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Supplementary Data from Identification of Novel Kinase Targets for the Treatment of Estrogen Receptor–Negative Breast Cancer

Purpose: Previous gene expression profiling studies of breast cancer have focused on the entire genome to identify genes differentially expressed between estrogen receptor (ER) α–positive and ER-α–negative cancers.Experimental Design: Here, we used gene expression microarray profiling to identify a distinct kinase gene expression profile that identifies ER-negative breast tumors and subsets ER-negative breast tumors into four distinct subtypes.Results: Based on the types of kinases expressed in these clusters, we identify a cell cycle regulatory subset, a S6 kinase pathway cluster, an immunomodulatory kinase–expressing cluster, and a mitogen-activated protein kinase pathway cluster. Furthermore, we show that this specific kinase profile is validated using independent sets of human tumors and is also seen in a panel of breast cancer cell lines. Kinase expression knockdown studies show that many of these kinases are essential for the growth of ER-negative, but not ER-positive, breast cancer cell lines. Finally, survival analysis of patients with breast cancer shows that the S6 kinase pathway signature subtype of ER-negative cancers confers an extremely poor prognosis, whereas patients whose tumors express high levels of immunomodulatory kinases have a significantly better prognosis.Conclusions: This study identifies a list of kinases that are prognostic and may serve as druggable targets for the treatment of ER-negative breast cancer. (Clin Cancer Res 2009;15(20):6327–40)

CCR Translation for the Article from Identification of Novel Kinase Targets for the Treatment of Estrogen Receptor–Negative Breast Cancer

OBJECTIVES/SCOPE: This abstract will describe the evolution of a traditional occupational health group into a department that supports Shell's strategy and improves business outcomes by intentionally focusing on care and human performance. Program details and learnings will be shared, as well as quantitative results demonstrating the positive impact of Health on the business. METHODS/PROCEDURES: Shell Health was historically a traditional occupational health group, performing fitness-to-work, return-to-work, and medical surveillance exams. Over the past two decades, there was additional focus on weight loss and weight maintenance. In more recent years, Shell Health broadened to a more holistic approach to health, including psychological and emotional wellbeing, and introduced its new strategy entitled Human Performance and Care (HP&C). HP&C optimizes the capabilities of individuals, teams and businesses to be at our best, even in the face of great uncertainty and stress. Formal programs focusing on Resilience and Care for People (CfP) have been developed, along with analytic strategies tying HP&C to business outcomes. RESULTS/OBSERVATIONS/CONCLUSIONS: The Resilience Program has reported a positive association between Resilience and Employee Engagement (p=0.04), Inclusion (p CfP uses a 15-question index that is analyzed to determine its association with Engagement, safety, and other business outcomes of interest. We have seen improved safety performance measured by Total Recordable Case Frequency (TRCF target was 0.80, attained 0.75). Significantly higher Hands on Tools Time, a marker of productivity, was demonstrated in CfP compared to non-CfP projects (67% vs. 32%, p In addition, we have collaborated to integrate HP&C with parts of our business such as Real Estate, Contracting and Procurement, Safety Leadership and Shell Learning. We have reached senior leaders across the organization, with HP&C incorporated into long-term business strategies globally. Overall, we have demonstrated that a holistic approach to health will lead to improved business outcomes and a high performing organization. NEW OR ADDED INFORMATION TO EXISTING LITERATURE: Shell Health is the first in the oil and gas industry to recognize the opportunity to improve human performance through the science of care. HP&C addresses the core needs of humans as a way to optimize human performance while improving business outcomes such as engagement, safety and productivity.

