Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection

Because CD4(+) T cells play a key role in aiding cellular immune responses, we wanted to assess whether increasing numbers of gene-engineered antigen-restricted CD4(+) T cells could enhance an antitumor response mediated by similarly gene-engineered CD8(+) T cells. In this study, we have used retroviral transduction to generate erbB2-reactive mouse T-cell populations composed of various proportions of CD4(+) and CD8(+) cells and then determined the antitumor reactivity of these mixtures. Gene-modified CD4(+) and CD8(+) T cells were shown to specifically secrete Tc1 (T cytotoxic-1) or Tc2 cytokines, proliferate, and lyse erbB2(+) tumor targets following antigen ligation in vitro. In adoptive transfer experiments using severe combined immunodeficient (scid) mice, we demonstrated that injection of equivalent numbers of antigen-specific engineered CD8(+) and CD4(+) T cells led to significant improvement in survival of mice bearing established lung metastases compared with transfer of unfractionated (largely CD8(+)) engineered T cells. Transferred CD4(+) T cells had to be antigen-specific (not just activated) and secrete interferon gamma (IFN-gamma) to potentiate the antitumor effect. Importantly, antitumor responses in these mice correlated with localization and persistence of gene-engineered T cells at the tumor site. Strikingly, mice that survived primary tumor challenge could reject a subsequent re-challenge. Overall, this study has highlighted the therapeutic potential of using combined transfer of antigen-specific gene-modified CD8(+) and CD4(+) T cells to significantly enhance T-cell adoptive transfer strategies for cancer therapy.

CLINICAL TUMOR DIMENSIONS MAY BE USEFUL TO PREVENT GEOGRAPHIC MISS IN CONVENTIONAL RADIOTHERAPY OF UTERINE CERVIX CANCER-A MAGNETIC RESONANCE IMAGING-BASED STUDY

Purpose: To evaluate the risk of geographic miss associated with the classic four-field ""box"" irradiation technique and to define the variables that predict this risk. Materials and Methods: The study population consisted of 80 patients with uterine cervix cancer seen between 2001 and 2006. Median age was 55 years (23-82 years), and 72 (90%) presented with squamous cell carcinoma. Most patients (68.7%) presented with locally advanced disease (IIb or more). Magnetic resonance imaging findings from before treatment were compared with findings from simulation of the conventional four-field ""box"" technique done with rectal contrast. Study variables included tumor volume; involvement of vagina, parametrium, bladder, or rectum; posterior displacement of the anterior rectal wall; and tumor anteroposterior diameter (APD). Margins were considered adequate when the target volume (primary tumor extension, whole uterine body, and parametrium) was included within the field limits and were at least 1 cm in width. Results: Field limits were inadequate in 45 (56%) patients: 29 (36%) patients at the anterior and 28 (35%) at the posterior border of the lateral fields. Of these, 12 patients had both anterior and posterior miss, and this risk was observed in all stages of the disease (p = 0.076). Posterior displacement of the anterior rectal wall beyond S2-S3 was significantly correlated with the risk of geographic miss (p = 0.043). Larger tumors (APD 6 cm or above and volume above 50 cm(3)) were also significantly correlated with this risk (p = 0.004 and p = 0.046, respectively). Conclusions: Posterior displacement of the anterior rectal wall, tumor APD, and volume can be used as guidance in evaluating the risk of geographic miss. (C) 2009 Elsevier Inc.

Use of a Minimally Invasive Uncalibrated Cardiac Output Monitor in Patients Undergoing Cesarean Section under Spinal Anesthesia: Report of Four Cases

Bliacheriene F, Carmona MJC, Barretti CFM, Haddad CMF, Mouchalwat ES, Bortlotto MRFL, Francisco RPV, Zugaib M - Use of a Minimally Invasive Uncalibrated Cardiac Output Monitor in Patients Undergoing Cesarean Section under Spinal Anesthesia: Report of Four Cases. Background and Objectives: Hemodynamic changes are observed during cesarean section under spinal anesthesia. Non-invasive blood pressure (BP) and heart rate (HR) measurements are performed to diagnose these changes, but they are delayed and inaccurate. Other monitors such as filling pressure and cardiac output (CO) catheters with external calibration are very invasive or inaccurate. The objective of the present study was to report the cardiac output measurements obtained with a minimally invasive uncalibrated monitor (LiDCO rapid) in patients undergoing cesarean section under spinal anesthesia. Case report: After approval by the Ethics Commission, four patients agreed to participate in this study. They underwent cesarean section under spinal anesthesia while at the same time being connected to the LiDCO rapid by a radial artery line. Cardiac output, HR, and BP were recorded at baseline, after spinal anesthesia, after fetal and placental extraction, and after the infusion of oxytocin and metaraminol. We observed a fall in BP with an increase of HR and CO after spinal anesthesia and oxytocin infusion; and an increase in BP with a fall in HR and CO after bolus of the vasopressor. Conclusions: Although this monitor had not been calibrated, it showed a tendency for consistent hemodynamic data in obstetric patients and it may be used as a therapeutic guide or experimental tool.

