Plasma estrone sulfate concentrations and genetic variation at the CYP19A1 locus in postmenopausal women with early breast cancer treated with letrozole.

Estrogen synthesis suppression induced by aromatase inhibitors in breast cancer (BC) patients may be affected by single nucleotide polymorphisms (SNPs) of the gene encoding aromatase enzyme, CYP19A1. We assessed the association between plasma estrone sulfate (ES), letrozole treatment, and four SNPs of CYP19A1 gene (rs10046 C>T, rs4646 G>T, rs749292 C>T, rs727479 T>G) which seem to be related to circulating estrogen levels. Patients were enrolled into a prospective, Italian multi-center clinical trial (Gruppo Italiano Mammella, GIM-5) testing the association of CYP19A1 SNPs with the efficacy of letrozole adjuvant therapy, in postmenopausal early BC patients. SNPs were identified from peripheral blood cell DNA. Plasma ES concentrations were evaluated by Radio Immuno Assay. Blood samples were obtained immediately before letrozole therapy (N = 204), at 6-weeks (N = 178), 6 (N = 152) and 12-months (N = 136) during treatment. Medians (IQR) of ES were 160 pg/mL (85-274) at baseline, 35 pg/mL (12-64) at 6-weeks, 29 pg/mL (17-48) at 6 months and 25 pg/mL (8-46) after 12 months treatment. No statistically significant association was evident between polymorphisms and ES circulating levels during letrozole therapy. Letrozole suppression of the aromatase enzyme function is not affected by polymorphisms of CYP19A1 gene in postmenopausal BC patients.

Killer immunoglobulin-like receptors (KIR) and their HLA-ligands in Italian paroxysmal nocturnal haemoglobinuria (PNH) patients.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic disorder characterized by expansion of phosphatidylinositol glycan-A-defective progenitor(s). Immune-dependent mechanisms, likely involving a deranged T cell-dependent autoimmune response, have been consistently associated with the selection/dominance of PNH precursors. Natural killer (NK) lymphocytes might participate in PNH pathogenesis, but their role is still controversial. NK activity is dependent on the balance between activating and inhibiting signals. Key component in such regulatory network is represented by killer immunoglobulin-like receptors (KIR). KIR are also involved in the regulation of adaptive cytotoxic T cell response and associated with autoimmunity. This study investigated on the frequency of KIR genes and their known human leukocyte antigen (HLA) ligands in 53 PNH Italian patients. We observed increased frequency of genotypes characterized by ≤2 activating KIR as well as by the presence of an inhibitory/activating gene ratio ≥3.5. In addition, an increased matching between KIR-3DL1 and its ligand HLA-Bw4 was found. These genotypes might be associated with lower NK-dependent recognition of stress-related self molecules; this is conceivable with the hypothesis that an increased availability of specific T cell targets, not cleared by NK cells, could be involved in PNH pathogenesis. These data may provide new insights into autoimmune PNH pathogenesis.

Overexpression of ETV4 is oncogenic in prostate cells through promotion of both cell proliferation and epithelial to mesenchymal transition.

The discovery of translocations that involve one of the genes of the ETS family (ERG, ETV1, ETV4 and ETV5) has been a major advance in understanding the molecular basis of prostate cancer (PC). Each one of these translocations results in deregulated expression of one of the ETS proteins. Here, we focus on the mechanism whereby overexpression of the ETV4 gene mediates oncogenesis in the prostate. By siRNA technology, we show that ETV4 inhibition in the PC3 cancer cell line reduces not only cell mobility and anchorage-independent growth, but also cell proliferation, cell cycle progression and tumor growth in a xenograft model. Conversely, ETV4 overexpression in the nonmalignant human prostate cell line (RWPE) increases anchorage-independent growth, cell mobility and cell proliferation, which is probably mediated by downregulation of p21, producing accelerated progression through the cell cycle. ETV4 overexpression is associated with changes in the pattern of E-cadherin and N-cadherin expression; the cells also become spindle-shaped, and these changes are characteristic of the so-called epithelial to mesenchymal transition (EMT). In RWPE cells overexpressing ETV4 EMT results from a marked increase in EMT-specific transcription factors such as TWIST1, SLUG1, ZEB1 and ZEB2. Thus, whereas ETV4 shares with the other ETS proteins (ERG, ETV5 and ETV1) a major role in invasiveness and cell migration, it emerges as unique in that it increases at the same time also the rate of proliferation of PC cells. Considering the wide spectrum in the clinical course of patients with PC, it may be highly relevant that ETV4 is capable of inducing most and perhaps all of the features that make a tumor aggressive.

