Issues with the Detection of Large Genomic Rearrangements in Molecular Diagnosis of 21-Hydroxylase Deficiency Abstract More than 95% of congenital adrenal hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the human leukocyte antigen (HLA) class III area on the short arm of chromosome 6p21.3. In the diagnosis of 21-hydroxylase deficiency, CYP21A2 genotyping is a valuable complement to biochemical investigations. Genotyping can confirm the diagnosis (or carrier state) and, at the same time, provide accurate phenotype prediction in patients carrying severe mutations. In addition, the use of genetic testing is also helpful in prenatal diagnosis where the goal of prenatal treatment is preventing genital virilization of the female fetus. An in-depth knowledge of CYP21A2 genetics is essential to assure the correct interpretation of results obtained. To date, more than 200 small pathogenic variants of the CYP21A2 gene have been reported, showing good agreement between clinical phenotype and patient genotype. Recently, novel CYP21A2 deletions, involving one or more exons, have been reported in different populations. Since these rearrangements have never been described before in the genetic history of 21-hydroxylase deficiency, these new deletions have aroused particular interest. However, it is possible that these novel rearrangements are the result of incorrect interpretation of multiplex ligation-dependent probe amplification (MLPA).

Salivary Total Protease Activity Based on a Broad-Spectrum Fluorescence Resonance Energy Transfer Approach to Monitor Induction and Resolution of Gingival Inflammation Abstract Objective Salivary total protease and chitinase activities were measured by a broad-spectrum fluorescence resonance energy transfer approach as predictors of induction and resolution of gingival inflammation in healthy individuals by applying an experimental human gingivitis model. Methods Dental biofilm accumulated (21 days, Induction Phase) by omitting oral hygiene practices followed by a 2-week Resolution Phase to restore gingival health in an experimental gingivitis study. Plaque accumulation, as assessed by the Turesky Modification of the Quigley-Hein Plaque Index (TQHPI), and gingival inflammation, assessed using the Modified Gingival Index (MGI), scores were recorded and unstimulated saliva was collected weekly. Saliva was analysed for total protein, albumin, total protease activity and chitinase activity (n = 18). Results The TQHPI and MGI scores, as well as total protease activity, increased until day 21. After re-establishment of oral hygiene, gingival inflammation levels returned to values similar to baseline (day 0). Levels of protease activity decreased significantly, but not to baseline values. Furthermore, ‘fast’ responders, who responded immediately to plaque, exhibited significantly higher proteolytic activity throughout the experimental course than ‘slow’ responders, who showed a lagged inflammatory response. Conclusion The results indicate that differential inflammatory responses encompass inherent variations in total salivary proteolytic activities, which could be further utilised in contemporary diagnostic, prognostic and treatment modalities for periodontal diseases.

Efficacy of Platinum-Based Adjuvant Chemotherapy on Prognosis of Pathological Stage II/III Lung Adenocarcinoma based on EGFR Mutation Status: A Propensity Score Matching Analysis Abstract Objective This study aimed to retrospectively evaluate the efficacy of platinum-based adjuvant chemotherapy (PBAC) for patients with pathological II/III pulmonary adenocarcinoma after curative resection based on epidermal growth factor receptor (EGFR) mutation status using propensity score matching (PSM) analysis. Methods Among the 304 patients who underwent curative resection of the lung for pathological II/III pulmonary adenocarcinoma from 2002 to 2016 at the Kanagawa Cancer Center, 176 and 128 patients were wild-type EGFR (Wt) and mutant EGFR (Mt), respectively. Seventy-one Wt patients (40.3%) and 60 Mt patients (46.9%) received PBAC. The prognoses of Wt and Mt patients who did and did not receive PBAC were compared using PSM analysis to reduce bias. Results The overall survival (OS) of both Wt and Mt patients who received PBAC was significantly better than that of patients who did not receive PBAC before PSM. By multivariate analysis, PBAC was an independent prognostic factor for OS among Wt patients, as were age, carcinoembryonic antigen (CEA) level, pleural invasion, and lymph node metastasis. Although age and CEA level were independent factors for OS among Mt patients, PBAC was not a prognostic factor. After PSM, Wt patients who received PBAC had better OS than those who did not, although Mt patients who did and did not receive PBAC had no difference in OS. Conclusions PBAC was associated with favorable prognosis after curative resection among Wt patients, but not among Mt patients. PBAC might not be necessary for Mt patients with pathological stage II/III pulmonary adenocarcinoma.

