Loss of Heterozygosity of the Long Arm of Chromosome 7 in Follicular ...

0021-972X/99/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 1999 by The Endocrine Society

Vol. 84, No. 9 Printed in U.S.A.

Loss of Heterozygosity of the Long Arm of Chromosome 7 in Follicular and Anaplastic Thyroid Cancer, but Not in Papillary Thyroid Cancer* MARIA TROVATO, FILIPPO FRAGGETTA, DANIELA VILLARI, DARIO BATOLO, KAROL MACKEY, FRANCESCO TRIMARCHI, AND SALVATORE BENVENGA Dipartimento di Patologia Umana (M.T., D.V., D.B.), Policlinico Universitario, 98125 Messina, Italy; Anatomia Patologica (F.F.), Universita` 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 ABSTRACT Papillary thyroid cancer (PTC), but neither the follicular nor the anaplastic histotype [follicular thyroid cancer (FTC), anaplastic thyroid cancer (ATC)], overexpresses simultaneously the protooncogene HGF (hepatocyte growth factor) and its receptor HGF-R (or c-met). Because 1) HGF and c-met map to chromosome 7q21 and 7q31, respectively, 2) FTC loses genetic material at multiple loci with a frequency much higher than PTC, and 3) loss of heterozygosity (LOH) on 7q has been previously found in various tumors, we tested the hypothesis that both FTC and ATC, but not PTC, could harbor LOH in segments of 7q encompassing the loci for HGF and c-met. We screened 6 normal thyroids, 10 colloid nodules, 10 follicular hyperplasias, 10 oncocytic adenomas, 10 follicular adenomas (FA), 10

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ANCER is a process of clonal expansion of cells whose growth advantage derives from the accumulation of acquired (somatic) genetic alterations (1, 2). These alterations consist of gain of functions of protooncogenes and/or loss of function of tumor suppressor genes (TSGs). Fagin et al. (3) proposed a multistep model of the genetic alterations that occur in thyroid neoplasms arising from the follicular cell (Fig. 1). 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 – 6), and HGF/ HGF-R (hepatocyte growth factor/HGF receptor) (or c-met) (7–9). 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. In the said model (3) (Fig. 1), it was inferred that loss of genetic material on the long arm of chromosome 11 (11q13)

Received February 18, 1999. Revision received May 24, 1999. Accepted June 1, 1999. Address all correspondence and requests for reprints to: Maria Trovato, Dipartimento di Patologia Umana, Padiglione D, Policlinico Universitario, Gazzi - 98125 Messina, Italy. * This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro.

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. (J Clin Endocrinol Metab 84: 3235– 3240, 1999)

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 –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 (21–30). Based on this finding, it was concluded that the segment 7q31–7q32 contains a minimum of one TSG.

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stitute of Pathology (35). Surgical samples were fixed in 10% phosphate buffered formalin, pH 7.4, for 12 h. After being embedded in paraffin, 5-mm sections were stained with hematoxylin-eosin, and representative blocks were chosen for the immunohistochemical and for the PCR studies.

DNA extraction

FIG. 1. A hypothetical model for the initiation and progression of thyroid neoplasms, based on the relative prevalence of genetic lesions in the various phenotypes (from Ref. 3).

Five studies (14, 19, 30 –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 5 13) or PTC (n 5 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.

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 mL of progressively diluted ethanol (100%, 90%, and 70%, respectively). After the third centrifugation, the pellet was resuspended in 430 mL 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 mL reaction mixture. The mixture consisted of 20 ng DNA or 2.0 mL extracted DNA (10 ng/mL), 20 mL of each primer, 25 mmol/L magnesium chloride, 1.0 mmol/L deoxynucleotide triphosphates, 103 Taq Polymerase Buffer, and 0.5 mL 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.

Materials and Methods The study group consisted of 6 normal thyroids harvested during autopsy and 68 surgically removed thyroids. These surgical specimens included 10 colloid nodules (CNs), 10 follicular hyperplasias (FHs), 10 FA, 10 oncocytic adenomas (OAs), 12 PTC, 10 FTC and 6 ATC. These 68 nodules, therefore, encompass the whole spectrum of thyroid follicular growth (from totally benign to highly malignant). The 68 nodules were studied paired with the unaffected lobe of the thyroid. All lesions were classified according to the criteria proposed by the Armed Forces In-

Immunohistochemistry Immunohistochemistry on 5-mm 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

