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Find video protocols related to scientific articles indexed in Pubmed.
Prostate adenocarcinomas aberrantly expressing p63 are molecularly distinct from usual-type prostatic adenocarcinomas.
Mod. Pathol.
PUBLISHED: 04-28-2014
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We have described a rare group of prostate adenocarcinomas that show aberrant expression of p63, a protein strongly expressed in prostatic basal cells and absent from usual-type acinar prostate cancers. The partial basal-like immunophenotype of these tumors is intriguing in light of the persistent debate surrounding the cell-of-origin for prostate cancer; however, their molecular phenotype is unknown. We collected 37 of these tumors on radical prostatectomy and biopsy and assessed subsets for a diverse panel of molecular markers. The majority of p63-expressing tumors were positive for the ?Np63 isoform (6/7) by immunofluorescence and p63 mRNA (7/8) by chromogenic in situ hybridization. Despite p63 positivity, these tumors uniformly expressed luminal-type cytokeratin proteins such as CK18 (13/13), CK8 (8/8), and markers of androgen axis signaling commonly seen in luminal cells, including androgen receptor (10/11), NKX3.1 (8/8), and prostein (12/13). Conversely, basal cytokeratins such as CK14 and CK15 were negative in all cases (0/8) and CK5/6 was weakly and focally positive in 36% (4/11) of cases. Pluripotency markers including ?-catenin, Oct4, and c-kit were negative in p63-expressing tumors (0/11). Despite nearly universal expression of androgen receptor and downstream androgen signaling targets, p63-expressing tumors lacked ERG rearrangements by fluorescence in situ hybridization (0/14) and ERG protein expression (0/37). No tumors expressed SPINK1 or showed PTEN protein loss (0/19). Surprisingly, 74% (14/19) of p63-expressing tumors expressed GSTP1 protein at least focally, and 33% (2/6) entirely lacked GSTP1 CpG island hypermethylation by bisulfite sequencing. In contrast to usual prostatic adenocarcinomas, prostate tumors with p63 expression show a mixed luminal/basal immunophenotype, uniformly lack ERG gene rearrangement, and frequently express GSTP1. These data strongly suggest that p63-expressing prostate tumors represent a molecularly distinct subclass and further study of this rare tumor type may yield important insights into the role of p63 in prostatic biology and the prostate cancer cell-of-origin.Modern Pathology advance online publication, 12 September 2014; doi:10.1038/modpathol.2014.115.
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t(6;11) renal cell carcinoma (RCC): expanded immunohistochemical profile emphasizing novel RCC markers and report of 10 new genetically confirmed cases.
Am. J. Surg. Pathol.
PUBLISHED: 03-13-2014
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Renal cell carcinomas (RCCs) harboring the t(6;11)(p21;q12) translocation were first described in 2001 and recently recognized by the 2013 International Society of Urological Pathology Vancouver Classification of Renal Neoplasia. Although these RCCs are known to label for melanocytic markers HMB45 and Melan A and the cysteine protease cathepsin K by immunohistochemistry (IHC), a comprehensive IHC profile has not been reported. We report 10 new t(6;11) RCCs, all confirmed by break-apart TFEB fluorescence in situ hybridization. A tissue microarray containing 6 of these cases and 7 other previously reported t(6;11) RCCs was constructed and immunolabeled for 21 different antigens. Additional whole sections of t(6;11) RCC were labeled with selected IHC markers. t(6;11) RCC labeled diffusely and consistently for cathepsin K and Melan A (13 of 13 cases) and almost always at least focally for HMB45 (12 of 13 cases). They labeled frequently for PAX8 (14 of 23 cases), CD117 (10 of 14 cases), and vimentin (9 of 13 cases). A majority of cases labeled at least focally for cytokeratin Cam5.2 (8 of 13 cases) and CD10 and RCC marker antigen (10 of 14 cases each). In contrast to a prior study's findings, only a minority of cases labeled for Ksp-cadherin (3 of 19 cases). The median H score (product of intensity score and percentage labeling) for phosphorylated S6, a marker of mTOR pathway activation, was 101, which is high relative to most other RCC subtypes. In summary, IHC labeling for PAX8, Cam5.2, CD10, and RCC marker antigen supports classification of the t(6;11) RCC as carcinomas despite frequent negativity for broad-spectrum cytokeratins and EMA. Labeling for PAX8 distinguishes the t(6;11) RCC from epithelioid angiomyolipoma, which otherwise shares a similar immunoprofile. CD117 labeling is more frequent in the t(6;11) RCC compared with the related Xp11 translocation RCC. Increased pS6 expression suggests a possible molecular target for the uncommon t(6;11) RCCs that metastasize.
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PTEN loss is associated with upgrading of prostate cancer from biopsy to radical prostatectomy.
Mod. Pathol.
PUBLISHED: 02-14-2014
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When distinguishing between indolent and potentially harmful prostate cancers, the Gleason score is the most important variable, but may be inaccurate in biopsies due to tumor under-sampling. This study investigated whether a molecular feature, PTEN protein loss, could help identify which Gleason score 6 tumors on biopsy are likely to be upgraded at radical prostatectomy. Seventy one patients with Gleason score 6 tumors on biopsy upgraded to Gleason score 7 or higher at prostatectomy (cases) were compared with 103 patients with Gleason score 6 on both biopsy and prostatectomy (controls). A validated immunohistochemical assay for PTEN was performed, followed by fluorescence in situ hybridization (FISH) to detect PTEN gene deletion in a subset. PTEN protein loss and clinical-pathologic variables were assessed by logistic regression. Upgraded patients were older than controls (61.8 vs 59.3 years), had higher pre-operative PSA levels (6.5 vs 5.3?ng/ml) and a higher fraction of involved cores (0.42 vs 0.36). PTEN loss by immunohistochemistry was found in 18% (13/71) of upgraded cases compared with 7% (7/103) of controls (P=0.02). Comparison between PTEN immunohistochemistry and PTEN FISH showed the assays were highly concordant, with 97% (65/67) of evaluated biopsies with intact PTEN protein lacking PTEN gene deletion, and 81% (13/16) of the biopsies with PTEN protein loss showing homozygous PTEN gene deletion. Tumors with PTEN protein loss were more likely to be upgraded at radical prostatectomy than those without loss, even after adjusting for age, preoperative PSA, clinical stage and race (odds ratio=3.04 (1.08-8.55; P=0.035)). PTEN loss in Gleason score 6 biopsies identifies a subset of prostate tumors at increased risk of upgrading at radical prostatectomy. These data provide evidence that a genetic event can improve Gleason score accuracy and highlight a path toward the clinical use of molecular markers to augment pathologic grading.Modern Pathology advance online publication, 4 July 2014; doi:10.1038/modpathol.2014.85.
