Prospective identification of high-risk polycythemia vera patients based on JAK2(V617F) allele burden.

The aim of this study was to determine whether the burden of JAK2(V617F) allele correlated with major clinical outcomes in patients with polycythemia vera (PV). To this end, we determined JAK2 mutant allele levels in granulocytes of 173 PV patients at diagnosis. The mean (+/-s.d.) mutant allele burden was 52% (+/-29); 32 patients (18%) had greater than 75% mutant allele. The burden of JAK2(V617F) allele correlated with measurements of stimulated erythropoiesis (higher hematocrit, lower mean cell volume, serum ferritin and erythropoietin levels) and myelopoiesis (higher white cell count, neutrophil count and serum lactate dehydrogenase) and with markers of neutrophil activation (elevated leukocyte alkaline phosphatase and PRV-1 expression). As compared to those with less than 25% mutant allele, patients harboring greater than 75% JAK2(V617F) allele were at higher relative risk (RR) of presenting larger spleen (RR 4.7; P<0.001) or suffering from pruritus (RR 3.1; P<0.001). In these patients, the risk of requiring chemotherapy (RR 1.8; P=0.001) or developing major cardiovascular events (RR 7.1; P=0.003) during follow up were significantly increased. We conclude that a burden of JAK2(V617F) allele greater than 75% at diagnosis points to PV patients with high-risk disease.

Recombinant human erythropoietin for treatment of myelodysplastic syndromes.

Nine patients with myelodysplastic syndromes and one patient with agnogenic myeloid metaplasia have been treated with recombinant human erythropoietin (rhEpo), at the dose of 150 U/kg/day. Although serum Epo levels were correlated with hemoglobin concentrations in the whole population of patients, they clearly appeared inadequate in some instances, if compared to those of a group of control subjects with iron deficiency anemia. Moreover, no correlation was found between serum Epo and reticulocytes. Six patients showed a partial or complete response to the treatment and the outcome was not correlated with the pre-therapy serum Epo levels; however, serum Epo was less than 100 mU/ml in three of four patients who achieved a complete response. The mechanism(s) by which Epo stimulated erythrocyte production in myelodysplastic patients is unclear, because the number of both the reticulocytes and erythroid progenitors remained unchanged during and at the conclusion of a three months' therapy. Further studies are needed to better define the optimal dosage required to correct anemia in myelodysplastic syndromes, and to clarify rhEpo mechanism of action in these diseases.

Mixed acute leukemia with genotypic lineage switch: a case report.

Morphologically well classifiable leukemias can reveal a mixed phenotype. A case of acute myeloid leukemia (CD13, CD33, CD14, CD11b) which at presentation showed a co-expression of B-lymphoid markers (CD19, CD10, CD20), at the time of the first relapse revealed a morphologic, phenotypic and genotypic switch of the blasts to a purely lymphoid form. Analysis of the immunoglobulin (Ig) H chain locus and of the T-cell receptor (TCR) genes showed at diagnosis a germline configuration of the IgH, TCR beta and tau genes, and a deletion of the TCR delta gene at the second chromosome. At relapse, monoclonal rearrangements of the IgH, TCR tau, and TCR delta were detected. At a subsequent relapse, the blasts re-expressed myeloid morphologic features and myeloid-associated antigens, while they retained the same rearranged configuration of the IgH and TCR beta and delta genes. The TCR delta gene configuration, which links each phase of the disease, may represent an early pathogenetic event and makes the emergence of a second malignancy unlikely. Each phenotypic change occurred after anti-myeloid and anti-lymphoid oriented chemotherapy. The close correlation between the progressive acquisition of different phenotypes and the switch at the genomic level represent the peculiar features of this unusual case.

Constitutive expression of a megakaryocytic functional property by murine erythroleukemia (Friend) cells: the incorporation of serotonin.

Murine Friend-derived erythroleukemia cells (MEL) are generally believed to be unipotential progenitors inducible to terminal erythroid differentiation. However, we found that MEL can constitutively incorporate significant amounts of radiolabeled serotonin ([3H]5-HT). Because this process is typical of cells belonging to the megakaryocytic lineage, we investigated the significance and mechanisms of 5-HT incorporation in the MEL system. We observed that: 1) normal murine erythroid cells and erythroid progenitors do not incorporate [3H]5-HT, as well as normal murine myeloid cells and the human myeloid cell line HL-60; on the other hand, the human erythroleukemia cell lines K562 and HEL, which have been shown to constitutively express megakaryocytic features, were able to incorporate [3H]5-HT; 2) MEL incorporated 5-HT by an active and saturable mechanism, dependent on temperature and sodium concentration in the medium; and 3) 5-HT uptake was very rapid. Moreover, because about 65% of cell-associated radioactivity was no longer displaced by the cold substrate, we assumed it to represent "true" cytoplasmic internalization. Finally, 5-HT incorporation by MEL was inhibited by clomipramine, ouabain, and reserpine, which are known inhibitors of 5-HT uptake in platelets. The commitment of MEL to terminal erythroid differentiation by hexamethylene bisacetamide or dimethyl sulfoxide greatly reduced the capacity to incorporate [3H]5-HT. These results seem to suggest that the MEL system, although mainly erythroid as regards its differentiation capability, constitutively expresses features of the megakaryocytic lineage, possibly disclosed by the ability to incorporate 5-HT. This hypothesis was further supported by the findings that 30%-40% of uninduced MEL were labeled by a polyclonal antibody raised against murine platelets that selectively recognized megakaryocytes in murine bone marrow smears.

