A novel HLA-C null allele, HLA-C*05:99N.

HLA-C*05:99N results from a single nucleotide loss compared with its closest allele HLA-C*05:01:01:01.

Outcome in patients with diffuse large B-cell lymphoma who relapse after autologous stem cell transplantation and receive active therapy. A retrospective analysis of the Lymphoma Working Party of the European Society for Blood and Marrow Transplantation (EBMT).

Autologous hematopoietic stem cell transplantation (auto-HSCT) is the standard of care for patients with diffuse large B-cell lymphoma (DLBCL) who relapse/progress after first line chemoimmunotherapy. Long-term outcome of those who relapse after transplant is poor. We present the results of a retrospective study of 256 adult patients reported to the EBMT registry with DLBCL who relapsed after auto-HSCT performed between 2003 and 2013, and who received active salvage strategies. One hundred and fifty-four (60%) were male; median age was 53 years. Median time to relapse was 7 months, 65% relapsed during the first year. Overall response rate after salvage therapy was 46%. Median follow-up after first salvage therapy was 40 months (IQR 23-63 months). Overall survival (OS) at 3 years was 27% (95% CI 22-33). OS at 3 years of patients relapsing longer than 1 year after auto-HSCT was 41% (95% CI 31-53) compared with 20% (95% CI 14-24) in those who relapsed in less than 1 year. Eighty-two patients (32%) had a second HSCT, an allogeneic HSCT (allo-HSCT) in 69 cases, at a median time of 6.5 months after relapse. OS at 3 years after allo-HSCT was 36% (95% CI 25-51). In conclusion, the prognosis of patients with DLBCL that relapse after auto-HSCT is dismal. Patients who relapse in less than 1 year remain an unmet need, and should be considered for CAR T cell therapy or clinical trials. Patients who relapse after 1 year can be rescued with salvage therapies and a second HSCT. These results provide a benchmark to compare data of new prospective studies.

Medical costs of treatment and survival of patients with acute myeloid leukemia in Belgium.

The advent of new cell-based immunotherapies for leukemia offers treatment possibilities for certain leukemia subgroups. The wider acceptability of these new technologies in clinical practice will depend on its impact on survival and costs. Due to the small patient groups who have received it, these aspects have remained understudied. This non-randomized single-center study evaluated medical costs and survival for acute myeloid leukemia between 2005 and 2010 in 50 patients: patients treated with induction and consolidation chemotherapy (ICT) alone; patients treated with ICT plus allogeneic hematopoietic stem cell transplantation (HCT), which is the current preferred post-remission therapy in patients with intermediate- and poor-risk AML with few co-morbidities, and patients treated with ICT plus immunotherapy using autologous dendritic cells (DC) engineered to express the Wilms' tumor protein (WT1). Total costs including post- consolidation costs on medical care at the hematology ward and outpatient clinic, pharmaceutical prescriptions, intensive care ward, laboratory tests and medical imaging were analyzed. Survival was markedly better in HCT and DC. HCT and DC were more costly than ICT. The median total costs for HCT and DC were similar. These results need to be confirmed to enable more thorough cost-effectiveness analyses, based on observations from multicenter, randomized clinical trials and preferably using quality-adjusted life-years as an outcome measure.

Autologous stem cell transplantation for relapsed/refractory diffuse large B-cell lymphoma: efficacy in the rituximab era and comparison to first allogeneic transplants. A report from the EBMT Lymphoma Working Party.

In the era of chemoimmunotherapy, the optimal treatment paradigm for relapsed and refractory diffuse large B-cell lymphoma has been challenged. We reviewed the outcome of standard salvage therapy with an autologous stem cell transplant (autoSCT) over the last two decades and the outcome of allogeneic SCT (alloSCT) in the most recent decade. AutoSCT recipients diagnosed between 1992 and 2002 (n=2737) were compared with those diagnosed between 2002 and 2010 (n=3980). Patients diagnosed after 2002 had a significantly lower non-relapse mortality (NRM) and relapse incidence (RI) and a superior PFS and overall survival (OS). A total of 4210 patients diagnosed between 2002 and 2010 underwent either an autoSCT or an alloSCT as their first transplant procedure. Two-hundred and thirty patients received an alloSCT (myeloablative (MACalloSCT) n=132, reduced intensity (RICalloSCT) n=98). The 4-year NRM rates were 7%, 20% and 27% for autoSCT, RICalloSCT and MACalloSCT, respectively. The 4-year RI was 45%, 40% and 38% for autoSCT, RICalloSCT and MACalloSCT, respectively (NS). The 4-year PFS were 48%, 52% and 35% for autoSCT, RICalloSCT and MACalloSCT, respectively. The 4-year OS was 60%, 52% and 38% for autoSCT, RIC alloSCT and MACalloSCT, respectively. After adjustment for confounding factors NRM was significantly worse for patients undergoing alloSCT whilst there was no difference in the RI.