Cautionary Note The companies in which Royal Dutch Shell plc directly and indirectly owns investments are separate legal entities. In this Report: "Sustaining a high-performance culture by leading with a ‘Lens of Care’" "Shell", "Shell Group" and "Royal Dutch Shell" are sometimes used for convenience where references are made to Royal Dutch Shell plc and its subsidiaries in general. Likewise, the words "we", "us" and "our" are also used to refer to Royal Dutch Shell plc and its subsidiaries in general or to those who work for them. These terms are also used where no useful purpose is served by identifying the particular entity or entities. "Subsidiaries", "Shell subsidiaries" and "Shell companies" as used in this Report: "Sustaining a high-performance culture by leading with a ‘Lens of Care’" refer to entities over which Royal Dutch Shell plc either directly or indirectly has control. Entities and unincorporated arrangements over which Shell has joint control are generally referred to as "joint ventures" and "joint operations", respectively. Entities over which Shell has significant influence but neither control nor joint control are referred to as "associates". The term "Shell interest" is used for convenience to indicate the direct and/or indirect ownership interest held by Shell in an entity or unincorporated joint arrangement, after exclusion of all third-party interest. This Report: "Sustaining a high-performance culture by leading with a ‘Lens of Care’" contains forward-looking statements (within the meaning of the U.S. Private Securities Litigation Reform Act of 1995) concerning the financial condition, results of operations and businesses of Royal Dutch Shell. All statements other than statements of historical fact are, or may be deemed to be, forward-looking statements. 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There are a number of factors that could affect the future operations of Royal Dutch Shell and could cause those results to differ materially from those expressed in the forward-looking statements included in this Report: "Sustaining a high-performance culture by leading with a ‘Lens of Care’" including (without limitation): (a) price fluctuations in crude oil and natural gas; (b) changes in demand for Shell’s products; (c) currency fluctuations; (d) drilling and production results; (e) reserves estimates; (f) loss of market share and industry competition; (g) environmental and physical risks; (h) risks associated with the identification of suitable potential acquisition properties and targets, and successful negotiation and completion of such transactions; (i) the risk of doing business in developing countries and countries subject to international sanctions; (j) legislative, fiscal and regulatory developments including regulatory measures addressing climate change; (k) economic and financial market conditions in various countries and regions; (l) political risks, including the risks of expropriation and renegotiation of the terms of contracts with governmental entities, delays or advancements in the approval of projects and delays in the reimbursement for shared costs; and (m) changes in trading conditions. No assurance is provided that future dividend payments will match or exceed previous dividend payments. All forward-looking statements contained in this Report: "Sustaining a high-performance culture by leading with a ‘Lens of Care’" are expressly qualified in their entirety by the cautionary statements contained or referred to in this section. Readers should not place undue reliance on forward-looking statements. Additional risk factors that may affect future results are contained in Royal Dutch Shell’s Form 20-F for the year ended December 31, 2018 (available at www.shell.com/investor and www.sec.gov). These risk factors also expressly qualify all forward-looking statements contained in this Report: "Sustaining a high-performance culture by leading with a ‘Lens of Care’" and should be considered by the reader. Each forward-looking statement speaks only as of the date of this Report: "Sustaining a high-performance culture by leading with a ‘Lens of Care’", 15-11-2019. Neither Royal Dutch Shell plc nor any of its subsidiaries undertake any obligation to publicly update or revise any forward-looking statement as a result of new information, future events or other information. In light of these risks, results could differ materially from those stated, implied or inferred from the forward-looking statements contained in this Report: "Sustaining a high-performance culture by leading with a ‘Lens of Care’". We may have used certain terms, such as resources, in this Report: "Sustaining a high-performance culture by leading with a ‘Lens of Care’" that the United States Securities and Exchange Commission (SEC) strictly prohibits us from including in our filings with the SEC. U.S. investors are urged to consider closely the disclosure in our Form 20-F, File No 1-32575, available on the SEC website www.sec.gov. Abstract Shell companies conducted a case study into the use of worker welfare strategies to drive engagement in major brownfield and greenfield construction projects and thereby improve: worker productivity, the quality of construction work and overall project HSE and financial performance. Two major brownfield capital projects (>$100 mil US) in The United States and The Netherlands developed and implemented a comprehensive worker welfare strategy. The components of the strategy were built on 14 identified core principles of worker welfare. It was recognized that an intentional care mindset can be developed and maintained in a workplace. To measure impact, surveys set at regular intervals were conducted to measure workers perception of care, engagement and welfare and then paralleled to several project metrics, such as: Total Record Case Frequency (TRCF), Hands on Tools-Time (HoTT), schedule performance and work-hour rates. The results from the two projects were compared to other Shell projects of similar size and scope that did not have a comprehensive worker welfare strategy. In the US, the implemented strategy showed a substantial positive impact in three areas: (a) productivity, (b) safety records and (c) engagement/participation/retention which corroborated the data from the Netherlands (NL). The productivity improvements were reflected in the US in top quartile results for both absolute and relative man power hours. Statistically significant higher HoTT was observed for NL (54%), compared to non-Care for People (CfP) projects (average 36%) indicating greater productivity and efficiency. Efficiency and productivity were also evidenced by project delivery 4 months ahead of schedule. Further, the safety record showed a TRCF of 0.75, beating the 0.80 target at NL and the TRCF for US at (0.9) was significantly lower compared to non-CfP projects similar in geography, complexity, risk and size indicating a better safety performance. Since a large part of annual project investment is man hour related and the Shell worker welfare approach requires relatively small investments, the ROI and productivity gains have been estimated to be high. Finally, in the US, scores of 80+% were recorded in many key areas of most subcontractor’s groups (e.g. Secure Workplace) resulting in a project saving of approximately $10M from a lack of per diem. This paralleled The Netherlands with engagement scores (86, 81, 80, 79%). These results demonstrate the importance of focus on outcomes and business impact and showed that "care", as defined by the 14 elements of worker welfare, is a key driver of engagement, having subsequent positive impact on project outcomes. The worker welfare strategies present a clear and replicable strategy to drive both worker wellbeing and overall project performance. Recognizing the learnings from cross-industry coalitions such as Building Responsibly, this work will also be used to support, inform and guide the development of a comprehensive wider-industry standard to deliver worker welfare and care for employees.

