bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2-12-04: Evaluation of BRCA 1/2 mutation-carrier probability models, BRCAPRO and myriad II, in the Japanese population
Authors: Ito M , Yasuda Y , Furuta A , Ohuchi N
Published: August 2014

Background:

It is important to select high-risk group with a BRCA1/2 mutation and recommend them to provide genetic counseling and genetic testing. BRCAPRO and Myriad II are widely used for predicting BRCA mutation-carrier probability and useful for selecting eligible person of genetic testing. However, It is reported that BRCAPRO tends to underestimate the risk of BRCA1/2 mutation probability in Asian. The validation of these models are not sufficiently known in Japanese population.

Methods:

Twenty-six hundred sixty-five people visited to Ishinomaki Red Cross Hospital between 2011 and 2012.They were surveyed family history as a risk factor of inheriting breast cancer according to NCCN guideline. We calculated BRCA1/2 mutation probability by BRCAPRO and Myriad II. Those who had more than 10% mutation probability and early-onset breast cancer, triple negative breast cancer were received genetic counseling and genetic testing for applicants. We analyzed association between family history of the probands and BRCA 1/2 mutation. We also calculated sensitivity, specificity and positive predictive value(PPV) at the10% threshold in BRCAPRO and Myriad II.

Results:

Two handred forty-three(9.1%) people were selected as a risk of inheriting breast cancer, of which sixty-eight people (2.5%) were constructed family tree. The number of people who had more than 10% and 30% mutation probability by BRCAPRO was eight (0.30%) and five (0.18%).Fourteen people performed BRCA1/2 genetic testing (proband : thirteen, relative: one). Seven people carried a deleterious BRCA mutation (BRCA1: five, BRCA2: two). In the cases which had more than 30% mutation probability, all of them carried a deleterious BRCA mutation. Most of their mutation site was L63X which was most frequent in Japan.

People who had personal or family history of breast and ovarian cancer, triple negative breast cancer were significantly associated with BRCA1/2 mutation.(p = 0.035,p = 0.015,p = 0.009)

Using a 10% threshold, sensitivity, specificity, positive predictive value in BRCAPRO was 75%,83%,85% and 63%,67%,71% in Myriad II respectively. When it comes to the cases which had more than 30% mutation probability, Sensitivity, specificity, positive predictive value were all 100% in BRCAPRO.

Conclusions:

It is recommended to perform genetic testing for especially those who have personal or family history of both breast and ovarian cancer, triple negative breast cancer. 10% threshold of BRCAPRO and Myriad II is generally applicable to selecting eligible person in the Japanese population. BRCAPRO is superior to Myriad II in terms of sensitivity, specificity, PPV in our study. The cases which have more than 30% mutation probability need to have genetic testing.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-12-04.
Abstract P2-13-01: The Brazilian founder TP53 p.R337H mutation is uncommon in Portuguese women diagnosed with breast cancer
Authors: Giacomazzi J , Correia RL , Palmero EI , Gaspar JF , Almeida M , Portela C , Camey SA , Reis RM , Ashton-Prolla P
Published: August 2014

Since the first studies reporting the TP53 p.R337H mutation as founder mutation in Southern and Southeastern regions of Brazil, both in the general population and in patients from high risk breast cancer families with adrenocortical, choroid plexus and breast carcinomas, there has been controversy on the origin of this mutation. Preliminary analysis of the small subset of Brazilian mutation carriers that defined the founder haplotype using 29 tag SNPs revealed that the haplotype incided on a Caucasian background. The vast majority of carriers identified today reside in Brazil or, if identified in other countries, are Brazilian immigrants. To our knowledge, the only two exceptions of carriers without a recognizable link with Brazil are two European families (one Portuguese and one German) (Chompret et al., 2000; Herrmann et al. 2012). Haplotype analysis in the Portuguese family revealed the same haplotype identified in Brazilian individuals, but in the German family, a distinct haplotype was found. Knowing that a significant proportion of women with breast cancer in Southern Brazil are p.R337H carriers, we initiated TP53 genotyping in a Portuguese cohort of women with breast cancer recruited from the cities of Lisboa and Braga. Median age at diagnosis of breast cancer among the first 573 patients tested was 60 years and 100 (17.4%) patients had been diagnosed at or under the age of 45 years. Mutation screening was performed using Real-Time PCR (taqman assays), and failed to identify the mutation in the 573 patients tested. These results are in contrast with the mutation frequency observed in a study of 815 breast cancer-affected women from Southern and Southeastern Brazil, which has reached frequencies of 12.1 and 5.1% in pre- and post-menopausal women, respectively (Ashton-Prolla et al. 2012; ASCO Annual Meeting, ref 1522). We conclude that there is a significant difference in mutation frequency observed between the two cohorts (p<0.001). These findings suggest that TP53 p.R337H is not a common molecular alteration in Portuguese breast cancer-affected patients.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-13-01.
Abstract P2-14-04: Effect of age at the time of diagnosis on overall and cancer-specific mortality in breast cancer: Analysis of SEER, 1990-2008
Authors: Bao J , Yu K-D , Jiang Y-Z , Di G-H
Published: August 2014

Background: As a critical factor, age at the time of diagnosis has a significant impact on cancer therapy and outcomes. The purpose of this study is to investigate the overall mortality (OM) and breast cancer-specific mortality (BCSM) in patients with breast cancer corresponding to age at the time of diagnosis, thus constructing an age-related pattern on breast cancer survival.

