Multiple Breast Cancer Subtypes Research Articles

Abstract IA18: Targeting the androgen receptor in multiple breast cancer subtypes

Abstract The androgen receptor (AR) is even more widely expressed in breast cancer (BC) than estrogen receptor alpha (ER) or progesterone receptor (PR), and recently AR has emerged as a useful marker to refine classification of breast cancer (BC). However, we still understand relatively little about the specific effects of AR or its potential as a therapeutic target in the different subtypes of BC. Our data from clinical samples suggests that some ER+ tumors can switch from growth driven by estrogens to growth driven by adrenal androgens, particularly when the estradiol (E2)/ER pathway is inhibited by standard ER-directed endocrine treatments. Indeed, we have evidence showing that a high ratio of AR to ER (≥2.0) leads to a > 4 fold increased risk for relapse while on tamoxifen (HR=4.43, P<0.0001) and is a strong independent predictor of disease-free survival (HR=4.04, P=0.002). Since approximately 30% of metastatic ER+ tumors exhibit de novo resistance to standard endocrine therapy and all patients with metastatic disease ultimately progress with acquired resistance, targeting AR in ER+ BC may be clinically useful. Thus, clinical trials evaluating the role of androgen receptor inhibition in BC are underway. Importantly, we find that new generation anti-androgens that block AR DNA binding affect BC differently than older generation anti-androgens (which allow DNA binding), leading to new insights into the role of AR. When AR is excluded from the nucleus, both androgen- and estradiol (E2)-stimulated proliferation is inhibited in ER+ breast cancers, suggesting that AR plays a previously-unknown role in E2-mediated ER activity. Both enzalutamide (ENZ) and bicalutamide inhibited androgen-mediated proliferation of breast cancer lines in vitro. Interestingly, ENZ uniquely inhibited E2-mediated proliferation of ER.+/AR+ breast cancer cells in vitro, and also inhibited E2-driven tumor growth as effectively as tamoxifen in vivo. When opposing androgen-stimulated tumor growth in ER+ BC in vivo models, ENZ inhibited proliferation and increased apoptosis, while in ER- models it increased apoptosis but did not alter proliferation. When opposing E2-stimulated tumor growth in vivo ENZ decreased proliferation but did not increase apoptosis. In Her2+ but non-amplified, BC models the AR inhibitor ENZ, inhibited proliferation as well or better than trastuzumab (TRAS), whereas TRAS showed a greater inhibitory effect than ENZ in Her2 amplified lines. In all models tested, the combination of ENZ and TRAS inhibited proliferation more effectively than either agent alone. In TRAS resistant lines, addition of ENZ to TRAS significantly inhibited proliferation. Liganded AR upregulates Her3 in some Her2+ BC lines; however, in other Her2+ lines, Her3 is not increased by androgen stimulation, yet anti-androgens still inhibit proliferation. Our data suggest that the AR and Her2 pathways are linked in ways not previously understood and that there are novel Her3-independent mechanisms whereby AR impacts Her2+BC. A subset of TNBC express AR and knock down of AR using lentiviral shRNAs significantly reduced proliferation in multiple TNBC lines. Propidium iodide cell cycle analysis demonstrated that knockdown of AR in TNBC decreased the percentage of cells in G2/M. Lastly, inhibition of AR with ENZ, blocked ligand-mediated nuclear translocation of AR as assessed by nuclear and cytosolic fractionation and significantly decreased proliferation of TNBC in vitro. In summary, our studies demonstrate that AR plays varying roles in the different BC subtypes and will serve to guide appropriate combinations with AR inhibitor and existing therapeutic strategies. Further, we find that assessment of the amount of AR relative to ER may be informative. Citation Format: Nicholas C. D'Amato, Dawn R. Cochrane, Valerie N. Barton, Liu Bolin, Susan M. Edgerton, Sebastian Bernales, Haihua Gu, Ann D. Thor, Jennifer K. Richer. Targeting the androgen receptor in multiple breast cancer subtypes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA18.

