Mutations of EGFR or KRAS and expression of chemotherapy-related genes based on small biopsy samples in stage IIIB and IV inoperable non-small cell lung cancer

Purposes

Epidermal growth factor receptor (EGFR) and KRAS mutations may predict the outcome of targeted drug therapy and also may be associated with the efficacy of chemotherapy in patients with non-small cell lung cancer (NSCLC). This report investigated the relation of EGFR or KRAS mutation and expression of chemotherapy-related genes, including excision repair cross-complementing 1 (ERCC1), thymidylate synthetase (TYMS), ribonucleotide reductase subunit M1 (RRM1) and class III β-tubulin (TUBB3), as a potential explanation for these observations.

Methods

A total of 143 patients with stage IIIB and IV NSCLC from bronchoscopy or percutaneous lung biopsy obtained tumor samples were analyzed concurrently for EGFR or KRAS mutations, and mRNA expression of ERCC1, TYMS, RRM1 and TUBB3. EGFR or KRAS mutations were detected with xTAG liquidchip technology (xTAG-LCT), and mRNA expression levels of four genes were detected by branched DNA-liquidchip technology (bDNA-LCT).

Results

Of 143 patients, 63 tumors were positive for EGFR-activating mutations, and 16 tumors were positive for KRAS mutations. EGFR-activating mutations are more frequent in females, adenocarcinoma and non-smokers patients, and KRAS mutations are more frequent in smoking patients. ERCC1 mRNA levels were significantly associated with histological type and tumor differentiation, whereas TYMS levels were significantly associated with age. NSCLC specimens that harboring EGFR-activating mutations are more likely to express low ERCC1 and high TUBB3 mRNA levels, whereas tumors from patients with NSCLC harboring KRAS mutation are more likely to express high ERCC1 mRNA levels.

Conclusions

Mutations and expression of chemotherapy-related genes may provide a basis for the selection of suitable molecular markers for individual treatment in a population with stage IIIB and IV NSCLC.     

Piperlongumine induces cell death through ROS-mediated CHOP activation and potentiates TRAIL-induced cell death in breast cancer cells

Purpose

Piperlongumine (PL) has been shown to selectively induce apoptotic cell death in cancer cells via reactive oxygen species (ROS) accumulation. In this study, we characterized a molecular mechanism for PL-induced cell death.

Methods

Cell viability and cell death were assessed by MTT assay and Annexin V-FITC/PI staining, respectively. ROS generation was measured using the H₂DCFDA. Small interfering RNA (siRNA) was used for suppressing gene expression. The mRNA and protein expression were analyzed by RT-PCR and Western blot analysis, respectively.

Results

We found that PL promotes C/EBP homologous protein (CHOP) induction, which leads to the up-regulation of its targets Bim and DR5. Pretreatment with the ROS scavenger N-acetyl-cysteine abolishes the PL-induced up-regulation of CHOP and its target genes, suggesting an essential role for ROS in PL-induced CHOP activation. The down-regulation of CHOP or Bim with siRNA efficiently attenuates PL-induced cell death, suggesting a critical role for CHOP in this cell death. Furthermore, PL potentiates TRAIL-induced cytotoxicity in breast cancer cells by upregulating DR5, as DR5 knockdown abolished the sensitizing effect of PL on TRAIL responses.

Conclusions

Overall, our data suggest a new mechanism for the PL-induced cell death in which ROS mediates CHOP activation, and combination treatment with PL and TRAIL could be a potential strategy for breast cancer therapy.     

Gastric cancer-derived MSC-secreted PDGF-DD promotes gastric cancer progression

Purpose

This study was designed to investigate the role of PDGF-DD secreted by gastric cancer-derived mesenchymal stem cells (GC-MSCs) in human gastric cancer progression.

