Modern Pathology (2008) 21, 4653

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Diagnostic and prognostic markers for

gastrointestinal stromal tumors in Norway
Sonja E Steigen1, Bodil Bjerkehagen2, Hans K Haugland3, Ivar S Nordrum4,5,
Else Marit Lberg6, Vidar Isaksen1, Tor J Eide2 and Torsten O Nielsen7
1

Department of Pathology, Institute of Medical Biology, University of Troms, Norway; 2Department of

Pathology, Rikshospitalet-Radiumhospitalet Medical Centre, Oslo, Norway; 3Department of Pathology,
University Hospital of Bergen, Norway; 4Department of laboratory medicine, childrens and womans health,
Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway; 5Department of
Pathology and Medical Genetics, St Olavs Hospital, Trondheim, Norway; 6Department of Pathology, Ullevaal
University Hospital, Oslo, Norway and 7Department of Pathology and Genetic Pathology Evaluation Centre,
University of British Columbia, Vancouver, BC, Canada

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in the gastrointestinal tract.
The diagnosis of GIST is based on histology together with a panel of immunohistochemical markers; the most
important is KIT (CD117). A total of 434 cases of GISTs were confirmed by histology and immunohistochemistry,
and incorporated into tissue microarrays. Validation of histological features as well as the prognostic value of
two immunohistochemical biomarkers (p16 and L1) was assessed. High mitotic rate, large tumor size, nuclear
atypia, and small bowel primary site were all validated as negative prognostic factors in GISTs. Expression of
p16 was significantly correlated with unfavorable prognosis, whereas L1 expression was not.
Modern Pathology (2008) 21, 4653; doi:10.1038/modpathol.3800976; published online 5 October 2007
Keywords: gastrointestinal stromal tumors; CD117 antigen; prognostic markers; p16; L1

Mesenchymal neoplasms arising in the digestive

tract are rare compared with epithelial neoplasms.
Several entities exist, among which gastrointestinal
stromal tumor (GIST) is the most common.1,2 The
diagnosis of GIST is often suspected histologically,
but such tumors can have a broad morphologic
spectrum. A panel of immunohistochemical markers
is used to aid in correct diagnosis. A total of 91 to
98% of GISTs stain positively for KIT (CD117),3,4
which is a major factor in the initial identification of
GIST and therefore is often the inclusion criterion
into many reported series. Staining of GISTs for
other standard laboratory immunomarkers is more
variable, including CD34 (E70%), smooth-muscle
actin (E35%), S-100 (E10%) and desmin
(E5%).2,5,6 PKC theta is an immunohistochemical
marker found in two studies to be expressed in
GISTs, KIT-negative GISTs and schwannomas, but
not in other mesenchymal tumors of the GI tract.7,8
Prognostic factors in GISTs are controversial, but the
Correspondence: Dr SE Steigen, MD, Department of Pathology,
Institute of Medical Biology, University of Troms, Sykehusveien,
Troms N-9037, Norway.
E-mail: [email protected]
Received 26 March 2007; revised and accepted 16 July 2007;
published online 5 October 2007

most accepted pathologic features are mitotic rate

and tumor size. Small tumors with low mitotic
activity usually have a benign behavior, and increasing the number of mitoses and increasing
tumor size have been shown to be statistically
significant.9 An evidence-based approach for defining the risk of aggressive behaviour in GISTs, based
on tumor size and mitotic count, has been presented
in a consensus document from multiple scientists in
the field.10 Other factors as anatomic location,
cellular atypia and necrosis have been shown to be
independent prognostic factors.
Tissue microarrays are useful for rapid testing of the
diagnostic and prognostic utility of antibodies in large
numbers of archival samples. The arrayed tissue cores
are histologically guided samples from representative
regions of standard formalin-fixed paraffin-embedded
tumor blocks. Kononen et al11 first described this
technique, and has since been used in many studies
requiring the evaluation of large series. Tissue microarrays have repeatedly been shown to be an efficient
method in assessing the sensitivity and specificity of
antibodies used in determining the origin or cell type
of human tumors.12,13
p16 is a tumor suppressor protein that inhibits
cell cycling by arresting cells in G1 before entry into
S phase. Genetic alterations resulting in diminished

