G. Di

Inhibition of EGFR pathway signaling and the metastatic potential of breast cancer cells by PA-MSHA mediated by type 1 fimbriae via a mannose-dependent manner

To identify more therapeutic targets and clarify the detailed mechanisms of Pseudomonas aeruginosa-mannose-sensitive hemagglutinin (PA-MSHA) on breast cancer cells both in vitro and in vivo. PA-MSHA was administered to epidermal growth factor receptor (EGFR)-positive human breast cancer cell lines MDA-MB-231HM and MDA-MB-468 in vitro and to mice bearing tumor xenografts. The mannose cocultured test was used to detect the effect of mannose on PA-MSHA-induced cell proliferation, cell cycle arrest, apoptosis, and EGFR pathway signaling. We found that cells stimulated with PA-MSHA exhibited a downregulation of EGFR signaling. The addition of mannose partially inhibited the PA-MSHA-stimulated cell anti-proliferative effect, cell apoptosis, cell cycle arrest, activation of apoptosis-associated caspases, and even downregulation of the EGFR signaling pathway. In vivo, PA-MSHA treatment significantly suppressed mammary tumorigenesis in xenografts in mice and decreased lung metastasis in MDA-MB-231HM cell-transplanted mice. Tumor sample analyses confirmed inhibition of the EGFR pathway in the PA-MSHA-treated mice. In conclusion, this study showed that the involvement of the mannose-mediated EGFR pathway has a critical function in the preclinical rationale for the development of PA-MSHA for the treatment of human breast cancer. It also suggests the potentially beneficial use of PA-MSHA in adjuvant therapy for breast tumors with EGFR overexpression.

Treatment outcomes and unfavorable prognostic factors in patients with occult breast cancer.

AIMS The purpose of this study was to evaluate the treatment outcomes and prognostic factors in patients with occult breast cancer (OBC). METHODS We retrospectively analyzed 95 patients with OBC who were treated at our facility between January 1998 and June 2010. Of the 95 patients, 64 underwent mastectomy plus axillary lymph node dissection (ALND) with or without post-mastectomy radiation (Mast + ALND group), 13 underwent ALND followed by ipsilateral breast radiotherapy (BR + ALND group) and the remaining 18 were treated with ALND (ALND group). RESULTS Patients who underwent Mast + ALND or BR + ALND had significantly improved rates of locoregional recurrence-free survival (LRFS) and recurrence/metastasis-free survival (RFS) than patients who only underwent ALND (p < 0.05). There were no significant differences in the LRFS (p = 0.718), RFS (p = 0.935) and breast cancer-specific survival (BCSS) (p = 0.991) rates between the patients who underwent Mast + ALND compared with those who received BR + ALND. Multivariate analysis revealed that patients with four or more involved lymph nodes had significantly worse outcomes (p = 0.042, HR = 4.63, 95% CI = 1.66-32.47 for BCSS and p = 0.038, HR = 3.62, 95% CI = 1.08-20.77 for RFS). CONCLUSIONS Patients with OBC who received ALND and subsequent breast radiotherapy had similar outcomes to patients who underwent mastectomy. The presence of four or more involved lymph nodes may independently predict poor outcomes of OBC.

Diffusion-Weighted MRI in Predicting the Efficacy of Neoadjuvant Chemotherapy of Breast Cancer.

