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32 CHAPTER 3 INTRODUCTION TO THE METHODS USED FOR STUDYING THE BIOLOGICAL BEHAVIOR OF PILOCYTIC ASTROCYTOMAS
32
CHAPTER 3
INTRODUCTION TO THE METHODS USED FOR STUDYING THE BIOLOGICAL BEHAVIOR
OF PILOCYTIC ASTROCYTOMAS
1. AgNOR-STAINING
Nucleolar organizer regions (NORs) are specific portions of DNA, called rDNA, that, by using theenzyme RNA-polymerase-1, code for the transcription of ribosomal RNA (rRNA). This rRNAinside the ribosomes is responsible for protein synthesis of the cell. Protein synthesis is a necessarystep in the process of cell proliferation. Therefore a relation between NORs and cell proliferation issuggested.The existence of NORs has been well-known to cytogeneticists for many years, this NOR-DNAwas visualized by in situ hybridization making use of radio-labeled rRNA, a reliable but time-consuming method. With the use of silver colloid impregnation, described by Goodpasture andBloom in 1975 (1) and modified by Ploton et al in 1986 (2), NORs can be identified much easier.By using the silver nucleolar organizer region (AgNOR) impregnation technique the number, sizeand shape of NORs can be studied in a fast and simple way, not only in fresh frozen tissuespecimens but also in formalin fixed paraffin embedded material. The amount of silver deposit in acell, reflecting the amount of NORs that are involved in protein-synthesis, is thought to be related tothe proliferative capacity of that cell. The exact relationship between proliferation, protein-synthesisand expression of AgNORs is, however, not yet well understood. But the expression of AgNOR iseither causally or indirectly coupled to DNA-synthesis and thus AgNOR can be considered as acell proliferation marker (3). Therefore, in recent years the AgNOR staining method has become anew tool among the diagnostic possibilities for histopathologists. NORs reside on the short arms of the acrocentric human chromosomes 13, 14, 15, 21 and 22,these areas are the sites which hybridize with rRNA and are of importance with respect to theultimate synthesis of protein. NOR-DNA, the associated proteins and rRNA are located in thenucleolus of the cell, where also the nucleolar chromosomes rest in interphase (3). In simple terms:NORs are parts of DNA in the nucleolus that encode for rRNA, this rRNA forms the ribosomes,the "protein-factories" of the cell.The NOR-associated proteins bind very well to silver. This argyrophilia is associated to the step ofphosphorylation of the protein nucleolin (protein C23), by which this protein is activated, and thetranscription of rDNA is made possible. Nucleolin is the major silver staining protein in this process.Nucleolin is a 92 kd nucleolar protein, which is thought to control rDNA transcription. Thephosphorylation of the protein nucleolin is probably performed by p34cdc2 kinase, which is asubunit of M phase kinase, an enzyme involved in bringing cells into mitosis (4). Another importantNOR-associated protein beside nucleolin is "protein B23". There exists a good correlation betweenmean AgNOR area count and amounts of nucleolin and protein B23 inside the same cell (5).However, stimulation of rRNA synthesis, and thus of proteins associated to rRNA, does notnecessarily give a quantitative increase in amounts of nucleolin and protein B23. This means thatincreased rRNA synthesis may occur without increase in NORs (6). The numerous previous reports on studies of AgNOR staining in human tumors and tissues couldnot unequivocally support a direct positive relation between number or size of AgNORs andbiological behavior of the tumor studied. The different and sometimes conflicting results of thesestudies may be partially attributed to differences in the used staining technique and to differences ininterpreting the results of the silver-staining. As for the technique used for silver staining, many variations and improvements are reported. Wet-autoclaving pretreatment gives better staining results(7). A staining period of longer duration has
34
been made possible by using polyethylene glycol-thiosulfate (PEG-Th), this improved the contrastbetween silver deposit and background (8). The use of microwave-irradiation, giving a shorterprocessing time, results in less unspecific silver deposits and increases the contrast of the deposit.Gelatin as a protective colloid and Farmer's solution to optimize the specificity, are recommended(9). Triton-X-100 staining prior to the usual AgNOR staining has been reported to give bettercontrast and improves the background, without influencing the total AgNOR count (10). By usingthe blue-toning technique, which consists of a mixture of FeCl3, hexacyanoferrate and oxalic-acid,the resolution and the number of counted AgNORs increases two- to threefold (11). Importantfactors during the staining procedure appeared to be the oxidation-reduction level and the type ofgelatin used (12). In another study, many types of fixatives were tested; acetone, ethanol, methanol,Bouin's solution, 4% glutaraldehyde, 10% formalin and 10% formol-salin. It appeared that none ofthese were a limitation in acquiring reliable AgNOR counts (13).For scoring the amount of silver deposit after staining of a tissue slice, many different methods arebeing used. In older studies the number of intranuclear "silver-dots" were hand-counted, making useof a light-microscope. After counting at least 100 cells a mean number of AgNOR dots per cellcould be calculated. In most studies this method is still used and AgNOR staining is expressed asmean number of silverdots or AgNORs per cell or per nucleus or per nucleolus. A major problem iscaused by the clustering of small deposits to one larger dot, which makes it difficult and sometimesimpossible to establish the total number of small dots. To overcome this problem the total surfacearea of the silver deposit can be measured, which has the advantage that an automated computer-assisted analysis can be performed. The disadvantage of the latter technique is that also extranuclearprecipitated silver particles will be counted. Counting the number of AgNORs is subject to intra- and interobserver variability, which is oftenregarded as a limitation to the reliability of the results. However, when the interobserver variationwas tested, it appeared that there was a statistical significant correlation between the results foundby two observers (14,15), but the correlation coefficient was higher in counting AgNOR-areas thanin AgNOR numbers (16,17). To make the counting more specific, the silver-deposit only inside thenucleolus instead of the whole nucleus can be regarded. In one study this led to the conclusion thatmeasurement of the whole nucleolar size has the same relevance as AgNOR scoring in thesenucleoli (18). Another study found a positive correlation between AgNOR number and nuclear size(19) but both had no value in predicting survival of the patient. These results were contradicted byTosi et al. who found that the number of AgNORs had a significant predictive value in patientoutcome, but form, shape and size of the nucleus had not (20). The mean number of AgNOR dots per cell, or per nucleus, is often referred to as mAgNOR. ThemAgNOR scores in cells with slow proliferation may be in the range between 0,5 and 1,5, howeverthere are only very few reports giving "normal-values" of mAgNOR for different types of humantissues. The mAgNOR scores are higher in fast proliferating tissues. Most AgNOR studies focus onthe difference in AgNOR counts among tumors of different pathological grades and tissues indifferent stages of neoplasia, i.e. dysplasia, in situ carcinoma or invasive carcinoma.