Methods: We analyzed data for 396,978 women aged 20-79 from 1990 to 2008 diagnosed with breast cancer from the population-based Surveillance, Epidemiology, and End Results (SEER) program of the U.S. national cancer institute. Clinicopathological factors and outcomes were compared using a chi-square test, the Kaplan-Meier method, and Cox proportional hazards regression model.

Results: In the multivariable analysis, a significant relationship was found between age at the time of diagnosis and OM (p<0.001) as well as BCSM (p<0.001). Using 70-79 years old as the reference, the hazard ratio (HR) of OM and BCSM presents a U-like shape with the bottom at the group 40-49 years old (OM: HR,0.56; P<0.001; BCSM: HR,0.58; p<0.001) and 50-59 years old (OM: HR,0.59; P<0.001;; BCSM: HR,0.60; p<0.001), increasing to both ends gradually. Analysis in subgroup according to ER and PgR status, group 20-29 years old showed no significant difference with group 70-79 years old on both OM and BCSM in all subgroups except on BCSM in ER+PR+ subgroup (HR,1.25; P = 0.002).

Conclusion: Patients at very young and very old age (20-29 years old and 70-79 years old) have the poorest outcomes while prognosis improves with age towards mean level. The very young age (20-29 years old) may be regarded as a significant hazard factor for BCSM in patients with ER+PR+ breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-14-04.
Abstract P2-14-06: Characterization of risk factors in breast cancer young adult patients
Authors: Encinas G , Diz MDPE , Lyra EC , Katayama MLH , Pasini FS , Brentani MM , Chammas R , Góes JCGS , Folgueira MAAK
Published: August 2014

Background: Breast cancer diagnosis in the age group of 20-34 years represents 4% of breast cancer cases diagnosed at all ages in São Paulo state, Brazil. Risk factors in this age group of patients are little understood, especially in those not reporting familial history of breast/ovarian cancer syndrome (BOCS). Identification of predisposing factors and BRCA1/2 mutations (mut) in young Brazilian patients may contribute to characterize women with increased risk of breast cancer.

Objective: Characterize familial history and risk factors for breast cancer in young adult Brazilian patients and germline mutations in BRCA1/2 genes.

Methodology: Forty-five patients diagnosed with breast cancer at ages 18-35 years were interviewed using a comprehensive questionnaire. Genomic DNA was extracted from peripheral blood and BRCA1/2 mut were screened by Sanger sequencing of coding sequences, analyzed through Mutation Surveyor v.3.20 and searched in BIC Database, LOVD, LOVD-IARC and UMD.

Results: Mean age of patients at time of diagnosis was 33 y (24-35y); mean age of menarche was 13 y (8-19y); 32 (71.1%) patients had at least one born child, median age of first pregnancy was 22 years; 86.7% never smoked; 71.1% were no regular alcoholic drinkers; mean of body mass index was 27.14 (overweight). Most patients were diagnosed with invasive ductal carcinoma (91.1%); grades II or III (95.5%); ER/PR positive (57.8%); triple-negative (TN: 31.1%) or HER2 positive (17.8%). Among them, 22.2% reported positive familial history (NCCN criteria for BOCS). Deleterious mutation in BRCA1/2 genes was found in 17.8% of the patients (BRCA1, n = 3; BRCA2, n = 5). Among 10 and 35 patients reporting or not BOCS familial history, 3 (30%) and 5 (14.3%) respectively, had BRCA1/2mut. All BRCA1mut patients had TN tumors; BRCA2mut patients had ER/PR positive (60%), HER2 positive (20%) or TN tumors (20%). Most patients reported one or two of the following risk factors: early menarche (<12y); current/previous use of oral contraceptive; no breast feeding and no parities. Among 30 patients reporting no familial history and presenting BRCA1/2 wild type, six (20%) and four (13.3%) reported three or four of these risk factors, respectively. Considering breast cancer protective factors, 17.8% of the patients breast fed >16 months and 24.4% were aged <20y at birth of first child. Diet habits are being analyzed.

Conclusion: In young adults with breast cancer there is a trend towards a higher rate of positive familial history among whom there is a trend towards a higher chance of BRCA1/2 mutation. A better characterization of patients reporting no familial history is urgently needed.

Financial Support: FAPESP.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-14-06.
Abstract P2-16-12: An exploratory analysis of the role of dasatinib in preventing progression of disease in bone in patients with metastatic breast cancer
Authors: Cadoo KA , Morris PG , Lake DE , D'Andrea GM , Dickler MN , Gilewski TA , Dang CT , McArthur HL , Bromberg JF , Goldfarb SB , Modi S , Robson ME , Seidman AD , Sklarin NT , Norton L , Hudis CA , Fornier MN
Published: August 2014

Introduction: The role of dasatinib, an oral SRC inhibitor is being explored for the treatment of metastatic breast cancer. In a phase I study, we previously established that the combination of dasatinib and weekly paclitaxel was feasible. The activity of this combination is currently being explored in an ongoing phase II trial. Since Src kinase has a major role in osteoclast function and dasatinib has established anabolic and anti-resorptive effects in bone in vitro, we hypothesized that patients receiving this combination would have good control of osseous metastases and primarily develop progression of disease in sites other than bone.

