bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2-01-08: Imaging overview of metaplastic carcinomas of the breast; a study of 46 cases
Authors: Langlands FE , Dall B , Dodwell D , Shabaan AM , Sharma N
Published: August 2014

Background: Metaplastic breast cancer (MBC) is a rare form of breast cancer affecting <1% of breast cancer patients. Previous studies report that the size of MBC is greater than conventional invasive ductal carcinoma at presentation. This may reflect the rapid growth rate of MBC or the difficulty in diagnosis. Imaging characteristics of MBC are not well described but the few published reports suggest that MBC often mimics benign lesions on imaging. The aim of this study therefore is to review the clinical presentation, baseline imaging features, surgical management and final surgical pathology of MBC in a large UK breast cancer unit.

Methods: All female patients diagnosed with MBC during the time period Jan 2005-2012 were identified from the pathology database at Leeds University Teaching Hospitals Trust. Pathology reports were reviewed and data on MBC type (following the WHO classification), size, grade, nodal and molecular marker status was extracted. Mammography, Ultrasound (USS), CT and MRI features were recorded. Follow-up was complete until May 2013.

Results: 46 patients were identified with a median age of 61yrs and a 5yr survival of 32.6% (median 50 months). Of the 46 cases, 33 presented symptomatically (71.7%), 37 (80.4%) showed a mass on mammography of which 28 (75.7%) was ill defined, 4 (10.8%) well defined and 5 (13.5%) spiculated. All 46 cases had a mass on USS.

The imaging score was Bi-rads (Breast Imaging-Reporting and Data System) 5 in 23 cases, Birads 4c in 17 cases and 4a,b in 6 cases.

Thirteen patients had MRI. This was performed either to determine disease extent or for neoadjuvant chemotherapy monitoring. All but one of the tumours was seen as a solid mass with some central necrosis the other was seen as an area of nodular enhancement. Nine cases had a staging CT scan for metastatic disease which was negative in all cases.The average size was 28mm (range 11-75mm), 8 (17.3%) were histological grade 3, 5 (10.9%) cases had involved lymph nodes and 28 (60.8%) cases were triple receptor negative. Only 2 cases were HER2 positive.

Conclusion: In this series metaplastic cancers were symptomatic in >70%, tended to be large at the time of presentation and only 11% of cases had nodal involvement which is consistent with previous reports. Review of our radiology revealed that all modalities used (mammogram, USS and MRI) reliably showed characteristics suggestive of malignancy. Features suggestive of malignancy include spiculated or irregular ill defined masses on mammography or ultrasound and on MRI large irregular masses with central necrosis and rim enhancement following contrast. These are characteristic or suggestive of malignancy which is contrary to many previous studies of MBC that suggest MBC is difficult to pick up on imaging and commonly exhibits benign features.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-01-08.
Abstract P2-04-06: Successful results of a population-based organized mammography screening program in a developing country: The Turkish experience
Authors: Ozmen V , Ozkan-Gurdal S , Kayhan A , Ozaydin N , Cabioglu N , Ozcinar B , Aribal E
Published: August 2014

Objective: This study aims to determine the feasibility of a population-based organized mammography screening in a developing country (Bahcesehir, Istanbul, Turkey) and to find out the starting age for screening.

Material and methods: Women aged 40–69 were invited for screening at 2-year intervals starting in January 2009. Digital 2-view mammograms were obtained, and mammograms were double read by two independent radiologists who were experienced in breast imaging. The women were recalled in consensus for additional work-up including spot compression/magnification mammograms or breast ultrasound. Outcomes were measured from final assessment or histopathologic confirmation. The participation rate for second round, recall rate, number of biopsies, the women diagnosed with cancer and patient and tumor characteristics were evaluated.

Results: Between January 2009 and January 2013, 5938 (89.4%) of 6640 women aged between 40-69 years old accepted mammographic invitation. Most of them (58%) were at 40-49 age group, and the average 2-year attendance for second rounds were 82.6% and 81.2%, respectively. Spot recall rates were 16.8% and 25.6% for the first and second rounds, respectively. Core biopsy was required in 108 patients (1.8%), whereas BC was diagnosed in 50 patients (0.84%). Twenty-four patients (48%) detected with BC were at 40-49 age group. There were 39 patients (78%) with invasive BC and 11 patients (22%) with ductal carcinoma in situ (DCIS). Stages of 39 patients with invasive BC were Stage I (n = 26; 52%), II (n = 10; 20%) and III (n = 3; 6%), respectively. The majority of patients (88%) had breast conserving surgery, and sentinel lymph node biopsy only (72%) as axillary procedure. Of invasive cancers, 92% were found to be hormone receptor positive, and 59% had low Ki67 levels (≤15). However, HER2-neu positivity (8%) along with triple negative cancers (3%) were less frequently detected.

