Miriam H.H. Federico

A Randomized, Double-Blind, Placebo-Controlled Phase II Study of Vandetanib Plus Docetaxel/Prednisolone in Patients with Hormone-Refractory Prostate Cancer

Vandetanib (ZACTIMA) is a once-daily oral anticancer drug that selectively inhibits vascular endothelial growth factor receptor, epidermal growth factor receptor, and rearranged during transfection signaling. This randomized (1:1), double-blind study evaluated vandetanib (100 mg/day) or placebo in combination with docetaxel (D; 75 mg/m(2) every 3 weeks) and prednisolone (P; 2 x 5 mg/day) in 86 patients with metastatic hormone-refractory prostate cancer (mHRPC). The primary assessment was prostate-specific antigen (PSA) response (confirmed reduction of >or=50% from baseline) and a greater number of patients showed a PSA response with placebo + DP (67%) versus vandetanib + DP (40%); hazard ratio = 2.23 (one-sided 80% confidence limit = 2.90; one-sided p = 0.99). More patients experienced progression events (disease progression or death from any cause) with vandetanib + DP (65%) versus placebo + DP (60%); hazard ratio = 1.13 (one-sided 80% confidence limit = 1.44; one-sided p = 0.67). The overall incidence of adverse events was similar in both groups, although more patients experienced adverse events, leading to permanent discontinuation with vandetanib + DP (28%) versus placebo + DP (12%). However, the safety and tolerability profile for vandetanib was similar to that previously reported; adverse events that occurred more frequently in the vandetanib + DP arm were hypertension (14% vs. 2%), erythematous rash (14% vs. 2%), and exfoliative rash (12% vs. 2%). In this study of patients with mHRPC, vandetanib + DP did not demonstrate any efficacy benefit, compared with placebo + DP.

Overexpression of Fos-related antigen-1 in head and neck squamous cell carcinoma

The activating protein‐1 (AP‐1) family of transcription factors has been implicated in the control of proliferation and differentiation of keratinocytes, but its role in malignant transformation is not clear. The aim of this study is to assess the pattern of mRNA expression of jun‐fos AP‐1 family members in 45 samples of head and neck squamous cell carcinomas (HNSCC) and matched adjacent mucosa by means of Northern blot analysis. Transcripts of all family members were identified, except for JunB that was detected only by means of reverse transcription polymerase chain reaction. Neither c‐Fos nor JunD or FosB mRNA differed between tumours and normal tissues. We observed a strong Fos‐related antigen‐1 (Fra‐1) and Fra‐2 expression, but only Fra‐1 mRNA densitometric values were higher in tumour, compared to normal adjacent mucosa (t‐test, P = 0.006). A direct relationship between the positive expression of Fra‐1 mRNA, above tumour median, was associated with the presence of compromised lymph nodes (Fischer exact test, P = 0.006). In addition, Fra‐1 protein staining was assessed in a collection of 180 tumours and 29 histologically normal samples adjacent to tumours in a tissue array. Weak reactivity, restricted to the basal cell layer, was detected in 79% of tumour adjacent normal tissues, opposed to the intense reactivity of cancer tissues. In the subgroup of oral cancers, we have observed a shift in Fra‐1 immunoreactivity, as long as the number of patients in each category, cytoplasmic or nuclear/cytoplasmic staining, was analysed (Fischer exact test, P = 0.0005). Thus, Fra‐1 gene induction and accumulation of Fra‐1 protein may contribute to the neoplastic phenotype in HNSCC.

Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

BackgroundTo test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC).Patients and MethodsWe analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGFβ1, TGFβ2, TGFβ3 in oral SCC.ResultsOur results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFβ isoforms, we found that Smad2 mRNA and TGFβ1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGFβ1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population.ConclusionSmad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGFβ signaling should be better clarified in the future.

Eosinophil Accumulation in Rat Uterus Following Estradiol Administration is Modulated by Laminin and its Integrin Receptors

Eosinophils accumulate into the uterus of ovariectomized rats, after treatment with estradiol (E2). We have investigated whether this feature is related to interactions of eosinophils with uterine extracellular matrix proteins: laminin (LM) and fibronectin (FN). Eosinophils isolated from the peritoneal cavity of ovariectomized rats displayed estrogen receptors measured at both binding activity and mRNA levels. An increased number of laminin binding sites, calculated by Scatchard analysis using iodinated LM was determined in E2-treated eosinophils (70,100 +/- 28,000 sites/cell vs 21,000 +/- 5,000 sites/cell in controls). Eo binding to 125I-LM- was inhibited by the E8-LM fragment. Estradiol up-regulated the expression in eosinophils of alpha 6 and beta 2 integrin subunits evaluated by flow-cytometry as well as by alpha 6 mRNA expression. After E2 treatment, eosinophils showed higher adhesiveness to LM-coated dishes (10 +/- 2 vs 56 +/- 3%) which was inhibited by monoclonal antibodies against alpha 6, beta 1 and beta 2 integrins and by the steroid antagonist tamoxifen. These monoclonal antibodies also blocked the attachment of stimulated eosinophils to uterine cryostat sections obtained from spayed rats previously treated with estradiol. We did not detect any apparent influence of E2 on basal eosinophil adherence or binding to FN although alpha 4 and alpha 5 integrin subunits were expressed in eosinophils. Expression of laminin and merosin in the uterus was determined immunohistochemically. Our results suggest that integrin-laminin interactions may contribute to the preferential eosinophil recruitment in vivo.

