bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P4-01-17: Ductolobular breast carcinoma and the role of preoperative magnetic resonance imaging
Authors: Postma EL , El Sharouni MA , Verkooijen HM , Witkamp AJ , van den Bosch MA , van Hillegersberg R , van Diest PJ
Published: August 2014

Introduction: Lobular carcinoma is the second most frequently diagnosed type of invasive breast cancer (about 10%) after the ductal NOS type (80%). In patients with lobular carcinoma, magnetic resonance imaging (MRI) plays an important role in the pre-operative work up, since mammography and ultrasound often underestimate the tumor extent and have a high false negative rate for additional foci in the ipsi- or contra lateral breast when compared to MRI. As the histological spectrum of invasive breast cancers is however a continuum with gradual transitions between types, mixed type invasive carcinomas with both ductal and lobular histologic features occur and are usually denoted ductolobular cancer. In our institution, MRI is part of the routine pre-operative work up in patients with ductolobular breast cancer, but the value of MRI in the pre-operative work up of ductolobular carcinoma has yet to be determined.

Purpose: We set out to determine the probability of detecting additional, clinically relevant, findings on MRI in patients diagnosed with ductolobular carcinoma.

Methods: We retrospectively searched the pathology database for all patients diagnosed with histologically proven ductolobular breast cancer at our institution between 2008-Sept 2012. Findings on mammography and ultrasound were compared with the findings on the pre-operative MRI. Additional findings on MRI (larger tumor size, additional ipsi- and contralateral lesions) were verified by pathology evaluation.

Results: Out of the 121 patients diagnosed with ductolobular cancer, 91 (75%) underwent MRI for pre-operative work-up. With MRI, 67 additional findings were detected in 56 (62%) patients. In 23 patients a contralateral additional lesion was seen, of which 12 turned out malignant. Ipsilateral lesions were seen in 28 patients, 20 of which were malignant at histology. In 16 patients, more extensive disease was seen on MRI compared to conventional imaging (mean difference was 18 mm, range 6–67 mm). MRI correlated better with the histological extent of these tumors (r = 0.97) than conventional imaging (r = 0.79). Of all MRI detected additional findings, 69% (46/67) was proven malignant after histology assessment. On patient-level, MRI led to the detection of one or more malignant finding(s) in 41/91 (45%) patients.

Conclusion: For patients diagnosed with invasive ductolobular breast cancer, the probability of detecting clinically relevant additional findings on MRI is high. Preoperative MRI should therefore be part of the routine work up of this breast cancer type.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-01-17.
Abstract P4-02-04: Tissue oxyhemoglobin dynamics measured with functional optical imaging immediately after starting chemotherapy correlates with markers of cellular proliferation and inflammation in a rat breast tumor model.
Authors: Ueda S , Roblyer D , Cerussi A , Saeki T , Tromberg B
Published: August 2014

We have reported that a transient increase of oxyhemoglobin level measured 24 hours after the start of neoadjuvant chemotherapy using noninvasive Diffuse Optical Spectroscopic Imaging (DOSI) may be useful in discriminating nonresponding from reponding breast cancer patients (PNAS. 2011 Aug 30;108(35):14626–31). To elucidate the underlying biological mechanism, we compared optical measured hemoglobin levels to tissue biomarkers in a rat tumor model.

Method: An orthotopic mammary tumor model using Fischer 344 rats and MAT3B cells was used for this experiment. 16 mg of cyclophosphamide was administered (i.p.) in 15 rats after tumors had grown to a significant size. A wide-field functional imaging device that utilizes structured near-infrared light was used to measure superficial absolute concentrations of oxyhemoglobin (ctO2Hb) and deoxyhemoglobin (ctHHb) of the tumors. Rats were imaged and sacrificed (two per timepoint) at each of the following timepoints: baseline, 2, 6, 12, 24, 48, 96 hours, and day 8 following the injection. Resected tumors were sectioned after paraffin-embedding and representative slides were used for immunohistochemistry. Cellular markers of proliferation (measured by Ki67), apoptosis (Caspase3), NK cells (ANK61), and macrophages (MAC387) were counted in each slide using Image J software.

Result: Average tumors baseline values of ctO2Hb and ctHHb were 81.4 µM (±9.2 SD) and 22.4 µM (±4.1 SD) respectively. In most rats, both levels gradually increased from baseline to 24 hours after the injection and reached a maximum at 48 hours and then declined until Day 8. At 24 hours, ctO2Hb was significantly higher than baseline [96.8 µM (±8.8 SD) vs. 81.4µM (±9.2 SD), p = 0.003 using Student's t-test]. This trend was mimicked in the tissue markers of cellular proliferation and apoptosis, which peaked at 24 through 48 hours and then dropped. The average NK cell count was also found to rise consistently through the course of monitoring and reached the highest at day 8, while macrophage counts reached a peak at 48 hours and then declined. The differences between baseline and 24 hours for these were statistically significant: Ki67 [294 (±107 SD) vs. 115 (±68 SD), p = 0.0002], Caspase3 [2916 (±1383 SD) vs. 136 (±150 SD), p < 0.0001], NK cells [635 (±329 SD) vs. 159 (±110 SD), p = 0.0001], and macrophage markers [281 (±260 SD) vs. 39 (±31 SD), p = 0.01].

