Comparison of methodologies for the detection of BRAF mutations in bone marrow trephine specimens.

AIMS: BRAF V600E detection assists in the diagnosis of hairy cell leukaemia (HCL); however, testing practices vary. We evaluated the clinical utility of 5 BRAF mutation testing strategies for use on bone marrow trephines (BMT). METHODS: 11 HCL, 5 HCL 'mimic', 2 treated HCL and 10 normal BMT specimens were tested for mutant BRAF, comparing Sanger sequencing, pyrosequencing, amplicon-based next generation sequencing (NGS), automated (Idylla) PCR and immunohistochemistry (IHC). RESULTS: PCR and IHC were cheaper and identified V600E in 100 % of HCL cases. Pyrosequencing detected the mutation in 91%, NGS in 55% of cases and Sanger sequencing in 27%. All assays gave wild-type BRAF results in HCL mimics and normal BMT samples. CONCLUSIONS: PCR and IHC were most sensitive and cost-effective, but these have limited scope for multiplexing and are likely to be replaced by NGS gene panels or whole genome sequencing in the medium to long term.

Real-time detection of BRAF V600E mutation from archival hairy cell leukemia FFPE tissue by nanopore sequencing.

The MinION is a miniaturized high-throughput next generation sequencing platform of novel conception. The use of nucleic acids derived from formalin-fixed paraffin-embedded samples is highly desirable, but their adoption for molecular assays is hurdled by the high degree of fragmentation and by the chemical-induced mutations stemming from the fixation protocols. In order to investigate the suitability of MinION sequencing on formalin-fixed paraffin-embedded samples, the presence and frequency of BRAF c.1799T > A mutation was investigated in two archival tissue specimens of Hairy cell leukemia and Hairy cell leukemia Variant. Despite the poor quality of the starting DNA, BRAF mutation was successfully detected in the Hairy cell leukemia sample with around 50% of the reads obtained within 2 h of the sequencing start. Notably, the mutational burden of the Hairy cell leukemia sample as derived from nanopore sequencing proved to be comparable to a sensitive method for the detection of point mutations, namely the Digital PCR, using a validated assay. Nanopore sequencing can be adopted for targeted sequencing of genetic lesions on critical DNA samples such as those extracted from archival routine formalin-fixed paraffin-embedded samples. This result let speculating about the possibility that the nanopore sequencing could be trustably adopted for the real-time targeted sequencing of genetic lesions. Our report opens the window for the adoption of nanopore sequencing in molecular pathology for research and diagnostics.

BRAF V600E mutation in hairy cell leukemia: from bench to bedside.

Hairy cell leukemia (HCL) is a distinct clinicopathological entity whose underlying genetic lesion has remained a mystery for over half a century. The BRAF V600E mutation is now recognized as the causal genetic event of HCL because it is somatic, present in the entire tumor clone, detectable in almost all cases at diagnosis (encompassing the whole disease spectrum), and stable at relapse. BRAF V600E leads to the constitutive activation of the RAF-MEK-extracellular signal-regulated kinase (ERK) signaling pathway which represents the key event in the molecular pathogenesis of HCL. KLF2 and CDNK1B (p27) mutations may cooperate with BRAF V600E in promoting leukemic transformation. Sensitive molecular assays for detecting BRAF V600E allow HCL (highly responsive to purine analogs) to be better distinguished from HCL-like disorders, which are treated differently. In vitro preclinical studies on purified HCL cells proved that BRAF and MEK inhibitors can induce marked dephosphorylation of MEK/ERK, silencing of RAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression profile signature, change of morphology from "hairy" to "smooth," and eventually apoptosis. The overall response rate of refractory/relapsed HCL patients to the BRAF inhibitor vemurafenib approached 100%, with 35% to 40% complete remissions (CRs). The median relapse free-survival was about 19 months in patients who had achieved CR and 6 months in those who had obtained a partial response. Future therapeutic perspectives include: (1) combining BRAF inhibitors with MEK inhibitors or immunotherapy (anti-CD20 monoclonal antibody) to increase the percentage of CRs and (2) better understanding of the molecular mechanisms underlying resistance of HCL cells to BRAF inhibitors.

