Breast Cancer Subpopulations Research Articles

Amplification and high-level expression of heat shock protein 90 marks aggressive phenotypes of human epidermal growth factor receptor 2 negative breast cancer

IntroductionAlthough human epidermal growth factor receptor 2 (HER2) positive or estrogen receptor (ER) positive breast cancers are treated with clinically validated anti-HER2 or anti-estrogen therapies, intrinsic and acquired resistance to these therapies appears in a substantial proportion of breast cancer patients and new therapies are needed. Identification of additional molecular factors, especially those characterized by aggressive behavior and poor prognosis, could prioritize interventional opportunities to improve the diagnosis and treatment of breast cancer.MethodsWe compiled a collection of 4,010 breast tumor gene expression data derived from 23 datasets that have been posted on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. We performed a genome-scale survival analysis using Cox-regression survival analyses, and validated using Kaplan-Meier Estimates survival and Cox Proportional-Hazards Regression survival analyses. We conducted a genome-scale analysis of chromosome alteration using 481 breast cancer samples obtained from The Cancer Genome Atlas (TCGA), from which combined expression and copy number data were available. We assessed the correlation between somatic copy number alterations and gene expression using analysis of variance (ANOVA).ResultsIncreased expression of each of the heat shock protein (HSP) 90 isoforms, as well as HSP transcriptional factor 1 (HSF1), was correlated with poor prognosis in different subtypes of breast cancer. High-level expression of HSP90AA1 and HSP90AB1, two cytoplasmic HSP90 isoforms, was driven by chromosome coding region amplifications and were independent factors that led to death from breast cancer among patients with triple-negative (TNBC) and HER2-/ER+ subtypes, respectively. Furthermore, amplification of HSF1 was correlated with higher HSP90AA1 and HSP90AB1 mRNA expression among the breast cancer cells without amplifications of these two genes. A collection of HSP90AA1, HSP90AB1 and HSF1 amplifications defined a subpopulation of breast cancer with up-regulated HSP90 gene expression, and up-regulated HSP90 expression independently elevated the risk of recurrence of TNBC and poor prognosis of HER2-/ER+ breast cancer.ConclusionsUp-regulated HSP90 mRNA expression represents a confluence of genomic vulnerability that renders HER2 negative breast cancers more aggressive, resulting in poor prognosis. Targeting breast cancer with up-regulated HSP90 may potentially improve the effectiveness of clinical intervention in this disease.

Characterization and Clinical Evaluation of CD10+ Stroma Cells in the Breast Cancer Microenvironment

There is growing evidence that interaction between stromal and tumor cells is pivotal in breast cancer progression and response to therapy. Based on earlier research suggesting that during breast cancer progression, striking changes occur in CD10(+) stromal cells, we aimed to better characterize this cell population and its clinical relevance. We developed a CD10(+) stroma gene expression signature (using HG U133 Plus 2.0) on the basis of the comparison of CD10 cells isolated from tumoral (n = 28) and normal (n = 3) breast tissue. We further characterized the CD10(+) cells by coculture experiments of representative breast cancer cell lines with the different CD10(+) stromal cell types (fibroblasts, myoepithelial, and mesenchymal stem cells). We then evaluated its clinical relevance in terms of in situ to invasive progression, invasive breast cancer prognosis, and prediction of efficacy of chemotherapy using publicly available data sets. This 12-gene CD10(+) stroma signature includes, among others, genes involved in matrix remodeling (MMP11, MMP13, and COL10A1) and genes related to osteoblast differentiation (periostin). The coculture experiments showed that all 3 CD10(+) cell types contribute to the CD10(+) stroma signature, although mesenchymal stem cells have the highest CD10(+) stroma signature score. Of interest, this signature showed an important role in differentiating in situ from invasive breast cancer, in prognosis of the HER2(+) subpopulation of breast cancer only, and potentially in nonresponse to chemotherapy for those patients. Our results highlight the importance of CD10(+) cells in breast cancer prognosis and efficacy of chemotherapy, particularly within the HER2(+) breast cancer disease.