The pAgNOR refers to the percentage of cells in a tumor or tissue slice that harbors more than acertain number of AgNORs per cell (mostly more than 5), this is also called the AgNOR distributionscore and sometimes the AgNOR proliferation index. Some studies, correlating the results of
35
flowcytometry and BrdU-labeling with AgNOR staining, demonstrated that pAgNOR correlateswith percentage of cells in S-phase of the cell cycle, or with proliferative activity, whereasmAgNOR correlates with ploidy (21,22,23).Many investigators have regarded the size and some of them also the shape and the localization ofthe AgNOR-dots. An inverse relationship between number of AgNORs and AgNOR size is oftenreported (24,25,26). However, in contradiction to this also an increase in AgNOR size togetherwith increasing malignancy is reported (27,28,29). In general it is assumed that malignant cells,showing more AgNORs per nucleus, have smaller AgNOR dots than benign cells, showing less andlarger AgNORs. While individual AgNOR dots become smaller as their number increases withincreasing malignancy, total AgNOR area per cell increases together with increasing malignancy(30,31). AgNOR expression is also related to cell maturation; aging cells from the anterior lobe ofrat-hypophysis show that the number of AgNORs and the total AgNOR area decrease, whereasthe size of the individual AgNOR particles increase (32,33,34). Furthermore, AgNOR expressiondepends on the level of cell-differentiation, resulting in a decrease of AgNOR number and size withincreasing differentiation (35). This decrease of AgNOR number and size during aging, maturationand cell-differentiation may reflect suppression of rDNA transcription. AgNOR scores are modified in different ways in various reports. These modifications mostly try toincorporate not only the AgNOR number, but also the AgNOR size and the nuclear size. This hasled to AgNOR scores being expressed as the ratio of mean AgNOR-number and mean AgNOR-size (26), the ratio of AgNOR-area and nuclear-area (36) or the ratio of number of small AgNORs(< 3 microns) and number of large AgNORs (> 3 microns) (20). Also the localization of theAgNOR inside the nucleus might be related to cell behavior; with increasing malignancy in astrocytictumors AgNORs moved from central to peripheral inside the nucleus (29,37).With the improvement of the AgNOR-staining technique over the years and the use of automatedcomputer-assisted surface-area measurement of the precipitated silver-colloid inside the nucleus,the AgNOR staining has become much more standardized and reliable. However it should berealized that the number of detectable NORs depends on several factors: the level of transcriptionalactivity, the number of NOR-bearing chromosomes in the karyotype and the stage of the cell-cyclein which they are sought (38). In an attempt to estimate the value of AgNOR staining in tumor pathology we reviewed all reportedstudies on AgNOR staining during the years 1992, 1993, 1994 and 1995. In a "med-line" search,under the keyword "AgNOR", 285 articles were produced. These were all screened forconclusions on the presence or absence of a statistical significant relation between AgNOR scoreand tumor type, or between AgNOR score and patient outcome. Furthermore, the type of AgNORscore used and a possible relation to other proliferation markers were noted. These criteria weremet in 109 articles. Table 1 presents the results of these studies.
In the 109 reviewed reports on AgNOR expression in tumors, 118 times a conclusion was drawnconcerning the relation to tumor type (histopathological grade, benign versus malignant) or to patientoutcome (survival and prognosis). Of the 75 conclusions relating AgNOR score to tumor type, 55(73%) showed a positive statistical significant correlation and 20 (27%) showed no relation. Theother 43 conclusions concerned the relation between AgNOR score and patient outcome; 26
36
(60%) showed a statistical significant positive correlation and 17 (40%) showed no relation (table2).Almost two-thirds of the reviewed papers studied the relation of AgNOR score to tumor type. Asmentioned before, the determination of AgNOR expression is a subjective method regarding thevariations in staining methods and in counting and interpretation of the results. Also thedetermination of the tumor grade, using various histopathological parameters, is based on subjectivevariables. Thus, it can be argued that the majority of the previously published reports (64%) seek todraw conclusions on the results of two subjective "measurements". Most of these reports, even afterhaving shown a positive correlation between AgNOR expression and tumor-type, doubt therefore,the clinical relevance of their conclusions.A good parameter for testing the relation between AgNOR score and tumor proliferation would bethe assessment of tumor growth during follow-up of the patient. One single study was found in ourreview, done on adenocarcinomas of the lung, where tumor size was radiologically determinedduring patient follow-up (39). Interestingly the results of this study showed lack of correlationbetween AgNOR score and degree of histological differentiation or pathologic staging, but a highinverse relation between AgNOR count and doubling time of the tumor.Still an indirect, but a clinical more useful parameter for determining the value of AgNOR staining isthe patient-outcome as expressed in survival percentage or prognosis, which is done in one-third(36%) of the reviewed studies. In a small majority of these studies (60%) a positive correlationbetween AgNOR expression and patient outcome was found. However among the 26 studies thatreported this positive correlation, in 9 this relation could only be established when separating thepatients into two groups, with high and low AgNOR scores, using a "cut-off" score. The level of thiscut-off score was rather high in most studies; 5 or 6 AgNOR dots per nucleus and in one studyeven 11. Furthermore, the number of AgNOR dots in the tumors showed such variation that often agreat overlap existed between groups with favorable and unfavorable outcome, preventing clinicalusefulness in the individual case.Almost all reports express the result of AgNOR staining in number of AgNORs per nucleus. Only29 (25%) of 109 reports express the result of AgNOR staining in area-measurement and often notonly the AgNOR area is measured but also the nuclear area. Considering these 29 reviewed studiesseparately, the same conclusions regarding AgNOR score in relation to tumor type or patientoutcome are drawn as in the whole group of reviewed articles. In 69% there is a positive relationbetween AgNOR score and tumor type or patient outcome, and in 31% this relation could not beproven. Thus in this respect AgNOR area measurement leads to the same conclusions as AgNORnumber measurement.
The relation of AgNOR scores to other proliferation markers is as controversial as to theaforementioned parameters. Among the 109 reviewed studies, in 27 the AgNOR score wascorrelated to one or more other proliferation markers. These results are listed in table 3. Only in 1of 4 studies dating from the period 1992-1995 a positive relation to Ki-67 labeling could be found(40). Another nucleolar antigen is PCNA; from the 15 studies comparing PCNA labeling index toAgNOR expression only 60% showed a clear positive relation between the two. The same wasfound for the mitotic index, but in only 5 studies this relation was studied. S-phase fraction and
37
ploidy did correlate with AgNOR score in 6 out of 8 studies. Possibly the lack of a positive relation between the aforementioned markers and AgNOR score liesin the fact that AgNOR is expressed as well in proliferating as in resting cells, whereas for examplePCNA and Ki-67 expression are absent in resting cells. Furthermore, AgNOR staining reflects onlythe process of protein synthesis in the cell and not necessarily cell proliferation. Since AgNOR expression is not an indicator for number of growing cells or growth fraction butreflects the rapidity of the cell cycle and is related with tumor doubling time, and the technique issimple, fast and reliable when using the computer assisted area measurement, it is felt that thistechnique might be an adjunct to the diagnostic possibilities for determining tumor behavior (5,39).
In order to test the clinical usefulness of AgNOR staining as a proliferation marker in pilocyticastrocytomas, AgNOR expression is studied in relation to behavior of residual tumor, as determinedby follow-up neuroimaging studies of the patient (chapter 4).
TABLE 1. Results of 109 reviewed studies relating AgNOR score to histopathological type or gradeof tumor, or to patient outcome.
ref nr. tumorloc.
nr. tumor-type histolgrade
outcome
Ag-NORscore
otherprol.markers
re-marks
41 pancr. 60 var. tumors + pAg>5%Agarea
+metas
42 thyroid 56 var. tumors - mAgAgarea
43 thyroid 22 var. tumors - mAgAgarea
44 thymus 60 thymomas + mAg>5,75
45 thymus 29 ca.vs.thymoma + mAg overl
46 lung 29 all tissues + mAg +PCNA
47 lung 111 adenoca. + mAg>5
48 lung 86 adenoca. + mAgAgarea
49 lung 21 mesotheliomas + mAg +PCNA
50 lung metaplasia vs. ca.
