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2284
Prognostic Significance of DNA Flow Cytometric
Analysis in Patients with Nasopharyngeal Carcinoma
Timothy T. C. Yip, Ph.D.1
W. H. Lau, M.B.B.S.1
John K. C. Chan, M.B.B.S.2
Roger K. C. Ngan, M.B.B.S.1
Y. F. Poon, M.B.B.S.1
C. W. Lung, B.Sc.2
T. Y. Lo, B.Sc.1
John H. C. Ho, M.B.B.S., M.D.,
BACKGROUND. Nasopharyngeal carcinoma (NPC) is a prevalent malignant tumor
3
D.Sc.
1
Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong.
2
Department of Pathology, Queen Elizabeth Hospital, Kowloon, Hong Kong.
3
Department of Radiotherapy, Baptist Hospital,
Kowloon Tong, Hong Kong.
Presented in part in the 17th International Congress of the International Society of Analytical
Cytology, Lake Placid, New York, October 16 –21,
1994.
Supported by grants provided by the Queen Elizabeth Hospital Cancer Research Fund, Hong Kong
Anti-Cancer Society, and Hong Kong Jockey Club.
Address for reprints: Timothy T. C. Yip, Ph.D.,
Radiobiology Unit, Department of Clinical Oncology, Queen Elizabeth Hospital, Room 1305, Block
R, 30, Gascoigne Road, Kowloon, Hong Kong.
Received September 8, 1997; revision received
April 27, 1998; accepted April 27, 1998.
© 1998 American Cancer Society
among Southern Chinese. Previously, the authors described the prognostic significance of a serum antibody assay to a recombinant Epstein-Barr virus Bam HI–Z
replication activator protein (ZEBRA) in NPC patients with long term follow-up. In
this study, the authors further reported the use of DNA flow cytometry (DNA-FCM)
as an additional technique for determining the prognosis of NPC patients in the
same series.
METHODS. One hundred and forty-three archival biopsies from 110 NPC patients
were deparaffinized and subjected to DNA-FCM analysis. DNA ploidy state and
various proliferative indices (PI) of the tumors were correlated with patient survival
and frequency of recurrence.
RESULTS. Among the biopsies analyzed, 119 were histologically positive NPC and
24 were negative. Fifty-one tumor biopsies that fulfilled the guideline criteria of the
DNA Cytometry Consensus Conference were correlated with the clinical manifestations of the patients. Among them, 43 tumors (84%) were DNA diploid and 8
(16%) were aneuploid. Two PI, S-phase fraction (SPF) and proliferation fraction
(PF), appear to be potentially useful prognostic indicators. For example, PF in
patients who developed locoregional recurrence (15.1%) and distant recurrence
(16.4%) after radiation therapy both were significantly higher than PF in patients
who were in complete remission (8.2%) (P ⫽ 0.0005 and P ⫽ 0.004, respectively).
Significant differences in SPF between patients with distant recurrence (10.6%) and
those in remission (5.7%) also was found (P ⫽ 0.005). Using Kaplan-Meier analysis,
patients with high PF, high SPF, and aneuploid tumors had significantly poorer
12-year survival rates (35%, 26%, and 28%, respectively) than those patients with
low PF, low SPF, and diploid tumors (77%, 67%, and 59%, respectively) (P ⬍ 0.0009,
P ⬍ 0.004, and P ⬍ 0.01, respectively).
CONCLUSIONS. Determination of tumor PI and DNA ploidy state by DNA-FCM at
diagnosis of NPC can be potentially useful in selecting a poor prognostic subgroup
of NPC patients. These parameters may enable oncologists to plan for more
stringent treatment strategies such as hyperfractionated and accelerated radiation
therapy or concomitant chemoradiotherapy for these patients. Cancer 1998;83:
2284 –92. © 1998 American Cancer Society.
KEYWORDS: DNA flow cytometry, proliferative fractions, S-phase fraction, DNA
ploidy, nasopharyngeal carcinoma, prognostic markers, survival analysis.
N
asopharyngeal carcinoma (NPC) is a common malignant tumor in
Southeast Asia but it is rare in Western countries.1,2 This neoplastic disease is prevalent in two Southern Chinese provinces, Guangdong and Guangxi. In Hong Kong, it is the third most prevalent cancer
among males.3 The incidence of disease stands at 20 –27.5 per 100,000
population, which is much higher than that of ⬍ 1 per 100,000
population found among whites in Western countries.
Prognostic Role of DNA-FCM in NPC/Yip et al.
The diagnosis of NPC relies on endoscopic examination of the nasopharynx of the patients followed by
histopathologic examination of nasopharyngeal biopsy.4
Serological tests for IgA antibodies to Epstein-Barr
virus (EBV) viral capsid antigen (VCA/IgA) and early
antigen (EA/IgA) also are useful as first-line diagnostic
tests for NPC.5,6 Radiation therapy (RT) has been the
most effective modality of treatment.7–10 However, so
far, prognosis of NPC, remains a major problem for
oncologists. Clinical staging by Ho’s classification has
been shown to be invaluable for predicting the outcome of NPC patients after RT.11–13 However, a fraction of the patients with early stage disease still develop either locoregional or distant recurrence and die
of disease.13,14 Conversely, a proportion of patients
with intermediate or late stage disease are cured by
RT. Therefore, additional parameters have been
sought to supplement clinical staging for the prognostication of NPC.
Recently, we have shown that an immunoglobulin
(IgG) to a recombinant EBV Bam HI–Z replication
activator protein (ZEBRA/IgG) can serve as a useful
prognostic marker for NPC.15–17 The antibody titers
tested after RT were elevated in NPC patients who
later developed distant metastasis. The antibody also
was correlated strongly with the survival rate. However, the survival correlation was found only for the
antibody titer determined at 10 months after treatment but not at the time of diagnosis. Thus, the initial
treatment cannot be tailored accordingly for the poor
prognostic group.
