Enrichment of epithelial cells for molecular studies.
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Cell Separation↗
Biomedical subjects
S Milchgrub
Publications and source records attributed to S Milchgrub.
Comparison of features of human lung cancer cell lines and their corresponding tumors.
Although human lung tumor-derived cell lines play an important role in the investigation of lung cancer biology and genetics, there is no comprehensive study comparing the genotypic and phenotypic properties of lung cancer cell lines with those of the individual tumors from which they were derived. We compared a variety of properties of 12 human non-small cell lung carcinoma (NSCLC) cell lines (cultured for a median period of 39 months; range, 12-69) and their corresponding archival tumor tissues. There was, in general, an excellent concordance between the lung tumor cell lines and their corresponding tumor tissues for morphology (100%), the presence of aneuploidy (100%), immunohistochemical expression of HER2/neu (100%) and p53 proteins (100%), loss of heterozygosity at 13 chromosomal regions analyzed (97%) using 37 microsatellite markers, microsatellite alterations (MAs, 75%), TP53 (67%), and K-ras (100%) gene mutations. In addition, there was 100% concordance for the parental allele lost in all 115 comparisons of allelic losses. Some discrepancies were found; more aneuploid subpopulations of cells were detected in the cell lines as well as higher incidences of TP53 mutations (4 of 10 mutations not found in the tumors) and microsatellite alterations (two cell lines with MAs not detected in the tumors). Similar loss of heterozygosity frequencies by chromosomal regions and mean fractional allelic loss index were detected between successfully cultured and 40 uncultured lung tumors (0.45 and 0.49, respectively), indicating that both groups were similar. Our findings indicate that the NSCLC cell lines in the large majority of instances retain the properties of their parental tumors for lengthy culture periods. NSCLC cell lines appear very representative of the lung cancer tumor from which they were derived and thus provide suitable model systems for biomedical studies of this important neoplasm.
Adult↗
Deletions of chromosome 4 at multiple sites are frequent in malignant mesothelioma and small cell lung carcinoma.
Recent allelotyping studies suggest that allelic losses at one or both arms of chromosome 4 are frequent in several tumor types. Cytogenetic studies of malignant mesothelioma (MM) and comparative genomic hybridization analyses of small cell lung carcinoma (SCLC) suggest that chromosome 4 deletions may also play a role in these tumor types, although these results have not been confirmed by allelotyping. In an effort to more precisely identify and map the locations of putative tumor suppressor gene(s) on chromosome 4 involved in the pathogenesis of these tumors, we performed loss of heterozygosity studies using 16 polymorphic microsatellite markers. After precise microdissection of archival surgical cases, we studied DNA obtained from 20 MMs, 21 SCLCs, and 20 non-SCLCs (NSCLCs). In addition, DNA from 14 SCLC and 17 NSCLC cell lines and corresponding B lymphoblastoid lines were studied. In MM and SCLC, we observed frequent losses at three nonoverlapping regions: (a) 4q33-34 (region R1; >80%); (b) 4q25-26 (region R2; >60%); and (c) 4p15.1-15.3 (region R3; >50%). Losses at these sites occurred at lower frequencies in NSCLC (>20-30%). Data from tumors and cell lines were similar. In MM and SCLC, the most frequently observed pattern was loss at all three regions. However, in NSCLC, the most frequent pattern was loss at R3 alone. Our study has delineated three nonoverlapping regions of frequent deletions on chromosome 4 in MM and SCLC, suggesting that there may be three putative suppressor genes on chromosome 4, the inactivation of which may be important in the pathogenesis of these tumor types.
Carcinoma, Non-Small-Cell Lung↗
Mutation analysis of the PTEN/MMAC1 gene in lung cancer.
We studied PTEN/MMAC1, a newly discovered candidate tumor suppressor gene at 10q23.3, for mutations in lung cancer. One hundred and thirty-six lung cancer cell line DNAs (66 small cell lung cancers, SCLC, 61 non-small cell lung cancers, NSCLC, four mesotheliomas, five extrapulmonary small cell cancers) were analysed for PTEN/MMAC1 homozygous deletions and five (8%) SCLC lines showed homozygous deletions interrupting the PTEN/MMAC1 gene. Using single stranded conformation polymorphism (SSCP) analysis, we screened the PTEN/MMAC1 open reading frame of 53 lung cancer cell line cDNAs for point mutations and found that 3/35 SCLCs and 3/18 NSCLCs contained homozygous amino acid sequence altering mutations. Northern blot analysis revealed that expression of the PTEN/MMAC1 gene was considerably lower in all the tumor cell lines with point mutations while no expression was detected for cell lines with PTEN/MMAC1 homozygous deletions. Mutation analysis of 22 uncultured, microdissected, primary SCLC tumors and metastases showed two silent mutations, and two apparent homozygous deletions. We also discovered a processed pseudogene (PTEN2) which has 98.5% nt identity to PTEN/MMAC1, that needs to be accounted for in cDNA mutation analysis. Our findings suggest that genetic abnormalities of the PTEN/MMAC1 gene are only involved in a relatively small subset of lung cancers.
