M30 Expression Demonstrates Apoptotic Cells, Correlates With in Situ End-Labeling, and Is Associated With Ki-67 Expression in Large Intestinal Neoplasms.Apoptosis, or programmed cell death pro·grammed cell death
programmed cell death
proposed system of cell death, often including poly(ADP)-ribosylation, ensures that a cell will not survive if it is so badly damaged that its recovery would harm the , is the death of individual cells by a genetically controlled mechanism.[1-3] In general, apoptosis performs a beneficial function for the organism and occurs in many physiological circumstances, including embryological morphogenesis morphogenesis /mor·pho·gen·e·sis/ (mor?fo-jen´e-sis) the evolution and development of form, as the development of the shape of a particular organ or part of the body, or the development undergone by individuals who attain the type to , the regulation of cell numbers in adult tissues (such as the removal of senescent se·nes·cent
Growing old; aging. cells in the gastrointestinal tract and the cyclic loss of endometrial endometrial /en·do·me·tri·al/ (en?do-me´tre-il) pertaining to the endometrium.
n relating to the end-ometrium or cavity of the uterus. cells), and the elimination of autoreactive lymphocytes. It also occurs in a wide variety of pathologic conditions. In the case of neoplastic neoplastic /neo·plas·tic/ (ne?o-plas´tik)
1. pertaining to a neoplasm.
2. pertaining to neoplasia.
pertaining to neoplasia or a neoplasm. disease, the role of apoptosis in the growth and development of tumors has been a subject of great interest during the past decade.[1-3] As part of this interest, the counting of apoptotic cells in tissue sections of tumors has been important. It is possible to identify apoptotic cells in sections prepared with routine stains, for example, hematoxylineosin, but this requires care and expertise on the part of the observer. Therefore, workers have attempted to find techniques that will demonstrate apoptotic cells in tissue sections clearly, allowing easy and unambiguous identification by light microscopy in paraffin sections.
The techniques most widely used to demonstrate apoptotic cells in paraffin sections are terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL TUNEL Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling ) and in situ end-labeling (ISEL ISEL Instituto Superior de Engenharia de Lisboa (Portugal)
ISEL Institute for Studies in Environmental Law ). Both methods detect the DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. strand breaks characteristic of apoptosis by adding nucleotides, including deoxyuridine triphosphate triphosphate /tri·phos·phate/ (tri-fos´fat) a salt containing three phosphate radicals.
A salt or ester containing three phosphate groups. labeled with biotin biotin: see vitamin; coenzyme.
Organic compound, part of the vitamin B complex, essential for growth and well-being in animals and some microorganisms. , to the ends of the broken DNA.[5-8] The only essential difference between the two is that TUNEL uses the enzyme terminal deoxyribonucleotidyl transferase transferase /trans·fer·ase/ (trans´fer-as) a class of enzymes that transfer a chemical group from one compound to another.
n. , whereas ISEL uses DNA polymerase I DNA polymerase I is an enzyme that mediates the process of DNA replication in prokaryotes. It is 928 residues long, and an example of a processive enzyme - an enzyme which catalyzes a series of polymerisations. or its Klenow fragment.[7-9] Therefore, TUNEL can label DNA fragments with blunt ends or with ends that are either 3' recessed or 5' recessed, whereas ISEL only detects 3' recessed ends. Although these techniques (particularly TUNEL) have been used extensively in the study of neoplastic and nonneoplastic disease, they are not without their disadvantages. They both mark necrotic and autolytic au·tol·y·sis
The destruction of tissues or cells of an organism by the action of substances, such as enzymes, that are produced within the organism. Also called self-digestion. cells in addition to apoptotic cells.[2,3,11] They require pretreatment pretreatment,
n the protocols required before beginning therapy, usually of a diagnostic nature; before treatment.
n See predetermination. steps that need careful optimization, and the results depend on how these steps are performed.[5,12,13] Some false-positive reactions appear to be produced by endogenous endonucleases released during proteinase proteinase /pro·tein·ase/ (pro´ten-as?) endopeptidase.
