Skip to main content

KI-67 heterogeneity in well differentiated gastro-entero-pancreatic neuroendocrine tumors: when is biopsy reliable for grade assessment?

An Erratum to this article was published on 19 August 2017

This article has been updated



Ki-67 heterogeneity can impact on gastroenteropancreatic neuroendocrine tumor grade assignment, especially when tissue is scarce. This work is aimed at devising adequacy criteria for grade assessment in biopsy specimens.


To analyze the impact of biopsy size on reliability, 360 virtual biopsies of different thickness and lengths were constructed. Furthermore, to estimate the mean amount of non-neoplastic tissue component present in biopsies, 28 real biopsies were collected, the non-neoplastic components (fibrosis and inflammation) quantified and the effective area of neoplastic tissue calculated for each biopsy.


Heterogeneity of Ki-67 distribution, G2 tumors and biopsy size all play an important role in reducing the reliability of biopsy samples in Ki-67-based grade assignment. In particular in G2 cases, 59.9% of virtual biopsies downgraded the tumor and the smaller the biopsy, the more frequent downgrading occurs. In real biopsies the presence of non-neoplastic tissue reduced the available total area by a mean of 20%.


By coupling the results from these two different approaches we show that both biopsy size and non-neoplastic component must be taken into account for biopsy adequacy. In particular, we can speculate that if the minimum biopsy area, necessary to confidently (80% concordance) grade gastro-entero-pancreatic neuroendocrine tumors on virtual biopsies ranges between 15 and 30 mm2, and if real biopsies are on average composed of only 80% of neoplastic tissue, then biopsies with a surface area not <12 mm2 should be performed; using 18G needles, this corresponds to a minimum total length of 15 mm.

This is a preview of subscription content, access via your institution.

Fig 1

Change history

  • 19 August 2017

    An erratum to this article has been published.


  1. I.M. Modlin, K. Oberg, D.C. Chung, R.T. Jensen, W.W. de Herder, R.V. Thakker, M. Caplin, G. Delle Fave, G.A. Kaltsas, E.P. Krenning, S.F. Moss, O. Nilsson, G. Rindi, R. Salazar, P. Ruszniewski, A. Sundin, Gastroenteropancreatic neuroendocrine tumors. Lancet. Oncol. 9, 61–72 (2008)

    CAS  Article  PubMed  Google Scholar 

  2. G. Rindi, G. Klöeppel, H. Alhman, M. Caplin, A. Couvelard, W.W. de Herder, B. Eriksson, A. Falchetti, M. Falconi, P. Komminoth, M. Koerner, J.M. Lopes, A.M. McNicol, O. Nilsson, A. Perren, A. Scarpa, J.Y. Scoazec, B. Wiedenmann, all other Frascati Consensus Conference participants; European Neuroendocrine Tumor Society (ENETS), TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows. Arch. 449, 395–401 (2006)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. G. Rindi, G. Klöppel, A. Couvelard, P. Komminoth, M. Koerner, J. M. Lopes, A. M. McNicol, O. Nilsson, A. Perren, A. Scarpa, J.Y. Scoazec, B. Wiedenmann, TNM staging of midgut and hindgut (neuro) endocrine tumors: a consensus proposal including a grading system. Virchows. Arch. 451, 757–762 (2007)

    CAS  Article  Google Scholar 

  4. A. Perren, A. Couvelard, J.Y. Scoazec, F. Costa, I. Borbath, G. Delle Fave, V. Gorbounova, D. Gross, A. Grossma, R.T. Jense, M. Kulke, K. Oeberg, G. Rindi, H. Sorbye, S. Welin all other Antibes Consensus Conference participants ENETS consensus guidelines for the standards of care in neuroendocrine tumors: pathology: diagnosis and prognostic stratification. Neuroendocrinology (2017). doi:10.1159/000457956. [Epub ahead of print]

