Next-generation sequencing on fine needle aspirates in neck recurrence of thyroid cancers

in European Thyroid Journal
Authors:
Hélène Théodon Department of Thyroid and Endocrine Tumors, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Erell Guillerm Department of Oncogenetic, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Johanna Wassermann Department of Oncology, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Gabrielle Deniziaut Department of Pathology, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Loïc Jaffrelot Department of Oncology, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Jérome Denis Department of Endocrine and Oncology Biochemistry, Sorbonne Université, Pitié-Salpêtrière Hospital, Paris, France

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Nathalie Chereau Department of Endocrine Surgery, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Claude Bigorgne Department of Pathology, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Wiame Potonnier Department of Pathology, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Florence Coulet Department of Oncogenetic, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Laurence Leenhardt Department of Thyroid and Endocrine Tumors, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Camille Buffet Department of Thyroid and Endocrine Tumors, Sorbonne Université, GRC n°16, GRC Tumeurs Thyroïdiennes, Pitié-Salpêtrière Hospital, Paris, France

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Correspondence should be addressed to C Buffet: camille.buffet@aphp.fr
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Objective

Tumor molecular genotyping plays a key role in improving the management of advanced thyroid cancers. Molecular tests are classically performed on formalin-fixed, paraffin-embedded (FFPE) carcinoma tissue. However alternative molecular testing strategies are needed when FFPE tumoral tissue is unavailable. The objective of our study was to retrospectively assess the performance of targeted DNA and RNA-based next-generation sequencing (NGS) on the fine needle aspirate from thyroid cancer cervical recurrences to determine if this strategy is efficient in clinical practice.

Design/Methods

A retrospective study of 33 patients who had had DNA and/or RNA-based NGS on ultrasound (US)-guided fine needle aspirates of cervical thyroid cancer recurrences in our Department from July 2019 to September 2022.

Results

In total, 34 DNA and 32 RNA-based NGS analyses were performed. Out of the 34 DNA-based NGS performed, 27 (79%) were conclusive allowing the identification of an oncogenic driver for 18 patients (53%). The most common mutation (n = 13) was BRAF c.1799T>A. Out of the 32 RNA-based NGS performed, 26 were interpretable (81%) and no gene fusion was found. The identification of a BRAFV600E mutation was decisive for one patient in our series, who was prescribed dabrafenib and trametinib.

Conclusion

NGS performed on fine needle aspirates of neck lymph node metastases enabled the identification of an oncogenic driver alteration in 53% of the cases in our series of advanced thyroid cancer patients and could significantly alter patient management.

Significance statement

This paper shows that thyroid cancer genotyping on the fine needle aspirate (FNA) of a metastatic neck lymph node recurrence can be performed efficiently. This strategy of genotyping appears particularly effective and safe when FFPE tissue is unavailable and when the spread of the disease requires systemic treatment. To the best of our knowledge, our data regarding DNA and RNA next generation sequencing on FNA of metastatic neck recurrences are the first ever published.

Abstract

Objective

Tumor molecular genotyping plays a key role in improving the management of advanced thyroid cancers. Molecular tests are classically performed on formalin-fixed, paraffin-embedded (FFPE) carcinoma tissue. However alternative molecular testing strategies are needed when FFPE tumoral tissue is unavailable. The objective of our study was to retrospectively assess the performance of targeted DNA and RNA-based next-generation sequencing (NGS) on the fine needle aspirate from thyroid cancer cervical recurrences to determine if this strategy is efficient in clinical practice.

Design/Methods

A retrospective study of 33 patients who had had DNA and/or RNA-based NGS on ultrasound (US)-guided fine needle aspirates of cervical thyroid cancer recurrences in our Department from July 2019 to September 2022.

Results

In total, 34 DNA and 32 RNA-based NGS analyses were performed. Out of the 34 DNA-based NGS performed, 27 (79%) were conclusive allowing the identification of an oncogenic driver for 18 patients (53%). The most common mutation (n = 13) was BRAF c.1799T>A. Out of the 32 RNA-based NGS performed, 26 were interpretable (81%) and no gene fusion was found. The identification of a BRAFV600E mutation was decisive for one patient in our series, who was prescribed dabrafenib and trametinib.

Conclusion

NGS performed on fine needle aspirates of neck lymph node metastases enabled the identification of an oncogenic driver alteration in 53% of the cases in our series of advanced thyroid cancer patients and could significantly alter patient management.

Significance statement

This paper shows that thyroid cancer genotyping on the fine needle aspirate (FNA) of a metastatic neck lymph node recurrence can be performed efficiently. This strategy of genotyping appears particularly effective and safe when FFPE tissue is unavailable and when the spread of the disease requires systemic treatment. To the best of our knowledge, our data regarding DNA and RNA next generation sequencing on FNA of metastatic neck recurrences are the first ever published.

Introduction

Advances in molecular genotyping with high-throughput sequencing have led to better understanding of the genetic landscape of thyroid cancer and ultimately to major improvements in the treatment of advanced thyroid cancers (1, 2). Today, tumor molecular genotyping plays a key role in the management of radioactive iodine refractory (RAIR) thyroid cancers, as patients with cancers harboring a specific mutation or fusion can be offered highly specific targeted therapies (3).

