bluuebunny/crossref_metadata_2025 huggingface dataset:
Breast Cancer Patients Receiving Neoadjuvant, Adjuvant and Palliative Chemotherapy Have Different DNA Oxidative Damage and Repair Profiles.
Authors: Laura V. , MCarmen R. , Pedro S. , Cesar R. , Sergio G. , Jose L. , Jose Q.
Published: December 2010

Background: Antineoplastic agents induce oxidative stress in biological systems leading to lipid, carbohydrate, protein and DNA damage and affecting cell structure and function. These adverse effects may fuel up the acquisition of new mutations and the development of treatment resistances and secondary malignancies.Methods: We selected 90 breast cancer patients receiving neoadjuvant, adjuvant or palliative chemotherapy, to test the effect of this treatment on the systemic oxidative stress. The patients were distributed in equal groups and paired blood samples, before and after chemotherapy, were extracted. DNA oxidative damage was assessed by alkaline comet assay and a specific inmnoenzymoassay was employed to determine RPA and Ku86 DNA repair activity.Results: We found that neoadjuvant patients presented a marked raise in DNA damage after chemotherapy (Figure 1A). DNA repair activity of KU86 was significantly higher in the adjuvant setting and significantly lower in the neoadjuvant and metastatic settings after chemotherapy. Before chemotherapy administration, KU86 activity was higher in the metastatic and neoadjuvant groups compared to the adjuvant group (Figure 1B).Discussion: Our results show that chemotherapy induces the production of free radicals to an extent that causes a severe DNA damage. High levels of DNA damage are maintained along successive clinical interventions, due to continued production of free radicals during breast cancer treatment, despite the activation of the repair mechanisms that are not sufficient to overcome the effects of the oxidative stress at this level. Our work shows that breast cancer treatment affects the redox status of the patients and, because of its effects into cellular signaling pathways and gene expression, it must be considered as a potential therapeutic target to improve breast cancer treatment and minimize the associated toxicity.Figure legend 1.- Oxidative DNA damage and DNA repair markers in neoadjuvant, adjuvant and metastatic patients, before and after chemotherapy. (A) Percentage of DNA in the tail of lymphocytes, as measured by comet assay. (B) DNA repair activity of KU86. Intragroup statistical differences owed to chemotherapy are indicated as ‡ (P<0.001) or * (P<0.05). Intergroup statistical differences for each period (before and after chemotherapy) (P<0.05) are indicated by the letters a and b, in such a way that the measures with different superscript letters, are statistically different.Figure legend 2.- Fluorescent microscope images of damaged lymphocytes from a neoadjuvant patient before (A) and after (B) chemotherapy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1123.
miR-206 Expression Is Downregulated in Cyclin D1 Amplified Breast Tumours.
Authors: Ahnström Waltersson M. , Karlsson E. , Nordenskjöld B. , Fornander T. , Skoog L. , Stål O.
Published: December 2010

Amplification in the 11q13 region has been found in around 15% of all breast cancers and is strongly correlated with oestrogen receptor (ER) positive tumours. We have previously found that amplification of at least one of the genes PAK1 or CCND1 is associated with decreased recurrence-free survival among ER+ patients. Other genes in the amplicon might also contribute to this effect and situated close to CCND1 are the FGF-3, -4 and -19 genes. The FGF-4 protein has been shown to inhibit the expression of the ER a regulator miR-206 in chicken embryo. In this study we analysed 23 tumours with and 27 tumours without previously detected 11q13 amplification to explore if 11q13 amplification is associated with decreased levels of miR-206 and if miR-206 is associated with ER expression. Using eal-time PCR, we found that miR-206 expression was inversely correlated to CCND1 and 11q13 amplification (P=0.016 and P=0.022 respectively). Tumours with low miR-206 expression had higher levels of ER a than tumours with intermediate and high expression (P=0.043). In vitro studies of the breast cancer cell lines SKBR3 and ZR751 showed that treatment with FGF-3, -4 or -19 lowered the expression of miR-206. We conclude that miR-206 might be an important regulator of ER a expression. Our results that low mir-206 is associated with CCND1 amplification and thereby also FGF-4 amplification in combination with the findings that FGF-3, -4, and -19 may regulate the expression of miR-206 in vitro points towards the possibility of a miR-206 regulator, FGF-4 or another FGF, present in the amplicon.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1127.
A Functional Role for CERT in Cancer Drug Induced Autophagy and Prognosis in Her2 Positive Breast Cancer.
Authors: Lee A. , East P. , Nicke B. , Jones N. , Downward J. , Gorman P. , Roylance R. , Murphy N. , Hanby A. , Swanton C. , Swanton C.
Published: December 2010

