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Original Studies |
Dipartimento di Patologia Umana (M.T., D.V., D.B.), Policlinico Universitario, 98125 Messina, Italy; Anatomia Patologica (F.F.), Università degli Studi di Catania, 95124 Catania, Italy; Molecular Research Center, Inc. (K.M.), Cincinnati, Ohio 45212; Cattedra di Endocrinologia (F.T., S.B.), Policlinico Universitario, 98125 Messina, Italy
Address all correspondence and requests for reprints to: Maria Trovato, Dipartimento di Patologia Umana, Padiglione D, Policlinico Universitario, Gazzi - 98125 Messina, Italy.
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Abstract |
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We screened 6 normal thyroids, 10 colloid nodules, 10 follicular hyperplasias, 10 oncocytic adenomas, 10 follicular adenomas (FA), 10 FTC, 6 ATC, 12 PTC using two microsatellite markers for HGF, and two for c-met. LOH for all 4 markers was found in 100% of FTC, 100% of ATC, and (for only 1 or 2 markers) in 10–29% of FA.
This is the first demonstration of an LOH that separates both FTC and ATC from PTC, in the best possible manner: 100% vs. 0%. Clearly, each of the two segments we have probed contains at least one tumor suppressor gene, whose inactivation is crucial for the establishment of the FTC (and ATC) phenotype. This loss of genetic material explains why FTC and ATC, but not PTC, fail to express both HGF and c-met. Our findings may also have immediate diagnostic application, in the context of assisting pathologists in the often difficult task of distinguishing FA from FTC.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In brief, this cell may enter three
neoplastic pathways that lead to the hyperfunctioning adenoma or the
papillary thyroid cancer (PTC) or the follicular adenoma (FA) through
follicular carcinoma [follicular thyroid cancer (FTC)] histotypes.
The distinct histology and clinical behavior substantiate that PTC and
FTC belong to different molecular pathways. Indeed, we now know that
peculiar to PTC is the activation of tyrosine-kinase signals,
such as the protooncogenes RET, TRK, and variants thereof (4, 5, 6), and
HGF/HGF-R (hepatocyte growth factor/HGF receptor) (or c-met) (7, 8, 9). We
also know that such activation is not operative in FTC [and anaplastic
thyroid cancer (ATC)], but we still ignore what is distinctive of
FTC.
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), it was inferred that loss of
genetic material on the long arm of chromosome 11 (11q13) was
involved in the transformation from the follicular cell into FA,
whereas the subsequent loss of genetic material on the short arm of
chromosome 3 (3p) caused FA to progress to FTC. Finally, the
inactivation of the TSG p53 would have caused a further malignant
transformation, viz. the passage from FTC to the
undifferentiate and rapidly fatal ATC (10, 11). Two aspects remained
not defined in the schema (3). First, the bold-faced question
mark indicates the need for a better elucidation of the molecular
events involved in the passage from follicular cell to FA and
confirmation of loss of genetic material on 11p. Second, the
dotted line between PTC and ATC indicates the possibility
(but not the certainty) that PTC may progress to ATC (12). As a continuation of our studies on thyroid oncogenesis (9, 13), we wished to ascertain whether the FA/FTC pathway was characterized by loss of genetic material on the long arm of chromosome 7 (7q). We had a number of reasons to suspect that. First, cytogenetic and molecular analyses on thyroid neoplasms had demonstrated loss of genetic material on chromosomes such as lq, 2p, 2q, 3p, l0p, 10q, 13q, 17p,17q (14, 15, 16, 17, 18, 19, 20), and it was inferred that FTC loses genetic material (and at multiple loci) with much greater frequency than PTC (20). As reviewed by Biece et al. (21), loss of heterozygosity (LOH) on chromosome 1, 2, 3, 10, 13, and 17 has been demonstrated for a wide variety of solid tumors. Second, LOH on 7q (particularly on the c-met region at 7q31.3) had been found in several cancers (21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Based on this finding, it was concluded that the segment 7q31–7q32 contains a minimum of one TSG.
Five studies (14, 19, 30, 31, 32), 2 of which (31, 32) appeared while our manuscript was being prepared, have evaluated LOH on 7q in thyroid neoplasms. Kubo et al. (14) investigated a total of 23 cases (8 FA and 15 PTC) by probing 7q31, and they found LOH in a single case of FA. Zedenius et al. (30) studied a total of 24 cases (22 FA and 2 FTC) and found LOH in two cases of FA. Both Kubo et al. (14) and Zedenius et al. (30) used the Southern blot technique approach. Califano et al. (19) studied a total of 30 PTC, 22 of which were informative for one marker on 7q22 (D7S 479) and for another one (D7S 486) on 7q31.2. They found LOH at at least one locus, without specifying which one, in 3 PTC. Segev et al. (31) used only one marker (D7S 1824) and found no LOH in informative cases of FA (n = 13) or PTC (n = 14). Zhang et al. (32) investigated 13 PTC, 14 FTC, and 15 FA with 11 markers spanning the region from 7q31.1 to 7q33–34. They found that LOH for 2 markers, which were separated by 2 centiMorgans, distinguished PTC from PTC (see Discussion).
