bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1-01-14: Effects of axillary lymph node dissection on survival of patients with sentinel lymph node metastasis of breast cancer in the Surveillance, Epidemiology and End Results (SEER) database using a propensity score matching analysis
Authors: Bendifallah S , Chereau E , Bezu C , Coutant C , Rouzier R
Published: August 2014

Background: In two randomized trials, axillary lymph node dissection (ALND) did not significantly affect overall or disease-free survival of patients with a positive sentinel lymph node (SLN). These two trials closed early without the targeted enrollment questioning a lack of sufficient power. In retrospective observational studies, patients receive treatment according to tumor and patient (age, health status) factors and thus biasing the comparisons. Propensity score matching (PSM) analysis has been proposed as an alternative method to adjust for confounding factors with a statistical advantage over the standard methods of confounder adjustment. The method involves generation of a propensity score for each subject which is an estimate of the conditional probability of receiving a treatment given a set of known covariates. Propensity scores are used to reduce selection bias by equating groups based on these covariates.

Objective: To evaluate whether patients with SLN metastasis of breast cancer who underwent complete ALND demonstrate improved survival in the Surveillance, Epidemiology and End Results database using a propensity score matching.

Methods: The study population comprised 21073 patients. The 5-year cause-specific survival was tested, in order to examine the survival impact of complete lymphadenectomy by using propensity score matching analysis. The propensity scores to determine the conditional probability of receiving ALND were generated using logistic regression model. Patients were then matched using the propensity score by an optimal matching algorithm.

Results: The PSM was based on age, race, region of diagnosis, tumor type and grade, tumor size, nodal status (micrometastasis vs macrometastasis) and hormone receptor status. It generated a balanced, matched cohort (3229 patients in each group) in which baseline characteristics were not significantly different. Five-year overall survival was 96.9% (95% CI 96.1–97.6%) in the ALND group and 94.0 (95% CI 92.6–95.4%) in the SLN biopsy alone group. The benefit of complete lymphadenectomy was significant: p = 0.028.

Conclusion: Using PSM analysis, our results show evidence of benefit for ALND in case of metastatic sentinel lymph node. The results of randomized trials demonstrating no benefit for ALND may not been generalizable.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-14.
Abstract P1-01-16: Intraoperatively-palpable “non-sentinel” nodes: should they be removed?
Authors: Crivello ML , Ruth K , Sigurdson ER , Egleston BL , Boraas M , Bleicher RJ
Published: August 2014

Background: Sentinel lymphadenectomy is the standard of care for evaluation of the axilla in breast cancer. Not infrequently, despite a clinically negative axilla, axillary nodes are found and removed that are only palpable intraoperatively at the time of sentinel lymph node biopsy. There is limited data suggesting that their resection is beneficial, and there is some controversy over whether these non-hot, non-blue nodes should also be called “sentinel nodes” (SNs).

Methods: A retrospective chart review was conducted of breast cancer patients who underwent sentinel lymph node biopsy from 2007 to 2011 at a single institution. Patients were only included if they had SNs removed (defined as blue and/or radioactive nodes), as well as additional non-sentinel nodes that were only palpable intraoperatively (pNSNs) and not part of an axillary dissection (ALND). Pathology of both SNs and pNSNs were reviewed. Histologic nodal evaluation was performed by H&E alone.

Results: From 2007 to 2011, a total of 59 patients had both SNs and pNSNs removed. Fifty-five patients (93.2%) were female and the average age was 55 years old. Fifty-two patients (88.1%) had invasive ductal carcinoma while 7 had lobular carcinoma (11.9%). Average tumor size was 2.0 cm. Among the 59 patients, a total of 109 SNs (mean 1.8, median 1, range 1–6) and 202 pNSNs (mean 3.4, median 2, range 1–30) were removed at the same surgery. Twenty patients (33.9%) had metastases in either the SNs and/or pNSNs with 16 (80.0%) of these having metastases in the SNs alone. Sixteen patients proceeded to an ALND. There were 4 patients (6.8%, 95% CI 1.9–16.5%) who had SNs negative for tumor but pNSNs positive for tumor, and 4 (6.8%, 95% CI 1.9–16.5%) separate patients who had extracapsular extension (ECE) in their pNSNs but no ECE in the SNs. The pNSNs thus provided information altering operative treatment by American College of Surgeons Oncology Group (ACOSOG) Z0011 trial criteria in 8 patients (13.6%). Two patients (3.4%, 95% CI 0.4–11.7%) had ECE present in ALND nodes, but not present in either their SNs or pNSNs.

Conclusion: pNSNs provide information that changes the surgical plan by ACOSOG Z0011 criteria in 14% of cases. Whether their removal changes patient outcomes remains unclear, especially as SNs and pNSNs may, together, be falsely-negative for ECE present within the axilla. While we recommend their removal at this time based upon the added information they provide, further study is required to determine whether the changes resulting from pNSN dissection (i.e. need for ALND) provide any outcome benefit in this era of effective systemic therapy.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-16.
Abstract P1-02-02: Receptor discordance in breast cancer recurrence: Is re-biopsy a necessity?
Authors: Fujita T , Sawaki M , Hattori M , Kondo N , Horio A , Ushio A , Gondo N , Hiroji I
Published: August 2014

Background: Decision making on systematic treatment of patients with metastatic breast cancer is based on features like estrogen receptor (ER), progesterone receptor (PgR), and HER2 status assessed on the primary tumor. Recent prospective studies have investigated discordance in receptor status between the primary and metastatic tumor. We evaluated the discordance of receptor status between the primary and metastatic tumor and assessed the impact of re-biopsy on patient management.