HER3, p95HER2, and HER2 protein expression levels define multiple subtypes of HER2-positive metastatic breast cancer

Trastuzumab is effective in the treatment of HER2/neu over-expressing breast cancer, but not all patients benefit from it. In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKT–mTOR pathway leading to trastuzumab insensitivity. We sought to investigate the potential of HER3 alone and in the context of p95HER2 (p95), a trastuzumab resistance marker, as biomarkers of trastuzumab escape. Using the VeraTag® assay platform, we developed a dual antibody proximity-based assay for the precise quantitation of HER3 total protein (H3T) from formalin-fixed paraffin-embedded (FFPE) breast tumors. We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumab-based therapy, and correlated the results with progression-free survival and overall survival using Kaplan–Meier and decision tree analyses that also included HER2 total (H2T) and p95 expression levels. Within the sub-population of patients that over-expressed HER2, high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumab-based therapy. Based on quantitative H3T, p95, and H2T measurements, multiple subtypes of HER2-positive breast cancer were identified that differ in their outcome following trastuzumab therapy. These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy, and that multiple subtypes of HER2-positive breast cancer may be defined by quantitative measurements of H3T, p95, and H2T.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2665-0) contains supplementary material, which is available to authorized users.

Androgen receptor-induced tumor suppressor, KLLN, inhibits breast cancer growth and transcriptionally activates p53/p73-mediated apoptosis in breast carcinomas

Androgen receptor (AR) expression by immunohistochemistry correlates with better prognosis and survival among breast cancer patients. We and others have shown that AR inhibits proliferation and induces apoptosis in breast cancer cells. However, the mechanism of AR's anti-tumor effect in breast cancer is still not fully understood. Our recent study indicates that AR upregulates expression of tumor suppressor gene PTEN by promoter activation in breast cancer. KLLN, encoding KLLN protein, is a newly identified gene, which shares a bidirectional promoter with PTEN and is transcribed in the opposite direction. So far, the function of KLLN has never been studied in tumorigenesis. Here, we define KLLN as a tumor suppressor in breast carcinomas, which inhibits tumor growth and invasiveness. After analyzing 188 normal breast and 1247 malignant breast cancer tissues, we observed the loss of KLLN in multiple breast cancer subtypes and this decreased KLLN expression associates with tumor progression and increasing histological grade in invasive carcinomas. We characterize KLLN, for the first time, as a transcription factor, directly promoting the expression of TP53 and TP73, with consequent elevated apoptosis and cell cycle arrest in breast cancer cells. We demonstrate, in vitro and in murine xenograph models, that both KLLN and PTEN are AR-target genes, mediating androgen-induced growth inhibition and apoptosis in breast cancer cells. Our observations suggest that KLLN might be used as a potential prognostic marker and novel therapy target for breast carcinomas.

Mining the tissue-tissue gene co-expression network for tumor microenvironment study and biomarker prediction

BackgroundRecent discovery in tumor development indicates that the tumor microenvironment (mostly stroma cells) plays an important role in cancer development. To understand how the tumor microenvironment (TME) interacts with the tumor, we explore the correlation of the gene expressions between tumor and stroma. The tumor and stroma gene expression data are modeled as a weighted bipartite network (tumor-stroma coexpression network) where the weight of an edge indicates the correlation between the expression profiles of the corresponding tumor gene and stroma gene. In order to efficiently mine this weighted bipartite network, we developed the Bipartite subnetwork Component Mining algorithm (BCM), and we show that the BCM algorithm can efficiently mine weighted bipartite networks for dense Bipartite sub-Networks (BiNets) with density guarantees.ResultsWe applied BCM to the tumor-stroma coexpression network and find 372 BiNets that demonstrate statistical significance in survival tests. A good number of these BiNets demonstrate strong prognosis powers on at least one breast cancer patient cohort, which suggests that these BiNets are potential biomarkers for breast cancer prognosis. Further study on these 372 BiNets by the network merging approach reveals that they form 10 macro bipartite networks which show orchestrated key biological processes in both tumor and stroma. In addition, by further examining the BiNets that are significant in ER-negative breast cancer patient prognosis, we discovered a ubiquitin C (UBC) gene network that demonstrates strong prognosis power in nearly all types of breast cancer subtypes we used in this study.ConclusionsThe results support our hypothesis that the UBC gene network plays an important role in breast cancer prognosis and therapy and it is a potential prognostic biomarker for multiple breast cancer subtypes.

