PIEZO1 activation may serve as an early tissue biomarker for the prediction of irradiation-induced salivary gland dysfunction.

Irradiation (IR)-induced xerostomia is the most common side effect of radiation therapy in patients with head and neck cancer (HNC). Xerostomia diagnosis is mainly based on the patient's medical history and symptoms. Currently, no direct biomarkers are available for the early prediction of IR-induced xerostomia. Here, we identified PIEZO1 as a novel predictive tissue biomarker for xerostomia. Our data demonstrate that PIEZO1 is significantly upregulated at the gene and protein levels during IR-induced salivary gland (SG) hypofunction. Notably, PIEZO1 upregulation coincided with that of inflammatory (F4/80) and fibrotic markers (fibronectin and collagen fibers accumulation). These findings suggest that PIEZO1 upregulation in SG tissue may serve as a novel predictive marker for IR-induced xerostomia.

Comparing nodal with primary tumor desmoplasia uncovers metastatic patterns in multiple endocrine neoplasia 2B.

BACKGROUND: While primary tumor desmoplasia is a powerful biomarker of node metastases in sporadic medullary thyroid cancer (MTC), information for hereditary MTC is sparse. METHODS: This proof-of-concept study, comprising 3 consecutive children with multiple endocrine neoplasia 2B, evaluated simultaneously the metastatic behavior of multiple primary thyroid tumors of disparate size and extent of desmoplasia within patients. RESULTS: Altogether, MTC typically involved the ipsilateral central neck before spreading to the ipsilateral lateral and the contralateral neck. Medullary thyroid cancer in the upper thyroid lobe leaped the ipsilateral central neck to invade the ipsilateral lateral neck. Unlike the desmoplasia-positive 6-mm high-grade and 7-mm low-grade primary thyroid tumors, the desmoplasia-negative 8-, 11-, and 16-mm low-grade primary thyroid tumors did not spread to ipsilateral neck nodes. With extranodal growth, the extent of nodal desmoplasia was greater than with intranodal growth. CONCLUSION: This proof-of-concept study suggests that primary tumor desmoplasia is an equally powerful biomarker of node metastasis in hereditary MTC.

Krüppel-like Factor 10 as a Prognostic and Predictive Biomarker of Radiotherapy in Pancreatic Adenocarcinoma.

The prognosis of pancreatic adenocarcinoma (PDAC) remains poor, with a 5-year survival rate of 12%. Although radiotherapy is effective for the locoregional control of PDAC, it does not have survival benefits compared with systemic chemotherapy. Most patients with localized PDAC develop distant metastasis shortly after diagnosis. Upfront chemotherapy has been suggested so that patients with localized PDAC with early distant metastasis do not have to undergo radical local therapy. Several potential tissue markers have been identified for selecting patients who may benefit from local radiotherapy, thereby prolonging their survival. This review summarizes these biomarkers including SMAD4, which is significantly associated with PDAC failure patterns and survival. In particular, Krüppel-like factor 10 (KLF10) is an early response transcription factor of transforming growth factor (TGF)-ß. Unlike TGF-ß in advanced cancers, KLF10 loss in two-thirds of patients with PDAC was associated with rapid distant metastasis and radioresistance; thus, KLF10 can serve as a predictive and therapeutic marker for PDAC. For patients with resectable PDAC, a combination of KLF10 and SMAD4 expression in tumor tissues may help select those who may benefit the most from additional radiotherapy. Future trials should consider upfront systemic therapy or include molecular biomarker-enriched patients without early distant metastasis.

Biomarker in Active Surveillance for Prostate Cancer: A Systematic Review.

