Host biomarkers and combinatorial scores for the detection of serious and invasive bacterial infection in pediatric patients with fever without source.

BACKGROUND: Improved tools are required to detect bacterial infection in children with fever without source (FWS), especially when younger than 3 years old. The aim of the present study was to investigate the diagnostic accuracy of a host signature combining for the first time two viral-induced biomarkers, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and interferon γ-induced protein-10 (IP-10), with a bacterial-induced one, C-reactive protein (CRP), to reliably predict bacterial infection in children with fever without source (FWS) and to compare its performance to routine individual biomarkers (CRP, procalcitonin (PCT), white blood cell and absolute neutrophil counts, TRAIL, and IP-10) and to the Labscore. METHODS: This was a prospective diagnostic accuracy study conducted in a single tertiary center in children aged less than 3 years old presenting with FWS. Reference standard etiology (bacterial or viral) was assigned by a panel of three independent experts. Diagnostic accuracy (AUC, sensitivity, specificity) of host individual biomarkers and combinatorial scores was evaluated in comparison to reference standard outcomes (expert panel adjudication and microbiological diagnosis). RESULTS: 241 patients were included. 68 of them (28%) were diagnosed with a bacterial infection and 5 (2%) with invasive bacterial infection (IBI). Labscore, ImmunoXpert, and CRP attained the highest AUC values for the detection of bacterial infection, respectively 0.854 (0.804-0.905), 0.827 (0.764-0.890), and 0.807 (0.744-0.869). Labscore and ImmunoXpert outperformed the other single biomarkers with higher sensitivity and/or specificity and showed comparable performance to one another although slightly reduced sensitivity in children < 90 days of age. CONCLUSION: Labscore and ImmunoXpert demonstrate high diagnostic accuracy for safely discriminating bacterial infection in children with FWS aged under and over 90 days, supporting their adoption in the assessment of febrile patients.

Host test based on tumor necrosis factor-related apoptosis-inducing ligand, interferon gamma-induced protein-10 and C-reactive protein for differentiating bacterial and viral respiratory tract infections in adults: diagnostic accuracy study.

OBJECTIVES: To assess the performance of a test (called BV), integrating the blood levels of three immune proteins into a score, to differentiate bacterial from viral infection among adults with suspected lower respiratory tract infection (LRTI). METHODS: Prospective diagnostic accuracy study, enrolling febrile adults >18 years with LRTI signs or symptoms for less than 7 days presenting to several hospitals' emergency departments in Israel. The main exclusion criterion was immunodeficiency. Reference standard diagnosis (bacterial/viral/indeterminate) was based on three experts independently reviewing comprehensive patient data including follow-up data. BV generated three results: viral infection or other nonbacterial condition (0 ≤ score < 35), equivocal (35 ≤ score ≤ 65) and bacterial infection including co-infection (65 < score ≤ 100). BV performance was assessed against the reference standard with indeterminate reference standard and equivocal BV cases removed. RESULTS: Of 490 enrolled patients, 415 met eligibility criteria (median age 56 years, interquartile range 35). The reference standard classified 104 patients as bacterial, 210 as viral and 101 as indeterminate. BV was equivocal in 9.6% (30/314). Excluding indeterminate reference standard diagnoses and equivocal BV results, BV's sensitivity for bacterial infection was 98.1% (101/103; 95% confidence interval 95.4-100), specificity 88.4% (160/181; 83.7-93.1) and negative predictive value 98.8% (160/162; 97.1-100). DISCUSSION: BV exhibited high diagnostic performance for febrile adults with suspected LRTI among patients with reference standard diagnoses of bacterial or viral LRTI.

An immune-protein score combining TRAIL, IP-10 and CRP for predicting severe COVID-19 disease.

