Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancerpatients

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1,MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1based trios, with NOTCH1 or NUP210. Both trios correctly separated 86 percent of tumors (87 percent sensitivity and 80 percent specificity for predicting response), according to their response to chemotherapy (82 percent in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71 percent samples from the biological validation set were also correctly classified by both trios (72 percent sensitivity; 66 percent specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93 percent of samples from the technical validation group (95 percent sensitivity and 80 percent specificity; 86 percent accuracy by the cross-validation method) and 79 percent from the biological validation group (72 percent sensitivity and 100 percent specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Profile of thyroid hormones in breast cancer patients

Estrogen involvement in breast cancer has been established; however, the association between breast cancer and thyroid diseases is controversial. Estrogen-like effects of thyroid hormone on breast cancer cell growth in culture have been reported. The objective of the present study was to determine the profile of thyroid hormones in breast cancer patients. Serum aliquots from 26 patients with breast cancer ranging in age from 30 to 85 years and age-matched normal controls (N = 22) were analyzed for free triiodothyronine (T3F), free thyroxine (T4F), thyroid-stimulating hormone (TSH), antiperoxidase antibody (TPO), and estradiol (E2). Estrogen receptor ß (ERß) was determined in tumor tissues by immunohistochemistry. Thyroid disease incidence was higher in patients than in controls (58 vs 18 percent, P < 0.05). Subclinical hyperthyroidism was the most frequent disorder in patients (31 percent); hypothyroidism (8 percent) and positive anti-TPO antibodies (19 percent) were also found. Subclinical hypothyroidism was the only dysfunction (18 percent) found in controls. Hyperthyroidism was associated with postmenopausal patients, as shown by significantly higher mean T3 and T4 values and lower TSH levels in this group of breast cancer patients than in controls. The majority of positive ERß tumors were clustered in the postmenopausal patients and all cases presenting subclinical hyperthyroidism in this subgroup concomitantly exhibited Erß-positive tumors. Subclinical hyperthyroidism was present in only one of 6 premenopausal patients. We show here that postmenopausal breast cancer patients have a significantly increased thyroid hormone/E2 ratio (P < 0.05), suggesting a possible tumor growth-promoting effect caused by this misbalance.

Normal HC11 and ras-transformed mouse mammary cells are resistant to the antiproliferative effects of retinoic acid

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR á mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 æM 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of á-casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR á, as well as RXR a, á and g expression was low in both HC11 and HC11ras cells. In addition, RAR á mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

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 ß1 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 percent 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 alpha2 (63.8 ± 11.3 percent positive cells), alpha3 (93.3 ± 7.0 percent), alpha5 (50.4 ± 12.0 percent) and alpha6 (34.1 ± 4.9 percent) integrins but not alpha1, alpha4, alphav or ß4. Cells adhered well to laminin-1 (73.4 ± 6.0 percent) and fibronectin (40.0 ± 2.0 percent) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha2, alpha3 and alpha6 mediated laminin-1 adhesion, but neither alpha3 nor alpha5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 ± 2.4 percent vs DMSO: 70.7 ± 2.5 percent) while simultaneously reducing alpha5 (24.2 ± 19 percent) and alpha6 (14.3 ± 10.8 percent) 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, alpha3 and alpha5 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 alpha6. 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

Presence of ductal carcinoma in situ confers an improved prognosis for patients with T1N0M0 invasive breast carcinoma

We have retrospectively analyzed a series of 155 sequential cases of T1N0M0 ductal carcinomas of which 51 tumors had a ductal carcinoma in situ (DCIS) component for correlation between the presence of DCIS and clinicopathological variables, recurrence and patient survival. No correlations between the presence of DCIS and age, menopausal status, size, estrogen or progesterone receptors were found. High-grade infiltrative tumors tended not to present a DCIS component (P = 0.08). Patients with tumors associated with DCIS form a subgroup with few recurrences (P = 0.003) and good survival (P = 0.008). When tumors were classified by size, an association between large tumors (>1.0 cm) and increased recurrence and shortened overall survival was found. The presence of DCIS in this subgroup significantly reduced the relative risk of death

