Expression of Vitamin D Receptor (VDR) Positively Correlates with Survival of Urothelial Bladder Cancer Patients

Vitamin D3 shows tumoristatic and anticancer effects by acting through the vitamin D receptor (VDR), while hydroxylation of 25-hydroxyvitamin D3 at position 1α by CYP27B1 is an essential step in its activation. The expression of both the VDR and CYP27B1 has been found in many normal and cancer tissues, but there is a lack of information about its expression in human bladder cancers. The aim of the present research was to examine whether the expression of the VDR and CYP27B1 in bladder cancer was related to the prognostic markers and disease outcome. We analyzed VDR and CYP27B1 in samples of tumor and normal tissues obtained from 71 urinary bladder cancer patients. The highest VDR immunostaining was found in normal epithelium and was significantly lower in bladder cancer cells (p < 0.001 with Mann–Whitney U test). VDR expression was lowest in more advanced (pT2b–pT4) (p = 0.005 with Mann–Whitney U test) and metastasizing cancers (p < 0.05 and p = 0.004 with Mann–Whitney U test for nuclear and cytoplasmic VDR immunostaining, respectively). The lack of cytoplasmic and nuclear VDR was also related to shorter overall survival (for cytoplasmic VDR immunolocalization 13.3 vs. 55.3 months of survival, HR = 1.92, p = 0.04 and for nuclear VDR immunostaining 13.5 vs. 55.3 months of survival, HR = 2.47, p = 0.002 with Mantel-Cox test). In cases with the lack of high cytoplasmic VDR staining the non-classic differentiations (NDs) was observed in higher percentage of tumor area. CYP27B1 expression was lower in cancer cells than in normal epithelial cells (p = 0.03 with Mann–Whitney U test), but its expression did not correlate with tumor stage (pT), metastasizing, grade, mitotic activity or overall survival. In conclusion, expression of the VDR and CYP27B1 are deregulated in urothelial bladder cancers. Although our results showing a relationship between the decreased VDR expression and prognostic markers and survival time indicate potential usefulness of VDR as a new indicator of a poorer prognosis, further studies are needed in different patient cohorts by independent groups to validate this hypothesis. We also suggest that vitamin D-based therapies may represent an adjuvant strategy in treatment for bladder cancers expressing VDR.     

Identification and Validation of Esophageal Squamous Cell Carcinoma Targets for Fluorescence Molecular Endoscopy

Dysplasia and intramucosal esophageal squamous cell carcinoma (ESCC) frequently go unnoticed with white-light endoscopy and, therefore, progress to invasive tumors. If suitable targets are available, fluorescence molecular endoscopy might be promising to improve early detection. Microarray expression data of patient-derived normal esophagus (n = 120) and ESCC samples (n = 118) were analyzed by functional genomic mRNA (FGmRNA) profiling to predict target upregulation on protein levels. The predicted top 60 upregulated genes were prioritized based on literature and immunohistochemistry (IHC) validation to select the most promising targets for fluorescent imaging. By IHC, GLUT1 showed significantly higher expression in ESCC tissue (30 patients) compared to the normal esophagus adjacent to the tumor (27 patients) (p < 0.001). Ex vivo imaging of GLUT1 with the 2-DG 800CW tracer showed that the mean fluorescence intensity in ESCC (n = 17) and high-grade dysplasia (HGD, n = 13) is higher (p < 0.05) compared to that in low-grade dysplasia (LGD) (n = 7) and to the normal esophagus adjacent to the tumor (n = 5). The sensitivity and specificity of 2-DG 800CW to detect HGD and ESCC is 80% and 83%, respectively (ROC = 0.85). We identified and validated GLUT1 as a promising molecular imaging target and demonstrated that fluorescent imaging after topical application of 2-DG 800CW can differentiate HGD and ESCC from LGD and normal esophagus.     