Methods: In breast cancer patients who underwent surgical resection of primary tumor and re-biopsy of metastatic tumor at Aichi Cancer Center hospital, we examined the discordance of ER status, PgR status, and HER2 status between the primary and metastatic tumor. For sampling of metastases, core needle biopsy (CNB), trans-bronchial lung biopsy (TBLB), or surgical resection was performed. And fine-needle aspiration biopsy was not used.

The ER and PgR status were assessed using Allreds scoring system by IHC. These statuses were categorized as positive when the total score was more than two. HER2 expression status was tested by IHC and FISH. HER2 3+ by IHC, or 2+ and FISH positive were judged as HER2 positive.

Results: 48 patients underwent re-biopsy from 2003 to 2012. Twenty-eight were loco-regional (local: 25, Supraclavicular: 2, Axilla: 1) and 21 were distant (Lung: 14, Liver: 6, Bone: 1). Discordance in ER, PgR, or HER2 between the primary and metastatic tumor were 6.2% (3/48), 16.7% (8/48), and 4.2% (2/48), respectively. One patient had discordance on re-biopsy with initial triple negative primary tumor (1/5, 20%). 11/48 patients (22.9%) changed the molecular subtype (Group A), and 37/48 patients (77.1%) did not change (Group B). Time to recurrence (TTR) was significantly longer in Group A than Group B (82.3 months vs 51.2 months, p = 0.04). No significant difference in re-biopsy techniques (CNB and TBLB vs surgical resection), metastatic lesions (loco-regional vs distant), and adjuvant therapy were observed between Group A and Group B.

Conclusions: Our results show the low level of discordance for ER and HER2 between the primary and metastatic tumor. But re-biopsy should be considered in the patients with long TTR, since it is likely to impact treatment choice. Further study is needed to determine the value of re-biopsy.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-02-02.
Abstract P1-03-01: Evidence for the Warburg effect in mammary atypia from high-risk African American women.
Authors: Seewaldt V , Hoffman A , Ibarra-Drendall C
Published: August 2014

Background: Aggressive cancers are known to consume glucose avidly and produce lactic acid (rather than fully metabolize glucose via the Tricarboxylic Acid (TCA) cycle). This shift toward lactate production, even in the presence of adequate oxygen, is termed the Warburg effect. The Warburg effect is thought to be a late event in breast cancer, however, our studies in high-risk women provide evidence that the Warburg effect occurs during cancer initiation. This is an important observation as glucose-signaling can be readily targeted for breast cancer prevention with minimal toxicity. Here we investigated the role of the Warburg effect in breast cancer initiation in young high-risk women.

Methods and Results: Similar to fluorodeoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) is a fluorescent glucose analog that can be used to track glucose uptake and glycolysis. 2-NBDG spectroscopy provides a means to track glucose metabolism in live mammary epithelial cells from high-risk women. We used 2-NBDG spectroscopy to measure glucose uptake in ER− breast cancer and live atypical mammary epithelial cells from high-risk premenopausal women. We observe that both triple-negative breast cancer and a subset of atypia exhibits accumulation of 2-NBDG.

There is growing recognition that phosphoprotein signaling networks (rather than single genes) play a key role in breast cancer initiation and progression. Our team used Reverse Phase Proteomic Microarray (RPPM) profiling to test for activation of phosphoprotein signaling networks in atypical RPFNA cytology from high-risk premenopausal women in our cohort. RPFNA were obtained from two independent sets of 39 and 38 high-risk premenopausal women; 45% of these women were African American. The signaling network most highly expressed in precancerous cells contained activated signaling proteins associated with the Warburg effect (AKT/mTOR/PI3K), insulin signaling (pACC, IRS1) and epithelial to mesenchymal transition (EMT) IL6/Stat3/vimentin.

Conclusions: This is the first evidence that abnormal glucose uptake and the Warburg effect occurs during breast cancer initiation in high-risk premenopausal women. These studies demonstrate our ability to identify abnormal glucose and activated signaling networks associated with the Warbug effect in atypical mammary cells from high-risk women.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-03-01.
Abstract P1-03-03: milk deposition in women's mammary duct has been a potential risk factor of breast tumor
Authors: Xu Z , Gu Y , Gong G , Zhang Y
Published: August 2014

Background: According to the pathology and general resection specimens, so many patients have breast tumors together with deposited milk. Therefore, we performed this retrospective study to determine the distribution and characteristics of breast diseases in women undergoing mammary ductoscopy and explore the clinical values and implications of mammary ductoscopy in the management of the breast diseases.

Materials & Methods: Total of 10,082 women(20,082 breasts) with a mean age of 39.2 years old (ranging from 12 to 84 years old) underwent mammary ductoscopy from 2002 to 2011. We studied NSND in contrast with SND duct, specially focused on the situation of duct with intraductal mass. Comparing with the pathology, we studied the relationship between duct milk deposition and epithelial hyperplasia.

