Immune activation of the monocyte-derived dendritic cells using patients own circulating tumor cells.

BACKGROUND: Dendritic cell (DC) therapy counts to the promising strategies how to weaken and eradicate cancer disease. We aimed to develop a good manufacturing practice (GMP) protocol for monocyte-derived DC (Mo-DC) maturation using circulating tumor cells lysates with subsequent experimental T-cell priming in vitro. METHODS: DC differentiation was induced from a population of immunomagnetically enriched CD14 + monocytes out of the leukapheresis samples (n = 6). The separation was provided automatically, in a closed bag system, using CliniMACS Prodigy® separation protocols (Miltenyi Biotec). For differentiation and maturation of CD14 + cells, DendriMACs® growing medium with supplements (GM-CSF, IL-4, IL-6, IL-1B, TNFa, PGE) was used. Immature Mo-DCs were loaded with autologous circulating tumor cell (CTCs) lysates. Autologous CTCs were sorted out by size-based filtration (MetaCell®) of the leukapheresis CD14-negative fraction. A mixture of mature Mo-DCs and autologous non-target blood cells (NTBCs) was co-cultured and the activation effect of mature Mo-DCs on T-cell activation was monitored by means of multimarker gene expression profiling. RESULTS: New protocols for mMo-DC production using automatization and CTC lysates were introduced including a feasible in vitro assay for mMo-DC efficacy evaluation. Gene expression analysis revealed elevation for following genes in NTBC (T cells) subset primed by mMo-DCs: CD8A, CD4, MKI67, MIF, TNFA, CD86, and CD80 (p ≤ 0.01). CONCLUSION: Summarizing the presented data, we might conclude mMo-DCs were generated using CliniMACS Prodigy® machine and CTC lysates in a homogenous manner showing a potential to generate NTBC activation in co-cultures. Identification of the activation signals in T-cell population by simple multimarker-qPCRs could fasten the process of effective mMo-DC production.

Molecular characterization and heterogeneity of circulating tumor cells in breast cancer.

INTRODUCTION: This study analyzes peripheral blood samples from breast cancer (BC) patients. CTCs from peripheral blood were enriched by size-based separation and were then cultivated in vitro. The primary aim of this study was to demonstrate the antigen independent CTC separation method with high CTC recovery. Subsequently, CTCs enriched several times during the treatment were characterized molecularly. METHODS: Patients with different stages of BC (N = 167) were included into the study. All patients were candidates for surgery, surgical diagnostics, or were undergoing chemotherapy. In parallel, 20 patients were monitored regularly and in addition to CTC presence, also CTC character was examined by qPCR, with special focus on HER2 and ESR status. RESULTS: CTC positivity in the cohort was 76%. There was no significant difference between the tested groups, but the highest CTC occurrence was identified in the group undergoing surgery and similarly in the group before the start of neoadjuvant treatment. On the other hand, the lowest CTC frequencies were observed in the menopausal patient group (56%), ESR+ patient group (60%), and DCIS group (44.4%). It is worth noting that after completion of neoadjuvant therapy (NACT) CTCs were present in 77.7% of cases. On the other hand, patients under hormonal treatment were CTC positive only in 52% of cases. DISCUSSIONS: Interestingly, HER2 and ESR status of CTCs differs from the status of primary tumor. In 50% of patients HER2 status on CTCs changed not only from HER2+ to HER2-, but also from HER2- to HER2+ (33%). ESR status in CTCs changed only in one direction from ESR+ to ESR-. CONCLUSIONS: Data obtained from the present study suggest that BC is a heterogeneous disease but CTCs may be detected independently of the disease characteristics in 76% of patients at any time point during the course of the disease. This relatively high CTC occurrence in BC should be considered when planning the long-term patient monitoring.

[AR-V7 Androgen Receptor Variant as a Predictor of Response to Androgen-receptor Targeting Agents Used to Treat Castration-refractory Metastatic Prostate Cancer]. / Testování varianty androgenového receptoru AR-V7 pro výber pacientu s kastracne refrakterním metastazujícím karcinomem prostaty k lécbe novými hormonálními léky.