Objective: To approach the apparent diffusion coefficient(ADC) obtained on diffusion weighted imaging (DWI) in predicting the response of breast cancer to neoadjuvant chemotherapy. Methods: 53 locally advanced breast cancer patients participated in this prospective study on neoadjuvant chemotherapy. All patients were treated with weekly neoadjuvant paclitaxel plus carboplatin for 4 cycles. DWI and ADC value were examined before chemotherapy(the first time point), after the first and the forth cycle of chemotherapy(the second and the third time point). Patients were categorized as high responders if their ADC at the second time point were higher by 2 times the standard deviation than the mean pre-chemotherapy ADC, and otherwise the patients were considered as low responders. Logistic regression was conducted to examine the independent factors predictive of clinical complete response(CR) and pathologic complete response(pCR). Results: CR was documented in 13 patients (24.5%); partial response in 36 patients (67.9%); stable disease in 4 patients (7.5%) and no patient had progressive disease. 11 patients(20.8%) have achieved pCR. There was a strong positive correlation between the percentage changes in ADC value at the third time points and the degree of the tumor reduction. A significant increase in ADC value was observed at the second time point compared with the first time point (1.104±0.117×10 -3 mm 2 /s, 1.220±0.158×10 -3 mm 2 /s, P P >0.05). Logistic regression revealed that percentage changes of ADC value at the second time point were significantly associated with CR( P =0.022, hazard ratio[HR] =1.08, 95% confidence interval[CI]: 1.01∼1.16) and pCR( P =0.022, HR =1.11, 95%CI: 1.02∼1.21). The AUC value of the receiver operating characteristic curve were 0.721(95%CI: 0.512∼0.931, P =0.017) and 0.775(95%CI: 0.598∼0.952, P =0.005) in the two diagnostic tests for CR and pCR, and the sensitivity and specificity were 76.9%, 63.6% and 77.5%, 90.5%, respectively. According to categorizing system described above, 10 patients(18.9%) were considered as high responders and 43 patients(81.1%) low responders. 7 patients(70%) of the high responders have achieved clinical CR and pCR while for the low responders there were only 6 patients(14.0%) and 4 patients (9.3%) proven to have CR and pCR. The difference have both reached statistical significance( P =0.001, P P =0.006, HR=42.26, 95%CI: 2.86∼624.08) and pCR( P =0.013, HR=25.19, 95%CI: 2.00∼318.91). Taking this categorizing system as a dignostic test, the sensitivity and specificity were 53.8% and 92.5% for CR and 63.6% and 92.9% for pCR, respectively. Conclusions: The change of ADC value obtained on DWI after the first cycle of neoadjuvant chemotherapy occured prior to the morphologic changes of the tumor and at this time ADC value could to some extent predict the efficacy of neoadjuvant chemotherapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4025.

Abstract P1-14-18: Postmastectomy radiotherapy improves the outcomes of stage III breast cancer patients with negative lymph nodes after neoadjuvant chemotherapy

Purpose: The aim of this study is to evaluate the role of postmastectomy radiotherapy (PMRT) in clinical stage II and III breast cancer patients who achieved negative node status (pN0) after neoadjuvant chemotherapy (NAC). Material and methods: We retrospectively analyzed the outcomes of 143 patients with pN0 after NAC and mastectomy at Fudan University Shanghai Cancer Center. In total, 103 (72%) patients received PMRT, and 40 (28%) patients did not. Univariate and multivariate survival analyses were performed to evaluate the effect of PMRT on locoregional recurrence-free survival (LRRFS) and overall survival (OS) of the two groups. Results: There were no differences between the two groups with respect to age, nuclear grade, estrogen receptor (ER) status, HER2/neu receptor status, lymphovascular space invasion (LVSI) status or pathological tumor size. However, a significantly higher proportion of patients in the irradiated group (64%) had clinical lymph node involvement than in the nonirradiated group (45%). After a median follow-up time of 49 months, 10 locoregional recurrence events occurred. For the entire cohort of patients, use of radiation therapy improved the 5-year LRRFS rate (94.5% vs. 80.2%; P =0.032) but not the 5-year OS rate (92.2% vs. 88.7%; P =0.617). In the subset of patients who presented with clinically stage II disease, the 5-year LRRFS and 5-year OS did not differ significantly between the PMRT and no-PMRT group (96.3% vs. 91.3%; P = 0.190 and 96.2% vs. 91.3%; P = 0.199, respectively). For patients with stage III disease at diagnosis, a trend was seen toward better local regional control with PMRT (the 5-year LRRFS rate was 92.7% vs. 64.2%; P = 0.063), although the benefit from radiation with respect to OS was not significant (5-year OS rate was 88.1% vs. 85.2%; P = 0.657). On multivariate Cox regression analyses, the clinical tumor size (hazard ratio [HR], 3.27; 95% confidence interval [CI], 1.05-10.18; P = 0.041), pathologic breast tumor response (HR, 1.82; 95% CI, 1,11-3.77; P = 0.046) and delivery of radiation therapy (HR, 1.27; 95% CI, 1.08-9.25; P = 0.047) were independent predictors of locoregional recurrence. Conclusions: For patients who achieved pN0 after NAC, PMRT seemed to provide a clinical benefit for breast cancer patients with stage III disease. Omission of PMRT in patients with stage II disease did not increase the risk of locoregional recurrence and death. Citation Format: He M, Li J, Ni X-J, Chen S, Jiang Y-Z, Di G-H, Shao Z-M. Postmastectomy radiotherapy improves the outcomes of stage III breast cancer patients with negative lymph nodes after neoadjuvant chemotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-14-18.

