Accepted Article
Received Date : 28-Mar-2014 Revised Date : 13-May-2014 Accepted Date : 14-May-2014 Article type
: Original Article
Next-generation sequencing improves the diagnosis of thyroid FNA specimens with indeterminate cytology
Short title: NGS improves thyroid FNA diagnosis
Marie Le Mercier1, Nicky D’Haene1, Nancy De Nève1, Oriane Blanchard1, Caroline Degand1, Sandrine Rorive1,2 and Isabelle Salmon1,2
1
Department of Pathology, Erasme University Hospital, Université Libre de Bruxelles (ULB),
Brussels, Belgium 2
DIAPath - Center for Microscopy and Molecular Imaging (CMMI); Académie Universitaire
Wallonie-Bruxelles, Gosselies, Belgium
Corresponding Author: Isabelle Salmon, MD, PhD. Laboratory of Pathology – Erasme Hospital 808 route de Lennik – B-1070 Brussels – Belgium Tel: +32 2 555 3115 Fax: +32 2 555 4790; e-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/his.12461 This article is protected by copyright. All rights reserved.
Accepted Article
Conflict of interest statement: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Abstract Aims: The assessment of thyroid nodules is a common clinical challenge. Fine-needle aspiration (FNA) is the standard preoperative tool for thyroid nodule diagnosis. However, up to 30% of the samples are classified as indeterminate. This often leads to unnecessary surgeries. In this study, we evaluated the added value of next-generation sequencing (NGS) for helping in the diagnosis of FNA samples. Methods and Results: We retrospectively analysed 34 indeterminate FNA samples for which surgical resection was performed. DNA was obtained from cell blocks or from stained smears and subjected to NGS to analyse mutations in 50 genes. Mutations in BRAF, NRAS, KRAS and PTEN that are known to be involved in thyroid cancer biology were detected in 7 FNA samples. The presence of a mutation in these genes was a strong indicator of cancer because 5 (71%) of the mutation-positive FNA samples had malignant diagnosis after surgery. Moreover, the cancer risk in nodules with indeterminate cytological diagnosis but with a negative molecular test was of only 8%. Conclusion: This study demonstrates that thyroid FNA can be successfully analysed by NGS. The detection of mutations known to be involved in thyroid cancer improves the sensitivity of thyroid FNA diagnosis.
Key words: Next-generation sequencing, Thyroid cancer, Thyroid FNA, Molecular diagnosis
This article is protected by copyright. All rights reserved.
Accepted Article
Introduction The assessment of thyroid nodules is a common clinical challenge. The frequency of thyroid nodules detected by ultrasound (US) has sharply increased in recent years to 67% of the adult population.1 In contrast, thyroid cancers are rare, accounting for only 1% of all cancers and occurring in approximately 5% of all thyroid nodules, independent of their size.1,
2
As the majority of thyroid
nodules are benign, the challenge for physicians who manage patients with thyroid nodules is to efficiently stratify patients according to their risk of malignancy to identify the best follow-up and therapeutic options. Fine-needle aspiration (FNA) followed by cytological assessment, has become the predominant method used for the primary diagnosis of benign and malignant thyroid nodules, resulting in the categorisation of patients as operative or non-operative candidates.3, 4 However, FNA has intrinsic limitations in distinguishing between benign and malignant follicular lesions. More particularly, the management of patients with indeterminate cytology (10-26% of FNA) remains problematic. As this category is associated with a 20% to 30% incidence of malignancy, most of these patients are referred for surgery.3, 5, 6 This approach induces a major health-care problem because it leads to unnecessary surgery for patients with benign lesions. In addition to the identification of additional clinical and US data that efficiently predict malignancy,5, 7-10
efforts to improve the management of these patients have focused on the development of
molecular markers.11, 12 The discovery of genetic alterations in thyroid cancer prompted the search for somatic mutations in material obtained by FNA to increase the diagnostic accuracy of traditional cytology. Several prospective studies have shown that testing for mutations in the BRAF, NRAS, HRAS and KRAS genes, as well as detecting RET/PTC1, RET/PTC3 and PAX8/PPARG rearrangements, is feasible for thyroid FNA and provides helpful diagnostic information.11,
13-17
Among the published guidelines for the diagnosis and management of thyroid nodules, the revised American Thyroid Association’s guidelines now recommend molecular testing for these markers for nodules with indeterminate FNA cytology. 3, 4
This article is protected by copyright. All rights reserved.