Conclusion: We observed an increase in oxyhemoglobin early after chemotherapy, closely linked to changes in cellular proliferation, apoptosis, NK cells and macrophage accumulation. Early optical changes may be linked to chemotherapy-induced tumor metabolic and acute inflammatory responses.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-02-04.
Abstract P4-02-08: Quantitative Characterization of 3D Vasculature Spatial Patterns Within Tumor Microenvironment of Breast Cancer Stem Cells
Authors: Zhan M , Li F , Zhu Y , Ma J , Landua J , Wei W , Vadakkan T , Zhang M , Dickinson M , Lewis M , Rosen J , Wong S
Published: August 2014

The sustainment characters of cancer stem cells (CSCs) such as self-renew and differentiation to other tumor cells greatly depend on the tumor microenvironment, which is composed of many components, e.g. vasculature, extracellular matrix, epithelial cells, stromal cells, as well as nutrients and oxygen. As vasculature is an important factor for the CSC and tumor development, the understanding of their spatial patterns is essential for calibrating the CSC-microenvironment interactions in mathematical modeling. In this study, we acquired the vasculature in side tumors and normal breast tissues by using two-photon fluorescence microscopy, which enables 3D in vivo imaging. We developed an advanced vasculature segmentation approach for an objective and accurate quantification of the vasculature in 3D image volumes. The approach integrates supervoxel analysis and the orientation guided hidden Markov random field (ori-HMRF) modeling together to compensate for low quality images, e.g., low signal-to-noise ratio (SNR) and uneven background. By constructing a new feature space that combines the CIELAB color space and the coordinates space, the supervoxel analysis divides an image volume into subregions with local similar intensity and restricted regular shape, boundaries of which can delineate the vasculature boundaries accurately even in low intensity contrast regions. We further designed a set of features for the separation of blood vessel regions from the background. To make use of the context information, i.e. the continuity of vasculature, the ori-HMRF model is used to incorporate the consistency of vasculatures' orientation in order to reduce the false positives and negatives. Experimental results on image volumes from both breast cancer and normal breast tissues show that the proposed method can effectively reconstruct the vasculature structure with the CSC embedded tumor microenvironment.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-02-08.
Abstract P4-02-09: Molecular Breast Imaging: the sensitivity of breast-specific gamma imaging (BSGI) as a diagnostic adjunct to mammography and ultrasound in a triple assessment protocol
Authors: Weigert JM , Kieper DA , Stern LH , Böhm-Vélez M
Published: August 2014

Purpose: BSGI is a diagnostic breast imaging procedure becoming more common in clinical breast practice. The goal of this work is to quantify its performance as an addition to mammography and ultrasound in detection of breast carcinoma when used in the community breast center setting.

Methods: A multi-center patient registry was maintained for all patients routinely sent to BSGI as part of their diagnostic work up. From the registry data, patients who had a mammogram followed by ultrasound and BSGI were selected for evaluation. The BIRADS rating schematic was used for mammography and sonography and a similar category system was used for the BSGI images. For each modality, the reports were classified as positive (categories 4–6) or Negative (categories 0–3). Needle and/or surgical biopsy were conducted as deemed clinically necessary and all patients who had a malignant diagnosis by pathology were entered into this analysis.

Results: 731 patients had all three imaging modalities as part of their diagnostic work up resulting in 180 malignancies confirmed by pathology: 29 ductal carcinoma in-situ, 110 infiltrating ductal carcinoma, 11 infiltrating lobular carcinoma, 9 papillary carcinoma and 21 mixed component malignancies. Mammography was positive in 130 (sensitivity = 72%) while ultrasound was positive in 114 (sensitivity = 63%) and BSGI was positive in 147 (sensitivity = 82%). The combination of mammography and ultrasound was positive in 163 cases (sensitivity = 90%) while the addition of BSGI provided positive findings for 177 malignancies resulting in a sensitivity of 98%.

A subsequent breast MRI detected one of the lesions missed by the three modalities while two lesions were found by pathology alone.

Conclusions: Of the three imaging modalities, BSGI provided the highest independent sensitivity and when added to the diagnostic workup, BSGI detected an additional 14 malignancies, increasing the sensitivity from 90% to 98%. Although it is beyond the scope of this work, it is interesting to note that the cost of the BSGI procedure is relatively low, about $320, and that in this population the BSGI specificity was 74%. In summary, when added to the diagnostic work up of patients in the community breast cancer, BSGI can improve the detection of breast malignancy when compared to mammography and ultrasound alone.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-02-09.
Abstract P4-03-03: Feasibility study of a new volume navigation system-guided breast biopsy method for incidental enhancing lesions detected by breast contrast-enhanced magnetic resonance imaging
Authors: Takahashi M , Jinno H , Hayashida T , Nemoto M , Tanimoto A , Kitagawa Y
Published: August 2014

Background: Incidental enhancing lesions (IELs) undetected by conventional imaging such as mammography and ultrasonography (US) have been reported in 16–29% of patients undergoing subsequent breast contrast-enhanced magnetic resonance imaging (CE-MRI). We recently assessed IELs by performing breast biopsies using a volume navigation system (V-Navi) which synchronizse real-time US images with reconstructed CE-MRI data side-by-side on the same monitor. The aim of this retrospective study was to evaluate the feasibility of V-Navi-guided biopsy method as an IEL diagnostic procedure.

