Calcaneotibial screws for immobilisation of the tarsocrural joint of dogs in extension: effect of the angle of screw placement on the force to failure in a canine cadaveric model.

AIMS: To compare the force to failure under axial loading of a calcaneotibial screw placed approximately perpendicular to the tibia with that of a screw placed perpendicular to the calcaneus, when used to immobilise the tarsus in an ex vivo canine model. METHODS: Twelve pairs of cadaveric hindlimbs from large breed dogs, without orthopaedic or soft tissue disease, were prepared by transecting the limb at the level of the stifle and stripping the limbs of all musculature from the stifle to mid-metatarsus, including removal of the common calcaneal tendon from all limbs. The limbs in each pair were randomly assigned to receive a calcaneotibial screw placed perpendicular to the long axis of either the calcaneus (C group) or the tibia (T group) with the tarsus in full extension. The distal limb was potted in resin and the proximal tibia was pinned to allow biomechanical testing in compressive loading. Testing was performed to apply an axial load using a material testing machine in a proximodistal direction through the tibia, advancing at a rate of 10 mm/second. The force to failure was recorded in kN and compared between groups. RESULTS: The median force to failure of the C group was 0.86 (min 0.50; max 1.64) kN which was higher than the T group which had a median force to failure of 0.74 (min 0.26, max1.05) kN (p = 0.004). All modes of failure were by screw pull-out. CONCLUSIONS: A calcaneotibial screw placed at an angle approximately perpendicular to the long axis of the calcaneus, has a higher force to failure under axial loading than a calcaneotibial screw that is placed at an angle approximately perpendicular to the tibia, in a canine cadaveric model. CLINICAL RELEVANCE: A temporary calcaneotibial screw is a common method of immobilising the tarsus in extension to protect primary repair of a common calcaneal tendon injury. Placing a calcaneotibial screw perpendicular to the calcaneus may be a more reliable option for immobilisation of the tarsus to protect a common calcaneal tendon repair compared to screws placed perpendicular to the tibia. However extrapolation of these results into a clinical setting requires caution.

Clonal gammopathies following alemtuzumab-based reduced intensity conditioning haematopoietic stem cell transplantation: association with chronic graft-versus-host disease and improved overall survival.

The presence of clonal gammopathies (CG) has been reported following both conventional myeloablative and autologous haematopoietic stem cell transplantation (HSCT). We monitored the occurrence of CG in a cohort of patients with myeloid malignancies receiving FBC (fludarabine-busulphan-alemtuzumab)-based reduced intensity conditioned (RIC) HSCT, and assessed its correlation with infections, graft-versus-host disease (GvHD) and survival. Serial serum protein electrophoresis was analysed in a total of 138 patients and CG were detected in 49 patients (36%). The predominant Ig isotype was IgG (82%). There was no difference in the incidence of viral infections between patient groups. However, patients with gammopathies were more likely to have had prior chronic GvHD (OR 2.7, 95% CI 1.3-5.5, P<0.001). On multivariate analysis, the only factors that were found to influence overall survival (OS) were presence of gammopathies, which was associated with an improved OS (OR 0.35 95% CI 0.14-0.86, P=0.02) as well as disease stage, patients with advanced disease having a higher risk of death (OR 2.20 95% CI 1.18-4.11, P=0.02). Disease stage was the only variable that influenced relapse incidence on multivariate analysis (OR 4.22 95% CI 1.82-9.78, P<0.01). Clonal gammopathies are a frequent but benign occurrence following alemtuzumab-based RIC HSCT, and their appearance may define a group of patients with a favourable overall outcome.

Progenitor-cell mobilization after low-dose cyclophosphamide and granulocyte colony-stimulating factor: an analysis of progenitor-cell quantity and quality and factors predicting for these parameters in 101 pretreated patients with malignant lymphoma.