Abstract P6-04-04: Comprehensive transcriptome analysis identifies novel molecular subtypes and subtype-specific lncRNAs of triple-negative breast cancer

Background: Triple-negative breast cancer (TNBC) is a highly heterogeneous group of cancers with no effective therapeutic targets hitherto. Thus molecular subtyping is necessary to better identify molecular-based therapies. While some classifiers have been established, no one has integrated the expression profiles of long-noncoding RNAs (lncRNAs) into such subtyping criterions. Considering the emerging important role of lncRNAs in gene regulation and other cellular processes, a novel classification integrating the transcriptome profiles of both messenger RNA (mRNA) and lncRNA would help us better understand the heterogeneity of TNBC and treat patients accordingly. Methods: Using human transcriptome microarray, we retrieved the transcriptome profiles of 165 consecutive TNBC samples. We used k-means clustering to classify the samples based on the most differentially expressed genes (standard deviation>0.65). Empirical cumulative distribution function was analyzed to determine the optimal number of subtypes. Then the new classifier was compared with the Lehmann/Pietenpol system, and survival analyses were performed to compare the recurrence-free survival (RFS) in different subtypes. Gene Ontology (GO) and pathway analyses were applied to determine the main function of the subtype-specific genes and pathways. We conducted co-expression network analysis to identify interactions between lncRNAs and mRNAs, and to predict possible functions of subtype specific lncRNAs. Results: All 165 TNBC tumors were classified into four distinct clusters, each displaying unique GOs and pathways. These include an immunomodulatory (IM) subtype, a luminal androgen receptor (LAR) subtype, a mesenchymal-like (MES) subtype and a basal-like and immune suppressed (BLIS) subtype, accounting for 17.0%, 17.6%, 33.3%, and 32.2% of the patients, respectively. The IM subtype had unique GOs and pathways involving immune cell process. The LAR subtype was highly enriched in hormonally regulated pathways. Enriched pathways in the MES subtype included ECM-receptor interaction, focal adhesion, and processes linked to growth factor signaling pathways. The BLIS subtype was characterized by downregulation of immune response gene and activation of cell cycle and DNA repair, and patients in this subtype experienced worse RFS compared to other subtypes (log-rank test,P=0.045), which was in concordance with the highly proliferative and immune-suppressed nature of these tumors. When analyzing the distribution of the Lehmann/Pietenpol subtypes in our classification system, we found that the two classification systems were significantly correlated (P=0.039). However, our novel classification was more concise and significantly connected with survival outcome. Subtype-specific lncRNAs were identified and their possible functions were predicted using co-expression network analysis. Conclusions: We developed a novel TNBC classification system integrating the expression profiles of both mRNAs and lncRNAs, and determined subtype-specific lncRNAs that are potential biomarkers and targets of TNBC. If further validated in larger population, our novel classification system could facilitate patient counseling and individualize treatment of TNBC. Citation Format: Liu Y-R, Jiang Y-Z, Xu X-E, Zuo W-J, Yu K-D, Jin X, Hu X, Wu J, Liu G-Y, Di G-H, Shao Z-M. Comprehensive transcriptome analysis identifies novel molecular subtypes and subtype-specific lncRNAs of triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-04.

