Non-invasive Breast Cancer Research Articles

Separate modal analysis for tumor detection with a digital image elasto tomography (DIET) breast cancer screening system

It is estimated that every year, 1 × 10(6) women are diagnosed with breast cancer, and more than 410,000 die annually worldwide. Digital Image Elasto Tomography (DIET) is a new noninvasive breast cancer screening modality that induces mechanical vibrations in the breast and images its surface motion with digital cameras to detect changes in stiffness. This research develops a new automated approach for diagnosing breast cancer using DIET based on a modal analysis model. The first and second natural frequency of silicone phantom breasts is analyzed. Separate modal analysis is performed for each region of the phantom to estimate the modal parameters using imaged motion data over several input frequencies. Statistical methods are used to assess the likelihood of a frequency shift, which can indicate tumor location. Phantoms with 5, 10, and 20 mm stiff inclusions are tested, as well as a homogeneous (healthy) phantom. Inclusions are located at four locations with different depth. The second natural frequency proves to be a reliable metric with the potential to clearly distinguish lesion like inclusions of different stiffness, as well as providing an approximate location for the tumor like inclusions. The 10 and 20 mm inclusions are always detected regardless of depth. The 5 mm inclusions are only detected near the surface. The homogeneous phantom always yields a negative result, as expected. Detection is based on a statistical likelihood analysis to determine the presence of significantly different frequency response over the phantom, which is a novel approach to this problem. The overall results show promise and justify proof of concept trials with human subjects.

Performance Comparison of Digital microRNA Profiling Technologies Applied on Human Breast Cancer Cell Lines

MicroRNA profiling represents an important first-step in deducting individual RNA-based regulatory function in a cell, tissue, or at a specific developmental stage. Currently there are several different platforms to choose from in order to make the initial miRNA profiles. In this study we investigate recently developed digital microRNA high-throughput technologies. Four different platforms were compared including next generation SOLiD ligation sequencing and Illumina HiSeq sequencing, hybridization-based NanoString nCounter, and miRCURY locked nucleic acid RT-qPCR. For all four technologies, full microRNA profiles were generated from human cell lines that represent noninvasive and invasive tumorigenic breast cancer. This study reports the correlation between platforms, as well as a more extensive analysis of the accuracy and sensitivity of data generated when using different platforms and important consideration when verifying results by the use of additional technologies. We found all the platforms to be highly capable for microRNA analysis. Furthermore, the two NGS platforms and RT-qPCR all have equally high sensitivity, and the fold change accuracy is independent of individual miRNA concentration for NGS and RT-qPCR. Based on these findings we propose new guidelines and considerations when performing microRNA profiling.

Identification of epithelial stromal interaction 1 as a novel effector downstream of Krüppel-like factor 8 in breast cancer invasion and metastasis

Krüppel-like factor 8 (KLF8) is a transcriptional factor critical for metastatic progression of breast cancer. Epithelial stromal interaction 1 (EPSTI1), a recently identified stromal fibroblast-induced gene in non-invasive breast cancer cells is highly overexpressed in invasive breast carcinomas. The function and regulation of EPSTI1, however, remain largely unknown. In this paper, we report a novel KLF8 to EPSTI1 signaling pathway in breast cancer. Using various expression analyses, we revealed a high co-overexpression of KLF8 and EPSTI1 in invasive human breast cancer cells and patient tumors. Ectopic overexpression of KLF8 in the non-invasive, MCF-10A cells induced the EPSTI1 expression, whereas KLF8 knockdown from the invasive, MDA-MB-231 cells decreased the EPSTI1 expression. Promoter activation and binding analyses indicated that KLF8 promoted the EPSTI1 expression by directly acting on the EPSTI1 gene promoter. EPSTI1 knockdown dramatically reduced the KLF8-promoted MCF-10A cell invasion and ectopic expression of EPSTI1 in the non-invasive, MCF-7 cells is sufficient to induce the cell invasion. Experiments using nude mice demonstrated that the ectopic EPSTI1 granted the MCF-7 cells capability of both invasive growth in the breasts and metastasis to the lungs. Using co-immunoprecipitation coupled with mass spectrometry, we discovered that EPSTI1 interacts with the valosin containing protein (VCP), resulting in the degradation of IκBα and subsequent activation of NF-κB in the nucleus. These findings suggest a novel KLF8 to EPSTI1 to VCP to NF-κB signaling mechanism potentially critical for breast cancer invasion and metastasis.

