Primary Ductal Breast Carcinomas Research Articles

Effects of neoadjuvant therapies on genetic regulation of targeted pathways in ER+ primary ductal breast carcinoma: A meta-analysis of microarray datasets

Breast cancer arises as a result of multiple interactions between environmental and genetic factors. Conventionally, breast cancer is treated based on histopathological and clinical features. DNA technologies like the human genome microarray are now partially integrated into clinical practice and are used for developing new “personalized medicines” and “pharmacogenetics” for improving the efficiency and safety of cancer medications. We investigated the effects of four established therapies—for ER+ ductal breast cancer—on the differential gene expression. The therapies included single agent tamoxifen, two-agent docetaxel and capecitabine, or combined three-agents CAF (cyclophosphamide, doxorubicin, and fluorouracil) and CMF (cyclophosphamide, methotrexate, and fluorouracil). Genevestigator 8.1.0 was used to compare five datasets from patients with infiltrating ductal carcinoma, untreated or treated with selected drugs, to those from the healthy control. We identified 74 differentially expressed genes involved in three pathways, i.e., apoptosis (extrinsic and intrinsic), oxidative signaling, and PI3K/Akt signaling. The treatments affected the expression of apoptotic genes (TNFRSF10B [TRAIL], FAS, CASP3/6/7/8, PMAIP1 [NOXA], BNIP3L, BNIP3, BCL2A1, and BCL2), the oxidative stress-related genes (NOX4, XDH, MAOA, GSR, GPX3, and SOD3), and the PI3K/Akt pathway gene (ERBB2 [HER2]). Breast cancer treatments are complex with varying drug responses and efficacy among patients. This necessitates identifying novel biomarkers for predicting the drug response, using available data and new technologies. GSR, NOX4, CASP3, and ERBB2 are potential biomarkers for predicting the treatment response in primary ER+ ductal breast carcinoma.

Expression of p27 and c-Myc by immunohistochemistry in breast ductal cancers in African American women.

Proteins p27 and c-Myc are both key players in the cell cycle. While p27, a tumor suppressor, inhibits progression from G1 to S phase, c-Myc, a proto-oncogene, plays a key role in cell cycle regulation and apoptosis. The objective of our study was to determine the association between expression of c-Myc and the loss of p27 by immunohistochemistry (IHC) in the four major subtypes of breast cancer (BC) (Luminal A, Luminal B, HER2, and Triple Negative) and with other clinicopathological factors in a population of 202 African-American (AA) women. Tissue microarrays (TMAs) were constructed from FFPE tumor blocks from primary ductal breast carcinomas in 202 AA women. Five micrometer sections were stained with a mouse monoclonal antibody against p27 and a rabbit monoclonal antibody against c-Myc. The sections were evaluated for intensity of nuclear reactivity (1-3) and percentage of reactive cells; an H-score was derived from the product of these measurements. Loss of p27 expression and c-Myc overexpression showed statistical significance with ER negative (p < 0.0001), PR negative (p < 0.0001), triple negative (TN) (p < 0.0001), grade 3 (p = 0.038), and overall survival (p = 0.047). There was no statistical significant association between c-Myc expression/p27 loss and luminal A/B and Her2 overexpressing subtypes. In our study, a statistically significant association between c-Myc expression and p27 loss and the triple negative breast cancers (TNBC) was found in AA women. A recent study found that constitutive c-Myc expression is associated with inactivation of the axin 1 tumor suppressor gene. p27 inhibits cyclin dependent kinase2/cyclin A/E complex formation. Axin 1 and CDK inhibitors may represent possible therapeutic targets for TNBC.

