Hairy Cell Leukemia (HCL) is a rare indolent disease affecting mature B lymphocytes. While general symptoms are rather nonspecific (splenomegaly, fatigue, pancytopenia), HCL cells show a characteristic morphologic, immunophenotypic, and mutational profile that features prominently in the diagnostic process. Malignant cells have oval nuclei, abundant cytoplasm, and typical cytoplasmatic projections. Flow cytometric immunophenotyping includes key antigens such as CD11c, CD25, CD103, and CD200, which are strongly expressed, in addition to the ordinary panel used for the detection of B cell lymphoproliferative diseases (LPD). Moreover, a recurrent V600E BRAF mutation has been recently uncovered in HCL but not in its mimickers and is now considered the molecular hallmark of the disease (Cross & Dearden, 2020).

Usually, LPD present one single clonal population, but bi- or multiclonal cases, where two or more distinct aberrant clones coexist in a single anatomic site, are well known and described. Multicolor flow cytometry (MFC) plays a key role in the detection of these cases for being both highly sensitive and specific (Mahdi et al., 2018). Although the association of HCL with other non-Hodgkin's lymphoma is a known phenomenon, among the so-called composite lymphoma, pure biclonal HCL is to be considered an exceptional finding.

We report a case of biclonal HCL found by MFC in a 80 y.o. male patient referred to our clinic because of new onset of peripheral blood leucopenia and thrombocytopenia. A full blood count showed WBC 2310/μl, with 800 neutrophils/μl. Hb was 13.8, and platelets 73,000/μl. Past medical history showed an ischemic stroke and a metastatic squamous cell carcinoma (SCC) of unknown primary origin, for which he underwent several surgical treatments but no systemic therapy. A routine CT scan of the thorax and a neck, and abdomen US performed for SCC follow-up showed multiple lung lesions and enlarged laterocervical and mediastinal lymph nodes, all interpreted as metastatic SCC sites, as well as a 15.5 cm spleen. The cytopenias and splenomegaly were interpreted as possible signs of myelophthisis or myelodysplastic syndrome. Therefore, a peripheral blood smear was performed, and a second sample was sent to the Flow Cytometry Laboratory. This sample was marked with a two-tube eight-color panel initially addressing monocyte and neutrophil maturation and myeloid blast count, in addition to a limited lymphocyte subpopulation analysis for total T, B, and NK subsets. The immunophenotypic evaluation was done using FACSDiva software on an eight-color FACSCanto II Flow Cytometer (BD Biosciences). The analysis confirmed neutropenia (40%) and an inversion of the leukocyte formula. A subset of CD20+ B lymphocytes with high Side Scatter (SSC), accounting for 15% of all lymphocytes, was noted. These cells overlapped monocytes in the CD45/SSC dot-plot suggesting an increased cellular complexity, as typically observed in HCL (Figure 1). To investigate this hypothesis, B lymphocytes were further analyzed using an eight-color panel including kappa FITC, lambda PE, CD5 PerCP-Cy5.5, CD19 PE-Cy7, CD11c APC, CD38 APC-H7, CD20 V450, CD45 V500 monoclonal antibodies, directed to identify a possible CD11c+ clonal subpopulation, coupled with a second tube including antibodies against the HCL-associated CD25 and CD103 markers labeled with FITC and PE respectively. Then CD10, CD22, CD200 and FMC7 were also tested (all purchased from BD Biosciences).

CD19+/SSC high-gated B lymphocytes turned strongly positive for CD19 and CD20 as well as for CD11c, CD25, CD103, CD22, CD200, and FMC7, while CD5, CD10, and CD38 were not detected. These results, consistent with HCL, were also supported by the blood smear analysis that identified a subset of lymphocytes with atypical protrusions. Unexpectedly, the ratio between the Ig Kappa and Lambda light chains was within the normal range (1.8) (Figure 1 panel (a)). However, due to the immunologic profile and the peculiar SSC values, a possible biclonal HCL with one kappa- and one lambda-restricted subclones was strongly suspected.

