Cytopathology and diagnostics of Warthin's tumour.

Warthin's tumour (WT) is a benign epithelial salivary tumour, one type of salivary adenoma. Histologically, WT is structured of two components, epithelial tissue that often lines cystic formations and lymphoid tissue in the tumour stroma. FNA is a reliable diagnostic approach in the diagnosis of salivary gland lesions allowing a highly accurate categorization of benign tumour-like lesions, benign tumours and malignant tumours. In the proposed Milan reporting system of salivary gland lesions, WT is categorized in the IVA group of benign neoplasms. Accurate cytological diagnosis is straightforward when three characteristic components are present: oncocytes, either isolated or associated in clusters, lymphocytes and lymphoid cells and often an inflammatory/necrotic-like substance. Also, specific features of scintigraphy and radiological imaging contribute to the diagnosis of WT. WT is categorized according to Seifert G. et al in 4 types, depending on the proportions of the epithelial component and lymphoid stroma. Differential cytopathological and pathohistological diagnosis include other salivary gland lesions with lymphoid, oncocytic epithelial and cystic components. In some cases, such as the metaplastic WT variant, there are additional cytopathological and histological diagnostic difficulties. Moreover, bilateral, multicentric or multiple and infrequently seen extra-salivary localizations of WT are associated with further cytopathological diagnostic difficulties. Also, a rare possibility of malignant transformation of the epithelial or lymphoid component of WT as well as possible association with other primary tumours remains a challenge in accurate cytopathological and histological diagnosis of WT.

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.

Apoptosis of leukemic cells: a case report.

Transformation of leukemic cells is associated with delay in maturation and in apoptosis, and to altered responsiveness to growth factors. However, some studies have revealed that Fas (CD95/APO1) which mediates apoptotic signal and decrease of anti-apoptotic Bcl-2 are frequently observed in acute myeloid leukemia (AML) M4/M5 leukemic cells. The aim of the study was to compare cytomorphology and cytochemistry of bone marrow (BM) apoptotic leukemic cells to preserved peripheral blood (PB) leukemic cells in our patient, a 76-year-old man with AML-M5b treated at Zagreb University Hospital Center. BM and PB of the AL patient were analyzed after Pappenheim and cytochemical stainings, and leukemic cells were classified according to FAB and WHO classification. Analysis of PB revealed leukocytosis and 80-90% monocytic cells (46% monoblasts, 29% promonocytes and 11% monocytes). Only a few preserved monoblasts and promonocytes were found in BM, together with numerous morphologically altered cells with characteristic chromatin condensation and pyknosis of nucleus, as well as nuclear fragmentation and formation of apoptotic bodies. Thus, cytomorphology of PB leukemic cells pointed to proliferation of immature monocytic cells, and cytomorphology of BM to cell apoptosis. Cytochemistry of PB monocytic cells and BM apoptotic cells confirmed monocytic cell lineage because esterase was strongly positive in almost all BM apoptotic leukemic cells and PB leukemic cells, and esterase was completely inhibited with sodium fluoride. On the basis of these findings, AML-M5b was diagnosed in our patient. There are many possible explanations for our observation of BM leukemic cell apoptosis in a patient with AML-M5. The most reliable one is that apoptosis was induced ex vivo after BM aspiration in course of the air drying of BM specimen before staining. Mass BM leukemic cell apoptosis that was recorded in contrast to numerous preserved leukemic cells in PK could be probably connected to unfavorable ratio of relatively low concentration of cytokines in relation to high leukemic cell number in BM aspirated cytologic specimen.

FNA based diagnosis of head and neck nodal lymphoma.

Fine-needle aspiration (FNA) biopsy has become a well established technique in the diagnosis, staging, and follow-up of patients with head and neck lesions. As in lymphoma diagnostics, FNA serves as a screening method in evaluating potentially affected lymph node for open or core biopsy. According to the World Health Organization classification of lymphoid neoplasms, today it is important to recognize cell morphology and reveal its phenotype, then combine it with different genotypic information and clinical data to provide appropriate therapy. The aim of this study was to assess the efficacy of FNA and immunocytochemistry based lymphoma diagnostic in head and neck region. We conducted a retrospective study during a period of three years where cases with either FNA diagnosis or clinical suspicion of newly recognized or relapsing lymphoma were reviewed. In the study were included patients that were referred to our laboratory from hematology department, in whom head and neck lymphadenopathia was found and lymph node FNA preceded other procedures. Two hundred eighty-five aspirations from 248 patients fulfilled study criteria. Adequate specimens were diagnosed as lymphoma in 100 cases (36%), in 65 male and 35 female patients, 76 in patients with newly discovered disease and 24 in patients with prior lymphoma diagnosis. Overall sensitivity of FNA specimens in the diagnosis of head and neck lymphomas was 90%, specificity 88%, predictive value of a positive result 97%, and predictive value of negative result 61%. Based on our results FNA corroborated with immunophenotyping by immunocytochemistry can be method of choice in primary lymphoma diagnosis as a method complementary to histopathology in lymphoma diagnostics.

