- Email: [email protected]
Genetic analysis of a hereditary medullary thyroid carcinoma case with normal preoperative serum calcitonin levels
Pathology - Research and Practice 215 (2019) 152529
Contents lists available at ScienceDirect
Pathology - Research and Practice journal homepage: www.elsevier.com/locate/prp
Case report
Genetic analysis of a hereditary medullary thyroid carcinoma case with normal preoperative serum calcitonin levels
T
Gang Zhanga, Yan Jianga, Shu Zhanga, Lianhua Zhaob, Jun Fana, Zhe Zhanga, Jianhui Mac,d, ⁎ Rongrong Chend, Yan Xua, a
Department of Breast and Thyroid Surgery, Daping Hospital, Army Military Medical University, Chongqing, 400042, China Department of Pathology, Daping Hospital, Army Military Medical University, Chongqing, 400042, China c Ludwig Institute for Cancer Research, La Jolla, CA, 92037, USA d Geneplus-Beijing Institute, Beijing, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Calcitonin DICER1 Hereditary medullary thyroid carcinoma RET Oncogene
Context: Serum calcitonin is often elevated in medulla thyroid carcinoma (MTC) and thus serves as an indicator of primary and recurrent disease. However, there are MTC patients with normal Serum calcitonin and the underlying mechanisms are largely unknown. Case Description: A 48-year-old female patient presenting with a right anterior cervical mass was diagnosed with medullary carcinoma. She had elevated carcinoembryonic antigen (CEA) but normal Serum calcitonin levels. Next generation sequencing (NGS) of paired tumor and peripheral blood revealed a germline pathogenic RET mutation, indicating the hereditary nature of MTC in this patient. Two somatic loss-of-function mutations in DICER1 gene were also found, which we postulated to account for the normal calcitonin levels found in this patient. To our knowledge, this is the first report of a hereditary MTC case displaying a normal Serum calcitonin. Conclusions: The case suggests NGS can be used in the diagnosis of hereditary MTC and exploring the reasons of normal Serum calcitonin in these patients.
1. Introduction
of “calcitonin-negative” MTC.
Thyroid medullary carcinomas (MTC) originate from neuroendocrine C-cells, accounting for approximately 4-5% of all thyroid cancers [1]. Synthesized by C-cells, elevated Serum calcitonin levels serves as a diagnostic biomarker for both sporadic and hereditary MTC [2,3]. Further histopathological diagnosis of MTC is confirmed by immunohistochemical staining with calcitonin. There are a few “calcitonin-negative” MTC cases described in literatures [4–8], in which Serum calcitonin was not elevated. However, the underlying mechanisms were unknown. Here, we report an inherited case of MTC with normal preoperative Serum calcitonin levels. Next Generation Sequencing (NGS) revealed a germline pathogenic RET mutation, indicating the hereditary nature of MTC in this patient. We further analyzed somatic mutations of the tumor tissue to investigate the potential cause of normal Serum calcitonin and identify two somatic loss of function mutations in DICER1, which was essential for differentiation of C-cells and thyroid functions [9]. Thus, our study revealed a potential mechanism in the occurrence
2. Case presentation A 48-year-old female patient presenting with a right anterior cervical mass for 6 months was admitted to our center. Ultrasound examination of the thyroid gland (Fig. 1A) suggested hypoechoic nodules on the right lateral lobe of the thyroid gland of about 3.2 cm*1.5 cm in size; a thyroid image reporting and data system (TI-RADS) score of 4b; hypoechoic nodules on the left lateral lobe of the thyroid gland about 1.0 cm * 0.6 cm in size. No flow signal was detected by color ultrasonography in the cervical lymph nodes. Cervical enhanced CT examination (Fig. 1B) found multiple bilateral thyroid nodules with clear boundaries and uneven densities. The largest one located on the right side was approximately 3.2 cm * 2.4 cm * 1.7 cm in size. The nodule on the left thyroid had a calcification shadow in it and the size was approximately 1.0 cm * 0.9 cm * 0.7 cm. Preoperative Serum tests showed elevated thyroglobulin (TG) 89.41 ng ml−1 (normal range: 1.59-50.03 ng ml−1) and
⁎
Corresponding author at: Breast and Thyroid Surgery Department, Daping Hospital, Army Medical University, Chongqing, 400042, China. E-mail addresses: [email protected] (G. Zhang), [email protected] (Y. Jiang), [email protected] (S. Zhang), [email protected] (L. Zhao), [email protected] (J. Fan), [email protected] (Z. Zhang), [email protected] (J. Ma), [email protected] (R. Chen), [email protected] (Y. Xu). https://doi.org/10.1016/j.prp.2019.152529 Received 18 April 2019; Received in revised form 12 June 2019; Accepted 5 July 2019 0344-0338/ © 2019 Published by Elsevier GmbH.
Pathology - Research and Practice 215 (2019) 152529
G. Zhang, et al.
Fig. 1. (a) Ultrasound of the thyroid gland identified hypoechoic nodules on both left (upper) and right thyroid (lower). (b) Computed tomography (CT) examination of the thyroid gland in coronal plane showed both nodules. (c-e) Hematoxylin and eosin (HE)-stained and IHC staining of CEA and calcitonin in the resected tumor. Original magnification 100 × .
