Benign Breast Epithelium Research Articles

Immunohistochemical findings and clinicopathological features of breast cancers with pathogenic germline mutations in Non-BRCA genes

Deleterious germline mutations in multiple genes confer an increased breast cancer (BC) risk. Immunohistochemical (IHC) expression of protein products of mutated high-risk genes has not been investigated in BC. We hypothesized that pathogenic mutations may lead to an abnormal IHC expression pattern in the tumor cells. BCs with deleterious germline mutations in CHEK2, ATM, PALB2 &PTEN were identified. Immunohistochemistry was performed using Dako staining platform on formalin fixed paraffin embedded tumor tissue. Primary antibodies for PALB2 (ab202970), ATM [2C1(1A10)}, CHK2 (EPR4325), and PTEN (138G6) proteins were used for BCs with respective deleterious mutations. IHC expression was assessed in tumor and adjacent benign breast tissue. Total 27 BCs with 10 CHEK2, 9 ATM, 6 PALB2 & 2 PTEN deleterious germline mutations were identified. IHC staining was performed on 8 CHEK2, 7 ATM, 6 PALB2 & 2 PTEN cases. Abnormal CHEK2 IHC staining was identified in 7/8(88%) BCs. Three distinct CHK2 IHC patterns were noted: 1) Strong diffuse nuclear positivity (5 BC), 2) Null-pattern (2 BC), & 3) Normal breast–like staining in 1 BC Four of 5 (80%) strong CHK2 staining BC had missense CHEK2 mutations. Null-pattern was present with a missense & a frameshift mutation. Normal breast-like CHEK2 IHC staining pattern was present in 1 BC with CHEK2 frameshift mutation. Loss of nuclear/cytoplasmic PTEN IHC expression was noted in 2 in-situ carcinomas. Abnormal PTEN and CHK2 IHC were present in atypical ductal hyperplasia and flat epithelial atypia. ATM and PALB2 IHC expression patterns were similar in tumor cells and benign breast epithelium: mild to moderate intensity nuclear and cytoplasmic staining. We report abnormal CHEK2 IHC expression in 88% of BCs with pathogenic CHEK2 mutations. With PTEN and CHEK2 pathogenic mutations, abnormal IHC patterns are seen in early atypical proliferative lesions. IHC may be applied to identify CHEK2 &PTEN mutated BCs and precursor lesions.

Abstract 2207: High-dose omega-3 fatty acid supplementation is associated with a decrease in FOXA1 in benign breast tissue

Abstract Background: FOXA1 is a pioneer transcription factor necessary for full expression of estrogen receptor activity. In collaboration with Grainyhead-like 2 (GRHL2), increased FOXA1 activity can result in endocrine resistance through increase of the endoplasmic reticulum protein AGR2 and its receptor LYPD3. Hyperglycemia and hyperinsulinemia increase FOXA1. Since eicosapentaenoic and docosahexaenoic omega-3 fatty acids (EPA and DHA) improve insulin sensitivity and reduce IGFR-related signaling, we explored the effect of EPA + DHA supplementation on FOXA1 and AGR2 expression in benign breast epithelium. Methods: Reserved excess cells from fine needle aspirations performed pre and post 6 months of supplementation given to postmenopausal women at increased risk for breast cancer were utilized. Cells had been stored at -20 C in a methanol fixative for ~10 y (Fabian et al, Ca Prev Res 2015). Of the 35 women entered, 28 had reserved excess cells available for blocks and after obtaining 4-5 sections per block, 12 had sufficient epithelial cells on both pre and post intervention slides as determined by H and E staining to proceed with immunohistochemistry assessment. Of the 12, median age was 53 and 7/12 were on low dose hormone replacement during the study. Slides were stained with FOXA1 (2F83 Mouse Monoclonal Antibody) at a 1:50 dilution and AGR2 rabbit polyclonal (Proteintech,12275-1-AP) at a 1:800 dilution. F0XA1 exhibits a nuclear staining pattern whereas AGR2 is predominately cytoplasmic. IHC staining was assessed individually by two readers (TM and TP). The most abnormal cell clusters were preferentially assessed. Up to 500 cells are evaluated for percent positive stain and staining intensity. An Allred score was computed as the sum of a categorical score for percent positive cells and a categorical score for the predominant staining intensity. Results: For FOXA1 there was a statistically significant decrease in percent positive cells (11/12, p=0.019) and Allred score (9/12, p=0.022) by Wilcoxon signed rank test. Of the 11 which showed a decrease in FOXA1, seven were on HRT throughout the study (usually estrogen alone). There was no statistically significant change in AGR2 IHC. However, there was a robust linear relationship between stain positivity for FOXA1 and AGR2 (p<0.001), as well as for the change in percent positive cells over the course of the intervention (p<0.003). There was no statistically significant correlation between change in FOXA1 and change in Ki-67 immunocytochemical staining (performed as part of the original study), nor for a variety of serum measures of insulin sensitivity or resistance including HOMA-IR and HOMA %B. Conclusions: If this finding is confirmed in other studies, FOXA1 protein expression may be a useful tissue response biomarker in breast cancer prevention trials of omega-3 fatty acids. Citation Format: Carol J. Fabian, Trina Metheny, Teresa A. Phillips, Marsha Danley, Bruce F. Kimler. High-dose omega-3 fatty acid supplementation is associated with a decrease in FOXA1 in benign breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2207.

