U.S. patent application number 10/455042 was filed with the patent office on 2004-01-08 for circulating epstein-barr virus dna in the serum or plasma of patients for the prediction and detection of epstein-barr virus associated cancers apart from head, neck and lymphoid malignancies.
This patent application is currently assigned to The Chinese University of Hong Kong. Invention is credited to Chan, Wing Yee, Lo, Yuk Ming Dennis, Ng, Kwok Wai.
Application Number | 20040005551 10/455042 |
Document ID | / |
Family ID | 23010993 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040005551 |
Kind Code |
A1 |
Lo, Yuk Ming Dennis ; et
al. |
January 8, 2004 |
Circulating epstein-barr virus DNA in the serum or plasma of
patients for the prediction and detection of epstein-barr virus
associated cancers apart from head, neck and lymphoid
malignancies
Abstract
The present invention features methods for diagnosing,
detecting, monitoring and determining the prognosis of Epstein Barr
virus associated cancers apart from head, neck and lymphoid
malignancies in a patient by detecting or measuring EBV DNA present
in the serum or plasma of the patient. The present invention also
features diagnostic kits comprising suitable reagents for detecting
EBV DNA in the serum or plasma of a patient.
Inventors: |
Lo, Yuk Ming Dennis;
(Kowloon, HK) ; Chan, Wing Yee; (Tuen Mun, HK)
; Ng, Kwok Wai; (Tai Po, HK) |
Correspondence
Address: |
Research & Technology Administration Office
The Chinese University of Hong Kong
Room 226
Pui Chiu Building
Shatin
HK
|
Assignee: |
The Chinese University of Hong
Kong
Shatin
HK
|
Family ID: |
23010993 |
Appl. No.: |
10/455042 |
Filed: |
June 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10455042 |
Jun 3, 2003 |
|
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10057579 |
Jan 25, 2002 |
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60265568 |
Jan 31, 2001 |
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Current U.S.
Class: |
435/5 ;
435/6.12 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 1/705 20130101 |
Class at
Publication: |
435/5 ;
435/6 |
International
Class: |
C12Q 001/70; C12Q
001/68 |
Claims
What is claimed is:
1. A method of determining the increased probability of a patient
with increased Epstein Barr virus DNA in blood for the prognosis
and diagnosis of Epstein Barr virus associated cancers apart from
head, neck and lymphoid malignancies by: (a) obtaining a sample of
non-cellular blood-derived fluid from the patient; and (b) assaying
the fluid for the presence or absence of Epstein Barr virus where
the presence of the virus is an indication of increased probability
of the patient to have Epstein Barr virus associated cancers apart
from head, neck and lymphoid malignancies.
2. A method of claim 1 where the patient can be diagnosed with
Epstein Barr virus associated cancers apart from head, neck and
lymphoid malignancies and the cancer cells are free of EBV nucleic
acid.
3. A method of claim 1 where the patient can be diagnosed with
Epstein Barr virus associated cancers apart from head, neck and
lymphoid malignancies and the cancer cells contain EBV nucleic
acid.
4. The method of claim 1 comprising the steps of: (1) obtaining a
blood sample from a patient; (2) obtaining a fluid fraction from
the blood sample; (3) extracting DNA from the fluid fraction; and
(4) measuring the amount of circulating EBV DNA present in the
fluid fraction.
5. The method of claim 4 further comprising the step of: (5)
comparing the amount of circulating EBV DNA present in the fluid
fraction to a control.
6. A kit for determining the increased probability of a patient
with increase EBV DNA in blood for the prognosis and diagnosis of
Epstein Barr virus associated cancers apart from head, neck and
lymphoid malignancies. (a) nucleic acid for detecting Epstein Barr
virus in the blood of patients suffering from; and (b) instructions
for use of the nucleic acid to determine the presence or absence of
Epstein Barr virus and an explanation of the increased probability
of the patient suffering from Epstein Barr virus associated cancers
apart from head, neck and lymphoid malignancies.