PURPOSE: To define parameters that predict for rapid engraftment after peripheral-blood stem-cell (PBSC) transplantation, progenitor thresholds, the proportion of patients who achieve these thresholds with a standardized mobilization regimen, and the factors that predict for mobilization efficiency. PATIENTS AND METHODS: One hundred and one patients with pretreated lymphoma were mobilized with cyclophosphamide 1.5 g/m2 and granulocyte colony-stimulating factor (G-CSF), with the first apheresis performed when the recovery WBC count was > or = 5.0 x 10(9)/L. The relationship between the number of progenitor cells collected and patient age, sex, diagnosis, prior radiotherapy, and time since last chemotherapy was determined by multivariate analysis. The relationship between these factors, progenitor numbers returned, post-PBSC G-CSF, and hematologic recovery was performed in 81 patients following chemotherapy with carmustine (BCNU), etoposide, cytarabine, and melphalan (BEAM protocol). RESULTS: No BEAM recipients had delayed neutrophil recovery beyond 28 days. Delayed platelet recovery occurred in 7.4% and minimum and optimum thresholds of 1 x 10(6) and 3.5 x 10(6) CD34+ cells/kg and 1 x 10(5) and 3.5 x 10(5) granulocyte-macrophage colony-forming cells (GM-CFC)/kg were established. Hematologic recovery was adversely affected by prior treatment with mini-BEAM, and neutrophil recovery was accelerated by post-PBSC G-CSF. The minimum GM-CFC threshold was achieved with a single apheresis in 83% of patients and in 90% with two aphereses. The optimal threshold was achieved with two leukaphereses in 69% of patients. Prior radiotherapy adversely affected mobilization. CONCLUSION: Hematopoietic recovery following PBSC is dependent on progenitor-cell number infused and affect of previous chemotherapy on progenitor quality. Progenitor-cell mobilization is adversely affected by prior radiotherapy. The minimum threshold of GM-CFC required is achieved in most patients with a single apheresis, but an optimal collection usually requires at least two harvests.

Back-up bone marrow is frequently ineffective in patients with poor peripheral-blood stem-cell mobilization.

PURPOSE: To assess hematologic recovery and procedure-related mortality in patients who received high-dose therapy with stem-cell support, in whom the peripheral-blood stem-cell (PBSC) collection fails (CD34+ cells < 1 x 10(6)/kg). The predictive value of granulocyte-monocyte colony-forming cell (GM-CFC) measurements and the value of bone marrow obtained after PBSC collection failure was assessed. PATIENTS AND METHODS: The study group comprised 324 consecutive patients mobilized with granulocyte colony-stimulating factor (G-CSF) and cyclophosphamide (273 patients), G-CSF with other chemotherapy (37 patients), and G-CSF alone (14 patients). Between one and four aphereses were performed. RESULTS: In 51 of 324 patients, there was failure to obtain 1 x 10(6)/kg CD34+ cells. Twenty-three patients had greater than 1 x 10(5)/kg GM-CFC; 22 patients proceeded to high-dose therapy. Neutrophil recovery occurred within 21 days, but platelet independence was delayed (> 28 days) in eight patients. Of 28 patients with less than 1 x 10(5)/kg GM-CFC, six received high-dose therapy with PBSC alone and five had delayed engraftment. Twelve patients with less than 1 x 10(5)/kg GM-CFC received high-dose therapy supported by bone marrow collected after PBSC collection failure. Eleven patients were assessable for engraftment; four patients had slow (> 21 days) or delayed (> 28 days) neutrophil recovery and eight patients had delayed platelet recovery. In the group of patients who received less than 1 x 10(5)/kg GM-CFC, there were five procedure-related deaths. CONCLUSION: This study shows that delayed hematologic recovery is frequent if less than 1 x 10(6)/kg CD34+ cells are infused after high-dose therapy, particularly with GM-CFC less than 1 x 10(5)/kg. The procedure-related mortality in this latter group is high. In most patients whose PBSC collection contains less than 1 x 10(5)/kg GM-CFC, the use of bone marrow cells does not improve engraftment, which suggests that poor PBSC mobilization usually indicates poor marrow function.

Analysis of mutations in the GM-CSF receptor alpha coding sequence in patients with acute myeloid leukaemia and haematologically normal individuals by RT-PCR-SSCP.

Mutations of signal transducing molecules such as Ras have been shown to confer a growth advantage in leukaemic blasts and contribute to the pathogenesis of the disease. Alterations of signal transducing molecules other than Ras may play a role in leukaemogenesis. Knowledge of such mutations could also further our understanding of the normal signalling processes. We have therefore studied the coding sequence of the GM-CSF receptor alpha chain (GM-CSFR alpha) in patients with acute myeloid leukaemia (AML) and non-AML controls using single strand conformation polymorphism (SSCP) analysis. Abnormalities were detected in 4/32 AML patients (13%) and 2/15 non-AML controls (13%). Direct sequencing of PCR products revealed five different base substitutions. Three were conservative, two caused amino acid changes. The base substitution leading to amino acid change alanine to glycine at position 17 was found in both an AML patient and a control. It lies in the signal sequence and does not affect the mature protein. The other base change altering arginine to glutamine at position 164 is unlikely to influence the receptor structure as this structural position in the chain is not well conserved in members of the cytokine receptor family. Both amino acid changes were constitutive alterations as they could be demonstrated in the patients' children. The base changes described in the AML patients thus represent polymorphisms and do not contribute to the pathogenesis of AML.