COVID-19 patients are oftentimes over- or under-treated due to a deficit in predictive management tools. This study reports derivation of an algorithm that integrates the host levels of TRAIL, IP-10, and CRP into a single numeric score that is an early indicator of severe outcome for COVID-19 patients and can identify patients at-risk to deteriorate. 394 COVID-19 patients were eligible; 29% meeting a severe outcome (intensive care unit admission/non-invasive or invasive ventilation/death). The score's area under the receiver operating characteristic curve (AUC) was 0.86, superior to IL-6 (AUC 0.77; p = 0.033) and CRP (AUC 0.78; p < 0.001). Likelihood of severe outcome increased significantly (p < 0.001) with higher scores. The score differentiated severe patients who further deteriorated from those who improved (p = 0.004) and projected 14-day survival probabilities (p < 0.001). The score accurately predicted COVID-19 patients at-risk for severe outcome, and therefore has potential to facilitate timely care escalation and de-escalation and appropriate resource allocation.

A point-of-need platform for rapid measurement of a host-protein score that differentiates bacterial from viral infection: Analytical evaluation.

The objective was to evaluate the analytical performance of a new point-of-need platform for rapid and accurate measurement of a host-protein score that differentiates between bacterial and viral infection. The system comprises a dedicated test cartridge (MeMed BV®) and an analyzer (MeMed Key®). In each run, three host proteins (TRAIL, IP-10 and CRP) are measured quantitatively and a combinational score (0-100) computed that indicates the likelihood of Bacterial versus Viral infection (BV score). Serum samples collected from patients with acute infection representing viral (0 ≤ score < 35), equivocal (35 ≤ score ≤ 65), or bacterial (65 < score ≤ 100) scores based on pre-defined score cutoffs were employed for the analytical evaluation studies as well as samples from healthy individuals. To assess reproducibility, triplicate runs were conducted at 3 different sites, on 2 analyzers per site over 5 non-consecutive days. Lower limit of quantitation (LLoQ) and analytical measurement range were established utilizing recombinant proteins. Sample stability was evaluated using patient samples representative of BV score range (0-100). MeMed Key® and MeMed BV® passed the acceptance criteria for each study. In the reproducibility study, TRAIL, IP-10 and CRP measurements ranged with coefficient of variation from 9.7 to 12.7%, 4.6 to 6.2% and 5.0 to 11.6%, respectively. LLoQ concentrations were established as 15 pg/mL, 100 pg/mL and 1 mg/L for TRAIL, IP-10 and CRP, respectively. In summary, the analytical performance reported here, along with diagnostic accuracy established in the Apollo clinical validation study (NCT04690569), supports that MeMed BV® run on MeMed Key® can serve as a tool to assist clinicians in differentiating between bacterial and viral infection.

The diagnostic value of nasal microbiota and clinical parameters in a multi-parametric prediction model to differentiate bacterial versus viral infections in lower respiratory tract infections.

BACKGROUND: The ability to accurately distinguish bacterial from viral infection would help clinicians better target antimicrobial therapy during suspected lower respiratory tract infections (LRTI). Although technological developments make it feasible to rapidly generate patient-specific microbiota profiles, evidence is required to show the clinical value of using microbiota data for infection diagnosis. In this study, we investigated whether adding nasal cavity microbiota profiles to readily available clinical information could improve machine learning classifiers to distinguish bacterial from viral infection in patients with LRTI. RESULTS: Various multi-parametric Random Forests classifiers were evaluated on the clinical and microbiota data of 293 LRTI patients for their prediction accuracies to differentiate bacterial from viral infection. The most predictive variable was C-reactive protein (CRP). We observed a marginal prediction improvement when 7 most prevalent nasal microbiota genera were added to the CRP model. In contrast, adding three clinical variables, absolute neutrophil count, consolidation on X-ray, and age group to the CRP model significantly improved the prediction. The best model correctly predicted 85% of the 'bacterial' patients and 82% of the 'viral' patients using 13 clinical and 3 nasal cavity microbiota genera (Staphylococcus, Moraxella, and Streptococcus). CONCLUSIONS: We developed high-accuracy multi-parametric machine learning classifiers to differentiate bacterial from viral infections in LRTI patients of various ages. We demonstrated the predictive value of four easy-to-collect clinical variables which facilitate personalized and accurate clinical decision-making. We observed that nasal cavity microbiota correlate with the clinical variables and thus may not add significant value to diagnostic algorithms that aim to differentiate bacterial from viral infections.

