[Primary cutaneous lymphoma-a case series of 163 patients]. / Primär kutane Lymphome ­ eine Fallserie von 163 Patienten.

BACKGROUND: In addition to a broad and clinically diverse spectrum of known primary cutaneous lymphomas, for which an incidence of 1-3:100,000 is postulated, each year further entities are specified and defined. The goal is the presentation of a case series from daily clinical routine. METHODS: Over a period of 6 years and 2 months, patients consulting the Department of Dermatology, Medical Center University of Freiburg, were registered. Subsequently, collectives of mycosis fungoides (MF), Sezary syndrome (SS), CD30+ lymphoproliferative diseases, single cases with rare primary cutaneous lymphomas, and subcollectives of B­cell lymphomas were examined. The high number of MF cases allowed the additional quantitative analyses of the types of therapies used in this group. RESULTS: Yearly 16-25 new diagnoses of primary cutaneous lymphoma are made. The evaluation of 163 primary cutaneous lymphoma revealed 111 cases with MF (68.1%), including 9 particular variants, 15 primary cutaneous CD30+ lymphoproliferative diseases (9.2%) dominated by 10 lymphomatoid papulosis (LyP), in addition to 5 primary cutaneous anaplastic large cell lymphoma (PCALCL), 6 SS (3.68%), and 24 cutaneous B­cell lymphomas (14-72%). Three cases with rare primary cutaneous T/NK cell lymphomas are addressed in detail. In all, 82% of MF cases were stage IA and IB. The descending use of therapies for stage I-III included steroids and diverse UV therapies followed by bexarotene, interferon-α, methotrexate, and extracorporal photophoresis. CONCLUSIONS: Diagnoses of cutaneous lymphomas belong to a vast spectrum of differential diagnoses. This registry describes frequent findings and shows rare variants. You can only diagnose what you know; accordingly, a collection of case reports, which we wish to encourage, can help in processing and specification of entities.

MicroRNA-146a reduces MHC-II expression via targeting JAK/STAT signaling in dendritic cells after stem cell transplantation.

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a-/- dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2-STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a-/- DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK-STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.

Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency.

The gene PIK3CD codes for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), and is expressed solely in leucocytes. Activating mutations of PIK3CD have been described to cause an autosomal dominant immunodeficiency that shares clinical features with common variable immunodeficiency (CVID). We screened a cohort of 669 molecularly undefined primary immunodeficiency patients for five reported mutations (four gain-of-function mutations in PIK3CD and a loss of function mutation in PIK3R1) using pyrosequencing. PIK3CD mutations were identified in three siblings diagnosed with CVID and two sporadic cases with a combined immunodeficiency (CID). The PIK3R1 mutation was not identified in the cohort. Our patients with activated PI3Kδ syndrome (APDS) showed a range of clinical and immunological findings, even within a single family, but shared a reduction in naive T cells. PIK3CD gain of function mutations are more likely to occur in patients with defective B and T cell responses and should be screened for in CVID and CID, but are less likely in patients with a pure B cell/hypogammaglobulinaemia phenotype.

Oncostatin M is a FIP1L1/PDGFRA-dependent mediator of cytokine production in chronic eosinophilic leukemia.

BACKGROUND: Chronic eosinophilic leukemia (CEL) is a myeloproliferative neoplasm characterized by expansion of neoplastic eosinophils, tissue infiltration, and organ damage. In a subset of these patients, the FIP1L1/PDGFRA (F/P) oncoprotein is detectable. F/P exhibits constitutive tyrosine kinase activity and activates a number of signaling pathways. So far, however, little is known about the role of F/P-dependent proteins in the pathogenesis of CEL. METHODS: A screen for F/P-dependent cytokines was performed in growth factor-dependent human cell lines lentivirally transduced with F/P. Signal transduction pathways were characterized in Ba/F3 cells with doxycycline-inducible expression of F/P and in EOL-1 cells. Cytokine expression was confirmed in patients' material by immunohistochemistry, immunofluorescence, and confocal microscopy. Gene expression analysis, proliferation assays, and chemotaxis assays were used to elucidate paracrine interactions between neoplastic eosinophils and stromal cells. RESULTS: We show that F/P upregulates expression of oncostatin M (OSM) in various cell line models in a STAT5-dependent manner. Correspondingly, neoplastic eosinophils in the bone marrow were found to overexpress OSM. OSM derived from F/P + cells stimulated proliferation of stromal cells. Moreover, OSM-containing supernatants from F/P + cells were found to upregulate production of stromal cell-derived factor-1 (SDF-1)/CXCL12 in human fibroblasts. SDF-1, in turn, induced migration of EOL-1 cells in a dose-dependent manner. CONCLUSIONS: We have identified a F/P-driven paracrine interaction between neoplastic eosinophils and stromal cells that may contribute to tissue fibrosis and accumulation of neoplastic eosinophils in CEL.