Results: From this study we concluded that duct milk deposition was a common disease in 25–59 years old women, it occupied 42.89% cases of the mammary ductal disease and almost 46% are NSND. The character of deposited milk became solid from liquid with the age growing up. Duct milk deposition 476/1431(33.26%)was mostly associated with intraductal masses(total 1431 cases), followed by duct dilatation 517/1431(36.13%). Pathology of patients diagnosed by FDS for duct milk deposition are epithelial hyperplasia(100%), adenosis(86.5%), chronic inflammation(8.8%), metaplasia(16.9), florid hyperplasia(10.8%), tumor-like hyperplasia (57.2%), intraductal tumor or fibroadenoma (27.8%), dysplasia (3.0%), breast cancer (11.7%).

Discussion: Diagnosis of duct milk deposition, which may not necessarily presented by nipple discharge, is the most common finding and associated with proliferation and thus intraductal mass, etc. we should noticed 30–50 years old women groups, cause they have have stopped breast-feeding for years but still had yellow or brown, liquid or solid milk-like things in their lactiferous ducts. We predict duct milk deposition may be related with breast carcinogenesis. It is a kind of long time mechanical stimulation in the duct, both the physico-chemical properties and the acid-alkaline environment had changes by years. Still, the deposited milk would be a best medium for microbial and virus, which would cause the genesis of hyperplasia even breast cancer. Therefore, we should go on the research of deposited milk and the changes of the microenvironment of the duct to find the pathogenesis of breast disease.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-03-03.
Abstract P1-04-03: Knocking down Suppressor of Cytokine Signaling 7 in breast cancer: The role in Insulin-like Growth Factor - I/Phospholipase Cγ-1 signaling
Authors: Sasi W , Ye L , Jiang WG , Mokbel K , Sharma A
Published: August 2014

BACKGROUND: Suppressor of Cytokine Signaling 7 (SOCS7) is a member of the SOCS family and is known to interact with Phospholipase Cγ-1 (PLCγ-1), one of the Insulin like Growth Factor - I (IGF-I) receptor downstream molecules. In the present study, we sought to examine the effect of knocking down SOCS7 gene on breast cancer cells growth and migration and to elucidate whether this has involved IGF-I — PLCγ-1 signaling using the PLCγ-1 blocker U73122.

METHODS: Suitable breast cancer cells (MCF7 and MDA-MB-231) were transfected with anti-SOCS7 ribozymal transgene, to create sublines with SOCS7 knockdown that was verified by RT-PCR. The growth and migration of the cells were evaluated in the presence or absence of IGF-I and PLCγ-1 inhibitor using in vitro growth assay and Electrical Cell Impedance Sensing (ECIS) migration assay.

RESULTS: MCF7ΔSOCS7 and MDA-MB-231ΔSOCS7 (SOCS7 knockdown) were constructed. Both sublines showed a higher rate of growth compared to control cells with and without IGF-I stimulation. U73122 treatment did not appear to change this growth outcome. Using ECIS migration assay, it was shown that knocking down SOCS7 had a significant positive effect on the migration of MCF7 and MDA-MB-231 cells, and that both IGF-I treatment and SOCS7 knockdown had a synergistic positive influence on their migration (p < 0.05). We further demonstrated that the impact of U73122 on the IGF-I migratory effect was dependent upon SOCS7 knockdown as it has significantly blocked the stimulatory effect of IGF-I on MCF7ΔSOCS7 and MDA-MB-231ΔSOCS7 migration but not that of the control cells. While SOCS7 has acted to control the IGF-I effect, it appeared to cancel the inhibitory function of U73122, indicating a specific anti - PLCγ-1 role for SOCS7 in IGF-I induced breast cancer cellular migration.

CONCLUSION: SOCS7 loss resulted in increased growth and migration of breast cancer cells and this had a synergistic effect on their response to IGF-I. This role could be related to its interaction with PLCγ-1 during the cellular migration but not the growth. It may be possible that SOCS7 acts through different mechanism to control the cellular growth.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-03.
Abstract P1-04-05: Role of the Rb and p53 Tumor Suppressor Pathways in Mammary Tumorigenesis
Authors: Jones RA , Liu JC , Zhe J , Schimmer AA , Eldad Z
Published: August 2014

The retinoblastoma (Rb) and p53 tumor suppressor pathways are frequently altered in human malignancies including breast cancer. To investigate the role of these pathways in mammary tumorigenesis, we crossed mice expressing Cre recombinase in the mammary epithelium (MMTV-Cre) with mice harboring Rb and p53 floxed alleles to generate MMTV-Cre:Rbf/f:p53f/f mutant mice. Combined somatic loss of Rb and p53 led to the formation of aggressive triple-negative tumors (ER−, PR−, HER2−) that often displayed features of an epithelial to mesenchymal transition (EMT) including sarcomatoid differentiation and high expression of the mesenchymal marker N-Cadherin. Molecular profiling revealed Rb/p53 deficient tumors shared similar gene expression profiles with mouse and human claudin-low breast cancers. Interstingly, limiting dilution transplantation analysis also suggests that Rb/p53 deficient claudin-low tumors are enriched for tumor-initiating cells (TICs).

To investigate the cellular origin of claudin-low tumors, the Rbf/f and p53f/f alleles were deleted within the luminal and basal compartments of the mammary gland using a Cre-expressing adenovirus (Ad-Cre) and CD24-CD49f-based FACS analysis. The different cellular fractions were then transplanted into the cleared mammary fat pad of recipient mice which are currently being monitored for tumor development.

Finally, primary cell lines isolated from Rb/p53 deficient mammary tumors were used in conjunction with high-throughput chemical and genetic screens to identify new therapeutic targets. A primary screen of 260 kinase inhibitors and 312 FDA-approved off-patent drugs has identified novel candidates and a genome wide negative selection (‘drop-out’) screen is currently in progress.

