Signet ring cell variant of follicular thyroid carcinoma: Report of two cases with focus on morphological, expressional and genetic characteristics
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Follicular thyroid carcinoma (FTC) is a neoplasm that presents with a micro-follicular growth pattern and a neutrally stained cytoplasm. Seldom, FTCs display unusual morphological characteristics – but given the rarity of these histological subtypes, little is known regarding the underlying genetics and the coupling to patient outcome.
We present two extremely rare cases of minimally invasive FTC with signet ring cell morphology (SRC-FTC) and describe the cytological, microscopic, immunohistochemical and molecular features for both tumors. Both were male patients, age 71 and 51 respectively. The preoperative cytology for both cases could not pinpoint a clear-cut signet ring cell morphology, but a tendency towards nuclear marginalization was seen. The tumors were 38 mm and 22 mm respectively and displayed evident signet ring cell features in subsets of tumor cells as well as degenerative stromal changes. The tumor cells were positive for TTF1, PAX8 and thyroglobulin, and the proliferation indexes were 4% and 1,9% respectively. Both tumors displayed capsular invasion, but not lymphovascular invasion. The tumors were sequenced for mutations in the TERT promoter and 22 additional cancer-related genes, interestingly; one patient was shown to carry a deleterious intronic variant in PTEN, a tumor suppressor gene coupled to thyroid tumorigenesis and Cowden syndrome. Both patients are alive and well awaiting postoperative radioiodine treatment.
The SRC-FTCs described herein were small, TERT promoter wildtype tumors exhibiting low proliferation, thereby suggesting that these exceedingly rare lesions probably carry a favorable prognosis – although the scarce availability regarding descriptions of this tumor entity nevertheless might justify careful clinical monitoring and mandate investigations in larger case series.
KeywordsSignet ring cell Follicular thyroid cancer PTEN
Combined Annotation Dependent Depletion
Deleterious Annotation of Genetic Variants using Neural Networks
Functional Analysis through Hidden Markov Models
Fine needle aspiration
Follicular thyroid adenoma
Follicular thyroid carcinoma
Genome-Wide Annotation of Variants
Noninvasive follicular thyroid neoplasm with papillary-like nuclear features
Papillary thyroid cancer
Single nucleotide polymorphisms
FTC with signet ring cell morphology
Follicular thyroid tumors constitute the most common thyroid neoplasia, and are categorized as follicular adenomas (FTAs), follicular tumors with uncertain malignant potential (FT-UMPs) and follicular carcinomas (FTCs). The FTCs are furthermore split into three different groups based on histological features: minimally invasive FTCs, encapsulated angio-invasive FTCs and widely invasive FTCs . Regularly, follicular thyroid tumors are seen with a neutrally stained cytoplasm, but subsets of tumors present with an eosinophilic cytoplasm (oncocytic cell tumors). More uncommonly encountered histological variants include follicular tumors with clear-cell or signet ring cell (SRC) morphology, two poorly characterized entities based on the scarce literature available [2, 3, 4, 5, 6, 7, 8].
The SRC appearance in tumors is most intimately coupled to gastric carcinoma, but the phenotype is also reported in adenocarcinomas from other primary sites . The morphological hallmark is defined as a vacuolization of the cytoplasm that marginalizes the nucleus. The causes underlying the SRC morphology include interactions between mucin 4 (MUC4) and ErbB2/Her2, leading to a constitutively active stimulation of cell growth as well as mucin vacuolization [10, 11].
In the thyroid gland, SRC features derive from either accumulations of mucin, lipids, glycogen and thyroglobulin, and have been described for secretory carcinoma, papillary thyroid cancer (PTC), noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), FTA and FTC [3, 4, 5, 6, 8]. The description stems from single case reports and small case series with less than 30 reported SRC cases in total - of which FTCs with this phenotype constitute seven cases.
The underlying molecular alterations of these lesions have only been partly dissected. In a previous study of thyroid SRC tumors, single PTEN and FGFR3 mutations were found in an FTA and NIFTP respectively, as well as an ETV6-NTRK3 fusion in a secretory carcinoma of the thyroid .
In this study, two exceedingly rare cases of minimally invasive follicular thyroid carcinomas presenting with SRCs are detailed from cytological, histological and molecular aspects.