CERT was recently identified in a functional genomic siRNA screen for regulators of multi-drug sensitivity. CERT encodes a ceramide transporter which shuttles the pro-apoptotic lipid ceramide from the endoplasmic reticulum to the golgi. Silencing CERT, promoted sensitivity to paclitaxel, doxorubicin, cisplatin and 5-FU in breast, colon and non-small cell lung cancer cell lines. Further analysis of CERT expression in 18 NCI-60 cancer cell lines together with drug sensitivity data for over 5000 drugs within the NCI database, revealed that elevated CERT expression is associated with multi-drug resistance to diverse cancer cytotoxics. CERT mRNA was significantly elevated in HER2 positive breast cancer cell lines. Consistent with these data, in a primary breast cancer tissue microarray cohort of 356 primary breast cancers with outcome data, CERT protein expression was significantly greater in Her2 positive disease (p<0.0001). Survival analysis indicates that increased CERT expression correlates with significantly poorer long-term survival across the whole cohort and in Her2 positive patients.In order to explore the downstream molecular targets of CERT activity, we used the Affymetrix GeneChip Exon Array to detect gene expression changes following siRNA mediated CERT silencing. Genes that were differentially expressed following CERT silencing were identified and validated by real-time PCR. Significant representation of genes involved in the autophagy pathway were identified. One of these genes, LAMP2, a protein involved in autophagosome-lysosome fusion, is up-regulated following CERT silencing. LAMP2 silencing promotes paclitaxel resistance when depleted from cancer cells. Consistent with a role for CERT silencing in mediating an autophagy response following drug exposure, CERT depletion together with Paclitaxel treatment induces alterations in the autophagy response marker-LC3 similar to those seen in starvation-induced autophagy and an increase in lysosomal activity as visualised by microscopy. These data indicate a role for autophagy in regulating cytotoxic drug response in cancer.The pharmacological targeting of CERT, a poor prognostic marker in breast cancer, may be a rational approach to enhance drug response and patient outcome.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1137.
Tau Expression and Efficacy of Paclitaxel Treatment in Metastatic Breast Cancer.
Authors: Tanaka S. , Nohara T. , Iwamoto M. , Sumiyoshi K. , Kimura K. , Takahashi Y. , Tanigawa N.
Published: December 2010

Purpose: Paclitaxel is widely used for the treatment of patients with metastatic breast cancer (MBC), such as in anthracycline-based combinations as a first-line chemotherapy. Although several mechanisms of paclitaxel resistance have been demonstrated, useful markers of paclitaxel resistance have not been available in clinical practice. A recent study revealed that low tau expression in cases of early breast cancer can predict susceptibility to paclitaxel administration in neoadjuvant therapy.Methods: In this study, the clinical significance of tau expression in MBC cases was established by identifying candidates with paclitaxel administration. Tissue specimens obtained from 35 patients who had received neither chemotherapy nor radiotherapy before surgery were examined. Status of tau expression was determined by immunohistochemistry.Results: Based on a previously reported classification scheme, 15 cases were classified as tau-negative (0, 1+) and 20 cases were classified as tau-positive (2+, 3+).Nine (60%) of 15 cases with tau-negative expression showed favorable response. Conversely, 17 (85%) of 20 cases with tau-positive expression showed progressive or stable disease after paclitaxel administration.Table 1 Relationship between tau expression and response to paclitaxel  nNumber of responses (%)PTaunegative159 (60%)  positive203 (15%)0.01 Time to disease progression in tau-negative and tau-positive groups was 9.4 ± 6.6 months and 6.0 ± 3.7 months, respectively. Although no significant differences were apparent, this period tended to be longer in the tau-negative group.Discussion: Patients with tau-positive expression may derive less benefit than tau-negative from paclitaxel therapy in MBC. This suggests that tau expression could be used at diagnosis to select patients for whom paclitaxel treatment is likely to be of most benefit. However, this study has some clear limitations in terms of the modest sample size and the heterogeneity of eligible patients (e.g., different numbers and regimens for adjuvant and MBC chemotherapy). Many more cases must be examined before tau expression can be applied to practical clinical treatment.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1138.
Knockout of Heregulin (HRG) Expression Reverts Paclitaxel-Resistance and Promotes Mesenchymal Epithelial Transition (MET) of Triple Negative Breast Cancer Cells.
Authors: Li J. , Espinoza I. , Lupu R.
Published: December 2010

The growth factor Heregulin (HRG) is expressed in about 30% of breast cancer tumors, and induces tumorigenicity and metastasis of breast cancer cells. We have demonstrated previously that HRG overexpression renders breast cancer cells resistance to the microtubule-interfering agent Taxol, a drug of choice for the treatment of metastatic breast cancer. The mechanism by which HRG induces Taxol resistance is largely unknown. It is also known that triple negative breast cancer tumors do express high levels of HRG and unfortunately they do not respond to HRG. Our studies were aimed at targeting HRG with the goal of achieving a therapeutic target as well as restoring the response to Taxol, while preventing the formation of metastasis. Thus, we knocked-down HRG expression in three different breast cancer cell lines: MDA-MB-23, HS578T and BT549. Our data demonstrates that HRG expression is an absolute requirement for the anchorage-independent growth for triple negative breast cancer cells, since none of the breast cancer cells MDA-MB-231, HS578T and BT549 stable expressing the silencing (shRNA) for HRG, were capable of forming colony in soft agar. Furthermore, these cells, not only no longer were not anchorage-independent were no longer resistant to Taxol, to the contrary the shRNA/HRG cells were exquisitely sensitive to Taxol, to induce growth inhibition and apoptosis. More importantly, we observed that the disorganized structured observed in 3D matrigel culture observed for triple negative cells, was completely abolished once HRG was knockdown and a very organized structure. These characteristics resembled an EMT (epithelial-mesenchymal epithelial transition (MET). This should be deemed a potential target in developing therapies for triple negative breast carcinomas.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1142.
Epigenetic Control of Estrogen Synthesis in Human Preadipocytes and Breast Cancer Cells.
Authors: Knower K. , To S. , To S. , Simpson E. , Simpson E. , Clyne C. , Clyne C.
Published: December 2010