Impact of 5-HT3 RA selection within triple antiemetic regimens on uncontrolled highly emetogenic chemotherapy-induced nausea/vomiting

Background: It is recommended that patients initiate triple antiemetic therapy with one of the 5-hydroxytryptamine receptor antagonists (5-HT3 RAs), aprepitant (or its intravenous prodrug fosaprepitant) and dexamethasone prior to the start of highly emetogenic chemotherapy (HEC). However, the impact of 5-HT3 RA selection within triple antiemetic regimens on the risk of uncontrolled chemotherapy-induced nausea and vomiting (CINV) with HEC has not been well studied. Aim: To assess the likelihood of an uncontrolled CINV event following antiemetic prophylaxis with the 5-HT3 RA palonosetron + aprepitant/fosaprepitant + dexamethasone (palonosetron cohort) versus any of the other 5-HT3 RAs + aprepitant/fosaprepitant + dexamethasone (other 5-HT3 RA cohort) among single-day HEC cycles. Methods: Single-day HEC cycles (a gap of at least 5 days between two administrations) among patients with a cancer diagnosis and receiving antiemetic prophylaxis with the aforementioned regimens between 1/1/2006 and 6/30/2010 were identified from the IMS LifeLink claims database. Uncontrolled CINV events were identified through ICD-9-CM codes (nausea and vomiting), Current Procedural Terminology codes (hydration), rescue medications and/or use of antiemetic therapy from days 2–5 following HEC administration. Risks for an uncontrolled CINV event among all patients, and within breast cancer and multiple cancer subpopulations, were analyzed at cycle level using logistic multivariate regression models. Results: A total of 8018 cycles for the palonosetron cohort and 1926 cycles for the other 5-HT3 RA cohort (3574 and 978 patients, respectively) were analyzed. Single-day HEC cycles received by the palonosetron cohort had a significantly lower unadjusted risk of an uncontrolled CINV event (17.5 vs 20.7% for the other 5-HT3 RA cohort; p = 0.0010), with a 17% lower adjusted risk for palonosetron-administered cycles (odds ratio: 0.83; 95% CI: 0.73–0.94; p = 0.0042). Results in the breast cancer and multiple cancer subgroups were consistent with those for the overall population. Conclusion: In this retrospective claims data analysis, single-day HEC cycles administered with palonosetron + aprepitant/fosaprepitant + dexamethasone had a lower risk for an uncontrolled CINV event versus other 5-HT3 RAs + aprepitant/fosaprepitant + dexamethasone.

Abstract LB-311: Combinatorial logic over breast cancer control modules predicts survival and chemotherapy response of ISPY1 breast cancer patients (CALGB 150007/150012; ACRIN 6657)

Abstract Background: Breast cancer is a heterogeneous disease. Clinically useful predictors for breast cancer management may therefore need to consider the variety of molecular processes that can impact chemotherapy response and survival. With the growing database of gene expression data, it is now possible to deduce co-expression modules active in breast cancer subpopulations. Viewed from a combinatorial perspective supporting ‘OR’ and ‘AND’ reasoning, the activities of these modules might provide insights into breast cancer biology and form the basis for improved predictors. Methods: We assembled 74 breast-cancer related gene expression datasets containing ∼5,500 samples altogether. Per dataset, we identified genes with bimodal expression analyzed using mixture-model clustering to ultimately find gene groups that are consistently co-regulated across multiple datasets. We scored the AFFY U133 ISPY1 dataset (117 pts) for module expression, and explored the relationships between module state, chemotherapy response, patient outcome, receptor status, intrinsic subtype, and other signatures. To derive logical functions consisting of nested ‘AND’ and ‘OR’ gates relating module expression to clinical variables, we discretized the module scores and applied a methodology in the spirit of Karnaugh maps for digital circuit design. Results: Our meta-analysis identified 11 modules ranging in size from ∼5–200 genes. The expression level of some modules were associated with known molecular markers such as intrinsic subtype or wound signature scores, whereas others – three immune related modules, two ECM related modules, a mixed immune/ECM module, and a small module encoding histones – do not. Disease free survival of patients with triple negative disease is predicted (p=1.93e-08) by the logical function fDFS_TN=[ER_module>LOW] OR [TN_Immune_ECM=HIGH] OR [(AURK/PROLIF_module=HIGH) AND ((Histone_module>LOW) OR (TN_Immune_ECM=HIGH)], where TN_Immune_ECM is a composite function over the three immune modules and the immune/ECM hybrid module. The best predictor of chemo-sensitivity for triple negatives is a hybrid over subtype and module state, fRCB01_TN=Subtype_basal AND TN_immune_ECM, correctly classifying 95% of the RCB01 sensitives and 90% of the RCBIII resistants. Conclusion: In triple negatives, high proliferation can be rescued by immune/ECM upregulation, though neither immune rescue nor low RCB is necessary for survival if the estrogen module is even slightly upregulated or if the histone module is high. Similar analyses of ER/Pr+ and Her2+ patients demonstrate receptor-subtype specificity in the logic predicting response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-311. doi:10.1158/1538-7445.AM2011-LB-311