+ mAg>5,34
51 lung 57 carcinoma carcinoid
+ + mAgAgarea
ref nr. tumorloc.
nr. tumor-type histolgrade
outcome
Ag-NORscore
otherprol.markers
re-marks
38
52 lung 111 carcinomas + mAg
39 lung 58 adenoca. - mAg + prol *1
53 colorec 61 all tissues + mAg
54 colorec 40 adenoca - mAg,Agarea
- BrdU- 3HdT
55 colorec 60 adenoca - Agind.
56 colorec 92 adenoca + mAgAgarea
57 colorec 116 hereditary vsspor. polyps
+ mAgAgarea
58 colorec 150 var polyps - mAg - Ki67
59 colorec 109 var biopsies - mAg +metas
60 colorec 164 var cancers + mAg
28 colorec 27 var cancers + Agarea
61 colorec adenomas + AgareaNuarea
62 colorec 95 var tumors - - mAg
63 gastint 60 var tumors + + mAg
64 gastint 24 var tumors - mAg + MI
65 gastint 101 biopsies + Agarea
66 gastint dysplastic tissue - Agarea -MIB1-PCNA
67 breast 131 var cancers + Agarea
15 breast 44 biopsies + mAg +metas overl
68 breast 112 biopsies + mAg
69 breast 62 hyperplasia - mAg -Ki67
70 breast 200 carcinomas Agarea +p53
71 breast 72 biopsies + mAg>11
ref nr. tumorloc.
nr. tumor-type histolgrade
outcome
Ag-NORscore
otherprol.markers
re-marks
39
72 breast 74 var tumors + Agarea
73 breast 44 biopsies - mAgAgarea
-C-erbB-2
74 breast 170 carcinomas + mAg>11
75 breast 230 var cancers - mAg +Spf
76 breast var cancers + ratioAgareaNuarea
+DNAc +MI
77 breast 75 var cancers - - mAg
78 breast 31 var cancers + mAg + TNM
79 skin 20 melanoma vsben. naevus
+ mAg
80 skin 228 biopsies + Agarea +PCNA
81 skin 41 melanoma vsben. naevus
+ mAg
82 skin 50 acrospiroma vssweatgl.ca
+ mAg
83 skin 43 melanomas - mAg -PCNA-MI
84 skin 36 keratoacant. vssq cell ca
+ mAg overl
85 skin 98 melanomas + mAg>3,6
+metas
86 eye 46 melanomas - mAg
87 brain 35 pineal tumors - mAg
29 brain 79 astrocytomas + mAg
88 brain 39 meningiomas + mAg -PCNA
89 brain 27 astrocytomas + mAg
30 brain 61 astrocytomasmeningiomas
+ mAgAgarea
ref nr. tumorloc.
nr. tumor-type histolgrade
outcome
Ag-NORscore
otherprol.markers
re-marks
40
90 brain 8 lymphomas + mAg>4
91 brain 28 gliosis vs low-gradeastro
+ mAg
92 uterus 78 all tissues + mAg
93 uterus 45 adenoca. + Agarea
94 uterus all tissues + mAg overl
95 uterus hyperplasia vsadenoca
+ mAg
96 cervixuteri
85 adenoca. + mAgAgarea
97 cervixuteri
46 invasive vs in situca
- mAg
98 oralcavity
26 var cancers + - mAg
99 oralcavity
dysplasia vs sq.cell ca.
+ mAg -PCNA-MI
100 oralcavity
41 verrucous ca vssq cell ca
- mAgpAg>5
40 oralcavity
var cancers + mAg +PCNA+KI67+Spf
101 oralcavity
24 hyperplasia vs sqcell ca
+ mAg
102 esophag 182 var tumors + mAg>5
103 esophag 50 var tumors + ?
104 esophag 45 sq cell ca + mAg
105 esophag 31 var cancers + mAg>6
106 esophag 91 var cancers + mAg *2
107 esophag 15 all tissues - mAg +PCNA
108 salivgland
30 adenoid cystic ca - - mAg
ref nr. tumorloc.
nr. tumor-type histolgrade
outcome
Ag-NORscore
otherprol.markers
re-marks
41
109 salivgland
25 adenoid cystic ca + mAg
110 maxil.sin 25 sq cell ca - mAg
111 headneck
15 paragangliomas + mAg overl
112 pharynx 45 carcinomas + mAg +PCNA
27 urin bl 24 carcinomas - mAgAgarea
+PCNA
113 urin bl 80 carcinomas + - mAg
14 urin bl hyperpl vs ca - mAg
26 urin bl 170 all tissues + ratiomAgarea
114 urin bl 50 carcinomas + mAg
115 prost. var tumors + mAg
116 prost 50 carcinomas - Agarea
117 prost 74 carcinomas + mAg
118 prost 20 all tissues - mAg -Spf
119 prost 78 adenoca - mAgNuarea
120 prost 28 adenoca + mAg -DNAc
121 blood/lymf
58 NHL + + mAg>2,9
122 blood/lymf
101 NHL vs hyper-plasia
- Agarea
123 blood/lymf
200 NHL + mAg +PCNA
124 blood/lymf
NHL mAg -PCNA
125 blood/lymf
35 NHL + + mAg
ref nr. tumorloc.
nr. tumor-type histolgrade
outcome
Ag-NORscore
otherprol.markers
re-marks
42
126 muscle/soft ti
151 Soft tissuesarcoma
+ mAg +Spf+DNAc
127 muscle/soft ti
194 Soft tissuesarcoma
+ mAg
14 muscle leiomyoma vsleiomyosarc
+ mAg +MI
128 blood 50 leukemia + mAgpAg>5
+DNAc
129 kidney 95 renal cell ca + mAg -PCNA
130 kidney 59 renal cell ca + Agind.
131 kidney 173 renal cell ca + ?
132 kidney 44 rcc vs sarco-matoid rcc
+ mAg +PCNA
133 liver 43 all tissues - mAg
134 liver 48 Cholangiocel-lularca
+ mAg
135 liver 89 hepatocel-lular ca + + mAg>3,04
136 galbl 53 normal vs ca + + mAgAgarea
137 testis 45 var tumors - mAg
138 bone 54 osteosarcoma - mAg
139 skull 36 chordomas - mAgAgarea
Legend table 1:
- Ref nr.= reference number of study cited. - Tumor loc. = location of tumor, pancr= pancreatic gland, thyroid= thyroid gland,colorec=
colorectal, gastint= gastrointestinal, esophag= esophagus, saliv= salivary gland, maxil= maxillary, sin= sinus, urin bl= urinary bladder, prost= prostate, galbl= galbladder
- Nr.= number of tumors studied with AgNOR staining; - type tumor= type of tumor or tissue studied, var.= various, ca= carcinoma, vs.= versus
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(when two types of tumors or tissues are compared to each other regarding AgNOR score),
spor.= sporadic, ben.= benign, sweatgl.= sweat gland, sq. cell ca= squamous cell carcinoma,
astro.= astrocytoma, hyperpl= hyperplasia, NHL= Non Hodgkin Lymphoma, leiomyosarc=
leimyosarcoma, rcc= renal cell carcinoma..
