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The development of DNA microarray and quantitative real-time polymerase chain reaction technologies has allowed for precise quantitation of RNA expression of hundreds to thousands of genes. These technologies have significantly impacted the study and understanding of cancer in terms of its molecular and genetic characteristics. In this review article, breast cancer, colon cancer, and adrenal carcinoma have been chosen to illustrate the principle and techniques of genomic profiling and to illustrate how such methods may be used to develop genomic signatures for personalized risk assessment and to individualize patient treatment.

Although RNA is isolated from archival fixed tissues routinely for reverse transcription polymerase chain reaction (RT-PCR) and microarray analyses to identify biomarkers of cancer prognosis and therapeutic response prediction, the sensitivity of these molecular profiling methods to variability in pathology tissue processing has not been described in depth. As increasing numbers of expression analysis studies using fixed archival tumor specimens are reported, it is important to examine how dependent these results are on tissue-processing methods.We carried out a series of studies to systematically evaluate the effects of various tissue-fixation reagents and protocols on RNA quality and RT-PCR gene expression profiles. Human placenta was selected as a model specimen for these studies since it is relatively easily obtained and has proliferative and invasive qualities similar to solid tumors. In addition, each specimen is relatively homogeneous and large enough to provide sufficient tissue to systematically compare a range of fixation conditions and reagents, thereby avoiding the variability inherent in studying collections of tumor tissue specimens. Since anatomical pathology laboratories generally offer hundreds of different tissue-fixation protocols, we focused on fixation reagents and conditions used to process the most common solid tumors for primary cancer diagnosis. Fresh placentas donated under an IRB-approved protocol were collected at delivery and immediately submerged in cold saline for transport to a central pathology laboratory for processing. RNA was extracted from each specimen, quantified, and analyzed for size distribution and analytical performance using a panel of 24 RT-PCR gene expression assays. We found that different tissue-fixation reagents and tissue-processing conditions resulted in widely varying RNA extraction yields and extents of RNA fragmentation. However, the RNA extraction method and RT-PCR assays could be optimized to achieve successful gene expression analysis for nearly all fixation conditions represented in these studies.

African American and Hispanic women develop more triple negative breast cancer at younger ages than Caucasian women. The frequently observed association between race and socioeconomic status (SES) has confounded our understanding of the outcomes disparities seen in these groups. Given the association between inflammatory cells and high-grade, triple negative tumors, we sought to investigate whether differences in the presence of these cells varies by race. We evaluated breast tumor specimens for the presence PCNA+ tumor-associated macrophages (TAMs) in consecutive cases from a county hospital serving primarily un- or under-insured patients. All patients in this cohort had elevated PCNA + TAM levels. Higher PCNA + TAM counts were associated with hormone receptor (HR) negative tumors and non-Caucasian ethnicity. Hispanic women specifically had significantly higher PCNA + TAM counts than Caucasian patients and shorter disease-free survival. These findings implicate immune function in the development of aggressive breast cancer and suggest a possible link between SES and the inflammatory response.

Breast cancer is a heterogeneous disease encompassing a variety of entities with distinct morphological features and clinical behaviors. Although morphology is often associated with the pattern of molecular aberrations in breast cancers, it is also clear that tumors of the same histological type show remarkably different clinical behavior. This is particularly true for basal-like cancer, which is an entity defined using gene expression analysis. The purpose of this article was to review the current state of knowledge of basal-like breast cancers, to discuss the relationship between basal-like and triple-negative breast cancers, and to clarify practical implications of these diagnoses for pathologists and oncologists. © 2011 USCAP, Inc.

Macrophages, a key cell in the inflammatory cascade, have been associated with poor prognosis in cancers, including breast cancer. In this study, we investigated the relationship of a subset of macrophages-proliferating macrophages (promacs)-with clinico-pathologic characteristics of breast cancer, including tumor size, grade, stage, lymph node metastases, hormone receptor status, subtype, as well as early recurrence, and survival. This study included a discovery and validation set that was conducted at two institutions and laboratories (University of California, San Francisco and University of Chicago) using two independent cohorts of patients with breast cancer. Formalin-fixed, paraffin-embedded sections and/or tissue microarrays were double-stained with anti-CD68 (a macrophage marker) and anti-PCNA (a proliferation marker) antibodies. The presence of intratumoral promacs was significantly correlated with high grade, hormone receptor negative tumors, and a basal-like subtype. In contrast, there was no correlation between promacs and tumor size, stage, or the number of the involved lymph nodes. These findings were consistent between the two study cohorts. Finally, promac numbers were a significant predictor of recurrence and survival. In the pooled analysis, elevated promac levels were associated with a 77% increased risk of dying (P = 0.015). The presence of promacs in human breast cancer may serve as a prognostic indicator for poor outcomes and early recurrence and serve as a potential cellular target for novel therapeutic interventions.

Purpose These studies were conducted to determine the relationship between quantitative tumor gene expression and risk of cancer recurrence in patients with stage II or III colon cancer treated with surgery alone or surgery plus fluorouracil (FU) and leucovorin (LV) to develop multigene algorithms to quantify the risk of recurrence as well as the likelihood of differential treatment benefit of FU/LV adjuvant chemotherapy for individual patients. Patients and Methods We performed quantitative reverse transcription polymerase chain reaction (RT-qPCR) on RNA extracted from fixed, paraffin-embedded (FPE) tumor blocks from patients with stage II or III colon cancer who were treated with surgery alone (n = 270 from National Surgical Adjuvant Breast and Bowel Project [NSABP] C-01/C-02 and n = 765 from Cleveland Clinic [CC]) or surgery plus FU/LV (n = 308 from NSABP C-04 and n = 508 from NSABP C-06). Overall, 761 candidate genes were studied in C-01/C-02 and C-04, and a subset of 375 genes was studied in CC/C-06. Results A combined analysis of the four studies identified 48 genes significantly associated with risk of recurrence and 66 genes significantly associated with FU/LV benefit (with four genes in common). Seven recurrence-risk genes, six FU/LV-benefit genes, and five reference genes were selected, and algorithms were developed to identify groups of patients with low, intermediate, and high likelihood of recurrence and benefit from FU/LV. Conclusion RT-qPCR of FPE colon cancer tissue applied to four large independent populations has been used to develop multigene algorithms for estimating recurrence risk and benefit from FU/LV. These algorithms are being independently validated, and their clinical utility is being evaluated in the Quick and Simple and Reliable (QUASAR) study.