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Recent studies of unconventional resource development (URD) and adverse health effects have been limited by distance-based exposure surrogates. Our study compared exposure classifications between air pollutant concentrations and &ldquo, well activity&rdquo, (WA) metrics, which are distance-based exposure proxies used in Marcellus-area studies to reflect variation in time and space of residential URD activity. We compiled Pennsylvania air monitoring data for benzene, carbon monoxide, nitrogen dioxide, ozone, fine particulates and sulfur dioxide, and combined this with data on nearly 9000 Pennsylvania wells. We replicated WA calculations using geo-coordinates of monitors to represent residences and compared exposure categories from air measurements and WA at the site of each monitor. There was little agreement between the two methods for the pollutants included in the analysis, with most weighted kappa coefficients between &minus, 0.1 and 0.1. The exposure categories agreed for about 25% of the observations and assigned inverse categories 16%&ndash, 29% of the time, depending on the pollutant. Our results indicate that WA measures did not adequately distinguish categories of air pollutant exposures and employing them in epidemiology studies can result in misclassification of exposure. This underscores the need for more robust exposure assessment in future analyses and cautious interpretation of these existing studies.

Background: Inflammatory breast cancer (IBC) is a rare form of aggressive breast cancer with no existing identifiers for screening or prevention strategies. Women with triple-negative (TNBC, ER–, PR–, Her2–) non-inflammatory breast cancer are less likely to breastfeed, and we have shown in adjacent normal breast tissue that this tissue has more foci of stem cells compared to non-TNBC cancers. A disproportionately higher percentage of women with IBC have TNBC relative to women with non-IBC. We hypothesized that adjacent normal tissue in TNBC IBC vs. TN non-IBC may also display unique biological features based on epidemiologic characteristics. Methods: We examined epidemiologic factors by breast cancer receptor subtype in 144 patients diagnosed with IBC in 1991–2011 at MD Anderson Cancer Center. Breast cancer risk factors including parity and breastfeeding were compared between patients with TN and non-TN IBC with chi-square or Wilcoxon rank sum tests. Normal adjacent tissues were stained for stem cell markers CD44+CD49f+CD133/2+ and macrophage marker CD68. Results: The mean age at diagnosis was 52.3 years (range = 23–80) and 83% of patients were non-Hispanic white, 80% were overweight or obese (BMI >25), and 36% were TN IBC. Patients with TN IBC had significantly lower frequency of breastfeeding compared with non-TN IBC, 28% vs. 55%, (p = 0.01). No differences were found in the frequency of other breast cancer risk factors. All 8 IBC adjacent tissue samples showed a distinct spatial distribution of stem cell staining, not limited to the triple negative patients. Compared with 0/60 non-IBC cases, 0/8 triple negative non-IBC, (p = 0.001 Pearson chi-square). Given the high BMI among IBC patients, we further examined normal tissues for the presence of CD68+ cells distributed individually or as clusters exhibiting a “crown-like” pattern (multiple CD 68+ macrophages found around dead adipocytes), and found that 7 of the 8 IBC adjacent tissues were CD68+. Benign biopsies collected from 2 patients at 10 years before diagnosis displayed similar staining, including both stem cell and CD68 staining. Compared with 12/60 non-IBC adjacent tissues were positive for CD68, with 1/8 TN non-IBC, (p = 0.001 Pearson chi-square). Conclusion: We describe for the first time a stem-cell staining pattern unique to IBC present in all IBC tissues examined, including pre-cancer biopsies. Tissue samples from additional patients will be examined to further explore the relationship between stem cells and CD68 positivity with IBC subtypes. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-10-01.

