STAT3 as a Chemoprevention Target in Carcinogen-Induced Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal disease due, in large part, to a high rate of second primary tumor (SPT) formation. The 4-nitroquinoline 1-oxide (4-NQO) mouse model of oral carcinogenesis provides a robust system in which to study chemopreventive agents in the context of chemically induced HNSCC tumors. STAT3 is a potent oncogene that is hyperactivated by tyrosine phosphorylation early in HNSCC carcinogenesis and is a rational therapeutic target. We recently reported that loss-of-function of the STAT3 phosphatase PTPRT promotes STAT3 activation in HNSCC tumors and preclinical models and may serve as a predictive biomarker of response to STAT3 inhibitors, including the small-molecule Stattic. We therefore investigated the hypothesis that Ptprt-knockout (KO) mice would be more susceptible to 4-NQO-induced oral carcinogenesis and more sensitive to Stattic-mediated chemoprevention compared with wild-type (WT) mice. Herein, we demonstrate that Ptprt WT and KO mice develop similar spectra of HNSCC disease severity upon 12 weeks of 4-NQO administration, with no apparent effect of Ptprt genotype on carcinogenesis or treatment outcome. Targeting of STAT3 with Stattic resulted in a chemopreventive effect against 4-NQO-induced oral cancer (P = 0.0402). While these results do not support a central role for PTPRT in 4-NQO-induced HNSCC carcinogenesis, further investigation of STAT3 as a chemoprevention target in this cancer is warranted. Cancer Prev Res; 9(8); 657-63. ©2016 AACR.

A comparative analysis of LEF-1 in odontogenic and salivary tumors.

LEF-1 is a nuclear transcription factor of the Wnt pathway that regulates multipotent skin stem cell differentiation. ß-Catenin is considered a transcriptional coactivator that interacts with LEF-1.This study evaluates LEF-1 in a variety of odontogenic and salivary tumors and determines the prevalence of ß-catenin coexpression. Ninety-eight salivary gland tumors and 51 odontogenic tumors were evaluated for LEF-1 and ß-catenin immunohistochemical staining. Positivity was defined as at least 2+ intensity in more than 50% of tumor cells, which required a composite score of 6 or more. LEF-1 was positive in 64% (7/11) of calcifying cystic odontogenic tumors (CCOT). Nuclear ß-catenin was present in 82% (9/11) of CCOT. Coexpression of LEF-1 and nuclear ß-catenin was noted in all LEF-1-positive CCOT. Strong and diffuse LEF-1 expression was seen in 69% (11/16) of basal cell adenocarcinomas (BCAC) and 63% (5/8) of basal cell adenomas (BA). Nuclear ß-catenin was present in 50% (4/8) of BA and 43% (6/14) of BCAC. For BA, 4 of 5 LEF-1-positive tumors showed coexpression of ß-catenin, and for BCAC, 5 of 9 LEF-1-positive tumors showed coexpression. In conclusion, this study documents for the first time the presence of LEF-1 expression and nuclear ß-catenin coexpression in select basaloid salivary gland tumors and various odontogenic tumors. We demonstrate LEF-1 expression in both BA and BCAC preferentially over other salivary gland tumors suggesting some utility as a diagnostic marker.

Proteomic analysis of patient tissue reveals PSA protein in the stroma of benign prostatic hyperplasia.

