Year: 1999
Type: article
Abstract: We agree with Drs. Kitamoto and Ide that the conventional telomeric repeat amplification protocol (TRAP) assay has two major limitations: 1) it is not quantitative, and 2) additional controls are needed for verification of the specificity and sensitivity of the results. To rule out false-negative results, we added to our samples (as a control for the polymerase chain reaction [PCR]) a diluted sample of IMR-32, a telomerase activity positive neuroblastoma cell line.1 In our opinion, this procedure, which has also been used by others,2 is an acceptable alternative to using internal telomerase assay standards (ITAS). To rule out false-positive results, we used RNase A, as generally accepted; further, to ensure that the PCR reaction would start at the same point in time in all samples, we performed a variant of ≪hot start≫ PCR by adding Taq polymerase and the CX primer after heating the samples to stop telomerase activity. These modifications of the conventional TRAP assay are described and discussed in sufficient detail in our article. However, an extensive discussion of TRAP assay methodologies was beyond the scope of our article. It is quite probable that, because of its sensitivity, the TRAP assay would be useful in clinical diagnostics, if adequately modified. In this sense, the method proposed by Hirose et al.3 is indeed very promising, because it avoids the use of radioactive compounds and electrophoresis and allows a relative quantitation of telomerase activity. The results of this quantitation3, 4 are in agreement with those of previous studies, in which the semiquantitative TRAP assay was used and showed higher telomerase activity in hepatocellular carcinomas (HCCs) than in noncancerous livers.5-7 However, according to every cutoff level used in Hirose et al.3 and Nakashio et al.4 (10 cell equivalent of the MKN-1 cell line, or 0.42–1 U representing an equivalent of K562 cell activity), there was still some overlap between the relative telomerase activity values in chronic liver disease and well- or moderately differentiated HCCs. Drs. Kitamoto and Ide state that the relative telomerase activity level in the precancerous nodules was much lower than that in the well-differentiated HCCs they studied. They also make reference to already published results of the same research group,4 mentioning a very high mean value (87 cell equivalent) for the well-differentiated HCCs. However, this value does not appear in the articles cited and, to our understanding, cannot be concluded from the presentation of the respective results. For example, in Figure 2 of Nakashio et al.4 the relative telomerase activity value was below a 65 MKN-1 cell equivalent in all but one of the well-differentiated HCCs, whereas in Figure 7B of Hirose et al.3 it was around or below 1 U (10 K562 cell equivalent) in several well- and even moderately differentiated HCCs. Most importantly, and in contrast to what is found in the sample groups of noncancerous livers, the range of the relative telomerase activity levels (corresponding either to MKN-1 or K562 cell equivalents) in the well- and moderately differentiated HCC groups is very broad.3, 4 This means that the standard deviations in the HCC groups, especially in the group of the well-differentiated tumors, will also be very broad, not allowing mean values to be used to describe telomerase activity data. Moreover, exactly because of the wide range of telomerase activity levels observed in well-differentiated HCCs, which extend into those of noncancerous chronic liver disease, overlap of levels between dysplastic nodules and well-differentiated HCCs is justified in the lower area of the scale. Thus, we do not think that there is any major discrepancy between our findings and those of Drs. Kitamoto and Ide, even though identifying the same value in all 3 positive precancerous nodules (21 cell equivalent for each of them) is quite surprising. In terms of the histologic material utilized in our study, all noncancerous nodules were detected on careful macroscopic examination of livers removed at transplantation. Therefore, these lesions may be thought of as incidental findings, although we cannot rule out the possibility that at least some of them would be detectable by radiologic means. A representative area of each nodule was snap-frozen for molecular studies, and the remaining tissue was submitted for histologic examination.1 It should be emphasized that, despite difficulties, histologic examination currently represents the gold standard for diagnosis and classification of nodular hepatic lesions.8 Examination of fine-needle aspirates, even if combined with radiologic and serologic information, as suggested by Drs. Kitamoto and Ide, should not be regarded as a substitute for histologic examination at this time. It should also be emphasized that a significant number of large regenerative and precancerous nodules should be evaluated by the method of Hirose et al.3 before any conclusions regarding its practical utility can be reached. Once the telomerase activity range in the various types of nodules is established, evaluation of such activity in fine-needle aspirates may become a useful auxiliary diagnostic method. In conclusion, there is no doubt that the rapidly developing methodologies of telomerase activity determination have significant potential for diagnostic applications. Nevertheless, until the usefulness of these procedures is validated in large clinical studies, enthusiasm should be moderated. Vassiliki Kotoula M.D.* , Prodromos Hytiroglou M.D.* , Swan N. Thung M.D. , Maria Tsokos M.D. , M. Isabel Fiel M.D. , Constantine S. Papadimitriou M.D.*, * Department of Pathology, Aristotle University Medical School, Thessaloniki, Greece, Laboratory of Pathology, Section for Pediatric Pathology and Electron, Microscopy, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, Lillian and Henry M. Stratton–Hans Popper, Department of Pathology, Mount Sinai School of Medicine, New York, New York
Source: Cancer
Language: English
Subfield: Physiology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W4244847533