Reduction In Ki67 Expression Research Articles

Abstract 1465: Studying basal cell adaptation in the airways in response to chronic tobacco exposure

Abstract Premalignant lesions in the airways are preceded by the accumulation of cells with a high mutational load, many of which carry driver mutations. Our previous work suggests this is a dynamic process: tobacco smoking increases the size of mutated clones, which contain shorter telomeres and higher Ki67, while smoking cessation appears to be associated with the expansion of clones with near-normal mutational burden and an overall reduction in Ki-67 expression. To understand how these long-term changes take place, we have performed ex vivo functional assays to quantify the competitive advantage imparted by smoke. We have then explored the transcriptomic landscape of these cultures to determine how differential smoke exposure modulates epithelial cell dynamics before and after smoking cessation. Primary basal cells from bronchial explants were co-cultured following different regimes of exposure to cigarette smoke extract (CSE). We find that, upon acute exposure, pre-exposed cells have a ~20% higher chance of proliferating per week than smoke-naïve cells. To test this in a more physiological model, long-term epithelioid cultures were established with chronic exposure to low-dose CSE for months. As before, we observe that over time, pre-exposed cells outcompete the smoke-naïve, suggesting that smoke imparts a beneficial adaptation on re-exposed cells even at low doses. To elucidate this mechanism, we performed RNA sequencing of 48 different combinations of acute and chronic exposure to CSE. Genes were classified into distinct modules according to their regulation following exposure, re-exposure and cessation. Differential expression analysis and gene set enrichment analysis showed that metabolic pathways, including both nutrient and detoxification pathways, are predominantly enriched in these modules. In particular, following cessation, cells with the highest cumulative exposure to CSE displayed senescent-like features which are suppressed with chronic re-exposure. Further analysis and in vivo validation of these results will help elucidate how these changes affect the likelihood of premalignancy and may provide valuable targets for cancer prevention. Citation Format: David Osuna de la Peña, Zornitsa Stoichkova, Moritz Jakob Przybilla, Charlotte Percival, Sarah E. Clarke, Kate H. Gowers, Peter J. Campbell, Sam M. Janes. Studying basal cell adaptation in the airways in response to chronic tobacco exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1465.

Environmental cadmium exposure facilitates mammary tumorigenesis via reprogramming gut microbiota-mediated glutamine metabolism in MMTV-Erbb2 mice

Cadmium (Cd) is a heavy metal that has been widely reported to be linked to the onset and progression of breast cancer (BC). However, the mechanism of Cd-induced mammary tumorigenesis remains elusive. In our study, a transgenic mouse model that spontaneously develops tumors through overexpression of wild-type Erbb2 (MMTV-Erbb2) was constructed to investigate the effects of Cd exposure on BC tumorigenesis. The results showed that oral exposure to 3.6 mg/L Cd for 23 weeks dramatically accelerated tumor appearance and growth, increased Ki67 density and enhanced focal necrosis and neovascularization in the tumor tissue of MMTV-Erbb2 mice. Notably, Cd exposure enhanced glutamine (Gln) metabolism in tumor tissue, and 6-diazo-5-oxo-l-norleucine (DON), a Gln metabolism antagonist, inhibited Cd-induced breast carcinogenesis. Then our metagenomic sequencing and mass spectrometry-based metabolomics confirmed that Cd exposure disturbed gut microbiota homeostasis, especially Helicobacter and Campylobacter abundance remodeling, which altered the gut metabolic homeostasis of Gln. Moreover, intratumoral Gln metabolism profoundly increased under Cd-elevated gut permeability. Importantly, depletion of microbiota with an antibiotic cocktail (AbX) treatment led to a significant delay in the appearance of palpable tumors, inhibition of tumor growth, decrease in tumor weight, reduction in Ki67 expression and low-grade pathology in Cd-exposed MMTV-Erbb2 mice. Also, transplantation of Cd-modulated microbiota decreased tumor latency, accelerated tumor growth, increased tumor weight, upregulated Ki67 expression and exacerbated neovascularization as well as focal necrosis in MMTV-Erbb2 mice. In summary, Cd exposure induced gut microbiota dysbiosis, elevated gut permeability and increased intratumoral Gln metabolism, leading to the promotion of mammary tumorigenesis. This study provides novel insights into environmental Cd exposure-mediated carcinogenesis.

Metformin pretreatment potentiates the antiproliferative action of doxorubicin against breast cancer

The present study aimed to evaluate the effect of metformin pretreatment on the potentiation of antiproliferative action of doxorubicin against breast cancer. Female Wistar rats were administered with 7,12-Dimethylbenz(a)anthracene (DMBA) (35 mg) in 1 ml olive oil subcutaneously beneath the mammary gland. Animals were pretreated with metformin (Met) 200 mg/kg two weeks before DMBA administration. DMBA control groups received doxorubicin (Dox) (4 mg/kg and 2 mg/kg), Met (200 mg/kg) alone and in combination with Dox (4 mg/kg). Met pre-treated DMBA control groups received Dox 4 mg/kg and 2 mg/kg. Met pre-treated groups treated with Dox exhibited a decrease in tumor incidence, tumor volume and increased survival rate than the DMBA group. Organ-to-body weight ratios and histopathology of heart, liver and lungs of Met pre-treated groups treated with Dox showed lesser toxicity than Dox treated DMBA control groups. There was a noteworthy decrease in malondialdehyde levels and a substantial increase in the levels of reduced glutathione together with a significant decrease in the levels of inflammatory markers like IL-6, IL-1β and NF-κB in Met pre-treated groups treated with Dox. Histopathology of breast tumors revealed better control of tumors in Met pre-treated groups treated with Dox than DMBA control group. Immunohistochemistry and real-time PCR data revealed a significant reduction in Ki67 expression in Met pre-treated groups treated with Dox as compared to the DMBA control group. The present study suggests that metformin pretreatment potentiates the antiproliferative action of doxorubicin against breast cancer.

