Systemic chemotherapy-induced microsatellite instability in the mononuclear cell fraction of women with breast cancer can be reproduced in vitro and abrogated by amifostine

Objectives Microsatellite instability (MSI) induction by alkylating agent-based chemotherapy (ACHT) may underlie both tumor resistance to chemotherapy and secondary leukaemias in cancer patients. We investigated if ACHT could induce MSI in tumor-derived plasma-circulating DNA (pfDNA) and in normal peripheral blood mononuclear (PBMN) cells. We also evaluated if amifostine could interfere with this process in an in-vitro model. Methods MSI was determined in pfDNA, PBMN cells and urine cell-free DNA (ufDNA) of 33 breast cancer patients before and after ACHT. MCF-7 cells and PBMN from normal donors were exposed in vitro to melphalan, with or without amifostine. Results We observed at least one MSI event in PBMN cells, pfDNA or ufDNA of 87, 80 and 80% of patients, respectively. In vitro, melphalan induced MSI in both MCF-7 and normal PBMN cells. In PBMN cells, ACHT-induced MSI occurred together with a significant decrease in the expression of the DNA mismatch repair gene hMSH2. Amifostine decreased hMSH2 expression and also prevented MSI induction only in normal PBMN cells. Conclusions ACHT induced MSI in PBMN cells and in tumour-derived pfDNA. Because of its protective effect against ACHT induction of MSI in normal PBMN cells in vitro, amifostine may be a potential agent for preventing secondary leukaemias in patients exposed to ACHT.

DOG1 for the Diagnosis of Gastrointestinal Stromal Tumor (GIST): Comparison Between 2 Different Antibodies

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. Discovered on GIST-1 (DOG1) is a recently described protein expressed in GISTs irrespective of mutation status. The aim of this study was to investigate the immunohistochemical expression of DOG1 using 2 different monoclonal antibodies (DOG1.1 and the commercially available K9 antibody) in 668 GIST cases and to compare the results with the expression of KIT. DOG1 and KIT expression also were studied in most human normal tissues and several nonmesenchymal and mesenchymal tumors other than GIST. KIT was expressed in 643 (96.3%) GISTs. DOG1.1 and K9 were positive in 538 (80.5%) and 642 (96.1%) GIST cases, respectively. In 25 (3.7%) KIT-negative GIST cases, DOG1 was expressed in 5 (20.0%) and 19 (76.0%) using DOG1.1 and K9 antibodies, respectively. Only 0.9% of GISTs were negative for KIT, DOG1.1, and K9. Most normal human tissues did not reveal KIT and DOG1 expression. DOG1.1 was positive in only 2 of 57 synovial sarcomas and 1 of 61 soft tissue leiomyosarcomas. K9 was positive in 5 of 57 synovial sarcomas, 1 of 14 angiosarcomas, 1 of 61 soft tissue leiomyosarcomas, 3 of 4 adenoid cystic carcinomas of the head and neck, and in myoepithelial cells of 9 of 11. broadenomas of the breast. In conclusion, the commercially available K9 is of great utility for the diagnosis of most KIT-negative GISTs, and the combination of both KIT and K9 antibody in a panel of immunohistochemistry can define the diagnosis of GIST in more than 99% of cases.

E-cadherin and beta-catenin Loss of Expression Related to Bone Metastasis in Prostate Cancer

