Allyson L. Valente

Aberrant methylation of imprinted genes is associated with negative hormone receptor status in invasive breast cancer.

Epigenetic regulation of imprinted genes enables monoallelic expression according to parental origin, and its disruption is implicated in many cancers and developmental disorders. The expression of hormone receptors is significant in breast cancer because they are indicators of cancer cell growth rate and determine response to endocrine therapies. We investigated the frequency of aberrant events and variation in DNA methylation at nine imprinted sites in invasive breast cancer and examined the association with estrogen and progesterone receptor status. Breast tissue and blood from patients with invasive breast cancer (n = 38) and benign breast disease (n = 30) were compared with those from healthy individuals (n = 36), matched with the cancer patients by age at diagnosis, ethnicity, body mass index, menopausal status and familial history of cancer. DNA methylation and allele‐specific expression were analyzed by pyrosequencing. Tumor‐specific methylation changes at IGF2 DMR2 were observed in 59% of cancer patients, IGF2 DMR0 in 38%, DIRAS3 DMR in 36%, GRB10 ICR in 23%, PEG3 DMR in 21%, MEST ICR in 19%, H19 ICR in 18%, KvDMR in 8% and SNRPN/SNURF ICR in 4%. Variation in methylation was significantly greater in breast tissue from cancer patients compared with that in healthy individuals and benign breast disease. Aberrant methylation of three or more sites was significantly associated with negative estrogen‐alpha (Fishers exact test, p = 0.02) and progesterone‐A (p = 0.02) receptor status. Aberrant events and increased variation in imprinted gene DNA methylation, therefore, seem to be frequent in invasive breast cancer and are associated with negative estrogen and progesterone receptor status, without loss of monoallelic expression.

Impact of lifestyle factors on prognosis among breast cancer survivors in the USA

Advances in diagnostic screening and adjuvant therapy have dramatically increased the number of breast cancer survivors in the USA, who may face changes in physical and mental health, social support, quality of life and economics. Women living with breast cancer are increasingly interested in lifestyle modification to decrease the risk of recurrence and mortality while increasing physical and emotional wellbeing. Although organizations such as the American Cancer Society support a healthy diet, frequent physical activity and stress reduction for decreasing breast cancer risk, studies examining the effects of lifestyle on clinical outcomes including survival and prognosis have been inconclusive. With the number of breast cancer survivors predicted to increase to 3.4 million by 2015, it is important to develop effective treatment paradigms that overcome barriers to behavioral modification to improve clinical outcomes and survivorship in breast cancer patients.

Leukocyte DNA as Surrogate for the Evaluation of Imprinted Loci Methylation in Mammary Tissue DNA

There is growing interest in identifying surrogate tissues to identify epimutations in cancer patients since primary target tissues are often difficult to obtain. Methylation patterns at imprinted loci are established during gametogenesis and post fertilization and their alterations have been associated with elevated risk of cancer. Methylation at several imprinted differentially methylated regions (GRB10 ICR, H19 ICR, KvDMR, SNRPN/SNURF ICR, IGF2 DMR0, and IGF2 DMR2) were analyzed in DNA from leukocytes and mammary tissue (normal, benign diseases, or malignant tumors) from 87 women with and without breast cancer (average age of cancer patients: 53; range: 31–77). Correlations between genomic variants and DNA methylation at the studied loci could not be assessed, making it impossible to exclude such effects. Methylation levels observed in leukocyte and mammary tissue DNA were close to the 50% expected for monoallellic methylation. While no correlation was observed between leukocyte and mammary tissue DNA methylation for most of the analyzed imprinted genes, Spearmans correlations were statistically significant for IGF2 DMR0 and IGF2 DMR2, although absolute methylation levels differed. Leukocyte DNA methylation levels of selected imprinted genes may therefore serve as surrogate markers of DNA methylation in cancer tissue.

Genomic (in)stability of the breast tumor microenvironment.