Bortezomib-induced Sweet's syndrome.

Sweet's syndrome is an uncommon acute skin disease, associated with a variety of medical problems. The drug-induced variant is even rarer. We describe two cases of this syndrome associated with the administration of the proteasome inhibitor bortezomib. The diagnostic criteria for drug-induced Sweet's syndrome as proposed by Walker and Cohen were fulfilled. Vasculitis and neutrophilic eccrine hidradenitis were excluded.

Decreasing antibiotic resistance of Enterobacteriaceae by introducing a new antibiotic combination therapy for neutropenic fever patients.

Prompt empiric antibiotic therapy is of critical importance for patients with neutropenic fever. However, a major concern with important clinical consequences is the emergence of bacterial resistance to antibiotics. After using ceftazidime with a glycopeptide as initial empiric therapy for neutropenic fever, we were confronted with a 75% reduced susceptibility rate to ceftazidime of inducible Enterobacteriaceae collected in 1994. The initial empiric therapy was therefore replaced in May 1995 by a combination of cefepime with amikacin, with addition of a glycopeptide after 48 h if necessary. After this change, we observed a significant decrease in reduced susceptibility of inducible Enterobacteriaceae, not only to ceftazidime, but also to amikacin, cotrimoxazole and ciprofloxacin. There was also a decrease in reduced susceptibility of non-inducible Enterobacteriaceae, such as Klebsiella spp, to ceftazidime. The reduction of resistance may be related at least in part to the combined use of cefepime together with an aminoglycoside. This study shows that it is possible to reverse bacterial resistance by modifying the antibiotic regimen used.

A critical appraisal of non-invasive diagnosis and exclusion of deep vein thrombosis and pulmonary embolism in outpatients with suspected deep vein thrombosis or pulmonary embolism: how many tests do we need?

The requirement for a safe diagnostic strategy should be based on an overall posttest incidence of venous thromboembolism of less than 1% during 3 month follow-up. Compression ultrasonography (CUS) has a negative predictive value (NPV) of 97% to 98% indicating the need of repeated CUS testing. Serial CUS testing is safe but you have to repeat 100 CUS to find 1 or 2 CUS positive for deep vein thrombosis (DVT), which is not cost-effective indicating the need to improve the diagnostic work-up of DVT by the use of clinical score assessment and D-dimer testing. The combination of a less sensitive D-dimer test (SimpliRed) and low clinical score does not, whereas the combination of a sensitive D-dimer test (ELISA VIDAS or Tinaquant) and low clinical score does safely exclude DVT without the need of CUS. The combination of a first negative CUS and a negative less sensitive D-dimer test (SimpliRed) or a sensitive ELISA D-dimer at a higher cut off level of 1,000 ng/ml safely excludes DVT with a NPV of > 99% without the need to repeated CUS in about 60%. The sequential use of a sensitive D-dimer and clinical score assessment will safely reduce the need for CUS testing by 40% to 60%. Large prospective outcome studies demonstrate that one negative examination with complete duplex color ultrasonography (CCUS) of the proximal and distal veins of the affected leg with suspected DVT is safe to withhold anticoagulant treatment with a NPV of 99.5%. This indicates that CCUS is equal or superior to serial CUS or the combined use of clinical score, D-dimer testing and CUS. Pulmonary angiography is the gold standard for segmental pulmonary embolism (PE) but not for subsegmental PE. A normal perfusion lung scan and a normal rapid ELISA VIDAS D-dimer test safely exclude PE. Helical spiral CT detects all clinically relevant PE and a large number of alternative diagnoses in symptomatic patients with a non-diagnostic ventilation perfusion scan (VP-scan) or a high probability VP-scan. Single-slice helical CT as the primary diagnostic test in patients with suspected PE in 5 retrospective studies and in 3 prospective management studies indicate that the NPV of a normal helical spiral CT, a negative CUS of the legs together with a low or intermediate pretest clinical probability is 99%. Helical spiral CT can replace both the VP-scan and pulmonary angiography to safely rule in and out PE. The combination of clinical assessment, a rapid ELISA VIDAS D-dimer followed by CUS will reduce the need for helical spiral CT by 40% to 50%.