Active surveillance (AS) in prostate cancer (PCa) represents a curative alternative for men with localised low-risk PCa. Continuous improvement of AS patient's selection and surveillance modalities aims at reducing misclassification, simplifying modalities of surveillance and decreasing need for invasive procedures such repeated biopsies. Biomarkers represent interesting tools to evaluate PCa diagnosis and prognosis, of which many are readily available or under evaluation. The aim of this review is to investigate the biomarker performance for AS selection and patient outcome prediction. Blood, urinary and tissue biomarkers were studied and a brief description of use was proposed along with a summary of major findings. Biomarkers represent promising tools which could be part of a more tailored risk AS strategy aiming to offer personalized medicine and to individualize the treatment and monitoring of each patient. The usefulness of biomarkers has mainly been suggested for AS selection, whereas few studies have investigated their role during the monitoring phase. Randomized prospective studies dealing with imaging are needed as well as larger prospective studies with long-term follow-up and strong oncologic endpoints.

Ceruloplasmin expression in renal cell carcinoma correlates with higher-grade and shortened survival.

Aim: We aimed to explore ceruloplasmin (CP) expression in clear cell renal cell carcinoma (ccRCC). Materials & methods: CP was analyzed in biofluid samples of 63 ccRCC patients, divided into three grading groups, and immunohistochemically, in 308 ccRCC. Results: Significant differences of mean plasma and urine CP levels in different grading groups were found. CP immunoreactivity was significantly linked to high-grade disease. Log rank tests showed a significant shorter overall survival rate in CP-positive cases (all p < 0.05). Conclusion: CP protein levels in biofluid samples confirmed differential CP expressions, depending on nuclear grade in ccRCC as previously seen in RNA expression analysis. CP expression was linked to high-grade disease and reduced survival rate in RCC.

Tissue-Based Biomarkers for the Risk Stratification of Men With Clinically Localized Prostate Cancer.

Risk stratification of men with clinically localized prostate cancer has historically relied on basic clinicopathologic parameters such as prostate specific antigen level, grade group, and clinical stage. However, prostate cancer often behaves in ways that cannot be accurately predicted by these parameters. Thus, recent efforts have focused on developing tissue-based genomic tests that provide greater insights into the risk of a given patient's disease. Multiple tests are now commercially available and provide additional prognostic information at various stages of the care pathway for prostate cancer. Indeed, early evidence suggests that these assays may have a significant impact on patient and physician decision-making. However, the impact of these tests on oncologic outcomes remains less clear. In this review, we highlight recent advances in the use of tissue-based biomarkers in the treatment of prostate cancer and identify the existing evidence supporting their clinical use.

New developments in the diagnosis of fibrillary glomerulonephritis.

Fibrillary glomerulonephritis is a glomerular disease historically defined by glomerular deposition of Congo red-negative, randomly oriented straight fibrils that lack a hollow center and stain with antisera to immunoglobulins. It was initially considered to be an idiopathic disease, but recent studies highlighted association in some cases with autoimmune disease, malignant neoplasm, or hepatitis C viral infection. Prognosis is poor with nearly half of patients progressing to end-stage renal disease within 4 years. There is currently no effective therapy, aside from kidney transplantation, which is associated with disease recurrence in a third of cases. The diagnosis has been hampered by the lack of biomarkers for the disease and the necessity of electron microscopy for diagnosis, which is not widely available. Recently, through the use of laser microdissection-assisted liquid chromatography-tandem mass spectrometry, a novel biomarker of fibrillary glomerulonephritis, DnaJ homolog subfamily B member 9, has been identified. Immunohistochemical studies confirmed the high sensitivity and specificity of DnaJ homolog subfamily B member 9 for this disease; dual immunofluorescence showed its colocalization with IgG in glomeruli; and immunoelectron microscopy revealed its localization to individual fibrils of fibrillary glomerulonephritis. The identification of this tissue biomarker has already entered clinical practice and undoubtingly will improve the diagnosis of this rare disease, particularly in developing countries where electron microscopy is less available. Future research is needed to determine whether DnaJ homolog subfamily B member 9 is an autoantigen or just an associated protein in fibrillary glomerulonephritis, whether it can serve as a noninvasive biomarker, and whether therapies that target this protein are effective in improving prognosis.

