Maria Regina Regis Silva

Comparison of cytogenetics with FISH in 40 myelodysplastic syndrome patients.

Karyotyping is important for diagnosis and prognosis of myelodysplastic syndrome (MDS). Using fluorescence in situ hybridization (FISH) either mitotic or interphase cells can be analyzed and a higher number of cells can be screened. This study evaluated the effectiveness of FISH in detecting the most common chromosomal abnormalities [-5/del 5q/-7/+8/del 11q23 and -Y] in 40 patients with MDS. Karyotype detected abnormalities in 35.2% of the patients and FISH in 35%, while some abnormalities remained undetected by each approach but the association of both methods increased the detection rate up to 40%.

Detection and Possible Prognostic Relevance of p53 Gene Mutations in Diffuse Large B-cell Lymphoma. An Analysis of 51 Cases and Review of the Literature

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkins lymphoma. Although the presence of p53 gene mutations has been considered as a bad prognostic feature in DLBCL, its clinical significance is still controversial. The aims of this study were: detect the presence of mutations in exons 5 to 9 of the p53 gene and correlate it to prognosis in DLBCL. Fifty-one DLBCL patients were enrolled in this study. Expression of p53 was evaluated by immunohistochemistry. The screening of p53 mutations was performed using PCR-SSCP methods. Cases showing a mobility shift on SSCP electrophoresis were analyzed by automatic sequencing. We could identify 8 missense mutations in 6 of 48 cases (12.5%). In addition, we found a known polymorphism at codon 213 and 2 instances of silent mutations. Of all mutations/polymorphisms found, 7 (64%) were localized in codons previously described as p53 hot spots in NHL cases. Of the remaining alterations (4 or 36%), 2 mutations were localized in codons previously described as hot spots for p53 in other tumors and 2 (codon 142 of the exon 5 and codon 195 of the exon 6), in codons not described as hot spots for p53 up to now. The presence of missense mutations in exons 5 to 9 of p53 gene had adverse impact on overall survival (P = 0.020). Coxs Regression Model identified that high-risk International Prognostic Index (IPI) and p53 gene mutations have independent negative impact on OS. Therefore, the association of IPI with cellular factors, such as p53 mutation, can be very helpful in deciding when we should indicate more aggressive therapies in patients with DLBCL, to somehow increase the chance of cure in these patients.

Prognostic significance of vascular endothelial growth factor immunoexpression in the context of adverse standard prognostic factors in multiple myeloma

Abstract:  Objectives: Vascular endothelial growth factor (VEGF) acts in several steps of multiple myeloma (MM) pathogenesis and it is an important mediator of tumor angiogenesis. The aim of this study was to examine the prognostic significance of VEGF immunoexpression in the context of standard prognostic factors present in a cohort of advanced MM patients. Methods: Fifty untreated MM patients were enrolled from May 2000 to December 2002. Bone marrow sections were subjected to morphologic assessment and immunohistochemical studies with antibodies against CD34 and VEGF. Angiogenesis was measured by microvessel density (MVD) and stratified into high (MVD ≥ 20) and low angiogenesis status (MVD < 20). VEGF immunoreactivity was examined on the basis of intensity and percentage of positive plasma cells (PC). Results: Ninety‐four percent of patients presented advanced disease at diagnosis. Median PC marrow infiltration was 80%. Twelve percent of patients presented plasmablastic morphology. Low angiogenesis was present in 27% of patients, while high angiogenesis was present in 73%. Twenty‐nine percent of patients had VEGF < 10% and 71% had VEGF ≥ 10%. Weak‐intensity VEGF was observed in 34% of cases, while 37% had moderate/strong VEGF intensity. Although VEGF had prognostic impact on overall survival (OS) and event‐free survival (EFS) in univariate analysis, multivariate analysis identified only plasmablastic morphology and elevated serum lactate dehydrogenase (LDH) level as independent prognostic factors to predict OS (P = 0.04 and P = 0.02, respectively). With regard to EFS, although VEGF showed statistical trend to influence survival (P = 0.08), the parameters of independent prognostic value were also plasmablastic morphology (P = 0.01) and elevated LDH level (P = 0.01). Conclusion: Our findings underline the frequent expression of VEGF in advanced‐stage MM and the greater prognostic information of simple and readily available factors, namely plasmablastic morphology and elevated LDH. Moreover, despite the absence of prognostic importance in multivariate analysis, VEGF and its receptors remain promising therapeutic targets in MM.

Fungal infections in marrow transplant recipients under antifungal prophylaxis with fluconazole

Fungal infection is one of the most important causes of morbidity and mortality in bone marrow transplant (BMT) recipients. The growing incidence of these infections is related to several factors including prolonged granulocytopenia, use of broad-spectrum antibiotics, conditioning regimens, and use of immunosuppression to avoid graft-versus-host disease (GvHD). In the present series, we report five cases of invasive mold infections documented among 64 BMT recipients undergoing fluconazole antifungal prophylaxis: 1) A strain of Scedosporium prolificans was isolated from a skin lesion that developed on day +72 after BMT in a chronic myeloid leukemic patient. 2) Invasive pulmonary aspergillosis (Aspergillus fumigatus) was diagnosed on day +29 in a patient with a long period of hospitalization before being transplanted for severe aplastic anemia. 3) A tumoral lung lesion due to Rhizopus arrhizus (zygomycosis) was observed in a transplanted patient who presented severe chronic GvHD. 4) A tumoral lesion due to Aspergillus spp involving the 7th, 8th and 9th right ribs and local soft tissue was diagnosed in a BMT patient on day +110. 5) A patient with a history of Ph1-positive acute lymphocytic leukemia exhibited a cerebral lesion on day +477 after receiving a BMT during an episode of severe chronic GvHD. At that time, blood and spinal fluid cultures yielded Fusarium sp. Opportunistic infections due to fungi other than Candida spp are becoming a major problem among BMT patients receiving systemic antifungal prophylaxis with fluconazole.

