5hmC Immunohistochemistry: A Predictor of TERT Promoter Mutational Status in Follicular Thyroid Carcinoma?

Telomerase reverse transcriptase (TERT) gene aberrancies correlate to adverse prognosis in follicular thyroid carcinoma (FTC). As loss of 5-hydroxymethylcytosine (5hmC) has been associated with TERT promoter mutations in papillary thyroid carcinoma, this study sought to analyze the levels of 5hmC in a cohort of follicular thyroid tumors with available TERT data. A total of 29 tumors (26 FTCs, 2 follicular thyroid tumors of uncertain malignant potential, and 1 oncocytic thyroid carcinoma) with known TERT promoter mutational status and TERT gene expression were assessed for 5hmC immunoreactivity using two antibodies (clones RM236 and 4D9.) Slides were analyzed using a semiquantitative scoring system. Of the 10 tumor cases with aberrant TERT, only 1 scored negative with both antibodies (1/10; 10%), whereas the remaining 9 cases (9/10; 90%) exhibited some positivity for at least one antibody. Of the 19 TERT wild-type tumors, no case was scored negative using RM236, and 2 cases (2/19; 11%) using 4D9. The differences between TERT promoter mutated and wild-type groups were non-significant. The sensitivity and specificity for 5hmC immunohistochemistry (IHC) to detect mutated cases were 10% and 100% (RM236) and 20% and 89% (4D9). Therefore, 5hmC IHC is not a sensitive marker for detecting TERT promoter mutations in follicular thyroid tumors.

Digital droplet PCR TERT promoter mutational screening in fine needle aspiration cytology of thyroid lesions: A highly specific technique for pre-operative identification of high-risk cases.

BACKGROUND: Despite the advent of comprehensive molecular testing in surgical pathology, most centers still rely on the morphological assessment of fine-needle aspiration cytology (FNAC) to triage patients with thyroid nodules for surgery. Subsets of patients could benefit from the inclusion of molecular testing to increase the diagnostic and/or prognostic properties of the cytology analysis, including the assessment of TERT promoter mutations, an event coupled with thyroid malignancy, and poor prognosis. METHODS: In this prospective study, preoperative FNAC material from 65 cases was assessed for TERT promoter hotspot mutations C228T and C250T using the digital droplet PCR (ddPCR) technique on frozen pellets and re-evaluated postoperatively. RESULTS: Our cohort consisted of 15 B-III (23%), 26 B-IV (40%), 1 B-V (2%), and 23 (35%) B-VI lesions according to the Bethesda System for Reporting Thyroid Cytopathology. TERT promoter mutations were detected in 7 cases; 4 papillary thyroid carcinomas (all with preoperative B-VI status), two follicular thyroid carcinomas (one B-IV and one B-V status), and one poorly differentiated thyroid carcinoma (with B-VI status). All mutated cases were verified by mutational analysis of tumor tissue derived from postoperative formalin-fixed paraffin-embedded tissue, while all cases identified as wild-type on FNAC remained wild-type postoperatively. Moreover, the occurrence of a TERT promoter mutation was significantly associated with malignant disease and higher Ki-67 proliferation indices. CONCLUSION: In the present cohort, we found that ddPCR is a highly specific method for detecting high-risk TERT promoter mutations on thyroid FNAC material that could guide different surgical approaches in subsets of indeterminate lesions if reproduced in larger materials.

Cyto-morphological features of parathyroid lesions: Fine-needle aspiration cytology series from an endocrine tumor referral center.

BACKGROUND: Descriptions of parathyroid cell cyto-morphology are limited. Fine-needle aspiration cytology (FNAC) with immunocytochemistry (ICC) or biochemical PTH measurements may help verify the parathyroid origin in extraordinary cases, although these methods are nowadays largely replaced by imaging techniques. METHODS: We collected all available FNAC reports of parathyroid lesions from our department spanning 20 years, to characterize the clinical use of parathyroid FNAC, and to assess morphological correlates between cytology and subsequent histopathology. RESULTS: Twenty-eight cases with assessable cytological smears were found, of which 21 cases were surgically resected and 20 available for histological review (15 adenomas, 2 carcinomas, 2 atypical tumors and a single case with secondary hyperplasia). FNAC was predominantly performed to localize the diseased gland in cases with inconclusive imaging, in cases with a suspicion of intrathyroidal localization or in cases with persistent hypercalcemia following unsuccessful surgery. The diagnosis was verified using either PTH ICC and/or PTH measurements for most cases, and the procedure affected the clinical decision-making for the majority of patients in this selected cohort. Cytological differences between parathyroid adenomas and carcinomas were found, as only carcinomas showed pleomorphism with irregular nuclei and prominent nucleoli. Morphologically, no correlations to predominant cell types or growth patterns visualized at histological investigations among adenomas were noted, and biopsy artifacts were evident in 40% of cases. CONCLUSIONS: Parathyroid FNAC could be considered as a complementary analysis for a small group of selected patients, but benefits have to be weighed against the risk of biopsy artifacts in histological preparations.