CHROMOSOME 7 LONG ARM AND THYROID CANCER

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TABLE 1. Loss of heterozygosity on 7q at the loci D7S 660, D7S 492, D7S 486, D7S 655 in normal and pathological thyroid tissues HGF region Thyroid tissue

c-met region

D7S 660

D7S 492

D7S 486

D7S 665

Informative

LOH

Informative

LOH

Informative

LOH

Informative

LOH

5 5 8 7 9 9 9 6

0/5 0/5 0/8 2/7 0/9 0/9 9/9 6/6

5 8 6 10 7 10 8 5

0/5 0/8 0/6 2/10 0/7 0/10 8/8 5/5

6 7 9 7 8 9 8 5

0/6 0/7 0/9 2/7 0/8 0/9 8/8 5/5

4 6 10 10 9 11 9 6

0/4 0/6 0/10 1/10 0/9 0/11 9/9 6/6

Normal (n 5 6) CN (n 5 10) FH (n 5 10) FA (n 5 10) OA (n 5 10) PTC (n 5 12) FTC (n 5 10) ATC (n 5 6)

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 3,39 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%.

Results Immunohistochemistry

With respect to our previous study (9), we have now 3 additional OA, 5 additional FTC, and 3 additional ATC; FH (n 5 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 5 30%; FA 5 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. Within the benign lesions, only FA (cases no. 7–10 in Table 2) 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

TABLE 2. Allele loss in the FA cases LOH Case no.

Histopathology

HGF

c-met

S660

S492

S486

S655

³ ³ ³ ³ X X d X ³ d

³ ³ ³ ³ ³ ³ ³ d d ³

³ ³ ³ X ³ X X d ³ d

³ ³ ³ ³ ³ ³ ³ ³ d ³

FA 1 2 3 4 5 6 7 8 9 10

Macrofol. Microfol. Toxic Toxic Macrofol. Follicular Microfol. Follicular Microfol. Hyalinizing trabecular

LOH was determined as described in Materials and Methods. ³, Retention of heterozygosity; d, LOH; X, homozygous alleles (i.e. marker not informative for the locus). Only cases no. 1 and 2 showed immunohistochemical expression of both HGF and c-met.

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

Loss of genetic material in thyroid neoplasms has been studied by cytogenetic and LOH analysis. Two recent studies (16, 20) have examined multiple chromosomes by the LOH approach. Grebe et al. (16) investigated 7 FA, 14 FTC, and 14 PTC, whereas Ward et al. (20) investigated 24 FA, l0 FTC, 1 ATC, and 30 PTC, but both papers overlooked 7q. As mentioned in the Introduction, 5 papers (14, 19, 30 –32) have dealt with LOH on 7q, but only 1 compared PTC (n 5 13) with FTC (n 5 14) and FA (n 5 15). The 11 markers used to analyze these 3 histotypes had an average rate of informativity lower than our markers (68% vs. 80%). The overall rate of infor-

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TABLE 3. Allele loss in the PTC, FTC, and ATC cases LOH Case no.

PTC

FTC

ATC

1–3 4 –7 8 9 10 11 12 1 2,3 4,5 6–8 9 10 1 2 3–5 6

Histopathology

Staging (pTNM score)

PTC/c PTC/c PTC/f PTC/f PTC/onc PTC/tall cells PTC/c Minimally invasive Minimally invasive Minimally invasive Widely invasive Widely invasive Widely invasive Spindle cells Spindle cells Spindle cells Giant cells

T2N0M0 T3N0M0 T3N0M0 T3N0M0 T3N0M0 T3N0M0 T3N0M0 T2N0M0 T2N0M0 T3N0M0 T3N0M0 T4N0M0 T3N0M0 T4N0M0 T4N0M0 T4N0Mx T4N0M0

HGF

c-met

S660

S492

S486

S655

³ ³ X ³ ³ X RER d d d d d RER d d d d

³ ³ ³ ³ X ³ RER X d d d d RER X d d d

³ ³ ³ X X ³ RER d d d d d RER X d d d

³ ³ ³ ³ ³ ³ RER d d d d d RER d d d d

LOH was determined as described in Materials and Methods. ³, Retention of heterozygosity; d, LOH; X, homozygous alleles (i.e. marker not informative for the locus); Abbreviations for PTC variants are: c, conventional; f, follicular; onc, oncocytic.

FIG. 2. Representative PCR amplification of the (C-A)n microsatellite repeats. Lanes N and T indicate DNA samples isolated from the unaffected lobe of thyroid and tumor tissue, respectively. The arrow indicates LOH. No LOH occurred in the shown PTC case.

mativity is similar (74% vs. 78%) for D7S 486, the only marker in common with our study. Additional comparisons with this paper (32) will be presented later. 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

CHROMOSOME 7 LONG ARM AND THYROID CANCER

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

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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. Acknowledgments We are appreciative of the kindness of Professor J. A. Fagin for having allowed use of his facilities at the Division of Endocrinology and Metabolism, University of Cincinnati, OH.

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