The left thyroid lobe measured 70 × 50 × 30 mm and displayed a weight of 50 g. At gross examination, a demarcated and encapsulated lesion measuring 38 × 30 × 36 mm with a tan cut surface, focal hemorrhages and a central cystic clearing of 25 mm was observed (Fig. 1b). Histological examination revealed a circumscribed lesion consisting of sparsely to densely cellular areas against a background of a fibrous and hemorrhagic stroma. The cells were growing in a predominant micro-follicular pattern, and exhibited enlarged, oval to irregularly shaped nuclei with a finely dispersed chromatin and prominent nucleolus. No nuclear features suggestive of papillary thyroid carcinoma were seen. In the majority of cells, the cytoplasm was observed with a balloon-like vacuolized dilation with an accompanied marginalization of the nucleus, corresponding to signet ring cell morphology (Fig. 1c). No mucinous deposits in the surrounding stroma were seen. Tumor necrosis was not present, and the mitotic count was not elevated. Several areas with capsular invasion were noted, but no focus with vascular invasion was observed (Fig. 1d).
Clinical and histopathological charateristics of the two patients with SRC-FTC
Age at surgery
SRC component (%)
Vascular or capsular invasion
Tumor size (mm)
The diagnosis was consistent with a minimally invasive follicular thyroid carcinoma with signet ring cell morphology according to the WHO 2017 classification of endocrine tumors . The tumor was excised with negative margins, and no extrathyroidal extension was seen. The pTNM stage was pT2Nx (Table 1). Cytological re-investigation of the preoperative FNA biopsy material did pinpoint scarce subsets of tumor cells exhibiting a tendency of nuclear marginalization, but noticeably no clear-cut signet ring cell phenotypes.
Genetic findings using the Oncomine panel for both SRC-FTCs
In silico prediction
Chromato-gram allele frequency
Location / transcript
Tumoral expression (IHC)*
c.1505-20C > T
c.955 + 58C > T
c.3451-30C > T
c.209 + 11 T > G
The patient was discussed at a tumor board conference and was planned for a right-sided completion lobectomy and subsequently radioiodine ablation (RAI) with a total dose of 1,1 G-Becquerel (GBq). The right thyroid lobe displayed a physiological C cell hyperplasia, but no tumors.
The histological examination revealed a circumscribed lesion consisting of tumor cells with a principal micro-follicular growth pattern. The tumor cells were seen with small, relatively monomorphic nuclei with a homogenous chromatin and a punctate nucleolus, and a large subset of tumor cells exhibited a signet ring cell phenotype with a prominent vacuolization of the cytoplasmic compartment with a peripheral marginalization of the tumor nuclei (Fig. 2b). Areas with central degeneration consisting of a fibrous and hypocellular stroma were also observed (Fig. 2c). No nuclear features of papillary thyroid carcinoma were seen. No mitoses or areas with tumor necrosis were observed. Several areas with capsular invasion were noted (Fig. 2d). The tumor was devoid of extrathyroidal extension, and surgical margins were negative.
Immunohistochemical analyses confirmed the tumor as thyroid-derived, as the cells were uniformly positive for cytokeratin MNF116, PAX8 and TTF1 (Fig. 2e, Table 1). The majority of the vacuoles stained positive for thyroglobulin, indicating that the distension of the cytoplasmic compartment at least to some extent stems from inappropriate thyroglobulin accumulation (Fig. 2f). PTEN immunohistochemistry was also positive (Fig. 2g). Negative immunoreactivity was seen for chromogranin A, calcitonin, MUC2, MUC4, MUC5AC, MUC6 and CDX2. The Ki-67 index was 1.9%. The cells were also negative when assessed with a periodic-acid-schiff (PAS) stain.
The final diagnosis was a minimally invasive follicular thyroid carcinoma with signet ring cell morphology, pT2Nx. Other areas of the thyroid lobe exhibited features of Hashimoto’s thyroiditis.
