ALBERT

Description: Accurate prediction of venous thromboembolic (VTE) risk remains a clinical challenge, and no prospectively validated risk scoring systems have been described. Recently, a computer-based alert to highlight hospitalised patients at risk of VTE that employed a simple risk score based on eight principal VTE risk factors was shown to improve patient outcome (N Engl J Med2005;352:969–977). The objective of this study was to assess the performance of this risk score in identifying those at risk of VTE. The study cohort consisted of all patients who were enrolled in ‘VERITY’ up to 30th November, 2004. VERITY is a UK, prospective VTE treatment registry that enrolls patients presenting to hospital with suspected VTE (www.verityonline.co.uk). The vast majority of patients enrolled have no history of hospitalization and therefore patients will not have been risk assessed or have been given thromboprophylaxis, so essentially reflecting an untreated population. The risk score was applied to the patient database and a score calculated for each patient for whom all 8 risk factors were known. Risk was scored as previously described: score=3 for cancer, prior VTE, hypercoagulability; score=2 for major surgery; score=1 for age〉70 years, BMI〉29, bed rest not related to surgery, use of hormone-replacement therapy or oral contraceptives. An increased risk was defined as a cumulative risk score of at least 4. Of 27,179 patients presenting with suspected VTE, 6,124 had a positive diagnosis of deep vein thrombosis, pulmonary embolism, or both; in 15,980 cases the diagnosis was not confirmed. All 8 risk factors were known for 5,692 cases (1880 with confirmed VTE [31%] and 3812 VTE-negative cases [24%]). An increasing risk score was associated with an approximately linear rise in the proportion of patients with confirmed VTE; more than 50% of patients with a risk score ≥4 were diagnosed with VTE, rising to more than 71% in those with a score 〉7 (see figure). Although this patient cohort is not identical to the population the risk score was developed for, these data suggest the value of this weighted risk score in identifying those at risk of VTE. Further refinement of this score, perhaps using more sophisticated modeling to increase the accuracy of the score to predict thromboembolic events, would be highly valuable for routine integration of VTE assessment into clinical practice. Final diagnosis of VTE and weighted risk score (n=5,692) Final diagnosis of VTE and weighted risk score (n=5,692)

Description: A failure of IL-2 transcription has been associated with tolerance induction. We hypothesized that inhibition of the NF-κB pathway in alloreactive T-cells, which is critical for IL-2 transcription, could lead to alloantigen-specific hyporesponsiveness and prevention of GVHD. PS1145, a potent inhibitor of IκB kinase, and hence NF-κB activation, was added to an MLR culture consisting of CD4+ T-cells and MHC class II-disparate stimulators. Inhibition of NF-κB activity was verified by EMSA and confocal microscopy. Global inhibition of cytokine production and T-cell hyporesponsiveness was observed which persisted after washing T-cells and re-exposure to alloantigen. Responses to non-specific mitogens remained largely intact and alloantigen hyporesponsiveness was reversed by exogenous IL-2. Treatment of T cells and stimulator cells with PS1145 was required for maximal effect. Depletion of CD4+CD25+ cells from the MLR indicated that these cells were not required for tolerance induction in this system. Using an MLR system containing alloreactive and non-alloreactive transgenic T cells indicated that PS1145 treatment increased the rate of T-cell apoptosis selectively in alloreactive cells. Data from each of 4 experiments showed that GVHD in recipients of ex vivo PS1145 treated cells was profoundly inhibited, whereas CD4+ T-cells recovered from a vehicle-treated 7-day MLR were uniformly fatal upon adoptive transfer into sublethally irradiated MHC class II-disparate recipients. Studies addressing non-alloreactive in vivo responses of PS1145 treated T cells will also be presented. Our studies indicate that the NF-κB pathway is a critical regulator of productive alloresponses and provide a novel ex vivo approach to induce alloantigen-specific tolerance as a means of preventing GVHD.