Objectives: E-cadherin and beta-catenin are adhesion molecules responsible for the maintenance of normal epithelial cell phenotype. A disturbance in epithelial cell adhesion, which leads to a more invasive and metastatic phenotype, is a hallmark of tumor progression. Several immunohistochemical studies have reported a strong correlation between loss of their expression to higher stage and grade in prostate carcinoma, but their influence in metastatic process is not yet known. The aim of this study is to verify the role of adhesion molecules in the progression of prostate cancer (PC), assessing the expression of E-cadherin and beta-catenin in bone metastasis. Materials and Methods: Twenty-eight bone metastases of prostate carcinoma were submitted to immunohistochemistry analysis for E-cadherin and beta-catenin expression. In 6 patients, we were able to assess the expression of the adhesion molecules in the primary tumors and their respective metastases. The definition of normal expression for both antibodies was strong and diffuse expression in more than 70% of tumor cells. Results: In bone metastases, there was loss of expression of E-cadherin and beta-catenin in 86% and 82%, respectively. Among the primary tumors, E-cadherin and beta-catenin expression was normal in 83% and 50% cases, respectively. Considering the 6 patients with paired primary and bone metastasis, we found loss of expression for both E-cadherin and beta-catenin in most of the cases. Conclusions: Comparing primary PC and its metastasis, we showed persistent loss of E-cadherin and beta-catenin expression. This phenomenon may be related to metastatic potential in PC, because we have shown underexpression for E-cadherin and beta-catenin in 86% and 82% of bone metastases.

Laparoscopic Enucleation of Liver Tumors. Corkscrew Technique Revisited

Background: Enucleation of small lesions located near the hepatic surface can be achieved with low morbidity and mortality. This article describes a simple laparoscopic technique for enucleation of liver tumors. Methods: After inspection and intraoperative ultrasonography, Glisson`s capsule is marked with eletrocautery 2 cm away from the tumor margin. Ultrasonography is used to ascertain surgical margin right before liver transection. Hemihepatic ischemia is applied and marked area is anchored by stitches. The suture is held together by metallic clips and upward traction is performed, facilitating the transection of the parenchyma and correct identification of vascular and biliary structures. Results: This technique has been successfully employed in six consecutive patients. There were four men and two women, mean age 50.3 years. Four patients underwent liver resection for malignant disease and two for benign liver neoplasm. Pathologic surgical margins were free in all cases and mean hospital stay was 2 days. No postoperative mortality was observed. Conclusion: This technique may facilitate laparoscopic nonanatomical liver resection and reduce risk of positive surgical margins. It is also useful in combination with anatomical laparoscopic liver resections such as right or left hemihepatectomies in patients with bilateral liver tumors as occurred in one of our patients.

Expression profiles of the glucose-dependent insulinotropic peptide receptor and LHCGR in sporadic adrenocortical tumors

Glucose-dependent insulinotropic peptide receptor (GIPR) and LHCGR are G-protein-coupled receptors with a wide tissue expression pattern. Aberrant expression of these receptors has rarely been demonstrated in adult sporadic adrenocortical tumors with a lack of data on pediatric tumors. We quantified the GIPR and LHCGR expression in a large cohort of 55 patients (25 children and 30 adults) with functioning and non-functioning sporadic adrenocortical tumors. Thirty-eight tumors were classified as adenomas whereas 17 were carcinomas. GIPR, and LHCGR expression were analyzed by real-time PCR and normal human pancreatic and testicular tissue samples were used as positive controls. Mean expression values were determined by fold increase in comparison with a normal adrenal pool. GIPR mRNA levels were significantly higher in adrenocortical carcinomas than in adenomas from both pediatric and adult groups. LHCGR expression was similar in both carcinomas and adenomas from the pediatric group but significantly lower in carcinomas than in adenomas from the adult group (median 0.06 and 2.3 respectively, P<0.001). GIPR was detected by immunohistochemistry in both pediatric and adult tumors. Staining and real-time PCR results correlated positively only when GIPR in RN A levels were increased at least two-fold in comparison with normal adrenal expression levels. In Conclusion, GIPR overexpression was observed in pediatric and adult adrenocortical tumors and very low levels of LHCGR expression were found in all adult adrenocortical carcinomas.

