Proliferation Marker (Ki67) in Sub-Categorization of Neuroendocrine Tumours of the Lung
Rashi GARG1, Amanjit BAL1, Ashim DAS1, Navneet SINGH2, Harkant SINGH3
1Departments of Histopathology, Post Graduate Institute of Medical Education & Research (PGIMER), CHANDIGARH, INDIA
2Departments of Pulmonary Medicine, Post Graduate Institute of Medical Education & Research (PGIMER), CHANDIGARH, INDIA
3Departments of Cardiovascular and Thoracic Surgery, Post Graduate Institute of Medical Education & Research (PGIMER), CHANDIGARH, INDIA
Keywords: Ki67, Lung, Neuroendocrine tumours
The 2015 WHO classification classifies neuroendocrine tumours (NET) of the lung into typical carcinoid, atypical carcinoid, large
cell neuroendocrine carcinoma and small cell carcinoma based on morphology alone. Mitosis is the major parameter for this classification, and
thus several studies have focused on the role of Ki67 in these tumours but without conclusive results. The aim of the study was to categorize
neuroendocrine tumours of the lung based on morphology and to assess the utility of Ki67 in diagnosis.
Material and Method: The study included 42 cases (23 biopsies and 19 lobectomy specimens) of neuroendocrine tumours (excluding small cell
carcinoma). Haematoxylin & eosin stained sections, immunohistochemistry for neuroendocrine markers and Ki67 were studied.
Results: Based on WHO criteria, cases were classified as typical carcinoids (83.3%), atypical carcinoids (12%) and large cell neuroendocrine
carcinomas (4.7%). The Ki67 index ranged between 1%-10% (mean 2.6%), 10%-30% (mean 19%), 35%-50% (mean 42.5%) in typical carcinoid,
atypical carcinoid and large cell neuroendocrine carcinoma respectively. Using the ROC curve, the cut off value of Ki67 for typical and atypical
carcinoids was 7.5% (P value<0.001), and for atypical carcinoid/large cell neuroendocrine carcinoma was 32.5% (P value=0.051). On comparing
the size and infiltration pattern (both local and lymphovascular invasion) of tumours in resected specimens, there was no association with the
proliferation index (P value >0.05).
Conclusion: Morphological features are the gold standard for subtyping of neuroendocrine tumours. Ki-67 is a potentially meaningful marker
for sub-categorization of lung NETs, especially in small biopsies. However, the size and infiltrative pattern of the tumours are independent of the
The lung is a common site of carcinoid tumours and
accounts for 30% of well differentiated neuroendocrine
tumours (NETs) in the body 1
. The 2015 World Health
Organization (WHO) classification recognizes 4 major
types of neuroendocrine tumours (NETs) of the lung:
typical carcinoid (TC), atypical carcinoid (AC), large
cell neuroendocrine carcinoma (LCNEC) and small
cell carcinoma (SCC). Excluding SCC, the remaining
NETs constitute 5% of lung tumours 2
WHO classifies these tumours based on the morphologic
features of which mitoses and necrosis are of paramount
importance. Several studies have tried to evaluate the role of
Ki67 similar to other organ sites such as the gastrointestinal
tract and pancreas in diagnosing these tumours but without
conclusive results. Since these subtypes of lung NETs have
markedly different prognostic implications and treatment
regimens, the importance of accurate pathologic diagnosis
is underscored. The aim of this study was to assess the utility of proliferation marker Ki67 in sub-categorization
of these tumours (excluding SCC).
Forty two cases (23 biopsies and 19 lobectomy specimens)
of NETs of the lung excluding SCC of lung were analyzed
retrospectively from Jan 1999 to Aug 2014. Sections prepared
from paraffin blocks were stained with hematoxylin and
eosin and the morphological features were studied.
Cases were classified based on morphological criteria used
by 2015 WHO classification. Tumours with carcinoid
morphology, <2 mitoses per 2mm2 and lacking necrosis
were classified as TC, those with carcinoid morphology
and 2-10 mitoses per 2mm2 or necrosis (often punctate)
were categorized as AC. The tumours with neuroendocrine
morphology, positive immunohistochemical staining
for one or more NE markers (other than neuron-specific
enolase), high mitotic rate: ≥11 per 2mm2, necrosis (often large zones) and cytologic features of a non-small-cell lung
carcinoma i.e. large-cell size, low nuclear to cytoplasmic
ratio, vesicular or fine chromatin, and/or frequent nucleoli
were classified as LCNEC.
Neuroendocrine Markers: Immunohistochemistry for
one of the neuroendocrine markers; chromogranin,
synaptophysin and CD56 was done in all cases.
