JOURNAL OF PATHOLOGY, VOL.
166: 329-330 (1 992)
EDITORIAL P53 IN TUMOUR PATHOLOGY: CAN WE TRUST IMMUNOCYTOCHEMISTRY? No single gene has made more impact on both reach detectability by ICC. There is accumulating fundamental and applied cancer biology than p53 evidence to support this. Thus, in our laboratory, (reviewed in ref. I). Indeed, the latest meeting of we have recently found that in the human thyroid the Pathological Society carried no fewer than cancer cell line FTC133, nuclear p53 is uniformly 17 presentations devoted to it. Tumour markers and strongly detectable in tissue culture, but have come and (mostly) gone, but p53 mutation is becomes weakly or undetectable when these cells different. First, it is a fundamental mechanism are grown as tumours in an immunodeficient host.* involved in disordered growth control, not a (That the mutation is still present is shown by secondary consequence of the neoplastic pheno- the return of full immunostaining when cells are type. Second, it is amazingly widespread, occurring replated in culture.) Whereas most p53-mutant at high frequency in tumours of all cell lineages. breast cancer lines are immunopositive, in one Of course, this would be of little interest to report as many as 7 out of 10 surgical cancer samples diagnostic pathology were it not for the fortuitous containing missense mutations were negative by fact that the underlying genetic lesion-point frozen section ICC3 (Table I). Most concerning is mutation-in addition to abolishing the tumour the suggestion that ICC detectability may require suppressor activity of the protein, also stabilizes it, not just mutation of the gene, but some additional resulting in its accumulation to levels detectable by secondary mechanism conferred by the neoplastic immunocytochemistry (ICC). Furthermore, most phenotype. In our model, for example, further studies to date suggest that p53 mutation arises spontaneous transformation of FTCl33 to a more relatively ‘late’ in neoplastic progression and may aggressive derivative-FTC23bcorrelates with correlate with increasing dysplasia, malignant full retention of ICC positivity in vivo. Likewise, transformation, or increased tumour aggressive- there are reports of increasing immunostaining ness depending on the cell/tissue in question. The intensity with increasing progression of neoplasia in potential clinical applications are obvious. surgical samples. If generally true, this phenomenon However, the current wave of enthusiasm is at would largely invalidate the use of ICC as an risk of turning to disappointment if we do not guard indicator of p53 mutation. against oversimplification. The rapidly expanding False positives-Until recently, failure to find experience in this field is now uncovering worrying mutations in ICC-positive tumour cases has been examples of a breakdown in the correlation between ascribed to insensitivity of the sequencing strategy ICC positivity and mutation-in both ‘directions’. (dilution by normal DNA, for example, is a com‘False negatives-Ignoring technical (e.g., fixa- mon problem with ‘direct’ sequencing methods). tion) artefact, there are two potential reasons why However, increasingly, false positives are being loss of p53 activity may not be accompanied by the found in clonal cell lines (Table I), in which this expected accumulation of the protein. First, the argument, of course, cannot apply. We have underlying lesion may be not a missense point observed that spontaneous transformation of rat mutation but a gross deletion which abolishes all thyroid cells expressing a mutant ras oncogene is p53 protein production. Fortunately, this is rare regularly associated with the appearance of strong except in some sarcomas (although the occurrence p53 ICC positivity, but that the stabilized p53 of ‘stop’ mutations having the same effect may well protein is not m ~ t a n t This, . ~ of course, raises the be more common). The second possibility, hitherto possibility that some of the human tumour cases largely ignored, is that the point mutation does may also indeed be genuine false positives. The not stabilize the protein sufficiently for its level to mechanism for such non-mutational stabilization 0022-341 7/92/04032942 $05.00 0 1992 by John Wiley & Sons, Ltd.
330
EDITORIAL
Table I-Some examples of discordance between immunocytochemistry and sequencing in p53 analysis False negatives FTC133 thyroid cancer line (grown in viva)* Breast cancer cases: T29, T59, T34, T50, T 104,T 106’ Leukaemia cell line MOLT45
False positives Spontaneously immortalized rat thyroid cell lines4 Lung cancer lines: A549, A427, HuT292DM, A2 182(‘ Colon cancer line JW’
False negatives: absent ICC staining despite the presence of mutation (excluding ‘stop’ mutations); false positives: presence of ICC staining in the absence of any p53 mutation.
