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1.  Multiple cores of Gleason score 6 correlate with favourable findings at radical prostatectomy 
BJU international  2013;111(8):E306-E309.
To establish whether the good prognosis of Gleason score 6 (GS6) is maintained in the setting of multiple involved cores.
Patients and Methods
In total, 6156 men (from 1 April 2000 to 30 April 2007) with GS6 on biopsy underwent radical prostatectomy (RP) at our institution.
The number of positive cores was correlated with the outcome at RP.
More positive cores correlated with less organ-confined disease (P < 0.001), positive margins (P < 0.012), increasing RP grade (P < 0.001) and increased seminal vesicles/lymph node involvement (P = 0.012).
For men with data available, the actuarial risk of being biochemically free of disease at 5 years was 93.2% when ≤6 cores were positive (812 men followed to 5 years) vs 89.1% if >6 cores were positive (41 men followed to 2 years) (P = 0.6).
Although the predicted ‘cure rate’ of >75% probability of a tumour showing no evidence of biochemical recurrence at 10 years after RP was statistically different between cases with ≤6 vs >6 positive cores (P < 0.0001), the outcome in both groups was still favourable (90.5% vs 84%).
Partin-like tables were generated factoring in the number of positive cores to predict organ-confined disease as a guide for urologists to perform nerve-sparing surgery.
For example, with T1c disease, there was a ≥75% probability of organ-confined disease with one to three positive cores regardless of prostate-specific antigen (PSA) level, and the same probability was present with four to six positive cores and a PSA level of 0–4 ng/mL.
A low Gleason score on biopsy is a powerful prognostic finding, such that this favourable outcome is maintained even in the setting of multiple positive cores with GS6.
PMCID: PMC3978182  PMID: 23350787
Gleason score 6; needle biopsy; radical prostatectomy
2.  Implication of Cell Kinetic Changes during the Progression of Human Prostatic Cancer1 
The daily percentage of cells proliferating and dying were determined for normal, premalignant, and cancerous prostatic cells within the prostate as well as for prostatic cancer cells in lymph node, sort tissue, and bone metastases from untreated and hormonally failing patients. These data demonstrate that normal prostatic glandular cells have an extremely low but balanced rate of cell proliferation and death (i.e., both <0.20%/day). This results in a steady-state, self-renewing condition in which there is no net growth, although the glandular cells are continuously being replaced (i.e., turnover) every 500 ± 79 days. Transformation of these cells into high-grade prostatic intraepithelial neoplastic cells initially involves an unbalanced increase in the daily percentage of cells proliferating versus dying, such that net continuous growth occurs (i.e., mean doubling time, 154 ± 22 days). As these early proliferation lesions continue to grow into late stage high-grade prostatic intraepithelial neo-plastic cells, the daily percentage of cells dying increases further to a point equaling the daily percentage of proliferation. This results in cessation of net growth while inducing a 6-fold increase in the turnover time of these cells (i.e., 56 ± 12 days), increasing their risk of further genetic changes. The transition of late stage high-grade prostatic intraepithelial neoplastic cells into localized prostatic cancer cells involves no further increase in proliferation but a decrease in death resulting in net continuous growth of localized prostatic cancers with a mean doubling time of ≥475 days. As compared to localized prostatic cancer cells, metastatic prostatic cancer cells within lymph nodes or bones of untreated patients have an increase in daily rate of proliferation coupled with a reduction in their daily percentage of cell death, producing net growth rates with a mean doubling time of 33 ± 4 days and 54± 5 days, respectively. Remarkably, there is no further increase in proliferation in hormonally failing patients, but instead an increase in the daily percentage of androgen-independent prostatic cancer cells dying within sort tissue or bone metastases. These changes result in doubling times which are two to three times longer (i.e., 126 ± 21 and 94 ± 15 days) in these lymph node and bone meta-static sites, respectively, compared to similar sites in hormonally untreated patients. These data demonstrate that the daily percentage of proliferation for either androgen-dependent or -independent metastatic prostatic cancer cells is remarkably low (i.e., <3.0%/day), consistent with why antiproliferative chemotherapy has been of such limited value against such metastatic cells. These results also suggest that prostatic carcinogenesis starts in the second to third decade of life and may require over 50 years for progression to pathologically detectable metastatic disease.
PMCID: PMC4086477  PMID: 9816006
3.  Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors 
Alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5mC) are a hallmark of cancer cells, including testicular germ cell tumors. Virtually all testicular germ cell tumors are believed to be derived from intratubular germ cell neoplasia unclassified (IGCNU), which is thought to arise from primordial germ cells. Prior studies revealed that seminomas contain reduced levels of global DNA methylation as compared with nonseminomatous germ cell tumors. Smiraglia et al have proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone 5mC erasure, and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation after the original erasure of methylation and contain normal 5mC levels. Yet the methylation status of IGCNU has not been determined previously. We used immunohistochemical staining against 5mC to evaluate global methylation in IGCNU and associated invasive testicular germ cell tumors. Strikingly, staining for 5mC was undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet there was robust staining in nonseminomatous germ cell tumors. The lack of staining for 5mC in IGCNU and seminomas was also found in mixed germ cell tumors containing both seminomatous and nonseminomatous components. Lack of 5mC staining was not related to a lack of the maintenance methyltransferase (DNA methyltransferase 1) protein. We conclude that testicular germ cell tumors are derived in most cases from IGCNU cells that have undergone developmentally programmed 5mC erasure and that the degree of subsequent de novo methylation is most closely related to the differentiation state of the neoplastic cells. That is, IGCNU cells and seminoma cells remain unmethylated, whereas all other histological types appear to arise after de novo methylation.
PMCID: PMC4086525  PMID: 18622385
seminoma; nonseminomatous germ cell tumors; IGCNU; global methylation
4.  Pathological Examination of Radical Prostatectomy Specimens in Men with Very Low Risk Disease at Biopsy Reveals Distinct Zonal Distribution of Cancer in Black American Men 
The Journal of urology  2013;191(1):60-67.
Of men with very low risk prostate cancer at biopsy recent evidence shows that black American men are at greater risk for adverse oncologic outcomes after radical prostatectomy. We studied radical prostatectomy specimens from black and white men at very low risk to determine whether there are systematic pathological differences.
