No evidence of the APC D1822V missense variant's pathogenicity in Tunisian patients with sporadic colorectal cancer

Objectives

Sporadic colorectal cancer is influenced by numerous single nucleotide polymorphisms (SNPs), each with minor effects on the cancer risk. This study seeks to determine whether there is any association of the I1307K, E1317Q and D1822V variants within the Adenomatous polyposis coli gene (APC) and risk to develop colorectal cancer in Tunisian population.

Methods

Direct sequencing was used to investigate three SNPs in the APC in 48 Tunisian sporadic colorectal cancer cases and 63 controls.

Results

There was no statistically significant association between the I1307K, E1317Q and D1822V variants investigated and colorectal cancer risk.

Conclusion

The lack of association may show that these variants selected for this study are not involved in the colorectal carcinogenic process. Otherwise, the eventual biological effect is so little to go undetected, unless increasing the sample size.     

Competitive allele specific TaqMan PCR for KRAS, BRAF and EGFR mutation detection in clinical formalin fixed paraffin embedded samples

Background

The development of targeted therapies has created a need for robust molecular characterization of cancer and it has become a challenge to validate methods to ensure accuracy in tumor mutation testing.

Methods

The current study was designed to evaluate KRAS, BRAF and EGFR genotyping by Competitive Allele Specific hydrolysis probes (TaqMan) PCR technology (CAST), on suboptimal formalin fixed paraffin embedded (FFPE) tumor samples. Assays were calibrated on FFPE samples and a minimal quantification cycle (Cq) cut-off was determined to standardize analyses and avoid over-interpretation of degraded material. Sensibility, specificity and blinded clinical sample screenings (n = 63) were evaluated.

Results

CAST PCR allowed efficient amplification of FFPE samples, probes were highly specific and all assays had a sensibility inferior to 1% except for the EGFR p.T790M assay. 60/63 samples were correctly typed. The three missed mutations were EGFR exon 19 deletions that were not recognized by the DEL19 assays that were used.

Conclusions

This technology is less laborious and prevent crossover of PCR products as compared to multistep methods. TaqMan® Mutation Detection assay is an important technology to consider in the field of mutation detection for KRAS, BRAF and EGFR point mutation screening. Assay calibration on FFPE samples may prevent erroneous interpretations that will ultimately harm clinical oncology practice.     

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.     

Implications of MGMT methylation status in pituitary adenoma

Background

Very little literature exists on frequency of MGMT methylation status in pituitary adenoma. We assessed the frequency of MGMT methylation and protein expression in pituitary adenoma and correlated with patients’ age group and prognosis.

Methods

Tumor tissues from 30 patients with pituitary adenoma were evaluated for MGMT promoter methylation status by methylation specific PCR method. All tumors were also immunostained with MIB-1, anti-p53 and anti-MGMT antibodies.

Results

MGMT methylation status was noted in 40% of cases (7/20 non-functioning adenomas and 5/10 functioning adenomas). MGMT protein expression on IHC was noted in 72.2% of unmethylated tumors, while only 41.6% of methylated tumors demonstrated protein expression. The mean labeling index of MGMT protein was higher in unmethylated tumors as compared to the methylated group, though the difference was not statistically significant (18.6% vs 8.8%; p = 0.131). Tumor regrowth occurred in four unmethylated tumors as compared to none in methylated group. Even though there was no correlation between patient age and MGMT methylation status (p = 0.823), we noted that the frequency of methylation in middle age patients (between 30 and 59 yrs) was 64.7%, which was higher compared to other age groups.

Conclusion

This is the first study from India showing MGMT promoter methylation status with protein expression in pituitary adenoma. We noted that tumor regrowth was higher in unmethylated tumors.     

Foxp3 methylation status in children with primary immune thrombocytopenia

Aim

To investigate the status of DNA methylation in the Foxp3 promoter in pediatric ITP patients and assess the role of DNA methylation of Treg cells in the pathogenesis of ITP.

Methods

Quantitative DNA methylation levels of Foxp3 promoter in pediatric ITP patients were detected by MassARRAY EpiTYPER. Methylation levels of Foxp3 promoter were analyzed in ITP patients and normal controls.

