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Standardized AgNOR analysis of the invasive tumour front in oral squamous cell carcinomas

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??????? ?? ?????????, ???. 182: 450?456 (1997)
STANDARDIZED AgNOR ANALYSIS OF THE INVASIVE
TUMOUR FRONT IN ORAL SQUAMOUS CELL
CARCINOMAS
??????? ???????1, ?????? ??????????2, ??????? ??????3, ?????? ?????2, ?????? ????1 ??? ???? ?. ??????2*
Department of Maxillo-Facial Surgery, University of M黱ster, Germany; 2Department of Pathology, University of M黱ster,
Germany; 3Department of Surgery I, University of Innsbruck, Austria
1
SUMMARY
In the last decade, silver staining of nucleolar organizer region-associated proteins (AgNORs) has been widely used in tumour
pathology both for diagnostic and for prognostic purposes. However, a reliable and reproducible assessment of these proteins on
routinely processed archival tissues has only become possible since the recent introduction of standardized staining method and
computer-aided morphometric analysis. In the present study, the AgNOR content at the invasive front of 80 squamous cell carcinomas
of the floor of the mouth/tongue was investigated using this novel approach, with regard to prognosis and a variety of clinico-pathological
parameters. All standardized AgNOR parameters [mean of AgNOR number, mean of AgNOR area, coefficients of variation (CV) of
both AgNOR number and area] were statistically significantly associated with the clinical course. The strongest correlation was found
for the AgNOR-area univariate analysis (P=0�6). In multivariate analysis, the mean of AgNOR number could independently predict
both overall (P=0�) and disease-free survival (P=0�1). It is concluded that standardized staining and computer-aided analysis of
AgNORs are prerequisites for an objective and reproducible AgNOR assessment, which has potential as a supplementary diagnostic and
prognostic tool in oral cancer. ? 1997 by John Wiley & Sons, Ltd.
J. Pathol. 182: 450?456, 1997.
No. of Figures: 4. No. of Tables: 3.
KEY WORDS?standardized
No. of References: 42.
AgNOR staining; image analysis; oral carcinoma; prognosis
INTRODUCTION
Nucleolar organizer regions (NORs) are segments of
DNA present in humans on the five acrocentric chromosomes (chromosomes 13, 14, 15, 21, and 22) that carry
ribosomal genes associated with a group of histone and
non-histone proteins. There have been at least 200
NOR-associated proteins identified until now, some of
which bind silver (AgNORs) in actively transcribing
NORs.1 Thus, NORs can be indirectly demonstrated
both in metaphase spreads and in histopathological
sections by virtue of the highly argyrophilic properties of
the AgNORs,2,3 mainly represented by C23 (nucleolin)
and B23 (numatrin) proteins.4,5 AgNORs are known to
reflect the activity of ribosomal biogenesis in a cell. Their
amount was demonstrated to be closely related to cellular proliferation and the rapidity of the cell cycle by
means of both molecular biological methods6 and in situ
morphometric analysis.7?10 There is also evidence that
AgNOR quantity can reflect the cellular differentiation
state.11
In squamous cell carcinomas of the head and neck,
visualization of interphase AgNORs has been shown to
be associated with stage of the disease,12 S-phase fraction, Ki-67 or MIB 1 labelling index,13 as well as tumour
progression14 and prognosis.15 Furthermore, AgNORs
were found to detect incipient cellular alterations in
*Correspondence to: Kurt Werner Schmid, MD, Department of
Pathology, Domagkstrasse 17, D-48149 M黱ster, Germany. E-mail:
schmikw@uni-muenster.de
CCC 0022?3417/97/080450?07 $17.50
? 1997 by John Wiley & Sons, Ltd.
tumour-adjacent epithelium within the oral cavity.16
However, all these studies were performed on routinely
processed paraffin sections, which results, as repeatedly
pointed out by our group, in poor overall staining
quality.17 Moreover, the assessment of AgNORs was
predominantly carried out by counting ?black intranuclear dots? by eye, which is known to be greatly influenced by staining quality.18 The results from all these
studies are thus hampered by their poor reproducibility
and reliability.