Recently, Liang et al. (33) reported LOH within 7q22 and 7q31 in myeloid neoplasms and concluded that there are three distinct critical loci that may contribute, alone or in combination, to the evolution of adult myelodysplasia and adult acute myelogenous leukemia. Finally, we have recently shown that HGF and c-met protooncogenes are simultaneously overexpressed in PTC, whereas FTC and ATC consistently fail to coexpress them (9). It is worth noting that both the ligand (HGF) and the receptor (HGF or c-met) map to 7q (precisely, 7q21.1 and 7q31.3, respectively) (34).
In brief, we hypothesized that FTC and ATC could harbor LOH for 7q21.1 and 7q31.3, but PTC would not. The evidence presented here validates our hypothesis and puts not only FA and FTC but also ATC on the same molecular pathway, a pathway which is distinct from that of PTC.
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Materials and Methods |
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DNA extraction
Formalin-fixed, paraffin-embedded specimens were cut with a sterile scalpel, and the scalpel was changed for each specimen. Each crushed specimen was placed in a sterile 1.7-mL tube. After addition of 1 mL xylene, the tube was centrifuged at 14,000 rpm for 5 min. In the subsequent three cycles, the supernatant was always discarded, and the pellet was resuspended in 500 µL of progressively diluted ethanol (100%, 90%, and 70%, respectively). After the third centrifugation, the pellet was resuspended in 430 µL of 0.05 mmol/L Tris-HCl (pH 8.5) containing 1 mmol/L EDTA, 0.5% SDS, and 0.5 mg/mL proteinase-K. After incubation at 55 C overnight, tubes were vortexed and DNA extracted with phenol/chloroform. The DNA concentration was quantitated by spectrophotometry (OD260/OD280 nm).
PCR and microsatellite analysis
LOH was detected by PCR-based (C-A)n polymorphic microsatellite markers. For the PCR amplification, we used appropriate primers obtained from MapPairs (Research Genetics, Inc., Huntsville, AL; http://www.resgen.com). Two primers for the HGF locus (the centromeric D7S 660 and the telomeric D7S 492) and two for the c-met locus (the centromeric D7S 486 and the telomeric D7S 655) were selected for their high polymorphic informative content. The anatomy of these markers within the pertinent segment of 7q was found at the worldwide web site http://www.nhgri.nih.gov/dir/gtb/chr7. The extracted DNA was amplified by PCR performed in 40 µL reaction mixture. The mixture consisted of 20 ng DNA or 2.0 µL extracted DNA (10 ng/µL), 20 µL of each primer, 25 mmol/L magnesium chloride, 1.0 mmol/L deoxynucleotide triphosphates, 10x Taq Polymerase Buffer, and 0.5 µL Taq DNA polymerase (Promega Corp., Madison, WI). The Hot-start PCR began with 5 min denaturation at 94 C, during which Taq DNA polymerase was added, and it was followed by 30 cycles of amplification. Each cycle consisted of one denaturation step at 94 C for 1 min, one annealing step at 55 C for 1 min, and one elongation step at 72 C for 1 min, with a final extension of 5 min at 72 C. The apparatus was the DNA Thermal Cycler by Perkin-Elmer Corp. (Norwalk, CT). The PCR products were separated by electrophoresis in a 3.5% Metaphor Agarose gel at 100 mV for 3 h using TBE buffer, pH 7.5 (89 mmol/L TRIS-borate/89 mmol/L boric acid/2 mmol/L EDTA) containing 0.5 mg/mL ethidium bromide.
Determination of LOH
Definition of the allelic loss was limited to the informative cases. In the LOH technique, a given case is considered informative for a given microsatellite marker when the corresponding genomic (nontumoral) DNA is polymorphic or heterozygous, and thus produces two bands in the gel, namely the maternal and the paternal allele. Homozygous are, therefore, noninformative. The signal intensity of the polymorphic alleles was evaluated visually by three reviewers in an independent and blind fashion. Thus, a nodule had LOH when one of the two bands visible in the simultaneously tested genomic DNA was absent or had a signal intensity reduced by more than 50%. To ensure that results were reproducible, we repeated LOH analysis starting from PCR. In the analysis of the results, we disregarded the two tumors (one PTC and one FTC) in which a replication error (RER) was observed. RER is the appearance of a band not seen in the paired normal tissue.