7. A diagnostic kit for detecting EBV DNA in the serum or plasma of
a patient comprising reagents suitable for detecting EBV DNA in the
serum or plasma.
8. A diagnostic kit according to claim 6 comprising a device for
obtaining a blood sample from a patient.
9. A diagnostic kit according to claim 6 comprising a means to
separate EBV DNA from a blood sample.
10. A diagnostic kit according to claim 6 comprising a means to
quantify the amount of EBV DNA present in the blood sample.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the discovery that Epstein Barr
virus may be found in the cell free fluid of a patient's blood and
when such virus is found, the patients may be suffering from
Epstein Barr virus associated cancers apart from head, neck and
lymphoid malignancies.
BACKGROUND OF THE INVENTION
[0002] It is known that tumour-derived DNA can be released by
cancer cells of a variety of tumours (Anker et al., Cancer
Metastasis Rev. 18: 65-73 (1999)). Examples include oncogene
mutations from pancreatic carcinoma (Anker et al.,
Gastroenterology. 112: 4-1120 (1997)), microsatellite alterations
in lung cancer (Chen et al., Nature Medicine. 2: 3-1035 (1996)) and
epigenetic changes from liver cancer (Wong et al., Cancer Res. 59:
3 (1999)). In addition, virus DNA has been found in the circulation
of a number of cancers known to be associated with virus infection.
Examples include Epstein-Barr virus (EBV) DNA from nasopharyngeal
cancer (Mutirangura et al., Clin Cancer Res. 4: 665-9 (1998); Lo et
al., Cancer Res. 59: 1188-91 (1999)) and certain lymphomas (Lei et
al., Br J Haematol. 111: 239-46 (2000); Gallagher et al., Int J
Cancer. 84: 442-8 (1999); Drouet et al., J Med Virol. 57: 383-9
(1999)), and human papillomavirus DNA from head and neck cancer
(Capone et al., Clin Cancer Res. 6: 4171-5 (2000)).
[0003] Recently, much interest has been focused on the presence of
tumor-derived DNA in the plasma and serum of cancer patients (Chen,
X. Q. et al., Nat. Med., 2: 1033-1035 (1996); Nawroz, H. et al.,
Nat. Med., 2: 1035-1037 (1996)). For virally-associated cancers,
cell-free tumor-associated viral DNA has been detected in the
plasma and serum of patients (Mutirangura, A. et al., Cancer Res.,
4: 665-669 (1998); Lo, Y. M. D. et al., Clin. Cancer Res. 59:
1188-1191 (1999); Capone, R. B. Clin. Cancer Res., 6: 4171-4175
(2000)). One important virus which has been associated with many
types of malignancy is the Epstein-Barr virus (EBV) (Cohen, J. I.
N. Engl. J. Med., 343: 481-492 (2000)). Epstein-Barr virus (EBV) is
a human herpesvirus that infects the majority of the human
population. EBV is commonly transmitted by saliva and established
latent infection in B lymphocytes where it persists for the
lifetime of the host. In this regard, circulating EBV DNA has been
detected in the plasma and serum of patients with nasopharyngeal
carcinoma (NPC) (Mutirangura, A. et al., Cancer Res., 4: 665-669
(1998); Lo, Y. M. D. et al., Clin. Cancer Res., 59: 1188-1191
(1999)) and certain lymphoid malignancies (Lei, K. I. et al., Br.
J. Haematol., 111: 239-246 (2000); Drouet, E. et al., J. Med.
Virol., 57: 383-389 (1999); Gallagher, A. et al., Int. J. Cancer,
84: 442-448 (1999)).
[0004] EBV infection has also been reported to be associated with a
proportion of gastric carcinomas (Shibata, D. et al., Am. J.
Pathol., 140: 769-774 (1992)). In Hong Kong, approximately 10% of
gastric carcinoma cases have been found to be associated with EBV
infection (Yuen, S. T. et al., Am. J. Surg. Pathol., 18: 1158-1163
(1994)).
