Hartmann, Lynn C.

Abstract Mini-Chromosome Maintenance protein 7 (MCM7) is involved in replicative licensing and synthesis of DNA. It is previously identified as over-expressed gene in high-grade serous carcinomas compared to serous borderline tumors of the ovary in cDNA microarray studies. In this study, we sought to validate MCM7 expression in ovarian tumors by applying immunohistochemical analysis on tissue microarrays consisting of 445 ovarian tumors. MCM7 expression was quantified as MCM7 labeling index by determining the tumor nuclei positive for MCM7 staining as a percentage of total tumor nuclei. The labeling index scores were independently generated by two methods: PATH score provided by a manual review of each samples by a pathologist and ACIS score provided by Automated Cell Imaging System. Analyses of both scores indicated high degree of concordance in score distribution between PATH and ACIS. MCM7 expression is significantly higher in high-grade serous carcinomas compared to serous borderline tumors, confirming our previous cDNA microarray studies. In both PATH and ACIS analysis, MCM7 expression is also significantly higher in high-grade serous carcinomas compared to other histology subtypes of ovarian cancer. Finally, to identify the clinical significance of MCM7 expression, we determined the association between MCM7 expression and disease-free survival. MCM7 labeling index was dichotomized into two groups (high and low) using median labeling index as cut-off. For both PATH or ACIS-derived scores, univariate analyses indicate significant association of high MCM7 labeling index with better disease-free survival in high-grade serous carcinomas. These results suggest the clinical significance of MCM7 expression in high-grade serous carcinomas of the ovary and the need for further evaluation of MCM7 as a potential theranostic biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4637.

Abstract Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients. Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00–2.24, P = 0.052). Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Clin Cancer Res; 17(6); 1591–602. ©2011 AACR.

Abstract Background: Epithelial proliferation in benign breast disease (BBD) is a well-established biomarker of breast cancer risk, and BBD tissues can be used as a platform to assess other biomarkers for breast cancer risk. Lobular involution (the degree of age-related atrophy in lobules) is a new biomarker, with greater degrees of involution associated with lower breast cancer risk. Successful application of tissue biomarkers of risk to breast tissues from normal women would be valuable for risk prediction in the general population. We hypothesized that normal breast tissue samples would differ from BBD tissues in these histologic features of breast cancer risk, with less epithelial proliferation but a similar degree of involution compared to BBD tissues. Methods: Core biopsy samples from the Mayo Benign Breast Disease Cohort Study were compared to core biopsy samples of normal breast tissue obtained from the Susan G. Komen for the Cure Tissue Bank at IU Simon Cancer Center (KTB), a unique resource of breast tissue from donor women without any known breast abnormalities. All core biopsies were assessed histologically by a single breast pathologist for degree of epithelial proliferation (classified as non-proliferative, proliferative without atypia, or atypical hyperplasia) and extent of lobular involution (classified as none, 1-24%, 25-74%, or >75% of lobules). Data were analyzed using chi-square tests and logistic regression. Results: Overall, 318 Mayo BBD and 303 normal (KTB) core breast biopsy samples were evaluated. As anticipated, significantly less epithelial proliferation was present in normal samples compared to BBD samples. Proliferative disease occurred in 9/303 (3%) of normals versus 79/318 (25%) of BBD, and atypical hyperplasia was seen in 2/303 (0.7%) of normal samples versus 24/318 (8%) of BBD core biopsies, p < 0.0001. This difference remained significant after adjusting for age (which was greater overall in the BBD samples). Complete involution was more frequent in normal compared to BBD tissues, although the difference did not reach statistical significance. The age-adjusted odds ratio of complete involution in normal versus BBD tissues was 1.46 (95% CI 0.98-2.17, p = 0.06), suggesting a possible delay or failure of involution in women with BBD. Conclusions: Histologic features of normal breast tissues are different from those of BBD tissues, with less epithelial proliferation and more lobular involution, consistent with a lower risk of breast cancer expected for women with normal breast tissue compared to those with benign breast disease. Normal breast tissues are a valuable and distinct resource that should be considered for biomarker validation studies. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B23.

