Histopathology 1992, 20, 2 5 1-2 5 5
Proliferating cell nuclear antigen (PCNA) expression in formalin-fixed tissue of non-small cell lung carcinoma P.H.M.H.THEUNISSEN, M.P.G.LEERS & E.C.M.BOLLEN* Departments of Pathologg arid *Srtrgerg, LIe Wever Hospital, Heerlen. The Netherlands Date of submission 2 5 July 1991 Accepted for publication 2 6 September 199 1
T H E U N I S S E N P . H . M . H . . LEERS M . P . G . & BOLLEN E . C . M .
(1992) Histopathologg 20, 251-255
Proliferating cell nuclear antigen (PCNA) expression in formalin-fixed tissue of non-small cell lung carcinoma An immunohistochemical study of non-small cell lung carcinoma using PCI 0. a monoclonal antibody against PCNA, was performed on tissues routinely processed with formalin fixation and paraffin embedding. The PCNA labelling index and mitotic index were determined from sections of these tissues. Tumours showed a high mean PCNA labelling index of 53.3%. The mean mitotic index was 10.3/1000 cells. Inter-examiner agreement of mitotic counting was good. A linear correlation between the PCNA labelling index and mitotic index was demonstrated (r=0.71, P < 0.00001).It is concluded that immunohistochemical nuclear labelling with anti-PCNA on routinely processed tissue is a simple technique for the assessment of proliferation in non-small cell lung carcinoma. Keywords: non-small cell lung carcinoma, PCNA labelling index, mitotic index
Introduction
Materials and methods
The light microscopic grading of non-small cell lung carcinomas (squamous cell carcinoma, adenocarcinoma and large cell undifferentiated carcinoma) is subjective and does not have a prognostic value'. Useful information about tumour biology has been gained by studying tumour cell kinetics in lung carcinoma'. Methods for assessing the state of cellular proliferation such as thymidine- or bromodeoxyuridine-labelling and flow cytometry are not easily practicable for routine diagnosis. On the other hand, immunohistochemical cell-cycle markers such as antibodies against Ki-67 and PCNA can easily be applied in routine surgical pathology laboratories. The advantage of anti-PCNA is that it can be used with routinely processed formalin-fixed and paraffinembedded tissue, in contrast to the Ki-67 antibody which requires frozen tissue sections. The aim of this study was to see whether PCNA labelling in non-small cell lung carcinoma could be considered as a n index of proliferation or whether there is a deregulated expression of PCNA in these tumours.
Histological sections from 39 consecutive cases of resected non-small cell lung carcinoma were studied for tumour classification. Because of its poor reproducibility tumour differentiation grade was not recorded. One formalin-fixed (fixation time 2 4 h at the most) and paraffin-embedded tissue block was selected from each tumour on the basis of maximum vital tumour content. Sections ( 5 pm thick) were de-waxed and immersed for 10 min in methanol with 0 . 3 % H'OL to block endogenous peroxidase activity. After pre-incubation with 1% bovine serum albumin-TBS for 1 5 min, sections were incubated with commercially available monoclonal IgG antibody against PCNA (clone PC10, Dakopatts, Denmark). A dilution of 1:200 of the primary antibody with overnight incubation at room temperature was chosen in accordance with the study of Hall and co-workers3. After rinsing in TBS, sections were incubated with a sheep anti-mouse bridging antibody (Amersham, Denmark) for 1 h at room temperature and a dilution of 1 : 300. The sections were then incubated for 1.5 h at room temperature with ABC complex (Dakopatts) in a dilution 1: 1 : 100 in Tween 20/PBS. Diamino-benzidinehydrogen peroxide was employed as a chromogen and a
Address for correspondence: Dr P.H.M.H. Theunissen. Department of Pathology. I k Wever Hospital. PO Box 4446. 6401 CX Heerlen, The Netherlands.
