Triple-negative Breast Cancer Disease Research Articles

Abstract CT062: NCT02456857: A phase II trial of liposomal doxorubicin, bevacizumab and everolimus (DAE) in patients with localized triple-negative breast cancer (TNBC) with tumors predicted insensitive to standard neoadjuvant chemotherapy

Abstract BACKGROUND: While targeted therapy has improved survival for ER+ and HER2+ breast cancers, no such therapy exists for TNBC. Nearly 50% of TNBC patients treated with neoadjuvant chemotherapy (taxane/anthracycline-based) will have insensitive disease manifested as extensive residual disease (RCB-II or III). This is associated with high chance of recurrence within 3 years of diagnosis. Through the Moon Shot program at MD Anderson, we have created a biomarker-driven drug development strategy (ARTEMIS: A Randomized, TNBC Enrolling trial to confirm Molecular profiling Improves Survival) to identify novel targeted therapies for chemo-insensitive disease in TNBC. In the ARTEMIS trial, patients with localized TNBC undergo biopsy for molecular profiling prior to the start of initial anthracycline-based chemotherapy. Results of molecular characterization along with diagnostic imaging is then used to identify patients with chemo-insensitive disease and inform second phase of neoadjuvant therapy to overcome intrinsic drug resistance. The mesenchymal subtype of TNBC is characterized by high incidence of PI3K pathway alterations. Consequently, preclinical models have demonstrated response to PI3K inhibitors in this subtype. Metaplastic breast cancers constitute approximately 30% of tumors characterized as ‘claudin-low/mesenchymal’ by gene expression profiling. A combination regimen consisting of liposomal doxorubicin, bevacizumab and mTOR inhibitors temsirolimus or everolimus (DAT or DAE) has demonstrated responses (including durable complete responses) in patients with advanced metaplastic breast cancers. PRIMARY OBJECTIVE: Determine the rate of pathologic complete response (pCR/RCB-0) or minimal residual disease (RCB-I) after 4 cycles of DAE for treatment of mesenchymal TNBC deemed to be chemo-insensitive after first phase of neoadjuvant therapy in the ARTEMIS trial TRIAL DESIGN AND STATISTICAL METHODS: Patients deemed to have chemo-insensitive mesenchymal TNBC identified through the ARTEMIS trial would be enrolled into this non-randomized phase II study. Considering that patients without response to initial chemotherapy have a very low chance (5%) of achieving pCR with additional cycles of chemotherapy, it would be clinically meaningful to see pCR improved to 20% in this population. Estimating pCR (RCB-0) or RCB-I as response, a two-stage Gehan-type design will be employed with 14 patients in the first stage. This design has a 49% chance of terminating after the first stage if the true response rate is 0.05, 23% chance if the true rate is 0.10, 10% chance if the true rate is 0.15 and 4% chance if the true rate is 0.20. If at least one patient responds, 23 more will be enrolled for a total of 37 patients. With 37 patients enrolled, the 95% confidence interval for a 0.20 response rate will extend from 0.10 to 0.35. BRIEF ELIGIBILITY CRITERIA: Inclusion: early stage TNBC patients enrolled in the ARTEMIS trial with adequate organ, bone marrow and cardiac function Exclusion: patients with metastatic disease, medical illness that increases chance of moderate to severe toxicity, pregnant or lactating. CORRELATIVE SCIENCE: correlate mesenchymal signatures and PI3K pathway aberrations and vimentin expression by IHC with response Citation Format: Senthil Damodaran, Ken Hess, Maia Rauch, Beatrice Astrada, Lumarie Santiago, Jennifer Litton, Elizabeth Mittendorf, Bora Lim, Naoto Ueno, Debu Tripathy, Alastair Thompson, Mike Gilcrease, Wei Yang, Helen Piwnica-Worms, W. Fraser Symmans, Stacy L. Moulder. NCT02456857: A phase II trial of liposomal doxorubicin, bevacizumab and everolimus (DAE) in patients with localized triple-negative breast cancer (TNBC) with tumors predicted insensitive to standard neoadjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT062. doi:10.1158/1538-7445.AM2017-CT062

Health-related quality of life in Black breast cancer survivors with and without triple-negative breast cancer (TNBC).