The breast tumor microenvironment plays an active role in tumorigenesis. Molecular alterations have been identified in tumor-associated stroma; however, there is considerable debate as to whether the stroma is characterized by genomic instability or whether detection of chromosomal alterations reflects technological artifact rather than the true genomic content of the tumor microenvironment. Thus, breast stroma specimens from 112 women undergoing reductive mammoplasty (n = 7), prophylactic mastectomy (n = 6), or mastectomy for a breast disease (n = 99) were frozen in optimal cutting temperature medium. Allelic imbalance (AI) analysis was conducted using a panel of 52 microsatellite markers in 484 stromal specimens from 98 women, of which 92% had no detectable AI events. When compared with previously generated AI data from 77 formalin-fixed, paraffin-embedded (FFPE) stroma specimens, 42% of which harbored at least one detectable AI event, the frequency of AI in the FFPE specimens (4.62%) was significantly higher (P < 0.001) than that found in frozen specimens (0.45%). This comparison of AI between FFPE and research-grade specimens suggests that past reports of AI in breast stroma reflect artifact in the archival specimens caused by formalin-fixation, paraffin-embedding and tissue storage. Furthermore, SNP data were generated from a subset of 86 stromal specimens using SNP arrays and copy number alterations were identified using Partek Genomics Suite. For 95% of the specimens, no detectable copy number alterations were found and the 11 changes that were detected were small and not shared between specimens. These data, therefore, support a model in which the tumor microenvironment is genetically stable. Mol Cancer Res; 10(12); 1526–31. ©2012 AACR.

Molecular response of the axillary lymph node microenvironment to metastatic colonization

Breast stroma plays an active role in tumorigenesis, undergoing both phenotypic and molecular changes that facilitate and promote tumor development and growth. The metastatic microenvironment also plays a role in successful colonization; however, genetic changes in these secondary microenvironments are not well described. To improve understanding of molecular changes associated with metastatic colonization, gene expression patterns from lymph node tissues from women with at least one positive, as well as one negative node, were compared. Lymph node tissue was microdissected and hybridized to U133A 2.0 gene expression arrays. Differential expression was detected using Partek® Genomics SuiteΤΜ 6.6 with FDR <0.05 and >2-fold change defining significance. Twenty-two genes were differentially expressed, 14 genes, including AZGP1, FOXA1 and PIP, were expressed at significantly higher levels in colonized lymph nodes and eight genes, such as CXCL2 and HPGDS, were expressed at significantly higher levels in non-metastatic lymph nodes. Thus, lymph node tissues harboring metastases have different gene expression patterns from those without metastases. Many differentially expressed genes are involved in cellular proliferation and survival, immune function and mesenchymal-epithelial transition, suggesting that repression of immune response and restoration of an epithelial phenotype in the host tissue are critical for successful establishment of lymph node metastases.

Sequence-based detection of mutations in cadherin 1 to determine the prevalence of germline mutations in patients with invasive lobular carcinoma of the breast

BackgroundLoss of cadherin 1 (CDH1) expression, which is normally involved in cell adhesion and maintenance of tissue architecture, is a hallmark of invasive lobular carcinoma (ILCA). Because hereditary cancers may require different risk reduction, counseling and treatment options than sporadic cancer, it is critical to determine the prevalence of germline CDH1 mutations in patients with ILCA.MethodsAll patients with ILCA (n = 100) previously enrolled in the Clinical Breast Care Project were identified. Genomic DNA was isolated from peripheral blood samples and DNA variants were detected for each exon of CDH1 using high-resolution melting technology followed by direct sequencing.ResultsWithin the 100 samples screened, four nonsynonymous variants were detected: A592T in one Hispanic patient, A617T in two patients, both African American, P825L in a Causasian patient whose grandmother had stomach cancer, and G879S in a Caucasian patient. Further evaluation of A617T in an additional 165 African American patients found that 11 patients, none with ILCA, carried this variant including one patient who was homozygous for the variant.ConclusionsCDH1 mutations are infrequent in patients with ILCA, and the variants that were detected have been classified as non-pathogenic. These data suggest that ILCA does not have a significant hereditary component and do not support CDH1 gene mutation testing in patients with ILCA.

