U.S. patent application number 12/725309 was filed with the patent office on 2010-12-30 for dna markers for management of cancer.
Invention is credited to Dave S.B. Hoon, Bret Taback.
Application Number | 20100330567 12/725309 |
Document ID | / |
Family ID | 33131682 |
Filed Date | 2010-12-30 |
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United States Patent
Application |
20100330567 |
Kind Code |
A1 |
Hoon; Dave S.B. ; et
al. |
December 30, 2010 |
DNA MARKERS FOR MANAGEMENT OF CANCER
Abstract
A method is provided for assessing allelic losses and
hypermethylation of genes in CpG tumor promotor region on specific
chromosomal regions in cancer patients, including melanoma,
neuroblastoma breast, colorectal, and prostate cancer patients. The
method relies on the evidence that free DNA and hypermethylation of
genes in CpG tumor promotor region may be identified in the bone
marrow, serum, plasma, and tumor tissue samples of cancer patients.
Methods of melanoma, neuroblastoma, colorectal cancer, breast
cancer and prostate cancer detection, staging, and prognosis are
also provided.
Inventors: |
Hoon; Dave S.B.; (Los
Angeles, CA) ; Taback; Bret; (Santa Monica,
CA) |
Correspondence
Address: |
PERKINS COIE LLP
POST OFFICE BOX 1208
SEATTLE
WA
98111-1208
US
|
Family ID: |
33131682 |
Appl. No.: |
12/725309 |
Filed: |
March 16, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10809965 |
Mar 25, 2004 |
7718364 |
|
|
12725309 |
|
|
|
|
60457395 |
Mar 25, 2003 |
|
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Current U.S.
Class: |
435/6.13 ;
436/94 |
Current CPC
Class: |
Y10T 436/143333
20150115; C12Q 2600/118 20130101; C12Q 1/6806 20130101; C12Q 1/6886
20130101; C12Q 2600/154 20130101; C12Q 2600/112 20130101 |
Class at
Publication: |
435/6 ;
436/94 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with support in part by grants from
NCI (Grant Nos. R21 CA100314, PO CA 29605 Project II, and PO CA
13917 Project II), Gonda Foundation, USA DOD Breast Cancer Research
Grant, California Breast Cancer Research Grant, and Roy E Coates
Foundation. Therefore, the U.S. government has certain rights in
the invention.
Claims
1-31. (canceled)
32. A kit, comprising one or more agents for detecting LOH, DNA
hypermethylation or gene mutation of one or more DNA markers in a
bodily fluid or tissue sample.
33. The kit of claim 32, wherein the bodily fluid or tissue sample
is a cell-free bone marrow sample
34. A packaged product, comprising a container; one or more agents
for detecting one or more DNA markers in a bodily fluid or tissue
sample from a subject; and an insert associated with the container,
the insert comprising a label or instruction sheet indicating the
diagnosis, stage, or prognosis of cancer in the subject based on
the detection of the one or more agents.
35. The packaged product of claim 34, wherein the bodily fluid or
tissue sample is a cell-free bone marrow sample
36. The packaged product of claim 34, wherein the DNA markers are
indicative of LOH, DNA hypermethylation or DNA mutation, or a
combination thereof.
37. The packaged product of claim 36, wherein the DNA markers
include one or more of the group consisting of D1S228, D8S321,
D4S175, D4S1586, D5S299, D8S133, D8S261, D8S262, D8S264, D9S171,
D10S197, D10S591, D10S532, D14S51, D14S62, D15S127, D16S421,
D16S422, D17S796, D17S849, D17S855, D18S58, D18S61, and D18S70
38. The packaged product of claim 36, wherein the DNA markers
indicative of DNA hypermethylation include one or more of the group
consisting of RASSF1A, MGMT, GSTP1, RAR-.beta., TWIST, APC, DAPK,
P16, or Cyclin D2 promoter.
39. The packaged product of claim 36, wherein the DNA markers
indicative of DNA mutation include KRAS gene muration or BRAF gene
mutation.
40. The packaged product of claim 34, wherein the insert indicates
that LOH, DNA hypermethylation or DNA mutation of the one or more
DNA markers is indicative of a cancer diagnosis in the subject.
41. The packaged product of claim 34, wherein the insert indicates
that a combination of LOH, DNA hypermethylation or DNA mutation of
the one or more DNA markers is indicative of a cancer diagnosis in
the subject.
42. The packaged product of claim 34, wherein the insert indicates
that LOH, DNA hypermethylation or DNA mutation of the one or more
DNA markers is indicative of an advanced stage of a cancer or a
poor prognosis of a cancer in the subject.
43. The packaged product of claim 34, wherein the insert indicates
that a combination of LOH, DNA hypermethylation or DNA mutation of
the one or more DNA markers is indicative of an advanced stage of a
cancer or a poor prognosis of a cancer in the subject.
44. A packaged product, comprising a container; one or more agents
for detecting LOH, DNA hypermethylation or gene mutation of one or
more DNA markers in a cell-free bone marrow sample from a subject;
and an insert associated with the container, the insert comprising
a label or instruction sheet indicating the diagnosis, stage, or
prognosis of cancer in the subject based on the detection of LOH,
DNA hypermethylation or gene mutation of the one or more DNA
markers.
45. The packaged product of claim 44, wherein the DNA markers
include one or more of the group consisting of D1S228, D8S321,
D4S175, D4S1586, D5S299, D8S133, D8S261, D8S262, D8S264, D9S171,
D10S197, D10S591, D10S532, D14S51, D14S62, D15S127, D16S421,
D16S422, D17S796, D17S849, D17S855, D18S58, D18S61, and D18S70
46. The packaged product of claim 44, wherein the DNA markers
indicative of DNA hypermethylation include one or more of the group
consisting of RASSF1 A, MGMT, GSTP1, RAR-.beta., TWIST, APC, DAPK,
P16, or Cyclin D2 promoter.
47. The packaged product of claim 44, wherein the DNA markers
indicative of DNA mutation include KRAS gene muration or BRAF gene
mutation.
48. The packaged product of claim 44, wherein the insert indicates
that LOH, DNA hypermethylation or DNA mutation of the one or more
DNA markers is indicative of a cancer diagnosis in the subject.
49. The packaged product of claim 44, wherein the insert indicates
that a combination of LOH, DNA hypermethylation or DNA mutation of
the one or more DNA markers is indicative of a cancer diagnosis in
the subject.
50. The packaged product of claim 44, wherein the insert indicates
that LOH, DNA hypermethylation or DNA mutation of the one or more
DNA markers is indicative of an advanced stage of a cancer or a
poor prognosis of a cancer in the subject.
51. The packaged product of claim 44, wherein the insert indicates
that a combination of LOH, DNA hypermethylation or DNA mutation of
the one or more DNA markers is indicative of an advanced stage of a
cancer or a poor prognosis of a cancer in the subject.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/809,965, filed Mar. 25, 2004, which claims
priority to U.S. Provisional Application Ser. No. 60/457,395, filed
Mar. 25, 2003. The content of both applications is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention is related to the fields of molecular
biology and oncology and provides methods for diagnosis, staging
and monitoring of cancer patients.
BACKGROUND OF THE INVENTION
[0004] Bone Marrow is the most frequent site of the systemic spread
of some types of cancer, including breast (Abrams et al 1950 and
Lee 1983), neuroblastoma, colorectal, and prostate cancer. Once
metastases are clinically apparent, overall prognosis is poor.
Undetected occult tumor cells contribute to disease recurrence and
therefore methods to identify subclinical disease (micrometastasis)
may improve staging and guide additional therapeutic decisions.
Historically, conventional cytologic assessment of blood and bone
marrow (BM) aspirates has been performed with limited success
(Molino et al 1991 and Beiske et al 1992). Immunocytochemical
techniques using antibodies specific to epithelial antigens have
improved sensitivity and can identifying a single tumor cell
amongst a background of >1 million normal cells (Osborne et al
1989 and Chaiwun et al 1992). Enrichment methods with
antibody-magnetic bead conjugates of BM aspirates have demonstrated
the presence of occult tumor cells in early stage breast cancer
patients (Osborne et al 1989 and Rye et al 1997).
[0005] Recently it has been shown that the detection of
micrometastasis in the BM of early stage breast cancer patients is
an independent prognostic risk factor (Braun et al 1998 and Diel et
al 1996). However, the accurate microscopic analysis of many
cytologic samples requires considerable cytopathologic expertise
and can be tedious, particularly if performed serially to assess
disease progression and/or response to treatment. Additionally, the
variable specificity of individual antibodies used to detect single
cells has been questioned (Braun et al 1998; Litle et al 1996; and
Moll et al 1982). Finally, these assay systems cannot characterize
the biologic behavior of the cells being detected and thus many may
represent dormant tumor cells, apoptotic cells, nonpathologic tumor
cells, or displaced normal breast epithelial cells.
[0006] A variety of serial genetic changes have been implicated in
the initiation and progression of solid tumors. One such event,
allelic imbalance (loss of heterozygosity; LOH) has been shown to
occur commonly in primary breast tumors and with additional
frequency in metastasis (Takita et al 1992; Hampl et al 1999;
Driouch et al 1997; and Silva et al 1999). Furthermore, there is
emerging evidence to suggest that microsatellite markers for
detecting LOH at specific chromosome loci may have important
clinical prognostic correlations (Takita et al 1992; Hampl et al
1999; Emi et al 1999; and Hirano et al 2001). However, the
examination of an excised primary tumor specimen may be of limited
value in that it provides information of those genetic events that
have occurred and not ongoing alterations which may be of clinical
relevance, either prognostically or for therapeutic decisions.
Additionally, because of the potentially long latent period that
may exist between early breast cancer diagnosis and clinically
detectable systemic recurrence, improved assessment methods are
needed for serial surveillance of occult disease progression and
monitoring response to therapy.
[0007] Recently it has been shown that free tumor-associated DNA
can be identified in the serum and plasma from patients with
melanoma, breast, lung, renal, gastrointestinal, and head and neck
tumors (Anker et al 1997; Chen et al 1999; Chen et al 1996; Silva
et al, Cancer Research 1999; Shaw et al, 2000; Taback et al,
Academy of Science 2001; Taback et al, Cancer Research 2001;
Sanchez-Cespedes et al 1998; Goessl et al, Cancer Research 58 1998;
Fujiwara et al 1999; Hibi et al 1998; Kopreski et al 1997; Mayall
et al 1999; Nawroz et al 1996; and Stroun et al 1987). Furthermore
a high-quality concordance has been shown to exist between the
genetic alterations (i.e., LOH, microsatellite instability,
mutations) found in circulating tumor DNA and those from the
primary tumor suggesting a potential surrogate tumor marker (Chen
et al 1996; Silva, Cancer Research 1999; Shaw et al 2000; Fujiwara
et al 1999; and Hibi et al 1998). Early studies have shown the
prognostic importance of circulating microsatellite markers for LOH
in blood (Silva, Cancer Research 1999 and Taback et al, Cancer
Research 2001). Although, BM is a common site for recurrence of
some types of cancer, such as breast and prostate cancer, to date,
BM has not been studied for the presence of suitable genetic
markers.
[0008] Recently, methylation of gene promoter regions and the role
that this epigenetic event plays in the development of various
cancers has become an important area of investigation in assessing
the mechanisms of tumor suppressor and regulatory gene
inactivation. The tumor suppressor genes (TSG) can be
transcriptionally silenced when their promoter region CpG islands
contain methylated cytosines located 5' to an adjacent guanine. The
utilization of methylation-specific PCR (MSP) assay has simplified
and significantly improved detecting hypermethylated CpG bases with
minimum amount of DNA. The methylation status of several TSG
promoter regions have been profiled for a number of cancers. The
hypermethylation of CpG islands of promoter regions of TSG is quite
common and is a significant genetic aberration for tumor cells to
shut off TSG expression.
[0009] Majority of these studies have been focused on carcinomas;
there are limited studies in cutaneous melanomas and other types of
cancers. The studies of epigenetic inactivation of TSG in melanomas
have been limited mostly to methylation of promoter regions of
p16.sup.INK4a and MGMT(O.sup.6-methylguanine-DNA
methyltransferase). Interestingly, the frequency of TSG
inactivation or mutations in oncogenes is reportedly low in
cutaneous melanomas. These observations have been a major enigma in
deciphering the genetic events occurring in melanoma
progression.
SUMMARY OF THE INVENTION
[0010] The present invention is based on the unexpected discovery
that DNA markers can be detected in cell-free bone marrow samples
and are useful for cancer diagnosis, staging, and prognosis.
[0011] Accordingly, the invention features a method of detecting
DNA markers in a sample, comprising providing a cell-free bone
marrow sample from a subject and detecting one or more DNA markers
in the sample. Examples of DNA markers include those in the 1p, 3p,
6p, 6q, 8p, 10q, 11q, 14q, 16q, or 17p region. In particular, the
DNA markers may be indicative of LOH, DNA hypermethylation, or DNA
mutation. Such DNA markers include D1S228, D8S321, D4S175, D4S1586,
D5S299, D8S133, D8S261, D8S262, D8S264, D9S171, D10S197, D10S591,
D10S532, D14S51, D14S62, D15S127, D16S421, D16S422, D17S796,
D17S849, D17S855, D18S58, D18S61, and D18S70; those indicative of
hypermethylation in RASSF1A, MGMT, GSTP1, RAR-.beta., TWIST, APC,
DAPK, P16, or Cyclin D2 promoter; and those indicative of mutation
in KRAS and BRAF gene (e.g. mutation at codon 12 of KRAS and BRAF
K600E mutation).
[0012] In one aspect, the invention provides a method of detecting
cancer, comprising providing a cell-free bone marrow sample from a
subject and detecting one or more DNA markers in the sample,
wherein LOH, hypermethylation, or mutation of the markers is
indicative of cancer (e.g., melanoma, neuroblastoma, colorectal,
breast, or prostate cancer) in the subject.
[0013] In another aspect, the invention provides a method of
staging cancer, comprising providing a cell-free bone marrow sample
from a subject suffering from cancer and detecting one or more DNA
markers in the sample, wherein LOH, hypermethylation, or motation
of the markers is indicative of an advanced stage of the cancer in
the subject.
[0014] The invention further provides a method of prognosing
cancer, comprising providing a cell-free bone marrow sample from a
subject suffering from cancer and detecting one or more DNA markers
in the sample, wherein LOH, hypermethylation, or mutation of the
markers is indicative of a poor prognosis of the cancer in the
subject.
[0015] The invention is also based on the unexpected discovery that
LOH and hypermethylation of DNA markers, when combined, provide
more sensitive and precise diagnosis, staging and prognosis of
cancer than when used individually. Therefore, the invention
provides a method of detecting LOH and DNA hypermethylation,
comprising providing a sample from a subject and detecting a
combination of LOH and DNA hypermethylation in the sample (e.g., a
serum, plasma or tissue sample). In one embodiment, the LOH is
detected through DNA markers including D1S228, D8S321, D4S175,
D4S1586, D5S299, D8S133, D8S261, D8S262, D8S264, D9S171, D10S197,
D10S591, D10S532, D14S51, D14S62, D15S127, D16S421, D16S422,
D17S796, D17S849, D17S855, D18S58, D18S61, or D18S70. In another
embodiment, the DNA hypermethylation is detected in RASSF1A, MGMT,
GSTP1, RAR-.beta., TWIST, APC, DAPK, P16, KRAS, BRAF, or Cyclin D2
promoter.
[0016] In one aspect, the invention features method of detecting
cancer, comprising providing a sample from a subject and detecting
one or more DNA markers in the sample, wherein a combination of LOH
and hypermethylation of the markers is indicative of cancer (e.g.,
melanoma, neuroblastoma, colorectal, breast, or prostate cancer) in
the subject.
[0017] In another aspect, the invention features a method of
staging cancer, comprising providing a sample from a subject
suffering from cancer and detecting one or more DNA markers in the
sample, wherein a combination of LOH and hypermethylation of the
markers is indicative of an advanced stage of the cancer in the
subject.
[0018] In still another aspect, the invention features a method of
prognosing cancer, comprising providing a sample from a subject
suffering from cancer and detecting one or more DNA markers in the
sample, wherein a combination of LOH and hypermethylation of the
markers is indicative of a poor prognosis of the cancer in the
subject.
[0019] Moreover, the invention provides kits and packaged products
for implementing the methods described above. For example, one
packaged product comprises a container, one or more agents for
detecting one or more DNA markers in a sample and an insert
associated with the container and indicating that the sample is a
cell-free bone marrow sample.
[0020] Another example of a packaged product comprises a container,
one or more agents for detecting one or more DNA markers in a
cell-free bone marrow sample from a subject, and an insert
associated with the container and indicating that LOH,
hypermethylation, or mutation of the markers is indicative of
cancer in the subject.
[0021] A packaged product may also comprise a container, one or
more agents for detecting one or more DNA markers in a cell-free
bone marrow sample from a subject suffering from cancer, and an
insert associated with the container and indicating that LOH,
hypermethylation, or mutation of the markers is indicative of an
advanced stage of the cancer or a poor prognosis of the cancer in
the subject.
[0022] The invention further provides (1) a kit comprising one or
more agents for detecting a combination of LOH and DNA
hypermethylation of one or more DNA markers in a sample; (2) a
packaged product, comprising a container, one or more agents for
detecting one or more DNA markers in a sample from a subject and an
insert associated with the container and indicating that a
combination of LOH and hypermethylation of the markers is
indicative of cancer in the subject; and (3) a packaged product,
comprising a container, one or more agents for detecting one or
more DNA markers in a sample from a subject suffering from cancer,
and an insert associated with the container and indicating that a
combination of LOH and hypermethylation of the markers is
indicative of an advanced stage of the cancer or a poor prognosis
of the cancer in the subject.
[0023] The methods of the present invention advantageously permit a
minimally invasive detection of tumor genetic changes that may
provide valuable prognostic and diagnostic information, which may
improve staging of the disease and monitoring of disease
progression and response to therapy. In addition, because the
methods of the present invention may be used to survey ongoing
genetic changes, they may also be used to identify potential
targets to individualize patient therapy. The method may also be
used to identify markers in BM aspirates, plasma, and serum for
other types of cancers.
[0024] The above-mentioned and other features of this invention and
the manner of obtaining and using them will become more apparent,
and will be best understood, by reference to the following
description, taken in conjunction with the accompanying drawings.
