Maximino Redondo

Overexpression of Clusterin in Human Breast Carcinoma

Clusterin has been implicated in numerous processes including active cell death, immune regulation, cell adhesion and morphological transformation. The purpose of this study was to examine clusterin expression in a large series of breast carcinomas by immunohistochemistry and in situ hybridization. The study included 40 samples of non-neoplastic glandular epithelia, 42 benign lesions, 15 atypical intraductal hyperplasias, 35 carcinomas in situ, 114 invasive carcinomas, and lymph node metastases from 40 patients. Epithelial normal cells were always negative for clusterin expression and only 19% of the benign lesions presented positive staining. In contrast to the benign lesions, however, the frequency of clusterin positive samples increased in atypical hyperplasias (47%, P = 0.08), intraductal carcinomas (49%, P = 0.01) and invasive carcinomas (53%, P < 0.001). Positive staining presented a cytoplasmic pattern, except in 3 cases of invasive carcinomas which had nuclear staining. Clusterin mRNA by in situ hybridization confirmed the specific cellular pattern of clusterin expression by immunohistochemistry. Clusterin expression was associated with large tumor size (P = 0.04), estrogen and progesterone receptor negative status (P = 0.02 and P = 0.001, respectively) and with the progression from primary carcinoma to metastatic carcinoma in lymph nodes (80% metastatic nodes had positive expression) (P = 0.004). Ten of 15 (67%) primary carcinomas without clusterin expression became positive in lymph node metastases, while most (22 of 25, 88%) of the clusterin-positive primary carcinomas were also immunoreactive in metastases. In survival analysis, clusterin expression did not represent a prognostic indicator by uni- or multivariate analysis. The increased clusterin expression in breast carcinomas tended to correlate inversely with the apoptotic index (P = 0.09) which indicates that clusterin gene expression is not a prerequisite to cellular death by apoptosis. From these results, we suggest that clusterin may have a role in tumorigenesis and progression of human breast carcinomas.

Expression of substance P and neurokinin‐1‐receptor in laryngeal cancer: linking chronic inflammation to cancer promotion and progression

Sir: Substance P (SP) is an undecapeptide that belongs to the tachykinin family and has been implicated in a myriad of physiological processes. In recent years it has been demonstrated that SP acts through the neurokinin-1 receptor (NK1R) as a mitogen on several human cancer cell lines. It seems that the SP ⁄ NK1R system could play an important role in the development of cancer, as SP may be a universal mitogen in NK1R-expressing tumour cell types. However, in their pioneer work on SP receptors in human primary neoplasms, Henning and coworkers did not study squamous cell carcinoma. Although NK1R has been recently described in the Hep-2 laryngeal carcinoma cell line, this is, to our knowledge, the first study of SP and NK1R expression in laryngeal cancer. We reviewed 114 consecutive cases of laryngeal carcinoma treated surgically from the Department of Otolaryngology. Surgical procedure, site of the primary tumour and TNM staging were recorded. None of the patients had received radiotherapy and ⁄ or chemotherapy prior to surgery. Of the 114 patients, 78 (68%) remained disease free at the end of the follow-up period (average 40 months, range 12–96 months). Tumour differentiation was recorded according to World Health Organization criteria. Cases were also carefully staged to conform to the American Joint Committee on Cancer Staging and End Results Reporting. The presence of adjacent non-neoplastic epithelium was also recorded. Laryngeal non-neoplastic epithelium consisted of pseudostratified squamous epithelium. The histological and immunohistochemical analyses were performed without any knowledge of the clinical stage, treatment or further course of the disease. For the detection of SP and NK1R we used monoclonal antibodies against SP (Sigma, St Louis, MO, USA) and NK1R (Sigma-Aldrich, Madrid, Spain), as previously described. The number of tumour cells showing immunoreactivity (brown staining) and its location were noted. Groups were formed according to the percentage of positive cells (0–24, 25–49, 50–74 and >75%). SP and NK1R expression in non-neoplastic epithelium adjacent to tumour was also recorded in the same way. The presence of basal and suprabasal expression was also recorded. The relationship between SP and NK1R expression and clinical and pathological parameters was determined by v analysis and Fisher’s exact test. All P-values corresponded to two-sided significance testing. The relationship of survival to the expression of proteins was examined using the Kaplan–Meyer method and multivariate survival analysis. SP was immunoreactive in 93 out of 97 samples of non-neoplastic epithelium close to the tumour. Reactivity was found mainly in the basal layers (Figure 1A), with >75% reactive cells. Out of 114 cases, 111 studied showed homogeneous SP expression, with 90 tumours scored as ++++ (Figure 1B). Only three cases were classified as negative. Six were considered +, seven ++ and eight +++. All 17 metastases studied were immunoreactive for SP (Figure1C). All cases showed the same expression as the primary tumour (+++ ⁄ ++++). Out of 97 cases, the basal layers were reactive for NK1R in 72 (Figure 2A). Ninety of 97 samples were positive for NK1R, with >25% of tumour cells reactive (Figure 2B). Reactivity was detected both in the membrane and cytoplasm. We did not find any relationship between expression in primary carcinomas and clinicopathological parameters or survival (data not shown). All cases of metastases (17 out of 17) were found to express NK1R in most tumour cells (+++ ⁄ ++++) (Figure 2C). SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such as mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis. We found both membranous and cytoplasmic SP immunoreactivity, which can be explained on the basis of NK1R internalization. Cytoplasmic reactivity has also been reported in lymphocytes of children who go on to develop acute lymphoblastic leukaemia. The ubiquitous presence of SP in laryngeal tumours may represent a common mechanism of tumour progression. In fact, an intriguing question is why some lymphocytes in normal mucosa express SP, whereas those in metastatic lymph nodes do not. We propose a SP link between chronic mucosal inflammation and cancer, with submucosal lymphocytes secreting SP, as has been described in neoplastic lymphocytes. In addition, it has been demonstrated that the activation of NK1R by SP or SP analogues induces

