Search

Maternal embryonic leucine-zipper kinase (MELK) regulates cell cycle progression and is highly expressed in many cancers. The molecular mechanism of MELK dysregulation has not been determined in aggressive forms of breast cancer, such as triple negative breast cancer (TNBC). To evaluate molecular markers of MELK aberrations in aggressive breast cancer, we assessed MELK gene amplification and expression in breast tumors. MELK mRNA expression is highly up-regulated in basal-like breast cancer (BLBC), the major molecular subtype of TNBC, compared to luminal or other subtypes of breast tumors. MELK copy number (CN) gains are significantly associated with BLBC, whereas no significant association of CpG site methylation or histone modifications with breast cancer subtypes was observed. Accordingly, the CN gains appear to contribute to an increase in MELK expression, with a significant correlation between mRNA expression and CN in breast tumors and cell lines. Furthermore, immunohistochemistry (IHC) assays revealed that both nuclear and cytoplasmic staining scores of MELK were significantly higher in invasive ductal carcinoma (IDC) tumors compared to ductal carcinoma in situ (DCIS) and normal breast tissues. Our data showed that upregulation of MELK in BLBC may be in part driven by CN gains, rather than epigenetic modifications, indicating a potential for overexpression and CN gains of MELK to be developed as a diagnostic and prognostic marker to identify patients who have more aggressive breast cancer.

We performed a pilot study in anticipation of using long-aged precut formalin-fixed paraffin-embedded tissue sections stored in real-world conditions for translational biomarker studies of topoisomerase 2A (TOP2A), Ki67, and human epidermal growth factor receptor 2 (HER2) in endometrial cancer. Formalin-fixed paraffin-embedded tissue blocks or unstained slides or both from GOG-0177 were collected centrally (1999-2000) and stored at room temperature. During 2004 to 2011 specimens were stored at 4°C. Matched pairs of stored slides and freshly cut slides from stored blocks were analyzed for TOP2A (KiS1), Ki67 (MIB1), and HER2 (HercepTest) proteins. To assess DNA stability (HER2 PathVision), fluorescence in situ hybridization (FISH) was repeated on stored slides from 21 cases previously shown to be HER2 amplified. Immunohistochemistry (IHC) staining intensity and extent, mean FISH copies/cell, and copy number ratios were compared using the κ statistic for concordance or signed rank test for differences in old cut versus new cut slides. IHC results reflected some protein degradation in stored slides. The proportion of cells with TOP2A staining was lower on average by 12% in older sections (P=0.03). The proportion of Ki67-positive cells was lower in stored slides by an average of 10% (P

This abstract was not presented at the conference. Citation Format: Hardeman A, Grushko T, Clayton W, Hurley I, Khramtsova G, Parker J, Perou C, Olopade O. Not presented [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-07-05.

Background: Biomarker testing in lung cancer is often limited by a lack of sufficient formalin fixed, paraffin embedded (FFPE) tissue for comprehensive genomic profiling. To promote personalized therapy for lung cancer, a multiplex FISH assay was developed to simultaneously assess aberrations in ROS1, RET, and MET on a single FFPE specimen slide. Methods: Specimens included primary tumor (N = 39) as well as biopsies from a variety of metastatic sites (N = 16). These included 12 samples with ROS1 rearrangements, 3 samples with RET rearrangements, and 11 samples with MET amplification reported by a previously validated laboratory test method. A probe mix contained 6 differentially labeled fluorescent probes: 3' ROS1, 5' ROS1, 3' RET, 5' RET, MET and CEP7. The probes were formulated in Vysis IntelliFISH Hybridization Buffer to allow for a 2 h hybridization time. BioView imaging platform and Duet software algorithm were used to perform automated slide scanning and digital analysis. Specimens were considered positive for ROS1 or RET rearrangement if >15% evaluated cells contained a break apart (rearranged) signal. Specimens were considered positive for MET amplification if >15% of cells had MET/CEP7 ratio >2 and positive for polysomy if >15% of cells had 5 or more MET signals copies. Results: The 6 color FISH assay was 97% concordant for ROS1 rearrangement and 100% concordant for RET rearrangement. The average background percentage of positive tumor cells in cases without known gene rearrangements was approximately 5%, yielding a negative cutoff threshold of approximately 15%, in accordance with cutoff thresholds reported in literature. The 6 color FISH assay was 91% concordant for MET amplification or polysomy. Results were interpretable for 98% of targets analyzed by the 6 color FISH method. Four samples failed on analysis for one of the targets due to lack of sufficient cells or lack of adequate hybridization signal. Conclusion: A newly developed 6-color FISH assay allows simultaneous detection of three genomic abnormalities using only 1 specimen slide. This feature combined with rapid hybridization in IntelliFISH buffer and automated BioView slide imaging and analysis can significantly increase the yield of molecular testing on limited lung cancer tissue samples. Careful pathologic correlation for tumor cell identification and careful assessment of hybridization quality are necessary to optimize the accuracy of this test method. Citation Format: Irina A. Sokolova, Patrick Bedroske, Tatyana A. Grushko, Amber R. Schneider, Kristine Jacobson, Jesse S. Voss, Yishay Tauber, Yossi Avisror, Vitaliy Shkolnik, Katerina Pestova, Katherine B. Geiersbach. Multiplex fast FISH assay for detecting ROS1, RET and MET aberrations in FFPE specimens using BioView image analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4256.

Gefitinib has activity in patients with advanced squamous cell carcinoma of the head and neck (SCCHN) and skin toxicity has been postulated to be a predictor of response and improved outcome.This open-label, multi-institution, phase II study evaluated the activity of gefitinib at individually escalated doses up to 750 mg to achieve the skin toxicity grade ≥2.Forty four patients were enrolled. Only twenty-three (52%) experienced skin rash grade ≥2. Of 44 patients, partial responses were noted in 3 (7%), stable disease in 8 (18%) and progressive disease in 33 patients. Median progression-free survival was 1.9 months (95% CI 1.6-2.2) and median overall survival was 5.1 months (95% CI 2.4-7.8). Grade of skin rash was not associated with response rate (p=0.169) nor tumor control rate (p=0.284); however, higher gefitinib trough levels were associated with disease control. Of the 11 tissue samples analyzed for EGFR gene copy by FISH, 7 were EGFR FISH positive, but this was not associated with improved tumor control or survival.Gefitinib has clinical activity as monotherapy in SCCHN. Dose escalation of gefitinib is feasible and may increase skin toxicity, but our data do not support increased activity.

