The association of Greig syndrome and mastocytosis reveals the involvement of the hedgehog pathway in advanced mastocytosis.

Mastocytosis is a heterogeneous disease characterized by an abnormal accumulation of mast cells (MCs) in 1 or several organs. Although a somatic KIT D816V mutation is detected in ∼85% of patients, attempts to demonstrate its oncogenic effect alone have repeatedly failed, suggesting that additional pathways are involved in MC transformation. From 3 children presenting with both Greig cephalopolysyndactyly syndrome (GCPS, Mendelian Inheritance in Man [175700]) and congenital mastocytosis, we demonstrated the involvement of the hedgehog (Hh) pathway in mastocytosis. GCPS is an extremely rare syndrome resulting from haploinsufficiency of GLI3, the major repressor of Hh family members. From these familial cases of mastocytosis, we demonstrate that the Hh pathway is barely active in normal primary MCs and is overactive in neoplastic MCs. GLI3 and KIT mutations had a synergistic, tumorigenic effect on the onset of mastocytosis in a GCPS mouse model. Finally, Hh inhibitors suppressed neoplastic MC proliferation in vitro and extend the survival time of mice with aggressive systemic mastocytosis (ASM). This work revealed, for the first time, the involvement of Hh signaling in the pathophysiology of mastocytosis and demonstrated the cooperative effects of the KIT and Hh oncogenic pathways in mice with ASM, leading to the identification of new promising therapeutic targets.

From hepatitis C virus infection to B-cell lymphoma.

In addition to liver disorders, hepatitis C virus (HCV) is also associated with extrahepatic immune manifestations and B-cell non-Hodgkin lymphoma (NHL), especially marginal zone lymphoma, de novo or transformed diffuse large B-cell lymphoma and to a lesser extent, follicular lymphoma. Epidemiological data and clinical observations argue for an association between HCV and lymphoproliferative disorders. The causative role of HCV in NHL has been further supported by the response to antiviral therapy. Pathophysiological processes at stake leading from HCV infection to overt lymphoma still need to be further elucidated. Based on reported biological studies, several mechanisms of transformation seem however to emerge. A strong body of evidence supports the hypothesis of an indirect transformation mechanism by which sustained antigenic stimulation leads from oligoclonal to monoclonal expansion and sometimes to frank lymphoma, mostly of marginal zone subtype. By infecting lymphocytes, HCV could play a direct role in cellular transformation, particularly in de novo large B-cell lymphoma. Finally, HCV is associated with follicular lymphoma in a subset of patients. In this setting, it may be hypothesized that inflammatory cytokines stimulate proliferation and transformation of IgH-BCL2 clones that are increased during chronic HCV infection. Unraveling the pathogenesis of HCV-related B-cell lymphoproliferation is of prime importance to optimize therapeutic strategies, especially with the recent development of new direct-acting antiviral drugs.

BCL2 expression but not MYC and BCL2 coexpression predicts survival in elderly patients with diffuse large B-cell lymphoma independently of cell of origin in the phase 3 LNH03-6B trial.

BACKGROUND: Our aim was to evaluate whether the cell of origin (COO) as defined by the Hans algorithm and MYC/BCL2 coexpression, which are the two main biological risk factors in elderly patients treated with rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine, and prednisolone (R-CHOP), maintain their prognostic value in a large prospective clinical trial. PATIENTS AND METHODS: We evaluated 285 paraffin-embedded samples from patients (60-80 years of age) enrolled in the Lymphoma Study Association trial LNH03-6B who were treated with R-CHOP. We correlated the COO defined by the transcriptome according to the Wright algorithm with that defined by the Hans algorithm in a subset of 62 tumors with available frozen tissue samples. RESULTS: The non-germinal center B-cell-like phenotype according to the Hans algorithm and BCL2 expression (but not MYC and BCL2 coexpression) predicted worse progression-free survival [hazard ratio (HR)=1.78, P = 0.003 and HR = 1.79, P = 0.003, respectively] and overall survival (HR = 1.85, P = 0.005 and HR = 1.67, P = 0.02, respectively) independently of the International Prognostic Index. The correlation between the Hans algorithm and the Wright algorithm was 91%, with an almost perfect concordance according to a kappa test (0.81). CONCLUSIONS: Our results suggest that immunohistochemically defined COO remains a useful tool for predicting prognosis in diffuse large B-cell lymphoma when performed under optimized standardized conditions and that BCL2 expression may help to identify elderly patients at risk for relapse and who could potentially respond to anti-BCL2 targeted agents. In this prospective phase III trial, the coexpression of MYC and BCL2 does not appear to predict worse survival. CLINICAL TRIAL NUMBER: NCT00144755.