FLT3 internal tandem duplication during myelodysplastic syndrome follow-up : a marker of transformation to acute myeloid leukemia

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and risk for evolving to acute leukemia. Some molecular abnormalities related to acute myeloid leukemia (AML) transformation have been reported, such as FLT3 (FMS-like tyrosine kinase 3) mutations. FLT3, a member of the class 3 receptor tyrosine kinase family, mediates stem cell proliferation and differentiation, and its mutations, internal tandem duplication (ITD) and Asp835, have been reported in rare MDS patients. We studied FLT3 ITD, prospectively, in 50 MDS patients at diagnosis, at 6 and 12 months follow-up, and at any other time-point if AML transformation was detected. FLT3 ITD was not observed at diagnosis, but during follow-up the mutation was present in 2 of 50 patients (4%). Of these, one case exhibited FLT3 ITD at the end of the 6 months of follow-up in approximately 8% of bone marrow cells; this case evolved into AML at 8 months, at which time FLT3 ITD was present in approximately 85% of bone marrow cells. The other case exhibited FLT3 ITD in 68% of bone marrow cells at 7 months, precisely at the time of AML transformation. Although rare in MDS, FLT3 ITD is associated with a high probability of evolution to AML.

Frequency and prognostic relevance of cancer testis antigen 45 expression in multiple myeloma.

OBJECTIVE This study aims to analyze the expression of cancer testis antigen 45 (CT45) in normal tissues and in plasma cell disorders and to identify possible associations with clinical data and prognosis in multiple myeloma (MM) patients. MATERIALS AND METHODS Expression of CT45 was studied in 20 normal tissues (testis, placenta, skeletal muscle, bladder, lung, spleen, heart, brain and fetal brain, thymus, uterus, stomach, mammary gland, pancreas, prostate, small intestine, kidney, adrenal gland, spinal cord, colon, and one pool of 10 normal bone marrow samples) and bone marrow aspirates from 3 monoclonal gammopathies of undetermined significance, 5 solitary plasmacytomas, 61 newly diagnosed MM patients and MM cell line U266 by reverse transcriptase polymerase chain reaction. RESULTS CT45 was positive in 3 of 20 (15%) normal tissues tested: lung, brain (both fetal and adult), and spinal cord. Among monoclonal gammopathies, CT45 was positive in 2 of 5 (40%) solitary plasmacytomas bone marrow aspirates, 10 of 61 (16%) MM bone marrow aspirates, and in the U266 MM cell line. CONCLUSIONS We did not find associations between bone marrow histology and CT45 expression. However, we demonstrated for the first time that positive expression of CT45 was associated with poor prognostic (International Staging System) and poor outcomes in MM patients, meaning that CT45-positive cases presented seven times more chance of worse evolution than the negative ones.

Clinical correlations and prognostic relevance of HGF, VEGF AND FGF expression in Brazilian patients with non-Hodgkin lymphoma

The aims of this study were to correlate HGF, VEGF and FGF serum levels and microvessel density (MVD) with cell origin, biological behavior, tumor load and prognosis in NHL. Eighty-seven consecutive previously untreated NHL patients had serum samples collected; 37 (42%) of them also had serum follow-up samples; the control group was composed of 10 healthy blood donors. Cytokine serum levels were determined by ELISA, and MVD was measured by CD34 staining in paraffin blocks. HGF mean serum level was significantly higher in both early and advanced NHL stages when compared with the control group. HGF was also significantly higher in aggressive and indolent NHL when compared with the control group. Also, mean serum level of HGF in aggressive NHL was significantly higher than in indolent NHL. Regarding International Prognostic Index (IPI), HGF mean serum level at diagnosis was significantly higher for patients with IPI >2 when compared to IPI ≤2. Sequential analyses of HGF, VEGF and FGF serum levels in NHL showed that serum HGF and VEGF levels decreased significantly after 6 months of treatment completion. Our findings suggest that HGF serum level is associated with tumor load and aggressiveness, and response to treatment results in a decrease in HGF serum levels in NHL patients.

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia

Background The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms. Objectives The aim of this study was to detect the following mutations: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases. Methods Twenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities. Results Chromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary myelofibrosis. The MPL W515L-positive patient with primary myelofibrosis had more severe anemia than other patients with primary myelofibrosis. Conclusions This study demonstrates that karyotyping for JAK2 and MPL mutations is useful in the diagnosis of myeloproliferative neoplasms. The precise pathogenetic contribution of these alterations is still unclear. However, this study adds more information about the pathophysiology of polycythemia vera, essential thrombocythemia and primary myelofibrosis.

t(4;12)(q11;p13): a rare chromosomal translocation in acute myeloid leukemia

We present a case of acute myeloid leukemia with t(4;12). This translocation is rare and has been observed in acute leukemias with different but immature phenotypes. To the best of our knowledge, there are around 15 descriptions of t(4;12) in AML, and most interesting, presenting morphological aspects of a pseudo-lymphoid cell with dysplasia of other series.

Experimental protothecosis in laboratory animals

Attempts were made to produce protothecosis in laboratory animals by intratesticular, intraperitoneal, intravenous and subcutaneous inoculation of Prototheca zopfii, P. wickerhamii and P. stagnorta. Only intratesticular injection successfully caused infection, and of the inoculated protothecae, only P. zopfii and P. wickerhamii were recovered in culture from testicular material.