Use of cholesterol-rich nanoparticles that bind to lipoprotein receptors as a vehicle to paclitaxel in the treatment of breast cancer: pharmacokinetics, tumor uptake and a pilot clinical study

Purpose In animal experiments paclitaxel oleate associated with a cholesterol-rich nanoemulsion concentrated in the neoplastic tissues and showed reduced toxicity and increased antitumor activity compared with paclitaxel-Cremophor EL. Here, a clinical study was performed in breast cancer patients to evaluate the tumoral uptake, pharmacokinetics and toxicity of paclitaxel associated to nanoemulsions. Methods Twenty-four hours before mastectomy [(3)H]paclitaxel oleate associated with [(14)C]-cholesteryl oleatenanoemulsion or [(3)H]- paclitaxel in Cremophor EL were injected into five patients for collection of blood samples and fragments of tumor and normal breast tissue. A pilot clinical study of paclitaxel-nanoemulsion administered at 3-week intervals was performed in four breast cancer patients with refractory advanced disease at 175 and 220 mg/m(2) dose levels. Results T(1/2) of paclitaxel oleate associated to the nanoemulsion was greater than that of paclitaxel (t(1/2) = 15.4 +/- 4.7 and 3.5 +/- 0.80 h). Uptake of the [(14)C]-cholesteryl ester nanoemulsion and [(3)H]- paclitaxel oleate by breast malignant tissue was threefold greater than the normal breast tissue and toxicity was minimal at the two dose levels. Conclusions Our results suggest that the paclitaxel-nanoemulsion preparation can be advantageous for use in the treatment of breast cancer because the pharmacokinetic parameters are improved, the drug is concentrated in the neoplastic tissue and the toxicity of paclitaxel is reduced.

Change in blood flow velocity demonstrated by Doppler ultrasound in upper limb after axillary dissection surgery for the treatment of breast cancer

The aim of this study was to evaluate the arterial and venous blood flow in women who underwent upper limb axillary dissection surgery for the treatment of breast cancer. Sixty women were divided into two groups: group 1 (G1)-30 women who underwent breast surgery with axillary dissection level II or III (55.6 +/- A 8.6 years); group 2 (G2)-control, 30 women with no breast cancer (57.4 +/- A 7.0 years). Blood flow profile was evaluated by a continuous wave ultrasound Doppler device (Nicolet Vascular Versalab SE(A (R))) with an 8 MHz probe. Axillary, brachial arteries and veins, arm circumference, volumes, and the ankle-brachial index (ABI) were examined. Wilcoxon test and Mann-Whitney tests were applied to analyze blood flow velocity intra-group and between G1 and G2, respectively. The G1 results showed no lymphedema and no peripheral arterial disease (ABI > 0.9). Moreover, the mean blood flow velocity of the vessels ipsilateral to the surgery was significantly higher than the contralateral ones for all vessels examined (P < 0.05). The mean velocity of blood flow of the vessels contralateral to surgery was significantly higher than the axillary artery in G2 (P < 0.05). It can be concluded that women who underwent axillary dissection due to breast cancer showed probable stenosis in the arterial and venous axillary and brachial vessels of the upper limb ipsilateral to the surgery, confirmed by the increase of blood flow velocity, and such obstruction might affect the limb contralateral to the operation site.

Surgical management of glomus jugulare tumors: a proposal for approach selection based on tumor relationships with the facial nerve Clinical article

