Recent advances in understanding and managing hairy cell leukemia

Introduction

Hairy cell leukemia (HCL) is a rare mature B-cell malignancy, which was initially described by Bouroncle et al. in 1958¹. HCL has an incidence of 0.3 cases per 100,000 individuals². It occurs about four times more often in men than in women, with a median age at diagnosis of 55 years³. HCL cells are characterized by thin cytoplasmic hair-like projections, giving the disease its name⁴. Leukemic hairy cells accumulate in the bone marrow and cause pancytopenia, which is the most common finding at initial presentation. HCL patients report symptoms of fatigue, infections, and, occasionally, left-sided abdominal pain caused by splenomegaly. In contrast to many B-cell malignancies, lymphadenopathy is rare in HCL patients⁵. A severe fibrotic reaction is commonly found in the bone marrow of HCL patients, which often complicates a diagnostic bone marrow aspiration. The diagnosis is usually based on the detection of typical morphological features and a unique immunophenotype with flow cytometry of peripheral blood and/or histological and immunohistochemical (IHC) analysis of trephine biopsies. HCL cells show a characteristic gene expression profile signature that points to their origin from memory B cells^6,7. Although standard treatment with purine analogues is very effective in the majority of patients with HCL, there is a small subset of relapsed and refractory HCL patients who qualify for investigational therapies with monoclonal antibodies or small molecule compounds⁸. In this review, we will discuss these novel therapeutic agents as well as recent advances in understanding the molecular pathogenesis of HCL.

The biology of HCL

In 2011, Tiacci et al. discovered that classical HCL is characterized by a gain-of-function mutation of the BRAF serine/threonine protein kinase (V600E)^9,10. In the initial validation series, all HCL patients showed this particular mutation, while a set of 195 B-cell lymphomas and leukemias did not harbor a mutated BRAF gene. The vast majority of BRAF-V600E mutations in HCL are heterozygous. Homozygous mutations are rare but have been suggested to be associated with a more aggressive disease course¹¹. Recurrent deletions of the BRAF gene locus on chromosome 7q34 have been described in HCL and lead to loss of heterozygosity¹². BRAF mutations, different from V600E, seem to be extremely rare in HCL and have been described in only two patients so far¹³. The incidence of BRAF mutations in nearly 100% of HCL cases at diagnosis (encompassing the whole disease spectrum), their somatic nature, their presence in the entire tumor clone, and their high stability at relapse strongly suggest that the pathogenesis of HCL critically depends on constitutively activated BRAF^10,14,15.

Chung et al. reported that BRAF-V600E mutations are already present in hematopoietic stem cells (HSCs) or B-cell lymphoid progenitors of HCL patients and that these patients exhibit marked alterations in hematopoietic stem/progenitor cell (HSPC) frequencies¹⁶. Transplantation of BRAF-V600E-mutant HSCs from an HCL patient into immunodeficient mice resulted in stable engraftment of BRAF-V600E-mutant human hematopoietic cells, revealing the functional self-renewal capacity of HCL HSCs. However, none of the transplanted mice developed typical HCL, strongly suggesting that the development of a full HCL phenotype may require a permissive epigenetic background (likely restricted to a particular stage of B-cell differentiation) and/or the acquirement of further genetic lesions¹⁶.

The BRAF-V600E mutation constitutively activates BRAF, providing oncogenic signaling through the MEK-ERK cascade^10,17 (Figure 1). Both in vitro and in vivo studies have demonstrated that BRAF-dependent phospho-ERK activation is a critical signaling event in HCL^10,18,19. Moreover, in vitro treatment of primary purified HCL cells with BRAF and MEK inhibitors has resulted in marked dephosphorylation of MEK/ERK, silencing of the RAF-MEK-ERK pathway transcriptional output, loss of the specific HCL gene expression profile signature, change of the characteristic morphology of the leukemic cells (from “hairy” to “smooth”), and eventually apoptosis^14,15,20.

Figure 1. RAF-MEK-ERK signaling pathway in hairy cell leukemia.

The figure shows the RAF-MEK-ERK signaling pathway in hairy cell leukemia and highlights targets for therapeutic intervention.