KIT Expression Is Regulated by DNA Methylation in Uveal Melanoma Tumors

Uveal melanoma (UM) is an ocular tumor with a dismal prognosis. Despite the availability of precise molecular and cytogenetic techniques, clinicopathologic features with limited accuracy are widely used to predict metastatic potential. In 51 UM tissues, we assessed a correlation between the expression of nine proteins evaluated by immunohistochemistry (IHC) (Melan-A, S100, HMB45, Cyclin D1, Ki-67, p53, KIT, BCL2, and AIFM1) and the presence of UM-specific chromosomal rearrangements measured by multiplex ligation-dependent probe amplification (MLPA), to find IHC markers with increased prognostic information. Furthermore, mRNA expression and DNA methylation values were extracted from the whole-genome data, achieved by analyzing 22 fresh frozen UM tissues. KIT positivity was associated with monosomy 3, increasing the risk of poor prognosis more than 17-fold (95% CI 1.53–198.69, p = 0.021). A strong negative correlation was identified between mRNA expression and DNA methylation values for 12 of 20 analyzed positions, five located in regulatory regions of the KIT gene (r = −0.658, p = 0.001; r = −0.662, p = 0.001; r = −0.816; p < 0.001; r = −0.689, p = 0.001; r = −0.809, p < 0.001, respectively). DNA methylation β values were also inversely associated with KIT protein expression (p = 0.001; p = 0.001; p = 0.015; p = 0.025; p = 0.002). Our findings, showing epigenetic deregulation of KIT expression, may contribute to understanding the past failure to therapeutically target KIT in UM.     

Impact of Spatially Heterogeneous Trop-2 Expression on Prognosis in Oral Squamous Cell Carcinoma

Oral cancer often presents with aggressive behavior and a high risk of recurrence and metastasis. For oral squamous cell carcinoma (OSCC), which is the most frequent histological subtype, therapy strategies include surgery, radiation therapy, chemotherapy, immune checkpoint inhibitors, and EGFR inhibitors. Recently, a Trop-2 antibody-drug conjugate (ADC) has been approved in the United States of America for the treatment of advanced triple-negative breast cancer. However, this ADC has also been tested in other solid tumors including head & neck squamous cell carcinoma. The prognostic impact of Trop-2 has already been reported for several cancers. We studied the prognostic influence of Trop-2 protein expression on OSCC patients’ survival. The cohort comprised n = 229 OSCC patients with available archived tumor tissue and corresponding non-neoplastic oral mucosa tissue. Using immunohistochemistry, we investigated Trop-2 expression in both the central and peripheral regions of each tumor and in corresponding non-neoplastic oral mucosa. In patients suffering from OSCC with combined high central and low peripheral Trop-2 expression, five-year overall survival (OS) was 41.2%, whereas 55.6% of OSCC patients who presented lower central and/or higher peripheral tumoral Trop-2 expression were alive after five years (p = 0.075). In multivariate Cox regression, the expression pattern of high central tumoral and lower peripheral Trop-2 expression was significantly correlated with impaired OS (HR = 1.802, 95%-CI: 1.134–2.864; p = 0.013) and recurrence-free survival (RFS) (HR = 1.633, 95%-CI: 1.042–2.560; p = 0.033), respectively, when adjusting for co-variables. Hence, Trop-2 may serve as an independent prognostic biomarker in OSCC. In subsequent studies, the pathophysiological meaning of downregulated Trop-2 expression in the OSCC periphery has to be analyzed.     