Nuclear-specific accumulation of telomerase reverse transcriptase (TERT) mRNA in TERT promoter mutated follicular thyroid tumours visualised by in situ hybridisation: a possible clinical screening tool?

AIMS: Upregulation of the telomerase reverse transcriptase (TERT) gene is a frequent finding in follicular thyroid carcinomas (FTCs) with metastatic features. The augmented expression is usually caused by TERT promoter mutations. As TERT protein immunohistochemistry might not correlate to TERT mRNA levels in follicular thyroid tumours, we therefore sought to determine if visualisation of TERT mRNA through in situ hybridisation could highlight high-risk cases. METHODS: We collected formalin-fixated paraffin-embedded tissues from 26 follicular thyroid tumours; 7 FTCs, 2 follicular thyroid tumours of uncertain malignant potential (FT-UMPs) and a single Hürthle cell carcinoma with established TERT promoter mutations and gene expression, as well as 16 FTCs with no TERT gene aberrancy or gene expression, and assessed them using RNA Scope in situ hybridisation (ISH) and TERT probes targeting the two main TERT transcripts (TERT1 and TERT2). RESULTS: TERT 1 and/or 2 mRNA was found by ISH in 8/10 cases with established promoter mutations and mRNA expression, whereas all 16 cases without TERT gene aberrancies or gene expression were negative (Fisher's exact p<0.001). Strikingly, TERT mRNA was visualised in the nuclear compartment only, thereby corroborating earlier studies suggesting a non-conventional role for TERT in tumour biology. Moreover, TERT mRNA expression was scattered across the tissue sections and only found in a few percentages of tumour nuclei. CONCLUSIONS: TERT mRNA seems to be focally expressed and localised exclusively to the nucleus in TERT promoter mutated follicular thyroid tumours, possibly reflecting a true biological and unorthodox phenomenon worthy of further investigations.

Macrofollicular Variant of Follicular Thyroid Carcinoma (MV-FTC) with a Somatic DICER1 Gene Mutation: Case Report and Review of the Literature.

Benign thyroid lesions such as multinodular goiter and adenomatoid nodules are well-circumscribed lesions displaying a macrofollicular growth pattern and lack of nuclear atypia. The highly unusual macrofollicular variant of follicular thyroid carcinoma (MV-FTC) mirrors these attributes and is thereby misclassified by cytological examination of fine-needle aspiration biopsies. The MV-FTC diagnosis is instead suggested following histological investigation, in which malignant attributes, most commonly capsular invasion, are noted. The bulk of MV-FTCs described in the literature arise in younger female patients and carry an excellent prognosis. A recent coupling to mutations in the DICER1 tumor suppressor gene has been proposed, possibly indicating aberrancies in micro-RNA (miRNA) patterns as responsible of the tumorigenic process. We describe the cytological, histological and molecular phenotype of a 35 mm large MV-FTC arising in the right thyroid lobe of a 33-year-old female with a family history of multinodular goiter. The tumor was encapsulated and strikingly inconspicuous in terms of cellularity and atypia, but nevertheless displayed multiple foci with capsular invasion. A next-generation molecular screening of tumor DNA revealed missense variants in DICER1 (p. D1709N) and MET (p. T1010I), but no established fusion gene events. After sequencing of germline DNA, the DICER1 mutation was confirmed as somatic, while the MET variant was constitutional. The patient is alive and well, currently awaiting radioiodine treatment. This MV-FTC mirrors previous publications, suggesting that these tumors carry a favorable prognosis and predominantly arise in younger females. Moreover, DICER1 mutations should be considered a common driver event in the development of MV-FTCs.

Spatial Distribution Patterns of Clinically Relevant TERT Promoter Mutations in Follicular Thyroid Tumors of Uncertain Malignant Potential: Advantages of the Digital Droplet PCR Technique.

