FLT3 overexpression in acute promyelocytic leukemia patients without detectable FLT3-ITD or codon 835-836 mutations: a pilot study.

BACKGROUND: Activating mutations of the FLT3 receptor tyrosine kinase are common in acute promyelocytic leukemia (APL) but have uncertain prognostic significance. Information regarding FLT3 expression levels in APL without FLT3 mutations is lacking. MATERIALS AND METHODS: Using RT-PCR, mutation analysis of the FLT3 gene, regarding internal tandem duplications (ITDs) and codon 835-836 point mutations, was performed and real-time PCR was carried out to determine the level of FLT3 expression in 11 APL patients at diagnosis and 5 in haematological remission with molecularly detectable disease. RESULTS: High levels of FLT3 transcript, at least a 10-fold increase compared to the normal controls, were found at diagnosis in all 3 mutated cases and in 2 patients without detectable FLT3 mutations. CONCLUSION: FLT3 overexpression can be documented in patients without FLT3 mutations. These patients might benefit from treatment using specific FLT3 tyrosine kinase inhibitors. Larger studies are needed to evaluate the clinical and biological significance of FLT3 overexpression in the absence of FLT3 mutations.

Origin and diversification of the clonogenic cell in multiple myeloma: lessons from the immunoglobulin repertoire.

The study of immunoglobulin genes in multiple myeloma over the last decade has provided important information regarding biology, ontogenetic assignment, disease evolution, pathogenic consequences and tumor-specific therapeutic intervention. Detailed analysis of VH genes has revealed the clonal relationship between switch variants expressed by the bone marrow plasma cell and myeloma progenitors in the marrow and peripheral blood. Regarding VH usage, a bias was found against the V4-34 gene encoding antibodies with cold agglutinin specificity (anti-I/i), thus explaining in part the absence of autoimmune phenomena in myeloma compared to other B cell lymphoproliferative disorders. However, in some studies a substantial number of cases analyzed were carrying the rearranged Humkappav325 Vkapppa gene, known to be over utilized by B cell chronic lymphocytic leukemia clones and possessing autoantibody binding activity. VH genes accumulate somatic hypermutations following a distribution compatible with antigen selection, but with no intraclonal heterogeneity. The analysis of Vkappa genes indicates a bias in usage of Vkappa family members; somatic hypermutation, in line with antigen selection, of the expressed Vkappa genes is higher than any other B cell lymphoid disorder. Similar conclusions were reached for Vlambda genes; in this case, the analysis raises the controversial issue of N nucleotide insertion at Vlambda-Jlambda junctions, apparently as a result of TdT activity. A complementary imprint of antigen selection as evidenced by somatic hypermutation of either the VH or VL clonogenic genes has been observed. The absence of ongoing somatic mutations in either VH or VL genes gives rise to the notion that the cell of origin in myeloma is a post-germinal center memory B cell.

Prognostic evaluation of the microvascular network in myelodysplastic syndromes.

Considering the recently stated suggestion of neovascularization being implicated in myelodysplastic syndromes (MDS) pathogenesis, we evaluated multiple morphometric microvascular characteristics in MDS, in relation to clinicopathologic factors and prognosis. Trephines from 50 newly diagnosed MDS patients were immunostained for factor VIII and compared to those from 20 controls, 10 chronic myelomonocytic leukemia (CMML) and 12 acute myeloid leukemia (AML) patients. Quantitation of microvessel density (MVD), area, total vascular area (TVA), major and minor axis length, perimeter, compactness, shape factor, Feret diameter, and the number of branching vessels was performed by image analysis. Overall, the MDS group had significantly higher MVD, TVA, minor axis and shape factor values and significantly lower compactness than the control group. AML was characterized by increased vascularity compared to MDS and CMML, as well as by the presence of flattened microvessels (lower values of shape factor). Hypercellular MDS showed higher MVD. RA/RARS displayed larger caliber vessels than RAEB, which explains the favorable prognostic effect of increased size-related parameters on progression and/or survival. Moreover, decreased compactness and MVD were independent predictors of longer progression-free survival. It is concluded that angiogenesis is involved in the conversion of normal marrow to MDS and ultimately to AML and that disease progression within MDS is accompanied by qualitative alterations of the microvascular network. Furthermore, size-related parameters affect survival, while shape-related parameters and MVD are more influential with regard to progression-free survival.