Low-Dose Metformin Treatment Reduces In Vitro Growth of the LL/2 Non-small Cell Lung Cancer Cell Line.

Lung cancer maintains a relatively small survival rate (~19%) over a 5-year period and up to 80-85% of all lung cancer diagnoses are Non-Small Cell Lung Cancer (NSCLC). To determine whether metformin reduces non-small cell lung cancer (NSCLC) LL/2 cell growth, cells were grown in vitro and treated with metformin for 48 h. qPCR was used to assess genes related to cell cycle regulation and pro-apoptotic markers, namely Cyclin D, CDK4, p27, p21, and HES1. Treatment with 10 mM metformin significantly reduced HES1 expression (p = 0.011). Furthermore, 10 mM metformin treatment significantly decreased REDD1 (p = 0.0082) and increased p-mTOR Ser2448 (p = 0.003) protein expression. Control cells showed significant reductions in phosphorylated p53 protein expression (p = 0.0367), whereas metformin treated cells exhibited reduced total p53 protein expression (p = 0.0078). There were no significant reductions in AMPK, PKB/AKT, or STAT3. In addition, NSCLC cells were treated for 48 h. with 10 mM metformin, 4 µM gamma-secretase inhibitor (GSI), or the combination of metformin (10 mM) and GSI (4 µM) to determine the contribution of respective signaling pathways. Metformin treatment significantly reduced total nucleus expression of the proliferation maker Ki-67 with an above 65% reduction in Ki-67 expression between control and metformin-treated cells (p = 0.0021). GSI (4 µM) treatment significantly reduced Ki-67 expression by ~20% over 48 h (p = 0.0028). Combination treatment (10 mM metformin and 4 µM GSI) significantly reduced Ki-67 expression by more than 50% over 48 h (p = 0.0245). As such, direct administration of metformin (10 mM for 48 h) proved to be an effective pharmaceutical agent in reducing the proliferation of cultured non-small cell cancer cells. These intriguing in vitro results, therefore, support the further study of metformin in appropriate in vivo models as an anti-oncogenic agent and/or an adjunctive therapy.

SNRPB promotes cervical cancer progression through repressing p53 expression

Cervical cancer is still a leading cause of tumor death in women across the world. Small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) gene encodes the components of the core spliceosomal machinery, and regulates the development of several types of cancers. However, its function in cervical cancer progression remains unclear. In the study, we found that SNRPB was highly expressed in human cervical cancer tissues and in cervical cancer cell lines. Meanwhile, SNRPB knockdown using shRNA in cervical cancer cells markedly reduced the cell proliferation, migration and invasion. Furthermore, the increased percentage of cells in G2/M phase and apoptotic cell death was detected in cervical cancer cells with SNRPB knockdown, suggesting that SNRPB might contribute to cervical cancer growth. Moreover, we found that SNRPB could directly interact with p53, and the interaction showed an essential role in modulating cervical cancer cell proliferation, migration, invasion and apoptosis. In xenograft model, the knockdown of SNRPB exerted effectively anti-cervical cancer ability characterized by the reduced tumor volume and weight, and a remarkable reduction in KI-67 expression. Improved expression of p53 validated the in vitro findings. Therefore, SNRPB might be a potent therapeutic target in cervical cancer through interacting with p53.

Abstract DP-008: SILICA GEL ENCAPSULATION AS A POTENTIAL TOOL TO IDENTIFY DORMANT AND DRUG-RESISTANT CELL POPULATIONS IN OVARIAN CANCER

Abstract Despite advances in standard of care treatment, a significant number of ovarian cancer patients will exhibit disease recurrence, either due to the presence of chemoresistant cells or the reawakening of dormant tumor cells. Currently there are no methods to predict which patients are likely to recur, and the mechanisms that allow a unique subpopulation of cancer cells to enter dormancy and survive chemotherapy remain unclear. Since chemotherapy is more effective at destroying highly proliferative cells, it is theorized that tumor cells capable of entering a dormant state are also the same population able to evade initial treatment and ultimately engender chemoresistance. A silica gel encapsulation platform that uses physical confinement to inhibit proliferation has previously demonstrated the ability to distinguish between dormancy-capable and dormancy-incapable breast cancer cell lines. In this work, we sought to apply this platform to an ovarian cancer cell line and determine if it could be used to select for the dormancy-capable subset of cells and to detect differences upon enrichment of dormant or chemoresistant cell populations. Upon encapsulation in silica gels, surviving OVCAR-3 cells demonstrated hallmarks of dormancy, including reduction in Ki67 expression, modulation of key cell cycle regulatory genes and dormancy-associated signaling pathways, and ability to resume proliferation when the stress of physical confinement was removed. Moreover, enrichment of the dormant subpopulation using hypoxia treatment or serum starvation prior to silica gel encapsulation resulted in enhanced survival in gels relative to cells grown in standard culture conditions. These results suggest that silica gel encapsulation may be used as a simple method to isolate dormancy-capable cells for further applications such as gene or protein expression analysis and drug screening. Similarly, cells already in a dormant state demonstrated lower susceptibility to cisplatin drug treatment relative to actively proliferating cells, further supporting the idea that dormant cancer cells are less susceptible to chemotherapy and that chemoresistance may stem from a subpopulation of dormant cancer cells. After extraction from silica gels and subsequent return to a proliferative state, cells surviving prolonged encapsulation also demonstrated enhanced survival after cisplatin treatment relative to control populations. This suggests that the dormancy-capable cells selected for within silica gels are indeed more chemoresistant, even when no longer dormant, and that the ability to enter dormancy and chemoresistance in ovarian cancer are likely somehow connected. Lastly, when the drug-resistant phenotype was selected for through cisplatin drug treatment prior to encapsulation, a significant difference in viability was observed upon encapsulation between cisplatin-treated and untreated cells. Overall, these results indicate that the silica gel technology and the behavior of ovarian cancer cells after encapsulation may be an indicator of cells inherently capable of entering dormancy and less susceptible to platinum-based chemotherapies. In future applications, silica gel encapsulation of patient ovarian cancer cells could potentially be used as a predictive clinical tool to identify patients at risk of early recurrence or as a facile in vitro platform to investigate mechanistic links between dormancy, chemoresistance, and recurrence. Citation Format: Tiffany Lam, Hak Rae Lee, Melissa A. Geller, Alptekin Aksan, Samira Azarin. SILICA GEL ENCAPSULATION AS A POTENTIAL TOOL TO IDENTIFY DORMANT AND DRUG-RESISTANT CELL POPULATIONS IN OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-008.