Patients and methods: Patients were included in this analysis if they participated in the phase I or II metastatic breast cancer studies and received dasatinib at or above the recommended phase II dose of 120mg with paclitaxel (80mg/m2 day 1 and 8 of each 21day cycle). Patients who discontinued therapy for reasons other than progression were excluded. Per protocol, patients were required to discontinue bisphosphonates or other bone modulating agents for the first 8 weeks of study due to the potential for hypocalcaemia. Thereafter, they were permitted to receive these agents at the discretion of their treating physician. Patients provided serum samples for correlative studies. Assessment of N-telopeptide of type 1 collagen (NTX), a product of mature bone collagen that reflects bone specific resorption, is planned.

Results: The median age of the 24 patients who met criteria for analysis was 50y (37 - 66y). Of these, 15 (63%) had ER+ disease, and 24 (100%) were negative for human epidermal growth factor receptor (HER2). At study entry, 17 (71%) patients had bone involvement. Following the initial eight week moratorium, 7 (29%) patients received a bisphosphonate or rank ligand inhibitor during treatment with dasatinib + paclitaxel. Patients received a median 2 months (range 1-23) of dasatinib + paclitaxel therapy. To date, 3 (13%) continue on therapy, and 21 (88%) have had progression of disease. Among patients who progressed, 18 (86%) have progressed in visceral sites and only 3 (14%) progressed in bone. Analyses of serum NTX levels are ongoing and will be compared by site of progression.

Conclusion: The potential role of serum NTX as a predictive biomarker of benefit from dasatinib and paclitaxel is being explored and updated results will be presented.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-16-12.
Abstract P2-18-06: Preservation or division of the intercostobrachial nerve in axillary dissection for breast cancer: Meta-analysis of randomised controlled trials
Authors: Warrier SK , Hwang S , Koh C , Shepherd H , Mak C , Carmalt H , Solomon M
Published: August 2014

Purpose: Management of the ICBN during axillary dissection is controversial and the division of ICBN is often trivialised.The effect of dividing the ICBN, and its association with sensory disturbance, is unclear. A systemic review and meta-analysis was performed to evaluate the effect of preserving the ICBN during axillary dissection.

Methods: A systemic literature review and meta-analysis was performed according to the PRISMA and Cochrane Collaboration guidelines. Two authors (SW and SH) independently searched Medline, Pubmed, Embase, Cochrane Database of Systemic Reviews and the Cochrane Controlled Trials Register from 1950 to December 2012 for studies comparing preservation or division of the ICBN in axillary dissection for breast cancer. The primary outcome of this meta-analysis was sensory disturbance, defined as objectively assessed change in sensation in the distribution of the ICBN after axillary dissection. The nature of sensory disturbance was assessed as a secondary outcome, categorised into two types: “hypersensitivity” and “hyposensitivity”.

Results: Three RCTs and four non-RCTs were reviewed. A meta-analysis demonstrated that the incidence of sensory disturbance was significantly lower with preservation of ICBN compared to division of the ICBN with Mantel-Haenzel combined odds ratio 0.31 (0.17-0.57, 95% CI). There was relatively low level of heterogeneity (I2 = 19%, c2 = 2.48, df = 2).

The sensory disturbance was more likely to be hyposensitivity when compared to hypersensitivity (p <0.0001). No difference on number of lymph nodes dissected or operating time was noted.

Conclusion: This meta-analysis demonstrates that division of the ICBN is associated with higher risk of sensory disturbance, and that the nature of this sensory disturbance is more likely to be hyposensitivity, attributable to reduced nerve function.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-18-06.
Abstract P2-18-07: Nipple sparing mastectomy-infectious complication risk. A preliminary assessment of the American Society of Breast Surgeons nipple sparing mastectomy registry
Authors: Mitchell SD , Feldman SM , Beitsch PD , Willey SC , Manasseh DME , Unzeitig GW
Published: August 2014

Objectives: The American Society of Breast Surgeons (ASBS) Nipple Sparing Mastectomy Registry (NSMR) is a prospective, IRB approved, non-randomized, multi-institutional registry assessing surgical techniques, utilized metrics, oncologic outcome, and aesthetic outcome of nipple sparing mastectomies. The registry has been open for enrollment for 26 months. The target accrual is 1000 individuals. This abstract specifically analyzes rates of post-operative infection in NSMs.

Methods: Fifty two investigators from 41 institutions are participating in the ASBS NSMR. A total of 373 patients underwent 631 mastectomies, with indications of prophylaxis (365), cancer (248), and unknown (18). We assessed a sub group of 449 mastectomies, with indications of prophylaxis (253) and cancer (196) that had all data sets completed. We assessed infection rates(characterized as needing oral or iv antibiotics with or without removal of implant/expander and/or debridement) in the entire group as well as by indication (cancer vs. prophylaxis). Factors such as smoking history, previous radiation therapy, previous surgery, incision type, reconstruction technique, and flap dissection technique were analyzed.

Results: In a subgroup of 449 mastectomies (253 prophylactic and 196 cancer):

Post operative infections were reported in 22 (4.9%) of patients: 7 (3.6%) of mastectomies with an indication of cancer and 15 (5.9%) of prophylactic mastectomies (p-value 0.3140). Infections were characterized as: treatment with oral antibiotics alone, treatment with i.v. antibiotics alone, iv antibiotics with washout or debridement, or antibiotics and implant/ tissue expander removal. No correlation was found in smoking history (p-value 1.000), previous breast surgery (p-value 0.1277) or previous radiation (p-value 0.6024). No correlation was found in incision utilized, reconstruction technique, or method of flap dissection either.