Black women are more likely to develop early-onset (≤50years) breast cancer (BC) and have the lowest five-year, cause-specific survival rate of any United States (U.S.) racial or ethnic group. These disparities can be attributed partially to the higher rate of triple-negative BC (TNBC) in Blacks. Yet, little is known about health-related quality of life (HRQOL) among Black women with TNBC. Black women with invasive BC≤50years were recruited via the Florida Cancer Data System as part of a population-based case-only study of etiology and outcomes of early-onset invasive BC. Of 460 consented participants, a subset of 355 self-reported sociodemographic, clinical, and psychosocial variables. Descriptive analyses included participants with known TNBC (n=85) or non-TNBC (n=245) disease. Univariable and multivariable analyses were conducted to examine differences in factors associated with HRQOL. In unadjusted analyses, TNBC participants had significantly lower FACT-B total scores (90.1±27.9) compared to non-TNBC (98.5±27.6) participants (p<0.05). For the TNBC group, multivariable analyses indicated five individual-level, and three systemic-level factors explain 80% of the response variation in HRQOL. For the non-TNBC group, seven individual-level factors and three systemic-level factors account for 76% of the variation in HRQOL scores. Compared to Black women with non-TNBC, TNBC women have worse HRQOL. There are key individual and systemic-level factors that are unique to both groups. Findings can inform future HRQOL interventions to support young Black BC survivors.

Abstract 584: Significant enhancement of efficacy of an anti-Trop-2 antibody-drug conjugate, sacituzumab govitecan (IMMU-132), in experimental triple-negative breast cancer (TNBC) when combined with microtubule or PARP inhibitors

Abstract Purpose: In current clinical trials (ClinicalTrials.gov, NCT01631552), TNBC patients treated with IMMU-132, which is composed of the active metabolite of irinotecan, SN-38, conjugated to an anti-Trop-2 antibody (drug:Ab ratio = 7.6), shows manageable toxicity and encouraging responses in relapsed/refractory cases. Preclinical studies were performed to determine the utility of combinations of IMMU-132 with either a poly(adenosine diphosphoribose) polymerase (PARP) inhibitor (olaparib) or microtubule inhibitors (paclitaxel or eribulin mesylate) in mice bearing BRCA1/2 defective (HCC1806)and wild-type (MDA-MB-468) TNBC tumor xenografts. Procedures: In vitro, human TNBC cell lines were incubated with IMMU-132 and olaparib to determine a combination index number and whether the interaction was synergistic, as well as incubating with SN-38 or IMMU-132 ± olaparib with analysis by western blot or flow cytometry (FACS) for double-stranded DNA breaks, as evidenced by increases in phosphorylated histone H2AX (p-H2AX). In vivo, mice bearing MDA-MB-468 or HCC1806 tumors were treated with either paclitaxel (qwklyx5wks) or eribulin mesylate (wks 1, 2, 4, &amp; 5) alone or in combination with IMMU-132 (wks 1, 2, 4, &amp; 5). Additionally, mice bearing TNBC tumors were treated with olaparib (qdx5d) plus IMMU-132 (qwkly) for 4 wks. Study survival endpoint was tumor progression to &amp;gt;1.0 cm3. Results: Treatment with IMMU-132 plus paclitaxel in HCC1806 or MDA-MB-468 tumor-bearing mice significantly inhibited tumor growth compared to monotherapy (P&amp;lt;0.0195 and &amp;lt;0.0328, respectively). IMMU-132 plus eribulin mesylate also resulted in significant tumor regressions when compared to all other treatments in these two disease models (P&amp;lt;0.0007 and &amp;lt;0.0432, respectively). In vitro, olaparib combined with SN-38 or IMMU-132 increased p-H2AX levels. Cytotoxicity assays revealed this interaction to be synergistic in both BRCA1/2 defective and wild-typeTNBC cell lines. In vivo, IMMU-132 plus olaparib had significant anti-tumor effects in both HCC1806 and MDA-MB-468 tumor-bearing mice when compared to single-agent responses (P&amp;lt;0.0017 and &amp;lt;0.004, respectively). In all studies, the combination of IMMU-132 with either microtubule inhibitors or olaparib was well tolerated, with no observable toxicities (e.g., weight loss). Conclusions: Combining IMMU-132 with a PARP inhibitor achieves synergistic growth inhibition in TNBC, regardless of BRCA1/2 status. The combination of IMMU-132 therapy with either microtubule or PARP inhibitors results in significant anti-tumor effects in TNBC disease models with no observable toxicity. These data provide the rationale for the clinical evaluation of IMMU-132 in combination with these chemotherapeutics in TNBC patients. Citation Format: Thomas M. Cardillo, Serengulam V. Govindan, Maria Zalath, Ali Mostafa, Roberto Arrojo, Robert M. Sharkey, David M. Goldenberg. Significant enhancement of efficacy of an anti-Trop-2 antibody-drug conjugate, sacituzumab govitecan (IMMU-132), in experimental triple-negative breast cancer (TNBC) when combined with microtubule or PARP inhibitors. [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 584.