Estrogen plays a significant role in the development and progression of breast cancer (BC). Cytochrome aromatase p450, encoded by the gene CYP19, is the key enzyme catalyzing the synthesis of estrogens from androgens. In post-menopausal women, adipose tissue becomes the major site for estrogen production and an increase in CYP19 expression in mammary adipose is strongly linked to the hormone-dependent progression of BC. Therefore, understanding CYP19 regulation in BC is critical for the development of therapeutic measures. Previous studies of CYP19 regulation in BC have mainly focussed on hormone-induced regulation of transcription via upstream tissue- and promoter-specific regulatory regions. While this is clearly important, it is increasingly evident that epigenetic regulation of expression, such as DNA methylation, is also a common factor in the progression of cancers. The aim of this study was to investigate whether CYP19 expression is under epigenetic regulation and to determine if such mechanisms contribute to the tissue- and promoter-specific expression of CYP19 observed in BC.CYP19 transcripts in cancer-free primary human breast adipose fibroblasts (BAFs) stimulated with cytokines and glucocorticoids (e.g. TNFα and dexamethasone) are derived from the distal promoter 1.4, whereas breast tumour-derived factors (e.g. prostaglandin E2) induce a regulatory switch to proximal promoters 1.3 and II. DNA from BAFs maintained under these two conditions was treated with sodium bisulfite allowing for methylation analysis of CpG dinucleotides. Methylation mapping revealed a stochastic heterogeneous level of methylation among 9 CpG sites (promoter 1.4) and 11 CpG sites (promoters 1.3 and II) in untreated BAFs.No correlation was observed between promoter methylation and promoter activation after response to stimuli. However, treatment of BAFs with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, increased total CYP19 mRNA expression up to 40-fold, primarily via promoter 1.4 thorugh up-regulation of trans-acting factors, suggesting CYP19 is under tonic inhibition by methylation in BAFs. Similar results were also obtained in four BC cell lines. Ongoing work has elucidated trans-acting factors of CYP19 epigenetically silenced in BAFs. These studies uncover a new layer of complexity in the regulation of aromatase expression in BC and may identify new targets for epigenetic therapy, including DNA methylation inhibitors. These findings will translate to BC by determining the methylation state of CYP19 in clinical BC specimens, and by correlation with clinicopathological parameters such as steroid receptor status, grade and stage.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1147.
Epigenetics Differences between Estrogen Receptor (ERS1) and Her2/Neu Status in Breast Cancer Patients.
Authors: Martínez-Galán J. , Torres-Torres B. , Ales I. , Durán G. , Gutierrez V. , Del Moral R. , Cobo M. , Nuñez M. , Benavides M. , Ruiz De Almodóvar M.
Published: December 2010

Background: The CpG island methylator phenotype is associated with distinct clinicophatological characteristics as Estrogen Receptor (ESR1) positive and amplification of HER2 in breast cancer.Objetive: To evaluate epigenetics differences in tumor-related genes to ERS1 and HER2/neu status in primary breast cancer.Material and Methods: We quantified methylation levels of promoter of 5 genes (ERS1, RAR- β, 14-3-3 sigma, APC, E-Cadherin) which are to confer growth adventage to cells, in 107 women with breast cancer and 108 control subjects. Real Time QMS-PCR SYBR green (methylation-specific PCR) was used to analyze the hypermethylation. Tumours were classified as phenotype basal, luminal A, Luminal B and phenotype HER2+.Results: Ours analyses revealed low or absent methylation ESR1and 14-3-3σ in healthy controls and significant differences between breast cancer patients (pts) and healthy controls in relative serum levels of methylated gene promoters ESR1 (p=0.0112) and 14-3-3s (p=0.0047). Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p=0.0179). Of the available cases, 60 pts (56%) were Luminal A, 10 pts (9.3%) Luminal B, 13 pts (12%) Basal like and 9 pts (8.4%) HER2+. We observed that methylated ERS1 was preferably associated with phenotype Basal Like and worse interval progression free and survival global though p>0.05 and the amplification HER2+ was correlation with significant more frequent methylation gene (p<0.05). Thet hypermethylation of normal ERS1 and 14-3-3σ combined differentiated between breast cancer patients and healthy controls (p=0.0001) with a sensitivity of 81% (95% CI: 72–88%) and specificity of 88% (95% CI: 78–94%).Conclusions: This study identifies the presence of variations in global levels of methylation promoters genes in healthy controls and breast cancer with different phenotype classes and shows that these differences have clinical significance. These showed that frequent methylation had a strong association with molecular phenotype of breast cancer and perhaps in the future can explain therapy resistance related to RE and HER2/neu status in breast cancer patients.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1151.
Critical Role of a Transcription Elongation Regulator in Mammary Gland Development and Mammary Tumorigenesis.
Authors: Nair S. , Li R.
Published: December 2010

