Marta Gómez

Myelofibrosis with myeloid metaplasia following essential thrombocythaemia: actuarial probability, presenting characteristics and evolution in a series of 195 patients

Summary. Myelofibrotic transformation is a known complication of essential thrombocythaemia (ET), but information on its incidence, presenting features and evolution is scarce. In a series of 195 patients with ET followed for a median of 7·2 years (range: 1·9–24), evolution into myelofibrosis with myeloid metaplasia (MMM) occurred in 13 cases, a median of 8 years (range: 3·6–20·2) from diagnosis. The actuarial probability of this complication was 2·7% (95% CI: 2·4–2·9) at 5 years, 8·3% (95% CI: 7·8–8·9) at 10 years, and 15·3% (95% CI: 6·1–24·5) at 15 years. Four patients had not been treated before developing MMM. The main features indicating this condition were the appearance of immature myeloid precursors in the peripheral blood, a decrease in the Hb value not related to treatment and increased serum lactate dehydrogenase levels, followed by a progressive decrease in the platelet count, increasing leucocytosis and progressive splenomegaly. No patient had constitutional symptoms, and none of five evaluable cases showed chromosome abnormalities in bone marrow or unstimulated blood. After a median the myelofibrotic transformation, three patients have died and four have not required treatment for MMM as yet.

Pfkfb3 is transcriptionally upregulated in diabetic mouse liver through proliferative signals.

The ubiquitous isoform of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (uPFK‐2), a product of the Pfkfb3 gene, plays a crucial role in the control of glycolytic flux. In this study, we demonstrate that Pfkfb3 gene expression is increased in streptozotocin‐induced diabetic mouse liver. The Pfkfb3/‐3566 promoter construct linked to the luciferase reporter gene was delivered to the liver via hydrodynamic gene transfer. This promoter was upregulated in streptozotocin‐induced diabetic mouse liver compared with transfected healthy cohorts. In addition, increases were observed in Pfkfb3 mRNA and uPFK‐2 protein levels, and intrahepatic fructose‐2,6‐bisphosphate concentration. During streptozotocin‐induced diabetes, phosphorylation of both p38 mitogen‐activated protein kinase and Akt was detected, together with the overexpression of the proliferative markers cyclin D and E2F. These findings indicate that uPFK‐2 induction is coupled to enhanced hepatocyte proliferation in streptozotocin‐induced diabetic mouse liver. Expression decreased when hepatocytes were treated with either rapamycin or LY 294002. This shows that uPFK‐2 regulation is phosphoinositide 3‐kinase–Akt–mammalian target of rapamycin dependent. These results indicate that fructose‐2,6‐bisphosphate is essential to the maintenance of the glycolytic flux necessary for providing energy and biosynthetic precursors to dividing cells.

Limitations of Gallium-67 SPECT in histological transformation of chronic lymphocytic leukaemia: an analysis of 13 patients with clinically suspected Richter's syndrome

Summary.  Gallium‐67 single photon emission computerized tomography (Ga‐67 SPECT) was performed in 13 chronic lymphocytic leukaemia (CLL) patients suspected of evolution into diffuse large B‐cell lymphoma (DLCL) or Richters syndrome (RS). Six positive and nine negative Ga‐67 SPECTs were observed. Ten patients were biopsied (five in each group). DLCL was not detected in any Ga‐67‐positive patient, including those in whom Ga‐67‐positive areas were biopsied. The only case of DLCL was demonstrated in a Ga‐67‐negative patient. The tumoral proliferative index (Ki67 antigen expression) was moderate and similar in both groups of patients. These results illustrate the limitations of Ga‐67 SPECT in identifying RS.

