Bcl-xL is translocated to the nucleus via CtBP2 to epigenetically promote metastasis.

Besides its mitochondria-based anti-apoptotic role, Bcl-xL also travels to the nucleus to promote cancer metastasis by upregulating global histone H3 trimethyl Lys4 (H3K4me3) and TGFß transcription. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates H3K4me3 modification are not understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced cell migration and metastasis in mouse models. Furthermore, knockout of CtBP2 suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGFß mRNA upregulation as well as cell invasion. Moreover, cleavage under targets and release using nuclease (CUT&RUN) coupled with next generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor region of genes encoding TGFß and its signaling pathway in the cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1.

Low-dose carbon monoxide suppresses metastatic progression of disseminated cancer cells.

Low-dose carbon monoxide (CO) is under investigation in clinical trials to treat non-cancerous diseases and has an excellent safety profile. Due to early detection and cancer awareness, an increasing number of cancer patients are diagnosed at early stages, when potentially curative surgical resection can be done. However, many patients ultimately experience recurrence. Here, we evaluate the therapeutic effect of CO on metastatic cancer progression. We show that 250 ppm CO inhibits the migration of multiple types of cancer cell lines, including breast, pancreatic, colon, prostate, liver, and lung cancer and reduces the ability to adhere to fibronectin. We demonstrate that in mouse models, 250 ppm inhaled CO inhibits lung metastasis of breast cancer and liver metastasis of pancreatic cancer. Moreover, low-dose CO suppresses recurrence and increases survival after surgical removal of primary pancreatic cancer in mice. Mechanistically, low-dose CO blocks transcription of heme importers, leading to diminished intracellular heme levels and a heme-regulated enzyme, cytochrome P4501B1 (CYP1B1). Either supplementing heme or overexpressing CYP1B1 reverses the anti-migration effect of low-dose CO. Taken together, low-dose CO therapy inhibits cell migration, reduces adhesion to fibronectin, prevents disseminated cancer cells from expanding into gross metastases, and improves survival in pre-clinical mouse models of metastasis.

High interobserver variability and frequent overdiagnosis of dysplasia in fundic gland polyps can be improved by detecting atypia on the surface epithelium and an abrupt transition to non-neoplastic cells.

AIMS: Fundic gland polyps (FGPs) arise sporadically and in combination with familial adenomatous polyposis (FAP). Criteria for distinguishing low-grade dysplasia (LGD) from regenerative atypia in FGPs are not well established. The aims of study were to determine: (i) interobserver variability in diagnosing LGD in FGPs; (ii) bias in diagnosing LGD in FAP patients; and (iii) stringent criteria for LGD in FGPs. METHODS AND RESULTS: Five senior pathologists who were blinded to the clinical history reviewed 72 FAP-associated FGPs and 34 sporadic FGPs. Cases were classified as negative (score = 0) or positive (score = 1) for LGD. Each case was assigned a 'combined dysplasia score' (CDS) ranging from 0 to 5 to reflect all five opinions. Fleiss' kappa showed only moderate interobserver agreement (κ = 0.46). Forty-one FGPs were classified as negative for dysplasia by consensus (CDS = 0-1), including 10 (24%) originally diagnosed as LGD. In contrast, all 37 cases classified as LGD by consensus (CDS = 4-5) were originally diagnosed as LGD, indicating that overdiagnosis of dysplasia is more common than underdiagnosis (P = 0.0012). Cytological atypia in the surface epithelium and an abrupt transition between atypical and normal-appearing epithelium were the most sensitive (97% and 100%, respectively) and specific (100% and 98%, respectively) features of dysplasia (P < 0.0001 for both comparisons). Very good agreement was achieved when a diagnosis of dysplasia was based on the presence of both features (κ = 0.85). CONCLUSIONS: There is high interobserver variability and a tendency to overdiagnose LGD in FGPs. Strict criteria requiring both surface atypia and abrupt transition for LGD in FGPs result in low interobserver variability.

RHAMMB-mediated bifunctional nanotherapy targeting Bcl-xL and mitochondria for pancreatic neuroendocrine tumor treatment.