These drawings depict only typical embodiments of the invention and
do not therefore limit its scope.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 shows representative examples of paired frozen and
paraffin-embedded melanoma tumors analyzed by CAE for determining
methylation status of (A) RAR-.beta.2, (B) MGMT, and (C) RASSF1A.
Methylated (M) and unmethylated (U) PCR products from frozen (F1)
or paraffin-embedded (P1) tumor specimens were analyzed
simultaneously and distinguished by size and fluorescence.
[0026] Also shown are representative examples of tumor
histopathology negative patients' paraffin-embedded lymph nodes
(PLN) analyzed by CAE for determining methylation status of (D)
RAR-.beta.2, (E) MGMT, and (F) RASSF1A.
[0027] FIG. 2 shows representative expression and re-expression of
RAR-.beta.2, RASSF1A and MGMT in two melanoma cell lines treated
with 5Aza-dC. The cells were treated for four days with different
concentrations of 5Aza-dC followed by 24 h treatment with ATRA
where indicated. Gene expression was analyzed by RT-PCR. The
house-keeping gene GAPDH was included as an RT-PCR control for all
assays.
[0028] FIG. 3 shows representative PCR analysis of promoter region
CpG island sequence of MGMT and RAR-.beta.2 from bisulfite-treated
DNA obtained from melanoma cell lines. Fully methylated CpGs are
indicated as solid black boxes and partially methylated CpGs are
shown as shaded boxes. All CpGs contained in the MSP products are
shown.
[0029] FIG. 4 provides representative images demonstrating LOH in
breast cancer patients' paired BM aspirate (BM) and primary tumors
(T) at D14S62, D14S51, and D8S321, respectively. Allelic loss is
represented by the arrows. The first lane of each panel exhibits
patients' lymphocyte DNA (L) allele pattern as a control.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Since BM is a common site for cancer recurrence in certain
types of cancer and because an application of conventional
histochemical techniques to BM has been limited due to sub-optimal
efficiency and sensitivity, it is one object of the present
invention to determine whether BM aspirates may be used as a source
of tumor-specific DNA associated with systemic metastasis from
cancer, including metastasis associated with neuroblastoma, breast,
prostate, and colorectal cancer. It is a further object of the
present invention to identify tumor-specific nucleic acid
alterations in the bone marrow, serum/plasma, and tumor tissue
samples of cancer patients as diagnostic and prognostic markers of
malignancy. Also, since the hypermethylation of CpG islands of
promoter regions of TSG appears to play a significant role in the
development of various cancers, it is another objective of the
present invention to identify TSG and tumor-related genes
methylation of which could indicate development of a cancer. It is
a further object of the present invention to develop a method of
using the identified methylation markers in the bone marrow,
serum/plasma, and tumor tissue samples of cancer patients to
diagnose malignancy.
[0031] It is a discovery of the present invention that LOH may be
detected in BM aspirates and that the advancement of AJCC stages is
associated with an increased incidence of LOH. In one study, the
inventors used a panel of microsatellite markers for LOH on
chromosomes 1p, 3p, 6p, 6q, 8p, 10q, 11q, 14q, 16q, and 17p to
demonstrate the association between the LOH identified in BM
aspirates with stage and tumor type in breast cancer. The inventors
believe that other cancers that metastasize preferentially in bone,
such as melanoma, prostate, and colorectal cancers, may be detected
and monitored using the same group of LOH markers that were
identified in breast cancer patients.
[0032] In another study, the inventors have demonstrated a
correlation of LOH identified in serum/plasma of prostate cancer
patients and AJCC staging. In still another study, the inventors
showed that the presence of certain circulating nucleic acids in
serum/plasma may assist in diagnosis of colorectal cancer.
Accordingly, the present invention provides tumor-related genetic
markers in BM aspirates, serum/plasma, and tumor tissue samples of
cancer patients and provides a unique approach for assessing the
subclinical systemic disease progression and the monitoring of
cancer patients. The present invention also provides molecular
techniques for the identification of genetic alterations on
circulating nucleic acids in the bone marrow aspirates, plasma,
serum, and tumor tissue of cancer patients.
[0033] One aspect of the present invention provides a detection
assay for detecting the loss of heterozygosity (LOH) in DNA from
BM, tumor tissue, plasma, and serum. The assay comprises the steps
of (a) amplifying nucleic acid from an LOH marker, if present, (b)
detecting the presence or absence of the LOH marker, and (c)
correlating the findings with the occurrence and/or progression of
a cancer. The determination of heterozygosity is well within the
skill of the art and includes examining the second sample of DNA,
which is isolated from non-neoplastic tissue. For example, U.S.
Pat. No. 6,465,177, which is assigned to the assignee of the
present invention and the content of which is incorporated herein
by the reference, describes the detection of the loss of
heterozygosity in the tumor and serum of melanoma patients.
[0034] Although any detection method may be used in association
with markers of the present invention, in one embodiment
amplification/detection methods used were PCR-based methods
selected from the group consisting of PCR and gel electrophoresis
using labeled primers (fluorescent or radioactive), RealTime PCR
using specific labeled primers Taqman and probes (labeled with
chromatographic dyes), and capillary array electrophoresis (CAE)
with labeled PCR primers (no probes).
[0035] In one embodiment, the detection is carried out in a sample
derived from bone marrow aspirates, plasma/serum, or tumor tissues.
In another embodiment, because the combination of assessing blood,
tumor tissue, and bone marrow is believed by the inventors to have
a better predictive and diagnostic value, LOH is assessed in
several different samples selected from the group consisting of BM
aspirates, tumor tissue, serum, and plasma.
[0036] In one embodiment of the present invention, the set of
alleles which are tested for LOH in BM aspirate, blood/plasma, or
tumor tissue sample are selected from the group consisting of
D1S228, D8S321, D4S175, D4S1586, D5S299, D8S133, D8S261, D8S262,
D8S264, D9S171, D10S197, D10S591, D10S532, D14S51, D14S62, D15S127,
D16S421, D16S422, D17S796, D17S849, D17S855, D18S58, D18S61, and
D18S70.
[0037] It is also a discovery of the present invention that the
methylation of DNA CpG tumor promoter regions is detectable in the
plasma/serum, tumor tissue, and bone marrow of breast melanoma
colon cancer patients for the following genes: RASSF1A, MGMT,
GSTP1, RAR-.beta., TWIST, APC, DAPK, P16, and Cyclin D2 (CCND2).
For example, although there are no reported comprehensive studies
on melanoma tumor methylation correlating with clinicopathology,
the inventors discovered an inactivation of a newly identified TSGs
RASSF1A and RAR-.beta. in melanomas, as well as methylation of
MGMT.
[0038] The inventors believe that methylation markers may be used
to provide significant prognostic and diagnostic information in
cancer patients, including melanoma, colorectal, breast, and
prostate cancer patients. The inventors also believe that
utilization of both LOH markers and DNA methylation markers will
allow establishing a comprehensive panel of human genetic
prognostic molecular markers (PMMs) for melanoma, colorectal,
breast, and prostate cancer. Primary tumors, metastatic tissue,
blood (plasma/serum) or/and BM may be tested for methylation and
microsatellite DNA markers for diagnosis and prognosis. For
example, blood (plasma/serum) and BM LOH markers may be used as PMM
in patient follow up to identify sub-clinical disease recurrence.
Assessment of tumor tissue may be used for prognosis of disease
outcome. Although it appears that LOH and methylation markers
somewhat overlap for breast, prostate, melanoma and colon cancer,
there are specific LOH and methylation markers that are more
frequent or exclusively in specific cancers. Thus, in one
embodiment, a panel of markers specific to the cancer suspected in
the patient is used. In another embodiment, a panel comprising a
broad range markers, including non-specific markers, is used to
conduct a broader screening for various types of cancer.
[0039] In another aspect of the present invention, BM aspirates,
blood, and tumor are assessed collectively for LOH and methylation
markers to obtain a comprehensive profile of cancer patients,
including prediction of metastasis to lymph nodes and disease
outcome. The obtained results may be used to predict metastasis to
lymph nodes (sentinel node) and disease outcome in cancer patients,
including breast cancer, prostate cancer, and melanoma
patients.
[0040] For example, in one embodiment, BM aspirates, serum, and/or
plasma samples are evaluated for the presence of microsatellite
markers selected from the group consisting of D1S228, D8S321,
D4S175, D4S1586, D5S299, D8S133, D8S261, D8S262, D8S264, D9S171,
D10S197, D10S591, D10S532, D14S51, D14S62, D15S127, D16S421,
D16S422, D17S796, D17S849, D17S855, D18S58, D18S61, and D18S70 and
methylation markers selected from the group consisting of RASSF1A,
MGMT, GSTP1, RAR-.beta., TWIST, APC, DAPK, P16, and Cyclin D2
(CCND2) to obtain a prognostic and diagnostic information in cancer
patients, including melanoma, breast, colorectal, and prostate
cancer patients. In another embodiment, BM aspirates, serum, and/or
plasma samples are evaluated for the presence of microsatellite
markers with LOH on chromosomes 1p, 3p, 6p, 6q, 9p, 10q, 11q, and
12q and methylation markers RASSF1A, MGMT, and RAR-.beta. to obtain
prognostic and diagnostic information in breast cancer
patients.
[0041] There is mounting evidence to suggest that the presence of
occult tumor cells in the BM of breast cancer patients may have
prognostic significance (Diel et a 1996; Mansi et al 1991; Berger
et al 1988; Cote et al 1991; Dearnaley et al 1991; Harbeck et al
1994; and Braun et al, N Engl J Med 2000). Furthermore, some have
shown these findings to be independent of pathologic lymph node
status (Braun et al, J Natl Cancer Inst 1998 and Diel et al 1996).
These studies are important because, historically, 20% of lymph
node negative patients will subsequently develop systemic disease
and therefore, early detection of BM micrometastasis may identify
high-risk patients for additional systemic therapy. More so, BM
provides a readily accessible source to serially monitor
subclinical disease progression and the potential impact of
adjuvant therapies early in the disease course. Conventional
histologic analysis of BM aspirates for tumor cells has proven
unreliable (Molino et al 1991 and Beiski et al 1992). More
recently, immunocytochemical techniques using antibodies to
epithelial antigens expressed on tumor cells have improved
detection sensitivity. However, assay reliability has been shown to
be highly dependent on the antibody selected as well as the
variability by which the tumor cell expresses the preferred epitope
(Braun et al, J Natl Cancer Inst 1998 and Moll et al 1982. Finally,
sample processing and antibody staining require considerable
attention to methodology and an experienced reviewer to interpret
the results.
[0042] With the implication of an accrual of aberrant genetic
events in tumor development and progression, and their potential
for clonality, these genetic markers may provide unique surrogates
for monitoring subclinical disease events, particularly in light of
the ease and widespread use of PCR techniques. Studies have
demonstrated the presence of circulating nucleic acids in the
plasma and serum of patients with various malignancies (Stroun et
al 1987). In breast cancer, LOH presence in plasma/serum has been
described to occur anywhere from 15% to 66% (Chen et al, Clin
Cancer Res 1999; Silva et al, Cancer Res 1999; Shaw et al 2000;
Taback et al, Ann NY Acad Sci 2001; and Mayall et al 1999). These
results may vary due to differences in the techniques of sample
collection and processing, DNA isolation, PCR methods, and scoring
of LOH. Furthermore, in the earlier work, the inventors have shown
that the presence of the circulating tumor DNA increases with the
advancing stage of disease (Taback et al, Ann NY Acad Sci 2001).
Since BM is a frequent site of melanoma, prostate, colorectal and
breast cancer relapse, it was an object of the present invention to
determine whether BM aspirates harbor tumor-specific DNA
alterations associated with early breast cancer progression.
[0043] The present invention provides highly sensitive methods of
detecting tumor-specific DNA in the BM aspirates, plasma/serum, and
tumor tissue of cancer patients, including melanoma, breast,
colorectal, and prostate cancer patients. The increased incidence
in the more advanced stages correlates with tumor burden and
therefore may have applicability as a surrogate marker for disease
detection, prognosis, and monitoring tumor progression and response
to therapy. The present invention demonstrates an association
between known prognostic factors in breast cancer (tumor
histopathology, tumor size, lymph node status, and AJCC stage) and
an incidence of LOH and methylation markers in BM, blood
(plasma/serum), and primary tumor and metastatic tissues.
[0044] Some advantages of the methods of the present invention over
conventional methods include the ease of their use, high
sensitivity and specificity, and their broad application to a
variety of malignancies. Additional tumor-specific genetic markers
or combinations thereof may be easily incorporated into methods of
the present invention to further enhance the assay's utility.
[0045] The methods of the present invention may provide a unique
alternative/supplement to optical systems for occult tumor
detection which can be technically demanding and viewer dependent.
The methods of the present invention may also provide an
alternative/supplement to RT-PCR methods that assess mRNA markers
which may have limited specificity as a result of unstable gene
products, variable expression levels, and nonspecific transcripts
(Zippelius et al 1997; Bostick et al 1998; Ko et al 1998; and Jung
et al 1998). The inventors believe that the detection of genomic
alterations in BM may offer more specificity than
immunohistochemical and/or current mRNA marker assays.
[0046] In one study, which is described in more detail in Example
5, the inventors observed LOH in BM ranging from 0% to 12% for
various microsatellite markers (see Table 10). A similar detection
of LOH has been described from the peripheral plasma/serum of early
stage breast cancer patients (Chen et al, Clinical Cancer Research
1999; Shaw et al 2000; and Taback, Ann NY Acad of Sci 2001). For 10
of the 11 patients whose BM contained LOH, primary tumor blocks
were available for assessment and in all cases, a similar
corresponding LOH pattern was identified in the respective primary
tumor specimens. The findings demonstrate the specificity of this
marker detection system.
[0047] In the study discussed in Example 5, no patients had
detectable tumors cells identified on routine histopathologic
examination. This demonstrates the relative ease and sensitivity
that the methods of the present invention provide in the
identification and diagnosis of subclinical disease. Because of the
earlier detection of breast cancers and the benefits of adjuvant
radiotherapy, immunotherapy, and chemotherapy in these stages, the
methods and microsatellite markers of the present invention provide
improved occult disease surveillance and ability to assess
individual patient risk more accurately. This allows modification
of treatment strategies before clinical manifestations occur.
[0048] Breast cancer recurrence is a result of undetected
metastasis at the time of primary patient treatment. More sensitive
methods are needed to identify subclinical disease progression to
better accompany those increasing advances in early breast cancer
screening. Aberrant hypermethylation of tumor-suppressor genes is
found frequently in primary breast tumors and has been implicated
in disease initiation and progression. The increased sensitivity
for the detection of methylated genes associated with a cancer
phenotype among a background of unmethylated genes from normal
cells offers a potential specific surrogate marker for molecular
detection of occult disease progression. We evaluated whether
tumor-associated methylated DNA markers could be identified
circulating in BM aspirates and paired serum samples from 33
early-stage patients undergoing surgery for breast cancer.
Methylation specific PCR was performed using a tumor-related gene
panel for RAR-.beta.2, MGMT, RASSF1A and APC. Tumor-associated
hypermethylated DNA was identified in 7 (21%) of 33 BM aspirates
and 9 (27%) serum samples. In three patients, the bone marrow and
serum were positive for hypermethylation. The most frequently
detected hypermethylation marker was RASSF1A occurring in 7 (21%)
patients. Concordance was present between gene hypermethylation
detected in BM/serum samples and matched-pair primary tumors.
Advanced AJCC stage was associated with an increased incidence of
circulating gene hypermethylation. This study demonstrates the
novel finding of tumor-associated epigenetic markers in BM
aspirates and their potential role as targets for molecular
detection and as an aid to early-stage breast cancer patient risk
identification.
[0049] Gene promoter region hypermethylation is a frequent event in
primary breast cancer. However its impact on tumor progression and
potential prognostic implications remain relatively unknown. We
conducted hypermethylation profiling of 151 primary breast tumors
with association to known prognostic factors in breast cancer using
methylation specific PCR for six known tumor suppressor and related
genes: RASSF1A, APC, Twist, CDH1, GSTP1 and RAR-.beta.2.
Furthermore correlation with sentinel lymph node tumor status was
assessed as it represents the earliest stage of metastasis that can
be readily detected. Hypermethylation for any one gene was
identified in 147 (97%) of 151 primary breast tumors. The most
frequently hypermethylated gene was RASSF1A (81%). Hypermethylation
of the CDH1 was significantly associated with primary breast tumors
demonstrating lymphovascular invasion (p=0.008), infiltrating
ductal histology (p=0.03), and negative for the estrogen receptor
(p=0.005), whereas RASSF1A and RAR-.beta.2 gene hypermethylation
were significantly more common in ER positive (p<0.001) and HER2
positive (p<0.001) tumors, respectively. In multivariate
analysis, hypermethylation of GSTP1 and/or RAR-.beta.2 was
significantly associated with patients have macroscopic sentinel
lymph node metastasis, odds ratio 4.59 (95% Cl, 2.02 to 10.4;
p<0.001). Hypermethylation profiling of primary breast cancers
may have clinical and pathologic utility for assessing patient
prognosis and predicting early lymph node regional metastasis.
[0050] Aberrant methylation of CpG islands in promoter regions of
tumor suppressor genes (TSG) has been demonstrated in epithelial
origin tumors. However, the methylation profiling of tumor-related
gene promoter regions in cutaneous melanoma tumors has not been
reported. Seven known or candidate TSGs that are frequently
hypermethylated in carcinomas were assessed by methylation-specific
polymerase chain reaction (MSP) in 15 melanoma cell lines and 130
cutaneous melanoma tumors. Four TSGs were frequently
hypermethylated in 86 metastatic tumor specimens: retinoic acid
receptor-.beta.2 (RAR-.beta.2) (70%), RAS association domain family
protein 1A (RASSF1A) (57%), and O.sup.6-methylguanine DNA
methylatransferase (MGMT) (34%), and death-associated protein
kinase (DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK
was significantly lower in primary melanomas (n=20) compared to
metastatic melanomas. However, hypermethylation of RAR-.beta.2 was
70% in both primary and metastatic melanomas. Cell lines had
hypermethylation profiles similar to those of metastatic melanomas.
The analysis of these four markers of metastatic tumors
demonstrated that 97% had .gtoreq.1 gene(s) and 59% had .gtoreq.2
genes hypermethylated, respectively. The methylation of genes was
verified by bisulfite sequencing. The mRNA transcripts could be
re-expressed in melanoma cell lines having hypermethylated genes
following treatment with 5'-aza 2'-deoxycytidine (5Aza-dC).
