Molecular Subgroups Of Breast Cancer Research Articles

Biological processes associated with breast cancer subtypes: A meta-analysis study

This study delves into the modular mechanisms underlying various breast cancer subtypes, including Basal-like, HER2, Luminal A, Luminal B, Normal-like, and the differences between Luminal A and B. Through microarrays meta-analyses, the research identifies potential biomarkers for these subtypes by comparing each with the normal state, revealing 408, 429, 531, 346, 113, and 1085 differentially expressed genes associated with Basal-like, HER2, Luminal A, Luminal B, Normal-like, and Luminal A vs Luminal B, respectively. Significant enrichment of top GO terms like 'nuclear-transcribed mRNA catabolic process nonsense-mediated decay', 'SRP-dependent cotranslational protein targeting to membrane', 'translational initiation', 'rRNA processing', and 'viral transcription and response to corticosteroid' was observed in different breast cancer subtypes. Specifically, in the comparison between Luminal A and B cancers, 'tumor necrosis factor-mediated signaling' was the most enriched pathway. The most differentially expressed genes in this comparison were 'TOP2A, AURKA, RRM2, CDK1, and MDA2L1' (up-regulated), and 'LTF and MYBPC1' (down-regulated). These insights could be pivotal in developing new clinical-genomic models and identifying novel therapeutic strategies for specific molecular subgroups of breast cancer. The present study aims to investigate the modular mechanisms underlying different breast cancer subtypes and identifies potential biomarkers for Basal-like subtype (Normal vs Basal-like), HER2 subtype (Normal vs HER2), Luminal A subtype (Normal vs Luminal A), Luminal B subtype (Normal vs Luminal B), Normal-like subtype (Normal vs Normal-like) and between Luminal A and B (Luminal A vs Luminal B) using microarrays meta-analyses. 408, 429, 531, 346, 113, and 1085 differentially expressed genes were associated with Basal-like, HER2, Luminal A, Luminal B, Normal-like subtypes, and ‘Luminal A vs Luminal B’, respectively. Top GO terms significantly enriched for different breast cancer subtypes include ‘nuclear-transcribed mRNA catabolic process nonsense-mediated decay’, ‘SRP-dependent cotranslational protein targeting to membrane’, ‘translational initiation’, ‘rRNA processing’, and ‘viral transcription and response to corticosteroid’. The comparison between Luminal A and B cancers found that ‘tumor necrosis factor-mediated signaling’ was the most enriched pathway and the most differentially expressed genes included ‘sTOP2A, AURKA, RRM2, CDK1 and MDA2L1 (up-regulated)’ and ‘LTF and MYBPC1 (down-regulated)’. These findings may contribute to defining new clinical-genomic models and identifying new therapeutic strategies in the specific molecular subgroups.

Tumor-infiltrating lymphocytes in triple negative breast cancer

The aim of this retrospective study is to assess the prognostic value of tumor-infiltrating lymphocytes in triple negative breast carcinomas that lack both estrogen, progesterone receptors and Her2 amplification.The group of triple negative carcinomas has worse survival rates than other molecular subgroups of breast cancer, but at the same time it is very heterogeneous, and therefore requires the search for additional prognostic factors to improve the prognosis of patients.Materials and methods. The study included 350 samples of biopsies and surgical material from patients with stage I–III triple negative breast cancer who had not previously received chemotherapy. The median follow-up was 3.8 years for overall survival and 3.2 years for relapse-free survival. The prognostic value of tumor-infiltrating lymphocytes was evaluated by Kaplan–Meyer survival curves with a log-rank test, and one- and multifactor regression analysis according to the proportional risks Cox model.Results. Kaplan–Meier survival curves showed statistically significant relationships between tumor-infiltrating lymphocytes and overall and recurrence-free survival with a general trend to improve data for tumors with high lymphocyte volume. Proportional risks Cox model showed that tumors with a high number of tumor-infiltrating lymphocytes have statistically significantly better overall and recurrence-free survival rates (compared with tumors with a low number of tumor-infiltrating lymphocytes). The Kappa coefficient for interobserver variability was 0.79. It indicates a high level of agreement between pathologists in the evaluation of histological material.Conclusions. The high number of tumor-infiltrating lymphocytes in triple negative breast cancers is statistically significantly associated with better overall and recurrence-free survival rates. Assessment of tumor-infiltrating lymphocytes is rapid and does not require additional training, which makes this prognostic marker convenient to use.

