bluuebunny/crossref_metadata_2025 huggingface dataset:
P4-19-02: A Population-Based Study of Contemporary Systemic Therapy for Male Breast Cancer.
Authors: Bradley KL , Villa D , Speers CH , Woods R , Tyldesley S
Published: June 2012

Purpose: To describe the contemporary utilization, effectiveness and tolerability of tamoxifen, aromatase inhibitors, and trastuzumab in the treatment of early and advanced male breast cancer at a population level in British Columbia.

Patients and Methods: Records of 158 consecutive referred cases of invasive male breast cancer diagnosed from January 2000 to March 2010 were analyzed in a population-based cancer program with respect to patient and tumour characteristics, usage of endocrine agents, toxicity and medication adherence, trastuzumab usage and outcome. This group was compared to a population-based random sample of 1000 contemporary female breast cancer patients for prognostic and demographic factors. Survival outcomes were also compared to a cohort of women, matched 2:1 on prognostic and treatment factors.

Results: Pathology was most commonly infiltrating ductal carcinoma (94.3%) and estrogen-receptor positive (98.7%). Males were older (median 69.5 years) compared to women (median 60 years). Stage distribution was I (25.3%), II (43.0%), III (24.7%) and IV (7.0%), reflecting more advanced presentation compared to female cases I (43.9%), II (37.9%), III (12.5%) and IV (5.3%). Tamoxifen was prescribed as adjuvant therapy to109 patients, and as palliative therapy to 11. Fourteen (11.7%) patients discontinued tamoxifen due to toxicity. Aromatase inhibitors were prescribed as adjuvant therapy for 16 patients and as palliative therapy for 24; these were discontinued due to toxicity in 5 patients (12.5%). Five of 9 patients known to over-express Her2 were treated with adjuvant chemotherapy with trastuzumab. Overall survival, breast cancer-specific survival and progression-free survival at 5 years were 72.4%, 85.5% and 79.7%, respectively. There was no statistical difference in outcomes compared to the matched female cases.

Conclusions: In this large population-based study of contemporary systemic therapy for male breast cancer, outcomes appear to be similar to those of women with the same diagnosis. Side effect profiles, tolerance, adherence and outcomes after tamoxifen, aromatase inhibitors and trastuzumab appear to be similar in men compared to that described in the literature for women.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-19-02.
P5-01-02: Wnt5a Is a Prognostic Biomarker in Estrogen Receptor-Positive Premenopausal Breast Cancer.
Authors: Sand-Dejmek JT , Ehrnstrom R , Andersson T , Ryden L
Published: June 2012

Background

Wnt proteins belong to a family of secreted growth factors, which are involved in a wide range of cellular processes. Wnt signaling can be broadly divided into two categories; the canonical, β-catenin-dependent pathway and the non-canonical β-catenin-independent pathway. Wnt5a is a non-canonical signaling member of the Wnt family. Studies in two smaller cohorts of breast cancer revealed that loss of Wnt5a was associated with early relapse, metastatic disease and poor survival. Moreover, reconstitution of Wnt5a signalling decreased the migratory capacity and invasiveness of cultured breast cancer cells and administration of a Wnt5a-mimicking peptide was proven to inhibit metastases in a mouse model.

Material and methods: Premenopausal women (n=564) were included in a controlled randomised trial and allocated to tamoxifen for two years versus no adjuvant treatment. A tumour tissue microarrays was constructed and 390/500 samples were evaluable for Wnt5a protein expression by immunohistochemistry. Only cases with at least 2 evaluable cores were included. Wnt5a levels were determined semiquantitatively (scores 0–3). Clinical and pathological data such as patient age, histological subtype, pT stage, tumor size, pN stage, ER, PR, Ki67 and HER2 status were available. The end-point was 10 year recurrence-free survival.

Results: Wnt5a protein expression was lost in 158/390 of the analyzed tumors and more common in younger (<45 yrs) women (p=0.032). Furthermore, loss of Wnt5a was significantly associated with ER and PR negativity (p=0.019 and p= 0.033, respectively). Loss of Wnt5a was significantly correlated to poor survival in the whole cohort, p=0.02. When analyzing the data according to breast cancer subtype, we found that Wnt5a was a strong independent prognostic factor in ER+ breast cancer p=0.009, but not in ER- breast cancer p=0.8. This was true both in the adjuvantely treated and untreated cohorts, verifying that Wnt5a indeed is a true prognostic factor and not a predictor of response to therapy.

Discussion: While associated with a better short-time prognosis than ER negative tumors, a subset of ER positive breast cancer patients are known to experience tumor recurrencies >5 years after primary treatment and hence have a dismal prognosis. Those patients need to be identified and their tumors targeted with more aggressive treatment regimens. We have identified and validated Wnt5a as a prognostic biomarker in ER positive breast cancer in premenopausal women. Wnt5a signalling could potentially be reconstituted by administration of a Wnt5a-mimicking peptide, shown to inhibit metastases in mice, thereby offering ER positive premenopausal patients a targeted adjuvant therapy that potentially could lower their risk of metastatic disease.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-02.
P5-01-12: Identification of an ATM Activation Subtype in PTEN Mutant Breast Tumours.
Authors: McCabe N , Walker SM , Goffard N , Wikstrom K , Greenan C , Delaney T , McCarthy M , McDyer F , Hill L , Deharo S , Proutski V , Keating K , Mullan P , Harkin DP , Kennedy RD
Published: June 2012

Background PTEN is frequently lost in cancer cells through genetic mutation or epigenetic silencing. Loss of PTEN function has been widely reported to cause up-regulation of the PI3K/AKT signalling pathway resulting in increased cell growth, proliferation and survival. More recently it has been reported that PTEN null cells demonstrate genomic instability through increased ROS and oxidative stress induced DNA damage. The aim of this study was to identify a biomarker for PTEN status in human breast cancers.

