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Supplementary Table S1. Patient characteristics genotyped cohort vs non-genotyped cohort. Supplementary Table S2. Patient characteristics according to FCGR3A genotype. Supplementary Table S3. Patient characteristics according to FCGR2A genotype. Supplementary Table S4. Patient characteristics according to FCGR2B genotype. Supplementary Figure S1. DFS was not improved for any genotypic group in the trastuzumab-containing regimens.

Patients with HER2+ breast cancer treated with trastuzumab and chemotherapy have superior survival compared with patients treated with chemotherapy alone. Polymorphisms within FCGR2A and FCGR3A are associated with binding affinity of natural killer cells to the IgG1 portion of trastuzumab, and a polymorphism in FCGR2B (I232T) is associated with impaired regulatory activity. The association of these polymorphisms with clinical response among trastuzumab-treated patients is equivocal, with both positive and negative associations. We performed genotyping analysis on the FCGR3A V158F, FCGR2A R131H, and FCGR2B I232T polymorphisms in 1,325 patients from the N9831 clinical trial. Patients in arm A (N = 419) received chemotherapy only. Patients in arms B (N = 469) and C (N = 437) were treated with chemotherapy and trastuzumab (sequentially in arm B and concurrently in arm C). Using log-rank test and Cox proportional hazard models, we compared disease-free survival (DFS) among genotypic groups within pooled arms B/C. We found no differences in DFS between trastuzumab-treated patients who had the FCGR3A 158 V/V and/or FCGR2A 131 H/H high-affinity genotypes and patients without those genotypes. Furthermore, there was no significant interaction between FCGR3A and FCGR2A and treatment. However, there was a difference in DFS for FCGR2B I232T, with I/I patients deriving benefit from trastuzumab (P < 0.001), compared with the T carriers who did not (P = 0.81). The interaction between FCGR2B genotype and treatment was statistically significant (P = 0.03). Our analysis did not reveal an association between FcγR high-affinity genotypes and outcomes. However, it seems that the FCGR2B inhibitory gene may be predictive of adjuvant trastuzumab benefit. Cancer Immunol Res; 2(10); 962–9. ©2014 AACR.

Supplementary Figure S1 describes the pre-existing antibody responses to tumor antigens in patients with surgically resected HER2+ breast cancer. Supplementary Figure S2 compares the proportion of either the metastatic or adjuvant patients that respond to therapy with increased antibody responses. Table S1 shows the demographic information for the patients in this study. Table S2 compares the levels of antibodies to tumor antigens and TT in metastatic patients and healthy controls. Table S3 compares the mean pre- and post-treatment antibody levels to all of the tumor antigens and TT in the patients with metastatic disease. Table S4 describes correlations of the antibody responses to the type of chemotherapy received in the metastatic patients. Table S5 shows the results of a cox univariate analysis correlating PFS and antibody responses. Table S6 shows the results of a cox univariate analysis correlating OS and antibody responses. Table S7 compares the mean pre- and post-treatment antibody levels to all of the tumor antigens and TT in patients with surgically-resected disease.

Supplementary Figures S1, S2, S3 and Tables S1, S2

Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2mut detection.Experimental Design: Tumor tissue positive for HER2mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2mut).Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2mut cases were identified locally. Twenty-one of these 22 HER2mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31–74) years and three (2–10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%–55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8–31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%–94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%–100%). In addition, ctDNA HER2mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression.Conclusions: Neratinib is active in HER2mut, nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687–95. ©2017 AACR.