In order to provide molecular evidence supporting our unusual MFC finding, DNA was extracted from peripheral blood leukocytes of the patient and subjected to multiplex polymerase chain reaction (PCR) for size discrimination of immunoglobulin heavy chain (IgH) and IgK light chain gene rearrangement with the IdentiClone™ IgH + IgK B-cell Clonality Assay-ABI Fluorescence Detection kit (InVivoScribe Technologies) according to the Biomed-2 assay protocol. The results were evaluated with the aid of GeneMapper™ Software (Applied Biosystems™). Two prominent peaks were generated by the rearrangement of both IgH (FR3-JH) and IgK (V-Kde and JC intron-Kde) genes, confirming the clonal origin of the “hairy” population (Figure 1 panels (b,c)). Moreover, the BRAF V600E gene mutation was demonstrated by Allele Specific PCR (AS-PCR) and confirmed by direct sequencing (Figure 1 panels (d,e)) and a diagnosis of HCL was finally rendered. Subsequently, an extended bone marrow localization was proved by histologic evaluation.

The possible HCL origin of the multiple adenopathies was ruled out by molecular analysis of biopic material of an axillary lymph node, which was negative for BRAF V600E and consistent with SCC involvement. In reason of the comorbidity described above and because cytopenias were not severe, we elected the patient for a watch and wait approach.

Our case highlights the fundamental role of MFC and molecular biology in discovering a rare biclonal HCL. MFC is the most available method for detecting bi- or multiclonal LPD (Mahdi et al., 2018), and an atypical immunologic profile, even in the presence of a normal kappa/lambda ratio, is highly suggestive of malignant proliferation and should drive further investigations.

Biclonality is a rare event among B-LPD; it presents either in the form of composite lymphoma or as an expansion of two or multiple concurrent subclones in a process of intraclonal evolution within the same disease, as frequently reported in B-CLL (Mahdi et al., 2018). Overall, HCL has been seldom detected in the setting of composite lymphoma and, to our knowledge, pure HCL with two simultaneous subclones is an extremely rare finding. The only report about a biclonal HCL, dated 1986, was based on Ig gene rearrangement studies. In this short article, Raghavachar et al. described two coexisting kappa and lambda clones, one of which was resistant to IFN-α therapy. In our cohort of 1090 newly diagnosed B-LPD detected in a 10-year period, including 26 HCL (2.4%), bi- or multiple clonality was found in 4.7% of all cases. Only one HCL turned out biclonal.

As part of an integrated diagnostic pathway, molecular confirmation through size discrimination or nucleotide sequencing of IgH and IgK gene rearrangements is generally recommended to support MFC finding of bi- or multiple clonal B-NHL, especially when Ig light chain restriction is not directly demonstrated by MFC. In our patient, the IgH gene rearrangement analysis detected two predominant peaks, most likely consistent with biclonality. Similarly, IgK amplification generated two signals (specifically IgK V/Intron-Kde). Since this recombination occurs following a non-functional rearrangement of an IgK allele, we could not univocally demonstrate whether the two detected IgK peaks belonged to different subclones (one expressing kappa and the other one lambda light chains) or we were just catching the two nonfunctional IgK alleles of the lambda subclone. Nonetheless, the whole Ig gene molecular findings together with the flow cytometric immunophenotypic profile strongly supported the biclonal nature of the “hairy” B-cell population in spite of an apparent normal kappa/lambda ratio.

The finding of BRAF mutation served as the last piece of such an intriguing diagnostic puzzle. Notably, BRAF inhibitors, used in V600E positive metastatic melanoma, are a new potential approach for HCL patients who experienced refractory or relapsed disease (Cross & Dearden, 2020) and may become a potential therapy option if the conditions of our HCL patient would not be suitable for standard chemotherapy regimens. From a biological point of view, our findings also raise the question as to whether the BRAF mutation arose before the VDJ specification in a pro-B lymphocyte, or two separate clones acquired the same mutation in an example of convergent evolution; further studies are needed to address this issue.

In conclusion, we highlight the value of MFC in detecting an asymptomatic HCL in a clinical setting complicated by comorbidities and in dissecting two different subclones in spite of a balanced kappa/lambda ratio. An equally relevant contribution to the final diagnosis was provided by the molecular demonstration of biclonality and the finding of the specific HCL genetic signature. These data reinforce the notion that an integrated approach is an added value to the diagnostic pathway of B-LPD, allowing a thoroughly accurate response to the diagnostic framing.

