Diminazene aceturate-induced cytotoxicity is associated with the deregulation of cell cycle signaling and downregulation of oncogenes Furin, c-MYC, and FOXM1 in human cervical carcinoma Hela cells.

Diminazene aceturate (DIZE) is an FDA-listed small molecule known for the treatment of African sleeping sickness. In vivo studies showed that DIZE may be beneficial for a range of human ailments. However, there is very limited information on the effects of DIZE on human cancer cells. The current study aimed to investigate the cytotoxic responses of DIZE, using the human carcinoma Hela cell line. WST-1 cell proliferation assay showed that DIZE inhibited the viability of Hela cells in a dose-dependent manner and the observed response was associated with the downregulation of Ki67 and PCNA cell proliferation markers. DIZE-treated cells stained with acridine orange-ethidium and JC-10 dye revealed cell death and loss of mitochondrial membrane potential (Ψm), compared with DMSO (vehicle) control, respectively. Cellular immunofluorescence staining of DIZE-treated cells showed upregulation of caspase 3 activities. DIZE-treated cells showed downregulation of mRNA for G1/S genes CCNA2 and CDC25A, S-phase genes MCM3 and PLK4, and G2/S phase transition/mitosis genes Aurka and PLK1. These effects were associated with decreased mRNA expression of Furin, c-Myc, and FOXM1 oncogenes. These results suggested that DIZE may be considered for its effects on other cancer types. To the best of our knowledge, this is the first study to evaluate the effect of DIZE on human cervical cancer cells.

Insights into the molecular roles of FOXR2 in the pathology of primary pediatric brain tumors.

Forkhead box gene R2 (FOXR2) belongs to the family of FOX genes which codes for highly conserved transcription factors (TFs) with critical roles in biological processes ranging from development to organogenesis to metabolic and immune regulation to cellular homeostasis. A number of FOX genes are associated with cancer development and progression and poor prognosis. A growing body of evidence suggests that FOXR2 is an oncogene. Studies suggested important roles for FOXR2 in cancer cell growth, metastasis, and drug resistance. Recent studies showed that FOXR2 is overexpressed by a subset of newly identified entities of embryonal tumors. This review discusses the role(s) FOXR2 plays in the pathology of pediatric brain cancers and its potential as a therapeutic target.

Extracellular vesicles of human diabetic retinopathy retinal tissue and urine of diabetic retinopathy patients are enriched for the junction plakoglo bin protein.

Introduction: Diabetic Retinopathy (DR) is a potentially blinding retinal disorder that develops through the pathogenesis of diabetes. The lack of disease predictors implies a poor prognosis with frequent irreversible retinal damage and vision loss. Extracellular Vesicles (EVs) present a novel opportunity for pre-symptomatic disease diagnosis and prognosis, both severely limited in DR. All biological fluids contain EVs, which are currently being studied as disease biomarkers. EV proteins derived from urine have emerged as potential noninvasive biomarkers. Methods: In this study, we isolated EVs from DR retinal tissue explants and from DR patients' urine, and characterized the vesicles, finding differences in particle number and size. Next, we performed proteomic analysis on human explanted DR retinal tissue conditioned media, DR retinal EVs and DR urinary EVs and compared to normal human retinal tissue, retinal EVs, and urinary EVs, respectively. Results: Our system biology analysis of DR tissue and EV expression profiles revealed biological pathways related to cell-to-cell junctions, vesicle biology, and degranulation processes. Junction Plakoglobin (JUP), detected in DR tissue-derived EVs and DR urinary EVs, but not in controls, was revealed to be a central node in many identified pathogenic pathways. Proteomic results were validated by western blot. Urinary EVs obtained from healthy donors and diabetic patient without DR did not contain JUP. Conclusion: The absence of JUP in healthy urinary EVs provide the basis for development of a novel Diabetic Retinopathy biomarker, potentially facilitating diagnosis.

Human retinal organoids release extracellular vesicles that regulate gene expression in target human retinal progenitor cells.