Purpose: Previous gene expression profiling studies of breast cancer have focused on the entire genome to identify genes differentially expressed between estrogen receptor (ER) α–positive and ER-α–negative cancers. Experimental Design: Here, we used gene expression microarray profiling to identify a distinct kinase gene expression profile that identifies ER-negative breast tumors and subsets ER-negative breast tumors into four distinct subtypes. Results: Based on the types of kinases expressed in these clusters, we identify a cell cycle regulatory subset, a S6 kinase pathway cluster, an immunomodulatory kinase–expressing cluster, and a mitogen-activated protein kinase pathway cluster. Furthermore, we show that this specific kinase profile is validated using independent sets of human tumors and is also seen in a panel of breast cancer cell lines. Kinase expression knockdown studies show that many of these kinases are essential for the growth of ER-negative, but not ER-positive, breast cancer cell lines. Finally, survival analysis of patients with breast cancer shows that the S6 kinase pathway signature subtype of ER-negative cancers confers an extremely poor prognosis, whereas patients whose tumors express high levels of immunomodulatory kinases have a significantly better prognosis. Conclusions: This study identifies a list of kinases that are prognostic and may serve as druggable targets for the treatment of ER-negative breast cancer. (Clin Cancer Res 2009;15(20):6327–40)

Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)–negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor–induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.

BACKGROUND. There is anecdotal evidence that the incidence of central nervous system (CNS) metastases in breast cancer patients is increasing. It is unclear whether specific tumor biological properties or the use of systemic therapies influence this risk. METHODS. Using a database of 10,782 patients, 2685 patients were identified who experienced recurrence distantly. Clinical and biological features were analyzed in 2 ways: 1) patients who ever had versus those who never had CNS metastases, and 2) CNS metastases as the first site of recurrence versus those who had other sites. Correlations of survival after CNS metastasis with clinical and biologic features were also analyzed. RESULTS. In the ever versus never analysis, CNS metastases were significantly associated with younger age, premenopausal status, infiltrating ductal carcinoma histology (IDC), estrogen receptor (ER) and progesterone receptor (PR) negativity, low Bcl-2, high S-phase, aneuploidy, and altered p53. Tumor size, lymph node status, and use of adjuvant systemic therapy played little role. HER-2 overexpression was not associated with an increased risk in these patients (none of whom were treated with trastuzumab) (P = .91). However, epidermal growth factor receptor (EGFR) overexpression was associated with increased risk (P = .02). A multivariate analysis revealed ER negativity (odds ratio [OR] 2.8, P

Introduction We hypothesized that cells present in normal tissue that bear cancer stem cell markers may represent a cancer cell of origin or a microenvironment primed for tumor development, and that their presence may correlate with the clinically defined subtypes of breast cancer that show increased tumorigenicity and stem cell features. Methods Normal tissues sampled at least 5 cm from primary tumors (normal adjacent tissue) were obtained from 61 chemotherapy-naive patients with breast cancer treated with mastectomy. Samples were stained simultaneously with immunofluorescence for CD44/CD49f/CD133/2 stem cell markers. We assessed the association between CD44+CD49f+CD133/2+ staining in normal adjacent tissue and breast cancer receptor subtype (defined by the expression of the estrogen (ER), progesterone (PR), or human epidermal growth factor-2 (Her2) receptors). We also examined the correlation between CD44+CD49f+CD133/2+ immunofluorescence and each of two previously published gene signatures, one derived from stem-cell enriched tissue and one from BRCA mutated tissue expected to have defective DNA repair. Results Patients with triple negative breast cancer (ER–/PR–/HER2–) expressed CD44+CD49f+CD133/2+ in 9 of 9 normal adjacent tissue samples compared with 7 of 52 ER+ and/or Her2+ tumors (P < 0.001). Further, expression of CD44+CD49f+CD133/2+ by normal adjacent tissue correlated positively with a stem cell-derived tumorigenic signature (P

