Carcinoembryonic antigen-related cell adhesion molecules as surrogate markers for EGFR inhibitor sensitivity in human lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LADCA) patients with epidermal growth factor receptor (EGFR) mutations are in general associated with relatively high clinical response rate to EGFR-tyrosine kinase inhibitors (TKIs) but not all responded to TKI. It has therefore become important to identify the additional surrogate markers regarding EGFR-TKI sensitivity. METHODS: We first examined the effects of EGFR-TKIs, gefitinib and erlotinib, upon cell proliferation of lung adenocarcinoma cell lines. We then evaluated the gene profiles related to EGFR-TKI sensitivity using a microarray analysis. Results of microarray analysis led us to focus on carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, CEACAM 3, 5, 6, 7, and 19, as potential further surrogate markers of EGFR-TKI sensitivity. We then examined the correlation between the status of CEACAM 3, 5, 6, 7, and 19 immunoreactivity in LADCA and clinicopathological parameters of individual cases. RESULTS: In the cases with EGFR mutations, the status of all CEACAMs examined was significantly higher than that in EGFR wild-type patients, but there were no significant differences in the status of CEACAMs between TKI responder and nonresponder among 22 patients who received gefitinib therapy. However, among 115 EGFR mutation-negative LADCA patients, both CEACAM6 and CEACAM3 were significantly associated with adverse clinical outcome (CEACAM6) and better clinical outcome (CEACAM3). CONCLUSION: CEACAMs examined in this study could be related to the presence of EGFR mutation in adenocarcinoma cells but not represent the effective surrogate marker of EGFR-TKI in LADCA patients. However, immunohistochemical evaluation of CEACAM3/6 in LADCA patients could provide important information on their clinical outcome.

First Report of Guinea Grass Smut Caused by Conidiosporomyces ayresii in Japan.

Guinea grass (Panicum maximum Jacq.) is an important C-4 perennial plant that grows in southern Japan. In March 2010, a smut disease was found in grass that is cultivated in the Ishigaki Islands, Okinawa, in southernmost Japan. Spikelets of susceptible cultivars were swollen and filled with gray spore masses and seed production was substantially reduced. Two single-spore isolates of a smut fungus were obtained from infected spikelets and deposited at the NIAS Genebank, Japan as MAFF511519 and 511520. The 28S rDNA sequences of the isolates were analyzed as described by Boekhout et al. (1). The 28S rDNA sequence (GenBank Accession No. AB647346) of isolate MAFF511519 matched that of Conidiosporomyces ayresii (GenBank Accession No. AY819017) isolated from P. maximum with 99.8% similarity. Spores were pale brown to brown, globose to subglobose, verrucose, and 14 to 16 × 15 to 18 µm in diameter with relatively thick walls of 2 to 3 µm. With scanning electron microscopy, warts appeared dense and short with pointed tips. Spores germinated under wet conditions and produced masses of basidiospores. Basidiospores were aseptate, long, cylindrical, straight to slightly curved, 20 to 37 × 2 to 3 µm, and often germinated into Y-shaped conidia. This description matches previous descriptions (3) of C. ayresii (Berk.) Vánky (Tilletia ayresii Berk.) of the smut pathogen of guinea grass (2). The smut fungus was identified as C. ayresii on the basis of morphology and molecular phylogenetic analysis. To produce inoculum, the isolates were grown on potato dextrose agar at 25°C in the dark for 7 days. Two plants of cv. Ryukyu 5-gou with half-flowering heads were grown in a greenhouse for approximately 1 month and then inoculated by atomizing them with conidial suspensions of each isolate (106 conidia/ml). A plant sprayed with sterilized distilled water served as the control. Inoculated heads were covered with plastic bags for 48 h at 25°C. After 30 days, all inoculated plants were symptomatic with swollen spikelets releasing gray spores. Controls remained asymptomatic. The smut fungus was reisolated from released gray spores, confirming Koch's postulates. To our knowledge, this is the first report of smut caused by C. ayresii on guinea grass in Japan. References: (1) T. Boekhout et al. Stud. Mycol. 38:175, 1995. (2) J. M. Lenné and P. Trutmann. Diseases of Tropical Pasture Plants. CAB International, Wallingford, UK, 1994. (3) K. Vánky and R. Bauer. Mycotaxon 43:427, 1992.

