An Introduction to the Relationship Between Lewis x and Malignancy Mainly Related to Breast Cancer and Head Neck Squamous Cell Carcinoma (HNSCC).

Lewis x functions as an adhesion molecule in glycolipids and glycoproteins since it mediates homophilic and heterophilic attachment of normal and tumoral cells. During malignancy, altered glycosylation is a frequent event; accumulating data support the expression of Lewis x in tumors although controversial results have been described including its relationship with patient survival. This report has been developed as an introduction to the relationship between Lewis x expression and breast cancer and head and neck squamous cell carcinoma (HNSCC). Results obtained in our laboratory are presented in the context of the literature.

Alternative splicing variant of RHBDD2 is associated with cell stress response and breast cancer progression.

RHBDD2 is an intramembrane pseudoprotease member of the Rhomboid superfamily. Our previous studies in breast and colorectal cancer indicate an association between RHBDD2 overexpression and advanced tumor stages. Two alternative transcriptional variants have been described for RHBDD2, which would be encoding for different RHBDD2 protein isoforms. The expression of these RHBDD2 variants/isoforms and its association with breast cancer was the focus of this study. First, expression of RHBDD2 splicing variants was evaluated in normal and breast tumor samples. RHBDD2 variant 2 overexpression was detected in tumors in respect to normal breast tissues at the mRNA and protein levels (P<0.05). Moreover, RHBDD2 variant 2 expression was associated with poor prognostic factors such as basal­like intrinsic subtype (P<0.05), high proliferation (P<0.01) and long­term risk­of­recurrence (P<0.01) scores. Second, the expression of both variants was evaluated under nutritional­deprived conditions in breast cancer cell lines. Results demonstrated that RHBDD2 splicing was switched from mRNA variant 1 to variant 2 in association with a significant increment of protein isoform B in response to glucose starvation treatment. Therefore, we propose that the switch from the RHBDD2 variant 1, expressed in normal epithelial cells, to variant 2 occurs as an adaptive phenotype to bypass the stressful tumor microenvironment and promote tumor progression. Finally, the RHBDD2 subcellular localization was corroborated at the Golgi apparatus and their associated v­SNARE transport vesicles, suggesting a putative new role for RHBDD2 in the protein trafficking of human breast cancer cells.

Nuclear localization of MUC1 extracellular domain in breast, head and neck, and colon cancer.

BACKGROUND: The glycoprotein MUC1 is overexpressed and underglycosylated in cancer cells. MUC1 is translated as a single polypeptide that undergoes autocleavage into 2 subunits (the extracellular domain and the cytoplasmic tail), and forms a stable heterodimer at the apical membrane of normal epithelial cells. The MUC1 cytoplasmic tail localizes to the cytoplasm of transformed cells and is targeted to the nucleus. AIMS: To study the expression of the MUC1 extracellular subunit in cell nuclei of neoplastic breast, head and neck, and colon samples. MATERIALS AND METHODS: 330 primary tumor samples were analyzed: 166 invasive breast carcinomas, 127 head and neck tumors, and 47 colon tumors; 10 benign breast disease (BBD) and 40 normal specimens were also included. A standard immunohistochemical method with antigen retrieval was performed. Nuclear fractions from tissue homogenates and breast cancer cell lines (ZR-75, MDA-MB-231, MCF7, and T47D) were obtained and analyzed by Western blotting (WB). The anti-MUC1 extracellular subunit monoclonal antibody HMFG1 was used for immunohistochemistry. RESULTS: 37/166 breast cancer specimens, 5/127 head and neck cancer specimens, 2/47 colon cancer samples, and 3/10 BBD samples showed immunohistochemical staining at the nuclear level. No nuclear reaction was detected in normal samples. By WB, breast and colon cancer purified nuclear fractions showed reactivity at 200 kDa in 3/30 breast and 3/20 colon cancer samples as well as purified nuclear fractions obtained from breast cancer cell lines. CONCLUSIONS: This study shows that the MUC1 extracellular domain might be translocated to the cell nucleus in breast, head and neck, and colon cancer as well as BBD.

High expression of sLex associated with poor survival in Argentinian colorectal cancer patients.