Materials and Methods
Radical prostatectomy specimens were evaluated in men with National Comprehensive Cancer Network® (NCCN) very low risk prostate cancer. At diagnosis all men underwent extended biopsy sampling (10 or more cores) and were treated in the modern Gleason grade era. We analyzed tumor volume, grade and location in 87 black and 89 white men. For each specimen the dominant nodule was defined as the largest tumor with the highest grade.
Compared to white men, black men were more likely to have significant prostate cancer (61% vs 29%), Gleason 7 or greater (37% vs 11%, each p <0.001) and a volume of greater than 0.5 cm3 (45% vs 21%, p = 0.001). Dominant nodules in black men were larger (median 0.28 vs 0.13 cm3, p = 0.002) and more often anterior (51% vs 29%, p = 0.003). In men who underwent pathological upgrading the dominant nodule was also more frequently anterior in black than in white men (59% vs 0%, p = 0.001).
Black men with very low risk prostate cancer at diagnosis have a significantly higher prevalence of anterior cancer foci that are of higher grade and larger volume. Enhanced imaging or anterior zone sampling may detect these significant anterior tumors, improving the outcome in black men considering active surveillance.
PMCID: PMC4042393  PMID: 23770146
prostate; prostatic neoplasms; African Americans; risk; neoplasm grading
5.  Low Prostate-specific Antigen and No Gleason Score Upgrade Despite More Extensive Cancer During Active Surveillance Predicts Insignificant Prostate Cancer at Radical Prostatectomy 
Urology  2012;80(4):883-888.
To identify parameters that predict insignificant prostate cancer in 67 radical prostatectomies after biopsy reclassification to worse disease on active surveillance.
Parameters evaluated at diagnosis and at biopsy reclassification included serum prostate-specific antigen, prostate-specific antigen density, number of positive cores, maximum percent involvement of cancer per core, and any interval negative biopsies. Gleason upgrading at biopsy reclassification was also assessed to predict insignificant cancer.
Mean time between diagnosis and radical prostatectomies was 30.3 months with a median of 3 biopsies (range 2–9). Nineteen of 67 (28.4%) had clinically insignificant cancer at radical prostatectomy. In the entire group, there were no variables significantly associated with insignificant cancer at radical prostatectomy. In a subgroup analysis of 37 patients without Gleason pattern 4/5 at biopsy reclassification, 16/37 (43.2%) showed insignificant cancer at radical prostatectomy. In this subgroup, prostate-specific antigen at diagnosis was significantly lower in men with insignificant cancer (3.7 ng/mL) vs significant cancer (5.4 ng/mL) (P = .0005). With prostate-specific antigen <4 ng/mL at diagnosis or at biopsy reclassification, 12/13 (92.3%) men showed insignificant cancer, whereas only 4/24 (16.7%) men with prostate-specific antigen >4 ng/mL both at diagnosis and at biopsy reclassification showed insignificant cancer.
Most men with biopsy reclassification while on active surveillance have significant disease at radical prostatectomy, justifying their treatment. Low prostate-specific antigen at diagnosis or at biopsy reclassification can predict a high probability of insignificant cancer in the absence of Gleason pattern 4/5 on biopsy. These men may be candidates for continuing active surveillance.
PMCID: PMC3715088  PMID: 22921697
6.  Prognostic Gleason grade grouping: data based on the modified Gleason scoring system 
BJU international  2013;111(5):753-760.
• To investigate pathological and short-term outcomes since the most recent Gleason system modifications by the International Society of Urological Pathology (ISUP) in an attempt to divide the current Gleason grading system into prognostically accurate Gleason grade groups.
Patients and Methods
• We queried the Johns Hopkins Radical Prostatectomy Database (1982–2011), approved by the institutional review board, for men undergoing radical prostatectomy (RP) without a tertiary pattern since 2004 and identified 7869 men.
• Multivariable models were created using preoperative and postoperative variables; prognostic grade group (Gleason grade ≤6; 3 + 4; 4 + 3; 8; 9–10) was among the strongest predictors of biochemical recurrence-free (BFS) survival.
• Significant differences were noted among the Gleason grade groups at biopsy; differences were noted in the race, PSA level, clinical stage, number of positive cores at biopsy and the maximum percentage of positive cores among the Gleason grade groups at RP.
• With a median (range) follow-up of 2 (1–7) years, 5-year BFS rates for men with Gleason grade ≤6, 3 + 4, 4 + 3, 8 and 9–10 tumours at biopsy were 94.6, 82.7, 65.1, 63.1 and 34.5%, respectively (P < 0.001 for trend); and 96.6, 88.1, 69.7, 63.7 and 34.5%, respectively (P < 0.001), based on RP pathology.
• The 2005 ISUP modifications to the Gleason grading system for prostate carcinoma accurately categorize patients by pathological findings and short-term biochemical outcomes but, while retaining the essence of the Gleason system, there is a need for a change in its reporting to more closely reflect tumour behaviour.
• We propose reporting Gleason grades, including prognostic grade groups which accurately reflect prognosis as follows: Gleason score ≤6 (prognostic grade group I); Gleason score 3+4=7 (prognostic grade group II); Gleason score 4+3=7 (prognostic grade group III); Gleason score 4+4=8 (prognostic grade group (IV); and Gleason score 9–10 (prognostic grade group (V).
PMCID: PMC3978145  PMID: 23464824
Gleason grade; prostate carcinoma; radical prostatectomy
7.  Serum prostate-specific antigen (PSA) concentration is positively associated with rate of disease reclassification on subsequent active surveillance prostate biopsy in men with low PSA density 
BJU international  2013;113(4):561-567.
To investigate the association between serum prostate-specific antigen (PSA) concentration at active surveillance (AS) entry and disease reclassification on subsequent AS biopsy (‘biopsy reclassification’) in men with low PSA density (PSAD).
To investigate whether a clinically meaningful PSA threshold for AS eligibility/ineligibility for men with low PSAD can be identified based on risk of subsequent biopsy reclassification.
Patients and Methods
We included men enrolled in the Johns Hopkins AS Study (JHAS) who had a PSAD of <0.15 ng/mL/g (640 men).
