P16 and HPV Genotype Significance in HPV-Associated Cervical Cancer—A Large Cohort of Two Tertiary Referral Centers

The expression of p16 is a good surrogate of human papillomavirus (HPV) infection in HPV-associated cancers. The significance of p16 expression, HPV genotype and genera in the outcome of patients with HPV-associated cervical cancer (CC) is unclear. Our aim is to ascertain the prognostic significance of these factors. Data from 348 patients (median age: 47.5 years old) with CC, diagnosed in two referral centers, were retrospectively collected. Advanced disease (FIGO2018 IB2-IV) was present in 68% of patients. A single HPV genotype was identified in 82.8% of patients. The most common HPVs were HPV16 (69%) and HPV18 (14%). HPV genera reflected this distribution. HPV16 tumors presented at an earlier stage. P16 was negative in 18 cases (5.2%), 83.3% of which were squamous cell carcinomas. These cases occurred in older patients who tended to have advanced disease. In the univariate analysis, HPV16 (HR: 0.58; p = 0.0198), α-9 genera (HR: 0.37; p = 0.0106) and p16 overexpression (HR: 0.54; p = 0.032) were associated with better survival. HPV16 (HR: 0.63; p = 0.0174) and α-9 genera (HR: 0.57; p = 0.0286) were associated with less relapse. In the multivariate analysis, only the International Federation of Gynecology and Obstetrics (FIGO) stage retained an independent prognostic value. HPV16, α-9 genera and p16 overexpression were associated with better survival, although not as independent prognostic factors. Patients with p16-negative HPV-associated CC were older, presented with advanced disease and had worse prognosis.     

Clinicopathologic and Prognostic Association of GRP94 Expression in Colorectal Cancer with Synchronous and Metachronous Metastases

Patients with advanced colorectal cancer (CRC) with distant metastases have a poor prognosis. We evaluated the clinicopathological relevance of GRP94 expression in these cases. The immunohistochemical expression of GRP94 was studied in 189 CRC patients with synchronous (SM; n = 123) and metachronous metastases (MM; n = 66), using tissue microarray; the association between GRP94 expression, outcome, and tumor-infiltrating lymphocytes (TILs) was also evaluated. GRP94 was expressed in 64.6% (122/189) patients with CRC; GRP94 positivity was found in 67.5% and 59.1% patients with SM and MM, respectively. In the SM group, high GRP94 expression was more common in patients with a higher density of CD4+ TILs (p = 0.002), unlike in the MM group. Survival analysis showed that patients with GRP94 positivity had significantly favorable survival (p = 0.030); after multivariate analysis, GRP94 only served as an independent prognostic factor (p = 0.034; hazard ratio, 0.581; 95% confidence interval, 0.351–0.961) in the SM group. GRP94 expression was detected in 49.4% of metastatic sites and showed significant heterogeneity between primary and metastatic lesions (p = 0.012). GRP94 is widely expressed in CRC with distant metastases; its expression was associated with favorable prognosis in the SM group, unlike in the MM group.     

Leptin Protein Expression and Promoter Methylation in Ovarian Cancer: A Strong Prognostic Value with Theranostic Promises

