Reduced HIC-1 gene expression in non-small cell lung cancer and its clinical significance.

BACKGROUND: HIC-1 (hypermethylated in cancer-1) is a candidate tumor suppressor gene, identified in a region of frequent loss of heterozygosity on chromosome 17p13.3, which is telomeric from TP53 and often deleted in surgically resected lung cancers. To determine the significance of HIC-1 in lung cancer, we assessed its expression status and prognostic association in 47 adenocarcinomas and squamous cell carcinomas. MATERIALS AND METHODS: RNA was extracted from tumors and corresponding normal tissues of surgically resected lungs, and the amount of HIC-1 mRNA was determined by means of semi-quantitative reverse transcriptase-polymerase chain reaction. RESULTS: HIC-1 expression in tumors was less than that in normal lung tissues in 40 of 47 patients (85%), indicating frequent partial silencing. Median tumor/normal lung tissue (T/L) ratios for HIC-1 expression were 0.51 and 0.75 for adenocarcinomas and squamous cell carcinomas, respectively. No significant difference of median T/L ratio was observed between the two histological types, or among clinical stages of the patients. However, the reduced expression of HIC-1 gene in the tumor had a direct link with the clinical outcome: lower T/L ratios (< 0.5) were significantly associated with short survival (P = 0.034), an association also observed in cases restricted to stage I (P = 0.047). CONCLUSIONS: The results suggest that low HIC-1 expression is involved in malignant progression of non-small cell lung cancer.

Different subtypes of human lung adenocarcinoma caused by different etiological factors. Evidence from p53 mutational spectra.

Human lung adenocarcinomas are only relatively weakly associated with tobacco smoke, and other etiological factors need to be clarified. These may also vary with the histopathology. Because the p53 mutation status (frequency and spectrum) of a carcinoma can provide clues to causative agents, we subclassified 113 adenocarcinomas into five cell types: hobnail, columnar/cuboidal, mixed, polygonal, and goblet (54, 23, 18, 13, and 5, respectively) and investigated relationships with p53 mutations and smoking history. In the hobnail cell type, a low mutational frequency (37%) and a high proportion of transitions (65%), especially G:C to A:T transitions at CpG dinucleotides (45%) associated with spontaneous mutations, were found with a weak relation to tobacco smoke. In contrast, a high mutation frequency (70%) with a higher proportion of transversions (50%), especially G:C to T:A (44%) on the nontranscribed DNA strand, caused by exogenous carcinogenic agents like tobacco smoke, were observed for the columnar cell type, as with squamous cell carcinomas. These results indicate that two major subtypes of lung adenocarcinoma exist, one probably caused by tobacco smoke, and the other possibly due to spontaneous mutations. For the prevention of lung adenocarcinomas, in addition to stopping tobacco smoking, the elucidation of endogenous mechanisms is important.

Prognostic value of genetically diagnosed lymph node micrometastasis in non-small cell lung carcinoma cases.

The predictive value of lymph node micrometastasis, detected by immunohistochemical or genetic methods, is well appreciated in terms of prognosis. However, a major problem is high false-positive rates, because most methods focus on cytokeratin, which is a component not only of carcinoma but also normal epithelial and nonepithelial cells. Mutant allele-specific amplification (MASA) can detect DNAs derived from cancer cells itself, reportedly with high sensitivity. It was, therefore, used with nested-PCR using p53 or K-ras mutation for analysis of lymph node micrometastasis in non-small cell lung carcinoma (NSCLC) patients in the present study, in comparison with the immunohistochemical method using an anti-cytokeratin reagent for the same samples. Lymph nodes from 31 NSCLC patients with p53 and K-ras mutated tumors (30 and 1, respectively) staged as pathological (p)-T1-4 N0-1 and M0 were examined. Genetic and immunohistochemical methods demonstrated positive reactions in 34 (15%) and 61 (27%) of 229 lymph nodes, respectively (9 cases, 29%, and 24 cases, 77%). The concordance with the two methods was 77%, but 13 (39%) of 34 genetically positive lymph nodes could not be detected by immunohistochemistry (IHC). Of 22 cases with p-N0 disease, 6 (27%) were genetically positive in hilar and/or mediastinal lymph nodes, and 4 (67%) of them died after cancer relapse. In contrast, none of the patients without micrometastasis died of cancer (P < 0.001, log rank analysis). Of the same p-N0 patients, 17 (77%) were positive by IHC, and 4 (24%) of them died of cancer, whereas 5 negative patients did not suffer cancer relapse. Survival did not significantly differ between cases positive and negative (P = 0.246) by IHC. According to the g-N (N factor restaged by a genetic method), patients with g-N1 and g-N2 disease had a shorter survival than those with g-N0 disease (P = 0.042 and P < 0.001, respectively). However, no significant difference was observed with grading by IHC. Thus, detection of micrometastasis in regional lymph nodes with the MASA method, in other words with a carcinoma-specific marker, is of greater prognostic significance for early stage NSCLC patients than immunohistochemical results. This approach should facilitate selection of patients for whom postoperative adjuvant chemotherapy should be performed.