Analysis of melanoma patients' plasma (preoperative blood; n=31)
demonstrated circulating hypermethylated MGMT, RAR-.beta.2, and
RASSF1A DNA for at least one of the markers in 29% of the patients.
Our findings indicate that the incidence of TSG hypermethylation
increases during tumor progression. Methylation of TSG may play a
significant role in cutaneous melanoma progression.
[0051] Cancer cells almost invariably undergo loss of genetic
material (DNA) when compared to normal cells. This deletion of
genetic material which almost all, if not all, varieties of cancer
undergo is referred to as "loss of heterozygosity" (LOH). The loss
of genetic material from cancer cells can result in the selective
loss of one of two or more alleles of a gene vital for cell
viability or cell growth at a particular locus on the chromosome.
All genes, except those of the two sex chromosomes, exist in
duplicate in human cells, with one copy of each gene (allele) found
at the same place (locus) on each of the paired chromosomes. Each
chromosome pair thus contains two alleles for any gene, one from
each parent. This redundancy of allelic gene pairs on duplicate
chromosomes provides a safety system. If a single allele of any
pair is defective or absent, the surviving allele will continue to
produce the coded protein.
[0052] Due to the genetic heterogeneity or DNA polymorphism, many
of the paired alleles of genes differ from one another. When the
two alleles are identical, the individual is said to be homozygous
for that pair of alleles at that particular locus. Alternatively,
when the two alleles are different, the individual is heterozygous
at that locus. Typically, both alleles are transcribed and
ultimately translated into either identical proteins in the
homozygous case or different proteins in the heterozygous case. If
one of a pair of heterozygous alleles is lost due to deletion of
DNA from one of the paired chromosomes, only the remaining allele
will be expressed and the affected cells will be functionally
homozygous. This situation is termed as "loss of heterozygosity"
(LOH) or reduction to homozygosity. Following this loss of an
allele from a heterozygous cell, the protein or gene product
thereafter expressed will be homogeneous because all of the protein
will be encoded by the single remaining allele. The cell becomes
effectively homozygous at the gene locus where the deletion
occurred. Almost all, if not all, cancer cells undergo LOH at some
chromosomal regions.
[0053] Through the use of DNA probes, DNA from an individual's
normal cells can be compared with DNA extracted from the same
individual's tumor cells and LOH can be identified using
experimental techniques well known in the art. Alternatively, LOH
can be assayed by demonstrating two polymorphic forms of a protein
in normal heterozygous cells, and only one form in cancer cells
where the deletion of an allele has occurred. See, for example,
Lasko et al, 1991, Annu. Rev. Genet. 25:281-314.
[0054] Recent advances in molecular biology have revealed that
genesis and progression of tumors follow an accumulation of
multiple genetic alterations, including inactivation of tumor
suppressor genes and/or activation of proto-oncogenes. There are
over 40 known proto-oncogenes and suppressor genes to date, which
fall into various categories depending on their functional
characteristics. These include, growth factors and growth factor
receptors, messengers of intracellular signal transduction
pathways, for example, between the cytoplasm and the nucleus, and
regulatory proteins influencing gene expression and DNA
replication. Frequent LOH on specific chromosomal regions has been
reported in many kinds of malignancies, which indicates the
existence of putative tumor suppresser genes or tumor-related genes
on or near these loci. LOH analysis is a powerful tool to search
for a tumor suppresser gene by narrowing and identifying the region
where a putative gene exists. By now, numerous LOH analyses,
combined with genetic linkage analysis on pedigrees of familial
cancer (Vogelstein et al 1988; Fearon et al 1990; and Friend et al
1986) or homozygous deletion analyses (Call et al 1990; Kinzler et
al 1991; and Baker 1989) have identified many kinds of candidate
tumor suppressor or tumor-related genes. Also, because allelic
losses on specific chromosomal regions are the most common genetic
alterations observed in a variety of malignancies, microsatellite
analysis has been applied to detect DNA of cancer cells in
specimens from body fluids, such as sputum for lung cancer and
urine for bladder cancer (Rouleau et al 1993 and Latif et al 1993).
Moreover, it has been established that markedly increased
concentrations of soluble DNA are present in plasma of individuals
with cancer and some other diseases, indicating that cell free
serum or plasma can be used for detecting cancer DNA with
microsatellite abnormalities (Kamp et al 1994 and Steck et al
1997). Two groups have reported microsatellite alterations in
plasma or serum of a limited number of patients with small cell
lung cancer or head and neck cancer (Hahn et al 1996 and Miozzo et
al 1996).
[0055] Recent developments in cancer therapeutics have demonstrated
the need for more sensitive staging and monitoring procedures to
ensure initiation of appropriate treatment, to define the end
points of therapy and to develop and evaluate novel treatment
modalities and strategies. In the management of cancer patients,
the choice of appropriate initial treatment depends on accurate
assessment of the stage of the disease. Patients with limited or
regional disease generally have a better prognosis and are treated
differently than patients who have distant metastases (Minna et al
1989). However, conventional techniques to detect these metastases
are not very sensitive, and these patients are often not cured by
primary tumor resection because they have metastases that are not
identified by standard methods during preoperative staging. Thus,
more sensitive methods to detect metastases in other types of
carcinomas would identify patients who will not be cured by local
therapeutic measures, for whom effective systemic therapies would
be more appropriate.
[0056] The strategy of the present invention is to utilize genetic
differences between normal and cancer cells for diagnosis and
monitoring of cancer patients. Many genes coding for proteins or
other factors vital to cell survival and growth that are lost, can
be identified through LOH analysis of microsatellite, single
nucleotide polymorphism (SNP) loci in cancer cells and mapped to
specific chromosomal regions. Gene expression may be suppressed due
to hypermethylation in the promoter region or mutation in the gene.
In melanoma, mutations of several already-known tumor suppresser
genes such as p53 gene, neurofibromatosis 1 (NF1) gene, and NF2
gene have been reported at a low frequency and deletions and/or
mutations of the cyclin dependent kinase 4 (CDK4) inhibitor gene,
which is a responsible tumor suppresser gene for a familial
melanoma, have been thought to be important genetic changes in
tumor development (Miozzo et al., 1996, Cancer Research
56:2285-2288). In addition to the locus of CDK4 inhibitor gene
(9p21), frequent chromosomal deletions have been reported on 1p36,
3p25, 6q22-q26, 10q24-q26, and 11q23. (Mao et al 1996; Stroun et al
1987; Chen et al, Nat Medicine 1996; and Nawroz et al 1996). An
efficient method of testing DNA microsatellite or SNP loci for LOH,
hypermethylation in the promoter region of a gene, and mutations in
a gene allows early diagnosis of melanoma patients and monitoring
of the progression of the disease as well as effectiveness of the
therapeutic regimen.
[0057] A cellular DNA can be obtained from a sample of a biological
fluid by deproteinizing the sample and extracting DNA according to
the procedures well known in the art. Examples of biological fluids
include urine, blood plasma or serum, sputum, cerebral spinal
fluid, peritoneal fluid, ascites fluid, saliva, stools, and bone
marrow plasma. The DNA to be tested may be a fraction of a larger
molecule or can be present initially as a discrete molecule. Where
the test DNA contains two strands, it may be necessary to separate
the strands of the nucleic acid before it can be used, e.g., as a
template for amplification. Strand separation can be effected
either as a separate step or simultaneously with synthesis of
primer extension products. This strand separation can be
accomplished using various suitable denaturing conditions,
including physical, chemical, or enzymatic means. If the nucleic
acid is single stranded, its complement is synthesized by adding
one or two oligonucleotide primers. If a single primer is utilized,
a primer extension product is synthesized in the presence of
primer, an agent for polymerization, and the four nucleoside
triphosphates. The product will be complementary to the
single-stranded nucleic acid and will hybridize with a
single-stranded nucleic acid to form a duplex of unequal length
strands that may then be separated into single strands to produce
two single separated complementary strands.
[0058] A DNA marker refers to a DNA sequence (e.g., a
microsatellite or SNP locus, a promoter region, or a gene sequence)
associated with a specific biological event (e.g., presence or
absence of a gene, hypermethylation of a promoter, mutation in a
gene, expression of a gene, and occurrence of a disease).
Microsatellites are short repetitive sequences of DNA widely
distributed in the human genome. Somatic alterations in the repeat
length of such microsatellites have been shown to represent a
characteristic feature of tumors. SNP is a common nucleotide
variant in DNA at a single site. Each individual has many single
nucleotide polymorphisms that together create a unique DNA
sequence. These markers can be tested either independently or in
combination with each other.
[0059] Detection of a DNA marker can be accomplished by a number of
means well known in the art. One means of detecting a DNA marker is
by digesting a test DNA sample with a restriction endonuclease.
Restriction endonucleases are well known in the art for their
ability to cleave DNA at specific sequences, and thus generate a
discrete set of DNA fragments from each DNA sample. The restriction
fragments of each DNA sample can be separated by any means known in
the art. For example, agarose or polyacrylamide gel electrophoresis
can be used to electrophoretically separate fragments according to
physical properties such as size. The restriction fragments can be
hybridized to nucleic acid probes which detect restriction fragment
length polymorphisms (RFLP). There are various hybridization
techniques known in the art, including both liquid and solid phase
techniques. One particularly useful method employs transferring the
separated fragments from an electrophoretic gel matrix to a solid
support such as nylon or filter paper so that the fragments retain
the relative orientation which they had on the electrophoretic gel
matrix. The hybrid duplexes can be detected by any means known in
the art, for example, by autoradiography if the nucleic acid probes
have been radioactively labeled. Other labeling and detection means
are well known in the art and may be used accordingly.
[0060] An alternative means for detecting a DNA marker is by using
PCR (polymerase chain reaction; see, e.g., U.S. Pat. Nos.
4,683,195, 4,683,202, and 4,683,194). This method allows
amplification of discrete regions of DNA containing microsatellite
sequences. Amplification is accomplished by annealing, i.e.,
hybridizing a pair of single stranded primers, usually comprising
DNA, to a target DNA. The primers embrace oligonucleotides of
sufficient length and appropriate sequence so as to provide
specific initiation of polymerization of a significant number of
nucleic acid molecules containing the target nucleic acid. In this
manner, it is possible to selectively amplify the specific target
nucleic acid sequence containing the nucleic acid of interest. More
specifically, the primers are designed to be substantially
complementary to each strand of target nucleotide sequence to be
amplified. Substantially complementary means that the primers must
be sufficiently complementary to hybridize with their respective
strands (i.e., with the flanking sequences) under conditions which
allow amplification of the nucleotide sequence to occur. The primer
is preferably single stranded for maximum efficiency in
amplification but may be double-stranded. If double-stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent for polymerization. The exact length of a primer
will depend on many factors, including temperature, buffer, and
nucleotide composition. The oligonucleotide primers for use in the
present invention may be prepared using any suitable method, such
as conventional phosphotriester and phosphodiester methods or
automated embodiments thereof. In one such automated embodiment,
diethylphosphoramidites are used as starting materials and may be
synthesized as described by Beaucage et al. One method for
synthesizing oligonucleotides on a modified solid support is
described in U.S. Pat. No. 4,458,066. The primers are annealed to
opposite strands of the DNA sequence containing a DNA marker, such
that they prime DNA synthesis in opposite but convergent directions
on a chromosome. Amplification of the region containing the DNA
marker is accomplished by repeated cycles of DNA synthesis.
Experimental conditions conducive to synthesis include the presence
of nucleoside triphosphates and an agent for polymerization, such
as DNA polymerase, and a suitable temperature and pH. Preferably,
the DNA polymerase is Taq polymerase which is relatively heat
insensitive. The amplification procedure includes a specified
number of cycles of amplification in a DNA thermal cycler. After an
initial denaturation period of 5 minutes, each amplification cycle
preferably includes a denaturation period of about 1 minute at
95.degree. C., primer annealing for about 2 minutes at 58.degree.
C., and an extension at 72.degree. C. for approximately 1 minute.
Following the amplification, aliquots of amplified DNA from the PCR
can be analyzed by techniques such as electrophoresis through
agarose gel using ethidium bromide staining. Improved sensitivity
may be attained by using labeled primers and subsequently
identifying the amplified product by detecting radioactivity or
chemiluminescense on film.
[0061] In a preferred embodiment, the assay involves labeling of
the PCR primers with multiple types of chromophore dyes. In another
embodiment, the PCR primers are labeled with an atom or inorganic
radical, most commonly using radionuclides, but also perhaps heavy
metals. Radioactive labels include .sup.32P, .sup.125I, .sup.3H,
.sup.14C, or any radioactive label which provides for an adequate
signal and has sufficient half-life. Other labels include ligands,
which can serve as a specific binding pair member for a labeled
ligand, and the like.
[0062] Another object of the invention is to provide a method of
detecting DNA markers in biological fluids, wherein the presence of
LOH, hypermethylation, or mutation is associated with the
occurrence of cancer. This method represents a significant advance
over such techniques as tissue biopsy by providing a non-invasive,
rapid, and accurate method for detecting DNA markers associated
with cancer. Thus, the present invention provides a method which
can be used to screen high-risk populations and to monitor high
risk patients undergoing chemoprevention, chemotherapy,
immunotherapy, surgical procedure, or other treatment.
[0063] According to the method of the present invention, DNA is
isolated from a biological fluid of a patient. For comparison, a
control DNA sample may be prepared, for example, from a
non-neoplastic tissue from the same patient, or from a biological
fluid or tissue from a normal person. It is desirable that the
alleles used in the allelotype loss analysis be those for which the
subject is heterozygous. Determination of heterozygosity is well
within the skill of the art. Loss of an allele is ultimately
determined by comparing the pattern of bands corresponding to the
allele in the control sample to the test sample and noting the
size, number of bands, or level of amplification of signal of
individual bands. For example, LOH may be defined when one allele
showed more than a threshold degree (e.g., .gtoreq.50%) reduction
of peak intensity for serum DNA as compared to the corresponding
allele identified in the control DNA. Methods of detecting
hypermethylation of DNA (see Examples below) and mutations in a
gene are well known in the art.
[0064] This invention also provides a logistically practical assay
to monitor the genetic changes during cancer progression. The
events of tumor progression are dynamic and the genetic changes
that concurrently occur also are very dynamic and complex. The most
significant advantage of this approach compared to other approaches
is the ability to monitor disease progression and genetic changes
without assessing the tumor. This is particularly important during
early phases of distant disease spread, in which subclinical
disease is undetectable by conventional imaging techniques. In
addition, in advance stage diseases or inoperable sites in which
tumor tissue is very difficult or impossible to obtain for genetic
analysis, the present invention provides an alternative for
assessing LOH, DNA hypermethylation, and gene mutation.
[0065] Because the methods described above require only DNA
extraction from bodily fluid such as blood, it can be performed at
any time and repeatedly on a single patient. Blood can be taken and
monitored for LOH, DNA hypermethylation, and gene mutation before
or after surgery; before, during, and after treatment, such as
chemotherapy, radiation therapy, gene therapy or immunotherapy; or
during follow-up examination after treatment for disease
progression, stability, or recurrence. The method of the present
invention also may be used to detect subclinical disease presence
or recurrence with a DNA marker specific for that patient since DNA
markers are specific to an individual patient's tumor. The method
also can detect if multiple metastases may be present using tumor
specific DNA markers.
[0066] Further, the invention provides predictive measures of
response to cancer therapies and mortality. The method comprises
providing a sample from the subject and detecting one or more DNA
markers in the sample, wherein the status of the DNA markers are
indicative of response to cancer therapies and mortality. More
specifically, the invention provides a method of predicting the
probability of survival of a subject suffering from a cancer. For
example, if LOH, DNA hypermethylation and/or gene mutation occur in
a cancer patient, the patient is expected to have a low probability
of survival.
[0067] LOH, DNA hypermethylation and gene mutation can also be
detected in a tissue sample (e.g., a tumor sample). For a tumor
sample, if a non-neoplastic tissue is used as a control sample, it
can be of the same type as the neoplastic tissue or from a
different organ source. It is desirable that the neoplastic tissue
contains primarily neoplastic cells and that normal cells be
separated from the neoplastic tissue. Ways for separating cancerous
from non-cancerous cells are known in the art and include, for
example, microdissection of tumor cells from normal cells of
tissues, DNA isolation from paraffin-embedded sections and cryostat
sections, as well as flow cytometry to separate aneuploid cells
from diploid cells. DNA can also be isolated from tissues preserved
in paraffin. Separations based on cell size or density may also be
used. Once the tissues have been microdissected, DNA can be
isolated from the tissue using any means known in the art. Frozen
tissues can be minced or homogenized and then the resulting cells
can be lysed using a mixture of enzyme and detergent, see, for
example, Maniatis, Molecular Cloning, a Laboratory Manual, Cold
Spring Harbor Laboratory, 1982. The nucleic acids can be extracted
using standard techniques such as phenol and chloroform extraction,
and ethanol precipitation.
[0068] It is another object of the invention to provide kits and
packaged products for diagnosing, staging and monitoring cancer
patients. Such a kit or product usually contains a set of reagents
for detecting LOH, DNA hypermethylation, and gene mutation. For
example, a kit or product may include nucleic acid probes for
specified alleles for which the patient is homozygous or
heterozygous to detect LOH in these specified alleles. This
provides a measure of the extent of genetic change in a neoplastic
tissue or a biological fluid which can be correlated with a
diagnosis or prognosis. In one specific embodiment, the presence or
absence of a specific allele or combination of alleles is tested by
amplification of regions of the DNA markers using pairs of primers
which bracket specific regions of the DNA markers on specific
chromosome arms containing repeat sequences with polymorphism.
Preferably, the assay uses fluorescent labeling of DNA with
multiple types of chromophores. However, radioactive and other
labeling techniques known in the art also may be used. Optionally,
the kit or product may include a container, and an insert
associated with the container. The insert may be a label or an
instruction sheet with the information as to, e.g., what sample to
use and what the indication is if LOH, DNA hypermethylation or gene
mutation is detected.
[0069] The kit or product may comprise a carrier means being
compartmentalized to receive in close confinement one or more
container means such as vials, tubes, and the like, each of the
container means comprising one of the separate elements to be used
for detecting DNA markers. Such elements include a labeled primer
pair for amplifying a DNA marker. The product also may include a
DNA polymerase for amplifying the target DNA, appropriate
amplification buffers and deoxyribonucleoside triphosphates. The
nucleic acids in the product may be provided in solution or
lyophilized form. Preferably, the nucleic acids will be sterile and
devoid of nucleases to maximize shelf-life.
