ORIGINAL
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E n d o c r i n e
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Trisomy 17 as a Marker for a Subset of Noninvasive Thyroid Nodules with Focal Features of Papillary Carcinoma: Cytogenetic and Molecular Analysis of 62 Cases and Correlation with Histological Findings Daniela V. Frau, Maria L. Lai, Paola Caria, Tinuccia Dettori, Pierpaolo Coni, Gavino Faa, Luca Morandi, Giovanni Tallini, and Roberta Vanni Dipartimento di Scienze e Tecnologie Biomediche (D.V.F., P.Ca., T.D., R.V.) and Citomorfologia (M.L.L., P.Co., G.F.), University of Cagliari, 09042 Monserrato (CA), Italy; and Dipartimento Clinico di Scienze Radiologiche e Istocitopatologiche (L.M., G.T.), University of Bologna, 40127 Bologna, Italy
Context: Differentiated carcinomas of the thyroid are divided into follicular thyroid carcinoma and papillary thyroid carcinoma (PTC), based on their propensity to invade and their cytological features [papillary carcinoma-type nuclear changes (PTC-NCs)]. PTC typically exhibits a diploid karyotype sometimes with inv10(q11.2q21.2), leading to rearranged RET gene. Follicular thyroid carcinomas are often aneuploid and may exhibit t(2;3)(q13;p25), resulting in PAX8-PPAR␥1 gene fusion. Isolated trisomy 17 has rarely been reported in thyroid lesions, and its significance is unknown. Objective/Design: Our objective was to determine whether isolated trisomy 17 corresponds to a specific histological or molecular thyroid tumor subset. Nine cases with isolated trisomy 17 were critically reviewed and investigated for RAS and BRAF mutations and for RET and PAX8-PPAR␥1 rearrangements. Results: All nine cases were noninvasive, exhibited follicular growth pattern, and showed PTC-NCs focally defined within the nodule: four were PTCs follicular variant within larger tumors, and five were follicular-patterned nodules with incomplete cytological features of papillary carcinoma (variable proportion of cells with PTC-NCs scattered inside the lesion). RAS, BRAFV600E mutation, RET or PAX8-PPAR␥1 rearrangements were not identified. One case had BRAFK601E mutation. Only two of the 53 control cases showed focal PTC-NCs. Conclusions: Isolated trisomy 17 is associated with focal papillary carcinoma changes in follicularpatterned thyroid nodules and may be a marker for this subset of thyroid lesions that are often difficult to classify. (J Clin Endocrinol Metab 93: 177–181, 2008)
D
ifferentiated carcinomas of the thyroid are divided into two categories, papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC), based on their propensity to invade and the cytological features of the neoplastic cell nuclei (1). PTC is by far the most common, representing more than 80% of all malignant thyroid tumors (2), and typically shows normal or simple rearranged karyotypes (http://cgap.nci.nih.gov/ Chromosomes/Mitelman). Although activating mutations of BRAF are now believed to represent the most common molecular
alteration in papillary carcinoma (3), activation of the RET protooncogene, via chromosome inversions or translocations (RET/ PTC), has been reported as a cancer causative event since 1990 (4, 5). Chromosome aberrations other than those leading to RET rearrangement may be found in PTC, however, they are not representative of specific morphological or cytogenetic subgroups. Simple chromosome translocations are also relevant for the development of FTC. The gene fusion PAX8-PPAR␥1 (6), secondary to a t(2;3)(q13;p25), is found in 50% of FTC and 8% follicular thyroid
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Abbreviations: FISH, Fluorescence in situ hybridization; FNA, fine-needle aspiration; FTA, follicular thyroid adenoma; FTC, follicular thyroid carcinoma; OC, oncocytic change; PTC, papillary thyroid carcinoma; PTC-Cl, classical PTC; PTC-FV, papillary carcinoma follicular variant; PTC-NC, papillary carcinoma-type nuclear change; WDT-UMP, well-differentiated tumor of uncertain malignant potential.