Age, invasive ventilatory support and outcomes in elderly patients admitted to intensive care units

Objective: to determine the relationship between age and in-hospital mortality of elderly patients, admitted to ICU, requiring and not requiring invasive ventilatory support. Design: prospective observational cohort study conducted over a period of 11 months. Setting: medical-surgical ICU at a Brazilian university hospital. Subjects: a total of 840 patients aged 55 years and older were admitted to ICU. Methods: in-hospital death rates for patients requiring and not requiring invasive ventilatory support were compared across three successive age intervals (55-64; 65-74 and 75 or more years), adjusting for severity of illness using the Acute Physiologic Score. Results: age was strongly correlated with mortality among the invasively ventilated subgroup of patients and the multivariate adjusted odds ratios increased progressively with every age increment (OR = 1.60, 95% CI = 1.01-2.54 for 65-74 years old and OR = 2.68, 95% CI = 1.58-4.56 for >= 75 years). For the patients not submitted to invasive ventilatory support, age was not independently associated with in-hospital mortality (OR = 2.28, 95% CI = 0.99-5.25 for 65-74 years old and OR = 1.95, 95% CI = 0.82-4.62 for >= 75 years old). Conclusions: the combination of age and invasive mechanical ventilation is strongly associated with in-hospital mortality. Age should not be considered as a factor related to in-hospital mortality of elderly patients not requiring invasive ventilatory support in ICU.

MiR-1 Is a Tumor Suppressor in Thyroid Carcinogenesis Targeting CCND2, CXCR4, and SDF-1 alpha

Context: Micro-RNA have emerged as an important class of short endogenous RNA that act as posttranscriptional regulators of gene expression and are constantly deregulated inhumancancer. MiR-1 has been found down-regulated in lung, colon, and prostate cancer. Objectives: In this study, we investigated the possible role of miR-1 in thyroid carcinogenesis. Design: We have analyzed miR-1 expression in a panel of thyroid neoplasias including benign and malignant lesions and searched for miR-1 targets. Results: Our results show that miR-1 expression is drastically down-regulated in thyroid adenomas and carcinomas in comparison with normal thyroid tissue. Interestingly, miR-1 down-regulation was also found in thyroid hyperproliferative nonneoplastic lesions such as goiters. We identified the CCND2, coding for the cyclin D2 (CCND2) protein that favors the G1/S transition, CXCR4, and SDF-1 alpha genes, coding for the receptor for the stromal cell derived factor-1 (SDF-1)/CXCL12 chemokine and its ligand SDF-1/CXCL12, respectively, as miR-1 targets. An inverse correlation was found between miR-1 expression and CXC chemokine receptor 4 (CXCR4) and SDF-1 alpha protein levels in papillary and anaplastic thyroid carcinomas. Consistent with a role of the CCND2 protein in cell proliferation and CXCR4 and SDF-1 alpha proteins in cell invasion and metastasis, functional studies demonstrate that miR-1 is able to inhibit thyroid carcinoma cell proliferation and migration. Conclusions: These results indicate the involvement of miR-1 in thyroid cell proliferation and migration, validating a role of miR-1 down-regulation in thyroid carcinogenesis. (J Clin Endocrinol Metab 96: E1388-E1398, 2011)

Molecular profiling of isolated histological components of Wilms tumor implicates a common role for the Wnt signaling pathway in kidney and tumor development

