Subset Of Breast Carcinomas Research Articles

Abstract 3030: Integrated in vitro and in silico screening of breast cancer reveals serine metabolism as a precision oncology target

Abstract Cellular metabolism sits at the forefront of growth and has been identified as an essential element for the progression of cancer. Large-scale chromosomal alterations and mutations incurred by cancerous cells can predispose cancer cells to dysfunctional metabolic programming independent of microenvironmental pressure. Cancer cells are adaptable to this reprogramming and find alternative measures to sustain elevated metabolic demands. However, this process reduces the number of functional metabolic pathways. Dysregulated metabolic pathways within cancerous cells create an opportunity for therapeutic intervention by targeting genetic chokepoints within these pathways. We analyzed metabolic landscape of breast cancers by combining genomic and transcriptomic data from cancer patient datasets, to identify metabolic pathways prone to dysregulation due to genomic influence. Our platform identified disruption in the serine biosynthesis, which some cancerous cells rely upon to sustain elevated growth in a subset of breast carcinoma. We utilized gain- and loss-of-function techniques in multiple breast cancer cell lines to recapitulate metabolic disruption observed in tumors. These in vitro screens are complemented by metabolic flux analysis and stable-isotope tracer analysis that reveal the vulnerable metabolic pathways in breast cancers with dysregulated serine metabolism. Our data demonstrate that specific metabolic targets can be identified through this integrated screening and validated empirically to confirm their significance as avenues for potential therapeutics. Citation Format: Mark Daniel Slayton, Abhinav Achreja, Jin Heon Jeon, Liwei Bao, Alisa Liu, Mason Collard, Deepak Nagrath, Sofia Merajver. Integrated in vitro and in silico screening of breast cancer reveals serine metabolism as a precision oncology target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3030.

Abnormal promoter DNA hypermethylation of the integrin, nidogen, and dystroglycan genes in breast cancer

Cell transmembrane receptors and extracellular matrix components play a pivotal role in regulating cell activity and providing for the concerted integration of cells in the tissue structures. We have assessed DNA methylation in the promoter regions of eight integrin genes, two nidogen genes, and the dystroglycan gene in normal breast tissues and breast carcinomas (BC). The protein products of these genes interact with the basement membrane proteins LAMA1, LAMA2, and LAMB1; abnormal hypermethylation of the LAMA1, LAMA2, and LAMB1 promoters in BC has been described in our previous publications. In the present study, the frequencies of abnormal promoter hypermethylation in BC were 13% for ITGA1, 31% for ITGA4, 4% for ITGA7, 39% for ITGA9, 38% for NID1, and 41% for NID2. ITGA2, ITGA3, ITGA6, ITGB1, and DAG1 promoters were nonmethylated in normal and BC samples. ITGA4, ITGA9, and NID1 promoter hypermethylation was associated with the HER2 positive tumors, and promoter hypermethylation of ITGA1, ITGA9, NID1 and NID2 was associated with a genome-wide CpG island hypermethylated BC subtype. Given that ITGA4 is not expressed in normal breast, one might suggest that its abnormal promoter hypermethylation in cancer is non-functional and is thus merely a passenger epimutation. Yet, this assumption is not supported by our finding that it is not associated with a hypermethylated BC subtype. ITGA4 acquires expression in a subset of breast carcinomas, and methylation of its promoter may be preventive against expression in some tumors. Strong association of abnormal ITGA4 hypermethylation with the HER2 positive tumors (p = 0.0025) suggests that simultaneous presence of both HER2 and integrin α4 receptors is not beneficial for tumor cells. This may imply HER2 and integrin α4 signaling pathways interactions that are yet to be discovered.

The role of TP53 pathogenic variants in early-onset HER2-positive breast cancer.

