Segmental chromosomal aberrations as the poor prognostic factor in children over 18 months with stage 3 neuroblastoma without MYCN amplification.

Introduction: Patients with stage 3 neuroblastoma (NBL) according to International Neuroblastoma Staging System (INSS) without MYCN amplification represent a heterogenous group with respect to disease presentation and prognosis. Methods: Retrospective analysis of 40 stage 3 patients with NBL without MYCN amplification was performed. The prognostic value of age at diagnosis (under 18 vs over 18 months), International Neuroblastoma Pathology Classification (INPC) diagnostic category and presence of segmental or numerical chromosomes aberrations were evaluated, as well as biochemical markers. Array comparative genomic hybridization (aCGH) for analyzing copy number variations and Sanger sequencing for ALK point mutations were done. Results: In 12 patients (two patients under 18 months), segmental chromosomal aberrations (SCA) were found and numerical chromosomal aberrations (NCA) were found in 16 patients (14 patients under 18 months). In children over 18 months SCA were more common (p=0.0001). Unfavorable pathology was significantly correlated with SCA genomic profile (p=0.04) and age over 18 months (p=0.008). No therapy failures occurred in children with NCA profile over or under 18 months or in children under 18 months, irrespective of pathology and CGH results. Three treatment failures occurred in the SCA group, in one patient CGH profile was not available. For the whole group at 3, 5 and 10-year OS and DFS were 0.95 (95% CI 0.81-0.99), 0.91 (95% CI 0.77-0.97) and 0.91 (95% CI 0.77-0.97), and 0.95 (95% CI 0.90-0.99), 0.92 (95% CI 0.85-0.98) and 0.86 (95% CI 0.78-0.97), respectively. DFS was significantly lower in the SCA group than in the NCA group (3-years, 5-years, and 10-years DFS 0.92 (95% CI 0.53-0.95), 0.80 (95% CI 0.40-0.95) and 0.60 (95% CI 0.16-0.87) vs 1.0, 1.0 and 1.0, respectively, p=0.005). Conclusions: The risk of treatment failure was higher in patients with SCA profile, but only in patients over 18 months. All relapses occurred in children having obtained the complete remission, with no previous radiotherapy. In patients over 18 months, SCA profile should be taken into consideration for therapy stratification as it increases the risk of relapse and this group may require more intensive treatment.

CD44+ cells determine fenofibrate-induced microevolution of drug-resistance in prostate cancer cell populations.

Combinations of metabolic blockers (incl. fenofibrate) with chemotherapeutic drugs interfere with the drug-resistance of prostate cancer cells. However, their effect on cancer stem cells-dependent microevolution of prostate cancer malignancy remains unaddressed. Here, we hypothesize that the combined docetaxel/fenofibrate treatment prompts the selective expansion of cancer stem cells that affects the microevolution of their progenies. Accordingly, we adapted a combined in vitro/in vivo approach to identify biological and therapeutic consequences of this process. Minute subpopulations of docetaxel-resistant CD133high and/or CD44high cancer stem cell-like (SCL) cells were found in prostate cancer DU145 and PC3 cell populations. When pretreated with docetaxel, they readily differentiated into docetaxel-resistant CD44negative "bulk" cells, thus accounting for the microevolution of drug-resistant cell lineages. Combined docetaxel/fenofibrate treatment induced the generation of poly(morpho)nuclear giant cells and drug-resistant CD44high SCL cells. However, the CD44negative offspring of docetaxel- and docetaxel/fenofibrate-treated SCLs remained relatively sensitive to the combined treatment, while retaining enhanced resistance to docetaxel. Long-term propagation of drug-resistant SCL-derived lineages in the absence of docetaxel/fenofibrate resulted in their reverse microevolution toward the drug-sensitivity and invasive phenotype. Consequently, prostate tumors were able to recover from the combined docetaxel/fenofibrate stress after the initial arrest of their expansion in vivo. In conclusion, we have confirmed the potential of fenofibrate for the metronomic treatment of drug-resistant prostate tumors. However, docetaxel/fenofibrate-induced selective expansion of hyper-resistant CD44high SCL prostate cells and their "bulk" progenies prompts the microevolution of prostate tumor drug-resistance. This process can limit the implementation of metabolic chemotherapy in prostate cancer treatment.