AIM: Colorectal cancer (CRC) is one of the most prevalent malignancies in Argentina with 11,043 new cases and 6,596 deaths estimated to have occurred in 2008. The present study was developed to clarify the differential expression of MUC1, MUC2, sLex, and sLea in colorectal cancer patients and their relationship with survival and clinical and histological features. METHODS: Ninety primary tumor samples and 43 metastatic lymph nodes from CRC patients were studied; follow-up was documented. Twenty-six adenoma and 68 histological normal mucosa specimens were analyzed. An immunohistochemical approach was applied and statistical analysis was performed. RESULTS: In tumor samples, MUC1, sLea, and sLex were highly expressed (94%, 67%, and 91%, respectively); also, we found a significantly increased expression of the 3 antigens in primary tumors and metastatic lymph nodes compared with normal mucosa and adenomas. MUC2 was expressed in 52% of both normal mucosa and CRC samples; this reactivity significantly decreased in metastatic lymph nodes (p<0.05). A multiple comparison analysis showed that MUC1 and sLex discriminated among 3 groups: normal, adenoma, and CRC tissues. The increase of sLex expression showed an association with recurrence, and survival analysis showed that a high sLex staining was significantly associated with a poor survival. By multivariate analysis MUC1 inmunoreactivity correlated positively and significantly with tumor size, while MUC2 expression showed the opposite correlation. CONCLUSIONS: The correlation of sLex overexpression in primary tumors and metastatic lymph nodes, the discrimination among the normal, adenoma, and CRC groups based on sLex expression, as well as its association with recurrence and survival, all suggest a prognostic role of sLex in Argentinian CRC patients.

Features related to breast cancer in an entire Argentine rural population.

AIM: A descriptive study was developed in an entire Argentine rural community considering breast cancer risk factors, preventive strategies and breast cancer incidence. PATIENTS AND METHODS: the study comprised of 83 women. A questionnaire of 34 items was employed; a mammogram and a breast ultrasound were performed. ANOVA and Pearson correlation were employed. RESULTS: Mean age was 54.5 years; 69% of women were postmenopausal; 96% had children; breastfeeding was X=10 months/child; Body Mass Index (BMI) was X=27.8 kg/m(2); 13% had first-degree relatives with breast cancer; 90% of women considered mammographic screening a necessary study. One woman had presented breast cancer. Argentine screening guidelines were not followed and an inverse relationship between education level and age of first mammogram was found (p<0.05). Mammographic and ultrasound studies did not reveal potential abnormalities. CONCLUSION: Peculiar social and cultural characteristics may be relevant to evaluate breast cancer risk factors in Argentina.

MUCI positive cutaneous metastasis with transepidermal elimination from a breast carcinoma.

Breast cancer is the most common cause of cutaneous metastases from internal malignancies. Generally, the neoplastic cells are located in the dermis or hypodermis, while a finding of transepidermal elimination on cutaneous metastases is exceptional. In this report we present a patient with perforating cutaneous metastases from breast cancer with mucin 1 expression. Cutaneous, bone, lung, and hepatic lesions were detected two years after the diagnosis of the primary tumor.

Invasive breast cancer in Argentine women: association between risk and prognostic factors with antigens of a peptidic and carbohydrate nature.

OBJECTIVE: In breast cancer, several tumor markers have been identified. The marker most extensively associated with breast cancer is MUC1. The objective of the study was to analyze prognostic and risk factors in relation to tumor markers in order to clarify breast cancer biology. A total of 349 primary tumor samples and lymph nodes from breast cancer patients were studied. Risk and prognostic factors were considered. An immunohistochemical approach was applied and an extensive statistical analysis was performed, including frequency analysis and analysis of variance. Correlation among variables was performed with principal component analysis. RESULTS: All the antigens showed an increased expression according to tumor size increment; moreover, sialyl Lewis x expression showed a significant increase in relation to disease stage, whereas Tn and TF presented a positive tendency. Vascular invasion was related to sialyl Lewis x expression and number of metastatic lymph nodes. Taking into account risk factors, when a patient had at least one child, Lewis antigens diminished their expression. In relation to breastfeeding, sialyl Lewis x expression diminished, although its apical expression increased. CONCLUSION: Associations between MUC1 and carbohydrate antigens and risk and prognostic factors show the complexity of the cellular biological behavior that these antigens modulate in breast cancer.

Comparative immunohistochemical study of MUC1 and carbohydrate antigens in breast benign disease and normal mammary gland.

The aim was to compare the expression of MUC1 and carbohydrate antigens in 124 tissue samples; 42 fibroadenoma (FA), 23 nonproliferative benign diseases (NPF), 25 usual epithelial hyperplasia (UEH), 7 atypical ductal hyperplasia (ADH), and 27 breast normal tissues. An immunohistochemical approach was adopted, using the following antibodies: reactive with MUC1 variable number of tandem repeats (C595, HMFG2, and SM3 monoclonal antibodies), anti-MUC1-cytoplasmic tail polyclonal antibody (CT33), and anti-carbohydrate antigens (sialyl Lewis x, Lewis x, Lewis y, Tn, and Thomsen-Friedenreich epitopes). Positive area of reaction, intensity, and pattern of expression were considered. A reactivity index was calculated as intensity (I) x 100+percentage of positive area (A). Statistical analysis comprised frequency analysis, P < 0.05, analysis of variance, and multiple correlation with principal component analysis. All samples expressed MUC1, detected by at least one anti-MUC1 antibody whereas Lewis x was the carbohydrate antigen most frequently found in all groups whereas variable number of tandem repeats MUC1 and Lewis x showed the highest correlation: 93% of normal samples, 62.5% of NPF, 87% of FA, 85% of UEH, and finally 80% of ADH. Although principal component analysis using reactivity indexes explained only 39% of data variability, normal samples appeared grouped and separated from benign breast diseases, which remained spread. Thomsen-Friedenreich was the only antigen that showed an increased tendency for positive expression and intensity from NPF through FA, UEH to ADH, whereas it was not detected in normals. With respect to the pattern of expression, an apical pattern was predominantly found in all the groups.

