BCR/ABL1 rearrangement or t(9;22)(q34.1;q11.2) is the most common recurrent cytogenetic abnormality seen in B/myeloid mixed phenotype acute leukemia (MPAL) but is also present in ~50% of adult B-ALL (Swerdlow et al., 2017). Both entities can demonstrate B lymphoblasts with aberrant myeloid expression; however, an important immunophenotypic finding that distinguishes MPAL from B-ALL with aberrant myeloid expression is reciprocal intensities of myeloid and lymphoid markers. This pattern is emphasized as the most recognizable feature of MPAL in the WHO classification but there are few examples in the literature demonstrating its practical application. Herein, we highlight this signature inverse expression characteristic using a case of MPAL B/myeloid with BCR/ABL1.

Detailed methodology and antibody panels have been previously described in Cytometry B (DOI: 10.1002/cyto.b.21979). In brief, sodium heparin anticoagulated bone marrow (BM) was processed within 6 h of collection. BM specimens were washed twice using 37°C phosphate-buffered saline and adjusted to ~10 × 10⁹/L. One hundred microliter were stained using antibody panels per DOI: 10.1002/cyto.b.21979. Specifically, the markers presented here included: CD10-APC-AF700 (ALB1), CD13-PE (SJ1D1) CD19-APC-AF700 (J3-119), CD19-APC-A750 (J3-119), CD19-ECD (J3-119), CD33-PC5.5 (D3HL60.251) CD34-PC7 (581), CD45-KO (J.33) CD117-APC (104D2D1) cCD79a-APC (HM47) and cMPO-PE (CLB-MPO-1), all antibodies from Beckman Coulter (BC). For surface staining, cells were incubated for 15 min in the dark at room temperature, and then lysed using VersaLyse (BC, ref. A09777) and IOTest-3 fixative (BC, ref. A07800). For intracellular staining BC IntraPrep™ reagent kit (BC, ref. A07802) was used according to manufacturer's instructions. After washing, samples were suspended in IOTest-3 Fixative (BC, ref. A07800) and PBS. A minimum of 50 × 10³ CD45+ events were acquired on a Navios™ Flow cytometer (BC) within 1 h of preparation. Listmode data files were analyzed using Kaluza analysis software (BC, v1.3). Fluorochrome abbreviations: Phycoerythrin (PE), Phycoerythrin Texas Red-X (ECD), Phycoerythrin Cyanin 7 (PC7), Allophycocyanin (APC), Allophycocyanin Alexa Fluor 700 (APC-AF700), Allophycocyanin Alexa Fluor 750 (APCAF750) and Krome Orange (KO).

A previously healthy 25-year-old male presented to medical attention with a 3-week history of progressive exertional dyspnea along with a new rash on his chest and shins. Physical examination was notable for mild tachycardia, pallor, and a petechial rash. Initial blood work revealed marked leukocytosis with a blast count of 134 × 10⁹/L (83% blasts), profound anemia and thrombocytopenia. BM studies revealed 91% blasts by morphology with minimal granulocyte maturation. Blasts showed variable morphology ranging from small blasts with high nuclear/cytoplasmic ratio to medium-sized blasts with prominent nucleoli and granules, but no Auer rods. The trephine biopsy showed almost 100% cellularity with normal hematopoiesis replaced by a dense blast infiltrate.