In thyroid carcinomas, telomerase reverse transcriptase (TERT) promoter mutations C228T and C250T predict an unfavorable clinical outcome. The analysis is particularly valuable when assessing histologically equivocal follicular thyroid tumors of uncertain malignant potential (FT-UMPs). Given recent findings of TERT promoter mutational heterogeneity in thyroid cancer, we determined the frequency of this phenomenon in FT-UMPs and minimally invasive follicular thyroid carcinomas. DNA was extracted from several tissue blocks from 16 FT-UMPs as well as 10 minimally invasive follicular thyroid carcinomas, and interrogated using Sanger sequencing as well as digital droplet PCR (ddPCR). Mutational heterogeneity was observed by Sanger sequencing in four of seven (57%) FT-UMPs. In two FT-UMPs with C228T mutations, analyses of additional blocks gave wild-type results using Sanger sequencing in one or several blocks interrogated, whereas ddPCR found low-frequency C228T mutations in one of these fractions. In two additional FT-UMPs with the C228T and C250T mutation, respectively, sequencing of additional blocks revealed the opposite mutation. Moreover, in the C250T mutated area in one of these tumors, the ddPCR displayed a co-occurring C228T mutation that failed detection through Sanger sequencing. To conclude, most TERT promoter mutated FT-UMPs display mutational heterogeneity when analyzed by Sanger sequencing, thereby emphasizing the importance of the tissue sampling process. The ddPCR technique might overcome this phenomenon because of increased sensitivity and should be considered for clinical screening purposes.

Metastatic Anaplastic Thyroid Carcinoma in Complete Remission: Morphological, Molecular, and Clinical Work-Up of a Rare Case.

Anaplastic thyroid carcinoma (ATC) exhibits an exceedingly poor prognosis, and the current treatment options are, for most cases, palliative by nature. Few reports of long-time survivors exist, although in these patients, tumors often were limited to the thyroid and/or regional lymph nodes. We describe a 64-year-old male who developed a rapidly growing mass in the left thyroid lobe. A fine-needle aspiration biopsy (FNAB) was consistent with ATC, and the patient underwent preoperative combined chemo- and radiotherapy followed by a hemithyroidectomy. The ensuing histopathological investigation was consistent with ATC adjoined by an oxyphilic well-differentiated lesion, likely a Hürthle cell carcinoma. Tumor margins were negative, and no extrathyroidal extension was noted. Focused next-generation sequencing analysis of the primary tumor tissue identified a TP53 gene mutation but could not identify any potential druggable targets. Additional Sanger sequencing detected a C228T TERT promoter mutation. The tumor was found to be microsatellite stable and displayed PDL1 expression in 80% of tumor cells. Following a CT scan 1 month postoperatively, metastatic deposits were suspected in the lung as well as in the left adrenal gland, of which FNAB verified the latter. Remarkably, upon radiological follow-up, the disease had gone into apparent complete remission. The patient is alive and well with no signs of residual disease after 12 months of follow-up. We here summarize the clinical, histological, and molecular data of this highly interesting patient case and review the literature for possible common denominators with other patients with disseminated ATC.

Signet ring cell variant of follicular thyroid carcinoma: Report of two cases with focus on morphological, expressional and genetic characteristics.

BACKGROUND: 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. CASE PRESENTATION: 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. CONCLUSIONS: 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.

Clinical Routine TERT Promoter Mutational Screening of Follicular Thyroid Tumors of Uncertain Malignant Potential (FT-UMPs): A Useful Predictor of Metastatic Disease.

Mutations of the Telomerase reverse transcriptase (TERT) gene promoter are recurrently found in follicular thyroid carcinoma (FTC) and follicular tumors of uncertain malignant potential (FT-UMP), but nearly never in follicular thyroid adenoma (FTA). We, therefore, believe these mutations could signify malignant potential. At our department, postoperative TERT promoter mutational testing of FT-UMPs was implemented in 2014, with a positive mutation screening leading to vigilant follow-up and sometimes adjuvant treatment. To date, we screened 51 FT-UMPs and compared outcomes to 40 minimally invasive FTCs (miFTCs) with known TERT genotypes. Eight FT-UMPs (16%) displayed TERT promoter mutations, of which four cases underwent a completion lobectomy at the discretion of the patient, and a single patient also opted in for radioiodine (RAI) treatment. Three mutation-positive patients developed distant metastases, registered in one patient receiving a completion lobectomy and in two patients with no additional treatment. Three out of four patients who received additional surgery, including the RAI-treated patient, are still without metastatic disease. We conclude that FT-UMPs with TERT promoter mutations harbor malignant potential and exhibit at least similar recurrence rates to TERT-promoter-mutated miFTCs. Mutational screening should constitute a cornerstone analysis in the histopathological work-up of FT-UMPs.

