Rapid Multiplex RT-PCR for Influenza A and B by Genesoc®, a Microfluidic PCR System.

Background: Rapid antigen tests are widely used to diagnose influenza. However, despite their simplicity and short turnover time, the sensitivity of these tests is relatively low, and molecular tests with greater sensitivity are being sought. In this study, we developed and clinically evaluated a protocol for the rapid multiplex testing of influenza A and B, using a rapid real-time PCR system, GeneSoC®, that is based on microfluidic thermal cycling technology. Methods: The specificity of the developed assay was validated using cultured viral strains of influenza A/B, human metapneumovirus, and respiratory syncytial virus. Analytical sensitivity was evaluated using serially diluted RNA synthesized via in vitro transcription and nasopharyngeal swab samples collected from consecutive patients seeking medical attention for a combination of upper respiratory and general symptoms. Cross-validation of GeneSoC® based on comparisons with conventional real-time RT-PCR and rapid antigen tests was performed by parallel testing of influenza-positive clinical specimens. Results: The GeneSoC® assay detected the target sequences of influenza A and B at minimum concentrations of 38 and 65 copies/µL in reaction, respectively. For the analysis of clinical specimens, the positive, negative, and overall agreement between GeneSoC® RT-PCR and a conventional real-time RT-PCR was in all cases 100%, whereas for the comparison between GeneSoC® RT-PCR and the rapid antigen test, the agreements for positive, negative, and overall findings were 100%, 90.9%, and 95.7%, respectively. The mean time for completing GeneSoC® RT-PCR was 16 min 29 s (95% confidence interval, 16 min 18 s to 16 min 39 s). Conclusion: The microfluidic real-time PCR system, GeneSoC®, has an analytical performance comparable to that of conventional real-time RT-PCR with rapid turnover time, and represents a promising alternative to rapid antigen tests for diagnosing influenza A and B.

Effect of Cetuximab and EGFR Small Interfering RNA Combination Treatment in NSCLC Cell Lines with Wild Type EGFR and Use of KRAS as a Possible Biomarker for Treatment Responsiveness.

BACKGROUND: The epidermal growth factor receptor (EGFR) is a therapeutic target for patients with non-small cell lung cancer (NSCLC). Cetuximab is an anti-EGFR monoclonal antibody that inhibits EGFR signaling and proliferation of colorectal cancer and head and neck cancers. Since only few NSCLC patients benefit from cetuximab therapy, we evaluated a novel combination treatment using cetuximab and EGFR small interfering RNA (siRNA) to strongly suppress EGFR signaling and searched for a biomarker in NSCLC cell lines harboring wild-type EGFR. METHODS: Alterations in EGFR and its downstream genes in five NSCLC cell lines (A549, Lu99, 86-2, Sq19 and Ma10) were assessed through sequencing. The protein expression levels of these molecules were assessed through western blotting. The effect of combination treatment was determined through cell proliferation assay, caspase-3/7 assay, invasion assay, and migration assay. RESULTS: All cell lines were harboring wild-type EGFR, whereas KRAS, PTEN, TP53 and TP53 were mutated in A549 and Lu99; Lu99 and Sq19; Lu99, 86-2, Sq19 and Ma10; and A549, 86-2, and Sq19 cell lines, respectively. PTEN was not expressed in Sq19, and LKB1 was not expressed in both A549 and Sq19. TP53 was not expressed in both A549 and Lu99. The combination of cetuximab and EGFR siRNA significantly suppressed cell proliferation in 86-2, Sq19 and Ma10, which express wild-type KRAS. It induced apoptosis in A549, 86-2 and Ma10 cells, which express wild type PTEN. The combination treatment had no effect either on cell invasion nor migration in all cell lines. CONCLUSION: EGFR targeted therapy using the combination of cetuximab and EGFR siRNA is effective in NSCLC cell lines harboring wild-type EGFR. Wild-type KRAS may act as a potential biomarker for response to combination treatment by the induction of apoptosis in cells with wild-type PTEN.

Evaluation of antigen-positive toxin-negative enzyme immunoassay results for the diagnosis of toxigenic Clostridium difficile infection.

