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1.  Influence of Biospecimen Variables on Proteomic Biomarkers in Breast Cancer 
PI3K/Akt/mTOR signaling is being actively pursued as a therapeutic target for breast cancer. We sought to determine if tumor heterogeneity and biospecimen variables affect the evaluation of PI3K/Akt/mTOR pathway markers.
Intraoperative image-guided core-needle biopsies (CNB), and central and peripheral surgical tumor specimens were prospectively collected in 53 patients with invasive breast cancer. Specimens were assessed with reverse phase protein arrays (RPPA) and immunohistochemistry (IHC).
There was a moderate or strong correlation between the expression of 149 (97%) of the 154 different RPPA markers in the center and periphery. Correlation was higher for smaller tumors, in patients who did not undergo neoadjuvant therapy, and with shorter cold ischemia time. Of 154 markers, 132 (86%) were not statistically different between the center and periphery, and 97 (63%) were not different between the CNB and the surgical specimen (average of the central and peripheral specimen). pAkt S473 and PTEN had a significant correlation between central and peripheral specimens, and between CNB and surgical specimen. However, pAkt S473, pS6 S235/236 and pS6 240/244 levels were significantly higher in CNB than the central specimens both by RPPA and by IHC.
Most individual proteomic biomarkers studied do not have significant intratumoral heterogeneity. However, protein and phosphoprotein levels are affected by biospecimen type and other preanalytic variables. PI3K pathway activation is greater in CNB compared to post-excision surgical samples suggesting a potential loss of phosphorylation during surgical manipulation, or with cold ischemia of surgical specimens.
PMCID: PMC4112583  PMID: 24895461
biopsy; proteomics; tumor heterogeneity; Akt
3.  Staging of Breast Cancer in the Neoadjuvant Setting 
Cancer research  2008;68(16):6477-6481.
The use of neoadjuvant chemotherapy has become more prevalent in the treatment of breast cancer patients. The finding of a pathologic complete response to neoadjuvant chemotherapy (no evidence of residual invasive cancer in the breast and lymph nodes at the time of surgical resection) has been shown to correlate with improved survival. The current version of the American Joint Committee on Cancer (AJCC) staging for breast cancer has a pretreatment clinical stage designation that is determined by clinical and radiographic examination of the patient and a postoperative pathologic stage classification based on the findings in the breast and regional lymph nodes removed at surgery. Pathologic staging has not been validated for patients receiving neoadjuvant chemotherapy; thus, prognosis is determined for these patients based on the pretreatment clinical stage. We hypothesized that clinical and pathologic staging variables could be combined with biological tumor markers to provide a novel means of determining prognosis for patients treated with neoadjuvant chemotherapy. Two scoring systems, based on summing binary indicators for clinical and pathologic substages, negative estrogen receptor status, and grade 3 tumor pathology, were devised to predict 5-year patient outcomes. These scoring systems facilitated separation of the study population into more refined subgroups by outcome than the current AJCC staging system for breast cancer, and provide a novel means for evaluating prognosis after neo-adjuvant therapy.
PMCID: PMC4441792  PMID: 18701468
4.  Concordance of Genomic Alterations Between Primary and Recurrent Breast Cancer 
Molecular cancer therapeutics  2014;13(5):1382-1389.
There is growing interest in delivering genomically-informed cancer therapy. Our aim was to determine the concordance of genomic alterations between primary and recurrent breast cancer. Targeted next generation sequencing was performed on formalin-fixed paraffin embedded (FFPE) samples, profiling 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes often rearranged in cancer. Point mutations, indels, copy number alterations and select rearrangements were assessed in 74 tumors from 43 patients (36 primary, 38 recurrence/metastases). Alterations potentially targetable with established or investigational therapeutics were considered “actionable”. Alterations were detected in 55 genes (mean 3.95 alterations/sample, range 1-12), including mutations in PIK3CA, TP53, ARID1A, PTEN, AKT1, NF1, FBXW7 and FGFR3 and amplifications in MCL1, CCND1, FGFR1, MYC, IGF1R, MDM2, MDM4, AKT3, CDK4, AKT2. In 33 matched primary and recurrent tumors, 97 of 112 (86.6%) somatic mutations were concordant. Of identified copy number alterations, 136 of 159 (85.5%) were concordant: 37 (23.3%) were concordant, but below the reporting threshold in one of the matched samples, and 23 (14.5%) discordant. There was an increased frequency of CDK4/MDM2 amplifications in recurrences, as well as gains and losses of other actionable alterations. 40 of 43 (93%) patients had actionable alterations that could inform targeted treatment options. In conclusion, deep genomic profiling of cancer-related genes reveals potentially actionable alterations in most breast cancer patients. Overall there was high concordance between primary and recurrent tumors. Analysis of recurrent tumors prior to treatment may provide additional insights, as both gains and losses of targets are observed.
PMCID: PMC4348062  PMID: 24608573
breast cancer; genomics; next generation sequencing; genomic evolution
5.  American College of Surgeons Oncology Group (ACOSOG) Z0011: Impact on Surgeon Practice Patterns 
Annals of surgical oncology  2012;19(10):3144-3151.
The ACOSOG Z0011 trial has been described as practice-changing. The goal of this study was to determine the impact of the trial on surgeon practice patterns at our institution.
