SUMMARYAnalysis of molecular genetic markers in biological fluids has been proposed as a useful tool for cancer diagnosis. MicroRNAs (miRNAs) are small regulatory RNAs that are frequently dysregulated in lung cancer and have shown promise as tissue-based markers for its prognostication. The aim of this study was to determine whether aberrant miRNA expression can be used as a marker in sputum specimen for the diagnosis of non-small cell lung cancer (NSCLC). Experimental Design: Expressions of mature miRNAs, mir-21 and mir-155, were examined by real-time reverse transcription polymerase chain reaction (RT-PCR) and normalized to that of control miRNA, U6B, in sputum of 23 patients with NSCLC and 17 cancer-free subjects. The data was compared with conventional sputum cytology for the diagnosis of lung cancer. All endogenous miRNAs were present in sputum in a remarkably stable form and sensitively and specifically detected by real-time RT-PCR. Mir-21 expression in the sputum specimens was significantly higher in cancer patients (76.32 ± 9.79) than cancer-free individuals (62.24±3.82) (p<0.0001). Furthermore, overexpression of mir-21 showed highly discriminative receiver-operator characteristic (ROC) curve profile, clearly distinguishing cancer patients from cancer-free subjects with areas under the ROC curve at 0.902 ± 0.054. Detection of mir-21 expression produced 69.66% sensitivity and 100.00% specificity in diagnosis of lung cancer, as compared with 47.82% sensitivity and 100.00% specificity by sputum cytology. The measurement of altered miRNA expression in sputum could be a useful noninvasive approach for the diagnosis of lung cancer.

The RB and p16 INK4A tumor suppressor genes function in the same pathway of cell cycle control. Previous evidence indicates that the p16 INK4A gene is transcriptionally repressed by the RB gene product, pRB. In this study using human ovarian cancer cell lines, we found that RB protein and mRNA were expressed at higher levels in cell lines lacking p16 than in those with normal p16. Since this suggests a potential role of p16 in regulating the cellular level of pRB, we studied the e ect of wild-type p16 INK4A on expression of the RB gene. Introduction of p16 INK4A , carried by an adenovirus vector, into p16-negative cell lines dramatically decreased expression of RB protein and mRNA. Nuclei run-o assays demonstrated that p16 expression induced transcriptional downregulation of the RB gene. These results indicate that expression of RB is inversely regulated by p16. The ®ndings reveal a new dimension of pRB-p16 interaction and should have implications for p16 INK4A -mediated gene therapy.

BACKGROUND Sputum is an easily accessible diagnostic material for lung cancer early detection by cytologic and molecular genetic analysis of exfoliated airway epithelial cells. However, the use of sputum is limited by its cellular heterogeneity, which includes >95% macrophages and neutrophils and only about 1% bronchial epithelial cells. We propose to obtain concentrated and purified bronchial epithelial cells to improve early detection of lung cancer in sputum samples. METHODS Sputum was collected from patients with stage I nonsmall-cell lung cancer, cancer-free smokers, and healthy nonsmokers. Magnetic-assisted cell sorting (MACS) with anti-CD14 and anti-CD16 antibody beads were used to enrich bronchial epithelial cells by depleting macrophages and neutrophils from sputum. Fluorescence in situ hybridization (FISH) analysis for detection of FHIT deletion and cytology were evaluated in the enriched specimens. RESULTS The bronchial epithelial cells were concentrated to 40% purity from 1.1% of the starting population, yielding an average of 36-fold enrichment and at least 2.3 × 105 cells per sample. Detecting FHIT deletions for lung cancer diagnosis produced 58% sensitivity in the enriched sputum, whereas there was 42% sensitivity in the unenriched samples (P = .02). Cytologic examination of the enriched sputum resulted in 53% sensitivity, as compared with 39% sensitivity in unenriched sputum (P = .03). Furthermore, only 2 cytocentrifuge slides of the unenriched sputum were needed for the analyses, as compared with up to 10 cytocentrifuge slides required from the unprocessed specimens. CONCLUSIONS The enrichment of bronchial epithelial cells could improve the diagnostic value of sputum and the efficiency of genetic and cytologic analysis of lung cancer.

Although umbilical cord stricture and umbilical cord overcoiling have been established as causes of intrauterine fetal demise, relatively few studies addressed this issue, most of them being case reports. We reviewed a total of 268 fetal autopsies during a 3-year period from 1998 to 2001. One hundred thirty nine cases of fetal demise including spontaneous abortion were identified. Nineteen percent (26 of 139) were associated with umbilical cord stricture, overcoiling, or a combination of both. Stricture of the umbilical cord was defined as a decrease in diameter in relation of the remaining umbilical cord; overcoiling as 0.3 coil/cm or greater. Fetal demise most commonly occurred in the second trimester, with a mean gestation age of 21 weeks. The average maternal age was 33 years; 15% had a prior fetal demise. We found that 77% (20 of 26) of these cases had umbilical cord stricture only or with overcoiling, 23% (6 of 26) had umbilical cord overcoiling alone. Localized deficiency of Wharton's jelly and increased collagen were found in all cases with umbilical cord stricture with or without overcoiling. In patients with umbilical cord overcoiling alone, 25% had Wharton's jelly deficiency; half of them had increased collagen deposition in the umbilical cords. The placenta was reviewed for secondary thrombosis of the vessels of the chorionic plate. Thrombosis of the vessels of the chorionic plate was noted in 54% of the patients. Our study suggests that umbilical cord stricture and cord overcoiling may represent two distinct pathological entities commonly causing fetal demise. This observation reinforces the importance of a fetal autopsy with careful examination of the placenta and umbilical cord with documentation of the cord coil index.