Renal cell carcinoma: the search for a reliable biomarker.

One particular challenge in the treatment of kidney tumors is the range of histologies and tumor phenotypes a renal mass can represent. A kidney tumor can range from benign (e.g., oncocytoma) to a clinically indolent malignancy (e.g., papillary type I, chromophobe) to aggressive disease [e.g., papillary type II or high-grade clear cell renal cell carcinoma (ccRCC)]. Even among various subtypes, kidney cancers are genetically diverse with variable prognoses and treatment response rates. Therefore, the key to proper treatment is the differentiation of these subtypes. Currently, a wide array of diagnostic, prognostic, and predictive biomarkers exist that may help guide the individualized care of kidney cancer patients. This review will discuss the various serum, urine, imaging, and immunohistological biomarkers available in practice.

MicroRNA profiling of ovarian granulosa cell tumours reveals novel diagnostic and prognostic markers.

BACKGROUND: The aim of this study was to explore the clinical utility of microRNAs (miRNAs) as improved markers of ovarian granulosa cell tumours (GCTs) for cancer diagnosis and prognosis prediction. Current histopathological and genetic markers, such as the presence of a FOXL2 gene mutation to distinguish between the two major subtypes are not wholly accurate and as such novel biomarkers are warranted. METHODS: The miRNA expression profiles of five formalin-fixed, paraffin-embedded (FFPE) adult-GCTs and five juvenile-GCTs were assessed using Affymetrix miRNA 3.0 Arrays and compared for differential expression. Ten miRNAs were assessed in an additional 33 FFPE tumours and four normal granulosa cell samples by quantitative RT-PCR, and their expression correlated to clinical information. RESULTS: MicroRNA array found 37 miRNAs as differentially expressed between the two GCT subtypes (p < 0.05, fold change ≥2 and among these, miRs -138-5p, -184, -204-5p, -29c-3p, -328-3p and -501-3p were validated by RT-qPCR as differentially expressed between the two GCT subtypes (p < 0.05). In addition, the expression of miR-184 was predictive of tumour recurrence in adult-GCTs, specifically for patients diagnosed with stage I and II and stage I only disease (p < 0.001 and p < 0.05, respectively). CONCLUSIONS: This study is the first to report on global miRNA expression profiles of human ovarian GCTs using FFPE tumour samples. We have validated six miRNAs as novel markers for subtype classification in GCTs with low levels of miR-138-5p correlating with early tumour stage. Low miR-184 abundance was correlated with tumour recurrence in early stage adult-GCT patients as a candidate predictive biomarker. Further studies are now needed to confirm the clinical utility of these miRNAs as diagnostic and recurrence markers, and understand their possible roles in the pathogenesis of GCTs.

Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis.

Here, we describe a multiplexed immunohistochemical platform with computational image processing workflows, including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination and revealed that response to therapy correlated with degree of mono-myelocytic cell density and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification and provide digital image processing pipelines to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to improve biomarker discovery and assessment.

Atheroprotective effects of pure tocotrienol supplementation in the treatment of rabbits with experimentally induced early and established atherosclerosis.