Methods: We performed breast biopsies on 333 patients after mammography, US and CE-MRI (GE Signa Excite HDx 1.5T, USA) examinations between January 2011 and May 2012 at Keio University Hospital. Patients with IELs detected by CE-MRI underwent a second-look US and a bilateral CE-MRI in the supine position in order to achieve the same position as in the US. CE-MRI data was then fused with real-time US images using a V-Navi (Logiq E9, GE Healthcare, USA). When an IEL was identified, we performed a V-Navi-guided vacuum-assisted needle biopsy (11G) or an excisional biopsy after V-Navi-guided marking.

Results: US-guided and stereo-guided breast biopsies were performed on 77.2% (257) and 16.8% (56) of the 333 patients, respectively. Twenty patients (6.0%) had IELs detected by CE-MRI and all such IELs were identified by the V-Navi. Sixteen (80%) of those 20 patients had IELs clearly detected by the V-Navi and underwent V-Navi-guided needle biopsies. The four remaining patients (20%) had IELs that were not clearly detected by the V-Navi and they underwent excisional biopsies. The median IEL size was 8.8mm (range, 5.0–48) and the median procedure time for V-Navi-guided needle biopsies was 42 minutes. No major complications were encountered in any of the cases. Histopathologically, nine IELs (45%) had malignant findings of invasive ductal carcinoma (5/20) and ductal carcinoma in situ (4/20) and 11 IELs (55%) had benign findings of fibrocystic disease (10/20) and intraductal papilloma (1/20).

Conclusion: The V-Navi-guided breast biopsy method was shown to be a safe and effective diagnostic method for the assessment of IELs detected by breast CE-MRI.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-03-03.
Abstract P4-03-04: The potential use of Optical Coherence Tomography for intraoperative breast tumour margin width estimation
Authors: Wilson BC , Akens MK , Niu CJ
Published: August 2014

Total mastectomy and lumpectomy with radiation have been shown to have equivalent patient outcomes, which has likely contributed to the more widespread adoption of breast conserving surgery (BCS) procedures. Assessment of breast lumpectomy margin widths in both an accurate and timely manner is essential to successful breast conservation procedures. Current BCS methodologies have been reported to result in reoperation rates of up to 20–60%, which represents a significant and unmet need for improved margin assessment. High reoperation rates present both increased treatment risk to patients and increased burden on healthcare systems. In the USA alone, over 150,000 lumpectomies are performed per year at an average cost between $11,000 and $19,000 USD per procedure. Assuming a relatively modest average repeat operation rate of 25%, potentially preventable repeat surgeries represent an approximate cost to the US healthcare system of $500M (USD) annually.

Reducing the prevalence of repeat surgeries may be accomplished by providing faster and more accurate intraoperative tools for assessing margin widths during the time of the first surgery. One such potential technique involves the use of Optical Coherence Tomography (OCT) imaging, which uses light to produce images in much the same way that ultrasound produces images with sound. Compared to ultrasound, OCT provides decreased depth of penetration, but increased resolution capabilities. The increased resolution that OCT provides allows for the visualization of the internal cellular structure within a tissue sample and therefore, provides the potential ability to differentiate cancerous from normal or benign cells. We propose the use of an intraoperative OCT imaging system to provide near real-time imaging information about the internal structure of tissue samples excised during BCS procedures. Our hypothesis is that the overall rate of repeat operations can be reduced by providing a tool to assist surgeons with the task of margin width estimation during the time of surgery.

We have developed an early stage prototype OCT imaging system that has completed laboratory phantom and preclinical studies. This paper will present the capabilities of an OCT imaging system to provide margin assessment information in biological breast tissue mimicking phantoms. The phantoms were designed to encompass imaging characteristics across a wide range of human breast densities. The paper will go on to describe preclinical imaging that was done in tumor specimens excised from human breast cancer rat models. The results obtained in the phantom and preclinical studies suggest the potential for OCT as a near-real time, intraoperative imaging tool to aid surgeons with breast lumpectomy margin width estimation. To help realize this potential, further research is required in to the use of OCT during BCS.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-03-04.
Abstract P4-03-05: The Clinical Trial of New Optical Mammography
Authors: Ogura H , Yoshimoto K , Nasu H , Hosokawa Y , Matsunuma R , Ide Y , Yamaki E , Yamashita D , Suzuki T , Oda M , Ueda Y , Yamashita Y , Sakahara H
Published: August 2014

Objectives: Optical mammography (O-MMG), which utilizes near-infrared light for the detection of abnormal breast tissue, can be a potentially useful and non-invasive tool for the screening of breast cancer patients. Here we report on the preliminary results of our clinical trial using a time-resolved O-MMG system developed by Hamamatsu Photonics (Hamamatsu, Japan). In our past examination, negative results included cases with mismatch for breast cup because of small breast, lesion outside of cup because of big breast, and bad positioning. So, size and shape of a gantry was improved (3 cup sizes; S, M and L cup). We reported the initial experience of the improved O-MMG systems.

Materials and Methods: In principle, the technique involves sequential irradiation of the breast tissue with a pulsed laser with a wavelength of 760, 800 and, 830nm, which is then detected by the variable capacity gantry at multiple sites after passing through the breast tissue. Measurement time has improved to about 7 minutes (a third of the conventional measurement time). The data are analyzed and tomographic images of the absorption coefficient of the breast are then reconstructed based on our tomographic reconstruction algorithm. Between October 2011 and May 2012, fifteen patients with breast cancer and one patient with fibroadenoma participated in the trial.