Two variants were detected in somatic DNA: an unusual intronic SNP in ALK (rs745932805) as well as a previously unreported intronic variant in PTEN (Table 2, Fig. 2h). The PTEN variant (chr10: 87925568 T > G, c.209 + 11 T > G) is located in intron 3 of the main PTEN transcript, a region in which similar alterations has been judged as pathogenic and coupled to the skipping of exons 3 and/or 4 in subsets of Cowden syndrome patients . Sanger sequencing analyses of histologically normal thyroid DNA from FFPE material as well as leukocyte DNA from blood preoperatively drawn from the patient confirmed the variant as constitutional. Since the variant is not listed in the SNP repositories (dbSNP, 1000 Genomes, and the Exome Variant Server), we consider this a PTEN intronic mutation. Further analysis with a panel of in silico prediction softwares verified the mutation as deleterious (Table 2). Using Mutation Taster, the mutation was not presumed to abrogate any potential splice sites, but the nucleotide change was predicted to create new transcription factor binding sites for 33 transcription factors (TFs) and disrupt the assumed binding site for 13 additional TFs (Additional file 1: Table S1). Several TFs with an altered binding site display a known coupling to thyroid tumorigenesis, including CREB1, FOXA1, YY1 and RXRA to name a few [13, 14, 15, 16]. As the auxiliary immunohistochemical analysis of the patient’s FTC displayed uniform PTEN immunoreactivity in the tumor cells, this indicates that the overall expressional level of PTEN is retained.
The tumor was wildtype for both positions C228 and C250 of the TERT promoter, as mutations at these sites have been noted as a reliable prognostic indicator of future recurrences in follicular thyroid carcinoma [17, 18]. Moreover, using real-time PCR, no ETV6-NTRK3 fusion transcripts were observed (data not shown).
In the ensuing tumor board conference, the patient was planned for a left-sided completion lobectomy and radioiodine ablation (RAI) with 1,1 G-Becquerel (GBq). The left thyroid lobe displayed features of Hashimoto’s thyroiditis and a cholesterol granuloma, but no tumors.
For both cases, the immunohistochemical stainings were performed in an accredited pathology laboratory using a Ventana Benchmark Ultra system (Ventana Medical Systems, Tucson, AZ, USA). All stainings were assessed by conventional light microscopy by experienced endocrine pathologists (AH and CCJ).
The Oncomine Solid Tumor Panel (Ion Torrent S5, Hi-Q Chef; Thermo Scientific) was used to screen DNA extracted from formalin-fixated paraffin-embedded (FFPE) tissues for > 1800 cancer related mutations within the following genes: EGFR, KRAS, NRAS, PIK3CA, BRAF. ALK, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, MET, DDR2, AKT1, PTEN, MAP 2 K1, STK11, NOTCH1, CTNNB1, SMAD4, FBXW7 and TP53. The input DNA required was 10 ng.
For TERT, bi-directional Sanger sequencing of the promoter region interrogating the two mutational hotspots C228T and C250T was performed using the Genetic Analyzer 3500, Applied Biosystems, CA, USA  and a conventional protocol in a clinically accredited setting.
For the ETV6-NTRK3 fusion analyses, RNA was extracted from the FFPE material using the Maxwell®16 MX3031 instrument with Maxwell®16 FFPE LEV RNA Purification Kit from Promega (Fitchburg, WI, USA). The quality and quantity of the RNA was estimated through Nanodrop technology (Nanodrop technologies, Wilmington, DE, USA). cDNA was subsequently synthesized using the High-Capacity cDNA reverse transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR (TaqMan) was performed in duplicate (ABI, SDS 7000 Applied Biosystems, Foster City, CA, USA) targeting the ETV6-NTRK3 fusion transcript corresponding to the chromosomal translocation t (12;15)(p13;q25).
Single nucleotide variants/polymorphisms for all cases were evaluated through the Predict SNP2 platform, in which the variant is classified as either “neutral”, “deleterious” or “unknown” using six different integrated prediction tools: Predict SNP2, Combined Annotation Dependent Depletion (CADD), Deleterious Annotation of Genetic Variants using Neural Networks (DANN), Functional Analysis through Hidden Markov Models (FATHMM), FunSeq2 and Genome-Wide Annotation of Variants (GWAVA) . Extended analyzes for the PTEN variant were performed using the Mutation Taster function .