Antiproliferative effects of 1, 25-dihydroxyvitamin D3 on breast cells: a mini review

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the active form of vitamin D3, is an important regulator of calcium homeostasis, exerts antiproliferative effects on various cell systems and can induce differentiation in some kinds of hematopoietic cells. These effects are triggered by its receptor, vitamin D receptor (VDR), a phosphoprotein member of the nuclear receptor superfamily, which functions as a transcriptional factor. VDR binds as a heterodimer with retinoid X receptor (R X R) to hexameric repeats, characterized as vitamin D-responsive elements present in the regulatory region of target genes such as osteocalcin, osteopontin, calbindin-D28K, calbindin-D9K, p21WAF1/CIP1, TGF-ß2 and vitamin D 24-hydroxylase. Many factors such as glucocorticoids, estrogens, retinoids, proliferation rate and cell transformation can modulate VDR levels. VDR is expressed in mammary tissue and breast cancer cells, which are potential targets to hormone action. Besides having antiproliferative properties, vitamin D might also reduce the invasiveness of cancer cells and act as an anti-angiogenesis agent. All of these antitumoral features suggest that the properties of vitamin D could be explored for chemopreventive and therapeutic purposes in cancer. However, hypercalcemia is an undesirable side effect associated with pharmacological doses of 1,25-(OH)2D3. Some promising 1,25-(OH)2D3 analogs have been developed, which are less hypercalcemic in spite of being potent antiproliferative agents. They represent a new field of investigation

Expression of c-erbB-2, p53 and c-myc proteins in male breast carcinoma: Comparison with traditional prognostic factors and survival

There are few data evaluating biological markers for men with breast cancer. The purpose of the present study was to analyze the expression of the oncogenes c-erbB-2 and c-myc and of the suppressor gene p53 by immunohistochemical techniques in archival paraffin-embedded tissue blocks of 48 male breast cancer patients, treated at the A.C. Camargo Cancer Hospital, Säo Paulo, SP, Brazil. The results were compared with clinicopathological prognostic features. Immunopositivity of c-erbB-2, p53 and c-myc was detected in 62.5, 16.7 and 20.8 percent of the cases analyzed, respectively. Estrogen and progesterone receptors were positive in 75 and 69 percent of the cases, respectively. Increasing staging was statistically associated with c-erbB-2 (P = 0.04) and weakly related to p53 positivity (P = 0.06). No significant correlation between specific survival rate (determined by the log rank test) and the molecular markers analyzed was found, whereas the number of compromised lymph nodes and advanced TNM (tumor, node, metastasis) staging were associated with diminished survival

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.

Integrin receptors and TGF-Beta expression in chronic myeloid leukemia cells

To understand relationiship between transforming growth factor beta-1 (TGF-ß1) and the integrin profile presented by chronic myeloid leukemia cells, we have studied, using Northen analysis, the expression of TGF-ß1 messenger RNA (TGF-ß mRNA) in myeloid cell lines and in patient with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In addition we determined the positivity for alfa4 and alfa5 integrin moleculas in those cell using specific monoclonal antibodies and flow cytometry. CML patients (N=3) presented mean values of alfa4 higher (alfa4: 60 ñ 20 per cent); alfa5: 70 ñ 41 per cent) than AML (N=10) blast cells (alfa4: 25 ñ 23 per cent); alfa5: 18 ñ 16 per cent). Northern analysis revealed an almost four-fold higher expression of TGF-ß mRNA in K562 (derived from a patient with chronic myeloid leukemia) compared to the myeloblastic cell line HL60. The highest TGF-ß mRNA levels were seen in the U937 lineage. CML leukemic cells (N=3) showed high TGF-ß mRNA levels comparable to the levels expressed by K562 which was paralleled by high ß1 integrin mRNA. AML blast cells presented a variable degree of expression of TGF-ß mRNA when compared to HL60. One patient with acute megakaryoblastic leukemia (FAB subtype M7), usually associated with myelofibrosis, presented the highest TGF-ß mRNA levels. We conclude that studing TGF-ß1 and its mechanisms of action will help in understanding fibrosis in leukemic patients, and perhaps to design treatments for such conditions

