bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1-01-06: Elaboration of a nomogram to predict the non sentinel node status (NSN) in breast cancer (BC) patients with positive SN, intra-operatively assessed with OSNA (one step nucleic acid amplification) method
Authors: Di Filippo F , Gianarelli D
Published: August 2014

BACKGROUND

Diagnostic axillary lymph node dissection (ALND) has been replaced by sentinel node biopsy (SNB) in nodal staging. Tumor-positive SNB results in a risk of NSN metastases in case of micro and macro metastases ranging from 10 to 50% respectively. Therefore most of the patients are submitted to an unnecessary ALND. Therefore the development of a mathematical model for predicting patient-specific risk of non sentinel node metastases is strongly warranted. We have developed an European Database with positive SN detected by OSNA molecular method, able to identify intra-operatively patients with micro (CK19 mRNA copies 250-5000) or macrometastases (number of copies > 5000).

MATERIAL AND METHODS

European OSNA database has been developed, only patients whose SN was totally assessed with OSNA were included. The following parameters were recorded:

- Clinical: hospital, age, record number

- Bio-patological: tumor size, grading, multifocality, histological type, LVI, ER-PR status, HER-2, KI67, molecular classification (luminal A, luminal B, HER2 like, triple negative)

- Sentinel and non-sentinel lymph node related: number of removed SNs, number of positive and negative SNs, copy number of positive sentinel node, ratio: number of positive SN to number of removed SN number of removed and number of positive nodes after ALND.

A total of 2351 patients have been included in the database (938 micrometastases and 1413 macrometastases) with the collaboration of 20 European and Italian OSNA centers.

Univariate analysis was performed to determine factors associated with NSN metastases. Chi-square test was used for categorigal variables. All variables with p values less than 0.05 were included in the logistic analysis using backward stepwise method.

RESULTS

Univariate and multivariate analysis for micro and macro metastases are reported. In patients with SN micrometastases, multivariate analysis showed that grading (p<0.006), Tsize (p<0.04) and the number of CK19 mRNA copies (p<0.0001) are strictly related with positivity of NSN status. In patients with macrometastases multivariate analysis selected KI 67 (p<0.04), Tsize (p<0.002), number ofcopies (p<0.0001) and multifocality (p<0.007) as parameters significantly correlated with NSN positivity.

CONCLUSIONS

In our analysis we have evaluated only parameters available at the time of operation because the final nomogram has to be employed by the surgeons as a guide for the surgical decision-making whether to perform or not ALND in BC women. The large population employed and the power of number of copies are the right prerequisites for a reliable nomogram that will be presented at the Meeting.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-01-06.
Abstract P1-01-08: Diagnostic accuracy of pre-operative ultrasonographic evaluation of axillary lymph nodes in breast cancer patients: Is there a place for axillary ultrasonography in future nomograms?
Authors: Moorman AM , Bourez R , de Leeuw D , Kouwenhoven EA
Published: August 2014

Background

During the years there has been a significant interest in the development of clinical prediction tools to estimate the risk of having positive lymph nodes in the axilla, and thereby making it possible to plan specific therapies. A non-invasive and widely available method is axillary ultrasonography.

Aim

The purpose of this study was to evaluate the diagnostic accuracy of axillary ultrasonography.

Patients and methods

Between January 2007 and July 2011, pre-operative axillary ultrasonography was performed in 1124 consecutive primary breast cancer patients who underwent surgery with sentinel node biopsy (SNB) and/or complete axillary lymph node dissection (ALND). Suspected lymph nodes (cortex thickness > 2.3 mm) were further analysed by cytology or histology. In case of a negative axillary ultrasonography, standard SNB was performed during operation. In case of suspected lymph nodes with axillary ultrasonography, confirmed by biopsy, routine complete ALND was performed.

Results

Of the 1124 patients, 922 (82%) had unsuspected lymph nodes and 202 (18%) had suspicious lymph nodes during axillary ultrasonography. The sensitivity and specificity of axillary ultrasonography was 46.6% and 93.0% respectively in case of a macrometastases. In all cases of suspected malignancy, axillary core biopsy was performed. The sensitivity and specificity of axillary ultrasonography in combination with core biopsy was 40.5% and 97.2% respectively. With increasing number of axillary nodes the sensitivity increases: 53.9% in case of 2 macrometastases and 83.3% in case of 3 macrometastases.

Conclusion

Ultrasonography and core biopsy are effective in predicting the presence of axillary metastases in breast cancer patients. Especially the high specificity might be of additional value for future nomograms in the surgical treatment of breast cancer patients. In addition, the sensitivity is of greater value in case of gross nodal disease.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-01-08.
Abstract P1-01-13: Intraoperative evaluation of sentinel lymph nodes using touch imprint cytology in patients with early-stage breast cancer: A series of 4367 procedures
Authors: Chen Y , Chen J , Chen J , Yang B , Li L , Huang X , Shao Z , Shen Z , Wu J
Published: August 2014

Background: As the screening for breast cancer becomes common in China, an increasing number of early-stage breast cancer has been detected. For those patients, especially the ones with cliniaclly negetive lymph nodes, sentinel lymph node biopsy (SLNB) plays an important role in the assessment of the axillary status, while minimizing the mobidity of axillary lymph node dissection(ALND). Accurate intraoperative assessment of SLN metastases enables the selection of patients for ALND during the same operation. Touch imprint cytology (TIC) serves as a main intraoperative evaluation of SLNs in our institute since Feb-2005. The purpose of this study is to evaluate its clinical value.