Carcinoma, Small Cell↗
Comparison of molecular changes in lung cancers in HIV-positive and HIV-indeterminate subjects.
CONTEXT: Human immunodeficiency virus (HIV) infection has been associated with an increasing incidence of malignancy, and HIV-infected persons have an increased incidence of primary lung carcinoma compared with the general population. OBJECTIVE: To investigate the molecular changes present in HIV-associated lung tumors and compare them with those present in lung carcinomas arising in HIV-indeterminate subjects ("sporadic tumors"). DESIGN: Convenience sample. SUBJECTS: Archival tissues from 11 HIV-positive persons and from 35 persons of indeterminate HIV status. SETTING: University-based medical centers and affiliated hospitals. MAIN OUTCOME MEASURES: Analysis of frequency of loss of heterozygosity (LOH) and microsatellite alteration (MA) using polymerase chain reaction and 16 polymorphic microsatellite markers at 8 chromosomal regions frequently deleted in lung cancer. Presence of HIV and human papillomavirus (HPV) sequences. RESULTS: The overall frequency of LOH at all chromosomal regions tested and the frequencies at most of the individual regions were similar in the 2 groups. Frequency of MA present in the HIV-associated tumors (0.18) was 6-fold higher than in sporadic tumors (0.03) (P<.001). At least 1 MA was present in 10 (91%) of 11 HIV-associated tumors vs 17 (48%) of 35 sporadic tumors (P=.02). Molecular changes were independent of tumor stage and gender. HIV and HPV sequences were not detected in the HIV-associated lung carcinomas. CONCLUSIONS: Microsatellite alterations, which reflect widespread genomic instability, occur at greatly increased frequency in HIV-associated lung carcinomas. Although the mechanism underlying the development of increased MAs is unknown, it may play a crucial role in the development of many HIV-associated tumors.
Adult↗
Telomerase activity and expression of its RNA component in cervical lesions.
BACKGROUND: The authors investigated telomerase enzyme activity and expression of its RNA component (hTR) during the multistage pathogenesis of cervical carcinomas, and correlated activation with histopathologic findings and human papillomavirus (HPV) infection. METHODS: The authors analyzed 180 cervical specimens for enzyme activity, and analyzed hTR expression in an additional 55 samples from archival carcinoma cases. Polymerase chain reaction-based assays were used to determine telomerase enzyme activity and HPV infection, whereas a radioactive in situ assay was used for hTR expression. RESULTS: Telomerase enzyme activity was present in some samples of histologically normal epithelium (18 of 138; 13%) and low grade squamous intraepithelial lesions (LSIL) (7 of 21; 33%), and in most high grade squamous intraepithelial lesions (HSIL) (13 of 21; 62%). The relative levels of telomerase activity were low in all preinvasive specimens except for three samples of HSIL with high activity. Although 21% of the brush samples had evidence of HPV infection, there was no obvious correlation between telomerase activity and HPV status. hTR expression was low in normal squamous/glandular epithelium and LSIL lesions, in which it was limited to the basal cells. In squamous and glandular in situ and invasive carcinomas, increased and dysregulated hTR expression was observed, although heterogeneity was noted. Intense focal up-regulation of hTR expression occurred in a subset of in situ lesions. CONCLUSIONS: Increased frequency and dysregulation of telomerase activation is correlated with increasing severity of histopathologic changes, but not with HPV infection. Whether dysregulated activity is a prognostic marker for development of invasive carcinoma remains to be determined.
Adenocarcinoma↗
Telomerase enzyme activity and RNA expression during the multistage pathogenesis of breast carcinoma.