For a long time transcriptional regulation was thought to primarily regulated at the stage of transcription initiation. However, recent genome-wide studies suggest that the majority of genes, both active and inactive, in human cells experience transcription initiation and many of them have RNA polymerase II (RNAPII) physically present at the promoter-proximal region. Thus, transcription elongation may represent a common but under-explored step for mammalian gene regulation. Cofactor of BRCA1 (COBRA1), which was first identified as a BRCA1-binding protein, acts as an integral subunit of the negative elongation factor (NELF) complex to stall RNAPII at the promoter proximal regions of multiple estrogen-responsive genes. COBRA1/NELF-B also shares a number of target genes for transcriptional regulation with BRCA1 in breast cancer cells. In support of its potential role in tumor suppression, its reduced expression is strongly correlated with metastatic breast cancer and knockdown of COBRA1 leads to elevated hormone-dependent transcription and accelerated cell proliferation both in vitro and in xenograft models.In order to get a better understanding of the molecular functions of Cobra1 in mammary gland, we employed Cre-lox system to delete mouse Cobra1 in the mammary gland. These mammary gland-specific Cobra1 knockout mice exhibit severe defects in alveologenesis and lactogenesis. These animals also show abnormal epithelial morphology specifically in the luminal compartment. Luminal epithelial population isolated from these mice using cell surface markers showed reduced colony forming ability both in 2-D and 3-D cultures. Molecular basis for the Cobra1-associated developmental defects and the possible impact of Cobra1 deletion on mammary tumorigenesis will be discussed. By linking regulation of transcription elongation with mammary gland-specific functions, our work establishes a new mode of gene regulation in mammary gland development.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1152.
Inhibition of Breast Cancer Stem Cells (CSCs) by Novel STAT3 Inhibitors.
Authors: Dave B. , Tweardy D. , Lewis M. , Chang J.
Published: December 2010

Background: Many patients with breast cancer relapse over time despite an initial response to systemic therapy. One explanation is that a rare sub-population of CSCs with tumorigenic potential is intrinsically resistant to therapy. Previously, we showed that residual tumors after chemotherapy are 1) enriched for the tumorigenic CD44+/CD24-/low population, 2) show enhanced mammosphere-forming efficiency (MSFE), and 3) display an increase in outgrowth potential in xenograft transplants in immunocompromised SCID/Beige mice. Gene expression profiling of CD44+/CD24-/low and mammosphere-forming cells derived from human breast cancer biopsies revealed that the top canonical pathways in CSC self-renewal included STAT3 signaling. We hypothesized that inhibition of STAT3 would lead to a decrease in the number of breast CSCs either alone, or in combination with systemic therapies. Methods: We established primary xenografts in immunocompromised SCID/Beige mice from biopsies of primary human breast cancers; that faithfully recapitulate biology similar to the primary breast cancers in patients. We tested two classes of drugs that target Stat3--G-quartet oligodeoxynucleotide (GQ-ODN, T40214) and small molecules (Cpd188 and its derivatives)--for their ability to inhibit human breast cancer xenograft MSFE.The GQ-ODN T40214 was previously shown to target the Stat3 Src homology (SH) 2 domain destabilizing Stat3 dimers while Cpd188 was identified using virtual ligand screening and was shown to competitively and selectively inhibit binding of the Stat3 SH2 domain to its phosphotyrosine (pY) peptide ligand. Cpd188 derivatives were identified by 2-D similarity screening using the scaffold of Cpd188 and 3-D pharmacophore analysis and their activity confirmed by pY peptide binding inhibition. A triple negative (ER-negative, PgR-negative, and HER-2) tumor line (n=3 mice/group) was treated with Cpd188 (125mcg or 250mcg), T40214 (125mcg or 250mcg), a control non-specific (NS) ODN (125 or 250 mcg) or vehicle alone. The animals were sacrificed after two days of treatment and MSFE determined. In addition, we tested the 39 Cpd188 derivatives for the ability to decrease MSFE in vitro. Results: In these short term experiments, a statistically significant reduction (p≤0.05) was observed in the MFSE with Cpd188 (250mcg) and T40214 (125 and 250 mcg) vs. control. Of the 39 Cpd188 derivatives, 11 derivatives significantly decreased MSFE in two triple negative cell lines (SUM159PT and HS578T). Conclusions: STAT3 inhibition using 2 classes of molecules that target its SH2 domain significantly reduced CSC in both in vivo and in vitro assays raising the possibility that breast cancer therapeutic regimens that contain agents that target Stat3 may reduce relapse rates.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1155.
Mammary Stem Cell-Like Gene Expression and Relation to Cancer.
Authors: Williams C. , Helguero L. , Edvardsson K. , Haldosén L. , Gustafsson J.
Published: December 2010

Mammary stem cells are bipotential and suggested to be the origin of breast cancer development, but are elusive and vaguely characterized.Breast tumors can be divided into subgroups, each one requiring specific treatment. To determine a possible association between mammary stem cells and breast cancer, we have previously made a detailed characterization of the transcriptome in murine mammary epithelial stem-like cell line (HC11) and made a thorough investigation of global gene-expression changes during stepwise differentiation using dual-color comparative microarray technique.Subsequently, we performed a cross-species comparison to reveal conserved gene expression between stem cells and subtype-specific and prognosis gene signatures, and correlated gene expression to in vivo mammary gland development. Our analysis provided a unique map of the transcriptomic changes and a number of novel mammary stem cell markers. Interestingly, our comparisons show that specific subtypes of breast cancers with poor prognosis and metastasizing capabilities show resemblance to stem-like gene expression. We are now investigating several marker candidates and regulations of miRNAs during differentiation.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1162.
Disease Related Outcome with Long Term Follow-Up: An Updated Analysis of the Intergroup Exemestane Study (IES).
Authors: Bliss J. , Kilburn L. , Coleman R. , Forbes J. , Coates A. , Jones S. , Jassem J. , Delozier T. , Andersen J. , Paridaens R. , Coombes R.
Published: December 2010

