bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1-01-12: Intraoperative detection of sentinel lymph nodes in breast cancer patients using ultrasonography-guided direct indocyanine green dye-marking by real-time virtual sonography constructed with three-dimensional computed tomography-lymphography
Authors: Yamamoto S , Maeda K , Maeda N , Yoshimura K , Oka M
Published: August 2014

Purpose: This study aims to determine the utility of ultrasonography (US)-guided direct dye-marking of sentinel lymph nodes (SLNs) by real-time virtual sonography (RVS) constructed with three-dimensional (3D) computed tomography (CT)-lymphography (LG).

Patients and Methods: We identified SLNs in 258 clinically node-negative breast cancer patients using an RVS system to display in real time a virtual multiplanar reconstruction CT image obtained from CT volume data corresponding to the same cross-sectional image from US. CT volume data were obtained using our original 3D CT-LG, which accurately detects SLNs in breast cancer. We then perform US-guided dye-marking close to SLNs using indocyanine green (ICG). Subsequently, indigo carmine blue dye was injected into the subareolar and peritumoral areas around each primary tumor. All patients underwent SLN biopsy and SLN metastases were examined pathologically.

Results: In all 258 patients, we were able to detect the same SLNs visualized on 3DCT-LG, using the RVS system. We detected ICG close to SLNs in 257 of 258 patients (99.6%) during SLN biopsy. In 25 patients (9%), we failed to follow the blue lymphatic route stained by indigo carmine and SLNs were not stained by indigo carmine, but easily detected SLNs by ICG marking.

Conclusion: US-guided direct ICG dye-marking of SLNs using this RVS system seems useful for the detection of SLNs, allowing easy detection of SLNs even when the stained lymphatic route is not followed.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-01-12.
Abstract P1-01-23: Intraoperative optical coherence tomography for the assessment of metastatic disease in human lymph nodes
Authors: Marjanovic M , Adie SG , Nolan RM , Chaney EJ , Shemonski ND , South FA , Erickson-Bhatt SJ , Shelton RL , Bower AJ , Simpson DG , Ray PS , Cradock KA , Brockenbrough J , Liu G , Boppart SA
Published: August 2014

The status of lymph nodes is an important factor in staging cancer since the transport of primary cancer cells via the lymphatic system is one of the main pathways of metastasis to distant organs. During cancer surgery, lymph node status is evaluated via sentinel lymph node biopsy (SLNB), which involves the removal and analysis of the first (or sentinel) nodes along the lymphatic chain of nodes draining the primary tumor. The sentinel nodes are identified through the accumulation of a radioactive agent (technetium-99) and/or isosulfan blue dye within the nodes, frequently resulting in the resection of multiple nodes for subsequent, often time-consuming, histopathological analysis. The majority of these resected nodes are found by histological analysis to be normal, leading to unnecessary complications, including increased risk of lymphedema. Thus, a method for the in situ assessment of node status could reduce the number of normal lymph nodes that are resected. In this study we evaluated the sensitivity and specificity of three-dimensional optical coherence tomography (OCT) for the intraoperative assessment of metastatic disease in lymph nodes.

OCT is the optical analogue to ultrasound imaging, except images are based on the optical scattering properties of near-infrared light. Real-time OCT with micron-scale resolution affords optical biopsies of tissue for immediate feedback. Intraoperative OCT imaging was conducted on human lymph nodes resected from 49 subjects during breast and, head and neck cancer surgeries. Three-dimensional OCT datasets were recorded ex vivo from one or more locations per node, and marked with surgical ink for subsequent correlation to histology. These lymph nodes then underwent the standard histological processing.

A double-blinded study was performed comparing the assessment of OCT datasets to the co-registered histological findings. Three-dimensional -OCT datasets from 206 sites were independently analyzed by six observers and classified as cancerous or non-cancerous. Seventy-nine sets were identified as unsuitable for OCT analysis due to insufficient nodal tissue within the imaged field-of-view. Early training classification results from three of the six observers resulted in a sensitivity of 64.8% and a specificity of 73.3% for identifying metastatic lymph nodes intraoperatively, in real-time, compared to the gold standard of post-operative histopathology. Final study results are expected to improve with observer training and a decision tree for interpreting OCT images.

Our initial imaging studies of resected lymph nodes in human cancer subjects demonstrate the potential of OCT as a technique for real-time optical biopsy of lymph nodes for the intraoperative staging of cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-01-23.
Abstract P1-04-08: Co-expression of putative stemness and epithelial-to-mesenchymal transition markers on single circulating tumour cells from patients with early and metastatic breast cancer
Authors: Papadaki MA , Agelaki S , Kallergi G , Zafeiriou Z , Theodoropoulos PA , Georgoulias V , Mavroudis D
Published: August 2014

Background: The detection of circulating tumor cells (CTCs) in peripheral blood (PB) of patients with breast cancer (BC) has been associated with poor disease outcome. Cancer cells with stemness and epithelial-to-mesenchymal transition (EMT) properties are considered to display enhanced metastatic potential. The presence of these features in CTCs could be an important determinant of increased metastatic behaviour. A new methodology was developed to investigate the co-expression of the stemness marker ALDH1 and the EMT marker TWIST on CTCs from patients with early and metastatic breast cancer, at the single-cell level.

Patients and Methods : PBMC’ cytospins were prepared from PB obtained from 80 patients with early and 50 with metastatic breast cancer. Triple immunofluorescence staining was performed using anti-pancytokeratin (A45-B/B3), anti-ALDH1 and anti-TWIST antibodies. Classification of ALDH1 expression levels (high or low) and TWIST subcellular localization (nuclear or cytoplasmic) was performed in HepG2 control cells and three representative BC cell lines, using the ARIOL system. A total of 500.000 PBMCs per patient were subsequently analyzed.