Fig. 2. (a) Germline mutation of RET in the patient and her son; (b) two somatic mutations of DICER1 in the tumor samples.
carcinoembryonic antigen (CEA) 5.8 pg ml−1 (normal range: 0-5pg ml−1). However, all other Serum biomarkers were in the normal range, including Serum calcitonin (CT) 7.12 pg ml−1 (normal range: 0-18pg ml−1), and other thyroid function parameters. The sampling of Fine needle aspiration (FNA) biopsy was not satisfactory due to large amount of coarse calcification, and only a small amount of follicular epithelial cells was noticed without malignant
tumor component. Intra-operative frozen section biopsy indicated bilateral thyroid malignant tumors. Total thyroidectomy plus bilateral central lymph node dissection were performed after comprehensive evaluation of the intraoperative findings. Postoperative pathological findings revealed a well differentiated MTC with calcification in both lobes of the thyroid gland. Metastatic lesions were found in the right paratracheal lymph nodes (3/3) and the
2
Pathology - Research and Practice 215 (2019) 152529
G. Zhang, et al.
unnecessary cervical lymphadenectomy. Early diagnosis of MTC is crucial, yet challenging for Serum calcitonin negative patients. In this case, multiple lymph nodes metastasis has occurred at the time of examination. The patient underwent total thyroidectomy, central lymph node dissection and a close postoperative follow-up especially for the lateral cervical lymph nodes. Postoperative monitoring for the calcitonin level and its doubling time may reflect the efficacy of surgery and recurrence of the residual lesions. Changes in CEA levels are also used to monitor MTC progression, especially for those with normal calcitonin. In this study, CEA was slightly elevated before surgery, and returned to normal in 1 month after surgery, indicating the achievement of a Serum biochemical cure. In addition, ctDNA was also monitored for relapse and it has been negative up to present. Germline DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 syndrome (OMIM 601,200), and multinodular goiter is frequently present in those affected families while differentiated thyroid carcinoma is less common [13–18]. In addition to germline mutations, somatic mutations of DICER1 have been identified in tumors such as benign or malignant thyroid tumors, pleuropulmonary blastoma, nonepithelial ovarian tumors, and Wilms tumors [11,19,20]. It has been reported in MTC that genes involved in miRNA biogenesis including DICER were significantly overexpressed in patients harboring RET mutations. And the authors suggested that expression of DICER1, is subjected to RET regulation in a RAS-independent manner [21]. Moreover, Dicer conditional knockout mice with Pax8-cre had a dramatic loss of the thyroid gland follicular architecture associated with down regulation of several differentiation markers, including calcitonin [9,22]. Taken together, in our patient, probably due to the loss of function mutations of DICER1, the RET mutation failed to increase the expression of DICER1, and subsequently no elevated calcitonin level. Thus, we speculated that the somatic mutations in DICER1 gene may be the underlying reason. The mechanisms by which DICER1 influence calcitonin production and secretion requires further study. In summary, with NGS based genetic analysis, we identified a case of hereditary MTC with normal Serum calcitonin for the first time. More importantly, we identified two somatic mutations of DICER1 which may account for normal Serum calcitonin levels in this case.
anterior tracheal lymph nodes (2/8). No metastasis was found in the prelaryngeal lymph nodes (0/1). Immunohistochemical staining of the lesions revealed: Syn (+), Galectin-3 (-), calcitonin (60% focal positive staining), CgA(+), CEA(+), TG (-), TTF-1 (+), S-100 (-), Ki-67 (2%) (Fig. 1C). This patient was diagnosed as MTC (T2N1 AM0, stage Ⅲ). No specific clinical manifestations of hypoparathyroidism (HPTH) or pheochromocytoma (PHEO) were found in the patient. Preoperative parathyroid hormone is in the normal range, and there is no abnormally enlarged parathyroid gland during surgery. No adrenal lesions were found in postoperative ultrasound or adrenal enhanced CT. Thus neither HPTH nor PHEO was considered in the patient. With informed consent, we took 10 formalin-fixed paraffin embedded tumor tissue sections and 10 ml of peripheral blood for tissue and postoperative ctDNA somatic mutation analysis (with DNA from peripheral blood cell as control) and germline mutation analysis with a targeted NGS panel covering 1021 genes (Supplementary Table 1, Geneplus-Beijing, Beijing, China) [10]. We identified a germline mutation of the RET proto-oncogene (NM_020,975.4, c.1832 G > A, p.C611Y), a known pathogenic mutation (Fig. 2A), which confirmed the diagnosis of hereditary MTC in the patient. We further examined the 23-year-old son of the proband for RET mutation with PCR-based Sanger DNA sequencing (Geneplus-Beijing, Beijing, China) and the same mutation was detected (Fig. 2A). In addition, multiple echogenic nodules in the right lobe of the thyroid gland with strong echo points were detected by color Doppler ultrasound examination, which were classified as TI-RADS3 (Supplementary Fig. 1). Thyroid function examination showed normal Serum calcitonin and CEA levels. The DICER1 c.2669 T > A p.L890* (NM_030,621.3) nonsense mutation located at the 18th exon with allele frequency of 26.4% (Fig. 2B), and the DICER1 c.5126A > G p.D1709 G missense mutation was in the 25th exon with allele frequency of 23.0% (Fig. 2C). The DICER1 p.D1709 N mutation had been reported as a hotspot mutation in many cancers, and shown to interfere with the production of 5p miRNAs by DICER1 due to the substitution of the key metal-ion binding amino acid D1709 within the catalytic RNase IIIb domain [11,12]. Thus, the DICER1 c.5126A > G p.D1709 G mutation led to loss of function of DICER1. Postoperative monitoring for Serum calcitonin and CEA levels as well as ctDNA showed Serum calcitonin remained in normal range, and CEA dropped back to normal range 44 d after surgery (Table 1). No somatic mutation was detected in the postoperative ctDNA analysis. The patient is in good condition after surgery up to present.