A protease that begins the hydrolytic breakdown of proteins usually by splitting them into polypeptide chains. digestion; endonuclease endonuclease /en·do·nu·cle·ase/ (-noo´kle-as) any nuclease specifically catalyzing the hydrolysis of interior bonds of ribonucleotide or deoxyribonucleotide chains. inhibition can abolish this effect. Studies into the effects of fixation and prefixation times have yielded variable results.[15-17] Prolonged fixation times can reduce the number of detectable apoptotic cells, while delayed fixation can increase their number; on the other hand, prolonged archival storage of paraffin blocks appears to have a negligible effect. There is good evidence that the physical act of cutting the tissue to produce sections creates TUNEL reactivity. Furthermore, interassay variability in TUNEL has been demonstrated. Some authors believe that morphology is superior to TUNEL or ISEL for the detection of apoptosis.[1,19]
Another means of identifying apoptotic cells has been described recently. It is a monoclonal antibody called M30, which recognizes a neoepitope of cytokeratin 18 (CK18) in paraffin-embedded tissue. This neoepitope is produced by caspase cleavage of CK18 during apoptosis and is not present in nonapoptotic cells. An advantage over TUNEL and ISEL is the lack of M30 expression in necrotic cells. Since CK18 has wide distribution, being present in virtually all simple, ductal, and pseudostratified epithelia ep·i·the·li·a
A plural of epithelium. , the demonstration of M30 immunoreactivity could be immensely useful in the investigation of apoptosis in tissue sections.
This study used adenomas and adenocarcinomas of the large intestine with 2 aims: to perform a direct comparison between M30 and ISEL to determine the relationship between them, and to compare the results with Ki-67 expression.
MATERIALS AND METHODS
One hundred cases of sporadic adenoma adenoma: see neoplasm. and adenocarcinoma of the large intestine from 100 different patients were selected from the archives of the Royal Air Force Institute of Pathology (Halton, Buckinghamshire, United Kingdom), the Royal Hospital Haslar The Royal Hospital Haslar in Gosport, is one of several hospitals serving the city of Portsmouth. It is owned and administered by the Portsmouth Hospitals NHS Trust. The Royal Hospital Haslar is the last military hospital in the UK and will be closed in 2009. (Gosport Gosport (gŏs`pôrt), city (1991 pop. 69,664) and district, Hampshire, S England. The city is a major port and shares its harbor with Portsmouth. There are ship- and yacht-building facilities and various light industries. , Hampshire, United Kingdom), and St Mark's Hospital (Harrow, Middlesex, United Kingdom). Five-micrometer-thick sections were cut from paraffin blocks of the formalin-fixed tissue.
In situ end-labeling was performed according to a protocol based on the techniques of Ansari et al and Wijsman et al.[9,12] After being dewaxed in xylene xylene (zī`lēn) or dimethylbenzene (dī'mĕthəlbĕn`zēn), C6H4(CH3)2 and hydrated hy·drat·ed
Chemically combined with water, especially existing in the form of a hydrate.
Adj. 1. hydrated - containing combined water (especially water of crystallization as in a hydrate)
hydrous in ethanol and water, endogenous peroxidase peroxidase /per·ox·i·dase/ (per-ok´si-das) any of a group of iron-porphyrin enzymes that catalyze the oxidation of some organic substrates in the presence of hydrogen peroxide.
n. activity was abolished by placing the slides in a 1% solution of hydrogen peroxide in distilled water for 10 minutes. The slides were rinsed and incubated at room temperature in a 0.5% solution of pepsin pepsin, enzyme produced in the mucosal lining of the stomach that acts to degrade protein. Pepsin is one of three principal protein-degrading, or proteolytic, enzymes in the digestive system, the other two being chymotrypsin and trypsin. . This solution was prepared by dissolving lyophilized ly·oph·i·lize
tr.v. ly·oph·i·lized, ly·oph·i·liz·ing, ly·oph·i·liz·es
To freeze-dry (blood plasma or other biological substances).