BACKGROUND: Several systemic treatment options are currently available for patients with metastatic castration-refractory prostate cancer (mCRPC), including the androgen-receptor targeting agents (ARTA) enzalutamide and abiraterone, the taxanes docetaxel and cabazitaxel, and the radioisotope drug 223-radium dichloride. In some patients with mCRCP, alternative splicing of androgen receptor (AR) mRNA occurs, resulting in the formation of a truncated AR lacking the androgen-binding domain. These receptors activate downstream signalling pathways even without the ligand. Recent studies show that the presence of the AR-V7 (ARV - AR variants) splicing variant is associated with resistance to ARTA. Bec>ause the presence of AR-V7 does not affect the efficacy of other systemic therapies used in mCRCPs, particularly taxanes, AR-V7 is a candidate predictive biomarker for the individualisation of mCRCP treatment. Two types of assays based on mRNA or abnormal protein detection are used to detect AR-V7 in circulating tumour cells. AIM: To describe the current status of AR-V7 testing in mCRPC and possible applications of this method for predicting outcomes of ARTA therapy. CONCLUSION: The percentage of CTC AR-V7+ in ARTA-naive men is relatively low at baseline, but in patients pretreated with ARTA, the prevalence of AR-V7 increases to 19-34%. Given the relatively high expected prevalence, AR-V7 testing may be economically feasible in this population. The proportion of AR-V7+ patients responding to ARTA retreatment appears to be very low, at only 4.8%. AR-V7 testing could thus be useful if an ARTA switch is considered in a patient progressing onto an ARTA drug. Both protein-based tests and mRNA-based tests are currently undergoing clinical validation in prospective studies, with results expected within a year.Key words: prostate cancer - abiraterone - enzalutamide - alternative splicing - drug resistanceSubmitted: 30. 8. 2017Accepted: 5. 11. 2017 doc. MUDr. Tomás Büchler, Ph.D. received honorary lectures and publications from Astellas and Janssen and a travel grant from Janssen. Supported by Ministry of Health, Czech Republic - conceptual development of research organization Thomayer Hospital - TN 0064190. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

Expression of Ceramide Galactosyltransferase (UGT8) in Primary and Metastatic Lung Tissues of Non-Small-Cell Lung Cancer.

Ceramide galactosyltransferase (UGT8) is an enzyme that regulates the synthesis of sphingolipids of the myelin sheath in nervous systems. The protein raises an increasing research interest as a potential marker of cancer progression in various organs. In the present study we seek to determine whether UGT8 could play a role of a therapeutic marker in non-small cell lung carcinoma (NSCLC). We addressed the issue by examining the intensity of UGT8 expression in tissue specimens of primary and corresponding metastatic lung tumors in 19 NSCLC patients undergoing surgery. The methodology was one of immunohistochemical tissue staining using light microscopy. The findings were that the majority of both lung primary and metastatic tumor tissues were positive in UGT8 signals. The cytoplasmic expression of UGT8 was found in 68.4 % of cases of primary tumors and 82.2 % of metastases, with a positive correlation between the UGT8 expression in both tumor tissues. The normal tissue adjacent to tumors showed no positive UGT8 staining. However, we failed to find any appreciable difference in UGT8 expression depending on the clinical stage of NSCLC or lymph node involvement. Nor was there any association between UGT8 expression in tumor tissues and patients' survival time. We conclude that it is unlikely that therapeutic targeting of UGT8 could inhibit cell proliferation and invasion of NSCLC. UGT8, although enhanced in NSCLC tissues, does not meet the criteria of a lung tumor marker. Thus, UGT8 cannot be considered as having diagnostic or therapeutic utility in NSCLC. The pathophysiological meaning of enhanced expression of UGT8 in lung cancer remains to be explored in further studies.

In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

The impact of platelet-rich plasma on chronic synovitis in hemophilia.