Aims To determine whether 0.6 mm cores of formalin-fixed paraffin-embedded (FFPE) tissue, as commonly used to construct immunohistochemical tissue microarrays, may be a valid alternative to tissue sections as source material for quantitative real-time PCR-based transcriptional profiling of breast cancer. Methods Four matched 0.6 mm cores of invasive breast tumour and two 10 μm whole sections were taken from eight FFPE blocks. RNA was extracted and reverse transcribed, and TaqMan ® assays were performed on the 21 genes of the Oncotype DX ® Breast Cancer assay. Expression of the 16 recurrence-related genes was normalised to the set of five reference genes, and the recurrence score (RS) was calculated. Results RNA yield was lower from 0.6 mm cores than from 10 μm whole sections, but was still more than sufficient to perform the assay. RS and single gene data from cores were highly comparable with those from whole sections (RS p=0.005). Greater variability was seen between cores than between sections. Conclusions FFPE sections are preferable to 0.6 mm cores for RNA profiling in order to maximise RNA yield and to allow for standard histopathological assessment. However, 0.6 mm cores are sufficient and would be appropriate to use for large cohort studies.

Breast cancer is a heterogeneous disease with varied morphological appearances, molecular features, behavior, and response to therapy. Current routine clinical management of breast cancer relies on the availability of robust clinical and pathological prognostic and predictive factors to support clinical and patient decision making in which potentially suitable treatment options are increasingly available. One of the best-established prognostic factors in breast cancer is histological grade, which represents the morphological assessment of tumor biological characteristics and has been shown to be able to generate important information related to the clinical behavior of breast cancers. Genome-wide microarray-based expression profiling studies have unraveled several characteristics of breast cancer biology and have provided further evidence that the biological features captured by histological grade are important in determining tumor behavior. Also, expression profiling studies have generated clinically useful data that have significantly improved our understanding of the biology of breast cancer, and these studies are undergoing evaluation as improved prognostic and predictive tools in clinical practice. Clinical acceptance of these molecular assays will require them to be more than expensive surrogates of established traditional factors such as histological grade. It is essential that they provide additional prognostic or predictive information above and beyond that offered by current parameters. Here, we present an analysis of the validity of histological grade as a prognostic factor and a consensus view on the significance of histological grade and its role in breast cancer classification and staging systems in this era of emerging clinical use of molecular classifiers.

In the last decade, the development of microarrays and the ability to perform massively parallel gene expression analysis of human tumours were received with great excitement by the scientific community. The promise of microarrays was of apocalyptic dimensions, with some experts envisaging that it would be a matter of a few years for this technology to replace traditional clinicopathological markers in clinical practice and treatment decision-making. The replacement of histopathology by high-tech and more objective approaches to cancer diagnosis, prognostication and prediction was, at that time, a foregone conclusion. Ten years after the initial publications of translational research studies using microarrays, one cannot deny that this technology has changed the way breast cancer is perceived. It has brought the concept of breast cancer heterogeneity to the forefront of cancer research, and the fact that distinct subtypes of breast cancer are completely different diseases that affect the same anatomical site. Furthermore, it has led to the development of prognostic and predictive 'gene signatures', which are yet to be fully incorporated into clinical practice. Importantly, though, the prognostic and predictive power of microarrays has been shown to be complementary to, rather than a replacement for, traditional clinicopathological parameters. Here we endeavour to provide a fair and balanced assessment of what microarray-based gene expression analysis has taught us in the last decade and its contribution to breast cancer classification, prognostication and prediction.

Purpose: To determine the prognostic significance of a multimarker assay incorporating expression levels of three molecular markers in primary cutaneous melanoma. Experimental Design: We assessed expression levels of NCOA3, SPP1, and RGS1 using immunohistochemical analysis in a tissue microarray cohort of 395 patients. For each marker, we identified optimal cut-points for expression intensity to predict disease-specific survival (DSS) and, as a secondary endpoint, sentinel lymph node (SLN) status. The cumulative overexpression of all three markers was embodied in a multimarker index, and its prognostic effect on DSS and SLN status was assessed using Cox regression, Kaplan-Meier analysis, and logistic regression. The prognostic effect of this multimarker assay on DSS was assessed in an independent cohort of 141 patients, in which marker expression levels were scored using immunohistochemical analysis of stained tissue sections. Results: Increasing multimarker index scores were significantly predictive of reduced DSS and increased SLN metastasis in the 395-patient cohort. Multivariate logistic regression analysis revealed multimarker expression scores as an independent predictor of SLN status (P = 0.001). Multivariate Cox regression analysis showed the independent effect of the multimarker index on DSS (P < 0.001). The multimarker index was the most significant factor predicting DSS when compared with other clinical and histologic factors, including SLN status (P = 0.002). Multimarker expression scores were also the most significantly predictive of DSS in the independent cohort (P = 0.01). Conclusions: These results describe a multimarker assay with independent prognostic effect on the prediction of survival associated with melanoma in two distinct cohorts. (Clin Cancer Res 2009;15(22):698792)

The roles of epithelial cells encompass both cellular- and tissue-level functions that involve numerous cell–cell and cell–matrix interactions, which ultimately mediate the highly structured arrangement of cells on a basement membrane. Although maintaining this basic structure is critical for preserving tissue integrity, plasticity in epithelial cell behavior is also critical for processes such as cell migration during development or wound repair, mitotic cell detachment, and physiological shedding. The mechanisms that mediate epithelial cell plasticity are only beginning to be understood. We previously identified Trask, a transmembrane protein that is phosphorylated by src kinases during mitosis. In this study, we report that the phosphorylation of Trask is associated with anchorage loss in epithelial cells. Phosphorylation of Trask is seen during the cell-detachment phase of mitosis, in experimentally induced interphase detachment, and during cell migration in experimental epithelial models. An analysis of human tissues shows that Trask is widely expressed in many epithelial tissues but not in most tissues of mesenchymal origin, except for a subset of early hematopoietic cells. Trask is not phosphorylated in epithelial tissues in vivo; however, its phosphorylation is seen in epithelial cells undergoing mitosis or physiological shedding. Trask is a novel epithelial membrane protein that is phosphorylated by src kinases when epithelial cells disengage from their tissue framework, identifying an important new regulator of epithelial tissue dynamics.

Purpose: The frequently elevated activities of the c-src and c-yes products in human epithelial tumors suggest that these activated tyrosine kinases have tumorigenic functions analogous to the v-src and v-yes oncogene products. Studies of v-src–transformed fibroblasts have identified many of the effectors of this potent oncogene; however, because c-src and c-yes lack the mutational and promiscuous activities of their retroviral oncogene homologues, their presumptive tumorigenic functions in human epithelial tumors are more subtle, less well-defined, and await identification of possible effectors more directly relevant to epithelial cells. Experimental Design: We recently identified a transmembrane glycoprotein named Trask that is expressed in epithelial tissues but not fibroblasts and is phosphorylated by SRC kinases in mitotic epithelial cells. In this study, we have surveyed the expression and phosphorylation of Trask in many human epithelial cancer cell lines and surgical tissues and tumors. Results: Trask is widely expressed in human epithelial tissues, but its phosphorylation is tightly regulated and restricted to detached mitotic cells or cells undergoing physiologic shedding. However, abberant Trask phosphorylation is seen in many epithelial tumors from all stages including preinvasive, invasive, and metastatic tumors. Trask phosphorylation requires SRC kinases, and is also aberrantly hyperphosphorylated in the SRC-activated PyMT mouse epithelial tumors and dephosphorylated by the SRC inhibitor treatment of these tumors. Conclusions: The widespread phosphorylation of Trask in many human epithlelial cancers identifies a new potential effector of SRC kinases in human epithelial tumorigenesis.