Abstract Estrogen receptor (ER) is expressed in normal and malignant breast epithelium, and expression levels have been found to increase with age in normal breast epithelium but not in atypical hyperplasia (AH) and carcinoma in situ. Here we assess ER expression in AH and its association with later breast cancer. ER expression was assessed immunohistochemically in archival sections from 246 women with AH who had open benign breast biopsy from 1967 to 1991. The ACIS III (Dako) was utilized to calculate ER expression in all atypical foci. Using multivariate linear regression, we examined associations of ER expression with age at biopsy, indication for biopsy, type of atypia, number of atypical foci, involution status, and family history. Breast cancer risk across levels of ER expression was also assessed compared with the Iowa SEER control population. Among 246 women, 87 (35%) had atypical ductal hyperplasia (ADH), 141 (57%) had atypical lobular hyperplasia (ALH), and 18 (7%) had both. Forty-nine (20%) developed breast cancer (median follow-up of 14.4 years). Multivariate analysis indicated that type of atypia and age at diagnosis were significantly associated with ER percent staining and intensity (P < 0.05). ER expression was increased in women with ADH and/or those over age 55. ER expression did not significantly impact breast cancer risk in patients diagnosed with atypia. We found increasing ER expression in AH with increasing age. ER expression in AH does not further discriminate breast cancer risk in women with atypia. Cancer Prev Res; 4(3); 435–44. ©2011 AACR.

Abstract Background: p16 is a nuclear protein encoded by the p16INK4a gene that regulates the G1-S cell cycle checkpoint. Its overexpression could reflect normal proliferative arrest, but also the evolution of carcinogenic processes that have overcome the p16 block, with progression from hyperplasic lesions to invasive breast cancer. Our aim with this study was to correlate p16 expression, both individually and in combination with expression of COX-2 and Ki67, with subsequent risk of breast cancer among women with atypical hyperplasia. Methods: p16 expression was assessed by immunohistochemical assays in archival paraffin-embedded, formalin-fixed sections from 233 women with atypia whose biopsy specimens were obtained via surgical excision at the Mayo Clinic from January 1, 1967, through December 31, 1991. p16 expression was scored as percent of cells positive and intensity of staining. Risk factor information and follow-up for breast cancer events were obtained via study questionnaire and the medical records. Standardized incidence ratios (SIRs) were used to compare observed numbers of breast cancer events to population-based expected counts, both at 10 years and over the entire course of follow-up. Results: Forty-seven patients (20%) developed breast cancer with a mean follow-up of 14.3 (SD=6.96) years. The overall risk of developing breast cancer was not modified by increasing p16 overexpression (low percent of staining with RR=3.89, 95% CI=2.72 to 5.56; high percent of staining with RR=3.91, 95% CI=2.43 to 6.28; similar results for intensity of staining). However, overexpression of both p16 and COX2 conveyed a stronger risk of breast cancer (SIR=4.01, 95% CI 2.28 to 7.07) as compared to dual low expression (SIR 1.78, 95% CI 0.67-4.74). Moreover, overexpression of both Ki67 and p16 was related to a higher 10-year breast cancer risk (SIR 6.25, 95% CI 2.35-16.6) as compared to low expression of both markers (SIR 0.87, 95% CI 0.22-3.49). Conclusions: although single expression of p16 does not stratify breast cancer risk, its overexpression in concert with biomarkers that reflect other carcinogenic processes (e.g. Ki67 and COX2) could help to define risk of tumor development. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 910.

