X-linked sideroblastic anaemia due to ALAS2 mutations in the Netherlands: a disease in disguise.

BACKGROUND: X-linked sideroblastic anaemia (XLSA; OMIM#300751) is the most common inherited form of sideroblastic anaemia and is associated with several mutations in the erythroid specific 5-aminolevulinate synthase gene (ALAS2). This gene encodes for aminolevulinic acid synthase 2 (ALAS2), the catalytic enzyme involved in the first en rate-limiting step of haem biosynthesis.1-3 The disorder is characterised by mostly mild hypochromic microcytic anaemia with bone marrow ring sideroblasts. Even untransfused patients with mild or no anaemia are at risk for severe systemic iron overload due to ineffective erythropoiesis. To date, 61 different ALAS2 mutations have been reported in 120 families with XLSA. Descriptions of molecularly confirmed case series from the Netherlands, however, are lacking. METHODS: We reviewed age of presentation, clinical and biochemical features, ALAS₋2 defects and treatment characteristics of 15 Dutch patients from 11 unrelated families diagnosed with XLSA. RESULTS AND CONCLUSIONS: In one family a novel pathogenic c.1412G>A (p.Cys471Tyr) mutation was found. All other families shared the previously described c.1355G>A (p.Arg452His) mutation. Haplotype analysis in seven probands with the p.Arg452His mutation strongly suggests that six of them were ancestrally related. Nevertheless, their phenotype was very different. Our patients illustrate the phenotypical heterogeneity in the presentation of XLSA patients, the effectiveness of treatment regimens and the various pitfalls associated with the diagnosis, follow-up and treatment of the disease. A timely diagnosis avoids unnecessary investigations and allows adequate treatment that can prevent systemic iron load with subsequent severe life-threatening complications. Therefore, we suggest considering XLSA in both male and female patients with unexplained iron overload and÷or (mild) microcytic anaemia, also at older age.

Short intensive sequential therapy followed by autologous stem cell transplantation in adult Burkitt, Burkitt-like and lymphoblastic lymphoma.

The feasibility and efficacy of up-front high-dose sequential chemotherapy followed by autologous stem cell transplantation (ASCT) in previously untreated adults (median age 33 years; range 15-64) with Burkitt lymphoma (BL), Burkitt-like lymphoma (BLL) or lymphoblastic lymphoma (LyLy), both without central nervous system or extensive bone marrow involvement was investigated in a multicenter phase II study. Treatment consisted of two sequential high-dose chemotherapy induction courses incorporating prednisone, cyclophosphamide, doxorubicin, etoposide and mitoxantrone, without high-dose methotrexate or high-dose cytarabine. Patients with at least PR went on with BEAM and ASCT. Protocol treatment was completed by 23/27 (85%) BL/BLL and 13/15 (87%) LyLy patients. Median treatment duration until BEAM was 70 (range: 50-116) days. No toxic deaths occurred. Response to treatment was complete response (CR) 81% and partial response (PR) 11% for BL/BLL, CR 73% and PR 20% for LyLy. At a median follow-up of 61 months of patients still alive, six BL/BLL and eight LyLy patients have died. The actuarial 5-year overall and event-free survival estimates are 81 and 73% for BL/BLL vs 46 and 40% for LyLy patients. In conclusion, this short up-front high-dose sequential chemotherapy regimen, followed by ASCT is highly effective in adults with BL/BLL with limited bone marrow involvement, but less so in patients with LyLy.

Follicular non-Hodgkin's lymphoma with refractory paraneoplastic pemphigus: case report with review of novel treatment modalities.

In this paper a patient with a non-Hodgkin's lymphoma (NHL) and paraneoplastic pemphigus (PNP) is described. PNP is a very rare, painful mucocutaneous intraepithelial blistering disease associated with occult or confirmed malignancy. Patients with PNP show severe, progressive mucocutaneous disease with a high mortality rate, because of drug-induced infectious complications. The patients sometimes benefit from high doses of oral corticosteroids. However, pulse therapy with high doses of prednisolone (or dexamethasone) in combination with other immunosuppressants induces variable and inconstant results. Intravenous immunoglobulin (IVIg) has been applied in different cases of PNP with encouraging results. Plasmapheresis or plasma exchange (PE) in combination with corticosteroids and/or cyclophosphamide or azathioprine showed similar rapid and beneficial results in association with decreasing auto-antibody levels in this group of refractory pemphigus. Another interesting therapeutic option is rituximab, a chimeric monoclonal antibody directed against the CD20 antigen, which is found on the surface of normal and malignant B-lymphocytes. Administration of rituximab for patients with PNP in combination with follicular NHL is not always successful regarding oral lesions as we report in this case. PE leading to prompt depletion of autoreactive antibodies combined with immunosuppressants or synchronisation of PE with IVIg seems the best treatment modality for this refractory group, but the therapeutic value and appropriate timing of rituximab obviously deserve further evaluation in patients with low grade NHL and PNP.