Printed in U.S.A. Copyright © 2008 by The Endocrine Society doi: 10.1210/jc.2007-0970 Received April 30, 2007. Accepted October 15, 2007. First Published Online October 23, 2007
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adenomas (FTAs) (7), although a variety of other aberrations have been reported, and aneuploidy is not uncommon in FTCs. PTC of the follicular variant (PTC-FV) is characterized by follicular growth pattern and the papillary carcinoma-type nuclear change (PTCNC), considered specific for papillary cancer. Recent studies have suggested that the cytogenetic and molecular profile of PTC-FV may be closer to that of FTC and FTA, rather than classical papillary cancer (PTC-Cl) (8, 9). In our database of over 300 thyroid nodules analyzed cytogenetically, we identified a small group of cases with trisomy 17 as the only karyotypic change. All were lesions with follicular architecture and often were diagnosed as PTC-FV. Because isolated trisomy 17 has rarely been reported in thyroid nodules (http://cgap.nci.nih.gov/Chromosomes/Mitelman) and never been associated with any specific morphological tumor subset, we reviewed these lesions investigating in depth their cytogenetic, molecular, and histological features.
Materials and Methods Case selection Nine nodules with trisomy 17 as the only chromosome change were identified among 329 thyroid lesions analyzed cytogenetically, including 219 nodules from surgical samples successfully karyotyped by conventional cytogenetics and/or studied by fluorescence in situ hybridization (FISH), as well as 110 fine-needle aspirations (FNAs) investigated by FISH. All thyroid nodules were diagnosed according to current criteria (1). PTC-NCs and oncocytic changes (OCs) were carefully analyzed on histology sections. A total of 38 PTC (including 14 PTC-FVs), five invasive FTCs, and 10 FTAs were reevaluated, both histologically and cytogenetically, as controls (their features can be found in supplemental Table 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). To identify previously undetected small clones of cells with trisomy 17, cytogenetic reevaluation was performed on nuclei from touch preparations kept at ⫺30 C or paraffin blocks. The study was conducted with the approval of local ethics committees and consent from patients, in accordance with the Declaration of Helsinki guidelines.
Molecular cytogenetics Cytogenetic preparations from in situ harvested cell cultures, touch preparations, cells obtained during the FNA procedure for conventional cytological diagnosis (10), and isolated nuclei from paraffin-embedded tissue, either from whole or partial sections, were used as described previously (11, 12). FISH and fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms were performed as described elsewhere (11). Touch preparations and nuclei from paraffin-embedded normal thyroid tissue (obtained from thyroidectomy specimens) were used as negative controls. Nuclei from a PTC positive for RET/PTC3 rearrangement by RT-PCR and from a FTC positive for PAX8-PPAR␥1 gene fusion by RT-PCR (gift from Yuri E. Nikiforov, Department of Pathology, University of Pittsburgh, Pittsburgh, PA) were used as positive FISH controls for gene rearrangement studies.
Probes Centromeric probes for chromosomes 7, 8, 10, 17, and X, and BAC clones RP11–199F11 (TP53 gene), RP11–351D16 (RET gene) (13), RP11–339F22, and RP11–167M22 (PAX8-PPAR␥1 fusion) (14) were used. FISH results were evaluated as previously reported (11). By definition, disomy and trisomy were diagnosed only when the percentage of cells with two or three FISH signals, respectively, was greater than the
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mean ⫹3 ⫻ SD of the controls. Statistical calculation was done using Statgrafics 2.1 (Statistical Graphics Corp., Herndon, VA). RET probe showed three signals in 0.5% nuclei of negative controls and in 33% nuclei of the positive control. Overlapping PAX8-PAPR␥1 signals were never observed in negative controls and were present in 21% of the positive control.
BRAF and RAS mutational analysis Genomic DNA was purified from paraffin-embedded neoplastic tissue using a commercial kit (Charge Switch DNA Tissue Kits; Invitrogen, Life Technologies, Paisley, Scotland, UK). BRAFV600E, BRAFK601E, and H-, K-, N-RAS codons 12, 13, and 15, and 61 mutations, were analyzed as described elsewhere (15, 16). A CEQ8000 instrument (Beckman Coulter, Inc., Fullerton, CA) was used for sequencing.