The molecular testing of advanced thyroid cancers usually involves using targeted DNA and RNA-based next-generation sequencing (NGS) with large panels to analyze numerous genes in the detection of somatic mutations or fusions. These molecular tests are classically performed on formalin-fixed, paraffin-embedded (FFPE) tumoral tissue. Alternatively, molecular genotyping on liquid biopsy, i.e. plasma samples, can also be performed, chiefly to search for somatic mutations (4, 5). Molecular genotyping on fine needle aspirates can be used to infer the risk of malignancy in indeterminate thyroid nodules (6, 7, 8) and for genetic analysis in other types of cancers such as pancreatic cancers (9).

For thyroid cancer patients, when FFPE tumoral tissue is unavailable, molecular testing on ultrasound (US)-guided fine needle aspirate (FNA) from a metastatic neck lymph node seems a suitable option as it is a minimally invasive procedure and neck lymph nodes are easily accessible. This genotyping strategy has been suggested in the literature but never tested in practice (10, 11). The dedicated biopsy or surgery of a neck recurrence or distant solid metastasis of the mediastinum, lung or liver performed to obtain FFPE tumoral tissue for genetic testing are invasive procedures with intrinsic risks and extended waiting times for molecular results. Such procedures seem even less safe when local treatment of the distant metastatic location is not planned and systemic therapy is warranted.

Molecular testing on the fine needle aspirate from a metastatic neck lymph node may also be of particular interest when FFPE tissue has been archived for more than 10 years making the risk of sequencing failure high (12, 13). This issue is frequent in clinical practice since the time from initial diagnosis of differentiated thyroid cancer to the occurrence of radioiodine refractory disease can vary considerably: with a median estimation of 3.3 years, ranging from less than a year to 22.3 years (14). Other specific situations limiting the possibility of molecular testing may be encountered in clinical practice such as initial surgery performed in a foreign country or no available archive of properly fixed tumoral tissue. Moreover, genomic analysis from FFPE tissues can produce artifacts as formalin fixation negatively impacts DNA and RNA quality and quantity compared to fresh frozen material (15).

The objective of our study was to retrospectively assess the performance of targeted DNA and RNA-based NGS on fine needle aspirate from thyroid cancer cervical recurrences to determine if this strategy of molecular testing could be applied in clinical practice.

Materials and methods

Patients

We retrospectively reviewed the files of patients who agreed to have DNA and/or RNA-based NGS on neck US-guided fine needle aspirates in our Department from July 2019 (beginning of NGS on fine needle aspirates in our center) until September 2022.

In total, 413 NGS analyses were performed on 385 patients on fine needle aspirates. NGS analyses performed on neck recurrences from thyroid cancer patients were included. The following analyses were excluded because NGS had been performed on:

  • A thyroid nodule aspirate (n = 355)

  • An aspirate from very rapidly evolving neck masses corresponding to anaplastic thyroid cancer (n = 3) (16)

  • A metastatic neck lymph node aspirate in a patient with a history of parathyroid carcinoma (n = 1)

  • A synchronous metastatic neck lymph node aspirate performed before thyroid surgery (n = 1)

  • A non-metastatic neck lymph node or mass (n = 19)

In total, 34 DNA and/or RNA-based NGS analyses were included, performed on cytologically proven recurrent thyroid cancer metastatic neck lymph nodes or masses (Fig. 1). Metastatic neck lymph node or mass was defined by the presence of metastatic cells in the cytology report and/or with an elevated concentration of the in situ thyroglobulin (TG) >10 µg/L or calcitonin >2000 ng/L.

Figure 1
Figure 1

Flowchart of the sample selection.

Citation: European Thyroid Journal 13, 1; 10.1530/ETJ-23-0164

Of the 34 samples included, 21 were from metastatic neck lymph nodes and 13 from metastatic masses, including 12 thyroid bed recurrences and 1 muscle metastasis.

One patient was submitted to the sampling of two different lesions: one was a right level VII metastatic tumor mass and the second was a right level VI metastatic tumor mass.

Sample collection and DNA and RNA extractions

FNA was performed with a 25 or 27 gauge fine needle under US guidance. Samples were collected for molecular analysis either by one dedicated pass or by washing out the residual material from at least two different passes made for cytological analysis purposes. The percentage of tumoral cells could not be estimated in the samples. The specimens were collected into a preservative solution in a Roche Cell-Free DNA Collection Tube® for molecular testing. The samples were transported and stored at room temperature. Extractions were carried out within 7 days of sample collection.

DNA and RNA extractions were performed on each sample separately. DNA extractions were made with Maxwell® RSC Blood DNA kit (Promega) and RNA extractions were made with Maxwell® RSC simplyRNA Blood kit for RNA (Promega) according to the manufacturer’s protocol. The quality control was performed by the dosage of DNA and RNA. The libraries were prepared for all samples and sequencing was systematically performed. Dilutions of DNA or RNA was not performed when the concentrations were inferior to the manufacturer’s recommendation.