Disseminated Juvenile Xanthogranuloma and Hemophagocytic Lymphohistiocytosis Developed During Treatment of Acute Lymphoblastic Leukemia: Case Report.

The association between acute lymphoblastic leukemia (ALL), non-Langerhans cell histiocytosis (non-LCH), and hemophagocytic lymphohistiocytosis (HLH), to the best of our knowledge, has not been published to date. Juvenile xanthogranuloma (JXG), as a type of non-LCH, is usually a benign disease limited to the skin. Systemic involvement is rarely reported. The present case report describes a 15-year-old boy diagnosed with disseminated JXG involving skin and bone marrow concurrent with severe symptoms of HLH during ALL therapy. Examination of immunoglobulin heavy chain genes in B-cell precursor leukemic blasts and histiocytes in the skin and bone marrow revealed identical rearrangements, confirming clonal relationship between both diseases. Implementation of corticosteroids, vinblastine, etoposide, cyclosporine, and tocilizumab resulted in partial skin lesion resolution with no improvement of bone marrow function; therefore, hematopoietic stem cell transplantation (HSCT) was eventually performed. The patient's hematological and general status has improved gradually; however, remarkable recovery of skin lesions was observed after empirical antitubercular therapy. Mycobacterium spp. infection should be considered as a possible secondary HLH trigger. Triple association of ALL, non-LCH, and HLH highlights heterogeneity of histiocytic disorders and possible common origin of dendritic and lymphoid cells.

Fenofibrate Augments the Sensitivity of Drug-Resistant Prostate Cancer Cells to Docetaxel.

Metronomic agents reduce the effective doses and adverse effects of cytostatics in cancer chemotherapy. Therefore, they can enhance the treatment efficiency of drug-resistant cancers. Cytostatic and anti-angiogenic effects of fenofibrate (FF) suggest that it can be used for the metronomic chemotherapy of drug-resistant prostate tumors. To estimate the effect of FF on the drug-resistance of prostate cancer cells, we compared the reactions of naïve and drug-resistant cells to the combined treatment with docetaxel (DCX)/mitoxantrone (MTX) and FF. FF sensitized drug-resistant DU145 and PC3 cells to DCX and MTX, as illustrated by their reduced viability and invasive potential observed in the presence of DCX/MTX and FF. The synergy of the cytostatic activities of both agents was accompanied by the inactivation of P-gp-dependent efflux, dysfunction of the microtubular system, and induction of polyploidy in DCX-resistant cells. Chemical inhibition of PPARα- and reactive oxygen species (ROS)-dependent pathways by GW6471 and N-acetyl-L-cysteine, respectively, had no effect on cell sensitivity to combined DCX/FF treatment. Instead, we observed the signs of adenosine triphosphate (ATP) deficit and autophagy in DCX/FF-treated drug-resistant cells. Furthermore, the cells that had been permanently propagated under DCX- and DCX/FF-induced stress did not acquire DCX/FF-resistance. Instead, relatively slow proliferation of DCX-resistant cells was efficiently inhibited by FF. Collectively, our observations show that FF reduces the effective doses of DCX by interfering with the drug resistance and energy metabolism of prostate cancer cells. Concomitantly, it impairs the chemotherapy-induced microevolution and expansion of DCX/FF-resistant cells. Therefore, FF can be applied as a metronomic agent to enhance the efficiency of palliative chemotherapy of prostate cancer.

CD44+ cytokeratin-positive tumor cells in blood and bone marrow are associated with poor prognosis of patients with gastric cancer.