- histol/grade= significant relation of AgNOR score to histological type or grade of tumor:+ =
positive correlation, - = no correlation. - Outcome= significant relation of AgNOR score to prognosis, survival or outcome of thepatient:
+ =positive correlation, - = no correlation. - AgNOR score= type of score used, being mAg= mean number of AgNOR per cell or pernucleus,
pAg= percentage of cells showing AgNORscore above a certain number; Agarea= meanarea of
AgNOR per cell or per nucleus, Nuarea= mean total nuclear area, Agind.= ratio of mAg intumor
cell to mAg in normal cell.- Other prol. markers= relation of AgNOR score to other tested proliferation markers(PCNA=
proliferating cell nuclear antigen, BrdU LI= BromodeoxyUridine labeling index, 3HdT LI=3H thymidine labeling index, MI= mitotic index, Spf= S-phase fraction, DNAc= DNA content(by flowcytometry) or relation to other tumor characteristics (metas= to metastases): + =positive
correlation, - = no correlation. - Remarks: overl= great overlap between AgNOR scores in subsets of groups, so that thereis no
diagnostic value to an individual AgNOR score.*1 Negative relation of AgNOR number to histological grade, but positive relation to tumor
proliferation rate as measured by tumor size on chest-radiographs.*2 Only prognostic value of mAgNOR in combination with DNA-content as measured by
flowcytometry.
TABLE 2. Results of 109 reviewed papers on the relation of AgNORscore to tumor type and topatient-outcome.
AgNOR SCORE RELATEDTO:
TUMOR TYPE PATIENT OUTCOME
total
positive relation 47% 22% 69%
no relation 17% 14% 31%
total 64% 36% 100%
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TABLE 3. Relation of AgNOR score to several other tumor proliferation markers in 109 reviewedpapers.
proliferationmarker
number of studies positive relation no relation
PCNA 15 60% 40%
Mitotic index 5 60% 40%
S-phase fraction 4 75% 25%
DNA-content 4 75% 25%
Ki-67 LI 4 25% 75%
p53 LI 1 1 -
BrdU LI 1 - 1
3HdT LI 1 - 1
Legend table 3.Number of studies: Among the 109 reviewed papers, the number of times the specificproliferation marker was studied in relation to AgNOR score.PCNA: Proliferating Cell Nuclear Antigen.S-phase fraction: percentage of cells in S-phase determined by flowcytometry.DNA-content: ploidy, as measured by flowcytometry.Ki-67 LI: Ki-67 or MIB-1 labeling index.p53 LI: Immunohistochemic p53 protein labeling index.BrdU LI: Bromodeoxy-Uridine labeling index.3HdT LI: radiolabeled Hydrogen-Thymidin labeling index.
2. KI-67 AND MIB-1 LABELING
In the search for antibodies against nuclear antigens specific to Hodgkin and Reed-Sternberg cells,Gerdes et al. in 1983, discovered a mouse-monoclonal antibody, named Ki-67, that recognized anuclear antigen present only in proliferating cells (140). The Ki-67 reactive nuclear antigen is onlyexpressed in the G1, G2, M and S-phase of the cell cycle, and not in the resting or Go-phase.Schonk et al. concluded that the gene involved in the expression of the Ki-67 antigen is localized onchromosome 10 (141). The immunoreaction between the Ki-67 antibody and the reactive antigencould only take place in fresh frozen tissues. Apparently, the routinely used formalin fixation processdestroyed the antigenic activity to the Ki-67 antibody. However, in 1991, by the same group as in1983, part of the gene, encoding for the Ki-67 protein was cloned and sequenced (142). By
45
immunizing mice with recombinant Ki-67 gene product a new monoclonal antibody was developed,named MIB-1. It appeared that this antibody reacted with the Ki-67 antigen in formalin fixed tissuesafter antigen retrieval. The deduced aminoacid sequence of the gene encoding for the Ki-67 proteindid not reveal homology to any known cell-cycle protein, thus the function of the Ki-67 proteinremains unknown (143). Many studies have been undertaken to use the Ki-67 antibody, or the later developed MIB-1antibody, for assessment of the amount of proliferating cells in all kinds of normal human tissues,tumors and cell lines. Especially in neoplastic diseases the quantifying of the fraction of proliferatingcells can be an adjunct to conventional histologic diagnosis for predicting the biological behavior ofthe tumor and the prognosis of the patient. The Ki-67 labeling index (LI), being the number ofpositive staining cells divided by all counted cells, expressed as a percentage and also called theproliferating cell index (PCI) or proliferation index (PI), correlates well with the behavior of manymalignant tumors, such as lymphomas and breast carcinoma (144).The reliability of the Ki-67 immunostaining technique in gliomas was confirmed when comparing itto other labeling techniques, such as incorporation of radiolabeled 3H-Thymidine in the tumor cellDNA or measurement of the uptake of the thymidine-analogue Bromodeoxyuridine (BrdU)(145,146,147). These older techniques have proven their usefulness in determining the number ofproliferating cells or the growth fraction, but have the great disadvantage of being invasive, sincethey make it necessary to administer, by intravenous injection, a tracer into the patient. Table 4 lists the results of 32 previous studies of Ki-67 labeling in gliomas. The earliest studiesmerely tried to establish "normal-values" of Ki-67 LI in different types and grades of gliomas.Others compared Ki-67 labeling to results of other methods of growth fraction assessment in thesetumors, such as Proliferating Cell Nuclear Antigen (PCNA) labeling, Bromodeoxyuridine (BrdU)labeling, p53 immunostaining, determination of S-phase fraction by flow-cytometry and countingnumber of mitoses. More recent studies investigated a possible relation between MIB-1 labelingand patient survival or outcome.
Values for Ki-67 LI are in general lower than for MIB-1 LI. In one study these 2 differentantibodies were compared using the same tumors to test both (148). It appeared that MIB-1 LI isapproximately 1.6 times higher than Ki-67 LI. Probably the antigenic activity is better preserved inparaffin embedded tissues than in fresh frozen tissues. Furthermore, cells in early G1 phase containvery little antigenic activity, possibly these cells are detected with MIB-1 but not with KI-67. In 17 of the 32 cited previous studies Ki-67 has a clear correlation with astrocytoma grade; LIvalues being higher in tumors of higher grade. Mean Ki-67 and MIB-1 LI values of 25 grade Iastrocytomas were 0.25%-6%, of 159 grade II astrocytomas 0.5%-10.7%, of 230 grade IIIastrocytomas 3.0%-18.4% and from 424 glioblastomas (grade IV) 5.2%-31.6%. Despite the goodcorrelation between tumor grade and Ki-67 expression, the clinical usefulness for individual tumorsis very limited, since the values overlap significantly. Furthermore, the Ki-67 LI values in pilocyticastrocytomas are not different from grade II astrocytomas; the first even have slightly higher valuesin some studies (149,150,151,152). MIB-1 labeling studies on pilocytic astrocytomas have notbeen performed before 1997. Ki-67 LI values of 23 pilocytic astrocytomas were found to rangefrom 0.7%-6% (145,149-154,162). Among 92 grade II astrocytomas Ki-67 LI values ranged
46
from 0.5%-2%, whereas MIB-1 LI values of 65 grade II astrocytomas ranged from 2.03%-10.7%. In one study dating from 1997 the mean MIB-1 LI of 2 pilocytic astrocytomas was 0.25%(155). The paradox of equal Ki-67 antigenic expression among grade I and grade II astrocytomasand different prognosis, being much better in grade I tumors than in grade II, has not been studiedyet. The 7 studies that showed a good correlation between MIB-1 LI and survival or prognosis of thepatient were all referring to astrocytomas grade II-IV (155-161). In 5 studies this correlation wasachieved by using a statistical method that sets a cut-off score for the labeling index at a level wherepatients scoring lower have a better prognosis than patients scoring higher. The level of the LI-cut-off score varied considerably, from 1.5%-15.3% (156,157,159,162,163). In the 3 studies that compared Ki-67 labeling to BrdU labeling or S-phase fraction, a goodcorrelation was found (146,148,164). A relation with PCNA labeling, performed in one study,could not be proven (161). Comparing Ki-67 labeling to p53 immunostaining, mitotic index andAgNOR staining, as done in several previous studies, gives equivocal results.