Methods: Early-stage breast cancer patients treated in our institute between Feb-2005 and Apr-2013 enrolled in this study. To avoid breast tissue contamination, all patients had an intraoperative SLNB performed before undergoing lumpectomy or mastectomy. The fresh SLNs were sectioned into 1.5-3.0mm pieces along the short axis, TIC was performed on both surfaces of each piece, its result was correlated with the histological assessments with Hematoxylin-Eosin (H&E) staining, which considered as the gold standard.

Results: A total of 4,367 early-stage breast cancer patients enrolled in the study, and 14,112 SLNs were successfully harvested. Among those, 917 (21.0%) patients were confirmed to have at least one SLN positive for metastases, and 1343 (9.5%) positive SLNs were removed. Based on the final H&E report, the sensitivity, specificity and overall accuracy of TIC was 71.4%, 99.2% and 93.6%, respectively on a per patient basis, and 69.6%, 99.5% and 96.6%, respectively on a per node basis. The sensitivity for macrometastasis and micrometastasis was 74.8% and 26.5%, respectively on a per patient basis, and 78.9% and 25.2%, respectively on a per node basis. To discuss the influence of error results on the surgery procedure, we further reviewed the detail medical records of the patients enrolled in the recent 3 years. Of all the 3290 patiens treated between Jun-2010 and Apr-2013, 207(6.5%) patients needed a second opereation to have the ALN dissected. Meanwhile, 20(0.6%) patients had undergone extra ALND because of the false positive results of TIC.

Conclusion: The sensitivity of TIC seems a little lower then the ones reported previously, but the overall accuracy still remains acceptable. In addition, only 7.1% patients were influenced by the error results of TIC, along with the advantages, such as the shorter preparation time and the less cost of cytological specimens, we consider TIC a feasible method, and believe it can serve well in the intraoperative evaluation of SLNs for early-stage breast cancer patients.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-01-13.
Abstract P1-01-17: Sentinel node biopsy for breast cancer: Intraoperative injection of radiocolloid
Authors: Vu HN , O'Connor PW , Shoemaker RR , Wan W , Fratkin MJ , Bear HD
Published: August 2014

INTRODUCTION. Preoperative injection of radiocolloid for axillary sentinel node biopsy for breast cancer is visibly uncomfortable for many patients. We sought to determine whether injection of the radiocolloid once the patient has been anesthetized would be reliable for sentinel lymph node identification when compared to preoperative injection.

METHODS. A cohort of patients with breast cancer who underwent intraoperative injection of radiocolloid was compared to those who received their injection preoperatively. All injections employed 1mCi 0.5 ml of filtered (0.22 micron) Tc-99m sulfur colloid. All operative surgeons directed injections into the retroareolar tissue; some also intradermally directly over the tumor.

RESULTS. 904 sentinel node biopsy procedures were performed in the six years study period. 759 sentinel node biopsies were analyzed, 664 with preoperative injection of radiocolloid for localization and ninety-five with intraoperative injection. The average age of the patients was 56.8 years: 57.3 years in the preoperative injection group and 53.8 years in the intraoperative injection group. 753 of the 759 patients were women.

Sentinel Node Identification. The overall sentinel lymph node identification rate was similar for the two groups: 94.1% for the preoperative injection group and 93.5% for the intraoperative injection group (P = NS). The identification rate remained equivalent for both groups when analyzed by T stage of the tumor. Similarly, the average number of lymph nodes removed did not differ between preoperative vs. intraoperative injection: 2.66 nodes and 2.55 nodes, respectively.

Sentinel Node Positivity. The overall rate of positive sentinel nodes was also similar for the two groups, 24.7% for the preoperative injection group and 26.4% for the intraoperative injection group (P = NS). When analyzed by T stage, the positivity rate remained similar and not statistically significant. T1 tumors were 21.6% positive in the preoperative group versus 23.9% in the intraoperative group, and T2 tumors were 40.1% vs. 37.0%, respectively.

DISCUSSIONS. Our sentinel lymph node identification rate of 93.5% for the intraoperative injection group appears similar to that of the published literature (96-100%). Our study also showed no statistical difference in positive sentinel node biopsy between preoperative injection (24.7%) and intraoperative injection (26.2%) and is comparable to that of others When analyzed by T stage, our study had 23.9% positive sentinel node biopsies for T1, and 37% for T2 with intraoperative injection.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-01-17.
Abstract P1-01-19: Role of axillary clearance with tumor positive sentinel node in mastectomy group: Is the results of ACOSOG Z0011 trial adaptable to mastectomy patient?
Authors: You JY , Song EJ , Lee MH , Jung SY , Lee SY , Kang HS , Lee ES
Published: August 2014

Background: Recent data from ACOSOG Z0011 trial or AMAROS trial suggest that axillary lymph node dissection(ALND) may be unnecessary for patients with positive sentinel lymph node biopsy(SLNB) receiving breast conserving surgery(BCS) with irradiation. However, consensus statements and guidelines until recently recommended that patients with mastectomy and tumor positive sentinel node undergo completion ALND. In this preliminary study, we compared these patients who did not undergo ALND with the patients who received BCS with SLNB only and irradiation and we analyzed the locoregional recurrence rate to show no differences of outcomes between two groups.