This is a review of practice patterns comparing the year before release of Z0011 to the year after an institutional multidisciplinary meeting discussing the results. Patients meeting Z0011 inclusion criteria were identified. Clinicopathologic data were compared between the cohorts.
There were 658 patients with clinical T1-2 tumors planned for breast conservation: 335 in the pre-Z0011 cohort and 323 post-Z0011. Sixty-two (19 %) patients were sentinel lymph node (SLN) positive in the pre-Z0011 group versus 42 (13 %) post-Z0011 (p = 0.06). Before Z0011, 85 % (53/62) of SLN-positive patients underwent axillary node dissection (ALND) versus 24 % (10/42) after Z0011 (p < 0.001). After Z0011, surgeons were more likely to perform ALND on patients with larger tumors (2.2 vs. 1.5 cm, p = 0.09), lobular histology (p = 0.01), fewer SLNs (1 vs. 3, p = 0.09), larger SLN metastasis size (4 vs. 2.5 mm, p = 0.19), extranodal extension present (20 vs. 6 %, p = 0.16), or a higher probability of positive non-SLNs (p = 0.03). Surgeons were less likely to perform intraoperative nodal assessment post-Z0011 (26 vs. 69 %, p < 0.001) resulting in decreased median operative times for SLN-negative patients (79 vs. 92 min, p < 0.001).
Surgeons at our institution have implemented Z0011 results for the majority of patients; however, clinicopathologic factors still impact the decision to perform ALND. Z0011 results have significantly impacted practice by decreasing rates of ALND, use of intraoperative nodal evaluation, and operative times.
PMCID: PMC4403637  PMID: 22847123
6.  Delays in Primary Surgical Treatment Are Not Associated with Significant Tumor Size Progression in Breast Cancer Patients 
Annals of surgery  2011;254(1):119-124.
We evaluated the effect of time to surgery on tumor growth by comparing initial imaging and pathologic tumor size estimates. We also determined predictors of delay to surgery.
Summary Background Data
Preoperative work-up, coordination of reconstructive surgery, and referral to tertiary care centers can delay surgical treatment of breast cancer. Whether these delays are associated with interim tumor progression is unknown.
We identified 818 clinically node-negative breast cancer patients at our cancer center who had undergone surgery as their first therapeutic modality for invasive breast cancer from 9/2003 to 12/2006. Baseline tumor size was determined by mammography and sonography; tumor size at surgery was determined from pathology reports.
The median time from imaging to surgery was 21 days (1 to 132 days). In multivariate analysis, increased time to surgery was associated with older age, total mastectomy versus breast-conserving surgery, and reconstructive surgery. The median difference from baseline mammographic tumor size to surgery was 0 cm (8.6 cm smaller to 7.3 cm larger at surgery). The median difference from baseline sonographic tumor size to surgery was 0.1 cm (7.5 cm smaller to 8.3 cm larger at surgery). Neither of these differences was significantly associated with time to surgery. Time to surgery was associated with positive lymph nodes at surgery; however, no association was found after controlling for other prognostic factors.
Modest time intervals from imaging to surgery are not significantly associated with change in tumor size; thus, patients may undergo preoperative work-up without experiencing significant disease progression.
PMCID: PMC4345121  PMID: 21494124
8.  Lymphovascular Invasion and Lobular Histology are Associated with Increased Incidence of Isolated Tumor Cells in Sentinel Lymph Nodes from Early-Stage Breast Cancer Patients 
Annals of surgical oncology  2008;15(12):3369-3377.
Isolated tumor cells (ITC) are more likely to be identified when serial sectioning and immunohistochemical staining are used to evaluate sentinel lymph nodes (SLN). Our goal was to identify clinicopathologic features associated with ITC in patients undergoing sentinel lymph node dissection (SLND).
We reviewed clinicopathologic data for 3557 patients with no clinical evidence of lymph node metastases undergoing SLND between November 1993 and March 2007. Patients were staged according to the 6th edition of the American Joint Committee on Cancer staging system, with metastasis ≤.2 mm classified as ITC.
A SLN was identified in 3475 patients (97.7%), including 2518 (72.4%) with negative nodes and 169 (4.9%) with ITC. A statistically significant association existed between lobular histology and the identification of ITC; 13.6% of patients with ITC had lobular histology versus 7.3% of patients with a negative SLN (P = .003). The presence of lymphovascular invasion (LVI) was also associated with ITC; 18.3% of patients with ITC had LVI in the primary tumor versus 8.5% of patients with a negative SLN (P < .001). No difference existed between patients with and without ITC with respect to T stage, grade, estrogen receptor, progesterone receptor, HER2/neu status, or biopsy method.
The association between ITC and LVI, a known predictor of poor outcome, suggests ITC may have clinical relevance. The relationship between lobular histology and ITC is consistent with the known pattern of lobular metastases, which frequently present as small foci requiring immunohistochemistry for detection. Longer follow-up is needed to determine whether ITC have prognostic significance.
PMCID: PMC4331098  PMID: 18815841
The aim of this study was to investigate the role of postmastectomy radiation therapy in women with breast cancer who achieved a pathologic complete response (pCR) to neoadjuvant chemotherapy.