Breast cancer humoral immune response: involvement of Lewis y through the detection of circulating immune complexes and association with Mucin 1 (MUC1).

BACKGROUND: In cancer patients, MUC1 glycoprotein may carry Lewis y which could be involved in immune response. PURPOSES: 1- to evaluate the presence of Lewis y and MUC1 in circulating immune complexes (Lewis y/CIC and MUC1/CIC, respectively) and their correlation; 2- to analyze the possible presence of Lewis y in carbohydrate chains of tumoral MUC1 glycoprotein and 3- to correlate serum and tissue parameters considered. METHODS: Pretreatment serum and tissue breast samples from 76 adenocarcinoma, 34 benign and 36 normal specimens were analyzed. Anti-MUC1 and anti-Lewis y MAbs were employed. To detect Lewis y/CIC and MUC1/CIC, ELISA tests were developed; serum samples containing MUC1 were previously selected by Cancer Associated Serum Antigen (CASA). Immunoprecipitation (IP) was performed in 9 malignant, benign and normal samples and analyzed by SDS-PAGE and Western blot. Lewis y and MUC1 expression was studied by immunohistochemistry (IHC). Statistical analysis was performed employing principal component analysis (PCA), ANOVA, Tukey HSD, Chi square test and classical correlation (p < 0.05). RESULTS: By ELISA, Lewis y/IgM/CIC levels showed statistically significant differences between breast cancer versus benign and normal samples; mean +/- SD values expressed in OD units were: 0.525 +/- 0.304; 0.968 +/- 0.482 and 0.928 +/- 0.447, for breast cancer, benign disease and normal samples, respectively, p < 0.05. Lewis y/IgG/CIC did not show any statistically significant difference. MUC1/IgM/CIC correlated with Lewis y/IgM/CIC. By CASA, 9 samples with MUC1 values above the cut off were selected and IP was performed, followed by SDS-PAGE and Western blot; bands at 200 kDa were obtained with each MAb in all the samples. By IHC, with C14 MAb, 47.5%, 31% and 35% of malignant, benign and normal samples, respectively, showed positive reaction while all the samples were positive with anti-MUC1 MAb; in both cases, with a different pattern of expression between malignant and non malignant samples. CONCLUSION: Our findings support that in breast cancer there was a limited humoral immune response through Lewis y/IgM/CIC levels detection which correlated with MUC1/IgM/CIC. We also found that Lewis y might be part of circulating MUC1 glycoform structure and also that Lewis y/CIC did not correlate with Lewis y expression.

The use of carbohydrate antigens for the preparation of vaccines for therapy in breast cancer.

Immunotherapy of human breast cancer is a rapidly growing experimental area based on peptidic as well as carbohydrate tumor-associated antigens. Cell surface carbohydrates are characteristic of different stages of normal development and differentiation; distinct carbohydrates are expressed in tissue- and cell-specific manners during those processes. Under pathological conditions such as neoplasia, changes in carbohydrate structures can almost always be present. In the successful development of carbohydrate anticancer vaccines, adjuvant and carrier molecules that promote the presentation of an antigen to the immune effector cells are prominent factors. At present, keyhole limpet hemocyanin (KLH) is the most widely used carrier protein in cancer immunotherapy, and QS-21 is the adjuvant most widely employed. In breast cancer, the immunogenicity of TF, Tn, STn and globo H antigens has been explored in different clinical trials. Approaches including vaccines against STn and globo H are presently being assayed with expectancy. From the experience obtained, it is possible to conclude that: 1) the employment of glycolipopeptide with clustered epitopes seems to be more effective than that of related structures with single epitopes in inducing antitumor cell antibodies; 2) the conjugation to carriers is best achieved using KLH; 3) totally synthetic constructs can be better immunogens in conjunction with a suitable adjuvant such as QS-21; in some cases, this adjuvant leads to a bypass in the need for specific T-cell help to stimulate IgG as well as IgM antibodies. In other cases, a T-cell mediated immune response is obtained, and (4) the development of a totally synthetic vaccine would greatly facilitate the production of the vaccine for large scale clinical trials with the attainment of regulatory approval.