Endocrine therapy in patients with breast cancer can be limited by the problem of resistance. Preclinical studies suggest that complete blockade of the estrogen receptor (ER) combined with inhibition of the epidermal growth factor receptor (EGFR) can overcome endocrine resistance. We tested this hypothesis in a phase II neoadjuvant trial of anastrozole and fulvestrant combined with gefitinib in postmenopausal women with newly diagnosed ER-positive breast cancer. After a baseline tumor core biopsy, patients were randomized to receive anastrozole and fulvestrant (AF) or anastrozole, fulvestrant, and gefitinib (AFG) for 3 weeks. After a second biopsy at 3 weeks, all patients received AFG for 4 months and surgery was done if the tumor was operable. The primary endpoint was best clinical response by RECIST criteria and secondary endpoints were toxicity and change in biomarkers. The study closed after 15 patients were enrolled because of slow accrual. Median patient age was 67 years and median clinical tumor size was 7 cm. Four patients had metastatic disease present. Three patients withdrew before response was assessed. In the remaining twelve patients, there were two complete clinical responses (17%), three partial responses (25%), five had stable disease (41%), and two (17%) had progressive disease. Most common adverse events were rash in four patients, diarrhea in four, joint symptoms in three, and abnormal liver function tests in three. There were no grade 4 toxicities and all toxicities were reversible. At 3 weeks, cell proliferation as measured by Ki-67 was significantly reduced in the AFG group (p value= 0.01) with a parallel reduction in the expression of the Cyclin D1 (p value=0.02). RNA microarray data showed a corresponding decrease in the expression of cell cycle genes. These results suggest that AFG was an effective neoadjuvant therapy and consistently reduced proliferation in ER-positive tumors.

Adjuvant chemotherapy in premenopausal women with breast cancer may induce amenorrhea, which can affect fertility, choice of hormonal therapy, and increase the risk of late toxicity. The incidence of chemotherapy-induced amenorrhea (CIA) resulting from doxorubicin and cyclophosphamide (AC) followed by a taxane (T) is poorly characterized.We retrospectively surveyed women who were premenopausal and less than age 50 at initiation of chemotherapy to determine the rates of CIA in women receiving AC followed by T compared with AC alone.One hundred ninety-one eligible women completed the survey. The rate of CIA in women who received AC followed by T was 64% (95% confidence interval [CI] = 55-72%) compared with 55% (95% CI = 43-66%) in AC alone. As expected, CIA rates were higher in older than younger women (82% vs. 55%, P = 0.004). Multivariate logistic regression analysis revealed that age40 was associated with a 4.6-fold increased risk of CIA (95% CI = 1.7-12.1, P = 0.002). It also revealed that receiving T after AC was associated with an odds ratio of 1.9 for CIA as compared with receiving AC alone (95% CI = 1.0-3.5, P = 0.05). Despiteor =6 months of amenorrhea, many womenor =40 resumed menses (40%). CIA was more likely to be irreversible in those40. The addition of taxanes did not alter the rate of reversibility for the group as a whole (P = 0.36).Older age and the addition of taxane to AC increased the risk of CIA and the amenorrhea was more likely to be irreversible for women40. Womenor =40 often resume menstruation even after 6 months of amenorrhea, and the addition of T does not play a role. Subsequent resumption of menstrual function must be considered when initiating appropriate hormonal therapy.

