Supportive Oligonucleotide Therapy (SOT) as a Potential Treatment for Viral Infections and Lyme Disease: Preliminary Results.

Antisense therapy is widely used as an alternative therapeutic option for various diseases. RNA interference might be effective in infections, through the degradation of messenger RNA and, therefore, translation process. Hence, proteins essential for microorganisms and viruses' proliferation and metabolism are inhibited, leading to their elimination. The present study aimed to evaluate the use of oligonucleotide in patients infected by Epstein-Barr (EBV) or Herpes Simplex Viruses 1/2 or with Lyme Disease caused by Borrelia burgdorferi. Blood samples were collected from 115 patients and the different species were characterized using molecular biology techniques. Then, SOT molecules (Supportive Oligonucleotide Therapy), which are specific small interfering RNA (siRNA), were designed, produced, and evaluated, for each specific strain. Oligonucleotides were administered intravenously to patients and then a quantitative Polymerase Chain Reaction was used to evaluate the effectiveness of SOT. This study revealed that for Lyme Disease, one or two SOT administrations can lead to a statistically significant decrease in DNA copies, while for viruses, two or three administrations are required to achieve a statistically significant reduction in the genetic material. These preliminary results indicate that antisense SOT therapy can be considered a potential treatment for viral as well as Lyme diseases.

A novel platform for the production of autologous human antibodies.

At Research Genetic Cancer Centre, we have developed a novel method for the production of human monoclonal antibodies against a specific antigen of our choice (c-met) using isolated human blood cells. By mimicking nature, dendritic, CD4 and CD19 cells from healthy volunteers were driven towards Th2 immunity. Cell activation was succeeded by a cytokine cocktail, and IgG production was promoted by IgG class switching factors. IgG secretion was determined using both enzyme linked immunosorbent assay (ELISA) and Western blot as well as immunoglobulin heavy chain gamma polypeptide gene expression. Secreted antibody was further purified by affinity column chromatography against c-met peptide. Anti-c-met activity was determined using the purified antibody as primary antibody for c-met detection by ELISA, Western blot and flow cytometry. Finally, anti-c-met antibody efficiency was determined by MCF-7 viability assay. Plasma cell formation and IgG secretion took place after 6 days of culture. Plasma cells produced anti-c-met IgG antibody that significantly decreased MCF-7 breast cancer cell proliferation. To our knowledge, this is the first platform of its kind, generating fully human antibodies-on-demand using patient's own cells, bringing personalized, targeted therapy for cancer one step closer.

Circulating tumour cell gene expression and chemosensitivity analyses: predictive accuracy for response to multidisciplinary treatment of patients with unresectable refractory recurrent rectal cancer or unresectable refractory colorectal cancer liver metastases.

BACKGROUND: Patients with unresectable recurrent rectal cancer (RRC) or colorectal cancer (CRC) with liver metastases, refractory to at least two lines of traditional systemic therapy, may receive third line intraarterial chemotherapy (IC) and targeted therapy (TT) using drugs selected by chemosensitivity and tumor gene expression analyses of liquid biopsy-derived circulating tumor cells (CTCs). METHODS: In this retrospective study, 36 patients with refractory unresectable RRC or refractory unresectable CRC liver metastases were submitted for IC and TT with agents selected by precision oncotherapy chemosensitivity assays performed on liquid biopsy-derived CTCs, transiently cultured in vitro, and by tumor gene expression in the same CTC population, as a ratio to tumor gene expression in peripheral mononuclear blood cells (PMBCs) from the same individual. The endpoint was to evaluate the predictive accuracy of a specific liquid biopsy precision oncotherapy CTC purification and in vitro culture methodology for a positive RECIST 1.1 response to the therapy selected. RESULTS: Our analyses resulted in evaluations of 94.12% (95% CI 0.71-0.99) for sensitivity, 5.26% (95% CI 0.01-0.26) for specificity, a predictive value of 47.06% (95% CI 0.29-0.65) for a positive response, a predictive value of 50% (95% CI 0.01-0.98) for a negative response, with an overall calculated predictive accuracy of 47.22% (95% CI 0.30-0.64). CONCLUSIONS: This is the first reported estimation of predictive accuracy derived from combining chemosensitivity and tumor gene expression analyses on liquid biopsy-derived CTCs, transiently cultured in vitro which, despite limitations, represents a baseline and benchmark which we envisage will be improve upon by methodological and technological advances and future clinical trials.

