Hairy cell leukemia: a chronic B-cell lymphoma with unique clinicopathological features and unresolved molecular mechanisms

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Abstract

Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder that is characterized by pancytopenia, splenomegaly, and hepatomegaly, resulting from the organ-specific infiltration of “hairy” leukemic cells. Despite tremendous therapeutic advances with purine analogs and interferon, approximately half of patients with HCL relapse after initial treatment. The discovery of valine-to-glutamic acid mutation at amino acid 600 on B-rapidly accelerated fibrosarcoma [BRAF(V600E)] in HCL, revealed an aberrant MAPK signaling pathway that drives the proliferation and survival of HCL B cells, provides a promising and effective target for treating patients who have developed resistance to myelotoxic and immune-suppressive drugs. More recently, 2 BRAF(V600E)-based HCL mouse models have been developed that could be extremely useful both for functional studies and for testing the activity of new drugs. This review aims to summarize current understanding of key pathogenic mechanisms underlying HCL development and discusses major hurdles that need to be overcome in the context of other BRAF-mutated malignancies.

Introduction

Hairy cell leukemia (HCL), first described in 1958, is named for the morphology of leukemic cells: an irregularly frayed surface with hair-like villi and membrane ruffles.¹ HCL is characterized by hairy cells homing to the bone marrow, spleen, and liver.² Hairy cells show a mature B-cell phenotype with bright CD11c, CD25, CD103, and CD123 and a low number in peripheral blood and lymph nodes.^2,3

HCL, constituting ∼2% of all leukemia cases, exhibits a sex bias, with a male-to-female ratio of 4:1.^4,5 The common clinical presentations include pancytopenia, splenomegaly, and an increased risk of infection.² Therapeutic approaches that significantly affect clinical outcomes of patients with HCL remained limited until introduction of interferon alfa (IFN-α) in 1984.⁶ In the 1990s, the application of purine analogs (pentostatin and cladribine) turned HCL into one of the most successfully treated cancers in history.^7,8 Even though significant progress has been achieved over past decades, ∼50% of patients with HCL relapse and exhibit progressively drug resistance.^7,9

HCL, together with HCL variant (HCLv) and splenic marginal zone lymphoma (SMZL), is characterized by overlapping features, including splenomegaly, absence of lymphadenopathy, and a presence of “hairy” B cells.^10,11 In 2011, a central genetic driver of HCL, a gain-of-function mutation of B-rapidly accelerated fibrosarcoma (BRAF) serine/threonine protein kinase (V600E [valine (V) is substituted by glutamic acid (E) at amino acid 600]), was found in all patients with HCL but not in other B-cell neoplasms, such as HCLv and SMZL.12, 13, 14, 15 This discovery further supports the fact that HCL is a distinct entity different from other lymphomas and provides a new therapeutic target for developing treatment. Indeed, BRAF(V600E) inhibitors have shown remarkable activity in patients with HCL.16, 17, 18

More recently, 2 genetically engineered mouse models of HCL have been established. By introducing BRAF(V600E) in combination with Trp53 or PTEN loss in B-lymphocytes, mice develop a chronic lymphoma phenotype that closely mirrors the pathological features of human HCL,¹⁹ providing a valuable platform for studying disease pathogenesis and evaluating potential therapeutics. In this review, we aim to provide a comprehensive summary of current biological outcomes associated with HCL and to discuss pertinent challenges in elucidating HCL pathology.

Hairy B cells with distinct features

One unusual characteristic of hairy cells is their “hairy” morphology: presenting undulating, irregular projections and short microvilli on cell surface.^20,21 This arises from cellular cytoskeletal reorganization induced by hyperactive Rho signaling.^22,23 In contrast to normal B cells, the hairy morphology of leukemic cells enlarges their interaction surface with the surroundings. Additionally, an abundance of cytoplasm rich in mitochondria, coupled with ribosomal-lamellar complexes, indicates hairy cells with high metabolic demands, which likely sustains a prolonged cell survival.²⁴

