Profiling molecular regulators of recurrence in chemorefractory triple-negative breast cancers.

BACKGROUND: Approximately two thirds of patients with localized triple-negative breast cancer (TNBC) harbor residual disease (RD) after neoadjuvant chemotherapy (NAC) and have a high risk-of-recurrence. Targeted therapeutic development for TNBC is of primary significance as no targeted therapies are clinically indicated for this aggressive subset. In view of this, we conducted a comprehensive molecular analysis and correlated molecular features of chemorefractory RD tumors with recurrence for the purpose of guiding downstream therapeutic development. METHODS: We assembled DNA and RNA sequencing data from RD tumors as well as pre-operative biopsies, lymphocytic infiltrate, and survival data as part of a molecular correlative to a phase II post-neoadjuvant clinical trial. Matched somatic mutation, gene expression, and lymphocytic infiltrate were assessed before and after chemotherapy to understand how tumors evolve during chemotherapy. Kaplan-Meier survival analyses were conducted categorizing cancers with TP53 mutations by the degree of loss as well as by the copy number of a locus of 18q corresponding to the SMAD2, SMAD4, and SMAD7 genes. RESULTS: Analysis of matched somatic genomes pre-/post-NAC revealed chaotic acquisition of copy gains and losses including amplification of prominent oncogenes. In contrast, significant gains in deleterious point mutations and insertion/deletions were not observed. No trends between clonal evolution and recurrence were identified. Gene expression data from paired biopsies revealed enrichment of actionable regulators of stem cell-like behavior and depletion of immune signaling, which was corroborated by total lymphocytic infiltrate, but was not associated with recurrence. Novel characterization of TP53 mutation revealed prognostically relevant subgroups, which were linked to MYC-driven transcriptional amplification. Finally, somatic gains in 18q were associated with poor prognosis, likely driven by putative upregulation of TGFß signaling through the signal transducer SMAD2. CONCLUSIONS: We conclude TNBCs are dynamic during chemotherapy, demonstrating complex plasticity in subclonal diversity, stem-like qualities, and immune depletion, but somatic alterations of TP53/MYC and TGFß signaling in RD samples are prominent drivers of recurrence, representing high-yield targets for additional interrogation.

A large microRNA cluster on chromosome 19 is a transcriptional hallmark of WHO type A and AB thymomas.

BACKGROUND: Thymomas are one of the most rarely diagnosed malignancies. To better understand its biology and to identify therapeutic targets, we performed next-generation RNA sequencing. METHODS: The RNA was sequenced from 13 thymic malignancies and 3 normal thymus glands. Validation of microRNA expression was performed on a separate set of 35 thymic malignancies. For cell-based studies, a thymoma cell line was used. RESULTS: Hierarchical clustering revealed 100% concordance between gene expression clusters and WHO subtype. A substantial differentiator was a large microRNA cluster on chr19q13.42 that was significantly overexpressed in all A and AB tumours and whose expression was virtually absent in the other thymomas and normal tissues. Overexpression of this microRNA cluster activates the PI3K/AKT/mTOR pathway. Treatment of a thymoma AB cell line with a panel of PI3K/AKT/mTOR inhibitors resulted in marked reduction of cell viability. CONCLUSIONS: A large microRNA cluster on chr19q13.42 is a transcriptional hallmark of type A and AB thymomas. Furthermore, this cluster activates the PI3K pathway, suggesting the possible exploration of PI3K inhibitors in patients with these subtypes of tumour. This work has led to the initiation of a phase II clinical trial of PI3K inhibition in relapsed or refractory thymomas (http://clinicaltrials.gov/ct2/show/NCT02220855).

Next-generation transcriptome sequencing of the premenopausal breast epithelium using specimens from a normal human breast tissue bank.