[Myelodysplastic syndromes. Epidemiology, molecular and morphological characteristics and risk stratification]. / Myelodysplastische Syndrome. Epidemiologie, molekulare und morphologische Merkmale, Risikostratifizierung.

Myelodysplastic syndromes (MDS) comprise a spectrum of clonal stem cell disorders which are currently defined according to the classification scheme of the revised 2008 WHO classification but which may be further refined in the future. The clinical presentation is often characterized by unexplained isolated or multiple peripheral blood cytopenias resulting in anemia, bleeding events or increased susceptibility to infections. The generally hypercellular, but rarely hypocellular and occasionally fibrotic bone marrow shows dysplastic features in ≥ 10 % of cells of at least one of the hematopoietic lineages. These features and enhanced apoptosis, stem cell senescence and immunologic dysregulation result in ineffective hematopoiesis. Diagnostics in MDS relies on complementary consideration of hematological, morphological and cytogenetic/molecular parameters. Methods include marrow and peripheral blood cytology, cytogenetics, fluorescence in situ hybridization (FISH), trephine bone marrow biopsy examination, immunophenotyping and the evaluation of molecular markers by established and new techniques. Mutations affecting growth factor receptors, cell cycle and apoptosis regulators, intracellular signaling, transcription factors, epigenetic regulation and the splicosome are involved in MDS pathogenesis and progression.

Remission of refractory Crohn's disease by high-dose cyclophosphamide and autologous peripheral blood stem cell transplantation.

BACKGROUND: Despite advances in immunosuppressive therapy, up to 10% of patients with severe Crohn's disease (CD) remain refractory to conventional treatment. Limited evidence from pilot trials suggests that high-dose immunosuppression and autologous peripheral blood stem cell transplantation (autoPBSCT) may induce remission in these patients, but there is substantial controversy regarding the safety and efficacy of this approach. AIM: To address this issue, a monocentre phase I/II trial of autoPBSCT was performed in patients with refractory CD in our hospital. METHODS: Here, we report on the outcome of 12 patients with refractory CD treated with autoPBSCT. Briefly, CD34(+) -selected PBSCs were harvested after mobilisation therapy with cyclophosphamide and granulocyte-colony stimulating factor. Later, immunoablative conditioning therapy with high-dose cyclophosphamide followed by autoPBSCT was applied and clinical and endoscopic responses were analysed after a mean follow-up of 3.1 years (range 0.5-10.3 years). RESULTS: PBSC harvest following mobilisation chemotherapy was successful in 11/12 patients and resulted in a clinical and endoscopic improvement in 7/12 patients. Subsequent conditioning and autoPBSCT were performed in nine patients and were relatively well tolerated. Among those, five patients achieved a clinical and endoscopic remission within 6 months after autoPBSCT. However, relapses occurred in 7/9 patients during follow-up, but disease activity could be controlled by low-dose corticosteroids and conventional immunosuppressive therapy. CONCLUSION: Immunoablation by cyclophosphamide and autologous peripheral blood stem cell transplantation is safe and effective to induce remission of refractory Crohn's disease, and should be further evaluated in randomised controlled trials.

Endoscopic diagnosis of acute intestinal GVHD following allogeneic hematopoietic SCT: a retrospective analysis in 175 patients.