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Rb Loss is Characteristic of Prostatic Small Cell Neuroendocrine Carcinoma.
Clin. Cancer Res.
PUBLISHED: 12-09-2013
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Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly common with recent advances in pharmacologic androgen suppression. Thus, developing molecular markers of small cell differentiation in prostate cancer will be important to guide diagnosis and therapy of this aggressive tumor.
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USP22 Regulates Oncogenic Signaling Pathways to Drive Lethal Cancer Progression.
Cancer Res.
PUBLISHED: 11-06-2013
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Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease. Cancer Res; 74(1); 1-15. ©2013 AACR.
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GABAA receptor transmembrane amino acids are critical for alcohol action: disulfide crosslinking and alkyl methanethiosulfonate labeling reveal relative location of binding sites.
J. Neurochem.
PUBLISHED: 09-10-2013
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Alcohols and inhaled anesthetics modulate GABAA receptor (GABAA R) function via putative binding sites within the transmembrane regions (TMs). The relative position of the amino acids lining these sites could be either inter- or intra-subunit. We introduced cysteines in relevant TM locations and tested the proximity of cysteine pairs using oxidizing and reducing agents to induce or break disulfide bridges between cysteines, and thus change GABA-mediated currents in wild-type and mutant ?1?2?2 GABAA Rs expressed in Xenopus laevis oocytes. We tested for: (1) inter-subunit crosslinking: a cysteine located in either ?1TM1 [?1(Q229C) or ?1(L232C)] was paired with a cysteine in different positions of ?2TM2 and TM3; (2) intra-subunit crosslinking: a cysteine located in either in ?2TM1 [?2(T225C)] or TM2 [?2(N265C)] was paired with a cysteine in different locations along ?2TM3. Three inter-subunit cysteine pairs and four intra-subunits crosslinked. In three intra-subunit cysteine combinations, the alcohol effect was reduced by oxidizing agents, suggesting intra-subunit alcohol binding. We conclude that the structure of the alcohol binding site changes during activation and that potentiation or inhibition by binding at inter- or intra-subunit sites is determined by the specific receptor and ligand. This article is protected by copyright. All rights reserved.
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Tissue microarray analysis of cyclin-dependent kinase inhibitors p21 and p16 in Fuchs dystrophy.
Cornea
PUBLISHED: 09-06-2013
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To investigate the novel application of tissue microarray (TMA) technology to corneal disease and to report altered protein expression of senescence-associated cyclin-dependent kinase inhibitors p21 and p16 in Fuchs endothelial corneal dystrophy (FECD).
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Prostate cancer cell telomere length variability and stromal cell telomere length as prognostic markers for metastasis and death.
Cancer Discov
PUBLISHED: 06-18-2013
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Current prognostic indicators are imperfect predictors of outcome in men with clinically localized prostate cancer. Thus, tissue-based markers are urgently needed to improve treatment and surveillance decision-making. Given that shortened telomeres enhance chromosomal instability and such instability is a hallmark of metastatic lesions, we hypothesized that alterations in telomere length in the primary cancer would predict risk of progression to metastasis and prostate cancer death. To test this hypothesis, we conducted a prospective cohort study of 596 surgically treated men who participated in the ongoing Health Professionals Follow-up Study. Men who had the combination of more variable telomere length among prostate cancer cells (cell-to-cell) and shorter telomere length in prostate cancer-associated stromal (CAS) cells were substantially more likely to progress to metastasis or die of their prostate cancer. These findings point to the translational potential of this telomere biomarker for prognostication and risk stratification for individualized therapeutic and surveillance strategies.
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Activation of mammalian target of rapamycin signaling pathway markers in minute adenocarcinoma of the prostate.
Urology
PUBLISHED: 05-30-2013
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To asses the mammalian target of rapamycin (mTOR) pathway in minute prostatic adenocarcinoma on the basis of the previously reported role of phosphatase and tensin homolog (PTEN) inactivation and mTOR pathway activation as a negative prognosticator in prostatic cancer.
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Dysregulation of mammalian target of rapamycin pathway in upper tract urothelial carcinoma.
Hum. Pathol.
PUBLISHED: 04-30-2013
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Upper tract urothelial carcinoma (UTUC) accounts for 5% to 10% of all urothelial carcinomas. Despite many shared features, key clinical and molecular genetic differences between upper tract and bladder urothelial carcinomas are becoming apparent. We have previously demonstrated alterations of mammalian target of rapamycin (mTOR) pathway in bladder carcinoma with a potential impact on biological behavior. In the current study, we evaluated the expression status and prognostic significance of mTOR pathway members in UTUC. Archival formalin-fixed and paraffin-embedded tissues from 99 primary UTUCs were retrieved from one of the authors institution. Tissue microarrays were constructed with triplicate tumor samples and paired nonneoplastic urothelium. Tissue microarrays were analyzed using immunohistochemistry for mTOR pathway members: PTEN, phos-AKT, phos-mTOR, phos-S6, phos-4EBP1, and related markers p27 and c-MYC; correlation with clinicopathologic parameters and outcome was performed. We found significantly lower expression of PTEN, phos-AKT, phos-mTOR, phos-S6, phos-4EBP1, p27, and c-MYC in UTUC compared with paired benign urothelium (P < .0005). We found a strong positive correlation between PTEN and phos-AKT. Moderate correlation was observed between phos-mTOR and phos-S6, PTEN and p27, phos-AKT and p27, phos-S6 and p27, phos-mTOR and c-MYC, phos-S6 and c-MYC, and p27 and c-MYC. None of the evaluated biomarkers were associated with increased hazard ratios for tumor recurrence or for cancer-specific mortality, when adjusting for relevant clinicopathologic variables. Dysregulation of the mTOR pathway was observed in UTUC compared with normal urothelium, implicating a potential pathogenic role in tumor development. In our cohort, expression of the evaluated biomarkers had no prognostic value.
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Relevance of the mammalian target of rapamycin pathway in the prognosis of patients with high-risk non-muscle invasive bladder cancer.
Hum. Pathol.