Monoclonal antibody-based immunoassay for carcinoembryonic antigen (CEA) measurement in human serum

Clinical oncology requires methods to detect tumor markers in patients sera and tissues Presently, monoclonal antibodies (MAbs based enzyme immunoassays are amongst the most advantageous techniques. Here we present a sensitive and specific double-antibody enzyme immunoassay for serum me surement of carcinoembryonic antigen (CEA). It has been developed with MAbs which recognize nonoverlapping peptide epitopes on the antigen molecule. The capture MAb 6C7 is GOLD 1 highly specific antibody while the tracer MAb 5.D11, labeled with biotin, is GOLD 4 that cross-reacts with nonspecific cross-reacting antigen (NCA) expressed on granulocytes. In addition the biotinylate MAb is shown to be also useful to detect CEA by immunohistochemistry.

Regulation of extracellular matrix-degrading proteases

Extracellular matrix-degrading enzymes have a very important role in many normal and pathological processes. Members of the matrix metalloproteinase and plasminogen activator families are the major modulators of extracellular matrix degradation. Here, we discuss some topics about these enzymes giving special attention to the transcriptional and extracellular regulation of their expression.

Steroid receptors in Brazilian patients with large bowel cancer

1. Twenty-two colorectal carcinomas were examined for the presence of estrogen (ER), progesterone (PR), androgen (AR) and glucocorticoid receptors (GR) by a charcoal dextran assay. 2. ER was detected in 4/13 (31 per cent ) and 5/9 (56 per cent ) of the rectum and colon carcinomas analyzed, and density values ranged from 10 to 14 and from 10 to 27 fm/mg protein, respectively. Normal distal or adjacent mucosa presented similar incidence and ER density values within the tumor ranges. 3. The incidence of PR-positive samples was also higher in colon than in rectal carcinomas (44 per cent vs 23 per cent ). Normal mucosa displayed significantly higher PR titers than the corresponding tumor tissue. It seems reasonable to assume that normal colorectal mucosa may be one of the target tissues of progesterone activity. Most tumor biopsies and normal mucosa were completely AR negative, whilst GR was present in a larger fraction (63 per cent ) of tumoral specimens, occurring more commonly in colon than in rectum carcinomas. GR incidence tended to be higher in neoplasms than in normal mucosa (54 per cent vs 38 per cent in rectum and 78 per cent vs 56 per cent in colon), suggesting that glucocorticoids may be involved in the control of tumor-cell proliferation. 4. Our findings which indicate low densities of ER, PR, and absence of AR in some large bowel cancers, suggest sex hormone and endocrine independence for those cancers. The role of glucocorticoid receptors in those forms of cancer remains to be elucidated

Regulaçäo dos receptores de glicocorticóide em leucócitos mononucleares humanos / Glucocorticoid receptores of human mononuclear leukocytes

O presente estudo foi realizado com a finalidade de analisar receptores de glicocorticóide em leucócitos mononucleares (MNL) de sangue periférico de indivíduos normais, näo tratados e de indivíduos normal aos quais foram administrados exogenamente os corticóides: prednisona e deflazacort. MNL de mulheres e homens normais apresentaram uma concentraçäo de receptores de glicocorticóide de 7,52 ñ 2,5fMol/10**6 cel e uma constante de dissociaçäo (Kd) de 9,5 ñ 2,3nM. Näo houve diferença em relaçäo ao sexo e as fases do ciclo menstrual. Em indivíduos tratados com predinisona, näo houve mudança nos parâmetros de ligaçäo. A administraçäo deflazacort determinou uma sensível reduçäo dos sítios de ligaçäo. Concluímos que certos glicocorticóides podem exercer um efeito regulatório sobre seus receptores