Results Cytogenetic and clinicopathological features of the thyroid nodules with trisomy 17 as the only karyotypic change are summarized in Table 1, and those of the control cases in supplemental Table 1. All nine nodules were resected after cytological examination of the FNA (examples in Fig. 1A, a and b). G banding showed trisomy 17 as the only change in cases 1 and 3 (Table 1 and Fig. 1B, a). In the remaining cases, it was demonstrated by FISH (Table 1, cases 2 and 4 –9, and Fig. 1B, b– d). Disomy 7 was used as the indicator for a diploid karyotype because several reports (11, 17) have shown that in follicular-patterned thyroid lesions, aneuploidies accumulate starting from trisomy 7. The correctness of this assumption was tested by probing additional chromosomes in the reevaluation study. To confirm and broaden the first initial results, all cases were reevaluated. Chromosomes 7, 8, 10, 17, X, and TP53 gene imbalances, integrity of RET gene, and PAX8-PPAR␥1 gene fusion were investigated by FISH on nuclei isolated from paraffin-embedded material and on case 5 on original FNA cells kept at ⫺30 C (Fig. 1B, e– h). In cases 3 and 6 –9, a circumscribed area with PTC-NCs was clearly distinguishable within the thyroid nodule. From this area and from an area without PTC-NCs, lying as far as possible from the first one, nuclei were isolated separately and separately analyzed. In all five cases, the highest percentage of cells with trisomy 17 was observed in the area with PTC-NCs, and in two of them (cases 3 and 9), it was detected only in the areas with PTC-NCs (Table 1). All cells with trisomy 17 lacked evidence of RET rearrangement (Fig. 1B, i) and PAX8-PAPR␥1 gene fusion (Fig. 1B, j). No cases had H-, K-, N-RAS, BRAFV600E mutations. Case 1 showed BRAFK601E (Table 1). Correlation with clinicopathological findings demonstrated that these cases were either PTC-FV within larger lesions (Table1, cases 3, 6, 8, and 9) (Fig. 1A, c), or were follicular-patterned lesions with poorly/incompletely developed cytological features of papillary carcinoma (Table 1, cases 1, 2, 4, 5, and 7), showing a variable proportion of cells with PTC-NCs scattered inside the resected nodule (Fig. 1A, d). Lesions 3, 5, and 7 were circumscribed but not encapsulated; the remaining lacked evidence of invasion of the tumor capsule or of blood vessels (Table 1). The histological diagnosis according to conventional criteria is reported in Table 1. The term well-differentiated tumor of uncer-
F/47
F/37
F/38
M/34
F/49
M/31
F/30
F/34
F/38
Case no.
1
2
3
4
5
6
7
8
9
Histology section: nuc ish cen17 ⫻ 3 (8%) Histology section: nuc ish cen17 ⫻ 3 (9%)
FNA: nuc ish cen17 ⫻ 3 (38%)
FNA: nuc ish cen17 ⫻ 3 (9%)
Cell culture: 47,XX,⫹17关13兴; touch preparations: nuc ish cen17d ⫻ 3 (60%) Cell culture: nuc ish cen17 ⫻ 3 (36%); touch preparations: nuc ish cen17 ⫻ 3 (67%) Cell culture: 47,XX,⫹17关2兴/46, XX关12兴/nuc ish cen17 ⫻ 3 (14%); touch preparations: nuc ish cen17 ⫻ 3 (25%) Cell culture: 46,XY关12兴/ nuc ish cen17 ⫻ 3 (45%); touch preparations: nuc ish cen17 ⫻ 3 (76%) FNA: nuc ish cen17 ⫻ 3 (32%)
Trisomy 17 (initial evaluation)
Area with PTC-NC: 40% Area without PTC-NC: 32% Area with PTC-NC: 24% Area without PTC-NC: 11% Area with PTC-NC: 19% Area without PTC-NC: 5% Area with PTC-NC: 12% Area without PTC-NC: 0%
NA
64%
Area with PTC-NC: 16% Area without PTC-NC: 0%
40%
57%
Trisomy 17 reevaluation in nuclei from 30-m histology sectionsa
Noe Noe Noe
Noe Noe
Noe
No
Noe
Noe
No
No
Noe
Noe
No
No
No
No
No
RET rearr
PAX8PPAR␥1 rearr
No
No
No
No
NA
No
NA
No
No
NA
NA
No
NA
No
NA
No
Thyr 4
Thyr 4
Thyr 3
Thyr 4
Thyr 3
Thyr 3
Thyr 3
Thyr 4
Thyr 3
BRAFK601E No
Cytology BTAb
H-, K-, BRAF N-RAS activating activating mutation mutation
PTC-NC: 15% OC: 5% PTC-NC: 30% OC: 10% PTC-NC: 30% OC: 10% PTC-NC: 25% OC: 50% PTC-NC: 25% OC: 10%
PTC-NC: 50% OC: 15%
PTC-NC: 25% OC: 30%
PTC-NC: 10% OC: 20%
PTC-NC:10% OC: 5%
Percentage of cells with PTC-NCs and OCs within the thyroid nodule
WDT-UMP 关hyperplastic nodule兴c (2.5 cm) PTC-FV (0.9 cm) within follicular adenoma (2.2 cm) WDT-UMP 关hyperplastic nodule兴c (2.6 cm) PTC-FV (0.9 cm) within follicular adenoma (2.5 cm) PTC-FV (1.0 cm) within follicular adenoma (4.0 cm)
WDT-UMP 关PTC-FV兴c (5 cm)
PTC-FV (0.3 cm) within hyperplastic nodule (0.8 cm)
WDT-UMP 关follicular adenoma兴c (2.2 cm)
WDT-UMP 关follicular adenoma兴c (3 cm)
Histological diagnosis (tumor dimension)
Reporting of FNA cytology according to the British Thyroid Association (BTA) guidelines (2007) (10).
