WNT5A: a motility-promoting factor in Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) has a typical clinical manifestation, with dissemination involving functionally neighboring lymph nodes. The factors involved in the spread of lymphoma cells are poorly understood. Here we show that cHL cell lines migrate with higher rates compared with non-Hodgkin lymphoma cell lines. cHL cell migration, invasion and adhesion depend on autocrine WNT signaling as revealed by the inhibition of WNT secretion with the porcupine inhibitors Wnt-C59/IWP-2, but did not affect cell proliferation. While application of recombinant WNT5A or WNT5A overexpression stimulates HL cell migration, neither WNT10A, WNT10B nor WNT16 did so. Time-lapse studies revealed an amoeboid type of cell migration modulated by WNT5A. Reduced migration distances and velocity of cHL cells, as well as altered movement patterns, were observed using porcupine inhibitor or WNT5A antagonist. Knockdown of Frizzled5 and Dishevelled3 disrupted the WNT5A-mediated RHOA activation and cell migration. Overexpression of DVL3-K435M or inhibition of ROCK (Rho-associated protein kinase) by Y-27632/H1152P disrupted cHL cell migration. In addition to these mechanistic insights into the role of WNT5A in vitro, global gene expression data revealed an increased WNT5A expression in primary HL cells in comparison with normal B-cell subsets and other lymphomas. Furthermore, the activity of both porcupine and WNT5A in cHL cells had an impact on lymphoma development in the chick chorionallantoic membrane assay. Massive bleeding of these lymphomas was significantly reduced after inhibition of WNT secretion by Wnt-C59. Therefore, a model is proposed where WNT signaling has an important role in regulating tumor-promoting processes.

Microenvironmental interactions between endothelial and lymphoma cells: a role for the canonical WNT pathway in Hodgkin lymphoma.

The interaction between vascular endothelial cells (ECs) and cancer cells is of vital importance to understand tumor dissemination. A paradigmatic cancer to study cell-cell interactions is classical Hodgkin Lymphoma (cHL) owing to its complex microenvironment. The role of the interplay between cHL and ECs remains poorly understood. Here we identify canonical WNT pathway activity as important for the mutual interactions between cHL cells and ECs. We demonstrate that local canonical WNT signaling activates cHL cell chemotaxis toward ECs, adhesion to EC layers and cell invasion using not only the Wnt-inhibitor Dickkopf, tankyrases and casein kinase 1 inhibitors but also knockdown of the lymphocyte enhancer binding-factor 1 (LEF-1) and ß-catenin in cHL cells. Furthermore, LEF-1- and ß-catenin-regulated cHL secretome promoted EC migration, sprouting and vascular tube formation involving vascular endothelial growth factor A (VEGF-A). Importantly, high VEGFA expression is associated with a worse overall survival of cHL patients. These findings strongly support the concept that WNTs might function as a regulator of lymphoma dissemination by affecting cHL cell chemotaxis and promoting EC behavior and thus angiogenesis through paracrine interactions.

A patient with severe respiratory failure caused by novel human coronavirus.

We report a case of a 45-year-old patient who developed severe acute respiratory distress syndrome accompanied by renal failure. An infection with a novel human coronavirus was confirmed and found to be the reason for rapidly progressive respiratory failure of our patient.

Contact investigation of a case of human novel coronavirus infection treated in a German hospital, October-November 2012.

On 24 October 2012, a patient with acute respiratory distress syndrome of unknown origin and symptom onset on 5 October was transferred from Qatar to a specialist lung clinic in Germany. Late diagnosis on 20 November of an infection with the novel Coronavirus (NCoV) resulted in potential exposure of a considerable number of healthcare workers. Using a questionnaire we asked 123 identified contacts (120 hospital and three out-of-hospital contacts) about exposure to the patient. Eighty-five contacts provided blood for a serological test using a two-stage approach with an initial immunofluorescence assay as screening test, followed by recombinant immunofluorescence assays and a NCoV-specific serum neutralisation test. Of 123 identified contacts nine had performed aerosol-generating procedures within the third or fourth week of illness, using personal protective equipment rarely or never, and two of these developed acute respiratory illness. Serology was negative for all nine. Further 76 hospital contacts also tested negative, including two sera initially reactive in the screening test. The contact investigation ruled out transmission to contacts after illness day 20. Our two-stage approach for serological testing may be used as a template for similar situations.

Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.

We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.