Markers for GISTs in Norway

SE Steigen et al

p16 levels are common in human cancers.14 Loss of

p16 protein correlated with high-risk GIST in a
small study by Sabah et al15 and later by SchneiderStock et al.13 In this second, larger study with 284
GIST patients, p16 loss was found in 50% of the
cases. These patients had a worse prognosis than
those with p16-positive tumors, with a 2.3-fold
increased risk of dying of disease. However, expression (rather than loss) of p16 correlates with
unfavorable prognosis in high-grade breast carcinomas16 and also in most high-grade cervical intraepithelial neoplasia (CIN) and cervical carcinomas
of squamous, glandular and small cell types.17 This
implies that p16 loss is not required for oncogenesis
and other mechanisms to dysregulate cell-cycle
control (such as overexpression of cyclin D or loss
of RB) may instead be employed.
L1 is a cell-surface adhesion molecule that is a
member of the immunoglobulin superfamily, with
six amino-terminal immunoglobulin-like domains
followed by three to five fibronectin type III
domains, a single transmembrane segment and a
highly conserved cytoplasmic tail.18 The L1 antigen
is expressed in neural cells and is involved in a
number of different cellular processes, including
neuronal cell migration, myelination, axon growth
and pathfinding.19,20 In addition, L1 has a role in the
development of the renal collecting duct system.21
Expression of L1 has been demonstrated on paraffinembedded tissue and in cell lines in a wide variety
of normal and neoplastic tissues.22 The presence of
L1 correlates with progression and metastatic spread
of melanomas,23 and is associated with poor prognosis in ovarian and endometrial carcinomas.24
Recently, Kaifi et al25 evaluated L1 as a potential
diagnostic marker and prognostic factor in a study
with 72 GISTs. They reported L1 to be expressed in
73% of the GISTs, and these patients trended toward
reduced survival.
The purpose of this study was to validate the
clinical and immunohistochemical prognostic markers on a large series of defined GISTs from Norway.

Materials and methods

Patients and Samples

The study material consisted of selected tissue

blocks from the archives of pathology departments
in Norway. Cases were selected by evaluating the
records of the Cancer Registry of Norway for
mesenchymal tumors and poorly differentiated
carcinomas in the gastrointestinal tract over a period
of 30 years (19732002). A total of 3672 reports were
evaluated, and all reports with clear evidence that
the origin of the tumor was not mesenchymal were
disqualified (based on the results of immunohistochemical staining and other methods reported at the
time of primary diagnosis). A total of 1192 cases of
candidate mesenchymal tumors were so identified,
and slides and blocks from all these cases requested.

47

The material was located in all of the 20 pathology

departments in Norway. At the largest departments,
a local pathologist examined the cases and a single
representative block was forwarded. From two
hospitals we received no material, which constituted 92 cases. In 129 cases (11%) no blocks were
found and in addition in 102 cases (10%) the blocks
did not contain enough material for further investigations. New slides of the remaining 869 cases were
made and stained with hematoxylin and eosin
(H&E), and histology re-reviewed by the first author.
A total of 187 cases with inconsistent histologic
features were additionally evaluated by the last
author, and 146 of these cases were excluded from
the study because they were almost certainly not
mesenchymal tumors (based on the H&E staining),
but rather carcinomas or lymphomas. Sixteen cases
underwent additional independent expert review
and three cases were thereby further excluded. A
total of 720 cases were evaluated as representing
true mesenchymal tumors of the GI tract, but for
making tissue microarrays an additional 69 cases
did not contain enough material for the required
duplicate core extractions (blocks containing only
small bite or core biopsies). The 651 remaining cases
from the Cancer Registry were assembled into five
tissue microarrays. For all cases, data were collected
on age, date of death (if applicable), surgical
treatment, location of primary tumor, size of tumor
and history of other malignancies. Overall survival
and metastatic status at presentation were recorded
at the time of evaluating the records. Eight patients
were treated with STI-571 (Glivecs), as this cohort
largely predates the use of this drug. The Regional
Committee for Medical Research Ethics, Northern
Norway approved the study.
Construction of Tissue Microarrays

A slide with representative, viable tumor was

selected from each case, and coupled to the
corresponding formalin-fixed paraffin-embedded
block. Duplicate 0.6-mm cores were taken from
representative tumor tissue and inserted into a
recipient paraffin block to create a tissue microarray,
using a Beecher Instruments Micro Tissue Arrayer.26
The completed recipient blocks were sectioned at
4 mm and transferred to silanized glass slides. A total
of five recipient blocks were made.
Histological Evaluation