Association of -318 C/T and +49 A/G cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms with a clinical subset of Italian patients with systemic sclerosis.

Systemic sclerosis (SSc) is a complex and heterogeneous autoimmune disorder with a multi-factorial pathogenesis. Like other autoimmune disorders, the possible role of specific cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms in predisposing to SSc has been hypothesized, but it remains controversial. CTLA-4 promoter (-318C/T) and exon 1 (+49 A/G) polymorphisms have been analysed in 43 Italian females with SSc and in 93 unrelated matched healthy controls by a newly designed tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method. No significant association has been found with either polymorphisms.Nevertheless, SSc patients without concomitant Hashimoto's thyroiditis (HT) were carrying both the -318T allele (P = 0.031) and the +49 G allele (P = 0.076) more frequently than SSc patients with HT [defined by positivity for anti-thyroperoxidase (TPO) and anti-thyroglobulin (TGA) autoantibodies] than controls. Haplotype analysis confirms this association (P = 0.028), and suggests the predominant role of the -318T, whereas that of the +49 G, if any, seems weak. Thus, in Italian SSc patients the CTLA-4 -318C/T promoter polymorphism appears to be associated with the susceptibility to develop SSc without thyroid involvement. Larger studies are needed to confirm these findings and to clarify whether the -318C/T polymorphism is the functional responsible or whether it reflects the presence of another linked genetic element in the same chromosomal region.

Dynamics of hematopoiesis in paroxysmal nocturnal hemoglobinuria (PNH): no evidence for intrinsic growth advantage of PNH clones.

PNH is characterized by expansion of one or more stem cell clones with a PIG-A mutation, which causes a severe deficiency in the expression of glycosylphosphatidylinositol (GPI)-anchored proteins. There is evidence that the expansion of PIG-A mutant clones is concomitant with negative selection against PIG-A wild-type stem cells by an aplastic marrow environment. We studied 36 patients longitudinally by serial flow cytometry, and we determined the proportion of PNH red cells and granulocytes over a period of 1-6 years. We observed expansion of the PNH blood cell population(s) (at a rate of over 5% per year) in 12 out of 36 patients; in all other patients the PNH cell population either regressed or remained stable. The dynamics of the PNH cell population could not be predicted by clinical or hematologic parameters at presentation. These data indicate that in most cases the PNH cell expansion has already run its course by the time of diagnosis. In addition, since in most cases no further expansion takes place, we can infer that the tendency to overgrow normal cells is not an intrinsic property of the PNH clone.

Cytogenetic and morphological abnormalities in paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by the expansion of a haematopoietic stem cell clone with a PIG-A mutation (the PNH clone) in an environment in which normal stem cells are lost or failing: it has been hypothesized that this abnormal marrow environment provides a relative advantage to the PNH clone. In patients with PNH, generally, the karyotype of bone marrow cells has been reported to be normal, unlike in myelodysplastic syndrome (MDS), another clonal condition in which cytogenetic abnormalities are regarded as diagnostic. In a retrospective review of 46 patients with a PNH clone, we found a karyotypic abnormality in 11 (24%). Upon follow-up, the proportion of cells with abnormal karyotype decreased significantly in seven of these 11 patients. Abnormal morphological bone marrow features reminiscent of MDS were common in PNH, regardless of the karyotype. However, none of our patients developed excess blasts or leukaemia. We conclude that in patients with PNH cytogenetically abnormal clones are not necessarily malignant and may not be predictive of evolution to leukaemia.

Glucose 6-phosphate dehydrogenase expression is less prone to variegation when driven by its own promoter.