TERT Promoter Mutation Spatial Heterogeneity in a Metastatic Follicular Thyroid Carcinoma: Implications for Clinical Work-Up.

Follicular thyroid carcinoma (FTC) is not routinely diagnosed by a preoperative fine needle aspiration biopsy (FNAB), and the final diagnosis relies on histopathological criteria visible upon microscopic examination of the excised tumor. Several markers have been proposed as helpful in the identification of follicular thyroid tumors with malignant potential and worse prognosis, of which the specific point mutations C250T and C228T in the Telomerase Reverse Transcriptase (TERT) promoter region seem to be particularly promising. We describe a patient presenting with a large pelvic mass, in which a core needle biopsy was consistent with follicular-patterned thyroid tissue positive for a Q61R NRAS mutation and the C228T TERT promoter mutation. Upon clinical investigation, a 60-mm lesion was detected in the right thyroid lobe. The ensuing FNAB was consistent with a follicular thyroid tumor, Bethesda IV, positive for the same NRAS mutation and both the C228T and C250T TERT promoter mutations. A total thyroidectomy was performed, and a widely invasive FTC was diagnosed. Tumor tissue samples from various parts of the primary lesion were investigated for TERT promoter mutations, displaying C228T in three samples and C250T in one. Interestingly, the C228T mutations showed a coupling to areas with high Ki-67 proliferation indexes. Our data indicate that TERT promoter mutations can exhibit spatial heterogeneity in FTCs, with implications for clinical management as well as providing insights into the molecular biology underlying the tumoral etiology.

TERT promoter mutational screening as a tool to predict malignant behaviour in follicular thyroid tumours-three examples from the clinical routine.

Follicular thyroid adenomas (FTAs) and carcinomas (FTCs), collectively the most common thyroid neoplasms, constitute a significant clinical challenge since histological evidence of invasive behaviour is required for a malignant diagnosis. Small subsets of FTAs relapse as manifest malignant FTCs, indicating that histology is not always adequate to predict malignant potential. Lately, recurrent mutations in the promoter of the Telomerase reverse transcriptase (TERT) gene have been coupled to FTCs, whereas FTAs usually lack this aberrancy. We describe three patients with follicular thyroid tumours in which TERT promoter mutational screening was employed as part of the clinical work-up to pinpoint malignant potential. In two retrospective analyses of seemingly benign lesions, the detected mutations predicted future skeletal metastases, and in one prospective case, the mutational screening led to a different clinical management of the afflicted patient. We therefore consider TERT promoter mutational screening an adjunct tool of value in equivocal cases.

Quality improvement in preoperative assessment by implementation of an electronic decision support tool.

OBJECTIVES: To evaluate the impact of the electronic decision support (eDS) tool 'PReOPerative evaluation' (PROP) on guideline adherence in preoperative assessment in statutory health care in Salzburg, Austria. MATERIALS AND METHODS: The evaluation was designed as a non-randomized controlled trial with a historical control group (CG). In 2007, we consecutively recruited 1363 patients admitted for elective surgery, and evaluated the preoperative assessment. In 2008, PROP was implemented and available online. In 2009 we recruited 1148 patients preoperatively assessed using PROP (294 outpatients, 854 hospital sector). Our analysis includes full blood count, liver function tests, coagulation parameters, electrolytes, ECG, and chest x-ray. RESULTS: The number of tests/patient without indication was 3.39 in the CG vs 0.60 in the intervention group (IG) (p<0.001). 97.8% (CG) vs 31.5% (IG) received at least one unnecessary test. However, we also observed an increase in recommended tests not performed/patient (0.05±0.27 (CG) vs 0.55±1.00 (IG), p<0.001). 4.2% (CG) vs 30.1% (IG) missed at least one necessary test. The guideline adherence (correctly tested/not tested) improved distinctively for all tests (1.6% (CG) vs 49.3% (IG), p<0.001). DISCUSSION: PROP reduced the number of unnecessary tests/patient by 2.79 which implied a reduction of patients' burden, and a relevant cut in unnecessary costs. However, the advantage in specificity caused an increase in the number of patients incorrectly not tested. Further research is required regarding the impact of PROP on perioperative outcomes.