A host signature based on TRAIL, IP-10, and CRP for reducing antibiotic overuse in children by differentiating bacterial from viral infections: a prospective, multicentre cohort study.

OBJECTIVES: Identifying infection aetiology is essential for appropriate antibiotic use. Previous studies have shown that a host-protein signature consisting of TNF-related apoptosis-induced ligand (TRAIL), interferon-γ-induced protein-10 (IP-10), and C-reactive protein (CRP) can accurately differentiate bacterial from viral infections. METHODS: This prospective, multicentre cohort study, entitled AutoPilot-Dx, aimed to validate signature performance and to estimate its potential impact on antibiotic use across a broad paediatric population (>90 days to 18 years) with respiratory tract infections, or fever without source, at emergency departments and wards in Italy and Germany. Infection aetiology was adjudicated by experts based on clinical and laboratory investigations, including multiplex PCR and follow-up data. RESULTS: In total, 1140 patients were recruited (February 2017-December 2018), of which 1008 met the eligibility criteria (mean age 3.5 years, 41.9% female). Viral and bacterial infections were adjudicated for 628 (85.8%) and 104 (14.2%) children, respectively; 276 patients were assigned an indeterminate reference standard outcome. For the 732 children with reference standard aetiology, the signature discriminated bacterial from viral infections with a sensitivity of 93.7% (95%CI 88.7-98.7), a specificity of 94.2% (92.2-96.1), positive predictive value of 73.0% (65.0-81.0), and negative predictive value of 98.9% (98.0-99.8); in 9.8% the test results were equivocal. The signature performed consistently across different patient subgroups and detected bacterial immune responses in viral PCR-positive patients. CONCLUSIONS: The findings validate the high diagnostic performance of the TRAIL/IP-10/CRP signature in a broad paediatric cohort, and support its potential to reduce antibiotic overuse in children with viral infections.

Differential Serum and Urine CRP, IP-10, and TRAIL Levels in Pediatric Urinary Tract Infection.

Background: It is estimated that clinical evaluation and urinalysis are unable to diagnose >10% of urinary tract infections (UTI) in young children. TNF-related apoptosis induced ligand (TRAIL), interferon gamma induced protein-10 (IP-10), and C-reactive protein (CRP) exhibit differential expression in the blood in response to bacterial vs. viral infection. We assessed if the urinary and serum levels of these host biomarkers discriminate UTI, nephronia, and response to antibiotic treatment. Methods: Hospitalized febrile children aged <18 years with suspected UTI based on abnormal urinalysis were recruited prospectively between 2016 and 2018; also, non-febrile controls were recruited. Following urine culture results and hospitalization course, participants were divided into three groups based on AAP criteria and expert adjudication: UTI, viral infection, and indeterminate. Results: Seventy-three children were enrolled, 61 with suspected UTI and 12 non-febrile controls. Of the 61 with suspected UTI, 40 were adjudicated as UTI, 10 viral infection, and 11 as indeterminate. Urinary CRP and IP-10 levels were significantly higher in the UTI group (p ≤ 0.05). Urinary CRP differentiated UTI from non-bacterial etiology in children under and over 3 months of age, with AUCs 0.98 (95% CI: 0.93-1.00) and 0.82 (0.68-0.95), respectively. Similarly, urinary IP-10 discriminated with AUCs of 0.80 (0.59-1.00) and 0.90 (0.80-1.00), respectively. Serum CRP and IP-10 levels were significantly higher in UTI cases with nephronia (p ≤ 0.03). UTI-induced changes in the levels of urinary and serum biomarkers resolved during recovery. Conclusions: CRP, IP-10, and TRAIL represent biomarkers with potential to aid the clinician in diagnosis and management of UTI.

Observational cohort study of IP-10's potential as a biomarker to aid in inflammation regulation within a clinical decision support protocol for patients with severe COVID-19.

