Human CD30+ B cells represent a unique subset related to Hodgkin lymphoma cells.

Very few B cells in germinal centers (GCs) and extrafollicular (EF) regions of lymph nodes express CD30. Their specific features and relationship to CD30-expressing Hodgkin and Reed/Sternberg (HRS) cells of Hodgkin lymphoma are unclear but highly relevant, because numerous patients with lymphoma are currently treated with an anti-CD30 immunotoxin. We performed a comprehensive analysis of human CD30+ B cells. Phenotypic and IgV gene analyses indicated that CD30+ GC B lymphocytes represent typical GC B cells, and that CD30+ EF B cells are mostly post-GC B cells. The transcriptomes of CD30+ GC and EF B cells largely overlapped, sharing a strong MYC signature, but were strikingly different from conventional GC B cells and memory B and plasma cells, respectively. CD30+ GC B cells represent MYC+ centrocytes redifferentiating into centroblasts; CD30+ EF B cells represent active, proliferating memory B cells. HRS cells shared typical transcriptome patterns with CD30+ B cells, suggesting that they originate from these lymphocytes or acquire their characteristic features during lymphomagenesis. By comparing HRS to normal CD30+ B cells we redefined aberrant and disease-specific features of HRS cells. A remarkable downregulation of genes regulating genomic stability and cytokinesis in HRS cells may explain their genomic instability and multinuclearity.

The Impact of Soy Isoflavones on MCF-7 and MDA-MB-231 Breast Cancer Cells Using a Global Metabolomic Approach.

Despite substantial research, the understanding of the chemopreventive mechanisms of soy isoflavones remains challenging. Promising tools, such as metabolomics, can provide now a deeper insight into their biochemical mechanisms. The purpose of this study was to offer a comprehensive assessment of the metabolic alterations induced by genistein, daidzein and a soy seed extract on estrogen responsive (MCF-7) and estrogen non-responsive breast cancer cells (MDA-MB-231), using a global metabolomic approach. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that all test compounds induced a biphasic effect on MCF-7 cells and only a dose-dependent inhibitory effect on MDA-MB-231 cells. Proton nuclear magnetic resonance (¹H-NMR) profiling of extracellular metabolites and gas chromatography-mass spectrometry (GC-MS) profiling of intracellular metabolites confirmed that all test compounds shared similar metabolic mechanisms. Exposing MCF-7 cells to stimulatory concentrations of isoflavones led to increased intracellular levels of 6-phosphogluconate and ribose 5-phosphate, suggesting a possible upregulation of the pentose phosphate pathway. After exposure to inhibitory doses of isoflavones, a significant decrease in glucose uptake was observed, especially for MCF-7 cells. In MDA-MB-231 cells, the glutamine uptake was significantly restricted, leading to alterations in protein biosynthesis. Understanding the metabolomic alterations of isoflavones represents a step forward in considering soy and soy derivates as functional foods in breast cancer chemoprevention.

Prognostic Impact of Port-Site Metastasis After Diagnostic Laparoscopy for Epithelial Ovarian Cancer.

BACKGROUND: This study was designed to evaluate the prevalence, morbidity, and prognostic impact of port-site metastasis (PSM) in patients with epithelial ovarian cancer (EOC) undergoing laparoscopy before subsequent primary debulking surgery (PDS). METHODS: All consecutive patients treated between 2000 and 2014, who had a laparoscopy followed by PDS, were extracted from our prospectively maintained database. All patients with histological examination of port-sites were included in this unicentric exploratory analysis. RESULTS: A total of 250 (25.5 %) of 982 patients with EOC underwent laparoscopy before PDS. Port-site resection was performed in those 214 (85.6 %) patients in whom a complete or almost complete resection with residuals ≤1 cm was achieved. Median interval between laparoscopy and PDS was 25 days. PSM was detected in 100 of 214 patients (46.7 %). Risk factors for PSM were higher tumor stage (odds ratio [OR] 13.5, 95 % confidence interval [CI] 2.9-62.0, p = 0.04), positive lymph node status (OR 3.0, 95 % CI 1.3-6.7, p = 0.009), and ascites >500 mL (OR 3.9, 95 % CI 1.5-10.0, p = 0.005). Wound healing disorders and postoperative morbidity were significantly higher in patients with PSM (Clavien-Dindo Classification grade 3-5: 41.0 vs. 14.9 %, p < 0.001). However, multivariate Cox-regression models did not identify PSM as independent prognostic factor. CONCLUSIONS: The prevalence of PSM after laparoscopy in EOC patients is considerably high. PSM had no impact on survival; however, PSM were associated with more postoperative complications and a higher surgical treatment burden. This should be balanced with the expected benefit when laparoscopy is considered for the management of EOC.

Soy Isoflavones and Breast Cancer Cell Lines: Molecular Mechanisms and Future Perspectives.