Mesenchymal Stem Cells Gene Signature in High-Risk Myeloma Bone Marrow Linked to Suppression of Distinct IGFBP2-Expressing Small Adipocytes

Introduction:Certain studies suggest that multiple myeloma (MM) induces expansion of bone marrow (BM) mesenchymal stem cells (MSCs), but others showed induction of MSC senescence. MM cells suppress MSC lineages such as osteoblasts, while their effects on adipocytes remain to be elucidated. Recent studies identified “regulated” adipocytes that are smaller in size than large adipocytes that are constitutively present (Scheller et al, Nature Commun 2015), and that function as an endocrine tissue and regulators of hematopoiesis (Cawthorn et al, Cell Metab 2014), suggesting that BM adipocytes are functionally heterogeneous.We established a MSC gene signature in whole bone biopsies and showed that it gradually changes in different disease stages and is associated with outcome (Schinke et al, CCR 2018).The aim of the study was to identify changes in expression of MSC genes in BM of patients with high risk MM and in focal lesions (FLs), and to elucidate whether these changes reflect altered proportion and function of MSCs and their lineages.Methods:MSC gene expression in whole bone biopsies from normal donors (n=68), and patients with MGUS/SMM (n=90) and MM (n=531) was analyzed using global gene expression profiles. Unexpanded single MSCs from normal donors (n=3; 175 single MSCs) and MM patients (n=3; 162 single MSCs) were sorted by FACSAria and expression of mesenchymal cell, proliferation and senescence markers were analyzed by qRT-PCR using Fluidigm Biomark HD. Functionally, single MSCs were tested for their ability to multiply using supportive serum and MSC-conditioned media. Cell senescence was analyzed by SA-βGal staining. IGFBP2 and adiponectin protein were detected using immunohistochemistry (IHC). Numbers of IGFBP2+ cells were analyzed in biopsies from patients with MGUS/SMM, low risk (LR) and high risk (HR) MM (10 biopsies/group). Cultured MSCs were differentiated to adipocytes by treatment with dexamethasone, insulin and indomethacin for 3-4 weeks. The effects of recombinant IGF1 and IGFBP2 on MM cell growth were performed on BM-dependent MM lines (n=3) cultured in serum-free conditions for 48 hrs.Results:We compared MSC gene expression levels in random interstitial BM biopsies of patients with LR and HR MM, and in paired biopsy samples from random BM and FLs; 41 of the 345 MSC genes were differentially expressed in both comparisons. Most overexpressed genes were related to angiogenesis and ECM, including several collagen genes (e.g., COL4A1, POSTN, and HSPG2). Several underexpressed genes were associated with adipocytes, including IGFBP2 and aldo/keto reductases.To unravel whether these differences in gene expression reflect changes in the proportion of MSCs we tested expression of genes associated with MSCs, osteoblastogenesis, adipogenesis, proliferation and senescence in unexpanded singleMSCs. MM single MSCs had significant reduction in expression of KI67 and increased expression of the senescence marker, CDKN2A/p16, whereas expression of other tested genes were modestly differentially expressed between the two groups. Functionally, unexpanded MM MSCs had increased SA-βGal expression, and only 65±8% of single MSCs divided at least once compared to 90±4% of their normal donor counterparts (p<0.03), suggesting that the in vivo MSC gene expression in MM reflects modifications in mesenchymal cell lineages that encompass most of the mesenchymal compartment in bone.Since IGFBP2 is involved in adipogenesis and bone homeostasis we traced the source of this factor using IHC. Double staining for IGFBP2 and adiponectin revealed that IGFBP2 was mainly expressed by small adipocytes. The number of IGFBP2+ cells was higher in BM biopsies from patients with MGUS/SMM than in those from patients with MM (p<0.02), and the number was lower in biopsies from patients with HR MM than with LR MM (p<0.01).Normal MSCs differentiated to adipocytes produced a high level of IGFBP2, while co-culturing MSCs with MM cells inhibits their differentiation to adipocytes by 6 folds (p<0.001) and reduced expression of IGFBP2 and adiponectin. Recombinant IGFBP2 effectively blocked IGF1-mediated growth of MM cells, indicating its role in controlling IGF1 bioavailability.Conclusions:Our data demonstrate that MM MSCs are less proliferative and that IGFBP2+ small adipocytes are a distinct mesenchymal cell population suppressed by MM, and their depletion may contribute to disease progression. DisclosuresEpstein:University of Arkansas for Medical Sciences: Employment. Davies:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; TRM Oncology: Honoraria. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