Supportive Oligonucleotide Therapy (SOT) as an Alternative Treatment Option in Cancer: A Preliminary Study.

BACKGROUND/AIM: An early evaluation concerning the effectiveness of supportive oligonucleotide therapy (SOT) in cancer as a monotherapy and in combination with other types of treatment. PATIENTS AND METHODS: This study evaluated the clinical condition and performance status (Karnofsky-Index) of 95 patients, post-SOT administration. Furthermore, circulating tumor cells (CTCs) from 47 patients' pre- and post-SOT administration were measured and analyzed by repeated-measures ANOVA. RESULTS: Improvement of the clinical condition was observed in all patients who used SOT (77.89%), SOT in combination with other therapy (69.77%) and SOT as a monotherapy or no information was given concerning another therapy (84.31%). Positive results for Karnofsky-Index were also observed in 71.58%, 61.36%, and 80.39%, respectively. Finally, statistically significant reductions in CTCs were observed for both SOT as a monotherapy and SOT as an adjunctive therapy. CONCLUSION: The preliminary results indicate that SOT therapy can be used both as monotherapy as well as in combination with other therapies for cancer.

Targeting SARS-CoV-2 Polymerase with New Nucleoside Analogues.

Despite the fact that COVID-19 vaccines are already available on the market, there have not been any effective FDA-approved drugs to treat this disease. There are several already known drugs that through drug repositioning have shown an inhibitory activity against SARS-CoV-2 RNA-dependent RNA polymerase. These drugs are included in the family of nucleoside analogues. In our efforts, we synthesized a group of new nucleoside analogues, which are modified at the sugar moiety that is replaced by a quinazoline entity. Different nucleobase derivatives are used in order to increase the inhibition. Five new nucleoside analogues were evaluated with in vitro assays for targeting polymerase of SARS-CoV-2.

Cancer Comprehensive Analysis in Gastric Carcinoma: Benefits and New Perspectives.

Gastric cancer is one of the most common and deadly cancers worldwide. Screening tests as well as tools for prediction of treatment outcomes and prognosis have been developed, but they have many limitations. The integration of liquid biopsy provided new aspects in screening and diagnosis of gastric cancer. In the present study, we used different techniques, studying the genetic and epigenetic profile of circulating tumor cells. We aimed to acquire all the available information, compare it with already existing studies, and evaluate the benefit of this approach. A blood sample was isolated from 2 gastric cancer patients at stages III-IV, followed by the isolation of CTCs. The circulating tumor cells were used for array comparative genomic hybridization, next-generation sequencing, and whole gene expression microarrays. Different variants were detected, while the microsatellite instability status was stable in both cases. The tumor mutational burden was low to medium. Gene expression assays revealed that >100 genes were overexpressed compared to noncancer samples. Amplifications of X chromosome were also observed in both cases, by using array comparative genomic hybridization. Although there are several techniques for cancer screening, prediction of therapy outcomes, and prognosis, the application of a complete comprehensive cancer panel, combining the study of variants, fusions, chromosomal abnormalities, and gene expression, is more appropriate. Information provided by the above techniques might contribute in designing more efficient treatment protocols and screening tools. Despite the limitation of samples, the data are encouraging, and further study is needed so that they can be used at clinical level.

Increased breast cancer cell sensitivity to cisplatin using a novel small molecule inhibitor.