Correlation Between Gamma-Glutamyl Transferase Activity and Glutathione Levels in Molecular Subgroups of Breast Cancer.

The gamma-glutamyl cycle catalyzed by gamma-glutamyl transferase (GGT) plays an important role in glutathione (GSH) homeostasis in the cell. In cells continuously exposed to the drug, the main phase of the enzymatic detoxification is the conjugation of the drug with GSH catalyzed by glutathione-S-transferase (GST). Conjugation of drugs with GSH is the first step in the development of chemotherapeutic drug resistance. In this study, we aimed to investigate the relationship between GGT and GSH in molecular subgroups of breast cancer patients. Serum GGT activity and GSH levels for patients diagnosed with breast cancer (n=58) and healthy controls (n=8) were measured by a spectrophotometric method and a colorimetric kit, respectively. GGT activity was significantly higher in the total patient group and in the molecular subgroups than those in the control groups (p<0.05). Serum GSH levels were higher in the patient groups compared to controls without reaching statistical significance (p>0.05). GGT activity was positively correlated with GSH levels in the total patients and healthy controls (p<0.001 and p<0.05, respectively). There was also a positive correlation between GGT activity and GSH levels in Luminal A, HER2-positive (Human epidermal growth factor receptor 2), and Triple-negative groups (p<0.05). This is the first study showing the relationship between GGT and GSH in molecular subgroups of breast cancer. An increase in GGT activity may affect intracellular GSH synthesis. Therefore, having a correlation between GGT and GSH in some molecular subgroups may affect the course of treatment in these patients.

Neoadjuvant Chemotherapy Plays an Adverse Role in the Prognosis of Grade 2 Breast Cancer.

Background: The role of neoadjuvant chemotherapy (NAC) in the prognosis of breast cancer among patients with grade 2 tumors remains unclear. As such, we aimed to explore the relationships between NAC and survival outcomes among patients with grade 2 breast cancer.Materials and Methods: We collected data on 726 breast cancer patients with grade 2 tumors and at least 5-years of follow-up from the date of diagnosis. We then conducted survival analyses to examine the association between NAC and disease-free survival (DFS) and overall survival (OS). The role of NAC in prognosis was further examined in subgroup analyses, with patients stratified according to molecular subtypes, histological grade, ER status, PR status, HER2 status and Ki67 index. We also determined the main sites of local recurrence, as well as these organs involved in distant metastasis among patients receiving NAC. Finally, we analyzed independent predictive factors for DFS and OS using Cox regression analyses.Results: Among patients who received NAC, the prevalence of pathologic complete response (pCR) was 9.87% (23/233), with 32.6% of patients (76/233) experiencing partial response. Survival analyses demonstrated that NAC had an overall adverse effect on DFS and OS. Subgroup analyses showed that patients who received NAC had shorter DFS in all molecular subgroups of breast cancer, with exception of triple negative breast cancer (TNBC) patients. NAC was also associated with shorter OS among patients with histological grade of 2 and a low Ki67 index. The main recurrence site was the chest well, while distant metastasis occurred in the bone, liver and lung. In Cox regression analyses, we found that NAC was an independent predictor for DFS, but not for OS.Conclusions: NAC may have an adverse effect on breast cancer prognosis among patients with grade 2 tumors. These patients need not receive NAC, except when the patient has a strong desire for breast conservation, and this is unlikely to be achieved in the absence of NAC.

Gene modules associated with breast cancer distant metastasis-free survival in the PAM50 molecular subtypes.