The incidence of pancreatic neuroendocrine tumor (PNET) has continued to rise. Due to their indolent feature, PNET patients often present with incurable, metastatic diseases. Novel therapies are urgently needed. We have previously shown that Receptor for Hyaluronic Acid-Mediated Motility isoform B (RHAMMB) and Bcl-xL are upregulated in PNETs and both of them promote PNET metastasis. Because RHAMM protein is undetectable in most adult tissues, we hypothesized that RHAMMB could be a gateway for nanomedicine delivery into PNETs. To test this, we developed a RHAMMB-targeting nanoparticle (NP). Inside this NP, we assembled small interfering RNA (siRNA) against Bcl-xL (siBcl-xL) and mitochondria-fusing peptide KLA. We demonstrated that RHAMMB-positive PNETs picked up the RHAMMB-targeting NPs. siBcl-xL or KLA alone killed only 30% of PNET cells. In contrast, a synergistic killing effect was achieved with the co-delivery of siBcl-xL and KLA peptide in vitro. Unexpectedly, siBcl-xL induced cell death before reducing Bcl-xL protein levels. The systemically injected RHAMMB-targeting NPs carrying siBcl-xL and KLA peptide significantly reduced tumor burden in mice bearing RHAMMB-positive PNETs. Together, these findings indicate that the RHAMMB-targeting nanotherapy serves as a promising drug delivery system for PNET and possibly other malignancies with upregulated RHAMMB. The combination of siBcl-xL and KLA peptide can be a therapy for PNET treatment.

High levels of truncated RHAMM cooperate with dysfunctional p53 to accelerate the progression of pancreatic cancer.

Pancreatic cancer has the lowest survival rate out of all types of cancer. Pancreatic cancer patients are often diagnosed at advanced stages, hence an urgent need for a better therapeutic development of this devastating disease. Receptor for hyaluronan-mediated motility (RHAMM), not expressed in adult normal pancreas, has been suggested as a prognostic factor and a potential therapeutic target for pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNET). In this study, we initially sought to determine whether genetic deletion of RHAMM would slow down pancreatic cancer progression using Rhamm-/- mice. However, we found that Rhamm-/- mice expressed a truncated HMMRΔexon8-16 protein at higher abundance levels than wild-type RHAMM. While HMMRΔexon8-16 did not enable malignant progression of pancreatic intraepithelial neoplasia in p48-Cre; LSL-KRASG12D mice, it accelerated the formation of invasive PDAC and shortened the survival of p48-Cre; LSL-KRASG12D mice with heterozygous p53 knockout. KrasG12D PDAC mice with homozygous p53 knockout mice died around 10 weeks, and the effect of HMMRΔexon8-16 was not apparent in these short lifespan mice. In addition, HMMRΔexon8-16 shortened the survival of PNET-bearing RIP-Tag mice, which had inactivated p53. In our analysis of TCGA dataset, pancreatic cancer patients with mutant TP53 or loss of one copy of TP53 had higher RHAMM expression, which, combined, predicted worse outcomes. Taken together, by collaborating with dysfunctional p53, high levels of HMMRΔexon8-16 , which lacks the centrosome targeting domain and degrons for interaction with the Anaphase-Promoting Complex (APC), accelerated pancreatic cancer progression.

Minocycline-induced black bone disease with synovial pigmentation in a patient undergoing revision anterior cruciate ligament surgery: A case report.

INTRODUCTION: Long-term use of minocycline at high doses is associated with hyperpigmentation with multiple sites of involvement. While the cutaneous organs and the oral cavity are most commonly affected, bone discoloration is a rare entity. CASE PRESENTATION: A 19-year-old male patient with a history of acne vulgaris and intermittent treatment with high dose minocycline for three years presented with recurrent anterior cruciate ligament (ACL) tear. During arthroscopic surgery, however, hyperpigmentation of the femur and synovium was observed. Abnormal tissue was biopsied and confirmed through histopathological examination to contain melanin-related minocycline pigmentation. Revision surgery was re-scheduled with no intraoperative complications and excellent long-term clinical outcomes. CLINICAL DISCUSSION: There are several possible causes of hyperpigmentation, including hemosiderin deposition, infection, aseptic necrosis, demineralization, and metastatic disease. Black bone disease, caused by minocycline-induced hyperpigmentation, is rare. While the appearance is grossly abnormal in black bone disease, there has been no evidence suggesting that tissue integrity is compromised. CONCLUSION: This case confirms that hyperpigmentation does not affect bone integrity and that surgical procedures can be performed safely. Knowing the adverse effects of minocycline administration could reduce inappropriate postponement of surgical procedures, thereby saving time and resources.