BACKGROUND: Although cisplatin is used for the treatment of more than half of cancer patients, its use is restricted by serious side effects as well as the development of cisplatin-resistant cancer cells, limiting its use. In RGCC we have synthesized an intermediate molecule in an ERK inhibitor synthesis process. AIMS AND OBJECTIVES: The aim of the study was to evaluate the effects of combined cisplatin plus RGCC molecule treatment on MCF-7 and MDAMB231 breast cancer cell viability, proliferation, ability to form clones and migrate, as well as the effects on cell cycle and gene expression. MATERIALS AND METHODS: Cell viability and proliferation were measured by Crystal violet exclusion dye and MTT respectively. Clone formation and wound healing assays were also used for clone formation and cell migration evaluation. Cell cycle was studied by flow cytometry, expression of genes was evaluated by PCR and protein expression was evaluated by western blot. RESULTS: It was found that combination therapy decreased cell viability and proliferation, caused growth arrest, decreased cancer cell invasiveness and the ability to form clones as well as perturbed the expression of genes involved in ERK, cell cycle and cell death pathways. Conclusion: Although the exact mechanism of action of the combination therapy remains to be investigated, it was found that it is more effective than cisplatin monotherapy. Our findings could potentially lead to a new therapeutic regime for the treatment of cancer.

Study and detection of potential markers for predicting metastasis into lymph nodes in prostate cancer.

Hormone-refractory prostate carcinoma has a different cell surface protein profile than hormone-sensitive prostate carcinoma, which provides migration ability and interactions with organs/tissues. Detection and association of these proteins with lymph node metastasis via lymphadenectomy might be beneficial for patients. Gene expression analysis in hormone-refractory and hormone-sensitive commercial cancer cell lines was performed and, after co-cultivation with osteoblasts or endothelial cells, knockdown experiments followed to validate potential biomarkers. "Myeloid-associated differentiation markers, myosin 1b and phosphatidylinositol-4-phosphate-5-kinase type 1 alpha are implicated in metastasis", their knockdown altered the expression of key regulators of endothelial-mesenchymal transition, invasion, motility and migration. In primary prostate tumors, these genes could be an indicator for future metastasis into lymph nodes.

A Pilot Study of the Predictive Potential of Chemosensitivity and Gene Expression Assays Using Circulating Tumour Cells from Patients with Recurrent Ovarian Cancer.

Circulating tumour cells (CTCs) from liquid biopsies are under current investigation in several cancers, including epithelial ovarian cancer (EOC) but face significant drawbacks in terms of non-standardised methodology, low viable cell numbers and accuracy of CTC identification. In this pilot study, we report that chemosensitivity assays using liquid biopsy-derived metastatic EOC CTCs, from 10 patients, nine with stage IIIC and one with stage IV disease, in progression after systemic chemotherapy, submitted for hypoxic isolated abdominal perfusion (HAP), are both feasible and useful in predicting response to therapy. Viable metastatic EOC CTCs (>5 cells/mL for all 10 blood samples), enriched by transient culture and identified by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence (IF), were subjected to flow cytometry-based Annexin V-PE assays for chemosensitivity to several chemotherapeutic agents and by RT-PCR for tumour gene expression profiling. Using a cut-off value of >80% cell death, CTC chemosensitivity tests were predictive of patient RECIST 1.1 responses to HAP therapy associated with 100% sensitivity, 50% specificity, 33% positive predictive, 100% negative predictive and 60% accuracy values. We propose that the methodology employed in this study is feasible and has the potential to predict response to therapy, setting the stage for a larger study.

Circulating tumour cell liquid biopsy in selecting therapy for recurrent cutaneous melanoma with locoregional pelvic metastases: a pilot study.

OBJECTIVES: Circulating tumour cells (CTCs) from liquid biopsies provide an exceptional opportunity to obtain real-time tumour information and are under current investigation in several cancers, including cutaneous melanoma, but face significant drawbacks in terms of non-standardised methodology, low viable cell numbers and accuracy of CTC identification. In this pilot study, we report that chemosensitivity assays using liquid biopsy-derived metastatic melanoma (MM) CTCs, from 7 patients with stage IIIC, BRAF wild-type metastatic melanomas, localized exclusively to the pelvic region, un-eligible for immunotherapy and treated with melphalan hypoxic pelvic perfusion (HPP), is both feasible and useful in predicting response to therapy. Viable MM CTCs (> 5 cells/ml for all 7 blood samples), enriched by transient culture, were characterised in flow cytometry-based Annexin V-PE assays for chemosensitivity to several drugs. RESULTS: Using melphalan as a standard, chemosensitivity cut-off values of > 60% cell death, were predictive of patient RECIST 1.1 response to melphalan HPP therapy, associated with calculated 100% sensitivity, 66.67% specificity, 33.33% positive predictive, 100% negative predictive, and 71.43% accuracy values. We propose that the methodology in this study is both feasible and has potential value in predicting response to therapy, setting the stage for a larger study. Trial registration Clinical Trials.gov Identifier NCT01920516; date of trial registration: August 6, 2013.

