A Very Rare Case -  Hairy Cell Leukemia in Patient with Sarcoidosis.

Although the coexistence of hairy cell leukemia with sarcoidosis has been reported in a few cases in the literature, in our case the patient had been diagnosed and followed about 10 years with sarcoidosis and massive splenomegaly. It has been demonstrated that T helper 1 cells exist in organs influenced by sarcoidosis. These cells produce IL-2 and IFN-γ and induce a nonspecific inflammatory response and granuloma formation. Also these cytokines may play a role in the development of hairy cell leukemia.Key words: hairy cell leukemia -  sarcoidosis - massive splenomegaly.

Class II human leucocyte antigen DRB1*11 in hairy cell leukaemia patients with and without haemolytic uraemic syndrome.

Frequencies of human leucocyte antigens (HLA) were determined in 287 classic hairy cell leukaemia (HCL) patients. With respect to both population (n = 287) and allele (2n = 574) frequency respectively, the most common HLA class I and II antigens expressed were HLA-A*02 (49·1% and 28·6%), HLA-B*07 (21·3% and 11·1%), HLA-C*07 (46·7 and 28·2%), HLA-DQB1*03 (62·7% and 37·3%), HLA-DRB1*11 (30·0% and 16·0%) and HLA-DRB4*01 (45·3% and 29·6%). In comparing 6-14 databases of control Caucasians to 267 Caucasian HCL patients, only HLA-DRB1*11 was consistently over-represented in HCL, 31·1% of patients vs. 17-19·9% of controls (P = 0·0055 to <0·0001) and 16·5% of alleles vs. 6·5-12·3% of control alleles (P = 0·022 to <0·0001). HLA-DRB1*11 is a known risk factor for acquired thrombotic microangiopathy. Anti-CD22 recombinant immunotoxin BL22 in HCL was associated with a 12% incidence of completely reversible grade 3-4 haemolytic uraemic syndrome (HUS), mainly during the second or third retreatment cycle. Of 49 HCL patients receiving ≥2 cycles of BL22, 7 (14%) had HUS and HLA-DRB1*11 was expressed in 71% of 7 with HUS compared with only 21% of 42 without (P = 0·015). These data suggest that DBR1*11 may be a marker for increased susceptibility to HCL and, among HCL patients, could be a risk factor for BL22-induced HUS.

Hairy cell leukemia accompanied by Evans syndrome.

We report a case of Western type hairy cell leukemia (HCL), a very rare leukemia in Japan. In this malignancy, leukemic cells in a peripheral blood film may be missed due in part to accompanying pancytopenia and in part to loss of typical cytoplasmic projections if prepared in a conventional Japanese way using forced air-drying. Our present patient also had a variety of autoantibodies and the clinical picture was primarily that of Evans syndrome (ES), suggesting disturbed immune responses associated with the HCL. Although HCL accompanied by either AIHA or ITP has been reported, the occurrence of ES in HCL is extremely rare.

Evidence of canonical somatic hypermutation in hairy cell leukemia.

To compare hairy cell leukemia (HCL) with chronic lymphocytic leukemia (CLL) and normal B cells with respect to their B-cell receptors, somatic hypermutation (SHM) features in HCL were examined in a series of 130 immunoglobulin gene heavy chain rearrangements, including 102 from 100 classic (HCLc) and 28 from 26 variant (HCLv) patients. The frequency of unmutated rearrangements in HCLc was much lower than that in HCLv (17% vs 54%, P < .001) or historically in CLL (17% vs 46%, P < .001), but HCLv and CLL were similar (P = .45). As previously reported for CLL, evidence of canonical SHM was observed in HCLc rearrangements, including: (1) a higher ratio of replacement to silent mutations in the complementarity determining regions than in the framework regions (2.83 vs 1.41, P < .001), (2) higher transition to transversion ratio than would be expected if mutations were random (1.49 vs 0.5, P < .001), and (3) higher than expected concentration of mutations within RGYW hot spots (13.92% vs 3.33%, P < .001). HCLv met these 3 criteria of canonical SHM to a lesser extent. These data suggest that, whereas HCLc cells may recognize antigen-like CLL and normal B cells before malignant transformation, HCLv cells from some patients may originate differently, possibly without undergoing antigen recognition.

Differential and tumor-specific expression of CD160 in B-cell malignancies.