Classifying Lung Neuroendocrine Neoplasms through MicroRNA Sequence Data Mining.

Lung neuroendocrine neoplasms (NENs) can be challenging to classify due to subtle histologic differences between pathological types. MicroRNAs (miRNAs) are small RNA molecules that are valuable markers in many neoplastic diseases. To evaluate miRNAs as classificatory markers for lung NENs, we generated comprehensive miRNA expression profiles from 14 typical carcinoid (TC), 15 atypical carcinoid (AC), 11 small cell lung carcinoma (SCLC), and 15 large cell neuroendocrine carcinoma (LCNEC) samples, through barcoded small RNA sequencing. Following sequence annotation and data preprocessing, we randomly assigned these profiles to discovery and validation sets. Through high expression analyses, we found that miR-21 and -375 are abundant in all lung NENs, and that miR-21/miR-375 expression ratios are significantly lower in carcinoids (TC and AC) than in neuroendocrine carcinomas (NECs; SCLC and LCNEC). Subsequently, we ranked and selected miRNAs for use in miRNA-based classification, to discriminate carcinoids from NECs. Using miR-18a and -155 expression, our classifier discriminated these groups in discovery and validation sets, with 93% and 100% accuracy. We also identified miR-17, -103, and -127, and miR-301a, -106b, and -25, as candidate markers for discriminating TC from AC, and SCLC from LCNEC, respectively. However, these promising findings require external validation due to sample size.

Characterizing and classifying neuroendocrine neoplasms through microRNA sequencing and data mining.

Neuroendocrine neoplasms (NENs) are clinically diverse and incompletely characterized cancers that are challenging to classify. MicroRNAs (miRNAs) are small regulatory RNAs that can be used to classify cancers. Recently, a morphology-based classification framework for evaluating NENs from different anatomical sites was proposed by experts, with the requirement of improved molecular data integration. Here, we compiled 378 miRNA expression profiles to examine NEN classification through comprehensive miRNA profiling and data mining. Following data preprocessing, our final study cohort included 221 NEN and 114 non-NEN samples, representing 15 NEN pathological types and 5 site-matched non-NEN control groups. Unsupervised hierarchical clustering of miRNA expression profiles clearly separated NENs from non-NENs. Comparative analyses showed that miR-375 and miR-7 expression is substantially higher in NEN cases than non-NEN controls. Correlation analyses showed that NENs from diverse anatomical sites have convergent miRNA expression programs, likely reflecting morphological and functional similarities. Using machine learning approaches, we identified 17 miRNAs to discriminate 15 NEN pathological types and subsequently constructed a multilayer classifier, correctly identifying 217 (98%) of 221 samples and overturning one histological diagnosis. Through our research, we have identified common and type-specific miRNA tissue markers and constructed an accurate miRNA-based classifier, advancing our understanding of NEN diversity.

Expression of the Receptor for Hyaluronic Acid-Mediated Motility (RHAMM) in Endometrial Cancer is Associated With Adverse Histologic Parameters and Tumor Progression.

Endometrial cancer is one of the most common gynecologic malignancies worldwide. Only 2 agents have been approved by Food and Drug Administration for endometrial cancer since 1971. There is a need to identify molecular targets to treat advanced endometrial cancer. The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various types of cancer. Here, we aimed to determine the clinical significance of RHAMM expression in endometrial cancer. Two hundred twenty-five cases of endometrial cancer, including serous and endometrioid types, and 8 cases of normal endometrium were used for studying RHAMM protein levels. The Cancer Genome Atlas database was also queried for RHAMM mRNA expression in endometrial cancer. Increased expression of RHAMM protein was seen in endometrial cancer compared with no or weak expression in normal endometrium. RHAMM expression positively correlated with tumor grade. RHAMM expression was significantly increased in endometrial serous carcinomas, which are high-grade, aggressive types of endometrial cancer, compared with the relatively less aggressive endometrioid carcinomas. RHAMM expression also correlated with the presence of lymphovascular invasion. RHAMM mRNA expression correlated with decreased survival in The Cancer Genome Atlas cohort. Therefore, increased RHAMM expression in endometrial cancer is associated with high-grade tumors and is indicative of more aggressive behavior. These findings suggest RHAMM as a prognostic factor in endometrial cancer and as a potential therapeutic target in advanced endometrial cancer for future studies.