Clostridium difficile (C. difficile)-associated diarrhea (CDAD) is a challenging nosocomial infectious disease. C. DIFF Quik Chek Complete assay is widely used to detect glutamate dehydrogenase (GDH) antigen and toxin A/B of C. difficile simultaneously. However, the interpretation of GDH positive/toxin negative results is problematic. We performed a retrospective study of patients with GDH positive/toxin negative results to determine the probability of detecting toxigenic C. difficile and its risk factors. Between April 2012 and March 2017, we investigated cultures of fecal specimens followed by toxin detection tests. The clinical histories of patients with and without toxigenic C. difficile were compared using univariate- and multivariate-analyses. In total, 2675 patients were examined using C. Diff Quik Chek Complete assay. Among 356 GDH positive/toxin negative patients, cultures were performed in 220 cases and toxigenic C. difficile was recovered from 139 (63.2%) specimens. Patients with toxigenic C. difficile had significantly lower body mass index than those without. Over half the GDH positive/toxin negative patients were infected with toxigenic C. difficile. Lower BMI was a CDAD risk factor in this patient population. These data can be utilized to initiate isolation and clinical interventions before confirmatory test results are available. J. Med. Invest. 65:131-135, February, 2018.

Splenic Infarction in Acute Cytomegalovirus and Human Parvovirus Concomitant Infection.

We present a case report of a 35-year-old woman who had splenic infarction. She had persistent high fever, systemic joint pain, and abnormal liver function. She was diagnosed with cytomegalovirus and human parvovirus B19 concomitant infection. Her coagulopathy test revealed no abnormal results. She was treated with intravenous ganciclovir for 13 days; consequently, her splenic infarction improved after 7 weeks. As per our knowledge, this is the first case of cytomegalovirus and parvovirus B19 coinfection complicated by splenic infarction. Cytomegalovirus and parvovirus B19 may induce a hypercoagulation state during the acute phase.

Effect of Clarithromycin on the Expression of UL16-Binding Protein 2 in Human Cells.

BACKGROUND: Clarithromycin is a macrolide antibiotic that possesses anti-inflammatory and immunomodulatory properties. Although recent data suggests that macrolide antibiotics enhance Pseudomonas aeruginosa clearance from the lung, involving natural killer (NK) T cells in this process by activating the NKG2D-NKG2D ligand system, the precise underlying mechanism is still unclear. In this study, we examined the effect of clarithromycin on a potent NKG2D ligand, UL16-binding protein 2 (ULBP2), in the lung and its shedding mechanism. METHODS: The gene expressions of ULBP2 and the shredder proteinases of ULBP2, a disintegrin and metalloproteinase domain 10 (ADAM10) and ADAM17, were measured using real-time PCR. The cell surface ULBP2 expression was measured by flow cytometry. The amount of solubilized ULBP2 (sULBP2) was measured using an ELISA. The activity of ADAM17 was examined by measurement of fluorescence intensity from the fluorescence resonance energy transfer peptide substrate cleaved by ADAM17. RESULTS: Clarithromycin significantly induced transcription of ULBP2 and ADAM17 in both A549 and LCSC #2 cells, which endogenously express minimal and abundant levels of ULBP2, respectively. However, there was no significant change on transcription of ADAM10. The same tendency was observed when LCSC #2 cells were treated with tumor necrosis factoralpha processing inhibitor-2 to inhibit ADAM17 activity. The amount of sULBP2 was significantly decreased in both A549 and LCSC #2 cells by treatment with clarithromycin. Finally, clarithromycin significantly inhibited the activity of ADAM17 in LCSC #2 cells. CONCLUSION: These findings suggest that clarithromycin induces ULBP2 expression and reduces the amount of sULBP2, by possibly inhibiting the activity of the potent ULBP2-shedding enzyme ADAM17. Because these changes in ULBP2 and sULBP2 levels could activate NKT cells, this finding might indicate a novel mechanism by which clarithromycin improves the clearance of P. aeruginosa in chronic respiratory diseases.