PUBLISHED: 04-23-2013
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High-risk non-muscle invasive bladder cancer (NMIBC) is associated with higher rates of recurrence and progression. Molecular markers within aberrant signaling pathways in cancer need further evaluation of their role as prognostic indicators and potential future targets for prevention of recurrence. Our objective was to investigate the role of the mammalian target of rapamycin (mTOR) signaling pathway on the stage and outcome of patients with high-risk NMIBC. Tissue microarrays were built from archival bladder tumor specimens (n = 142). Various clinicopathologic variables were collected retrospectively from patients treated with transurethral resection. Immunohistochemical staining was performed for phosphatase and tensin homolog, phosphorylated Akt, phosphorylated mTOR, phosphorylated S6 (p-S6), eukaryotic translation initiation factor 4E-binding protein-1, and p27. Multivariate analysis using Cox regression models addressed recurrence-free survival (RFS), progression-free survival, and worsening-free survival. In multivariate analysis, p-S6 was an independent predictor of shorter RFS (hazard ratio, 3.55; 95% CI, 1.31-9.64). Expression of p27 was inversely correlated with RFS (hazard ratio, 0.27; 95% CI, 0.10-0.74). Low levels of phosphatase and tensin homolog expression were associated with worsening-free survival (P < .03). None of the markers showed correlation with progression-free survival. Our results demonstrate that activation of the mTOR pathway, as assessed by p-S6 and expression of p27, might be used to provide prognostic information, particularly as a predictor of recurrence among patients with high-risk NMIBC.
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Dysregulation of the mammalian target of rapamycin pathway in chromophobe renal cell carcinomas.
Hum. Pathol.
PUBLISHED: 04-17-2013
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Targeted therapy in advanced clear cell renal cell carcinomas (RCC) is now an established modality. The latter is in stark contrast to non-clear cell subtypes. We explored the translational support for the use of antagonists of the mammalian target of rapamycin (mTOR) and the vascular endothelial growth factor pathways in chromophobe RCC. The immunoexpression of PTEN, phos-AKT, phosphorylated S6 (phos-S6), 4EBP1, p27, c-MYC, and HIF-1? was evaluated in 33 patients with chromophobe RCC who were treated by partial/radical nephrectomy without adjuvant therapy. PTEN was lower in tumor than in normal kidney (P<.001), and loss of PTEN expression was found in 67% of the tumors. In tumor tissues, phos-S6 and 4EBP1 were higher than in normal kidney (P?.005). Conversely, scores of p27 were lower in tumor than in normal kidney (P<.001). Finally, scores of phos-AKT, c-MYC, and HIF-1? were not significantly different in tumor and in normal kidney. Overall mortality and cancer-specific mortality were 9% and 0%, respectively. Multifocal tumors had higher levels of PTEN, phos-AKT, and HIF-1? (P?.01). None of the clinicopathologic variables (age, ethnicity, gender, pT stage, tumor size, multifocality, and positive surgical margins) was associated with outcome. Similarly, none of the tested biomarkers predicted overall mortality, either in unadjusted or adjusted Cox regression models. In summary, our study provides new evidence of dysregulation of the mTOR pathway in chromophobe RCC. Immunohistochemistry for mTOR pathway and hypoxia-induced pathway members lacked prognostic significance in our cohort.
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Tracking the clonal origin of lethal prostate cancer.
J. Clin. Invest.
PUBLISHED: 04-09-2013
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Recent controversies surrounding prostate cancer overtreatment emphasize the critical need to delineate the molecular features associated with progression to lethal metastatic disease. Here, we have used whole-genome sequencing and molecular pathological analyses to characterize the lethal cell clone in a patient who died of prostate cancer. We tracked the evolution of the lethal cell clone from the primary cancer to metastases through samples collected during disease progression and at the time of death. Surprisingly, these analyses revealed that the lethal clone arose from a small, relatively low-grade cancer focus in the primary tumor, and not from the bulk, higher-grade primary cancer or from a lymph node metastasis resected at prostatectomy. Despite being limited to one case, these findings highlight the potential importance of developing and implementing molecular prognostic and predictive markers, such as alterations of tumor suppressor proteins PTEN or p53, to augment current pathological evaluation and delineate clonal heterogeneity. Furthermore, this case illustrates the potential need in precision medicine to longitudinally sample metastatic lesions to capture the evolving constellation of alterations during progression. Similar comprehensive studies of additional prostate cancer cases are warranted to understand the extent to which these issues may challenge prostate cancer clinical management.
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A subset of malignant phyllodes tumors harbors alterations in the Rb/p16 pathway.
Hum. Pathol.
PUBLISHED: 04-09-2013
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Breast phyllodes tumors are fibroepithelial neoplasms with variable risk of aggressive local recurrence and distant metastasis, and the molecular pathogenesis is unclear. Here, we systematically study p16 and Rb expression in 34 phyllodes tumors in relation to proliferation. Tissue microarrays were constructed from 10 benign, 10 borderline, and 14 malignant phyllodes (5 cores/tumor) and from 10 fibroadenomas (2 cores/tumor). Tissue microarrays were labeled by immunohistochemistry for p16, Rb, and Ki-67 and by in situ hybridization for high-risk human papillomavirus. Cytoplasmic and nuclear p16 were scored by percentage labeling (0%-100%, diffuse >95%) and intensity. Nuclear Rb was scored by percentage labeling (0%-100%, diffuse >75%) and intensity. p16 and Rb labeling were repeated on whole sections of cases with Rb loss on the tissue microarray. Twenty-nine percent (4/14) malignant phyllodes showed diffuse strong p16 labeling with Rb loss in malignant cells (diffuse p16+/Rb-), whereas 21% (3/14) malignant phyllodes showed the reverse pattern of p16 loss with diffuse strong Rb (p16-/diffuse Rb+). Results were consistent between tissue microarrays and whole sections. No borderline phyllodes, benign phyllodes, or fibroadenoma showed diffuse p16+/Rb- or p16-/diffuse Rb+ phenotypes. No cases contained high-risk human papillomavirus. Average Ki-67 proliferation indices were 15% in malignant phyllodes, 1.7% in borderline phyllodes, 0.5% in benign phyllodes, and 0% in fibroadenoma. Ki-67 was highest in malignant phyllodes with diffuse p16+/Rb- labeling. In summary, 50% malignant phyllodes display evidence of Rb/p16 pathway alterations, likely reflecting p16 or Rb inactivation. These and other mechanisms may contribute to the increased proliferation in malignant phyllodes relative to other fibroepithelial neoplasms.
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Nucleotide resolution analysis of TMPRSS2 and ERG rearrangements in prostate cancer.