BACKGROUND: The phenotypic heterogeneity of circulating tumor cells (CTC) in peripheral blood and disseminated tumor cells (DTC) in bone marrow is an important constraint for clinical decision making. Here, we investigated the implications of two different subpopulations of these cells in gastric cancer (GC). METHODS: GC patients (n = 228) who underwent elective gastric resections were prospectively examined for CTC/DTC. The cells obtained from peripheral blood and bone marrow aspirates were sorted by flow cytometry and CD45- cells expressing cytokeratins (8, 18, and 19) and CD44 were identified by immunofluorescent double staining. RESULTS: Ninety-three (41%) patients had cytokeratin-positive tumor cells in either blood or bone marrow, while cells expressing CD44 were found in 22 (10%) cases. CK+CD44+ cells were significantly more common among patients with distant metastases (50 vs 19%, P = 0.001), while no such correlations were demonstrated for CK+CD44- cells. Detection of CK+CD44+ cells, but not CK+CD44-, was associated with significantly shortened survival. Moreover, the Cox proportional hazards model identified CK+CD44+ cells as a negative prognostic factor with an odds ratio of 2.38 (95% CI 1.28-4.41, P = 0.006). CONCLUSION: CD44+ phenotype of cytokeratin-positive cells in blood and bone marrow is an independent prognostic factor in patients with gastric cancer.

Characterization of human gastric adenocarcinoma cell lines established from peritoneal ascites.

The three cell lines, designated as gastric cancer (GC)1401, GC1415 and GC1436 were derived from peritoneal effusions from patients with gastric adenocarcinoma. Cell lines were established in tissue culture and in immunodeficient, non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. These cell lines were grown as an adherent monolayer with doubling time ranging between 25 h (GC1436 cell line) and 30-34 h (GC1401 and GC1415, respectively). All cells showed morphological features of epithelial-like cells, forming sheets of polygonal cells. Chromosomal analysis showed that the modal numbers ranged from 52 (GC1401), 51-56 (GC1415) and 106 (GC1436). High heterogeneity, resulting from several structural and numerical chromosomal abnormalities were evident in all cell lines. The surface marker expression suggested a tumor origin of the cells, and indicated the intestinal phenotype of a GC (CD10+, MUC1). All three cell lines were tumorigenic but not metastatic, in vivo, in NOD/SCID mice. The lack of metastatic potential was suggested by the lack of aldehyde dehydrogenase 1A1 activity. In conclusion, these newly established GC cell lines widen the feasibility of the functional studies on biology of GC as well as drug testing for potential therapeutic purposes.

Heme oxygenase-1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells.

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1-/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2-/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018.

Novel insights in ultrasound evaluation of thyroid gland in children with papillary thyroid carcinoma.

BACKGROUND: The coincidence of autoimmune thyroiditis (AIT) in patients with papillary thyroid carcinoma (PTC) is ranging between 10 and 58% in the general population. MATERIAL AND METHODS: In the present study retrospective ultrasound, clinical and autoimmune assessment of 24 patients diagnosed with papillary thyroid carcinoma between 2000-2016 was performed. RESULTS: The coexistence of PTC and AIT was found in 50% of patients with PTC. Patients were divided into two groups. PTC AIT (+) group involved 12 children at the mean age 14.9 years (range 11-20 years, 9 girls) and PTC AIT (-) 12 children at the mean age 12.9 years (range 7-18 years, 5 girls). Papillary thyroid microcarcinoma (PTMC) was diagnosed in 6 patients (in 5 with AIT). US characteristics of PTC was heterogenous: hypoechogenic with/without increased vascularisation, normoechogenic with halo, with/without microcalcifications. In 70% PTC AIT (+) and in all PTC AIT (-) patients ultrasound analysis revealed that the thyroid tissue of the whole gland was normoechogenic. Local metastases in lymph nodes were found in 40% of PTMC AIT (+). CONCLUSION: Lack of increased vascularization and microcalcifications and presence of``halo`in the nodule does not exclude malignancy. Due to the presence of lymph node involvement in PTMC in all children with PTC total thyroidectomy should be performed with lymph nodes verification.