Abstract Background: Uncontrolled proliferation is a defining feature of the malignant phenotype. Ki67 is a marker for proliferating cells and is overexpressed in many breast cancers. Atypical hyperplasia is a premalignant lesion of the breast (relative risk ∼ 4.0). Here we asked if Ki67 expression could stratify risk in women with atypia.Methods: Ki67 expression was assessed immunohistochemically by digital image analysis in archival sections from 192 women with atypia diagnosed at the Mayo Clinic 1/1/67-12/31/91. Risk factor and follow-up data were obtained via study questionnaire and medical records. Observed breast cancer events were compared to population expected rates (Iowa SEER) using standarized incidence ratios (SIRs). We examined both short-term (within 10 years) and long-term (after 10 years) risk of breast cancer (BC) following atypia biopsy.Results: The median value for percent positive cells for Ki67 was 1.0%; the 75th percentile value was 2.3%. Based on the empirical distribution of staining values in our cohort, we selected a cutoff of 2% cells positive to separate high from low staining. There were no differences in Ki67 levels by age at biopsy, type of atypia (ADH, ALH, or both), number of foci of atypia, or family history. 32 women developed BC over a median of 14.6 years. Among them, those with ≥2% Ki67 expression had a shorter time to breast cancer (median 5.5 years, IQR=3.2-7.2) than those with <2% cells positive (median=13.8 years, IQR=11.6-20.3). There was a positive association between Ki67 overexpression (≥2% of cells positive) and risk of BC in the first 10 years of follow-up (SIR=4.42 [95% CI 2.21-8.84]). This excess risk resulted in a 10-year cumulative incidence of 14.1%. In contrast, in the women with low Ki67 expression, we found no increased risk of BC in the first 10 years, with SIR 1.01 (95% CI 0.38-2.70), which was significantly lower than the women with high expression (p=0.01). The 10-year cumulative incidence of BC was 3% for the low Ki67group, in line with population averages. After 10 years, risk increased significantly in the low Ki67 group [SIR 5.69 (3.63-8.92)] vs. no increased risk in the high Ki67 group [SIR 0.78 (0.11-5.55)]. We formally examined this apparent time-dependent difference in incidence patterns by Ki67 staining levels via Poisson regression analysis, and found a statistically significant interaction between pre- vs. post-10 years incidence and high vs. low staining levels (p<0.001).Conclusions: Ki67 appears to be a time-varying biomarker of risk of breast cancer in women with atypical hyperplasia. Women whose atypias show higher proliferation rates have an increased risk for an earlier BC (within 10 years). Those with lower proliferation rates tend to develop BCs later, after 10 years. Ki67 levels may help to define the time period of greatest risk for women with atypia. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 909.

Abstract Background: Risk prediction models are important clinical tools for counseling and decision making for women with an elevated risk of breast cancer, such as women with atypical lobular or ductal hyperplasia (atypia). The Gail model has been shown to underestimate the risk of breast cancer in women with atypia. We sought to evaluate the performance of the Tyrer-Cuzick model in a well-defined cohort of women with atypia.Methods: The Mayo Benign Breast Disease (BBD) Cohort includes 9,376 women aged 18 to 85 who had an open breast biopsy with benign pathological findings between 1967 and 1991. The subset of women with atypia was identified by our study pathologists. Risk factor data required by the Tyrer-Cuzick model were collated for each woman, and the model was used to predict each woman's risk of developing invasive breast cancer within ten years. The predicted numbers of breast cancers were compared to the observed numbers to assess overall model calibration, and the concordance between breast cancer outcomes and predicted probabilities was used to measure the accuracy of the model on the individual level. Subset analysis of women with strong family history was performed. Strong family history was defined as: at least one first-degree relative with breast cancer before the age of 50 years or two or more relatives with breast cancer, at least one being a first degree relative.Results: Three hundred and thirty one women with atypia were identified. Over a mean follow up of 13.7 years, 62 (19%) women have developed invasive breast cancer. Thirty one women developed invasive cancer in the first ten years after biopsy. The Tyrer-Cuzick model predicted that 64.6 invasive breast cancers would occur in the first ten years after biopsy. Thus, the predicted to observed ratio was 2.08 [95% CI: 1.47 – 2.96], p < 0.001).The concordance statistic was 0.51, revealing that the Tyrer-Cuzick model did not accurately distinguish, on an individual level, between women who were diagnosed with invasive breast cancer within ten years of biopsy and those who were not.Performance was somewhat better in the subset of 68 women with a strong family history of breast cancer. The model predicted 12.3 invasive breast cancers, and 7 were observed, for a predicted to observed ratio of 1.76 [95% CI: 0.84 – 3.70]. The concordance statistic was 0.57.Conclusion: The Tyrer-Cuzick model significantly overestimated the risk of breast cancer in the first ten years after biopsy for this group of women with atypia. Individual risk estimates showed poor concordance. Performance was slightly improved in women with a strong family history of breast cancer. Physicians should be cautious when using the Tyrer-Cuzick model to predict ten-year risk in patients with atypia. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6063.