Conclusion:

The rate of postoperative infections in nipple sparing mastectomies is comparable if not lower than non-nipple sparing mastectomies. No statistically significant difference in infection rate was found between mastectomies completed for prophylaxis or cancer. No correlation was found between factors such as smoking history, history of radiation therapy, prior breast surgery, incision utilized, reconstruction technique, or method of flap dissection. Improved aesthetics with a nipple sparing approach (technically more demanding and typically through a smaller incision) does not come at the cost of a higher rate of infectious complications.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-18-07.
Abstract P2-18-10: High risk of late in-breast tumour recurrence after BRCA1/2-associated breast cancer
Authors: Nilsson M , Hartman L , Kristoffersson U , Olsson H , Borg Å , Henriksson K , Lanke E , Loman N
Published: August 2014

Background: In the general population, partial mastectomy followed by postoperative radiotherapy (PMRT) reduces the risk of local recurrences of breast cancers to a level comparable to the risk after mastectomy (M). However, for BRCA1/2 mutation carriers, a potentially increased risk of a new primary breast cancer in the ipsilateral breast must be taken into account when recommendations about type of surgery are given. To address this question, we conducted a retrospective study on the risk of in-breast tumor recurrence (IBTR) after PMRT in BRCA1/2 mutation carriers.

Methods: Women with invasive breast cancer from families with a pathogenic mutation in BRCA1 or BRCA2 identified at a single institution were included in the study (n = 174). Median age at diagnosis was 43 years and median follow-up was 10.8 years for patients alive at end of follow-up. Patients who were treated with PMRT (n = 45) were compared with patients who had M as primary surgery (n = 118). Endpoints were local recurrence as first failure (LR [IBTR or thoracic wall recurrence]), overall survival (OS), breast cancer-specific survuval (BCSS) and distant disease-free survival (DDFS). LR, OS, BCSS and DDFS were estimated using the Kaplan-Meier method and compared by the log-rank test. For calculation of hazard ratios and for multivariable analysis, the Cox proportional hazards model was used.

Results: In univariate analysis, no difference in OS, BCSS or DDFS was seen between the PMRT and M groups (HR 0.9 [95% CI 0.5-1.4], HR 0.9 [CI 0.5-1.7] and HR 0.8 [CI 0.5-1.5], respectively). In multivariable analysis, adjusting for tumor stage, age and use of adjuvant chemotherapy, the hazard ratios were lower (HR 0.8 [95% CI 0.4-1.3], HR 0.6 [CI 0.3-1.3] and HR 0.6 [CI 0.3-1.1], respectively), but remained inconclusive.

Compared to M, PMRT was associated with an increased risk for LR in univariate analysis (HR 4.0 [CI 1.6-9.8]). In the M group, all local recurrences were seen in the first 5 years. As opposed to this, in the PMRT group the rate of IBTR was rather constant over 15 years of follow-up. The cumulative risk for IBTR in the PMRT group was 19%, 32% and 43% after 5, 10 and 15 years, respectively.

Conclusion: BRCA1/2 mutation carriers treated with PMRT have a high risk for IBTR, many of which are new primary breast cancers. This must be thoroughly discussed with the patient and is an example of how rapid treatment-focused genetic testing already could influence choice of treatment.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-18-10.
Abstract P2-19-05: Oncoplastic breast conserving surgery with perforator flap in breast cancer patients
Authors: Park HY , Kang JG , Choi HH , Hwang SO , Kim WW , Jung JH , Lee YH , Yang JD , Lee SJ
Published: August 2014

Purpose : Oncoplastic breast surgery has been used widely as a treatment protocol for breast cancer. Thoracodorsal artery perforator (TDAP) flap and intercostal artery perforator (ICAP) flap can provide adequate cover without sacrificing any muscle and allow closing of the donor sites in inconspicuous sites. Therefore, among the oncoplastic volume replacement techniques indicated using local flap that can adequately cover the volume of breast, TDAP and ICAP are especially suggested. This study describes the use of TDAP and ICAP flap techniques after breast conserving surgery. Methods : From March 2010 to August 2012, 38 patients with breast cancer received breast reconstruction. All patients who were selected had small- to moderate-sized defects on breasts, middle aged, and were not sensitive to scars. The TDAP flap is the first choice for performing the surgery, but if the perforator of the TDAP flap is not found, a dissection toward the anterior area to find an adequate perforator is made and the serratus anterior artery perforator is normally used. Otherwise after the dissection is performed more anterior, ICAP can be used. If the perforator penetrates the LD muscle, the TDAP flap can be used. The perforator that penetrates from the serratus anterior muscle is also used in the anterior area. Otherwise, ICAP can be used in partial breast reconstruction. The TDAP flap can be applied to any defect site regardless of the size of the defect area. Results : The mean age was 44.9 years and the average follow-up interval was 6 months. The average specimen weight was 98g. Complications developed in 4 cases including 3 cases of venous congestion, though self-limited, and 1 case of wound disruption on the inframammary fold suture area. Majority of the patients were satisfied with the cosmetic result.

Conclusion : Thoracodorsal artery perforator flap (TDAP) and intercostal artery perforator flap (ICAP) techniques can be reliable and useful in correcting breast deformity after breast conservation surgery, especially in patients with small- to moderate-sized defects on breasts.