[0005] The present invention provides methods for detecting EBV DNA
in the sera of patients with EBV associated cancers apart from
head, neck and lymphoid malignancies and correlating the amount of
EBV DNA so detected into clinical diagnosis or prognosis.
BRIEF SUMMARY OF THE INVENTION
[0006] In a first aspect, the present invention features methods
for diagnosing, detecting, monitoring and determining the prognosis
of EBV associated cancers apart from head, neck and lymphoid
malignancies in a patient. The methods feature detecting or
determining the amount of Epstein Barr Virus DNA (EBV DNA) present
in the serum or plasma of such patients. Accordingly, the present
invention have broad applicability in clinical medicine especially
latent EBV infection occur over 90% of some populations.
[0007] The methods according to the present invention are also
applicable for diagnosing, detecting, monitoring and determining
the prognosis of EBV associated cancers apart from head, neck and
lymphoid malignancies, such as gastric, breast, liver, lung and
colon cancers. These neoplasms have been associated with EBV
infection.
[0008] The methods according to the present invention generally
comprise the steps of (1) obtaining a blood sample from a patient,
(2) extracting DNA from the blood sample, (3) measuring the amount
of circulating EBV DNA present in the blood sample, and (4)
comparing the amount of circulating EBV DNA present in the blood
sample to a control.
[0009] Preferably, the blood sample is a non-cellular fluid sample.
By non-cellular we mean that the sample is either blood sera where
the cells are extracted by clotting and separation of the cells
from the remaining fluid or by inhibiting clotting and centrifuging
the fluid fraction (plasma). The EBV DNA is measured from the fluid
fraction. When EBV is found in the fluid of a non-cellular sample,
it is understood that the infection is active and infected cells
releasing EBV.
[0010] In a second aspect, the present invention features
diagnostic kits comprising suitable reagents for detecting EBV DNA
in the serum or plasma of patients. The kits according to the
present invention may further comprise one or more of a device for
obtaining a blood sample from a patient, a means to separate the
EBV DNA from the blood sample and a means to quantify the amount of
EBV DNA present in the blood sample. Such kits are useful for
diagnosing, detecting, monitoring and determining the prognosis for
EBV associated cancers apart from head, neck and lymphoid
malignancies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1A depicts gastric adenocarcinoma with small EBV
encoded RNA (EBER)-positive tumor cells. EBER in-situ
hybridization, .times.200 magnification. FIG. 1B depicts gastric
adenocarcinoma with occasional EBER-positive tumor infiltrating
lymphocytes. The tumor cells are negative. EBER in-situ
hybridization, .times.400 magnification.
[0012] FIG. 2 illustrates the difference in the level of
circulating EBV DNA amongst three patient groups. Serum EBV DNA was
detected in every one of the EBER-positive cases (median serum EBV
DNA concentration: 1063 copies/mL; interquartile range: 485-5141
copies/mL). No serum EBV DNA was detected in any of the 32 negative
cases. Thirteen out of the 14 cases (93%) demonstrating
`background` EBER positivity had detectable serum EBV DNA. These
cases had an intermediate median serum EBV DNA concentration of 50
copies/mL (interquartile range: 42 to 98 copies/mL).
[0013] FIG. 3 demonstrates a comparison between cases with
detectable serum EBV DNA in the gastric carcinoma cases with
`background` EBER-positivity, gastritis cases and control subjects.
The serum EBV DNA concentrations of these three groups are
presented. There is no statistically significant difference in
circulating EBV DNA levels amongst these three groups
(Kruskal-Wallis test, p=0.296).
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention features methods for diagnosing,
detecting, monitoring and determining the prognosis of EBV
associated cancers apart from head, neck and lymphoid malignancies
in a patient. The methods feature detecting or determining the
amount of EBV DNA present in the serum of these patients. The
methods according to the present invention have broad applicability
in clinical medicine.