Ultimately, the results of this research will provide important insight into the biology of triple-negative breast cancer and will lead to the identification of novel therapeutic targets for the treatment of patients living with this disease.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-05.
Abstract P1-04-10: PDGFRA signaling in Inflammatory breast cancer.
Authors: Joglekar M , van Golen K
Published: August 2014

Inflammatory breast cancer is the most lethal form of locally advanced breast cancer. It is clear from the recent research advancements in IBC that it is entirely a different entity, not only in its clinical presentation but also in its molecular expression profile. Therefore, it is essential to carry out molecular expression studies in IBC compared to non-IBC as well as to pursue these differences to study their effects on IBC phenotype.

In collaboration with the Translational Cancer Research group in Antwerp Belgium, our lab has recently found that PDGFRA is significantly over expressed in IBC patient samples (between 15–36%) compared to non-IBC patient samples. This project focuses on identifying the PDGFRA activation signature in SUM149 IBC cells. Upon ligand stimulation, PDGFR can either homo or heterodimerize (AA, AB, BB) and produce an array of signaling events responsible for cell growth, cell proliferation, cell differentiation in normal cells as well as in several cancer cells. SUM149 and SUM190 cells lack ER, PR and HER-2 expression and hence are named triple negative IBC cells. Triple negative breast cancers are highly aggressive in nature and therefore suffer from poor prognosis. We seek to determine if signaling via over expressed PDGFRA could be a potential surviving factor in producing such fast growing and highly invasive IBC tumors. Preliminary data on immunostaining of PDGFRA with and without permeabilization suggests intracellular localization of PDGFRA. We also know that SUM149s and SUM190s express all four PDGF ligands (A, B, C, D). This unusual expression of all the PDGF lignads suggests the self-sufficiency of these cells for PDGFRA signaling. In other words, these cells maintain constitutively active PDGFRA via intracrine/autocrine loop. Protein expression of active PDGFRA was studied by using two different phospho specific PDGFRA antibodies (Tyr 1018 and Tyr 849) without any prior stimulation by PDGF ligands. Moreover our data shows that PDGFR- activation in IBC, unlike in n-IBC, is PDGF-C driven.

Possibility of such intracrine/autocrine loop could significantly increase the overall PDGFRA signaling in IBC cells and hence can produce fast growing aggressive IBC tumors. Thus targeting PDGFRA, an important biomarker in IBC could stand as a novel and systematic therapeutic approach.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-10.
Abstract P1-05-02: Epithelial-to-epithelial transition precedes collective breast cancer invasion
Authors: Cheung KJ , Ewald AJ
Published: August 2014

BACKGROUND: Invasion represents a fundamental step in tumor progression and is a driving force for metastasis and cancer-related mortality. Invasion has been studied largely in the context of single cells, but in vivo, most tumor cells are not found in isolation, but rather are found as ensembles of cells with substantial molecular heterogeneity. It remains poorly understood in this context which cells are invasive, how these cells arise or how these cells interact collectively to accomplish invasion.

RESULTS: We developed an ex-vivo 3D culture assay enabling us to distinguish invasive from non-invasive cells and to interrogate their molecular phenotype. In two mouse models of luminal breast cancer, we find that invasive cells express markers of basal epithelium including cytokeratin K14, p63 and P-cadherin. Invasive K14+ cells were also molecularly and functionally distinct from normal myoepithelial cells and co-expressed luminal K8 and E-cadherin. In vivo, K14+ cells were localized to tumor-stromal interfaces in the primary tumor and in lung micro and macro-metastases. Using a molecular biosensor and time-lapse microscopy, we observed that K14+ invasive cells were derived from K14-negative luminal tumor cells by direct phenotypic conversion. Interestingly, the capacity of luminal tumor cells to acquire basal markers varied by model, with few K14+ cells in MMTV-Her2 and many K14+ cells in MMTV-PyMT mice, correlating with the known in vivo kinetics of tumor progression in these models. To block this epithelial to epithelial transition, we tested the activity of an inhibitor of ribosome S6 kinase (RSK), which has been shown previously to inhibit myoepithelial differentiation in normal mammary epithelium. RSK inhibition caused relative depletion of K14+ tumor cells in 3D culture and abrogation of invasion. Finally, our assay revealed that in fresh primary tissue from human ER+ luminal breast cancers, invasive cells also expressed basal epithelial markers.

CONCLUSIONS: Taken together, these data suggest that an epithelial-to-epithelial transition precedes collective invasion in luminal breast cancers. Because we observe K14+ leader cells independent of tumor estrogen receptor status, these data may be generally applicable to breast cancer invasion across subtypes. Because many epithelial tissues have basal populations, K14+ tumor cells may also lead invasion in other cancers.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-02.
Abstract P1-05-04: Expression of Quiescin Sulfhydryl Oxidase 1 is associated with a highly invasive phenotype and correlates with a poor prognosis in Luminal B breast cancer.
Authors: Katchman BA , Ocal T , Hostetter G , Cunliffe HE , Wantanabe A , Lake DF
Published: August 2014