Breast cancer is the most frequent event in Li-Fraumeni syndrome associated with germline TP53 variants. Some studies have shown that breast cancers in women with Li-Fraumeni syndrome are commonly HER2-positive, suggesting that HER2 amplification or over-expression in a young woman may be a useful criterion to test for germline variants in the TP53 gene. We assessed the prevalence of germline TP53 variants by Sanger sequencing or next-generation sequencing in 149 women with HER2-positive breast cancer diagnosed until age 40. The pattern of HER2 amplification was evaluated with dual-probe FISH in a subset of breast carcinomas from patients with germline TP53 variants as compared with those of noncarriers. Among 149 women tested, three presented a deleterious TP53 germline variant (2%), with one patient diagnosed at age 31 and the other two with bilateral breast cancer at ages 29/33 and 28/32, respectively. Three of the 36 patients (8.3%) with the first breast cancer diagnosed at age 31 or younger presented a pathogenic TP53 variant. Additionally, all TP53 deleterious variant carriers had a first degree relative diagnosed with different early-onset cancers (frequently not belonging to the Li-Fraumeni syndrome tumor spectrum) diagnosed at age 45 or younger. Higher levels of HER2 amplification were found in breast carcinomas of TP53 pathogenic variant carriers than in those of noncarriers. Deleterious germline TP53 variants account for a small proportion of early-onset HER2-positive breast cancers, but these seem to have higher HER2 amplification ratios. All TP53 pathogenic variant carriers found in this study had the first breast carcinoma diagnosed at age 31 or younger and a first-degree relative with early-onset cancer. Further studies are needed to clarify if HER2 status in early-onset breast cancer patients, in combination with other personal and/or familial cancer history, is useful to update the TP53 testing criteria.

Abstract 5184: N-CoR2 epigenetically regulates adhesion-dependent branching morphogenesis and its loss correlates with malignant progression of the mammary gland

Abstract Branching morphogenesis depends upon extracellular matrix remodeling and dynamic cell-cell and cell-extracellular matrix interactions, although how this developmental program is coordinated remains unclear. Expression profiling of mammary tissue at different stages of branching morphogenesis combined with histological and functional studies identified the histone deacetylase (HDAC) regulator, nuclear receptor co-repressor 2 (N-CoR2), as a key orchestrator of adhesion-dependent branching morphogenesis in the mammary gland. Molecular studies revealed that N-CoR2 actively represses fibronectin (FN) and thrombospondin-1 (THBS-1) promoter accessibility in an HDAC-dependent manner. Consistently, histological analysis revealed that N-CoR2 is abundantly expressed in the luminal epithelial cells localized along the outer rim of invading terminal end buds (TEB) and that its expression is lowest at sites of TEB branching bifurcation. In addition, reducing N-CoR2 expression drove the scattering of human mammary acini in vitro and promoted promiscuous branching of humanized mouse mammary tissue in vivo, suggesting that downregulation of N-CoR2 is required for branching morphogenesis in mammary gland. Furthermore, downregulation of N-CoR2 in breast cancer cells increased the expression of both FN and THBS-1 and reduced the expression of E-cadherin and enhanced the migratory capability of breast cancer cells. Intriguingly, immunohistochemical and clinical correlative analysis indicated that loss of N-CoR2 correlates with perturbed cell-cell and enhanced cell-ECM adhesion and acquisition of an invasive phenotype and local lymph node metastasis in a subset of breast carcinomas. These findings suggest N-CoR2 may epigenetically regulate adhesion-dependent branching morphogenesis in the mammary gland and that its loss could promote malignant progression of some breast cancers. Citation Format: Shenq-Shyang Huang, Valerie M. Weaver, Kelvin K.C. Tsai. N-CoR2 epigenetically regulates adhesion-dependent branching morphogenesis and its loss correlates with malignant progression of the mammary gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5184.

MicroRNAs and Androgen Receptor: Emerging Players in Breast Cancer.

Breast cancer (BC) is the most common cause of cancer among women, with a high incidence rate occurrence every year worldwide despite advances in its management. BC is characterized by a spectrum of subtypes which respond differently to treatments due to their biological features, representing the main issue in the control of this type of malignancy. Androgen receptor (AR) is emerging as a target to investigate among hormone receptors, since it seems to play a role at various stages of development of specific BC subsets. For this reason, in recent years AR has become very important in the clinical practice, although its role remains controversial. A number of studies have proposed a correlation between microRNAs (miRNAs), a class of gene expression modulators, and AR in prostate cancer (PC), but there are still few evidences about the relationship between miRNAs and AR in BC. The purpose of this review is to present a state of the art scenario with consideration to the most recent discoveries about miRNAs involved in the AR associated pathogenesis of BC, in order to provide new insights into the role of miRNAs as key drivers in the modulation of AR, and possible actors in the development and progression of BC. Moreover, we consider findings about involvement of AR signaling in all stages of BC, highlighting its association with different subsets of breast carcinomas and with pre- and postmenopausal state of patients.