Abstract 1062: Identification of flotilin-1 as an H-Ras-interacting lipid raft protein: Its implications in breast epithelial cell invasion

Abstract Metastatic spread is the major cause of death from breast cancer. We previously showed that H-Ras, but not N-Ras, induces invasive/migratory phenotypes of MCF10A human breast epithelial cells, while both H-Ras and N-Ras induce proliferation/transformation. Here, we identified flotillin-1 as an H-Ras-induced lipid raft protein through comparative proteome profiling of lipid raft proteins. Flotillin-1 interacts with H-Ras in lipid raft at a higher affinity than with N-Ras, elucidating a molecular nature of the crucial link between lipid rafts and the function of H-Ras. Small interfering RNA (siRNA)-mediated flotillin-1 knockdown significantly reduced H-Ras activation and H-Ras-mediated motility/invasion in MCF10A cells engineered to express H-Ras and in highly invasive human breast carcinoma Hs578T cells that express endogenous H-Ras. These results suggest a positive signal amplification loop between flotillin-1 and H-Ras for the invasive signaling program in breast epithelial cells. Importantly, tissue microarrays of 289 patients with invasive breast cancer revealed that flotillin-1 expression positively correlates with lymph node metastases (p=0.027), HER2/neu oncogene expression (p<0.001), and high histologic grade (p=0.014). Although infrequent, the membranous flotillin-1 expression is significantly associated with poor disease-free survival of patients (p=0.005), suggesting the clinical importance of predicting the characteristics of a small subpopulation of malignant breast cancer. Taken together, our findings provide new insight into the molecular basis of the Ras isoform-specific signaling mechanism that depends on the lipid-based sorting platforms. [This work was supported by the Korea government (Nos.ROA-2008-000-20070-0)] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1062. doi:10.1158/1538-7445.AM2011-1062

Abstract 3489: In silico analysis of angiogenesis pathway as a prognostic tool in breast cancer

Abstract Gene expression profiling studies have enabled us to study breast cancer biology at a molecular level. Studies have been geared at identifying better and comprehensive prognostic markers for the development of targeted therapeutics. With the growing use of anti-angiogenic agents as second line agents, we aim to study the prognostic role of angiogenesis related genes through retrospective analyses of publicly available breast cancer gene expression datasets. Three gene expression datasets from studies by van't Veer et al, Sorlie et al., and Sotiriou et al were analyzed. The data was downloaded from the BRB Array Tools archives and analyzed using BRB Array Tools. Since, we were particularly interested in genes related to angiogenesis; we selected the (CGAP) angiogenesis subset for the purpose of survival analyses. Two endpoints, overall survival (OS) and relapse free survival (RFS), were analyzed. Data for OS was not available for van't Veer et al. A subset analysis for estrogen receptor positive cases was performed. HIF1A was prognostic for OS and RFS in all datasets. VEGF was associated with survival only in ER+ cases. EGFR is prognostic for RFS but not OS. Several other angiogenesis genes were noted to affect prognosis in terms of OS and RFS (Table 1). Analysis of other publically available datasets will be undertaken to further validate these findings in ER+ and negative subgroups of breast cancer. These analyses highlight the importance of VEGF as an important marker among ER+ breast cancer patients. Thus, anti-angiogenic agents targeting VEGF may appear to play an important therapeutic role in this subset of patients. The analysis provides additional evidence of the importance of HIF1A in breast cancer. This may help identifying sub-populations of breast cancer that are most likely to respond to targeted therapies. Further immunohistochemical validation in in-vitro models will be performed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3489. doi:10.1158/1538-7445.AM2011-3489