The whole-section slides that were used for selecting

representative tissue microarray cores were used for
histological evaluation. Mitotic figures were counted
in 50 consecutive high-power fields. Additional
parameters were recorded in all cases and included
the presence of spindled and/or epithelioid cell
morphology. Focal or diffuse atypia was included in
the review and defined as previously described by
Modern Pathology (2008) 21, 4653

Markers for GISTs in Norway

SE Steigen et al
48

Miettinen et al.4 Other parameters such as necrosis,

ulceration, mucosal invasion and hemorrhage were
also assessed. The tumors were classified according
to the published consensus risk-group stratification
system based on maximum tumor size and mitotic
count per 50 high-power fields.10
Immunohistochemical Staining and Scoring

Sections from the arrays were stained with H&E to

confirm the presence of a representative tumor in
each core. Further sections were stained with a
panel of antibodies using a Ventana (Tucson, AZ,
USA) automated immunohistochemical stainer according to the manufacturers guidelines.
The antibodies used were the following: c-KIT
(polyclonal, dilution 1:200; Dako), smooth-muscle
actin (clone 1A4, dilution 1:200; Dako), S100
(polyclonal, dilution 1:2000; Dr A Marks, University
of Toronto), CD34 (QB End/10, dilution 1:25; Cell
Marque), desmin (D33, dilution 1:200; Dako), vimentin (V9, dilution 1:10 000; Biogenex), cytokeratin cocktails CK (AE1/AE3, dilution 1.200; Dako
CAM 5.2, dilution 1.50; Becton/DickinsonPolyclonal, dilution 1:1000; Dako), MIB1(SP6, dilution
1:200; Labvision), L1 (IgG, Clone UJ127, dilution
1:50; NeoMarkers), p16 (dilution 1:500; MTM
Laboratories AG, Heidelberg, Germany) and PKC
theta (pT538 clone 19, dilution 1:20; BD Transduction Labs). The immunostaining was performed
with an avidinbiotin detection system. Diaminobenzidine hydrochloride solution with hydrogen
peroxide (Ventana Gen II, Dab basic) was the
chromogen. Clinical positive control cases for all
markers were included in every staining batch.
Tissue cores were scored on the basis of the
percentage of positive tumor cells staining above
background, as completely negative, weakly stained
(o10%), moderate positive (1050%) or strongly
positive (450%).
Scores were entered into a Microsoft Excel
spreadsheet. Scoring results were dichotomized into
either negative (score of 0 or 1) or positive (score of 2
or 3) categories, and uninterpretable results were
eliminated from further considerations (ie, they
were not used for calculations for sensitivity and
specificity). Discrepant score results for duplicate
cores, when present, were consolidated as the
higher interpretable score.27 To aid the analysis of
the numerous tissue cores stained by immunohistochemistry, digital images were collected using a
BLISS instrument (Bacus Laboratories, Lombard, IL,
USA; http://bacuslabs.com). Scoring results were
combined using Deconvoluter and represented in
Treeview.28
Statistical Analysis

SPSS for Windows (version 14.0) was used for

statistical analysis. Survival curves were plotted
Modern Pathology (2008) 21, 4653

using KaplanMeier methods, with significance

assessed using log-rank tests and based on overall
survival over 20 years. Multivariate analyses were
analyzed using Cox regression model. For correlations, the Pearson w2-test was used.

