Nucleolin Staining May Aid in the Identification of Circulating Prostate Cancer Cells

Circulating tumor cells (CTCs) have great potential as circulating biomarkers for solid malignancies. Currently available assays for CTC detection rely on epithelial markers with somewhat limited sensitivity and specificity. We found that the staining pattern of nucleolin, a common nucleolar protein in proliferative cells, separates CTCs from white blood cells (WBCs) in men with metastatic prostate cancer. Whole peripheral blood from 3 men with metastatic prostate cancer was processed with the AccuCyte CTC system (RareCyte, Seattle, WA). Slides were immunostained with 4',6-diamidino-2-phenylindole (DAPI), anti-pan-cytokeratin, anti-CD45/CD66b/CD11b/CD14/CD34, and anti-nucleolin antibodies and detected using the CyteFinder system. DAPI nucleolin colocalization and staining pattern wavelet entropy were measured with novel image analysis software. A total of 33,718 DAPI-positive cells were analyzed with the novel imaging software, of which 45 (0.13%) were known CTCs based on the established AccuCyte system criteria. Nucleolin staining pattern for segmentable CTCs demonstrated greater wavelet entropy than that of WBCs (median wavelet entropy, 6.86 × 10(7) and 3.03 × 10(6), respectively; P = 2.92 × 10(-22); approximated z statistic = 9.63). Additionally, the total nucleolin staining of CTCs was greater than that of WBCs (median total pixel intensity, 1.20 × 10(5) and 2.55 × 10(4) integrated pixel units, respectively; P = 2.40 × 10(-21); approximated z statistic = 9.41). Prostate cancer CTCs displayed unique nucleolin expression and localization compared to WBCs. This finding has the potential to serve as the basis for a sensitive and specific CTC detection metho

Elevated Prostate Health Index (phi) and Biopsy Reclassification During Active Surveillance of Prostate Cancer

The Prostate Health Index (phi) has been FDA approved for decision-making regarding prostate biopsy. Phi has additionally been shown to positively correlate with tumor volume, extraprostatic disease and higher Gleason grade tumors. Here we describe a case in which an elevated phi encouraged biopsy of a gentleman undergoing active surveillance leading to reclassification of his disease as high risk prostate cancer

Characterization of Urothelial Cancer Circulating Tumor Cells with a Novel Selection-Free Method

To investigate circulating tumor cells (CTCs) as biomarkers of urothelial carcinoma (UC). The majority of this work to date has utilized the CellSearch test, which has limited sensitivity due to reliance on positive selection for the cell surface protein EpCAM. We used a novel selection-free method to enumerate and characterize CTCs across a range of UC stages. Blood samples from 38 patients (9 controls, 8 non-muscle invasive bladder cancer [NMIBC], 12 muscle-invasive bladder cancer [MIBC] and 9 metastatic UC) were processed with the AccuCyte-CyteFinder system. Slides were stained for the white blood cell (WBC) markers CD45 and CD66b and the epithelial markers EpCAM and pancytokeratin (CK). CTCs were defined as any CK+/WBC- cell. Separately, the more restrictive CellSearch definition was applied, with the additional requirement of EpCAM positivity. The Kruskal-Wallis ANOVA test compared CTC counts by stage. ≥1 CTC was detected in 2/8(25%) patients with NMIBC, 7/12(58%) with MIBC, and 6/9(67%) with metastatic disease. No control had CTCs. Comparing CTC counts between groups, the only statistically significant comparison was between controls and patients with metastatic UC (p=0.009). With EpCAM positivity as a CTC requirement, no CTCs were detected in any NMIBC patient, and only 2(17%) MIBC patients had CTCs. CTCs tended to be larger in metastatic patients. CTCs were detected at all UC stages and exhibited phenotypic diversity of cell size and EpCAM expression. EpCAM negative CTCs that would be missed with the CellSearch test were detected in NMIBC and MIBC patient

Analogous detection of circulating tumor cells using the AccuCyte®—CyteFinder® system and ISET system in patients with locally advanced and metastatic prostate cancer