Heat shock protein 90 (Hsp90) inhibitors tethered to radioiodinated or optical probes for noninvasive breast cancer imaging

Heat shock protein 90 (Hsp90) inhibitors tethered to radioiodinated or optical probes for noninvasive breast cancer imaging

Secondary cancers after breast-conserving therapy in Tokyo database.

139 Background: The more patients overcome early breast cancer and become cancer survivors as a result of modern sophisticated approach, the more secondary cancers inevitably arise. The second malignancies after breast conserving therapy (BCT) are well-known sticky dilemma because of additional anxiety and need for medical care for longer-time. However, it is unclear whether secondary cancers negatively affect prognosis of breast cancer survivors. Methods: We performed a retrospective study of long-term cancer survivors after BCT for locoregional invasive or noninvasive breast cancer diagnosed between 1982 and mid-2012. Actuarial rates of overall (OS) and cause-specific survival (CSS) were calculated by using the Kaplan-Meier method. We compared between-group differences using the log-rank test. Results: Eight hundred sixty patients (32%) were followed-up for more than 10 years. At a median follow-up of 90 months, 146 patients had developed a second malignancy. The greatest increases in risk were for leukemia (Standardized incidence ratio (SIR): 4.24 (1.52–8.31)), ovarian cancer (SIR: 4.12 (2.40–6.31)), reno-ureteral cancer (SIR: 3.18 (1.14–6.23)), endometrial cancer (SIR: 2.48 (1.27–4.08)), and pancreatic cancer (SIR: 2.32 (1.11–3.99)). No increased risk was observed for other gastrointestinal and genitourinary cancer, malignant melanoma, lymphoma, thyroid or head and neck cancer. Overall 10-year cumulative incidence of OS without secondary cancer was 93.3%, and 10-year cumulative incidence of OS with secondary cancer was 81.5%. (p<0.001)Overall 10-year cumulative incidence of CSS without secondary cancer was 94.2%, and 10-year cumulative incidence of CSS with secondary cancer was 92.8%. (p=0.749). This likelihood of survival disadvantage is similar to that with ipsilateral breast tumor recurrecnce. Conclusions: Secondary cancers after BCT negatively impact on OS. Given the life-threatening nature to cancer survivors, lifetime caution such as smoking cessation, alcohol intake abstention, weight control, physical activity, and other healthy lifestyle must be paid.

Synergistic anti-breast cancer effect of a combined treatment with the methyl donorS-adenosyl methionine and the DNA methylation inhibitor 5-aza-2′-deoxycytidine

DNA-demethylating agents activate tumor suppressor genes that are silenced by DNA methylation in cancer and are therefore emerging as a novel approach to cancer therapy. 5-azacytidine (VIDAZA), the first representative of this class of drugs was approved for treatment of myelodysplastic syndromes and is currently being tested on other cancers including solid tumors. However, 5-azacytidine or its deoxy-analog, 5-aza-2'-deoxycytidine (5-azaCdR) could also induce methylated prometastatic genes by DNA demethylation and induce cancer cell invasiveness. Since 5-azacytidine is a potent cancer growth inhibitor, we tested whether combining it with a DNA-methylating agent, the methyl donor S-adenosyl methionine (SAM), would block the adverse demethylating activity of 5-azaCdR while maintaining its growth suppression effects. We show here using several invasive and non-invasive breast cancer cell lines that SAM inhibits global- and gene-specific demethylation induced by 5-azaCdR, prevents 5-azaCdR activation of prometastatic genes uPA and MMP2, resulting in inhibition of cell invasiveness while augmenting the growth inhibitory effects of 5-azaCdR and its effects on tumor suppressor genes. Combination of drugs acting on the DNA methylation machinery at different levels is proposed as a new strategy for epigenetic therapy of cancer.

