Summary
5‐Azacitidine has been used before stem cell transplantation in juvenile myelomonocytic leukaemia (JMML) patients. Recently, we have described immunophenotypic features in JMML at diagnosis. ...Here, our aim was to examine the changes in the immunophenotypic features during azacitidine treatment, correlating it with clinical response. Patients treated with 5‐azacitidine were evaluated at diagnosis and after three and six cycles of medication. Among 32 patients entering the study, 28 patients were examined after three cycles and 25 patients after six. Patients showed a reduction in CD34/CD117+ cells: median 3.35% at diagnosis, 2.8% after three cycles and 1.63% after six. B‐cell progenitors were decreased at diagnosis and decreased after treatment. Monocytes decreased: 11.91% to 6.4% and 4.18% respectively. Complete response was associated with increase in classical monocytes. T lymphocytes, reduced at diagnosis, increased in patients responding to 5‐azacitidine. Immunophenotypic aberrancies including expression of CD7 in myeloid progenitors remained after treatment. This feature was associated with a worse response to treatment, as well as presence of NF1. Immunophenotyping was feasible in all patients. Clinical response was associated with a decrease of myeloid progenitors and monocytes and a rise in T lymphocytes although phenotypic aberrancies persisted. The largest effect was observed after three cycles.
The parameter intensity of bone involvement (IBI) was recently proposed to quantitatively assess patients with multiple myeloma using 18F-fluorodeoxyglucose-PET combined with computed tomography ...(18F-FDG PET/CT) images. Here, we aimed to calculate IBI variation (ΔIBI) between two consecutive PET/CT of the same patient and verified its relationship with a subjective visual analysis of the images and with clinical outcome.
Consecutive whole-body 18F-FDG PET/CT performed to assess the outcomes of 29 patients diagnosed with multiple myeloma were retrospectively evaluated. ΔIBI was calculated after bone segmentation, using liver standardized uptake value as a threshold to determine metabolically active volumes in the skeleton. For each pair of consecutive PET/CTs, two nuclear medicine physicians classified visually the most recent image as PET-remission, PET-progression or PET-stable when compared to the previous examination.
The lowest ΔIBI was -1.27 and the highest was 0.29. PET-remission was related to ΔIBI <0 (median = -0.10; -1.27 to +0.03), while PET-progression was related to ΔIBI >0 (median = 0.02; -0.07 to +0.29). ΔIBI around zero was found in images classified as PET-stable (median = 0.00; -0.08 to +0.06). Significant difference in ΔIBI was found between the three groups. Multivariate stepwise analysis showed that IBI value at diagnostic PET/CT, serum calcium and percentage of plasma cells in the bone marrow are independent prognostic factors.
Delta IBI provides quantitative data for variations of 18F-FDG uptake in the bone marrow during the follow-up of the patients. In addition, higher IBI values at diagnosis are associated with a higher risk of patient's death.
The diagnosis of juvenile myelomonocytic leukaemia (JMML) is based on clinical, laboratory and molecular features but immunophenotyping multiparametric flow cytometry (MFC) has not been used ...routinely. In the present study, we describe the flow cytometric features at diagnosis with special attention to the distribution of monocytic subsets and the relation between MFC and molecular subgroups. MFC was performed with an eight‐colour platform based on Euroflow. We studied 33 JMML cases. CD34+/CD117+/CD13+ cells >2% was found in 25 cases, and 51·5% presented an aberrant expression of CD7. A decrease of CD34+/CD19+/CD10+ cells was seen in eight cases and in four they were absent. The granulocytic population had a decreased side scatter in 29 cases. Bone marrow monocytic precursors were increased in 28 patients, with a decrease in classical monocytes (median 80·7%) and increase in CD16+ (intermediate and non‐classical). A more pronounced increase in myeloid CD34+ cells was seen in patients with Neurofibromatosis type 1 (NF1) and tyrosine‐protein phosphatase non‐receptor type 11 (PTPN11), with aberrant CD7 expression in four of six and 10/12 patients respectively. Thus, JMML shows an immunophenotypic profile similar to myelodysplastic syndromes, and a different monocyte subset distribution when compared with chronic MML. MFC proved to be an important diagnostic tool that can help in differential diagnosis with other clonal diseases with monocytosis.