BACKGROUND: Treatment of severely ill COVID-19 patients requires simultaneous management of oxygenation and inflammation without compromising viral clearance. While multiple tools are available to aid oxygenation, data supporting immune biomarkers for monitoring the host-pathogen interaction across disease stages and for titrating immunomodulatory therapy is lacking. METHODS: In this single-center cohort study, we used an immunoassay platform that enables rapid and quantitative measurement of interferon γ-induced protein 10 (IP-10), a host protein involved in lung injury from virus-induced hyperinflammation. A dynamic clinical decision support protocol was followed to manage patients infected with severe acute respiratory syndrome coronavirus 2 and examine the potential utility of timely and serial measurements of IP-10 as tool in regulating inflammation. RESULTS: Overall, 502 IP-10 measurements were performed on 52 patients between 7 April and 10 May 2020, with 12 patients admitted to the intensive care unit. IP-10 levels correlated with COVID-19 severity scores and admission to the intensive care unit. Among patients in the intensive care unit, the number of days with IP-10 levels exceeding 1,000 pg/mL was associated with mortality. Administration of corticosteroid immunomodulatory therapy decreased IP-10 levels significantly. Only two patients presented with subsequent IP-10 flare-ups exceeding 1,000 pg/mL and died of COVID-19-related complications. CONCLUSIONS: Serial and readily available IP-10 measurements potentially represent an actionable aid in managing inflammation in COVID-19 patients and therapeutic decision-making. TRIAL REGISTRATION: Clinicaltrials.gov, NCT04389645, retrospectively registered on May 15, 2020.

Antibiotic misuse in respiratory tract infections in children and adults-a prospective, multicentre study (TAILORED Treatment).

Respiratory tract infections (RTI) are more commonly caused by viral pathogens in children than in adults. Surprisingly, little is known about antibiotic use in children as compared to adults with RTI. This prospective study aimed to determine antibiotic misuse in children and adults with RTI, using an expert panel reference standard, in order to prioritise the target age population for antibiotic stewardship interventions. We recruited children and adults who presented at the emergency department or were hospitalised with clinical presentation of RTI in The Netherlands and Israel. A panel of three experienced physicians adjudicated a reference standard diagnosis (i.e. bacterial or viral infection) for all the patients using all available clinical and laboratory information, including a 28-day follow-up assessment. The cohort included 284 children and 232 adults with RTI (median age, 1.3 years and 64.5 years, respectively). The proportion of viral infections was larger in children than in adults (209(74%) versus 89(38%), p < 0.001). In case of viral RTI, antibiotics were prescribed (i.e. overuse) less frequently in children than in adults (77/209 (37%) versus 74/89 (83%), p < 0.001). One (1%) child and three (2%) adults with bacterial infection were not treated with antibiotics (i.e. underuse); all were mild cases. This international, prospective study confirms major antibiotic overuse in patients with RTI. Viral infection is more common in children, but antibiotic overuse is more frequent in adults with viral RTI. Together, these findings support the need for effective interventions to decrease antibiotic overuse in RTI patients of all ages.

Clinically significant sub-clonality for common drivers can be detected in 26% of KRAS/EGFR mutated lung adenocarcinomas.

Genetic sub-clonality has been described in multiple malignancies, however the presence of sub-clonality for major drivers in lung adenocarcinoma and its clinical significance is a subject under debate. Using molecular and morphometric approach, 347 lung adenocarcinoma samples were analyzed for KRAS and EGFR sub-clonality, which was further correlated with clinical and pathological variables.KRAS and EGFR mutations were identified in 100 (29%) and 82 (23%) cases, respectively. One hundred and forty four KRAS or EGFR positive cases were also available for morphometric analysis, among which 37 (26%) were defined as sub-clonal. The presence of sub-clonality was associated with shorter survival time (p=0.02). Interestingly, cases with sub-clonality were also associated with earlier disease stage (89% vs 66% stage I disease in sub-clonal vs clonal cases, respectively, p=0.01) and less lymph node involvement (8% vs 25% in sub-clonal vs clonal cases, respectively, p=0.02). Our findings demonstrate the presence of sub-clonality for mutations in common drivers in lung adenocarcinoma and link it both to earlier disease stage and to poor survival. These findings are in line with the different evolutionary models that can present with genetic sub-clonality.

