Mirna Sučić

Azithromycin modulates neutrophil function and circulating inflammatory mediators in healthy human subjects

Effects on human neutrophils and circulating inflammatory mediators were studied in 12 volunteers who received azithromycin (500 mg/day, p.o.) for 3 days. Blood was taken 1 h before treatment, 2.5, 24 h and 28 days after the last dose. An initial neutrophil degranulating effect of azithromycin was reflected in rapid decreases in azurophilic granule enzyme activities in cells and corresponding increases in serum. The oxidative response to a particulate stimulus was also acutely enhanced. These actions were associated with high plasma and neutrophil drug concentrations. A continuous fall in chemokine and interleukin-6 serum concentrations, within the non-pathological range, accompanied a delayed down-regulation of the oxidative burst and an increase in apoptosis of neutrophils up to 28 days after the last azithromycin dose. Neutrophils isolated from blood at this time point still contained detectable drug concentrations. Acute neutrophil stimulation could facilitate antibacterial effects of azithromycin, while delayed, potentially anti-inflammatory activity may curtail deleterious inflammation.

Acute promyelocytic leukemia M3: cytomorphologic, immunophenotypic, cytogenetic, and molecular variants.

Acute promyelocytic leukemia (APL) M3 is an acute myeloid leukemia (AML) subtype characterized by proliferation of malignant promyelocytes with mature myeloid immunophenotype and the translocation t(15;17)(q22;q11), which results in the fusion of retinoic acid receptor-alpha (RARalpha) gene on chromosome 17 and the gene PML on chromosome 15. There are three M3 morphologic variants: the typical hypergranular form and the microgranular and basophilic variants. Although most leukemic cells in M3 patients express t(15;17), other cytogenetic abnormalities have also been reported. Also, there are three molecular variants of the PML/RARalpha transcript (bcr1, bcr2, bcr3). Blasts had typical hypergranular appearance (13 patients) with a mature myeloid immunophenotype (HLA-DR(-),CD13(+), and/or CD33(+)) (10 patients) in the majority of patients with M3 followed in this study. The typical translocation [t(15;17)(q22;q11)] was detected by cytogenetic analysis in 5 M3 patients, but PML/RARalpha was positive in 13 out of 15 patients, as assessed by RT-PCR (8 patients with bcr1 and 5 with bcr3 subtype). Cytogenetic diversity was found in three patients (1 with t(17;17), 1 with +8, and 1 with add (7)(q22); -7; +8). According to many studies, leukemic cell heterogeneity in APL influences the clinical outcome of disease. The analysis of certain leukemic cell characteristics on the clinical outcome in our study revealed that patients with bcr3 had shorter medians of first remission and survival in comparison to patients with the bcr1 isoform of PML/RARalpha. Also, the clinical relapse of disease in 4 APL patients with reverted PML/RAR alpha positivity is consistent with the view that detection of PML/RARalpha by RT-RCR in patients in remission implies a poor prognosis. On the contrary, lack of detection of PML/RARalpha by RT-PCR at least three times is a sign of long remission and survival.

Biological features and outcome of biphenotypic acute leukemia: a case series

BACKGROUND Biphenotypic acute leukemia (BAL) is a distinct entity that is immunophenotypically defined by the European Group for the Immunological Classification of Leukemia (EGIL) scoring system and accounts for less than 5% of all acute leukemia cases. Since it is a rare and heterogeneous form of acute leukemia with an allegedly poor outcome, there is no consensus on the best treatment approach in these patients. Our objective was to analyze the biological features and outcome of patients diagnosed with BAL in our institution. PATIENTS AND METHODS Using the EGIL system, we identified 21 cases (3.9%) of BAL from 535 newly diagnosed acute leukemia patients in an 11-year period. RESULTS There were ten cases of myeloid+B-lymphoid leukemia, eight cases of myeloid+T-lymphoid, one case of B+T-lymphoid and two cases of trilineage (myeloid+B+T-lymphoid leukemia). The complete remission (CR) rate with high-dose chemotherapy was 72% and overall survival at 5 years was 21%. Patients that received acute lymphoblastic leukemia-oriented chemotherapy had a higher CR rate compared with those who received acute myeloid leukemia-oriented chemotherapy (100% vs. 60%, P = .007). The white blood cell count at diagnosis was found to have statistically significant impact on survival. CONCLUSION Despite the progress in the treatment of acute leukemia, the prognosis of BAL remains poor and treatment protocols devised explicitly for this entity should be investigated in prospective collaborative studies.