The mechanisms underlying retinal development have not been completely elucidated. Extracellular vesicles (EVs) are novel essential mediators of cell-to-cell communication with emerging roles in developmental processes. Nevertheless, the identification of EVs in human retinal tissue, characterization of their cargo, and analysis of their potential role in retina development has not been accomplished. Three-dimensional retinal tissue derived from human induced pluripotent stem cells (hiPSC) provide an ideal developmental system to achieve this goal. Here we report that hiPSC-derived retinal organoids release exosomes and microvesicles with small noncoding RNA cargo. EV miRNA cargo-predicted targetome correlates with Gene Ontology (GO) pathways involved in mechanisms of retinogenesis relevant to specific developmental stages corresponding to hallmarks of native human retina development. Furthermore, uptake of EVs by human retinal progenitor cells leads to changes in gene expression correlated with EV miRNA cargo predicted gene targets, and mechanisms involved in retinal development, ganglion cell and photoreceptor differentiation and function.

Plumbagin Induces Cytotoxicity via Loss of Mitochondrial Membrane Potential and Caspase Activation in Metastatic Retinoblastoma.

BACKGROUND/AIM: We investigated the cytotoxic effects of plumbagin on metastatic retinoblastoma, using the highly metastatic cell line Y79. MATERIALS AND METHODS: Effect of plumbagin on cell growth was assessed with water-soluble tetrazolium 1 (WST-1) cell proliferation assay and automated hemocytometry with trypan blue-exclusion assay. Cell death was studied with acridine orange/ethidium bromide live-dead assay and annexin-V-fluorescein isothiocyanate/propidium iodide microscopy. Loss of mitochondrial membrane potential was studied with JC-10 dye and caspase activation was investigated using CellEvent Caspase-3/7 Green detection reagent. RESULTS: Plumbagin highly significantly reduced the growth of Y79 cells treated for 24 h with 2.5 µM or more. Plumbagin also induced significantly high levels of cell death which was associated with loss of mitochondrial membrane potential and caspase activation. CONCLUSION: At very low concentration (2.5 µM), plumbagin potently induced cytotoxicity in metastatic retinoblastoma cells via loss of mitochondrial membrane potential and caspase activation.

Immunohistochemical localization of prolactin receptor (PRLR) to Hodgkin's and Reed-Sternberg cells of Hodgkin's lymphoma.

Prolactin receptor (PRLR), a type-1 cytokine receptor, is overexpressed in a number of cancer types. It has attracted much attention for putative pro-oncogenic roles, which however, remains controversial in some malignancies. In this study, we reported the localization of PRLR to the Hodgkin's and Reed-Sternberg (HRS) cells of Hodgkin's lymphoma (HL), a neoplasm of predominantly B cell origin. Immunohistochemistry performed on 5-µm thick FFPE sections revealed expression of PRLR in HRS cells. Cellular immunofluorescence experiments showed that the HL-derived cell lines, Hs445, KMH2 and L428 overexpressed PRLR. The PRLR immunofluorescent signal was depleted after treating the cell lines with 10 µM of siRNA for 48 h. We also tested whether PRLR is involved in the growth of HL, in vitro. One-way analysis of variance (ANOVA) on cell growth data obtain from WST-1 cell proliferation assay and trypan blue exclusion assay and hemocytometry showed that siRNA-depletion of PRLR expression resulted in decreased growth in all three cell lines. These results offered only a short insight into the involvement of PRLR in HL. As a result, further investigation is required to decipher the precise role(s) of PRLR in the pathogenesis of HL.

Luteolin Induces Cytotoxicity in Mix Cellularity Classical Hodgkin's Lymphoma via Caspase Activated-cell Death.

BACKGROUND/AIM: We investigated the effects of luteolin (LUT) on classical Hodgkin's lymphoma (cHL), since such studies in malignant lymphomas are lacking. MATERIALS AND METHODS: Effect of LUT on cell growth was assessed with water-soluble tetrazolium 1 (WST-1) cell proliferation assay and automated hemocytometry on trypan blue-exclusion assay. Cell death was investigated with acridine orange/ethidium bromide live-dead assay, propidium iodide (PI) flow cytometry, and Annexin-V-PI microscopy. Caspase activation was studied using CellEvent Caspase-3/7 Green detection reagent. High resolution immunofluorescence microscopy was used to detect cleaved-PARP-1. RESULTS: LUT induced a dose-dependent decrease in the growth of KMH2 and L428 cells, cellular models of mix-cellularity (MC) and nodular sclerosis (NS) cHL, respectively. However, LUT induced cell death only in KMH2, at a higher concentration, and this was associated with caspase activation and cleaved PARP-1. CONCLUSION: LUT induces cytotoxicity in the MC-cHL cellular model KMH2 via caspase activation.

Effects of vitamin D3 and its chemical analogs on the growth of Hodgkin's lymphoma, in vitro.