Results

Significantly higher expression of CpG-2, CpG-3 and CpG-11.12 was observed in ITP patients compared to the controls. A subgroup analysis revealed that persistent and chronic ITP patients exhibited significantly higher CpG-6 expression than in the subgroup of newly diagnosed ITP patients. All patients who represented newly diagnosed ITP at admission were reclassified at later follow-up. In this follow-up subgroup analysis, there were significantly higher levels of CpG-6 in the persistent ITP group than that in the newly diagnosed ITP group.

Conclusions

Our results indicate that defective Treg cell activity identified in ITP might be partially mediated through hypermethylation of CpG sites in the promoter region of Foxp3.     

Comparison of different tissue sampling methods for protein extraction from formalin-fixed and paraffin-embedded tissue specimens

Aims

Protein extracts from formalin-fixed and paraffin-embedded (FFPE) tissue for proteomic analysis has recently gained attention. In this study, we explored the possibility to standardize tissue sampling from paraffin blocks and compared the protein extracts with those obtained from fresh frozen material.

Materials and methods

Fresh frozen and FFPE material was obtained from five patients with pancreatic ductal adenocarcinoma either by cutting sections with a microtome or by stamping a cylinder with tissue micro-array technology. All samples were weighed, forwarded to protein extraction and analyzed by polyacrylamide gel electrophoresis and Western blotting. Immunohistochemistry allocated proteins in tissue sections.

Results

Sampling of tissue was highly reproducible, as assessed by sample weight. While protein concentrations were significantly higher in fresh frozen material compared to FFPE material, equal amounts of protein were extracted from FFPE using either paraffin sections or core cylinders in SDS-PAGE, all three procedures showed comparable protein patterns. In Western blotting, annexin I had the same molecular weight independent of the sample source and sampling procedure.

Conclusions

The sampling of FFPE specimens for protein extraction and analysis can be standardized, uncovering equal amounts of tissue and protein. In addition, the proteins extracted from FFPE tissue seem to be the same compared with those extracted from fresh frozen tissue.     

Traceability and distribution of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> DNA in archived post mortem tissue samples from patients with systemic meningococcal disease

Background

The pathophysiology and outcome of meningococcal septic shock is closely associated with the plasma level of N. meningitidis lipopolysaccharides (LPS, endotoxin) and the circulating level of meningococcal DNA. The aim of the present study was to quantify the number of N. meningitidis in different formalin-fixed, paraffin-embedded (FFPE) tissue samples and fresh frozen (FF) tissue samples from patients with systemic meningococcal disease (SMD), to explore the distribution of N. meningitidis in the body.

Methods

DNA in FFPE and FF tissue samples from heart, lungs, liver, kidneys, spleen and brain from patients with meningococcal shock and controls (lethal pneumococcal infection) stored at variable times, were isolated. The bacterial load of N. meningitidis DNA was analyzed using quantitative real-time PCR (qPCR) and primers for the capsule transport A (ctrA) gene (1 copy per N. meningitidis DNA). The human beta-hemoglobin (HBB) gene was quantified to evaluate effect of the storage times (2-28 years) and storage method in archived tissue.

Results

N. meningitidis DNA was detected in FFPE and FF tissue samples from heart, lung, liver, kidney, and spleen in all patients with severe shock. In FFPE brain, N. meningitidis DNA was only detected in the patient with the highest concentration of LPS in the blood at admission to hospital. The highest levels of N. meningitidis DNA were found in heart tissue (median value 3.6 × 10⁷ copies N. meningitidis DNA/μg human DNA) and lung tissue (median value 3.1 × 10⁷ copies N. meningitidis DNA/μg human DNA) in all five patients. N. meningitidis DNA was not detectable in any of the tissue samples from two patients with clinical meningitis and the controls (pneumococcal infection). The quantity of HBB declined over time in FFPE tissue stored at room temperature, suggesting degradation of DNA.