Abstract PD7-05: Correlation of CDH1 alterations and aberrant E-cadherin expression in lobular carcinomas

Abstract Background: E-cadherin (ECAD), encoded by the CDH1 gene, is expressed in the majority of invasive ductal carcinomas (IDC). In contrast, ECAD expression is lost in ˜90% of invasive lobular carcinomas (ILC) due to genomic alterations of CDH1. Immunohistochemical (IHC) staining for ECAD has thus increasingly been utilized to differentiate between ductal and lobular lesions. This study examines the correlation between CDH1 mutation profile, morphology and ECAD IHC status in invasive breast carcinomas (BC). Methods: The Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) platform is a hybrid capture next-generation sequencing assay to detect somatic alterations in exons and selected introns of 468 cancer-related genes. Invasive BC tested from 1/2014 – 5/2018 which yielded a CDH1 alteration were identified and only cases with ECAD IHC results were included. Histologic features were extracted from pathology reports. All available H&E/ECAD slides were reviewed by two pathologists to confirm the histology and immunophenotype. Results: CDH1 alterations were identified in 455 out of 4103 BC analyzed (11%) and ECAD IHC were available for 209 (46%) tumors. Most of the tumors were reported as ILC (70%, 147/209). The majority (77%, 161/209) were ECAD-negative but 23% (48/209) showed some ECAD staining. Table 1 summarizes the histologic subtypes and IHC results. Upon review of H&E/ECAD slides in cases with ECAD staining, the histologic subtype was re-classified in 52% (25/48) of cases (Table 2). For ECAD-negative BC, at least 61% (98/161) of CDH1 alterations were truncating mutations (frameshift deletion 47/161, nonsense 51/161) and 39% (63/161) were variants of unknown significance (VUS). In cases with ECAD staining, 48% (23/48) of CDH1 alterations were truncating mutations (frameshift deletion 12/48, nonsense 11/48) however 52% (25/48) were VUS. Conclusion: In this study, 88% (141/161) of cases with negative ECAD staining and CDH1 alteration were classified as ILC, confirming the known observation that CDH1 alteration and loss of ECAD expression is relatively specific for a lobular phenotype. The correlation is, however, imperfect. A subset of BC (23%; 48/209) with CDH1 alteration still exhibits ECAD expression. Morphologically this subset consists of ILC in 60% (29/48), IMC in 25% (12/48) and IDC in 15% (7/48). Our preliminary findings suggest that aberrant ECAD expression can occur in BC with morphologic features of ILC due to certain CDH1 alterations that most likely result in non-functional ECAD complex. BC with histologic features of ILC should therefore not be re-classified as IDC/IMC based solely on the status of ECAD expression. Given that ILC is clinically distinct from IDC, correct classification based on morphological and molecular features is prudent. Table 1:Histologic subtypes and ECAD IHC ILC (n = 147)IDC (n = 13)IMC (n = 49)ECAD staining Negative141020Strong, diffuse4135Heterogenous0020Reduced/weak204IMC: invasive mammary with mixed ductal/lobular features Table 2:Morphologic review of cases with ECAD stainingECAD stainingOriginal DX (n)Morphology Review DX ILCIDCIMCStrong, diffuseILC (4)301 IDC (13)670 IMC (5)500HeterogenousIMC (20)10010Reduced/weakILC (2)200 IMC (4)301 Citation Format: Grabenstetter A, Mohanty AS, DeLair DF, Tan LK, Ross DS. Correlation of CDH1 alterations and aberrant E-cadherin expression in lobular carcinomas [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-05.

Molecular features of adenoid cystic carcinoma with an emphasis on microRNA expression.

Molecular features of adenoid cystic carcinoma with an emphasis on microRNA expression.

Neuroendocrine Tumors of the Breast.