Materials and Methods A metagene representing ATM activation was generated from public cell line data of AT fibroblasts treated with gamma-irradiation. This was used to perform hierarchical clustering analysis of a public DNA microarray profiling dataset with known PTEN IHC status. The metagene was validated in PTEN wildtype and null breast cancer cell lines.

Results We found that PTEN null cells have elevated levels of ROS and furthermore activation of the DNA damage signalling kinase, ATM. In agreement with this, the ATM metagene signature correlated with PTEN mutation in breast cancer tumours. Scoring of PTEN wildtype and null breast cancer cell lines using the metagene correlated with ATM activation and sensitivity to inhibition of ATM. Furthermore we show that inhibition of ATM caused DNA damage, cell cycle arrest and apoptosis in PTEN deficient cells suggesting a novel therapeutic strategy.

Conclusion These observations suggest that ATM may represent a therapeutic target in PTEN deficient tumours and furthermore ATM activation may also be an important biomarker of PTEN mutation or loss in breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-12.
P5-06-04: Mechanisms of Acquired Resistance to Insulin-Like Growth Factor 1 Receptor Inhibitor in MCF-7 Breast Cancer Cell Line.
Authors: Ekyalongo RC , Mukohara T , Kataoka Y , Kiyota N , Fujiwara Y , Minami H
Published: June 2012

Background: No studies have yet clarified the mechanism of acquired resistance to insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitor (TKI). Our previous study of 16 breast cancer cell lines found that only MCF-7 expressed high levels of insulin receptor substrate (IRS)-1 and was sensitive to the IGF-1R-TKI NVP-AEW541.

Material and Methods: We developed a model of acquired resistance to NVP-AEW541 by continuously exposing MCF-7 cells to NVP-AEW541, naming the model MCF-7-NR. To explore the mechanism of acquired resistance to NVP-AEW541, the effects of NVP-AEW541 on cell growth and IGF-1R signaling in MCF-7 and MCF-7-NR cells were examined.

Results: With Western blot analysis, we found that MCF-7-NR had much lower levels of IRS-1 than parental MCF-7. While phosphorylation of Akt was completely inhibited by administration of NVP-AEW541 (3 μM) in both cell lines, phosphorylation of S6K remained high only in MCF-7-NR. The notion of Akt-independent S6K phosphorylation in MCF-7-NR was further supported by the fact that cell growth and phosphorylation of S6K was affected by administration of the Akt inhibitor perifosine to a lesser degree in MCF-7-NR than in MCF-7. Further, the mTOR inhibitor everolimus inhibited phosphorylation of S6K and cell growth equally in both lines. Screening of MCF-7 and MCF-7-NR lines for phosphorylation of 42 receptor tyrosine kinases with and without 3μM NVP-AEW541 showed that Tyro3 phosphorylation remained high only in MCF-7-NR cells. Gene silencing of Tyro3 using siRNA resulted in reduced cell growth, decreased phosphorylation of phosphoinositide-dependent kinase-1 (PDK-1) and protein kinase C α/βII, reduced expression of cyclin D1 in the MCF-7-NR line, with minimal effects evident in the MCF-7 line.

Discussion: Findings from our current study support the possibility of predicting sensitivity to NVP-AEW541 by measuring IRS-1 expression, as hypothesized in our previous study. Akt-independent activation of mTOR/S6K by an as-yet-undefined mechanism appears to induce acquired resistance to NVP-AEW541, and as such mTOR inhibitors may have therapeutic value. Tyro3 upregulation and migration of control of cellular growth and cyclin D1 expression may also induce resistance to NVP-AEW541. Although the validity of these findings should be evaluated in further preclinical studies and eventually in clinical trials, our observations may lead to individualized use of NVP-AEW541 and the development of “backup drugs” against tumors that acquire resistance to NVP-AEW541.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-04.
P5-06-11: Quercetin-3-Methyl Ether Inhibits Lapatinib-Sensitive and Lapatinib-Resistant Breast Cancer Cell Growth by Inducing G2/M Arrest and Apoptosis.
Authors: Bode AM , Li J , Zhu F , Normanno NE , Ericson ME , Lubet RA
Published: June 2012

Background: Lapatinib, an oral, small-molecule, reversible inhibitor of both EGFR and HER2, is highly active in HER2 positive breast cancer as a single agent and in combination with other therapeutics. However, resistance against lapatinib is an unresolved problem in clinical oncology. Recently, interest in the use of natural compounds to prevent or treat cancers has gained increasing interest because of presumed low toxicity. Quercetin-3-methyl ether, a naturally occurring compound present in various plants, has potent anticancer activity.

