Comparison of ACINUS, caspase-3, and TUNEL as apoptotic markers in determination of tumor growth rates of clinically localized prostate cancer using image analysis.

BACKGROUND: The balance between apoptotic and proliferative processes determines the enlargement of a tumor. Accurate measurement of apoptotic and proliferative rates from diagnostic prostate biopsies would allow calculation of tumor growth rates in a population-based prostate cancer (CaP) study. Automated image analysis may be used if proliferation and apoptotic biomarkers provide clearly resolved immunostained images. METHODS: Clinical CaP aggressiveness was assigned as low, intermediate or high using clinical criteria for 46 research subjects with newly diagnosed CaP. Diagnostic biopsy sections from the research subjects were dual-labeled for proliferation biomarker, Ki-67 and apoptotic biomarker, apoptotic chromatin condensation inducer in the nucleus (ACINUS). Apoptotic biomarkers, caspase-3 and terminal deoxyribonucleotidyltransferase mediated dUTP-biotin nick end labeling (TUNEL) were labeled separately. Images from immunostained sections were analyzed using automated image analysis and tumor growth rates computed. Association between clinical CaP aggressiveness and tumor growth rates was explored. RESULTS: Sixteen subjects had high, 17 had intermediate, and 13 had low clinical CaP aggressiveness. Positive immunostaining was localized to the nucleus for Ki-67, ACINUS, and TUNEL. A statistically significant linear trend across clinical CaP aggressiveness categories was found when tumor growth rates were calculated using ACINUS (P = 0.046). Logistic regression and ROC plots generated showed ACINUS (AUC = 0.677, P = 0.048) and caspase-3 (AUC = 0.694, P = 0.038) to be better predictors than TUNEL (AUC = 0.669, P = 0.110). CONCLUSIONS: ACINUS met the criteria for automated image analysis and for calculation of apoptotic rate. Tumor growth rates determined using automated image analysis should be evaluated for clinical prediction of CaP aggressiveness, treatment response, recurrence, and mortality.

Java Web Start based software for automated quantitative nuclear analysis of prostate cancer and benign prostate hyperplasia.

BACKGROUND: Androgen acts via androgen receptor (AR) and accurate measurement of the levels of AR protein expression is critical for prostate research. The expression of AR in paired specimens of benign prostate and prostate cancer from 20 African and 20 Caucasian Americans was compared to demonstrate an application of this system. METHODS: A set of 200 immunopositive and 200 immunonegative nuclei were collected from the images using a macro developed in Image Pro Plus. Linear Discriminant and Logistic Regression analyses were performed on the data to generate classification coefficients. Classification coefficients render the automated image analysis software independent of the type of immunostaining or image acquisition system used. The image analysis software performs local segmentation and uses nuclear shape and size to detect prostatic epithelial nuclei. AR expression is described by (a) percentage of immunopositive nuclei; (b) percentage of immunopositive nuclear area; and (c) intensity of AR expression among immunopositive nuclei or areas. RESULTS: The percent positive nuclei and percent nuclear area were similar by race in both benign prostate hyperplasia and prostate cancer. In prostate cancer epithelial nuclei, African Americans exhibited 38% higher levels of AR immunostaining than Caucasian Americans (two sided Student's t-tests; P < 0.05). Intensity of AR immunostaining was similar between races in benign prostate. CONCLUSION: The differences measured in the intensity of AR expression in prostate cancer were consistent with previous studies. Classification coefficients are required due to non-standardized immunostaining and image collection methods across medical institutions and research laboratories and helps customize the software for the specimen under study. The availability of a free, automated system creates new opportunities for testing, evaluation and use of this image analysis system by many research groups who study nuclear protein expression.

Variability in visual segmentation of digitized prostate tissue microarray cores.