Next-generation sequencing

Deoxyribonucleic acid

A library was prepared from 8 ng of DNA by PCR multiplex (Kit AmpliSeq™ for Illumina Focus Panel) of a part of the exonic regions covering hotspot mutations in 40 genes of interest for DNA mutations with 269 amplicons: AKT1, ALK, AR, BRAF, CCND1, CDK4, CDK6, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, GNA11, GNAQ, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KIT, KRAS, MAP2K1, MPA2K2, MET, MTOR, MYC, MYCN, NRAS, PDGFRA, PIK3CA, RAF1, RET, ROS1, SMO.

Library amplification with bridge-PCR and high rate sequencing were performed on an lllumina (MiSeq or MiniSeq) platform.

The bioinformatic analysis (detection sensitivity >99%) was made using the Sequence Pilot/module SeqNext (JSI Medical Systems) software and LRM (local run manager, Illumina software).

Assured variant detection threshold was 2% with >99% coverage at 300× on hotspots. On hotspots when the coverage is greater, the detection threshold can be lowered to 1%, for example, the coverage on the BRAF location c.1799T>A is always greater than 1000×. When cover was lower, the threshold could be up, but when the quality cover was too poor, i.e. lower than 300× for all region of interest or on hotspot loci, the samples were considered not interpretable.

Ribonucleic acid

A library was prepared from 10.5 ng of RNA by PCR multiplex (Kit AmpliSeq™ for Illumina Focus Panel) for fusions with 284 amplicons with the following 23 target genes: ABL1, ALK, AKT3, AXL, BRAF, EGFR, ERBB2, ERG, ETV1, ETV4, ETV5, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK2, NTRK3, PDGFRA, PPARG, RAF1, RET, ROS1.

Library amplification with bridge-PCR and high rate sequencing were performed on an lllumina (MiSeq or MiniSeq) platform.

The bioinformatic analysis (detection sensitivity >99%) was made using the Sequence Pilot/module SeqNext (JSI Medical Systems) software LRM (local run manager, Illumina software).

Whenever the NGS performed on fine needle aspirate showed no mutation or gene fusion and/or was not interpretable, FFPE tumoral tissue from the primary tumor or additional neck surgery, if available, was submitted to DNA and RNA extraction and to NGS.

The result of the molecular test was declared positive if the molecular alteration identified fell into one of the following two categories ‘pathogenic’, ‘likely pathogenic’, and declared negative otherwise (i.e. categories ‘variant of unknown significance’ or ‘likely benign’ or ‘benign’) (17).

Results

The clinical and histological characteristics of the 33 patients included in our series are summarized Table 1. Most patients were male (n = 18, 55%) and had papillary thyroid cancer (n = 23; 70%).

Table 1

Patients’ characteristics. Values are presented as n (%).

Characteristics Values
Sex
 Female 15 (45)
 Male 18 (55)
Mean age (years) at diagnosis 57
pTNM (OMS 2017) at diagnosis
 pT
  1a 2 (6)
  1b 4 (12)
  2 9 (27)
  3a 3 (9)
  3b 3 (9)
  4a 6 (18)
  4b 0 (0)
  x 6 (18)
 N
  0 8 (24)
  1a 1 (3)
  1b 20 (61)
  x 4 (12)
 M
  0 15 (45)
  1 12 (36)
  x 6 (18)
Histological type of DTC
 Papillary 23 (70)
 Follicular 4 (12)
 Poorly differentiated 2 (6)
 Medullary 4 (12)

DTC, differentiated thyroid cancer.

The mean DNA and RNA concentrations were 1.7 ng/µL (0.01; 23.00) and 8.92 ng/µL (2.00; 21.60), respectively (Fig. 2 and Supplementary Table 1, see the section on supplementary materials given at the end of this article). Samples were collected by one dedicated pass for 5 out of 34 (15%), by washing out the residual material from at least 2 different passes made for cytological analysis purposes for 15 out of 34 (44%), and by combining both techniques for 2 out of 34 (6%). For the remaining 12, (35%) the sampling technique was not specified.

Figure 2
Figure 2

DNA and RNA concentration repartition.

Citation: European Thyroid Journal 13, 1; 10.1530/ETJ-23-0164

Out of the 34 DNA-based NGS, 7 were uninterpretable for point mutations and 27 (79%) were interpretable regarding the presence or not of an oncogenic driver. Out of these 27 samples with interpretable DNA-based NGS, 18 (67%) showed at least one mutation: 16 harbored one point mutation and 2 harbored 2 point mutations (Supplementary Table 1).