Conclusions

High levels of N. meningitidis DNA were detected in the different tissue samples from meningococcal shock patients, particularly in the heart and lungs suggesting seeding and major proliferation of meningococci in these organs during the development of shock, probably contributing to the multiple organ failure. The age of archived tissue samples appear to have an impact on the amount of quantifiable N. meningitidis DNA.

     

Detection of extended-spectrum ß-lactamase-producing Enterobacteriaceae in rectal swabs with the Mastdiscs™ ID AmpC ßLSE detection set

Objectives

The double-disk synergy test was compared to the Mastdiscs™ ID AmpC and ESßL method for detection of ESßL production in rectal swab.

Methods

Two hundred and forty-nine rectal swabs were directly inoculated onto Mueller-Hinton plates and analyzed according to both methods.

Results

A total of 41 (16%) and 208 (84%) were positive and negative for ESßL, respectively. Twelve (29%) and 20 (49%) of the 41 rectal swabs positive for ESßL were detected after 24 h of incubation with the double-disk synergy test and the Mastdiscs™ method, respectively (P = 0.013). One hundred fifty-eight (76%) et 183 (88%) of the 208 rectal swabs were detected negative for ESßL after 24 h of incubation with the double-disk synergy test and the Mastdiscs™ method, respectively (P < 0.001). Finally, 79 (32%) and 46 (18%) rectal swabs respectively inoculated according to the double-disk synergy test and the Mastdiscs™ method were inconclusive after 24 h of incubation. The better performance of the Mastdiscs™ method was due to an easier detection of cephalosporinase producing bacteria.

Conclusions

The Mastdiscs™ method is a simple phenotypic method that detects more easily ESßL and non-ESßL producing bacteria in rectal swab.     

An evidence base for patient-centered cancer care: A meta-analysis of studies of observed communication between cancer specialists and their patients

Objective

In the context of patients visiting cancer specialists, the objective is to test the association between both patient-centered communication (including Affective Behavior and Participation Behavior) and Instrumental Behavior and patients’ post-visit satisfaction with a variety of visit phenomena.

Methods

Meta-analysis of 25 articles representing 10 distinct data sets.

Results

Both patient-centered- and instrumental behavior are significantly, positively associated with satisfaction, with patient-centered communication having a relatively stronger association.

Conclusion

There is an evidence base for the efficacy of patient-centered care.

Practice implications

Cancer specialists need to train to improve their patient-centered communication.     

Estrogen receptor β promoter methylation: a potential indicator of malignant changes in breast cancer

Introduction

Estrogen receptor β (ERβ) always lacks expression in estrogen-dependent tumors, which may result from gene inactivation by methylation. In this study, we aimed to determine whether aberrant methylation of the ERβ promoter is associated with decreased ERβ gene expression in breast cancer.

Material and methods

ERβ methylation status was determined for 132 pairs of breast cancer and adjacent normal tissues via the MethyLight method. Additionally, mRNA relative expression was quantified by real-time polymerase chain reaction (RT-PCR) to determine whether aberrant methylation had a negative correlation with expression. The correlation of ERβ promoter methylation and clinical parameters is also discussed.

Results

Methylation was observed in 96 (72.7%) breast cancer samples, and the median percentage of fully methylated reference (PMR) among methylated tissues was 0.83. Meanwhile, 94 (71.2%) adjacent normal tissues were methylated and the median PMR was 0.48. Compared to adjacent normal tissues, the methylation level of breast cancer was significantly higher (p < 0.001) and mRNA expression was much lower (p < 0.001). There was a significant correlation between ERβ methylation and mRNA expression in adjacent normal breast tissues (p = 0.004). In addition, the methylation rate of cancer tissues whose maximum diameter < 3 cm was significantly higher than those > 3 cm (p = 0.025).

Conclusions

ERβ promoter methylation level varies between cancerous and adjacent normal breast tissues. There was significant downregulation of ERβ methylation expression in pre-cancerous stages of breast cancer. Therefore, demethylation drugs may offer a potential strategy for preventing the development of pre-cancerous cells.     

