Circulating tumor cells in patients with breast cancer: monitoring chemotherapy success.

UNLABELLED: Circulating tumor cells (CTCs) are an independent prognostic factor for patients with metastatic breast cancer (MBC). However, the role of CTCs in early breast cancer management is not yet clearly defined. The aim of this study was to assess the CTC-positivity rate in patients undergoing chemotherapy depending on breast cancer stage in the adjuvant and neoadjuvant setting. We evaluated the ability to confirm therapy response by CTC analysis. PATIENTS AND METHODS: CTCs isolated from blood by means of immunomagnetic separation were further characterized by means of reverse transcriptase - polymerase chain reaction (RT-PCR) for epithelial cell adhesion molecule (EPCAM), mucin 1 (MUC1) and v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (HER2) transcripts with the AdnaTest™. This prospective study included 179 patients; altogether 419 blood samples were evaluated. Patients with primary tumors were divided into neoadjuvant (n=38), and adjuvant (n=100) groups. Forty-one patients with MBC were evaluated under palliative treatment. RESULTS: CTC positivity was described in 35% of patients with early breast cancer without detected metastases before neoadjuvant chemotherapy; similarly, a 26% positivity rate was found in the adjuvant group. In patients with MBC, we detected CTCs in 43% of them. After completing the therapy, the CTC positivity rate decreased to 5% in the neoadjuvant group, to 13% in the adjuvant group and to 12% in the MBC group. CTC positivity after the therapy may classify a subgroup of patients at high risk of developing metastatic disease. This was even true when a patient was evaluated as being CTC-negative before chemotherapy. The multivariate analysis evaluating the correlation of CTC positivity with clinicopathological characteristics such as tumor size, nodal involvement, hormone receptor status, HER2 expression and number of metastatic sites revealed no statistically significant relationships. CONCLUSION: CTC status may have a significant impact on early BC management.

heterogeneity of dna ploidy in endometrial carcinoma: comparison of different tissue samples obtained during diagnosis and treatment.

AIM: Comparison of DNA ploidy status of different tumour tissue samples (fresh/frozen vs. paraffin-embedded; curettage vs. hysterectomy samples) obtained during diagnosis and treatment of patients with endometrial carcinoma. PATIENTS AND METHODS: DNA ploidy status and conventional prognostic parameters were recorded for 74 patients with endometrial carcinoma prospectively. RESULTS: In 59 (79.7%) patients the DNA status was described as diploid in all analyzed tissue samples. The remaining 15 (20.3%) cases were described as DNA aneuploid in at least one of the corresponding tissue samples. The concordance between DNA ploidy status in fresh vs. paraffin-embedded hysterectomy samples as well as curettage vs. hysterectomy paraffin-embedded samples was high (kappa coefficient κ=0.6348, 95% confidence interval CI=0.3673-0.9023, and p=0.6408, 95% CI=0.3977-0.8838), however, the methods are not interchangeable. CONCLUSION: The DNA ploidy discordance observed in our study group seems to document intratumoral heterogeneity that should be expected when applying DNA ploidy status in the clinical management of endometrial carcinoma.

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.

Tail spontaneous metastatic mouse model: comparison of metastatic potential of orthotopic and heterotopic models imaged by GFP and RFP protein.

Studies over the past decade have clearly shown that s.c. implant of primary and cultured tumor cells rarely leads to the occurrence of metastatic disease. Orthotopic transplantation of cell suspensions, surgical orthotopic implantation (SOI) of cancer tissue fragments resulted in metastases in many cancer types reaching 100% successful rate. We compared two metastatic models - heterotopic model of Lewis lung cancer and orthotopic B16 mouse melanoma. Both models were syngeneic with high metastatic ratio in C57BL/6 mice after transplantation of cancer cells, by injection into subcutaneous region of mice tail and without surgical intervention. The conclusion is that the localisation of cancer cell injection is a crucial condition for metastatic potential. The site with 100% haematogenous and lymph metastasis rate, after simple injection of cancer cells only, has been defined in mice, without dependence on the genetically predisposition and tumor cell line.

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.

Circulating human prostate cancer cells from an orthotopic mouse model rapidly captured by immunomagnetic beads and imaged by GFP expression.