Results: In patients with breast cancer, S cup was used for one patient, M cup for 12 patients and L cup for two patients. Five patients after neo-adjuvant chemotherapy were excluded from the analysis. The lesions were depicted as an area of high hemoglobin concentration in 7 of 10 patients with cancer. One lesion of fibroadenoma was also identified as an area of high hemoglobin concentration.

Conclusion: Further examination with our new O-MMG systems will be of interest.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-03-05.
Abstract P4-03-07: Angiogenic effect of bevacizumab and paclitaxel in metastatic breast cancer: evaluation by contrast-enhanced ultrasonography using Sonazoid®
Authors: Ito T , Mizuno H , Iiboshi Y , Yamamura N , Fujii H , Hitora T , Fujii R , Ohashi T , Nakagawa T , Izukura M
Published: August 2014

Agents targeting vascular endothelial growth factor (VEGF) have been validated as breast cancer therapeutics, yet efficacy can differ between tumor types and individual patients. Rapid noninvasive determination of response could provide significant benefits. We tested if response to the VEGF antibody bevacizumab and chemo drug paclitaxel could be detected using contrast-enhanced ultrasound imaging (CEUS).

The objective of this study was to evaluate contrast-enhanced ultrasound (CEUS) with contrast agent Sonazoid® injection as a predictor of tumor response.

Patients and methods: Ten patients with advanced and metastatic breast carcinomas treated were evaluated. Examinations were performed at baseline, after 2 and 4 weeks and every one month on antiangiogenic drug bevacizumab and chemo drug paclitaxel in tumor targets that were accessible to CEUS. Histological findings were obtained in all cases by ultrasound-guided 14 gauge core needle biopsy or 10 gauge vacuum-assisted biopsy. Each examination included a bolus injection of Sonazoid® and 40–60 seconds of raw linear data with an Aplio (Toshiba), Logiq (GE) and Ascendus (Hitachi-ALOKA) with a 8–12 MHz linear transducer. A low reduced mechanical index (MI) of less than 0.24 was chosen to avoid early gas bubble destruction.

The ethic committee of our institution approved this study. An informed consent was signed by each patient for the ultrasound examination and before administering for the contrast medium.

Results: A total of 60 examinations were performed. Various intratumoral perfusion parameters changed over time, and the signal intensity was significantly impaired in the treated tumors before the treatment efficacy on tumor size could be observed.

Conclusion: CEUS has advantages for real-time visualization and is a new noninvasive imaging technique which might be an effective tool for evaluating antiangiogenic drugs and chemo drugs in breast cancer.

The use of a noninvasive ultrasound approach may allow for earlier and more effective determination of efficacy of antiangiogenic therapy.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-03-07.
Abstract P4-03-08: A new real-time image fusion technique, a coordinated sonography and MRI using magnetic position tracking system, improves the sonographic identification of enhancing lesions in breast MRI
Authors: Nakano S , Fujii K , Yoshida M , Kousaka J , Mouri Y , Fukutomi T , Ishiguchi T
Published: August 2014

Breast magnetic resonance imaging (MRI) is a method that can detect clinically and mammographically occult cancer foci. For lesions suspected to be malignant on the basis of breast MRI, targeted sonography can help determine whether the lesion is amenable to tissue biopsy using sonographic guidance. However, the reproducibility of sonography as well as interexaminer reliability in targeted sonography is still a clinical issue. Recently, we have developed real-time virtual sonography (RVS) that can coordinate a sonographic image and a MRI multiplanar reconstruction (MRI-MPR) of the same section in real time. Although RVS has been reported to be useful for breast cancers, only a few studies have investigated the size and positioning error between a real-time sonographic image and a MRI-MPR image. The purpose of this study was to evaluate the accuracy of RVS to sonographically identify enhancing lesions by breast MRI. Between December 2008 and May 2009, RVS was performed in 51 consecutive patients with 63 enhancing lesions. MRI was performed with the patients in the supine position using a 1.5-T imager with a body surface coil to achieve the same position as with sonography. To assess the accuracy of the RVS, the following three issues were analyzed: (i) The sonographic detection rate of enhancing lesions, (ii) the comparison of the tumor size measured by sonography and the MRI-MPR and (iii) the positioning errors as the distance from the actual sonographic position to the expected MRI position in 3-D. Among the 63 enhancing lesions, 42 (67%) lesions were identified by conventional B-mode, whereas the remaining 21 (33%) initial conventional B-mode occult lesions were identified by RVS alone. The sonographic size of the lesions detected by RVS alone was significantly smaller than that of lesions detected by conventional B-mode (p < 0.001). The mean tumor size provided by RVS was 12.3 mm for real-time sonography and 14.1 mm for MRI-MPR (r = 0.848, p < 0.001). The mean positioning errors for the transverse and sagittal planes and the depth from the skin were 7.7, 6.9 and 2.8 mm, respectively. The overall mean 3D positioning error was 12.0 mm. Our results suggest that RVS has good targeting accuracy to directly compare a sonographic image with MRI results without operator dependence.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-03-08.
Abstract P4-04-01: Combination of intratumoral CpG with systemic anti-OX40 and anti-CTLA4 mAbs eradicates established triple negative breast tumors in mice
Authors: Li J , Tandon V , Levy R , Esserman L , Campbell M
Published: August 2014