OBJECTIVE: Vitamin D receptor (VDR) activities have been noted for a number of B cell malignancies which showed varying sensitivities to vitamin D3 (1,25-dihydroxyvitamin D3, VD3, calcitriol) and its synthetic analogs. The objective of this study was to address the potential effects of VD3 and vitamin D3 analogs (VDAs) on the growth of Hodgkin's lymphoma (HL), a malignant pathology of B cell origin, in vitro. RESULTS: Immunofluorescence staining showed the expression of VDR by primary Hodgkin's (H) and Reed-Sternberg (RS)-HRS-tumor cells in HL histological sections. Western blot analyses revealed expression of VDR in the HL cell lines Hs445, HDLM2, KMH2, and L428. One-way analysis of variance (ANOVA) on data obtained from water-soluble tetrazolium 1 (WST-1) cell proliferation assay showed decreased cell growth in HDLM2 and L428, 72 h after treatment with 10 µM of either VD3 of VDAs. Western blot analyses showed that treatment of L428 cells with the VDAs (calcipotriol and EB1089) resulted in modest increases in nuclear accumulation of VDR (nuVDR) compared to either dimethyl sulfoxide (DMSO) or VD3 treatments. nuVDR for DMSO control and VD3 was comparable. These results suggest that VD3 or VDAs may affect growth of HL.

Putative Cellular and Molecular Roles of Zika Virus in Fetal and Pediatric Neuropathologies.

Although the World Health Organization declared an end to the recent Zika virus (ZIKV) outbreak and its association with adverse fetal and pediatric outcome, on November 18, 2016, the virus still remains a severe public health threat. Laboratory experiments thus far supported the suspicions that ZIKV is a teratogenic agent. Evidence indicated that ZIKV infection cripples the host cells' innate immune responses, allowing productive replication and potential dissemination of the virus. In addition, studies suggest potential transplacental passage of the virus and subsequent selective targeting of neural progenitor cells (NPCs). Depletion of NPCs by ZIKV is associated with restricted brain growth. And while microcephaly can result from infection at any gestational stages, the risk is greater during the first trimester. Although a number of recent studies revealed some of specific molecular and cellular roles of ZIKV proteins of this mosquito-borne flavivirus, the mechanisms by which it produces it suspected pathophysiological effects are not completely understood. Thus, this review highlights the cellular and molecular evidence that implicate ZIKV in fetal and pediatric neuropathologies.

Author Correction: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins.

A range of cell types, including embryonic stem cells, neurons and astrocytes have been shown to release extracellular vesicles (EVs) containing molecular cargo. Across cell types, EVs facilitate transfer of mRNA, microRNA and proteins between cells. Here we describe the release kinetics and content of EVs from mouse retinal progenitor cells (mRPCs). Interestingly, mRPC derived EVs contain mRNA, miRNA and proteins associated with multipotency and retinal development. Transcripts enclosed in mRPC EVs, include the transcription factors Pax6, Hes1, and Sox2, a mitotic chromosome stabilizer Ki67, and the neural intermediate filaments Nestin and GFAP. Proteomic analysis of EV content revealed retinogenic growth factors and morphogen proteins. mRPC EVs were shown to transfer GFP mRNA between cell populations. Finally, analysis of EV mediated functional cargo delivery, using the Cre-loxP recombination system, revealed transfer and uptake of Cre+ EVs, which were then internalized by target mRPCs activating responder loxP GFP expression. In summary, the data supports a paradigm of EV genetic material encapsulation and transfer within RPC populations. RPC EV transfer may influence recipient RPC transcriptional and post-transcriptional regulation, representing a novel mechanism of differentiation and fate determination during retinal development.

Dextran hydrogels by crosslinking with amino acid diamines and their viscoelastic properties.

Amine functionalized polysaccharide hydrogels such as those based on chitosan are widely examined as biomaterials. Here we set out to develop a facile procedure for developing such hydrogels by crosslinking dextran with amino acid diamines. The dextran-amino acid gels were formed by the addition of the amino acid diamines to a dextran and epichlorohydrin solution once it became homogeneous. This was demonstrated with three amino acid diamines, lysine, lysine methyl ester, and cystine dimethyl ester. Hydrogel networks with albumin entrapped were also demonstrated. These hydrogels were characterized by FTIR, SEM, rotational rheometry, swelling studies and cell biocompatibility analysis. These hydrogels showed the unexpected pH-responsive behavior of greater swelling at more basic pH, similar to that of an anionic hydrogel. This is uncharacteristic for amine functionalized gels as they typically exhibit cationic hydrogel behavior. All hydrogels showed similar biocompatibility to that of dextran crosslinked without amino acids.

