Development of a new flow cytometric method to study chronic lymphocytic leukemia.

Background: B-cell chronic lymphocytic leukaemia (B-CLL) is a B-cell neoplasm that in many patients shows an indolent course and survive for a prolonged period without therapy, but in others, CLL progresses rapidly and they die of the disease. Because of the difficulty in identifying such patients at diagnosis, only patients with progressive or symptomatic disease currently are recommended for therapy. Although easily characterized by diagnostic markers, the evaluation of an individual CLL patient’s prognosis remains a problematic issue. . Methods: We have performed a simple six colours flow cytometry assay for ZAP-70 in peripheral blood of 5 healthy donors and 20 B-CLL patients. Samples were incubated with the following monoclonal antibodies: anti CD3-PerCP, anti CD5 FITC anti CD38-APC, anti CD45 APC Cy-7, anti CD19 PE Cy-7, treated with the Intrasure kit and finally stained with the anti-human/mouse ZAP-70-PE conjugate. Samples were acquired on a FACSCanto flow cytometry and analyzed with FacsDiva software (Becton-Dickinson). Quantification of CLLU1 expression in CD19+ cells of donors and buffy coat of patients was performed with the use of CLLU-1 ProfileQuant Kit (Ipsogen) by RQ-PCR. Conclusion: About ZAP-70, the ratio between B-MFI/ T-MFI could be used to discriminate positive and negative patients instead of T-cells residual method. About CLLU-1 expression we have analyzed 5 healthy donors and 20 patients, donors showed very low levels of expression of the gene while CLL patients showed variable levels of expression

Prethymic Cytoplasmic CD3 Negative Acute Lymphoblastic Leukemia or Acute Undifferentiated Leukemia: A Case Report

Acute undiffentiated leukemia (AUL) is an acute leukemia with no more than one membrane marker of any given lineage. Blasts often express HLA-DR, CD34, and/or CD38 and may be positive for terminal deoxynucleotidyl transferase (TdT). The expression of CD34, HLA-DR, and CD38 has been shown in pro-T-ALL, although in this case, blasts should also express CD7 and cyCD3. However, some cases of T-ALL without CD3 in the cytoplasm and all TCR chain genes in germ line configuration are reported, features that fit well with a very early hematopoietic cell. We report a case of acute leukemia CD34+/−HLADR+CD7+CD38+cyCD3− in which a diagnosis of AUL was considered. However the blasts were also positive for CD99 and TCR delta gene rearrangement which was found on molecular studies. Therefore a differential diagnosis between AUL and an early cyCD3 negative T-ALL was debated

Prethymic cytoplasmic CD3 negative acute lymphoblastic leukemia or acute undifferentiated leukemia: a case report

Acute undiffentiated leukemia (AUL) is an acute leukemia with no more than one membrane marker of any given lineage. Blasts often express HLA-DR, CD34, and/or CD38 and may be positive for terminal deoxynucleotidyl transferase (TdT). The expression of CD34, HLA-DR, and CD38 has been shown in pro-T-ALL, although in this case, blasts should also express CD7 and cyCD3. However, some cases of T-ALL without CD3 in the cytoplasm and all TCR chain genes in germ line configuration are reported, features that fit well with a very early hematopoietic cell. We report a case of acute leukemia CD34+/-HLADR+CD7+CD38+cyCD3- in which a diagnosis of AUL was considered. However the blasts were also positive for CD99 and TCR delta gene rearrangement which was found on molecular studies. Therefore a differential diagnosis between AUL and an early cyCD3 negative T-ALL was debated

A High Performance Gel Filtration Chromatography Method for gamma-Glutamyltransferase Fraction Analysis

The clinical relevance of serum c-glutamyltransferase (GGT) activity, in areas other than hepatic function, has recently been increased by several epidemiological associations. Still, GGT remains a nonspecific test because of the influence of various pathophysiological factors. We devised a procedure based on gel filtration chromatography, followed by postcolumn injection of fluorescent GGT substrate (cglutamyl- 7-amido-4-methylcoumarin), permitting the quantification of GGT fractions in serum or plasma. Plasma GGT molecular weight distribution was analyzed in healthy volunteers (20 males; mean ± SD age 38 ± 10 years; 20 females; age 44 ± 13; total GGT 21 ± 11 for males vs 13 ± 7 for females; P < 0.01). The method is highly sensitive (determination limit: 0.5 U GGT/L), with a linear dynamic range between 0.5 and 150 U/L for each fraction. Four GGT fractions of different molecular weight were detected in all subjects of both genders: b-GGT, m-GGT, s-GGT (likely lipoprotein-bound, molecular masses >2000, 940, and 140 kDa, respectively), and a free fraction (f-GGT, 70 kDa). f-GGT and s-GGT were the main fractions in subjects with lower and higher total GGT activity, respectively. Higher total GGT activity in males is related mainly to f-GGT (P < 0.01). GGT fraction analysis may increase the sensitivity and specificity of the GGT activity test