Ovarian cancer (OC) is the deadliest among all gynecological cancers. Epidemiological studies showed that obesity might influence many cancers including OC. One of the key factors that may link obesity and OC is leptin (LEP), known as an adipokine with pleiotropic effects on body homeostasis. This study aims to investigate the expression pattern of LEP, assess the methylation profiles of LEP and their associations with clinicopathological features including survival outcomes of OC patients. The protein expression of LEP was evaluated in 208 samples using both tissue microarray and immunohistochemistry techniques. The methylation profiles of LEP were measured in 63 formalin-fixed, paraffin-embedded tumor tissues by quantitative polymerase chain reaction using a MethyLight assay. Our results showed a significant association of LEP protein overexpression with several clinicopathological variables, mainly tumor subtype, LVI, age of menarche, tumor size and stage (p < 0.04). Kaplan–Meier analysis (using low expression versus high expression as a discriminator) indicated that LEP protein overexpression is a powerful positive prognosticator of both OC recurrence (DFS) and disease-specific survival (DSS) in our OC cohort (log-rank p = 0.01 and p = 0.002, respectively). This implies that patients with high LEP expression profiles live longer with less recurrence rates. Methylation analysis results demonstrated a clear association between no/low LEP protein expression pattern (38%) and LEP promoter CpG island hypermethylation (43%). Results of this study suggest that LEP is a powerful prognosticator of OC recurrence and DSS. LEP expression in OC seems to be regulated by its promoter hypermethylation through gene partial/total silencing. Further multi-institutional studies using larger cohorts are required to demystify the intricate molecular functions of this leptin-driven effects in OC pathophysiology and to accurately assess its theranostic potential and validate its prognostic/predictive power in OC onset, progression towards more effective and personalized management of OC patients.     

Metalloproteinases 1 and 3 as Potential Biomarkers in Breast Cancer Development

Breast cancer continues to be one of the main causes of morbidity and mortality globally and was the leading cause of cancer death in women in Spain in 2020. Early diagnosis is one of the most effective methods to lower the incidence and mortality rates of breast cancer. The human metalloproteinases (MMP) mainly function as proteolytic enzymes degrading the extracellular matrix and plays important roles in most steps of breast tumorigenesis. This retrospective cohort study shows the immunohistochemical expression levels of MMP-1, MMP-2, MMP-3, and MMP-9 in 154 women with breast cancer and 42 women without tumor disease. The samples of breast tissue are assessed using several tissue matrices (TMA). The percentages of staining (≤50%–>50%) and intensity levels of staining (weak, moderate, or intense) are considered. The immunohistochemical expression of the MMP-1-intensity (p = 0.043) and MMP-3 percentage (p = 0.018) and intensity, (p = 0.025) present statistically significant associations with the variable group (control–case); therefore, expression in the tumor tissue samples of these MMPs may be related to the development of breast cancer. The relationships between these MMPs and some clinicopathological factors in breast cancer are also evaluated but no correlation is found. These results suggest the use of MMP-1 and MMP-3 as potential biomarkers of breast cancer diagnosis.     

New Circulating Circular RNAs with Diagnostic and Prognostic Potential in Advanced Colorectal Cancer

Circular RNAs (circRNAs) are a group of special endogenous long non-coding RNAs which are highly stable in the circulation, and, thus, more suitable as new biomarkers of colorectal cancer (CRC). The aim of our study was to explore the plasma expression levels of four circRNAs: has_circ_0001445, hsa_circ_0003028, hsa_circ_0007915 and hsa_circ_0008717 in patients with CRC and to evaluate their associations with clinicopathological characteristics and the clinical outcome of the patients. CircRNAs were extracted from patients’ plasma obtained prior to chemotherapy. Their expression levels were measured by qPCR and calculated applying the 2^−ΔΔCt method. The levels of all four circRNAs were significantly increased in the plasma of CRC patients. At the optimal cut-off values hsa_circ_0001445 and hsa_circ_0007915 in plasma could significantly distinguish between patients with or without metastatic CRC with 92.56% sensitivity and 42.86% specificity, and with 86.07% sensitivity and 57.14% specificity, respectively. The mean overall survival (OS) of patients with high/intermediate expression of hsa_circ_0001445 was 30 months, significantly higher in comparison with the mean OS of the patients with low expression—20 months (log-rank test, p = 0.034). In multivariate Cox regression analysis, the low levels of hsa_circ_0001445 were also associated with shorter survival (HR = 1.59, 95% CI: 1.02–2.47, p = 0.040). A prognostic significance of hsa_circ_0001445 for patients with metastatic CRC was established.     

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.     