In contrast, the AgNOR analysis of the present study
was performed using a recently developed standardized
method for AgNOR staining and computer-assisted
morphometric analysis on paraffin-embedded archival
material, which was proposed by the ?Committee on
AgNOR Quantification? of the European Society of
Pathology as the ?gold standard? of AgNOR analysis.19,20 In a recently performed study on oral squamous
cell carcinomas (OSCCs) and adjacent non-neoplastic
mucosa, we could clearly demonstrate that tumour
heterogeneity is reflected by standardized AgNOR
analysis.21 The substantial difference of the invasive
tumour front from central tumour areas of OSCCs was
also supported by the findings of other immunohistochemical investigations (Ki-67, p53, etc.) performed on
the same series of OSCCs.22,23
The prognostic relevance of the invasive tumour front
has been established in classical histopathological
studies24,25 and reconfirmed by a previous international
reproducibility study (Bryne et al., unpublished data).
The present study was performed in order to quantify
Received 7 October 1996
Accepted 6 March 1997
451
STANDARDIZED AgNOR ANALYSIS OF THE INVASIVE TUMOUR-FRONT IN OSCCs
the AgNOR content at the invasive tumour front in a
series of 80 archival OSCCs; the findings were correlated
with established grading and staging parameters, as well
as the clinical course [overall survival and disease-free
survival (DFS)].
MATERIALS AND METHODS
Tumour tissues of 80 squamous cell carcinomas of the
floor of the mouth or tongue (13 female and 67 male
patients; mean age 54 years) were included in this study.
All patients had been operated with radical intent
between 1985 and 1990 at the Department of MaxilloFacial Surgery, University of M黱ster, Germany.
Routinely processed (formalin-fixed and paraffinembedded) tumour tissues were retrieved from the files
of the Department of Pathology, University of M黱ster,
Germany. The histopathological grading of the tumours
was based on the WHO criteria,26 and the malignancy
grading of the invasive tumour front was performed
according to Bryne et al.24 with minor modification. The
invasive tumour front was defined as the most progressed 3?6 tumour cell layers or detached tumour cell
groups at the advancing edge of the OSCCs. Patients
were meticulously followed up for a maximum of 120
months. The mean follow-up time of this series was 68
months. Detailed clinico-pathological data are given in
Table I. The patient group presented has already been
the subject of other prognostic studies.22,23
Silver staining of AgNORs
Paraffin sections 2 靘 thick were mounted on silanecoated glass slides. After routine dewaxing and rehydration, sections were immersed in sodium citrate buffer
(0� ? Na-citrate monohydrate, pH 6� and incubated in a G鰏sner autoclave (GLA 40-2, Hamburg,
Germany) at 120)C for 20 min. Silver staining was
performed using a freshly prepared silver solution containing 1 part by volume of 2 per cent gelatine in 1 per
cent formic acid and 2 parts by volume of 25 per cent
aqueous silver nitrate solution for 25 min at room
temperature.18 Following thorough rinsing of the
sections in de-ionized water, dehydration through
graded ethanol, clearing in xylene, and mounting
with Vitroclud (all chemicals purchased from Merck,
Darmstadt, Germany) were performed.
Quantification of AgNOR
The AgNOR content of at least 100 tumour cell nuclei
per area (at 400-fold magnification) was evaluated at
one focal plane independently by two of us. 繠, DR) by
means of a semi-automated image analysing system
(Zeiss Axioscop microscope, JVC TK 1070E colour
video camera, 80486-based microcomputer equipped
with a digitizer board, and software application based
on VIDAS release 25 from Kontron Elektronik GmbH,
Germany). The area and number of AgNORs per
nucleus were measured simultaneously and the respective coefficients of variation were calculated in each
? 1997 by John Wiley & Sons, Ltd.
Table I?Frequency distribution of the prognostic parameters
evaluated in 80 carcinomas of the oral cavity
Parameter
Clinical course
Uneventful
Poor
Lost
pT stage
PT1
pT2
pT3
pT4
pNstage
pN0
pN1
pN2
pNx
pM stage
pM0
pMx
R stage
R0
R1
Histological grading (WHO)
I
II
III
Tumour front score
4?8
9?10
10?14
Unclassified
Irradiation therapy
No
Yes
Age
� years
>54 years
Sex
Male
Female
N
%
39
33
8
49
41
10
18
47
4
11
22
59
5
14
47
25
7
1
59
31
9
1
70
10
88
12
75
5
94
6
25
42
11
32
54
14
26
35
14
5
32
44
18
6
44
36
55
45
42
38
52
48
67
13
84
16
section investigated. All data are given as the mean; the
area of AgNORs was calibrated to 靘2. The coefficient
of variation (CV) is defined as the standard deviation
divided by the respective mean value and has been
proven to provide the highest reproducibility among all
AgNOR parameters.19,27 Thirty randomly selected cases
were independently measured by the two assessors in
order to determine inter-observer variability.