Abstract P3-04-12: Pathological and molecular effects of lack of PR expression in ER positive breast tumors

Background: Evaluation of hormone receptor status is standard in breast cancer diagnosis; however, prognosis and treatment decisions are frequently based on the status of the estrogen receptor (ER) but not the progesterone receptor (PR). To determine how PR status affects etiology and outcome of ER positive tumors, pathological and molecular characteristics of ER+PR+ and ER+PR- tumors were assessed. Methods: ER and PR positivity were defined as >10% staining and all patients with ER+PR+ and ER+PR- were identified. Differences in pathological characteristics were evaluated using Chi-square tests with P 2.0-fold difference to define significance. Results: Of the 1,139 ER+ tumors, 21% were PR-. Age at diagnosis, ethnicity, tumor size, lymph node and Ki67 status did not differ between groups; however, patients with PR- tumors were significantly more likely to be diagnosed at an advanced stage, with high-grade, HER2 positive tumors, and more likely to die of disease. Thirty genes were differentially expressed including significantly higher expression levels of APOBEC3B and significantly lower expression of GREB1, MAPT and SCUBE2. Discussion: Expression of PR significantly alters tumor biology and clinical outcomes of ER positive tumors. Genes with altered expression in PR negative tumors are associated with more aggressive characteristics, such as increased invasion, kataegis, regulation of ER and response to tamoxifen. Thus, PR status is critical in the diagnosis and treatment of patients with ER positive breast tumors. Citation Format: Rachel E Ellsworth, Allyson L Valente, Matthew T Hueman, Craig D Shriver. Pathological and molecular effects of lack of PR expression in ER positive breast tumors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-12.

Abstract P4-10-01: Evaluation of the role of EBV in breast cancer

Background: A number of factors, both modifiable and non-modifiable, are associated with increased risk of developing breast cancer. Although these factors may help predict risk and prognosis, these known factors are imperfect and suggest that additional factors exist that promote the development and behavior of breast cancer. The Epstein-Barr virus (EBV), which is associated with tumor types such as Burkitt’s lymphoma and gastric cancer, may contribute to breast cancer development and/or progression, however, current research has led to mixed results, with some groups finding a positive correlation between EBV and breast cancer and others failing to find an association. Methods: The Clinical Breast Care Project database was queried to identify patients with high-quality, research-grade frozen tumor specimens and serum samples available. Previous exposure to EBV was determined using the Epstein-Barr virus (EBNA) IgG Human ELISA Kit. EBV, B-cells and macrophages were detected by immunofluorescence (IF) in seropositive patients using probes for EBNA1, CD-68 and CD-20, respectively. EBV and B-cell status was scored as present or absent while macrophages were classified as sparse, focal (weak, moderate or strong expression) or disseminated (macrophages detected throughout the tumor and stroma). Results: ELISA analysis on 211 serum samples found that 202 (96%) patients were seropositive for past EBV exposure. IF data has been generated for 31 tumors, from which six had no detectable B-cells. Of the remaining B-cell positive tumors, 32% (8/25) were positive for EBNA1 within the B-cells. Distribution of intrinsic subtypes was similar between both EBV+ and EBV- tumors, however, diagnosis with a late-stage tumor and lymph node metastasis was higher in EBV+ (50% stage III/IV, 88% lymph node positive) compared to EBV- tumors (24% stage III/IV, 47% lymph node positive). In addition, EBV+ tumors were more likely to have sparse/focal(weak) macrophage staining (63%) compared to EBV- tumors (47%). Conclusions: These data demonstrate that latent EBV is present within B-cells in a significant number of invasive breast tumors. Decreased detection of macrophages within the tumor component supports the theory that expression of EBV proteins impairs the function and maturation of macrophages, thus impairing immune surveillance and creating a pro-tumorigenic environment. This altered immune response may contribute to the later stage and increased frequency of metastasis in patients with EBV positive B-cells within the tumor microenvironment. Citation Format: Rachel E Ellsworth, Jill D Henning, Nikki Oakes, Allyson L Valente, Brednda Deyarmin, Jeff Meyer, Matthew T Hueman, Craig D Shriver. Evaluation of the role of EBV in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-10-01.

Abstract 3270: Gene expression profiling of breast tumors from African American and Caucasian women: Are molecular differences meaningful