Twist-1 Up-Regulation in Carcinoma Correlates to Poor Survival

Epithelial-to-mesenchymal transition (EMT) facilitates tumor metastasis. Twist is a basic helix-loop-helix protein that modulates many target genes through E-box-responsive elements. There are two twist-like proteins, Twist-1 and Twist-2, sharing high structural homology in mammals. Twist-1 was found to be a key factor in the promotion of metastasis of cancer cells, and is known to induce EMT. Twist-1 participation in carcinoma progression and metastasis has been reported in a variety of tumors. However, controversy exists concerning the correlation between Twist-1 and prognostic value with respect to carcinoma. A systematic review and meta-analysis were performed to determine whether the expression of Twist-1 was associated with the prognosis of carcinoma patients. This analysis included 17 studies: four studies evaluated lung cancer, three evaluated head and neck cancer, two evaluated breast cancer, two evaluated esophageal cancer, two evaluated liver cancer and one each evaluated osteosarcoma, bladder, cervical and ovarian cancer. A total of 2006 patients were enrolled in these studies, and the median trial sample size was 118 patients. Twist-1 expression was associated with worse overall survival (OS) at both 3 years (hazard ratio “HR” for death = 2.13, 95% CI = 1.86 to 2.45, p < 0.001) and 5 years (HR for death = 2.01, 95% CI = 1.76 to 2.29, p < 0.001). Expression of Twist-1 is associated with worse survival in carcinoma.     

High Expression of LAMP3 Is a Novel Biomarker of Poor Prognosis in Patients with Esophageal Squamous Cell Carcinoma

Lysosomal-associated membrane protein 3 (LAMP3), identified as a molecular marker of mature dendritic cells, is one of the LAMP family members. Its expression was induced by hypoxia, and was associated with hypoxia mediated metastasis in breast and cervical cancers. However, epithelial expression of LAMP3 and its prognostic value in esophageal squamous cell carcinoma (ESCC) is still unknown. In the current study, mRNA expression of LAMP3 in 157 ESCC tissues and 50 adjacent normal tissues was detected by quantitative real-time PCR (qRT-PCR). LAMP3 protein expression in 46 paired cancerous and normal tissues was detected by immunohistochemistry (IHC). Then, DNA copy number was examined to observe its potential correlation with mRNA expression. The results showed that both mRNA and protein expression level of LAMP3 was significantly higher in cancerous tissues compared with normal controls (p < 0.001). LAMP3 DNA copy number was amplified in 70% of ESCC tissues and positive correlated with mRNA expression (p = 0.037). Furthermore, patients with higher LAMP3 expression had worse overall survival (HR = 1.90, 95% CI = 1.17–3.09, p = 0.010) and disease-free survival (HR = 1.80, 95% CI = 1.18–2.74, p = 0.006). In conclusion, our results suggest that epithelial LAMP3 expression is an independent prognostic biomarker for ESCC.     

Impact of Oncogenic Targets by Tumor-Suppressive miR-139-5p and miR-139-3p Regulation in Head and Neck Squamous Cell Carcinoma

We newly generated an RNA-sequencing-based microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC). Analysis of the signature revealed that both strands of some miRNAs, including miR-139-5p (the guide strand) and miR-139-3p (the passenger strand) of miR-139, were downregulated in HNSCC tissues. Analysis of The Cancer Genome Atlas confirmed the low expression levels of miR-139 in HNSCC. Ectopic expression of these miRNAs attenuated the characteristics of cancer cell aggressiveness (e.g., cell proliferation, migration, and invasion). Our in silico analyses revealed a total of 28 putative targets regulated by pre-miR-139 (miR-139-5p and miR-139-3p) in HNSCC cells. Of these, the GNA12 (guanine nucleotide-binding protein subunit alpha-12) and OLR1 (oxidized low-density lipoprotein receptor 1) expression levels were identified as independent factors that predicted patient survival according to multivariate Cox regression analyses (p = 0.0018 and p = 0.0104, respectively). Direct regulation of GNA12 and OLR1 by miR-139-3p in HNSCC cells was confirmed through luciferase reporter assays. Moreover, overexpression of GNA12 and OLR1 was detected in clinical specimens of HNSCC through immunostaining. The involvement of miR-139-3p (the passenger strand) in the oncogenesis of HNSCC is a new concept in cancer biology. Our miRNA-based strategy will increase knowledge on the molecular pathogenesis of HNSCC.     