INTRODUCTION: Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. In order to provide the specimens that will facilitate such an understanding, The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is, to our knowledge, the only biorepository in the world prospectively established to collect normal, healthy breast tissue from volunteer donors. As a first initiative toward a molecular understanding of the biology and developmental genetics of the normal mammary gland, the effect of the menstrual cycle and hormonal contraceptives on DNA expression in the normal breast epithelium was examined. METHODS: Using normal breast tissue from 20 premenopausal donors to KTB, the changes in the mRNA of the normal breast epithelium as a function of phase of the menstrual cycle and hormonal contraception were assayed using next-generation whole transcriptome sequencing (RNA-Seq). RESULTS: In total, 255 genes representing 1.4% of all genes were deemed to have statistically significant differential expression between the two phases of the menstrual cycle. The overwhelming majority (221; 87%) of the genes have higher expression during the luteal phase. These data provide important insights into the processes occurring during each phase of the menstrual cycle. There was only a single gene significantly differentially expressed when comparing the epithelium of women using hormonal contraception to those in the luteal phase. CONCLUSIONS: We have taken advantage of a unique research resource, the KTB, to complete the first-ever next-generation transcriptome sequencing of the epithelial compartment of 20 normal human breast specimens. This work has produced a comprehensive catalog of the differences in the expression of protein-coding genes as a function of the phase of the menstrual cycle. These data constitute the beginning of a reference data set of the normal mammary gland, which can be consulted for comparison with data developed from malignant specimens, or to mine the effects of the hormonal flux that occurs during the menstrual cycle.

Characterizing the heterogeneity of triple-negative breast cancers using microdissected normal ductal epithelium and RNA-sequencing.

Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER, PR, and HER-2). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90 % of TNBCs revealing an over-expressed central network. In conclusion, use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos.

Initial Phase I Safety Study of Gedatolisib plus Cofetuzumab Pelidotin for Patients with Metastatic Triple-Negative Breast Cancer.

PURPOSE: The PI3K pathway is dysregulated in the majority of triple-negative breast cancers (TNBC), yet single-agent inhibition of PI3K has been ineffective in TNBC. PI3K inhibition leads to an immediate compensatory upregulation of the Wnt pathway. Dual targeting of both pathways is highly synergistic against TNBC models in vitro and in vivo. We initiated a phase I clinical trial combining gedatolisib, a pan-class I isoform PI3K/mTOR inhibitor, and cofetuzumab pelidotin, an antibody-drug conjugate against the cell-surface PTK7 protein (Wnt pathway coreceptor) with an auristatin payload. PATIENTS AND METHODS: Participants (pt) had metastatic TNBC or estrogen receptor (ER) low (ER and PgR < 5%, HER2-negative) breast cancer, and had received at least one prior chemotherapy for advanced disease. The primary objective was safety. Secondary endpoints included overall response rate (ORR), clinical benefit at 18 weeks (CB18), progression-free survival (PFS), and correlative analyses. RESULTS: A total of 18 pts were enrolled in three dose cohorts: gedatolisib 110 mg weekly + cofetuzumab pelidotin 1.4 mg/kg every 3 weeks (n = 4), 180 mg + 1.4 mg/kg (n = 3), and 180 mg + 2.8 mg/kg (n = 11). Nausea, anorexia, fatigue, and mucositis were common but rarely reached ≥grade 3 severity. Myelosuppression was uncommon. ORR was 16.7% (3/18). An additional 3 pts had stable disease (of these 2 had stable disease for >18 weeks); CB18 was 27.8%. Median PFS was 2.0 months (95% confidence interval for PFS: 1.2-6.2). Pts with clinical benefit were enriched with genomic alterations in the PI3K and PTK7 pathways. CONCLUSIONS: The combination of gedatolisib + cofetuzumab pelidotin was well tolerated and demonstrated promising clinical activity. Further investigation of this drug combination in metastatic TNBC is warranted.

BRE12-158: A Postneoadjuvant, Randomized Phase II Trial of Personalized Therapy Versus Treatment of Physician's Choice for Patients With Residual Triple-Negative Breast Cancer.