Fluorescence lifetime imaging (FLIM) has been used in living cells to measure metabolic activity and demonstrate cell differentiation. The aim of this study was to investigate whether the FLIM ...technique could be able to demonstrate cell maturation during myelopoiesis and erythropoiesis in unlabeled routine bone marrow (BM) preparations. Air‐dried, unstained smears of BM aspiration samples of 32 patients without BM disease and a normal morphology on May–Grünwald–Giemsa (MGG) stained smears entered the study. FLIM images were captured with a Zeiss LSM 780 NLO multiphoton microscope equipped with a Becker & Hickl SPC‐830 TCSPC FLIM module and HPM‐100‐40 hybrid detector. The samples were irradiated by two‐photon excitation at 800 nm with a titanium‐sapphire laser of the LSM 780 NLO. FLIM images were compared with those obtained by autofluorescence high resolution imaging. FLIM images of unstained smears were highly contrasted. Different cell types could be easily recognized as they were similar to those seen in MGG stained preparations. Cytoplasm of cells from the erythroid lineage revealed relatively short fluorescence lifetimes due to the presence of hemoglobin, and therefore could easily be distinguished from granulocytic precursors. Nuclear fluorescence lifetimes of all cell types were higher than those of the corresponding cytoplasm. So, FLIM of unstained BM smears obtained under routine real‐life conditions permits an easy identification of BM cells, by highlighting differences of their physicochemical properties.
Background
Immunophenotyping of bone marrow (BM) hemopoietic precursors is useful for diagnosis of adult myelodysplastic syndrome (MDS), but data concerning pediatric patients are limited. We ...analyzed immunophenotypic features of BM cells at diagnosis of children who were referred to the Brazilian Pediatric Cooperative Group of Myelodysplastic Syndromes.
Methods
Diagnosis was based on clinical information, peripheral blood counts, BM cytology and cytogenetics. Patients with Down syndrome were excluded. Children with deficiency anemias or transitory neutropenias were used as controls (CTRLs). Immunophenotyping was performed on an eight‐color antibody platform evaluating myelomonocytic maturation and progenitor cells.
Results
A total of 32 patients were examined: 6 refractory cytopenia of childhood RCC; 5 refractory anemia with excess of blasts RAEB; 8 refractory anemia with excess of blasts in transformation RAEB‐t; 13 juvenile myelomonocytic leukemia JMML and 10 CTRLs. Median age was 66 months (RCC), 68 months (RAEB/RAEB‐t), 29 months (JMML) and 70 months (CTRLs). Median number of phenotypic alterations was 4 (range 1–6) in RCC; 6 (range 2–11) in RAEB/RAEB‐t and 6 (range 2–11) in JMML (P = 0.004). The percentage of CD34+/CD117+/CD13+ cells was 0.5% (range 0.1–2.8) in RCC; 4.2% (range 0.3–10.1) in RAEB/RAEB‐t and 3.7 % (range 0.5–8.6) in JMML cases, compared with 0.7% (0.5–1.2) in CTRLs (P < 0.0005). Aberrancies in antigen expression of myeloid progenitors were seen in 63% of JMML and in 45% of RAEB/RAEB‐t. CD34+/CD19+/CD10+ cells were decreased or absent in patients compared with age‐matched controls. T lymphocytes were decreased in JMML.
Conclusions
Phenotypic abnormalities were similar to those found in adult MDS. A decrease in B‐cell precursors was observed especially in RAEB/RAEB‐t. JMML and RAEB showed a similar pattern.
Bone marrow (BM) blast count is an essential parameter for classification and prognosis of myelodysplastic syndromes (MDS). However, a high degree of cell atypias in bone marrow hemopoietic cells may ...be found in this group of clonal disorders, making it difficult to quantify precisely myeloblasts, and to distinguish them from promyelocytes and atypical immature myeloid precursors. Our aim was to investigate whether computerized image analysis of routine cytology would help to characterize these cells.
In May-Grünwald-Giemsa stained BM smears of 30 newly diagnosed MDS patients and 19 cases of normal BM, nuclei of blasts and promyelocytes were digitalized and interactively segmented. The morphological classification of the cells was done by consensus of two observers. Immature granulocytic precursors, which could not be clearly classified either as blasts or promyelocytes, were called "atypic myeloid precursors". Nuclear morphometry and texture features derived from the co-occurrence matrix and fractal dimension (FD) were calculated.
In normal BM, when compared to myeloblasts, nuclei of promyelocytes showed significant increase in perimeter and local texture homogeneity and a decrease in form factor, chromatin gray levels, Haralick's entropy, inertia, energy, contrast, diagonal moment, cluster prominence, the fractal dimension according to Minkowski and its goodness-of-fit. Compared to normal myeloblast nuclei, the chromatin texture of MDS myeloblasts revealed higher local homogeneity and goodness-of-fit of the FD, but lower values of entropy, contrast, diagonal moment, and fractal dimension. The same differences were found between nuclei of normal promyelocytes and those of MDS. Nuclei of atypical myeloid precursors showed intermediate characteristics between those of blasts and promyelocytes according to the quantitative features (perimeter, form factor, gray level and its standard deviation), but were similar to promyelocytes according to the texture variables inertia, energy, contrast, diagonal moment, cluster prominence, and Minkowski's fractal dimension.
BM atypical immature myeloid precursors are difficult to be correctly classified in routine cytology. Although their cytoplasm is more similar to that of myeloblasts, computerized texture analysis indicates a nuclear chromatin remodeling more close to the promyelocyte, thus indicating an asynchronous intermediate maturation stage between blast and promyelocyte.
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