Next generation sequencing in bleeding disorders: two novel variants in the F5 gene (Valencia-1 and Valencia-2) associated with mild factor V deficiency.

Inherited bleeding coagulation disorders (IBCDs) have a powerful diagnostic tool in next generation sequencing (NGS) that not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and helps to predict the clinical course and follow-up of a disease. In our group, targeted-NGS using a Custom SureSelect QXT Panel (Agilent Technologies, Inc., Santa Clara, CA, USA) was designed to screen for causal variants in 40 genes related with the coagulation cascade. In this work, we used NGS for screening all the coding and intronic boundary regions of F5 gene in two patients affected by factor V (FV) deficiency (parahemophilia). Two new mutations were found: c.4745A>G (p.Tyr1582Cys, NM_000130.4) and c.1999_2002dupAATT (p.Ser668ter; NM_000130.4), both located in exon 13 of the F5 gene. We designated them Valencia-1 and Valencia-2 respectively. Valencia-1 could provoke loss of the fifth cupredoxin domain of the FV, and would be responsible for its defective activity. Valencia-2 prematurely stops the translation of mRNA, resulting in a truncated FV protein which lacks completely the B domain and the light chain. NGS has permitted to describe an increasing number of FV deficiency-causing mutations and a better understanding of FV's structure and function. The description of deficiency-causing mutations will continue to increase our knowledge of the functional residues of FV, as well as those which are involved in the correct folding of the protein. In this sense, NGS is a useful tool for studying IBCDs, as permits studying the whole coagulation cascade at once and gives a global view of the patient's genetic background.

Cutaneous metastasis of inflammatory breast carcinoma mimicking an erythema annulare centrifugum: a sign of locally recurrent cancer.

Erythema annulare centrifugum (EAC) is a clinical reaction pattern that includes lupus erythematosus, spongiotic dermatitis (particularly pityriasis rosea), pseudolymphoma and cutaneous B-cell lymphoma. However, it can be the result of cutaneous metastasis by an internal carcinoma. We present the case of a 38-year-old woman with bilateral inflammatory breast cancer following multimodal therapy. After chemotherapy, the patient developed EAC on her back, clinically suspect of subacute cutaneous lupus erythematosus. A skin biopsy of annular lesion revealed dermal lymphatic infiltration by inflammatory breast carcinoma. Immunohistochemically, HER2 overexpression and negativity for hormone receptor are the hallmarks of this disease. Cutaneous metastasis by inflammatory breast carcinoma mimicking EAC is rare, and it has not been described in extramammary locations. Its recognition by the dermatologist is important because it can be a clinical manifestation of locally recurrent cancer.

Chylothorax as Rare Manifestation of Pleural Involvement in Waldenström Macroglobulinemia: Mechanisms and Management.

Here we report the clinical, pathological, and immunological features of a rare case of Waldenström macroglobulinemia (WM) with pleural infiltrations. An atypical chylothorax, successfully treated by videothoracoscopy, represented the main clinical feature of this case of low-grade lymphoplasmacytic lymphoma. Pleuropulmonary manifestations are rare (from 0 to 5% of cases) in WM, with chylothorax observed in just seven patients worldwide. In addition to describing this uncommon clinical presentation, we investigate hypothetical pathogenetic mechanisms causing chylothorax and through an up-todate review of available literature furnish helpful suggestions for diagnosis and management of chylothorax in WM patients.

Trichoderma species fungemia after high-dose chemotherapy and autologous stem cell transplantation: a case report.

We present a case of Trichoderma fungemia with pulmonary involvement in a multiple myeloma patient, who was severely immunocompromised and heavily treated with high-dose melphalan, and underwent autologous hematopoietic cell transplantation. This is the first report, to our knowledge, of proven Trichoderma fungemia, defined by published criteria, successfully treated with voriconazole.

Reticular variant of mid-dermal elastolysis after insertion of a pacemaker.