Results
A total of 401 of the 651 samples represented in the
tissue microarrays were positive for CD117 and
regarded as definite GISTs. A total of 71% of these
CD117-positive tumors stained positive for CD34,
19% for smooth muscle actin, 4% for desmin, o1%
for S100 and 97% for vimentin. In addition, 37 cases
not staining positive for CD117 or with cores
missing from the CD117 tissue microarray slide
(four cases) were positive for PKC theta. Four of
these cases stained positive for S100 and were
regarded to be true schwannomas. The remaining
33 cases were grouped together with the CD117positive cases as immunohistochemically confirmed
GISTs. Results of immunohistochemical staining are
shown in Table 1. Clinicopathological results concerning GIST patients are presented in Table 2. No
size was stated in 128 cases, but for the remaining
cases, 20% of the tumors were classified into the
very-low-risk and the low-risk groups, 34% in the
intermediate-risk group and 47% in the high-risk
group according to Fletcher et al.5 The standard
markers (CD34, smooth-muscle actin, desmin or
S100) did not correlate significantly with outcome.
A total of 182 of the 434 defined GISTs had 0 or
1 mitosis/50 high-power fields, 91 had 25, 45
patients had 610 and 116 had more than 10
mitoses/50 high-power fields. There was no significant difference in overall survival between the two
groups with fewest mitoses, but for the group with
610 mitoses/50 high-power fields and again for the
tumors with more than 10 mitoses/50 high-power
fields, there was a significant less favorable overall
survival (P 0.009 and 0.002). Median survival time
for the patients with five mitoses or less was 6.5
years, for the group with 610 mitoses 3.2 years and
for the patients with more than 10 mitoses 1.6 years.
There was no significant correlation between the
number of mitoses and location in the stomach or
small bowel. Tumors with low mitotic index (o5/50
high-power fields) had a favorable overall survival
(Po0.001) in both the locations.
Size of tumor has a close to linear curve, with a
decrease in overall survival with increasing size. No
significant difference was found however between
tumors up to 2 cm and from 2 to 5 cm. Between
groups of tumors from 5 to 10 cm and larger than
10 cm, there was likewise no difference. Patients
with tumors 5 cm or less (108 cases) had a
significantly better overall survival than those with
tumors larger than 5 cm (198 cases), with a median
survival time of 7.3 vs 3.2 years, respectively
(Po0.001). There was a correlation between the

Markers for GISTs in Norway

SE Steigen et al
49

Table 1 Tissue microarray immunostaining results

No. of cases

kit

PKC

cd34

sma

desmin

s100

CK

Vimentin

401
33
24
4
80
0a
4
8
15
61
21
651

401
0
0
0
0
0
0
0
0
0

328
33
0
4
0

286
8
24
0
0
0
0
0
0
0

77
7
3
2
80
40
0
0
0
0

14
5
2
1
40
0
4
0
0
0

1
0
0
4
1
1
0
8
0
0

4
4
1
0
6
3
0
1
15
0

390
26
22
4
68
35
4
8
7
61

Interpretation
Unequivocal GIST
kit-negative GIST
Possible GIST
Schwannomas
LM/LMS
Probable LM/LMS
LM/LMS
Melanomas/clear cell sarcomas
Carcinomas
Probable desmoid-type fibromatosis
Missing values
Total

CK, cytokeratin; PKC, protein kinase C theta; LM, leiomyoma; LMS, leiomyosarcoma.
a
Included in the 80 cases above.

Table 2 Clinicopathological characteristics of 434 Norwegian

patients with confirmed GIST
Gender
Male
Female
Age at primary diagnosis, mean (range)
Localization of primary tumor
Esophagus
Stomach
Small bowel
Colon
Rectum
Omentum/mesentery
Unknown locations
Size, mean (based on 306 cases
with available data) (range)
Cellular features
Spindle cells
Epithelioid cells
Spindle and epithelioid cells
Focal atypia
Diffuse atypia
Necrosis
Mitosis per 50 high-power field,
mean (range)

219 (51%)
215 (49%)
64.8 years (1994)
1
212
148
10
23
7
33
8.9 cm

320
45
69
118
71

(o1%)
(49%)
(34%)
(2%)
(5%)
(2%)
(8%)
(1.030)

(74%)
(10%)
(16%)
(27%)
(16%)

43 (10%)
10.6 (0276)

number of mitoses and size, as tumors with more

than five mitoses were found almost four times
more frequently among tumors larger than 5 cm
(Po0.001). There was a significant correlation
between size and location, with 60% of the gastric
tumors being 5 cm or larger compared with 75% of
the small bowel tumors (P 0.011). The mean size of
the gastric tumors was 8.7 cm and that of the small
bowel tumors was 9.4 cm. Smaller size tumors gave
a significantly better overall survival in gastric
tumors (Po0.001), but not in the small bowel
tumors (P 0.974).
For patients with tumors without evident cellular
atypia, the median survival time was 6.5 years
compared with the group with focal or diffuse atypia