Abstract ES8-2: Breast Cancer Stroma: A Predictor of Clinical Outcome and Tumour Heterogeneity

Abstract Breast cancer heterogeneity is one of the principal obstacles both to predicting outcome and to determining an effective course of treatment for this disease. Individual cases demonstrate heterogeneity at multiple levels, including those parameters assessed by studies of gene expression, chromosomal aberrations, and classical and immuno-pathology. Although genomic technologies have been used to gain a better understanding of the impact of gene expression heterogeneity on breast cancer outcome by identifying gene expression signatures associated with clinical outcome, histopathological breast cancer subtypes, and a variety of cancer–related pathways and processes, relatively little is known about the effects of heterogeneity in the tumor microenvironment. We have addressed changes in stroma by analyzing changes in gene expression in stromal tissue associated with breast tumors when compared to normal breast tissue. We have integrated gene expression data from laser capture microdissected breast tumor stroma with matched normal stroma. Using this approach we have identified that the microenvironment of a breast tumor can be classified into one of six distinct molecular phenotypes exhibiting distinct biological functions and carrying prognostic information independent of existing therapeutic biomarkers and tumor subtypes. A trained predictor of 23 genes was developed and contains new information to stratify breast cancer subtypes. This is independent of clinical parameters and published predictors of outcome and identifies patients with poor outcome in multiple breast cancer expression data generated using using whole tissue. The stromal predictor selects poor outcome patients from multiple clinical subtypes of breast cancer and contains genes representing distinct biological features, including differential immune response, angiogenic response, as well as a hypoxic response. Elements of this signature are present in murine models of breast cancer. These results highlight the complex relationship between the tumor and its microenvironment, and underline the role that the stroma plays in tumor progression. These results demonstrate an important role for the tumor microenvironment in defining breast cancer heterogeneity, with a consequent impact upon clinical outcome. Novel therapies could be targeted at the processes that define the stroma classes, suggesting new avenues for the development of individualized treatment regimens. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr ES8-2.

Abstract P5-10-06: A functional role for miR-150 in breast cancer

Abstract Background: During mammary gland development, massive coordinated changes in protein expression govern the progression through pregnancy to lactation and involution. These dramatic changes are likely regulated in part at the translational level by changes in microRNAs (miRNAs). We profiled miRNA expression in mammary epithelial cells (MECs) isolated from mice at pregnancy day 14 (P14) or lactation day 2 (L2) and found that miR-150 is the most significantly downregulated miRNA between pregnancy and lactation. Interestingly, miR-150 was recently discovered to be decreased in mouse mammary tumors compared to normal mammary tissue in numerous transgenic models. However, a causal role for miR-150 has yet to be studied in human breast cancer and little is known about its functional role and relevant targets in the normal breast or breast cancer. Hypothesis: We hypothesized that miR-150 may be a tumor suppressor whose loss in breast cancer cells is an important event that allows for expression of multiple pro-tumorigenic genes. Methods: miR-150 levels in human breast cancers were evaluated in specimens of both ER+ and triple-negative breast cancer (TNBC) as compared to adjacent, non-involved normal breast epithelium by in situ hybridization. miR-150 levels were also measured by qRT-PCR in cell lines representative of multiple breast cancer subtypes. We stably restored miR-150 in TNBC cell lines by lentiviral infection and evaluated its effects on clonogenicity, growth in 3D culture, migration/invasion, and tumorigenicity. Expression of predicted miR-150 targets was assessed by immunoblot. Crossing of miR-150fl/fl mice with BLG-Cre or MMTV/NIC transgenic mice will be utilized to determine the effects of MEC-specific loss of this miRNA on mammary tumorigenesis. Results: In clinical samples, in situ hybridization reveals that miR-150 levels are lower in both ER+ and TNBC tumor specimens compared to adjacent normal epithelium, with triple-negative tumors having the lowest expression. All breast cancer cell lines tested also have low miR-150 expression as compared to normal mammary epithelial tissue, with TNBC cell lines expressing the lowest levels. Exogenous expression of miR-150 in breast cancer cell lines caused a dramatic decrease in migration and invasion in vitro, and we are testing predicted miR-150 targets for their role in this phenotype. Experiments with transgenic models will determine if loss of miR-150 in mammary epithelial cells in the MMTV/NIC mouse model results in decreased latency or increased tumor formation and metastasis, or if miR-150 loss during pregnancy results in alterations in lactation, hyperplasia, or tumor formation. Conclusions: TNBC specimens and cell lines have the lowest expression of miR-150, though decreased miR-150 expression compared to normal mammary epithelium is a common feature of breast cancers regardless of subtype. miR-150 expression dramatically inhibits breast cancer cell migration and invasion in vitro, and ongoing experiments will determine the relevant target genes. Supported by Susan G. Komen Grant KG090415 and NIH NICHD P01 PAR-10-245 (Project 3) to JKR Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-06.

What Are the Most Important New Targets in Breast Cancer?

