Significance of trisomy 7 in thyroid tumors

Significance of Trisomy 7 in Thyroid Tumors Marille E. Herrmann and Peter A. Lalley

ABSTRACT: Standard cytogenetic studies of a multifocal metastasizing papillary thyroid carcinoma revealed two clonal chromosome aberrations: rearranged lOq and trisomy 7. Trisomy 7 seemed to be restricted to tumor nodule A, whereas era (lOq) was detected in tumor nodule B and in a metastatic lymph node. We applied fluorescent in situ hybridization to ask whether trisomy 7 was a feature of the original tumor nodule or an in vitro phenomenon changing quantitatively during early passages and to see whether trisomy 7 was restricted to tumor nodule A. We used the biotinylated chromosome 7 alphasatellite probe D7Z1 on freshly dropped slides from metaphase harvests from tumor nodule A,B, and the lymph node and on touch preparations from the frozen specimen of tumor nodule A. Trisomy 7 was present in the original tumor nodule (6% of cells), as well as in early passages (P1-3) from both tumor nodules and the metastatic lymph node with a frequency of 10.7-13.2%. The detection of trisomy 7 as a stable component in short-term cell culture and its presence in the original tumor material indicates that this common numerical aberration is an in viva phenomenon.

INTRODUCTION The majority of thyroid cancers, either papillary or follicular, are well differentiated. Papillary thyroid cancers rarely recur after operative treatment, but when they do, generally they do so as locally well differentiated tumors. Very few recur as undifferentiated anaplastic carcinomas. These well-differentiated tumors are often detected at an early stage (pT1 or pT2) and show multifocal occurrence. Whether such a multifocal tumor of small size represents intrathyroidal metastases or whether these tumors arise from multiple events is of clinical relevance. Molecular genetic techniques, such as fluorescent in situ hybridization (FISH) using centromeric probes for numerical aberrations, can be applied in screening these multifocal tumors for genetic aberrations. Trisomy 7 represents one of the most common genetic findings in solid tumors [1], including thyroid tumors [2-6]. The occurrence of trisomy 7 in tissues from different organs, from highly malignant to well differentiated, benign and normal tissues, has led to an interpretation of this aberration as a cell culture artefact. Quantification of this numerical aberration using FISH techniques on cytogenetic harvests and tumor touch preparations may help to clarify the significance of trisomy 7. Case Report A 29-year-old female was diagnosed as having thyroid nodules. Aspiration cytology revealed papillary thyroid cancer. A total thyroidectomy with central neck dissection was

From the Center for Molecular Biology, Wayne State University, Detroit, Michigan. Address reprint requests to: Marille E. Hermann, M.D., Ph.D., Centerfor Molecular Biology, Wayne State University, 5047 Gullen Mall, Detroit MI 48202. Received December 23, 1991; accepted February 18, 1992. 144 Cancer Genet Cytogenet62:144-149 (1992) 0165-4608/92/$05.00

performed. Papillary thyroid cancer was found in three separate nodules from the right gland as well as in two lymph nodes from the central compartment. Material from two of the nodules (A and B) and one metastatic lymph node were supplied for cytogenetic investigation. The tumor was staged pT2b Nlb MO. Two months later a local recurrence in the right scalene region was treated with modified neck dissection. MATERIALS AND METHODS The papillary thyroid cancer nodules A and B and the metastatic lymph node were grown in RPMI 1640 (GIBCO) supplemented with fetal bovine serum (15%), antibiotics, epidermal growth factor (20 ng/ml) (Sigma), insulin (10 ~g/ ml), transferrin (5 ~g/ml), selenium (5/~g/ml) (Collaborative Research), and cholera toxin (100 ng/ml) (Sigma). Metaphase harvests were performed according to standard procedures, applying 0.03 ~g/ml Colcemid for metaphase arrest and using 50 mM KCI as hypotonic solution. For chromosome banding, slides were dried 12-36 hours at 56°C and G or Q banded. FISH was performed either on freshly dropped slides or on tumor touch preparations from frozen specimens after storage in Carnoy's fixative. We used the biotinylated alpha-satellite probe D7Z1 (Oncor) for chromosome 7 staining and followed the protocol from the commercial supplier with an additional step of proteinase K pretreatment (0.6 ~g/ml) (BRL) for 7,5 min at 37°C and paraformaldehyde fixation for 10 minutes at RT before the denaturation step. We also used blood metaphase harvests as controls. For evaluation of FISH data we applied the following categories: number of fluorescent dots ("1", "2", "3", "4"), "UC", and "NS". "UC" (uncertain) was used when two fluorescent signals appeared close or were connected by a fluorescent line, so that two chromosomes could not be distinguished from one in the G2 phase of © 1992 Elsevier SciencePublishing Co., Inc. 655 Avenueof the Americas, New York, NY 10010