Results : CTCs were detected in 13 out of 80 (16%) and in 25 out of 50 (50%) patients with early and metastatic BC, respectively. Although co-expression of ALDH1 and TWIST was observed on almost all CTCs in both patient groups, CTCs expressing the phenotype ALDH1high/TWISTnuclear were more frequently detected among patients with metastatic compared to early disease (76% vs. 15% of patients, p = 0.0004). Moreover, a higher proportion of ALDH1high/TWISTnuclear CTCs was confirmed in the metastatic setting (62% vs. 13% of CTCs, p<0.0001). On the contrary, the phenotype ALDH1low/TWISTcytoplasmic was more prevalent among CTCs detected in early disease (32% vs. 8% of CTCs, p = 0.0006).

Conclusions : A new assay is provided for the evaluation of ALDH1 and TWIST co-expression at the single-CTC level. CTCs bearing the ALDH1high/TWISTnuclear phenotype were more commonly detected in metastatic compared to early BC, suggesting that this phenotype prevails during disease progression. The characterization of CTCs according to these markers could be used to improve risk stratification in BC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-08.
Abstract P1-07-02: Significance of RB-ZEB axis in EMT phenotype of breast cancer and inhibition of ZEB by CDK4/6 inhibitor
Authors: Arima Y , Hosonaga M , Saya H
Published: August 2014

The retinoblastoma protein (pRB) is a key tumor suppressor that regulates the progression of cell cycles from G1 to S phase. pRB is often inactivated due to mutation, very low level expression or hyperphosphorylation by cyclin-dependent kinases (CDKs) in many cancers. We found that knockdown of pRB induced the expression of ZEB1 (zinc finger E-box binding homeobox 1), which is one of the transcriptional repressors of the E-cadherin gene, and led to epithelial-mesenchymal transition (EMT) through the ZEB-dependent pathways in breast cancer cells. EMT is defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype, and is associated with an invasive and metastatic potential in cancers. We found that siRNA-mediated depletion of ZEB in pRB-inactive cells suppressed cell invasiveness and proliferation and induced epithelial-cell marker expressions. We also found that knockdown of ZEB1 reduced the dissemination of breast cancer cells in bone marrow from primary tumors growing in mammary fat pads of nude mice. We then conducted a screen with an existing drug library for inhibitors of ZEB1 expressions and identified several hits, including CDK inhibitors. We confirmed that the CDK4/6 inhibitor, Palbociclib (PD0332991), suppressed breast cancer cell proliferation and invasiveness in vitro, and the treatment with the PD0332991 suppressed tumor growth in vivo using an orthotopic xenograft breast cancer model. In terms of cancer stemness, we found that the treatment of PD0332991 decreased the CD44+/CD24Low/- breast cancer stem cell population. We also determined that CDK4 inhibitors blocked tumorsphere formations, which are a characteristic of stem-like cancer cells. p16INK4a (p16) is an endogenous CDK4/6 inhibitor, and we found that knockdown of p16 increased the CD44+/CD24Low/- breast cancer stem cell population in estrogen receptor-negative breast cancer. CDK4/6 inhibitors appear to substitute for the p16 functions which suppress cancer stemness. Our findings indicate that the CDK4/6 inhibitor affects tumor progression, not only through cell cycle control, but also through the suppression of EMT and stem-like properties of cancer cells.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-02.
Abstract P1-13-04: A comparison of cardiovascular outcomes in postmenopausal women treated with aromatase inhibitors versus tamoxifen in breast cancer
Authors: Fiorino LH , Pitz M
Published: August 2014

OBJECTIVE: Breast cancer treatment Phase III studies show that Aromatase Inhibitors (AI) provide a longer disease free survival period compared to tamoxifen although overall survival is not consistently improved. This suggests an increase in a competing risk. The purpose of this study is to determine if there is an increase in cardiovascular morbidity and mortality in postmenopausal women treated with AI versus those treated with tamoxifen.

METHODS: Using a retrospective cohort study design all postmenopausal women with breast cancer from January 1, 2002 to December 31, 2010 were identified from the Manitoba Cancer Registry (MCR). Data from these patients was requested from the Manitoba Health administrative databases for exposure, comorbidity, and outcome variables. Specifically, Drug Program Information Network (DPIN) data from 2002 to the most recent data available to determine prescriptions filled for tamoxifen and AI as a measure of exposure to these drugs. Hospital separation abstracts, physician claims data, and DPIN were used to determine comorbidity as well as new events for cardiovascular disease. Four specific groups were identified for comparison with respect to the outcomes of interest: tamoxifen exposed, AI exposed, exposure to both and no exposure. The primary end point was cardiovascular outcomes, including incident coronary heart disease and stroke.

RESULTS: There were 4729 postmenopausal women diagnosed with Stage I – III breast cancer within previously defined years. The numbers for those treated with AI, Tamoxifen or both were 1590, 1015 and 607 respectively. Excluding women with preexisting comorbidity of coronary heart disease (CHD) and stroke, a competing risk regression model found that tamoxifen at 1 year of use had a statistically significantly lower rate of CHD endpoints compared to AI (p 0.040). There was no difference in stroke incidence between the two groups.

