Pilot Study: Immune Checkpoints Polymorphisms in Greek Primary Breast Cancer Patients.

Background: Breast cancer is the most prevalent and second leading cause of cancer-related death in women worldwide. Despite early detection and better treatment therapies, 30% of early-stage breast cancer patients still develop recurrent disease. Breast cancer is a heterogeneous disease comprising several molecular subtypes, commonly classified into clinical subtypes based on the hormone receptor status. These subtypes included luminal A and luminal B, which have different prognoses. Breast cancer development and progression involve many factors. Polymorphisms of PD-1, PD-L1, and PD-L2 genes have been previously associated with high risk and prognosis of cancer. However, no studies have associated PD-1, PD-L1, and PD-L2 polymorphisms with primary breast cancer subtypes. Hence, this study evaluated functional single nucleotide polymorphisms of PD-1, PD-L1, and PD-L2 with primary breast cancer subtypes, luminal A, and luminal B. In addition, we evaluated the PD-L1 protein expression in relation to primary breast cancer subtypes and stages. Results: There were no significant differences in the allele frequencies of PD-1 polymorphisms (rs2227981 G>A, rs7421861 A>G, and rs11568821 C>T) and PD-L1 polymorphisms (rs10815225 C>T and rs2282055 T>G) when compared with the general European population. However, a significant difference was detected in one of the PD-L2 polymorphisms (rs1009759 A>G), with the G allele higher in breast cancer patients than in the general European population. A higher prevalence of the T allele of PD-L1 polymorphism rs2282055 T>G was observed in luminal B breast cancer patients compared with luminal A. No significant difference was detected in other polymorphisms. We also observed that the PD-L1 rs2282055 TT genotype was more prevalent in luminal B breast cancer patients compared with luminal A. Our results found no association of the selected SNPs in the PDCD1 gene with breast cancer risk. Similarly, the protein expression data showed that PD-L1 and PD-L2 are associated with an aggressive phenotype, Luminal B, and advanced breast cancer stage. Conclusion: These findings suggest that immune checkpoint polymorphisms are associated with the risk and subtypes of breast cancer.

Estrogen receptors ß1 and ß2 are associated with distinct responses of estrogen receptor α-positive breast carcinoma to adjuvant endocrine therapy.

Our purpose was to assess whether and how ERß1 and/or ERß2 expression status could predict response of early stage ERα-positive breast carcinoma to adjuvant endocrine therapy (AET). ERß1 and ERß2 expression were determined using immunohistochemistry. ERß1- and ERß2-positivity were derived from receiver operating characteristic analysis and the median percentage of immunostained tumor cells, respectively. Patients with recurrent disease were grouped according to whether they relapsed within 4 years or after 4 years from surgery. The predictive significance of ERß1 and ERß2 was determined using Kaplan-Meier survival analysis and Cox proportional hazards regression analysis. ERß1-positivity in the first-4-year relapse patient group was lower and ERß2-positivity in the post-4-year relapse group was higher compared with no-relapse group. ERß1-positivity was associated with lower tumor size and longer first-4-year disease-free survival, while ERß2-positivity was associated with shorter post-4-year disease-free survival. Cox multivariate analysis including ERß1, ERß2 and established clinico-pathological variables showed that ERß1-positivity was an independent predictor of lower first-4-year risk of relapse. Thus, low ERß1 expression and high ERß2 expression are markers for identification of AET-treated ERα-positive breast carcinoma patients at risk of early and late relapse, respectively.

Dendritic cell immunotherapy: clinical outcomes.

The use of tumour-associated antigens for cancer immunotherapy studies is exacerbated by tolerance to these self-antigens. Tolerance may be broken by using ex vivo monocyte-derived dendritic cells (DCs) pulsed with self-antigens. Targeting tumour-associated antigens directly to DCs in vivo is an alternative and simpler strategy. The identification of cell surface receptors on DCs, and targeting antigens to DC receptors, has become a popular approach for inducing effective immune responses against cancer antigens. Many years ago, we demonstrated that targeting the mannose receptor on macrophages using the carbohydrate mannan to DCs led to appropriate immune responses and tumour protection in animal models. We conducted Phase I, I/II and II, clinical trials demonstrating the effectiveness of oxidised mannan-MUC1 in patients with adenocarcinomas. Here we summarise DC targeting approaches and their efficacy in human clinical trials.