Risk for molecular contamination of tissue samples evaluated for targeted anti-cancer therapy.

With the increasing usage of sensitive PCR technology for pharmacogenetics, cross contamination becomes a significant concern. Researchers employed techniques which basically include replacing laboratory equipment after each sample preparation; however, there are no recommended guidelines. In the present work we wanted to evaluate the risk of cross contamination during tissue processing using the routine precaution measures. Twenty-one surgical samples of lung adenocarcinoma were used, of which 7 contained EGFR exon 19 mutation, 7 contained EGFR exon 21 mutation (p.L858R) and 7 were EGFR wild-type. The samples were ordered by alternating the mutation group to maximize the potential for cross contamination and underwent tissue sectioning and de-paraffinization. The entire process was performed using the same tools. Following DNA extraction all samples underwent PCR amplification and were scrutinized for small fractions of EGFR mutation using deep sequencing with the Ion torrent PGM technology. Twenty samples yielded results. The fraction of mutated copies was 41 ± 23% (range 11-66) for the cases with known exon 19 mutation and 48±24% (range 0-65) for the cases with known exon 21 mutations. No in-frame exon 19 deletion mutations were identified in the wild-type (WT) and exon 21 groups. The fraction of EGFR exon 21 (codon 858) mutations was 0.018±0.014% (range 0-0.05%) in the WT and exon 19 groups, which was not statistically different than the background sequencing artifact noise for the same base-pair alteration (p = 0.21). Our results suggest that standard precautions are sufficient for molecular pathology diagnosis of surgical samples and are not associated with increased risk of cross contamination.

Subclonality for BRAF Mutation in Papillary Thyroid Carcinoma Is Associated With Earlier Disease Stage.

CONTEXT: The presence of driver mutations only in a subset of tumor cells within a single lesion, defined as subclonality, is being appreciated as a clinically significant factor. BRAF mutation is the most common driver mutation in papillary thyroid carcinoma (PTC). There are conflicting data in the literature regarding the presence of BRAF mutation subclonality in PTC and its clinical significance. OBJECTIVE: The purpose of the present study was to use a molecular and morphometric approach to determine BRAF clonality status and its clinical-pathological correlates. DESIGN: Fifty-nine cases of PTC were studied. DNA extracted from the tumors underwent deep sequencing to determine the percentage of BRAF mutant allele copies. Additionally, we used computerized morphometry to determine the fraction of tumor cells in each sample. Using both variables, we were able to determine the presence or absence of subclonality for BRAF mutation, which was further correlated with clinical, pathological, and prognostic data. RESULTS: BRAF mutation was found in 49 (83%) cases. The average percentage of tumor cells and of BRAF mutant alleles in the samples were 68.1 ± 9.8 and 26 ± 6.7, respectively. Based on the molecular and morphometric analysis, 11 (24%) cases were found to be subclonal for BRAF mutation. Tumors with subclonal BRAF mutations were significantly smaller compared to tumors with clonal mutation (0.82 ± 0.38 cm vs 1.37 ± 0.57 cm, P = .005) and were less likely to have lymph node metastasis (0% vs 32%, P = .03). CONCLUSIONS: In PTC, subclonality for BRAF mutation is associated with earlier stage. Molecular-morphometric analysis of PTC can provide clonality information with potential clinical significance.

Variation in KRAS driver substitution distributions between tumor types is determined by both mutation and natural selection.

Different tumor types vary greatly in their distribution of driver substitutions. Here, we analyzed how mutation and natural selection contribute to differences in the distribution of KRAS driver substitutions between lung, colon and pancreatic adenocarcinomas. We were able to demonstrate that both differences in mutation and differences in selection drive variation in the distribution of KRAS driver substitutions between tumor types. By accounting for the effects of mutation on the distribution of KRAS driver substitutions, we could identify specific KRAS driver substitutions that are more favored by selection in specific tumor types. Such driver substitutions likely improve fitness most when they occur within the context of the tumor type in which they are preferentially favored. Fitting with this, we found that driver substitutions that are more favored by natural selection in a specific type of tumor tend to associate with worse clinical outcomes specifically in that type of tumor.