Abstract 3335: Self-renewal, tumorigenicity and metastatic potential of CD44+ cells in ER-negative lines and dissociated primary human breast cancers is increased in CD24+ subsets

Abstract Primary human breast cancer subpopulations with a CD44+ CD24NEG/LOW ESA+ phenotype exhibit self-renewal and generate xenograft tumors with as few as 100 cells in immunodeficient mice. While CD44 expression is strongly associated with self renewal in several cancers, the relative contributions of CD24 negative versus low subpopulations to stemness is poorly defined. Early reports have not distinguished negative from low CD24 expression in human lines and primary tumors, and both, together with CD44+ status have been linked to breast cancer stem cell phenotypes. In contrast, certain reports have associated CD24 expression with tumor progression and metastasis. Here, we show increased mitogenic kinase activation, expression of ESC and EMT genes in the CD24LOW subpopulation of CD44+ cells in both triple negative MDA-MB-231 breast cancer cells and primary dissociated breast cancers (DTs) compared to the CD24NEG cells. Moreover, tumorigenicity was increased and metastasis arose exclusively from orthotopic xeno-implantation of the CD44+CD24LOW cells. MDA-MB-231 and two different primary DTs were live sorted into CD44+CD24NEG and CD44+CD24LOW enriched subpopulations. Cells expressing other putative stem cell markers, ALDH1 and ESA were almost entirely CD44+CD24LOW. CD44+CD24LOWcells showed higher PI3-kinase/Akt, MAPK, and Src activites compared to CD44+ CD24NEG cells. While both subpopulations formed soft agar colonies and/or mammospheres, those arising from CD44+CD24LOW were greater in number and size than from CD44+CD24NEG cells. When cultured in 2D after sorting, CD44+ CD24LOW cells gave rise to both CD44+ CD24LOW and CD44+ CD24NEG progeny while CD44+ CD24NEG yielded only CD44+ CD24NEG progeny. CD44+CD24LOW expressed higher levels of ESC genes and EMT-associated genes than CD44+CD24NEG cells and uniquely expressed stem cell-associated miRNAs. On orthotopic injection into BalbC nude mice, both subpopulations initiated tumors with as few as 100 cells, however tumors arising from CD44+CD24LOW cells had a shorter latency and grew more rapidly than those arising from CD44+CD24NEG cells. In vivo imaging of luciferase positive cells showed CD44+CD24LOW cells yield metastasis while CD44+CD24NEG cells did not. Our data demonstrate, for the first time, distinct biological properties between the CD44+CD24LOW and CD44+CD24NEG enriched subpopulations of MDA-MB-231 and primary human breast cancer DTs. Low surface expression of CD24 is associated with ALDH1 and ESA+ status, increased self-renewal as shown by mammosphere and colony formation, tumorigenicity and metastatic potential of CD44+ cells. The ability to isolate and characterize such TSC-enriched subpopulations will be necessary for development of therapeutic strategies that preferentially target breast cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3335. doi:10.1158/1538-7445.AM2011-3335

Elimination of the chemotherapy resistant subpopulation of 4T1 mouse breast cancer by haploidentical NK cells cures the vast majority of mice

Metastatic breast cancer is currently incurable despite initial responsiveness, assumingly due to the presence of chemoresistant subpopulations that can be characterized as label retaining cells (LRC). In the 4T1 mouse breast cancer model, we previously achieved cure after Cyclophosphamide and Total Body Irradiation (CY + TBI) followed by haploidentical bone marrow and spleen transplantation (BMSPLT). CY + TBI without transplantation induced only transient impaired tumor growth indicating a critical role of donor immune cells. As it remained unknown if the 4T1 model resembles human disease with respect to the presence of subpopulations of chemoresistant LRC, we now demonstrate this is indeed the case. Chemoresistance of 4T1 LRC was demonstrated by in vitro co-incubation of fluorescently labeled 4T1 cells in limiting dilution with cyclophosphamide, doxorubicin or cisplatinum, after which only LRC containing colonies remained. LRC also remain in vivo after treatment with CY + TBI. Succeeding experiments set up to identify the haploidentical effector cell responsible for cure and, therefore, for the elimination of chemoresistant LRC designate donor NK cells crucial for the anti-tumor effect. NK cell depletion of the haploidentical graft fully abrogated the anti-tumor effect. Increased disease-free survival retained after transplantation of haploidentical bone marrow and NK cell-enriched spleen cell grafts, even in the absence of donor T-cells or of donor bone marrow. Tumor growth analysis indicates the anti-tumor effect being immediate (days). Based on these data, it is worthwhile to explore alloreactive adoptive NK cell therapy as consolidation for patients with metastasized breast cancer.