Material and Methods: Quercetin-3-methyl ether was obtained from Analyticon Discovery (Potsdam, Germany). Lapatinib-resistant SK-Br-3 (SK-Br-3-Lap R) cells were isolated in the laboratory of Cell Biology and Biotherapy at the Istituto Nazionale dei Tumori, Naples, Italy. Parental SK-Br-3 and SK-Br-3-Lap R cells were cultured in monolayers at 37 °C in a 5% CO2 incubator in 10% FBS/McCoy supplemented with penicillin/streptomycin (100 units/ml; Invitrogen). SK-Br-3 Lap R cells were routinely maintained in 1 μM lapatinib. Anchorage-dependent and -independent growth and cell cycle were assessed in the presence and absence of quercetin-3-methyl ether. Results: Here, we found that quercetin-3-methyl ether caused in a significant growth inhibition of lapatinib-sensitive and -resistant breast cancer cells. Western blot data showed that quercetin-3-methyl ether had no effect on Akt or MAPKs signaling in resistant cells. However, quercetin-3-methyl ether caused a pronounced G2/M block mainly through the Chk1-Cdc25c-cyclin B1/Cdk1 pathway in lapatinib-sensitive and -resistant cells. In contrast, lapatinib produced an accumulation of cells in the G1 phase mediated through cyclin D1, but only in lapatinib-sensitive cells. Moreover, quercetin-3-methyl ether induced significant apoptosis, accompanied with an increase the levels of in cleaved caspase 3, caspase 7 and poly(ADP-ribose) polymerase (PARP) in both cell lines.

Conclusion: Overall, these results suggested that quercetin-3-methyl ether might be a novel and promising therapeutic agent in lapatinib-sensitive or -resistant breast cancer patients.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-11.
P5-07-02: Could the Combination of COX-2 Inhibitor and Calcitriol Be a New Chemopreventive Approach To Decrease the Incidence of Breast Cancer?
Authors: Thill M , Cordes T , Fischer D , Hoellen F , Friedrich M , Diedrich K , Dittmer C
Published: June 2012

Background

COX-2 is a potential molecular prognostic factor for breast cancer and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is described as a tumour suppressor in cancer. The antiproliferative effects of calcitriol (1,25(OH)2D3) mediated via the vitamin D receptor (VDR) render vitamin D a promising target in breast cancer therapy.

Material and Methods: The expression of the prostaglandin (PG) metabolising enzymes (COX-2, 15-PGDH), the PG receptors, the vitamin D metabolising enzymes (1-alpha-hydroxylase, 24-hydroxylase) and the VDR were determined in benign and malignant breast cell lines as well as in normal and malignant breast tissue. Measurement of PGE2 and 25(OH)2D3 serum level in healthy women and breast cancer patients was performed in winter and summertime. Additionally, the influence of calcitriol on cell proliferation was determined. In addition we examined the effect of calcitriol on the enzyme expression.

Results: We found an inverse correlation between the expression of the PG metabolising enzymes with the VDR as well as with the vitamin D metabolising enzymes by investigating the tissue samples. Moreover, we detected an inverse correlation between the PGE2 and 25(OH)2D3 serum level in breast cancer patients during wintertime. Furthermore the PG receptors were associated with an increased carcinogenesis. The breast cell experiments presented a dysregulated vitamin D metabolism, especially in the invasive breast cell line. Calcitriol showed an antiproliferative effect only in the benign but not in the malignant cell lines, and the expression of COX-2 and 15-hydroxyprostaglandindehydrogenase was influenced by calcitriol only in the benign breast cell line.

Conclusions: These results suggest a link between vitamin D and PG metabolism and therefore a possible synergism between COX-2 inhibition and calcitriol in breast cancer cells. For the first time, we could show an inverse correlation between the two metabolisms in breast cancer on different levels. Thus, the chemopreventive combination of calcitriol and/or vitamin D analogue with a COX-2 inhibitor might decrease the incidence of breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-07-02.
P5-07-07: Follistatin Suppresses In Vitro Growth of Breast Cancer Cells and Its Reduced Expression in Breast Cancer Associated with Poor Differentiation and Prognosis.
Authors: Ye L , Mansel RE , Jiang WG
Published: June 2012

Background: Bone morphogenetic protein (BMP) belongs to TGF-β superfamily, being widely involved in prenatal and postnatal development and homeostasis of various tissues and organs. BMP signaling has been implicated in tumorigenesis and disease progression of certain malignancies. Recent studies demonstrated a profound role played Noggin, a BMP antagonist in bone metastasis from breast cancer. Current study examined expression of Follistatin, another BMP antagonist in a breast cancer cohort, and its relevance to pathological and clinical parameters.

Methods: Expression of Follistatin was examined in breast cancer tissues (n=93) and normal background tissues (n=30) using quantitative real time PCR and immunhistochemistry (IHC). The breast tissue samples were collected immediately after surgery. The clinical follow-up was routinely performed after surgery. The median follow-up period was 120 months (June 2004). Full-length human Follistatin coding sequence was cloned into a plasmid vector. Following transfection and verification, effect of Follistatin overexpression on cell growth was examined using an in vitro growth assay.

Results: IHC revealed cytoplamic staining of Follistatin in mammary epithelial cells in breast tissues. The staining appeared to be weaker in breast cancer, but there was no significant difference in its transcripts levels compared with that of normal background tissues. Follistatin transcripts were decreased in both moderately differentiated and poorly differentiated tumors, p=0.0165 and p=0.0169 in comparison with well differentiated tumors respectively. Transcript levels of Follistatin in primary tumors tended to be lower in the patients with poor prognosis (33.8±14.7) including those patients had local recurrence, metastases and died of breast cancer, p=0.09 when compared with that of patients remained disease free (117.0±46.0). Its expression appeared particularly lower in the patients died of the disease (21.8±12.0), p=0.05 v.s. disease free. Over-expression of Follistatin exerted inhibitory effect on in vitro growth of MDA-MB-231 cells.