Differences in Ki67 expressions between pre- and post-neoadjuvant chemotherapy specimens might predict early recurrence of breast cancer

The prognosis of breast cancer patients not obtaining a pathological complete response (pCR) with neoadjuvant chemotherapy (NAC) is poorer than that of pCR patients. Identifying new prognostic factors for non-pCR patients is important because fractions of this population might benefit from novel adjuvant treatments currently under development. High Ki67 expression in remnant disease after NAC has been described as a poor prognostic factor. Studies have shown that a reduction in Ki67 expression is more often observed in good responders to chemotherapy. We hypothesized that the change in Ki67 expression might be useful for predicting patient outcomes and thus retrospectively examined pairs of biopsy and surgical specimens of breast tissue from individual patients. One hundred sixteen patients with remnant invasive disease in the breast, who received NAC and underwent surgery at our institution, were retrospectively examined. Differences in Ki67 expression between pre- and post-NAC specimens were analyzed in relation to patient outcomes. The mean Ki67 expression value after NAC was higher in patients who developed metastasis than in those without metastasis (P<.01). Tumors showing higher Ki67 expression in the surgical than in the biopsy specimen were more frequent in patients with metastasis (P<.01). This trend was more obvious in patients who developed metastasis within 1 year after surgery. Our results indicate that a difference in Ki67 expressions after versus before NAC might be an important predictor of early metastasis. Evaluating not only absolute Ki67 values, but also any changes in response to NAC, may improve the prediction of patient outcomes.

Downregulation of CXCR7 inhibits proliferative capacity and stem cell-like properties in breast cancer stem cells.

Breast cancer stem cells (bCSCs) are considered an obstacle in breast cancer therapy because they exhibit long-term proliferative potential, phenotypic plasticity and high resistance to the current therapeutics. CXC chemokine receptor type 7 (CXCR7), which provides a growth advantage to breast cancer cells, has recently been demonstrated to play an important role in the maintenance of stem cell-like properties in the CSCs of glioblastoma and lung cancer, yet its role in bCSCs remains elusive. In this study, CD44+/CD24low bCSC-enriched cells (bCSCs for short) were isolated from MCF-7 cells, and CXCR7 was stably knocked down in bCSCs via lentivirus-mediated transduction with CXCR7 short hairpin RNA (shRNA). Knockdown of CXCR7 in bCSCs decreased the proportion of CD44+/CD24low cells, and markedly reduced the clonogenicity of the cells. Moreover, silencing of CXCR7 downregulated the expression of stem cell markers, such as aldehyde dehydrogenase 1 (ALDH1), Oct4, and Nanog. In addition, CXCR7 silencing in bCSCs suppressed cell proliferation and G1/S transition in vitro, and delayed tumor growth in vivo in a xenograft mouse model. In situ immunohistochemical analysis revealed a reduction in Ki-67 expression and enhanced apoptosis in the xenograft tumors as a result of CXCR7 silencing. Furthermore, combined treatment with CXCR7 silencing and epirubicin displayed an outstanding anti-tumor effect compared with either single treatment. Our study demonstrates that CXCR7 plays a critical role in the maintenance of stem cell-like properties and promotion of growth in bCSCs, and suggests that CXCR7 may be a candidate target for bCSCs in breast cancer therapy.

Young Barley Indicates Antitumor Effects in Experimental Breast Cancer In Vivo and In Vitro

ABSTRACTThe effect of dietary administered young barley containing a mixture of phytochemicals to female rats for the prevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis was evaluated. After carcinogen administration (14 wk), mammary tumors were removed and prepared for histopathological and immunohistochemical analysis. Moreover, in vitro evaluation of possible mechanisms in MCF-7 breast cancer cell line was performed. Barley (0.3%) demonstrated mild antitumor effect in mammary carcinogenesis, yet 3% barley did not further improve this effect. Immunohistochemical analysis of rat tumor cells in treated groups showed significant increase in caspase-3 expression and significant reduction in Ki67 expression. In addition, 3% barley significantly decreased dityrosine levels versus control. Barley in higher dose significantly decreased serum low-density lipoprotein-cholesterol in rats. In vitro studies showed that barley significantly decreased survival of MCF-7 cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and significantly decreased 5-bromo-20-deoxyuridine incorporation versus control. Barley prevented cell cycle progression and extended incubation with barley showed significant increase in the percentage of annexin V/propidium iodide-positive MCF-7 cells. Our results propose an antitumor effect for the mixture of phytochemicals present in young barley in a breast cancer model.

Haem oxygenase 1 expression is associated with prognosis in cholangiocarcinoma patients and with drug sensitivity in xenografted mice.