[0015] Clinically, circulating EBV DNA is applicable in diagnosing
and monitoring gastric carcinoma patients who have EBER-positive
tumors, similar to what has been achieved for nasopharyngeal
cancers (Lo, Y. M. D. et al., Clin. Cancer Res., 59: 1188-1191
(1999); Lo, Y. M. D. et al., Cancer Res., 59: 5452-5455 (1999)) and
certain lymphomas (Lei, K. I. et al., Br. J. Haematol., 111:
239-246 (2000); Drouet, E. et al., J. Med. Virol., 57: 383-389
(1999); Gallagher, A. et al., Int. J. Cancer, 84: 442-448 (1999)).
The recent demonstration of the prognostic significance of
circulating EBV DNA in nasopharyngeal cancers (Lo, Y. M. D. et al.,
Cancer Res., 60: 6878-6881) suggests that EBV DNA measurement has
prognostic importance for gastric carcinoma.
[0016] The methods according to the present invention are also
applicable for detecting, monitoring and determining the prognosis
of EBV associated cancers apart from head, neck and lymphoid
malignancies where those cancers are associated with EBV. Some of
these neoplasms have been shown previously to be associated with
EBV infection (Bonnet et al., J Natl Cancer Inst. 91: 1376-81
(1999)), as have certain liver cancers (Sugawara et al., Virology.
256: 196-202 (1999)).
[0017] Any of the conventional DNA amplification or signal
amplification methods may be used for detection of EBV DNA. In most
instances, it is desirable to amplify the target sequence using any
of several nucleic acid amplification procedures which are well
known in the art. Specifically, nucleic acid amplification is the
enzymatic synthesis of nucleic acid amplicons (copies) which
contain a sequence that is complementary to a nucleic acid sequence
being amplified. Examples of nucleic acid amplification procedures
practiced in the art include the polymerase chain reaction (PCR),
strand displacement amplification (SDA), ligase chain reaction
(LCR), and transcription-associated amplification (TAA). Nucleic
acid amplification is especially beneficial when the amount of
target sequence present in a sample is very low. By amplifying the
target sequences and detecting the amplicon synthesized, the
sensitivity of an assay can be vastly improved, since fewer target
sequences are needed at the beginning of the assay to better ensure
detection of nucleic acid in the sample belonging to the organism
or virus of interest.
[0018] Methods of nucleic acid amplification are thoroughly
described in the literature. PCR amplification, for instance, is
described by Mullis et al. in U.S. Pat. Nos. 4,683,195 Methods of
nucleic acid amplification are thoroughly described in the
literature. PCR amplification, for instance, is described by Mullis
et al. in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in
Methods in Enzymology, 155: 335-350 (1987). Examples of SDA can be
found in Walker, PCR Methods and Applications, 3: 25-30 (1993),
Walker et al. in Nucleic Acids Res., 20: 1691-1996 (1992) and Proc.
Natl Acad. Sci., 89: 392-396 (1991). LCR is described in U.S. Pat.
Nos. 5,427,930 and 5,686,272. And different TAA formats are
provided in publications such as Burg et al. in U.S. Pat. Nos.
5,437,990; Kacian et al. in U.S. Pat. Nos. 5,399,491 and 5,554,516;
and Gingeras et al. in International Application No. PCT/US87/01966
and International Publication No. WO 88/01302, and International
Application No. PCT/US88/02108 and International Publication No. WO
88/10315.
[0019] Real-time quantitative PCR is a preferred means to monitor
EBV DNA and is based on the continuous optical monitoring of the
progress of a fluorogenic PCR reaction (Heide et al. Genome Res. 6:
986-694, 1996 and Lo et al. Am J Hum. Genet. 62: 768-775, 1998). In
this system, in addition to the two amplification primers used in
conventional PCR, a dual-labeled fluorogenic hybridization probe is
also included (Livak, et al. PCR Methods Appl., 4357-362, 1995).
