Assessment of response to therapy in multiple myeloma by multiparameter flow cytometry. Usefulness of an eight-color single tube with monoclonal antibodies in dried formulation.

OBJECTIVE: Multiparameter flow cytometry is considered the gold standard to evaluate minimal residual disease in multiple myeloma (MM) and patients in complete remission can achieve "Flow MRD-negative" status (i.e. immunophenotypically abnormal plasma cells not detectable). In the current study we report the usefulness of an eight-color flow cytometric method with a 10-5 sensitivity, using monoclonal antibodies in dried formulation. MATERIALS AND METHODS: Forty-six patients with MM were treated with bortezomib-based regimens and, when eligible, with autologous stem cell transplantation. Response to therapy was assessed according to the criteria validated by the International Myeloma Working Group. Multiparameter flow cytometry was carried out with an 8-color panel validated by the Euroflow Consortium. A commercially available single 8-color tube in dried formulation was used and almost 2,000,000 events were acquired, in order to obtain a 10-5 sensitivity. RESULT: Sixteen patients achieved stringent complete remission and another three patients achieved complete remission. In these groups of patients, the "Flow MRD-negative" status was achieved in sixteen cases. In patients who had a different degree of response (very good partial response, partial response, minimal response) immunophenotypically abnormal plasma cells were always detected. CONCLUSION: Using a single eight-color tube in dried formulation, and an acquisition strategy able to obtain a 10-5 sensitivity, not only is it possible to detect a deep response to modern therapy in patients who obtained at least complete remission, but it is also always possible to detect minimal residual disease in patients with either complete remission or stringent complete remission.

Multiparameter immunophenotyping by flow cytometry as a diagnostic tool in multiple myeloma and monoclonal gammopathy of undetermined significance.

BACKGROUND: Immunophenotyping by multiparameter flow cytometry (MFC) provides relevant information about prognosis and minimal residual disease detection in multiple myeloma (MM) and might be used to distinguish MM from monoclonal gammopathies of undetermined significance (MGUS). MATERIALS AND METHODS: We evaluated a possible usage of MFC to predict the differential diagnosis between MM and MGUS. One hundred consecutive patients were studied at diagnosis and underwent conventional diagnostic procedures. We carried out a double-blind study. Immunophenotyping was performed on samples from myeloaspirates before establishing diagnosis, while the final clinical diagnosis was established independently from MFC results. A five- or six-color method was carried out by means of monoclonal antibody combinations able to identify abnormal plasma cells (CD19-) and the most relevant immunophenotypic aberrations (loss of CD27; overexpression of CD117, CD56, CD28; asynchronous expression of CD20). MFC was applied following the indications of the European Myeloma Network. When abnormal plasma cells were /= 3.1%, MGUS was predicted. RESULTS: MFC results predicted 63 cases of MM and 37 cases of MGUS. At the end of our study, 61 cases of MM and 39 cases of MGUS were diagnosed. Therefore, 4% of patients were misdiagnosed by MFC parameters alone, with sensitivity and specificity of 0.983 and 0.92, respectively. CONCLUSIONS: Only a small proportion of patients with MM and MGUS were misdiagnosed by MFC alone and a possible systematic application of MFC in all patient with MM and MGUS at diagnosis might be proposed. Novel additional criteria could be necessary to improve the diagnostic impact of MFC in monoclonal gammopathies.

Diagnosis and classification of B-cell non-Hodgkin lymphomas. The role of multiparameter flow cytometry.

Immunophenotyping is a routine method to evaluate B-cell non-Hodgkin lymphomas. Flow cytometry plays a complementary role in diagnosis and classification of these types of lymphomas, since combination of morphologic, immunophenotypic and genotypic features is needed to correctly classifying each disease entity. Multiparameter flow cytometry, which is now carried out with routine combinations of six to eight monoclonal antibodies, allows identifying even small lymphomatous cell populations on the basis of aberrant B-cell marker expression and clonality. The immunophenotypic patterns obtained by multiparameter flow cytometry are useful to correctly diagnose most of cases of specific subtypes of B-cell non-Hodgkin lymphomas and to discover peculiar clinical presentations, such as discordant and composite lymphomas. Immunophenotypic variability, however, characterizes B-cell lymphomas. Therefore, flow cytometry should always be used in combination with other techniques to correctly classify each disease entity. Finally, multiparameter flow cytometry is characterized by high sensitivity in detecting residual disease.

Combination of morphology, flow cytometry and PCR assay to detect bone marrow infiltration in B-cell non-Hodgkin's lymphomas.