Loss of PTEN in high grade advanced stage triple negative breast ductal cancers in African American women

IntroductionPTEN is a tumor suppressor gene that inhibits cell proliferation by inhibiting the phosphoinositide 3-kinase (PI3 K) signaling pathway. The significance of PTEN mutations resulting in variable PTEN expression and their impact on prognosis of breast cancer is not well established. The objective of our study was to correlate the immunohistochemical expression of PTEN in the four major subtypes of breast carcinoma (Luminal A, Luminal B, HER2 positive, and Triple Negative) in a population of 202 African-American (AA) females with other clinicopathological factors. Materials and methodsTissue microarrays (TMAs) were constructed from FFPE tumor blocks from primary ductal breast carcinomas in 202 African-American females. Five micrometer sections were stained with a mouse monoclonal antibody against PTEN. The sections were evaluated for the intensity of cytoplasmic and nuclear reactivity. Bivariate analysis was done via χ2 analysis and survivability data was calculated via the generation of Kaplan-Meier curves (SPSS v19). ResultsLoss of PTEN expression was associated with ER negative (p = 0.021), PR negative (p = 0.024) and triple negative (p = 0.0024) breast ductal cancers. It was marginally associated with distant metastasis (p = 0.074). There was no association between PTEN loss and recurrence-free survival or overall survival. ConclusionIn our study, a statistically significant association between PTEN loss and the triple negative breast cancers (TNBC) was found in AA women. PTEN inhibits PI3 K resulting in decreased activation of downstream effector, mammalian target of rapamycin (mTOR). Loss of PTEN results in cell proliferation through activation of mTOR. Targeted therapy with mTOR inhibitors might be useful in the treatment of TNBC.

Abstract 607: PDL1 expression associated with triple negative breast ductal carcinomas in African American women

Abstract Background: Tumor cells avoid host immune response through expression of inhibitory T cell regulator, Programmed cell death ligand (PDL1).Our objective was to evaluate PDL1 expression by immunohistochemistry in the four major subtypes of breast carcinoma (Luminal A, Luminal B, HER2, and Triple Negative) in a population of 202 African-American (AA) women with other clinicopathological factors. Design: Tissue microarrays (TMAs) were constructed from FFPE tumor blocks from primary ductal breast carcinomas in 202 African-American females. Two separate 1 mm cores represented each case. Five micrometer sections were stained with rabbit monoclonal antibody against PDL1. The sections were evaluated for the percentage and intensity of membrane staining in both tumor and immune cells. Cut off was &amp;gt; 1%. Bivariate analysis was done via χ2 analysis and survivability data was calculated via the generation of Kaplan-Meier curves (SPSS v19). Statistical significance was assumed if p &amp;lt; 0.05. Results: PDL1 expression in only tumor cells, only immune cells and in combination was associated with ER negative (p&amp;lt;0.0001), PR negative (p&amp;lt;0.0001), Triple negative subtype (p&amp;lt;0.0001) and high grade (p&amp;lt;0.0001) breast ductal cancers. Conclusion: Our study finding of selective expression of PDL1 in triple negative breast ductal cancers (TNBC) in AA women suggests that inhibition of adaptive immune response is involved in the progression of these cancers. PDL1/PD1 is the inhibitory check point of immune response resulting in decreased T cell activation and cytokine production. Some studies have shown that PDL1 expression is associated with loss of PTEN and phosphatide inositol 3 kinase pathway (PI3K) activation. Our previous study showed loss of PTEN in TNBC. Combining PDL1 inhibitors with PI3K inhibitors may be useful therapy for aggressive TNBC. Citation Format: Farhan Khan, Yasmine Kanaan, Luisel J. Ricks-Santi, Rabia Zafar, Hagos Aymut, Tammey Naab. PDL1 expression associated with triple negative breast ductal carcinomas in African American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 607. doi:10.1158/1538-7445.AM2017-607

The nuclear pool of tetraspanin CD9 contributes to mitotic processes in human breast carcinoma.