Real-life multidisciplinary treatment for unresectable colorectal cancer liver metastases including hepatic artery infusion with chemo-filtration and liquid biopsy precision oncotherapy: observational cohort study.

BACKGROUND: Hepatic artery infusion (HAI) and drug selection by liquid biopsy precision oncotherapy are under investigation for the multidisciplinary treatment of unresectable colorectal liver metastases (CRCLM) in progression after systemic therapy. Here, we compare the safety and efficacy of third-line HAI followed by target therapy with drug regimes selected by liquid biopsy precision oncotherapy to third-line systemic therapy with drug regimes selected partly by tissue biopsy precision oncotherapy, in a retrospective real-life study of 106 unresectable CRCLM patients. METHODS: Drug regimens for HAI/target therapy were selected by assessing the sensitivity of purified circulating tumor cell (CTCs) to 5-fluorouracil, carboplatin, cisplatin, oxaliplatin, irinotecan, doxorubicin, mitomycin, raltitrexed, and melphalan in-vitro and by real-time qRT-PCR gene expression assays, and for the Systemic therapy cohort were selected by age, comorbidity, performance status, and absence of RAS mutations. Therapeutic responses, adverse events, and quality of life were evaluated by RECIST 1.1, CTCAE 4.03, and ECOG criteria, respectively, and chemo-filtration performed following HAI to reduce systemic toxic effects. RESULTS: HAI/target therapy with drugs selected by liquid biopsy precision oncotherapy (44 patients), resulted in 2.27% CRs, 38.63% PRs, 56.81% SD,s and 2.27% PDs; ECOG 2 to 1 improvement, but no infusion-related technical or vascular complications, or deaths. Systemic therapy (62 patients) resulted in 1.6% CRs, 17.74% PRs, 37.09% SDs, and 45.16% PDs; more grade 1-2 adverse events and 4.84% ECOG 1 to 2 worsening. The median 5 month PFS in the HAI/target therapy cohort was significantly longer than 3 months in the systemic cohort (P < 0.007) and the median 14 month survival in the HAI/target therapy cohort was longer than 8.5 months in the systemic therapy cohort but not statistically significant. Multivariate analysis identified ECOG grade 2 as the most unfavourable survival prognostic factor in both cohorts. CONCLUSIONS: HAI plus chemo-filtration followed by target therapy, with drug regimens selected by liquid biopsy precision oncotherapy, is a safe and efficacious alternative therapeutic strategy for unresectable CRCLM in progression after two lines of systemic therapy and should be considered for a multicentre prospective phase III study, to fully confirm this potential.

Precision oncotherapy based on liquid biopsies in multidisciplinary treatment of unresectable recurrent rectal cancer: a retrospective cohort study.

BACKGROUND: Third line innovative systemic treatments and loco-regional chemotherapy by hypoxic pelvic perfusion (HPP) have both been proposed for the treatment of unresectable not responsive recurrent rectal cancer (URRC). In the present study, we have compared the safety and efficacy of HPP/target therapy, using drug regimens selected by liquid biopsy precision oncotherapy, to third-line systemic therapy based on tissue specimens precision oncotherapy. METHODS: HPP/target therapy regimens were selected based on precision oncotherapy, including assays for chemosensitivity and viability, and qRT-PCR for tumor-related gene expression. In the control group, systemic third-line and further lines of therapy were defined according to clinical and biological parameters. RESULTS: From 2007 to 2019, 62 URRC patients were enrolled, comprised of 43 patients in the HPP/target-therapy group and 19 patients in the systemic therapy control group. No HPP related complications were reported and the most common adverse events were skin and bone marrow toxicity. In the HPP/target-therapy group, the ORR was 41.8% whereas in the systemic therapy control group was 15.8%. DCR of the HPP/target-therapy group was significantly improved over the systemic therapy group (P = 0.001), associated with a PFS of 8 vs 4 months (P = 0.009), and OS of 20 vs 8 months (P = 0.046). CONCLUSIONS: The present data indicate that in URCC patients, the integration of HPP/target-therapy and precision oncotherapy based upon liquid biopsy is as effective and efficacious as third-line treatment in local disease control and, therefore, deserves to be further assessed and compared to conventional systemic treatments in future prospective randomized trials.