Abstract P2-07-01: Polymorphism A247S of XRCC4 contributes to the risk of breast cancer and impairs DNA damage repair pathway

Genetic variations in a network of genes involved in DNA double strand breaks (DSB) repair pathway have been shown to increase the risk of breast cancer. XRCC4 is identified as a pivotal modulator in non-homologous end joining (NHEJ), a predominant DSB repair pathway. In this study, we evaluated the associations between single nucleotide polymorphisms (SNPs) in XRCC4 and breast cancer, and investigated the impact of XRCC4A247S variant on nuclear localization and DNA damage response functions of XRCC4. A hospital-based case-control study, comprising 412 breast cancer patients and 458 cancer-free controls recruited from Shanghai Cancer Center, was conducted to investigate the relationship between common polymorphisms in XRCC4 and the risk of breast cancer. Our results showed that XRCC4 A247S polymorphism (rs3734091), located near the putative nuclear localization signal (NLS) of XRCC4, was associated with a significantly increased risk of breast cancer in a recessive genetic model (Adjusted odds ratio (OR) = 1.47, 95% confidence interval (CI) = 1.09-4.52; P = 0.042). This homogeneous variant was more often observed in triple negative subtype (Adjusted OR = 2.07, 95% CI = 1.34 - 3.58 in the recessive model; P = 0.036). Although XRCC4 A247S variant did not harm its interactions with XLF and ligase IV (the other two components of DNA ligase complex), we revealed that A247S alteration partially impaired the nuclear localization of XRCC4 in mammary cells. Remarkably, more evidences implied that XRCC4 A247S variant significantly reduced the in vivo NHEJ repair efficiency compared to the wild-type XRCC4 via colony formation assay and NHEJ reporter assay, suggesting that reduced nuclear accumulation by XRCC4 A247S variant impaired its functions in response to DNA damage. Importantly, XRCC4 A247S alternation promoted cellular sensitivity to ionizing radiation and DNA damaging agents in chemotherapy. Taken together, this study demonstrates that genetic variation of XRCC4 A247S could impair NHEJ mediated DSB repair and provide further evidences that genetic variants involved in DNA repair pathways contribute to breast cancer susceptibility. In addition, our results also indicate that XRCC4 A247S variant might be a potential predictive biomarker and therapeutic target in breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-01.

Abstract P2-14-04: Effect of age at the time of diagnosis on overall and cancer-specific mortality in breast cancer: Analysis of SEER, 1990-2008

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 P2-07-06: Detection of germline mutations in 196 genes in Chinese breast cancer families using targeted capture and next-generation sequencing

Background : Germline mutations in BRCA1, BRCA2 and other genes predispose to high risks of breast cancer. Genetic testing for germline mutations in BRCA1 and BRCA2 has been widely used in women with severe family history of breast or ovarian cancer in many western countries. However, in China, hereditary breast cancer associated genes and their mutation types remain unclear. So far, theres no recommended guidelines for high-risk populations. Next-Generation Sequencing (NGS) technology has been proved to detect all classes of mutations cost-effectively. Here we describe the first study of germline mutations in 196 genes in Chinese breast cancer families using NGS. Methods : Breast cancer patients were consecutively enrolled at diagnosis in our hospital between 2011-2012. Inclusion criteria were as follows: Firstly, breast cancer patients are Shanghai natives; secondly, breast cancer patients aged 35 or younger had at least another family member with any type of cancer or those between 35 and 50 years had ≥2 family members with cancer on same side of family or those above 50 years had ≥3 family members with cancer on same side of family. A total of 196 genes were tested and they included two parts. One hundred and twenty-two genes selected were associated with 54 hereditary cancer syndromes and another 74 genes were cancer susceptibility genes proved by researches published on Pubmed up to 2012. Blood samples were collected and genomic DNA were subjected to targeted capture using the Agilent SureSelect DNA kit and sequencing of exons and flanking regions corresponding to 196 genes using the Illumina HiSeq 2000 platform. All classes of genomic alterations were detected. The study was approved by the Scientific and Ethical Committee of the Cancer Hospital of Fudan University. All participants provided informed consent. Results : Eighty-six breast cancer patients met our inclusion criteria were enrolled in our study. The 86 families totally harbored 456 cancers, mainly including 206(45.2%) breast cancers, 45(9.9%) colorectal carcinomas, 38(8.3%) lung cancers, 36(7.9%) gastric cancers, 28(6.1%) hepatic carcinomas and 17(3.7%) ovarian cancers. A total of 66 germline mutations were detected in 29 genes of 46 probands, including 18 BRCA1 or BRCA2 mutations and one recurrent mutation in each gene, one novel TP53 mutations, one germline mutation in RAD50, PALB2, FANCD2, FANCI, RAD51C, SLX4/FANCP, etc. All of these genes were previously reported in hereditary breast cancer. Of women with germline mutations, the mean age at first primary breast cancer diagnosis was 39.6 y and 10(21.7%) of them had bilateral breast cancer. As for all genomic rearrangements, bioinformatics analysis is in process. Conclusion: This is the first study to report the spectrum of germline mutations in 196 hereditary cancer associated genes in Chinese familial breast cancer patients regardless of cancer types of family members. It will give us a reference for carrying out genetic counseling in Chinese breast cancer families. At the same time, NGS is an effective method to test many genes simultaneously at low cost. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-06.