Trisomy 7 in Thyroid Tumors

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Partialkaryogram in (a) of three cells showing rea(lOq) in the right-hand member of each pair of chromosomes 10, in (b) of one cell containing trisomy 7.

the cell cycle. "NS" was used for nonspecific staining. In particular, touch preparations included areas of overlapping cells. These areas were excluded from evaluation. RESULTS

Evaluation of cell culture and standard cytogenetic findings of this case were described previously [7]. Short-term cultures from specimens of tumor nodule B were harvested on day 10. First harvests were achieved on day 21 from tumor nodule A and from the lymph node. Cell morphology in each of these cultures was similar and revealed a heterogenous population of cells with epithelial-like and spindlelike appearance. In each culture, cells had lost contact inhibition. Cytogenetic evaluation detected two clonal aberrations: trisomy 7 and rearranged 10q with a breakpoint at 10qll (Fig. la,b). Each of the tumor specimens had either one of the aberrations. Tumor nodule B and the lymph node showed the rearranged chromosome 10q (26/33 cells) together with a normal karyotype and tumor nodule A contained trisomy 7 (4/14 cells) together with a normal karyotype [7]. Employing fluorescent in situ hybridization with the chromosome-7-specific probe D7Z1 enabled us to quantitate the prevalence of trisomy 7 in cytogenetic harvests from early cell cultures of all three tumor samples and from the original tumor nodule A. Figure 2a-d demonstrates the criteria employed in scoring the cells for trisomy 7. Figure 2a shows a nucleus from nodule A with three fluorescent signals and was scored as trisomic for chromosome 7. Figure 2b is a nucleus categorized "UC". Figure 2c illustrates typical findings in blood lymphocytes (metaphase as inset) normal for chromosome 7. Figure 2d is a typical touch preparation. The cell in the middle (arrow) was scored as trisomy 7, one neighboring cell (arrow) as "UC", and several overlapping cells were excluded from evaluation. Overall, the frequency of nuclei with a three-signal score did not change considerably from the original tumor to early passages, and remained stable between 6% and 13% when compared with tumor nodules and the lymph node. In Figure 3a-d the distribution of nuclei with 1, 2, 3, or 4 fluorescent signals or nuclei from the category "UC" or " N S " is illustrated in column graphics. In each graph the