Key Words : intercostal artery perforator flap, thoracodorsal artery perforator flap Oncoplastic surgery, ICAP, TDAP.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-19-05.
Abstract P3-01-01: Targeting glutamine metabolism in breast cancer for therapy
Authors: Korangath P , Sadik H , Mori N , Wildes F , Zhang Z , Bhujwalla Z , Sukumar S
Published: August 2014

Metabolic reprogramming of cancer cells is observed in different types of tumors including breast. Oncogenic signals aid changes in metabolism that provide selective advantage to the cancer cells to meet their energy requirements to accomplish rapid proliferation. The increased dependence on the glycolytic pathway for energy called the “Warburg effect” was reported by Otto Warburg several decades ago. Our recent understanding of cancer metabolism has thrown light on alternative energy sources, especially glutamine and other branched chain amino acids. The role of glutamine in breast cancer cell growth has not yet been studied extensively.

In this study we found that a number of breast cancer cell lines, especially those negative for ER, PR, HER2, display a high dependence on glutamine for their survival and growth. Interestingly, most of these glutamine-dependent cell lines express high levels of c-myc protein. Consistent with their growth dependency on glutamine, transaminases responsible for entry of glutamine into the tricarboxylic acid cycle are transcriptionally up regulated under low glutamine conditions. Consequently, growth of these cancer cell lines was found to be specifically inhibited by the transaminase inhibitor, amino oxyacetate (AOA). Moreover, the AOA mediated cytotoxic effect was partially c-myc dependent. Through 1H-NMR studies of AOA-treated cells we show that in addition to glutamine, AOA treatment decreases the aspartate and alanine content in the cells. In line with these findings, exogenous supplementation with aspartate partially rescued the cells from the growth inhibitory effects of AOA. Flow cytometry analysis showed that AOA causes cell cycle arrest in the S phase. AOA also had significant inhibitory effect on in vivo growth of rapidly growing SUM 149 and SUM159 xenografts in immunodeficient mice. When combined with chemotherapeutic agents, doxorubicin and carboplatin, AOA inhibited growth of MDA-MB-231 xenograft tumors more effectively than AOA alone. Lastly we present evidence that the cytotoxic effect of AOA is mediated through activation of the ER stress pathway, combined with depletion of key amino acids and likely, a reduction in the nucleoside pool in the cells. Our preclinical studies, both in vitro and in vivo, combined with development of intermediate markers of response, provide a strong rationale for testing AOA for therapy in Phase 0 clinical trials.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-01.
Abstract P3-02-01: Pregnancy-associated plasma protein A regulates mitosis and is epigenetically silenced in breast cancer
Authors: Sainsbury R , Loddo M , Andryszkiewicz J , Widschwendter M , Stoeber K , Williams G
Published: August 2014

Using Phosphohistone-H3, a marker of cells in mitosis, we identified mitotic delay in 95% of invasive breast cancers with a significant proportion of cells in prophase/prometaphase (58%). This delay was seen in breast cancer tissues only with other malignancies and normal tissues showing a normal mitotic phase distribution (23% of mitotic cells in prophase/prometaphase). We could detect a clear mitotic delay phenotype already in 80% (55 out of 69 evaluable patients) of precursor ductal carcinoma in situ (DCIS) lesions. This strong phenotype has been concealed for over a century because tinctorial stains routinely used in diagnostic practice do not allow cells in prophase or prometaphase to be distinguished from interphase cells.

Promoter methylation analysis of candidate genes linked this defect to epigenetic silencing of the gene encoding Pregnancy-Associated Plasma Protein-A (PAPPA), a secreted protease. PAPPA silencing was highly prevalent in precursor lesions and invasive breast cancer. Experimental manipulation of PAPPA protein levels in human mammary epithelial cells and in breast cancer cell lines demonstrates that progression through early mitosis is dependent on PAPPA function, and that breast cancer cells become more invasive after PAPPA down regulation. We discovered that PAPPA regulates mitotic progression through its known role in modulating the IGF-1 signalling pathway, resulting in activation of the forkhead transcription factor FoxM1 which drives a transcriptional cluster of essential mitotic genes.

Our results identify PAPPA as a candidate tumour suppressor in breast cancer. PAPPA loss is an early event in breast tumourigenesis increasing tumour cell invasiveness and may contribute to the transition from precursor lesions to invasive disease.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-02-01.
Abstract P3-03-02: Na+,HCO3--cotransport is the major mechanism of cellular acid extrusion in human and murine breast cancer
Authors: Mele M , Lee S , Moreira J , Vahl P , Wielenga V , Jensen V , Pedersen S , Christiansen P , Aalkjær C , Boedtkjer E
Published: August 2014

High metabolism and insufficient blood supply are characteristics of cancer tissue, which in combination with biochemical changes favor glycolytic metabolism and result in prominent intracellular acid production. Although extracellular pH at the core of malignant tumors is as low as one unit below normal, intracellular pH (pHi) in tumor cells is typically normal or even slightly alkaline. Thus, cancer cells must possess efficient mechanisms of acid extrusion to eliminate the excess acid load.

We investigated the role of the Na+,HCO3–cotransporter NBCn1 (SLC4A7), which in recent genome-wide association studies has been linked to human breast cancer. Based on immunohistochemistry of tumor slices and immunoblotting of enzymatically isolated epithelial organoids, we found that NBCn1 expression is upregulated in human and murine primary breast carcinomas and metastases compared to normal breast tissue. The upregulation of NBCn1 was of similar or greater magnitude than that observed for the Na+/H+-exchanger NHE1, which has previously been implicated in cell migration, proliferation and malignancy. Measurements of pHi from slices of human and murine breast cancers and from malignant and normal breast epithelial organoids showed that Na+,HCO3–cotransport is the major mechanism of acid extrusion in the near-physiological pHi range. Na+/H+-exchange was important for acid extrusion only at very low pHi values. We furthermore found that Na+,HCO3–cotransport activity was substantially greater in malignant compared to normal breast epithelial organoids of both human and murine origin, while no apparent difference in Na+/H+-exchange activity was detected between cancer and normal breast tissue. Steady-state pHi was higher in the breast cancer tissue compared to normal breast epithelium in the presence of CO2/HCO3- but not in its nominal absence.