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. Informatic analysis using the newly available “Gene Expression Based Outcome for Breast Cancer Online” (GOBO) tool indicated high levels of QSOX1 expression in Estrogen Receptor positive (ER+) subtypes of breast cancer. We confirmed this finding by evaluation of QSOX1 protein expression in breast tumors and in a panel of breast cancer cell lines. In addition, Kaplan Meyer analyses revealed QSOX1 as a significant predictive marker for both relapse and poor overall survival in Luminal B tumors, but not in other intrinsic subtypes. To investigate malignant cell mechanisms in which QSOX1 might play a key role, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in ER+ MCF7 and ER− BT549 breast cancer cell lines. Suppression of QSOX1 protein dramatically slowed cell proliferation but did not significantly effect apoptosis or cell cycle regulation. Inhibition of QSOX1 did however dramatically inhibit MCF7 and BT549 breast tumor cells from invading through Matrigel in a modified Boyden chamber assay. Inhibition of invasion could be rescued by the exogenous addition of recombinant QSOX1. Gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-9 a key mediator of breast cancer invasive behavior. Taken together, our results suggest that QSOX1 is a novel biomarker for risk of relapse and poor survival in Luminal B breast cancer, and has a pro-invasive role in malignant progression through post-translational activation of MMP-9.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-04.
Abstract P1-05-05: Podocalyxin is a key regulator of breast cancer progression and metastasis.
Authors: Snyder KA , Hughes MR , Graves M , Roskelly C , McNagny KM
Published: August 2014

Podocalyxin (gene name Podxl) is a CD34-related sialomucin that has been shown to be important in regulating cell adhesion, migration, and polarity of hematopoietic progenitors and vascular endothelia. In breast cancer, we have shown that podocalyxin is overexpressed on a subset of node-negative tumors and its expression is strongly associated with poor overall survival. To determine whether podocalyxin directly regulates breast cancer cell behavior, we ectopically expressed podocalyxin in the human breast cancer cell line, MCF7, which expresses low levels of endogenous podocalyxin, is minimally invasive, and non-metastatic. Upon ectopic expression of podocalyxin, MCF7 “bulge apically”, produce apical and lateral microvilli, are less adhesive in vitro, and are delayed in targeting integrins to the basolateral surface. In contrast to MCF7, MDA-MB-231 is a basal-like breast cancer cell-line, which expresses high levels of endogenous podocalyxin, exhibits poorly polarized monolayer and mammosphere architecture in vitro and forms “metastatic” lung tumors in vivo. Using Podxl-targeted shRNA vectors we show that expression of podocalyxin is required for MDA-MB-231 cells to form distant metastases in a competitive in vivo assay utilizing humanized NOD/SCIDIL-2rγ−/− (NSG) mice. Previously, we observed that Podxl-deficient hematopoietic cells have impaired CXCR4-mediated chemotaxis to CXCL12, a known axis for tumor metastasis. Our current hypothesis is that podocalyxin regulates chemotaxis of tumor-initiating cells (TICs) to the CXCL12-rich tissues of the lungs, liver, and bone marrow. Up-regulation of podocalyxin on breast cancer cells may be a key event in tumor progression and likely enables these cells to sense chemokines and metastasize to peripheral sites.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-05.
Abstract P1-05-08: Targeting integrin signaling suppresses invasive recurrence in a three-dimensional model of radiation treated ductal carcinoma in situ
Authors: Nam J-M , Ahmed KM , Costes S , Zhang H , Sabe H , Shirato H , Park CC
Published: August 2014

Background: DCIS (ductal carcinoma in situ) is comprised of cancerous cells that are contained within the milk duct and separated from the stroma by a basement membrane. Akt activation is implicated in breast cancer progression and also upregulated in ∼30% of DCIS lesions. We have previously shown that β1-integrin inhibition enhanced the efficacy of ionizing radiation (IR) in invasive breast cancer, via an Akt-mediated effect. Radiation therapy (RT) is commonly used to treat DCIS, resulting in a decrease in recurrence risk by ∼50%. However, DCIS still recurs in a substantial number of patients, and as invasive disease half of the time. This study aimed to identify the effects of IR on Akt-driven DCIS, and possible mechanisms underlying invasive progression in surviving cells.

Materials and Methods: To model DCIS, we took advantage of the ability of non-invasive mammary epithelial cells, MCF10A that form duct-like structures in 3-dimensional laminin-rich extracellular matrix (3D lrECM), and overexpressed an activated myristoylated form of the serine/threonine kinase, Akt. The 3D lrECM cultured MCF10A-Akt cells show filled lumen and retained basal polarity, characteristic of DCIS lesions in vivo. After DCIS structures formed in 3D lrECM, cultures were exposed to 8 Gy IR. Confocal microscopy analysis, western blot analysis, matrigel chemoinvasion assay and matix degradation assay were performed to characterize the IR effect on luminal or basal cells of the DCIS like structures.

Results: MCF10A-Akt structures post-8 Gy IR shows a significant increase in apoptosis measured by cleaved caspase-3 (n = 3, P < 0.05). When we propagated cells post-IR in 3D lrECM, an invasive phenotype emerged in a sub-population of survivors (n = 3, P < 0.01). We also confirmed that inhibitory antibodies to β1-integrin suppressed the invasive phenotype that was induced by 8 Gy IR. In addition to this, invasion activity emerged by 8 Gy IR was inhibited by nuclear factor-kappaB (NF-κB) inhibitor JSH-23 (n = 3, P < 0.01). Finally, inhibition of β1-integrins or NF-κB translocation completely suppresses the formation of invasive colonies.