Tetraspanin-29 (CD9) is an integral membrane protein involved in several fundamental cell processes and in cancer metastasis. Here, characterization of a panel of breast cancer cells revealed a nuclear pool of CD9, not present in normal human mammary epithelial cells. Antibody binding to surface CD9 of breast cancer cells resulted in increased nuclear CD9 fluorescence. CD9 was also found, along with a plasma membrane-associated pool, in the nuclei of all primary ductal breast carcinoma patient specimens analyzed. In all patients, about 40% of the total CD9 cellular fluorescence was nuclear. CD9 colocalized at the nuclear level with CEP97, a protein implicated in centrosome function, and with the IGSF8, an established CD9 partner in the plasma membrane. Co-immunoprecipitation of CEP97 and IGSF8 with CD9 was shown in nuclear extracts from breast cancer cells expressing a CD9-GFP fusion protein. However, by fluorescence resonance energy transfer (FRET) analysis, no direct binding of CD9 with either protein was observed, suggesting that CD9 is part of a larger nuclear protein complex. CD9 depletion or exposure of parental breast cancer cells to anti-CD9 mAb resulted in polynucleation and multipolar mitoses. These data indicate that the nuclear CD9 pool has an important role in the mitotic process. The discovery of a nuclear pool of CD9 has prognostic and/or therapeutic potential for patients with ductal carcinoma of the breast.

Pituitary Tumor-Transforming Gene 1 Is Expressed in Primary Ductal Breast Carcinoma, Lymph Node Infiltration, and Distant Metastases

Despite the advances that have been made in the fields of molecular and cell biology, there is still considerable debate explaining how the breast cancer cells progress through carcinogenesis and acquire their metastatic ability. The lack of preventive methods and effective therapies underlines the pressing need to identify new biomarkers that can aid early diagnosis and may be targets for effective therapeutic strategies. In this study we explore the pituitary tumor-transforming gene 1 (PTTG1) expression in primary ductal breast carcinoma, lymph node infiltration, and distant metastases. Three human cell lines, 184B5 derived from normal mammary epithelium, HCC70 from a primary ductal carcinoma, and MDA-MB-361 from a breast metastasis, were used for quantifying PTTG1 mRNA expression. The PTTG1 immunohistochemical expression was carried out on specimens taken from eight patients with invasive ductal breast cancer who underwent surgical treatment and followup for five years retrospectively selected. The study demonstrated that PTTG1 is expressed gradually in primary ductal breast carcinoma, lymph node infiltration, and distant metastases. Our findings suggest that the immunohistochemical evaluation of PTTG1 expression might be a powerful biomarker of recognition and quantification of the breast cancer cells in routine pathological specimens and a potential target for developing an effective immunotherapeutic strategy for primary and metastatic breast cancer.

Bone metastatic process of breast cancer involves methylation state affecting E-cadherin expression through TAZ and WWOX nuclear effectors

We investigated the involvement of Hippo-related pathways in bone metastasis from breast cancer, by evaluating E-cadherin expression downstream of WWdomain-containing oxidoreductase (Wwox) and transcriptional co-activator with PDZ-binding motif (TAZ). These nuclear effectors functioned in a context-specific fashion on transcriptome, depending on breast-cancer aggressiveness and methylation state. Wwox and E-cadherin were found in human specimens of bone metastasis but not in primary-ductal breast carcinoma, while TAZ showed a characteristic localisation in metastasis nuclei. Wwox and E-cadherin were higher in 1833-metastatic clone with bone avidity than in parental-MDA-MB231 cells, while only metastatic cells presented TAZ. In 1833 cells, a complex interplay of transcriptional signalling controlled E-cadherin transactivation. Wwox and TAZ activated Hypoxia inducible factor-1 (HIF-1) binding to E-cadherin promoter, while Peroxisome proliferator-activated receptor γ (PPARγ) intervened in E-cadherin transactivation favouring and preventing Wwox and TAZ functions, respectively. Methylation impinged on Hippo-related pathways through Wwox and TAZ, modifying metastatic phenotype. The protract exposure to 5-azacytidine (Aza), by affecting methylation state modified the shape of 1833 cells, becoming mesenchymal as that of MDA-MB231 cells and reduced spontaneous-Matrigel invasion. The underlying-molecular mechanisms were diminutions of E-cadherin, Wwox, matrix metalloproteases 2 and 9, HIF-1- and PPARγ-activities, inversely correlated to Snail and nuclear-TAZ accumulations. Exogenous WWOX restored 1833-Aza invasion. Thus, 1833-Aza cells permitted to study the role played by methylation in metastasis plasticity, being E-cadherin loss part of an entire-gene reprogramming. Of note, bone-metastasis formation in 1833-Aza xenograft was partially impaired, prolonging mice survival. In conclusion, the methylation-heritable changes seemed important for cancer progression to establish bone metastasis engraftment/growth, by affecting steps requiring homotipic and/or heterotypic-adhesive properties and matrix degradation.