(examples in Figure 1), where median survival was

2.4 and 1.8 years (Po0.001). Patients with gastric
GISTs survived for 5.3 years compared with patients
with small bowel tumors who survived for a median
of 3.2 years (P 0.011). Females had an overall
significantly better survival than men (median
survival time 4.5 years vs 2.9 years, P 0.009).
Other assessed parameters (ulceration, mucosal
invasion, hemorrhage or necrosis) had no impact
on overall survival.
In our series, 180 (42%) of the defined GIST cases
stained for p16, and these patients had a significantly worse overall survival (P 0.013) than
patients not expressing p16 (Figure 2). Location of
p16-expressing tumors is shown in Table 3. When
evaluating only gastric tumors and tumors from the
small bowel, the expression of p16 was still a
negative prognostic factor compared with the group
not expressing p16 (P 0.002). The distribution
between gastric and small bowel tumors in the
p16-expressing group and the group not expressing
p16 shows significant differences (P 0.004). The
number of gastric vs small bowel tumors was equal
in the p16-expressing group, and with twice as
many gastric tumors as small bowel tumors in the
group not expressing p16. When investigating p16
in the group with gastric tumors, the patients not
expressing p16 had an overall better survival
(median survival time 6.5 years) compared with
those who express p16 (median survival time 3.8
years, P 0.033). For patients with small bowel
tumors, the median survival time for patients not
expressing p16 was 5.2 years and for those expressing p16 was 3.2 years (P 0.065).
More than five mitoses/50 high-power fields
correlated with tumors expressing p16, as 43% of
these tumors had a high mitotic index. A total of
33% of the tumors not expressing p16 had more
than five mitoses/50 high-power fields (P 0.050).
Tumors expressing p16 were larger than 5 cm in
72% of cases, compared with those not expressing
p16, where 59% of tumors were larger than 5 cm
(P 0.021).
Modern Pathology (2008) 21, 4653

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50

Figure 1 GIST tumor without atypia (a) and showing diffuse atypia (b).

Table 3 Location of CD117-positive positive mesenchymal

tumors within the gastrointestinal tract, stratified by p16 and L1
immunostaining

1.0

p16positive
tumors
(%)

0.8

p = 0.013

Survival

0.6

0.4
p16 negative cases, N = 254

Localization of primary tumor

Esophagus
1 (1)
Stomach
74 (41)
Small bowel
74 (41)
Colon
4 (2)
Rectum
9 (5)
Omentum/
2 (1)
mesentery
Other locations/
16 (9)
unknown
Number of patients

180

p16negative
tumors
(%)

L1positive
tumors
(%)

0
138 (55)
74 (29)
6 (2)
14 (6)
5 (2)

1
166
48
4
20

(1)
(65)
(19)
(2)
(7)

L1negative
tumors
(%)

45
99
6
3
7

0
(25)
(56)
(3)
(2)
(4)

17 (6)

16 (6)

17 (10)

254

255

177

0.2

p16 positive cases, N = 180

0.0
0.00

5.00

10.00

15.00

20.00

Years

Figure 2 Overall survival for patients with GISTs expressing p16

(bottom gray line, N 180) and not expressing p16 (top black line,
N 254).

In a multivariate model evaluating all cases with

complete data (N 306), the expression of p16 was a
significant factor when mitotic index and size were
accounted for (P 0.034).
A total of 255 (59%) of the GISTs expressed L1,
and there was no difference in overall survival
between the groups expressing or not expressing L1
(P 0.943) (Figure 3). This was also true for tumors
located in the stomach or in the small bowel
Modern Pathology (2008) 21, 4653

(P 0.074 and 0.184). L1 expression was more than

three times as frequent among tumors located in the
stomach compared with the small bowel tumors. For
the tumors not expressing L1, only half of them were
located in the stomach (Po0.001).
There was a significant correlation between
tumors with more than five mitoses and the
expression of L1 (P 0.043), with more than twice
as many L1-expressing tumors having a high mitotic
index than tumors not expressing L1. No correlation
was found with tumor size. Location of L1-expressing tumors is shown in Table 3.

Discussion
This tissue microarray series was built using
material from Norwegian pathology department
archives. The series was based on tumors reported
to the Cancer Registry of Norway and therefore

Markers for GISTs in Norway

SE Steigen et al
51
1.0

0.8

Survival

p = 0.943
0.6

0.4
L1 negative cases, N = 177

0.2
L1 positive cases, N = 255
0.0
0.00

5.00

10.00
Years

15.00

20.00

Figure 3 Overall survival for patients expressing L1 (gray line,

N 255) compared with patients not expressing L1 (black line,
N 177). Two cases had no interpretable L1 stain.

