Kathryn Edmiston

IMP3 is a novel biomarker for triple negative invasive mammary carcinoma associated with a more aggressive phenotype

IMP3, an oncofetal protein, is a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein family. Its relevance as a novel biomarker in lung, pancreatic, renal, and cervical adenocarcinoma was recently revealed. However, its role in breast carcinogenesis and tumor progression is not yet established. Basal-like carcinoma was initially identified by gene expression profiling. It accounts for 15% to 30% of all breast cancers. These tumors express basal epithelial markers including cytokeratin 5 but lack expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), therefore, are often referred to as triple negative breast cancer. They have been found to be associated with a worse overall and disease-free survival. In this retrospective study, we examined the IMP3 expression in invasive ductal carcinoma of the breast and correlated its expression with morphological and biologic prognostic factors. The study group comprised 138 cases of invasive ductal carcinoma retrieved from the surgical pathologic files for a 10-year period from 1997 to 2006. Survival data and clinical stage were available on all 138 patients. Tumor characteristics including size, grade, lymphovascular invasion, necrosis, lymph node metastasis, estrogen receptor, progesterone receptor, and HER2 status were obtained from pathologic reports. Immunohistochemistry was performed on formalin-fixed paraffin-embedded tissue using mouse monoclonal antibody against IMP3 and CK5/6. Of the 138 breast cancer cases, IMP3 expression was seen in 45 (33%). Twenty-five of the IMP3+ cases were triple negative. We found significant correlation between IMP3 expression and higher grade (P = .001), necrosis (P< .0001) triple negative, and CK5/6 expression (P < .0001 for each). Cox multivariate analysis showed a hazard ratio of IMP3 expression at 3.14 (P = .05). IMP3 is a novel biomarker for triple negative (basal-like) invasive mammary carcinoma, and its expression is associated with a more aggressive phenotype and decreased overall survival.

HER-2 status in breast cancer: correlation of gene amplification by FISH with immunohistochemistry expression using advanced cellular imaging system.

It has become important to accurately evaluate the status of HER-2/neu in invasive breast cancer, especially when one is considering the use of anti-HER-2 monoclonal antibody therapy (Trastuzumab). Almost one third of invasive breast carcinomas overexpress the HER-2/neu protein, so the use of the anti-HER-2/neu monoclonal antibody Herceptin (trastuzumab) to block the protein has become important in the management of and in prolonging the survival for patients with metastatic breast cancer. The effectiveness of this therapy is dependent on accurately evaluating the HER-2 status in these tumors, which can be done either by studying the expression of HER-2 protein by immunohistochemistry (IHC) or by evaluating HER-2 gene amplification by fluorescent in situ hybridization (FISH). Since interobserver variability may occur in manually grading HER-2 protein expression by IHC, the aim of this study was to compare the HER-2/neu expression by IHC using a computer-based image analysis system with that of the gene amplification by FISH. Formalin-fixed paraffin-embedded archival tissue from 108 primary infiltrating ductal carcinomas were immunostained using the HercepTest (DAKO). To reduce interobserver variability, membrane staining was evaluated using the Automated Cellular Imaging System (ACIS) by ChromaVision, and the cases were divided into four groups: group 1 (n=23) with HER-2/neu expression ACIS score less than or equal to 1.5; group 2 (n=17) with a score ranging from 1.6 to 1.9; group 3 (n=46) with a score 2.0 to 2.5; and group 4 (n=22) with a score greater than or equal to 2.6. FISH was performed on all of the 108 cases using the PathVysion HER-2/neu DNA probe kit from Vysis Inc. All cases were also manually reviewed and graded as negative, 1+, 2+, and 3+ according to the DAKO HercepTest grading scheme. Cases with negative and 1+immunostaining were considered as HER-2 not overexpressed, and cases with 2+ and 3+ staining were classified as showing HER-2 overexpression. In group 1, 1 of 23 (4%), in group 2, 2 of 17 (12%), in group 3, 5 of 46 (11%), and in group 4, 19 of 22 (86%) cases showed gene amplification by FISH. Furthermore, in group 4 all 15 (100%) cases with an ACIS score of 3 or greater were FISH positive. Correlation with manual IHC score and FISH showed that 2 of the 23 (9%) IHC negative (0 and 1+) cases and 25 of the 85 (29%) IHC positive (2+ and 3+) cases showed gene amplification by FISH. This study shows that the amplification of the HER-2/neu gene correlates better with overexpression of the HER-2/neu protein by IHC when the score is either less than 1.5 or greater than 2.6 by ACIS. Therefore, FISH may be useful to better evaluate HER-2/neu status in breast cancer in cases where the ACIS score by immunohistochemistry is 1.6 to 2.5, and since the correlation is so good, FISH may not be needed for HER-2 evaluation in cases with ACIS scores less than 1.5 and greater than 2.6.