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.     

Reduced Gonadotrophin Receptor Expression Is Associated with a More Aggressive Ovarian Cancer Phenotype

Follicle-stimulating hormone (FSH) and luteinising hormone (LH) play important roles in regulating cell growth and proliferation in the ovary. However, few studies have explored the expression of FSH and LH receptors (FSHR and LHCGR) in ovarian cancer, and their functional roles in cancer progression remain inconclusive. This study investigated the potential impact of both mRNA (FSHR, LHCGR) and protein (FSHR, LHCGR) expression on ovarian cancer progression using publicly available online databases, qRT-PCR (high grade serous ovarian cancers, HGSOC, n = 29 and benign ovarian tumors, n = 17) and immunohistochemistry (HGSOC, n = 144). In addition, we investigated the effect of FSHR and LHCGR siRNA knockdown on the pro-metastatic behavior of serous ovarian cancer cells in vitro. High FSHR or high LHCGR expression in patients with all subtypes of high-grade ovarian cancer was significantly associated with longer progression-free survival (PFS) and overall survival (OS). High FSHR protein expression was associated with increased PFS (p = 0.050) and OS (p = 0.025). HGSOC patients with both high FSHR and high LHCGR protein levels had the best survival outcome, whilst both low FSHR and low LHCGR expression was associated with poorest survival (p = 0.019). Knockdown of FSHR significantly increased the invasion of serous ovarian cancer cells (OVCAR3 and COV362) in vitro. LHCGR knockdown also promoted invasion of COV362 cells. This study highlights that lower FSHR and LHCGR expression is associated with a more aggressive epithelial ovarian cancer phenotype and promotes pro-metastatic behaviour.     

Assessment of the Efficacy and Clinical Utility of Different Circulating Tumor Cell (CTC) Detection Assays in Patients with Chemotherapy-Naïve Advanced or Metastatic Non-Small Cell Lung Cancer (NSCLC)

We herein investigated the detection frequency and clinical relevance of circulating tumor cells (CTCs) in chemotherapy-naïve stage IIIB/IV non-small cell lung cancer (NSCLC), by using the CellSearch and real-time CEACAM5mRNA assays. Blood samples from 43 patients were obtained at different time points during first-line chemotherapy. CellSearch revealed the detection of ≥1 CTCs in 41.9%, 40.9%, and 16.7% of patients at baseline, post-1st, and post-2nd treatment cycle, respectively, and of ≥5 CTCs in 11.6%, 9.1%, and 5.6%, respectively. CEACAM5mRNA+ CTCs were detected in 29.3% and 16% of patients pre- and post-treatment, respectively. The positivity concordance between the two assays was 2.2%. CTC-detection by CellSearch (≥5 CTCs: p = 0.004), CEACAM5mRNA (p = 0.010), or by any assay (p = 0.000) was associated with disease progression. Reduced survival was demonstrated for patients harboring ≥5 CTCs (progression-free survival; PFS: p = 0.000; overall survival; OS: p = 0.009), CEACAM5mRNA+ CTCs (PFS: p = 0.043; OS: p = 0.039), and CTCs by any assay (PFS: p = 0.005; OS: p = 0.006, respectively). CTC-detection by any assay independently predicted for increased risk of relapse (hazard ratio; HR: 3.496; p = 0.001) and death (HR: 2.866; p = 0.008). CellSearch-positivity either pre-, post-1st, or post-2nd cycle, was predictive for shorter PFS (p = 0.036) compared to negativity in all time points. Persistent CEACAM5mRNA-positivity pre- and post-treatment was associated with reduced PFS (p = 0.036) and OS (p = 0.026). In conclusion, CTC detection and monitoring using the CellSearch and CEACAM5mRNA assays provides valuable and complementary clinical information for chemo-naïve advanced or metastatic NSCLC.     