[lyophil(ic) + -ize. pepsin 1:60 000 (2500-3500 units/mg protein) (Sigma, Poole, Dorset, United Kingdom) in 0.1 mol/L hydrochloric acid. Following a thorough rinsing, the slides were allowed to dry (about 20 minutes). The solution for the nucleotide-polymerase reaction was 50 mmol/L Tris-HCl, pH 7.4 (Sigma); 5 mmol/L magnesium chloride; 10 mmol/L 2-mercaptoethanol; 0.005% bovine serum albumin; 0.02 mmol/L deoxyadenosine triphosphate, deoxycytosine triphosphate, and deoxyguanosine triphosphate (Sigma); 0.004 mmol/L bio-21-dUTP (Clontech, Palo Alto, Calif); and 30 units/mL of DNA polymerase I (Sigma) in distilled water. The sections were covered with this solution and incubated for 90 minutes at 37 [degrees] C. The biotin-labeled nucleotide was detected with streptavidin-biotin (Dakopatts, Glostrup, Denmark) and 3,3'-diaminobenzidine-hydrogen peroxide. The sections were then counterstained with hematoxylin hematoxylin /he·ma·tox·y·lin/ (he?mah-tok´si-lin) an acid coloring matter from the heartwood of Haematoxylon campechianum; used as a histologic stain and also as an indicator. , dehydrated de·hy·drate
v. de·hy·drat·ed, de·hy·drat·ing, de·hy·drates
1. To remove water from; make anhydrous.
2. To preserve by removing water from (vegetables, for example). , and mounted. Each batch of slides included a positive control (a colonic adenocarcinoma) and a negative control (the same carcinoma treated in the same way, except that the polymerase was omitted from the nucleotide solution). The sections were examined microscopically at high power. At least 2000 cells per case were counted; positive cells were defined as cells with nuclei, whole or fragmented, showing morphology consistent with apoptosis. Zones of necrosis and apoptotic fragments in glandular glandular /glan·du·lar/ (glan´du-ler)
1. pertaining to or of the nature of a gland.
1. lumina were not counted. The number of positive cells was expressed as a percentage of the total number of epithelial cells counted.
M30 immunostaining was performed as follows. Sections were dewaxed in xylene and hydrated in ethanol. Antigen retrieval was performed by placing the slides in plastic Coplin jars containing target retrieval solution (Dakopatts) and heating in a microwave oven (Energy Beam Sciences model H2800, The Laboratory Microwave Company, Agawam, Mass). The temperature of the retrieval solution was raised to 98 [degrees] C, and then this temperature was maintained for 15 minutes. The sections were allowed to cool in this solution for about 15 minutes and then washed. Hydrogen peroxide solution from the EnVision kit (Dakopatts) was applied for 7 minutes, then the slides were washed and incubated with serum-free protein block (Dakopatts). The primary antibody solution was a 1:150 dilution of M30 (Roche Diagnostics, Lewes, Sussex, United Kingdom) in Tris-buffered saline (Dakopatts) with 0.05% Tween 20; it was applied for 1 hour at room temperature. After rinsing, labeled EnVision polymer (Dakopatts) was added for 30 minutes, and the presence of bound antibody was demonstrated with the substrate-chromogen solution (hydrogen peroxide and 3,3'-diaminobenzidine) from the EnVision kit. The slides were counterstained with hematoxylin, dehydrated, and mounted. Each batch of slides included a positive control (a colonic adenocarcinoma). The negative control was the same carcinoma treated identically, except that the primary antibody was omitted. In an additional experiment, the primary antibody was replaced with isotypic mouse immunoglobulin IgG2b (Sigma) at the same concentration. The sections were examined at high power, with at least 1000 cells being counted in each case. Cells exhibiting immunoreactivity for M30 were expressed as a percentage of the total number of epithelial cells counted.
Immunohistochemistry for Ki-67 followed standard procedures. Tissue sections were dewaxed and hydrated as described for M30. Antigen retrieval was performed by placing the slides in a citrate citrate /cit·rate/ (sit´rat) a salt of citric acid.
citrate phosphate dextrose (CPD) anticoagulant citrate phosphate dextrose solution. buffer (0.01 mol/L citric acid with 80 g of sodium hydroxide per liter, adjusted to pH 6.0) and heating in a microwave for 15 minutes at 95 [degrees] C. After cooling and washing, a 3% solution of hydrogen peroxide in distilled water was applied, followed by washing and incubation with the blocking reagent from the LSAB LSAB London Society of Air-Britain (London Branch of UK-based Air-Britain aviation historical society)
LSAB Lock, Stock, and Barrel
LSAB Lets Start A Band (Amy Macdonald song) kit (Dakopatts) for 5 minutes. The primary antibody solution was a 1:25 dilution of Ki-67 monoclonal antibody (Dakopatts) in phosphate-buffered saline; slides were incubated in antibody solution for 3 hours at room temperature. The slides were rinsed, incubated with the link antibody, and then incubated with the streptavidin peroxidase solution from the LSAB kit. The presence of bound antibody was demonstrated with 3,3'-diaminobenzidine-hydrogen peroxide solution. The slides were counterstained with hematoxylin, dehydrated, and mounted. Each batch of slides included a positive control and a negative control, as for M30 and ISEL. At least 1000 cells were counted in each case. Cells with nuclei showing immunoreactivity for Ki-67 were expressed as a percentage of the total number of epithelial cells counted.