is as yet unknown but is most likely the result of functional status of the p53 control pathway in a cell interruption to the normal degradative pathway of than does D N A sequencing. We need then to consider not one but several p53. A key question must now be whether such false correlations, i.e., (i) p53 mutation (DNA sequence) negatives and positives are common enough in prac- vs. stabilization (ICC positivity), (ii) p53 mutation tice to make p53 ICC too unreliable an indicator vs. tumour behaviour, and (iii) ICC positivity vs. of underlying mutation. Good correlative studies tumour behaviour. It would be ideal, of course, if should resolve this quickly (provided attention is they were all concordant, but even if (i) is not, p53 paid to the analytical techniques). However, the ICC will still be useful, provided that it correlates answer, even if unfavourable, may not decide the with tumour behaviour. Only case-by-case analyses fate of p53 as a diagnostic marker. The problem is of all three parameters will decide. While we await these results (and hopefully also further advances in more subtle. To take first the ‘false negatives’, the conven- the basic biology), we should retain an open mind. tional assumption is that even if a mutant p53 DAVID WYNFORD-THOMAS protein is insufficiently stabilized to be detectable CRC Thyroid Tumour Biology Research Group by ICC, it has nevertheless lost all biological Department of Pathology activity-but is this true? We must consider the University of Wales College of Medicine alternative-that incomplete stabilization correCardiflCF4 4 X N , U.K. lates not only with weak detection, but also with incomplete inactivation. If so, the aditional stabilization which seems to occur with tumour progression (whatever its mechanism) may result in REFERENCES further loss of function of the ‘p53 pathway’ and hence further loss of growth control. Likewise, ‘false positives’ may well be ‘false’ in 1. Lane DP, Benchimol S. p53. oncogene or anti-oncogene? Genes Dev 1990:4 1-8. that there is no mutation, but may not be misleading 2. Wyllie FS, Bond JA, Burns JS, ef a/. Immunocytochernical detection of p53 mutations: false positives and False negatives. J Pufho/(in press). at all in the biological sense if the secondary stabiliBorresen A-L, Hovig E, Smith-Sorensen B, et a/. Constant denaturant zation of p53 occurs by a mechanism which also 3. gel electrophoresis as a rapid screening technique for p53 mutations. abolishes its function. There may be, for example, P r o c N u f / A c u d S c i USA 1991;88:8405-8409. a requirement for p53 to complex with its target 4. Burns JS, Blaydes JP, Wright PA, ef a/. Step-wise transformation of primary thyroid epithelial cells by a mutant H-ms oncogene: a n in vifro protein(s) before degradation can take place, so model of tumour progression. Mol Carcinogen (submitted). that loss of the target would secondarily lead to 5. Rodrigues NR. Rowan A, Smith M E F , e f a/. p53 mutations in colorectal cancer. Pror Nut1 AcudSci U S A 1990; 87: 7555-7559. stabilization. 6. Lehman TA, Benedict WP, Metcalf RA, el ul. p53 mutations, rus If such mechanisms prove to be commonplace, mutations, and p53-heat shock 70 protein complexes in human lung carcinoma cell lines. Cunrer Res 1991; 51: 4090-4096. ICC analysis may often tell us more about the
166: 329-330 (1 992)
EDITORIAL P53 IN TUMOUR PATHOLOGY: CAN WE TRUST IMMUNOCYTOCHEMISTRY? No single gene has made more impact on both reach detectability by ICC. There is accumulating fundamental and applied cancer biology than p53 evidence to support this. Thus, in our laboratory, (reviewed in ref. I). Indeed, the latest meeting of we have recently found that in the human thyroid the Pathological Society carried no fewer than cancer cell line FTC133, nuclear p53 is uniformly 17 presentations devoted to it. Tumour markers and strongly detectable in tissue culture, but have come and (mostly) gone, but p53 mutation is becomes weakly or undetectable when these cells different. First, it is a fundamental mechanism are grown as tumours in an immunodeficient host.* involved in disordered growth control, not a (That the mutation is still present is shown by secondary consequence of the neoplastic pheno- the return of full immunostaining when cells are type. Second, it is amazingly widespread, occurring replated in culture.) Whereas most p53-mutant at high frequency in tumours of all cell lineages. breast cancer lines are immunopositive, in one Of course, this would be of little interest to report as many as 7 out of 10 surgical cancer samples diagnostic pathology were it not for the fortuitous containing missense mutations were negative by fact that the underlying genetic lesion-point frozen section ICC3 (Table I). Most concerning is mutation-in addition to abolishing the tumour the suggestion that ICC detectability may require suppressor activity of the protein, also stabilizes it, not just mutation of the gene, but some additional resulting in its accumulation to levels detectable by secondary mechanism conferred by the neoplastic immunocytochemistry (ICC). Furthermore, most phenotype. In our model, for example, further studies to date suggest that p53 mutation arises spontaneous transformation of FTCl33 to a more relatively ‘late’ in neoplastic progression and may aggressive derivative-FTC23bcorrelates with correlate with increasing dysplasia, malignant full retention of ICC positivity in vivo. Likewise, transformation, or increased tumour aggressive- there are reports of increasing immunostaining ness depending on the cell/tissue in question. The intensity with increasing progression of neoplasia in potential clinical applications are obvious. surgical samples. If generally true, this phenomenon However, the current wave of enthusiasm is at would largely invalidate the use of ICC as an risk of turning to disappointment if we do not guard indicator of p53 mutation. against oversimplification. The rapidly expanding False positives-Until recently, failure to find experience in this field is now uncovering worrying mutations in ICC-positive tumour cases has been examples of a breakdown in the correlation between ascribed to insensitivity of the sequencing strategy ICC positivity and mutation-in both ‘directions’. (dilution by normal DNA, for example, is a com‘False negatives-Ignoring technical (e.g., fixa- mon problem with ‘direct’ sequencing methods). tion) artefact, there are two potential reasons why However, increasingly, false positives are being loss of p53 activity may not be accompanied by the found in clonal cell lines (Table I), in which this expected accumulation of the protein. First, the argument, of course, cannot apply. We have underlying lesion may be not a missense point observed that spontaneous transformation of rat mutation but a gross deletion which abolishes all thyroid cells expressing a mutant ras oncogene is p53 protein production. Fortunately, this is rare regularly associated with the appearance of strong except in some sarcomas (although the occurrence p53 ICC positivity, but that the stabilized p53 of ‘stop’ mutations having the same effect may well protein is not m ~ t a n t This, . ~ of course, raises the be more common). The second possibility, hitherto possibility that some of the human tumour cases largely ignored, is that the point mutation does may also indeed be genuine false positives. The not stabilize the protein sufficiently for its level to mechanism for such non-mutational stabilization 0022-341 7/92/04032942 $05.00 0 1992 by John Wiley & Sons, Ltd.