Personalized Medicine, by the utilization of powerful gene expression technologies, is rapidly revolutionizing the clinical management of treatment decisions in patients with breast cancer. In addition to powerful traditional clinicopathologic metrics the new technologies offer the personalized assessment of individual patient tumor samples. By accurately quantitating gene expression and integrating these precise measurements into powerful biostatistical models, the risk of distant recurrence and the potential benefit of chemotherapy may be assessed for individual patients and their health-care providers. This personalized medical information allows for actionable, individualized treatment decisions. In addition to a detailed review of the traditional clinicopathologic metrics, this chapter will review current molecular assessment technologies as well as review landmark trials that use these tools in a prespecified manner in clinical validation trials and that demonstrate their clinical utility in the management of individual patient treatment decisions.

Estrogen receptor (ER) status in breast cancer is currently the most important predictive biomarker that determines breast cancer prognosis after treatment with endocrine therapy. Although immunohistochemistry has been widely viewed as the gold standard methodology for ER testing in breast cancer, lack of standardized procedures, and lack of regulatory adherence to testing guidelines has resulted in high rates of "false-negative" results worldwide. Standardized testing is only possible after all aspects of ER testing--preanalytical, analytical, and postanalytical, have been closely controlled. A meeting of the "ad-hoc committee" of expert pathologists, technologists, and scientists, representing academic centers, reference laboratories, and various agencies, issued standardization testing recommendations, aimed at optimization of clinical ER testing environment, as a step toward improved standardized testing.

Purpose Adjuvant! is a standardized validated decision aid that projects outcomes in operable breast cancer based on classical clinicopathologic features and therapy. Genomic classifiers offer the potential to more accurately identify individuals who benefit from chemotherapy than clinicopathologic features. Patients and Methods A sample of 465 patients with hormone receptor (HR) –positive breast cancer with zero to three positive axillary nodes who did (n = 99) or did not have recurrence after chemohormonal therapy had tumor tissue evaluated using a 21-gene assay. Histologic grade and HR expression were evaluated locally and in a central laboratory. Results Recurrence Score (RS) was a highly significant predictor of recurrence, including node-negative and node-positive disease (P < .001 for both) and when adjusted for other clinical variables. RS also predicted recurrence more accurately than clinical variables when integrated by an algorithm modeled after Adjuvant! that was adjusted to 5-year outcomes. The 5-year recurrence rate was only 5% or less for the estimated 46% of patients who have a low RS (< 18). Conclusion The 21-gene assay was a more accurate predictor of relapse than standard clinical features for individual patients with HR-positive operable breast cancer treated with chemohormonal therapy and provides information that is complementary to features typically used in anatomic staging, such as tumor size and lymph node involvement. The 21-gene assay may be used to select low-risk patients for abbreviated chemotherapy regimens similar to those used in our study or high-risk patients for more aggressive regimens or clinical trials evaluating novel treatments.

Purpose Central and local laboratory concordance for hormone receptor measurement is therapeutically important. This study compares estrogen receptor (ER) and progesterone receptor (PR) measured by local laboratory immunohistochemistry (IHC), central IHC, and central reverse-transcriptase polymerase chain reaction (RT-PCR) using a proprietary 21-gene assay. Patients and Methods A case-control sample of 776 breast cancer patients from Eastern Cooperative Oncology Group (ECOG) study E2197 was evaluated. Central IHC Allred score for ER and PR was obtained using tissue microarrays and 1D5 ER antibody and 636 PR antibody. Quantitative RT-PCR for ER and PR in whole sections was performed using the 21-gene assay. Results For ER, the concordance between local and central IHC was 90% (95% CI, 88% to 92%), between local IHC and central RT-PCR was 91% (95% CI, 89% to 93%), and between central IHC and central RT-PCR was 93% (95% CI, 91% to 95%). For PR, the concordance between local IHC and central IHC was 84% (95% CI, 82% to 87%), between local IHC and central RT-PCR was 88% (95% CI, 85% to 90%), and between central IHC and central RT-PCR was 90% (95% CI, 88% to 92%). Although concordance was high, IHC ER-negative cases that were RT-PCR positive were more common than IHC ER-positive cases that were RT-PCR negative. In ER-positive patients, ER expression by central IHC Allred score was marginally associated with recurrence (P = .091), and ER expression by central RT-PCR was significantly associated with recurrence (P = .014). However, recurrence score, which incorporates additional genes/pathways, was a highly significant predictor of recurrence (P < .0001). Conclusion There is a high degree of concordance among local IHC, central IHC, and central RT-PCR by the proprietary gene assay for ER and PR status. Although ER expression is marginally associated with relapse in ER-positive patients treated with chemohormonal therapy, recurrence score is a highly significant predictor of recurrence.

581 Background: In the Ontario population based study, the DCIS Score was significantly associated with 10 year risk of an ipsilateral local recurrence (LR - in situ or invasive carcinoma) in women...

1013 Background: The DCIS Score (DS) was validated as a predictor of ipsilateral breast recurrence (IBR; DCIS or invasive) in E5194 pts tx by breast-conserving surgery (BCS) w/o RT. The Ontario pop...

Statins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation reactions and reduction of signals driving cell proliferation and survival responses. The objectives of this study were to examine the effects of statins on breast cancer cells, both in vitro and in vivo, and to begin to determine their mechanism of action. We evaluated the effects of statins on breast cancer cell growth, phosphoprotein signaling intermediates, survival/apoptosis regulators, cell cycle regulators, and activated transcription factors. We also examined the in vivo effect of statin administration in a mouse ErbB2+ breast cancer model. Only lipophilic statins had direct anticancer activity in vitro. Breast cancer cells with activated Ras or ErbB2 pathways seemed to be more sensitive than those overexpressing estrogen receptor, and this correlated with endogenous levels of activated nuclear factor κB (NF-κB). Key intermediates regulating cell survival by NF-κB activation, as well as cell proliferation by the mitogen activated protein kinase cascade, were among the earliest phosphoproteins influenced by statin treatment. These early effects were followed by declines in activator protein-1 and NF-κB activation and concordant changes in other mediators of proliferation and apoptosis. In vivo results showed that oral dosing of statins significantly inhibited the growth of a mouse mammary carcinoma. Lipophilic statins can exert direct anticancer activity in vitro by reducing proliferation and survival signals in susceptible breast cancer phenotypes. Tumor growth inhibition in vivo using a clinically relevant statin dose also seems to be associated with reduced tumor cell proliferation and survival. These findings provide supporting rationale for future statin trials in breast cancer patients. (Cancer Res 2006; 66(17): 8707-13)