Breast cancer is a heterogeneous disease with known expression-defined tumor subtypes. DNA copy number studies have suggested that tumors within gene expression subtypes share similar DNA Copy number aberrations (CNA) and that CNA can be used to further sub-divide expression classes. To gain further insights into the etiologies of the intrinsic subtypes, we classified tumors according to gene expression subtype and next identified subtype-associated CNA using a novel method called SWITCHdna, using a training set of 180 tumors and a validation set of 359 tumors. Fisher’s exact tests, Chi-square approximations, and Wilcoxon rank-sum tests were performed to evaluate differences in CNA by subtype. To assess the functional significance of loss of a specific chromosomal region, individual genes were knocked down by shRNA and drug sensitivity, and DNA repair foci assays performed. Most tumor subtypes exhibited specific CNA. The Basal-like subtype was the most distinct with common losses of the regions containing RB1, BRCA1, INPP4B, and the greatest overall genomic instability. One Basal-like subtype-associated CNA was loss of 5q11–35, which contains at least three genes important for BRCA1-dependent DNA repair (RAD17, RAD50, and RAP80); these genes were predominantly lost as a pair, or all three simultaneously. Loss of two or three of these genes was associated with significantly increased genomic instability and poor patient survival. RNAi knockdown of RAD17, or RAD17/RAD50, in immortalized human mammary epithelial cell lines caused increased sensitivity to a PARP inhibitor and carboplatin, and inhibited BRCA1 foci formation in response to DNA damage. These data suggest a possible genetic cause for genomic instability in Basal-like breast cancers and a biological rationale for the use of DNA repair inhibitor related therapeutics in this breast cancer subtype. Electronic supplementary material The online version of this article (doi:10.1007/s10549-011-1846-y) contains supplementary material, which is available to authorized users.

Purpose Assess efficacy and toxicity of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, added to, and in maintenance after, concurrent chemoradiotherapy (CCRT) in locally advanced head and neck cancer (LA-HNC) and correlate outcomes with EGFR gene copy number alterations. Patients and Methods Patients with stage III to IV LA-HNC received two cycles of carboplatin/paclitaxel induction chemotherapy (IC) followed by split-course CCRT with fluorouracil, hydroxyurea, twice daily radiotherapy (FHX), and gefitinib (250 mg daily) followed by continued gefitinib for 2 years total. The primary end point was complete response (CR) rate after CCRT. EGFR gene copy number was assessed by fluorescent in situ hybridization. Results Sixty-nine patients (66 with stage IV disease, 37 with oropharynx primary tumors, and 67 with performance status 0 to 1) were enrolled with a median age of 55 years. Predominant grade 3 or 4 toxicities during IC and CCRT were neutropenia (n = 20) and in-field mucositis (n = 59) and dermatitis (n = 23), respectively. CR rate after CCRT was 90%. After median follow-up of 3.5 years, 4-year overall, progression-free, and disease-specific survival rates were 74%, 72%, and 89%, respectively. To date, one patient has developed a second primary tumor in the aerodigestive tract. In 31 patients with available tissue, high EGFR gene copy number was associated with worse overall survival (P = .02). Conclusion Gefitinib can be administered with FHX and as maintenance therapy for at least 2 years, demonstrating CR and survival rates that compare favorably with prior experience. High EGFR gene copy number may be associated with poor outcome in patients with LA-HNC treated with this regimen.

Triple-negative breast cancers represent a subset of breast cancers with a particularly aggressive phenotype and poor clinical outcomes. Recent molecular profiling of these tumors has revealed a high frequency of epidermal growth factor receptor (EGFR) dysregulation, among other abnormalities. EGFR status correlates negatively with survival in patients with triple-negative breast cancers, and thus focus has turned on this receptor as a potential clinical target. Two classes of EGFR inhibitors are currently in clinical use: the monoclonal antibodies and the small molecule tyrosine kinase inhibitors. Trials of these drugs in breast cancer, however, have been largely disappointing. It remains to be seen whether advances in our understanding of the mechanisms of EGFR dysregulation and effects of multiple compensatory pathways in breast cancer, coupled with improved targeting to appropriate patient populations, will yield meaningful improvements in clinical outcomes.

There is increasing evidence that metformin, a commonly used treatment for diabetes, might have the potential to be repurposed as an economical and safe cancer therapeutic. The aim of this study was to determine whether stage III-IV or recurrent endometrial cancer patients who are using metformin during treatment with chemotherapy have improved survival. To test this we analyzed a retrospective cohort of subjects at two independent institutions who received chemotherapy for stage III-IV or recurrent endometrial cancer from 1992 to 2011. Diagnosis of diabetes, metformin use, demographics, endometrial cancer clinico-pathologic parameters, and survival duration were abstracted. The primary outcome was overall survival. The final cohort included 349 patients, 31 (8.9%) had diabetes and used metformin, 28 (8.0%) had diabetes but did not use metformin, and 291 (83.4%) did not have diabetes. The results demonstrate that the median overall survival was 45.6 months for patients with diabetes who used metformin compared to 12.5 months for patients with diabetes who did not use metformin and 28.5 months for patients without diabetes (log-rank test comparing the three groups P = 0.006). In a model adjusted for confounders, the difference in survival between the three groups remained statistically significant (P = 0.023). The improvement in survival among metformin users was not explained by better baseline health status or more aggressive use of chemotherapy. Overall, the findings in this retrospective cohort of endometrial cancer patients with stage III-IV or recurrent disease treated with chemotherapy indicate that patients with diabetes who were concurrently treated with metformin survived longer than patients with diabetes who did not use metformin.

Epidermal growth factor receptor (EGFR) inhibitors have demonstrated efficacy in squamous cell carcinoma of the head and neck (SCCHN). In addition to EGFR, other ErbB family members are expressed and activated in SCCHN. Afatinib is an ErbB family blocker that has been approved for treating patients with EGFR-mutated nonsmall cell lung cancer. We sought to determine the efficacy of afatinib in preclinical models and compare this to other EGFR-targeted agents. Afatinib efficacy was characterized in a panel of ten SCCHN cell lines and found to be most effective against cell lines amplified for EGFR. Afatinib had lower IC(50) values than did gefitinib against the same panel. Two EGFR-amplified cell lines that are resistant to gefitinib are sensitive to afatinib. Cetuximab was not found to have a synergistic effect with afatinib either in vitro or in vivo. Both afatinib and cetuximab were effective in tumor xenograft model. Afatinib is an effective agent in SCCHN especially in models with EGFR amplification.