One fluorescent dye serves as a reporter (FAM), and its emission
spectrum is quenched by a second fluorescent dye (TAMRA). During
the extension phase of PCR, the 5' to 3' exonuclease activity of
the Taq DNA ploymerase (9) cleaves the reporter from the probe,
thus releasing it from the quencher and resulting in an increase in
fluorescence emission at 518 nm.
[0020] The methods according to the present invention generally
comprise the steps of (1) obtaining a blood sample from a patient,
(2) extracting DNA from the blood sample, (3) measuring the amount
of circulating EBV DNA present in the blood sample, and (4)
comparing the amount of circulating EBV DNA present in the blood
sample to a control. Preferably, the blood sample is centrifuged, a
fluid fraction is obtained, and the EBV DNA is measured from the
fluid fraction.
[0021] Those of skill in the art will understand that the DNA may
be extracted from a blood sample by many means known in the art.
One preferred means is using a QlAamp Blood Kit. Also, the amount
of circulating EBV DNA may be measured using one of many known or
novel protocols. A protocol comprising a real time PCR
amplification system is particularly preferred. Standard procedures
for comparing the levels of EBV DNA so detected to a control may
easily be devised so as to statistically assess the significance of
the values obtained.
[0022] The number of copies of EBV DNA may be measured over time
and correlated to disease progression or regression. Thereby, the
present invention provides a non-invasive method that allows
diagnosis and subsequent monitoring of gastric carcinoma, gastritis
and certain other cancers.
[0023] In a second aspect, the present invention features
diagnostic kits comprising suitable reagents for detecting EBV DNA
in the serum or plasma of patients. The kits according to the
present invention may further comprise one or more of a device for
obtaining a blood sample from a patient, a means to separate the
EBV DNA from the blood sample and a means to quantify the amount of
EBV DNA present in the blood sample. Such kits are useful for
diagnosing, detecting, monitoring and determining the prognosis of
EBV associated cancers apart from head, neck and lymphoid
malignancies.
[0024] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0025] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
[0026] EXAPMPLES
[0027] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill will readily
recognize a variety of noncritical parameters which could be
changed or modified to yield essentially similar results.
Example 1
Materials and Methods
[0028] Fifty-one patients with gastric carcinoma were recruited
with informed consent from the Prince of Wales Hospital, Hong Kong.
Blood samples were taken before surgical resection of the tumor.
Following operation, sections of the tumor were taken for in-situ
hybridization analysis for EBER (small EBV encoded RNA). Blood
samples were also taken from 30 individuals with gastritis, without
evidence of cancer carcinoma, and 197 apparently healthy control
subjects.
[0029] DNA Extraction from Plasma Samples. Peripheral blood (5 ml)
can be collected from each subject into an EDTA tube for the
isolation of plasma. Blood samples are centrifuged at 1600.times.g,
and plasma carefully removed from the EDTA-containing tubes and
transferred into plain polypropylene tubes. The samples are stored
at -20.degree. C. until further processing. DNA form plasma samples
are extracted using a QIAamp Blood Kit (Qiagen, Hilden, Germany)
using the blood and body fluid protocol as recommended by the
manufacturer (2). Plasma samples (130-800 .mu.l/column) are used
for DNA extraction. The exact amount is documented for the
calculation of the target DNA concentration. A final elution volumn
of 50 .mu.l is used from the extraction columns.
[0030] Circulating EBV DNA concentrations were measured using a
real time quantitative PCR system towards the BamHI-W fragment
region of the EBV genome (Lo, Y. M. D. et al., Cancer Res., 59:
1188-1191 (1999)). The principals of real time quantitative PCR and
reaction set-up procedures were as previously described (Lo, Y. M.
D. et al., Cancer Res., 59: 1188-1191 (1999)). Data were collected
using an ABI Prism 7700 Sequence Detector and were analyzed using
the Sequence Detection System software (version 1.6.3) developed by
Applied Biosystems. Results were expressed as copies of EBV genomes
per millititer of serum.
