Abstract
Classic hairy cell leukemia (HCL), comprising 2% of leukemias, is an indolent B-cell malignancy with malignant lymphocytes expressing B-cell antigens CD20 and CD22, CD103, CD11c, CD25, Annexin A1, BRAF V600E mutation, and monoclonal immunoglobulin (Ig) rearrangement. HCL variant (HCLv), which is ~10% as common as HCL, has much poorer response to therapy and more aggressive course, lacks CD25 and Annexin A1, and is wild-type for BRAF. HCLv is considered separate from HCL by the World Health Organization in the unclassifiable splenic B-cell leukemia/lymphomas. Another poor-prognosis group overlaps HCL and HCLv in which unmutated IGHV4-34 Ig rearrangement is expressed. IGHV4-34+ leukemic cells can resemble classic HCL with CD25 and Annexin A1 expression, but are BRAF wild-type. No uniform mutation has been identified for HCLv and IGHV4-34+ HCL, although MAP2K1 (MEK1) mutations have recently been identified in half of cases. Thus HCLv and IGHV4-34+ HCL are less indolent leukemias with few therapy options and no known molecular target.
To study HCLv and IGHV4-34+ HCL, leukemic samples were purified by negative B-cell isolation followed by positive CD11c sorting. Following extraction (Qiagene, AllPrep DNA/RNA Kit), RNA samples from patients were analyzed in microarray studies (Human HT-12 v4 BeadChips, Illumina, Inc.). Expression data were compared by unpaired nonparametric analysis using Mann-Whitney. MYC expression using one probe (log2 values, mean +/- standard deviation) was 7.30 +/- 1.51 for 37 HCL vs 9.77 +/- 1.15 for 32 HCLv or IGHV4-34+ HCL (2-sided p<0.0001). For the other probe, expression was 7.07 +/- 1.51 vs 9.44 +/- 1.17 (p<0.0001). Expression data for MYC had previously been submitted for 31 chronic lymphocytic leukemia (CLL) and 16 HCL samples (Dataset GSE2350, Basso et al, Nat Genet, 37:382, 2005). By 1 probe for MYC, expression was 8.07 +/- 0.55 for CLL vs 9.31 +/- 1.19 for HCL (p=0.0023). By another MYC probe, expression was 9.28 +/- 0.47 for CLL vs 10.22 +/- 0.98 for HCL (p=0.0032).
To investigate potential therapeutic relevance of aberrant MYC expression in HCL, HCLv and IGHV4-34+ HCL, the bromodomain and extra terminal (BET) protein inhibitor JQ1, which has been associated with down-regulation of c-Myc via Brd4, was incubated with primary leukemic cells and ATP incorporation was measured. JQ1 inhibited 12 samples of HCL (IC50s 214 +/- 217 nM) more potently than 14 samples of CLL (IC50s 1.77uM +/-2.62 uM, p=0.020), and also inhibited 14 samples of HCLv or IGHV4-34+ HCL (IC50s 221 +/- 234 nM) more potently than the 14 CLL samples (p=0.0079). However, JQ1 inhibition was similar comparing HCL and HCLv or IGHV4-34+ HCL (p=0.89). To exclude non-specific inhibition of the cells, the inactive control molecule JQ1R was tested and was only 6.0% +/- 4.0% as active as JQ1 toward HCL or HCLv or IGHV4-34+ HCL samples. Normal peripheral blood mononuclear cells were resistant (IC50 > 20 uM).
In conclusion, our results show that MYC expression is higher in HCLv and IGHV4-34+ HCL than in classic HCL and higher in classic HCL than CLL. Moreover, JQ1 inhibits HCL or its variants more potently than CLL, although the inhibition assay used does not detect a difference between the variants and classic HCL. Further experiments with other inhibitors will be needed to determine if the increased expression of MYC in HCL and its poor-prognosis variants can be exploited for treatment.
Disclosures
No relevant conflicts of interest to declare.
Hairy cell leukemia cell lines expressing annexin A1 and displaying B-cell receptor signals characteristic of primary tumor cells lack the signature BRAF mutation to reveal unrepresentative origins