Insights into the molecular roles of Zika virus in human reproductive complications and congenital neuropathologies.

The recent upsurge in the association of congenital neurological disorders and infection by the Zika virus (ZIKV) has resulted in increased research focus on the biology of this flavivirus. Studies in animal models indicate that ZIKV can breach the placental barrier and selectively infect and deplete neuroprogenitor cells (NPCs) of the developing fetus, resulting in changes of brain structures, reminiscent of human microcephaly. In vitro and ex vivo studies using human cells and tissues showed that human NPCs and placental cells are targeted by ZIKV. Also of concern is the impact of ZIKV on human reproductive structures, with the potential to cause infertility, as the virus appears to remain in the genital tract for extended periods of time. This review discusses the putative roles of ZIKV on human reproductive complications and congenital neuropathologies.

Insights into the molecular roles of heparan sulfate proteoglycans (HSPGs-syndecans) in autocrine and paracrine growth factor signaling in the pathogenesis of Hodgkin's lymphoma.

Syndecans (SDC, SYND) comprise a group of four structurally related type 1 transmembrane heparan sulfate proteoglycans (HSPGs) that play important roles in tumorigenic processes. SDCs exert signaling via their protein cores and their conserved transmembrane and cytoplasmic domains or by forming complexes with growth factors (GFs). In classical Hodgkin's lymphoma (cHL), a lymphoid neoplasm of predominantly B cell origin, SDC1 and SDC4 are the active SDCs, and a number of GF (vascular endothelial growth factor, fibroblast growth factor, etc.) signaling pathways have been studied. However, despite extensive pre-clinical and clinical research on SDC-mediated GF signaling in many cancer types, there is very limited data for this interaction in cHL. Thus, this review highlights the relevant literature focusing on the potential interactions of SDCs and GFs in cHL pathogenesis. Also discussed are the pre-clinical and clinical studies targeting signaling through these pathways.

Advances in the molecular functions of syndecan-1 (SDC1/CD138) in the pathogenesis of malignancies.

Syndecan-1 (SDC1, synd, CD138) is the most widely studied member of four structurally related cell surface heparan sulfate proteoglycans (HSPG). Although SDC1 has been implicated in a wide range of biological functions, its altered expression often produces malignant phenotypes, which arise from increased cell proliferation and cell growth, cell survival, cell invasion and metastasis, and angiogenesis. Recent studies revealed much about the underlying molecular roles of SDC1 in these processes. The changes in SDC1 expression also have a direct impact on the clinical course of cancers, as evident by its prognostic significance. Accumulating evidence suggest that SDC1 is involved in stimulation of cancer stem cells (CSC) or tumor initiating cells (TIC) and this may affect disease relapse, and resistance to therapy. This review discusses the progress on the pro-tumorigenic role(s) of SDC1 and how these roles may impact the clinical aspect of the disease. Also discussed, are the current strategies for targeting SDC1 or its related signaling.

Kallikrein family proteases KLK6 and KLK7 are potential early detection and diagnostic biomarkers for serous and papillary serous ovarian cancer subtypes.

BACKGROUND: Early detection of ovarian cancer remains a challenge due to widespread metastases and a lack of biomarkers for early-stage disease. This study was conducted to identify relevant biomarkers for both laparoscopic and serum diagnostics in ovarian cancer. METHODS: Bioinformatics analysis and expression screening in ovarian cancer cell lines were employed. Selected biomarkers were further validated in bio-specimens of diverse cancer types and ovarian cancer subtypes. For non-invasive detection, biomarker proteins were evaluated in serum samples from ovarian cancer patients. RESULTS: Two kallikrein (KLK) serine protease family members (KLK6 and KLK7) were found to be significantly overexpressed relative to normal controls in most of the ovarian cancer cell lines examined. Overexpression of KLK6 and KLK7 mRNA was specific to ovarian cancer, in particular to serous and papillary serous subtypes. In situ hybridization and histopathology further confirmed significantly elevated levels of KLK6 and KLK7 mRNA and proteins in tissue epithelium and a lack of expression in neighboring stroma. Lastly, KLK6 and KLK7 protein levels were significantly elevated in serum samples from serous and papillary serous subtypes in the early stages of ovarian cancer, and therefore could potentially decrease the high "false negative" rates found in the same patients with the common ovarian cancer biomarkers human epididymis protein 4 (HE4) and cancer antigen 125 (CA-125). CONCLUSION: KLK6 and KLK7 mRNA and protein overexpression is directly associated with early-stage ovarian tumors and can be measured in patient tissue and serum samples. Assays based on KLK6 and KLK7 expression may provide specific and sensitive information for early detection of ovarian cancer.