The fluorescence in situ hybridization (FISH) technique is a sensitive method for detecting gene amplification in tumor cells. However, FISH on formalin-fixed and paraffin-embedded tissue sections is time-consuming and labor-intensive (1). Tissue processing and cross-linking fixatives such as formalin may prevent the probe from accessing its target sequence, prolonging the FISH processing time for fixed tissues compared to cells in suspension or frozen material (2,3). For example, the standard FISH procedure for fixed tissue takes about 2 days and contains several acid/chaotrope-based pretreatment steps and an overnight hybridization (www.vysis.com/ PretreatingParaffinSpecimens_32217. asp). Despite its sensitivity and specificity, FISH on paraffin sections is therefore not the method of choice in many clinical and research laboratories. Here we propose a simplified and rapid FISH protocol for fixed tissues using microwave irradiation. Using the commercially available probe mixture of HER-2 LSP (locusspecific probe) and CEP17 (centromere enumeration probe for chromosome 17, α-satellite DNA; Vysis/Abbott, Downers Grove, IL, USA), FISH was conducted on 4 μm thick formalinfixed, paraffin-embedded tissue sections from 9 breast cancer and 7 endometrial cancer cases. Normal human lymphocytes and 4 breast cancer cell lines, all embedded in paraffin, were used as controls for hybridization and scoring efficiency (4). In total, 21 specimens were analyzed. Sections from the same block were treated either with the commonly used FISH procedure developed by Vysis or the new microwave procedure. For each case, an average of 62 (30–173) cells were scored and the mean copy number for each probe, the HER-2/CEP17 ratio, and the proportion of aneusomic cells were compared between the methods (for statistical analysis, see Supplementary Table S1 available online at www.BioTechniques.com). In this study, a Samsung Microwave 1100W (Model no. MW1080STA, with 10 power levels; Samsung Electronics America, Ridgefield Park, NJ, USA) was used. The microwave settings for other brands of microwaves can be determined by measuring the temperatures in the buffer (pretreatment step) and water (hybridization step). The optimal temperatures are written in parentheses after each irradiation step in Protocol 1. Keep in mind that irradiation time must be optimized to prevent the loss of tissue morphology, but the tissue has to be irradiated long enough for the LSP probe to hybridize. The mean copy number results from the two procedures were highly comparable (see Supplementary Table S1). The microwave FISH procedure performed equally well for both nonamplified and amplified cases as well as controls, with two exceptions. In BT474 cells, the mean absolute HER-2 signal per cell ±sd was slightly higher after microwave treatment (34.0 ± 11.0) compared to standard treatment (28.1 ± 10.5), and the difference reached significance (P = 0.002). We ascribe the observed difference to the complex nature of the HER-2 amplification in these cells, rather than to the type of treatment. In BT-474 cells, due to the formation of compact heterogeneous clusters of HER-2 signals, the identification of individual signals can be difficult and may result in slightly variable numbers of the mean HER-2 signals per cell in repetitions (4,5). However, the other FISH parameters showed comparable values, and the results from both treatments meet the criteria for HER-2 amplification status in the BT-474 cells (4,5). The same explanation is applicable to the one cancer case (see Supplementary Table S1, case 14). Our rapid microwave protocol showed high hybridization efficiency and gave us signals that were as bright (Figure 1, B, D, and F) as the traditional method (Figure 1, A, C, and E). In Figure 1, A and B, adjacent tissue sections from the same breast cancer case are shown. The HER-2/CEP17 ratios were 4.2 (standard treatment) and 4.5 (microwave) (P = 0.448). By

The differentiation of copper cells, which secrete stomach acid in Drosophila larvae, has been shown previously to be sensitive to the labialk3 mutation. Here we found that stomach acid secretion in adults was insensitive to labk3. The basis for this stage-specific effect was elucidated by characterizing the development, structure, and function of the adult midgut. First, we demonstrated by copper-dependent fluorescence and morphology that copper cells were present in the adult stomach. Fine-structure analysis of adult copper cells led to the identification of a previously unrecognized plasma membrane domain: apicolateral contacts between copper cells and their neighbors consisted of smooth septate junctions that were enriched in alphabeta-spectrin and ankyrin. Second, we demonstrated that adult copper cells were present in labk3/labvd1 (conditional/null) adults. The labial protein was expressed in adult labk3/labvd1 copper cells, but not in larvae. Thus the labk3 mutation had a stage-specific effect on midgut labial expression, but did not appear to affect protein function. Surprisingly, stomach acidification was dispensable during larval development, since labk3/labvd1 mutant larvae that lacked midgut acidification developed into fertile adults.

Spectrin, together with actin and a number of other accessory proteins, forms a submembrane cytoskeletal network in the human erythrocyte ghost. Through an elegant combination of structural, biochemical, and genetic studies, spectrin was shown to be an important determinant of erythrocyte shape and membrane stability. Genetic studies of a novel nonerythroid spectrin (βH) in Drosophila and Caenorhabditis elegans now reveal that spectrin can influence the shape and stability of whole organisms.(1,2) Nonerythroid spectrins are proposed to have roles in cell adhesion, establishment of cell polarity, and attachment of other cytoskeletal structures to the plasma membrane. The phenotypes of the βH spectrin mutations provide an exciting biological context in which to evaluate these roles and perhaps to uncover new ones. BioEssays 20:875–878, 1998. © 1998 John Wiley & Sons, Inc.

Despite nearly universal expression of the wild-type epidermal growth factor receptor (EGFR) and reproducible activity of EGFR inhibitors in patients with squamous cell carcinoma of the head and neck (SCCHN), the majority of patients will not have objective responses. The mechanisms of this intrinsic resistance are not well established. We hypothesized that sensitivity to EGFR inhibitors can be predicted based on the inhibitors' effects on downstream signaling. Cell viability assays were used to assess sensitivity to the EGFR inhibitor gefitinib (ZD1839) in 8 SCCHN cell lines. Fluorescence in-situ hybridization showed the two most sensitive lines to be highly gene-amplified for EGFR. Western blotting confirmed that phosphoEGFR was inhibited at low concentrations of gefitinib in all lines tested. Phosphorylation of downstream signaling protein AKT was inhibited in sensitive lines while inhibition of phosphoERK displayed no relationship to gefitinib efficacy. Phosphatase and tensin homolog (PTEN) expression was evident in all cell lines. Activating PIK3CA mutations were found in two resistant cell lines where pAKT was not inhibited by gefitinib. In resistant cell lines harboring PIK3CA mutations, a PI3K inhibitor, LY294002, or AKT siRNA reduced cell viability with an additive effect demonstrated in combination with gefitinib. Additionally, LY294002 alone and in combination with gefitinib, was effective at treating PIK3CA mutated tumors xenografted into nude mice. Taken together this suggests that constitutively active AKT is a mechanism of intrinsic gefitinib resistance in SCCHN. This resistance can be overcome through targeting of the PI3K/AKT pathway in combination with EGFR inhibition.