Method: We identified 6,163 women with invasive breast cancer who underwent surgical resection at the National Cancer Center (Goyang,Gyeonggi-do,Korea) between January 2000 to December 2011. Clinico-pathological data obtained from prospective collecting medical database of our institution were analyzed retrospectively. The mastectomy with positive SLNB group was 47 patients and BCS with positive SLNB and irradiation group was 172 patients. The primary end point was loco-regional recurrence rate.

Result : Clinical and tumor characteristics were similar between two groups except T stage and receptor status. The mean tumor size was 5.5cm with mastectomy group and 3.5cm with BCS group. The median number of nodes removed was three. There was not a single case of locoregional recurrence in both groups. At a median follow-up of 53.5 months (last follow-up, May 2013), 5-year overall survival was 85.7% with mastectomy and 97.3% with BCS group.

Conclusion : In our study, there was no case of locoregional recurrence as above. This results lend weight to the argument that SLNB without ALND may be reasonable management for selected patients with appropriate surgery and adjuvant systemic therapy. This study can be regarded as a preliminary study with a sufficient value despite of the prognosis showed some statistical differences between two groups. It resides in the difference of initial stage of patients of two groups. We will present additional data compared with the mastectomy with axillary clearance group at the meeting.

Acknowledgement This work was supported by grant from the National Cancer Center Korea (1210331-2).

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-01-19.
Abstract P1-02-05: Identification of HER-2/neu amplification using amplicon based benchtop next generation sequencing
Authors: Portier BP , Patel K , Medeiros LJ , Singh R , Aldape KJ , Hamilton S , Luthra R , Routbort M
Published: August 2014

Introduction

Aplicon based next generation sequencing (NGS) assays have the potential to incorporate simultaneous assessment of somatic mutations and copy number variation into a single platform. Although not specifically designed for this purpose, we noted numerous examples of apparent HER-2/neu gene amplification in analyzing results from our mutation profiling 46-gene cancer NGS panel (Life Technologies, South San Francisco, CA).

Material and Methods

Existing amplicon coverage data from the TorrentSuite 2.0 (Life Technologies) pipeline was accessed and used to generate a HER-2/neu coverage proportion (ratio between all HER-2/neu amplicon reads and all other amplicon reads). This ratio was calculated for 170 unique cases of breast carcinoma. A total of 4 cases were eliminated from analysis due to indeterminate IHC/FISH results. Using a total of 166 cases, we performed ROC curve analysis to determine the sensitivity and specificity of NGS based HER-2/neu amplification compared to that of traditional IHC and FISH testing (MedCalc v8.0).

Results

The NGS based HER-2/neu coverage proportions in the 166 cases showed a clearly non-normal distribution with an outlier cluster of cases with an elevated NGS HER-2/neu ratio (6 cases with ratios >0.045). The distribution of NGS based HER-2/neu ratios for the entire population ranged from 0.01 to a maximum of 0.34. ROC curve analysis of NGS HER-2/neu proportions compared to IHC/FISH data showed a maximal sensitivity of 75.0% and specificity of 100% at an NGS ratio cut off of ≥0.045.

In the 166 cases, 6 showed a HER-2/neu coverage proportion of > 0.045 (range 0.045-0.34). Utilizing the 0.045 NGS ratio cut off value, the population frequency of cases positive for HER2 expression was 3.4%. Of the 160 samples with NGS coverage proportion < 0.045, only two cases were positive by IHC/FISH testing.

The correlation between NGS HER-2/neu coverage proportion and IHC/FISH testing was highly significant (Spearman rank correlation r = 0.67, p < 0.0001 CI: 0.58-0.75).

Conclusions

Even utilizing a limited somatic cancer panel that includes less than 200 amplicons in 46 genes, clinically significant HER-2/neu amplification can be readily identified. This is the first study to describe the functional utility of HER-2/neu amplification detection using a NGS based amplicon assay. This study shows that in addition to mutation analysis, amplification data can be simultaneously obtained, which has striking implications for potential clinical utility and HER-2/neu amplification detection outside of traditional IHC and HER2 testing modalities. Furthermore, utilizing this assay as a primary screening modality could limit the additional expense of multiple single gene testing.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-05.
Abstract P1-02-07: Discordance for low and intermediate levels (1+ or 2+ by IHC) of HER2 protein expression in the phase III PRESENT trial
Authors: Rugo HS , Melisko M , Ben-Baruch NE , Price Hiller J , Dahkil S , Sutton L , Mazanet R , Mittendorf E
Published: August 2014

Background

Significant discordance has been described between different laboratories and between testing methodologies for HER2 overexpression and gene amplification. Concern has focused on missing patients who might benefit from treatment with HER2 targeted therapy. Concordance rates between local and central testing for tumors selected for HER2 0-2+ is not well described. We describe discordance rates between local and central testing performed to identify tumors with 1 and 2+ HER2 expression.