Predictive and Prognostic Role of Tumor-Infiltrating Lymphocytes for Early Breast Cancer According to Disease Subtypes: Sensitivity Analysis of Randomized Trials in Adjuvant and Neoadjuvant Setting.

The role of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is still an issue for clinical research. Toward this end, a sensitivity analysis of neoadjuvant and adjuvant randomized clinical trials was performed according to disease subtypes. Pathological complete responses (pCRs) after neoadjuvant treatment according to the presence or absence of lymphocyte-predominant BC (LPBC) were extracted and cumulated as odds ratios (ORs) by adopting a random-effects model by subtype. Overall survival hazard ratios as a function of 10% incremental values of stromal TILs (sTILs) in adjuvant trials were extracted. The interaction test was adopted to determine the differential effect according to the subtype. Eight trials (5,514 patients) were identified. With regard to neoadjuvant setting (4 studies), a significant interaction (p < .0001) according to LPBC was found. The presence of LPBC was associated with a 29.5% increase in pCR rate compared with non-LPBC (p < .0001). The pCR rate was significantly higher in patients with LPBC in triple-negative BC (TNBC) and HER2-positive BC settings, with an absolute difference of 15.7% (95% confidence interval [CI], 4.9%-26.2%) and 33.3% (95% CI, 23.6%-42.7%), respectively. With respect to the adjuvant setting (4 studies), a significant interaction (p < .0001) according to sTILs was found. A survival benefit was more likely to be determined for HER2-positive BC (p = .025) and TNBC (p < .0001), with no statistically significant difference for estrogen receptor-positive/HER2-negative disease. Despite the retrospective nature of this analysis, the presence of TILs may represent a robust predictive and prognostic marker for BC, particularly for TNBC and HER2-positive disease.

Abstract PR09: Creating a comprehensive patient-derived xenograft (PDX) bank to represent racial disparities in triple-negative breast cancer (TNBC)

Abstract Background: Triple negative breast cancer (TNBC) is negative for estrogen receptor (ER), progesterone receptor (PR), and HER2 (human epidermal growth factor receptor 2) and known to be highly aggressive. TNBC carries poor prognosis in all ethnicities but it is especially prevalent in women of African descent, next most prevalent in African American women, and then to a lesser degree in Caucasian women. Cancer cell lines from various ethnicities have been used to study the nature of these tumors, however, PDX models more accurately reflect the characteristics of the original human tumors. To study this aggressive form of breast cancer amongst ethnicities, including up-regulated and down-regulated genes that play major roles across these tumors, we created a multi-ethnic cohort of TNBC PDXs which allows us to model TNBC disease disparities among these populations. Methods: Several trips to Ghana, Africa (Komfo Anoyke Teaching Hospital (KATH)) were made to procure tumors from African women presenting with breast cancer. We created a protocol to successfully freeze and transport tumors back to the University of Michigan, with very limited research supplies available at KATH. Once back at the University of Michigan, tumor pieces were quickly thawed, minced into small fragments and implanted orthotopically into the fat pads of NOD/SCID IL2R gamma null (NSG) mice. For African American and Caucasian PDX development, we collected tumor specimens from lumpectomies performed at the University of Michigan hospital, Baylor College of Medicine, and Van Andel Research Institute and implanted freshly minced tumor pieces into the fat pads of NSG mice. Once the tumors reached at least one centimeter in size, mice were humanely euthanized and their tumors and organs were removed. Samples of each PDX and all organs were formalin-fixed and paraffin-embedded so expression of ER, PR and HER2 receptor status could be confirmed and cancer stem cell populations assessed, and organ metastasis analyzed, by immunohistochemistry. Results: To date we have successfully established (defined by three or more passages) six African, five African American, and five Caucasian TNBC PDXs. We collected TNBC tumors from fifty-six women, of which forty-two tumors were implanted into mice and twenty four PDXs were generated (this total also includes other ethnicities in addition to the three we are looking at in this particular study). Furthermore, we created an inclusive database which indicates those PDXs that grow as tumorspheres to not only study the characteristics and cancer stem cell properties of each of these tumors and compare them based on ethnicity, but also to determine which models are best for investigational drug studies. Conclusions: We developed a method of orthotopic implantation amenable to PDX generation, even for tumor samples collected abroad, which allow us to accurately model TNBC in different ethnicities. These TNBC PDXs afford a more thorough examination of cancer stem cell properties driving aggressiveness of TNBC among different ethnicities and can inform potential drug therapies to treat TNBC and eliminate metastases. This abstract was also presented as Poster A69. Citation Format: Tahra Kaur Luther, Evelyn Jiagge, Michael T. Lewis, David Monsma, Craig Webb, Suling Liu, Hasan Korkaya, Sean McDermott, Shawn G. Clouthier, Lisa Newman, Dafydd Thomas, Max S. Wicha. Creating a comprehensive patient-derived xenograft (PDX) bank to represent racial disparities in triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr PR09.