Identification and Validation of a New Set of Five Genes for Prediction of Risk in Early Breast Cancer

Molecular tests predicting the outcome of breast cancer patients based on gene expression levels can be used to assist in making treatment decisions after consideration of conventional markers. In this study we identified a subset of 20 mRNA differentially regulated in breast cancer analyzing several publicly available array gene expression data using R/Bioconductor package. Using RTqPCR we evaluate 261 consecutive invasive breast cancer cases not selected for age, adjuvant treatment, nodal and estrogen receptor status from paraffin embedded sections. The biological samples dataset was split into a training (137 cases) and a validation set (124 cases). The gene signature was developed on the training set and a multivariate stepwise Cox analysis selected five genes independently associated with DFS: FGF18 (HR = 1.13, p = 0.05), BCL2 (HR = 0.57, p = 0.001), PRC1 (HR = 1.51, p = 0.001), MMP9 (HR = 1.11, p = 0.08), SERF1a (HR = 0.83, p = 0.007). These five genes were combined into a linear score (signature) weighted according to the coefficients of the Cox model, as: 0.125FGF18 − 0.560BCL2 + 0.409PRC1 + 0.104MMP9 − 0.188SERF1A (HR = 2.7, 95% CI = 1.9–4.0, p < 0.001). The signature was then evaluated on the validation set assessing the discrimination ability by a Kaplan Meier analysis, using the same cut offs classifying patients at low, intermediate or high risk of disease relapse as defined on the training set (p < 0.001). Our signature, after a further clinical validation, could be proposed as prognostic signature for disease free survival in breast cancer patients where the indication for adjuvant chemotherapy added to endocrine treatment is uncertain.     

DDX3X Biomarker Correlates with Poor Survival in Human Gliomas

Primary high-grade gliomas possess invasive growth and lead to unfavorable survival outcome. The investigation of biomarkers for prediction of survival outcome in patients with gliomas is important for clinical assessment. The DEAD (Asp-Glu-Ala-Asp) box helicase 3, X-linked (DDX3X) controls tumor migration, proliferation, and progression. However, the role of DDX3X in defining the pathological grading and survival outcome in patients with human gliomas is not yet clarified. We analyzed the DDX3X gene expression, WHO pathological grading, and overall survival from de-linked data. Further validation was done using quantitative RT-PCR of cDNA from normal brain and glioma, and immunohistochemical (IHC) staining of tissue microarray. Statistical analysis of GEO datasets showed that DDX3X mRNA expression demonstrated statistically higher in WHO grade IV (n = 81) than in non-tumor controls (n = 23, p = 1.13 × 10⁻¹⁰). Moreover, DDX3X level was also higher in WHO grade III (n = 19) than in non-tumor controls (p = 2.43 × 10⁻⁵). Kaplan–Meier survival analysis showed poor survival in patients with high DDX3X mRNA levels (n = 24) than in those with low DDX3X expression (n = 53) (median survival, 115 vs. 58 weeks, p = 0.0009, by log-rank test, hazard ratio: 0.3507, 95% CI: 0.1893–0.6496). Furthermore, DDX3X mRNA expression and protein production significantly increased in glioma cells compared with normal brain tissue examined by quantitative RT-PCR, and Western blot. IHC staining showed highly staining of high-grade glioma in comparison with normal brain tissue. Taken together, DDX3X expression level positively correlates with WHO pathologic grading and poor survival outcome, indicating that DDX3X is a valuable biomarker in human gliomas.     

Significant Overexpression of DVL1 in Taiwanese Colorectal Cancer Patients with Liver Metastasis

Undetected micrometastasis plays a key role in the metastasis of cancer in colorectal cancer (CRC) patients. The aim of this study is to identify a biomarker of CRC patients with liver metastasis through the detection of circulating tumor cells (CTCs). Microarray and bioinformatics analysis of 10 CRC cancer tissue specimens compared with normal adjacent tissues revealed that 31 genes were up-regulated (gene expression ratio of cancer tissue to paired normal tissue > 2) in the cancer patients. We used a weighted enzymatic chip array (WEnCA) including 31 prognosis-related genes to investigate CTCs in 214 postoperative stage I–III CRC patients and to analyze the correlation between gene expression and clinico-pathological parameters. We employed the immunohistochemistry (IHC) method with polyclonal mouse antibody against DVL1 to detect DVL1 expression in 60 CRC patients. CRC liver metastasis occurred in 19.16% (41/214) of the patients. Using univariate analysis and multivariate proportional hazards regression analysis, we found that DVL1 mRNA overexpression had a significant, independent predictive value for liver metastasis in CRC patients (OR: 5.764; 95% CI: 2.588–12.837; p < 0.0001 on univariate analysis; OR: 3.768; 95% CI: 1.469–9.665; p = 0.006 on multivariate analysis). IHC staining of the immunoreactivity of DVL1 showed that DVL1 was localized in the cytoplasm of CRC cells. High expression of DVL1 was observed in 55% (33/60) of CRC tumor specimens and was associated significantly with tumor depth, perineural invasion and liver metastasis status (all p < 0.05). Our experimental results demonstrated that DVL1 is significantly overexpressed in CRC patients with liver metastasis, leading us to conclude that DVL1 could be a potential prognostic and predictive marker for CRC patients.     