Fc gamma RII-mediated superoxide production by phagocytes is augmented by GM-CSF without a change in Fc gamma RII expression.

Freshly purified neutrophils and monocytes respond to multiple cross-linking of Fc gamma RII with the IgG1 monoclonal antibody, CIKM5, with a rapid rise in Ca(2+)i, but not with a respiratory burst, although superoxide is generated by these cells when stimulated with the chemotactic peptide, FMLP, or phorbol ester (TPA). Incubation in vitro for 30-60 min at 37 degrees C in medium + 0.1% FCS had no effect on the neutrophil superoxide response to CIKM5 but induced a weak monocyte response in 11/13 experiments. However, incubation with rhGM-CSF (10 ng/ml) under similar conditions induced a neutrophil respiratory burst in response to cross-linking Fc gamma RII in 12/14 experiments and enhanced the monocyte response by 181%. GM-CSF also enhanced the response of neutrophils and monocytes to FMLP by 308% and 165%, respectively. The response to TPA was not significantly enhanced by GM-CSF. rhIFN-gamma (100 mu/ml) was ineffective as a priming agent for all agonists tested in short-term incubations but augmented the monocyte response to CIKM5 after 5 d exposure in vitro. Whilst GM-CSF induced neutrophil superoxide production in response to cross-linking Fc gamma RII, there was no concomitant change in Fc gamma RII expression either in in vitro studies of neutrophils from healthy individuals or in in vivo studies of patients receiving GM-CSF. Stimulation of unprimed neutrophils with CIKM5 induced a rapid transient increase in intracellular calcium levels to 181% of resting levels. However, incubation with GM-CSF did not further augment the calcium transients above the stimulated level. The mechanism by which GM-CSF induces an enhanced respiratory burst in response to cross-linking of Fc gamma RII remains to be elucidated, but is not related to receptor expression or increases in receptor mediated calcium mobilization.

Effect of changes in expression of the amphotropic retroviral receptor PiT-2 on transduction efficiency and viral titer: implications for gene therapy.

The aim of this study was to quantify the impact of amphotropic retroviral receptor (PiT-2) levels on susceptibility to transduction and to determine whether the low level of PiT-2 found on CD34+ hematopoietic cells is within the range likely to compromise gene transfer. Receptor-deficient Chinese hamster ovary (CHO) cells were transfected with a PiT-2 construct that could be induced by the removal of tetracycline. The level of PiT-2 expression measured by virus binding in uninduced and in fully and partially induced transfectants correlated with the efficiency of transduction by an amphotropic retroviral reporter vector. Fully induced CHO-PiT-2 cells gave apparent viral titers similar to NIH 3T3 fibroblasts while addition of tetracycline reduced titers by up to 140-fold. Binding of the same vector preparation to purified CD34+ peripheral blood stem cells (PBSCs) was always less than to uninduced CHO-PiT-2 transfectants even after preincubation in 10-ng/ml concentrations of IL-3, IL-6, and stem cell factor, which increased retroviral binding by an average of 35%. The level of expression of the amphotropic retroviral receptor PiT-2 thus significantly limits transduction efficiency within the range observed in target cells of importance in human gene therapy.

Therapy associated leukaemia.

A dose related risk of acute leukaemia, myelodysplasia and other cancers is seen in patients treated with certain drugs, particularly alkylating agents, and radiotherapy either alone or in combination. Treatment associated acute myeloid leukaemia (tAML) and myelodysplasia have biological and clinical features in common and are distinct from the corresponding de-novo disorders. tAML generally occurs between 2-11 years from administration of chemo/radiotherapy with few cases thereafter. Patients may present with myelodysplasia and severe cytopenia with abnormalities in all cell lines or as an acute leukaemia which may be difficult to classify because of multi-lineage involvement. Clonal cytogenetic abnormalities usually including either loss or interstitial deletion of the long arm of chromosomes 5 or 7 are frequently identified. Critical regions deleted in all patients with these lesions have been localised at 5q23-32 and 7q22-31; regions carrying the genes for several haemopoietic growth factors, receptors and oncogenes. The prognosis of patients with tAML is poor with low remission rates and a median survival of 6 months. Complex karyotypes or lesions of chromosomes 5 or 7 seen in the majority of cases are associated with a particularly poor outlook. The risks of this most serious complication of therapy should be weighed carefully against possible benefits.