Conclusion: These findings suggest the prevailing view that mammographic screening increases early breast cancer detection rate which has a less aggressive biology. Screening mammography program can be successfully implemented in a developing country, Turkey effectively. However, more experienced dedicated breast cancer screening team and continuous increasing efforts are required to decrease our relatively high recall rates.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-04-06.
Abstract P2-05-03: Role of tumor immune environment in tumor initiation and growth rates in mouse mammary tumor models
Authors: Stanton S , Gadd E , Rastetter L , Slota M , Koehnlein M , Treuting P , Disis M
Published: August 2014

The role of the tumor immune environment is critical in human breast cancer tumorigenesis and response to therapy: tumors with a Th1 immune activating environment (high cytotoxic CD8+ T cells and low FoxP3 and myeloid derived suppressor cells (MDSC)) show improved response to chemotherapy and improved disease free survival and prognosis, where tumors with a Th2 immune suppressive environment (increased FoxP3, increased MDSC, and decreased CD8 T cells) shows poor response to chemotherapy and worse disease free survival and prognosis. There has not been good correlation with animal models of mammary tumorigenesis and human response to chemotherapy because in mouse models (1) tumor implant models do not show the tumor immune infiltration seen in human tumors (2) xenograph models are not immunocompetent (3) the tumor immune infiltration has not been evaluated the spontaneous mouse mammary models. We evaluated the tumor immune environment in relation to time to tumor development and rate of tumor growth in two commonly used transgenic mouse models: TgMMTV-neu and C3(1)Tag to better define role of the tumor immune environment in tumorigenesis in spontaneous mouse mammary tumor models.

Spontaneous tumorigenesis was studied in 80 TgMMTV-neu and 58 C3(1)Tag mice who were observed for tumor development from 6 weeks until tumor ∼1000 mm3. Differences in (1) time to tumor development (2) rate of tumor growth (3) tumor immune environment were evaluated. Between the transgenic mouse models, TgMMTV-neu mice (n = 10) developed tumors later, around 35.0 weeks, and the tumors grew slower with an average growth rate of 47.9 mm3/week than C3(1)Tag mice which developed tumors around 18.2 weeks with an average growth rate of 88.1 (p = 0.006). The tumor infiltration of MDSC inhibitory cells were higher in the C3(1)Tag mice than the TgMMTV-neu mice and approaches significance (17.4% of all lymphocytes in C3(1)Tag and 4.1% in TgMMTV-neu p = 0.08). Furthermore, there are increased CD8+ T cells in TgMMTV-neu mouse tumors than in C3(1)Tag (34.08% CD3+ cells in TgMMTV-neu mice and 22.38% in C3(1)Tag mice, p = 0.07) with similar FoxP3 levels (6.4% in TgMMTV-neu and 10.2% in C3(1)Tag mice p = 0.2) therefore the CD8/FoxP3 ratio in TgMMTV-neu mice is 5.3 where the CD8/FoxP3 ratio in C3(1)Tag mice is 2.2. These data demonstrate that C3(1)Tag mice have more aggressive tumorigenesis than the TgMMTV-neu mice and a more immunosuppressive tumor environment (higher MDSC, lower CD8, and smaller CD8/FoxP3 ratio) supporting the tumor immune environment plays an important role in tumorigenesis.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-03.
Abstract P2-05-05: Canine invasive mammary carcinoma as a spontaneous model for insulin-like growth factor-1 receptor-overexpressing triple negative breast cancer
Authors: Jaillardon L , Nguyen F , Loussouarn D , Godard T , Abadie J , Siliart B , Campone M
Published: August 2014

Background: Dogs spontaneously develop invasive mammary carcinoma with a high prevalence of the triple-negative subtype, making this model relevant for the investigation of new therapeutic pathways. Insulin-like growth factor-1 receptor (IGF-1R) is extensively involved in malignant transformation, proliferation and metastasis in human breast cancer and multiple clinical trials have used IGF-1R inhibitors with encouraging results.

Aims: To quantify IGF-1R expression in canine triple-negative invasive mammary carcinoma (TN-CMC) and assess its prognostic value (overall, specific and disease-free survival). To validate the dog as a spontaneous animal model for preclinical trials targeting IGF-1R in triple-negative breast cancer.

Material and methods: 104 triple-negative canine invasive mammary carcinomas (Estrogen receptor negative, Progesterone receptor negative, not HER2 overexpressing) were included. Bitches were followed for at least 2 years after mastectomy. TN-CMC were classified as basal-like (Cytokeratin-CK 5/6 positive and/or Epidermal Growth Factor Receptor-EGFR positive) and non-basal-like (CK 5/6 and EGFR negative). IGF-1R expression (clone G11) was quantified using the same scoring system as for HER2 in breast cancer.

Results: 68.3% (n = 71/104) of the TN-CMC were classified as basal-like and 31.7% (n = 33/104) as non-basal-like. 45.2% (n = 47/104) of TN-CMC overexpressed IGF-1R (score 3+), of which 70.2% (n = 33/47) were basal-like and 61.7% (n = 29/47) were grade III. Univariate and multivariate analyses revealed that IGF-1R overexpression (score 3+) was associated with significantly shorter overall, specific and disease-free survivals (median: 163, 332 and 278 days respectively) than weak (score 1+) IGF-1R expression (median: 842 days, not achieved and 1011 days respectively).