Breast cancer is a remarkably heterogeneous disease. Data clearly demonstrate the presence of multiple molecular subtypes of breast cancer, and alterations in cell physiology shared by most tumors have been identified. These recent developments in our understanding of the molecular events involved in breast cancer have led to the identification of novel targets as well as anticancer therapies based on these targets. These therapies are referred to as targeted therapy in that the agent has a high specificity for molecules involved in molecular events responsible for the cancer phenotype. This review highlights some of the most promising future targets in breast cancer.

ES2-3: Patient-Derived Xenograft Models for Preclinical Breast Cancer Research: Not Just "Basal" Anymore.

Abstract Translational breast cancer research is hampered by difficulties in obtaining and studying primary human breast tissue, and by the lack of in vivo preclinical models that reflect the range of patient tumor biology accurately. In an effort to overcome these limitations, several groups have recently succeeded in propagating patient-derived breast cancer xenografts representing multiple breast cancer subtypes, including estrogen and progesterone receptor positive, and HER2 positive tumors. In our laboratory, we have developed a diverse set of human breast tumors grown as xenografts in the mammary fat pad of SCID/Beige and NOD/SCID/IL2γ-receptor null (NSG) mice, two relatively new immunocompromised mouse models, under various transplant conditions. In all cases, xenograft lines were established directly from breast cancer patient samples, without intervening culture in vitro, using the epithelium-free mammary fat pad as the transplantation site. Of the conditions tested, xenograft take rate was highest in the presence of a low-dose estradiol pellet without exogenous human fibroblasts. Thirty five stably transplantable xenograft lines representing 27 patients were established, using pre-treatment, mid-treatment, and/or post-treatment samples. Most patients yielding xenografts were “triple-negative” (ER−PR-HER2−) (n=21). However, we were able to establish lines from three ER−PR-HER2+ patients, one ER+PR+HER2−, one ER+PR-HER2−, and one “triple-positive” (ER+PR+HER2+) patients. Serially passaged xenografts show phenotypic consistency with the tumor of origin at the histopathology level, and remarkable stability across multiple transplant generations at both the genomic, transcriptomic, and proteomic levels. Of 27 lines evaluated fully, thirteen xenografts showed metastasis to the mouse lung. These models, in conjunction with others like them, thus serve as renewable, quality-controlled tissue resources, and are proving useful for preclinical evaluation of experimental therapeutics. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr ES2-3.

Elevated PI3K signaling drives multiple Breast Cancer subtypes

Most human breast tumors have mutations that elevate signaling through a key metabolic pathway that is induced by insulin and a number of growth factors. This pathway serves to activate an enzyme known as phosphatidylinositol 3' kinase (PI3K) as well as to regulate proteins that signal in response to lipid products of PI3K. The specific mutations that activate this pathway in breast cancer can occur in genes coding for tyrosine kinase receptors, adaptor proteins linked to PI3K, catalytic and regulatory subunits of PI3K, serine/threonine kinases that function downstream of PI3K, and also phosphatidylinositol 3' phosphatase tumor suppressors that function to antagonize this pathway. While each genetic change results in net elevation of PI3K pathway signaling, and all major breast cancer subtypes show pathway activation, the specific mutation(s) involved in any one tumor may play an important role in defining tumor subtype, prognosis and even sensitivity to therapy. Here, we describe mouse models of breast cancer with elevated PI3K signaling, and how they may be used to guide development of novel therapeutics.

Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

PIK3CA, which codes for the p110α catalytic subunit of phosphatidylinositol 3-kinase, is one of the most frequently mutated genes in human breast cancer. Here, we describe a mouse model for PIK3CA-induced breast cancer by using the ROSA26 (R26) knock-in system, in which targeted Pik3ca alleles can be activated through transgenic expression of Cre recombinase. We mated Pik3ca(H1047R) and Pik3ca(wt) knock-in lines with MMTV-Cre transgenics, which express Cre in mammary epithelium. Starting at approximately 5 months of age, female R26-Pik3ca(H1047R);MMTV-Cre mice, but not control R26-Pik3ca(wt);MMTV-Cre mice, developed mammary tumors, as well as lymphoid and skin malignancies. R26-Pik3ca(H1047R);MMTV-Cre mammary tumors were typically either adenosquamous carcinoma or adenomyoepithelioma. As p53 is the most commonly mutated gene in breast cancer, we tested for genetic interaction between Pik3ca(H1047R) and p53 loss-of-function mutations in R26-Pik3ca(H1047R);p53(loxP/+);MMTV-Cre mice. This led to decreased survival of double-mutant animals, which developed lymphoma and mammary tumors with rapid kinetics. Mammary tumors that formed in p53(loxP/+);MMTV-Cre conditional mutants were either poorly differentiated adenocarcinoma or spindle cell/EMT, whereas R26-Pik3ca(H1047R);p53(loxP/+);MMTV-Cre mammary tumors were mostly adenosquamous carcinoma or spindle cell/EMT indicating that double-mutant mice develop a distinct spectrum of mammary tumors. Thus, an oncogenic variant of PIK3CA implicated in multiple human breast cancer subtypes can induce a very diverse spectrum of mammary tumors in mice. Furthermore, Pik3ca(H1047R) shows cooperation with p53, which altered the specific tumors that formed. Thus, the two most frequently mutated genes in human breast cancer show cooperation in mammary tumor formation.