Discussion and conclusions
Rare morphological variants of thyroid cancer are important to characterize from both a clinical and molecular perspective, so that an eventual coupling to underlying genetics and patient prognosis can be made. This is exemplified by the association between specific histological subtypes in papillary thyroid carcinoma and prognostic features . Although the current WHO classification of endocrine tumors recognizes the SRC subtype of FTC, no information regarding the underlying genetic events and prognosis is presented – making it hard for clinicians to recognize if the SCR-FTC diagnosis should imply specific considerations in the clinical management of the individual patient.
SRC-FTC is an extremely rare entity – and drawing conclusions based on the scarce amount of cases presented in the scientific literature is challenging. Even so, a few similarities between our two cases and previously published thyroid tumors with SRC features were observed. Firstly, it seems as not all preoperative FNA biopsies are able to pinpoint the SRC morphology, including our two cases [5, 6]. Instead, the pinpointing of the SRC features was only evident after histopathological examination. This is possibly due to the fact that the SRC morphology was only observed in approximately half of the tumor cell population (Table 1), which mirrors previous findings in which SRC cells only lined parts of the follicles . Indeed, only after reinvestigation of the cytology specimen, a tendency of marginalization of the nuclei was seen for a subset of tumor cells. Moreover, the cell-sparse, degenerative stromal features seen in both cases might also complicate the analysis. On histological investigation, the distended cytoplasm was positive for thyroglobulin for both cases, indicating aberrant accumulation of this protein as part of the underlying cause of the signet ring phenotype. Secondly, both our cases displayed stromal degeneration, with central areas of the tumor presenting with a dense fibrosis, sparsely populated by stromal cells and few tumor cells. Findings of a desmoplastic tumoral stroma have been previously reported in SRC tumors of the thyroid, indicating this phenomenon as a general part of the tumoral phenotype .
We found a previously unreported, germline mutation in the intron 3 of the tumor suppressor gene PTEN in case 2. Mutations in this intronic region has previously been coupled to Cowden syndrome, as a result of aberrant splicing and exon skipping . Our variant is not thought to disrupt a splice site, but still predicted to be deleterious by in silico analyses. The retained tumoral PTEN immunoreactivity partly argues against an inactivating genetic event, but since the mutation was intronic, the true pathogenicity of this mutation could remain unnoticed by immunohistochemistry. The patient has no previous family history indicative of Cowden syndrome-related tumors, suggesting that the variant might not cause this disease phenotype, alternatively occurring de novo or with a reduced penetrance. DNA from additional family members was not available. Interestingly, an FTA with SRC features harboring a deleterious PTEN missense mutation has previously been reported in a case series of four SRC-FTAs . Therefore, it seems likely that PTEN mutations could constitute a molecular aberrancy underlying the development of subsets of SRC thyroid tumors.
Regarding prognostication of SRC-FTC, little is known. A previous case of SRC-FTC with capsular and angio-invasive properties displayed a 12-month disease-free survival . In our study, both SCR-FTCs displayed capsular invasion, but no areas with lymphovascular invasion were observed – representing a minimally invasive subtype. Moreover, the Ki-67 proliferation indexes were fairly low and no TERT promoter mutations were detected. In all, these factors might indicate a low risk of future recurrences – but careful monitoring is nevertheless justified given the lack of scientific literature covering this very infrequent histological subtype.
We conclude that SRC-FTCs are exceedingly rare, with subsets of cases displaying mutations in genes involved in thyroid tumorigenesis. The tumors may elude correct characterization on preoperative cytological examination based on the tumoral blend of SCR cells and conventional tumor cells. Although based on few cases, the prognosis is believed to be favorable, or at least on par with conventional FTCs.
The authors are indebted to Ms. Lisa Ånfalk for her excellent technical assistance.
MH, AH and CCJ reviewed the histology, KJ and AS performed the molecular genetic analyses, EDR performed cytological investigations, JZ reviewed the clinical information and patient follow-up and CCJ performed the in silico analyses. MH and CCJ wrote the paper, and all authors read and approved the final manuscript.
The study was financially supported by grants generously provided by the Swedish Cancer Society and the Swedish Society for Medical Research.
Ethics approval and consent to participate
Ethical approval has been acquired by the local ethical committee, and informed consent has been obtained from the individual patients.
Consent for publication
Both patients have given consent for publication.
The authors declare that they have no competing interests.
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