Abstract 4293: Evaluation of activated CD44 as a biological target in the eradication of breast cancer stem cells

Abstract Introduction: Cancer therapies are designed to kill rapidly proliferating cells where initial therapeutic benefits are usually transient resulting in tumor relapse and metastatic disease. Current treatment failure is due to a small population of slow-growing and drug resistant cells known as cancer stem cells (CSCs). To effectively treat breast cancer it is necessary to develop anti-cancer agents that concomitantly kill the rapidly growing breast cancer cells and the dormant CSCs. CD44 is a protein over-expressed on rapidly dividing breast cancer cells where it plays a critical role in the formation and progression of the tumor. CD44 is used as a reliable marker for breast cancer stem cells, therefore, through the high expression of this protein on both rapidly proliferating and CSCs there is strong rationale to use CD44 as a therapeutic target. CD44 mediates the majority of its biological processes through its affinity for the polysaccharide hyaluronan (HA). HyACT® anti-cancer drugs (HA drug delivery vehicle containing cytotoxic drugs) are specifically designed to target and transport chemotherapeutic agents to activated intra-tumoral CD44, where preclinical studies have strongly correlated the efficacy of the HyACT® platform with enhanced therapeutic responsiveness in CD44-positive tumor cells. This study evaluated the activation status of CD44 on CSC and non-CSC breast cancer cell sub-populations and then validated it as a therapeutic target in the treatment of breast cancer CSC. Methodology: Four breast cancer cell lines were separated into CSC (CD44+CD24-ALDH+/−) and non-stem cell (CD44+CD24+ ALDH+/−, CD44-CD24+ ALDH+/−) sub-populations using FACS. Attachment and proliferation characteristics of the breast cancer sub-populations were established using real time electronic microsensory array assays. The activation status of CD44 was determined using a functional HA internalization assay. After confirming the CD44 activation status cell sub-populations were treated with two HyACT® drugs, HA/Doxorubicin or HA/Gemcitabine where efficacy was determined using clonogenic and microsensory array assays. Results: CD44s was the predominant CD44 isoform expressed on all CD44+ve breast cancer sub-populations but high levels of the CD4v6 and v9 variant isoforms were uniquely identified on CSC populations. All isoforms of CD44 were shown to be activated. When CSC and non-CSC breast cancer sub-populations were treated the CD44-targeted drugs, the EC50 was reduced 2 to 40-fold when compared to drug alone. This increase in efficacy was negated when CD44-ve breast cancer sub-populations were treated with the CD44-targeted drug. Conclusions: This preliminary study has demonstrated breast cancer CD44 variant isoforms to be valid targets for hyaluronan encapsulated chemotherapeutic drugs that can be used in the eradication of all CD44+ve breast cancer populations including cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4293.

Selective regain of egfr gene copies in CD44+/CD24-/lowbreast cancer cellular model MDA-MB-468

BackgroundIncreased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies.MethodsThe basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique.ResultsLow and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies.ConclusionProgressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.

International Breast Cancer Research: Launching an Expedition to the Moon.

Abstract In his State of the Union address in 1971, President Nixon launched a "war on cancer" by calling for the "same kind of concentrated effort that split the atom and took man to the moon" to be turned towards conquering cancer. Since this landmark address, great strides have been made in the war on breast cancer. Lumpectomy with adjuvant radiotherapy, sentinel lymph node dissection, and adjuvant systemic therapy in the form of chemotherapy, hormonal therapy and trastuzumab have improved the quality of life and long-term survival of women diagnosed with early breast cancer. International collaboration involving research groups and initiatives such as the Early Breast Cancer Trialists' Collaborative Group quinquennial overviews has provided the clinical trials infrastructure and framework necessary to synthesize the evidence-based data upon which these gains have been achieved. Despite these successes, the pathway to new drug development and improved medical effectiveness today is costly, labor-intensive, and far too slow for our patients. Over the last decade, the advent of molecular technologies has dramatically improved our understanding of the biology of breast cancer. We now have targeted therapies available to inhibit specific signaling pathways that are aberrant in subpopulations of breast cancer. To further improve breast cancer care, we must fundamentally rethink how translational research is performed in clinical trials. The challenge of making personalized medicine a reality demands the launch of an expedition taking us to and beyond the moon: it will involve clinicians, basic scientists, bioinformaticians, statisticians, industry sponsors, regulators, and patients in new partnership that is committed to changing the vision, culture and methodology of breast cancer research. The Breast International Group's efforts in this direction will be presented. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr W1-1.