Favorable impact of the interleukin-4 receptor allelic variant I75 on the survival of diffuse large B-cell lymphoma patients demonstrated in a large prospective clinical trial.

BACKGROUND: Recently published data indicate that host germline variations in immune genes can influence the outcome of lymphoma patients. Interleukin (IL)-4 and IL13 are crucial immune factors and may influence the course of the disease. Both cytokines signal through the interleukin-4 receptor (IL4R). Therefore, we investigated whether polymorphisms of IL4, IL13 and IL4R genes could predict the outcome of diffuse large B-cell lymphoma (DLBCL) patients. METHODS: In 228 DLBCL samples of the German High-Grade Non-Hodgkin's Lymphoma Study Group, the polymorphisms of IL4 (-524CT, rs2243250), IL13 (-1069CT, rs1800925) and IL4R (I75V, rs1805010; S503P, rs1805015; Q576R, rs1801275) were analyzed and the soluble interleukin-4 receptor (sIL4R) serum level was measured before the start of chemotherapy. RESULTS: Patients harboring IL4R V75 (IL4R(I75V-AG) and IL4R(I75V-GG)) had shorter overall survival (OS) (P = 0.044) and event-free survival (EFS) (P = 0.056) periods compared with I75 carriers (IL4R(I75V-AA)). Multivariate analysis adjusted to the International Prognostic Index revealed a relative risk of 1.9 for carriers of the IL4R V75 (P = 0.011) in relation to OS. DLBCL patients homozygous for the IL4R I75 and low sIL4R serum levels have the most favorable OS and EFS. CONCLUSIONS: These data support the role for host germline gene variations of immunologically important factors like the IL4R I75V gene variation to predict the survival in DLBCL patients.

Distal and proximal interleukin (IL)-10 promoter polymorphisms associated with risk of cutaneous melanoma development: a case--control study.

Inherited promoter polymorphisms of the interleukin (IL)-10 gene resulting in altered IL-10 production may contribute to a genetic susceptibility for melanoma. We investigated the role of a haplotype from distal as well as proximal polymorphic sites [-7400InDel, -6752AT (rs6676671), -3538AT (rs1800890), -1087AG (rs1800896), -597AC (rs1800872)] of the IL-10 5'-flanking region in a hospital-based case-control study of 165 Caucasian patients with cutaneous melanoma from Germany in comparison with 162 healthy cancer-free Caucasian control participants from the same area matched by age. Using multivariate analysis for the number of nevi and skin type, the IL-10 'higher producing' haplotype ITAGC was found to be significantly associated with a reduced risk of developing melanoma (adjusted P=0.02). Although our findings need to be confirmed by independent and larger multicenter studies, we have described for the first time the association of distal gene variants of the IL-10 gene as an independent risk factor for melanoma.

MDM2 gene amplification and lack of p53 point mutations in Hodgkin and Reed-Sternberg cells: results from single-cell polymerase chain reaction and molecular cytogenetic studies.

Hodgkin's disease (HD) is the most common haematological malignancy after chronic lymphocytic leukaemia, but very little is known about its pathogenesis or the genetic events that contribute to the malignant phenotype of the tumour cells. p53 is assumed to play an important role in the pathogenesis of HD, based on the observation that p53 protein is frequently accumulated in Hodgkin and Reed-Sternberg (H & RS) cells. We investigated single H & RS cells from five different HD patients for point mutations at the genomic level using multiplex polymerase chain reaction amplification and subsequent sequencing. No point mutations were detected in 50 single H & RS cells analysed. Hence, accumulation of p53 protein cannot be explained by mutations within the gene. A genome-wide screening for genomic imbalances using comparative genomic hybridization revealed gain on chromosome 12q14, i.e. the mapping position of the MDM2 gene in several HD cases. Therefore, we assessed the copy number of the MDM2 gene using fluorescence in situ hybridization. In four out of six HD cases analysed, the copy number of the MDM2 gene was found to be increased. As gene amplification is frequently associated with protein overexpression, the observed accumulation of p53 in the nuclei of H & RS cells could be as a result of elevated MDM2 protein levels resulting in stabilization of p53 protein.

Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma.