A small but increasingly recognized and studied subset of breast carcinomas are characterized by neuroendocrine (NE) differentiation. As with nearly all forms of breast neoplasia, NE tumors are characterized by considerable heterogeneity in microscopic appearance and clinical aggressiveness. About half of NE breast carcinomas recapitulate the histological spectrum typical of their counterparts in other organ systems, varying from "carcinoid-like" to small cell carcinoma, with most representing intermediate grade tumors. Despite NE morphology, these tumors have a high frequency of estrogen receptor expression. Clinical outcomes of women with NE breast carcinomas are reliably grade and stage dependent. Tumors associated with "solid papillary" differentiation comprise the remaining cases of NE breast neoplasia. Solid papillary carcinoma is an intrinsically low grade/favorable prognosis class of breast neoplasia that usually presents in post-menopausal age groups. About half of solid papillary carcinoma present as a distinctive pattern of ductal carcinoma in situ that may be difficult to recognize owing to its close resemblance to florid proliferative lesions. Invasive solid papillary carcinomas are characterized by a variety of histological patterns and often show mucinous differentiation. Future studies are necessary to better define the histogenesis, optimal classification, and improved directed therapies for NE breast neoplasia.

Immunoexpression of lactoferrin in triple-negative breast cancer patients: A proposal to select a less aggressive subgroup.

Triple-negative breast cancer (TNBC) indicates a subset of breast carcinomas that does not express estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). According to the literature, TNBCs are aggressive tumors, characterized by a high incidence of recurrence and a high risk of disease progression. Lactoferrin (LF) is a single-chain, iron-binding glycoprotein of ~700 amino acids, which is involved in a wide range of biological activities, including iron-trafficking and carcinogenesis. The present study aimed to assess LF expression in human TNBC samples and the possible correlation with clinico-pathological parameters associated with biological aggressiveness. LF immunohistochemical expression was investigated in formalin-fixed, paraffin-embedded samples of human TNBC. Cases were analyzed according to an intensity distribution (ID) score, and only those showing an ID score of >2 were considered as positive for LF. LF immunostaining was encountered in 26.15% cases. A significant correlation was found between LF expression and a low Ki-67 labeling index (P=0.040), the absence of recurrence (P=0.010) and alive status (P=0.020). LF may assist in identifying a subset of TNBC with less aggressive biological behavior. The meaning of LF expression in TNBC remains unclear and is controversial. The present findings indicated that LF expression is correlated with a low growth fraction in these tumors. Thus, it is possible that the inhibition of the LF axis may be a valid therapeutic target for TNBC, and this should be confirmed by future studies.

Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer.

Estrogen receptor (ER) expression and Her-2 amplification define specific subsets of breast tumors for which specific therapies exist. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. Using microarray analysis and immunohistochemistry, we determined that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases. Interestingly, the subset of ER-negative breast carcinomas overexpressing Skp2 are also characterized by high tumor grade, negativity for Her-2, basal-like phenotype, high expression of certain cell cycle regulatory genes, and low levels of p27 protein. We also found that Skp2 expression is cell adhesion-dependent in normal human mammary epithelial cells but not in breast cancer cells and that an inhibition of Skp2 induces a decrease of adhesion-independent growth in both ER-positive and ER-negative cancer cells. Finally, forced expression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens. We conclude that Skp2 has oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (ER- and Her-2 negative) for which Skp2 inhibitors may represent a valid therapeutic option.

A newly developed mouse monoclonal SOX10 antibody is a highly sensitive and specific marker for malignant melanoma, including spindle cell and desmoplastic melanomas.

Recent immunohistochemical studies have demonstrated Sry-related HMG-Box gene 10 (SOX10) expression in malignant melanomas, malignant peripheral nerve sheath tumors, a subset of breast carcinomas, and gliomas. SOX10 has shown important clinical utility in its ability to detect desmoplastic and spindle cell melanomas. To date, most publications have employed a research use-only goat polyclonal SOX10 antibody for immunohistochemical staining. To describe the development of a new mouse monoclonal SOX10 antibody (BC34) and evaluate its immunohistochemical staining profile in a wide range of normal and neoplastic tissues, with an emphasis on melanoma. SOX10 antibody was optimized for staining using a polymer detection system and visualization with diaminobenzidine. In normal tissues, SOX10 was expressed in skin melanocytes and eccrine cells, breast myoepithelial and lobular epithelial cells, salivary gland myoepithelial cells, peripheral nerve Schwann cells, and central nervous system glial cells. SOX10 was expressed in 238 of 257 melanomas (92.6%), including 50 of 51 of both spindle cell and desmoplastic melanomas (98%). SOX10 was expressed in 100% of nevi (20 of 20) and schwannomas (28 of 28). In other neoplasms, SOX10 was expressed in 18 of 109 invasive ductal breast carcinomas (16.5%). All other carcinomas were negative for SOX10. SOX10 was identified in 25 of 52 central nervous system neoplasms, primarily in astrocytomas (22 of 41; 53.7%), and in 4 of 99 various sarcomas examined (4.0%). The newly developed mouse monoclonal SOX10 antibody BC34 is highly sensitive and specific for malignant melanoma, including desmoplastic and spindle cell variants, and appears highly suitable for clinical use.