Haem oxygenase-1 (HO-1) plays important roles in cytoprotection and tumour growth. Cholangiocarcinoma (CCA) is a deadly malignancy with very poor prognosis. The role of HO-1 in tumour progression in CCA up to now has been relatively unexplored, thus, its possible therapeutic implications in CCA have been investigated here. HO-1 expression in tumour tissues from 50 CCA patients was determined by immunohistochemical analysis and its association with survival time was evaluated using the Kaplan-Meier method. Its role in CCA cells in vitro was evaluated by transwell and wound healing assays and suppression of HO-1 expression by siRNA. Effects of HO-1 inhibition on gemicitabine (GEM)-mediated tumour suppression was evaluated in nude mice xenografted with CCA cells. HO-1 expression was inversely associated with median overall survival time. Hazard ratio of patients with high HO-1 expression was 2.42 (95% CI: 1.16-5.08) with reference to low expression and HO-1 knock-down expression inhibited transwell cell migration. Suppression of HO-1 by Zn-protoporphyrin (ZnPP) enhanced cytotoxicity to GEM in CCA cells, validated in CCA xenografts. Treatment with GEM and ZnPP almost completely arrested tumour growth, whereas treatment with only a single reagent, retarded it. Tumour inhibition was associated with reduction in expression of Ki-67 and microvascular density, and enhanced p53 and p21 immunohistochemical staining. High HO-1 expression was associated with poor prognosis of CCA. Synergistic role of HO-1 inhibition in chemotherapy of CCA is a promising insight for treatment of this tumour and warrants further investigation.

Combination of letrozole, metronomic cyclophosphamide and sorafenib is well-tolerated and shows activity in patients with primary breast cancer.

Purpose:To assess whether the combination of letrozole, metronomic cyclophosphamide and sorafenib (LCS) is well tolerated and shows activity in primary breast cancer (BC).Methods:Thirteen oestrogen receptor-positive, postmenopausal, T2-4, N0-1 BC patients received the LCS combination for 6 months. In these patients we examined the pharmacokinetics of sorafenib and cyclophosphamide, toxicity of the regimen, the clinical response to therapy and changes in the levels of biologically relevant biomarkers.Results:Adequate plasma concentrations of sorafenib were achieved in patients when it was dosed in combination with L+C. The mean plasma concentrations of C were consistently lower following administration of LCS, compared with administration of L+C only. The most common drug-related grade 3/4 adverse events were skin rash (69.3%), hand-foot skin reaction (69.3%) and diarrhoea (46.1%). According to RECIST Criteria, a clinical complete response was observed in 6 of 13 patients. A significant reduction in tumour size, evaluated with MRI, was also observed between baseline and 14 days of treatment in all 13 patients (P=0.005). A significant reduction in SUV uptake, measured by 18FDG-PET/CT, was observed in all patients between baseline and 30 days of treatment (P=0.015) and between baseline and definitive surgery (P=0.0002). Using modified CT Criteria, a response was demonstrated in 8 out of 10 evaluable patients at 30 days and in 11 out of 13 evaluable patients at the definitive surgery. A significant reduction in Ki67 expression was observed in all patients at day 14 compared with baseline (P<0.00001) and in 9 out of 13 patients at the definitive surgery compared with baseline (P<0.03). There was also a significant suppression of CD31 and VEGF-A expression in response to treatment (P=0.01 and P=0.007, respectively).Conclusions:The LCS combination is feasible and tolerable. The tumour response and target biomarker modulation indicate that the combination is clinically and biologically active.

Abstract OT3-2-02: PreOperative window of endocrine therapy provides information to increase compliance: POWER PIINC: A feasibility study

Abstract Background: The addition of systemic therapy to the surgical treatment of breast cancer has improved survival of patients. A mainstay of systemic therapy in the setting of hormone receptor positive breast cancer is endocrine therapy. Despite the known advantages, the compliance with short and long term systemic endocrine therapy is less than ideal. Several studies report significant rates of non-compliance with endocrine therapy, either in patients never starting the medication or not completing the recommended multi-year course. It is estimated that as many as 10% of patients per year discontinue their therapy. We wondered why women would forgo such an important part of their treatment and how we might intervene to improve compliance. Using information gleaned from POWER PIINC, we hope to design a behavioral intervention study to see if the patient knowing her tumor's preoperative response to endocrine therapy will improve long term compliance. In order to do such a study we would need to define the minimal length of preoperative endocrine therapy needed to detect measurable changes in the tumor. After 14 days of endocrine therapy, significant decreases in Ki67 can be seen in most hormone sensitive breast cancers. In fact two current studies are using this information (POETIC and ADAPT trials). Both of these trials are being conducted outside of the U.S. where operative therapy does not typically occur within a week or two of seeing the surgeon. POWER PIINC is a feasibility study to determine if we can detect changes in Ki67 with only 7 days of therapy. The results of POWER PIINC will inform the behavioral intervention trial. Trial Design: This is a prospective, single-arm feasibility study. Participants take tamoxifen for 7 days prior to surgery. Ki67 is measured pre (core biopsy) and post (surgical specimen) tamoxifen. Eligibility critieria: Non-pregnant women age 18 or older with a hormone positive (&amp;gt;1% ER or PR) clinical Stage 1 or 2 breast cancer who are candidates for surgical therapy of their breast cancer. No concurrent CYP2D6 inhibitors or other contraindications to tamoxifen. Specific Aims: Primary Objective: Demonstrate a significant reduction in Ki67 expression in tumors with 7 days of pre-surgical tamoxifen. Secondary Objectives: - Evaluate symptom patterns from baseline through 18 months of follow-up (presence, severity, and bother) -Evaluate 18-month endocrine therapy adherence -Evaluate change in attitude regarding endocrine therapy -Evaluate correlation between changes in Ki67 expression and symptom scores -Evaluate additional changes in proliferative markers (subset of PAM 50) Statistical Methods: A one-sample t-test will be applied to the log-ratio of Ki67 at resection to pre therapy. If this ratio is not normally distributed we will use a non-parametric Wilcoxon test. Secondary objectives will be analyzed using a variety of appropriate statistical tests. Accrual: Target accrual is 52 patients over 18 months. We opened this study in August of 2012. To date (9 months) we have accrued 23 patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT3-2-02.