Abstract LB-224: Tyrosine phosphorylation of p27Kip1 associates with Palbociclib responsiveness in breast cancer

Abstract PURPOSE: Cdk4 inhibitors (CDK4i), such as palbociclib, are approved in combination with hormonal therapy as a front line treatment for metastatic HR+, Her2- breast cancer. However, despite these advances, many patients demonstrate either de novo or acquired resistance. This highlights the need to identify biomarkers to pinpoint patients who would respond to this therapy. In Rb+ tumors, which comprise the majority of HR+ breast cancers, Ki67, Cyclin D, cdk4, and p16 levels do not identify responsive subgroups. Cyclin D or cdk4 levels themselves are not reliable because tyrosine phosphorylated p27Kip1 (pY88) is required for activation of the cyclin D-cdk4 (DK4) complex. We have previously shown that the level of pY88 correlates with cdk4 activity: the more pY88 detected, the greater the specific activity of cdk4 (Patel, et. al. MCR, 2018). We developed dual immunohistochemical staining for p27 and pY88 and demonstrated that pY88 status, which is negative in benign breast epithelium, stratified tumors across all hormone receptor groups (Gottesman, et.al, MCR, 2018). Lack of pY88 suggested that DK4 was inactive, and that these samples would not have the active cdk4 target. We have grown tumor resection material in explant culture, treated with palbociclib, and stained with Ki67 as a marker of response. Explants from the no pY88 group were non-responsive, while explants from the low or high pY88 group responded to drug. Thus, we hypothesized the pY88-p27 status may serve as a biomarker for patients that can respond to cdk4i therapy. RESULTS: We analyzed paraffin-embedded archival breast cancer biopsies from a 13 patient cohort of HR+, Her2- patients who had received Palbociclib/Letrozole in the front line metastatic setting. 62% of these patients progressed within 8 months of treatment, while 38% were on drug for 12-38 months. Using our staining method, we stratified patients into three groups based on pY88 status: 23% had no staining (Group 0), 38% had a low percentage of pY88+ cells (Group 1), and 38% had a high percentage of pY88+ cells (Group 2, >30% of cells pY88+). We then compared the time until disease progression, when they were removed from drug, for each group. Patients with a pY88 score of 0 had disease progression within 6-8 months, suggesting that they exhibited primary resistance. Patients with a pY88 score of 2 also showed disease progression within 6 months. This suggested that the Y88 high group may have too much active cdk4 target to respond to pharmacological drug dose, a hypothesis supported by our breast cancer cell line data. However, patients with a low pY88 score were all still on drug after 12+ months with either reduced tumor or stable disease. Additional patients continue to be recruited. CONCLUSION: Our data suggest that pY88-p27 status, as a surrogate marker for cdk4 activity, associates with responsiveness to CDK4i treatment. Clinical use of the pY88 biomarker may identify patients responsive or resistant to Cdk4 targeting drugs. Citation Format: Stacy W. Blain, Susan R.S. Gottesman, Jonathan Somma, Vladislav Tsiperson, Julia McGuinness, Matthew Ingham, Kevin Kalinsky, Gary Schwartz, Elina Tress, Hanina Hibshoosh, Mohamed Mh Kahila, Bachar Samra Samra, Evelyn Taiwo, Steve Xie. Tyrosine phosphorylation of p27Kip1 associates with Palbociclib responsiveness in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-224.

The Impact of Partial Weak Staining in Normal Breast Epithelium on the Reliability of Immunohistochemistry Results in HercepTest-positive Breast Cancer

IntroductionAlthough normal epithelial cells do not show human epidermal growth factor receptor-2 (HER2) gene amplification and should lack membrane staining by HER2 immunohistochemistry (IHC), HER2 staining in benign breast epithelium is occasionally encountered. The significance of this occurrence has not yet been adequately studied, and its associated American Society of Clinical Oncology/College of American Pathologists recommendations are vague. Our objective is to assess the correlation between HER2 IHC 3+ breast cancer cases with normal epithelium staining (NES) and their corresponding fluorescence in situ hybridization (FISH) results, and to suggest recommendations for interpretation. Materials and MethodsA total of 154 breast cancer cases with HER2 IHC 3+ were reviewed. NES, along with other clinicopathologic characteristics, were recorded. NES was scored as present or absent. All study cases were sent for FISH testing. All cases, and particularly those that showed false positivity for IHC (positive IHC, negative FISH) were examined for NES. ResultsOf the 154 cases, 146 cases were FISH-positive (94.8%) and 2 failed FISH testing (1.3%). Conversely, 22% (34/154) of the cases showed NES for HER2. Of these 34 cases, 23 (67%) were FISH-amplified, 9 (26%) were FISH not amplified, and 2 failed FISH testing. Notably, all of the false-positive (FISH-negative) breast cancer cases showed some degree of positivity in normal breast epithelium. ConclusionsOur findings, though descriptive, show a very strong association between NES and false-positive HER2 IHC. This confirms the need to carefully evaluate IHC-positive breast cancers for NES, and to have a low threshold for confirmatory testing by FISH.

Tyrosine Phosphorylation of p27Kip1 Correlates with Palbociclib Responsiveness in Breast Cancer Tumor Cells Grown in Explant Culture.

Cdk4-targeting drugs, such as palbociclib, are approved for metastatic ER/PR+, Her2- breast cancer. However, other than loss of retinoblastoma, which is very rare in this subset, there are no biomarkers to predict response. Cyclin D or cdk4 levels are not by themselves indicative, because p27Kip1 is required for cyclin D-cdk4 complex activation. Tyrosine phosphorylation of p27, including modification on residue Y88 (pY88), activates DK4-p27, and the pY88 level correlates with palbociclib responsiveness in cell lines. We developed dual IHC staining for p27 and pY88, and found that benign breast epithelium was negative, while breast cancer biopsies (of varied hormonal status) could be stratified for pY88 status. Lack of pY88 suggested that DK4 was inactive, and that these samples would not have the target required for palbociclib response. Tumor resection material was grown in explant culture, treated with palbociclib, and stained with Ki67 as a marker of response. Explants from the no pY88 group were nonresponsive, while explants from the low or high pY88 group responded to drug. IMPLICATIONS: Use of the pY88 biomarker, as a surrogate for cdk4 activity, may identify patients responsive to cdk4-targeting drugs and expand use of this therapy.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/3/669/F1.large.jpg.