Accepted Article
Several molecular tests have been shown to offer significant diagnostic improvement to FNA cytology.11,
14-16, 18-20
However, these molecular tests are not commonly used in Europe or are
restricted to a few labs. This can be explained by the fact that the number of markers to test is high and that the sequential analysis of all these markers is too expansive and time consuming for most pathology labs to apply them in daily practice. In the last decade, a new technology called nextgeneration sequencing (NGS) has emerged.21, 22 This technology enables the simultaneous sequencing of large panels of genes (targeted sequencing) with high sensitivity and in a cost-effective manner compared to traditional Sanger sequencing- and PCR-based methods.22-24 Among these platforms, the Ion Torrent PGM has the advantage that it requires as little as 10 ng of input DNA, allowing the sequencing of small formalin-fixed and paraffin-embedded (FFPE) samples.24 The present pilot study aims to evaluate the potential impact of molecular screening by NGS for the management of patients with thyroid indeterminate FNA using a commercially available 50-gene panel (AmpliSeq Cancer Hotspot Panel v.2).
Materials and Methods NGS panel Validation The performance of the AmpliSeq Cancer Hotspot panel was evaluated using 14 FFPE tumour tissues with known mutations, 5 commercial FFPE reference standards (Horizon Diagnostics, Cambridge, UK) carrying mutations at 50% allelic frequency and 1 FFPE multiplex reference standard (Horizon Diagnostics) carrying 11 different mutations at defined allelic frequencies varying from 0.9 to 24.4% (Supporting information and supplementary Table 1 and 2).
Cytological evaluation Cytological evaluation was done using our own grading system as described previously.5 cytological
This article is protected by copyright. All rights reserved.
Accepted Article
diagnoses were classified into four major categories: i) Unsatisfactory or non-diagnostic samples; ii) Benign diagnosis; iii) Follicular proliferation (FP) diagnosis (used for cytology indeterminate for malignancy) and iv) Malignant diagnosis. The FP category was subclassified into three grades associated with an increased malignancy risk (FP1, FP2 and FP3).5 This classification is similar to the Bethesda classification but does not include an “atypia of undetermined significance”category (AUS/FLUS; category III). As already debatted by others.25-27, we consider that this category is very heterogeneous and is not sufficiently informative to allow an adequate therapeutic decision by the clinicians
Sample selection Thirty-four FNA samples obtained between January 2010 and December 2012 at the Erasme University Hospital (Brussels, Belgium) were analysed retrospectively. The selection criteria consisted of patients with an FNA of FP diagnosis (FP1, FP2 and FP3) followed by surgery and for which sufficient material was available for DNA extraction (Table 1). The clinical data collected for each patient include age and sex. The US examination allowed the assessment of a series of features characterizing multinodularity, nodule size, solid or cystic nodule (with 2 levels of cystic changes: +, ++), calcification, echogenicity, echogenicity pattern and cytological diagnosis. Their distributions are detailed in Table 1. Histological diagnoses, taken as the gold standard, were assessed according to the criteria of WHO classification. Nevertheless, the differential diagnosis of thyroid tumours with follicular patterns remains difficult for a few cases that do not perfectly satisfy the criteria. For such cases, we therefore decided to use the terminology of “follicular tumour with uncertain malignant potential” (FT-UMP). The histological diagnoses were established prior to molecular testing. This work was approved by the ethical committee of the Erasme University Hospital (Brussels, Belgium) in May 2013 (n° P2013/173).
This article is protected by copyright. All rights reserved.
Accepted Article
Next-generation sequencing Genomic DNA was extracted from cell blocks using the QIAamp FFPE tissue kit or from smears using the QIAamp DNA mini kit (Qiagen, Antwerp, Belgium) according to the manufacturer’s instructions. For library construction, 10 ng of DNA was amplified using the Cancer hotspot panel v2 (AmpliSeq™, Life Technologies, Gent, Belgium) and Ion AmpliSeq™ HiFi Master Mix (Ion AmpliSeq™ Library kit 2.0). An amplicon library was thus generated for sequencing 2850 hotspot mutations in 50 genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL. The amplicons were then digested, barcoded and amplified using the Ion AmpliSeq™ Library kit 2.0 and Ion Xpress™ barcode adapters kit (Life Technologies) according to the manufacturer’s instructions. The library was then quantified using the Qubit® fluorometer and the Qubit® dsDNA HS assay kit (Life Technologies). A total of 8 pM of each library was multiplexed and clonally amplified on Ion sphere™ particles (ISPs) by emulsion PCR performed using the Ion One Touch 2 instrument with the Ion PGM™ template OT2 200 kit (Life Technologies) according to the manufacturer’s instructions. Quality control was performed using the Ionsphere™ quality control kit (Life Technologies) to ensure that 10-30% of template-positive ISPs were generated in the emulsion PCR. Finally, the template ISPs were enriched loaded onto an Ion 318™ chip and sequenced using a PGM™ sequencer with the Ion PGM™ sequencing 200 kit v2 according to the manufacturer’s instructions.