Disclosure summary The authors have nothing to disclose.
3. Discussion Funding
We presented a hereditary MTC case displaying normal Serum calcitonin here. NGS helped in the diagnosis of familial medullary thyroid carcinoma (FMTC), which is helpful for family members to determine appropriate clinical management. RET mutations in codon 611 is typically associated with later age-of-onset and less aggressive MTC compared with codon 634 mutation [3]. The proband was diagnosed at 48 years old, while the son of the proband, currently 23 years old, who is also a carrier of the RET 611 mutation, had a nodule on the right thyroid gland lobe with a TI-RADS score of 3. At present, his calcitonin and CEA levels are normal. Prophylactic thyroidectomy should be performed as soon as possible with the patient’s consent to avoid
This work was supported by the National Natural Science Foundation of China under Grant81472482; and the Key Laboratory of the Ministry of Education of China under Grant No. 2013jszl13.
Acknowledgments We are thankful to all referring surgeons, pathologists, and specialists for their contributions to this study.
Table 1 Changes in Serum calcitonin and carcinoembryonic antigen levels.
−1
Serum calcitonin (0-18pg ml ) carcinoembryonic antigen (CEA) (0-5pg ml−1)
Preoperative
8 days postoperative
44 days postoperative
7.12 5.8
7.77 5.66
8.82 1.57
Note: Parathyroid hormone levels were decreased postoperatively. Considering the potential occurrence of transient parathyroid shock, postoperative administration of calcium supplementation might have a certain impact on the fluctuation of the calcitonin levels. 3
Pathology - Research and Practice 215 (2019) 152529
G. Zhang, et al.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.prp.2019.152529.
[12]
References [13]
[1] M. Xing, Molecular pathogenesis and mechanisms of thyroid cancer, Nat Rev Cancer 13 (3) (2013) 184–199, https://doi.org/10.1038/nrc3431. [2] G. Costante, D. Meringolo, C. Durante, D. Bianchi, M. Nocera, S. Tumino, U. Crocetti, M. Attard, M. Maranghi, M. Torlontano, S. Filetti, Predictive value of serum calcitonin levels for preoperative diagnosis of medullary thyroid carcinoma in a cohort of 5817 consecutive patients with thyroid nodules, J Clin Endocrinol Metab 92 (2) (2007) 450–455, https://doi.org/10.1210/jc.2006-1590. [3] S.A. Wells Jr., S.L. Asa, H. Dralle, R. Elisei, D.B. Evans, R.F. Gagel, N. Lee, A. Machens, J.F. Moley, F. Pacini, F. Raue, K. Frank-Raue, B. Robinson, M.S. Rosenthal, M. Santoro, M. Schlumberger, M. Shah, S.G. Waguespack, American Thyroid Association Guidelines Task Force on Medullary Thyroid C, Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma, Thyroid 25 (6) (2015) 567–610, https://doi.org/10.1089/thy.2014. 0335. [4] K. Frank-Raue, A. Machens, G. Leidig-Bruckner, S. Rondot, C. Haag, E. Schulze, A. Lorenz, M.C. Kreissl, H. Dralle, F. Raue, K.W. Schmid, Prevalence and clinical spectrum of nonsecretory medullary thyroid carcinoma in a series of 839 patients with sporadic medullary thyroid carcinoma, Thyroid 23 (3) (2013) 294–300, https://doi.org/10.1089/thy.2012.0236. [5] E.F. Brutsaert, A.J. Gersten, A.B. Tassler, M.I. Surks, Medullary thyroid cancer with undetectable serum calcitonin, J Clin Endocrinol Metab 100 (2) (2015) 337–341, https://doi.org/10.1210/jc.2014-3095. [6] P. Trimboli, L. Giovanella, Serum calcitonin negative medullary thyroid carcinoma: a systematic review of the literature, Clin Chem Lab Med 53 (10) (2015) 1507–1514, https://doi.org/10.1515/cclm-2015-0058. [7] M.T. Sama, R. Rossetto Giaccherino, M. Gallo, F. Felicetti, F. Maletta, N. Bonelli, A. Piovesan, N. Palestini, E. Ghigo, E. Arvat, Clinical challenges with calcitoninnegative medullary thyroid carcinoma, J Cancer Res Clin Oncol 142 (9) (2016) 2023–2029, https://doi.org/10.1007/s00432-016-2169-5. [8] Q. Zhou, S. Yue, Y. Cheng, J. Jin, H. Xu, Clinical and pathological analysis of 19 cases of medullary thyroid carcinoma without an increase in calcitonin, Exp Toxicol Pathol 69 (8) (2017) 575–579, https://doi.org/10.1016/j.etp.2017.05.003. [9] D. Frezzetti, C. Reale, G. Cali, L. Nitsch, H. Fagman, O. Nilsson, M. Scarfo, G. De Vita, R. Di Lauro, The microRNA-processing enzyme Dicer is essential for thyroid function, PLoS One 6 (11) (2011) e27648, , https://doi.org/10.1371/journal.pone. 0027648. [10] Y. Wang, C. Zhao, L. Chang, R. Jia, R. Liu, Y. Zhang, X. Gao, J. Li, R. Chen, X. Xia, A. Bulbul, H. Husain, Y. Guan, X. Yi, J. Xu, Circulating tumor DNA analyses predict progressive disease and indicate trastuzumab-resistant mechanism in advanced gastric cancer, EBioMedicine 43 (2019) 261–269, https://doi.org/10.1016/j.ebiom. 2019.04.003. [11] A. Heravi-Moussavi, M.S. Anglesio, S.W. Cheng, J. Senz, W. Yang, L. Prentice, A.P. Fejes, C. Chow, A. Tone, S.E. Kalloger, N. Hamel, A. Roth, G. Ha, A.N. Wan, S. Maines-Bandiera, C. Salamanca, B. Pasini, B.A. Clarke, A.F. Lee, C.H. Lee, C. Zhao, R.H. Young, S.A. Aparicio, P.H. Sorensen, M.M. Woo, N. Boyd, S.J. Jones,