Recent studies have demonstrated a role for telomerase in driving tumor progression, but its mechanism of action remains unclear. Here we show that stable, ribozyme-mediated suppression of mouse telomerase RNA reduced telomerase RNA expression, telomerase activity, and telomere length, which significantly reduced tumor invasion and metastatic potential. Our studies reveal that previously unidentified effects of telomerase may mediate its tumor-promoting effects. First, reducing telomerase activity induced a more dendritic morphology, accompanied by increased melanin content and increased expression of tyrosinase, a key enzyme in melanin biosynthesis. Second, gene expression profiling revealed that telomerase targeting down-regulated expression of several glycolytic pathway genes, with a corresponding decrease in glucose consumption and lactate production. Thus, telomerase activity controls the glycolytic pathway, potentially altering the energy state of tumor cells and thereby modulating tyrosinase activity and melanin production. These studies have important implications for understanding the mechanisms by which telomerase promotes tumor invasion and metastasis.

Adult perineal soft tissue sarcomas are rare. Fewer than 30 cases have been reported, and all were diagnosed after surgical resection by histologic examination. Below we report a case in which the diagnosis was established preoperatively by fine needle aspiration (FNA).A 27-year-old man presented with a firm, midline, perineal mass. Magnetic resonance imaging showed a 3-cm, enhancing mass that was considered neoplastic. FNA biopsy, followed by cytologic examination, revealed moderately cellular aspirates composed of discohesive, small, blue cells with scant cytoplasm, high nuclear/cytoplasmic ratios and pleomorphic nuclei with irregular nuclear contours; uniform, hyperchromatic chromatin; and occasional mitotic figures. Frequent naked nuclei and scattered cells with more abundant, dense cytoplasm and eccentric nuclei were also noted. The diagnosis of rhabdomyosarcoma was favored on FNA and was corroborated by immunohistochemical stains for desmin, myogenin and CD56. Upon surgical resection, the diagnosis of alveolar rhabdomyosarcoma was confirmed histologically and immunophenotypically.FNA is a useful tool in diagnosing soft tissue lessions of the perineum, including rare primary tumors, such as adult rhabdomyosarcoma. In this case, early identification avoided incisional biopsy and directed appropriate extirpative surgery and reconstruction considerations.

The protean morphology of malignant melanoma is diagnostically challenging. Balloon cell melanoma is a histologic variant composed predominantly or entirely of large cells with abundant, vacuolated cytoplasm. It shares the cytologic features of the other subcategories of malignant melanoma, such as discohesion, nuclear pleomorphism and intranuclear cytoplasmic pseudoinclusions, but generally lacks melanin pigment and, as the name would suggest, is characterized by the presence of numerous cytoplasmic vacuoles.A 55-year-old man presented with an enlarged right cervical lymph node. Clinically and radiographically this mass was considered to be metastatic; however, the patient had no known primary neoplasm. Fine needle aspiration biopsy (FNAB) and cytologic examination showed numerous discohesive, variably sized, malignant cells with abundant, vacuolated cytoplasm and pleomorphic nuclei with irregular nuclear contours, macronucleoli and frequent intranuclear cytoplasmic pseudoinclusions. Pigment was not identified. These features, along with strong immunohistochemical positivity for S-100, HMB-45 and Melan-A, suggested the diagnosis of balloon cell melanoma. A right parotidectomy and lymph node dissection were performed, and histologic tissue evaluation confirmed the diagnosis.This case of lymph node balloon cell melanoma metastasis was diagnosed by FNAB.

background The likelihood of distant recurrence in patients with breast cancer who have no involved lymph nodes and estrogen-receptor–positive tumors is poorly defined by clinical and histopathological measures. methods We tested whether the results of a reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay of 21 prospectively selected genes in paraffin-embedded tumor tissue would correlate with the likelihood of distant recurrence in patients with node-negative, tamoxifen-treated breast cancer who were enrolled in the National Surgical Adjuvant Breast and Bowel Project clinical trial B-14. The levels of expression of 16 cancerrelated genes and 5 reference genes were used in a prospectively defined algorithm to calculate a recurrence score and to determine a risk group (low, intermediate, or high) for each patient. results Adequate RT-PCR profiles were obtained in 668 of 675 tumor blocks. The proportions of patients categorized as having a low, intermediate, or high risk by the RT-PCR assay were 51, 22, and 27 percent, respectively. The Kaplan–Meier estimates of the rates of distant recurrence at 10 years in the low-risk, intermediate-risk, and high-risk groups were 6.8 percent (95 percent confidence interval, 4.0 to 9.6), 14.3 percent (95 percent confidence interval, 8.3 to 20.3), and 30.5 percent (95 percent confidence interval, 23.6 to 37.4). The rate in the low-risk group was significantly lower than that in the high-risk group (P

Withdrawn by Author Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-01.

Background: High grade in situ (DCIS) features associated with high recurrence include large size (>5cm), comedo necrosis, palpable mass, hormone receptor (HR) negativity, and HER2 positivity. Given that high grade, HR-neg invasive breast cancers have an inflammatory component (significant macrophage infiltration) we sought to characterize the immune microenvironment of DCIS to assess patterns of immune cell infiltrates associated with high risk lesions. Methods: 48 cases of high grade DCIS were age matched with 64 cases of non-high grade DCIS. Immunohistochemical analyses were performed as single color stains for the following antigens: CD115, FoxP3, ALDH, Ki-67, HER2. Two color IHC was performed for the following antigen pairs: CD68/PCNA; CD68/Mac 387; CD8/HLA-DR; CD68/MRC1, and CD24/CD44. HR status was determined from ER and PR staining results in pathology reports. For each case, 3 hot spots were identified and marked on 10 consecutive sections. Nuance multispectral imaging software was used to image each hot spot. Protocols for automated image analysis were developed using CellProfiler software. Clinical parameters of interest included tumor palpability, recurrence, and Van Nuys score, (12 point scale-margins, age, size, grade). Associations were identified with non-parametric Spearman correlation test. Results: High numbers of macrophages were associated with high Van Nuys score, palpability, and high Ki-67. High CD115 (CSF-1 receptor/c-fms) was associated with HER2+, high Ki-67, and recurrence. Mac387+ cells and FoxP3+ regulatory T cells (Treg) were significantly associated with high Van Nuys score, comedo necrosis, high Ki-67, HR- and HER2+. Interestingly, both Mac387 and CD115 were expressed on tumor cells as well as macrophages and high CD115 staining on tumor cells was associated with recurrence. The presence of CD8+HLA-DR negative T cells throughout a section was associated with high Van Nuys score, HR-, HER2+, and recurrence. In contrast, CD8+ T cells within the nests of tumor cells were negatively associated with Van Nuys score, palpability, and comedo necrosis. Conclusions: Suppressed immunity (high Treg and CD8+HLA-DR neg T cells) and upregulated CD115 and Mac387 expression on both tumor cells and macrophages were strongly correlated with high risk DCIS features (increased recurrence, palpability, high Van Nuys scores, high proliferation, HR- and HER2+). These results suggest that manipulation of the immune microenvironment in DCIS, via local stimulation of the immune system, depletion of Treg, and/or manipulation of macrophages could potentially alter disease progression. Targeted agents are in trials and could be tested in preoperative window trials using these biomarkers to monitor the impact of presurgical immunotherapy. Citation Format: Michael J. Campbell, Rita Mukhtar, Ekene Obi-Okoye, Booyeon Han, Vick Tandon, Sarah Zheng, Zelos Zhu, Max Endicott, Max Wicha, Linda Lindstrom, Alfred Au, Frederick Baehner, Joe Gray, Laura Esserman. Suppressed immunity and macrophages characterize high risk high grade DCIS. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 165. doi:10.1158/1538-7445.AM2014-165