Methods and Materials
We retrospectively identified 226 patients treated at our institution who achieved a pCR at surgery after receiving neoadjuvant chemotherapy. Of these, the 106 patients without inflammatory breast cancer who were treated with mastectomy were analyzed. The patients’ clinical stages at diagnosis were I in 2%, II in 31%, IIIA in 30%, IIIB in 25%, and IIIC in 11% (American Joint Committee on Cancer 2003 system). Of the patients, 92% received anthracycline-based chemotherapy, and 38% also received a taxane. A total of 72 patients received postmastectomy radiation therapy, and 34 did not. The actuarial rates of local-regional recurrence (LRR) and survival of the two groups were compared using the log-rank test.
The median follow-up of surviving patients was 62 months. Use of radiation therapy did not affect the 10-year rates of LRR for patients with Stage I or II disease (the 10-year LRR rates were 0% for both groups). However, the 10-year LRR rate for patients with Stage III disease was significantly improved with radiation therapy (7.3% ± 3.5% with vs. 33.3% ± 15.7% without; p = 0.040). Within this cohort, use of radiation therapy was also associated with improved disease-specific and overall survival.
Postmastectomy radiation therapy provides a significant clinical benefit for breast cancer patients who present with clinical Stage III disease and achieve a pCR after neoadjuvant chemothearpy.
PMCID: PMC4329732  PMID: 17418973
Neoadjuvant chemotherapy; Pathologic complete response; pCR; Breast cancer; Postmastectomy radiation
10.  Increased gene copy number of the vesicle SNARE VAMP7 disrupts human male urogenital development through altered estrogen action 
Nature medicine  2014;20(7):715-724.
Vesicle transport is intimately connected with key nuclear functions and transcriptional regulation. Here, children born with congenital genitourinary tract masculinization disorders were analyzed by array-Comparative Genomic Hybridization, which revealed the presence of de novo copy number gains on Xq28 encompassing the VAMP7 gene encoding a vesicle-trafficking protein. Humanized VAMP7 BAC transgenic mice displayed cryptorchidism, urethral defects, and hypospadias. Mutant mice exhibited reduced penile length, focal spermatogenic anomalies, diminished sperm motility, and subfertility. VAMP7 colocalized with estrogen receptor alpha (ESR1) in the presence of ligand. Elevated levels of VAMP7 markedly intensified ESR1 transcriptional activity by increasing ESR1 protein cellular content upon ligand stimulation and up-regulated the expression of estrogen-responsive genes including ATF3, CYR61, and CTGF, all of which are implicated in human hypospadias. Hence, increased gene dosage of the SNARE protein, VAMP7, enhances estrogen receptor action in male genitourinary tissues, affects the virilization of the reproductive tract, and results in genitourinary birth defects in humans.
PMCID: PMC4283218  PMID: 24880616
11.  Genomic Copy Number Imbalances Associated with Bone and Non-bone Metastasis of Early-Stage Breast Cancer 
To identify and validate copy number aberrations in early-stage primary breast tumors associated with bone or non-bone metastasis.
Patients and Methods
Whole-genome molecular inversion probe arrays were used to evaluate copy number imbalances (CNIs) in breast tumors from 960 early-stage patients with information about site of metastasis. The CoxBoost algorithm was used to select metastasis site-related CNIs and to fit a Cox proportional hazards model.
Gains at 1q41 and 1q42.12 and losses at 1p13.3, 8p22, and Xp11.3 were significantly associated with bone metastasis. Gains at 2p11.2, 3q21.3–22.2, 3q27.1, 10q23.1, and 14q13.2–3 and loss at 7q21.11 were associated with non-bone metastasis. To examine the joint effect of CNIs and clinical predictors, patients were stratified into three risk groups (low, intermediate, and high) based on the sum of predicted linear hazard ratios (HRs). For bone metastasis, the hazard (95% confidence interval) for the low-risk group was 0.32 (0.11–0.92) compared to the intermediate-risk group and 2.99 (1.74–5.11) for the high-risk group. For non-bone metastasis, the hazard for the low-risk group was 0.34 (0.17–0.66) and 2.33 (1.59–3.43) for the high-risk group. The prognostic value of loss at 8p22 for bone metastasis and gains at 10q23.1 for non-bone metastasis, and gain at 11q13.5 for both bone and non-bone metastases were externally validated in 335 breast tumors pooled from four independent cohorts.
Distinct CNIs are independently associated with bone and non-bone metastasis for early-stage breast cancer patients across cohorts. These data warrant consideration for tailoring surveillance and management of metastasis risk.
PMCID: PMC3993091  PMID: 24305980
Breast cancer; bone metastasis; non-bone metastasis; copy number imbalances; molecular inversion probe array
12.  Elafin is downregulated during breast and ovarian tumorigenesis but its residual expression predicts recurrence 
Breast Cancer Research : BCR  2014;16(6):3417.
Elafin is an endogenous serine protease inhibitor. The majority of breast cancer cell lines lack elafin expression compared to human mammary epithelial cells. In this study, we hypothesized that elafin is downregulated during breast and ovarian tumorigenesis.
We examined elafin expression by immunohistochemistry (IHC) in specimens of normal breast tissue (n = 24), ductal carcinoma in situ (DCIS) (n = 54), and invasive breast cancer (n = 793). IHC analysis of elafin expression was also performed in normal fallopian tube tissue (n = 20), ovarian cystadenomas (n = 9), borderline ovarian tumors (n = 21), and invasive ovarian carcinomas (n = 216). To understand the significance of elafin in luminal breast cancer cell lines, wild-type or M25G elafin (lacking the protease inhibitory function) were exogenously expressed in MCF-7 and T47D cells.