Results of treatment with azacitidine in patients aged ≥ 75 years included in the Spanish Registry of Myelodysplastic Syndromes

Abstract The tolerability of azacitidine (AZA) allows its administration in elderly patients. The objective of this study was to analyze the clinical and biological characteristics, transfusion independence (TI), overall survival (OS) and toxicity in a series of 107 patients ≥ 75 years of age from the Spanish Registry of Myelodysplastic Syndromes (MDS) treated with AZA. The median age (range) was 78 (75–90) years. According to the World Health Organization (WHO) classification, 86/102 (84%) had MDS, 10/102 (10%) had mixed myeloproferative/myelodysplastic disorder and 6/102 (6%) had acute myeloblastic leukemia. Regarding MDS by the International Prognostic Scoring System on initiation of AZA, 38/84 (45%) were low–intermediate-1 risk and 46/84 (55%) were intermediate-2–high risk. Ninety-five patients (89%) were red blood cell or platelet transfusion dependent. The AZA schedule was 5-0-0 in 39/106 (37%) patients, 5-2-2 in 36/106 (34%) patients and 7 consecutive days in 31/106 (29%) patients. The median number of cycles administered was 8 (range, 1–30). Thirty-eight out of 94 (40%) patients achieved TI. Median OS (95% confidence interval [CI]) was significantly better in patients achieving TI (n = 38) compared to patients who did not (n = 56) (22 [20.1–23.9] months vs. 11.1 [4.8–17.5] months, p = 0.001). No significant differences were observed in TI rate and OS among the three different schedules. With a median follow-up of 14 (min–max, 1–50) months, the median OS (95% CI) of the 107 patients was 18 (12–23) months and the probability of OS (95% CI) at 2 years was 34% (22–46%). Cycles were delayed in 31/106 (29%) patients and 47/101 patients (47%) were hospitalized for infection. These results show that treatment with AZA was feasible and effective in this elderly population, with 40% achieving TI, having a better OS than patients not achieving it. The schedule of AZA administration did not affect efficacy and toxicity.

Switches in 6-phosphofructo-2-kinase isoenzyme expression during rat sperm maturation.

Here we analyzed Pfkfb3 and Pfkfb4 gene expression in rat testis development, isolated testicular cells and spermatozoa. Real time RT-PCR analysis during testis development showed the maximum expression of Pfkfb3 in pre-puber samples and of Pfkfb4 in adult samples. Western blot analysis showed that uPFK-2 protein, a product of Pfkfb3 gene, was present in all the cell types forming the seminiferous epithelium (Sertoli, interstitial and spermatogenic cells). In contrast, tPFK-2, a product of Pfkfb4 gene, was restricted to spermatogenic cells. Confocal analyses by indirect immunofluorescence also corroborated this expression pattern. Immunoblotting studies of isolated spermatozoa demonstrated the presence of uPFK-2 only in immature sperm and once spermatozoa became fully functional this isozyme was replaced by the testicular isozyme tPFK-2. Moreover, immunostaining confirmed that tPFK-2 was localized mainly in the acrosomal region of the sperm head and in the mid-piece of the flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found.

Characterization of a new liver- and kidney-specific pfkfb3 isozyme that is downregulated by cell proliferation and dedifferentiation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P(2)), a signalling molecule that controls the balance between glycolysis and gluconeogenesis in several cell types. Four genes, designated Pfkfb1-4, code several PFK-2 isozymes that differ in their kinetic properties, molecular masses, and regulation by protein kinases. In rat tissues, Pfkfb3 gene accounts for eight splice variants and two of them, ubiquitous and inducible PFK-2 isozymes, have been extensively studied and related to cell proliferation and tumour metabolism. Here, we characterize a new kidney- and liver-specific Pfkfb3 isozyme, a product of the RB2K3 splice variant, and demonstrate that its expression, in primary cultured hepatocytes, depends on hepatic cell proliferation and dedifferentiation. In parallel, our results provide further evidence that ubiquitous PFK-2 is a crucial isozyme in supporting growing and proliferant cell metabolism.