Gene expression profiling as a potential predictor between normal and cancer samples in gastrointestinal carcinoma.

Analysis and comparison of gene expression profile among molecules, correlated with essential and crucial biological processes, is of primary importance in cancer research, since it provides significant info regarding the resistance to chemo/radiotherapy, risk for relapse or prediction of metastasis etc. In this study, gene expression profile is used for discriminating efficiently colon cancer cell lines from normal cells and cancer cells in blood samples of colon cancer patients and categorizing different types of gastrointestinal cancer. In particular, blood samples were collected from normal donors as well as from colon cancer patients. Peripheral blood mononuclear cells were isolated and gene expression analysis was performed for more than fifty genes. The same assays were performed for commercial cancer cell lines representing different types of gastrointestinal cancer. In order to examine whether the comparison of gene expression profile can lead to a thorough discrimination between cancer and normal states as well as between different cancer types, we performed clustering analysis based on hierarchical, and k-means algorithms. The clustering analysis efficiently separated: a) colon cancer cell lines from colon patients' samples, b) normal from the colon cancer samples, c) gastric and pancreatic cancer from liver and colon types based. The exploitation of gene expression profile can be successfully used for the discrimination between normal vs cancer samples and/or for categorizing various types of cancer. This of course has important implications in cancer management since it enables the quick discrimination based on cells, isolated from bloodstream, needless of tissue examination or protocols requiring specialized equipment.

Novel small molecule decreases cell proliferation, migration, clone formation, and gene expression through ERK inhibition in MCF-7 and MDA-MB-231 breast cancer cell lines.

The Ras-Raf-MEK1/2-ERK1/2 pathway is an attractive target for the development of anticancer agents because of the high prevalence of ERK activation in human cancers. However, resistance is often developed despite initial clinical response, most likely because of activation of cross-talk pathways. In Research Genetic Cancer Center (RGCC), we are in the process of synthesizing a novel ERK inhibitor, targeting the final stage of the pathway, thus minimizing cross-talk. We have synthesized an intermediate molecule -RGCC416 - and tested its biological activity. MCF-7 and MDA-MB-231 cells were used. Cell viability was measured by crystal violet and cell proliferation by the methyl tetrazolium assay using various compound concentrations. Cell migration and colony formation were determined to assess the ability of invasion and single cancer cell growth, respectively. Expression of genes linked to MAPK/PI3K pathways was determined by PCR. ERK and phospho-ERK levels were determined in both the cytoplasm and the nucleus by western blot. It was found that although the compound did not affect viability, it significantly decreased cell proliferation, migration, and colony formation in both cell lines. In MDA-MB-231, this is possibly through retaining phospho-ERK to the cytoplasm, where it cannot activate cancer-associated genes. There was no difference in ERK levels in MCF-7 cells. This could be because of the different pathways that these cells utilize for survival. We have synthesized a molecule, which could be a promising ERK inhibitor, leading to possible novel treatment options for breast cancer patients.

Folic Acid-Functionalized Gold Nanorods for Controlled Paclitaxel Delivery: In Vitro Evaluation and Cell Studies.