Conclusion and perspectives: IGF-1R overexpression is common and related to a poor outcome in canine triple-negative invasive mammary carcinoma. Preclinical studies using the dog as a spontaneous animal model for triple-negative breast cancer could be considered to investigate new therapies targeting IGF-1R.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-05.
Abstract P2-05-08: Combined neoadjuvant iniparib and carboplatin in locally advanced or metastatic canine mammary tumors (MT) to support human clinical studies
Authors: Morio F , Becavin S , Gogny A , Nguyen F , Albaric O , Labrut S , Séveno C , Vrignaud P , Juin P , Campone M , Abadie J , Ibisch C
Published: August 2014

INTRODUCTION

Among breast cancers, the triple negative subtype (negative for hormone receptors and not overexpressing HER2) has the worst prognosis and its response to Iniparib has been investigated in clinical trials. Further investigations are needed to optimize drug schedule and patient selection criteria. Iniparib antitumor mechanism is not completely understood, as well as iniparib diffusion kinetic into tumoral tissues. We address these questions in spontaneous canine invasive mammary carcinomas, which are a good model for this cancer subtype (Ibisch et al., World Veterinary Cancer Congress 2012), in a neoadjuvant setting. To our knowledge, this is the first study of iniparib administration in cancer-bearing dogs.

MATERIAL AND METHODS

Twenty female dogs with spontaneous MT with malignant criteria (tumor size, speed of growth, ulceration, relapse, or metastasis) were included. All tumors were described as rapidly growing. Dogs received a first infusion of iniparib at day 0 and a combination of carboplatin and iniparib at day 7. Biological materials (tumor biopsies and blood) were collected before and 5 minutes after iniparib infusion for pharmacokinetic and metabolism studies. Tumor response was evaluated by caliper measurements and histopathological analysis of mammary tumors and draining lymph nodes. A chain mastectomy was performed 3 or 4 weeks later. Histological records included the subtype of carcinoma (WHO 1999), Elston & Ellis grade, presence of emboli, lymph node metastasis and IHC stainings using ER, PR, Her2 (scored according to Wolff et al.2007), CK5/6, EGF-R and Ki67. Intensity of necrosis and apoptosis was evaluated using PAS coloration and immunohistochemistry for caspase 3, at DO on tumor biopsies and at surgery. Toxicity of the protocol was evaluated and its efficiency on invasive carcinomas was compared to surgery alone (control group of 27 female dogs with invasive mammary carcinomas treated by chain mastectomy alone).

RESULTS

Treated and control groups shared similar features concerning animal breeds, age, neutering status and tumor location. 75% of the treated MT were malignant. Necrosis and apoptosis were significantly increased in respectively 63 and 56% of iniparib treated tumors. Clinical evidence of toxicity was minimal (15% of dogs with nausea, 60% with transient polyuria-polydipsia). Tumor stabilization was observed before surgery in all dogs but one. Median survival has not been reached.

CONCLUSION

Iniparib at 35mg/kg combined with carboplatin at 300 mg/m2 seemed well tolerated in this study and deserves further investigations. The degree of necrosis and apoptosis in the treated tumors can be evaluated with these techniques. Iniparib pharmacokinetic and metabolism studies in cancer-bearing dogs are ongoing.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-08.
Abstract P2-06-03: The BRCA1 promoter methylation is one of the mechanisms of th BRCA1 dysfunction of triple-negative breast cancer
Authors: Yamashita N , Tokunaga E , Tanaka K , Saeki H , Oki E , Kitao H , Morita M , Maehara Y
Published: August 2014

Background: Triple negative breast cancer (TNBC) is typically associated with aggressive tumor phenotype and poor prognosis. TNBC is also considered highly heterogeneous disease. A better understanding of molecular and histopathological features of TNBC is of great importance, in order to develop a new therapeutic strategy and to improve the prognosis of TNBC. TNBC has many similarities with basal-like breast cancer, and is also associated with BRCAness. The major role of BRCA1 is to respond to DNA damage by participating in cellular pathways for DNA repair, mRNA transcription, cell cycle regulation, and protein ubiquitination. BRCA1 function loss leads to impaired homologous recombination mediated DNA repair. A loss of BRCA1 may thus be a biomarker of responsiveness to DNA damaging agents such as PARP inhibitor and cis/carboplatin. Germline mutations in BRCA1, however, account for approximately 5% of breast cancer cases, somatic mutations in BRCA1 rarely occur. Instead, lower than normal expression of BRCA1 is reported to be an important contributing factor in sporadic tumors. The BRCA1 promoter methylation may thus play an important role in the BRCA1 function loss in sporadic breast cancer.

Aim: To evaluate the clinical importance of BRCA1 promoter methylation in breast cancer.

Materials and Methods: Specimens were obtained from 71 TNBC and 163 non-TNBC patients who underwent surgery without neoadjuvant therapy in our department between 1990 and 2011. BRCA1 promoter methylation was investigated by using combined bisulfate and restriction analysis (COBRA). The BRCA1 mRNA expression was evaluated by quantitive RT-PCR. The BRCA1 protein level was assessed by immnohistochemistry. Loss of heterozygosity(LOH) at the BRCA1 locus was analyzed with microsatellite markers (D17S855 and D17S579) using our microsatellite analysis system. Copy number variations(CNVs) were analysed by SNP-CGH array (Illumina, HumanOmni2.5-8).

Results: We found 12 patients with BRCA1 promoter methylation and all of them were TNBC (p<0.0001). BRCA1 promoter methylation was associated with lymphovessel invasion (p = 0.009), high nuclear grade (p = 0.03), low BRCA1 mRNA expression (p = 0.002) and low BRCA1 protein expression (p<0.0001). CNVs were observed more frequently in TNBC(p = 0.012). High CNVs significantly correlated with BRCA1 LOH (p = 0.0025), but not with BRCA1 promoter methylation among TNBC patients.