Abstract LB-263: Enhancing of CD8+ tumor-infiltrating lymphocyte expansion from triple-negative breast cancer patients using of 41BB costimulation

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with few treatment options. Recently, it was shown that infiltration of TNBC with CD8+ tumor-infiltrating lymphocytes (TIL) is associated with improved prognosis, suggesting that T-cell responses at the tumor site can be harnessed as an autologous T-cell therapy for TNBC. Although TIL therapy has been developed for solid tumors such as melanoma, cervical, and ovarian cancer, whether TIL, especially CD8+ cells, with effector-memory properties can be expanded with reasonable yields from primary TNBC is not known. Given the high rates of relapse and refractory nature of recurrent TNBC disease, autologous TIL therapy may offer a life-saving option for these patients. Moreover, methods to facilitate CD8+ TIL expansion from TNBC are also desirable given the cytotoxic potential of these cells. One approach to address this need is to manipulate TNF-R family member signaling, such as 41BB/CD137, to provide potent costimulatory signals for CD8+ T-cell activation and division. In this study, we established a method of isolating and generating TIL from patients with primary TNBC surgical specimens and core biopsies using an agonistic 41BB/CD137 antibody. Patients and methods: Four primary human TNBC tumor samples were obtained by surgical resection or core biopsy after neoadjuvant chemotherapy. Small tumor fragments were cultured in 24-well plates in medium containing 3000 IU/ml IL-2, or 3000 IU/ml IL-2 plus 10 μg/ml added agonistic anti-41BB IgG4 (BMS663513). Viable cell numbers and the expression of CD3, CD8, CD4, CD27, CD28, CCR7, CD45RA, CD56, CD16, Granzyme B, and Perforin was determined by flow cytometry on days 7, 14, 21, 28, and 35 after culture set-up. Cytotoxic function of the TIL was evaluated by measuring Caspase 3 cleavage in target cells. Results: Although TIL were successfully expanded from the tumor fragments in 4/4 tumor samples, IL-2 alone yielded fewer cells than IL-2 together with 41BB costimulation provided early during the culture. Anti-41BB markedly increased the percentage and yield of CD8+CD3+ T cells at all times (6.34x106 vs , 202x106, P&amp;lt;0.01). However, the resulting CD8+ cells had decreased CD27 expression coincident with increased CD70 when anti-41BB was added. 41BB costimulation also increased the cytotoxic T-cell activity of the expanded TIL. The expanded TIL could also be cryopreserved and later expanded using anti-CD3 and IL-2. Conclusions: We have established a method to expand CD8+ TIL with cytotoxic activity with high yield from small samples of remaining primary tumor after neoadjuvant chemotherapy using IL-2. Provision of 41BB costimulation immediately upon tumor fragment culture initiation was critical in enhancing the rate and yield of CD8+ T cells with increased effector function; these could be cryopreserved and later thawed with high viability for secondary expansion. Our results support further development of an autologous TIL expansion protocol after neoadjuvant therapy for use in an adoptive cell therapy approach to treat TNBC recurrence or metastasis. Citation Format: Michiko Harao, Elizabeth A. Mittendorf, Gildy V. Babiera, Naoto T. Ueno, Laszlo G. Radvanyi. Enhancing of CD8+ tumor-infiltrating lymphocyte expansion from triple-negative breast cancer patients using of 41BB costimulation. [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 LB-263. doi:10.1158/1538-7445.AM2014-LB-263

