JAK2V617F activating mutation is associated with the myeloproliferative type of chronic myelomonocytic leukaemia.

BACKGROUND: Chronic myelomonocytic leukaemia (CMML) is a haematopoietic malignancy with heterogeneous clinical and morphological features. It is classified in the World Health Organization myeloproliferative-myelodysplastic overlap category. JAK2(V617F) mutation can be found in a large percentage of patients with myeloproliferative neoplasms. AIMS: To investigate the association between JAK2(V617F) mutation and clinical, haematological and bone marrow histological features in CMML and to verify whether the mutation is associated with the myeloproliferative type of the disease. METHODS: 78 consecutive patients with newly diagnosed CMML from 2004 to 2008 were included in the study. JAK2(V617F) mutation was assessed using direct sequencing of exon 14 or by allele-specific PCR from total peripheral blood or bone marrow samples. RESULTS: JAK2(V617F) mutation was identified in eight cases (10.2%). All patients with the mutation presented with splenomegaly and had a significantly higher haemoglobin level and neutrophil count than patients without the mutation. All bone marrow biopsies of JAK2(V617F)-mutated CMML showed increased erythropoiesis, a marked myeloid and megakaryocytic hyperplasia with occasionally clustered megakaryocytes, and a mild or moderate (grade 1 or 2) fibrosis; six cases showed an increased number of dilated sinusoids and reactive lymphoid nodules. CONCLUSIONS: The results indicate that JAK2(V617F) mutation is associated with clinical and morphological features of the myeloproliferative type of CMML. Therefore, JAK2 mutation analysis together with bone marrow morphology could help in a more appropriate classification of the disease.

Increased frequency of HLA-DR6 allele in Italian patients with hepatitis C virus-associated oral lichen planus.

BACKGROUND: Recent controlled studies have confirmed that hepatitis C virus (HCV) is the main correlate of liver disease in patients with lichen planus (LP), mainly in southern Europe and Japan. However, a low prevalence of HCV infection has been found in LP patients in England and northern France, and significant differences in serum HCV RNA levels or HCV genotypes have not been found between LP patients and controls. Thus host rather than viral factors may be prevalent in the pathogenesis of HCV-related LP. The HLA-DR allele may influence both the outcome of HCV infection and the appearance of symptoms outside the liver. OBJECTIVES: To assess whether major histocompatibility complex class II alleles play a part in the development of HCV-related LP. METHODS: Intermediate-resolution DRB typing by hybridization with oligonucleotide probes was performed in 44 consecutive Italian oral LP (OLP) patients with HCV infection (anti-HCV and HCV RNA positive), in an age, sex and clinically comparable disease control group of 60 Italian OLP patients without HCV infection (anti-HCV and HCV RNA negative), and in 145 healthy unrelated Italian bone marrow donors without evidence of liver disease or history of LP and with negative tests for HCV. RESULTS: Patients with exclusive OLP and HCV infection possessed the HLA-DR6 allele more frequently than patients with exclusive OLP but without HCV infection (52% vs. 18%, respectively; Pc (Pcorrected) = 0.028, relative risk = 4.93). We did not find any relationship between mucocutaneous LP, HCV infection and HLA-DR alleles. CONCLUSIONS: HCV-related OLP therefore appears to be a distinctive subset particularly associated with the HLA class II allele HLA-DR6. This could partially explain the peculiar geographical heterogeneity of the association between HCV and LP.

Interleukin-8 induces the accumulation of B-cell chronic lymphocytic leukemia cells by prolonging survival in an autocrine fashion.