Conclusions: Decreased expression of Follistatin in primary tumors of breast cancer correlates with poorer differentiation and mortality. Follistatin suppresses in vitro growth of breast cancer cells. It suggests that Follistatin is a negative regulator for tumor growth and disease progression of breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-07-07.
P5-08-04: Mammographic Microcalcifications and Breast Cancer Tumorigenesis: A Radiologic-Pathologic Analysis.
Authors: Naseem M , Murray J , Hilton JF , Han D , Hogeveen S , Heersink RL , Muradali D , Simmons C , Bell D , Haq R , Brezden-Masley C
Published: June 2012

Background

Microcalcifications (MCs) are tiny deposits of calcium in breast soft tissue. They serve as key diagnostic radiological features for localization of malignancy. Approximately 30% of early invasive breast cancers have fine, granular MCs detectable on mammography; however, their role in breast cancer tumorigenesis is currently unknown. The purpose of this study was to investigate the relationship between mammographic MCs and breast cancer pathology.

Methods: A retrospective chart review was performed for 882 women treated for breast cancer between 2000–2010 at St. Michael's Hospital. Demographic information (age and menopausal status), tumor pathology (size, histology, grade, nodal status and lymphovascular invasion), hormonal status (ER and PR), HER-2 overexpression and presence of MCs were collected for breast cancer patients. Chi-square tests were performed for categorical variables and t-tests were performed for continuous variables. All tests were two-sided and p-values less than 0.05 were considered statistically significant.

Results: A total of 826 patient charts were included; 56 (6.4%) patients had metastatic carcinoma and were excluded from analysis. Only 37.0% (326/882) of the patients presented with mammographic MCs. Patients were more likely to have MCs if they were HER-2 positive (51%) as opposed to being HER-2 negative (33.4%) (p=0.001). There was a significant association between MCs and being perimenopausal with a mean age of 50 (65.2%) (p=0.012). Patients with invasive ductal carcinomas (39.7%) were more likely to present with MCs than were patients with other tumor histology (p=0.001). There was a positive correlation between MCs and tumor grade (p=0.051), with grade III tumors (41.85%) presenting with the most MCs, followed by grade II (37.95%) and grade I (29.8%). There was no significant association between mean age, mean tumor size, ER and PR status with the presence of MCs.

Conclusion: This is the largest study that suggests the appearance of MCs on mammograms is strongly associated with HER-2 overexpression, invasive ductal carcinoma and perimenopausal status. Since HER-2 is implicated in mediating aggressive tumor growth and metastasis, future studies should investigate the molecular pathways underlying HER-2 overexpression and MC development. This would help better understand the role of MCs in breast cancer tumorigenesis.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-08-04.
P5-10-01: Metabolic Pharmacodynamic Effect Evidenced by 18FDG-PET as a Tool for Early Prediction of Iniparib Efficacy in Metastatic Triple Negative Breast Cancer (MTNBC): A Proof of Concept Study.
Authors: Kerrou K , Gligorov J
Published: June 2012

Rationale: It was hypothesised that Iniparib or 4-iodo-3-nitrobenzamide acts as an irreversible inhibitor of PARP1 and possibly other enzymes. It has shown successively promising then confusing results in MTNBC in combination with chemotherapy. To date, its mechanism of action has not been completely elucidated but since 1992, inhibition of glycolysis by iniparib and subsequent necrotic cell death has been described. We hypothesised that early inhibition of glycolysis evidenced by in-vivo imaging with 2-((18)F)Fluoro-2-Deoxy-D-Glucose Positron-Emission Tomography (18FDG-PET) may be a predictor of efficacy to iniparib, as it was previously reported for other anticancer drugs.

Objectives: To detect metabolic pharmacodynamic effect of iniparib plus chemotherapy by means of 18FDG PET and proof the concept of its use as an early predicitive tool of Iniparib efficacy in MTNBC.

Patients & Methods: From March to November 2011, 8 pts were included in our institution in a randomized phase II study comparing bi-weekly versus weekly iniparib treatement in combination with gemcitanine and carboplatin. All patients had 18FDG-PET at baseline, at day 7 (D7). The maximum value of standardized uptake value (SUVmax) of all measurable metastatic lesions was measured and the median SUVmax was calculated for all patients at baseline and D7. The metabolic response was defined as the percentage of reduction of SUVmax (SUVmax Baseline — SUVmax D7) / SUVmax Baseline and the median SUVmax response was correlated to RECIST 1.1 response at six weeks and to disease-free survival (DFS).

Results: The median decrease in SUVmax of evaluable lesions measured at D7 was predictive of RECIST 1.1 response at six weeks and was correlated to the best obtained response during the treatment phase. Furthermore, it showed a linear correlation with DFS (y = −0,0007x - 0,1325; R2 = 0,3402).

Conclusion: Metabolic pharmacodynamic effect as evidenced by 18FDG PET as soon as one week of treatement act as an early predicitive tool of Iniparib plus chemotherapy efficacy in MTNBC.