毛细胞白血病 (HCL) 是一种影响成熟 B 淋巴细胞的罕见惰性疾病。虽然一般症状是相当非特异性的（脾肿大、疲劳、全血细胞减少），但 HCL 细胞表现出特征性的形态学、免疫表型和突变特征，在诊断过程中具有显着特征。恶性细胞有椭圆形的细胞核、丰富的细胞质和典型的细胞质突起。除了用于检测 B 细胞淋巴增生性疾病 (LPD) 的普通 panel 外，流式细胞免疫表型分析还包括强表达的关键抗原，如 CD11c、CD25、CD103 和 CD200。此外，最近在 HCL 中发现了一个复发性 V600E BRAF 突变，但在其模拟物中没有发现，现在被认为是该疾病的分子标志（Cross & Dearden，  2020）。

通常，LPD 呈现一个单一的克隆群体，但双克隆或多克隆病例，其中两个或多个不同的异常克隆共存于一个解剖部位，是众所周知的和描述的。多色流式细胞仪 (MFC) 在检测这些病例中发挥着关键作用，因为它具有高度敏感性和特异性（Mahdi 等人，  2018 年）。虽然 HCL 与其他非霍奇金淋巴瘤的关联是一种已知现象，但在所谓的复合淋巴瘤中，纯双克隆 HCL 被认为是一个例外的发现。

我们报告了一例由 MFC 在一名 80 岁男性患者中发现的双克隆 HCL 病例，该患者因新发外周血白细胞减少症和血小板减少症而转诊到我们诊所。全血细胞计数显示 WBC 2310/μl，中性粒细胞 800/μl。Hb 为 13.8，血小板为 73,000/μl。既往病史显示缺血性中风和原发性不明的转移性鳞状细胞癌 (SCC)，为此他接受了多次手术治疗，但未进行全身治疗。为 SCC 随访而进行的胸部、颈部和腹部 US 的常规 CT 扫描显示多处肺部病变和扩大的侧颈和纵隔淋巴结，所有这些都被解释为转移性 SCC 部位，以及 15.5 厘米的脾脏。血细胞减少和脾肿大被解释为可能是骨髓炎或骨髓增生异常综合征的迹象。所以，进行了外周血涂片检查，并将第二份样本送到流式细胞术实验室。除了对总 T、B 和 NK 亚群进行有限的淋巴细胞亚群分析外，该样本还标有一个两管八色面板，最初涉及单核细胞和中性粒细胞成熟以及髓母细胞计数。使用 FACSDiva 软件在八色 FACSCanto II 流式细胞仪 (BD Biosciences) 上进行免疫表型评估。分析证实了中性粒细胞减少症（40%）和白细胞公式的倒置。注意到具有高侧向散射 (SSC) 的 CD20+ B 淋巴细胞亚群，占所有淋巴细胞的 15%。这些细胞在 CD45/SSC 点图中与单核细胞重叠，表明细胞复杂性增加，这通常在 HCL 中观察到（图 1）。为了研究这个假设，使用八色板进一步分析 B 淋巴细胞，包括 kappa FITC、λ PE、CD5 PerCP-Cy5.5、CD19 PE-Cy7、CD11c APC、CD38 APC-H7、CD20 V450、CD45 V500 单克隆抗体，用于鉴定可能的 CD11c+ 克隆亚群，再加上第二管，其中包括分别用 FITC 和 PE 标记的 HCL 相关 CD25 和 CD103 标记的抗体。然后还测试了 CD10、CD22、CD200 和 FMC7（均购自 BD Biosciences）。

CD19+/SSC 高门控 B 淋巴细胞对 CD19 和 CD20 以及 CD11c、CD25、CD103、CD22、CD200 和 FMC7 呈强阳性，而未检测到 CD5、CD10 和 CD38。这些结果与 HCL 一致，也得到了血液涂片分析的支持，该分析确定了具有非典型突起的淋巴细胞亚群。出乎意料的是，Ig Kappa 和 Lambda 轻链之间的比率在正常范围内 (1.8)（图 1 面板 (a)）。然而，由于免疫学特征和特殊的 SSC 值，强烈怀疑具有一个 kappa 和一个 lambda 限制性亚克隆的可能双克隆 HCL。