Several cytokines have been suggested to play a regulatory action on the neoplastic clone of patients with B-cell chronic lymphocytic leukemia (B-CLL) by interfering in the differentiation, proliferation, or death/survival pathways. Interleukin-8 (IL-8) is a chemoattractant protein constitutively expressed at the mRNA level and released by B-CLL cells. In view of the presence of the IL-8 receptor mRNA and of specific IL-8 binding, confirmed also by Scatchard analysis using 125I-IL-8, the study was extended to evaluate the possible regulatory role of this cytokine on B-CLL cells. IL-8 failed to show any in vitro proliferative effect on leukemic B-CLL cells. By contrast, the propidium iodide (PI) staining of the DNA content showed that IL-8 could prolong the survival of resting B-CLL cells in 11 of 16 cases studied. In the remaining 5 cases, 90.6% +/- 4.39% SD of the cells after culture remained viable and IL-8 could exert a significant death protection action after pretreatment with 10(-4) mol/L hydrocortisone, which reduced the percentage of viable B-CLL cells. The dose range of IL-8 capable of inducing the prolonging survival effect is comparable with the levels of IL-8 released constitutively by B-CLL cells, indicating that the death protection action is exerted at physiologic doses. The in vitro rescue from death induced by IL-8 is reflected by an increased expression of bcl-2 mRNA in B-CLL cases incubated in the presence of IL-8. These findings were further confirmed at the protein level, because in B-CLL cells that displayed a bimodal bcl-2 intracytoplasmatic protein expression IL-8 was capable of upmodulating the bcl-2high expression peak. The potential autocrine regulatory action exerted by IL-8 is supported by the evidence that exogenous IL-8 can upregulate IL-8 mRNA in B-CLL cells. These results, together with the demonstration that antibody-mediated neutralization of endogenous IL-8 could induce a significant in vitro reduction in the number of living cells, further support the hypothesis that, in B-CLL, the physiologic doses of IL-8 released constitutively by the leukemic clone may play an autocrine role in the process of cell accumulation characteristic of this disease.

The IL-2 receptor complex: expression and function on normal and leukemic B cells.

The interleukin 2 receptor (IL-2R) is composed of at least three different chains that may be variably expressed on normal and neoplastic lymphocytes. While considerable knowledge has been gained on the expression of the IL-2R alpha chain on human leukemic cells of different origin, less is known of the beta and gamma chains. In view of the highly pleiotropic functions exerted by IL-2, the IL-2-IL-2R interactions may play an important role in various leukemias. In this review we focus on the expression and function of the IL-2R complex on human normal and leukemic B cells. Possible implications in the expansion of the neoplastic clone and in the clinical course of the disease are discussed.

A potential role for antigen selection in the clonal evolution of Burkitt's lymphoma.

Burkitt's lymphoma (BL) is a monoclonal lymphoproliferative disorder characterized by the presence of specific chromosomal translocations that involve the c-myc proto-oncogene. Two subtypes of BL exist (endemic and sporadic) that differ in the prevalence of EBV genome expression. Although EBV infection may promote cellular proliferation in endemic BL, little is known about the forces that drive clonal expansion and evolution in the majority of EBV-negative sporadic BL. This study on an EBV-negative sporadic BL cell line derived from an AIDS patient provides evidence that antigenic stimulation may play a role in the development and/or expansion of such tumors. This cell line (BRG-P) contained a series of cellular clones that elaborated both IgM and IgA. Southern blot analyses of the line and its sublines indicated that both the IgM+ and IgA+ cells had identical c-myc and Ig JH gene rearrangements, indicating that they were derived from a common precursor, some of which eventually underwent an isotype switch. Ig VH gene sequence analyses of 21 molecular clones derived from the parental BRG BL line and two of its sublines demonstrated that all the clones used the same VH3 gene. Five unique intraclonal variants were identified at four distinct nucleotide positions (125, 161, 355, 375), which undoubtedly represented somatic mutations. Four of these five mutations occurred within complementary determining regions; all resulted in amino acid replacements. Moreover, an identical G to A nucleotide substitution that resulted in an identical amino acid change occurred at two distinct points in clonal evolution that were separated by the isotype class switch. Thus, the locations and types of the VH gene mutations, together with the occurrence of an isotype switch, are highly suggestive of an ongoing role for Ag stimulation and selection in the evolution of the malignant clone.