Diagnosis of acute intestinal GVHD (aGVHD) following allogeneic hematopoietic cell transplantation is based on clinical symptoms and histological lesions. This retrospective analysis aimed to validate the 'Freiburg Criteria' for the endoscopic grading of intestinal aGVHD. Grade 1: no clear-cut criteria; grade 2: spotted erythema; grade 3: aphthous lesions; and grade 4: confluent defects, ulcers, denudation of the mucosa. Having excluded patients with infectious diarrhea, we evaluated 175 consecutive patients between January 2001 and June 2009. Setting a cutoff between grade 1 (no change in therapy) and grade 2 (intensification of immunosuppression), macroscopy had a sensitivity of 89.2% (95% confidence interval (CI): 80.4-94.9%), a specificity of 79.4% (95% CI: 69.6-87.1%), a positive-predictive value of 79.6% (95% CI: 70.0-87.2%) and a negative-predictive value of 89.0% (95% CI: 80.2-94.9%). In all, 20% of patients with aGVHD in the lower gastrointestinal tract (GIT) had lesions only in the terminal ileum. In all patients with aGVHD ≥2 of the upper GIT, typical lesions were also found in the lower GIT. Ileo-colonoscopy showed the highest diagnostic yield for aGVHD. In conclusion, the 'Freiburg Criteria' for macroscopic diagnosis of intestinal aGVHD provide high accuracy for identifying aGVHD ≥2.

[Chronic myeloid neoplasms. Diagnostic criteria and current therapeutic concepts]. / Chronische myeloische Neoplasien. Diagnostische Kriterien und Ausblick auf aktuelle Theapiekonzepte.

Myeloproliferative neoplasms (MPNs) and related chronic disorders constitute a subgroup of myeloid malignancies which are defined according to clinical, morphological and molecular features by the actual World Health Organization classification of tumors of the haematopietic system. Screening procedures for a BCR-ABL fusion gene, JAK2, thrombopoietin receptor and KIT mutations are formally included in the diagnostic approach. Myelodysplastic/MPN overlap syndromes include rare entities such as refractory anemia with ringed sideroblasts characterized by a high proportion of JAK2V617F mutated cases. The paradigm of targeted treatment of chronic myeloid leukemia with imatinib has now been extended to eosinophilia-associated myeloid neoplasms with PDGFRA, PDGFRB or FGFR1 gene mutations. Pegylated interferon-alpha has convincingly been proved to reduce the JAK2 allele burden. JAK2 inhibitor drugs are currently being tested in clinical trials. The development of pathogenesis-targeted diagnostic and therapeutic approaches to the various MPNs will continue in the future.

Alternative implication of CXCR4 in JAK2/STAT3 activation in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive, rapidly metastasising tumour. Previously, we demonstrated the influence of CXCL12-CXCR4 interaction on processes involved in metastasis and chemoresistance in SCLC. We show here that STAT3 is expressed in both primary SCLC tumour tissues and SCLC cell lines. We investigated the function of STAT3 upon CXCL12 stimulation in SCLC cell lines. Small cell lung cancer cell lines present constitutive phosphorylation of STAT3, and in the reference cell lines NCI-H69 and NCI-H82 constitutive phosphorylation was further increased by CXCL12 stimulation. Further investigating this signalling cascade, we showed that it involves interactions between CXCR4 and JAK2 in both cell lines. However CXCL12-induced adhesion to VCAM-1 could be completely inhibited by the JAK2 inhibitor AG490 only in NCI-H82. Furthermore, CXCR4 antagonist but not AG490 inhibited cell adhesion whereas both antagonisms were shown to inhibit growth of the cells in soft agar, indicating the central involvement of this signalling in anchorage-independent growth of SCLC cells. Most interestingly, while using primary tumour material, we observed that in contrast to non-small-cell lung cancer samples from primary tumour tissues, all analysed samples from SCLC were strongly positive for tyrosine-phosphorylated STAT3. Taken together, these data indicate that STAT3 is constitutively phosphorylated in SCLC and is important in SCLC growth and spreading thus presenting an interesting target for therapy.