Mycobacterial hypersensitivity pneumonitis requires TLR9-MyD88 in lung CD11b+ CD11c+ cells.

Mycobacteria are among the most common causes of hypersensitivity pneumonitis (HP), but controversy persists with regard to the involvement of the infectious potency of the organism in mycobacterial HP (hot tub lung). This study aimed to establish a mouse model of hot tub lung to clarify its pathophysiology. Mice were exposed intranasally to formalin-killed Mycobacterium avium from a patient with hot tub lung (HP strain) or chronic pulmonary infection (non-HP strain), and bronchoalveolar lavage fluids and lung tissues were evaluated for allergic inflammation. Dead M. avium HP strain, but not non-HP strain, elicited marked HP-like pulmonary inflammation in wild-type mice. Although the inflammation was induced in mice lacking CD4 or CD8, the induction of HP-like responses was prevented in mice lacking myeloid differentiation factor (MyD)88 or Toll-like receptor (TLR)9. Cultured lung CD11c+ cells responded to M. avium in a TLR9-dependent manner, and reconstitution of TLR9-/- mice with lung CD11c+ cells from wild-type mice restored the inflammatory responses. Further investigation revealed that pulmonary exposure to M. avium HP strain increased the number of lung CD11b+ CD11c+ cells (dendritic cells) through TLR9 signalling. Our results provide evidence that hot tub lung develops via the mycobacterial engagement of TLR9-MyD88 signalling in lung CD11b+ dendritic cells independent of the mycobacterial infectious capacity.

Rapid improvement of osseous sarcoidosis after the treatment of pulmonary aspergillosis by itraconazole.

Osseous sarcoidosis is relatively uncommon, and treatment with corticosteroids is not always effective. Moreover, patients with an advanced stage of pulmonary sarcoidosis are sometimes infected with aspergillus in the cavities of the pulmonary lesions, and long-term use of corticosteroids should be prohibited to prevent the development of fatal invasive pulmonary aspergillosis. Here, we described a unique case of osseous sarcoidosis with pulmonary aspergillosis, showing a rapid improvement of the osseous symptoms just after the administration of the antifungal agent, itraconazole. Itraconazole is likely to become a candidate among new therapeutic agents for osseous sarcoidosis.

Pirfenidone in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease without proven effective therapy. A multicentre, double-blind, placebo-controlled, randomised phase III clinical trial was conducted in Japanese patients with well-defined IPF to determine the efficacy and safety of pirfenidone, a novel antifibrotic oral agent, over 52 weeks. Of 275 patients randomised (high-dose, 1,800 mg x day(-1); low-dose, 1,200 mg x day(-1); or placebo groups in the ratio 2:1:2), 267 patients were evaluated for the efficacy of pirfenidone. Prior to unblinding, the primary end-point was revised; the change in vital capacity (VC) was assessed at week 52. Secondary end-points included the progression-free survival (PFS) time. Significant differences were observed in VC decline (primary end-point) between the placebo group (-0.16 L) and the high-dose group (-0.09 L) (p = 0.0416); differences between the two groups (p = 0.0280) were also observed in the PFS (the secondary end-point). Although photosensitivity, a well-established side-effect of pirfenidone, was the major adverse event in this study, it was mild in severity in most of the patients. Pirfenidone was relatively well tolerated in patients with IPF. Treatment with pirfenidone may decrease the rate of decline in VC and may increase the PFS time over 52 weeks. Additional studies are needed to confirm these findings.