The addition of trastuzumab to chemotherapy extends survival among patients with HER2+ breast cancer. Prior work showed that trastuzumab and chemotherapy augments HER2 extracellular domain (ECD)-specific antibodies. The current study investigated whether combination therapy induced immune responses beyond HER2-ECD and, importantly, whether those immune responses were associated with survival. Pretreatment and posttreatment sera were obtained from 48 women with metastatic HER2+ breast cancer on NCCTG (now Alliance for Clinical Trials in Oncology) studies, N0337 and N983252. IgG to HER2 intracellular domain (ICD), HER2-ECD, p53, IGFBP2, CEA, and tetanus toxoid were examined. Sera from 25 age-matched controls and 26 surgically resected HER2+ patients were also examined. Prior to therapy, some patients with metastatic disease had elevated antibodies to IGFBP2, p53, HER2-ICD, HER2-ECD, and CEA, but not to tetanus toxin, relative to controls and surgically resected patients. Treatment augmented antibody responses to HER2-ICD in 69% of metastatic patients, which was highly associated with improved progression-free survival (PFS; HR = 0.5, P = 0.0042) and overall survival (OS; HR = 0.7, P = 0.038). Augmented antibody responses to HER2-ICD also correlated (P = 0.03) with increased antibody responses to CEA, IGFBP2, and p53, indicating that treatment induces epitope spreading. Paradoxically, patients who already had high preexisting immunity to HER2-ICD did not respond to therapy with increased antibodies to HER2-ICD and demonstrated poorer PFS (HR = 1.6, P < 0.0001) and OS (HR = 1.4, P = 0.0006). Overall, the findings further demonstrate the importance of the adaptive immune system in the efficacy of trastuzumab-containing regimens. Cancer Res; 76(13); 3702–10. ©2016 AACR.

Table S3

Introduction: It is well established that multidisciplinary tumor boards improve the decision-making process for cancer patients. Tumor boards have been shown to improve the accuracy of diagnosis and staging, optimize patient outcomes, increase adherence to guidelines, and educate our peers and trainees. However, over 80% of patients in the United States receive their cancer care in the community setting, where access to multi-disciplinary tumor boards may not be readily available. This may particularly impact underserved populations who often lack the resources to travel to an academic center for second opinions or treatment. The problem is worse in low-resource countries. Virtual expert tumor boards could provide an effective solution. Methods: Preeminent breast oncology faculty from around the Unites States were assembled into virtual tumor board panels via an online platform to discuss challenging cases submitted by community providers and trainees. These tumor boards consisted of a moderator, a breast radiologist, a breast medical oncologist, a breast surgeon, and a breast radiation oncologist. The purpose of this ongoing endeavor is to educate community oncologists on how to best manage challenging cases. Following tumor board discussions, written recommendations were shared with submitting providers within 48 hours and recordings of the discussions were also later provided. After submitting providers watched the recording of their case discussion, we conducted a survey to determine their perceived benefit of the expert panel discussion. Results: From Sept 2020 to June 2021, ten breast cancer panels were virtually convened with 17 expert faculty panelists. During that time, 21 providers submitted 94 cases from the U.S. and around the world to be discussed by the expert panel. Thirty-three percent of the providers who submitted a case to be discussed have subsequently submitted an additional case to a later panel. Surveys were sent to all submitting providers and responses were recorded from 16/21 submitters (76.2%). Conclusion: With more than three out of four submitters responding, we learned that not only is it feasible to convene virtual expert breast tumor boards to discuss challenging cases, but the vast majority of respondents learned new information, changed management of their patients, and wanted to submit additional cases. This effort could raise the level of breast cancer care around the world. Ongoing assessment of educational and patient care impacts will be necessary. QuestionNumber answered (n)Number who answered yes (%)Number who answered no (%)Did you learn something new from the PrecisCa discussion of your case scenario?1614 (87.5)2 (12.5)Will anything you learned from the PrecisCa discussion of your case scenario change the management of this or future patients?1615 (93.8)1 (6.2)Are you likely to submit a future challenging case scenario to PrecisCa?1616 (100)0 (0) Citation Format: Naomi Dempsey, Lauren Chiec, Mikala Lodder, Erin Shonkwiler, Kayla Haines, Reshma Mahtani, William Gradishar, Thomas Buchholz, Anne O'Dea, Norman Wolmark, Sara Hurvitz, Joyce O'Shaughnessy, Maxine Jochelson, Reni Butler, Eleftherios Mamounas, Frank Vicini, Mark Pegram, Chirag Shah, Tari King, Ruth O'Regan, Monica Morrow, Mohammad Jahanzeb. Raising the level of cancer care around the world: The feasibility and perceived benefit of a virtual breast tumor board [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-17-03.

