Annals of the Royal College of Surgeons of England (1992) vol.

74, 172-177

The prognostic value of nucleolar

organiser regions in colorectal cancer:
a 5-year follow-up study
William P Joyce MCh FRCSI Peter Dervan MRCPath
Surgical Registrar Consultant Pathologist
Michelle Fynes BSc Thomas F Gorey MCh FRCSI
Medical Student Senior Lecturer/Consultant Surgeon
Kevin T Moran MCh FRCSI John M Fitzpatrick MCh FRCSI
Surgical Registrar Professor of Surgery
David B Gough MCh FRCSI
Surgical Registrar
Department of Surgery, Mater Misericordiae Hospital and University College Dublin and Department
of Pathology, Mater Misericordiae Hospital, Dublin, Ireland

Key words: Colorectal cancer; Staging; Prognosis

Nucleolar organiser regions (AgNORs) are loops of riboso-
mal DNA which reflect the cellular activity or malignant
potential of the cell and are identified by a specific staining
technique. The purpose of this study was to assess the
prognostic value of AgNORs in colorectal cancer and to
compare it with other accepted prognostic methods.
We studied 164 patients who were surgicaily staged for
colorectal cancer and who had complete follow-up data
available for 5 years. Using a highly specific silver staining
and counting technique each patient was given an AgNOR
score. There were 5 Dukes' C tumours, 108 were Dukes' B
and 5 were Dukes' A. No cancer deaths occurred in patients
with Dukes' A tumours. The incidence of well-differentiated,
moderately-differentiated and poorly-differentiated tumours
was 37.2%, 53.7% and 9.1%, respectively. Non-survivors had
significantly higher AgNOR scores compared with survivors
(mean value ± SD, 14.2 ± 0.9 vs 8.2 ± 0.6, P <0.0001). In a
regression analysis model AgNOR score was the most
significant individual variable for predicting survival (x2= 15,
P < 0.01) when compared with Dukes' classification, histolo-
gical grade, tumour depth or vascular invasion.
Correspondence to: Mr William P Joyce MCh FRCSI,
Department of Surgery, St Vincent's Hospital, Dublin 4,
Ireland
Many problems exist with the standard methods avail-
able in determining prognosis after resection of colorectal
cancer. Dukes' classification (1), and the degree of
histological grade (2) remain the accepted standards in
determining survival in colorectal cancer. Despite the
widespread use of these determinants, one-third of
patients with lymph node metastases at presentation
become long-term survivors, while one-third of patients
with Dukes' B tumours die from their cancer irrespective
of histological grade (3,4).
Recent studies (3,5,6) have assessed the prognostic
significance of DNA ploidy in colorectal cancer as
measured by flow cytometry and have found it to be no
better than the standard Dukes' classification or histolo-
gical grade. Nucleolar organiser regions are loops of
ribosomal DNA which occur in the nuclei of all cells
directing ribosome and protein formation (7) and are
currently being evaluated in a variety of tumours (7-10).
These proteins are accurately identified by a specific
silver staining technique and can be counted under light
microscopy, and are thought to represent the cellular
activity or malignant potential of the cell (7,8).
It has been suggested that an increase in the cellular
AgNOR content in breast (10) and prostate carcinoma
(9), malignant melanoma (7), and certain lymphomas (8)
represents a more malignant type of tumour process. It
was therefore the purpose of this study to assess the
 Nucleolar organiser regions in colorectal cancer 173
Table I. Tumour location and operative procedures performed (n = 164)
Tumour location n (%) Operative procedure n (%)
Caecum 22 (13.4) Right hemicolectomy or 41 (25)
extended right hemicolectomy
Ascending colon 9 (5.5) Transverse colectomy 2 (1.2)
Hepatic flexure 6 (3.7) Left hemicolectomy 19 (11.6)
Transverse colon 9 (5.5) Sigmoid colectomy 22 (13.4)
Splenic flexure 4 (2.4) Anterior resection 39 (23.8)
Descending colon 11 (6.7) Abdominoperineal resection 42 (25.6)
Sigmoid colon 49 (29.9)
Rectum 54 (32.9)