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
4
M. Hirst, M.A. Marra, B. Gilks, S.P. Shah, W.D. Foulkes, G.B. Morin, D.G. Huntsman, Recurrent somatic DICER1 mutations in nonepithelial ovarian cancers, New England J. Med. 366 (3) (2012) 234–242, https://doi.org/10.1056/ NEJMoa1102903. J. Chen, Y. Wang, M.K. McMonechy, M.S. Anglesio, W. Yang, J. Senz, S. MainesBandiera, J. Rosner, G. Trigo-Gonzalez, S.W. Grace Cheng, J. Kim, M.M. Matzuk, G.B. Morin, D.G. Huntsman, Recurrent DICER1 hotspot mutations in endometrial tumours and their impact on microRNA biogenesis, J Pathol 237 (2) (2015) 215–225, https://doi.org/10.1002/path.4569. A.M. Poma, V. Condello, M. Denaro, L. Torregrossa, R. Elisei, P. Vitti, F. Basolo, DICER1 somatic mutations strongly impair miRNA processing even in benign thyroid lesions, Oncotarget 10 (19) (2019) 1785–1797, https://doi.org/10.18632/ oncotarget.26639. J.D. Wasserman, N. Sabbaghian, S. Fahiminiya, R. Chami, O. Mete, M. Acker, M.K. Wu, A. Shlien, L. de Kock, W.D. Foulkes, DICER1 Mutations Are Frequent in Adolescent-Onset Papillary Thyroid Carcinoma, J. Clin. Endocrinology Metabolism 103 (5) (2018) 2009–2015, https://doi.org/10.1210/jc.2017-02698. M.M. Rutter, P. Jha, K.A. Schultz, A. Sheil, A.K. Harris, A.J. Bauer, A.L. Field, J. Geller, D.A. Hill, DICER1 Mutations and Differentiated Thyroid Carcinoma: Evidence of a Direct Association, J. Clin. Endocrinology Metabolism 101 (1) (2016) 1–5, https://doi.org/10.1210/jc.2015-2169. L. de Kock, N. Sabbaghian, D.B. Soglio, R.P. Guillerman, B.K. Park, R. Chami, C.L. Deal, J.R. Priest, W.D. Foulkes, Exploring the association Between DICER1 mutations and differentiated thyroid carcinoma, J. Clin. Endocrinology Metabolism 99 (6) (2014) E1072–1077, https://doi.org/10.1210/jc.2013-4206. L. de Kock, I. Bah, T. Revil, P. Berube, M.K. Wu, N. Sabbaghian, J.R. Priest, J. Ragoussis, W.D. Foulkes, Deep Sequencing Reveals Spatially Distributed Distinct Hot Spot Mutations in DICER1-Related Multinodular Goiter, J. Clin. Endocrinology Metabolism 101 (10) (2016) 3637–3645, https://doi.org/10.1210/jc.2016-1328. T. Rio Frio, A. Bahubeshi, C. Kanellopoulou, N. Hamel, M. Niedziela, N. Sabbaghian, C. Pouchet, L. Gilbert, P.K. O’Brien, K. Serfas, P. Broderick, R.S. Houlston, F. Lesueur, E. Bonora, S. Muljo, R.N. Schimke, D. Bouron-Dal Soglio, J. Arseneau, K.A. Schultz, J.R. Priest, V.H. Nguyen, H.R. Harach, D.M. Livingston, W.D. Foulkes, M. Tischkowitz, DICER1 mutations in familial multinodular goiter with and without ovarian Sertoli-Leydig cell tumors, Jama 305 (1) (2011) 68–77, https://doi.org/10.1001/jama.2010.1910. D.R. Stewart, Y. Messinger, G.M. Williams, J. Yang, A. Field, K.A. Schultz, L.A. Harney, L.A. Doros, L.P. Dehner, D.A. Hill, Nasal chondromesenchymal hamartomas arise secondary to germline and somatic mutations of DICER1 in the pleuropulmonary blastoma tumor predisposition disorder, Human Genetics 133 (11) (2014) 1443–1450, https://doi.org/10.1007/s00439-014-1474-9. M.K. Wu, N. Sabbaghian, B. Xu, S. Addidou-Kalucki, C. Bernard, D. Zou, A.E. Reeve, M.R. Eccles, C. Cole, C.S. Choong, A. Charles, T.Y. Tan, D.M. Iglesias, P.R. Goodyer, W.D. Foulkes, Biallelic DICER1 mutations occur in Wilms tumours, J. Pathology 230 (2) (2013) 154–164, https://doi.org/10.1002/path.4196. C. Puppin, C. Durante, M. Sponziello, A. Verrienti, V. Pecce, E. Lavarone, F. Baldan, A.F. Campese, A. Boichard, L. Lacroix, D. Russo, S. Filetti, G. Damante, Overexpression of genes involved in miRNA biogenesis in medullary thyroid carcinomas with RET mutation, Endocrine 47 (2) (2014) 528–536, https://doi.org/10. 1007/s12020-014-0204-3. M. Bouchard, A. Souabni, M. Busslinger, Tissue-specific expression of cre recombinase from the Pax8 locus, Genesis 38 (3) (2004) 105–109, https://doi.org/10. 1002/gene.20008.