Object. The goal of this paper is to analyze the extension and relationships of glomus jugulare tumor with the temporal bone and the results of its surgical treatment aiming at preservation of the facial nerve. Based on the tumor extension and its relationships with the facial nerve, new criteria to be used in the selection of different surgical approaches are proposed. Methods. Between December 1997 and December 2007, 34 patients (22 female and 12 male) with glomus jugulare tumors were treated. Their mean age was 48 years. The mean follow-up was 52.5 months. Clinical findings included hearing loss in 88%, swallowing disturbance in 50%, and facial nerve palsy in 41%. Magnetic resonance imaging demonstrated a mass in the jugular foramen in all cases, a mass in the middle ear in 97%, a cervical mass in 85%, and an intradural mass in 41%. The tumor was supplied by the external carotid artery in all cases, the internal carotid artery in 44%, and the vertebral artery in 32%. Preoperative embolization was performed in 15 cases. The approach was tailored to each patient, and 4 types of approaches were designed. The infralabyrinthine retrofacial approach (Type A) was used in 32.5%; infralabyrinthine pre- and retrofacial approach without occlusion of the external acoustic meatus (Type B) in 20.5%; infralabyrinthine pre- and retrofacial approach with occlusion of the external acoustic meatus (Type C) in 41 W. and the infralabyrinthine approach with transposition of the facial nerve and removal of the middle ear structures (Type D) in 6% of the patients. Results. Radical removal was achieved in 91% of the cases and partial removal in 9%. Among 20 patients without preoperative facial nerve dysfunction, the nerve was kept in anatomical position in 19 (95%), and facial nerve function was normal during the immediate postoperative period in 17 (85%). Six patients (17.6%) had a new lower cranial nerve deficit, but recovery of swallowing function was adequate in all cases. Voice disturbance remained in all 6 cases. Cerebrospinal fluid leakage occurred in 6 patients (17.6%), with no need for reoperation in any of them. One patient died in the postoperative period due to pulmonary complications. The global recovery, based on the Karnofsky Performance Scale (KPS), was 100% in 15% of the patients, 90% in 45%, 80% in 33%, and 70% in 6%. Conclusions. Radical removal of glomus jugulare tumor can be achieved without anterior transposition of the facial nerve. The extension of dissection, however, should be tailored to each case based on tumor blood supply, preoperative symptoms, and tumor extension. The operative field provided by the retrofacial infralabyrinthine approach, or the pre- and retrofacial approaches. with or without Closure of the external acoustic meatus, allows a wide exposure of the jugular foramen area. Global functional recovery based on the KPS is acceptable in 94% of the patients. (DOI: 10.3171/2008.10.JNS08612)

Gene expression profile analysis of primary glioblastomas and non-neoplastic brain tissue: identification of potential target genes by oligonucleotide microarray and real-time quantitative PCR

The prognosis of glioblastomas is still extremely poor and the discovery of novel molecular therapeutic targets can be important to optimize treatment strategies. Gene expression analyses comparing normal and neoplastic tissues have been used to identify genes associated with tumorigenesis and potential therapeutic targets. We have used this approach to identify differentially expressed genes between primary glioblastomas and non-neoplastic brain tissues. We selected 20 overexpressed genes related to cell cycle, cellular movement and growth, proliferation and cell-to-cell signaling and analyzed their expression levels by real time quantitative PCR in cDNA obtained from microdissected fresh tumor tissue from 20 patients with primary glioblastomas and from 10 samples of non-neoplastic white matter tissue. The gene expression levels were significantly higher in glioblastomas than in non-neoplastic white matter in 18 out of 20 genes analyzed: P < 0.00001 for CDKN2C, CKS2, EEF1A1, EMP3, PDPN, BNIP2, CA12, CD34, CDC42EP4, PPIE, SNAI2, GDF15 and MMP23b; and NFIA (P: 0.0001), GPS1 (P: 0.0003), LAMA1 (P: 0.002), STIM1 (P: 0.006), and TASP1 (P: 0.01). Five of these genes are located in contiguous loci at 1p31-36 and 2 at 17q24-25 and 8 of them encode surface membrane proteins. PDPN and CD34 protein expression were evaluated by immunohistochemistry and they showed concordance with the PCR results. The present results indicate the presence of 18 overexpressed genes in human primary glioblastomas that may play a significant role in the pathogenesis of these tumors and that deserve further functional investigation as attractive candidates for new therapeutic targets.