Summarizing the information as given above, leads to the following conclusions:
- Ki-67 labeling and MIB-1 labeling are methods used to determine the fraction ofproliferating cells in tumors.
- MIB-1 LI values have a statistically significant relation to tumor grade among gliomasgrade II, III and IV.
- When using a cut-off score, that separates two groups, one with lower and one withhigher MIB-1 LI's in high grade gliomas, the MIB-1 labeling index has a predictive valueconcerning patient-outcome.
- MIB-1 LI values for pilocytic astrocytomas have hardly been assessed.- Ki-67 LI values of pilocytic astrocytomas are in the same range as for astrocytomas grade
II, despite their totally different prognoses.
The following questions remain:
- What are "normal-values" of MIB-1 LI in pilocytic astrocytomas ?- Are MIB-1 LI values of pilocytic astrocytomas and grade II astrocytomas in the same
range?- Has MIB-1 labeling a predictive value towards outcome or tumor behavior in the
relatively homogenous group of pilocytic astrocytomas ?
In an attempt to answer these questions a MIB-1 labeling study was performed among 39 pilocyticand 5 low grade astrocytomas in children and the results were analyzed in relation toneuroradiological follow-up of the patients (chapter 5).
47
TABLE 4. results of previous Ki-67 and MIB-1 labeling studies on gliomas, in chronological order.
1study
2anti-body
3gradeI
nr.LI
4gradeII
nr.LI
5grade III
nr.LI
6gradeIV
nr.LI
7relation tosurvival(S)or tumorgrade(G)
8 remarks
153Ki-67
11%
7 10-40%
G+
145Ki-67
130.9%
121.8%
64.3%
539.8%
149Ki-67
14.5%
150.7%
83.5%
2711.1%
G+ S-
"grade II piloc. astr."in a 53 y. old patient
165Ki-67
28 1-8.5%
138.6-14.2%
181-22.1%
166Ki-67
50.5%
53%
35.2%
G+ AgNOR: +
154Ki-67
10.7%
61.5%
45.1%
189.9%
AgNOR: -
167 Ki-67 45 gliomas, various grades AgNOR: +
168Ki-67
260.5%
264.1%
386.4%
G+
150Ki-67
16%
4<1%
84.4%
721%
G+ Ki-67 superior toPCNA
169Ki-67
21 20 32 G+ MI: +
151Ki-67
15.6% <1% 8% 10.1%
G+
170 Ki-67 S- only GBMs
162Ki-67
30.9%
71.2%
137.5%
2011.1%
G+S+(5%)
p53: -
163Ki-67
205.2%
S+(1.5%)
MI: -
171Ki-67
70.8%
9 7.2%
G+
48
146 Ki-67 200 brain tumors, various grades
G+ BrdU (S-phase): +AgNOR: -
152Ki67
22.1%
102%
56.1%
2018.5%
G+
147MIB-1
242.03%
2612.8%
914.6%
148MIB-1
227.3%
3023.9%
BrdU: +Mib-1= 1.6x Ki-67
156 MIB-1 50 astrocytomas, various grades
G+ S+(15,3%)
157 Ki-67 24 31 68 S+(2%) p53: +
172 MIB-1 137 brain tumors G+
173MIB-1
11<10%
2<10%
80-15%
80-15%
p53 mutation: +pediatric astrocytomas
164 MIB-1 31 glial tumors G+ S-phase: +
174 MIB-1 48 astrocytomas,various gr. G+
158 MIB-1 19 intramedullary spinal cord tumors S+
159MIB-1
1011.9%
1927.3%
S+(12%)
pediatric non- brain-stem gliomas
160MIB-1
193.8%
2518.4%
2831.6%
S+
161MIB-1
159.0%
1917.9%
8623.3%
S+ G+
p53: -PCNA: -
175 MIB-1 75 astrocytomas,various gr. G+
155MIB-1
20.25%
710.7%
76.3%
3715.8%
S+
176 MIB-1 46 astrocytomas,various gr. telomerase RNA: +(associated withtumorigenesis)
Legend Table 4:1= reference number of study. 2= Type of antibody used. 3= Astrocytoma grade I, or pilocyticastrocytoma, number of tumors studied and mean Ki-67 LI. 4= Astrocytoma grade II. 5=Astrocytoma grade III. 6= Astrocytoma grade IV or glioblastoma multiforme; various gr.=various grades. 7= Relation between Ki-67 LI and tumor grade(G) or survival (S), if present: +, the value of the "cut-off" Ki-67 LI score that discriminates between good and poor outcome is
49
given in brackets. 8= Remarks concerning relations found between Ki-67 LI and otherproliferation markers. MI= Mitotic Index.3. THE TP53 GENE AND p53 PROTEIN
The TP53 gene is known as a tumor suppressor gene located on the short arm of chromosome17.The p53 protein was discovered in 1979, and in the mid-1980's the gene was cloned. Itappeared that parts of the gene remained identical during evolution among species and this suggeststhat these highly conserved regions of the gene play a vital role for protein function (177). The genespans 20 kilobases of genomic DNA and consists of 11 exons (178). The TP53 gene and its product, being the wildtype p53-protein, have a key function in theregulation of the cell cycle. Overwhelming evidence exists for a pivotal role of p53 in regulating cellproliferation, differentiation, DNA-repair and apoptosis (177,180-184) (figure 1). Numerousmutations in the p53 encoding gene have been described that strongly associate with carcinogenesis.The TP53 gene is the most frequently mutated gene in human tumors. In it’s normal function p53 is up regulated in case of anoxia, DNA damage or disturbances in thereplicative process of the cell. This leads to several possible p53 mediated actions in the cell, alldirected towards “guarding” the genome (183). The p53 protein induces p21 (WAF1, WildtypeActivating Factor), which inhibits the cdk (cyclin dependent kinase) mediated signal transductionpathway resulting in a block of the cell-cycle into a G1-arrest. By stimulating the GADD (GrowthArrest and DNA Damage)-gene, DNA-repair mechanisms become active. When thesemechanisms are insufficient, p53 can accelerate the transcription of the bax-gene and inhibit bcl-2,which both lead to increased apoptosis, or programmed cell death (185).