Chronic colitis due to an epithelial barrier defect: the role of kindlin-1 isoforms.

Kindlin-1 is an epithelium-specific phosphoprotein and focal adhesion adaptor component. Mutations in the corresponding gene (KIND1) cause Kindler syndrome (KS), which is manifested by skin blistering, poikiloderma, photosensitivity and carcinogenesis. Some patients also exhibit gastrointestinal symptoms, but it has remained unclear whether these represent a feature of Kindler syndrome or a coincidence. We examined kindlin-1 in human gastrointestinal epithelia and showed that it is involved in the aetiopathology of Kindler syndrome-associated colitis. Kindlin-1 expression was assessed by indirect immunofluorescence, western blot and RT-PCR. Kindlin-1 is expressed in oral mucosa, colon and rectum. Both the full-length 74 kDa kindlin-1 protein and a 43 kDa isoform were detected in CaCo2 cells, the latter resulting from alternative splicing. In the first months of life, patients (homozygous for null mutations) had severe intestinal involvement with haemorrhagic diarrhoea and showed morphological features of severe ulcerative colitis. Later in childhood, histopathology demonstrated focal detachment of the epithelium in all segments of the colon, chronic inflammation and mucosal atrophy. These findings define an intestinal phenotype for Kindler syndrome as a consequence of a primary epithelial barrier defect. The different clinical intestinal manifestations in Kindler syndrome patients may be explained by partial functional compensation of kindlin-1 deficiency by the intestinal isoform or by the presence of truncated mutant kindlin-1.

The Ki67+ proliferation index correlates with increased cellular retinol-binding protein-1 and the coordinated loss of plakophilin-1 and desmoplakin during progression of cervical squamous lesions.

AIMS: To investigate the modulation of cellular retinol-binding protein (CRBP)-1 and the desmosomal plaque proteins plakophilin (PKP)-1 and desmoplakin (DP) in correlation with the Ki67+ proliferation index (PI) during the progression of cervical squamous intraepithelial lesions (SIL) to squamous cell carcinoma (SCC). METHODS: Using in situ imaging by brightfield and confocal laser scanning microscopy, the expression of CRBP-1 protein and transcripts, PKP-1, DP and the Ki67 PI were analysed in 38 low-grade (L) SIL, 56 high-grade (H) SIL, 49 SCC, 30 control cervices and 10 human papillomavirus-positive condylomatous lesions. RESULTS: CRBP-1+ cells increased from 11.4% in the normal cervix to 80.3% in LSILs, 92.3% in HSILs and slightly decreased to 78.3% in invasive SCCs (P = 0.0001) in close association with the Ki67 PI (r =0.41; P < 0.0001). PKP-1+ and DP+ cells were correlated (0.32; P < 0.0001) and decreased from normal (81% versus 92.3%) to LSIL (53.1% versus 85.3%), to HSIL (46.4% versus 67.5%) and SCC (35.1% versus 35.9%). The Ki67+ PI was inversely correlated with DP (-0.20, P = 0.0014) and PKP-1 (-0.19, P = 0.015). Condylomata retained low CRBP-1 and high expression of PKP-1 and DP. CONCLUSIONS: The gain of CRBP-1 and the loss of desmosomal proteins occur early in cervical carcinogenesis.

CD117 (c-kit) expression in human hepatocellular carcinoma.

AIMS: Although various methods of treatment have been tried, treatment options for advanced hepatocellular carcinoma (HCC) remain limited. Expression of the platelet-derived growth factor has been shown in HCC, which may derive from hepatic stem cells that express the c-kit proto-oncogene. Because of the promising results of imatinib and the key role played by c-kit in gastrointestinal stromal tumours and other solid tumours, the aim of this study was to determine the prevalence of c-kit (CD117) overexpression in patients with HCC. MATERIALS AND METHODS: A retrospective study of 258 archival specimens of subjects with histologically confirmed HCC was carried out. Expression of the c-kit proto-oncogene was evaluated by immunohistochemistry using rabbit anti-CD117 antibody A4502. RESULTS: The overall percentage of positive immunohistochemical staining of HCCs was 2.3% (6/258). CONCLUSIONS: Our results suggest that CD117 is not significantly overexpressed in HCC and there seems to be no role for the use of imatinib.