Discussion: In present study, we found that inhibition of β1-integrins abrogates the emergence of an invasive phenotype among surviving clones. Our current data suggest that regulation of β1-integrin signaling via NF-κB plays an important role in the emergence of invasive disease after radiation, and may be an important clinical target.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-08.
Abstract P1-05-19: Modeling Cancer Recurrence and the Therapeutic Effect of Adjuvant Systemic Therapy
Authors: Ganesan S , Bhanot G , Boemo M , Lee JH
Published: August 2014

Despite complete surgical removal of early stage cancers, recurrence can occur at distant locations in time spans ranging from months to decades. This is due to the presence of disseminated cancer cells that can ultimately transition into active growth leading to recurrence. Systemic adjuvant therapy can decrease the incidence of distant relapse and lead to improved survival in some cancers. In this study, data on natural recurrence patterns of different breast cancer subtypes is used to build a probabilistic model of distant recurrence. The central hypothesis of this model is that after initial local therapy, a fraction of patients will have the presence of one or more disseminated dormant metastatic foci (DMF). The transition of DMF into actively growing metastatic foci (AMF) is described using a stochastic model. DMF of individual breast cancer subclasses each have a distinct probability per unit time of entering active growth. Fitting of this model to clinical trial data of early stage breast cancers treated with surgery alone demonstrates that DMF present in estrogen receptor negative (ER−) cancers and Luminal B cancer have greater probabilities of transition to active growth per unit time than Luminal A breast cancers. Clinical trial data were then used to determine the effect of adjuvant therapy on the dynamics of tumor recurrence. The data strongly support a model in which adjuvant chemotherapy works by eliminating only a fraction of DMF that have transitioned into AMF during the time of chemotherapy treatment. Thus short duration adjuvant chemotherapy would have greater efficacy in ER−, HER+ and Luminal B breast cancers, which have fast dynamics of recurrence, and little effect in Luminal A breast cancers with far slower dynamics of recurrence. Similarly, analysis of adjuvant hormonal therapy trial data, demonstrate that hormonal therapy also affects only DMF that transition into active growth during the period of hormonal therapy. This model can explain why extended duration therapy of up to 5 years is more effective than shorter duration of adjuvant hormonal therapy. Interestingly some adjuvant treatments, such as trastuzumab for HER2+ breast cancers, affect recurrence rates in a way that suggest that these interventions directly kill DMF. This model of tumor recurrence has several clinical implications that could inform future clinical trial design.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-19.
Abstract P1-05-22: Breast cancer cells that undergo an Epithelial-to-Mesenchymal transition co-opt LPP, a regulator of mesenchymal cell migration and invasion.
Authors: Ngan E , Northey JJ , Ursini-Siegel J , Siegel PM
Published: August 2014

Transforming Growth Factor β (TGFβ) promotes breast cancer cell metastasis to multiple sites, including the bone and lungs. TGFβ is a strong inducer of Epithelial-to-Mesenchymal transitions (EMT) and breast cancers that exhibit features of an EMT acquire stem cell-like characteristics, are highly aggressive, are resistant to therapy and are refractory to tumor suppressive processes. While TGFβ itself is non-oncogenic, it is a potent modifier of the malignant phenotype in breast cancer and is capable of enhancing the migration, invasion and metastasis of ErbB2 expressing breast cancer cells.

We have identified Lipoma Preferred Partner (LPP) as an indispensable regulator of TGFβ-induced migration and invasion of ErbB2 expressing breast cancer cells. LPP is ubiquitously expressed in smooth muscle cells where it mediates cell adhesion, migration and the formation of lamellipodial extensions. We hypothesize that breast cancer cells capable of undergoing an EMT can utilize novel mediators that are engaged in promoting the migration and invasion of mesenchymal cells. We propose that LPP is one such example, which promotes the migration and invasion of ErbB2 expressing breast cancer cells that have undergone a TGFβ-induced EMT.

We demonstrate that ErbB2 expressing breast cancer cells display significant increases in cell migration and invasion upon TGFβ stimulation, and such responses are dependent on LPP expression. We show that LPP re-localizes to focal adhesion complexes following TGFβ-induced EMT and it is a critical determinant in focal adhesion turnover. Furthermore, we determined that LPP targeting to focal adhesions through its LIM1 domain requires the cooperation of ErbB2 and TGFβ signaling pathways. Finally, we demonstrate that LPP promotes TGFβ-induced migration and invasion of ErbB2 expressing breast cancer cells through recruitment of α-Actinin, an actin cross-linking protein.

Overall, we have identified LPP as a novel mediator that integrates TGFβ and ErbB2 signaling to promote the migration and invasion of breast cancer cells that undergo an EMT. Our data reveal that breast cancer cells, which can transition from an epithelial to mesenchymal phenotype, can engage a regulator of mesenchymal cell migration and invasion.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-22.
Abstract P1-05-23: Blockade of mTORC1 decreases CXCR4-mediated migration and metastasis
Authors: Dillenburg Pilla P , Patel VT , Dorsam R , Masedunskas A , Amornphimoltham P , Weigert R , Molinolo A , Gutkind JS
Published: August 2014

Tumor cells can co-opt the promigratory activity of chemokines and their cognate G protein–coupled receptors (GPCRs) to metastasize to regional lymph nodes or distant organs. Indeed, the migration toward SDF-1 (stromal cell–derived factor 1) of tumor cells bearing CXCR4 [chemokine (C-X-C motif) receptor 4] has been implicated in the lymphatic and organ-specific metastasis of various human malignancies, including breast cancer. The downstream mechanisms of CXCR4-mediated migration are not fully elucidated. The study of the precise signaling pathways involved in cell migration and metastasis in each tumor type may help identify novel therapeutic targets for improving cancer therapy. Here we show that blockade of mTOR complex 1 (mTORC1) by Rapamycin or knocking down Raptor decreases CXCR4-Gαi mediated cell migration. Moreover, using a spontaneous lymph node metastasis model, we were able to show that pharmacological blockade of mTORC1 decreases lymph node metastasis in vivo. Taken together our data suggest that Rapamycin, a FDA-approved drug, could be beneficial to prevent tumor cells spreading from the primary tumor and metastasize. Molecular events underlying the effects of Rapamycin on CXCR4-induced migration will be presented.