Expression of 6 Common Antigenic Markers in Invasive Ductal Breast Carcinoma

Expression of estrogen (ER) and progesterone receptors, c-erbB-2 oncogene, mutant p53 antioncogene (mp53), e-cadherin adhesion, and apoptotic caspase-8 antigens in tumor relative to matched normal tissue specimens from 102 unselected patients with primary ductal breast carcinoma of various tumor grades was assessed by immunohistochemistry and correlated with patient's biologic and clinical features, such as age, menstrual status, age of menarche, tumor grade and diameter, the presence or absence of metastases, and number of infiltrated lymph nodes. We observed association of e-cadherin adhesion, ER and progesterone antigen marker expression with low histologic grade tumors and limited number of lymph node metastases and of c-erbB-2, mp53, and casp-8 antigen marker expression with high histologic grade tumors and increased number of lymph node metastases. We also observed strong correlation (P<0.05) between 4 of the 6 biomarkers and 4 of the 7 patient/tumor parameters examined. Our findings support the hypothesis of independent expression of these 4 strong biomarkers and reveal that nearly 40% of all breast tumor cases studied express similar proportions of 2 major phenotypic combinations [ER/c-erbB-2/mp53/casp-8: +/+/-/+ (19.6%) & +/-/-/+ (17.8%)]. We conclude that, in agreement with earlier reports, our findings support the diagnostic and potential prognostic value of these markers in the clinical assessment of breast cancer.

Distinct expression profiles of Notch-1 protein in human solid tumors: Implications for development of targeted therapeutic monoclonal antibodies

Biological therapies, such as monoclonal antibodies (mAbs) that target tumor-associated antigens have been considered an effective therapeutic approach in oncology. In considering Notch-1 receptor as a potential target, we performed immunohistochemistry on tissue microarrays to determine 1) whether the receptor is overexpressed in tumor cells as compared to their corresponding normal tissues and 2) the clinical significance of its expression levels in human breast, colorectal, lung and prostate cancers. We found that the expression of Notch-1 protein was overexpressed in primary colorectal adenocarcinoma and nonsmall cell lung carcinoma (NSCLC), but not in primary ductal breast carcinoma or prostate adenocarcinoma. Further analysis revealed that higher levels of Notch-1 protein expression were significantly associated with poorer differentiation of breast and prostate tumors. Strikingly, for NSCLC, the expression levels of Notch-1 protein were found to be inversely correlated with tumor differentiation and progression. For colorectal tumors, however, no correlation of Notch-1 protein expression was found with any tumor clinicopathological parameters, in spite of its overexpression in tumor cells. Our data demonstrated the complexity of Notch-1 protein expression in human solid tumors and further supported the notion that the roles of Notch-1 expression in tumorigenesis are highly context-dependent. The findings could provide the basis for development of distinct therapeutic strategies of Notch-1 mAbs for its applications in the treatment of suitable types of human cancers.

Transduction of E2F-1 TAT fusion proteins represses expression of hTERT in primary ductal breast carcinoma cell lines

BackgroundTelomerase expression is detectable in 81–95% of breast carcinomas and may serve as a therapeutic target. The objective of this study was to investigate repression of telomerase activity in primary ductal breast cancer cells through transcriptional regulation of the catalytic subunit hTERT. We hypothesized that inhibition of telomerase expression could be achieved via Tat mediated protein transduction of the repressor protein E2F-1.MethodsProtein purification techniques were refined to yield biologically active Tat fusion proteins (TFPs) capable of transducing the breast cancer cell lines HCC1937 and HCC1599. Cell lines were treated with wildtype E2F-1 (E2F-1/TatHA), mutant E2F-1 (E132/TatHA) and a control Tat peptide (TatHA) for 24 hours. Total RNA was isolated from treated cells, reverse transcribed and fold changes in gene expression for hTERT determined via real-time RT-qPCR.ResultsSignificant repression of the catalytic subunit of telomerase (hTERT) was present in both HCC1937 and HCC1599 cells following treatment with E2F-1/TatHA. In HCC1937 cells, hTERT was repressed 3.5-fold by E2F-1/TatHA in comparison to E132/TatHA (p < 0.0012) and the TatHA peptide controls (p < 0.0024). In HCC1599 cells, hTERT was also repressed with E2F-1/TatHA treatment by 4.0-fold when compared to the E132/TatHA control (p < 0.0001). A slightly lower hTERT repression of 3.3-fold was observed with E2F-1/TatHA in the HCC1599 cells when compared to the TatHA control (p < 0.0001).ConclusionThese results suggest that transduction of E2F-1/TatHA fusion proteins in vitro is an effective repressor of hTERT expression in the primary ductal breast cancer cell lines HCC1937 and HCC1599.