Elafin expression was downregulated in 24% of DCIS and 83% of invasive breast tumors when compared to elafin expression in the normal mammary epithelium. However, the presence of elafin-positive cells in invasive breast tumors, even at low frequency, correlated with poor recurrence-free survival (RFS), reduced overall survival (OS), and clinicopathological markers of aggressive tumor behavior. Elafin-positive cells were an especially strong and independent prognostic marker of reduced RFS in IHC-defined luminal A-like tumors. Elafin was also downregulated in 33% of ovarian cystadenomas, 43% of borderline ovarian tumors, and 86% of invasive ovarian carcinomas when compared to elafin expression in the normal fallopian tube. In ovarian tumors, elafin-positive cells were correlated with reduced RFS, OS and disease-specific survival (DSS) only in stage I/II patients and not in stage III/IV patients. Notably, exogenous expression of elafin or elafin M25G in the luminal breast cancer cell lines MCF-7 and T47D significantly decreased cell proliferation in a protease inhibitory domain-independent manner.
Elafin predicts poor outcome in breast and ovarian cancer patients and delineates a subset of endocrine receptor-positive breast cancer patients susceptible to recurrence who could benefit from more aggressive intervention. Our in vitro results suggest that elafin arrests luminal breast cancer cells, perhaps suggesting a role in tumor dormancy.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0497-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4326485  PMID: 25551582
13.  Breast Cancer Molecular Signatures as Determined by SAGE: Correlation with Lymph Node Status 
Molecular cancer research : MCR  2007;5(9):881-890.
Global gene expression measured by DNA microarray platforms have been extensively used to classify breast carcinomas correlating with clinical characteristics, including outcome. We generated a breast cancer Serial Analysis of Gene Expression (SAGE) high-resolution database of ~2.7 million tags to perform unsupervised statistical analyses to obtain the molecular classification of breast-invasive ductal carcinomas in correlation with clinicopathologic features. Unsupervised statistical analysis by means of a random forest approach identified two main clusters of breast carcinomas, which differed in their lymph node status (P = 0.01); this suggested that lymph node status leads to globally distinct expression profiles. A total of 245 (55 up-modulated and 190 down-modulated) transcripts were differentially expressed between lymph node (+) and lymph node (−) primary breast tumors (fold change, ≥2; P < 0.05). Various lymph node (+) up-modulated transcripts were validated in independent sets of human breast tumors by means of real-time reverse transcription-PCR (RT-PCR). We validated significant overexpression of transcripts for HOXC10 (P = 0.001), TPD52L1 (P = 0.007), ZFP36L1 (P = 0.011), PLINP1 (P = 0.013), DCTN3 (P = 0.025), DEK (P = 0.031), and CSNK1D (P = 0.04) in lymph node (+) breast carcinomas. Moreover, the DCTN3 (P = 0.022) and RHBDD2 (P = 0.002) transcripts were confirmed to be overexpressed in tumors that recurred within 6 years of follow-up by real-time RT-PCR. In addition, meta-analysis was used to compare SAGE data associated with lymph node (+) status with publicly available breast cancer DNA microarray data sets. We have generated evidence indicating that the pattern of gene expression in primary breast cancers at the time of surgical removal could discriminate those tumors with lymph node metastatic involvement using SAGE to identify specific transcripts that behave as predictors of recurrence as well.
PMCID: PMC4186709  PMID: 17855657
14.  DEAR1 is a Chromosome 1p35 Tumor Suppressor and Master Regulator of TGFβ-Driven Epithelial-Mesenchymal Transition 
Cancer discovery  2013;3(10):1172-1189.
Deletion of chromosome 1p35 is a common event in epithelial malignancies. We report that DEAR1 (annotated as TRIM62) is a chromosome 1p35 tumor suppressor that undergoes mutation, copy number variation and loss of expression in human tumors. Targeted disruption in the mouse recapitulates this human tumor spectrum with both Dear1−/− and Dear1+/− mice developing primarily epithelial adenocarcinomas and lymphoma with evidence of metastasis in a subset of mice. DEAR1 loss of function in the presence of TGFβ results in failure of acinar morphogenesis, upregulation of EMT markers, anoikis resistance, migration and invasion. Furthermore, DEAR1 blocks TGFβ-SMAD3 signaling resulting in decreased nuclear phosphorylated SMAD3 by binding to and promoting the ubiquitination of SMAD3, the major effector of TGFβ-induced EMT. Moreover, DEAR1 loss increases levels of SMAD3 downstream effectors, SNAI1 and SNAI2, with genetic alteration of DEAR1/SNAI2 serving as prognostic markers of overall poor survival in an 889 invasive breast cancer cohort.
PMCID: PMC4107927  PMID: 23838884
DEAR1; tumor suppressor; EMT; TGFβ; Signaling; SMAD3 ubiquitination
15.  From Mice to Humans: Identification of Commonly Deregulated Genes in Mammary Cancer via Comparative SAGE Studies 
Cancer research  2004;64(21):7748-7755.