BACKGROUND: Benign prostatic hyperplasia (BPH) is an age-related disease frequently associated with lower urinary tract symptoms (LUTS) that involves hyperplasia of both epithelial and stromal cells. Stromal fibrosis is a distinctive feature of BPH, but the exact mechanisms underlying this phenomenon are poorly understood. METHODS: In the current study, proteomics analyses were utilized to identify proteins altered in the BPH stromal compartment from patients with symptomatic BPH. Stromal cells were isolated from histological nodules of BPH by laser capture microdissection (LCM) and subjected to liquid chromatography/mass spectrometry. RESULTS: Proteins identified included several stromal-specific proteins involved in extracellular matrix (ECM) remodeling, focal adhesion, and cellular junctions. Additionally, the proteomics array identified the presence of luminal epithelial secretory protein PSA. Immunostaining, ELISA, and in situ hybridization analyses of BPH tissues verified the presence of PSA protein but absence of PSA mRNA in the stromal compartment. E-cadherin was down-regulated in BPH epithelial cells compared to normal adjacent tissues, suggesting that alteration of cellular junctions could contribute to the presence of luminal epithelial secreted proteins PSA and KLK2 in the stromal compartment. CONCLUSIONS: The above findings suggest that the presence of secreted proteins PSA and KLK2 from prostate luminal epithelial cells in BPH stroma is a hallmark of BPH nodules, which could in part be due to alterations in cellular junction proteins and/or increased epithelial barrier permeability. Elucidating the cause and consequence of these secreted proteins in the stromal compartment of BPH may lead to new understanding of BPH pathogenesis as well as approaches to prevent and/or treat this common disease.

Amylase α-1A (AMY1A): a novel immunohistochemical marker to differentiate chromophobe renal cell carcinoma from benign oncocytoma.

Chromophobe renal cell carcinoma (ChRCC) and oncocytoma present with a perplexing overlap of morphologic and immunohistochemical features. ChRCC have deletions in the 1p21.1 region including the amylase α-1A gene (AMY1A). No such deletions are found in oncocytoma. Instead, oncocytomas shared other deletions on chromosome 1: 1p31.3, 1q25.2, and 1q44. We performed AMY1A immunostaining on 75 oncocytomas (57 tissue microarray [TMA] cores, 18 whole slides) and 54 ChRCCs (20 TMA cores, 34 whole slides). Staining was assessed using the H-score method. The intensity was graded as follows: no staining=0, weak=1, moderate=2, and strong=3. The AMY1A immunostain preferentially stained the distal tubules and collecting ducts of normal kidney. All oncocytomas (100%) expressed AMY1A with an H-score that varied from 100 to 300 (mean 205). Mild to moderate heterogeneity in staining intensity was noted within a given oncocytoma. For oncocytomas, 87% (65/75) cases had H-scores of at least 120 with a mean score of 221. Notably, the 13% (10/75) of oncocytoma cases that had an H-score of 100 were derived from the TMA. A total of 87% (47/54) of the ChRCC cases were negative for the AMY1A immunostain. Of the ChRCC cases, 4% (2/54) showed very weak cytoplasmic staining (H-score of 70 each), which was less than the lowest H-score of oncocytoma cases. All 5 cases of ChRCC, which showed an H-score of 100 or more, were referred to as eosinophilic variants of ChRCC. Three of these 5 cases showed a very nondescript, diffuse staining of the cytoplasm. Two of these 5 cases showed an H-score of 130. We think that as the staining pattern of these 2 cases is similar to that of oncocytoma, they should be put in a category of renal oncocytic neoplasms favoring oncocytoma. This result shows that AMY1A staining could be very helpful in further classifying even a subset of the eosinophilic variants of ChRCC. The difference between ChRCC and oncocytoma was statistically significant (χ test, P<0.0001). All cases of clear cell RCC and papillary RCC were negative for AMY1A expression. Overall, sensitivity and specificity of AMY1A staining for oncocytoma was 100% (95% confidence interval, 0.95-1.00) and 96.75% (95% confidence interval, 0.93-0.99), respectively. Similarly, the sensitivity and specificity for distinguishing oncocytoma from ChRCC was 100% (95% confidence interval, 0.95-1.00) and 90.74% (95% confidence interval, 0.80-0.97), respectively. These data show that the novel marker AMY1A can be of great diagnostic utility when trying to differentiate ChRCC (classic and eosinophilic variant) and oncocytoma.

Primary bladder adenocarcinoma versus metastatic colorectal adenocarcinoma: a persisting diagnostic challenge.