Evaluating gastroenteropancreatic neuroendocrine tumors through microRNA sequencing.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) can be challenging to evaluate histologically. MicroRNAs (miRNAs) are small RNA molecules that often are excellent biomarkers due to their abundance, cell-type and disease stage specificity and stability. To evaluate miRNAs as adjunct tissue markers for classifying and grading well-differentiated GEP-NETs, we generated and compared miRNA expression profiles from four pathological types of GEP-NETs. Using quantitative barcoded small RNA sequencing and state-of-the-art sequence annotation, we generated comprehensive miRNA expression profiles from archived pancreatic, ileal, appendiceal and rectal NETs. Following data preprocessing, we randomly assigned sample profiles to discovery (80%) and validation (20%) sets prior to data mining using machine-learning techniques. High expression analyses indicated that miR-375 was the most abundant individual miRNA and miRNA cistron in all samples. Leveraging prior knowledge that GEP-NET behavior is influenced by embryonic derivation, we developed a dual-layer hierarchical classifier for differentiating GEP-NET types. In the first layer, our classifier discriminated midgut (ileum, appendix) from non-midgut (rectum, pancreas) NETs based on miR-615 and -92b expression. In the second layer, our classifier discriminated ileal from appendiceal NETs based on miR-125b, -192 and -149 expression, and rectal from pancreatic NETs based on miR-429 and -487b expression. Our classifier achieved overall accuracies of 98.5% and 94.4% in discovery and validation sets, respectively. We also found provisional evidence that low- and intermediate-grade pancreatic NETs can be discriminated based on miR-328 expression. GEP-NETs can be reliably classified and potentially graded using a limited panel of miRNA markers, complementing morphological and immunohistochemistry-based approaches to histologic evaluation.

Assessing colorectal cancer mismatch repair status in the modern era: a survey of current practices and re-evaluation of the role of microsatellite instability testing.

Results of DNA mismatch repair testing are used to detect Lynch syndrome and have prognostic and therapeutic implications among patients with sporadic colorectal carcinomas. Immunohistochemistry for mismatch repair proteins (MLH1, PMS2, MSH2, MSH6) and PCR for microsatellite instability are two established methods for assessing mismatch repair function. Older literature suggested a discordance rate of approximately 5% between these assays, leading some institutions to perform dual testing on all cases. Although universal mismatch repair testing is now recommended by multiple professional organizations, none provide guidelines regarding preferred assays. We surveyed 96 academic and nonacademic institutions to assess Lynch syndrome screening practices and evaluated discordance rates between immunohistochemistry and PCR among 809 colorectal cancers tested in our own institution. Our survey demonstrated no significant differences between academic and nonacademic practices with respect to testing strategies. Eighty six percent performed universal screening, and usually (76%) employed immunohistochemistry on initial biopsy samples. Only 20% employed PCR; these were mostly academic practices that used both immunohistochemistry and PCR (p < 0.01 compared with the nonacademic groups). Loss of MLH1/PMS2 staining was often (90%) followed by either BRAF mutational analysis or MLH1 methylation assays. Only 24% adhered to WHO recommendations to assign histologic grade based on mismatch repair status. We found only 3 cases (0.4%) with discordant immunohistochemistry and PCR results in our own practice: 1 reflected decreased MSH-6 staining in a neoadjuvantly treated microsatellite stable tumor, 1 MLH1-deficient tumor showed diminished MLH1/PMS2 in the tumor compared with internal control, and 1 case reflected an error in the molecular laboratory. Overall, our results showed extremely low discordance between methods assessing mismatch repair status and would suggest immunohistochemistry as the preferred single screening test. PCR can be reserved for cases that show equivocal immunostaining patterns.

Immunohistochemical analysis of RHAMM expression in normal and neoplastic human tissues: a cell cycle protein with distinctive expression in mitotic cells and testicular germ cells.