[0070] The following examples are intended to illustrate, but not
to limit, the scope of the invention. While such examples are
typical of those that might be used, other procedures known to
those skilled in the art may alternatively be utilized. Indeed,
those of ordinary skill in the art can readily envision and produce
further embodiments, based on the teachings herein, without undue
experimentation.
EXAMPLES
Example 1
Identification of Circulating Tumor-Associated Epigenetic
Alterations in the Bone Marrow from Breast Cancer Patients Using a
Hypermethylation Gene Panel
Introduction
[0071] A variety of genetic alterations including microsatellite
instability, allelic loss, and mutation have been described in
primary breast cancers. These events result in loss of gene
function and have been implicated in tumor development and
progression. Clinical tools (i.e., radiographic) used to detect
breast cancer progression have been limited particularly in the era
of earlier disease diagnosis. The most sensitive method for the
identification of breast cancer progression at the time of patient
diagnosis is histopathologic lymph node evaluation. However 20-30%
of node-negative breast cancer patients will develop recurrent
disease within 10 years (Fisher et al 1989 and Rosen et al 1989).
Therefore, recurrence may be considered a consequence of occult
metastasis not detected at the time of patient diagnosis and
treatment. The most frequent site of breast cancer metastasis is
bone (Goldhirsch et al 1988). Identification of patients at
increased risk for systemic metastasis may improve prognostic
staging and provide selection for additional therapy that may have
a significant impact on disease outcome. Assessment of body fluids
for circulating tumor cells using microscopy has shown poor results
(Molino et al 1991). This technique is labor-intensive, insensitive
and subjective. Furthermore, the rapid circulation and turbulent
environment of blood may contribute to the low yield. In contrast,
detection of occult tumor cells in BM using immunocytokeratins has
been associated with the subsequent development of systemic
metastasis and shows promise marker of a poorer prognostic outcome
(Braun et al, N Engl J Med 2000). Regardless, identification of a
few tumor cells among a background of one million normal BM cells
can be difficult and tedious. Automated techniques such as RT-PCR
can facilitate identification with improved sensitivity but may
have diminished specificity as tumor cell specific mRNA markers are
uncommon and expression levels may vary substantially affecting
results.
[0072] Recently cell-free DNA has been identified in the serum and
plasma from patients with various cancers (Sidransky et al 1997).
These circulating nucleic acids have demonstrated similar genetic
alterations and characteristics as those found in the primary
tumor. Their presence in blood can be readily identified using
common PCR techniques and appear to be elevated during disease
progression (Silva et al 1999; Taback et al 2001; Muller et al
2003; and Silva et al 2003).
[0073] Alternatively, promoter region hypermethylation has been
described as a common genetic abnormality occurring in various
cancers. Aberrant methylation of CpG islands in promoter regions of
putative tumor-suppressor and related genes resulting in their
silencing has been implicated in oncogenesis. Identification of
these additional genetic events may offer a more accurate molecular
portrait accounting for a tumor's metastatic potential and provide
unique tumor-specific surrogate markers for monitoring occult
disease progression. Methylation-specific real-time PCR provides a
highly sensitive DNA based assay for the detection of methylated
alleles associated with breast cancer (Esteller et al, Cancer Res
2001).
[0074] Because BM is the most common site for systemic relapse
following breast cancer diagnosis, we attempted to determine
whether BM aspirate plasma could provide a viable source to detect
tumor-specific epigenetic alterations associated with systemic
metastasis from early stage breast cancer patients.
Methods and Materials
[0075] Surgical Specimens and DNA Isolation. BM aspirates were
collected prospectively in 4.5 ml sodium citrate tubes (Becton
Dickinson, Franklin Lakes, N.J.) through bilateral anterior iliac
approach from 33 consecutive patients as follows: 17 American Joint
Committee on Cancer (AJCC) stage I patients, 14 AJCC stage II
patients, and 2 AJCC stage III patients; undergoing surgical
resection of their primary breast cancer at the Saint John's Health
Center/John Wayne Cancer Institute. In addition, BM aspirates were
obtained from five healthy female volunteer donors to serve as
controls. Institutional Review Board approved consent forms were
signed by all patients prior to participation in the study. BM was
drawn and (cell-free supernatant) plasma was separated, filtered
and cryopreserved as previously described (Taback et al 2003) In
addition, match-paired peripheral venous blood was drawn
pre-operatively and DNA was extracted from one ml of both
peripheral blood serum and BM aspirate plasma using QIAamp
extraction kit (Qiagen, Valencia, Calif.) as previously described
(23).
[0076] To determine the correlation of gene hypermethylation found
in the primary breast tumor, DNA was isolated from ten 10 .mu.m
sections cut from paraffin-embedded tissue blocks. Samples were
deparaffinized, microdissected from normal tissue using laser
capture microscopy (Arcturus, Mountain View, Calif.) and incubated
in lysis buffer and proteinase K at 37.degree. C. overnight as
described previously.
[0077] Gene hypermethylation in BM of tumor DNA was analyzed as
described below. Additionally, each BM aspirate was assessed for
the presence of occult tumor cells by standard histologic staining
methods using hematoxylin and eosin (H&E).
[0078] Gene Hypermethylation Markers and MSP. Sodium bisulfite
modification was performed on 1 ug of genomic DNA as previously
described (Hoon et al 2004). Primer sets were used for the
detection of four genes frequently hypermethylated in breast
cancer: RAS association domain family protein 1 A protein
(RASSF1A), adenomatous polyposis coli (APC), retinoic acid binding
receptor-.beta.2 (RAR-.beta.2) and MGMT. In addition, MYOD was
assessed as an internal control to confirm DNA presence in the
final reaction. MSP was performed with an initial incubation for 15
min at 95.degree. C. followed by 35 cycles (40 cycles for BM and
plasma aspirate samples) of denaturation at 94.degree. C. for 30 s,
annealing at 50-56.degree. C., and extension for 90 s at 72.degree.
C., followed by a final extension step of 72.degree. C. for 5 min.
For each MSP reaction, normal donor lymphocyte DNA served as a
negative control, SssI treated lymphocyte served as a positive
control, and water served as a control for contamination.
[0079] Clinical and pathologic data was obtained from John Wayne
Cancer Institute's Breast Tumor Computer Database. Chi-Square and
Wilcoxon Rank Sum tests were performed for statistical evaluation
for the association of BM methylation status and known prognostic
parameters in breast cancer.
Results
[0080] Circulating tumor DNA containing gene promoter
hypermethylation for any one marker was identified in the BM of 7
(21%) of 33 patients. The most frequently detected hypermethylated
gene marker was RASSF1A occurring in 5 (15%) patients BM, followed
by MGMT in 2 (6%) patients, RAR-.beta.2 and APC in 1 (3%) patient
each Table 1. Five patients demonstrated one hypermethylated gene
in their BM, whereas two patients had two hypermethylated genes
identified and in twenty-six patients no hypermethylated DNA
sequences could be detected for any of the genes assessed. No
hypermethylation was detected in the BM from five healthy female
donors.
TABLE-US-00001 TABLE 1 Frequency of gene hypermethylation in
patient's serum and bone marrow Frequency in Patients' Body Fluid
(n = 33) Marker Serum Bone Marrow RASSF1A 5 (15%) 7 (21%) MGMT 2
(6%) 2 (6%) RAR.beta. 1 (3%) 2 (6%) APC 1 (3%) 0
There was an increased association between the presence of gene
hypermethylation detected in the BM and advanced disease stage.
Three (18%) of 17 AJCC stage I patients demonstrated
hypermethylated DNA for at least one marker in their BM, in
contrast to 3 (21%) of 14 AJCC stage II patients, and 1 (50%) of 2
AJCC stage III patients (Table 2).
TABLE-US-00002 TABLE 2 Gene hypermethylation detection in breast
cancer patient's serum and bone marrow according to AJCC stage
Patients with hypermethylation AJCC Stage Serum Bone Marrow I (n =
17) 4 (24%) 3 (18%) II (n = 14) 4 (29%) 3 (21%) III (n = 2) 1 (50%)
1 (50%)
[0081] Hypermethylation was detected in paired peripheral blood
serum in 9 (27%) of 33 patients. Again RASSF1A was most frequently
identified occurring in 7 (21%) patients serum samples followed by
RAR-.beta.2 (6%) and MGMT (6%) (Table 1). Eight patients
demonstrated hypermethylation in serum for one gene and one patient
for 3 genes. Similarly, there was an increased association between
the presence of gene hypermethylation in serum and advanced AJCC
stage. Hypermethylation for any one gene was identified in 4 (24%)
of 17 AJCC stage I patients serum, whereas 4 (29%) of 14 AJCC stage
II patients, and 1 (50%) of 2 AJCC stage III patients had these
findings (Table 2).
[0082] Twelve clinicopathologic prognostic factors were assessed
for correlation with BM methylation status: patient age, histologic
tumor type, size, grade, Bloom-Richardson score, lymph node
involvement, AJCC stage, receptor status (estrogen (ER),
progesterone (PR), HER2), Ki-67 and p53 status. A trend towards
increased circulating methylated DNA in serum from patients with PR
negative tumors was identified: 5 (50%) of 10 patients as compared
to 4 (18%) of 23 patients with PR positive tumors. In multivariate
analysis, patients with PR positive tumors were less likely to have
methylation markers in their BM and serum, odds ratio 0.04, 95% Cl:
0.00-0.82 (p<0.04). However, due to the small sample size, no
other correlations were identified with BM or serum methylation
status.
[0083] Concordance between the presence of serum and/or BM
hypermethylation status among patients is shown in Table 3.
However, identification of the same gene hypermethylated between
the BM and serum occurred in only 2 patients, with one additional
patient having the same gene hypermethylation profile in BM for two
of the three genes detected in serum.
TABLE-US-00003 TABLE 3 Hypermethylation status: concordance between
patient's serum and bone marrow Serum Yes No Bone Marrow Yes 3 4 No
6 20 Yes: presence of hypermethylation detected for any one gene
No: absence of hypermethylation detected for any genes
[0084] To determine whether a correlation existed between the gene
hypermethylation detected in patients BM and their primary tumor,
DNA was isolated from primary tumors and evaluated with the same
hypermethylation markers. Of 13 patients with BM and/or serum
positive for gene hypermethylation, 8 had primary tumor blocks
available for assessment. In all eight patients, the
hypermethylated gene(s) identified in the BM/serum was also
hypermethylated respectively in the primary tumor.
[0085] Conventional histologic analysis of all specimens using
standard H&E staining did not demonstrate occult tumor cells in
any of the BM samples.
Discussion
[0086] Advances in breast imaging modalities and greater awareness
for early detection has resulted in a dramatic increase in the
number of smaller breast cancer diagnosed. Concurrently, these
tumors are less likely to be associated with readily identified
metastasis. However, patients with small primary tumors are not
exempt from developing recurrent disease, and these relapses are
most likely a consequence of occult metastasis present at the time
of initial diagnosis and treatment. Thus, improved methods are
needed to detect submicrscopic disease which can identify patients
at increased risk for recurrence sooner in their treatment course.
Additionally, techniques that detect occult metastasis may better
stratify those patients with subclinical disease that may benefit
from adjuvant therapy while more accurately recognizing patients
who do not require additional treatment.
[0087] MSP provides a highly sensitive and quantitative technique
that can identify 1 methylated allele among a background of 1000
normal alleles (Herman et al 1996) and therefore may prove useful
for assessing the presence of occult disease and increased patient
risk. Additionally, this approach allows for the identification of
novel aberrantly hypermethylated genes, which may be associated
with breast cancer tumor growth and metastasis and thereby
distinguish additional potential targets for therapy. In this
study, we identified tumor-associated epigenetic alterations
circulating in the blood and BM from 4 (18%) of 22 early stage
breast cancer patients without evidence of lymph node metastasis.
It is estimated that 20-30% of node-negative patients will develop
a systemic recurrence by 10 years and younger patients remain at
risk for relapse many decades after diagnosis (Brenner et al 2004).
Thus longer-term follow-up to determine whether these findings are
associated with disease recurrence in this group of patients will
be needed. However, these findings provide a promising potential
for analyzing circulating nucleic acids in body fluids from
patients with breast cancer for assessing the earliest stages of
disease progression. Recently, investigators have shown DNA
methylation in breast cancer patients serum to correlate with a
worse survival (Muller et al 2003). We have previously demonstrated
the presence of LOH in the BM from patients with breast cancer
(Taback et al, 2003). These findings are significant because bone
is the most frequent site of systemic metastasis. Therefore,
performing a comprehensive assessment of a patient's body fluids,
particularly the location most common for relapse, may yield highly
informative information, improve risk assessment and allow for a
more accurate method for monitoring treatment responses earlier in
the disease course. Innovative techniques are needed to detect and
characterize subclinical disease progression in this new era of
early breast cancer diagnosis.
Example 2
Distinct Hypermethylation Profile of Primary Breast Cancer is
Associated with Sentinel Lymph Node Metastasis
Introduction
[0088] Improved access to mammography and increased patient
awareness in breast cancer screening have resulted in a dramatic
increase in the detection of early breast cancers (Cady et al 1996;
Shapiro 1982; Tabar 1985; and Miller et al 1993). As important, at
the time of breast cancer diagnosis, is the identification of
concurrent metastatic disease for accurate patient staging and
therapeutic decision making. Axillary lymph node dissection (ALND)
has provided an invaluable approach to assess for the presence of
tumor cell metastasis, particularly in early disease states where
standard radiographic imaging is less sensitive. However, ALND can
be associated with considerable morbidity including lymphedema and
reduced shoulder mobility (Ivens et al 1992 and Warmuth et al
1998). In addition, ALND often requires general anesthesia, in
patient hospitalization and a postoperative drain. Evidence from
prospective randomized trials has questioned the therapeutic value
of routine ALND in patients without palpable disease (1980 #44;
Fisher et al 1985; and Cabanes et al 1992).
[0089] Sentinel lymph node (SLN) biopsy provides an effective
alternative approach to the identification of regional nodal
metastasis, and is associated with reduced morbidity when compared
to standard ALND (Giuliano et al 2000). This procedure, although
less invasive, is not entirely risk-free as it still requires an
axillary incision and general anesthesia, subjects patients to
lymphatic mapping reagents and its success is dependent on the
skill of the surgeon (Giuliano et al 1999 and Borgstein et al
1998). The main advantage of the technique is that it provides a
more cost-effective, less labor-intensive process for focused
detection of metastasis, particularly when assessing for the
presence of occult tumor cells which are more likely to be
associated with earlier disease states (Giuliano et al 1998). The
addition of immunohistochemical (IHC) analysis has further improved
their identification (Turner et al 1999). The clinical implications
of these findings remain to be conclusively determined by
historical reviews and therefore prospective multicenter studies
are currently underway to evaluate their significance (Wilke et al
2003 and Grube et al 2001). Regardless, a greater number of
patients with small primary tumors are being treated with adjuvant
chemotherapy and hormonal therapy. This may be a result of recent
studies demonstrating a survival advantage in breast cancer
patients without lymph node metastasis [, 1998 #62; 1998 #133; and
Eifel et al 2001). It must be cautioned that patients with lymph
node disease derive the greatest benefit and widespread application
of such an aggressive approach may not prove necessary for all
cases of early stage breast cancers, as only 20-30% of patients
without histopathologic evidence of lymph node metastasis will
subsequently develop a recurrence (Winchester et al 1991 and Cooper
et al 1991). Adjuvant chemotherapy is associated with potential for
patient toxicity, and its added healthcare costs and resources for
its administration must be considered (Hillner et al 1991 and Smith
et al 1993). Consequently, improved methods are needed to better
identify patients at increased risk for disease recurrence and
systemic metastasis, which would provide a more appropriate
utilization of patient care resources.
[0090] Breast cancer development is a consequence of a serial
accumulation of genetic alterations ultimately resulting in the
ability of epithelial cells to proliferate uncontrollably, invade
tissues and avoid apoptosis. These genetic events lead to gene
activation/inactivation through the mechanisms of mutation,
amplification and deletion (Sidransky et al 1997). More recently,
it has been shown that different cancers demonstrate significant
CpG island hypermethylation in the promoter regions(s) of specific
tumor-suppressor and related genes regulating cellular function and
contributing to their transcription silencing when compared to
normal cells (Baylin et al 2001 and Esteller et al 2001). These
epigenetic events have been suggested to play a significant role in
cancer progression (Widschwendter et al 2002).
[0091] Despite the descriptive profile studies of various genes
hypermethylated in breast cancer, relatively little is known of
their impact on tumor development and progression (Muller et al
2003). Even more important is whether these tumor genetic
aberrations have clinicopathologic utility. In breast cancer where
outcome data such as recurrence and survival can only adequately be
obtained after a relatively prolonged follow-up period, correlation
with established clinical and pathologic prognostic factors may
serve as an interim surrogate (Stearns et al 2003). Because lymph
node metastasis remains the most significant prognostic factor in
patients with early stage breast cancer, we sought to determine
whether a hypermethylation marker panel comprising six tumor
suppressor and cancer related genes: RAS association domain family
protein 1 A protein (RASSF1A), adenomatous polyposis coli (APC),
Twist gene of a basic-helix-loop-helix family of transcription
factors, E-cadherin (CDH1), glutathione S-transferase pi 1 (GSTP1)
and retinoic acid binding receptor-.beta.2 (RAR-.beta.2), detected
in primary breast tumors could predict the likelihood of SLN
metastasis.
[0092] The SLN has been shown to represent the first site of
drainage from a primary breast tumor and is most likely to harbor
detectable metastasis in patients with early stage disease. Thus,
the characterization of an epigenetic tumor profile that is
associated with SLN metastasis would not only provide better
insight into the biology of breast cancer progression by defining
those genetic events associated with the earliest of tumor
spreading but may also provide prognostic information from primary
tumor assessments. In this study, we developed a
methylation-specific PCR (MSP) assay to assess archived
paraffin-embedded primary breast tumors for hypermethylation
profiles of known tumor-suppressor and related genes with
clinicopathologic correlation.
Methods
[0093] Patients. A total of 151 patients were identified from the
Breast Cancer Database at the John Wayne Cancer Institute who
underwent surgery for their primary breast cancer with SLN biopsy
alone or with ALND from August 1992 to May 2001. Two thirds of the
patients were postmenopausal and mean patient age was 55 years
(range: 27-86 years) with a mean tumor size of 3.1 cm (range:
0.1-10 cm). Additional primary tumor characteristics are listed in
Table 4. The study was approved by the joint Saint John's Health
Center/John Wayne Cancer Institute's institutional review board
with all patients providing informed written consent.