Immunohistochemistry
Immunohistochemistry on 5-µm sections of the paraffin-embedded blocks was performed as described in detail previously (9). We used either a goat polyclonal antibody against HGF (Sigma, St. Louis, MO) or a rabbit polyclonal antibody against c-met (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and the antigen retrieval technique of Gown et al. (36). Tissue sections were deparaffinized in xylene. Slides were microwaved for 15 min (Whirlpool AVM 300, power set at 500 watts). Staining was obtained with a streptavidin-biotin peroxidase method (LSAB kit from DAKO Corp., Carpinteria, CA). To avoid a nonspecific staining in oncocytic cells, we used a second antibody conjugated with peroxidase (Envision system, DAKO Corp.). The color reaction was developed using 3,3' diaminobenzidine as chromogen. Specificity was assessed by omitting the primary antiserum or by replacing the primary antiserum with normal goat or normal rabbit serum, or by preincubating the primary antiserum with either HGF (Sigma) or c-met (Santa Cruz Biotechnology, Inc.). In each of these omissions, replacements, or preincubations, no staining was evident. Immunohistochemical staining was evaluated twice and independently by three pathologists (M. Trovato, D. Villari, and D. Batolo). At the end of these six readings, reproducibility was 100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With respect to our previous study (9), we have now 3 additional OA, 5 additional FTC, and 3 additional ATC; FH (n = 10) were not studied before. To avoid interstudy differences, we repeated immunohistochemistry on the remaining specimens (7 OA, 5 FTC, and 3 ATC) taken from our previous series (9). Of the 11 additional tumoral tissues and their adjacent nontumoral counterparts, only 2 OA stained positive for both HGF and c-met. The corresponding prevalence in FH was 3/10 (30%), which is congruent with that we reported previously (9) for other benign lesions (CNs = 30%; FA = 20%). Compared with our previous data (9), the prevalence of coexpression of HGF and c-met in OA is somewhat higher (4/10 or 40% vs. 2/7 or 29%) because 2/3 of the new cases of OA were immunostained by both HGF and c-met antibodies. We do confirm that neither the ligand (HGF) nor the receptor (c-met) was expressed in FTC (0/11) or ATC (0/6). In sharp contrast to all of the above, there was concurrent (over-)expression of these two protooncogenes in l2/12 PTC.
LOH
Data are summarized in Tables 1, 2, and 3; illustrative cases are shown in
Fig. 2. The frequency of informative
cases was similar for each marker: 77% for D7S 660, 78% for both D7S
492 and D7S 486, and 85% for D7S 655. Among the informative cases,
heterozygosity at all four loci was retained (i.e. LOH was
absent) in all normal tissues, all CNs, all FH, all OA and, most
importantly, all PTC.
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) showed
LOH, and with this increasing order of frequency: D7S 655 (l/l0 or
l0%), D7S 492 (2/l0 or 20%), D7S 486 or D7S 660 (2/7 or 29% for
both). As shown in Table 2, the most frequent pattern was LOH for one
marker of c-met and for one of HGF in variable combinations. Only cases
no. 1 and 2 in Table 2 expressed c-met and HGF. From Table 2, it seems
that the macrofollicular and the toxic (hyperfunctioning) variants are
spared by loss of genetic material on the loci we have probed (0/2 both
variants), whereas the microfollicular variant has a particular
propensity for such LOH (2/3 or 67%). Within the malignant lesions,
none of the four histological variants of PTC showed LOH for any of the
four markers (Table 3). In sharp contrast to PTC, 8/8 FTC and 5/5 ATC
had an unambiguously uniform pattern: LOH for all four markers (Table 3). In either FTC or ATC, LOH was unrelated to the clinical stage of
the malignancy (Table 3). |
Discussion |
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The major result of the studies by Grebe et al. (16) and Ward et al. (20) is that PTC have exceedingly low rates of LOH. Particularly, metaanalysis (20) showed that the average rate of LOH per chromosome arm in PTC (2.5%) is statistically different from that of FA (5.8%) and FTC (19.7%). In addition, in FTC, there is a significantly higher frequency of LOH on both arms of a given chromosome, suggesting relatively high frequency of whole chromosome losses. LOH, on at least one site, was detected in 60% of FTC, 33% of FA, and 23% of PTC. In FTC, a LOH prevalence greater than 40% concerned 3p, 10q, 13q, and 17p (16); on the same chromosomes, PTC had a lower prevalence. For instance, the 86% and 72% prevalence of LOH on 3p and 17p in FTC compared with 29% and 22%, respectively, in PTC (16). The crucial point is, therefore, that the literature reports no clear-cut differences between FTC and PTC. Such differences have been shown by Zhang et al. (32), and they are attributable to two markers on 7q31.2, separated by 2 centiMorgans: D7S 480 and D7S 490. Indeed, LOH, at the first and second loci, was absent in 2/2 informative PTC and in 4/4 informative PTC, respectively. This contrasted with a corresponding prevalence of 5/9 and 6/10 in FTC, and 2/4 and 0/4 in FA. The most centromeric marker (D7S 479 on 7q22) and the most telomeric marker (D7S 1805) used by these authors (32) had a 90% informativity rate, and no case of the three histotypes harbored LOH at either locus. Our four markers operate a far better distinction. LOH was detected in 8/8 to 9/9 informative cases of FTC, but 0/9 to 0/11 informative cases of PTC; in informative FA, LOH ranged from 1/10 (D7S 655) to 2/7 (D7S 486 and D7S 660).