Data analysis The raw data were analysed using Torrent Suite v3.6.2 software (Life Technologies). The coverage analysis was performed using the Coverage Analysis plugin v3.6. Cases for which the number of mapped reads was
Received Date : 28-Mar-2014 Revised Date : 13-May-2014 Accepted Date : 14-May-2014 Article type
: Original Article
Next-generation sequencing improves the diagnosis of thyroid FNA specimens with indeterminate cytology
Short title: NGS improves thyroid FNA diagnosis
Marie Le Mercier1, Nicky D’Haene1, Nancy De Nève1, Oriane Blanchard1, Caroline Degand1, Sandrine Rorive1,2 and Isabelle Salmon1,2
1
Department of Pathology, Erasme University Hospital, Université Libre de Bruxelles (ULB),
Brussels, Belgium 2
DIAPath - Center for Microscopy and Molecular Imaging (CMMI); Académie Universitaire
Wallonie-Bruxelles, Gosselies, Belgium
Corresponding Author: Isabelle Salmon, MD, PhD. Laboratory of Pathology – Erasme Hospital 808 route de Lennik – B-1070 Brussels – Belgium Tel: +32 2 555 3115 Fax: +32 2 555 4790; e-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/his.12461 This article is protected by copyright. All rights reserved.
Accepted Article
Conflict of interest statement: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Abstract Aims: The assessment of thyroid nodules is a common clinical challenge. Fine-needle aspiration (FNA) is the standard preoperative tool for thyroid nodule diagnosis. However, up to 30% of the samples are classified as indeterminate. This often leads to unnecessary surgeries. In this study, we evaluated the added value of next-generation sequencing (NGS) for helping in the diagnosis of FNA samples. Methods and Results: We retrospectively analysed 34 indeterminate FNA samples for which surgical resection was performed. DNA was obtained from cell blocks or from stained smears and subjected to NGS to analyse mutations in 50 genes. Mutations in BRAF, NRAS, KRAS and PTEN that are known to be involved in thyroid cancer biology were detected in 7 FNA samples. The presence of a mutation in these genes was a strong indicator of cancer because 5 (71%) of the mutation-positive FNA samples had malignant diagnosis after surgery. Moreover, the cancer risk in nodules with indeterminate cytological diagnosis but with a negative molecular test was of only 8%. Conclusion: This study demonstrates that thyroid FNA can be successfully analysed by NGS. The detection of mutations known to be involved in thyroid cancer improves the sensitivity of thyroid FNA diagnosis.
Key words: Next-generation sequencing, Thyroid cancer, Thyroid FNA, Molecular diagnosis
This article is protected by copyright. All rights reserved.
Accepted Article
Introduction The assessment of thyroid nodules is a common clinical challenge. The frequency of thyroid nodules detected by ultrasound (US) has sharply increased in recent years to 67% of the adult population.1 In contrast, thyroid cancers are rare, accounting for only 1% of all cancers and occurring in approximately 5% of all thyroid nodules, independent of their size.1,
2
As the majority of thyroid
nodules are benign, the challenge for physicians who manage patients with thyroid nodules is to efficiently stratify patients according to their risk of malignancy to identify the best follow-up and therapeutic options. Fine-needle aspiration (FNA) followed by cytological assessment, has become the predominant method used for the primary diagnosis of benign and malignant thyroid nodules, resulting in the categorisation of patients as operative or non-operative candidates.3, 4 However, FNA has intrinsic limitations in distinguishing between benign and malignant follicular lesions. More particularly, the management of patients with indeterminate cytology (10-26% of FNA) remains problematic. As this category is associated with a 20% to 30% incidence of malignancy, most of these patients are referred for surgery.3, 5, 6 This approach induces a major health-care problem because it leads to unnecessary surgery for patients with benign lesions. In addition to the identification of additional clinical and US data that efficiently predict malignancy,5, 7-10
efforts to improve the management of these patients have focused on the development of
molecular markers.11, 12 The discovery of genetic alterations in thyroid cancer prompted the search for somatic mutations in material obtained by FNA to increase the diagnostic accuracy of traditional cytology. Several prospective studies have shown that testing for mutations in the BRAF, NRAS, HRAS and KRAS genes, as well as detecting RET/PTC1, RET/PTC3 and PAX8/PPARG rearrangements, is feasible for thyroid FNA and provides helpful diagnostic information.11,
13-17
Among the published guidelines for the diagnosis and management of thyroid nodules, the revised American Thyroid Association’s guidelines now recommend molecular testing for these markers for nodules with indeterminate FNA cytology. 3, 4
This article is protected by copyright. All rights reserved.
