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.

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.

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.

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.

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.

Induction of features characteristic of hairy cell leukemia in chronic lymphocytic leukemia and prolymphocytic leukemia cells.

In vitro maturation was induced with 12-O-tetradecanoylphorbol-13-acetate in leukemic cell samples from patients with chronic lymphocytic leukemia (n = 10) and prolymphocytic leukemia (n = 4). The cells were studied for morphology, for immunological markers using the fluorescence activated cell sorter, and for acid phosphatase isoenzymes using both cytochemistry and isoelectrofocusing. Morphologically the induced changes included appearance of cells with an excentric nucleus and basophilic cytoplasm and eventually of cells with many fine cytoplasmic projections ("hairs"). Analysis of immunological markers by flow cytometry revealed that the monoclonal antibody defined cell surface molecule HD6 (CD22), which is strongly expressed on hairy cell leukemia (HCL) but absent from plasmacytoma and plasma cells, can be induced or enhanced in the leukemic samples. In the study of acid phosphatase isoenzymes using cytochemistry we observed the induction of the tartrate resistant isoenzyme. Further, using isoelectrofocusing we could demonstrate the induction of the same band of tartrate resistant acid phosphatase with an isoelectric point of 9.0-9.7 as detected also in HCL. This particular isoenzyme is considered characteristic of HCL but is absent in plasmacytoma. Our data demonstrate that chronic lymphocytic leukemia and prolymphocytic leukemia cells can be induced to realize a common genetic program which bears characteristics of HCL, indicating that these three entities are much more closely related than previously thought.

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.

beta-N-acetylhexosaminidases in the spleen of a patient with hairy-cell leukaemia.

The spleen from a patient with hairy-cell leukaemia had beta-N-acetylhexosaminidase activity that could be resolved into three isoenzymes by chromatography on phenyl boronate agarose. Two of these were the major forms, A and B, found in normal tissues but, in addition, there was an 'extra' form that accounted for 15% of total activity. The 'extra' form hydrolysed the synthetic substrate 4-methylumbelliferyl-beta-N-acetylglucosamine 6-sulphate, indicating the presence of alpha-subunits. It was more acidic than A, was less heat-stable and showed no generation of B on denaturation under a variety of conditions. These findings and the immunoblot (Western blotting) analysis demonstrate that the 'extra' form is entirely composed of alpha-subunits, and most closely resembles S, the residual activity in Sandhoff's disease.

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%.

Cytochemistry of tartrate-resistant acid phosphatase: 15 years' experience.

Tartrate-resistant acid phosphatase (T-AcP) has been used in the past 15 years as a specific test for the diagnosis of hairy cell leukemia (HCL). However, enzyme activity has been reported to be absent from the hairy cells of rare cases of HCL and to be present in the neoplastic cells of diseases other than HCL. In order to fully utilize T-AcP for the diagnosis of HCL, it is necessary to maximize the sensitivity and specificity of the staining method, to have adequate quality control to ensure technical and interpretative accuracy, and to prepare optimal cytologic and histologic materials for study. In blood, only the presence of cells with intense T-AcP activity is diagnostic of HCL (positive T-AcP test). In tissues other than blood, the presence of cells with intense enzyme activity may not be diagnostic for HCL; assessment of cell morphology and appreciation of the pattern of enzyme localization are also important. In studying the blood of over 1,000 patients, we have found a negative T-AcP test in two of 200 cases of HCL and a positive T-AcP test in three of 800 patients with diseases other than HCL. We believe that the T-AcP test, when performed and interpreted properly, is a useful diagnostic test for HCL.

Demonstration of the increase in serine esterase-positive T cells in hairy-cell leukemia patients undergoing alpha-interferon therapy.

An increased number of serine esterase non-B lymphocytes including CD4+ helper/cytotoxic cells were observed in hairy-cell leukemia patients during (56 +/- 18) and immediately post (52 +/- 16) alpha-interferon therapy compared with pretreatment status (17 +/- 5) and normal donors (29 +/- 3). This increase in lymphocytes bearing one of the putative cytotoxicity-linked proteins was paralleled by a higher cytotoxicity towards K562 cells in the NK and LDCC assays during therapy and similarly, but to a much reduced level, against the hairy-cell derived JOK-1 target cells. Phenotypically, a higher percentage of non-B cells also expressing DR or CD11 antigens were seen in the HCL patients during IFN therapy. In lymphocyte target binding assays, a high affinity of effector to target conjugate formation was observed when either of the hairy cell lines JOK-1 or Hair-M was chosen as targets for IFN-therapy effector cells. Although predominantly CD4+ cells exhibited the in vitro affinity for effector/target conjugation with autologous HCs, the polarisation of serine esterase activity towards the E/T membrane contact area was shown by both CD4+ and CD8+ cells during a short incubation period and before target cell death.

Predominance of a distinct subtype of hairy cell leukemia in Japan.