Specific expression of pfkfb4 gene in spermatogonia germ cells and analysis of its 5′‐flanking region

The results presented demonstrate the expression of pfkfb4 gene in adult testis and in a mouse spermatogonia germ cell line (GC‐1spg). The genomic organization of the human pfkfb4 gene shows the existence of 14 exons and 13 introns, spanning 45 kb. A detailed analysis of the 5′‐flanking region by transient transfection assays with different 5′‐deletion promoter constructs in GC‐1spg and mouse sertoli cells (TM‐4), allows us to define the minimal promoter unit, containing several GC‐rich and ETF sequences along the first −141 nucleotides involved in basal expression. This gene is activated by serum and chemical hypoxia (CoCl2 treatment) whereas β‐estradiol decreases its expression.

Sertoli-secreted FGF-2 induces PFKFB4 isozyme expression in mouse spermatogenic cells by activation of the MEK/ERK/CREB pathway

Sertoli cells play a central role in the control and maintenance of spermatogenesis by secreting growth factors, in response to hormonal stimulation, that participate in the paracrine regulation of this process. In this study, we investigated how the hormonal regulation of spermatogenesis modulates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) isozyme expression in two mouse spermatogenic cell lines, GC-1 spg and GC-2 spd (ts). For this purpose, TM4 Sertoli cells were used to obtain conditioned medium that was treated or not with dihydrotestosterone for 2 days [dihydrotestosterone conditioned medium (TCM) and basal conditioned medium (BCM), respectively]. We observed an increase in the expression of PFKFB4 along with a decrease in PFKFB3 in spermatogenic cell lines treated with TCM. These effects were inhibited by the antiandrogen drug flutamide and by heat-inactivated TCM, indicating the protein nature of the TCM mediator and its dependence on Sertoli cell stimulation by dihydrotestosterone. In addition, adult rat testes treated with the GnRH antagonist Degarelix exhibited a reduction in the expression of PFKFB4 in germ cells. Addition of exogenous FGF-2 mimicked the changes in the Pfkfb gene expression, whereas neutralizing antibodies against FGF-2 abolished them. Interestingly, similar effects on Pfkfb gene expression were observed using different MAPK inhibitors (U-0126, PD-98059, and H-89). Luciferase analysis of Pfkfb4 promoter constructs demonstrated that a putative CRE-binding sequence located at -1,463 relative to the transcription start site is required to control Pfkfb4 gene expression after TCM treatment. Pulldown assays showed the binding of the CREB transcription factor to this site. Altogether, these results show how the paracrine regulation orchestrated by Sertoli cells in response to testosterone controls glycolysis in germ cells.

Allogenic stem cell transplantation as salvage therapy for patients relapsing after autologous transplantation: experience from a single institution

The prognosis of patients relapsing after an autologous transplant (autoSCT) is very poor. Allogenic stem cell transplantation (alloSCT) offers the possibility of curing some of these patients, at the cost, however, of a high transplant related mortality (TRM). The aim of this study was to analyze the outcome of 14 consecutive patients with hematologic malignancies, from a single institution, who underwent alloSCT for progressive disease after autoSCT. Patients had relapsed at a median of 11.5 months (range 2-72) after autoSCT and they underwent alloSCT at a median of 25.5 months (range 7-73) from the first transplant. Ten patients received HLA-identical related peripheral blood progenitor cells, three patients underwent matched-unrelated donor marrow transplants, and one patient received a mismatched related transplant. Conditioning regimens consisted of total body irradiation plus cyclophosphamide (n=5) or melphalan (n=1), or high-dose combination chemotherapy (n=8). Cyclosporin A and methotrexate were administered as graft-versus-host disease (GVHD) prophylaxis. Eight patients (57%) developed grade II-IV acute GVHD. All evaluable patients (n=6) presented extensive chronic GVHD. Overall survival at 1 year was 16% (median 3.5 months, 95% CI 0.7-10.3). Ten patients (71%) died from transplant related complications at a median of 3.5 months (range 0.7-11). Only one patient died of recurrent disease. Three patients remain alive and in complete remission at the time of this report (4, 20 and 20 months, respectively). In conclusion, alloSCT offers the possibility of a sustained control of the disease in some patients who relapse after an autoSCT. However, the procedure is associated with a high transplant-related mortality. Better results might be obtained by carefully selecting patients and by reducing the intensity of the preparative regimen.