In the 24 neck recurrences of papillary thyroid cancers, the most frequent mutation was the BRAF c.1799T>A mutation (BRAFV600E) found alone in 11 samples (46%), and in association with a PIK3CA mutation in 2 samples (8%) - one PIK3CA c.3140A>G (p.H1047L) and one PIK3CA c.3140A>T (p.H1047R) mutation. One sample harbored a BRAF c.1795_1797dup (T599dup) mutation (4%) and another a KIT (V560del) mutation (4%) (Fig. 3). In the four patients with recurrent follicular thyroid cancers, only one showed a molecular alteration – an NRAS mutation – among the 3 interpretable samples. Regarding the NGS performed on the two poorly differentiated thyroid cancers, one showed no mutation and one was not interpretable. The most frequent molecular alteration found in the four recurrent medullary thyroid cancers patients was a RET mutation found in two out of the three interpretable samples (Fig. 3).

Figure 3
Figure 3

NGS results according to the historical subtype.

Citation: European Thyroid Journal 13, 1; 10.1530/ETJ-23-0164

The allelic frequencies found on DNA-based NGS ranged from 72% to 1% (Fig. 4).

Figure 4
Figure 4

Results of allelic frequencies found by FNA.

Citation: European Thyroid Journal 13, 1; 10.1530/ETJ-23-0164

RNA-based NGS was not performed for two samples that harbored a BRAFV600E mutation (BRAF c.1799T>A). Out of the 32 RNA-based NGS performed, 26 were interpretable (81%) and 6 were uninterpretable for gene fusions. Among the 26 interpretable RNA-based NGS, no gene fusion was found.

NGS was repeated on available FFPE tissue whenever possible when no molecular alteration was identified on the fine needle aspirates NGS (n = 9 out of 9 for DNA, n = 10 out of 12 for RNA) or when the result was uninterpretable (n = 5 out of 7 for DNA, n = 4 out of 4 for RNA) (Table 2 and 3). NGS was performed on FFPE tissue from the primary thyroid cancer (n = 4), from a lung (n = 1) or a neck lymph node (n = 9) metastasis. No FFPE tissue was available for the remaining two samples.

Table 2

Comparison of DNA-based NGS results on FNA samples and FFPE tumoral tissues.

NGS result on FNA samples
No mutation (n = 9) Not interpretable (n = 7)
FFPE tissue available, n 9 5
NGS on FFPE tissue
 No mutation 7 samples 3 samples
 Not interpretable 0 0
 Mutation found 2 samples 2 samples
c.1799T>A BRAF; VAF : 21% c.1799T>A BRAF; VAF : 15%
c.182A>G NRAS; VAF : 43% c.1799T>A BRAF; VAF : 37% and c.241G>A PIK3CA; VAF : 4.3%

VAF, variant allelic frequency.

Table 3

Comparison of RNA-based NGS results on FNA samples and FFPE tumoral tissues.

NGS result on FNA samples
No fusion (n = 12) Not interpretable (n = 4)
FFPE tissue available, n 10 4
NGS on FFPE tissue
 No fusion 8 samples 4
 Not interpretable 1 0
 Fusion found 1 sample RET::NCOA4

The identification of an oncogenic driver on the fine needle aspirate from a neck recurrence in a patient with a history of radioiodine refractory papillary thyroid cancer significantly altered the patient management. This 67-year-old male patient had been diagnosed with a 30 mm classic papillary thyroid carcinoma pT2N1b. He had undergone total thyroidectomy with central and lateral homolateral neck dissection in a foreign country. The patient was re-operated on his neck 1 year later revealing four persistent metastatic lymph nodes in the lateral left compartment. The surgery was followed by 3.7 Gbq adjuvant radioiodine therapy. A cervicothoracic–abdominal–pelvic computed tomography scan performed 1 year later revealed multiple neck lymph nodes, mediastinal, lung and bone metastases and a right subscapularis muscle lesion, all presenting fluorodeoxyglucose (FDG) uptake. The patient was considered radioiodine refractory because of significant disease progression within 12 months after radioiodine therapy. Because of painful vertebral epiduritis, the patient was treated with high doses of corticosteroid before percutaneous cementoplasty and external beam radiation therapy of the 11th thoracic vertebra. Additionally, a threatening left acetabulum lytic metastasis was also treated with percutaneous cementoplasty and a painful skull metastasis with external beam radiotherapy. Because of diffuse distant metastatic progression associated with symptomatic epiduritis, the patient qualified for systemic therapy. Antiangiogenic tyrosine kinase inhibitors were considered unsafe because of multiple hypervascular supra-centimetric metastatic lymph nodes in direct contact with the jugular vein. FFPE tissues from the primary thyroid cancer or neck lymph node metastases were not available. NGS performed on one of the fine needle aspirates of a metastatic neck lymph node found a BRAFV600E mutation. Dabrafenib and trametinib were finally prescribed off-labeled after decision in a multidisciplinary staff meeting. At the 3 month follow-up, CT showed morphological partial response according to the RECIST 1.1 criteria with a 50% decrease of the target lesions.

Discussion

Although molecular genotyping of advanced thyroid cancers is of upmost importance for the clinical management of these patients, finding available tumoral samples for molecular testing is a common issue in clinical practice. When no tumoral FFPE tissue is available, molecular testing on fine needle aspirate from accessible metastatic neck lymph nodes or masses is an interesting option. An alternative could be a core-needle biopsy of a neck lymph node metastasis. However, this procedure may present a risk, especially in case of small metastases close to the carotid and/or jugular axis.