A novel point-of-care system for high-speed real-time polymerase chain reaction testing for epidermal growth factor receptor mutations in bronchial lavage fluids after transbronchial biopsy in patients with non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) gene mutation testing is essential for choosing appropriate treatment options in patients with advanced non-small cell lung cancer (NSCLC). However, a time delay occurs between histological diagnosis and molecular diagnosis in clinical situations. To minimize this delay, we developed a novel point-of-care test for EGFR mutations, based on a high-speed real-time polymerase chain reaction (PCR) system designated here as ultrarapid PCR combined with highly accurate bronchoscopic sampling. We investigated whether our system for detecting EGFR mutations was valid by comparing test results with those obtained using a commercialized EGFR mutation test. We obtained small amounts of bronchial lavage fluids after transbronchial biopsies (TBBs) were performed on enrolled patients (n=168) who underwent endobronchial ultrasonography using a guide sheath (EBUS-GS). EGFR mutation analysis was performed by ultrarapid PCR immediately after EBUS-GS-TBBs were obtained (on the same day). After pathological diagnoses of NSCLC, EGFR mutation status in formalin-fixed, paraffin- embedded samples was confirmed by the PCR-invader method, and the concordance rates between the PCR methods were compared. The total diagnostic yield of EBUS-GS-TBB was 91.0%. The positive concordance rates for detecting 19del and L858R with the ultrarapid PCR and PCR-invader methods were both 100%. Negative concordance rates were 97.2 and 98.1%, respectively. We also demonstrated a dramatic effect of early erlotinib administration, based on ultrarapid PCR results, for a 52-year-old woman suffering from respiratory failure due to severe intrapulmonary metastases with poor performance status. In conclusion, ultrarapid PCR combined with EBUS-GS-TBB enabled rapid and reliable point-of-care testing for EGFR mutations.

A new rapid method for detecting epidermal growth factor receptor mutations in non-small cell lung cancer.

Mutations in the epidermal growth factor receptor (EGFR) gene are associated with a favorable clinical response to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib in non-small cell lung cancer (NSCLC). We present here, a new method for the rapid detection of the two most common EGFR mutations (delE746-A750 and L858R) from clinical samples. The methodology involves the combination of newly designed mutation-specific primers and a novel real-time PCR machine with an innovative thermo-control mechanism that enables ultrarapid PCR. We evaluated this method using a cell mixture composed of various ratios of lung cancer cells harboring mutated or wild-type EGFR, lung cancer tissues obtained by surgery, and a cytology sample obtained by bronchoscopy from a lung cancer patient. In the cell mixture analysis, our method detected 0.1% of cells with delE746-A750 and 1% of cells with L858R among cells with wild-type EGFR. In 143 lung cancer tissues, the result of this assay was concordant with those of direct sequencing in 138 samples. The five samples with discordant results were tested using a PCR-Invader assay and the result matched those of our method at 100%. We also successfully detected EGFR mutations in the lavage obtained from a lung cancer patient. The turnaround time for this method was <10 min, and all steps could be accomplished in <50 min after sample collection. Thus, our novel PCR method offers a rapid, simple, and less expensive test for EGFR mutations and can be applied as a point-of-care diagnostic test.

[A case of lung abscess due to Aspergillus viridinutans in a patient with aplastic anemia].

A 75-year-old woman with aplastic anemia was admitted to our university hospital because of a dry cough that had persisted for a month. Chest computed tomography showed a mass shadow with a central low attenuation area in the lower lobe of the left lung. Filamentous fungus resembling Aspergillus fumigatus was cultured from the specimens obtained by transthoracic needle aspiration biopsy and bronchoalveolar lavage. The initial diagnosis was a lung abscess due to A. fumigatus, although the patient did not respond well to antifungal agents. Subsequently, the filamentous fungus was identified as Aspergillus viridinutans by sequence analysis of the ß-tubulin gene, and the patient was successfully treated with combination therapy along with granulocyte colony-stimulating factor. The incidence of A. viridinutans infection is very rare. A. viridinutans is morphologically similar to A. fumigatus; however, the response to antifungal agents is generally worse than that observed in A. fumigatus infections. Therefore, the selection of agents and supplemental therapy is of vital importance in cases of A. viridinutans infection.