Circulating tumor cells (CTCs) are potential precursors of metastasis. They are also of use in diagnosing malignancy and for prognostic purposes. Our laboratory has previously isolated CTCs from orthotopic nude mouse models of human prostate cancer cells where the PC-3 cancer cells express green fluorescent protein (GFP). It was found that orthotopic tumors produced CTCs and not subcutaneous tumors, which may explain why orthotopic tumors metastasize and subcutaneous tumors do not. However, in this previous study, CTCs were observed only after culture. In the present study, using the GFP-expressing PC-3 orthotopic model and immunomagnetic beads coated with anti-epithelial cell adhesion molecule (EpCAM) and anti-prostate specific membrane antigen (PSMA), GFP-expressing CTC were isolated within 15 minutes and were readily visualized by GFP fluorescence. It was possible to immediately place the immunomagnetic-bead-captured GFP-expressing PC-3 CTCs in 3-dimensional sponge cell culture, where they proliferated. The combination of GFP expression and the use of immunomagnetic beads is a very powerful method to obtain CTCs for either immediate analysis or for biological characterization in vivo or in 3-dimensional culture.

Cimetidine: an anticancer drug?

Cimetidine, H(2) receptor antagonists, is commonly prescribed for gastric and duodenal ulcer disease. Additionally, cimetidine has been shown to have anticancer effects. This review describes the mechanism of antitumor action of cimetidine including its ability to interfere with tumor cell adhesion, angiogenesis and proliferation; its effect on the immune system; as well as inhibition of postoperative immunosuppression. Its anticancer effect is also compared to that of the other H(2) receptor antagonists as well as outcomes of cimetidine in clinical studies in cancer patients.

A clinically relevant, syngeneic model of spontaneous, highly metastatic B16 mouse melanoma.

We report a syngeneic model of spontaneous metastatic B16-F10 mouse melanoma in C57/BL6 mice with a very high metastatic frequency that mimics clinical metastatic melanoma. The B16 melanoma cells were injected between the skin and cartilage on the dorsal side of the ear. The model generated lymphatic and visceral metastases in all of the tested animals. In mice with large primary tumors, tumor weight correlated with the tumor growth time and also with the number of metastases in lymph nodes and organs. The dorsal ear space between the skin and cartilage enables both lymphatic and hematogenous metastatic spread. The model should be useful to study the mechanism of melanoma metastasis and to develop therapy for this currently untreatable disease.

Autoimmune diabetes mellitus with adult onset and type 1 diabetes mellitus in children have different genetic predispositions.

Type 1 diabetes with manifestation after 35 years of age is defined by CP <200 pmol/L and institution of insulin therapy within 6 months after diagnosis. Latent autoimmune diabetes mellitus in adults (LADA) manifesting after 35 years of age is defined by minimum 6 months after diagnosis without insulin therapy and C peptide (CP) >200 pmol/L and antiGAD > 50 ng/mL. We aimed to find a possible genetic discrimination among different types of autoimmune diabetes. To accomplish this goal, we analyzed DNA samples from 31 LADA patients, 75 patients with adult-onset type 1 diabetes mellitus, 188 type 1 diabetic children, and 153 healthy adult individuals. We studied five genetic loci on chromosomes 6, 11, 4, and 14: HLA DRB1 and DQB1 alleles, major histocompatibility complex (MHC) class I-related gene-A (MIC-A) microsatellite polymorphism, interleukin (IL)-18 single nucleotide polymorphism, the microsatellite polymorphism of nuclear factor kappa B gene (NF-kappaB1), and the single nucleotide polymorphism of a gene for its inhibitor (NF-kappaBIA). HLA-DR3 was detected as the predisposition allele for LADA (OR = 4.94, P < 0.0001). Further we found a statistically significant increase of NF-kappaBIA AA genotype (OR = 2.68, P < 0.01). On the other hand, DRB1*04, which is linked with DQB1*0302, was observed as a risk factor in patients with type 1 diabetes mellitus (T1DM) onset after 35 years of age (OR = 10.47, P < 0.0001 and OR = 9.49, P < 0.0001, respectively). There was also an association with MIC-A5.1 (OR = 2.14, P < 0.01). Statistically significant difference was found in the distribution of IL-18 promoter -607 (C/A) polymorphism between LADA and T1DM in adults (P < 0.01). We conclude that all subgroups of autoimmune diabetes have partly different immunogenetic predisposition.