DNA methylation profiling reveals novel diagnostic biomarkers in renal cell carcinoma

Background

Renal cell carcinoma (RCC) is the tenth most commonly diagnosed cancer in the United States. While it is usually lethal when metastatic, RCC is successfully treated with surgery when tumors are confined to the kidney and have low tumor volume. Because most early stage renal tumors do not result in symptoms, there is a strong need for biomarkers that can be used to detect the presence of the cancer as well as to monitor patients during and after therapy.

Methods

We examined genome-wide DNA methylation alterations in renal cell carcinomas of diverse histologies and benign adjacent kidney tissues from 96 patients.

Results

We observed widespread methylation differences between tumors and benign adjacent tissues, particularly in immune-, G-protein coupled receptor-, and metabolism-related genes. Additionally, we identified a single panel of DNA methylation biomarkers that reliably distinguishes tumor from benign adjacent tissue in all of the most common kidney cancer histologic subtypes, and a second panel does the same specifically for clear cell renal cell carcinoma tumors. This set of biomarkers were validated independently with excellent performance characteristics in more than 1,000 tissues in The Cancer Genome Atlas clear cell, papillary, and chromophobe renal cell carcinoma datasets.

Conclusions

These DNA methylation profiles provide insights into the etiology of renal cell carcinoma and, most importantly, demonstrate clinically applicable biomarkers for use in early detection of kidney cancer.

     

JK1 (FAM134B) gene and colorectal cancer: A pilot study on the gene copy number alterations and correlations with clinicopathological parameters

Aims

The aims of the study are to characterize changes in JK-1 (FAM134B) at the DNA level in colorectal adenocarcinoma and adenoma and exploring the possible correlations with clinical and pathological features.

Method

JK-1 gene DNA copy number changes were studied in 211 colorectal carcinomas, 32 colorectal adenoma and 20 colorectal non-cancer colorectal tissue samples by real-time quantitative polymerase chain reaction. The results were correlated with clinical and pathological parameters.

Results

Colorectal adenomas were more likely to be amplified than deleted with regard to JK-1 (FAM134B) DNA copy number change. The copy number level of JK-1 (FAM134B) DNA in colorectal adenocarcinomas was significantly lower in comparison to colorectal adenomas. Changes in JK-1 (FAM134B) DNA copy number were associated with histological subtypes, and cancer stage. Lower copy numbers were associated with higher tumor stage, lymph node stage and overall pathological stage of cancer. Conversely, higher DNA copy numbers were detected more often in the mucinous adenocarcinoma.

Conclusions

This is the first study showing significant correlations of the JK-1 (FAM134B) gene copy number alterations with clinical and pathological features in a large cohort of pre-invasive and invasive colorectal malignancies. The changes in DNA copy number associated with progression of colorectal malignancies reflect that JK-1 (FAM134B) gene could play a role in controlling some steps in development of the invasive phenotypes.     

Implementing patient decision support tools: Moving beyond academia?

Objective

To ascertain the feasibility of implementing three decision support tools (DSTs) for people with rectal cancer within the surgical consultation.

Methods

Twenty colorectal surgeons participated in a focus group or individual interviews. Colorectal surgeons were also asked to complete a self-administered questionnaire.

Results

All surgeons responded encouragingly to the concept of DSTs. However, for every positive statement an accompanying caveat was made and these were either a criticism of each tool or a barrier to their implementation. Surgeons stated DSTs should be used by patients and surgeons together (80%). The majority (70–75%) thought each tool was ‘useful’ or ‘extremely useful’. However, there were strong views that in their current form the DSTs would not feasible to be used within the surgical consultation. Time restraints, personal and clinical characteristics of the patient, the content of each tool, the potential negative impact on the doctor–patient relationship were noted as real barriers to their implementation.

Conclusion

Surgeons have identified a number of barriers that may limit implementation of DSTs into routine clinical practice.

Practice implications

Feasibility and implementation studies have the potential to provide important information to help guide development, evaluation and implementation of DSTs.     