Diagnosis of deep vein thrombosis: how many tests do we need?

The requirement for a safe diagnostic strategy should be based on an overall post-test incidence of venous thromboembolism (VTE) of less than 1% during 3 month follow-up. Compression ultrasonography (CUS) has a negative predictive value (NPV) of 97 to 98% indicating a post-CUS incidence of deep vein thrombosis (DVT) of 2 to 3%. A post-CUS DVT incidence of 3% implicates that 90 to 120 DVTs per 1 million inhabitants will be overlooked each year indicating the need to improve the diagnostic work-up of DVT as much as possible. The qualitative D-dimer test (SimpliRed) has a sensitivity of 82 to 89% and a negative predictive value of 94 to 95% indicating a 5 to 6% post-test incidence of DVT, which is not sensitive enough for venous thrombosis exclusion. The post-test DVT incidence could be reduced from 3.2% to 0.6% in one study and from 11% to 2% in another study by the combination of a normal CUS and low clinical score and from 4.5% to 1.6% by the combination of low clinical score and a negative SimpliRed test in one study. The combination of a negative CUS and a negative SimpliRed test reduced the post-test incidence of DVT from 2.6% to < 1% or even < 1% in two management studies without the need of a repeated CUS on the basis of which anticoagulant therapy can safely be withheld. The rapid quantitative turbidimetric D-dimer assay (Tinaquant) has a sensitivity and a negative predictive value (NPV) of 97.7% with a 2.3% post-test incidence of DVT. The combination of a normal Tinaquant D-Dimer test result plus a low to moderate clinical score reduces the post-test incidence of DVT from 2.3 to 0.6% without the need of CUS testing in 29% of patients with suspected DVT. The rapid ELISA VIDAS D-dimer assay has a sensitivity and NPV of 98.6 and 99.5% in two management studies for the exclusion of DVT irrespective of clinical score. The combination of a normal ELISA VIDAS D-Dimer test with clinical score assessment will reduce the post-test DVT incidence of less than 0.5% and the need for CUS testing by 40 to 50%. It is concluded that the sequential use of a rapid quantitative D-dimer test, clinical score and CUS appears to be safe and the most cost-effective diagnostic work-up of DVT.

The influence of the protease inhibitor aprotinin on tumor invasion of three cell lines in vitro.

The production of protease by malignant cells has been held responsible for invasion. The effect of aprotinin, a broad-spectrum protease inhibitor, on malignant invasion was examined histologically in organotypical cocultures of precultured embryonic chick heart fragments with two human melanoma cell lines and one virus-transformed fetal mouse carcass cell line. In the presence of 400 KIU/ml aprotinin, invasion was still permissive, while in the comparison with 0.1 microgram/ml vincristine, a microtubule and directional motility inhibitor and a well-documented anti-invasive agent, invasion was completely stopped. Aprotinin remained stable in the culture medium so that the presence of invasion cannot be explained by low drug concentration. It is concluded that aprotinin-sensitive proteases are not implicated in invasion in vitro in the cell types investigated, and that this in vitro technique deserves further interest for the study of protease inhibitors in the mechanism of invasion.

Resistance of the embryonic notochord to invasion by malignant neurogenic tumor cells in vitro.

Very few tissues in vivo are resistant to invasion by malignant cells. In this paper, confrontation cultures consisting of chick embryonic notochord fragments and cell aggregates from three malignant neurogenic cell lines showed a resistance of the embryonic organ to malignant invasion by tumor cells. The mechanism of resistance remains unclear.

In vitro invasiveness of human bladder cancer from cell lines and biopsy specimens.