Therapeutic CDK4/6 inhibition in breast cancer: key mechanisms of response and failure

A hallmark of cancer is the deregulation of cell-cycle machinery, ultimately facilitating aberrant proliferation that fuels tumorigenesis and disease progression. Particularly, in breast cancers, cyclin D1 has a crucial role in the development of disease. Recently, a highly specific inhibitor of CDK4/6 activity (PD-0332991) has been developed that may have efficacy in the treatment of breast cancer. To interrogate the utility of PD-0332991 in treating breast cancers, therapeutic response was evaluated on a panel of breast cancer cell lines. These analyses showed that the chronic loss of Rb is specifically associated with evolution to a CDK4/6-independent state and, ultimately, resistance to PD-0332991. However, to interrogate the functional consequence of Rb directly, knockdown experiments were performed in models that represent immortalized mammary epithelia and multiple subtypes of breast cancer. These studies showed a highly specific role for Rb in mediating the response to CDK4/6 inhibition that was dependent on transcriptional repression manifest through E2F, and the ability to attenuate CDK2 activity. Acquired resistance to PD-03322991 was specifically associated with attenuation of CDK2 inhibitors, indicating that redundancy in CDK functions represents a determinant of therapeutic failure. Despite these caveats, in specific models, PD-0332991 was a particularly effective therapy, which induced Rb-dependent cytostasis. Combined, these findings indicate the critical importance of fully understanding cell-cycle regulatory pathways in directing the utilization of CDK inhibitors in the clinic.

Abstract 3888: Therapeutically activating the RB pathway in breast cancer: Key mechanisms of response and failure

Abstract A hallmark of cancer is the deregulation of cell cycle machinery, ultimately providing aberrant proliferation that fuels tumorigenesis and disease progression. Particularly, in breast cancers, cyclin D1 plays a crucial role in the development of disease. Recently, a highly-specific inhibitor of CDK4/6 activity (PD-0332991) has been developed that may have efficacy in the treatment of breast and additional cancers. To interrogate the utility of PD-0332991 in treating breast cancers, therapeutic response was evaluated in panel of breast cancer cell lines. These analyses demonstrated that the chronic loss of Rb is specifically associated with evolution to a CDK4/6-independent state and, thus, resistance to PD-0332991. However, to interrogate the functional consequence of Rb directly, knockdown experiments were performed in models that represent immortalized mammary epithelia and multiple subtypes of breast cancer. These studies demonstrated a highly-specific role for Rb in mediating the response to CDK4/6 inhibition that was dependent on transcriptional repression manifest through E2F and the ability to attenuate CDK2 activity. In spite of these caveats, in specific models, PD-0332991 was a particularly effective therapy, which induced Rb-dependent cytostasis. Combined, these findings indicate the critical importance of fully understanding cell cycle regulatory pathways in directing the utilization of CDK inhibitors in the clinic. 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 3888.

New Molecular Classifications of Breast Cancer

Traditionally, pathologic determinations of tumor size, lymph node status, endocrine receptor status, and human epidermal growth factor receptor 2 (HER2) status have driven prognostic predictions and adjuvant therapy recommendations for patients with early stage breast cancer. However, these prognostic and predictive factors are relatively crude measures, resulting in many patients being overtreated or undertreated. As a result of gene expression assays, there is growing recognition that breast cancer is a molecularly heterogeneous disease. Evidence from gene expression microarrays suggests the presence of multiple molecular subtypes of breast cancer. The recent commercial availability of gene expression profiling techniques that predict risk of disease recurrence as well as potential chemotherapy benefit have shown promise in refining clinical decision making. These techniques will be reviewed in this article.