We estimated the incidence rates (IRs; per 100 person years) and hazard ratios (HR) of biopsy reclassification (Gleason score ≥ 7, any Gleason pattern 4 or 5, ≥3 positive cores, or ≥50% cancer involvement/biopsy core) for categories of serum PSA concentration at the time of entry into AS.
We generated predicted IRs using Poisson regression to adjust for age and prostate volume, mean percentage free PSA (ratio of free to total PSA) and maximum percentage biopsy core involvement with cancer.
The unadjusted IRs (per 100 person years) of biopsy reclassification across serum PSA concentration at entry into JHAS showed, in general, an increase; however, the pattern was not linear with higher IRs in the group ≥ 4 to <6 ng/mL (14.2, 95% confidence interval [CI] 11.8–17.2%) when compared with ≥ 6 to <8 ng/mL (8.4, 95% CI 5.7–12.3%) but almost similar IRs when compared with the group ≥ 8 to <10 ng/mL (14.8, 95% CI 8.4–26.1%).
The adjusted predicted IRs of reclassification showed a similar non-linear increase in IRs, whereby the rates around 4 ng/mL were similar to the rates around 10 ng/mL.
Risk for biopsy reclassification increased non-linearly across PSA concentration in men with low PSAD, whereby no obvious clinically meaningful threshold could be identified. This information could be incorporated into decision-making for AS. However, longer follow-up times are needed to warrant final conclusions.
PMCID: PMC3978167  PMID: 23746233
active surveillance; PSA; patient selection
8.  Pathological Outcomes in Men with Low Risk and Very Low Risk Prostate Cancer: Implications on the Practice of Active Surveillance 
The Journal of urology  2013;190(4):1218-1222.
We assessed oncologic outcomes at surgery in men with low risk and very low risk prostate cancer who were candidates for active surveillance.
Materials and Methods
In a prospectively collected institutional database, we identified 7,486 subjects eligible for active surveillance who underwent radical retropubic prostatectomy. Candidates were designated as being at low risk (stage T1c/T2a, prostate specific antigen 10 ng/ml or less, and Gleason score 6 or less) or very low risk (stage T1c, prostate specific antigen density 0.15 or less, Gleason score 6 or less, 2 or fewer positive biopsy cores, 50% or less cancer involvement per core) based on preoperative data. Adverse findings were Gleason score upgrade (score 7 or greater) and nonorgan confined cancer on surgical pathology. The relative risk of adverse findings in men at low risk with very low risk disease was evaluated in a multivariate model using Poisson regression.
A total of 7,333 subjects met the criteria for low risk disease and 153 had very low risk disease. The proportion of subjects at low risk found to have Gleason score upgrade or nonorgan confined cancer on final pathology was 21.8% and 23.1%, respectively. Corresponding values in those at very low risk were 13.1% and 8.5%, respectively. After adjusting for age, race, year of surgery, body mass index, and prostate specific antigen at diagnosis, the relative risk of Gleason score upgrade in men with low risk vs very low risk disease was 1.89 (95% CI 1.21–2.95). The relative risk of nonorgan confined cancer was 2.06 (95% CI 1.19–3.57).
Men with very low risk prostate cancer were at significantly lower risk for adverse findings at surgery compared to those with low risk disease. These data support the stratification of low risk cancer when selecting and counseling men who may be appropriate for active surveillance.
PMCID: PMC3978170  PMID: 23643603
prostate; prostatic neoplasms; risk; disease progression; treatment outcome
9.  Multiparametric magnetic resonance imaging findings in men with low-risk prostate cancer followed using active surveillance 
BJU international  2013;111(7):1037-1045.
To assess the performance of multiparametric magnetic resonance imaging (MRI) in identifying pathological-index (path-index) lesions, defined as cancer present in the same prostate sextant in two separate surveillance biopsies, in men followed within an active surveillance (AS) programme for low-risk prostate cancer (CaP) with extended follow-up.
Materials and Methods
A total of 50 men, representing >215 person-years of follow-up in an AS programme, who were referred for prostate MRI were randomly chosen to have their images reviewed by a radiologist with expertise in prostate MRI, who was blinded to biopsy results.
Index lesions on MRI were defined as a single suspicious lesion ≥10 mm or >2 lesions in a given prostate sextant. Lesions on MRI were considered suspicious if ≥2 abnormal parameters co-registered anatomically. Path-index lesions were defined as cancer present in a given prostate sextant on two separate biopsy sessions.
Sensitivity and specificity were calculated to test the performance of MRI for identifying path-index lesions.
Clinical and pathological features were compared between men with and without a MRI-index lesion.
A total of 31 path-index and 13 MRI-index lesions were detected in 22 and 10 patients, respectively.
Multiparametric MRI demonstrated excellent specificity and negative predictive value (0.974 and 0.897, respectively) for the detection of path-index lesions. Sensitivity (0.19) and positive predictive value (0.46) were considerably lower.
Patients with an index lesion on MRI were younger and less likely to have met the ‘Epstein’ criteria for very low-risk CaP.
Compared with men without an MRI lesion, a significant increase in biopsy reclassification was noted for men with a MRI lesion (40 vs 12.5%, P = 0.04).
A non-suspicious MRI was highly correlated with a lack of path-index lesions in an AS population.
Multiparametric MRI may be useful in both the selection and monitoring of patients undergoing AS.
PMCID: PMC3978179  PMID: 23464904
prostate cancer; surveillance; magnetic resonance imaging; sensitivity and specificity
10.  Association of [-2]proPSA with Biopsy Reclassification During Active Surveillance for Prostate Cancer 
The Journal of urology  2012;188(4):1131-1136.
Previous studies have suggested an association between [-2]proPSA expression and prostate cancer detection. Less is known about the utility of this marker in following prostate cancer patients on active surveillance. Thus, our objective was to examine the relationship between [-2]proPSA and biopsy results in men enrolled in an active surveillance program.
Materials and Methods
In 167 men from our institutional active surveillance program, we used Cox proportional hazards models to examine the relationship between [-2]proPSA and annual surveillance biopsy results. The outcome of interest was biopsy reclassification (Gleason score ≥7, or >2 positive biopsy cores, or >50% involvement of any core with cancer). We also examined the association of biopsy results with total PSA, %fPSA, [-2]proPSA/%fPSA, and the Beckman Coulter Prostate Health Index [phi=([-2]proPSA/fPSA) x (tPSA)½].
