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Researchers Trace Origin of Hairy Cell Leukemia to Hematopoietic Stem and Progenitor Cells

NEW YORK (GenomeWeb) — Researchers have traced the BRAFV600E mutation in hairy cell leukemia back to hematopoietic stem and progenitor cells, as they reported in Science Translational Medicine today.

Through a series of sequencing analyses and animal model experiments, the researchers led by Omar Abdel-Wahab at Memorial Sloan Kettering Cancer Center found that the mutation was present in a subset of hematopoietic stem cells from hairy cell leukemia patients and that the expression of this mutation in mouse hematopoietic stem and progenitor cells led the mice to exhibit classic symptoms of human hairy cell leukemia. Additionally, they noted that a BRAF inhibitor might be a possible therapeutic for hairy cell leukemia.

"Our results predict that therapeutic mutant BRAF inhibition will have the capability to induce durable remissions and restoration of normal hematopoiesis in HCL," Abdel-Wahab and colleagues wrote in their paper

Hairy cell leukemia, the researchers noted, has been characterized as a mature B-cell malignancy because it expresses CD19, surface immunoglobulin, and has clonal rearrangements of immunoglobulin heavy and light chain genes, all of which are hallmarks of mature B cells. However, HCL cells also express cell surface markers not typically found on B cells, HCL patients exhibit symptoms that differ from those linked to other B-cell malignancies, and gene expression microarray studies have been unable to pinpoint which B cell is the source of HCL. And so, the researchers said, the cellular origin of HCL has been unclear.

To search for the cells along the hematopoietic cell lineage in which the BRAFV600E mutation arises and leads to hairy cell leukemia, the researchers purified hematopoietic stem cells, hematogones, myeloid progenitor cells, and hairy cell leukemia cells from the bone marrow of 14 HCL patients and age-matched controls. After double FACS sorting, the researchers used an allele-specific assay to search for the BRAFV600E mutation in those purified cell types.

From this, Abdel-Wahab and his colleagues found the mutation in hematopoietic stem cells, pro-B cells, and HCL cells of HCL patients, suggesting that the leukemia is initiated within the hematopoietic stem and progenitor cells rather than in committed lymphoid cells, as had been thought.

The researchers also quantitatively sequenced the BRAFV600E mutation in cDNA from purified HCL cells, hematogones, and hematopoietic stem cells from five additional patients and, at a mean sequencing depth of more than 1,000x, they reported a mean variant allele frequency of 63.1 percent, 23.0 percent, and 4.97 percent, respectively, in those cells.

Using a panel of 300 known cancer genes, the researchers sought to determine whether there were other mutations present alongside the BRAFV600E mutation that might also have a role in hematopoietic transformation. In the three patients analyzed, one only had the BRAFV600E mutation, one had the BRAFV600E mutation plus an ARID1A mutation, and one had the BRAFV600E mutation plus a MLL3 mutation in their HCL cells. Using MiSeq, the researchers expanded their scope to additional cell types, but still mainly found the BRAFV600E mutation.

The researchers also transplanted 3,000 hematopoietic stem cells from the bone marrow of an untreated HCL patient into mice. These mice developed an HCL immunophenotype and immunophenotypic hematopoietic stem cells. Unfractionated hCD45+/ cells also had a variant allele frequency of 4 percent at three months and 9 percent at six months after transplant.

"These data indicate that HSCs from HCL patients bearing the BRAFV600E mutation have functional self-renewal capacity, and strongly suggest that these cells give rise to HCL," the researchers said.

In a series of mouse models, Abdel-Wahab and his colleagues examined how the BRAFV600E mutation affected the HSC-progenitor cell compartment or the B-cell lineage.

For instance, in their model of postnatal HSPCs, mutant BRAF expression led to a lethal hematopoietic disorder marked by splenomegaly and hepatomegaly as well as anemia and thrombocytopenia by three weeks of age. Additionally, expression of the mutant BRAF in fetal hematopoietic cells led to 100 percent embryonic lethality.

In B-lineage cells, though, expression of mutant BRAF did not lead to a change in survival or to a hematopoietic phenotype.

"These data demonstrate that restricting BRAFV600E expression to committed B lymphoid cells does not result in malignant transformation, suggesting that the phenotype of HCL is driven by alterations in HSPCs," the researchers said.

Additionally in their transgenic mouse models, the researchers found that the BRAFV600E mutation led to changes in hematopoiesis in HSCs, but not B-lineage cells. For example, bone marrow expressing the mutant BRAF could be serially re-plated, indicating an increased self-renewal capability.

"The above genetic and functional data suggest that BRAF V600E mutations alter the differentiation and self-renewal potential of HSPCs and that the ensuing lymphoid transformation and block in myeloid and erythroid differentiation result in phenotypic HCL," the researchers said

Vemurafenib, a BRAF inhibitor, may be a possible therapy for hairy cell leukemia, the researchers noted.

Abdel-Wahab and his colleagues examined how the drug affected both a mouse model of the disease and patients with relapsed or refractory HCL participating in a phase II study. Treatment of mice led to improved anemia, reduced hepatosplenomegaly, and attenuated B cell clonogenic capacity. Meanwhile, patients given the drug experienced normalized HSPC frequencies.

"More genetic analysis will be required to confirm that BRAFV600E-bearing HSCs are eliminated in these patients, but the preliminary results are promising," added John Dick from the Princess Margaret Cancer Centre at the University of Toronto in an accompanying Science Translational Medicine article.