Environmental signals influencing expression of the IFN-gamma receptor on human T cells control whether IFN-gamma promotes proliferation or apoptosis.

IFN-gamma R expression is subject to contrasting modulation on human T cells. IFN-gamma R constitutive expression is low on three human malignant T cells (ST4, PF382, and Jurkat) growing in medium supplemented with serum. The addition of IFN-gamma down-modulates IFN-gamma R expression and increases both proliferation and MHC class I Ag expression. By contrast, when malignant T cells are cultured in medium without serum, IFN-gamma R expression dramatically increases and the cells undergo a slow apoptotic death. The addition of IFN-gamma enhances apoptosis and inhibits cell rescue in serum-supplemented medium. This opposite ability of IFN-gamma to stimulate malignant T cell proliferation or death correlates with the intensity of IFN-gamma R cell expression, high expression being a marker for cell apoptosis. IFN-gamma R up-modulation also occurs on malignant T cells undergoing apoptosis after treatment with dexamethasone, on irradiated normal CD3+ PBL, and on cultured normal CD3+ thymocytes. Moreover, the ability of IFN-gamma to augment apoptosis of highly IFN-gamma R-positive thymocytes suggests that its role in promoting T cell apoptosis is also important in physiologic conditions.

Mixed acute leukemia with genotypic lineage switch: a case report.

Morphologically well classifiable leukemias can reveal a mixed phenotype. A case of acute myeloid leukemia (CD13, CD33, CD14, CD11b) which at presentation showed a co-expression of B-lymphoid markers (CD19, CD10, CD20), at the time of the first relapse revealed a morphologic, phenotypic and genotypic switch of the blasts to a purely lymphoid form. Analysis of the immunoglobulin (Ig) H chain locus and of the T-cell receptor (TCR) genes showed at diagnosis a germline configuration of the IgH, TCR beta and tau genes, and a deletion of the TCR delta gene at the second chromosome. At relapse, monoclonal rearrangements of the IgH, TCR tau, and TCR delta were detected. At a subsequent relapse, the blasts re-expressed myeloid morphologic features and myeloid-associated antigens, while they retained the same rearranged configuration of the IgH and TCR beta and delta genes. The TCR delta gene configuration, which links each phase of the disease, may represent an early pathogenetic event and makes the emergence of a second malignancy unlikely. Each phenotypic change occurred after anti-myeloid and anti-lymphoid oriented chemotherapy. The close correlation between the progressive acquisition of different phenotypes and the switch at the genomic level represent the peculiar features of this unusual case.

Persistent polyclonal lymphocytosis in human immunodeficiency virus-1-infected patients.

In this study we describe the clinical, morphologic, immunologic, and genetic features of a chronic peripheral blood lymphocytosis associated with posttraumatic splenectomy in patients with human immunodeficiency virus-1 (HIV-1) infection. Among a series of 2,365 consecutive HIV-1 seropositive cases investigated, eight patients were selected for the presence of more than 4,000 lymphocytes/mm3. All cases were characterized by a lymphocytosis with cytoplasmic azurophilic granules; in three patients the hematologic picture was superimposable with that of lymphoproliferative disease of granular lymphocytes. Phenotypic analysis of lymphocytes showed a prevalent CD3+CD8+ pattern. In vitro evaluations, including the response to mitogens and interleukin-2 and the cytotoxic assays, showed an unimpaired lymphocyte function in the majority of our patients, even in those with advanced stages of the syndrome. The analysis of the configuration of the T-cell receptor (TCR) beta and gamma genes showed a polyclonal pattern of rearrangement. At the mean follow-up time of 45 +/- 8 months, one patient died of overdose when the clinical conditions were stable; all the other patients are alive, although disease progression was documented in two. Our results indicate that a chronic polyclonal lymphocytosis may be associated with HIV-1 infection; this finding seems to be restricted to patients who have undergone splenectomy. The demonstration of a still uncompromised immune system together with a silent clinical course in the patients under study also suggest that splenectomy per se does not favor an aggressive clinical behavior of HIV-1 infection.