ImportanceIt is estimated that up to 50% of patients with ERBB2 (HER2)-positive metastatic breast cancer (MBC) will develop brain metastases (BMs), which is associated with poor prognosis. Previous reports of the HER2CLIMB trial have demonstrated that tucatinib in combination with trastuzumab and capecitabine provides survival and intracranial benefits for patients with ERBB2-positive MBC and BMs.ObjectiveTo describe overall survival (OS) and intracranial outcomes from tucatinib in combination with trastuzumab and capecitabine in patients with ERBB2-positive MBC and BMs with an additional 15.6 months of follow-up.Design, Setting, and ParticipantsHER2CLIMB is an international, multicenter, randomized, double-blind, placebo-controlled clinical trial evaluating tucatinib in combination with trastuzumab and capecitabine. The 612 patients, including those with active or stable BMs, had ERBB2-positive MBC previously treated with trastuzumab, pertuzumab, and trastuzumab emtansine. The study was conducted from February 23, 2016, to May 3, 2019. Data from February 23, 2016, to February 8, 2021, were analyzed.InterventionsPatients were randomized 2:1 to receive tucatinib (300 mg orally twice daily) or placebo (orally twice daily), both in combination with trastuzumab (6 mg/kg intravenously or subcutaneously every 3 weeks with an initial loading dose of 8 mg/kg) and capecitabine (1000 mg/m2 orally twice daily on days 1-14 of each 3-week cycle).Main Outcomes and MeasuresEvaluations in this exploratory subgroup analysis included OS and intracranial progression-free survival (CNS-PFS) in patients with BMs, confirmed intracranial objective response rate (ORR-IC) and duration of intracranial response (DOR-IC) in patients with measurable intracranial disease at baseline, and new brain lesion–free survival in all patients. Only OS was prespecified before the primary database lock.ResultsAt baseline, 291 of 612 patients (47.5%) had BMs. Median age was 52 years (range, 22-75 years), and 289 (99.3%) were women. At median follow-up of 29.6 months (range, 0.1-52.9 months), median OS was 9.1 months longer in the tucatinib-combination group (21.6 months; 95% CI, 18.1-28.5) vs the placebo-combination group (12.5 months; 95% CI, 11.2-16.9). The tucatinib-combination group showed greater clinical benefit in CNS-PFS and ORR-IC compared with the placebo-combination group. The DOR-IC was 8.6 months (95% CI, 5.5-10.3 months) in the tucatinib-combination group and 3.0 months (95% CI, 3.0-10.3 months) in the placebo-combination group. Risk of developing new brain lesions as the site of first progression or death was reduced by 45.1% in the tucatinib-combination group vs the placebo-combination group (hazard ratio, 0.55 [95% CI, 0.36-0.85]).Conclusions and RelevanceThis subgroup analysis found that tucatinib in combination with trastuzumab and capecitabine improved OS while reducing the risk of developing new brain lesions, further supporting the importance of this treatment option for patients with ERBB2-positive MBC, including those with BMs.Trial RegistrationClinicalTrials.gov Identifier: NCT02614794

