Citations

Abstract Epigenetic silencing of tumor suppressing genes by promoter-specific DNA methylation is common in many types of cancer. As an early event, this process has been well shown to promote tumor initiation and progression; however little is known how such epigenetic silencing can contribute to tumor metastasis. The PRKD1 gene encodes Protein Kinase D1 (PKD1), a serine/threonine kinase expressed in epithelial cells of the normal mammary gland that maintains the epithelial phenotype of normal breast cells and prevents epithelial-to-mesenchymal transition (EMT). PKD1 is also a critical suppressor of tumor cell invasion and is silenced in expression and activity during breast tumor progression. Here, we show that aberrant methylation of PRKD1 promoter region is not only correlated with the silencing of its expression but is also associated with invasiveness of breast cancer cell lines and with aggressiveness of breast tumors. Using the highly invasive MDA-MB-231 cells, we show that the inhibition of PRKD1 promoter methylation with the DNA methyltransferase inhibitor decitabine restores PKD1 expression and significantly decreases their invasive abilities in vitro. More importantly, in a tumor xenograft model it dramatically blocks tumor spread and metastasis to the lung in a PKD1-dependent fashion. Our data suggest that the status of epigenetic regulation of the PRKD1 promoter can provide valid information on the invasiveness of breast tumors, and therefore could serve as an early diagnostic marker. Moreover, targeted upregulation of PKD1 expression may be used as a therapeutic approach to reverse the invasive phenotype of breast cancer cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-24.

Abstract 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 Background: Metaplastic breast carcinoma (MBC) accounts for <5% of all breast malignancies. This entity represents a heterogenous group of tumors in which the adenocarcinomatous element is admixed with one or more neoplastic mesenchymal (spindle, squamous, chondroid, or osseous) elements. Patients typically present with large triple negative tumors and have a poor prognosis. The likelihood of lymph node (LN) involvement has been reported to be low in MBC patients. Due to the paucity of data, we undertook this study to explore the role of axillary ultrasound (US) and sentinel LN biopsy (SLNB) in the management of MBC. Methods: With IRB approval, we retrospectively identified patients diagnosed with MBC from 2001–2011 from the surgical pathology database. Histopathology and imaging were reviewed. Demographic, treatment and outcome data were obtained by clinical chart review. Data were analyzed using JMP 9.0 software. Results: We identified 41 women with MBC. Median age was 60 years (range 33–90). Histologic subtypes were spindle cell (46%), mixed adenocarcinoma and mesenchymal elements (20%), squamous cell (17%) chondroid/osseous (10%) and adenosquamous (7%). Tumor stage was T1 (24%), T2 (44%), T3 (12%) and T4 (20%). 26 patients (63%) were treated by mastectomy and 15 (37%) by wide excision. Of the 38 patients who underwent LN surgery (6 low-grade MBC, 32 intermediate/high-grade MBC), 10 (26%) were LN positive. All low-grade MBC patients were LN negative while 10 of 32 intermediate/high-grade MBC patients (31%) had LN metastasis. 22 patients had a preoperative axillary US. 14 patients had a negative axillary US and none had LN metastasis. 4 of 8 patients with suspicious axillary US findings had a preoperative axillary LN fine needle aspiration biopsy (FNAB) and 3 were positive for cancer. These patients proceeded directly to axillary LN dissection (ALND). 24 patients had a SLNB of whom one was SLN positive and underwent completion ALND. LN metastasis was associated with larger tumor size (p = 0.003), higher tumor grade (p = 0.04), angioinvasion (p = 0.07) and abnormal axillary US (p = 0.003). Surviving patients were followed for a mean (median) of 41 (30) months during which 13 (32%) recurred at a median of 6 months (IQR 3–17 months) and 11 (27%) subsequently died of disease. One SLN negative patient developed an axillary recurrence at 8 months, was successfully treated by ALND and is disease-free at 38 months. There were no axillary LN relapses after ALND. Conclusions: For clinically node-negative MBC patients, our contemporary data series suggests the incidence of occult LN metastasis is sufficiently high to warrant LN staging especially for patients with intermediate and high-grade tumors. This can be accomplished in a minimally invasive fashion with reasonable accuracy (∼97%) with axillary US, FNAB of sonographically suspicious LNs, and SLNB for patients with negative axillary US or FNAB. Axillary LND should be performed for patients with clinically and/or FNAB-positive LN for enhanced disease control. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-22.