Clinicopathologic correlations of bone marrow angiogenesis in chronic myeloid leukemia: a morphometric study.

Various morphometric characteristics of microvessels, highlighted by means of anti-CD34 immunohistochemical staining, were evaluated in the bone marrow of 52 patients with chronic myeloid leukemia (CML) in chronic phase, in relation to several clinicopathologic parameters. Twenty control bone marrows and 15 cases of CML in blastic phase were also studied. Microvessel density (MVD), total vascular area (TVA) and several size- and shape-related parameters were quantitated in the region of most intense vascularization using image analysis. Overall, the group of chronic phase CML had higher MVD and size-related parameters and more branching microvessels than controls. Blastic phase was characterized by increased numbers of microvessels with a rounder shape and smaller caliber than chronic phase. A positive correlation emerged between marrow fibrosis and MVD as well as between white blood cell counts and rounder vessel sections. No relationship existed between microvascular parameters and Hasford or Sokal prognostic scores. In univariate analysis, overall and progression-free survival were adversely affected by MVD, size-related parameters, increased platelet count, age and spleen size. Multivariate analysis indicated that microvessel area was related to progression-free survival, whereas both MVD and area were significant prognosticators of overall survival, even when Hasford or Sokal scores are introduced into the model. Our data suggest that changes in angiogenic parameters may participate in the conversion of normal marrow to CML and ultimately to blastic transformation. More importantly, MVD and microvessel caliber are significant predictors of patient survival and progression.

Quantitative cytochemistry of glycogen in blood cells. Methods and clinical application.

Quantitative glycogen determinations can be made in single blood and bone marrow cells, using microspectrophotometry or microfluorometry after staining with variants of the periodic acid--Schiff (PAS) reaction. These PAS variant reactions generally do not indicate the presence of non-glycogen PAS-positive substances, known to be prevalent in various hematopoietic cells, possibly due to masking of reactive groups. The specificity of the reaction in blood cells was ascertained by alpha-amylase digestion, which removed more than 95% of the PAS-positive material. Calibration of the PAS reaction was undertaken with a microdroplet model of pure leukocyte glycogen. The glycogen amounts in the droplets were determined by microinterferometry, the droplets were stained with a variant PAS reaction, and the total extinction of the reaction product in the stained droplets was determined by microspectrophotometry. The extinction coefficient (k) was obtained from the equation k equals Etot divided by M where (Etot) is the total extinction as determined by microspectrophotometry and (M) the dry glycogen amount as determined by microinterferometry. The microinterferometric dry mass determinations were calibrated by X-ray absorption in order to obtain the absolute amounts of glycogen. For practical purposes a reference system was made of normal neutrophil leukocytes. The glycogen content in the reference neutrophils was first determined with the micromodel. These neutrophils, now with a known glycogen amount, were stained with the PAS reagents and measured microspectrophotometrically in parallel with cells containing an unknown glycogen amount. Alternatively, the staining was made with a fluorescent PAS reaction, and the glycogen content determined by microfluorometry. Both methods appeared suitable for determining the glycogen content of blood cells from patients with various diseases, though the microfluorometric method was preferable for measurements of small amounts of inhomogeneously distributed glycogen. The mean glycogen content of normal neutrophil leukocytes was found to be 13.6 times 10(-12) g. The content was increased in infectious diseases such as pneumonia and tonisillitis, as well as in polycythemia vera and myelofibrosis, while low amounts were found in untreated chronic myelocytic leukemia. In chronic myelocytic leukemia in remission, the glycogen content of mature neutrophils had completely normalized. Erythroblasts normally do not contain detectable amounts of glycogen. However, in certain diseases such as beta-thalassemia and Di Guglielomo's syndrome, appreciable amounts of glycogen accumulate in the erythropoietic precursor cells. In beta-thalassemia this was associated with an arrest in the proliferation of early polychromatic erythroblasts, which accumulate glycogen in the G1 phase of the cell cycle. In all these diseases quantitative glycogen determinations in the blood cells have diagnostic importance.

alpha Interferon treatment of essential thrombocythaemia and other myeloproliferative disorders with excessive thrombocytosis.

