Gaetano Bergamaschi

Role of the JAK2 mutation in the diagnosis of chronic myeloproliferative disorders in splanchnic vein thrombosis

The diagnosis of an underlying chronic myeloproliferative disorder (CMPD) is often problematic in patients with primary extrahepatic portal vein obstruction (EHPVO) or Budd‐Chiari syndrome (BCS); indeed, conventional clinical and hematological parameters usually yield insufficient information. To assess the diagnostic contribution of the gain‐of‐function mutation V617F of the JAK2 gene, 93 patients with EHPVO or BCS were investigated. JAK2 V617F was identified in 35.6% of 73 patients with EHPVO and in 40% of 20 patients with BCS. Taking the JAK2 mutation as a test with the highest positive predictive value for the diagnosis of CMPD, conventional clinical‐hematological parameters had a sensitivity for CMPD lower than 48%. Bone marrow (BM) histology provided a diagnosis of CMPD in 41/74 (55.4%) patients, with a sensitivity of 93.5%. Clonality of hematopoiesis as assessed by granulocyte X‐chromosome inactivation was present in 65.1% of 43 informative female patients, with a sensitivity of 86.6%. By resolving the sensitivity bias of the JAK2 mutation with the results of BM histology and clonality assay, CMPD was diagnosed in 53% of patients with EHPVO or BCS. In conclusion, CMPD is the major cause of primary EHPVO or BCS. JAK2 V617F is a very reliable and noninvasive molecular marker for CMPD and should be used as a first test for diagnosis. (HEPATOLOGY 2006;44:1528–1534.)

Anaemia characterises patients with myelofibrosis harbouring MplW515L/K mutation

The clinical and haematological phenotype of patients with myelofibrosis harbouring MPLW515L/K mutation has not been thoroughly investigated. Of 217 myelofibrosis subjects, 18 (8·2%) had an MPL mutation, four of which (22%) co‐existed with JAK2V617F mutation. When compared with MPL wild‐type patients, irrespective of JAK2V617F status, those with MPLW515L/K, were more frequently female, were older (61 years vs. 57 years; P = 0·02), presented with more severe anaemia (haemoglobin, 101 g/l vs. 121 g/l; P = 0·002) and were more likely to require regular transfusional support (P = 0·012). These data indicate that MPL mutation in myelofibrosis characterises patients with more severe anaemic phenotype.

Evidence for a polyclonal nature of the cell infiltrate in sinus histiocytosis with massive lymphadenopathy (Rosai‐Dorfman disease)

Summary. Sinus histiocytosis with massive lymphadenopathy (SHML), or Rosai‐Dorfman disease, is rare histiocytic disorder of known origin which shares several cell markers with Langerhans’cell histiocytosis (LCH). Although Rosai‐Dorfman cells exhibit an aberrant immunophenotype, the indolent clinical course of SHML suggests a reactive disorder rather than a neoplastic process. Until recently this was prevailing opinion concerning LCH also, but recent studies have detected clonal histiocytes in all forms of this latter condition, which is therefore considered a clonal neoplastic disorder with highly variable biologial behaviour. To determine whether the histiocytic proliferation in SHML is polyclonal or clonal we used X‐linked polymorphic loci to assess clonality in lesional tissues in two women. Polymorpic regions of the human androgen receptor (HUMARA) locus were amplified by polymerase chain reaction (PCR) analysis. The HUMARA locus was informative in both cases and, following digestion with methylation‐sensitive enzymes, typical polyclonal X‐inactivation patterns were observed. Since abnormal cells accounted for >90% lesional tissue cells, we conclude that Rosai‐Dorfman histiocytic proliferation was polyclonal in the women studied.

Prevalence and pathogenesis of anemia in inflammatory bowel disease. Influence of anti-tumor necrosis factor-α treatment

Background Anemia is a common complication of inflammatory bowel disease, but its epidemiology may be changing due to earlier diagnosis and improved treatments. We investigated the prevalence and pathogenesis of anemia in patients with inflammatory bowel disease. Design and Methods In a cross-sectional study 263 out-patients with inflammatory bowel disease (165 with Crohn’s disease, 98 with ulcerative colitis) were investigated. The influence of time from diagnosis, disease activity, inflammation and the status of iron and hematinic vitamins on the level of hemoglobin and prevalence of anemia were evaluated. In a second group of 27 patients with Crohn’s disease, undergoing anti-tumor necrosis factor-α treatment with infliximab because of refractory or fistulizing disease, we determined the effects of infliximab on disease activity, hemoglobin, serum erythropoietin levels, iron status and inflammation. Results In all, 104 of the 263 patients with inflammatory bowel disease were anemic. Age, gender and azathioprine treatment had no influence on anemia. The prevalence of anemia was highest at diagnosis (65%), decreased during the first 4 years after disease onset, and was stable thereafter. Active disease was associated with higher rates of anemia. At diagnosis most anemic patients had anemia of chronic disease; during follow-up iron deficiency and multifactorial forms of anemia became more prevalent. Eighteen of 27 patients undergoing treatment with infliximab were anemic; most of them had anemia of chronic disease. Infliximab reduced disease activity and improved anemia in 12 patients. This was mediated by an increased production of erythropoietin for the degree of anemia. In vitro infliximab increased the growth of erythroid progenitors from the peripheral blood of patients with active disease. Conclusions Anemia is a common problem in out-patients with inflammatory bowel disease; the prevalence and severity of anemia are related to the activity of the bowel disorder. The pathogenesis of anemia changes during the course of the disease, with anemia of chronic disease having a major role at diagnosis and iron deficiency and multifactorial forms of anemia during follow-up. In patients requiring anti-tumor necrosis factor-α treatment, response to therapy improves erythropoiesis.