Intratumor Heterogeneity of KRAS Mutation Status in Pancreatic Ductal Adenocarcinoma Is Associated With Smaller Lesions.

OBJECTIVES: Recent studies have demonstrated intratumor heterogeneity (ITH) for genetic mutations in various tumors and suggest that ITH might have significant clinical impact. Because KRAS is the most commonly mutated oncogene in pancreatic ductal adenocarcinoma and has an important role in pancreatic carcinogenesis, the purpose of this study was to evaluate ITH for KRAS gene mutation and its clinical significance. METHODS: Deep sequencing of 47 pancreatic ductal adenocarcinoma cases was used to determine the fraction of KRAS mutant alleles. In addition, computerized morphometry was used to calculate the fraction of tumor cells. After analysis of both results, cases were classified as ITH or as having amplification of mutant KRAS. The presence of ITH was correlated with clinical and pathological factors. RESULTS: KRAS mutation was found in 38 (81%) cases, of which 12 (32%) showed ITH and 9 (23%) were found to have KRAS mutant allele amplification. The presence of ITH was associated with smaller tumors, whereas amplification was associated with higher tumor diameter. CONCLUSIONS: In pancreatic ductal adenocarcinoma, ITH for KRAS gene mutation was associated with smaller tumors. It is possible that, as the tumor progresses, more cells carry this mutation, which leads to more aggressive tumor features.

A deep penetrating facial congenital melanocytic tumor with bone involvement and ipsilateral eye blindness.

Bone involvement has been described in tumors with melanocytic differentiation such as melanotic neuroectodermal tumor of infancy, and very rarely in cellular blue nevi and neurocristic cutaneous hamartoma. We present an unusual case of facial congenital melanocytic tumor that involved the underlying bones and maxillary sinus and led to unilateral blindness. A newborn with a large red bluish patch with peripheral brown and black macules overlying marked swelling on the left side of his face was presented. The tumor was shown by magnetic resonance imaging, scintigraphy, and histopathology to invade the underlying bones and maxillary sinus and to compress the left eyeball resulting in blindness. Histopathology, immunohistochemistry, morphometric computerized microscopy, molecular genetic mutation analysis, and fluorescent in situ hybridization studies were more congruent with a melanocytic nevus. An 8.5-year follow-up was uneventful, with spontaneous partial shrinkage of the tumor.

Adenoma and carcinoma components in colonic tumors show discordance for KRAS mutation.

Activating mutations in KRAS are common events in the pathogenesis of colorectal carcinoma and predict response to treatment with anti-EGFR antibodies. Molecular pathology testing for KRAS mutations has become the standard of practice for patients with metastatic colorectal carcinoma. Despite the known histologic and molecular differences between adenomas and carcinomas, the concordance of KRAS mutation between adenomas and carcinomas has not been established leaving some open questions regarding the appropriate choice of tissue for KRAS mutation analysis and correct interpretation of the test results. To address these questions, we analyzed the concordance of KRAS mutation in 70 tumors that contained both adenoma and carcinoma components (2 cases of intramucosal carcinoma, 66 cases with invasion of the submucosa, and 2 invading the muscularis propria). For each case, DNA was separately isolated from the adenoma and the carcinoma component and analyzed for KRAS mutation using direct sequencing. Overall, 30 (43%) of the adenoma cases and 36 (51%) of the carcinoma cases were positive for KRAS mutation. Of the 70 cases, 16 (23%) showed discordant results. Interestingly, the fraction of discordant cases went down as the depth of carcinoma invasion increased. In summary, we identified significant KRAS mutation discordance between the adenoma and carcinoma component of the lesion. Our results suggest that effort should be made to analyze only the invasive component of the lesion and that caution should be taken when interpreting a result based on DNA extracted from noninvasive elements.