Prognostic significance of activated cytotoxic T-lymphocytes in primary nodal diffuse large B-cell lymphomas.

Clinical outcome in diffuse large B-cell lymphoma (DLBCL) remains unpredictable, despite the identification of clinical prognostic parameters. Here, we investigated in pretreatment biopsies of 70 patients with DLBCL whether numbers of activated cytotoxic T-lymphocytes (CTLs), as determined by the percentage of CD3-positive lymphocytes with granzyme B (GrB) expression, have similar prognostic value as found earlier in Hodgkin's lymphoma and anaplastic large-cell lymphoma and whether loss of major histocompatibility complex (MHC)-I molecules or expression of the GrB antagonist protease inhibitor 9 (PI9) may explain immune escape from CTL-mediated cell death. Independent of the International Prognostic Index (IPI), the presence of >/=15% activated CTLs was strongly associated with failure to reach complete remission, with a poor progression-free and overall survival time. Downregulation of MHC-I light- and/or heavy-chain expression was found in 41% of interpretable cases and in 19 of 56 interpretable cases PI9 expression was detected. We conclude that a high percentage of activated CTLs is a strong, IPI independent, indicator for an unfavorable clinical outcome in patients with primary nodal DLBCL. Although in part of DLBCL expression of PI9 and loss of MHC-I expression was found, providing a possible immune-escape mechanism in these cases, no correlation with clinical outcome was found.

Pseudoplatelets: a retrospective study of their incidence and interference with platelet counting.

AIMS: Spurious platelet counts can be found in acute leukaemias, as a result of the fragmentation of blood cells. Microscopic examination of a blood smear should be performed to detect the presence of these so called pseudoplatelets. When present, the platelet count should be corrected because of the important clinical consequences that a lower platelet count may have in these patients. METHODS: K(3)EDTA anticoagulated blood was measured on an automated blood cell counter, and a blood smear was made and stained according the May Grünwald-Giemsa method for microscopic observation. A 500 cell/particle differentiation was performed and the automated platelet count was corrected. RESULTS: The incidence of pseudoplatelets in 169 patients with acute leukaemia was studied. Pseudoplatelets were detected in 43 patients (25.4%), and seven patients (4.1%) were re-classified as having a major bleeding risk (platelet count, < 15 x 10(9)/litre). CONCLUSIONS: Platelets should be determined morphologically in patients with acute leukaemia and a routine screening method for the detection of pseudoplatelets should be developed.

Dosimetric verification of inverse planned step and shoot multileaf collimator fields from a commercial treatment planning system.

An inverse treatment planning (ITP) module on a commercial treatment planning system (TPS) (Helax AB, Uppsala, Sweden) is being used for an in-house clinical trial for treatment of nasopharyngeal cancer with contralateral parotid sparing. Intensity modulated radiation therapy (IMRT) fields are delivered by step and shoot multileaf collimator (MLC) with a DMLC enabled Varian 2300 CD (Varian Associates, Palo Alto, CA). A series of testing procedures have been devised to quantify the modeling and delivery accuracy of routine clinical inverse planned IMRT using Helax TMS and the Varian step and shoot MLC delivery option. Testing was done on specific aspects of the TPS modeling germane to DMLC. Measured relative dose factors (head scatter plus phantom scatter) for small MLC fields, normalized to a 10x10 cm2 non-MLC field, were found to differ by 2-3% from the TPS values for the smallest of the fields tested. Relative distributions for small off axis fields were found to be in good agreement. A process for the routine clinical verification of IMRT fields has been implemented. Each IMRT field in an inverse plan is imported into a flat water tank plan and a "beam's eye view" (BEV) dose distribution is generated. This is compared to the corresponding measured BEV dose distribution. The IMRT verification process has also been performed using an anthropomorphic phantom. Large clinical fields (i.e., greater than 14.5 cm in the leaf direction) caused difficulties due to a vendor specific machine restriction, and several techniques for dealing with these were examined. These techniques were (i) the use of static stepping of closed junctions, (ii) the use of two separate IMRT fields for a given gantry angle, and (iii) restricting the overall maximum field size used. The overall process has allowed implementation of an in-house protocol for IMRT use on an initial clinical site. Results of the verification measurements for the first ten patients treated at this center reveal an average maximum dose per IMRT field delivered of 71.0 cGy, with a mean local deviation from the planned dose of -1.2 cGy, and a standard deviation of 2.4 cGy.