DNA flow cytometry (DNA-FCM) involves staining
of tumor cell DNA content by nucleic acid binding
dyes followed by flow cytometric determination of the
DNA ploidy state and proliferative indices (PI) of the
17,18
The technique has been shown to be a
tumors.
useful tool in the diagnosis and prognosis of a variety
of malignant tumors.19 –25 In this study, we examined
whether DNA-FCM was able to provide prognostic
information for NPC in 143 nasopharyngeal biopsies
from 110 NPC patients.
MATERIALS AND METHODS
NPC Patients
One hundred and ten patients with newly diagnosed
NPC treated at the Department of Clinical Oncology
(formerly called Institute of Radiology and Oncology)
at the Queen Elizabeth Hospital, Hong Kong between
1978 –1983 were recruited into this study. They were
staged clinically according to the classification system
of Ho26 and the International Union Against Cancer
27
(UICC). The patients were followed for ⬎ 12 years or
until death. Detailed clinical characteristics of each
patient such as disease stage; extent of primary dis-
2285
ease; treatment modalities; time, site, and extent of
recurrence; modes of salvage treatment; survival periods; and causes of death all were recorded using Foxplus database software (Microsoft Corporation, Redmond, WA) for analysis.
Sample Preparation for DNA Flow Cytometric Analysis
One hundred and forty-three archival paraffin blocks
of nasopharyngeal biopsies from 110 patients were
retrieved. Of these, 58 were undifferentiated carcinomas of the nasopharyngeal type (UCNT), 61 were
poorly differentiated squamous cell carcinomas (PDSCC), and 24 were histologically negative biopsies.27–30
All positive and negative biopsies were obtained
from NPC patients at the time of first presentation.
Histologic diagnosis confirmed no malignancy in the
24 negative biopsies. Repeated sections from these
biopsies then were subjected to both hematoxylin and
eosin (H & E) staining and in situ hybridization (ISH)
for Epstein-Barr virus small RNAs (EBERs) (which
have been found to be expressed preponderantly in
the NPC cells but rarely in the normal cells31,32) prior
to DNA-FCM analysis. These sections were determined by one of us (J.K.C.C.) to be devoid of any
malignant cells. Moderately or well differentiated
squamous cell carcinomas (MDSCC or WDSCC) were
rare among Chinese NPC patients. No patient with
such histologic types was entered into this study.
Consecutive 5-␮m and 50-␮m biopsy sections
were cut from each block. The tumor cell ratio was
assessed from the 5-␮m H & E stained section by one
of us (J.K.C.C.). This ratio also was judged from a
consecutive 5-␮m section after being subjected to ISH
labeling for EBERs. These sections were scanned further by a Vidas microscopic imaging system (Kontron
Bildanalyze, Munich, Germany) followed by quantitative analysis in a “Medlab” computer program that
had been tailor-made for image quantitation of ISH
positive cells in the Electronic Engineering Department of Hong Kong Polytechnic University.33
Two to three 50-␮m biopsy sections were pooled
together and processed into nuclear suspension for
DNA-FCM analysis by a modified Hedley’s method.34
Briefly, the thick sections were deparaffinized twice in
8 mL of xylene for 10 minutes. They then were rehydrated in 100%, 95%, 70%, and 50% ethanol and then
in water for 10 minutes each time. Nuclear suspension
was prepared by digesting the tissues in 5 mL of 0.5%
pepsin (Sigma Chemical Company, St. Louis, MO) in
citrate buffer (pH 6.5) (Sigma Chemical Company) for
2 hours at 37 °C with constant mixing. The resultant
nuclear suspension then was filtered through 45-␮m
nylon mesh to remove cell clumps. It was stained
according to the supplier’s instructions overnight us-
2286
CANCER December 1, 1998 / Volume 83 / Number 11
ing a DNA preparation kit (Coulter Corporation, Hialeah, FL) that contained propidium iodide, Triton
X-100, and ribonuclease A.
guidelines drawn up in the DCCC. Only 51 samples
that met such stringent criteria were correlated with
the clinical outcome of the patients.
Flow Cytometric Analysis
Statistical Analysis
The DNA stained nuclear preparation was analyzed in
a Profile II flow cytometry analyzer (Coulter Corporation) that was equipped with a 15-milliwatt air-cooled
argon ion laser operating at 488 nanometers (nm) blue
light. To achieve optimal running, the flow cytometer
was precalibrated with fluorescent DNA-Check Beads
(Coulter Corporation) to obtain a percentage of halfpeak coefficient of variation (%HPCV) of ⬍ 2.0 for
either forward angle light scatter and red fluorescence
at 635 nm. Chick red blood cells, peripheral blood
lymphocytes, and cells derived from two paraffin
blocks of reactive lymph nodes served as controls to
calibrate the fluorescent channel for the normal DNA
diploid peak further. This DNA diploid peak also was
assessed from the normal cells contained in the histologically negative or NPC biopsies. A DNA aneuploid
population is defined as a DNA peak that differs from
the DNA diploid peak by at least 10% in DNA content
(i.e., with a DNA index of ⱕ 0.9 or ⱖ 1.1). Cellular
debris were excluded from analysis by gating in a
two-dimensional (2-D) plot of forward angle light
scatter against a 90° light side scatter. A large proportion of the cell clumps were excluded by a doublet gate
in a 2-D plot of integral and peak fluorescent signals.