OBJECTIVE: To examine bias associated with human-interactive semi-automated systems key components with machine vision used in quantitative histometry. STUDY DESIGN: A standard image set of 20 images was created using 5 nuclei sampled from hematoxylin-eosin-stained sections of benign tissue within a prostate tissue microarray that were rotated through the cardinal directions. Four trained technicians performed segmentation of these images at the start, then at the end, of 3 daily sessions, creating a total analytic set of 480 observations. Measurements of nuclear area (NA), nuclear roundness factor (NRF), and mean optical density (MOD) were compared by segmenter, time, and rotational orientation. RESULTS: NA varied significantly among sessions (p < 0.0009) and session variance differed within segmenter (p < 0.0001). NRF was significant among segmenters (p < 0.001) and sessions (p < 0.0001), and in session (p < 0.0001) and intra-session differences (p = 0.026). Differences in MOD varied among sessions (p < 0.0001) and within sessions (p < 0.049). CONCLUSION: Imaging systems remain vulnerable to statistical inter-segmenter variation, in spite of extensive efforts to eliminate variation among individual segmenters. As statistical significance often guides decision-making in morphometric analysis, statistically significant effects potentially produce bias. Current practices and quality assurance methods require review to eliminate individual operator effects in semiautomated machine systems.

Manual and automated systems in the analysis of images from prostate tissue microarray cores.

OBJECTIVE: To compare manual and automated image analysis systems in morphologic analysis of nuclei from benign prostate, high-grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (CaP). Morphologic features derived using automated image analysis systems may be more objective and reproducible than manual systems, which require humans to segment nuclei from histologic images. STUDY DESIGN: Images of hematoxylin-eosin-stained sections of prostate tissue microarray were analyzed independently using the automated and manual systems. Mean optical density (MOD), nuclear area (NA), and nuclear roundness factor (NRF) were the morphologic features studied. The ability to differentiate between tissue types using morphologic features derived from an automated and a manual system was compared. RESULTS: Nuclei from 17 benign prostate hyperplasia (BPH), 4 HGPIN, and 8 aggressive CaP were analyzed. The manual system distinguished better between BPH and HGPIN (p < 0.0001), whereas the automated system distinguished better between BPH and CaP (p = 0.01) in multivariate models. The manual system distinguished better BPH and HGPIN using NA (p < 0.0001) and MOD (p < 0.0001), whereas the automated system distinguished better BPH and CaP using MOD (p < 0.0001) and NRF (p = 0.004). CONCLUSION: The minimal human effort required for automated image analysis makes it superior to the manual system.

Thioredoxin Reductase 1 Expression and Castration-recurrent Growth of Prostate Cancer.

INTRODUCTION: Many genes are differentially expressed between androgen-dependent and androgen-independent prostate cancer (CaP). Differential expression analysis and subtractive hybridization previously identified nine genes expressed in intact mice bearing CWR22 tumors and castrated mice bearing recurrent CWR22 tumors but not in regressed tumors. The objectives of this study were to develop an immunostaining method to dual-label foci of proliferating tumor cells [the origin of castration-recurrent CaP (CR-CaP)], to determine which of the nine candidate proteins were differentially expressed in proliferating versus nonproliferating cells at the onset of growth after castration, and to test preclinical findings using clinical specimens of androgen-stimulated benign prostate (AS-BP) and CaP (AS-CaP) and CR-CaP. METHODS: Paraffin-embedded, bromodeoxyuridine-injected CWR22 tumors were hydrated, antigen-retrieved using high heat and high pressure, labeled for each of the nine antigens of interest, visualized using peroxidase, and counterstained with hematoxylin. Mean optical density was calculated for proliferating and nonproliferating areas using automated (nuclear staining) or manual (cytoplasmic staining) image analysis. Prostate tissue microarray sections were immunostained and visually scored. RESULTS: Immunohistochemistry revealed higher nuclear expression of thioredoxin reductase 1 (TrxR1) in proliferating cells than nonproliferating cells (P < .005). There were no statistical differences between cell types in the expression of other proteins. TrxR1 expression was higher (P < .01) in CR-CaP compared with AS-BP or AS-CaP. CONCLUSIONS: Increased TrxR1 expression in CR-CaP was consistent with increased TrxR1 and BrdU expression at the onset of growth in the CWR22 model. Thioredoxin reductase 1 should be targeted in an attempt to delay or prevent CaP recurrence after castration.

Evaluation of heart rate variability indices using a real-time handheld remote ECG monitor.