Aggregates prepared from cell lines established from a human transitional cell carcinoma of the urothelium (Hu 456) or from apparently normal urothelium before (Hu 609) and after phenotypic transformation (Hu 609T) were confronted with fragments of embryonic chick cardiac muscle in organ culture. In this assay a correlation was found between in vitro invasiveness of animal cell lines and their capacity to produce invasive tumours in syngeneic animals. The invasiveness of cells from established human urothelial lines was compared to the invasiveness of cells from established human urothelial lines was compared to the invasiveness of cells from fresh biopsy specimens of a normal urothelium, a non-invasive papilloma, and a metastasizing transitional cell carcinoma. Cells from all established lines (Hu 609, Hu 609T and HU 456) and from the biopsy specimens of the transitional cell carcinoma occupied and eventually replaced the cardiac muscle by contrast with cells from the normal urothelium or from the non-invasive papilloma. We concluded that the organ culture assay for invasiveness might be used to define malignancy of human bladder cell lines and to follow the various steps during the acquisition of invasiveness in vitro.

Validation of clinical predictive value of in vitro colorimetric chemosensitivity assay in head and neck cancer.

For chemosensitivity testing, a rapid in vitro colorimetric method (MTT assay) was used. Eleven head and neck cancer cell lines were investigated to distinguish five known active agents from five compounds inactive in phase II studies. Evaluation of the reliability of the assay for assessing drug sensitivity in this tumor cell population was done by correlating the in vitro results with reported in vivo response data. Methotrexate and cisplatin (clinically active) and vindesine and doxorubicin (less active clinically) were recognized in vitro as active and correlated well with clinical experience. Bleomycin (clinically active) was ineffective against some cell lines. The in vitro findings for the clinically inactive drugs (deoxyazacytidine, lomustine, and carmustine) also corresponded. Amsacrine and etoposide, contrary to clinical experience, showed activity in vitro. Further comparison of MTT assay results with clinical data is warranted and essential before its use in large-scale drug screening studies.

Acquired von Willebrand syndromes: clinical features, aetiology, pathophysiology, classification and management.

Acquired von Willebrand syndrome (AVWS) associated with hypothyroidism is of type I, results from a decreased synthesis of factor VIII and von Willebrand factor (VWF), responds to desmopressin with normal half-life times for factor VIII and VWF parameters, and disappears after treatment with I-thyroxine. AVWS type I or III, which occurs in a minority of patients with Wilms' tumour in the complete absence of an inhibitor against VWF and no absorption of factor VIII or VWF onto nephroblastoma cells, responds to chemotherapy and/or tumour resection. Hyaluronic acid produced by nephroblastoma cells may be the causative factor in atypical AVWS in Wilms' tumour. AVWS associated with thrombocythaemia of various myeloproliferative disorders is characterized by normal factor VIII and von Willebrand factor antigen (VWF: Ag) levels and a selective deficiency of functional ristocetin co-factor activity (VWF: RCo) and collagen-binding activity (VWF: CBA). AVWS type II in thrombocythaemia is caused by a platelet-dependent proteolysis of large VWF multimers, given the inverse relationship between platelet count and large VWF multimers in plasma and specific increases in the number of proteolytic VWF fragments in plasma. The laboratory findings of AVWS associated with systemic lupus erythematosus or IgG benign monoclonal gammopathy are characterized by a prolonged bleeding time and activated partial thromboplastin time, decreased or absent ristocetin-induced platelet activity, low to very low levels of factor VIII coagulant activity (mean 15%), VWF: Ag (mean 10.7%) and VWF: RCo (mean 6.2%), and a type II multimeric pattern of VWF. Neutralizing and non-neutralizing anti-VWF autoantibodies, usually IgG, have been detected in patient plasma either free or tightly bound to the intermediate and high molecular weight VWF factor VIII particles. The bound auto antibody-antigen complex is rapidly cleared from the circulation, resulting in low levels of factor VIII, VWF parameters as documented by a poor response to desmopressin and VWF factor VIII concentrate. High-dose intravenous immunoglobulin transiently corrects the factor VIII coagulant and VWF levels, lasting for a few weeks in AVWS type II associated with systemic lupus erythematosus or IgG benign monoclonal gammopathy. Prednisolone is effective in AVWS associated with autoimmune disorder. Prednisolone and chemotherapy will not affect AVWS associated with IgG benign monoclonal gammopathy because the monoclonal IgG protein remains to act as an anti-VWF autoantibody. An absorption of VWF to malignant cells has been documented in a few patients with various lymphoproliferative disorders or adrenal carcinoma and suggested to result in a depletion of VWF. The clinical picture of AVWS associated with early-stage IgG multiple myeloma, chronic lymphocytic leukaemia or non-Hodgkin's lymphoma without a paraprotein or no detectable underlying disorder is similar to that of AVWS type II in IgG benign monoclonal gammopathy but poorly documented with regard to the underlying immune mechanism of AVWS. The mechanical destruction of large VWF multimers may be of relevance in conditions in which the shear rate of flowing blood is increased, as may occur in cases of aortic stenosis, other heart valve defects or stenosed vessels. Drug-induced AVWS has been described in association with the use of pesticides valproic acid, ciprofloxacin, griseofulvin, tetracycline, thrombolytic agents and hydroxyethyl starch.