CONCLUSION: Tamoxifen appears to provide a protective affect against CHD. Aromatase Inhibitors may lead to an increase in cardiovascular morbidity and mortality which may explain the lack of overall survival benefits with it's use. In the future, Tamoxifen may be preferentially used in women with a significant cardiac history.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-13-04.
Abstract P1-13-08: Arthralgia and changes in serum levels of IGF-I, its binding protein and estrogen in breast cancer patients on endocrine agents
Authors: Lintermans A , Van Asten K , Wildiers H , Paridaens R , Leunen K , Smeets A , Amant F , Christiaens M-R , Verhaeghe J , Vermeersch P , Vanderschueren D , Neven P
Published: August 2014

Objective Aromatase inhibitors (AIs) frequently induce or enhance musculoskeletal problems, also known as the AI-induced musculoskeletal syndrome (AIMSS). Underlying mechanisms are unknown, likely multiple and induced by low estrogen levels. We hypothesized a role for the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis (Lintermans et al, Ann of Oncol 2011). Recently, Gallicchio et al. (J Cancer Res Clin Oncol 2013) confirmed our hypothesis. Here we report the effect of tamoxifen and AIs on AIMSS, IGF-I, its binding protein and estrogen levels from a prospective study.

Material & Methods In this prospective cohort study postmenopausal women with an early breast cancer scheduled to start adjuvant endocrine therapy with any of the third generation AIs or tamoxifen were included. After providing informed consent, a rheumatologic questionnaire was completed and serum was collected. AI-induced musculoskeletal pain was defined as an increase in joint or muscle pain relative to baseline; AIMSS presence was defined as the presence of ≥ 3 out of five features (arthralgia, myalgia, joint stiffness, tingling, carpal tunnel syndrome).

IGF-I levels were determined in batch with an inhouse radio-immunoassay; levels of IGF binding protein 3 (IGFBP-3) with a commercial EASIA from DiaSource. A sensitive liquid chromatography dual mass spectrometry was used for assessment of estradiol (E2) and estrone (E1) values, with a limit of quantification of 1.3 and 1.2 ng/l, respectively. SHBG levels were determined with the Roche Modular E170 analyzer. Re-evaluation was done after 3, 6 and 12 months of therapy.

Results 84 patients started on tamoxifen (n = 42) or an AI (n = 42; anastrozole n = 6, letrozole n = 36). In the AI group, 4 patients discontinued due to AIMSS. In the tamoxifen group, 1 patient developed a deep vein thrombolic event and discontinued tamoxifen therapy.

In all BMI categories, AI and not tamoxifen decreased E1 and E2 levels below limit of quantification from month 3 onwards. SHBG values dropped on AI while they increased on tamoxifen.

AI therapy resulted in elevated IGF-I and IGF-I/IGFBP-3 ratio levels, whereas tamoxifen users had a decrease. AI-induced musculoskeletal symptoms were characterized by a higher increase from baseline in IGF-I levels and IGF-I/IGFBP-3 ratio than AI users without any complaints. Patients experiencing AIMSS showed a significant decrease in IGFBP-3 levels (p = 0.0007) and an increase in IGF-I/IGFBP-3 ratio (p = 0.07) compared to patients without AIMSS. This divergence was most pronounced at 3 months following AI start.

Conclusion These findings provide preliminary evidence that AI-induced musculoskeletal symptoms are associated with changes in the GH/IGF-I axis.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-13-08.
Abstract P2-02-10: Evaluation of apparent diffusion coefficient to predict grade, micro-invasion and invasion in DCIS
Authors: Hussein HM , Chung C , Moshonov H , Freitas V , Miller N , Kulkarni SR , Scaranelo A
Published: August 2014

PURPOSE: To evaluate the role of apparent diffusion coefficient (ADC) values in distinguishing DCIS grades and identifying the presence of micro-invasive/invasive disease.

METHODS AND MATERIALS:

REB approved study with informed consent obtained from 70 women (age 36-84) scheduled for core-biopsy with results of 71 non-invasive/high-risk breast lesions. All patients underwent surgery and were assessed pre-operatively using diffusion weighted (DWI)-MRI. Lesion size, morphology and ADC values were recorded. The Kruskal Wallis or one-way ANOVA test and Pearson correlation coefficient were used to study the association between ADC values and the analyzed MRI lesion characteristics. Logistic regression analysis was used to evaluate the ability of ADC values to predict the presence of invasion.

RESULTS:

Of 71 cases, 45.1% were imaged on a 3T magnet and 54.9% on 1.5 T. Final pathology demonstrated invasive cancer in 26.8%, micro-invasion in 18.3% and pure DCIS in 59.2%. On 3T, mean ADC value was 1.20 ×10-3 mm2/s ± 0.48 (SD) (range, 0.47 - 1.78 ×10-3 mm2/s) for non-high grade DCIS, 1.23 × 10-3 mm2/s ± 0.40 (SD) (range, 0.26 - 1.77 ×10-3 mm2/s) for high-grade DCIS, and 1.15 × 10-3 mm2/s ± 0.45 (SD) (range, 0.26 - 1.75 ×10-3 mm2/s) for invasive/microinvasive disease. On 1.5T, mean ADC value was 1.04 × 10-3 mm2/s ± 0.41 (SD) (range, 0.15 - 1.85 ×10-3 mm2/s) for non-high grade DCIS, 1.01 × 10-3 mm2/s ± 0.37 (SD) (range, 0.06 - 1.76 ×10-3 mm2/s) for high-grade DCIS, and 1.11 × 10-3 mm2/s ± 0.30 (SD) (range, 0.64 - 1.76 ×10-3 mm2/s) for invasive/microinvasive disease. Based on logistic regression analysis, mean ADC value was not a significant predictor for invasiveness using 1.5 T [OR = 2.6 (95% CI (0.409, 17.12)), p = 0.3] or 3T [OR = 0.4 (95% CI (0.076, 2.399)), p = 0.3]