CALGB 150002: Correlation of HER2 and chromosome 17 (ch17) copy number with trastuzumab (T) efficacy in CALGB 9840, paclitaxel (P) with or without T in HER2+ and HER2- metastatic breast cancer (MBC)

1009 Background: Accurate assessment of HER-2 is critical in MBC and predicts benefit from T. We have previously shown low level amplification of HER-2 associated with ch17 polysomy is common in IHC 2+ cases that are FISH(-). However, the clinical relevance of this is unknown. Methods: CALGB 150002, a correlative companion to 9840, was designed to assess the predictive role of HER-2 in MBC pts treated with weekly vs q 3W P ± T. Pts HER-2(-) locally were randomized to ±T. 585 pts were enrolled in 9840; 304 blocks were available for central analysis with DAKO HercepTest (IHC) and Pathvysion (FISH). Logistic regression was used to test HER-2:ch17 ratio and HER-2 copy # as predictor of response rate (RR) to T in HER-2(+) pts. 1-sided Fisher’s Exact Test was used to compare RR of P vs P+T in pts with ch17 polysomy on central testing (>2.2 copies ch17/cell), but defined as HER-2(-) locally and randomized to T. Results: In pts HER-2(+) locally, FISH is a significant predictor of RR to P+T. A higher HER-2:ch17 ratio is associated with a higher RR by logistic regression (p=0.033, n=95). No interaction is seen between HER-2 and P schedule, p=0.71. On central testing of cases IHC(-) locally, 16/140 (11%) were IHC 3+ and 5/133 (4%) HER-2 amplified. In 21 HER-2(-) cases reclassified as HER-2 3+ or FISH (+) centrally, we do not find a difference in RR to P vs P+T. However among 133 cases HER-2(-) locally and central FISH(-) we find 32 with ch17 polysomy (copy # ch17 = 2.2); 12 treated with P alone, and 19 with P+T (see table for RR). Conclusions: These data suggest a higher RR to P+T in HER-2(+) pts with a higher HER-2:ch17 ratio, consistent with a relationship between RR to T and HER-2 copy #. In FISH(-) cases we note an increased RR to P+T vs P in cases with ch17 polysomy, typically reported clinically as HER-2(-). This analysis suggests that T might be effective in a subpopulation of breast cancer conventionally defined as HER-2(-), but in fact displaying low level HER-2 amplification. [Table: see text] [Table: see text]

Breast cancer subpopulation with high risk of internal mammary lymph nodes metastasis: analysis of 2,269 Chinese breast cancer patients treated with extended radical mastectomy

The selective treatment of internal mammary lymph nodes (IMNs) in breast cancer is controversial. The purpose of this research was to determine the subpopulation patients with high risk of internal mammary lymph nodes metastasis who received extended radical mastectomy without any preoperative treatment from 1956 to 2003 in China. 1,679 Chinese patients were underwent extended radical mastectomy (ERM) between 1956 and 2003. Four individual variables were selected (tumor site, tumor size, the number of ALNs involvement, patient age),then classified by following standards: tumor site(lateral, central, medial),tumor size(T1:<or=2 cm; T2:2 < T <or= 5 cm, T3:>5 cm), ALNs(0,1-3,4-6, >or=7), age(<or=35 y, 36-50 y, >50 y). Chi-square and binary logistic regression were used to analysis relationship of these variable and IMMs. The four individual variables were significantly associated with IMNs metastasis using univariate analysis. However, three individual variables except for tumor size independently impact the IMNs metastasis using multivariate analysis. The incidence of IMNs metastasis in patients with 4-6 and >or=7 positive ALNs was 28.1%, 41.5%. Within subgroup patients with medial tumor and positive ALNs, the incidence of IMNs metastasis was 23.6% for patients with 1-3 positive ALNs, and 47.5% for 4-6 positive ALNs, 38.7% for patients with >or=7 positive ALNs. The incidence of IMNs metastasis was 25.4% for patients with T3 tumor and younger than 35 y. Patients with following conditions had high risk of IMNS metastasis: (1) patients with 4 or more positive ALNs. (2) patients with medial tumor and positive ALNs.(3) patients with T3 tumor and younger than 35 y. (4) patients with T2 tumor and positive ALNs.(5) patients with T2 tumor and medial tumor .The incidences of IMNS metastasis for those patients were more than 20%.