Comprehensive flow cytometry was performed using four 10 color panels, routine at our institution for evaluation of new acute leukemia. Panel 1 showed a large CD34 positive blast population in the CD45-dim low side scatter region with partial expression of CD117 alongside variable expression of CD13 and CD33. Such strong expression of myeloid markers initially suggested a myeloid leukemia (Figure 1a–d). Uniformly positive CD19, however, then broadened the differential diagnosis to B-ALL with aberrant myeloid expression, AML with aberrant CD19 (e.g., AML with t(8;21)(q22;q22.1), or B/myeloid MPAL (Figure 1e). The next sequence of markers demonstrated a large proportion of the blasts expressed CD10 and CD79a (Figure 1f,i), meeting WHO criteria for assigning at least a B lineage with a small proportion of the blasts being MPO positive (2.95%; Figure 1g). Cytoplasmic CD3 was negative (not shown), narrowing the differential to B-ALL with aberrant myeloid expression or B/myeloid MPAL. While MPO was only weakly/partially expressed, importantly MPAL-defining inverse co-expression patterns between myeloid and lymphoid markers were evident. MPO was only expressed in the cells brightest for the myeloid marker CD33 and dimmest for the B lineage associated marker CD79a (Figure 1i,j, black backgating on MPO+). A similar negative inverse correlation could be demonstrated for CD10 and CD33 (Figure 1k,l, black backgating on CD117+); however, CD19 intensity did not vary. Similar to MPO, CD117 was only expressed in cells that were strongly positive for CD33, but negative for CD10. Given these phenotypic associations, we inferred that MPO was expressed in CD19+ cells positive for the myeloid markers CD33 and CD117, but negative for B cell markers CD10 and CD79a. Based on this characteristic inverse B-lymphoid and myeloid expression pattern, a preliminary diagnosis of MPAL, B/myeloid was communicated to the treatment team the day of the BM biopsy.

Subsequent cytogenetics results demonstrated t(9;22)(q34.1;q11.2) in 19/20 metaphases and a p210 BCR/ABL1 transcript by molecular. A final WHO classification of MPAL with t(9;22)(q34.1;q11.2) was made. Next generation sequencing results detected a variant of uncertain significance (VUS) in RUNX1 leading to the duplication of a valine residue at position 119 (RUNX1c.356_358dupTGG/p.Val119dup), variant allele frequency 47%. The patient was started on a tyrosine kinase inhibitor based B-ALL regimen and eventually went on to receive an allogeneic stem cell transplant and is currently in remission.

Acute leukemias are increasingly classified by recurrent molecular/cytogenetic abnormalities. In this case, t(9;22)(q34.1;q11.2); BCR/ABL1 did not assist with distinguishing MPAL from B-ALL since it a common finding in both entities. In fact, BCR/ABL1 in cases such as this one may create more ambiguity since it is known to be associated with aberrant myeloid expression in B-ALL. The presence of a BCR/ABL1 p210 isoform and mixed phenotype blasts also raises the possibility of chronic myeloid leukemia (CML) presenting in a mixed blast crisis. In our case this was unlikely based on the acute clinical history and absence of other suggestive features of CML such as splenomegaly, peripheral granulocytosis, or residual granulocyte maturation. Given the controversies related to isolated MPO expression in otherwise typical B-ALL presentations, this case helps emphasize the value of additional aberrant myeloid markers, particularly when the pattern of B-lymphoid and myeloid markers are inversely correlated. The fact that lineage defining MPO expression clearly followed this pattern made it highly unlikely that the MPO staining was non-specific. Other phenotypic support for MPAL included CD117 positivity, which in contrast to CD13 and CD33, is rare in B-ALL.

It is likely that additional genetic events occur that ultimately determine the dominant blast lineage(s) at presentation since BCR/ABL1 may be detected in de novo ALL, AML, MPAL and blast phase CML. The added finding of a RUNX1 mutation in this case (albeit classified as a VUS) is interesting as emerging evidence suggests that RUNX1 can play a role in the pathogenesis of MPAL, and specifically seems to be enriched in B/myeloid MPAL (Takahashi et al., 2018). In the tyrosine kinase inhibitor era the clinical impact of distinguishing MPAL, B-myeloid with BCR/ABL1, and B-ALL with BCR/ABL1 remains unclear; however, the categorization as MPAL in a young patient may favor allotransplant consolidation where feasible, regardless of measurable residual disease. This highlights the therapeutic importance of accurate flow cytometric interpretation and awareness of the distinguishing features between these two subtypes of acute leukemia.