Massive pleural effusion and bronchopleural fistula in Wegener's granulomatosis.

Wegener's granulomatosis (WG) is characterized by systemic granulomatous necrotizing vasculitis, primarily affecting the respiratory tract and kidneys. We describe a rare case in a 28-year-old woman with WG, presenting with a massive lateral pleural effusion, accompanied by an aseptic bronchopleural fistula formed during immunosuppressive treatment. Although any organ can be involved in WG, only left pleuritis and a purpuric lesion on the neck were detected in this case. The pleural effusion and bronchopleural fistula resolved following immunosuppressive treatment for six months. Thus, WG should be considered in the differential diagnosis of a massive pleural effusion, and fistula formation is a possible complication of treatment. Moreover, immunosuppressive treatment was sufficient to resolve the massive pleural effusion and fistula formation without infection (120 words).

Association between mycobacterial genotypes and disease progression in Mycobacterium avium pulmonary infection.

BACKGROUND: Non-tuberculous mycobacterial lung disease, most commonly caused by Mycobacterium avium infection, tends to show variable disease progression, and significant disease predictors have not been adequately established. METHODS: Variable numbers of tandem repeats (VNTR) were evaluated in 16 mycobacterial interspersed repetitive unit (MIRU) loci from M avium isolates cultured from respiratory specimens obtained from 2005 to 2007. Specifically, the association between VNTR profiles and disease progression was assessed. RESULTS: Among the 37 subjects who provided positive respiratory cultures for M avium during the 2005-6 period, 15 subjects were treated within 10 months following a microbiological diagnosis of progressive M avium lung disease. Nine subjects underwent long-term follow-up (>24 months) without treatment for stable M avium lung disease. Based on a neighbour-joining cluster analysis used to classify M avium-positive subjects according to the VNTR profile, subjects with progressive versus stable lung disease were found to be grouped together in distinct clusters. Further analysis using logistic regression modelling showed that disease progression was significantly associated with the genetic distance of the M avium isolate from an appropriately selected reference (age-adjusted odds ratio 1.95; 95% confidence interval 1.16 to 3.30; p = 0.01 for the most significant model). A best-fit model could be used to predict the progression of M avium lung disease when subjects from the 2005-6 period were combined with those from 2007 (p = 0.003). CONCLUSION: Progressive lung disease due to M avium infection is associated with specific VNTR genotypes of M avium.

Relationship of p53 overexpression and up-regulation of proliferating cell nuclear antigen with the clinical course of non-small cell lung cancer.

p53 is known to play a central role in the control of cell proliferation and carcinogenesis. In non-small cell lung cancer, however, the clinicopathological studies of p53 have yielded conflicting results. In the current study, we examined 123 non-small cell lung cancers with detailed clinical information, 71 primary and 52 metastatic tumors, using formalin-fixed and paraffin-embedded surgical specimens to show the clinicopathological correlation of the immunohistochemical (DO-7) overexpression of p53. Nuclear specific p53 overexpression appeared in 48 (39%; any number of tumor cells positive) of 123 tumors (35% of primary and 44% of metastatic tumors). The distribution and intensity of staining were variable. Ninety-eight % of all tumors also expressed nuclear immunoreactivity for proliferating cell nuclear antigen (PCNA; PC 10) to a varying degree. In a univariate analysis, both p53 (> 10% of tumor cells positive; n = 11) and PCNA (> 50% of tumor cells positive; n = 32) were associated with shorter survival in the curative intent group (stages I, II, and IIIA) of 63 patients. In a multivariate analysis including all clinicopathological variables, the overexpression of p53 (but not PCNA) was found to be an independent prognostic factor (P2 = 0.0011) in the curative intent group. No correlation was detected between the immunoreactivities and patient characteristics, such as age, gender, or smoking. Double immunohistochemistry of both p53 and PCNA revealed a distinct pattern, whereas its clinicopathological correlation remained elusive. We conclude that p53 overexpression in non-small cell lung cancer (but not PCNA) is independently associated with a shortened survival and may be of prognostic significance in selected patients with earlier stage cancer.

p53 and Ki-ras gene mutations in epithelial ovarian neoplasms.