The function of the TP53 gene can be studied in different ways, either by analyzing the structure ofthe short arm of chromosome 17 and the TP53 gene itself by molecular techniques, or by studyingthe presence or absence of the gene product, being the p53 protein by immunohistochemicalmethods. Currently, molecular techniques like Single Strand Conformation Polymorphism (SSCP)determination and Denaturing Gradient Gel Electrophoresis (DGGE) in combination withsubsequent DNA sequencing, allow rapid mutation screening. It appears that tumor relatedmutations in TP53 are predominantly present in exons 5,7 and 8, which are the evolutionary highlyconserved regions of the gene. Mutations outside these “hot spots” have also been reported though.The p53 protein in it's normal appearance is called "wildtype". The half life of this wildtype protein isvery short, in the range of 15 to 30 minutes and present only in very low concentrations in normalcells (177). Therefore it is thought to be undetectable by monoclonal antibodies used in even themost recent immunostaining techniques. Positive immunostaining with these monoclonal antibodiestherefore, reflects the presence of an altered p53 protein, with a longer half life, or an abundantpresence of the wildtype, which is normally not the case. The existence of such an altered p53protein is thought to be the result of a structurally changed TP53 gene, for example because of amutation. Depending on the severity of damage that is caused in the encoding process to the protein,which is dependant on the type of mutation, the function of the p53 protein may be impaired.
50
Mutation of the TP53 gene is an early event in the development of malignant gliomas of the brain(186,187). High grade astrocytic tumors of adulthood show over expression of the p53 protein ormutations in the TP53 gene in 32%-90% of cases (188-192). In adult low-grade astrocytomas thisfrequency varies from 5%- 60 % (186,187,188,189,191,193,194,195). Mutations of TP53 arestrongly associated with progression of low grade astrocytomas to high grade astrocytomas. Weberet al. studied 5 astrocytomas grade II that recurred as an anaplastic astrocytoma, all had a mutationof the TP53 gene (196). Different pathways have been distinguished in the development of a gradeIV astrocytoma (glioblastoma) (197). Some astrocytomas grade IV are the result of malignantprogression of a lower grade astrocytoma, these tumors showed disturbed function of the TP53gene, either immunohistochemically or genetically. Other grade IV astrocytomas arise “de novo”,those are p53-immunonegative and have no mutations in the TP53 gene.
In malignant gliomas of adulthood TP53 mutations are more often found in younger patients than inthe older (198). However, the reported incidence of these mutations in pediatric astrocytomas isvery variable. Litofsky et al. found no mutation among 35 pediatric astrocytomas of all grades(199), Rasheed et al. found only 1 mutation among 48 astrocytomas of all grades in patients undereighteen years of age (200). Neither did Felix et al. find a mutation among 17 low gradeastrocytomas of childhood, but 2 of 3 glioblastomas of childhood appeared to have a TP53mutation, of which one also had a germline TP53 mutation (201). Among 29 malignant brainstemgliomas of childhood 18 were p53-immunopositive and 11 showed a TP53 mutation (202). Theprognosis of the children with TP53 mutations was less favorable. It was concluded that TP53mutation frequency among high grade gliomas of childhood is in the same range as for adults,however the frequency among children under 4 years of age was lower, and the prognosis of thesevery young children was better. In another study among 21 malignant gliomas in childhood almost50 % was p53 immunopositive (203). However, in this study the p53 immunostatus had no relationwith survival (203).
In pilocytic astrocytomas p53 dysfunction is very rare. In the 19 previously published papers a totalof 218 pilocytic astrocytomas were studied for p53 abnormalities, either immunohistochemically, orby molecular analysis techniques. Results of these studies are listed in table 5. From the 77 tumorsimmunohistochemically studied 19 showed p53 protein over expression (25%). Chromosomalanalysis, performed in 75 tumors, showed in 8 structural changes (LOH, Loss of Heterozygosity) onthe short arm of chromosome 17 (11%), but in 4 cases these changes occurred just outside theregion of the TP53 gene. Furthermore, the one tumor with LOH of chromosome 17p in the study ofWu et al. is probably an adult low grade astrocytoma, which leaves only 3 pilocytic astrocytomaswith LOH of 17p on the site of the TP53 gene (204). From the 107 tumors studied by geneticanalysis techniques, mainly being SSCP and sequencing, only 3 showed a mutation in the gene.These 3 mutations were found on 3 different localizations: At codon 47 in exon 4, codon 248 inexon 7 and codon 324 in exon 9. Most of these genetic analyses have screened the geneincompletely, they were restricted to the known frequent mutation hot-spots of other tumors, whichare exons 5 to 8. Willert et al. demonstrated one of these mutations in a study of 20 juvenilepilocytic astrocytomas (JPA's), exons 4 to 9 were analyzed, after PCR, denaturing gradient gel
51
electrophoresis and sequencing (205). The mutation was found in exon 7 at codon 248, which is aknown hot-spot for TP53 mutations in many other malignancies. However, they found other placeson the short arm of chromosome 17 where loss of heterozygosity was detected, i.e. in two of 20JPA's telomeric to TP53 and in 6 of 20 JPA's centromeric to the TP53 region. Of those 6 JPA'swith centromeric loss, 4 tumors (66%) behaved rather aggressively, whereas only 5 of the remaining14 tumors (36%) showed this behavior. One of the conclusions of the authors is that the TP53 genemay have a role in the formation of JPA's.
Immunohistochemical analysis of the p53 protein may show immunopositiveness without mutation ofthe gene (186). The frequency of p53-immunopositiveness among astrocytic tumors is much higherthan of TP53 mutations. In the study of Lang et al. p53 immunopositiveness was found in 71% ofpilocytic astrocytomas (n=7), 63% of grade II astrocytomas (n=8) and in 63% of grade IIIastrocytomas (n=16) (186). However, DNA sequencing after SSCP revealed among the sametumors TP53 mutations in only 14%, 25% and 19% respectively. Another study found similar highincidences of p53 immunopositiveness, i.e. 54% in grade II, 75% in grade III and 90% in grade IVastrocytomas (189). TP53 mutation frequency determined by SSCP and DNA sequencing in thestudy of Patt et al. (187) was again low: in pilocytic astrocytomas 14% (1 out of 7), in grade IIastrocytomas 5.6%, in grade III astrocytomas 25% and none in glioblastomas. Although it wasbelieved that immunostaining identified only mutant forms of the p53 protein, since the wildtypeprotein has a half-life too short to be detected by the monoclonal antibody, these results led to theassumption that the antibody also reacts with the wildtype p53 protein but only when present in anexcessive amount, which means an over expression. Such an over expression of wildtype p53 canbe caused by cell-cycle specific variations, amplification of the MDM2 gene resulting in binding ofthe gene product to the p53 protein and thus accumulation, DNA damage and anoxia leading tostress-induced increases of wildtype p53 expression (202,206).Rubio et al. demonstrated that the often used monoclonal antibody PAb 1801 binds to mutant aswell as to wildtype p53 protein, whereas the antibody PAb 240 only binds to the mutant form(207). In this study it was proved that over expression of wildtype p53 protein can occur without aTP53 mutation. Another study showed the same discrepancy between results of immunostainingand DNA analysis in gliomas grade II-IV (190). However, in this study there was a correlationbetween the results of the two techniques among tumors that showed more than 5% of positive p53immunostaining cells. Most of the highly immunopositive tumors showed indeed a high mutationfrequency, whereas in many of the tumors that were immunopositive for less than 5% of the totalamount of cells a DNA mutation could not be detected. This was attributed to the low sensitivity ofthe DNA analysis technique used, being SSCP followed by direct sequencing of DNA. Theconclusion of the authors was that the immunostaining technique might be more useful to detect p53alterations than the DNA analysis technique. Furthermore, it was stated that genetic analysistechniques using the PCR method, only screen a very small sample of tumor DNA, mostly from avery small area of the whole tumor. Knowing that astrocytomas are heterogenous tumors, this maylead to sample errors giving rise to false-negative results.