Background: The Breast Cancer Index (BCI) is a validated gene expression assay that provides a quantitative individualized risk of late recurrence and predicts the likelihood of benefit from extended endocrine therapy (EET) in HR+ early-stage breast cancer. The objective of this analysis was to assess the impact of BCI on clinical decision-making regarding EET in the BCI Registry Study. Methods: The BCI Registry Study is an ongoing, prospective, large-scale study to investigate the long-term clinical outcome, decision impact, and medication adherence in HR+ early-stage breast cancer patients receiving BCI testing as part of routine clinical care. Per protocol, physicians and patients completed pre- and post-BCI test questionnaires to assess the following: physician decision-making regarding EET; physician confidence with decision-making; patient preferences for EET; patient concerns about the cost, side effects, drug safety and benefit of EET; and patient satisfaction regarding treatment recommendation. Pre- and post-BCI responses were compared using McNemar’s test and Wilcoxon signed rank test. Results: Pre- and post-BCI test physician and patient questionnaires were completed for 843 and 823 patients, respectively. For the patients included in this analysis, mean age at enrollment was 65y and 88.4% of patients were postmenopausal. 74.7% of tumors were T1, 53.4% were G2, 75.3% N0, and 13.8% HER2-positive. Following BCI testing, physicians changed treatment recommendations for EET in 40.1% (338/843) of patients (p< 0.0001). In cases in which physicians recommended EET prior to BCI testing, 44.7% (214/479) changed their recommendation not to extend endocrine therapy. Of these cases, 98.1% (210/214) were BCI Prognostic low risk and/or BCI (H/I) Predictive low likelihood of benefit from EET. In cases in which physicians did not recommend EET prior to BCI testing, 34.6% (124/358) changed their recommendation in favor of EET. In such cases, 87.1% (108/124) of cases were BCI Prognostic high risk and/or BCI (H/I) Predictive high likelihood of benefit from EET. Further, following BCI testing, 38.8% (327/843) of physicians felt more confident in their recommendation (p< 0.0001). The percentage of physicians having high confidence levels (confident or strongly confident) increased from 58.1% (490/843) before BCI testing to 80.5% (679/843) after BCI testing. The percentage of physicians having low confidence levels (not at all confident, not confident, or ambivalent) decreased from 39.1% (330/843) before BCI testing to 18.7% (158/843) after BCI testing. In addition, 40.5% (341/843) of patients felt more comfortable with their EET decision (p< 0.0001) following BCI testing. Notably, changes in patient preferences for EET correlated with BCI test results. In BCI (H/I)-Low patients, 46.9% (241/514) showed a decreased preference for EET (p< 0.0001) while in BCI (H/I)-High patients, 28.2% (87/309) showed an increased preference for EET (p< 0.0001). Compared with baseline, after BCI testing, significantly more patients were less concerned about cost (20.9%, p< 0.0001), drug safety (25.4%, p=0.0014), and the benefits of EET (29.3%, p=0.0002). No significant change in concern regarding side-effects was observed (p=0.1486). Conclusions: This first analysis in a large patient cohort of the BCI Registry confirms previous findings on the significant clinical decision-making impact of BCI for extending adjuvant endocrine therapy. Incorporating BCI into clinical practice resulted in changes in physician recommendations for EET in over 40% of cases, while at the same time increasing physician confidence in their recommendations. Knowledge of the BCI result also improved patient satisfaction and reduced patient concerns regarding cost, drug safety and benefit of EET. Citation Format: Tara B. Sanft, Jenna Wong, Brandon O’Neal, Natalia Siuliukina, Rachel C. Jankowitz, Mark Pegram, Jenny Fox, Yi Zhang, Kai Treuner, Joyce O’Shaughnessy. Prospective assessment of the decision-making impact of the Breast Cancer Index in the BCI Registry Study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-02.