Reduced-intensity rituximab-BEAM-CAMPATH allogeneic haematopoietic stem cell transplantation for follicular lymphoma is feasible and induces durable molecular remissions.

The indolent non-Hodgkin's lymphomas are theoretically curable through allogeneic haematopoietic stem cell transplantation (allo-HSCT). The applicability of standard conditioning allo-HSCT is, however, restricted by its toxicity. Recently, reduced-intensity conditioning regimens have demonstrated efficacy with significantly reduced early morbidity and mortality. We examined the safety and efficacy of rituximab-BEAM-CAMPATH as reduced-intensity conditioning for allo-HSCT in follicular lymphomas. Minimal residual disease was assessed by polymerase chain reaction (PCR) for bcl-2/IgH translocations, and chimerism by X,Y-FISH or PCR amplification of short tandem repeat sequences. At a median follow-up of 521 days (371-719), four of five patients were alive and three were in complete molecular remission. Three patients required pre-emptive treatment for CMV reactivation. One succumbed to a perforated viscus and one had slowly progressive disease. A graft-versus-lymphoma effect was demonstrable and quantitative PCR for bcl-2/IgH may allow better accuracy in scheduling post-allograft rituximab and/or donor lymphocyte infusions. Although follow-up is short, reduced-intensity allo-HSCT with BEAM-CAMPATH conditioning appears to be safe, effective in inducing a molecular remission and is potentially curative. Further recruitment and much longer follow-up will be necessary to determine if this impacts favourably upon disease-free and overall survival.

Fatal donor-derived Epstein-Barr virus-associated post-transplant lymphoproliferative disorder following reduced intensity volunteer-unrelated bone marrow transplant for myelodysplastic syndrome.

Post-transplant lymphoproliferative disorders (PTLD) are a well recognised complication of conventional haemopoietic stem cell transplantation (HSCT). Reduced intensity HSCT involves intensive immunosuppression to permit engraftment. Thirty reduced intensity transplants with the FBC (fludarabine 150 mg/m2, busulphan 8 mg/m2, CAMPATH-1H 100 mg) protocol have been performed at our centre, with one confirmed EBV-positive PTLD. The female recipient developed a perforated viscus day +191 following HSCT from a volunteer unrelated male donor. A large caecal mass and a retroperitoneal abscess were excised, revealing an EBV-positive diffuse large B cell lymphoma confirmed by FISH to be of donor origin. More experience is required before the risk of PTLD in this setting can be assessed.

An unusual case of intrapulmonary granulocytic sarcoma presenting as interstitial pneumonitis following allogeneic bone marrow transplantation for acute myeloid leukaemia and responding to donor lymphocyte infusion.

We report a 45-year-old female with AML who underwent a T cell-depleted sibling allograft and relapsed a year later with extramedullary disease involving the lung parenchyma and presenting with the clinical and radiological features of interstitial pneumonitis. The patient was treated with donor lymphocyte infusion (DLI) resulting in complete resolution of the radiological signs. The unusual presentation and the management options are discussed.

Evaluation of clinical scale CD34+ cell purification: experience of 71 immunoaffinity column procedures.

Seventy-one mobilised PBSC collections were subject to CD34+ cell purification using the CEPRATE SC stem cell concentration system. The overall median purity of CD34+ cells was 69% (6-93%). CD34+ cell, and GM-CFC recoveries were 52% (8-107%) and 36% (3-118%). Purity was logarithmically related to the input percentage of CD34+ cells and starting requirements were established of 1% CD34 cell content for optimal purity and a minimum of 2 x 10(6)/kg CD34+ cells to ensure recovery of our minimum engraftment threshold of 1 x 10(6)/kg CD34+ cells. Reduction of the washing steps reduced non-specific cell losses and shortened the procedure but did not affect progenitor cell recovery. Purified CD34+ cells were reinfused following high-dose therapy in 35 patients. The median time to neutrophil recovery of 0.5 x 10(9)/l was 12 (10-23) days and to the attainment of platelet independence was 13 (7-100) days. The risks of delayed platelet recovery were related to the CD34+ cell dose infused and were identical to the risks when non-purified PBSC collections were used. In conclusion, purification of CD34+ cells using the CEPRATE device is reliable and the purified product results in prompt engraftment. The cell losses that occur do however restrict its use in many patients.