Estrogen receptor ß2 is inversely correlated with Ki-67 in hyperplastic and noninvasive neoplastic breast lesions.

PURPOSE: The purpose of the study is to compare expression levels of ΕRα, ERß1, ERß2 and cell proliferation marker Ki-67 in normal breast and hyperplastic and noninvasive neoplastic breast lesions. MATERIALS AND METHODS: Routinely processed breast tissue from 55 patients provided 65 cases of noninvasive lesions, namely, epithelial hyperplasia of usual type (HUT), apocrine metaplasia (AM), atypical hyperplasia (AH) and ductal carcinoma in situ (DCIS) and 14 cases of adjacent normal breast tissue. Expression of ERα, ERß1 and ERß2 were evaluated using immunohistochemistry and correlated with Ki-67 labeling index (Ki-67 LI) and menopausal status of the patients. RESULTS: Compared with normal breast, ERα expression increased in low to intermediate-grade DCIS (DCIS1/2) and tended to decrease in high-grade DCIS, while ERß1 expression decreased in DCIS irrespective of grade. Mean Ki-67 LI in HUT, low to intermediate-grade DCIS and high-grade DCIS was higher than in normal breast. Higher than normal Ki-67 LI correlated with low ERß2 expression in the whole set of cases and with high ERα expression and low ERß2 expression in the postmenopausal cases of the subset that is generated by excluding AM and high-grade DCIS. Postmenopausal status correlated with low ERß1 expression in the whole set and with higher than normal Ki-67 LI, high ERα expression and low ERß1 expression in the subset. CONCLUSIONS: These findings are in accordance with an ERα-opposing oncosuppressive role of ERß2 in mammary carcinogenesis along the HUT-AH-DCIS1/2 pathway.

Up to 15-year clinical follow-up of a pilot Phase III immunotherapy study in stage II breast cancer patients using oxidized mannan-MUC1.

BACKGROUND: Targeting antigens to dendritic cell receptors has recently become a popular approach to inducing effective immune responses against cancer antigens. Almost 20 years ago, however, we demonstrated that targeting the mannose receptor on macrophages and dendritic cells leads to strong cellular immune responses. We conducted numerous human clinical trials demonstrating the effectiveness of oxidized mannan-MUC1 (M-FP) in MUC1(+) adenocarcinoma patients. In one trial, the 5-8-year follow-up of breast cancer patients vaccinated with M-FP was published previously; we now report here the 12-15-year follow-up. Details regarding the preparation of the vaccine, inclusion and exclusion criteria, immunotherapy and follow-up schedule, were published previously. RESULTS: The follow-up at 12-15 years showed that the recurrence rate in patients receiving placebo was 60% (nine of 15). In those receiving immunotherapy (M-FP), the rate was 12.5% (two of 16). The time of recurrence in the placebo group ranged from 7 to 180 months (mean: 65.8 months) and in the two patients of the vaccine group, the recurrence appeared at 95 and 141 months (mean: 118 months) after surgery. These findings are statistically significant (p = 0.02 for survival and p = 0.009 for percentage of patients cancer-free). All patients injected with M-FP showed no evidence of toxic effects or signs of autoimmunity during the 12-15-year follow-up. DISCUSSION: The preliminary evidence indicates that M-FP is beneficial in the overall survival of early-stage breast cancer patients. This long-term clinical follow-up of patients strongly supports the necessity for a large Phase III study of direct M-FP injection in early-stage breast cancer patients, to evaluate immunotherapy as an adjuvant treatment for breast cancer.

Estrogen receptor beta 2 is associated with poor prognosis in estrogen receptor alpha-negative breast carcinoma.