Detection of Serum Protein Biomarkers for the Diagnosis and Staging of Hepatoblastoma

The present study aimed to identify serum biomarkers for the detection of hepatoblastoma (HB). Serum samples were collected from 71 HB patients (stage I, n = 19; stage II, n = 19, stage III, n = 19; and stage IV, n = 14) and 23 age- and sex-matched healthy children. Differential expression of serum protein markers were screened using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), and the target proteins were isolated and purified using HPLC and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), SEQUEST, and bioinformatics analysis. Differential protein expression was confirmed by enzyme-linked immunosorbent analysis (ELISA). SELDI-TOF-MS screening identified a differentially expressed protein with an m/z of 9348 Da, which was subsequently identified as Apo A–I; its expression was significantly lower in the HB group as compared to the normal control group (1546.67 ± 757.81 vs. 3359.21 ± 999.36, respectively; p < 0.01). Although the expression level decreased with increasing disease stage, pair-wise comparison revealed significant differences in Apo A–I expression between the normal group and the HB subgroups (p < 0.01). ELISA verified the reduced expression of Apo A–I in the HB group. Taken together, these results suggest that Apo A–I may represent a serum protein biomarker of HB. Further studies will assess the value of using Apo A–I expression for HB diagnosis and staging.     

A Keratin 7 and E-Cadherin Signature Is Highly Predictive of Tubo-Ovarian High-Grade Serous Carcinoma Prognosis

During tubo-ovarian high-grade serous carcinoma (HGSC) progression, tumoral cells undergo phenotypic changes in their epithelial marker profiles, which are essential for dissemination processes. Here, we set out to determine whether standard epithelial markers can predict HGSC patient prognosis. Levels of E-CADH, KRT7, KRT18, KRT19 were quantified in 18 HGSC cell lines by Western blot and in a Discovery cohort tissue microarray (TMA) (n = 101 patients) using immunofluorescence. E-CADH and KRT7 levels were subsequently analyzed in the TMA of the Canadian Ovarian Experimental Unified Resource cohort (COEUR, n = 1158 patients) and in public datasets. Epithelial marker expression was highly variable in HGSC cell lines and tissues. In the Discovery cohort, high levels of KRT7 and KRT19 were associated with an unfavorable prognosis, whereas high E-CADH expression indicated a better outcome. Expression of KRT7 and E-CADH gave a robust combination to predict overall survival (OS, p = 0.004) and progression free survival (PFS, p = 5.5 × 10⁻⁴) by Kaplan–Meier analysis. In the COEUR cohort, the E-CADH-KRT7 signature was a strong independent prognostic biomarker (OS, HR = 1.6, p = 2.9 × 10⁻⁴; PFS, HR = 1.3, p = 0.008) and predicted a poor patient response to chemotherapy (p = 1.3 × 10⁻⁴). Our results identify a combination of two epithelial markers as highly significant indicators of HGSC patient prognosis and treatment response.     