Methods

The PRESENT study is a multicenter, multinational, prospective, randomized, double-blind, controlled Phase 3 study assessing efficacy and safety of the peptide vaccine NeuVax, in HLA-A2 and/or A3 positive patients with early stage, node positive breast cancer expressing low and intermediate levels (1/2+ by IHC) of HER2 protein. PRESENT 2-step screening includes HLA testing followed by central lab confirmation of HER2 1 or 2+ expression using the DAKO HercepTest.

Results

215 patients underwent central IHC testing for HER2. Discordance between local and central testing was seen in 54% (117/215); 34% (65/192) of those eligible by local results were ineligible on central testing, and 48% (11/23) of those not eligible by local results were eligible on central testing.

Local testingCentral testing (IHC (%))IHC (n)01+2+3+0 (15)5 (33%)7 (47%3 (20%)—1+ (127)39 (31%)68 (53%)15 (12%)5 (4%)2+ (65)11 (17%)23 (35%)21 (32%)10 (16%)3+ (8)3 (38%)—1 (12%)4 (50%)

Of those with 1 or 2+ local results, 8% (15/192) were found to be 3+ on central testing. Discordant rates varied by geographic location with higher rates seen in Eastern Europe and Russia.

Conclusion

Significant discordance exists between local and central laboratory test results for HER2 expression by IHC even at lower levels of expression, and current tests are defined by their ability to determine 3+ positivity. In order to improve accuracy of testing and to develop a companion diagnostic for NeuVax, the Leica Bond Oracle HER2 IHC System will be incorporated into future HER2 screening for the PRESENT trial.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-07.
Abstract P1-02-08: Papillary lesions of the breast on core biopsies: Should routine surgical excision be performed?
Authors: Ho CS , Yap WM , Chong BK
Published: August 2014

Background: The optimal management of papillary lesions, particularly benign papillomas, detected on core needle biopsies remains debatable. The purpose of this study was to determine the risk of upgrade to malignancy of core biopsy diagnosed papillary lesions on the subsequent surgical excision.

Methods: Following institutional ethics review board approval, the pathology database was searched for all papillary lesions diagnosed on core needle biopsies from Jun 2005 to June 2010. The papillary lesions were histologically categorized as benign, atypical, suspicious for malignancy and definite for malignancy. The core biopsy findings were compared with the follow-up excision biopsy material to identify any upgrade to malignancy (i.e. ductal carcinoma in situ or invasive carcinoma). The presence of any associated significant risk lesions such as atypical ductal hyperplasia (ADH) and lobular neoplasia (LN) were also recorded.

Results: A total of 113 papillary lesions were diagnosed on core needle biopsies of 98 patients, with a mean age of 57 years. A median of 5 cores of breast tissue were obtained per papillary lesion. Of these papillary lesions, 73 were benign, 25 atypical, 8 suspicious for malignancy and 7 definite for malignancy. The overall excision rate was 83%. Of the 58 benign papillary lesions excised, only one (1.7%) showed malignancy (3mm ductal carcinoma in situ) - of interest, the papillary lesion per se was confirmed benign on excision, while an adjacent focus of flat epithelial atypia was present in the initial core biopsy. Nine papillomas were associated with adjacent ADH and/or LN on excision and the rest of the excisions (n = 48) showed no residual papillary lesion or benign papillomas. Among the 24 excised atypical papillary lesions, the positive predictive value (PPV) for malignancy was 42% (10 in situ papillary carcinomas, 4 of these associated with invasive carcinoma). The remaining excisions disclosed ADH and/or LN (n = 3) or benign breast tissue/papillomas (n = 11). None of the 4 papillary lesions with the apocrine atypia were malignant on excision. Of the 5 excised papillary lesions which were suspicious for malignancy on the cores, all showed malignancy (5 in situ carcinomas; 2 associated with invasive carcinoma) on open surgical biopsy. The PPV for malignancy of the 7 core biopsy diagnosed malignant papillary lesions was 100%.

Conclusions: Papillary lesions diagnosed as atypical or suspicious for malignancy on core needle biopsies should be completely excised to exclude malignancy. Our findings suggest, however, that routine surgical excision may not be necessary for core biopsy detected benign papillomas which are not associated with atypia.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-08.
Abstract P1-02-10: Variation in HER2 positive rates in California by geographic region: Implications for setting pathology laboratory benchmarks
Authors: Allison KH , Jensen KC , West RB , Clarke CA , Gomez SL , Kurian AW
Published: August 2014

Background: It is unclear how much regional variation there is for HER2 positive rates in breast cancer. By looking at regional variation, we can begin to examine the impact of both demographic variation and pathology laboratory practice on HER2 positive rates so that valid laboratory benchmarks can be established.