Aberrant expression of cell cycle-related proteins such as cyclin D1 has been shown to be reversible using inhibitors of activated BRAF signaling, suggesting that expression is not a constitutive disease trait but elicited by MEK/ERK signaling and oncogenic BRAF mutations, respectively¹⁸. This could have a significant effect on the assessment of minimal residual disease (MRD) when considering inhibitor treatment because the profile of the marker cyclin D1 might be dynamic, as well as on targeted drug therapy, which may be shortened due to the on-target effect of inhibitors.

Differential diagnosis of HCL

Historically, there were two different forms of HCL: the more-common classical HCL (90%) and the less-frequent HCL variant (10%). HCL variant is characterized by a more aggressive disease course and poor response to purine analogs²¹. Most importantly, HCL variant cases are commonly negative for BRAF-V600E mutation, indicating that HCL variant is a biologically distinctive entity. A small subset of patients with bona fide classical (HCL) who also do not harbor any BRAF mutation has been reported only in a single study²². However, these cases are often characterized by an IGHV4-34 immunoglobulin rearrangement, which is in general absent in classical HCL and is associated with as poor a prognosis as HCL variant²².

Almost 50% of HCL-variant and IGHV4-34-expressing HCL cases were found to harbor activating mutations in the MAP2K1 gene encoding MEK1²³. All but one of the identified mutations (n=15) have been described and are known to strongly increase phospho-ERK levels and consequently cell proliferation²³. These findings underline the importance of constitutive MEK-ERK signaling, even in this HCL-like disorder.

HCL cells typically show a distinctive immunophenotype co-expressing CD19, CD20, CD11c, CD25, CD103, and CD123. In contrast, HCL variant lacks the expression of CD25 and CD123²⁴. Moreover, HCL cells strongly express CD200, which can also be used as another distinctive marker to differentiate HCL^25,26. BRAF-V600E is now regarded as a specific oncogenic mutation occurring only in HCL²⁷. Another distinctive feature of HCL is the expression of annexin A1, which is easily accessible by immunohistochemical staining²⁸. In addition to HCL variant, the 2016 revision of WHO classification of lymphoid neoplasms recognizes two other entities resembling HCL: splenic marginal zone lymphoma (SMZL), usually associated with NOTCH2 mutations, and splenic diffuse red pulp small B-cell lymphoma (SDRPBCL), still listed as a provisional entity, whose genomic landscape has not been yet clarified²⁷. Table 1 summarizes the most important differential diagnoses of HCL and their characteristic markers.

Testing for the BRAF-V600E mutation in routine clinical practice can be helpful as an additional marker if there is any diagnostic uncertainty. For relapsed and refractory patients, we strongly recommend evaluating the BRAF mutation status, since this may serve as a therapeutic target. The limited number of HCL cells present in the peripheral blood requires highly sensitive molecular assays to detect BRAF mutations (e.g. allele-specific polymerase chain reaction)⁹. Alternatively, BRAF-V600E mutation-specific antibodies can be used for immunohistochemical staining in bone marrow biopsies^29,30. However, further validation of the diagnostic utility of these reagents in a larger number of cases is required.

Cooperating mutations of BRAF-V600E in HCL

In addition to the BRAF-V600E mutation, the most common genetic alteration in classical HCL was a copy number loss of chromosome 7q. The minimally deleted region of this copy number alteration includes the wild-type locus of BRAF. This genetic lesion subdivides individuals with classical HCL into those with hemizygous versus heterozygous mutations of BRAF¹². A whole-exome sequencing study of relapsed and refractory HCL patients revealed known cancer-associated genes, such as EZH2 and ARID1A, as well as novel inactivating mutations of the cell cycle inhibitor CDKN1B (p27)³¹. In a cohort of 81 mostly untreated HCL patients, the incidence of CDKN1B mutations was 16%³¹. While a clinical impact of CDKN1B mutations was not found, the data identify CDKN1B as the second most commonly mutated gene in HCL. CDKN1B is a critical element of cell-cycle control and a known tumor suppressor in different solid cancers³². CDKN1B prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes and thereby regulates cell-cycle progression in the G1 phase. Interestingly, BRAF-induced senescence in premalignant naevi is circumvented by deletion or mutation of CDKN2A in invasive melanoma³³. In BRAF-mutated hairy cell leukemia, CDKN1B loss may serve as a mechanism to escape oncogene-induced senescence³¹. In addition to CDKN1B mutations cooperating with BRAF-V600E, recurrent, inactivating mutations in KMT2C (MLL3) were identified in 15% and 13% of classical HCL and HCL variant, respectively¹². Another study described somatic mutations or deletions of the Krüppel-like factor 2 (KLF2) in 4 of 24 (16%) HCL patients examined³⁴, but KLF2 mutations are more frequent in other B-cell malignancies, such as SMZL (31%) and diffuse large B-cell lymphoma (26%)^34,35. Although we have better described the genetic landscape of HCL during recent years, the function of mutations cooperating with BRAF-V600E remains to be elucidated.