Abstract Experiences at time of breast cancer (BC) diagnosis are associated with patient psychological health several months to years later; however, little investigation of this relationship during initial period following diagnosis has been undertaken.METHODS:This prospective study was designed to characterize the experience of receiving cancer diagnosis in women initiating treatment for BC (Stage 0 to III) and to identify associations between this experience and early psychological adjustment, hopefulness, and satisfaction with medical care. 121 women who recently received BC diagnosis prompting referral to specialty center responded to questionnaires assessing their experiences and coping styles:1. Experience Receiving Breast Cancer Diagnosis (ERBCD), categorizing patient demographic, medical, and psychosocial factors; context factors (location, privacy); clinician factors and behaviors.2. Cancer Diagnostic Interview Scale (CDIS) for perceived psychotherapeutic techniques used by clinican conveying cancer diagnosis.3. Mental Adjustment to Cancer Scale (MAC) for coping styles of fighting spirit, hopelessness/helplessness, anxious preoccupation, fatalism, and avoidance.Subjects completed the Profile of Mood States (POMS), Spielberger State-Trait Anxiety Inventory (STAI) and ratings of hopefulness and satisfaction. Multivariate regression analysis was employed with scores from POMS, STAI, hopefulness and satisfaction as dependent variables and values from ERBCD, CDIS and MAC as independent variables.RESULTS:Patients received diagnosis from general physicians, surgeons, radiologists, nurses, NP/PAs, gynecologists, breast specialists, family members, and secretaries. BC diagnosis was disclosed by telephone (59%), face-to-face (40%), letter (1%) and spouse (1%). Mean CDIS-Caring score was 3.4 ± 1.1 and CDIS-Competence was 4.0 ± 0.9 of 5.0. Results of multivariate regression analysis are shown:POMS-Total Mood DisturbanceIndependent VariableParameterSEP ValueAge-0.6600.2760.0183MAC-Fighting Spirit-1.1550.4360.0093MAC-Anxious Preoccupation5.2720.780< 0.001 STAI-State AnxietyIndependent VariableParameterSEP ValueRecent Stress Level0.9420.3710.0127MAC-Fighting Spirit-0.3670.1480.0147MAC-Anxious Preoccupation1.9740.271<0.0001MAC-Avoidant4.4671.4950.0036 Satisfaction was predicted by CDIS-Caring, individual conveying diagnosis, type of information helpful, feeling concerns were understood.CONCLUSIONS:Experiences receiving BC diagnosis are variable and associated with hopefulness and satisfaction. Age, recent stress level and predominant coping style appear more predictive of psychological adjustment (POMS, STAI) than experience receiving BC diagnosis. Understanding these relationships provides direction for future research and clinical interventions to identify patients at risk for early mood disturbance and anxiety following BC diagnosis and to modify aspects of conveying BC diagnosis that influence hopefulness and satisfaction with medical care.HopefulnessIndependent VariableParameterSEP ValueCDIS-Caring0.4270.088<0.0001Diagnosis by Telephone0.3810.1770.0338Opportunity to Ask Questions0.4440.2270.0535Recent Stress Level-0.0820.0290.0057MAC-Fighting Spirit0.0430.0120.0005MAC-Anxious Preoccupation-0.0610.0230.0098 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1069.