Hodgkin lymphoma (HL) is characterized by the abnormal expression of multiple cytokines, accounting for its unique clinicopathologic features. We have previously shown that interleukin-13 (IL-13) is secreted by HL cell lines and may serve as an autocrine growth factor. To determine the frequency of IL-13 expression in lymphoma patients, tissue sections from 36 patients with classical HL, 5 patients with nodular lymphocyte predominance HL (NLPHL), and 23 patients with non-Hodgkin lymphoma (NHL) were subjected to in situ hybridization. In 31 of 36 cases (86%) of classical HL patients of all histologic subtypes, between 25% to almost 100% of Hodgkin and Reed Sternberg (HRS) cells were positive for IL-13 expression. In contrast, in no case of NLPHL and in only 4 of 23 NHL cases (1 of 5 T-cell-rich B-cell lymphomas, 2 of 5 anaplastic large cell lymphomas, and 1 of 5 peripheral T-cell lymphomas) did the neoplastic cells express IL-13. The expression of the IL-13 receptor chain alpha1 (IL-13Ralpha1) was also analyzed by in situ hybridization. In 24 of 27 (89%) cases of classical HL, between 25% to 75% of HRS cells, as well as a high frequency of lymphocytes and histiocytes, were positive for IL-13Ralpha1 expression. These results were confirmed by the construction of complementary DNA libraries from single HRS cells, followed by polymerase chain reaction analysis, in which IL-13Ralpha1 transcripts were found to be present in all 6 cases of HL. These data indicate that expression of IL-13 and IL-13Ralpha1 is a common feature of HRS cells in HL, consistent with the hypothesis that IL-13 may play a role in autocrine growth in classical HL.

Assessment of clonality of rosetting T lymphocytes in Hodgkin's disease by single-cell polymerase chain reaction: detection of clonality in a polyclonal background in a case of lymphocyte predominance Hodgkin's disease.

Rosetting of CD4+ T cells around the neoplastic Hodgkin and Reed-Sternberg (H&RS) cells is a characteristic feature of Hodgkin's disease (HD). To answer the question whether this phenomenon is solely due to chemokine-mediated attraction of T cells or whether the rosetting T cells in addition recognize antigens presented by the H&RS cells, we examined the T cells adherent to H&RS cells. Cells from five cases of HD [four classic HD and one lymphocyte-predominant (LP) HD] were examined by single-cell analysis for the T-cell receptor (TCR) gamma gene. Between 5 and 17 rosettes containing one to ten rosetting lymphocytes and the corresponding H&RS cells were amplified in separate plastic tubes. Of the resulting 119 TCRgamma polymerase chain reaction (PCR) products, 87 were sequenced. While no evidence of a clonal expansion was obtained in the lymph nodes from four of five patients with classic HD, clonal TCRgamma sequences were found in the lymph node from the patient within LPHD in two independent experiments analyzing seven and ten different rosetting complexes, respectively. Of 13 products, 11 showed identical Vgamma9 sequences. Unrelated products were found in all other TCRgamma family subgroups in this case. Single H&RS cells picked as controls were negative for TCRgamma rearrangements. Our results demonstrate that clonal proliferations on a polyclonal background can occur among the T cells forming rosettes with Hodgkin cells and lend support to the view that Hodgkin cells may also function as cells presenting antigens to the adhering T cells.

Genomic imbalances including amplification of the tyrosine kinase gene JAK2 in CD30+ Hodgkin cells.

Comparative genomic hybridization was applied for a comprehensive screening of frequently occurring net gains and losses of chromosomal subregions in small populations of CD30+ Hodgkin cells and their morphological variants. In 12 Hodgkin's lymphomas, recurrent gains were detected on chromosomal arms 2p, 9p, and 12q (in six, four, and five tumors, respectively) and distinct high-level amplifications were identified on chromosomal bands 4p16, 4q23-q24, and 9p23-p24. In Hodgkin cells with 9p23-p24 amplification, fluorescence in situ hybridization revealed an increased copy number of chromosomal sequences spanning the tyrosine kinase gene JAK2. Several of the imbalances described, in particular a gain in chromosomal arm 9p that includes JAK2 amplification, are similar to the genomic changes detected in primary mediastinal B-cell lymphoma.

Detection of the t(2;5)-associated NPM/ALK fusion cDNA in peripheral blood cells of healthy individuals.