Untreated chronic haemophiliac synovitis leads to the development of haemophilic arthropathy (HA) by affecting the metabolism of chondrocytes. Symptoms are progressive and often surgical intervention is required to prevent total loss of joint function. The focus of our study was to influence the chronic haemophiliac synovitis by means of autologous platelet-rich plasma (PRP) injection. Six patients with hemophilia (PWH), aged between 9 and 45 and manifesting chronic synovitis of the ankle joint on one or on both sides (8 joints in total) were included into the PRP-study. The patients were classified depending on their joint status using the Hemophilia Joint Health Score (HJHS) prior to and again two months after treatment. Three to five ml of PRP was injected into the joint cavity within 30 seconds. In all of the tested PWH pain relief has been reported subjectively by means of the HJHS and VAS scoring systems, comparing the pain intensity before PRP injection and two months after. The difference of pain perception has been found statistically significant for the VAS-scores. Considering the objective synovitis signs shown on MRI before and after PRP injection we recorded a decrease in the volume of free synovial fluid after PRP. All of the tested patients reported benefit of the PRP therapy.

Radiosynoviorthesis in hemophilic joints with yttrium-90 citrate and rhenium-186 sulfide and long term results.

Repeated bleeding in the joint cavities is the most annoying symptom and often has disabling effects in patients with hemophilia (PWH). Our aim was to study the effect of radiosynovectomy (RSO) with beta particle-emitting radiocolloids in the treatment of hemorhagic arthropathy. We have treated 22 joints from 18 patients with hemophilia A, from April 2008 to February 2012, 5 knees, 11 elbows and 6 ankles. Joints were divided into two Groups, those treated with yttrium-90-citrate ((90)Y-C) (5 knees, 2 of them twice)-Group I and those with rhenium-186-sulfide ((186)Re-S) (11 elbows, 1 of them treated twice and 6 ankles)-Group II. A total of 25 treatments. Follow-up period was 3 months, 1 year and 3 years. Results showed a favourable subjective and a better objective result in all 5 joints of Group I and in 15/17 joints of Group II, respectively. Follow-up after 3 months showed significant improvement in Hemophilia Join Health Score (HJHS) after 20 treatments and steady score after 5 treatments. After 1 year, 19 treated joints had improved for the first time, 3 remained steady and 3 were not examined. After 3 years, 9 treated joints were HJHS steady, while 16 were not examined. One year after treatment, 13/14 joints of patients, aged 6-23 years showed better HJHS score, while 9/11 joints of patients aged 26-51 years, showed better HJHS. Synovial membrane thickness as measured by MRI in 8 joints, before and 3 months after treatment was not related to prognosis. In conclusion, in a small group of hemophilic patients with hemorrhagic arthropathy treated with (90)Y-C and with (186)Re-S, our study showed good results irrespective of age in 22/25 treatments after 3 months or 1 year. The thickness of synovial membrane in the 8 joints studied was not related to prognosis.

Mitochondrially targeted tamoxifen as anticancer therapy: case series of patients with renal cell carcinoma treated in a phase I/Ib clinical trial.

Mitochondrially targeted anticancer drugs (mitocans) that disrupt the energy-producing systems of cancer are emerging as new potential therapeutics. Mitochondrially targeted tamoxifen (MitoTam), an inhibitor of mitochondrial respiration respiratory complex I, is a first-in-class mitocan that was tested in the phase I/Ib MitoTam-01 trial of patients with metastatic cancer. MitoTam exhibited a manageable safety profile and efficacy; among 37% (14/38) of responders, the efficacy was greatest in patients with metastatic renal cell carcinoma (RCC) with a clinical benefit rate of 83% (5/6) of patients. This can be explained by the preferential accumulation of MitoTam in the kidney tissue in preclinical studies. Here we report the mechanism of action and safety profile of MitoTam in a case series of RCC patients. All six patients were males with a median age of 69 years, who had previously received at least three lines of palliative systemic therapy and suffered progressive disease before starting MitoTam. We recorded stable disease in four, partial response in one, and progressive disease (PD) in one patient. The histological subtype matched clear cell RCC (ccRCC) in the five responders and claro-cellular carcinoma with sarcomatoid features in the non-responder. The number of circulating tumor cells (CTCs) was evaluated longitudinally to monitor disease dynamics. Beside the decreased number of CTCs after MitoTam administration, we observed a significant decrease of the mitochondrial network mass in enriched CTCs. Two patients had long-term clinical responses to MitoTam, of 50 and 36 weeks. Both patients discontinued treatment due to adverse events, not PD. Two patients who completed the trial in November 2019 and May 2020 are still alive without subsequent anticancer therapy. The toxicity of MitoTam increased with the dosage but was manageable. The efficacy of MitoTam in pretreated ccRCC patients is linked to the novel mechanism of action of this first-in-class mitochondrially targeted drug.