Contents lists available at ScienceDirect
Pathology - Research and Practice journal homepage: www.elsevier.com/locate/prp
Case report
Genetic analysis of a hereditary medullary thyroid carcinoma case with normal preoperative serum calcitonin levels
T
Gang Zhanga, Yan Jianga, Shu Zhanga, Lianhua Zhaob, Jun Fana, Zhe Zhanga, Jianhui Mac,d, ⁎ Rongrong Chend, Yan Xua, a
Department of Breast and Thyroid Surgery, Daping Hospital, Army Military Medical University, Chongqing, 400042, China Department of Pathology, Daping Hospital, Army Military Medical University, Chongqing, 400042, China c Ludwig Institute for Cancer Research, La Jolla, CA, 92037, USA d Geneplus-Beijing Institute, Beijing, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Calcitonin DICER1 Hereditary medullary thyroid carcinoma RET Oncogene
Context: Serum calcitonin is often elevated in medulla thyroid carcinoma (MTC) and thus serves as an indicator of primary and recurrent disease. However, there are MTC patients with normal Serum calcitonin and the underlying mechanisms are largely unknown. Case Description: A 48-year-old female patient presenting with a right anterior cervical mass was diagnosed with medullary carcinoma. She had elevated carcinoembryonic antigen (CEA) but normal Serum calcitonin levels. Next generation sequencing (NGS) of paired tumor and peripheral blood revealed a germline pathogenic RET mutation, indicating the hereditary nature of MTC in this patient. Two somatic loss-of-function mutations in DICER1 gene were also found, which we postulated to account for the normal calcitonin levels found in this patient. To our knowledge, this is the first report of a hereditary MTC case displaying a normal Serum calcitonin. Conclusions: The case suggests NGS can be used in the diagnosis of hereditary MTC and exploring the reasons of normal Serum calcitonin in these patients.
1. Introduction
of “calcitonin-negative” MTC.
Thyroid medullary carcinomas (MTC) originate from neuroendocrine C-cells, accounting for approximately 4-5% of all thyroid cancers [1]. Synthesized by C-cells, elevated Serum calcitonin levels serves as a diagnostic biomarker for both sporadic and hereditary MTC [2,3]. Further histopathological diagnosis of MTC is confirmed by immunohistochemical staining with calcitonin. There are a few “calcitonin-negative” MTC cases described in literatures [4–8], in which Serum calcitonin was not elevated. However, the underlying mechanisms were unknown. Here, we report an inherited case of MTC with normal preoperative Serum calcitonin levels. Next Generation Sequencing (NGS) revealed a germline pathogenic RET mutation, indicating the hereditary nature of MTC in this patient. We further analyzed somatic mutations of the tumor tissue to investigate the potential cause of normal Serum calcitonin and identify two somatic loss of function mutations in DICER1, which was essential for differentiation of C-cells and thyroid functions [9]. Thus, our study revealed a potential mechanism in the occurrence
2. Case presentation A 48-year-old female patient presenting with a right anterior cervical mass for 6 months was admitted to our center. Ultrasound examination of the thyroid gland (Fig. 1A) suggested hypoechoic nodules on the right lateral lobe of the thyroid gland of about 3.2 cm*1.5 cm in size; a thyroid image reporting and data system (TI-RADS) score of 4b; hypoechoic nodules on the left lateral lobe of the thyroid gland about 1.0 cm * 0.6 cm in size. No flow signal was detected by color ultrasonography in the cervical lymph nodes. Cervical enhanced CT examination (Fig. 1B) found multiple bilateral thyroid nodules with clear boundaries and uneven densities. The largest one located on the right side was approximately 3.2 cm * 2.4 cm * 1.7 cm in size. The nodule on the left thyroid had a calcification shadow in it and the size was approximately 1.0 cm * 0.9 cm * 0.7 cm. Preoperative Serum tests showed elevated thyroglobulin (TG) 89.41 ng ml−1 (normal range: 1.59-50.03 ng ml−1) and
⁎
Corresponding author at: Breast and Thyroid Surgery Department, Daping Hospital, Army Medical University, Chongqing, 400042, China. E-mail addresses: [email protected] (G. Zhang), [email protected] (Y. Jiang), [email protected] (S. Zhang), [email protected] (L. Zhao), [email protected] (J. Fan), [email protected] (Z. Zhang), [email protected] (J. Ma), [email protected] (R. Chen), [email protected] (Y. Xu). https://doi.org/10.1016/j.prp.2019.152529 Received 18 April 2019; Received in revised form 12 June 2019; Accepted 5 July 2019 0344-0338/ © 2019 Published by Elsevier GmbH.
Pathology - Research and Practice 215 (2019) 152529
G. Zhang, et al.
Fig. 1. (a) Ultrasound of the thyroid gland identified hypoechoic nodules on both left (upper) and right thyroid (lower). (b) Computed tomography (CT) examination of the thyroid gland in coronal plane showed both nodules. (c-e) Hematoxylin and eosin (HE)-stained and IHC staining of CEA and calcitonin in the resected tumor. Original magnification 100 × .