In an effort to define the pathogenic relationship between ovarian neoplasms spanning the clinicopathological spectrum from benign to malignant, the incidence of Ki-ras and p53 mutations was determined in 20 ovarian cystadenomas, 20 low malignant potential (LMP) tumors of the ovary, and 23 ovarian carcinomas. Using DNA extracted from paraffin embedded tissue, polymerase chain reaction amplification, designed restriction fragment length polymorphism analysis, and DNA sequencing, 1 cystadenoma (5%), 6 LMP tumors (30%), and 1 ovarian carcinoma (4%) demonstrated an activated Ki-ras gene. All of the Ki-ras mutations identified except one were GGT to GAT transversions at codon 12. One LMP tumor demonstrated a CAA to CAC transversion at codon 61. Using polymerase chain reaction/single strand conformational polymorphism, DNA sequencing, and immunohistochemistry, 11 ovarian carcinomas (48%) demonstrated a p53 mutation. These mutations included 5 missense, 2 nonsense, and 1 frameshift mutation located within exons 6-8 and 3 mutations that were identified only by immunohistochemical staining. No p53 mutations could be identified in cystadenomas or LMP tumors. Clinically, the presence of either a Ki-ras or p53 mutation was associated with advanced stage disease. The pattern of Ki-ras and p53 mutations appears to distinguish LMP tumors from invasive carcinomas and suggests that they may be separate biological entities.

In situ detection of unexpected patterns of mutant p53 gene expression in non-small cell lung cancers.

Many solid tumors, including non-small cell lung cancers (NSCLCs), are characterized by heterogenous expression of p53 protein in the neoplastic cells. To analyse the molecular implications of this finding, we examined topographic distribution of p53 mutations using in situ polymerase chain reaction (PCR) in primary NSCLCs, showing distinct patterns of variable p53 overexpression by immunohistochemistry. Unique sets of primers for each mutation were designed, and optimal PCR conditions were determined by standard PCR using DNA from cloned mutants or cell lines established from these tumors. All tumor cell nuclei, regardless of the status of p53 overexpression, demonstrated homogeneous distribution of mutant p53 with specific primers, indicating that only subgroups of the mutated cells overexpressed p53 protein. In situ reverse transcription (RT)-PCR was applied to detect mutant mRNA in the individual tumor cells using specific primers. We found that in each case the distribution of mutant p53 mRNA coincided with that of immunohistochemical overexpression of p53 protein. Our results suggest that the regulation of mutant p53 expression, but not the genotype, is heterogeneous in the neoplastic cells. The topographic genomapping of p53 in NSCLC using in situ PCR provides a novel approach to view molecular mechanisms of lung carcinogenesis.

Non-radioactive localization of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections.

Technological developments have made possible extension of polymerase chain reaction (PCR) analysis to individual cells to localize DNA/RNA with non-radioactive labels at the light microscopic level. This approach, in situ PCR, is particularly useful in resolving low-frequency message expression in mixed populations of cells and tissues. We have established a working protocol for direct in situ PCR and have utilized several controls to validate our results. In this report we outline the procedures for detecting either DNA or RNA in a rapid and reproducible manner. We evaluate the sequential steps required for this analysis, such as protease hydrolysis, DNAse digestion, "hot start" capabilities, and detection methods. We have applied these methods in several applications, including detection of the p53 gene in human tumor samples, localization of insulin-like growth factor-IA mRNA in cell lines with low levels of expression, and distribution of transferrin mRNA in lung cancer cell lines and tumors. We demonstrate from this study that the in situ PCR technique is an investigative approach capable of detecting specific DNA/RNA sequences at the cellular level and of identifying cells with low levels of mRNA expression.