TABLE-US-00004 TABLE 4 Patient characteristics Factors n = 151
Menopausal pre 51 (33.8) post 100 (66.2) T Stage T1a 1 (0.7) T1b 4
(2.6) T1c 13 (8.6) T2 118 (78.2) T3 15 (9.9) N Stage N0 71 (47.0)
N1 74 (49.0) N2 6 (4.0) M Stage M0 147 (97.4) M1 4 (2.6) AJCC Stage
I 1 (0.7) IIa 86 (57.0) IIb 43 (28.5) IIIa 17 (11.3) IV 4 (2.7)
Histology Ductal 118 (78.2) Lobular 33 (21.8) Differentiation Well
30 (20.1) Moderate 62 (41.6) Poor 57 (38.3) [Unknown] [2] Invasion
No 102 (70.3) Yes 43 (29.7) [Unknown] [6] SLN status Negative 70
Micro 40 Macro 41
[0094] DNA Extraction and MSP. Paraffin-embedded primary tumor
specimen blocks were sectioned at 10 .mu.m deparaffinized in 100%
xylene, followed by 100% ethanol incubation and stained with
hematoxylin and eosin (H&E). Tumor tissue was microdissected in
comparison to a similarly stained and cover-slipped reference slide
cut in sequence from each tissue block. The samples were incubated
in buffer containing SDS-proteinase K for 48 hr at 50.degree. C.
with an additional 1 .mu.g proteinase K added twice within each 24
hr period. DNA was extracted and bisulfite modification was
performed using the agarose bead technique as previously described
(Spugnardi et al 2003). Briefly, following extraction, DNA was
quantitated using Picogreen (Molecular Probes, Eugene, Oreg.) and 1
.mu.g of genomic DNA was mixed with, 0.3 M NaOH, 2 vols of 2% LMP
agarose dissolved in molecular grade water, heated at 80.degree. C.
for 10 min and then added to 2-3 drops of chilled mineral oil to
create an agarose bead. Sodium bisulfite conversion of DNA
suspended in the agarose bead was achieved by adding 2.5 M sodium
metabisulfite and 125 mM hydroquinone and incubating at 50.degree.
C. for 14 hr. Subsequently, desulphonation was performed by
evacuating residual mineral oil and adding 0.2 M NaOH.times.2 for
15 min each, followed by neutralization with 1/5 vol 1 M HCL for 5
min and then the bead was washed in Tris-EDTA buffer and stored in
molecular grade water at 4.degree. C. until analysis. A panel of
six genes was assessed for their methylation status: RASSF1A, APC,
Twist, CDH1, GSTP1 and RAR-.beta.2. MSP was performed on each bead
in a 100 .mu.l reaction containing 200 .mu.M each of dNTP and
AmpliTaq Gold DNA polymerase (Perkin Elmer, Norwalk, Conn.) and 50
pmol of each forward (F) and reverse (R) primer set for methylated
(M) and unmethylated (U) sets as follows: RAR-.beta.2, (M)
F-GAACGCGAGCGATTCGAGT (SEQ ID NO:1) and R-GACCAATCCAACCGAAACG (SEQ
ID NO:2), (U) F-GGATTGGGATGTTGAGAATGT (SEQ ID NO:3) and
R-CAACCAATCCAACCAAAACAA (SEQ ID NO:4); CDH1, (M)
F-TTAGGTTAGAGGGTTATCGCGT (SEQ ID NO:5) and
R-TAACTAAAAATTCACCTACCGAC (SEQ ID NO:6), (U)
F-TAATTTTAGGTTAGAGGGTTATTGT (SEQ ID NO:7) and
R-CACAACCAATCAACAACACA (SEQ ID NO:8); APC, (M) F-TATTGCGGAGTGCGGGTC
(SEQ ID NO:9) and R-TCGACGAACTCCCGACGA (SEQ ID NO:10), (U)
F-GTGTTTTATTGTGGAGTGTGGGTT (SEQ ID NO:11) and
R-CCAATCACAAACTCCCAACAA (SEQ ID NO:12); RASSF1A, (M)
F-GTGTTAACGCGTTGCGTATC (SEQ ID NO:13) and R-AACCCCGCGAACTAAAAACGA
(SEQ ID NO:14), (U) F-TTTGGTTGGAGTGTGTTAATGTG (SEQ ID NO:15) and
R-CAAACCCCACAAACTAAAAACAA (SEQ ID NO:16); GSTP1, (M)
F-TTCGGGGTGTAGCGGTCGTC (SEQ ID NO:17) and R-GCCCCAATACTAAATCACGACG
(SEQ ID NO:18), (U) F-GATGTTTGGGGTGTAGTGGTTGTT (SEQ ID NO:19) and
R-CCACCCCAATACTAAATCACAACA (SEQ ID NO:20); Twist, (M)
F-TTTCGGATGGGGTTGTTATCG (SEQ ID NO:21) and R-GACGAACGCGAAACGATTTC
(SEQ ID NO:22), (U) F-TTGGATGGGGTTGTTATTGT (SEQ ID NO:23) and
R-ACCTTCCTCCAACAAACACA (SEQ ID NO:24). PCR was carried out after
optimizing annealing temperatures for each primer set to include 40
timed cycles of denaturation at 94.degree. C. for 30 sec, annealing
for 30 sec, and extension at 72.degree. C. for 30 sec. Post-MSP
product analysis was performed using capillary array
electrophoresis (CEQ 8000XL Genetic Analysis System, Beckman
Coulter, Fullerton, Calif.) as described previously (Spugnardi et
al 2003).
[0095] Sequencing analysis. Sixteen primary breast tumor samples
were randomly selected and analyzed by sequencing to validate the
accuracy of the MSP assay for individualized genes. Briefly, PCR
was performed on bisulfite modified DNA in 40 .mu.l reactions with
forward and reverse primers for specific genes as previously
described (Spugnardi et al 2003) and (Hoon et al 2004). Fifteen
.mu.l of post-PCR products were resolved on 2% Tris-borate
EDTA-agarose gels and target bands were isolated and purified using
the Qiagen Gel purification kit (Qiagen Inc., Valencia, Calif.).
Sequencing reactions were performed with the dye terminator cycle
sequencing kit on the CEQ 8000XL.
[0096] Statistical analysis. Descriptive statistics, such as mean,
standard deviation, median, frequency and percentage were used to
summarize patient's characteristics and gene hypermethylation
status. T-test (for continuous variables) and chi-square test (for
categorical variables) were used for comparing clinical factors
between tumors demonstrating hypermethylation versus no
hypermethylation.
[0097] A logistic regression model was developed to investigate the
correlation of gene methylation status with lymph node metastasis
status while the effects of clinical factors on node metastasis
were taken into account. First, a stepwise procedure was used to
select clinical factors that significantly related with lymph node
metastasis status. Tumor size and estrogen receptors status (ER)
were selected in the model, a stepwise procedure was used again to
select genes that predict node metastasis status. The statistical
analysis was carried out using SAS software (SAS, Cary, N.C.) and
all tests are two-sided with significant p values.ltoreq.0.05.
Results
[0098] Promoter region CpG hypermethylation was identified in 147
(97%) of 151 primary breast tumors when evaluated for any one
marker using the following panel of genes: RASSF1A, APC, Twist,
E-cadherin, GSTP1 and RAR-.beta.2. The most frequently
hypermethylated gene detected was RASSF1A occurring in 122 (81%)
patients' tumors; this was followed by E-cadherin (53%), APC (49%),
Twist (48%), RAR-.beta.2 (24%) and GSTP1 (21%). Forty-five (30%) of
151 tumors demonstrated hypermethylation for three genes, 43 (28%)
tumors for two genes, 25 (17%) for four genes, 20 (13%) for one
gene, 10 (7%) for five genes, and 4 (3%) for all six genes. In only
four patient's tumors, hypermethylation was not detected for any of
the six genes assessed. Sequence analysis was performed on 16
primary tumors to verify the hypermethylated or unmethylated
status. In all cases, direct sequencing of the PCR product
correlated with the methylation status as initially detected by
MSP. Ten normal breast tissue samples demonstrated no promoter
hypermethylation for any of the genes assessed.
[0099] The individual gene hypermethylation status for each
patient's tumor was assessed to determine whether any clinical or
pathologic correlation could be identified for any of the following
prognostic parameters associated with breast cancer: patient's age,
menopause status, tumor size, histology, degree of differentiation,
DNA index, the presence of lymphovascular invasion, T stage, nodal
involvement, AJCC stage, hormone receptor (estrogen and
progesterone) status and HER2 receptor presence. GSTP1 methylation
was significantly more frequent in primary breast tumors
demonstrating lymph node metastasis occurring in 22 (28%) of 81
patients, as compared to 10 (14%) of 70 patients without evidence
of lymph node involvement (p=0.044). Hypermethylation of the CDH1
was more frequent in primary tumors demonstrating lymphovascular
invasion, 31 (72%) of 43 patients versus 49 (48%) of 102 patient
tumors without lymphovascular invasion (p=0.008); those with an
infiltrating ductal histology, 68 (58%) of 118 tumors as compared
to infiltrating lobular histology 12 (36%) of 33 tumors (p=0.03);
and in ER negative tumors, 27 (73%) of 37 patients' tumors versus
53 (47%) of 114 patients' tumors (p=0.005). In contrast, RASSF1A
hypermethylation was more frequently associated with ER positive
tumors occurring in 99 (87%) of 114 patients versus 23 (62%) of 37
patients with ER negative tumors (p<0.001). RAR-.beta.2
hypermethylation was more common in HER2 positive than negative
tumors, 15 (48%) of 31 cases versus 21 (19%) of 112 cases,
respectively (p<0.001). No clinical or pathologic correlations
were identified for APC or Twist hypermethylation.
[0100] In a similar manner, the combination of hypermethylated
genes was assessed to determine whether there was any predictive
correlation. The presence of hypermethylation for GSTP1 and/or
RAR-.beta.2 was more frequently associated with the presence of
lymph node metastasis and HER2 receptor positive tumors. Thirty-six
(44%) of 81 primary tumors with corresponding lymph node
involvement demonstrated hypermethylation for one or both of these
markers, whereas this event was only detected in 18 (25%) of 70
primaries without lymph node metastasis (p<0.02). Additionally,
hypermethylation for either one or both of these genes was more
often found in HER2 positive breast cancers than those that were
HER2 negative: 19 (61%) of 31 primary tumors versus 33 (30%) of 112
primary tumors, respectively (p=0.001).
[0101] It has been suggested that the amount of regional lymph node
involvement is associated with a worse patient prognosis (Rosen et
al 1981; Nasser et al 1993; and Goldstein et al 1999). To determine
whether hypermethylation profiling of the primary tumor was
predictive of lymph node tumor burden, patients were categorized
according to the size of the SLN metastasis: macro, >2.0 mm
(n=41); micro, .ltoreq.2.0 mm (n=40); and none (n=70), absence of
tumor identification following H&E and IHC staining. Among
these three groups there was a statistically significant
association between increasing SLN tumor burden and larger primary
tumor size (p<0.015). Correlation with primary tumor
hypermethylation status found a greater frequency of GSTP1
hypermethylation associated with macro-SLN metastasis, 13 (32%) of
41 patients, as compared to those without tumor cells in the SLN,
10 (14%) of 70 patients, p<0.029. RAR-.beta.2 hypermethylation
was more common in those tumors having macro-SLN metastasis, 17
(42%) of 41 patients, versus micro-SLN metastasis, 6 (15%) of 40
patients, or no SLN metastasis, 13 (19%) of 70 patients,
(p<0.009 for each, respectively). Similarly, the presence of
either GSTP1 hypermethylation, RAR-.beta.2 hypermethylation, or
both was more frequently observed in primary tumors having
macro-SLN metastasis, 23 (56%) of 41 patients, than micro-SLN
metastasis, 12 (30%) of 40 patients, or no SLN metastasis, 18 (26%)
of 70 patients, p values<0.018 and 0.002, respectively.
[0102] A logistic regression model was developed to investigate the
correlation of gene methylation status with SLN tumor status while
the effects of clinical factors on node metastasis were taken into
account. Only tumor size and RAR-.beta.2 gene hypermethylation were
significantly associated with a greater risk for a macro-SLN
metastasis as compared to micro- or no SLN involvement, odds ratio
of 1.5 (95% Cl, 1.16 to 1.93; p<0.002) and 3.86 (95% Cl, 1.65 to
9.00; p<0.002). Similarly, in multivariate analysis, the
presence of either GSTP1 hypermethylation, RAR-.beta.2
hypermethylation or both markers in primary tumors correlated with
an increased risk of having a macroscopic SLN metastasis, odds
ratio 4.59 (95% Cl, 2.02 to 10.4; p<0.001). Increasing primary
tumor size was also associated with a greater risk for macro-SLN
metastasis, odds ratio 1.57 (95% Cl, 1.21 to 2.05; p<0.001). No
clinical, pathologic or hypermethylation gene marker variables
could discriminate between microscopic SLN metastasis and
histologically tumor-free SLN.
TABLE-US-00005 TABLE 5 Correlation between primary tumor gene
hypermethylation marker and patient tumor characteristics Gene
hypermethylation and tumor histopathology P-value GSTP1 Lymph node
positive 0.044 CDH1 Lymph node positive 0.008 Infiltrating ductal
histology 0.03 Estrogen receptor positive 0.005 RASSF1A Estrogen
receptor positive <0.001 RAR-.beta.2 Her2 receptor positive
<0.001 GSTP1 and/or RAR-.beta.2 Lymph node positive <0.02
Her2 receptor positive 0.001
TABLE-US-00006 TABLE 6 Correlation between primary tumor gene
hypermethylation marker and SLN histology status Gene
hypermethylation and SLN histopathology P-value GSTP1 Macro vs.
None <0.015 RAR-.beta.2 Macro vs. Micro <0.009 Macro vs. None
<0.009 GSTP1 and/or RAR-.beta.2 Macro vs. Micro <0.018 Macro
vs. None <0.002 Macro, metastasis >2 mm; Micro, metastasis
.ltoreq.2 mm; None, no metastasis detected by H&E and IHC
Discussion
[0103] Breast cancer remains the most frequently diagnosed
malignancy in women (Jemal et al 2003), the incidence of which
continues to rise yearly. As of lately, many of these detected
cases of breast cancer are smaller in size than those reported in
previous decades and therefore less likely to be associated with
overt lymph node metastasis (Chu et al 1996). Because prospective
randomized trials have not demonstrated a survival advantage with
up-front ALND, its therapeutic utility in this new era of breast
cancer diagnosis will only further diminish (Fisher et al 2002).
Regardless, at present, tumor status of the axillary lymph nodes is
the single most important clinically used predictor of patient
outcome to date (Mansour et al 1994). Furthermore, lymph node
evaluation remains a mainstay for disease staging, as a treatment
stratification factor, and for assessing overall patient
prognosis.
[0104] The more frequent utilization of chemotherapy in patients
with node-negative breast cancer may further contribute diminishing
role of SLN biopsy. In addition there has been a dramatic increase
in breast conserving therapy, which entails local radiation. The
additional effects of these local and systemic therapies on minimal
residual disease in axillary lymph nodes is at present unknown, but
may prove beneficial and further reduce the need for lymph node
surgery in patients with early stage breast cancer.
[0105] Discrimination of primary tumors based on molecular
characteristics may prove useful for predicting lymph node
metastasis, risk of recurrence, and improving our understanding of
the etiologic events that promote disease spreading (Isaacs et al
2001). Advantages of gene-based assays are their rapidity for
assessment, widespread use of currently implemented technology,
objectivity of results and requirement of only a minimal amount of
sample without imposing excessive demands on stringent collection
and processing techniques. Additionally, DNA assay studies allow
for easy evaluation of available paraffin-embedded tissue
specimens. Finally, DNA-based assays offer an alternative to
RNA-based approaches, which may be affected by heterogeneity,
variations in the levels of gene expression, and most importantly
RNA degradation. These factors have proven problematic in
large-scale studies.
[0106] This study provides the largest series to date with
correlation to known prognostic factors in breast cancer to
determine the role of gene promoter hypermethylation status as a
molecular predictor of disease progression. We found GSTP1
methylation to correlate strongly with increasing tumor size and a
greater likelihood of SLN metastasis. This finding is important, as
LSTs are a family of enzymes that detoxify hydrophobic
electrophiles, which include carcinogens that have been implicated
in a variety of cancers (Henderson et al 1998). GSTP1 loss appears
to be an early event in the development of prostate cancer and loss
of this enzyme may impair cellular defenses leading to increasing
genome instability and cancer progression (Nelson et al 2002).
Because breast cancer is similarly a hormone mediated malignancy
and epidemiologic studies have shown diet and its components as
potential contributing factors to its development, this same enzyme
may be critical in this disease as well (Clavel-Chapelon et al 1997
and Krajinovic et al 2001). Additional studies using large-scale
populations will better identify these risks and characterize the
potential impact of gene-environment interactions.
[0107] Hypermethylation of RAR-.beta.2 was shown to correlate more
frequently with HER2 positive tumor, which is over-expressed in
25-30% of all breast cancers and when identified is associated with
a poorer patient prognosis. Retinoids have been shown to inhibit
the growth of breast cancer cell lines in culture and breast tumors
in animal models [Lacroix et al 1980; Fraker et al 1984; and
Gottardis et al 1996). RAR-.beta.2 has been proposed as a tumor
suppressor gene and loss of expression has been found in variety of
tumors as well as premalignant lesions resulting in uncontrolled
cellular proliferation (Martinet et al 2000 and Sun et al 2002).
Detection of RAR-.beta.2 hypermethylation may identify additional
therapeutic targets of interest in these groups of patients with
more aggressive tumors. The correlation of RAR-.beta.2 with the
presence of macroscopic SLN metastasis is significant. Tumor burden
in the lymph nodes is a significant prognosticator of patient
outcome. However, the clinical implication of occult tumor cells in
lymph nodes remains a controversial issue (Dowlatshahi et al 1997
and Cote et al 1999). We have demonstrated that patients with SLN
micrometastasis (.ltoreq.2.0 mm) have equivalent overall survival
rate as those without SLN metastasis and both groups have a better
outcome than those with SLN macrometastasis (>2.0 mm) (Hansen et
al 2001). Genetic markers that predict for lymph node metastasis
may avoid further surgery in patients with clinically insignificant
disease in their axilla and better identify those more likely to
benefit from the addition of systemic therapy. Methylation status
of the RAR-.beta.2 may identify patients suitable for enrollment
into clinical trials employing retinoids (Lawrence et al 2001 and
Singletary et al 2002).