Characterization of a new cytogenetic subtype of ductal breast carcinomas.

About 50% of ductal breast carcinomas do not yield analysable karyotypes after short-term culturing. Comparison of the cytogenetic subset to the whole data set of tumors revealed that slightly hyperdiploid tumors, that is, with DNA index between 1.05 and 1.3, were under-represented in tumors for which cytogenetic analysis was successful. The purpose of this study was to determine whether the pattern of chromosome imbalances in this subset differs from that generally reported. Comparative genomic hybridization (CGH) was used on 43 primary ductal breast carcinomas selected for slight hyperdiploidy. Microsatellite instability (MSI), TP53 mutation and expression were also investigated. All tumors were MSI negative. In all, 18 tumors (42%) presented mostly unbalanced chromosome rearrangements and DNA amplifications, with only few or no whole chromosome gains (WCG). This pattern of chromosome imbalances corresponds to that described in most breast tumors by previous cytogenetic and CGH analyses. It was associated with TP53 mutation in 17% of tumors. Another subset of 17 tumors (39%) displayed different and new features, characterized by recurrent gains of whole chromosomes 5, 7 and 8 with few chromosome rearrangements, rare DNA amplifications and no TP53 mutation. Eight tumors with as many rearrangements as WCG were left unclassified. We propose that, beside a major pathway characterized by multiple chromosome rearrangements, there is a minor pathway mainly characterized by WCG.

The expression of proliferating antigen Ki67 and PCNA and products of suppressor gene p53 in primary invasive ductal carcinoma of female breast

The aim of the study was to examine the consecutive series of primary ductal invasive tumoure and fincl out: a) the expression of some biological cellular parameters as proliferating antigens Ki67 and PCNA and products of suppressor gene ~53; b) correlation between the levels of expression of those factors and classical prognostic factors. In 1994 eighty eight woman were operated for primary ductal carcinoma of the breast. The material used in the study: a) tissue samples fixed in 10% buffer formaline and paraffin embedded; b) tissue samples of the same turnours to estimate levels of steroids receptors. We used monoclonal antibodies (Dakopatts). The examination of steroids receptors was done with the usage of immunoenzymatic ELISA method. The kinetics for nuclear antigens: Ki67, PCNA, ~53, defined as index, has been estimated with half-quantitative method with the usage of Hogg’s grating (number of positive cells/1000 cells in 10 microscopic fields). The estimation has been done by two pathologists and the final result has been reported as arithmetical mean. Results: 1. High index IP PCNA, Ki67, I p53 and low levels of steroids receptors correlates with high histological malignancy (Bloom Ill); 2. Low index IP PCNA, Ki67, I ~53 correlates with high levels of steroids receptors; 3. The estimation of high levels of index IP PCNA, IP Ki67 can be helpful for separate tumours of high proliferating activity; 4. High index of I ~53 do not correlate with the diameter of tumour and with involvement of axillary lymph nodes; 5. It seems that the estimation of proliferating antigens together with products of suppressor gene ~53 might have greater prognostic value than the estimation of single of those factors.