Since the discovery of HCL, many studies have attempted to elucidate the origin of hairy cells. Although hairy cells express a series of mature B-cell markers, such as CD19 and CD25, their normal counterpart remains unclear. Currently, there are 2 plausible hypotheses regarding the origin of hairy cells: late-activated post–germinal center (post-GC) B cells and splenic marginal zone B cells. The diversification of intraclonal immunoglobulin variable gene in hairy cells suggests that these cells may have proliferated within GC.25, 26, 27 Moreover, hairy cells express activation-induced cytidine deaminase, an enzyme normally expressed in GC B cells.²⁶ In comparative expressed sequence hybridization studies, a “spleen signature” was identified in hairy cells, suggesting that they may arise from the spleen.²⁸ However, hairy cells do not express BCL6, a crucial GC marker.² Alternatively, hairy cells may originate from late-activated post-GC memory B cells. Hairy cells exhibit switched immunoglobulin isotypes, and in >85% of cases, their rearranged immunoglobulin variable genes bear somatic mutations.25, 26, 27^,29,30 Furthermore, genome-wide expression profile and DNA methylation profile from patient samples strongly indicate that hairy cells must have transited through GC.^30,31 Despite these considerable efforts, the origin of hairy cells still remains uncertain, because they do not share similarities in immunophenotype or morphology with any established B-cell subpopulations.

Hairy cells selectively accumulate in bone marrow, splenic red pulp, and hepatic sinusoids rather than lymph nodes, a process fine-tuned by the expression of chemokines, adhesion receptors, and cytokines in tissue microenvironments, as well as their receptors or ligands on hairy cells. Overexpression of CXCR4 (CD184) and CD9, as well as underexpression of L-selectin (CD62L), CXCR5, and CCR7 on hairy cells, may explain the minimal involvement of lymph nodes.³² In bone marrow, mesenchymal stromal cells of endosteal niche persistently secret chemokines and express ligands for different adhesion molecules, which promotes survival and growth of hairy cells (Figure 1A).³³ Moreover, the CXCR4 on hairy cells interacts with its ligand, chemokine ligand 12 (CXCL12) secreted by mesenchymal bone marrow stromal cells, which directs hairy cells from circulation into the bone marrow. Hairy cells also express diverse integrins, including integrin α4β1 (VLA-4), which bind to vascular cell adhesion molecule 1 (VCAM-1). This interaction occurs specifically in bone marrow and in hepatic and splenic sinusoids, contributing to a favored homing of hairy cells to these sinusoidal regions and protecting hairy cells from apoptosis when exposed to IFN-α or BRAF inhibitors.34, 35, 36 In addition, HCL-associated splenomegaly is also caused by pseudosinuses forming through VLA-4/-VCAM-1 interaction.³⁷ Accumulation of hairy cells in splenic red pulp could be related to their expression of vitronectin receptor CD51 (αvβ3 integrin), which engages with CD31 (PECAM-1) and regulates this homing (Figure 1B).³⁷ In addition, interactions between laminin on hairy cell surface and basement membrane could mediate the replacement of endothelial cells by hairy cells, which could be responsible for hepatic hemangiomatous lesions and splenic pseudosinuses.^38,39 In contrast, an association of CD44 isoforms, CD44v3 and CD44H, on hairy cells with hyaluronan promotes bone marrow fibrosis by activating basic fibroblast growth factor–mediated production of fibronectin.^40,41 Hairy cells also produce transforming growth factor β1 (TGFβ1), which contributes to bone marrow fibrosis by activating bone marrow fibroblasts.⁴¹

Figure 1.