Adaptive Conjoint Analysis as individual preference assessment tool: Feasibility through the internet and reliability of preferences

Objective

Patient values are not routinely assessed in clinical practice. Adaptive Conjoint Analysis (ACA) is increasingly applied in studies assessing treatment preferences, and could provide a means to routinely assess individual patients’ treatment preferences.

Methods

An ACA-questionnaire was administered three times (7–10 days apart) to 98 long-term rectal cancer survivors either on a portable computer or through internet, to assess whether (a) responses differ according to administration mode, (b) relative importances of rectal cancer treatment outcomes (survival, local control, incontinence, sexual problems) consolidate over time, (c) ACA-outcomes are sufficiently reliable (ICC) for use in individual decision-making. We also evaluated patients’ acceptance of ACA.

Results

Mode did not affect ACA-completion or evaluation. Importance scores did not consolidate over time. ICCs were poor for sexual problems and fair for the other outcomes, and were at least equal or higher from first to second retest. Most participants valued completing the ACA-questionnaire and learning their results.

Conclusion

Values did not show consolidation over time. ACA-derived preferences should not determine which treatment patients should choose.

Practice implications

Findings extend ACA-validation studies to the health care setting and suggest that ACA-questionnaires might be appreciated as adjuncts to treatment decision-making in newly diagnosed patients.     

Correlation of microRNA-375 downregulation with unfavorable clinical outcome of patients with glioma

Background and aim

MicroRNA-375 (miR-375) is frequently demonstrated to be frequently dysregulated and functions as a tumor suppressor or an oncogene in different cancer types. However, its roles in human gliomas have not been reported. The aim of this study was to investigate the expression pattern and clinical significance of miR-375 in patients with gliomas.

Methods

Real-time quantitative RT-PCR assay was performed to detect miR-375 expression in human gliomas and non-neoplastic brain tissues. Then, the association of miR-375 expression with clinicopathological factors and prognosis of glioma patients was also statistically analyzed.

Results

miR-375 expression was significantly decreased on average in glioma tissues relative to non-neoplastic brain tissues (P < 0.0001) with ascending pathological grade. Then, the low miR-375 expression in glioma tissues was significantly associated with advanced pathological grade (P = 0.003) and low Karnofsky performance score (KPS, P = 0.01). Moreover, both univariate and multivariate Cox regression analyses determined that loss of miR-375 expression effectively predicted the decreased overall survival in patients with gliomas.

Conclusions

These findings offer the first convinced evidence that the downregulation of miR-375 expression in human gliomas may play an inhibitory role during the tumor development. This miRNA might function as a candidate unfavorable prognostic marker for human gliomas.     

BDNF promoter methylation and suicidal behavior in depressive patients

Introduction

Suicide is a major health problem, and depression is a major psychiatric cause of suicide. Suicide is influenced by the multifactorial interaction of many risk factors. Therefore, epigenetic research may lead to understandings that are applicable to suicide. This study investigated whether epigenetic changes are associated with suicidal behavior and evaluated the treatment outcome of suicidal ideation in depressive patients.

Methods

In 108 patients with major depression, the promoter methylation of the gene encoding brain-derived neurotrophic factor (BDNF) was measured. Sociodemographic and clinical characteristics including a history of previous depressive episodes, age at onset, duration of illnesses, family history of depression, and number of stressful life events as well as subjective perception of stress and assessment scales for depression (HAMD), anxiety (HAMA), function (SOFAS), disability (WHODAS-12), and quality of life (WHOQOL-BREF) were evaluated at baseline. Suicidal behavior was ascertained using a semistructured clinical interview with questions about severity and intent. Beck Scale for Suicide Ideation (BSS) was administered during 12 weeks of treatment with antidepressants.

Results

A higher BDNF promoter methylation status was significantly associated with a previous suicidal attempt history, suicidal ideation during treatment, and suicidal ideation at last evaluation as well as with higher BSS scores and poor treatment outcomes for suicidal ideation.

Limitations

Methylation status was investigated with limited area of the BDNF gene and sample size was relatively small.

Conclusions

BDNF methylation status could be a proxy marker for previous suicidal attempts and a clinical biomarker for poor treatment outcomes of suicidal ideation in depression.