BCR/ABL1重排或 t(9;22)(q34.1;q11.2) 是 B/髓混合表型急性白血病 (MPAL) 中最常见的复发性细胞遗传学异常，但也存在于约 50% 的成人 B -ALL（Swerdlow 等人，  2017 年）。两种实体都可以证明 B 淋巴母细胞具有异常的骨髓表达；然而，将 MPAL 与具有异常骨髓表达的 B-ALL 区分开来的一个重要免疫表型发现是骨髓和淋巴标志物的强度倒数。这种模式在 WHO 分类中被强调为 MPAL 最可识别的特征，但文献中很少有例子证明其实际应用。在此，我们使用带有BCR/ABL1的 MPAL B/髓样病例强调了这一标志性的反向表达特征。

详细的方法学和抗体组先前已在 Cytometry B (DOI: 10.1002/cyto.b.21979) 中进行了描述。简而言之，肝素钠抗凝骨髓 (BM) 在采集后 6 小时内进行处理。BM 样本使用 37°C 磷酸盐缓冲盐水洗涤两次，并调整至 ~10 × 10 ⁹/L。每个 DOI 使用抗体组对 100 微升进行染色：10.1002/cyto.b.21979。具体来说，此处介绍的标记包括：CD10-APC-AF700 (ALB1)、CD13-PE (SJ1D1) CD19-APC-AF700 (J3-119)、CD19-APC-A750 (J3-119)、CD19-ECD (J3 -119)、CD33-PC5.5 (D3HL60.251)、CD34-PC7 (581)、CD45-KO (J.33) CD117-APC (104D2D1) cCD79a-APC (HM47) 和 cMPO-PE (CLB-MPO- 1)，所有来自 Beckman Coulter (BC) 的抗体。对于表面染色，细胞在室温下在黑暗中孵育 15 分钟，然后使用 VersaLyse（BC，参考 A09777）和 IOTest-3 固定剂（BC，参考 A07800）裂解。对于细胞内染色，根据制造商的说明使用 BC IntraPrep™ 试剂盒（BC，参考号 A07802）。洗涤后，将样品悬浮在 IOTest-3 Fixative (BC, ref. A07800) 和 PBS 中。至少 50 × 10 ³在准备后 1 小时内，在 Navios™ 流式细胞仪 (BC) 上采集了 CD45+ 事件。使用 Kaluza 分析软件 (BC, v1.3) 分析 Listmode 数据文件。荧光染料缩写：藻红蛋白 (PE)、藻红蛋白 Texas Red-X (ECD)、藻红蛋白蓝蛋白 7 (PC7)、别藻蓝蛋白 (APC)、别藻蓝蛋白 Alexa Fluor 700 (APC-AF700)、别藻蓝蛋白 Alexa Fluor 750 (APCAF750) 和 Krome Orange (高）。

一名既往健康的 25 岁男性因进行性劳力性呼吸困难 3 周而就诊，同时胸部和胫骨上出现新的皮疹。体格检查发现轻度心动过速、苍白和瘀点皮疹。最初的血液检查显示显着的白细胞增多，原始细胞计数为 134 × 10 ⁹ /L（83% 原始细胞）、严重贫血和血小板减少症。BM 研究显示 91% 的胚细胞在形态上具有最小的粒细胞成熟度。胚细胞表现出不同的形态，从具有高核/细胞质比率的小胚细胞到具有突出核仁和颗粒但没有 Auer 棒的中型胚细胞。环钻活检显示几乎 100% 的细胞结构，正常的造血功能被密集的原始细胞浸润所取代。