The effect of recombinant interferon alfa-2b on platelet count, thrombocytosis-associated symptoms and marrow fibrosis was studied in 18 patients with myeloproliferative diseases and associated thrombocytosis (nine with essential thrombocythaemia, three with polycythaemia vera, three with myelofibrosis and three with chronic myelogenous leukaemia). A reduction of the platelet count below 600 x 10(9)/L was achieved in 94%, and below 400 x 10(9)/L in 77% of the patients within 8 to 330 days of treatment. The selective thrombocytosis-reducing effect of alpha interferon was maintained for long periods of time in most patients without serious side effects. Thrombocytosis-associated symptoms were relieved once the number of platelets was reduced to near normal levels. Marrow reticulin content was found to be reduced after treatment in two of the seven patients studied. Side effects of alpha interferon were flu-like symptoms, which usually subsided within 7 days of treatment.

Evidence for sinoatrial blockade associated with high dose cytarabine therapy.

Cytarabine therapy is rarely complicated by cardiotoxicity. The present report describes the clinical course of a 35-year-old female patient with acute myelogenous leukemia in complete remission, who developed sinus bradycardia while on high dose cytarabine as a consolidation therapy. The electrocardiographic findings suggested that bradycardia was most probably the result of sinoatrial blockade. The available information regarding a possible association of cytarabine with disturbances of cardiac rhythm is reviewed.

'Fetal' erythropoiesis following bone marrow transplantation as estimated by the number of F cells in the peripheral blood.

The aim of this study was to define factor(s) influencing fetal erythropoiesis following bone marrow transplantation. Thirty-one transplanted patients (14 males, 17 females) were studied. The underlying diseases were chronic myelogenous leukaemia (CML, 18 patients), acute myeloblastic leukaemia (AML, 7 patients) and acute lymphoblastic leukaemia (ALL, 6 patients). Reticulocyte and peripheral F cell estimation was carried out in donors and patients before transplantation and repeatedly during recovery. For F cell estimation, an indirect immunofluorescence assay was utilized. A significant increase above pre-BMT values in the percentage of F cells was observed in all patients from days 11 to 40 after transplantation. The increase of F cells on days 15, 18, 25, 32, 40 and 50 after transplantation was statistically significant in 14 patients who had shown an increase of F cells following chemotherapy (high responders) compared with the remaining 17 patients who did not respond so significantly. This finding supports the influence of the host bone marrow micro environment. The nature of the mechanisms operating remains to determined.

Beta-glucuronidase activity of lymph node imprints from malignant lymphomas and chronic lymphocytic leukaemia.

beta-Glucuronidase activity was semiquantitatively estimated in the cells of lymph node (LN) imprints from patients with Hodgkin's disease (HD), diffuse non-Hodgkin's lymphomas, chronic lymphocytic leukaemia (CLL), normal lymph nodes, and benign lymphadenopathies. In addition, in some of these cases beta-glucuronidase activity was semiquantitatively determined in peripheral blood smear lymphocytes. The beta-glucuronidase score (betaGS) was very low in the cells of the LN imprints from patients with diffuse non-Hodgkin's lymphomas. The LN lymphocytes of HD had a normal betaGS independently of the histological subtype of the disease, while in the LN imprint of CLL the enzyme activity was low, normal, or high. The betaGS of the lymphocytes in LN imprints of normal controls and HD were in general significantly lower compared with he lymphocytes of the peripheral blood smears in the same cases. The relation of our findings to the B and T cell origin of malignant lymphomas and chronic lymphocytic leukaemia is discussed.