Fig. 2. (a) Germline mutation of RET in the patient and her son; (b) two somatic mutations of DICER1 in the tumor samples.
carcinoembryonic antigen (CEA) 5.8 pg ml−1 (normal range: 0-5pg ml−1). However, all other Serum biomarkers were in the normal range, including Serum calcitonin (CT) 7.12 pg ml−1 (normal range: 0-18pg ml−1), and other thyroid function parameters. The sampling of Fine needle aspiration (FNA) biopsy was not satisfactory due to large amount of coarse calcification, and only a small amount of follicular epithelial cells was noticed without malignant
tumor component. Intra-operative frozen section biopsy indicated bilateral thyroid malignant tumors. Total thyroidectomy plus bilateral central lymph node dissection were performed after comprehensive evaluation of the intraoperative findings. Postoperative pathological findings revealed a well differentiated MTC with calcification in both lobes of the thyroid gland. Metastatic lesions were found in the right paratracheal lymph nodes (3/3) and the
2
Pathology - Research and Practice 215 (2019) 152529
G. Zhang, et al.
unnecessary cervical lymphadenectomy. Early diagnosis of MTC is crucial, yet challenging for Serum calcitonin negative patients. In this case, multiple lymph nodes metastasis has occurred at the time of examination. The patient underwent total thyroidectomy, central lymph node dissection and a close postoperative follow-up especially for the lateral cervical lymph nodes. Postoperative monitoring for the calcitonin level and its doubling time may reflect the efficacy of surgery and recurrence of the residual lesions. Changes in CEA levels are also used to monitor MTC progression, especially for those with normal calcitonin. In this study, CEA was slightly elevated before surgery, and returned to normal in 1 month after surgery, indicating the achievement of a Serum biochemical cure. In addition, ctDNA was also monitored for relapse and it has been negative up to present. Germline DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 syndrome (OMIM 601,200), and multinodular goiter is frequently present in those affected families while differentiated thyroid carcinoma is less common [13–18]. In addition to germline mutations, somatic mutations of DICER1 have been identified in tumors such as benign or malignant thyroid tumors, pleuropulmonary blastoma, nonepithelial ovarian tumors, and Wilms tumors [11,19,20]. It has been reported in MTC that genes involved in miRNA biogenesis including DICER were significantly overexpressed in patients harboring RET mutations. And the authors suggested that expression of DICER1, is subjected to RET regulation in a RAS-independent manner [21]. Moreover, Dicer conditional knockout mice with Pax8-cre had a dramatic loss of the thyroid gland follicular architecture associated with down regulation of several differentiation markers, including calcitonin [9,22]. Taken together, in our patient, probably due to the loss of function mutations of DICER1, the RET mutation failed to increase the expression of DICER1, and subsequently no elevated calcitonin level. Thus, we speculated that the somatic mutations in DICER1 gene may be the underlying reason. The mechanisms by which DICER1 influence calcitonin production and secretion requires further study. In summary, with NGS based genetic analysis, we identified a case of hereditary MTC with normal Serum calcitonin for the first time. More importantly, we identified two somatic mutations of DICER1 which may account for normal Serum calcitonin levels in this case.
anterior tracheal lymph nodes (2/8). No metastasis was found in the prelaryngeal lymph nodes (0/1). Immunohistochemical staining of the lesions revealed: Syn (+), Galectin-3 (-), calcitonin (60% focal positive staining), CgA(+), CEA(+), TG (-), TTF-1 (+), S-100 (-), Ki-67 (2%) (Fig. 1C). This patient was diagnosed as MTC (T2N1 AM0, stage Ⅲ). No specific clinical manifestations of hypoparathyroidism (HPTH) or pheochromocytoma (PHEO) were found in the patient. Preoperative parathyroid hormone is in the normal range, and there is no abnormally enlarged parathyroid gland during surgery. No adrenal lesions were found in postoperative ultrasound or adrenal enhanced CT. Thus neither HPTH nor PHEO was considered in the patient. With informed consent, we took 10 formalin-fixed paraffin embedded tumor tissue sections and 10 ml of peripheral blood for tissue and postoperative ctDNA somatic mutation analysis (with DNA from peripheral blood cell as control) and germline mutation analysis with a targeted NGS panel covering 1021 genes (Supplementary Table 1, Geneplus-Beijing, Beijing, China) [10]. We identified a germline mutation of the RET proto-oncogene (NM_020,975.4, c.1832 G > A, p.C611Y), a known pathogenic mutation (Fig. 2A), which confirmed the diagnosis of hereditary MTC in the patient. We further examined the 23-year-old son of the proband for RET mutation with PCR-based Sanger DNA sequencing (Geneplus-Beijing, Beijing, China) and the same mutation was detected (Fig. 2A). In addition, multiple echogenic nodules in the right lobe of the thyroid gland with strong echo points were detected by color Doppler ultrasound examination, which were classified as TI-RADS3 (Supplementary Fig. 1). Thyroid function examination showed normal Serum calcitonin and CEA levels. The DICER1 c.2669 T > A p.L890* (NM_030,621.3) nonsense mutation located at the 18th exon with allele frequency of 26.4% (Fig. 2B), and the DICER1 c.5126A > G p.D1709 G missense mutation was in the 25th exon with allele frequency of 23.0% (Fig. 2C). The DICER1 p.D1709 N mutation had been reported as a hotspot mutation in many cancers, and shown to interfere with the production of 5p miRNAs by DICER1 due to the substitution of the key metal-ion binding amino acid D1709 within the catalytic RNase IIIb domain [11,12]. Thus, the DICER1 c.5126A > G p.D1709 G mutation led to loss of function of DICER1. Postoperative monitoring for Serum calcitonin and CEA levels as well as ctDNA showed Serum calcitonin remained in normal range, and CEA dropped back to normal range 44 d after surgery (Table 1). No somatic mutation was detected in the postoperative ctDNA analysis. The patient is in good condition after surgery up to present.
Disclosure summary The authors have nothing to disclose.