Vitamin D3 binding activity during leukemic cell differentiation

In this paper we report that differentiation of the human promyelocytic leukemia cell line, HL60, along the myelocytic pathway, induced by retinoic acid (RA), or monocytic pathway, induced by phorbol-myristate acetate (PMA) and gamma interferon (IFN), was accompanied by a significant decline in 1,25-dihydroxycholecalciferol (1,25(OH)2D3) binding (control: 30.3 +/- 3.0 fM/10(6) cells; RA treated: 6.8 + 2.5 fM/10(6) cells; PMA treated: 12.3 +/- 6.7 fM/10(6) cells and IFN treated: 16.0 +/- 5.0 fM/10(6) cells). When differentiation and proliferation were uncoupled, by incubation with IFN or by inhibition of proliferation by cell density saturation, 1,25(OH)2D3 binding was better related to differentiation than to proliferation. Additionally we have compared 1,25(OH)2D3 binding levels in blasts from acute lymphocytic leukemia (ALL) patients (25.4 +/- 18.1 fM/10(6) cells) and normal, mature lymphocytes (10.6 +/- 2.1 fM/10(6) cells). Receptor binding was significantly higher (p < 0.05) in the immature blasts. Our data suggest that 1,25(OH)2D3 receptor levels could be considered a marker of functional immaturity, in these cells.

Expression of vitamin D receptor (VDR) in HL-60 cells is differentially regulated during the process of differentiation induced by phorbol ester, retinoic acid or interferon-γ

The effects of three inducers of differentiation, phorbol myristate acetate (PMA), retinoic acid (RA) and interferon-gamma (IFN-gamma), on the temporal regulation of vitamin D receptor (VDR) expression in HL-60 cells were analyzed by Northern blotting and immunofluorescence assays. VDR, at the protein level, expressed by 81% of uninduced cells, was reduced to 57% after 48 h of PMA or 96 h of RA treatment, preceded by growth inhibition and cell differentiation, evaluated by CD11b expression. Sorted CD11b positive cells in G0/G1 phase exhibited 53% the VDR content of CD11b negative cells (distributed throughout the cell cycle). PMA also induced an increase in PKC beta and PKC alpha mRNA and protein. Simultaneous exposure to PMA and sphingosine blocked stimulation of CD11b and PKC expression without affecting growth arrest and VDR down regulation. Similar effects were observed during sphingosine treatment. In IFN-gamma differentiated cells, the proportion of cells in G0/G1 phase was unchanged and VDR protein was unaltered as compared to uninduced cells. Control cells in G0/G1 expressed less VDR than cells in S and G2/M phases (74% and 59% respectively). All results suggest that in HL-60 cells, reduction of VDR expression is related to growth inhibition rather than to the differentiation process.

Four-gene expression model predictive of lymph node metastases in oral squamous cell carcinoma

Abstract Background. Previous knowledge of cervical lymph node compromise may be crucial to choose the best treatment strategy in oral squamous cell carcinoma (OSCC). Here we propose a set four genes, whose mRNA expression in the primary tumor predicts nodal status in OSCC, excluding tongue. Material and methods. We identified differentially expressed genes in OSCC with and without compromised lymph nodes using Differential Display RT-PCR. Known genes were chosen to be validated by means of Northern blotting or real time RT-PCR (qRT-PCR). Thereafter we constructed a Nodal Index (NI) using discriminant analysis in a learning set of 35 patients, which was further validated in a second independent group of 20 patients. Results. Of the 63 differentially expressed known genes identified comparing three lymph node positive (pN +) and three negative (pN0) primary tumors, 23 were analyzed by Northern analysis or RT-PCR in 49 primary tumors. Six genes confirmed as differentially expressed were used to construct a NI, as the best set predictive of lymph nodal status, with the final result including four genes. The NI was able to correctly classify 32 of 35 patients comprising the learning group (88.6%; p = 0.009). Casein kinase 1alpha1 and scavenger receptor class B, member 2 were found to be up regulated in pN + group in contrast to small proline-rich protein 2B and Ras-GTPase activating protein SH3 domain-binding protein 2 which were upregulated in the pN0 group. We validated further our NI in an independent set of 20 primary tumors, 11 of them pN0 and nine pN + with an accuracy of 80.0% (p = 0.012). Conclusions. The NI was an independent predictor of compromised lymph nodes, taking into the consideration tumor size and histological grade. The genes identified here that integrate our “Nodal Index” model are predictive of lymph node metastasis in OSCC.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Cost-effectiveness analysis of cisplatin-based chemoradiation to treat patients with unresectable, nonmetastatic head and neck cancer in Brazil.

The purpose of this study was to analyze the cost‐effectiveness of cisplatin‐based chemoradiation compared to radiation therapy (RT) alone to treat patients with advanced head and neck cancer in Brazil.

Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1)

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of beta 1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha 2 (63.8 11.3% positive cells), alpha 3 (93.3 7.0%), alpha 5 (50.4 12.0%) and alpha 6 (34.1 4.9%) integrins but not alpha1, alpha 4, alpha v or 4. Cells adhered well to laminin-1 (73.4 6.0%) and fibronectin (40.0 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha 2, alpha 3 and alpha 6 mediated laminin-1 adhesion, but neither alpha 3 nor alpha 5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 2.4% vs DMSO: 70.7 2.5%) while simultaneously reducing alpha 5 (24.2 19%) and alpha 6 (14.3 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha 3 and alpha 5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 2 cells vs DMSO: 64 6 cells), was blocked by an antibody against alpha 6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.