To identify PAM50 subtype–specific associations between distant metastasis-free survival (DMFS) in breast cancer (BC) patients and gene modules describing potentially targetable oncogenic pathways, a comprehensive analysis evaluating the prognostic efficacy of published gene signatures in 2027 BC patients from 13 studies was conducted. We calculated 21 gene modules and computed hazard ratios (HRs) for DMFS for one-unit increases in module score, with and without adjustment for clinical characteristics. By comparing gene expression to survival outcomes, we derived four subtype-specific prognostic signatures for BC. Univariate and multivariate analyses showed that in the luminal A subgroup, E2F3, PTEN and GGI gene module scores were associated with clinical outcome. In the luminal B tumors, RAS was associated with DMFS and in the basal-like tumors, ER was associated with DMFS. Our defined gene modules predicted high-risk patients in multivariate analyses for the basal-like (HR: 2.19, p=2.5×10−4), luminal A (HR: 3.03, p=7.2×10−5), luminal B (HR: 3.00, p=2.4×10−10) and HER2+ (HR: 5.49, p=9.7×10−10) subgroups. We found that different modules are associated with DMFS in different BC subtypes. The results of this study could help to identify new therapeutic strategies for specific molecular subgroups of BC, and could enhance efforts to improve patient-specific therapy options.

Genomic profiling of breast cancers.

To describe recent advances in the application of advanced genomic technologies towards the identification of biomarkers of prognosis and treatment response in breast cancer. Advances in high-throughput genomic profiling such as massively parallel sequencing have enabled researchers to catalogue the spectrum of somatic alterations in breast cancers. These tools also hold promise for precision medicine through accurate patient prognostication, stratification, and the dynamic monitoring of treatment response. For example, recent efforts have defined robust molecular subgroups of breast cancer and novel subtype-specific oncogenes. In addition, previously unappreciated activating mutations in human epidermal growth factor receptor 2 have been reported, suggesting new therapeutic opportunities. Genomic profiling of cell-free tumor DNA and circulating tumor cells has been used to monitor disease burden and the emergence of resistance, and such 'liquid biopsy' approaches may facilitate the early, noninvasive detection of aggressive disease. Finally, single-cell genomics is coming of age and will contribute to an understanding of breast cancer evolutionary dynamics. Here, we highlight recent studies that employ high-throughput genomic technologies in an effort to elucidate breast cancer biology, discover new therapeutic targets, improve prognostication and stratification, and discuss the implications for precision cancer medicine.

Role of urokinase plasminogen activator and plasminogen activator inhibitor mRNA expression as prognostic factors in molecular subtypes of breast cancer.

BackgroundProtein levels of urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) determined by enzyme-linked immunosorbent assay from fresh-frozen tumor tissue have been evaluated as prognostic factors in prospectively randomized trials in breast cancer. However, the role of uPA and PAI-1 in the context of breast cancer subtypes and for mRNA expression of these factors is less clear.MethodsWe evaluated uPA and PAI-1 mRNA expression using the Affymetrix HG-U 133A array within molecular subgroups of breast cancer in cohorts of patients with systemic treatment (cohort A, n=362) and without systemic treatment (cohort B, n=200). We validated mRNA expression in a cohort of HER2-positive breast cancer patients (cohort C, n=290). Luminal, triple-negative, and HER2-positive subcohorts were defined by ESR1 and ERBB2 mRNA expression using predefined cutoffs.ResultsIn the entire cohort A, elevated PAI-1 but not uPA mRNA expression was associated with shorter disease-free survival (P=0.007 for PAI and 0.069 for uPA). Regarding different molecular subgroups, 67% (n=244) of tumors were luminal, 14% (n=49) were HER2-positive, and 19% (n=69) were triple-negative. Elevated PAI-1 mRNA expression was associated with shorter disease-free survival only in the HER2-positive subgroup (P=0.031). The same disease-free survival results were found for uPA in HER2-positive patients (P=0.011). In contrast, no association between either marker and survival was observed in the luminal or triple-negative subgroups. In the HER2-positive validation cohort C, elevated uPA and PAI-1 mRNA expression also showed strong associations with shorter disease-free survival (P=0.014 for PAI-1, P<0.001 for uPA).ConclusionIn this study, the prognostic impact of uPA and PAI-1 expression was mainly observed in patients with HER2-positive tumors.