Adhesion and Invasion of Breast and Oesophageal Cancer Cells Are Impeded by Anti-LRP/LR-Specific Antibody IgG1-iS18

Adhesion and invasion have been identified as the two key components of metastasis. The 37 kDa/67 kDa laminin receptor (LRP/LR) is thought to enhance these two processes thus endorsing the progression of cancer. Here we report on LRP/LR and the metastatic potential of MDA-MB 231 breast and WHCO1 oesophageal cancer cells. Western blot analysis revealed a significant increase in total laminin receptor precursor (LRP) levels of breast and oesophageal cancer cells in comparison to non-invasive MCF-7 breast cancer cells, whereas LRP/LR cell surface levels in both cell lines were not significantly different to those of MCF-7 cells as analysed by flow cytometry. Incubation of breast and oesophageal cancer cells with the anti-LRP/LR specific antibody, IgG1-iS18, resulted in significant reduction in the adhesive potential of WHCO1 and MDA-MB 231 cells by 92% and 16%, respectively. Moreover, invasion was significantly impeded by 98% and 25% for WHCO1 and MDA-MB 231 cells, respectively. Pearson’s correlation coefficients proved a positive correlation between total LRP/LR levels and invasive potential as well as between the adhesive and invasive potential of breast and oesophageal cancer cells. Our findings suggest that through interference of the LRP/LR-laminin-1 interaction, anti-LRP/LR specific antibody IgG1-iS18 may act as a possible alternative therapeutic tool for metastatic breast and oesophageal cancer treatment.

TLR4 Ligand/H2O2 Enhances TGF-β1 Signaling to Induce Metastatic Potential of Non-Invasive Breast Cancer Cells by Activating Non-Smad Pathways

TGF-β1 has the potential to activate multiple signaling pathways required for inducing metastatic potential of tumor cells. However, TGF-β1 was inefficient in inducing metastatic potential of many non-invasive human tumor cells. Here we report that the enhancement of TGF-β1 signaling is required for inducing metastatic potential of non-invasive breast cancer cells. TGF-β1 alone could not efficiently induce the sustained activation of Smad and non-Smad pathways in non-invasive breast cancer cells. TLR4 ligand (LPS) and H2O2 cooperated with TGF-β1 to enhance the sustained activation of non-Smad pathways, including p38MAPK, ERK, JNK, PI3K, and NF-κB. The activation of MAPK and PI3K pathways resulted in a positive feed-back effect on TGF-β1 signaling by down-regulating Nm23-H1 expression and up-regulating the expression of TβRI and TβRII, favoring further activation of multiple signaling pathways. Moreover, the enhanced TGF-β1 signaling induced higher expression of SNAI2, which also promoted TβRII expression. Therefore, the sustained activation levels of both Smad and non-Smad pathways were gradually increased after prolonged stimulation with TGF-β1/H2O2/LPS. Consistent with the activation pattern of signaling pathways, the invasive capacity and anoikis-resistance of non-invasive breast cancer cells were gradually increased after prolonged stimulation with TGF-β1/H2O2/LPS. The metastatic potential induced by TGF-β1/H2O2/LPS was sufficient for tumor cells to extravasate and form metastatic foci in an experimental metastasis model in nude mice. The findings in this study suggested that the enhanced signaling is required for inducing higher metastatic capacity of tumor cells, and that targeting one of stimuli or signaling pathways might be potential approach in comprehensive strategy for tumor therapy.

Intraductal Therapy of Ductal Carcinoma In Situ: A Presurgery Study

IntroductionDuctal carcinoma in situ (DCIS) is a noninvasive breast cancer wherein malignant cells are confined within a ductal lobular unit. Although less than half the cases of DCIS will progress to invasive disease, most women are treated aggressively with surgery, radiation, and/or hormone therapy due to the inability to clinically evaluate the extent and location of the disease. Intraductal therapy, in which a drug is administered directly into the mammary duct through the nipple, is a promising approach for treating DCIS, but the feasibility of instilling drug into a diseased duct has not been established. Patients and MethodsFour to 6 weeks before their scheduled surgery, 13 women diagnosed with DCIS were subjected to cannulation of the affected duct. After both the absence of perforation and presence of dye in the duct were confirmed by ductogram, pegylated liposomal doxorubicin was instilled. Histopathologic assessment was performed after surgery to assess the treatment effects. ResultsOf the 13 women enrolled in the study, 6 had their DCIS duct successfully cannulated without perforation and instilled with the drug. The treatment was well tolerated, and no serious adverse events have been reported. Biomarker studies indicated a general decrease in Ki-67 levels but an increase in annexin-1 and 8-hydroxydeoxyguanosine in the lumen of DCIS-containing ducts, which suggests a local response to pegylated liposomal doxorubicin treatment. ConclusionsIntraductal therapy offers a nonsurgical strategy to treat DCIS at the site of disease, potentially minimizing the adverse effects of systemic treatment while preventing development of invasive cancer.