Expression of Receptor for Hyaluronic Acid Mediated Motility (RHAMM) increases cellular motility and RHAMM overexpression promotes invasive phenotype and metastasis of cancer cells. RHAMM has been suggested as a biomarker for poor prognosis in several tumor types, including lung, breast, colorectal, gastric, pancreatic ductal, and ovarian cancers. RNA studies showed restricted RHAMM expression in normal tissues, but its protein expression data in tissues were limited. In light of its potential as a prognostic marker and a therapeutic target, we performed immunohistochemical analysis to systematically characterize RHAMM expression in normal and neoplastic human tissues. Among 29 normal adult tissues, RHAMM protein showed restricted expression and was observed in the thymus, lymph node/tonsil, small intestine, colon, skin, bone marrow, placenta, and testis. The cellular distribution patterns of RHAMM in these normal tissues were consistent with RHAMM being a G2/M cell cycle protein, and this was further supported in comparison to the expression of cyclin B2, another G2/M protein. However, unlike the subcellular localization of cyclin B2, RHAMM decorated mitotic spindles in both anaphase and metaphase. RHAMM expression in tumor tissues is variable; and higher RHAMM protein expression is associated with histologically higher-grade tumors in general. Distinct from its expression in somatic tissues, RHAMM showed diffuse, strong, stage-specific expression in the spermatocyte stage of germ cells in adult testis. The neoplastic counterpart, spermatocytic tumor, also showed strong RHAMM expression. This unique expression in testis suggests that RHAMM may function during normal testicular germ cell maturation.

Beyond the Percentages of PD-L1-Positive Tumor Cells: Induced Versus Constitutive PD-L1 Expression in Primary and Metastatic Head and Neck Squamous Cell Carcinoma.

Anti-PD1 antibody has been approved for metastatic squamous cell carcinoma of the head and neck (SCCHN) and objective response rates of approximately 20% have been reported. Defining PD-L1 expression at ≥ 1% tumor cells as positive, PD-L1-positive tumors showed a higher response rate. However, it is unclear whether 1% is the optimal cutoff, and studies on lung cancer suggested 50% cutoff as a stronger predictive biomarker. 96 primary SCCHN from oropharynx and oral cavity and 34 corresponding metastatic lesions were typed for membranous PD-L1 expression. p16 immunohistochemistry was used as a surrogate marker for HPV status in SCCHN from the oropharynx. Fifty-two of 96 (54%) tumors were PD-L1-positive, 72% if PD-L1 expression in tumor-infiltrating immunocytes was also included as positive. Fifteen of 34 primary-metastasis tumor pairs differed in PD-L1 expression, and p16(+) cases more frequently showed PD-L1 expression in immunocytes than p16(-) cases (82 vs. 45%, p < 0.05). PD-L1-positive SCCHN showed two distinct patterns of expression. In the induced pattern of expression, PD-L1-positive tumor cells were limited to the periphery of tumor nests at the tumor-immunocyte interface, comprising < 5% of tumor cells, and were almost always associated with PD-L1-positive immunocytes. In contrast, tumors with constitutive PD-L1 expression had a higher percentage of positive tumor cells, often diffusely distributed throughout the tumor, and often were not accompanied by PD-L1-positive immunocytes. We propose that distinguishing these two biologically distinctive patterns of PD-L1 expression and typing metastatic instead of primary lesions might better predict immunotherapeutic response to anti-PD1/PD-L1 regimens beyond just the percentage of PD-L1-positive tumor cells.

Bcl-xL promotes metastasis independent of its anti-apoptotic activity.

Bcl-xL suppresses mitochondria-mediated apoptosis and is frequently overexpressed in cancer to promote cancer cell survival. Bcl-xL also promotes metastasis. However, it is unclear whether this metastatic function is dependent on its anti-apoptotic activity in the mitochondria. Here we demonstrate that Bcl-xL promotes metastasis independent of its anti-apoptotic activity. We show that apoptosis-defective Bcl-xL mutants and an engineered Bcl-xL targeted to the nucleus promote epithelial-mesenchymal transition, migration, invasion and stemness in pancreatic neuroendocrine tumour (panNET) and breast cancer cell lines. However, Bcl-xL proteins targeted to the mitochondria or outside of the nucleus do not have these functions. We confirm our findings in spontaneous and xenograft mouse models. Furthermore, Bcl-xL exerts metastatic function through epigenetic modification of the TGFß promoter to increase TGFß signalling. Consistent with these findings, we detect nuclear Bcl-xL in human metastatic panNETs. Taken together, the metastatic function of Bcl-xL is independent of its anti-apoptotic activity and its residence in the mitochondria.