Considering the lack of a predicitive biomarker of response and if confirmed on larger study, 18FDG PET could be used as a surrogate predictive biomarker is this setting. These results may be considered as an in-vivo proof of concept for the inhibition of glycolysis by iniparib.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-10-01.
P5-11-09: High Concordance for Microarray Based Determination of ER, PR and HER2 Receptor Status and Local IHC/FISH Assessment Worldwide in 749 Patients.
Authors: Wesseling J , Cusumano G , Tinterri C , Sapino A , Zanconati F , Lutke-Holzik M , Nguygen B , Deck K , Querzoli P , Perin T , Giardina C , Seitz G , Guinebretiere J , Barone J , Watanabe T
Published: June 2012

Background The level of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression is predictive for prognosis and/or treatment response in breast cancer patients. However, differences in fixation and IHC and subjective interpretation can substantially affect the accuracy and reproducibility of the results. The commercially available TargetPrint test measures the mRNA expression level of ER, PR and HER2 and provides high quality second opinion for local IHC/FISH assessment. This study compares results from the microarray-based TargetPrint with IHC and FISH (for HER2 IHC2+) generated by local standard procedures.

Methods Prospective tumor samples were collected for 749 patients diagnosed with breast cancer stage I to IV between February 2008 and January 2011. The mRNA level of ER, PR and HER2 (TargetPrint) was assessed in the Agendia laboratories (Agendia Inc, Irvine, CA; Agendia BV, Amsterdam, the Netherlands) in fresh tumor samples submitted from 22 hospitals from Europe, New Zealand, Japan and US. The results of the IHC/FISH assessments performed according to the local standards at the hospitals were compared to the quantitative gene expression readouts.

Results Of the 749 samples, IHC assessment was unknown for 5 ER samples and 4 PR samples; FISH was unknown for 24 samples. TargetPrint read out was not assessed for HER2 for 11 samples.

Median age of these patients was 61 years. Comparison of IHC and gene expression read out by TargetPrint showed a concordance of 95% for ER; 82% for PR and 91% for HER2.

In this study, only 4% of all IHC ER positive samples were classified negative by microarray. In contrast, 14% of IHC ER negative samples were classified positive by microarray. However for HER2, 28% of IHC/FISH HER2 positive samples were classified negative by microarray and 5% of IHC/FISH HER2 negative samples were classified positive by microarray.

Samples with discordant classifications for TargetPrint and local assessment are being reviewed in greater detail by a central pathologist.

Conclusions Microarray based readout of ER, PR and HER2 status using TargetPrint is highly comparable to local IHC and FISH analysis in 749 analyzed samples in various hospitals worldwide. The results indicate mRNA expression read out for ER, PR and HER2 by TargetPrint provides high quality second opinion for local IHC/FISH assessment.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-09.
P5-11-10: Reproducibility and Robustness of the FDA Approved INFORM HER2 Dual ISH DNA Probe Cocktail Assay.
Authors: Loftin IR , McElhinny AS , Miller R , Garcia C , Bai I , Ranger-Moore J , Ostrem J , Padilla M , Grogan T
Published: June 2012

Background: Amplification and/or HER2 overexpression is associated with poor clinical outcome for patients with invasive breast carcinoma. Determination of HER2 status dictates eligibility of patients for trastuzumab (Herceptin) therapy, which has been shown to improve prognosis. We present data from the inter-observer reproducibility and robustness studies of the FDA approved INFORM HER2 Dual ISH DNA Probe Cocktail (HER2 Dual ISH) assay, which is an alternative to FISH. The assay is fully automated and is scored using light microscopy providing morphological context.

Methods: Ninety-five human invasive breast carcinoma specimens (a mix of amplified and non-amplified cases) were stained in a study, using the HER2 Dual ISH assay. The slides were enumerated by two qualified readers. The same cohort was stained using Path Vysion FISH and read by one qualified reader.

To verify the robustness of the HER2 Dual ISH assay, customer selectable options for factors known to impact the staining quality and intensity, which can be varied for troubleshooting, were tested on human invasive breast carcinoma cases and xenografts. The slides were evaluated by one qualified reader.

For both studies all assay steps were fully automated on a VENTANA BenchMark XT automated slide stainer using silver metallographic and red colorimetric detections for Dual ISH.

Results: In the inter-observer study the first pass staining success rate was >93% for the HER2 Dual ISH assay, while the FISH first pass staining success rate was 59%. Some cases were rescued after a second attempt, which yielded a second pass staining success rate of 80% for FISH and 97% for HER2 Dual ISH. The negative, positive and overall agreement rates and 95% confidence intervals for the 61 clinical samples of this cohort that were enumerable with both FISH and the HER2 Dual ISH assay are 94.7 (75.4−99.1), 95.2 (84.2−98.7), 95.1 (86.5−98.3) and 89.5 (68.6−97.1), 97.6 (87.7−99.6), 95.1 (86.5−98.3) for reader A vs. FISH and reader B vs. FISH respectively. The positive and negative agreement rates for the inter-observer reproducibility of the HER2 Dual ISH assay were 94.7 and 97.6 %, respectively. In robustness testing, the recommended staining protocol was shown to be effective. All conditions tested resulted in appropriate staining (except for mildest cell conditioning and protease options). Conclusion: The fully automated FDA approved INFORM HER2 Dual ISH assay is robust, reproducible and concordant with the manual FISH assay in determining HER2 status in invasive breast carcinoma.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-10.
P5-13-02: Prediction of Dasatinib Sensitivity of Breast Cancer Based on a Novel Tyrosine Kinase-Activity Profiling Assay.
Authors: Kawai M , Torikoshi Y , Notoya M , Gohda K , Ueno NT , Ishihara H
Published: June 2012