Methods: Utilizing data from the California Cancer Registry (CCR), we examined the HER2 positive rates sorted by 58 California counties. Counties were grouped into 10 geographic regions for analysis. Stage at presentation and total numbers of breast cancer cases were studied at as variables. Results: 97,248 breast cancer cases were included in the CCR data. Overall, the HER2 positive rate was 16.5%. HER2 positive rates increased with increasing stage at presentation both overall and by region. Overall they were 12.9% for stage 1, 17.4% for stage 2, 23.2% for stage 3, 25.9% for stage 4 and 25.2% for unstaged cases. There was significant regional variation in overall HER2 positive rates ranging from 13.2-18.9%. 4 regions had overall rates within 0.5% of each other, 3 regions had rates > 0.5% below average and 3 regions had rates >0.5% above average. In the regions with overall rates below the average, one region (region 4, N = 4,636) had lower rates across all stages, and two regions (region 7, N = 8,623 and region 8, N = 14,152) had lower rates only for stages 1-2. Of regions with overall rates higher than the average, one region had increased rates across all stages (region 9, N = 24,279) and two regions had increased rates only in stages 1-3 (region 5, N = 9,176 and region 6, N = 4,888).

Conclusions: In California, there is significant variation in HER2 positive rates by geographic region, even when controlling for stage. The possible influence of differences in regional population characteristics versus differences in laboratory practices to explain this variation should be further examined so that valid regional laboratory benchmarks can be established.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-10.
Abstract P1-04-03: Genome-wide analysis of copy number variations and mutation profiles of single circulating tumour cells using massively parallel paired-end sequencing
Authors: Peeters DJE , Kumar P , Van der Aa N , Rothé F , Theunis K , Op de Beeck K , Van Laere SJ , Vermeulen PB , van Dam PA , Vincent D , Desmedt C , Sotiriou C , Dirix LY , Ignatiadis M , Voet T
Published: August 2014

INTRODUCTION

Recent advances in single cell isolation techniques and next generation sequencing (NGS) have paved the way for the genome-wide molecular analysis of individual circulating tumour cells (CTCs) in patients with metastatic carcinomas. Here we present the results of a pilot study evaluating the feasibility and reliability of NGS of single CTC from whole blood samples.

MATERIALS & METHODS

Single cells of the human breast cancer cell line HCC38 were harvested from spiked blood samples in a semi-automated workflow consisting of immunomagnetic enrichment using the CellSearch system and dielectrophoretic cell sorting using the DEPArray system. DNA was isolated and amplified using the Ampli1 whole genome amplification (WGA) kit and subjected to low-coverage genome-wide paired-end sequencing for copy number variation (CNV) analysis and targeted re-sequencing of 200 cancer-related genes for somatic mutation analysis.

RESULTS

Single-cell WGA products of four HCC38 cells were subjected to whole genome sequencing for CNV analysis. Average coverage depth was 0,68x. At a binning window of 50 kb, detection results of CNVs in single-cell samples were highly consistent (>81% copy number concordance per bin genome wide) with CNV profiles from non-amplified multi-cell samples of the same cell line. We could demonstrate that part of the discordance was due to the acquisition of novel DNA-rearrangements in the single cells. Three of the single-cell WGA products were additionally subjected to targeted re-sequencing for mutation analysis of 200 selected genes, of which the analysis is currently ongoing.

DISCUSSION

Our study demonstrates the feasibility of a comprehensive genome-wide CNV analysis and targeted mutation analysis using NGS of single tumour cells isolated from whole blood samples in a highly automated isolation workflow. This approach provides a robust framework for the study of intercellular heterogeneity within the CTC population in blood samples of patients with (metastatic) breast cancer. In addition, our results document the extent of WGA-induced bias of a recently commercialized PCR-based WGA kit.

These authors contributed equally to the data presented in this abstract.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-03.
Abstract P1-04-09: Extended characterization and target analysis of circulating epithelial cells in early and late breast cancer
Authors: Habets L , Bettina F , El-Ghali I , Körber W , Danaei M , Kusche M , Pachmann K
Published: August 2014

The MAINTRAC based (RBC lysis immunofluoretic detection and analysis on the Olympus ScanR system) finds more cells compared to enrichment based systems. The relative cheapness and low amounts of blood (1ml) allows frequent monitoring during early and late disease therapy steps. The clinical relevance of rising or falling counts of living (EPCAM+CD45negDAPIneg) CEC has been demonstrated in early and late disease by Pachmann. In addition we could show that these cells express stem cell, EMT and hypoxia markers. To our surprise we found high counts of this cell type in 90% of patients with fatty liver disease (NAFLD) in a non healthy non malignant control population. In a healthy control population in only 2% very few cells were found. Our conclusion so far CEC are a phenomenon driven by a hypoxic hyperacidic tissue situation shared by malignant and non malignant disease. The detectable cell population showed considerable morphologic heterogeneity urging us to introduce extended characterization and more comprehensive analysis. We therefore slightly adapted the basic MAINTRAC procedure to allow four color flowcytometric detection and analysis on the Amnis (EMD-Millipore) FlowSight device. The basic panel consists of (CD45PE, DAPI- blue, ALDH-FITC and EPCAMAF660). Extended Characterization is possible with different other panels including (HIF1, betacatenin, several pan- and single cytokeratins and CD44/CD24) and targets like ER, HER2, PDL-1 (immune checkpoint target) and phosphatidylserine. With this new approach we examined so far 66 patients with early and 53 patients with late disease. In early disease we found living CEC in 32 of 66 cases, with cell counts from 200 to 21642 per ml. In late disease in 27of 53 cases, cell counts varying from 481 to 97339 cells per ml. The percentage of living CEC varied from 0 to 60%. In all cases we found PDL-1 and ALDH1 expressed nearly exclusively on EPCAM+ cells demonstrating their suitability as a CEC specific marker. In cases with early relapse and rapid progression only few or none CEC are found especially in triple negative and HER2+ER negative cases. In these situations we find however EPCAM negative cell clusters. The IDEAS software allows comprehensive analysis of all visible phenomena including single cells, cell aggregates and exosomes. So far we still see no differences between “real” tumor derived CEC and “false positive” liver derived CEC. There is however preliminary evidence that CK20+ cells counts are much higher than EPCAM +counts in real compared with liver caused disease. A conclusive picture of these intruiging phenomena is not possible it seems however that the chosen way of extended characterization including new issues like cell clusters and exosomes should lead to a further elucidation of this biology and the use of CEC as a reliable “fluid biopsy”.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-09.
Abstract P1-04-12: Prognostic value of CTC detection by RT-PCR and the CellSearch system before first-line chemotherapy in metastatic breast cancer
Authors: Agelaki S , Mavroudis D , Politaki E , Apostolaki S , Kallergi G , Spiliotaki M , Georgoulias V
Published: August 2014

