Lisa Yee

Morphologically normal-appearing mammary epithelial cells obtained from high-risk women exhibit methylation silencing of INK4a/ARF.

Purpose: p16(INK4a) has been appreciated as a key regulator of cell cycle progression and senescence. Cultured human mammary epithelial cells that lack p16(INK4a) activity have been shown to exhibit premalignant phenotypes, such as telomeric dysfunction, centrosomal dysfunction, a sustained stress response, and, most recently, a dysregulation of chromatin remodeling and DNA methylation. These data suggest that cells that lack p16(INK4a) activity would be at high risk for breast cancer development and may exhibit an increased frequency of DNA methylation events in early cancer. Experimental Design: To test this hypothesis, the frequencies of INK4a/ARF promoter hypermethylation, as well as four additional selected loci, were tested in the initial random periareolar fine needle aspiration samples from 86 asymptomatic women at high risk for development of breast cancer, stratified using the Masood cytology index. Results:INK4a/ARF promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia and, importantly, in morphologically normal-appearing mammary epithelial cells; 29 of 86 subjects showed INK4a/ARF promoter hypermethylation in at least one breast. Importantly, INK4a/ARF promoter hypermethylation was not associated with atypia, and the frequency of hypermethylation did not increase with increasing Masood cytology score. The frequency of INK4a/ARF promoter hypermethylation was associated with the combined frequency of promoter hypermethylation of retinoic acid receptor-β2, estrogen receptor-α, and breast cancer-associated 1 genes (P = 0.001). Conclusions: Because INK4a/ARF promoter hypermethylation does not increase with age but increases with the frequency of other methylation events, we predict that INK4a/ARF promoter hypermethylation may serve as a marker of global methylation dysregulation.

Retinoic Acid Receptor-β2 Promoter Methylation in Random Periareolar Fine Needle Aspiration

Methylation of the retinoic acid receptor-β2 (RARβ2) P2 promoter is hypothesized to be an important mechanism for loss of RARβ2 function during early mammary carcinogenesis. The frequency of RARβ2 P2 methylation was tested in (a) 16 early stage breast cancers and (b) 67 random periareolar fine needle aspiration (RPFNA) samples obtained from 38 asymptomatic women who were at increased risk for breast cancer. Risk was defined as either (a) 5-year Gail risk calculation ≥1.7%; (b) prior biopsy exhibiting atypical hyperplasia, lobular carcinoma in situ, or ductal carcinoma in situ; or (c) known BRCA1/2 mutation carrier. RARβ2 P2 promoter methylation was assessed at two regions, M3 (−51 to 162 bp) and M4 (104-251 bp). In early stage cancers, M4 methylation was observed in 11 of 16 (69%) cases; in RPFNA samples, methylation was present at M3 and M4 in 28 of 56 (50%) and 19 of 56 (38%) cases, respectively. RPFNAs were stratified for cytologic atypia using the Masood cytology index. The distribution of RARβ2 P2 promoter methylation was reported as a function of increased cytologic abnormality. Methylation at both M3 and M4 was observed in (a) 0 of 10 (0%) of RPFNAs with Masood scores of ≤10 (nonproliferative), (b) 3 of 20 (15%) with Masood scores of 11 to 12 (low-grade proliferative), (c) 3 of 10 (30%) with Masood scores of 13 (high-grade proliferative), and (d) 7 of 14 (50%) with Masood scores of 14 of 15 (atypia). Results from this study indicate that the RARβ2 P2 promoter is frequently methylated (69%) in primary breast cancers and shows a positive association with increasing cytologic abnormality in RPFNA.