AIM: This study attempted to distinguish primary bladder adenocarcinoma (PBA) from metastatic colonic adenocarcinomas (MCA), which is a difficult diagnostic and clinical problem. METHODS: Twenty-four cases of bladder adenocarcinomas (12 primary & 12 metastatic colorectal) were included in the study with urothelial carcinoma (UC) and colonic adenocarcinoma (CA) as controls. A panel of immunohistochemical (IHC) stains along with fluorescence in-situ hybridization (FISH), using the UroVysion probe set, was performed. RESULTS: The majority of the PBAs presented with advanced disease. Enteric histologic subtype was the most common morphological variant. Strong nuclear with cytoplasmic-membranous staining of ß-catenin was seen in 75% of MCA and only 16.7% PBA (<10% staining cells). Although abnormal nuclear staining with E-cadherin was seen in both PBA and MCA, it was more frequent in former. CK-7, CK-20, villin and CDX-2 stains were not helpful in distinguishing the two entities. FISH did not reveal any unique differences in chromosomal abnormality between the two groups. CONCLUSION: Although there was a statistically significant difference in ß-catenin and E-cadherin staining between two groups, we did not find any IHC or FISH marker that was specific for PBA. Distinction between PBA and MCA remains a diagnostic problem and clinical correlation is vital before rendering a diagnosis. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1393156268152357.

Partial mesenchymal to epithelial reverting transition in breast and prostate cancer metastases.

Epithelial to mesenchymal transition (EMT) is an oft-studied mechanism for the initiation of metastasis. We have recently shown that once cancer cells disseminate to a secondary organ, a mesenchymal to epithelial reverting transition (MErT) may occur, which we postulate is to enable metastatic colonization. Despite a wealth of in vitro and in vivo studies, evidence supportive of MErT in human specimens is rare and difficult to document because clinically detectable metastases are typically past the micrometastatic stage at which this transition is most likely evident. We obtained paired primary and metastatic tumors from breast and prostate cancer patients and evaluated expression of various epithelial and mesenchymal markers by immunohistochemistry. The metastases exhibited increased expression of membranous E-cadherin compared to primary tumors, consistent with EMT at the primary site and MErT at the metastatic site. However, the re-emergence of the epithelial phenotype was only partial or incomplete. Expression of epithelial markers connexins 26 and/or 43 was also increased on the majority of metastases, particularly those to the brain. Despite the upregulation of epithelial markers in metastases, expression of mesenchymal markers vimentin and FSP1 was mostly unchanged. We also examined prostate carcinoma metastases of varied sizes and found that while E-cadherin expression was increased compared to the primary lesion, the expression inversely correlated with size of the metastasis. This not only suggests that a second EMT may occur in the ectopic site for tumor growth or to seed further metastases, but also provides a basis for the failure to discern epithelial phenotypes in clinically examined macrometastases. In summary, we report increased expression of epithelial markers and persistence of mesenchymal markers consistent with a partial MErT that readily allows for a second EMT at the metastatic site. Our results suggest that cancer cells continue to display phenotypic plasticity beyond the EMT that initiates metastasis.

Combined analysis of estrogen receptor beta-1 and progesterone receptor expression identifies lung cancer patients with poor outcome.