Background: IES is an investigator-led study in 4724 postmenopausal patients with early breast cancer (sponsored by Pfizer Ltd; ISRCTN11883920) that has already demonstrated significant, clinically important benefits from switching adjuvant endocrine therapy after 2-3 years tamoxifen (T) to exemestane (E) (NEJM 2004, Lancet 2007). With longer follow-up the sensitivity of a disease free survival analysis may be reduced by the increasing number of non breast cancer-related deaths. Analysis of breast cancer free survival (BCFS), censoring intercurrent deaths, provides a sensitive estimate of the true impact of an adjuvant intervention on breast cancer outcome.Materials and methods: Patients who were disease-free after 2-3 years of adjuvant T, were randomized to continue T or switch to E to complete a total of 5 years of adjuvant endocrine therapy. At the time of this planned analysis in June 2009, the median follow-up was 91 months, and 91% of surviving patients had ≥6 years followup. The analysis focuses on 4052 pts known to be ER positive (+) and 547 with ER unknown (unk) tumors. The remaining 125 patients now known to be ER negative are excluded from this analysis.Results: 933 BCFS events have been reported (E=425 T=508) giving an unadjusted hazard ratio (HR) of 0.81 (95%CI: 0.71, 0.92); p=0.001 in favor of E in the ER+ and ER unk groups. With mature follow-up, the benefits of E appear similar in the ER unk and ER+ sub-groups (HR=0.85 95%CI (0.60, 1.20); p=0.36 and HR=0.80 95%CI (0.70, 0.92); p=0.002 respectively). Similarly, all other prognostic subgroups show consistency with the overall BCFS effect. An analysis partitioned at 2.5 years post randomization showed the on-treatment BCFS benefit of switching to E (HR=0.60 95%CI (0.48, 0.75); p<0.0001) was maintained post-treatment (HR=0.94 95%CI (0.80, 1.10); p=0.46). Continuing improvement in OS was demonstrated, with E=352 versus T=405 deaths (HR=0.86, 95%CI (0.75, 0.99); p=0.04). Of these, 219 were reported as intercurrent deaths (E=105, T=114).Conclusions: The protective effect of switching to E, compared with continuing on T, on relapse rate is maintained for at least 5 years post treatment completion. Associated with this is a continuing benefit on overall survival.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 12.
Lack of Correlation between Genetic Polymorphisms in Tamoxifen Metabolizing Enzymes with Primary Endpoints in the IMPACT Trial.
Authors: Rae J. , Thibert J. , Sikora M. , Johnson M. , Skene A. , A'Hern R. , Smith I. , Dowsett M.
Published: December 2010

Background. Several studies aimed at correlating genetic variants in the enzymes responsible for tamoxifen metabolism and disposition with therapeutic outcomes have provided equivocal and conflicting data. The IMPACT trial compared the preoperative used of tamoxifen with anastrozole alone or in combination in postmenopausal breast cancer patients and provided strong evidence that tamoxifen-induced changes in the nuclear proliferation marker Ki67 can act as a surrogate marker of tamoxifen response. An advantage of this study design is that all patients contribute data to the end-point analyses which included changes in Ki67 and objective clinical response as defined by the WHO criteria. We therefore genotyped patients from IMPACT for variants in the enzyme (cytochrome p450 2D6 (CYP2D6)) responsible for the conversion of tamoxifen to its potent anti-estrogenic metabolite endoxifen, as well as the enzyme responsible for the detoxification and inactivation of endoxifen via conjugation (UDP-glucuronosyltransferase 2B7 (UGT2B7).Results: Depending on their CYP2D6 variant genotype, patients were assigned an 'activity score' based on predicted allele activities from 0 (no activity) to 2 (high activity). Patients were also tested for the UGT2B7*2 variant, which has lower enzymatic activity and is therefore correlated with higher serum endoxifen concentrations. Of the 135 patient samples available for genotype determinations, 123 and 134 we evaluable for CYP2D6 and UGT2B7, respectively. There was no evidence of an association between CYP2D6 score, as determined by genotype alone, with suppression of Ki67 after 2 or 12 weeks, or with objective clinical response after 16 weeks of therapy in the tamoxifen alone, anastrozole alone or combination groups. Furthermore, this lack of association was not altered by adjustment of the CYP2D6 score for concomitant medications, known to inhibit CYP2D6 and to correlate with lower serum endoxifen concentrations. Lastly, while a statistical test for a potential interaction between CYP2D6 genotype and UGT2B7 genotype was negative, there was a marginally statistically significant association between UGT2B7 genotype alone with suppression of Ki67 after 2 weeks in the tamoxifen only group.Conclusion: Although this was a small study, these data do not support the hypothesis that patients must have active CYP2D6 in order to benefit from tamoxifen therapy. Larger more definitive studies are underway to test the effects of drug metabolizing enzymes on anti-estrogen therapies.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2011.
Early Assessment of Proliferation by the Genomic Grade Index (GGI) Predicts Response to Neo-Adjuvant Letrozole.
Authors: Singhal S. , Bedard P. , Ignatiadis M. , Haibe-Kains B. , Desmedt C. , Loi S. , Evans D. , Dixon J. , Miller W. , Sotiriou C.
Published: December 2010