[0108] CDH1 hypermethylation was highly associated with ER negative
tumors. E-cadherin is involved in cell-to-cell adhesion and the
metastasis process. Loss of heterozygosity for this gene with near
complete absence of CDH1 protein expression is highly common for
invasive lobular breast cancers, whereas tumors of ductal histology
often present with varying levels of expression (Asgeirsson et al
2000 and Cleton-Jansen et al 2002) Promoter region hypermethylation
may provide an alternative mechanism to account for this finding in
ductal carcinomas.
[0109] Promoter region hypermethylation is a common epigenetic
event that has been shown to occur among a variety of different
tumor types affecting multiple genes that regulate cell cycling,
signal transduction, gene transcription, angiogenesis, adhesion and
metastasis. Hypermethylation profiling of specific genes in cancers
can characterize those genetic alterations associated with
tumorigenesis and metastasis (Krassenstein et al 2004).
Identification of specific tumor-associated genetic events can
potentially account for the pathobiology of tumor progression and
provide molecular markers for assessing patient risk, monitoring
tumor progression and predicting response to therapy. In addition,
this approach allows for the detection of alterations in pathways
critical to maintaining cell integrity, stability, survival and
chemoresistance which can identify unique patient-specific targets
to customize therapies for improved treatment response.
Furthermore, development of epigenetic therapeutic protocols may
prove useful in the future as preventatives for individuals at high
risk for breast cancer. Molecular events associated with the
primary tumor that predict for metastasis and patient outcome
offers the desired opportunity to avoid additional surgical
interventions for staging and will prove more suitable in this new
era of earlier cancer detection.
Example 3
Profiling Epigenetic Inactivation of Tumor Suppressor Genes in
Tumors and Plasma From Cutaneous Melanoma Patients
Introduction
[0110] Epigenetic events in the form of hypermethylation of TSG
promoter region(s) CpG islands can play a role in the development
and progression of various cancers (Baylin et al 2000; Esteller et
al 2001; Jones et al 2002; and Sidransky et al 2002). The detection
of hypermethylated genes in tumors has become important in
assessing the mechanisms of known and candidate TSG inactivation.
Genes can be transcriptionally silenced when their promoter
region(s) CpG islands are hypermethylated (Jones et al 2002).
Recent studies have shown this is a significant mechanism whereby
TSG expression is shut off in cancer cells (Baylin et al 2000;
Esteller et al 2001; Jones et al 2002; Sidransky et al 2002). The
hypermethylation status of several known or candidate TSG promoter
regions has been profiled for a number of cancers (Esteller et al
2002; Harden et al 2003; Jeronimo et al 2001; Jones et al 2002; Lo
et al 2001; Pfiefer et al 2002; Rosas et al 2001; Toyooka et al
2003; Widschwendter et al 2002). This epigenetic regulation of TSG
can provide a selective advantage for cells undergoing
transformation or progressing to a more malignant phenotype. In the
past, considerable effort was devoted to correlating known or
candidate TSG deletions and mutations to phenotypic properties.
Recent studies have indicated that inactivation of specific TSGs
significantly influences tumor promotion and progression in
carcinomas (Harden et al 2003; Jeronimo et al 2001; Jones et al
2002; Lo et al 2001; Rosas et al 2001; Toyooka et al 2003; and
Widschwendter et al 2002).
[0111] Most studies on hypermethylation of gene promoter regions
have focused on carcinomas; no major study has addressed
hypermethylation of TSG in cutaneous melanomas. The genetic
mechanisms involved in melanoma tumor progression are poorly
understood. BRAF mutation V599 (Davies et al 2002) and 9p21 region
chromosome deletions (Fujiwara et al 1999) are the major genetic
aberrations frequently (>40%) found so far in sporadic primary
or metastatic cutaneous melanomas. The frequency of other
tumor-related gene mutations or deletions is less than 25% in
melanomas. This suggests that there are potential genetic
aberrations that have yet to be identified. We recently reported on
the frequent hypermethylation (>40%) of RASSF1A in melanoma cell
lines and frozen metastatic melanoma specimens (Spugnardi et al
2003). Although its function remains uncertain, RASSF1A is
considered a strong candidate as a TSG.
[0112] To date, there has been no major study profiling
hypermethylation of known or potential TSGs of cutaneous melanomas.
We assessed the hypermethylation status of several known or
candidate TSG promoter regions in melanoma cell lines and in frozen
and paraffin-embedded melanoma tissues. Several major TSG were
frequently hypermethylated in primary tumors and more so in
metastatic tumors. Some prominently methylated genes in carcinomas
were infrequently methylated in melanomas.
[0113] Tumor-related DNA circulates in serum/plasma of patients
with melanoma and other types of tumors (Fujiwara et al 1999;
Johnson et al 2002; Sidransky et al 2002; and Usadel et al 2002).
Studies in melanoma patients have shown that specific
microsatellites with loss of heterozygosity (LOH) on different
chromosomes are frequent with disease progression (Fujiwara et al
1999). Recent studies have shown that hypermethylated tumor-related
DNA can be detected circulating in blood (Sidransky et al 2002 and
Usadel et al 2002). We examined the feasibility of detecting
hypermethylated TSG in plasma of melanoma patients. Circulating DNA
of three hypermethylated genes was demonstrated in plasma of
melanoma patients.
Materials and Methods
[0114] Cell Lines and Tissues. Fifteen established melanoma cell
lines were cultured in growth medium and prepared for DNA
extraction as previously described (Fujiwara et al 1999). Frozen
metastatic melanoma tumor specimens (n=53) were obtained from 44
patients who underwent elective surgery at John Wayne Cancer
Institute, Saint John's Health Center, Santa Monica, Calif. Frozen
tumor-draining lymph nodes (n=10), histopathology tumor negative by
immunohistochemistry, were obtained from melanoma patients having
elective surgery. Paraffin-embedded metastatic tumor tissues (n=33)
and primary tumors (n=20) from melanoma patients were obtained from
the Division of Surgical Pathology at Saint John's Health Center.
Paraffin-embedded melanoma and breast cancer tumor-draining lymph
nodes (n=12) that were histopathology (immunohistochemistry)
negative were assessed. For the studies on paired tumors and plasma
from the same patients there were additional 24 metastatic tumors
assessed; tumors from seven pairs overlapped the initial 86
metastatic patients assessed. All patients had given signed
informed consent to participate in the studies. Human subjects IRB
approval was obtained for the use of human subjects in this study
from Saint John's Health Center and John Wayne Cancer Institute
joint committee.
[0115] Bisulfite Treatment. DNA was isolated from cell lines and
frozen tissues using DNAzol Genomic DNA Isolation Reagent
(Molecular Research Center, Inc., Cincinnati, Ohio) according to
the manufacturer's recommendations. Paraffin-embedded tumor DNA was
extracted as previously described (Fujiwara et al 1999). Following
extraction, DNA was subjected to sodium-bisulfite modification
(Spugnardi et al 2003). One .mu.g of DNA was denatured at
100.degree. C. for 10 min and quickly chilled on ice. Sodium
hydroxide (Sigma, St. Louis, Mo.) was then added to a final
concentration of 0.3 M, and the DNA incubated at 50.degree. C. for
15 min. The DNA was then mixed with 2 vol 2% LMP agarose
(BioWhittaker Molecular Applications, Rockland, Me.) and pipetted
into chilled mineral oil, forming a single bead. Four hundred .mu.l
of a 5 M bisulfite solution (pH 5.0) consisting of 2.5 M sodium
metabisulfite and 125 mM hydroquinone (Sigma) was added, and the
reaction incubated at 50.degree. C. for 14 h. The modification was
stopped by equilibrating the beads with 0.5 ml 1.times. Tris-EDTA
(TE) (6.times.15 min) followed by desulphonation using 0.2 M NaOH
(500 .mu.l, 2.times.15 min). The reactions were neutralized using
100 .mu.l (1/5 vol) 1 N hydrochloric acid (Sigma). One final TE
rinse was followed by equilibration in molecular grade H.sub.2O
(2.times.15 min). The beads were then used for MSP.
[0116] Genomic Sequencing. DNA sequences were amplified by mixing
100 ng of bisulfite treated melanoma cell line DNA with 100 pmoles
of each respective primer: MGMT, M1
5'-GGGTTATTTGGTAAATTAAGGTATAGAG-3' (SEQ ID NO:25) and M2
5'-CACCTAAAAATAAAACAAA AACTACCAC-3' (SEQ ID NO:26); RASSF1A,
R35'-GGGAGTTTGAGTTTATTGAGTTG-3' (SEQ ID NO:27) and R25'-CAC
CTCTACTCATCTATAACCCAAATAC-3' (SEQ ID NO:28); RAR-.beta.2, RA3
5'-GTGTGATAGAAGTAGTA GGAAGTGAGTTGT-3' (SEQ ID NO:29) and RA2
5'-ACTCCATCAAACTCTACCCCTTTTTTAAC-3' (SEQ ID NO:30) in a 50 .mu.l
reaction containing buffer, of dNTP and AmpliTaq gold polymerase
(Applied Biosystems, Foster City, Calif.) at 95.degree. C. for 45
s, 55.degree. C. for 45 s and 72.degree. C. for 2 min for 40
cycles. PCR products were gel purified using the QIAquick Gel
Extraction Kit (Qiagen, Valencia, Calif.) and sequenced using an
automated DNA sequencer (CEQ 8000XL DNA Analysis System, Beckman
Coulter, Fullerton, Calif.) with the respective internal primer:
MGMT, M3 5'-GTTGT(c/t)GGAGGATTAGGGT-3' (SEQ ID NO:31); RASSF1A,
R45'-TACCCCTTAACTACCCCTTCC-3' (SEQ ID NO:32), and RAR-.beta.2, RA4
5'-AATCATAAATTATAACAAACAAACCAACT-3' (SEQ ID NO:33).
[0117] Fluorescent MSP Analysis. Methylation status was assessed
for each gene using two sets of fluorescent labeled primers
specifically designed to amplify methylated or unmethylated DNA
sequence. Primer sequences are listed as methylated sense and
antisense followed by unmethylated sense and antisense, with
annealing temperatures and PCR product size:
TIMP-3,5'-CGTTTCGTTATTTTTTGTTTTCGGTTTC-3' (SEQ ID NO:34) and
5'-CCGAAAACCCCGCCTCG-3' (SEQ ID NO:35) (59.degree. C., 116 bp)
5'-TTTTGTTTTGTTATTTTTTGTTTTTGGTTTT-3' (SEQ ID NO:36) and
5'-CCCCCAAAAA CCCCACCTCA-3' (SEQ ID NO:37) (59.degree. C., 122 bp)
(19); RASSF1A, 5'-GTGTTAACGCGTTGCGTATC-3' (SEQ ID NO:13) and
5'-AACCCCGCGAACTAAAAACGA-3' (SEQ ID NO:14) (60.degree. C., 93 bp),
5'-TTTGGTTGGAGTGTGTTAATG TG-3' (SEQ ID NO:15) and
5'-CAAACCCCACAAACTAAAAACAA-3' (SEQ ID NO:16) (60.degree. C., 105
bp) (11,16); RAR-.beta.2, 5'-GAACGCGAGCGATTCGAGT-3' (SEQ ID NO:1)
and 5'-GACCAATCCAACCGAAACG-3' (SEQ ID NO:2) (59.degree. C., 142
bp), 5'-GGATTGGGATGTTGAGAATGT-3' (SEQ ID NO:3) and
5'-CAACCAATCCAACCAAAACAA-3' (SEQ ID NO:4) (59.degree. C., 158 bp)
(Evron et al 2001); MGMT, 5'-TTTCGACGTTCGTAGGTTTTC GC-3' (SEQ ID
NO:38) and 5'-GCACTCTTCCGAAAACGAAACG-3' (SEQ ID NO:39) (66.degree.
C., 81 bp), 5'-TTTGTGTTTTGA TGTTTGTAGGTTTTTGT-3' (SEQ ID NO:40) and
5'-AACTCCACACTCTTCCAAAA ACAAAAC (SEQ ID NO:41) (66.degree. C., 93
bp) (Esteller et al 1999); DAPK 5'-GGATAGTCG GATCGAGTTAACGTC (SEQ
ID NO:42) and 5'-CCCTCCCAAACGCCGA (SEQ ID NO:43) (64.degree. C., 98
bp), 5'-GGAGGATA GTTGGATTGAGTTAATGTT-3' (SEQ ID NO:44) and
5'-CAAATCCCTCCCAAACACCAA-3' (SEQ ID NO:45) (64.degree. C., 106 bp)
(Goessl et al 2000); GSTP1, 5'-TTCGGGGTGTAGCGGTCGTC-3' (SEQ ID
NO:17) and 5'-GCCCCAATACTAAATCACGACG-3' (SEQ ID NO:18) (59.degree.
C., 91 bp), 5'-GATGTTTGGG GTGTAGTGGTTGTT-3' (SEQ ID NO:19) and
5'-CCACCCCAATACTAAATCACA ACA-3' (SEQ ID NO:20) (59.degree. C., 97
bp) (Esteller et al 1999 and Zochbauer-Muller at al 2001);
p16.sup.INK4a, 5'-TTATTAGAG GGTGGGGCGGATCGC-3' (SEQ ID NO:46) and
5'-GACCCGAACCGCGACCGTAA-3' (SEQ ID NO:47) (65.degree. C., 150 bp),
5'-TTATTAGAGGGTGGGGTGGATTGT-3' (SEQ ID NO:48) and
5'-CAACCCCAAACCACAACCATAA-3' (SEQ ID NO:49) (65.degree. C., 151 bp)
(21,24); and MYOD1, 5'-CCAACTCCAAATCCCCTCTCTAT-3' (SEQ ID NO:50)
and 5'-TGATTAATTTAGATTGGGTTTAGAGAAGGA-3' (SEQ ID NO:51) (60.degree.
C., 162 bp) (Eads et al 1999). One hundred ng of bisulfite-modified
DNA was subjected to PCR amplification in a final reaction volume
of 20 .mu.l containing PCR buffer, 2.5-4.5 mM MgCl.sub.2, dNTPs,
0.3 .mu.M primers, BSA and 0.5 U of AmpliTaq gold polymerase
(Applied Biosystems). PCR was performed with an initial 10 min
incubation at 95.degree. C., followed by 40 cycles of denaturation
at 95.degree. C. for 30 s, annealing for 30 s, and extension at
72.degree. C. for 30 s, and a final seven min hold at 72.degree. C.
Sodium-bisulfite modified lymphocytes from healthy donors were used
as positive unmethylated control, SssI Methylase (New England
BioLabs, Beverly, Mass.) treated modified lymphocytes were used as
a positive methylated control, and unmodified lymphocytes were used
as a negative control for methylated and unmethylated reactions.
PCR products were visualized using capillary array electrophoresis
(CAE; CEQ 8000XL). The assay was set up in a 96-well microplate
format. Multiple PCR products can be assayed in the same well for
comparison. Methylated and unmethylated PCR products from each
sample were assessed simultaneously by labeling forward primers
with a choice of three Beckman Coulter WeIIRED Phosphoramidite
(PA)-linked dyes (Genset Oligos, Boulder, Colo.). Forward
methylated specific primer was labeled with D4pa dye and forward
unmethylated specific primer was labeled with D2pa dye. One .mu.l
of methylated PCR product and one .mu.l of unmethylated PCR product
were mixed with 40 .mu.l loading buffer and 0.5 .mu.l dye-labeled
size standard (Beckman Coulter Inc). The CAE analysis detects the
different dyes and displays them in respective colors.
[0118] RT-PCR Analysis. Total cellular RNA was extracted from
melanoma cell lines using TriReagent (Molecular Research Center,
Cincinnati, Ohio). RT-PCR was performed as previously described
(Takeuchi et al 2003). Briefly, all RT reactions were carried out
with oligo-dT priming using 1 .mu.g of total RNA. Resulting cDNA
was subjected to PCR conditions of 95.degree. C. for 30 s,
annealing for 30 s, and 72.degree. C. for 1 min, for 35 cycles for
MGMT, RASSF1A and RAR-.beta.2 and 25 cycles for the GAPDH
housekeeping-gene. All samples were assessed for presence of GAPDH
mRNA. All PCR products were separated on 2% Tris-borate EDTA
agarose gels and stained with ethidium bromide as previously
described (Spugnardi et al 2003).
[0119] Reexpression of MGMT, RAR-.beta.2, RASSF1A genes. Several
cell lines were grown for four days in T75 cm.sup.2 tissue culture
flasks in the presence of 0, 3, or 5 .mu.M of 5Aza-dC (Sigma) as
previously described (Spugnardi et al 2003). An additional flask of
cells was grown in the presence of 5 .mu.M 5Aza-dC for four days
followed by treatment with 1 .mu.M ATRA (Sigma) for 24 h. RNA was
isolated and RT-PCR was performed as described above to analyze for
RASSF1A, MGMT and RAR-.beta.2 gene reexpression.
[0120] Plasma DNA Isolation and Methylation Analysis. DNA was
extracted from plasma as previously described (Taback et al 2001).
Briefly, 500 .mu.l of plasma was diluted with 0.9 M NaCl.sub.2,
SDS, and proteinase K (QIAGEN) and incubated at 50.degree. C. for 3
h. An equal volume of phenol-chloroform isoamyl alcohol (25:24:1)
was then added and the sample was vortexed vigorously. After
centrifugation at 1000.times.g for 10 min the aqueous layer was
collected and phenol-chloroform isoamyl alcohol was again added.
DNA was precipitated using pellet paint NF co-precipitant (Novagen,
Madison, Wis.) and isopropanol.
[0121] Extracted DNA was subjected to sodium bisulfite
modification. Briefly, DNA extracted from 500 .mu.l of plasma and
supplemented with 1 .mu.g salmon sperm DNA (Sigma) was denatured in
0.3 M NaOH for 15 min at 37.degree. C. Five hundred fifty .mu.l of
a 2.5 M sodium bisulfite/125 mM hydroquione solution was then added
and samples were incubated under mineral oil in the dark for 3 h at
60.degree. C. Salts were removed using the Wizard DNA Clean-Up
System (Promega, Madison, Wis.) and samples were then desulfonated
in 0.3 M NaOH at 37.degree. C. for 15 min. Modified DNA was
precipitated with ethanol using Pellet Paint NF (Novagen) as a
carrier and then resuspended in molecular grade H.sub.2O.