Immunoexpression of 5-methylcytosine (5mc) in Bone Marrow Haematopoietic Cells in Patients with Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are biologically and clinically heterogenous groups of clonal haematopoietic stem cell diseases characterized by ineffective haematopoiesis and peripheral blood cytopenia, with a variable tendency to transform within acute leukaemia (AL). DNA hypermethylation and hypo-methylation are associated with cancer. Thus, the hypermethylation of DNA is essential for the molecular pathophysiology of MDS by inactivating genes involved in cell growth, differentiation and apoptosis. It was documented that the 5-methylcytosine (5mc) immunostaining score of BM haematopoietic mononuclear cells is higher in MDS patients than in a normal control group, and that the prognosis of the disease significantly correlated with global DNA methylation, age and IPSS score. In our study, we analysed the immunocytochemical expression of 5mc in bone marrow (BM) mononuclear cells from 13 MDS patients and a control group consisting of 13 patients with anaemia of chronic disease. The immunopositivity of 5mc BM mononuclear cells was statistically significantly higher in our MDS patients than in patients with anaemia of chronic disease. In most MDS patients (11 out of 13), a higher 5mc immunopositivity of BM mono-nuclear cells (above 10%) was found. Our results are in concordance with data from literature observing that a higher percentage of 5mc immunopositive BM mononuclear cells is documented in MDS patients.

[Cytomorphology of acute mixed leukemia]. / Citomorfologija akutnih mjesovitih leukemija.

UNLABELLED: Biphenotypic acute leukemias (AL) with blasts expressing both myeloid and lymphoid antigens are grouped with undifferentiated AL and bilineal AL in the group of AL of ambiguous lineage. Not all AL with myeloid and lymphoid antigens (ALMy+Ly) are true biphenotypic AL. According to EGIL scoring system, true biphenotypic ALMy+Ly are those with a sum of antigens 2 or more points for both myeloid and lymphoid lineage or for B and T lineage. The aim of this study was to compare cytomorphology and immunophenotype of AL to better understand the relation of certain AL morphology, immunophenotype, cytogenetics and molecular biology of biphenotypic AL. PATIENTS AND METHODS: The study included a group of 169 AL patients treated from 1985 till 1991, and a group of 102 AL patients treated from 1993 till 1996 at Zagreb University Hospital Center. Bone marrow and peripheral blood of the two groups of AL patients were analyzed according to Pappenheim (May-Grunwald-Giemsa), cytochemical and alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunocytochemical staining. Flow cytometry immunophenotyping of bone marrow was also done in both patient groups. RESULTS AND DISCUSSION: In the group of 169 adult AL patients, 116 were cytomorphologically classified as acute myeloblastic leukemias (AML), 35 as acute lymphoblastic leukemias (ALL) and 18 as acute undifferentiated leukemias (ANLM). In 6 (3.4%) of 169 AL patients, blasts expressed both myeloid and lymphoid antigens. In the group of 102 AL patients there were 19 (18.6%) ALMy+Ly. In 64 patients cytomorphologically classified into AML subgroup out of 102 AL patients, there were 15 (14.7%/102; 23.4%/64) AML with lymphoid antigens (AMLLy+). In 35 patients cytomorphologically diagnosed as ALL and 3 as ANLM out of 102 AL, there were 4 (3.9%/102; 10.5%/38) ALL with myeloid antigens (ALLMy+). The incidence of mixed AL in 102 AL was more consistent with other studies, pointing to the necessity of myeloperoxidase (MPO), CD7 and TdT determination as part of standard immunophenotyping for better recognition of mixed AL. CONCLUSION: In both groups of 169 and 102 AL patients, the majority of AL cases were cytomorphologically classified as AML. In the group of 169 patients there were 5 AMLLy+ and in the group of 102 patients there were 15 AMLLy+. In one ANLM,My+ out of 169 AL and also one ANLM,My+ out of 102 AL, blasts were cytomorphologically undifferentiated; in 3 ALLMy+ of 102 AL blasts expressed lymphoid morphology. According to EGIL scoring system, among 15 AMLLy+ of 102 AL there were 4 true biphenotypic ALMy+Ly (1 M1, 2 M3, 1 M4), and in 4 ALMy+Ly with undifferentiated and lymphoid morphology there were 2 true biphenotypic AL (1 L2; 1 ANLM). In 3 ALLB+T out of 35 ALL, one was interlineal biphenotypic AL. These observations are consistent with other studies and WHO determinations indicating that the majority of true biphenotypic leukemias are associated with immature monoblastic or myeloid cytomorphology or with lymphoid or undifferentiated characteristics, but may also express any AML cytomorphology type. Thus, there is no direct correlation of leukemic cell cytomorphology and biphenotypic AL immunophenotype.