BACKGROUND: Atherosclerosis is the main cause of coronary artery disease -related deaths worldwide. The atheroprotective properties of pure tocotrienols (T3) in the absence of alpha-tocopherol (α-TCP) in vitamin E has not been extensively examined. AIM: To determine the atheroprotective properties of T3 in early and established atherosclerosis rabbits. METHODS: Thirty New Zealand white rabbits were divided into two groups, B1 and B2 which represent early [fed 1% high cholesterol diet (HCD) for 2 weeks] and established (fed 1% HCD for 8 weeks) atherosclerosis. Each group was subdivided into three intervention arms: 1) T3-4 mg/kg, 2) T3-15 mg/kg and 3) vehicle without T3 (T3 negative) for 8 weeks. Serial fasting blood samples were obtained for lipid profile, and whole lengths of aorta were used to determine tissue markers of endothelial activation, inflammation and plaque stability. RESULTS: In B1, atherosclerotic lesion in T3-4 mg/kg group was significantly reduced (p=0.008), while aortic tissue expression of vascular cellular adhesion molecule 1 (VCAM-1), interleukin-6 (IL-6) and matrix metalloproteinase (MMP-12) was reduced in T3-4 mg/kg compared to T3-negative rabbits group (0.2±0.1 vs. 28.5±3.1%; 3.0±1.6 vs. 14.0±1.7%; and 5.2±2.2 vs. 27.7±0.8%, respectively, p<0.05). T3-15 mg/kg group showed reduction in VCAM-1, E-selectin, IL-6 and MMP-12 (3.9±1.9 vs. 28.5±3.1%; 10.3±0.5 vs. 59.8±8.5%; 2.6±1.7 vs. 14.0±1.7%; and 16.2±3.2 vs. 27.7 0.8%, respectively, p<0.05). In B2, T3-4 mg/kg group reduced aortic tissue expression of intercellular adhesion molecule 1 (ICAM-1), E-selectin, IL-6, MMP-12 and MMP-9 compared to T3-negative rabbits group (29.9±2.4 vs. 55.3±1.3%; 26.7±1.5 vs. 60.5±7.6%; 15.7±0.7 vs. 27.7±4.8%; 34.8±2.7 vs. 46.5±3.4%; and 25.89±3.9 vs. 45.9±1.7%, respectively, p<0.05). T3-15 mg/kg group showed reduced VCAM-1, ICAM-1, E-selectin, IL-6, MMP-12 and MMP-9 (20.5±3.3 vs. 35.6±2.5%; 24.9±1.3 vs. 55.3±1.3%; 29.9±6.7 vs. 60.5±7.6; 11.3±2.2 vs. 27.7±4.8%; 23.0±1.7 vs. 46.5±3.4%; and 17.6±1.9 vs. 45.9±1.7%, respectively, p<0.05. CONCLUSION: These findings suggest the possible atheroprotective role T3 plays as an adjunct supplementation to standard treatment in the prevention of CAD.

TRPM4 protein expression in prostate cancer: a novel tissue biomarker associated with risk of biochemical recurrence following radical prostatectomy.

BACKGROUND: Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level with biochemical recurrence (BR) following radical prostatectomy (RP). MATERIAL AND METHODS: The study included 614 patients who had undergone RP. TRPM4 immunohistochemical staining was performed on samples of benign tissue, tissue containing PIN glands and PCa tissue using a commercially available polyclonal antibody. Staining intensity was recorded by two independent observers using a four-tired semi-quantitative grading system (0, 1+, 2+, 3+) converted into H-scores. Interobserver agreement was calculated by linear weighted kappa statistics. The association between staining intensity and BR was analysed using the Kaplan-Meier estimator and uni- and multiple Cox proportional hazard regression models. RESULTS: Significantly higher staining intensity was found in PCa glands compared to benign glands (p < 0.001). The concordance rate in TRPM4 staining intensities for benign, PIN and PCa tissue ranged from 86.0 to 91.5 %, corresponding to linear weighted kappa values of 0.566-0.789. After adjusting for patient and tumour characteristics, patients with a higher staining intensity in PCa glands compared to matched benign glands and an H-score equal to or above the median had an increased risk of BR (HR 1.79-2.62; p = 0.01-0.03 for the two observers) when compared to patients with a lower staining intensity. CONCLUSIONS: TRPM4 protein expression is widely expressed in benign and cancerous prostate tissue, with highest staining intensities found in PCa. Overexpression of TRPM4 in PCa (combination of high staining intensity and a high H-score) is associated with increased risk of BR after RP.

