Craig A. Beam

Persistent Activation of Stat3 Signaling Induces Survivin Gene Expression and Confers Resistance to Apoptosis in Human Breast Cancer Cells

Purpose: Signal transducer and activator of transcription 3 (Stat3) protein is persistently activated in breast cancer and promotes tumor cell survival. To gain a better understanding of the role of constitutive Stat3 signaling in breast cancer progression, we evaluated the expression profile of potential Stat3-regulated genes that may confer resistance to apoptosis. Experimental Design: Stat3 signaling was blocked with antisense oligonucleotides in human MDA-MB-435s breast cancer cells and Affymetrix GeneChip microarray analysis was done. The candidate Stat3 target gene Survivin was further evaluated in molecular assays using cultured breast cancer cells and immunohistochemistry of breast tumor specimens. Results: Survivin, a member of the inhibitor of apoptosis protein family, was identified as a potential Stat3-regulated gene by microarray analysis. This was confirmed in Survivin gene promoter studies and chromatin immunoprecipitation assays showing that Stat3 directly binds to and regulates the Survivin promoter. Furthermore, direct inhibition of Stat3 signaling blocked the expression of Survivin protein and induced apoptosis in breast cancer cells. Direct inhibition of Survivin expression also induced apoptosis. Increased Survivin protein expression correlates significantly (P = 0.001) with elevated Stat3 activity in primary breast tumor specimens from high-risk patients who were resistant to chemotherapy treatment. Conclusions: We identify Survivin as a direct downstream target gene of Stat3 in human breast cancer cells that is critical for their survival in culture. Our findings suggest that activated Stat3 signaling contributes to breast cancer progression and resistance to chemotherapy by, at least in part, inducing expression of the antiapoptotic protein, Survivin.

Activation of stat3 in primary tumors from high-risk breast cancer patients is associated with elevated levels of activated SRC and survivin expression.

Purpose: Constitutive activation of signal transducer and activator of transcription 3 (Stat3) protein has been observed in a wide variety of tumors, including breast cancer, and contributes to oncogenesis at least in part by prevention of apoptosis. In a study of 45 patients with high-risk breast cancer enrolled in a phase II neoadjuvant chemotherapy trial with docetaxel and doxorubicin, we evaluated the levels of Stat3 activation and potentially associated molecular biomarkers in invasive breast carcinoma compared with matched nonneoplastic tissues. Experimental Design: Using immunohistochemistry and image analysis, we quantified the levels of phospho-Stat3 (pY-Stat3), phospho-Src (pY-Src), epidermal growth factor receptor, HER2/neu, Ki-67, estrogen receptor, Bcl-2, Bcl-xL, Survivin, and apoptosis in formalin-fixed, paraffin-embedded sections from invasive carcinomas and their paired nonneoplastic parenchyma. The levels of molecular biomarkers in nonneoplastic and tumor tissues were analyzed as continuous variables for statistically significant correlations. Results: Levels of activated pY-Stat3 and pY-Src measured by immunohistochemistry were significantly higher in invasive carcinoma than in nonneoplastic tissue (P < 0.001). In tumors, elevated levels of pY-Stat3 correlated with those of pY-Src and Survivin. Levels of pY-Stat3 were higher in partial pathologic responders than in complete pathologic responders. In partial pathologic responders, pY-Stat3 levels correlated with Survivin expression. Conclusions: Our findings suggest important roles for elevated activities of Stat3 and Src, as well as Survivin expression, in malignant progression of breast cancer. Furthermore, elevated Stat3 activity correlates inversely with complete pathologic response. These findings suggest that specific Stat3 or Src inhibitors could offer clinical benefits to patients with breast cancer.