Abstract Abstract #MS1-1 In this mini-symposium, we will address the major risk factors for breast cancer, current risk prediction tools, and measures to reduce risk in those women deemed at high risk. Those factors that convey a significantly increased risk of breast cancer (e.g. relative risk greater than 4.0) include: strong family history of breast cancer (i.e. early onset of disease, multiple relatives with disease, including BRCA1/2 carriers); atypical hyperplasia on benign biopsy; older age; and dense breast tissue. Multiple other features are recognized as contributing to breast cancer risk but to a lesser degree. These include having a family history of breast cancer but not with a hereditary pattern (RR approximately 2.0), benign breast disease (RR 1.5), multiple reproductive factors including early menarche, late menopause, current or recent estrogen plus progesterone use, current OC use (all with relative risks in the 1.2-1.3 range); and alcohol use (e.g. 3 drinks per day) (RR∼1.3). Numerous other features such as induced abortion and sedentary lifestyle have been reported to increase risk of breast cancer but to a very minor degree and results are not consistent.
 Several risk prediction models for breast cancer focus on an individual's likelihood of carrying a hereditary predisposition to the disease (e.g. Frank, Couch, Shattuck-Eidens, Parmigiani, BRCAPRO, BOADICEA, Claus). Outside of the hereditary setting, the most widely used tool is the Gail model. Features incorporated in the Gail model include age, family history, number of breast biopsies, age at menarche, age at first live birth, and the presence of atypia on a benign biopsy. While the Gail model has been shown to be calibrated in predicting number of breast cancers likely to develop in groups of women, its discriminatory accuracy in predicting risk for individual women is limited. This limitation is not well appreciated by many clinicians.
 Regarding options for surgical risk reduction in high risk individuals, several studies examined the efficacy of both bilateral and contralateral mastectomy in women at increased risk for breast cancer, including BRCA1/2 carriers. Results consistently show a greater than 90% reduction in risk of subsequent breast cancer. A study by the Cancer Research Network (6 HMOs) examined the impact of contralateral PM on mortality from breast cancer and showed reduced death from breast cancer with CPM (HR 0.57 [0.45-0.72]).
 Studies of risk-reducing salpingo-oophorectomy (RRSO) in BRCA carriers have shown dramatic reductions in risk of subsequent ovarian/peritoneal cancer, providing approximately 90% protection. The reduction in breast cancer risk after RRSO is seen only when the oophorectomy is performed in premenopausal women. Recent data on 368 BRCA1 and 229 BRCA2 carriers from a multicenter study show a differential effect on breast cancer risk following RRSO by mutation type. The hazard ratio for risk of breast cancer in BRCA2 carriers was 0.28 (0.08-0.92) (p 0.04) but for BRCA1 carriers was 0.61 (0.30-1.22) (p 0.16). This appeared to be due to the preponderance of ER-negative breast cancers in BRCA1 carriers, where no risk reduction with RRSO was seen. (N Kauff et al, JCO, 3/10/08) Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr MS1-1.

Abstract Abstract #4033 Background: Aromatase activity within the breast is a source of estrogen that may cause breast cancer. Mammographic density (MD) is a risk factor for breast cancer whose biologic basis is unknown. Our study compared aromatase expression in tissue from dense and non-dense areas of the breasts of healthy volunteers.
 Methods: Participants were 40+ yrs, had a screening mammogram with visible MD, no history of cancer and were not on endocrine therapy. Ultrasound-guided core biopsies were done within 6 months of mammography to obtain three paired cores from mammographically dense and non-dense regions of the breast. Immunostaining for aromatase expression employed the streptavidin-biotin amplification method using the recently developed 677 mouse monoclonal antibody. Immunoreactivity (IR) was scored in terms of proportion of cells staining positive for aromatase (PPC) (0=<1%, 1=1-25%, 2=26-50%, 3=51+% (and 4=75%+ for adipocytes)) and relative immunointensity (0=none, 1=weak, 2=moderate, 3=intense) for each cell type (stroma, normal ductal epithelium, adipocytes). A composite score weighting the PPC by their relative intensity (range 0-9; 0-12 for adipocytes) was also calculated. The sum of the composite score across the three cell types provided a global assessment of aromatase IR. Repeated measures analyses evaluated differences in aromatase IR for dense compared to non-dense tissue within and across cell types. Parameter estimates (β) indicated the average difference between dense and non-dense IR.
 Results: 18 (37%) of the 49 participants were premenopausal (median age 46 yrs). Summing across cell types, the global composite score showed increased aromatase IR on sections sampled from dense vs. non-dense regions (β=5.3,p<0.001). This global difference reflected increased aromatase IR in the stroma and normal ductal epithelium from dense sections (vs. non-dense). For 42 women with >1% stromal cells on each section, there was evidence for increased IR on dense sections as indicated by the greater PPCs (β=0.9), relative intensity (β=0.7) and composite score (β=2.7)(p's<0.001). Similarly, for 20 women with >1% normal ductal epithelium in both dense and non-dense sections, there was a greater composite score (β=1.4,p=0.004) for cells on dense sections although differences for PPC (β=0.3,p=0.11) and relative intensity (β=0.3,p=0.09) did not reach statistical significance. No differences were seen in IR for adipocytes from the two density regions (46 women). Findings were unchanged with adjustment for covariates, including proportions of each cell type per section.
 Conclusions: There is strong evidence for increased aromatase expression in the stromal and normal ductal epithelium of dense vs. non-dense tissue. These results support ongoing research into mammographic density as a biomarker of effect of aromatase inhibitors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4033.