Background: Expression of the proliferation antigen Ki-67 after short-term pre-surgical endocrine treatment is predictive of long-term relapse free survival in post-menopausal women with estrogen-receptor (ER) positive breast cancer (Dowsett et al., JNCI 2007). There are no established predictive markers of clinical response to neo-adjuvant endocrine therapy.Methods: Whole genome expression profiles (Affymetrix HG-U133A) from paired tumor biopsies before and after 10 to 14 days of neo-adjuvant letrozole were compared to tumor volume changes over 3 months of treatment for 52 post-menopausal women with hormone receptor positive breast cancer to evaluate early predictive markers of clinical response.Results: Clinical response was observed in 80% Luminal A (24/30) and 59% Luminal B (13/22) tumors (p=0.1). Baseline tumor proliferation assessed by GGI or Ki-67 mRNA expression were not predictive of clinical response (p=0.29 and p=0.73 respectively). The absolute value of GGI at Day 10 to 14 was predictive of clinical response (p=0.001), particularly for luminal B (p=0.001) but not luminal A (p=0.35) tumors. The change in GGI from baseline to Day 10 to 14 was marginally predictive of response (p=0.063). Absolute day 10 to 14 Ki67 mRNA expression and the change in Ki67 mRNA were not associated with clinical response (p=0.54 and p=0.58 respectively). Baseline, absolute Day 10 to 14, and change in the expression of the ER gene (ESR1) or an integrative gene module reflecting ER signaling were not predictive of clinical response.Conclusion: Expression of GGI after 10 to 14 days of neo-adjuvant letrozole and the change in GGI with short-term treatment are predictive of clinical response observed after 3 months of therapy. Early assessment of GGI may be useful to determine which post-menopausal patients with highly-proliferative ER-positive disease benefit from endocrine therapy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2016.
p53 Status Splits Triple-Negative Breast Cancers in Subgroups with Distinct Predictive and Prognostic Potential Value.
Authors: Alberti S. , Ambrogi F. , Pedriali M. , Piantelli M. , Querzoli P. , Nenci I. , Ellis I. , Boracchi P. , Coradini D. , Biganzoli E.
Published: December 2010

Background: Triple-negative breast cancers largely encompass basal-like breast cancers, which demonstrate an aggressive clinical behaviour and poor prognosis. However, triple-negatives also include normal breast-like cancers, as identified by gene expression profiling, which have a better prognosis than basal-like cancers, but do not respond as well to neoadjuvant chemotherapy. Thus, a clear-cut distinction between the two subgroups is urgently needed. p53 is heterogeneously expressed in triple-negative cases, suggesting association to subgroups with diverse biological profiles. Hence, we comparatively analyzed p53 expression in triple-negative breast cancers from two independent case series.Material and Methods: Two independent breast tumor case series (633 cases from Ferrara University, Italy and 1076 cases from Nottingham University, UK) were analysed. Applying non-hierarchical algorithms, we have previously identified four breast tumor clusters, that possess markedly different prognosis: cluster [1], characterized by high values of ER/PR (good prognosis); cluster [2], with intermediate / heterogeneous ER/PR values (good prognosis); cluster [3], with low-to-nil ER (bad prognosis) and cluster [4] with low-to-nil PR and high HER2 values (bad prognosis) (Clin Cancer Res 2006;12:781-90). p53 expression in triple-negative cases belonging to the different clusters was analyzed.Results: p53 protein expression sharply subdivided the triple-negative Ferrara cases into two distinct subsets, that were tightly associated to clusters [2] or [3], respectively. Low-to-nil p53 levels were only observed in cluster [2] while overexpression of p53 was only seen in cluster [3].The triple-negative tumors of the Nottingham dataset were comparatively evaluated. Consistently with the data of the Ferrara case series, p53 similarly split this case series in p53-overexpressing and p53 low-to-nil triple-negative cases. p53 expression was associated with shorter relapse-free (Figure 2) and overall survival.Discussion: Our findings indicate that p53 expression can split triple negative breast cancers into sharply distinct subgroups, with critically different prognosis and prediction of drug response potential.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2018.
A Systems Analysis of Mitotic Apparatus Inhibitors Defines a Response Network for Breast Cancer.
Authors: Hu Z. , Mao J. , Huang G. , Kuo W. , Lenburg M. , Ziyad S. , Korkola J. , Bayani N. , Wang N. , Gu S. , Weber B. , Wooster R. , Gray J.
Published: December 2010

Deregulation of aspects of the mitotic apparatus leads to increased genome instability, carcinogenesis and aggressive tumor behavior in human and rodent model systems1. This knowledge has stimulated development of inhibitors of elements of the mitotic apparatus as anticancer agents including PLK1, CENPE, and AURKB and several are now being tested for efficacy clincially2-6. These trials and eventual clinical use will benefit from molecular markers that predict response. In order to identify such markers, we assessed quantitative responses to the agents GSK461364, GSK923295 and GSK1070916 that target PLK1, CENPE and AURKB; respectively, in a panel of 50 breast cancer cell lines. This analysis showed that basal subtype cell lines were preferentially sensitive to all three agents and that responses among the lines to the three agents were strongly correlated. This may be explained by our discovery that components of the mitotic apparatus including PLK1, CENPE and AURKB form a transcriptionally co-regulated network comprised of more than 50 genes that is preferentially active in basal subtype of breast cell lines and primary tumors. Remarkably, this network also is activate in subsets of cancers of the lung, ovarian, prostate and brain, Wilms tumor, human blood malignancies and selected normal tissues. We then defined a mitotic apparatus network index (MANI) and showed that high MANI was associated with poor outcome clinically and with preferential responsive to GSK461364, GSK923295 and GSK1070916 in preclinical models. This suggests that measures of the MANI will identify poor outcome tumors that will likely respond well to mitotic apparatus network gene inhibitors as well as potential dose limiting normal tissues.Reference1. Quigley, D.A. et al. Nature 458, 505-8 (2009).2. Strebhardt, K. & Ullrich, A. Nat. Rev. Cancer 6, 321-330 (2006).3. Toyoshima-Morimoto, F., Taniguchi, E., Shinya, N., Iwamatsu, A. & Nishida, E. Nature 410, 215-20 (2001).4. Barr, F.A., Sillje, H.H. & Nigg, E.A. Nat. Rev. Mol. Cell. Biol. 5, 429–440 (2004).5. McInnes, C. et al. Nat. Chem. Biol. 2, 608–617 (2006).6. Yamada, S. et al. Oncogene 23, 5901-5911(2004).