Impacts of donor-specific anti-HLA antibodies and antibody-mediated rejection on outcomes after intestinal transplantation in children.

AMR is a risk factor for graft failure after SBTx. We studied impact of DSAs and AMR in 22 children transplanted between 2008 and 2012 (11 isolated SBTx, 10 liver inclusive Tx, and one modified multivisceral Tx). Three patients never developed DSA, but DSAs were found in seven in the pre-Tx period and de novo post-Tx in 19 children. Pathology revealed cellular rejection (15/19), with vascular changes and C4d+. Patients were treated with IV immunoglobulins, plasmapheresis, and steroids. Rescue therapy included antithymocyte globulins, rituximab, eculizumab, and bortezomib. Pathology and graft function normalized in 13 patients, graft loss occurred in two, and death in seven. At the end of the follow-up, 15 children were alive (68%), 13 with functioning graft (59%). Prognosis factors for poor outcome after Tx were the presence of symptoms at AMR suspicion (P +.033). DSAs were often found following SBTx, mostly de novo. Resistant ACR or severe AMR is still difficult to differentiate, with a high need for immunosuppression in both. DSAs may precede development of severe disease and pathology features on the graft: relationship and correlation need to be better investigated with larger groups before and after Tx.

Decreased tryptophan and increased kynurenine levels in mastocytosis associated with digestive symptoms.

The main metabolism pathway of tryptophan is protein formation, but it can also be metabolized into serotonin and kynurenine. Indoleamine 2,3-dioxygenase (IDO) is the enzyme that catalyzes the degradation of tryptophan into kynurenine. Mastocytosis is a heterogeneous disease characterized by mast cell accumulation in various tissues with 57% of patients having gastrointestinal involvement. We studied tryptophan metabolism in mastocytosis patients displaying or not gastrointestinal features and healthy subjects (n = 26 in each group). Mastocytosis patients with digestive symptoms displayed significantly increased kynurenine level and IDO activity as compared to healthy controls and mastocytosis patients without digestive symptoms. This could be linked to mast cell-mediated digestive inflammation among patients with mastocytosis. This work is the first focusing on kynurenine pathway in a mast cell disease and could help to understand the pathogenesis of digestive features in mastocytosis as well as in other mast cell-mediated diseases.

FDG-PET/CT findings in systemic mastocytosis: a French multicentre study.

INTRODUCTION: Mastocytosis is a clonal haematological disease characterized by uncontrolled proliferation and the activation of mast cells. The value of FDG-PET/CT (FDG-PET) in mastocytosis has yet to be determined. METHODS: We retrospectively identified patients with an established diagnosis of systemic mastocytosis (SM), according to the WHO criteria, who underwent PET using the French Reference Centre for Mastocytosis database. Semi-quantitative and visual analysis of FDG-PET was performed and compared to the clinico-biological data. RESULTS: Our cohort included 19 adult patients, median age 65 years [range 58-74], including three with smouldering SM (SSM), three with aggressive SM (ASM), 10 with an associated clonal haematological non-mast-cell lineage disease (SM-AHNMD), and three with mast cell sarcoma (MCS). FDG-PET was performed at the time of the SM diagnosis (15/19), to evaluate lymph node (LN) activity (3/19) or the efficacy of therapy (1/19). FDG uptake was observed in the bone marrow (BM) (9/19, 47%), LN (6/19, 32%), spleen (12/19, 63%), or liver (1/19, 5%). No significant FDG uptake was observed in the SSM and ASM patients. A pathological FDG uptake was observed in the BM of 6/10 patients with SM-AHNMD, appearing as diffuse and homogeneous, and in the LN of 5/10 patients. All 3 MCS patients showed intense and multifocal BM pathological uptake, mimicking metastasis. No correlation was found between the FDG-PET findings and serum tryptase levels, BM mast cell infiltration percentage, and CD30 and CD2 expression by mast cells. CONCLUSIONS: FDG uptake does not appear to be a sensitive marker of mast cell activation or proliferation because no significant FDG uptake was observed in most common forms of mastocytosis (notably purely aggressive SM). However, pathological FDG uptake was observed in the SM-AHNMD and in MCS cases, suggesting a role of FDG-PET in their early identification and as a tool of therapeutic assessment in this subgroup of patients.