While on active surveillance (median time from diagnosis 4.3 years), 63 (37.7%) men demonstrated biopsy reclassification based on the above criteria, including 28 (16.7%) of whom had reclassification based on Gleason score upgrading (Gleason score≥7). Baseline and longitudinal %fPSA, %[-2]proPSA, [-2]proPSA/%fPSA, and phi measurements were significantly associated with biopsy reclassification, and %[-2]proPSA and phi provided the greatest predictive accuracy for high-grade cancer.
In men on active surveillance, measures based on [-2]proPSA such as phi, appear to provide improved prediction of biopsy reclassification during follow-up. Additional validation is warranted to determine whether clinically useful thresholds can be defined, and to better characterize the role of %[-2]proPSA and phi in conjunction with other markers in monitoring patients enrolled in active surveillance.
PMCID: PMC3976250  PMID: 22901577
proPSA; PSA; prostate cancer; biopsy; active surveillance
11.  A Broad Survey of Cathepsin K Immunoreactivity in Human Neoplasms 
Cathepsin K is consistently and diffusely expressed in alveolar soft part sarcoma (ASPS) and a subset of translocation renal cell carcinomas (RCCs). However, cathepsin K expression in human neoplasms has not been systematically analyzed. We constructed tissue microarrays (TMA) from a wide variety of human neoplasms, and performed cathepsin K immunohistochemistry (IHC). Only 2.7% of 1,140 carcinomas from various sites exhibited cathepsin K labeling, thus suggesting that among carcinomas, cathepsin K labeling is highly specific for translocation RCC. In contrast to carcinomas, cathepsin K labeling was relatively common (54.6%) in the 414 mesenchymal lesions studied, including granular cell tumor, melanoma, and histiocytic lesions, but not paraganglioma, all of which are in the morphologic differential diagnosis of ASPS. Cathepsin K IHC can be helpful in distinguishing ASPS and translocation RCC from some but not all of the lesions in their differential diagnosis.
PMCID: PMC3957187  PMID: 23355199
Cathepsin K; TFE3; TFEB; Immunohistochemistry
12.  Interobserver Variability in Histologic Evaluation of Radical Prostatectomy Between Central and Local Pathologists: Findings of TAX 3501 Multinational Clinical Trial 
Urology  2010;77(5):1155-1160.
To determine the agreement between the local pathologist findings and central pathologist findings using data from the TAX 3501 trial. TAX 3501 was a randomized, multinational trial comparing the outcomes of patients with high-risk prostate cancer treated with androgen deprivation with or without docetaxel after radical prostatectomy (RP). Patient eligibility was determined by a minimal 5-year progression-free survival estimate of 60% using Kattan’s nomogram.
The pathologic findings were reassessed in 257 consecutive RP specimens by 2 central pathologists and compared with the local pathologist data.
For the Gleason score, agreement was found in 181 (70%) of 257 cases, upgrading in 57 (75%), and downgrading in 25% of the RP specimens The most frequent upgrade was from Gleason score 7 to 8 or 9 and downgrading from Gleason score 8 to 7. Of the upgrades and downgrades, 37% and 21% were of 2 Gleason score points, respectively. For the tumor extent, agreement was found in 179 (70%) of 256 specimens, with upstaging in 70 (91%) and downstaging in 9%. The most frequent upstage was from focal to extensive extraprostatic extension (45%). For seminal vesicle invasion, agreement was found for 238 (93%) of 256 RP specimens Almost equal rates of underdiagnosing and overdiagnosing seminal vesicle invasion was observed. For margin status, agreement was present for 229 (89%) of 256 cases. The central pathologist review led to reclassification as a positive margin in 17 cases and a negative margin in 10. For lymph node status, 2 (1%) of 210 RP specimens had positive nodes identified only by the central pathologist. Agreement was observed in 154 negative and 54 positive cases.
Significant interobserver variations were found between the central and local pathologists. From the central pathologist review, the progression-free survival estimates were altered in 31 patients (13%), including 22 who were reassigned a greater risk estimate, rendering them study eligible. Thus, interobserver variability affected prognostication and trial accrual.
PMCID: PMC3449146  PMID: 21146858
13.  An updated prostate cancer staging nomogram (Partin tables) based on cases from 2006 to 2011 
BJU international  2012;111(1):10.1111/j.1464-410X.2012.11324.x.
To update the 2007 Partin tables in a contemporary patient population.
Patients and Methods
The study population consisted of 5,629 consecutive men who underwent RP and staging lymphadenectomy at the Johns Hopkins Hospital between January 1, 2006 and July 30, 2011 and met inclusion criteria.
Polychotomous logistic regression analysis was used to predict the probability of each pathologic stage category: organ-confined disease (OC), extraprostatic extension (EPE), seminal vesicle involvement (SV+), or lymph node involvement (LN+) based on preoperative criteria.
Preoperative variables included biopsy Gleason score (6, 3+4, 4+3, 8, and 9–10), serum PSA (0–2.5, 2.6–4.0, 4.1–6.0, 6.1–10.0, greater than 10.0 ng/mL), and clinical stage (T1c, T2c, and T2b/T2c).
Bootstrap re-sampling with 1000 replications was performed to estimate 95% confidence intervals for predicted probabilities of each pathologic state.
The median PSA was 4.9 ng/mL, 63% had Gleason 6 disease, and 78% of men had T1c disease.
73% of patients had OC disease, 23% had EPE, 3% had SV+ but not LN+, and 1% had LN+ disease. Compared to the previous Partin nomogram, there was no change in the distribution of pathologic state.
The risk of LN+ disease was significantly higher for tumours with biopsy Gleason 9–10 than Gleason 8 (O.R. 3.2, 95% CI 1.3–7.6).
The c-indexes for EPE vs. OC, SV+ vs. OC, and LN+ vs. OC were 0.702, 0.853, and 0.917, respectively.
Men with biopsy Gleason 4+3 and Gleason 8 had similar predicted probabilities for all pathologic stages.