Abstract Purpose: The stage IB designation was added to the AJCC 7th edition staging system to denote micrometastasis in patients with T1 tumors. We have previously examined this group and found that receptor status and nuclear grade were better survival discriminates than the presence of micrometastases. The current study was undertaken to validate this finding in a larger cohort from the American College of Surgeons Oncology Group (ACOSOG) Z0010 study. Methods: Clinicopathologic and outcomes data from patients enrolled on ACOSOG Z0010 were recorded. All patients underwent breast conserving surgery, sentinel lymph node biopsy and whole breast radiation for clinical T1-2, N0 breast cancer. Sentinel lymph nodes were evaluated by H&E locally and if negative, by immunohistochemistry (IHC) at a central laboratory. Patients were staged according to the 7th edition AJCC system and recurrence-free (RFS), disease-specific (DSS) and overall survival (OS) were determined using the Kaplan-Meier method and compared using the log-rank test. Results: There were 5210 eligible and evaluable patients enrolled on ACOSOG Z0010. AJCC stage distribution was known in 4590 and included: 2849 (62.0%) stage IA, 376 (8.2%) stage IB, 878 (19.1%) stage IIA, 322 (7.0%) stage IIB, and 170 (3.7%) stage III. Median follow-up for the cohort was 9.0 years (range 0–12.6). Five and 10-year RFS, DSS and OS rates for patients with stage IA versus IB disease are shown in table 1. There were no significant differences between groups. When all stage I patients (stage IA and IB) were evaluated by ER status (positive vs negative) or grade (grade 1 vs 2 vs 3), these biologic factors were able to significantly discriminate patients with respect to RFS, DSS and OS (table 2). Conclusion: Differentiating patients with micrometastases (stage IB) from node negative patients (stage IA) does not stratify patients well with respect to survival. Biologic factors including ER status and grade are better discriminants of survival than the presence of small volume nodal metastases in patients with early stage breast cancer. Breast cancer staging should include biologic factors from the primary tumor. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-06.

Abstract Background: 1/3 of women with metastatic HER2+ breast cancer will develop central nervous system (CNS) metastases yet evidence-based treatments for women with progressive CNS disease are limited. Neratinib is an irreversible inhibitor of erbB1, HER2, and erbB4 which has promising activity in HER2+ breast cancer. Preclinical evidence suggests it may cross the blood brain barrier. Trial Design: This is a multicenter, phase II, open-label study of neratinib for patients with HER2+ breast cancer and brain metastases. Neratinib is administered at 240 mg orally daily during a 28 day cycle. Two cohorts will be enrolled: Cohort 1 will enroll 40 patients with progressive CNS disease; cohort 2 will enroll ≤5 patients who are candidates for surgical excision of intracranial disease. Surgical candidates receive neratinib 7–21 days preoperatively and resume postoperatively. All patients are re-staged every 2 cycles. Those who develop non-CNS progression have an option to extend therapy with trastuzumab+neratinib. Circulating tumor cells (CTC) are collected at baseline and progression; neurocognitive testing, HADS and EORTC QLQ30/BN20 measures are administered at baseline, cycle 2, cycle 3, and progression (cohort 1). Intracranial tumor, cerebrospinal fluid (CSF), and plasma are collected at surgery (cohort 2). Specific Aims: The primary endpoint is CNS objective response rate (ORR) by composite criteria. Additional endpoints include: non-CNS ORR, progression-free survival, overall survival (OS), site of 1st progression, and toxicity. Correlative and exploratory endpoints include association of CTC count and OS and longitudinal neurocognitive function and quality of life. In an exploratory analysis (cohort 2), we will quantify neratinib concentrations in CSF, intracranial tissue, and plasma and examine associations with response. Eligibility: Patients must have confirmed HER2+ metastatic disease with ≥1 parenchymal brain lesion measuring ≥10 mm that is new or progressed after completing ≥1 line of standard CNS-directed treatment (cohort 1) or CNS disease that is amenable for surgery, including those without prior CNS treatments (cohort 2). Additional eligibility criteria (cohorts 1,2) include: adequate performance status and end organ/marrow function, and ejection fraction ≥50%. Any number of prior lines of therapy is allowed, including prior lapatinib. Statistical Methods: Cohort 1 has a 2-stage design with up to 40 patients. CNS ORR is defined as ≥50% reduction in sum volume of CNS target lesions, without evidence of new lesions, progression of non-target CNS lesions, non-CNS disease progression, worsening neurological symptoms, or increase in corticosteroids. CNS lesion measurements are performed centrally by the Harvard Tumor Imaging Metrics Core. If 1/18 patients have a CNS response in the 1st stage, another 22 patients will enroll. With this design, if ≥5 of 40 patients achieve a CNS response, the drug will be deemed worthy of future study. This 2-stage design has 92% power to distinguish between a true CNS ORR of 20% and a null of 6% (one-sided type I error rate=9%). Accrual: Accrual has begun. Target=45 (cohort 1=40, cohort 2=5) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT1-1-11.