Methods

Gastric cancer cells were indirectly co-cultured with GC-MSCs in a transwell system. The growth and migration of gastric cancer cells were evaluated by cell colony formation assay and transwell migration assay, respectively. The production of PDGF-DD in GC-MSCs was determined by using Luminex and ELISA. Neutralization of PDGFR-β by su16f and siRNA interference of PDGF-DD in GC-MSCs was used to demonstrate the role of PDGF-DD produced by GC-MSCs in gastric cancer progression.

Results

GC-MSC conditioned medium promoted gastric cancer cell proliferation and migration in vitro and in vivo. Co-culture with GC-MSCs increased the phosphorylation of PDGFR-β in SGC-7901 cells. Neutralization of PDGFR-β by su16f blocked the promoting role of GC-MSC conditioned medium in gastric cancer cell proliferation and migration. Recombinant PDGF-DD duplicated the effects of GC-MSC conditioned medium on gastric cancer cells. Knockdown of PDGF-DD in GC-MSCs abolished its effects on gastric cancer cells in vitro and in vivo.

Conclusions

PDGF-DD secreted by GC-MSCs is capable of promoting gastric cancer cell progression in vitro and in vivo. Targeting the PDGF-DD/PDGFR-β interaction between MSCs and gastric cancer cells may represent a novel strategy for gastric cancer therapy.     

Quantitative and immunocytochemical studies of APUD cells in airways and gut

The amine precursor uptake and decarboxylase (APUD) cells in airways and small intestine of adult hamsters have been studied by quantitative, histochemical and immunocytochemical methods for the presence of argyrophilia, ACTH, hGH, calcitonin, bombesin and prolactin in control animals and following priming with two amine precursors: L-dopa and 5-hydroxytryptophane (5-HTP). APUD cells have been defined by the presence of intracytoplasmic argyrophilic granules. A method that related them to airway or gut perimeter length or total epithelial cell numbers has been applied. Calcitonin-like-immunoreactivity (CLIR) is present in epithelial cells in the tracheal epithelium and glands. The presence of ACTH, hGH, bombesin or prolactin is not detected. Priming with L-Dopa and 5-HTP results in up to 12 fold increases in densities of argyrophilic APUD cells in airways and ileum when ratios of APUD cells/100 nuclei are compared to those in control animals. Exposure to 5-HTP induces no significant changes in densities of cells with CLIR in the trachea but CLIR is not detected in animals exposed to L-DOPA. Ratios of APUD cells/100 epithelial cells appear to be more sensitive than those of APUD cells/mm in detecting significant changes in APUD cell densities. Priming with amine precursors is a valuable tool in quantitative studies of neuroendocrine cells.     

Cell mediated immunity and suppressor T cell function in children with cystic fibrosis

The relationship of general and suppressor T cell function to IgE sensitization was investigated in a group of 12 children with cystic fibrosis (CF) and 10 non-atopic control children. Increased IgE sensitization was noted in the cystic fibrosis group, as measured by increased group mean serum IgE levels and an increased incidence of multiple IgE skin sensitivities. Studies of active E rosettes, lymphocyte stimulation, delayed-type skin responsiveness, and Concanavalin A (Con A) induced suppressor cell function revealed no statistically significant group differences. The results of this study may implicate multiple mechanisms for IgE sensitization.     

Diabetes mellitus among outpatients with COPD attending a university hospital

Type 2 diabetes mellitus is a common comorbidity of COPD, but there are still many doubts about the relation among diabetes and COPD. We retrospectively collected data from patients afferent to our Respiratory Diseases outpatient clinic at the Tor Vergata University Hospital between 2010 and 2012. The study population was analyzed by clusters of age, gender, body mass index (BMI), smoking status, lung function, concomitant pharmacologic therapies and comorbidities. The values of the association between variables were expressed as odds ratio. Data were adjusted for gender, age and possible confounding variables by Mantel–Haenszel method. We identified 493 patients with COPD. Ninety-two (18.7 %) patients were affected by type 2 diabetes mellitus, with no significant gender differences. The prevalence distribution was similar among the different age clusters, but the association was stronger in patients younger than 65 years. The association was present only in obese subjects in whom it was significant only in patients with moderate-to-severe COPD, but not mild COPD. The presence of cardiovascular diseases was significantly associated with diabetes mellitus in patients with COPD. There was a slight association of inhaled corticosteroid (ICS) use with the presence of diabetes mellitus in COPD, but the combination of an ICS with a β₂-agonist apparently reduced this association. The association with type 2 diabetes mellitus was greater in patients with COPD respect to general population, and correlated with the increase in BMI and the presence of other comorbidities, suggesting that both diseases may be targets of systemic inflammation.     