PURPOSE: Steroid hormones and growth factors affect lung cancer, and it is possible they act in concert to influence patient outcome. EXPERIMENTAL DESIGN: Primary lung tumors and normal lung tissue were analyzed for expression and localization of estrogen receptor α and ß-1 (ERα and ERß), aromatase, progesterone receptor (PR), and epidermal growth factor receptor (EGFR). RESULTS: Tumors expressed higher levels of ERß compared to matched normal lung, whereas the reverse was true of PR. High cytoplasmic ERß expression was identified as an independent negative prognostic predictor of overall survival (OS; HR = 1.67), and low total PR was identified as an independent negative predictor of time to progression (TTP; HR = 1.59). After adjusting for stage, age, sex, and smoking, combined high cytoplasmic ERß and low total PR showed enhanced effects on OS (HR = 2.64) and on TTP (HR = 6.02). Further effects on OS were observed when EGFR expression was included (HR = 5.32). Patients with low cytoplasmic ERß, low aromatase, low EGFR, and high total PR had shorter OS than patients with the opposite pattern (HR = 6.60). Contribution of these markers to survival showed no significant sex differences in a multivariable model. ERα was elevated in tumors but was not predictive of survival, and appears to represent a variant ERα protein that is only recognized by a C-terminal antibody. CONCLUSIONS: Hormonal and EGFR pathways together may contribute to lung cancer prognosis. Lung tumors with high ERß-1/low PR may define patients with aggressive biology. A validation study is necessary to fully assess the predictive value of these markers.

Expression analysis of Ubc9, the single small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, in normal and malignant tissues.

Unlike ubiquitination, which targets proteins for degradation, sumoylation modulates protein-protein interactions of target proteins. Although there are multiple E2 enzymes required for ubiquitination, there is only one E2-conjugating enzyme for sumoylation, which is Ubc9. In line with increasing evidence that sumoylation plays an important role in tumorigenesis, we recently demonstrated that Ubc9 is expressed at high levels in advanced melanomas and that blocking expression of Ubc9 sensitizes melanomas to the cytotoxic effects of chemotherapeutic drugs. To determine whether and to what extent Ubc9 is expressed in other malignancies and their normal tissue counterparts, we undertook a detailed analysis of colon, lung, prostate, and breast cancer tissue microarrays. The findings, presented here, document that in primary colon and prostate cancer, Ubc9 expression is increased compared with their normal tissue counterparts, whereas in metastatic breast, prostate, and lung cancer, it is decreased in comparison with their corresponding normal and primary adenocarcinoma tissues. We also provide evidence that Ubc9 expression correlates positively with Dukes' stage and negatively with the Gleason score as well as breast cancer grade and that Ubc9 expression is substantially higher in the luminal than in the nonluminal type of breast cancer.

Renal oncocytoma: a comparative clinicopathologic study and fluorescent in-situ hybridization analysis of 73 cases with long-term follow-up.

Clinical studies have confirmed that renal oncocytoma (RO) is a benign neoplasm with excellent prognosis. In diagnostically challenging cases of renal oncocytic epithelial neoplasms, fluorescent in-situ hybridization (FISH) is increasingly being used and its ability to distinguish RO from chromophobe renal cell carcinoma (ChRCC) has been documented. In this study, we evaluated the differential diagnostic contribution of FISH in cases of RO.Clinicopathologic data and glass slides from 73 patients with RO were reviewed; 20 cases of ChRCC were included for comparison. FISH analysis of formalin-fixed, paraffin-embedded sections was performed using centromeric probes for chromosomes 1, 2, 7 and 17. FISH analysis revealed ROs had frequent loss of signal for chromosome 1 (56%) and 17 (44%). Tumors with more than one loss were common (41%) and 10% cases showed loss of all chromosomes examined. A total of 18% cases did not show any abnormality.Our study shows that chromosomal abnormalities in both ROs and ChRCCs are common with frequent loss of chromosomes 1 and 17. No association was found between overall patient survival and the extent of chromosomal abnormalities. FISH results, even those showing significant chromosomal abnormalities, should not alter the primarily morphology-based diagnosis of RO.

Immunodetection of DNA repair endonuclease ERCC1-XPF in human tissue.