Aiming at improving the therapeutic outcome of sequential taxane and anthracycline front line chemotherapy in breast cancer, G and N were incorporated into the taxane arm; GNT x 4 courses (G 1800-2500mg/m2, N 20-30mg/m2, T 100mg/m2, I.V. on day 1. GNT was administered sequentially with L-FAC x 4 courses; 72hr infusion of 400mg/m2/d 5-fluorouracil modulated by I.V. bolus 200mg/m2/d x 3 leucovorin. This was given concomitantly with 24hr d1 I.V. infusion 600mg/m2 cyclophosphamide, followed by 48hr I.V. infusion d2 + d3 60mg/m2 doxorubicin (ASCO 2006# 10741). Chemotherapy was given in a dose dense pattern with filgrastim support. As data became available, transtuzumab (for 1≤ year) was incorporated in HER2+ subset (15 patients) as of 11/2004, cis-platin (P) 70mg/m2/d1; GNTP in HER2+ and Triple negative (TN) subsets (20 patients) as of 5/2007 and bevacizumab 5mg/kg/wk I.V. in inflammatory and Stage IV HER2- subsets (6 patients) as of 10/2007. Hormone receptor positive (HOR+) subset (18 patients) received post chemotherapy hormone maintenance. Ten patients also received additional therapies. From 3/2001 to 2/2012, 52 patients (Median age 48) participated including the following stages: I; 3 (6%), II; 24 (46%), III; 17 (33%), IV; 8 (15%). The following subsets were represented; Triple negative 22 (42%), HER2+ 19 (37%), HER2-/HOR+ 11 (21%). Modified Blacks nuclear grade 3 was noted in 71% and Ki67 > 14% in 91%. 83% had breast conservation and 23 (44%) neoadjuvant chemotherapy; 87% achieved major pathologic response (14 PCR, 2 RCB1, 4 PET/CR/negative biopsy) at a median follow up of 75 months (m). Adjuvant chemotherapy follow up was longer at 116 m. At 81 m, 8/8 Stage IV patients achieved complete remission, though 1 patient (HER2+) had successfully treated intracranial relapse and another developed a second breast primary. At an overall follow up of 85 m (29-154), 4 relapses were observed; 2 HER+ (Stage III/without transtuzumab; Stage IV intracranial relapse only), 1 Inflammatory TN without PCR, 1 HOR+ late relapse > 10 year with short hormonal therapy. There were 3 deaths; 2 breast cancer related. Freedom from relapse for TN was 95% at 82 m, HER2+ 89% at 84 m, HER-/HOR+ 91% at 116 m. Dose dense limiting events included thrombocytopenia, muco-cutaneous reactions, and patient desired breaks. Other side effects noted were; decreased cardiac ejection fraction (2), decreased hearing (1), chronic peripheral neuropathy (2), AML (1), and MDS (1). Despite the limited study population number, this treatment approach resulted in high pathologic remission, complete remission in Stage IV and substantial freedom from relapse in high-risk and metastatic breast cancer subsets. This is highly suggestive that the natural history of aggressive breast cancer can be favorably influenced with this treatment approach. Citation Format: Khaled Jabboury, Lena Rogers, Krystal Sexton, Odelia Garcia. Can potential curability be reached in high risk/metastatic breast cancer patients with a treatment optimization strategy that includes a novel GNT (gemcitabine, vinorelbine, docetaxel) in front line combination chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT127. doi:10.1158/1538-7445.AM2015-CT127

Premenopausal women are diagnosed with 25% of all invasive breast cancers;adjuvant chemotherapy given to many of this population may induce menopause and increase the risk of osteoporosis development. Guidelines issued by the American Society of Clinical Oncology recommend regular assessment of bone health in such women. To assess appropriate attention to bone health, we performed a retrospective, cross-sectional survey of young women at high risk of osteoporosis secondary to chemotherapy-induced premature menopause. In all, 102 women with chemotherapy-induced menopause, 75% of whom were 40 years of age or younger, were asked whether they underwent screening and preventive measures for osteoporosis. Only 56% had discussed bone health with their healthcare providers; age at diagnosis, race, and use of tamoxifen were not linked to the likelihood of such discussions. Regular exercise was recommended to 73% of the women, calcium supplementation to 56%, and bone mineral density (BMD) testing to 40%. Approximately one half of the women regularly exercised and took a calcium supplement; however, over 37% of those using a supplement took less calcium than that recommended to prevent osteoporosis. Further, 32% reported having had BMD testing;women 40 years of age or younger were less likely to have had such tests (27%) than were older women (48%;P = 0.05). More emphasis must be given to educating breast cancer survivors with chemotherapy-induced menopause about bone health and its maintenance. Approved therapies to prevent osteoporosis probably are underused in this population.