Summary Mid-dermal elastolysis is an unusual process characterized by the absence of the elastic fibres within the mid-dermis. The disappearance of these fibres causes fine wrinkles (type 1) or perifollicular protrusions (type 2) affecting the trunk, neck and arms. There is a female predilection, and most patients have a history of intense sun exposure. Recently, some cases of mid-dermal elastolysis presenting as reticular erythema have been reported. We report a case of reticular erythema with mid-dermal elastolysis that occurred in a 70-year-old man after insertion of a pacemaker.

Four-step high-dose sequential chemotherapy with double hematopoietic progenitor-cell rescue for metastatic breast cancer.

PURPOSE: High-dose chemotherapy produces high complete remission (CR) rates and some survival advantage in patients with metastatic breast cancer (BC). A current issue is the possibility that these patients may have an even better prognosis with multiple high-dose treatments. In this study, we evaluated the feasibility of a four-step, high-dose sequential chemotherapy (HDSC) with double autologous hematopoietic progenitor-cell rescue. We also tested the hypothesis that peripheral-blood progenitor cells (PBPCs) harvested following a single recruitment with cyclophosphamide (CY) and granulocyte-macrophage colony-stimulating factor (GM-CSF) allow the safe administration of the whole HDSC with closely timed repeated courses of several non-cross-resistant agents. PATIENTS AND METHODS: The treatment plan included CY 7 g/m2, followed by GM-CSF 5 to 7 micrograms/kg/d administered by continuous intravenous (i.v.) infusion on days 2 to 14; PBPCs with or without bone marrow (BM) harvest; mitoxantrone (NOV) 60, 75, or 90 mg/m2 plus melphalan (L-PAM) 140 to 180 mg/m2 with hematopoietic rescue; methotrexate (MTX) 8 g/m2 plus vincristine (VCR) 1.4 mg/m2; and etoposide (VP-16) 1.5 g/m2 plus carboplatin (PP) 1.5 g/m2 with hematopoietic rescue. RESULTS: All 15 patients enrolled completed the entire treatment and there were no toxic deaths. Hematologic reconstitution was good at each step. The median number of days with an absolute neutrophil count (ANC) less than 100/microL and platelet count less than 20,000/microL were 8 and 3, respectively, after NOV plus L-PAM, and 7 and 4, respectively, after VP-16 plus PP. The main non-hematologic toxicity was mucositis, while organ toxicity was mild and reversible. CONCLUSION: This regimen is feasible, with acceptable toxicity. GM-CSF and PBPCs have a pivotal role, as they hasten hematologic reconstitution, abate toxicity, and allow rapid recycling.

Comparative effects of three cytokine regimens after high-dose cyclophosphamide: granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and sequential interleukin-3 and GM-CSF.

PURPOSE: To compare the toxicity and effects on hematologic recovery and circulating progenitor cell mobilization of three cytokine regimens administered after high-dose cyclophosphamide (HD-CTX; 6 g/m(2)), given as the first step of a high-dose sequential chemotherapy. PATIENTS AND METHODS: Forty-eight patients with breast cancer or non-Hodgkin's lymphoma were randomized to receive granulocyte colony-stimulating factor (G-CSF) alone (arm 1), granulocyte-macrophage colony-stimulating factor (GM-CSF) alone (arm 2), or sequential interleukin-3 (IL-3) and GM-CSF (arm 3). Cytokines were administered as a single daily subcutaneous injection at a dose of 5 to 6 microg/kg/d. Progenitor cells were evaluated in peripheral blood as well as in apheretic product as both CD34(+) cells and granulocyte-macrophage colony-forming units (CFU-GM). RESULTS: Neutrophil recovery was faster in arm 1 as compared with arms 2 and 3 (P <.0001); no significant differences were observed between arms 2 and 3. In arm 3, a moderate acceleration of platelet recovery was observed, but it was statistically significant only as compared with arm 1 (P =.028). The peak of CD34(+) cells was hastened in a median of 2 days in arm 1 compared with arms 2 and 3 (P =.0002), whereas the median peak value of CD34(+) cells and CFU-GM was similar in the three patient groups. Administration of IL-3 and GM-CSF resulted in more significant toxicity requiring pharmacologic treatment in 90% of patients. CONCLUSION: The three cytokine regimens administered after HD-CTX are comparably effective in reducing hematologic toxicity and mobilizing the hematopoietic progenitor cells. G-CSF accelerates leukocyte recovery and progenitor mobilization. Although G-CSF-treated patients have somewhat slower platelet recovery, they definitely have fewer side effects.