Primary pulmonary histiocytosis X immunological analysis in two cases

Several immunological markers were studied in two patients with primary pulmonary histiocytosis X diagnosed by open-lung biopsy. The population of T- and B-lymphocytes from peripheral blood and the response of peripheral lymphocytes to phytohemagglutinin were within the normal ranges in two patients. The concentrations of serum IgG, IgA, and IgM in two patients were normal. However, Patient 1 showed a decreased serum IgE level and Patient 2 showed a lower border-line level of serum IgE as well. The selective low serum IgE level was the only immunological abnormality seen in primary pulmonary histiocytosis X.     

The humoral immune system in inflammatory bowel disease

As there have been reports of differences in mean levels of serum immunoglobulins between patients with ulcerative colitis and Crohn's disease, serum IgG, IgA, and IgM were estimated in 158 patients with inflammatory bowel disease and the results correlated with the clinical features of the patients. Although a higher mean IgG level in ulcerative colitis compared to Crohn's disease was confirmed, no difference was found when the comparison was limited to patients with colonic Crohn's disease. Patients with either disease had higher mean IgM levels than controls, and the IgM levels were higher on treatment with corticosteroids and showed a tendency to rise in remission. IgG and IgM levels were also higher in both diseases if extraintestinal manifestations were present. It is concluded that if clinical features, particularly disease site, are taken into account, the overall immunoglobulin responses in these two diseases show no differences.     

Different efficacy of EGFR tyrosine kinase inhibitors and prognosis in patients with subtypes of EGFR-mutated advanced non-small cell lung cancer: a meta-analysis

Background

Nearly 85 % of lung-cancer-specific epidermal growth factor receptor (EGFR) sensitive mutations comprise a substitution at position 858 (21L858R) and deletion mutants in exon 19 (19del). The aim of this study was to assess the role of EGFR mutation subtypes in predicting the efficacy of EGFR tyrosine kinase inhibitors (EGFR TKIs) and the prognosis of patients with advanced non-small cell lung cancer (NSCLC).

Method

We systematically searched for eligible articles investigating the association between EGFR mutation subtypes and the efficacy of EGFR TKIs and the prognosis of patients with NSCLC. The summary risk ratio (RR) and mean difference (MD) were calculated using meta-analysis. In addition, we used variance analysis for the progression-free survival data (PFS) and used the rank sum test for the overall survival data.

Results

We identified 22 eligible trials involving 1,082 patients. The objective response rate of the 19del mutation group was significantly higher than the 21L858R mutation group (RR 1.23; 95 % CI 1.12–1.36; P < 0.0001). The PFS (MD 3.55; 95 % CI 0.90–6.20; P = 0.009; MD 2.57; 95 % CI 0.51–4.62; P = 0.01) and overall survival (OS) (MD 10.52; 95 % CI 5.10–15.93; P = 0.0001) of the 19del mutation group were significantly longer than the 21L858R mutation group; the same results were observed in the variance analysis and rank sum test.

Conclusion

The 19del mutation may be a more efficient clinical marker for predicting the response of patients with NSCLC to EGFR TKIs. Furthermore, patients with the 19del mutation have both a longer PFS and OS. The 19del mutation is also the prognostic factor for patients with NSCLC.     