Telomerase, an RNA-containing enzyme, is associated with cellular immortality and malignancy. We investigated the role of telomerase during the multistage pathogenesis of breast cancer. We used the semiquantitative, PCR-based telomeric repeat amplification protocol assay for enzyme activity (42 specimens from 42 patients) and a radioactive in situ assay for expression of its RNA component (human telomerase RNA; hTR) for the identification of telomerase-positive cells in archival resection samples (n = 67 from 39 patients). Low telomerase activity was detected in 1 (14%) of 7 samples of benign breast disease, in 4 (67%) of 6 fibroadenomas, in 11 (92%) of 12 carcinoma in situ (CIS) lesions, and in 16 (94%) of 17 invasive breast cancers. There was a progressive increase in the mean telomerase levels with progressive increase in severity of histopathological change (P < 0.05). Almost all of 67 resection samples expressed hTR, irrespective of histology. Expression was low to moderate in some samples of normal epithelium and nonproliferative fibrocystic changes. hTR expression was limited to epithelial cells; expression in stromal cells, including those in fibroadenomas, was negative. Increased hTR expression was observed in some foci of apocrine metaplasia and atypical hyperplasia. Increased hTR expression was also observed in all CIS and invasive lesions, although considerable heterogeneity was noted. Focal up-regulation was frequently noted in CIS lesions in the vicinity of invasive tumors. Thus, up-regulation of hTR may be a predictive marker for invasive tumor development.
Breast↗
Comparison of features of human breast cancer cell lines and their corresponding tumors.
Although human tumor-derived cell lines play an important role in the investigation of cancer biology and genetics, there is no comprehensive study comparing tumor cell line properties with those of the individual tumors from which they were derived. We compared the properties of a series of 18 human breast cancer cell lines that were cultured for a median period of 25 months (range, 9-60 months) and their corresponding archival tumor tissues. We compared morphological characteristics, ploidy, and immunohistochemical expression of estrogen receptors, progesterone receptors, and HER2/neu and p53 proteins. For 17 of these cases, we also tested for allelic losses at 18 chromosomal regions frequently deleted in breast tumors using 51 polymorphic microsatellite markers, and we determined the TP53 gene mutation status in exons 5 to 10. There was an excellent correlation between the breast tumor cell lines and their corresponding tumor tissues for morphological features (100%); presence of aneuploidy (87%); immunohistochemical expression of estrogen receptors (87%), progesterone receptors (73%), and HER2/neu (93%) and p53 proteins (100%); allelic loss at all of the chromosomal regions analyzed (82-100% concordance); and TP53 gene mutations (75%). The same parental allele was lost in 279 (99%) of 281 of the comparisons of allelic losses. The fractional allelic loss indices (a reflection of the total allelic loss) of the cell lines and their corresponding tumor tissues were identical or similar in 15 (88%) of 17 paired comparisons. Although our previous studies (A. Gazdar et al., Int. J. Cancer, in press) indicated that only a subset of primary breast carcinomas that have several features indicative of advanced tumors with poor prognosis can be successfully cultured, the cell lines retain the properties of their parental tumors for lengthy culture periods and, thus, provide suitable model systems for biomedical studies.
Adult↗
Deletions of chromosome 3p are frequent and early events in the pathogenesis of uterine cervical carcinoma.
To study the molecular abnormalities involved in the multistage development of cervical carcinoma (CC), we investigated the presence of oncogenic human papillomavirus (HPV) sequences, loss of heterozygosity (LOH), and microsatellite alterations at several genes/loci at 3p (3p14.2 at the FHIT gene, 3p14.3-21.1, 3p21, and 3p22-24.2), 9p21, RB and P53, and P53 gene point mutations in precisely microdissected archival tissues from 20 CCs and their accompanying precursor lesions (cervical intraepithelial neoplasia, CIN; n = 40) and normal epithelia (n = 20). In all HPV-positive cases (90% of CCs), HPV sequences were detected as the earliest appearing molecular change or simultaneously with other changes. LOH at any 3p region was found in 70% of CCs, and 3p14.2 (FHIT gene/FRA3B fragile site) (56%) and 3p21 (57%) were the most frequent 3p sites of loss. LOH at some 3p region was in the CIN I stage, and the 3p deletions in precursor CIN lesions were smaller than the 3p losses found in the associated invasive CC. LOH at the other regions studied and P53 gene mutations were less frequent and later events. Microsatellite alterations were detected in 35% of CCs, and identical abnormalities were detected in the associated precursor lesions. Although infection with oncogenic HPV strains is the earliest and most frequent molecular event, progressive deletions at one or more 3p regions (particularly at 3p14.2, and 3p21) are also frequent events occurring early in the pathogenesis of CC.
Acid Anhydride Hydrolases↗
Telomerase expression in respiratory epithelium during the multistage pathogenesis of lung carcinomas.