prognostic value of AgNORs in terms of 5-year survival known as Type 1 or simple AgNORs (Fig. 1) and Type 2
and to compare them with other established prognostic or clusters (Fig. 2). Type 1 AgNORs were each individu-
indices in patients with surgically resected colorectal ally given a score of 1. Type 2 AgNORs were allocated a
cancer. numerical score according to size by using a graticule in
the objective lens. Type 2 AgNORs measuring up to
4 x 4 [im were awarded a score of 3, from 4 x 4 ,tm to
Patients and methods 6 x 6 [tm a score of 5, and from 6 x 6 to 8 x 8 Cum a score
of 7. A single numerical value was therefore obtained for
From 1978 to 1983, 224 consecutive cases of colorectal each section by combining Type 1 and Type 2 scores.
cancer were treated surgically at our institution.
Fifty-eight patients were excluded from the study due to
inadequate archival tissue remaining in the pathology
laboratory, incomplete 5-year follow up data, or causes of
death occurring during the 5-year follow-up period
unrelated to colorectal cancer. A total of 164 patients
were eligible for study: 88 were male and 76 were female;
ages ranged from 33 to 84 years (median 62.7 years) and
complete follow-up data were available on all these
patients. All patients had their tumours resected and
tumour locations and operative procedures performed
are listed in Table I.
Archival tissue was available on all 164 patients.
Sections from primary tumours which had previously
been fixed in 10% formalin and processed to paraffin wax
were cut at 4 [tm thickness. These sections were then
taken to water via xylene and graded ethanols. Staining
was performed at room temperature for 60 min, using a
reaction mixture comprised of 2% gelatin in 1% formic
acid which had been mixed in a proportion of 1:2
volumes with 50% aqueous silver nitrate under darkroom
conditions. A 1% methyl green counterstain was applied
to each section which was then dehydrated to xylene and
mounted.
After staining, representative areas in each section
were selected by a pathologist and AgNORs were
counted by a single observer who was unaware of tumour
classification or patient fate. A total of 50 nuclei in each
selected area were counted under oil immersion micro-
scopy at a magnification of x 1000 and a median AgNOR
score and range were calculated. This technique was
validated by examining interobserver and intraobserver
variations in AgNOR counts and was found to be less
than 5%. Two types of staining patterns were noted as Figure 1. Survivor, low AgNOR count-a few Type 2
previously described (6,11). Nucleolar organiser regions AgNORs and an occasional Type 1 AgNOR in each nucleus
were visualised as black dots arranged individually ( x 1000).
 -~ ~ .
174 W P Joyce et al.
Correlations between AgNORs and clinical and patho-
logical features were examined using the Mann-Whitney
U test. The independent significance of individual prog-
nostic variables was determined using Cox's multivariate
regression analysis.

Results
Of the 164 tumours studied, 51 were Dukes' C, 108 were
Dukes' B and 5 were Dukes' A. Thirty-five patients with
Dukes' C tumours died (31.2%, 5-year survival); all of
these patients died with metastatic disease. Forty-six
patients with Dukes' B tumours died (57.4%, 5-year
survival): 26% of these deaths were from recurrent
disease, while 74% died with metastatic disease. No
cancer-related deaths occurred in the Dukes' A group.
The AgNOR score of the Dukes' A, B, and C tumours
are compared with survivors and non-survivors in Table
II. A significantly higher AgNOR score was found in
non-survivors when compared with survivors, irrespec-
tive of Dukes' classification (P<0.01). Histological
grade of the survivors and non-survivors and AgNOR
scores are summarised in Table III. A high AgNOR
score is consistently seen in the non-survivors irrespec-
tive of the degree of histological differentiation. Tables
IV and V compare the depth of tumour invasion through
the bowel wall and the presence or absence of vascular
...
...
invasion of the survivors and non-survivors. A signfi-
cantly higher AgNOR score is seen in the non-survivors
Figure 2. Non-survivor, high AgNOR count numerous Type irrespective of the depth of tumour invasion or the
1 and Type 2 AgNORs in each nucleus (x 1000). presence or absence of vascular invasion. In a regression

Table II. AgNOR score and Dukes' staging in survivors and

non-survivors (n = 164)
Survivors AgNOR Non-survivors AgNOR P Value*
Dukes' A 5 5.9
(n= 5) (4-9)
Dukes' B 62 6.9 46 11 <0.01
(n= 108) (5-8) (9-14)
Dukes' C 15 7.5 32 13.1 <0.001
(n= 51) (6-9) (10-16)
Values are median (range) * Mann-Whitney U test

Table III. AgNOR score and histological grade in survivors and

non-survivors (n = 164)
Grade Survivors AgNOR Non-survivors AgNOR P Value*

WD 31 6.1 29 11.5 <0.01

(4-7) (9-15)
MD 50 7.2 38 11 <0.01
(4-8) (9-13)
PD 5 7.9 11 13.7 <0.001
(5-9) (8-15)
WD: Well-differentiated; MD: Moderately differentiated; PD: Poorly differentiated
Values are median (range) * Mann-Whitney U test