Detection of cancer/testis antigens as a diagnostic tool in routine pathology practice.

Most of the studies on cancer/testis (CT) antigens performed to date have focused on their potential value as targets for immunotherapy. Several recent studies, however, revealed that CT antigens might represent useful tools for diagnostic pathology, in particular for the identification of squamous cell carcinoma and related pre-malignant lesions, as well as specific types of sarcoma.

Cancer-testis antigen expression in digestive tract carcinomas: frequent expression in esophageal squamous cell carcinoma and its precursor lesions.

Cancer-testis (CT) antigens are attractive tumor antigens for cancer immunotherapy. They comprise a group of proteins normally expressed in germ cells and aberrantly activated in a variety of human cancers. The protein expression of eight cancer-testis antigens [MAGEA, NY-ESO-1, GAGE, MAGEC1 (CT7), MAGEC2 (CT10), CT45, SAGE1, and NXF2] was evaluated by immunohistochemistry in 61 esophageal carcinomas (40 adenocarcinoma and 21 squamous cell carcinoma), 50 gastric carcinomas (34 diffuse and 16 intestinal type), and 141 colorectal carcinomas. The highest frequency of expression was found in esophageal squamous cell carcinomas: Positive staining for MAGEA, CT45, CT7, SAGE1, GAGE, NXF2, NY-ESO-1, and CT10 was observed in 57%, 38%, 33%, 33%, 29%, 29%, 19%, and 14% of squamous cell carcinomas, respectively. Similar staining patterns were observed in squamous dysplasias. Expression frequencies of cancer-testis antigens were seen in 2% to 24% of gastroesophageal adenocarcinomas and were not significantly different between adenocarcinomas of the stomach versus the esophagus, or between diffuse and intestinal types of gastric adenocarcinomas. Colorectal cancers did not express NY-ESO-1, CT7, CT10, or GAGE, and only infrequently expressed SAGE1 (0.7%) MAGEA (1.4%), CT45 (3.5%), and NXF2 (8.5%). We conclude that cancer-testis antigens are frequently expressed in esophageal squamous neoplasms. Although cancer-testis antigens are generally considered to be expressed later in tumor progression, they are found in squamous dysplasias, suggesting a potential diagnostic role for cancer-testis antigens in the evaluation of premalignant squamous lesions.

Association of oncofetal protein expression with clinical outcomes in patients with urothelial carcinoma of the bladder.

PURPOSE: Oncofetal proteins are expressed in the developing embryo. Oncofetal protein expression correlates with the clinical outcome of nonmuscle invasive bladder urothelial carcinoma. IMP3, MAGE-A, glypican-3 and TPBG are oncofetal proteins that have not been well characterized in urothelial carcinoma of the bladder. MATERIALS AND METHODS: We investigated the expression of these 4 proteins and their association with clinical outcomes using tissue microarrays from 384 consecutive patients treated with radical cystectomy between 1988 and 2003 at 1 academic center. We stained for IMP3, MAGE-A, glypican-3 and TPBG. Univariable and multivariable Cox regression analyses were done to evaluate the association of oncofetal protein expression with disease recurrence and cancer specific mortality. RESULTS: IMP3, MAGE-A, glypican-3 and TPBG were expressed in 39.5%, 45%, 6% and 85% of urothelial bladder carcinomas, respectively. Expression was tumor specific and did not correlate with pathological features except for TPBG. At a median followup of 128 months 176 patients (46%) experienced disease recurrence, 175 (45.5%) had died of the disease and 96 (27.5%) had died of another cause. On univariable analysis IMP3 and MAGE-A expression was associated with an increased risk of disease recurrence (p <0.001 and 0.03) and cancer specific mortality (p = 0.004 and 0.03, respectively). On multivariable Cox regression analysis adjusted for the effects of standard clinicopathological features IMP3 and MAGE-A expression was independently associated with disease recurrence (p = 0.004, HR 1.55, 95% CI 1.15-2.11 and p = 0.02, HR 1.44, 95% CI 1.05-1.99, respectively) but not with cancer specific mortality. CONCLUSIONS: Oncofetal proteins are commonly and differentially expressed in urothelial carcinoma of the bladder compared to normal urothelium. IMP3 and MAGE-A expression was associated with disease recurrence and cancer specific mortality but glypican-3 and TPBG expression was not.