Melanoma and non-melanoma skin cancers in hairy cell leukaemia: a Surveillance, Epidemiology and End Results population analysis and the 30-year experience at Memorial Sloan Kettering Cancer Center.

Few studies have examined melanoma and non-melanoma skin cancer (NMSC) incidence rates after a diagnosis of hairy cell leukaemia (HCL). We assessed 267 HCL patients treated at Memorial Sloan Kettering Cancer Center (MSKCC) and Surveillance, Epidemiology and End Results (SEER) data for melanoma and NMSC incidence rates after HCL. Incidence data from MSKCC patients demonstrated a 10-year combined melanoma and NMSC skin cancer rate of 11·3%, melanoma 4·4% and NMSC 6·9%. Molecular analysis of skin cancers from MSKCC patients revealed activating RAS mutations in 3/9 patients, including one patient with melanoma. Of 4750 SEER patients with HCL, 55 (1·2%) had a subsequent diagnosis of melanoma. Standardized incidence ratios (SIRs) did not show that melanoma was more common in HCL patients versus the general population (SIR 1·3, 95% CI 0·78-2·03). Analysis of SEER HCL patients diagnosed before and after 1990 (approximately before and after purine analogue therapy was introduced) showed no evidence of an increased incidence after 1990. A better understanding of any potential association between HCL and skin cancer is highly relevant given ongoing trials using BRAF inhibitors, such as vemurafenib, for relapsed HCL, as RAS-mutant skin cancers could be paradoxically activated in these patients.

Phospho-ERK(THR202/Tyr214) is overexpressed in hairy cell leukemia and is a useful diagnostic marker in bone marrow trephine sections.

BRAF V600E mutations are present in virtually all cases of hairy cell leukemia (HCL). We hypothesized that detection of phospho-ERK (pERK) in tissue sections may be a useful marker for diagnosis of HCL. pERK/CD20 double immunostaining was performed on 90 formalin-fixed bone marrow trephine samples affected with small B-cell lymphoproliferative disorders, including 28 cases of HCL. pERK staining was observed in all 28 cases of HCL and in 1 of 62 cases of non-HCL B-cell lymphoproliferative disorders. By allele-specific polymerase chain reaction, all 11 cases of HCL with available DNA were positive for BRAF V600E, as was the 1 pERK non-HCL case. The remaining 31 non-HCL cases tested were negative for BRAF V600E. The sensitivity and specificity of pERK for diagnosis of HCL was 100% and 98%, respectively. We conclude that the presence of pERK as detected by immunohistochemical staining is a useful surrogate marker for BRAF V600E in the diagnosis of HCL.

Application of a BRAF V600E mutation-specific antibody for the diagnosis of hairy cell leukemia.

In recent times BRAF V600E mutations have emerged as a genetic hallmark of hairy cell leukemia (HCL). This specific point mutation is present in virtually all cases of HCL but is exceedingly rare in other peripheral B-cell neoplasms. In this study we investigated the application of a BRAF V600E mutation-specific antibody (clone VE1) to differentiate HCL from HCL mimics, such as HCL variant and splenic marginal zone lymphoma. A total of 52 routinely processed formalin-fixed paraffin-embedded tissue specimens were investigated (bone marrow, n=46; spleen, n=6) for expression of V600E-mutated BRAF protein. All 32 cases of HCL were scored positive, and all non-HCL cases were scored negative. In 28 of 30 HCL cases the presence of a BRAF V600E mutation could be confirmed by direct sequencing, whereas no BRAF mutations were detected among 20 HCL mimics. We further screened 228 mature B-cell neoplasms with VE1 and detected 1 positive case of chronic lymphocytic leukemia. Sequencing confirmed the presence of a BRAF V600E mutation. In conclusion, we demonstrate that VE1 immunohistochemistry can be used to reliably differentiate HCL from HCL-mimicking entities. This on-slide technique might be particularly helpful in interpreting challenging biopsies with low tumor content.