CONCLUSION: Mean ADC acquired using a 1.5T or 3T MRI was unable to predict high-grade or invasive disease in biopsy-proven DCIS lesions. Further work is exploring voxel-based approaches that may better appreciate tumor heterogeneity and identify sub-regions of tumor with these higher risk features.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-02-10.
Abstract P2-03-05: Relationship between 64Cu-DOTA-trastuzumab positron emission tomography uptake and assessment of HER2 by immunohistochemistry in women with advanced breast cancer
Authors: Mortimer JE , Colcher D , Frankel P , Raubitschek A , Carroll M , Conti P , Tong S , Poku K , Miles J , Bading J
Published: August 2014

Background: We have utilized 64Cu-DOTA-trastuzumab with PET imaging to assess the in vivo expression of HER2 in women with advanced breast cancer. We have demonstrated that a preadministered dose of trastuzumab 45 mg prior to injection of 64Cu-DOTA-trastuzumab resulted in a 75% decrease in the hepatic uptake of Cu-64, resulting in improved image quality.

Methods: Patients with biopsy confirmation of recurrent disease located outside the breast and axilla considered for study. Complete staging workup included CT of the chest, abdomen, and pelvis, bone scintigraphy and 18F FDG PET. At least 1 non-hepatic site of metastasis that was > 2 cm separate from the biopsy site was also required. HER2 status was assessed by both IHC and FISH. Index lesions were identified on CT imaging. After the first two patients, all patients received a cold dose of 45mg of trastuzumab immediately prior to 64Cu-DOTA-trastuzumab to decrease liver uptake. 64Cu-DOTA-trastuzumab PET imaging was performed at 24 and 48 hours. Uptake on 64Cu-DOTA-trastuzumab was correlated with HER2 status by IHC and FISH.

Results: Fifteen women have undergone 64Cu-DOTA-trastuzumab PET imaging and quantitative image analysis. 10 patients were HER2+ (7 IHC 3+, 3 IHC 2+/FISH+), 3 patients were IHC 2+/FISH-, and 2 patients were IHC 1+. Tumor uptake by 64Cu-DOTA-trastuzumab PET max SUV was higher in HER2+ positive than HER2- patients (1.9-fold higher on day1, p<0.02, and 1.7-fold higher on day2, p<0.05). However, the lowest max SUV was in a HER2+ patient (HER2 2+/FISH+), demonstrating considerable heterogeneity.

Conclusion: 64Cu-DOTA-trastuzumab PET correlates with HER2+ status. However, due to high within and between patient variability, 64Cu-DOTA-trastuzumab PET imaging could potentially enrich for HER2+ patients that respond to HER2-targeted therapy, and could also suggest some HER2- patients that may benefit from HER2-targeted therapy. This hypothesis needs to be further explored in patients undergoing HER2-targeted therapy. This work was supported by the Department of Defense grant # BC095002.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-03-05.
Abstract P2-04-02: Overdiagnosis in mammography screening in two randomised trials in Malmö, Sweden – An age specific analysis
Authors: Jonsson H , Johansson R , Andersson I , Zackrisson S
Published: August 2014

OBJECTIVES: The aim of the current study was to estimate age-specific overdiagnosis due to mammography screening in the two randomized trials in Malmö, Sweden: The Malmö Mammographic Screening Trial I and II (MMST I and MMST II).

SETTING: Women living in Malmö were randomized in a 1:1 proportion to a screening group or a control group. The MMST I included 42,283 women born between 1908 and 1932 and the MMST II included 17,793 women born between 1933 and 1945. Women in the study group were invited from 1976 until 1990 when the control group was invited. The age at randomization was 45-70 years in MMST I and 43-48 years in MMST II.

METHODS: Data from the two trials were merged and overdiagnosis was estimated using age specific incidence rates excluding the first years of screening. The age specific relative breast cancer incidence rate for the screening group versus the control group was estimated and adjusted for lead time. Only the first breast cancer diagnosis was considered and results for invasive cancer and carcinoma in situ combined as well as for invasive cancer were calculated.

RESULTS: The number of breast cancer cases, including carcinoma in situ, for the period 1976-1989 was 867 and 676 for the study group and the control group, respectively. The rate ratio for overdiagnosis in age 43-78 years adjusted for lead time was 1.04 (95% CI, 0.93-1.16). No overdiagnosis from subsequent screening was found for women below the age of 60. In women aged 60-69 and 70-78 years overdiagnosis was estimated at 1.13 (0.95-1.33) and 1.15 (0.85-1.54), respectively. The corresponding results for invasive cancer only was lower and no overdiagnosis was seen except for the age group 60-69 years where the rate ratio was 1.10 (0.92-1.32). For age 43-78 the result was 0.96 (0.85-1.08).

CONCLUSIONS

For the total material overdiagnosis from subsequent screening, including carcinoma in situ, was 4%. This is somewhat lower than an earlier evaluation of MMST I where age were higher and another method was used. For women below the age of 60 no overdiagnosis was seen and indicated that the possible problem with overdiagnosis concern women 60+. However none of the results were statistically significant at the 5% level.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-04-02.
Abstract P2-05-04: Comparative expression profiling of patient samples and preclinical models of inflammatory breast cancer reveals gene signatures of epithelial plasticity and suppression of TGFb signaling
Authors: Van Laere SJ , Marsan M , Vermeulen PB , Viens P , Barsky SH , Cristofanilli M , Dirix LY , Bertucci F , Robertson FM
Published: August 2014

Introduction: Genome-wide expression profiling of samples from patients with and without Inflammatory Breast Cancer (IBC) has revealed novel insights into the biology of IBC. The present study was undertaken to compare these novel insights with data obtained from all available preclinical IBC models including 2 new models that we have recently developed that recapitulate the characteristics of IBC including retention of E-cadherin, formation of tumor emboli and encircling lymphoangiogenesis

Materials and Methods: Five replicates of 7 preclinical IBC models (SUM149, SUM190, FC-IBC-01, FC-IBC-02, MDA-IBC-03, KPL-4, and Mary-X) were profiled using Affymetrix HGU133plus2 GeneChips. Using a nearest shrunken centroid algorithm, each expression profile was classified according to an IBC-specific signature identified in patient samples. Available expression profiles were further queried for expression patterns related to Epithelial-to-Mesenchymal Transition (EMT), TGFβ-signaling and IBC-specific patterns of transcription factor activation.