Hypermethylation of the Breast Cancer–Associated Gene 1 Promoter Does Not Predict Cytologic Atypia or Correlate with Surrogate End Points of Breast Cancer Risk

Mutation of the breast cancer–associated gene 1 (BRCA1) plays an important role in familial breast cancer. Although hypermethylation of the BRCA1 promoter has been observed in sporadic breast cancer, its exact role in breast cancer initiation and association with breast cancer risk is unknown. The frequency of BRCA1 promoter hypermethylation was tested in (a) 14 primary breast cancer biopsies and (b) the initial random periareolar fine-needle aspiration (RPFNA) cytologic samples obtained from 61 asymptomatic women who were at increased risk for breast cancer. BRCA1 promoter hypermethylation was assessed from nucleotide −150 to nucleotide +32 relative to the transcription start site. RPFNA specimens were stratified for cytologic atypia using the Masood cytology index. BRCA1 promoter hypermethylation was observed at similar frequency in nonproliferative (normal; Masood ≤10: 18%, 2 of 11), hyperplastic (Masood 11-13: 15%, 6 of 41), and atypical cytology (Masood 14-17: 22%, 4 of 18; P = 0.79). BRCA1 promoter hypermethylation was not associated with (a) family history of breast or ovarian cancer or (b) calculated Gail or BRCAPRO risk score. BRCA1 promoter hypermethylation was associated with (a) age (P = 0.028) and (b) the combined frequency of promoter hypermethylation of the retinoic acid receptor-β2 (RARB) gene, estrogen receptor-α (ESR1) gene, and p16 (INK4A) gene (P = 0.003). These observations show that BRCA1 promoter hypermethylation (a) is not associated with breast cancer risk as measured by mathematical risk models and (b) does not predict mammary atypia in RPFNA cytologic samples obtained from high-risk women. (Cancer Epidemiol Biomarkers Prev 2007;16(1):50–6)

CBP/p300 induction is required for retinoic acid sensitivity in human mammary cells.

The coactivators CBP and p300 are recruited by retinoic acid receptors (RARs) during retinoid mediated transcriptional regulation. To assess the role of CBP/p300 in all-trans-retinoic acid (ATRA)-mediated growth arrest in mammary epithelial cells, two systems were tested: (1) ATRA resistant MCF-7 cells were transduced with a functional RAR-beta 2; (2) normal human mammary epithelial cells (HMECs) were transduced with a pan-RAR dominant negative, RAR-alpha 403. Expression of RAR-beta 2 in MCF-7 cells resulted in increased sensitivity to ATRA-induced growth arrest and correlated with induction of CBP/p300 mRNA and protein. Inhibition of RAR function in HMECs resulted in resistance to ATRA-induced growth arrest and loss of CBP/p300 induction. Antisense suppression of CBP/p300 in HMECs resulted in decreased retinoic acid response element reporter trans-activation and decreased ATRA-mediated growth arrest. Thus, in human mammary epithelial cells, CBP/p300 were both modulated by an ATRA signaling pathway and were required for a normal response to ATRA.

Suppression of pRB expression in normal human mammary epithelial cells is associated with resistance to all-trans-retinoic acid but not N-(4-hydroxylphenyl)-retinamide

Despite the widespread clinical use of synthetic and naturally occurring retinoids, the down stream targets of retinoids have not been fully characterized. We observe that G1/0-phase arrest induced by all-trans-retinoic acid (ATRA) in normal human mammary epithelial cells (HMECs) is temporally associated with a significant decrease in the levels of hyperphosphorylated retinoblastoma protein (pRB). Suppression of pRB protein expression in HMECs by retroviral-mediated expression of the E7 protein of the human papillomavirus strain 16 (HPV-16) was associated with resistance to ATRA- mediated growth arrest but not to the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR or fenretinide). 4-HPR but not ATRA induced apoptosis in HMECs independent of the level of pRB protein expression. These observations suggest that ATRA- but not 4-HPR-mediated growth arrest may be dependent on the coordinated expression of pRB and emphasize the chemotherapeutic potential of 4-HPR, particularly for suppressing growth of tumors lacking pRB function.