Mitochondrially targeted tamoxifen in patients with metastatic solid tumours: an open-label, phase I/Ib single-centre trial.

Background: Mitochondria present an emerging target for cancer treatment. We have investigated the effect of mitochondrially targeted tamoxifen (MitoTam), a first-in-class anti-cancer agent, in patients with solid metastatic tumours. Methods: MitoTam was tested in an open-label, single-centre (Department of Oncology, General Faculty Hospital, Charles University, Czech Republic), phase I/Ib trial in metastatic patients with various malignancies and terminated oncological therapies. In total, 75 patients were enrolled between May 23, 2018 and July 22, 2020. Phase I evaluated escalating doses of MitoTam in two therapeutic regimens using the 3 + 3 design to establish drug safety and maximum tolerated dose (MTD). In phase Ib, three dosing regimens were applied over 8 and 6 weeks to evaluate long-term toxicity of MitoTam as the primary objective and its anti-cancer effect as a secondary objective. This trial was registered with the European Medicines Agency under EudraCT 2017-004441-25. Findings: In total, 37 patients were enrolled into phase I and 38 into phase Ib. In phase I, the initial application of MitoTam via peripheral vein indicated high risk of thrombophlebitis, which was avoided by central vein administration. The highest dose with acceptable side effects was 5.0 mg/kg. The prevailing adverse effects (AEs) in phase I were neutropenia (30%), anaemia (30%) and fever/hyperthermia (30%), and in phase Ib fever/hyperthermia (58%) together with anaemia (26%) and neutropenia (16%). Serious AEs were mostly related to thromboembolic (TE) complications that affected 5% and 13% of patients in phase I and Ib, respectively. The only statistically significant AE related to MitoTam treatment was anaemia in phase Ib (p = 0.004). Of the tested regimens weekly dosing with 3.0 mg/kg for 6 weeks afforded the best safety profile with almost all being grade 1 (G1) AEs. Altogether, five fatalities occurred during the study, two of them meeting criteria for Suspected Unexpected Serious Adverse Events Reporting (SUSAR) (G4 thrombocytopenia and G5 stroke). MitoTam showed benefit evaluated as clinical benefit rate (CBR) in 37% patients with the largest effect in renal cell carcinoma (RCC) where four out of six patients reached disease stabilisation (SD), one reached partial response (PR) so that in total, five out of six (83%) patients showed CBR. Interpretation: In this study, the MTD was established as 5.0 mg/kg and the recommended dose of MitoTam as 3.0 mg/kg given once per week via central vein with recommended preventive anti-coagulation therapy. The prevailing toxicity included haematological AEs, hyperthermia/fever and TE complications. One fatal stroke and non-fatal G4 thrombocytopenia were recorded. MitoTam showed high efficacy against RCC. Funding: Smart Brain Ltd. Translation: For the Czech translation of the abstract see Supplementary Materials section.

Enhancement of musculocutaneous nerve reinnervation after vascular endothelial growth factor (VEGF) gene therapy.