Heterogeneous immunoglobulin gene rearrangement in a B-chronic lymphocytic leukemia progressing into non-Hodgkin lymphoma (Richter syndrome).

BACKGROUND: The relationship between chronic lymphocytic leukemia (CLL) and supervening non-Hodgkin lymphoma is debated, as is whether a particular genomic pattern is related to the emergence of the terminal lymphoma. To investigate these features, the molecular organization of the immunoglobulin (Ig) gene region in a case during both the B-CLL and Richter transformation phase was studied. METHODS: B-CLL and non-Hodgkin lymphoma cells were processed for Southern blot analysis of Ig heavy- and light-chain gene configuration. RESULTS: Molecular studies of B-CLL cells revealed the presence of a single Ig heavy-chain rearrangement with both kappa and lambda light-chain rearranged genes, which was consistent with the occurrence of multiple mutational events during the development of the B-CLL clone. Molecular analysis of the lymphoma DNA showed new Ig heavy- and kappa light-chain rearrangements in addition to the original ones related to the CLL phase, indicating that the lymphoma tissue consisted of two genotypically distinct populations of cells. CONCLUSIONS: On the basis of the overall molecular configuration, this heterogeneous pattern of Ig gene rearrangement was interpreted as an inherent genetic instability of the CLL clone, in which multiple mutational events allowed a selective pressure toward more aggressive subclones, resulting in the emergence of the terminal lymphoma.

Constitutive production of tumor necrosis factor-alpha in hairy cell leukemia: possible role in the pathogenesis of the cytopenia(s) and effect of treatment with interferon-alpha.

PURPOSE: In view of the pleomorphic role cytokines play in human lymphoproliferative disorders, we investigated the possible involvement of tumor necrosis factor-alpha (TNF) in hairy cell leukemia (HCL). PATIENTS AND METHODS: The levels of TNF were measured in the serum of untreated patients, and in the culture supernatants of unstimulated and stimulated enriched hairy cells (HC). Furthermore, the presence of TNF mRNA transcripts in HC was analyzed. The possibility that HC could inhibit the in vitro growth of normal erythroid progenitors via the release of TNF was also investigated. Finally, in an attempt to correlate the circulating levels of TNF with the course of the disease, these were retested during and after treatment with interferon-alpha (IFN). RESULTS: Significantly increased levels of TNF were found in the sera of untreated HCL patients compared with normal control sera were seen from patients with other diseases (P less than .001), with values greater than 10 pg/mL in 21 of 42 samples tested. A significant decrease (P less than .01) of TNF levels was recorded following IFN-2a administration in 16 cases with detectable pretreatment serum levels of TNF. In two cases, an increase in TNF values was associated with persistence or progression of disease. The likelihood that the circulating levels of TNF were caused by the pathologic cells is supported by the evidence that purified HC may release TNF spontaneously. The values can be markedly increased following in vitro activation with the phorbol ester 12-0-tetradecanoylphorbol-13 acetate (PMA), with B-cell growth factor (BCGF), and, to a further extent, with the combination of PMA and BCGF. Furthermore, the constitutive mRNA for TNF was found in seven of eight HC samples analyzed. Although supernatants of enriched HC, were capable of reducing the growth of normal bone marrow erythroid progenitors by 50%, duplicate experiments using an anti-TNF antibody produced an almost complete disappearance of the inhibitory effect. CONCLUSION: The results of this study suggest that TNF plays an important role in the pathogenesis of the cytopenia(s) characteristically associated with HCL.

Phenotypic and genotypic switch in Philadelphia-positive, BCR-positive blast crisis of chronic myeloid leukemia.