In addition, NGS may produce better results when performed on fine needle aspirates rather than on FFPE archived specimens since the FFPE preservation may create mutational artifacts (18). Another advantage of this strategy is to provide genotyping results rapidly.

To the best of our knowledge, no study has been published on the application of targeted DNA and RNA-based NGS on the fine needle aspirate of neck recurrences for thyroid cancer patients. Our work showed that this sequencing strategy was efficient and interpretable for 27 out of 34 samples (79%). DNA-based sequencing identified an oncogenic driver in 18 out of these 27 samples (67%) and RNA-based NGS was either negative or uninterpretable in those 27 cases. However, oncogenic drivers are known to be mutually exclusive in thyroid cancers and if a mutation is identified in an oncogene, the absence of oncogenic fusion can be expected.

This genotyping strategy is efficient, practical and fast to detect a molecular alteration and could be particularly useful for progressive distant metastatic radio-iodine refractory thyroid cancer patients, as illustrated by the prescription of highly targeted therapy, namely, dabrafenib and trametinib for one of our patients.

Despite the small size of our sample, the molecular alterations identified were in line with what is expected, including high prevalence of BRAFV600E (19) and RET (20) mutations in recurrent papillary and medullary thyroid cancers, respectively, and one RAS mutation identified in a recurrent follicular thyroid cancer (21) out of 3 samples with interpretable NGS for this histological type. Interestingly, 2 of the recurrent papillary thyroid cancers harbored both BRAF c.1799 T>A and PIK3CA mutations, the association of these mutations being described as associated with worse prognosis in BRAF V600E-driven thyroid cancer patients (22). The BRAF T599dup mutation is also known to be oncogenic, located in the kinase domain in exon 15, similarly to the BRAFV600E mutation and has been found in other malignancies such as melanoma (23).

DNA-based NGS on FNA allowed the detection of allelic frequencies as low as 1% in our study. Even with low DNA concentrations in some samples (Supplementary Table 1, e.g. sample 10) we managed to detect low allelic frequency.

One of the limitations of this study is that our DNA panel did not include the search for mutations either on the TERT promoter gene or on the P53 gene, which are genes involved in aggressive thyroid carcinomas (24, 25). In addition, our panel gave no information regarding copy number alterations.

Out of 16 samples with no alteration found or uninterpretable results, 14 NGS were performed for DNA and RNA extracted from FFPE tissues (primary thyroid carcinoma, neck or distant metastasis) 4 were positive for an oncogenic driver mutation and 1 was positive for a fusion. Genotyping on FFPE tumoral tissue revealed RAS, BRAF or PIK3CA mutations while no mutation was found on the FNA. Discordance between the primary tumor genotype and lymph node or distant metastases have already been described for BRAF or RAS mutations, albeit with very low frequencies (26). We have no clear explanation for these discrepancies. The impossibility to check the thyroid tumoral cells content with the routine NGS panel used in our institution, in the analyzed sample is an important limit to understand these discrepancies. We cannot formally exclude sampling errors or sample handling issues, despite that, on the one hand cytological analysis found thyroid tumoral cells for these samples and on the other hand, several processes for identity vigilance have been set up in our oncogenetic Department.

Our strategy (i.e. to look for a driver molecular alteration in the neck recurrence to treat metastatic patients) relies on the hypothesis that the driver oncogene is conserved during tumor progression and also present in the distant metastases. This is in agreement with the oncogenic addiction concept in thyroid oncology.

DNA- and RNA-based NGS performed on the fine needle aspirate was uninterpretable for 7 out of 34 samples (21%) and 6 out of 32 samples (19%), respectively. One limit of this strategy is the lack of opportunity to assess the quantity or type of cells collected during the procedure at the patient’s bedside.

In conclusion, NGS performed on the fine needle aspirate of metastatic neck lymph nodes or masses allowed the identification of an oncogenic driver alteration in 53% of the cases in our series of advanced thyroid cancer patients. Such a molecular testing strategy is a reasonable alternative when no FFPE tumoral tissue is available or when FFPE tissue has been archived for more than 10 years, making the risk of sequencing failure high (12, 13). However, when the NGS performed on the fine needle aspirate of easily accessible neck metastases does not found any molecular alteration, NGS on FFPE material obtained from microbiopsy remained useful to avoid missing the presence of an oncogenic driver not detected on the liquid biopsy.

Identification of oncogenic alteration in these advanced thyroid cancer patients may alter their management and allow the prescription of highly specific targeted therapy.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/ETJ-23-0164.

Declaration of interest

The authors declare that they have no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Camille Buffet is on the editorial board of European Thyroid Journal. Camille was not involved in the review or editorial process for this paper, on which she is listed as an author.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Statement of ethics

The authors confirm that all of the research meets the ethics guidelines, including adherence to the legal requirements of France. This retrospective study does not fall within the scope of the French Jardé law and therefore cannot benefit from the approval of ‘committees for the protection of individuals’ (’comités de protection des personnes (CPP)’).