Abstract 4185: Analysis of sequencing data to identify potential drug targets for an individual newly diagnosed with basal breast cancer who failed to respond to current standard neoadjuvant chemotherapy

Abstract INTRODUCTION Next generation sequencing (NGS) of patients has significantly changed our ways to study cancer genomics as it provides precise estimates of gene expression, fusion transcripts, expressed single nucleotide variants (eSNVs), splice variants and copy number variants. The Breast Cancer Genome Guided Therapy (BEAUTY) is an ongoing clinical study in which RNA sequencing (RNAseq) and whole exome sequencing (WES) are performed prior to, during and after neoadjuvant chemotherapy. Here we report the use of these sequencing technologies to investigate gene expression levels and mutational profiles in a triple negative breast cancer (TNBC) patient enrolled in BEAUTY whose disease did not respond to neoadjuvant paclitaxel and anthracycline/cycphosphamide. METHODS Computational approaches were used to integrate WES and RNAseq data obtained before therapy (V1T), after 12 weekly paclitaxel treatments (V2T) and after anthracycline-based regimen at surgical resection (V3T) to study a single patient with persistent TNBC disease after neoadjuvant chemotherapy. RESULTS Using RNA-Seq data, we identified an inter-chromosomal fusion transcript between chromosome 20 and 22 (GNAS-TTC38) that was highly expressed at V1T, V2T and V3T. We also identified intra-chromosomal fusion transcripts that were expressed at two time points, such as fusion transcript (KANSL1-ARL17A) on chromosome 17 for V1T and V2T and fusion transcript (RBM12B-LINC00535) on chromosome 8 for V2T and V3T time points. Several gene expression changes were also observed. Gene expression analysis of V1T, V2T and V3T tumors was performed. Differential temporal gene expression profiles of 9884 genes that were significant and varying at different time points were obtained for pathway analysis. Pathway analysis of 9884 genes identified up regulation and down regulation of several transcription factors with a fold change of 2x or more. When compared to blood, DNA tumor and RNA-Seq data, we identified 81 common somatic eSNVs that were expressed in both V1T and V2T time points and we are in the process of investigating V3T data. We found alterations of key transporter domains (CD225, Coatamer_beta_C, DUF2435, Dynamitin, EI24, GLTP, LMF1, Porin_3, V-ATPase_C) in our V1T and V2T SNV data. Similar to gene expression analysis, we are in the process of obtaining the list of mutations at various time points to identify driver and passenger mutation candidate genes for this specific TNBC patient. CONCLUSIONS Our initial time-series analysis of eSNV, fusion transcripts and gene expression data demonstrate that intensive analysis for individual patients is feasible. Further investigation of drug transporters and transcription regulators may help develop personalized treatment strategies for patients with disease resistant to current regimens. Citation Format: Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Douglas W. Mahoney, Poulami Barman, Jason P. Sinnwell, Hugues Sicotte, Peter Vedell, Steven N. Hart, Travis J. Dockter, Katie N. Jones, Amy L. Conners, Ann M. Moyer, Daniel W. Visscher, Jia Yu, Bowen Gao, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Jean-Pierre A. Kocher, Eric D. Wieben, Gianrico Farrugia, Cloann G. Schultz, James N. Ingle, Richard Weinshilboum, Matthew P. Goetz, Liewei Wang, Judy C. Boughey. Analysis of sequencing data to identify potential drug targets for an individual newly diagnosed with basal breast cancer who failed to respond to current standard neoadjuvant chemotherapy. [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 4185. doi:10.1158/1538-7445.AM2014-4185

Chemotherapy Use and Surgical Treatment by Receptor Subtype in Node-Negative T1a and T1b Female Breast Cancers, Iowa SEER Registry, 2010- to 2012