PURPOSE: Patients with triple-negative breast cancer (TNBC) with residual disease after neoadjuvant chemotherapy (NAC) have high risk of recurrence with prior data suggesting improved outcomes with capecitabine. Targeted agents have demonstrated activity across multiple cancer types. BRE12-158 was a phase II, multicenter trial that randomly allocated patients with TNBC with residual disease after NAC to genomically directed therapy versus treatment of physician choice (TPC). PATIENTS AND METHODS: From March 2014 to December 2018, 193 patients were enrolled. Residual tumors were sequenced using a next-generation sequencing test. A molecular tumor board adjudicated all results. Patients were randomly allocated to four cycles of genomically directed therapy (arm A) versus TPC (arm B). Patients without a target were assigned to arm B. Primary end point was 2-year disease-free survival (DFS) among randomly assigned patients. Secondary/exploratory end points included distant disease-free survival, overall survival, toxicity assessment, time-based evolution of therapy, and drug-specific outcomes. RESULTS: One hundred ninety-three patients were randomly allocated or were assigned to arm B. The estimated 2-year DFS for the randomized population only was 56.6% (95% CI, 0.45 to 0.70) for arm A versus 62.4% (95% CI, 0.52 to 0.75) for arm B. No difference was seen in DFS, distant disease-free survival, or overall survival for the entire or randomized populations. There was increased uptake of capecitabine for TPC over time. Patients randomly allocated later had less distant recurrences. Circulating tumor DNA status remained a significant predictor of outcome with some patients demonstrating clearance with postneoadjuvant therapy. CONCLUSION: Genomically directed therapy was not superior to TPC for patients with residual TNBC after NAC. Capecitabine should remain the standard of care; however, the activity of other agents in this setting provides rationale for testing optimal combinations to improve outcomes. Circulating tumor DNA should be considered a standard covariate for trials in this setting.

Profiling of Adipose and Skeletal Muscle in Human Pancreatic Cancer Cachexia Reveals Distinct Gene Profiles with Convergent Pathways.

The vast majority of patients with pancreatic ductal adenocarcinoma (PDAC) suffer cachexia. Although cachexia results from concurrent loss of adipose and muscle tissue, most studies focus on muscle alone. Emerging data demonstrate the prognostic value of fat loss in cachexia. Here we sought to identify the muscle and adipose gene profiles and pathways regulated in cachexia. Matched rectus abdominis muscle and subcutaneous adipose tissue were obtained at surgery from patients with benign conditions (n = 11) and patients with PDAC (n = 24). Self-reported weight loss and body composition measurements defined cachexia status. Gene profiling was done using ion proton sequencing. Results were queried against external datasets for validation. 961 DE genes were identified from muscle and 2000 from adipose tissue, demonstrating greater response of adipose than muscle. In addition to known cachexia genes such as FOXO1, novel genes from muscle, including PPP1R8 and AEN correlated with cancer weight loss. All the adipose correlated genes including SCGN and EDR17 are novel for PDAC cachexia. Pathway analysis demonstrated shared pathways but largely non-overlapping genes in both tissues. Age related muscle loss predominantly had a distinct gene profiles compared to cachexia. This analysis of matched, externally validate gene expression points to novel targets in cachexia.

Resequencing of the vascular endothelial growth factor promoter reveals haplotype structure and functional diversity.

Our group has previously reported that SNPs in the VEGF promoter are strongly associated with efficacy and toxicity to the anti-VEGF antibody bevacizumab in breast cancer. In order to better understand the biologic mechanism for our previously reported biomarkers, we embarked on a comprehensive evaluation of genetic variation in the VEGF promoter coupled with a study of its intrinsic function. We resequenced 48 Caucasians and 48 African-Americans for the VEGF promoter to identify SNPs and elucidate its haplotype structure. We further cloned the haplotypes into reporter constructs and assessed the role of SNPs on promoter function in breast cancer cell lines. SNPs that were identified included twenty previously reported SNPs/insertions/deletions, one novel SNP, and one novel deletion. Among these variants, we identified five SNPs that tag six haplotypes capturing 74% of the genetic variation of the promoter. Subsequently, assessment of the haplotypes in reporter constructs demonstrates significant variation in promoter induced expression among the haplotypes. In particular, two haplotypes had higher expression and one haplotype had lower expression across cell lines. Haplotypes containing SNPs previously reported to be associated with increased survival with the use of bevacizumab are high-expressing haplotypes, thus lending putative functional evidence to the prior clinical finding.