Aim: The 12-gene DCIS Score has been validated to predict 10yr risk of local recurrence (DCIS or invasive) (Solin et al, JNCI, 2013). Here we report the Clinical Laboratory experience in the first 2 yrs of assay availability for the score results, quantitative gene expression and results of the assay across histologic subtypes. Methods: 3045 patient (pt) samples from 12/11 to 1/14 were processed for DCIS score. The DCIS score is based on the validated algorithm using 7 of 16 cancer-related genes from the 21-gene assay. Low, intermediate, and high risk groups are defined as scores of Results: Of 3045 samples, 2264 (74%) were excisions and 781 (26%) were cores; median age was 60y (21-94). The mean DCIS Score was 29 (0-97) with a mean score result of 29 for excisions and 29 for cores. 2064 (68%) were low, 495 (16%) were intermediate, and 486 (16%) were high. Mean ER, PR, and HER2 levels were 9.6, 7.5, and 9.9, respectively, and similar between excisions and cores. A range of DCIS Score results were observed for each histologic subtype with the highest mean values observed in samples with >50% comedo necrosis (mean = 47) and solid (mean = 34) subtypes, although none of the scores were in the high risk category. Table: 259PD . Tumor type Distribution (%) Mean DCIS Score (range) Mean ER Mean PR Mean HER2 Cribriform 1202 (39) 22 (0-90) 9.9 7.8 9.7 Solid 1053 (35) 34 (0-96) 9.4 7.2 9.9 DCIS with ≥50% comedo necrosis 345 (11) 47 (0-97) 8.4 6.3 10.2 Papillary 281 (9) 17 (0-90) 10.9 8.6 9.6 Micropapillary 146 (5) 26 (0-88) 9.4 7.3 10.0 Apocrine 11 (0.4) 26 (4-53) 7.6 6.0 9.7 Conclusions: In the first 2 yrs, there were > 3000 samples submitted for the 12-gene DCIS score assay. The proportion of score results that were low, intermediate and high was similar to the validation study. The mean DCIS score result between core bx and excision was also similar supporting assay use on cores. The overall experience shows that the score results reflect underlying biology differently than histology and are informative for guiding treatment decisions. Disclosure: All authors have declared: Genomic Health, Inc. - employee and stock ownership

Aim: Identification of molecular determinants predicting late recurrence (>5 yrs) in stage I and II breast cancer has become clinically important in light of data demonstrating a benefit for 10 yrs of tamoxifen administration. Since the 21-gene Recurrence Score (RS) is commonly utilized in early stage BC, we wished to determine its utility in predicting distant recurrences beyond 5 yrs as a function of quantitative ER expression. Methods: The 21-gene RS was assessed in 1065 chemo and tam-treated, ER + , node-positive pts from NSABP B-28 and 668 tam-treated, ER + , node-negative pts from NSABP B-14. Cox PH models, KM estimates and log rank statistics were used to assess the association of the RS with risk of DR by quantitative ER expression, using the 21-gene assay, in pts event-free after 5 yrs. We established an ER cut-point (high vs low) in B-28, and tested the cut-point in B-14, formally evaluating the interaction of RS and ER. Results: Median follow-up was 11.2 yrs (B-28) and 14.5 yrs (B-14). 832 B-28 pts and 564 B-14 pts were DR-free after 5 yrs. A reference normalized ER cut-point of 9.1 CT was established in B-28 based on the association of the RS with DR after 5 yrs. Of the event-free pts at 5 yrs, 68% in B-28 and 88% in B-14 had ER > 9.1. In B-28 the RS result was strongly associated with DR after 5 yrs in the higher ER expressing pts (log rank P = 0.001), but not in the lower ER expressing pts (log rank P = 0.87). It was confirmed in the B-14 data that RS was associated with DR after 5 yrs in higher ER pts (Table) but not in the lower ER pts (interaction P = 0.03). The association of RS risk groups within clinicopathologic subgroups for the higher ER patients still at risk at 5 years will also be presented. Table 177P . DR Risk after 5 yrs in B-14 by RS Risk Group for pts with ER > 9.1 C T % DR KM estimate (95% CI) RS Risk Group N(%) pts 5 to 10 yrs (%) 5 to 15 yrs % Low 289 (58%) 4.7 (2.8 – 8.0) 6.8 (4.4 – 10.6) Intermediate 111 (22%) 4.1 (1.6 – 10.6) 11.2 (6.2 – 19.9) High 97 (20%) 12.6 (7.4 – 21.2) 16.4 (10.2 – 25.7) Log rank P = 0.01 Conclusions: For late recurrences (beyond 5 yrs), the RS is strongly prognostic in pts with higher quantitative ER expression (>9.1). The findings suggest that extending tamoxifen beyond 5 yrs may be most beneficial in pts with high (and intermediate) RS with higher quantitative ER expression and of limited benefit in pts with a low RS (>50% of population under study). Disclosure: F. Baehner, S. Butler, F. Jamshidian and A. Sing have decalred: I am an employee of Genomic Health, Inc. I receive a salary and company stock. S. Shak: I am an employee of Genomic Health, Inc and I serve in a leadership position. I receive a salary and I have company stock.All other authors have declared no conflicts of interest.

and high risk in 31 (38%), 43 (53%), and 7 (9%) pts, respectively. Median prescription dose was 50.4 Gy in 1.8 Gy fractions. The observed 10-year rate of IHD was 1.4% (95% CI Z 0.2-9.4). MRE for IHD with ABC and FB were 3.56% (95% CI Z 2.78-4.33) and 4.31% (95% CI Z 3.35-5.28), respectively [Table]. Cardiac testing was performed in 15 (19%) pts due to signs and symptoms of IHD: 1 of 3 pts had occlusive CAD on angiography, 1 of 7 pts had ischemic signs on stress testing, and 8 pts had normal stress myocardial perfusion single photon emission tomography (SPECT) imaging. Conclusions: The rate of IHD at 10 years was 1.4% in this first report of cardiac outcomes following RT with ABC. While limited by small study size, the low prevalence of abnormal cardiac testing corroborates findings. Using MRE, RT with ABC reduces the risk of IHD when compared to FB with the largest benefit observed for AHA high-risk pts. In appropriately selected pts, RT with ABC can provide a clinically significant reduction in the risk of IHD in survivors of LBC. Author Disclosure: H.B. Eldredge-Hindy: None. D. Duffy: None. K. Yamoah: None. A.P. Dicker: None. J. Skowronski: None. N.L. Simone: None. P.R. Anne: None.