251
252
P.H.M.H.Theunissen. M.P.G.Leers and E.C.M.Bollen
light haematoxylin counterstain was used. The specificity of the reaction was demonstrated in non-neoplastic control tissues (colon mucosa. tonsillar tissue, skin). Negative controls were provided by omitting the primary antibody and replacing it with non-immune serum. The distribution of PCNA immunoreactivity within the tumours was recorded and scored by counting the percentage of cells showing positive nuclear staining. The overall staining (mild, moderate or marked) in each tumour was estimated at low power, then 10 different high-power fields representative of the overall staining pattern were examined by scoring 1000 morphologically vital cells. Mitoses were identified according to the criteria defined by Baak4 and the number of mitoses was counted in the same fields ( 1000 cells) as those selected for determination of PCNA labelling index. The interobserver variability of mitotic counting was evaluated using Pearson's product moment correlation coefficient r and the one-way ANOVA intraclass correlation coefficient (ICC) as reliability indices5. The correlation between the PCNA labelling index and the mitotic index was assessed using a linear regression analysis program (Statgraphics) on a personal computer.
Results In total, 74.6% (29/39) of the non-small cell lung carcinomas were squamous cell carcinomas and 20.5% (8/39)were adenocarcinomas. The remaining two were a large cell undifferentiated carcinoma and a carcinosarcoma, although the latter is strictly speaking not a nonsmall cell lung carcinoma. The distribution of nuclear PCNA staining in non-neoplastic control tissues was entirely consistent with the results of earlier reports3'. The numerical data of mitotic index and PCNA labelling index are summarized in Table 1. Inter-
Table 1 . Summary of numerical data
Variable Sample size Minimum Maximum Mean Median Kange Standard deviation
Mitotic index (no. of mitoses/ 1000 cells)
PCNA labelling index (% positively staining nuclei)
39 1 22 10.3 9 21 5.6
39 10.1 81.4 53.4 58.5 71.3 19.2
301 24
I
I
0
6
12
18
24
30
Observer 1 Figure 1 . Inter-observer variability of mitotic counting in 39 nonsmall cell lung carcinomas ( r = 0 . 8 3 . P
Proliferating cell nuclear antigen (PCNA) expression in formalin-fixed tissue of non-small cell lung carcinoma P.H.M.H.THEUNISSEN, M.P.G.LEERS & E.C.M.BOLLEN* Departments of Pathologg arid *Srtrgerg, LIe Wever Hospital, Heerlen. The Netherlands Date of submission 2 5 July 1991 Accepted for publication 2 6 September 199 1
T H E U N I S S E N P . H . M . H . . LEERS M . P . G . & BOLLEN E . C . M .
(1992) Histopathologg 20, 251-255
Proliferating cell nuclear antigen (PCNA) expression in formalin-fixed tissue of non-small cell lung carcinoma An immunohistochemical study of non-small cell lung carcinoma using PCI 0. a monoclonal antibody against PCNA, was performed on tissues routinely processed with formalin fixation and paraffin embedding. The PCNA labelling index and mitotic index were determined from sections of these tissues. Tumours showed a high mean PCNA labelling index of 53.3%. The mean mitotic index was 10.3/1000 cells. Inter-examiner agreement of mitotic counting was good. A linear correlation between the PCNA labelling index and mitotic index was demonstrated (r=0.71, P < 0.00001).It is concluded that immunohistochemical nuclear labelling with anti-PCNA on routinely processed tissue is a simple technique for the assessment of proliferation in non-small cell lung carcinoma. Keywords: non-small cell lung carcinoma, PCNA labelling index, mitotic index
Introduction
Materials and methods
The light microscopic grading of non-small cell lung carcinomas (squamous cell carcinoma, adenocarcinoma and large cell undifferentiated carcinoma) is subjective and does not have a prognostic value'. Useful information about tumour biology has been gained by studying tumour cell kinetics in lung carcinoma'. Methods for assessing the state of cellular proliferation such as thymidine- or bromodeoxyuridine-labelling and flow cytometry are not easily practicable for routine diagnosis. On the other hand, immunohistochemical cell-cycle markers such as antibodies against Ki-67 and PCNA can easily be applied in routine surgical pathology laboratories. The advantage of anti-PCNA is that it can be used with routinely processed formalin-fixed and paraffinembedded tissue, in contrast to the Ki-67 antibody which requires frozen tissue sections. The aim of this study was to see whether PCNA labelling in non-small cell lung carcinoma could be considered as a n index of proliferation or whether there is a deregulated expression of PCNA in these tumours.