Correlation of umbilical cord blood haematopoietic stem and progenitor cell levels with birth weight: implications for a prenatal influence on cancer risk

We examined the relation with birth weight and umbilical cord blood concentrations of haematopoietic stem and progenitor populations in 288 singleton infants. Across the whole range of birth weight, there was a positive relation between birth weight and CD34+CD38− cells, with each 500 g increase in birth weight being associated with a 15.5% higher (95% confidence interval: 1.6–31.3%) cell concentration. CD34+ and CD34+c-kit+ cells had J-shaped relations and CFU-GM cells had a U-shaped relation with birth weight. Among newborns with ⩾3000 g birth weights, concentrations of these cells increased with birth weight, while those below 3000 g had higher stem cell concentrations than the reference category of 3000–3499 g. Adjustment for cord blood plasma insulin-like growth factor-1 levels weakened the stem and progenitor cell–birth weight associations. The positive associations between birth weight and stem cell measurements for term newborns with a normal-to-high birth weight support the stem cell burden hypothesis of cancer risk.

Novel measurements of mammary stem cells in human umbilical cord blood as prospective predictors of breast cancer susceptibility in later life

BACKGROUND The size of the breast stem-cell pool could underlie the intrauterine roots of breast cancer. We studied whether breast stem cells exist in umbilical cord blood and if they correlate with hematopoietic stem-cell measurements that have been positively associated with perinatal risk factors for breast cancer. SUBJECTS AND METHODS We isolated mononuclear cells from umbilical cord blood of 170 singleton full-term pregnancies and determined, by reverse transcription polymerase chain reaction, the presence of genes of putative breast epithelial stem-cell/progenitor markers [including epithelial cell adhesion molecule (EpCAM), CD49f (α6-integrin), CD117 (c-kit receptor), CD24, and CD29 (β1-integrin)]. By immunocytochemistry, we colocalized protein expressions of EpCAM+CD49f+, CD49f+CD24+, and CD24+CD29+. We correlated concentrations of putative breast stem-cell/progenitor subpopulations, quantified by flow cytometry, with concentrations of hematopoietic stem cells. RESULTS Mammary stem-cell phenotypes were identified in umbilical cord blood. The measured EpCAM+ subpopulation was positively correlated with concentrations of CD34+ and CD34+CD38- hematopoietic stem cells (both P=0.006). Additionally, EpCAM+CD49f+ and CD49f+CD24+ subpopulations were positively correlated to the CD34+ cells (P=0.03 and 0.008, respectively). CONCLUSION The positive association between measurable breast and hematopoietic stem cells in human umbilical cord blood suggests plausible mechanisms for a prenatal influence on breast cancer risk.BACKGROUND The size of the breast stem-cell pool could underlie the intrauterine roots of breast cancer. We studied whether breast stem cells exist in umbilical cord blood and if they correlate with hematopoietic stem-cell measurements that have been positively associated with perinatal risk factors for breast cancer. SUBJECTS AND METHODS We isolated mononuclear cells from umbilical cord blood of 170 singleton full-term pregnancies and determined, by reverse transcription polymerase chain reaction, the presence of genes of putative breast epithelial stem-cell/progenitor markers [including epithelial cell adhesion molecule (EpCAM), CD49f (α6-integrin), CD117 (c-kit receptor), CD24, and CD29 (β1-integrin)]. By immunocytochemistry, we colocalized protein expressions of EpCAM+CD49f+, CD49f+CD24+, and CD24+CD29+. We correlated concentrations of putative breast stem-cell/progenitor subpopulations, quantified by flow cytometry, with concentrations of hematopoietic stem cells. RESULTS Mammary stem-cell phenotypes were identified in umbilical cord blood. The measured EpCAM+ subpopulation was positively correlated with concentrations of CD34+ and CD34+CD38- hematopoietic stem cells (both P = 0.006). Additionally, EpCAM+CD49f+ and CD49f+CD24+ subpopulations were positively correlated to the CD34+ cells (P = 0.03 and 0.008, respectively). CONCLUSION The positive association between measurable breast and hematopoietic stem cells in human umbilical cord blood suggests plausible mechanisms for a prenatal influence on breast cancer risk.