Background: Evidence is mounting across diverse datasets and scientific techniques implicating immune dysregulation in poor outcomes of high grade and/or triple negative breast cancers. A multimodal strategy to stimulate the immune system could be an effective therapeutic approach for breast cancer. CpG oligodeoxynucleotides are single-stranded DNA that stimulate dendritic cells and B cells by binding to Toll-like receptor 9 (TLR9). OX40 (CD134) is predominantly expressed on activated T cells and the binding of OX40 with an agonistic antibody (anti-OX40) leads to the proliferation and survival of T cells. Agonistic anti-OX40 antibodies can also reverse the suppressive effects of Treg cells. A third avenue is to target Cytotoxic T lymphocyte associated antigen (CTLA4), which plays a negative regulatory role in T cell activation and is also constitutively expressed on Treg cells. Blocking CTLA4 with an antagonistic antibody (anti-CTLA4) has been shown to enhance anti-tumor responses. In this study, we tested the combination of intratumoral (IT) CpG with systemic anti-OX40 plus anti-CTLA4 in a mouse model of triple negative breast cancer.

Methods: 4T1, an aggressive, triple-negative mammary carcinoma, was implanted orthotopically into syngeneic BALB/C mice. Treatment was initiated when tumors were ∼0.5 cm in the largest dimension. Mice were divided into 8 groups. Various combinations of PBS control, CpG, taxol, and anti-OX40+anti-CTLA4 mAbs were tested (Tables 1 and 2). CpG, 100 µg, was injected IT for 5 consecutive days (days 1–5); Taxol, 16 mg/kg, was administered IP on day 1; anti-OX40 (400 µg) and anti-CTLA4 (100 µg) were given IP on days 1 and 5. Mice were sacrificed once tumors became ulcerated or reached a diameter >2 cm.

Results: As shown in Table 1, CpG alone, taxol alone, or the anti-OX40 + anti-CTLA4 combination each delayed the growth of established 4T1 tumors when compared to the untreated control group. However, the majority of the mice (except one in CpG treated group) died from tumor progression. The combination of CpG + anti-OX40 + anti-CTLA4 had the best anti-tumor effects, with 4 out of 5 mice having demonstrated complete regression of their tumors (Table 2). The addition of taxol to the combination of CpG + anti-OX40 + anti-CTLA4 did not improve the anti-tumor effect, and while there was a substantial delay in growth, only one animal achieved a complete regression.

Conclusion: Our study demonstrated that the combination of IT CpG with systemic anti-OX40 + anti-CTLA4 mAbs can cure mice with established triple negative breast tumors, supporting the possibilities of applying immune modulation approach in human triple negative tumors. Studies are on-going to further dissect the immunological mechanisms involved.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-01.
Abstract P4-04-04: Immunohistochemical analysis of Cancer Testis antigens and Topoisomerase 2-alpha expression in triple negative breast carcinomas: a retrospective study
Authors: Juretic A , Mrklic I , Spagnoli GC , Pogorelic Z , Tomic S
Published: August 2014

Aim. The aim of this study is to assess the expression of cancer testis (C/T) antigens in the TNBC group, expression of topoisomerase 2-alpha (TOPO2A), basal-like (BL) immunophenotype defined by basal markers (CK5/6, CK14, and EGFR), BL morphology and conventional clinicopathologic factors as well as to demonstrate their prognostic relevance with respect to this group of tumours.

Methods. The study includes 83 patients who underwent surgery between January 2003 and December 2009. Slides were stained immunohistochemically with CK5/6, CK14, EGFR, Ki-67, TOPO2A, MAGE-A1, MAGE-A10, NY-ESO and multi-MAGE. Survival-time and multivariate survival analyses were performed for the purpose of identifying prognostic markers for tumours with more aggressive behaviour.

Results. Of the 83 triple negative tumours, 55 (66.3%) have BL immunophenotype and 40 (48.2%) BL morphology. MAGE-A1 show the most frequent expression (69.0%), followed by multi-MAGE (58.0%), NY-ESO (27.1%) and MAGE-A10 (13, 2%). MAGE-A10 expression is significantly correlated with tumour size (p = 0.026). The expressions of MAGE-A1, MAGE-A10 and multi-MAGE are significantly correlated with clinical stage (p = 0.024, p = 0.041, p = 0.031, respectively). A significant correlation has been found between the expressions of MAGE-A10 and NY-ESO and the expression of TOPO2A (p = 0.005, p = 0.013). There is no significant correlation between the expression of C/T antigens and disease-free survival (DFS) or overall survival (OS).

Conclusions. High expression levels of MAGE and NY-ESO observed in TNBC are of potential clinical relevance, especially in the adjuvant setting of treatment, and may be of therapeutic value as a vaccine-based treatment in the TN group of breast tumours for which therapeutic options are limited.