Background: Molecularly targeted therapies such as tamoxifen, aromatase inhibitors, and trastuzumab are useful for the treatment of breast cancer. However, these targeted therapies do not treat all breast cancers, especially estrogen receptor alpha (ER)-negative, progesterone receptor (PR)-negative, HER2-negative (“triple-negative”) breast cancer. Our lab previously used gene expression profiling of human breast cancers to identify kinases overexpressed in ER-negative breast cancers. One of the kinases highly expressed in a poor prognosis group of triple negative breast cancers is maternal embryonic leucine-zipper kinase (MELK). The purpose of this study was to demonstrate that MELK expression is upregulated in ER-negative breast cancers, and to test the hypothesis that MELK is a critical regulator of ER-negative breast cancer cell growth. Methods: RNA from breast cancer cell lines and 60 human breast tumors was isolated for Q-PCR analysis of MELK. To determine if MELK regulates cell proliferation, ER-positive and ER-negative breast cancer cell lines were transfected with siRNA targeting MELK. Cells were replated 36 hours after transfection in 96-well plates at a density of 2,000/well. Cell number was measured by the MTS assay every day for 6 days. To determine if MELK levels are independently prognostic, we performed uni- and multivariate Cox regression analysis, including all available biological characteristics of the tumor in the model. Results: MELK mRNA levels were significantly higher (p Conclusions: MELK is highly expressed in ER-negative breast cancer, essential for ER-negative breast cancer cell growth, and is independently prognostic. These findings suggest that MELK is a promising target for the treatment of ER-negative breast cancer. Supported by a Susan G. Komen for the Cure Promise Grant. (KG081694). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1664. doi:10.1158/1538-7445.AM2011-1664

Abstract #6039 Background: We used a previously published gene expression signature that can identify tumors from BRCA1 mutation carriers to evaluate its predictive value in triple-negative breast cancer as a marker for chemosensitivity to anthracycline-based chemotherapy. We proposed that based on preclinical evidence suggesting that BRCA1-deficient breast cancer cells are sensitive to DNA damaging drugs such as cisplatin and anthracyclines this gene expression profile may identify tumors with anthracycline chemosensitivity. Two previously published studies defined a gene expression signature associated with BRCA1 germline mutation.(1,2) In these studies, sporadic tumors were misclassified as BRCA1 tumors and further analysis revealed methylation of the BRCA1 promoter region and decreased BRCA1 gene expression. This finding suggests the possibility of identifying sporadic tumors with decreased BRCA1 activity. Methods: We selected from our database of a locally advanced breast cancer neoadjuvant trial all cases of triple negative breast cancer that received 4 cycles of doxorubicin/cyclophosphamide(AC, 60/200 mg/m2, every 3 weeks) prior to surgery. Pathologic response to chemotherapy was disappearance of all invasive cancer or microscopic residual disease. Tumoral gene expression profile previously obtained using Affymetrix U133A Chip was analyzed for an optimal set of 100 most differentially expressed genes distinguishing BRCA1 and sporadic triple negative tumors according to the previously identified gene signature by van't Veer et al.1 We performed unsupervised clustering to determine if this signature could classify a subtype of triple-negative tumors with "BRCAness" and to test our hypothesis that BRCA1-like tumors are more sensitive to AC. We then performed a supervised analysis to determine the most differentially expressed genes that could prospectively identify triple-negative sporadic tumors with “BRCAness” and tumors from BRCA1 germline carriers that are sensitive to anthracyclines. Results: Of the 66 patients enrolled in our neoadjuvant trial, 12 patient's tumors were triple negative and received preoperative AC. By unsupervised clustering, the gene expression pattern associated with BRCA1 cancers subdivided these sporadic cancers in to two groups: Group A(6/7 pathologic responders), and group B(5/5 non-pathologic responders). By supervised analysis, the most differentially overexpressed gene from the BRCA1 profile for AC sensitivity was YWHAH(14-3-3 eta polypeptide), while DKK3(Inhibitor of Wnt and Notch signaling pathway) and RPL23A were most overexpressed in all cases with adriamycin-resistance(p Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6039.

21141 Background: Black and Hispanic breast cancer patients have a worse outcome when compared to Caucasians. This could be due to socioeconomic, cultural or biologic factors. We hypothesized that host and tumor biologic characteristics associated with a poor outcome may be found more often in minority women. Methods: Race/ethnicity, menopausal status, tumor histological features, and patient characteristics including age and body mass index (BMI) were reviewed from a prospective neoadjuvant trial of docetaxel vs. doxorubicin/cyclophosphamide at Baylor College of Medicine Breast Center, from September 2002 to September 2006. The data were analyzed using Chi-square and Fisher's exact tests, while the Kruskal-Wallis method was used to analyze BMI. Results: Of the 167 patients, 63% (n=105) were Caucasian, 15% (n=26) were Hispanic and 22% (n=36) were Black. The mean age was 47.6 years (range: 30–72). Fifty-nine percent were premenopausal. Overall, mean BMI was 29, with Caucasians having a mean BMI of 27.5, Hispanics with 29.8 and Blacks with a BMI of 34.6 (P No significant financial relationships to disclose.