Acyl CoA synthetase from rat lung purification and properties

Long chain fatty acyl-CoA synthetase, a key enzyme in fatty acid metabolism, occurs in liver, brain, muscle, adipose tissue and bacteria. In our study, rat lung was found to have an acyl-CoA synthetase very active for the synthesis of palmitoyl CoA. The lung microsomal and cell membrane fractions were the principle sources of this enzyme, with mitochondria the next most active fraction. Palmitoyl CoA synthetase was isolated from rat lung microsomes and purified 100-fold. Enzyme activity was determined either by spectrophotometric or by radioactive assay methods. Kinetic parameters and properties were determined using purified microsomal palmitoyl CoA synthetase. The assay system required ATP and CoA as substrates. Maximal activation was reached with palmitate as substrate.     

Accumulation of histamine and cyclic nucleotides in lung tissue during chronic hypobaric hypoxia

Rats exposed to hypobaric hypoxia (P_B = 440 Torr) from 1 to 9 days showed a rise of the total lung histamine content which reached a maximum of 1.138 ng/µg DNA = 274%; (control values were 0.415 ng/µg DNA = 100%) after 2 days of hypoxia. The cyclic AMP (cAMP) concentrations were elevated during the first 3 days of hypobaric hypoxia and returned almost to control values later on in the time course. Cyclic GMP (cGMP) increased after an initial delay, reaching plateau values from the 6th to the 9th day. Histamine and cAMP concentrations were highly correlated during the first 3 days of hypoxia. 6-Methylprednisolone increased lung tissue concentrations of both histamine and cAMP in hypoxic as well as nonhypoxic animals; the level of cGMP was unchanged. The results demonstrate an accumulation of histamine in lung tissue and suggest that histamine is stored rather than released from rat lungs during chronic hypoxia. The possible role of cyclic nucleotides in the regulation of histamine lung tissue content is discussed.     

Serum and intestinal fluid immunoglobulins in patients with giardiasis

To test the possibility that a factor predisposing to symptomatic giardiasis in the general population is immunoglobulin deficiency, we measured immunoglobulins in serum and intestinal fluids of 9 giardiasis patients (8 after eradication ofGiardia lamblia) and 12 healthy individuals. The concentrations of IgG, IgA, IgM, and IgE in serum and of IgA and IgM in intestinal fluids from the 2 groups were similar. Intestinal fluid IgG levels were significantly higher (P<0.1), however, in the patients (5.8±6.3 mg/100 ml) than in the control subjects (1.1±1.9 mg/100 ml). This difference was unexplained and is of uncertain biological significance. We conclude that giardiasis in generally healthy individuals is not etiologically related to unsuspected immunoglobulin deficiencies, even thoughG. lamblia infection is known to be very common in grossly immunoglobulin-deficient patients.     

Stimulation of human pepsin output by topical hydrochloric acid

Pepsin secretion is stimulated by the back-diffusion of acid across the mucosa of the vagally denervated canine pouch. If back-diffusion is enhanced by damage, pepsin secretion increases. The current study investigates whether this mechanism exists in man. The stomachs of normal human volunteers were irrigated for 1 hour with either buffer or 0.01 N HCl, 1 hour with 0.2 N HCl, and a final hour with buffer or 0.01 N HCl. During the middle hour both the concentration and output of pepsin increased three- or fourfold. From these studies it appears that the human gastric mucosa contains a mechanism similar to the dog's which results in the stimulation of pepsin secretion when exposed to acid. This mechanism could be of etiologic significance in gastric ulcer disease, which has been shown to be associated with increased gastric-musocal permeability.     

The significance of cyclic 3′5′-AMP for the euler-liljestrand mechanism

Changes in the pulmonary vascular resistance and the cAMP levels in the lung parenchyma of four pigs were recorded during normoxia and hypoxia and following the administration of orciprenaline under hypoxia. Additional measurements recorded were pulmonary artery pressure, left-ventricular pressure, dp/dt_max, mean arterial pressure, arterial oxygen saturation, blood gas values. The rise of the pulmonary vascular resistance during hypoxia is not preceded by a decrease in intracellular cAMP levels; extreme cAMP concentrations, however, block the Euler-Liljestrand mechanism. The findings suggest that alveolar hypoxia and/or intracellular metabolic acidosis suppresses the inhibiting action of cAMP on the Ca⁺⁺ influx in the smooth vascular muscles or affect the Ca⁺⁺-binding process of the sarcoplasmic reticulum of the vascular musculature, resulting in vascoconstriction.     