Conclusions: The BRCA1 promoter methylation is considered to be a specific feature of aggressive phenotype of TNBC and to be one of the mechanisms of BRCA1 dysfunction in breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-06-03.
Abstract P2-07-02: A novel megadeletion mouse model for mechanistic studies on the 8q24 non-coding breast cancer susceptibility locus
Authors: Smits BMG , Gould MN
Published: August 2014

Genome-wide association studies (GWAS) have identified over 80 variants associated with breast cancer susceptibility. These loci have low-penetrance and are common in the population. Some of the alleles are associated with specific types of breast cancer, and/or associate differentially between certain ancestral populations. The vast majority of common, low-penetrance breast cancer susceptibility variants are located in non-protein coding genomic regions. It is hypothesized that the polymorphisms along the risk-associated alleles alter regulatory elements to modulate gene expression regulation. How such gene regulatory effects affect the susceptibility to breast cancer is completely unknown. To begin to study in vivo mechanisms underlying such non-protein coding breast cancer-associated loci, novel mammalian genetic models are indispensable.

A prototype non-coding breast cancer susceptibility locus is the gene desert (i.e. a large genomic region devoid of known protein-coding genes) on human chromosomal band 8q24. The gene desert is flanked by MYC, PVT1 and FAM84b. It harbors 2 common, low-penetrance breast cancer susceptibility variants. We developed a novel megadeletion mouse model lacking a 450-Kb non-coding region orthologous to the breast cancer-associated 8q24 gene desert region. Mice homozygous for the megadeletion are viable. Analysis of the transcript levels of the flanking genes reveal organ-specific effects of the megadeletion mutation. Through genetic crosses with well-known transgenic mouse breast cancer models, we will examine the region's effect on mammary cancer multiplicity, size, latency and metastasis. Additional phenotyping and gene expression analysis will reveal novel insight in the mechanisms underlying this non-protein coding breast cancer-associated gene desert locus. The conclusions will extend beyond the 8q24 locus and will aid in understanding the influence of common, low-penetrance breast cancer loci in the etiology of breast cancer.

[The mouse model was generated with help from University of Wisconsin Paul P. Carbone Comprehensive Cancer Center funds. The work is supported by a DoD MUSC-Baylor BRIDGE scholarship awarded to BMGS].

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-02.
Abstract P2-07-04: HER2 overexpressing breast cancers are more frequent in carriers of TP53 DNA-binding domain mutations than in carriers of oligomerization-site mutations
Authors: Fitarelli-Kiehl M , Giacomazzi J , Santos-Silva P , Ashton-Prolla P
Published: August 2014

Germline TP53 mutations are associated with Li-Fraumeni Syndrome (LFS), an autosomal dominant disorder characterized by predisposition to multiple early-onset cancers including breast cancer (BC), the most prevalent tumor among women. The majority of germline TP53 mutations are missense substitutions clustered within the DNA-binding domain of the gene that disrupt the structure and function of the protein. Recently, the germline mutation p.R337H (c.1010G>A), lying in the oligomerization domain of p53, was reported at a high frequency in Southern and Southeastern Brazil. This mutation appears to result in a more subtle defect in the protein, which becomes functionally deficient only under particular conditions and specific tissues. Recent reports suggest that BC are HER2-postive in a high proportion (63-83%) of TP53 mutation carriers. Histopathologic features of breast cancers among carriers of oligomerization domain mutations have not been reported. Our objective was to analize HER2 status in breast cancers from carriers of germline TP53 mutations, both in the DNA-binding and oligomerization domains. We retrospectively reviewed the immunohistochemistry data of breast cancers from 64 carriers of TP53 p.R337H and 6 carriers of nonfunctional germline TP53 mutations (p.G245S, p.R273H, p.G244D or c.672+1G>T). All patients were recruited from the cancer genetics clinic at Hospital de Clínicas de Porto Alegre, Brazil. Germline TP53 mutations were identified by Sanger sequencing of the entire coding sequence and flanking intronic regions. Immunohistochemistry (IHC) was performed using standard procedures. HER2 expression was assessed using a 0 to 3+ scale according the ASCO-CAP Guidelines. All of the six patients with nonfunctional TP53 mutations showed strong (3+) HER2 staining by IHC in their breast tumors. In the group of p.R337H carriers, 23.4% showed significant HER2 overexpression (3+), 21.9% presented ambiguous HER2 results (2+), and 54.7% showed weak or no HER2 expression (1+ or 0). Thus, HER2 overexpressing breast cancers were more frequent in carriers of TP53 nonfunctional (DNA binding domain) mutations (p≤0.001) than in those carrying the oligomerization domain mutation. Preliminary results from this study suggest that germline mutations in different domains of the TP53 gene may predispose to breast cancer through different mechanisms.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-04.
Abstract P2-08-05: Functional characterization of three novel breast cancer-related somatic mutations in metalloproteinase-disintegrin ADAM12
Authors: Qi Y , Duhachek-Muggy S , Li H , Zolkiewska A
Published: August 2014