Anticlusterin treatment of breast cancer cells increases the sensitivities of chemotherapy and tamoxifen and counteracts the inhibitory action of dexamethasone on chemotherapy-induced cytotoxicity

IntroductionOverexpression of the apoptosis-related protein clusterin is associated with breast cancer development and tumor progression. We describe the use of clusterin-specific antisense oligonucleotides and antibodies to sensitize breast carcinoma cells to anticancer drugs routinely used in breast cancer therapy.MethodsMCF-7 and MDA-MB-231 cells were treated with the oligonucleotide or antibody, chemotherapeutic agents (doxorubicin or paclitaxel), tamoxifen, or with combinations of these.ResultsTreatments that include antisense clusterin oligonucleotide or antibody to clusterin have been shown to reduce the number of viable cells more effectively than treatment with the drugs alone. We also demonstrate that dexamethasone pretreatment of breast cancer cell lines inhibits chemotherapy-induced cytotoxicity and is associated with the transcriptional induction of clusterin. However, anticlusterin treatment increases chemotherapy-induced cytotoxicity, even in the presence of glucocorticoids, suggesting a possible role for these proteins in glucocorticoid-mediated survival.ConclusionThese data suggest that combined treatment with antibodies to clusterin or antisense clusterin oligodeoxynucleotides and paclitaxel, doxorubicin, or tamoxifen could be a novel and attractive strategy to inhibit the progression of breast carcinoma by regulation of the clusterin function. Moreover, glucocorticoid activation in breast cancer cells regulates survival signaling by the direct transactivation of genes like clusterin which encode proteins that decrease susceptibility to apoptosis. Given the widespread clinical administration of dexamethasone before chemotherapy, understanding glucocorticoid-induced survival mechanisms is essential for achieving optimal therapeutic responses.

Aggressiveness features and outcomes of true interval cancers: comparison between screen-detected and symptom-detected cancers.

The question of whether screen detection confers an additional survival benefit in breast cancer is unclear and subject to several biases. Our aim was to examine the role of the diagnostic method (screen-detected, symptom-detected, and true interval cancers) and the clinical–pathological features in relapse-free survival and overall survival in breast cancer patients. We included 228 invasive breast cancers diagnosed in Barcelona from 1996 to 2008 among women aged 50–69 years. Ninety-seven patients were screen detected within the screening, 34 truly arose between 2-year screening mammograms (true interval cancers), and 97 were symptom detected outside the screening. The clinical–pathological features at diagnosis were compared. The overall and disease-free survival probabilities were computed using the Kaplan–Meier method. Cox proportional hazard models were applied, with adjustment by clinical–pathological variables. At diagnosis, symptom-detected and true interval cancers were in more advanced stages and were less differentiated. The highest proportion of triple-negative cancers was detected among true interval cancers (P=0.002). At 5 years of follow-up, the disease-free survival rates for screen-detected, true interval, and symptom-detected cancers were 87.5% (95% confidence interval, 80.5–95.2%), 64.1% (46.4–88.5%), and 79.4% (71.0–88.8%), respectively, and the overall survival rates were 94.5% (89.3–99.9%), 65.5% (47.1–91.2%), and 85.6% (78.3–93.6%), respectively. True interval cancers had the highest hazard ratio for relapse prediction (1.89; 0.67–5.31) and a hazard ratio of death of 5.55 (1.61–19.15) after adjustment for tumor–node–metastasis stage and phenotype. Clinically detected tumors, especially true interval cancers, more frequently showed biological features related to worse prognosis and were associated with poorer survival even after adjustment for clinical–pathological characteristics.