Three new regions on chromosome 17p13.3 distal to p53 with possible tumor suppressor gene involvement in lung cancer.

We investigated loss of heterozygosity (LOH) at the distal portion of the p53 gene on the short arm of chromosome 17 in lung cancers in order to search for new tumor suppressor genes. The roles of the putative genes were also studied in terms of pathological features. One hundred and forty-five resected non-small cell lung cancers were examined for LOH using 11 markers mapped on, and distal to the p53 locus, and deletion maps were constructed. Four commonly deleted regions were found: one from TP53 to ENO3, where the p53 gene resides, and three others from ENO3 to D17S1566, D17S379 to D17S1574 and distal to ABR, with LOH frequencies almost the same as, or higher than, at the TP53 locus. Examination of the relationship between LOH of the latter three regions and histopathological parameters of adenocarcinomas (genetically negative for p53 mutation) revealed allelic losses on D17S379 to be associated with advanced lesions, while D17S513 was more frequently deleted in poorly differentiated tumors. These results indicate that new tumor suppressor gene(s) may reside on these three distinctly deleted regions on chromosome 17p13.3 distal to the p53 gene in lung cancer, with possible roles in progression and differentiation of adenocarcinomas.

Abnormalities of the FHIT gene in human oral carcinogenesis.

The abnormalities of the fragile histidine triad (FHIT) gene in tissue samples of oral squamous cell carcinomas (SCCs) along with several leukoplakias and an erythroplakia were examined to determine whether the FHIT gene is actually a frequent target in vivo for alteration during oral carcinogenesis. Abnormal transcripts of the FHIT gene were found in eight of 15 oral SCCs. Although these abnormal transcripts varied widely, deletion patterns incorporating a deletion of exon 5 were the most common. Loss of heterozygosity (LOH) analysis demonstrated that the abnormal FHIT transcripts found in cancer cells were attributable to abnormalities of the FHIT gene. Abnormal FHIT transcripts were also observed in two of seven premalignant lesions. Interestingly, in the case of one patient with a premalignant lesion showing an abnormal FHIT transcript, subsequent oral SCC developed during a 3-year follow-up period. On the other hand, in the two patients from whom both leukoplakia and SCC samples were taken simultaneously, abnormal FHIT transcripts were found only in the SCCs. Although the functional role of FHIT remains to be clarified, these results suggest that the FHIT alteration is actually involved in carcinogenesis of the oral epithelium.

[A case of non-small-cell lung cancer with intramedullary spinal cord metastasis diagnosed pre-mortem].

The patient was a 54-year-old man who in May 1999 received a diagnosis of squamous cell carcinoma, T4 N2 M1, stage IV. Systemic chemotherapy and stereotactic radiosurgery were performed only to result in further progression of the disease. In August 1999, he experienced gait disturbance due to lumbar pain. Rehabilitation improved the gait disturbance and he was discharged. In October, since the pain reappeared and there was numbness in the right leg, he was readmitted. Brain MRI revealed multiple brain metastasis and whole brain irradiation was performed. But his symptoms deteriorated, and palsy of the right leg ensued. Later, bladder dysfunction also developed. Since spinal cord MRI revealed intramedullary metastasis at Th 12 and L1 levels, we performed radiotherapy for the lumbar medullary lesion, together with systemic chemotherapy. After chemoradiotherapy the tumor size decreased and the pain improved. Cases of lung cancer with intramedullary metastasis are rare, especially those diagnosed before death.

p53 null mutations undetected by immunohistochemical staining predict a poor outcome with early-stage non-small cell lung carcinomas.