Forty Japanese patients with hairy cell leukemia (HCL) were reviewed. Nine cases were diagnosed as typical HCL, and two cases had the features of HCL variant (prolymphocytic variant). The remaining 29 cases (72.5%) differed morphologically and hematologically from the other two groups in that they usually had a moderately high leukocyte count (average 27.9 x 10(3)/microliters), and abnormal cells showing a densely stained round nucleus and an inconspicuous nucleolus. Tartrate-resistant acid phosphatase reaction was weak, and their cells exhibited generally smooth or slightly irregular, cellular outlines in smears. The cells showed weak expression of surface immunoglobulin G (IgG) with kappa-chain predominance. CD25 antigen was not detected. Some of these findings resemble those of B-cell chronic lymphocytic leukemia, but the patients also had several important features of HCL. They had splenomegaly without significant lymphadenopathy. The abnormal cells were CD20+, CD11c+ and showed typical 'hairy morphology' under phase-contrast and scanning electron microscopy. Furthermore, spleen sections revealed diffuse infiltration by the abnormal cells in the red pulp. From these findings, we speculated that this group of patients constitute a distinct subtype of HCL which is commonly seen in Japan. We propose to term the disease as HCL Japanese variant.

Protein-tyrosine phosphatase activity of hairy cell tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAcP) is a reliable cytochemical marker for the diagnosis of hairy cell leukemia (HCL). The enzyme has been the subject of much biochemical investigation yet its function in the hairy cells (HC) is still unknown. Two TRAcPs have been purified from HCL spleen tissues by a series of chromatographic separations. The two enzymes, provisionally called peak 1 and peak 2, had specific activities of greater than 600 U/mg and 800 U/mg respectively when p-nitrophenyl phosphate (p-NPP) was used as substrate and had Km values in the range of 1 to 5 mM p-NPP. The two TRAcPs had the same substrate specificities and inhibitor sensitivities, therefore could be isoforms of the same enzyme. Their pH optima were between 5 and 6 for all substrates tested including the phosphotyrosine-containing peptide, Raytide, which was still hydrolyzed efficiently at neutral pH. Neither phosphoserine nor phosphoserine-containing casein were hydrolyzed by either enzyme. The TRAcPs of HC may thus be capable of functioning as protein-tyrosine phosphatases (PTP). High activity of a PTP could regulate the activities of protein-tyrosine kinases and thereby influence the growth and differentiation of the hairy cells.

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.

Induction of intracellular and plasma 2',5'-oligoadenylate synthetase by pentostatin.

Similar to interferon alpha, pentostatin is highly effective in hairy cell leukemia and moderately active in other chronic lymphoid malignancies. In ten patients with hairy cell leukemia (HCL) and seven patients with other B-cell chronic leukemias (BCL), we have studied the intracellular 2',5'-oligoadenylate synthetase (2,5OAS) activity of the mononuclear cells before, 4 h, 24 h, and 48 h after pentostatin administration. In patients with HCL the median level of intracellular 2,5OAS increased 4.6-fold at 4 h and 11.5-fold at 24 h compared to the pretreatment value. Among the other seven patients, the median intracellular 2,5OAS remained unchanged in three patients and rose slightly by 2 to 14 times in four patients. Eleven patients (eight with HCL and two with BCL) responded to pentostatin. The median increase in 2,5OAS among the responders was 13.0-fold (range 4.8-30.0) whereas that among non-responders was 2.2-fold (range 0.2-6.3). The difference was highly significant (p less than 0.0001). In five of the total seventeen patients, the plasma levels of 2,5OAS activity were also determined and changes in plasma levels paralleled those measured intracellularly. To determine if the elevation of 2,5OAS is mediated by induction of interferon alpha, the expressions of mRNA for interferon alpha and beta were investigated by means of reverse transcription and polymerase chain reaction using the corresponding sense primers. In none of the five patients thus studied could we find an induction of mRNA for interferon alpha or beta in the leukemic cells during treatment with pentostatin. Thus, response to pentostatin correlates with induction of 2,5OAS directly and the 2',5'-oligoadenylate system seems to be involved in cytotoxicity.

Development of an immunoassay for human serum osteoclastic tartrate-resistant acid phosphatase.