PURPOSE: Our aim was to examine the prognostic significance of ERbeta1 and ERbeta2 expression in ERalpha-negative breast carcinomas. MATERIALS AND METHODS: We evaluated nuclear and cytoplasmic expression of ERbeta1 and ERbeta2 by immunohistochemistry in a group of 95 patients with long follow-up. ERbeta1 and ERbeta2 status was correlated with clinicopathological parameters and disease outcome. Univariate and multivariate analyses of ERbeta1 and ERbeta2 as independent markers of disease-free survival (DFS) were carried out using the Cox proportional hazards model. RESULTS: Nuclear ERbeta1 (nERbeta1) and nERbeta2 status was positively correlated (p = 0.01). nERbeta1 positivity was associated with low histological grade (p = 0.01) in all patients and in the nERbeta2-positive subgroup (p = 0.03) but not in the nERbeta2-negative (p = 0.27). nERbeta2 positivity was associated with lymph node involvement and tumor relapse in all cases (p < 0.00 and p < 0.00, respectively) and in the nERbeta1-negative subgroup (p < 0.00 and p < 0.00, respectively) but not in the nERbeta1-positive (p = 0.09 and p = 0.20, respectively). nERbeta2 positivity was associated with poor DFS in all patients (log-rank p <0.00), in the post-menopausal patient subgroup (log-rank p = 0.02) and in the HER2-negative (triple-negative) subgroup (log-rank p = 0.04). Cox multivariate analysis including ERbeta1, ERbeta2 and established clinicopathological variables highlighted ERbeta2 as an independent marker of early disease recurrence (hazard ratio 4.87; 95 % confidence interval 1.07-22.3; p = 0.04). CONCLUSION: High nERbeta2 is an independent marker of early relapse in ERalpha-negative breast carcinoma, and in particular, in the nERbeta1-negative, the post-menopausal patient and the triple-negative subgroups. These findings suggest that inhibition of expression and/or function of ERbeta2 could improve disease outcome.

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer.

BACKGROUND: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting. RESULTS: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells. CONCLUSION: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

Nuclear localization of cytokeratin 8 and the O-linked N-acetylglucosamine-containing epitope H in epithelial cells of infiltrating ductal breast carcinomas: a combination of immunogold and EDTA regressive staining methods.

In a previous study, the authors have shown cytokeratin 8 (CK8) and epitope H ultrastructural localization in breast cancer cell nuclei. Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H. In this study, double immunogold labeling of CK8 and epitope H combined with the EDTA regressive staining method was applied in biopsy material from infiltrating ductal breast carcinomas and fibroadenomas, to localize both antigens in correlation to RNPs distribution in the nuclear subcompartments of cancer cells. CK8 and epitope H were localized mostly over condensed chromatin, whereas staining was weaker over interchromatin granule clusters and perichromatin fibers. These results revealed, the distribution of CK8 in the nucleus as MAR-binding protein, contributing in the organization of the nuclear DNA in the neoplastic cell, as well as the distribution of O-GlcNAc glycosylated polypeptides bearing the epitope H. The latter finding indicates that these polypeptides might play a significant role in the neoplastic behavior of breast cancer cells because they colocalize in the same nuclear subcompartments with proteins modified by O-GlcNAc, such as hnRNPs G and A1, RNA polymerase II, its transcription factors, and the oncogene product of c-myc. These proteins are known to participate in coordinated transcription/RNA processing events, contributing in the neoplastic behavior of breast cancer cells.

Pilot phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1 [ISRCTN71711835].

INTRODUCTION: Mucin 1 (MUC1) is a high molecular weight glycoprotein overexpressed on adenocarcinoma cells and is a target for immunotherapy protocols. To date, clinical trials against MUC1 have included advanced cancer patients. Herein, we report a trial using early stage breast cancer patients and injection of oxidized mannan-MUC1. METHOD: In a randomized, double-blind study, 31 patients with stage II breast cancer and with no evidence of disease received subcutaneous injections of either placebo or oxidized mannan-MUC1, to immunize against MUC1 and prevent cancer reoccurrence/metastases. Twenty-eight patients received the full course of injections of either oxidized mannan-MUC1 or placebo. Survival and immunological assays were assessed. RESULTS: After more than 5.5 years had elapsed since the last patient began treatment (8.5 years from the start of treatment of the first patient), the recurrence rate in patients receiving the placebo was 27% (4/15; the expected rate of recurrence in stage II breast cancer); those receiving immunotherapy had no recurrences (0/16), and this finding was statistically significant (P = 0.0292). Of the patients receiving oxidized mannan-MUC1, nine out of 13 had measurable antibodies to MUC1 and four out of 10 had MUC1-specific T cell responses; none of the placebo-treated patients exhibited an immune response to MUC1. CONCLUSION: The results suggest that, in early breast cancer, MUC1 immunotherapy is beneficial, and that a larger phase III study should be undertaken.

Transcriptional deregulation of VEGF, FGF2, TGF-beta1, 2, 3 and cognate receptors in breast tumorigenesis.