The high incidence of resistance to DNA-damaging chemotherapeutic drugs and severe side effects of chemotherapy have led to a search for biomarkers able to predict which patients are most likely to respond to therapy. ERCC1-XPF nuclease is required for nucleotide excision repair of helix-distorting DNA damage and the repair of DNA interstrand crosslinks. Thus, it is essential for several pathways of repair of DNA damage by cisplatin and related drugs, which are widely used in the treatment of non-small cell lung carcinoma and other late-stage tumors. Consequently, there is tremendous interest in measuring ERCC1-XPF expression in tumor samples. Many immunohistochemistry studies have been done, but the antibodies for ERCC1-XPF were not rigorously tested for antigen specificity. Herein, we survey a battery of antibodies raised against human ERCC1 or XPF for their specificity using ERCC1-XPF-deficient cells as a negative control. Antibodies were tested for the following applications: immunoblotting, immunoprecipitation from cell extracts, immunofluorescence detection in fixed cells, colocalization of ERCC1-XPF with UV radiation-induced DNA damage in fixed cells, and immunohistochemistry in paraffin-embedded samples. Although several commercially available antibodies are suitable for immunodetection of ERCC1-XPF in some applications, only a select subset is appropriate for detection of this repair complex in fixed specimens. The most commonly used antibody, 8F1, is not suitable for immunodetection in tissue. The results with validated antibodies reveal marked differences in ERCC1-XPF protein levels between samples and cell types.

Polymeric immunoglobulin receptor-negative tumors represent a more aggressive type of adenocarcinomas of distal esophagus and gastroesophageal junction.

CONTEXT: Polymeric immunoglobulin receptor (PIgR) expression has been found in gastric mucosa and gastric cancers, but it is not known whether PIgR expression is related to background intestinal metaplasia nor the patterns of PIgR expression in tumors arising in the distal esophagus and gastroesophageal (GE) junction. OBJECTIVES: To identify clinicopathologic features of tumors associated with PIgR expression and to determine whether PIgR expression is associated with intestinal differentiation of tumors and intestinal metaplasia in background mucosa in 3 groups of upper gastrointestinal adenocarcinomas. These groups are (1) gastric adenocarcinomas, (2) adenocarcinomas of the distal esophagus and GE junction with background intestinal metaplasia, and (3) adenocarcinomas of the distal esophagus and GE junction without background intestinal metaplasia. DESIGN: Expression of PIgR and CDX2 in nonneoplastic mucosa, intestinal metaplasia, and adenocarcinomas was examined by immunohistochemistry in 42 cases: 14 gastric and 28 from the distal esophagus and GE junction, including 13 with esophageal or GE junction intestinal metaplasia. RESULTS: PIgR and CDX2 were expressed in all cases of intestinal metaplasia. PIgR expression was positive in 40% of group 3 versus 77% of group 2 and 71% of gastric adenocarcinomas (P = .06), and the expression of CDX2 was similar in all tumor groups (80%-83%). Metastatic-positive lymph nodes were more frequent in PIgR-negative tumors (94% vs 58%, P = .01). CONCLUSIONS: PIgR is uniformly expressed in intestinal metaplasia and in a subgroup of adenocarcinomas of the distal esophagus, GE junction, and stomach. Esophageal and GE junction adenocarcinomas that do not express PIgR show more frequent lymph node metastasis, suggesting that lack of expression of PIgR identifies a subgroup of more aggressive adenocarcinomas.

Up-regulation of dicer, a component of the MicroRNA machinery, in prostate adenocarcinoma.

MicroRNAs are small noncoding 18- to 24-nt RNAs that are predicted to regulate expression of as many as 30% of protein-encoding genes. In prostate adenocarcinoma, 39 microRNAs are up-regulated, and six microRNAs are down-regulated. Production and function of microRNA requires coordinated processing by proteins of the microRNA machinery. Dicer, an RNase III endonuclease, is an essential component of the microRNA machinery. From a gene array analysis of 16 normal prostate tissue samples, 64 organ-confined, and four metastatic prostate adenocarcinomas, we identified an up-regulation of major components of the microRNA machinery, including Dicer, in metastatic prostate adenocarcinoma. Immunohistochemical studies on a tissue microarray consisting of 232 prostate specimens confirmed up-regulation of Dicer in prostatic intraepithelial neoplasia and in 81% of prostate adenocarcinoma. The increased Dicer level in prostate adenocarcinoma correlated with clinical stage, lymph node status, and Gleason score. Western blot analysis of benign and neoplastic prostate cell lines further confirmed Dicer up-regulation in prostate adenocarcinoma. Dicer up-regulation may explain an almost global increase of microRNA expression in prostate adenocarcinoma. The presence of up-regulated microRNA machinery may predict the susceptibility of prostate adenocarcinoma to RNA interference-based therapy.

