Grading and Ki67/Mitotic Count

Grade is the aggressiveness of cancer – basically, how fast it’s growing. Neuroendocrine Neoplasms (NENs) are divided into 3 Grades depending on what range the Ki-67% measurement number lands in. With NENs, the differentiation (well or poorly) is very important when dealing with grade 3 as specialists now regard a well-differentiated tumour (i.e. a NET) with a Ki67 of less than 55% as important for outcomes and therapy decisions.

So, what is KI-67? It’s a nuclear protein associated with and may be necessary for cellular proliferation. It’s, therefore, an excellent marker to determine the growth fraction of a given cell population. The fraction of Ki-67-positive tumour cells (the Ki-67 labelling index) is often correlated with the clinical course of cancer. Pathologists normally need to count about a thousand cells to determine the percentage of cells that are Ki67 positive – thus why you see Ki67 expressed as a percentage. Zero percent is the lowest, 100% is the highest. Often, they add greater or less than signs depending on the sample involved, i.e. >5% or <5%. There are other measurement systems in place, mainly Mitotic Count which tends to be more frequently used for Lung and Thymic NENs. Even in cases of small biopsies, the Ki-67 index can usually be determined but Mitotic rate counting requires a moderate amount of tumour tissue (at least 50 HPFs or 10 mm) and that may not be feasible for small biopsies. The Mitotic Count method may be preferred or used instead or in addition to Ki-67 for certain Lung and Thymic NET scenarios as it is said to be more helpful in distinguishing atypical from typical Lung and Thymic NETs, and for small and large cell lung Neuroendocrine Carcinomas (NEC).

Please note there are major differences in how Pheochromocytomas and Paragangliomas are graded and staged, see below.

Why is differentiation important?

To fully understand grading, you also need to understand the concept of ‘differentiation’. In the most basic of terms, ‘differentiation’ refers to the extent to which the cancerous cells resemble their non-cancerous counterparts. This is an important point for high grade NENs because the differentiation of a high grade NEN could have an impact on both prognostics and treatment regimes.

Although there are 3 grades of NEN, there are two classifications of differentiation, and these become really important and only applicable at Grade 3.

Some of you may have heard the term ‘moderately differentiated’ which tended to align with an intermediate grade or Grade 2. However, please note that the term moderately differentiated as a classification was phased out in 2010 by WHO reducing from 3 differentiation levels to 2. Grade 2 is also defined as well-differentiated but based on different proliferative rate (see table).

Grading – Key WHO 2019 Changes (GEPNENs)

WHO Classifications of Cancer are published in something known in the medical world as “The Blue Book”. It’s important to understand the source of how all types of cancer (including Neuroendocrine) are named and graded, including any associated terms. This mainly comes from a World Health Organisation agency known as the International Agency for Research on Cancer (IARC). The “Blue Book” is in fact several books. Each book covers an anatomical system with the human body and Neuroendocrine Neoplasms are covered within each particular system – this is a topical point for Neuroendocrine Neoplasms given that they can appear anywhere in the body and often this causes confusion when describing cancers (e.g. we know the issues behind Pancreatic Cancer for example). There is a proposal for a single “Neuroendocrine” blue book but that has yet to come to fruition. However, the latest publication is “Digestive System” which covers most Neuroendocrine types so it’s a very useful update, and as you will see a milestone in resolving past nomenclature issues with our disease. The work behind the 2019 Digestive System follows on from the excellent work in the 2017 Endocrine update where the significant change from 2 to 3 grades of well-differentiated pancreatic NETs was a significant change. In fact, the 2017 pancreatic neuroendocrine update has been copied and pasted right into the pancreatic section of the 2019 digestive system book, effectively delivering a blue book that contains the vast majority of Gastroenteropancreatic Neuroendocrine Neoplasms (GEPNENs) including those in the hepatopancreatobiliary organs (pancreas/bile duct/liver/gallbladder). Important to also point out that a very similar structure was presented in the 2017 Endocrine Organ blue book which included (but was not limited to) Neuroendocrine related tumours of the Pituitary, Thyroid, Parathyroid, Pheochromocytoma, Paraganglioma, and their associated hereditary syndromes. ENETS and many other regions adopted this for all types of Neuroendocrine Cancer (as a result of a proposal to do so), so the 2019 Digestive System is really a rubber-stamping of this change, but it also confirms one very important difference – the word “carcinoid” has been removed from the vocabulary, not before time and something I’ve been quite vocal about since 2015. Unfortunately many doctors and healthcare establishments still remain out of date (2023!). 

