Early and reliable diagnosis of non-Hodgkin lymphoma in childhood and adolescence: contribution of cytomorphology and flow cytometric immunophenotyping.

Non-Hodgkin lymphoma (NHL) represents one of the most rapidly growing malignancies in childhood and adolescence. About 80% of patients now are cured with adequate treatment. Serious complications at presentation due to tumor lysis syndrome or local tumor effects are commonly observed. Thus, a rapid diagnosis with the least invasive procedure enabling the initiation of early and specific therapy is necessary to diminish early fatality or persistent impairment. In 56 centrally registered patients with NHL, cytomorphologic analyses (FAB criteria) of May-Grünwald-Giemsa-stained touch imprints or malignant effusions and flow cytometric immunophenotyping (EGIL criteria) of fresh cell suspensions with a standardized panel of monoclonal antibodies were performed. The authors identified 23 patients with Burkitt lymphoma by the combination of FAB L3 morphology and a mature B-cell phenotype and 22 patients with lymphoblastic lymphoma by FAB L1/L2 morphology and a T-/B-cell precursor phenotype. They also found 11 patients with large cell lymphomas, 3 of them with anaplastic large cell lymphoma (T-cell phenotype; NPM/ALK-positive). In the remaining 8 patients diffuse large B-cell lymphoma was suspected by the combined use of cytologic and immunophenotypic findings (mature B-cell phenotype). In all cases with available solid tumor material (n = 42/56) the preliminary diagnosis was confirmed by histopathology. Burkitt lymphoma, lymphoblastic lymphoma, and, in a few cases, some large cell lymphomas could be classified reliably by cytomorphology and immunophenotyping of freshly obtained tumor cell material, enabling an early start of specific lymphoma treatment.

Cyclin D3 is a predictive and prognostic factor in diffuse large B-cell lymphoma.

Cyclin D3 is an important regulator for transition from G(1) to the S phase of the cell cycle. Cyclin D3 expression is associated with cell proliferation in lymphoid tissues, but its impact on clinical outcome in non-Hodgkin's lymphomas has not been studied. Therefore, we determined the clinical relevance of cyclin D3 expression in patients with diffuse large B-cell lymphoma. We examined the relation between cyclin D3 expression at diagnosis and response to conventional polychemotherapy and overall survival in 81 previously untreated patients with diffuse large B-cell lymphoma. Cyclin D3 expression was assessed by immunohistochemistry. Cyclin D3 immunostaining ranged from 0-100% (median, 30%) of the lymphoma cells. Patients with high (>or=50% cyclin D3-positive lymphoma cells) cyclin D3 expression had a more advanced clinical stage (P = 0.003) and more often had extranodal disease in more than one site (P = 0.007) than patients with low cyclin D3 expression. Patients with high cyclin D3 expression had a significantly lower complete response rate (17% versus 74%; P < 0.001) and a shorter overall survival (3-year survival rate, 18% versus 74%; P < 0.001) than those with low cyclin D3 expression. Multivariate analyses that included cyclin D3 and the International Prognostic Index demonstrated that cyclin D3 expression had independent effects on the complete response rates and overall survival of the patients. In conclusion, high cyclin D3 expression is an independent predictive and prognostic factor associated with poor clinical outcome in patients with diffuse large B-cell lymphoma.

Truncated ALK derived from chromosomal translocation t(2;5)(p23;q35) binds to the SH3 domain of p85-PI3K.

The chromosomal translocation t(2;5)(p23;q35) is associated with "Anaplastic large cell lymphomas" (ALCL), a Non Hodgkin Lymphoma occurring in childhood. The fusion of the tyrosine kinase gene-ALK (anaplastic lymphoma kinase) on chromosome 2p23 to the NPM (nucleophosmin/B23) gene on chromosome 5q35 results in a 80 kDa chimeric protein, which activates the "survival" kinase PI3K. However, the binding mechanism between truncated ALK and PI3K is poorly understood. Therefore, we attempted to elucidate the molecular interaction between ALK and the regulatory p85 subunit of PI3K. Here we provide evidence that the truncated ALK homodimer binds to the SH3 domain of p85. This finding may be useful for the development of a new target-specific intervention.

CD25 indicates the neoplastic phenotype of mast cells: a novel immunohistochemical marker for the diagnosis of systemic mastocytosis (SM) in routinely processed bone marrow biopsy specimens.