Abstract P6-06-44: Role of uPA and PAI-1 mRNA expression as prognostic factors in molecular subtypes of breast cancer

Abstract Background: Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) determined by ELISA from fresh-frozen tumor tissue have been evaluated as prognostic factors in prospectively randomized trials in node-negative breast cancer. However, uPA and PAI-1 mRNA expression might reveal new clinically relevant information using paraffin-embedded tumor tissue. Methods: We evaluated uPA and PAI-1 mRNA expression with the Affymetrix HG-U 133A array also within molecular subgroups of breast cancer in a finding cohort of breast cancer patients (cohort A; n = 548). We validated mRNA expression in a cohort of HER2 positive breast cancer patients (cohort B, n = 347). For uPA and PAI-1, mRNA values below and above the median were compared in two different probesets. “Luminal”, “triple-negative” and “HER2 positive” subcohorts were defined by ESR1 and ERBB2 mRNA expression using pre-defined cut-offs. Results: In cohort A, elevated PAI-1 mRNA expression was associated with shorter disease-free survival (DFS) and overall survival (OS), elevated uPA mRNA expression was associated with shorter OS. Regarding different molecular subgroups, 71% (n = 388) of tumors had a luminal, 13% (n = 69) a HER2 positive and 17% (n = 91) a triple-negative subtype. Only in the HER2 positive subgroup, elevated PAI-1 mRNA expression was associated with shorter disease-free survival and overall survival (p = 0.003 for DFS and p = 0.012 for OS). Same results were found for uPA in HER2 positive patients (DFS, p = 0.027 and OS, p = 0.011). In contrast, no association between both markers and DFS and OS could be seen in the luminal or triple-negative subgroups. In the HER2 positive validation cohort B elevated uPA and PAI-1 mRNA expression showed a strong association with shorter DFS (p = 0.013 for PAI-1, p = 0.001 for uPA). Conclusion: Results of this study demonstrate that the prognostic impact of uPA and PAI-1 mRNA expression was observed mainly in patients with HER2 positive tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-44.

Validity of the proliferation markers Ki67, TOP2A, and RacGAP1 in molecular subgroups of breast cancer

High proliferation rates are characteristic of cancer, and proliferation markers make up the majority of genes included in RNA-based prognostic gene signatures applied for breast cancer patients. Based on prior data on differences in molecular subgroups of breast cancer, we hypothesized that the significance of single proliferation markers might differ in luminal, Her2-positive and triple-negative subtypes. Therefore, we compared mRNA expression data of Ki67, TOP2A, and RacGAP1 using a pool of 562 Affymetrix U133A microarrays from breast cancer samples. "Luminal," "triple-negative," and "Her2-positive" subcohorts were defined by ESR1 and ERBB2 mRNA expression using pre-defined cut-offs. The analysis of the three potential proliferation markers revealed subtype-specific differences: in luminal carcinomas, expression of all three markers was a significant indictor of early recurrence in univariate and multivariate analysis, but RacGAP1 was superior to Ki67 and TOP2A in significance. In triple-negative tumors, only Ki67 was a significant and independent marker, whereas none of the markers showed a significant prognostic impact in Her2-positive cases. Within the group of luminal carcinomas, the proliferation markers had different impact depending on the treatment of patients: in untreated patients, Ki67, TOP2A, and RacGAP1 were significant and independent prognostic markers. In chemotherapy-treated patients, overexpression of all three markers was predictive for early recurrence, but only RacGAP1 retained significance in multivariate analysis. In contrast, RacGAP1 was the only predictive proliferation marker in the endocrine treatment group. These data point to subtype-specific differences in the relevance of proliferation-associated genes, and RacGAP1 might be a strong prognostic and predictive marker in the luminal subgroup.

Dacomitinib (PF-00299804), an Irreversible Pan-HER Inhibitor, Inhibits Proliferation of HER2-Amplified Breast Cancer Cell Lines Resistant to Trastuzumab and Lapatinib

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC(50) values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G(0)-G(1) arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib.

Vitamin D and the mammary gland: a review on its role in normal development and breast cancer

