Jin Jen

Molecular characteristics of non-small cell lung cancer

We used hierarchical clustering to examine gene expression profiles generated by serial analysis of gene expression (SAGE) in a total of nine normal lung epithelial cells and non-small cell lung cancers. Separation of normal and tumor, as well as histopathological subtypes, was evident by using the 3,921 most abundant transcript tags. This distinction remained when only 115 highly differentially expressed tags were used. Furthermore, these 115 transcript tags clustered into groups suggestive of the unique biological and pathological features of the different tissues examined. Adenocarcinomas were characterized by high-level expression of small airway-associated or immunologically related proteins, whereas squamous cell carcinomas overexpressed genes involved in cellular detoxification or antioxidation. The messages of two p53-regulated genes, p21WAF1/CIP1and 14-3-3σ, were consistently underexpressed in the adenocarcinomas, suggesting that the p53 pathway itself might be compromised in this cancer type. Gene expression patterns observed by SAGE were consistent with results obtained by quantitative real-time PCR or cDNA array analyses by using a total of 43 lung tumor and normal samples. Thus, although derived from only a few tissue libraries, gene expression profiles obtained by using SAGE most likely represent an unbiased yet distinctive molecular signature for the most common forms of human lung cancer.

PGP9.5 As a Candidate Tumor Marker for Non-Small-Cell Lung Cancer

PGP9.5 is a neurospecific peptide that functions to remove ubiquitin from ubiquitinated proteins and prevents them from targeted degradation by proteasomes. Using the serial analysis of gene expression method (SAGE), we observed that the PGP9.5 transcript was highly expressed in primary lung cancers and lung cancer cell lines but was not detectable in the normal lung. Here we examined the expression of PGP9.5 protein in normal lung epithelium, lung tumor cell lines, and 98 resected primary non-small-cell lung carcinomas (NSCLCs). We found PGP9.5 reactivity in normal lung in a pattern compatible with K-cells of the diffuse neuroendocrine system. However, the PGP9.5 was present in both small-cell lung cancer (SCLC) and NSCLC cell lines (22/24) independent of neuronal differentiation. In primary NSCLCs, 54% (53/98) of the cases had positive PGP9.5 staining, and the expression of protein was strongly associated with pathological stage of the cancer. It was present in 44% (29/66) of stage I NSCLCs and in 75% (24/32) of stage II and IIIA NSCLCs (p = 0.0032). These results suggest that the increased expression of PGP9.5 is specifically associated with lung cancer development and may serve as a potential marker for the detection of lung cancer.

Genomic imbalances in human lung adenocarcinomas and squamous cell carcinomas

Comparative genomic hybridization analysis was performed on 67 non‐small‐cell lung cancers (NSCLCs), including 32 squamous cell carcinomas (SCCs) and 35 adenocarcinomas (ACs), to identify differences in the patterns of genomic imbalance between these two histologic subtypes. Among the entire tumor set, the chromosome arms most often overrepresented were 1q, 3q, 5p, and 8q, each detected in 50–55% of cases. The most frequently underrepresented arms were 9q, 3p, 8p, and 17p. The number of imbalances was similar in SCCs and ACs (median number/case: 12 and 11, respectively). Moreover, many imbalances, such as gains of 1q, 5p, and 8q, occurred at a high frequency in both histologic subgroups. Several statistically significant differences, however, were found. The most prominent difference was gain of 3q24‐qter, seen in 81% of SCCs compared with 31% of ACs (P < 0.0001), with amplification at 3q25‐26 being detected in eight of 32 (25%) SCCs but in only two of 35 (6%) ACs. Gain of 20p13 and loss of 4q also were seen at a significantly higher rate in SCCs than in ACs, whereas overrepresentation of 6p was more common in ACs. Gains of 7q and 8q each were associated with higher‐stage tumors and either positive nodal involvement or higher tumor grade. These data suggest that genes located in several chromosomal regions, particularly 3q25‐26, may be associated with phenotypic properties that differentiate lung SCCs from ACs. Furthermore, certain imbalances, prominent among them gains of 7q and 8q, may be indicative of tumor aggressiveness in NSCLCs.