The potential benefit of soy isoflavones in breast cancer chemoprevention, as suggested by epidemiological studies, has aroused the interest of numerous scientists for over twenty years. Although intensive work has been done in this field, the preclinical results continue to be controversial and the molecular mechanisms are far from being fully understood. The antiproliferative effect of soy isoflavones has been commonly linked to the estrogen receptor interaction, but there is growing evidence that other pathways are influenced as well. Among these, the regulation of apoptosis, cell proliferation and survival, inhibition of angiogenesis and metastasis or antioxidant properties have been recently explored using various isoflavone doses and various breast cancer cells. In this review, we offer a comprehensive perspective on the molecular mechanisms of isoflavones observed in in vitro studies, emphasizing each time the dose-effect relationship and estrogen receptor status of the cells. Furthermore, we present future research directions in this field which could provide a better understanding of the inner molecular mechanisms of soy isoflavones in breast cancer.

Alterations of the CD58 gene in classical Hodgkin lymphoma.

Immune evasion plays a central role in the pathophysiology of classical Hodgkin lymphoma (cHL). As mutations of the CD58 gene contribute to immune evasion of diffuse large B cell lymphoma tumor cells, we studied whether alterations of the CD58 gene also occur in Hodgkin and Reed/Sternberg (HRS) cells of cHL. Single nucleotide polymorphism chip analysis revealed homozygous deletions within the CD58 gene in two cHL cell lines (SUP-HD1 and U-HO1). Sequencing of the CD58 gene in seven cHL cell lines disclosed in addition a homozygous splice site mutation in cell line KM-H2. None of the three mutated lines expressed CD58 protein on their surface. Thus, three of seven cHL cell lines analyzed harbor destructive CD58 mutations. Molecular analysis of isolated HRS cells from 10 primary cases of cHL; however, did not reveal any case with a CD58 mutation. A FICTION study indicated heterozygous deletions of CD58 in 3 of 13 cHL analyzed. Overall, we report frequent inactivating mutations of CD58 in cHL cell lines, but their rare occurrence in primary HRS cells. As the three cHL cell lines with CD58 mutations were all established from HRS cells located in pleural effusions, i.e., outside the normal lymph node microenvironment, in end-stages of the disease, CD58 inactivation in cHL might be predominantly prevalent to such situations.

Subclonal evolution of a classical Hodgkin lymphoma from a germinal center B-cell-derived mantle cell lymphoma.

Composite lymphomas (CL) represent the occurrence of two distinct lymphomas in the same patient. Often, CL share a common cellular origin, thus representing a unique model to investigate the multistep genetic path leading to lymphomagenesis in general and to the specific development of each distinct lymphoma component in particular. Here, we present the molecular analysis of a case consisting of an unusual Hodgkin lymphoma (HL) and a mantle cell lymphoma (MCL), intimately admixed within one another in lymph nodes and bone marrow yet phenotypically distinct, in a patient who first presented with splenic/leukemic MCL two years earlier. MCL and Hodgkin and Reed/Sternberg (HRS) cells harbored identical immunoglobulin (Ig) VH gene rearrangements with shared somatic mutations, proving their common clonal origin from a (post-)germinal center (GC) B cell. This also demonstrates the (post-)GC origin of MCL with mutated IgV genes. Both lymphomas carried the same CCND1/IGH translocation and, unexpectedly for HL, expressed cyclin D1 and OCT2. Thus, HRS cells are able to preserve IGH locus activity (otherwise usually silenced in HL) to promote expression of an oncogene translocated into this locus. Both lymphoma populations further showed an identical TP53 function-impairing mutation, and later acquired a TP53 heterozygous deletion independently from one another (convergent evolution). The surprisingly close genetic relationship of the lymphomas, together with their histological intermingling and the clinical history of the patient, suggests subclonal evolution of HL from MCL as a plausible pathway in alternative to that so far described in CL, i.e. separate development from a common precursor.

Early use of cardiac troponin-I and echocardiography imaging for prediction of myocardial infarction size in Wistar rats.

AIMS: Evaluating myocardial infarct (MI) size prior to intervention is fundamental to ensure accurate results in experimental studies. However, this assessment is performed at late time points. We aimed to evaluate whether measuring plasma cardiac troponin I (cTnI) and performing echocardiographic assessment at earlier time points can predict the occurrence of MI and infarct size. MAIN METHODS: Male Wistar rats were subjected to MI (n=40) or sham surgery (n=11). cTnI levels were measured 2 and 8h after MI. Echocardiographic evaluations were performed at 48h and 14days post-MI. After 14days, the animals were euthanized, and the hearts were removed and paraffin-embedded for Sirius red staining. KEY FINDINGS: cTnI plasma levels increased in the MI group relative to the sham group at 2h after MI (7.2±9.4ng/mL vs. 2.3±1.0ng/mL; p<0.01) with a further increase at 8h after MI (22.2±13.5ng/mL vs. 1.5±1.7ng/mL; p<0.001). cTnI levels (8h) and echocardiographic outcomes correlated with histological infarct size 14days after MI (r=0.74, p<0.001 and r=0.84, p<0.001, respectively), but only echocardiography could confidently identify small, medium, and large infarcts. Additionally, using a cutoff value of 4.8ng/mL we achieved 100% specificity and 91% sensitivity in detecting MI. SIGNIFICANCE: A cutoff value of 4.8ng/mL for cTnI could be used as early as 8h after MI to accurately identify infarct in this model, whereas echocardiographic images taken 48h after MI predicted the infarcted area 14days after MI.