[0031] All serum DNA samples were also subjected to real time PCR
analysis for the (beta-globin gene (Lo, Y. M. D. et al, Cancer
Res., 59: 1188-1191 (1999)), which gave a positive signal on all
tested samples, thus demonstrating the quality of the extracted
DNA. Multiple negative water blanks were included in every
analysis.
[0032] More specifically, two real-time quantitative PCR systems
have been developed for EBV DNA detection: (a) one toward the
BamHI-W region; and (b) the other toward the EBNA-I region (Baer,
et al Nature, 310: 207-211, 1984). The BamHI-W system consisted of
the amplification primers (SEQ ID NO: 1) W-44F
(5'-CCCAACACTCCACCACACC-3') and (SEQ ID NO: 2) W-119R (5'-TCTT
AGGAGCTGTCCGAGGG-3') and the dual-labeled fluorescent probe (SEQ ID
NO: 3) W-67T (5'-FAM)CACACACTACACACACCCAC-CCGTCTC(TAMRA)-3']. The
EBNA-1 system consisted of the amplification primers (SEQ ID NO: 4)
EBNA-1162F (5'-TCATCATCATCCGGGTCTCC-3') and (SEQ ID NO: 5)
EBNA-1229R (5'-CCTACAGGGT-GGAAAAATGGC-3') and the dual-labeled
fluorescent probe (SEQ ID NO: 6) EBNA-1186T
[5'-(FAM)CGCAGGCCCCCTCCAGGTA-GAA(TAMRA)-3']. The fluorescent probes
contained a 3'-blocking phosphate group to prevent probe extension
during PCR. Primer/probe combinations were designed using Primer
Express software (Perkin-Elmer Corp., Foster City, Calif.).
Sequence data for the EBV genome were obtained from the GenBank
Sequence Database (accession number V01555). Real-time quantitative
PCR for the .beta.-globin gene consisted of primers and probe, as
described previously in Lo, et al. Am J Hum Genet 62: 768-775,
1998), and was used as a control for the amplifiability of plasma
DNA.
[0033] Fluorogenic PCR reactions are set up in a reaction volume of
50 .mu.L using components (except for the fluorescent probes and
amplification primers) supplied in a TaqMan PCR Core Reagent Kit
(Perkin-Elmer Corp.). Fluorescent probes are custom-synthesized by
Perkin-Elmer Applied Biosystems. PCR primers were synthesized by
Life Technologies, Inc. (Gaithersburg, Md.). Each reaction
contained 5 .mu.l of 10.times. buffer A; 300 nM of each of the
amplification primers; 25 nM (for the EBV probes) or 100 nM (for
the .beta.-globin probe) of the corresponding fluorescent probe; 4
MM MgCl.sub.2; 200 .mu.m each of dATP, dCTP, and dGTP; 400 .mu.M
dUTP; 1.25 units of AmpliTaq Gold; and 0.5 unit of AmpErase uracil
N-glycosylase.
[0034] DNA amplifications are carried out in a 96-well reaction
plate format in a Perkin-Elmer Applied Biosystems 7700 Sequence
Detector. Each sample are analyzed in duplicate. Multiple negative
water blanks were included in every analysis.
[0035] A calibration curve is run in parallel and in duplicate with
each analysis, using DNA extracted from the EBV-positive cell line
Namalwa (American Type Culture Collection CRL-1432; See Klein et
al., Int J. Cancer, 10: 44-57, 1972) as a standard. Namalwa is a
diploid cell line that contains two integrated viral genomes/cell.
A conversion factor of 6.6 pg of DNA/diploid cell was used for copy
number calculation (Saiki et al., Science, 239: 487-491, 1988).
Concentrations of circulating cell-free EBV DNA were expressed as
copies of EBV genome/ml plasma.