Association of Circulating Tumor DNA and Circulating Tumor Cells After Neoadjuvant Chemotherapy With Disease Recurrence in Patients With Triple-Negative Breast Cancer: Preplanned Secondary Analysis of the BRE12-158 Randomized Clinical Trial.

Importance: A significant proportion of patients with early-stage triple-negative breast cancer (TNBC) are treated with neoadjuvant chemotherapy. Sequencing of circulating tumor DNA (ctDNA) after surgery, along with enumeration of circulating tumor cells (CTCs), may be used to detect minimal residual disease and assess which patients may experience disease recurrence. Objective: To determine whether the presence of ctDNA and CTCs after neoadjuvant chemotherapy in patients with early-stage TNBC is independently associated with recurrence and clinical outcomes. Design, Setting, and Participants: A preplanned secondary analysis was conducted from March 26, 2014, to December 18, 2018, using data from 196 female patients in BRE12-158, a phase 2 multicenter randomized clinical trial that randomized patients with early-stage TNBC who had residual disease after neoadjuvant chemotherapy to receive postneoadjuvant genomically directed therapy vs treatment of physician choice. Patients had blood samples collected for ctDNA and CTCs at time of treatment assignment; ctDNA analysis with survival was performed for 142 patients, and CTC analysis with survival was performed for 123 patients. Median clinical follow-up was 17.2 months (range, 0.3-58.3 months). Interventions: Circulating tumor DNA was sequenced using the FoundationACT or FoundationOneLiquid Assay, and CTCs were enumerated using an epithelial cell adhesion molecule-based, positive-selection microfluidic device. Main Outcomes and Measures: Primary outcomes were distant disease-free survival (DDFS), disease-free survival (DFS), and overall survival (OS). Results: Among 196 female patients (mean [SD] age, 49.6 [11.1] years), detection of ctDNA was significantly associated with inferior DDFS (median DDFS, 32.5 months vs not reached; hazard ratio [HR], 2.99; 95% CI, 1.38-6.48; P = .006). At 24 months, DDFS probability was 56% for ctDNA-positive patients compared with 81% for ctDNA-negative patients. Detection of ctDNA was similarly associated with inferior DFS (HR, 2.67; 95% CI, 1.28-5.57; P = .009) and inferior OS (HR, 4.16; 95% CI,1.66-10.42; P = .002). The combination of ctDNA and CTCs provided additional information for increased sensitivity and discriminatory capacity. Patients who were ctDNA positive and CTC positive had significantly inferior DDFS compared with those who were ctDNA negative and CTC negative (median DDFS, 32.5 months vs not reached; HR, 5.29; 95% CI, 1.50-18.62; P = .009). At 24 months, DDFS probability was 52% for patients who were ctDNA positive and CTC positive compared with 89% for those who were ctDNA negative and CTC negative. Similar trends were observed for DFS (HR, 3.15; 95% CI, 1.07-9.27; P = .04) and OS (HR, 8.60; 95% CI, 1.78-41.47; P = .007). Conclusions and Relevance: In this preplanned secondary analysis of a randomized clinical trial, detection of ctDNA and CTCs in patients with early-stage TNBC after neoadjuvant chemotherapy was independently associated with disease recurrence, which represents an important stratification factor for future postneoadjuvant trials. Trial Registration: ClinicalTrials.gov Identifier: NCT02101385.

Exploratory study evaluating the association of polymorphisms of angiogenesis genes with hot flashes.