Arterial stiffness, atherosclerosis and cardiovascular risk: Pathophysiologic mechanisms and emerging clinical indications.

Arterial stiffness results from a degenerative process affecting mainly the extracellular matrix of elastic arteries under the effect of aging and risk factors. Changes in extracellular matrix proteins and in the mechanical properties of the vessel wall related to arterial stiffening may activate number of mechanisms involved also in the process of atherosclerosis. Several noninvasive methods are now available to estimate large artery stiffness in the clinical setting, including carotid-femoral pulse wave velocity, the reference for aortic stiffness estimate, and local distensibility measures of superficial arteries, namely carotid and femoral. An independent predictive value of arterial stiffness for cardiovascular events has been demonstrated in general as well as in selected populations, and reference values adjusted for age and blood pressure have been established. Thus, arterial stiffness is emerging as an interesting tissue biomarker for cardiovascular risk stratification and estimation of the individual "biological age". This paper overviews the mechanisms accounting for development and progression of arterial stiffness and for associations between arterial stiffness, atherosclerotic burden and incident cardiovascular events, summarizes the evidence and caveat for clinical use of stiffness as surrogate marker of cardiovascular risk, and briefly outlines some emerging methods for large artery stiffness characterization.

XPF protein levels determine sensitivity of malignant melanoma cells to oxaliplatin chemotherapy: suitability as a biomarker for patient selection.

As the options for systemic treatment of malignant melanoma (MM) increase, the need to develop biomarkers to identify patients who might benefit from cytotoxic chemotherapy becomes more apparent. In preclinical models, oxaliplatin has activity in cisplatin-resistant cells. In this study, we have shown that oxaliplatin forms interstrand crosslinks (ICLs) in cellular DNA and that loss of the heterodimeric structure-specific endonuclease XPF-ERCC1 causes hypersensitivity to oxaliplatin in mammalian cells. XPF deficiency resulted in late S-phase arrest and persistence of double-strand breaks following oxaliplatin treatment. In a panel of 12 MM cell lines, oxaliplatin sensitivity correlated with XPF and ERCC1 protein levels. The knockdown of ERCC1 and XPF protein levels by RNA interference increased sensitivity of cancer cells to oxaliplatin; overexpression of exogenous ERCC1 significantly decreased drug sensitivity. Following immunohistochemical optimization, XPF protein levels were quantified in MM tissue samples from 183 patients, showing variation in expression and no correlation with prognosis. In 57 patients with MM treated with cisplatin or carboplatin, XPF protein levels did not predict the likelihood of clinical response. We propose that oxaliplatin should not be discarded as a potential treatment for MM on the basis of the limited activity of cisplatin in unselected patients. Moreover, we show that XPF-ERCC1 protein levels are a key determinant of the sensitivity of melanoma cells to oxaliplatin in vitro. Immunohistochemical detection of XPF appears suitable for development as a tissue biomarker for potentially selecting patients for oxaliplatin treatment in a prospective clinical trial.

Protein expression of kidney and liver bilitranslocase in rats exposed to mercuric chloride--a potential tissular biomarker of toxicity.

Bilitranslocase (BTL) is a plasma membrane carrier that transports organic anions of physiological and pharmacological interest. It is expressed in basolateral plasma membrane of kidney and liver. BTL has been recently described as a marker of transition from normal tissue to its neoplastic transformation in human kidney. Inorganic mercury is a major environmental contaminant that produces many toxic effects. Previous reports have described an interaction between BTL and mercuric ions. This study was designed to evaluate the renal and hepatic expression of BTL in rats exposed to a nephrotoxic and hepatotoxic dose of HgCl2. Male rats were treated with a single injection of HgCl2 at a dose of 4mg/kg body wt, i.p. (HgCl2 group). Control rats received the vehicle alone (Control group). Studies were carried out 18h after injection. Afterwards, the kidneys and livers were excised and processed for histopathological studies or immunoblot (homogenates and crude membranes) techniques. In rats treated with HgCl2, immunoblotting showed a significant decrease in the abundance of BTL in homogenates and plasma membranes from kidney and liver. BTL decrease of expression might reflect the grade of damage in renal tubule cells and in hepatocytes. Thus, BTL might be postulated as a new biomarker of tissue toxicity induced by mercury.