Abstract P2-05-07: Comparison of the Xpert breast cancer stratifier mRNA assay with central ER, PR, HER2, and Ki67 immunohistochemistry (IHC) for rapid biomarker analysis in developing countries

Abstract Breast cancer care in the developing world is limited by access to quality ER and HER2 IHC diagnostic assays needed to justify hormone and HER2 therapeutics. Shipping pathology specimens to a central testing site often out of country delays therapy and is costly. The Xpert Breast Cancer Stratifier assay makes quantitative measurements of ESR1, PGR, ERBB2, and MKi67 mRNAs from FFPE specimens in <2 hours on an easy-to-use automated diagnostic platform, the GeneXpert (GX). 10,000 GX machines are currently in use in 182 countries offering the possibility of a point-of-care solution. We compared concordance in tumor samples between IHC and mRNA intending to challenge the limits of the GX mRNA assay. 83 breast tumor samples were chosen including those with low cellularity, small volume disease, unusual subtypes, ER- tumors with surrounding benign epithelium, and low level HER2+ tumors. mRNA, IHC and FISH assays were performed. Slides were tested following macrodissection of invasive carcinoma and as non-macrodissected whole sections. GX measurements for Ki67 were compared with mitotic rate as an alternative to Ki67 IHC. Overall percent agreement following macrodissection was 95% for ER, 89% for HER2, 76% for PR, and 80% for Ki67 (>20% positive cut), and using whole section, 99% for ER, 80% for PR, 92% for HER2, and 73% for Ki67. Concordance was 92% for both macrodissection and whole section using mitotic rate to assess proliferation. Ignoring HER2 2+ calls which represented low level amplified tumors by FISH, the concordance rates were 95% for macrodissection and 99% for whole section. Discordance when testing long-term stored 4μm sections was resolved in a number of cases by using a fresh cut from the FFPE block. Half the ER discrepancies were in very small volume tumors ≤25mm2 and 75% were classified as ER-ve by IHC, and positive by Stratifier. 80% of ER IHC- cases were appropriately identified as ER- by the Stratifier in the presence of benign breast epithelium. HER2+ DCIS adjacent to HER2- invasive tumor resulted in a discrepant HER2 mRNA result even with macrodissection. No ER or HER2 discrepancies occurred in low cellularity tumors (≤30% cellularity) nor in lobular and mucinous subtypes. In a study intended to challenge an mRNA breast biomarker assay, concordance between mRNA results and IHC was high for ER and HER2, the two most important prognostic markers needed for therapeutic decision making. Use of whole sections rather than tumor macrodissection did not decrease concordance. Discrepant ER cases were more prevalent when analyzing low volumes of tumor and in this setting were seen in ER IHC- tumors surrounded by ER+ normal epithelium, or with weak IHC expression, highlighting predictable limitations of the assay. Concordance was better between Ki67 mRNA and mitotic rate than with IHC. Re-test data suggested that a fresh cut of the FFPE block yields the best results by GX, perhaps due to mRNA degradation in stored 4μm sections. The Xpert Breast Cancer Stratifier may provide a rapid, cost-effective solution to the problem of obtaining accurate diagnostic results at the point-of-care in low resource settings, and deserves further evaluation in developing countries. Citation Format: Brock JE, Milner DA, Ho K, Natalie W, Victor C, Annaliza R, Teresa B, Kathryn G-F, Edwin LW, Jodi W, Wendy W, Michael B. Comparison of the Xpert breast cancer stratifier mRNA assay with central ER, PR, HER2, and Ki67 immunohistochemistry (IHC) for rapid biomarker analysis in developing countries [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-07.

Abstract P6-01-07: Mitochondria-nuclear communication regulates epithelial-mesenchymal transition and metastasis in triple negative breast cancer

Abstract For triple negative breast cancer (TNBC), the driver pathways are still poorly understood. Advances in cancer metabolism research over the last decade have enhanced and modified our understanding on Warburg effect. It is now known that mitochondria in tumors are not always defective in their ability to carry out oxidative phosphorylation. Instead, in proliferating cells, mitochondrial energy pathways are reprogrammed to meet the challenges of macromolecular synthesis and to escape from apoptosis. Tumor initiating cells (TICs) maintain cancer stem cell properties and are known to play significant role in TNBC metastasis. Mitochondrial retrograde regulation (MRR) is a bidirectional communication between mitochondria and nucleus. MRR is triggered by mitochondrial functional demands and it responds in a continuous manner to change metabolic needs of the cell. Using transmitochondrial cybrid (cybrid) technology, we generated different cybrid models under common nuclear backgrounds of benign breast epithelium or TNBC. Mitochondria from cells with different cancer potential such as benign breast epithelium, moderately metastatic and highly metastatic breast cancer cell lines were studied under the common nuclear background to understand MRR-regulated TIC properties and cancer pathways. Using genomic, metabolomic, and proteomic approaches, we confirmed the significance of mitochondrial character in the regulation of epithelial mesenchymal transition (EMT), TIC and metastatic properties. Altogether, our results suggest that MRR is critical in TNBC TIC character and stemness. Citation Format: Park JH, Jung KH, Sirupangi T, Vithayathil S, Jin F, Putluri V, Piyarathna DWB, Yotnda P, Bhat VB, Sreekumar A, Lewis MT, Coarfa C, Putluri N, Creighton CJ, Wong L-JC, Kaipparettu BA. Mitochondria-nuclear communication regulates epithelial-mesenchymal transition and metastasis in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-07.