We propose that NBCn1 plays a major role for cellular acid extrusion and pHi regulation in human and murine breast cancer. The upregulated expression of NBCn1 and the functional importance of Na+,HCO3–cotransport for pHi regulation support a causative role for NBCn1 in breast cancer development or progression.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-02.
Abstract P3-03-05: Modulation of the metastatic propensity of triple-negative breast cancer cells harboring p53 mutations
Authors: Morrison BL , Tai JW , Bernal F
Published: August 2014

The mutation of p53 is implicated in the increased metastatic potential of triple-negative (ER—/PR—/Her2—) breast cancer (TNBC). The low survival rates of advanced metastatic cancer make it critical not only to understand the mechanisms underlying the functional implications of p53 mutations, but also to identify ways to regulate their impact in cancer cell physiology. A common gain-of-function activity of the mutant p53 protein is the enhancement of cell migration and invasion. We have found that a hydrocarbon-stapled p53 transactivation domain peptide (SAH-p53) designed to disrupt the interaction with p53 negative regulators HDM2 and HDMX also has the ability to inhibit the interaction between mutant p53 (mtp53) and HDM2/HDMX. In addition, SAH-p53 was found to inhibit the migration and invasion of MDA-MB-231 cells (mtp53), while exhibiting no alteration in their proliferative ability. The capacity of SAH-p53 to inhibit MDA-MB-231 cell migration was not altered in the absence of HDMX, suggesting an alternative mechanism of action for SAH-p53. The propensity of MDA-MB-231 cells to migrate and invade through Matrigel in response to transforming growth factor b (TGF-β), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF) was decreased by treatment with SAH-p53. Furthermore, increased concentrations of these growth factors displayed a synergistic effect with SAH-p53 in the inhibition of cell migration, suggesting cross-talk between receptor signaling and SAH-p53 activity. This effect was accompanied by an alteration in stress fiber formation and dissolution of focal adhesions. While the knockdown of mutant p53 in MDA-MB-231 cells significantly decreased migration on its own, inhibition of migration by SAH-p53 was not seen in the EGFR/p53-null MDA-MB-157 cells, suggesting a need for either the p53 or EGFR protein for the inhibitory activity of SAH-p53. Moreover, a SAH-p53-induced decrease in the total levels of EGFR and integrin beta1, together with alterations in actin stability, suggests a defect in receptor recycling, a process required for cell migration known to be modulated by an interaction between mutant p53 and p63. These data show that the use of the SAH-p53 peptide provides a novel avenue to study the molecular interactions that drive metastatic behavior originated by p53 gain-of-function mutations.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-05.
Abstract P3-03-12: Gamma-tocotrienol induces autophagy in malignant mammary tumor cells
Authors: Tiwari RV , Parajuli P , Sylvester PW
Published: August 2014

γ-Tocotrienol, a member of the vitamin E family of compounds, displays potent antiproliferative and apoptotic activity in a variety of cancer cell types at treatment doses that have little or no effect on normal cell viability or growth. Autophagy is a tightly regulated lysosomal self-digestive process that can either promote cell survival or be involved in type II cell death. Autophagy is induced in several diseases such as liver disease, neurodegeneration, Crohn's disease, aging, cancer, and metabolic syndrome. Autophagy is accompanied by the progressive development of vesicle structures from autophagomsomes (not acidic) to amphisome and autolysosomes (acidic). The execution and regulation of the autophagic program relies on several highly conserved autophagy-related genes (the Atg genes) that regulate the cannibalism of intracellular cytoplasm, proteins and organelles. The potential role of autophagy in mediating γ-tocotrienol-induced cytotoxicity is not currently understood. Therefore, studies were conducted to determine the effects of γ-tocotrienol on the induction of autophagy in various breast cancer cell lines. Mouse (+SA) and human (MCF-7 and MDA-MD-231) mammary tumor cells lines were exposed for an acute 24 h period to 20 or 40 μM γ-tocotrienol. Results showed that γ-tocotrienol treatment caused a relatively large increase in the accumulation of monodansylcadaverine (MDC)-labeled vacuoles, an early marker of autophagic compartment formation, in all mammary tumor cell lines. These cells also displayed an increased conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I, the cytosolic form) to LC3-II (lapitated form associated with autophagosome), which is associated with the increased formation of autophagic vacuoles, followed by upregulation of Beclin-1. Treatment with γ-tocotrienol also caused a significant increase in the LC3II/LC3I ratio, a characteristic marker of autophagy, as compared to vehicle-treated control groups. Additional flow cytometery analysis showed that γ-tocotrienol treatment caused an increase in acridine orange staining, an indicator of autophagy, in all mammary cancer cell lines. In contrast, similar treatment of γ-tocotrienol on immortalized mouse (CL-S1) and human (MCF10A) normal mammary epithelial cell lines did not show significant accumulation of MDC–labeled vacuoles or the conversion of LC3-I to LC3-II. In addition, γ-tocotrienol-induced autophagy was also associated with a suppression in PI3K/Akt/mTOR signaling and the initiation of apoptosis as indicated by caspase activation in these mammary cancer cells. In summary, these finding suggest that γ-tocotrienol selectively induces autophagy in mouse and human mammary cancer cell lines and support a novel function of autophagy in promoting breast cancer cells death. This study was supported by grants from First Tech International Ltd., and the Malaysian Palm Oil Council.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-12.
Abstract P3-04-01: Discovery of distinct sub-groups of ER positive breast cancers by DNA repair expression profiling: Implications for patient stratification and personalization of therapy
Authors: Madhusudan S , Abdel-Fatah TMA , Russell R , Moseley P , Agarwal D , Mukherjee A , Ball G , Abbotts R , Perry C , Caldas C , Rakha EA , Chan S , Ellis IO
Published: August 2014