Statistical analysis
All data of patients and tumour variables were converted to a Macintosh PowerPC and statistical analysis
was carried out using a SYSTAT statistical package28
including a SURVIVAL supplementary module.29 The
prognostic value of all parameters was studied in univariate analysis. Survival probabilities were estimated by
the Kaplan?Meier method;30 survival curves were compared by the log-rank test (Mantel?Haenszel method).31
??????? ?? ?????????, ???. 182: 450?456 (1997)
452
J. PIFFK� ET AL.
Fig. 1?AgNOR staining after the wet autoclave method of a well-differentiated OSCC. Note the
multiple AgNORs which can be distinguished within nucleoli, as well as the complete absence of
staining artefacts
Multivariate analysis was used to determine the independent prognostic value of selected variables, using
Cox?s proportional hazards linear regression model
with a forward stepwise regression.32 All analyses were
performed for both overall and disease-free survival.
Descriptive statistics for continuous measures are
given as the mean; for discrete data frequency counts
and percentages are tabulated. Non-parametric tests
(Kruskal?Wallis or Mann?Whitney U whenever appropriate) were carried out when AgNOR parameters were
compared with other prognostic markers. Simple regression analysis was computed to assess inter-observer
variability.
RESULTS
AgNOR staining
Wet autoclave pretreated silver staining of AgNORs
resulted in an excellent staining quality of routinely
formalin-fixed and paraffin-embedded sections, with
clearly distinguishable, sharply delineated single intranucleolar AgNOR dots without evidence of staining
artefacts (Fig. 1). An obvious difference was observed in
the distribution pattern of AgNORs between the invasive front and central parts of the OSCCs. Central,
usually more differentiated, tumour parts contained
large, tightly clustered AgNOR dots, whereas at the
invasive zone, predominantly dispersed small scattered
AgNORs were found throughout the whole nucleus
(Fig. 2).
Inter-observer reproducibility
The inter-observer variability in the 30 randomly
selected carcinomas was low. The most reproducible
parameters were the CV of AgNOR number
? 1997 by John Wiley & Sons, Ltd.
(Spearman?Rank correlation coefficient R=0�
P=0�01; Fig. 3) and the CV of AgNOR area (R=0�
P=0�03), whereas area and number measurements
were more variable (R=0�.
Relationship between AgNOR parameters and
clinico-pathological features
All AgNOR parameters except the CV of area were
statistically significantly associated with the clinical
course. The strongest relationship was indicated by the
mean value of AgNOR area (P=0�1). The presence of
lymph node metastases was best revealed by the CV of
AgNOR number (P=0�). The relationship between
AgNOR parameters and classical WHO grading, the pT
stage of the tumours, and the age and gender of the
patients did not reach statistical significance, whereas
the histopathological malignancy score of the tumour
front showed a significant association with the mean
area of AgNORs (P=0�) (Table II).
Uni- and multivariate long-term survival analysis
The possible impact of patients and tumour variables
was investigated by univariate analysis with respect to
overall and disease-free (DFS) survival (Table III). The
highest prognostic value concerning overall survival was
revealed by the histopathological malignancy grade of
the tumour front, followed by the CV of AgNOR area
and the presence versus absence of lymph node involvement at the time of diagnosis (pN0 versus pN1?2). Age
and sex of the patients did not reveal any statistically
significant effect on survival.The most outstanding prediction of DFS likelihood was associated with AgNOR
mean area, followed by the histopathological malignancy grade of the tumour front. All other AgNOR
??????? ?? ?????????, ???. 182: 450?456 (1997)
STANDARDIZED AgNOR ANALYSIS OF THE INVASIVE TUMOUR-FRONT IN OSCCs
453
Fig. 2?Different distribution of AgNORs in central (clustered) and peripheral, invasive (dispersed)
parts of a well-differentiated OSCC
Fig. 3?Scatter-plot interpretation of the inter-observer reproducibility of the CV of AgNOR number
measurements in 30 of the cases studied
parameters and the histopathological WHO grading
lacked significant impact on DFS, whereas classical
prognostic parameters, such as pT and pN stages and
the age and gender of the patients, did not achieve
significance in the univariate analysis. Figure 4 illustrates the overall survival curves for all patients of
the present study with regard to AgNOR parameters.