Abstract 184: A gemini vVitamin D analog, BXL0124, inhibits mammary tumorigenesis by delaying the transition of ductal carcinoma in situ to iInvasive breast carcinoma.

Abstract The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a crucial event in the progression of breast tumors. DCIS is a noninvasive breast cancer that harbors various grades of lesions ranging from non life threatening to invasive type lesions. The disappearance and breakdown of the myoepithelial cell layer and basement membrane in DCIS has been identified as a major event in the progression from DCIS to invasive carcinoma. We have previously reported that a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), inhibits the growth of MCF10DCIS.com xenografted tumors in immunodeficient mice without hypercalcemic toxicity. Based on this knowledge, we have investigated whether BXL0124 blocks or delays the progression of DCIS. In the present study, MCF10DCIS.com cells (1x106 cells/mouse) were injected into the mammary fat pad of nu/nu mice. The mice were then treated with BXL0124 (0.1μg/kg) over weeks 1, 2, and 3. Mammary glands and tumors were analyzed over the course of three weeks to determine the effects of BXL0124. The myoepithelial marker, smooth muscle actin (SMA), and the luminal marker, cytokeratin 18 (CK18), were used to analyze tumor histology. The inhibitory effect of BXL0124 treatment on cell proliferation was determined by the cell proliferation marker, proliferating cell nuclear antigen (PCNA). BXL0124 showed a 53% and 60% reduction in the tumor volume by week 2 and 3, respectively. The myoepithelial structure of DCIS was maintained in the treatment group, whereas loss of the myoepithelial layer was evident in the control group. The treatment with BXL0124 also decreased cell proliferation of DCIS luminal cells compared to the control group, suggesting that growth potential of luminal cells was reduced by BXL0124. In conclusion, the Gemini vitamin D analog BXL0124 inhibits the progression of MCF10DCIS.com xenografted mammary tumors in part by maintaining the vital myoepithelial layer within the tumors and by reducing proliferation rate of the luminal cells. Our results suggest a novel mechanism in the cancer preventive action of BXL0124 during the progression of DCIS like lesions to invasive carcinoma. (This work was supported in part by the National Institutes of Health National Cancer Institute R01-CA127645 and the National Institute of Environmental Health Sciences Grant ES005022). Citation Format: Joseph Wahler, Jae Young So, Amanda Smolarek, Hubert Maehr, Milan Uskokovic, Nanjoo Suh. A gemini vVitamin D analog, BXL0124, inhibits mammary tumorigenesis by delaying the transition of ductal carcinoma in situ to iInvasive breast carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 184. doi:10.1158/1538-7445.AM2013-184

Breast Irradiation and Cardiac Toxicity

Breast irradiation is a critical element in breast conservation therapy for both early-stage invasive and noninvasive breast cancer (ductal carcinoma in situ). Large clinical trials have clearly and consistently demonstrated that breast irradiation reduces local disease recurrence, but it can also increase the risk for cardiac events, particularly for patients receiving left-sided irradiation. To define this risk, investigators conducted a population-based, case-control study of major coronary events (defined as myocardial infarction, coronary …

Can we select individuals with low risk ductal carcinoma in situ (DCIS)? A population-based outcomes analysis

Ductal carcinoma in situ (DCIS), a non-invasive breast cancer, is usually treated by breast-conserving surgery (BCS). Randomized trials prove that the addition of radiotherapy (XRT) leads to lower rates of recurrence. Despite the evidence, half of women do not receive XRT after BCS. It is unknown how well clinicians identify women with low risk DCIS for treatment by BCS alone or to what extent women with DCIS develop recurrent cancer due to the omission of radiotherapy. We report the outcomes of a population of women with DCIS treated with BCS, alone or with radiotherapy, and evaluate the effectiveness of each therapeutic approach. All women diagnosed with DCIS and treated with BCS, alone or with radiotherapy in Ontario from 1994 to 2003 were identified. Treatments and outcomes were validated by chart review. Survival analyses were used to study the development of local recurrence (LR) in relation to patient and tumor characteristics and the use of radiotherapy. The cohort included 3,762 women treated with breast-conserving therapy; 1,895 of whom (50%) also received radiation. At 10years median follow-up, LR developed in 233 (12%) women who received radiotherapy and in 363 (19%) of women who did not (p<0.0001). The 10-year actuarial LR rate for women who did and did not receive radiotherapy was 12.7 and 20.0% (p<0.0001). Differences were significant for both for invasive LR (7.0 vs. 10.0%, p<0.0001) and for DCIS recurrence (6.1 vs. 10.8%, p<0.0001). We estimate that 22% of recurrences diagnosed in Ontario women treated for DCIS between 1994 and 2003 would have been prevented if all patients had received radiotherapy. The omission of radiotherapy after BCS for DCIS resulted in substantive recurrences that might have been avoided with treatment. Additional markers are needed to identify a low risk group in whom radiation can be safely omitted.