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2020.
PI3K Pathway Activity and Response to First-Line Chemotherapy in Combination with Trastuzumab in Patients with HER2-Positive Metastatic Breast Cancer.
Authors: Vorkas P. , Agelaki S. , Poumpouridou N. , Poumpouridou N. , Kroupis C. , Kroupis C. , Mavroudis D. , Stathopoulos E. , Georgoulias V. , Lianidou E.
Published: December 2010

Background. Not all patients (pts) with HER2-overexpressing metastatic breast cancer (MBC) respond to trastuzumab and most that initially respond, ultimately develop resistance to treatment. Activated PI3K signalling has been proposed to predict trastuzumab resistance.Patients and Methods. PI3K pathway activity was evaluated in tumor samples from pts with HER2-overexpressing MBC treated with first-line chemotherapy in combination with trastuzumab by: a) assessing PIK3CA mutation status by our recently developed High Resolution Melting Analysis (HRMA) for the detection of mutations in exons 9 and 20, performed in the HR-1 High Resolution Melter (Idaho Technology, USA) and b) PTEN expression levels using immunohistochemical analysis. All samples with mutant melting transitions were verified by DNA sequencing. PI3K pathway activation defined by the presence of PIK3CA hotspot mutations and/or low or absent PTEN was correlated to the response to trastuzumab-based therapy.Results. Fifty-six pts received trastuzumab in combination with vinorelbine (n=15) or taxane-based chemotherapy (n=41). Median age was 62 years, 55 pts (98.2%) had PS 0-1, 10 (17.9%) were premenopausal, 27 (48.2%) had hormone receptor negative tumors and 37 (66.1%) had visceral metastases. HRMA identified the presence of PIK3CA mutations in 22 (39.3%) pts [exon 9 mutations in 5 (8.9%) and exon 20 mutations in 17 (30.4%) pts]. Subsequent PIK3CA sequence analysis demonstrated exon 9 mutations (E542K and E545K) in 4 (7.1%) pts and exon 20 mutations in 17 (30.4%; H104R in 16, and H1047L in 1) corresponding to a PIK3CA hotspot mutation frequency of 35.7%. Only in one sample that showed a mutant melting transition by HRMA, DNA sequencing did not confirm the presence of a PIK3CA mutation in exon 9, possibly due to lower sensitivity. Absent or reduced PTEN expression was detected in 22 (39.3%) of 47 pts. In 9 (16.1%) pts, PTEN score could not be retrieved. A trend for lower response rates was observed for patients with PI3K hotspot mutations compared to pts with wild-type tumors (35% vs 60%, p= 0.074). No difference in response rates was identified for pts with low or absent compared to normal PTEN expression (p=0.920). However, activated PI3K pathway (presence of PI3K mutations and/or low or absent PTEN expression) was associated with significantly lower response rates compared to non-activated one (40.6% vs 72.2%; p=0.032).Conclusions. The activation of PI3K pathway is predictive of response to first-line chemotherapy plus trastuzumab in pts with HER2-overexpressing MBC.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2022.
Multiple Subtypes of HER-2/Neu-Positive Metastatic Breast Cancer.
Authors: Lipton A. , Leitzel K. , Koestler W. , Fuchs E. , Singer C. , Ali S. , Huang W. , Sperinde J. , Goodman L. , Jin X. , Banerjee J. , Weston J. , Mukherjee A. , Larson J. , Weidler J. , Paquet A. , Williams S. , Winslow J. , Parry G. , Bates M.
Published: December 2010

Background: Using IHC or FISH to select patients for trastuzumab-based therapy, only half of HER2-positive patients show evidence of response. In vitro data implicate HER2:HER3 heterodimers and p95HER2 (p95), the truncated 95-kilodalton C-terminal fragment of HER-2 lacking the trastuzumab binding site, as mediators of resistance to trastuzumab at the receptor level. We have previously reported that central FISH-positive patients with low HER2 protein expression by VeraTag had significantly reduced response to trastuzumab compared to patients who had FISH-positive tumors with high HER2 protein expression (Lipton, SABCS 2008). Adding quantitative measurements of HER3 and p95, we offer evidence for the existence of multiple sub-types of HER2-positive tumors that respond differently to trastuzumab.Methods: Using the VeraTag assay, quantitative protein measurements of HER2, HER3, and p95 were made in FFPE specimens from a cohort of patients with metastatic breast cancer (MBC) and correlated with time to progression (TTP) and overall survival (OS) following treatment with first-line trastuzumab using Kaplan-Meier (KM) and Cox proportional hazards regression analyses.Results: Measurements of HER2 (H2T), HER3 (H3T) and p95 were made in FFPE tumor samples from 95 patients treated with first-line trastuzumab for metastatic breast cancer. Within the group that overexpressed HER2 by the VeraTag Assay (n=60), a group with highly overexpressed HER2 (n=15) had shorter TTP and OS than those that had moderate HER2 overexpression (median TTP 4.6 vs. 12 mos, HR=2.1; p=0.011; median OS 29 vs. 40 mos, HR=2.0; p=0.047). Within the subgroup with moderate H2T overexpression (n=45), bivariate Cox analyses demonstrated that p95 and H3T were independent predictors of TTP (p95 HR=2.1; p=0.031; H3T HR=3.5; p=0.0037). For OS, p95 was significant and H3T showed a strong trend (p95 HR=2.5; p=0.025, H3T HR=2.2; p=0.089). Univariate KM analysis with the p95+ and H3T+ groups combined, gives the results in the table below. These data suggest that HER2-positive breast cancer patients can be classified into at least 4 sub-groups with different outcomes following trastuzumab treatment.Conclusions: These data suggest the existence of multiple subgroups of HER2-positive patients expressing varying HER2, p95, and HER3 levels that experience different clinical outcomes following treatment with trastuzumab. Furthermore, the association of HER3 and p95 overexpression with poor response to trastuzumab in otherwise HER2-positive tumors suggests possible treatment approaches with combinations of targeted therapies.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2030.
Titration of Serum p53 Antibodies Predicts Pathological Response in Women with Operable Early Stage Breast Cancer Receiving Preoperative Sequential FEC and Docetaxel Chemotherapy.
Authors: Hideo S. , Yoshiaki N. , Hideotoshi K. , Kimihiro T. , Satoko S. , Chinami K. , Emiko M. , Sumiko N. , Kenichi N. , Shinji O.
Published: December 2010