Serotonin incorporation as a marker of murine megakaryocyte proliferation in liquid cultures.

We have developed a new in vitro method for the quantitation of murine megakaryocyte proliferation that is based on the unique property of megakaryocytes to incorporate and store [14C]serotonin in cytoplasmic dense granules. The specificity of the assay was demonstrated by autoradiography of whole bone marrow cell suspensions, which showed evidence of grain accumulation only in megakaryocytes. Bone marrow cells were cultured in liquid cultures in the presence of a stimulator of megakaryocyte growth before the addition of 2.5 microM [14C]serotonin. The amount of serotonin incorporated in cells was evaluated after 3 h. Radioactivity peaked at days 6 and 7 and remained high until day 10; there was a linear relationship between the incorporation of serotonin and the number of cells plated. A dose-response curve between the incorporation of serotonin and the concentration of pokeweed mitogen spleen-conditioned medium (PWM-SCM) was observed, with inhibitory effects becoming predominant at the highest concentrations. The proliferation of megakaryocyte progenitors was also stimulated by partially purified interleukin 3, whereas both human recombinant erythropoietin and human recombinant granulocyte colony-stimulating factor (rG-CSF) failed to modify the incorporation of serotonin in comparison with unstimulated cultures. Finally, in parallel experiments we observed a significant correlation between the number of megakaryocytic colonies grown in agar and the radioactivity in liquid cultures. The method described herein is reproducible, sensitive, and easy to perform; it should be useful for the study and purification of factors affecting megakaryocyte proliferation.

The risk of second malignant tumors and its consequences for the overall survival of Hodgkin's disease patients and for the choice of their treatment at presentation: analysis of a series of 1524 cases consecutively treated at the Florence University Hospital.

PURPOSE: To quantify the incidence of second malignant tumors (SMT) as a whole and that of second "solid" tumors (SST) and leukemia (L) in a large series of 1524 Hodgkin's disease (HD) patients (pts) treated at the Florence University Hospital (UFH); to define the clinical and therapeutic features possibly related with SMT occurrence; to evaluate the consequences of SMT for the overall survival of the series studied and for the choice of the treatment of HD at presentation. METHODS AND MATERIALS: From 1960 to 1991, 1524 pts with HD, Clinical Stage (CS) I--IV have been treated at the UFH. Overall treatment consisted of radiation alone (RT, 36%), chemotherapy alone (CHT, 21%), or both (RT + CHT, 43%). The cumulative probability (CP) of SMT, SST, and L was calculated for the whole series and for the different clinical and therapeutic subgroups, and the results compared with uni- and multivariate analysis ("internal" comparison, IC). Standardized incidence ratios (SIR) for different SMT types (estimated on the basis of gender, age, period specific incidence rates of the general population) have been also calculated ("external" comparison, EC). The impact of the SMT-related mortality on the survival of the entire series has been estimated. RESULTS: A 14.9% 20-year CP of SMT was registered, along with a SIR of 2.04 (95% confidence interval [CI]: 1.2--2.5). Both IC and EC showed a statistically significant relationship between L incidence and treatment with CHT, alone or in combination with RT. A significant excess of breast cancers has been observed in RT-treated patients with longer follow-up (SIR, 2.9); an excess of other common SST (lung, non-Hodgkin's lymphomas) is evident in pts treated with either RT, RT + CHT, or CHT. The actuarial long-term survival of the series would have been better of about 3%, in absence of the SMT mortality possibly due to HD treatment, which is almost equally divided between patients treated with RT alone, CHT alone, and RT + CHT. CONCLUSIONS: SMT represent an important late event in HD long-term survivors. The relationship between L and treatment with CHT seems to be the most clearly defined. The effect of SMT on the survival of the entire series, although not negligible, does not seem to justify by itself substantial alterations in the current standards for the treatment of HD at presentation.

Hepatitis-free interval after clotting factor therapy in first infused haemophiliacs.