Abstract A03: ERβ expression and breast cancer risk prediction for women with atypias

Abstract Introduction: Estrogen receptor beta (ERβ), which is highly expressed in benign breast epithelium, is postulated to function as a tumor suppressor of breast cancer. Atypias of the breast increase the lifetime risk of breast cancer four-fold. However, little is known about ERβ expression in association with atypias or whether ERβ plays a role in mitigating breast cancer risk in high-risk individuals. We therefore undertook this study to examine the relationship of ERβ expression in atypias and adjacent normal lobules with the risk of subsequent breast cancer. Methods: We studied women in a well-characterized benign breast disease cohort with atypical ductal hyperplasia (ADH) or atypical lobular hyperplasia (ALH) diagnosed from 1967 to 1991. Nuclear and cytoplasmic ERβ expression was assessed via immunohistochemistry in both the atypical hyperplasia epithelium (atypia) and background normal lobules for percent (scores 0-4 for <1%, 1-25%, 26-50%, 51-75%, >75%, respectively) and intensity (scores 0-3 for negative, weak, intermediate, strong, respectively) using a highly specific ERβ monoclonal antibody (PPG5/10). For nuclear staining, an ERβ sum score (percent + intensity, range 0-7) was created and grouped as low (0-2), moderate (3-5) or high (6-7). Competing risks regression was used to assess the association of ERβ expression with future breast cancer risk while also accounting for the competing risk of death from other causes. Results: 171 women, median age 56, were studied (79 ADH and 92 ALH). Median follow-up was 15 years overall, and 36 women developed breast cancer at a median of 13 years after their atypia biopsy. ERβ expression was lower in the atypia versus normal lobules whether evaluated by nuclear percent stained, nuclear intensity or cytoplasmic intensity (all p < 0.001). For nuclear staining specifically, ERβ expression in the atypia was low in 44 (26%), moderate in 117 (68%) and high in only 10 (6%) based on the sum score. In contrast, ERβ was higher in the normal lobules with a majority showing high expression (n = 96, 56%), moderate expression in 74 (43%) and low expression in only 1 (0.6%). Lower ERβ nuclear expression in the atypia was associated with increased breast cancer risk, whether evaluated by percent staining, intensity or sum score; the same was true for ERβ nuclear expression in the background normal lobules. Low ERβ expression in the atypia (sum score 0-2) was associated with a 2-fold increased risk of subsequent breast cancer compared to atypia with moderate-high expression (sum score 3-7), HR 2.0, 95% CI: 1.02-3.8, p = 0.04. Lower ERβ expression in the normal lobules was also associated with an increased risk of future breast cancer: HR = 2.5 (95% CI: 1.3-5.1, p = 0.009) for low-moderate versus high expression. Conclusions: Increased ERβ expression in normal and atypical lobules was associated with a significantly decreased risk of breast cancer in our high-risk patient cohort. These data suggest that in women with atypical hyperplasia, ERβ expression is protective against the future development of breast cancer. ERβ may serve as a useful biomarker to further stratify breast cancer risk in women with benign breast disease. Citation Format: Tina J. Hieken, Jodi M. Carter, John R. Hawse, IV, Tanya L. Hoskin, Marlene Frost, Lynn C. Hartmann, Derek C. Radisky, Daniel W. Visscher, Vernon S. Pankratz, Amy C. Degnim. ERβ expression and breast cancer risk prediction for women with atypias. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A03.

Abstract 3055: Regulation of mitochondria-nuclear crosstalk in triple negative breast cancer

Abstract Driver pathways of triple negative breast cancer (TN BCa) is still poorly understood. Thus, it is important to identify the underlying mechanisms of TN BCa progression. Importantly, African American and Hispanic patients are more likely to be diagnosed of BCa at a younger age, have a higher probability of developing TN BCa. They also diagnosed with a more advanced stage of the disease compared to Caucasian women. Src oncognenic pathway is one of the major tumor driving mechanisms in TN BCa. Src pathway is frequently over-expressed in TN BCa and Src inhibitors are one of their current treatments of choices. However, recent single drug therapy Phase-II trials with Src inhibitors failed to control unselected metastatic TN BCa. This raises some important questions on the underlying mechanism in the regulation of Src in TN BCa especially in aggressive metastatic BCa. Mitochondria, a semiautonomous organelle in cells, play an important role in cellular energy metabolism, free radical generation, and apoptosis. Mitochondria-nuclear crosstalk is a bidirectional pathway of communication between mitochondria and nucleus that influences many cellular and organismal activities. This crosstalk can regulate several oncogenic pathways involved in tumorigenesis. Using transmitochondrial cybrid (cybrid) technology, we generated different cybrid models under common nuclear background of TN BCa. Mitochondria from cells of different cancer potential including benign breast epithelium, moderately and highly metastatic breast cancer cell lines were used to understand cancer mitochondria regulated tumor pathways. Extensive analysis of cybrid models as well as confirmation in parental cell lines and other tumor models suggested that autophosphorylation of c-Src is regulated by mitochondrial tumor characteristics. Ongoing studies using knock-down and over expression approaches focus on the role of specific mitochondrial proteins that are responsible for mitochondria-nuclear crosstalk and the regulation oncopathways in TN BCa. Citation Format: Jun H. Park, Sajna A. Vithayathil, Nagireddy Putluri, Efrosini Tsouko, Taraka R. Donti, Daniel E. Frigo, Chad J. Creighton, Michael T. Lewis, Arun Sreekumar, Lee-Jun Wong, Benny A. Kaipparettu. Regulation of mitochondria-nuclear crosstalk in triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3055. doi:10.1158/1538-7445.AM2015-3055