MicroRNA-132 is frequently down-regulated in ductal carcinoma in situ (DCIS) of breast and acts as a tumor suppressor by inhibiting cell proliferation

Ductal carcinoma in situ (DCIS) is the most common type of non-invasive breast cancer. The currently accepted step-wise model suggests that breast cancer progressed in the following manner: normal breast cell→usually ductal hyperplasia (UDH)→atypical ductal hyperplasia (ADH)→DCIS→invasive ductal carcinoma (IDC). Therefore, DCIS can serve as a good model to analyze the mechanism underlying invasive breast cancer occurrence. MicroRNAs (miRNAs) are a novel class of small non-coding RNAs (∼22nt) involved in the regulation of various biological processes. Altered miRNA expression could also contribute to the origination of cancer, including breast cancer. Here, by using miRNA microarray and real time PCR, we analyzed the miRNA expression profile in 21 DCIS and the corresponding normal tissues. miR-10b, miR-125b, miR-132, miR-145, miR-154-3p, miR-382-5p and miR-409-3p were found to be significantly deregulated in DCIS. Results from CCK-8 assay showed that the overexpression of miR-132 could inhibit the proliferation of breast cancer cell line. High expression of miR-132 could also inhibit the colony formation. Our findings will lead to further understanding of the development of breast cancer.

Abstract C13: The role of cancer heterogeneity in metastasis

Abstract Breast cancer doesn't contain only one colony of tumor cells. It appears to have heterogeneous cell composition of characteristically different subtypes with distinct molecular and gene expression signatures. Due to the tumor heterogeneity, which determines the risk of metastatic progression and drug resistance, it is difficult to predict progression patterns of cancer and provide the most suitable treatment to patients. Most research has focused on molecular definition and genetic association for the cancer heterogeneity studies. However, lack of in vitro models to precisely mimic the heterogeneous conditions of tumors has made it difficult to understand the role of heterogeneity in metastasis, especially invasion. So, to precisely regulate heterogeneous microenvironment of tumors and evaluate the role of the heterogeneity on invasion &amp; metastasis, we developed a microfluidic in vitro assay incorporating 3D ECM scaffolds that emulate in vivo tumor microenvironment in 3D. We cultured highly invasive breast cancer cell, MDA-MB-231, and non-invasive breast cancer cell, MCF-7, together in a mixed population and let the cells interact with other cells and ECMs. As a result, we found that heterogeneity in cancer cell population promotes metastatic potential. Highly invasive breast cancer cells, MDA-MB-231, made microtracks in ECM and leaded collective migration of non-invasive breast cancer cells, MCF-7, into the microtracks. When MCF-7 were cultured alone, MCF-7 cells didn't invade into ECM. It has been widely announced that only stromal cells such as fibroblast remodel ECM and enhance the invasion potential of cancer cells. However, from the result of our experiment, cancer cells that have different characteristics in single tumor can promote the metastatic potential, result from that ECM remodeling (generating microtracks) by highly invasive cancer cells. It was precisely investigated by IF, rt-PCR and TEM analysis in microfluidic environments. Understanding about not only genetic and molecular definition of cancer heterogeneity, but also the role of heterogeneous cancer cell population in metastatic dissemination, could help new drugs and therapeutic strategies development. Citation Format: Yoojin Shin, Hyo Eun Jeong, Sewoon Han, Jihee Won, Seok Chung. The role of cancer heterogeneity in metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C13.