Production of matrix metalloproteinases in specific subpopulations of human-patient breast cancer invading in three dimensional culture system

This article aims at investigating the effect of production of matrix metalloproteinases (MMP) in human breast cancer tissues by means of three dimensional culture system. Thirty-nine tumour samples were taken from breast cancer patients. The tumour blocks were cultured on sponge gel using the three dimensional culture system. Breast cancer cells began shedding into the culture medium after 24 hours of culture. The cells were stained with trypan blue dye to assess viability on days 2, 4, 6 and 8. The culture medium was collected at these time points and tested for matrix metalloproteinases (MMP) 1,2,3 and 9 activities. There was a progressive increase in migration of cancer cells into the gel and culture medium from day 2 to day 8 and the interval difference was statistically significant (F ratio = 4.06; p = 0.008). The levels of all the MMPs tested were also significantly raised (P<0.05 for all the MMPs tested). When the levels of MMPs were correlated with the metabolic activities in the gel, medium and tumour block, cells in block show no correlation whereas cells in gel correlated significantly with MMP-1 and MMP-3. Cancer cells in the culture medium correlated with MMP-9. In conclusion, there is a progressive migration of cancer cells outside the tumour block. The migration into the gel and culture medium is associated with progressive and differential production of MMPs. It is likely that the three dimensional culture model assists in the selection of different subpopulations of cancer cells with different invasion properties as exemplified by the differential production of MMP.

Efficacy of Epoetin Alfa in a Retrospective Non-stratified Subgroup Analysis of a Breast Cancer Cohort Receiving Non-platinum Chemotherapy

More than 60% of patients with metastatic breast cancer receiving non-platinum-based chemotherapy experience anemia, which is associated with fatigue and impaired quality of life. Epoetin alfa treatment in patients with a variety of malignancies has been shown to decrease transfusion requirements and improve hemoglobin levels and quality-of-life efficacy parameters. Retrospective subgroup analyses were performed in patients with breast cancer who were part of a multinational, randomized (2:1), double-blind, placebo-controlled trial of anemic cancer patients (n = 375) undergoing non-platinum-based chemotherapy. In the breast cancer subpopulation (n = 114, 48% with stage IV disease at baseline), the hemoglobin increase was greater for epoetin alfa patients than placebo patients (2.3 versus 0.9 g/dL). Epoetin alfa patients had lower transfusion requirements (28.2% versus 33.3%), improvement or preservation versus deterioration of quality of life, and a higher proportion of responders (patients achieving a > or = 2 g/dL increase in hemoglobin levels unrelated to transfusion) (68.0% versus 22.9% for placebo). The results were similar to those observed in the full study cohort, where statistical analyses showed the differences to be significant (P <0.05 for all). Epoetin alfa treatment was well tolerated. Although the study was not designed or powered for survival as an endpoint, Kaplan-Meier estimates for the full cohort showed a trend in overall survival favoring epoetin alfa treatment (P = 0.13, log rank test); a similar benefit was seen in the breast cancer subpopulation. In the full study cohort and the breast cancer subpopulation, epoetin alfa effectively treated anemia (increased hemoglobin levels and decreased transfusion requirements) and improved or preserved quality of life. Results concerning potential survival benefits support further study of epoetin alfa in anemic cancer patients.