Most men presenting with Gleason 6 disease or Gleason 3+4 disease have <2% risk of harboring LN+ disease and may have lymphadenectomy omitted at RP.
The distribution of pathologic stages did not change at our institution between 2000–2005 and 2006–2011.
The updated Partin nomogram takes into account the updated Gleason scoring system and may be more accurate for contemporary patients diagnosed with prostate cancer.
PMCID: PMC3876476  PMID: 22834909
prostate cancer; prostatectomy; prostage-specific antigen; nomograms; staging
Distinguishing invasive high-grade urothelial carcinoma (UC) from other carcinomas occurring in the genitourinary tract may be difficult. The differential diagnosis includes high-grade prostatic adenocarcinoma, spread from an anal squamous cell carcinoma (SCC), or spread from a uterine cervical SCC. In terms of metastatic UC, the most common problem is differentiating spread of UC to the lung versus a primary pulmonary SCC. Immunohistochemistry (IHC) for GATA binding protein 3 (GATA3), thrombomodulin (THROMBO), and Uroplakin III was performed on a tissue microarray (TMA) containing 35 cases of invasive high-grade UC. GATA3 IHC was also performed on TMAs containing 38 high-grade (Gleason score 8) prostatic adenocarcinomas, representative tissue sections from 15 invasive anal SCCs, representative tissue sections from 19 invasive cervical SCCs, and TMAs with 12 invasive cervical carcinomas of the cervix [SCC (n=10), SCC with neuroendocrine features (n=1), adenosquamous carcinoma (n=1)]. Additionally, GATA3 IHC was performed on representative tissue sections from 15 pulmonary UC metastases and a TMA with 25 SCCs of the lung and 5 pulmonary non-small cell carcinomas with squamous features. GATA3, THROMBO, and Uroplakin III were positive in 28 (80%), 22 (63%), and 21 (60%) cases of high-grade UC, respectively. All GATA3 positive staining was non-focal, 25 (89%) cases demonstrated moderate-strong staining, and 3 (11%) cases demonstrated weak staining. Of the 7 cases that failed to express GATA3, 5 were positive for THROMBO and/or Uroplakin III, while 2 cases were negative for all 3 markers. None of 38 high-grade prostatic adenocarcinomas were positive for GATA3. Weak GATA3 staining was present in occasional basal cells of benign prostate glands, in a few benign atrophic glands, and in urothelial metaplasia. Of the 15 cases of anal SCCs, 2 (7%) cases showed focal weak staining and 1 (3%) case showed focal moderate staining. Weak staining was also rarely observed in the benign anal squamous epithelium. Of the 31 uterine cervical carcinomas, 6 (19%) showed weak GATA3 staining (3 non-focal, 3 focal) and 2 (6%) demonstrated focal moderate staining. Twelve (80%) of the metastatic UC to the lung were positive for GATA3 with 11 cases showing diffuse moderate or strong staining and 1 case showing focal moderate staining. None of the pulmonary SCC or non-small cell carcinomas with squamous features were GATA3 positive. GATA3 IHC is a sensitive marker for UC and positive staining in UC is typically non-focal and moderate or strong in intensity. GATA3 is also highly specific in excluding high-grade prostate adenocarcinoma. Although some cervical and anal SCCs can be GATA3 positive, unlike in UC, staining is more commonly focal and weak. GATA3 is also a useful maker when diagnosing metastatic UC to the lung.
PMCID: PMC3444740  PMID: 22982890
16.  Cytoplasmic PTEN Protein Loss Distinguishes Intraductal Carcinoma of the Prostate from High Grade Prostatic Intraepithelial Neoplasia 
Intraductal carcinoma of the prostate is a marker of aggressive disease. However, intraductal carcinoma exists on a morphologic continuum with high grade prostatic intraepithelial carcinoma (PIN) and distinguishing intraductal carcinoma from PIN is a common diagnostic dilemma with significant clinical implications. We evaluated whether immunostains for PTEN and ERG can sensitively identify intraductal carcinoma and accurately distinguish it from high grade PIN. A combined immunostain for PTEN, ERG, p63 and CK903 was developed and validated. Radical prostatectomy specimens with lesions meeting criteria for intraductal carcinoma (n=45), intraductal cribriform proliferations falling short of intraductal carcinoma (n=15), and PIN lesions (n=39) were retrospectively identified and assessed for PTEN and ERG. Cytoplasmic PTEN loss was identified in 84% (38/45) of the intraductal carcinoma and 100% (15/15) of intraductal cribriform proliferation cases. In contrast, cytoplasmic PTEN loss was never observed in PIN (0/39) (p<0.0001). Of the 53 cases of intraductal carcinoma or intraductal cribriform proliferation with cytoplasmic PTEN loss, it was homogeneously lost in 42 cases (79%). Weak, focal nuclear positivity for PTEN was retained in 31 of these 42 cases (74%). ERG expression was identified in 58% (26/45) of intraductal carcinoma and 67% (10/15) of intraductal cribriform proliferations compared to 13% (5/39) of PIN. Concordance between the PTEN/ERG status of the intraductal carcinoma lesions and the concurrent invasive carcinoma was high (>95% and p<0.0001 for each), and substantially less for PIN and the concurrent invasive tumor (83% for PTEN and 67% for ERG; p=NS for each). Cytoplasmic PTEN loss occurs in the majority of intraductal carcinoma and intraductal cribriform proliferation cases. Cytoplasmic PTEN loss was never observed in PIN (100% specificity). Our study identifies PTEN loss as a potentially useful marker to distinguish intraductal carcinoma from PIN and provides a plausible molecular explanation for why intraductal carcinoma is associated with poor prognosis.
