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.

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.

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.

Pharmacogenetics of acenocoumarol in patients with extreme dose requirements.

SUMMARY BACKGROUND: There is currently intense debate as to whether pharmacogenetic algorithms for estimating the initial dose of coumarins provide a more accurate dose than the fixed-dose approach. Recently, it has been suggested that the greatest benefit of pharmacogenetic algorithms is observed in patients with extreme dose requirements. OBJECTIVES: To identify clinical and genetic factors that better characterize patients who need extreme acenocoumarol doses for steady anticoagulation state. PATIENTS/METHODS: We reviewed 9538 patients with a steady acenocoumarol dose from three Spanish hospitals, selecting 83 who took or= 30.00 mg week(-1) (p95). We also selected patients matched by gender and age taking 13.50-14.00 mg week(-1) (p50). We genotyped VKORC1 (rs9923231), CALU (rs1043550), GGCX (rs699664), CYP2C9 (rs1799853; rs1057910), CYP4F2 (rs2108622) and F7 (rs5742910) single-nucleotide polymorphisms (SNPs). RESULTS: Comparison between p5 and p95 revealed five parameters with significant differences: body surface area (BSA) (P = 0.006), age, VKORC1, CYP2C9 and CYP4F2 genotypes (all P < 0.001). First VKORC1, and second, CYP2C9 SNPs played a strong effect by determining extreme doses, particularly in p95. Only one out of 203 p95 had the VKORC1 A-1639A genotype, but this subject was CYP2C9*1/*1. In contrast, nine out of 83 p5 carried the VKORC1 G-1639G genotype, although six of them were CYP2C9*3 homozygotes and another two were heterozygotes. Surprisingly, CYP4F2 V433M SNP displayed prevalences that suggest that its influence might only be evident when patients are treated with high doses. CONCLUSION: Two clinical data, age and BSA, and three SNPs in the VKORC1, CYP2C9 and CYP4F2 genes strongly predict outlier patients treated with acenocoumarol.

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.

Preparation and structural study of the enantiomers of alpha,alpha'-bis(trifluoromethyl)-9,10-anthracenedimethanol and its perdeuterated isotopomer, highly effective chiral solvating agents.

Enantiopure forms of alpha,alpha'-bis(trifluoromethyl)-9,10-anthracenedimethanol and the corresponding perdeuterated isotopomers were prepared. The conformational study was carried out by (1)H NMR, and the absolute configuration was determined by the X-ray study of the crystallized diastereoisomeric carbamate derivative. This compound was tested as a chiral solvating agent (CSA). The results showed very good discrimination for several racemic mixtures that improved other classical methods. The study of diastereomeric complexes was carried out by determination of the stoichiometry of the complex and the binding constant of the equilibrium.

[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.

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.

Reverse-transcriptase polymerase chain reaction of the maspin gene in the detection of bone marrow breast carcinoma cell contamination.

BACKGROUND: Maspin is a molecular marker used for the detection of contaminating breast carcinoma (BC) cells in peripheral blood and lymph nodes. However, its specificity has been questioned recently. The objective of this study was to verify the specificity of this marker and to determine the incidence of positive bone marrow results in patients with BC who are eligible for high-dose chemotherapy (HDT) both in early and advanced disease stages and before and after treatment. METHODS: Bone marrow specimens from 41 patients with BC as well as from 35 normal volunteers and 17 patients with hematologic tumors were examined for maspin transcript expression by a modified nested reverse transcriptase-polymerase chain reaction technique. RESULTS: Maspin transcript was found in all normal and neoplastic breast tissues and in none of the 35 normal bone marrow specimens (specificity, 100%; 95% confidence interval, 90-100%). However, the transcript was found in 40% of the bone marrow samples from patients with hematologic malignancies. Thus, this marker appears very specific for discriminating between normal controls and patients with BC, but it cannot be considered disease specific. Among patients with BC, bone marrow was positive for the maspin transcript in 32% of patients with early-stage disease and in 75% of patients with metastatic disease before chemotherapy. After treatment, in 75% of patients with early-stage disease and in 50% of patients with metastatic disease, the bone marrow results became maspin negative. CONCLUSIONS: On the basis of the current data, although it is not disease specific, maspin is a reliable marker for detecting bone marrow molecular disease in patients with BC and should be considered for prospective studies as a prognostic indicator and as an assay for monitoring residual disease.

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.

Adjuvant chemotherapy with high-dose cyclophosphamide, etoposide and cisplatin intensification without progenitor cell support in breast cancer patients with ten or more involved nodes: 5-year results of a pilot trial.

OBJECTIVES: The purpose of this study was to evaluate the clinical efficacy and tolerability of high-dose (HD) chemotherapy with growth factor support in primary breast cancer with extensive nodal involvement. PATIENTS AND METHODS: Fifty-three patients with ten or more involved nodes were recruited and were given three cycles of standard-dose fluorouracil, epidoxorubicin and cyclophosphamide followed by one single course of high-dose CEP (cyclophosphamide, etoposide and cisplatin). No autologous progenitor support was used. RESULTS: Five-year actuarial disease-free and overall survival were 40 and 60%, respectively. High-dose CEP required a median of 22 days of hospitalization and was associated with grade G3--4 nausea and vomiting in two thirds of the cases. Hematological toxicity was comparable to that of high-dose therapies delivered with autologous progenitor support. No therapy-related mortality was observed. CONCLUSIONS The efficacy of treatment was comparable to the best results of conventional therapy, with only a trend for improved survival. High-dose CEP was feasible with acceptable toxicity. Although this regimen does not require stem cell harvesting and storage, it requires clinical support comparable to autotransplantation procedures and side effects are not so manageable to recommend its use outside specialized units.