Proliferation Index: The Ki-67 proliferative index was
determined in all cases on formalin-fixed paraffinembedded
tissue sections using a mouse anti-Ki-67
monoclonal antibody (1:50; clone MIB-1; Dako). Five hot
spots i.e. areas of highest proliferation were counted. A
total of 1000 cells in each spot were counted and results
were expressed in percentage.
Microsoft Excel was used for all the calculations. The
Ki67 distribution among WHO 2015 categories (TC,
AC, LCNEC) was investigated using non parametric test:
the Kruskal-Wallis equality of population rank test and
Mann-Whitney tests. Receiver Operating Characteristic
(ROC) curve was used to decide the cutoff value of Ki67 in
distinguishing these tumours with maximum sensitivities
The patients were of an age group ranging from 18 to
72 years. Mean age was 37.56 (median 38) for TC, 34.40
(median 31) for AC and 56 years for LCNEC. Patients with
LCNEC presented in the 5th
decades whereas patients
with AC and TC presented from the 2nd
decades (Table I
). The male-female ratio was 1.15:1 (Table I
). All the
cases were showing neuroendocrine architecture-organoid
nests, trabeculae and rosettes and all were positive for one
or more neuroendocrine markers (CD56, synaptophysin
and Chromogranin). Based upon morphology, of the 42
cases 35 (83.3%) were TC (Figure 1A-D
), 5 (12%) were AC
) and 2 (4.7%) cases were LCNEC (Figure 3AC
Click Here to Zoom
|Figure 1: Typical Carcinoid. Photomicrographs showing A-B) Organoid pattern of arrangement (H&E; x100 and x200). C) Cytoplasmic
positivity with chromogranin (IHC; x200). D) Ki67 staining nuclei of <1% of cells (IHC; x400).
Click Here to Zoom
|Figure 2: Atypical Carcinoid. A) Photomicrographs showing focal areas of necrosis. (H&E; x10X). B) Ki67 shows nuclear staining in 20%
of the tumour cells (IHC; x40).
Click Here to Zoom
|Figure 3: LCNEC. Photomicrographs showing A) extensive areas of necrosis with organoid pattern (H&E; x10). B) Large cell size,
vesicular nucleus, prominent nucleolus and frequent mitosis (H&E; x40). C) Cytoplasmic positivity with synaptophysin (IHC; x20).
D) Ki67 staining nuclei of 35% of cells (IHC; x40).
Ki67 index ranged between 1%-10% (mean 2.6%), 10%-
30% (mean 19%), and 35%-50% (mean 42.5%) in TC, AC
and LCNEC respectively (Figure 1D, 2B, 3D respectively)
(Table II). The Kruskal-Wallis equality of population rank
test showed a statistically significant difference between
the three categories of tumours with a P value <0.001.
Using the Mann-Whitney test, a significant difference was
found for pairwise comparison between TC and AC with
a P value <0.001. However, the results were not found
to be significant between AC and LCNEC with a P value
0.051. Using the ROC curve, the cut off value of Ki67 for
typical/atypical carcinoids was 7.5% with sensitivity and
specificity of 91.4% and 100% (area under curve is 0.9685)
(Figure 4A,B), whereas for atypical carcinoid/large cell
neuroendocrine carcinoma, the cut off value was 32.5%
with sensitivity and specificity of 100% (Figure 4C,D) (area
under curve is 1). Overall the Ki67 cutoffs discriminated
with optimal discrimination power between 3 classes.
Click Here to Zoom
|Figure 4: A-B) ROC-Ki67 cutoff values with different sensitivities and specificities for TC/AC. C-D) ROC-Ki67 cutoff values with
different sensitivities and specificities for AC/LCNEC.
The pneumonectomy/lobectomy specimens were available
in 19 cases. Of these, three cases were AC and remaining 16
cases were TC. The size of the tumours ranged from 1 to 9 cm
(in the largest dimension). On gross examination, all cases
were well circumscribed except four cases (Figure 5). On
microscopic examination, 9 cases had infiltrative margins,
of which two showed lymphovascular involvement; both
were TCs. Of the three ACs, two showed infiltrative
margins. Lymph nodes (hilar/peribronchial/subcarinal)
were available in 6 cases which were free of tumour. On
comparing the size and infiltration pattern (both local and
lymphovascular invasion), using the T-test and Pearson
Correlation, there was no association with the proliferation
index (P value>0.05) as well as tumour type.
Click Here to Zoom
|Figure 5: Gross photographs of
endobronchial carcinoid tumour; a
well circumscribed grayish yellow
NETs of the lung include four different histologic subtypes
as defined by WHO; Typical carcinoid (TC-1-2%), atypical
carcinoid (AC-0.1-0.2%), large cell neuroendocrine
carcinoma (LCNEC-3%) and small cell carcinoma (SCC-
. TCs are associated with a fairly benign
behavior and are classified as low grade NET/Carcinoma
(Grade 1), ACs are classified as intermediate grade tumour
(Grade 2), whereas LCNEC and SCC are grouped together
under high grade NET/Carcinoma (Grade 3) 2
. Of the high
grade tumours, the SCCs have distinct nuclear morphology
posing less diagnostic problems, and thus were excluded
from the study. The main concern lies in distinguishing low
and intermediate grade tumours (TC/AC).