BackgroundPatients with small node-negative breast tumors who are younger or who have human epidermal growth factor receptor 2–positive (HER2+) or triple-negative breast cancer (TNBC) subtypes are at increased risk of recurrence. Concurrently, systemic treatment recommendations have evolved. Less is known about how frequently cytotoxic chemotherapy is given to these patients. Mastectomy rates have also increased. This study reports the recent incidence of T1a,bN0M0 breast cancer and the characteristics associated with chemotherapy delivery and the surgery selected. Patients and MethodsThis retrospective cohort is composed of invasive female breast cancers diagnosed with American Joint Committee on Cancer (AJCC) stage T1a,bN0M0 during 2010 to 2012 from the Iowa Surveillance, Epidemiology, and End Results (SEER) Cancer Registry. Chemotherapy use and surgery were identified by the registry. Univariate and multivariate analyses were performed to determine patient differences across subtype and factors associated with treatment. ResultsThe study included 1687 patients. This represented 27.6% of all AJCC stages I(a-c) to III breast cancer in 2010 to 2012, up from 18% in 1990 (P < .0001). Of 1456 patients with known subtype, 8.8% and 6.4% had HER2+ and TNBC disease, respectively. Chemotherapy was given to 7.5% of women with T1aN0M0 and 12.7% of T1bN0M0 tumors. The likelihood of systemic treatment was associated with breast cancer subtype, tumor differentiation, and age in a multivariate model. The mastectomy rate was 31.8%. ConclusionSmall node-negative breast cancers continue to grow significantly as a percentage of invasive breast cancer diagnoses. In 2010 to 2012 in Iowa, systemic chemotherapy correlated with risk factors associated with recurrence: age, subtype, and tumor differentiation. Relatively high rates of mastectomy were seen.

Phenotypic Diversity of Breast Cancer-Related Mutations in Metalloproteinase-Disintegrin ADAM12

Six different somatic missense mutations in the human ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the corresponding D299H and G477E mutants of mouse ADAM12 described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity - R612Q and L792F - occur in triple-negative breast cancers, while loss-of-function mutations - D301H, G479E, T596A, and G668A - are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of an active ADAM12-L in the triple negative breast cancer disease.

Abstract P5-12-10: Pathological characteristics and patterns of recurrence and mortality among young Mexican breast cancer patients

Abstract Background: In Mexico, it is recognized that breast cancer (BC) is diagnosed at younger ages than that reported in the US and other populations. Although national data is not available regarding BC age of diagnosis distribution, data from the INCAN, the main referral center in Mexico, showed that 17% of the total 893 newly diagnosed BC patients seen in 2012 were younger than 40 years old (which is more than twice that -7%- reported for US BC patients). We conducted a retrospective study in order to describe the frequency of BC among young Mexican BC patients, as well as their pathological characteristics at diagnosis and patters of recurrence. Methods: Clinical and pathologic data from young pts (≤42 years old) who were newly diagnosed with invasive BC between January 2007 and December 2010 at the INCAN in Mexico and were followed for at least 2 years were identified. Clinical stage at diagnosis was recorded and estrogen receptor, progesterone receptor and HER2 expression were determined by immunohistochemistry and/or FISH. Proportion differences were tested using the Chi-square test. Results: 445 young patients were identified from a total of 2919 BC patients (15%). A total of 320 patients met the inclusion criteria for analysis. Median age of diagnosis was 36 years (19-42). 49% of patients were very young patients (≤ 35 years). 19% were diagnosed as early disease, 67.5% as locally advanced BC and 13.5% as metastatic. 32% of patients had triple negative BC disease, while 27% were HER2 positive. From the non-metastatic patients at diagnosis, 31% developed recurrence (65% systemic, 21% loco regional and 14% both). After a median follow-up of 26 months, 18% of the 320 patients died secondary to BC disease progression. Conclusions: Although this series corresponds to patients treated at a reference national center, the data reported in this study suggests that BC among young women is very prevalent in Mexico compared to other populations. The causes underlying the striking difference in age at diagnosis in Mexican BC patients are unknown and have not been investigated, including the genetic contributing factors. As reported in non-Hispanic population studies, BC among young women is diagnosed in more advanced stages, and triple-negative and HER2 positive diseases are more frequent. It is associated with a high proportion of systemic and loco regional recurrence, with a dismal prognosis. Due to the substantial number of BC cases among young women in Mexico, its prospective study is more than justified. In order to meet with this goal, our group is preparing the establishment of the Mexican Young Women's Breast Cancer Cohort Program at the INCAN in Mexico and to launch the related scientific projects related to this plan. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-12-10.