The diagnosis of systemic mastocytosis (SM) is based primarily on the histologic and immunohistochemical evaluation of a bone marrow trephine biopsy specimen. Although mast cell (MC) specific antigens like tryptase and chymase are detectable in routinely processed tissue, no immunohistochemical markers that can be used to discriminate between normal and neoplastic MCs are yet available. We have investigated the diagnostic value of an antibody against CD25 for the immunohistochemical detection of MCs in bone marrow sections in 73 patients with SM and 75 control cases (reactive marrow, n = 54; myelogenous neoplasms, n = 21) and correlated the results with the presence of c-kit mutations. While MCs in almost all patients with SM (72 of 73) expressed CD25, none of the control samples contained CD25-positive MCs. Irrespective of the SM subtype, most of neoplastic MCs expressed CD25. In 3 patients with advanced MC disease, pure populations of neoplastic MCs were obtained and found to express CD25 mRNA by RT-PCR analysis. In addition, all patients with CD25-positive MCs contained c-kit mutations, while all control cases exhibited wild type c-kit. CD25 therefore appears to be a reliable immunohistochemical marker for the discrimination of neoplastic from normal/reactive MCs, with potential as a diagnostic tool in SM.

Effective treatment of primary plasma cell leukemia with thalidomide and dexamethasone - a case report.

Plasma cell leukemia (PCL) is an aggressive disease with poor prognosis and a median survival of only 2-6 months. Currently, no standard therapy is available, but intensive polychemotherapy appears to be more effective than the conventional melphalan plus prednisone. However, the efficacy of thalidomide in PCL has not yet been widely evaluated. Recently, treatment with thalidomide has been reported to yield promising results in refractory multiple myeloma. Here, we report on a patient with primary PCL in whom first-line treatment with thalidomide/dexamethasone resulted in a rapid response and achievement of a very good partial remission.

The immunophenotype of 325 adult acute leukemias: relationship to morphologic and molecular classification and proposal for a minimal screening program highly predictive for lineage discrimination.

Bone marrow cells of 325 adults with acute leukemia were immunophenotyped using a panel of monoclonal antibodies proposed by the European Group for the Immunological Characterization of Leukemias (EGIL). Of these, 97.2% could be assigned clearly to myeloid or lymphoid lineage (254 acute myeloid leukemias [AMLs], 48 B-cell lineage acute lymphoblastic leukemias [ALLs], 14 T-cell lineage ALLs), 1.8% as biphenotypic, and less than 1% as undifferentiated. Immunologic subtyping of ALLs revealed an association between early precursor phenotypes and coexpression of myeloid antigens, particularly CD15/CD65s coexpression and pre-pre-B cell-specific phenotypes and genotypes. The common ALL phenotype was associated with BCR-ABL translocation. Among AMLs, CD2 coexpression was almost exclusively restricted to French-American-British subtypes M3 variant and M4Eo and related molecular aberrations. The most valuable markers to differentiate between myeloperoxidase-negative AML subtypes M0 and ALLs were CD13, CD33, and CD117, typical of M0, and intracytoplasmic CD79a, intracytoplasmic CD3, CD10, and CD2, typical of B cell- or T cell-lineage ALL. Our results confirm excellent practicability of the EGIL proposalfor immunologic classification of acute leukemias. For myeloperoxidase-negative AMLs, we suggest a scoring system based on markers most valuable to distinguish between AML-M0 and ALLs.

Immunohistochemical detection of VEGF in the bone marrow of patients with chronic myeloid leukemia and correlation with the phase of disease.

We studied expression of vascular endothelial growth factor (VEGF) in paraffin-embedded bone marrow sections obtained from 15 patients with chronic myeloid leukemia (CML) in chronic phase (CP), 3 in accelerated phase (AP), 7 in myeloid blast phase (BP(M)), 6 in lymphoid blast phase (BP(L)), and in 3 normal bone marrow samples. VEGF expression was determined immunohistochemically by using an anti-VEGF antibody. In CML-CP, the distribution of VEGF showed a pattern similar to that of normal marrow. VEGF was expressed in myeloid progenitors and megakaryocytes and less abundantly in mature granulomonocytic cells, whereas erythroid cells did not stain positively for VEGF. In CML-BP(M), myeloblasts expressed substantial amounts of VEGF. By contrast, little if any VEGF was detectable in blast cells in CML-BP(L). VEGF messenger RNA (mRNA) was detected in leukemic cells in CML-BP(M) by reverse transcriptase-polymerase chain reaction, whereas blast cells in CML-BP(L) did not express substantial amounts of VEGF mRNA. Our data show that VEGF is expressed in immature myeloid cells in CML. The extent of VEGF expression depends on the phase of disease and the cell type involved in disease progression.

Immunohistochemical detection of VEGF in the bone marrow of patients with acute myeloid leukemia. Correlation between VEGF expression and the FAB category.