Syndecan Family Gene and Protein Expression and Their Prognostic Values for Prostate Cancer

Prostate cancer (PCa) is the leading cause of cancer-associated mortality in men, and new biomarkers are still needed. The expression pattern and protein tissue localization of proteoglycans of the syndecan family (SDC 1–4) and syntenin-1 (SDCBP) were determined in normal and prostatic tumor tissue from two genetically engineered mouse models and human prostate tumors. Studies were validated using SDC 1–4 and SDCBP mRNA levels and patient survival data from The Cancer Genome Atlas and CamCAP databases. RNAseq showed increased expression of Sdc1 in Pb-Cre4/Pten^f/f mouse Pca and upregulation of Sdc3 expression and downregulation of Sdc2 and Sdc4 when compared to the normal prostatic tissue in Pb-Cre4/Trp53^f/f-;Rb1^f/f mouse tumors. These changes were confirmed by immunohistochemistry. In human PCa, SDC 1–4 and SDCBP immunostaining showed variable localization. Furthermore, Kaplan–Meier analysis showed that patients expressing SDC3 had shorter prostate-specific survival than those without SDC3 expression (log-rank test, p = 0.0047). Analysis of the MSKCC-derived expression showed that SDC1 and SDC3 overexpression is predictive of decreased biochemical recurrence-free survival (p = 0.0099 and p = 0.045, respectively), and SDC4 overexpression is predictive of increased biochemical recurrence-free survival (p = 0.035). SDC4 overexpression was associated with a better prognosis, while SDC1 and SDC3 were associated with more aggressive tumors and a worse prognosis.     

Elevated Circulating and Placental SPINT2 Is Associated with Placental Dysfunction

Biomarkers for placental dysfunction are currently lacking. We recently identified SPINT1 as a novel biomarker; SPINT2 is a functionally related placental protease inhibitor. This study aimed to characterise SPINT2 expression in placental insufficiency. Circulating SPINT2 was assessed in three prospective cohorts, collected at the following: (1) term delivery (n = 227), (2) 36 weeks (n = 364), and (3) 24–34 weeks’ (n = 294) gestation. SPINT2 was also measured in the plasma and placentas of women with established placental disease at preterm (<34 weeks) delivery. Using first-trimester human trophoblast stem cells, SPINT2 expression was assessed in hypoxia/normoxia (1% vs. 8% O2), and following inflammatory cytokine treatment (TNFα, IL-6). Placental SPINT2 mRNA was measured in a rat model of late-gestational foetal growth restriction. At 36 weeks, circulating SPINT2 was elevated in patients who later developed preeclampsia (p = 0.028; median = 2233 pg/mL vs. controls, median = 1644 pg/mL), or delivered a small-for-gestational-age infant (p = 0.002; median = 2109 pg/mL vs. controls, median = 1614 pg/mL). SPINT2 was elevated in the placentas of patients who required delivery for preterm preeclampsia (p = 0.025). Though inflammatory cytokines had no effect, hypoxia increased SPINT2 in cytotrophoblast stem cells, and its expression was elevated in the placental labyrinth of growth-restricted rats. These findings suggest elevated SPINT2 is associated with placental insufficiency.     

HOXB9 Overexpression Promotes Colorectal Cancer Progression and Is Associated with Worse Survival in Liver Resection Patients for Colorectal Liver Metastases

As is known, HOXB9 is an important factor affecting disease progression and overall survival (OS) in cancer. However, its role in colorectal cancer (CRC) remains unclear. We aimed to explore the role of HOXB9 in CRC progression and its association with OS in colorectal liver metastases (CRLM). We analysed differential HOXB9 expression in CRC using the Tissue Cancer Genome Atlas database (TCGA). We modulated HOXB9 expression in vitro to assess its impact on cell proliferation and epithelial-mesenchymal transition (EMT). Lastly, we explored the association of HOXB9 protein expression with OS, using an institutional patient cohort (n = 110) who underwent liver resection for CRLM. Furthermore, HOXB9 was upregulated in TCGA-CRC (n = 644) vs. normal tissue (n = 51) and its expression levels were elevated in KRAS mutations (p < 0.0001). In vitro, HOXB9 overexpression increased cell proliferation (p < 0.001) and upregulated the mRNA expression of EMT markers (VIM, CDH2, ZEB1, ZEB2, SNAI1 and SNAI2) while downregulated CDH1, (p < 0.05 for all comparisons). Conversely, HOXB9 silencing disrupted cell growth (p < 0.0001). High HOXB9 expression (HR = 3.82, 95% CI: 1.59–9.2, p = 0.003) was independently associated with worse OS in CRLM-HOXB9-expressing patients after liver resection. In conclusion, HOXB9 may be associated with worse OS in CRLM and may promote CRC progression, whereas HOXB9 silencing may inhibit CRC growth.     