    Google Scholar 

  5. G. Rindi, R. Arnold, F.T. Bosman, C. Capella, D.S. Klimstra, G. Klöppel, P. Komminoth, E. Solcia, in Nomemclature and classification of neuroendocrine neoplasms of the digestive system. WHO Classification of Tumors of the Digestive System. eds. by F .T. Bosman, F. Carneiro, R. H. Hruban, N. D. Theise (IARC Press, Lyon, 2010), p. 13–14

    Google Scholar 

  6. C.M. McCall, C. Shi, T.C. Cornish, D.S. Klimstra, L.H. Tang, O. Basturk, L.J. Mun, T.A. Ellison, C.L. Wolfgang, M.A. Choti, R.D. Schulick, B.H. Edil, R.H. Hruban, Grading of well-differentiated pancreatic neuroendocrine tumors is improved by the inclusion of both Ki67 proliferative index and mitotic rate. Am. J. Surg. Pathol. 37, 1671–1677 (2013)

    Article  PubMed Central  Google Scholar 

  7. K. Öberg, U. Knigge, D. Kwekkeboom, A. Perren; on behalf of ESMO Guidelines Working Group, Neuroendocrine gastro-entero-pancreatic tumors: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 23, vii124–vii130 (2012)

    PubMed  Google Scholar 

  8. D.S. Klimstra, Reassessing the grade of gastroenteropancreatic neuroendocrine neoplasms. Endocrine 53, 4–6 (2016)

    CAS  Article  PubMed  Google Scholar 

  9. M. Milione, Prognostic factors for gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs): what’s better? Endocrine (2017). doi:10.1007/s12020-017-1299-0. [Epub ahead of print]

    Google Scholar 

  10. A. Couvelard, L. Deschamps, P. Ravaud, G. Baron, A. Sauvanet, O. Hentic, N. Colnot, V. Paradis, J. Belghiti, P. Bedossa, P. Ruszniewski, Heterogeneity of tumor prognostic markers: a reproducibility study applied to liver metastases of pancreatic endocrine tumors. Mod. Pathol. 22, 273–281 (2009)

    CAS  Article  PubMed  Google Scholar 

  11. Z. Yang, L.H. Tang, D.S. Klimstra, Effect of tumor heterogeneity on the assessment of Ki67 labeling index in well-differentiated neuroendocrine tumors metastatic to the liver: implications for prognostic stratification. Am. J. Surg. Pathol. 35, 853–860 (2011)

    Article  PubMed  Google Scholar 

  12. A. Larghi, G. Capurso, A. Carnuccio, R. Ricci, S. Alfieri, D. Galasso, F. Lugli, A. Bianchi, F. Panzuto, L. De Marinis, M. Falconi, G. Delle Fave, G.B. Doglietto, G. Costamagna, G. Rindi, Ki-67 grading of nonfunctioning pancreatic neuroendocrine tumours on histologic samples obtained by EUS-guided fine-needle tissue acquisition: a prospective study. Gastrointest. Endosc. 76, 570–577 (2012)

    Article  Google Scholar 

  13. V. Rebours, J. Cordova, A. Couvelard, M. Fabre, L. Palazzo, M.P. Vullierme, O. Hentic, A. Sauvanet, A. Aubert, P. Bedossa, P. Ruszniewski, Can pancreatic neuroendocrine tumour biopsy accurately determine pathological characteristics? Dig. Liver. Dis. 47, 973–977 (2015)

    Article  PubMed  Google Scholar 

  14. F. Grillo, M. Albertelli, M.P. Brisigotti, T. Borra, M. Boschetti, R. Fiocca, D. Ferone, L. Mastracci, Grade increases in gastroenteropancreatic neuroendocrine tumor metastases compared to the primary tumor. Neuroendocrinology. 103, 452–459 (2015)

    Article  PubMed  Google Scholar 

  15. F. Grillo, S. Pigozzi, P. Ceriolo, P. Calamaro, R. Fiocca, L. Mastracci, Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections. Histochem. Cell. Biol. 144, 93–99 (2015)

    CAS  Article  Google Scholar 

  16. A. Gambella, L. Porro, S. Pigozzi, R. Fiocca, F. Grillo, L. Mastracci, Section detachment in immunohistochemistry: causes, troubleshooting, and problem-solving. Cell. Biol. 148, 95–101 (2017)