This research was supported [in part] by the Intramural Research Program of the NIH, NIDCR.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-23.
Abstract P1-06-02: A newly angiogenic biomarker Vasohibin-1 expression in ductal carcinoma in situ of the breast.
Authors: Tamaki K , Tamaki N , Kamada Y , Uehara K , Miyashita M , Ishida T , Ohuchi N , Sasano H
Published: August 2014

Vasohibin-1 is a recently identified negative feedback regulator of angiogenesis induced by VEGF-A and bFGF. Our previous study was the first study demonstrating the status of vasohibin-1 in human breast lesions, which indicates that vasohibin-1 is associated with neovascularization and may especially play important roles in the regulation of intratumoral angiogenesis in human breast cancer. In this study, we first evaluated mRNA expression of vasohibin-1 and CD31 in 39 Japanese emale breast carcinoma specimens including 22 invasive ductal arcinoma (IDC) and 17 ductal carcinoma in situ (DCIS) using a real-time quantitative RT-PCR (QRT-PCR) with LightCycler system. Table shows the primer sequences used in real-time PCR in this study.

In addition, we also immunolocalized vasohibin-1 and CD31 and compared their immunoreactivity to nuclear grades and histological grades of 100 carcinoma cases (50 IDC and 50 DCIS). There were no statistically significant differences of CD31 mRNA expression and the number of CD31 positive vessels between DCIS and IDC (P = 0.250 and P = 0.191, respectively), whereas there was a statistically significant difference in vasohibin-1 mRNA expression and the number of vasohibin-1 positive vessels in DCIS and IDC (P = 0.022 and P < 0.001, respectively). There was a significant positive correlation between vasohibin-1 mRNA level and Ki-67 labeling index in DCIS (r2 = 0.293, P < 0.001). In addition, vasohibin-1 mRNA expression was correlated with high nuclear and histological grades in DCIS cases and a significant positive correlation was detected between the number of vasohibin-1 positive vessels and Ki-67 labeling index or nuclear grade or Van Nuys classification of carcinoma cells (P <0.001, respectively). These results all indicate the possible correlation between aggressive biological features in DCIS including increased tumor cell proliferation and the status of neovascularization determined by vasohibin-1 immunoreactivity.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-06-02.
Abstract P1-07-16: Liver derived epithelial cells as source of false positive circulating tumor cells in early breast cancer
Authors: Habets L , Körber W , Frenken B , Danaei M , Kusche M , Peisker U , Kroll T , Pachmann K
Published: August 2014

The MAINTRAC technique as introduced by our coworkers from Jena (RBC lysis, fluorometric detection and analysis on Olympius ScanR) detects more circulating epithelial cells than techniques using enrichment. Also cells with a low EPCAM expression are detected and not only the typical cells with bright expression found after immunomagnetic enrichment. The relative cheapness and reproducibility allows frequent monitoring during and after therapy Using 3 colour detection (EPCAMfitc, DAPI, Vimentin PE) living and dead circulating epithelial cells in EMT, or cells in EMT with stemcell markers (EPCAMfitc, Vimentin-PE, CD44PacBlue) can be detected. In early breast cancer (n = 135) cells can be found in 60% of patients and in 40% higher cell counts (>100 ml are detectable. A control population(n = 100) showed low numbers in 98% (e.g (<100 CECin 1 ml blood). Expression of the mesenchymal marker (vimentin) ranges between 10 and 40% with different expression. CD44 shows also a wide range of expression. Two main cell types can be distinguished: type 1 shows generalized but weaker expression patterns and a second type with very bright dotted expression. The clinical relevance of these subsets is not known and their behavior under therapy has not been analysed in depth yet. In advanced breast cancers high cell counts were detectable in most patients with a less agressive disease course. In the rapidly progressing unfavorable subtypes (TN and Her2+. HRneg) less or none cells were found. During crossvalidation in non cancer patients we found high cell numbers in several forms of liver affections (n = 108). The expression patterns of markers on these cells were not differing from those in cancer patients. So this same cell type merging EMT, stemcell an hypoxic stress markers is detectable in advanced and early breast cancer (n = 40) and in benign disease. These cells disappear or decrease after response to chemo or anti-hormonal therapy in cancer or antioxidant therapy in NAFLD. We believe that the evasion of these cells is driven by the same force in cancer as in non cancer conditions. We suggest that this are the wellknown hypoxic and hyperacidic conditions causing epithelial mesenchymal transition. Cancer cell hijack this functions occurring normally under these conditions to survive and to facilitate evasion. More comprehensive analysis (four colour analysis on the AMNIS Flowsight) is needed and should show differences in expression patterns of liver derived epithelial cells (LDEC) and real tumor derived epithelial cells (TDEC). Further clarification of these phenomena should give new insights of the biological events in early disease and the possibilities and reliability of “fluid biopsy”.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-16.
Abstract P1-07-18: Association between Bone Turnover Markers in patients with breast cancer and bone metastases on treatment with bisphosphonates (ZOMAR study)
Authors: Tusquets I , De la Piedra C , Manso L , Crespo C , Gómez P , Calvo L , Ruiz M , Martínez P , Perelló A , Antón A , Codes M , Margelí M , Murias A , Salvador J , Seguí MA , De Juán A , Gavilá J , Luque M , Pérez D , Zamora P , Arizcum A , Chacón JI , Heras L , Barnadas A
Published: August 2014