The endocytic uptake pathways of targeted toxins are influenced by synergistically acting Gypsophila saponins.

The expression of the epidermal growth factor (EGF) receptor is upregulated in many human tumors. We developed the targeted toxin SE, consisting of the plant toxin saporin-3 and human EGF. The cytotoxic effect of SE drastically increases in a synergistic manner by a combined treatment with Saponinum album (Spn), a saponin composite from Gypsophila paniculata L. Here we analyzed which endocytic pathways are involved in the uptake of SE and which are mandatory for the Spn-mediated enhancement. We treated HER14 cells (NIH-3T3 cells transfected with human EGF receptor) with either chlorpromazine, dynasore, latrunculin A, chloroquine, bafilomycin A1 or filipin and analyzed the effect on the cytotoxicity of SE alone or in combination with Spn. We demonstrated that SE in combination with Spn enters cells via clathrin- and actin-dependent pathways and the acidification of the endosomes after endocytosis is relevant for the cytotoxicity of SE. Notably, our data suggest that SE without Spn follows a different endocytic uptake pathway. SE cytotoxicity is independent of blocking of clathrin or actin, and the decrease in endosomal pH is irrelevant for SE cytotoxicity. Furthermore, Spn has no influence on the retrograde transport. This work is important for the better understanding of the underlying mechanism of Spn-enhanced cytotoxicity and helps to describe the role of Spn better.

A lysine-free mutant of epidermal growth factor as targeting moiety of a targeted toxin.

AIMS: Elevated levels of epidermal growth factor (EGF) receptor are observed on several human tumors, e.g. cervical carcinoma and mamma carcinomas. The natural ligand EGF is an alternative to established antibodies and tyrosine kinase inhibitors for targeting EGF receptor-overexpressing tumor cells for therapy. Conjugations of compounds to EGF lack the necessary homogeneity for an intended application, since several amino acids may react with the chemical linker. MAIN METHODS: We designed an EGF variant (EGF(RR)) in which the two lysines were substituted with arginine (K28R and K48R). EGF(RR) was fused to the protein toxin saporin to obtain a model protein for detailed analyses on EGF receptor binding and on both the enzymatic activity of saporin and the cytotoxicity of the fusion protein. KEY FINDINGS: The mutation decreased the enzymatic activity of saporin 2.3-fold and the binding of EGF(RR) retained its specificity for EGF receptor while increasing the Kd 5.5-fold. In spite of these differences the cytotoxicity of the fusion protein was unchanged in comparison to a fusion protein with EGF both when applied alone and in combination with cytotoxicity augmenting saponin. SIGNIFICANCE: We conclude that EGF(RR) retained its ability to bind with high specificity to EGF receptor and is thus suitable for a number of chemical linkage applications such as targeting drugs or dyes to EGF receptor-expressing cells.

Epidermal growth factor receptor expression affects the efficacy of the combined application of saponin and a targeted toxin on human cervical carcinoma cells.

Cervical cancer is the second most common cancer in women worldwide. Targeting the epidermal growth factor receptor (EGFR) is a very promising approach since it is overexpressed in about 90% of cervical tumors. Here, we quantified the toxic effect of SE, a targeted toxin consisting of epidermal growth factor (EGF) as targeting moiety and the plant toxin saporin-3, on 3 common human cervical carcinoma cell lines (HeLa, CaSki and SiHa) and recently established lines (PHCC1 and PHCC2) from 2 different individuals. A human melanocytic and a mouse cell line served as negative control. Additionally, we combined SE with saponinum album, a saponin composite from Gypsophila paniculata, which exhibited synergistic properties in previous studies. The cell lines, except for SiHa cells, revealed high sensitivity to SE with 50% cell survival in the range of 5-24.5 nM. The combination with saponin resulted in a remarkable enhancement of cytotoxicity with enhancement factors ranging from 9,000-fold to 2,500,000-fold. The cytotoxicity of SE was clearly target receptor specific since free EGF blocks the effect and saporin-3 alone was considerably less toxic. For all cervical carcinoma cell lines, we evinced a clear correlation between EGFR expression and SE sensitivity. Our data indicate a potential use of targeted toxins for the treatment of cervical cancer. In particular, the combination with saponins is a promising approach since efficacy is drastically improved.