bcl-2 Expression and Apoptosis in Primary and Metastatic Breast Carcinomas

Objectives: To evaluate the role of bcl-2 and apoptotic index in the progression from primary to metastatic breast carcinoma and their influence on prognosis. Methods:bcl-2 expression was examined by immunohistochemistry and apoptotic index by in situ end-labelling in 116 surgical breast carcinomas and lymph node metastases from 50 patients. Results:bcl-2 was observed in 69 cases (59.4%) of primitive carcinomas and 26 cases (65%) of metastatic breast carcinomas and there was agreement of bcl-2 expression between primary and metastatic sites except in 3 cases. bcl-2 expression was significantly associated with several favourable prognostic features, such as small tumour size (p = 0.03) and oestrogen and progesterone-receptor positivity (p < 0.01 and p < 0.001, respectively). A high apoptotic index was significantly associated with a number of poor prognostic factors, including poorly differentiated carcinomas, large tumour size, high Ki67 expression and high mitotic count (p < 0.001 in all cases). The mean apoptotic index was higher in lymph node metastasis than in primary carcinomas (1.19 vs. 0.69, p < 0.01). A low bcl-2 expression and a high apoptotic index were significantly associated with short-relapse free survival rates (p = 0.02 and p < 0.01, respectively), but only apoptotic extent provided independent prognostic information by multivariate analysis. Conclusions: The evaluation of bcl-2 expression and extent of apoptosis may provide useful prognostic information on breast cancer patients; however while increased apoptosis is strongly associated with the progression from primary carcinomas to lymph node metastases, bcl-2 does not seem to play a significant role in this process.

Expression of the Antiapoptotic Proteins Clusterin and Bcl-2 in Laryngeal Squamous Cell Carcinomas

Bcl-2 and clusterin genes have been related to the inhibition of apoptosis, an event that plays a key role in malignant transformation and in invasive disease. In this work, we determine the significance of clusterin and bcl-2 expression in a large series of laryngeal carcinomas. We used immunohistochemical methods and in situ hybridization to examine the expression of these proteins. Nontumoral epithelial laryngeal tissues did not express clusterin and bcl-2 proteins. However, 9% (14 out of 154) and 25% of these tumors (39 of 154) had positive clusterin and bcl-2 staining, respectively. Clusterin expression was significantly related to the degree of local invasion and higher bcl-2 expression was found in these clusterin-positive tumors (p < 0.05). Bcl-2 expression was significantly correlated with supraglottic localization, nodal metastases, invasion in depth, and poorly differentiated tumors. However, by multivariate analysis, bcl-2 was shown to be an independent predictor of good prognosis in these tumors (OR = 0.12, 95% CI = 0.02–0.91). These findings indicate that clusterin and bcl-2 are upregulated in laryngeal carcinomas and their expression is related to the invasiveness of these tumors.

High Frequency of HLA-B44 Allelic Losses in Human Solid Tumors

Human leukocyte antigen (HLA) class I downregulation, a frequent phenomenon observed in a variety of human tumors, favors tumor immune escape from T-lymphocyte recognition. However, it is not known whether a particular HLA class I allele is lost more frequently than others. To address this question we analyzed HLA class I expression in tumor tissues derived from 300 patients diagnosed as having breast, colorectal, or laryngeal carcinomas. Cryostatic tumor sections and a broad panel of anti-HLA class I monoclonal antibodies were used. We found that the HLA-B44 allele was lost more frequently than other HLA class I alleles, and that the difference was not related with changes in HLA-B44 allele frequencies between patients and controls. In addition, we observed that 35% of the HLA-B44 negative tumors presented HLA haplotype loss associated with loss of heterozygosity. These tests were performed on DNA samples obtained from microdissected tumor tissues. The results seem to indicate that HLA class I allelic losses are not randomly distributed during tumor development but that some HLA class I alleles, and HLA-B44 in particular, are more frequently downregulated and may play an important role in immune escape mechanisms.

Upregulation of glial clusterin in brains of patients with AIDs

Since clusterin (CLU) production in reactive astrocytes may be neuroprotective, we examined its distribution in AIDS brains where brain injury and reactive astrocytosis are common. The relative area and number of CLU-positive astrocytes, as well as their percent total of all white matter glia, significantly increased in AIDS brains with and without HIV encephalitis (P<0.05). Proliferation markers were absent. In contrast, the relative area and number of GFAP-positive astrocytes and their percent of all white matter glia, increased in some cases but the mean increases were not significant. Clusterin is sensitive marker of glial reactivity in AIDS brains and its enhanced expression was not dependent on increases in GFAP.

Clusterin expression is associated with decreased disease‐free survival of patients with colorectal carcinomas

Redondo M, Rodrigo I, Alcaide J, Tellez T, Roldan M J, Funez R, Diaz‐Martin A, Rueda A & Jiménez E
(2010) Histopathology 56, 932–936
Clusterin expression is associated with decreased disease‐free survival of patients with colorectal carcinomas

The Role of Clusterin (CLU) in Malignant Transformation and Drug Resistance in Breast Carcinomas

Breast cancer is the main cause of cancer-related death among women in Western countries. Current research is focused on identifying antiapoptotic proteins which could be a possible target for novel chemotherapeutic drugs. Secretory clusterin (sCLU) is an extracellular chaperone that has been functionally implicated in DNA repair, cell-cycle regulation, apoptotic cell death and tumorigenesis. The implication of sCLU in carcinogenesis and the progression of breast carcinomas make it an interesting gene, worthy of investigation. It has been reported to present powerful antiapoptotic activity and to perform a prosurvival function with most therapeutic treatments for breast cancer. This review summarizes our current understanding of the role of CLU in tumorigenesis, progression, and response to treatment in breast carcinomas.