Background: Receptor tyrosine kinases and other membrane-associated tyrosine kinases are frequently overexpressed, activated, or mutated in cancer cells and cause aberrant signal transduction, which leads to cellular proliferation, angiogenesis, metastasis, and antiapoptosis. Blockage of these signals is considered an efficient strategy in cancer therapy, and to date, several tyrosine kinase inhibitors (TKIs) and antibody drugs targeting such kinases have been developed and are routinely used in clinics. However, the efficacy rates of the drugs are, unfortunately, limited. To provide optimum care for individual patients, an accurate prediction method for drug efficacy is now strongly demanded. Recent studies have shown that alteration of multiple kinases is involved in both primary and acquired drug resistance. Advantages of multi-targeted TKIs have also been reported. These findings indicate the need for a comprehensive evaluation of multiple kinases for predicting drug response. To achieve this, we developed a novel method of comprehensively profiling kinase activity.

Materials and methods: We characterized the membranous tyrosine kinases of breast cancer cell lines using a newly established profiling assay. Briefly, crude membrane fractions were prepared and directly subjected to the assay with nonspecific substrate (Poly(Glu4-Tyr)). The profile of the kinases in the crude membrane fraction was obtained by inhibiting the total activity with 13 selected kinds of adenosine triphosphate antagonists, independently. The residual activity (RA) of membranous kinases was defined as the percentage of activity with/without TKI.

Results: Nineteen breast cancer cell lines were classified as “sensitive” (n = 6) and “resistant” (n = 13) to dasatinib according to the definition in the publication (Cancer Res., 67 2226–38. 2007). The RA of tyrosine kinases targeted by different types of TKIs was determined by our assay, and we found that two RAs (src inhibitor 1 and PP1) showed statistically significant differences between the “sensitive” and “resistant” groups (p=0.017, and p=0.002, respectively, Student's t-test). The RA of the two TKIs were also significant predictors for dasatinib sensitivity in a receiver operating characteristic curve analysis (area under the curve: 0.846 for src inhibitor 1 and 0.910 for PP1). Since the major target of dasatinib are src family kinases, protein expressions of the src family of the cell lines were quantified by a Western blotting and compared between the groups. We found that the protein expression is not a statistically significant predictor of dasatinib sensitivity.

Conclusion: We have shown that a comprehensive tyrosine kinase activity profiling assay of cancer cells can predict dasatinib sensitivity. We plan to validate this assay prospectively in patients with breast cancer who receive dasatinib.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-02.
P5-13-10: Discordance of Estrogen Receptor and HER-2/neu Status Can Be Seen between First and Subsequently Biopsied Metastatic Lesions in Breast Cancer.
Authors: Lower EE , Kennedy D
Published: June 2012

Background

We have previously reported discordance between primary and first metastasis for both estrogen receptor (ER) and HER-2/neu (HER 2) status (Breast Cancer Res Treat 2005;90:65–70 and Breast Cancer Res Treat 2009;113:301–6). Because biomarker status is the primary determinant for targeted therapy, changes in biomarkers can impact recommendations for systemic treatment.

Materials and Methods: In order to evaluate the possibility of further discordance with the development of additional metastatic sites, we examined the ER and HER 2 status of primary and first and subsequently biopsied breast cancer metastasis. In most cases, the subsequent site of metastasis was the second detected metastasis. Pathology reports were reviewed from 65 patients with biopsy proven first and additional metastasis; however, immunostain results were not available for all three timepoints for all patients. Sites of biopsied metastasis included lung, liver, bone, chest wall, and central nervous system.

Results: The time between the initial diagnosis and first metastasis was 102 (3-676) months [Median (Range)], and the time between the first and subsequent metastasis was 60 (6-273) months. For ER, there was discordance between the primary lesion and either the first or subsequent metastasis in 18 of 65 cases (27.7%). Discordance of the ER occurred between the first and subsequent metastasis in 12 (18%) of cases. Three patterns of ER discordance occurred in these 12 cases: 4 cases of both primary and first metastasis negative with subsequent metastasis positive; 3 cases of primary positive, first metastasis negative, and subsequent metastasis positive, and 5 cases of primary and first metastasis positive and subsequent metastasis negative. For HER 2, discordance was detected between the primary and either first or subsequent metastasis in 11 of 36 cases (30.6%). Further discordance between the first and subsequent metastatic lesions occurred in 8 of 51 (16%) of cases.

Conclusions: A significant level of discordance for either ER or HER 2 was detected between first and subsequent metastatic breast cancer lesions. For breast cancer patients with new metastasis, consideration of reevaluating ER and HER-2/neu status may be worthwhile.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-10.
P5-13-22: Gene Expression Profiles Predict Pathological Complete Response to Standard Neoadjuvant Fluorouracil, Doxorubicin, and Cyclophosphamide and Paclitaxel with or without Trastuzumab in Early Breast Cancer.
Authors: Tamura K
Published: June 2012

Background

To examine the feasibility of gene expression signature as a predictor of pathological complete response (pCR) to sequential fluorouracil, doxorubicin, and cyclophosphamide (FEC) and weekly paclitaxel (P) with or without trastuzumab (T) neoadjuvant chemotherapy.