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: To identify molecular factors associated with survival disparities, multiple studies have identified differentially expressed genes in tumors from African American women (AAW) compared to Caucasian women (CW), however, it is not clear whether these changes, such as increased expression of PHSPL, SOS1 and CRYBB2P1 in tumors from AAW, are clinically meaningful or represent population artifact. Gene expression data were thus evaluated in breast tumors with defined intrinsic subtypes, non-malignant breast tissue and blood from patients without breast disease. Methods: Gene expression data was generated using U133 2.0 microarrays for 184 breast tumors from AAW and CW matched by subtype, grade, age and presence of local or distant metastases, from 44 benign biopsy specimens, and from 143 peripheral blood samples from patients without current or past invasive breast disease. Principal component analysis (PCA) was performed using Partek Genomics Suite 6.6 and differentially expressed genes were detected using ANOVA with FDR<0.05, 2-fold difference defining significance. Results: When all tumors were analyzed together, only PSPHL, CRYBB2P1 and SOS1 were differentially expressed, each at significantly higher levels in tumors from AAW. When analyzed by subtype, PSPHL, CRYBB2P1, SOS1 and AMFR were expressed at higher levels in luminal A tumors (n=86) and SOS1 at higher levels in basal-like tumors (n=66) from AAW; no differentially expressed genes were detected in HER2-enriched (n=18) or luminal B (n=14) tumors. AMFR, CRYBB2P1, PSPHL and SOS1 were also expressed at significantly higher levels in both non-malignant breast tissue from AAW patients, and in blood specimens from disease-free AAW patients compared to non-malignant and blood specimens from CW. Conclusions: PCA demonstrates that molecular profiles of tumors from AAW are highly similar to those from CW; the few genes that are differentially expressed in tumor specimens are not associated with breast cancer as the same changes are also found in disease-free breast tissues as well as blood from patients without breast disease. Lack of meaningful genetic differences, even within specific tumor subtypes, suggests that disparity differences between AAW and CW with breast cancer are not attributable to molecular differences inherent to the tumors, but likely stem from other factors such as genetic predisposition to the development of basal-like tumors or altered provision of or response to treatment. Citation Format: Allyson L. Valente, Craig D. Shriver, Rachel E. Ellsworth. Gene expression profiling of breast tumors from African American and Caucasian women: Are molecular differences meaningful. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3270. doi:10.1158/1538-7445.AM2014-3270

Abstract 4129: Outcome disparities in breast cancer of the young: Is young age at diagnosis associated with unique disease biology

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Although breast cancer in young women (YW) only accounts for ∼7% of cases diagnosed annually, tumors in young patients have more aggressive characteristics and higher mortality rates. The cost of breast cancer increases significantly with more aggressive tumors in terms of treatment costs, physical and psychosocial effects on the patient and her family, and lost productivity. Improved understanding of factors associated with etiology and outcome of breast cancer in YW is critical to developing more effective prevention strategies and improved treatments, reducing the burden of breast cancer in this population. Methods: The Clinical Breast Care Project (CBCP) database was queried to identify all female patients with invasive breast cancer diagnosed before 40 years (YW) and between 50-65 years (post-menopausal women, PMW). Pathological characteristics from both groups were compared using chi-square analysis with P<0.05 defining significance. Gene expression data was generated using U133 2.0 arrays from tumors from a subset of 76 patients matched by subtype, grade and stage. ANOVA analysis was used to detect differentially expressed genes with a False Discovery Rate <0.05, 2-fold difference used to define significance. Results: YW represented 6% (89/1434) of the female invasive cancer population of the CBCP. YW were significantly more likely to be of African American ancestry (27%) compared to PMW (14%). Tumors from YW were significantly more likely to be hormone receptor negative, higher stage and grade, larger and more likely to have positive lymph node status and significantly less likely to be ER+/HER2-. Cancer mortality rates were significantly higher in YW (12%) compared to PMW (3%). Gene expression analysis, however, failed to identify any differentially expressed genes in subtype, grade and stage-matched tumors between YW and PMW. Conclusions: Outcomes in YW with breast cancer are less favorable than those in PMW. Gene expression analysis suggests that tumors from YW are not molecularly different from those from PMW and thus tumors from YW do not represent a unique disease entity. Rather, higher stage and tumor size at diagnosis due to lack of screening in combination with increased incidence of more aggressive subtypes is likely driving higher mortality rates. Identification of epidemiological and/or genetic factors contributing to the etiology of aggressive tumors in conjunction with evaluation of possible treatment differences may reduce outcome disparities by decreasing the incidence of and improving treatment for breast cancer in young women. Citation Format: Allyson L. Valente, Matthew T. Hueman, Craig D. Shriver, Rachel E. Ellsworth. Outcome disparities in breast cancer of the young: Is young age at diagnosis associated with unique disease biology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4129. doi:10.1158/1538-7445.AM2014-4129