Highlighting the Role of Biomarkers of Inflammation in the Diagnosis and Management of Complex Regional Pain Syndrome Abstract Complex regional pain syndrome (CRPS) is characterized by continuous pain that is often accompanied by sensory, motor, vasomotor, sudomotor, and trophic disturbances. If left untreated, it can have a significant impact on the quality of life of patients. The diagnosis of CRPS is currently based on a set of relatively subjective clinical criteria: the New International Association for the Study of Pain clinical diagnostic criteria for CRPS. There are still no objective laboratory tests to diagnose CRPS and there is a great need for simple, objective, and easily measurable biomarkers in the diagnosis and management of this disease. In this review, we discuss the role of inflammation in the multi-mechanism pathophysiology of CRPS and highlight the application of potential biomarkers of inflammation in the diagnosis and management of this disease.

MicroRNAs: Key Players in Bladder Cancer Abstract Bladder cancer (BC) is the second highest morbid malignancy of the urinary tract and the fifth most common cancer worldwide. BC is highly malignant with significant morbidity and mortality, especially muscle-invasive BC (MIBC), which has a poor prognosis and frequently recurs after the first resection. Therefore, more sensitive diagnostic tools and effective therapeutic methods are urgently needed. MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of protein-coding genes by repressing their translation or cleaving RNA transcripts in a sequence-specific manner. miRNAs play very important roles in regulating genes related to tumorigenesis, tumor development, progression, metastasis and angiogenesis. With the rapid development of high-throughput sequencing technology, an increasing number of miRNAs with aberrant expression between either BC patients and healthy volunteers or between BC tumor tissues and matched peripheral control tissues have been recently examined. The tumor etiopathogenesis must be determined to promote the development of new markers as diagnostic and prognostic tools and targets for bladder tumor therapy, it is therefore vital to elucidate the function of miRNAs with aberrant expression in BC. In the present study, we examined the published data of BC-related miRNAs by reviewing their expression levels, possible functions, potential target genes, related molecular regulatory networks, candidate markers for prognosis and diagnosis, and prospective therapeutic cases, and we summarized the status of research on BC-related miRNAs in recent years.

Evolving Concepts in Chronic Obstructive Pulmonary Disease Blood-Based Biomarkers Abstract In recent years, there has been a great deal of interest in the identification and validation of blood-based biomarkers for clinical use in chronic obstructive pulmonary disease (COPD). We now have panels of blood biomarkers that potentially hold great promise as they show statistically significant associations with COPD, but biomarkers for the diagnosis of COPD remain elusive. In fact, they are yet to demonstrate sufficient accuracy to be accepted in clinical use, and many are not specific to COPD but more related to inflammation (e.g. interleukin-6) or associated with other chronic diseases such as diabetes (e.g. soluble receptor for advanced glycation endproducts [sRAGE]). Although no single blood-based biomarker has demonstrated clinical utility for either the diagnosis or progression of COPD, it has been suggested that combinations of individual markers may provide important diagnostic or prognostic information; however, the interpretation of COPD biomarker results still requires thought and many questions remain unanswered.

Clinical Evaluation of IntelliPlex™ KRAS G12/13 Mutation Kit for Detection of KRAS Mutations in Codon 12 and 13: A Novel Multiplex Approach Abstract Background Colorectal cancer (CRC) is among the most frequently occurring cancers worldwide and its incidence is forecasted to increase. Testing for KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations in colorectal tissue biopsy samples has become a crucial tool to guide therapeutic decisions for personalized treatment. Objective The objective of this study was to determine the diagnostic sensitivity and specificity of the IntelliPlex™ KRAS G12/13 Mutation Kit using clinical specimens compared to Sanger sequencing as the reference method. Methods A total of 248 formalin-fixed paraffin-embedded (FFPE) tissue samples, with CRC tumors comprising more than 10% of the whole tissue sample, were included in the study and analyzed for specific KRAS mutations in codons 12 and 13. For samples with discordant results between Sanger sequencing and the IntelliPlex™ KRAS G12/13 Mutation Kit, Pyrosequencing was utilized to resolve the KRAS mutational status. Results Sequencing determined 153 specimens as KRAS wild-type genotype while the IntelliPlex™ KRAS G12/13 Mutation Kit confirmed 139 of the wild-type cases, resulting in a clinical specificity of 90.8% (95% confidence interval (CI) 85.12–94.91). All 95 specimens with a reported mutation in codons 12 or 13 of KRAS by sequencing were also reported as non-wild-type by the IntelliPlex™ KRAS G12/13 Mutation Kit, resulting in a clinical sensitivity to detect KRAS mutations of 100% (95% CI 96.19–100). Conclusions The IntelliPlex™ KRAS G12/13 Mutation Kit demonstrates suitable specificity and sensitivity for use in clinical laboratories to determine the mutational status of KRAS codons 12 and 13.