Aim: The Recurrence Score (RS) predicts outcome in node- and node + , ER+ pts treated with endocrine therapy and chemotherapy (CT) benefit. We studied the prognostic impact of the RS in node + , HR+ pre and post-menopausal pts treated with adjuvant chemohormonal therapy in PACS01 and here present an analysis of the performance of the RS in pre- and post-menopausal pts. Methods: PACS01 compared FEC with FEC-D in 1999 pts. The current study included 530 pts from the PACS01 parent trial who were central IHC HR+ with sufficient tissue for Oncotype DX. Primary objective was to estimate the association between RS and distant recurrence free interval (DRFI). Secondary endpoints included disease free survival (DFS) and overall survival (OS). Herein we explore the association of the RS in pre- and post-menopausal women. Median follow-up time was 7.7 yrs. Results: Of 530 pts, 317 were pre-menopausal and 205 were post-menopausal - 209 (39.4%) had low; 159 (30.0%) intermediate; and 162 (30.6%) high RS. Of pre-menopausal, 124 (39.1%) had low; 89 (28.1%) intermediate & 104 (32.8%) high RS. Of post-menopausal 84 (41.0%) had low, 68 (33.2%) intermediate & 53 (25.9%) high RS. 74.2% were treated with tam. In the primary analysis, RS was a significant predictor of DRFI (HR= 4.1 for a 50 point difference, P RS Low RS Int RS High Log-rank p-value All pts n = 530 93.7 (89.4–96.3) 87.3 (81.0–91.6) 69.3 (61.5–75.8) p Pre-meno n = 317 93.5 (87.4–96.7) 89.8 (81.3–94.5) 70.9 (61.1–78.7) p Post-meno n = 205 94.0 (86.2–97.5) 85.2 (74.2–91.7) 64.9 (50.2–76.3) p Conclusions: The 21-gene RS was prognostic in HR + , node+ pts treated with FEC or FEC-D adjuvant CT and the performance was similar in pre- and post-menopausal pts. The data is consistent with those presented from B28 providing further supportive evidence for the assay's validity in pre-menopausal, node+ pts. Disclosure: F. Penault-Llorca: I have an advisory relationship with Genomic Health Inc, and receive speaker's honoraria from Genomic Health, Inc.; F. Baehner: Frederick Baehner is an employee of Genomic Health, Inc.; P. Fumoleau: Dr Fumoleau has an consultant/advisory relationship with Sanofi, Roche and Novartis. S. Butler: Mr. Butler is an employee of Genomic Health, Inc.; F. Jamshidian: Mr Jamshidian is an employee of Genomic Health, Inc.; D. Cherbavaz: Mrs Cherbavaz is an employee of Genomic Health, Inc. All other authors have declared no conflicts of interest.

11003 Background: The Oncotype DX 21-gene Recurrence Score assay was developed in endocrine-treated patients (pts) and validated as a predictor of 10-yr distant recurrence risk and chemotherapy ben...

11024 Background: Identification of molecular determinants predicting late recurrence (>5 yrs) in stage I and II breast cancer has become clinically important in light of data demonstrating a benef...

11052 Background: The Recurrence Score (RS) predicts outcome in node- and node+, ER+ pts treated with endocrine therapy and predicts chemotherapy benefit. We studied the prognostic impact of RS in ...

11 Background: Classic lobular carcinoma is characterized by a distinctive morphology, loss of E-cadherin commonly due to mutation or deletion of CDH1 on chromosome 16q, and a variable clinical course. Variants (pleomorphic, solid and alveolar) with distinct morphologies and potential differences in outcome have been described (Rosen 2009). Herein we provide an 8-year update of the patterns of quantitative gene expression as measured by the 21 gene Oncotype DX assay observed between ductal NOS (DC) and classic and variant lobular carcinomas. Methods: All tumors analyzed in the Genomic Health laboratory from 6/1/04-5/31/12 were included. Central path used WHO criteria for classification of classic lobular (CL), solid and alveolar lobular (SAL), and pleomorphic lobular (PL) carcinomas. Quantitative expression of 16 cancer related genes was measured on a scale from 2 to 15 (relative to reference genes) where a 1 unit increment is associated with an ~2-fold change in expression. Descriptive statistics for RS & individual genes [ER, PR, invasion gene group (IGG) and proliferation gene group (PGG)] among the subtypes were obtained. Comparisons of means among the subtypes were adjusted to control the overall error rate under any complete or partial null hypothesis. Results: DC accounted for 81.8% of 286,726 cases, CL 7.3%, SAL 0.4% and PL 0.4%. For all types a continuous range of RS was observed. DC had the greatest percentage of high risk RS followed by SAL, PL and CL. DC had the highest mean RS and PL and CL had the lowest RS. SAL had the highest mean ER expression and CL and PL had the lowest ER expression. These results may reflect a submission bias and are not population based. The proportion with ER+/PR- phenotype was slightly different among the subtypes: SAL (24.0%) and PL (19.4%) had a higher incidence compared to DC (14.1%) and CL (15.2%). SAL had the highest PGG expression; CL had the lowest. DC had the highest IGG; CL had the lowest. Conclusions: CL and the lobular carcinoma variants are characterized by differential patterns of gene expression. Outlier cases are not infrequent within each of the special subtypes in this large observational cohort. The variation in gene expression, noted by histologic subtype, will be presented in detail.