We report a case of Ph1-positive, bcr-positive chronic myeloid leukemia blast crisis (CML-BC) which at presentation showed a mixed myeloid/B-lymphoid immunophenotype along with TdT positivity and, at the molecular level, an oligoclonal rearrangement of the immunoglobulin heavy chain (IgH) gene region. After obtaining a successful remission, at the time of relapse the patient underwent a phenotypic and genotypic switch from mixed to myeloid phenotype, characterized by the loss of the lymphoid markers and TdT expression and by a germline configuration of the IgH gene region. The same bcr rearrangement was, however, found in both phases of the disease, supporting the suggestion of a true phenotypic and genotypic conversion. This report confirms that the neoplastic event in CML may take place at an early multipotent stem-cell level, prior to a well-defined phenotypic and genotypic lineage expression. Moreover, it is suggested that different factors (chemotherapy? growth factors?) may have either eradicated the bcr+/IgH+ clone and promoted the growth of bcr+/IgH- leukemic cells or, alternatively, supported the lymphoid differentiation program and induced a myeloid lineage shift.

Immunoglobulin and T-cell receptor gene analysis in lymphoproliferative disorders.

The analysis of the configuration of the immunoglobulin (Ig) and T-cell receptor (TCR) gene regions has been of great relevance in defining conclusively the nature of several lymphoproliferative disorders in man. Furthermore, this technological tool has also helped to dissect between a monoclonal and polyclonal pattern of proliferation. The major results obtained, the potential use of molecular studies for the detection of minimal residual disease and the relevance of these techniques in the understanding of the processes of leukemogenesis and lymphomagenesis are discussed.

Cytokines in B-Cell Chronic Lymphocytic Leukemia.

The pleomorphic action of different cytokines in regulating the growth and proliferation of normal B- and T-lymphocyte populations is becoming progressively more apparent. Thus, the possibility that some cytokines, either alone or in variable combination, may play a role in different lymphoproliferative disorders has been increasingly suggested and potential autocrine or paracrine loops hypothesized. Here, we shall discuss some of the known B-cell growth factors which have been postulated to be involved in the hematological progression, clinical course and complications in B-cell lymphocytic leukemia.

High prevalence of T-cell receptor V delta 2-(D)-D delta 3 or D delta 1/2-D delta 3 rearrangements in B-precursor acute lymphoblastic leukemias.

Rearrangement of the immunoglobulin (Ig) and T-cell receptor (TcR) genes generally has been considered a useful marker of B- and T-cell lineage in lymphoproliferative disorders. However, concomitant rearrangements of Ig and TcR genes have been commonly reported in the most immature lymphoid malignancies, mainly in B-cell precursor acute lymphoblastic leukemia (ALL). To better characterize the nature of this lineage promiscuity, we have analyzed the configuration of the TcR delta locus in 75 B-precursor ALL. The large majority of cases (87%) displayed a rearrangement or deletion at the delta locus. Among the 57 nondeletional rearrangements, two patterns were predominant and both appeared to derive from partial joining: a V delta-(D)-D delta 3 (32/57) and a D delta 1/2-D delta 3 (11/57) type. A single V delta gene (V delta 2) appeared to be involved in the first type of rearrangement. These findings are at variance with T-ALL, where rearrangements 5' to V delta 2 are usually found. It remains to be elucidated whether this incomplete attempt of V delta 2 gene assembly is related to the neoplastic event or represents a physiologic predisposition occurring during early stages of the normal lymphocyte differentiation.

Establishment of an EBV-positive lymphoblastoid cell line that grows as a lymphoma in nude mice and expresses membrane CD2 molecules.