PMCID: PMC3610824  PMID: 23222491
Prostatic adenocarcinoma; intraductal carcinoma; PTEN; ERG; immunohistochemistry
Although rare, there are cases within reported series of men with GS≤6 in radical prostatectomies that have pelvic lymph node (LN) metastases. However, there are no studies as to whether pelvic LN metastases occur in tumors GS≤6 using the International Society of Urological Pathology (ISUP) updated Gleason scoring system. We performed a search of the radical prostatectomy databases at 4 large academic centers for cases of GS≤6. Only prostatectomies submitted and embedded in entirety with pelvic lymph node dissections were included. A combined total of 14,123 cases were identified out of which 22 cases had a positive LN. Histopathology review of 19 cases (3 cases unavailable for review) showed higher grade than originally reported by the pathologists in all cases. Of the 17 pre-ISUP reviewed cases, 2 were upgraded to 4+3=7 with both cribriform and poorly formed glands. One case was upgraded to 4+3=7 with tertiary pattern five displaying cribriform glands, poorly formed glands, and cords of single cells. Eleven cases were upgraded to 3+4=7 with glomeruloid structures and small to large cribriform glands (1 of these also had ductal adenocarcinoma features). Two cases had tertiary pattern 4 with small cribriform glands. One case had a prominent colloid component that would currently be graded as 4+5=9 due to large cribriform glands and solid sheets of cells within mucin. Of the two post-ISUP cases, one demonstrated tertiary pattern 4 and the other showed Gleason score 3+4=7 with irregular cribriform glands. Under-grading primarily accounts for LN positivity with GS ≤6, which has decreased significantly since the adoption of the ISUP grading system in 2005. Out of over 14,000 totally embedded RPs from multiple institutions, there was not a single case of a GS≤6 tumor with LN metastases. In contrast to prevailing assumptions, Gleason score ≤6 tumors do not appear to metastasize to lymph nodes. Rather, Gleason patterns 4 or 5, as better defined by the current ISUP updated grading system, is required for metastatic disease.
PMCID: PMC3421030  PMID: 22531173
Gleason score; radical prostatectomy; lymph node metastases
18.  Dysregulation of mammalian target of rapamycin pathway in plasmacytoid variant of urothelial carcinoma of the urinary bladder☆,☆☆ 
Human pathology  2012;44(4):612-622.
Plasmacytoid urothelial carcinoma is a rare but aggressive variant of bladder cancer with no clear therapeutic guidelines. Dysregulation of the mammalian target of rapamycin (mTOR) pathway has been linked to oncogenesis in conventional bladder cancer. Several antineoplastic agents targeting mTOR pathway are currently available. This study assesses mTOR pathway status as well as c-myc and p27 expression. We retrieved 19 archival cases of plasmacytoid urothelial carcinoma from two institutions. Whole tissue sections were evaluated for immunoexpression of phosphatase and tensin homolog (PTEN), phosphorylated mTOR, phosphorylated protein kinase B (AKT), phosphorylated S6, c-myc, and p27. We evaluated intensity (0 to 3+) and extent (0%–100%) of expression for all markers. An H score was calculated as the sum of products of intensity and extent for each marker and used during analysis. In addition, PTEN loss was defined as absence of expression in >10% of tumor cells. We encountered PTEN loss in 28%. Higher H score for nuclear phosphorylatedAKT and a lowerHscore for phosphorylated S6 was encountered in muscle invasive tumors compared to non-muscle invasive tumors (P = .007 and P = .009, respectively). Although a trend for negative prognostic impact on overall survival for higher phosphorylated mTOR expression was noted (P = .051), markers expression levels failed to predict survival in our cohort. We found dysregulation of mTOR pathway members in urinary bladder plasmacytoid urothelial carcinoma, suggesting that the use of mTOR pathway inhibitors might be beneficial for patients with this aggressive tumor.
PMCID: PMC3742093  PMID: 23084634
Mammalian target of rapamycin; PTEN; Plasmacytoid urothelial carcinoma; Bladder
The Prostate  2011;72(10):1133-1139.
To evaluate the prognostic significance of six epigenetic biomarkers (AIM1, CDH1, KIF1A, MT1G, PAK3 and RBM6 promoter hypermethlation) in a homogeneous group of prostate cancer patients, following radical prostatectomy.
Patients and Methods
Biomarker analyses were performed retrospectively on tumors from 95 prostate cancer patients all with a Gleason score of 3+4=7 and a minimum follow up period of 8 years. Using Quantitative Methylation Specific PCR (QMSP), we analyzed the promoter region of six genes in primary prostate tumor tissues. Time to any progression was the primary endpoint and development of metastatic disease and/or death from prostate cancer was a secondary endpoint. The association of clinicopathological and biomolecular risk factors to recurrence was performed using the Log-rank test and Cox proportional hazards model for multivariate analysis. To identify independent prognostic factors, a stepwise selection method was used.
At a median follow-up time of 10 years, 48 patients (50.5%) had evidence of recurrence: biochemical/PSA relapse, metastases, or death from prostate cancer. In the final multivariate analysis for time to progression, the significant factors were: older age, HR=0.95 (95% CI: 0.91, 1.0) (P=0.03), positive lymph nodes HR=2.11 (95%CI: 1.05, 4.26) (P=0.04) and decreased hypermethylation of AIM1 HR=0.45 (95%CI: 0.2, 1.0) (P=0.05).
Methylation status of AIM1 in the prostate cancer specimen may predict for time to recurrence in Gleason 3+4=7 patients undergoing prostatectomy. These results should be validated in a larger and unselected cohort.
PMCID: PMC3360823  PMID: 22127895
20.  Evaluation of GSTP1 and APC methylation as indicators for repeat biopsy in a high-risk cohort of men with negative initial prostate biopsies 
Bju International  2011;110(1):56-62.
To evaluate the performance of DNA methylation biomarkers in the setting of repeat biopsy in men with an initially negative prostate biopsy but a high index of suspicion for missed prostate cancer.
We prospectively evaluated 86 men with an initial histologically negative prostate biopsy and high-risk features.
All men underwent repeat 12-core ultrasonography-guided biopsy.
DNA methylation of glutathione-S-transferase P1 (GSTP1) and adenomatous polyposis coli (APC) was determined using tissue from the initially negative biopsy and compared with histology of the repeat biopsy.
The primary outcome was the relative negative predictive value (NPV) of APC compared with GSTP1, and its 95% confidence interval (CI).
On repeat biopsy, 21/86 (24%) men had prostate cancer.
APC and GSTP1 methylation ratios below the threshold (predicting no cancer) produced a NPV of 0.96 and 0.80, respectively. The relative NPV was 1.2 (95% CI: 1.06–1.36), indicating APC has significantly higher NPV.