Description: Platelet-type Von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder. It results from an abnormally high affinity interaction between the platelet membrane glycoprotein Ib/IX/V complex and Von Willebrand factor (VWF), leading to characteristic platelet hyperaggregability. The condition shares most of the clinical and laboratory features of type 2B VWD and the final discrimination between these two diseases requires either platelet mixing studies or a molecular genetic approach. Five members of a British family with PT-VWD were studied; three affected and two unaffected. The proposita, had a long history of bleeding with recurrent epistaxis, postoperative and dental bleeding and menorrhagia. An initial series of hematological investigations lead to a diagnosis of type 2B VWD but a significant fall in her platelet count following therapy with a VWF/FVIII concentrate resulted in the consideration of PT-VWD as the correct diagnosis. This diagnosis was subsequently confirmed with appropriate FVIII and VWF assays. Affected individuals had VWF:Ag values in the range 0.34–0.47 U/mL, FVIII 0.45–0.64 U/mL and VWF:RCo ranging from 0.18–0.42 U/mL. These subjects showed loss of the HMW VWF multimers from plasma and enhanced ristocetin-induced platelet agglutination (RIPA). Also, washed normal platelets did not show enhanced RIPA when mixed with the patient’s plasma. On molecular genetic analysis of the GPIbα locus, the three affected family members were heterozygous for a 27 bp in-frame deletion (nucleotides 4374–4400). This corresponds to residues 421–429 in the macroglycopeptide region of GPIbα. This deletion was not identified in any of 50 normal controls. Flow cytometric quantitative analysis of the platelet GPIb/IX/V complex showed that the number of molecules for each of GPIb, GPIX and GPV, in each of the affected and non-affected family members fell within the normal range. Wild type (WT) GPIbα and the 27bp deletion mutant GPIba were then expressed in CHO b/IX cells. To examine the effect of the 27bp deletion on the conformation of the receptor, the cells were incubated with monoclonal antibodies that bind to different sites on GPIbα. We demonstrated that each of the three monoclonal antibodies bound WT and Mut GPIba to a similar extent and that the mutant protein was expressed at a normal level on the CHO cell surface. Finally, the effect of the 27bp deletion on VWF binding was analysed in CHO b/IX cells expressing the mutant and WT GPIbα receptor with 125I-VWF in the presence of varying ristocetin concentrations. We showed that the mutant receptor binds significantly more VWF than the WT GPIbα in the absence of ristocetin and also displays heightened sensitivity to low ristocetin concentrations compared to the WT cells. This study reports the first mutation resulting in PT-VWD that does not directly involve the VWF binding region of GPIbα. Our functional studies with the mutant receptor confirm that the deletion of 9 amino acids from the macroglycopeptide region of the protein enhances its interaction with VWF.

Description: The association between venous thromboembolism (VTE) and cancer is well-recognised, but the thrombosis risk factor profile of patients with cancer-associated VTE is poorly characterised; it is unclear if additional risk factors contribute to the risk of thrombosis beyond the cancer itself, or if the risk factor profile is tumour-specific. Our aim was to compare the thrombosis risk factor profiles of cancer patients with or without symptomatic VTE enrolled in VERITY, an ongoing UK prospective VTE registry. The VERITY registry records data on patients with VTE and those in whom the diagnosis of VTE is excluded. Between Jun 05 and Mar 08, 49044 patient entries were made. Individual case data for patients with cancer were extracted. Using available risk factor data, univariate analysis of 9 established risk factors for VTE (medical in-patient history/immobilisation 〉3 days within last 4 weeks; major surgery in the last 4 weeks; hormonal risk factor; previous history of VTE; family history of VTE; known thrombophilia; intravenous drug abuse; current smoking; and leg paralysis), comparing VTE and non-VTE cancer cases, was performed for the 4 most common cancers using SPSS. To account for the potential impact of age and sex on VTE risk, age-adjusted values were calculated for breast and prostate cancer, and age- and sex-adjusted values for colorectal and lung. A nominal level of 5% statistical significance was assumed. Of 2825 cancer cases, 1382 had an objectively confirmed diagnosis of VTE and in 1443 the diagnosis of VTE was excluded. Breast (n=498), prostate (n=374), colorectal (n=343) and lung cancer (n=275) accounted for 53% of cancer cases. Univariate associations between risk factors and symptomatic VTE were found only for prostate cancer: history of VTE (odds ratio [OR] = 3.48; 95% CI, 2.01, 6.02; p 〈 0.00001), family history of VTE (OR = 2.56; 95% CI, 1.02, 6.44; p = 0.046), hormonal risk factor (OR = 2.22; 95% CI, 1.00, 4.92; p = 0.049). In colorectal cancer, smoking was less likely in VTE cases (OR = 0.54; 95% CI, 0.34, 0.87; p = 0.012). Adjusting for age (and sex), univariate associations between risk factors and symptomatic VTE were again found only for prostate cancer: history of VTE (OR = 3.23; 95% CI, 1.56, 6.68; p = 0.002), with smoking less likely in age-adjusted VTE cases (OR = 0.50; 95% CI, 0.28, 0.91; p = 0.022). Our analysis of a registry population found few associations between known thrombosis risk factors and symptomatic VTE in patients with common cancers, suggesting these factors impact little on thromboembolic risk in these cancers.