The importance of p53 mutations in the pathogenesis of human lung carcinoma is well established, but it is still controversial whether the presence of p53 mutations or overexpression of p53 protein adversely affects an individual patient's chances of survival. The controversy may be partially due to the methodological differences in examination for p53 alterations: gene analysis or immunohistochemical staining. Furthermore, recent studies have suggested that different types of mutations of the p53 tumor suppressor gene confer different biological properties. To clarify the relationship between immunohistochemical staining and prognosis, we investigated mutations using single-strand conformation polymorphism followed by sequencing for exons 4-8 and 10 in 144 surgically treated non-small cell lung carcinoma patients with intensive clinical follow-up. Of 144 cases, 107 adenocarcinomas were examined for immunohistochemical staining with RSP53 antibody. p53 gene mutations were observed in 65 tumors (45%), including 44 missense and 21 null mutations, the latter comprising 7 nonsense mutations, 8 deletions, 2 insertions, and 4 splicing junction mutations. Presence of p53 mutations was an independent prognostic factor with a statistical trend (P = 0.14) in stage I patients but not in all cases. When examined by mutational pattern, null mutation was a significant indicator of poor outcome by multivariate analysis (P = 0.03) in stage I patients, whereas cases with missense mutations and without mutations did not differ (P = 0.76). Forty (37%) tumors demonstrated overexpression of the p53 protein but without any survival difference. Most tumors (76%) with missense mutations were immunopositive, but those with null mutations with one exception (93%) were not, and the concordance between the mutations and immunohistochemical staining was rather low at 65%. These data suggest that the type of p53 mutation is important for prediction of outcome in early-stage non-small cell lung carcinoma patients, whereas immunohistochemical staining for abnormal p53 gene products is nonpredictive. Furthermore, null mutations causing loss of function of the gene product may play more important roles than missense mutations in tumor progression.

The expression of p73 is increased in lung cancer, independent of p53 gene alteration.

p73 gene, a new p53 homologue, has been identified: it supposedly acts as tumour suppressor gene in neuroblastoma. To clarify whether p73 might be involved in lung carcinogenesis, we examined p73 expression in resected lung cancer and paired normal lung in 60 cases using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We also examined p73 gene status in three representative cases using Southern blot, and p53 gene alteration in 49 cases using PCR-single-strand conformation polymorphism (PCR-SSCP) and direct sequence. In 87% of the cases (52/60) p73 expression in tumour was more than twice as high as that in paired normal lung tissues, and the difference between p73 expression in tumour and normal lung tissue was significant (P < 0.0001). However, Southern blot analysis revealed that none of the cases showed p73 gene amplification. Compared with clinicopathological characteristics, p73 expression correlates significantly with histological differences and age of patient, independently (P < 0.05). Concerning p53 gene status, 43% (21/49) showed p53 gene alteration, but there was no correlation between p73 overexpression and p53 gene alteration. Our results suggest that need for further functional analysis of the role of p73 in lung carcinogenesis.

[A clinical application of laser scanning cytometer--the significance in the cytology as an additional diagnostic technique].

Laser scanning cytometer (LSC) is a new machine similar to flow cytometer but with advantages for certain clinical and research applications. LSC is a microscope based and measures cells on a slide with the position of each cell on the slide. This new technique of LSC can be utilized on extremely small specimens and enables direct correlation of all of the measured fluorescent parameters with light microscopic cytologic morphology. To date, LSC has been successfully used to perform DNA content analysis of numerous specimen types and automated analysis of fluorescence in situ hybridization specimens. In this report, we describe characteristics of LSC comparison with flow cytometry and a clinical application of LSC focused on DNA content analysis in clinical specimens with pulmonary disorders. LSC provides a number of benefits that may make it more suitable for clinical laboratories than FCM.

Abnormal FHIT transcripts found in both lung cancer and normal lung tissue.

Occurrence of abnormal transcripts of the FHIT (fragile histidine triad) gene has been reported in various types of cancer. On the other hand, aberrant transcripts are sometimes found in non-neoplastic tissues, so the relationship between the presence of abnormal transcripts of the FHIT gene and cancer pathogenesis is controversial. We investigated alterations in the FHIT locus, detected by nested reverse transcription-polymerase chain reaction and/or allelic status, in 88 primary lung cancers and normal lung tissues, and 22 normal lung tissues with metastatic lung cancer as a control. The frequencies of abnormal transcripts were 59% in lung cancer, 35% in paired normal lung, and 64% in normal control lung; the difference in frequencies between lung cancer and paired normal lung was significant, while that between lung cancer and normal control lung was not. Sequence analysis revealed that there were no cancer-specific abnormal transcripts entirely missing two or more exons, nor were the abnormal transcripts of lung cancer identical with those of paired normal lung in the same individual. Furthermore, we found no correlation between loss of heterozygosity in the FHIT locus and occurrence of abnormal FHIT transcripts. These results suggest that the presence of abnormal FHIT transcripts, in terms of their frequency and variety, is not cancer-specific in lung carcinogenesis, and the abnormality may be mainly due to abnormal splicing and processing of the transcripts. To estimate the precise function of the FHIT gene, further study of the FHIT protein in lung carcinogenesis is needed.

Intracellular accumulation of thallium as a marker of cisplatin cytotoxicity in nonsmall cell lung carcinoma: an application of inductively coupled plasma mass spectrometry.