Six different somatic missense mutations in the human ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the effect of the corresponding D229H and G477E mutations in mouse ADAM12 protein described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity - R612Q and L792F - occur in triple-negative breast cancers, while loss-of-function mutations - D301H, G479E, T596A, and G668A - are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of ADAM12-L in the triple negative breast cancer disease.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-08-05.
Abstract P2-09-07: Inhibition of aurora-A by MLN8237 decreases SMAD5 expression and increases effectiveness of chemotherapeutic agents in breast cancer cells
Authors: Opyrchal M , Iankov I , Ingle JN , Galanis E , D'Assoro AB
Published: August 2014

BACKGROUND: Breast cancer affects thousands of women each year. Epithelial to Mesenchymal transition (EMT) has been associated with increased metastatic potential of cancer cells as well as resistance to chemotherapy. Predicatively, presence of EMT leads to worse prognosis in patients with breast cancer. We have recently showed that Aurora-A plays a key role in development of EMT and increased ability of breast cancer cells for self renewal. Therefore we hypothesized that inhibition of Aurora-A in breast cancer cells will lead to increased sensitivity to chemotherapy.

RESULTS: Increased Aurora-A expression, as assessed by IHC staining, in patients with locally advanced disease was associated with worse prognosis. We show that in ER+ cell line, MCF7, activating MAPK pathway through overexpression of Raf1 leads to increase in Aurora-A activity. These cells have decreased sensitivity to treatment with paclitaxel when compared to untransfected cells in MTT assays. This effect is even more exacerbated with over-expression of Aurora-A. The sensitivity to paclitaxel was restored with inhibition of Aurora-A by specific inhibitor, Alisertib. This was further explored in triple negative (MDA-MB-231) or Her-2/neu expressing (SUM149) cell lines. These cells express higher levels of Aurora-A. Inhibition of Aurora-A activity in combination with paclitaxel was superior when compared to either therapy alone. Similar effect was seen with use of anthracyclines. Inhibition of Aurora-A resulted in decreased SMAD5 expression as well as decreased Akt phosphorylation. Current studies are looking at a role of Aurora-A in activating SMAD5 and its role in resistance to chemotherapy. In vivo experiments evaluating combination therapies in breast cancer animal model are ongoing.

CONCLUSIONS: Inhibition of Aurora-A by a specific inhibitor, MLN8237, resulted in decreased SMAD5 expression and increased effectiveness of chemotherapeutic agents in breast cancer cells. These results contribute to better understanding of signaling pathways involved in resistance of breast cancer cells to chemotherapy. This knowledge could be very useful in developing more effective treatments for breast cancer patients in neo-adjuvant, adjuvant and metastatic settings.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-07.
Abstract P2-09-11: Identification of a novel inhibitor of triple-negative breast cancer cell growth by screening of a small molecule library
Authors: Fujita T , Mizukami T , Okawara T , Inoue K , Fujimori M
Published: August 2014

Background: Triple-negative breast cancers (TNBC) are defined as not having amplification of the estrogen receptor, progesterone receptor, or epidermal growth factor receptor 2. At present, recovery in patients is severely limited following diagnosis of metastatic TNBC, with less than 30% of patients surviving more than 5 years.. The most effective therapy to date is chemotherapy, which has been unsuccessful due to the lack of therapeutic targets for these aggressive cancers. To identify new molecular targets for TNBC, we have developed a novel method for drug discovery using active compounds for identification of pharmacodynamic biomarkers.

Methods: We used chemical informatics to design a small molecule library that has structural diversity. This library was used to screen for compounds that selectively inhibit the proliferation in TNBC cell lines. The differences in gene expression profiles in cell lines before and after the addition of selected compounds were analyzed and compared with control cells.

Results: We identified (E)-3-(3,4-dihydroxybenzylidene)-benzofuran-2(3H)-one (DBBF) that exhibited specific inhibition of the proliferation in a TNBC cell line, MDA-MB-468 with IC50 of 2.4 μM. Microarray analysis identified several signaling pathways, including the irinotecan pathway, which changed specifically in the TNBC cell lines by the addition of DBBF.

Conclusion: We have developed a novel research strategy that involves screening of selective inhibitors of TNBC cell line proliferation that can be used for identification of pharmacodynamic biomarkers for TNBC. The discovery of new pathways by this technique should lead to the identification of new therapeutic targets for this aggressive cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-11.
Abstract P2-09-16: Pre-clinical evaluation of novel anti-angiogenic agents as breast cancer therapeutics
Authors: McGarrigle SA , Murphy A , Kennedy BN , Reynolds A , O'Sullivan JN , Kennedy MJ , Connolly EM
Published: August 2014

Background

There is a need to develop novel drugs that will improve survival in breast cancer patients. Angiogenesis is essential for breast tumour progression. To date, the most promising approach to inhibit angiogenesis in breast cancer patients has been the drug bevacizumab which targets the pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF). Recently, there has been controversy regarding the efficacy of bevacizumab for breast cancer treatment. In clinical trials, bevacizumab failed to establish an overall-survival benefit and was associated with serious toxicities. This resulted in the FDA revoking approval for the drug for the first line treatment of advanced breast cancer. Due to this uncertainty surrounding the efficacy of bevacizumab for the treatment of breast cancer it is clear that there is a clinical need for more effective novel anti-angiogenic drugs with better toxicity profiles for the treatment of breast cancer.

Aim

This study aimed to identify novel small-molecule anti-angiogenic agents with therapeutic potential in human breast cancer.