Abstract P1-07-06: Activation of oncogenic pathways by mitochondrial reprogramming in triple negative breast cancer

Abstract Triple negative breast cancer (TN BCa) Driver pathway is still poorly understood. Thus, it is important to identify the underlying mechanisms of triple negative breast cancer progression. Mitochondria, a semiautonomous organelle in cells, play an important role in cellular energy metabolism, free radical generation, and apoptosis. Mitochondria-nuclear crosstalk is a bidirectional pathway of communication between mitochondria and nucleus that influences many cellular and organismal activities. This crosstalk can regulate several oncogenic pathways involved in tumorigenesis. Using transmitochondrial cybrid (cybrid) technology, we generated different cybrid models under common nuclear background of TN BCa. Mitochondria from cells of different cancer potential including benign breast epithelium, moderately and highly metastatic breast cancer cell lines were used to understand cancer mitochondria regulated tumor pathways. Tumor and gene expression analysis suggested that among different cancer pathways, c-Src signaling pathway is one of the most consistently activated pathways in cybrids with TN BCa cancer mitochondria. Further analysis in parental cells and other tumor models suggested that autophosphorylation of c-Src is regulated by mitochondrial tumor characteristics. Our preliminary analysis also suggest that mitochondria targeted drugs are promising combination therapy for the management of Src-driven TN BCa. This finding is particularly important while considering the poor response rate observed after single drug therapy with Src family tyrosine kinase inhibitor Dasatinib in unselected TN BCa patients. Citation Format: Jun Hyoung Park, Santhosh Kumar, Sajna Vithayathil, Kavisha Arora, Nagireddy Putluri, Efrosini Tsouko, Taraka R Donti, Daniel E Frigo, Chad J Creighton, Michael T Lewis, Arun Sreekumar, Lee-Jun Wong, Benny Abraham Kaipparettu. Activation of oncogenic pathways by mitochondrial reprogramming in triple negative 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 P1-07-06.

Expression and clinical significance of matrix metalloproteinase-9 in lymphatic invasiveness and metastasis of breast cancer.

BackgroundMatrix metalloproteinase 9 (MMP-9) is a type-IV collagenase that is highly expressed in breast cancer, but its exact role in tumor progression and metastasis is unclear.Methods MMP-9 mRNA and protein expression was examined by real-time reverse transcriptase PCR and immunohistochemical staining, respectively, in 41 breast cancer specimens with matched peritumoral benign breast epithelial tissue and suspicious metastatic axillary lymph nodes. Lymph vessels were labeled with D2-40 and lymphatic microvessel density (LMVD) was calculated. Correlation of MMP-9 protein expression with clinicopathological parameters and LMVD was also evaluated.ResultsMMP-9+ staining in breast cancer specimens (35/41, 85.4%) was higher than in matched epithelium (21/41, 51.2%; P<0.05) and lymph nodes (13/41, 31.7%; P<0.001). Higher MMP-9 mRNA expression was also detected in tumor specimens compared with matched epithelial tissues and lymph nodes (P<0.05). Elevated MMP-9 expression was correlated with lymph node metastasis and LMVD (P<0.05).ConclusionMMP-9 was overexpressed in breast cancer specimens compared with peritumoral benign breast epithelium and lymph nodes. Moreover, its expression in the matched epithelium and lymph nodes was positively associated with lymph node metastasis, and its expression in lymph nodes was positively associated with lymphangiogenesis in breast cancer. Thus, MMP-9 is a potential marker for breast cancer progression.

Abstract 1696: Mitochondria-nuclear cross talk regulate cancer properties by activating oncogenic pathways.

Abstract Mitochondria are semiautonomous organelles within cells that play an important role in cellular energy metabolism, free radical generation, and apoptosis. Mitochondria contain their own genome (mtDNA), which encodes a number of proteins critical for energy metabolism, particularly in oxidative phosphorylation. They are also the major source of generating reactive oxygen species (ROS). So far, only limited studies (mostly focused on specific mtDNA mutations) analyzed the significance of mitochondria in cancer progression. However, it is not clear whether mitochondrial genomic status or functional defects affect nuclear genome stability and whether proteins involved in inter-genomic cross talk are involved in tumorigenesis. Transmitochondrial cybrids (cybrids) are a great utility for the study of the functional effects of mitochondria in a defined nuclear background. Cybrids are constructed by fusing enucleated cells harboring mitochondria of interest with ρ0 cells (mtDNA-depleted cells). In our cybrid model, we used ρ0 cells from moderately metastatic breast cancer cell line SUM159 as the common nuclear background. Cybrids were generated using mitochondria from benign breast epithelium (A1N4 &amp; MCF10A), moderate (SUM159) and highly metastatic (MDA-MB231) breast cancer cells. Mitochondrial functional studies suggest that cybrids maintain the mitochondria characteristics of the mitochondria donor cells with decreased electron transport chain (ETC) function and increased ROS production in cybrids containing cancer mitochondria. In vitro tumorigenic studies suggest that even under the common moderate cancerous nuclear background, mitochondria from benign cells decreased and metastatic cell increased oncogenic properties. Metastatic mitochondria increased the expression of metastatic and EMT marker proteins and decreased the epithelial cell markers. Cybrids with mitochondria from benign breast cells showed an opposite protein expression pattern. Interestingly autophosphorylation of tyrosine residue (Tyr416) which results in Src activation was markedly increased in cybrids with metastatic mitochondria. Further analysis suggested that Src kinase phosphorylation depends on the mitochondrial characteristics and phosphorylated Src located in mitochondria. Treatment with inhibitors of Src kinases could abolish the oncogenic property of cybrids with metastatic mitochondria. Our results demonstrated that mitochondria can initiate cross-talk by regulation of nuclear gene expression or by post-translational modification of regulatory proteins such that some cellular properties of the origin of the mitochondria can be carried on to next generation. Citation Format: Santosh Kumar, Vineet Gupta, Sajna Vithayathil, Junhyoung Park, Taraka R. Donti, Chad J. Creighton, Michael T. Lewis, Arun Sreekumar, Lee-Jun Wong, Benny A. Kaipparettu. Mitochondria-nuclear cross talk regulate cancer properties by activating oncogenic pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1696. doi:10.1158/1538-7445.AM2013-1696