Fall in C-Peptide During First 2 Years From Diagnosis: Evidence of at Least Two Distinct Phases From Composite Type 1 Diabetes TrialNet Data

Interpretation of clinical trials to alter the decline in β-cell function after diagnosis of type 1 diabetes depends on a robust understanding of the natural history of disease. Combining data from the Type 1 Diabetes TrialNet studies, we describe the natural history of β-cell function from shortly after diagnosis through 2 years post study randomization, assess the degree of variability between patients, and investigate factors that may be related to C-peptide preservation or loss. We found that 93% of individuals have detectable C-peptide 2 years from diagnosis. In 11% of subjects, there was no significant fall from baseline by 2 years. There was a biphasic decline in C-peptide; the C-peptide slope was −0.0245 pmol/mL/month (95% CI −0.0271 to −0.0215) through the first 12 months and −0.0079 (−0.0113 to −0.0050) from 12 to 24 months (P < 0.001). This pattern of fall in C-peptide over time has implications for understanding trial results in which effects of therapy are most pronounced early and raises the possibility that there are time-dependent differences in pathophysiology. The robust data on the C-peptide obtained under clinical trial conditions should be used in planning and interpretation of clinical trials.

Fall in C-peptide During First 2 Years From Diagnosis Evidence of at Least Two Distinct Phases From Composite TrialNet Data

Interpretation of clinical trials to alter the decline in β-cell function after diagnosis of type 1 diabetes depends on a robust understanding of the natural history of disease. Combining data from the Type 1 Diabetes TrialNet studies, we describe the natural history of β-cell function from shortly after diagnosis through 2 years post study randomization, assess the degree of variability between patients, and investigate factors that may be related to C-peptide preservation or loss. We found that 93% of individuals have detectable C-peptide 2 years from diagnosis. In 11% of subjects, there was no significant fall from baseline by 2 years. There was a biphasic decline in C-peptide; the C-peptide slope was −0.0245 pmol/mL/month (95% CI −0.0271 to −0.0215) through the first 12 months and −0.0079 (−0.0113 to −0.0050) from 12 to 24 months (P < 0.001). This pattern of fall in C-peptide over time has implications for understanding trial results in which effects of therapy are most pronounced early and raises the possibility that there are time-dependent differences in pathophysiology. The robust data on the C-peptide obtained under clinical trial conditions should be used in planning and interpretation of clinical trials.

Magnetic resonance imaging of blood flow with a phase subtraction technique: In vitro and in vivo validation

RATIONALE AND OBJECTIVES.One promising approach to flow quantification uses the velocity-dependent phase change of moving protons. A velocity-encoding phase subtraction technique was used to measure the velocity and flow rate of fluid flow in a phantom and blood flow in volunteers. METHODS.In a model, the authors measured constant flow velocities from 0.1 to 270.0 cm/second with an accuracy (95% confidence intervals) of ±12.5 cm/second. There was a linear relationship between the magnetic resonance imaging (MRI) measurement and the actual value (r2 = .99; P = .0001). RESULTS.Measuring mean pulsatile flow from 125 to 1,900 mL/minute, the accuracy of the MRI pulsatile flow measurements (95% confidence intervals) was ±70 mL/minute. There was a linear relationship between the MRI pulsatile flow measurement and the actual value (r2 =.99;P = .0001). In 10 normal volunteers, the authors tested the technique in vivo, quantitating flow rates in the pulmonary artery and the aorta. The average difference between the two measurements was 5%. In vivo carotid flow waveforms obtained with MRI agreed well with the shape of corresponding ultrasound Doppler waveforms. CONCLUSIONS.Velocity-encoding phase subtraction MRI bears potential clinical use for the evaluation of blood flow. Potential applications would be in the determination of arterial blood flow to parenchymal organs, the detection and quantification of intra- and extra-cardiac shunts, and the rapid determination of cardiac output and stroke volume.

Making the diagnosis of acute appendicitis: Do more preoperative CT scans mean fewer negative appendectomies? A 10-year study