Assessment and outcome of children with intestinal failure referred for intestinal transplantation.

BACKGROUND & AIMS: Chronic intestinal failure (CIF) requires long term parenteral nutrition (PN) and, in some patients, intestinal transplantation (ITx). Indications and timing for ITx remain poorly defined. In the present study we aimed to analyze causes and outcome of children with CIF. METHODS: 118 consecutive patients referred to our institution were assessed by a multidisciplinary team and four different categories were defined retrospectively based on their clinical course: Group 1: patients with reversible intestinal failure; group 2: patients unsuitable for ITx, group 3: patients listed for ITx; group 4: patients stable under PN. Analysis involved comparison between groups for nutritional status, central venous catheter (CVC) related complications, liver disease, and outcome after transplantation by using non parametric tests, Mann-Whitney tests, Kruskal-Wallis, Wilcoxon signed rank tests and chi square distribution for percentage. RESULTS: 118 children (72 boys) with a median age of 15 months at referral (2 months-16 years) were assessed. Etiology of IF was short bowel syndrome [n = 47], intractable diarrhea of infancy [n = 37], total intestinal aganglionosis [n = 18], and chronic intestinal pseudoobstruction [n = 17]. Most patients (89.8%) were totally PN dependent, with 48 children (40.7%) on home-PN prior to admission. Nutritional status was poor with a median body weight at -1.5 z-score (ranges: -5 to +2.5) and median length at -2.0 z-score (ranges: -5.5 to +2.3). The mean number of CVC inserted per patient was 5.2 (range 1-20) and the mean number of CRS per patient was 5.5 (median: 5; range 0-12) Fifty-five patients (46.6%) had thrombosis of ≥2 main venous axis. At admission 34.7% of patients had elevated bilirubin (≥50 µmol/l), and 19.5% had platelets <100,000/ml, and 15% had both. Liver biopsy performed in 79 children was normal (n = 4), or showed F1 or F2 fibrosis (n = 29), bridging fibrosis F3 (n = 20), or cirrhosis (n = 26). Group 1 included 10 children finally weaned from PN (7-years survival: 100%). Group 2 included 12 children with severe liver disease and associated disorders unsuitable for transplantation (7-years survival: 16.6%). Group 3 included 66 patients (56%) who were listed for small bowel or liver-small bowel transplantation, 62/66 have been transplanted (7 years survival: 74.6%). Factors influencing outcome after liver-ITx were body weight (p < .004), length (p < .001), pre-Tx bilirubin plasma level (p < .001) and thrombosis (p < .01) for isolated ITx, Group 4 included 30 children (25.4%) with irreversible IF considered as potential candidates for isolated ITx. Four children were lost from follow up and 3 died within 2 years (survival 88.5%). Among potential candidates, the following parameters improved significantly during the first 12 months of follow up: Body weight (p.0001), length (p < .0001) and bilirubin (p < .0001). CONCLUSIONS: many patients had a poor nutritional status with severe complications especially liver disease. PN related complications were the most relevant indication for ITx, but also a negative predictor for outcome. Early patient referral for Tx-assessment might help to identify and separate children with irreversible IF from children with transient IF or uncomplicated long-term PN, allowing to adapt a patient-based treatment strategy including or not ITx.