[Patient with refractory celiac disease and secondary lymphoma]. / Een patiënt met refractaire coeliakie en een secundair lymfoom.

A 50-year-old woman who had suffered from well-regulated coeliac disease for 16 years, presented with weight loss, soft stools and abdominal cramps. She had ulcers in the oesophagus and stomach, and in biopsies localisations of so-called enteropathy-associated T-cell lymphoma (EATL) were detected. During a staging investigation she suffered an enteric perforation and later on repeated haemorrhages, from which she eventually died. Patients with coeliac disease who do not respond to a gluten free diet or who relapse after an initial response should be investigated for the presence of a gastrointestinal malignancy. Weight loss is an important symptom. The most frequently occurring malignant complication is an EATL. This is often difficult to diagnose and the prognosis is poor, with frequent complications such as haemorrhages and perforations.

A case of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma presenting with spontaneous splenic rupture: an extremely unusual presentation.

In a 22-year-old male with a 10-day history of fever, painful swelling in the left groin, and abdominal complaints, emergency surgery was performed because of spontaneous splenic rupture. At histology, a cellular infiltrate of intermediate-sized atypical lymphocytes was seen in the splenic white pulp, staining for T-cell markers. In addition, CD30 and anaplastic lymphoma kinase 1 (ALK) were diffusely positive, thus, representing a case of anaplastic large cell lymphoma (ALCL), T-cell, ALK-positive, small cell monomorphic variant. ALK-positive ALCL patients generally bear a much better prognosis than patients with T-cell lymphomas, unspecified, or ALK-negative ALCL. Therefore, besides the very unusual clinical presentation, this case highlights the importance of immunostaining for CD30 and ALK in all T-cell lymphomas. This report is the first extensive description of ALK-positive ALCL involvement of the spleen.

A polymorphic tetranucleotide repeat in the CYP19 gene and male breast cancer.

The CYP19 gene codes for the aromatase enzyme that is involved in the synthesis of oestrogens. This case-control study examines the relationship between a tetranucleotide repeat sequence in the CYP19 gene and the development of male breast cancer. No significant differences were found between male breast cancer cases and controls.

Field isolates of fowlpox virus contaminated with reticuloendotheliosis virus.

The polymerase chain reaction (PCR) method was used to examine samples from field cases of fowlpox for the presence of reticuloendotheliosis virus (REV). The S-strain fowlpox vaccine, known to be contaminated with REV, served as a positive control. Fowlpox virus was grown from field samples and vaccines by inoculation of embryonated hen eggs by the chorioallantoic membrane (CAM) route. DNA was extracted from the CAM lesions and examined for REV proviral sequences using primers specific for the long terminal repeats of REV. Amplicons of the expected length were detected in all the 45 field samples from poultry and in the S strain vaccine. Two other vaccines and two isolates from wild birds contained no detectable REV sequences. The PCR products from the vaccine and one field isolate were sequenced and were identical. These products showed 81 to 87.5% homology with the published sequences for the long terminal repeats of REV. It was not determined whether the REV proviral DNA was integrated with cellular DNA, fowlpox DNA or both. Inoculation of day-old chickens with the S-strain vaccine resulted not only in the production of fowlpox lesions but also feathering defects and proventriculitis. This suggests that the REV present in the vaccine is replication competent. Problems being encountered with protection from fowlpox following vaccination in Australia might be attributed to simultaneous challenge with fowlpox virus and REV.

[Clinical thermometry. I. Historical developments]. / Klinische thermometrie. I. Historische ontwikkelingen.