Key interactions between hairy cells and various components in the bone marrow and spleen that facilitate organ-specific homing of hairy cells. (A) In bone marrow, hairy cells express high levels of chemokine receptor CXCR4, which is activated by CXCL12 secreted by BMSC. This CXCR4/CXCL12 cross talk directs hairy cells from circulation into bone marrow. Hairy cells also express high levels of integrin, VLA-4, which binds to its ligand VCAM-1 in bone marrow and hepatic and splenic sinusoids, accounting for their preferential homing to sinusoids of these organs. In addition, an association of CD44 isoforms, CD44v3 and CD44H, on hairy cells with hyaluronan drives bone marrow fibrosis. (B) In the spleen, VLA-4/VCAM-1 interaction facilitates pseudosinus formation, which eventually causes splenomegaly, whereas the vitronectin receptor CD51 on hairy cells, which engages with CD31, mediates their accumulation in spleen red pulp. An engagement of CD40 (on hairy cells) by CD154 (on T cells), in both the bone marrow and spleen, also contributes to HCL progression. BMSC, mesenchymal bone marrow stromal cells; CAR cell, CXCL12-abundant reticular cell; CXCL12, CXC chemokine ligand 12; ECM, extracellular matrix; MSC, mesenchymal stromal cell; Treg, regulatory T cells; VCAM-1, vascular cell adhesion molecule; VLA-4, integrin α4β1.

Hairy cells express CD40 and are activated by CD40 ligand (CD154) on T cells. Thus, T cells in microenvironment could contribute to HCL progression. Various cytokines are elevated in serum from patients with HCL, affecting the biology of HCL, which include tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), IL-2Rα (CD25), TGFβ, IFN-α, and granulocyte-macrophage colony-stimulating factor (GMCSF).^35,42, 43, 44, 45 Hairy cells also overexpress CD11c, CD103, CD200, CD1d, cyclin D1, and annexin A1 (ANXA1), which are diagnostic markers of HCL, although their functional significance in HCL biology remains ambiguous.^46,47

Genetic alterations and dysregulated signaling

Except for the origin of hairy cells, genetic drivers in HCL have also been obscured for a long time due to lack of recurrent chromosomal aberrations and genetic events. It was not until 2011 that genomic studies identified a gain-of-function mutation, BRAF(V600E), present in all patients with HCL.¹² This mutant aberrantly activates downstream extracellular signal-regulated kinase (ERK) MAPK signaling, which is responsible for molecular pathogenesis and “hairy” morphology of HCL.^48,49 It is worth noting that BRAF(V600E) can be either heterozygous or hemizygous, resulting from a heterozygous loss of chromosome 7q.⁵⁰ However, the biological implications of hemizygous vs heterozygous BRAF-mutated HCL are not fully understood. Apart from BRAF(V600E), other BRAF mutations are also described in HCL, including BRAF(F468C), BRAF(D449E), BRAF(S602T), BRAF(F595L), and BRAF(W604L),^51,52 whereas all these instances are reported as singular cases for respective mutations.

BRAF(V600E) has been recognized as a central genetic driver in HCL, largely due to its consistent presence across the entire tumor cell population and detection in all anatomical sites associated with disease.^13,53, 54, 55, 56, 57 This unique characteristic enables reliable detection, even during instances of relapse. Since being reported in 2011, BRAF(V600E) has been detected in >97% patients with HCL from different cohorts,^49,57, 58, 59, 60 except for 1 study, in which a lower frequency (79%) of BRAF(V600E) was shown, with 5 patients negative for BRAF(V600E) presented a IGHV4-34 rearrangement that is usually absent in HCL.⁶⁰ IGHV4-34–positive patients with HCL could be another subset of HCL different from patients carrying BRAF(V600E) mutation and are usually associated with a poor prognosis including poor response to standard therapy and shorter overall survival.¹⁸ Interestingly, downstream effector of BRAF, MEK1, was found to be mutated in IGHV4-34–positive HCL,^18,61 suggesting a critical role of ERK signaling in HCL pathology.