Because Zhang et al. (32) reported no LOH at the two most centromeric markers of 7q31.1 and at the sole marker of 7q22 for any thyroid tumor, but we found LOH at two loci of 7q21 for all informative FTC and two informative FA, we believe there must be at least one TSG in the vicinity of the c-met locus and a second TSG in the vicinity of HGF locus. This interpretation goes along with that of Liang et al. (33). As mentioned in the Introduction, these authors (33) concluded that 7q contains at least three noncontiguous TSG and that there should be none distal to the c-met locus. In brief, 7q21 and 7q31.2–31.3 contains the long sought molecular markers for FTC. These are TSG, which, upon their inactivation, are crucially involved in directing thyroid oncogenesis toward the follicular histotype.
LOH at the four sites we have probed is not necessary for the
establishment of the FA histotype because less than 50% of the FA are
LOH positive. However, by starting losing genetic material, FA become
more genetically instable. It then takes a more extensive loss (as that
observed in all of our FTC) to cause greater genetic instability and
ultimately to lead to the transformation from FA to FTC. In the context
of such passage (Fig. 1), we propose that 7q should replace 11q and,
most importantly, 3p. Indeed, the involvement of putative TSG at 11q or
other chromosomes (such as VHL at 3p or Rb at 13q) are being ruled-out
(16, 20). If one admits that the FA stage can be preceded by the FH
stage and that the FH stage, in turn, is preceded by the CN stage, then
LOH on 7q is not involved in these two passages because all CN and all
FH retained homozygosity.
We would like to underscore two other data. First, we found the same
100% prevalence of LOH on 7q in FTC and ATC. This observation is
crucial because, taken together with the absent coexpression of the
HGF/c-met system (9) and other systems (M. Trovato et al.,
manuscript in preparation) in FTC and ATC, it indicates that FTC and
ATC share genetic abnormalities, but PTC do not. The major implication
is therefore that ATC is correctly placed after FTC (Fig. 1); but the
dotted line between PTC and ATC might be eliminated because
FTC and ATC seem to be two subsequent stages of the same pathway,
whereas PTC should be on a separate pathway. It takes some additional
genetic abnormality (i.e. inactivation of p53) to cause FTC
to progress to the highly malignant ATC histotype. In brief, the
FA/FTC/ATC pathway is characterized by subsequent loss of functional
TSG, whereas the PTC pathway is characterized by activation of
tyrosine-kinase protooncogenes. Second, LOH on 7q was detected in 0/7
to 0/9 informative cases of OA but in 2/7 to 2/10 informative cases of
FA. This data goes along with recent evidence (31) (also based on LOH)
that oncocytic neoplasms differ from follicular neoplasms at the
molecular level, even though they are considered variants of the latter
at the histological level (31).
In agreement with others (31, 32), we believe that our findings may have a major diagnostic application. Unless there are evident features of malignancy, the histological differentiation between FA and FTC [as well as between OA and oncocytic carcinomas (OC)] may prove difficult. For the latter distinction, LOH for a combination of three markers on 1q was observed in 92% of OC vs. 30% of OA, and LOH for two markers on 2p was observed in 50% of OC vs. 12% of OA (31). As to the FTC vs. FA distinction, we have already stressed that the LOH pattern given by our four markers is totally different from that seen in FA. Instead, using the two markers proposed by Zhang et al. (32), one FTC had the same pattern as one FA, and another FTC had the same pattern as a second FA; only three FTC had LOH for both markers. Together with Segev et al. (31), we propose that LOH could be used also at the cytological level, with the advantage of planning the correct therapeutic strategy well ahead of operation.
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Acknowledgments |
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Footnotes |
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Received February 18, 1999.
Revised May 24, 1999.
Accepted June 1, 1999.
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References |
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