Description: Accurate estimation of risk for venous thromboembolism (VTE) may help clinicians assess prophylaxis needs. Only empirical algorithms and risk scores have been described; an empirical risk score (‘Kucher’) based on 8 VTE risk factors (cancer, prior VTE, hypercoagulability, surgery, age〉75 yrs, BMI〉29, bed rest, hormonal factor) using electronic alerts improved hospitalized patient outcome (NEJM2005;352:969–77). We wished to develop a multivariate regression model for VTE risk, based on Kucher, and validate its performance. The initial derivation cohort consisted of patients enrolled in ‘VERITY’, a multicentre VTE treatment registry for whom the endpoint of VTE and all 8 risk factors were known. Initial univariate analysis (n=5928; 32.4% with diagnosis of VTE) suggested VTE risk was not accounted for by the 8 factors; an additional 3 were added (leg paralysis, smoking, IV drug use [IVD]). The final derivation cohort was 5241 patients (32.0% with VTE) with complete risk data. The validation cohort (n=915) was derived from a database of 928 consecutively enrolled patients at a single DVT clinic. Model parameters were estimated using the statistical package ‘R’ using a stepwise selection procedure to choose the optimal number of main effects and pair-wise interactions. This showed that advanced age (estimated odds ratio [OR]=2.8, p

Description: Cumulative thrombocytopenia is a dose-limiting toxicity of dose-intensive chemotherapy for advanced breast cancer. In this phase I study, we have studied the hematologic toxicity associated with sequential interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF; molgramostim) administration after multiple cycles of FLAC (5-fluorouracil, leucovorin, doxorubicin, cyclophosphamide) chemotherapy compared with that after concurrent cytokine administration or to each cytokine administered alone. Ninety- three patients with advanced breast cancer were treated with five cycles of FLAC chemotherapy and either IL-3 alone, GM-CSF alone, sequential IL-3 and GM-CSF administered by schedule A (5 days of IL-3 followed by 10 days of GM-CSF) or schedule B (9 days of IL-3 followed by 6 days of GM-CSF), or concurrent administration of IL-3 and GM-CSF for 15 days. Cohorts of patients were treated with one of four dose levels of IL-3 (1,2.5, 5, and 10 micrograms/kg) administered subcutaneously for each schedule of cytokine administration. The GM-CSF dose in all schedules was 5 micrograms/kg/day. Sequential IL-3 and GM- CSF (schedule B) was associated with higher platelet nadirs, shorter durations of platelet counts less than 50,000/microL, and the need for fewer platelet transfusions over five cycles of FLAC chemotherapy compared with concurrent cytokines, sequential IL-3 and GM-CSF schedule A, and GM-CSF alone. Concurrent IL-3 and GM-CSF was associated with unexpected platelet toxicity. The duration of granulocytopenia after FLAC chemotherapy was significantly worse with IL-3 alone compared with each of the GM-CSF-containing cytokine regimens. Although no cycle 1 maximum tolerated dose for IL-3 was defined in this study, 5 micrograms/kg was well tolerated over multiple cycles of therapy and is recommended for future studies. The data from this phase I study suggest that sequential IL-3 and GM-CSF with IL-3 administered for 9 days before beginning GM-CSF may be superior to shorter durations of IL- 3 administered sequentially with GM-CSF, to concurrent IL-3 and GM-CSF, and to either colony-stimulating factor alone in ameliorating the cumulative hematologic toxicity associated with multiple cycles of FLAC chemotherapy. Additional studies of sequential IL-3 and GM-CSF are warranted.