The ability to transfer permanently genes into mammalian cells makes retroviruses suitable vectors for the ultimate purpose of treating inherited genetic disease. However, expression of the retrovirally transferred genes is variable (position effect and expression variegation) because retroviruses are highly susceptible to the influence of the host genome sequences which flank the integration site. We have investigated this phenomenon with respect to the human housekeeping enzyme, glucose 6-phosphate dehydrogenase (hG6PD). We have constructed retroviral vectors in which the hG6PD cDNA is driven by either of two conventional retroviral promoters and enhancers from the Moloney Murine Leukemia Virus (MMLV) and the Myeloproliferative Sarcoma Virus (MPSV) long terminal repeats (LTR) or by the hG6PD own promoter replacing most of enhancer and promoter LTR (GRU5). We have compared the activity of retrovirally transferred hG6PD driven by these promoters after retroviral integration in bulk cultures and in individual clones of murine fibroblasts. The level of hG6PD expressed by the hG6PD promoter of GRU5-G6PD was significantly lower than that expressed by conventional retroviral vectors. However, analysis of the single copy clones showed less variation of expression with GRU5-G6PD (coefficient of variation, CV, 35.5%) than with conventional vectors (CV, 58.9%). Thus we have several vectors competent for reliable transfer and expression of hG6PD. The hG6PD promoter provides reproducible expression of hG6PD and limits the variability of expression. This decreased variability is important in order to help ensuring a consistent level of delivery of the needed gene product in future therapeutic protocols.

Association of clonal T-cell large granular lymphocyte disease and paroxysmal nocturnal haemoglobinuria (PNH): further evidence for a pathogenetic link between T cells, aplastic anaemia and PNH.

There is mounting evidence to suggest that T-cell-mediated suppression of haemopoiesis is a pathogenetic mechanism in three bone marrow failure syndromes: aplastic anaemia (AA), paroxysmal nocturnal haemoglobinuria (PNH) and myelodysplasia (MDS). T-cell microclones can be detected by sensitive polymerase chain reaction (PCR)-based methods in all three disorders. Recently, larger clonal populations of T-cell large granular lymphocytes (T-LGLs) have been observed in some patients with AA and MDS. Here, we report the development of a large clonal T-LGL population in a patient with bona fide PNH. In this patient, we defined part of the sequence of the T-cell receptor (TCR) beta-chain gene, and we have shown that the large T-LGL population emerged from a background of multiple smaller T-cell clones. Thus, T-LGL clones in AA, MDS and PNH probably expand as a result of antigenic stimulation. It is postulated that the antigen driving clonal T-cell proliferations in these disorders exists on haemopoietic stem cells.

Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer.

Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

Abnormal T-cell repertoire is consistent with immune process underlying the pathogenesis of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of the hematopoietic stem cell (HSC). Somatic mutations in the PIG-A gene result in the deficiency of several glycosylphosphatidylinositol-linked proteins from the surface of blood cells. This explains intravascular hemolysis but does not explain the mechanism of bone marrow failure that is almost invariably seen in PNH. In view of the close relationship between PNH and idiopathic aplastic anemia (IAA), it has been suggested that the 2 disorders might have a similar cellular pathogenesis, namely, that autoreactive T-cell clones are targeting HSCs. In this paper, we searched for abnormally expanded T-cell clones by size analysis of the complementarity-determining region 3 (CDR3) in the beta variable chain (BV) messenger RNA (mRNA) of the T-cell receptor (TCR) in 19 patients with PNH, in 7 multitransfused patients with hemoglobinopathy. and in 11 age-matched healthy individuals. We found a significantly higher degree of skewness in the TCR BV repertoire of patients with PNH, compared with controls (R(2) values 0.82 vs 0.91, P <.001). The mean frequency of skewed families per individual was increased by more than 2-fold in patients with PNH, compared with controls (28% +/- 19.6% vs 11.4% +/- 6%, P =.002). In addition, several TCR BV families were significantly more frequently skewed in patients with PNH than in controls. These findings provide experimental support for the concept that PNH, like IAA, has an immune pathogenesis. In addition, the identification of expanded T-cell clones by CDR3 size analysis will help to investigate the effect of HSC-specific T cells on normal and PNH HSCs.

Human mutations in glucose 6-phosphate dehydrogenase reflect evolutionary history.

Glucose 6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions. Inherited G6PD deficiency is associated with either episodic hemolytic anemia (triggered by fava beans or other agents) or life-long hemolytic anemia. We show here that an evolutionary analysis is a key to understanding the biology of a housekeeping gene. From the alignment of the amino acid (aa) sequence of 52 glucose 6-phosphate dehydrogenase (G6PD) species from 42 different organisms, we found a striking correlation between the aa replacements that cause G6PD deficiency in humans and the sequence conservation of G6PD: two-thirds of such replacements are in highly and moderately conserved (50-99%) aa; relatively few are in fully conserved aa (where they might be lethal) or in poorly conserved aa, where presumably they simply would not cause G6PD deficiency. This is consistent with the notion that all human mutants have residual enzyme activity and that null mutations are lethal at some stage of development. Comparing the distribution of mutations in a human housekeeping gene with evolutionary conservation is a useful tool for pinpointing amino acid residues important for the stability or the function of the corresponding protein. In view of the current explosive increase in full genome sequencing projects, this tool will become rapidly available for numerous other genes.