Methylation ratios above the threshold yielded a sensitivity of 0.95 for APC and 0.43 for GSTP1.
Combining both methylation markers produced a performance similar to that of APC alone.
APC methylation patterns were consistent with a possible field effect or occurrence early in carcinogenesis.
APC methylation provided a very high NPV with a low percentage of false-negatives, in the first prospective study to evaluate performance of DNA methylation markers in a clinical cohort of men undergoing repeat biopsy.
The potential of APC methylation to reduce unnecessary repeat biopsies warrants validation in a larger prospective cohort.
PMCID: PMC3397791  PMID: 22077694
prostate cancer; biopsy; methylation; APC; GSTP1
21.  Evolution of the clinical presentation of men undergoing radical prostatectomy for high-risk prostate cancer 
Bju International  2011;109(7):988-993.
To investigate the outcomes and potential effect of improved longitudinal screening in men presenting with high-risk (advanced clinical stage [> T2b], Gleason score 8–10 or prostate-specific antigen [PSA] level > 20 ng/mL) prostate cancer (PC).
Patients and methods
The Institutional Review Board approved, Institutional Radical Prostatectomy Database (1992–2010) was queried for men with high-risk PC based on D’Amico criteria.
Year of surgery was divided into two cohorts: the Early PSA Era (EPE, 1992–2000) and the Contemporary PSA Era (CPE, 2001–2010).
PC features and outcomes were evaluated using appropriate comparative tests.
In total, 667 men had high-risk PC in the EPE and 764 in the CPE.
In the EPE, 598 (89.7%) men presented with one high-risk feature; 173 (29.0%) men had a Gleason score of 8–10 on biopsy. In the CPE, 717 (93.9%) men presented with one high-risk feature (P = 0.004) and 494 (68.9%) men had a Gleason score of 8–10.
At 10 years, biochemical-free survival (BFS) was 44.1% and 36.4% in the EPE and CPE, respectively (P = 0.04); metastases-free survival (MFS) was 77.1% and 85.1% (P = 0.6); and PC-specific survival (CSS) was 83.3% and 96.2% (P = 0.5).
BFS, MFS and CSS were worse for men with more than one high-risk feature in both eras.
Over the PSA era, an increasing percentage of men with high-risk PC were categorized by a biopsy Gleason score of 8–10.
The accumulation of multiple high-risk features increases the risk of biochemical recurrence, the development of metastases and death from PC.
BFS, MFS and CSS are stable over the PSA era for these men. The balance between a greater proportion of men having high Gleason disease and a greater proportion with small, less advanced tumours may explain the stability in MFS and CSS over time.
PMCID: PMC3235267  PMID: 21880104
high-risk; outcomes; prostate cancer
22.  Topoisomerase II α Status in Renal Medullary Carcinoma: Immuno-Expression and Gene Copy Alterations of a Potential Target of Therapy 
The Journal of urology  2009;182(2):735-740.
Renal medullary carcinoma is an aggressive renal neoplasm without currently available effective therapy to our knowledge. Topoisomerase II α is a gyrase involved in cell proliferation, and DNA maintenance and repair. Topoisomerase II α is a target of inhibiting agents such as anthracyclines. Triggered by a recent response to topoisomerase II α inhibitors in a patient with renal medullary carcinoma, we evaluated topoisomerase II α expression in relation to the proliferation index and topoisomerase II α gene copy number status in a larger series of patients with renal medullary carcinoma.
Materials and Methods
Archival tissues from 14 renal medullary carcinomas were retrieved from our 3 institutions. Immunohistochemistry was performed using monoclonal antibodies for topoisomerase II α and Ki67. The percent of cells with positive nuclear staining was assessed in the highest area of expression for each marker. A previously suggested greater than 5% cutoff was used for topoisomerase II α over expression. The topoisomerase II α gene copy number was evaluated using fluorescence in situ hybridization. Locus specific topoisomerase II α gene and chromosome 17 centromere probes were used. The total number of topoisomerase II α and chromosome 17 centromere signals was counted in 150 cells per tumor and a topoisomerase II α-to-chromosome 17 centromere signal ratio was calculated in each tumor. A topoisomerase II α-to-chromosome 17 centromere ratio of 2.0 or greater and less than 0.8 was used as a cutoff for amplification and deletion, respectively. The percent of tumor cells with polysomic, eusomic or monosomic chromosome 17 status was also determined.
On immuno-expression analysis topoisomerase II α immunohistochemistry was technically inconclusive in 1 renal medullary carcinoma. Topoisomerase II α was over expressed in 11 of 13 renal medullary carcinomas (85%) (median 50%, range 1% to 80%). As expected, a high Ki67 proliferation index was noted in 13 of 14 tumors (median 87.5%, range 2% to 100%). Ki67 expression was greater than topoisomerase II α expression in all 13 informative tumors. A strong, statistically significant correlation was found for topoisomerase II α and Ki67 expression (pairwise CC 0.9, p = 0.0000). Topoisomerase II α over expression was associated with shorter survival (p = 0.000). On fluorescence in situ hybridization no topoisomerase II α amplification was detected in any of the 14 renal medullary carcinomas, including the 11 with topoisomerase II α over expression. Topoisomerase II α gene deletions were noted in 4 tumors. Two of 4 deletions were associated with chromosome 17 monosomy and 2 were in eusomic chromosome 17 tumors.
Topoisomerase II α is over expressed in 85% of renal medullary carcinomas, potentially supporting the use of topoisomerase II α inhibitor agents to treat this aggressive renal tumor. Our findings suggest that topoisomerase II α over expression in our renal medullary carcinoma cohort was not due to gene amplification, but rather to transcriptional or post-transcriptional modifications. The significance of the incidentally found topoisomerase II α deletions in 28% of renal medullary carcinomas requires further evaluation.
PMCID: PMC3505671  PMID: 19539329
kidney; carcinoma; kidney medulla; DNA topoisomerase II alpha; gene deletion
23.  PAX8 (+)/p63 (−) Immunostaining Pattern in Renal Collecting Duct Carcinoma (CDC) 
Collecting duct carcinoma (CDC) is a relatively rare but aggressive type of renal malignancy with variable morphologic features. One of the World Health Organization diagnostic criteria for CDC is the exclusion of urothelial carcinoma of renal pelvis from the differential diagnosis. PAX8 is a novel lineage restricted transcription factor expressed in renal tubules. We investigated the expression pattern of PAX8 in CDC and its utility, in combination with p63, in resolving the differential diagnosis of CDC versus upper tract urothelial carcinoma (UUC).