RON (MST1R) is one of two members of the MET receptor tyrosine kinase family, along with parent receptor MET. RON has a putative role in several cancers, but its expression and function is poorly characterized in gastroesophageal adenocarcinoma. A recognized functional role of MET tyrosine kinase in gastroesophageal cancer has led to early phase clinical trials using MET inhibitors, with unimpressive results. Therefore, the role of RON in gastroesophageal cancer, as well as its role in cooperative signaling with MET and as a mechanism of resistance to MET inhibition, was studied in gastroesophageal tissues and cell lines. By IHC, RON was highly overexpressed in 74% of gastroesophageal samples (n = 94) and overexpression was prognostic of poor survival (p = 0.008); RON and MET co-expression occurred in 43% of samples and was prognostic of worst survival (p = 0.03). High MST1R gene copy number by quantitative polymerase chain reaction and confirmed by fluorescence in situ hybridization and/or array comparative genomic hybridization, was seen in 35.5% (16/45) of cases. High MST1R gene copy number correlated with poor survival (p = 0.01), and was associated with high MET and ERBB2 gene copy number. a novel somatic MST1R juxtamembrane mutation R1018G was found in 11% of samples. RON signaling was functional in cell lines, activating downstream effector STAT3, and resulted in increased viability over controls. RON and MET co-stimulation assays led to enhanced malignant phenotypes over stimulation of either receptor alone. Growth inhibition as evidenced by viability and apoptosis assays was optimal using novel blocking monoclonal antibodies to both ROn and MET, versus either alone. SU11274, a classic MET small molecule tyrosine kinase inhibitor, blocked signaling of both receptors and proved synergistic when combined with STAT3 inhibition (combination index

Purpose Compared with white women, black women experience a disproportionate burden of aggressive breast cancer for reasons that remain unknown and understudied. In the first study of its kind, we determined the distribution of molecular subtypes of invasive breast tumors in indigenous black women in West Africa. Patients and Methods The study comprised 507 patients diagnosed with breast cancer between 1996 and 2007 at six geographic regions in Nigeria and Senegal. Formalin-fixed and paraffin-embedded sections were constructed into tissue microarrays and immunostained with 15 antibodies. Five molecular subtypes were determined, and hierarchical cluster analysis was conducted to explore subgroups for unclassified cases. Results The mean (± standard deviation) age of 378 patients in the first cohort was 44.8 ± 11.8 years, with the majority of women presenting with large (4.4 ± 2.0 cm) high-grade tumors (83%) in advanced stages (72% node positive). The proportions of estrogen receptor (ER) –positive, progesterone receptor–positive, and human epidermal growth factor receptor 2 (HER2) –positive tumors were 24%, 20%, and 17%, respectively. Triple negativity for these markers was predominant, including basal-like (27%) and unclassified subtype (28%). Other subtypes were luminal A (27%), luminal B (2%), and HER2 positive/ER negative (15%). The findings were replicated in the second cohort of 129 patients. The unclassified cases could be grouped into a bad prognosis branch, with expression of vascular endothelial growth factor, B-cell lymphoma extra-large protein, and Cyclin E, and a good prognosis branch, with expression of B-cell lymphoma protein 2 and Cyclin D1. Conclusion These findings underscore the urgent need for research into the etiology and treatment of the aggressive molecular subtypes that disproportionately affect young women in the African diaspora.

Breast cancer is a complex disease caused by the progressive accumulation of multiple gene mutations combined with epigenetic dysregulation of critical genes and protein pathways. There is substantial interindividual variability in both the age at diagnosis and phenotypic expression of the disease. With an estimated 1,152,161 new breast cancer cases diagnosed worldwide per year, cancer control efforts in the postgenome era should be focused at both population and individual levels to develop novel risk assessment and treatment strategies that will further reduce the morbidity and mortality associated with the disease. The discovery that mutations in the BRCA1 and BRCA2 genes increase the risk of breast and ovarian cancers has radically transformed our understanding of the genetic basis of breast cancer, leading to improved management of high-risk women. A better understanding of tumor host biology has led to improvements in the multidisciplinary management of breast cancer, and traditional pathologic evaluation is being complemented by more sophisticated genomic approaches. A number of genomic biomarkers have been developed for clinical use, and increasingly, pharmacogenetic end points are being incorporated into clinical trial design. For women diagnosed with breast cancer, prognostic or predictive information is most useful when coupled with targeted therapeutic approaches, very few of which exist for women with triple-negative breast cancer or those with tumors resistant to chemotherapy. The immediate challenge is to learn how to use the molecular characteristics of an individual and their tumor to improve detection and treatment, and ultimately to prevent the development of breast cancer. The five articles in this edition of CCR Focus highlight recent advances and future directions on the pathway to individualized approaches for the early detection, treatment, and prevention of breast cancer.

Over the past two decades, we have come to an understanding of cancer as a genetic disorder caused by the progressive accumulation of multiple genetic changes, which include point mutations, chromosomal rearrangements, viral insertions, and genomic amplifications and deletions (1,2). Gene amplifications, point mutations, viral insertions, and chromosomal rearrangements are dominant genetic damages that primarily target oncogenes whose gain of function (overexpression) leads to dysregulation of cell growth and transformation. Recessive point mutations and deletions mainly cause loss of function in tumor suppressor genes (TSGs) that control cell-cycle progression and DNA repair mechanisms (2).

Introduction: TNBC has the highest mortality rate amongst all other breast cancer types due to its complex tumor heterogeneity and lack of well-defined molecular targets. It is known that women of African descent are two to three times more likely to develop TNBC compared to women of European ancestry, yet wide-scale genomic studies of African and African American breast tumors are limited. To elucidate genotypes and molecular subtypes associated with the most aggressive forms of breast cancer, we used the PAM50 NanoString platform to reclassify Nigerian (NG), African American (AA) and Caucasian (CA) tumors previously annotated by Immunohistochemistry (IHC), and correlated our findings to their germline genotype data obtained using high-throughput technologies. Methods: RNAs were isolated from formalin-fixed, paraffin embedded (FFPE) tumor tissues using the High Pure Paraffin Kit (Roche) following manufacturer's protocol, and assayed on NanoString nCounter Analysis System using a custom Nano110 (PAM50 + claudin-low & VEGF signatures) probe set. Intrinsic subtyping and gene-expression data were evaluated using R statistical software. All study samples were previously annotated and subtyped by the ER/PR/HER2 IHC classifier. Genotypes were obtained from next generation sequencing or Illumina Human2.5M BeadChip platform using germline DNA from more than 2000 breast cancer cases and 2000 controls were studied. Results: To date, Intrinsic molecular subtyping by Nano110 has been completed on 69 NG, 81 AA and 74 CA tumors. Concordance between IHC and PAM50 was 59%, which is adequate and comparable to previous studies. Basal-like subtype was overrepresented and accounted for nearly 30% of NG and AA cases, compared to 17% in CA cases. HER2-enriched subtype was overrepresented only in NG cases (9%). The proportion with Luminal A tumors were 44% NG, 56% AA and 68% CA, respectively. Conclusions: PAM50 NanoString assay is reliable and high-throughput for molecular subtyping breast cancer using RNA extracted from FFPE tumors. Ongoing work will correlate PAM50 intrinsic subtypes to genotype data. Citation Format: Olayiwola OA, Ogundiran TO, Hardeman A, Yoshimatsu TF, Clayton W, Adeoye A, Ademola A, Ajani MA, Khramtsova G, Grushko TA, Huo D, Zheng Y, Parker J, Perou C, Olopade OI. Genotype-phenotype classification of triple negative breast cancers (TNBC) in women of African descent using the PAM50 NanoString platform and genomic data. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-05.