Background: The role of DNA repair in estrogen receptor (ER) positive breast cancer is largely unknown. We hypothesized that DNA repair dysregulation could lead to evolution of aggressive phenotypes with increasing resistance to endocrine therapy.

Methods: We immuno-profiled 1120 early stage ER positive breast tumours for Pol b, FEN1, APE1, XRCC1, SMUG1, PARP1, ATR, ATM, DNA-PK, BRCA1, CHK1, CHK2, TOPO2, and p53. Multivariate analysis was used to calculate a DNA repair prognostic index after controlling for other validated prognostic factors, confounders and adjuvant therapy. For additional validation, genomic expression as well as array CGH evaluation of key DNA repair genes was performed [discovery cohort (n = 128), external validation in Uppsala (n = 249) and METABRIC cohort (n = 1950)]. Neural network analysis was conducted in 249 tumours to map genetic interactions.

Results: DNA repair immuno-profiling stratified ER positive breast tumours into four distinct sub-groups with increasing aggressiveness and endocrine resistance (p = 1.6 × 10 −27). Subgroup 1 had the best breast cancer specific survival, subgroups 2 & 3 have intermediate breast cancer specific survival and sub-group 4 had the worst breast cancer specific survival (Table).

Survival analysis HR (95% CI)P ValuesSub-group 2 versus. Sub-group 11.6 (1.1-2.1)0.006Sub-group 3 versus. Sub-group 13.1 (2.3-4.3)1.5 × 10-12Sub-group 4 versus. Sub-group 15.7 (4.0-8.1)9.0 × 10−21

These distinct subgroups manifest progressively increasing tumour size, level of proliferation, de-differentiation, lymphovascular invasion, metastasis, altered apoptosis and increasing level of genomic instability (ps<0.001). At the genomic level DNA repair gene dysregulation remains independently associated with poor outcome and resistance (ps<0.001). Neural network analysis confirms interaction with genes involved in ER response.

Conclusion: This is the first and largest study with 20 years long term follow-up data to provide compelling evidence that DNA repair dysregulation impacts ER positive breast cancer pathogenesis and drives evolution of aggressive phenotypes. Our data could transform stratification and the personalization landscape for ER positive tumours.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-01.
Abstract P3-04-04: Amplification and overexpression of TLK2 promote genomic instability in invasive breast cancer
Authors: Kim J-A , CAO X , Tan Y , Wang X , Schiff R , Wang X
Published: August 2014

The accumulation of numerous chromosomal aberrations is a hallmark characteristic of invasive breast cancer (IBC). It is thus critical to find specific chromosomal aberrations that may drive breast cancer initiation and progression, particularly those that are drivers of the genomic instability. By integrative analysis of genomic data from The Cancer Genome Atlas (TCGA), we have nominated Tousled-Like Kinase 2 (TLK2) as a candidate oncogene target for IBC. Ultra-high resolution copy-number data from TCGA suggest that TLK2 was amplified in approximately 10% of IBC, which are more frequent in luminal B than luminal A tumors. These data also revealed that TLK2-high breast tumors harbor significantly more global copy-number aberrations than the TLK2 low-tumors. TLK2 is a nuclear serine/threonine kinase that serves as a cell cycle checkpoint regulating chromatin assembly, and in response to DNA damage, TLK2 can be inactivated by phosphorylation through the ATM-dependent pathway. We thus postulated that up-regulation of TLK2 may help cancer cells to surpass the cell cycle checkpoint upon DNA damage, which may promote genomic instability in breast cancer cells. To analyze the effect of TLK2 overexpression, we introduced an inducible expression vector containing the TLK2 open-reading frame into the MCF10A benign epithelial cells and the T47D breast cancer cells. As shown by fluorescence activated cell sorting (FACS), ectopic expression of TLK2 in these cells overcame the cell cycle checkpoint in response to the DNA damage induced by gamma irradiation. gamma-H2AX foci formation revealed increased DNA double strand breaks and prolonged DNA repair window after irradiation in the TLK2 overexpressing cells. Interestingly, immunofluorescence localized TLK2 to the nucleus of breast cancer cells, which was markedly enriched in DNA double-strand break foci (or gamma-H2AX foci). To examine the effect of TLK2 knockdown, the MCF7 and MDAMB361 breast cancer cells overexpressing TLK2 were transfected with different TLK2 siRNAs. TLK2 inhibition led to G1/S cell cycle arrest (FACS analysis), profound decrease in cell growth (MTT assay), and enhanced apoptosis (Annexin V assay) in the MCF7 and MDAMB361 breast cancer cells over-expressing TLK2. Together, this study supports the role of TLK2 deregulation in the increased genomic instability of TLK2-high breast cancers, and suggests its potential as an attractive therapeutic target.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-04.
Abstract P3-04-05: Amplification driven expression of KIAA0020, a PARP1 interacting gene, facilitates repair of replication associated DNA damage in triple-negative breast cancers
Authors: Grigoriadis A , Noel E , Weekes D , Shah V , Sidhu B , Gazinska P , Watkins J , Marra P , Tutt A
Published: August 2014

Approximately 25% of triple-negative breast cancers display increased copy number in the region of 9p24. Since several genes mapping to this genomic locus have been shown to be involved in the biology of triple-negative breast cancers, a copy number-dependent transcriptional influence of genes at 9p24 on the malignant phenotypes has been suggested. Given the limited options of targeted therapy for triple-negative breast cancer patients, further studies of genes located at this locus could therefore provide potential novel targets or companion biomarkers for this disease.