Histological sections from 39 consecutive cases of resected non-small cell lung carcinoma were studied for tumour classification. Because of its poor reproducibility tumour differentiation grade was not recorded. One formalin-fixed (fixation time 2 4 h at the most) and paraffin-embedded tissue block was selected from each tumour on the basis of maximum vital tumour content. Sections ( 5 pm thick) were de-waxed and immersed for 10 min in methanol with 0 . 3 % H'OL to block endogenous peroxidase activity. After pre-incubation with 1% bovine serum albumin-TBS for 1 5 min, sections were incubated with commercially available monoclonal IgG antibody against PCNA (clone PC10, Dakopatts, Denmark). A dilution of 1:200 of the primary antibody with overnight incubation at room temperature was chosen in accordance with the study of Hall and co-workers3. After rinsing in TBS, sections were incubated with a sheep anti-mouse bridging antibody (Amersham, Denmark) for 1 h at room temperature and a dilution of 1 : 300. The sections were then incubated for 1.5 h at room temperature with ABC complex (Dakopatts) in a dilution 1: 1 : 100 in Tween 20/PBS. Diamino-benzidinehydrogen peroxide was employed as a chromogen and a
Address for correspondence: Dr P.H.M.H. Theunissen. Department of Pathology. I k Wever Hospital. PO Box 4446. 6401 CX Heerlen, The Netherlands.
251
252
P.H.M.H.Theunissen. M.P.G.Leers and E.C.M.Bollen
light haematoxylin counterstain was used. The specificity of the reaction was demonstrated in non-neoplastic control tissues (colon mucosa. tonsillar tissue, skin). Negative controls were provided by omitting the primary antibody and replacing it with non-immune serum. The distribution of PCNA immunoreactivity within the tumours was recorded and scored by counting the percentage of cells showing positive nuclear staining. The overall staining (mild, moderate or marked) in each tumour was estimated at low power, then 10 different high-power fields representative of the overall staining pattern were examined by scoring 1000 morphologically vital cells. Mitoses were identified according to the criteria defined by Baak4 and the number of mitoses was counted in the same fields ( 1000 cells) as those selected for determination of PCNA labelling index. The interobserver variability of mitotic counting was evaluated using Pearson's product moment correlation coefficient r and the one-way ANOVA intraclass correlation coefficient (ICC) as reliability indices5. The correlation between the PCNA labelling index and the mitotic index was assessed using a linear regression analysis program (Statgraphics) on a personal computer.
Results In total, 74.6% (29/39) of the non-small cell lung carcinomas were squamous cell carcinomas and 20.5% (8/39)were adenocarcinomas. The remaining two were a large cell undifferentiated carcinoma and a carcinosarcoma, although the latter is strictly speaking not a nonsmall cell lung carcinoma. The distribution of nuclear PCNA staining in non-neoplastic control tissues was entirely consistent with the results of earlier reports3'. The numerical data of mitotic index and PCNA labelling index are summarized in Table 1. Inter-
Table 1 . Summary of numerical data
Variable Sample size Minimum Maximum Mean Median Kange Standard deviation
Mitotic index (no. of mitoses/ 1000 cells)
PCNA labelling index (% positively staining nuclei)
39 1 22 10.3 9 21 5.6
39 10.1 81.4 53.4 58.5 71.3 19.2
301 24
I
I
0
6
12
18
24
30
Observer 1 Figure 1 . Inter-observer variability of mitotic counting in 39 nonsmall cell lung carcinomas ( r = 0 . 8 3 . P