Background: CTCs have significant prognostic role in patients with breast cancer. Several techniques developed for CTC detection might help in monitoring treatment efficacy. The CellSearch System is the only clinically validated for use in patients with metastatic breast cancer (MBC). We have previously shown a concordance between RT-PCR for cytokeratin 19 (CK-19) and CellSearch System in the detection of CTCs in MBC. In this study we aimed to evaluate the prognostic value of CTC detection using these assays in the same cohort of patients. Methods: Blood was obtained from patients with MBC before the initiation of first-line chemotherapy. Different aliquots of the same blood sample were evaluated for the presence of CTCs by RT-PCR for CK-19 mRNA and by the CellSearch System. Disease progression and response to treatment were determined using standard clinical and imaging criteria. Results: In 142 patients with available clinical data, CTC evaluation was performed using both methods. Median age was 60.7 years (range, 23-82), 74.6% were post-menopausal and 16.9% were HER2-positive. CTCs were detected in 52.1% and 36.6% of patients by the CellSearch System (cut-offs ≥2 and ≥5 CTCs/7.5 ml, respectively) and in 38.7% by RT-PCR; 27.5% (cut-off ≥2) and 20.4% (cut-off ≥5) of patients were CTC-positive by both methods. Response rate was 40% in patients with CTCs detected by CellSearch (cut-off ≥2) vs 62% in CTC-negative patients; there was no difference in objective responses using the ≥5 cut-off, or using RT-PCR for CTC detection. Median PFS was 9.0 (cut-off ≥2) and 7.5 months (cut-off ≥5) for CTC-positive versus 20.0 (cut-off ≥2) and 18.7 months (cut-off ≥5) for CTC-negative patients using the CellSearch System (p = 0.0001). No significant difference in PFS was evident according to CTC detection by RT-PCR (10.0 vs 13.1 months, p = 0.253). Median survival was 24.7 vs 57.3 (cut-off ≥2) and 18.5 vs 53.7 (cut-off ≥5) months for CTC-positive vs CTC-negative patients using the CellSearch System (p = 0.0001); by RT-PCR, median survival was 30.3 and 50.1 months for CTC-positive and CTC-negative patients, respectively (p = 0.021). Conclusions: The detection of CTCs before first-line chemotherapy using either the CellSearch System or RT-PCR for CK-19 mRNA has significant prognostic value in patients with MBC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-12.
Abstract P1-07-01: Histone deacetylase inhibitor entinostat reverses epithelial to mesenchymal transition of breast cancer cells by reversing the repression of E-cadherin
Authors: Sabnis GJ , Shah P , Kazi A , Gau Y , Chumsri S , Brodie A
Published: August 2014

Loss of ERa in breast cancer correlates with poor prognosis, increased recurrence rates and higher incidence of metastasis. In our previous studies, we have shown that histone deacetylase (HDAC) inhibitor entinostat (ENT) can upregulate ERα and aromatase in ER-negative cells and tumors, making them sensitive to aromatase inhibitors (AIs). In the current study, we are showing that ENT can also reverse epithelial to mesenchymal transition (EMT), which is considered to be a first step in the process of metastases formation. EMT is characterized by loss of intracellular adhesion (loss of E-cadherin); loss of epithelial markers such as cytokeratins and upregulation of mesenchymal markers such as vimentin; acquisition of fibroblast-like spindle morphology and increased motility. Various carcinomas undergo varying degrees of EMT and capacity to undergo EMT correlates inversely with levels of E-Cadherin. It is widely accepted that loss of E-cadherin is associated with more invasive phenotype. Epigenetic silencing of E-cadherin has been implicated in metastatic cell lines and invasive breast cancers.

Triple negative breast cancer cells such as MDA-MB-231 and Hs578T show a basal phenotype characterized by loss of E-cadherin expression and higher expression of mesenchymal markers such as N-cadherin, vimentin along with transcriptional repressors such as twist and snail.