Abstract P1-09-03: Prevention of aromatase Inhibitor (AI)-induced joint symptoms with omega-3 fatty acid supplementation: A randomized placebo-controlled pilot study

Introduction: AI-induced joint symptoms negatively impact drug adherence and quality of life. Based on observations that n-3 polyunsaturated fatty acids (PUFAs) have anti-inflammatory effects and that the mechanism of AI-induced joint symptoms may be partly due to inflammation, we hypothesized that women taking more n-3 PUFAs are less likely to develop AI-induced joint symptoms. Methods: We conducted a randomized, double-blind, placebo-controlled study comparing n-3 PUFA vs placebo in postmenopausal breast cancer patients starting adjuvant AIs. Participants were randomized to n-3 supplements [2.58 g eicosapentaenoic acid + 1.74 g docosahexaenoic acid/day; Marine Nutriceuticals, Mt. Bethel, PA] vs matched placebo for 24 weeks (wks). Primary endpoints was feasibility; secondary outcomes were self-reported symptoms as assessed by the Brief Pain Inventory short form (BPI-SF), Functional Assessment of Cancer Treatment, Breast & Endocrine Symptoms (FACTB-ES), and Stanford9s Health Assessment and Disability Index (HAQ) at baseline prior to AI receipt, 12 and 24 wks. Compliance and toxicity were evaluated monthly. Serial peripheral blood n-3 PUFA levels and inflammatory cytokines (IL-6, TNFR2, IL-17) were drawn. MRI of hands/wrists was performed in selected patients using a 3 Tesla dedicated wrist coil at baseline and treatment end. Results: Forty-four women were enrolled and randomized to study drug; 42 received ≥1 cycle (4 wks) of treatment; 36 had ≥1 post treatment evaluation at wk 12 or 24. Median age was 59.5 (range 43-76); history of prior taxane (n=15, 34%). The two groups’ baseline characteristics were similar. Overall, 93% and 88% of patients took >80% of the placebo and n-3 PUFA doses, respectively. Baseline erythrocyte n-3 PUFA was similar for both groups (6.6% ± 1.6%, 7.2% ±1.9%, p=0.20), but higher in the n-3 PUFA arm by wk 24 (6.5%±1.0% vs 15.0%±3.3%, p Conclusions: This is the first randomized pilot study to show that n-3 PUFA supplementation to prevent AI-induced joint symptoms is feasible and well tolerated. There is preliminary evidence that this intervention may help reduce the burden of AI-induced arthralgias. OSU Study #11022; ClinicalTrials.gov Identifier: NCT01478477. Grants from the National Cancer Institute (CA037447-26) to the Alliance for Clinical Trials in Oncology supported this pilot study. Citation Format: Maryam B Lustberg, Tonya Orchard, Xueliang Pan, Raquel Reinbolt, Amanda Logan, Joanne Lester, Rachel M Layman, Erin Macrae, Ewa Mrozek, Bhuvaneswari Ramaswamy, Robert Wesolowski, Michael Berger, Michael Knopp, Charles Loprinzi, Charles L Shapiro, Lisa Yee. Prevention of aromatase Inhibitor (AI)-induced joint symptoms with omega-3 fatty acid supplementation: A randomized placebo-controlled pilot study [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 P1-09-03.

Abstract P1-09-30: Relationship of dietary and red blood cell polyunsaturated fatty acids to inflammatory markers in breast cancer survivors taking aromatase inhibitors