Clinical impact of immunophenotypic remission after allogeneic hematopoietic cell transplantation in multiple myeloma.

Immunophenotypic remission (IR) is a strong prognostic factor in myeloma patients. The combination of IR and conventional CR was retrospectively evaluated in 66 patients after allografting. IR was defined as the absence of monoclonal plasma cells in BM aspirates by multiparameter flow cytometry. Conditioning was non-myeloablative in 55 patients; reduced-intensity in 10 and myeloablative in 1 patient. The allograft was given upfront in 35/66 (53%) patients. After a median follow-up of 7.1 years, 24 patients achieved both CR and IR (CR/IR group), 21 achieved IR but not CR with persistence of a urine/serum M-component (no CR/IR group) and 21 did not achieve either CR or IR (no CR/no IR group). Median OS and EFS were 'not reached' and 59 months in the CR/IR group; 64 and 16 months in the no CR/IR; and 36 and 6 months in the no CR/no IR, respectively (P<0.001). Cumulative incidence of extramedullary disease was 4.4% in the CR/IR, 38.1% in the no CR/IR and 14.3% in the no CR/no IR groups, respectively, at 4 years (P<0.001). IR was a valid tool to monitor residual disease after allografting, and allowed definition of a cohort of patients at higher incidence of extramedullary relapse.

Hereditary homozygous heparin cofactor II deficiency and the risk of developing venous thrombosis.

Heparin cofactor II (HCII) is a specific inhibitor of thrombin in the presence of heparin or dermatan sulphate. Although there have been reports on families in which a heterozygous HCII deficiency is associated with thromboembolic events, several epidemiological studies revealed that heterozygous HCII deficiency is as prevalent among healthy subjects as it is among patients with deep venous thrombosis (DVT). It is therefore not yet clear whether HCII is or is not a thrombotic risk factor. We analyze and describe in an extended family the biochemical and genetic thrombophilic risk factors and evaluate the potential thrombotic risk involved in homozygous and heterozygous HCII deficiency, either alone or associated with other thrombotic or circumstantial risk factors. The propositus has had three episodes of DVT and a pulmonary embolism. During the first episode of DVT the patient was diagnosed as having AT deficiency. Later, a functional and antigenic HCII deficiency, compatible with the homozygous form, was detected. The family study shows that both the propositus and her sister have homozygous HCII deficiency and that 12 of the 27 family members have heterozygous HCII deficiency. This is possibly the first case report on a homozygous phenotype for the HCII deficiency with. in addition, partial AT deficiency. The propositus has suffered several thrombotic events, unlike the other 12 family members with heterozygous HCII deficiency and her sister, who is also homozygous for this disorder. We suggest that HCII deficiency may play a limited in vivo role as a thrombotic risk factor unless associated with AT deficiency or another congenital thrombotic risk factor.

Elevated high molecular weight fibrinogen in plasma is predictive of coronary ischemic events after acute myocardial infarction.

This study investigates the association between the concentration and function of plasma fibrinogen molecules measured at the time of hospital admission in patients with acute myocardial infarction (AMI), with reference to the risk of new coronary ischemic events during a three-day follow-up period of. Before starting fibrinolytic and anticoagulant treatment plasma fibrinogen, high molecular weight fibrinogen (HMW-fibrinogen), fibrin formation rate (FbFR) and phosphorous content in fibrinogen were determined in 90 AMI patients. During a three-day follow-up period 12 patients suffered new ischemic events. The 12 patients with coronary ischemia had higher concentrations of plasma fibrinogen (312+/-23 vs. 270+/-73 mg/dl, p<0.05) and HMW-fibrinogen (246+/-35 vs. 189+/-23 mg/dl, p<0.001) and a higher FbFR (65+/-30 vs. 40+/-25, p<0.001) than patients without these events. No association was found between the phosphorous content in fibrinogen and new coronary ischemic events. We conclude that after myocardial infarction an elevated plasma level of HMW-fibrinogen and a high FbFR value at the time of hospital admission are associated with new coronary ischemic events during a three-day follow-up period.