CD160 is a human natural killer (NK)-cell-activating receptor that is also expressed on T-cell subsets. In the present study, we examined 811 consecutive cases of B-cell lymphoproliferative disorders (B-LPDs), and demonstrated CD160 expression in 98% (590 of 600) of chronic lymphocytic leukemia (CLL) cases, 100% (32 of 32) of hairy cell leukemia (HCL) cases, 15% (5 of 34) of mantle cell lymphoma (MCL) in the leukemic phase, and 16% (23 of 145) of other B-LPD cases. CD160 transcript and protein were absent in the normal B-cell hierarchy, from stem cells, B-cell precursors, maturing B cells in the germinal center, and circulating B cells, including CD5(+)CD19(+) B1 cells in umbilical cord. CD160 positivity was significantly higher in CLL and HCL in terms of percentage (65.9% and 67.8%, respectively, P < .0001) and median fluorescence intensity (552 and 857, respectively, P < .0001) compared with all other B-LPD cases. Lymph node CLL samples were also CD160(+). Using the disease-specific expression of CD5, CD23, and CD160, a score of 3 characterized CLL (diagnostic odds ratio, 1430); a score of 0 excluded CLL, MCL, and HCL; and the CD23/CD5 ratio differentiated CLL from leukemic CD23(+) MCL. In the B-cell lineage, CD160 is a tumor-specific antigen known to mediate cellular activation signals in CLL, and is a novel target for therapeutic manipulation and monitoring of minimal residual disease.

Lack of allelic exclusion by secondary rearrangements of tumour B-cell receptor light chains in hairy cell leukaemia.

Analyses of the tumour immunoglobulin (Ig) gene (IG) heavy (H) and light chains show heterogeneity of mutational status, but reveal common features of ongoing IGH isotype-switching with multiple IGH isotype expression and preference of IG lambda (IGL) light chain with selective use of IGLJ3. Phenotypic and immunogenetic analyses were performed in a series of 105 HCL patients to estimate prevalence of multiple IG light chain expression by the tumour cells. By phenotype, 3/105 HCL (2.9%) expressed double tumour-related Ig kappa (K) and L light chain proteins. By immunogenetic analysis, functional mutated double IGK(I) /IGK(II) , IGK(I) /IGL(I) and IGL(I) /IGL(II) transcripts were cloned and sequenced in 3/71 (4.2%) HCL. These latter three HCL expressed multiple IGH isotypes with mutated IGHVDJ rearrangements at the time of AID transcript expression. Most interestingly, the three cases had reinduced RAG1 transcript. In the double IGL expresser, single-cell analysis documented co-expression of the tumour-related IGLs in 5/6 cells (83%). In the IGK/IGL co-expresser, evidence of surface IgK/IgL isotype proteins confirmed functionality of the tumour-derived transcripts. The evidence of double light chain expression in single HCs and the new observation of RAG re-induction suggest ongoing selective influences on the BCR that may promote or maintain the HCL clone in the periphery.

Hairy cell leukaemia: biological and clinical overview from immunogenetic insights.

Hairy cell Leukaemia (HCL) is a rare neoplasm of peripheral B cells which represents a paradox in oncology. Despite its largely unknown origin and behaviour, HCL is one of the few example of dramatic success in the treatment of a malignancy. The recent steps forward to understanding the biology of HCL from immunogenetic and genomic studies have recently provided new insight into diagnosis and prognosis. Several data from immunoglobulin gene (IG) analysis have provided hints regarding the cell of origin and the ongoing selective interactions of the tumour BCR with environmental stimuli. It has also recently emerged that an unmutated status of the HCL IG can be associated with failure to respond to cladribine, genetic abnormalities indicative of poor outcome and aggressive disease. These observations suggest a central role of the tumour B-cell receptor in defining the outcome of HCL and that that IG gene analysis may have biological and prognostic relevance. Hopefully, IG analysis will help tailor treatment strategies for the most aggressive cases.

An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes.

Recombinant immunotoxins are hybrid proteins composed of an Fv that binds to a tumor antigen fused to a bacterial or plant toxin. Immunotoxin BL22 targets CD22 positive malignancies and is composed of an anti-CD22 Fv fused to a 38-kDa fragment of Pseudomonas exotoxin A (PE38). BL22 has produced many complete remissions in drug-resistant Hairy cell leukemia, where many treatment cycles can be given, because neutralizing antibodies do not form. In marked contrast, only minor responses have been observed in trials with immunotoxins targeting solid tumors, because only a single treatment cycle can be given before antibodies develop. To allow more treatment cycles and increase efficacy, we have produced a less immunogenic immunotoxin by identifying and eliminating most of the B cell epitopes on PE38. This was accomplished by mutation of specific large hydrophilic amino acids (Arg, Gln, Glu, Lys) to Ala, Ser, or Gly. The new immunotoxin (HA22-8X) is significantly less immunogenic in three strains of mice, yet retains full cytotoxic and anti-tumor activities. Elimination of B-cell epitopes is a promising approach to the production of less immunogenic proteins for therapeutic purposes.