Studies on retrospective electrocardiogram (ECG) recordings of patients during cardiac arrest have shown significant changes in heart rate variability (HRV) indices prior to the onset of cardiac arrhythmia. The early detection of these changes in HRV indices increases the chances for a successful medical intervention by increasing the response time window. A portable, handheld remote ECG monitor designed in this research detects the QRS complex and calculates short-term HRV indices in real-time. The QRS detection of the ECG recordings of subjects from the MIT-Arrhythmia database yielded a mean sensitivity of 99.34% and a specificity of 99.31%. ECG recordings from normal subjects and subjects with congestive heart failure were used to identify the differences in HRV indices. An increase in heart rate, high-frequency spectral power (HFP), total spectral power, the ratio of HFP to low-frequency spectral power (LFP), and a decrease in root mean square sum of RR differences were observed. No difference was found on comparison of the standard deviation of normal to normal interval between adjacent R-waves, LFP, and very-low-frequency spectral power. Based on these, additional analytical calculations could be made to provide early warnings of impending cardiac conditions.

Feasibility of constructing tissue microarrays from diagnostic prostate biopsies.

OBJECTIVES: The ability to construct prostate tissue microarrays (TMAs) using prostate needle biopsies could allow high throughput molecular profiling to search for prostate cancer prognostic biomarkers. MATERIALS AND METHODS: Diagnostic prostate biopsies from 13 patients (diagnosed 1996-2000) were obtained from the University of North Carolina (UNC) to construct one prostate TMA under uniform conditions. A second prostate TMA was attempted using diagnostic prostate biopsies from 45 patients (diagnosed 2004) obtained from the North Carolina-Louisiana Prostate Cancer Project (PCaP). RESULTS: Eleven men had sufficient prostate cancer in their diagnostic prostate biopsy blocks to construct a UNC TMA that yielded six-micron sections that provided an average of 76% of cores (maximum 99%) to a depth of 360 microm. Diagnostic prostate biopsy blocks from 35 PCaP research subjects were unsuitable for TMA construction as a result of no remaining prostate cancer in 4, insufficient prostate cancer in 9, and insufficient prostate tissue in 22. The PCaP TMA constructed from 10 men yielded an average of 37%, and a maximum of 45%, of cores when sectioned to a depth of 360 microm. CONCLUSIONS: TMAs may be constructed from diagnostic prostate biopsies obtained at an academic center under uniform conditions. However, excessive facing of blocks and the large number of re-cuts ordered by many community pathology laboratories deplete diagnostic prostate biopsy tissue. The experience of a population-based study of men with newly diagnosed prostate cancer in Louisiana and North Carolina suggests that TMAs may not be constructed using diagnostic prostate biopsies from men diagnosed with prostate cancer throughout the United States.

Development of a remote handheld cardiac arrhythmia monitor.

In this paper we present the design and development of a real-time remote handheld cardiac arrhythmic monitoring system (RCAM). A client-server model based on Internet protocols was used. ECG data was transmitted from the remote handheld client to a centralized server, where the QRS and premature ventricular contraction detection algorithms were implemented and graded depending on the number and pattern of PVCs present. The QRS sensitivity and specificity on ECG records from Physionet archives in absence of arrhythmia was 100% and 99.62%, while in presence of arrhythmia was 99.34% and 99.31%. The average 'negative time' measured on ventricular tachyarrhythmia records was 92 seconds. The RCAM can provide remote detection of cardiac abnormalities and give specific diagnosis and recommendations of actions to be taken immediately. The limitation due to the inability of the PDA to perform complex computations was overcome by the use of the remote server.

Sampling strategy for prostate tissue microarrays for Ki-67 and androgen receptor biomarkers.

OBJECTIVE: To develop an optimal sampling strategy for tissue microarrays using automated digital analysis for androgen receptor (heterogeneous expression) and the cellular proliferation marker Ki-67 (homogeneous expression and evaluated by others using nonautomated methods). STUDY DESIGN: Tissue microarrays were constructed from 23 radical prostatectomy specimens and immunostained for androgen receptor expression and cellular proliferation. Automated digital image analysis was used, and the minimum number of cores necessary to capture variance change <3% was determined. Androgen receptor immunostaining was described by percent positive nuclei (PPN) and mean optical density (MOD). RESULTS: Androgen receptor PPN variance measurements showed that 5 cores should be obtained when a single block of a radical prostatectomy specimen contained cancer. If all of 15 blocks contained cancer, 2 cores should be obtained from each of 6 blocks. An optimal sampling strategy was developed for androgen receptor PPN, androgen receptor MOD and Ki-67 PPN. CONCLUSION: The selection of the number of cores to sample is a tradeoff between the number of cores available that contain cancer and the amount of work involved in the analysis. Sampling no fewer than 5 but no more than 12 cores per radical prostatectomy specimen can capture tissue heterogeneity.