Simalikalactone E (SkE), a new weapon in the armamentarium of drugs targeting cancers that exhibit constitutive activation of the ERK pathway.

Simalikalactone E (SkE) is a quassinoid extracted from a widely used Amazonian antimalarial remedy. Although SkE has previously been shown to have cytostatic and/or cytotoxic activities in some tumor cell lines, its mechanism of action has not yet been characterized. We show here that SkE in the high nanomolar range inhibited the growth of various leukemic and solid tumor cell lines. Importantly, SkE was highly efficient at inhibiting chronic myelogenous leukemia (CML) cells that exhibit constitutive activation of the MAPK pathway and, accordingly, it impaired the phosphorylation of ERK1/2. SkE also abrogated MEK1/2 and B-Raf phosphorylation but had no effect on Ras activity. Moreover, SkE was particularly effective against melanoma cell lines carrying the B-Raf-V600E mutation. Importantly, SkE resensitized the PLX-4032-resistant 451Lu melanoma cell line (451Lu-R) and was more efficient than U0126, a MEK inhibitor, and PLX-4032 (PLX) at inducing the apoptosis of two hairy cell leukemia (HCL) patient samples carrying the B-Raf-V600E mutation. Finally, SkE was as efficient as imatinib at inhibiting tumor formation in a xenograft model of CML cells in athymic mice. In conclusion, we show that SkE, a very potent inhibitor of B-Raf-V600E, is highly effective against cancer cell lines that exhibit constitutive activation of the ERK1/2 pathway.

Protein-tyrosine phosphatase activity maintains the viability of hairy cells and modulates their response to interferon-alpha.

Hairy-cell leukemia is characterised by a marked sensitivity of the malignant cells to the cytotoxic effects of therapeutically administered interferon-alpha. The aim of this study was to assess the role of protein tyrosine phosphatases in the maintenance of hairy-cell (HC) viability and their sensitivity to interferon-alpha. The selective tyrosine phosphatase inhibitor mpV(pic) killed HCs, but not normal B lymphocytes or chronic lymphotic leukemia (CLL) cells. HCs displayed increased expression of the phosphatases SHP-1 and SHP-2 when compared with normal B lymphocytes. Phosphatase inhibition also enhanced the cytotoxic effect of interferon-alpha against HCs in four of the five cases tested. Therefore, HCs, but not normal B cells or CLL-cells, require tyrosine phosphatase activity for preservation of their viability. In addition, HC sensitivity to interferon is down-regulated by this activity.

Central role of protein kinase Cepsilon in constitutive activation of ERK1/2 and Rac1 in the malignant cells of hairy cell leukemia.

We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCepsilon in the constitutive activation of Rac1 and ERK in HCs. PKCepsilon and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCepsilon was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCepsilon plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia.

Purine analogues: rationale for development, mechanisms of action, and pharmacokinetics in hairy cell leukemia.

Cladribine is effective therapy for HCL, and there are several ways to achieve the adequate concentrations of the active metabolites in relevant cells, without the need for long-term continuous infusions. This simplifies therapy, although careful control of patients is required during and after treatment in most instances because of the significant activity of the drug on leukemia cells of various types and also on lymphoid cells and normal stem cells.

The value of tartrate resistant acid phosphatase (TRAP) immunoreactivity in diagnosis of hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder characterized by splenomegaly, pancytopenia, and circulating atypical lymphocytes with circumferential cytoplasmic projections. We investigated the specificity and the sensitivity of anti-TRAP antibody immunoreactivity in 57 cases of HCL. We found that there is a statistically highly significant difference between TRAP immunoreactivities of the study and the control groups, and HCL can be diagnosed by TRAP immunoreactivity in bone marrow trephine biopsy materials with a specificity of 98.27 % and a sensitivity of 100%.

Disruption of a novel ectodermal neural cortex 1 antisense gene, ENC-1AS and identification of ENC-1 overexpression in hairy cell leukemia.