[0122] The methylation status of the bisulfite-treated DNA was
determined using primers and probes specifically designed to
amplify methylated gene promoter regions. Quantitative RealTime PCR
was preformed as previously described (Takeuchi et al 2003).
RealTime PCR reactions were run on the iCycler iQ RealTime
thermocycler (Bio-Rad Laboratories, Hercules, Calif.). Analysis
involved 25 .mu.L PCR reaction containing sodium bisulfite treated
DNA template, 0.8 .mu.M of each primer, 0.4 fluorescence resonance
energy transfer probe, AmpliTaq gold polymerase (Applied
Biosystems), dNTPs, MgCl.sub.2, BSA, and AmpliTaq PCR buffer.
Amplification conditions were 95.degree. C. for 10 min followed by
55 cycles of denaturation at 95.degree. C. for 1 min, annealing at
60.degree. C. for MYOD1 and RASSF1A (annealing at 59.degree. C. for
RAR-.beta.2 and 66.degree. C. for MGMT) and extension at 72.degree.
C. for one min. Each PCR plate contained positive controls
including melanoma cell lines shown to be methylated for the gene
being assessed as well as SssI treated healthy donor lymphocytes.
Negative controls included healthy donors plasma DNA and reactions
which contained no template DNA.
[0123] Realtime PCR assay was performed to obtain the approximate
number of methylated gene copies present in a sample. The internal
reference gene MYOD1 was used to amplify sodium bisulfite treated
DNA independent of methylation status to confirm presence of
modified DNA (9). In addition, a standard curve was constructed
with serial dilutions of 10.sup.1 to 10.sup.5 copies of the
targeted TSG promoter region template. Copy numbers for the
individual samples were established using the standard curve.
Primer sequences for the realtime PCR were the same as for the CAE
analysis while the probe sequences were as follows: MYOD,
5'-CCCTTCCTATTCCTAAATCCAACCTA-3' (SEQ ID NO:52); MGMT,
5'-CGTTTGCGATTTGGTGAGTGTT TGGG-3' (SEQ ID NO:53); RASSF1A,
5'-CAACTACCGTATAAAATTACACGCGATACCCCG-3' (SEQ ID NO:54); and
RAR-.beta.2, 5'-CCGAATACGTTCCGAATCCTACCCCG-3'(SEQ ID NO:55).
Results
[0124] Methylation profiling of melanoma cell lines. Initially,
seven known or candidate TSG were assessed for aberrant methylation
of CpG promoter regions in melanoma cell lines. Methylation status
was analyzed using MSP and assessed by gel electrophoresis
initially to verify specific bands. The assay was converted for
automated CAE analysis which provided a more objective detection
system for methylation status, allowing both methylated and
unmethylated MSP products to be assessed simultaneously in the same
analysis. A comparison of CAE versus gel electrophoresis
demonstrated >95% concordance of results. All subsequent tissue
analyses were performed by CAE. The frequency of promoter
hypermethylation in the genes DAPK, GSTP1, MGMT, p16.sup.INK4a,
RAR-.beta.2, RASSF1A, and TIMP-3 was assessed by MSP in 15
established melanoma cell lines. The most frequent hypermethylated
gene was RASSF1A followed by RAR-.beta.2 and MGMT (Table 7).
Overall, 14 (93%) of the lines were hypermethylated for one or more
of the seven genes. Eight (53%) cell lines had two or more
hypermethylated genes, and two (13%) cell lines had three
hypermethylated genes. Positive controls for methylated gene
promoter regions included known hypermethylated cell lines and
bisulfite-modified SssI treated normal donor PBLs. Negative
controls for both methylated and unmethylated primer sets included
unmodified (wild-type) DNA and reagent controls. Under the
conditions used normal (histopathology tumor negative) frozen lymph
nodes (n=10) and healthy donor PBLs did not show methylation for
any gene except DAPK, which was positive in PBLs from two healthy
donors.
TABLE-US-00007 TABLE 7 Methylation of Gene Promoter CpG Islands in
Melanoma Cell Lines Melanoma cell lines RAR-.beta.2 RASSF1A MGMT
DAPK GSTP1 TIMP3 p16.sup.INK4a MA - + - - - - - MB - - - - - - - MC
- + - - - - - MD + - + - - - - ME + + - - - - - MF - + - - - - - MG
- + - - - - - MH + + + - - - - MI + + - - - - - MJ + + - - - - - MK
+ + + - - - - ML + - - - - - - MM + + - - - - - MN - + - - - - - MO
- + + - - - - Total 8/15 12/15 4/15 0/15 0/15 0/15 0/15 (53%) (80%)
(27%) (0%) (0%) (0%) (0%)
[0125] Methylation profiling of melanoma tumors. We next assessed
53 frozen metastatic melanoma tumor tissues obtained from 44 AJCC
stage III/IV melanoma patients (FIG. 1, Table 8). Hypermethylation
was detected in one or more genes of 51 (96%) tumors, two or more
genes of 34 (64%) tumors, and three or more genes of 13 (25%)
tumors (Table 9). The four most frequently hypermethylated genes
were RAR-.beta.2, RASSF1A, MGMT, and DAPK, respectively (Table 8).
The other three genes were hypermethylated in less than 10% of the
tumors.
TABLE-US-00008 TABLE 8 Detection of Hypermethylated Genes in
Melanoma Tumors MGMT RAR-.beta.2 RASSF1A DAPK Primary tumors
Paraffin n = 20 2 (10) 14 (70) 3 (15) 0 (0) Metastatic tumors
Frozen n = 53 20 (38) 38 (72) 31 (58) 13 (25) Paraffin n = 33 9
(27) 22 (67) 18 (55) 3 (9) Total n = 86 29 (34) 60 (70) 49 (57) 16
(19) ( ) percentage
TABLE-US-00009 TABLE 9 Frequency of Hypermethylated Genes in
Melanoma Tumors Primary tumors Genes.sup.a Frozen n = 0 Paraffin n
= 20 Total N = 20 0 N/A 5 (25) 5 (25) .gtoreq.1 N/A 15 (75) 15 (75)
.gtoreq.2 N/A 4 (20) 4 (20) .gtoreq.3 N/A 0 (0) 0 (0) Metastatic
tumors Genes* Frozen n = 53 Paraffin n = 33 Total N = 86 0 2 (4) 1
(3) 3 (3) .gtoreq.1 51 (96) 32 (97) 83 (97) .gtoreq.2 34 (64) 17
(52) 51 (59) .gtoreq.3 13 (25) 3 (11) 16 (19) 4 4 (8) 0 (0) 4 (5) (
) percentage .sup.aGenes assessed were: RAR-.beta.2, MGMT, DAPK,
and RASSF1A.
[0126] Because molecular assessment of frozen tumor tissues is
hampered by availability, size of lesion surgically removed,
logistics of tissue procurement, not knowing the level of
contamination with hemopoietic cell infiltrates and normal cells,
the MSP assay was adapted for analysis of paraffin-embedded tumor
tissue. We focused on the four genes most frequently
hypermethylated in frozen melanomas: RAR-.beta.2, RASSF1A, MGMT,
and DAPK. We initially assessed 11 paired frozen and
paraffin-embedded melanomas to verify that the sensitivity of the
assay was equivalent for both types of tissues. There was 100%
concordance for all four markers between frozen and
paraffin-embedded melanomas.
[0127] Primary melanomas were assessed only from paraffin-embedded
tissues opposed to fresh frozen tissues due to the lesion size, and
logistics in procurement for pathology analysis. Paraffin-embedded
primary melanomas (n=20) and metastatic melanomas (n=33) were
assessed (FIG. 1, Table 8). The most frequently hypermethylated
genes for primary tumors were RAR-.beta.2, RASSF1A, and MGMT,
respectively. Surprisingly, RAR-.beta.2 was hypermethylated in 70%
of the primary tumors. Fifteen (75%) tumors had one or more genes
methylated, and only four (20%) had two or more genes methylated.
Of the metastatic tumors 32 (97%) had one or more genes methylated,
and 17 (52%) had two or more genes methylated (Table 9). Overall,
metastatic tumors had a higher frequency of hypermethylated genes.
Paraffin-embedded histopathology tumor-negative lymph nodes (n=12)
were negative for hypermethylation of RAR-.beta.2, RASSF1A, and
MGMT (FIG. 1). Analysis of the combination of metastatic (frozen
and paraffin-embedded) melanoma tumors (n=86) is shown in Table 9.
There was no significant association of hypermethylation between
any individual genes, nor was gene hypermethylation correlated with
disease outcomes. However, RAR-.beta.2 significantly (p=0.009)
correlated with primary tumor Breslow thickness. This correlation
is important in that Breslow thickness is a major prognostic factor
in early stage melanoma patients with localized disease.
[0128] Gene reexpression by demethylation. Expression of MGMT,
RASSF1A, and RAR-.beta.2 was assessed in eight melanoma cell lines
by RT-PCR. No gene transcripts were detected in cell lines
exhibiting hypermethylation. Gene expression was detected in the
non-methylated cell lines and in some partially hypermethylated
cell lines.
[0129] Melanoma cell lines that were hypermethylated were treated
with the DNA methylation inhibitor 5Aza-dC to reverse epigenetic
transcriptional silencing caused by methylation (16). Melanoma cell
lines were treated with 0, 3, and 5 .mu.M of 5Aza-dC for four days.
Treatment with 5Aza-dC induced significant elevation of gene
expression in all methylated cell lines, of which mRNA expression
for was absent or very minimal prior to drug treatment (FIG. 2).
Melanoma cell lines exhibiting no hypermethylation of the gene
promoter region for MGMT and RASSF1A demonstrated significant
elevation of mRNA levels. For assessment of hypermethylated
RAR-.beta.2 in cell lines, cells were treated with 5Aza-dC for four
days followed by treatment with 1 .mu.M ATRA for 24 h. Methylation
was reversed in all treated cell lines and RAR-.beta.2 mRNA
expression elevation was detected. Cell lines demonstrating
hypermethylated product by MSP showed no mRNA expression. For some
of the genes in the cell lines, there was moderate to low levels of
hypermethylated product produced by MSP with minimal gene
expression before drug treatment. Gene expression was significantly
elevated in all cell lines after drug treatment. These in in vitro
studies are suggestive that hypermethylation of the promoter region
of the genes assessed is an active mechanism of silencing gene
expression.
[0130] Bisulfite sequencing was carried out on multiple cell lines
to confirm the methylation status of MGMT, RASSF1A, and RAR-.beta.2
CpG island promoter regions and confirm the MSP results.
Representative examples of sequencing is shown in FIG. 3.
Sequencing and MSP of individual gene hypermethylation were
concordant.
[0131] Analysis of circulating methylated DNA in plasma. We
examined the presence of methylated DNA circulating in plasma of
melanoma patients using realtime PCR. A realtime MSP assay was
developed for detection of methylated DNA in plasma because the
level of DNA is significantly lower in plasma than tissues. An
assay using a reference gene (MYOD1) and standard curves for
individual markers was developed to assess realtime MSP results.
The average DNA recovered from normal donor controls was 18.15 ng
DNA/500 .mu.l (range 5.31-42.92) and from melanoma patients 29.24
ng DNA/500 .mu.l (range 9.97-166.87). In this study, we assessed
RAR-.beta.2, RASSF1A, and MGMT, the three genes most frequently
hypermethylated in tumors. Thirty-one AJCC stage III/IV patients in
whom we had paired plasma and melanoma tumor tissues comprised the
study group. Selection was based on availability of paired samples.
Plasma was obtained from a preoperative blood specimen. The most
frequently methylated gene in plasma was RASSF1A (n=6; 19%)
followed by MGMT (n=6; 19%) and RAR-.beta.2 (n=4; 13%). Of the 31
patients, 29% had at least one gene hypermethylated and 16% of the
patients had a least two genes hypermethylated in their plasma.
Plasma from 33 healthy normal donors was negative for
hypermethylation of all three genes. Analysis of methylation for
MYOD1 gene was run on all samples for verification of modification
and detection of DNA. Concordance of plasma gene hypermethylation
status to respective paired tumors was as follows: MGMT (6 of 17;
33%); RASSF1A (5 of 20; 24%); and RAR-.beta.2 (4 of 20; 18%). This
suggests that there may be degradation or limited release of these
DNA markers. In two patients, hypermethylation of RASSF1A was
absent in tumors but present in plasma. This may be due to other
metastases not surgically excised or subclinical disease. These
preliminary studies suggest hypermethylated genes can be detected
in accellular plasma of melanoma patients. Further detailed studies
will validate the clinical utility of these DNA markers.
Discussion
[0132] Hypermethylation of gene promoter regions silences genes in
many types of carcinomas. Profiling studies have shown gene
hypermethylation frequency and specific genes for tumors of
different histological origins (Chen et al 2003; Esteller et al
2001; Maruyanma et al 2001; Pfeifer et al 2002; Toyooka et al 2003;
and Widschwendter et al 2002). Patterns of gene hypermethylation in
primary tumors versus metastases, and their association with
clinicopathological factors are not well described. However, there
is clear indication that hypermethylation of TSG promoter regions
is a significant mechanism by which gene transcription is turned
off in cancer cells. Studies of tumor cell lines may not be
accurate as to the actual frequency of hypermethylation of gene
promoter regions in tumor specimens (Paz et al 2003; Smiraglia et
al 2001; and Ueki et al 2000). Hypermethylation of specific genes
in cell lines may represent clonal selection during culture
adaptation and passaging. Our major finding is that
hypermethylation of promoter regions of known and candidate TSGs in
melanomas is quite frequent. Hypermethylation of a gene can be
potentially used as a surrogate of altered gene expression patterns
to characterize phenotypic behavior.
[0133] At least one of seven genes was hypermethylated in 93% of
cell lines, a frequency that suggest a relation between
hypermethylation and melanoma progression. The study demonstrated
that several frequently methylated TSGs in carcinomas were also
found in primary and metastatic cutaneous melanomas. Interestingly,
the three genes most commonly hypermethylated in cell lines, two
were slightly more hypermethylated and one was more hypomethylated
in metastatic tumors. Recent studies have reviewed the comparison
of cell lines and tumors and have come up with different
conclusions (Paz et al 2003; Smiraglia et al 2001; and Ueki et al
2000). However, one has to be careful in comparing cell lines to
tumors; an important consideration is whether the tumor is a
primary or metastatic lesion or one of multiple lesions. In our
study, hypermethylation was less marked in primary tumors compared
to cell lines and metastases. One exception was RAR-.beta.2 where
both primary and metastatic tumors demonstrated higher levels of
hypermethylation than cell lines.
[0134] RAR-.beta.2 was the most frequent hypermethylated gene in
the panel of seven genes assessment. This is the first major report
in describing hypermethylation of RAR-.beta.2 in melanoma in a
large series of tumor specimens. Previous studies have demonstrated
the frequent hypermethylation of this gene in breast and lung
carcinomas (Paz et al 2003; Sirchia et al 2002; and Widschwendter
et al 2000). RAR-.beta.2 is a member of the nuclear retinoid
receptor of genes, family referred as retinoic acid receptors (RAR)
(Mangelsdorf et al 1995), which are frequently turned off or not
expressed in a number of carcinomas. The loss of RAR-.beta.2 has
been implicated in tumorigenesis. Interestingly, the frequency
(70%) of RAR-.beta.2 hypermethylation was similar among primary and
metastatic tumors. This is one of the highest frequencies of
genetic aberration reported for sporadic primary melanomas. BRAF
mutation in primary tumors is about 31% (Shinozaki et al;
unpublished data). The inhibition of transcription of RAR-.beta.2
may be a key factor in sporadic cutaneous melanoma tumor
development. RAR-.beta. loss has been demonstrated as a biomarker
of bronchial preneoplasia (Kurie et al 2003). We demonstrated a
significant correlation between hypermethylation of RAR-.beta.2 and
increasing primary tumor Breslow thickness which is a major
prognostic factor for early-stage melanoma (Bostick et al 1999).
Silencing of RAR-.beta.2 may be a key epigenetic factor in
melanocyte transformation and primary lesion progression. Further
studies are needed to identify RAR-.beta.2 loss during melanocyte
and nevus transformation to melanoma.
[0135] Retinoic acid treatment can induce differentiation and
inhibition of proliferation in selective melanoma cells (Demary et
al 2001). The variable responsiveness of melanomas has not been
understood. It has also been shown that retinoic acid can activate
RAR-.beta. receptors (Spanjaard et al 1997). Further studies may be
warranted to examine strategic molecular targeting of therapeutics
based on RAR-.beta. status in melanoma.
[0136] The second most frequently hypermethylated gene was RASSF1A.
This larger study supported our previous report that RASSF1A is
frequently methylated in metastatic melanomas (Spugnardi et al
2003). The 42% higher rate of RASSF1A hypermethylation in
metastatic versus primary tumors suggests that hypermethylation of
RASSF1A is likely to be acquired during tumor progression. Few
published studies have compared hypermethylation of genes in
primary and metastatic tumors of the same tumor type. The
functional role of RASSF1A is still not clear. However, its
inactivation as a TSG in multiple types of cancers has been
demonstrated (Damann et al 2000; Dammann et al 2001; Lo et al 2001;
Pfiefer et al 2002; and Spugnardi et al 2003).
[0137] The third most commonly gene hypermethylated gene was MGMT;
its rate of hypermethylation was 24% higher in metastases than in
primary tumors. MGMT, a DNA repair gene, serves as a key regulator
of genome integrity. Studies have shown that MGMT expression
protects mammalian cell lines from spontaneous G:C to A:T
transitions (Christmann et al 2001). Melanoma is known to have
acquired resistance to antineoplastic agents such as alklylating
drugs exhibiting methylating and chlorethylating properties such as
dacarbazine, procarbazine, and temozolomide (Christmann et al
2001). The overall frequency of DAPK, p16.sup.Ink4a and GSTP1 gene
often found hypermethylated in carcinomas, was quite low in
melanoma. The three major genes we assessed were frequently
hypermethylated in both primary and metastatic melanomas. There are
likely other TSGs and tumor-related genes inactivated through
hypermethylation during melanoma progression. A more global
screening approach is needed such as DNA methylation microarray
analysis (Shi et al 2003) that will allow assessment of multiple
genes for multiple specimens.