Wilms tumor (WT), a tumor composed of three histological components - blastema (BL), epithelia and stroma - is considered an appropriate model system to study the biological relationship between differentiation and tumorigenesis. To investigate molecular associations between nephrogenesis and WT, the gene expression pattern of individual cellular components was analyzed, using a customized platform containing 4,608 genes. WT gene expression patterns were compared to genes regulated during kidney differentiation. BL had a closer gene expression pattern to the earliest stage of normal renal development. The BL gene expression pattern was compared to that of fetal kidney (FK) and also between FK and mature kidney, identifying 25 common de-regulated genes supposedly involved in the earliest events of WT onset. Quantitative RT-PCR was performed, confirming the difference in expression levels for 13 of 16 genes (81.2%) in the initial set and 8 of 13 (61.5%) in an independent set of samples. An overrepresentation of genes belonging to the Wnt signaling pathway was identified, namely PLCG2, ROCK2 and adenomatous polyposis coli (APC). Activation of the Wnt pathway was confirmed in WT, using APC at protein level and PLCG2 at mRNA and protein level. APC showed positive nuclear immunostaining for an independent set of WT samples, similarly to the FK in week 11. Lack of PLCG2 expression was confirmed in WT and in FK until week 18. Taken together, these results provided molecular evidence of the recapitulation of the embryonic kidney by WT as well as involvement of the Wnt pathway in the earliest events of WT onset. Copyright (C) 2008 S. Karger AG, Basel.

NFAT1 transcription factor is central in the regulation of tissue microenvironment for tumor metastasis

Members of the nuclear factor of activated T cell (NFAT) family of transcription factors were originally described in T lymphocytes but later shown to be expressed in several immune and non-immune cell types. NFAT proteins can modulate cellular transformation intrinsically, and NFAT-deficient (NFAT1-/-) mice are indeed more susceptible to transformation than wild-type counterparts. However, the contribution of an NFAT1-/- microenvironment to tumor progression has not been studied. We have addressed this question by inoculating NFAT1-/- mice with B16F10 melanoma cells intravenously, an established model of tumor homing and growth. Surprisingly, NFAT1-/- animals sustained less tumor growth in the lungs after melanoma inoculation than wild-type counterparts. Even though melanoma cells equally colonize NFAT1-/- and wild-type lungs, tumors do not progress in the absence of NFAT1 expression. A massive mononuclear perivascular infiltrate and reduced expression of TGF-beta in the absence of NFAT1 suggested a role for tumor-infiltrating immune cells and the cytokine milieu. However, these processes are independent of an IL-4-induced regulatory tumor microenvironment, since lack of this cytokine does not alter the phenotype in NFAT1-/- animals. Bone marrow chimera experiments meant to differentiate the contributions of stromal and infiltrating cells to tumor progression demonstrated that NFAT1-induced susceptibility to pulmonary tumor growth depends on NFAT1-expressing parenchyma rather than on bone marrow-derived cells. These results suggest an important role for NFAT1 in radio-resistant tumor-associated parenchyma, which is independent of the anti-tumor immune response and Th1 versus Th2 cytokine milieu established by the cancer cells, but able to promote site-specific tumor growth.

Coordinated expression of galectin-3 and galectin-3-binding sites in malignant mammary tumors: implications for tumor metastasis

Galectin-3 is a glycan-binding protein that mediates cell-cell and/or cell-extracellular matrix (ECM) interactions. Although galectin-3 is implicated in the progression of various types of cancers, the mechanisms by which galectin-3 enhances metastasis remain unclear. In order to elucidate the role of galectin-3 in the complex multistage process of cancer metastasis, we examined galectin-3 and galectin-3-binding site expression in a series of 82 spontaneous canine mammary tumors (CMT) and two CMT cell lines. Benign CMT tumors exhibited strong nuclear/cytoplasmic galectin-3 immunostaining, whereas malignant CMT tumors and metastases exhibited dramatically decreased galectin-3 expression with the majority of the immunostaining confined to the cytoplasm. Interestingly, intravascular tumor cells overexpressed galectin-3 regardless of their location. CMT-U27 xenografts displayed the same pattern of galectin-3 expression found in spontaneous malignant CMT. In parallel with the downregulation of galectin-3, malignant CMT displayed an overall loss of galectin-3-binding sites in the ECM and focal expression of galectin-3-binding sites mainly detected in intravascular tumor cells and endothelium. Furthermore, loss of galectin-3-binding sites was correlated with the downregulation of GLT25D1, a beta (1-O) galactosyltransferase that modifies collagen, and upregulation of stromal galectin-1. Finally, GLT25D1 mRNA expression was strikingly downregulated in malignant CMT-U27 compared with the benign cell line, and its expression was further de-creased in a galectin-3 knockdown CMT-U27 cell line. We therefore hypothesized that the loss of galectin-3-binding sites in the ECM in conjunction with the overexpression of galectin-3 in specific tumor cell subpopulations are crucial events for the development of mammary tumor metastases.