Key words: triple negative breast cancer, basal-like breast cancer, cancer-testis antigen, topoisomerase 2-alpha, prognosis, survival analysis.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-04.
Abstract P4-04-05: Listeria monocytogenes-based bivalent Lm-LLO immunotherapy for the treatment of HER2/neu positive and triple negative breast cancer and its impact on immunosuppression
Authors: Wallecha A , Ramos K , Malinina I , Singh R
Published: August 2014

Overexpression of tumor associated antigens (TAA) such as HER2/neu and high molecular weight melanoma associated antigen (HMW-MAA) are associated with aggressive high-grade tumors leading to disease progression and reduced survival. HMW-MAA has been reported as a TAA in triple negative breast tumors and is also expressed at high levels by activated pericytes and pericytes in tumor angiogenic vasculature that are associated with neovascularization in vivo. Previously published reports have shown that bioengineered Listeria monocytogenes (Lm) secreting either a chimeric HER2/neu antigen (cHER2), or HMW-MAA fragment, fused to a truncated highly immunogenic fragment of the adjuvant protein listeriolysin O (LLO) were found to impact the growth of both transplantable and transgenic mouse breast tumor models. Therefore, we hypothesized that an Lm-LLO immunotherapy capable of delivering two different antigens would likely have a synergistic effect of decreasing tumor growth by targeting two independent mechanisms that support tumor growth; 1) tumor angiogenesis, and 2) tumor cell surface marker, thus improving the therapeutic efficacy of the agent. Here we report the construction of a bivalent recombinant Lm-LLO immunotherapy that expresses and secretes both the cHER2 and HMW-MAA antigens concomitantly as fusion proteins. Initial characterization of the bivalent immunotherapy indicates that both the antigens cHER2 and HMW-MAA are stably expressed and secreted after two in vivo mouse passages. Preliminary studies support the hypothesis that bivalent immunotherapy causes efficient reduction of tumor growth in both HER2 expressing and triple negative breast tumors. Further, mechanistic studies demonstrate that treatment with Lm-LLO immunotherapy resulted in a decreased proportion of regulatory T cells (Tregs) as well as Myeloid Derived Suppressor Cells (MDSC) particularly in the tumor microenvironment and not in spleen, tumor draining lymph nodes, or peripheral blood. Interestingly, both residual Tregs and MDSCs isolated from the tumor microenvironment were found to have a decreased ability to suppress the division of T cells after Lm-LLO immunotherapy, a finding which warrants further investigation. We will present data on the efficacy of bivalent Lm-LLO immunotherapy and the mechanisms that are likely responsible for the observed tumor regression. Currently Lm-LLO immunotherapies are being evaluated in Phase 2 clinical trials for HPV-associated dysplasia and malignancies such as CIN2/3, cervix cancer and head & neck cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-05.
Abstract P4-04-07: Tartrate-resistant Acid Phosphatase (TRAcP)-Expressed Tumor-Associated Macrophages Promote Breast Cancer Progression
Authors: Dai M-S , Wu C-C , Chang P-Y , Ho C-L , Hsieh Y-F , Lo K-Y , Kao W-Y , Chao T-Y , Yu J-C
Published: August 2014

Purpose: Tumor-associated macrophages (TAMs) can promote or dampen breast cancer progression, depending on phenotypic changes within the cancer microenvironment. Two contrasting activation states were described in the literature: the M1 anti-tumoral and M2 pro-tumoral subtype of TAMs. In clinical observation, we analyzed the TRAcP expression in breast cancer TAM and correlated to patient's chemotherapy responses and survival. In animal models, we aim to investigate the tumor-promoting or inhibiting properties among tartrate-resistant acid phosphatase (TRAcP) expressed TAMs.

Methods: TRAcP expression, T lymphocytes, and other macrophage relevant markers were determined by immunohistochemical staining in the human breast cancer tissue from the patients before neoadjuvant chemotherapy. The expression of TRAcP within the TAMs were calculated and correlated to the patient's chemotherapy responsiveness and outcome. The polarized M1/M2 macrophages and breast cancer hematogenous metastasis were created in the animal model. Activated macrophages from TRAcP+/+ or TRAcP−/− mice were injected with JC (Balb/c breast cancer) cells in the metastatic model.

Results: The presence of Foxp3+ regulatory T cell (Treg) correlates to the tumor proliferation (Ki-67 and Her2 expression) and higher Foxp3+ Treg prevalence and tumor Ki-67 expression inferred poorer chemotherapy response. TRAcP staining within the CD68+ macrophages were observed with better chemotherapy response. In animal model, M2 polarized macrophages have higher TRAcP expression, promote breast cancer metastasis, and attract Treg infiltrating in tumor, consistent with the clinical observation.

Conclusion: TRAcP-expressing TAMs, representing M2-polarized macrophage, demonstrat local tumor-promoting and immune-suppressive effects, and chemotherapy resistance. TRAcP expression within TAM can be a potential prognostic marker for breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-07.
Abstract P4-04-08: A Potential non-viral vector to transfect dendritic cell and thereby MHC-Class I antigen presentation might be a potential use in carcinoma
Authors: Shaheen S , Akita H , Souichirou I , Miura N , Harashima H
Published: August 2014