In line with the discovery of BRAF(V600E), its inhibitors have proven effective in treating patients with HCL, including vemurafenib and dabrafenib.^16,62,63 Meanwhile, hairy cells undergo loss of characteristic gene expression signature and hairy morphology, ultimately succumbing to cell death after treatment with BRAF and MEK inhibitors.⁶² However, immunodeficient mice reconstituted with hematopoietic stem cells, carrying BRAF(V600E) or MEK1 mutation, did not present full phenotypic symptoms of HCL, implying that other concurrent genetic alterations are required for driving HCL ontogeny.^63,64

Indeed, other genes are found recurrently mutated together with BRAF(V600E) and play a role in disease progression. The tumor suppressor CDKN1B gene, encoding the cyclin-dependent kinase inhibitor p27, has been shown as the second most mutated gene in patients with HCL, accounting for 16% of cases.⁴² Furthermore, p27 expression is absent or weak in all HCL cases, indicating that additional mechanisms of CDKN1B silencing exist in HCL.⁶⁵ p27 downregulation in HCL may facilitate cloning expansion. Another cyclin-dependent kinase inhibitor that promotes cellular senescence, CDKN1A/p21, is also disrupted in some patients with HCL,^66,67 suggesting that cell cycle disruption plays an important role in driving hairy cell growth. The transcription factor KLF2 has been shown mutated in 4 of 24 patients with HCL (16%), which mainly affects cell differentiation and B-cell homing to lymph nodes.^68,69 Although Trp53 mutation seems infrequent in HCL, it serves as an important indicator for disease relapses or refractory to standard purine analog therapy.^49,70, 71, 72 Other recurrent mutations in patients with HCL with lower frequency include CCND1, KMT2C, and NOTCH1.^58,73, 74, 75

Except for a stable genomic profile of BRAF(V600E) in HCL, epigenetic alterations also significantly contribute to HCL ontogeny. In patients with HCL, genome-wide promotor methylation profile was different from that of normal B cells and consistent with a constitutive activation of ERK signaling.^31,75 Notably, PTEN was shown downregulated by promoter methylation.³¹ Moreover, the microRNA expression profile was altered in HCL, including miR-221/miR-222 family, which targets tumor suppressor CDKN1B (p27).⁶⁷ Altogether, these discoveries suggest that cooperating genetic and epigenetic events are needed for BRAF(V600E) to drive HCL ontogeny.

The discovery of BRAF(V600E) unveils a critical role of MAPK signaling in HCL ontogeny. This signaling pathway is initiated by receptor tyrosine kinase engagement that activates Ras small GTPases. In turn, active Ras recruits RAF/MEK complexes to the plasma membrane, where the RAF/MEK/ERK kinase cascade is turned on through complex molecular interactions and sequential phosphorylations (Figure 2).75, 76, 77, 78, 79, 80, 81, 82 Dysfunction of MAPK signaling often causes human cancers and developmental disorders.76, 77, 78, 79 As for BRAF(V600E), it is a constitutively active mutant that phosphorylates MEK independent of upstream signaling. The high levels of MEK and ERK phosphorylation induced by BRAF(V600E) in hairy cells can be effectively blocked by BRAF inhibitors, such as vemurafenib, dabrafenib, and encorafenib.^62,80 Coupled with this, BRAF inhibitors downregulate HCL-specific signatures, including typical hairy morphology, immunophenotype, and clinal presentation in patients.⁶² Targeting MEK directly by inhibitors, such as trametinib and binimetinib, is also verified to be effective for treating HCL, indicating that ERK signaling is critical for HCL ontogeny.⁶²

Figure 2.