Abstract A107: Epigenetic markers for tissue-based breast cancer risk stratification

Abstract Effective breast cancer risk management requires accurate breast cancer risk stratification. The Gail model is the most widely used and best validated epidemiological risk assessment model. Though well calibrated, this model is not ideal for individualized risk counseling as it is only 8% better than chance for discriminating between women with breast cancer and unaffected women. Biologically based approaches, such as cytological assessment of nipple aspirate fluid or assessment of mammographic density, only marginally improve the discrimination of the Gail model. Promoter region methylation is one of the earliest identifiable molecular changes during breast carcinogenesis. We have previously shown that RASSF1A methylation, detected in benign breast epithelium obtained by random periareolar fine needle aspiration biopsy (RP-FNA), is associated with increased breast cancer risk, but this was based primarily on correlation with Gail risk and not cancer case prediction. A clinically useful epigenetic risk stratification panel would be ideally suited to benign RP-FNA samples and would provide a high degree of discrimination between benign samples from women with cancer and unaffected women. We performed a genome-wide 5-aza-2’-deoxycytidine (5-aza) induced gene re-expression screen in 6 breast cancer cell lines and 6 early passage primary benign breast epithelial cell cultures to identify potential epigenetic markers of breast cancer risk. MSP assays were designed for the 286 genes that were 1) expressed in benign breast epithelium, 2) not expressed in cancer, and 3) induced by 5-aza in cancer, or 4) had previously shown some potential for breast cancer risk stratification. 20 genes were identified that were frequently methylated in primary breast cancer, rarely methylated in benign RP-FNA samples and never methylated in lymphocytes. Quantitative multiplex methylation-specific PCR assays were designed and optimized for the 17 best genes and then assessed in an archival RP-FNA sample set that included 146 samples from unaffected women, 59 benign samples from breast cancer patients, and 52 primary breast cancers. A three marker panel including PSAT1, HS3ST2, and GNE provided the best discrimination between benign samples from cancer patients and unaffected women. One or more of these markers was scored as positive in 46% of benign samples from cancer patients, 8% of samples from unaffected patients, and 72% of primary cancers. The odds ratio for cancer case prediction based on assessment of benign RP-FNA samples was 9.42 (95% CI 4.31 — 20.59, p &amp;lt; 0.0001). These observations will be validated in an independent set of 160 sample prospectively acquired for this purpose. These data suggest that a limited epigenetic marker panel can detect a small population of breast cells accessible by RP-FNA whose presence is associated with significantly increased breast cancer risk. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A107.

Human Calmodulin‐Like Protein (CLP) Expression in Oral Squamous Mucosa and in Malignant Transformation

The purpose of this study was to test whether calmodulin-like protein (CLP) is expressed in normal human oral mucosal cells and if downregulation of CLP occurs in malignant transformation. Oral mucosal tissue was taken from three individuals in a double-blind manner. The samples were cut, measured, and homogenized. Total RNA was extracted and reverse transcribed. Each cDNA sample was subjected to polymerase chain reaction (PCR). PCR fragments were purified, cloned, and sequenced to verify the presence of CLP. Three oral mucosal tissue samples with biopsy-confirmed squamous cell carcinoma were obtained. These samples demonstrated regions of normal epithelial cells as well as invasive squamous cell carcinoma. One normal breast epithelial sample was also obtained for positive control. Sections were stained with an affinity-purified CLP antibody and counterstained with a diluted hematoxylin. Two observers evaluated the specimens for expression of CLP. Staining patterns and intensity were noted in normal oral mucosa, comparing them to the normal breast epithelium sample. Staining patterns and intensity were then observed in squamous tumor cells, comparing them to the patterns of benign squamous mucosa. CLP coding sequences were positively identified from the normal oral mucosal tissue samples by reverse transcription and polymerase chain reaction (RT-PCR) with 100% identity to the published CLP sequence (accession #M58026). In the three oral mucosa tissue samples with known squamous cell carcinoma, expression of CLP was readily detected in areas of normal oral mucosa, while a notable downregulation of CLP expression occurred in areas of malignant transformation. The staining intensity was equivalent to the staining seen in the benign breast epithelium used as a control. In the areas of squamous cell carcinoma, a decrease in CLP immunoreactivity occurred. There was a sharp contrast in staining quality and clarity between benign and malignant tissue. In the majority of the carcinoma regions, a complete lack of immunoreactivity was noted. The RNA for human CLP is found in normal oral mucosa. CLP expression is seen in normal oral mucosa with a downregulation of CLP expression in malignant transformation.