The efficacy of exclusive nutritional therapy in paediatric Crohn's disease, comparing fractionated oral vs. continuous enteral feeding.

BACKGROUND: Nutritional therapy has an established role as induction therapy in paediatric Crohn's disease. However, compliance is the main difficulty and may be greatly influenced by the administration route. AIM: To analyse the efficiency of exclusive nutrition to induce remission in children with Crohn's disease comparing fractionated oral vs. continuous enteral feeding. METHODS: The medical records of 106 patients treated by exclusive nutritional therapy [Modulen IBD (R)] by either oral or continuous enteral route were reviewed retrospectively. Comparative analyses of remission rates, changes in anthropometry, Paediatric Crohn's disease Activity Index (PCDAI), laboratory indices and compliance rates were performed. RESULTS: On exclusive enteral nutrition, at 8 weeks, 34/45 patients achieved remission in the oral group (75% on intention-to-treat analysis) and 52/61 (85%) in the enteral nutrition group (P = 0.157). All patients showed a significant decrease in disease severity assessed by PCDAI (P < 0.0001) and significant improvements in anthropometric measures and inflammatory indices. No difference was observed whether Modulen IBD was administered orally or by continuous enteral feeding, apart from weight gain, which was greater in the enteral group (P = 0.041). In a subgroup of patients, mucosal healing was evidenced on follow-up endoscopies showing a clear correlation to remission. Compliance rates (87% and 90%) were similar. Nevertheless, noncompliant patients had lower mucosal healing and remission rates. CONCLUSIONS: These retrospective data suggest that the use of fractionated oral nutritional therapy might be as efficacious as continuous enteral administration to induce remission and mucosal healing in children with Crohn's disease. However, appropriate prospective clinical trials are needed to confirm these findings.

Systemic mastocytosis and bone involvement in a cohort of 75 patients.

OBJECTIVES: To investigate bone involvement in a large cohort of systemic mastocytosis (SM) patients, and evaluate the efficacy of bisphosphonate therapy. PATIENTS AND METHODS: From 2000 to 2004, 75 patients with SM according to WHO criteria underwent skeletal x-rays and bone mineral density (BMD) assessment. Sequential BMD assessments were performed in nine patients treated with bisphosphonate (mean follow-up 65 months). RESULTS: 37 patients (49%) had bone involvement according to both x-rays and BMD evaluations: osteoporosis (23 patients, 31%, mean lumbar spine T score: -3 SD), with vertebral fracture (13 patients, 17%), axial skeleton osteosclerosis (six patients, 8%), mixed patterns (three patients), osteopenia with pre-existing fractures (four patients) and focal osteolytic lesion (one patient). Blood count abnormalities were associated with osteosclerosis (p=0.005). In nine patients with osteoporosis and bisphosphonate therapy, mean lumbar spine BMD increased from 0.83 to 0.92 g/cm(2) (+11.1%; ie, +2.05% per year) without recurrence of vertebral fracture. CONCLUSION: Half of adult patients with SM have bone involvement. Osteoporosis is the most prevalent bone manifestation in SM (31%). Bisphosphonate therapy seems efficient to improve lumbar spine BMD during SM-related osteoporosis. Spine x-ray and BMD should be performed in all SM patients to detect those who may benefit from anti-osteoporotic therapy.

Evaluation of c4d deposition and circulating antibody in small bowel transplantation.

Antibody-mediated rejection (AMR) consensus criteria are defined in kidney and heart transplantation by histological changes, circulating donor-specific antibody (DSA), and C4d deposition in affected tissue. AMR consensus criteria are not yet identified in small bowel transplantation (SBTx). We investigated those three criteria in 12 children undergoing SBTx, including one retransplantation and four combined liver-SBTx (SBTx), with a follow-up of 12 days to 2 years. All biopsies (91) were evaluated with a standardized grading scheme for acute rejection (AR), vascular lesions and C4d expression. Sera were obtained at day 0 and during the follow-up. C4d was expressed in 37% of biopsies with or without AR, but in 50% of biopsies with severe vascular lesions. In addition, vascular lesions were always associated with AR and a poor outcome. All children with AR (grade 2 or 3) observed before the third month died or lost the graft. DSA were never found in any studied sera. We found no evidence that C4d deposition was of any clinical relevance to the outcome of SBTx. However, the grading of vascular lesions may constitute a useful marker to identify AR that is potentially resistant to standard treatment, and for which an alternative therapy should be considered.