Proteomics identified nuclear N-myc downstream-regulated gene 1 as a prognostic tissue biomarker candidate in renal cell carcinoma.

The aim of this study was to identify proteins with aberrant expression in clear cell renal cell carcinoma (ccRCC), and elucidate their clinical utilities. The protein expression profiles of primary ccRCC tumor tissues and neighboring non-tumor tissues were obtained from 9 patients by two-dimensional difference gel electrophoresis and mass spectrometry. Comparative analysis of 3771 protein spots led to the identification of 73 proteins that were expressed at aberrant levels in tumor tissues compared with non-tumor tissues. Among these 73 proteins, we further focused on N-myc downstream-regulated gene 1 protein (NDRG1). NDRG1 expression is regulated by members of myc family as well as by p53, HIF1A, and SGK1. The biological and clinical significance of NDRG1 is controversial for various malignancies and no detailed studies on NDRG1 have been reported in ccRCC until our study. For the 82 newly enrolled ccRCC patients, immunohistochemical analysis revealed a significant association between nuclear NDRG1 and favorable prognosis (p<0.05). Multivariate analysis demonstrated the role of NDRG1 as an independent factor of progression-free survival (p=0.01). Subsequent in vitro gene suppression assay demonstrated that NDRG1 silencing significantly enhanced cell proliferation and invasion of RCC cells. The cytotoxic effects of NDRG1 up-regulation induced by an iron chelator were also confirmed. These findings suggest that nuclear NDRG1 has tumor suppressive effects, and the NDRG1 expression may have clinical values in ccRCC. Nuclear NDRG1 may provide additional insights on molecular backgrounds of ccRCC progression, and contribute to the development of novel therapeutic strategy.

MALDI-MS tissue imaging identification of biliverdin reductase B overexpression in prostate cancer.

New biomarkers are needed to improve the specificity of prostate cancer detection and characterisation of individual tumors. In a proteomics profiling approach using MALDI-MS tissue imaging on frozen tissue sections, we identified discriminating masses. Imaging analysis of cancer, non-malignant benign epithelium and stromal areas of 15 prostatectomy specimens in a test and 10 in a validation set identified characteristic m/z peaks for each tissue type, e.g. m/z 10775 for benign epithelial, m/z 6284 and m/z 6657.5 for cancer and m/z 4965 for stromal tissue. A 10-fold cross-validation analysis showed highest discriminatory ability to separate tissue types for m/z 6284 and m/z 6657.5, both overexpressed in cancer, and a multicomponent mass peak cluster at m/z 10775-10797.4 overexpressed in benign epithelial tissue. ROC AUC values for these three masses ranged from 0.85 to 0.95 in the discrimination of malignant and non-malignant tissue. To identify the underlying proteins, prostate whole tissue extract was separated by nano-HPLC and subjected to MALDI TOF/TOF analysis. Proteins in fractions containing discriminatory m/z masses were identified by MS/MS analysis and candidate marker proteins subsequently validated by immunohistochemistry (IHC). Biliverdin reductase B (BLVRB) turned out to be overexpressed in PCa tissue. BIOLOGICAL SIGNIFICANCE: In this study on cryosections of radical prostatectomies of prostate cancer patients, we performed a MALDI-MS tissue imaging analysis and a consecutive protein identification of significant m/z masses by nano-HPLC, MALDI TOF/TOF and MS/MS analysis. We identified BLVRB as a potential biomarker in the discrimination of PCa and benign tissue, also suggesting BVR as a feasible therapeutic target.