Dissection of the bone marrow microenvironment in hairy cell leukaemia identifies prognostic tumour and immune related biomarkers.

Hairy cell leukaemia (HCL) is a rare CD20+ B cell malignancy characterised by rare "hairy" B cells and extensive bone marrow (BM) infiltration. Frontline treatment with the purine analogue cladribine (CDA) results in a highly variable response duration. We hypothesised that analysis of the BM tumour microenvironment would identify prognostic biomarkers of response to CDA. HCL BM immunology pre and post CDA treatment and healthy controls were analysed using Digital Spatial Profiling to assess the expression of 57 proteins using an immunology panel. A bioinformatics pipeline was developed to accommodate the more complex experimental design of a spatially resolved study. Treatment with CDA was associated with the reduction in expression of HCL tumour markers (CD20, CD11c) and increased expression of myeloid markers (CD14, CD68, CD66b, ARG1). Expression of HLA-DR, STING, CTLA4, VISTA, OX40L were dysregulated pre- and post-CDA. Duration of response to treatment was associated with greater reduction in tumour burden and infiltration by CD8 T cells into the BM post-CDA. This is the first study to provide a high multiplex analysis of HCL BM microenvironment demonstrating significant immune dysregulation and identify biomarkers of response to CDA. With validation in future studies, prospective application of these biomarkers could allow early identification and increased monitoring in patients at increased relapse risk post CDA.

Gene expression profiling of hairy cell leukemia reveals a phenotype related to memory B cells with altered expression of chemokine and adhesion receptors.

Hairy cell leukemia (HCL) is a chronic B cell malignancy characterized by the diffuse infiltration of bone marrow and spleen by cells displaying a typical "hairy" morphology. However, the nature of the HCL phenotype and its relationship to normal B cells and to other lymphoma subtypes remains unclear. Using gene expression profiling, we show here that HCL displays a homogeneous pattern of gene expression, which is clearly distinct from that of other B cell non-Hodgkin lymphomas. Comparison with the gene expression profiles of purified normal B cell subpopulations, including germinal center (GC), pre-GC (naive), and post-GC (memory) B cells, shows that HCL cells are more related to memory cells, suggesting a derivation from this B cell population. Notably, when compared with memory cells, HCL cells displayed a remarkable conservation in proliferation, apoptosis, and DNA metabolism programs, whereas they appeared significantly altered in the expression of genes controlling cell adhesion and response to chemokines. Finally, these analyses have identified several genes that are specifically expressed in HCL and whose expression was confirmed at the protein level by immunocytochemical analysis of primary HCL cases. These results have biological implications relevant to the pathogenesis of this malignancy as well as clinical implications for its diagnosis and therapy.

Purified lymphocyte function-associated antigen 3 binds to CD2 and mediates T lymphocyte adhesion.

CD2 is a T lymphocyte glycoprotein that functions in adhesion of T lymphocytes and also as a putative receptor for activation signals. Functional data suggest that LFA-3, a widely distributed cell surface glycoprotein, may be the biological ligand of CD2. We have purified LFA-3 from human erythrocytes and characterized the purified protein functionally. LFA-3 bound specifically to CD2+ cells, and this binding was inhibited by CD2 mAb. Conversely, purified LFA-3 inhibited binding of CD2 mAb to cells, and the concentration required for this effect suggests that LFA-3 half-saturated CD2 at 1-5 nM LFA-3. Purified LFA-3 inhibited rosetting of human and sheep erythrocytes with CD2+ T lymphoma cells and T lymphocytes, and mediated aggregation of a CD2+ T lymphoma cell line. Purified LFA-3 reconstituted into planar membranes mediated efficient CD2-dependent adhesion of T lymphoblasts. These data demonstrate that LFA-3 is a ligand for CD2 and that LFA-3 can mediate T lymphocyte adhesion.

Human T cell gamma genes are frequently rearranged in B-lineage acute lymphoblastic leukemias but not in chronic B cell proliferations.