PURPOSE: Hot flashes are a common symptom and an important cause of decreased quality of life in women with breast cancer. Hot flashes involve vasodilatation and flushing, however, their complex etiology is not fully understood. We evaluated the association between germline polymorphisms in genes important to angiogenesis and subjective reporting of hot flashes. EXPERIMENTAL DESIGN: We recruited 1,244 subjects; 520 were breast cancer cases, 715 were documented healthy controls, and nine were of unknown status. Subjects were asked to provide a blood specimen and complete a questionnaire which included whether they had recently or had ever experienced hot flashes. We evaluated candidate polymorphisms in the following genes: hypoxia inducible factor-1 alpha (HIF1alpha), vascular endothelial growth factor (VEGF), VEGF-receptor 2 (VEGFR-2), endothelial nitric oxide synthase (eNOS), neuropilin-1 (NRP-1), and NRP-2. Testing for an association between polymorphisms and a history of current flashes or ever having hot flashes was performed. RESULTS: 441 premenopausal and 533 postmenopausal, Caucasian women were evaluable for hot flash analysis. For premenopausal women the eNOS-786 CT and TT genotypes were significantly associated with a greater likelihood of a subject reporting current hot flashes than the CC genotype (P = 0.03). After adjusting for clinical variables, the genotype association was no longer significant (P = 0.08). For postmenopausal women, the HIF1alpha 1744 CT and TT genotypes were significantly associated with a greater likelihood of a subject reporting current hot flashes (P = 0.05) and this remained significant after consideration of established clinical variables (P = 0.04). CONCLUSION: Hot flashes may be regulated by genes that control angiogenesis.

Next-generation sequencing of circulating tumor DNA to predict recurrence in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy.

Next-generation sequencing to detect circulating tumor DNA is a minimally invasive method for tumor genotyping and monitoring therapeutic response. The majority of studies have focused on detecting circulating tumor DNA from patients with metastatic disease. Herein, we tested whether circulating tumor DNA could be used as a biomarker to predict relapse in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy. In this study, we analyzed samples from 38 early-stage triple-negative breast cancer patients with matched tumor, blood, and plasma. Extracted DNA underwent library preparation and amplification using the Oncomine Research Panel consisting of 134 cancer genes, followed by high-coverage sequencing and bioinformatics. We detected high-quality somatic mutations from primary tumors in 33 of 38 patients. TP53 mutations were the most prevalent (82%) followed by PIK3CA (16%). Of the 33 patients who had a mutation identified in their primary tumor, we were able to detect circulating tumor DNA mutations in the plasma of four patients (three TP53 mutations, one AKT1 mutation, one CDKN2A mutation). All four patients had recurrence of their disease (100% specificity), but sensitivity was limited to detecting only 4 of 13 patients who clinically relapsed (31% sensitivity). Notably, all four patients had a rapid recurrence (0.3, 4.0, 5.3, and 8.9 months). Patients with detectable circulating tumor DNA had an inferior disease free survival (p < 0.0001; median disease-free survival: 4.6 mos. vs. not reached; hazard ratio = 12.6, 95% confidence interval: 3.06-52.2). Our study shows that next-generation circulating tumor DNA sequencing of triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy can predict recurrence with high specificity, but moderate sensitivity. For those patients where circulating tumor DNA is detected, recurrence is rapid.

Dual PI3K and Wnt pathway inhibition is a synergistic combination against triple negative breast cancer.

Triple negative breast cancer accounts for 15-20% of all breast cancer cases, but despite its lower incidence, contributes to a disproportionately higher rate of mortality. As there are currently no Food and Drug Administration-approved targeted agents for triple negative breast cancer, we embarked on a genomic-guided effort to identify novel targeted modalities. Analyses by our group and The Cancer Genome Atlas have identified activation of the PI3K-pathway in the majority of triple negative breast cancers. As single agent therapy is commonly subject to resistance, we investigated the use of combination therapy against compensatory pathways. Herein, we demonstrate that pan-PI3K inhibition in triple negative breast cancers results in marked activation of the Wnt-pathway. Using the combination of two inhibitors currently in clinical trial as single agents, buparlisib(pan-PI3K) and WNT974(WNT-pathway), we demonstrate significant in vitro and in vivo synergy against triple negative breast cancer cell lines and xenografts. Taken together, these observations provide a strong rationale for testing dual targeting of the PI3K and WNT-pathways in clinical trials.

