Absence of BRAF-V600E in the human cell lines BONNA-12, ESKOL, HAIR-M, and HC-1 questions their origin from hairy cell leukemia

To the editor:

Hairy cell leukemia (HCL) shows distinct clinicopathologic, immunophenotypic, and gene expression features.^1,–3  We previously identified the BRAF-V600E mutation as the disease-defining genetic event in HCL.⁴  This mutation is present in virtually all cases of HCL but rarely in other B-cell lymphomas, remains stable over time (being consistently detectable at relapse), and leads to the constitutive activation of the mitogen–activated protein kinase (MAPK) pathway⁴  that is potentially druggable with BRAF-V600E inhibitors. These findings have been confirmed in 2 studies recently published in this journal.^5,6 

Functional assays in HCL have been hampered by the scarcity of leukemic cells available for analysis because of frequent pancytopenia and/or punctio sicca at marrow aspiration. Moreover, no animal models of HCL are available.³  To overcome these problems, cell lines have been established from HCL patients^7,,–10  and used for functional studies. However, there is no definitive evidence that they are of authentic HCL origin.

To clarify this issue, we searched for BRAF-V600E (the genetic hallmark of HCL) in the human HCL cell lines BONNA-12, ESKOL, HAIR-M, and HC-1 obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). None of them carried BRAF-V600E (Figure 1). However, HAIR-M carried a clonal heterozygous T-to-A transversion at position 1782 of the BRAF coding sequence (not shown), leading to the replacement of aspartic acid with glutammic acid (D594E). This variant has been previously reported in only 1 case (a primary cutaneous melanoma¹¹ ) of > 19 000 BRAF-mutated cancer samples listed in COSMIC-v56 (Catalog of Somatic Mutations In Cancer). Although this missense variant (D594E) is not described as a germ line polymorphism in single-nucleotide polymorphism (SNP) databases (dbSNP135 and 1000-genomes), its somatic origin in that melanoma patient was not evaluated.¹¹  Thus the functional consequences of BRAF-D594E in the HAIR-M cell line remain unclear. Taken together, these results cast doubts on the HCL origin of the BONNA-12, ESKOL, HAIR-M, and HC-1 cell lines.

Doubts were also reinforced by the morphologic appearance of these cell lines (similar to lymphoblastoid cells), their EBV-positivity (except for HAIR-M),^7,,–10  and their lack of the typical HCL immunophenotype (coexpression of annexin-1/CD25/CD11c/CD103; not shown). Similarly, ESKOL and HC-1 cells xenotransplanted in severely immunodeficient NSG mice showed the morphology of diffuse large B-cell lymphoma and were annexin-1 negative (Figure 1).

Because these cell lines do not represent a reliable HCL model either in vitro or in vivo, we sought to engraft in NSG mice MACS-purified (> 90% pure) patients' hairy cells. No engraftment was observed in a mouse that was injected intravenously with 3.8 million HCL cells and died 1 year later. Engraftment was not achieved even in another mouse that died 10 months after injection of 15 million leukemic cells from a different HCL patient harboring a hemizygous/homozygous BRAF-V600E mutation and presenting with markedly high hairy cell count (WBC 39000/mmc). Conversely, as few as 1.2 million ESKOL cells and 0.98 million HC-1 cells were enough to kill the animals as early as 3 weeks after intravenous injection. These results suggest that primary HCL cells appear difficult to engraft even in NSG mice.

Altogether our findings call for the development of conditional knock-in mice expressing BRAF-V600E in specific mature B-cell subsets as appropriate models for studying HCL.