Abstract PD07-03: INCREASED PATHOLOGIC COMPLETE RESPONSE RATE AFTER A LONG TERM NEOADJUVANT LETROZOLE TREATMENT IN POSTMENOPAUSAL ESTROGEN AND/OR PROGESTERONE RECEPTOR-POSITIVE BREAST CANCER

Abstract Background: Neoadjuvant endocrine therapy has been increasingly employed in clinical practice to improve surgical options for postmenopausal women with bulky hormone receptor-positive breast cancer. Recent studies indicate that tumor response and in particular the pathologic complete response (CR) in this setting may predict long-term outcome. The letrozole approval in neoadjuvant setting is based on a study that included 4 months of letrozole treatment only. Prospective studies have not investigated treatment duration beyond 6 months, and retrospective studies suggest that useful responses can occur after this period. The purpose of this study was to determine the clinical and pathologic response of 2.5 mg daily letrozole used with different schedule in breast cancer patients (pts) unfit for chemotherapy. Patients and Methods: This single Institution retrospective study comprised 102 postmenopausal women with primary breast cancer (clinical stage ≥T2, N0-1), who were divided into 3 subgroups (34 each in single subgroup) according to the different duration of neoadjuvant letrozole treatment: 4 months (subgroup A), 8 months (subgroup B) or 12 months (subgroup C) of treatment. The subgroups were comparable in terms of baseline characteristics. Results: Pts and tumor characteristics included: median age 77.3 years (range 53.9–95.4); 100% estrogen receptor-positive, 81.4% progesterone receptor-positive; median Ki67 13.5% (range 2–90%). All pts were evaluable for clinical and pathologic response. A total of 77 pts (75.5%) achieved an objective response at individual end: 40 (39.2%) clinical CR and 37 (36.3%) partial responses (PR). The clinical CR were significantly confined to subgroups C (20/34 pts, 58.8%) and B (16/34 pts, 47.1%) than to subgroup A (4/34 pts, 11.8%) (p for trend = 0.00001). Objective response rate was 46.1% (47/102 pts) at month 4, 83.8% (57/68 pts) at month 8, and 94.1% (32/34 pts) at month 12 compared with baseline. As expected, letrozole induced a significant reduction of Ki67 expression after treatment in any single subgroup, without significant differences between them. With very interest, the pathologic CR rate was significantly higher in subgroup C (7/34 pts, 20.6%) than in subgroups A (1/34 pt, 2.9%) and B (1/34 pt, 2.9%) (p for trend &amp;lt;0.02). Conclusions: One-year neoadjuvant letrozole therapy can lead to a high pathologic CR rate. This observation suggests that pathologic CR may be a reliable endpoint after endocrine therapy, but a long-term drug exposure is needed. The optimal letrozole treatment duration in neoadjuvant setting is a matter of future research. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD07-03.

Safety and activity of nonpegylated liposomal doxorubicin (nPLD) combined with oral metronomic cyclophosphamide (mC) as preoperative treatment for locally advanced breast cancer (BC) patients (pts).

e11546 Background: nPLD has the same clinical benefits but less toxicity than the traditional doxorubicin. It’s well reported mC produces anti-proliferative and anti-angiogenic effect in tumours. We conducted a retrospective study to evaluate the safety and activity of the association of nPLD and mC in pts with locally advanced BC. Methods: A total of 41 female pts with HER2-negative locally advanced BC unfit for standard chemotherapy received nPLD 25 mg/mq biweekly for eight courses along with mC 50 mg daily as primary systemic treatment. Toxicity and clinical-radiological response with Ki67 and CD31 expression were evaluated in tumour specimens obtained before and after treatment. Results: Pts characteristics included: median age 47.5 years; 31.7% Luminal A (LA), 56.1% Luminal B (LB), and 12.2% Triple Negative BC subtype classified by immunohistochemistry. The rate of breast-conserving surgery was 59.5%. All pts were evaluable for response. Objective response rate (ORR) was 58.5% (95% confidence interval, 42.1% to 73.7%), with 4 (9.7%) clinical complete responses (CR) and 20 (48.8%) partial responses. Sixteen pts (39.0%) achieved a stable disease and 1 pt progressed. One pt experienced a pathological CR. LA subtype was significantly associated with better ORR than others subtypes (p=0.049) and especially than LB (p=0.040). There was a borderline significant reduction of Ki67 expression after treatment (median, 15.5% to 9%; p=0.056), but no significant variation in CD31 expression (p=0.865). The majority of adverse events (AEs) were mild or moderate. Grade ≥3 AEs included neutropenia (12.2%), vomiting (4.9%) and hyperglycemia (2.44%). Temporary treatment suspension due to toxicity occurred in 8 (19.5%) pts; discontinuation was necessary in 1 pt. No symptoms were related to impairment of cardiac function. Mean left ventricular ejection fraction levels did not change substantially before and after treatment: 58.7% and 57.1%, respectively. Conclusions: The combination of nPLD and mC is well tolerated, with a promising activity as preoperative therapy in unfit pts with locally advanced BC.

P4-16-03: Proof of the Anti-Tumour Effect of Zoledronic Acid (ZA) in Naive Bone-Only Metastatic and Locally Advanced Breast Cancer: Results from the Biological Window Therapy.