BACKGROUND: Vascular endothelial growth factor (VEGF) is not only a potent angiogenic factor but it also promotes axonal outgrowth and proliferation of Schwann cells. The aim of the present study was to quantitatively assess reinnervation of musculocutaneous nerve (MCN) stumps using motor and primary sensory neurons after plasmid phVEGF transfection and end-to-end (ETE) or end-to-side (ETS) neurorrhaphy. The distal stump of rat transected MCN, was transfected with plasmid phVEGF, plasmid alone or treated with vehiculum and reinnervated following ETE or ETS neurorrhaphy for 2 months. The number of motor and dorsal root ganglia neurons reinnervating the MCN stump was estimated following their retrograde labeling with Fluoro-Ruby and Fluoro-Emerald. Reinnervation of the MCN stumps was assessed based on density, diameter and myelin sheath thickness of regenerated axons, grooming test and the wet weight index of the biceps brachii muscles. RESULTS: Immunohistochemical detection under the same conditions revealed increased VEGF in the Schwann cells of the MCN stumps transfected with the plasmid phVEGF, as opposed to control stumps transfected with only the plasmid or treated with vehiculum. The MCN stumps transfected with the plasmid phVEGF were reinnervated by moderately higher numbers of motor and sensory neurons after ETE neurorrhaphy compared with control stumps. However, morphometric quality of myelinated axons, grooming test and the wet weight index were significantly better in the MCN plasmid phVEGF transfected stumps. The ETS neurorrhaphy of the MCN plasmid phVEGF transfected stumps in comparison with control stumps resulted in significant elevation of motor and sensory neurons that reinnervated the MCN. Especially noteworthy was the increased numbers of neurons that sent out collateral sprouts into the MCN stumps. Similarly to ETE neurorrhaphy, phVEGF transfection resulted in significantly higher morphometric quality of myelinated axons, behavioral test and the wet weight index of the biceps brachii muscles. CONCLUSION: Our results showed that plasmid phVEGF transfection of MCN stumps could induce an increase in VEGF protein in Schwann cells, which resulted in higher quality axon reinnervation after both ETE and ETS neurorrhaphy. This was also associated with a better wet weight biceps brachii muscle index and functional tests than in control rats.

Wound healing gene therapy: cartilage regeneration induced by vascular endothelial growth factor plasmid.

AIMS: The identification of growth factors and cytokines with angiogenic activity has enabled new therapeutic treatments for a variety of diseases; this concept is called therapeutic angiogenesis. The vascular endothelial growth factor (VEGF) is the most critical regulator of vascular formation. In the present study, we were interested in the therapeutic angiogenesis effect using plasmid transfer of human complementary DNA VEGF(165) (phVEGF(165)) in experimental skin and cartilage trauma. METHODS: Ten BALB/c mice were used for cartilage injury model. At 6 weeks of age, all mice were ear-punched, resulting in 2-mm-diameter puncture through the center of both pinnae. Each mouse got phVEGF(165) injection into the first ear and vector without insert or saline injection into the second one. The healing process was followed. The hollow diameter was measured on days 0, 14, and 42. Histological sections of experimental and control pinnae were taken from days 1, 3, 5, 7, 9, 11, 13, 15, 20, and 30 after experimental injury for hematoxylin and eosin and periodic acid Schiff staining and for human VEGF immunocytochemistry. The expression of human VEGF was also checked by real-time polymerase chain reaction in formalin-fixed, paraffin-embedded tissue sections. KEY FINDINGS: In BALB/c mouse strain, a significant angiogenesis promotion and cartilage repair were observed after phVEGF(165) injection into the punched ear area. SIGNIFICANCE: We suggest that administering phVEGF(165) leads to faster cartilage regeneration even if not only on the angiogenic basis.

Tissue repair driven by two different mechanisms of growth factor plasmids VEGF and NGF in mice auricular cartilage: regeneration mediated by administering growth factor plasmids.

The focus of this study was to compare the role of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) in the regeneration of experimental skin and cartilage trauma. The role of VEGF in this process is known since decade; the NGF participation on this process has been first discussed within the spinal cord injury repair. We hypothesized that both VEGF and NGF induce angiogenesis and take part on the repair process. The angiogenesis response and the cartilage regeneration after phVEGF(165) plasmid and rat pcNGF plasmid administration were investigated using BALB/c mice. PhVEGF(165) and pcNFG were injected into the right mice ear and plain vector injection into the left ear the day before trauma. The next day, all mice were ear-punched, resulting in 2-mm diameter puncture through the center of both pinnae. In BALB/c mouse strain, a significantly faster cartilage repair was observed after phVEGF(165) and pcNGF injection into punched ear area in comparison to the control group. It has been shown that the healing process is after VEGF and NGF injection driven differentially. In case of VEGF is the cartilage wound repaired by induction of new chondrocytes differentiation. In the case of NGF, the regeneration is supported by immature leukocytes attracted into the punched area. The leukocytes induct angiogenesis so far indirectly by inflammation. The NGF-induced inflammation environment may be a part of mosaic creating the complete picture of regeneration.

Development of new spontaneous metastatic heterotopic model of lewis lung carcinoma imaged by GFP expression.