Post-infusion hepatitis is known to occur very frequently in haemophiliacs after treatment with unheated commercial clotting factor concentrates, obtained from large plasma donation pool. On the contrary, single-donor cryoprecipitate is likely to carry a lower risk of transmitting hepatitis. To evaluate this hypothesis, we retrospectively reviewed the medical records of 25 first infused haemophiliacs (from 1981 to 1984) treated with unheated commercial clotting factor concentrates (n = 19) or cryoprecipitate (n = 6). The hepatitis-free interval after the beginning of therapy was expressed as exposure days. The end point of each patient, i.e. the hepatitis occurrence, was defined as an increase of amino-transferases (ALT and AST) and/or the seroconversion of HBV-markers, which were checked every three months. The life-table method and log-rank test showed that cryoprecipitates had a significantly longer hepatitis-free interval (p = 0.0131, log-rank test) and a lower risk of transmitting hepatitis (p = 0.01-0.05, life-table method) than the commercial concentrates. However, the safety of cryoprecipitate therapy was shown to cover only a few exposure days, and so the real advantage of this product depends on the bleeding frequency of the patient concerned. We believe that these methods and our findings may be useful to assess and compare the safety of the new "heat-treated" clotting factor concentrates.

Differential sensitivity to (dl)-5-methyltetrahydrofolate of normal CFU-GM and HL-60 cells.

We studied the effect of (dl)-5-methyltetrahydrofolate on clonogenic growth of HL-60 cells in comparison with human normal CFU-GM. Seven normal bone marrow samples were tested for CFU-GM assay with or without (dl)-mTHF at concentrations ranging from 1.25 X 10(-4) to 5 X 10(-4) M. (dl)-mTHF only slightly affected CFU-GM formation, while, at the same concentrations, it showed a dose related inhibition of HL-60 colony formation, up to a complete arrest of growth at the doses of 5 X 10(-4) and 1 X 10(-3) M. The same impairment of proliferation was observed in liquid culture. These results are in keeping with reported observations, describing a different membrane system mediating "folate" transport in normal and leukemic cells.

Erythropoietin upregulates the expression of its own receptor in TF-1 cell line.

In the erythroleukemia cell line TF-1, recombinant human erythropoietin (rHEpo), but not c-kit ligand, enhanced the number of cells expressing the erythropoietin receptor (EpoR), as measured by flow-cytometric analysis of binding of the biotin-labeled Epo. Moreover, 125I-Epo binding and Scatchard analyses, indicated that TF-1 cells, maintained in standard conditions with IL-3, and those stimulated with c-kit ligand, bear a single class of EpoR. On the other hand, cells cultured in the presence of rHEpo had a higher number of receptors than IL-3 or c-kit ligand-stimulated cells, and had two binding sites with different affinities for the ligand. EpoR mRNA expression was higher in cells exposed to rHEpo than in IL-3 or c-kit-stimulated cells. This difference may have been dependent on either a higher level of transcription or an increased stability of mRNA. The observed changes of EpoR in rHEpo-stimulated TF-1 cell line could cooperate, together with the alteration of the gene (3' end deletion), in the occurrence of the erythroleukemic process. Changes induced in EpoR by rHEpo were not accompanied by an increase in the expression of glycophorin A or globin chain mRNAs. This may suggest that rHEpo is unable to induce erythroid differentiation in TF-1 cells. The results also indicate that this cell line could be a model for the investigation of the role of transcription factor(s) in the expression of EpoR, and for the study of the mechanism(s) underlying the changes in the number and affinity of the cell receptors.

6p+ and 9q- in two chromosomally distinct clones occurring in a case of myelodysplastic syndrome evolving to acute nonlymphocytic leukemia.

Different and unrelated chromosome changes were found to occur in a patient with a myelodysplastic syndrome with rapid evolution to acute nonlymphocytic leukemia. A 6p anomaly was found during the chronic phase and a del(9q) characterized the cells in the leukemic phase. Deletions with a breakpoint in 9q31 appeared to be associated with more aggressive disease.

Continuous-infusion cyclophosphamide in combination with teniposide and dexamethasone in refractory myeloma.

Forty-three consecutive patients with refractory myeloma, median age 60, received monthly courses of teniposide 30 mg/m2 i.v. on days 1-2, dexamethasone 40 mg i.v. on days 1-7 and cyclophosphamide 200 mg/m2 by continuous i.v. infusion for seven days. Major response (decrease > 50% of M-protein) was achieved in 18 of 37 evaluable patients and minor response in 9, with an overall response rate of 73%. Response was irrespective of disease status, time from diagnosis and previous treatments, while beta 2 microglobulin > 6 mg/l was a powerful prognostic factor. All patients experienced transient granulocytopenia but extramedullary toxicity was negligible. Median survival of the whole group is 20 months, with 74% of responding patients projected to be alive at 30 months. In refractory myeloma cyclophosphamide appears to be more active when given by continuous infusion.