Genetically engineered mouse mammary cancer models have been used over the years as systems to study human breast cancer. However, much controversy exists on the utility of such models as valid equivalents to the human cancer condition. To perform an interspecies gene expression comparative study in breast cancer we used a mouse model that most closely resembles human breast carcinogenesis. This system relies on the transplant of p53 null mouse mammary epithelial cells into the cleared mammary fat pads of syngeneic hosts. Serial analysis of gene expression (SAGE) was used to obtain gene expression profiles of normal and tumor samples from this mouse mammary cancer model (>300,000 mouse mam-mary-specific tags). The resulting mouse data were compared with 25 of our human breast cancer SAGE libraries (>2.5 million human breast-specific tags). We observed significant similarities in the deregulation of specific genes and gene families when comparing mouse with human breast cancer SAGE data. A total of 72 transcripts were identified as commonly deregulated in both species. We observed a systematic and significant down-regulation in all of the tumors from both species of various cytokines, including CXCL1 (GRO1), LIF, interleukin 6, and CCL2. All of the mouse and most human mammary tumors also displayed decreased expression of genes known to inhibit cell proliferation, including NFKBIA (IKBα), GADD45B, and CDKN1A (p21); transcription-related genes such as CEBP, JUN, JUNB, and ELF1; and apoptosis-related transcripts such as IER3 and GADD34/PPP1R15A. Examples of overexpressed transcripts in tumors from both species include proliferation-related genes such as CCND1, CKS1B, and STMN1 (oncoprotein 18); and genes related to other functions such as SEPW1, SDFR1, DNCI2, and SP110. Importantly, abnormal expression of several of these genes has not been associated previously with breast cancer. The consistency of these observations was validated in independent mouse and human mammary cancer sets.
This is the first interspecies comparison of mammary cancer gene expression profiles. The comparative analysis of mouse and human SAGE mammary cancer data validates this p53 null mouse tumor model as a useful system closely resembling human breast cancer development and progression. More importantly, these studies are allowing us to identify relevant biomarkers of potential use in human studies while leading to a better understanding of specific mechanisms of human breast carcinogenesis.
PMCID: PMC4170686  PMID: 15520179
16.  Identification of Novel Amplification Gene Targets in Mouse and Human Breast Cancer at a Syntenic Cluster Mapping to Mouse Identification of Novel Amplification Gene Targets in Mouse and Human Breast Cancer at a Syntenic Cluster Mapping to Mouse ch8A1 and Human ch13q34 
Cancer research  2007;67(9):4104-4112.
Serial analysis of gene expression from aggressive mammary tumors derived from transplantable p53 null mouse mammary outgrowth lines revealed significant up-regulation of Tfdp1 (transcription factor Dp1), Lamp1 (lysosomal membrane glycoprotein 1) and Gas6 (growth arrest specific 6) transcripts. All of these genes belong to the same linkage cluster, mapping to mouse chromosome band 8A1. BAC-array comparative genomic hybridization and fluorescence in situ hybridization analyses revealed genomic amplification at mouse region ch8A1.1. The minimal region of amplification contained genes Cul4a, Lamp1, Tfdp1, and Gas6, highly overexpressed in the p53 null mammary outgrowth lines at preneoplastic stages, and in all its derived tumors. The same amplification was also observed in spontaneous p53 null mammary tumors. Interestingly, this region is homologous to human chromosome 13q34, and some of the same genes were previously observed amplified in human carcinomas. Thus, we further investigated the occurrence and frequency of gene amplification affecting genes mapping to ch13q34 in human breast cancer. TFDP1 showed the highest frequency of amplification affecting 31% of 74 breast carcinomas analyzed. Statistically significant positive correlation was observed for the amplification of CUL4A, LAMP1, TFDP1, and GAS6 genes (P < 0.001). Meta-analysis of publicly available gene expression data sets showed a strong association between the high expression of TFDP1 and decreased overall survival (P = 0.00004), relapse-free survival (P = 0.0119), and metastasis-free interval (P = 0.0064). In conclusion, our findings suggest that CUL4A, LAMP1, TFDP1, and GAS6 are targets for overexpression and amplification in breast cancers. Therefore, overexpression of these genes and, in particular, TFDP1 might be of relevance in the development and/or progression in a significant subset of human breast carcinomas.
PMCID: PMC4166497  PMID: 17483321
17.  Src family kinases as novel therapeutic targets to treat breast cancer brain metastases 
Cancer research  2013;73(18):5764-5774.
Despite better control of early stage disease and improved overall survival of patients with breast cancer, the incidence of life-threatening brain metastases continues to increase in some of these patients. Unfortunately, other than palliative treatments there is no effective therapy for this condition. In this study, we reveal a critical role for Src activation in promoting brain metastasis in a preclinical model of breast cancer, and we show how a Src-targeting combinatorial regimen can treat HER2+ brain metastases in this model. We found that Src was hyperactivated in brain-seeking breast cancer cells derived from human cell lines or from patients’ brain metastases. Mechanistically, Src activation promoted tumor cell extravasation into the brain parenchyma via permeabilization of the blood-brain barrier. When combined with the EGFR/HER2 dual-targeting drug lapatinib, a Src-targeting combinatorial regimen prevented outgrowth of disseminated breast cancer cells_through the induction of cell cycle arrest. More importantly, this combinatorial regimen inhibited the outgrowth of established experimental brain metastases, prolonging the survival of metastases-bearing mice. Our results provide a rationale for clinical evaluation of Src-targeting regimens to treat breast cancer patients suffering from brain metastasis.
