Michael Grever
The Ohio State University

In patients who are having symptoms from this disease, there is often an enlarged spleen felt on physical examination. In the original series published by Dr. Bouroncle, more than 90% had an enlarged spleen. Subsequently over the years, the finding on an enlarged spleen is more often identified in about 80% of the patients. Therefore, the failure to identify an enlarged spleen does not preclude the diagnosis of hairy cell leukemia. Most patients will have reduced hemoglobin, neutrophiles, and monocytes. Thrombocytopenia is also quite common. A careful examination of the peripheral blood smear will identify the circulating leukemic cells with a serrated cytoplasmic border.

While a bone marrow aspirate is difficult to obtain in about 75% of the patients, the bone marrow biopsy will be important to establish the diagnosis. Many patients have evidence of bone marrow fibrosis, and there is an infiltrating presence of widely spaced leukemic cells. Many patients will have a hypercellular bone marrow, but approximately 10% of patients will have a hypocellular bone marrow. In general, granulocyte precursors are more reduced than erythroid elements. In those patients with a hypocellular bone marrow, the diagnosis of aplastic anemia may be inappropriately entertained in particular if there is no splenomegaly appreciated on physical examination. Therefore, immunophenotyping is essential to search for evidence of a monoclonal population of hairy cells.

Immunohistochemical staining of the bone marrow biopsy with antibodies directed against CD20 and DBA.44 will assist in establishing the correct diagnosis. Furthermore, immunophenotyping of the peripheral blood and the cells aspirated from the bone marrow will identify the characteristic profile consistent with hairy cell leukemia including: CD19, CD20, and CD22. Furthermore, they are negative for CD5, CD10, and CD23. Typical hairy cells strongly express CD11c, FMC7, CD25, and CD103. It is essential to distinguish hairy cell leukemia from other lymphoid malignancies that can masquerade as this disease (e.g., hairy cell variant; splenic marginal zone lymphoma, chronic lymphocytic leukemia, prolymphocytic leukemia, other low grade lymphomas, and systemic mastocytosis).

In establishing the correct diagnosis, therapy can be appropriately selected. The outstanding results that have been achieved with single agent purine agents in hairy cell leukemia will not likely reproduce the same benefits in the other related lymphoid malignancies. In addition to selecting the appropriate therapy for the correct disease, identification of the correct diagnosis is important in providing accurate prognostic information to the patient.

What is the rationale for “watch and wait” rather than embarking directly on treatment?

The purine agents that are used to treat hairy cell leukemia have clearly been highly effective in achieving a complete remission. However, the disease is not cured by the achievement of a complete remission using standard single agent therapy. We understand that there is substantial remaining disease called “minimal residual disease” that can be observed by using histochemical stains after observing a complete remission by the usual light microscope. There is no evidence that early intervention in an asymptomatic patient with marginal reduction in peripheral blood counts improves survival. Because the single agent therapy is not curative, it is difficult to recommend therapy in a truly asymptomatic patient. Furthermore, we know that the purine analogs can induce prolonged immunosuppression. Their use in a non-protocol setting for a stable patient with decent counts must be weighed against the potential side-effects.

Experience, however, has indicated that initiation of treatment should not be delayed when the peripheral counts are beginning to decline below specific numbers or if the patient is experiencing symptoms. Patients with minimal bone marrow involvement that is interstitial and not diffuse with a minimal enlargement of the spleen may be candidates for close observation. If the absolute neutrophile count declines below 1,500/µL, or if the hemoglobin declines below 11 grams/dL, or the platelet count falls below 100,000/µL, then consideration should be given to initiating therapy irrespective of active symptoms.

Patients who have a new diagnosis of hairy cell leukemia may be uncomfortable with a “watch and wait” strategy, and a thorough discussion of the goals of therapy must be pursued. In addition, we need to explain that ongoing clinical trials will answer important questions of whether the purine agents should be used in combination with Rituxan either administered concomitantly or sequentially in an effort to evaluate the importance of eradicating minimal residual disease. The benefit of adding additional therapy on the long-term outcome must be carefully assessed. Furthermore, clinical investigation must define the optimal strategy of administering the therapeutic agents. The value of participating in organized clinical trials should also be discussed so that the patient and their family understand what we know and what questions remain.