为了提供支持我们不寻常的 MFC 发现的分子证据，从患者的外周血白细胞中提取 DNA，并进行多重聚合酶链式反应 (PCR) 以区分免疫球蛋白重链 (IgH) 和 IgK 轻链基因重排的大小。 IdentiClone™ IgH + IgK B 细胞克隆性检测-ABI 荧光检测试剂盒 (InVivoScribe Technologies)，符合 Biomed-2 检测方案。借助 GeneMapper™ 软件 (Applied Biosystems™) 评估结果。IgH (FR3-JH) 和 IgK (V-Kde 和 JC 内含子-Kde) 基因的重排产生了两个突出的峰，证实了“多毛”种群的克隆起源（图 1 面板（b，c）） . 而且，BRAF V600E 基因突变通过等位基因特异性 PCR (AS-PCR) 得到证实，并通过直接测序证实（图 1 面板 (d,e)），最终诊断为 HCL。随后，通过组织学评估证明了扩展的骨髓定位。

腋窝淋巴结活检材料的分子分析排除了多发性淋巴结病可能的 HCL 起源，BRAF V600E 为阴性，与 SCC 受累一致。由于上述合并症和血细胞减少并不严重，我们选择患者进行观察和等待方法。

我们的案例突出了 MFC 和分子生物学在发现罕见的双克隆 HCL 中的基本作用。MFC 是检测双克隆或多克隆 LPD 的最可用方法（Mahdi 等人，  2018 年），即使存在正常的 kappa/lambda 比率，非典型免疫学特征也高度提示恶性增殖，应进一步推动调查。

双克隆性在 B-LPD 中是罕见的事件；它以复合淋巴瘤的形式出现，或者在同一疾病的克隆内进化过程中作为两个或多个并发亚克隆的扩展，正如 B-CLL 中经常报道的那样（Mahdi 等，  2018）。总体而言，HCL 在复合淋巴瘤中很少被发现，据我们所知，具有两个同时亚克隆的纯 HCL 是极其罕见的发现。1986 年有关双克隆 HCL 的唯一报告是基于 Ig 基因重排研究。在这篇短文中，Raghavachar 等人。描述了两个共存的 kappa 和 lambda 克隆，其中一个对 IFN-α 治疗具有抗性。在我们在 10 年期间检测到的 1090 例新诊断的 B-LPD 队列中，包括 26 例 HCL（2.4%），在所有病例中发现双克隆或多克隆的占 4.7%。只有一种 HCL 是双克隆的。

作为综合诊断途径的一部分，通常建议通过 IgH 和 IgK 基因重排的大小区分或核苷酸测序进行分子确认，以支持 MFC 发现双克隆或多克隆 B-NHL，特别是当 Ig 轻链限制不能通过以下方法直接证明时MFC。在我们的患者中，IgH 基因重排分析检测到两个主要峰，很可能与双克隆一致。同样，IgK 扩增产生两个信号（特别是 IgK V/Intron-Kde）。由于这种重组发生在 IgK 等位基因的非功能性重排之后，我们无法明确证明检测到的两个 IgK 峰是否属于不同的亚克隆（一个表达 kappa，另一个表达 lambda 轻链），或者我们只是捕获了 lambda 亚克隆的两个非功能性 IgK 等位基因。尽管如此，整个 Ig 基因分子发现以及流式细胞术免疫表型谱强烈支持“多毛”B 细胞群的双克隆性质，尽管 kappa/lambda 比率明显正常。

BRAF 突变的发现是这个有趣的诊断难题的最后一块。值得注意的是，用于 V600E 阳性转移性黑色素瘤的 BRAF 抑制剂对于患有难治性或复发性疾病的 HCL 患者是一种新的潜在方法（Cross & Dearden，  2020），如果我们的 HCL 患者的病情不会适用于标准化疗方案。从生物学的角度来看，我们的研究结果还提出了一个问题，即 BRAF 突变是否出现在前 B 淋巴细胞中的 VDJ 规范之前，或者两个独立的克隆在趋同进化的例子中获得了相同的突变。需要进一步研究来解决这个问题。

总之，我们强调了 MFC 在临床环境中检测无症状 HCL 并伴有合并症的价值，以及在解剖两种不同的亚克隆（尽管 kappa/lambda 比率平衡）方面的价值。双克隆性的分子证明和特定 HCL 遗传特征的发现为最终诊断提供了同样相关的贡献。这些数据强化了这样一种观念，即综合方法是 B-LPD 诊断途径的附加值，可以对诊断框架做出彻底准确的反应。