Carmen S. P. Lima

Fractal Characteristics of May-Grünwald-Giemsa Stained Chromatin Are Independent Prognostic Factors for Survival in Multiple Myeloma

Background The use of computerized image analysis for the study of nuclear texture features has provided important prognostic information for several neoplasias. Recently fractal characteristics of the chromatin structure in routinely stained smears have shown to be independent prognostic factors in acute leukemia. In the present study we investigated the influence of the fractal dimension (FD) of chromatin on survival of patients with multiple myeloma. Methodology We analyzed 67 newly diagnosed patients from our Institution treated in the Brazilian Multiple Myeloma Study Group. Diagnostic work-up consisted of peripheral blood counts, bone marrow cytology, bone radiograms, serum biochemistry and cytogenetics. The International Staging System (ISS) was used. In every patient, at least 40 digital nuclear images from diagnostic May-Grünwald-Giemsa stained bone marrow smears were acquired and transformed into pseudo-3D images. FD was determined by the Minkowski-Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R2 values in the log-log plots. The influence of diagnostic features on overall survival was analyzed in Cox regressions. Patients that underwent autologous bone marrow transplantation were censored at the day of transplantation. Principal Findings Median age was 56 years. According to ISS, 14% of the patients were stage I, 39% were stage II and 47% were stage III. Additional features of a bad prognosis were observed in 46% of the cases. When stratifying for ISS, both FD and its goodness-of-fit were significant prognostic factors in univariate analyses. Patients with higher FD values or lower goodness-of-fit showed a worse outcome. In the multivariate Cox-regression, FD, R2, and ISS stage entered the final model, which showed to be stable in a bootstrap resampling study. Conclusions Fractal characteristics of the chromatin texture in routine cytological preparations revealed relevant prognostic information in patients with multiple myeloma.

Minimal doses of hydroxyurea for sickle cell disease

The use of hydroxyurea (HU) can improve the clinical course of sickle cell disease. However, several features of HU treatment remain unclear, including the predictability of drug response and determination of adequate doses, considering positive responses and minimal side effects. In order to identify adequate doses of HU for treatment of sickle cell disease, 10 patients, 8 with sickle cell anemia and 2 with S beta thalassemia (8SS, 2S beta), were studied for a period of 6 to 19 months in an open label dose escalation trial (10 to 20 mg kg-1 day-1). Hemoglobin (Hb), fetal hemoglobin (Hb F) and mean corpuscular volume (MCV) values and reticulocyte, neutrophil and platelet counts were performed every two weeks during the increase of the HU dose and every 4 weeks when the maximum HU dose was established. Reduction in the number of vasoocclusive episodes was also considered in order to evaluate the efficiency of the treatment. The final Hb and Hb F concentrations, and MCV values were significantly higher than the initial values, while the final reticulocyte and neutrophil counts were significantly lower. There was an improvement in the concentration of Hb (range: 0.7-2.0 g/dl) at 15 mg HU kg-1 day-1, but this concentration did not increase significantly when the HU dose was raised to 20 mg kg-1 day-1. The concentration of Hb F increased significantly (range: 1.0-18.1%) when 15 mg HU was used, and continued to increase when the dose was raised to 20 mg kg-1 day-1. The final MCV values increased 11-28 fl (femtoliters). However, reticulocyte (range: 51-205 x 10(9)/l) and neutrophil counts (range: 9.5-1.3 x 10(9)/l) obtained at this dose were significantly lower than those obtained with 15 mg kg-1 day-1. All patients reported a decrease in frequency or severity of vasoocclusive episodes. These results suggest that a hydroxyurea dose of 15 mg kg-1 day-1 seems to be adequate for treatment of sickle cell disease in view of the minimal side effects observed and the improvement in laboratory and clinical parameters.

Increased metabolic activity detected by FLIM in human breast cancer cells with desmoplastic reaction: a pilot study

Introduction: In breast cancer (BC), desmoplastic reaction, assembled primarily by fibroblasts, is associated with unfavorable prognosis, but the reason of this fact remains still unclear. In this context, nonlinear optics microscopy, including Fluorescence Lifetime Imaging Microscopy (FLIM), has provided advancement in cellular metabolism research. In this paper, our purpose is to differentiate BC cells metabolism with or without contact to desmoplastic reaction. Formalin fixed, paraffin embedded samples were used at different points of hematoxylin stained sections. Methodology: Sections from 14 patients with invasive ductal breast carcinoma were analyzed with FLIM methodology to NAD(P)H and FAD fluorescence lifetime on a Confocal Upright LSM780 NLO device (Carl Zeiss AG, Germany). Quantification of the fluorescence lifetime and fluorescence intensity was evaluated by SPC Image software (Becker &Hickl) and ImageJ (NIH), respectively. Optical redox ratio was calculated by dividing the FAD fluorescence intensity by NAD(P)H fluorescence intensity. Data value for FLIM measurements and fluorescence intensities were calculated using Wilcoxon test; p< 0.05 was considered significant. Results: BC cells in contact with desmoplastic reaction presented a significantly lower NAD(P)H and FAD fluorescence lifetime. Furthermore, optical redox ratio was also lower in these tumor cells. Conclusion: Our results suggest that contact of BC cells with desmoplastic reaction increase their metabolic activity, which might explain the adverse prognosis of cases associated with higher peritumoral desmoplastic reaction.