52
In summary, immunohistochemical p53 positiveness can occur in the following situations:1. There is over expression of wildtype p53 protein: the p53 protein is present in an increasedamount but has a normal function. This reaction occurs as a physiological response to DNA-damage (185). An example of this are the elevated levels of wildtype p53 found in skin-cells after asunburn (208). 2. There is accumulation of p53 protein: the p53 protein is bound in the cell to other proteins whichcause an increased stability of the protein and a prolonged half-life. The function of the p53 proteinis impaired in these circumstances. Proteins that are able to bind to p53 are the product of theMDM-2 gene, viral oncoproteins such as those from SV-40 ,HPV E6 and adenovirus E1B. Alsosequestration of the p53 protein in the cytoplasm, away from the target DNA in the nucleus of thecell may render the p53 protein to become dysfunctional (178).3. There is mutant p53 protein: the TP53 gene is altered and depending on the type of mutation theprotein produced is full-length with an amino acid change, truncated or the protein is absent. Thefirst form of protein leads to impaired function and a prolonged half-life, which makes the proteindetectable for the used antibody, in the remaining two situations the protein will not be detectable byan antibody (178). In the situations as described above under 1. and 2. p53 immunopositiveness may occur without thepresence of a TP53 gene mutation.
The involvement of the TP53 gene in the development of high grade astrocytic tumors is notdebated, but the majority of studies trying to establish a relation between p53 function and patientsurvival fail to prove this relation (Table 6). The reason for a lack of correlation between p53 statusand survival among high grade glioma patients might be that in these gliomas so many othercarcinogenic genetic events have taken place that p53 dysfunction by itself only plays a minor role.Assuming a relatively more important role for the TP53 gene in low grade astrocytomas, where notso many other carcinogenic genetic events have occurred yet, p53 status in these tumors may havea predictive value. This is supported by the study of Chozick et al. who did find a relation betweenp53 function and survival among 24 patients with low grade astrocytomas. However, possibly dueto the low number of patients, this relation was not significant (p=0,08) (198). Another study among52 grade II astrocytomas showed a difference in survival after 5 years of follow-up between p53immunopositive and immunonegative patients (193). The group of Kraus et al. could not establish arelation between p53 mutations and survival among grade II astrocytoma patients (209). From the38 grade II astrocytomas studied, 10 had a malignant recurrence. In 6 out of those 10 a TP53mutation was found, however in 5 this mutation was already present in the precursory grade IIastrocytoma. In 2 cases the TP53 mutation of the grade II astrocytoma could not be found in thecorresponding malignant recurrence. These authors conclude that TP53 plays no role in themalignant progression of grade II astrocytomas and has no prognostic significance. In contrast,Bodey et al. have found 100% immunopositiveness among 5 studied JPA's (210). These authorsstate that p53 immunopositiveness without TP53 gene mutation is one of the primary steps inastrocytoma formation. Furthermore, they assume that this p53 accumulation may prohibit thenormal function of the p53 protein. When the primary step of p53 protein dysfunction has occurred,which might be the case in those immunopositive pilocytic astrocytomas, and the genome becomes
53
altered, subsequently a cascade of further disturbances in DNA replication may be initiated, sincethe DNA-repairing and protective role of p53 is then missing. In this way dedifferentiated cell-clones may develop and cause the malignant degeneration of grade I to grade II, and of grade II tograde III or IV astrocytomas. The observation that increased levels of wildtype p53 in normalfibroblasts enhance the tumorigenic potential of these cells is in line with this hypothesis (211).Also the observation that transfection of the human wildtype p53 in a pilocytic astrocytoma-derivedcell line resulted in a growth suppressor effect on the cell line and the induction of importantmorphological changes in the cells, resembling those of differentiated astrocytes, suggests a role forp53 in the formation of pilocytic astrocytomas (195).
In summary, the above mentioned information leads to the following conclusions:
- The TP53 gene: The most frequently mutated gene in human neoplasms. p53 is a cell cycleregulator and "guards the genome". In case of DNA damage or disturbances in DNA replication,p53 induces G1-arrest with DNA-repair or triggers apoptosis. Transfer of TP53 gene products intop53-mutant glioma-cells (including a cell line derived from pilocytic astrocytoma) results in agrowth suppressor effect and rapid cell death via apoptosis.
- Malignant gliomas: TP53 mutation frequency varying from 30%-90%, in adults as well as inchildren. The TP53 gene is certainly involved in the formation of these tumors, probably whenarising from malignant progression of low grade astrocytomas. But, p53 status,immunohistochemically established, has no predictive value for patient survival.
- Low grade gliomas: TP53 mutation frequency varying from 5%-60% in adults. In childrenprobably a much lower fre quency. The role of the TP53 gene in the formation of these tumors isunclear. This role is accepted in malignant progression of low to high grade astrocytomas. Twostudies reported a positive relation between p53 status and survival, whereas one rejected thisrelation. The TP53 gene seems to play no role in pediatric low grade gliomas.
- Pilocytic astrocytomas: TP53 mutation frequency is very rare (<3%). But in most studies only thewell known "hot-spots" of the gene (exons 5-8) were examined. Of the 3 mutations reported, 1 ison exon 7, while the other 2 are on exons 4 and 9. Results of p53-immunostaining are veryconflicting: in 4 studies negative, but strongly positive in another 3 studies. In 2 of these 3 studiesp53 dysfunction is supposed to be a major initiating event in astrocytoma formation and subsequentdedifferentiation to higher grades. The relation between p53 status and patient survival or prognosishas not been studied before.
54
Figure 1. Tumor suppressor functions of p53.
55
TABLE 5. Results of previous p53 studies in pilocytic astrocytomas.
immunohistochemical structural(LOH)
genetic analysis(SSCP and sequencing)
stu-dy
antibody nr.pos/tot
study nr.pos/tot
study
exonsstudied
nr.pos/tot
mutation
210 PAb-1801 5/5 204 1/2(*1) 218 5,7,8 0/8
212 PAb-1801CM-1
8/21 205 6/20 219 5-8 0/12
192 DO-7CM-1
0/12 200 0/28 (*2)
199 5-8 0/17
191 DO-7 0/8 216 1/20 187 5-9 1/7 exon 9co.324
213 PAb-1801DO-1
0/11 217 0/5 213 5-8 0/11
214 PAb-1801PAb-421
0/4 201 4-8 0/17
186 PAb-1801BP 53-12
5/7 205 4-9 1/20 exon 7co.248
215 DO-1 1/9 220 0/3
217 1-11 0/5
186 2-11 1/7 exon 4cod.47
TOTAL 19/77 (25%) TOT 8/75 (11%) TOTAL 3/107 (3%)
*1: the positive tumor is a grade I astrocytoma in an adult, but the authors use a grading system of I toIII, in which grade III refers to a glioblastoma multiforme, so probably this tumor is actually a lowgrade astrocytoma.*2: The 28 tumors investigated are "low grade astrocytomas of childhood".