T cell rearranging gene gamma (TRG gamma) and T cell antigen receptor beta (TCR beta) chain gene rearrangement and transcription were studied in a series of patients with B-lineage acute lymphoblastic leukemia (ALL), in which the Ig H chain genes are rearranged and the surface phenotype reproduces the stages of normal pre-B maturation. For comparison, polyclonal T cells from peripheral blood of healthy donors and blast cells from 19 cases of T lineage ALL were also studied. In this study we demonstrate the presence of a clonal rearrangement of the TRG gamma in 18 of the 22 B-lineage ALL cases and establish that this rearrangement, which generally involves the J gamma 1 region, is often monoallelic and appears different from the biallelic J gamma 2 rearrangement frequently seen in T-cell ALLs. In 9 of 22 cases, we found rearrangement of the genes of the TCR beta chain, which never involved the J beta 1 region. Conversely, the TRG gamma were seen in germline configuration in all 19 cases of B chronic lymphoid malignancies. In none of the 9 AML cases studied was TRG gamma and TCR beta chain gene rearrangement found. The TCR beta chain genes were rearranged in one B cell chronic lymphocytic leukemia (CLL). We also show that in B-lineage ALL, the cells probably use the same V gamma genes for TRG gamma rearrangements as the malignant cells in T-ALL and the polyclonal T cells. In none of the 13 B-lineage ALL cases investigated by Northern analysis was TCR beta mRNA expression detected, whereas a weak expression of TRG gamma transcripts was found in two of these cases. The correlations between surface phenotype, rearrangement of TRG gamma, TCR beta, and Ig H chain genes were analyzed. The significance of rearrangement of TRG gamma and TCR beta chain genes in B or pre-B cells is also discussed.

Effects of interferon alpha on autocrine growth factor loops in B lymphoproliferative disorders.

The B lymphoproliferative disorders B chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) produce a number of autocrine growth factors, including tumor necrosis factor (TNF), interleukin 6 (IL-6), and IL-1, all of which may induce positive feedback growth loops. If such malignancies depend on these autocrine growth loops for survival, their interruption may be therapeutically valuable. Interferon alpha (IFN-alpha) abrogates TNF- or IL-6-induced proliferation of HCL and B-CLL cells in vitro and has therapeutic activity in these diseases. We have investigated the possibility that IFN-alpha may act by interrupting autocrine growth factor loops. If purified B-CLL or HCL cells are cultured in the presence of TNF, there is induction of mRNA for TNF, IL-1 alpha, IL-1 beta, and IL-6. However, culture in the presence of IFN-alpha in addition to TNF reduced the level of mRNA for all these cytokines, compared with cells cultured in TNF alone. While cytokine mRNA levels were diminished, levels of mRNA for the ribonuclease activator 2-5A synthetase were increased. Analysis of the kinetics of cytokine mRNA production showed that levels fall shortly after the rise of 2-5A synthetase mRNA. IFN-alpha may produce these effects by shortening the half-life of cytokine mRNA, since TNF mRNA half-life in B-CLL and HCL cells is substantially reduced when the cells are cultured with IFN-alpha. These data suggest that IFN-alpha may mediate its therapeutic effects in these malignancies by blocking autocrine growth factor loops.

[Case with hairy cell leukemia-Japanese variant following radiotherapy for orbital adnexal MALT lymphoma].

We report a patient with hairy cell leukemia Japanese variant (HCL-Jv) that developed after radiotherapy for orbital adnexal MALT lymphoma. A 78-year-old man was diagnosed as having MALT lymphoma in the left conjunctiva in December 2003. The patient was treated by local radiotherapy and the tumor disappeared. Thereafter, he gradually developed leukocytosis and mild splenomegaly. In May 2009, the leukocyte count was 34,300 with 80% lymphoid cells. A diagnosis of HCL-Jv was made since the lymphoid cells showed a hairy morphology with round nuclei and indistinct nucleoli. These cells expressed CD11c, CD19, CD20, CD103 and showed weak reaction for tartrate-resistant acid phosphatase (TRAP). Bone marrow was infiltrated by atypical cells with an intrasinusoidal pattern. No treatment was needed as the patient was asymptomatic without anemia, thrombocytopenia or lymphadenopathy. Results of the immunoglobulin light chain expression and the heavy chain rearrangement in the tumor cells indicated that the two mature B-lymphoid neoplasms, MALT lymphoma and HCL-Jv, in this patient were derived from independent clones. This appears to be the first reported case of HCL-Jv associated with other lymphoid tumor. Further analysis is needed to clarify the risk of secondary malignancy in HCL-Jv.