Unbalanced X‐chromosome inactivation in haemopoietic cells from normal women

We studied X‐chromosome inactivation patterns in blood cells from normal females in three age groups: neonates (umbilical cord blood), 25–32 years old (young women group) and >75 years old (elderly women). Using PCR, the differential allele methylation status was evaluated on active and inactive X chromosomes at the human androgen receptor (HUMARA) and phosphoglycerate kinase (PGK) loci. A cleavage ratio (CR) ⩾ 3.0 was adopted as a cut‐off to discriminate between balanced and unbalanced X‐chromosome inactivation. In adult women this analysis was also performed on hair bulbs. The frequency of skewed X‐inactivation in polymorphonuclear (PMN) cells increased with age: CR ⩾ 3.0 was found in 3/36 cord blood samples, 5/30 young women and 14/31 elderly women. Mathematical analysis of patterns found in neonates indicated that X‐chromosome inactivation probably occurs when the total number of haemopoietic stem cell precursors is 14–16. The inactivation patterns found in T lymphocytes were significantly related to those observed in PMNs in both young (P < 0.001) and elderly women (P < 0.01). However, the use of T lymphocytes as a control tissue for distinguishing between skewed inactivation and clonal proliferation proved to be reliable in young females, but not in elderly women, where overestimation of the frequency of clonal myelopoiesis may appear.

Effects of the R216Q mutation of GATA-1 on erythropoiesis and megakaryocytopoiesis

The transcription factor GATA-1, together with its cofactor FOG-1, regulates erythropoiesis and megakaryocytopoiesis. Mutations in the DNA or FOG-1 binding sites of its N-terminal zinc finger result in different illnesses. Alterations of the FOG-1 face are responsible for dyserythropoietic anemia with thrombocytopenia while R216Q, the only mutation identified in the DNA face, induces X-linked thrombocytopenia with thalassemia (XLTT). The former disorder has been studied in detail whereas little is known about the latter since only one family has been investigated. We studied a second family with an R216Q, showing that XLTT and dyserythropoietic anemia with thrombocytopenia, even if different clinical entities, are closely related disorders. In both cases, patients present mild dyserythropoiesis, red cell hemolysis, severely defective maturation of megakaryocytes, macrothrombocytopenia with alpha-granule deficiency, and abnormalities of the cytoplasmic membrane system. However, a thalassemia minor phenotype has only been described in patients with XLTT whereas severe anemia and thrombocytopenia with evident defects of platelet composition and function may be observed only in dyserythropoietic anemia with thrombocytopenia.

Saporin, a ribosome-inactivating protein used to prepare immunotoxins, induces cell death via apoptosis.

The plant toxin saporin is a ribosome‐inactivating protein which inhibits protein synthesis and growth of both normal and tumour cells. Its cytotoxic activity can be increased by coupling with antibodies recognizing cell surface antigens. In this work we performed experiments to test the hypothesis that saporin induces cell death via apoptosis. Exposure to saporin induced apoptosis in different cellular models, such as human peripheral blood B lymphocytes and neutrophils, in the Daudi B‐cell line, and in the haemopoietic cell lines HL‐60 and TF‐1. This was indicated by: (a) the appearance of typical morphological features such as chromatin condensation, nuclear fragmentation and blebbing of plasma membranes; (b) DNA degradation into oligonucleosomal fragments; (c) the appearance of apoptotic cells on DNA flow cytometry as a cell population with reduced DNA content (A0 region). The fraction of cells showing features of apoptosis ranged from 19 ± 5% for TF‐1 cells to 35 ± 8% for neutrophils. In experiments with normal peripheral blood B lymphocytes or with Daudi cells, we compared the activity of native saporin with that of an immunotoxin hybrid molecule obtained by binding the toxin to two bispecific antibodies recognizing both saporin and the B lymphocyte‐specific antigen CD22 (Sap/BsAb complexes). Saporin bound to the antibodies was 2–3 logs more effective than native saporin in inducing apoptosis, with maximal inhibitions being observed at concentrations of 10−6 M for native saporin and 10−9–10−8 M for the hybrid molecules. These findings indicate that treatment with saporin results in apoptosis of target cells and suggest that this may be relevant to the therapeutic use of saporin‐containing immunotoxins. In fact, if used in vivo as an immunotoxin, its cytotoxic activity could be devoid of more extensive and non‐specific tissue damaging effects as would be the case if saporin induced necrosis of target cells.