DNA histograms of the tumor samples without debris
gating were assessed using Multicycle DNA-FCM analysis software (Phoenix Flow Systems, San Diego, CA)
to determine the S-phase fraction (SPF), G2/M-phase
fraction (G2/MF), proliferative fraction (PF) (which is
the sum of SPF and G2/MF), cellular debris level, and
%HPCV. Guideline criteria drawn up in the DNA Cytometry Consensus Conference (DCCC)35 for tumor
paraffin samples were adopted in the data analysis;
samples were excluded from correlation for prognosis
if they contained ⬍ 20% tumor cells, if the DNA histogram had a %HPCV ⬎ 8.0%, or if cell count was ⬍
10,000. Of the 119 histologically positive biopsies analyzed from 110 patients, 9 patients required repeated
biopsies for confirmation of diagnosis. Although later
confirmed to be histologically positive by reviewing
the H & E stained slides, five of the nine initial biopsies
from these patients had an insufficient tumor cell ratio
for DNA analysis, one had insufficient total cell
counts, one had unacceptably high %HPCV, and two
had an unacceptably high content of cellular debris,
most likely due to severe necrosis in the tumors. These
9 biopsies in conjunction with another 59 from the
remaining 110 biopsies (i.e., a total of 68 biopsies)
were excluded from further analysis according to the
PI in the two histologic types and in different clinical
stages and sizes of metastatic lymph nodes (LNs) of
NPC patients were compared by the Mann-Whitney U
test36 whereas the DNA ploidy state in these groups
were compared by the Chi-Square (␹2) test.39 The PI
between tumor positive and negative biopsies and
between each of the three clinical groups (the distant
metastasis [MET] group, the locoregional recurrence
[LR-RC] group, and the complete remission [REM]
group) also were compared by the Mann-Whitney U
test. Survival analysis in patients with different DNA
ploidy states or PI was performed using the KaplanMeier method38 and significant differences between
these groups were analyzed by the log rank test.39
RESULTS
DNA-FCM parameters (namely PF, SPF, G2/MF, and
DNA ploidy state) in 51 histologically positive biopsies
that met the stringent criteria of the DCCC37 were
correlated with tumor histologic types, patients’ clinical stages, and sizes of the NPC metastatic LNs in the
neck (Table 1). No significant difference was found in
these parameters between the two histologic types
(UCNT and PDSCC), Ho’s or UICC stages, and size of
the metastatic LNs. Only DNA aneuploid tumors had a
significantly higher SPF than the diploid tumors (P ⫽
0.01). Although there was a gradual increase of DNA
aneuploidy frequency from UICC Stage I-IV, this relation was not statistically significant.
The three PI in NPC biopsies were compared with
those in the histologically negative biopsies. The PF,
SPF, and G2/MF of NPC biopsies (mean values: 12.4%,
7.8%, and 4.6%, respectively) were all significantly
higher than those of the negative biopsies (mean values: 5.8%, 3.7%, and 2.1%, respectively) (P ⬍ 0.000004,
0.0003, and 0.004, respectively) (Fig. 1).
However, the PI of NPC biopsies showed a wide
scattering, with values overlapping with those of negative biopsies. The PI then were correlated with the
clinical outcome by categorizing the patients into
three groups. Groups 1 (MET) and 2 (LR-RC) were
comprised of patients who developed distant metastases and locoregional recurrence, respectively, after
RT, whereas Group 3 (REM) was comprised of those
patients who were in complete remission after treatment. Patients in the REM group generally had low
tumor PI (8.2%, 5.7%, and 2.7% for mean PF, SPF, and
G2/MF, respectively) (Fig. 2). In contrast, the patients
in the 2 recurrence groups (MET and LR-RC) all had
Prognostic Role of DNA-FCM in NPC/Yip et al.
2287
TABLE 1
Correlation of Proliferative Indices and DNA Ploidy States with Certain Clinical Parameters in NPC Patients
Clinical parameters
Histology
UCNT
PDSCC
Ho’s stage
I
II
III
IV
UICC stage
I
II
III
IV
Metastatic LN size (cm)
0–6
7–9
⬎9
DNA ploidy
DP
AN
No. of
patients
Mean PF
(%) ⴞ SD
Mean SPF
(%) ⴞ SD
Mean G2/MF
(%) ⴞ SD
DNA ploidy
ratio (AN/DP)
23a
24a
12.1 ⫾ 6.7
13.2 ⫾ 6.0
7.0 ⫾ 4.3
9.0 ⫾ 5.9
5.2 ⫾ 5.2
4.3 ⫾ 2.6
3/20
5/19
8
11
18
14
10.4
10.4
14.3
12.7
5.3 ⫾ 1.4
6.7 ⫾ 4.2
10.2 ⫾ 6.9
7.0 ⫾ 2.9
5.1
3.7
4.2
5.6
⫾ 3.1
⫾ 3.5
⫾ 2.7
⫾ 5.9
1/7
0/11
4/14
3/11
8
0
15
28
10.4 ⫾ 4.1
0
13.8 ⫾ 7.0
12.3 ⫾ 6.3
5.3 ⫾ 1.4
0
9.8 ⫾ 7.0
7.5 ⫾ 4.2
5.1 ⫾ 3.1
0
4.0 ⫾ 4.3
4.8 ⫾ 4.1
1/7
0
2/13
5/23
35
8
8
12.1 ⫾ 5.4
12.6 ⫾ 7.6
13.3 ⫾ 8.5
7.7 ⫾ 4.9
9.2 ⫾ 7.5
7.2 ⫾ 2.5
4.5 ⫾ 3.5
3.4 ⫾ 3.6
6.4 ⫾ 6.1
5/30
2/6
1/7
43
8
11.5 ⫾ 5.6
17.1 ⫾ 7.5
6.7b ⫾ 3.4
13.8b ⫾ 8.2 (bDP/AN P ⫽ 0.01)
4.8 ⫾ 4.2
3.3 ⫾ 2.8
/
/
⫾ 4.1
⫾ 4.5
⫾ 6.5
⫾ 7.5
NPC: nasopharyngeal carcinoma; PF: proliferative fractions; SD: standard deviation; SPF: S-phase fraction; G2/MF: G2/M-phase fraction; AN: aneuploid; DP: diploid; UCNT: undifferentiated carcinoma of the
nasopharyngeal type; PDSCC: poorly differentiated squamous cell carcinoma; UICC: International Union Against Cancer; LN: lymph node.