Somatic mutations of the PTEN tumor suppressor gene in sporadic follicular thyroid tumors

The PTEN (MMAC1/TEP1) tumor suppressor gene was recently isolated and mapped to human chromosome band 10q23. Homozygous deletions and mutations of PTEN were observed in cell lines and sporadic cancers of the breast, kidney, and central nervous system. Germline mutations in PTEN were recently found in Cowden disease, an autosomal dominant inherited syndrome, previously mapped to chromosome bands 10q22–23. This disease is associated with a wide variety of malignancies and hamartomas of ectodermal, mesodermal, and endodermal origin. The most common neoplasms in Cowden disease patients arise in the breast, skin, and thyroid (follicular subtype). To determine the involvement of PTEN in sporadic follicular thyroid tumors, we first analyzed sporadic follicular adenomas and carcinomas for deletions of the PTEN gene. Loss of heterozygosity was found in 7/26 (27%) follicular carcinomas and 2/27 (7%) follicular adenomas, one of which was a small hemizygous deletion (∼3 cm). Sequence analysis of the entire PTEN coding region revealed two mutations in carcinomas with 10q loss. Our findings suggest that the PTEN tumor suppressor gene is occasionally inactivated in sporadic follicular thyroid tumors. Genes Chromosomes Cancer 23:239–243, 1998.

Microsatellite instability at AAAG repeat sequences in respiratory tract cancers

We surveyed the occurrence of novel alleles at microsatellite sequences in non‐small cell lung cancers (NSCLC) using 61 tetranucleotide repeat markers. The presence of at least one new allele, consistent with microsatellite instability (MSI), was observed in 26 of 61 (43%) markers involving 30 of 47 (64%) NSCLC. Twelve of the 26 markers detected new alleles in 2 or more tumors and 11 of these 12 markers contained an AAAG repeat sequence. Using this panel of 12 markers, MSI was detected in 24 of 47 (51%) NSCLC and 10 of 18 (56%) head and neck cancers but was only observed in 8 of 38 (21%) bladder cancers and 3 of 25 (12%) kidney cancers. Our results suggested that about 50% of respiratory tract cancers exhibited microsatellite instability predominantly at AAAG sequences. This distinct type of instability was termed EMAST for elevated microsatellite alterations at selected tetranucleotide repeats. The identification of markers with EMAST should have potential application for the molecular detection of respiratory tract cancers.

Loss of chromosome arms 3p and 9p and inactivation of P16INK4a in normal epithelium of patients with primary lung cancer

The accumulation of genetic alterations in the respiratory epithelium may give rise to cancer and often is accompanied by a series of histologic alterations over a period of several years. Recent studies have identified some molecular alterations in histologically normal‐appearing epithelium among patients with lung cancer. To extend these observations, we investigated clonal genetic alterations by using fluorescence in situ hybridization (FISH) analysis and immunohistochemistry in 69 biopsy samples of histologically normal‐appearing bronchial epithelium from 22 patients with or without lung cancer. Thirty‐seven biopsy specimens from 13 patients were examined for loss of 3p14, and 48 biopsy specimens from 18 patients were examined for loss at 9p21 by FISH. P16INK4a expression was analyzed in 54 biopsy samples from 19 patients. In at least one biopsy specimen from five of the13 patients with primary lung cancer, FISH or immunohistochemistry detected loss of the 3p14 or 9p21 region. In contrast, no alterations were detected for the same regions in the nine patients without primary lung cancer. Our results support the concept that the normal epithelial surface of large bronchi of patients with lung cancer has molecular changes suggestive of the outgrowth of numerous clonal foci.

Molecular detection of cervical intraepithelial neoplasia and cervical carcinoma by microsatellite analysis of Papanicolaou smears

Carcinoma of the uterine cervix is one of the most common malignancies worldwide, yet it is clearly preventable by population screening. The Papanicolaou (Pap) smear has proved to be the most successful test for the detection of precancerous lesions and is largely responsible for the reduction of cervical cancer mortality and morbidity rates. However, the Pap smear is not perfect; false‐negative results of various rates are reported. To improve the diagnostic efficacy of cervical cytology, we performed microsatellite analysis on paired Pap smear samples from cervical lesions. Nine microsatellite markers were chosen from chromosomal regions commonly displaying loss of heterozygostity (LOH) in cervical cancer and those displaying microsatellite instability (MI) in other squamous cell cancer. Microsatellite alterations were detected in 16/21 (76%) Pap smear DNA samples including 11 of 13 (85%) smears from invasive squamous cell carcinomas (SCCs) and 5 of 8 (63%) from squamous intraepithelial lesions (SILs). Microsatellite alterations detected in the Pap smear DNA were identical to those identified in seven paired primary tumors available for analysis. Moreover, this molecular approach detected genetic alterations in two cases apparently negative by cytologic examination. None (0/25) of the control patients displayed microsatellite alterations in paired Pap smears. Microsatellite analysis of cervical cytologic samples may provide a complementary method to analyze suspicious but not diagnostic cytologic samples further.