Marked goblet cell hyperplasia with mucus accumulation in the airways of patients who died of severe acute asthma attack.

To examine the changes in airways in bronchial asthma (BA) during an asthma attack causing death, we performed morphometric analysis of autopsied lungs from three outpatients who died of severe acute asthma attacks (group A) and compared these to five patients who died of non-status asthmaticus (group B). Controls (group NL) were four patients who died of diseases other than respiratory disorders. Area proportions of bronchial glands to bronchial wall (gland [percent]) and of goblet cells to total epithelial layer (goblet [percent]) and the intraluminal amount of mucus in the airways (MOR) were measured in a paraffin section. There were no significant differences in age, sex, smoking history, duration of BA history, and dosage of glucocorticoids received between groups A and B. Although both groups A and B showed significantly larger values of gland (percent) in the central airways and of inflammatory cell numbers in the airway walls than did group NL, no significant differences were observed between groups A and B. In contrast, markedly significant increases in goblet (percent) and in MOR were observed in group A compared to groups B and NL. These increases in group A were more dominant in the peripheral airway: 30-fold and threefold increases of group B in goblet (percent) and MOR, respectively. Furthermore, MOR significantly correlated with goblet (percent) in the peripheral airways (p less than 0.05). These findings suggest that a marked increase in goblet cells of the airways is a feature characteristic of patients with BA who die of a severe acute attack.

Detection of K-ras point mutation by in situ PCR in cell suspensions: comparison of the indirect and direct methods.

In situ PCR is a new technique for the localization of low copy number sequences. We report here a method for the in situ visualization of a point mutation in K-ras codon 12 by indirect in situ PCR. Twenty-five primers were examined to select mutant-specific primers. Harvested cell lines were fixed and suspended in PCR mixture. Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method. Cells were cytocentrifuged onto slides, and post-fixation was performed. The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction. Both Calu-1 (mutated: TGT) and NCI-H460 (wild type: GGT) cells had strong hybridization signals in the nuclei with general primers. But with mutant-specific primers, only Calu-1 cells had hybridization signals. No signal was observed without primers or Taq DNA polymerase. Southern blotting of the same preparation confirmed desired amplification. We also applied direct in situ PCR, but this method failed to detect the point mutation. We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations.

Bilateral pneumothoraces with multiple bullae in a patient with asymptomatic bronchiolitis obliterans 10 years after bone marrow transplantation.

A 16-year-old boy developed bronchiolitis obliterans (BO) 10 years after BMT for myelodysplastic syndrome. Although the patient complained of almost no dyspnea on exertion, he had mild hypercapnea with a markedly reduced forced expiratory volume of 0.32 l. Chest X-rays showed occasional bilateral minimal pneumothoraces, which is in accordance with the existence of multiple small bullae found on the pleural surface at video-assisted thoracic surgery. Histologic examination of the biopsied lung revealed BO. This case indicates that BO in adolescence following BMT and possible chronic GVHD may be masked because of lung immaturity at BMT, and BO after BMT may be associated with multiple pleural bullae.

Papillary serous carcinoma of the peritoneum: analysis of clonality of peritoneal tumors.

Papillary serous carcinoma of the peritoneum (PSCP) is a primary neoplasm of peritoneal origin, and is histologically difficult to differentiate from papillary serous carcinoma of the ovary (PSCO). PSCP is frequently accompanied by many peritoneal tumors, and has been managed as a disseminated disease. In previous reports, however, the clonality of the tumors has not been fully discussed. Recently, the significant roles of the p53 and BRCA1 genes in PSCP have been reported. In this study, we investigated immunohistochemical staining for p53 proteins, and investigated p53 gene mutations, using DNA sequencing analysis, to clarify the clonality of PSCP tumors. Immunohistochemically, all the tumor samples demonstrated nuclear overexpression of p53 proteins, and the DNA sequencing analysis of the p53 gene showed diverse point mutations at codons 167 and 192 in two of four anatomically different tumors. In conclusion, the possibility of polyclonality of PSCP tumors is suggested.