Schematic diagram depicting signaling pathways in HCL. The MAPK signaling pathway (left) is physiologically activated when a ligand binds to a RTK on cell surface. This interaction triggers the activation of RAS, which subsequently turns on the RAF-MEK-ERK kinase cascade through complex molecular interactions and sequential phosphorylations. The PI3K-AKT-mTOR pathway (left) runs parallel to the MAPK pathway downstream of RTK and RAS. Upon activation, PI3K converts PIP2 into PIP3, which recruits PDK1 and ATK to the plasma membrane via their PH domains and triggers the PDK1-AKT-mTOR kinase cascade. The BCR signaling pathway (middle) is activated upon antigen loading, inducing LYN and SYK phosphorylation, which in turn activates BTK. Active BTK phosphorylates PLCγ, which catalyzes the production of IP3 and induces calcium mobilization, eventually turns on NF-κB and ERK. BTK also involved chemokine receptor CXCR4 signaling, which is important in cell trafficking. The NF-κB pathway (right) is activated when inflammatory receptors are engaged by cytokines or pathogenic molecules; adapter molecules are recruited to assemble signaling complexes that activate inflammatory kinases such as TAK1. These inflammatory kinases then phosphorylate IKK and turns on NF-κB by promoting ubiquitin-dependent degradation of IκB. Alterations in these signaling pathways have been significantly implicated in HCL pathology. LYN, Lck/yes novel tyrosine kinase; mTOR, mammalian target of rapamycin; PDK1, phosphoinositide-dependent kinase-1; PIP₂, phosphatidylinositol 4,5-bisphosphate; PH, pleckstrin homology; PLCγ2, phospholipase C gamma 2; RTK, receptor tyrosine kinase; SYK, spleen tyrosine kinase; TAB1, TGFβ-activated kinase 1-binding protein 1; TAK1, TGFβ-activated kinase 1; TLR, toll-like receptor; TNF-α, tumor necrosis factor α; TRAF6, tumor necrosis factor receptor-associated factor 6; Ub, ubiquitin.

B-cell receptor (BCR) signaling has also been highlighted in HCL. This signaling is essential for B-cell survival and mediates antigen-driven activation, proliferation, and differentiation. Upon antigen loading, BCR signaling drives phosphorylation of various kinases and adapter proteins, including spleen tyrosine kinase (Syk), Bruton tyrosine kinase (BTK), and phospholipase C gamma 2 (PLCγ2), which leads to intracellular calcium mobilization and nuclear factor κB (NF-κB) activation (Figure 2).⁸³ In hairy cells, BCR signaling promotes cell viability by inducing phosphorylation of ERK and BTK, as well as secretion of chemokines CCL3 and CCL4.^84,85 Nevertheless, BCR crosslinking may also result in diverse responses in hairy cells, including apoptosis.⁸⁴ As a key regulator of BCR signaling, BTK has been shown to be overexpressed in hairy cells and to promote cell survival and proliferation.⁸⁶ Blocking BTK effectively inhibits hairy cell growth, proliferation, and secretion of CCL3 and CCL4.⁸⁵ The BTK inhibitors, ibrutinib and zanubrutinib, have exhibited promising efficacy in relapsed or refractory HCL, with overall response rates (ORR) of 54% and 58%, respectively.^87,88 These agents have shown durable remissions and manageable safety profiles, highlighting their potential as novel therapeutics for this difficult-to-treat population.

Parallel to ERK signaling, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling is also critical for various cellular processes, including growth, survival, proliferation, metabolism, and angiogenesis.⁸⁹ Downstream of receptor tyrosine kinase and RAS, PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which recruits and activates phosphoinositide-dependent protein kinase 1 (PDK1) and AKT, ultimately turning on mTOR signaling (Figure 2). Dysregulation of this pathway is commonly observed in cancers, including HCL.90, 91, 92 Similar to ERK signaling, this pathway is often overactive in HCL due to oncogenic mutations or constitutive receptor signaling. Therapeutically, PI3K inhibitors such as idelalisib, which specifically target PI3Kδ, have shown promising efficacy for treating B-cell malignancies,^93,94 suggesting that these new agents may serve as alternative therapeutics for certain subgroups of HCL.

As a master regulator of inflammation, NF-κB activated by BCR signaling plays a pathogenic role in HCL and other B-cell lymphomas, by promoting leukemic cell survival and proliferation.95, 96, 97 Apart from BCR signaling, NF-κB can be also activated by cytokines (eg, tumor necrosis factor α and IL-1), pathogen-associated molecular patterns, and stress-induced signals (Figure 2).⁹⁸ Given its critical role in B-cell malignancies, targeting NF-κB signaling represents a promising therapeutic strategy.⁹⁹ Blocking IκB kinase protein complex (IKK) complex or NF-κB directly disrupts leukemic cell survival. Therapeutically targeting NF-κB is exemplified by bortezomib, a proteasome inhibitor that prevents IκB degradation and subsequent NF-κB activation, demonstrating efficacy in B-cell malignancies.⁹⁹ Similarly, the BTK inhibitor ibrutinib could indirectly inhibit NF-κB in B-cell leukemias.^93,94,99