56
TABLE 6. Results of previous studies among gliomas on the relation between p53 status and patientsurvival.
studyref.nr.
type ofastrocytoma
nr. oftumors
method of p53analysis
relationp53 to survival
221 grade 4 ? immuno CM-1 yes
222 all grades 53 immuno DO-1 yes
202 pediatricmalign.brainstem
29 immuno DO-1
SSCP, seq.
yes
yes
198 grade 2 24 immuno PAb1801 yes (p=0,08)
200 all grades 126 immuno no
193 grade 2 52 immuno DO-7 no
223 all grades 105 immuno no
194 grades 2,3,4 95 immuno PAb1801 no
214 grade 3,4 63 immuno PAb1801 PAb 421
no
191 all grades 56 immuno DO-7 no
224 grades 2,3,4 123 immuno DO-7 no
206 grade 3 54 immuno CM-1 no
198 grade 3,4 125 immuno PAb1801 no
209 grade 2 38 immunoSSCP
no no
225 grade 2,3childhood
21 immuno PAb1801PAb240,DO1,DO7BP53-12, CM-1
no
57
4. THE NF1 GENE
Neurofibromatosis 1 (NF1), the von Recklinghausen or peripheral form of neurofibromatosis, isclosely associated with pilocytic astrocytomas. NF1 patients develop in 15% of cases pilocyticastrocytomas and one third of all patients with pilocytic astrocytomas of the optic nerve have NF1(226).NF1 is one of the most frequently occurring genetic disorders as it affects 1 in 3500 individualsworldwide. It is inherited in an autosomal dominant fashion, but half of the cases are new mutationsin the germline without prior family history of the disease. The disease is characterized by multiplecafe-au-lait spots, neurofibromas, Lisch noduli, axillary freckling, optic nerve gliomas, distinctosseous lesions, and sometimes development of malignant tumors (227). However, the diseaseshows a very variable expression, meaning that patients from the same family and presumablycarrying the same mutation, may exhibit a wide range of symptoms.The NF1 gene locus was mapped in 1987 within band q11.2 on the long arm of chromosome 17(228,229). It is a large gene, spanning approximately 300.000 nucleotides, but with an open readingframe of 8454 nucleotides (230).The gene product is a 250 kDa protein called neurofibromin; a small part of this protein ishomologous to human GTPase-activating proteins (GAPs). The part of the NF1 gene that encodesfor the GAP is called the GAP-related domain (GRD). GAPs are involved in the regulation of thep21ras proto-oncogene, in such a way that GAPs inactivate p21ras. P21ras is involved in cellproliferation and differentiation via the tyrosine kinase signal transduction pathway, in which theepidermal, nerve and platelet derived growth factors (EGF, NGF and PDGF) play a role. In normalresting cells p21ras is in its inactive state. Activation of p21ras leads to the development of tumors.It is suggested that GAPs regulate the p21ras mediated growth and differentiation pathways bykeeping p21ras in its inactive state. Assuming that neurofibromin has a function homologous toGAP, by analogy, a mutation in the NF1-GRD would cause a dysfunction of neurofibromin andsubsequently dysregulation of p21ras leading to unlimited cell proliferation and the formation oftumors. In this way the NF1 gene can be regarded as a tumor-suppressor gene.To test the hypothesis that NF1 acts as a tumor suppressor gene, molecular genetic analyses havebeen performed on tumors occurring in NF1 patients. The "two-hit" model, established by Knudsonafter studying the sporadic and familiar occurrence of retinoblastomas and the retinoblastoma (Rb)-gene, can also be applied to NF1 patients (231). In case of inherited loss of one NF1-allele in thegermline DNA, the most frequently occurring symptoms would arise, including the formation ofneurofibromas. The formation of other tumors in these patients, such as the optic glioma orneurofibrosarcomas, would be caused by loss of the remaining NF1-allele, due to a somaticmutation, being the "second hit". Therefore, studying these tumors, one would expect to find loss ofheterozygosity near or at the NF1-gene locus or mutation of the gene itself. Some studies have found results supporting this hypothesis: Genetic analysis of neuro-fibrosarcomasfrom NF1 patients showed a deletion of chromosome 17, including the NF1 gene (232,233).Homozygous deletions of chromosome 17 were found in a malignant melanoma cell line and inneuroblastoma cell line (234,235). Li et al. have found mutations in at least one allele of the NF1gene in 1 anaplastic astrocytoma, in a colon carcinoma and in myelodysplastic syndrome (236).
58
NF1 mutations were found in 1 recurrent low grade astrocytoma, 1 ependymoma, 2 glioblastomasmultiforme and 1 primitive neuroectodermal tumor (PNET) out of a group of 31 gliomas and 3PNETs from non-NF1 patients (237). In the study of von Deimling et al. 4 of 20 pilocyticastrocytomas showed LOH of chromosome 17, in 3 of the long arm and in 1 of the wholechromosome 17 (226). One of these pilocytic astrocytomas occurred in a NF1 patient; in this tumorthe LOH included the NF1 locus. It is tempting to assume that in all these 4 tumors the responsiblegene for pilocytic astrocytoma development would be the NF1 gene, however, evidence for thiswas not established, since the gene itself was not analyzed.
Other findings however, oppose the theory of NF1 being a tumor suppressor gene: Inneurofibromas, the hallmark tumor of NF1 patients, LOH on or near 17q11.2 could not be found(238). Among the 31 gliomas in the study of Thiel et al. there were 3 pilocytic astrocytomasincluding 1 from a NF1 patient, however, none of those 3 showed DNA abnormalities in the regionof the NF1 gene. In 51 pediatric brain tumors, including 16 pilocytic astrocytomas, the part of theNF1 gene that encodes for the part of the protein with the GAP-like function, which is called theFLR exon of the NF1 gene, was screened for mutations by SSCP, but these were not found (239).In this study only the FLR exon of the NF1 gene was screened, which forms less than 2% of thewhole gene. Neither were mutations found in the FLR exon among 18 glioblastomas in anotherstudy (240). This leaves however the possibility that mutations are present in other parts of the gene.These data, especially the lack of LOH 17q in neurofibromas of NF1 patients and the enormoussize of the NF1 gene, which makes it very difficult to screen for mutations, cause that the status ofthe NF1 gene as a tumor suppressor gene remains to be proven. Moreover, the NF1 gene carries adominant inheritance, whereas the typical tumor suppressor genes are recessive.Other studies were performed to look more specifically at the function of the gene product,neurofibromin. Structural studies of this protein disclosed that it has 3 isoforms due to alternativesplicing of the gene product before transcription. The levels of GAP activity of these isoforms arenot identical and the expression of the 3 isoforms differs among cells of certain tissues and in variousstages of development and differentiation of these tissues (241). From this it is suggested that thevarious isoforms of neurofibromin have specific functions, mainly related to the stage of embryonicdevelopment of the certain tissues. Nevertheless, in relation to the supposed tumor suppressorfunction of NF1, neurofibromin expression can be studied by using a specific antibody to thisprotein, or by studying the amount of RNA transcript. In the case of DNA damage at the level ofthe NF1 gene one would expect a lowered or absent expression of neurofibromin. In many of thetumors that showed NF1 mutations, being melanomas, neuroblastomas, pheochromocytomas andneurofibrosarcomas the levels of neurofibromin were indeed absent or reduced (234,235,242).
To evaluate the possible tumor-suppressor function of the NF1 gene neurofibromin expression in 6pilocytic astrocytomas was analyzed by studying the amount of RNA-transcript and by antibody,directed against neurofibromin, detection. (Chapter 7).
59
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