Acute promyelocytic leukemia relapsing into FAB-M2 acute myeloid leukemia with trisomy 8.

Acute promyelocytic leukemia was diagnosed in a 48-year-old man; the karyotype was normal, whereas reverse transcriptase polymerase chain reaction (RT-PCR) analysis identified PML/RAR alpha chimeric transcripts of the bcr3 type. Rather unexpectedly, the patient did not respond to alltrans retinoic acid administration; he attained complete remission with conventional chemotherapy and became PML/RAR alpha negative. Two years later, while PML/RAR alpha negative on RT-PCR, he presented with thrombocytopenia. Bone marrow examination was compatible with myelodysplasia of the RAEB type; the karyotype was normal. Then, after 10 months, he developed overt acute myeloid leukemia with PML/RAR alpha negative, French-American-British M2 blasts; karyotypic analysis revealed mosaicism for trisomy 8.

Evaluation at single cell level of residual Philadelphia negative hemopoietic stem cells in chronic phase CML patients.

In chronic myeloid leukemia, accurate determination of Ph(-) Hemopoietic stem cells (HSC) in peripheral blood (PB), bone marrow (BM) and leukapheresis products is important for the selection of patients for whom mobilization, collection, and autografting of Ph(-) HSC are envisaged. To this effect, the BCR/ABL fusion was assessed at the single cell level in 25 sets of PB and BM samples using dual-color I-FISH in immunophenotyped CD34(+) cells and RT-PCR of individual CFU-GM colonies. In 15 cases found to be 100% Ph(+), the respective BCR/ABL gene was absent in 30% of CD34(+) cells, while the respective transcripts could not be identified in 17% of CFU-GM. The mean percentage of BCR/ABL(-) CD34(+) cells and CFU-GM cells was higher (38% and 29%, respectively) in untreated patients than in treated patients (24% and 7%, respectively). In eight cases with cytogenetic response (CgR), the percentage of Ph(-) metaphases correlated with the level of BCR/ABL(-) colonies in BM and PB and with the proportion of BCR/ABL(-) CD34(+) cells in the BM. Immunophenotyping and FISH was fast, easy, always informative, and quantitative for the BCR/ABL(-) CD34(+) cells. Our results show that (a) at early diagnosis a high frequency of BCR/ABL(-) HSC circulate in the PB and that Ph(-) hematopoiesis is not completely suppressed; (b) although normal clonogenic cells decline rapidly within a few months after diagnosis, appreciable numbers of normal CD34(+) cells survive in chronic phase, especially in patients with CgR.

Reverse transcription polymerase chain reaction for the diagnosis and molecular monitoring of the PML/RAR alpha fusion gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) translocation and a high rate of response to all-trans retinoic acid. The translocation generates a PML/RAR alpha chimeric gene which is transcribed in a fusion PML/RAR alpha mRNA. In this study, by using RT-PCR, we examined 14 APL patients for PML/RAR alpha fusion gene transcripts. Eight patients were studied at diagnosis, 2 at relapse, 1 both at relapse and after reinduction, 1 both at diagnosis and after three cycles of consolidation chemotherapy, and 2 patients were examined for minimal residual disease (MRD) 4 months after completing treatment. A positive result was observed in all 14 cases. Two patients who were in complete hematologic remission had evidence of hematologic relapse soon after the positive test. We conclude that RT-PCR for APL yields important diagnostic and prognostic information for the APL patients.

A novel chromosomal abnormality involving chromosomes 2 and 18 in a patient with myelodysplastic syndrome.

Cytogenetic analysis of bone marrow cells from a patient with myelodysplastic syndrome associated with eosinophilia showed a complex translocation with a 46,XY,t(2;18;2)(p23;q11;q32) karyotype. The patient has refractory anemia (RA) according to the French-American-British Cooperative Group (FAB) classification, and after 90 months of follow-up he shows no evidence of leukemic transformation. This chromosomal abnormality has not been previously described in myelodysplastic syndromes and may be associated with good prognosis as the patient has been stable for a long time.