Comparative effect of cisplatin, spiroplatin, carboplatin and iproplatin in a human tumor clonogenic assay.

A modified double-layer Hamburger and Salmon cloning assay was used to test cisplatin and its analogs (spiroplatin, carboplatin and iproplatin) on fresh tumor samples from 63 patients with a variety of non-hematological malignancies. Among them were 18 breast cancers, 17 ovarian cancers and 7 of unknown primaries. Half the patients received prior chemotherapy. Cisplatin regimens were given in 16 cases. When possible, cells were exposed for 1 h to each drug in concentrations of 0.1 microgram/ml and 1.0 microgram/ml for cisplatin and spiroplatin, 1.0 microgram/ml and 10 micrograms/ml for carboplatin and iproplatin. A greater than or equal to 50% cell kill with at least one drug was found in 20 samples including 8 ovarian cancers, 3 breast cancers and 1 unknown primary. A greater than or equal to 70% cell kill was seen in 2 samples with cisplatin, 3 with spiroplatin and carboplatin, and 6 with iproplatin. There was only partial cross-resistance between cisplatin and its analogs. Among 57 paired comparisons of cisplatin with spiroplatin, 2 showed drug sensitivity to cisplatin alone, 6 to spiroplatin alone, and 6 to both. The same sort of observation was made with carboplatin. The lack of cross-resistance between cisplatin and iproplatin was particularly striking: among 53 pairs, 6 were sensitive to cisplatin alone, 8 to iproplatin alone, and 2 to both. About 20% of the samples that were resistant to cisplatin were sensitive to iproplatin. Our data show hints of activity in breast and ovarian cancers with all analogs and suggest that they will achieve clinical antitumor activity similar to that they will achieve clinical antitumor activity similar to that of cisplatin. The in vitro evidence of incomplete cross-resistance between cisplatin and its analogs should be investigated further.

Blastic phase chronic myeloid leukemia with a four-break rearrangement: t(11;9)(9;22)(q23;p22q34;q11).

Chromosome analysis of bone marrow (BM) aspirate from a 36-year-old man with chronic myeloid leukemia (CML) in blastic phase (BP) showed a four-break rearrangement t(11;9)(9;22)(q23; p22q34;q11), which can be considered a t(9;22)(q34;q11) and a secondary t(9;11)(p22;q23). It is not surprising that additional chromosome abnormalities occur in patients with Ph-positive CML in BP, but it is of interest that t(9;11)(p22;q23), characteristic of acute myeloid leukemia French-American-British (FAB) type M5 (ANLL-M5) was observed. The possible meaning of this additional change in BP of CML is discussed.

Different invasion capacity of NBT II and MDCK in the chick embryo chorioallantois.

Questions have been raised as to the validity of the chick embryo chorioallantoic membrane for studying malignant invasion. Two cell lines, the invasive and non-invasive character of which we have documented in another system, were used in the CAM model to test for similar behaviour. Results showed that NBT II cells, an invasive tumour in the rat and in the confrontation assay, penetrated the ectoderm and grew within the mesoderm. MDCK, of low tumourigenicity in nude mice and non-invasive in the confrontation assay, did not form morphologically defined tumours and no MDCK cells were seen in the mesoderm.

Distal deep venous thrombosis in a hemophilia A patient with inhibitor and severe infectious disease, 18 days after recombinant activated factor VII transfusion.