Therapeutic targeting of ERBB2 in breast cancer: understanding resistance in the laboratory and combating it in the clinic.

ERBB2 gene amplification occurs in about one quarter of breast carcinomas (BCs) and identifies a distinct clinical subset of BC. The introduction in the clinic of Trastuzumab, a humanized monoclonal antibody (mAb) directed to the ERBB2 extracellular domain, has had a great impact on the therapeutic management of ERBB2+ BC. Yet, not all patients respond to Trastuzumab and resistance develops also among patients that initially benefit from Trastuzumab-based regimens. Pre-clinical studies have discovered several mechanisms through which tumor cells may escape from Trastuzumab-mediated ERBB2 inhibition. These include rewiring of the ErbB signaling network, loss of ERBB2 expression, expression of ERBB2 isoforms refractory to Trastuzumab inhibition, vicarious signaling by non-ErbB tyrosine kinases and constitutive activation of downstream signaling routes, such as the PI3K pathway. While the relative contribution of each of these mechanisms to establishing Trastuzumab resistance in the clinical setting is not fully understood, much attention has been focused on abating resistance by achieving complete blockade of ERBB2-containing dimers. This approach, propelled by the development of novel anti-ERBB2 therapeutics, has led to the recent approval of Lapatinib, Pertuzumab and T-DM1 as additional anti-ERBB2 therapeutics in BC. However, full success is far from being achieved and resistance to ERBB2 targeting remains a relevant problem in the clinical management of BC. Herein, we provide an overview of biological and molecular bases underpinning resistance to ERBB2 therapeutics in BC, discuss outstanding issues in the field of ERBB2 therapeutic targeting and elaborate on future directions of translational research on ERBB2+ breast cancer.

Carcinoma arising in microglandular adenosis of the breast: triple negative phenotype with variable morphology

Carcinoma arising in microglandular adenosis (MGACA) is an extremely rare subtype of breast carcinoma. In this study, clinicopathological analysis of MGACA from 11 Chinese patients was conducted. Microscopically, all cases showed a spectrum of structure and glandular proliferations ranging from MGA to atypical MGA (AMGA) to MGACA. Carcinoma components were composed of high grade ductal carcinoma in situ (DCIS) in one case and invasive carcinoma in 10 cases. Invasive carcinomas were grade 3 in 10 tumors and grade 2 in one. Invasive components in five of 10 cases were composed of invasive carcinoma of no special type (NST), and one case showed prominent oncocytic change in a considerable portion of epitheliums. In five cases, invasive components were mixed of NST and matrix-producing carcinoma (MPC). All epitheliums in 11 cases were triple negative (ER-, PR-, HER2-), and diffuse positive for CK and S-100 protein. Ki-67 proliferation index ranged from 30% to 70%. No myoepithelial cells were demonstrable from MGA to invasive components with immunohistochemical staining for P63 and calponin. PAS or reticulin stain showed the presence of a basement membrane around glands in MGA, AMGA, DCIS, and its absence in invasive components. Follow-up time ranged from 8 to 62 months. One patient developed a lung metastasis 24 months after surgery, 10 patients have been alive without recurrence. Our study revealed that MGACA is a distinct subset of breast carcinoma, with triple negative phenotype, high grade nuclear and variable morphology. Despite histopathologic and immunohistochemical features usually associated with a poor prognosis, MGACA seems to have a relatively favorable outcome.