The data were analyzed using Stat-100 (Biosoft, Cambridge, United Kingdom). P values are quoted to 2 decimal places; P [is less than or equal to] .05 was considered significant.
Nineteen of the 100 cases (12 adenomas, 7 carcinomas) failed to give satisfactory results with ISEL, either because of nonspecific nonspecific /non·spe·cif·ic/ (non?spi-sif´ik)
1. not due to any single known cause.
2. not directed against a particular agent, but rather having a general effect.
1. reactions in normal nuclei or lack of reaction despite the presence of apoptotic cells morphologically. These cases were excluded from the study, leaving 81 cases for analysis. The mean age of the 81 patients was 62 years. Thirty-one cases involved adenomas; the mean age of patients in this group was 60 years, and the male-female ratio was 18:13. Sixteen adenomas were from the right side of the large intestine, and 15 were from the left. Mild dysplasia was present in 17 adenomas, moderate dysplasia was noted in 11, and severe dysplasia was identified in 3. Fifty cases represented carcinomas; the mean age of patients with carcinomas was 63 years, and the male-female ratio was 34:16. Twenty-three carcinomas were from the right side, and 27 were from the left. Five cases were Dukes stage A, 19 were Dukes stage B, and 21 were Dukes stage C. In 5 patients the Dukes stage was unknown. Histologically, all the carcinomas were adenocarcinomas.
The results of the ISEL and M30 experiments are given in the Table and a scatterplot of the counts is shown in Figure 1. Simple linear regression Simple linear regression
A regression analysis between only two variables, one dependent and the other explanatory. showed a strong positive correlation between ISEL and M30 counts (t = 7.44, P = .00). The residuals around the regression line were normally distributed. The distributions of the counts were skewed markedly to the right, and this skewness persisted even after logarithmic logarithmic
pertaining to logarithm.
when the logs of two variables plotted against each other create a straight line. transformation. Therefore, nonparametric tests of correlation were used. The Spearman rank correlation coefficient (p) was 0.80 (2-sided probability P = .00). The Kendall correlation coefficient ([Tau]), adjusted for ties and continuity corrected, was 0.60 (P = .00).
[Figure 1 ILLUSTRATION OMITTED]
Counts of In Situ End Labeling (ISEL) and M30 All Cases Adenomas ISEL M30 ISEL M30 n 81 81 31 31 Mean 0.98 1.30 0.64 0.98 95% CI for mean 0.47-1.49 0.36-2.24 0.34-0.94 0.06-1.91 Carcinomas ISEL M30 n 50 50 Mean 1.19 1.50 95% CI for mean 0.28-2.01 0.06-2.90
Immunoexpression of M30 was generally easier to interpret than ISEL, since cells giving ambiguous signals were rare with M30, but were common with ISEL. The background tended to be "clean" with M30 (Figure 2), although some nonspecific reactivity in mucin mucin: see glycoprotein. caused slight problems in mucinous mucinous /mu·ci·nous/ (mu´si-nus) resembling, or marked by formation of, mucin.
relating to, resembling or containing mucin. adenocarcinomas. The range of appearances was similar to that described by Leers et al. Cells with condensed chromatin chromatin: see chromosome. and cell fragments with pyknotic nuclear material showed granular or diffuse cytoplasmic staining. Some apoptotic bodies apparently at an advanced stage of degradation were negative. Granular reactivity was seen in the cytoplasm of some cells with normal nuclei; these cells were assumed to be at an early stage of apoptosis and were counted as positive.
[Figure 2 ILLUSTRATION OMITTED]
The negative control slides showed no cytoplasmic staining, but a weak nonspecific nuclear signal was observed in 1% to 2% of cells. This signal did not prove troublesome, because only cytoplasmic reactivity is significant in this context. The slides treated with mouse IgG2b showed identical results to the negative control slides.