Introduction: A large body of literature supports modulation of inflammation by polyunsaturated fatty acids (PUFA). Inflammation may play a role in the painful joint symptoms commonly experienced by breast cancer survivors taking aromatase inhibitors (AI). AI-induced joint symptoms negatively impact drug adherence and quality of life in many breast cancer survivors. N-3 PUFAs, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have anti-inflammatory effects; in contrast, excess very long chain n-6 PUFAs may promote peripheral and joint inflammation. We hypothesized that breast cancer survivors with greater EPA+DHA or n-3/n-6 PUFA in their diet would have decreased inflammation and be less likely to develop AI-induced joint symptoms. Materials and methods: This is a secondary analysis of PUFA intake self-reported from food frequency questionnaires and red blood cell (RBC) PUFA data from a randomized placebo-controlled pilot study comparing n-3 PUFA supplementation vs placebo in 44 postmenopausal women with breast cancer initiating first line adjuvant AIs. Primary outcomes were feasibility and tolerability; secondary outcomes were differences in clinical symptoms and inflammatory markers. Participants were randomized to 4.8 g/day DHA+EPA vs matched placebo for 24 weeks (wks). Serial peripheral blood samples were drawn for RBC PUFA levels and inflammatory cytokines (IL-6, TNFα-R2 and IL-17). Clinical symptoms were assessed by the Brief Pain Inventory short form (BPI-SF), FACTB-ES, and Stanford Health Assessment -Disability Index (HAS) at 12 and 24 wks. Compliance and toxicity were evaluated monthly. Results: Median age of all 44 women enrolled and randomized was 60 years (range 43-76); breast cancer stage I (n=29), II (n=10), III (n=5); prior taxane (n=15, 34%).Baseline mean dietary intakes of n-3 PUFAs were: total n-3 PUFAs = 1.43 ± 0.86 g/d; EPA = 0.05 ± 0.05g/d; and DHA = 0.10 ± 0.11 g/d. There was a trend toward lower TNFα-R2 with higher dietary intake of EPA (p=0.09) and DHA (p=0.10). Baseline mean RBC EPA+DHA (n-3 Index) was 4.36% and the RBC n-3/n-6 ratio was 0.20. RBC total n-3 PUFAs were similar at baseline between n-3 treatment and placebo groups (6.6 ± 1.6%, 7.2 ±1.9%, p=0.20). RBC PUFAs were not predictive of baseline IL-6 or IL-17. However, the baseline RBC n-3 Index trended toward an inverse relationship with TNFα-R2 (r = -0.23, p 0.14) Mean dietary intake of n-3 PUFAs did not change over 24 wks for the 30 women with complete FFQ data and blood samples: total n-3 = 1.38 ± 0.70 g/d; EPA = 0.06 ± 0.07 g/d; and DHA = 0.13 ± 0.14 g/d. Dietary EPA and DHA were significantly associated with lower TNFα-R2 (r = -0.40, p=0.02 for both) at 24 wks. RBC total n-3 PUFAs were significantly higher in the n-3 treatment vs placebo group at wk 24 (6.5%±1.0% vs 15.0%±3.3%, p Conclusions: There was a trend toward lower TNFα-R2 in breast cancer survivors with higher dietary and RBC EPA and DHA before starting AIs, but only dietary intake of EPA and DHA was significantly related to lower TNFα-R2 after 24 wks of AI therapy. Citation Format: Tonya S Orchard, Xueliang Pan, Joanne Lester, Amanda Logan, Charles L Shapiro, Rebecca D Jackson, Martha A Belury, Lisa Yee, Maryam B Lustberg. Relationship of dietary and red blood cell polyunsaturated fatty acids to inflammatory markers in breast cancer survivors taking aromatase inhibitors [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 P1-09-30.

A Mentored Cooperative Group Pilot Study: Atrophic Vaginitis

OBJECTIVES To review nursing research initiatives from two cooperative groups and outline a pilot study performed by a junior nurse researcher mentored by cooperative group nurse researchers and institutional physicians. DATA SOURCES PubMed, Cochrane Library, Scopus, World Wide Web. CONCLUSION Nursing research can be initiated and led by nurses in the cooperative group setting. The team approach model of research includes several disciplines to examine multiple facets of the same problem, or of multiple problems that a cancer patient may face. This new model will enable a greater number of nurse researchers to investigate symptom management, survivorship, and quality-of-life issues. IMPLICATIONS FOR NURSING PRACTICE Nurse researchers should be included in every cooperative group study to investigate nurse-sensitive outcomes and issues related to symptom management, survivorship, and quality of life.