Introduction: Circulating tumor cells (CTCs) can provide important information on patient's prognosis and treatment efficacy. Currently, a plethora of methods is available for the detection of these rare cells. We compared the outcomes of two of those methods to enumerate and characterize CTCs in patients with locally advanced and metastatic prostate cancer (PCa). First, the selection-free AccuCyte® − CyteFinder® system (RareCyte®, Inc., Seattle, WA) and second, the ISET system (Rarecells Diagnostics, France), a CTC detection method based on cell size-exclusion. Methods: Peripheral blood samples were obtained from 15 patients with metastatic PCa and processed in parallel, using both methods according to manufacturer's protocol. CTCs were identified by immunofluorescence, using commercially available antibodies to pancytokeratin (PanCK), EpCAM, CD45/CD66b/CD34/CD11b/CD14 (AccuCyte® − CyteFinder® system), and pancytokeratin, vimentin (Vim) and CD45 (ISET system). Results: The median CTC count was 5 CTCs/7.5 mL (range, 0-20) for the AccuCyte® − CyteFinder® system and 37 CTCs/7.5 mL (range, 8-139) for the ISET system (P < 0.001). Total CTC counts obtained for the two methods were correlated (r = 0.750, P = 0.001). When separating the total CTC count obtained with the ISET system in PanCK+/Vim− and PanCK+/Vim+ CTCs, the total CTC count obtained with the AccuCyte® − CyteFinder® system was moderately correlated with the PanCK+/Vim− CTCs, and strongly correlated with the PanCK+/Vim+ CTCs (r = 0.700, P = 0.004 and r = 0.810, P < 0.001, respectively). Conclusion: Our results highlight significant disparities in the enumeration and phenotype of CTCs detected by both techniques. Although the median amount of CTCs/7.5 mL differed significantly, total CTC counts of both methods were strongly correlated. For future studies, a more uniform approach to the isolation and definition of CTCs based on immunofluorescent stains is needed to provide reproducible results that can be correlated with clinical outcomes

Prevalence and Prognostic Significance of PTEN Loss in African-American and European-American Men Undergoing Radical Prostatectomy.

African-American (AA) men have a higher risk of lethal prostate cancer (PCa) compared to European-American (EA) men. However, the molecular basis of this difference, if any, remains unclear. In EA PCa, PTEN loss, but not ERG rearrangement, has been associated with poor outcomes in most studies. Although ERG rearrangement is less common in AA compared to EA PCa, the relative frequency of PTEN loss and the association of PTEN/ERG molecular subtypes with outcomes is unknown for AA PCa. We examined PTEN/ERG status by immunohistochemistry in self-identified AA patients undergoing radical prostatectomy at Johns Hopkins with tumor tissue available on tissue microarray (TMA; n = 169) and matched these cases by pathologic parameters to 169 EA patients from the same TMAs. The rate of PTEN loss was significantly lower in AA compared to EA PCa (18% vs 34%; p = 0.001), similar to the lower rate of ERG expression (25% vs 51%; p < 0.001). To examine the association of PTEN/ERG status with oncologic outcomes, we created an additional TMA of 87 AA tumors with Gleason score > 4 + 3 = 7. Among the total population of AA men with outcome data from all TMAs (n = 222), PTEN loss was associated with higher risk of biochemical recurrence (hazard ratio [HR] 2.25, 95% confidence interval [CI] 1.33–3.82) and metastasis (HR 3.90, 95% CI 1.46–10.4) in multivariable models

Clinical Validation of the 2005 ISUP Gleason Grading System in a Cohort of Intermediate and High Risk Men Undergoing Radical Prostatectomy.

In 2005, the International Society of Urological Pathology (ISUP) introduced several modifications to the original Gleason system that were intended to enhance the prognostic power of Gleason score (GS). The objective of this study was to clinically validate the 2005 ISUP Gleason grading system for its ability to detect metastasis. We queried our institutional RP database for men with NCCN clinically localized intermediate to high-risk disease undergoing radical prostatectomy (RP) between 1992 and 2010 with no additional treatment until the time of metastatic progression. A case-cohort design was utilized. A total of 333 available RP samples were re-reviewed and GS was reassigned per the 2005 ISUP Gleason system. Cumulative incidence of metastasis was 0%, 8.4%, 24.5% and 44.4% among specimens that were downgraded, unchanged, had one point GS increase and two point GS increase, respectively. The hazard ratio for metastasis raised in GS 8 and 9 compared to GS 7 from 2.77 and 5.91 to 3.49 and 9.31, respectively. The survival c-index of GS increased from 0.70 to 0.80 when samples were re-graded at 5 years post RP. The c-index of the reassigned GS was higher than the original GS (0.77 vs 0.64) for predicting PCSM at 10 years post RP. The regraded GS improved the prediction of metastasis and PCSM. This validates the updated Gleason grading system using an unambiguous clinical endpoint and highlights the need for reassignment of Gleason grading according to 2005 ISUP system when considering comparisons of novel biomarkers to clinicopathological variables in archival cohorts

(A) Fused glands (400x). In the original Gleason grading this pattern was considered pattern 3. According to the 2005 ISUP Gleason system it would be graded as pattern 4; B) Adenocarcinoma with glomeruloid features currently assigned a Gleason pattern 4 (100x); C) Ill-defined glands (100x). This pattern would be graded as Gleason pattern 4 by the ISUP 2005 Gleason grading; and D) Individual cells (100x).

<p>This pattern was originally accepted under Gleason pattern 3 and would be assigned a Gleason pattern 5 according to the 2005 ISUP Gleason system.</p

Cumulative incidence of metastasis at 5 years post radical prostatectomy.

<p>A) Difference in re-graded 2005 ISUP modified and original Gleason score; B) Difference in primary re-graded 2005 ISUP modified and original Gleason score; and C) Difference in secondary re-graded 2005 ISUP modified and original Gleason score.</p