The translocation t(2;5), which leads to the fusion of the nucleophosmin gene (NPM) on chromosome 5q35 to the receptor kinase ALK on chromosome 2p23, is found in CD30+ anaplastic large cell lymphomas and some cases of B-cell lymphoma. Hodgkin's disease (HD) is a malignant lymphoma characterized by large multinucleated tumour cells, Hodgkin and Reed-Sternberg (H&RS) cells, surrounded by a dense lymphohistiocytic infiltrate. Our group recently demonstrated NPM/ALK fusion cDNAs by single-cell RT-PCR in < 3% of CD30+ tumour cells in 2/9 cases of HD. To further delineate the relevance of this finding for HD, we studied the occurrence of NPM/ALK fusion genes in peripheral blood cells of healthy donors by RT-PCR. NPM/ALK fusion cDNAs were found by RT-PCR in 14/29 healthy individuals and confirmed by hybridization with a breakpoint-specific oligonucleotide. Due to the low rate of NPM/ALK-positive cells in the peripheral blood of positive individuals, an assignment to a defined cellular subpopulation was not possible. We conclude that NPM/ALK fusion genes are present in peripheral blood cells of healthy donors. After t(14;18) and t(9;22), t(2;5) represents the third example of tumour-associated translocation products in blood cells of apparently healthy donors. The implications of this finding are discussed.

NPM/ALK fusion mRNA expression in Hodgkin and Reed-Sternberg cells is rare but does occur: results from single-cell cDNA analysis.

BACKGROUND: The translocation t(2;5)(p23;q35) leads to the fusion of the nucleophosmin gene (NPM) on chromosome 5q35 to the recently described receptor kinase ALK on 2p23. It is characteristic of a subgroup of CD30+ large-cell anaplastic non-Hodgkin's lymphoma (ALCL). Since some cases of Hodgkin's disease (HD) and ALCL share common features, a common pathogenesis has been proposed in a report of the expression of NPM/ALK fusion mRNA in 11/13 Hodgkin's lymphomas. PATIENTS AND METHODS: We approached this question by micro-manipulatory isolation of single Hodgkin and Reed-Sternberg (H-RS) cells and subsequent RT-PCR amplification of NPM/ALK fusion cDNA from these single cells. RESULTS: Specificity of cell selection was shown by the HD-specific pattern of EBV-gene expression in single H-RS cells. In 4 out of 7 cases, NPM/ALK fusion cDNA was detected in the RNA from whole lymph node tissue. In 2 out of 9 cases, NPM/ALK fusion sequences were amplified from single H-RS cells, albeit in a very low frequency (< 5%). CONCLUSIONS: These data indicate that NPM/ALK fusion transcripts do not play an early role in the pathogenesis of HD. Whether the rare expression of NPM/ALK is the result of clonal heterogeneity or an indication for clonal evolution and progression toward ALCL can only be answered by the repeated analysis of indicator cases during the course of the disease.

N-ras genes are not mutated in Hodgkin and Reed-Sternberg cells: results from single cell polymerase chain-reaction examinations.

Ras mutations play an important role in many human tumors. They usually occur at only three codons (12, 13 and 61) of the three ras gene family members and lead to altered proteins resulting in a constitutively activated downstream signal cascade. We have examined the N-ras gene status in Hodgkin's disease (HD). Little is known about the pathogenetic events leading to malignant phenotype in HD. Since Hodgkin and Reed-Sternberg (H and RD) cells comprise only a minority of the cellular infiltrate in HD-lymph nodes, molecular studies concerning the status of oncogenes have been difficult to perform and have yielded conflicting results. We have established a single cell PCR assay for N-ras analysis and have examined H and RS cells from 12 cases of HD by PCR amplification and direct sequencing. None of the single H and RS cells examined carried N-ras mutations at either codons 12/13 or 61. Therefore, N-ras mutations are not involved in the pathogenesis of HD.

Single cell PCR for the analysis of Hodgkin's disease: four years later.

BACKGROUND: Single cell-based studies represent a promising alternative to conventional molecular approaches in the study of Hodgkin's disease since the malignant Hodgkin and Reed-Sternberg cells (H & RS) represent only a small minority of the cellular infiltrate in affected nodes. METHODS: Single cell polymerase chain reaction (PCR) assays were developed for the analysis of specific genomic DNA sequences and the detection of gene expression. Single H & RS cells were isolated by micromanipulation from cytospin slides or fresh cell suspensions after staining with an anti-CD 30 MoAB. RESULTS: The status of oncogenes and immune receptor genes was examined by DNA-PCR. So far, no IgH or TCR gamma rearrangements were detected in H & RS cells of T- and B-antigen negative classical Hodgkin's cases but were detected in two cases of nodular paragranuloma. Global cDNA amplification was successfully performed from single H & RS cells, and specific gene transcripts were detected with a novel PCR method. CONCLUSION: Single cell PCR is a novel and promising method that will help to elucidate many of the open questions in the biology of Hodgkin's disease. In the case of contradictory results, collaborations between different groups utilizing similar approaches have to be performed.