Serial analysis of gene expression of lobular carcinoma in situ identifies down regulation of claudin 4 and overexpression of matrix metalloproteinase 9

IntroductionAlthough lobular carcinoma in situ (LCIS) has traditionally been viewed as a marker of breast cancer risk, recent clinical, pathological and genetic analyses have supported the concept that LCIS is a low risk, direct precursor of invasive lobular carcinoma. Global gene expression profiling of LCIS has not been performed.MethodsWe analysed the comprehensive gene expression profile of a unique case of mass-forming LCIS using serial analysis of gene expression (SAGE). This SAGE library is publicly available online. By comparing the gene expression profile of LCIS to that of benign breast epithelium and stroma, we identified several genes up and down regulated in LCIS. Differential expression of selected genes not previously studied in LCIS was validated at the protein level by immunohistochemistry and at the RNA level by quantitative reverse transcriptase PCR (RT-PCR).ResultsWe identified down regulation of claudin 4 and overexpression of matrix metalloproteinase 9 in LCIS relative to normal breast epithelium and stroma. We validated these findings by immunohistochemistry in a separate series of 11 and 19 LCIS cases, respectively. Overexpression of matrix metalloproteinase 9 was further confirmed by quantitative RT-PCR analysis of the index case.ConclusionsWe have created the first global gene expression profile of LCIS, and demonstrated down regulation of cell junction proteins (an expected result) and overexpression of matrix metalloproteinase 9 (an unexpected result). Additional analysis of this data made available as an online resource should facilitate further molecular characterisation of LCIS.

Estrogen and progesterone receptor levels in nonneoplastic breast epithelium of breast cancer cases versus benign breast biopsy controls

BackgroundPrevious studies and biological mechanisms of carcinogenesis suggest that the steroid receptor content of benign breast epithelium may be related to breast cancer risk. The objective in this study was to compare the levels of estrogen receptor-α (ER) and progesterone receptor (PR) in nonneoplastic breast epithelium between breast cancer cases and biopsy controls.MethodsBetween 1995 and 1997 at two sites (Women's College Hospital in Toronto and Kingston General Hospital), 667 women who were scheduled for diagnostic excisional breast biopsies completed a questionnaire providing personal information and agreed to allow analysis of routinely resected tissue. Histological slides with nonneoplastic epithelium were available for 101 cancer cases and 200 biopsy controls in Toronto and for 105 cancer cases and 119 controls in Kingston. Nonneoplastic epithelium was examined with immunohistochemical assays to determine the percent of epithelial cells staining for ER and PR. Unconditional logistic regression was used to calculate odds ratios (OR) stratified by study site.ResultsThe ER content of nonneoplastic tissue was higher in cases than biopsy controls in unadjusted analyses; after adjustment for age, however, a weak association remained in only one of the study sites. After adjustment for age, the PR content of nonneoplastic tissue was slightly lower in breast cancer cases than controls in one study site. Furthermore, this inverse association was confined to women with PR negative breast cancer in comparison to the controls. No interaction between ER and PR content of nonneoplastic tissue was observed in relation to the odds of having breast cancer.ConclusionThe results of this study are consistent with only a slight indication of increased ER levels in nonneoplastic tissue in breast cancer cases relative to controls. This study contributes to the understanding of breast cancer by examining both ER and PR in nonneoplastic tissue. Limitations remain, however, such as the necessity of using as controls women with benign breast changes, difficulties in selecting the appropriate tissue for analysis, and tissue sampling concurrent to diagnosis.

DNA Methylation in Benign Breast Epithelium in Relation to Age and Breast Cancer Risk

Many established breast cancer risk factors are related to the timing and duration of exposure to reproductive hormones, which are known to drive breast epithelial cell proliferation. The epigenetic molecular clock hypothesis suggests that CpG island methylation records the cell division history of benign epithelium. In proliferative epithelium, such as breast, this may provide an individualized cell-based measure of cancer risk. Methylation of cyclin D2, APC, HIN1, RASSF1A, and RAR-beta2 was measured by quantitative multiplex methylation-specific PCR in 290 benign and malignant breast epithelial cell samples obtained by palpation-directed fine-needle aspiration biopsy from 164 women. Univariate, multivariate, and unsupervised cluster analysis was used to establish the relationship between TSG methylation and a personal history of breast cancer, predicted breast cancer risk, and specific breast cancer risk factors. RASSF1A methylation was highly correlated with breast cancer risk [odds ratio (OR), 5.28; 95% confidence interval (95% CI), 1.95-14.32; P = 0.001], atypical cytology (OR, 4.11; 95% CI, 1.30-12.98; P = 0.016), and benign breast disease requiring biopsy (OR, 6.12; 95% CI, 1.41-26.51; P = 0.016). RASSF1A methylation increased linearly between ages 32 and 55. Increasing parity was associated with decreased APC methylation. TSG methylation increases in benign breast epithelium with increasing age. Because it is independently related to a personal history of benign or malignant breast disease and to predicted breast cancer risk, it may have value for breast cancer risk stratification and as a surrogate endpoint marker in prevention trials.