10741 Background: Front-line dose-intensive L-FAC has demonstrated a favorable 5-year relapse free survival pattern (ASCO 2004 #739). Tamoxifen was given for 5 years to ER+ patients after L-FAC completion. We evaluated the impact of adding tamoxifen to L-FAC. By design, this pilot study excluded low-risk patients not candidates for chemotherapy. Methods: 109 breast cancer patients were enrolled (4 excluded due to treatment violations) from 6/1989 to 1/2003: 20 Stage I (S), 52 S-II, 22 S-III, for a total of 94 patients. 11 S-IV patients were excluded from survival analysis. Adverse tumor presentations included: ER- 49, PgR- 60, P53+ 24, non-diploid 39, histological grade III 37, CerbB2+ 33. L-FAC included 72 hour (h) iv infusion 400mg/m2/day (d) 5-fluorouracil (F) modulated by iv bolus 200mg/m2/d X3 leucovorin (L), concomitantly with 24h iv d1 600–1000mg/m2 cyclophosphamide (C), 48h iv d2 + d3 60mg/m2 doxorubicin (A). S-I and S-II were given 6 courses and 8 for S-III. Increasing A + C dose level and/or shortening treatment intervals < 3 weeks with growth factors provided intensification. 40 patients received tamoxifen. Results: At a median follow-up of 74 months (range 9–214), 73 (78%) are alive (1 with relapse). Relapse free survival was: S-I 95%, S-II 81%, S-III 78%. At average course intervals of 18 days, dose intensity A/C mg/m2/wk was 24.2 / 335.4 with evidence of WHO grade III/IV stomatitis in 43%, neutropenia 59%, cumulative thrombocytopenia 50%, hand-foot syndrome 32% of patients. Aside from delayed relapse associated with tamoxifen, relapse-free survival >82 months was similar with and without tamoxifen. No relapse was observed after >53 months in ER- tumors despite showing higher frequency of adverse tumor risk factors. Conclusion: The impact of adding tamoxifen appears quite limited in a patient population with adverse tumor presentation treated with dose-intensive L-FAC. No significant financial relationships to disclose.

538 Background: Docetaxel has one of the highest response rates as a single agent in breast cancer, but de novo resistance is frequent. Previously, we had identified a 92-gene expression pattern that predicted response to neoadjuvant docetaxel. Other studies have validated that a high Recurrence Score (RS) by the 21-gene RT-PCR assay is predictive of worse prognosis (Paik, NEJM 2004) but better response to chemotherapy (Gianni, JCO 2005). We investigated whether tumor gene expression of these 21 genes and other candidate genes can predict response to docetaxel. Methods: Core biopsies from 97 patients were obtained before treatment with neoadjuvant docetaxel (4 cycles, 100 mg/m2 q3 weeks). Baseline and post-treatment measurements of the primary breast cancers were recorded. Three 10-micron FFPE sections were submitted for quantitative RT-PCR assays of 192 genes that were selected from our previous work and the literature. Results: Of the 97 patients, 81 (84%) had sufficient invasive breast cancer, 80 (82%) had sufficient RNA for assay of 192 genes, and 72 (74%) had clinical response data. Mean age was 48.5 years, and the median tumor size was 6 cm. Clinical complete responses (CR) by RECIST were observed in 12 (17%), partial responses in 41 (57%), stable disease in 17 (24%), and progressive disease in 2 patients (3%). The concordance of IHC and RT-PCR results was >80% for ER, PgR, and HER2. By univariate logistic regression, a significant correlation (p [Table: see text]

651 Background: 25% of invasive breast cancers occur in premenopausal women. Adjuvant chemotherapy may induce amenorrhea, resulting in infertility, influencing hormonal therapy options, and potentially increasing risks of late toxicity. Methods: We retrospectively surveyed women who were premenopausal and 40 but < 50. 77 patients received AC, 118 received AC → T. In patients ≤ 40, the rate of CIA was greater for those who received AC → T (61%) as compared to AC (44%), p=0.04 (OR...