tumors such as leiomyomas considered totally

benign at the time of primary diagnosis were not
registered. When comparing the numbers of patients
in the different risk groups, our results showed less
than half the number of cases in the very-low and
low-risk groups, compared with a Swedish study
described as being population based.9 The intermediate-risk group contained around 30% more
patients in our study, but the high-risk group
contained 25% more patients than in the Swedish
study. Most other series have confined analyses to
KIT-positive tumors, but we have also added PKC
theta-positive cases in our study, based on recent
reports by others,7,8 which suggest that KIT-negative
GISTs express PKC theta. Some of the KIT and
PKC-double-negative cases can be categorized as
leiomyomas, leiomyosarcomas, schwannomas or
carcinomas based on other immunostains.
In GISTs, tumor size and mitotic activity are the
most consistent clinicopathological features indicative of aggressive clinical behavior, features not
readily assessable from typical bite or core biopsies.
Here we have studied the immunohistochemical
stainings for the proposed biomarkers p16 and L1 in
GISTs to see if they can aid in the clinical prognostic
assessment.
Decreased expression of p16 by immunohistochemical staining was previously reported to have a
negative association with survival in GIST. The
largest previous study on prognostic value of p16 in
GISTs used patient material from five different
pathology departments.13 Patients with tumors
showing loss of p16 expression in their study had
worse prognosis than those expressing p16. Our
current larger study did not validate these findings,

as patients expressing p16 were found to do worse

than those not expressing this biomarker. Loss of
p16 expression is biologically expected to contribute
to malignancy. There are however many other
oncogenic changes that can achieve the same net
effect of increased proliferation, including loss of RB
or TP53, and aberrant activation of cyclin D by gene
amplification or by mutations activating the receptor
tyrosine kinase-ras-raf-ERK-signaling cascade. Indeed, such alternative changes in cell-cycle control
could lead to a compensatory upregulation of p16,
and in support of this hypothesis we found p16
expression was actually more common among GISTs
with higher mitotic counts. In cervical neoplasms,
p16 is found to be increased in carcinoma in situ
relative to benign lesions, and advanced carcinomas
retain positivity for p16.29 In breast cancer, expression of p16 (rather than lack of expression) was
found to correlate with poorer outcome.16
L1 is a protein with several interesting biological
roles. In L1-mutant mice, corticospinal axons fail to
project to the spinal cord.20 Anti-L1-antibodies
cause alterations of branching morphogenesis in
renal epithelium, leading to marked reduction of
kidney size.21 L1 is expressed at the invasive front of
colon cancers.30 Overexpression of L1 is associated
with metastasis in cutaneous malignant melanomas,
whereas inactivation of L1 significantly reduces the
invasion and migration of melanoma cells.31 In the
work of Keifi et al,25 L1 was evaluated both as a
diagnostic and as a prognostic marker for GIST.
Their study included 72 cases, with little information provided about the patient population from
which the series was derived. A total of 73% of their
GIST cases were L1 positive, and survival analysis
was performed on 55 cases. Their analysis demonstrated only a trend to poorer survival in L1-positive
vs L1-negative tumors (P 0.13), but has nevertheless been cited as supporting L1 as a factor for
less favorable prognosis.22 We found no difference in
survival between GISTs expressing L1 vs those not
expressing L1.
Initially reported studies of a biomarker may show
great promise, but subsequent studies on the same or
related markers can yield inconsistent conclusions
or sometimes even be in direct contradiction to the
original reports. This has been recently discussed in
a paper by McShane et al, where recommendations
for tumor marker prognostic studies have been
made.32 This article was simultaneously published
in five journals, and presents guidelines for complete
reporting so that relevant information is available for
the readers to be able to judge the usefulness of the
data. In retrospective studies, patient population is
often biased toward patients with available tumor
specimens. Specimen availability may be related to
tumor size and patient outcome. In prospective
studies the validation of prognostic factors can be
confounded by patient selection. These problems are
suggested as being surmountable by improvements
in study design and reporting.33
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52

We use a large series comprised of cases completely independent of the previous reports to be
validated. Our results validate the established
clinical prognostic markers, but do not validate the
prognostic values of p16 and L1 immunohistochemical biomarkers presented in other series.

10

Acknowledgements

12

We thank Dr David Owen for helpful advice and for

providing additional expert review on 16 cases.
TON is a Scholar of the Michael Smith Foundation
for Health Research. The tissue microarray facility at
the Genetic Pathology Evaluation Centre is supported in part by an unrestricted educational grant
from sanofi-aventis. We also thank Kristin Smistad
Myrmel for help with immunohistochemical scoring
of the tissue microarray slides.

11

13

14

15

Conflict of interest statement

None declared.
16

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