J. Pathol.
PUBLISHED: 02-08-2013
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TMPRSS2-ERG rearrangements occur in approximately 50% of prostate cancers and therefore represent one of the most frequently observed structural rearrangements in all cancers. However, little is known about the genomic architecture of such rearrangements. We therefore designed and optimized a pipeline involving target capture of TMPRSS2 and ERG genomic sequences coupled with paired-end next-generation sequencing to resolve genomic rearrangement breakpoints in TMPRSS2 and ERG at nucleotide resolution in a large series of primary prostate cancer specimens (n = 83). This strategy showed > 90% sensitivity and specificity in identifying TMPRSS2-ERG rearrangements, and allowed identification of intra- and inter-chromosomal rearrangements involving TMPRSS2 and ERG with known and novel fusion partners. Our results indicate that rearrangement breakpoints show strong clustering in specific intronic regions of TMPRSS2 and ERG. The observed TMPRSS2-ERG rearrangements often exhibited complex chromosomal architecture associated with several intra- and inter-chromosomal rearrangements. Nucleotide resolution analysis of breakpoint junctions revealed that the majority of TMPRSS2 and ERG rearrangements (~88%) occurred at or near regions of microhomology or involved insertions of one or more base pairs. This architecture implicates non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) pathways in the generation of such rearrangements. These analyses have provided important insights into the molecular mechanisms involved in generating prostate cancer-specific recurrent rearrangements.
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Immunohistochemical evaluation of TMPRSS2-ERG gene fusion in adenosis of the prostate.
Hum. Pathol.
PUBLISHED: 01-30-2013
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Adenosis (atypical adenomatous hyperplasia) is a benign lesion that morphologically mimics prostate adenocarcinoma, although the relationship between these 2 lesions is still debated. The TMPRSS2-ERG fusion is a common chromosomal rearrangement that occurs early in the development of invasive adenocarcinoma of the prostate and results in the expression of a truncated ERG protein. This fusion is present in 50% of adenocarcinomas and in 20% of high-grade prostate intraepithelial lesions. Until recently, fluorescent in situ hybridization was the only method available to detect these rearrangements. A specific anti-ERG antibody is now available for detecting ERG protein expression and serves as a useful marker for ERG rearrangements. Formalin-fixed, paraffin-embedded tissue sections of adenosis from cases of prostate biopsies (n = 30), transurethral resections of the prostate (n = 12), and radical prostatectomies (n = 3) were analyzed via immunohistochemistry for ERG. None (0%) of the foci of adenosis were positive for ERG protein expression. Of 40 cases of Gleason score 6 adenocarcinoma on a tissue microarray, 22 (55%) were positive for ERG protein. Of the positive cases, 14 (63.6%) were moderate in intensity, with the remaining 36.4% being weak. The lack of ERG expression in adenosis supports the notion that it is not a precursor lesion of adenocarcinoma. Moreover, it suggests that immunohistochemistry for ERG expression could be a useful tool to differentiate adenosis from adenocarcinoma.
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High levels of phosphatase and tensin homolog expression are associated with tumor progression, tumor recurrence, and systemic metastases in pT1 urothelial carcinoma of the bladder: a tissue microarray study of 156 patients treated by transurethral resect
Urology
PUBLISHED: 01-01-2013
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To evaluate immunohistochemical expression of phosphatase and tensin homolog (PTEN) and mammalian target of rapamycin (mTOR) pathway members in pT1 urothelial carcinomas treated by transurethral resection and to determine if immunohistochemistry can be used to predict prognosis.
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MYC gene amplification is often acquired in lethal distant breast cancer metastases of unamplified primary tumors.
Mod. Pathol.
PUBLISHED: 11-04-2011
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In breast cancer, amplification of MYC is consistently observed in aggressive forms of disease and correlates with poor prognosis and distant metastases. However, to date, a systematic analysis of MYC amplification in metastatic breast cancers has not been reported. Specifically, whether the MYC amplification status may change in metastases in comparison to the corresponding primary breast tumor, and potential variability among different metastases within the same patient have also not been assessed. We generated single patient tissue microarrays consisting of both primary breast carcinomas and multiple matched systemic metastases from 15 patients through our previously described rapid autopsy program. In total, the 15 tissue microarrays contained 145 primary tumor spots and 778 spots derived from 180 different metastases. In addition, two separate tissue microarrays were constructed composed of 10 matched primary breast cancers and corresponding solitary metastases sampled not at autopsy but rather in routine surgical resections. These two tissue microarrays totaled 50 primary tumor spots and 86 metastatic tumor spots. For each case, hormone receptor status, HER2/neu, EGFR and CK5/6 expression were assessed, and the cases were characterized as luminal, basal-like or HER2 based on published criteria. Both fluorescence in situ hybridization and immunohistochemistry for MYC was performed on all cases. Of the 25 cases, 24 were evaluable. While 4 of 24 primary tumors (16%) demonstrated MYC amplification, an additional 6 (25% of total evaluable cases) acquired MYC amplification in their systemic metastases. Of note, there was remarkably little heterogeneity in MYC copy number among different metastases from the same patient. MYC immunoreactivity was increased in metastases relative to matched primaries in the surgical cohort, although there was no perfect correlation with MYC amplification. In conclusion, amplification of MYC is a frequent event in breast cancer, but occurs more frequently as a diffuse, acquired event in metastatic disease than in the corresponding primary. These observations underscore the importance of MYC in breast cancer progression/metastasis, as well as its relevance as a potential therapeutic target in otherwise incurable metastatic disease.
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Immunoexpression status and prognostic value of mTOR and hypoxia-induced pathway members in primary and metastatic clear cell renal cell carcinomas.
Am. J. Surg. Pathol.
PUBLISHED: 09-02-2011
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The need for effective targeted therapies for renal cell carcinomas (RCCs) has fueled the interest for understanding molecular pathways involved in the oncogenesis of kidney tumors. Aiming to analyze the expression status and prognostic significance of mTOR and hypoxia-induced pathway members in patients with clear cell RCC (ccRCC), tissue microarrays were constructed from 135 primary and 41 metastatic ccRCCs. Immunoexpression levels were compared and correlated with clinicopathologic parameters and outcome. PTEN levels were significantly lower in primary and metastatic ccRCCs compared with benign tissues (P<0.001). Levels of phos-AKT, phos-S6, and 4E-binding protein-1 (4EBP1) were higher in metastatic ccRCC (P?0.001). For phos-S6 and 4EBP1, levels were higher in primary ccRCC compared with benign tissues (P<0.001). c-MYC levels were higher in metastatic ccRCC (P<0.0001), and incremental p27 levels were observed in benign, primary ccRCC, and metastatic ccRCC (P<0.0001). HIF-1? levels were significantly higher in primary and metastatic ccRCCs compared with benign tissues (P<0.0001). In primary ccRCC, levels of all mTOR and hypoxia-induced pathway members were significantly associated with pT stage (P?0.036), p27 levels with Fuhrman grade (P=0.031), and 4EBP1, p27, and HIF-1? levels with tumor size (P?0.025). Tumor size, HIF-1?, and phos-S6 levels were associated with disease-specific survival (DSS) (P?0.032) and tumor progression (P?0.043). In conclusion, both mTOR and hypoxia-induced pathways were activated in primary and metastatic ccRCC. PTEN loss seems to be an early event during tumorigenesis. Tumor size, HIF-1?, and phos-S6 expression were found to be independent predictors of both DSS and tumor progression in primary ccRCC.
