Molecular alterations of Ras‐Raf‐mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase‐Akt signaling pathways in colorectal cancers from a tertiary hospital at Kuala Lumpur,Malaysia

Molecular alterations in KRAS, BRAF, PIK3CA, and PTEN have been implicated in designing targeted therapy for colorectal cancer (CRC). The present study aimed to determine the status of these molecular alterations in Malaysian CRCs as such data are not available in the literature. We investigated the mutations of KRAS, BRAF, and PTEN, the gene amplification of PIK3CA, and the protein expression of PTEN and phosphatidylinositol 3‐kinase (PI3K) catalytic subunit (p110α) by direct DNA sequencing, quantitative real‐time PCR, and immunohistochemistry, respectively, in 49 CRC samples. The frequency of KRAS (codons 12, 13, and 61), BRAF (V600E), and PTEN mutations, and PIK3CA amplification was 25.0% (11/44), 2.3% (1/43), 0.0% (0/43), and 76.7% (33/43), respectively. Immunohistochemical staining demonstrated loss of PTEN protein in 54.5% (24/44) of CRCs and no significant difference in PI3K p110α expression between CRCs and the adjacent normal colonic mucosa (p = 0.380). PIK3CA amplification was not associated with PI3K p110α expression level, but associated with male cases (100% of male cases vs 56% of female cases harbored amplified PIK3CA, p = 0.002). PI3K p110α expression was significantly higher (p = 0.041) in poorly/moderately differentiated carcinoma compared with well‐differentiated carcinoma. KRAS mutation, PIK3CA amplification, PTEN loss, and PI3K p110α expression did not correlate with Akt phosphorylation or Ki‐67 expression. KRAS mutation, PIK3CA amplification, and PTEN loss were not mutually exclusive. This is the first report on CRC in Malaysia showing comparable frequency of KRAS mutation and PTEN loss, lower BRAF mutation rate, higher PIK3CA amplification frequency, and rare PTEN mutation, as compared with published reports.     

Tandem pore domain K+‐channel TASK‐3 (KCNK9) and idiopathic absence epilepsies

Recently, the gene coding for the tandem pore domain K⁺‐channel TASK‐3 (KCNK9) has been localized to the chromosomal region 8q24. Because mutations in ion channel genes have been recognized as an important factor in the etiology of abnormal neuronal excitability, TASK‐3 is an interesting candidate gene for epilepsies linked to 8q24. We therefore performed a mutation analysis of the TASK‐3 gene in 65 patients with childhood and juvenile absence epilepsy. Only one silent nucleotide exchange (636C/T) was detected in exon 2 of the TASK‐3 coding region. No evidence for an allelic association was found between the exon 2 polymorphism and absence epilepsy. Accordingly, genetic variation of the TASK‐3 coding region does not play a major role in the etiology of idiopathic absence epilepsies. © 2002 Wiley‐Liss, Inc.     

SPINK5 is associated with early‐onset and CHI3L1 with late‐onset atopic dermatitis

We have recently showed that filaggrin (FLG) mutations are associated only with early‐onset of AD, but not with late‐onset of AD. Consequently, other susceptibility genes should receive attention, especially in patients with late‐onset of AD. Our aim was to assess the associations between development of AD and the polymorphisms rs2303067 in SPINK5 and rs490928 in CHI3L1. A study population of 241 AD patients and 164 healthy controls was genotyped for two polymorphisms (rs2303067 in SPINK5 and rs490928 in CHI3L1). Rs2303067 in SPINK5 was significantly associated with early‐onset AD (≤8 years: p = .003; OR = 2.57) and was characterized by the need for hospitalization (p = .006; OR = 2.76), prolonged duration (≥10 years; p = .008; OR = 2.32) and more body parts affected (p = .015; OR = 2.01). In contrast, rs490928 in CHI3L1 was associated with late‐onset AD (>8 years: p = .048; OR = 1.65) and was characterized by no need for hospitalization (p = .049; OR = 1.59), shorter duration (<10 years; p = .017; OR = 1.94) and fewer body parts affected (p = .049; OR = 1.75). Our results confirmed that different AD phenotypes, specifically early‐ and late‐onset AD, have different genetic backgrounds. Early‐onset AD was associated with rs2303067 in SPINK5, which is involved in skin barrier functioning, and late‐onset was associated with rs4950928 in CHI3L1, which is involved in the immune response. Future studies should examine the early‐ versus late‐onset subgrouping more closely.     