Anemia of chronic disease and defective erythropoietin production in patients with celiac disease

Anemia due to hematinic deficiencies is common in paients with untreated celiac disease. This study shows that, in addition to iron and vitamin deficiencies, anemia of chronic disease has a significant role in some patients with celiac disease. See related perspective article on page 1761. Background Anemia due to hematinic deficiencies is common in patients with untreated celiac disease. Although celiac disease is a chronic condition characterized by an intense inflammatory response of the intestinal mucosa, scant data are available about the prevalence of anemia of chronic disease in celiac disease. Design and Methods One hundred and fifty-two patients with celiac disease at presentation were studied. Anemia was investigated by determining complete blood counts, body iron status, serum levels of the soluble transferrin receptor, erythropoietin, prohepcidin and interferon-γ. Genotyping for HFE mutations associated with hereditary hemochromatosis was performed. Fifty-three anemic patients were re-evaluated for hematologic response after 1 year on a gluten-free diet. Results At the time of diagnosis of celiac disease the prevalence of anemia was 34%. Fifty-three out of 65 anemic patients had either iron and/or vitamin deficiency (folate, vitamin B12). Hereditary hemochromatosis mutations did not affect the prevalence of anemia. In 11 cases iron status parameters were indicative of anemia of chronic disease, sometimes in association with iron deficiency (6 patients). Patients with anemia of chronic disease had low levels of erythropoietin for the degree of anemia and increased serum interferon-γ. In most cases anemia improved following a gluten-free diet, response rates being similar in anemia of chronic disease and in anemia due to hematinic deficiencies. Conclusions Our study shows that, in addition to iron and vitamin deficiencies, anemia of chronic disease has a significant role in some patients with celiac disease. Suppression of intestinal inflammatory changes as a result of a gluten-free diet improves anemia by correcting iron and vitamin malabsorption as well as mechanisms contributing to anemia of chronic disease.

Prevalence of Anemia in Inflammatory Bowel Diseases in European Countries: A Systematic Review and Individual Patient Data Meta-analysis.

Background:The main objective is to determine the overall prevalence of anemia in inflammatory bowel diseases (IBD) in Europe. Methods:A systematic literature search in PubMed and Embase was performed for studies published between January 2007 and May 2012. Eligible studies were included if they were original full-paper publications originated from Europe and if the authors agreed to provide their data. An overall prevalence of anemia in IBD, disease specific, and age–gender stratified basis prevalences were estimated. The influence of disease entity (Crohns disease/ulcerative colitis), gender, age, disease activity (remission/active disease), and IBD-specific treatment strategies on the prevalence of anemia was analyzed by a mixed logistic regression model. Thereby, the factor country of origin was included as a random effect. Results:Data were available for 2192 patients, mainly treated in tertiary referral centers. The overall prevalence of anemia in IBD patients was 24% (95% confidence interval, 18–31). Age–gender stratified prevalences were estimated for the age strata 18 to 29, 30 to 39, 40 to 49, 50 to 64, 65 to 74, >74 years and ranged from 18% to 35%. Patients receiving IBD-specific medication (P = 0.0002, odds ratio 1.54), and patients with active disease status (P < 0.0001, odds ratio 2.72) were significantly more likely to have anemia compared with patients not receiving IBD-specific medication or being in remission. Patients with ulcerative colitis tended to have anemia less likely than patients with Crohns disease (P = 0.01, odds ratio 0.77). Conclusions:The overall prevalence of anemia in patients with Crohns disease was 27% (95% confidence interval, 19–35) and 21% (95% confidence interval, 15–27) in patients with ulcerative colitis. Thereby, 57% of the anemic patients were iron deficient.

Soluble transferrin receptor as a potential determinant of iron loading in congenital anaemias due to ineffective erythropoiesis

Congenital anaemias due to ineffective erythropoiesis may be associated with excessive iron absorption and progressive iron loading. We investigated whether the soluble transferrin receptor (TfR) level was related to the degree of iron overload in 20 patients with thalassaemia intermedia, six patients with congenital dyserythropoietic anaemia type II (CDA II) and four patients with X‐linked congenital sideroblastic anaemia (XLSA). All but two patients had increased serum ferritin levels (median 601 μg/l, range 105–2855 μg/l). Multiple regression analysis showed that 62% (P < 0.0001) of the variation in serum ferritin was explained by age and by changes in soluble TfR.