Human breast tumor slices: A model for identification of vitamin D regulated genes in the tumor microenvironment

While many studies have addressed the direct effects of 1 alpha,25(OH)(2)D(3) on breast cancer (BC) cells, stromal-epithelial interactions, which are important for the tumor development, have been largely ignored. In addition, high concentrations of the hormone, which cannot be attained in vivo, have been used. Our aim was to establish a more physiological breast cancer model, represented by BC tissue slices, which maintain epithelial-mesenchymal interactions, cultured with a relatively low 1 alpha,25(OH)(2)D(3) concentration, in order to evaluate the vitamin D pathway. Freshly excised human BC samples were sliced and cultured in complete culture media containing vehicle, 0.5 nM or 100 nM 1 alpha,25(OH)(2)D(3) for 24 h. BC slices remained viable for at least 24 h, as evaluated by preserved tissue morphology in hematoxylin and eosin (HE) stained sections and bromodeoxyuridine (BrdU) incorporation by 10% of tumor cells. VDR mRNA expression was detected in all samples and CYP24A1 mRNA expression was induced by 1 alpha,25(OH)(2)D(3) in both concentrations (but mainly with 100 nM). Our results indicate that the vitamin D signaling pathway is functional in BC slices, a model which preserves stromal-epithelial interactions and mimics in vivo conditions. (C) 2010 Elsevier Ltd. All rights reserved.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

Purpose We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the antihypertensive drug losartan (LOS). Results We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumorassociated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C. Conclusions Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

Concomitant expression of epithelial-mesenchymal transition biomarkers in breast ductal carcinoma: Association with progression

Epithelial to mesenchymal transition (EMT) is a process implicated in cancer progression in which the underlying cellular changes have been identified mainly using in vitro models. We determined the expression of some putative EMT biomarkers including E-cadherin, beta-catenin, zinc finger factor Snail (Snail), transforming growth factor beta 1 (TGF beta 1), TGF beta type II receptor (TBRII) and the HGF receptor (c-met) and their possible correlation to progression and overall survival in a series of breast ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDC). Biomarkers were immunohistochemically determined in 55 IDC specimens from which 21 had lymph node metastases and in 95 DCIS specimens, 46 of these cases associated to invasive carcinoma, in a tissue microarray (TMA). Positive cytoplasmic staining of TGF beta 1 (78.2%), c-met (43.6%), Snail (34.5%), TBRII (100%), membranous E-cadherin (74.5%) and membranous/cytoplasmic beta-catenin (71%) were detected in the IDC samples. Metastatic lymph node samples displayed similar frequencies. A significant increase of c-met and TGF beta 1 positivity along DCIS to IDC progression was noted but only TGF beta 1 positivity was associated with presence of lymph node metastases and advanced stages in IDC. The evaluation of the other EMT markers in DCIS did not show differences in positivity rate as compared to invasive carcinomas. DCIS either pure or associated to IDC showed similar expression of the analyzed biomarkers. All the carcinomas exhibited positive expression of TBRII. Associations between the markers, determined by Spearman`s correlation coefficient, showed a significant association between TGF beta 1 and respectively E-cadherin, beta-catenin and cmet in DCIS cases, but in invasive carcinomas only cadherin and catenin were positively correlated. Kaplan-Meier survival curves revealed that none of the EMT biomarkers analyzed were correlated with survival, which was significantly determined only by clinical and hormone receptor parameters.

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family Hp1(Hs alpha), Hp1(Hs beta) and Hp1(Hs gamma) which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1(Hs alpha), HP1(Hs beta) and HP1(Hs gamma) was determined by real-time RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow. (Int J Biol Markers 2008; 23: 219-24)

