New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation

Autoři: Suzan Schwertheim aff001;  Sarah Theurer aff001;  Holger Jastrow aff002;  Thomas Herold aff001;  Saskia Ting aff001;  Daniela Westerwick aff001;  Stefanie Bertram aff001;  Christoph M. Schaefer aff001;  Julia Kälsch aff001;  Hideo A. Baba aff001;  Kurt W. Schmid aff001
Působiště autorů: Institute of Pathology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany aff001;  Institute of Anatomy and Electron Microscopy Unit of Imaging Center Essen, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany aff002;  Department of Gastroenterology and Hepatology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226199



Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function.


NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing.


In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14–29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins.


NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.

Klíčová slova:

Cytoplasm – Cytoplasmic staining – Immunostaining – Lamins – Membrane staining – Nuclear membrane – Thyroid carcinomas – Cytoplasmic inclusions


1. Ehrlich P. Ueber das Vorkommen von Glykogen im diabetischen und im normalen Organismus. Z Klin Med. 1883;6:33–46.

2. Schiller E. Kerneinschluesse und Amitose. Z Zellforsch Mikrosk Anat. 1949;34:356–361.

3. Schiller E. Variationsstatistische Untersuchungen ueber Kerneinschluesse und Kristalle der menschlichen Leber. Z Zellforsch Mikrosk Anat. 1949;34:337–355.

4. Christ ML, Haja J. Intranuclear cytoplasmic inclusions (invaginations) in thyroid aspirations. Frequency and specificity. Acta Cytol. 1979;23:327–331. 231366

5. Oyama T. A histopathological, immunohistochemical and ultrastructural study of intranuclear cytoplasmic inclusions in thyroid papillary carcinoma. Virchows Arch A Pathol Anat Histopathol. 1989;414:91–104. doi: 10.1007/bf00718588 2536977

6. Kleinfeld RG, Greider MH, Frajola WJ. Electron microscopy of intranuclear inclusions found in human and rat liver parenchymal cells. J Biophys Biochem Cytol. 1956;2:435–439. doi: 10.1083/jcb.2.4.435 13357582

7. Ip Y-T, Dias Filho MA, Chan JKC. Nuclear inclusions and pseudoinclusions: friends or foes of the surgical pathologist? Int J Surg Pathol. 2010;18:465–481. doi: 10.1177/1066896910385342 21081532

8. Schwertheim S, Westerwick D, Jastrow H, Theurer S, Schaefer CM, Kälsch J et al. Intranuclear inclusions in hepatocellular carcinoma contain autophagy-associated proteins and correlate with prolonged survival. J Pathol Clin Res. 2019;

9. Jaskolski D, Papierz T, Liberski PP, Sikorska B. Ultrastructure of meningiomas: autophagy is involved in the pathogenesis of “intranuclear vacuoles”. Folia Neuropathol. 2012;50:187–193. 22773465

10. Arora SK, Dey P. Intranuclear peudoinclusions: Morphology, pathogenesis, and significance. Diagn Cytopathol. 2012;40:741–744. doi: 10.1002/dc.21714 21548120

11. Asioli S, Bussolati G. Emerin immunohistochemistry reveals diagnostic features of nuclear membrane arrangement in thyroid lesions. Histopathology. 2009;54:571–579. doi: 10.1111/j.1365-2559.2009.03259.x 19302538

12. Carcangiu ML, Zampi G, Rosai J. Papillary thyroid carcinoma: a study of its many morphologic expressions and clinical correlates. Pathol Annu. 1985;20:1–44.