Angiogenesis is an important event during the neoplastic process and is induced by the secretion of numerous growth factors from endothelial cells. Vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (FGF2), and transforming growth factor-beta1, beta2, beta3 (TGF-beta1, 2, 3) and cognate receptors (TGF-betaRI, II, III) mRNA expression pattern was evaluated by RT-PCR in 25 breast cancer tissue samples and adjacent normal tissues, and correlated to clinicopathological features. Western blot analysis was performed to evaluate VEGF and TGF-beta1 protein levels. TGF-beta1 and TGF-beta3 mRNA levels were significantly different in breast cancer specimens of differing histology (ductal, lobular, other) (P=0.020 and P=0.043). No statistically significant difference was observed at the mRNA level of VEGF between normal and tumor tissues while elevated VEGF protein levels in tumors were associated with patients' menopausal status. A strong hormonal influence of ER and PR on TGF-beta mRNA expression was established. FGF2 transcript levels were substantially decreased in cancer compared to adjacent normal specimens (P=0.031). A disruption of mRNA co-expression patterns was observed in malignant breast tissues compared to controls. Western blot analysis revealed differences between VEGF and TGFbeta1 mRNA and their corresponding protein levels. A substantial negative correlation of TGF-beta1 protein and TGF-beta1 mRNA levels (P=0.016) was demonstrated by breast tissue-pair analysis. Summarizing, our findings suggest that transcript levels of the examined markers in breast cancer are associated with menopausal and hormonal status, while their co-expression pattern is altered in malignant tissues compared to controls. In addition the difference between VEGF and TGF-beta1 mRNA and protein levels observed, indicates that post-transcriptional mechanisms may regulate expression of these molecules in breast cancer.

Ultrastructural immunostaining of infiltrating ductal breast carcinomas with the monoclonal antibody H: a comparative study with cytokeratin 8.

The monoclonal antibody H (mAbH) detects an epitope consisting of an O-linked N-acetyl glucosamine (O-GlcNAc) and neighboring amino acids. This epitope has been found by using extracts from the MCF-7 human breast carcinoma cell line in immunoblotting experiments, on cytokeratin 8 (CK8) and 5 other polypeptides. In the present study, a double immunogold method was applied for the colocalization of CK8 and mAbH epitope on epoxy thin sections in 18 cases of infiltrating ductal breast carcinomas (IDBC) and in 6 cases of fibroadenomas, to study the accurate subcellular distribution of CK8 in breast cancer cells, as compared to the 5 polypeptides, recognized by mAbH. Furthermore, a detailed quantitative evaluation of the double immunolocalization over the cellular compartments of cancer cells was undertaken with the aid of a computerized image analysis system and the results were assessed statistically. The distribution pattern of CK8 and the mAbH epitope in the neoplastic mammary epithelial cells was similar in IDBC as compared to fibroadenomas, while the gold labeling intensity of these epitopes differed over the cellular compartments between malignant and benign biopsies. The results reveal the significance of the role of CK8 and O-GlcNAc glycosylation in the biology of the neoplastic mammary cells in vivo, determining their malignant potential.

Frequent LOH at hMLH1, a highly variable SNP in hMSH3, and negligible coding instability in ovarian cancer.

BACKGROUND: Molecular alterations such as DNA microsatellite instability (MSI/RER), single nucleotide polymorphism (SNP) and loss of heterozygosity (LOH) can occur throughout the genome and be associated with different types of cancer. In the present study, we aimed at detecting molecular alterations within the mismatch DNA repair genes in ovarian cancer (OC), using a sensitive, accurate and reliable protocol we have developed. MATERIALS AND METHODS: A combination of high-resolution GeneScan software analysis and automated DNA cycle sequencing was used. RESULTS: Negligible coding MSI was observed in selected sequences of mismatch DNA repair genes in our series of sixty-two ovarian tumors and matched blood DNAs. Unlike MSI, loss of one hMLH1 allele was scored in almost half (47%) of the informative cases. In addition, an SNP in hMSH3/intron 5 was found to be highly variable in OC patients. CONCLUSION: 1) Coding DNA instability is likely to be a very rare event in OC and, therefore, may not significantly contribute to the development of OC, and 2) the high frequency of LOH at hMLH1 observed in our ovarian tumors suggests that further investigation is needed to determine if such a trend exists in other mismatch DNA repair and/or critical genes.