Vertically transmitted HPV-dependent squamous cell carcinoma of the external auditory canal : Case report of a child.

BACKGROUND: There is much evidence that high-risk human papillomavirus (HPV) plays a causative role in a subset of head and neck squamous cell cancer (HNSCC) in adults. HPV-positive tumors behave differently even in their response to treatment and are therefore a distinct subset. Both HPV-positive and HPV-negative tumors of the head and neck region are usually in the domain of adults and cases in children are rare; thus when a 2­year-old child was diagnosed with this cancer in the external auditory canal, an in-depth assessment of the tumor was considered necessary. CASE REPORT: A 2­year-old girl was born to a HPV-positive mother who was diagnosed with cervical cancer during pregnancy. The child was delivered by caesarean section and the mother died of her cancer 7 months after delivery. After the diagnosis of locally invasive HPV-positive squamous cell cancer of the external auditory canal, the child was treated surgically, and with chemotherapy and radiotherapy. Full remission was obtained lasting up to 325 weeks since treatment was started, resulting in over 6 years of disease-free survival. CONCLUSION: This is the first case of advanced, HPV-related HNSCC in a 2­year-old child, in whom the tumor was located in the external auditory canal and who made a dramatic recovery after treatment with nonradical surgery, chemotherapy and radiotherapy. The child has currently been disease free for 6 years. This case supports the observation that HPV-related HNSCC tumors appear to respond favorably to treatment despite the patient's young age and the clinically advanced stage of the tumor.

Interactions of tumour-derived micro(nano)vesicles with human gastric cancer cells.

BACKGROUND: Tumour cells release membrane micro(nano)fragments called tumour-derived microvesicles (TMV) that are believed to play an important role in cancer progression. TMV suppress/modify antitumour response of the host, but there is also some evidence for their direct interaction with cancer cells. In cancer patients TMV are present in body fluid and tumour microenvironment. The present study aimed at characterization of whole types/subpopulations, but not only exosomes, of TMV from newly established gastric cancer cell line (called GC1415) and to define their interactions with autologous cells. METHODS: TMV were isolated from cell cultures supernatants by centrifugation at 50,000×g and their phenotype was determined by flow cytometry. The size of TMV was analysed by dynamic light scattering and nanoparticle tracking analysis, while morphology by transmission electron microscopy and atomic force microscopy. Interactions of TMV with cancer cells were visualized using fluorescence-activated cell sorter, confocal and atomic force microscopy, biological effects by xenografts in NOD SCID mice. RESULTS: Isolated TMV showed expression of CD44H, CD44v6 (hyaluronian receptors), CCR6 (chemokine receptor) and HER-2/neu molecules, exhibited different shapes and sizes (range 60-900 nm, highest frequency of particles with size range of 80-120 nm). TMV attached to autologous cancer cells within 2 h and then were internalized by them at 24 h. CD44H, CD44v6 and CCR6 molecules may play a role in attachment of TMV to cancer cells, while HER-2 associated with CD24 be involved in promoting cancer cells growth. Pre-exposure of cancer cells to TMV resulted in enhancement of tumour growth and cancer cell-induced angiogenesis in NOD SCID mice model. CONCLUSIONS: TMV interact directly with cancer cells serving as macro-messengers and molecular cargo transfer between gastric cancer cells resulting in enhancement of tumour growth. TMV should be considered in future as target of anticancer therapy.

Atypical cardiac myxoma in the course of Carney complex in the child. Case Report.

Cardiac tumors in infants and children are rare. Myxomas are the second (after rhabdomyomas) most common primary cardiac tumors in pediatric patients. Cardiac, cutaneous and mucous myxomas are likewise the second most frequent manifestation of the Carney complex, an autosomal dominant multi neoplasia syndrome, which consists of myxomas in different locations, spotty skin pigmentation and endocrine overactivity. We present a case of 13-years-old boy send to our department from a district hospital because of the large tumor in the right atrium discovered in the echo study. On admission he presented discrete signs of Cushing's syndrome and scarse pigmented nevi on the face and trunc. The detailed echo examination showed the large right atrial tumor with features of myxoma, protruding across the tricuspid valve into the right ventricle during diastolic period. Atypical location of cardiac myxoma as well as the signs of Cushing's syndrome suggested Carney's complex. Detailed endocrine studies confirmed the hypothesis. Thus two-step bilateral adrenalectomy was planned. The histopathologic study confirmed primary pigmented nodular adrenocortical disease.