PMCID: PMC3781592  PMID: 23913825
Src; Breast cancer; Brain metastasis; Blood brain barrier; Lapatinib; Saracatinib
18.  Genitourinary Defects Associated with Genomic Deletions in 2p15 Encompassing OTX1 
PLoS ONE  2014;9(9):e107028.
Normal development of the genitourinary (GU) tract is a complex process that frequently goes awry. In male children the most frequent congenital GU anomalies are cryptorchidism (1–4%), hypospadias (1%) and micropenis (0.35%). Bladder exstrophy and epispadias complex (BEEC) (1∶47000) occurs less frequently but significantly impacts patients' lives. Array comparative genomic hybridization (aCGH) identified seven individuals with overlapping deletions in the 2p15 region (66.0 kb-5.6 Mb). Six of these patients have GU defects, while the remaining patient has no GU defect. These deletions encompass the transcription factor OTX1. Subjects 2–7 had large de novo CNVs (2.39–6.31 Mb) and exhibited features similar to those associated with the 2p15p16.1 and 2p15p14 microdeletion syndromes, including developmental delay, short stature, and variable GU defects. Subject-1 with BEEC had the smallest deletion (66 kb), which deleted only one copy of OTX1. Otx1-null mice have seizures, prepubescent transient growth retardation and gonadal defects. Two subjects have short stature, two have seizures, and six have GU defects, mainly affecting the external genitalia. The presence of GU defects in six patients in our cohort and eight of thirteen patients reported with deletions within 2p14p16.1 (two with deletion of OTX1) suggest that genes in 2p15 are important for GU development. Genitalia defects in these patients could result from the effect of OTX1 on pituitary hormone secretion or on the regulation of SHH signaling, which is crucial for development of the bladder and genitalia.
PMCID: PMC4159299  PMID: 25203062
19.  Frequent loss of WWOX expression in breast cancer: correlation with estrogen receptor status 
WWOX is a cancer gene, spanning the common chromosomal fragile site 16D. Genomic and expression aberrations affecting this gene and locus are common in various neoplasias including breast cancer. The aim of the present study was to evaluate the relationship between WWOX expression at the protein level with respect to clinico-pathological characteristics. We performed immunohistochemical analyses on breast specific tissue microarrays representing, human normal breast epithelium (n=16), ductal carcinoma in situ (n=15) and invasive breast cancer cases (n=203). Staining intensity measurements were objectively determined utilizing an image analysis system. Western blot analyses were also performed on an independent set of 23 invasive breast carcinomas. All normal breast epithelial samples express WWOX protein abundantly while 34% (69/203 cases) of invasive breast carcinomas were ‘completely negative’ for WWOX expression and an additional 26% (52/203) of cases expressed WWOX very weakly. For DCIS samples five out of 15 (33%) were negative or weak for WWOX staining. Interestingly, we found a statistically significant correlation between WWOX expression and estrogen receptor (ER) status, 27% of ER+ breast carcinomas were completely negative for WWOX expression versus 46% for ER−cases ( p = 0.0054). Furthermore, when negative plus weakly WWOX stained cases were considered the difference became more significant with 51% of ER+ cases and 73% for the ER − group, with a p=0.003. These data indicate that loss of WWOX expression is a common event in breast cancer. It is unclear at this point whether loss of WWOX expression is a consequence of tumor progression or represents a subclass of breast carcinomas. The strong association of WWOX expression with ER status reinforces the suggested role of this protein as an enzyme involved in sex steroid metabolism.
PMCID: PMC4145848  PMID: 15692750
breast cancer; estrogen receptor; FRA16D; tumor suppressor; WWOX
20.  Age and Survival Estimates in Patients Who Have Node-Negative T1ab Breast Cancer by Breast Cancer Subtype 
Clinical breast cancer  2011;11(5):325-331.
The treatment of tumors ≤1 cm are difficult to treat as recurrence rates are difficult to assess. The purpose of this study was to assess recurrence by underlying triple receptor subtype and by age, both of which had significant impact on outcomes.
This article evaluates the risk of recurrence for patients who have small node-negative breast cancer by age and tumor subtype.
One thousand twelve patients with a T1a,bN0 breast cancer diagnosed between 1990 and 2002 who did not receive chemotherapy or trastuzumab were included. Patients and tumor characteristics were compared using the χ2 or Wilcoxon’s rank sum tests. Survival outcomes were estimated with the Kaplan-Meier method and compared using the log-rank statistic. Cox proportional hazards models were used to determine association of breast cancer subtypes and age at diagnosis with other covariates.
Median age was 51.5 years. There were 771 hormone receptor (HR)-positive, 98 HER2-positive, and 143 triple-negative breast cancers (TNBC). Six hundred ninety-three patients were > 50 years, and 33 patients were ≤ 35 years. For 5-year survival estimates, there were 118 deaths and overall survival was 94.6% (95% confidence interval [CI] = 93.2%, 96.1%). After adjusting for breast cancer subtype and other tumor characteristics, patients ≤ 35 had 2.51 (95% CI = 1.21–5.22) times greater risk of worse recurrence-free survival (RFS), and 2.60 (95% CI = 1.05–6.46) times greater risk of worse distant RFS (DRFS) compared to patients > 50 years old. Compared to patients with HR-positive disease, patients with HER2-positive breast cancer had 4.98 (95% CI = 2.91–8.53) times the risk of worse RFS and 4.70 (95% CI = 2.51–8.79) times greater risk of worse DRFS, and patients with TNBC had 2.71 (95% CI = 1.59–4.59) times greater risk of worse RFS and 2.08 (95% CI = 1.04–4.17) times greater risk of worse DRFS.
