Human telomerase reverse transcriptase expression in Diff-Quik-stained FNA samples from thyroid nodules.

Fine-needle aspiration (FNA) is a highly sensitive method in the differential diagnosis of thyroid nodules. However, 10% of thyroid FNAs are indeterminate for cancer, and thus additional markers may be useful diagnostically. The authors have demonstrated previously that human telomerase reverse transcriptase (hTERT) gene expression is useful in the distinction of benign lesions from malignant lesions. They therefore wondered whether the detection of hTERT gene expression was feasible using archival slides. To establish an experimental system, ribonucleic acid was extracted from human anaplastic thyroid carcinoma cell line (ARO) in cytologic specimens, and reverse transcription-polymerase chain reaction (RT-PCR) for hTERT expression was performed. RT-PCR analysis for hTERT gene detection was then performed using 58 Diff-Quik-stained archival FNA samples collected retrospectively. RT-PCR for human thyroglobulin (hTg) or beta-actin gene expression served as a positive control. Successful PCR results were obtained from 48 of the 58 cases. All 10 slides in which no RT-PCR products were noted were older than 3 years. hTERT gene expression was demonstrated in FNAs from two of seven cases (29%) of hyperplastic nodule, one of one case (100%) of Hashimoto's thyroiditis, three of eight cases (38%) of follicular adenoma, three of eight cases (38%) of Hürthle cell adenoma, three of four cases (75%) of follicular carcinoma, two of two cases (100%) of Hürthle cell carcinoma, and 11 of 18 cases (61%) of papillary carcinoma. All but one of the available 33 corresponding frozen samples exhibited the same RT-PCR results. This study demonstrates that Diff-Quik-stained thyroid FNA specimens less than 3 years old can be used for the detection of hTERT gene expression by RT-PCR. This test, along with careful cytopathologic examination, may improve our ability to differentiate benign lesions from malignant lesions in indeterminate FNA samples from thyroid nodules.

Human telomerase reverse transcriptase (hTERT) gene expression in thyroid neoplasms.

Ten percent of fine-needle aspirations (FNAs) of the thyroid are deemed "indeterminate" or "suspicious" for malignancy by the cytopathologist, but most of these lesions are benign. Therefore, additional markers of malignancy may prove to be a useful adjunct. The catalytic component of telomerase, human telomerase reverse transcriptase (hTERT), has been found to be reactivated in immortalized cell lines. Reverse transcription-PCR of the hTERT gene revealed expression in 15 (79%) of 19 malignant thyroid neoplasms, including 6 of 6 follicular carcinomas and 9 of 13 papillary carcinomas. In contrast, hTERT gene expression was detected in only 5 (28%) of 18 benign thyroid nodules, including 2 of 7 follicular adenomas and 3 of 11 hyperplastic nodules. All five benign thyroids exhibiting hTERT gene expression had lymphocytic thyroiditis. No normal thyroids exhibited hTERT gene expression. Telomerase enzyme activity was examined in all 37 nodules and was found to correlate with hTERT gene expression in 35 (95%) nodules. The two cases in which telomerase activity and hTERT expression results were discrepant were in two papillary carcinomas that were telomerase activity negative and hTERT positive. Finally, we have demonstrated that hTERT gene expression can be measured in in vivo FNA samples. These results suggest that hTERT expression may be more accurate than telomerase activity in distinguishing benign from malignant and may be measured in FNA samples from suspicious thyroid lesions.

Effect of methylation on the electrophoretic mobility of chromosomal DNA in pulsed-field agarose gels.

Factors other than molecular weight are known to affect DNA electrophoretic mobility. DNA methylation has been found to affect the curvature of DNA, causing anomalous mobility in polyacrylamide gels; the effect of methylation on the mobility of large DNA molecules in agarose gels was unknown. Chromosomal DNA from Mycoplasma capricolum, a wall-less prokaryote which has a low intrinsic methylation rate, was methylated in agarose blocks by SssI methylase, a de novo methylase with a CpG recognition sequence. (A surprising finding was that SssI methylase altered the structure of InCert, but not SeaKem Gold, agarose.) Restriction enzyme analysis was used to estimate the extent of CpG methylation. DNA methylation was found to have no effect on the electrophoretic mobility of full-length chromosomal DNA (1,120 kbp) in agarose gels. Therefore, methylation is not a source of error in PFGE-based size estimation for chromosomal DNA molecules less than 1.12 Mbp in agarose gels.

Induction of avian cardiac myogenesis by anterior endoderm.

An experimental system was devised to study the mechanisms by which cells become committed to the cardiac myocyte lineage during avian development. Chick tissues from outside the fate map of the heart (in the posterior primitive streak (PPS) of a Hamburger & Hamilton stage 4 embryo) were combined with potential inducing tissues from quail embryos and cultured in vitro. Species-specific RT-PCR was employed to detect the appearance of the cardiac muscle markers chick Nkx-2.5 (cNkx-2.5), cardiac troponin C and ventricular myosin heavy chain in the chick responder tissues. Using this procedure, we found that stage 4-5 anterior lateral (AL) endoderm and anterior central (AC) mesendoderm, but not AL mesoderm or posterior lateral mesendoderm, induced cells of the PPS to differentiate as cardiac myocytes. Induction of cardiogenesis was accompanied by a marked decrease in the expression of rho-globin, implying that PPS cells were being induced by anterior endoderm to become cardiac myocytes instead of blood-forming tissue. These results suggest that anterior endoderm contains signaling molecules that can induce cardiac myocyte specification of early primitive streak cells. One of the cardiac muscle markers induced by anterior endoderm, cNkx-2.5, is here described for the first time. cNkx-2.5 is a chick homeobox-containing gene that shares extensive sequence similarity with the Drosophila gene tinman, which is required for Drosophila heart formation. The mesodermal component of cNkx-2.5 expression from stage 5 onward, as determined by in situ hybridization, is strikingly in accord with the fate map of the avian heart. By the time the myocardium and endocardium form distinct layers, cNkx-2.5 is found only in the myocardium. cNkx-2.5 thus appears to be the earliest described marker of avian mesoderm fated to give rise to cardiac muscle.