Hairy Cell Leukemia Variant (HCL-V)

Estella Matutes
Royal Marsden Hospital

Disease features

Hairy cell leukemia-variant (HCL-V) is a very rare B-cell disorder accounting for c.10% of HCL cases. The two conditions are recognized in the World Health Organization (WHO) classification and are distinguishable by their clinical manifestations, morphology and immunophenotype while both share very similar histology (Piris et al, 2008). HCL-V affects elderly patients (median age: 71 years) with only a slight male predominance (M/F: 1.5). Main presenting features are splenomegaly (85%), high white blood count (median: 35x109/l) with lymphocytosis and no monocytopenia. (Matutes et al, 2001; Sainati et al,1990; Zinzani et al, 1990). The circulating cells have a morphology intermediate between prolymphocytes and hairy cells and, unlike prolymphocytic leukemia, the lymphocyte doubling time is >6months. As in typical HCL, the cells often express IgG heavy chain, are CD11c+ and frequently CD103+ and DBA44+; unlike typical HCL, the cells are CD25, CD123 (anti-IL-3 receptor) and HC2 negative. The bone marrow often can be aspirated and the pattern of infiltration in the trephine biopsy is similar or identical to HCL. The pattern of spleen infiltration in HCL-variant resembles that of typical HCL with expansion of the red pulp and is different from that seen in B-PLL and splenic marginal zone lymphoma/splenic lymphoma with villous lymphocytes (SMZL/SLVL) (Matutes et al, 2001 and 2003). There is no recurrent chromosomal abnormality but complex karyotypes and monoallelic TP53 deletion, that can detected by fluorescence in situ hybridization (FISH), have been documented. The high incidence of TP53 deletions in HCL-variant may account for the resistance to therapy. So far there are no data on gene profiling expression in HCL-V and information on IGVH mutational status and VH usage is very scanty.

Diagnosis

The diagnosis of HCL-variant is established by compounding the clinical features with cell morphology, immunophenotype and histology. Problems on differential diagnosis may arise not only with typical HCL but also and more frequently with SMZL/SLVL and B-PLL. Distinction among these disorders is relevant from the clinical point of view due to the different response to therapy and outcome of patients.

Response to therapy and outcome

Despite of the designation of HCL-variant, the disease has a natural history and outcome different from typical HCL. The clinical course of HCL-v is usually chronic and a small proportion of patients may not require immediate treatment. In contrast to typical HCL, HCL-variant patients are resistant to alkylating agents and to Interferon alpha and usually to pentostatin and/or cladribine (Matutes et al, 2001; 2003; Sainati et al, 1990; Robak et al 1999; Tetrault et al, 1999). Combinations such as CHOP do not yield better responses.

Our experience based on 52 patients has shown that splenectomy is a good palliative treatment that results in long lasting partial responses (median 4 years, range 1-10+) by debulking disease and improving blood counts in over two thirds (13/19; 74%) of the HCL-variant patients (Matutes et al, 2001). Splenic irradiation may be an alternative in patients with high surgical risks (Sgaraboto et al, 1997). In our series of patients, only 14% (2/14) and 52% (12/23) achieved partial transient responses to Interferon-alpha and the purine analogs pentostatin and/or cladribine respectively. Data are scanty on the use of monoclonal antibody therapy with Rituximab and/or BL22, a recombinant immunotoxin containing an anti-CD22 variable domain fused to truncated pseudomonas toxin, but the efficacy of these agents has been documented in a few case reports (Kreitman et al., 2001; Narat et al, 2005).

The median survival of our HCL-v patients was 9 years and 15% survived beyond 17 years. Further, 42% of patients died from unrelated causes, namely non-hemopoietic malignancies and cardiovascular disease. Transformation to large cell was documented in a minority (6%) of patients.

Conclusion

HCL-V is a disease different from HCL which appears to be resistant to Interferon alpha and the purine analogs. There are data suggesting that antibody therapy with Rituximab or BL22 is effective and studies with these agents alone or in combination with purine analogs are warranted. At present, splenectomy is a valuable approach particularly in patients with hypersplenism and bulky spleens. The worse survival in HCL-variant, compared with typical HCL cases, may reflect the chemotherapy resistance.

Morphological Characteristics and Immunophenotyping

Gerald Lozanski
Assistant Professor in Pathology
The Ohio State University

Diagnosis of Hairy Cell Leukemia:

Accurate diagnosis of hairy cell leukemia (HCL) is important since very effective therapy used for treatment of HCL is much less effective in other types of chronic B cell lymphoproliferative disorders. Diagnosis of hairy cell leukemia is established based on a combination of morphologic and immunophenotypic findings. Blood smear, bone marrow aspirate smears, bone marrow touch preparations and bone marrow biopsy are most often used for diagnosis of HCL. If available; spleen, liver biopsy or rarely other tissue involved by HCL may be used for diagnosis of HCL as well.