Abstract P3-01-05: The Role of Mammographic Calcification in the Neo-adjuvant Therapy of Breast Cancer Imaging Evaluation

Aim: To evaluate the relationship between malignant appearance of mammographic calcification and breast cancer pathological features, and to explore the predictive value of calcification appearances and morphology changes in neo-adjuvant setting. Methods: 419 patients with operable breast cancer received neo-adjuvant therapy between 2008.2 and 2011.8 in Shanghai Cancer Hospital. Core needle biopsy was conducted before preoperative therapy to determine cancer and achieve pathological features. Mammogram (MG), ultrasound and breast MRI were routinely done prior to therapy and prior to surgical operation. We conducted a detailed analysis of MG images in patients with malignant calcification, recorded the morphology, distribution, range, density, diameter and number of the calcification. Results: 419 patients enrolled, 108 patients (25.8%) showed malignant calcification in MG, 6 patients missed the first MG before therapy. 214 patients were Luminal A, 95 were Luminal B, 64 were Her2 positive and 46 were triple negative, the pCR rate was 14%, 30.5%, 53%, 43.5% respectively. Patients with malignant calcification have more ER positive (81.5% vs. 71.7%, p = 0.045) and HER2 positive (51.8% vs. 33%, p = 0.001) diseases. The pCR rate was 26% in patients with malignant calcification and 28% in patients without, p = 0.8. Different morphology shapes showed similar pCR rate, p = 0.89. Casting-type had a higher pCR rate 45.8%, compared with 20% in crushed stone-like and 16.7% in powderish, p = 0.031. Range more than 5cm had a higher pCR rate, 40.7% vs. 20%, p = 0.034. Density, diameter and number of the calcification did not reach statistical difference, however high density, diameter >1mm and number >20 per cm 2 showed a trend of higher pCR rate. Patients with diameter ≤0.5mm had a higher lymphatic vascular invasive rate 51.4%, compared to diameter≤1mm (26.8%) and diameter >1mm (22.7%), p = 0.03. Morphology and distribution of calcification did not change obviously. Less than 30% patients showed changes in range, number or density, no relationship with pCR rate. Conclusion: Patients with malignant calcification are more likely to have ER positive and Her2 positive diseases. MG should be considered the standard prior to the start of therapy, the distribution and range of the calcification may predict pCR rate. Calcification appearance does not change significantly after neo-adjuvant therapy, therefore MG is not an appropriate method for efficacy evaluation. But MG before surgery is still useful to identify the extent of surgery, especially in breast conserve therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-01-05.

P5-13-13: The Role of Topoisomerase IIa in Predicting Sensitivity to Anthracyclines in Breast Cancer Patients: A Meta-Analysis of Published Literatures.

Topoisomerase IIα is not only a proliferation marker of tumor cells but also a target for anthracycline-based chemotherapy. Both in vitro and in vivo studies have shown that there was a relationship between topo IIα and chemosensitivity to anthracyclines, but the predictive role of topo IIα is still controversial in breast cancer patients. A meta-analysis based on published studies was performed with the aim of obtaining an accurate evaluation of the association between topo IIα and sensitivity to anthracycline-based chemotherapy. A total of 13 eligible studies including 2,633 cases and 2,118 controls were identified. Topo IIα was associated with sensitivity to anthracyclines in locally advanced breast cancer patients who received neoadjuvant chemotherapy (RR = 1.93, 95%CI: 1.27−2.94, P=0.002; RR =1.98, 95%CI: 1.37−2.86, P Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-13.