To date almost no remarkable potential non-viral vector was developed to transfect promisingly primary dendritic cell like Bone Marrow Derived Dendritic Cell (BMDC) so that it might generate antigen presentation while epitopic-plasmid was delivered. Here we have introduced a potential non-viral vector named Stearyl-KALA MEND (100–130 nm, zeta potential 35–40 mV), which expressed to date the highest transgene expression while firefly luciferase was introduced (sub-cloned), and thereby promising antigen presentation in vitro while OVA plasmid containing MHC Class-I restricted SIINFEKL epitope was introduced and co-cultured with B3Z T-cell hybridoma. On the basis of these results, we used this vector to transfect BMDC and harvested the DC to chase the corresponding EG7-OVA induced tumor ex vivo. Later we further immunized mice directly with STR-KALA MEND containing OVA plasmid and challenged against the tumor (EG7-OVA induced). In this in vivo study we found also a significant antitumor activity. To evaluate the promptness of our vector we further sub-cloned Mart-1 gene, in our sub-cloned plasmid vector and immunized the mice as before. Thereafter inserting B16F10 melanoma cells to the immunized mice we found also a significant antitumor activity after 24 days of inoculation. Thus the vector, STR-KALA MEND might be a future potential use as DNA vaccine in anti-tumor methodology.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-08.
Abstract P4-05-01: Oncolytic Herpes Simplex Virus Vector G47Δ Effectively Targets Breast Cancer Stem Cells
Authors: Liu R , Zeng W , Hu P , Wu J , Li J , Wang J , Lei L
Published: August 2014

Breast tumors can be classified according to their gene-expression profiles into different molecular subtypes with distinct biological and clinical features. Moreover, different subtypes show distinct responses to different therapeutic regimens, thereby resulting in markedly different outcomes. Recently, accumulating evidence has suggested that breast cancer originates from cancer stem cells (CSCs), which comprise a small percentage of the overall tumor but are highly tumorigenic and pluripotent with unlimited proliferation potential. Furthermore, cancer stem cells are highly resistant to conventional treatment, which potentially explains certain difficulties in treating cancer with current therapy options. The use of oncolytic viruses is a relatively new strategy in cancer therapy, oncolytic herpes simplex virus (oHSV) has been shown to be a safe and effective therapeutic approach for a variety of different cancers.

In this study, different types of breast cancer cell lines were cultured in anchorage-independent conditions, the cells typically form mammospheres within 10 days of anchorage-independent culture, which showed properties of CSCs. The mammospheres are capable of being passaged indefinitely and single dissociated mammosphere cells were able to regenerate spheres. Moreover, compared to the parental cell lines, mammoshpere cells were enriched for CD44+CD24− cells and highly expression of the alternative breast cancer stem cell marker ALDH-1.

The third generation oHSV vector G47Δ effectively killed different subtypes of breast cancer cells in vitro, with more than 98% of the tumor cells killed by day 5, even at multiplicities of infection(MOI) 0.01. Moreover, G47Δ equally targeted non-cancer stem cells (NCSCs) and CSCs which showed resistance to paclitaxel. We also demonstrated that G47Δ effectively replicated and spread among CSCs. Moreover, G47Δ impaired the self-renewal ability of CSCs, as the viable cells unable to form secondary tumor spheres. We also showed that G47Δ was able to induce the regression of tumor xenografts in BALB/c nude mice and demonstrated the ability of G47Δ to synergize with paclitaxel for killing both NCSCs and CSCs. This is the first report that oHSV effectively targets different breast cancer NCSCs and CSCs, thereby suggesting that oHSV may be an effective treatment modality for patients with breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-05-01.
Abstract P4-06-06: RNAi screen of the breast cancer genome identifies KIF14 and TLN1 as genes that modulate chemosensitivity in breast cancer
Authors: Singel SM , Cornelius C , Batten K , Fasciani G , Wright WE , Lum L , Shay JW
Published: August 2014

We conducted a RNA-interference screen for genes whose loss-of-function enhanced doxorubicin and docetaxel chemosensitivity in a “triple-negative,” estrogen receptor negative, progesterone receptor negative, and Her2 negative (ER− PR− Her2−) breast cancer cell line, MDA-MB-231. From ranking chemosensitivity of 334 short-hairpin RNA (shRNA) MDA-MB-231 cell lines (targeting 134 genes with known somatic mutations in breast cancer), we focused on two genes, kinesin family member 14 (KIF14) and talin (TLN1) that not only enhanced chemosensitivity but also have oncogenic annotations. KIF14 has robust expression in breast cancer cells compared to normal mammary cells. TLN1 expression is important for migration of breast cancer cells. In TLN1-deficient cells, CK19 and ZO-1 are upregulated while CK14 and snail are downregulated, suggesting that TLN1 is important for the maintenance of epithelial-mesenchymal transition in MDA-MB-231 cells. KIF14 and TLN1 loss-of-function also enhanced chemosensitivity in 3 other triple negative breast cancer cell lines (HCC1937, HCC38, and Hs578T) but not in normal human mammary epithelial cells (HMECs). Examining protein-protein-interaction networks, we identified RIP2 as a target whose inhibition via SB203580 or PP2 can further enhance chemosensitization in KIF14-deficient cells. Knock-down of a number of other known protein-protein interaction partners of KIF14 and TLN1, including FAK, CIT, ARRB2, PSTPiP1, PRC1, SVIL, ITGA2B, ITGB3, VCL and PXN, do not significantly alter doxorubicin or docetaxel chemosensitivity in MDA-MB-231 cells. Mammary fat pad xenografts of KIF14- and TLN1- deficient MDA-MB-231 cells into NOD/SCID mice demonstrated significantly less tumor mass compared to control MDA-MB-231 cells after chemotherapy. Expressions of KIF14 and TLN1 from breast cancer expression arrays improve prognostic predictions compared to clinicopathological features alone. In summary, screenings for therapeutic targets using chemotherapy and genes with known somatic mutations in breast cancer not only provide a rational targeted screen, but also present possible up-front novel treatment combinations for patients with triple negative breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-06.
Abstract P4-06-11: Expression of genes spanning a breast cancer susceptibility locus on 6q25.1 is modulated by epigenetic modification
Authors: Dunbier AK , White J , Van Huffel S
Published: August 2014