occurrence of each category in the tumor samples is compared to the frequencies in blood lymphocytes. Figure 3a illustrates the distribution in tumor nodule A (passage 1), Figure 3b in tumor nodule B (passage 3), Figure 3c in the lymph node (passage 1), and Figure 3d in the tumor nodule A (touch preparation]. We found that 12.6% of the nuclei from nodule A (326 nuclei evaluated), 13% of the nuclei from nodule B (371 nuclei evaluated), 10.7% of the nuclei from the metastatic lymph node (289 nuclei evaluated), and 6% of the nuclei from the original tumor nodule A (536 nuclei evaluated) were trisomic for chromosome 7. The classification "UC" in tumor node A ranged from 4.2% in passage 1 to 10.6% in the touch preparation. According to the strict application of this category, trisomic cells may be included. The increase of nuclei scored "UC" (10.6%) in the touch preparation and the decrease of nuclei with the three-signal score (6%) may be related. Another possible interpretation of the somewhat lower frequency of trisomy 7 cells in the touch preparation could be due to the phenomenon of truncation, which was evaluated in detail for FISH studies on pathologic tumor sections by Hopman et al. [8]. FISH studies allowed us to detect small populations of trisomic cells in all samples tested in this tumor. This finding is in contrast to the results obtained using standard cytogenetic techniques, where trisomy 7 was found in only one tumor nodule (A). This observation indicates that the FISH method may be more sensitive for detecting numerical aberrations than standard cytogenetic approaches. DISCUSSION

Trisomy 7 is one of the most common findings in solid tumors [1]. It is frequently found in thyroid tumors [2, 4-6], renal tumors [9-12], prostate tumors [13], bladder tumors [14-16], brain tumors [17-19], colorectal carcinomas [20, 21], and in pheochromocytomas [22-24]. It is also found in benign lesions such as multinodular goiter [6], Dupuytren's contracture [25], in colonic polyps [26, 27], Barret's esophagus [28], and atherosclerotic plaques [29, 30]. Because it was found in many different tumors as the only aberration, it was originally interpreted as a primary change. It is also found as an indicator of poor prognosis in bladder cancer, where it occurs in later stages and poorer graded tumors [31], and in malignant recurrent gliomas after irradiation treatment and chemotherapy [32]. Yet, trisomy 7 has also been demonstrated in normal lung [33], normal brain [18], and normal kidney tissues [34, 35]. Thus, the attribution of trisomy 7 as an indicator of a neoplastic process has been questioned. It appears, therefore, that trisomy 7 opens a box of very contradictory findings. The obvious explanation seemed to be that trisomy 7 represents a cell culture artefact giving trisomic cells a selective advantage during in vitro culture conditions [34]. However, some bladder tumors, where trisomy 7 was found in direct harvests, cast doubt on this hypothesis [15]. Fluorescent in situ hybridization techniques using repetitive chromosome-specific probes allow for quantitative studies of numerical aberrations. This study clearly shows that the percentage of cells harboring trisomy 7 does not dramatically change in this papillary

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F i g u r e 2 Micrographs of nuclei of tumor nodule A after application of FISH with alpha-satellite probe D7Z1 demonstrating in (a) nucleus with three-signal score, in (b) nucleus categorized "uncertain", in (c) blood control with interphase nuclei and metaphase (inset), in (d) cell with three-signal score (arrow) and neighboring cell categorized "uncertain" (arrow) in the touch preparation of tumor nodule A.

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F i g u r e 3 Quantification of trisomy 7 using FISH with the centromeric probe D7Z1 (a) in tumor nodule A, (b) in tumor nodule B, (c) in the metastatic lymph node, and (d) in the touch preparation of tumor nodule A. Controls from patients blood lymphocytes.