Morphologic characteristics of hairy cell leukemia.

Peripheral blood findings:

Review of Wright-Giemsa stained peripheral blood smears from patients with hairy cell leukemia show pancytopenia. Very characteristic for HCL, absolute monocytopenia is invariably present. Circulating hairy cells (HC) are often rare and finding them may require meticulous search in multiple high power fields. In approximately 10% of HCL patients with marked leukopenia, hairy cells are too rare to be found on even carefully reviewed regular peripheral blood smear. In such cases preparation of buffy coat smears is required for HC identification. It is important to note that in the thicker portion of blood smear hairy cell like projections may be induced as an artifact even in normal lymphocytes and therefore the review of smear in search for HCs should focus on the thin portion of blood smear. Low number of malignant cells in blood is characteristic for this disease and a leukemic picture with a high number of circulating malignant cells is rare. In fact, the finding of a high number of leukemic cells, especially without absolute monocytopenia, should prompt differential diagnosis of other forms of B cell lymphoproliferative disorders including a variant form of hairy cell leukemia.

Hairy cells are small to medium sized lymphocytes with moderately abundant pale blue cytoplasm and centrally to slightly eccentrically placed nuclei. Supravital observation under contrast phase microscope shows cells with a fully circumpherantial corona of prominent hairy projections, giving hairy cells their name. However, on air dried Wright Giemsa stained blood smears hairy cells show a spectrum of cytoplasmic edge irregularities ranging from classical hairy projections (often in a minor population of cells) through shaggy to serrated borders. Due to pale blue staining cytoplasmic borders, hairy cells often are indistinct and seem to blend with the background of stained blood smear. In a small subset of cases blue-gray; round or rod shaped discrete cytoplasmic inclusion are present. These inclusions represent ribosome-lamellar complexes that are unique for HCL and can be seen under scanning electron microscopy. Nuclear shape of HC may be variable, however, in the majority of cases the nuclei are oval to slightly indented with relaxed “spongy” chromatin and absent nucleoli. Prominent nucleoli are very atypical for classical hairy cells and such findings should question diagnosis of HCL. In rare cases HCL may present with atypical large cells with bi-lobed or complex nuclei and variably clumped chromatin. In such cases morphologic evaluation is of limited utility and correct diagnosis of HCL relies on characteristic immunophenotypic profile of such cells.

Peripheral Blood Findings

Bone marrow findings:

Obtaining an adequate bone marrow aspirates in HCL is difficult and often impossible due to bone marrow fibrosis induced by HCL. In the majority of cases touch preparations of fresh core biopsy yield cellular samples that can be used for morphologic evaluation. However, due to HC fragility touch preparation often show numerous naked nuclei and extensive crush artifact that obscure characteristic morphologic features of HCs limiting utility of touch preparations in diagnosis of HCL. Therefore, an adequate trephine biopsy of the bone marrow is essential for correct diagnosis of HCL.

Considering the patchy distribution of HCL in the bone marrow, an adequate biopsy should be obtained for all cases investigated for HCL. Unlike other forms of chronic B cell lymphoproliferative disorders, HCL does not form discrete lymphoid aggregates but infiltrates the bone marrow interstitium diffusely with preservation of bone marrow architecture and stromal fat. In fact, due to fibronectin deposited by HCs, the bone marrow appears “super organized” or “stiff” due to uniformly distributed pericellular reticulin that can be readily highlighted by silver stain.

Bone marrow with extensive involvement by HCL shows sheets of very monotonous medium sized cells with abundant clear cytoplasm and centrally placed widely spaced nuclei. Nuclei vary in shape from round through oval to reinform and show moderately condensed chromatin and inconspicuous nucleoli. This well organized infiltrate with regular cell size, shape and spacing of nuclei is often compared to “fried eggs” in appearance. While easy to identify when abundant in cases with a low number of malignant cells, HCs are notorious for blending in with residual hematopoietic elements. Such rare HCs are often obscured by the residual bone marrow cells to the point where even an experienced hematopathologist can not distinguish them from normal cells on H&E stained bone marrow biopsy sections.