Multivariate analysis by the Cox regression model
revealed that only the tumour front score and the mean
of AgNOR number were significantly correlated both to
overall survival (P=0�1 and 0�, respectively) and
DFS (P=0�0 and 0�1, respectively), whereas other
significant factors in the univariate analysis pT, pN,
? 1997 by John Wiley & Sons, Ltd.
WHO grade, AgNOR mean area, the CV of AgNOR
area, and the CV of AgNOR number) did not yield
independent prognostic information (data not shown).
DISCUSSION
Deregulated proliferation is considered to be a prime
characteristic of malignancies. It is thought to reflect the
biological aggressiveness of a given tumour, exerting,
therefore, a putative impact on the clinical course of
different cancers. However, the estimation of proliferation in clinical material is still problematic. In contrast
??????? ?? ?????????, ???. 182: 450?456 (1997)
454
J. PIFFK� ET AL.
between the simplicity needed for a wide application and
the high discriminative power required for differentiation in treatment options approaching individual patient
management. The results of the present study contribute
to the fulfilment of the above requirements, as follows.
With regard to standardization, the application of the
principles of salt-mediated autoclave pretreatment for
immunohistochemistry35 for silver staining of AgNORs
has solved the repeatedly reiterated problem of unsatisfactory and unreliable staining of routinely
formalin-fixed and paraffin-embedded tissues, providing
a standardized AgNOR staining method.18
With regard to reproducibility, the use of computeraided morphometric analysis of standardized AgNOR
parameters (proposed by the Committee on AgNOR
Quantitation19) eliminates the poor or failing reproducibility of results obtained by counting AgNORs by
eye. Furthermore, the introduction of the coefficient of
variation (CV) of simultaneously measured number
and area of AgNORs per nucleus provides a highly
reproducible parameter of AgNOR analysis.36
With regard to clinical significance, the outstanding
performance of standardized AgNOR analysis in predicting the clinical course of the respective patients
independently from established prognostic factors has
been proven so far in colorectal,37 breast,38 and lung
cancer,39 as well as in OSCCs (if assessed at the invasive
tumour front), as demonstrated in the present and
previous works.23,40
Finally, the cost of silver staining is lower than that of
most immunohistochemical reactions generally used in
tumour diagnostics. However, extra costs arise for essential requirements of standardization and reproducibility,
such as the cost of an autoclave for pretreatment and an
image analyser for assessment.
Concerning the simplicity of AgNOR number estimations by eye, it has been repeatedly proven that the area
of silver-stained NOR-associated proteins represents a
more important factor in AgNOR evaluation than
their number.18,19,41 These studies indicate that in situ-
Table II?Relationship of morphometric AgNOR parameters
to clinical course and various staging and grading parameters
AgNORs per nucleus
Area (靘2)
Mean
Clinical course
pT stage
pN stage?
pN0 vs. pN1?2?
Tumour grade?
Tumour front score
Sex
Age (� >54 years)
3�**
2�0�0�0�5�
0�0�
Number
CV
Mean
0�3�5�
1�
0�1�1�0�
2�
1�5�
2�
0�0�1�1�
CV
2�
1�6�
2�*
0�0�1�0�
Values represent Kruskal?Wallis or Mann?Whitney statistics
corrected for tied groups whenever appropriate.
*P<0�; **P<0�; ***P<0�1.
?pNx cases (N=1) excluded.
?Grade IV case (N=1) grouped with grade III cases (N=11).
to several techniques which merely estimate static
parameters of proliferation, such as the percentage of
cycling or S-phase cells, AgNORs seem rather to reflect
dynamic aspects of the cell cycle, i.e., the rapidity of cell
duplication.7,9,33,34 Although the assessment of proliferation markers such as Ki-67 is much easier and quicker
since far fewer ?events? are scored, AgNOR analysis
apparently provides a much better insight into the
proliferation kinetics of malignancies than the abovementioned static parameters alone, with additional
information on the metabolic activity of the cell
population investigated.