Background: Genome wide association studies have identified single nucleotide polymorphisms around C6ORF97, an open reading frame (ORF) immediately upstream of the gene encoding ERα (ESR1) on 6q25.1, to be associated with increased risk of breast cancer1,2. C6ORF97 and two neighbouring genes, RMND1 and C6ORF211, are also co-expressed with ESR1 in oestrogen receptor positive (ER+ve) breast cancers and C6ORF97 expression is inversely associated with proliferation and recurrence free survival in a tamoxifen-treated patient cohort3. Epigenetic modification represents one potential mechanism through which expression of co-localised genes could be modulated.

Aim: To investigate the role of epigenetic modification in the co-expression of ESR1, RMND1, C6ORF211 and C6ORF97.

Methods: Methylation status and expression of genes in the ESR1/C6ORFs locus was assessed in postmenopausal breast cancers and ER+ve cell lines. Two ER+ve cell lines, MCF7 and T47D, were cultured in oestrogen-depleted media and samples were taken fortnightly for 6 months. DNA and RNA from ER+ve tumours was also analysed in addition to control ER-ve cell lines. Methylation analysis of CpG Islands within the ESR1/C6ORFs locus was carried out using methylation sensitive sequencing analysis. Expression levels of ESR1, RMND1, C6ORF211 and C6ORF97 were determined using quantitative real-time PCR.

Results: Variation in methylation patterns was observed at a number of key CpG islands in the ESR1/C6ORFs region. ER+ve cell lines and highly ER+ve tumours were hypomethylated. Hypomethylation was associated with increased expression of ESR1, RMND1, C6ORF211 and C6ORF97. Temporal changes in methylation and expression of these genes were also observed over the period of oestrogen deprivation in both MCF7 and T47D.

Conclusions: Methylation at the ESR1/C6ORFs locus is associated with expression levels of ESR1, RMND1, C6ORF211 and C6ORF97. This suggests that methylation is involved in modulating the expression of these four genes and that it could partially explain the previously observed co-expression of the genes. Our finding that hypomethylation in this region occurs in tumours with high ERα expression suggests that it could be a mechanism through which tumours increase ERα levels to maximise growth stimulation from oestradiol. Our observation that RMND1, C6ORF211 and C6ORF97 also increase in expression suggests that the proteins encoded by these genes may contribute to the phenotype exhibited by ER+ve breast tumours.

References:

1. Turnbull C et al. Nature Genetics 2010;42:504–7. 2. Zheng W et al. Nature Genetics 2009;41:324–8. 3. Dunbier A et al. PLoS Genetics 2011;7:e1001382.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-11.
Abstract P4-06-16: TGF-β2, A Novel Target of CD44-Promoted Breast Cancer Invasion
Authors: Gupta I , Madani S , Abdraboh M , Al Riyami H , Muzumdar S , AbdElmageed Z , Shanmuganathan S , Bhat S , Ramkumar A , Hakkim L , Ouhtit A
Published: August 2014

Background: Hyaluronan (HA) mediates communication between cancer cells and the environment via interactions with the cell surface receptor CD44. We have previously shown that CD44-HA interaction in BC cells promotes adhesion to bone marrow endothelial cells. This suggests that HA/CD44 signalling may be correlated with breast cancer (BC) metastasis. The long-term objective of this study is to increase our understanding of the mechanisms by which CD44-HA interaction promotes BC metastasis, and further identify and validate CD44-downstream transcriptional targets for anti-metastatic therapy.

Materials & Methods: Pursuant to this goal, we have developed a tetracycline (tet)-regulated expression of CD44 gene in the BC cell line MCF-7 (B5 clone) and identified TGF-β2 (Transforming Growth Factor beta-2; 3 fold induction) as a potential CD44s-downstream transcriptional target by microarray analysis. To further validate this finding, the same RNA samples, used for microarray analysis and their corresponding protein lysates collected from the BC cell line MCF-7-B5, were examined for CD44 expression in the presence of HA for 18, 24, and 48 hr post-tet withdrawal. Expression of TGF-β2 was examined using RT-PCR and western-Blot analyses.

Results: Our results showed that TGF-β2 mRNA levels were significantly elevated following the removal of tet at 18, 24, and 48 h post-HA stimulation compared to the parental cells. Furthermore, the TGF-β2 precursor protein increased in a time-dependent pattern upon HA-stimulation and in the absence of tet. More interestingly, inhibition of CD44 gene by RNAi method decreased TGF-β2 expression upon HA-stimulation and in the absence of tet.

Conclusion: Our data strongly support the hypothesis that TGF-β2 is a potential target of HA/CD44- downstream-signaling mediating BC cell invasion. Ongoing investigation aims to elucidate the signal transduction pathways coupling CD44 to the regulation of TGF-β2 expression, and further validate CD44/TGF-β2 axes in breast tumor invasion and metastasis.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-16.