High-dose chemotherapy with tandem autologous transplantation as part of the initial therapy for aggressive non-Hodgkin's lymphoma.

The purpose of the present study was to evaluate the feasibility and the efficacy of employing a high-dose chemotherapy (HDT) regimen with tandem peripheral blood progenitor cells (PBPC) supported transplantation in the initial treatment of aggressive non-Hodgkin's lymphoma (NHL). HDT was preceded by a standard course of conventional dose chemotherapy in 17 out of the 25 patients treated, while in 8 cases it was delivered after only one or two cycles. HDT was a three-step procedure which included high-dose (6-7 g/m2) cyclophosphamide (CY) supported by haematopoietic growth factors, the first myeloablative course with mitoxantrone (NOV) 60, 75 or 90 mg/m2 plus melphalan (L-PAM) 140-180 mg/m2 with haematopoietic rescue, and the second myeloablative course with etoposide (VP) and carboplatin (CARBO) given at 1.5 g/m2 each with haematopoietic rescue. PBPC were collected after CY administration. Twenty-two patients (88%) completed the HDT, haematological reconstitution was rapid and complete at each step and there were no toxic deaths. The activity of the treatment was high with a CR rate over 90% in the entire patient population. The 2-year overall survival (OS) and failure-free survival (FFS) rates of patients in both Age-Adjusted International Prognostic Index (A-AIPI) groups 2 and 3 are 79% and the disease-free survival (DFS) rate for the CRs is 85%. In A-AIPI group 1 the 2-year OS and FFS rates are both 91%.

The use of sample rotation for minimizing convection effects in self-diffusion NMR measurements.

Undesirable temperature gradients in a NMR sample tube are usually generated by an inappropriate temperature regulation system. We have shown that such convection effects can greatly distort the measurement of translational self-diffusion coefficients. The use of sample spinning helps to minimize such undesirable effects by disruption of convection fluxes due to resulting Coriolis forces that have a strongly stabilizing effect on the conducting state of the system (J. Lounila et al., J. Magn. Reson. A 118, 50 (1996)). This simple trick allows the accurate measurement of diffusion coefficients for a wide range of temperatures and solvents without the need for a convection-compensated NMR pulse sequences or more sophisticated temperature control units. Experimental data obtained for some target compounds dissolved in several common deuterated solvents at different temperatures are reported and discussed.

Standard-dose recombinant human granulocyte colony-stimulating factor (rhG-CSF) allows safe and repeated administration of high-dose cyclophosphamide, etoposide, and cisplatin (CEP).

High-dose chemotherapy often requires hematopoietic progenitor cell reinfusion, but drugs with extramedullary dose-limiting toxicity may be administered in the high-dose range by simple growth factor support. In this study, we evaluated the feasibility and toxicity of a three-drug high-dose regimen supported by recombinant human granulocyte colony-stimulating factor (rhG-CSF). Ten patients with histologically proven malignancy were enrolled. Eight had breast cancer, one non-Hodgkin's lymphoma, and one a mediastinal tumor of unknown origin. The regimen included cyclophosphamide (C) 5 g/m2, etoposide (E) 1.5 g/m2, and cisplatin (P) 150 mg/m2 (CEP), administered in a 3-day schedule followed by rhG-CSF, 300 micrograms once a day, beginning from day +5 (36 h after the end of chemotherapy). The cycle was repeated as clinically needed up to three times. After the first course, hematologic recovery was rapid and complete without documented infections, and no relevant extramyeloid toxicities were observed. Eight of 10 patients received a second course with comparably low toxicity, and three of them received a third course. We concluded that CEP therapy can be administered safely and even repeatedly, by simple growth factor support, in good performance status cancer patients.

Tumor cell lysis by activated human neutrophils: analysis of neutrophil-delivered oxidative attack and role of leukocyte function-associated antigen 1.