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thyroid carcinoma during early passages, arguing against a selective advantage. The presence of trisomy 7 in one of the original nodules is d e m o n s t r a t e d by a p p l y i n g the FISH techniques to a tumor touch preparation. Similar to that finding, W o l m a n et al. [36], a p p l y i n g the FISH technique to cells released from paraffin-embedded k i d n e y tumors, confirmed trisomy 7 in two of three cases where it had been found using standard cytogenetics. The view that trisomy 7 is tumor related is further substantiated by a recent report a p p l y i n g interphase cytogenetics to normal brain tissues from autopsies, where occurrence of trisomy 7 could not be verified [37]. The studies on normal brain tissues w h i c h i n c l u d e d cell p o p u l a t i o n s with trisomy 7 need to be evaluated critically. The tissues were taken 2 to 3 cm from the tumor and might have been contaminated by microscopic tumor cells. A second line of evidence that trisomy 7 may not be neoplasia related is found in a colorectal carcinoma with a mosaic karyotype i n c l u d i n g a clone with trisomy 7 and one with structural and n u m e r i c a l c h r o m o s o m e aberrations. In culture, cell m o r p h o l o g y was mixed, exhibiting both separate colonies w i t h fibroblast-like and epithelial-like cells. Because the trisomy 7 clone was found in the fibroblast-like colony [38], the authors suggested that trisomy 7 may be a feature of stromal cells representing an immunologic response to the disease process. However, in one report on prostate cancer the authors claim to have removed all fibroblast-like cells and still found one tumor with trisomy 7 in a harvest of epithelial-like cells [13]. There is evidence for a different interpretation of the fibroblastoid cells in culture: It is well k n o w n that differentiated epithelial cells (such as t h y r o i d follicles) can transform into mese n c h y m a | cells, d e p e n d i n g on their extracellular environment [39, 40]. What role this transformation plays in malignancy has to be s t u d i e d in more detail. Preliminary observations by the authors [41] show that in thyroid adenomas and two p a p i l l a r y thyroid cancers cells with fibroblastoid a p p e a r a n c e stain positive for the p r o h o r m o n e thyroglobulin, indicating that an e p i t h e l i a l - m e s e n c h y m a l transition can take place in vitro in early passages of these tumors. Further investigations of trisomy 7 could involve double staining techniques either using standard cytogenetics c o m b i n e d with i m m u n o c y t o l o g y or FISH with i m m u n o c y tology to d e t e r m i n e w h e t h e r trisomy 7 is restricted to a specific cell type. A d d i t i o n a l studies are necessary to define w h i c h gene products are preferentially expressed in tumors with trisomic cells. In a recent report on Barret's esophagus, involvement of the EGFR receptor, w h i c h is located on chromosome 7, was studied. One case with trisomy 7 revealed i n c r e a s e d c-erbB mRNA and increased EGF binding [28], whereas in a m e n i n g i o m a w i t h trisomy 7, increased EGF b i n d i n g could not be demonstrated, despite the fact that a series of m e n i n g i o m a s d i d express variable amounts of the EGFR receptor [19]. This m a y indicate that trisomy 7 represents very different m o l e c u l a r events in different tumors. If trisomy 7 is indicative of a neoplastic process, the multifocal t u m o r w o u l d consist of several nodules, all related to each other, w h i c h most likely arose through intra-

M . E . H e r r m a n n and P. A. Lalley thyroidal metastasis. If this h y p o t h e s i s holds true, one w o u l d expect to find the second aberration, a rearranged 10q, in the tumor n o d u l e A and in t h e . l y m p h node. Cytogenetic analysis did show the rearranged lOq in cells from the l y m p h node harvested after 3 weeks in culture. Unfortunately, the presence of rea(10q) could not be d e m o n s t r a t e d in cells from tumor nodule A due to the poor quality of the metaphase cells harvested from this culture. To clarify multifocality in this tumor, the rearrangement of 10q, w h i c h may involve the ret proto-oncogene [42, 43], has to be studied with m o l e c u l a r techniques such as PCR or Southern analysis to determine w h e t h e r the ret proto-oncogene participates in the d e v e l o p m e n t of this tumor. Presently, the significance of trisomy 7 is not known, but it should not be d i s m i s s e d as a neoplasia-related phen o m e n o n until further m o l e c u l a r genetic studies have been performed.

M.E.H. is a fellow of the Deutsche Forschungsgemeinschaft (He 1627/3-2). We thank Dr. Tina Trevor for helpful discussions concerning the epithelial-mesenchymal transitions, Dr. Sandra Wolman for critical reading of the manuscript, and Dr. Gary Talpos for supplying the tumor materials.

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