Long-term outcome, growth and digestive function in children 2 to 18 years after intestinal transplantation.

OBJECTIVE: Small bowel (SB) transplantation (Tx), long considered a rescue therapy for patients with intestinal failure, is now a well recognised alternative treatment strategy to parental nutrition (PN). In this retrospective study, we analysed graft functions in 31 children after SBTx with a follow-up of 2-18 years (median 7 years). PATIENTS: Twelve children had isolated SBTx, 19 had combined liver-SBTx and 17 received an additional colon graft. Growth, nutritional markers, stool balance studies, endoscopy and graft histology were recorded every 2-3 years post-Tx. RESULTS: All children were weaned from PN after Tx and 26 children remained PN-free. Enteral nutrition was required for 14/31 (45%) patients at 2 years post-Tx. All children had high dietary energy intakes. The degree of steatorrhoea was fairly constant, with fat and energy absorption rates of 84-89%. Growth parameters revealed at transplantation a mean height Z-score of -1.17. After Tx, two-thirds of children had normal growth, whereas in one-third, Z-scores remained lower than -2, concomitant to a delayed puberty. Adult height was normal in 5/6. Endoscopy and histology analyses were normal in asymptomatic patients. Chronic rejection occurred only in non-compliant patients. Five intestinal grafts were removed 2.5-8 years post-Tx for acute or chronic rejection. CONCLUSIONS: This series indicates that long-term intestinal autonomy for up to 18 years is possible in the majority of patients after SBTx. Subnormal energy absorption and moderate steatorrhoea were often compensated for by hyperphagia, allowing normal growth and attainment of adult height. Long-term compliance is an important pre-requisite for long-term graft function.

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Variable expression of Epstein-Barr virus-induced gene 3 during normal B-cell differentiation and among B-cell lymphomas.

Epstein-Barr virus (EBV)-induced gene 3 (EBI3) is expressed by tumour cells in several EBV-associated malignancies. EBI3 was recently found to associate with a novel peptide, p28, to form a new heterodimeric cytokine, called interleukin-27. In this study, we investigated EBI3 and p28 expression in normal human B lymphocytes and in non-EBV-associated B-cell lymphomas. Low levels of EBI3 were detected in purified tonsillar B cells and expression was upregulated upon anti-CD40 or anti-micro stimulation via NF-kappaB activation. In non-neoplastic tissues, EBI3 expression by lymphocytes was largely restricted to a subset of germinal centre (GC) B cells located at the margin of the light zone, in close contact with CD3+ T lymphocytes. Over 50% of EBI3+ GC B cells were engaged in cell proliferation as assessed by Ki67 expression, and 10-30% expressed MUM1, an early marker of plasma cell differentiation expressed by late centrocytes. Many EBI3+ GC B cells had downregulated bcl-6 expression, which further suggests that these cells correspond to late CD40-activated centrocytes. Immunohistochemical analysis of 64 B-cell lymphomas showed that the highest EBI3 levels were detected in follicular lymphomas and in diffuse large B-cell lymphomas of both GC B-cell-like or non-GC B-cell-like types. No or rare p28 expression was detected in normal or tumour B cells. This constitutive expression of EBI3 by neoplastic B cells may be involved in lymphomagenesis, and may be a useful marker for lymphoma diagnosis.

Reversibility of pulmonary arterial hypertension in HIV/HHV8-associated Castleman's disease.