Results: Application of our IBC-specific signature (posterior probabilities exceeded 0.50 in at least 4/5 replicates) revealed that out of 7 pre-clinical models of IBC, 3 of these robustly classified as IBC (FC-IBC-01, FC-IBC-02, and KPL-4). All preclinical IBC models were characterized by retention of E-Cadherin expression, absence of ZEB1 expression, attenuated expression of specific components of the TGFβ pathway (TGFβR2, SMAD3, SMAD7, and TGFβ1), and ambiguous activation patterns of several transcription factors involved in regulating cellular plasticity and cell fate decisions (Up in IBC: NR4A2, RARB/RXRA, PTX3, GSC2, and ZEB1; Down in IBC: SOX10, PAX5, and SMAD2). For each of the molecular alterations described above, Z-scores greater than 2 were achieved in at least 4/5 replicates.

Conclusions: The observations that we have made using IBC patient tumor samples with regards to EMT, cell plasticity and TGFβ-signaling are corroborated in pre-clinical models of IBC using current analytic approaches, despite the fact that expression patterns of the majority of preclinical models of IBC deviate from the IBC-specific expression patterns observed in patient samples. Our data suggest that despite their highly invasive nature, IBC cancer cells retain an epithelial cell phenotype characterized by E-cadherin expression and loss of ZEB1 which appears to be mediated by, amongst others, attenuated TGFβ-signaling. This study strengthens our hypothesis that cancer cells from IBC exhibit cohesive invasion, and invade as a unit, possibly explaining the presence of florid tumour emboli which is a primary characteristic observed in IBC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-04.
Abstract P2-07-01: Polymorphism A247S of XRCC4 contributes to the risk of breast cancer and impairs DNA damage repair pathway
Authors: He M , Hu X , Cao A-Y , Yu K-D , Shao Z-M , Di G-H
Published: August 2014

Genetic variations in a network of genes involved in DNA double strand breaks (DSB) repair pathway have been shown to increase the risk of breast cancer. XRCC4 is identified as a pivotal modulator in non-homologous end joining (NHEJ), a predominant DSB repair pathway. In this study, we evaluated the associations between single nucleotide polymorphisms (SNPs) in XRCC4 and breast cancer, and investigated the impact of XRCC4A247S variant on nuclear localization and DNA damage response functions of XRCC4.

A hospital-based case-control study, comprising 412 breast cancer patients and 458 cancer-free controls recruited from Shanghai Cancer Center, was conducted to investigate the relationship between common polymorphisms in XRCC4 and the risk of breast cancer. Our results showed that XRCC4A247S polymorphism (rs3734091), located near the putative nuclear localization signal (NLS) of XRCC4, was associated with a significantly increased risk of breast cancer in a recessive genetic model (Adjusted odds ratio (OR) = 1.47, 95% confidence interval (CI) = 1.09-4.52; P = 0.042). This homogeneous variant was more often observed in triple negative subtype (Adjusted OR = 2.07, 95% CI = 1.34 - 3.58 in the recessive model; P = 0.036). Although XRCC4A247S variant did not harm its interactions with XLF and ligase IV (the other two components of DNA ligase complex), we revealed that A247S alteration partially impaired the nuclear localization of XRCC4 in mammary cells. Remarkably, more evidences implied that XRCC4A247S variant significantly reduced the in vivo NHEJ repair efficiency compared to the wild-type XRCC4 via colony formation assay and NHEJ reporter assay, suggesting that reduced nuclear accumulation by XRCC4A247S variant impaired its functions in response to DNA damage. Importantly, XRCC4A247S alternation promoted cellular sensitivity to ionizing radiation and DNA damaging agents in chemotherapy.

Taken together, this study demonstrates that genetic variation of XRCC4A247S could impair NHEJ mediated DSB repair and provide further evidences that genetic variants involved in DNA repair pathways contribute to breast cancer susceptibility. In addition, our results also indicate that XRCC4A247S variant might be a potential predictive biomarker and therapeutic target in breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-01.
Abstract P2-07-03: High-throughput germline mutation screening for hereditary breast cancer in southern Chinese patients by massively parallel DNA sequencing
Authors: Kwong A , Au CH , Law FB , Ho DN , Ip BK , Wong AT , Shin VY , Chan TL , Ma ES
Published: August 2014

Introduction: Breast cancer is the most common malignancy and 3rd leading cause of deaths among the female population in Hong Kong. Since the establishment of The Hong Kong Hereditary Breast Cancer Family Registry in 2007, 1344 patients with breast and/or ovarian cancer who met the selection criteria were recruited for genetic testing in Hong Kong. Since 2011 we started to employ next-generation DNA sequencing (NGS) to expedite the analysis workflow and expand the panel of genes for sequencing.