The lysis of tumor cells, and other nucleated mammalian cells, by neutrophilic polymorphonuclear leukocytes (PMNs) triggered by phorbol myristate acetate (PMA) represents a widely used model system to dissect the PMN cytolytic armamentarium, potentially responsible for the cell damage at tissue sites of PMN activation. Although oxidants are generally considered to be instrumental in the target lysis by PMNs, the mediators actually involved remain a matter of controversy. Moreover, other factors potentially crucial to the lysis have not been clearly identified. In order to reexamine the determinants of the cytolytic process, we studied the events underlying the PMA-triggered PMN-delivered attack against two different targets, selected on the basis of preliminary experiments (B lymphoblastoid Daudi cells and erythroleukemic K 562 cells). The results suggest that the lysis is promoted by hypochlorous acid (HOCl) or a compound with characteristics very similar to HOCl itself. No evidence was obtained for the intervention or contribution of hydrogen peroxide (H2O2), hydroxyl (OH.) radicals, and the major HOCl-derived chloramines. PMNs appeared to use 35% of the generated H2O2 to produce HOCl, while the remainder appears to be consumed by PMNs themselves and target cells as well. Moreover, PMNs and target cells coaggregated at an early step of the cytolytic reaction, through a process efficiently prevented by a monoclonal antibody (MoAb J-90) directed against leukocyte function-associated antigen-1 (LFA-1). The inhibition of the PMN-target aggregation by the MoAb J-90 resulted in the impairment of the lysis, despite a normal generation of HOCl. Thus, the data demonstrate that the PMA-triggered lysis of tumor target cells by PMNs requires at least two events, occurring simultaneously: the LFA-1-mediated effector-target adherence and the PMN production of HOCl. The intervention of the LFA-1-mediated PMN-target adherence in the PMA-triggered lysis is likely to allow PMNs to focus HOCl on the target cell surface and suggests that the process requires a sort of molecule to molecule recognition at the effector-target surface level.

Schnitzler's syndrome: report of a case with bone osteolysis.

A case of chronic urticaria with macroglobulinemia (Schnitzler's syndrome) is described. To our knowledge, this is only the second case reported in the literature with osteolysis and the presence of cryoglobulins in sera. The characteristics of the previous 21 cases of this syndrome are reviewed. The pathogenesis of this peculiar entity is discussed.

Congenital and acquired thrombotic risk factors in women using oral contraceptives: clinical aspects.

We describe the thrombophilic and clinical characteristics of a group of patients who suffered venous thrombosis (VT) (n = 36) and ischemic stroke (n = 8) while taking oral contraceptives (OC). Our purpose is to ascertain whether there are differences between users of second and third generation progestogen and to investigate the influence of concurrent congenital and acquired risk factors (other than OC) on the onset of the thrombosis. The group of patients included 36 women with VT and eight with ischemic stroke. The patients' recognized predisposing factors were recorded. We also considered age, length of time on OC, types of OC, rethrombosis, family history of VT, and the presence of thrombophilic genetic defects. In the group of patients with VT, 54% were treated with second generation OC (n = 23), and 30% (n = 11) were treated with third generation OC. We found no significant statistical differences with respect to age and length of time on OC between the two types of OC. The prevalence of genetic defects in these patients--factor V (FV) Leiden, prothrombin G20210A mutation and protein S deficiency--was 19% (n = 7), 17% (n = 6), and 8% (n = 3) respectively. We observed the shortest time lapse between initiating OC and the first thrombotic event in carriers of FV Leiden and in patients with combined defects, but the differences were not significant. In patients with ischemic stroke, 50% were treated with second generation OC and 50% were treated with third generation OC. Prothrombin G20210A mutation was detected in two patients. In both patients,the stroke occurred earlier than in the rest of the patients, but these differences were not statistically significant. With respect to preventing thrombotic events in these patients, our data suggest that OC therapy should be avoided in patients with a previous history of thrombosis and in patients with an evident thrombotic tendency in the family. In patients in whom the family history of thrombosis is not very evident, it would be recommended to screen for FV Leiden, prothrombin G20210A mutation, and protein S, and to rule out OC if the patient does in fact have one of these risk factors. Moreover, if a patient develops a thrombotic complication while taking OC, an evaluation to search for a thrombophilic defect is warranted, and at the same time, alternative methods of contraception should be considered.