A novel approach to assess domain specificity of anti-drug antibodies to moxetumomab pasudotox, an immunotoxin with two functional domains.

Biologics are potentially immunogenic and can elicit immune response. Complex biologics, such as bispecific antibodies or multi-domain molecules can induce anti-drug antibodies (ADA) with specificity to different domains. Domain specific ADAs may differently affect drug efficacy and safety, and thus, characterization of ADA domain specificity has become a regulatory expectation for multi-domain biologics. Unlike well-established methods for screening, confirmation, titer and neutralizing ADA detection, characterization of ADA domain specificity is an emerging field. The conventional approach for determination of ADA domain specificity is a competitive inhibition with domain-containing molecules. When developing a conventional domain specificity assay for moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin, comprised of two functional domains (CD22-binding fragment and truncated Pseudomonas exotoxin A (PE38), we encountered a bioanalytical challenge. The method was able to detect immunodominant anti-PE38 (ADA-PE) but generated false negative results for low abundant CD22-binding domain ADA (ADA-BD) in a polyclonal sample. Troubleshooting experiments using control samples with varying levels of each ADA subtype demonstrated that a major factor for successful ADA identification was the ratio of the ADA signals contributed by each ADA subtype. To overcome this unique bioanalytical challenge, we developed a novel approach, which ensures detection of a domain-specific ADA subtype regardless of its relative level in a polyclonal ADA sample by evaluating signal inhibition by a respective domain-containing molecule at the condition when signals from all other ADAs are fully blocked. The method has been used for characterization of ADA domain specificity in moxetumomab pasudotox clinical trials, including study 1053, the pivotal Phase III study in hairy cell leukemia patients. It allowed for successful detection of ADA-BD in the presence of immunodominant ADA-PE, enabling accurate determination of domain specificity for moxetumomab pasudotox. The results demonstrated that the method was superior than the conventional approach. The method could be applied broadly to other biologics with two or more domains when there is a need to detect a minor ADA subtype in polyclonal samples.

Development of Recombinant Immunotoxins for Hairy Cell Leukemia.

Hairy cell leukemia (HCL) is an indolent B-cell malignancy with excellent initial response to purine analogs pentostatin or cladribine, but patients are rarely, if ever, cured. Younger patients will usually need repeat chemotherapy which has declining benefits and increasing toxicities with each course. Targeted therapies directed to the BRAF V600E mutation and Bruton's tyrosine kinase may be helpful, but rarely eradicate the minimal residual disease (MRD) which will eventually lead to relapse. Moxetumomab pasudotox (Moxe) is an anti-CD22 recombinant immunotoxin, which binds to CD22 on HCL cells and leads to apoptotic cell death after internalization and trafficking of the toxin to the cytosol. Phase I testing achieved a complete remission (CR) rate of 57% in relapsed/refractory HCL. Most CRs were without MRD and eradication of MRD correlated with prolonged CR duration. Patients were often MRD-free after five years. Important mild-moderate toxicities included capillary leak and hemolytic uremic syndromes which could be prevented and managed conservatively. A phase 3 trial met its endpoint of durable CR with acceptable toxicity, leading to FDA approval of Moxe for relapsed/refractory HCL, under the name Lumoxiti. Moxe combined with rituximab is currently being evaluated in relapsed/refractory HCL to improve the rate of MRD-free CR.

Synthetic peptide immunoassay distinguishes HIV type 1 and HIV type 2 infections.

Efforts to solve the epidemiologic puzzle of AIDS in Africa are complicated by the presence of multiple human retroviruses. Simple serologic tests that unambiguously distinguish among infections by these retroviruses are essential. To that end, a partially conserved immunoreactive epitope was identified in the transmembrane glycoproteins of human immunodeficiency viruses (HIV) types 1 and 2. Synthetic peptides derived from these conserved domains were used in sensitive and specific immunoassays that detect antibodies in sera from patients infected with HIV-1 or HIV-2. By making single amino acid substitutions in the HIV-1 peptide, it was possible to demonstrate HIV-1 strain-specific antibody responses to this epitope. Such custom-designed peptides synthesized from this domain are likely to detect newly discovered HIV types, define infection with specific HIV strains, and allow detection of group-common antibodies.