Immunophenotype as prognostic factor for diffuse large B-cell lymphoma in patients undergoing clinical risk-adapted therapy.

BACKGROUND: For patients with diffuse large B-cell lymphoma (DLBCL), the International Prognostic Index (IPI) predicts the likelihood for cure with chemotherapy. Biological parameters, including expression of Bcl-6, Bcl-2, CD10, major histocompatibility complex class II, and categorization as germinal center (GC) type have been described as IPI-independent prognostic factors. PATIENTS AND METHODS: Biological parameters were evaluated retrospectively by immunohistochemistry in 60 consecutive DLBCL patients of the prerituximab era. Forty-one of 60 patients underwent a risk-adapted treatment strategy including autologous stem-cell transplantation for high-risk patients (age-adjusted IPI = 2-3; slow response to chemotherapy). RESULTS: Bcl-6 expression was associated with superior overall survival (OS) independently of the IPI. Inferior progression-free survival (PFS) was independently correlated with high expression of Bcl-2 and low positivity for HLA-DR and CD10. Distinction into GC and non-GC DLBCL on the basis of Bcl-6, CD10, and IRF-4 expression had no independent prognostic value. Within the risk-adapted treatment strategy, only HLA-DR retained a prognostic impact on OS (P = 0.0058) and PFS (P = 0.0002). CONCLUSIONS: In 60 patients with DLBCL treated with risk-adapted therapy, immunohistochemical subcategorization of DLBCL into GC and non-GC type has little clinical value. The IPI-associated risk appears to be mitigated by intensified upfront therapy. Low HLA-DR expression is associated with poor outcome after intensified upfront therapy. Therefore, additional treatment modalities appear to be required.

Polymyositis--an unusual presentation of cGvHD in children.

Polymyositis is a rare manifestation of cGvHD in adult patients following allogeneic BMT. Here, we report on a 2.1-yr-old girl who presented with a facial swelling and rapidly developing respiratory failure eight months after BMT for severe combined immunodeficiency. Possible infectious agents and autoimmune origin other than cGvHD were excluded. The girl responded promptly to steroid therapy and remains well without other signs of cGvHD four months later.

[Hematological side effects of tyrosine kinase inhibition using imatinib]. / Hämatologische Nebenwirkungen der Tyrosinkinaseinhibition mit Imatinib.

Imatinib (STI571, Gleevec/Glivec) and other small-molecule tyrosine kinase inhibitors are highly effective in the treatment of chronic myeloid leukemia (CML), gastrointestinal stromal tumors and, for example, eosinophilia-associated chronic myeloproliferative disorders. This molecularly targeted approach disrupts abnormal tyrosine kinase dependent signalling pathways, thus providing a preferred treatment option for selected neoplastic disorders with activating mutations of Abelson-, Abl-related-, Kit-, and platelet-derived growth factor receptor A and B genes. Loss of response to imatinib may be due to an acquired resistance of emerging mutant tumor cell clones. Therapy is generally well tolerated. However, toxicities including edema, skin rashes, fatigue, nausea and myelosuppression have been reported. Philadelphia/Bcr-Abl-negative clonal chromosomal abnormalities may develop. Bone marrow trephines obtained from CML patients in complete remission with prolonged pancytopenia secondary to imatinib generally show marrow hypoplasia. Morphological features may be in keeping with either aplastic anemia or myelodysplasia developing in Philadelphia-negative hematopoiesis. Single or multilineage myelodysplasia may be accompanied by an excess of blasts and rarely evolves into acute leukemia in CML patients. Severe adverse hematological effects of imatinib are extremely rare. Current questions involve the molecular mechanisms of hematological side effects of tyrosine kinase inhibitors with special regard to the emergence of distinct aberrant clones.