Breast cancer is a heterogeneous disease associated with diverse biological behaviours and clinical outcome. Although some molecular subgroups of breast cancer have a targeted therapy, the most aggressive tumours still lack a molecular target. Despite vitamin D being classically associated with the physiological role of calcium regulation and phosphate transport in bone metabolism, several studies have demonstrated a wide range of functions for this hormone, which are particularly important in the field of cancer. The mechanisms underlying the protective actions of vitamin D in cancer development are only sparsely understood, but evidence shows that vitamin D participates in cell growth regulation, apoptosis and cell differentiation. In addition, it has been implicated in the suppression of cancer cell invasion, angiogenesis and metastasis. Most of vitamin D biological actions are mediated by the vitamin D receptor and the synthesis and catabolism of this hormone are regulated by the enzymes CYP27B1 and CYP24A1. In the present review we highlight research data concerning the function of this hormone in the mammary gland, with a special focus on breast carcinogenesis. Hence, and although the available data are controversial, we consider not only updated information on the epidemiology of vitamin D in breast cancer and its potential value as a therapeutic agent or prophylactic (with an emphasis on molecular mechanisms and effectors of vitamin D action), but include data on its role in other stages of breast cancer progression as well. Accordingly, we review data on the influence of vitamin D in the development of normal breast and the expression of vitamin D-related proteins (VDR, CYP27B1 and CYP24A21) in benign mammary lesions and ductal carcinomas in situ.

Delineation of molecular subgroups of breast cancer with distinct genomic profiles and different clinical courses: a novel definition of disease subclasses

Delineation of molecular subgroups of breast cancer with distinct genomic profiles and different clinical courses: a novel definition of disease subclasses

799 Molecular subgroups of breast cancer show distinct genomic profiles and different clinical courses: a novel definition of disease subclasses

Objectives: To examine the utility of a risk-approach model in identifying infants at greater risk of postneonatal mortality (PNM) and to determine whether there is a relationship between PNM and linkage of at-risk infants to primary health care. Methods: The Sheffield Birth Score instrument was applied to 90,846 newborn infants to determine high-score (HS) and low-score (LS) infant risk groups. Health care visit data were collected on all HS infants who were referred for primary pediatric care. Mortality rates were calculated for both HS and LS infant groups and for HS infants who were linked and not linked to care. Results: The HS infant group was at significantly greater risk of PNM (p < 0.0001). The linked group had a lower PNM rate (p < 0.05), and linked/not-linked group differences were noted for 11 of 20 variables. When these 11 variables and the linked/not-linked variable were entered into a logistic regression analysis, linkage was the only significant variable (p < 0.01) in predicting PNM. Conclusions: The Sheffield Birth Score differentiated at birth those infants who were at greater risk of PNM. The lower incidence of PNM among linked HS infants suggests a promising argument for early pediatric intervention. Further research to clarify specific factors that influence health care participation decisions is suggested. (J Pediatr 1998;132:822-6)

Identification of molecular subtypes within a formalin-fixed, paraffin-embedded breast cancer tumour cohort

Breast cancer is not a single disease but is highly heterogeneous at both the molecular and clinical levels. Gene expression profiling of breast tumours by multiple independent groups and technologies have revealed five major molecular subtypes of breast cancer. These molecular differences result in distinct clinical outcomes and responses to treatment. The gene expression profiling studies that have defined the molecular subgroups of breast cancer to date were performed using fresh frozen tissue. Routine clinical practice dictates the preservation of surgical specimens via paraffin embedding of formalin-fixed tissue. Therefore, derivation of molecular subgroups of breast cancer from formalin-fixed, paraffin-embedded (FFPE) preserved tissue would have more application in the clinical setting as such profiles could be applied to routinely collected specimens. The Almac Diagnostics' Breast Cancer DSA™ has been optimised for analysis of FFPE samples enabling the use of these valuable archived tissue banks. We have demonstrated the ability to identify the molecular subgroups previously defined within a cohort of sporadic, BRCA1 mutant and BRCA2 mutant FFPE breast tumours as well as defining two novel subgroups within this tumour set. This study demonstrates that it is possible to derive biologically meaningful data from a cohort of archived FFPE tumour samples using the Almac Breast DSA™. We demonstrate that there is considerable molecular diversity within BRCA mutant and sporadic breast tumours, suggesting that traditional assumptions of the behaviour of tumours based on their immunohistochemistry status may not always be correct. At present, a number of clinical trials are stratifying patients for poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor therapy based on BRCA mutation and triple-negative status. The data presented here would suggest that not all BRCA1 mutant, BRCA2 mutant and indeed triple-negative patients are similar at the molecular level and as such will not respond equally to PARP-1 inhibitor or indeed other therapeutics in the same manner.

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro

IntroductionAlterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines.MethodsForty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response.ResultsCell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade.ConclusionsThese studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991.