MicroRNAs Differentially Expressed in ACTH-Secreting Pituitary Tumors

Context: MicroRNAs (miRNAs) are small noncoding RNAs, functioning as antisense regulators of gene expression by targeting mRNA and contributing to cancer development and progression. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites of the genome. Objective: The aim of the study was to analyze the differential expression of let-7a, miR-15a, miR-16, miR-21, miR-141, miR-143, miR-145, and miR-150 in corticotropinomas and normal pituitary tissue and verify whether their profile of expression correlates with tumor size or remission after treatment. Material and Methods: ACTH-secreting pituitary tumor samples were obtained during transphenoidal surgery from patients with Cushing disease and normal pituitary tissues from autopsies. The relative expression of miRNAs was measured by real-time PCR using RNU44 and RNU49 as endogenous controls. Relative quantification of miRNA expression was calculated using the 2(-Delta Delta Ct) method. Results: We found underexpression of miR-145 (2.0-fold; P = 0.04), miR-21 (2.4-fold; P = 0.004), miR-141 (2.6-fold; P = 0.02), let-7a (3.3-fold; P = 0.003), miR-150 (3.8-fold; P = 0.04), miR-15a (4.5-fold; P = 0.03), miR-16 (5.0-fold; P = 0.004), and miR-143 (6.4-fold; P = 0.004) in ACTH-secreting pituitary tumors when compared to normal pituitary tissues. There were no differences between miRNA expression and tumor size as well as miRNA expression and ratio of remission after surgery, except in patients presenting lower miR-141 expression who showed a better chance of remission. Conclusion: Our results support the possibility that altered miRNA expression profile might be involved in corticotrophic tumorigenesis. However, the lack of knowledge about miRNA target genes postpones full understanding of the biological functions of down-regulated or up-regulated miRNAs in corticotropinomas. (J Clin Endocrinol Metab 94: 320-323, 2009)

The A61 G EGF polymorphism is associated with development of extraaxial nervous system tumors but not with overall survival

Epidermal growth factor can activate several signaling pathways, leading to proliferation, differentiation, and tumorigenesis of epithelial tissues by binding with its receptor. The EGF protein is involved in nervous system development, and polymorphisms in the EGF gene on chromosome band 4q25 are associated with brain cancers. The purpose of this study was to investigate the association between the single-nucleotide polymorphism of EGF + 61 G/A and extraaxial brain tumors in a population of the southeast of Brazil. We analyzed the genotype distribution of this polymorphism in 90 patients and 100 healthy subjects, using the polymerase chain reaction restriction fragment length polymorphism technique. Comparison of genotype distribution revealed a significant difference between patients and control subjects (P < 0.001). The variant genotypes of A/G and G/G were associated with a significant increase of the risk of tumor development, compared with the homozygote A/A (P < 0.0001). When the analyses were stratified, we observed that the genotype GIG was more frequent in female patients (P = 0.021). The same genotype was observed more frequently in patients with low-grade tumors (P = 0.001). Overall survival rates did not show statistically significant differences. Our data suggest that the EGF A61 G polymorphism can be associated with susceptibility to development of these tumors. (C) 2010 Elsevier Inc. All rights reserved.

Polymorphisms of Lewis and Secretor genes are related to breast cancer and metastasis in axillary lymph nodes

ABH and Lewis antigen expression has been associated with cancer development and prognosis, tumor differentiation, and metastasis. Considering that invasive ductal breast carcinoma (IDC) presents multiple molecular alterations, the aim of the present study was to determine whether the polymorphism of ABO, Lewis, and Secretor genes, as well as ABO phenotyping, could be associated with tumor differentiation and lymph nodes metastasis. Seventy-six women with IDC and 78 healthy female blood donors were submitted to ABO phenotyping/genotyping and Lewis and Secretor genotyping. Phenotyping was performed by hemagglutination and genotyping by the polymerase chain reaction with sequence-specific primers. ABO, Lewis, and Secretor genes were classified by individual single nucleotide polymorphism at sites 59, 1067, 202, and 314 of the Lewis gene, 428 of the Secretor gene, and 261 (O1 allele), 526 (O2 and B allele), and 703 (B allele). No association was found between breast cancer and ABO antigen expression (P = 0.9323) or genotype (P = 0.9356). Lewis-negative genotype was associated with IDC (P = 0.0126) but not with anatomoclinical parameters. Nonsecretor genotype was associated with axillary lymph node metastasis (P = 0.0149). In conclusion, Lewis and Secretor genotyping could be useful to predict respectively breast cancer susceptibility and axillary lymph nodes metastasis.