Modulation of neutrophil and inflammation markers in chronic obstructive pulmonary disease by short-term azithromycin treatment.

The anti-inflammatory potential of azithromycin in chronic obstructive pulmonary disease (COPD) patients was explored following a standard oral dosing regimen. Patients with moderate and severe COPD were treated with azithromycin (500 mg, n=16) or placebo (n=8) once daily for 3 days in a randomized, double blind design, to compare effects on inflammation markers with those seen in a previous study in healthy volunteers. A battery of tests was made on serum, blood neutrophils and sputum on days 1 (baseline), 3, 4, 11, 18 and 32. In comparison to placebo, azithromycin resulted in an early transient increase in serum nitrites plus nitrates (day 3), associated with a tendency towards an increase in the blood neutrophil oxidative burst to phorbol myristic acetate. Subsequently, prolonged decreases in blood leukocyte and platelet counts, serum acute phase protein (including C reactive protein) and soluble E-selectin and blood neutrophil lactoferrin concentrations and a transient decrease in serum interleukin-8 were observed. Blood neutrophil glutathione peroxidase activity showed a prolonged increase after azithromycin treatment. The biphasic facilitatory-then-inhibitory response to azithromycin seen in healthy volunteers is not so clearly detectable in COPD patients, only potential anti-inflammatory effects. Treatment for longer periods may give therapeutic anti-inflammatory benefit in these patients.

Patient with immunoproliferative disease and lung carcinoma: a case report.

There are a number of reports on collision occurrence of non-hematologic cancers and Non-Hodgkin's lymphoma (NHL) and multiple myeloma. In this report we present a case of patient with immunoproliferative disease, extramedullary plasmocytoma and NHL-lymphoplasmocytoid lymphoma (LPL) and squamous cell carcinoma of the lung. After diagnosis of extramedullary plasmocytoma cytostatic therapy was commenced and the patient was well. Five years after patient was clinically worse and diagnostic evaluation this time revealed lymphoplasmocytoid cells in bone marrow. Five months later malignant morphologically undifferentiated cells were found in bone marrow which were by immunocytochemistry established as CD38 positive. After the patient's death, disseminated NHL-LPL and squamous cell carcinoma of lung was confirmed. In the report, we compared clinical course and diagnostic findings of our patient with literature data. We have also discussed the possible relationship of multiple B-cell lymphoid tumors and squamous cell carcinoma concluding that multidiscplinary diagnostic tools are essential not only for carcinoma diagnosis and follow-up, but also for further understanding of carcinogenesis.

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.

[Cytodiagnosis in irradiated breast cells--its value and pitfalls]. / Citodijagnostika ozracenih stanica dojke--vrijednost i moguce pogreske.

Morphologic changes in glandular epithelium of the breast after quadrantectomy of the breast carcinoma and radiotherapy are described. The aim of the study was to establish the possibilities of cytologic assessment of morphological changes in palpable lesions in the residual breast tissue. Fine needle aspirates of 50 patients after surgery and radiotherapy of the breast carcinoma were analyzed. Biopsy and pathohistologic verification were performed in 12 patients. Carcinoma was cytologically found and confirmed pathohistologically in two cases. Recurrence was pathohistologically confirmed in 3/10 (33.3%) cytologically suspect aspirates, while in the remaining 7/10 (66.7%) suspect findings benign changes were established (2 mild ductal proliferations, 2 florid ductal proliferations, 3 cases of adenosis). The patients were followed-up clinically and cytomorphologically. No relapse was noted in the follow up period. Although its role is limited, fine needle aspiration cytology is useful in the assessment and follow-up of palpable changes after irradiation. A cytologist should be informed on whether and when a patient underwent irradiation. The finding of the changed cell after the period without changes points to the relapse of the disease.