A Hybrid Model of Tumor–Stromal Interactions in Breast Cancer

Ductal carcinoma in situ (DCIS) is an early stage noninvasive breast cancer that originates in the epithelial lining of the milk ducts, but it can evolve into comedo DCIS and ultimately, into the most common type of breast cancer, invasive ductal carcinoma. Understanding the progression and how to effectively intervene in it presents a major scientific challenge. The extracellular matrix (ECM) surrounding a duct contains several types of cells and several types of growth factors that are known to individually affect tumor growth, but at present the complex biochemical and mechanical interactions of these stromal cells and growth factors with tumor cells is poorly understood. Here we develop a mathematical model that incorporates the cross-talk between stromal and tumor cells, which can predict how perturbations of the local biochemical and mechanical state influence tumor evolution. We focus on the EGF and TGF-β signaling pathways and show how up- or down-regulation of components in these pathways affects cell growth and proliferation. We then study a hybrid model for the interaction of cells with the tumor microenvironment (TME), in which epithelial cells (ECs) are modeled individually while the ECM is treated as a continuum, and show how these interactions affect the early development of tumors. Finally, we incorporate breakdown of the epithelium into the model and predict the early stages of tumor invasion into the stroma. Our results shed light on the interactions between growth factors, mechanical properties of the ECM, and feedback signaling loops between stromal and tumor cells, and suggest how epigenetic changes in transformed cells affect tumor progression.

Restoration of the methylation status of hypermethylated gene promoters by microRNA-29b in human breast cancer: A novel epigenetic therapeutic approach

It is well established that transcriptional silencing of critical tumor-suppressor genes by DNA methylation is a fundamental component in the initiation of breast cancer. However, the involvement of microRNAs (miRNAs) in restoring abnormal DNA methylation patterns in breast cancer is not well understood. Therefore, we investigated whether miRNA-29b, due to its complimentarity to the 3’- untranslated region of DNA methyltransferase 3A (DNMT3A) and DNMT3B, could restore normal DNA methylation patterns in human breast cancers and breast cancer cell lines. We demonstrated that transfection of pre-miRNA-29b into less aggressive MCF-7 cells, but not MDA-MB-231 mesenchymal cells, inhibited cell proliferation, decreased DNMT3A and DNMT3B messenger RNA (mRNA), and decreased promoter methylation status of ADAM23 , CCNA1, CCND2, CDH1, CDKN1C, CDKN2A, HIC1, RASSF1, SLIT2, TNFRSF10D, and TP73 tumor-suppressor genes. Using methylation polymerase chain reaction (PCR) arrays and real-time PCR, we also demonstrated that the methylation status of several critical tumor-suppressor genes increased as stage of breast disease increased, while miRNA-29b mRNA levels were significantly decreased in breast cancers versus normal breast. This increase in methylation status was accompanied by an increase in DNMT1 and DNMT3B mRNA in advanced stage of human breast cancers and in MCF-7, MDA-MB-361, HCC70, Hs-578T, and MDA-MB-231 breast cancer cells as compared to normal breast specimens and MCF-10-2A, a non-tumorigenic breast cell line, respectively. Our findings highlight the potential for a new epigenetic approach in improving breast cancer therapy by targeting DNMT3A and DNMT3B through miRNA-29b in non-invasive epithelial breast cancer cells.

MMP1, MMP9, and COX2 Expressions in Promonocytes Are Induced by Breast Cancer Cells and Correlate with Collagen Degradation, Transformation-Like Morphological Changes in MCF-10A Acini, and Tumor Aggressiveness

Tumor-associated immune cells often lack immune effector activities, and instead they present protumoral functions. To understand how tumors promote this immunological switch, invasive and noninvasive breast cancer cell (BRC) lines were cocultured with a promonocytic cell line in a Matrigel-based 3D system. We hypothesized that if communication exists between tumor and immune cells, coculturing would result in augmented expression of genes associated with tumor malignancy. Upregulation of proteases MMP1 and MMP9 and inflammatory COX2 genes was found likely in response to soluble factors. Interestingly, changes were more apparent in promonocytes and correlated with the aggressiveness of the BRC line. Increased gene expression was confirmed by collagen degradation assays and immunocytochemistry of prostaglandin 2, a product of COX2 activity. Untransformed MCF-10A cells were then used as a sensor of soluble factors with transformation-like capabilities, finding that acini formed in the presence of supernatants of the highly aggressive BRC/promonocyte cocultures often exhibited total loss of the normal architecture. These data support that tumor cells can modify immune cell gene expression and tumor aggressiveness may importantly reside in this capacity. Modeling interactions in the tumor stroma will allow the identification of genes useful as cancer prognostic markers and therapy targets.