PURPOSE To determine the frequency of preoperative computed tomography (CT) in the evaluation of patients suspected of having appendicitis at one institution during the past 10 years and to determine whether changes in CT utilization were associated with changes in the negative appendectomy rate. MATERIALS AND METHODS Institutional review board approval was obtained, and a waiver of informed consent was granted for this HIPAA-compliant study. A surgical database search yielded medical record numbers of 925 patients (526 [ 56.9%] men and 399 [43.1%] women; mean age, 38 years (range, 18-95 years]) who underwent urgent appendectomy between January 1998 and September 2007. Patients who were younger than 18 years of age at the time of surgery were excluded. CT, pathology, and surgery reports were reviewed. By using logistic regression, changes in the proportion of patients undergoing CT and in the proportion of patients undergoing each year appendectomy in which the appendix was healthy were evaluated. Subgroup analyses based on patient age (<or= 45 years or > 45 years) and sex also were performed. RESULTS Prior to urgent appendectomy, 18.5% of patients underwent preoperative CT in 1998 compared with 93.2% of patients in 2007. The negative appendectomy rate for women 45 years of age and younger decreased from 42.9% in 1998% to 7.1% in 2007. However, the timing of the decline in negative appendectomy rates for women 45 years and younger could not be proved to be associated with the increase in CT use. There was no significant trend toward a lower negative appendectomy rate for men regardless of age or for women older than 45 years of age with increased use of preoperative CT. The shift from single-detector CT to multidetector CT and the use of decreasing section thickness also correlated with a reduction in false-positive diagnoses. CONCLUSION Rising utilization of preoperative CT and advances in technology coincided with a decrease in the negative appendectomy rate for women 45 years and younger but not in men of any age or women older than 45 years.