AIMS: Morphology on bone marrow biopsy (BMB) samples has historically been the primary method used to detect bone marrow (BM) infiltration in B-cell non-Hodgkin's lymphoma (NHL), while fl ow cytometry (FC) and PCR assays have been generally used as ancillary methods. In this study we evaluated a combined approach utilizing all three methods to detect BM infiltration in patients with NHL both at diagnosis and after therapy. MATERIALS AND METHODS: We analyzed 193 patients with NHL, who received simultaneous BMB, FC and PCR assays. Morphology on histologic specimens was used to assess infiltration pattern and immunohistochemistry, FC to analyze immunophenotype, PCR to identify IgH rearrangement, BCL-1/JH and BCL-2/JH translocation. RESULTS: Morphology, FC and PCR assays agreed in 142 cases (73.5%) with more concordance at initial diagnosis than during postchemotherapy follow-up. PCR was the single best-performing test, while combination of morphology and PCR yielded a higher sensitivity than individual methods and was similar to PCR + FC. We observed little added benefit using a third approach. CONCLUSION: Given the initial importance of histological information evident by morphology, our data suggest that combination of morphology and PCR should be considered the gold standard for evaluation of BM infiltration at diagnosis, while combination of PCR + FC should be employed during post-treatment follow-up.

Association of B-chronic lymphocytic leukemia and acute myeloid leukemia.

A 62-year-old man presented with fatigue, pallor and mild weight loss. Laboratory studies showed Hb 7.6 g/dl, Hct 21.8%, WBC 108x10(9)/1, PLT 143x10(9)/1. At morphological examination, circulating cells appeared as 60% blasts and 40% lymphocytes, with smudge cells. A bone marrow aspirate showed infiltration by blasts (50%) and lymphocytes (40%); alpha-naphtyl-acetate esterase was positive in 90% of blasts, while myeloperoxidase was positive in 10%. The immunologic phenotype of blasts was characterized by the co-expression of CD13, CD33, CD14, CD4, CD15, CD64, CD117, HLA-DR, CD11b. Lymphocytes were characterized by a B-CLL immunophenotype: CD19+, CD5+, CD23+, CD20+(dim), FMC7+(dim), K light chain+(dim). Karyotype was normal and PCR assays for AML-ETO, CBFbeta-MYH11, PML-RARalpha, BCR-ABL and bcl-1/JH translocation were negative. Coexistence of CLL and AML with monoblastic features was diagnosed. Simultaneous appearance of CLL and AML has rarely been described and represents a peculiar biological phenomenon.

Recombinant human granulocyte colony-stimulating factor administration in a case of neutropenia due to increased neutrophil sequestration.

A 55-year-old female was admitted with fever which followed an episode of pseudomembranous colitis. Despite an accurate clinical investigation, there was no evidence for specific sites of infection. Remission of fever was not obtained with antibiotic therapy (gentamycin plus carbepenem) and progressive neutropenia was observed. Neutrophils fell to 0.3 x 10(9)/1. The diagnostic approach, including a bone marrow aspirate, excluded mechanisms leading to impaired neutrophil production, and in the suspect of increased neutrophil sequestration/destruction, whole-body scintigraphy with (99m)technetium-hexamethylpropyleneamineoxime ((99m)Tc-HMPAO)-labeled autologous leukocytes was performed. As a result, a site of leukocyte sequestration localized at the medium lobe of the right lung was detected. In an attempt to enhance neutrophil functions and achieve remission of infection, recombinant human granulocyte colony-stimulating factor (Filgrastim, Granulokine 30, Roche) at the dosage of 300 microg/day, subcutaneously, was added. As a results, fever disappeared in three days, but neutrophil recovery was slower, and normalization of the absolute neutrophil count (ANC) was obtained on day +7. The results obtained in this peculiar case of neutropenia, and the kinetics of both fever and ANC, suggest the possible combination of neutrophil function enhancement and an anti-inflammatory effect of rhG-CSF.

Cell clonality in hypereosinophilic syndrome: what pathogenetic role?