Scattered HCs can mimic erythroid elements, left shifted mononuclear myeloid cells or monocytes. In such cases the IHC stains for pan B cell antigens such as CD20 or CD79a are invaluable for identification of insidious interstitial HCL infiltrates. Moreover, due to myelosuppressive cytokines produced by HCs in a subset of patients, the bone marrow may appear “aplastic” with marked trilineage hypoplasia, preserved stromal empty fat and rare nucleated cells. In such aplastic cases scattered leukemic cells never form discrete lesions and can be easily confused with residual bone marrow cells. Proper identified of such rare HCs can be achieved by IHC stains using pan B cell antigens. For that reason pan B IHC staining should be routinely used for all adult patients investigated for bone marrow aplasia.


A. Bone marrow involvement by HCL H&E stain 20x magnification.

B. Bone marrow involved by HCL CD20 IHC stain highlights numerous hairy cells.

C. Bone marrow involved by HCL DBA44 IHC stain highlights numerous hairy cells.

Spleen findings:

Splenomegaly is present in 90% of patients with HCL, with the spleen often weighing more than 1000g. Therapeutic splenectomy is no longer routinely performed in HCL patients since the discovery of purine analogues as effective treatment for HCL. Infrequent splenectomy may be performed in HCL patients with marked hypersplenismor resistant disease. Careful examination for HCL should always be performed in every splenectomy specimen submitted for pathologic evaluation. HCL involvement of red pulp with sparing of the white pulp is very characteristic for HCL. However, it is not absolutely specific since similar red pulp infiltration is present in hairy cell leukemia variant (HCLv) and in recently described diffuse red pulp small B-cell lymphoma. Low power review of spleen sections demonstrates expanded red pulp and markedly atrophic white pulp. On higher power the red pulp shows altered architecture with blurred demarkation between cords of Billroth and sinuses. Closer examination shows diffuse lymphoid infiltration comprised of small to medium sized cells with abundant pale to clear cytoplasm with centrally placed round to reniform (sometimes irregular) nuclei with open chromatin and indistinct nucleoli. The nuclei show wide spacing due to abundant cytoplasm. Another characteristic finding in the spleen with HCL involvement is the formation of “blood lakes” that represent dilated sinusoidal spaces lined by hairy cells. This change occurs as a consequence of basement membrane degeneration that is required for support of endothelial cells with secondary red cell extravasation and formation of “blood lakes”.


A. Normal spleen distinct white pulp and unremarkable red pulp with visible sinuses and cords. (4 x magnification)

B. Spleen involved by HCL infiltrate with characteristic “ blood lakes” (10x magnification)

C. Spleen with complete effacement of white pulp and replacement of the red pulp by HCL infiltrate. Characteristic for HCL “ blood lake” ( 20 x magnification)

D. Blood lake – characteristic morphologic finding in spleen involved by HCL ( 40x magnification)

E. Spleen involved by HCL (40 x magnification)

HCL immunophenotype:

Classical HCL expresses a very characteristic repertoire of surface antigens that allows reliable HCL distinction form other forms of low grade B cell lymphoproliferative disorders. It is important to note that since the individual antigens show variability of expression both in HCL and the other forms of B cell disorders their expression should not be interpreted individually but rather in the context of a broad panel of antigens. This approach enables pathologist to define disease specific immunophenotype. Comprehensive immunophenotype can be determined by two methods; flow cytometric analysis that uses viable unfixed cells in blood or bone marrow aspirates or by immunohistochemistry (IHC) that can use formalin fixed and paraffin embedded tissue. The advantage of flow cytometric analysis is its ability to determine simultaneously the co-expression of different antigens in a single cell. Provided adequate cellular specimen this method allows accurate immunoprofiling of thousands of cells in a short period of time. Unfortunately in HCL, blood and bone marrow aspirates often are hypocellular with only rare malignant cells available for flow studies. The advantage of the IHC method is the fact that it can be used in formalin and paraffin embedded variety of tissues including bone marrow core biopsy, spleen, liver and rarely other tissues involved by HCL. The disadvantage of this method is that in its most popular form used currently in clinical laboratories it can only determine individual antigens separately and is difficult to demonstrate co-expression of multiple antigens. Moreover, since some antigens undergo irreversible changes induced by fixation not all antigens of interest may be investigated by IHC. Considering advantages and limitations of these two methods pathologists use them both in a complementary manner to determine the immunophenotype that is critical for accurate diagnosis of HCL. Antigens expressed by classical hairy cell leukemia cells are listed in the table 1.

: characteristic positive antigen that as a panel are specific for the diagnosis of HCL.