Many studies have demonstrated the importance of spontaneous metastases in cancer research. Until now, we still had only a few spontaneous metastatic models with high occurrence rate of metastasis in distant lymph and visceral tissues. We report a syngeneic heterotopic metastatic model using the Lewis lung cancer cell line with high metastatic ratio in C57BL/6 mice after transplantation by injection of cancer cells and without surgical intervention. Metastatic process was declared for each mouse in two groups ?sacrificed 3 or 5 weeks after subcutaneous (s.c.) injection of the tumor cells into the dorsal side of the tail. The total number of metastases was counted as the sum of observed macrometastases. Our model produced produced a 100% rate of spontaneous lymphatic and visceral metastases after a simple injection transplantation into the heterotopic site. In mice with large primary tumors which are non-lethal, visceral and lymph macrometastases were observed. Tumor volume correlated linearly not only with the tumor growth time, but also with the number of metastases in lymph nodes and organs. This new metastatic model could be useful for studying the metastasis mechanism and for developing therapy for lymph and visceral metastases.

Next generation sequencing of glioblastoma circulating tumor cells: non-invasive solution for disease monitoring.

Treatment of aggressive glioblastoma multiforme (GBM) must be based on very precise histological and molecular diagnostic of GBM type. According to the WHO guidelines, only tissue biopsy is a relevant source of cellular material evaluated in the diagnostic process to specify the tumor features. Nevertheless, obtaining a GBM biopsy is complicated and relies mostly on resection surgery. Evaluating circulating free DNA and/or circulating tumor cells (CTCs) in the clinic, using a liquid biopsy could represent a non-invasive cancer care optimization. In the present study, the peripheral blood of patients undergoing GBM resection (n = 18) was collected and examined for CTCs. The feasibility of GBM molecular diagnostics from a simple non-invasive peripheral blood withdrawal was evaluated. The size-based enriched CTCs were analyzed using cytomorphology and their origin confirmed based on mutational analysis. In addition, shared DNA mutations in CTCs and in primary tumor tissue were searched. For the identification of CTCs, next generation sequencing (NGS) was used. The GeneReader™ sequencing platform enables targeted sequencing of a 12-gene panel and direct evaluation of detected gene variations using QIAGEN Clinical Insight Analyze (QCI-A) software with a special algorithm for liquid biopsy sequencing analysis. Herein, we present a standard operating procedure for CTC enrichment in GBM patients, CTC in vitro culture, CTC cytomorphological evaluation, and NGS analysis of CTCs using the QIAGEN Actionable Insights Tumor (ATP) Panel. CTCs were present in all tested patients (18/18). The NGS data generated for formalin-fixed paraffin-embedded (FFPE) primary tumor tissues and CTCs reached significantly high-quality parameters. The comparisons between different sample types (CTCs vs. primary tumors) and sampling area (different primary tumor regions) showed a significant level of concordance, indicating CTC testing could be used for patient monitoring and recurrence awareness. Notably, more mutations were detected when analyzing CTC samples compared with the paired primary tumors (n = 3). The results confirm the feasibility of using CTCs as a source of tumor DNA in a diagnostic process, especially when evaluating the molecular characteristics of GBMs. A major advantage of the presented NGS approach for detecting CTCs is the simultaneous identification of several markers relevant for GBM diagnostics, allowing molecular diagnostics on cytological specimens and potential administration of innovative targeted therapies.

Characterization of circulating tumor cells in early breast cancer patients receiving neoadjuvant chemotherapy.