Estrogen receptor alpha (ER) and its ligand estrogen play vital roles in the development, progression and treatment of breast cancer. An increasing number of studies have also provided evidence linking disruption of the Fanconi anemia/BRCA cascade to breast cancer. Our objectives were to examine the methylation status and expression profiles of ER, correlate the findings with BRCA1 and FANCF methylation and map the critical CpGs for ER expression. We found that the CpG islands in the 5' region of the ER gene are methylated in 59 of 120 (49.2%) primary breast cancers, including 45 of 59 ER-negative tumors (76.3%, P0.00001). In addition, we observed a strong correlation between ER promoter and BRCA1 promoter methylation (odds ratio 3.12, 95% confidence interval 1.10-9.68, P = 0.02). In contrast, FANCF methylation was rare in breast tumors: one of 120 (0.8%). ER methylation was associated with high tumor grade (60.4% methylated vs. 39.6% unmethylated in grade 3 tumors, P = 0.04) and tumor subtype (P = 0.03). Though small in number, all tumors of the medullary subtype were ER methylated. In contrast, the lobular subtype had the least methylation (23.1% methylated vs. 76.9% unmethylated). After treatment of MDA-MB-231 cells with 5-aza-cytidine (5-aza-dC) and trichostatin, which resulted in re-expression of ER mRNA, we localized dramatic demethylation effects to CpG islands in positions +68, +165, +192, +195, +337, +341 and +405 from transcription start site of the ER promoter. These data suggest that unlike FANCF, both ER and BRCA1 are specifically targeted for methylation in sporadic breast cancers, a phenomenon that should be explored for development of novel diagnostic and therapeutic approaches.

Objectives. To investigate the frequency and potential prognostic or predictive value of HER-2 amplification or overexpression in advanced and recurrent endometrial cancers. Methods. Immunohistochemical staining (IHC; DAKO Herceptest®) and fluorescence in situ hybridization (FISH; Vysis Inc. PathVysion® DNA Probe Kit) were performed on specimens collected on a randomized Gynecologic Oncology Group (GOG) protocol testing the addition of paclitaxel to doxorubicin/cisplatin. Results. HER-2 overexpression (either 2+ (moderate) or 3+ (strong) immunostaining) and HER-2 gene amplification (a ratio of HER-2 copies to chromosome 17 ( CEP17 ) copies ≥2) were detected in 44% (104 of 234; 58 were 2+ and 46 were 3+) and 12% (21 of 182) of specimens, respectively. There was a significant increased frequency of overexpression in serous tumors vs. all others (23 of 38, 61% vs. 81 of 196, 41%, respectively, P =0.03). HER-2 amplification also appeared to be more common in serous tumors, but results were not significant (6 of 28, 21% vs. 15 of 141, 11%, P =0.12). There was a significant association between grade and HER-2 amplification among nonserous tumors, with grades 1, 2, and 3 cancers demonstrating 3%, 2%, and 21% amplification, respectively ( P =0.003). Neither overexpression nor amplification predicted overall survival (OS) after adjusting for treatment and performance status. Conclusions. HER-2 amplification was more common in high grade tumors with a trend to being more common in serous tumors. There was no clear evidence for a survival difference or a difference in benefit from the addition of paclitaxel for women with HER-2 amplified or overexpressed tumors; however, power to detect clinically meaningful differences was low.

e16509 Background: Nuclear enzyme TOP2A controls the topologic states of DNA, and is a major target for anthracyclines. In the adjuvant trials of breast cancer, TOP2A is likely a marker of anthracy...

Background: Topoisomerase IIα (topoIIα) is an essential enzyme gene in regulating DNA structure and cell proliferation and is encoded by the TOP2A. Using cDNA microarray analysis, TOP2A has been reported to be one of the top genes overexpressed in Wilms’ tumour. Aim: To evaluate the role of TopoIIα in Wilms’ tumorigenesis and its prognostic value. Methods: TOP2A gene copy numbers were determined using the fluorescence in situ hybridisation technique, and protein expression levels of TopoIIα by immunostaining in 39 samples of primary and 18 samples of metastatic Wilms’ tumour. Results: TOP2A gene amplification was detected only in anaplastic Wilms’ tumours, and none of the Wilms’ tumours showed deletion of the TOP2A gene. TopoIIα protein overexpression was detected in 97% of Wilms’ tumours, and correlated strongly with proliferation, as measured by Ki-67 (r = 0.85). The high TopoIIα expression was associated with the presence of vascular invasion, prominent apoptosis, metastases and adverse clinical outcomes (p Conclusions: Our findings suggest that TopoIIα overexpression in Wilms’ tumours is caused by a change at the transcription level, except for anaplastic Wilms’ tumours, in which gene amplification was present. High levels of TopoIIα protein are correlated with tumour aggressiveness. The assessment of TopoIIα expression in Wilms’ tumour may have prognostic value.

To explore the molecular mechanisms for the similarities between inherited and noninherited forms of breast cancer, we tested the hypothesis that inactivation of BRCA1 by promoter hypermethylation is associated with reduced gene copy number and chromosome 17 aneusomy as observed in tumors from BRCA1 mutation carriers. Using a combination of methylation-specific PCR analysis and fluorescence in situ hybridization, we observed varying degrees of promoter methylation in 39 of 131 (29.8%) primary tumors. Despite significant tumor heterogeneity, mean copy numbers of BRCA1 and CEP17 per cell were lower in methylated cases compared with unmethylated cases [1.78 versus 2.30 (P = 0.001) and 1.85 versus 2.29 (P = 0.005), respectively]. Methylation was more frequently observed in younger women (P = 0.05) with high-grade (P = 0.001), estrogen receptor–negative (P = 0.04), and progesterone receptor–negative (P = 0.01) tumors. Moreover, methylation was associated with reduced or absent BRCA1 transcripts, which was reversible in the heavily BRCA1-methylated cell line UACC3199 following treatment with 5-aza-2′-deoxycytidine and trichostatin A. We identified five CpGs at positions −533, −355, −173, −21, and +44 as critical in the reexpression of BRCA1. We conclude that BRCA1 methylation contributes to a subset of sporadic breast cancers with the resulting molecular and clinicopathologic phenotype similar to that of hereditary BRCA1-associated breast cancers. Our data support a model of carcinogenesis in which BRCA1 promoter methylation may serve as a “first hit,” much like an inherited germ line mutation, and promote tumor progression down a restricted set of molecular pathways.