Karyotypical alteration of chromosome 5 and in particular band 5q13 is a frequent finding in hairy cell leukemia (HCL). We have previously identified a number of candidate genes localized in close proximity to a constitutional inv(5)(p13.1q13.3) breakpoint in one HCL patient. These included beta-hexosaminodase HEXB, frequently mutated in the lysosomal storage disorder Sandhoff disease. We now report that the 5q13.3 breakpoint disrupts a novel evolutionary conserved alternative isoform of HEXB. This isoform directly overlaps, in a cis-antisense fashion, exon 1 of the gene for ectodermal neuronal cortex 1 ENC-1, and was thus named ENC-1AS. ENC-1 has previously been shown to be overexpressed in several malignancies, and is believed to play a critical regulatory role in malignant transformation of various tumors. Importantly, subsequent analysis of ENC-1 in purified primary HCL tumor cells revealed a striking upregulation of ENC-1 in all 26 patients examined, compared with normal peripheral blood lymphocytes from healthy donors. Upon further analysis of the ENC-1/ENC-1AS locus, we identified a complex 5' regulatory mechanism involving an inverse expression of the ENC-1 sense and the ENC-1AS transcripts in several tissues supporting the hypothesis that expression of ENC-1AS regulates ENC-1 levels. In addition, we have also found tissue-specific methylation of a 1.2 kb segment encompassing the overlapping ENC-1/ENC-1AS 5' exons, adding to the complexity of the regulation of this locus. Altogether, these results suggest that upregulation of ENC-1 contributes to the development of HCL and provides new information on the possible dysregulation of ENC-1 including expression of a novel antisense gene, ENC-1AS.

Regulation of hairy-cell survival through constitutive activation of mitogen-activated protein kinase pathways.

The hairy cells (HCs) of hairy-cell leukemia are intrinsically activated mature clonal B cells. The aims of this study were to gain further insights into the nature of this activation and to assess its importance for the prolonged HC survival in this chronic disease. We show that HCs contain phosphorylated/activated p38 MAPK, JNK and ERK1/ERK2 (ERK1/2). PKC inhibitors increased the activation of p38 and JNK, but reduced the phosphorylation of ERK1/2. Moreover, PKC inhibition resulted in cell death; cell death was also observed when the activation of ERK1/2 in HCs was abrogated with an inhibitor of MEK1/2 activation. In addition to PKC, active Src kinase was also shown to be involved in the maintenance of Raf-independent ERK activation in HCs. During cell culture on a nonadherent surface, ERK phosphorylation was sustained, while phosphorylation of p38 and JNK decreased. This decrease was not observed in HCs cultured on vitronectin (VN), indicating that p38/JNK activation is probably a consequence of in vivo HC interaction with VN present in abundance in the red pulp of the spleen. Taken together, these results suggest that active p38/JNK make HCs susceptible to apoptosis, but the cells are effectively rescued by ERK activation involving constitutively active PKC and Src. These findings are relevant for the understanding of the prolonged cell survival of HCs and their selective sensitivity to some chemotherapeutic agents.

Expression of a functional inducible nitric oxide synthase in hairy cell leukaemia and ESKOL cell line.

The expression of nitric oxide synthase (NOS) isoforms was investigated in the established ESKOL hairy cell line and in leukemic cells of patients with hairy cell leukemia (HCL). By reverse transcription-polymerase chain reaction (RT-PCR), these cells were found to spontaneously express inducible NOS (iNOS)-specific mRNA, but not endothelial constitutive NOS (ecNOS) mRNA. The iNOS protein was detected by immunofluorescence in the cytoplasm of permeabilized leukemic cells and ESKOL cells, using different anti-iNOS monoclonal antibodies. A protein of 135 kDa was identified by Western blotting in ESKOL and HCL lysates, confirming the presence of an iNOS in these cells. Cytosolic homogenates displayed NOS catalytic activity, as measured by the conversion of 14C-labelled L-arginine into 14C L-citrulline and by detection in situ using the DAF-2DA (diaminofluorescein diacetate) NO-sensitive fluorescent probe. Ligation of CD23 (low affinity IgE receptor) was found to increase iNOS expression in ESKOL and conversely to decrease the percentage of cells undergoing apoptosis, as measured by the percentage of cells expressing annexin V. These results indicate that, as in chronic B cell lymphocytic leukemia cells (B-CLL) a functional iNOS is expressed constitutively in hairy cells that contributes to protecting these tumoral cells from apoptosis.