Methods

Compounds with physiochemical properties consistent with drug-like compounds were screened for anti-angiogenic activity by high-throughput screening involving zebrafish larvae. Human breast tumour explants were treated with the lead compound and secretion of angiogenic factors was assessed by ELISA.

Results

We have identified a novel small-molecule agent ‘SMG1’ that significantly inhibited inter-segmental blood-vessel development in zebrafish and showed no toxicity. Treatment of breast tumour explants with SMG1 significantly inhibited secretion of the potent pro-angiogenic cytokine VEGF (p = 0.01). Furthermore, SMG1 inhibited VEGF secretion more than the standard targeted breast cancer therapies tamoxifen and Herceptin® which have been reported to inhibit angiogenesis.

Conclusion

Continuing pre-clinical work will determine if SMG1 has potential as a therapeutic agent for human breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-16.
Abstract P2-09-17: Allosteric inhibitors of the proteasome: A new concept for breast cancer treatment
Authors: Osmulski PA , Gaczynska M
Published: August 2014

Proteasome is an essential protease of the proteasome-ubiquitin pathway, responsible for maintaining intracellular proteostasis. It is a multifunctional, multisubunit enzyme built from interchangeable modules and intricately regulated on the molecular and cellular levels. Proteasome inhibitors display proapoptotic properties, preferentially aimed at cancerous cells. Consequently, targeted therapy with bortezomib, the competitive inhibitor of the proteasome, has been very successful with refractory/relapsed multiple myeloma and with lymphomas. Unfortunately, breast cancers performed disappointingly in clinical trials with bortezomib. We propose that a new generation of small-molecule proteasome inhibitors distinct from bortezomib may prove successful with breast cancers. In contrast to bortezomib which directly blocks proteasome active sites, the new inhibitors are allosteric ligands binding to noncatalytic sites and disrupting the enzyme's function by targeting long-distance transfer of structural signals within the enzyme's molecule. The advantages of allosteric ligands, now rapidly gaining popularity in targeted drug design, include versatility of molecular actions, defying drug resistance mechanisms, and usually high specificity with low toxicity. For example, one of the small-molecule allosteric ligands we created compromises interactions between the catalytic core proteasome and its regulatory sub-assembly 19S (“cap”, regulatory particle) responsible for recognizing and processing the majority of intracellular protein substrates tagged for degradation by polyubiquitin. We found that treatment with nanomolar concentration of the compound induced cell death in Mcf7 human breast cancer cultured cells. Interestingly, this cell line is known for its resistance to bortezomib treatment. Even more, treatment with the allosteric ligand together with bortezomib produced a synergistic effect and sensitized the Mcf7 cells to the actions of competitive inhibitor. Remarkable, the cytotoxic effects of allosteric ligand or its combination with bortezomib were not observed with the noncancerous human breast cell line, Mcf10A. Summarizing, allosteric regulation of the proteasome by small-molecule ligands, alone or in combination with established drugs competitively inhibiting proteasome, seem to be a promising strategy to utilize proteasome as a drug target in breast cancers.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-17.
Abstract P2-10-03: Metastasis-associated in colon cancer 1 predicts poor outcomes in patients with breast cancer
Authors: Lee JS , Cho JS , Park MH , Yoon JH
Published: August 2014

Background: Metastasis-associated in colon cancer 1 (MACC1) plays a role in cancer invasion and metastasis through the upregulation of hepatocyte growth factor/mesenchymal epithelial transition-pathway, which is a known critical regulator of the mitogen-activated protein kinase (MAPK) cascades. However, the potential involvement of MACC1 in breast cancer has not been assessed.

Materials and Methods: To investigate whether MACC1 expression is a prognostic marker in breast cancer, we performed immunohistochemical staining for MACC1 expression in tissue microarrays consisting of 198 invasive breast carcinomas. To demonstrate the potential correlation between MACC1 and MAPK cascades, phospho-p44/42 MAPK (ERK1/2) expression was also analyzed.

Results: Expression of MACC1 was detected in 109 (55.1%) of 198 invasive breast carcinomas. MACC1 expression was significantly associated with several clinicopathologic parameters, including ER negativity (P < 0.01), PR negativity (p < 0.01), and HER-2 positivity (p < 0.01). The rate of metastatic relapse was significantly higher in the MACC1-positive group (87.8%, 36/41) than in the MACC1-negative group (46.5%, 73/157) (p < 0.001). MACC1 expression was significantly correlated with phospho-p44/42 MAPK expression (p < 0.05). On univariate survival analysis, a significant association was observed between MACC1 expression and decreased disease-free survival (p < 0.001) and overall survival (p = 0.001). On multivariate analysis, MACC1 expression was one of the statistically significant independent risk factors for disease-free survival (p = 0.001).

Conclusion: Our results suggest that MACC1 may serve as a new parameter for the prognostic prediction in patients with invasive breast carcinoma. MACC1 is likely to be involved in the regulation of MAPK cascades in invasive breast carcinoma.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-10-03.
Abstract P2-10-06: Serum protein peak 3144m/z is a potential prognostic factor for postmenopausal breast cancer patients
Authors: Gao Y , Wang X-J , Xu S-H , Yang H-J , Chen Z-H , Cao W-M
Published: August 2014

Background: Serum protein profiles have been investigated frequently to discover predicted or prognostic biomarkers for breast cancer. Our previous studies have shown that serum protein peak 3144m/z is significantly elevated in metastatic ovarian cancer and gastric cancer. The receiver operating characteristic (ROC) curve obtained from metastatic ovarian cancer patients for differentially expressed protein peak 3144m/z showed when the cut-off value is 1.15, the sensitivity and specificity would be 65.4% and 91.4% respectively. Therefore, we investigated whether serum protein peak 3144m/z could be used as a potential prognostic tool for postmenopausal breast cancer, taking into account clinicopathological features.