To investigate the role of telomerase in the multistage pathogenesis of lung cancer, we examined 205 fresh and archival tissue samples obtained from 40 patients, 34 of whom had invasive lung carcinoma, 5 with carcinoma in situ (CIS) without invasion, and 1 without lung carcinoma. We analyzed samples for telomerase enzyme activity using the semiquantitative PCR-based telomeric repeat amplification protocol assay (131 samples) or by a radioactive in situ hybridization method for expression of the RNA component of human telomerase (hTR; 74 samples). A subset of samples was assayed by both methods, and the correlation was excellent (30 of 36; 83%). With the exception of a carcinoid tumor and a necrotic squamous cell carcinoma, all tumor cells were moderate to strongly positive for both hTR and telomerase activity, except for foci of keratinization in squamous cell carcinomas. Telomerase positivity, with weak enzyme activity and/or low hTR expression, was present in basal epithelial cells of large bronchi, both histologically normal (26%) and hyperplastic (71%), and in 23% of peripheral lung samples (in epithelium of small bronchi and bronchioles or lymphoid aggregates). More advanced epithelial changes (metaplasia, dysplasia, and CIS) were associated with telomerase dysregulation. Dysregulation in preneoplasia was manifested in three ways: almost all such lesions expressed hTR, although enzyme activity levels were several-fold lower than in the corresponding invasive tumors; cells throughout these multilayered processes expressed hTR; and intense, focal up-regulation of hTR occurred in CIS foci in the vicinity of invasive cancers. Alveolar cells and areas of atypical adenomatous hyperplasia (possible precursor lesions for peripheral adenocarcinomas) were negative. Our studies demonstrate that dysregulation of telomerase occurs early in the multistage pathogenesis of bronchogenic lung carcinomas and that intense focal localized hTR expression in CIS may indicate imminent invasion.
Carcinoma↗
Mucinous adenocarcinoma in defunctionalized bladders.
Primary adenocarcinoma of the bladder accounts for less than 2% of all bladder cancers. There is no report of such a case in a defunctionalized bladder. All reported cases of carcinoma in defunctionalized bladders were either squamous cell, signet ring cell, or transitional cell carcinoma, detected within an average of 5 years after urinary diversion, and all have been associated with chronic inflammation of the bladder. We report on 2 cases of adenocarcinoma that developed in defunctionalized bladders 30 and 8 years after ileal loop diversion for prune belly syndrome and neurogenic bladder, respectively.
Adenocarcinoma, Mucinous↗
The tubular variant of adenoid cystic carcinoma of the Bartholin's gland.
The authors report two examples of the tubular variant of adenoid cystic carcinoma of the Bartholin's gland. Each occurred in young women (both aged 34 years) and presented with a painful indurated mass, clinically thought to be of inflammatory nature. On microscopic examination, the most distinctive feature was the haphazard proliferation of bland-appearing tubular structures often lined by two cell layers. A thin, periodic acid-Schiff-positive basement membrane immunoreactive for collagen IV surrounded the tubules, but hyaline globules were virtually absent. The inner cell layer was strongly and diffusely cytokeratin positive, whereas epithelial membrane antigen reactivity was seen only along the luminal borders. Focal positivity for S100 protein identified the peripheral myoepithelial cells, which were confirmed by electron microscopy. Focal perineural invasion was seen. The histologic, ultrastructural, and immunohistochemical features of these tumors are similar to those of the tubular variant of adenoid cystic carcinoma arising in major and minor salivary glands. In one of the cases, which was followed for 6 years, the tumor metastasized, despite the low histologic grade, and the patient died. Similarities and differences between the tubular, cribriform, and solid variants and other vulvar tumors are discussed.
Adult↗
Hyalinizing clear cell carcinoma of salivary gland.
We describe 11 patients with a distinctive salivary gland neoplasm. Most of the patients were adult women who presented with a painless mass. Nine tumors arose in minor salivary glands of the oral cavity (82%). Microscopically, they were characterized by the formation of trabeculae, cords, islands, and/or nests of monomorphic clear cells that were glycogen rich and mucin negative and were surrounded by hyalinized bands with foci of myxohyaline stroma. Cells with eosinophilic and granular cytoplasm were also noted. Both cell types showed minimal nuclear pleomorphism and a very low mitotic index. The neoplasms all had infiltrative borders. Immunohistochemically, the tumor cells expressed cytokeratins and epithelial membrane antigen, but not S-100 protein and smooth muscle actin. Ultrastructurally, the tumor cells contained abundant glycogen, desmosomes, peripheral tonofilaments, and prominent interdigitating microvilli without actin myofilaments or dense bodies. These immunohistochemical and ultrastructural findings provide evidence of epithelial differentiation without myoepithelial differentiation. For these tumors, we propose the name, hyalinizing clear cell carcinoma (HCCC). These are low-grade malignant neoplasms. Two patients had ipsilateral cervical lymph node metastases at presentation, but with surgical excision and either preoperative or post-operative radiotherapy in three cases, eight of 10 patients with clinical follow-up are alive and well without evidence of recurrence. The mean clinical follow-up is 3.6 years, with a range of 6 months to 11 years. One patient died as a result of surgery, another died of unrelated causes, and one patient was lost to follow-up.