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PTEN protein loss by immunostaining: analytic validation and prognostic indicator for a high risk surgical cohort of prostate cancer patients.
Clin. Cancer Res.
PUBLISHED: 08-30-2011
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Analytically validated assays to interrogate biomarker status in clinical samples are crucial for personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome FISH spot counting assays, is associated with poor prognosis, few studies have validated immunohistochemistry (IHC) assays to determine whether loss of PTEN protein is associated with unfavorable disease.
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Humanizing ?-class glutathione S-transferase regulation in a mouse model alters liver toxicity in response to acetaminophen overdose.
PLoS ONE
PUBLISHED: 07-22-2011
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Glutathione S-transferases (GSTs) metabolize drugs and xenobiotics. Yet despite high protein sequence homology, expression of ?-class GSTs, the most abundant of the enzymes, varies significantly between species. In mouse liver, hepatocytes exhibit high mGstp expression, while in human liver, hepatocytes contain little or no hGSTP1 mRNA or hGSTP1 protein. ?-class GSTs are known to be critical determinants of liver responses to drugs and toxins: when treated with high doses of acetaminophen, mGstp1/2+/+ mice suffer marked liver damage, while mGstp1/2-/- mice escape liver injury.
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Androgen receptor expression is usually maintained in initial surgically resected breast cancer metastases but is often lost in end-stage metastases found at autopsy.
Hum. Pathol.
PUBLISHED: 06-30-2011
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Androgen receptor (AR) is expressed in approximately 70% of primary breast carcinomas (PBCs) and is a promising therapeutic target for metastatic breast carcinoma (MBC). Here, we examine AR expression in a population of initial surgically resected metastases and a separate cohort of end-stage metastases harvested at autopsy compared with their matched PBCs. Tissue microarrays of matched PBC and MBC were labeled by immunohistochemistry for AR, estrogen receptor (ER), progesterone receptor (PR), and Her2 and classified into the following previously described categories: luminal (ER/PR+/Her2-), triple negative (ER/PR/Her2-), Her2 (ER/PR-/Her2+), and luminal loss (ER/PR loss from primary to metastasis). In the cohort of surgically resected metastases (n = 16), AR was expressed in 12 of 16 PBC and maintained in 11 of 12 corresponding MBCs. Of these, 36% showed stronger AR labeling in the metastases and none showed a decrease. In the cohort of metastases harvested at autopsy (n = 16), AR was expressed in 11 of 16 primary carcinomas and maintained in only 5 of 11 corresponding metastases. Of these, none showed increased AR and 80% showed decreased AR labeling. AR expression is overwhelmingly concordant between matched PBC and MBC at initial presentation. These findings validate AR as a therapeutic target in MBC and suggest that AR may need to be reevaluated in metastases even if the primary is negative. However, similar to ER/PR, AR expression is often decreased with a trend toward complete loss in end-stage metastases, suggesting a shift of AR expression between initial and end-stage metastases. This suggests an opportunity for targeted antiandrogen therapy at an earlier stage of disease progression.
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Immunohistochemistry for ERG expression as a surrogate for TMPRSS2-ERG fusion detection in prostatic adenocarcinomas.
Am. J. Surg. Pathol.
PUBLISHED: 06-17-2011
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TMPRSS2-ERG fusions have been identified in about one-half of all prostatic adenocarcinomas (PCas). Fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction have been the most commonly used techniques in this setting. The aim of this study was to evaluate the utility of ERG immunoexpression as a surrogate for TMPRSS2-ERG fusion in a large series of PCa cases.
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Alterations in nucleolar structure and gene expression programs in prostatic neoplasia are driven by the MYC oncogene.
Am. J. Pathol.
PUBLISHED: 03-26-2011
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Increased nucleolar size and number are hallmark features of many cancers. In prostate cancer, nucleolar enlargement and increased numbers are some of the earliest morphological changes associated with development of premalignant prostate intraepithelial neoplasia (PIN) lesions and invasive adenocarcinomas. However, the molecular mechanisms that induce nucleolar alterations in PIN and prostate cancer remain largely unknown. We verify that activation of the MYC oncogene, which is overexpressed in most human PIN and prostatic adenocarcinomas, leads to formation of enlarged nucleoli and increased nucleolar number in prostate luminal epithelial cells in vivo. In prostate cancer cells in vitro, MYC expression is needed for maintenance of nucleolar number, and a nucleolar program of gene expression. To begin to decipher the functional relevance of this transcriptional program in prostate cancer, we examined FBL (encoding fibrillarin), a MYC target gene, and report that fibrillarin is required for proliferation, clonogenic survival, and proper ribosomal RNA accumulation/processing in human prostate cancer cells. Further, fibrillarin is overexpressed in PIN lesions induced by MYC overexpression in the mouse prostate, and in human clinical prostate adenocarcinoma and PIN lesions, where its expression correlates with MYC levels. These studies demonstrate that overexpression of the MYC oncogene increases nucleolar number and size and a nucleolar program of gene expression in prostate epithelial cells, thus providing a molecular mechanism responsible for hallmark nucleolar alterations in prostatic neoplasia.
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Identification of replication competent murine gammaretroviruses in commonly used prostate cancer cell lines.
PLoS ONE
PUBLISHED: 02-24-2011
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A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral "contamination", much like routine mycoplasma testing.
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ERG gene rearrangements are common in prostatic small cell carcinomas.
Mod. Pathol.
PUBLISHED: 02-18-2011
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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.
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Xp11 translocation renal cell carcinoma (RCC): extended immunohistochemical profile emphasizing novel RCC markers.
Am. J. Surg. Pathol.