Triple-negative breast cancer (TNBC) is a pathological term used to identify invasive breast cancers that lack expression of estrogen and progesterone receptors and do not have pathologic overexpression of the HER2 receptor or harbor ERBB2 gene amplification. TNBC includes a collection of multiple distinct disease entities based upon genomic, transcriptomic and phenotypic characterization. Despite improved clinical outcomes with the development of novel therapeutics, TNBC still yields the worst prognosis among all clinical subtypes of breast cancer. We will systematically review evidence of the genomic evolution of TNBC, as well as potential mechanisms of disease progression and treatment resistance, defined in part by advances in next-generation DNA sequencing technology (including single cell sequencing), providing a new perspective on treatment strategies, and promise to reveal new potential therapeutic targets. Moreover, we review novel therapies aimed at homologous recombination deficiency, PI3 kinase/AKT/PTEN pathway activation, androgen receptor blockade, immune checkpoint inhibition, as well as antibody-drug conjugates engaging novel cell surface targets, including recent progress in pre-clinical and clinical studies which further validate the role of targeted therapies in TNBC. Despite major advances in treatment for TNBC, including FDA approval of 2 PARP inhibitors for metastatic TNBC, the crossing of the superiority boundary in a phase 3, placebo-controlled study of adjuvant olaparib in early-stage patients with germline BRCA-mutated high-risk HER2-negative early breast cancer, the FDA approval of 2 PD-(L)1 checkpoint antibodies for metastatic TNBC, and the FDA approval of the first antibody drug conjugate for TNBC, significant challenges remain. For example, despite the dawn of immunotherapy in metastatic TNBC, durable responses are limited to a small subset of patients, definitive biomarkers for patient selection are lacking, and the Oncology Drug Advisory Committee to the FDA has recently voted against approval of an anti-PD-1 checkpoint antibody high risk early-stage TNBC in the neoadjuvant setting. Also, despite early positive randomized phase 2 studies of AKT inhibition in metastatic TNBC, a recent phase 3 registration trial failed to validate earlier phase 2 data. Finally, we note that level one evidence for clinical efficacy of androgen receptor blockade in TNBC is still lacking. To meet these and other challenges, we will catalogue the ongoing exponential increase in interest in basic, translational, and clinical research to develop new treatment paradigms for TNBC.

Background: Tucatinib is an oral tyrosine kinase inhibitor highly specific for HER2 that is approved for use in combination with trastuzumab and capecitabine in adults with advanced or metastatic HER2+ breast cancer, including patients with brain metastases, who have received one or more prior anti-HER2-based regimens in the metastatic setting. In the HER2CLIMB trial, the tucatinib regimen significantly prolonged progression-free survival (PFS) and overall survival (OS) in patients with HER2+ metastatic breast cancer (Murthy, NEJM 2020), including in patients with untreated, treated stable, and treated progressing brain metastases (Lin, J Clin Oncol, 2020). With an additional 15.6 months of follow-up, addition of tucatinib continued to show clinically meaningful prolongation of PFS and OS in the total study population (Curigliano, ASCO Meeting, 2021). We report updated results of exploratory efficacy analyses in patients with brain metastases. Methods: All patients in HER2CLIMB had a baseline brain MRI. Patients with brain metastases were eligible and classified as untreated, treated stable, or treated progressing. Patients were randomized 2:1 to receive tucatinib 300 mg twice daily or placebo, in combination with trastuzumab and capecitabine. Following the primary analysis, the protocol was amended to unblind sites to treatment assignment and allowed crossover from the placebo regimen to the tucatinib regimen. Efficacy analyses in patients with brain metastases at baseline were performed at approximately 2 years from the last patient randomized by applying RECIST 1.1 to the brain based on investigator evaluation. OS and CNS-PFS (progression in the brain or death) were evaluated in all patients with brain metastases. Patients without CNS-PFS events were censored at the last brain MRI. Confirmed intracranial (IC) objective response rate (ORR-IC) was evaluated in patients with measurable IC disease. Results: At a median follow-up of 29.6 months, median OS was 21.6 months vs 12.5 months in all patients with brain metastases (HR: 0.60; 95% CI: 0.44, 0.81), 21.4 months vs 11.8 months in patients with untreated/treated progressing brain metastases (HR: 0.52; 95% CI: 0.36, 0.77), and 21.6 months vs 16.4 months in patients with treated stable brain metastases (HR: 0.70; 95% CI: 0.42, 1.16). Median CNS-PFS was 9.9 months vs 4.2 months in all patients with brain metastases (HR: 0.39; 95% CI: 0.27, 0.56), 9.6 months vs 4.0 months in patients with untreated/treated progressing brain metastases (HR: 0.34; 95% CI: 0.22, 0.54), and 13.9 months vs 5.6 months in patients with treated stable brain metastases (HR: 0.41; 95% CI: 0.19, 0.85). ORR-IC was higher in the tucatinib arm (47.3%; 95% CI: 33.7, 61.2) vs the placebo arm (20.0%; 95% CI: 5.7, 43.7) for patients with brain metastases, and median duration of response (DOR) was 8.6 months (95% CI: 5.5, 10.3) vs 3.0 months (95% CI: 3.0, 10.3). Conclusions: With 15.6 months of additional follow-up, the tucatinib-trastuzumab-capecitabine regimen resulted in a robust and durable prolongation of OS for all patients with HER2+ metastatic breast cancer and brain metastases. Additionally, this benefit was maintained in patients with untreated/treated progressing and treated stable brain metastases. Treatment with tucatinib continued to show clinically meaningful benefit in CNS-PFS consistent with the primary analysis. Citation Format: Nancy U Lin, Rashmi K Murthy, Vandana Abramson, Carey Anders, Thomas Bachelot, Philippe Bedard, Virginia Borges, David Cameron, David Cameron, Lisa Carey, A Jo Chien, Giuseppe Curigliano, Michael DiGiovanna, Karen Gelmon, Gabriel Hortobagyi, Sara Hurvitz, Ian Krop, Sherene Loi, Sibylle Loibl, Volkmar Mueller, Mafalda Oliveira, Elisavet Paplomata, Mark Pegram, Dennis Slamon, Amelia Zelnak, Jorge Ramos, Wentao Feng, Eric Winer. Updated results of tucatinib vs placebo added to trastuzumab and capecitabine for patients with previously treated HER2-positive metastatic breast cancer with brain metastases (HER2CLIMB) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD4-04.