10 Background: ER+ special histologic breast cancer subtypes are prognostically significant. Here we report the special histologic subtypes of ER+ breast carcinoma and associated patterns of observed gene expression as measured by the 21 gene OncotypeDX assay. Methods: All tumors from 6/1/04-5/31/12 were included in analyses. Central path used WHO criteria. Ductal NOS (DC), tubular (TC), cribriform (CC), mucinous (MC) and papillary (PC) carcinomas were included. Quantitative expression of 16 cancer related genes was measured on a scale from 2 to 15 (relative to reference genes) where a 1 unit increment is associated with ~2-fold change in expression. Recurrence Score (RS) was calculated as published. Descriptive stats for the RS and individual genes [ER, PR, invasion gene group (IGG) and proliferation gene group (PGG)] were obtained. Comparisons of means were adjusted to control the overall error rate under any complete or partial null hypothesis. Results: DC accounted for 95.1%, TC 0.7% , CC 0.3%, MC 3.1% and PC 0.7% of 246,555 cases. For all types a wide continuous range of RS was noted. DC had the highest mean RS, followed in decreasing order by MC, TC, CC and PC. PC had the highest ER and PC and CC the highest PR. TC had the lowest ER. ER was not different between CC and MC but PR was. The proportion with ER+/PR- phenotype was different among the subtypes: TC (8.1%) and CC (7.1%) had the lowest incidence whereas MC (12.9%) and PC (10.3%) were more similar to DC (14.1%). TC had lowest PGG expression. MC and PC had lower IGG expression compared to other subtypes. Conclusions: The special subtypes had a wide continuous range of RS: DC had the highest mean RS, followed in decreasing order by MC, TC, CC and PC. PC had the highest ER and PC and CC the highest PR. TC had the lowest ER (may reflect submission bias). ER was not different between CC and MC but PR was. The proportion with ER+/PR- phenotype was different among the subtypes: TC and CC had the lowest incidence, MC and PC were more similar to DC. TC had the lowest PGG expression. MC and PC had lower IGG expression. [Table: see text]

520 Background: There is considerable interest in developing HER2 testing criteria for adjuvant H. We used the 21-gene assay to examine the relationship of HER2 mRNA to benefit from H. Methods: N9831 compared adjuvant chemotherapy AC-T to concurrent chemotherapy-trastuzumab AC-TH in stage I-III HER2+ breast cancer. Recurrence Score (RS) and HER2 mRNA expression were determined by Oncotype DX (neg2 expression units). Cox regression was used to assess the association of HER2 expression with H benefit for distant recurrence. Results: Median follow-up: 7.4 yrs. Of 1,940 total pts, 901 had consent and sufficient tissue. HER2 by RT-PCR was neg in 130 (14%), equiv in 85 (9%), and pos in 686 (76%) pts. Concordance between HER2 assessments was 95% for RT-PCR vs central IHC (>10% + cells = +), 91% for RT-PCR vs central FISH (≥2.0 = pos) and 94% for central IHC vs central FISH. In the primary analysis, the association of HER2 expression with H benefit was marginally non-significant (P=0.057). In hormone receptor pos pts (local IHC) the association was significant (P=0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 mRNA expression levels. The observed treatment benefit in low HER2 pts was not due to imbalance between arms in RS and individual gene expression values. Conclusions: Concordance among HER2 assessments by central IHC, FISH, and RT-PCR was high. Association of HER2 mRNA expression with H benefit was marginally non-significant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in the pts with either IHC+ or FISH+ tumors. Benefit was observed in pts with high HER2 by RT-PCR but also observed for the small groups of pts with negative results by quantitative RT-PCR or FISH (Table). Plausible mechanisms for this observation will be discussed. [Table: see text]

1 Background: RS predicts outcome in node- and node+, ER+ pts treated with adjuvant endocrine therapy as well as benefit from adjuvant chemotherapy, with high RS receiving most of the benefit. We studied the prognostic impact of RS in node+, ER+ pts treated with adjuvant chemotherapy plus endocrine therapy as part of the NSABP B-28 trial. Methods: B-28 compared doxorubicin/cyclophosphamide (ACX4) with ACX4 followed by paclitaxel X4. Pts >50 yrs and those

190 Background: In selected low-risk patients with DCIS treated with wide local excision without radiation, the DCIS score was validated as a predictor of 10 year risk of an ipsilateral breast event (IBE - recurrence of in situ or invasive carcinoma) (p = 0.02) (Solin; SABCS 2011). As part of the development of the DCIS score, scaling from 0 to 100 and determination of risk group cutoff values was done using 100 patient DCIS samples from Marin General Hospital (MGH) selected to have a wide range of tumor characteristics. Methods: 100 patient specimens diagnosed with DCIS were provided by MGH. The Oncotype DX assay was performed, normalized expression levels for the 16 cancer related genes were determined, the DCIS Score and Recurrence Score (RS) were calculated. Distributions of the DCIS score, RS, and individual gene expression levels were described overall and by tumor characteristics. Treatment and pt outcome data were not available. Results: Samples for 96 pts had sufficient tumor and were evaluable; 47% had high nuclear grade, 52% comedo necrosis, 32% tumor size >10 mm, and 9% were ER-negative by IHC. After scaling and risk group cutoff determination, the DCIS score was low risk (0-38) for 49%, intermediate risk (39-54) for 27%, and high risk (≥55) for 24% of pts (Table). The DCIS score was widely distributed within subgroups defined by each of the clinical and pathology characteristics. Proliferation gene expression levels were low in DCIS, on average, relative to prior studies in invasive breast cancer. 92% had a proliferation gene group score

1021 Background: At 5 years, AT did not improve disease free survival or overall survival and RS was a more accurate predictor of relapse than standard clinicopathologic characteristics for patients with hormone receptor (HR) positive tumors. Methods: A Phase III Intergroup trial tested adjuvant AT vs. AC. Women with 1-3 N + or N - and T-size > 1cm were randomized to 4 cycles of AT (60 mg/m2/60 mg/ m2) or AC (60 mg/m2/600 mg/m2) q 3 wk x 4. Patients(pts) with ER + and/ or PR + tumors received tam for 5 yrs. Pts were stratified by nodal, HR (ER+ PR+, ER+PR-, ER-PR+, ER-PR-, ER/PR unk) and menopausal status. The primary endpoint was DFS. A sample of 465 pts with HR + breast cancer with 0 to 3 positive axillary nodes who did (N =116) or did not have a recurrence had tumor tissue evaluated using the 21- gene assay. Grade and HR expression were evaluated locally and centrally. Results: 2952 pts were randomized between 7/30/98 and 1/21/00. 2883 were eligible and analyzable. Arms were balanced for age, HR, menopause, nodes, surgery, grade and T-size: median age 51; 64% ER +; 65% LN-; grade: 10% low, 38% int., 46% high; and median T-size - 2.0 cm. At a median follow-up of 11.5 years the DFS/OS results are shown in the table below. RS was a highly significant predictor of recurrence including node negative and node positive disease (P < .0001) and predicted recurrence more accurately than clinical variables. Conclusions: At 11.5 yrs. median follow-up, there remains no difference in DFS or OS, although there continue to be fewer events in the AT arm in the prespecified ER/PR negative subgroup. At 10 years, the RS continues to be a more accurate predictor of relapse than standard clinical features. [Table: see text]

