Quantification of collagen content and stromal cellularity within reactive stroma is predictive of prostate cancer biochemical recurrence and specific death.

Semiquantitative reactive stromal grading has been shown to be a predictor of biochemical recurrence and prostate cancer (PCa) specific death. It has been extensively validated. In this study we tested novel technologies to introduce quantitative measures of host response, in particular collagen content and stromal cellularity. We use 3 large retrospective cohorts, the Baylor College of Medicine cohort, the Brady cohort and the Pound cohort. Slides were stained and digitized using image deconvolution and analyzed using image segmentation and image analyses. PicroSirius red stain histochemical stains were used for collagen quantification. Area of cancer and stroma were measured independently, without regard to quality of stroma. Cellularity, in each compartment, was measured using image deconvolution, image segmentation and image analysis. Two biomarkers were tested in 3 independent cohorts with two endpoints, biochemical recurrence and prostate cancer specific death. Stromal cellularity (qCollCell) and stromal collagen area (qCollArea) are independently predictive biochemical recurrence in the Hopkins Brady cohort, particularly in Gleason 6-7 patients. Multivariate analysis demonstrated that increased stroma cellularity (qCollCell) was a significant predictor of PCa specific death, when compared to an established model of PCa, in the Baylor cohort. Stromal collagen (qCollArea) independently predicts PCa-specific death in the Hopkins Pound cohort. The introduction of a computerized quantitative test of the host response increases the probability that this test will be reproducible in other cohorts. The ability to improve prediction of prostate cancer specific death might lie in the study of the host and its response.

Development and validation of a quantitative reactive stroma biomarker (qRS) for prostate cancer prognosis.

To develop and validate a new tissue-based biomarker that improves prediction of outcomes in localized prostate cancer by quantifying the host response to tumor. We use digital image analysis and machine learning to develop a biomarker of the prostate stroma called quantitative reactive stroma (qRS). qRS is a measure of percentage tumor area with a distinct, reactive stromal architecture. Kaplan Meier analysis was used to determine survival in a large retrospective cohort of radical prostatectomy samples. qRS was validated in two additional, distinct cohorts that include international cases and tissue from both radical prostatectomy and biopsy specimens. In the developmental cohort (Baylor College of Medicine, n = 482), patients whose tumor had qRS > 34% had increased risk of prostate cancer-specific death (HR 2.94; p = 0.039). This result was replicated in two validation cohorts, where patients with qRS > 34% had increased risk of prostate cancer-specific death (MEDVAMC; n = 332; HR 2.64; p = 0.02) and also biochemical recurrence (Canary; n = 988; HR 1.51; p = 0.001). By multivariate analysis, these associations were shown to hold independent predictive value when compared to currently used clinicopathologic factors including Gleason score and PSA. qRS is a new, validated biomarker that predicts prostate cancer death and biochemical recurrence across three distinct cohorts. It measures host-response rather than tumor-based characteristics, and provides information not represented by standard prognostic measurements.

Moving Beyond Gleason Scoring.

CONTEXT.­: The combination of grading and staging is the basis of current standard of care for prediction for most cancers. D. F. Gleason created the current prostate cancer (PCa) grading system. This system has been modified several times. Molecular data have been added. Currently, all grading systems are cancer-cell based. OBJECTIVE.­: To review the literature available on host response measures as reactive stroma grading and stromogenic carcinoma, and their predictive ability for PCa biochemical recurrence and PCa-specific death. DATA SOURCES.­: Our own experience has shown that reactive stroma grading and the subsequently binarized system (stromogenic carcinoma) can independently predict biochemical recurrence and/or PCa-specific death, particularly in patients with a Gleason score of 6 or 7. Stromogenic carcinoma has been validated by 4 other independent groups in at least 3 continents. CONCLUSIONS.­: Broders grading and Dukes staging have been combined to form the most powerful prognostic tools in standard of care. The time has come for us to incorporate measures of host response (stromogenic carcinoma) into the arsenal of elements we use to predict cancer survival, without abandoning what we know works. These data also suggest that our current definition of PCa might need some revision.

Histologic features of stromogenic carcinoma of the prostate (carcinomas with reactive stroma grade 3).