Positive Predictive Value of True Bacteremia according to the Number of Positive Culture Sets in Adult Patients.

BACKGROUND: Performing multiple blood culture sets simultaneously is a standard blood culture methodology, although it is often difficult to distinguish true bacteremia from contamination when only one of several blood culture sets is positive. This study clarified the relationship between the number of positive blood culture sets and clinical significance in patients with positive blood culture. METHODS: Patients aged 18 years and over with at least 1 positive blood culture were enrolled. Positive blood culture episodes were categorized from clinical records as true bacteremia, contamination, or unknown clinical significance. The associations among episodes of true bacteremia, isolated bacteria, the number of positive blood culture sets from among the performed sets, and the clinical background of patients were analyzed. RESULTS: Among a total of 407 episodes, 262, 67 and 78 were true bacteremia, contamination and unknown clinical significance, respectively. The positive predictive values (PPVs) of 1 out of 1, 1 out of 2 and 2 out of 2 positive sets in cases of Staphylococcus aureus, were 81.3%, 50% and 100% respectively; those in cases of coagulase-negative Staphylococci were 20.5%, 10.8% and 63.5%, respectively. Almost all cases of Escherichia coli, Pseudomonas aeruginosa, Klebsiella species and Candida species were true bacteremia. The probability of true bacteremia was strongly associated with recent surgery in multivariate analysis (P < 0.05). CONCLUSION: The probability of true bacteremia based on the number of positive culture sets from among the performed sets varies by microorganism. Therefore, PPVs calculated using this method may help physicians distinguish true bacteremia from contamination.

Evaluation of a noise reduction procedure for chest radiography.

BACKGROUND: The aim of this study was to evaluate the usefulness of noise reduction procedure (NRP), a function in the new image processing for chest radiography. METHODS: A CXDI-50G Portable Digital Radiography System (Canon) was used for X-ray detection. Image noise was analyzed with a noise power spectrum (NPS) and a burger phantom was used for evaluation of density resolution. The usefulness of NRP was evaluated by chest phantom images and clinical chest radiography. We employed the Bureau of Radiological Health Method for scoring chest images while carrying out our observations. RESULTS: NPS through the use of NRP was improved compared with conventional image processing (CIP). The results in image quality showed high-density resolution through the use of NRP, so that chest radiography examination can be performed with a low dose of radiation. Scores were significantly higher than for CIP. CONCLUSION: In this study, use of NRP led to a high evaluation in these so we are able to confirm the usefulness of NRP for clinical chest radiography.

Rapamycin induces p53-independent apoptosis through the mitochondrial pathway in non-small cell lung cancer cells.

The mammalian target of rapamycin (mTOR) is a key kinase acting downstream of growth factor receptor PI3K and AKT signaling, leading to processes resulting in increased cell size and proliferation through translation control. Rapamycin, a specific inhibitor of mTOR, results predominately in G1 cell cycle arrest through translation control and occasionally, cell type-dependent apoptosis by an unknown mechanism. In this study, we investigated the effect and mechanism of action of rapamycin on non-small cell lung cancer (NSCLC) cell lines with p53 mutations. Cell proliferation was evaluated by modified MTT assay. The apoptotic effect of rapamycin was measured by caspase-3 activation and flow cytometric analysis of Annexin V binding. The expression of Bcl-2 and the release of cytochrome c from mitochondria were evaluated by western blotting. We found that rapamycin induced apoptosis in NSCLC cell lines with p53 mutations. Western blot analysis demonstrated that rapamycin downregulates the expression levels of Bcl-2, which leads to increased cytochrome c release from mitochondria and subsequent activation of caspase cascades. These findings suggest that rapamycin induces p53-independent apoptosis through downregulation of Bcl-2 and the mitochondrial pathway in NSCLC cell lines as a novel antitumor mechanism.

Diagnostic and prognostic impact of serum-soluble UL16-binding protein 2 in lung cancer patients.