Abstract P2-08-05: Functional characterization of three novel breast cancer-related somatic mutations in metalloproteinase-disintegrin ADAM12

Abstract Six different somatic missense mutations in the human ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the effect of the corresponding D229H and G477E mutations in mouse ADAM12 protein described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity - R612Q and L792F - occur in triple-negative breast cancers, while loss-of-function mutations - D301H, G479E, T596A, and G668A - are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of ADAM12-L in the triple negative breast cancer disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-08-05.

Analyzing Residual Disease in Triple-Negative Breast Cancer

Abstract Next-generation sequencing of 81 triple-negative breast cancer residual-disease tumors has shown that about 90% had an aberration in at least one gene targeted by PI3K/mTOR inhibitors, DNA-repair targeting agents, RAF/MEK inhibitors, cell-cycle inhibitors, or growth factor receptor tyrosine kinase inhibitors.

Understanding the biology of triple-negative breast cancer

Greater understanding of the biology of triple-negative breast cancer (TNBC) is needed to discern the roughly 60% of node-negative patients who are already cured with locoregional therapy from the 40% who need adjuvant systemic therapy to be cured. Recent evidence suggests that patients with TNBC whose tumours have an activated immune response gene signature have a more favourable outcome than TNBC patients without this signature. For the group who needs additional systemic therapy, the challenge remains to choose the right systemic drug combination for the right TNBC sub-type. Significant heterogeneity exists within the TNBC class that is exemplified by differing chemotherapeutic sensitivity observed for some sub-types. This heterogeneity establishes the need for identifying differentiating molecular markers within the overall class of TNBC disease, which may help refine therapeutic management. In this review, we discuss some of these promising predictive molecular markers for tailoring therapy. In addition, several gene expression profiling and functional studies employing genetic screens that help to establish TNBC sub-groups with varying sensitivities to a variety of targeted therapies currently under clinical investigation are conferred. It is anticipated that a greater understanding of the biology of TNBC and its complex heterogeneity will reveal novel targets or identify markers around which clinical trials in molecularly well-defined sub-groups can be designed.

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.

Genotypic characterization of phenotypically defined triple-negative breast cancer

500 Background: Triple negative breast cancer (TNBC) is associated with a higher risk of recurrence and earlier recurrences than other breast cancer phenotypes. We evaluated the genotypic features of TNBC compared with hormone receptor (HR)-positive disease, and also evaluated genotypic features associated with recurrence. Methods: RNA extracted from tumor samples obtained from 764 patients with stage I-III breast cancer was analyzed by RT-PCR for 371 genes. All patients received adjuvant chemotherapy (plus hormonal therapy in HR-positive disease) in trial E2197; HR and HER2 expression were evaluated by immunohistochemistry (IHC) in a central lab (J Clin Oncol 26:2473–2481). An unsupervised clustering analysis was performed in all samples (N=764). Cox proportional hazard models were used to identify differences in gene expression in TNBC versus HR-positive disease, and with recurrence in phenotypically defined (by IHC) TNBC (N=246) and HR-positive (N=465) disease. Results: Unsupervised analysis revealed two major clusters that differed with regard to HR expression by IHC. Supervised analysis comparing the TNBC vs. HR-positive phenotypes revealed 269 genes (73%) with significantly different expression (p&lt;0.0001). The top 10% of genes exhibiting higher expression the TN group included genes associated with nucleosome assembly (CENPA), kinase activity (TTK), cell division (KIFC2), proliferation (BUB1), intracellular signaling (DEPDC1), DNA repair (CHK1), anti-apoptosis (GSTP1), and transcriptional regulation (MYBL2). There was increased expression of genes for which inhibitors are currently being evaluated, including AURKB and CHK1 in TNBC, and IGF1R and RhoC in HR-positive disease. Although GRB7 expression was significantly lower in the TN group, increased expression of GRB7 was the only gene in the TNBC group (but not the HR-positive group) associated with increased recurrence (p=0.04), and did not correlate with nodal status, tumor size, or grade. Conclusions: We genotypically characterized breast cancers that have also undergone rigorous phenotypic characterization.. There were significant differences in gene expression between the TN and HR-positive groups, including genes for which targeted agents are currently being evaluated in the clinic. [Table: see text]