MET receptor is a potential therapeutic target in high grade cervical cancer.

Cervical cancer is one of the leading causes of death among women suffering from tumors. Current treatment options are insufficient. Here, we investigated the MET receptor as a potential molecular target in advanced cervical cancer. Downregulation of MET receptor expression via RNA interference in different cervical carcinoma cell lines dramatically decreased tumor growth and forced tumor differentiation in vivo. MET receptor silencing also led to a dramatic decrease in cell size and a decrease in proliferation rate under normal and stress conditions. MET receptor downregulation also resulted in decreased cyclin D1 and c-myc levels but did not increase apoptosis. Subsequent experiments showed that downregulation of the MET receptor decreased the expression of a key regulator of the epithelial-to-mesenchymal transition, SLUG. and increased the expression of E-cadherin, a hallmark of the epithelial phenotype. Moreover, MET downregulation impairs expression and signaling of CXCR4 receptor, responsible for invasive phenotype. Taken together, our results strongly suggest that the MET receptor influences the oncogenic properties of cervical carcinoma cells in vitro and in vivo. These findings highlight a unique role of the MET receptor in cervical carcinoma cells and indicate the MET receptor as a potential therapeutic target for advanced cervical carcinoma.

Novel mutation-deletion in the PHOX2B gene of the patient diagnosed with Neuroblastoma, Hirschsprung's Disease, and Congenital Central Hypoventilation Syndrome (NB-HSCR-CCHS) Cluster.

INTRODUCTION: Neuroblastoma (NB), Hirschsprung disease (HSCR), Congenital Central Hypoventilation Syndrome (CCHS), clinically referred as the NB-HSCR-CCHS cluster, are genetic disorders linked to mutations in the PHOX2B gene on chromosome 4p12. SPECIFIC AIM: The specific aim of this project is to define the PHOX2B gene mutations as the genomic basis for the clinical manifestations of the NB-HSCR-CCHS cluster. PATIENT: A one day old male patient presented to the Jagiellonian University Medical College (JUMC), American Children Hospital, neonatal Intensive Care Unit (ICU) due to abdominal distention, vomiting, and severe apneic episodes. With the preliminary diagnosis of the NB-HSCR-CCHS, the blood and tissue samples were acquired from the child, as well as from the child's parents. All procedures were pursued in accordance with the Declaration of Helsinki, with the patient's Guardian Informed Consent and the approval from the Institutional Review Board. GENETIC/GENOMIC METHODS: Karyotyping was analyzed based upon Giemsa banding. The patient's genomic DNA was extracted from peripheral blood and amplified by polymerase chain reaction. Direct microfluidic Sanger sequencing was performed on the genomic DNA amplicons. These procedures were pursued in addition to the routine clinical examinations and tests. RESULTS: G-banding showed the normal 46 XY karyotype. However, genomic sequencing revealed a novel, heterozygous deletion (8 nucleotides: c.699-706, del8) in exon 3 of the PHOX2B gene on chromosome 4. This led to the frame-shift mutation and malfunctioning gene expression product. CONCLUSION: Herein, we report a novel PHOX2B gene mutation in the patient diagnosed with the NB-HSCR-CCHS cluster. The resulting gene expression product may be a contributor to the clinical manifestations of these genetic disorders. It adds to the library of the mutations linked to this syndrome. Consequently, we suggest that screening for the PHOX2B mutations becomes an integral part of genetic counseling, genomic sequencing of fetal circulating nucleic acids and / or genomes of circulating fetal cells prenatally, while preparing supportive therapy upon delivery, as well as on neonates' genomes of intubated infants, when breathing difficulties occur upon extubation. Further, we hypothesize that PHOX2B may be considered as a potential target for gene therapy.