Proliferative fractions, S-phase fraction, and G2/M-phase fraction in different clinical groups were compared using the Mann–Whitney U test.
a
Of 51 samples analyzed, 23 were undifferentiated carcinoma of the nasopharyngeal type, 24 were poorly differentiated squamous cell carcinoma, and the remaining 4 cases were reported as anaplastic carcinomas
(2 cases) and nonkeratinized carcinomas (2 cases); these 4 cases were excluded in the statistical analysis because of their small number.
b
No statistically significant difference was found in any of the groups with the exception of the S-phase fraction of DNA diploid and aneuploid tumors (P ⫽ 0.01). DNA ploidy states in different clinical groups were
compared using the chi-square test. No statistically significant difference was found in any of the groups analyzed.
significantly higher PF (16.4% and 15.1%, respectively)
than those in the REM group (P ⫽ 0.0005 and 0.004,
respectively). A higher tumor proliferation rate in the
two recurrence groups also was demonstrated by the
higher SPF and G2/MF, although a significant difference was found only between the SPF in the MET and
REM groups (P ⫽ 0.005). Setting a cutoff value of ⬎
11% PF for rapidly proliferating tumors, 78% (18 of 23)
of the tumors in the MET and LR-RC groups were
proliferating rapidly whereas only 26.3% (5 of 19) of
the REM group were fast-growing tumors (Table 2). If
the cutoff value was set at 7% SPF, 61% of the patients
in the 2 recurrence groups had rapidly proliferating
tumors compared with 26.3% in the REM group. Furthermore, patients with recurrence also had a higher
frequency of DNA aneuploid tumors (37.5% and 27%,
respectively) than those with complete remission
(5.2%) (Fig. 3).
All three PI (PF, SPF, and G2/MF) were correlated strongly with the survival rate of NPC patients.
This was demonstrated by the significantly lower
12-year survival rates in patients with high PF, SPF,
and G2/MF (35%, 26%, and 34%, respectively) compared with those with low PF, SPF, and G2/MF (77%,
67%, and 65%, respectively) (p ⬍ 0.0009, ⬍ 0.004,
and 0.004, respectively, by the log rank test) (Fig. 4).
Furthermore, patients with DNA aneuploid tumors
also had a significantly lower 12-year survival rate
(28%) than patients with DNA diploid tumors (59%)
(P ⬍ 0.01) (Fig. 4). The results of this retrospective
analysis demonstrated the importance of tumor PI
and ploidy states in determining the outcome of
NPC patients after treatment.
DISCUSSION
Previous studies have shown the applicability of the
DNA-FCM technique in the diagnosis and prognosis
of tumors.17,18 Various parameters such as DNA ploidy
states and PI (such as SPF and PF) have been found to
correlate with the survival and recurrence rate for
various tumors including carcinomas of the breast,
colon, rectum, lung, bladder, ovary, cervix, and the
head and neck.19 –25 In the current study, the prognostic significance of DNA-FCM parameters was demonstrated in NPC patients using archival biopsy materials. The recurrence groups more frequently had DNA
aneuploid tumors or rapidly proliferating tumors
compared with the REM group. Furthermore, using
2288
CANCER December 1, 1998 / Volume 83 / Number 11
TABLE 2
Distribution of Fast Proliferating Tumors in Patients Who Developed
Distant Metastasis or Locoregional Recurrence or Who Remained in
Complete Remission after Radiation Treatment
No. of fast proliferating tumorsa/total
no. of tumors analyzed (%)
FIGURE 1. Comparison of three proliferative indices (proliferative fractions
Category of patients
PF > 11%
SPF > 7%
Locoregional recurrence
Distant metastasis
All recurrences
Remission
6/8 (75%)
12/15 (80%)
18/23 (78%)
5/19 (26.3%)
4/8 (50%)
10/15 (66.7%)
14/23 (61%)
5/19 (26.3%)
PF: proliferative fractions; SPF: S-phase fraction.
a
Fast proliferating tumors were defined arbitrarily as tumors with proliferative fractions ⬎ 11% or
S-phase fraction ⬎ 7%.
[PF], S-phase fraction [SPF], and G2/M-phase fraction [G2/MF]) in nasopharyngeal carcinoma and histologically negative biopsies. Statistical differences
in the indices of the two groups of biopsies were analyzed by the MannWhitney U test. TUM: tumor biopsies; NEG: histologically negative biopsies; SD:
standard deviation.
FIGURE 2. Comparison of three proliferative indices (proliferative fractions
[PF], S-phase fraction [SPF], and G2/M-phase fraction [G2/MF]) in biopsies from
nasopharyngeal carcinoma patients who developed distant metastasis (MET) or
locoregional recurrence (LR-RC) or who remained in complete remission (REM)
after radiation treatment. Statistical differences in the indices of the different
groups of biopsies were analyzed by the Mann-Whitney U test. SD: standard
deviation.
Kaplan-Meier analysis, DNA ploidy state, PF, SPF, and
G2/MF all were significantly correlated with the survival rate.
Advantages of DNA-FCM for Prognostication
We previously reported the prognostic role of serum
ZEBRA/IgG antibody in NPC patients.15,16 Titers of this
antibody frequently were elevated in patients who
developed metastases to the lung and liver 1– 6
months before the clinical diagnosis of distant spread
FIGURE 3.