Novel[1n]-meta-heterophane frameworks with a bis-betaine nature.

A convergent "5 + 1" and "5 + 3" synthetic strategy allowed the synthesis of the first examples of bis-betaines 2 and 3, a prototype of phanes that incorporate heterocyclic betaines. The structure of the quadrupolar macrocyclic systems 2 and 3 together with the dicationic [1(6)]- and [1(8)] meta-heterophane precursors 5*2X and 6*2X were examined by spectroscopy using 1H and 13C NMR techniques together with 1H-DNMR studies and electrospray ionization mass spectrometry.

Technetium-99m-sestamibi scintigraphy in multiple myeloma and related gammopathies: a useful tool for the identification and follow-up of myeloma bone disease.

BACKGROUND AND OBJECTIVES: Technetium-99m 2-methoxy-isobutyl-isonitrile ((99m)Tc-sestamibi) has recently been proposed as a potential tracer in patients with multiple myeloma (MM), as its increased uptake in the bone marrow has been reported as indicator of myeloma activity. We evaluated the role of (99m)Tc-sestamibi scintigraphy in the detection of myeloma bone disease in MM and related gammopathies, and also assessed its relationship with clinical status and stage of the disease, focusing in particular on the early follow-up of a small series of MM patients treated with high-dose therapy. DESIGN AND METHODS: Forty-six consecutive patients affected by MM or monoclonal gammopathy of undefined significance (MGUS) were studied by whole body scans obtained 20 minutes after administration of 740 MBq of (99m)Tc-sestamibi. A semiquantitative uptake score was used and scintigraphic findings were correlated with clinical and laboratory data. RESULTS: All the MGUS patients showed a negative (99m)Tc-sestamibi scan. Among the 32 MM patients (25 with active disease and 7 in clinical remission) 24 showed a positive scan, while 8 presented only a physiologic uptake of the tracer. The uptake score correlated significantly with all the most relevant clinical variables. In the follow-up of 8 MM patients treated with high-dose chemotherapy (99m)Tc-sestamibi closely paralleled the activity of myeloma bone disease. Comparison with X-ray skeletal survey showed discordant results in 14 out of the overall 56 scans obtained (27%), with 10 cases of negative (99m)Tc-sestamibi scans but lytic bone lesions revealed by X-ray (7 of them were in clinical remission), and 4 negative X-ray surveys in patients with positive (99m)Tc-sestamibi scans. Overall sensitivity and specificity of (99m)Tc-sestamibi scintigraphy in detecting myeloma bone disease were 90% and 88%, respectively. INTERPRETATION AND CONCLUSIONS: This study provides additional evidence indicating that (99m)Tc-sestamibi scintigraphy closely reflects myeloma disease activity in bone marrow, with very high sensitivity and specificity. (99m)Tc-sestamibi scintigraphy is therefore suggested as a reliable new tool for the staging and follow-up of myeloma bone disease.

Risk of venous thrombosis in carriers of the prothrombin G20210A variant and factor V Leiden and their interaction with oral contraceptives.

BACKGROUND AND OBJECTIVES: The prothrombin G20210A mutation and factor V Leiden have been found to be associated with an increased risk of venous thrombosis, but the reported prevalences of the prothrombin gene variant both in the normal population and in patients with deep venous thrombosis (DVT) vary greatly in the literature. Moreover, the influence of oral contraceptives (OC) on thrombotic events in patients with the prothrombin G20210A variant has not been well established. In this study we evaluate both circumstances. DESIGN AND METHODS: A case-control study was run on 229 patients with DVT and 246 healthy controls. The patients' history of thrombosis and acquired thrombotic risk factors, especially OC, were recorded. Prothrombin G20210A mutation, factor V Leiden, antithrombin, heparin II cofactor, plasminogen and proteins C and S were evaluated. RESULTS: Seven and a half percent of the patients and 2.9% of the controls were carriers of the prothrombin mutation, while 12.2% of the patients and 1.6% of the controls had factor V Leiden. Among the 229 DVT patients there were 130 patients with clinically suspected thrombophilia (first thrombotic event occurring before the age of 45 years or positive family history of thrombosis or recurrent venous thrombosis). Ten percent of these 130 patients were carriers of the prothrombin G20210A mutation and 18.5% had the factor V Leiden mutation. The odds ratios (OR) for DVT risk were: 2.4 (95% CI, 1.0-6.3) for the total DVT patients and 5.2 (95% CI, 1.4-19.5) for the patients with clinically suspected thrombophilia with the prothrombin mutation. The risk of thrombosis was 6.9 (95% CI, 2.3-20.6) for the DVT patients and 14.3 (95% CI, 3.3-64.6) for the patients with clinically suspected thrombophilia with factor V Leiden. Fifty-five percent of the patients with combined congenital defects (prothrombin mutation G20210A plus another congenital defect) had recurrent thrombosis. In women receiving OC the risk of DVT was 3.5 (95% CI, 1.5-8.2) that of the patients not receiving OC. When women with combined defects were also taking OC, the risk of thrombosis increased significantly. INTERPRETATION AND CONCLUSIONS: The prevalence of the prothrombin G20210A mutation in the healthy population in our study is similar to that observed in other southern European countries. The prothrombin G20210A mutation does not by itself seem to be a high thrombotic risk factor. However, when it is present together with other thrombotic risk factors, the predicted risk of thrombotic events increases. The use of OC by women with the prothrombin G20210A variant or FV Leiden, either alone or combined with other thrombotic risk factors, was associated with a significant increase in the risk of venous thrombosis.