We studied vascular endothelial growth factor (VEGF) expression in bone marrow sections obtained from 3 healthy donors and 41 patients with acute myeloid leukemia (AML) of various French-American-British (FAB) subtypes by immunohistochemical analysis using an anti-VEGF antibody. In normal bone marrow, the anti-VEGF antibody reacted with myeloid progenitor cells and megakaryocytes but not with erythroid cells or mature granulocytic cells. High levels of VEGF were found in the bone marrow in patients with AML-M1, -M2, -M3, -M4, -M4Eo, and -M5. In these leukemias, the vast majority of myeloblasts (> 90%) expressed VEGF. By contrast, in AML-M0, the percentage of VEGF-positive blasts was lower in most cases (median, 42%), and if at all detectable, these blast cells contained only trace amounts of VEGF. In AML-M3 and -M4Eo, maturing granulocytes failed to express VEGF similar to granulocytes in normal bone marrow. In AML-M6, myeloblasts exhibited VEGF, whereas erythroid cells did not. In AML-M7, blast cells and megakaryocytes were identified as major sources of VEGF. In summary, VEGF expression in the bone marrow is restricted to certain stages of differentiation and maturation of myeloid cells and correlates with the FAB category.

Burkitt lymphoma following splenic marginal zone lymphoma. evidence for two independent B-cell clones.

We report the progression of splenic marginal zone lymphoma (SMZL) with circulating villous lymphocytes to Burkitt lymphoma with the presence of a t(8;14)(q24;q32) followed by a highly aggressive course. While the initial indolent lymphoma had an IgM lambda immunophenotype the Burkitt lymphoma was IgM kappa-positive. Immunoglobulin heavy chain gene (IGH) sequence analysis showed no identity between the two clones. We conclude that Burkitt lymphoma can occur in patients with SMZL, although not necessarily of identical clonal origin.

Splenic mastocytosis: report of two cases and detection of the transforming somatic C-KIT mutation D816V.

In the vast majority of patients with systemic mastocytosis (SM), the bone marrow is the primary extracutaneous site of disease. In addition to bone marrow involvement, other visceral organs such as the spleen, liver or the gastrointestinal tract, may also be affected. However, isolated involvement of a single extramedullary organ is rarely seen in SM. We report on two patients with SM with splenic involvement, lack of 'diagnostic' mast cell (MC) infiltrates in the bone marrow, and absence of skin lesions. In one patient, a myelodysplastic syndrome was diagnosed prior to the detection of SM. Both patients presented with massive splenomegaly and multifocal MC infiltrates in splenic tissues. These MCs also expressed CD25 as well as the C-KIT mutation D816V. In consecutive examinations, the mutation was also detected in the bone marrow in both patients suggesting diffuse infiltration with neoplastic cells. In summary, our data show that the spleen can be a primary site of disease in rare cases of SM. Mastocytosis should therefore be considered as a (rare) differential diagnosis in patients with splenomegaly of unknown etiology.

Acquired immune-mediated thrombophilia in lymphoproliferative disorders.

We analyzed 66 cases of immune-mediated thrombophilia in patients with lymphoma reported in the literature. Sixty-one cases had a lupus anticoagulant, three an antibody to protein S, one to protein C, and one to ADAMTS 13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). Lupus anticoagulants occurred in all histological subtypes of non-Hodgkin lymphoma, except mantle cell lymphoma, MALT (mucosa-associated lymphoid tissue) lymphoma, and angioimmunoblastic T-cell lymphoma, and rarely in Hodgkin lymphoma. The largest number of cases was described in splenic marginal zone and lymphoplasmacytic lymphoma. Lupus anticoagulants were highly associated with immunoglobulin M (IgM) paraproteinemia, autoimmune hemolytic anemia, and immune thrombocytopenia. About half of the patients had thrombotic events (antiphospholipid antibody syndrome). Venous thromboembolism was more than twice as common as arterial thrombosis; 6.5% had a catastrophic antiphospholipid antibody syndrome. The lupus anticoagulant could be eliminated by lymphoma treatment (chemoimmunotherapy or splenectomy) in more than one-third of patients. It is suggested that a search for lupus anticoagulant should be done in all patients with lymphoma, because a diagnosis of lupus anticoagulant may influence the management of lymphomas in some patients.

A variant of lymphomatoid papulosis simulating primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma. Description of 9 cases.