Abstract Background: Pre-clinical studies have demonstrated an anti-tumour activity of ZA in several cancers. However, the clinical evidence of the ZA anti-tumour effect is still uncertain. Our study aimed to prove that ZA has anti-tumour activity administered alone as a biological window of 14 days therapy on naïve bone-only metastatic and locally advanced breast cancer. Material and Methods: 27 patients with locally advanced breast cancer (Group 1) and 12 patients at their first relapse with bone metastasis only (Group 2) received 4 mg single dose of ZA before starting with any treatment. In Group 1, Ki67 expression was evaluated in tumour specimens obtained before and after ZA administration (basal, day 14). In Group 2, circulating tumour cells (CTCs) were evaluated at baseline, 48 hrs and day 14. In Group 1 and 2, the apoptosis and necrosis of tumour cells were tested on blood samples by M30 and M65 ELISA assay respectively. Parallel, the anti-antitumor activity of ZA, given at the dose of 250 mg/mouse every 4 days for 6 cycles, in MDA-Mb-231 bearing mice was evaluated. Results: A significant reduction of Ki67 expression after 14 days (mean, 25.33% to 21.78%; p=0.035) was observed in Group 1. In Group 2, we detected a significant reduction of CTC number after 48 hrs (median, 16 to 7; p=0.045), followed by a tendency to increase on day 14 (p=0.093). The M30/M65 analysis performed on paired blood samples in Group 2 showed after 14 days a significant increase in M65 (median, 97 to 112 U/L; p=0.018), more accentuated than the concomitant increase in M30 (p=0.308). In MDA-MB-231 bearing mice, ZA treatment caused a significant inhibition of tumor growth. Conclusions: These results are the first prospective clinical data showing a direct anti-tumour effect (either on the tumour or on CTCs) of ZA, confirming the in vitro data. By directly affecting the proliferation, ZA could have an anti-tumour effect via induction of necrosis, suggesting its possible use in combination with conventional oncologic treatments of breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-16-03.

Abstract 4788: Modulation of T lymphocytes by tumor-released survivin

Abstract Introduction: The role of tumor infiltrating lymphocytes (TILs) in the progression of cancer has become an important area of research. Recent studies have shown that T lymphocytes within the tumor microenvironment have decreased activation and proliferation. Our lab has shown survivin is released from tumor cells within exosomes, and is capable of altering the phenotype of malignant cells. We hypothesize that this exosomally-released survivin prevent TIL activation and proliferation in T cell subsets and that Th2 type cytokines will be released in higher levels than Th1 cytokines. Methods: HeLa-S-WT cells that produce Flag-HA tagged survivin were growth in a bioreactor and survivin isolated from the media using Flag-HA tandem affinity purification. T lymphocytes from normal donors were cultured in control or survivin-containing media and activated with anti-CD28-anti-CD3 microbeads (32 ul/ml). Flow cytometric analysis of activation marker CD69 and the proliferation marker Ki-67 were performed at 24h and 6 days, respectively. Cytokine release was evaluated using a Cytokine Bead Array (BD Bioscience) for IFN-gamma, IL-2, IL-4 and IL-10. Results: CD69 expression was decreased significantly in CD8+ T cells after incubation with survivin (Control = 72 MFU, Treated = 57 MFU; p&amp;lt;0.05) while no significant change was seen in CD4+ T cells (Control = 65 MFU, Treated = 68 MFU). CD8+ T cells showed a significant reduction in Ki-67 expression after incubation with survivin when compared to controls (Control = 218 MFU, Treated = 139 MFU; p &amp;lt; 0.001), while CD4+ T cells showed little difference between control and treated groups (Control = 74 MFU, Treated = 81 MFU). Cells treated with survivin in activating conditions released higher amounts of IL-4 (Control = 4.8 pg/ml, Treated = 28.3 pg/ml) and decreased amounts of IFN-gamma (Control = 32.4 pg/ml, Treated = 16.9 pg/ml) compared to control activating conditions. IL-2 and IL-10 showed little difference between treatment groups. Conclusions: This data suggests that survivin in the tumor microenvironment is capable of negatively affecting cytotoxic T cell activation and proliferation. While survivin treatment does not affect the activation or proliferation of CD4+ subsets, the Th1: Th2 cytokine balance is altered when survivin is present. Thus, survivin in the tumor microenvironment could play a role in the suppression of immune surveillance by inhibiting the cytotoxic T cell response and preventing tumor death by increasing levels of Th2 cytokines and decreasing levels of Th1 cytokines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4788.

Abstract A11: Phase II trial of letrozole (L), cyclophosphamide (C) and sorafenib (S) as neoadjuvant therapy in breast cancer patients (BC): Pharmacokinetic (PK) and pharmacodinamic (PD) analysis