Melanoma risk in women with previous breast cancer

7553 Background: Four cases of double primaries (melanoma and breast cancer), which were treated in our institute, raised our attention to the risk of both tumors in women. Our objective was to determine the risk for development of melanoma or breast cancer as second primary tumor in women with either one of them. Methods: We considered 51,000 breast cancer women patients and 6248 malignant melanoma women registered by the Israel Cancer Registries (6,000,000 inhabitants) and followed up from 1960 to 2000. Results: Overall, 276 malignant melanoma women were observed in the subpopulation of breast cancer versus 157 expected [standardized incidence ratio (SIR): 1.76; 95% confidence interval (CI) 1.50–2.05]. The incidence for breast cancer (276) in the sub population of melanoma females was as expected (267) with SIR 1.03 (95% CI 0.91 -1.17). Interestingly most women had been diagnosed with the second primary within the 12 months after diagnosing the first primary. The SIR for melanoma in the sub population of females with breast cancer (43 observed versus 19 expected) during 1 year after diagnosis of breast cancer was 2.28 (95% CI 1.39 –3.50). The SIR for breast cancer (44 observed versus 20 expected) in subpopulation of females up to one year after diagnosing of melanoma was 2.21 (95% CI 1.36 –3.35). Conclusions: We report excess risk of melanoma in women with breast cancer. Most of the cuetaneous tumors were discovered during the first year after diagnosis of the breast cancer. Although there is no excess risk for breast cancer in melanoma patients we observed that during the first year after diagnosing the melanoma there were double observed cases than expected. These findings suggest the possibility that more intense follow up lead to diagnosis of both second primary tumors. Genetic susceptibility might be responsible for the excess risk. Recommendation: Women with either breast cancer or melanoma and their clinicians should be aware of the reciprocal higher risk for both tumors. Skin and breast examination should be performed after diagnosing one of the primaries. No significant financial relationships to disclose.

Prediction of prognosis of estrogen receptor-positive breast cancer with combination of selected estrogen-regulated genes.

Estrogen receptor (ER)-positive breast cancer is a distinct subpopulation of breast cancer exhibiting a high response rate to endocrine therapy. However, not all ER-positive patients respond to the therapy, and a subgrouping of ER-positive patients based on the physiology of estrogen signaling is expected to be useful for predicting the prognosis. This study has revealed that selected estrogen-regulated genes (ERGs) are useful in identification of a poor-prognosis population among ER-positive breast cancer patients. First, the expression levels of 11 ERGs, selected based on our earlier microarray study in cultured cells, were analyzed by means of real-time reverse transcription-PCR in 14 ER-positive human breast cancer tissues. The patients were clearly divided into two groups in cluster analysis. Then, we examined the expression levels of two representative ERGs, histone deacetylase 6 (HDAC6) and insulin-like growth factor binding protein 4 (IGFBP-4), in 62 ER-positive patients with immunohistochemistry to assess the impact of ERG expression on prognosis (median follow-up 4409 days). Positive HDAC6 staining was significantly correlated with a lower disease-free survival rate. Moreover, when the expression level of HDAC6 was assessed in combination with IGFBP-4 expression in the nucleus, the poor-prognosis patients were more accurately identified. This study has identified new candidate ERGs for prediction of prognosis, and we suggest that combined assessment of the expression levels of these ERGs will contribute to the clinically useful stratification of ER-positive breast cancer patients.

Determination of DNA synthesis, estrogen receptors, and carcinoembryonic antigen in isolated cellular subpopulations of human breast cancer.

Primary breast adenocarcinomas obtained from ten patients were enzymatically digested using collagenase (1 mg/ml), hyaluronidase (1 mg/ml), elastase (0.1 mg/ml) and DNAse (0.2 mg/ml). The tumor cells were labeled with 3H-thymidine and, in some cases, with 3H-estradiol. The isolated cells were submitted successively to a Ficoll-Hypaque and a bovine serum albumin gradient, from which 12 fractions were obtained. In each fraction, several characteristics were determined: carcinoembryonic antigen (CEA), thymidine (dThd) incorporation, and estrogen receptors (ER). Three main cellular subpopulations were characterized: An intermediate density subpopulation (1.046-1.054 g/ml), in which the proliferating cells are concentrated. In this subpopulation a small number of CEA-positive cells are present, but ER containing cells are virtually absent. A high-density, small cell subpopulation that concentrates most of the ER-containing cells. This subpopulation lacks proliferating cells, but CEA-containing cells are abundant. A low-density subpopulation, lacking proliferating cells and with scarce ER-positive cells, although CEA-positive cells are frequent. These findings strongly suggest that proliferating cells lack ER.