Expression of miR‐155 and miR‐126 in situ in cutaneous T‐cell lymphoma

Recently, miR‐155 has been implicated in cutaneous T‐cell lymphoma (CTCL). Thus, elevated levels of miR‐155 were observed in skin lesions from CTCL patients as judged from qPCR and micro‐array analysis and aberrant, high miR‐155 expression was associated with severe disease. Moreover, miR‐155 promoted proliferation of malignant T cells in vitro. Little is, however, known about which cell types express miR‐155 in vivo in CTCL skin lesions. Here, we study miR‐155 expression using in situ hybridization (ISH) with a miR‐155 probe, a negative control (scrambled), and a miR‐126 probe as a positive control in nine patients with mycosis fungoides, the most frequent subtype of CTCL. We provide evidence that both malignant and non‐malignant T cells stain weakly to moderately positive with the miR‐155 probe, but generally negative with the miR‐126 and negative control probes. Reversely, endothelial cells stain positive for miR‐126 and negative for miR‐155 and the control probe. Solitary T cells with a malignant morphology display brighter staining with the miR‐155 probe. Taken together, our findings suggest that both malignant and non‐malignant T cells express miR‐155 in situ in CTCL. Moreover, they indicate heterogeneity in miR‐155 expression among malignant T cells.     

Neutrophilic superoxide production can assess pharmacological and pharmacogenetic β‐adrenoreceptor effects

Asthma can be controlled well in most patients by inhaled β‐adrenoreceptor (β₂AR) agonists and steroids. Poor response to β₂AR agonists is difficult to predict, especially in young children and by lung function testing, which may be affected by multiple influences. As an alternative approach, we analyzed ex vivo neutrophilic superoxide inhibition in response to β₂AR stimulation. In 60 healthy volunteers, this assay was unaffected by sex, age, smoking, atopy or asthma status. Furthermore, we assessed effects of genetic variants in β₂AR by sequencing the ADRB2 gene in our cohort and relating genotypes to β₂AR‐mediated neutrophilic superoxide inhibition. Gly16Arg genotypes correlated with minor decrease in overall adrenoresponse in this small study population. Taken together, ex vivo testing of the β₂AR response in human neutrophils represents a robust tool with good signal‐to‐noise ratio at physiological β₂AR agonist concentrations, and this assay may be useful to complement future pharmacogenetic studies in asthma.     

Well‐differentiated pancreatic neuroendocrine tumours (PanNETs) and poorly differentiated pancreatic neuroendocrine carcinomas (PanNECs): concepts,issues and a practical diagnostic approach to high‐grade (G3) cases

With increasing accessibility and advancements in abdominal imaging modalities, the incidence of pancreatic neuroendocrine neoplasms has increased steadily during the past few decades. By definition, neuroendocrine neoplasms of the pancreas show neuroendocrine differentiation, but they represent a broad and heterogeneous group of neoplasms with diverse clinical and pathological characteristics. The majority of pancreatic neuroendocrine neoplasms can be classified as well‐differentiated pancreatic neuroendocrine tumours (PanNETs) or poorly differentiated pancreatic neuroendocrine carcinomas (PanNECs). While PanNETs and PanNECs are distinct entities with respect to clinical presentation, outcome and therapeutic approach, they may exhibit overlapping histopathological features. Moreover, the frequent modifications in nomenclature and prognostic grading systems over the years of not only pancreatic neuroendocrine neoplasms, but neuroendocrine neoplasms from other organ sites, has created confusion for both pathologists and clinicians as to the appropriate use of terminology and grading when evaluating these neoplasms. This review examines the current concepts and issues of nomenclature and grading of PanNETs and PanNECs. In addition, considering the morphological overlap between high‐grade (G3) PanNETs and PanNECs, we discuss an integrative and practical diagnostic approach to aid in discriminating challenging cases.     

Amomum tsao‐ko fruit extract suppresses lipopolysaccharide‐induced inducible nitric oxide synthase by inducing heme oxygenase‐1 in macrophages and in septic mice

Amomum tsao‐ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti‐inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao‐ko (AOM) in lipopolysaccharide (LPS)‐induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS‐induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase‐1 (HO‐1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO‐1) and silencing HO‐1 using siRNA prevented the AOM‐mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF‐E2‐related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N‐acetyl‐l ‐cysteine (NAC; a ROS scavenger) diminished the AOM‐induced phosphorylation of ERK and JNK and AOM‐induced HO‐1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM‐induced signalling of HO‐1 expression. In LPS‐induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO‐1 expression and survival rates. These results indicate that AOM strongly inhibits LPS‐induced NO production by activating the ROS/MAPKs/Nrf2‐mediated HO‐1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.