Archival tissues from 21 CDC and 34 UUC were retrieved from our institutional files. Immunohistochemistry for PAX8 and p63 were performed on routine and tissue microarray sections using standard immunohistochemistry protocol. Intensity of nuclear staining was evaluated for each marker and assigned an incremental 0, 1+, 2+, and 3+ score. Extent of staining was categorized as focal (<25%), nonfocal (25% to 75%), or diffuse (>75%).
CDC: All 21 (100%) CDC were positive for PAX8. Intensity of expression was moderate to strong (2+/3+) in 19 cases (90%). Extent of staining was diffuse in 13 of 21 tumors. The p63 was positive in 3 of 21 (14%) CDC cases (PAX8+/p63+). UUC: The 34 UUC included 5 pT1, 4 pT2, and 25 pT3/pT4 tumors. Thirty-one of 34 (91.2%) UUC were negative for PAX8, whereas 33 of 34 (97%) were p63 positive. Staining intensity was moderate in 15 cases (44%), of which 12 were nonfocal or diffuse. The unique p63-negative UUC was a pT1 tumor that was also negative for PAX8 (PAX8−/p63−).
We propose the use of the combination of PAX8 and p63 in the diagnosis of poorly differentiated renal sinus epithelial neoplasms where the differential diagnosis includes CDC versus UUC. The immunoprofile of PAX8+/p63− supports the diagnosis of CDC with a sensitivity of 85.7% and a specificity of 100%. In contrast, a (PAX8−/p63+) profile supports the diagnosis of UUC with a sensitivity of 88.2% and a specificity of 100%. The inverse PAX8/p63 expression seen in CDC and UUC supports a renal tubular rather than an urothelial differentiation in CDC given the nephric lineage restriction of PAX8.
PMCID: PMC3505675  PMID: 20463571
collecting duct carcinoma; urothelial carcinoma; PAX8; p63
24.  ERG gene rearrangements are common in prostatic small cell carcinomas 
Small cell carcinoma of the prostate is a rare subtype with an aggressive clinical course. Despite the frequent occurrence of ERG gene rearrangements in acinar carcinoma, the incidence of these rearrangements in prostatic small cell carcinoma is unclear. In addition, molecular markers to distinguish prostatic small cell carcinomas from lung and bladder small cell carcinomas may be clinically useful. We examined the occurrence of ERG gene rearrangements by fluorescence in situ hybridization in prostatic, bladder and lung small cell carcinomas. We also examined the expression of ERG, androgen receptor (AR) and NKX3-1 by immunohistochemistry in prostatic cases. Overall, 45% (10/22) of prostatic small cell carcinoma cases harbored ERG rearrangements, whereas no cases of bladder or lung small cell carcinomas showed ERG rearrangement (0/12 and 0/13, respectively). Of prostatic small cell carcinoma cases, 80% (8/10) showed ERG deletion and 20% (2/10) showed ERG translocation. In 83% (5/6) of prostatic small cell carcinoma cases in which a concurrent conventional prostatic acinar carcinoma component was available for analysis, there was concordance for the presence/absence of ERG gene rearrangement between the different subtypes. ERG, AR and NKX3-1 protein expression was detected in a minority of prostatic small cell carcinoma cases (23, 27 and 18%, respectively), while these markers were positive in the majority of concurrent acinar carcinoma cases (66, 83 and 83%, respectively). The presence of ERG rearrangements in nearly half of the prostatic small cell carcinomas is a similar rate of rearrangement to that found in prostatic acinar carcinomas. Furthermore, the high concordance rate of ERG rearrangement between the small cell and acinar components in a given patient supports a common origin for these two subtypes of prostate cancer. Finally, the absence of ERG rearrangement in bladder or lung small cell carcinomas highlights the utility of detecting ERG rearrangement in small cell carcinomas of unknown primary for establishing prostatic origin.
PMCID: PMC3484363  PMID: 21336263
androgen receptor; ERG; gene fusion; NKX3-1; prostatic adenocarcinoma; small cell carcinoma; TMPRSS2-ERG
25.  Immunohistochemical Panel to Identify the Primary Site of Invasive Micropapillary Carcinoma 
Invasive micropapillary carcinoma (IMC) is generally an aggressive morphologic variant that has been described in the bladder, lung, breast, salivary gland, gastrointestinal tract, and ovary. Given the morphologic similarities between IMCs arising from different organ systems and the high propensity of this histologic subtype for lymphatic metastasis, it may be necessary to use immunohistochemical (IHC) markers to determine the primary site of an IMC. Few studies have compared the IHC profiles of IMCs originating from different sites. We tested a panel of 11 IHC markers for their ability to distinguish urothelial, lung, breast, and ovarian IMC using a tissue microarray constructed with primary tumor tissue from 47 patients with IMC (13 bladder, 6 lung, 16 breast, and 12 ovarian). For each tumor, correct classification as IMC was verified by reverse polarity MUC1 expression. We found that immunostaining for uroplakin, CK20, TTF-1, estrogen receptor (ER), WT-1 and/or PAX8, and mammaglobin was the best panel for determining the most likely primary site of IMC. The best markers to identify urothelial IMC were uroplakin and CK20, whereas p63, high molecular weight cytokeratin, and thrombomodulin were less sensitive and specific. Lung IMC was uniformly TTF-1 positive. Breast IMC was ER positive, mammaglobin positive, and PAX8/WT-1 negative, while ovarian IMC was ER positive, mammaglobin negative, and PAX8/WT-1 positive. In the metastatic setting, or when IMC occurs without an associated in situ or conventional carcinoma component, staining for uroplakin, CK20, TTF-1, ER and WT-1, and/or PAX8, and mammaglobin is the best panel for accurately classifying the likely primary site of IMC.
PMCID: PMC3484367  PMID: 19238079
micropapillary carcinoma; urothelial; bladder; breast; lung; ovary; immunohistochemistry

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