Before a novel diagnostic method can be accepted and
widely used in routine clinical application, at least four
requirements have to be met: it should be standardized;
it should be reproducible; it should assess a clinically
significant parameter; and costs should be proportionate
to benefits. Furthermore, a compromise has to be found
Table III?Prognostic factors examined in 80 carcinomas of the oral cavity: a univariate approach to
cancer-specific mortality
Overall survival
Parameter
pT stage
pN stage*
pN0 vs. pN1?2*
Tumour grade?
Tumour front score
Sex
Age (cp: 54 years)
AgNOR mean area (cp: 1�靘2)
AgNOR area CV (cp: 0�)
AgNOR mean number (cp: 3�
AgNOR number CV (cp: 0�)
�
2
8�7�7�6�15�0�0�5�6�6�4�
DFS
DF
P
�
3
2
1
2
2
1
1
1
1
1
1
0�
0�
0�8
0�
0�0
NS
NS
0�
0�9
0�
0�
5�2�2�7�12�0�0�7�4�4�3�
2
DF
P
3
2
1
3
2
1
1
1
1
1
1
NS
NS
NS
0�
0�2
NS
NS
0�6
0�
0�
0�
DFS=disease-free survival; NS=not significant; DF=degrees of freedom; cp=cut-off point.
*pNx cases (N=1) excluded.
?Grade IV case (N=1) grouped with grade III cases (N=11).
? 1997 by John Wiley & Sons, Ltd.
??????? ?? ?????????, ???. 182: 450?456 (1997)
STANDARDIZED AgNOR ANALYSIS OF THE INVASIVE TUMOUR-FRONT IN OSCCs
455
Fig. 4?Kaplan?Meier survival curves of all the AgNOR parameters evaluated
determination of the AgNOR content by means of
morphometric evaluation of the mean area of AgNORs
per nucleus correlates with the quantity of AgNOR
proteins assessed by molecular biological methods and
is strictly related to the proliferative activity of those
tumours.6?8 These measurements can be carried out only
by computerized morphometry. The enumeration of the
number of AgNORs per nucleus, which can also be
performed by counting ?black dots? by eye, is more
likely to be subject to intra-/inter-observer variations,
depending on differences in material and/or staining
? 1997 by John Wiley & Sons, Ltd.
quality.41,42 Thus, the simplicity of the AgNOR counting method by eye cannot overcome the negative effect
caused by the ambiguity of the results obtained. In this
study we were able clearly to demonstrate that optimal
staining quality is associated with a high correlation of
both mean AgNOR area and number. Thus, it has to be
emphasized again that standardized NOR assessment
crucially depends not only on the improved staining
method, but also on image analysis, which implicates
a farewell to the simple technique of counting black
dots.
??????? ?? ?????????, ???. 182: 450?456 (1997)
456
J. PIFFK� ET AL.
In the OSCC cases of the present study, all the
AgNOR parameters showed a significant impact on the
clinical course of the patients; the most prominent
influence was exerted by the CV of AgNOR area.
However, high CV values of AgNOR number were
particularly associated with early tumour recurrence/
metastasis and death (see details in Piffk� et al.23).
We conclude that standardized AgNOR analysis of
the invasive tumour front of OSCCs is a highly reliable
and reproducible method for assessing histobiological
features in routinely formalin-fixed and paraffinembedded oral carcinoma tissues and provides a functional background to the well-established clinical
relevance of the histopathological tumour front grading.
It yields powerful and independent prognostic information concerning tumour-related death and DFS,
irrespective of common prognostic markers, and it offers
a very promising simple and reliable additional diagnostic tool with the potential of identifying (a) subgroup(s)
of patients with aggressive disease who could benefit
from extended and/or novel therapeutic approaches.
ACKNOWLEDGEMENTS
We are grateful to Ms Birgit Kunk and Ms Alice
Muhmann for performing the standardized AgNOR
staining and to Mrs Heidi Gerdes-Funnek鰐ter for
photographic assistance. Special thanks are due to
Dr Magne Bryne (Department of Pathology, The
Norwegian Radium Hospital, Oslo, Norway) for helpful
discussion.
REFERENCES
1. Crocker J. Molecular and biochemical aspects of interphase nucleolar
organizer regions. J Clin Pathol: Mol Pathol 1996; 49: M8?M11.
2. Goodpasture C, Bloom SE. Visualization of nucleolar organizer regions in
mammalian chromosomes using silver staining. Chromosoma 1975; 31:
260?262.
3. Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnet JJ.
Improvement in the staining and in the visualization of the argyrophilic
proteins of the nucleolar organizer region at the optical level. Histochem J
1986; 18: 5?14.
4. Roussel P, Belenguer P, Amalric F, Hernendez-Verdun D. Nucleolin is an
Ag-NOR protein; this property is determined by its amino-terminal domain
independently of its phosphorylation state. Exp Cell Res 1992; 203:
259?261.
5. Hernandez-Verdun D. The nucleolar organizer regions. Biol Cell 1983; 49:
191?202.
6. Derenzini M, Sirri V, Trer� D, Ochs RL. The quantity of nucleolar proteins
nucleolin and protein B23 is related to cell doubling time in human cancer
cells. Lab Invest 1995; 73: 497?502.
7. Trere D, Pession A, Derenzini M. The silver-stained proteins of interphasic
nucleolar organiser regions as a parameter of cell duplication rate. Exp Cell
Res 1989; 184: 131?137.
8. Derenzini M, Pession A, Trere D. The quantity of nucleolar silver stained
proteins is related to proliferating activity in cancer cells. Lab Invest 1990;
63: 137?140.
9. 謋ner D, Hittmair A, Marth C, et al. Relationship between quantity of
silver stained nucleolar organizer regions associated proteins (AgNORs)
and population doubling time in ten breast cancer cell lines. Pathol Res
Pract 1989; 188: 742?746.
10. Derenzini M, Trere D. AgNOR proteins as a parameter of the rapidity of
cell proliferation. Zentralbl Pathol 1994; 140: 7?10.
11. Jewell SC, Cordial CR. Silver staining of nucleolar organizer regions.
J Histotechnol 1996; 19: 241?256.
12. Hirsch SM, DuCanto BA, Caldarelli DD, Hutchinson JC, Coon JS.
Nucleolar organizer regions in squamous cell carcinoma of the head and
neck. Laryngoscope 1992; 102: 39?44.
13. Z鰈ler J, Flentje M, Sinn P, Born IA. ?Nukleolar Organizer Region? und
?Ki-67? als zellkinetische Parameter f黵 orale Dysplasien und Plattenepithelkarzinome. Dtsch Z Mund Kiefer Gesichts Chir 1993; 17: 185?190.
? 1997 by John Wiley & Sons, Ltd.
14. Raveendran Pillai K, Sujathan K, Kannan S, et al. Argyrophilic nucleolar
organizer regions in the evaluation of tumour progression in the oral
mucosa: correlation with tissue pathology. J Cancer Res Clin Oncol 1994;
120: 723?726.
15. Sano K, Takahashi H, Fujita S, et al. Prognostic implication of silverbinding nucleolar organizer regions (AgNORs) in oral squamous cell
carcinoma. J Oral Pathol Med 1991; 20: 53?56.
16. Schwint AE, Savino TM, Lanfranchi HE, Marschoff E, Cabrini RL, Itoiz
ME. Nucleolar organizer regions in lining epithelium adjacent to squamous
cell carcinoma of human oral mucosa. Cancer 1994; 73: 2674?2679.
17. Bankfalvi A, 謋ner D, Piffko J, Schmid KW. Nucleolar organizer regions in
lining epithelium adjacent to squamous cell carcinoma of human oral
mucosa (Letter to the Editor). Cancer 1994; 74: 3245?3246.
18. 謋ner D, Bankfalvi A, Riehemann K, Bier B, B鯿ker W, Schmid KW. Wet
autoclave pretreatment improves visualisation of silver stained nucleolar
organizer region associated proteins (AgNORs) in routinely formalin-fixed
and paraffin-embedded tissues. Mod Pathol 1994; 7: 946?950.
19. 謋ner D, Aubele M, Biesterfeld S, et al. Guidelines of AgNOR
quantification?first update. Virchows Arch 1995; 427: 341?342.
20. Aubele M, Biesterfeld S, Derenzini M, et al. Guidelines of AgNOR
quantitation. Zentralbl Pathol 1994; 140: 107?108.
21. Piffk� J, B鄋kfalvi A, 謋ner D, et al. Standardized demonstration of silver
stained nucleolar organizer regions (AgNORs) associated proteins in
archival oral squamous cell carcinomas and adjacent non-neoplastic
mucosa. Mod Pathol 1997; 10: 98?104.