Purpose: Germ-line mutations in the BRCA1 tumor suppressor gene predispose to early onset breast cancers with a distinct phenotype characterized by high tumor grade, aneuploidy, high proliferation rate, and estrogen receptor-negativity. The molecular mechanisms and cooperative oncogenes contributing to multistep tumor progression in cells lacking BRCA1 are not well defined. To examine whether C-MYC (MYC), a transforming oncogene associated with genetic instability, contributes to multistep tumor progression in BRCA1-associated breast cancer, we have analyzed tumors from women with hereditary BRCA1-mutated and sporadic breast cancers. Experimental Design: We performed fluorescence in situ hybridization using a MYC:CEP8 assay on formalin-fixed paraffin-embedded tumor tissues from 40 women with known deleterious germ-line BRCA1 mutations and 62 sporadic cases, including 20 cases with hypermethylation of the BRCA1 gene promoter. Results: We observed a MYC:CEP8 amplification ratio ≥2 in 21 of 40 (53%) BRCA1-mutated tumors compared with 14 of 62 (23%) sporadic tumors (P = 0.003). Of the 14 sporadic cases with MYC amplification, 8 (57%) were BRCA1-methylated. In total, MYC amplification was found in a significantly higher proportion of tumors with BRCA1 dysfunction (29 of 60, 48% versus 6 of 42, 14%; P = 0.0003). In a multivariable regression model controlling for age, tumor size, and estrogen receptor status, BRCA1-mutated tumors demonstrated significantly greater mean MYC:CEP8 ratio than sporadic tumors (P = 0.02). Conclusions: Our data indicate that MYC oncogene amplification contributes to tumor progression in BRCA1-associated breast cancers. Thus, we conclude that the aggressive histopathological features of BRCA1-associated tumors are in part due to dysregulated MYC activity.

The BRCA1 tumor suppressor gene and the HER-2/neu oncogene are located in close proximity on the long arm of chromosome 17 (17q11-21). Absence of BRCA1 or functional overexpression of the HER-2/neu gene presumably contributes to the somatic phenotype of breast cancer in premenopausal women, characterized by unfavorable prognostic features such as high tumor grade, hormone receptor negativity, and high proliferation rate. To examine whether amplification of HER-2/neu contributes to the aggressive biology of BRCA1-associated tumors, we have performed fluorescence in situ hybridization on formalin-fixed paraffin-embedded breast tumor tissue sections from 53 BRCA1 mutation carriers and 41 randomly selected, age-matched sporadic breast cancer cases. Although BRCA1-associated and sporadic tumors were equally likely (19% versus 22%) to exhibit HER-2/neu amplification [defined as a ratio of HER-2/neu copies to chromosome 17 centromere (CEP17) copiesor = 2], 6 (15%) of the sporadic tumors were highly amplified (defined as a ratio greater-than-or-equal 5) versus none of the BRCA1-associated tumors (P = 0.048). HER-2 protein overexpression as measured by immunohistochemical analysis was not observed among the BRCA1-associated cases (P = 0.042). Four of 21 (19%) sporadic tumors exhibited strong membranous staining of HER-2 (intensity level of 3+) as compared with 0 of 39 BRCA1-associated tumors. Our data suggest that a germ-line mutation in the BRCA1 tumor suppressor gene is associated with a significantly lower level of HER-2/neu amplification. Thus, it is possible that BRCA1-associated and HER-2/neu-highly amplified tumors progress through distinct molecular pathways, and the aggressive pathological features of BRCA1-associated tumors appear unrelated to amplification of the adjacent HER-2/neu oncogene.

Introduction: The MELK gene is located on chromosome 9p13.2 and encodes a serine/threonine kinase that is involved in cell cycle regulation and apoptosis. The MELK protein is abundantly expressed in various tumors including breast cancer, and is associated with poor patient survival. MELK is an attractive molecular target due to its critical roles in cancer stem cell maintenance. However, the function and mechanism of MELK overexpression remain elusive. Gene amplification/copy number gain is one of the potential mechanisms underlying MELK overexpression. In this pilot study, we used fluorescence in situ hybridization (FISH) to detect MELK gene amplification and analyzed other MELK gene copy number alterations (CNA) in endometrial, ovarian, and breast cancer cell lines. Methods: To date, 3 endometrial, 1 ovarian and 13 breast cancer cell lines of different subtypes were screened for MELK CNA. Of these, six breast cancer cell lines (BT20, MCF7, MDAMB231, SKBR3, BT549, and T47D) had known status of MELK: all carried high levels of protein expression by Western Blot. Cell harvest and metaphase slides were prepared according to standard protocols. MELK FISH probe (BAC RP11-450B8) directly labeled with SpectrumGreen was developed and validated in our laboratory. The chromosome 9 centromere enumeration probe (CEP9) labeled with SpectrumOrange (Abbott Molecular) was used to distinguish gene amplification from gene polysomy (gene and chromosome copy number gain ≥ 3). Mean copies of each signal per cell and copy number ratios per cell were calculated. Ratio of MELK to CEP9 ≥ 2.0 was a cut off point for MELK amplification. Results: Across all cell lines, the ratios of MELK to CEP9 ranged between 0.5 to 1.7, showing no amplification. However, 10 cell lines (59%) displayed 3-8 copies of MELK due to polysomy for chromosome 9, 4 (24%) harbored both gene polysomy and structural alterations (duplications and translocations); only two cell lines exhibited normal MELK and CEP9 copies and one presented with heterozygous deletion of MELK. Interestingly, all 6 MELK- overexpressed cell lines showed either gene polysomy or complex chromosomal alterations or both. Conclusions: Our pilot study suggests that amplification of MELK gene does not occur or is a rare event in human cancer cells in vitro. However, recurrent MELK structural alterations (duplications and translocations) and gene polysomy can cause elevated protein expression in breast cancer cell lines. MELK FISH study in primary tumors from breast cancer patients will be presented. Supported by: American Cancer Society Citation Format: Ashley Hardeman, Tatyana A. Grushko, Maria J. Gomez, Mariann Coyle, Yusuke Nakamura, Olufunmilayo I. Olopade. Molecular-cytogenetic analysis of the maternal embryonic leucine-zipper kinase (MELK) oncogene in cancer. [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 2386. doi:10.1158/1538-7445.AM2014-2386

5607 Background: Pre-clinical data suggests that the diabetic medication, metformin, has anti-cancer effects. We hypothesized that advanced stage endometrial cancer (EC) patients who used metformin...

The cytoskeletal proteins spectrin and ankyrin colocalize with sites of E-cadherin-mediated cell-cell adhesion in mammalian cells. Here we examined the effects of Drosophila DE-cadherin expression on spectrin and ankyrin in Drosophila S2 tissue culture cells. DE-cadherin caused a dramatic change in the cytoplasmic concentration and distribution of armadillo, the Drosophila homolog of beta catenin. However, DE-cadherin expression had no detectable effect on the quantity or subcellular distribution of ankyrin or spectrin. In reciprocal experiments, recruitment of ankyrin and alphabeta spectrin to the plasma membrane by another cell adhesion molecule, neuroglian, had no effect on the quantity or distribution of armadillo. The results indicate that DE-cadherin-catenin complexes and neuroglian-spectrin/ankyrin complexes form by nonintersecting pathways. Recruitment of spectrin does not appear to be a conserved feature of DE-cadherin function.