Short gold nanorods were synthesized (average length 28.08 nm, average aspect ratio 3.54), which were functionalized with folic acid (FA) and 8-mercaptooctanoic acid (MOA) or 11-mercaptoundecanoic acid (MDA) and loaded with paclitaxel (PCT). FA was conjugated to the nanorods in order to render them targetable for cancer cells overexpressing folate receptors whereas MOA or MDA was attached on the nanorods in order to generate extra hydrophobic areas for entrapment of hydrophobic drugs such as PCT in the nanorods and in order to provide free carboxylic groups, which would allow for the conjugation of drug or other biofunctional molecules to the nanorods. The functionalized gold nanorods (GNRs-MOA-FA and GNRs-MDA-FA) did not exhibit any significant degree of aggregation in cell culture medium and blood plasma even after a prolonged incubation period of 7 days, indicating the adequate colloidal stability of the nanorods in these media. The functionalized nanorods exhibited satisfactory entrapment efficiency (around 40%) for PCT and released less than 25% of their PCT content in phosphate buffer pH 7.4 in 48 h. PCT entrapment efficiency was a little higher and PCT release rate a little lower in the GNRs-MOA-FA. Molecular analysis (qPCR) was used to find out that the MDA-MB-231 cancer cell line expresses the folate receptor (FL1R) whereas the MCF-7 cancer cell line does not. The PCT-loaded GNRs-MOA-FA were more cytotoxic than the PCT-loaded GNRs-MOA nanorods against the MDA-MB-231 cells, which probably relates to the higher uptake of the GNRs-MOA-FA nanorods by these cells. The opposite was true in the case of the MCF-7 cells.

Evaluation of a simple method for storage of blood samples that enables isolation of circulating tumor cells 96 h after sample collection.

BACKGROUND: Minimizing the effects of transportation on the properties of biological material is a major challenge for the scientific community. The viability of cells is important in cases where their study is urgent for evaluation of treatment response or for the study of cancer progression. Circulating tumor cells (CTCs) constitute a cell subpopulation with great importance for oncologists, because of their prognostic value. Detection and isolation of CTCs from blood samples is a routine activity in many laboratories, but concerns exist with regard to the maintenance of the cells during transportation. In this study, experiments were conducted to determine the stability of gene and protein expression in CTCs over a period of 96 h. RESULTS: Blood samples collected from healthy individuals and patients with cancer were each divided into five aliquots, which were stored at 2-8 °C and analyzed after 0, 24, 48, 72 and 96 h of storage. CTCs from patients and CD45-negative cells from healthy individuals were isolated each day using enrichment protocols, and qPCR was performed to determine expression levels of genes encoding specific biological markers. In addition, cells from breast and colon cancer cell lines were spiked into blood samples from healthy individuals, and these samples were stored and analyzed over a period of 96 h by PCR and by flow cytometry. The markers that were studied included housekeeping genes and genes associated with the response to chemotherapy, as well as genes encoding transcription factors. The results demonstrated that the expression profiles of specific genes and proteins in CTCs were not significantly affected by 72 h of storage. After 96 h of storage, expression of some genes was altered. CONCLUSION: The transportation of blood at low temperature (2-8 °C) in the presence of the anticoagulant EDTA can protect CTCs from alteration of gene and protein expression for at least 72 h. Furthermore, under these conditions, CTCs can be detected and isolated 96 h after blood collection.

Current perspectives on CHEK2 mutations in breast cancer.

Checkpoint kinase 2 (CHEK2) is a serine/threonine kinase which is activated upon DNA damage and is implicated in pathways that govern DNA repair, cell cycle arrest or apoptosis in response to the initial damage. Loss of kinase function has been correlated with different types of cancer, mainly breast cancer. CHEK2 functionality is affected by different missense or deleterious mutations. CHEK2*1100delC and I157T are most studied in populations all over the world. Although these variants have been identified in patients with breast cancer, their frequency raises doubts about their importance as risk factors. The present article reviews the recent advances in research on CHEK2 mutations, focusing on breast cancer, based on the latest experimental data.

L1 retrotransposon expression in circulating tumor cells.