PUBLISHED: 08-04-2010
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Xp11 translocation renal cell carcinoma (RCC) harbor various TFE3 gene fusions, and are known to underexpress epithelial immunohistochemical (IHC) markers such as cytokeratin and EMA relative to usual adult type RCC; however, their profile in reference to other IHC markers that are differentially expressed in other subtypes of RCC has not been systematically assessed. Few therapeutic targets have been identified in these aggressive cancers. We created 2 tissue microarrays (TMA) containing five 1.4-mm cores from each of 21 Xp11 translocation RCC (all confirmed by TFE3 IHC, 6 further confirmed by genetics), 7 clear cell RCC (CCRCC), and 6 papillary RCC (PRCC). These TMA were labeled for a panel of IHC markers. In contrast to earlier published data, Xp11 translocation RCC frequently expressed renal transcription factors PAX8 (16/21 cases) and PAX2 (14/21 cases), whereas only 1 of 21 cases focally expressed MiTF and only 5 of 21 overexpressed p21. Although experimental data suggest otherwise, Xp11 translocation RCC did not express WT-1 (0/21 cases). Although 24% of Xp11 translocation RCC expressed HIF-1alpha (like CCRCC), unlike CCRCC CA IX expression was characteristically only focal (mean 6% cell labeling) in Xp11 translocation RCC. Other markers preferentially expressed in CCRCC or PRCC, such as HIG-2, claudin 7, and EpCAM, yielded inconsistent results in Xp11 translocation RCC. Xp11 translocation RCC infrequently expressed Ksp-cadherin (3/21 cases) and c-kit (0/21 cases), markers frequently expressed in chromophobe RCC. Using an H-score that is the product of intensity and percentage labeling, Xp11 translocation RCC expressed higher levels of phosphorylated S6, a measure of mTOR pathway activation (mean H score=88), than did CCRCC (mean H score=54) or PRCC (mean H score=44). In conclusion, in contrast to prior reports, Xp11 translocation RCC usually express PAX2 and PAX8 but do not usually express MiTF. Although they may express HIF-1alpha, they only focally express the downstream target CA IX. They inconsistently express markers associated with other RCC subtypes, further highlighting the lack of specificity of the latter markers. TFE3 and Cathepsin K remain the most sensitive and specific markers of these neoplasms. Elevated expression of phosphorylated S6 in Xp11 translocation RCC suggests the mTOR pathway as an attractive potential therapeutic target for these neoplasms.
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NKX3.1 as a marker of prostatic origin in metastatic tumors.
Am. J. Surg. Pathol.
PUBLISHED: 07-01-2010
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NKX3.1 is a prostatic tumor suppressor gene located on chromosome 8p. Although most studies have shown that staining for NKX3.1 protein is positive in the majority of primary prostatic adenocarcinomas, it has been shown to be downregulated in many high-grade prostate cancers, and completely lost in the majority of metastatic prostate cancers (eg, in 65% to 78% of lesions). A recent study showed that NKX3.1 staining with a novel antibody was highly sensitive and specific for high-grade prostatic adenocarcinoma when compared with high-grade urothelial carcinoma. This raised the question that this antibody may perform better than earlier used antibodies in metastatic prostate tumors. However, the sensitivity and specificity for prostate carcinomas for this antibody in metastatic lesions was not determined. Although prostate-specific antigen (PSA) and prostatic-specific acid phosphatase (PSAP) are excellent tissue markers of prostate cancer, at times they may be expressed at low levels, focally, or not at all in poorly differentiated primary and metastatic prostatic adenocarcinomas. The purpose of this study was to determine the performance of NKX3.1 as a marker of metastatic adenocarcinoma of prostatic origin. Immunohistochemical staining against NKX3.1, PSA, and PSAP was carried out on a tissue microarray (TMA) (0.6-mm tissue cores) of hormone naïve metastatic prostate adenocarcinoma specimens from lymph nodes, bone, and soft tissue. To determine the specificity of NKX3.1 for prostatic adenocarcinoma, we used TMAs that contained cancers from various sites including the urinary bladder, breast, colon, salivary gland, stomach, pancreas, thyroid, and central nervous system, and standard paraffin sections of cancers from other sites including the adrenal cortex, kidney, liver, lung, and testis. Overall 349 nonprostatic tumors were evaluated. Any nuclear staining for NKX3.1 was considered positive and the percentage of cells with nuclear staining and their mean intensity level were assessed visually. Sensitivity was calculated by considering a case positive if any TMA core was positive. The sensitivity for identifying metastatic prostatic adenocarcinomas overall was 98.6% (68/69 cases positive) for NKX3.1, 94.2% (65/69 cores positive) for PSA, and 98.6% (68/69 cores positive) for PSAP. The specificity of NKX3.1 was 99.7% (1/349 nonprostatic tumors positive). The sole positive nonprostatic cancer case was an invasive lobular carcinoma of the breast. NKX3.1 seems to be a highly sensitive and specific tissue marker of metastatic prostatic adenocarcinoma. In the appropriate clinical setting, the addition of IHC staining for NKX3.1, along with other prostate-restricted markers, may prove to be a valuable adjunct to definitively determine prostatic origin in poorly differentiated metastatic carcinomas.
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PAX8 (+)/p63 (-) immunostaining pattern in renal collecting duct carcinoma (CDC): a useful immunoprofile in the differential diagnosis of CDC versus urothelial carcinoma of upper urinary tract.
Am. J. Surg. Pathol.
PUBLISHED: 05-14-2010
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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).
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Mammalian target of rapamycin (mTOR) regulates cellular proliferation and tumor growth in urothelial carcinoma.
Am. J. Pathol.
PUBLISHED: 04-15-2010
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Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive tumor growth in many cancer models, although its role in urothelial carcinoma (UCC) has not been extensively explored. Expression of phosphorylated mTOR (P-mTOR) and a downstream target, ribosomal S6 protein (P-S6), was identified in 74% (90/121) and 55% (66/121) of muscle-invasive UCCs, respectively. P-mTOR intensity and %positive cells were associated with reduced disease-specific survival (P = 0.04, P = 0.08, respectively). Moreover, P-mTOR intensity corresponded to increased pathological stage (P < 0.01), and mTOR activity was associated with cell migration in vitro. In addition, mTOR inhibition via rapamycin administration reduced cell proliferation in UCC cell lines RT4, T24, J82, and UMUC3 in a dose-dependent manner to 6% of control levels and was significant at 1 nmol/L in J82, T24, and RT4 cells (P < 0.01, P < 0.01, P = 0.03, respectively) and at 10 nmol/L in UMUC3 cells (P = 0.03). Reduced proliferation corresponded with reduced P-S6 levels by Western blot, and effects were ablated by pretreatment of cells with mTOR-specific siRNA. No effects of rapamycin on apoptosis were identified by TUNEL labeling or PARP cleavage. Administration of rapamycin to T24-xenografted mice resulted in a 55% reduction in tumor volume (P = 0.03) and a 40% reduction in proliferation (P < 0.01) compared with vehicle-injected mice. These findings indicate that mTOR pathway activation frequently occurs in UCC and that mTOR inhibition may be a potential means to reduce UCC growth.