Aim: To evaluate the workflow of NGS in mutation screening of BRCA1, BRCA2, TP53 and PTEN genes, and compared with the sequence data obtained by Sanger sequencing.

Methods: We sequenced BRCA1, BRCA2, TP53 and PTEN genes in peripheral blood samples of 410 patients, 53 positive controls and 107 healthy local individuals using 454 GS Junior System. Generation of barcoded amplicon libraries was streamlined by microfluidic PCR using Fluidigm Access Array System. Sequencing data were analyzed by an in-house developed fully automatic bioinformatics pipeline, which mainly consists of GS Amplicon Variant Analyzer, SAMtools and Ensembl Variant Effect Predictor. All putative mutations identified were validated by Sanger sequencing. Furthermore, the frequency of BRCA1, BRCA2 and PTEN missense variants of unknown significance (VUS) identified in the cohort were compared among 107 healthy local individuals and 1000 Genomes project samples. The VUS were also subjected to a panel of in silico prediction methods including PolyPhen and SIFT.

Results: Among 410 patients, there were 7 in BRCA1, 6 in BRCA2 and 1 in TP53 mutations found, including 1 novel recurrent BRCA2 (c.7007G>T) and 1 novel founder BRCA2 (c.5164_5165delAG) mutations. Based on multiple criteria, 12 in BRCA1, 12 in BRCA2 and 1 in PTEN VUS could be prioritized for further investigation. The bioinformatics pipeline was extensively evaluated with Sanger-validated controls. The evaluation determined minimum sequencing coverage needed in this sequencing platform for accurate analysis. The pipeline accuracy was demonstrated by successful detecting mutations from 53 positive controls, including single nucleotide variants, insertions and deletions in different sequence context.

Conclusion: BRCA1, BRCA2, TP53 and PTEN mutation screening of 410 patients were expedited by high-throughput DNA sequencing. This method could detect 14 positive cases, including recurrent mutations, in a shorter period of time when compared with Sanger full gene sequencing. High-risk patients who are negative for the gene panel may need further investigation other than screening for BRCA1/2. The in-house developed bioinformatics pipeline was validated to detect various types of mutations and potentially become a conventional platform for genetic screening.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-03.
Abstract P2-07-05: Prevalence of germline BRCA1 and BRCA2 mutations in triple-negative breast cancer patients unselected for family history
Authors: Scott RJ , Wong-Brown M , Meldrum CJ , Avery-Kiejda K
Published: August 2014

Triple-negative breast cancer (TNBC) is a histological classification of a breast cancer subtype that is negative for the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBCs represent an important subtype of breast cancer as they have a lower recurrence-free and overall survival compared to receptor-positive disease, regardless of disease staging at time of diagnosis. It has been suggested that TNBCs may harbour mutations in genes involved in the BRCA-associated DNA repair pathway (i.e. BRCAness). Recent studies have shown that up to 20% of tumours classified as TNBC present with BRCA mutations.

Additionally, TNBCs are over-represented in patients who harbour BRCA1 or BRCA2 mutations and are predominantly associated with early onset breast cancer. This suggests a link between the BRCA-associated DNA repair pathway and TNBC.

Genomic DNA was obtained from a series of 347 consecutively-collected patients with histopathologically-diagnosed TNBC, irrespective of family history. BRCA1 and BRCA2 coding sequence (including the intron/exon boundaries) was amplified using Fluidigm 48.48 Access Arrays (184 primer sets designed to cover the coding regions of both BRCA1 and BRCA2 genes) were used to prepare the libraries. The libraries were paired-end sequenced on Illumina MiSeq and the data analyzed with NextGENe 2nd Generation Sequencing Software.

Overall the number of pathogenic BRCA mutations detected was 30 (8.6%): 18 (5.2%) in BRCA1 and 12 (3.4%) in BRCA2. The proportion of pathogenic mutations observed in our cohort is not too dissimilar to the number of mutations detected in a familial breast cancer setting, which in our experience is approximately 12%.

Investigation of the family histories of the TNBC patients revealed that 13/30 (43.3%) had a confirmed family history of disease. Examination of the families revealed that the disease spectrum was as expected for hereditary breast and ovarian cancer. The age of diagnosis of breast cancer in the TNBC BRCA mutation carriers (Mean = 51, SD = 16) was significantly different (p = .03) to those with wild-type BRCA (Mean = 58, SD15) suggesting a different disease trajectory.

This study confirms that there a substantial proportion of BRCA1 and BRCA2 mutation carriers reside in populations of patients diagnosed with TNBC. This adds to the growing body of evidence suggesting that breast cancer with a triple-negative phenotype should be considered as an additional criterion for genetic testing of both BRCA genes.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-05.
Abstract P2-08-03: Patterns of somatic mutations in metaplastic carcinoma of the breast
Authors: O'Toole SA , Yu B , Ng CC , Selinger CI , Cooper WA , Carmalt H , Mak C , Chan C , Lee S , Karim R , Beith J , Cooper C
Published: August 2014

Introduction

Metaplastic carcinoma of the breast (MCB) is a heterogeneous group of malignant tumors comprising less than 1% of all breast carcinomas encompassing tumors with purely epithelial, as well as epithelial and mesenchymal components, which may be entirely metaplastic or admixed with carcinoma. There is no agreed upon optimal treatment regime for these tumors, which overall have a worse prognosis than similar stage invasive ductal carcinoma (IDC). Research into MCB has been limited by the rarity of this tumor; however as the majority of these tumors do not express ER, PR or Her2 (triple negative) and response to traditional chemotherapy is limited, there is a need for further research to identify predictive biomarkers and new targets for treatment. We report our findings of multigene mutation profiling in a series of 21 MCB.