JMJD1C demethylates MDC1 to regulate the RNF8 and BRCA1–mediated chromatin response to DNA breaks

Chromatin ubiquitylation flanking DNA double-strand breaks (DSBs), mediated by RNF8 and RNF168 ubiquitin ligases, orchestrates a two-branch pathway, recruiting repair factors 53BP1 or the RAP80-BRCA1 complex. We report that human demethylase JMJD1C regulates the RAP80-BRCA1 branch of this DNA-damage response (DDR) pathway. JMJD1C was stabilized by interaction with RNF8, was recruited to DSBs, and was required for local ubiquitylations and recruitment of RAP80-BRCA1 but not 53BP1. JMJD1C bound to RNF8 and MDC1, and demethylated MDC1 at Lys45, thereby promoting MDC1-RNF8 interaction, RNF8-dependent MDC1 ubiquitylation and recruitment of RAP80-BRCA1 to polyubiquitylated MDC1. Furthermore, JMJD1C restricted formation of RAD51 repair foci, and JMJD1C depletion caused resistance to ionizing radiation and PARP inhibitors, phenotypes relevant to aberrant loss of JMJD1C in subsets of breast carcinomas. These findings identify JMJD1C as a DDR component, with implications for genome-integrity maintenance, tumorigenesis and cancer treatment.

Molecular Detection of Epstein-Barr virus in Breast Cancer

Background: The association of Epstein Barr virus (EBV) with breast carcinomas (BCs) is still in controversy. The aim of this study was to clarify the association of EBV & BC in Egyptian females and to assess its role as potential contributor to the development and behavioral alteration of BC. Subjects & methods: EBV-DNA was detected using PCR on breast tissue from 40 female patients with primary invasive BC; ductal (n=32) and lobular (n=8) and 20 age matched females undergoing reduction mammoplasties as control. Results: EBV-DNA was detected in 8/40 (20%) BC specimens. On the other hand all control specimens were negative As regards prognostic factors, no association was observed between EBV-DNA and patients' age, menopausal status and steroid receptor expression. However, significant associations were detected between the presence of EBV-DNA and other poor prognostic factors. All of the EBV-DNA positive BC were significantly associated with positive nodal status, where 7/8 cases showed more than three tumor-positive LN involvement. In spite of the small number of invasive lobular carcinoma included in this study there was a significant correlation between this histological type of poor prognosis and EBV-DNA detection rate where 4/8 (50%) of them were positive for EBV-DNA compared to 4/32 (12.5%) detection rate in invasive ductal carcinoma. A significant correlation was found between EBV-DNA detection rate in BC and high tumor grade of invasive ductal carcinoma; (100%, 1/1) association with grade III versus (9.67%; 3/ 31) with grade II. Conclusions: our results demonstrated the presence of the EBV genome in a considerable subset of BC in Egyptian patients. The virus was more frequently associated with bad prognostic factors. This indicates that EBV may play a role in the development and behavioral alteration of some aggressive BC.

Molecular Detection of Epstein-Barr virus in Breast Cancer

Background: The association of Epstein Barr virus (EBV) with breast carcinomas (BCs) is still in controversy. The aim of this study was to clarify the association of EBV & BC in Egyptian females and to assess its role as potential contributor to the development and behavioral alteration of BC. Subjects & methods: EBV-DNA was detected using PCR on breast tissue from 40 female patients with primary invasive BC; ductal (n=32) and lobular (n=8) and 20 age matched females undergoing reduction mammoplasties as control. Results: EBV-DNA was detected in 8/40 (20%) BC specimens. On the other hand all control specimens were negative As regards prognostic factors, no association was observed between EBV-DNA and patients' age, menopausal status and steroid receptor expression. However, significant associations were detected between the presence of EBV-DNA and other poor prognostic factors. All of the EBV-DNA positive BC were significantly associated with positive nodal status, where 7/8 cases showed more than three tumor-positive LN involvement. In spite of the small number of invasive lobular carcinoma included in this study there was a significant correlation between this histological type of poor prognosis and EBV-DNA detection rate where 4/8 (50%) of them were positive for EBV-DNA compared to 4/32 (12.5%) detection rate in invasive ductal carcinoma. A significant correlation was found between EBV-DNA detection rate in BC and high tumor grade of invasive ductal carcinoma; (100%, 1/1) association with grade III versus (9.67%; 3/ 31) with grade II. Conclusions: our results demonstrated the presence of the EBV genome in a considerable subset of BC in Egyptian patients. The virus was more frequently associated with bad prognostic factors. This indicates that EBV may play a role in the development and behavioral alteration of some aggressive BC.