Telomerase activity in chronic lymphoproliferative disorders of B-cell lineage.

The progressive shortening of telomeres at each cell division is a key mechanism in controlling cell proliferative capacity. The activation of telomerase, a reverse transcriptase that extends telomere length, potentially leads to unlimited cell proliferation, and is believed to play a critical role in the neoplastic process. High levels of telomerase activity have been demonstrated in almost all solid tumours; however, little data is available concerning its expression in chronic B-cell neoplasms. By using a quantitative polymerase chain reaction-based method we quantified telomerase activity in normal B lymphocytes, and in various B-cell malignancies, including chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL) and hairy cell leukaemia (HCL). Compared to normal B cells, which expressed very low levels of telomerase activity, malignant cells from most of the patients showed a significant increase in telomerase activity, with highest values observed in HCL samples. Moreover, among the CLL and HCL cases, significantly higher levels of telomerase activity were found in patients with progressive disease at 1 year follow-up versus patients with stable disease. These data suggest that telomerase activity might correlate with disease progression.

Species specificity of monoclonal antibodies to human tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is expressed abundantly by osteoclasts and is required for bone resorption. This enzyme is emerging as an important biomarker in bone pathology, both for histochemical identification of osteoclasts and as a serum marker of osteoclast activity and increased bone turnover. Rat and mouse models are becoming popular systems for studying osteoclast development, bone physiology and morphogenesis, and bone diseases such as osteoporosis. We have developed two unique antibodies to human TRAP purified from hairy cell leukemia spleen. Both antibodies (9C5 and 14G6) are suitable for immunohistochemistry of osteoclasts and macrophages. Only one (14G6) is capable of immunoprecipitating active TRAP from human cell lysates. Antibody 9C5 reacts with a denatured epitope of TRAP while antibody 14G6 probably reacts with a native, conformational determinant. The high degree of homology among TRAPs of various species predicts that these antibodies should be suitable for work in experimental animals as well as humans. Immunohistochemical staining, electrophoretic analyses, immunoprecipitation and immunoblotting assays of human rat and mouse TRAP were carried out to test the validity of these antibodies as cell markers in rodents. Both antibodies were suitable for immunohistochemistry in all species. Antibody 9C5 was suitable for immunoblotting of denatured TRAP of all species tested. Antibody 14G6 reacted with the native TRAP of humans only and failed to immunoprecipitate mouse or rat TRAP activity. Although TRAP is a phylogenetically conserved protein, subtle, species-specific determinants exist. Care should be exercised when anti-TRAP antibodies are used for immunoassay in experimental animals.

Immunohistochemical demonstration of acid phosphatase isoenzyme 5 (tartrate-resistant) in paraffin sections of hairy cell leukemia and other hematologic disorders.

The demonstration of tartrate-resistant acid phosphatase (TRAP) activity has long been a cornerstone in the diagnosis of hairy cell leukemia (HCL). Recently a monoclonal antibody to this enzyme has been developed that can be used in an immunoperoxidase method on paraffin-embedded tissues. By using a peroxidase-labeled streptavidin biotin method, paraffin sections of B5 and formalin-fixed tissue from 86 cases of HCL (41 bone marrow, 36 spleen, 9 liver) were stained with the antibody to TRAP and compared against staining for CD20 (L26) and DBA.44 (DAKO, Carpinteria, Calif). In addition, 193 specimens (127 bone marrow, 42 lymph node, 19 spleen, 5 other) from a variety of neoplastic and nonneoplastic hematologic conditions were stained using the monoclonal antibody to TRAP. For comparison, these cases were also stained with DBA.44. In the cases of HCL, 80 of 86 specimens were immunoreactive for TRAP. While the antibody to TRAP generally stained less than 50% of the hairy cells, CD20 and DBA.44 stained 90% and 50% to 60% of hairy cells, respectively. Two of three cases of marginal zone lymphoma showed weak immunoreactivity to the TRAP antibody. Two specimens from a patient with Gaucher's disease and 8 of 13 cases of mastocytosis also showed positivity to the TRAP antibody in the macrophages and mast cells, respectively. In contrast, staining for DBA.44 was positive in 3 of 9 cases of B-cell large cell lymphoma, 1 of 4 cases of mantle cell lymphoma, and in the paraimmunoblasts of 1 of 7 cases of small lymphocytic lymphoma. Only HCL was TRAP and DBA.44 positive. This antibody to TRAP is a useful addition to the diagnosis of HCL but should be used in conjunction with CD20 and DBA.44. The use of this antibody to determine minimal residual disease after chemotherapy was not addressed.