Author contribution statement

HT: Investigation, writing – original draft; EG: resources, writing – review and editing; JW: resources, writing – review and editing; GD: resources, visualization, writing-review and editing; LJ: resources, writing-review and editing; JD: resources, writing – review and editing; NC: resources, writing – review & editing; CB: resources; WP: inclusion of patients; FC: resources, writing – review and editing; LL: conceptualization, writing – review and editing; CB: conceptualization, supervision, visualization, project administration, resources, writing – review and editing.

Acknowledgement

The authors would like to thank Liz Atzel for editing the English version of the manuscript.

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    Iwaya H, Tanimoto A, Toyodome K, Kojima I, Hinokuchi M, Tanoue S, Hashimoto S, Kawahira M, Arima S, Kanmura S, et al.Next-generation sequencing analysis of pancreatic cancer using residual liquid cytology specimens from endoscopic ultrasound-guided fine-needle biopsy: a prospective comparative study with tissue specimens. Diagnostics 2023 13 1078. (https://doi.org/10.3390/diagnostics13061078)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10

    Krane JF, Cibas ES, Endo M, Marqusee E, Hu MI, Nasr CE, Waguespack SG, Wirth LJ, & Kloos RT. The afirma Xpression atlas for thyroid nodules and thyroid cancer metastases: insights to inform clinical decision-making from a fine-needle aspiration sample. Cancer Cytopathology 2020 128 452459. (https://doi.org/10.1002/cncy.22300)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11

    Ali SZ, Siperstein A, Sadow PM, Golding AC, Kennedy GC, Kloos RT, & Ladenson PW. Extending expressed RNA genomics from surgical decision making for cytologically indeterminate thyroid nodules to targeting therapies for metastatic thyroid cancer. Cancer Cytopathology 2019 127 362369. (https://doi.org/10.1002/cncy.22132)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12

    Carrick DM, Mehaffey MG, Sachs MC, Altekruse S, Camalier C, Chuaqui R, Cozen W, Das B, Hernandez BY, Lih CJ, et al.Robustness of next generation sequencing on older formalin-fixed paraffin-embedded tissue. PLoS One 2015 10 e0127353. (https://doi.org/10.1371/journal.pone.0127353)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    Hussain M, Corcoran C, Sibilla C, Fizazi K, Saad F, Shore N, Sandhu S, Mateo J, Olmos D, Mehra N, et al.Tumor genomic testing for >4,000 men with metastatic castration-resistant prostate cancer in the Phase III trial PROfound (olaparib). Clinical Cancer Research 2022 28 15181530. (https://doi.org/10.1158/1078-0432.CCR-21-3940)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Wassermann J, Bernier MO, Spano JP, Lepoutre-Lussey C, Buffet C, Simon JM, Ménégaux F, Tissier F, Leban M, & Leenhardt L. Outcomes and prognostic factors in radioiodine refractory differentiated thyroid carcinomas. Oncologist 2016 21 5058. (https://doi.org/10.1634/theoncologist.2015-0107)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    Guo Q, Lakatos E, Bakir IA, Curtius K, Graham TA, & Mustonen V. The mutational signatures of formalin fixation on the human genome. Nature Communications 2022 13 4487. (https://doi.org/10.1038/s41467-022-32041-5)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    Buffet C, Allard L, Guillerm E, Ghander C, Mathy E, Lussey-Lepoutre C, Julien N, Touma E, Quilhot P, Godiris-Petit G, et al.Detection of BRAFV600E by digital PCR on fine-needle aspirate enables rapid initiation of dabrafenib and trametinib in unresectable anaplastic thyroid carcinoma. European Journal of Endocrinology 2022 187 K33K38. (https://doi.org/10.1530/EJE-22-0366)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, et al.Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the association for molecular pathology. Genetics in Medicine 2015 17 405424. (https://doi.org/10.1038/gim.2015.30)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18

    Williams C, Pontén F, Moberg C, Söderkvist P, Uhlén M, Pontén J, Sitbon G, & Lundeberg J. A high frequency of sequence alterations is due to formalin fixation of archival specimens. American Journal of Pathology 1999 155 14671471. (https://doi.org/10.1016/S0002-9440(1065461-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19

    Cancer Genome Atlas Research Network. Integrated genomic characterization of papillary thyroid carcinoma. Cell 2014 159 676690. (https://doi.org/10.1016/j.cell.2014.09.050)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20

    Viola D, & Elisei R. Management of medullary thyroid cancer. Endocrinology and Metabolism Clinics of North America 2019 48 285301. (https://doi.org/10.1016/j.ecl.2018.11.006)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21

    Fagin JA, & Wells SA. Biologic and clinical perspectives on thyroid cancer. New England Journal of Medicine 2016 375 2307. (https://doi.org/10.1056/NEJMc1613118)

  • 22

    Pappa T, Ahmadi S, Marqusee E, Johnson HL, Nehs MA, Cho NL, Barletta JA, Lorch JH, Doherty GM, Lindeman NI, et al.Oncogenic mutations in PI3K/AKT/mTOR pathway effectors associate with worse prognosis in BRAFV600E -Driven papillary thyroid cancer patients. Clinical Cancer Research 2021 27 42564264. (https://doi.org/10.1158/1078-0432.CCR-21-0874)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23