Investigation of human JC and BK polyomaviruses in breast carcinomas

We have previously showed the presence of the simian virus 40 (SV40) and the mouse mammary tumor virus (MMTV)-like in a significant proportions of Tunisian breast carcinomas. However, to date there are no published studies concerning evaluation of the possible implication of the human polyomaviruses JC (JCV) and BK (BKV) in breast carcinomas. The presence of JCV and BKV DNA was investigated by PCR in a 123 primary breast carcinomas and matched adjacent non-tumor breast tissues. The results were correlated to clinicopathological and virological parameters. JCV T-antigen DNA was detected in 23% of breast carcinoma cases; however, all cases were negative for BKV. JCV T antigen PCR products were further confirmed as authentic JCV genome by direct sequencing. JCV was found in invasive ductal carcinomas (28/112 cases) but not in invasive lobular carcinomas (0/5) or medullary carcinomas (0/6). JCV DNA presence correlates inversely with the expression of estrogen (P = 0.022) and progesterone (P = 0.008) receptors. JCV DNA presence correlates also with "triple negative" phenotype (P = 0.021). With regard to virological data, a trend toward an inverse correlation was noted between the presence of JCV and SV40 (P = 0.06). Moreover, significant correlation was found between multiple viral infection (JCV, and/or SV40, and/or MMTV-like in the same tumor) and "triple negative" phenotype (P = 0.001) and also with p53 accumulation (P = 0.028). To the best of our knowledge, this is the first study demonstrating the presence of JCV in a subset of breast carcinomas. Also our results suggest that "triple negative" breast carcinomas are viral-related tumors.

Abstract 3581: Characterization of human breast cancer cells with acquired resistance to the EGFR/ErbB-2 tyrosine kinase inhibitor lapatinib

Abstract The epidermal growth factor receptor (EGFR) and the ErbB-2 receptor are co-expressed in a subset of breast carcinomas that show a more aggressive phenotype. The dual EGFR/ErbB-2 tyrosine kinase inhibitor lapatinib has been recently approved for the treatment of metastatic breast cancer patients whose tumors overexpress ErbB-2. We investigated the mechanisms of acquired resistance to lapatinib in breast cancer cells. For this purpose, we used as model the SK-Br-3 human breast carcinoma cell line that express high levels of ErbB-2, and moderate levels of EGFR and ErbB-3. We selected lapatinib-resistant SK-Br-3 cells by stepwise dose escalation of lapatinib. After six months of selection, we isolated SK-Br-3 cells that were able to grow in 1 μM lapatinib. The IC50 of lapatinib for parental SK-Br-3 cells was 140 nM, whereas the IC50 for the lapatinib-resistant SK-Br-3 derived cell line (Lap-R) was > 4 μM. Treatment with lapatinib significantly inhibited the activation of EGFR, ErbB-2 and ErbB-3 in both parental and Lap-R SK-Br-3 cells. However, following treatment with lapatinib, Lap-R cells showed a persistent activation of AKT and MAPK, which in contrast were completely inhibited in parental SK-Br-3 cells, as measured by both western blotting and a Luminex-based phosphoprotein array. In addition, treatment with lapatinib produced a slight activation of c-Src in parental cells, whereas significant levels of c-Src activation were detected in Lap-R cells cultured in presence of lapatinib. Lap-R cells also showed higher levels of expression of the chemokine receptor CXCR-4 than parental cells. In agreement with these findings, Lap-R cells showed a significantly higher ability to invade a matrigel-coated membrane as compared with parental SK-Br-3 cells, as measured with a Boyden-chamber based colorimetric invasion assay. Preliminary findings demonstrate that inhibition of c-Src with the selective src kinase inhibitor saracatinib produces a more significant inhibition of Lap-R cell invasion as compared with parental SK-Br-3 cells. Taken together, these findings suggest that activation of c-Src signalling might play a role in acquired resistance to lapatinib in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3581. doi:10.1158/1538-7445.AM2011-3581