Smectite as a Preventive Oral Treatment to Reduce Clinical Symptoms of DSS Induced Colitis in Balb/c Mice

Natural smectites have demonstrated efficacy in the treatment of diarrhea. The present study evaluated the prophylactic effect of a diosmectite (FI5pp) on the clinical course, colon damage, expression of tight junction (TJ) proteins and the composition of the gut microbiota in dextran sulfate sodium (DSS) colitis. Diosmectite was administered daily to Balb/c mice from day 1 to 7 by oral gavage, followed by induction of acute DSS-colitis from day 8 to 14 (“Control”, n = 6; “DSS”, n = 10; “FI5pp + DSS”, n = 11). Mice were sacrificed on day 21. Clinical symptoms (body weight, stool consistency and occult blood) were checked daily after colitis induction. Colon tissue was collected for histological damage scoring and quantification of tight junction protein expression. Stool samples were collected for microbiome analysis. Our study revealed prophylactic diosmectite treatment attenuated the severity of DSS colitis, which was apparent by significantly reduced weight loss (p = 0.022 vs. DSS), disease activity index (p = 0.0025 vs. DSS) and histological damage score (p = 0.023 vs. DSS). No significant effects were obtained for the expression of TJ proteins (claudin-2 and claudin-3) after diosmectite treatment. Characterization of the microbial composition by 16S amplicon NGS showed that diosmectite treatment modified the DSS-associated dysbiosis. Thus, diosmectites are promising candidates for therapeutic approaches to target intestinal inflammation and to identify possible underlying mechanisms of diosmectites in further studies.     

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.     

High Cytoplasmic Expression of Krüppel-like Factor 4 Is an Independent Prognostic Factor of Better Survival in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality in the world. Hepatocarcinogenesis is complex, with an extraordinary molecular heterogeneity. Krüppel-like factor 4 (KLF4) plays an important role in cell proliferation and differentiation, and it can function as a tumor suppressor or an oncoprotein, depending on tissue type. The role of KLF4 in HCC remains controversial. The aim of this study was to explore the clinical significance of KLF4 expression in HCC. The study included 205 patients with surgical resection. We performed immunostaining for KLF4 and Ki-67 to investigate the correlations of the clinicopathological parameters of HCC and to examine the proliferative index. KLF4 staining was observed in the cytoplasm of non-tumorous hepatocytes and tumor cells. We subdivided the immunohistological staining results for KLF4 into low expression (Staining 0 and 1+) and high expression (Staining 2+ and 3+) subgroups. The expression of KLF4 was significantly correlated with tumor differentiation (p = 0.001). The Ki-67 proliferative index was significantly lower in well-differentiated HCCs (0.781% ± 1.02% vs. 2.16% ± 3.14%, p = 0.012), but not significantly different between low-KLF4 expression and high-KLF4 expression (1.87% ± 2.93% vs. 2.51% ± 3.28%, p = 0.32). Kaplan–Meier analysis showed that a high expression of KLF4 was significantly correlated with a longer disease-specific survival (p = 0.019). Univariate and multivariate analyses showed that high KLF4 expression was an independent predictor of a better disease-specific survival (p = 0.017; hazard ratio = 0.398; 95% confidence interval: 0.19–0.85). High cytoplasmic expression of KLF4 was associated with better disease-specific survival and was an independently favorable prognostic factor in hepatocellular carcinoma. These promising results suggest that KLF4 may play an anti-oncogenic role in hepatocarcinogenesis.