By integrating microarray-based DNA copy number and gene expression of genes located on 9p24 in 111 triple-negative breast cancers, we identified among a wider set of genes three hypothetical genes, KIAA0020, KIAA1432 and KIAA2026 whose expression was highly correlated with their copy number status. External validation through analysis of several comprehensive breast cancer cohorts, confirmed KIAA0020 as being highly abundant in basal-like/ triple-negative breast cancers. Involvement of KIAA0020 in PARP1 activity and the DNA damage response has previously been suggested. To elucidate KIAA0020's functional involvement in cell growth, cell cycle progression, apoptosis and DNA damage response in this subtype of breast cancer, breast cancer cell lines with and without increased 9p24 copy number levels were used as in vitro models. In cell lines with increased DNA copy number at 9p24, depletion of KIAA0020 expression selectively impaired growth causing an accumulation in S-phase and a decrease in cell proliferation. Furthermore, KIAA0020-silencing in such cell lines resulted in decreased repair of hydroxyurea induced replication associated DNA damage, accumulation of DNA double strand breaks and decreased occurrence of RAD51 and PAR foci, all pointing to a decreased repair of inactivated replication forks.

Taken together, our data supports the notion that genes of unknown function, residing in the 9p24 copy number aberrant region are involved in DNA repair and may thereby also contribute to the tumourigenesis of triple-negative breast cancers. A subset of such triple-negative tumours seemed to have developed a dependency on the expression of KIAA0020, a PARP1 interacting gene, for replication fork associated repair. Thus, greater understanding of KIAA0020's molecular function may provide additional information for patient selection with regards to DNA damaging chemotherapeutics or PARP inhibitors within triple-negative breast cancers.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-05.
Abstract P3-04-06: Damage-induced BRCA1 phosphorylation contributes to the timing of end resection
Authors: Parameswaran B , Chiang H-C , Lu Y , Hu Y
Published: August 2014

Germline mutations of BRCA1 predispose women to breast and ovarian cancers. BRCA1 functions as a tumor suppressor. A wealth of evidence has established that BRCA1 plays an important role in DNA repair and cell cycle checkpoint control. Efficient repair of double-strand-breaks (DSBs) by BRCA1 is essential to its tumor suppression function. DSBs can be repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ). BRCA1 is hyperphosphorylated upon DSBs. Although phosphorylation of BRCA1 is among the earliest events documented upon DNA damage, the role of BRCA1 phosphorylation in DNA repair remains unclear after more than a decade. It has been proposed that BRCA1 serves as a scaffolding protein that recruits multiple repair proteins to the break sites. One of the BRCA1-containing complexes, MRN (Mre11-Rad50-Nbs1)-BRCA1-CtIP, promotes HR repair by initiating DNA end-resection, the first step in the HR pathway. Mre11 possesses intrinsic nuclease activity, which likely facilitates initial processing of DNA ends for subsequent extensive end-resection. Believed to stimulate the nuclease activity of the MRN complex, CtIP is recruited to the complex by BRCA1.

In spite of these useful molecular models, the precise function of BRCA1 in DSB repair, especially the role of its phosphorylation, remains elusive. BRCA1 is phosphorylated by Chk2 kinase upon g-irradiation at serine 988 (human) or serine 971 (mouse). Our data shows that a point mutation in mouse BRCA1 (S971A) that abolishes the Chk2 phosphorylation site impairs end-resection after DSBs. The S971A mutation does not appear to affect the assembly of MRN and CtIP at DSBs, but specifically delays dispersion of BRCA1 from DSBs. Interestingly, BRCA1 is ubiquitinated and degraded by SCFSkp2, and loss of Chk2 phosphorylation at S988 of human BRCA1 impairs its ubiquitination. Previous work has revealed BRCA1 as a scaffold protein for the assembly of HR proteins at DSBs. Paradoxically, BRCA1 also inhibits Mre11 nuclease activity. Our study suggests that BRCA1 plays a dual role in HR. Although BRCA1 is required for initial assembly of the MRN-BRCA1-CtIP complex at DSBs, the exit of BRCA1 from the MRN-BRCA1-CtIP complex is required to relieve its inhibitory effect on Mre11 nuclease activity. Mechanistically, this critical “exit” of BRCA1 from the MRN-BRCA1-CtIP complex is regulated through phosphorylation catalyzed by Chk2 and then degraded by SCFSkp2, which is expressed at S/G2 phases, thus ensuring the initiation of end resection/HR to S/G2 phases of the cell cycle. Our study explains two seemingly paradoxical observations: BRCA1 is required for assembly of the MRN-BRCA1-CtIP complex, but it inhibits Mre11 nuclease activity required for end-resection.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-06.