Methods

DNA was extracted from tumors of twenty one patients with MCB, genotyped using the Oncocarta v1.0 kit a sensitive, high throughput mass spectrometry based assay which detects a panel of 238 specific mutations in 19 oncogenes including KRAS, BRAF, PIK3CA, MET, AKT1 & ERBB2 and processed on the Sequenom compact MassARRAY platform. In the case with heterologous differentiation, separate cores were taken from the spindle cell, osteo- and chrondrosarcomatous areas.

Results

Histological review revealed three pure spindle cell cases with the remainder showing mixed patterns of spindle, squamous and IDC, no special type. A single case showed heterologous osteosarcomatous and chondrosarcomatous differentiation. Ten mutations were detected in these 21 MCB. PIK3CA gene mutations (six H1047L and one E545K mutation) were seen in seven patients; and KRAS gene mutations (two G12V and one G12A) were observed in three patients. No mutations were detected in the case with heterologous elements in the separate areas of spindle, osteo- and chondrosarcomatous differentiation.

Conclusions

PIK3CA mutations were seen in 33% and KRAS mutations were seen in 14% of MCB cases in this series. Given the general lack of effective chemotherapies in MCB, these findings suggest that the PI3K pathway may be a promising therapeutic target worthy of further investigation. We plan to also undertake detailed mutational analyses using next generation sequencing to identify further potential therapeutic targets in these rare and difficult to treat malignancies.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-08-03.
Abstract P2-09-22: Synthesis of nitroxyl radical-containing nanoparticles with pH-sensitive behavior and their application for cancer therapy
Authors: Yu A , Wang H , Zhang T , Wang Y
Published: August 2014

Extensive research has documented that redox dysregulation is a causative involvement in tumor progression, and reactive oxygen species (ROS) are involved in cancer initiation and progression. ROS are mutagenic and may promote cancer. Early studies observed that the increased ROS production was in response to oncogenic activity. It is now widely accepted that redox chemotherapeutics represents a novel class of promising anticancer agents. Small molecule nitroxides are stable free radicals which are able to scavenge radicals and act as radical chain reaction terminators. Nitroxyl radical, 2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPO), is capable of reacting with ROS and shows chemotherapeutic efficacy in various cancer cell lines and xenograft models. However, low-molecular-weight TEMPO derivatives are hard to utilize in the clinic, owing to a preferential renal clearance and side effects such as antihypertensive effect.

To address these issues, we have designed and developed nitroxyl radical-containing nanoparticles with pH-sensitive behavior using the first generation PAMAM (G1 PAMAM) partially modified with 3,4,5,6-tetrahydrophthalic anhydride (THPA) and PROXYL. The synthesis of the modified PAMAM was carried out by a two-step reaction. Firstly, part of the primary amine end group of PAMAM dendrimers reacted with anhydride THPA to produce a THPA-modified PAMAM. The b-carboxylic acid amides between G1 PAMAM and THPA are acid-sensitive. Therefore, the THPA-modified PAMAM are stable and negatively charged at physiological pH, thus preventing blood serum aggregation. However, the THPA-modified PAMAM degrades at acidic pH, converting its surface to positive charge which is benefit for cellular uptake. Secondly, the THPA-modified PAMAM reacted subsequently with 3-carboxy-PROXYL whose carboxyl group was activated by N-hydroxysuccinimide. The EPR signal of the product was analyzed by EPR measurement and confirmed the PROXYL group covalently conjugated onto THPA-modified PAMAM. The obtained THPA-PROXYL-modified PAMAM was denoted as TP-PAMAM. Experimental results showed that TP-PAMAM was stable at pH 7.4, and hydrolyzed at pH 6.0. The rate of hydrolysis was increased with pH decreasing. In vitro studies indicated that cell proliferation of MDA-MB-231 was greatly inhibited by TP-PAMAM compared to low-molecular-weight TEMPO. This may due to the fact that cellular uptake is easy for nanoparticles with pH-sensitive behavior. Therefore, the nitroxyl radical conjugated onto the pH-sensitive nanoparticles scavenges ROS more effectively. As a result, these new nitroxyl radical-containing nanoparticles have a significantly high therapeutic efficacy in the application for cancer therapy. Hence, engineered nitroxyl radical-containing nanoparticles with pH-sensitive behavior may enable therapeutic selectivity and to overcome drug resistance and metastatic cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-22.
Abstract P2-11-21: Ipsilateral breast tumor recurrence in patients with operable breast cancer who undergo breast-conserving treatment after receiving neoadjuvant chemotherapy: Validation of the MD Anderson prognostic index
Authors: Rouzier R , Bailleux M , Guinebretiere J-M , Delalande B , Lasry S , Lerebours F , Stevens D
Published: August 2014

BACKGROUND:

A prognostic index, the MD Anderson Prognostic Index (MDAPI), has been developed to help refine selection criteria for breast-conserving therapy after neoadjuvant chemotherapy. This score is based on initial nodal status N2/3, residual pathologic tumor size>2cm, a multifocal pattern of residual disease, and lymphovascular space invasion and ranged from 0 to 4. The objective of the current study was to validate the MDAPI for ipsilateral breast tumor recurrence.

METHODS:

Our study was a single-center retrospective analysis of a large prospectively collected breast cancer database. We identified data from 355 patients who received neoadjuvant chemotherapy and underwent breast-conserving therapy between 2002 and 2010. Predictive factors of ipsilateral breast tumor recurrence and the usefulness of the MDAPI were analyzed. Discrimitaion of models was also calculated.