Pulse-chase analysis to measure protein degradation.

In order to measure protein half-life by pulse-chase analysis, cells are incubated with labeled precursors, [(35)S]-methionine and [(35)S]-cysteine, for a short time (pulse), after which they are washed from the labeled compound and incubated with excess cold precursor (chase). The decay of the amount of protein labeled during the pulse period is measured during the chase with cold precursor.

Phosphorylation of the cyclin CaPcl5 modulates both cyclin stability and specific recognition of the substrate.

The Candida albicans cyclin CaPcl5 activates the cyclin-dependent kinase Pho85 and induces phosphorylation of the transcription factor CaGcn4, leading to its degradation. The high substrate specificity of the CaPcl5/Pho85 complex provides the opportunity to study the determinants of substrate selectivity of cyclins. Mutational analysis of CaPcl5 suggests that residues in a predicted α-helix at the N-terminal end of the cyclin box, as well as in helix I of the cyclin box, play a role in specific substrate recognition. Similar to Saccharomyces cerevisiae Pcl5, we show here that CaPcl5 induces its own phosphorylation at two adjacent sites in the N-terminal region of the protein and that this phosphorylation causes degradation of the cyclin in vivo via the SCF(CDC4) ubiquitin ligase. Remarkably, however, in vitro studies reveal that this phosphorylation also results in a loss of specific substrate recognition, thereby providing an additional novel mechanism for limiting cyclin activity.

Normal colon tissue and colon carcinoma show no difference in heparanase promoter methylation.

INTRODUCTION: Heparanase, the sole heparan sulfate degrading enzyme, has a role in cellular invasion. Accordingly, a large number of studies have demonstrated an association between heparanase expression and tumor stage and patients' prognosis. In colon carcinoma, heparanase shows increased expression in tumor compared to normal tissue and its expression correlates with the presence of metastasis. One of the regulatory mechanisms of heparanase expression is de-methylation on its promoter. In the present study we evaluated the role of heparanase promoter methylation in colon carcinoma. MATERIAL AND METHODS: Analysis of heparanase promoter methylation was done on 32 samples of colon carcinoma as well as 30 samples of normal colonic mucosa. DNA was extracted from FFPE tissue and subjected to bisulfite conversion. The relative fraction of methylated and unmethylated DNA was evaluated using quantitative real-time PCR. RESULTS: The fraction of methylated DNA was 1 ± 3.4% in the colon carcinoma group, and 2.5 ± 3.3% in the normal colon group (P=0.11). Only one case in the normal group and one case in the tumor group showed more than 10% methylation in the heparanase promoter. CONCLUSION: We did not find any significant difference in heparanase promoter methylation between colon carcinoma and normal colonic mucosa, suggesting that heparanase overexpression in colon carcinoma is mediated by other mechanisms.

KRAS mutation positive mucinous adenocarcinoma originating in mature ovarian teratoma: case report and review of literature.

Mature ovarian teratomas rarely undergo transformation into malignancy. Carcinomas, mostly squamous cell carcinoma, are the most common malignancy arising in mature cystic teratoma. In the present report we describe a 13-year-old girl who developed a large mass in her ovary. Fine needle biopsy identified intestinal type mucinous adenocarcinoma, which was also identified in the full surgical specimen. Extensive sampling of the surgical specimen also identified areas of mature cystic teratoma. Interestingly, molecular analysis of DNA extracted from various components of the lesion identified KRAS mutation in the carcinoma, borderline mucinous tumor and benign intestinal-type epithelium but not in the epidermal component of the teratoma. To the best of our knowledge this is the first report of KRAS mutation in mucinous carcinoma originating in mature cystic teratoma. We discuss the importance of extensive tissue sampling to differentiate between carcinoma originating in teratoma and metastatic colorectal carcinoma to the ovary. Additionally, the identification of KRAS mutation in the morphologically benign intestinal-type epithelium indicated that it is an early event in the carcinogenic sequence and that the molecular pathway of carcinogenesis in teratoma is similar to that in the carcinogenic process of somatic tissue.