Methods: Two hundred and two postmenopausal breast cancer patients from June 2006 to December 2009 were involved in this retrospective study at Zhejiang Cancer Hospital. Menopausal status of the cases was obtained through examination of the medical records. The histopathological type, lymph node metastasis, and hormone receptor status of the cases were determined by pathologists. All patients underwent conventional postsurgical treatment (chemotherapy, radiotherapy, endocrinotherapy and target therapy) and the serum samples were collected before treatment. Serum level of protein peak 3144m/z was assessed by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Prognostic factors (lymph node metastasis status, stage, hormone receptor status and molecular subtype) were compared across subgroups of patients depending on the protein peak 3144m/z levels. Cox proportional hazards regression was used for statistical analysis.

Results: The precentage of cases with higher 3144m/z protein peak were 32.7%(66/202) in postmenopausal breast cancer patients. The serum protein peak 3144m/z was positively related to lymph node metastasis. However, there were no significant correlations between serum protein peak 3144m/z and other clinicopathological features, such as hormone receptor status, molecular subtype, clinical stage, pregnancy times and abortion history. Importantly, it was shown that patients with higher protein peak 3144m/z had significantly poorer overall survival compared with patients with lower serum protein peak 3144m/z (P = 0.0053). Furthermore, Cox multivariate regression analysis also revealed that protein peak 3144m/z was an independent prognostic factor in postmenopausal breast cancer patients (P = 0.008).

Conclusions: The protein peak 3144m/z was a poor prognostic factor in postmenopausal breast cancer patients. Further prospective and longitudinal studies are needed to evaluate whether serum protein peak 3144m/z could be used as a prognostic tool in postmenopausal breast cancer patients monitoring and management.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-10-06.
Abstract P2-11-08: IHC 2+ FISH (-) breast cancer (BC): Patient characteristics and outcomes: A comparison to HER2 IHC 0, 1+ and HER2 + BC diagnosed 2000-2006
Authors: Fehrenbacher L , Capra A , Fulton R , Habel L
Published: August 2014

Background: HER2 IHC 2+ results are equivocal and lead to FISH testing. FISH(+) results lead to HER2(+) treatment often with trastuzumab. FISH(-) results lead to treatment without trastuzumab. We examined the characteristics and breast cancer-specific mortality of HER2 2+ FISH (-) BC in consecutive patients in a large health care delivery system. Methods: Using the KPNC cancer registry and electronic medical record, we identified 13,845 consecutive stage I-III BC patients diagnosed between 1/2000 and 12/2006 not treated with adjuvant trastuzumab. Patient demographics, disease stage, IHC and FISH results, and breast cancer mortality were obtained. IHC and FISH testing were performed in a single central laboratory. Results: After excluding any adjuvant trastuzumab, 10,760 (78%) patients had HER2 IHC scores of 0,1+, 1,533 (11%) were IHC2+/FISH (-), and 1,552 (11%) were IHC3+ or FISH (+). Compared to patients with IHC 0,1+ tumors, those with IHC2+FISH (-) tumors were younger (mean age 61 vs 62 years, p = 0.03) and more often node+ (36% vs 30%, p = <0.0001); their tumors were more often poorly differentiated (29% vs 22%, p = <0.0001),) and T2 vs T1 (31% vs 24%, p = <0.0001), and less frequently T1ab (20% vs 29%, p = <0.0001), ER+ (81% vs 85%, p = 0.0003), or PR+ (70% vs 74%, p = <0.0002). Among the ER+ patients, the 10-year BC-specific mortality was higher for those with IHC2+/FISH (-) than IHC 0, 1+ tumors (11.4% vs 8.9%, p = 0.0014). Within disease stages, there were trends but no statistical difference in BC-specific mortality. IHC3+/FISH (+) ER+ patients had greater differences in the above characteristics and significant increased mortality among middle, and higher stages. In multivariable analysis adjusting for age, tumor size, differentiation, hormonal therapy and chemotherapy, risk of BC death among ER+ patients was similar for IHC2+FISH (-) and IHC 0,1+ disease; but risk was higher for IHC3+/FISH (+) disease. Table 1: 10-year breast cancer (BC) survival of women with stage I-III ER-positive BC by HER2 subtype, 2000-2006.