13. Kaneko C, Shamoto M, Niimi H, Osada A, Shimizu M, Shinzato M. Studies on intranuclear inclusions and nuclear grooves in papillary thyroid cancer by light, scanning electron and transmission electron microscopy. Acta Cytol. 1996;40:417–422. doi: 10.1159/000333892 8669172

14. Söderström N, Biörklund A. Intranuclear cytoplasmic inclusions in some types of thyroid cancer. Acta Cytol. 1973;17:191–197. 4349305

15. Rezk S, Brynes RK, Nelson V, Thein M, Patwardhan N, Fischer A et al. Beta-Catenin expression in thyroid follicular lesions: Potential role in nuclear envelope changes in papillary carcinomas. Endocr Pathol. 2004;15:329–337. doi: 10.1385/ep:15:4:329 15681857

16. Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 2016;12:1–222. doi: 10.1080/15548627.2015.1100356 26799652

17. Schmid KW. Molecular pathology of thyroid tumors. Pathologe. 2010;31:229–233. doi: 10.1007/s00292-010-1321-2 20717681

18. Maddodi N, Huang W, Havighurst T, Kim K, Longley BJ, Setaluri V. Induction of autophagy and inhibition of melanoma growth in vitro and in vivo by hyperactivation of oncogenic BRAF. J Invest Dermatol. 2010;130:1657–1667. doi: 10.1038/jid.2010.26 20182446

19. Wang Y, Guo Q, Zhao Y, Chen J, Wang S, Hu JUN et al. BRAF-activated long non-coding RNA contributes to cell proliferation and activates autophagy in papillary thyroid carcinoma. Oncol Lett. 2014;8:1947–1952. doi: 10.3892/ol.2014.2487 25289082

20. Lloyd RV, Osamura RY, Klöppel G, Rosai J, Bosman FT, Jaffe ES et al. WHO classification of tumours of endocrine organs. 4 th ed. Lyon, France: International Agency for Research on Cancer (IARC); 2017

21. Schmid KW. Pathogenese, Klassifikation und Histologie von Schilddrüsenkarzinomen. Der Onkologe. 2010;16:644–656.

22. Shurbaji MS, Gupta PK, Frost JK. Nuclear grooves: a useful criterion in the cytopathologic diagnosis of papillary thyroid carcinoma. Diagn Cytopathol. 1988;4:91–94. doi: 10.1002/dc.2840040202 2468463

23. Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci. 2004;117:2805–2812. doi: 10.1242/jcs.01131 15169837

24. Pinto HC, Baptista A, Camilo ME, Valente A, Saragoca A, de Moura MC. Nonalcoholic steatohepatitis. Dig Dis Sci. 1996;41:172–179. doi: 10.1007/bf02208601 8565753

25. Papotti M, Manazza AD, Chiarle R, Bussolati G. Confocal microscope analysis and tridimensional reconstruction of papillary thyroid carcinoma nuclei. Virchows Arch. 2004;444:350–355. doi: 10.1007/s00428-003-0962-4 14758551

26. Leduc EH, Wilson JW. An electron microscope study of intranuclear inclusions in mouse liver and hepatoma. J Biophys Biochem Cytol. 1959;6:427–430. doi: 10.1083/jcb.6.3.427 14415166

27. Hagemann S, Wohlschlaeger J, Bertram S, Levkau B, Musacchio A, Conway EM et al. Loss of Survivin influences liver regeneration and is associated with impaired Aurora B function. Cell Death Differ. 2013;20:834–844. doi: 10.1038/cdd.2013.20 23519077

28. Odagiri S, Tanji K, Mori F, Kakita A, Takahashi H, Kamitani T et al. Immunohistochemical analysis of Marinesco bodies, using antibodies against proteins implicated in the ubiquitin-proteasome system, autophagy and aggresome formation. Neuropathology. 2012;32:261–266. doi: 10.1111/j.1440-1789.2011.01267.x 22118216

29. Dou Z, Ivanov A, Adams PD, Berger SL. Mammalian autophagy degrades nuclear constituents in response to tumorigenic stress. Autophagy. 2016;12:1416–1417. doi: 10.1080/15548627.2015.1127465 26654219

30. Shin H-JR, Kim H, Oh S, Lee J-G, Kee M, Ko H-J et al. AMPK–SKP2–CARM1 signalling cascade in transcriptional regulation of autophagy. Nature. 2016;534:553–557. doi: 10.1038/nature18014 27309807

31. Wei X, Li X, Yan W, Zhang X, Sun Y, Zhang F. SKP2 Promotes Hepatocellular Carcinoma Progression Through Nuclear AMPK-SKP2-CARM1 Signaling Transcriptionally Regulating Nutrient-Deprived Autophagy Induction. Cell Physiol Biochem. 2018;47:2484–2497. doi: 10.1159/000491622 29991055