We describe a human lymphoblastoid cell line (LCL), called ZS, that originated spontaneously from the cultures of gamma-irradiated (50 Gy) peripheral-blood mononuclear cells of a normal donor. When injected subcutaneously in sublethally irradiated, splenectomized and anti-asialo-GM1-treated nude mice, ZS cells invaded the lymph nodes, that appeared 10 to 50-fold enlarged in all of the mice tested. Furthermore, ZS cells expressed a typical T-cell surface structure, the CD2 molecule, detectable by a variety of different anti-CD2 monoclonal antibodies (MAbs). However, other T-cell markers were not found, with the possible exception of a truncated messenger of the beta chain of the T-cell receptor and ZS cells could be identified as B cells since they (i) expressed a battery of markers of the resting and activated B cells, (ii) displayed a monoclonal rearrangement of the IgH chain locus and (iii) synthesized IgM K molecules. The Epstein-Barr virus (EBV) genome was detected in ZS cells in approximately ten copies per cell by DNA hybridization techniques. Furthermore, the cells were positive for EBV nuclear antigens (EBNA). Western blotting analysis of EBV encoded antigens demonstrated clear differences with those present in the B 95.8 virus-producer cell line, indicating that ZS cells were not infected by EBV in vitro and that they already harbored the virus in vivo. ZS cells formed colonies in vitro with a high cloning efficiency and displayed chromosomal abnormalities in all of the mitoses (karyotype 47, xy, +13, -14, 8p+, 21p+, +m). In spite of these malignant features, ZS cells expressed the full range of EBV latent proteins as usually do "normal" LCSs and did not have any of the chromosomal abnormalities that juxtapose the c-myc oncogene to one of the genes coding for immunoglobulin molecules.

Immunologic and molecular evaluation of residual disease in B-cell chronic lymphocytic leukemia patients in clinical remission phase.

This study evaluates residual disease in 28 B-cell chronic lymphocytic leukemia (B-CLL) patients who obtained a clinicohematologic remission after intensive chemotherapy. Sixteen of 28 patients (57%) showed a normal number of circulating B-lymphocytes, as demonstrated by the low percentage of mouse rosette-forming cells (M-RFC), surface immunoglobulins (SIg), and CD24-positive cells. Clinically, a lower number of relapses occurred in this group of patients compared to those with a persistent expansion of peripheral B-cells (P less than 0.05). In order to assess monoclonality of the residual peripheral B-cell population, the distribution of SIg light chains was investigated on the B-cell-enriched fraction of 15 of these 16 cases. Only six of them had a kappa/lambda ratio which ranged between 1.7:1 and 3:1, whereas the remaining patients still displayed a clearly imbalanced kappa/lambda Ig light chain distribution. On the other hand, the analysis of the configuration of the Ig heavy chain gene region, performed in nine cases (including five of the above six cases), showed the persistence of a rearranged pattern in all cases tested but one. Therefore, residual monoclonal B-cells were found also in the majority of cases which displayed the lowest kappa/lambda ratio, a normal bone marrow lymphocytosis and a long-lasting clinical remission. Studies at the DNA level confirm that a remission is rarely achieved in this disease in spite of intensive and prolonged chemotherapy. Nonetheless, the follow-up of B-CLL patients by conventional immunologic markers may be helpful to better define response to therapy and to predict the occurrence of clinical relapse.

Acute lymphoblastic leukemia with the 4;11 translocation exhibiting early T cell features.

We describe a case of ALL with the t(4;11) (q21;q23) translocation in which both surface markers and molecular analyses suggest an unusual early T cell involvement. While the morphologic and cytochemical studies showed an undifferentiated pattern, immunophenotypic data were suggestive of a very immature cell population which stained only for TdT and CD7. Moreover, in contrast to previous reports but in agreement with the immunologic findings, the IgH gene region retained a germline configuration. T cell receptor beta and gamma chain gene loci also showed a germline pattern, in accordance with the expansion of immature CD7+, TdT+ T cells.

Ontogeny, gene rearrangements and immunophenotype of acute leukaemias.

The progressive availability of more sophisticated technologies has over the last few years allowed a more precise definition of the biological properties of acute leukaemia cells. This, in turn, has enabled to recognize the ontogeny of practically all cases, with particular emphasis to acute lymphoblastic leukaemia, the lineage affiliation of which had, for many years remained uncertain in over half of the cases. Here, we shall review the main achievements, obtained with extensive immunotyping coupled to the use of probes for the immunoglobulin and T-cell receptor genes, which have led to these important clinico-biological acquisitions, and discuss specific situations in which this combined phenotypic and genotypic approach (as well as response to cloned growth factors) may be of particular value.