In this study, we measured the effect of entinostat on the EMT. When MDA-MB-231 and Hs578T cells were treated with ENT, E-cadherin transcription was increased along with reduction in N-cadherin mRNA expression. Similar results were also seen in tumors of MDA-MB-231 and Hs578T xenografts treated with ENT (for 5 weeks and 2 weeks respectively). A dose dependent increase in E-cadherin was seen along with a dose dependent decrease in N-cadherin mRNA. Although, we did not observe any reduction in vimentin protein, phosphorylation of vimentin was increased and vimentin remodeling was changed as seen by immunofluorescence. We performed chromatin immunoprecipitation (ChIP) assay to measure the activation of E-cadherin promoter. Treatment of MDA-MB-231 and Hs578T cells increased the activation of E-cadherin promoter as seen by increased acetyl histones at the promoter region of E-cadherin. Twist and snail are known repressors of E-cadherin gene and we saw that ENT treatment reduced the association of twist and snail with the E-cadherin promoter. ENT was also able to downregulate twist, which may be responsible for reduced twist association with the E-cadherin promoter.

In summary, these findings suggest that HDAC inhibitor ENT can reverse EMT and may help reduce the formation of metastasis.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-01.
Abstract P1-07-04: A novel bioactive approach to inhibit leptin-induced epithelial-mesenchymal transition in breast cancer
Authors: Sharma D , Avtanski D , Nagalingam A , Kuppusamy P , Saxena N
Published: August 2014

Introduction: Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Recent studies from our lab reveal that leptin induces epithelial-mesenchymal transition (EMT) and tumorsphere formation via concomitant activation of Akt/GSK3b and MTA1/Wnt1 signaling axes leading to β catenin activation. Given the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of leptin are much desired. We showed previously that honokiol, a bioactive polyphenol from Magnolia grandiflora, inhibits breast carcinogenesis. The present study provides first evidence for the efficacy of honokiol against oncogenic effects of leptin including EMT.

Methods: Efficacy of honokiol to inhibit oncogenic effects of leptin was evaluated by using clonogenicity, anchorage–independent growth, matrigel-invasion and spheroid-migration assays. RT-PCR, western blot and immunofluorescence analyses were used to examine the molecular changes associated with EMT as well as underlying molecular pathways. Functional importance of MTA1/Wnt1/ β catenin axis was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immuniohistochemical, RT-PCR and western blot analysis of tumors was used.

Results: Honokiol treatment circumvents leptin-induced EMT-associated phenotypic changes. A biochemical hallmark of EMT is loss of expression of epithelial markers with a concurrent increase in mesenchymal marker expression. Honokiol elicits increased expression of E-cadherin, occludin and keratin-18 (epithelial markers) and decreased expression of vimentin, fibronectin and N-cadherin (mesenchymal markers) in leptin-treated cells providing molecular evidence for reversal of EMT. Honokiol also inhibits expression and nuclear translocation of transcriptional effectors of EMT; Snail, Slug, Zeb1 and Zeb2. Analysis of underlying molecular mechanisms reveals that Honokiol effectively inhibits leptin-induced MTA1/Wnt1/ β catenin axis. Furthermore, using nontoxic doses of honokiol, we show that honokiol treatment effectively inhibits leptin-induced breast tumorigenesis in vivo. Analysis of breast tumors show that honokiol treatment reverses leptin-associated signaling (MTA1/Wnt1/ β catenin axis) along with decreased expression of mesenchymal markers and increased expression of epithelial markers.

Conclusions: In this study, we demonstrate for the first time that HNK is able to abolish leptin-induced EMT and provide in vitro and in vivo evidence for the integral role of a previously unrecognized crosstalk between honokiol and MTA1/Wnt1/ β catenin axis. Thus, HNK may be used as a non-toxic and non-endocrine rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-04.
Abstract P1-07-05: The cell fate factor DACH1 represses YB-1-mediated oncogenic transcription and translation
Authors: Pestell RG , Wu K , Chen K , Wang C , Jiao X , Wang J , Cai S , Addya S , Sorensen PH , Lisanti MP , Quong A , Ertel A
Published: August 2014

The epithelial-mesenchymal transition (EMT) enhances cellular invasiveness and confers tumor cells with cancer stem cell like characteristics, through transcriptional and translational mechanisms. The mechanisms maintaining transcriptional and translational repression of EMT and cellular invasion are poorly understood. The Drosophila homologue of DACH1, the Dac gene is a key member of the retinal determination gene network that specifies organismal development. The dachshund (dac), eya1, eyes-absent (eya), twin of eyeless (toy), teashirt (tsh) and sinoculues (so) are expressed in progenitor cells, contributing to development of the eye and genitalia. Loss of DACH1 expression contributes to the expansion of neural progenitors, muscle satellite cell differentiation and breast cancer stem cells. In recent studies Dachshund repressed breast cancer stem cell expansion. DACH1 expression is reduced in a variety of human cancers including prostate, ovarian and human breast cancer.