NFKBIZ and CW6 in Adalimumab Response Among Psoriasis Patients: Genetic Association and Alternative Transcript Analysis Abstract Background Nuclear factor (NF)-κB is an essential mediator of the tumor necrosis factor (TNF) pathway, and has been implicated in psoriasis. NFKBIZ is a nuclear inhibitor of NF-κB with a prominent role in the pathogenesis of psoriasis. The genetic variation at the NFKBIZ gene has been associated with the risk of developing psoriasis, and could also contribute to defining the response to anti-TNF biological drugs. Objectives The objectives of this study were to determine the association of a common NFKBIZ insertion/deletion (indel) polymorphism (rs3217713) with the response to adalimumab and determine the differences in the relative expression of a NFKBIZ alternative transcript in patients with a positive versus negative response. Methods We genotyped a common NFKBIZ polymorphism in 169 psoriasis patients treated with adalimumab classified as responders (n = 120) and non-responders (n = 49), according to whether they had a 75% reduction in the Psoriasis Area and Severity Index score (PASI75) at week 24. The Cw6 polymorphism was also determined and allele and genotype frequencies were compared between the groups. We also determined the rate of the expression of a NFKBIZ transcript lacking exon 10 relative to the normal transcript in 60 patients (27 non-responders). In addition, because the intron indel could affect RNA splicing, we investigated whether the level of the alternative transcript was related to the intronic genotype. Results The NFKBIZ polymorphism was associated with adalimumab response, with carriers of the deletion allele significantly more frequent among responders (odds ratio = 2.76, 95% confidence interval 1.19–6.43; p = 0.015). The presence of the HLA-CW6 allele was also associated with a positive response in our cohort (p = 0.018). The alternative transcript was amplified in all the samples. We found higher but non-significant values of normal to alternative transcript in responders as well as in NFKBIZ insertion homozygotes. Conclusion Our study supported a significant effect of a common NFKBIZ polymorphism on the response to adalimumab. This result could help to optimize the prescription of this anti-TNF, but requires confirmation in other cohorts.

Combined Fascin-1 and MAP17 Expression in Breast Cancer Identifies Patients with High Risk for Disease Recurrence Abstract Background and Objective Breast cancer stem cells are considered to be a major cause of disease recurrence in breast cancer as they appear to be chemoresistant. Fascin-1 and MAP17 are stem cell markers whose excessive expression in tumors is associated with aggressive tumor phenotypes. The aim of the present study was to investigate the expression patterns of fascin-1 and MAP17 in breast cancer and to assess their clinical significance. Methods Expression of fascin-1 and MAP17 was assessed via immunohistochemistry in surgical specimens of a cohort comprised of 127 patients with resectable breast cancer. Results were correlated with clinicopathological characteristics and survival data. Progression-free survival (PFS) was defined as the primary outcome of the present study. Results Fascin-1 and MAP17 expression were strongly associated with the presence of triple-negative cancers (p < 0.0001). Tumors displaying high expression of fascin-1 presented correlations with high tumor grade (p = 0.002) and high expression of Ki-67 (p = 0.004). PFS of patients exhibiting high expression of fascin-1 and MAP17 in cancer cells in the first 5 years after surgery was significantly worse than in patients with low expression of the two markers (47.8%, 95% confidence interval [CI] 33–51 vs. 80.5%, 95% CI 47–56; p = 0.012) and independent of other clinicopathological characteristics (hazard ratio 0.171, 95% CI 0.034–0.869; p = 0.033). Conclusion Combined expression of fascin-1 and MAP17 in breast cancer cells is associated with a significantly worse 5-year PFS, therefore recognizing a group of patients with high risk for early disease recurrence.

A Meta-Analysis Evaluating Clinical Outcomes of Patients with Renal Cell Carcinoma Harboring Chromosome 9P Loss Abstract Context 9p loss appears a reliable and promising marker able to differentiate specific categories of patients with renal cell carcinoma associated with a worse prognosis. Objective The aim was to systematically evaluate relative risk of death, cancer-specific survival (CSS) and disease-free survival (DFS) among patients harboring 9p loss. Evidence Synthesis We found a total of 92 potentially relevant articles focused on the detection of 9p loss in patients with renal cell carcinoma and clinical outcomes of this population. Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines were employed to carry out this work. Fourteen studies resulted to be eligible for this analysis; 11 of these reported data on 5-year overall survival, six on CSS and four on DFS. An increased risk of death has been observed in patients harboring 9p loss (pooled relative risk of 3.965; 95% confidence interval [CI] 2.647–5.940, p < 0.001). Similarly, worse CSS (hazard ratio [HR] 6.776; 95% CI 3.824–12.009; p < 0.001) and DFS (HR 2.914; 95% CI 1.245–6.819; p = 0.014) have been observed in this population. Heterogeneity was significant in survival analysis, while no significant heterogeneity was observed in the CSS and DFS analyses. Conclusions Patients harboring chromosome 9p loss have worse clinical outcomes in terms of overall survival, CSS and DFS.