Anemia of chronic disease: illness or adaptive mechanism.

The anemia of chronic disease (ACD) is the most prevalent anemia after iron deficiency anemia. It is associated with infectious, inflammatory and neoplastic disease. ACD is a medical condition caused by the release of cytokines which mediate inflammatory and immune response (tumor necrosis factor, interleukins 1 and 6, and interferon). Abnormal iron metabolism with iron trapping in reticuloendothelial cells is primarily the cause of this condition, making iron unavailable for erythropoiesis although iron tissue reserves are elevated. Disorder in erythropoietin secretion and shortening of red cell life span also play a role in the pathogenesis of ACD. The main therapy is treatment of the underlying disorder and red cell transfusions in severe anemia. In more severe (protracted) anemias that lead to impaired quality of life and have an impact on the mortality and survival rate, erythropoiesis stimulating agents are used. Recently, new possibilities are being evaluated in terms of therapy for ACD in defined conditions, such as chelating agents, as well as hepcidin antagonist and other erythropoiesis stimulating agents.

Expression of Ki-67 and p27(Kip1) 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 p27(Kip1) , 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 p27(Kip1) 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 p27(Kip1) 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 p27(Kip1) were found in most patients with breast carcinoma, while majority of FCC with atypia were characterized by low Ki-67 and moderate to high p27(Kip1) cell immunoexpression. Majority of fibroadenomas were associated with low Ki-67 and low to moderate p27(Kip1) 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 p27(Kip1) 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 p27(Kip1) was noticed. Poorly differentiated carcinoma cells had mostly high Ki-67 and low p27(Kip1) cell immunoexpression.

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.A 16-year-old female patient presented with fever, lymphadenopathy, and high white blood cell count. Diagnosing a CD13+ ALCL with leukemic presentation with additional cytogenetic abnormality (duplication 5q35) was a significant diagnostic challenge.This combination of features, unusual for lymphoma, should be considered in differential diagnosis of myeloid neoplasms and fatal infections.

Cytological detection of lymphoma in Douglas aspirate.

Except in primary effusion lymphoma (PEL), serous effusions with lymphomatous cells in non-Hodgkin lymphoma (NHL) are not frequently seen as first manifestation of disease. In NHL lymphoplasmacytic lymphoma (LPL) the spleen, lymph nodes, and bone marrow are frequently sites of disease and this type of NHL is usually associated with a serum paraprotein of IgM type accompanied by the clinical syndrome of Waldenström macroglobulinemia. Our patient with NHL LPL type presented in this report had less frequently seen involvement of gastrointestinal tract and clinically was first manifested as effusion in Douglas space. Cytological evaluation as well immunoanalyses of effusion in such cases is essential, and various ancillary studies, because of differential diagnostic problems of lymphomas in serous effusions, first include reactive lymphocytoses and small round-cell tumors (SRCT). In our patient, cytology of effusion revealed cytomorphologically atypical lymphomatous cells with plasmocytoid differentiation. Immunocytochemical and flow cytometry analysis confirmed lymphoid cell differentiation and pathohistological diagnosis of CD20(+) LPL was set after pathohistological analysis of resected ileum.

Malignant epithelial tumors and hematologic malignancy.

The aim of this study was to analyze the value of cytology in differentiation between malignant epithelial tumor metastases and hematologic malignancy. The follow-up of ten (10) patients who underwent diagnosis and treatment of two malignant diseases, i.e. carcinoma and hematologic malignancy, was performed in the 2000-2005 period. The median of age of our patients was 72 years (range: 49-79). Cytological examination included epithelial tumors, lymph nodes and bone marrow standard Pappenheim and immunocytochemically stained smears. Carcinoma was initially diagnosed in 40% (4/10) patients and hematologic malignancy in 50% (5/10) patients, while both diseases co-occurred in one patient (1/10). Most of hematologic malignancy cases (4/10) were diagnosed as lymphoma. Multiple myeloma was diagnosed in 3 out of 10 patients (30%). Individual cases of acute myeloblastic leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia were diagnosed in the remaining three patients. Most carcinomas were breast cancer (8/10), while prostate and thyroid gland cancer were diagnosed each in one patient, respectively.

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.

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.