NFkappaB and its inhibitor IkappaB in relation to type 2 diabetes and its microvascular and atherosclerotic complications.

Nuclear factor kappa B (NFkappaB) is an important transcription factor that together with its inhibitor (IkappaB) participates in the activation of genes involved in immune responses. We examined the CA repeat polymorphism of the NFKB1 gene (encoding for NFkappaB) and A/G point variation in the 3'UTR region of the nuclear factor kappa B inhibitor alpha (NFKBIA) gene (encoding for IkappaB) in Czech and German patients with type 2 diabetes. The sample consisted of 211 patients, both with and without kidney complications, and 159 controls. Additionally, 152 patients with systemic lupus erythematosus (SLE) were genotyped for NFKBIA polymorphism. We observed a significant increase in the homozygous AA genotype of the NFKBIA gene when compared with the control group (the highest value was in diabetics without diabetic nephropathy [p(c)* = 0.0015, odds ratio = 3.59]). No differences were seen between the SLE and control groups. With regard to the polymorphism of the NFKB1 gene, we did not observe any significant differences between the groups. Since the AA genotype of the NFKBIA gene presents a risk for type 2 diabetes development but not for diabetic nephropathy alone, we believe that the NFkappaB gene polymorphism can influence the pathogenesis of diabetes mellitus and affect its complications. Negative findings relative to other inflammatory autoimmune diseases, such as SLE, suggest a specific relationship between NFkappaB and type 2 diabetes mellitus.

Gene therapy of the ischemic lower limb--Therapeutic angiogenesis.

The limitations of surgical revascularisation and pharmacological treatment in peripheral arterial occlusive disease (PAOD) are well recognized. Therapeutic options for critical leg ischemia are consequently limited to percutaneous transluminal angioplasty (PTA) or surgical revascularisation. Unfortunately, many patients with critical leg ischemia are poor candidates for either procedure. Therapeutic angiogenesis is a novel promising tool to treat these patients. Experimental and clinical and trials of gene transfer for therapeutic angiogenesis have already shown some clinical efficacy. This review is focused on gene transfer techniques in preclinical and clinical therapeutic angiogenesis, angiogenic growth factors, vectors, delivery methods and routes. The results of clinical and experimental studies, safety and side effects of gene therapy, and the perspectives of future research are also discussed.

Streptokinase increases the sensitivity of colon cancer cells to chemotherapy by gemcitabine and cis-platine in vitro.

The aim was to determine the effect of fybrinolytic therapy by streptokinase on chemotherapy and radiation response in human colon cancer cells. The cells were treated with different concentrations of gemcitabine, cis-platine and streptokinase, at a single use or in combinations. Radiation was tested at a dose 0.5, 5 and 15 Gy in three different schedules. The chemotherapy showed higher cytotoxic effect in combination with streptokinase. On the other hand, the combination of chemotherapy with streptokinase and radiotherapy provide no improvement in sensitivity of cancer cells to treatment. The data suggest that fybrinolytic therapy could influence the effect of chemotherapy.

Interleukin IL-18 gene promoter polymorphisms in adult patients with type 1 diabetes mellitus and latent autoimmune diabetes in adults.

Interleukin-18 (IL-18) gene promoter polymorphism is known as a genetic risk factor for child type 1 diabetes mellitus development. To test the role of IL-18 gene polymorphism in predisposition to adult type 1 diabetes (T1DM) and latent autoimmune diabetes in adults (LADA), we analysed SNPs at position -607 (C/A) and -137 (G/C) in the promoter region of IL-18 gene by sequence-specific PCR in 49 T1DM, 66 LADA patients and 139 healthy controls. We found differences in allele, genotype or haplotype distribution in tested patients when compared to frequencies found in control group but these differences did not reach statistical significance. However, there was a difference in -607 (C/A) allele and genotype distribution found in LADA and T1DM patients that reached statistical significance. These results suggest that the IL-18 gene promoter polymorphisms are not associated with adult type 1 diabetes or LADA susceptibility, and according to our findings genes involved in onset and progression of LADA and T1DM are probably different.

Development of a green fluorescent protein metastatic-cancer chick-embryo drug-screen model.

The chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents.