Erin Macrae

Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients

IntroductionCirculating tumor cells (CTCs) are commonly isolated from the blood by targeting the epithelial cell adhesion molecule (EpCAM) through positive selection. However, EpCAM can be downregulated during metastatic progression, or it can be initially not present. We designed the present prospective trial to characterize CTCs as well as other circulating cell populations in blood samples from women with metastatic breast cancer without EpCAM-dependent enrichment and/or isolation technology.MethodsA total of 32 patients with metastatic breast cancer were enrolled, and blood samples were processed using a previously described negative depletion immunomagnetic methodology. Samples from healthy volunteers were run as controls (n = 5). Multistep sequential labeling was performed to label and fix cell-surface markers followed by permeabilization for cytokeratins (CK) 8, 18 and 19. Multiparametric flow cytometry (FCM) analysis was conducted using a BD LSR II flow cytometer or a BD FACSAria II or FACSAria III cell sorter. Immunocytochemical staining on postenrichment specimens for DAPI, EpCAM, CD45, CK, epidermal growth factor receptor and vimentin was performed. Expression of these markers was visualized using confocal microscopy (CM).ResultsCD45-negative/CK-positive (CD45− CK+) populations with EpCAM + and EpCAM − expression were identified with both FCM and CM from the negatively enriched patient samples. In addition, EpCAM + and EpCAM − populations that were CK + and coexpressing the pan-hematopoietic marker CD45 were also noted. There were more CK + EpCAM − events/ml than CK + EpCAM + events/ml in both the CD45− and CD45+ fractions (both statistically significant at P ≤ 0.0005). The number of CK + CD45− and CK + CD45+ events per milliliter in blood samples (regardless of EpCAM status) was higher in patient samples than in normal control samples (P ≤ 0.0005 and P ≤ 0.026, respectively). Further, a significant fraction of the CK + CD45+ events also expressed CD68, a marker associated with tumor-associated macrophages. Higher levels of CD45-CK + EpCAM − were associated with worse overall survival (P = 0.0292).ConclusionsMetastatic breast cancer patients have atypical cells that are CK + EpCAM − circulating in their blood. Because a substantial number of these patients do not have EpCAM + CTCs, additional studies are needed to evaluate the role of EpCAM − circulating cells as a prognostic and predictive marker.

Neoadjuvant Dual HER2-Targeted Therapy With Lapatinib and Trastuzumab Improves Pathologic Complete Response in Patients With Early Stage HER2-Positive Breast Cancer: A Meta-Analysis of Randomized Prospective Clinical Trials

BACKGROUND Randomized clinical trials (RCT) that evaluated the addition of lapatinib to trastuzumab plus neoadjuvant chemotherapy (NAC) in patients with HER2-positive, operable breast cancer revealed a questionable improvement in pathologic complete response (pCR) rate. We performed a meta-analysis of prospective RCTs that examined the effect of adding lapatinib to trastuzumab and NAC on pCR rate. METHODS PubMed databases and abstracts from the proceedings of the American Society of Clinical Oncology and the San Antonio Breast Cancer Symposium were searched for RCTs that compared lapatinib plus trastuzumab and NAC with trastuzumab in combination with NAC and that included pCR as the primary outcome. Our main objective was to estimate the effect of adding lapatinib to trastuzumab plus NAC on pCR rate, defined as no residual invasive cancer in breast and axillary lymph nodes. RESULTS In total, 1,017 patients with early stage breast cancer from 5 trials were included. Four trials examined the addition of lapatinib to trastuzumab plus NAC; this resulted in statistically significant improvement in pCR, defined as no residual carcinoma in breast and lymph nodes. The pCR rate was 55.76% and 38.36% in the lapatinib plus trastuzumab and the trastuzumab plus NAC arms, respectively (odds ratio [OR]: 1.94; 95% confidence interval [CI]: 1.44-2.60). In three trials, the rates of pCR, defined as no residual invasive carcinoma in breast only, for the lapatinib plus trastuzumab and trastuzumab-alone groups were 55.01% and 40.70%, respectively, also resulting in significant improvement (OR: 1.78; 95% CI: 1.27-2.50). CONCLUSION The addition of lapatinib to trastuzumab in combination with neoadjuvant chemotherapy significantly improves pCR rates in patients with HER2-positive breast cancer.

Role of trastuzumab emtansine in the treatment of HER2-positive breast cancer.

Trastuzumab is a monoclonal antibody that is used in the treatment of breast cancer. Trastuzumab targets the human epidermal growth factor receptor 2 (HER2) receptor on breast cancer cells that express this tyrosine kinase receptor. These cancers are referred to as HER2-positive breast cancer. The original studies of trastuzumab showed improved survival in metastatic breast cancer; however, resistance often develops. In the adjuvant setting, women often progress despite therapy that includes trastuzumab. Antibody–drug conjugates are a new class of powerful drugs designed to target high-dose chemotherapy directly to the cancer cells. Trastuzumab emtansine is one of these antibody–drug conjugates and was the first Food and Drug Administration approved drug for a solid tumor. Emtansine is a potent antimicrotubule agent. Trastuzumab is used to target this potent chemotherapy agent directly to the HER2-expressing cancer cells. This review article will summarize the evidence from the preclinical studies, summarize evidence from the clinical trials, discuss current clinical trials, discuss current approval of trastuzumab emtansine, and discuss future directions of research.

Noncatalytic PTEN missense mutation predisposes to organ-selective cancer development in vivo

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.

Treatment-Related Mortality With Everolimus in Cancer Patients

INTRODUCTION The overall incidence and odds of fatal adverse events (FAEs) after exposure to everolimus are not well defined. We performed a comprehensive meta-analysis of published randomized controlled trials (RCTs) to determine the role of everolimus in treatment-related mortality in patients with cancer. METHODS PubMed databases and abstracts from the proceedings of the American Society of Clinical Oncology and the San Antonio Breast Cancer Symposium were searched for RCTs of everolimus either alone or in combination with another agent compared with the control arm without everolimus and that reported deaths from an adverse event from January 1966 to July 2013. The primary objective was to determine the difference of FAEs between everolimus-treated patients and control group patients. RESULTS In total, 2,997 patients with multiple solid tumors from nine RCTs were included. The overall incidence of FAEs in cancer patients treated with everolimus was 0.7% (95% CI 0.3%-1.1%) compared with 0.4% (95% CI 0.0%-0.7%) in cancer patients who did not receive everolimus. The odds ratio of FAEs was greater in everolimus-treated patients (Peto odds ratio = 3.80, 95% CI 1.59-9.07, p = .003). In subgroup analyses, no significant difference was found in the incidence or odds of FAEs by everolimus administration (alone or in combination) or tumor type (breast cancer vs. nonbreast cancer; p = .63). CONCLUSION In patients with cancer, everolimus is associated with a small but significant increase in the odds of a treatment-related fatal events.

JUNB promotes the survival of Flavopiridol treated human breast cancer cells

Chemotherapy resistance is a major obstacle to achieving durable progression-free-survival in breast cancer patients. Identifying resistance mechanisms is crucial to the development of effective breast cancer therapies. Immediate early genes (IEGs) function in the initial cellular reprogramming response to alterations in the extracellular environment and IEGs have been implicated in cancer cell development and progression. The purpose of this study was to investigate the influence of kinase inhibitors on IEG expression in breast cancer cells. The results demonstrated that Flavopiridol (FP), a CDK9 inhibitor, effectively reduced gene expression. FP treatment, however, consistently produced a delayed induction of JUNB gene expression in multiple breast cancer cell lines. Similar results were obtained with Sorafenib, a multi-kinase inhibitor and U0126, a MEK1 inhibitor. Functional studies revealed that JUNB plays a pro-survival role in kinase inhibitor treated breast cancer cells. These results demonstrate a unique induction of JUNB in response to kinase inhibitor therapies that may be among the earliest events in the progression to treatment resistance.

Abstract P4-09-18: Characterization of circulating myeloid derived suppressor cells and cytokines in patients undergoing neo-adjuvant chemotherapy for breast cancer

Myeloid derived suppressor cells (MDSC) are immature immune cells that expand in patients (pts) with cancer and suppress anti-tumor immunity. MDSC are also known to support angiogenesis. Higher circulating MDSC levels are seen in patients with greater tumor burden. Therefore, circulating MDSC levels could be affected by chemotherapy and could correlate with response. In this prospective pilot trial, peripheral blood (PB) levels of granulocytic (G-MDSC) and monocytic (M-MDSC) MDSC were measured in pts with operable breast cancer (BC) treated with neo-adjuvant chemotherapy (NAC) to study their association with pathologic complete response. It was hypothesized that MDSC % would show an association with complete pathologic response (pCR). The association of 10 different cytokine levels (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, TNF-α) with pCR was also explored. Linear mixed models tested the associations between MDSC % or cytokines across time points with pCR. Levels of MDSC were measured by flow cytometry as a % of PB mononuclear cells prior to cycle (C) 1 and 2 of doxorubicin and cyclophosphamide (AC) and 1st and last administration of paclitaxel (T) or T and anti-HER2 therapy (in HER2+ pts). For other regimens, MDSC were measured prior to 1st, 2nd and last cycle. MDSC were defined as HLA-DR-, CD11b+, CD33+ cells with G-MDSC and M-MDSC cells expressing CD15 and CD14, respectively. Plasma cytokine levels were measured using a multiplex assay (Bio-Rad). Of 24 enrolled pts, 1, 20 and 3 had clinical stage I, II, IIIA, respectively. Median age was 48 (range 32-70). 11, 6 and 7 pts were triple negative (TN), HER2+ and hormone receptor (HR)+, respectively. PCR rate was 45.8% (46%, 50%, 43%, 20% for TN, HER2+, HR+ and >10% HR+ pts). Rate of residual cancer burden (RCB) class 0-1 was 58.3% (63.6%, 50%, 57.1%, 40% in TN, HER2+, HR+ and >10% HR+ pts). Mean M-MDSC % were Citation Format: Wesolowski R, Duggan M, Stiff A, Trikha P, Schoenfield L, Abdel-Rasoul M, Layman R, Ramaswamy B, Macrae E, Lustberg MB, Mrozek E, Carson WE. Characterization of circulating myeloid derived suppressor cells and cytokines in patients undergoing neo-adjuvant chemotherapy for breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-18.

Abstract LB-216: NCI 9455: Phase II study of trametinib followed by trametinib plus AKT inhibitor,GSK2141795 in patients with advanced triple negative breast cancer

Background: Preclinical data demonstrates that activation of RAS/MEK/ERK pathway in basal-like breast cancer (BLBC) leads to resistance to chemotherapy. Inhibiting MEK pathway was highly effective in BLBC models with intact PTEN. Conversely activated PI3K/AKT pathway (loss of PTEN which occurs in ∼ 30% of TNBC) led to resistance to MEK inhibition. We proposed to test the hypothesis that single agent trametinib (T) will demonstrate efficacy in a subset of TNBC and that addition of AKTi, GSK2141795 (G) will overcome resistance to T. Methods: This is a single arm, multicenter study through the ETCTN. Eligible patients (pts) with advanced TNBC, measurable disease with 1-3 prior chemotherapies received mandatory research biopsy and entered Part I of the study with T alone (2 mg Q day-28 days). At the point of progression pts received a mandatory second biopsy and moved to PART II of study with 1.5 mg of T + 50 mg of G. Optional research biopsy was performed at the point of progression on the combination. Restaging scans were done every 2 cycles. Blood samples were collected at baseline, cycle 2 and at progression. Results: 33 pts with median age 55 (35-71) from six cancer centers were enrolled to Part I (T alone) and 17 pts entered part II (T +G). Most common toxicities for pts on T alone included Gr1: diarrhea, rash, transaminitis, Gr 2: fatigue; Gr 3: thromboembolism. Of 31 evaluable pts on T alone, two pts (1 still on study after 8 cycles) had a partial response (PR) and one pt has stable disease after 8 cycles and remains on study. Of the sixteen evaluable patients on part II (T+G) one patient has an unconfirmed PR (34% reduction on RECIST). Common toxicities for the combination include: Gr1: diarrhea, nausea, vomiting, myelosuppression, rash, hypertension, transaminitis, Gr2: Rash, fatigue, mucositis, diarrhea, Gr 3: diarrhea. Eleven patients died on study (10-progression, 1-adverse event). Blood and tissue samples are being analyzed for whole genome sequencing, IHC for PTEN, Quantitative targeted absolute proteomics (QTAP) for kinome assay and RPPA for PTEN, PI3KCA, AKT, ERK and MEK. This study is currently closed to accrual for interim analysis for efficacy on part II. Conclusion: Trametinib alone and in combination with AKTi, GSK2141795 has limited efficacy in TNBC. Proposed correlatives in serial biopsies may identify biomarkers in the few responders and help identify other novel targets. Citation Format: Bhuvaneswari Ramaswamy, Ewa Mrozek, Maryam Lustberg, Robert Wesolowski, Rachel Layman, Mahmoud Abdel-Rasoul, Cynthia Timmers, Robyn Patrick, Jennifer Sexton, Erin Macrae, Charles Shapiro, Thomas Budd, Lyndsay Harris, Claudine Isaacs, Roohi Ismail-Khan, Claire Dees, Andrew Poklepovic, Micheal Grever, Helen Chen, Miguel Villalona, William Carson. NCI 9455: Phase II study of trametinib followed by trametinib plus AKT inhibitor,GSK2141795 in patients with advanced triple negative breast cancer. [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 LB-216.