PURPOSE: Based on promising predinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2(mut) nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2(mut) detection. EXPERIMENTAL DESIGN: Tumor tissue positive for HER2(mut) was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2(mut)). RESULTS: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2(mut) (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2(mut) cases were identified locally. Twenty-one of these 22 HER2(mut) cases were estrogen receptor positive. Sixteen patients [median age 58 (31–74) years and three (2–10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI),13%–55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8–31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2(mut) in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%–94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%–100%). In addition, ctDNA HER2(mut) variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression. CONCLUSIONS: Neratinib is active in HER2(mut), nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2(mut) cancers for clinical trial participation.

Breast cancer (BC) is the second leading cause of cancer death among African-American (AA) women, with mortality 20% greater than that in Caucasians (Cauc). However, the basis for such disparity remains an enigma. Recent observations from our laboratory suggest the involvement of unidentified genes contributing to AA BC risk. Matched tumor and normal FFPE samples from Cauc and AA patients were obtained from the UM /Sylvester Breast Tissue Bank (UM/S BTB) under an IRB-approved protocol. Based on analysis of 22,000 transcripts, ethnic specific gene expression patterns were identified that may provide important new insights into molecular mechanisms of ethnic subtype differences in clinical outcomes. We propose to extend these preliminary findings to a large African tumor bank [available via collaboration between Drs. Peter A. Bird (Kijabe, Kenya) and Mark Pegram (UM Sylvester).] Additionally, we propose to analyze chromosomal alterations associated with gene expression differences utilizing array cGH (in collaboration with Alan Ashworth, England). This work will contribute to development of rationale designs of preventive, predictive and therapeutic measures for BC in different ethnicities, and thus, a significant reduction in current ethnic-specific disparities in BC incidence, morbidity and mortality.

This review has stressed the common hereditary and acquired blood protein defects associated with thrombosis. The most common of the hereditary defects appear to be antithrombin, protein C, and protein S deficiency and the most common acquired defects are anticardiolipin antibodies and the lupus anticoagulant. Therefore these are the defects that should first be looked for in an individual with unexplained thrombosis. If these more common defects are not found, then the rarer defects, including heparin cofactor II, plasminogen or tissue plasminogen activator deficiency, dysfibrinogenemia, or elevated PAI-1 should next be sought. The importance of finding these defects has significant implications for therapy of the individual patient and for institution of family studies to identify, inform, and possibly treat others at risk. It is expected that as knowledge of hemostasis expands, more hereditary and acquired defects, such as elevated lipoprotein(a) or defects of extrinsic (tissue factor) pathway inhibitor may be associated with enhanced risks of thrombosis.