Accepted Article
Several molecular tests have been shown to offer significant diagnostic improvement to FNA cytology.11,
14-16, 18-20
However, these molecular tests are not commonly used in Europe or are
restricted to a few labs. This can be explained by the fact that the number of markers to test is high and that the sequential analysis of all these markers is too expansive and time consuming for most pathology labs to apply them in daily practice. In the last decade, a new technology called nextgeneration sequencing (NGS) has emerged.21, 22 This technology enables the simultaneous sequencing of large panels of genes (targeted sequencing) with high sensitivity and in a cost-effective manner compared to traditional Sanger sequencing- and PCR-based methods.22-24 Among these platforms, the Ion Torrent PGM has the advantage that it requires as little as 10 ng of input DNA, allowing the sequencing of small formalin-fixed and paraffin-embedded (FFPE) samples.24 The present pilot study aims to evaluate the potential impact of molecular screening by NGS for the management of patients with thyroid indeterminate FNA using a commercially available 50-gene panel (AmpliSeq Cancer Hotspot Panel v.2).
Materials and Methods NGS panel Validation The performance of the AmpliSeq Cancer Hotspot panel was evaluated using 14 FFPE tumour tissues with known mutations, 5 commercial FFPE reference standards (Horizon Diagnostics, Cambridge, UK) carrying mutations at 50% allelic frequency and 1 FFPE multiplex reference standard (Horizon Diagnostics) carrying 11 different mutations at defined allelic frequencies varying from 0.9 to 24.4% (Supporting information and supplementary Table 1 and 2).
Cytological evaluation Cytological evaluation was done using our own grading system as described previously.5 cytological
This article is protected by copyright. All rights reserved.
Accepted Article
diagnoses were classified into four major categories: i) Unsatisfactory or non-diagnostic samples; ii) Benign diagnosis; iii) Follicular proliferation (FP) diagnosis (used for cytology indeterminate for malignancy) and iv) Malignant diagnosis. The FP category was subclassified into three grades associated with an increased malignancy risk (FP1, FP2 and FP3).5 This classification is similar to the Bethesda classification but does not include an “atypia of undetermined significance”category (AUS/FLUS; category III). As already debatted by others.25-27, we consider that this category is very heterogeneous and is not sufficiently informative to allow an adequate therapeutic decision by the clinicians
Sample selection Thirty-four FNA samples obtained between January 2010 and December 2012 at the Erasme University Hospital (Brussels, Belgium) were analysed retrospectively. The selection criteria consisted of patients with an FNA of FP diagnosis (FP1, FP2 and FP3) followed by surgery and for which sufficient material was available for DNA extraction (Table 1). The clinical data collected for each patient include age and sex. The US examination allowed the assessment of a series of features characterizing multinodularity, nodule size, solid or cystic nodule (with 2 levels of cystic changes: +, ++), calcification, echogenicity, echogenicity pattern and cytological diagnosis. Their distributions are detailed in Table 1. Histological diagnoses, taken as the gold standard, were assessed according to the criteria of WHO classification. Nevertheless, the differential diagnosis of thyroid tumours with follicular patterns remains difficult for a few cases that do not perfectly satisfy the criteria. For such cases, we therefore decided to use the terminology of “follicular tumour with uncertain malignant potential” (FT-UMP). The histological diagnoses were established prior to molecular testing. This work was approved by the ethical committee of the Erasme University Hospital (Brussels, Belgium) in May 2013 (n° P2013/173).
This article is protected by copyright. All rights reserved.
Accepted Article
Next-generation sequencing Genomic DNA was extracted from cell blocks using the QIAamp FFPE tissue kit or from smears using the QIAamp DNA mini kit (Qiagen, Antwerp, Belgium) according to the manufacturer’s instructions. For library construction, 10 ng of DNA was amplified using the Cancer hotspot panel v2 (AmpliSeq™, Life Technologies, Gent, Belgium) and Ion AmpliSeq™ HiFi Master Mix (Ion AmpliSeq™ Library kit 2.0). An amplicon library was thus generated for sequencing 2850 hotspot mutations in 50 genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL. The amplicons were then digested, barcoded and amplified using the Ion AmpliSeq™ Library kit 2.0 and Ion Xpress™ barcode adapters kit (Life Technologies) according to the manufacturer’s instructions. The library was then quantified using the Qubit® fluorometer and the Qubit® dsDNA HS assay kit (Life Technologies). A total of 8 pM of each library was multiplexed and clonally amplified on Ion sphere™ particles (ISPs) by emulsion PCR performed using the Ion One Touch 2 instrument with the Ion PGM™ template OT2 200 kit (Life Technologies) according to the manufacturer’s instructions. Quality control was performed using the Ionsphere™ quality control kit (Life Technologies) to ensure that 10-30% of template-positive ISPs were generated in the emulsion PCR. Finally, the template ISPs were enriched loaded onto an Ion 318™ chip and sequenced using a PGM™ sequencer with the Ion PGM™ sequencing 200 kit v2 according to the manufacturer’s instructions.
Data analysis The raw data were analysed using Torrent Suite v3.6.2 software (Life Technologies). The coverage analysis was performed using the Coverage Analysis plugin v3.6. Cases for which the number of mapped reads was