Involvement of sialic acid in high-affinity binding of quaternary ammonium compounds by brush border membrane from rat intestine.

As one approach to clarify the absorption mechanisms of quaternary ammonium compounds (QACs), their binding characteristics have been studied using brush border membrane vesicles isolated from rat small intestine and liposomes composed of phospholipid and GM3 ganglioside. The binding of propantheline was significantly decreased when the vesicles were pretreated with neuraminidase. Propantheline and methochlorpromazine bound to the liposomes, the binding for the latter drug being significantly greater than that for propantheline. When GM3, isolated from rat small intestine, was incorporated into the liposomes their binding capacity for both drugs increased significantly. It is suggested that the binding of QACs to the lipid layer and sialic acid play a role in the high binding of drugs to the intestinal brush border membrane. Furthermore, a sensitive and reproducible high-performance liquid chromatographic method for sialic acid has been developed.

[Evaluation of the effective use of the "health notebook"].

Since 1983, with the institution of the "Health Service Law for the Aged", the "health notebook" has been issued to people aged 40 years and over in order to aid in management of their health. Few people actually fill their health data in notebook by themselves. In order to develop effective use of the health notebook by residents and health professionals, the uses of the health notebooks by residents aged 40 years and over, public health nurses, and physicians were investigated. Three hundred and fifty four residents aged 40 and over, 41 public health nurses, and 18 physicians were studied in 1990, in Yamagata city. A majority of residents took their health notebooks with them to health consultations, and public health nurses used the notebooks to provide advice to them. Public health nurses effectively issued the health notebooks to residents using occasions where residents gathered. Some physicians reported that health notebooks were useful for motivating the people to maintain their health, while others preferred using a health card media. When comparing the health notebooks to the maternity passbooks, health notebooks need to be more easily utilized by users for recording information, and their value should be effectively explained to them. Furthermore, in order to promote self-care behaviors, greater use of health notebooks by all health professionals in indicated.

[Preanesthetic meals in elective surgical patients].

This study investigated the effect of preanesthetic meals on the volume and pH of gastric contents in forty elective surgical patients ranging in ages from 20 to 60 years. Twenty patients who were given either isotonic beverage 250 ml or apple juice 250 ml on the morning of the operative day were subjected as control group and twenty patients of the breakfast group took two slices of bread with the above drink. About seven hours following drinking and feeding, the mean values of gastric volume were 20.9 +/- 18.3 ml in the control group and 19.2 +/- 16.3 ml in the breakfast group. The mean values of gastric pH were 4.3 +/- 2.3 in the drink group and 4.6 +/- 2.3 in the breakfast group. There were no significant differences in the gastric volume and pH between the two groups. However, very small amount of the bread was detected in the gastric fluid of three patients in the breakfast group. As preanesthetic drinking and feeding are advantageous for reducing the anxieties of preoperative patients and also for their nutrition during operation, it is encouraging that eating two slices of bread did not induce a significant effect of gastric volume or pH. The minute fragment of bread seems to have no clinically significant effect.

[Topographic genomapping of p53 mutations in non-small cell lung cancer using in situ PCR].

We examined topographic distribution of p53 mutations using in situ PCR in three primary non-small cell lung cancers, showing distinct patterns of variable p53 overexpression by immunohistochemistry (Ebina, et al:Cancer Res. 54:2496, 1994). All tumor cell nuclei, regardless of the status of p53 overexpression, demonstrated homogeneous distribution of mutant p53 with specific primers, suggesting only subgroups of the mutated cells overexpressed p53 protein. Topographic genomapping using in situ PCR is a useful method to reveal the possible correlation between cell-morphology and molecular abnormality in cancer cells.