Hypereosinophilia associated with monosomy 7.

Cytogenetic analysis of bone marrow cells from a patient with anemia, marked leukocytosis with eosinophilia, and thrombocytopenia showed monosomy 7 in all metaphases examined. The patient has refractory anemia (RA) according to FAB classification. Because of the hypereosinophilia of the patient, PCR technique was performed and no bcr-abl mRNA, specific for chronic myelogenous leukemia, was detected. Monosomy 7 has not been previously described in cases with hypereosinophilia. We assume, according to previous reports, that multiple genetic lesions can be involved in the pathogenesis of hypereosinophilia in this patient.

Trisomy 11 with loss of the Y chromosome and trisomy 13 in a case of de novo acute myeloid leukemia.

We report a man with de novo acute myeloid leukemia (M4 of the FAB classification) bearing two abnormal clones in the bone marrow cells. The clones showed trisomy 11 with loss of the Y chromosome and trisomy 13, respectively.

Cytogenetic analysis and RAS mutations in primary myelodysplastic syndromes.

Cytogenetic analysis was performed in 60 patients with primary myelodysplastic syndromes--diagnosed, treated, and followed in our department. In 41 cases, the presence of the NRAS mutation was also evaluated. The aim of this study was to evaluate the prognostic value of chromosomal abnormalities and NRAS mutation. The median age of the patients was 67 years (18-88 years), and the French-American-British classification was as follows: refractory anemia 26, refractory anemia with ring sideroblasts 4, refractory anemia with excess of blast cells 15, refractory anemia with excess of blast cells in transformation 3, and chronic myelomonocytic leukemia 12. Survival analysis was performed for the patients with a normal (n = 35), an abnormal (n = 25) karyotype and with a single (n = 15) or multiple (n = 10) cytogenetic abnormalities. Abnormal karyotypes were detected in 25 of the 60 patients (41.6%). Fifteen of these patients had a single and 10 had two or more lesions. The median survival of the patients with a normal (33.1 months) and with an abnormal (36.5 months) karyotype was not significantly different. Patients with multiple lesions had a reduced median survival compared with patients with single anomalies (19.2 versus 39.7 months, p = 0.5). Patients with an abnormal karyotype progressed to acute leukemia more frequently compared with patients without lesions (36 versus 28.6%, p = 0.5). NRAS mutation was detected in 2 of 10 CMMoL patients studied and in none of the 31 patients with other types of myelodysplastic syndrome. Marrow blasts more than 10% significantly affected survival.

Correlation of the site of M-bcr breakpoint with chronic phase duration, blastic crisis lineage and thrombocytosis in Ph1-positive chronic myelogenous leukemia.

The breakpoints on chromosome 22 in CML occur within a 5.8 kb region called the Major breakpoint cluster region (M-bcr). DNA mapping within the M-bcr region was performed in 41 long term followed Ph1-positive CML patients using the Southern blot technique. The purpose of this study was to determine whether localization of M-bcr breakpoint correlates with the length of chronic phase of the disease, blastic crisis lineage and thrombocytosis at the time of initial diagnosis. Our results fail to indicate any correlation between breakpoint localization and duration of chronic phase, blastic crisis lineage and platelet count at diagnosis.

Molecular analysis of immunoglobulin genes in multiple myeloma.