Background: The presence of bone metastases (BMe) alters the balance of bone remodeling and consequently, levels of bone turnover markers (BTM). Increased levels of these biomarkers are related to the risk of skeletal-related events (SREs), disease progression and death. Treatment with bisphosphonates reduces the probability of SREs through osteoclastic activity inhibition. The aim of this study was to determine the relation between BTM, bone metastasis development and SREs, disease progression and death in patients with breast cancer (BC) and BMe.

Patients and methods: Observational, prospective and multicenter study. Patients with BC and BMe; no previous bone treatment in the last 6 months prior to study entry. Urinary aminoterminal telopeptide of collagen I (NTX, Osteomark NTx Urine, Wampole Laboratories, USA); urinary alpha-alpha-isomer of carboxyterminal telopeptide of collagen I (αα-CTX, ALPHA Crosslaps EIA, ids, UK) and serum bone alkaline phosphatase (BALP, OSTASE BAP, ids, UK) were determined at baseline (V0) and every 3 mo along 18 months (V6). Patients were treated with zoledronic acid (ZA) at inclusion and every 3–4 weeks.

Results: 234 patients with BC and BMe were analyzed. BTM results were available for 219 patients at basal visit (V0) and every 3 mo of treatment along 18 months (V6). Population basal characteristics (234 patients): mean age: 59.8 years; ER+: 80.3%; PR+: 64.9%; HER 2+: 18.3%. Patients with pathologic baseline levels were: 49.8% NTX, 39.6% αα-CTX and 83.4% BALP. A significant decrease was observed in BTM at V2 vs V0 after 6 months: 13.7%, 8.4% and 58.4% presented pathologic values of NTX, αα-CTX and BALP respectively. Normalized levels remained steady throughout 18 mo follow-up, finding significant decrease for each BMT for each time point except at V6 for αα-CTX. Regarding association between BTM and SREs, progression and exitus, a significant association was observed between pathologic levels of BTM throughout follow-up: with SRE at V3, V4 for NTX; with disease progression at V3, V4, V5, V6 for NTX, at V2 for αα–CTX and at each follow up visit for BALP; and with death at V1,4,5 for NTX, at V5 for αα–CTX and at V1,2,3,4,5 for BALP.

Conclusions: Addition of ZA to standard systemic therapy reduced BTM levels during the first 3 months of treatment and normalized levels remained steady throughout 18 months follow up except at Month 18 for αα-CTX. Pathological levels of BTM were significantly associated with SRE, disease progression and death.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-18.
Abstract P1-08-01: Effects Of An Allosteric AKT Inhibitor (MK2206) Administered With Or Without The Aromatase Inhibitor Vorozole In An ER+ Rat Mammary Cancer Model: Preventive And Therapeutic Effects
Authors: Lubet RA , Ellis MJ , Grubbs CJ
Published: August 2014

The AKT inhibitors appear to hold some promise with regards to inhibition of ER+ breast cancers which have relapsed. It has been hypothesized that the combination of an aromatase inhibitor plus an AKT inhibitor may be particularly effective. We, therefore, tested whether an allosteric AKT inhibitor (MK2206) alone or together with the aromatase inhibitor vorozole would be effective in a preventive and/or therapeutic setting. Female Sprague-Dawley rats were administered a single IV dose of methylnitrosourea (MNU) at 50 days of age. Beginning 5 days later, rats were administered the AKT inhibitor. In a preliminary study, two treatment regimens were employed: (1) dosing with inhibitor (100 mg/Kg BW/day, 7X/week) or (2) dosing with inhibitor (700 mg/Kg BW, 1X/week). These treatments minimally affected tumor multiplicity, but did significantly decrease tumor weights. We then examined the effects of combining the AKT inhibitor once weekly with a suboptimal dose of vorozole (0.12 mg/Kg BW/day) in a therapeutic setting. In a separate study, rats were treated with MNU and were allowed to develop their first palpable cancer. They were then treated with the combination of vorozole plus the AKT inhibitor or vehicle for 6 weeks. While tumors in vehicle treated rats grew an average of 600%, tumors treated with the inhibitor grew an average of only 95%; and, in fact, 5/12 showed a decrease in tumor size. Finally, we performed a similar combined study in a prevention setting. Dosing was: (a) AKT inhibitor (700 mg/Kg BW, 1X/week), (b) Vorozole (0.12 mg/Kg BW/day, 7X/week), (c) a plus b combined, and (d) vehicle. A, b, and c reduced tumor multiplicity by 26, 48 and 77%, respectively. Thus, combining an AKT inhibitor with an aromatase inhibitor showed synergy in prevention and treatment of ER+ tumors.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-08-01.