PNH cells are as sensitive to T-cell-mediated lysis as their normal counterparts: implications for the pathogenesis of paroxysmal nocturnal haemoglobinuria.

The mechanism responsible for the bone marrow failure that is almost invariable in paroxysmal nocturnal haemoglobinuria (PNH) is unknown. Based on the close association between PNH and idiopathic aplastic anaemia, a plausible pathogenetic model predicts that, in PNH, autoreactive T cells specific for haemopoietic stem cells (HSCs) cause depletion of normal HSCs, whereas PNH HSCs escape this T-cell-mediated attack. In this study, we addressed the hypothesis that PNH HSCs are resistant to the cytotoxic effect of T cells because they lack surface expression of one or more glycosylphosphatidylinositol (GPI)-linked molecules. We tested the sensitivity of normal and PNH Epstein-Barr virus (EBV)-transformed B-cell lymphoblastoid cell lines (BLCLs) to the cytotoxic effect of autologous EBV-specific T-cell lines and clones from a patient with PNH in an in vitro experimental system. We found that the PNH BLCLs were no less sensitive to T-cell-mediated cytotoxicity than non-PNH isogenic BLCLs, indicating that GPI-linked molecules on the surface of target cells are not required for killing by T cells. This suggests that the mechanism whereby PNH HSCs survive T-cell attack is not because of the lack of surface expression of one or more GPI-linked molecules. By implication, other mechanisms become more probable.

Acute promyelocytic leukemia after treatment for non-Hodgkin's lymphoma with drugs targeting topoisomerase II.

We report a patient who developed acute promyelocytic leukemia (APL) concomitantly with a second relapse of non-Hodgkin's lymphoma (NHL), intermediate grade, WF type E. At diagnosis and at first NHL relapse, the patient had received the same chemotherapy regimen, which included drugs targeting DNA topoisomerase II, i.e., etoposide (total dose 5,760 mg) and idarubicin (total dose 180 mg). Thirty-eight months after initial treatment, the patient showed pancytopenia associated with lymphoma recurrence. Bone marrow examination revealed the presence of atypical promyelocytes with Auer rods; cytogenetics showed t(15;17), and molecular analysis detected promyelocytic leukemia-retinoic acid receptor alpha rearrangement. APL reached complete remission after all trans retinoic acid therapy, whereas NHL did not respond to further chemotherapy. In the literature, five other patients developed APL after treatment for lymphoma, from a total of 59 patients developing sAPL after treatment for any type of neoplasia.

Fanconi's anemia cells are relatively resistant to H2O2-induced damage.

BACKGROUND AND OBJECTIVE: Fanconi's anemia (FA) is a rare autosomal recessive syndrome characterized by skeletal abnormalities, late onset bone marrow failure and susceptibility to neoplasias. Reduced defense against oxidative stress is thought to be one of the cell damaging mechanisms. We investigated in vitro the effects of oxidative stress on red blood cells (RBC) and on hematopoietic progenitor growth of normal donors and of FA patients. DESIGN AND METHODS: The effects of hydrogen peroxide (H2O2) on RBC and hematopoietic progenitors were studied in vitro by erythrophagocytosis assay and by hematopoietic progenitor colony assay, respectively. RESULTS: In an erythrophagocytosis assay using normal monocytes, RBC from nine FA patients showed increased binding index (defined as the percentage of monocytes with adherent or phagocytosed RBC) compared to that obtained with RBC from nine normal controls. Upon exposure to H2O2, the binding index of normal RBC increased, while that of FA RBC remained unchanged. In a set of different experiments, H2O2 treatment of peripheral blood mononuclear cells (PBMNC) caused a significant decrease of the number of colonies from circulating progenitor cells in all normal subjects; the inhibition was dose-dependent and direct as proven by using normal purified CD34+ cells. In nine FA patients colony assays from intact cells showed a decreased number of circulating progenitors as compared to normal subjects; however, H2O2 treatment of FA PBMNC did not cause any further decrease of the plating efficiency. INTERPRETATION AND CONCLUSIONS: Untreated FA cells behave as normal cells after exposure to the toxic effects of H2O2. However, since H2O2 exposure is inoffensive to circulating FA RBC and hematopoietic progenitors, it seems that a selection for cells resistant to further oxidative stress has taken place in the residual hematopoiesis of FA patients. We may surmise that the survival of cells that have suffered from oxidative damage may have increased the risk of their leukemic transformation.