IMP3 expression in gastric cancer: association with clinicopathological features and HER2 status

Background

The aim of this study was to evaluate the expression of IMP3, an independent poor prognostic factor for many cancers, and its association with clinicopathological features and HER2 status.

Methods

Gastrectomy specimens from 106 patients were evaluated by immunohistochemistry and fluorescence in situ hybridization.

Results

HER2 overexpression was found in 4.71 % of the samples. A negative association was observed between HER2 overexpression and grade of differentiation. No association was observed between HER2 overexpression and status of surgical margins, vascular invasion, perineural invasion, nodal metastasis and depth of invasion. Among all specimens of gastric cancer, 67.92 % were positive for IMP3. Expression of IMP3 was significantly higher in specimens with vascular invasion, perineural invasion, nodal metastasis and higher depth of invasion. HER2 overexpression was detected in only 5.55 % of IMP3 positive specimens.

Conclusions

IMP3 expression was frequently observed in gastric cancer and was associated with poor prognostic clinicopathological features. A survival benefit with HER2 therapy should be expected for the minority of patients with IMP3 positive specimens. Studies should be conducted to evaluate the response to HER2 therapy of gastric cancer expressing IMP3.     

The BRAF mutation is associated with the prognosis in colorectal cancer

Background

Two members of the Ras/Raf signaling pathway, KRAS and B-raf, are suspected to be involved in the stepwise progression of colorectal cancer (CRC) tumorigenesis.

Objective

We compared the KRAS and BRAF mutation status of CRC patients with their clinicopathological characteristics and examined the effect of mutation status on survival rates.

Methods

DNA was extracted from 164 samples, and the mutation statuses of KRAS and BRAF were assessed using peptide PNA clamp real-time PCR method. The presences of mutation were compared with clinicopathological factors and 5-year survival rate.

Results

Among the 164 CRC cases, KRAS mutation as detected in 71 cases (43.3 %), respectively, with no relationship with clinicopathological factors of the patients. On Kaplan–Meier survival analysis, KRAS mutation was not significantly associated with survival (p = 0.971). BRAF mutation was detected in 26 cases (15.9 %) and not associated with clinicopathological factors of the patients. However, the 5-year survival rate of BRAF mutations was significantly decreased (p = 0.02).

Conclusions

The presence of KRAS mutation did not correlate with the various clinicopathological factors of CRC patients or the survival rate. However, the survival rate was reduced in BRAF-mutated CRC patients. Therefore, BRAF mutation could be an important prognostic factor in CRC patients.     

Type 2 diabetes mellitus is associated with the fibrosis severity in patients with nonalcoholic fatty liver disease in a large retrospective cohort of Japanese patients

Background

The prevalence of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome have been increasing worldwide. The associations between metabolic factors and the histologic severity of NAFLD have not yet been clarified. Therefore, we studied the relationships between relevant metabolic factors and the histological severity of NAFLD.

Methods

In a cross-sectional multicenter study conducted in Japan, we examined 1,365 biopsy-proven NAFLD patients. The frequencies of underlying lifestyle-related diseases and their relationships to the NAFLD histology were investigated.