    Google Scholar 

  17. M.D. Reid, P. Bagci, N. Ohike, B. Saka, I. Erbarut Seven, N. Dursun, S. Balci, H. Gucer, K.T. Jang, T. Tajiri, O. Basturk, S.Y. Kong, M. Goodman, G. Akkas, V. Adsay, Calculation of the Ki67 index in pancreatic neuroendocrine tumors: a comparative analysis of four counting methodologies. Mod. Pathol. 28, 686–694 (2015)

    Article  PubMed  Google Scholar 

  18. F. Grillo, M. Albertelli, F. Annunziata, M. Boschetti, A. Caff, S. Pigozzi, D. Ferone, L. Mastracci, Twenty years of gastroenteropancreatic neuroendocrine tumors: is reclassification worthwhile and feasible? Endocrine 53, 58–62 (2016)

    CAS  Article  PubMed  Google Scholar 

  19. S.M. Remes, V.J. Tuominen, H. Helin, J. Isola, J. Arola, Grading of neuroendocrine tumors with Ki-67 requires high-quality assessment practices. Am. J. Surg. Pathol. 36, 1359–1363 (2012)

    Article  PubMed  Google Scholar 

  20. B. Weynand, I. Borbath, V. Bernard, C. Sempoux, J.F. Gigot, C. Hubert, V. Lannoy, P.H. Deprez, A. Jouret-Mourin, Pancreatic neuroendocrine tumour grading on endoscopic ultrasound-guided fine needle aspiration: high reproducibility and inter-observer agreement of the Ki-67 labelling index. Cytopathology 25, 389–395 (2014)

    CAS  PubMed  Google Scholar 

  21. L.H. Tang, M. Gonen, C. Hedvat, I.M. Modlin, D.S. Klimstra, Objective quantification of the Ki67 proliferative index in neuroendocrine tumors of the gastroenteropancreatic system: a comparison of digital image analysis with manual methods. Am. J. Surg. Pathol. 36, 1761–1770 (2012)

    Article  PubMed  Google Scholar 

  22. T. Hasegawa, K. Yamao, S. Hijioka, V. Bhatia, N. Mizuno, K. Hara, H. Imaoka, Y. Niwa, M. Tajika, S. Kondo, T. Tanaka, Y. Shimizu, T. Kinoshita, T. Kohsaki, I. Nishimori, S. Iwasaki, T. Saibara, W. Hosoda, Y. Yatabe, Evaluation of Ki-67 index in EUS-FNA specimens for the assessment of malignancy risk in pancreatic neuroendocrine tumors. Endoscopy 46, 32–38 (2014)

    Article  PubMed  Google Scholar 

  23. D.L. Aslan, H.E. Gulbahce, S.E. Pambuccian, J.C. Manivel, J. Jessurun, Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact. Am. J. Clin. Pathol. 123, 874–878 (2005)

    CAS  Article  PubMed  Google Scholar 

Download references


We thank Miss Simona Pigozzi for technical support in immunohistochemistry.


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

Author contributions

Dr. Grillo devised the work, performed the Ki-67 counting and wrote the manuscript. Dr. Valle performed the Ki-67 counting and wrote the manuscript. Prof. Ferone, Dr. Albertelli, and Dr. Cittadini aided in devising the study. Dr. Brisigotti collected samples and performed Ki-67 counting. Prof. Fiocca and Dr. Vanoli critically reviewed the work and manuscript. Dr. Mastracci wrote and critically reviewed the work.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Federica Grillo.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

The original version of this article was revised: the first and surname of all the authors are corrected.

Federica Grillo and Luca Valle contributed equally to this work.

An erratum to this article is available at

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Grillo, F., Valle, L., Ferone, D. et al. KI-67 heterogeneity in well differentiated gastro-entero-pancreatic neuroendocrine tumors: when is biopsy reliable for grade assessment?. Endocrine 57, 494–502 (2017).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Gastroenteropancreatic neuroendocrine tumors
  • Grade
  • Ki-67
  • Biopsy