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Loss of Nkx3.1 expression in bacterial prostatitis: a potential link between inflammation and neoplasia.
Am. J. Pathol.
PUBLISHED: 04-02-2010
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NKX3.1 is a homeodomain protein that functions as a dosage sensitive prostate-specific transcription factor. Diminished NKX3.1 expression is associated with prostate epithelial cell proliferation in vitro and with increasing Gleason grade in patient samples. Mouse Nkx3.1 also functions as a negative regulator of prostate cell growth in prostate cancer models. Identifying biological and environmental factors that modulate NKX3.1 accumulation is therefore central to efforts aimed at elucidating prostate growth control mechanisms. To determine the effect of inflammation on Nxk3.1 accumulation, bacterial prostatitis was induced by intraurethral inoculation of a uropathogenic E. coli strain in mice. Nkx3.1 expression was profoundly reduced in infected prostate lobes and correlated with increased expression of a proliferation marker. Androgen receptor levels were also reduced in concert with Nkx3.1, and a marked increase in the basal cell marker p63 was observed. Analyses of the inflammatory infiltrate revealed a classic acute inflammatory response that attained characteristics of a chronic state within fourteen days postinoculation. Comparison of the four prostate lobes revealed clear differences in the extent of inflammation. These data demonstrate that acute inflammation in response to a bacterial agent in the prostate is associated with a significant diminution in the level of a key regulator of prostate cell proliferation. These observations provide a plausible mechanism whereby prostate inflammation may establish a local environment conducive to epithelial cell growth.
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MYC overexpression induces prostatic intraepithelial neoplasia and loss of Nkx3.1 in mouse luminal epithelial cells.
PLoS ONE
PUBLISHED: 01-26-2010
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Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina-features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These "Super-Lo-MYC" mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a "paradoxical" increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas.
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Expression status and prognostic significance of mammalian target of rapamycin pathway members in urothelial carcinoma of urinary bladder after cystectomy.
Cancer
PUBLISHED: 01-13-2010
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Bladder urothelial carcinoma has high rates of mortality and morbidity. Identifying novel molecular prognostic factors and targets of therapy is crucial. Mammalian target of rapamycin (mTOR) pathway plays a pivotal role in establishing cell shape, migration, and proliferation.
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Shared TP53 gene mutation in morphologically and phenotypically distinct concurrent primary small cell neuroendocrine carcinoma and adenocarcinoma of the prostate.
Prostate
PUBLISHED: 01-07-2009
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Small cell carcinoma of the prostate is an uncommon neoplasm, the origin of which has been controversial. To address this, we performed transcriptome profiling and TP53 sequencing of concurrent small cell and prostatic adenocarcinoma to determine the relationship between these entities.
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Cytoplasmic PTEN protein loss distinguishes intraductal carcinoma of the prostate from high-grade prostatic intraepithelial neoplasia.
Mod. Pathol.
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Intraductal carcinoma of the prostate is a marker of aggressive disease. However, intraductal carcinoma exists on a morphologic continuum with high-grade prostatic intraepithelial neoplasia (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 with 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.
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Dysregulation of mammalian target of rapamycin pathway in plasmacytoid variant of urothelial carcinoma of the urinary bladder.
Hum. Pathol.
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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 phosphorylated AKT and a lower H score 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.
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Global levels of H3K27me3 track with differentiation in vivo and are deregulated by MYC in prostate cancer.
Am. J. Pathol.
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Cancer cells and stem cells share a number of biological characteristics including abundant amounts of decondensed chromatin. However, the molecular correlates and the factors involved in altering chromatin structure in cancer cells are not well known. Here, we report that less differentiated stem-like cells in the basal compartment of human and mouse prostate contain lower levels of the polycomb heterochromatin marker H3K27me3 than more differentiated luminal cells. This link to differentiated normal cells is also found in a number of other human and rodent tissues characterized by hierarchical differentiation. In addition to MYCs traditional role as a gene-specific transcription factor, recent studies indicate that MYC also affects global chromatin structure where it is required to maintain "open" or active chromatin. We now demonstrate that in both MYC-driven prostate cancers in mice and human prostate cancers, global levels of H3K27me3 are reduced in prostatic intraepithelial neoplasia and invasive adenocarcinoma lesions. Moreover, decreased levels of H3K27me3 correlate with increased markers of disease aggressiveness (eg, Gleason score and pathological stage). In vitro, experimentally forced reductions in MYC levels result in increased global levels of H3K27me3. These findings suggest that increased levels of decondensed chromatin in both normal progenitor cells and cancer cells are associated with global loss of H3K27me3, which is linked to MYC overexpression.
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Immunohistochemical evidence of dysregulation of the mammalian target of rapamycin pathway in primary and metastatic pheochromocytomas.
Urology
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To characterize the status of the mammalian target of rapamycin pathway using formalin-fixed, paraffin-embedded specimens from patients with primary and metastatic pheochromocytoma.
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Loss of PTEN expression is associated with increased risk of recurrence after prostatectomy for clinically localized prostate cancer.
Mod. Pathol.
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PTEN (phosphatase and tensin homolog on chromosome 10) is one of the most frequently lost tumor suppressor genes in human cancers and it has been described in more than two-thirds of patients with advanced/aggressive prostate cancer. Previous studies suggest that, in prostate cancer, genomic PTEN loss is associated with tumor progression and poor prognosis. Thus, we evaluated whether immunohistochemical PTEN expression in prostate cancer glands was associated with higher risk of recurrence, using a nested case-control study that included 451 men who recurred and 451 men who did not recur with clinically localized prostate cancer treated by radical prostatectomy. Recurrence was defined as biochemical recurrence (serum prostate-specific antigen >0.2?ng/ml) or clinical recurrence (local recurrence, systemic metastases, or prostate cancer-related death). Cases and controls were matched on pathological T stage, Gleason score, race/ethnicity, and age at surgery. Odds ratios of recurrence and 95% confidence intervals were estimated using conditional logistic regression to account for the matching factors and to adjust for year of surgery, preoperative prostate-specific antigen concentrations, and status of surgical margins. Men who recurred had a higher proportion of PTEN negative expression (16 vs 11%, P=0.05) and PTEN loss (40 vs 31%, P=0.02) than controls. Men with markedly decreased PTEN staining had a higher risk of recurrence (odds ratio=1.67; 95% confidence intervals 1.09, 2.57; P=0.02) when compared with all other men. In summary, in patients with clinically localized prostate cancer treated by prostatectomy, decreased PTEN expression was associated with an increased risk of recurrence, independent of known clinicopathological factors.