11021 Background: PTK6 gene on chromosome 20q13 encodes the intracellular non-receptor tyrosine kinase. Studies in vivo and in vitro revealed a role for PTK6 in cell proliferation and survival, particularly in HER2+ breast cancer cells suggesting that PTK6 may associate with the HER2 pathway and confer resistance to HER2-targeted therapy. PTK6 protein is frequently overexpressed in breast cancer, however, the mechanism(s) underlying PTK6 overexpression and its role in cancer remains unclear. To address this problem, we analyzed the frequency of PTK6 gene copy number variation (CNV) and expression in association with breast cancer subtypes. Methods: Retrospectiveparaffinsamples of invasive tumor and normal epithelium, and matching DCIS and metastases were mounted on TMA. PTK6 CNV was determined using PTK6:CEP20 FISH assay. Tumor subtypes were defined using the five-marker IHC classifier. The correlation between PTK6 CNV and mRNA expression and association of both with the intrinsic PAM50 tumor subtype were studied using TCGA database (547 cases) and publicly available seven breast cancer data sets (1005 cases). Data were normalized, gene median centered and standardized for the purpose of the study. Results: By FISH, 20% of 41 invasive tumors carried PTK6 CNV: amplification (10%) and gene polysomy (10%). The proportion of PTK6 amplified cases differed by subtype, with the largest proportion in HER2-enriched (17%) and LumB (14%). Strikingly, amplified invasive cases also showed amplification in matching DCIS and metastases. Analysis of the public datasets confirmed the frequent PTK6 amplification in breast cancer. Both low and high levels of amplification were detected with the largest proportion in HER2+ tumors (HER2-enriched and LumB; p=2.05e-26). None of the basal-like tumors showed high levels of PTK6 amplification. A high correlation between PTK6 gene copies and mRNA expression was observed (p=1.13e-08). Conclusions: PTK6 gene is amplified early in breast cancer progression, particularly in HER2+ tumors. Further studies on PTK6 biology may help clinicians to understand its potential role in HER2 resistance. Supported by BREAST CANCER SPORE, NCI K12CA139160 and CTSA-ITM CS UL1 RR024999.

10510 Background: Triple-negative (ER-, PR-, and HER2-) breast cancer (TNBC) is the most aggressive form of breast disease and is overrepresented in young women and women of African ancestry. Molec...

Abstract #4050 Triple-negative breast cancers are tumors characterized by their lack of hormone receptors (ER and PR) and HER2. They are the most aggressive form and account for 10–17% of all breast carcinomas. A subgroup of triple negative tumors with a basal-like phenotype share morphological features and similar gene expression profiles with tumors from BRCA1 mutation carriers. It has recently been shown that inactivation of BRCA1 in breast epithelial stem cells restricts subsequent progenitor cells to a basal cell phenotype and promotes expansion of ER negative cells (Liu et al 2008, PNAS). While mechanisms of BRCA1 inactivation in sporadic tumors are not completely elucidated, methylation of the BRCA1 promoter is one important mechanism that contributes to loss of BRCA1 expression in sporadic breast cancer. We hypothesize that inactivation of BRCA1 by epigenetic mechanisms such as promoter methylation contributes to the triple negative phenotype. Materials and Methods: Using a combination of methylation specific PCR and Immunohistochemistry, 120 primary breast cancers were analyzed for methylation of the BRCA1 promoter and protein expression of ER, PR and HER2. All tumors with negative staining for ER, PR and HER2 were classified as triple negative and all others were classified as non triple-negative. Results: We were able to classify 111 of the 120 (%) tumors by IHC. We found that 30 out of 111 (27%) tumors were ER, PR, and HER2 negative (triple negative). BRCA1 methylation was detected in 14 out of 30 (47%) triple negative tumors, compared with 10 out of 81 (12%) other tumor phenotypes (p Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4050.

9567 Background: African American (AA) women have a lower risk of breast cancer (BC) than Caucasian(C) women, but tumors in AAs are more likely to be poorly differentiated and estrogen receptor neg...

9509 Background: DNA microarray studies have identified 3 distinct subtypes of breast cancers (BC) based on “Intrinsic Gene Expression” patterns: basal-like tumors (ER-, HER2-), tumors with amplifi...

9513 Background: Although the incidence of breast cancer in African Americans (AA) is lower than that of their Caucasian (C) counterparts, the biology of the disease is more aggressive in these wom...

Mutations in Drosophila alpha spectrin cause larval lethality and defects in cell shape and adhesion (J. Lee et al., 1993, J. Cell Biol. 123, 1797-1809). Here we examined the effects of two lethal alpha spectrin alleles (alpha-specrg41 and alpha-specrg35) on development and function of the larval midgut. Homozygous null alpha-specrg41-mutant larvae exhibited a striking defect in middle midgut acidification. In contrast, many homozygous alpha-specrg35 mutants were capable of acidification, indicating partial function of the truncated alpha-specrg35 product. Acidification was also blocked by a mutation in the labial gene, which is required for differentiation of cuprophilic cells in the midgut, suggesting that these cells secrete acid. We found that two isoforms of spectrin (alphabeta and alphabetaH) are segregated within the basolateral and apical domains of cuprophilic cells, respectively. The most conspicuous defect in cuprophilic cells from labial and alpha spectrin mutants was in morphogenesis of the invaginated apical domain, although basolateral defects may also contribute to the acidification phenotype. Acid secretion in vertebrate systems is thought to involve the polarized activities of apical proton pumps and basolateral anion exchangers, both of which interact with spectrin. We propose that the alpha-specrg41 mutation in Drosophila interferes with the polarized activities of homologous molecules that drive acid secretion in cuprophilic cells.

Introduction This Phase I trial aimed to determine the maximum-tolerated-dose of erlotinib administered with two standard chemoradiotherapy regimens for non-small cell lung cancer. Methods Unresectable stage III non-small cell lung cancer patients were enrolled in this 2-arm dose-escalation study. Erlotinib, given only during chemoradiotherapy, was escalated from 50 to 150 mg/d in 3 to 6 patient cohorts. Arm A: erlotinib with cisplatin (50 mg/m 2 IV days 1, 8, 29, 36), etoposide (50 mg/m 2 IV days 1-5, 29-33) and chest radiotherapy (66 Gy, 2 Gy/d) followed by docetaxel (75 mg/m 2 IV Q21 d) for 3 cycles. Arm B: induction carboplatin (AUC 6) and paclitaxel (200 mg/m 2 ) for two 21-d cycles then radiotherapy with erlotinib, carboplatin (AUC = 2/wk) and paclitaxel (50 mg/m 2 /wk). Results Seventeen patients were treated in each arm. Patient characteristics: performance status 0 to 24 patients, 1 to 10 patients, median age 63 years, adenocarcinoma 21% and female 14 patients. Dose-escalation of erlotinib to 150 mg/d was possible on both chemoradiotherapy regimens. Grade 3/4 leukopenia and neutropenia were predominant toxicities in both arms. Grade 3 chemoradiotherapy toxicities in arm A were esophagitis (3 patients), vomiting (1), ototoxicity (1), diarrhea (2), dehydration (3), pneumonitis (1); and arm B was esophagitis (6). Seven patients (21%) developed rash (all grade 1/2). Median survival times for patients on Arm A and B were 10.2 and 13.7 months, respectively. Three-year overall survival in patients with and without rash were 53% and 10%, respectively (log-rank P = 0.0807). Epidermal growth factor receptor IHC or FISH positive patients showed no significant overall survival difference. Conclusion Addition of standard-dose erlotinib to chemoradiotherapy is feasible without evident increase in toxicities. However, the survival data are disappointing in this unselected patient population and does not support further investigation of this approach.