Cell-based breast cancer risk stratification based on DNA methylation in fine needle aspiration samples

1508 Background: Tumor suppressor gene (TSG) methylation is identified in nearly all breast cancers, but rarely in histologically normal breast tissue from wonen unaffected with breast cancer. Its occurrence in high risk preneoplasia and in benign breast tissue adjacent to breast cancer suggests that it may represent a high risk field change that could be exploited for cell-based breast cancer risk stratification. Methods: TSG methylation was measured by quantitative methylation-specific real time PCR in 53 breast tumor fine needle aspiration (FNA) biopsies, 84 cellular random periareolar FNAs (RP-FNA) ipsilateral or contralateral to these cancers, 36 cellular RP- FNAs from unaffected women at high risk for breast cancer by the Gail model, and 95 cellular RP-FNAs from unaffected women at lower risk by the Gail model. Results: The breast tumors showed a high frequency of TSG methylation: RASSF1A 80%, HIN-1 65%, Cyclin D2 60%, RAR-β2 53%, and APC 47%. In general, RP-FNA samples from cancer patients and Gail high risk patients showed a greater frequency of methylation than samples from Gail lower risk patients: RASSF1A 43% vs. 21%, P = 0.001, HIN-1 32% vs. 20%, P = 0.05; Cyclin D2 18% vs. 9%, P = 0.10; RAR-β2 21% vs. 18%, P = 0.68; and APC 25% vs. 16%, P = 0.17. Twelve of 215 RP-FNA samples (5%) showed very high levels of methylation (&gt;10% methylation for two or more genes). Only two of these samples were from women classified as lower risk by the Gail model. Methylation frequencies were entirely independent of cell yields but the frequency of RASSF1A methylation increased with increasing Masood scores (P = 0.05). Methylation of RASSF1A in one breast was highly predictive of RASSF1A methylation in the opposite breast (P &lt; 0.0001). Conclusions: TSG methylation appears to be a breast cancer risk-associated field change that can be quantified in RP-FNA samples. RASSF1A methylation occurs frequently in benign breast epithelium, provides reasonable discrimination between high and lower risk breasts (O.R. = 2.0), is related to cytological atypia, and may be an early marker of a methylator phenotype. Quantification of TSG methylation in RP-FNA samples may provide a valuable surrogate endpoint biomarker for Phase II prevention trials. No significant financial relationships to disclose.

Immunolocalisation of members of the polypeptide N-acetylgalactosaminyl transferase (ppGalNAc-T) family is consistent with biologically relevant altered cell surface glycosylation in breast cancer

An extensive family of UDP-N-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferases (polypeptide N-acetylgalactosaminyltransferases, ppGalNAc-T's) catalyse the attachment of the first N-acetylgalactosamine (GalNAc) monosaccharide to the polypeptide at the initiation of O-linked glycosylation of proteins. Some members of the family are broadly expressed while others are more restricted in their distribution, their expression and activity being confined to certain cells or tissues, being associated with physiological states or differentiation. Their careful regulation, which is not well understood, may mediate the synthesis of varied glycoforms of cellular proteins with different biological activities. Disruptions in glycosylation are a common feature of cancer and may have functional significance. Immunocytochemistry with confocal scanning laser microscopy was employed to detect members of the ppGalNAc-T family, ppGalNAc-T1, -T2, -T3, -T4 and -T6 in a range of breast cell lines. The cells were chosen to represent a range of phenotypes from 'normal'/benign (HMT 3,522), primary, non-metastatic breast cancer (BT 474), to aggressive, metastatic breast cancer (ZR75-1, T47D, MCF-7, DU 4,475). They stably synthesise varying levels, consistent with origin and phenotype, of aberrantly glycosylated glycoproteins featuring exposed, terminal GalNAc residues, including the cancer-associated Tn antigen, which, in numerous studies, have been associated with metastatic competence and poor cancer prognosis. GalNAc-T1 and -T2 were detectable at low levels in all cell lines studied. ppGalNAc-T4, which has never been described in breast, was very weakly detectable in BT 474, MCF7 and T47D. ppGalNAc-T3 and -T6 were weakly detectable or undetectable, respectively, in the cell line HMT 3,522 derived from 'normal'/benign breast epithelium, but were readily detectable in all malignant cell lines. Thus, a broader range of ppGalNAc-T's were detectable in the malignant cell lines in comparison to the 'normal'/benign cells, where only the 'housekeeping' ppGalNAc-T1 and -T2 were present. Expression of normally tightly restricted ppGalNAc-T's may result in initiation of O-linked glycosylation at normally unoccupied potential glycosylation sites leading to altered glycoforms of proteins with changed biological activity which may contribute to the pathogenesis of cancer.

Human Breast Fibroblasts Inhibit Growth of the MCF10AT Xenograft Model of Proliferative Breast Disease

Stromal fibroblasts are important for normal breast homeostasis and regulation of epithelial growth; however, this regulatory function is altered during carcinogenesis. To study the role of fibroblasts in the development of breast cancer, fibroblasts derived from normal breast (NAFs) were incorporated into the MCF10AT xenograft model of progressive proliferative breast disease. The persistence of human NAFs in xenografts was established by intracellular labeling and tyramide-coupled fluorescent in situ hybridization. Overall, the number of MCF10AT epithelial structures was decreased, and the rate of epithelial cell apoptosis was increased in xenografts containing NAFs. However, these changes were primarily in low-grade epithelial structures, corresponding to normal or mildly hyperplastic ductal epithelium. The level and rate of apoptosis of high-grade epithelial structures, corresponding to in situ and invasive carcinoma, were not consistently altered by NAFs. In addition, there was variability in the growth-inhibitory capacity of NAFs derived from different individuals. NAFs induced changes in the morphology of high-grade MCF10AT structures and in xenograft stroma, including the composition of extracellular matrix, and increased angiogenesis and lymphocytic infiltration. These findings imply that NAFs can inhibit the growth of normal and hyperplastic epi-thelium but are less able to regulate the more transformed epithelial cells that arise during carcino-genesis.