Expression of p16 protein in acral lentiginous melanoma

Background Acral lentiginous melanoma (ALM) is a clinicopathologic subtype of cutaneous malignant melanoma. ALM is the most common type of melanoma amongst Asians, Africans, and patients with mixed ancestry. In Brazil, ALM comprises 10% of cutaneous melanoma. ALM develops on palmar, plantar, and subungual skin, and its biology is different from that of other cutaneous melanomas, where sunlight is the major known environmental determinant. Alterations and inactivation of the p16INK4 gene that encodes a specific inhibitor of cyclin-dependent kinase have been related to melanoma genesis and progression. Few studies, however, have addressed p16 expression in ALM. Methods In order to verify and compare p16 protein expression, 32 paraffin-embedded ALM specimens were subjected to a immunohistochemical technique using a monoclonal anti-p16 antibody. The tumors were classified according to thickness (up to 1.0 mm and thicker than 1.0 mm) and the presence of ulceration. Results Twenty-five (78%) ALMs displayed positive p16 protein expression: 21 of the 25 (84%) with a thickness of more than 1.0 mm, and four of the seven (57%) with a thickness of 1.0 mm or less. Thirteen of the 17 (76%) nonulcerated lesions and 12 of the 15 (80%) ulcerated lesions displayed positive p16 protein expression. Conclusion The data obtained suggest that p16 protein expression per se may not represent a marker of retinoblastoma protein (pRb) pathway disturbance in ALM tumorigenesis.

Factors affecting management decisions in rectal cancer in clinical practice: results from a national survey

Management of rectal cancer has become increasingly complex and a multidisciplinary approach is considered of key importance for improving outcomes. A national survey among specialists involved in this multidisciplinary setting was performed. A web-based survey containing 11 questions regarding rectal cancer management was sent to surgeons and medical oncologists registered by their corresponding societies as members. Statistical analysis was performed using the chi-square and Fisher`s exact tests for all categorical variables according to response to individual questions. Multivariate analysis was performed using Cox`s logistic regression. Overall, 418 email recipients responded the survey. Local staging was performed without either magnetic resonance imaging or endorectal ultrasound by 64% of responders. Seventy-two percent considered that final management decision should be made after neoadjuvant chemoradiation therapy. Additionally, 46% considered that an alternative procedure (local excision or observation) was appropriate in a patient with a complete clinical response. Colorectal surgeons were more frequently in favor of longer intervals after completion of chemoradiation therapy (P = 0.001) and of alternative management procedures after a complete clinical response (P = 0.02). After multivariate analysis, the choice of a watch and wait approach after a complete clinical response following neoadjuvant chemoradiation therapy was significantly more frequent among surgeons (OR 3.5, 95% CI 1.8-7.1). Surgeons seem to be more in favor of tailoring management of rectal cancer according to tumor response after neoadjuvant chemoradiation therapy, with longer intervals after chemoradiation therapy, decisions about treatment strategy being made after chemoradiation therapy instead of before, and the use of alternative surgical procedures after a complete clinical response following neoadjuvant therapy.

Complete Clinical Response After Neoadjuvant Chemoradiation Therapy for Distal Rectal Cancer: Characterization of Clinical and Endoscopic Findings for Standardization

BACKGROUND: Complete tumor regression may develop after neoadjuvant chemoradiation therapy for distal rectal cancer. Studies have suggested that selected patients with complete clinical response may avoid radical surgery and close surveillance may provide good outcomes with no oncologic compromise. However, definition of complete clinical response is often imprecise and may vary between different studies. The aim of this study is to provide a clear definition for a complete clinical response after neoadjuvant chemoradiation therapy in patients with distal rectal cancer in addition to actual endoscopic videos from patients managed nonoperatively. METHODS: Patients with nonmetastatic distal rectal cancer treated by neoadjuvant chemoradiation therapy, including 50.4 Gy and concomitant 5-fluorouracil and leucovorin, were assessed for tumor response at least 8 weeks after chemoradiation therapy completion. Complete and incomplete clinical responses were defined based on clinical and endoscopic findings. Patients with complete clinical response were not immediately operated on and were closely followed. Early and late endoscopic findings were recorded. RESULTS: Definition of a complete clinical response should be based on very strict clinical and endoscopic criteria. The finding of any residual superficial ulceration, irregularity, or nodule should prompt surgical attention, including transanal full-thickness excision or even a radical resection with total mesorectal excision. Standard or incisional biopsies should be avoided in this setting. Complete clinical responders should harbor no more than whitening of the mucosa, teleangiectasia with mucosal integrity to be considered for a nonoperative approach. In the presence of these findings, regularly scheduled reassessments may provide a safe alternative to these patients with early detection of recurrent disease. CONCLUSION: Strict definition of the clinical and endoscopic findings of patients experiencing complete clinical response after neoadjuvant chemoradiation therapy may provide a useful tool for the understanding of outcomes of patients managed with no immediate surgery allowing standardization of classifications and comparison between the experiences of different institutions.