32. Chiosea S, Nikiforova M, Zuo H, Ogilvie J, Gandhi M, Seethala RR et al. A novel complex BRAF mutation detected in a solid variant of papillary thyroid carcinoma. Endocr Pathol. 2009;20:122–126. doi: 10.1007/s12022-009-9073-3 19370421

33. Cohen Y, Xing M, Mambo E, Guo Z, Wu G, Trink B et al. BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst. 2003;95:625–627. doi: 10.1093/jnci/95.8.625 12697856

34. Katoh H, Yamashita K, Enomoto T, Watanabe M. Classification and general considerations of thyroid cancer. Ann Clin Pathol. 2015;3:1045.

35. Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res. 2003;63:1454–1457. 12670889

36. Nikiforova MN, Kimura ET, Gandhi M, Biddinger PW, Knauf JA, Basolo F et al. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab. 2003;88:5399–5404. doi: 10.1210/jc.2003-030838 14602780

37. Jin L, Chen E, Dong S, Cai Y, Zhang X, Zhou Y et al. BRAF and TERT promoter mutations in the aggressiveness of papillary thyroid carcinoma: a study of 653 patients. Oncotarget. 2016;7:18346–18355. doi: 10.18632/oncotarget.7811 26943032

38. Guan H, Ji M, Bao R, Yu H, Wang Y, Hou P et al. Association of high iodine intake with the T1799A BRAF mutation in papillary thyroid cancer. J Clin Endocrinol Metab. 2009;94:1612–1617. doi: 10.1210/jc.2008-2390 19190105

39. Giorgadze TA, Scognamiglio T, Yang GCH. Fine-needle aspiration cytology of the solid variant of papillary thyroid carcinoma: A study of 13 cases with clinical, histologic, and ultrasound correlations. Cancer Cytopathol. 2015;123:71–81. doi: 10.1002/cncy.21504 25572906

40. Lim JY, Hong SW, Lee YS, Kim B-W, Park CS, Chang H-S et al. Clinicopathologic implications of the BRAF V600E mutation in papillary thyroid cancer: a subgroup analysis of 3130 cases in a single center. Thyroid. 2013;23:1423–1430. doi: 10.1089/thy.2013.0036 23496275

41. Smith RA, Salajegheh A, Weinstein S, Nassiri M, Lam AK-y. Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant. Hum Pathol. 2011;42:500–506. doi: 10.1016/j.humpath.2009.09.023 21167555

42. Finkelstein A, Levy GH, Hui P, Prasad A, Virk R, Chhieng DC et al. Papillary thyroid carcinomas with and without BRAF V600E mutations are morphologically distinct. Histopathology. 2012;60:1052–1059. doi: 10.1111/j.1365-2559.2011.04149.x 22335197

43. Rossi ED, Bizzarro T, Martini M, Capodimonti S, Cenci T, Fadda G et al. Morphological features that can predict BRAFV600E-mutated carcinoma in paediatric thyroid cytology. Cytopathology. 2017;28:55–64. doi: 10.1111/cyt.12350 27256275

44. Rossi ED, Bizzarro T, Martini M, Capodimonti S, Fadda G, Larocca LM et al. Morphological parameters able to predict BRAFV600E-mutated malignancies on thyroid fine-needle aspiration cytology: Our institutional experience. Cancer Cytopathol. 2014;122:883–891. doi: 10.1002/cncy.21475 25156883

45. Virk RK, Theoharis CGA, Prasad A, Chhieng D, Prasad ML. Morphology predicts BRAF V600E mutation in papillary thyroid carcinoma: an interobserver reproducibility study. Virchows Arch. 2014;464:435–442. doi: 10.1007/s00428-014-1552-3 24549591

46. Cui Y, Borysova MK, Johnson JO, Guadagno TM. Oncogenic B-RafV600E induces spindle abnormalities, supernumerary centrosomes, and aneuploidy in human melanocytic cells. Cancer Res. 2010;70:675–684. doi: 10.1158/0008-5472.CAN-09-1491 20068179