WDT-UMP (18). Histological diagnosis according to conventional criteria is shown in brackets.
cen17 probe, pZ17-14.
In both areas with and without PTC-NCs and OCs.
c
d
e
Nuclei were isolated from 30-m paraffin-embedded sections of the entire nodule if not otherwise indicated.
b
a
PTC-NCs are clearing, overlapping, and irregularities of the nuclear contour in the form of indentations, grooves, and pseudoinclusions. OCs are cells with abundant granular eosinophilic cytoplasm. F, Female; M, male; NA, not available; nuc ish, nuclear in situ hybridization; rearr, rearrangement.
Sex/ age (yr)
TABLE 1. Clinicopathological and molecular cytogenetic data of the patients with trisomy 17 as the only karyotypic change
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tain malignant potential (WDT-UMP), advocated when only minor PTC changes are present (18), has been used to describe some of our cases (Table 1, cases 1, 2, 4, 5, and 7). Only two cases in the control group (supplemental Table 1) had incomplete PTCNCs, with features similar to cases 1, 2, 4, 5, and 7 in Table 1. Limited follow-up data (available for cases 2, 3, 5, and 7) show no evidence of disease after an average of 42 months.
Discussion
FIG. 1. A, Cytology (a and b) and histology (c and d) of a PTC-FV occurring within an adenoma (case 8, Table 1) (a and c), and of an adenoma with focal nuclear changes of papillary carcinoma (case 2, Table 1) (b and d); histology of a PTC-FV (e) (case 25, supplemental Table 1) and PTC-Cl (f) (case 10, supplemental Table 1) without trisomy 17. B, Trisomy 17 (three red signals) in metaphase from cell culture (a), in cells from 5-m microdissection preparation (b), in nuclei from FNA preparation (c), and in nuclei from touch preparation together with disomy 7 (green signals) (d). Nuclei from paraffin-embedded material showing trisomy 17 (three green signals) accompanied by disomy 7 (two red signals) (e), disomy 8 (two red signals) (f), and three copies of TP53 gene on chromosome 17 (three red signals) (g), oncocytic cells (antimitochondrial antibody, green fluorescence) from FNA showing trisomy 17 (three red signals) (h). Lack of RET gene disruption (two RET red signals) in nuclei with chromosome 17 trisomy (three green signals) (i), lack of PAX8/PPAR␥1 gene fusion (two red and two green signals) (j), RET gene disruption (three red signals) and disomy 10 (two centromere green signals) in a RET/PTC3 positive PTC control (k), PAX8PPAR␥1 gene fusion (red and green overlapping signal) in a FTC positive control (l). Nuclei are counterstained with 4⬘,6-diamidino-2-phenylindole. BAC clones: RP11–199F11 (TP53), RP11–351D16 (a 207-kb BAC clone for the RET gene), RP11–339F22 (centromeric of PAX8), RP11–167M22 (PPAR␥1 gene).