Abstract P1-05-06: A novel mutation in the tyrosine kinase domain of ErbB2: molecular and proteomic investigation of its role in breast cancer invasion

Abstract Introduction: A landmark genomic study revealed 17 somatic coding mutations in a lobular metastatic breast tumor, none of which were present in the patient's primary tumor. Among these was a novel mutation in the tyrosine kinase domain of ErbB2, a gene often amplified in breast cancer. Other mutations in this domain have been shown to stimulate transformation, growth and invasion in breast cancer cells. We hypothesize that an accumulation of mutations in the primary tumor over time preceded the development of an invasive clone, which allowed it to metastasise. Therefore, we investigated the specific effects of ErbB2 on cellular signalling and invasion, in conjunction with proteomic analyses to show its effects on global protein expression. Methods: Wild-type and mutant ErbB2 cDNAs were cloned into V180 pLP 3X FLAG-tagged expression vectors. T47D breast cancer cells were transfected with one of these constructs. Protein was detected by immunoblotting with an anti-ErbB2 antibody. Membranes were then immunoblotted with an anti-phospho-ERK1/2 antibody. Transfected cells were seeded onto Matrigel; cells that migrated through the matrix were stained and counted after 48h. To identify global proteomic changes caused by mutant ErbB2, T47D cells transfected with mutant/wild-type ErbB2 or vector control, were differentially labeled by growing them in medium containing various lysine/arginine isotopes (known as stable isotope labeling of amino acids in cell culture; SILAC). Protein lysates were mixed and subjected to LC-MS/MS mass spectrometry. Proteomic changes were analysed using MaxQuant and DAVID software. Results: Overexpression of the wild-type or mutant ErbB2 genes produced protein expression in T47D, a non-invasive breast cancer cell line, confirmed by western blotting. To measure signalling downstream of ErbB2, we measured levels of the MAP kinase phospho-ERK1/2. Increased phosphorylation of ERK1/2 was demonstrated in cells expressing mutated ErbB2, compared with cells transfected with wild-type. This indicates increased activation of mutant ErbB2, demonstrating distinct phenotypic effects between wild-type and mutant ErbB2 in breast cancer cells. To analyze whether increased activation was accompanied by enhanced invasive properties, invasion assays were performed using Matrigel. A greater number of cells (1.5-fold increase) expressing mutant ErbB2 migrated across this matrix compared with wild-type-expressing cells. Global proteomic analysis of T47D cells overexpressing wild-type or mutant ErbB2 yielded 1,357 total proteins, of which 543 could be quantified. Two subsets of proteins were identified, whose levels were either increased or decreased, in the mutant versus wild-type-transfected lysates (compared to control ratios). Proteins elevated in the mutant-expressing cells included candidates involved in cell migration, inhibition of apoptosis and promotion of cellular survival. Proteins showing reduced expression were involved in pathways including adhesion and DNA damage response. Conclusion: This previously undescribed mutation in the tyrosine kinase domain of ErbB2 differentially effects downstream signalling pathways and may play a role in invasion of breast cancer cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-06.

Quality assurance in cancer. Acceptable delays in deciding the therapeutic plan in breast cancer and colorectal cancer

To determine recommended delays for treatment once there is well-founded clinical suspicion in care processes for breast and colorectal cancer, taking into account resources and the psychological well-being of the patients. A qualitative study among professionals. The study was conducted in two phases. Firstly, during 4 sessions (N=19) we conducted a revision of the care processes of breast cancer and colorectal cancer and fixed desirable times. Secondly, through a modification of the Delphi technique (N=49), the proposed times for each care process were validated. Delphi response rates of 69% and 58% for colorectal and breast processes respectively. The recommended time in the case of non-invasive breast cancer was 5 to 6 weeks. If the cancer was invasive and nuclear medicine was used for sentinel node study or axillary clearance 5 to 7 weeks were recommended. In the case of cancer of the colon and rectum 7 to 13 weeks were considered necessary. Breast cancer treatment should be started before six weeks. In colorectal cancer up to three months could be required. An interdisciplinary review of care processes relying on professionals is useful for establishing realistic quality standards.

P83. An audit and survey of ductal carcinoma in situ upgrading to invasive cancer and axillary staging

Introduction: Ductal carcinoma in situ (DCIS) of the breast is the most common form of non-invasive breast cancer. It is primarily treated by surgery with or without adjuvant radiotherapy. NICE guidelines (2012) state axillary staging in patients undergoing a mastectomy for DCIS. The aim of this audit and survey was to assess local incidence of DCIS upgrading to invasive cancer and current practice of axillary staging in DCIS in the UK.