Risk Factors for Coronary Heart Disease in Men 18 to 39 Years of Age

Coronary heart disease remains the leading cause of death in the United States (1). Autopsy studies (2-5) show that coronary atherosclerosis begins as early as 20 years of age, and a recent study found severely stenotic coronary arteries (narrowing 40%) in 19% of men in their early thirties (6). On the basis of these observations, the National Cholesterol Education Program recommended cholesterol screening in all adults 20 years of age or older (7). Similarly, the Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure recommends screening for hypertension in all persons 18 years of age or older (8). However, guidelines on prevention of coronary heart disease in young adults have not been uniformly accepted, in part because data on risk prediction and coronary disease prevention in adults younger than 40 years of age are limited (9-12). Numerous studies in middle-aged (40 to 65 years of age) and, to a lesser extent, older persons have shown that the major risk factors for coronary heart disease (which include cholesterol level, blood pressure, and cigarette smoking) are predictive of long-term outcomes in these age groups (13-16). In addition, primary and secondary prevention trials have convincingly shown benefits of reduction of certain risk factors, such as dyslipidemia and hypertension, in middle-aged and older adults (7, 8, 17-19). However, equally compelling data from observational studies or clinical trials are almost nonexistent in young adults (4, 5, 13, 20). The Chicago Heart Association Detection Project in Industry, one of the largest and longest cardiovascular disease studies, is a unique resource. The study cohort comprises 11 016 young men 18 to 39 years of age at baseline (mean age, 29 years), and more than 20 years of follow-up have been completed for coronary and all-cause mortality. We report data on risk prediction in young men from the Project. We hypothesized that the risk factors typically related to death from coronary heart disease in middle-aged cohorts are also related to death from coronary disease in younger cohorts. Methods Study Sample The Chicago Heart Association Detection Project in Industry is a prospective cohort study of long-term risks for coronary, total cardiovascular, and all-cause mortality. Demographic characteristics of participants, selection criteria, and standardized methods have been reported elsewhere (14, 21, 22). Briefly, approximately 75 000 employees of 84 Chicago-area companies and organizations were invited to participate in assessments of cardiovascular risk factors; the response rate was 53%. The cohort screened between November 1967 and January 1973 included 39 573 men and women 18 to 74 years of age from various ethnic backgrounds (white, Asian, African American, Hispanic, and other). Our report focuses on the 11 016 men who were 18 to 39 years of age at baseline. All age-eligible participants were included in the analyses, except diabetic persons; at that young age, fewer than 1% of the cohort members had diabetes, and those who did were presumed to have type 1 diabetes. A group of nondiabetic men who were 40 to 59 years of age at baseline was used for comparison. The study has been reviewed on a regular basis by the institutional review board at Northwestern University and has repeatedly received approval for ongoing follow-up. Because the study participants are not contacted directly in follow-up and are never identified by name in any report, this research has been granted an exemption from the need to obtain informed consent of the participants after the baseline examination. Risk Factor Assessment Baseline screening for risk factors was performed by trained personnel. Questionnaires were used to record age, sex, ethnicity, years of education, number of cigarettes smoked per day, medical history, and history of medical treatment for diabetes or hypertension. Measurements included height and weight to calculate body mass index, a single measurement of supine casual blood pressure obtained by using a standard stethoscope and mercury sphygmomanometer, resting electrocardiography, and a single measurement of total serum cholesterol. Standardized methods were used for laboratory determinations of total serum cholesterol (22), uric acid (23), and plasma glucose (24). Criteria from the Pooling Project (25) and Hypertension Detection and Follow-up Program (26) were used to classify electrocardiographic abnormalities as minor (such as nonspecific ST or T-wave abnormalities) or major (such as major Q-wave or ventricular hypertrophy). Outcomes Death from coronary heart disease was the primary outcome of interest. Before 1979, vital status was ascertained by periodic follow-up by using both local records and the Social Security Administration; from 1979 onward, the National Death Index was used. Ascertainment of vital status is more than 99% complete for all study participants. Death certificates were acquired for known decedents and were coded by trained physicians according to the International Classification of Diseases, Eighth Revision (ICD-8) (27). Study team members who were blinded to baseline data cross-checked all coding decisions. For this report, only underlying cause of death was used. Coronary heart disease deaths were those assigned ICD-8 codes 410.0 through 414.9. Cardiovascular disease mortality was defined by ICD-8 codes 400.00 through 445.9. All-cause mortality was also assessed. Follow-up was complete at 20 years in all participants except those who were deceased and 356 participants (3.2% of the cohort) with less than 20 years of follow-up. Statistical Analysis Cox proportional-hazards regression was used to assess the association of baseline risk factors with death from coronary heart disease. Risk factors assessed were age, serum cholesterol level, systolic blood pressure, diastolic blood pressure, number of cigarettes smoked per day, body mass index, major electrocardiographic abnormalities, minor electrocardiographic abnormalities, education, and black ethnicity. To determine whether associations of each risk factor with coronary heart disease mortality differed between young and middle-aged men, an interaction model was formulated that included a dummy variable representing the middle-aged group and the interaction of each risk factor with the dummy variable. Rejection of a Wald test (28) that the interaction equaled zero indicated that the relative risks differed in the young and middle-aged groups. This part of the analysis assessed whether risk factors that are typically related to death from coronary heart disease in a middle-aged cohort are also related to death from coronary heart disease in a young cohort. To assess whether risk factors that are significantly associated with death from coronary heart disease are also accurate in predicting death from coronary heart disease in a young cohort, we constructed receiver-operating characteristic (ROC) curves for selected models in the young cohort and calculated the corresponding c-statistic, which represents the area under the ROC curve (AUC) (29). A nonparametric statistical test developed by DeLong and colleagues (30) was performed to determine whether the AUCs for various models differed significantly from one another. The ROC curve is a graphical method of depicting the trade-off between the true-positive rate (sensitivity) and the false-positive rate (1 specificity) of a test (29). The AUC represents the discriminating ability of that particular test. An AUC of 0.50 indicates that a test has no discriminatory capacity; the true-positive rate equals the false-positive rate, and the value represents the probability of prediction by chance alone. As the AUC increases and the curve migrates closer toward the upper left margin of the graph, the predictive capability of that test increases. Previous studies have used ROC curves to identify and assess the predictive capacity of risk assessment strategies in persons 30 to 74 years of age who are at risk for subsequent coronary disease (15, 31). Role of the Funding Sources The current funding source, the National Heart, Lung, and Blood Institute, has had no role in the collection, analysis, or interpretation of the data or in the decision to submit the paper for publication. Previous funding sources have included the American Heart Association of Metropolitan Chicago, which contributed personnel who were involved in data collection at baseline. The American Heart Association of Metropolitan Chicago has not been involved in analysis or interpretation of the current data or in the decision to submit the paper for publication. Results Baseline Characteristics Table 1 shows baseline characteristics of the two age groups. Mean age in the 11 016 younger men was 29.7 years; mean total serum cholesterol level in this group was 4.92 mmol/L (189.9 mg/dL), and mean systolic blood pressure was 134.6 mm Hg. Approximately half of the younger participants were current smokers, and they consumed an average of 21.2 cigarettes per day. Mean formal education was 13.8 years, and black persons made up 8.1% of participants. The prevalence of major and minor electrocardiographic abnormalities was 3.3% and 5.5%, respectively. As expected, all average values for risk factors were lower (less adverse) in younger adults than in men 40 to 59 years of age at baseline. Table 1. Baseline Characteristics of Young and Middle-Aged Men in the Chicago Heart Association Detection Project in Industry, 19671973 Cardiovascular Disease and All-Cause Mortality Four hundred fifty-five men died during 20 years of follow-up. One third of these mortality events (n = 155) were due to cardiovascular disease, and coronary heart disease was the leading cause of death (n = 123). Relative Risk for Death from Coronary Heart Disease in Young Adult Men Table 2 shows the relative risk for death from coronary heart disease over

Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans

The study of regulatory networks in human pathogens such as Cryptococcus neoformans provides insights into host-pathogen interactions that may allow for correlation of gene expression patterns with clinical outcomes. In the present study, deletion of the cryptococcal copper-dependent transcription factor 1 (Cuf1) led to defects in growth and virulence factor expression in low copper conditions. In mouse models, cuf1Delta strains exhibited reduced dissemination to the brain, but no change in lung growth, suggesting copper is limiting in neurologic infections. To examine this further, a biologic probe of available copper was constructed using the cryptococcal CUF1-dependent copper transporter, CTR4. Fungal cells demonstrated high CTR4 expression levels after phagocytosis by macrophage-like J774.16 cells and during infection of mouse brains, but not lungs, consistent with limited copper availability during neurologic infection. This was extended to human brain infections by demonstrating CTR4 expression during C. neoformans infection of an AIDS patient. Moreover, high CTR4 expression by cryptococcal strains from 24 solid organ transplant patients was associated with dissemination to the CNS. Our results suggest that copper acquisition plays a central role in fungal pathogenesis during neurologic infection and that measurement of stable traits such as CTR4 expression may be useful for risk stratification of individuals with cryptococcosis.

Gene Expression Patterns in the Human Breast after Pregnancy

Epidemiologic studies have established that pregnancy has a bidirectional, time-dependent effect on breast cancer risk; a period of elevated risk is followed by a long-term period of protection. The purpose of the present study was to determine whether pregnancy and involution are associated with gene expression changes in the normal breast, and whether such changes are transient or persistent. We examined the expression of a customized gene set in normal breast tissue from nulliparous, recently pregnant (0-2 years since pregnancy), and distantly pregnant (5-10 years since pregnancy) age-matched premenopausal women. This gene set included breast cancer biomarkers and genes related to immune/inflammation, extracellular matrix remodeling, angiogenesis, and hormone signaling. Laser capture microdissection and RNA extraction were done from formalin-fixed paraffin-embedded reduction mammoplasty and benign biopsy specimens and analyzed using real-time PCR arrays containing 59 pathway-specific and 5 housekeeping genes. We report 14 of 64 (22%) of the selected gene set to be differentially regulated (at P < 0.05 level) in nulliparous versus parous breast tissues. Based on gene set analysis, inflammation-associated genes were significantly upregulated as a group in both parous groups compared with nulliparous women (P = 0.03). Moreover, parous subjects had significantly reduced expression of estrogen receptor α (ERα, ESR1), progesterone receptor (PGR), and ERBB2 (Her2/neu) and 2-fold higher estrogen receptor-β (ESR2) expression compared with nulliparous subjects. These initial data, among the first on gene expression in samples of normal human breast, provide intriguing clues about the mechanisms behind the time-dependent effects of pregnancy on breast cancer risk. Cancer Prev Res; 3(3); 301–11

Effect of human variability on independent double reading in screening mammography

RATIONALE AND OBJECTIVES To demonstrate the range of gains and losses that radiologists might experience from independent double reading in screening mammography. METHODS From a national random sample of radiologists, the authors formed 131 pairs. For each radiologist, the authors analyzed the increase relative to his or her individual true-positive rate (TPR) or false-positive rate (FPR), number of additional cancers detected, and change to negative biopsy rate that would result from independent double reading after pairing. RESULTS The average radiologist can expect an 8%-14% gain in TPR and a 4%-10% increase in FPR with pairing. For some radiologists, double reading increased the TPR with a small concomitant increase in FPR. Other radiologists, however, realized small gains in TPR with large increases in FPR. Adding the reading from a more experienced radiologist did not necessarily improve the TPR of a radiologist with less experience. CONCLUSION Radiologists can form complementary and noncomplementary pairs for double reading. Use of this procedure must be decided on an individual basis.