Fetus in fetu: a medical curiosity--considerations based upon an intracranially located case.

INTRODUCTION: "Fetus in fetu" (FIF) is defined as the abnormal monozygotic twin inside the body of its "host twin." Intracranial FIFs are extremely rare. CASE PRESENTATION: A male premature newborn was admitted to the hospital due to a large intracranial tumor diagnosed in the 31st week of gestation. The child died before surgical treatment because of failure of the respiratory system due to fetal respiratory distress syndrome. During general autopsy, a large intracranial tumor with four relatively well-developed limbs was found. Microscopically, apart from relatively well-formed musculoskeletal structures of limbs that were covered with skin, there were haphazardly distributed different tissues or fragments of organs. However, various neuroectodermal derivatives were dominant. CONCLUSION: We believe that intracranial FIFs, theoretically with poor prognosis, can be successfully curable in cases revealed prenatally, provided that optimal treatment is introduced and the achievement of proper pulmonary maturity of the host is accomplished prior to the operation of the tumor.

Multifunctional protein APPL2 contributes to survival of human glioma cells.

Some endocytic proteins have recently been shown to play a role in tumorigenesis. In this study, we demonstrate that APPL2, an adapter protein with known endocytic functions, is upregulated in 40% cases of glioblastoma multiforme, the most common and aggressive cancer of the central nervous system. The silencing of APPL2 expression by small interfering RNAs (siRNAs) in glioma cells markedly reduces cell survival under conditions of low growth factor availability and enhances apoptosis (measured by executor caspase activity). Long-term depletion of APPL2 by short hairpin RNAs (shRNAs), under regular growth factor availability, suppresses the cell transformation abilities, assessed by inhibited colony formation in soft agar and by reduced xenograft tumor growth in vivo. At the molecular level, the negative effect of APPL2 knockdown on cell survival is not due to the alterations in AKT or GSK3ß activities which were reported to be modulated by APPL proteins. Instead, we attribute the reduced cell survival upon APPL2 depletion to the changes in gene expression, in particular to the upregulation of apoptosis-related genes, such as UNC5B (a proapoptotic dependence receptor) and HRK (harakiri, an activator of apoptosis, which antagonizes anti-apoptotic function of Bcl2). In support of this notion, the loss of glioma cell survival upon APPL2 knockdown can be rescued either by an excess of netrin-1, the prosurvival ligand of UNC5B or by simultaneous silencing of HRK. Consistently, APPL2 overexpression reduces expression of HRK and caspase activation in cells treated with apoptosis inducers, resulting in the enhancement of cell viability. This prosurvival activity of APPL2 is independent of its endosomal localization. Cumulatively, our data indicate that a high level of APPL2 protein might enhance glioblastoma growth by maintaining low expression level of genes responsible for cell death induction.

Causes and consequences of abandoning one-stage bilateral adrenalectomy recommended in primary pigmented nodular adrenocortical disease--case presentation.

We present a 7-year-old girl with a 2-year history of decelerated growth rate and cushingoidal obesity, upon admission presenting with fixed hypertension. Cyclic hypercortisolemia with inhibited baseline and post-CRH stimulation ACTH level pointed to primary adrenal hypercortisolemia. Ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) showed normal adrenal glands. 131J-labeled cholesterol scintiscan showed a weak but slightly more expressed tracer uptake in the left adrenal gland. Cushing syndrome concomitant with isolated primary pigmented nodular adrenocortical disease (PPNAD) was diagnosed. After hypotensive pretreatment, a left adrenalectomy was performed, resulting in normalization of corticoadrenal function, blood pressure, Cushing features and growth rate. Histopathology confirmed PPNAD. In the course of infection, corticoadrenal function showed absence of adrenal reserve, and adrenal crisis. Hydrocortisone (HC) therapy, followed by HC supplementation was introduced. Four years later, a contralateral adrenalectomy was performed and total HC supplementation was introduced. Causes and consequences of abandoning one-stage bilateral adrenalectomy recommended in PPNAD are reviewed.