3. Discussion Funding
We presented a hereditary MTC case displaying normal Serum calcitonin here. NGS helped in the diagnosis of familial medullary thyroid carcinoma (FMTC), which is helpful for family members to determine appropriate clinical management. RET mutations in codon 611 is typically associated with later age-of-onset and less aggressive MTC compared with codon 634 mutation [3]. The proband was diagnosed at 48 years old, while the son of the proband, currently 23 years old, who is also a carrier of the RET 611 mutation, had a nodule on the right thyroid gland lobe with a TI-RADS score of 3. At present, his calcitonin and CEA levels are normal. Prophylactic thyroidectomy should be performed as soon as possible with the patient’s consent to avoid
This work was supported by the National Natural Science Foundation of China under Grant81472482; and the Key Laboratory of the Ministry of Education of China under Grant No. 2013jszl13.
Acknowledgments We are thankful to all referring surgeons, pathologists, and specialists for their contributions to this study.
Table 1 Changes in Serum calcitonin and carcinoembryonic antigen levels.
−1
Serum calcitonin (0-18pg ml ) carcinoembryonic antigen (CEA) (0-5pg ml−1)
Preoperative
8 days postoperative
44 days postoperative
7.12 5.8
7.77 5.66
8.82 1.57
Note: Parathyroid hormone levels were decreased postoperatively. Considering the potential occurrence of transient parathyroid shock, postoperative administration of calcium supplementation might have a certain impact on the fluctuation of the calcitonin levels. 3
Pathology - Research and Practice 215 (2019) 152529
G. Zhang, et al.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.prp.2019.152529.
[12]
References [13]
[1] M. Xing, Molecular pathogenesis and mechanisms of thyroid cancer, Nat Rev Cancer 13 (3) (2013) 184–199, https://doi.org/10.1038/nrc3431. [2] G. Costante, D. Meringolo, C. Durante, D. Bianchi, M. Nocera, S. Tumino, U. Crocetti, M. Attard, M. Maranghi, M. Torlontano, S. Filetti, Predictive value of serum calcitonin levels for preoperative diagnosis of medullary thyroid carcinoma in a cohort of 5817 consecutive patients with thyroid nodules, J Clin Endocrinol Metab 92 (2) (2007) 450–455, https://doi.org/10.1210/jc.2006-1590. [3] S.A. Wells Jr., S.L. Asa, H. Dralle, R. Elisei, D.B. Evans, R.F. Gagel, N. Lee, A. Machens, J.F. Moley, F. Pacini, F. Raue, K. Frank-Raue, B. Robinson, M.S. Rosenthal, M. Santoro, M. Schlumberger, M. Shah, S.G. Waguespack, American Thyroid Association Guidelines Task Force on Medullary Thyroid C, Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma, Thyroid 25 (6) (2015) 567–610, https://doi.org/10.1089/thy.2014. 0335. [4] K. Frank-Raue, A. Machens, G. Leidig-Bruckner, S. Rondot, C. Haag, E. Schulze, A. Lorenz, M.C. Kreissl, H. Dralle, F. Raue, K.W. Schmid, Prevalence and clinical spectrum of nonsecretory medullary thyroid carcinoma in a series of 839 patients with sporadic medullary thyroid carcinoma, Thyroid 23 (3) (2013) 294–300, https://doi.org/10.1089/thy.2012.0236. [5] E.F. Brutsaert, A.J. Gersten, A.B. Tassler, M.I. Surks, Medullary thyroid cancer with undetectable serum calcitonin, J Clin Endocrinol Metab 100 (2) (2015) 337–341, https://doi.org/10.1210/jc.2014-3095. [6] P. Trimboli, L. Giovanella, Serum calcitonin negative medullary thyroid carcinoma: a systematic review of the literature, Clin Chem Lab Med 53 (10) (2015) 1507–1514, https://doi.org/10.1515/cclm-2015-0058. [7] M.T. Sama, R. Rossetto Giaccherino, M. Gallo, F. Felicetti, F. Maletta, N. Bonelli, A. Piovesan, N. Palestini, E. Ghigo, E. Arvat, Clinical challenges with calcitoninnegative medullary thyroid carcinoma, J Cancer Res Clin Oncol 142 (9) (2016) 2023–2029, https://doi.org/10.1007/s00432-016-2169-5. [8] Q. Zhou, S. Yue, Y. Cheng, J. Jin, H. Xu, Clinical and pathological analysis of 19 cases of medullary thyroid carcinoma without an increase in calcitonin, Exp Toxicol Pathol 69 (8) (2017) 575–579, https://doi.org/10.1016/j.etp.2017.05.003. [9] D. Frezzetti, C. Reale, G. Cali, L. Nitsch, H. Fagman, O. Nilsson, M. Scarfo, G. De Vita, R. Di Lauro, The microRNA-processing enzyme Dicer is essential for thyroid function, PLoS One 6 (11) (2011) e27648, , https://doi.org/10.1371/journal.pone. 0027648. [10] Y. Wang, C. Zhao, L. Chang, R. Jia, R. Liu, Y. Zhang, X. Gao, J. Li, R. Chen, X. Xia, A. Bulbul, H. Husain, Y. Guan, X. Yi, J. Xu, Circulating tumor DNA analyses predict progressive disease and indicate trastuzumab-resistant mechanism in advanced gastric cancer, EBioMedicine 43 (2019) 261–269, https://doi.org/10.1016/j.ebiom. 2019.04.003. [11] A. Heravi-Moussavi, M.S. Anglesio, S.W. Cheng, J. Senz, W. Yang, L. Prentice, A.P. Fejes, C. Chow, A. Tone, S.E. Kalloger, N. Hamel, A. Roth, G. Ha, A.N. Wan, S. Maines-Bandiera, C. Salamanca, B. Pasini, B.A. Clarke, A.F. Lee, C.H. Lee, C. Zhao, R.H. Young, S.A. Aparicio, P.H. Sorensen, M.M. Woo, N. Boyd, S.J. Jones,
