Thyroid Cancer

Diagnosis

Histopathology and characteristics of NTRK+ tumours

One study of 351 cases of thyroid cancer identified NTRK gene fusions in 11 cases, all of which were PTC [14]. The main histopathological characteristics of these NTRK-positive tumours were:

• Multiple infiltrative tumour nodules and extensive lymphovascular spread within intrathyroidal and extrathyroidal vessels of variable calibre (n=11). Direct extrathyroidal extension, at least microscopic (n=9).
• Similar cellular architectural properties in cases with the same gene fusion
  • Predominantly follicular pattern associated with ETV6-NTRK3 (n=4) and RBPMS-NTRK3 (n=2) fusions. Multiple irregularly shaped tumour nodules infiltrating the thyroid.
  • Predominantly insular pattern associated with SQSTM1-NTRK3 fusion (n=1). Scattered microfollicles and increased mitotic activity. Multifocal features of PTC.
  • Predominantly papillary pattern associated with TPR-NTRK1 (n=2), and SQSTM1-NTRK1 fusions, displaying scattered glomeruloid structures. The former also showing multiple nodules of packeted papillae divided by fibrotic septa and  the latter numerous psammomatous calcifications.
  • Immunoreactivity, in 10 out of 11 cases, for all TTF1, PAX8, thyroglobulin, and HBME1. The other case only expressed low levels of TTF1 and there was diffuse immunopositivity for S100, mammaglobin, and GATA3. This latter case was initially diagnosed as PTC but PTC features such as nuclear crowding and clearing were lacking; hence, the final diagnosis was primary thyroid secretory carcinoma of the salivary type.

Along similar lines a study of a cohort of 525 consecutive PTC cases, identified three useful morphologic features, including non-infiltrative tumor border, clear cell change, and reduced nuclear elongation and irregularity, for the diagnosis of NTRK1/3-rearranged PTC [15]. A reduced nuclear elongation and irregularity of tumor cells as a novel pathological feature for NTRK1/3-rearranged PTC in contrast to the oval-shaped nuclei in the classical PTC, was also described.

Another study of 27 cases of paediatric PTC identified NTRK1/3 fusions in 7 cases, with the ETV6-NTRK3 fusion being the most prevalent (n=5). All NTRK-positive PTCs had a nodular, infiltrative architecture and included 3 tumours of the follicular type, 3 of the solid type, and 1 classic type PTC. All PTC types were represented among ETV6-NTRK3-positive tumours [2].

In cases of radiation-induced PTC, a study showed that most ETV6-NTRK3 fusion-positive tumours have a follicular growth pattern, while the remaining have a significant papillary component and are classified as classic papillary PTC [7].

It should be noted however that several reports have confirmed that from a cytological point of view, NTRK-rearranged thyroid carcinomas do not show unique features [16, 17, 18]

Current testing algorithms

Methods used in various studies to assess the presence of NTRK fusions in thyroid cancer include pan-TRK IHC, NGS (whole-genome sequencing and RNA-sequencing), targeted NGS (DNA- and RNA-based), and FISH. The use of the ThyroSeq v2 NGS assay was shown to detect additional molecular alterations in paediatric cases compared with a limited gene panel [19]. In a study including around 34,000 cancer cases (including thyroid carcinomas), MSK-IMPACT, a DNA-based NGS method, has shown a sensitivity of 81.1% (60/74) and specificity of 99.9% (33,877/33,923). The same study found a sensitivity of 81.8% (9/11) and a specificity of 100% (27/27) for pan-TRK IHC in 571 thyroid cancer cases of which 13 had NTRK fusions [9]. However, TRK IHC it is not acceptable as a stand-alone technique, and results should be confirmed by sequencing.

For neoplasms with a low-to-intermediate and intermediate frequency of NTRK fusions, such as thyroid cancers, it is suggested that NGS for biomarker testing would be sufficient if the overall screening approach includes NGS [20]; however, it should be noted that a solely DNA-based approach may miss up to 20% of NTRK fusions [9]. If the overall approach to biomarker testing does not include NGS, then testing with RNA-based NGS would be optimal. FISH may also be acceptable. Nevertheless, despite the faster turnaround time and lower cost of this technique, FISH may miss up to 20% of NTRK fusions [20].