Fatty acid synthase gene overexpression and copy number gain in prostate adenocarcinoma.

Cancer cells frequently exhibit a significant increase in overexpression and activity of fatty acid synthase (FASN). Elevated FASN pathway activity also occurs in prostate cancer, the second leading cause of cancer-related death in men in the United States. Studies show that genes associated with an increase in protein expression, such as HER2/neu in breast cancer, are associated with an increase in gene copy number as well as an increase in transcription. In the present study, we evaluated whether FASN follows a similar paradigm in prostate cancer. To date, elevated FASN expression in prostate cancer has not been correlated with gene copy number alterations. Using immunohistochemistry and fluorescence in situ hybridization analysis in paraffin-embedded tissue microarrays, we observed gene copy gain in 24% of all prostate adenocarcinoma specimens examined with concurrent increased FASN protein expression. Immunohistochemistry alone showed 59% of prostate cancer specimens in the same tissue microarray with high FASN expression. Increased FASN gene was observed in 53% of all prostate tissues expressing elevated FASN protein levels and in 2 of 5 prostate tumor cell lines tested. These findings suggest that FASN gene copy number increases may be involved in the resultant increase in FASN protein expression observed in prostatic disease.

Increased apoptosis of periprostatic adipose tissue in VDR null mice.

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors that controls mineral ion homeostatis and has potential tumor-suppressive functions for various cancer types, specifically prostate cancer. A VDR ablated transgenic animal model (VDDRII, vitamin D-dependent rickets type II) has been developed and the animals typically have various diseases including, hypocalcemia, hyperparathyroidism, rickets, osteomalacia, and alopecia. This transgenic mouse system provides us with a model to decipher the influences of the VDR on prostatic growth and function. VDRs are abundant both in prostatic epithelial and stromal cells, and vitamin D signaling can be studied in this model. Although, there were no gross differences between the prostate tissue of the experimental and control groups, VDR null mice showed fat necrosis and individual cell apoptosis in the periprostatic adipose tissue. This indicates a possible role of VDR in the signaling pathways resulting the prostate. This may be particularly attractive for VDR targets for the inhibition of cancer progression using VD(3) and its analogs as potential chemo-preventive agents.

Early identification of individuals with prostate cancer in negative biopsies.

PURPOSE: The use of prostate specific antigen (PSA) to screen individuals for prostate cancer has changed the management of the disease, permitting early detection in men. Repeat biopsies for persistently increased PSA are not uncommon with prostate cancer never being detected in many of the men. The novel prostate cancer marker, EPCA, is expressed throughout the prostate of individuals with prostate cancer but not in those without the disease. MATERIALS AND METHODS: The expression of ECPA was studied to identify those men with initially negative biopsies who were ultimately found to have prostate cancer. Negative biopsies, subsequent biopsies and prostatectomy specimens were evaluated by quantitative immunohistochemistry. RESULTS: The EPCA staining intensity in the samples analyzed from the patient cohort with prostate carcinoma were significantly different compared to controls (p <0.001) with almost no overlap in staining results. Sensitivity of the EPCA immunohistochemistry is 84% and specificity is 85%. CONCLUSIONS: An immunohistochemical test for EPCA could serve as an adjunct to the current diagnostic approach to a patient who undergoes prostate needle biopsy, and could identify individuals with prostate cancer as much as 5 or more years earlier than the current diagnostic approach and algorithms. In addition, this test might also help limit the numbers of biopsies performed as part of subsequent routine evaluation for increased PSA.