Desferioxamine increases iron depletion and apoptosis induced by ara-C of human myeloid leukaemic cells.

We investigated whether changes in iron metabolism and the transferrin receptor (TRF-R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara-C). Treatment with 100 nM ara-C for 48h reduced thymidine uptake and increased the surface expression of the TRF-R on leukaemic blasts derived from 13/16 (81%) patients and on the HL-60 and U-937 cell lines. Whereas intracellular non-haem iron was strongly depleted 24 h after ara-C addition, TRF-R up-regulation and recovery of intracellular non-haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara-C and the iron chelator desferioxamine (DSF) on the growth of HL-60 and U-937 cells. We found that desferioxamine strongly potentiated the effects of ara-C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara-C and an iron chelator in terms of antileukaemic effects.

Etoposide and idarubicin in a modified CHOP-like regimen (VICED) for aggressive non-Hodgkin's lymphomas.

The superiority of intensive versus standard chemotherapy for aggressive (I: intermediate; H: high grade) NHL is still debated; increased antitumor activity may be counterbalanced by increased toxicity. We have designed a first-line five-drug regimen (vincristine, idarubicin, cyclophosphamide, etoposide and deflazacort), with the aim of potentiating the CHOP protocol without losing tolerability and ease of administration. Seventy-one patients (33% aged > or = 65) entered the study. CR was obtained in 66.7% of patients (I: 74%; H: 56%), PR in 19.7%: overall response rate was 86.4%. Six patients were resistant, two died during treatment. With a median follow up of two years, relapse has occurred in 14 patients (8 I, 6 H). At 3 years, overall survival was projected to be 62.5% (I 73.5%; H 31.4%), disease free survival 66% (I 71%, H 56.3%). No organ toxicity occurred. Myelosuppression was moderate, with a nadir on the 14th day. Febrile episodes occurred in 16% of courses, dose delay in 19% of courses; dose reduction in 3% of patients. No patient required hospitalization. G-CSF was only occasionally used. This regimen has shown a potent antitumor effect with an excellent tolerance, even in elderly patients.

High prevalence of hepatitis C virus infection in patients with B-cell lymphoproliferative disorders in Italy.

Starting from the observation that a number of consecutive patients with non-Hodgkin's lymphoma (NHL) resulted positive for hepatitis C virus (HCV) antibodies on routine testing, we set up a survey for HCV contact prevalence in all patients with lymphoproliferative disorders (LPD) followed in our institution. We searched for HCV antibodies by a third-generation ELISA technique, followed by a confirmation test (RIBA III); serum viral RNA and HCV genotype were investigated by a RT-PCR technique. We screened a total of 315 patients suffering from B-NHL (91), multiple myeloma (56), MGUS (48), chronic lymphocytic leukemia (57), Waldentrom's macroglobulinemia (13), Hodgkin's disease (HD)(43), and T-NHL (9). While only 1 of 52 patients with a non-B-LPD (HD or T-NHL) had signs of HCV contact (i.e., 1.9%, which is in the range of the normal population in the South of Italy), 59 of 263 patients with a B-LPD (22.4%) had HCV antibodies or RNA, or both, with no major differences among the various types of disorders, except for WM, in which the rate was higher (61.5%). The same prevalence was found for patients tested at diagnosis or during the follow-up, and in transfused or never-transfused patients. Only a few patients were aware of having a liver disease; one-half of HCV-positive patients never had transaminase increase. A review of data from Central and Northern Italy is included, showing similar findings; a report from Japan has confirmed such an association, while limited surveys in England have not revealed any correlation. These findings may have important biological and clinical implications.

Small bowel infarction by Aspergillus.

Primary gut involvement by Aspergillus is a rare and often fatal complication of intensive antileukemic therapy. We describe the case of an adult patient affected by acute leukemia who developed a small bowel fungal thromboembolism without radiographic evidence of lung involvement during the post-induction aplastic phase. The diagnosis was made histologically at laparotomy performed for small bowel perforation. The patient died a week later in spite of amphotericin-B treatment and neutrophil recovery. Anti-Aspergillus prophylaxis and early introduction of amphotericin-B in the treatment of febrile neutropenia is probably advisable in all cases of AML.