Other signaling pathways that are dysregulated and significantly affect HCL pathogenesis include Rho signaling, which alters cellular cytoskeletal architecture and maintains a distinctive cellular morphology100, 101, 102, 103, 104, 105, 106, 107, 108, 109; and CXCR4-CXCL12 chemotactic signaling, which controls trafficking and retention of hairy cells in the bone marrow and spleen, supporting cell survival and proliferation.110, 111, 112, 113, 114 Drugs targeting these signaling pathways could have potential efficacy in patients with HCL.

However, the biological impact of these HCL-specific alterations is still poorly understood, highlighting a need for additional studies to clarify their contributions to HCL ontogeny. To overcome this hurdle, we recently investigated whether BRAF(V600E) mutation together with loss of tumor suppressors such as Trp53, p27, or PTEN in murine B cells could induce a leukemic syndrome resembling human HCL.¹⁹ Our results showed that mice with conditional BRAF(V600E) knockin and Trp53 or PTEN knockout simultaneously developed splenomegaly and hepatomegaly, along with hematopoietic abnormalities such as decreased hemoglobin levels and thrombocytopenia. Moreover, we found that NF-κB signaling was activated in murine spleens with genetic modifications, suggesting its potential role in disease progression. These 2 mouse models also highlight distinct features of HCL subtypes. The BRAF(V600E) knockin and Trp53 knockout mice displayed indolent disease with prolonged survival, closely resembling classical HCL in humans. By contrast, BRAF(V600E) knockin PTEN knockout mice developed rapidly progressive splenomegaly and hepatomegaly, modeling an aggressive HCL subtype driven by hyperactive PI3K/AKT signaling and associated with poor prognosis.¹¹⁵ Both models provide valuable resources for studying HCL pathogenesis and for evaluating novel therapeutic strategies.

HCL diagnosis

HCL presents as an indolent form of leukemia. Approximately 25% of patients remain asymptomatic at diagnosis, with disease often detected incidentally during routine blood tests revealing cytopenias, most commonly anemia, neutropenia, thrombocytopenia, and occasionally monocytopenia.^2,3 Nevertheless, symptomatic patients commonly present with disease-related manifestations. Anemia typically manifests as fatigue and exertional dyspnea, whereas thrombocytopenia presents with hemorrhagic features including ecchymoses and mucocutaneous bleeding.3, 4, 5 Splenomegaly often causes abdominal fullness or left upper quadrant discomfort. Constitutional symptoms such as malaise and unintentional weight loss may also occur. HCL-associated immune dysfunction predisposes patients to recurrent infections, particularly with herpesviruses, common bacterial pathogens, and opportunistic organisms including Pneumocystis jirovecii.⁴ Furthermore, patients have an increased risk of developing secondary lymphoid and nonlymphoid malignancies.^6,8

HCL is very similar to another 2 HCL-like B-cell malignancies: HCLv and SMZL, which also present with marked splenomegaly, as well as no lymph node involvement and circulating leukemic cells.^10,13 However, HCLv and SMZL respond poorly to purine analogs and interferon, highlighting a need for more specific diagnostic markers to distinguish them from classic HCL as well as distinct therapeutic strategies. Advancements in diagnostic techniques have enhanced accuracy and efficiency of HCL diagnosis. At present, immunophenotyping using flow cytometry has become a cornerstone in HCL diagnosis, which detects characteristic surface markers such as CD11c, CD25, CD103, and CD123.