UL16-binding protein 2 (ULBP2) is one of the ligands for NKG2D (NKG2DL). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Soluble forms of NKG2DL have been reported in the serum of patients with several types of cancer. The present study investigated the diagnostic and prognostic significance of serum-soluble ULBP2 (sULBP2) in lung cancer patients. We used flow cytometry to evaluate the surface expression of NKG2DL by various lung cancer cells, while sULBP2 was measured using our original ELISA. In addition, the immunological effect of sULBP2 on peripheral blood mononuclear cells (PBMC) was examined by the (51) Cr release assay. We found that ULBP2 was highly expressed and that the sULBP2 level was elevated in supernatants of cultured non-small-cell lung cancer (NSCLC) cells as well as in the serum of NSCLC patients. ULBP2 levels were especially high in squamous cell carcinoma (SQ) patients. Clinical stage IIIB and IV NSCLC patients with a sULBP2 level ≥ 8.7 pg/mL showed significantly shorter survival than patients with sULBP2 <8.7 pg/mL. In multivariate analysis, a sULBP2 level ≥ 8.7 pg/mL (hazard ratio [HR], 2.13; P = 0.038) and clinical stage IV (HR, 2.65; P = 0.019) were independent determinants of a poor outcome. As a possible mechanism, we demonstrated that sULBP2 directly suppresses the cytolytic activity of PBMC. In conclusion, ULBP2 is the most significant NKG2DL for lung cancer, and sULBP2 is useful in the diagnosis of SQ and as a prognostic indicator for patients with advanced NSCLC.

Influence of Asian Desert Dust on Lower Respiratory Tract Symptoms in Patients with Asthma over 4 Years.

The Asian Dust Storm (ADS) aggravates symptoms and pulmonary dysfunction in adult asthma patients. Our objective was to investigate the association of air pollutants and metals in desert dust with worsening of asthma symptoms during the ADS. A telephone survey was performed to investigate the upper and lower respiratory tract symptoms, ocular symptoms and skin symptoms of asthma patients during the ADS in March between 2007 and 2010. Four surveys were conducted in 46 patients. Two patients noted worsening of lower respiratory tract symptoms in all four surveys, as well as 2 patients in three surveys, 7 patients in two surveys, and 9 patients in one survey. There was no worsening of lower respiratory tract symptoms in 26 patients. In each patient, the influence of the ADS on lower respiratory tract symptoms varied between surveys. In 2010, the level of suspended particulate matter was highest in all four years, but the smallest number of patients noted worsening of lower respiratory tract symptoms. Among pollutants, only the maximum concentration of nitrogen dioxide during the ADS was significantly associated with the worsening of lower respiratory tract symptoms. The influence of the ADS on lower respiratory tract symptoms of adult asthma patients is variable.

Pollen augments the influence of desert dust on symptoms of adult asthma patients.

BACKGROUND: East Asian desert dust storms that occur during mainly spring are called Asian dust storms (ADS). Our objective was to study the association of pollen and ADS with symptoms of adult asthma patients in Japan. METHODS: We designed a telephone survey to investigate the upper and lower respiratory, ocular, and skin symptoms of asthma patients during ADS in February, March, and December on 2009. Peak expiratory flow (PEF) was also measured from February to May. RESULTS: We surveyed 106 patients in February, 101 patients in March, and 103 patients in December. In February and March, Japanese cedar and/or cypress pollen was also in the atmosphere during ADS, but no pollen was identified during December survey. Worsening of upper or lower respiratory, ocular, or skin symptoms was noted by 20.8% of patients in February, 33.7% in March, and 16.5% in December. Worsening of symptoms was significantly more common in March than in February or December. Two patients needed emergency treatment for exacerbation during ADS in March, but no patient needed hospitalization in any period. There was no significant difference of the daily morning PEF/personal best PEF ratio between ADS days and control days. However, in patients with worsening of upper and/or lower respiratory tract symptoms, the daily morning PEF/personal best ratio was significantly associated with the atmospheric level of particulate matter, but not with levels of pollen or other air pollutants. CONCLUSIONS: Pollen augmented symptoms in adult asthma patients, but ADS on its own also were able to aggravate symptoms and pulmonary function.

Correlation between Asian dust storms and worsening asthma in Western Japan.