Simultaneous presentation of Waldenström macroglobulinemia and multiple myeloma: multidisciplinary diagnosis, treatment and 30-month follow-up.

Waldenström macroglobulinemia and multiple myeloma are mature B-cell neoplasms deriving from post-germinal cells at different stages of differentiation. The simultaneous presentation of Waldenström macroglobulinemia and multiple myeloma in the same patient is a very rare phenomenon and, so far, only two cases have been described. We report the case of a 75-year Caucasian female patient, with a silent clinical history, who presented with anemia and two different monoclonal proteins (IgMκ and IgGκ). The trephine biopsy showed the presence of a dual population, represented by small lymphoplasmacytoid cells and by plasma cells, which infiltrated the bone marrow with a clearly different pattern. Both immunohistochemistry and flow cytometry demonstrated the biclonal origin such neoplastic cells, since lymphoplasmacytoid cells resulted IgMκ while plasma cells were IgGκ. This biclonal pattern was further confirmed by the demonstration of a different IgH gene rearrangement of the two neoplasms. The patient was treated with bortezomib, dexamethasone and rituximab, achieving partial remission of both Waldenström macroglobulinemia and multiple myeloma. After a 30-month follow-up, she is in stable disease. Multiple myeloma has been described in association with other indolent B-cell neoplasms, mostly chronic lymphocytic leukemia, while Waldenström macroglobulinemia can be followed by diffuse large B-cell lymphoma in some instances, after chemotherapy. The association of Waldenström macroglobulinemia and multiple myeloma seems to be very rare. Our study shows that an integrated diagnostic work-up is very useful in such cases, with an interesting role for flow cytometry. [J Clin Exp Hematop 53(1): 29-36, 2013]

A rare case of de novo CD5+ diffuse large B-cell lymphoma in leukemic phase and positive for CD13

We report a case of de novo diffuse large B-cell lymphoma (DLBCL) in leukemic phase, positive for both CD5 and CD13. Morphologic evaluation, flow cytometric immunophenotyping, karyotyping and polymerase chain reaction studies were performed. Neoplastic lymphocytes appeared as blast-like cells, positive for CD19, CD20, CD5, CD13, CD79a, HLADR, and with restriction for surface immunoglobulin K light chains. Rearrangement of IgH gene, BCL2/IgH translocation and complex karyotype were found. The patient was treated with RCOMP regimen and achieved complete remission. However, only one month after the first restaging of disease, the patient presented with symptoms attributable to central nervous system involvement and her clinical conditions worsened rapidly. While both CD5 expression and leukemic presentation are uncommon findings in DLBCL, positivity for CD13 is very rare. The outcome of our patient shows the poor prognosis of CD5+ DLBCL with leukemic presentation. The possible role of CD13 coexpression is discussed

Morphologic and immunophenotypic features of a case of acute monoblastic leukemia with unusual positivity for Glycophorin-A

Acute monoblastic leukemia (AMoL) is characterized by cells with highly undifferentiated morphology. Cytochemistry with non-specific esterases is negative in up to 20% of cases. Immunophenotyping by flow cytometry has an essential role in diagnosing such a subtype of leukemia and a multiparametric approach with a wide monoclonal antibody panel is necessary. We describe a case of AMoL with morphology resembling either plasma blasts or very immature erythroblasts. Diagnosis was made by alpha-naphtyl-acetate esterase staining and with immunophenotyping, which was made with a wide monoclonal antibody panel. Blasts were positive for monocytic markers. Most of leukemic cells, however, were positive for Glycophorin-A. The presence of Glycophorin-A, which is considered as a specific marker of the erythroid lineage, has never been reported previously in cases of AMoL. This peculiar immunophenotype might be interpreted as deriving from a common myelo-erythroid precursor undergone leukemic transformation

Mitochondrial Pathway Mediates the Antileukemic Effects of Hemidesmus Indicus, a Promising Botanical Drug