BACKGROUND: Thallium-201 (201Tl) scintigraphy has been used to detect malignant pulmonary disease. The mechanism of Tl influx in tumor cells is believed to be similar to that of cisplatin (CDDP) mediated by sodium- and potassium-activated adenosine triphosphatase (Na-K ATPase), and the Na-K ATPase activity may determine the cellular CDDP accumulation and sensitivity to CDDP. The objective of this study was to determine the accumulation of CDDP and Tl in vitro by using inductively coupled plasma mass spectrometry (ICP-MS), a new analytic technique for detecting ultra trace elements, and to evaluate the correlations between cellular CDDP and Tl accumulation, between CDDP 50% inhibitory concentration (IC50) values and cellular CDDP accumulation, and between CDDP IC50 values and cellular Tl accumulation. METHODS: Eight nonsmall cell lung carcinoma (NSCLC) cell lines were used (five adenocarcinomas and three squamous cell carcinomas). The cell lines were exposed to CDDP or Tl for 1 hour, and the resulting cellular accumulation of platinum and Tl was determined by ICP-MS. CDDP IC50 values were determined by a soluble tetrazolium/formazan assay. RESULTS: The authors were able to measure cellular CDDP and Tl accumulation precisely, and heterogeneity in the cellular accumulation of CDDP and Tl existed among the NSCLC cell lines. A significant inverse correlation was observed between CDDP IC50 values and the cellular accumulation of both CDDP and Tl. CONCLUSIONS: ICP-MS is suitable for the determination of cellular CDDP and Tl accumulation in NSCLC cell lines. Cellular Tl accumulation determined by ICP-MS may reflect CDDP cytotoxicity rather than cellular CDDP accumulation.

Predicting chemotherapeutic response to small-cell lung cancer of platinum compounds by thallium-201 single-photon emission computerized tomography.

Thallium-201 single-photon emission computerized tomography (SPECT) was used to clarify the relationship between 201Tl uptake and the response in chemotherapy to platinum compounds in 21 patients with small-cell lung cancer. 201Tl-SPECT scans were obtained twice: at 15 min (early scan) and 120 min (delayed scan) after an intravenous injection of 111 MBq (3 mCi) of thallium-201 chloride. We obtained the uptake ratio from each scan and calculated the retention index:uptake ratio = region of interest uptake/contralateral normal lung uptake; retention index = (delayed ratio - early ratio)/early ratio. After 201Tl scintigraphy, 12 patients received chemotherapy consisting of platinum compounds and nine were treated with chemoradiation. Among patients receiving only chemotherapy, the retention index correlated with the responses to chemotherapy. In an in vitro study, ouabain, an inhibitor of the Na,K-ATPase pump, reduced sensitivity to cisplatin and inhibited intracellular thallium uptake in the small-cell lung cancer cell line. These studies suggest that 201Tl-SPECT is a useful indicator of response to chemotherapy with platinum compounds in small-cell lung cancer, and that Na,K-ATPase is commonly involved in transporting both thallium and platinum compounds into cancer cells.

Bronchogenic cyst with high carbohydrate antigen 19-9 in the cyst fluid and the serum.

A mass of 8 cm in diameter was revealed in the right upper lung field of a 46-year-old female patient. The chest X-ray film taken one year previously revealed only a linear shadow in the same position, which was thought to be a vacant cyst. The levels of carbohydrate antigen (CA) 19-9 in cyst fluid and serum were elevated, at 410,000 and 130 U/ml, respectively. After surgical resection, serum CA19-9 returned to normal. Pathologically, the cyst wall was lined with bronchial epithelium with no evidence of malignancy. Immunohistochemical study revealed CA19-9 positivity in the bronchial epithelium of the cyst wall.

Quantification of glycated hemoglobin by electrospray ionization mass spectrometry.

Glycated hemoglobin, considered to be the best index for the treatment of diabetes mellitus, was measured by electrospray ionization mass spectrometry (ESI/MS) according to the method proposed by Morris et al. at the 44th ASMS Conference on Mass Spectrometry and Allied Topics, 1996. They compared the values obtained by MS and affinity chromatography. Here, the values obtained by ESI/MS were compared with those obtained by high-performance liquid chromatography and by latex agglutination immunoassay. Whole blood samples were diluted 500 fold with 0.2% formic acid-50% acetonitrile solution and 5 microliters of the diluted solution was injected with the ESI/MS system (TSQ 7000) via a sample loop. The within-run and between-run relative standard deviations of the ratio of glycated and non-glycated beta-chain were less than 5%. The correlation coefficients between ESI/MS and conventional methods were higher than 0.96. However, considerable discrepancies were observed among methods. ESI/MS will allow reproducible measurements of glycated hemoglobin and will be useful in the quality control of HbA1c measurement by other principles and also in routine clinical laboratory tests.