[0036] An identical thermal profile was used for the EBV BamHI-W
and EBNA-I PCR systems. Thermal cycling was initiated with a 2-min
incubation at 50.degree. C. for the uracil N-glycosylase to act,
followed by an initial denaturation step of 10 min at 95.degree.
C., and then 40 cycles of 95.degree. C. for 15 s and 56.degree. C.
for 1 min were carried out.
[0037] Amplification data collected by the 7700 Sequence Detector
and stored in a Macintosh computer (Apple Computer, Cupertino,
Calif.) is then analyzed using the Sequence Detection System
software developed by Perkin-Elmer Applied Biosystems. The mean
quantity of each duplicate is used for further concentration
calculation. The plasma concentration of EBV DNA or the
.beta.-globin gene (expressed in copies/ml) is calculated using the
following equation: 1 C = Q .times. V DNA V PCR .times. 1 V ext
[0038] in which C represents the target concentration in plasma
(copies/ml), Q represents the target quantity (copies) determined
by a sequence detector in a PCR, V.sub.DNA represents the total
volume of DNA obtained after extraction (typically 50 .mu.l/Qiagen
extraction), VPCR represents the volume of DNA solution used for
PCR (typically 5 .mu.l, and V.sub.ext represents the volume of
plasma/serum extracted (typically 0.13-0.80 ml)).
[0039] The presence of EBV in tumor cells was assessed by in-situ
hybridization on paraffin-embedded tissue sections using a
fluorescein-conjugated oligonucleotide probe for EBER (Novocastra,
U.K.) as previously described (Hui, P. K. et al., Hum. Pathol., 25:
947-952 (1994)).
Results
[0040] A total of 51 gastric carcinoma patients were recruited. In
this cohort, 5 gastric carcinomas were EBER-positive (FIG. 1A). In
14 cases, the tumor cells were EBER-negative, but there were
occasional infiltrating lymphocytes which were EBER-positive (FIG.
1B). These 14 cases were classified as having `background`
positivity. FIG. 2 illustrates the difference in the level of
circulating EBV DNA amongst these three patient groups. Serum EBV
DNA was detected in every one of the EBER-positive cases (median
serum EBV DNA concentration: 1063 copies/mL; interquartile range:
485 to 5141 copies/mL). No serum EBV DNA was detected in any of the
32 negative cases (FIG. 2). Thirteen out of the 14 cases (93%)
demonstrating `background` EBER positivity had detectable serum EBV
DNA. These cases had an intermediate median serum EBV DNA
concentration of 50 copies/mL (interquartile range: 42 to 98
copies/mL). The difference between these three groups is
statistically significant (p<0.001, Kruskal-Wallis test).
Pairwise multiple comparison analysis indicates significant
difference between the EBER-positive and EBER-negative groups
(p<0.05, Dunn's method) and between the EBER-background and
EBER-negative groups (p<0.05, Dunn's method).
[0041] EBV DNA was detectable in the serum of 7 of the 30 gastritis
samples (23%) and 7 of the 197 healthy controls (3.6%). The
proportions of serum EBV DNA positive cases between these groups
are significantly different (chi-square test, p=0.028). Even in the
cases with detectable circulating EBV DNA, the actual srum EBV DNA
concentrations were generally lower than those in the EBER-positive
gastric carcinoma cases.
[0042] A comparison was made for the cases with detectable serum
EBV DNA in the gastric carcinoma cases with `background`
EBER-positivity, gastritis cases and control subjects. The serum
EBV DNA concentrations of these three groups are plotted in FIG. 3.
There is no statistically significant difference in circulating EBV
DNA levels amongst these three groups (Kruskal-Wallis test,
p=0.296).