The study of immunoglobulin genes in multiple myeloma over the last five years has provided important information regarding biology, ontogenetic location, disease evolution, pathogenic consequences and tumor-specific therapeutic intervention with idiotypic vaccination. Detailed analysis of V(H) genes has revealed clonal relationship between switch variants expressed by the bone marrow plasma cell and myeloma progenitors in the marrow and peripheral blood. V(H) gene usage is biased against V4-34 (encoding antibodies with cold agglutinin specificity; anti-l/i) explaining the absence of autoimmune phenomena in myeloma compared to other B-cell lymphoproliferative disorders. V(H) genes accumulate somatic hypermutations following a distribution compatible with antigen selection, but with no intraclonal heterogeneity. V(L) genes indicate a bias in usage of VkappaI family members and somatic hypermutation, in line with antigen selection, of the expressed Vkappa genes is higher than any other B-cell lymphoid disorder. A complementary imprint of antigen selection as evidenced by somatic hypermutation of either the V(H) or V(L) clonogenic genes has been observed. The absence of ongoing somatic mutations in either V(H) or V(L) genes gives rise to the notion that the cell of origin in myeloma is a post-germinal center memory B-cell. Clinical application of sensitive PCR methods in order to detect clonal immunoglobulin gene rearrangements has made relevant the monitoring and follow-up of minimal residual disease in stem cell autografts and after myeloablative therapy. The fact that surface immunoglobulin V(H) and V(L) sequences constitute unique tumor-specific antigenic determinants has stimulated investigators to devise strategies aiming to generate active specific immunity against the idiotype of malignant B-cells in myeloma by constructing vaccines based on expressed single-chain Fv fragments, DNA plasmids carrying V(H)+V(L) clonogenic genes for naked DNA vaccination, or dendritic cell-based vaccination armed with the tumor-specific idiotype.

Detection of "PNH red cell" populations in hematological disorders using the Sephacryl Gel Test micro typing system.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder characterised by an unusual sensitivity of abnormal red cell population(s) to complement lysis, due to a complete or incomplete defect of various surface molecules, including CD55 and CD59. PNH has been associated with various hematological disorders. Using a newly introduced method, the Sephacryl gel test microtyping system, we investigated the presence of CD55 or CD59 defective red cell populations in several hematological disorders. It was also found that a large proportion of such patients possess CD55 deficient populations, while a smaller but still significant proportion possess CD59 deficient populations. Defective red cell populations were detected in normal subjects as well. These findings need further investigation. Nevertheless the Sephacryl Gel Test microtyping system although non specific, seems to be useful in screening for the PNH and/or "PNH-like" red cell defect in several hematological disorders.

Defective mitogen-induced cellular cytotoxicity in myelodysplastic syndromes. Recovery after alpha-interferon administration.

Peripheral blood mitogen - induced cellular cytotoxicity (MICC) and natural killer- cell cytotoxicity (NKCC) were assessed in 25 patients with myelodysplastic syndromes (MDS). Both MICC and NKCC were examined under the same experimental conditions using the 18 hr chromium release assay, except that cultures for MICC were stimulated in vitro by the addition of phytohemagglutinin (PHA). Patients' MICC was found significantly reduced, in relation to controls (p less than 0.001), but significantly higher than patients' NKCC (p less than 0.001). Furthermore, patients CD3+ cells and CD4+ cells, as well as the CD4+/CD8+ ratio, were significantly decreased (p less than 0.01, p less than 0.001 and p less than 0.001, respectively), while CD8+ cells and CD16+ cells were within normal limits. No relationship was noted between patients' MICC and total lymphocyte count or any lymphocyte subpopulation. In eleven patients who were subsequently subjected to a-interferon (a-IFN) administration, MICC values were found within normal range one month after the cessation of alpha-IFN, while NKCC values were significantly increased (p less than 0.01), but they still remained below the lower limit of the control (p less than 0.001). Percentages of CD3+, CD4+ and CD8+ cells, as well as the CD4+/CD8+ ratio, did not change after alpha-IFN, but the absolute numbers of CD3+ cells and CD8+ cells were significantly reduced. A statistically significant rise was noted in CD16+ cells. Post- IFN rises in MICC did not correlate with lymphocyte subpopulations. The findings indicate that MDS patients display very low MICC, which can be restored by alpha-IFN administration. The cause of this disturbance and the mechanism of its restoration by alpha-IFN remain unclear.