Abstract Differences in the relative amounts of fat, connective and epithelial tissues in the breast lead to variations in mammographic appearance. Breast density is a measure that reflects this variation in tissue composition and is most commonly assessed as the absolute amount or proportion of the mammogram comprised of fibroglandular or non-fatty tissue. Breast density has been characterized using both qualitative and quantitative estimates. The most widely used clinical density measure is the American College of Radiology's Breast Imaging Reporting and Data System (BI-RADS) four-category tissue composition measure. Quantitative estimates include user-assisted thresholding methods, and more recently, automated area and volumetric density measures. Increased breast density is common, with 26–32% of women in the general population having dense tissue occupying over half of their breast. Breast density has been shown to be a strong risk factor for breast cancer at any age of mammography, in both Caucasian and non-Caucasian populations, and years prior to the breast cancer. Women with over 50% of their breast comprised of dense tissue have a 3–5 fold increased risk of breast cancer relative to those with low or no density. Further, up to one third of all breast cancers are found in women with density over 50%. Breast density decreases with age, with the greatest declines seen between ages 45–55. This decline has been hypothesized to reflect the reduction of hormones and involution of the breast tissue with onset of menopause. Several risk factors demonstrate associations with percentage breast density that are consistent with their breast cancer associations, suggesting these factors may potentially affect breast cancer risk through their influence on breast density. Positive associations are seen between breast density and nulliparity, late age at first birth, never having breast fed, postmenopausal hormone use (particularly combination therapy), and alcohol intake. Inverse associations are seen with tamoxifen use as well as body mass index (BMI), but BMI and breast density have been shown to be independent risk factors for breast cancer, and likely act through different pathways on risk. Taken together, the epidemiologic risk factors account for only 20–30% of the variation in breast density in the population. Genetics also contribute to the variability in breast density. Twin and family studies have shown that percentage breast density is highly heritable, and that inherited factors explain between 30–60% of the variance. Some of these genetic factors are shared between breast density and breast cancer. Of the established breast cancer loci, single nucleotide polymorphisms in LSP1, ZNF365, ESR1 and RAD51L1, are also associated with breast density. Additional genetic loci for breast density (including a variant at 12q24) have been validated in large consortia. However, the loci identified to date together account for less than 5% of the variance in breast density. Identifying additional genetic loci for breast density will not only help inform the biology of breast density but also contribute to understanding breast cancer and risk prediction efforts. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr MS2-1.

Abstract Background: We investigated the association of germline genetic variation in hormone biosynthesis and metabolism genes with prostate cancer specific mortality in a cohort of advanced prostate cancer patients. Methods: We genotyped 852 single nucleotide polymorphisms (SNPs) from 97 genes in a cohort of 267 advanced prostate cancer patients at the time of progression to castration recurrence (CRPC) during on-going androgen ablation. Tagging SNPs with minor allele frequency (MAF) of >5% and r2 ≥0.8 were selected from HapMap, NIEHS and Seattle SNP databases. Medical records were queried for cause of death. The primary endpoint of time to prostate cancer specific mortality (PCSM), was pre-defined as time from development of CRPC to death from prostate cancer progression. Principle components analysis was used for gene-levels tests, and to account for multiple testing, we calculated the false discovery rate (FDR). For SNP level results, Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using cox regression. Results: The median age of the cohort was 72 years at CRPC. 43% had a Gleason score (GS)=8-10, 33% a GS=7, and 24% a GS<7. After a median follow up of 1.8 years (IQ range: 0.8-3.3 years), 139 patients died, of which 107 were due to prostate cancer progression. In unadjusted gene level analyses, UGT1A7 (p=0.0059; FDR=0.19), UGT1A10 (p=0.0017; FDR=0.17) and UGT1A3 (p=0.0037; FDR=0.18) were associated with PCSM. After adjusting for age and GS, SNPs strongly associated with PCSM are listed in the Table. Conclusions: Variation in UGT genes involved in hormone metabolism yield prognostic information in CRPC. Further validation is needed to develop these as prognostic biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4174. doi:10.1158/1538-7445.AM2011-4174