Follicular-patterned nodules with incomplete PTC-NCs and follicular variant papillary carcinoma represent a heterogeneous group of lesions sometimes difficult to characterize. They can be interpreted according to the pathologist’s bias as carcinomas, adenomas, or adenomatous lesions, including hyperplasia (19, 20), with important clinical consequences. Although evidence for distinct molecular pathways characterizing the classical forms of PTC and FTC have been substantiated both in vitro and in tumor samples (3), the clinicopathological and molecular profile of PTC-FV and noninvasive nodules with incomplete cytological signs of PTC are still poorly understood. RET/PTC and PAX8-PAPR␥1 gene fusions, RAS and BRAFK601E mutations have all been reported (8, 20), with RAS mutations and/or PAX8-PAPR␥1 rearrangement present in approximately two thirds of PTC-FV (8). To the best of our knowledge, karyotypes showing trisomy 17 as single aneuploidy have been reported in only three thyroid lesions: one classical papillary carcinoma, one macrofollicular adenoma, and one case of hyperplastic follicular cell nodule (http:// cgap.nci.nih.gov/Chromosomes/Mitelman). The present study reports nine additional nodules with isolated trisomy 17, shows that they lack evidence of RET/PTC and PAX8-PPAR␥1 rearrangement, of RAS mutations, and of the BRAF mutations typical of PTC-Cl, and that they exhibit a consistent set of histological features. These are the follicular growth pattern, the lack of invasion (either of the tumor capsule or the blood vessels), the finding of cytological alterations of the type observed in papillary carcinoma (either focally arranged or diffusely scattered within the nodule), and the presence
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within the nodule of a variable proportion of cells with OC. The strength of the association between trisomy 17 and the focality of the PTC-NCs is supported by the finding in this series of only two cases with incomplete papillary carcinoma features lacking trisomy 17. Incidentally, in one of the cases, RET/PTC rearrangement was identified in some of the tumor foci by RT-PCR (previously reported in Ref. 20), and the presence of the rearrangement was confirmed by FISH (data not shown). Interestingly, in all nodules with trisomy 17 where it was possible to analyze separately the areas with and without the PTC-NCs, the highest percentage of trisomic cells was observed in the area with PTC-NCs. The finding in several cases of trisomic cells in the areas of the tumor without the PTC-NCs points to a biological continuum between the area of the nodule with cytologically benign follicular architecture and the area with the PTC-NCs, indicating a sort of field effect phenomenon and suggesting that the acquisition of trisomy 17 is biologically related to the development of the “atypical” features. Thus, our data indicate that trisomy 17 may be a specific cytogenetic marker for follicular-patterned thyroid nodules featuring those “atypical” cytological changes that make them difficult to classify, to the point that the controversial term “well-differentiated tumors of uncertain malignant potential” has been proposed. Trisomy 17, extremely rare in PTC, and part of an orderly sequence of polysomies that starts from trisomy 7 when present in FTC, seems to be acquired as an early event and does not appear to promote additional chromosome changes (11). The absence of molecular markers for either classical papillary (RET rearrangement BRAFV600E mutation) or follicular (PAX8-PPAR␥1) carcinoma differentiates our cases with trisomy 17 from other tumors, including the PTC-FV with RAS mutations and/or PAX8-PPAR␥1 rearrangement (8). BRAFK601E has been associated with follicular variant but not with classical papillary cancer (15), and not surprisingly, was identified in one of our lesions with scattered PTC-NCs. In conclusion, our findings demonstrate the strong association between a peculiar genetic profile, isolated trisomy 17, and the occurrence of focal papillary carcinoma changes in follicular-patterned nodules. This cytogenetic marker may provide a very useful tool to define a poorly characterized subset of thyroid lesions.
Acknowledgments We thank Mariano Rocchi for the centromeric and gene-specific probes, and Sandra Serra for technical assistance. The cytogenetic file was assembled, over an extended period of time, thanks to the collaboration of staff surgeons and endocrinologists at the University of Cagliari Medical School. Address all correspondence and requests for reprints to: Roberta Vanni, Dipartimento di Scienze e Tecnologie Biomediche, Universita` di Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy. E-mail:
[email protected]. This study was supported by Assessorato Igiene Sanita` Regione Autonoma Sardegna, MIUR (Ministero Universita` Ricerco)-FIRB (Fondo Ricerca di Base) Project no. RBIP0695BB, and Italian Association Cancer Research through a fellowship to D.V.F.
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Disclosure Statement: The authors have nothing to declare.
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