10-year BC survival of stage I-III ER+ patients by IHC FISH statusHER2 SubtypeNo of PatientsBC Deathsat Risk%BC Survival95% CILogRank P-valueIHC 0,1+9,1616212,45491.190.4-91.8-IHC2+FISH(-)1,24811222388.686.3-90.60.0014IHC3+ and/or FISH(+)93512523783.680.6-86.2<0.0001

Conclusions: In ER+ patients, those with IHC2+FISH (-) tumors have prognostic characteristics that are intermediate between those with IHC 0,1+ and IHC3+/FISH(+) tumors. Compared to IHC 0,1+ disease, they are at increased risk of BC mortality overall but not within stages.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-08.
Abstract P2-11-18: Immunohistochemical expression of ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) as a significant prognostic marker in postmenopausal invasive breast cancer patients
Authors: Kurozumi S , Yamaguchi Y , Matsumoto H , Takei H , Kobayashi Y , Hayashi S-I , Yanagisawa J , Horiguchi J , Takeyoshi I , Kurosumi M
Published: August 2014

Background:

CHIP is a U box type ubiquitin ligase that induces ubiquitination and degradation of its target proteins, which include several oncogenic proteins. We clarified that CHIP suppresses tumor growth and metastasis in human breast cancer by inhibiting oncogenic pathways (Nat Cell Biol 2009;11:312-9). In this study, we investigated the relationship between immunohistochemical CHIP expression and several biomarkers, and evaluated the correlation between CHIP expression and prognosis of patients with invasive breast cancer.

Patients and Methods:

In this study, 272 consecutive patients with invasive carcinoma of no special type with invasion >5 mm, treated in 2000-2001, were enrolled. We performed immunohistochemical staining with antibodies against CHIP, ER, PgR, and HER2. Cytoplasmic expression patterns of CHIP were scored as 0 (no stain), 1 (weak stain), 2 (moderate stain), and 3 (strong stain), and divided into high CHIP (score 3) and low CHIP (score 2, 1, 0) groups. We investigated the relationship between CHIP expression and ER, PgR, HER2, histological grade (HG) and lymph node status (pN). We also evaluated correlation between CHIP expression and prognosis (RFS) of patients.

Results:

CHIP expression was as follows: score 0, 49 patients (18.0%); score 1, 91 (33.5%); score 2, 83 (30.5%); score 3, 49 (18.0%). High CHIP correlated significantly with positive ER (p<0.001), positive PgR (P<0.001), negative HER2 (p = 0.02), and low HG (p = 0.01). RFS was relatively better in the high CHIP group (49 patients) than the low CHIP group (223 patients) (p = 0.09). In addition, in postmenopausal patients, RFS was significantly better in the high CHIP group (23 patients) than the low CHIP group (133 patients) (p = 0.04); this was not so in premenopausal cases (p = 0.68). Furthermore, RFS was significantly better in CHIP score 3 and ER positive cases (42 patients) than in CHIP score 0 and ER negative cases (34 patients) (p = 0.03).

Conclusions:

CHIP expression correlated with status of hormone receptors and HER2 and might be a potent prognostic factor in invasive breast cancer patients, especially postmenopausal cases. Immunohistochemical examination of CHIP is thought to be useful for evaluating prognosis of breast cancer patients.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-18.
Abstract P2-11-19: Expression of folate receptor alpha in ductal carcinoma in situ of the breast and comparison with that of invasive ductal carcinoma
Authors: Zhang Z , Wang J , Tacha D , Li P , Bremer R , Skinner K , Hicks DG , Bu H , Tang P
Published: August 2014

Background: We have previously shown that the expression of Folate receptor alpha (FRA) is associated with high grade ER-/PR- or triple negative (TN) invasive ductal carcinoma of the breast. The aim of the current study is to investigate the expression pattern of FRA in a cohort of ductal carcinoma in situ (DCIS), analyze its relationship with different clinicopathological features and expression of several key biomarkers, and compare these findings to our previous data on invasive ductal carcinoma (IDC).

Methods: Ninety-eight cases of DCIS of the breast diagnosed between 1997 and 2008 in our institution were identified, reviewed and 6 blocks of tissue microassays (TMA) were constructed. The association between the expression of FRA (≥10% designed as positive expression) and the clinicopathologic features, the expression of ER, PR, HER2 and Ki-67, and the clinical outcome were evaluated, and these results were compared to those from IDC (399/430 cases with informative data). Results: Among the 98 cases of DCIS in TMA, 88 cases were informative. The expression rate of FRA was 21% for DCIS, less than the 29% seen in IDC. Expression of FRA was associated with high nuclear grades (42%), though less frequently than with IDC (52%). FRA expression was also associated with ER negativity (46%), PR negativity (33%) and high Ki-67 expression (50%) in DCIS, although significantly less than in IDC (75%, 63%, and 56%, respectively for negative ER and PR, and high Ki-67). Also, only 50% of ER/PR negative, TN and basal-like DCIS expressed FRA, significantly lower than that of IDC (74%, 80% and 80%, respectively). Due to the limited cases of DCIS, we did not demonstrate a significant difference in disease-free survival between FRA positive and negative tumors (p-value = 0.1792), although we did observe that IDC with FRA expression was significantly associated with a worse disease-free survival (p-value = 0.04).

Comparison between FRA expression between DCIS and IDC FRA≥10% in DCISFRA≥10% in IDCp-value 88 cases399 cases Nuclear 342%52%0.0185ER negative46%75%0.0442PR negative33%63%0.0214HER2 3+31%37%0.7003ER-/PR-50%74%0.0973Triple Negative50%80%0.1622Basal-Like50%80%0.1739

Conclusion: Our data demonstrates that FRA expression is associated with high nuclear grade, ER/PR negative and TN DCIS, though at a significantly lower rate than was observed in IDC, suggesting a possible role for this receptor in early breast carcinogenesis. There are differences in the rates of FRA expression between DCIS and IDC, suggesting that FRA may be one of the key molecules involved in progression of DCIS to IDC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-19.