Conventional therapeutic strategies in HCL

At initial diagnosis, most patients will require treatment owing to hematopoietic insufficiency. Accepted indications to start treatment are hemoglobin <11 g/dL, platelet count <100,000/µL, or absolute neutrophil count <1,000/µL. Less frequently, increased susceptibility to infections or symptomatic splenomegaly may also serve as criteria to start treatment⁵. The introduction of the purine analogs cladribine and pentostatin into the treatment landscape of HCL significantly improved the outcome of HCL patients³⁶. Prospective randomized studies comparing pentostatin and cladribine as first-line treatment have not been conducted, but retrospective studies suggested equivalent activity of both drugs with induction of complete remission (CR) in approximately 85% of untreated patients^37–39. The median treatment-free survival for patients with CR after treatment with purine analogs was more than 10 years in most studies^40–42. In contrast, patients with a partial remission (PR) had a significantly shorter treatment-free survival of 3 years^43,44. Although achievement of CR, beyond first-line treatment, is associated with similar good outcome, the proportion of patients with insufficient response and early relapse increases with each treatment round^38,44. Apart from pentostatin and cladribine, there are hardly any effective, approved treatment options: interferon alpha, splenectomy, and rituximab monotherapy should be considered only in a small subset of patients⁵. Recently, multiple biology-based treatment options have become available for refractory HCL patients, which will be discussed below.

Therapeutic targeting of BRAF-V600E

Based on the discovery of the BRAF-V600E mutation in virtually all patients with classical HCL¹⁰, as well as the success of BRAF inhibitor treatment in melanoma⁴⁵, it was intuitive that BRAF inhibition is a promising treatment strategy for patients with HCL. The first patient exposed to the BRAF inhibitor vemurafenib indeed showed an immediate and striking response, proving oncogene-dependence and clinical activity^18,46. The dynamics of the response were notable, with a spleen size reduction of more than 6 cm in only 6 days and improvement of blood count (hemoglobin, platelets, and granulocytes) within 1 month⁴⁶. Soon after the initial report, multiple studies confirmed the efficacy of both vemurafenib^47–49 and (because of availability) later dabrafenib⁵⁰. One report also presented the use of vemurafenib in a primary refractory patient with severe pulmonary aspergillosis⁵¹, where the avoidance of myelotoxicity may be particularly advantageous. Individual dosing regimens of vemurafenib with a minimum of 240 mg twice daily were reported in a series of 21 patients with refractory and relapsed HCL. Both antitumor and side effects were found to be independent of vemurafenib dosing⁵². Indeed, the initial melanoma study demonstrated response at the 2 × 240 mg level of dosing. Dose finding studies often pick dose levels based on the incidence of side effects rather than target inhibition. Therefore, the data suggest that individual dosing regimens in BRAF-driven cancers warrant reassessment in trials with implications for cost of cancer care.

Perspective

There is compelling evidence that BRAF inhibitors are clinically highly efficacious in HCL. However, the high percentage of incomplete responses and the lack of sustained remissions off drug call for the development of combination approaches. BRAF inhibitors could, for instance, be combined with anti-CD20 monoclonal antibodies to potentially eradicate BRAF inhibitor-resistant hairy cell leukemia cells. Patients with metastatic melanoma have been successfully treated with combined BRAF and MEK blockade⁵⁶, and the discovery of the reactivation of the MAPK pathway as a probable mode of resistance to vemurafenib in some patients also validates the use of this therapy. Studies for both approaches are on their way.