  = .07)。另一方面，与 Mayo Clinic 队列的病例不同，PV 诊断时或之前的 VTE 与 ANC ( p  = .4) 或 AMC ( p = .2)。由接受者操作特征 (ROC) 图分析确定的无 VTE 生存风险分层的最佳 ANC 截止水平为 Mayo Clinic 队列的 22 × 10 (9)/L（图 1A）和 10 × 10 (9)/ L 为佛罗伦萨队列（图 1B）。

ET 患者队列包括 349 名患者（中位年龄 57 岁，范围 18-89；女性 61%）：46% JAK2、34 % CALR、16% 三阴性和 4% MPL突变；国际 ET 血栓形成预后系统 (IPSET) 风险类别为高 24%、中 41% 和低 35%；呈现中位数（范围）值为血红蛋白 13.8 g/dL (11.1-16.4)，白细胞计数 8.2 × 10 (9)/L (3.2-29.8) 和 859 × 10 (9)/L (451-3460)用于血小板计数；48 例（14%）患者可触及脾肿大，56% 患者存在心血管危险因素；中位随访时间为 10 年（范围 0-47）。诊断时记录了 38 例（11%）静脉事件，诊断后记录了 31 例（9%），诊断时记录了 42 例（12%）AT 事件，诊断后记录了 64 例（18%）。

ANC ( p  = .8) 和 AMC ( p  = .38) 在诊断时或诊断前均与 AT 不相关；相反，显着性参数包括高龄 ( p  = .003)、JAK2突变 ( p  = .01)、男性 ( p  = .03) 以及具有临界意义的心血管危险因素 ( p  = .07)。同样，无 AT 生存不受 ANC (0.1) 或 AMC (0.1) 的影响；在多变量分析中，显着变量包括高龄 ( p  = .03)、既往 AT 事件 ( p  = .04) 和心血管危险因素 ( p = .04)。与 PV 一样，诊断时或诊断前的 VTE 与 AMC (0.003) 和 ANC ( p  = .06) 相关，后者具有临界意义；在这方面具有临界意义的其他参数包括JAK2突变 ( p  = .06)、高龄 ( p  = .08) 和女性 ( p  = .08)。在多变量分析中，无 VTE 生存率仅受高龄的影响 ( p  = .001)。

当前研究的观察结果表明，嗜中性粒细胞和可能的单核细胞是 PV 和 ET 中静脉血栓形成的潜在因素。这与中性粒细胞在静脉血栓形成中的作用的新证据一致，其中潜在机制还可能涉及血小板、单核细胞、内皮、促凝血蛋白和中性粒细胞胞外陷阱 (NET)，从而导致高凝状态和免疫介导的交互状态炎。^{8, 9}正如他们较差的无 VTE 生存率所表明的那样，高风险和低风险 PV 患者的 ANC 增加的持续 VTE 风险引发了对当前治疗在预防静脉血栓形成方面的充分性的质疑。先前发表的一项针对 ET 患者的对照研究也做出了类似的表示，其中接受羟基脲的患者与阿那格雷相比，VTE 的风险更高，两者都与阿司匹林联合治疗。¹⁰在涉及 PV 患者的两项独立协作工作中，我们还显示了较高的JAK2 V617F 等位基因负荷¹¹和中性粒细胞 - 淋巴细胞比率^{12对无 VTE 生存的不利影响}这进一步支持了与中性粒细胞增多和相关炎症状态相关的促血栓形成克隆负荷的有害作用，以淋巴细胞计数降低为标志。总之，包括当前工作在内的三项平行研究^11、12确定了与 AT 不同的 VTE 风险因素。在当前治疗的背景下，这些信息是否可行，目前无法推测，但表明需要对未来的前瞻性和回顾性研究进行以静脉血栓形成为重点的分析。