Characterization of monoclonal antibodies specific to human tartrate-resistant acid phosphatase.

A major product of osteoclasts, tartrate-resistant acid phosphatase (TRAP) is an essential but insufficient enzyme for bone resorption. TRAP is an excellent cell marker for osteoclasts and macrophages and is being investigated as a serum marker for osteoclast activity in diseases of bone destruction. For decades, TRAP has also been used as a marker for hairy cell leukemia. Immunoassays for TRAP are sought to increase the sensitivity and specificity of the TRAP test for bone and hairy cells. Our laboratory recently developed a monoclonal antibody to TRAP (9C5) useful for immunohistochemical identification of TRAP-positive cells in paraffin sections. Herein, we characterize 9C5 in greater detail and report production of another anti-TRAP monoclonal antibody antibody (14G6) reactive with native, active enzyme antigen. Enzyme immunoassay, immunoprecipitation, western blot, and immunohistochemical analyses revealed the contrasting properties of 9C5 and 14G6. Antibody 9C5 reacts with a heat-denatured epitope and is suitable for denaturing western blot analysis and for immunohistochemistry. Antibody 14G6 reacts with a conformational determinant destroyed by heat and is suitable for immunoprecipitation of active TRAP, although 20% to 30% of activity is inhibited in the immune complexes. Having characterized several properties of these anti-TRAP antibodies, 9C5 and 14G6 may be useful for development of TRAP-specific immunoassays in bone pathology and hematology. They will certainly be of use for the study of biosynthesis, regulation, expression, and function of TRAP.

Epitope enhancement for immunohistochemical demonstration of tartrate-resistant acid phosphatase.

We have developed a monoclonal antibody (9C5) for immunohistochemical localization of tartrate-resistant acid phosphatase (TRAcP). This antibody reacts with a denatured epitope of TRAcP and requires enhancement methods to promote antigenicity in paraffin-embedded tissues. We used this antibody to systematically examine proteolytic digestion and heat denaturation conditions for epitope enhancement in both paraffin sections and fixed smears. The goal was to increase the sensitivity of the immunohistochemical stain for TRAcP. Optimal conditions for proteolytic digestion were established. Denaturation in a conventional boiling water bath was compared to microwave irradiation in several commonly used solutions. Immunohistochemistry was compared directly to TRAcP cytochemistry in fixed smears from hairy cell leukemia specimens to gauge the level of sensitivity of our improved method. Attempts were made to "retrieve" the 9C5 epitope from overfixed tissues and aged smears. Maximal immunoreactivity of TRAcP was achieved by microwave irradiation in a citrate or Tris buffer of pH 6.0-8.0 without the need for a subsequent protease digestion step. With this method of epitope enhancement, immunohistochemistry with antibody 9C5 was as sensitive as direct cytochemical staining of TRAcP activity. However, once a tissue specimen had been overfixed or a smear stored for a year or more, the 9C5 epitope was no longer retrievable. The key element in epitope enhancement for 9C5 immunohistochemistry is heat denaturation of the target epitope. Immunohistochemistry of TRAcP in paraffin sections would be a great asset to the study of specialized forms of the monocyte/macrophage lineage and to the process of macrophage activation. It would also provide another means for more precise evaluation of residual disease in bone marrow of patients treated for hairy cell leukemia.