Correlation of Morphological FAB Classification and Immunophenotyping: Value in Recognition of Morphological, Cytochemical and Immunological Characteristics of Mixed Leukaemias

Correlation between the FAB classification and immunophenotype was studied in 169 consecutive adult patients with acute leukaemia (AL). The lineage of leukaemic cells could be determined in the majority of cases, whereas 3 patients (1.8%) remained unclassified. In 22 out of 71 patients (31%) with acute myeloid leukaemia (AML) FAB M1 and M2 types, and in 5 out of 16 patients (31%) with chronic myeloid leukaemia (CML) in myeloid blast crisis, leukaemic cells did not express myeloid lineage-related markers, indicating asynchronous expression of cell markers in a substantial proportion of patients. Flow cytometric two-colour immunofluorescence revealed mixed AL immunophenotype in 6 out of 169 patients (3.4%). This group included five CD2+AML (5% of AML tested) and one undifferentiated AL expressing CD10(CALLA), CDw65(VIM-2). The former group included FAB M1, M2, M3 and M4 forms of AML with a single cell population, and an AML M2 patient with both cytochemically and immunologically two separate populations of leukaemic cells. This further illustrates the heterogeneity of the target cell(s) for leukaemogenesis and the level of differentiation of AML cells. However, there was no difference in the treatment response and the remission duration between AML patients and patients with mixed phenotype AML.

Expression of Ki-67 and p27Kip1 in fine-needle aspirates from breast carcinoma and benign breast diseases

Cell atypia in breast fine needle aspiration (FNA) can introduce some diagnostic difficulties. Molecules reflecting proliferative cell potential, such as Ki‐67 and p27Kip1, can help in recognizing the true biological nature of a cell. Thus, the objective of the study was to analyze the difference in Ki‐67 and p27Kip1 cell immunoexpression in breast FNA specimens between fibroadenomas, fibrocystic changes (FCC) with atypia, and breast carcinoma. Microscopic analyses of cell cytomorphology and Ki‐67 and p27Kip1 breast cell immunoexpression were done after standard Pappenheim and immunocytochemical staining (labeled streptavidin‐biotin, LSAB) method in autostainer DakoCytomation TechMate™. The study included 50 patients with breast carcinoma, 20 patients with fibroadenoma, and 20 patients with FCC with atypia. High Ki‐67 and low or absent p27Kip1 were found in most patients with breast carcinoma, while majority of FCC with atypia were characterized by low Ki‐67 and moderate to high p27Kip1 cell immunoexpression. Majority of fibroadenomas were associated with low Ki‐67 and low to moderate p27Kip1 cell immunoexpression indicating progressive decrease in cell cycle inhibition, but still not so high proliferative activity as in carcinoma. However, although statistically significant difference for Ki‐67 and p27Kip1 was found between breast lesions in our study, the large ranges observed for each marker make them essentially useless for better cytological diagnosis in a single case. Regarding their opposite role in cell cycle, inverse correlation of Ki‐67 and p27Kip1 was noticed. Poorly differentiated carcinoma cells had mostly high Ki‐67 andlow p27Kip1 cell immunoexpression. Diagn. Cytopathol. 2011;39:333–340.

Prognostic significance of cytochemical analysis of leukemic M2 blasts

Cytochemical analysis of leukemic blasts from 46 patients with acute myeloblastic M2 leukemia (according to the FAB classification) was performed before and after cytostatic therapy, and compared with findings obtained in 20 age- and sex-matched control subjects. Cytochemical findings for myeloperoxidase (MPO), Sudan black B, acid phosphatase and alpha-naphthyl-acetate esterase (ANAE) were related to the achievement of the first complete remission (CR),i.e. data were compared after the patients had been divided into CR and non-CR groups. The analysis clearly showed that a high proportion of myeloperoxidase- and, to a lesser extent, Sudan black B-positive blasts before treatment may have constituted a significantly unfavourable prognostic factor.

A patient with prostate cancer and multiple myeloma-diagnostics and possible association of both diseases.