Since NTRK fusions have been associated with higher disease stage [2, 14] and radiation-induced thyroid tumours [5,7], it is recommended to screen such cases first by pan-TRK IHC. The ETV6-NTRK3 fusion shows characteristic cytoplasmic and nuclear positivity by IHC; in contrast, variable non-nuclear staining patterns are observed for other NTRK1 and NTRK3 fusions. Thus, heterogenous IHC staining should be expected in resection specimens [21].

At the time of diagnosis, DTC is frequently more advanced in pre-pubertal children when compared to adolescents and adults, although the long-term prognosis is highly favourable for both children and adolescents [22]. For paediatric patients with advanced DTC in the pre-operative setting (invasive cervical disease, bulky lymphadenopathy, or evidence of metastasis to the lungs, bones, or other distal sites), the Canadian Consensus recommends preserving snap-frozen surgical specimens for subsequent molecular analysis. The proposed molecular technique is NGS (either whole-transcriptome sequencing or targeted RNA-based panels) in all patients with unresectable or progressive and/or symptomatic distal disease who are unresponsive to standard surgery and radioactive iodine. This testing should include targetable oncogenic drivers including: NTRK1-3 gene fusions, BRAF mutations, RET gene fusions, ALK gene fusions, and MET overexpression/fusion [22]. These recommendations are in line with the algorithm for paediatric patients suggested by the Singapore Task Force [23].

Recently the use of the mRNA profile of ten genes was reported that can classify thyroid cancer in relation to the presence of driver NTRK-chimeric TRK genes with acceptable sensitivity and specificity [24]. RT-PCR was used to identify samples of papillary thyroid cancer carrying a EVT6-NTRK3 rearrangement (7/215, 3.26%) and subsequently machine learning applied to data extracted from TCGA identified a recognition function for predicting the presence of rearrangement in NTRK genes based on the expression of AUTS2, DTNA, ERBB4, HDAC1, IGF1, KDR, NTRK1, PASK, PPP2R5B and PRSS1.

Challenges

The genetic landscape of NTRK-rearranged thyroid carcinomas is characterised by multiple fusion combinations and otherwise low mutation burden [14]. Hence, DNA/RNA gene panels capable of detecting point mutations and the increasing number of gene fusions identified in paediatric patients are essential. A major caveat of these techniques is that amplifiable DNA/RNA samples are not always available. Especially if samples are obtained from formalin-fixed paraffin-embedded tumour blocks, the quality is usually poor. Although DNA-based NGS panels may detect multiple oncogenic genomic events from one sample, not all DNA-based NGS platforms can identify all NTRK gene fusions, especially those involving NTRK2 and NTRK3 where detection of gene fusions is complicated by the presence of large introns that are typically inadequately sequenced and difficult to analyse [25]