Abstract Introduction: This trial assessed the PK and PD of letrozole-cyclophosphamide-sorafenib combination as neoadjuvant setting in patients (pts) with operable breast cancer (BC). Methods: 13 estrogen receptor-positive postmenopausal BC pts with T2-4 N0-1 were enrolled to receive oral letrozole (L) 2.5 mg daily, 50 mg daily metronomic cyclophosphamide (C) and 400 mg twice daily sorafenib (S). S was introduced after 6 days of LC therapy. For PK analysis blood samples were collected on day 5 of cycle 1 and on day 1 of cycle 2 (day 29). At basal time and after 14 days, tumour size evaluation, Ki67 expression and MRI were performed. Results: A significant reduction in Ki67 expression was observed in all the 13 patients after 14 days only (median 20.7% (range 3-36%) and 9.75% (range 0-20%) at 15th respectively, p&amp;lt;0.0001) with a concomitant reduction in tumour size detected by MRI (median 37 mm (range 15-90 mm) and 31 mm (range 13.4-90 mm) respectively, p=0.047). Three patients attained a partial response at MRI (RECIST criteria) after 15 days. The changes in ki67 expression did not correlate with changes in tumor size evaluated by MRI (Spearman r: −0.37). 10 patients out of 13 showed a clinical complete response at the end of the treatment. The most common drug-related grade 2 (G2) adverse events were skin rush (46%), hand-foot syndrome (46%), anorexia (38%), fatigue (15%), and G3 diarrhea (23%). Mean plasma concentrations of C were consistently lower following concomitant administrations of L, S and C compared to administration of L and C. This trend was confirmed by the comparison of corresponding AUC (0-24) and Cmax values from Day 5, Cycle 1 and Day 1 of Cycle 2. No influence was detected by co-administration with L. Conclusions: These toxicity and PK results indicated that the combination feasible but sorafenib interpheres in non significant manner with the bioavailability of C. Moreover, the PD data indicated that the association of the already tested LC combination (Bottini at al, JCO 2006) with S is promising. A randomised study of LC vs LCS is ongoing. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A11

Evaluation of Ki-67 measured in benign breast tissue acquired from premenopausal women treated with a flaxseed derivative

1507 Background: The lignans enterolactone and enterodiol are derived from the action of gut bacteria on ingested secoisolariciresinol diglycoside (SDG) which is commonly found in flaxseed. Enterolactone and enterodiol are thought to impair mammary carcinogenesis via reduction in aromatase activity and the mid-cycle surge of luteinizing hormone. We assessed the modulatory activity of 1 year of SDG on a number of risk biomarkers for breast cancer in a prospective phase II pilot study. We report the effect of SDG on the primary endpoint, proliferation in benign breast tissue as measured by Ki-67 immunocytochemistry, in the first 35 women completing study. Methods: Premenopausal women age 21 to 55 at increased risk for breast cancer underwent a baseline random periareolar fine needle aspiration (RPFNA) between the first and tenth days of their menstrual cycle. Those with RPFNA evidence of hyperplasia and Ki-67 greater than or equal to 2% were invited to participate. Women taking flaxseed or oral contraceptives were ineligible. All women took one Brevail (lignan research) capsule containing 50 mg of SDG daily. Ki-67 staining was performed with DAKO M7240 antibody on hematoxylin counterstained slides and the number of cells staining positive in 500 cells within hyperplastic clusters was counted. Results: Forty-nine women were enrolled on study between February 2006 and June 2008. Of these, four stopped prematurely, 10 women have not completed, and 35 have completed study and undergone follow-up RPFNA. Baseline characteristics of the 35 women completing study are as follows: median age 44 (range 29–50), median baseline 5-year Gail model risk 1.6% (range 0.1%-5.7%), median Ki-67 4.2% (range 2.0%-16.8%). Thirty seven percent had hyperplasia without atypia, and 63% had atypia. At repeat RPFNA, Ki-67 expression was reduced (median value of 2.0%, range 0%-15.2%); with 29 of the 35 women demonstrating a decrease (median relative reduction of 0.70). Conclusions: Based upon reduction in Ki-67 expression in hyperplastic benign breast tissue after 12 months, 50 mg of SDG administered daily as Brevail appears promising as a preventive. Supported in part by grant R21 CA117847 from the National Cancer Institute. No significant financial relationships to disclose.

Phase II Randomized Study of Neoadjuvant Everolimus Plus Letrozole Compared With Placebo Plus Letrozole in Patients With Estrogen Receptor–Positive Breast Cancer

Cross-talk between the estrogen receptor (ER) and the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways is a mechanism of resistance to endocrine therapy, and blockade of both pathways enhances antitumor activity in preclinical models. This study explored whether sensitivity to letrozole was enhanced with the oral mTOR inhibitor, everolimus (RAD001). Two hundred seventy postmenopausal women with operable ER-positive breast cancer were randomly assigned to receive 4 months of neoadjuvant treatment with letrozole (2.5 mg/day) and either everolimus (10 mg/day) or placebo. The primary end point was clinical response by palpation. Mandatory biopsies were obtained at baseline and after 2 weeks of treatment (ie, day 15). Samples were assessed for PI3K mutation status (PIK3CA) and for pharmacodynamic changes of Ki67, phospho-S6, cyclin D1, and progesterone receptor (PgR) by immunohistochemistry. Response rate by clinical palpation in the everolimus arm was higher than that with letrozole alone (ie, placebo; 68.1% v 59.1%), which was statistically significant at the preplanned, one-sided, alpha = 0.1 level (P = .062). Marked reductions in progesterone receptor and cyclin D1 expression occurred in both treatment arms, and dramatic downregulation of phospho-S6 occurred only in the everolimus arm. An antiproliferative response, as defined by a reduction in Ki67 expression to natural logarithm of percentage positive Ki67 of less than 1 at day 15, occurred in 52 (57%) of 91 patients in the everolimus arm and in 25 (30%) of 82 patients in the placebo arm (P < .01). The safety profile was consistent with historical results of everolimus monotherapy; grades 3 to 4 adverse events occurred in 22.6% of patients who received everolimus and in 3.8% of patients who received placebo. Everolimus significantly increased letrozole efficacy in neoadjuvant therapy of patients with ER-positive breast cancer.