Basal levels of DNA damage detected by micronuclei and comet assays in untreated breast cancer patients and healthy women

Breast cancer is the second most frequent type of cancer worldwide and is the most common malignant disease among women. Risk factors for breast cancer include early menarche, late menopause, hormonal therapies, exposure to environmental pollutants, smoking and alcohol use. However, increased or prolonged exposure to estrogen is the most important risk factor. It has been suggested that accumulation of DNA damage may contribute to breast carcinogenesis. Epidemiological studies suggest that cytogenetic biomarkers such as micronuclei in peripheral blood lymphocytes may predict cancer risk because they indicate genomic instability in target tissues. The objective of the present study was to evaluate the frequencies of micronuclei and the extent of DNA damage detected by comet assay in peripheral blood lymphocytes of untreated breast cancer patients and healthy women. The study was conducted using peripheral blood lymphocytes from 45 women diagnosed for Ductal ""in situ"" or invasive breast carcinoma and 85 healthy control women. Micronuclei and comet assays were performed to detect spontaneous DNA damage. The results showed that micronuclei frequencies and tail intensity, detected by comet assay, were significantly higher in the breast cancer group than in controls. The levels of DNA damage were similar in smokers and non-smokers, and aging did not influence the frequencies of micronuclei or tail intensity values observed in either group. In conclusion, the present work demonstrates higher levels of DNA damage in untreated breast cancer patients than in healthy women.

Determination of Cyclophosphamide Enantiomers in Plasma by LC-MS/MS: Application to Pharmacokinetics in Breast Cancer and Lupus Nephritis Patients

This article describes the enantioseleclive analysis of cyclophosphamide (CPA) in human plasma using LC-MS/MS. CPA enantiomers were extracted from plasma using a mixture of ethyl acetate and chloroform (75:25, v/v). The enantiomers were separated on a Chiralcel(R) OD-R column, with the mobile phase consisting of a mixture of acetonitrile and water (75:25, v/v) plus 0.2% formic acid. The protonaled ions and their respective product ions were monitored using two functions, 261 > 141 for CPA enantiomers and 189 > 104 for the internal standard (antipyrine). Recovery rates were higher than 95% and the quantification limit was 2.5-ng/ml plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of intra- and interassay precision and accuracy were less than 10%. The method was applied for the investigation of the enantioselective pharmacokinetics of CPA in a lupus nephritis patient treated with 1 g CPA infused over 2 h and in a breast cancer patient treated with 0.9 g infused over 1 h. No stereoselectivity in the pharmacokinetic parameters was observed for either patient. Clearance values of 2.63 and 2.93 l/h and of 3.36 and 3.61 l/h for (-)-(S) and (+)-(R)-CPA were obtained for the breast cancer and lupus nephritis patient., respectively. Chirality 21:383-389, 2009. (C) 2008 Wiley-Liss, Inc.

Breast conserving surgery after neoadjuvant therapy for large primary breast cancer

Aim: The aim of this study was to evaluate the safety of breast conserving surgery ill patients with breast tumours satisfactorily downstaged after neoadjuvant therapy. Methods: A retrospective cohort study was undertaken to analyze the loco-regional recurrence (LRR) after breast conserving surgery. We enrolled 88 patients with breast cancer subjected to neoadjuvant therapy (NAT group) who achieved an objective response due to neoadjuvant treatment and compared them with 191 patients with early breast cancer (EBC group) who were submitted to primary conserving surgery. Lumpectomy or quadrantectomy with axillary lymph node dissection was performed in all patients who received adjuvant radiotherapy. Systemic adjuvant therapy was offered to all patients. The mean periods of observation were 61.3 months in the NAT group and 67.5 months in the EBC group. Results: The mean age was 53 years in the NAT group and 56 years in the EBC group (p = 0.04). There was no histological type and histological grade difference between groups. In the NAT group, the mean diameter of residual tumour was lower and the mean volume of breast tissue resection was higher than in the EBC group (p = 0.01 and p = 0.002, respectively). The ipsilateral recurrence rate was 7.9% in the NAT group and 7.8% in the EBC group (p = 0.9). The most important predictive factor of recurrence in the NAT group was the age of patient. Conclusion: Breast conserving therapy is a safe procedure in satisfactorily downstaged breast cancer after neoadjuvant therapy. (c) 2008 Elsevier Ltd. All rights reserved.