High Levels of KAP1 Expression Are Associated with Aggressive Clinical Features in Ovarian Cancer

KAP1 is an universal corepressor for Kruppel-associated box zinc finger proteins in both normal and tumor cells. In this study, the biological function and clinical significance of KAP1 expression in ovarian cancer were investigated. Immunohistological staining of KAP1 was evaluated in 111 patients with ovarian epithelial cancer, 15 with ovarian borderline tumor, and 20 normal ovarian tissue. The correlations of KAP1 expression with clinicopathological features were studied. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess overall survival to analyze the effect of KAP1 expression on the prognosis of ovarian cancer. The positive rates of KAP1 were significantly higher in ovarian epithelial cancer (55.7%) and borderline tumor (20.0%) than in normal ovarian tissue (5.0%) (all p < 0.01). KAP1 expression correlated significantly with clinical stage (χ² = 14.57, p < 0.0001), pathological grade (χ² = 6.06, p = 0.048) and metastases (χ² =10.38, p = 0.001). Patients with high KAP 1 levels showed poor survival (p < 0.0001). Multivariate analysis showed that KAP1 high expression was an independent predictor for ovarian cancer patients (hazard ratio = 0.463; 95% confidence interval = 0.230–0.9318, p = 0.031). Functionally, depletion of KAP1 by siRNA inhibited ovarian cancer cell proliferation, cell migration. KAP1 expression correlated with aggressive clinical features in ovarian cancer. High KAP1 expression was a prognostic factor of ovarian cancer.     

Application of Immunoprofiling Using Multiplexed Immunofluorescence Staining Identifies the Prognosis of Patients with High-Grade Serous Ovarian Cancer

Immunoprofiling has an established impact on the prognosis of several cancers; however, its role and definition in high-grade serous ovarian cancer (HGSOC) are mostly unknown. This study is to investigate immunoprofiling which could be a prognostic factor in HGSOC. We produced tumor microarrays of 187 patients diagnosed with HGSOC. We performed a multiplexed immunofluorescence staining using Opal Multiplex IHC kit and quantitative analysis with Vectra-Inform system. The expression intensities of programmed death-ligand 1 (PD-L1), CD4, CD8, CD20, FoxP3, and CK in whole tumor tissues were evaluated. The enrolled patients showed general characteristics, mostly FIGO stage III/IV and responsive to chemotherapy. Each immune marker showed diverse positive densities, and each tumor sample represented its immune characteristics as an inflamed tumor or noninflamed tumor. No marker was associated with survival as a single one. Interestingly, high ratios of CD8 to FoxP3 and CD8 to PD-L1 were related to the favorable overall survival (77 vs. 39 months, 84 vs. 47 months, respectively), and CD8 to PD-L1 ratio was also a significant prognostic factor (HR 0.621, 95% CI 0.420–0.917, p = 0.017) along with well-known clinical prognostic factors. Additionally, CD8 to PD-L1 ratio was found to be higher in the chemosensitive group (p = 0.034). In conclusion, the relative expression levels of CD8, FoxP3, and PD-L1 were significantly related to the clinical outcome of patients with HGSOC, which could be a kind of significant immunoprofiling of ovarian cancer patients to apply for treatment.