P53 Protein Expression in Benign and Malignant Breast Lesions

To investigate p53 protein expression in imprints from benign and ductal breast carcinoma cases in relation to the histologic grade of malignancy and clinical stage. The study group consisted of 60 cases of primary ductal breast carcinomas and 20 benign lesions. For the demonstration of p53 protein expression, an immunocytochemical avidin-extravidin complex technique was applied. Monoclonal antibody p53 was used as the primary antibody, diaminobenzidine as the chromogen and hematoxylin as the counterstain. Forty-five percent of breast cancer cases showed positive expression of p53. A statistically significant difference in p53 protein expression was observed between grade 1, 2 and 3 carcinomas and stage I, II and III cases. All benign lesions were negative for p53 protein expression. Immunocytochemical p53 protein expression in cytologic material is a simple method that can be applied in routine cytologic laboratories for the identification of genetic alterations in primary ductal breast cancer.

Epidermal growth factor receptor expression in patients with ductal breast carcinoma. Comparison with clinicopathological factors and estrogen receptor status

ABSTRACT Epidermal growth factor receptor (EGFR) and estrogen receptor (ER) status were immunohistochemically evaluated in 80 primary ductal breast carcinomas. The comparison between EGFR status and tumor grading, tumor size, lymph node involvement and age of patients was performed. EGFR expression was found in 87.5% of carcinoma samples and 46.3% of tumors were positive for ER staining. The inverse relationship between EGFR and ER expression was confirmed and it was evident that a high concentration of EGFR exceeding 25% of a tissue section may inhibit or significantly limit the ability to express ERs. EGFR presence was significantly associated with poorly differentiated tumors. No correlation was found between EGFR positivity and tumor size, lymph node involvement or age of patients. Our results indicate that EGFR status does not seem to be a valuable independent prognostic indicator, but that the combination of EGFR positivity (above 25% of a tissue section) accompanied by a low or undetectable level of ER expression should be considered as a potential marker of a poor prognosis in patients with ductal breast carcinoma.

A simplified immuno‐enzymetric assay of the epidermal growth factor receptor in breast tumors: Evaluation in 282 cases

The epidermal growth factor receptor (EGF-R) is currently being investigated in human clinical oncology, and particularly in breast cancer, as a potential prognostic factor and a biological target for therapy. As an alternative to the 125I-EGF binding assay, we propose a sensitive immuno-enzymetric assay (IEMA) suitable for EGF-R assay in breast cancer. The assay is performed on solubilized extracts of the 105,000 g pellet of a tumor homogenate, allowing estrogen (ER) and progesterone (PR) assays to be made on the cytosol. The IEMA is performed on 96-well plates coated with the monoclonal anti-EGF-R antibody RI, through an anti-mouse IgG2b bridge. Trapped EGF-R in the samples is covered by a second monoclonal antibody (MAb), 528, and revealed by an anti-IgG2a-peroxidase complex. The sensitivity is 1 fmol/mg membrane protein, and the asay can be performed on tissue samples down to 50 mg. Two hundred and twenty primary ductal breast carcinomas assayed by this method showed a log normal distribution with a modal value of 8 fmol/mg prot., a mean at 18 and a median at 13 fmol/mg prot. EGF-R-rich tumors (greater than 20 fmol/mg prot.) were highly correlated with the absence of estrogen receptors and/or with a high histological grade (SBR III). Our data demonstrate the validity of the IEMA assay of EGF-R in human breast tumors.

In vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine.

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In vitro immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections. In this paper, a method for the detection of the labeling index of S-phase cells in normal and neoplastic tissues with in vitro BrdU labeling and standard immunohistochemical techniques using anti-BrdU-MAb and avidin-biotin peroxidase complex is described. We have employed this method in 47 human solid tumor samples, including squamous cell carcinomas of head and neck and cervix uteri, adenocarcinomas and malignant lymphomas, and also evaluated the possible application of the BrdU labeling index to estimate the cycling S-phase cells in neoplastic cell populations. In our data, the in vitro labeling index varied greatly in an individual case (3.56-29.2%) and from an area to an area within the same case. Squamous cell carcinomas of the head and neck showed higher LI than those of the cervix uteri. A case of metastatic carcinoma to the lung from ductal carcinoma of the breast had the highest LI (29.2%), in contrast to the low LI (3.6%) in the primary ductal carcinoma of breast.