Distribution of DNA aneuploid (AN) and diploid (DP) tumors in
nasopharyngeal carcinoma patients who developed distant metastasis (MET) or
locoregional recurrence (LR-RC) or who remained in complete remission (REM)
after radiation treatment.
of disease. Posttreatment antibody titers also were
correlated significantly with patient survival. However,
the titers frequently were not elevated in patients who
developed bone metastasis or locoregional recurrence, and only the titers tested at 10 months after RT
but not at the time of diagnosis were correlated with
survival. In contrast, the DNA-FCM parameters could
be more useful clinically because they were correlated
with patient survival at the time of diagnosis and the
PI also were elevated in a significant proportion of
Prognostic Role of DNA-FCM in NPC/Yip et al.
FIGURE 4. Survival analysis of nasopharyngeal carcinoma patients in two
ploidy states and in high and low proliferative indices (proliferative fractions
[PF], S-phase fraction [SPF], and G2/M-phase fraction [G2/MF]) using the
Kaplan-Meier method. The statistical difference was analyzed by the log rank
test. AN: DNA aneuploid tumor; DP: DNA diploid tumor.
patients with either locoregional or distant recurrence,
including some bone recurrences.
Currently, the most effective treatment modality
for NPC is RT, which conventionally is performed by
delivering 30 –35 daily fractions to the tumor at approximately 2–2.5 grays (Gy) per fraction for 5 days
per week in an overall treatment time of approximately 6 –7 weeks.7–10 Although effective, some patients still develop recurrences even after a full
course of such treatment. If the current results can
be confirmed by large-scale studies, DNA-FCM
analysis could help to select a group of patients with
poor prognosis. Intensification of therapy or adjuvant treatment schemes planned for this group
hopefully may reduce the recurrence rate and improve survival.
Intensified therapy can be achieved by a continuous hyperfractionated and accelerated radiotherapy (CHART) scheme, which aims to complete
the whole course of RT within 12 consecutive days
(rather than 6 –7 weeks as in conventional RT) by
delivering 3 fractions of 1.5 Gy per day,40,41 or an
European Organization for Research and Treatment
of Cancer (EORTC) hyperfractionated and accelerated
radiotherapy scheme, which gives 3 daily fractionated
doses of 1.6 Gy each for the first week, followed by a
1-week rest gap and a further 2 weeks of similar treat-
2289
ment.42– 44 Both schemes intend to shorten the overall
treatment time to combat the accelerated proliferation rate of tumors during the treatment period.45
Previous analysis has shown that the EORTC accelerated scheme could result in ⬎ 60% of local control in
head and neck tumors with a rapid tumor growth rate
compared with the conventional scheme, which resulted in ⬍ 30% local control.44 The tumor control
rates for the two RT schemes in patients with slowgrowing tumors do not differ from each other significantly. A recent report also confirmed that the accelerated fractionation arm resulted in a 13% gain in
locoregional control over the conventional fractionation arm.44
These accelerated schemes potentially can be useful for NPC patients with rapid tumor growth rate.
However, they are costly, labor-intensive, and require
clinical and allied health staff continuously working
overtime. Thus it is more cost-effective to reserve
these treatment schemes for the poor prognostic
group. Furthermore, both acute and late toxicity are
more severe in patients treated by these stringent
treatment schemes. A selection using the DNA-FCM
technique perhaps could spare the good prognostic
group with slowly proliferating tumors from unnecessary side effects induced by these schemes. Furthermore, modification of the protocols in the future such
as reducing the 3 daily fractions to 2 with an even
longer interfractional period than the 6 hours currently used plus adding booster doses in small irradiation fields also may help to reduce the toxicity.
Despite earlier nonrandomized trials showing a
higher complete response rate and/or improved survival in NPC patients with adjuvant chemotherapy
using platinum regimens compared with historic controls,46 – 49 subsequent prospective randomized trials50,51 (except one52) did not show a survival benefit.
Recent concomitant chemoradiotherapy trials also
have shown some benefit in improving survival in
patients with advanced NPC.53,54 If the benefits can be
confirmed in the future by larger randomized trials,
the group selected by DNA-FCM to have a poor prognosis also can be given this treatment in the hope of
reducing the frequency of distant recurrence and improving survival. However, further study regarding
how to fit the chemotherapy within the radiotherapy
scheme to prevent unacceptable toxicity and the issue
of adding agents to reduce the specific types of toxicity
that inevitably occur with such a combination also
should be considered.
DNA-FCM and Clinical Stages
DNA-FCM and the prior ZEBRA/IgG antibody studies
were performed in the same series of NPC patients.
2290
CANCER December 1, 1998 / Volume 83 / Number 11
ZEBRA/IgG appeared to be tumor load-associated because the antibody titers were correlated with clinical
stage and the size of the metastatic cervical LNs.1 NPC
cells contained abundant EBV. EBV load and, subsequently, ZEBRA/IgG antibody response gradually
could accumulate as the tumor mass of NPC increases
with stage or size of the metastatic LNs. However, PI
did not correlate significantly with either one of these
clinical parameters (Table 1). The tumor proliferation
rate usually starts off exponentially in the initial phase
of tumor development but gradually slows down as
the size of the primary tumor reaches a substantially
high level.55 This can be due to overgrowth of the
tumor cells over the surrounding blood vessels, impairing the diffusion of nutrients and oxygen.56,57
Comparison with Previous Studies
Flow cytometric analysis previously has been performed in Australian NPC patients.58 Unfortunately,
various PI were not reported. The investigators did not
find any significant difference in 5-year survival between the patients with DNA diploid and aneuploid
tumors. These results differ from ours, most likely due
to the difference in the histology of the tumors analyzed. In our study, all 51 Chinese NPC patients analyzed had either UCNT (n ⫽ 25) or PDSCC (n ⫽ 26).