The utilization of radiomics (high-throughput extraction and analysis of imaging features) to abstract underlying genomic features of an evolving malignancy is an emerging field that has the potential to detail biological information of a tumor in a non-invasive and more accessible manner. Furthermore, applying radiomics confers a comprehensive spatial view of the tumor, a potential advantage over the limitations of sampling a small region of tissue that may not accurately represent the underlying complexity of the entire tumor. Most studies to date that incorporate radiogenomics in the analysis of breast cancers have focused on few basic clinical data or individual genetic mutations such as BRCA or HER2 status. Here, we use an automated quantitative radiomics analysis platform developed at the University of Chicago that enables computerized feature extraction of tumors to analyze magnetic resonant imaging scans of 50 breast cancer patients (mean age of diagnosis [range]: 54 [24-89]; receptor status: HER2+: 14, Triple negative: 7; stage [1 through 4]: 10%, 40%, 42%, 8%) who have had comprehensive gene expression profiling performed using Agilent Human Gene Expression arrays. Our imaging platform extracts 38 features across six major phenotypes (size, shape, morphology, enhancement texture, kinetic curve assessment, and enhancement variance kinetics) (see Table for listing of 24 selected features). Existing radiomic analysis derived from a TCGA/TCIA dataset suggests that there are many correlations between imaging phenotypes and various genetic pathways, such as VEGF signaling and volume of enhancing voxels, base excision repair and enhancement texture entropy, and TGF-beta signaling and enhancement texture variance. We confirm these relationships as well as establish novel associations using a robust imaging dataset. By associating specific radiomic features with gene expression profile of tumors, we have the opportunity to extract detailed biological information non-invasively through clinical imaging. Selected imaging phenotypes extracted from MRI scansPhenotype categoryImage phenotypeDescriptionSizeVolumeVolume of lesionSizeEffective diameterDiameter of a sphere with the same volume as the lesionSizeSurface areaLesion surface areaShapeSphericitySimilarity of the lesion shape to a sphereShapeIrregularityDeviation of the lesion surface from the surface of a sphereShapeSurface area / volumeRatio of surface area to volumeMorphologyMargin sharpnessMean of the image gradient at the lesion marginMorphologyVariance of margin sharpnessVariance of the image gradient at the lesion marginMorphologyVariance of radial gradient histogramDegree to which the enhancement structure extends in a radial pattern originating from the center of the lesionEnhancement TextureContrastLocal image variationsEnhancement TextureEntropyRandomness of the gray-levelsEnhancement TextureDifference varianceVariations of difference of gray-levels between voxel-pairsEnhancement TextureAngular second momentImage homogeneityEnhancement TextureMaximum correlation coefficientNonlinear gray-level dependenceEnhancement TextureSum averageOverall brightnessEnhancement TextureSum of squaresSpread in the gray-level distributionKinetic Curve AssessmentMaximum enhancementMaximum contrast enhancementKinetic Curve AssessmentTime to peakTime at which the maximum enhancement occursKinetic Curve AssessmentUptake rateUptake speed of the contrast enhancementKinetic Curve AssessmentCurve shape indexDifference between late and early enhancementKinetic Curve AssessmentTotal rate variationHow rapidly the contrast will enter and exit from the lesionEnhancement-Variance KineticsMaximum variance of enhancementMaximum spatial variance of contrast enhancement over timeEnhancement-Variance KineticsTime to peak maximum varianceTime at which the maximum variance occursEnhancement-Variance KineticsEnhancement variance increasing rateRate of increase of the enhancement-variance during uptake Citation Format: Albert C. Yeh, Stephanie McGregor, Hui Li, Yuan Ji, Yitan Zhu, Tatyana Grushko, Alexandra Edwards, Fan Lui, Jing Zhang, Qiu Niu, Yonglan Zheng, Toshio Yoshimatsu, Galina Khramtsova, Karen Drukker, Gregory Karczmar, Hiroyuki Abe, Jeffrey Mueller, Maryellen Giger, Olufunmilayo Olopade. Radiogenomics of breast cancer using DCE-MRI and gene expression profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2633.

Abstract #4039 Introduction: The importance of hormone receptor status in assigning treatment and the potential use of HER2 targeted therapy have made it imperative for laboratories to improve detection techniques. As inter-laboratory variability in immunohistochemical (IHC) tests may also affect epidemiologic studies of breast cancer subtypes in different countries, we conducted a validation study of breast cancer biomarkers between a well-established laboratory in the US and a field laboratory at the Institute for Medical Research and Training at the University College Hospital in Ibadan Nigeria. Method: 232 breast tumor blocks were evaluated for ER, PR, and HER2 at both laboratories using tissue micro arrays (TMA) technique. Web-based conferences were held periodically to discuss IHC staining protocols, standardize scoring systems and to resolve discrepant cases. Pathologists used whole slide imaging for joint review and kappa statistic (κ) was used to indicate concordance between the two laboratories. Fluorescence in situ hybridization was carried out to confirm HER2 status in all cases. Results: Initially, concordance analysis revealed an agreement of 91% (κ=0.52) for ER, 85% (κ=0.49) for PR, and 80% (κ=0.39) for HER2 between the two labs. Antigen retrieval techniques and scoring methods were identified as important reasons for discrepancy. After quality assurance and training, the agreement improved to 92% (κ=0.53) for ER, 88% (κ=0.64) for PR, and 94% (κ=0.75) for HER2. To date, florescence in situ hybridization (FISH) has been completed for 67 cases to confirm HER2 status, out of which 16 (24%) were shown to amplify the HER2 gene, 6 out of the 12 discordant HER2 results were resolved by FISH. Conclusion: We found web-based conference with TMA and digital microscopy a useful and cost-effective tool for quality assurance of IHC, consultation and collaboration between distant laboratories. Quality improvement exercises in testing of tumor biomarkers will reduce misclassification in epidemiologic study of breast cancer subtypes and provide much needed capacity building in resource poor field sites. Acknowledgement This study was carried out with the support of Breast SPORE NCI P50 CA125183, Breast Cancer Research Foundation and the Lee Jeans Entertainment Industry Fund. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4039.