A tartrate-resistant acid phosphatase (TrACP), which has been suggested to be very similar to the osteoclastic TrACP, was partially purified from the spleen of a patient with hairy cell leukemia. The purification procedure consisted of carboxymethyl-Sepharose, phosphocellulose, Sephacryl S-200, and phenyl-Sepharose chromatographies. Polyclonal antibodies were generated in guinea pigs with a titer of at least 1:6000. Immunohistochemical staining of fetal rat tibia with the antisera revealed that only the lysosomes of osteoclasts, but not osteoblasts, were stained. An enzyme-linked immunosorbent assay (ELISA) was developed with the antisera. There was no cross-reactivity with 1) partially purified acid phosphatases (ACPs) from normal human and beef spleens, 2) ACPs in extracts of human osteoblastic cells, 3) purified bovine bone matrix TrACP, or 4) commercial prostatic ACP. However, extracts of giant cell bone tumors, containing large amounts of bona fide osteoclasts, showed large amounts of cross-reactive material, which diluted in parallel with the partially purified hairy cell leukemic TrACP in the ELISA. Commercial serum band 5b TrACP also displaced in parallel with the partially purified hairy cell leukemic TrACP. Immunoblotting studies revealed that the antiserum, but not nonimmune guinea pig serum, reacted with the homogeneous hairy cell leukemia splenic band 5 TrACPs, which were recently purified by our laboratory. Preliminary application of the ELISA to sera of patients with metabolic bone diseases revealed that normal healthy individuals had measurable amounts of the immunoreactive material, and patients with Paget's disease or hyperparathyroidism, who should have high bone turnover, had elevated levels of this immunoreactive material in their sera. In contrast, the level of serum osteoclastic TrACP in a patient with an acute lymphatic leukemia was normal. In summary, 1) we have shown that hairy cell leukemia splenic TrACP shares significant immunological similarity with the osteoclastic TrACP and with the serum band 5b TrACP, and 2) the ELISA holds promise for a sensitive and specific assay for bone resorption.

Expression of deoxycytidine kinase and phosphorylation of 2-chlorodeoxyadenosine in human normal and tumour cells and tissues.

Deoxycytidine kinase (dCK) activates several clinically important drugs, including the recently developed antileukaemic compound 2-chlorodeoxyadenosine (CdA). The distribution of dCK in cells and tissues has previously been determined by activity measurements, which may be unreliable because of the presence of other enzymes with overlapping substrate specificities. Therefore we have measured dCK polypeptide levels in extracts of normal and malignant human peripheral blood mononuclear cells, gastrointestinal tissues and sarcomas, using a specific immunoblotting technique, as well as the phosphorylation of CdA in the same extracts. High levels of dCK were found in all major subpopulations of normal mononuclear leucocytes (120 +/- 19 ng dCK/mg protein) and in B-cell chronic lymphocytic leukaemia (81 +/- 30 ng/mg, n = 23). Hairy-cell leukaemia contained lower levels (28 +/- 23 ng/mg, n = 7), as did three samples of T-cell chronic lymphocytic leukaemia (18 +/- 14 ng/mg). Phytohaemagglutinin stimulation of normal lymphocytes did not lead to any substantial increase in either dCK activity or protein expression (less than 2.5-fold). The human CEM wt T-lymphoblastoid cell line contained 56 +/- 1 ng/dCK/mg protein, while in the CEM ddC50 and AraC8D mutants that lack dCK activity, no dCK polypeptide could be detected. In colon adenocarcinomas, the dCK content was significantly higher (20 +/- 9 ng/mg, n = 20) than in normal colon mucosa (8 +/- 3.5 ng/mg, n = 19, P < 0.05). A similar pattern of dCK expression was found in gastric adenocarcinomas (21 +/- 13 ng/mg, n = 5) and normal stomach mucosa (6 +/- 5 ng/mg, n = 5, P < 0.15). One leiomyosarcoma and one extra-skeletal osteosarcoma showed dCK levels comparable with those found in normal lymphocytes (84 +/- 6 and 109 +/- 4 ng/mg, respectively), while other sarcoma samples contained lower levels, comparable to the gastrointestinal adenocarcinomas (20 +/- 7 ng/mg, n = 12). Thus, dCK is expressed constitutively and predominantly in lymphoid cells, but it is also found in solid non-lymphoid tissues, with increased levels in malignant cells. The phosphorylation of CdA in crude extracts showed a close correlation to the dCK polypeptide level.

Ultrastruct and tartrate-resistant acid phosphatase localization in a T-cell hairy-cell leukemia cell line.

Hairy-cell leukemia is characterized clinically in splenomegaly and pancytopenia and pathologically by the proliferation in hematopoietic tissue of cells containing the tartrate-resistant isozyme 5 of acid phosphatase. We have described a patient with a T-lymphocyte variant of this disease. A permanent cell line obtained from the spleen of this patient has the biological and enzymatic characteristics of the fresh leukemic cells. We have used this line to study the surface morphology, ultrastructure, and ultrastructural localization of acid phosphatase in defined T-lymphoid hairy cells. The surface of the cells of the permanent line was smooth but many hair-like projections appeared after exposure to phytohemagglutinin (PHA). There was little acid phosphatase reaction produce visualized when beta-glycerophosphate was used as a substrate. With sodium haphthol AS-BI phosphoric acid heavy deposits were seen in the perinuclear membrane, mitochondria, and rough endoplasmic reticulum. Exposure to PHA and pokeweed mitogen resulted in increased reaction product, suggesting increased enzyme synthesis. Tartrate-resistant acid phosphatase was localized in the same organelles.

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.