1005 Background: We previously reported that in the E5194 clinical trial patients with ductal carcinoma in situ (DCIS) treated with wide local excision (WLE) without radiation (RT), the DCIS Score was significantly associated with 10 year risk of an ipsilateral breast event (IBE - recurrence of in situ or invasive carcinoma), whether evaluated as a continuous or categorical variable (P=0.02 for both). Here we evaluate correlation between DCIS Score and clinicopathologic (CP) features and if DCIS Score provides independent recurrence risk information. Methods: The study population included 327 women with DCIS prospectively selected for treatment with WLE without RT, including low-intermediate grade tumors ≤2.5 cm or high-grade ≤1 cm. CP variables included age, menopausal status, tamoxifen treatment (used in 29%) and expert centrally determined tumor size, grade, comedo necrosis, tumor type, and margin status. The association between DCIS Score and CP variables was examined by spearman rank correlation, and proportional hazards regression models were used to determine variables significantly associated with IBE. Results: Lesion size (p=0.009) and menopausal status (p=0.03) were significantly associated with IBE, while grade (p=0.69) and comedo necrosis (p=0.47) were not. DCIS Score was significantly associated with IBE after adjustment for CP features and tamoxifen use (p=0.02). DCIS Score was moderately correlated with grade (rs=0.46; 95% CI 0.37,0.54), percentage comedo necrosis (rs=0.49; CI 0.41,0.57), and lesion size (rs=0.18; CI 0.08,0.29) but not other features. Exploratory analyses in all CP subgroups, including the multicomponent Van Nuys Prognostic Index, showed a wide range of DCIS Scores in each subgroup. Concordance of the grades among readers was low: local vs parent central, 68%; local vs central nuclear grade, 45%; parent central vs central nuclear grade, 37%. Conclusions: DCIS Score is moderately correlated with grade, comedo necrosis, and tumor size. DCIS Score provides recurrence risk information independent of these features and identifies subjects with DCIS who are at high risk for local invasive and in-situ local recurrence after WLE alone.

526 Background: High tumor grade is included in practice guidelines as a marker of higher recurrence risk in stage II colon cancer. Multiple systems for tumor grading exist, without a standardized approach. We characterize agreement of 2 methods for tumor grading and association with recurrence in the context of clinical, pathologic, and genomic tumor features including mismatch repair (MMR) and the 12-gene recurrence score (RS). Methods: Colon tumors were graded independently by 2 academic GI pathologists (P1, P2). Grade was defined by % tumor with gland-like structures: well (> 95%), moderately (50-95%) and poorly (< 50%) differentiated. P1 used this 3-tier system while P2 used 2-tier scheme with well and moderately differentiated tumors defined as low grade and poorly differentiated as high grade. All mucinous tumors were high-grade by P2 but not P1. Relationship to recurrence-free interval (RFI) was assessed by Cox regression. Results: Primary tumors from 504 stage II colon cancer treated with surgery alone were included. 18% (P1) and 31% (P2) were high grade. High grade tumors were more likely right-sided (≥ 60%) and MMR deficient (≥ 35%) for P1 and P2 (all p < 0.001). Proportion of mucinous tumors was similar for high and low grade by P1 (25% vs. 21%, p = 0.39). P2 high grade trended to lower recurrence (HR = 0.63, p = 0.10) while P1 grade was not associated w/ RFI (p = 0.46). In multivariate analyses including grade and RS, P2 high grade was associated with lower recurrence (p = 0.007) but P1 grade was not associated w/ RFI (p = 0.30). Neither grade was associated w/ RFI (p > 0.30) after controlling for RS, MMR, tumor location and mucinous histology. Using the two-tier scheme, agreement b/w the 2 pathologists was low (kappa = 0.30, 95% CI 0.21-0.39) in all pts and moderate if mucinous tumors were excluded (kappa = 0.52, 95% CI 0.40-0.64). Conclusions: High tumor grade was not found to be a marker of higher recurrence risk in stage II colon cancer. Other markers validated in stage II colon cancer, such as MMR and RS, should be considered. Interpathologist agreement on colon tumor grade is modest, even with central expert review. [Table: see text]

403 Background: Colorectal cancer patients with deficient mismatch repair (dMMR) have significantly fewer recurrences and may respond less well to chemotherapy. Immunohistochemical (IHC) determination of MMR status is therefore recommended to identify patients in whom adjuvant therapy is not indicated, but little is known regarding the variability of assay results. We aimed to define MMR IHC assay variability in formalin-fixed, paraffin-embedded (FFPE) material from the QUASAR-1 colorectal cancer trial contained within heterogeneity-prone tissue microarrays (TMAs) Methods: TMA sections of FFPE material from the QUASAR-1 trial were distributed to 2 independent laboratories (Leeds, UK and Vitro Molecular Laboratories [VML], FL, USA) for MMR IHC assays. Serial TMA sections were stained with MLH1/MSH2 using techniques blinded to the other laboratory. Each stained section was double-scored independently and results compared to determine inter-assay variability. Results: Matched MMR data were available for 1224 cases of MLH1 and 1223 cases of MSH2. Of these, loss of expression of MMR (dMMR) was reported in 160 cases (13.1%) by VML and 179 cases (14.6%) by Leeds. 140 (11.4%) were dMMR in both labs, 20 cases (1.6%) by VML alone and 39 (3.2%) by Leeds alone. Discordant dMMR status was observed in 56/166 (34%) dMMR cases identified with MLH1 and 10/34 (29%) cases identified with MSH2. Kappa coefficients for inter-assay agreement were 0.798 (95% CI = 0.748 - 0.848) for MMR status overall, 0.778 (95% CI = 0.722 - 0.835) for MLH1 and 0.823 (95% CI = 0.714 - 0.932) for MSH2. 85% (33/39) of discordant Leeds dMMR colon cancers were in the right colon where dMMR is more common, compared to 47% of VML discordant dMMR cases. Conclusions: Independent determination of MMR status by IHC on TMAs is associated with excellent inter-assay agreement. The reasons for MMR case discordance are under further investigation. These results further support routine MMR testing by IHC. No significant financial relationships to disclose.

Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPARγ has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARγ has been validated through two supporting biological assays., Author Summary Cell culture models are an important vehicle for understanding biological processes and evaluation of therapeutic reagents. More importantly, the literature suggests that tumor cells grown in 3D exhibit pronounced drug and radiation resistances that are remarkably similar to that of tumors in vivo. Therefore, the needs for quantifying 3D assays continue to grow. In this paper, we develop robust computational methods to integrate morphometric and molecular information for a panel of breast cancer cell lines that are grown in 3D. Specifically, morphometric traits are imaged through microscopy, and then quantified computationally. We then show that these morphometric traits can identify subtypes within this panel of breast cancer cell lines, and that the subtypes are clinically relevant in terms of being ERBB2 positive or triple negative. These subtypes and their representations are then associated with their molecular data to reveal PPARG as an important marker for triple-negative breast cancer. Finally, we design two independent experiments to show the validity of this marker in both 3D cell culture models and human breast cancer tissue.