Prostatic carcinoma, like many other carcinomas, generates a stromal reaction. This phenomenon is well established in the scientific literature. The normal parenchymal smooth muscle phenotype switches to a myofibroblastic phenotype in response to the presence of cancer cells, with an expansion of the extracellular matrix compartment. The amount of reactive stroma is a predictor of biochemical recurrence in both radical prostatectomies and biopsies. It is a predictor of prostate cancer-specific death in prostatectomies. The aim of this study is to improve our histologic understanding of reactive stroma in prostate cancer and to determine the histologic features of the malignant epithelium found in stromogenic carcinomas or carcinomas with reactive stromal grade 3. Tissue microarrays of 800 patients and hematoxylin and eosin-stained sections of 120 radical prostatectomies, previously determined to contain a high proportion of areas with stromogenic carcinoma, were evaluated and findings systematically recorded. We identified 3 histologic patterns of reactive stroma: extracellular matrix-rich, cellular variant and edematous/myxoid variant. The most common pattern of carcinoma in stromogenic areas is of the acinar type with angulated glands and periglandular halos. The nuclei are enlarged, opened, with prominent nucleoli. Luminal borders are undulated, and amorphous pink secretion is often seen. Perineural invasion is frequently identified. Because of the clinical relevance, identification and quantification of areas with high reactive stromal grade by pathologists and reproducibility of our findings by others become essential. We believe that with the previously proposed grading system and the present morphologic description, both can be achieved.

Non-Cell-Autonomous Regulation of Prostate Epithelial Homeostasis by Androgen Receptor.

Prostate inflammation has been suggested as an etiology for benign prostatic hyperplasia (BPH). We show that decreased expression of the androgen receptor (AR) in luminal cells of human BPH specimens correlates with a higher degree of regional prostatic inflammation. However, the cause-and-effect relationship between the two events remains unclear. We investigated specifically whether attenuating AR activity in prostate luminal cells induces inflammation. Disrupting luminal cell AR signaling in mouse models promotes cytokine production cell-autonomously, impairs epithelial barrier function, and induces immune cell infiltration, which further augments local production of cytokines and chemokines including Il-1 and Ccl2. This inflammatory microenvironment promotes AR-independent prostatic epithelial proliferation, which can be abolished by ablating IL-1 signaling or depleting its major cellular source, the macrophages. This study demonstrates that disrupting luminal AR signaling promotes prostate inflammation, which may serve as a mechanism for resistance to androgen-targeted therapy for prostate-related diseases.

Determining prostate cancer-specific death through quantification of stromogenic carcinoma area in prostatectomy specimens.

We previously reported that reactive stroma grading in prostate cancer (PCa) is predictive of biochemical recurrence in prostatectomies and biopsies. In this study, we tested whether quantifying the percentage of reactive stromal grade 3 (RSG 3; stromogenic carcinoma pattern) in the entire tumor is predictive of PCa-specific death. Whole-mount prostatectomies operated by a single surgeon obtained between 1983 and 1998 were reviewed. Reactive stroma was evaluated as described previously, and areas of RSG 3 in the entire tumor were registered as percentages of total tumor. Statistical analysis was performed using Spearman, Kaplan-Meier, and Cox analyses. In all, 872 cases were evaluable. Quantification of RSG 3 percentage was an independent predictor of biochemical recurrence, analyzed as a continuous or grouped variable. Patients with higher RSG 3 percentages (larger tumor areas with RSG 3) had a significantly decreased biochemical recurrence-free survival than those with a lower RSG 3 percentage, even within the Gleason score 7 subset of patients. A nomogram introduced this new variable to the model. Furthermore, quantification of RSG 3 percentage was significantly predictive of PCa-specific death. Quantification of the RSG 3 (stromogenic carcinoma) area in PCa provides additional novel information on prognosis. These data substantiate the concept that the tumor microenvironment holds significant predictive information, as well as biological significance.

Cytoplasmic accumulation of glycogen synthase kinase-3beta is associated with aggressive clinicopathological features in human prostate cancer.

BACKGROUND: Activation of glycogen synthase kinase-3 (GSK-3) is involved in the regulation of cell growth, differentiation, mobility, proliferation and survival. However, its clinicopathologic significance remains unclear in prostate cancer (PCa). MATERIALS AND METHODS: A tissue microarray was produced from 640 samples. Sections were immunostained with an antibody against the non-phosphorylated form of GSK-3(GSK-3beta) and were digitized. Spearman correlation test was processed for correlations between GSK-3beta and biological and clinicopathological variables. The prognostic value of GSK-3beta was analyzed by Cox Regression model. RESULTS: Cytoplasmic GSK-3beta was higher in PCa than in normal prostate (mean expression index 4.55 vs. 3.50, p<0.0001). Conversely, nuclear expression was higher in normal prostate than that in PCa (3.38 vs. 2.04, p<0.0001). Cytoplasmic levels of GSK-3beta were correlated with clinical stage (rho=0.095, p=0.0337), lymph node metastasis (rho=0.116, p=0.0096), extracapsular extension (rho=0.092, p=0.0392), and Gleason score (rho=0.167, p=0.0002). Increased cytoplasmic GSK-3beta expression was correlated with high Ki-67 labeling index (rho=0.319, p<0.0001), low apoptotic index by TUNEL (rho=-0.118, p=0.0134), high levels of androgen receptor (rho=0.292, p<0.0001) and p-Akt (rho=0.396, p<0.0001). Patients with higher cytoplasmic levels of GSK-3beta had a two-fold risk of biochemical recurrence-free survival compared to those with lower levels of GSK-3beta [HR 1.934 (1.020-3.667), p=0.043]. CONCLUSION: Cytoplasmic accumulation of GSK-3beta is potentially associated with a pro-survival mechanism that promotes PCa development and progression.