So, what about other areas not included in GEPNENs? Please note there are still loose ends in some of the blue books, mostly Thoracic (Lung/Thymus). Hopefully, these blue books will be updated in the same way as the others for Neuroendocrine Neoplasms (or will be subsumed into the proposed Neuroendocrine blue book). But in the meantime, experts are proposing the nomenclature above be extended in advance of the blue book updates – this is covered in Neuroendocrine neoplasm update: toward universal nomenclature, © 2020 Society for Endocrinology 2020, Guido Rindi and Frediano Inzani.  What this effectively means is that the term “carcinoid” is effectively now totally defunct other than the loose ends of Carcinoid Syndrome, Carcinoid Heart Disease and Carcinoid Crisis (problems I could resolve in 10 minutes if they would listen to me). In fact, one well known pathologist has been campaigning for the removal of the terms ‘carcinoid‘ since 2007

I’ll summarise the key changes from 2019 below for your convenience, but I need to cite the article here:

The 2019 WHO classification of tumours of the digestive system

Iris D Nagtegaal, Robert D Odze, David Klimstra, Valerie Paradis, Massimo Rugge, Peter Schirmacher, Kay M Washington, Fatima Carneiro, Ian A Cree, for the WHO Classification of Tumours Editorial Board. First published: 21 August 2019 – https://doi.org/10.1111/his.13975

• • All well-differentiated GEPNETs now include Grade 3 alongside Grades 1 and 2.
  • All poorly differentiated GEPNENs are now Neuroendocrine Carcinomas and are by default Grade 3 (the inference in the guidance is that there is no need to use the term high grade or Grade 3 in reference to Neuroendocrine Carcinomas as they have those attributes by default).
  • Neuroendocrine Neoplasm is now the formal umbrella term for the grouping of well and poorly differentiated tumours.
  • The term ‘carcinoid’ is no longer used in nomenclature for Neuroendocrine Neoplasms within the Digestive System blue book.
  • Pancreatic Neuroendocrine Neoplasms will now appear in the Digestive System blue book, previously they were listed in the Endocrine Organ book.
  • The molecular differences of well and poorly differentiated tumours have been added to assist pathologists in identifying them, e.g. Mutations in MEN1, DAXX and ATRX are entity defining for well differentiated tumours while Neuroendocrine Carcinomas (poorly differentiated) usually have TP53 or RB1 mutations.
  • Poorly differentiated tumours are divided into Small Cell type (SCNEC) and Large Cell type (LCNEC).
  • Last year the term “Goblet Cell Carcinoid” was changed to “Goblet Cell Carcinoma”. However, this type of appendiceal neoplasm has effectively been removed from the Neuroendocrine family as it’s now recognised to have a minor neuroendocrine component. This cancer type is now called Goblet Cell Adenocarcinoma of the Appendix.
  • As per the previous content of this article, mixed tumours were renamed from Mixed AdenoNeuroendocrine Carcinomas (MANEC) are now named Mixed Neuroendocrine-Non-Neuroendocrine Neoplasms (MiNENs) – clearly they now have variable grades, variable mitotic rate/Ki67 and each component can be graded separately.

Grading – Key WHO 2021 Changes – Lung Neuroendocrine Neoplasms

Following the publication of the WHO 5th Edition of Thoracic (Lung/Thymic) Neuroendocrine Neoplasms in 2021, a new section will be added here – click here to see the differences 

Pheochromocytoma/Paraganglioma

Previously, Pheochromocytoma/ Paraganglioma (PHEO/PGL) did not have an official grading regime, i.e. they were just benign or malignant. However, since 2017 and as reflected in the fourth edition WHO classification of Endocrine Systems, on the basis that all PHEO/PGL have some metastatic potential and have their own grading system, there are different classification systems for Pheochromocytoma and Paraganglioma These grades are based on molecular profiles (the future way for many cancers and the first time for a NET). They have a system of grading called GAPP (grading of adrenal pheochromocytoma and paraganglioma) and they are clustered into the metastatic risk (this is the beauty of molecular methods). So, they are different than regular NETs and they now have 3 differentiations, loosely related to grade. This change is complex and is covered in depth here.

Misc Grading Issues

The proliferative rate can be diverse in NENs, so sampling issues can limit the accuracy of grading. More substantial samples of tumour are therefore preferable for grading thus why the Ki-67 index is preferred for biopsies where large amounts of tissue may not be available. The distinction of low-grade from intermediate grade can be challenging when using small samples. A couple of interesting observations about NET grading which I spotted during my research and ‘forum watching’. You can have multiple primary tumours, and these might have different Ki-67 scores. Additionally, on larger tumours, Ki-67 scores can be different on different parts of the tumour. And something I know from my own experience; secondary tumours can have different Ki-67 scores than primary – even a different grade. In my own case, my liver secondary tumours were graded higher than my primary which according to my surgeon is in keeping with a clone of the disease having become more aggressive over time. Royal Free Hospital NET Centre indicates a person’s grade should be taken from the highest biopsy grade taken. This is a fairly complex area but a recent study published by the US National Institute of Health and many anecdotal comments made by NET specialists indicates that is a fairly common scenario.