BACKGROUND: Severe wind storms during spring in East Asia, called Asian dust storms (ADS), have been assessed in the past for their effect on health in Asian countries. Our objective was to study the ADS association with asthma symptoms in adult patients in Japan. METHODS: We designed a telephone survey to assess ADS influence on upper and lower respiratory, ocular and cutaneous symptoms in 98 patients with adult asthma from April to May 2007. Peak expiratory flow (PEF) was also measured from February to May. RESULTS: Worsening lower respiratory symptoms were noted by 22 of 98 patients during ADS in April, when Japanese cedar pollen levels also increased. During ADS in May, however, Japanese cedar and cypress pollen levels were not elevated, 11 patients had worsening of lower respiratory symptoms. None required emergency treatment for the exacerbation. Lower respiratory symptoms worsening most were cough and sputum; this was more common in patients with allergic rhinitis or atopy than in those without (P < 0.05). Min%Max differed significantly at 88.7 ± 6.6% during dust dispersion period, defined as the ADS day plus the next 6 days, versus 92.0 ± 5.3% during the 7-day period before a dust storm. CONCLUSIONS: We found that ADS aggravated lower respiratory symptoms in adult patients with asthma, but this influence was mild.

Antibody-dependent cellular cytotoxicity mediated by cetuximab against lung cancer cell lines.

PURPOSE: Epidermal growth factor receptor (EGFR) is commonly overexpressed in lung cancer. Cetuximab is a chimeric mouse-human antibody targeted against EGFR. Compared with its inhibitory properties, its immunologic mechanisms have not been well studied. In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC) activity of cetuximab against lung cancer cell lines. EXPERIMENTAL DESIGN: We studied the correlation between EGFR expression in lung cancer cell lines and the ADCC activity of cetuximab as well as the influence of interleukin-2 and chemotherapy on the ADCC activity. EGFR expression was measured by a quantitative flow cytometric analysis and immunohistochemistry. The ADCC activity was assessed by a 4-h (51)Cr release assay. Peripheral blood mononuclear cells, purified T cells, natural killer (NK) cells, and monocytes from healthy donors or lung cancer patients were used as effector cells. RESULTS: Fresh peripheral blood mononuclear cells exhibited cetuximab-mediated ADCC activity against lung cancer cell lines at a low concentration of cetuximab (0.25 microg/mL). A logarithmic correlation was observed between the number of EGFRs and ADCC activity. Even low EGFR expression, which was weakly detectable by immunohistochemistry, was sufficient for maximum ADCC activity, and further increases in EGFR expression on the target cells had no further effect on the ADCC activity. In addition, ADCC activity was enhanced by interleukin-2 mainly through activation of NK cells and was less susceptible to immunosuppression by chemotherapy than NK activity in lung cancer patients. CONCLUSIONS: These observations suggest the importance of ADCC activity as an immunologic mechanism of cetuximab in biological therapy for lung cancer patients.

Diverse activation states of RhoA in human lung cancer cells: contribution of G protein coupled receptors.

Rho GTPases play an essential role in the control of various cellular functions. Accumulating evidence suggests that RhoA overexpression contributes to human cancer development. However, the activation states of RhoA are poorly defined in cancer cells. In this study, we examined both the expression levels and the activation states of RhoA in various lung cancer cells by quantitative real-time reverse transcriptase-polymerase chain reaction and in vivo Rho guanine nucleotide exchange assay, respectively. Moreover, we dissected the signaling pathway from the cell surface receptors to RhoA using a broad-spectrum G protein coupled receptor (GPCR) antagonist, [D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP), and a recently reported Galphaq/11-selective inhibitor, YM-254890. We found that RhoA was expressed highly in large cell carcinoma cells but only weakly in adenocarcinoma cells. The activation states of RhoA are considerably different from its expression profiles. We found that four of six small cell lung carcinoma (SCLC) cell lines exhibited a moderate to high activation rate of RhoA. The addition of [D-Arg1,D-Trp5,7,9,Leu11]SP reduced RhoA activity by almost 60% in H69 SCLC cells. The addition of YM-254890 had no effect on RhoA activity in H69 cells. Our results suggest that RhoA is activated in various lung cancer cells independent of its expression levels, and the high activation state of RhoA in SCLC cells mainly depends on a neuroendocrine peptide autocrine system which signals through Galpha12 coupled GPCR to RhoA. This study provides new insights into RhoA signaling in lung cancer cells and may help in developing novel therapeutic strategies against lung cancer.