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
4
M. Hirst, M.A. Marra, B. Gilks, S.P. Shah, W.D. Foulkes, G.B. Morin, D.G. Huntsman, Recurrent somatic DICER1 mutations in nonepithelial ovarian cancers, New England J. Med. 366 (3) (2012) 234–242, https://doi.org/10.1056/ NEJMoa1102903. J. Chen, Y. Wang, M.K. McMonechy, M.S. Anglesio, W. Yang, J. Senz, S. MainesBandiera, J. Rosner, G. Trigo-Gonzalez, S.W. Grace Cheng, J. Kim, M.M. Matzuk, G.B. Morin, D.G. Huntsman, Recurrent DICER1 hotspot mutations in endometrial tumours and their impact on microRNA biogenesis, J Pathol 237 (2) (2015) 215–225, https://doi.org/10.1002/path.4569. A.M. Poma, V. Condello, M. Denaro, L. Torregrossa, R. Elisei, P. Vitti, F. Basolo, DICER1 somatic mutations strongly impair miRNA processing even in benign thyroid lesions, Oncotarget 10 (19) (2019) 1785–1797, https://doi.org/10.18632/ oncotarget.26639. J.D. Wasserman, N. Sabbaghian, S. Fahiminiya, R. Chami, O. Mete, M. Acker, M.K. Wu, A. Shlien, L. de Kock, W.D. Foulkes, DICER1 Mutations Are Frequent in Adolescent-Onset Papillary Thyroid Carcinoma, J. Clin. Endocrinology Metabolism 103 (5) (2018) 2009–2015, https://doi.org/10.1210/jc.2017-02698. M.M. Rutter, P. Jha, K.A. Schultz, A. Sheil, A.K. Harris, A.J. Bauer, A.L. Field, J. Geller, D.A. Hill, DICER1 Mutations and Differentiated Thyroid Carcinoma: Evidence of a Direct Association, J. Clin. Endocrinology Metabolism 101 (1) (2016) 1–5, https://doi.org/10.1210/jc.2015-2169. L. de Kock, N. Sabbaghian, D.B. Soglio, R.P. Guillerman, B.K. Park, R. Chami, C.L. Deal, J.R. Priest, W.D. Foulkes, Exploring the association Between DICER1 mutations and differentiated thyroid carcinoma, J. Clin. Endocrinology Metabolism 99 (6) (2014) E1072–1077, https://doi.org/10.1210/jc.2013-4206. L. de Kock, I. Bah, T. Revil, P. Berube, M.K. Wu, N. Sabbaghian, J.R. Priest, J. Ragoussis, W.D. Foulkes, Deep Sequencing Reveals Spatially Distributed Distinct Hot Spot Mutations in DICER1-Related Multinodular Goiter, J. Clin. Endocrinology Metabolism 101 (10) (2016) 3637–3645, https://doi.org/10.1210/jc.2016-1328. T. Rio Frio, A. Bahubeshi, C. Kanellopoulou, N. Hamel, M. Niedziela, N. Sabbaghian, C. Pouchet, L. Gilbert, P.K. O’Brien, K. Serfas, P. Broderick, R.S. Houlston, F. Lesueur, E. Bonora, S. Muljo, R.N. Schimke, D. Bouron-Dal Soglio, J. Arseneau, K.A. Schultz, J.R. Priest, V.H. Nguyen, H.R. Harach, D.M. Livingston, W.D. Foulkes, M. Tischkowitz, DICER1 mutations in familial multinodular goiter with and without ovarian Sertoli-Leydig cell tumors, Jama 305 (1) (2011) 68–77, https://doi.org/10.1001/jama.2010.1910. D.R. Stewart, Y. Messinger, G.M. Williams, J. Yang, A. Field, K.A. Schultz, L.A. Harney, L.A. Doros, L.P. Dehner, D.A. Hill, Nasal chondromesenchymal hamartomas arise secondary to germline and somatic mutations of DICER1 in the pleuropulmonary blastoma tumor predisposition disorder, Human Genetics 133 (11) (2014) 1443–1450, https://doi.org/10.1007/s00439-014-1474-9. M.K. Wu, N. Sabbaghian, B. Xu, S. Addidou-Kalucki, C. Bernard, D. Zou, A.E. Reeve, M.R. Eccles, C. Cole, C.S. Choong, A. Charles, T.Y. Tan, D.M. Iglesias, P.R. Goodyer, W.D. Foulkes, Biallelic DICER1 mutations occur in Wilms tumours, J. Pathology 230 (2) (2013) 154–164, https://doi.org/10.1002/path.4196. C. Puppin, C. Durante, M. Sponziello, A. Verrienti, V. Pecce, E. Lavarone, F. Baldan, A.F. Campese, A. Boichard, L. Lacroix, D. Russo, S. Filetti, G. Damante, Overexpression of genes involved in miRNA biogenesis in medullary thyroid carcinomas with RET mutation, Endocrine 47 (2) (2014) 528–536, https://doi.org/10. 1007/s12020-014-0204-3. M. Bouchard, A. Souabni, M. Busslinger, Tissue-specific expression of cre recombinase from the Pax8 locus, Genesis 38 (3) (2004) 105–109, https://doi.org/10. 1002/gene.20008.