We describe a 38 year old hemophilia A patient with a factor VIII inhibitor who was admitted to our Hematology Department in January 2001 with a seriously infected and bleeding perianal ulcer. To treat infection and bleeding the patient received broad spectrum antibiotics and recombinant activated factor VII (rFVIIa) (Novoseven(R)) for about 1 month (see detailed time of administration and dosing schedule of rFVIIa further in text). Eighteen days after his last rVIIa infusion the patient developed an ultrasound proven right calf vein thrombosis. In the whole period of admission, preceding the thrombotic event the patient biologically showed a picture of severe systemic inflammatory disease as indicated by persistent increased levels of D-dimer and fibrinogen (table). It is an interesting point of discussion whether the calf thrombosis was provoked as a consequence of rFVIIa infusion (with symptoms 18 days after the last infusion) or as a consequence of long-standing immobilization and severe inflammatory disease immobilization and severe infection are conditions well known for promoting venous thromboembolic disease.

Folate and Vitamin B(12) deficiency presenting as pancytopenia in pregnancy: a case report and review of the literature.

We present a case of extreme pancytopenia in a 27-year-old pregnant woman. The initial picture was compatible with a severe hematological problem in the category of aplastic anemia, paroxysmal nocturnal hemoglobinuria or even acute leukemia. The further biochemical investigations revealed, however, a folate deficiency. Nowadays this is a very rare cause of pancytopenia. Next to this she also had a Vitamin B(12) deficiency due to intrinsic factor failure. The recent literature is discussed.

Atypical variant of acquired von Willebrand syndrome in Wilms tumor: is hyaluronic acid secreted by nephroblastoma cells the cause?

Acquired von Willebrand syndrome (AvWS) has been reported in eight children with Wilms tumor (nephroblastoma in four boys and four girls) at a mean age of 3.3 years (range, 0.33-9 years). Only three of eight patients with AvWS in Wilms tumor presented with mild mucocutaneous bleeding symptoms. The AvWS in seven children with Wilms tumor featured either undetectable or very low von Willebrand factor antigen (vWF.Ag) levels (mean, 3%) and decreased values for vWF ristocetin cofactor (RCF) activity (mean, 20%) and factor VIII coagulant (VIIIc) activity (mean, 16%). The response to 1-desamino-8-arginine vasopressin (DDAVP) was good in two and poor in one patient. Multimeric analysis of the vWF showed a normal pattern of type I von Willebrand disease (vWD) in three patients and an absence of multimers consistent with type III vWD in two patients. The higher functional levels, as compared with antigen levels, with increased ratios for factor VIIIc/vWFAg (mean, 5.3) and vWF.RCF/vWF.Ag (mean, 6.6) in seven patients with Wilms tumor are unexplained physiologically and are not consistent with type I vWF deficiency. The absence of vWD in the patient's family, and the return of factor VIII-vWF parameters to normal after chemotherapy or surgical removal of the Wilms tumor, support the diagnosis of AvWS causally related to the Wilms tumor. The causative agent is thought to be hyaluronic acid secreted by nephroblastoma cells of the Wilms tumor. Prospective studies to determine the nature of AvWS in children with Wilms tumor are warranted.

Acquired von Willebrand syndrome in systemic lupus erythematodes.

Acquired von Willebrand syndrome (AvWS) in systemic lupus erythematodes (SLE) is caused by autoantibodies directed against the circulating von Willebrand factor (vWF)/factor VIII (FVIII) complex. The autoantibody-vWF/FVIII antigen complex is cleared rapidly from the circulation, leading to a moderate to severe quantitative and qualitative deficiency of both vWF and FVIIIc. Consequently, AvWS in SLE is featured by a prolonged bleeding time and normal platelet count, a prolonged activated partial thromboplastin time (APTT) and normal prothrombin time (PT), decreased or absent ristocetin-induced platelet aggregation (RIPA), and type II vWF deficiency on multimeric analysis of the vWF protein. Acquired von Willebrand syndrome type II in SLE responds poorly to 1-desamino-8-D-arginine vasopressin (DDAVP) and FVIII concentrate, but responds transiently well to high-dose gammaglobulin given intravenously. All reported cases of AvWS in SLE were cured by appropriate treatment of the underlying autoimmune disease with prednisone or immunosuppression.