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An immunohistochemical signature comprising PTEN, MYC, and Ki67 predicts progression in prostate cancer patients receiving adjuvant docetaxel after prostatectomy.
Cancer
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Loss of the tumor suppressor PTEN is common in prostate cancer and may have prognostic significance. The authors examined PTEN and additional protein markers in primary tumors from patients with high-risk, localized prostate cancer who received adjuvant docetaxel in a prospective multicenter trial (TAX2501).
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Immunohistochemical expression of minichromosome maintenance complex protein 2 predicts biochemical recurrence in prostate cancer: a tissue microarray and digital imaging analysis-based study of 428 cases.
Hum. Pathol.
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Prostate cancer remains a major health problem in the United States. Established clinicopathologic parameters such as Gleason score, T stage, and prostate-specific antigen levels are currently the guiding tools for prognostication and disease management. The addition of biomarkers could increase the accuracy of these parameters for predicting disease progression, response to therapy, and survival. In this regard, the goal of this study was to evaluate minichromosome maintenance complex protein 2 and Ki-67 immunohistochemical expression as predictors of outcome in prostate cancer. For this purpose, 11 tissue microarrays were constructed using tumor and nontumor samples from 428 patients. Patients were divided into short-term (mean, 2.9 years) and long-term (mean, 14.1 years) follow-up groups. End points were biochemical recurrence for the short-term follow-up group and prostate cancer-related death for the long-term follow-up group. All men in the long-term follow-up group had biochemical recurrence at the time of recruitment. Expression of both markers was higher in tumor than in nontumor glands. Percentage of minichromosome maintenance complex protein 2 was associated with Gleason score in both groups. Percentage of Ki-67 was associated with Gleason score and pathologic stage only in the short-term follow-up group. Higher minichromosome maintenance complex protein 2 percentages were associated with biochemical recurrence in the short-term follow-up group. In the long-term follow-up group, neither minichromosome maintenance complex protein 2 nor Ki-67 levels predicted prostate cancer death. In conclusion, our results suggest that in patients treated by radical prostatectomy for clinically localized prostate cancer, immunohistochemistry for minichromosome maintenance complex protein 2 expression could be used to predict biochemical recurrence, independent of other known clinicopathologic factors.
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Immunoexpression status and prognostic value of mammalian target of rapamycin and hypoxia-induced pathway members in papillary cell renal cell carcinomas.
Hum. Pathol.
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Dysregulation of the mammalian target of rapamycin and hypoxia-induced pathways has been consistently identified in clear cell renal cell carcinomas. However, experience with non-clear cell renal cell carcinoma subtypes is scant. In this study, we evaluated the immunohistochemical expression of upstream (PTEN and phosphorylated AKT) and downstream (phosphorylated S6 and 4EBP1) effectors of the mammalian target of rapamycin pathway, as well as related cell-cycle proteins (p27 and c-MYC), and a member of the hypoxia-induced pathway (HIF-1?) in 54 patients with papillary renal cell carcinoma treated by nephrectomy. PTEN was lower in tumor than in normal kidney, and loss of PTEN expression was found in 48% of the patients. In tumor tissues, phosphorylated S6, 4EBP1, and HIF-1? were higher than in normal kidney. Conversely, scores of p27 were lower in tumor than in normal kidney. Finally, scores of c-MYC and phosphorylated AKT were similar in tumor and in normal kidney. Overall mortality and cancer-specific mortality were 24% and 11%, respectively. Tumor progression was observed in 17% of the patients. None of the tested biomarkers predicted cancer-specific mortality or tumor progression. As expected, patients with high T-stage tumors had higher hazard ratios for cancer-specific mortality (hazard ratio, 6.9) and tumor progression (hazard ratio, 6.7). Patients with higher Fuhrman grades also had higher risks for cancer-specific mortality (hazard ratio, 11.4) and tumor progression (hazard ratio, 4.5). In summary, our study provides evidence of dysregulation of the mammalian target of rapamycin and hypoxia-induced pathways in papillary renal cell carcinoma. Immunohistochemistry for members of the mammalian target of rapamycin pathway and for HIF-1? lacked prognostic significance in our cohort.
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Small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and distinct from well-differentiated pancreatic neuroendocrine tumors.
Am. J. Surg. Pathol.
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Poorly differentiated neuroendocrine carcinomas (NECs) of the pancreas are rare malignant neoplasms with a poor prognosis. The aim of this study was to determine the clinicopathologic and genetic features of poorly differentiated NECs and compare them with other types of pancreatic neoplasms. We investigated alterations of KRAS, CDKN2A/p16, TP53, SMAD4/DPC4, DAXX, ATRX, PTEN, Bcl2, and RB1 by immunohistochemistry and/or targeted exomic sequencing in surgically resected specimens of 9 small cell NECs, 10 large cell NECs, and 11 well-differentiated neuroendocrine tumors (PanNETs) of the pancreas. Abnormal immunolabeling patterns of p53 and Rb were frequent (p53, 18 of 19, 95%; Rb, 14 of 19, 74%) in both small cell and large cell NECs, whereas Smad4/Dpc4, DAXX, and ATRX labeling was intact in virtually all of these same carcinomas. Abnormal immunolabeling of p53 and Rb proteins correlated with intragenic mutations in the TP53 and RB1 genes. In contrast, DAXX and ATRX labeling was lost in 45% of PanNETs, whereas p53 and Rb immunolabeling was intact in these same cases. Overexpression of Bcl-2 protein was observed in all 9 small cell NECs (100%) and in 5 of 10 (50%) large cell NECs compared with only 2 of 11 (18%) PanNETs. Bcl-2 overexpression was significantly correlated with higher mitotic rate and Ki67 labeling index in neoplasms in which it was present. Small cell NECs are genetically similar to large cell NECs, and these genetic changes are distinct from those reported in PanNETs. The finding of Bcl-2 overexpression in poorly differentiated NECs, particularly small cell NEC, suggests that Bcl-2 antagonists/inhibitors may be a viable treatment option for these patients.
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Immunohistochemical Expression of the Mammalian Target of Rapamycin Pathway in Penile Squamous Cell Carcinomas: A Tissue Microarray Study of 112 Cases.
Histopathology
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The aim of this study is to evaluate the immunohistochemical expression of mTOR pathway-related biomarkers in penile carcinomas, and to assess the association with histologic type, histologic grade, and human papillomavirus (HPV) infection.
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