Materials and Methods: We have conducted consecutive two prospective phase II, establishing training and validation sets, with similar eligible criteria include, stage IIA-IIIC, chemotherapy-naïve, measurable disease, age ≥ 20, PS 0/1, and adequate organ function. Patients were treated preoperatively with 4 cycles of FEC (500/100/500 mg/m2) followed by 12 cycles of weekly P (80 mg/m2) with or without T (2mg/kg). Patients underwent pretreatment fine-needle biopsy for cDNA microarray using Affimetrix Gene Chip U133 plus 2.0 arrays with 30,000 differential expressions of various genes. We ranked gene probes from training sets according to a predictive power concerning pCR by Wilcoxon, and validated them using validation sets by SVM.

Results: Between July 2007 and December 2010, 122 patients were enrolled in the two consecutive prospective studies (training: 89 pts, validation: 33 pts). Median age was 51. PS 0/1: 115/7; Stage IIA/IIB/IIIA/IIIB/IIIC: 30/57/20/14/1; Histological subtype: ER+HER2− (LA) / ER+HER2+ (LB) / ER-HER2− (TN) / ER-HER2+ (enrich-HER): 51/18/24/29. All patients have received curable operations. pCR rate was 31.1% (LA; 2.0%, LB; 44.4%, TN; 37.5%, enrich-HER; 69.0%). 104 (85.2%) sufficient mRNA for cDNA microarray from individual primary breast cancer tissues fine-needle biopsy are available. As reported previously, the breast cancers were classified into a Luminal A/B, Basal-like, HER2−enriched, Claudin-low intrinsic subtypes, indicating a high quality of the representative method. In HER2 positive breast cancer, HER2−enriched subtype was a reproductive predictive marker. In contrast, In HER2 negative breast cancer, three genes (N-myc and STAT interacter, Tryptophanyl-tRNA synthetase, and IQCE) and basal-like subtype were validated as the predictors of pCR. The three genes were also identified as predictors of pCR in the triple negative population.

Conclusions: Specific gene expression profiles predict pCR to standard neoadjuvant regimen, especially in triple negative breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-22.
P5-14-03: Genomic Comparison of Paired Primary Breast Carcinomas and Macrometastatic Lymph Node Metastases Using Quantitative RT-PCR by Oncotype DX: Assessment of the Recurrence Score and Quantitative Single Genes.
Authors: Boolbol SK , Kirstein L , Harshan M , Klein P , Cohen J-M , Chadha M , Baehner FL , Malamud SC
Published: June 2012

Background

NCCN guidelines now include consideration of the 21 gene RT-PCR assay in node negative, hormone sensitive breast cancers greater than 0.6 cm. Recent data suggests a potential role for testing of node positive(N1), hormone sensitive patients as well. Currently, no data exists on testing of metastatic lymph nodes. We sought to establish the feasibility of Oncotype DX® testing in the metastatic lymph nodes and, furthermore, to evaluate the genomic concordance between the primary tumor and the nodal deposit. These results may further our understanding of tumor heterogeneity and biological selectivity in the process of lymph node metastasis and ultimately systemic metastases.

Methods: We examined the formalin fixed paraffin embedded tumor tissues (FPET) from 100 breast cancer patients from our institution's pathology database with available paired primary tumor and macrometastic nodal deposits by Oncotype Dx® testing. Inclusion criteria included patients with macrometastatic lymph node disease, hormone receptor positive, HER2 negative primary breast cancer. The testing laboratory was blinded from the clinical outcomes data available on these patients. All FPET samples had H&E slides made which were reviewed by board certified surgical breast pathologists to determine if there was sufficient invasive tumor and to direct dissection of all lymph node samples. The Recurrence Scores and quantitative single gene values from the paired samples will be examined descriptively with scatterplots and Pearson correlation coefficients and reported at the meeting.

Results: All 100 paired specimens were sent for Oncotype DX testing. 24 samples were found during standard H&E review to not have sufficient tumor for the assay. Of the 176 samples created for RNA extraction 173 samples had sufficient RNA for the Oncotype DX assay: 85 lymph node and 88 primary breast carcinoma samples. Recurrence Scores and quantitative single gene values from the paired primary breast carcinoma and lymph node samples will be examined descriptively and reported at the meeting.

Conclusion: Previous comparisons of paired primary and metastatic samples have used immunohistochemistry and FISH which are susceptible to variability in preanalytic variability such as delay to fixation, choice of fixative and duration in fixative. This study, using quantitative RT-PCR, will be one of the largest comparisons of tumor biology in paired samples yet reported in the era of genomic subtyping and may have implications for systemic adjuvant treatment.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-14-03.
P5-14-13: Favorable Prognosis in Patients with T1a,b Node-Negative Triple Negative Breast Cancers Treated with Multimodality Therapy.
Authors: Ho AY , Gupta G , Perez CA , King TA , Patil SM , Rogers KH , Brogi E , Morrow M , Hudis C , Traina T , McCormick B , Powell SN , Robson ME
Published: June 2012

Purpose: To evaluate the clinical characteristics, natural history and outcomes in patients with ≤1cm, node-negative triple negative breast cancer (TNBC).

Materials and Methods: After excluding patients who received neoadjuvant therapy, 1,022 TNBC patients who received definitive breast surgery from 1999 to 2006 were identified from an institutional database. Among these, 194 patients had node-negative tumors ≤1cm and comprise the study population. Clinical data was abstracted and survival outcomes were analyzed.