Inhibition of rhabdomyosarcoma's metastatic behavior through downregulation of MET receptor signaling.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma usually diagnosed in children. In advanced and metastatic stages the prognosis is often poor. RMS cell lines were used for evaluation of the role of MET receptor inhibition on chemotaxis and invasion. In vivo studies were performed using NOD-SCID xenograft model. This study shows that blocking of MET expression has strong influence on metastatic behavior of RMS. MET negative cells possess a reduced potential to migrate and to invade. Downregulation of MET suppressed the ability of RMS cells to populate bone marrow. Inhibition of MET negative tumor cells engraftment into bone marrow was observed. MET negative tumors were also two to four times smaller than their wild type counterparts. Since MET receptor plays a very important role in facilitating metastasis of RMS cells, blocking of HGF-MET axis might be considered as a therapeutic option for RMS patients, at more advanced and metastatic stages.

Detection of isolated tumour cells in the blood and bone marrow of patients with gastric cancer by combined sorting, isolation and determination of MAGE-1, -2 mRNA expression.

The detection of isolated (circulating or disseminated) tumour cells (ITC) in patients with cancer requires very sensitive methods, as such cells are very rare. In the present study, the method that combines the negative isolation of CD45- leukocytes from the blood and bone marrow of patients with gastric cancer by flow cytometry, followed by the positive isolation of single cytokeratin-positive (CK+) cells by a Laser Capture Microdissection System for the determination of MAGE-1, -2 mRNA expression was used to detect ITC. This study shows that this method is highly sensitive as it allows to determine beta-actin-mRNA expression in a single CK+ cell. Using > or =5 CK+ cells as a cut-off level, the MAGE-1 mRNA expression was detected in 100% of CK+ cells in the peripheral blood and in 75% of bone marrow samples of patients with gastric cancer. The MAGE-2 mRNA expression was observed in 40 and 58% of samples, respectively. Furthermore, an analysis of primary tumours and locoreginal lymph nodes with respect to the mRNA expression of the two genes showed that MAGE-1 mRNA expression was detected in 88% of the primary tumours and in 67% of the lymph node samples, whereas the MAGE-2 mRNA expression was observed in 72 and 67% of the cases, respectively. Thus, the method described here allows the precise and sensitive determination of tumour-associated gene expression in single ITC present in the blood and bone marrow of patients with gastric cancer.

Immunophenotype of isolated tumour cells in the blood, bone marrow and lymph nodes of patients with gastric cancer.

Immunophenotype of isolated (disseminated or circulating) tumour cells (ITC) in the blood, bone marrow and lymph nodes were studied in patients with gastric cancer. Coexpression of metalloproteinases inducer (EMMPRIN), chemokine receptors (CCR6, CXCR4) and adhesion molecules (Ep-CAM, CD44) was determined on cytokeratin positive (CK+) cells in CD45- cell population sorted out from the blood and/or bone marrow. Eight cytospin samples of blood and 69 samples of bone marrow containing CK+ cells from patients with gastric cancer were included into study. Expression of EMMPRIN and CCR6 were noted in a half of CK+ samples (of blood/bone marrow) whereas the expression of CXCR4 and Ep-CAM was much lower. Analysis of paired data of these determinants expression on CK+ cells showed no association between them. Expression of EMMPRIN, Ep-CAM, CCR6, CCR7, CXCR1, and CXCR4 on ITC in lymph nodes was determined by flow cytometry. In 18 lymph nodes (out of 36 assayed) CK+ cells were found. The expression of CCR6 and Ep-CAM on CK+ cells was observed in almost all studied lymph nodes, CXCR1--in half of them. The expression of EMMPRIN and CCR7 cells was lower. These results suggest that ITC of gastric cancer express variably several molecules that may be involved in metastasis formation.