Results

The hepatic fibrosis stages (Stage 0/1/2/3/4) were 22.6/34.1/26.7/14.5/2.1 (%) in the male patients, and 16.2/31.7/23.9/21.6/6.6 (%) in the female patients. Dyslipidemia was present in 65.7 % (hypertriglyceridemia, 45.3 %; increased low-density lipoprotein cholesterol, 37.5 %; decreased high density lipoprotein cholesterol, 19.5 %) of patients. Hypertension was present in 30.2 %, and diabetes mellitus (DM) in 47.3 %. The fibrosis stage increased with age, especially in postmenopausal females. The body mass index was positively correlated with the fibrosis stage. Deterioration of glucose control was positively correlated with the fibrosis stage, this correlation being more prominent in females. Multivariate analysis identified age and DM as significant risk factors for advanced fibrosis. No significant correlation of the fibrosis stage was observed with hypertension. There was a negative correlation between the serum triglyceride levels and the fibrosis stage.

Conclusions

DM appeared to be a significant risk factor for advanced fibrosis in patients with NAFLD, and would therefore need to be properly managed to prevent the progression of NAFLD.     

Assessment of arterial stiffness in chronic obstructive pulmonary disease by a novel method

Background

Patients with chronic obstructive pulmonary disease (COPD) have an increased risk of cardiovascular morbidity and mortality. Increased arterial stiffness is associated with the presence and severity of cardiovascular disease. The cardio-ankle vascular index (CAVI) is a new method for assessment of arterial stiffness that is not influenced by blood pressure at the time of measurement and is significantly correlated with the presence and severity of cardiovascular disease. The aim of the present study was to evaluate whether there is an association between the spirometric severity of COPD, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria, with arterial stiffness as assessed by CAVI.

Methods

We enrolled 123 patients with COPD (102 men) followed up by the chest medicine outpatient clinics and 35 healthy subjects (26 men). All patients were assessed with spirometry, CAVI, and clinical history.

Results

Patients with COPD had significantly increased CAVI values compared with control subjects (10.37?±?2.26 vs. 6.74?±?1.42, p?1?% predicted, FEV₁/FVC, and COPD stage (r: ??0.54, p?Conclusion In this study, we have shown that increased arterial stiffness assessed by CAVI is associated with the spirometric severity of COPD.     

Suppression of Placental Metallothionein 1 and Zinc Transporter 1 mRNA Expressions Contributes to Fetal Heart Malformations Caused by Maternal Zinc Deficiency

Zinc has been implicated to have a protective role against heart malformations during fetal development. Metallothionein 1 (MT-1) and zinc transporter 1 (ZnT-1) are two major metabolic factors that are associated with zinc metabolism. The present work aimed to investigate the association of placental MT-1 and ZnT-1 expressions with fetal heart malformations resulting from maternal zinc deficiency. Sprague–Dawley female rats were randomly divided into five groups of extremely low-zinc, low-zinc, moderately low-zinc, marginally low-zinc and normal zinc (n = 9–12), and were fed diets with controlled zinc content at 1.0 ± 0.3, 8.4 ± 1.8, 15.4 ± 2.8, 22.4 ± 4.1 and 29.4 ± 5.3 [mean ± standard deviation (SD)] mg of zinc/kg, respectively, from day 25 of preconception until day 19 of gestation. The female rats were bred, their fetuses were harvested at day 19 of gestation after killing the dams, and fetal hearts were morphologically examined. Zinc concentration and alkaline phosphatase (ALP) activity in maternal venous blood sera were tested, and MT-1 and ZnT-1 mRNA expressions in the placenta were assayed. Zinc concentrations and ALP activities in the blood were low in all zinc-deficient diet groups in a dose-dependent fashion. The incidences of heart malformations were increased, and the levels of placental MT-1 and ZnT-1 mRNA expressions were decreased in the extremely low-zinc, low-zinc and moderately low-zinc groups compared with the normal zinc group. Specifically, mRNA levels of placental MT-1 or ZnT-1 were significantly decreased and were lower than the specific threshold values in the fetuses with heart malformations but not in the fetuses without heart malformations in all the groups. These data indicate that maternal zinc deficiency resulted in an elevated incidence of fetal heart malformations, which was associated with significant decreases in placental MT-1 and ZnT-1 mRNA expressions to the levels below the threshold values that may be a crucial factor to determine the presence of fetal heart malformations.