Abstract RATIONALE: The inability to deliver viruses homogeneously throughout solid tumors is a universal obstacle encountered in oncolytic virotherapy. The initial intratumoral viral dispersion volume is a critical determinant of virotherapy efficacy, and depends not only on the number of infected cells, but also on the spatial distribution of the infected cells. In this study, we determined the feasibility and usefulness of monitoring the initial intratumoral delivery of oncolytic viruses to solid tumors using contrast-enhanced computed tomography (CT). EXPERIMENTAL PROCEDURES: Human BxPC-3 pancreatic tumor xenografts were established in nude mice. An engineered oncolytic measles virus expressing the sodium iodide symporter reporter (MV-NIS) was mixed with a 1:10 dilution of Omnipaque-300 contrast material and injected directly into mouse tumors. Mice were imaged with micro-CT immediately pre and post-injection of the virus + Omnipaque solution and on day 3 post-injection with 99mTcO4 micro-SPECT/CT. Tumors were harvested immediately after micro-SPECT/CT imaging for autoradiography and immunistochemistry analysis of radioactivity uptake and viral infection. Linear regression and Spearman correlation analyses were performed to determine the relationship between intratumoral contrast enhancement and subsequent intratumoral viral infection. RESULTS: This dilution of Omnipaque was found to have no effect on viral infectivity or cell viability in vitro, yet was more than adequate for CT imaging of the virus + Omnipaque injectate distribution following intratumoral injection. Ex vivo autoradiography and immunohistochemistry confirmed a strong correlation between intratumoral CT contrast enhancement, SPECT activity, viral infection, and sodium iodide symporter activity. CONCLUSIONS: This injection method is translatable to the clinic and should allow optimization of intratumoral injection in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4145. doi:10.1158/1538-7445.AM2011-4145

Abstract Biomarkers that select patients for therapeutics would benefit clinical trial design and patient care. The Akt pathway is a therapeutic target in Glioblastoma Multiforme (GBM) and an important determinant of patient outcome. However, it is not known whether activity of this pathway varies among GBM tumors. To investigate we used Akt pathway genes from published GBM expression datasets. We detected 5 patterns of Akt pathway gene expression and these Akt subgroups correlated with prognosis. We analyzed networks within subgroups using both gene set enrichment analysis and a novel method that scores relevance based on both expression and local network connectivity (Komurov et al., PLoS Comput Biol. 2010 6:8, 1-10). The results suggest therapeutic targets within the individual subgroups. Furthermore, preliminary analysis indicates that human GBM xenograft models and primary human GBM manifest similar Akt subgroups. We are investigating rodent xenograft models of Akt subgroups to evaluate subgroup-specific drug sensitivity, and will present a method and examples of analysis integrating gene expression data from human GBM xenografts and primary human GBM. We hypothesize that Akt subgroups will help personalize treatment for GBM therapeutics. Supported by Barrow Neurological Foundation, Halle Family Foundation, and NIH 1 K01 NS064952-01A1 (AMJ). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4138. doi:10.1158/1538-7445.AM2011-4138

Abstract Purpose: Polymorphisms in genes involved in the angiogenesis pathway have been investigated as predictive markers for efficacy and tolerability to anti-angiogenesis treatment, such as BEV. This study examines single nucleotide polymorphisms (SNPs) in VEGF, VEGFR2, and HIF-1α for their potential association with outcome and safety in metastatic colorectal cancer (mCRC) patients treated with the combined vertical blockade of VEGF signaling (i.e., sorafenib and BEV) as salvage therapy. Experimental procedures: Genomic DNA was extracted from peripheral blood for 78/79 evaluable patients in the North Central Cancer Treatment Group (NCCTG) Phase II study, N054C (Grothey, et al., ASCO 2010). TaqMan or direct sequencing was used for genotyping VEGF (rs25648, rs3025039, rs2010963, rs1005230, rs833061, rs699947, rs1570360), VEGFR2 (rs2305948, rs2219471, rs2071559, rs1870377), and HIF-1α (rs11549465, rs11549467) SNPs. Chi-square or Fisher's exact test was used to determine genotype associations with the study's primary endpoint, progression-free rate at 3 months of follow-up, as well as genotype associations with the most common grade 3+ adverse events at least possibly related (AE rels) to treatment (i.e., fatigue, hypertension, skin reaction). Results summary: The presence of a mutant T allele in the 5’ UTR of VEGF (rs25648; C534T) was associated with fewer successes in progression-free rate at 3 month follow-up (Fisher's exact test, p=0.0187). In addition, the presence of a C allele in the promoter region of VEGFR2 (rs2071559, T-604C) was associated with fewer grade 3+ hypertension AE rels (Fisher's exact test, p=0.0101). However, after correcting for multiple comparisons using the method of Benjamini & Hochberg none of the p-values were significant at a 0.05 level. At the same time, no differences were observed between the genotype subgroups and either progression-free rate at 3 months or the most common grade 3+ AE rels for the remaining SNPs that were analyzed, before or after correction. Conclusions: This study provides preliminary evidence that mCRC patients with the VEGF C534T (rs25648) variant may not benefit from combined treatment of sorafenib and BEV, whereas patients with the VEGFR2 T-604C (rs2071559) polymorphism may experience fewer grade 3+ AE rels. Further prospective validation of these SNPs and their association with this treatment combination in mCRC is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4131. doi:10.1158/1538-7445.AM2011-4131