Herein, the cell fate-determination factor Dachshund (DACH1), suppressed EMT via repression of cytoplasmic translational induction of Snail by inactivating the Y box-binding protein (YB-1). In the nucleus, DACH1 antagonized YB-1-mediated oncogenic transcriptional modules governing cell invasion. DACH1 blocked YB-1-induced mammary tumor growth and EMT in mice. In basal-like breast cancer (BLBC) the reduced expression of DACH1 and increased YB-1, correlated with poor metastasis free survival. The loss of DACH1 suppression of both cytoplasmic translational and nuclear transcriptional events governing EMT and tumor invasion may contribute to poor prognosis in BLBC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-05.
Abstract P1-07-06: E2F-1 directly induces HOXB9 accelerating breast cancer progression
Authors: Zhussupova A , Hayashida T , Takahashi M , Hiromitsu J , Kitagawa Y
Published: August 2014

Homeobox B9 (HOXB9) is a member of the class I HOX genes with highly conserved 61 amino acid homedomain motif, known to be overexpressed in breast cancer and related to tumor angiogenesis, EMT (Hayashida et al., PNAS 2010), and radio-resistance (Chiba et al., PNAS 2011), being significant prognostic factor in breast cancer patients (Seki et al., Ann Surg Oncol 2012). In this study, we investigated how HOXB9 expression is regulated in breast cancer cells.

First, we analyzed the upstream promoter region of HOXB9 gene to determine the important sequence for HOXB9 transcription. The multiple lengths of HOXB9 promoter region from -2898 to +1 were cloned into pGL3 luciferase reporter vector. Luciferase activities in MDA-MB231 cells which overexpress HOXB9 were analyzed and determined that the region from -404 to -392 played the critical role for the transcription of HOXB9 gene. Computer prediction system for the binding ability to critical sequence raised several candidate genes including E2F-1, PAX-5, P53, Sp-I, ETS-1 and NFY-A which are known to be related with cancer progression. It was confirmed that all these genes could induce HOXB9 overexpression, and especially E2F-1 was the strong inducer. Then, E2F-1-induced cell lines from MCF7, MCF10A and MDA-MB231were established and the correlation between E2F-1 and HOXB9 mRNA expression was determined. ChIP assay revealed that E2F-1 could directly bind to the critical sequence of HOXB9 promoter. Electrophoretic Mobility Shift Assay (EMSA) also indicated the binding ability of E2F-1. Finally, the tissue samples of 141 breast cancer patients, who undertook breast cancer surgery as a primary treatment, were stained with E2F-1 and HOXB9 antibodies, and correlation between HOXB9 and E2F-1 expression was confirmed with statistical significance (p<0.001).

There are many publications describe the E2F-1 relation to the breast cancer patients prognosis. The context of this research displays the direct dependence of HOXB9 gene's expression through E2F-1, which might be suppressed by specific treatment, for example CDK4/6 inhibitor, and improve breast cancer patients’ survival.

To confirm these genes might demonstrate novel factors and/or signaling pathways playing role in breast cancer progression through HOXB9 axis.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-06.
Abstract P1-08-05: The prognostic significance of Ki-67 expression before and after neoadjuvant anthracycline-taxane-based chemotherapy in different biological breast cancer phenotypes
Authors: Zhang J , Zhang X , Yu F , Liu J , Zhang S , Hao X , Hou G
Published: August 2014

Purpose

This study was conducted to analyze the Ki-67 expression before and after neoadjuvant chemotherapy and clinical pathology characteristics of different biological breast cancer phenotypes at our center. A correlation study was performed between Ki-67 index change and the prognosis of different biological breast cancer phenotypes and prognosis in Northern China.

Methods

A regression analysis was performed on 213 patients with invasive breast carcinoma accepted NAC admitted to Tianjin Medical University Cancer Institute and Hospital of Breast Surgery from January 2007 to April 2008. These patients were subtyped by hormone receptor status and HER2 status.The Ki67 index (percentage of Ki67-positive cancer nuclei) were determined immunohistochemically. The prognostic value of the Ki-67 index for different biological breast cancer phenotypes disease-free survival (DFS) and overall survival (OS) was investigated by use of Kaplan–Meier curves and multivariable Cox regression.

Results

The overall pathologic CR (pCR) rate, defined as no invasive residuals in breast and axilla, was 17.8%. The highest pCR rate of 32.1% was observed in patients with HR-/HER2+ tumors, which is 11.4%, 20.0% and 26.2% in HR+/HER2- HR+/HER2-,HR+/HER2+and HR-/HER2- tumor respectively(P = 0.024).The Ki-67 expression of pre-NAC and post-NAC have prognostic significance in HER2- breast cancer, which have not significance different in HER2+ breast cancer:The best Ki-67 idex cut point of pre-NAC and post-NAC to predict long-term survival was respectively 20% and 10% in HR+/HER2- breast cancer. Moreover, patients with a higher Ki-67 index (> cut-off point) showed significantly lower 5year-DFS/OS rates compared with those whose Ki-67 index were below cut-off point. In addition, Ki-67 index of post-NAC were independent prognostic factors for 5 year disease-free survival of patients with HR+/HER2- breast cancer. Ki-67 change between pre- and post-NAC as an independent prognostic factor independently predict prognosis in the patients who have not achieved pCR, while the best cut-off point was 18%.

Conclusion

The Ki-67 index of pre- and post-NAC could predict the prognosis in the patients with HER2- breast cancer. Moreover, the Ki-67 change between pre- and post-NAC was an independent prognostic factor in the patients who have not achieved pCR.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-05.