使用四个 10 色板进行综合流式细胞术，在我们的机构中​​常规用于评估新的急性白血病。图 1 显示 CD45-dim 低侧向散射区域中有大量 CD34 阳性胚细胞，CD117 部分表达以及 CD13 和 CD33 的可变表达。骨髓标志物的这种强烈表达最初表明是骨髓性白血病（图 1a-d）。然而，CD19 一致阳性随后将鉴别诊断扩大到具有异常骨髓表达的 B-ALL、具有异常 CD19 的 AML（例如，具有 t(8;21)(q22;q22.1) 的 AML 或 B/骨髓 MPAL（图1e). 下一个标记序列显示大部分原始细胞表达 CD10 和 CD79a（图 1f，i），符合 WHO 的标准，即分配至少一个 B 谱系，其中一小部分原始细胞为 MPO 阳性（2.95%；图 1g)。细胞质 CD3 为阴性（未显示），将差异缩小到具有异常骨髓表达的 B-ALL 或 B/骨髓 MPAL。虽然 MPO 仅微弱/部分表达，但重要的是，髓样和淋巴样标记物之间定义 MPAL 的反向共表达模式是明显的。MPO 仅在骨髓标记 CD33 最亮的细胞中表达，而 B 谱系相关标记 CD79a 最暗的细胞中表达（图 1i，j，MPO+ 上的黑色背栅）。对于 CD10 和 CD33，可以证明类似的负负相关（图 1k，l，CD117+ 上的黑色背景）；然而，CD19 强度没有变化。与 MPO 类似，CD117 仅在 CD33 强阳性但 CD10 阴性的细胞中表达。鉴于这些表型关联，我们推断 MPO 在骨髓标志物 CD33 和 CD117 阳性的 CD19+ 细胞中表达，但对 B 细胞标志物 CD10 和 CD79a 呈阴性。基于这种特征性的逆 B 淋巴和骨髓表达模式，在 BM 活检当天将 MPAL、B/骨髓的初步诊断传达给治疗团队。

随后的细胞遗传学结果表明 t(9;22)(q34.1;q11.2) 在 19/20 中期和 p210 BCR/ABL1分子转录本。制定了具有 t(9;22)(q34.1;q11.2) 的 MPAL 最终 WHO 分类。下一代测序结果在RUNX1中检测到一个不确定显着性 (VUS) 的变异，导致第 119 位缬氨酸残基的重复 (RUNX1c.356_358dupTGG/p.Val119dup)，变异等位基因频率为 47%。该患者开始使用基于酪氨酸激酶抑制剂的 B-ALL 方案，最终继续接受异基因干细胞移植，目前处于缓解期。

急性白血病越来越多地按复发性分子/细胞遗传学异常分类。在这种情况下，t(9;22)(q34.1;q11.2); BCR/ABL1无助于区分 MPAL 和 B-ALL，因为它是两个实体的共同发现。事实上，在这种情况下， BCR/ABL1可能会产生更多的歧义，因为已知它与 B-ALL 中的异常骨髓表达有关。BCR/ABL1的存在p210 同种型和混合表型原始细胞也增加了慢性粒细胞白血病 (CML) 出现混合原始细胞危机的可能性。在我们的案例中，这不太可能是基于急性临床病史和缺乏 CML 的其他提示性特征，如脾肿大、外周粒细胞增多或残余粒细胞成熟。鉴于在其他典型 B-ALL 表现中与孤立 MPO 表达相关的争议，该案例有助于强调其他异常骨髓标志物的价值，特别是当 B 淋巴和骨髓标志物的模式呈负相关时。事实上，定义 MPO 表达的谱系显然遵循这种模式，这使得 MPO 染色极不可能是非特异性的。MPAL 的其他表型支持包括 CD117 阳性，与 CD13 和 CD33 相比，这在 B-ALL 中很少见。

由于 BCR/ABL1 可能在新发 ALL、AML、MPAL 和急变期 CML 中检测到，因此可能会发生额外的遗传事件，最终确定出现时的主要母细胞谱系。在这种情况下发现RUNX1突变（尽管被归类为 VUS）很有趣，因为新出现的证据表明RUNX1可以在 MPAL 的发病机制中发挥作用，特别是似乎富含 B/骨髓 MPAL（Takahashi 等人） .,  2018）。在酪氨酸激酶抑制剂时代，区分 MPAL、B 骨髓与 BCR/ABL1 和 B-ALL 与 BCR/ABL1 的临床影响仍不清楚。然而，在年轻患者中归类为 MPAL 可能有利于在可行的情况下进行同种异体移植巩固，无论可测量的残留疾病如何。这突出了准确的流式细胞术解释和了解这两种急性白血病亚型之间的区别特征的治疗重要性。