Among the 55 Australian patients studied by Costello
et al.,58 65% had keratinizing squamous cell carcinoma (SCC) and only 35% had either UCNT or nonkeratinizing carcinoma (NKC) (NKC being equivalent
to PDSCC). SCC is substantially different from UCNT
or PDSCC (NKC) biologically. The latter two types
usually are more radio-sensitive and hence generally
have better prognosis after RT.59 In addition, the latter
more frequently express EBV DNA, RNA, and a variety
of viral latent antigens and in a greater quantity as
well.60 In flow cytometric analyses of various head and
neck tumors, PDSCC generally has a higher DNA aneuploidy rate than SCC (WDSCC or MDSCC).61– 63 Although survival analysis between the two ploidy types
also was performed in individual UCNT (i.e., lymphoepithelioma), NKC, and SCC in the Australian study,
the number of patients after further stratification was
too small for statistical significance to be achieved.
Future Approaches
Despite the prognostic significance of the DNA-FCM
study, it has certain drawbacks. Among 119 tumor
biopsies investigated in the current study, ⬎ 50% had
to be excluded from the prognostic analysis according
to the guideline criteria set in the DCCC,35 such as
exceptionally high %HPCV, scanty number of tumor
cells, or unacceptably high cellular debris. These criteria can improve the accuracy and consistency of
DNA-FCM study substantially. In this study, the major
reason for excluding the samples was insufficient tumor cells. The greatest problem with the study of NPC
is the frequent presence of large numbers of intratumoral and peritumoral lymphoid cells resulting in a
low tumor cell ratio in the biopsy samples. This problem potentially can be solved by enriching the carcinoma cells using anticytokeratin antibody or EBER
probes,32,33 which can enable more samples to be
analyzed and enhance the accuracy of DNA-FCM
analysis.
We currently are embarking on a prospective
study to increase the sample size to ⬎ 800 NPC patients so that the prognostic role of DNA-FCM parameters in determining individual clinical stage, especially the early disease stages, can be analyzed further.
Multivariate analysis also can be performed in such a
large study to compare the prognostic significance of
these parameters with other potentially useful laboratory prognostic markers. It is anticipated that in the
near future a number of laboratory prognostic indicators could be combined for the prognosis of NPC to
enable one marker to supplement the shortcomings of
the other.
REFERENCES
1.
Ho JHC. Nasopharyngeal carcinoma. Adv Cancer Res 1972;
15:57–92.
2. Huang DP. Epidemiology and aetiology. In: van Hasselt CA,
Gibb AG, editors. Nasopharyngeal carcinoma. Hong Kong:
Chinese University Press, 1991:23–35.
3. Hong Kong Cancer Registry. Cancer incidence and mortality
in Hong Kong 1992. Hong Kong: Hospital Authority Hong
Kong, 1996:12.
4. Wei WI, Sham JS, Zong YS, Choy D, Ng MH. The efficacy of
fiberoptic endoscopic examination and biopsy in the detection of early nasopharyngeal carcinoma. Cancer 1991;67:
3127–30.
5. Henle G, Henle W. Epstein-Barr virus specific IgA serum
antibody as an outstanding feature of nasopharyngeal carcinoma. Int J Cancer 1976;17:1–7.
6. Ho JHC, Ng MH, Kwan HC, Chau JCW. Epstein-Barr virus
specific IgA and IgG serum antibodies in nasopharyngeal
carcinoma. Br J Cancer 1976;34:655–9.
7. Ho JHC, Lau WH, Fong M. Treatment of nasopharyngeal
carcinoma (NPC). In: Grundmann E, Krueger GRF, Ablashi
DV, editors. Nasopharyngeal carcinoma, cancer campaign.
Volume 5. New York: Gustav Fischer Verlag, 1981:279 – 85.
8. Ho JHC. Nasopharynx. In: Halnan KE, Boak JL, Crowther D,
von Essen CE, Orr JS, Peckham MJ, editors. Treatment of
cancer. London: Chapman and Hall, 1982:249 – 67.
9. Mesic JB, Fletcher GH, Geopfert H. Megavoltage irradiation
of epithelial tumors of the nasopharynx. Int J Radiat Oncol
Biol Phys 1981;7:447–53.
10. Huang SC, Chu GL. Nasopharyngeal cancer: study II. Int J
Radiat Oncol Biol Phys 1981;7:713– 6.
Prognostic Role of DNA-FCM in NPC/Yip et al.
11. Ho JHC. Stage classification of nasopharyngeal carcinoma: a
review. In: De-The G, Ito Y, editors. Nasopharyngeal carcinoma: etiology and control. Lyon: International Agency for
Research on Cancer Science Publication, 1978:99 –114.
12. Ho JHC. Stage classification of nasopharyngeal carcinoma: a
review. In: De-The G, Ito Y, eds. Nasopharyngeal carcinoma:
etiology and control. Lyon: International Agency for Research on Cancer Science Publication, 1978:99 –114.
13. Teo PM, Leung SF, Yu P, Tsao SY, Foo W, Shiu W. A comparison of Ho’s, International Union Against Cancer and
American Joint Committee stage classifications for nasopharyngeal carcinoma. Cancer 1991;67:434 –9.
14. Lee AWM, Poon YF, Foo W, Law SCK, Cheung FK, Chan
DKK, et al. Retrospective analysis of 5037 patients with
nasopharyngeal carcinoma treated during 1976 –1985: overall survival and patterns of failure. Int J Radiat Oncol Biol
Phys 1992;23:261–70.
15. Yip TTC, Ngan RKC, Lau WH, Poon YF, Joab I, Cochet C, et
al. A possible prognostic role of immunoglobulin-G antibody against recombinant Epstein-Barr virus BZLF-1 transactivator protein ZEBRA in patients with nasopharyngeal
carcinoma. Cancer 1994;74:2414 –24.