Determination of human core temperature has a long history. Since Antiquity, the significance of normal and abnormal body temperatures has been the subject of various interpretations. In this respect, theories based on humoral pathology were replaced by more scientific concepts in the 19th and 20th centuries. Objective measurement and comparison could be performed only after the invention of the thermometer and the introduction of temperature scales. Sanctorius and subsequently Boerhaave and others emphasized the use of measurement of body temperature in the clinic, but its importance was not accepted generally until the late 19th century. Many physicians and scientists have contributed to the progress of thermometry; however, the creation of a firmer scientific basis for clinical thermometry is usually attributed to Wunderlich.

[Clinical thermometry. II. Current dilemmas]. / Klinische thermometrie. II. Huidige dilemma's.

The relevance of measuring core temperature for diagnosis and treatment of various diseases is generally acknowledged nowadays. Despite introduction of new techniques and markedly improved understanding of body temperature regulation and the pathophysiology of fever, several dilemmas remain to be elucidated in clinical thermometry. In the measurement and interpretation of body temperature, the many variables that influence core temperature, the site of temperature registration and the significance of an elevated or decreased core temperature should be taken into account. With every type of thermometer, good calibration and an adequate recording technique remain pivotal to obtain reliable and reproducible results.

Activation of the receptor tyrosine kinase Kit is required for the proliferation of melanoblasts in the mouse embryo.

The development of neural crest-derived melanocytes, as well as haematopoietic and germ cells, is affected by mutations of the Kit and Mgf genes, which lead to dominant spotting (W) or steel (Sl) phenotypes. Mgf codes for the ligand of the receptor tyrosine kinase encoded by the Kit locus. KitW-v, a point mutation exerting a dominant negative effect, causes a substantial reduction in tyrosine kinase activity of the Kit receptor and leads to a characteristic pigmentation phenotype, namely dilute coat colour and a white ventral and head spot with reduced pigmentation of the feet and tail in the heterozygous animal, as well as slight anaemia. Homozygous animals lack coat pigmentation and are severely anaemic and infertile. Dct is a marker for cells of the melanoblast lineage. In order to study these cells in detail we have generated transgenic mouse lines carrying the lacZ reporter under the control of the Dct promoter and have used the embryonic expression of the reporter to identify early melanoblasts before they begin to produce pigment. Our transgenic lines have simplified the study of melanoblasts in the mouse embryo, and by crossing our mice with KitW-v mutants we have been able to identify the midgestation stages at which melanoblasts rely critically on Mgf/Kit interactions. We conclude that the survival of immature melanoblasts depends crucially upon Kit signalling up until E11, and later in development Kit plays a vital role in melanoblast proliferation. Our data do not describe a dependence upon Kit for melanoblast migration or differentiation.

Skin blood flow and autonomic reactivity in human poikilothermia.

Autonomic reactivity is pivotal in maintaining a constant body core temperature. Skin vasomotor reflexes and cardiovascular reactivity were investigated in four women (aged 28-37 years) with acquired poikilothermia, during steady-state spontaneous hypothermia (rectal temperature (Tr) = 33.7 +/- 1.0 degrees C [mean +/- SD]) and steady-state normothermia (Tr = 36.7 +/- 0.3 degrees C), as well as in 12 normothermic control subjects. Baseline finger temperature (Tf) during hypothermia was significantly lower than during normothermia (Tf = 32.4 +/- 1.2 compared with 36.2 +/- 0.3 degrees C, respectively), and than in the controls (Tf = 34.8 +/- 0.8 degrees C). No significant differences in baseline skin blood flow and forearm blood flow were found between subjects during hypothermia or normothermia and controls, suggesting a failure of sympathetic drive to counter-regulate hypothermia in the subjects. Skin vasoconstrictor responses to the contralateral cooling test and neck cooling test were markedly attenuated in three subjects, and to the finger cooling test in two subjects, during normothermia compared with hypothermia. Blood pressure responses to the Valsalva manoeuvre and head-up tilting were normal in all subjects, whereas the heart rate response to head-up tilting was blunted in three subjects during hypothermia. The responses of blood pressure and forearm blood flow to the cold pressor test in the subjects during both thermal conditions were comparable with the controls. We conclude that in our subjects, without generalized autonomic failure, poikilothermia has to be attributed predominantly to disorders of the central thermoregulatory pathways. Our findings during hypothermia and normothermia indicate that variations in core and skin temperature significantly affect skin vasomotor reactivity.