Effect of preeclampsia on umbilical cord blood stem cells in relation to breast cancer susceptibility in the offspring

Women born from a preeclamptic (PE) pregnancy are associated with a lower risk of breast cancer. Prenatal and early-life exposures are hypothesized to influence breast cancer susceptibility through their effect on stem cells. We examined stem cell populations in umbilical cord blood from PE pregnancies and compared with those from pregnancies without this condition. We isolated mononuclear cells from 58 PE and 197 normotensive (non-PE) umbilical cord blood samples and examined the different stem cell populations. Hematopoietic (CD34(+) and CD34(+)CD38(-)), endothelial (CD34(+)CD133(+), CD34(+)VEGFR2(+), CD133(+)VEGFR2(+) and CD34(+)CD133(+)VEGFR2(+)), and putative breast (EpCAM(+), EpCAM(+)CD49f(+), EpCAM(+)CD49f(+)CD117(+), CD49f(+)CD24(+), CD24(+)CD29(+) and CD24(+)CD29(+)CD49f(+)) stem/progenitor cell subpopulations were quantified by flow cytometry and compared between PE and non-PE samples. Hematopoietic CD34(+) cell counts were significantly lowered in PE compared with non-PE samples (P = 0.039, Kruskal-Wallis test). Levels of CD34(+)CD133(+) endothelial progenitor cells were also lower in PE samples (P = 0.032, multiple regression analysis). EpCAM(+) and EpCAM(+)CD49f(+) putative breast stem cell levels were significantly lowered in PE subjects (multiple regression analysis: P = 0.038 and 0.007, respectively). Stratifying by newborn gender, EpCAM(+) and EpCAM(+)CD49f(+) stem cells were significantly lowered in PE samples of female, but not male, newborns. Umbilical cord blood samples from pregnancies complicated by preeclampsia thus had significantly lower levels of hematopoietic, endothelial, and putative breast stem cells than non-PE controls. With a lowered breast cancer risk for offspring of a PE pregnancy, our findings provide support to the hypothesis that susceptibility to breast oncogenesis may be affected by conditions and processes during the prenatal period.

Prenatal Maternal Physical Activity and Stem Cells in Umbilical Cord Blood.

PURPOSE Early life processes, through influence on fetal stem cells, affect postnatal and adult health outcomes. This study examines the effects of physical activity before and during pregnancy on stem cell counts in umbilical cord blood. METHODS We isolated mononuclear cells from umbilical cord blood samples from 373 singleton full-term pregnancies and quantified hematopoietic (CD34(+), CD34(+)CD38(-), and CD34(+) c-kit(+)), endothelial (CD34(+)CD133(+), CD34(+)CD133(+)VEGFR2(+), CD34(+)VEGFR2(+), and CD133(+)VEGFR2(+)), and putative breast (EpCAM(+), EpCAM(+)CD49f(+), EpCAM(+)CD49f(+)CD117(+), CD49f(+)CD24(+), CD24(+)CD29(+), and CD24(+)CD29(+)CD49f(+)) stem/progenitor cell subpopulations by flow cytometry. Information on physical activities before and during pregnancy was obtained from questionnaires. Weekly energy expenditure was estimated based on metabolic equivalent task values. RESULTS Prepregnancy vigorous exercise was associated positively with levels of endothelial CD34(+)CD133(+), CD34(+)CD133(+)VEGFR2(+), CD34(+)VEGFR2(+), and CD133(+)VEGFR2(+ )progenitor cell populations (P = 0.02, P = 0.01, P = 0.001, and P = 0.003, respectively); positive associations were observed in samples from the first births and those from the second or later births. Prepregnancy moderate and light exercises and light exercise during the first trimester were not significantly associated with any stem/progenitor cell population. Light exercise during the second trimester was positively associated with CD34(+)VEGFR2(+) endothelial progenitor cells (P = 0.03). In addition, levels of EpCAM(+)CD49f(+) and CD49f(+)CD24(+) breast stem cells were significantly lower among pregnant women who engaged in vigorous/moderate exercise during pregnancy (P = 0.05 and P = 0.02, respectively). CONCLUSIONS Vigorous exercise before pregnancy increases the number of endothelial progenitor cells in umbilical cord blood and thus could potentially enhance endothelial function and improve cardiovascular fitness in the offspring. Findings of lower levels of putative breast stem cell subpopulations could have implications on exercise and breast cancer prevention. Prenatal effects of exercise on fetal stem cells warrant further studies.