BACKGROUND AND AIMS: The aim of this study was to characterize circulating tumor cells (CTCs) during neoadjuvant chemotherapy (NACT) in early and locally advanced breast cancer (LABC) patients. Using ultrasound, tumor volume measurement was compared with the presence and the molecular nature of CTCs over multiple time intervals corresponding to treatment periods. METHODS: A total of 20 patients diagnosed with breast cancer (BC) of different histotypes were monitored during the NACT period and in the follow-up period (~5 years). Peripheral blood for CTCs (n = 115) was taken prior to NACT, after two to three chemotherapy cycles, after the completion of NACT (before surgery) and at some time points during adjuvant therapy. CTCs were enriched using a size-based filtration method (MetaCell®) capturing viable cells, which enabled vital fluorescence microscopy. A set of tumor-associated (TA) genes and chemoresistance-associated (CA) genes was analyzed by qPCR in the enriched CTC fractions. RESULTS: The analysis of tumor volume reduction after administration of anthracyclines (AC) and taxanes (TAX) during NACT showed that AC therapy was responsive in 60% (12/20) of tumors, whereas TAX therapy was responsive in 30% (6/20; n.s.). After NACT, CTCs were still present in 70.5% (12/17) of patients (responders versus non-responders, 61.5% versus 100%; not significant).In triple-negative BC (TNBC) patients (n = 8), tumor volume reduction was observed in 75% cases. CTCs were significantly reduced in 42.9% of all HER2-negative BC patients. In HER2+ tumors, CTC reduction was reported in 16.6% only. Relapses were also more prevalent in the HER2-positive patient group (28.5 versus 66.6%).During NACT, the presence of CTCs (three tests for each patient) identified patients with relapses and indicated significantly shorter progression-free survival (PFS) rates (p = 0.03). Differentiation between progressive disease and non-progressive disease was obtained when the occurrence of excessive expression for CA genes in CTCs was compared (p = 0.024). Absence of tumor volume reduction was also significantly indicative for progressive disease (p = 0.0224).Disseminated CTCs in HER2-negative tumors expressed HER2 in 29% of samples collected during the overall follow-up period (16/55), and in 32% of samples during the follow-up of NACT (10/31). The change accounted for 78.5% of HER2-negative patients (11/14) in total, and 63.6% of the conversion cases occurred during NACT (7/11). For the remaining four patients (36.3%), conversion to HER2+ CTCs occurred later during adjuvant therapy. We believe there is the possibility of preventing further progression by identifying less responsive tumors during NACT using CTC monitoring, which could also be used effectively during adjuvant therapy.

Circulating tumor cells in gynaecological malignancies.

New non-invasive approaches have developed for diagnosis and treatment of malignant diseases. Cells shed from the primary tumor circulating in the bloodstream with metastasis potential are called Circulating Tumor Cells (CTCs). These cells are easily acquired from the peripheral blood of patients, while several enrichment and isolation methods are available nowadays with different benefits and positive detection rates. A brief characterization of three major categories of detection is described (nucleic acid-based, physical properties-based, antibody-based). In this review we concentrate on gynecological malignancies and how CTCs could be used in the diagnosis of cancer, treatment management and its effective prognosis and early recurrence detection. Presence of CTCs in endometrial cancer patients show worse overall survival, while gene analysis could identify patients in need of systemic therapy after surgical treatment to prevent metastasis and recurrence. Based on the influence of human papillomavirus (HPV) in the etiology of cervical cancer, viral oncogene transcripts could be used as an ideal marker for cervical cancer cells detection. In ovarian cancer, CTCs could help in the differentiation from benign adnexal masses and show a high independence from other biomarkers such as CA125 and HE4. While detection of CTC after complete cytoreductive surgery could indicate invisible lesions, combination of tumor associated genes rises the specificity of CTC detection.

Detection of Circulating Tumor Cells in Renal Cell Carcinoma: Disease Stage Correlation and Molecular Characterization.

The presence of circulating tumor cells (CTCs) in patients with solid tumors is associated with poor prognosis. However, there are limited data concerning the detection of CTCs in renal cell cancer (RCC). The aim of this study is to evaluate the presence of CTCs in peripheral blood of patients with RCC undergoing surgery (n = 186). CTCs were tested before and after surgery as well as during the follow-up period afterwards. In total 495 CTC testing in duplicates were provided. To enrich CTCs, a size-based separation protocol and tube MetaCell® was used. CTCs presence was evaluated by single cell cytomorphology based on vital fluorescence microscopy. Additionally, to standardly applied fluorescence stains, CTCs viability was controlled by mitochondrial activity. CTCs were detected independently on the sampling order in up to 86.7% of the tested blood samples in patients undergoing RCC surgery. There is higher probability of CTC detection with growing tumor size, especially in clear cell renal cell cancer (ccRCC) cases. Similarly, the tumor size corresponds with metastasis presence and lymph node positivity and CTC detection. This paper describes for the first-time successful analysis of viable CTCs and their mitochondria as a part of the functional characterization of CTCs in RCC.