Description: We conducted a prospective randomized trial to evaluate the ability of the interleukin-3/granulocyte-macrophage colony-stimulating factor (GM- CSF) fusion protein, PIXY321, to ameliorate cumulative thrombocytopenia after multiple cycles of 5-fluorouracil, leucovorin, doxorubicin, cyclophosphamide (FLAC) chemotherapy compared with GM-CSF in patients with advanced breast cancer. Fifty-three patients were randomized to receive either PIXY321. 375 microg/m2 twice a day subcutaneously, or GM- CSF, 250 microg/m2 daily subcutaneously after FLAC chemotherapy. PIXY321 was less well tolerated than GM-CSF, with more patients developing chills and local skin reactions and more patients stopping PIXY321 due to intolerance. While no difference in the neutrophil nadirs was seen with the two cytokines, the duration of the absolute neutrophil count less than 1,000/muL for all cycles was significantly longer with PIXY321 than with GM-CSF. Fifty percent of patients treated with multiple cycles of FLAC chemotherapy on both study arms developed dose-limiting thrombocytopenia. No differences in platelet nadirs, duration of thrombocytopenia, or need for platelet transfusions were observed with PIXY321 versus GM-CSF. The average delivered doses of FLAC chemotherapy were somewhat higher in the GM-CSF study arm. PIXY321 was not superior to GM-CSF in ameliorating the cumulative thrombocytopenia observed with multiple cycles of FLAC chemotherapy and was less well tolerated.

Description: Cumulative thrombocytopenia is a dose-limiting toxicity of dose-intensive chemotherapy for advanced breast cancer. In this phase I study, we have studied the hematologic toxicity associated with sequential interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF; molgramostim) administration after multiple cycles of FLAC (5-fluorouracil, leucovorin, doxorubicin, cyclophosphamide) chemotherapy compared with that after concurrent cytokine administration or to each cytokine administered alone. Ninety- three patients with advanced breast cancer were treated with five cycles of FLAC chemotherapy and either IL-3 alone, GM-CSF alone, sequential IL-3 and GM-CSF administered by schedule A (5 days of IL-3 followed by 10 days of GM-CSF) or schedule B (9 days of IL-3 followed by 6 days of GM-CSF), or concurrent administration of IL-3 and GM-CSF for 15 days. Cohorts of patients were treated with one of four dose levels of IL-3 (1,2.5, 5, and 10 micrograms/kg) administered subcutaneously for each schedule of cytokine administration. The GM-CSF dose in all schedules was 5 micrograms/kg/day. Sequential IL-3 and GM- CSF (schedule B) was associated with higher platelet nadirs, shorter durations of platelet counts less than 50,000/microL, and the need for fewer platelet transfusions over five cycles of FLAC chemotherapy compared with concurrent cytokines, sequential IL-3 and GM-CSF schedule A, and GM-CSF alone. Concurrent IL-3 and GM-CSF was associated with unexpected platelet toxicity. The duration of granulocytopenia after FLAC chemotherapy was significantly worse with IL-3 alone compared with each of the GM-CSF-containing cytokine regimens. Although no cycle 1 maximum tolerated dose for IL-3 was defined in this study, 5 micrograms/kg was well tolerated over multiple cycles of therapy and is recommended for future studies. The data from this phase I study suggest that sequential IL-3 and GM-CSF with IL-3 administered for 9 days before beginning GM-CSF may be superior to shorter durations of IL- 3 administered sequentially with GM-CSF, to concurrent IL-3 and GM-CSF, and to either colony-stimulating factor alone in ameliorating the cumulative hematologic toxicity associated with multiple cycles of FLAC chemotherapy. Additional studies of sequential IL-3 and GM-CSF are warranted.