: HCL characteristic negative antigens. If B cells express any of these strongly diagnosis of HCL should be questioned.
*: Antigens that usually are negative in HCL. However, a subset of HCL may show variable expression of these antigens

A.Space for composite flow diagram illustrating classical HCL immunophenotype

HCL differential diagnosis:

Based on cell morphology, histologic features and immunophenotypic profile HCL can be easily distinguished from more common B cell lymphoproliferative disorders such as chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma and mantle cell lymphoma all of which express a distinct immunophenotypic profile and/or cytogenetic/chromosomal changes. A subset of B cell malignancies may mimic HCL with isolated involvement of spleen and bone marrow and may present diagnostic challenges are; variant of HCL, splenic marginal zone lymphoma with villous lymphocytes, splenic diffuse red pulp small B cell lymphoma and B cell prolymphocytic leukemia. Distinguishing these B cell malignancies from true HCL cases is important since their response to specific HCL therapy is poor. In general careful review of clinical, laboratory, radiology and morphologic findings in the context of complete immunophenotypic profile leads to proper diagnosis. The finding of moderate or high leukocyte count with absolute lymphocytosis without monocytopenia should prompt the question of hairy cell leukemia diagnosis. Morphologic features such as prominent nucleoli, clumped chromatin, very irregular nuclear outlines of lymphoid cells and formation of discrete lymphoid aggregates in bone marrow or involvement of white pulp in spleen are not c/w HCL. Furthermore, clinical or radiological evidence of lymphadenopathy or involvement of extranodal tissue other than spleen and liver is vary rare in HCL. Difficulty arises when a patient presents with low lymphocyte count, small lymphocytes without nucleoli and clinical/radiological evidence of involvement of spleen and bone marrow but no evidence of lymphadenopathy. Splenectomy is no longer used as a routine therapy option for HCL and therefore the diagnosis is often rendered based on blood and bone marrow samples. The combination of morphologic and comprehensive immunophenotypic studies using flow cytometric analysis for viable leukemic cells and IHC analysis of bone marrow core biopsy material is used to determine HCL specific antigenic profile. Table 2 lists the most useful markers that allow for the distinction between HCL and other low grade B cell lymphoproliferative disorders that may mimic HCL.

: characteristic positive antigen that as a panel are specific for the diagnosis of HCL.

: HCL characteristic negative antigens. If B cells express any of these strongly diagnosis of HCL should be questioned.

*: Antigens that usually are negative in HCL. However, a subset of HCL may show variable expression of these antigens

**: Limited data is available.

**: Conflicting data is available

SDRPSBL: This is a new category of B cell lymphoproliferative disorder that involves spleen red pulp and mimics HCL but biologically is more related to splenic marginal zone lymphoma with villous lymphocytes than to HCL. This is a rare disorder and reported here immunophenotypic data is limited ** or not available.

TRAP (immuno)

Tartrate resistant acid phosphatase (TRAP) in HCL: Hairy cells are uniquely rich in the tartrate resistant acid phosphatase (TRAP) isoenzyme 5. Detection of TRAP activity using freshly prepared air dried smears of blood is achieved by cytochemical reaction where enzymatic activity that produces color cytoplasmic granules resists inhibition with tartaric acid. Properly performed TRAP stain is fairly specific for HCL and shows strong reactivity with numerous cytoplasmic granules that often obscure nuclei. Since other B cell malignancies may show weak TRAP reactivity a minimum of two cells with at least 41 cytoplasmic positive TRAP granules is required for definitive identification of hairy cells on TRAP cytochemical stained slide. (Gerard: Do people really count these granules??) Multistep TRAP cytochemical staining is technically difficult and requires arcane knowledge that is not easily reproducible from laboratory to laboratory. Moreover, cytochemical TRAP reaction requires fresh air dried cells and can not be performed in formalin fixed cells. This limits the utility of this stain since in many cases bone marrow aspirates are acellular due to HCL associated bone marrow fibrosis and the only material available for evaluation is formalin fixed trephine biopsy. Recently, developed TRAP specific monoclonal antibody renewed interest in TRAP detection using immunohistochemical (IHC) staining. The IHC staining for TRAP allows detection of TRAP in formalin fixed samples such as bone marrow biopsies, clot preparations and tissue sections. The IHC TRAP stain is fairly specific and shows cytoplasmic granular staining in 100% of HCL cases tested and only in 9% of non HCL B cell malignancies when using formalin fixed and paraffin embedded sections.