Metformin induces ultrastructural alterations in hepatocytes of spontaneously hypertensive rats / La metformina induce alteraciones ultraestructurales en hepatocitos de ratas espontáneamente hipertensas

Metformin, an oral biguanide approved for the treatment of type II diabetes is widely prescribed for other clinical conditions. Currently, metformin is being investigated as potential anti-tumor agent. However, there have been recent concerns about hepatotoxicity associated with the use of metformin. This study, by means of high resolution transmission electron microscopy (TEM) and morphometry, investigated potential ultrastructural changes induced by metformin treatment on the hepatocytes of spontaneously hypertensive rats (SHR). Morphometric analysis was carried out on images of randomly selected cells from sectioned gluteraldehyde-osmium-fixed, Epon embedded liver tissue. One-way analysis of variance (ANOVA) on morphometric data showed statistically significant differences in the mean volume density (MVD) of lipid bodies (F=136.48, P<0.0001)and mean surface density (MSD) of endoplasmic reticulum (ER) (F=12.45, P<0.003) between hepatocytes of control (n=8) and metformin-treated (MT) (n=8) animals. MVD for control group was 5.42% (±0.36 SEM) but decreased significantly in the MT group (1.13%, ±0.04 SEM). Similarly, MSD of ER for control was 24.7 µm2/µm3 (±1.64 SEM) but decreased for MT animals (18.90 µm2/µm3, ±0.28 SEM). These data are most likely consistent with the effects of metformin on lipid metabolism, and may not reflect on hepatotoxicity induced by the drug, in SHRs.

La metformina, una biguanida oral aprobada para el tratamiento de la diabetes tipo II, es también ampliamente prescrita para otros cuadros clínicos. Actualmente, la metformina está siendo investigada como posible agente anti-tumoral. Sin embargo, ha habido recientes preocupaciones acerca de la hepatotoxicidad asociada con el uso de metformina. En este estudio, por medio de la microscopía electrónica de transmisión (MET) de alta resolución y morfometría, se investigaron los posibles cambios ultraestructurales, inducidos por el tratamiento con metformina, en los hepatocitos de ratas espontáneamente hipertensas (REH). El análisis morfométrico se llevó a cabo en imágenes de células seleccionadas al azar a partir de tejido hepático seccionado, fijado con glutaraldehído-osmio e inmerso en Epon. El análisis de la varianza (ANOVA) de los datos morfométricos mostró diferencias significativas en la densidad de volumen medio (DVM) de cuerpos lipídicos (F=136,48, P<0,0001) y la densidad de superficie media (DSM) del retículo endoplasmático (RE) (F=12,45, P<0,003) entre los hepatocitos control (n=8) y los animales tratados con metformina (MT) (n=8). La DVM para el grupo control fue de 5,42% (±0,36 EEM), pero disminuyó significativamente en el grupo MT (1,13%, ±0,04 EEM). Del mismo modo, la DSM del RE para el grupo control fue de 24,7 µm2/µm3 (±1,64 EEM), pero disminuyó para los animales MT (18,90 µm2/µm3, ±0,28 EEM). Estos datos están probablemente más relacionados con los efectos de la metformina sobre el metabolismo de los lípidos, y no se relacionarían con la hepatotoxicidad por inducción de la droga, en REH.

Regional and segmental differences in the embryonic expression of a putative leech Hox gene, Lox2, by central neurons immunoreactive to FMRFamide-like neuropeptides.