Hodgkin and Reed-Sternberg cells do not carry T-cell receptor gamma gene rearrangements: evidence from single-cell polymerase chain reaction examination.

Hodgkin and Reed-Sternberg (H&RS) cells are generally accepted to be the neoplastic cells of Hodgkin's disease (HD), even though they represent only a minority of the cellular infiltrate in affected tissues. Recent immunologic studies and Southern blot analyses of DNA extracted from whole lymph node tissue favored, but did not convincingly prove a lymphoid origin of H&RS cells. To detect rearrangements of the T-cell receptor gamma chain (TCR gamma) genes at the single-cell level as an indication of early T-cell lymphoid differentiation, we isolated H&RS cells by micromanipulation from cytospin preparations of fresh biopsy material. TCR gamma chain rearrangement was detected by polymerase chain reaction using four "forward primers" that were constructed corresponding to all four V families and two "reverse primers" corresponding to consensus sequences of J segments. Rearrangements of all V families in combination with the different J segments were detected in human peripheral blood and tonsillar T cells. Although rearrangements of TCR gamma chain genes were shown in single cells of 10 of 10 T-cell leukemias, no rearrangement of these genes was found in single H&RS cells from 13 consecutive patients with HD. Our results indicate that H&RS cells from the vast majority of cases are not derived from T cells. This finding may have implications for the pathogenesis of HD and the development of more effective treatment regimens.

Diagnosis of pancreatic adenocarcinoma by polymerase chain reaction from pancreatic secretions.

As mutations at codon 12 of the Ki-ras oncogene have been shown to occur in 90% of pancreatic adenocarcinomas, a novel strategy for the detection of these mutations in pancreatic secretions obtained at routine endoscopies was developed. Ki-ras DNA was amplified and screened for the presence of mutations at codon 12 with a combination of different rapid, non-radioactive molecular biology techniques. Examination of DNA from cell lines and paraffin-embedded tumour samples was used to establish and test the strategy employed. Pancreatic secretions from 27 patients were examined for the presence of Ki-ras mutations. Mutations at codon 12 were detected in 16/16 secretions from patients with histologically confirmed carcinoma and from one patient with carcinoma of the bile duct. In six patients a mutation identical to the one found in the pancreatic secretions was also demonstrated in paraffin-embedded fine-needle biopsy or surgical samples. Of the remaining ten patients (who had pancreatitis or cholelithiasis) mutations were not found in nine. Ki-ras codon 12 mutation was identified in one of these patients however, and mucous cell hyperplasia of pancreatic ducts was found upon histological examination. These findings establish Ki-ras polymerase chain reaction from pancreatic secretions as a valuable new diagnostic procedure for the demonstration of malignant cells, possibly at an early stage of the disease.

[Pure coronary spasm: autonomic disease or form of onset of atheroma? Contribution of repeat coronarography in 23 patients]. / Angor spastique pur: maladie autonome ou forme de début de l'athérome? Apport de la coronarographie répétée chez 23 malades.

Coronary spasm has often been blamed for facilitating the development of atheroma, but some authors regard it as a separate disease. In order to form an opinion on these two theories, we performed repeat coronary arteriography at an interval of 4 years on average in 23 patients: 19 men and 4 women aged from 38 to 62 years (mean: 49,4 years). At the initial examination the coronary vessels were normal in 11 patients and showed irregular arterial walls without significant stenosis in 12 patients. Coronary spasm was demonstrated directly in 17 cases (6 spontaneous spasms during arteriography and 11 induced spasms) and indirectly in 6 cases (ECG signs of ischaemia during the anginal attack). At the second coronary arteriography we found that the spasms persisted, with positive response to a challenge test in 17 out of the 19 patients tested. The challenge test was not performed in 4 patients who had developed significant lesions. The vessels themselves were altered in 6 patients, with images of occlusion (2 cases), stenosis (2 cases), parietal irregularities (1 case) and aneurysm (1 case) appearing on spastic arteries, and images of stenosis in 2 patients with apparently non-spastic arteries. There was no difference in age, sex, risk factors, initial coronary status and time interval between arteriographies between these 6 patients and the 17 patients whose coronary arteries had remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)