Circulating Endometrial Cells in Women With Spontaneous Pneumothorax.

BACKGROUND: The occurrence of catamenial pneumothorax (CP) is rare, and the awareness of this diagnosis among physicians is insufficient. CP is highly correlated with pelvic endometriosis and remains the most common form of thoracic endometriosis syndrome. Circulating endometrial cells (CECs) have been previously detected in patients with pelvic endometriosis. Could CECs bring new insights into pneumothorax management? METHODS: This study aims to describe the occurrence and molecular characteristics of CECs in women with spontaneous pneumothorax (SP) (N = 20) with high suspicion of its catamenial character. CECs were enriched from peripheral blood by size-based separation (MetaCell). In addition to cytomorphology, gene expression profiling of captured cells was performed for 24 endometriosis-associated genes. RESULTS: CECs were present in all 20 patients with SP. Enriched CECs exhibited four character features: epithelial, stem cell-like, stroma-like, and glandular. However, not all of them were present in every sampling. Gene expression profiling revealed two distinct phenotypes of CECs in SP and/or CP: one of them refers to the diaphragm openings syndrome and the other to endometrial tissue pleural implantations. Comparisons of the gene expression profiles of CECs in pneumothorax (CECs-SP group) with CECs in pelvic endometriosis (CECs-non-SP group) have revealed significantly higher expression of HER2 in the CECs-SP group compared with the CECs-non-SP group. CONCLUSIONS: This proof-of-concept study demonstrates successful isolation and characterization of CECs in patients with SP. Identification of CECs in SP could alert endometriosis involvement and help early referral to gynecologic consultation for further examination and treatment.

Circulating Tumor Cells in Diagnosis and Treatment of Lung Cancer.

Circulating tumor cells (CTCs), detached from the primary tumor or metastases and shed in the patient's bloodstream, represent a relatively easily obtainable sample of the cancer tissue that can indicate the actual state of cancer, and their evaluation can be repeated many times during the course of treatment. As part of liquid biopsy, evaluation of CTCs provides a lot of clinically relevant information, which reflects the actual, real-time conditions of the disease. CTCs can be used in cancer diagnosis or screening, real-time long-term disease monitoring and even therapy guidance. Their analysis can include their number, morphology, and biological features by using immunocytochemistry and all "-omic" technologies. This review describes methods of CTC isolation and potential clinical utilization in lung cancer.

Circulating Endometrial Cells: A New Source of Information on Endometriosis Dynamics.

The focus of the presented work was to isolate and characterize circulating endometrial cells (CECs) enriched from peripheral blood (PB) of patients with diagnosed endometriosis. The molecular characteristics of CECs could be supportive for an understanding of endometriosis pathogenesis and treatment decisions in the future. MATERIAL AND METHODS: Blood samples (n = 423) were tested for CECs presence. Subsequently, gene expression analysis (GEA) was carried out for CECs. In parallel, CECs presence and characteristics were tested during menstrual cycle (MC) phases in 11 patients. CECs were enriched by size-based separation. RESULTS: CECs were present in 78.4% of the tested blood samples. In line with the revised American Fertility Society (rAFS) classification, CECs presence was confirmed in all the acknowledged endometriosis stages: minimal, mild, moderate, and severe. Surprisingly, CECs negativity rate was also reported for severe disease in 21.1% of cases. The CECs captured during MC phases displayed different cytomorphology, including epithelial, stromal, and stem cell-like characteristics. The highest CECs numbers were detected in the mid-secretory phase of MC, which corresponds to uterine lining decidualization. CECs captured during mid-secretory periods expressed genes KRT18, NANOG, and VIM in higher amounts when compared to the proliferative phase of MC, where genes KRT19 and ESR1 were mostly elevated. GEA of the super-positive CECs samples (1000 CECs/8 mL PB) revealed high expression of genes KRT18, VIM, NANOG, and FLT1. The expression of these genes was also elevated in the endometriosis tissue samples and endometrioma. CONCLUSION: The panel of the identified CEC genes could be tested in a prospective manner to confirm the role of CECs in endometriosis pathogenesis and diagnostics.