Pre-clinical activity of the PARP inhibitor AZD2281 in human breast cancer cell lines and in combination with DNA damaging agents.

Abstract Abstract #1038 Background: Deficiencies in DNA repair mechanisms have been associated with breast cancer. AZD2281, a potent, oral, PARP inhibitor has been shown to have clinical activity in patients with BRCA mutant breast cancers. Laboratory studies have suggested that non-BRCA mutant breast cancers may also be sensitive to PARP inhibition in tumors as a consequence of deficiencies in other homologous recombination (HR) repair components. Using a large panel of human breast cancer cell lines we tested the hypotheses that (1) there may be a subset of non-BRCA mutant breast cancers that are sensitive to single-agent AZD2281 and (2) AZD2281 would potentiate the cytotoxic effects of the DNA damaging agent cisplatin. Methods: 43 human cell lines representing known molecular subgroups of breast cancer (i.e. ER+, HER2 amplified, “triple-negative”), and 3 immortalized breast lines were treated in duplicate in adherent plates with AZD2281 using two-fold dilutions over 6 concentrations for 6 days. Dose response curves were generated using a cell count assay to calculate the IC50 of AZD2281. In addition, a subset of cell lines that grow under anchorage independent conditions were grown in triplicate in the presence and absence of 1 µM AZD2281 in soft agar for at least 3 weeks and growth inhibition was calculated as per cent of untreated control. Cell lines (both sensitive and resistant to single agent AZD2281) were also evaluated in combination with cisplatin in a cell count assay to assess the interaction between the two agents.&amp;#x2028; Results: The majority of breast cancer cell lines evaluated in the short term 2-D growth assay did not show significant growth inhibition (IC50 &amp;lt; 1 µM) following AZD2281 treatment, including a known BRCA mutant cell line, suggesting this assay may not be ideal for determining sensitivity to AZD2281. However, in the longer term anchorage independent clonogenic assay, approximately half of the cell lines evaluated demonstrated an IC50 &amp;lt; 1 µM. Of note, the majority of the cell lines representing a “triple-negative” phenotype appeared sensitive to AZD2281 in this assay. In addition, pre-treatment with AZD2281 prior to cisplatin, potentiated the growth inhibition seen with cisplatin in both AZD2281 sensitive and resistant cell lines. Additional studies evaluating predictive markers other than BRCA status are ongoing. Conclusion: The PARP inhibitor AZD2281 has significant pre-clinical activity in human breast cancer cell lines. In a clonogenic assay, cell lines representing the “triple negative” subtype were especially sensitive to AZD2281 supporting clinical development in this population, regardless of BRCA status. In addition, these pre-clinical data support the hypothesis that PARP inhibition may potentiate the effects of chemotherapy induced DNA damage and provide further rationale for clinical development. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1038.

Panobinostat (LBH589), a pan-DAC inhibitor, induces cell death in ER+ and HER2 amplified cell lines in vitro and is synergistic in vivo with trastuzumab.