ANXA1 also served as a potential marker for HCL diagnosis, given its specific upregulation in HCL.⁴⁷ Immunostaining of bone marrow with anti-ANXA1 monoclonal antibody (mAb) is highly specific for HCL at first diagnosis.⁴⁹ However, monitoring ANXA1 is not suitable for relapsing cases because of the presence of macrophages and T cells that express ANXA1.¹¹⁶ Under this situation, the BRAF(V600E) mutation can serve as a promising marker for HCL diagnosis, which is detected in fresh bone marrow and peripheral blood samples by polymerase chain reaction and Sanger sequencing.¹¹⁷ Alternatively, BRAFV600E could be detected in routine paraffin sections by immunostaining with anti-BRAF(V600E) mAb.^118,119

Although some patients with HCL are completely asymptomatic, with their diagnosis made incidentally, symptomatic patients exhibit clinical manifestations associated with splenomegaly or cytopenias, including recurrent infections and asthenia. Therapy is generally initiated when hemoglobin level is <11 g/dL, platelet counts are <1.0 × 10⁹/L, and/or neutrophil counts are <1.0 × 10⁶/L.⁷

HCL treatment

The initial standard treatment for HCL was splenectomy, which achieved 5-year survival rates of 86%.^120,121 Although largely supplanted by newer therapies, splenectomy remains clinically relevant for patients with symptomatic splenomegaly, severe thrombocytopenia with bleeding, or during pregnancy.¹²² A therapeutic breakthrough occurred in 1984 with the introduction of IFN-α, which transformed HCL from an untreatable condition to a manageable disease, demonstrating significant hematologic improvement in anemia, leukopenia, and thrombocytopenia.⁷ However, IFN-α2b yields complete response rates (CRR) of only ∼10%.^123,124

A major therapeutic advance in HCL management came with the introduction of purine analogs, which currently serve as first-line treatment. The first such agent pentostatin (2'-deoxycoformycin) has demonstrated remarkable efficacy, with an ORR of 96% and a CRR of 59%.^125,126 It is given by IV at a dose of 4 mg/m² every 2 weeks until complete remission or maximum response is achieved.¹²⁷ Long-term follow-up studies reveal durable responses, with relapse-free survival rates ranging from 80% to 88% at 5 years and from 67% to 76% at 10 years.128, 129, 130 However, pentostatin therapy is associated with significant toxicities including fever, cytopenias, gastrointestinal disturbances (vomiting), and neurological effects (paresthesias and dysgeusia). Cladribine (2-chlorodeoxyadenosine), another purine analog, shows comparable efficacy, with CRR ranging from 75% to 98% in long-term studies.131, 132, 133 It is administered in several different schedules and by different routes, such as a single 5- to 7-day course via IV infusion or alternatively subcutaneously on a once-per-day regimen.^48,134 Cladribine is generally avoided in patients with active infections, whereas pentostatin has been used effectively in this setting.^7,48 In patients without active infection, cladribine is preferred as first-line therapy for HCL due to its convenient and straightforward administration schedule. Despite their effectiveness, ∼30% to 40% of patients eventually relapse after either cladribine or pentostatin therapy.

For patients with relapsed HCL, retreatment with a purine analog, with or without rituximab, remains the standard of care for first relapses. With a single-agent purine analog, it achieves an ORR of >95%, although the CRR is typically lower than that with initial therapy.^8,48,122 Combining cladribine with rituximab has demonstrated superior efficacy in relapsed HCL, achieving a 100% CRR and 100% overall survival.¹³⁵ In 2018, the US Food and Drug Administration approved moxetumomab pasudotox (Lumoxiti) for patients with HCL who have failed ≥2 prior systemic therapies.^136,137 However, it was withdrawn from the US market in 2023 not due to efficacy or safety concerns but rather minimal clinical adoption, administrative and monitoring challenges, and competition from simpler, effective alternatives.