Results: Median follow-up time was 71 months (range 2–143). Median age at diagnosis was 55.5 years (range 27–84). T stage was T1mic in 16 (8.2%), T1a in 49 (25.3%), T1b in 129 (66.5%). The majority of tumors were poorly differentiated (N= 142, 73%), lacked lymphovascular invasion (N= 170, 87.6%) and were screening-detected (69%, N=134). Breast-conserving surgery (BCS) was employed in 129 (66.5%) and mastectomy in 65 (33.5%) patients. 113 (58%) patients received adjuvant chemotherapy and 123 (63%) received whole breast radiation. Patients who received chemotherapy tended to have more adverse clinical and disease features (younger age,T1b, poor tumor grade; all p<0.05). For the entire group, 5 year local recurrence-free survival was 96% and distant metastasis-free survival was 95%, with no difference in distant relapse rates between T1mic/T1a vs. T1b patients (94.5% vs 95.5%, p=0.81 )or by receipt of chemotherapy (95.9% vs 94.5%, p=0.63).

Conclusion: Excellent 5-year locoregional and distant control rates were achievable in patients with TNBC tumors ≤ 1.0 cm, 58% of whom received chemotherapy. These results identify a group of TNBC patients with favorable outcomes following early detection and multimodality treatment.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-14-13.
P5-14-18: Today's Estrogen Receptor Positive/Her-2-neu Receptor Negative Breast Cancer Patients Do Significantly Better Than Yesterday's Estrogen Receptor Positive Breast Cancer Patients.
Authors: Madsen EV , Elias SG , Gobardhan PD , van Oort PM , van der Ent FW , Nieweg OE , Valdés Olmos RA , Smidt M , van Dalen T
Published: June 2012

Background: Her-2-neu receptor (Her2) positive and triple negative breast cancer patients have a poor prognosis. The majority of cancers are characterized as estrogen receptor (ER)+/Her2- and these patients may now have a better prognosis compared to before the introduction of the Her-2-neu receptor.

Material and methods: Since 1997 3424 patients were treated for cT1-2N0 breast cancer in three hospitals. Determination of Her2-neu status was introduced between 1999 and 2004. Trastuzumab treatment has been given routinely since 2005. Survival was evaluated for the different groups; ER+/Her2-, ER+/Her2+, ER+/Her2 unknown (status not determined).

Results: 2284 patients had ER+/Her2- tumors, 259 had ER+/Her2+ tumors and 262 had ER+/Her2 unknown tumors. Systemic treatment was given to 48.4%, 71.0% and 43.9% respectively. Estimated 5-and 10-year overall survival was 92.0% and 82.2% for ER+/Her2-, 91.6% and 70.8% ER+/Her2+ and 83.4% and 72.2% for ER+/Her2 unknown (p < 0.001). The outcome differences between ER+/Her-2- and ER+/Her-2 unknown tumors remained following adjustment for tumor malignancy grade, nodal status and adjuvant systemic treatment (OR 0.8: CI 0.72 — 0.88; p<0.001). For patients with ER+/Her2+ tumors 5 year overall survival was comparable with ER+/Her2- tumors but 10 year overall survival was comparable with ER+/Her2 unknown tumors.

Discussion: Patients with ER+/Her2- tumors have a significantly better outcome than patients who were classified as ER+ before the assessment of Her-2-neu over-expression. Current prognostic models do not take this effect into account.

The branching off of the survival curve for patients with ER+/Her2+ tumors can be explained by the standard use of trastuzumab during the last five years.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-14-18.
P5-20-08: Multicenter Results of Scalp Cooling To Prevent Chemotherapy-Induced Alopecia in 1500 Breast Cancer Patients.
Authors: van den Hurk C , Peerbooms M , Komen M , Nortier H , Breed W
Published: June 2012

Background Chemotherapy-induced alopecia (CIA) is a frequent occurring side effect of cancer treatment that has high psychological impact on many patients and their relatives. CIA may be prevented by scalp cooling.

Methods Breast cancer patients who received scalp cooling could participate in this registration study from 2006 and onwards. Nurses and patients completed questionnaires. Scalp cooling was performed using the Paxman PSC1 or PSC2 system and was considered satisfying when patients did not wear a wig or head cover. Logistic regression analyses will be used to examine determinants of the scalp cooling result, including age, type/length/thickness/chemical manipulation (dyeing, waving, colouring) of hair, type/dose of chemotherapy, cytostatic infusion time, post-infusion cooling time, dampening hair or use of conditioner before scalp cooling and previous treatment with chemotherapy.

Results The use of scalp cooling increased from 4 hospitals in 2005 to 60 out of 100 Dutch hospitals in 2011. The registration comprised about 1500 scalp cooled breast cancer patients of whom 79% were treated in the adjuvant setting. Overall, patients’ satisfaction with the result of scalp cooling was 50%, but varied for different chemotherapy schemes and dosages from 8% in docetaxel 75 mg/m2, doxorubicin 50 mg/m2, cyclophosphamide 500 mg/m2 (TAC) to 81% in paclitaxel 70–90 mg/m2. The results of the regression analyses will be presented at the conference.

Discussion Scalp cooling is effective in most commonly used chemotherapy regimens for breast cancer patients. Therefore, scalp cooling should be offered more often. Efficacy depends on the type and dose of chemotherapy. Other factors influencing the result have never been studied in multivariate analyses before. Scalp cooling is ineffective in patients treated with a combination of a taxane and an anthracyclin. The frequent use of scalp cooling in the adjuvant setting indicates minimal fear for an increased incidence of scalp skin metastases after scalp cooling in breast cancer patients. Multicenter registration of results improves information for medical professionals and patients and will lead to more frequent use of scalp cooling and improvement of the method.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-20-08.