Immediate side effects of large fraction radiotherapy.

The use of hypofractionated radiotherapy regimens is becoming more widely recognized in the palliation of non-small cell lung carcinoma (NSCLC). Anecdotal reports of chest pain, rigors and fevers in the hours that follow radiotherapy led us to perform a survey estimating the frequency and severity of these symptoms following treatment to the thorax. One hundred and eighteen patients completed questionnaires 24 hours after palliative radiotherapy treatment; 84 were male. The median age was 67 years. One hundred and seven had histologically confirmed NSCLC. A parallel opposed technique was used in 113 patients. Doses ranged from 8 Gy in a single fraction to 60 Gy in 30 fractions. Chest pain was reported by 54 (45.8%) patients after the first radiotherapy fraction; in 42 it commenced within 12 hours of treatment. The pain varied in site, nature and duration; on 23 occasions, it lasted under 2 hours. Systemic symptoms (rigors, sweating, fevers) were documented on 43 questionnaires, starting within 12 hours of treatment in 33 patients and on 30 occasions lasting less than 2 hours. Chest pain and systemic symptoms occurred together in 28 patients. Only 49 (41.5%) patients reported no immediate side effects. We conclude that patients receiving palliative radiotherapy for bronchial carcinoma often develop significant symptoms in the hours following treatment. The timing and duration suggest a relationship with the radiotherapy, and we feel that patients should be warned of the possible occurrence of these symptoms.

Leukaemia complicating treatment for Hodgkin's disease: the experience of the British National Lymphoma Investigation.

OBJECTIVE: To determine the incidence of and risk factors for the development of secondary acute leukaemia and myelodysplasia in patients treated in British National Lymphoma Investigation's studies of Hodgkin's disease since 1970. PATIENTS: 2676 Patients entered into Hodgkin's disease studies between February 1970 and November 1986. Data accrued up to November 1988 were analysed, ensuring a minimum follow up period of two years. DESIGN: Retrospective analysis of multicentre trial data by case-control and life table methods. RESULTS: 17 Cases of secondary leukaemia were recorded in this group of 2676 patients, giving an overall risk at 15 years of 1.7%. The risks of leukaemia after chemotherapy alone and chemotherapy with radiotherapy were not significantly different. The risk of leukaemia increased sharply with the amount of treatment given as measured by the number of attempts at treatment. The 15 year risks of leukaemia were 0.2%, 2.3%, and 8.1% for patients receiving one, two, or three or more attempts at treatment. The highest risk, 22.8% at 15 years, was observed in patients treated with lomustine (CCNU), and a case-control study suggested that this was an independent risk factor. The risk of secondary leukaemia was largely related to the overall quantity of treatment, although exposure to lomustine seemed to be an important risk factor. Treatment with both drugs and radiation was not more leukaemogenic than treatment with drugs alone. The greatest risk of secondary leukaemia was seen in multiply treated patients who were unlikely to be cured of Hodgkin's disease. CONCLUSIONS: Avoidance of secondary leukaemia should be a minor factor in the choice of treatment for Hodgkin's disease.

Analysis of a potential cluster of rhinovirus infections in patients and staff on two haemato-oncology wards.

BACKGROUND: Human rhinoviruses (HRV) cause the common cold, increased mortality in patients attending elderly care facilities and significant morbidity as well as mortality in the post-transplantation setting. OBJECTIVES: The aim of the study was to determine if there had been a breakdown in infection control practice in a large haemato-oncology centre. Molecular techniques had detected increased numbers of HRV in respiratory samples from patients and staff over a 6-week period. Typing was performed to investigate the possibility of transmission between individuals. STUDY DESIGN: This was a retrospective study having detected HRV RNA in combined nose and throat swab samples that were collected from 13 individuals: 8 patients and 5 staff members, in the haemato-oncology wards of a tertiary referral centre in January and February 2011. The 5'NTR and the VP4/VP2 region were used for HRV typing. RESULTS: All 3 HRV species were detected with 7 HRV-A, 1 HRV-B, 4 HRV-C and 1 untyped. None of the individuals were infected by the same HRV serotype. Three individuals had multiple samples collected: 1 patient had an HRV-B infection over a 4-week period, 1 patient had an HRV-A infection over 3 months and 1 staff member had an HRV-C infection over 1 week, each shedding an unchanged serotype throughout the whole period. CONCLUSION: Nucleotide sequence analysis confirmed that there was no breakdown in infection control measures. No transmission incidents had occurred between patients and/or between staff and patients.