In this report, we describe a case of a patient with prostate cancer and multiple myeloma as the second metachronous malignant disease. To our knowledge, synchronous occurrence of bone marrow prostate cancer metastases and multiple myeloma-as it was found in the clinical disease course of our patient-has not been documented in the literature. Among other diagnostic procedures, cytomorphology and immunocytochemistry analyses contribute to detection of metastases of epithelial cells and synchronous plasma cell proliferation in bone marrow. Occurrence of multiple myeloma and prostate cancer in our patient adds to other similar reports and points to possible association between both diseases and also to other factors involved in the development of a second malignant disease. Further studies are needed to confirm and clarify this association, because prostate cancer is a relatively common malignant disease.

CD13+ anaplastic large cell lymphoma with leukemic presentation and additional chromosomal abnormality

Anaplastic large cell lymphoma (ALCL) is a highly malignant neoplasm characterized by pleomorphic appearance, different immunophenotypes and variable sites of involvement. Expression of myeloid‐associated markers in anaplastic large cell lymphomas may mislead the medical team and result in delay of diagnosis due to unusual phenotype. It is important to diagnose this type of tumors and distinguish it from myeloid neoplasms (extramedullary myeloid cell tumors and histiocytic tumors) since therapy and prognosis are significantly different.

Double Immunoenzymatic APAAP Staining for the Detection of Leukemia-Associated Immunophenotypes

Detection of unusual or aberrant cell immunophenotype with flow cytometry is the basis for the immunologic recognition of minimal residual disease (MRD) in patients with acute leukemia (AL). In this study, we have shown that the double immunocytochemical alkaline phosphatase antialkaline phosphatase (APAAP) staining technique also makes possible the detection of leukemic cells with unusual (leukemic) combinations of antigens (ULCA) both at diagnosis and during follow-up of patients with ULCA+ AL. The applicability of double APAAP was analyzed on bone marrow (BM) samples obtained from 12 patients (8 with AML, 3 with ALL, and 1 with undifferentiated acute leukemia [AUL]) randomly chosen from a larger group of 22 ULCA+ patients treated at our center in a 3-year period (22% observed ULCA+ AL frequency). The percentages of ULCA+ BM cells before chemotherapy were in the range of 5%-60%, which dropped to 0%-7% in 10 patients who achieved remission (range 0%-7%, p < 0.01). However, these cells could also be found 60 days after the initiation of therapy, ranging from 0%-2% of all nucleated cells. In 2 of 10 patients who achieved remission, 2% ULCA+ BM cells were found on days 35 and 60 after initiation of chemotherapy, and this finding was followed by relapse on days 110 and 270. However, the other 8 patients remained in remission despite positive finding of ULCA+ BM cells ranging from 0.2%-2% on at least one occasion. In 2 patients with AML FAB-M3 and cytomorphologic remission, the finding of ULCA+ cells by double APAAP correlated with the molecular finding of PML/RARalpha junction. These results indicate that double APAAP staining can identify leukemic cells in samples with a cytomorphologic pattern consistent with remission, but its applicability in detection of MRD awaits additional studies on a larger number of patients with ULCA+ AL.

Fine-needle aspiration and nipple discharge cytology in the diagnosis of breast lesions in adolescent and young women: Cytologic findings as compared with those obtained in older women

Study Objective: To analyze fine needle biopsy of the breast and nipple discharge cytology in young women (aged ≤30), and compare it to the findings obtained in a group of older women (aged ≥31). Design: From January 1989 through December 1990, 258 adolescent and young women (aged ≤30) were examined for breast disease by fine needle aspiration (FNA) and nipple discharge cytology. Results were compared with those obtained from a group of 3,063 older women (aged ≥31). Results: Of the 137 nipple discharge smears from young women, foam cells were found in 97% of the samples. Suspect or malignant cells were only found in nipple discharge from older women. FN As of the breast from young women were compared with the findings from the older group. Fibroadenoma was found to be more frequent (p < 0.01), atypical epithelial proliferation was not observed, and carcinoma was rare (1.5%) in the group of young women. Fibrocystic changes with epithelial proliferation without atypia were found in the same proportion in both age groups. Conclusion: Criteria for biopsy of all epithelial proliferation should be very carefully considered in young women.