16. Yip TTC, Lau WH, Ngan RKC, Poon YF, Joab I, Cochet C, et
al. Role of Epstein-Barr virus serology in the prognosis of
nasopharyngeal carcinoma: the present and the future. Epstein-Barr Virus Rep 1996;3:25–33.
17. Merkel DE, Dressler LG, McGuire WL. Flow cytometry, cellular DNA content and prognosis in human malignancy.
J Clin Oncol 1987;5:1690 –703.
18. Merkel DE, McGuire WL. Ploidy, proliferative activity and
prognosis: DNA flow cytometry of solid tumors. Cancer
1990;65:1194 –205.
19. Witzig TE, Ingle JN, Schaid DJ, Wold LE, Barlow JF, Gonochoroff NJ, et al. DNA ploidy and percent S phase as prognostic factors in node-positive breast cancer: results from
patients enrolled in two prospective randomized trials.
J Clin Oncol 1993;11:351–9.
20. Ensley JF, Maciorowski Z. Clinical applications of DNA content parameters in patients with squamous cell carcinomas
of the head and neck. Semin Oncol 1994;21:330 –9.
21. Jones DJ, Moore M, Schofield PF. Refining the prognostic
significance of DNA ploidy status in colorectal cancer: a
prospective flow cytometric study. Int J Cancer 1988;41:206 –
10.
22. Isobe H, Miyamoto H, Shimizu T, Haneda H, Hashimoto M,
Inoue K, et al. Prognostic and therapeutic significance of the
flow cytometric nuclear DNA content in non-small cell carcinoma. Cancer 1990;65:1391–5.
23. Lopez-Beltran A, Croghan GA, Croghan I, Matilla A, Gaeta
JF. Prognostic factors in bladder cancer: a pathologic, immunohistochemical and DNA flow-cytometric study. Am J
Clin Pathol 1994;102:109 –14.
24. Braly PS, Klevecz RR. Flow cytometric evaluation of ovarian
cancer. Cancer 1993;71:1621– 8.
25. Lutgens LCHW, Schutte B, Jong JMA, Thunnissen FBJM.
DNA content as prognostic factor in cervix carcinoma stage
IB-III treated with radiotherapy. Gynecol Oncol 1994;54:275–
81.
26. Lee AW, Foo W, Poon YF, Lew CK, Chan DK, O SK, et al.
Staging of nasopharyngeal carcinoma: evaluation of N-staging by Ho and UICC/AJCC systems. Union Internationale
Contre le Cancer, American Joint Committee for Cancer.
Clin Oncol (R Coll Radiol) 1996;8:146 –54.
27. Shanmugaratnam K, Chan SH, de-The G, Goh JEH, Khor TH,
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
2291
Simons MJ, et al. Histopathology of nasopharyngeal carcinoma. Correlations with epidemiology, survival rates and
other biologic characteristics. Cancer 1979;44:1029 – 44.
Krueger GRF, Kottaridis SD, Wolf H, Ablashi DV, Sesterhenn
K, Bertram G. Histological types of nasopharyngeal carcinomas as compared to EBV serology. Anticancer Res 1981;1:
187–94.
Hsu HC, Chen CL, Hsu MM, Lynn TC, Tu SM, Huang SC.
Pathology of nasopharyngeal carcinoma. Proposal of a new
histologic classification correlated with prognosis. Cancer
1987;59:945–51.
McGuire LJ, Lee JCK. The histopathologic diagnosis of nasopharyngeal carcinoma. Ear Nose Throat J 1990;69:229 –36.
Wu TC, Mann RB, Epstein JI, MacMahon E, Lee WA,
Charache P, et al. Abundant expression of EBER1 small
nuclear RNA in nasopharyngeal carcinoma: a morphologically distinctive target for detection of Epstein-Barr virus in
formalin-fixed paraffin-embedded carcinoma specimens.
Am J Pathol 1991;138:1461–9.
Yip TTC, Chan JKC, Poon YF, Lau WH, Ngan RKC, Ma VWS.
Usefulness of in situ hybridization (ISH) for Epstein-Barr
virus encoded RNAs (EBERs) in assisting the histopathological diagnosis of nasopharyngeal carcinoma (NPC) [abstract]. Int J Surg Pathol 1995;2:259.
Yip TTC, Chan JKC, Poon YF, Lau WH, Ngan RKC, Ma VWS.
Delineation of various factors affecting the sensitivity and
specificity of in situ hybridization (ISH) for Epstein-Barr
virus encoded RNAs (EBERs) [abstract]. Int J Surg Pathol
1995;2:381.
Hedley DW. Flow cytometry using paraffin-embedded tissue: five years on. Cytometry 1989;10:229 – 41.
Shankey TV, Rabinovitch PS, Bagwell B, Bauer KD, Duque
RE, Hedley DW, et al. Guideline for implementation of clinical DNA cytometry. Cytometry 1993;14:472–7.
Whitney DR. A bivariate extension of the U statistic. Ann
Math Stat 1951;22:274 – 82.
Samuels ML. Statistics for the life sciences, 1st edition. San
Francisco and London; Collier Macmillan Publishers, 1989.
Breslow N. Statistical methods for censored survival data.
Environ Health Perspective 1979;32:182–92.
Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard
SV, et al. Design and analysis of randomized clinical trials
requiring prolonged observation of each patient. Br J Cancer
1977;35:1–39.
Saunders MI, Dische S, Hong A, Grosch EJ, Fermont DC,
Ashford RF, et al. Continuous hyperfractionated accelerated
radiotherapy in locally advanced carcinoma of the head and
neck region. Int J Radiat Oncol Biol Phys 1989;17:1287–93.