Prognostic value of Akt-1 in human prostate cancer: a computerized quantitative assessment with quantum dot technology.

BACKGROUND: Akt/protein kinase B signaling pathway has been implicated in tumorigenesis and progression. Previous studies showed the predictive potential of p-Akt-1, but total Akt-1 could provide more reliable information. We used image deconvolution, nanotechnology (quantum dots), and image analysis to improve Akt-1 quantification. DESIGN: This tissue microarray study included 840 radical prostatectomy cases. Slides were incubated with primary antibody against nonphosphorylated Akt-1 (Akt-1) followed by biotinylated secondary antibody and then by Qdot655 streptavidin conjugate. Slides were imaged under fluorescence microscopy and spectral deconvolution (Nuance) and quantified using plug-in image analysis software. Average intensity of Akt-1 signal was measured and subject to statistical analysis. Multivariate analysis (Cox regression) was applied to assess the prognostic value of Akt-1 for biochemical recurrence and prostate cancer-specific death. Akt-1 expression was also examined for correlations with Ki-67 index and apoptotic index in our database. RESULT: Akt-1 was inversely correlated with apoptotic index (rho = -0.203; P = 0.004) but not with Ki-67 index. The correlation between Akt and p-Akt is significant but weak (P = 0.0496; R(2) = 0.118). On multivariate analysis Akt-1 was independently predictive of biochemical recurrence [hazard ratio, 2.863 (95% confidence interval, 1.127-7.271); P = 0.0270]. Akt-1 level is also predictive of prostate cancer-specific death (P = 0.0376). CONCLUSION: High levels of Akt-1, assessed by quantum dots, deconvolution imaging, and image analysis, are associated with a higher risk of biochemical recurrence and prostate cancer-specific death.

Biological correlates of p27 compartmental expression in prostate cancer.

PURPOSE: As one of the cyclin-dependent kinase inhibitors, p27 has been associated with biological behavior and disease progression in malignancies. MATERIALS AND METHODS: Tissue microarrays were constructed using 640 radical prostatectomy specimens. Normal prostate tissue, benign prostatic hyperplasia and index tumor were cored in triplicate at 0.6 mm. Slides were stained with an antibody to p27 and digitized. The expression of p27 was quantified. SPSS 11.0 (SPSS, Chicago, Illinois) was used to perform the Spearman correlation test, Kaplan-Meier analysis and Cox proportional hazard regression. RESULTS: We noted p27 expression predominately located in the epithelial nuclei of benign and cancer tissues. In PCa p27 was decreased compared to that in normal tissue, while p27 cytoplasmic expression was increased in PCa. Moderate nuclear p27 and cytoplasmic displacement of p27 were associated with worse disease-free survival compared with high or low p27. The cytoplasmic presence of p27 was an independent indicator for predicting the probability of biochemical recurrence in PCa. CONCLUSIONS: We report that p27 expression is consistently high in the normal prostate. Moderate levels of nuclear p27 and/or cytoplasmic entrapment of p27 are associated with increased proliferation and worse biochemical recurrence-free survival. These findings suggest that PCa may enhance its growth and progression by decreasing nuclear p27 via cytoplasmic displacement and other molecular mechanisms.

Protein expression profiling of endometriosis: validation of 2-mm tissue microarrays.

Tissue microarrays (TMAs) are useful tools for studying protein expression in endometriosis. Tissue microarray analyses of immunohistochemical profiles of estrogen receptor-alpha and P receptor corroborate previously published results from the use of conventional immunohistochemistry, thus validating TMA use in endometriosis.

Metastatic cancer DNA phenotype identified in normal tissues surrounding metastasizing prostate carcinomas.

Fourier transform-infrared statistical models have the proven ability to identify subtle structural changes in DNA at various stages of tumor development. Using these models, we show evidence for a metastatic cancer DNA phenotype in histologically normal prostate tissues surrounding metastasizing tumors. Strikingly, the DNA base and backbone structures of the metastatic phenotype are indistinguishable from those of the metastasizing prostate tumors but distinctly different from the structure recently reported for the primary cancer DNA phenotype. These findings suggest that the DNA structure linked to the development of metastasis is preordained in progenitor cells relatively early in multistep tumorigenesis. The substantial structural differences found between the primary and metastatic cancer DNA phenotypes suggest that each evolves through a separate pathway. The metastatic phenotype is potentially an early predictor of metastatic disease. Interventions that inhibit its formation would be expected to also inhibit the development of metastatic tumors.