OBJECTIVE: Idiopathic hypereosinophilic syndrome (HES) is a heterogeneous disorder, including either a myeloproliferative or a lymphoproliferative variant (l-HES). In l-HES, T-lymphocytes could be involved in the pathogenesis through several cytokines, including IL5. METHODS: We assayed both TCR Beta- and delta-rearrangements by fluorescent PCR, characterizing 14 patients affected by HES. Lyn activation (a src-kinase involved in the IL5 pathway) was also tested in 6 cases. RESULTS: FIP1L1-PDGFRa was detected in 4 cases (28.6%); a clonal TCR was found in 10 cases (71.4%), including cases FIP1L1-PDGFRalpha-positive; four cases did not show any molecular marker. In this series, levels of IL5, IL4, IL2 and gammaIFN were measured, without any significant difference among different subgroups. All pathological samples tested did not show Lyn activation. Immunophenotype was also characterized: only one case showed an atypical CD3-/CD4+ population in the bone marrow. CONCLUSION: This study would suggest that a real distinction between m- and l-HES is not wholly convincing and that clonal T-cell expansion could not be the "primum movens" but an epiphenomenon in HES.

Glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factor differently modifies actin polymerization in neutrophils.

AIM: Several neutrophil functions can be modified by rhG-CSF administration. Neutrophil morphology changes in the course of treatment with Filgrastim (nonglycosylated rhG-CSF), along with impairment of chemotaxis. Both morphology and chemotaxis are not affected by treatment with Lenograstim (glycosylated rhG-CSF). Thus, we evaluated actin polymerization in neutrophils induced by treatment with the two forms of rhG-CSF. In fact, actin polymerization is crucial for neutrophil motility. MATERIALS AND METHODS: We evaluated twelve healthy subjects undergoing peripheral blood stem cells (PBSC) mobilization for allogeneic transplantation to HLA-identical siblings. Neutrophils were isolated by peripheral venous blood before and after administration of either Filgrastim (six PBSC donors) or Lenograstim (six PBSC donors). Actin polymerization was investigated by a flow cytometric assay, using FITC-phalloidin as a specific probe for F-actin, and two parameters were measured: spontaneous actin polymerization in resting neutrophils; fMLP-stimulated actin polymerization. Results were expressed as relative F-actin content. Fifteen blood donors were studied as a control group. RESULTS: Filgrastim administration induced an increased relative F-actin content in resting neutrophils; however, no further actin polymerization was observed after fMLP stimulation. Neutrophils from subjects treated with Lenograstim showed a normal behaviour in terms of both spontaneous and stimulated actin polymerization. CONCLUSIONS: Glycosylated and nonglycosylated rhG-CSF differently affect actin polymerization in newly generated neutrophils. Such effects may explain some previous findings concerning both morphology and chemotactic properties and may be due to different effects of the two forms of rhG-CSF on proteins involved in neutrophil motility regulation.

Screening of an Echinococcus granulosus cDNA library with IgG4 from patients with cystic echinococcosis identifies a new tegumental protein involved in the immune escape.

The worldwide problem of chronic Echinococcus granulosus disease calls for new parasite-derived immunomodulatory molecules. By screening an E. granulosus cDNA library with IgG4 from patients with active cystic echinococcosis, we identified a cDNA that encodes a predicted partial protein that immunofluorescence studies localized in the protoscolex tegument and on the germinal layer of cyst wall. We named this protein EgTeg because the 105 amino acid sequence scored highest against a family of Schistosoma tegumental proteins. Evaluating the role of EgTeg in the human early inflammatory response we found that EgTeg significantly inhibited polymorphonuclear cell (PMN) chemotaxis. Cytometric analysis of intracellular cytokines disclosed a significantly higher percentage of cells producing IL-4 than IFN-gamma (P = 0.001, Student's t-test) in T lymphocytes from patients with cystic echinococcosis stimulated with EgTeg. EgTeg induced weak Th1-dependent proliferation in 42% of patients' peripheral blood mononuclear cells. In immunoblotting (IB) analysis of total IgG and IgG subclass responses to EgTeg in patients with cystic echinococcosis, patients with other parasitoses, patients with cystic lesions and healthy controls, total IgG specific to EgTeg yielded high sensitivity (73%) but low specificity (44%) precluding its use in immunodiagnosis. Conversely, IgG4 specific to EgTeg gave acceptable sensitivity (65%) and high specificity (89%) suggesting its use in immunodiagnosis to confirm ultrasound documented cysts suggestive of E. granulosus. Because the new tegumental antigen EgTeg inhibits chemotaxis, induces IL-4-positive T lymphocytes and noncomplement fixing antibodies (IgG4) it is an immunomodulatory molecule associated with chronic infection.

Significant co-expression of WT1 and MDR1 genes in acute myeloid leukemia patients at diagnosis.