The discovery of the BRAF(V600E) mutation marked a major breakthrough in HCL, because it constitutively activates the MAPK/ERK pathway, driving leukemic cell proliferation and survival.¹² Targeting this oncogenic pathway with BRAF or downstream MEK inhibitors has demonstrated promising efficacy.78, 79, 80 Clinical studies have shown that monotherapies with the BRAF inhibitor vemurafenib exhibited remarkable activity in two phase 2 trials conducted in Italy and the United States, achieving an ORR of 96% to 100% and a CRR of 35% to 42% in patients with relapsed or refractory HCL.^16,138 Nonetheless, drug response is not always durable, and disease may relapse after drug discontinuation, with a median relapse-free survival of 19 to 25 months. In addition, drug-related adverse events were observed, including rash, arthralgia, and secondary cutaneous tumors.^124,139 To address resistance mechanisms, combination therapies targeting multiple nodes of this pathway have been explored. Vemurafenib plus the MEK inhibitor cobimetinib improves response durability by suppressing compensatory MAPK pathway reactivation.¹⁴⁰ Notably, a combination of anti-CD20 mAb with BRAF inhibitor has also been investigated in HCL treatment. Combining time-limited vemurafenib with obinutuzumab has achieved a CR rate of 95% in previously untreated HCL, with 96% of patients attaining minimal residual disease negativity.¹³⁹

Allogeneic hematopoietic cell transplantation (allo-HCT) has been reported as a treatment option for patients with refractory or relapsed HCL.141, 142, 143 A case study showed that a patient with refractory HCL received high-dose chemoradiotherapy followed by bone marrow transplantation. Four years after treatment, no hairy cells were detected, and the patient’s peripheral blood count remained normal. Additionally, this patient showed no evident predisposition to infections or significant long-term toxicities from therapy.¹⁴² Consistently, other studies on patients with HCL or splenic B-cell lymphoma/leukemia with prominent nucleoli undergoing their first allo-HCT for refractory or relapsed disease reported a CR rate of 59%, with a nonrelapse mortality rate of 14%.¹⁴¹ Although allo-HCT has a potential to induce long-term remission in various hematologic malignancies, including HCL, it should be noted that successful cases remain limited so far. In addition, some novel therapeutics are currently under investigation, including chimeric antigen receptor T cells and mAbs targeting ROR1 and ROR2.¹⁴³

Conclusions and perspectives

Gene expression profiling and related molecular studies have significantly advanced our understanding of HCL's origin, pathogenic mechanisms, and characteristic features, including its unique survival pathways, morphological properties, and adhesive behavior. Although these investigations have yielded valuable insights, findings require further experimental validation and systematic integration to fully elucidate their biological significance. Several critical questions remain unresolved, which we address below.

First of all, genetics and pathogenesis of HCL remain incompletely understood. Although the BRAF(V600E) mutation is a hallmark of classic HCL, it does not fully explain disease's complexity, heterogeneity, and resistance mechanisms observed in patients. Moreover, the interplays among genetic mutations, epigenetic factors, tumor microenvironment, and immune system dysregulation remain poorly characterized. Future research may focus on uncovering these gaps, which may result in novel and precise therapeutics, ultimately improving outcomes for patients with HCL.

Although HCL responds well to frontline chemotherapeutic purine analogs,^7,48 half of patients experience at least 1 relapse, after which they become progressively resistant. Given the ubiquitous BRAF(V600E) mutation in HCL, BRAF/MEK inhibitors are used for treating HCL and have turned out to be effective for relapsed/refractory patients.¹⁴⁴ However, their efficacy is also abrogated eventually by acquired resistance. Although several mechanisms underlying BRAF/MEK inhibitor resistance have been uncovered in solid tumors harboring BRAF(V600E),145, 146, 147, 148, 149 knowledge is scarce regarding patients with HCL relapsing after treatment. Overcoming therapeutic resistance in HCL may require integrated strategies. Rational combination regimens targeting BRAF/MEK alongside PI3K, BTK, or CD20 pathways may preempt compensatory signaling. Alternatively, sequential incorporation of targeted agents with purine analogs or mAbs could prolong remissions while limiting selective pressure on individual pathways. In addition, immunomodulatory interventions, including checkpoint blockade, engineered T cells, or tumor microenvironment–modifying agents, may restore immune surveillance and suppress resistant clones. Nevertheless, functional evaluation with BRAF(V600E)-driven HCL mouse models provides an appropriate preclinical framework to optimize these approaches. Together, these strategies would hold promise for durable disease control and improved long-term outcomes.

Conflict-of-interest disclosure: The authors declare no competing financial interests.