22. Piffk� J, B鄋kfalvi A, 謋ner D, et al. In situ assessment of cell proliferation
at the invasive front of oral squamous cell carcinomas. Virchows Arch 1996;
429: 229?234.
23. Piffk� J, B鄋kfalvi A, 謋ner D, et al. Prognostic value of histobiological
factors (malignancy grading and AgNOR content) assessed at the invasive
tumour front of oral squamous cell carcinomas. Br J Cancer 1997; 75:
1543?1546.
24. Bryne M, Koppang HS, Lilleng R, Kjaerheim A. Malignancy grading of the
deep invasive margins of oral squamous cell carcinomas has high prognostic
value. J Pathol 1992; 166: 375?381.
25. Bryne M, Jenssen B, Boysen M. Histological grading in the deep invasive
front of T1 and T2 glottic squamous cell carcinomas has high prognostic
value. Virchows Arch 1995; 427: 277?281.
26. Wahi PN. Histological Typing of Oral and Oropharyngeal tumours.
Geneva: WHO, 1971: 17?18.
27. Derenzini M, Trer� D. The coefficient of variation of Ag-NOR protein
values: a standardized parameter for cell kinetics evaluation. Proc
Histochem Soc 1992; 40: 600?604.
28. Wilkinson L. SYSTAT: The System of Statistics. Evanston, IL: SYSTAT, 1988.
29. Steinberg D, Colla P (eds) SURVIVAL: A Supplementary Module for
SYSTAT. Evanston, IL: SYSTAT, 1988.
30. Kaplan EL, Meier P. Nonparametric estimation from incomplete
observations. J Am Stat Assoc 1958; 53: 457?481.
31. Kalbfleisch JD, Prentice RL (eds). The Statistical Analysis of Failure Time
Data. New York: John Wiley, 1980.
32. Cox DR. Regression models and life tables. J R Stat Soc VB 1972; 34:
187?220.
33. Hittmair A, 謋ner D, Offner F, et al. In vitro investigations of interphase
and metaphase argyrophilic nucleolar organizer regions and cellular
proliferation in the human urothelial cancer cell line HOK-1. Virchows Arch
1994; 424: 149?154.
34. Dervan PA, Gilmartin LG, Loftus BM, Carney DN. Breast carcinoma
kinetics. Argyrophilic nucleolar organizer region counts correlate with Ki67
scores. Am J Clin Pathol 1989; 92: 401?407.
35. Bankfalvi A, Navabi H, Bier B, B鯿ker W, Jasani B, Schmid KW. Wet
autoclave pretreatment for antigen retrieval in diagnostic immunohistochemistry. J Pathol 1994; 174: 223?228.
36. Derenzini M, Trer� D. The coefficient of variation of AgNOR protein
values: a standardised parameter for cell kinetics evaluation. Proc
Histochem 1992; 40: 600.
37. 謋ner D, Riedmann B, Maier H, et al. Standardized staining and analysis of
argyrophilic nucleolar organizer region associated proteins (AgNORs) in
radically resected colorectal adenocarcinoma?correlation with tumour
stage and long-term survival. J Pathol 1995; 175: 441?448.
38. 謋ner D, Bier B, Heinrichs S, et al. Demonstration of silver-stained
nucleolar organizer regions associated proteins (AgNORs) after wet
autoclave pretreatment in breast carcinoma. Breast Cancer Res Treatment
1996; 39: 165?167.
39. T鰐sch M, 謋ner D, Maier H, Watzka SBC, Salzer M, Schmid KW.
Argyrophilic nucleolar organizer regions associated proteins (AgNORs) in
adenocarcinoma and squamous cell carcinoma of the lung?correlation
with tumour stage and long-term survival. Pathol Res Pract 1995; 191: 800.
40. 謋ner D, Schmid KW,. Standardized AgNOR analysis: its usefulness in
surgical oncology. Histochem Cell Biol 1996; 106: 193?196.
41. Trer� D. Technical and methodological aspects of silver staining and
measurement of nucleolar organizer region (NOR). Zentralbl Pathol 1994;
140: 11?14.
41. 謋ner D, Hittmair A, Maier H, et al. Sequential quantification of AgNOR
area and number during silver staining by means of an image analysing
system. Zentralbl Pathol 1994; 140: 37?40.
??????? ?? ?????????, ???. 182: 450?456 (1997)
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