A high expression of Wilms' tumor gene (WT1) in acute myeloid leukemia (AML) seems to correlate with a poor outcome and its increased levels can be predictive of an impending relapse. WT1 has been shown in vitro to interact with the promoter of the MDR1, a gene involved in the multidrug resistance phenomenon. The aim of this study was to measure, by real-time polymerase chain reaction, levels of WT1 and MDR1 expression, in order to find a possible association between these genes, in a series of 50 newly diagnosed AML cases. Twenty-five percent of patients carried very high (>75 degrees percentile) MDR1- and 23.3%WT1-mRNA levels. Interestingly, high levels of WT1 were significantly correlated with correspondent high levels of MDR1 gene. Nevertheless, the co-expression of these genes did not significantly influence the complete response rate to the induction therapy. Reported data confirm the existence of a co-expression of WT1 and MDR1 genes even in vivo; this may be relevant because one consequence could be the positive selection by chemotherapeutic regimens of cells with higher MDR1 levels already present before treatment. Thus, the association between these genes could suggest avoiding the use of drugs involved in the multidrug resistance (MDR) phenomenon in patients carrying high levels of WT1 at diagnosis.

Modulation of human immune response by Echinococcus granulosus antigen B and its possible role in evading host defenses.

By directly suppressing the function of certain immune cell subsets and by stimulating other cell populations related to immunopathology, parasite-derived substances play an important role in the chronic establishment of parasitic disease. Our objective was twofold: (i) to investigate further the role of Echinococcus granulosus antigen B (AgB) in the human early inflammatory response by determining its effect on polymorphonuclear cell (PMN) random migration, chemotaxis, and oxidative metabolism and (ii) to determine its action in acquired immunity by evaluating AgB and sheep hydatid fluid (SHF)-driven Th1 (gamma interferon [IFN-gamma] and interleukin 12 [IL-12]) and Th2 (IL-4 and IL-13) cytokine production by peripheral blood mononuclear cells (PBMC) from 40 patients who had cured or stable or progressive cystic echinococcosis. AgB significantly inhibited PMN recruitment but left their random migration and oxidative metabolism unchanged. Patients' PBMC stimulated with AgB produced IL-4 and IL-13 but did not produce IL-12. They also produced significantly lower IFN-gamma concentrations than did PBMC stimulated with SHF (P = 10(-5)). AgB skewed the Th1/Th2 cytokine ratios towards a preferentially immunopathology-associated Th2 polarization, predominantly in patients with progressive disease. AgB-stimulated patients' PBMC also proliferated less than SHF-stimulated PBMC (P = 9 x 10(-3)). In vitro Th2 cytokine production was reflected in vivo by elevated specific immunoglobulin E (IgE) and IgG4 antibodies binding to AgB. These findings confirm that AgB plays a role in the escape from early immunity by inhibiting PMN chemotaxis. They also add new information on the host-parasite relationship, suggesting that AgB exploits the activation of T helper cells by eliciting a nonprotective Th2 cell response.

Inhibitory effects of human neutrophil functions by the 45-kD glycoprotein derived from the parasitic nematode Trichinella spiralis.

AIM: We evaluated the effect of the 45-kD protein of Trichinella spiralis (gp45), purified by affinity chromatography, on random migration and chemotaxis, the oxidative metabolism of human neutrophils and on the CD11b upregulation induced by formyl-methionyl-leucyl-phenylalanine (f-MLP). METHODS: Donor neutrophils incubated with different amounts of gp45 (0.5, 1, 1.5, 2 microg/ml) or buffer and the random migration and chemotaxis, evaluated by means of a special technique of image analysis, and the chemiluminescence response to f-MLP or phorbol myristate acetate (PMA) were analyzed. The effect on CD11b upregulation was assessed incubating cells with the protein, when activating them with f-MLP. RESULTS: The results showed that gp45 inhibited both random and stimulated migrations, and reduced the response to f-MLP and PMA. Furthermore, gp45 significantly reduced the upregulation of the CD11b induced by f-MLP. CONCLUSION: The results show that gp45 inhibits PMN in different functions, suggesting an anti-inflammatory action.

Pseudoreticulocytosis in a case of myelodysplastic syndrome.

A patient with normocytic anemia and marked and persistent reticulocytosis is presented. Causes responsible for blood loss and hemolytic diseases were excluded and, after bone marrow examination, myelodysplastic syndrome (refractory anemia) was diagnosed. In vitro reticulocyte survival studies suggested that reticulocytosis was a consequence of delayed maturation of the reticulocytes. Pseudoreticulocytosis may be an unusual presentation of myelodysplastic syndromes, because only 4 patients with such a finding have previously been reported.