Lymphomatoid papulosis (LyP) is a recurrent, self-healing eruption belonging to the spectrum of cutaneous CD30+lymphoproliferative disorders. Three main histologic subtypes of LyP are recognized: type A (histiocytic), type B (mycosis fungoides-(MF)-like), and type C (anaplastic large cell lymphoma-like). We reviewed 26 biopsies from 9 patients (M:F=6:3, median age: 29; mean age 27,2; age range 10 to 38) who presented with clinical features typical of LyP but with histopathologic aspects that resembled primary cutaneous aggressive epidermotropic CD8+cytotoxic T-cell lymphoma. In all but 1 case atypical lymphoid cells showed expression of CD30, and in 8 of 9 cases a T-cell cytotoxic phenotype could be observed (betaF1+, CD3+, CD4-, CD8+). Expression of at least 1 cytotoxic marker (TIA-1, granzyme B) was observed in all cases. Polymerase chain reaction analysis of the T-cell receptor genes revealed a monoclonal rearrangement in 2 of 5 cases tested. Follow-up data available for 8 patients (mean follow-up time: 84 mo, median: 32.5 mo; range: 1 to 303 mo) revealed that none of them developed systemic involvement or signs of other cutaneous lymphomas. This cytotoxic variant of LyP may be histopathologically indistinguishable from primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma, and may be the source of pitfalls in the diagnosis and classification. We propose the term LyP type D for this unusual variant of the disease. Accurate clinicopathologic correlation is required in this setting, with crucial implications regarding prognosis and management of patients.

Extramedullary plasmacytoma of the adenoids.

Extramedullary plasmacytomas are extremely rarely diagnosed in children. We report two cases of extramedullary plasmacytoma detected coincidentally in the adenoidectomy specimens of children younger than 4 years. We show that these children are disease-free after local excision as the only treatment. Surgical treatment without anti-neoplastic therapy in pediatric plasma cell dyscrasias of the pharyngeal lymphoid tissue may be a sufficient curative therapy.

The spectrum of cutaneous lymphomas in patients less than 20 years of age.

Cutaneous lymphomas are rare in young patients and are mostly represented by mycosis fungoides and its variants and CD30+ lymphoproliferative disorders (lymphomatoid papulosis [LYP] and anaplastic large T-cell lymphoma). We report our observations in a series of 69 patients less than 20 years of age who presented either with primary cutaneous lymphoma (n = 62) or with secondary manifestations of extracutaneous disease (n = 7). Clinicopathologic features permitted classification of the cases into the following diagnostic categories: mycosis fungoides (n = 24, all primary cutaneous), anaplastic large T-cell lymphoma (n = 13, all primary cutaneous), LYP (n = 11, all primary cutaneous), subcutaneous "panniculitis-like" T-cell lymphoma (n = 1, primary cutaneous), small-medium pleomorphic T-cell lymphoma (n = 2, all primary cutaneous), natural killer (NK)/T-cell lymphoma, nasal-type (n = 1, secondary cutaneous), follicle center cell lymphoma (n = 1, primary cutaneous), marginal zone B-cell lymphoma (n = 7, all primary cutaneous), B-lymphoblastic lymphomas (n = 6, 3 primary and 3 secondary cutaneous), specific cutaneous manifestations of Hodgkin disease (n = 1, secondary cutaneous), and acute myeloid leukemia (n = 2, both secondary cutaneous). Cutaneous lymphoma in children should be differentiated from benign skin disorders that may simulate them. In particular, mycosis fungoides and LYP in this age group may present with clinicopathologic features reminiscent of inflammatory disorders such as pityriasis alba, vitiligo, pityriasis rosea, and pityriasis lichenoides et varioliformis acuta. Even in secondary cutaneous lymphomas, skin manifestations may be the first sign of the systemic disease, and a diagnosis may be achieved on examination of histopathologic specimens of a cutaneous lesion. Our study illustrates the wide spectrum of cutaneous lymphomas and leukemias in patients less than 20 years of age and underlines the need for proper interpretation of these lesions by dermatologists and dermatopathologists.

Fatty acid composition and preservation of the Tyrolean Iceman and other mummies.

In anthropology, objective parameters to adequately describe storage conditions and the preservation of mummies have yet to be identified. Considering that fatty acids degrade to stable products, we analysed their profile in human mummies and in control samples by gas chromatography coupled to mass spectrometry (GC/MS). Originating from different epochs and civilizations, samples of the Tyrolean Iceman, other glacier corpses, a freeze dried mummy, corpses from a permafrost region, a corpse mummified immersed in water, and a desert mummy were evaluated. Chemometric analysis based on the concentrations of 16 fatty acids revealed the degree of similarity between anthropologic and fresh corpse samples, which was mainly influenced by the content of palmitic acid, oleic acid, and 10-hydroxystearic acid. The presence of 10-hydroxystearic acid was associated with immersion in water, whereas dry mummification was accompanied by high contents of oleic acid. Samples of the Tyrolean Iceman clustered between fresh tissue and those of other glacier corpses indicating the good preservation of this mummy. Thus, environmental post-mortem conditions were associated with characteristic fatty acid patterns suggesting that chemometric analysis of fatty acid contents may add to our knowledge about post-mortem storage conditions and the preservation of human corpses.