Association of a myelodysplastic syndrome with hairy cell leukaemia

To the Editor: Peripheral blood cytopenias are common in hairy cell leukaemia (HCL). However, nearly 40% of patients show a myeloid hypoplasia in bone marrow, which is not proportional to the neoplastic infiltration (1). Some cases of HCL that mimic aplastic anaemia have been reported (2). Several CFU inhibitors may be responsible for this suppression of haemopoiesis. On the other hand, the association of several lymphoproliferative disorders and myelodysplastic syndrome (MDS) has been observed (9). However, to our knowledge, association of HCL and MDS has never been reported. A 55-year-old woman was referred to our hospital in September 1985, because of the incidental finding of leukopenia (leukocytes = 3.2 x 109/1) and thrombocytopenia (platelets = 93 x 109/l). She had no complaints and physical examination was normal. Bone marrow histology and cytology revealed a slightly hypocellular haemopoiesis with a predominant decrease of the granulopoiesis, resulting in an M:E ratio of 0.5. Both erythroblasts and granulocytic precursors showed atypic forms. No ring syderoblasts were found. Megakaryocytes were few in number, and some elements were also atypical (Fig. 1A). Reticulin fibres were normal. The patient received no treatment. In May 1986 another bone marrow biopsy and smears were taken, showing essentially unchanged features. On this occasion, a myelodysplastic syndrome (refractory anaemia) was diagnosed. The patient received folic acid 15 mg/ day, vitamin B6 600 mg/day and prednisone 60 mg/ day. One month later peripheral blood counts were normal (Table 1). Prednisone was then lowered to 20 mg/day, and vitamin B6 and folic acid were discontinued. In December 1986, the patient complained of severe pain in both hip joints. Radiographic features were consistent with bilateral aseptic necrosis of the femoral heads. Prednisone was then discontinued. One month later, peripheral blood counts showed anaemia and leukopenia (Table 1). In June 1994, anaemia became transfusiondependent. On this occasion, bone marrow histology revealed hypercellular areas. There were patchy infiltrates of mononuclear cells presenting features of hairy cells. These cells were positive for immunostaining with the monoclonal antibody DBA44 (Fig. 1C) as well as for CD45 and CD20 (L26), while negative for CD45RO (UCHL1). Reticulin fibres were diffusely increased. The dysplastic features of the haemopoietic precursors remained unchanged, but M:E ratio was 0.13. All former bone marrow biopsies were then reviewed. Immunohistochemical staining with the monoclonal antibody DBA44 disclosed small groups of positive cells in all biopsies after the diagnosis (Fig. 1B). Hairy cells were not observed in the peripheral blood smears. Treatment with a interferon 5 x lo6 U three times a week was started in September 1994. Blood counts soon improved (Table 1). During the whole course of the disease splenomegaly was not found on physical examination or on abdominal ultrasonography. In the present case, several remarkable features were observed. Initially, the finding of leukopenia and thrombocytopenia in the peripheral blood, together with a cellular marrow and atypias in all three haemopoietic lines led to the diagnosis of refractory anaemia. The patient responded well to prednisone, presenting a recovery of the peripheral blood counts after 4 weeks. Four months later, when she developed aseptic necrosis of both femoral heads, treatment was discontinued. Then, she slowly developed pancytopenia. Although 10% of patients with MDS respond to glucocorticoids (3), improvement of blood cell counts in HCL using this therapy has not been reported (4). The possibility of bone involvement by HCL causing aseptic necrosis has also been raised. This feature has been reported in some cases of HCL (5-7). In one of these reports, corticosteroids relieved bone pain (6). The occurrence of aseptic necrosis of femoral heads was first attributed to the use of corticosteroids. Radiologic lesions remained unchanged for 8 years. After the diagnosis of HCL was established in our patient, the hip lesions were also re-evaluated, in a search for possible bone infiltration. The gallium scan did not help to elucidate this point. Bone mar-

Comparison of blood neoangiogenesis and lymphatic vascularization in colorectal adenomas from patients with and without concomitant colorectal cancer

Blood and lymphatic vessel proliferation is essential for tumor growth and progression. Most colorectal carcinomas develop from adenomas (adenoma-carcinoma sequence) in a process due to accumulation of molecular genetic alterations. About 5% of adenomatous polyps are expected to become malignant, but data on the differential angiogenic patterns of these lesions in patients with and without concomitant cancer are missing. The aim of the present study is to compare the angiogenic and lymphatic patterns of adenomatous polyps from patients with and without sporadic cancer. Thirty adenomatous polyps (15 from patients with another principal malignant lesion, and 15 from patients without cancer) were submitted to immunohistochemical staining for CD105 (marker for neoangiogenesis) and D2-40 (marker for lymphatic endothelium). Microvessel density and total vascular area were determined by computer image analysis to quantify the immunostained and total areas, and to assess the number of microvessels. Adenomas from patients with carcinoma showed significantly higher values of total vascular area determined by immunostaining for CD105 (cutoff value = 4386 microm(2); P = 0.019) and of lymphatic microvessel density determined by immunostaining with D2-40 (cutoff value = 11.5; P = 0.041) when compared with those from patients without cancer. The present data indicate a significant increase in blood microvascular area and in lymphatic microvascular counts in adenomas removed from patients with cancer.