Combined surgery and chemotherapy in primary gastric non-Hodgkin's lymphoma: a retrospective study in sixty-six patients.

Sixty-six consecutive patients with primary gastric non-Hodgkin's lymphoma are reported. All patients underwent surgery which consisted of radical resection in 23 patients (36%) and partial or palliative excision in the remaining 43 cases (36 and 7 respectively). Three patients died before starting chemotherapy, two refused the treatment and 61 completed the postoperative chemotherapeutic programme. We analysed this group of patients in order to assess the efficacy of chemotherapy following surgery. Chemotherapy included either CVP or the original protocols from our institution. Excluding patients who underwent radical resection, postoperative chemotherapy induced complete remission in 87% of the remaining 39 patients. After a median follow-up of 84 months (range 6-216), the 10-year cause-specific survival was 90% with a stable curve plateau after about 25 months. The survival was only influenced by response to therapy (p < 0.0001). The disease-free survival for patients who were not radically resected was 93%. We encountered only two relapses after 15 and 32 months. One of these was local and the other systemic. Our results indicate that chemotherapy following surgery induces long-term remission and survival in primary gastric lymphoma and in particular improves remission and survival, in stage II. In our opinion, surgery may also be fundamental for the treatment of gastric lymphoma in the majority of cases.

A new protocol (MiCEP) for the treatment of intermediate or high-grade non-Hodgkin's lymphoma in the elderly.

Age has proved to be an important prognostic factor in patients with advanced non-Hodgkin lymphoma (NHL) and these patients require intensive and extensive therapy. Dose-reduction and therapy attenuation have reduced treatment-related toxicity, but have also decreased therapeutic efficacy. Between January 1990 and December 1992, 41 previously untreated patients, 65 years with stage 2-4 intermediate- or high-grade NHL were treated with a new therapeutic scheme which included Mitoxantrone, Etoposide, Cyclophosphamide and Prednisone (MiCEP). Twenty-eight patients achieved a complete remission, ten patients partial remission (overall response rate of 93%) and two cases were resistant. The overall survival was 66% with a median follow-up of 24 months from diagnosis: three patients relapsed after a median period of 7 months. The relapse-free survival was 92% after a median follow-up of 18 months. Blood and other organ toxicity was acceptable and 12% of patients experienced a grade 4 (WHO) neutropenia. In conclusion, MiCEP was effective in inducing a good remission rate with moderate toxic effects in elderly patients with intermediate- or high-grade NHL and appears to be a useful combination to use in this group of patients.

Multispectral imaging autofluorescence microscopy for the analysis of lymph-node tissues.

Although histochemical and immunohistochemical methods are the standard procedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the introduction of tissue autofluorescence analyses. We used microspectrofluorometry and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of different origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interference filters (50 nm bandwidth) peaked at 450, 550 and 658 nm. Monochrome images were combined together in a single red-green-blue color image. Most of the fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as from reduced form of intracellular nicotinamide adenine dinucleotide (phosphate). Autofluorescence imaging shows morphological differences between neoplastic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated by high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, MIAM permits to discriminate between non-neoplastic and neoplastic tissues on the basis of their autofluorescence pattern. Multispectral imaging of tissue autofluorescence may present some advantages with respect to standard histochemical microscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon specimen resection; and (3) supplies a representation of the biological structure organization linked to endogenous fluorophores.

Natural fluorescence of white blood cells: spectroscopic and imaging study.

Autofluorescence has been proved to be an intrinsic parameter of biological substrates that may aid in both the characterization of the physiological state and the discrimination of pathological from normal conditions of cells, tissues and organs. In this work, the fluorescence properties of human white blood cells have been studied in suspension and on single cells at microscopy. The results indicate that suspensions of agranulocytes and granulocytes differ in the amplitude of the fluorescence signal on excitation at wavelengths in the range 250-370 nm. The differences are particularly enhanced when excitation is performed in the 250-265 nm range. Microspectrofluorometric analysis, performed on single cells, allows several leukocyte families to be characterized. Lymphocytes, monocytes, neutrophils and eosinophils can be distinguished according to the intensity and spectral shape of the autofluorescence emission in the visible range from 440 to 580 nm. Both the nature and extent of the differences change when the excitation wavelength is moved from 366 to 436 nm. Differences in the intrinsic metabolic engagement, rather than in the cell dimensions, seem to be responsible for the differences observed between the leukocyte populations. The results identify interesting perspectives for autofluorescence as a discriminating parameter in the differential counting of human white blood cells.