Although cancers are characterized by the deregulation of multiple signalling pathways, most current anticancer therapies involve the modulation of a single target. Because of the enormous biological diversity of cancer, strategic combination of agents targeted against the most critical of those alterations is needed. Due to their complex nature, plant products interact with numerous targets and influence several biochemical and molecular cascades. The interest in further development of botanical drugs has been increasing steadily and the FDA recently approved the first new botanical prescription drug. The present study is designed to explore the potential antileukemic properties of Hemidesmus indicus with a view to contributing to further development of botanical drugs. Hemidesmus was submitted to an extensive in vitro preclinical evaluation.A variety of cellular assays and flow cytometry, as well as a phytochemical screening, were performed on different leukemic cell lines. We have demonstrated that Hemidesmus modulated many components of intracellular signaling pathways involved in cell viability and proliferation and altered the protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential and increased Bax/Bcl-2 ratio. ADP, adenine nucleotide translocator and mitochondrial permeability transition pore inhibitors did not reverse Hemidesmus-induced mitochondrial depolarization. Hemidesmus induced a significant [Ca(2+)](i) raise through the mobilization of intracellular Ca(2+) stores. Moreover, Hemidesmus significantly enhanced the antitumor activity of three commonly used chemotherapeutic drugs (methotrexate, 6-thioguanine, cytarabine). A clinically relevant observation is that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemic patients.These results indicate the molecular basis of the antileukemic effects of Hemidesmus and identify the mitochondrial pathways and [Ca(2+)](i) as crucial actors in its anticancer activity. On these bases, we conclude that Hemidesmus can represent a valuable tool in the anticancer pharmacology, and should be considered for further investigations

Survival of patients with metastatic melanoma and brain metastases in the era of MAP-kinase inhibitors and immunologic checkpoint blockade antibodies: A systematic review

Background: The incidence of brain metastases (BM) in melanoma patients is common and associated with poor prognosis. MAP-kinase inhibitors and immunologic checkpoint blockade antibodies led to improved survival of metastatic melanoma patients; however, patients with BM are under-represented or excluded from the majority of clinical trials and the impact of new drugs on their survival is less clear. With the present systematic review, we aimed to analyze outcomes of patients with melanoma BM treated with the new drugs, both in the setting of I-II-III phase clinical trials and in the "real world".Methods: An electronic search was performed to identify studies reporting survival outcomes of patients with melanoma BM treated with MAP-kinase inhibitors and/or immunologic checkpoint blockade antibodies, regardless of study design.Results: Twenty-two studies were included for a total of 2153 patients. Median OS was 7.9 months in phase I-II-III trials and 7.7 months in "real world" studies. In clinical trials, median OS was 7.0 months for patients treated with immunotherapy and 7.9 months for patients treated with BRAF inhibitors. In "real world" studies, median OS was 4.3 months and 7.7 months for patients treated with immunotherapy and BRAF inhibitors, respectively. Evidence of clinical activity exists for both immunotherapy and MAP-kinase inhibitors.Conclusions: MAP-kinase inhibitors and immunologic checkpoint blockade antibodies have clinical activity and may achieve improved OS in patients with metastatic melanoma and BM. These results support the inclusion of patients with BM in investigations of new agents and new treatment regimens for metastatic melanoma. (C) 2016 Elsevier Ltd. All rights reserved

Multiparameter flow cytometry to detect hematogones and to assess B-lymphocyte clonality in bone marrow samples from patients with non-Hodgkin lymphomas

Hematogones are precursors of B-lymphocytes detected in small numbers in the bone marrow. Flow cytometry is the most useful tool to identify hematogones and, so far, 4-color methods have been published. In addition, flow cytometry is used in the diagnosis and follow-up of lymphomas. We developed a flow cytometric 7-color method to enumerate hematogones and to assess B-lymphocyte clonality for routine purposes. We evaluated 171 cases of B-cell non-Hodgkin lymphomas, either at diagnosis or in the course of follow-up. By our diagnostic method, which was carried out by the combination K/l/CD20/CD19/CD10/CD45/CD5, we were able to detect hematogones in 97.6% of samples and to distinguish normal B-lymphocytes, neoplastic lymphocytes and hematogones in a single step. The percentage of hematogones showed a significant inverse correlation with the degree of neoplastic infiltration and, when bone marrow samples not involved by disease were taken into consideration, resulted higher in patients during follow-up than in patients evaluated at diagnosis