Mitogen-activated protein kinase signaling controls basal and oncostatin M-mediated JUNB gene expression

The mitogen-activated protein kinase (MAPK) pathway is aberrantly activated in many human cancers, including breast cancer. Activation of MAPK signaling is associated with the increased expression of a wide range of genes that promote cell survival, proliferation, and migration. This report investigated the influence of MAPK signaling on the regulation and expression of JUNB in human breast cancer cell lines. JUNB has been associated with tumor suppressor and oncogenic functions, with most reports describing JUNB as an oncogene in breast cancer. Our results indicated that JUNB expression is elevated in MCF10Amet, SKBR3, and MDA-MB-231 human breast cancer cell lines compared to nontransformed MCF10A mammary epithelial cells. Increased RAS/MAPK signaling in MCF10Amet cells correlates with the increased association of RNA polymerase II (Pol II) phosphorylated on serine 5 (Pol IIser5p) with the JUNB proximal promoter. Pol IIser5p is the “transcription initiating” form of Pol II. Treatment with U0126, a MAPK pathway inhibitor, reduces Pol IIser5p association with the JUNB proximal promoter and reduces JUNB expression. Oncostatin M (OSM) enhances MAPK and STAT3 signaling and significantly induces JUNB expression. U0126 treatment reduces OSM-induced Pol IIser5p binding to the JUNB proximal promoter and JUNB expression, but does not reduce pSTAT3 levels or the association of pSTAT3 with the JUNB proximal promoter. These results demonstrate that the MAPK pathway plays a primary role in the control of JUNB gene expression by promoting the association of Pol IIser5p with the JUNB proximal promoter.

Abstract P2-03-03: Incidence of actionable genomic alterations in metastatic breast cancer: A prospective study

Introduction: Foundation Medicine Incorporated (FMI) uses next generation sequencing (NGS) to simultaneously identify oncogenic molecular events and match targeted therapies to these alterations. We conducted a prospective study of the FMI test to describe the incidence of actionable genomic alterations found in advanced breast cancer patients. Methods: This is a prospective, single center, single-arm trial in advanced breast cancer patients designed to assess FMI testing’s feasibility and impact. Adult metastatic breast cancer patients with an estimated survival of ≥ 3 months were included, had tumor sample available for testing, and were within 10 weeks of starting their current therapy line. A massively-parallel sequencing platform (FoundationOne™) used the patient’s tumor (FFPE tissue) to sequence for 236 cancer-related genes plus 47 introns of 19 genes often rearranged in cancer to high depth (>500x). Genomic alterations were categorized as actionable if linked to an approved therapy in the solid tumor of study or another malignancy, a known or suspected contraindication to a given therapy, or a clinical trial linked to the alteration. The number of genomic alterations was quantified; genes with > 1 alteration were counted once. Results: Forty-nine patients were consented to FMI testing. Forty patients (82%) successfully completed testing; analysis was insufficient in 6 tissue samples, 2 failed DNA extraction, and 1 patient’s tissue was not received. Of the 8 unsuccessful tests, sampling from the bone (50%) and liver (38%) were the most common sites of failure. Thirty-seven reports have been received; 27 (73%) samples were taken from metastatic tissues, 10 (27%) from the primary breast tumor. A total of 192 actionable alterations were detected in tumors tested, with a median of 5 (range 1-11) alterations found per patient. Amplifications comprised 87 alterations, 105 were a base substitution, insertion/deletion or other copy number alteration. Of the 192 alterations, the TP53 gene was the most often altered [n=22 (11%)] and most frequent in ER/PR/HER2-neu negative breast tumors. Alterations in PIK3CA [n=16 (8%)] and ZNF217 [n=8 (4%)] were the 2nd and 3rd most commonly altered genes. The NGS report provided 24 of the 37 patients (65%) with a recommended FDA approved breast therapy; an additional 24 patients (65%) were suggested a non-breast FDA approved therapy. At least 1 potential treatment or clinical trial was proposed to 36 patients (97%) with everolimus (n=30) and temsirolimus (n=30) the most commonly recommended FDA approved interventions, and trametinib (n=6), regorafenib (n=6), and pazopanib (n=6) the next most commonly suggested interventions. Patients had a median of 3 (range 0-7) genomic alterations paired with a recommended clinical trial. Conclusions: This prospective study shows NGS testing in advanced breast cancer patients is successful in over 80% of patients screened. Two-thirds of patients were recommended a FDA approved therapy and nearly all patients were suggested at least one potential clinical trial. Further studies to assess barriers to patients’ access to these recommended targeted therapies are currently underway. Citation Format: Raquel E Reinbolt, Katlyn Tolliver, Mahmoud Abdel-Rasoul, Cynthia Timmers, Bhuvaneswari Ramaswamy, Rachel M Layman, Robert Wesolowski, Maryam B Lustberg, Ewa Mrozek, Susan Ottman, James Chen, Charles Shapiro, Michael C Ostrowski, Gustavo W Leone, Erin Macrae. Incidence of actionable genomic alterations in metastatic breast cancer: A prospective study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-03.