In this cohort, patients with T1a,bN0 breast cancer, young age and breast cancer subtype were significantly associated with RFS and DRFS.
PMCID: PMC4104010  PMID: 21764391
Distant recurrence-free survival; HER2; Hormone receptor; Recurrence-free survival; Triple-negative breast cancer
21.  The Relationship Between Eight GWAS-Identified Single-Nucleotide Polymorphisms and Primary Breast Cancer Outcomes 
The Oncologist  2013;18(5):493-500.
Several single-nucleotide polymorphisms (SNPs) associated with breast cancer risk have been identified through genome-wide association studies. This study investigated the association of eight risk SNPs with breast cancer disease-free survival and overall survival rates. Results suggest that two previously identified breast cancer risk susceptibility loci may influence breast cancer prognosis or comorbid conditions associated with overall survival.
Learning Objectives
Describe the results of genome-wide association studies (GWAS) that have identified genetic variants associated with breast cancer risk.Discuss whether genetic risk variants identified through genome-wide association studies (GWAS) are also associated with breast cancer prognosis.Describe molecular mechanisms through which germline genetic variants may influence breast cancer survival.
Several single-nucleotide polymorphisms (SNPs) associated with breast cancer risk have been identified through genome-wide association studies (GWAS). We investigated whether eight risk SNPs identified in GWAS were associated with breast cancer disease-free survival (DFS) and overall survival (OS) rates.
Patients and Methods.
A cohort of 739 white women with early-stage breast cancer was genotyped for eight GWAS-identified SNPs (rs2981582, rs1219648 [FGFR2], rs3803662, rs12443621, rs8051542 [TOX3], rs999737 [RAD51L1], rs6504950 [17q23], and rs4973768 [3p24]). Relationships between SNPs and breast cancer outcomes were evaluated using Cox proportional hazard regression models. The cumulative effects of SNPs on breast cancer outcomes were assessed by computing the number of at-risk genotypes.
At a median follow-up of 121 months (range: 188–231 months) for survivors, 237 deaths (32%) and 186 breast cancer events (25%) were identified among the 739 patients. After adjusting for age, clinical stage, and treatment, rs12443621 (16q12; p = .03) and rs6504950 (17q23; p = .008) were prognostic for OS but not DFS. A higher risk for death was also found in the multivariable analysis of patients harboring three or four at-risk genotypes of the GWAS SNPs compared to patients carrying two or less at-risk genotypes (hazard ratio: 1.60, 95% confidence interval: 1.23–2.24; p = .0008).
The study results suggest that previously identified breast cancer risk susceptibility loci, rs12443621 (16q12) and rs6504950 (17q23), may influence breast cancer prognosis or comorbid conditions associated with overall survival. The precise molecular mechanisms through which these risk SNPs, as well as others that were not included in the analysis, influence clinical outcomes remain to be determined.
PMCID: PMC3662839  PMID: 23635555
Breast cancer; Prognosis; Single-nucleotide polymorphisms; TNRC9; 17q23
22.  Prognostic Value of Single Nucleotide Polymorphisms of Candidate Genes Associated with Inflammation in Early Stage Breast Cancer 
To examine the role of germline genetic variations in inflammatory pathways as modifiers of time to recurrence (TTR) in patients with early stage breast cancer (BC), DNA from 997 early stage BC patients was genotyped for 53 tagging single nucleotide polymorphisms (SNPs) in 12 genes involved in inflammation. SNPs were analyzed separately for Caucasians versus African Americans and Hispanics. Cox proportional hazards models were used to evaluate the association between SNPs in the inflammatory genes and time to recurrence (TTR), adjusted for clinical and pathologic covariates. In univariable analyses of Caucasian women, the homozygous genotype of 12 SNPs, including 6 NFKB1 SNPs, 4 IL4 SNPs, and 2 IL13 SNPs, were significantly associated with a decrease in TTR compared with the heterozygous and/ or corresponding homozygous genotype (P <0.05). The significant NFKB1 and IL4 SNPs were in an area of high linkage disequilibrium (D'>0.8). After adjusting for stage, age, and treatment, carriage of the homozygous genotypes for NFKB1rs230532 and IL13rs1800925 were independently associated with a shorter TTR (P=0.001 and p=0.034, respectively). In African-American and Hispanic patients, expression of NFKB1rs3774932, TNFrs1799964, and IL4rs3024543 SNPs were associated with a shorter TTR in univariable model. Only NFKB1 rs3774932 (P=0.02) and IL4Rrs3024543 (P=0.03) had independent prognostic value in the multivariable model These data support the existence of host genetic susceptibility as a component in recurrence risk mediated by pro-inflammatory and immune factors, and suggest the potential for drugs which modify immune responses and inflammatory genes to improve prognosis in early stage BC.
PMCID: PMC3746974  PMID: 23529385
gene polymorphisms; inflammation; breast cancer
23.  Genetic variants in the vitamin D pathway and breast cancer disease-free survival 
Carcinogenesis  2012;34(3):587-594.