Abstract Abstract #4047 Background: Histone modifications have been investigated as causing altered gene expression resulting in activation of oncogenic pathways. Panobinostat (LBH589) is a potent pan-deacetylase (DAC) inhibitor in clinical development. Given the potentially broad biological effects of this class of agent, we assessed the in vitro effects of panobinostat on a large panel of molecularly profiled human breast cancer cell lines to better define the differential effects of panobinostat across subtypes and to help guide clinical development. In addition, preclinical studies evaluated the effects of panobinostat and the HER2-targeted antibody, trastuzumab.&amp;#x2028; Methods: 43 human cell lines representing known molecular subgroups of breast cancer, and 3 immortalized breast lines were treated in duplicate with panobinostat using two-fold dilutions over 12 concentrations. Dose response curves were generated using a cell count assay to calculate IC50 and LD50 of panobinostat. Data were analyzed against baseline gene expression data (Agilent microarray) to identify genes associated with sensitivity (S) and resistance (R) to panobinostat. 12 cell lines (6 S; 6 R) were then treated with clinically relevant concentrations of panobinostat; RNA was isolated after 48 hrs. Agilent expression profiles compared pre-and post-treatment cell lines to identify differentially expressed genes (DEGs) that change in S and R cell lines. In vivo, nude mice bearing HER2+ and pathway-dependent BT-474 xenografts were treated with panobinostat alone and in combination with trastuzumab. Tumor growth inhibition and delay were measured.&amp;#x2028; Results: In vitro panobinostat has potent anti-proliferative effects across all breast cancer subtypes with most IC50 values &amp;lt;100nM. When analyzed by effects on cell death, luminal ER+ and HER2 amplified cell lines were more sensitive to lethality than non-luminal subtypes using a cut-off of LD50 50nM (Chi-square&amp;lt;0.05 for subtype). Of interest, the non-malignant, immortalized lines were very resistant (LD50 &amp;gt;1000nM), which suggests some differential activity in malignant cell lines versus those that are more “normal”. When gene expression profiles were analyzed pre-and post-treatment, a remarkable overlap was seen in number and specific genes changing after panobinostat exposure in both S and R lines. However, the degree of gene changes was greater in S lines than R lines. In vivo, panobinostat acted synergistically with trastuzumab to inhibit growth of the HER2 amplified breast cancer cell line, BT-474.&amp;#x2028; Conclusion: These studies suggest that ER+ and HER2 amplified breast cancers may be more likely to respond to the pan-DAC inhibitor panobinostat. Profiling studies identified 218 DEGs that were significantly changed between S and R lines. These data support the ongoing development of panobinostat in breast cancer and provide rationale for patient selection. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4047.

PD 0332991, a selective CDK 4/6 inhibitor, preferentially inhibits proliferation of luminal ER+ breast cancer cells and acts synergistically with tamoxifen and trastuzumab in vitro.

Abstract Abstract #5064 Background: Cell cycle dysregulation is a common molecular finding in malignancy and the cyclin-D kinases (CDK) represent an attractive target in this pathway. PD 0332991 (Pfizer Inc.) is an orally active potent and highly selective inhibitor of CDK 4 and CDK6 kinases with the ability to block pRb phosphorylation at serine 780 and 795 in the low nanomolar range. To identify predictors of response to PD 0332991, we determined the in vitro sensitivity to PD 0332991 of a large panel of molecularly characterized breast cancer cell lines, and then used baseline expression profiles to determine genes associated with response. Methods: 44 human breast cancer cell lines representing the known molecular subgroups of breast cancer (i.e. luminal, HER2, basal, etc) and 3 immortalized breast lines were treated in duplicate with PD 0332991 using two-fold dilutions over 12 concentrations. Dose response curves were generated using a cell count assay to calculate the IC50 across the panel. These data were then analyzed against baseline gene expression data (Agilent microarray) to identify genes associated with sensitivity and resistance to PD 0332991. ANOVA analysis identified genes associated with response and/or resistance to PD 0332991. Western blot analysis was performed analyzing the effects of PD 0332991 on pRb phosphorylation. Cell cycle analysis was performed using NIM-DAPI staining and flow-cytometry. In addition, combination studies were performed to analyze the interaction between PD 0332991 and tamoxifen and trastuzumab. These data were analyzed using Calcusyn software to generate a combination index (CI) to define the interaction as additive (CI=1), synergisitc (CI&amp;lt;1), or antagonistic (CI&amp;gt;1). Results: Cell lines representing the luminal ER+ subtype (including HER2 amplified) were most sensitive to inhibition by PD 0332991 and non-luminal/basal (triple-negative) were most resistant. ANOVA analysis identified 563 differentially expressed genes between the sensitive and resistant groups. While, several of these genes are associated with breast cancer subtype, pRb was elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell lines treated with PD 0332991 showed clear G0/G1 arrest and a decrease in S-phase fraction in sensitive but not in resistant cell lines. In addition, Western blot showed that pRb phosphorylation is blocked in a time dependent manner. Finally, the combination of tamoxifen and PD 0332991 was strongly synergistic in three ER+ cell lines evaluated and appeared synergistic with trastuzumab as well. Conclusion: These studies suggest that a subgroup of breast cancers may be more likely to benefit from treatment with the PD 0332991 CDK4/6 inhibitor than others. This group represents ER+ luminal breast cancer and is associated with elevated pRb and low p16. The combination of PD 0332991 and tamoxifen and trastuzumab shows promising biologic activity in ER+ and HER2 amplified breast cancer cell lines, respectively. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5064.