Saunders MI, Dische S. Continuous, hyperfractionated, accelerated radiotherapy (CHART) in non small-cell carcinoma of the bronchus. Int J Radiat Oncol Biol Phys 1990;19:
1211–5.
Begg AC, Hofland I, Van Glabekke M, Bartlelink H, Horiot
JC. Predictive value of potential doubling time for radiotherapy of head and neck tumour patients: results from
the EORTC Cooperative Trial 22851. Semin Radiat Oncol
1992;2:22–5.
Horiot JC, Bontemps P, Begg AC, Le Fur R, van den Bogaert
W, Bolla M, et al. Hyperfractionated and accelerated radiotherapy in head and neck cancers: results of the EORTC
trials and impact on clinical practice. Bull Cancer Radiother
1996;83:314 –20.
2292
CANCER December 1, 1998 / Volume 83 / Number 11
44. Horiot JC, Bontemps P, van den Bogaert W, Le Fur R, van
den Weijngaert D, Bolla M, et al. Accelerated fractionation
(AF) compared to conventional fractionation (CF) improves
loco-regional control in the radiotherapy of advanced head
and neck cancers: results of the EORTC 22851 randomized
trial. Radiother Oncol 1997;44:111–21.
45. Hall EJ, editor. Radiobiology for the radiologist. 4th edition.
Philadelphia: J.B. Lippincott Co., 1994:211–30.
46. Azli N, Armand JP, Rahal M, Wibault P, Boussen H, Eschwege F, et al. Alternating chemo-radiotherapy with cisplatin and 5-fluorouracil plus bleomycin by continuous infusion for locally advanced undifferentiated carcinoma of
the nasopharyngeal type. Eur J Cancer 1992;28A:1792–7.
47. Tsujii H, Kamada T, Tsuji H, Takamura A, Matsuoka Y,
Usubuchi H, et al. Improved results in the treatment of
nasopharyngeal carcinoma using combined radiotherapy
and chemotherapy. Cancer 1989;63:1668 –72.
48. Dimery IW, Peters LJ, Goepfert H, Morrison WH, Byers RM,
Guillory C, et al. Effectiveness of combined induction chemotherapy and radiotherapy in advanced nasopharyngeal
carcinoma. J Clin Oncol 1993;11:1919 –28.
49. Al-Sarraf M, Pajak TF, Cooper JS, Mohiuddin M, Herskovic
A, Ager PJ. Chemo-radiotherapy in patients with locally advanced nasopharyngeal carcinoma: a Radiation Therapy
Oncology Group Study. J Clin Oncol 1990;8:1342–51.
50. Chan ATC, Teo PML, Leung WT, Leung TWT, Leung SF, Lee
WY, et al. A prospective randomized study of chemotherapy
adjunctive to definitive radiotherapy in advanced nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys 1995;33:569 –77.
51. Rossi A, Molinari R, Boracchi P, Del Vechio M, Marubini E,
Nava M, et al. Adjuvant chemotherapy with vincristine, cyclophosphamide, and doxorubicin after radiotherapy in local-regional nasopharyngeal cancer: results of a 4-year multicentre randomized study. J Clin Oncol 1988;6:1401–10.
52. Cvitkovic E. Neoadjuvant chemotherapy with epirubicin,
cisplatin, bleomycin (BEC) in undifferentiated nasopharyngeal cancer: preliminary results of an international phase III
trial. Proc Am Soc Clin Oncol 1994;13:283.
53. Cheng SH, Liu TW, Jian JJ, Tsai SY, Hao SP, Huang CH, et al.
Concomitant chemotherapy and radiotherapy for locally
advanced nasopharyngeal carcinoma. Cancer J Sci Am 1997;
3:100 – 6.
54. Lin JC, Jan JS, Hsu CY. Pilot study of concurrent chemotherapy and radiotherapy for stage IV nasopharyngeal cancer.
Am J Clin Oncol 1997;20:6 –10.
55. Steel GG. Tumor growth kinetics. In: Steel GG, Adams GE,
Horwich A, editors. The biological basis of radiotherapy. 3rd
edition. Amsterdam: Elsevier Science Publishers, 1995:8 –13.
56. Tannock IF. The relation between cell proliferation and the
vascular system in a transplanted mouse mammary tumor.
Br J Cancer 1968;22:258 –73.
57. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and
nutrient supply, and metabolic micro-environment of human tumors: a review. Cancer Res 1989;49:6449 – 65.
58. Costello F, Mason BR, Collins RJ, Kearsley JH. A clinical and
flow cytometric analysis of patients with nasopharyngeal
cancer. Cancer 1990;66:1789 –95.
59. Reddy SP, Raslan WF, Gooneratne S, Kathuria S, Marks JE.
Prognostic significance of keratinization in nasopharyngeal
carcinoma. Am J Otolaryngol 1995;16:103– 8.
60. Liebowitz D. Nasopharyngeal carcinoma: the Epstein-Barr
virus association. Semin Oncol 1994;21:376 – 81.
61. Kokal WA, Gardine RL, Sheibani K, Zak IW, Beatty JD, Riihimaki DU, et al. Tumor DNA content as a prognostic indicator in squamous cell carcinoma of the head and neck region.
Am J Surg 1988;156:276 – 80.
62. Hemmer J, Kriedler J. Flow cytometric DNA ploidy analysis
of squamous cell carcinoma of the oral cavity. Comparison
with clinical staging and histologic grading. Cancer 1990;66:
317–20.
63. Rua S, Comino A, Fruttero A, Cera G, Semeria C, Lanzillotta
L, et al. Relationship between histologic features, DNA flow
cytometry, and clinical behavior of squamous cell carcinomas of the larynx. Cancer 1991;67:141–9.
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