The present study describes a case of pulmonary arterial hypertension (PAH) associated with multicentric Castleman's disease in a patient infected with HIV type 1 and human herpes virus 8. Therapy included highly active antiretroviral therapy, warfarin, diuretics, continuous i.v. epoprostenol and 12-monthly pulses of cyclophosphamide. The patient's condition improved dramatically with complete reversibility of PAH, allowing weaning of continuous i.v. epoprostenol therapy. After 5 yrs, both Castleman's disease and PAH have not relapsed. This supports the hypothesis that control of inflammation and retroviral replication may be of interest in the context of PAH, complicating the course of an inflammatory condition associated with viral infection. In conclusion, further studies should help in characterising the best candidates for anti-inflammatory treatment in the setting of pulmonary arterial hypertension.

Fecal tumor necrosis factor-alpha and calprotectin as differential diagnostic markers for severe diarrhea of small infants.

OBJECTIVE: The etiology of early-onset diarrhea of neonates and small infants that persists despite bowel rest is heterogeneous. Two different categories of disorders presenting with diarrhea in the first weeks of life can be distinguished: constitutive intestinal epithelial disorders (microvillus atrophy [MVA] or epithelial dysplasia [ED]) and immune-inflammatory disorders, (autoimmune enteropathy [AIE] or inflammatory colitis [IC]). We aimed to evaluate in a prospective manner the use of fecal inflammatory markers in the differential diagnosis of severe persistent diarrhea. MATERIAL AND PATIENTS: Twenty-five patients (17 males) were enrolled in this study (median age 8 months). Fourteen children had a constitutive enterocyte disorder (group 1: MVA = 8, ED = 6), and 11 patients had an immuno-inflammatory disease (group 2: AIE = 5, IC = 6). Stool samples were collected at the time of diagnosis and stored at -80 degrees until tumor necrosis factor (TNF)-alpha and calprotectin were measured by enzyme-linked immunoadsorbent assay. RESULTS: No significant differences in age at onset of diarrhea or in stool volumes were observed between both groups. In group 1, fecal TNF-alpha was undetectable/normal in 14 of 14 children, whereas group 2 showed dramatically elevated TNF-alpha levels (mean 3,104, range 237-18,078 pg/g) in 8 of 11 patients. Similarly, calprotectin levels were undetectable/normal in 14 of 14 patients in group 1 and highly raised in 11 of 11 patients in group 2 (median 1,145, range 375-3,095 mug/g), P < 0.01. Under therapy, these inflammatory parameters normalized. CONCLUSIONS: Determination of fecal inflammatory markers is a simple method helping to distinguish constitutive from immuno-inflammatory etiologies of severe persistent diarrhea. These data also suggest that constitutive enterocyte disorders are not accompanied by an inflammatory mucosal reaction.

Angio-immunoblastic T cell lymphoma (AILD-TL) rich in large B cells and associated with Epstein-Barr virus infection. A different subtype of AILD-TL?

We studied eight patients with characteristic features of angio-immunoblastic T cell lymphoma (AILD-TL) associated with more than 25% of large B cells. Polymerase chain reaction (PCR) analysis showed a clonal rearrangement of the T cell receptor (TCR)-gamma chain gene in all cases. One additional case showed a clonal rearrangement of the TCR-beta chain gene by Southern blot hybridization. PCR analysis showed a clonal immunoglobulin rearrangement in three cases presenting with more than 50% of large B cells whereas the other cases had a germline configuration. In 6/8 cases, double-labeling immunohistochemistry and in situ hybridization demonstrated that Epstein-Barr virus (EBV) was mostly present in the large B cells but also detected in some T cells. We further evaluated the frequency of AILD-TL with more than 25% of large B cells in the 106 cases collected by the French GELA group and found an incidence of 18%. The outcome of these patients did not differ significantly from those with less than 25% of B cells. With this approach we confirm the heterogeneity of AILD-TL features and the possible association with a substantial numbers of CD20(+), EBV(+) large B cells. We propose to denominate these cases as 'AILD-TL rich in large B cells' and to consider them as a different entity which can be misdiagnosed as a reactive process or as T cell rich B cell lymphoma.