    Jones DTW, Kocialkowski S, Liu L, Pearson DM, Ichimura K, & Collins VP. Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549: BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma. Oncogene 2009 28 21192123. (https://doi.org/10.1038/onc.2009.73)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24

    Liu R, Bishop J, Zhu G, Zhang T, Ladenson PW, & Xing M. Mortality risk stratification by combining BRAF V600E and tert promoter mutations in papillary thyroid cancer: genetic duet of BRAF and tert promoter mutations in thyroid cancer mortality. JAMA Oncology 2017 3 202208. (https://doi.org/10.1001/jamaoncol.2016.3288)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25

    Li Z, Zhang Y, Wang R, Zou K, & Zou L. Genetic alterations in anaplastic thyroid carcinoma and targeted therapies. Experimental and Therapeutic Medicine 2019 18 23692377. (https://doi.org/10.3892/etm.2019.7869)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26

    Melo M, Gaspar da Rocha A, Batista R, Vinagre J, Martins MJ, Costa G, Ribeiro C, Carrilho F, Leite V, Lobo C, et al.TERT, BRAF, and NRAS in Primary Thyroid Cancer and Metastatic Disease. Journal of Clinical Endocrinology and Metabolism 2017 102 18981907. (https://doi.org/10.1210/jc.2016-2785)

    • PubMed
    • Search Google Scholar
    • Export Citation

Supplementary Materials

 

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  • 1

    Singh A, Ham J, Po JW, Niles N, Roberts T, & Lee CS. The genomic landscape of thyroid cancer tumourigenesis and implications for immunotherapy. Cells 2021 10 1082. (https://doi.org/10.3390/cells10051082)

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  • 2

    Lamartina L, Grani G, Durante C, & Filetti S. Recent advances in managing differentiated thyroid cancer. F1000Research 2018 7 86. (https://doi.org/10.12688/f1000research.12811.1)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3

    de la Fouchardière C, Wassermann J, Calcagno F, Bardet S, Al Ghuzlan A, Borget I, Borson Chazot F, Do Cao C, Buffet C, Zerdoud S, et al.Molecular genotyping in refractory thyroid cancers in 2021: when, how and why? A review from the TUTHYREF network. Bulletin du Cancer 2021 108 10441056. (https://doi.org/10.1016/j.bulcan.2021.06.009)

    • PubMed
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    • Export Citation
  • 4

    Romano C, Martorana F, Pennisi MS, Stella S, Massimino M, Tirrò E, Vitale SR, Di Gregorio S, Puma A, Tomarchio C, et al.Opportunities and challenges of liquid biopsy in thyroid cancer. International Journal of Molecular Sciences 2021 22 7707. (https://doi.org/10.3390/ijms22147707)

    • PubMed
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    • Export Citation
  • 5

    Pogliaghi G. Liquid biopsy in thyroid cancer: from circulating biomarkers to a new prospective of tumor monitoring and therapy. Minerva Endocrinology 2021 46 4561. (https://doi.org/10.23736/S2724-6507.20.03339-8)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6

    Hu MI, Waguespack SG, Dosiou C, Ladenson PW, Livhits MJ, Wirth LJ, Sadow PM, Krane JF, Stack BC, Zafereo ME, et al.Afirma genomic sequencing classifier and Xpression atlas molecular findings in consecutive Bethesda III-VI thyroid nodules. Journal of Clinical Endocrinology and Metabolism 2021 106 21982207. (https://doi.org/10.1210/clinem/dgab304)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7

    Nishino M, & Nikiforova M. Update on molecular testing for cytologically indeterminate thyroid nodules. Archives of Pathology and Laboratory Medicine 2018 142 446457. (https://doi.org/10.5858/arpa.2017-0174-RA)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8

    Nikiforov YE. Role of molecular markers in thyroid nodule management: then and now. Endocrine Practice 2017 23 979988. (https://doi.org/10.4158/EP171805.RA)

  • 9

    Iwaya H, Tanimoto A, Toyodome K, Kojima I, Hinokuchi M, Tanoue S, Hashimoto S, Kawahira M, Arima S, Kanmura S, et al.Next-generation sequencing analysis of pancreatic cancer using residual liquid cytology specimens from endoscopic ultrasound-guided fine-needle biopsy: a prospective comparative study with tissue specimens. Diagnostics 2023 13 1078. (https://doi.org/10.3390/diagnostics13061078)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10

    Krane JF, Cibas ES, Endo M, Marqusee E, Hu MI, Nasr CE, Waguespack SG, Wirth LJ, & Kloos RT. The afirma Xpression atlas for thyroid nodules and thyroid cancer metastases: insights to inform clinical decision-making from a fine-needle aspiration sample. Cancer Cytopathology 2020 128 452459. (https://doi.org/10.1002/cncy.22300)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11