We performed immunofluorescence experiments using a rat polyclonal antibody on formaldehyde-fixed whole-mount embryos to characterize the expression of a putative leech Hox gene, Lox2, during embryonic development. The main goal was to determine whether the differentiation of subsets of FMRFamide-like immunoreactive (FLI) neurons coincide with the expression domain of Lox2. The earliest expression of Lox2 was detected in relatively large, prominent nuclei in the posterior region at embryonic day 4, a very early stage. Lox2 expression was also detected in subsets of central neurons (neurons located in the CNS) located in midbody ganglia 6 (M6)-M21. In addition, Lox2 was expressed by a number of segment-specific and segmentally repeated central FLI neurons. Lox2-positive FLI neurons of interest included some of those previously identified: the rostral most ventral (RMV) neurons, the circular ventral (CV) neurons, and cell 261. The paired RMVs, which are located in all midbody ganglia, expressed Lox2 only in M7-M19. The CV neurons, specialized motor neurons that innervate the circular ventral muscles of the body wall, expressed Lox2 in M7-M19. The putative cell 261 expressed Lox2 in M7-M12, where Lox1 is also expressed. FMRFamide staining in putative segmental homologs of cell 261 was not detected in other segmental ganglia. Our results suggest a role for Lox2 in very early embryonic development (before the formation of the CNS), and in the differentiation of segmentally repeated and region-specific FLI neurons.

Circulating tumor cells in Hodgkin's lymphoma - a review of the spread of HL tumor cells or their putative precursors by lymphatic and hematogenous means, and their prognostic significance.

About 15% of patients diagnosed with classical Hodgkin's lymphoma (cHL) are considered high risk with unfavorable prognosis. The biology of the disease bears a direct relationship to its clinical course. However, some aspects of the disease are still being debated. Related topics include origin of neoplastic cells as circulating precursor versus germinal center B cell, and disease metastasis via hematogenous routes and the effect of HL circulation on relapse potential and further spread of the disease. The terminally differentiated giant neoplastic Hodgkin Reed-Sternberg (HRS) cells (HRSC) have limited proliferation and lack mobility. Therefore, they are unable to penetrate epithelium. Thus, the clinical aggressiveness of HRSCs that disseminate via both lymphatic and hematogenous may be determined by their molecular composition. This review discusses in detail the historical perspectives on scientific and clinical evidences of precursors of circulating HL cells and the prognostic importance of these circulating cells for predicting outcome.

Fibroblast growth factor-2 (FGF2) and syndecan-1 (SDC1) are potential biomarkers for putative circulating CD15+/CD30+ cells in poor outcome Hodgkin lymphoma patients.

BACKGROUND: High risk, unfavorable classical Hodgkin lymphoma (cHL) includes those patients with primary refractory or early relapse, and progressive disease. To improve the availability of biomarkers for this group of patients, we investigated both tumor biopsies and peripheral blood leukocytes (PBL) of untreated (chemo-naïve, CN) Nodular Sclerosis Classic Hodgkin Lymphoma (NS-cHL) patients for consistent biomarkers that can predict the outcome prior to frontline treatment. METHODS AND MATERIALS: Bioinformatics data mining was used to generate 151 candidate biomarkers, which were screened against a library of 10 HL cell lines. Expression of FGF2 and SDC1 by CD30+ cells from HL patient samples representing good and poor outcomes were analyzed by qRT-PCR, immunohistochemical (IHC), and immunofluorescence analyses. RESULTS: To identify predictive HL-specific biomarkers, potential marker genes selected using bioinformatics approaches were screened against HL cell lines and HL patient samples. Fibroblast Growth Factor-2 (FGF2) and Syndecan-1 (SDC1) were overexpressed in all HL cell lines, and the overexpression was HL-specific when compared to 116 non-Hodgkin lymphoma tissues. In the analysis of stratified NS-cHL patient samples, expression of FGF2 and SDC1 were 245 fold and 91 fold higher, respectively, in the poor outcome (PO) group than in the good outcome (GO) group. The PO group exhibited higher expression of the HL marker CD30, the macrophage marker CD68, and metastatic markers TGFß1 and MMP9 compared to the GO group. This expression signature was confirmed by qualitative immunohistochemical and immunofluorescent data. A Kaplan-Meier analysis indicated that samples in which the CD30+ cells carried an FGF2+/SDC1+ immunophenotype showed shortened survival. Analysis of chemo-naive HL blood samples suggested that in the PO group a subset of CD30+ HL cells had entered the circulation. These cells significantly overexpressed FGF2 and SDC1 compared to the GO group. The PO group showed significant down-regulation of markers for monocytes, T-cells, and B-cells. These expression signatures were eliminated in heavily pretreated patients. CONCLUSION: The results suggest that small subsets of circulating CD30+/CD15+ cells expressing FGF2 and SDC1 represent biomarkers that identify NS-cHL patients who will experience a poor outcome (primary refractory and early relapsing).