Background: In breast cancer, emerging dates demonstrate that p53 mutation is associated with response to preoperative chemotherapy, indicating that p53 mutation may be a predictive factor for response to preoperative chemotherapy. The accumulation of mutant p53 protein has been found to induce the formation of serum p53 IgG antibodies in breast cancer patients. A highly specific quantitative enzyme-linked immunosorbent assay (ELISA) kit has been developed to detect p53 antibody titers. However, whether this titration of serum p53 antibody reflects response to preoperative chemotherapy is unknown. The aim of this study was to evaluate the clinical implication of serum p53 antibody titers as a predictive factor for preoperative chemotherapy in breast cancer.Patinets and Methods: In this study, we analyzed 103 women with operable early stage breast cancer treated by preoperative chemotherapy and definitive curative surgery. Patients received four cycles of FEC (fluorouracil 500 mg/m2, epirubicin 100 mg/m2, cyclophosphamide 500 mg/m2 q3w) followed by four cycles of docetaxel (75 mg/m2 q3w). The pretreatment serum samples were used to determine the p53 antibody titers. The test's cut-off value was determined to be 1.3 U/ml based on reference distribution in healthy control individuals.Results: Twenty-three of 103 patients (22.3%) were positive for serum p53 antibody. The frequency of pathological complete response (pCR) was significantly higher in cases with positive for p53 antibody than in patients with negative for p53 antibody (9/23 or 39% vs. 15/80 or 23%; p=0.04). According to intrinsic subtypes, the tendency was most emphasized in cases with HER2-positive cases (6/9 or 67% vs. 8/25 or 32%). Interestingly, among 8 patients with high p53 antibody titers (>100 IU/ml) 7 patients experienced pCR and remaining one patients showed only foci invading areas in primary tumor and complete remission in axillary lymph node metastasis, suggesting that the value of p53 antibody titers may more precisely predict response to preoperative chemotherapy.Conclusion: p53 mutation analysis using titration of serum p53 antibody is useful predictive test for response to preoperative chemotherapy in patients with breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2038.
Chemosensitivity Testing of Circulating Epithelial Cells (CETC) in Breast Cancer Patients and Correlation to Clinical Outcome.
Authors: Pachmann K. , Stein E. , Spitz G. , Schill E. , Pachmann U.
Published: December 2010

In spite of ample prognostic markers available in breast cancer, still a considerable proportion of patients with good prognostic markers suffers relapse whereas patients with poor prognostic markers may remain disease free. It would, therefore, be desirable to control, at the individual patient level, whether the applied therapy is effective. Our previous work indicates, that in cancer patients most of the epithelial cells circulating in peripheral blood (CETC) are part of the tumor and that the response of these cells reflects the response of the tumor to the applied therapies.Therefore, these cells were used to assay chemosensitivity in short time cultures analyzing the percent of cell killing during short time incubation and monitoring the decrease or increase in numbers of these cells during treatment with the respective agents providing a unique tool for therapy surveillance. Patients were prospectively analysed for the number of CETC before each new combination of chemotherapy. 1ml of blood was drawn into EDTA vials, red blood cells lysed and the white blood cell pellet stained with FITC-labelled anti-Epcam. Green fluorescent cells were detected by image analysis and dead cells excluded due to red PI fluorescence. Activity of individual compounds was determined using three different concentrations of each compound at 3h, 6h and 12hs and displayed as % cell killing. The in vitro results were then compared to in vivo reduction of CETCs and to the reduction of a marker lesion.215 patients have been investigated so far. Cell killing was dose and time dependent. The highest killing rates were observed with Epirubicin and Taxanes, agents which are known for their high activity in breast cancer. Less than 20% killing activity was termed marginal activity. In vitro sensitivitity was highly significantly predictive of in vivo CETC reduction. In some cases an increasing resistance could be shown to develop during repeated cycles of the same combination therapy. CETC reduction was correlated with a prolonged progression free survival.Thus this approach can in the future be used to test in advance the sensitivity of the circulating tumor cells to chemotherapy and at the same time monitor the current response of the cells to therapy in vivo in order to optimize and individualize therapy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2044.