Pancreatic Cyst Fluid Vascular Endothelial Growth Factor A and Carcinoembryonic Antigen: A Highly Accurate Test for the Diagnosis of Serous Cystic Neoplasm.

BACKGROUND: Accurate differentiation of pancreatic cystic lesions is important for pancreatic cancer early detection and prevention as well as avoidance of unnecessary surgical intervention. Serous cystic neoplasms (SCN) have no malignant potential, but may mimic premalignant mucinous cystic lesions: mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm (IPMN). We recently identified vascular endothelial growth factor (VEGF)-A as a novel pancreatic fluid biomarker for SCN. We hypothesize that combining cyst fluid carcinoembryonic antigen (CEA) with VEGF-A will improve the diagnostic accuracy of VEGF-A. METHODS: Pancreatic cyst/duct fluid was collected from consenting patients undergoing surgical cyst resection with corresponding pathologic diagnoses. Pancreatic fluid VEGF-A and CEA levels were detected by ELISA. RESULTS: One hundred forty-nine patients with pancreatic cystic lesions met inclusion criteria. Pathologic diagnoses included pseudocyst (n=14), SCN (n=26), MCN (n=40), low/moderate grade IPMN (n=34), high grade IPMN (n=20), invasive IPMN (n=10) and solid pseudopapillary neoplasm (n=5). VEGF-A was significantly elevated in SCN cyst fluid compared to all other diagnoses (p<0.001). With a threshold of >5,000 pg/ml, VEGF-A alone has 100% sensitivity and 83.7% specificity to distinguish SCN from other cystic lesions. With a threshold of ≤10ng/ml, CEA alone identifies SCN with 95.5% sensitivity and 81.5% specificity. Sensitivity and specificity of the VEGF-A/CEA combination are 95.5% and 100% respectively. The c-statistic increased from 0.98 to 0.99 when CEA was added to VEGF-A alone in the ROC analysis. CONCLUSIONS: Although VEGF-A alone is a highly accurate test for SCN, the combination of VEGF-A with CEA approaches the gold-standard of pathologic diagnosis, thus importantly avoiding false positives. Patients with a positive test indicating benign SCN can be spared a high risk surgical pancreatic resection.

Association of polymorphisms of angiogenesis genes with breast cancer.

BACKGROUND: Few studies have systematically explored a pathway approach: to test the association of multiple polymorphisms from multiple genes important to angiogenesis simultaneously with risk of breast cancer. We report our preliminary data evaluating the association of polymorphisms from seven genes known to influence angiogenesis with the likelihood of having breast cancer. METHODS: We recruited 715 controls and 520 subjects with breast cancer. Subjects provided a blood specimen and completed a questionnaire that included common breast cancer risk factors and breast cancer status. We evaluated candidate polymorphisms in the following genes: Hypoxia Inducible Factor-1 alpha (HIF1alpha), Vascular Endothelial Growth Factor (VEGF), VEGF Receptor 1 (VEGFR-1), VEGFR-2, endothelial Nitric Oxide Synthase (eNOS), Neuropilin-1 (NRP-1) and Neuropilin-2 (NRP-2). Testing for associations between each polymorphism and the presence or absence of breast cancer was performed. RESULTS: VEGF-2578 AA and -1498 CC genotypes were more common in cancer cases than controls (P = 0.06 and P = 0.04, respectively). These two genotypes remained significant predictors of breast cancer status after adjusting for non-genetic risk factors estimated by the Gail model (P = 0.03 and P = 0.03, respectively). When comparing women with invasive versus pre-invasive breast cancer, the eNOS-786 TT and eNOS 894 GG genotypes were associated with a greater likelihood of invasive disease and the eNOS 894 GG genotype was associated with a greater likelihood of having metastatic disease. CONCLUSION: There is an association of the VEGF-2578A and -1498C alleles with increased breast cancer risk. This association remains significant when adjusted for Gail score-related risk factors.