Long interspersed nuclear element 1 (LINE-1 or L1) belongs to the non-long terminal repeat (non-LTR) retrotransposon family, which has been implicated in carcinogenesis and disease progression. Circulating tumor cells (CTCs) are also known to be involved in cancer progression. The present study aimed to compare the L1 expression between circulating tumor cells and non-cancerous samples. Blood samples were collected from 10 healthy individuals and 22 patients with different types of cancer. The whole blood cells were isolated using enrichment protocols and the DNA and RNA were extracted. RT-qPCR was performed for L1-ORF1 (open reading frame 1) and L1-ORF2, using 18S rRNA as the reference gene. The data were analyzed with the Livak method and statistical analyses were carried out with the Mann-Whitney and Kruskal-Wallis tests. In parallel with the above molecular biology experiments, FISH experiments were performed on the interphase nuclei of the cells for the detection of ORF2 RNA. DNA analysis revealed the presence of both ORF1 and ORF2 in all samples. RNA expression experiments demonstrated that ORF1 was not expressed in all samples, while ORF2 was expressed at varying levels in the non-cancer samples and the samples representing the different cancer types. A significant difference in ORF2 expression was observed between the CTCs and non-cancer samples (p = 0,00043), and significant differences were also observed between normal and lung (p = 0,034), pancreatic (p = 0,022), prostate (p = 0,014), and unknown primary of origin (p = 0,0039) cancer samples. Cytogenetic analysis revealed higher levels of ORF2 in the nuclei of CTCs than in normal samples. This study highlights the significant difference in L1-ORF2 expression between CTCs and normal samples. The increased expression levels observed for CTCs may be correlated with the characteristic features of these cells.

Identification of genes involved in breast cancer and breast cancer stem cells.

Breast cancer is the most frequent type of cancer in women. Great progress has been made in its treatment but relapse is common. One hypothesis to account for the high recurrence rates is the presence of cancer stem cells (CSCs), which have the ability to self-renew and differentiate into multiple malignant cell types. This study aimed to determine genes that are expressed in breast cancer and breast CSCs and to investigate their correlation with stemness. RNA was extracted from established breast cancer cell lines and from CSCs derived from five different breast cancer patients. DNA microarray analysis was performed and any upregulated genes were also studied in other cancer types, including colorectal and lung cancer. For genes that were expressed only in breast cancer, knockdown-based experiments were performed. Finally, the gene expression levels of stemness transcription factors were measured. The outcome of the analysis indicated a group of genes that were aberrantly expressed mainly in breast cancer cells with stemness properties. Knockdown experiments confirmed the impact of several of these on NANOG, OCT3/4, and SOX2 transcription factors. It seems that several genes that are not directly related with hormone metabolism and basic signal transduction pathways might have an important role in relapse and disease progression and, thus, can be targeted for new treatment approaches for breast cancer.

Tumor-induced osteomalacia due to a recurrent mesenchymal tumor overexpressing several growth factor receptors.

UNLABELLED: Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome caused primarily by benign mesenchymal tumors. These tumors typically follow a benign clinical course and local recurrence occurs in <5% of cases. We investigated a 49-year-old man with a recurrent mesenchymal phosphaturic tumor showing no signs of malignancy. The patient suffered from chronic muscle weakness, myalgia and cramps. His medical record included the diagnosis of oncogenic osteomalacia, for which he was submitted to tumor resection in the left leg three times before. Laboratory examination showed hypophosphatemia, hyperphosphaturia and an elevated serum FGF23 level. A radical surgical approach (amputation) was advised, however, complete biochemical and clinical remission was not reached. Molecular analysis of the tumor cells demonstrated overexpression of growth factor receptors implicated in tumor angiogenesis and metastatic potential (platelet derived growth factor type A (PDGFRA), PDGFRB and vascular endothelial growth factor receptor) together with increased expression of FGF23, x-linked-phosphate-regulating endopeptidase and KLOTHO. TIO is usually associated with benign phosphauturic tumors and, when identified, resection of the tumor leads to complete remission in the majority of cases. The underlying pathophysiology of recurrences in these tumors is not known. This is the first report showing increased expression of growth factor receptors in a locally aggressive but histopathologically benign phosphaturic mesenchymal tumor. LEARNING POINTS: TIO is usually associated with benign soft tissue or bone neoplasms of mesenchymal origin.These tumors typically follow a benign clinical course and even in the rare malignant cases local recurrence occurs in <5%.Successful identification and removal of the tumor leads to full recovery in the majority of cases.