Platinum-based therapy has not traditionally played a significant clinical role in breast cancer in the metastatic, adjuvant, or neoadjuvant settings. Recent clinical studies, however, suggest that regimens of taxane/platinum/trastuzumab are effective option for first-line treatment of HER2-overexpressing metastatic disease, and that docetaxel/carboplatin is viable in first-line treatment of HER2-negative metastatic disease, as well as in neoadjuvant therapy of locally advanced breast cancer. Further evaluation of platinum-based regimens in large multicenter studies is warranted, and several such studies are currently in progress.

elvin J Silverstein, MD, FACS, Abram Recht, MD, FASTRO, Michael D Lagios, MD, Ira J Bleiweiss, MD, eter W Blumencranz, MD, FACS, Terri Gizienski, MD, Steven E Harms, MD, FACR, Jay Harness, MD, FACS, oger J Jackman, MD, V Suzanne Klimberg, MD, FACS, Robert Kuske, MD, Gary M Levine, MD, ichael N Linver, MD, FACR, Elizabeth A Rafferty, MD, Hope Rugo, MD, Kathy Schilling, MD, ebu Tripathy, MD, Pat W Whitworth, MD, FACS, Shawna C Willey, MD, FACS

NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, is a novel small molecule anticancer agent. NB1011 is selectively active against tumor cells expressing high levels of thymidylate synthase (TS), a critical enzyme in DNA biosynthesis. NB1011 is different from the current TS-targeted drugs, which require inhibition of TS to be effective, because NB1011 cytotoxicity depends upon activation by TS. Here we report a dose-dependent, antitumor activity of NB1011 against established Tomudex-resistant breast cancer (MCF7TDX) xenografts in athymic mice. Against 5-fluorouracil-resistant colon carcinoma (H630R10) xenografts, NB1011 was as efficacious as irinotecan, a drug recently approved for the treatment of 5-fluorouracil-resistant colon cancer. To gain insight into the mechanisms NB1011 uses to suppress cellular growth, we analyzed the downstream molecular events in the high TS-expressing MCF7TDX and RKOTDX cell lines upon NB1011 treatment. NB1011 treatment increased the mRNA levels of p21, Bax, and GADD45. Furthermore, NB1011 induced p53, p21, and Bax proteins specifically in high TS-expressing tumor cells, whereas no induction was observed in low TS-expressing tumor cells (MCF7) or normal cells (WI38). Cell cycle analysis demonstrated that NB1011 treatment of MCF7TDX and RKOTDX cells resulted in an accumulation of cells in the G2-M phase of the cell cycle. Altogether, our data indicate that the induction of the p53 target genes p21, bax, and GADD45, with a concomitant deregulation of the cell cycle, may represent one of the mechanisms by which NB1011 exerts its growth-suppressive effects.

Background: Cancer is caused by mutations in oncogenes and tumor suppressor (TS) genes resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Methods: In order to identify functionally important TS genes we have conducted the first human whole genome in vivo RNA interference (RNAi) screen. Partially transformed human mammary epithelial cells (HMLEs), which do not form tumors in immunodeficient mice, were infected with the Expression Arrest™ GIPZ lentiviral shRNA library consisting of 62,000 shRNAs targeting the whole human genome, and injected into the mammary fat pad of immunodeficient mice. shRNAs that silenced TS genes fully transformed the mammary epithelial cells resulting in tumor formation. Candidate TS genes were identified by PCR amplification and sequencing of tumor integrated shRNAs. For validation, candidate TS genes were silenced in HMLEs and ectopically expressed in fully transformed breast cancer cells. The effect of modifying gene expression on the transformed phenotype was assessed using soft agar colony formation and mammary fat pad xenograft tumor formation assays. Clinical significance was determined by comparing expression in normal and cancerous human breast tissue using Oncomine Research. Results and Discussion: Using our novel approach, we identify previously validated TS genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). Silencing LIFR expression with multiple shRNA constructs fully transformed human mammary epithelial cells resulting in enhanced colony formation in soft agar (p 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 4979. doi:10.1158/1538-7445.AM2011-4979