47. Liu J, Cheng X, Zhang Y, Li S, Cui H, Zhang L et al. Phosphorylation of Mps1 by BRAF V600E prevents Mps1 degradation and contributes to chromosome instability in melanoma. Oncogene. 2013;32:713–723. doi: 10.1038/onc.2012.94 22430208

48. Fischer AH. The diagnostic pathology of the nuclear envelope in human cancers. Adv Exp Med Biol. 2014;773:49–75. doi: 10.1007/978-1-4899-8032-8_3 24563343

49. Fischer AH, Bond JA, Taysavang P, Battles OE, Wynford-Thomas D. Papillary thyroid carcinoma oncogene (RET/PTC) alters the nuclear envelope and chromatin structure. Am J Pathol. 1998;153:1443–1450. doi: 10.1016/S0002-9440(10)65731-8 9811335

50. Fischer AH, Taysavang P, Jhiang SM. Nuclear envelope irregularity is induced by RET/PTC during interphase. Am J Pathol. 2003;163:1091–1100. doi: 10.1016/S0002-9440(10)63468-2 12937150

51. Fischer AH, Zhao C, Li QK, Gustafson KS, Eltoum I-E, Tambouret R et al. The cytologic criteria of malignancy. J Cell Biochem. 2010;110:795–811. doi: 10.1002/jcb.22585 20564180

52. Knauf JA, Ma X, Smith EP, Zhang L, Mitsutake N, Liao X-H et al. Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res. 2005;65:4238–4245. doi: 10.1158/0008-5472.CAN-05-0047 15899815

53. Bozler J, Nguyen HQ, Rogers GC, Bosco G. Condensins exert force on chromatin-nuclear envelope tethers to mediate nucleoplasmic reticulum formation in Drosophila melanogaster. G3 (Bethesda). 2015;5:341–352.

54. Lammerding J, Fong LG, Ji JY, Reue K, Stewart CL, Young SG et al. Lamins A and C but Not Lamin B1 Regulate Nuclear Mechanics. J Biol Chem. 2006;281:25768–25780. doi: 10.1074/jbc.M513511200 16825190

55. Maniotis AJ, Chen CS, Ingber DE. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc Natl Acad Sci U S A. 1997;94:849–854. doi: 10.1073/pnas.94.3.849 9023345

56. Grbovic OM, Basso AD, Sawai A, Ye Q, Friedlander P, Solit D et al. V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc Natl Acad Sci U S A. 2006;103:57–62. doi: 10.1073/pnas.0609973103 16371460

57. Chiappetta G, Basile A, Arra C, Califano D, Pasquinelli R, Barbieri A et al. BAG3 down-modulation reduces anaplastic thyroid tumor growth by enhancing proteasome-mediated degradation of BRAF protein. J Clin Endocrinol Metab. 2012;97:E115–E120. doi: 10.1210/jc.2011-0484 22072743

58. Galdiero F, Bello AM, Spina A, Capiluongo A, Liuu S, De Marco M et al. Identification of BAG3 target proteins in anaplastic thyroid cancer cells by proteomic analysis. Oncotarget. 2018;9:8016–8026. doi: 10.18632/oncotarget.23858 29487711

59. Samant RS, Clarke PA, Workman P. E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells. Proc Natl Acad Sci U S A. 2014;111:6834–6839. doi: 10.1073/pnas.1322412111 24760825

60. Saei A, Palafox M, Benoukraf T, Kumari N, Jaynes PW, Iyengar PV et al. Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies. J Exp Med. 2018;215:1913–1928. doi: 10.1084/jem.20171960 29880484

61. Saei A, Eichhorn PJA. Ubiquitination and adaptive responses to BRAF inhibitors in Melanoma. Mol Cell Oncol. 2018;5:e1497862. doi: 10.1080/23723556.2018.1497862 30263945

62. Kim H, Kim E-S, Koo J. Expression of autophagy-related proteins in different types of thyroid cancer. Int J Mol Sci. 2017;18:E540. doi: 10.3390/ijms18030540 28257096

Článek vyšel v časopise


2019 Číslo 12