Identifying women at high risk for invasive breast cancer at the time of screening mammography.

6 Background: Most mammography centers collect reproductive, family, and environmental data on patients presenting for screening mammograms. These data, if entered into a Gail Model Risk Assessment Tool, can help identify those patients at increased risk. These patients can then be referred to high-risk centers that will focus on risk-reduction strategies. METHODS This study is an observational prospective cohort of 1,000 women presenting for mammographic screening or diagnostic evaluation at mammography clinics affiliated with a single institution. Women at the time of their mammogram were asked to fill out a standard intake sheet by the institutional clinic. These data sheets were gathered and Gail model risk scores were calculated. Women with a 5-year risk of invasive breast cancer of 1.7% or greater were identified. Patients with a history of breast cancer or who underwent diagnostic mammography were excluded. A high risk subgroup was identified and risk factors were analyzed. Women at particularly high risk ( ≥ 3.4% or double the 5-year risk) were analyzed separately. RESULTS Of 1,000 women screened, 366 had ≥ 1.7% 5-year risk of invasive breast cancer. 26% (96) of these women were under 60 years old while 74% (270) were ≥ 60 years old. Among the latter group, 19.6% (53) were found to have ≥ 3.4% of developing invasive cancer. In these women with double the 5-year risk, 96.2% had a family history of breast cancer and 92.4% had a prior biopsy. Similarly, in women under 60, greater than half were high risk secondary to a prior biopsy or family history. CONCLUSIONS 1/3 of patients who receive annual screening are at high risk for breast cancer. These patients can be identified from data routinely obtained at the time of screening mammography. Many were found to be at increased risk due to a strong family history or prior biopsy. Mammography centers may be the ideal setting in which to alert these patients of their risk and refer them to high risk centers for genetic counseling and consideration of chemoprevention.

Abstract P2-04-04: Breast cancer risk reduction in high risk women identified at the time of screening mammography. Integrating data from an established high risk clinic

Background: At the time of screening mammography, patient data can be collected and entered into a Modified Gail Model Risk Assessment Tool to identify patients at increased risk of invasive breast cancer. In turn, these patients can be referred to high risk centers where risk reduction strategies and chemoprophylaxis can be considered. The objective of this study is to establish how many women are at high risk of invasive breast cancer in a screening population who should be referred to a high risk center for counseling. Methods: This is an observational prospective study evaluating 1,000 women at a single institution presenting for mammographic screening or diagnostic evaluation. At the time of mammography, these women routinely complete a standard intake questionnaire addressing breast cancer risk factors put forward by the institutional clinic. Modified Gail model risk scores were calculated from these data sheets. Women with a 5 year risk of invasive breast cancer of 1.7% or greater were identified as well as women at particularly high risk (≥3.4% or double the 5-year risk). At the time of analysis patients with a history of breast cancer or who underwent diagnostic mammography were excluded. Retrospective data from our institutional high risk clinic from January 2007 to December 2009 was analyzed in respect to patient age at referral, Gail Model Score, personal history of breast cancer, history of benign breast biopsies, and recommendation for and acceptance of chemoprophylaxis. Results: Of 1,000 women screened in the prospective analysis, 366 had ≥1.7% 5-year risk of invasive breast cancer. 26% (96) of these women were under 60 years old while 74% (270) were ≥60 years old. Among the latter group, 19.6% (53) were found to have ≥ 3.4% of developing invasive cancer. In these women with double the 5-year risk, 96.2% had a family history of breast cancer and 69.8% had a prior biopsy. Similarly, in women under 60, greater than half were high risk secondary to a prior biopsy or family history. In the retrospective analysis of 600 patients evaluated in the high risk clinic, 38.8% were eligible for chemoprevention based on age >35, Gail model risk score, and no contraindications to treatment. 45.7% accepted recommendation for chemoprophylaxis as opposed 54.3% who had declined. Age was not found to be a predictor of acceptance of treatment. Conclusions: 1/3 of patients who receive annual screening are at high risk for breast cancer. These patients can be identified from data routinely obtained at the time of screening mammography. These women should be referred for high-risk counseling and consideration of chemoprevention at the time of screening mammography. Further prospective studies may be warranted to determine if this is an effective way to provide risk reduction strategies for high risk women. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-04-04.