    Ali SZ, Siperstein A, Sadow PM, Golding AC, Kennedy GC, Kloos RT, & Ladenson PW. Extending expressed RNA genomics from surgical decision making for cytologically indeterminate thyroid nodules to targeting therapies for metastatic thyroid cancer. Cancer Cytopathology 2019 127 362369. (https://doi.org/10.1002/cncy.22132)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12

    Carrick DM, Mehaffey MG, Sachs MC, Altekruse S, Camalier C, Chuaqui R, Cozen W, Das B, Hernandez BY, Lih CJ, et al.Robustness of next generation sequencing on older formalin-fixed paraffin-embedded tissue. PLoS One 2015 10 e0127353. (https://doi.org/10.1371/journal.pone.0127353)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    Hussain M, Corcoran C, Sibilla C, Fizazi K, Saad F, Shore N, Sandhu S, Mateo J, Olmos D, Mehra N, et al.Tumor genomic testing for >4,000 men with metastatic castration-resistant prostate cancer in the Phase III trial PROfound (olaparib). Clinical Cancer Research 2022 28 15181530. (https://doi.org/10.1158/1078-0432.CCR-21-3940)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Wassermann J, Bernier MO, Spano JP, Lepoutre-Lussey C, Buffet C, Simon JM, Ménégaux F, Tissier F, Leban M, & Leenhardt L. Outcomes and prognostic factors in radioiodine refractory differentiated thyroid carcinomas. Oncologist 2016 21 5058. (https://doi.org/10.1634/theoncologist.2015-0107)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    Guo Q, Lakatos E, Bakir IA, Curtius K, Graham TA, & Mustonen V. The mutational signatures of formalin fixation on the human genome. Nature Communications 2022 13 4487. (https://doi.org/10.1038/s41467-022-32041-5)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    Buffet C, Allard L, Guillerm E, Ghander C, Mathy E, Lussey-Lepoutre C, Julien N, Touma E, Quilhot P, Godiris-Petit G, et al.Detection of BRAFV600E by digital PCR on fine-needle aspirate enables rapid initiation of dabrafenib and trametinib in unresectable anaplastic thyroid carcinoma. European Journal of Endocrinology 2022 187 K33K38. (https://doi.org/10.1530/EJE-22-0366)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, et al.Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the association for molecular pathology. Genetics in Medicine 2015 17 405424. (https://doi.org/10.1038/gim.2015.30)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18

    Williams C, Pontén F, Moberg C, Söderkvist P, Uhlén M, Pontén J, Sitbon G, & Lundeberg J. A high frequency of sequence alterations is due to formalin fixation of archival specimens. American Journal of Pathology 1999 155 14671471. (https://doi.org/10.1016/S0002-9440(1065461-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19

    Cancer Genome Atlas Research Network. Integrated genomic characterization of papillary thyroid carcinoma. Cell 2014 159 676690. (https://doi.org/10.1016/j.cell.2014.09.050)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20

    Viola D, & Elisei R. Management of medullary thyroid cancer. Endocrinology and Metabolism Clinics of North America 2019 48 285301. (https://doi.org/10.1016/j.ecl.2018.11.006)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21

    Fagin JA, & Wells SA. Biologic and clinical perspectives on thyroid cancer. New England Journal of Medicine 2016 375 2307. (https://doi.org/10.1056/NEJMc1613118)

  • 22

    Pappa T, Ahmadi S, Marqusee E, Johnson HL, Nehs MA, Cho NL, Barletta JA, Lorch JH, Doherty GM, Lindeman NI, et al.Oncogenic mutations in PI3K/AKT/mTOR pathway effectors associate with worse prognosis in BRAFV600E -Driven papillary thyroid cancer patients. Clinical Cancer Research 2021 27 42564264. (https://doi.org/10.1158/1078-0432.CCR-21-0874)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23

    Jones DTW, Kocialkowski S, Liu L, Pearson DM, Ichimura K, & Collins VP. Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549: BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma. Oncogene 2009 28 21192123. (https://doi.org/10.1038/onc.2009.73)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24

    Liu R, Bishop J, Zhu G, Zhang T, Ladenson PW, & Xing M. Mortality risk stratification by combining BRAF V600E and tert promoter mutations in papillary thyroid cancer: genetic duet of BRAF and tert promoter mutations in thyroid cancer mortality. JAMA Oncology 2017 3 202208. (https://doi.org/10.1001/jamaoncol.2016.3288)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25

    Li Z, Zhang Y, Wang R, Zou K, & Zou L. Genetic alterations in anaplastic thyroid carcinoma and targeted therapies. Experimental and Therapeutic Medicine 2019 18 23692377. (https://doi.org/10.3892/etm.2019.7869)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26

    Melo M, Gaspar da Rocha A, Batista R, Vinagre J, Martins MJ, Costa G, Ribeiro C, Carrilho F, Leite V, Lobo C, et al.TERT, BRAF, and NRAS in Primary Thyroid Cancer and Metastatic Disease. Journal of Clinical Endocrinology and Metabolism 2017 102 18981907. (https://doi.org/10.1210/jc.2016-2785)

    • PubMed
    • Search Google Scholar
    • Export Citation