Epidemiological studies have investigated the association between vitamin D pathway genes and breast cancer risk; however, little is known about the association between vitamin D pathway genes and breast cancer prognosis. In a retrospective cohort of 1029 patients with early-stage breast cancer, we analyzed the association between 106 tagging single nucleotide polymorphisms (SNPs) in eight vitamin D pathway genes and breast cancer disease-free survival (DFS) using Cox regression analysis adjusted for known prognostic variables. Using a false discovery rate of 10%, six intronic SNPs were significantly associated with poorer DFS: retinoid-X receptor alpha (RXRA) SNPs (rs881658, rs11185659, rs10881583, rs881657 and rs7864987) and plasminogen activator and urokinase receptor (PLAUR) SNP (rs4251864). Treatment received (no systemic therapy, hormone therapy alone or chemotherapy) was an effect modifier of the RXRA SNPs association with DFS (P < 0.05); therefore, we stratified further analysis by treatment group. Among patients who did not receive systemic therapy, RXRA SNP [rs10881583 (P = 0.02)] was associated with poorer DFS, and among patients who received chemotherapy, RXRA SNPs (rs881658, rs11185659, rs10881583, rs881657 and rs7864987) were associated with poorer DFS (P < 0.001 for all SNPs). However, RXRA SNPs: rs10881583 (P < 0.001) and rs881657 (P = 0.02) were associated with improved DFS in patients treated with hormone therapy alone. Our results suggest that SNPs in the RXRA and PLAUR genes in the vitamin D pathway may contribute to breast cancer DFS. In particular, SNPs in RXRA may predict for poorer or improved DFS in patients, according to type of systemic treatment received. If validated, these markers could be used for risk stratification of breast cancer patients.
PMCID: PMC3581599  PMID: 23180655
24.  Outcomes of Sentinel Lymph Node Dissection Alone vs. Axillary Lymph Node Dissection in Early Stage Invasive Lobular Carcinoma: A Retrospective Study of the Surveillance, Epidemiology and End Results (SEER) Database 
PLoS ONE  2014;9(2):e89778.
The American College of Surgeons Oncology Group (ACOSOG) Z0011 trial demonstrated no difference in local-regional recurrence (LRR), disease-specific survival (DSS) or overall survival (OS) for sentinel lymph node dissection (SLND) and completion axillary lymph node dissection (ALND) among patients undergoing breast-conserving therapy for clinical T1–T2, N0 breast cancer with 1 or 2 positive SLNs. However, Only 7% of study participants had invasive lobular carcinoma (ILC). Because ILC has a different pattern of metastases, frequently presenting as small foci requiring immunohistochemistry for detection, the applicability of ACOSOG Z0011 trial data to ILC patients is unclear.
Study Design
We identified all ILC patients in the Surveillance, Epidemiology, and End Results (SEER) database (1998–2009) who met the ACOSOG Z0011 eligibility criteria. Patients were evaluated on the basis of the extent of axillary surgery (SLND alone or ALND), and the clinical outcomes of these 2 groups were compared.
1269 patients (393 SLND and 876 ALND) were identified from the SEER database. At a median follow-up time of 71 months, there were no differences in OS or disease-specific survival between the two groups.
SLND alone may result in outcomes comparable to those achieved with ALND for patients with early-stage ILC who meet the ACOSOG Z0011 eligibility criteria.
PMCID: PMC3934955  PMID: 24587029
25.  Hormone Receptor Status Influences the Locoregional Benefit of Trastuzumab in Patients with Non-metastatic Breast Cancer 
Cancer  2012;118(20):4936-4943.
Previous studies have shown that hormone receptor (HR) and HER2 status influence the outcome of locoregional treatments. However, the interrelationship of these factors with trastuzumab is unclear. We sought to determine the role of HR and HER2 status on the locoregional benefit of trastuzumab treatment in patients with non-metastatic breast cancer.
Locoregional outcomes of 5683 women treated in our institution from 2000–2008 for invasive breast cancer were analyzed using Kaplan-Meier and Cox regression methods to compare 6 subgroups: HR+/HER2−, HR−/HER2− (triple-negative), HR+/HER2+ with or without trastuzumab, and HR−/HER2+ with or without trastuzumab.
Overall, LRR was 5% at 5 years among patients with HER2+ disease. Patients with HR+/HER2+ disease treated with trastuzumab had half the rate of LRR as patients that did not receive trastuzumab, whereas patients with HR−/HER2+ disease had similar rates of LRR regardless of trastuzumab treatment. On Cox regression analysis comparing LRR risk to the cohort with HR+/HER2− disease, only the HR+/HER2+ cohort treated with trastuzumab had similar LRR risk (HR 1.24, 95% CI 0.56–2.73, p=0.591). All other subgroups, including the HR+/HER2+ cohort who did not receive trastuzumab, had significantly worse outcomes. LRR risk was highest among patients with triple-negative disease (HR 4.73, 95% CI 3.42–6.54, p<0.001).
Among patients with HR+/HER2+ disease, treatment with trastuzumab reduces LRR risk to the more favorable outcome of patients with HR+/HER2− disease. In contrast, the increased LRR risk among patients with HR−/HER2+ disease remains despite treatment with trastuzumab. Additional locoregional strategies are needed in this subgroup of patients.
PMCID: PMC3888990  PMID: 22511276
Hormone receptor; locoregional; trastuzumab; non-metastatic; breast cancer

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