Despite the significant correlation between M30 expression and ISEL, inspection of Figure 1 shows a wide scatter around the regression line. There is a particularly prominent outlier outlier /out·li·er/ (out´li-er) an observation so distant from the central mass of the data that it noticeably influences results.
an extremely high or low value lying beyond the range of the bulk of the data. ; it was a signet ring adenocarcinoma from a 73-year-old woman.
Four cases did not have satisfactory Ki-67 immunoreactivity (failure of internal positive controls to react) and were excluded. The mean Ki-67 counts of the remaining 77 cases were 26.8 for adenomas, 25.0 for carcinomas, and 25.7 for all cases combined. Since the set of Ki-67 results had a different number of items than the ISEL and M30 results, the Mann-Whiney U test was used to assess the correlation between Ki-67 counts and the apoptotic indices. There was a strong positive correlation in each case (P = .01 for Ki-67 vs ISEL and for Ki-67 vs M30).
No relationship between M30 count and age, sex, site (left vs right colon), Dukes stage, degree of dysplasia (adenomas), or degree of differentiation (carcinomas) was found. Cases in which the M30 count was more than twice the ISEL count or less than half the ISEL count were considered separately, but there were no correlations with any of the variables studied.
The results show that ISEL and M30 immunoexpression are correlated in adenomas and adenocarcinomas of the large intestine. However, there is a considerable scatter around the regression line (Figure 1), and it is clear that in any individual case the ISEL count does not necessarily predict the M30 count with any degree of accuracy. There are 2 possible explanations for differences between M30 and ISEL values in a given case. First, the techniques recognize different aspects of the apoptotic pathway. Second, the results of both tests depend on technique. In situ end-labeling in particular has been shown to be sensitive to the pretreatment step, which requires optimization for best results. The overall counts are consistent with previous reports in the literature, which describe mean values of around 1% to 2% for adenomas and adenocarcinomas of the large intestine.[21-25] Some studies have shown higher[25-27] or lower[28,29] values, at least for some types of lesion; this phenomenon might be explained by differences in case selection, technical procedures, or interpretation of the histologic appearances.
In their report on M30, Leers et al compared M30 expression with TUNEL and found that M30 expression was an earlier event in the apoptotic pathway than TUNEL positivity, and that TUNEL positivity persisted in late apoptosis with complete nuclear disintegration, although M30 became negative. However, a quantitative comparison between TUNEL counts and M30 counts was not presented in their article. My results are consistent with the onset of M30 expression preceding ISEL positivity, the latter remaining positive after M30 expression is no longer detectable.
Ki-67 is expressed in proliferating cells and is widely used to determine the growth fraction in tissue samples. Although Ki-67 is superior to other proliferation markers in that it is not expressed during excision-repair of DNA, it can be expressed in cells even when DNA synthesis is blocked, and can thus overestimate the proportion of actively cycling cells. Studies have not shown any relationship between Ki-67 expression and prognosis in colorectal carcinoma.[30,33-35] Like other studies,[23,24,29] my results showed a correlation between the expression of Ki-67 and the apoptotic count, using both ISEL and M30.
In conclusion, M30 immunoexpression and ISEL positivity correlate strongly. Since M30 immunohistochemistry is technically simpler and easier to interpret than ISEL, it has the potential to become the method of choice for demonstrating apoptosis in formalin-fixed tissue for cells containing cytokeratin 18.
This study was supported by the Education and Research Fund of the Royal Hospital Haslar (Gosport, Hampshire, United Kingdom). The author is an Honorary Research Fellow at St Mark's Hospital (Harrow, Middlesex, United Kingdom) and thanks Ian Talbot, MD, FRCPath, for his comments and support.
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1. Wearing or supplied with a cowl; hooded.
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The reduction or elimination of government power in a particular industry, usually enacted to create more competition within the industry.
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A person with numerical and computer skills who carries out quantitative analyses of companies.
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See Table at currency.
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Accepted for publication July 14, 2000.
From the Department of Pathology, Royal Hospital Haslar, Gosport, Hampshire, United Kingdom.
Reprints: Norman John Carr, FRCPath, Department of Cellular Pathology, Level E, South Block, Southampton General Hospital Southampton General Hospital is a large District General Hospital (DGH) in Southampton, operated by the Southampton University Hospitals NHS Trust. The hospital was the location for the daytime TV fly-on-the-wall documentary series, The General. , Tremona Road, Southampton, Hampshire SO16 6YD, United Kingdom.