Lung cancer arising in association with middle lobe syndrome.

Middle lobe syndrome, caused mainly by benign inflammatory diseases, such as chronic bronchitis and bronchiectasis, is manifested clinically as a chronic cough with sputum production. The prognosis associated with this syndrome is considered good in most cases which are caused by chronic inflammatory diseases. A patient who developed lung cancer in the course of long-term treatment for right middle lobe syndrome is described. A 63-year-old woman was admitted to our hospital with complaints of right iliac bone pain. She had been treated for chronic bronchitis and bronchiectasis associated with middle lobe syndrome for 16 years before admission. Work-up of a lung adenocarcinoma originating from the right middle lobe disclosed bone metastasis to the illium. Tumorigenesis in association with middle lobe syndrome has not yet been reported, but this first reported case suggests the need to be alert to the possibility.

Loss of imprinting of PEG1/MEST in lung cancer cell lines.

Paternally expressed imprinted gene 1/mesoderm-specific transcript (PEG1/MEST) is an imprinted gene expressed from the paternal allele. Recently, frequent loss of imprinting (LOI) of PEG1/MEST has been reported in lung adenocarcinomas. It is suggested that the LOI may be involved in pathogenesis of lung adenocarcinoma. In the present study, incidence of LOI of PEG1/MEST was examined in lung cancer cell lines, including small cell lung cancer (SCLC). Among 50 cell lines tested, 20 cell lines were heterozygous for the AflIII site of the PEG1/MEST gene. In these heterozygotes, biallelic expression was observed in 9 cell lines (45%), monoallelic in 11 (55%). In cell lines of non-small cell lung cancer (NSCLC), 62% (8 of 13) exhibited biallelic expression. In SCLC, only 1 of 7 cell lines (14%) showed biallelic expression. LOI of PEG1/MEST in the NSCLC cell line is significantly frequent compared with that in SCLC cell lines (p=0.043). This result supports the possibility that LOI may be related to tumorigenesis and malignant transformation, especially in NSCLC.

Identification of genes whose expression is upregulated in lung adenocarcinoma cells in comparison with type II alveolar cells and bronchiolar epithelial cells in vivo.

To identify genes whose expression is upregulated in lung adenocarcinoma (AdC) cells in comparison with noncancerous peripheral lung epithelial cells, type II alveolar cells and bronchiolar epithelial cells, as well as AdC cells, were isolated by laser capture microdissection, and subjected to cDNA microarray analysis of 637 human cancer-related genes. Each of the component cells was obtained from several different individuals and analysed independently. As a comparison, two lung AdC cell lines and two primarily cultured normal lung epithelial cell lines were also subjected to cDNA microarray analysis. Four genes, TOP2A, MMP15, MX2 and KOC1, were commonly upregulated in microdissected AdC cells in comparison with microdissected epithelial cells. Hierarchical clustering analysis revealed that differences in gene-expression profiles were more evident between cultured and uncultured cells than between cancerous and noncancerous cells. To further identify the common molecular targets of AdC cells in vivo, quantitative real-time RT-PCR was performed against the four genes upregulated by cDNA microarray analysis. The TOP2A, MMP15, MX2 and KOC1 genes were overexpressed in 10/10 (100%), 8/10 (80%), 5/10 (50%) and 3/10 (30%) microdissected AdC cell samples, respectively, in comparison with any of nine independently microdissected noncancerous epithelial cell samples. The TOP2A gene was commonly overexpressed in lung AdC cells, as previously reported. In addition, the MMP15 and MX2 genes were identified, for the first time, as being commonly overexpressed in lung AdC cells. These results strongly indicate that the MMP15 and MX2 genes could be novel markers for molecular diagnosis and therapy of lung AdC.