Adenocarcinoma, Clear Cell↗
The characteristic features of chondrolipoangioma on magnetic resonance imaging. A report of three cases.
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Adolescent↗
Transitional cell carcinoma of the ureteral stump 23 years after radical nephrectomy for adenocarcinoma.
We report a case of simultaneous invasive transitional cell carcinoma in a ureteral stump and superficial bladder tumor occurring 23 years after ipsilateral radical nephrectomy for adenocarcinoma of the kidney. We review the literature on similar cases and discuss potential etiologies of tumor formation in the ureteral stump.
Adenocarcinoma↗
Synovial sarcoma with extensive osteoid and bone formation.
Four cases of synovial sarcoma with extensive calcification and osteoid and bone formation are reported. Ages ranged from 21 to 38 years. Two tumors were located in the foot and two in the thigh. Because of a well-circumscribed, densely calcified soft tissue mass, radiologically three patients were thought to have a benign lesion. The fourth patient was thought to have a paraosteal osteosarcoma because of an accompanying bone defect. Tumor size varied from 4.0 to 9.0 cm. Histologically, three tumors were biphasic and one predominantly monophasic. All showed amorphous calcifications with extensive ossification sometimes in a ribbon-like pattern of osteoid, simulating osteosarcoma. The extensive bone formation with abundant osteoid deposition may lead to a misdiagnosis of osteosarcoma. It is important to recognize this variant of synovial sarcoma with ossification and bone formation and distinguish it from extraskeletal osteosarcoma because of the difference in clinical behavior and course. Although the most important point in the recognition of this variant of synovial sarcoma is its biphasic pattern, this may not be apparent in a small tissue sample. Points that aid in the diagnosis include the uniform nuclear appearance of both the epithelial and the spindle cells versus the pleomorphism of osteosarcoma and in some cases the presence of amorphous concretions in sheets and small calcospherites within spaces surrounded by flat or conspicuous epithelial cells. These cells are immunoreactive for cytokeratin and epithelial membrane antigen.
Adult↗
Sweat gland adenomas: immunohistochemical study with emphasis on myoepithelial differentiation.
Thirty-one dermal appendage tumors of sweat gland differentiation including 7 spiradenomas (SPA), 8 cylindromas (CYL), 8 acrospiromas (ACS), and 8 chondroid syringomas (CS) were analyzed using antibodies to epithelial membrane antigen (EMA), cytokeratin (AE1, AE3, CAM 5.2, 34BE12), S-100 protein, actin (ACT), and desmin (DES) to characterize the immunocytochemical profile of benign sweat gland tumors. Cytokeratin expression was variable; AE1, 34BE12, AE3, and CAM 5.2 were present in 31, 24, 23, and 22 tumors respectively; 29 tumors contained EMA. Seventeen tumors, (6 SPA, 8 CYL, 2 ACS, 1 CS) stained with antibody to alpha smooth muscle actin, and 26 (7 SPA, 7 CYL, 4 ACS, 8 CS) expressed S-100 protein. Although some prior studies had reported actin filaments on electron microscopy in both spiradenoma and cylindroma, these tumors have previously been considered to be negative for myoepithelial differentiation. All spiradenomas and cylindromas we studied demonstrated actin and/or S-100 protein positivity in basal epithelial cells, consistent with myoepithelial differentiation. The organization of actin and S-100 protein positivity displayed by the spiradenomas and cylindromas we studied suggests that the tumors are differentiated towards the secretory portion of the eccrine sweat gland.
Acrospiroma↗
Intraductal carcinoma of the pancreas.
Four multicentric intraductal papillary carcinomas arising in the main pancreatic duct are presented. Three of the neoplasms showed stromal invasion and metastasized to regional lymph nodes. Three patients had a long history of epigastric pain, confirming the progressive slow growth and less aggressive nature of this clinicopathologic entity. Histologically, all tumors were papillary, and three also showed a pseudocribriform pattern. Individual cells exhibited a range of atypia from mild to overt malignant change. Focal intestinal differentiation was recognized in two tumors. Despite the well-differentiated appearance of these tumors, two patients died within 1 year of surgery. One patient with an entirely intraductal carcinoma is alive and well 3 years after surgical treatment. The fourth patient who had lymph node metastasis is alive 6 months after a Whipple's procedure.
Adult↗