RESULTS:

The median follow-up was 65 months, and the 5-year ipsilateral breast tumor recurrence-free survival rate was 90.7%. 5-year IBTR-free survival were 91.3% and 87.4% in MDAPI 0/1 and MDAPI 2+ categories (p = 0.07). (Multivariate analysis demonstrated that MDAPI (p = 0.01), Ki67 (p = 0.0004) and HER2 status (p = 0.0006) were associated significantly with ipsilateral breast tumor recurrence-free survival. Concordance index of the MDAPI was fair (0.6) but adding HER2 and ki67 status in the model improved concordance index : 0.75.

CONCLUSIONS:

The MDAPI was barely adaptable to the study patients in terms of predicting ipsilateral breast tumor recurrence. Biological factors such as ki67 and HER2 status should be included in the prognostic model.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-21.
Abstract P2-18-04: No further axillary dissection in sentinel lymph node-negative breast cancer after neoadjuvant chemotherapy in patients with initial cytologically-proven axillary node metastasis
Authors: Lee HC , Lee JH , Kim MK , Kil WH , Kim J , Lee JE , Kim SW , Nam SJ , Kim SM , Bae SY , Lee SK , Kim J
Published: August 2014

Background

In patients with fine needle aspiration (FNA)-proven axillary lymph node metastasis at diagnosis (cN+), the current standard surgical procedure is axillary lymph node dissection (ALND) at definitive surgery after neoadjuvant chemotherapy (NAC). However, growing evidences suggest that SLNB after NAC is feasible and may demonstrate acceptable performance in selected patients. We performed sentinel lymph node biopsy in patients treated with cytologically-confirmed axillary lymph nodes metastases at presentation, who converted to a clinically negative axillary status after NAC (ycN0).

Patients and methods

We retrospectively evaluated 240 patients with invasive breast cancer with ultrasound-guided FNA-proven axillary nodal metastases at the time of diagnosis. All patients received NAC and underwent surgery at Samsung medical center between October 2007 and May 2013. Among these patients, 75 patients underwent SLNB. These patients converted to clinically node-negative disease (ycN0) after NAC on breast MRI or PET/CT scan. A combined detection technique was used with radioisotope and blue dye for the detection of SLN. Patients with negative SLN on frozen pathology and low clinical suspicion of metastasis during operation were not performed further ALND.

Results

The detection rate of SLNB was 93.3% (70/75), and median number of retrieved sentinel lymph nodes was 3.0 (range 1-8). False negative rate was 6.7% (1/15). Of these 75 patients, 35 (46.6%) patients had positive sentinel lymph nodes (ypN+) and underwent ALND. Thirty-five (46.6%) patients had tumor-free sentinel lymph nodes (ypN0sn) and 20 patients of them were followed without subsequent ALND. In these SLN-negative patients without further ALND, 9 patients were HER2-enriched subtypes and 9 patients, TNBC subtypes. Only two of them were Luminal B subtypes. The median follow-up period was 12.0 months (range 0-26 months) with 2 events; 1 regional recurrence in ipsilateral supraclavicular node and 1 systemic recurrence in brain on postoperative 7 months and 5 months, respectively. There has not occurred an ipsilateral axillary recurrence so far.

Conclusions Although the follow-up was not long enough to conclude, this study tried to demonstrate that SLNB after NAC was feasible and further ALND may not be necessary in patients with SLN-negative disease (ypN0sn).

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-18-04.
Abstract P3-01-07: Metformin has an additive effect to cisplatin in triple negative breast cancer cells with high lactate dehydrogenase B (LDHB) expression
Authors: Harada N , Varghese V , Tharayil IA , Kenny L
Published: August 2014

Background;

Glycolysis is one of the major metabolic pathways in cancer cells. The conversion of glucose to lactate in the presence of oxygen is a critical aerobic pathway that allows cancer cells to proliferate rapidly and the amount of lactate produced is correlated with tumor aggressiveness. Moreover, the increase in glycolysis is partially mediated by hypoxia-inducible transcription factor (HIF-1) and a metabolic enzyme, lactate dehydrogenase B (LDHB). However, the mechanism behind this metabolic shift is not well understood. Metformin reduces the overall risk of cancer incidence by 31%, improves cancer-related mortality and enhances response to chemotherapy in diabetic patients pre-operatively. This study aimed to evaluate metabolism in triple negative breast cancer.

Methods;

Breast cancer cell lines (estrogen receptor positive MCF7 and triple negative MDA-MB 231 and MDA-MB468) were analysed for protein expression by western blotting and mRNA expression by qRT-PCR with or without cisplatin and metformin. To study the biological mechanism, we evaluated cell growth using proliferation assay in the presence of metformin and cisplatin. The contribution of LDHB to glycolysis was evaluated using MDA-MB-231 and MDA-MB468 cell lines.

Results;

We first examined the basal expression levels of LDHB and HIF1a in these cancer cell lines. LDHB expression was high in MDA-MB 231, and HIF1α was high in MDA-MB468. Both metformin and cisplatin treatment resulted in the down-regulation of LDHB and HIF1α in MDA-MB231, metformin led to downregulation of LDHB in MDA-MB 468, while MCF7 demonstrated no significant changes. Inhibition of cell proliferation was dose-dependent and significant at low concentrations of cisplatin and metformin in only MDA-MB468 but not in MCF7 and MDA-MB231. The combination of cisplatin in with metformin had an added inhibitory effect on cell proliferation in these MD-MB231 cells.

Conclusion;

These studies suggest that combination treatment of metformin and cisplatin have potent anticancer activity in LDHB highly expressed triple negative breast cancer cells. Identification of a new targeted therapeutic approach could contribute to the new therapy to the breast cancer patients.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-07.