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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
Address all correspondence and requests for reprints to: Roberta Vanni, Dipartimento di Scienze e Tecnologie Biomediche, Università di Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy. E-mail: vanni{at}unica.it.
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Abstract |
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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-PPAR1 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-PPAR1 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 follicular-patterned thyroid nodules and may be a marker for this subset of thyroid lesions that are often difficult to classify.
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Introduction |
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1 (6), secondary to a t(2;3)(q13;p25), is found in 50% of FTC and 8% follicular thyroid 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 (PTC-NC), 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.
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Materials and Methods |
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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-PPAR1 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-PPAR1 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 mean +3 x 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-PAPR1 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.
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Results |
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, and those of the control cases in supplemental Table 1.
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, 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.
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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-PAPR1 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 (Table 1, 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 uncertain 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 PTC-NCs, 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.
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Discussion |
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1 gene fusions, RAS and BRAFK601E mutations have all been reported (8, 20), with RAS mutations and/or PAX8-PAPR1 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-PPAR1 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 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-PPAR1) carcinoma differentiates our cases with trisomy 17 from other tumors, including the PTC-FV with RAS mutations and/or PAX8-PPAR1 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.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online October 23, 2007
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.
Received April 30, 2007.
Accepted October 15, 2007.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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fusion oncogene in both follicular thyroid carcinomas and adenomas. J Clin Endocrinol Metab 88:354–357 rearrangement is frequently detected in the follicular variant of papillary thyroid carcinoma. J Clin Endocrinol Metab 91:213–220 fusion mRNA? Endocr J 51:361–366[CrossRef][Medline]This article has been cited by other articles:
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  S Nemolato, T Dettori, P Caria, D V Frau, G Faa, and R Vanni Would a morphomolecular approach help in defining pseudosarcomatous myofibroblastic proliferations? A study of a heart polypoid lesion J. Clin. Pathol., April 1, 2009; 62(4): 377 - 379. [Abstract] [Full Text] [PDF]
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 E. K. Alexander Approach to the Patient with a Cytologically Indeterminate Thyroid Nodule J. Clin. Endocrinol. Metab., November 1, 2008; 93(11): 4175 - 4182. [Abstract] [Full Text] [PDF]
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