Factor V Leiden and prothrombin G20210A in relation to arterial and/or vein rethrombosis: two cases.

The factor V Leiden (FV Leiden) and prothrombin G20210A mutations, are the most common established genetic risk factors for deep vein thrombosis (DVT). However, the relationship between these mutations and arterial thrombotic syndromes (coronary heart disease, myocardial infarction, stroke) has not been established. Some studies have suggested a relationship between them, but other authors have considered it unlikely that these anomalies are a major risk factor for arterial thrombosis. From the clinical point of view, a question arises concerning the risk of repeated thrombosis in patients carrying one of these two mutations. The question is whether the recurrence is attributable to the mutations or to the presence of additional circumstantial risk factors. As the risk of repeated thrombosis varies considerably from one patient to another, decisions about long-term treatment require weighing the persistence of risk factors for vascular disease (venous and arterial), especially in selected cases such as young patients or patients with thrombosis of unusual localization.

Hemorheological alterations and hypercoagulable state in deep vein thrombosis.

Deep vein thrombosis (DVT) seems to be related to a hypercoagulation and definite hemorheological alterations, but the importance of these alterations in the development of thrombotic events in the deep vein system has not been established. The present study examines both aspects in a group of 55 patients with DVT; the presence of a hypercoagulable state was assessed by quantifying the prothrombin fragment 1+2 (F1+2) and the thrombin-antithrombin III complex (T-AT), and the main hemorheological parameters were evaluated in the acute state and 6 and 12 months later. The results show marked hemorheological, F1+2, and TAT alterations in the acute phase. After 12 months the pattern shows a modest improvement, but erythrocyte aggregation, fibrinogen, F1+2 and T-AT remain increased with respect to the control group (8.51 +/- 1.43; 331 +/- 81 mg/dl; 1.33 +/- 0.60 nmol/l; 3.54 +/- 1.71 ng/ml vs. 8.10 +/- 1.40; 230 +/- 38; 0.94 +/- 0.40; 1.56 +/- 0.59, respectively). These data suggest that the thrombotic event could be influenced by the previous rheological situation and hypercoagulable state.

Inactivation of neutrophil-derived hypochlorous acid by nimesulide: a potential mechanism for the tissue protection during inflammation.

The anti-inflammatory drug nimesulide was found to effectively reduce the availability of hypochlorous acid, the most potent chlorinated oxidant generated by the myeloperoxidase system of activated neutrophils. Such an effect was observed at concentrations achievable in vivo after the oral administration of the drug. Higher concentrations of nimesulide were also found to limit both the oxygen consumption and the superoxide anion/hydrogen peroxide production by neutrophils. Taken together, the results suggest that nimesulide is endowed with a high potential to efficiently control the harmful effects of oxidants produced by neutrophils at inflammatory tissue sites.

[Value of power Doppler ultrasonography with intravenous contrast medium (Levograf) in the diagnosis of acute cholecystitis]. / Valor de la ecografía power Doppler con contraste intravenoso (Levograf) en el diagnóstico de colecistitis aguda.

The aim of this study was to evaluate the potential capacity of color and power Doppler sonography with intravenous contrast medium in the diagnosis of acute cholecystitis. We examined 18 patients with acute cholecystitis, 5 patients with chronic cholecystitis and a control group of 11 patients without gallbladder disease. In these patients, vascularization of the gallbladder wall was evaluated by color and power Doppler sonography before and after intravenous administration of contrast medium (Levograf). Vascularization was evaluated with a 3-point scale (grades 0, 1 and 3) according to the intensity and localization of signs of color. In the diagnosis of acute cholecystitis, basal examination with power Doppler had a sensitivity of 38.8%. After administration of intravenous contrast medium, sensitivity was 100%. In conclusion, the use of sonographic contrast media in the diagnosis of acute cholecystitis showed a sensitivity and specificity of 100%. This technique represents a viable diagnostic alternative to other complementary or imaging studies.