Table IV. AgNOR score and depth of tumour invasion through the wall
in survivors and non-survivors (n = 164)
Survivors AgNOR
Incomplete 85 6.8
(5-8)
Complete 23 6.5
(4-9)
Values are median (range) * Mann-Whitney U test
Table V. AgNOR score and the presence or absence of vascular invasion in survivors
and non-survivors (n = 164)
Survivors
Vascular invasion present 50
Vascular invasion absent 58
Values are median (range) * Mann-Whitney U test
analysis model the AgNOR score was found to be the
most significant individual variable for predicting survi-
val (X= 15, P<0.01) when compared with Dukes'
classification, histological grade, tumour depth or the
presence of vascular invasion.
Discussion
It is of significant value to predict accurately the preoper-
ative stage, the probability of tumour recurrence and
survival time in patients with colorectal cancer. These
data would help to identify the high-risk patient for
intense postoperative surveillance and in planning future
treatment modalities. The low-risk patient could then be
spared the time-consuming and costly routine follow-up
programmes in patients with colorectal cancer. With this
in mind many methods have been used over the years
with varying effectiveness. Dukes' classification (1) intro-
duced in the 1930s relies on the depth of tumour
penetration and location and number of involved nodes
and is the most commonly used method today. Dukes'
classification and its modifications (12,13) are a crude
estimation of survival and do not take into account the
preoperative stage or the biological behaviour of an
individual tumour. This classification may also vary with
intraoperative staging as a proportion of patients thought
to have a Dukes' A, B or C lesion at the time of surgery
and believed by the surgeon to have undergone a curative
resection have occult hepatic metastases from which they
ultimately die (14). Broder's histological grade (2),
patient's age, sex, tumour location and fixation, and
carcinoembryonic antigen levels (15) are also currently
used in assessing prognosis. They seem to have limited
value and at best can only predict recurrence in non-
metastatic colon cancer in 30-60% of cases (15,16).
Nucleolar organiser regions in colorectal cancer 175
Non-survivors AgNOR P Value*
16 10.9 <0.01
(8-15)
40 9.8 <0.01
(7-14)
AgNOR Non-survivors AgNOR P Value*
6.5 21 12.7 <0.01
(3-7) (9-14)
6.8 35 9.7 <0.01
(4-8) (8-13)
The newer more objective TNM classification of colo-
rectal tumours also has disadvantages, as Wolmark et al.
(17) have shown that a subset of patients with Dukes' C
tumours, ie patients with partial tumour penetration and
1-4 positive nodes, had a prognosis at least as good as
patients with Dukes' B lesions. These data raise serious
doubts about the value of the TNM classification that
fails to include the number of positive nodes as a
predictive discriminant.
The DNA content of tumour cells as assessed by
ploidy may reflect the biological behaviour of certain
cancers and have recently been associated with a poor
prognosis in colorectal and other cancers (18,19). The
biological and clinical significance of ploidy in colorectal
cancer is confused by the varied and sometimes contra-
dictory findings of different authors. Most studies have
shown some correlation with clinical outcome, but these
have been inconclusive regarding the correlation with
conventional morphological factors such as Dukes' classi-
fication and Broders' histological grade. Recently
(3,20,21) it has been shown that ploidy values were no
better than Dukes' classification in determining progno-
sis after colorectal cancer resection. We (6) have also
recently demonstrated in a pilot study of 51 patients with
advanced colorectal cancer that the AgNOR score was
the single best discriminant of postoperative survival
when compared with ploidy values, Dukes' classification
or Broders' histological grade. These 51 patients are
included for analysis in this study as they formed part of
the cohort of all patients surgically treated for colorectal
cancer at our institution over the study period.
In this consecutive series of all grades and stages of
colorectal cancer, the AgNOR score provided better
prognostic information than Dukes' classification, histo-
logical grade, tumour depth and penetration, and the
presence or absence of vascular invasion. As for all
176 W P Joyce et al.
techniques that may have an application in diagnostic
pathology the results must be reproducible and inter-
preted with ease. This study and others from this
department (6,9) have standardised the counting tech-
nique with only minor interobserver and intraobserver
variations. The staining technique described is easy to
perform and relatively inexpensive, but counting
AgNORs at present is labourious and time consuming.
The introduction of a computer-based automated count-
ing system for AgNORs would allow objective evaluation
and standardisation of the counting technique and would
decrease the labour involvement and improve reproduci-
bility.
In conclusion, it is unlikely that a single prognostic
indicator which can be applied to all cases will emerge in
colorectal cancer. At present Dukes' classification is still
the most widely used. We believe that the AgNOR score
is superior to prexisting modalities and a high AgNOR
score will identify those patients who warrant close
surveillance. Nucleolar organiser regions may now be
counted preoperatively from biopsy specimens and cyto-
logy and may be potentially important for physicians
staging and treating patients with colorectal cancer.
Adjuvant modes of therapy such as preoperative radio-
therapy may be an option in selected patients with high
AgNOR scores.
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Received 16 July 1991
series of patients can be made. It has been disappointing
that more modern technological advances such as the
assessment of the degree of aneuploidy of a particular
tumour by flow cytometry has not added more to the