The WHO 2015 classifies these tumours based on
morphological features only, which includes carcinoid morphology, mitosis and/or necrosis. All these tumours have
positive immunohistochemical staining for one or more NE
markers. The difficulties in classifying these tumours arise
in rare tumours that show mitotic activity just slightly or
focally exceeding 2/2mm2 or 10/2mm2 which otherwise
would qualify as AC or LCNEC respectively 4. Also, in
small biopsies with extensive crush artifact it is difficult to
count mitotic figures and when the amount of tumour tissue
is limited 5,6. To overcome these difficulties, an effective
grading system for digestive NETs was recently introduced
by the European Neuroendocrine Tumour Society and
endorsed by the WHO and the American Joint Cancer
Committee 7. This system largely relies on the assessment
of the proliferative marker Ki67 which proved accurate and
predictive 8. Different studies have assessed the role of
proliferation marker Ki67 in lung neuroendocrine tumours
but the results are not conclusive 9,10.
Table III shows average Ki67 values across different studies;
however, the results vary 11-14.
The mean values of Ki67 index in the present study are
2.6%, 19%, 42.5% in TC, AC and LCNEC respectively and
the cutoff values by ROC curve analysis are 7.5% for TC/AC
and 32.5% for AC/LCNEC. Of the 35 cases of TC, 3 cases
have Ki67 > 7.5% (10%). The discrepancy in Ki67 index and
mitotic count could also arise from the fact that the mitotic
phase represents the smallest portion of the cell cycle and
Ki-67 which detects cells from mid-G1 through S and G2
phases will detect proliferating cells that do not show mitotic
figures. The mean Ki67 proliferative index significantly
increases from TC to AC and poorly differentiated NETs
15. Ki67 is thus capable of distinguishing the 3 classes in
a significant manner. However, in the present study, pair
wise comparison between AC and LCNEC is not significant
with a P value of 0.051. This was probably due to very low
number of cases of each subtype.
Pelosi et al. 14 reported cases where TC and AC were over
diagnosed as SCC on biopsy specimen. In such cases, the
Ki67 labeling index is emerging as the most useful ancillary
technique. It supports mitotic count to discriminate
between low grade versus high grade tumours and also
helps to distinguish these cases by immunohistochemical
staining of cytological smears 16,17. It may reflect the
tumour grade and predicts survival 18.
These tumours have different treatment modalities and
survival rates, and a definite and accurate diagnosis
is therefore necessary. Though TCs are low grade
malignancies, they are capable of regional lymph node
metastasis in 10-15% of the cases. ACs have 50% nodal and
20% distant metastases 19. Patients with TC, AC, LCNEC
have a survival of 87%, 60% and 15-57% respectively
20-22. Surgery is the mainstay of the treatment of
carcinoids. A study by Ducrocq et al. 23 showed that
TCs have equivalent survival rate for limited resection
versus lobectomy/pneumonectomy. A mediastinal lymph
node dissection is recommended in clinical N0 central
AC. Patients of AC with a consequent higher rate of nodal
involvement and an adequate pulmonary reserve should
receive lobectomy 24. Some reports showed that patients
with ACs and regional lymph node metastasis have a high
likelihood of developing recurrent disease, and if treated
with surgical resection alone have a significantly worse
outcome. Thus adjuvant treatment, chemotherapy or
radiotherapy should be considered in such cases 25. Most
LCNEC are poor candidates for surgical resection 14.
To have accurate treatment modalities for these cases,
accurate and early diagnosis is very important. Ki67 can act
as an important supportive tool along with morphological
features in classifying these tumours. However, extensive
studies with larger case series to establish the appropriate
cutoff values and follow up studies to establish its
correlation with nodal and distant metastasis and overall
survival are required in order to assess its validity.
In conclusion, morphological features are the gold
standard for subtyping of neuroendocrine tumours. Ki-67
is a potentially meaningful marker for sub-categorization
of lung NE tumours, especially in small biopsies. The
mean values of Ki67 index in the present study were 2.6%,
19%, 42.5% in TC, AC and LCNEC respectively and the
cutoff values for differentiating TC/AC and AC/LCNEC
were 7.5% and 32.5% respectively. However, the size and
infiltrative pattern of these tumours are independent of the
CONFLICT of INTEREST
The authors have no funding, financial relationships, or
conflicts of interest to disclose.
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