Clinicopathological features and sites of recurrence according to breast cancer subtype in the National Comprehensive Cancer Network (NCCN)

543 Background: Gene expression profiling has defined multiple breast cancer subtypes which can approximated using standard immunohistochemical markers. Methods: We assessed clinicopathological features and sites of recurrence for patients (pts) presenting to NCCN sites with stage I-III breast cancer from Jan 2000 to Dec 2006 where estrogen receptor (ER), progesterone receptor (PR), and HER2 status were known. Tumors were grouped as luminal A (ER+ and/or PR+, and HER2-), HER2+ (any ER or PR, and HER2+), or triple-negative (ER-, PR-, and HER2-). Chi-square compared proportions across tumors; univariate logistic regression estimated risk of first site of recurrence. Results: 12,858 pts met inclusion criteria. Median follow-up from NCCN presentation was 3.2 years. Subtype distribution was: triple-negative (TN) 17%; HER2+ 18%; luminal A 66%. Compared to pts with luminal A and HER2+ tumors, TN were younger (p<0.0001), more likely African-American (p<0.0001) and overweight (p=0.0006). TN and HER2+ tumors were less often detected by screening mammography (TN, 28.9%; HER2+, 33.6%; luminal A, 48.4%) and less likely to present as T1 (TN, 46.5%; HER2+, 50.5%; luminal A, 67.0%) or diagnosed as stage I (TN, 32.6%; HER2+ 33.2%; luminal A, 49.4%) than luminal A (all p<0.0001). Rate of node positivity was lowest in TN (TN, 37.1%; HER2+, 44.9%; luminal A, 38.1%; p<0.0001). 83% of TN tumors were high grade; 93% were invasive ductal histology. Extensive intraductal component and lymphovascular invasion were more often associated with HER2+, compared to TN or luminal A (p<0.0001). Recurrences were recorded for 1,235 pts. Relative to luminal A, TN and HER2+ were more likely to experience lung (TN, odds ratio [OR] 2.27, 95% confidence interval [CI] 1.50, 3.43; p=0.0001; HER2+, OR 1.65, 95% CI 1.05, 2.60; p=0.03) and brain (TN, OR 5.32, 95% CI 2.85, 9.91; p<0.0001; HER2+, OR 5.53, 95% CI 2.93, 10.43; p<0.0001) as first site of recurrence; bone was less likely (TN, OR 0.23, 95% CI 0.16, 0.33; p<0.0001; HER2+, OR 0.38, 95% CI 0.28, 0.53; p<0.0001). Conclusions: Clinicopathological features and patterns of recurrence differed significantly by subtype and may inform the design of future clinical trials. No significant financial relationships to disclose.

Gene expression variation between distinct areas of breast cancer measured from paraffin-embedded tissue cores

BackgroundDiagnosis and prognosis in breast cancer are mainly based on histology and immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) material. Recently, gene expression analysis was shown to elucidate the biological variance between tumors and molecular markers were identified that led to new classification systems that provided better prognostic and predictive parameters. Archived FFPE samples represent an ideal source of tissue for translational research, as millions of tissue blocks exist from routine diagnostics and from clinical studies. These should be exploited to provide clinicians with more accurate prognostic and predictive information. Unfortunately, RNA derived from FFPE material is partially degraded and chemically modified and reliable gene expression measurement has only become successful after implementing novel and optimized procedures for RNA isolation, demodification and detection.MethodsIn this study we used tissue cylinders as known from the construction of tissue microarrays. RNA was isolated with a robust protocol recently developed for RNA derived from FFPE material. Gene expression was measured by quantitative reverse transcription PCR.ResultsSixteen tissue blocks from 7 patients diagnosed with multiple histological subtypes of breast cancer were available for this study. After verification of appropriate localization, sufficient RNA yield and quality, 30 tissue cores were available for gene expression measurement on TaqMan® Low Density Arrays (16 invasive ductal carcinoma (IDC), 8 ductal carcinoma in situ (DCIS) and 6 normal tissue), and 14 tissue cores were lost. Gene expression values were used to calculate scores representing the proliferation status (PRO), the estrogen receptor status and the HER2 status. The PRO scores measured from entire sections were similar to PRO scores determined from IDC tissue cores. Scores determined from normal tissue cores consistently revealed lower PRO scores than cores derived from IDC or DCIS of the same block or from different blocks of the same patient.ConclusionWe have developed optimized protocols for RNA isolation from histologically distinct areas. RNA prepared from FFPE tissue cores is suitable for gene expression measurement by quantitative PCR. Distinct molecular scores could be determined from different cores of the same tumor specimen.