References

1. Ferlay J, Ervik M, Lam F et al. Global Cancer Observatory: Cancer Today. Lyon: International Agency for Research on Cancer; 2020 (https://gco.iarc.fr/today/fact-sheets-cancers)
2. Prasad M.L., Vyas M., Horne M.J et al. NTRK Fusion Oncogenes in Pediatric Papillary Thyroid Carcinoma in Northeast United States. Cancer. 2016; 122:1097–1107.
3. Rossi ED, Pantanowitz L, Hornick JL. A worldwide journey of thyroid cancer incidence centred on tumour histology. Lancet Diabetes Endocrinol. 2021;9(4):193-194.
4. Bongarzone I, Pierotti MA, Monzini N et al. High frequency of activation of tyrosine kinase oncogenes in human papillary thyroid carcinoma. Oncogene. 1989;4(12):1457-1462.
5. Ricarte-Filho JC, Li S, Garcia-Rendueles ME et al. Identification of kinase fusion oncogenes in post-Chernobyl radiation-induced thyroid cancers. J Clin Invest. 2013;123(11):4935-4944.
6. Kong Y, Bu R, Parvathareddy SK et al. NTRK fusion analysis reveals enrichment in Middle Eastern BRAF wild-type PTC. Eur J Endocrinol. 2021;184(4):503-511.
7. Leeman-Neill RJ, Kelly LM, Liu P et al. ETV6-NTRK3 is a common chromosomal rearrangement in radiation-associated thyroid cancer. Cancer 2014;120(6):799-807.
8. Rosen EY, Goldman DA, Hechtman JF et al. TRK Fusions are enriched in cancers with uncommon histologies and the absence of canonical driver Mutations. Clin Cancer Res. 2020;26(7):1624-1632.
9. Solomon JP, Linkov I, Rosado A et al. NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls. Mod Pathol. 2020;33(1):38-46.
10. Okamura R, Boichard A, Kato S et al. Analysis of NTRK Alterations in pan-cancer adult and pediatric malignancies: implications for NTRK-targeted therapeutics. JCO Precis Oncol. 2018;2018:PO.18.00183.
11. Gatalica Z, Xiu J, Swensen J, Vranic S. Molecular characterization of cancers with NTRK gene fusions. Mod Pathol. 2019;32(1):147-153.
12. Wajjwalku W, Nakamura S, Hasegawa Y et al. Low frequency of rearrangements of the ret and trk proto-oncogenes in Japanese thyroid papillary carcinomas. Jpn J Cancer Res. 1992;83(7):671-675.
13. Said S, Schlumberger M, Suarez HG. Oncogenes and anti-oncogenes in human epithelial thyroid tumors. J Endocrinol Invest. 1994;17(5):371-379.
14. Chu YH, Dias-Santagata D, Farahani AA et al. Clinicopathologic and molecular characterization of NTRK-rearranged thyroid carcinoma (NTRC). Mod Pathol. 2020;33(11):2186-2197.
15. Lee YC, Chen JY, Huang CJ et al. Detection of NTRK1/3 Rearrangements in Papillary Thyroid Carcinoma Using Immunohistochemistry, Fluorescent In Situ Hybridization, and Next-Generation Sequencing. Endocr.Pathol. 2020;31(4):348-358.
16. Viswanathan K, Chu YH, Faquin WC, Sadow PM. Cytomorphologic features of NTRK-rearranged thyroid carcinoma. Cancer Cytopathol. 2020;128(11):812-827.
17. Lee YC, Hsu CY, Lai CR, Hang JF. NTRK-rearranged papillary thyroid carcinoma demonstrates frequent subtle nuclear features and indeterminate cytologic diagnoses. Cancer Cytopathol. 2022;130(2):136-143.
18. Abi-Raad R, Prasad ML, Adeniran AJ, Cai G. Fine-needle aspiration cytomorphology of papillary thyroid carcinoma with NTRK gene rearrangement from a case series with predominantly indeterminate cytology. Cancer Cytopathol. 2020;128(11):803-811.
19. Picarsic JL, Buryk MA, Ozolek J et al. Molecular characterization of sporadic pediatric thyroid carcinoma with the DNA/RNA ThyroSeq v2 next-generation sequencing assay. Pediatr Dev Pathol. 2016;19(2):115-122.
20. Weiss LM, Funari VA. NTRK fusions and Trk proteins: what are they and how to test for them. Hum Pathol. 2021; 112:59-69.
21. Conde E, Hernandez S, Sanchez E et al. Pan-TRK immunohistochemistry: an example-based practical approach to efficiently identify patients with NTRK fusion cancer. Arch Pathol Lab Med. 2021;145(8):1031-1040.
22. Perreault S, Chami R, Deyell RJ et al. Canadian consensus for biomarker testing and treatment of TRK fusion cancer in pediatric patients. Curr Oncol. 2021;28(1):346-366.
23. Lim KHT, Kong HL, Chang KTE et al. Recommended testing algorithms for NTRK gene fusions in pediatric and selected adult cancers: Consensus of a Singapore Task Force. Asia Pac J Clin Oncol. 2021;18(4):394-403.
24. Kechin AA, Ivanov AA, Kel AE et al Prediction of EVT6-NTRK3-Dependent Papillary Thyroid Cancer Using Minor Expression Profile. Bull Exp Biol Med. 2022;173(2):252-256.
25. Penault-Llorca F, Rudzinski ER, Sepulveda AR. Testing algorithm for identification of patients with TRK fusion cancer. J Clin Pathol. 2019; 72:460-467.