Discussion
[0043] These data demonstrate that cell-free EBV DNA can be
detected in serum samples obtained from a proportion of gastric
carcinoma patients. Since gastric carcinoma is not classified as a
lymphoma or lymphocyte associated cancer like nasopharyngeal
carcinoma or related head and neck cancers, this is the first time
that cell-free EBV DNA is shown effective for the detection and
diagnosis of EBV associated cancers apart from head, neck and
lymphoid malignancies. In addition, these data demonstrate an
interesting correlation between the detectability of serum EBV DNA
and tumoral EBER status. Thus, EBER-positive gastric carcinoma
cases were associated with high levels of serum EBV DNA; gastric
carcinoma cases with `background` EBER-positivity were associated
with intermediate levels; and no serum EBV DNA was seen in
EBER-negative cases. This observation lends further demonstrate
that plasma and serum represent noninvasive sources of materials
for monitoring cancer (Anker, P. et al., Cancer Metastasis Rev.,
18: 65-73 (1999)).
[0044] Clinically, circulating EBV DNA may have application in the
diagnosis and monitoring in the proportion of gastric carcinoma
patients who have EBER-positive tumors, similar to what has been
achieved for NPC (Lo, Y. M. D. et al., Cancer Res., 59: 1188-1191
(1999); Lo, Y. M. D. et al., Cancer Res., 59: 5452-5455 (1999)) and
certain lymphomas (Lei, K. I. et al., Br. J Haematol., 111: 239-246
(2000); Drouet, E, et al., J. Med. Virol. 57: 383-389 (1999);
Gallagher, A. et al., Int. J. Cancer, 84: 442-448 (1999)).
Recently, the value of circulating EBV DNA in nasopharyngeal cancer
prognosis has been demonstrated. The present data (Lo, Y. M. D. et
al., Cancer Res., 60: 6878-6881) indicate that EBV DNA measurement
also has prognostic importance for gastric carcinoma.
[0045] The detection of circulating EBV DNA in gastric carcinomas
demonstrating `background` EBER-positivity is interesting. The
EBER-positive lymphocytes infiltrating the tumor tissues may be the
origin of the low levels of serum EBV DNA that are detectable in
these cases. If this is correct then further work may elucidate the
mechanism of EBV liberation by these EBER-positive lymphocytes.
Mechanisms include active release of DNA (Rogers, J. C. et al.,
Proc. Natl. Acad. Sci. USA., 69: 1685-1689 (1972)) and activation
of lytic EBV infection in a proportion of these cells.
[0046] The long-term significance of the presence of low levels of
circulating EBV DNA in the blood of apparently healthy individuals
remains to be elucidated. Importantly, future studies should
address the possibility that these individuals might be at
increased risk of developing EBV-associated diseases. This issue
would be of tremendous public health and biological importance.
[0047] The present data also suggest that circulating EBV DNA may
be useful in many other cancer types that are associated with EBV.
Examples of such cancers include breast cancer (Bonnet, M. et al.,
J. Natl. Cancer Inst., 91:-1376-1381 (1999)) and hepatocellular
carcinoma (Sugawara, Y. et al., Virology, 256: 196-202 (1999)). As
the association between some tumor types and EBV is still
controversial, the possible detection of EBV DNA in the plasma of
patients with such tumors may contribute towards resolving these
issues.
Sequence CWU 1
1
6 1 19 DNA Artificial BamHI-W system amplification primer W-44F 1
cccaacactc caccacacc 19 2 20 DNA Artificial BamHI-W system
amplification primer W-119R 2 tcttaggagc tgtccgaggg 20 3 27 DNA
Artificial BamHI-W system dual - labeled fluorescent probe W-67T 3
nacacactac acacacccac ccgtctn 27 4 20 DNA Artificial EBNA-1 system
amplification primer EBNA-1162F 4 tcatcatcat ccgggtctcc 20 5 21 DNA
Artificial EBNA-1 system amplification primer EBNA-1229R 5
cctacagggt ggaaaaatgg c 21 6 22 DNA Artificial EBNA-1 system
dual-labeled fluorescent probe EBNA-1186T 6 ngcaggcccc ctccaggtag
an 22
* * * * *