Abstract 5142: VEGFA gene amplification and protein expression in breast cancer

Abstract Background: The vascular endothelial growth factor A (VEGFA) protein is a chemical signaling molecule that is known to be a major factor in the induction of angiogenesis during tumor initiation and progression, and is also a target of anti-angiogenic therapies. Recently, we discovered the genomic amplification of the VEGFA gene in a small subset of colorectal cancers. Aim of this study was to investigate the presence of VEGFA gene amplification in breast cancer and to determine its potential impact on VEGFA protein expression. Methods: VEGFA gene amplification was evaluated by fluorescence in situ hybridization (FISH) on a multitumor tissue microarray (MTMA) comprising 3417 tissue samples from more than 100 different tumor types. Further, a small tissue microarray was constructed from breast carcinoma samples whose VEGFA protein concentration had been previously quantified by chemiluminescence immunoassay. In order to interrogate tissue heterogeneity, VEGFA gene amplification was also analyzed on large tissue sections from 70 primary breast cancers. Results: We detected VEGFA gene amplification in 2% of the breast cancer samples from the MTMA and in 5% of the breast cancer samples with known VEGFA protein concentration. In addition, 8% of the samples (5 out of 70) were characterized by a high polysomy. Interestingly, elevated VEGFA gene copy number was strongly correlated with higher VEGFA protein levels (p<0.0001). Conclusions: VEGFA gene amplification defines a small subset of breast carcinomas with elevated VEGFA protein expression. Our data suggest that FISH analysis of VEGFA could represent an additional evaluation system for the identification of breast cancer patients who might benefit from anti-VEGFA therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5142. doi:10.1158/1538-7445.AM2011-5142

Abstract 3135: Notch 1 and notch 4 biomarker expression in triple-negative invasive breast cancer

Abstract BACKGROUND: Triple-negative breast cancer (TNBC) is a high-grade tumor showing lack of expression of estrogen and progesterone receptors and human epidermal growth factor receptor (HER-2/neu). Unlike hormone receptor and HER-2/neu positive breast carcinomas, there is no targeted therapy for TNBC. The objective of this study is to investigate Notch 1 and Notch 4 biomarker expression in TNBC and to correlate the expression with known prognostic factors. DESIGN: We performed a retrospective chart review of 29 TNBC during 1998-2007. Data collected included age at diagnosis, histological type and grade, Ki67 proliferation rate, and patient survival status. Formalin-fixed paraffin embedded tissue blocks from each TNBC was selected. Notch 1 and Notch 4 expression were analyzed by immunohistochemical method (Santa Cruz Biotechnology Inc, USA). The intensity of staining for Notch 1 and 4 expressions and the sub-cellular localization of these receptors (cytoplasmic or nuclear) in tumor epithelial cells and vascular endothelial cells were noted. Only strong cytoplasmic and/or nuclear staining was documented as positive for Notch expression. Student's t test and chi-square test were applied for data analysis. RESULTS: Age of the patients ranged from 37 to 83 years (mean 57.7). Twenty-seven tumors were infiltrating ductal carcinomas (93.1%) and 2 were infiltrating papillary carcinomas (6.9%). Twenty-one cases were grade 3 (72.4%), 7 were grade 2 (24.2%) and one was grade 1 (3.4%). Unfavorable Ki67 proliferation rate (>20%) was seen in 26 (90%), borderline rate (10-20%) in 2 (6.9%) and favorable rate (<10%) in one (3.1%). Notch 1 expression was evaluable in 25 and Notch 4 in 29 cases. Notch 1 expression was present in 25/25 (100%) cases in tumor cells and in vascular endothelial cells. Notch 4 was expressed in tumor cells in 21/29 (73%) and in vascular endothelial cells in 29/29 (100%). Notch 4 expression was negative in tumor cells in 8/29 (27%) cases. Notch 1 and Notch 4 expression in tumor cells and in vascular endothelial cells are correlated with tumor grade (p<0.05) and Ki67 proliferation rate (p<0.05) in TNBC. Follow-up period ranged from 4-9 years (mean 5.3). Twenty-one patients are alive and well (72.4%), 2 are alive with disease (6.9%), 4 patients died of disease (13.8%), and 2 patients died of causes unrelated to disease (6.9%). CONCLUSION: This study demonstrates that (1) majority of triple-negative invasive breast cancers are morphologically high grade infiltrating ductal carcinomas with high Ki 67 proliferation rate, (2) both Notch 1 and Notch 4 receptors are over-expressed in tumor cells and in vascular endothelial cells in majority of this molecular subset of breast carcinoma, and (3) this subtype is associated with high overall survival rate with the currently available treatment. Targeting Notch signaling with gamma secretase inhibitors needs to be explored to further improve the survival rate of TNBC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3135. doi:10.1158/1538-7445.AM2011-3135