330
EDITORIAL
Table I-Some examples of discordance between immunocytochemistry and sequencing in p53 analysis False negatives FTC133 thyroid cancer line (grown in viva)* Breast cancer cases: T29, T59, T34, T50, T 104,T 106’ Leukaemia cell line MOLT45
False positives Spontaneously immortalized rat thyroid cell lines4 Lung cancer lines: A549, A427, HuT292DM, A2 182(‘ Colon cancer line JW’
False negatives: absent ICC staining despite the presence of mutation (excluding ‘stop’ mutations); false positives: presence of ICC staining in the absence of any p53 mutation.
is as yet unknown but is most likely the result of functional status of the p53 control pathway in a cell interruption to the normal degradative pathway of than does D N A sequencing. We need then to consider not one but several p53. A key question must now be whether such false correlations, i.e., (i) p53 mutation (DNA sequence) negatives and positives are common enough in prac- vs. stabilization (ICC positivity), (ii) p53 mutation tice to make p53 ICC too unreliable an indicator vs. tumour behaviour, and (iii) ICC positivity vs. of underlying mutation. Good correlative studies tumour behaviour. It would be ideal, of course, if should resolve this quickly (provided attention is they were all concordant, but even if (i) is not, p53 paid to the analytical techniques). However, the ICC will still be useful, provided that it correlates answer, even if unfavourable, may not decide the with tumour behaviour. Only case-by-case analyses fate of p53 as a diagnostic marker. The problem is of all three parameters will decide. While we await these results (and hopefully also further advances in more subtle. To take first the ‘false negatives’, the conven- the basic biology), we should retain an open mind. tional assumption is that even if a mutant p53 DAVID WYNFORD-THOMAS protein is insufficiently stabilized to be detectable CRC Thyroid Tumour Biology Research Group by ICC, it has nevertheless lost all biological Department of Pathology activity-but is this true? We must consider the University of Wales College of Medicine alternative-that incomplete stabilization correCardiflCF4 4 X N , U.K. lates not only with weak detection, but also with incomplete inactivation. If so, the aditional stabilization which seems to occur with tumour progression (whatever its mechanism) may result in REFERENCES further loss of function of the ‘p53 pathway’ and hence further loss of growth control. Likewise, ‘false positives’ may well be ‘false’ in 1. Lane DP, Benchimol S. p53. oncogene or anti-oncogene? Genes Dev 1990:4 1-8. that there is no mutation, but may not be misleading 2. Wyllie FS, Bond JA, Burns JS, ef a/. Immunocytochernical detection of p53 mutations: false positives and False negatives. J Pufho/(in press). at all in the biological sense if the secondary stabiliBorresen A-L, Hovig E, Smith-Sorensen B, et a/. Constant denaturant zation of p53 occurs by a mechanism which also 3. gel electrophoresis as a rapid screening technique for p53 mutations. abolishes its function. There may be, for example, P r o c N u f / A c u d S c i USA 1991;88:8405-8409. a requirement for p53 to complex with its target 4. Burns JS, Blaydes JP, Wright PA, ef a/. Step-wise transformation of primary thyroid epithelial cells by a mutant H-ms oncogene: a n in vifro protein(s) before degradation can take place, so model of tumour progression. Mol Carcinogen (submitted). that loss of the target would secondarily lead to 5. Rodrigues NR. Rowan A, Smith M E F , e f a/. p53 mutations in colorectal cancer. Pror Nut1 AcudSci U S A 1990; 87: 7555-7559. stabilization. 6. Lehman TA, Benedict WP, Metcalf RA, el ul. p53 mutations, rus If such mechanisms prove to be commonplace, mutations, and p53-heat shock 70 protein complexes in human lung carcinoma cell lines. Cunrer Res 1991; 51: 4090-4096. ICC analysis may often tell us more about the