[0138] Previously, we have demonstrated circulating DNA in plasma
in the form of LOH of microsatellites in melanoma patients
(Fujiwara et al 1999 and Taback et al 2001). In the present study,
we demonstrated that melanoma patients have circulating
hypermethylated DNA in their plasma. The three most common genes in
melanoma tumors were detected in plasma at a lower frequency. A
quantitative realtime PCR assay was developed to improve
sensitivity and accuracy of methylated DNA in plasma. The assay was
100% specific as no normal donors' plasma under the assay
conditions was positive. Future studies need to optimize the assay
to obtain high sensitivity for early disease diagnosis. This is the
first major study demonstrating the presence of a significant
number of melanoma patients with circulating methylated DNA
markers. The half-life of individual genes will play a significant
role as to the value of detection of these circulating DNA.
Circulating methylation DNA markers may be used as surrogates of
subclinical disease recurrence or progression. Detailed studies on
larger cohorts of patients are needed to determine whether these
circulating methylation markers have clinical utility in predicting
disease outcome. Nevertheless, it is intriguing that circulating
methylated DNA is present in plasma and released by tumor cells.
Whether this DNA is from established metastases or circulating
tumor cells in blood needs to be determined. Further studies on
defined cohorts of melanoma patients need to be studied to
determine the potential clinicopathological utility in assessment
of melanoma patients plasma for circulating DNA.
Example 4
Prognostic Significance of Hypermethylated Tumor Suppressor Genes
in Metastatic Melanomas
Introduction
[0139] Hypermethylation (HM) of CpG islands of promoter regions of
tumor suppressor genes (TSG) silences gene expression and promotes
tumor progression. Among AJCC stage III melanoma patients who have
palpable nodes and undergo complete lymph node dissection (CLND),
there are patients who have better prognosis than others even when
matched for prognostic factors. We have discovered that the TSG RAS
association domain family protein 1 (RASSF1A) and retinoic acid
receptor .beta.2 (RARB) are HM in cutaneous melanomas. We
hypothesized that regional lymph node metastasis with HM TSGs is
predictive of a poorer disease outcome.
Methods
[0140] AJCC stage III melanoma patients (n=37) who underwent CLND
with palpable nodes were selected by the biostatistician. HM of the
promoter regions of RASSF1A and RARB using quantitative realtime
methylation-specific PCR from DNA isolated of paraffin-embedded
metastatic tumors was analyzed. Genomic copy numbers of gene
methylation were normalized and quantified with copy numbers of the
MYOD gene.
Results
[0141] The study group consisted of 10 females and 27 males (mean
age 55.7 yrs; mean Breslow thickness 2.32 mm.+-.1.46). Primary
lesions were located on the trunk (14), extremities (15), head and
neck (5), or an unknown site (3). All patients had at least 2
palpable nodes (mean 11.4; range 3-33). HM was detected for RASSF1A
alone (16%), RARB alone (28%), and both TSGs (14%). HM RARB alone
correlated with overall survival and disease-free survival by
multivariate analysis, Wald test; p=0.008 and p=0.009,
respectively. The median overall survival was 27.7 mos for nonHM
RARB vs 9.5 mos for HM RARB. The median disease-free survival was
8.5 mos for nonHM RARB vs 3.9 mos for HM RARB. RARB did not
correlate with any of the 7 prognostic factors.
Conclusions
[0142] HM of RARB in regional lymph node metastasis has prognostic
significance in prediction of disease outcome in melanoma patients.
This pilot study demonstrates that epigenetic inactivation of TSG
can be used as genomic predictive marker of disease outcome.
Example 5
Blood, Bone Marrow, and Tumor Markers for Various Types of
Cancer
Materials and Methods
[0143] BM Sample Preparation. Bone marrow was drawn and (cell-free
supernatant) plasma was immediately separated by centrifugation
(1000.times.g, 15 min), filtered through a 13-mm serum filter
(Fisher Scientific, Pittsburgh, Pa.) to remove any potential
contaminating cells, aliquoted and cryopreserved at -30.degree. C.
For normal genomic DNA controls, whole blood was collected from
each patient spotted and stored on FTA blood cards (Fitzco,
Minneapolis, Minn.) prior to DNA isolation. DNA was extracted from
one ml of BM aspirate plasma using QIAamp extraction kit (Qiagen,
Valencia, Calif.) using conventional methods (Taback et al
2001).
[0144] Preparation of Samples from Primary Tumor Tissues and LOH
Analysis of the Obtained Samples. DNA was isolated from 10 .mu.m
sections cut from paraffin-embedded tumor tissue blocks. Samples
were deparaffinized and microdissected using laser capture
microscopy (Arcturus, Mountain View, Calif.) from normal tissue.
Microdissection may also be carried out using a scalpel or needle
and a microscope or a precision laser cutting instrument. Then, DNA
was isolated, processed, purified, and quantitated.
[0145] For example, in one study, DNA was isolated by incubating
the samples with proteinase K in lysis buffer (50 mM Tris-HCl, 1 mM
EDTA and 0.5% Tween 20) at 37.degree. C. overnight and then heated
at 95.degree. C. for 10 min. The obtained samples were amplified
and analyzed for LOH using PCR methods. The amplification/detection
methods used were PCR and gel electrophoresis using labeled primers
(fluorescent or radioactive); RealTime PCR using specific labeled
primers Taqman and probes (labeled with chromatographic dyes); or
capillary array electrophoresis (CAE) with labeled PCR primers (no
probes). All of these methods are known to those skilled in the art
and will not be described here in detail.
[0146] LOH Analysis in Blood and BM Samples. LOH analysis of blood
(plasma/serum) and bone marrow were performed as described in
papers by B. Taback (Cancer Res 2001) and Y. Fujiwara (Cancer Res
1999), the content of which is incorporated herein by the
reference. DNA was isolated, processed, purified, and analyzed for
the presence of LOH as generally described in B. Taback and Y.
Fujiwara references. The isolation procedure was the same
regardless the type of cancer (breast, melanoma, prostate, colon
cancer) being detected.
[0147] The amplification/detection methods used were PCR and gel
electrophoresis using labeled primers (fluorescent or radioactive);
RealTime PCR using specific labeled primers Taqman and probes
(labeled with chromatographic dyes); or capillary array
electrophoresis (CAE) with labeled PCR primers (no probes). All of
these methods are known to those skilled in the art and will not be
described here in detail.
[0148] Methylation Analysis of Blood, BM, and Tumor Tissue Samples.
The samples of blood, BM, and tumor tissue were prepared as the
samples for LOH analysis. Then, the samples were treated with
bisulphite and proteinase K to separate out methylated from
unmethylated DNA. Methylation specific PCR (MSP) was performed. A
more detailed description of this method follows.
[0149] DNA was isolated from cell lines and tissues using DNAzol
Genomic DNA Isolation Reagent (Molecular Research Center, Inc.,
Cincinnati, Ohio) according to the manufacturer's recommendations.
The methylation status of the marker promoter region was determined
by a bisulfite modification protocol.sup.60,61. Briefly, 1 mg of
genomic DNA was denatured in NaOH at 37.degree. C. Cytosines were
sulfonated in the presence of sodium bisulfite and 5 mM
hydroquinone (Sigma) in a water bath for 16-18 h at 55.degree. C.
The DNA samples were desalted using the Wizard DNA Clean-Up System
(Promega, Madison, Wis.) and desulfonated in NaOH at 37.degree. C.
Treated DNA samples were precipitated with ethanol and resuspended
in 10 mM Tris-Cl, 1 mM EDTA, pH 7.6. DNA sequences were amplified
by mixing 100 ng of bisulfite treated DNA with 50 pmoles of
individual primer sets; reaction buffer containing each dNTP and
Taq polymerase at 95.degree. C. for 1 min, 55.degree. C. for 1 min
and 74.degree. C. for 2 min for 30 cycles.
[0150] For MSP (methylation specific PCR), two methods were used to
assess the different regions of the marker CpG promoter island. In
the first method, PCR was performed and assessed on 2% Tris-borate
EDTA agarose gel. In the second method, one hundred ng of
bisulfite-modified DNA was amplified in a final reaction volume of
20 ul containing 0.8 mM dNTPs, and Taq polymerase. PCR was
performed with an initial 10 min incubation at 95.degree. C.,
followed by 40 cycles of denaturation at 95.degree. C. for 30 sec,
annealing at 60.degree. C. for 30 sec, and extension at 72.degree.
C. for 30 sec, and a final 7 min hold at 72.degree. C. PCR products
were visualized using capillary array electrophoresis (CAE; CEQ
2000XL DNA Analysis System, Beckman Coulter, Fullerton,
Calif.).
[0151] The assay was set up in a 96-well microplate format.
Multiple PCR products can be run in each well for comparisons.
Multiple PCR products were visualized simultaneously by labeling
forward primers with a choice of three Beckman Coulter WeIIRED
Phosphoramidite (PA)-linked dyes. Forward methylated specific
primer was labeled with D4pa dye (blue) and forward unmethylated
specific primer was labeled with D2pa dye (black). One ml of
methylated PCR product and one ml of unmethylated PCR product were
mixed with 40 ml loading buffer and 0.5 ul dye-labeled size
standard (Beckman Coulter Inc.). Labeling forward primers specific
for methylated or unmethylated modified DNA distinguishes the
respective products so that they may be analyzed simultaneously.
Other methods for detection of methylation markers, such as ligase
PCR and Realtime PCR with specific marker probe, may also be
used.
[0152] Methylation Site Sequencing to Prove the Marker's Site is
Methylated. Bisulfite sequencing was carried out to confirm the
methylation status of the CpG island promoter region, which
regulates the marker gene transcription. Extracted DNA was treated
with sodium bisulfite, which converts unmethylated cytosines to
uracil. Thymine is then substituted for uracil during subsequent
PCR. Methylated cytosines (5-methylcytosine) are protected from
this process and remain unchanged. Accordingly, all cytosines
present following sequence analysis represent methylated cytosines.
All markers used for methylation were confirmed by methylation
sequencing.
Results
[0153] Determining LOH in the BM of Breast Cancer Patients. BM
aspirates were collected in 4.5 ml sodium citrate tubes (Becton
Dickinson, Franklin Lakes, N.J.) through bilateral anterior iliac
approach from 48 consecutive patients as follows: ductal carcinoma
in situ (DCIS), 1 patient; American Joint Committee on Cancer
(AJCC) stage I, 32 patients; AJCC stage II, 13 patients; and AJCC
stage III, 2 patients; undergoing surgical resection of their
primary breast cancer at the Saint John's Health Center/John Wayne
Cancer Institute. In addition, five healthy female volunteer donors
contributed BM aspirate samples for controls. To assess the
correlation of LOH found in the BM and that of the primary breast
tumor, DNA was isolated from 10 .mu.m sections cut from
paraffin-embedded tumor tissue blocks. Additionally, each BM
aspirate was assessed for the presence of occult tumor cells by
conventional histologic staining methods using Hematoxylin and
Eosin (H&E).
[0154] Eight polymorphic microsatellite markers which correspond to
regions that have been shown to demonstrate significant LOH
suggesting sites of putative tumor suppressor and/or metastasis
related genes were selected: D1S228 at 1p36; D8S321 at
8qter-8q24.13; D10S197 at 10p12; D14S51 at 14q32.1-14q32.2; D14S62
at 14q32; D16S421 at 16q22.1; D17S849 at 17pter-17qter, and D17S855
at 17q. All primer sets were obtained from Research Genetics
(Huntsville, Ala.) and sense primers were labeled with a
fluorescent dye: 5-(and-6)-carboxyfluoroscein, FAM.
[0155] Approximately 20 ng of genomic DNA was amplified by PCR in
25 .mu.l reactions containing 1.times.PCR buffer (Perkin Elmer,
Foster City, Calif.), 6 pmol of each primer, 1 unit of Taq DNA
polymerase, 2.5 .mu.M deoxynucleotide triphosphates, and 1.5 mM
MgCl.sub.2. Forty PCR cycles were performed with each cycle
consisting of 30 at 94.degree. C., 30 s at 50-56.degree. C., and 90
s at 72.degree. C., followed by a final extension step of
72.degree. C. for 5 min as previously described (Taback et al
2001).
[0156] PCR products were electrophoresed on 6% denaturing
polyacrylamide gel containing 7.7 M urea at 1600V for 2 h. Genomyx
SC scanner (Beckman Coulter, Fullerton, Calif.) was used to image
the fluorescent-labeled PCR products and densitometric analysis was
performed with ClaritySC software (Media Cybernetics, Silver
Spring, Md.). Intensity calculations and comparisons of the
specific alleles in patients' normal control and respective BM DNA
were performed to evaluate for LOH. The LOH was defined if a
greater than 50% reduction of intensity was noted in one allele
from tumor or BM DNA when compared with the respective allele in
the matched-paired lymphocytes (Taback et al 2001).
[0157] Clinical and histopathologic data was obtained from patient
chart review and the Breast Tumor Registry at the John Wayne Cancer
Institute. Chi-Square and Wilcoxon Rank Sum tests were performed
for statistical evaluation of association of BM LOH status and
known prognostic parameters.
[0158] LOH was identified in 11 (23%) of 48 patients' BM aspirates.
LOH was most commonly identified at microsatellite marker D14S62
occurring in 4 (12%) of 34 informative patients. Microsatellite
markers demonstrating LOH at D1S228 and D14S51 occurred in 3 (8%)
of 38 informative patients each, followed by LOH at D8S321 (5%),
D10S197 (4%), and D17S855 (3%). No LOH was detected for
microsatellite markers D16S421 and D17S849 (Table 10). Eight of the
11 patients with detectable LOH in their BM demonstrated this event
at only one of the chromosome loci assessed and three patients (1
stage I, 2 stage II patients) contained LOH for two microsatellite
markers. No LOH was detected for any of the microsatellite markers
assessed in the patient with DCIS or the BM aspirates collected
from five healthy female donors.
TABLE-US-00010 TABLE 10 LOH Frequency in Breast Cancer Patients
Bone Marrow Aspirates Microsatellite marker LOH in BM
aspirates/informative cases (%) D14S62 4/34 (12%) D14S51 3/38 (8%)
D1S228 3/38 (8%) D8S321 2/39 (5%) D10S197 1/26 (4%) D17S855 1/37
(3%) D17S849 0/31 (0%) D16S421 0/28 (0%)
[0159] The inventors found LOH on chromosome 14q as the most
frequent event identified on circulating DNA in BM. However, in
another study, LOH on 14q has been shown to occur more commonly in
primary tumors without lymph node metastasis, suggesting a site for
a possible metastasis-related gene; however, the metastasis itself
was not assessed for LOH(O'Connell et al 1999). While not wanting
to be bound by a theory, the inventors believe that metastatic
clones at different sites may demonstrate different LOH profiles.
Additionally, differences in these results may reflect the
stability of this marker as detected from various sources (blood,
BM, tumor tissues) or it may be uniquely associated with
site-specific metastasis. Molecular markers that are specific for
the metastatic phenotype and/or sites of metastasis may prove
useful for focusing clinical assessments.
[0160] There was an increased association between the presence of
LOH in the BM and advanced disease stage. Six (19%) of 32 AJCC
stage I patients demonstrated LOH for at least one marker, in
contrast to 4 (31%) of 13 AJCC stage II patients, and 1 (50%) of 2
AJCC stage III patients (Table 11).
TABLE-US-00011 TABLE 11 Association of LOH in Patients' BM
Aspirates with AJCC Stage AJCC Stage Patients with LOH in BM/total
patients (%) I 6/32 (19%) II 4/13 (31%) III 1/2 (50%)
[0161] Ten clinicopathologic prognostic factors were assessed for
correlation with BM LOH status: histologic tumor type, size, grade,
Bloom-Richardson score, lymph node involvement, presence of
lymphovascular invasion in the primary tumor, receptor status
(estrogen, progesterone, HER2), and p53 status. There was an
association between larger tumor size and BM LOH positivity: 2.46
cm versus 1.81 cm, mean tumor sizes, respectively. There was also a
trend towards an increased incidence of BM LOH in lymph node
positive patients as compared with lymph node negative patients: 3
(33%) of 9 patients versus 8 (21%) of 38 patients, respectively. No
significant correlation existed between any prognostic factor and
BM LOH status in this study except histology. Lobular carcinomas
were more likely associated with increased LOH in BM aspirates than
infiltrating ductal tumors: 6 (60%) of 10 patients versus 5 (14%)
of 37 patients, respectively (Chi-Square test P=0.006). Larger
populations with long-term follow-up are warranted to evaluate the
clinical and prognostic utility of this assay.
[0162] To determine whether a correlation existed between the LOH
detected in patients BM and their primary tumor, DNA was isolated
from primary tumors and evaluated with identical microsatellite
markers. Ten of the eleven patients demonstrating LOH in their BM
had primary tumors available for assessment. In all ten patients,
the LOH identified in the BM was also present respectively in the
primary tumor (FIG. 4). Conventional histological analysis of all
specimens using standard H&E staining did not demonstrate
occult tumor cells in any of the BM samples.
[0163] Determining LOH and CpG Hypermethylation in Blood
(Serum/Plasma) of Prostate Cancer Patients. To evaluate the
clinical utility of assessing circulating nucleic acids containing
tumor-associated genetic alterations in serum/plasma of prostate
cancer, patients' blood was collected from 23 prostate cancer
patients American Joint Committee on Cancer (AJCC) stages I-IV. A
panel of 7 microsatellite markers (D8S261, D8S262, D9S171, D10S591,
D10S532, D16S422 and D18S70) on 6 chromosome arms pertaining to
putative tumor suppressor gene regions was utilized to assess
prostate cancer patient's serum/plasma for LOH on circulating
nucleic acids.
[0164] In addition, methylation specific PCR and capillary array
electrophoresis was performed on the same samples to evaluate the
presence of CpG island hypermethylation in the promoter regions of
three known tumor suppressor genes: RASSF1A, RAR-.beta. and GSTP1.
The obtained results are shown in Table 12 below.
TABLE-US-00012 TABLE 12 Association of LOH and Methylation in
Prostate Cancer Patients' Blood Samples with AJCC Stage AJCC Stage
Methylation LOH Molecular Positive (%) I 0/3 0/3 0% II 0/5 2/5 40%
III/IV 8/15 5/15 60%
[0165] A correlation of an increased combination of promoter region
hypermethylation and LOH identified on circulating nucleic acids
was associated with advancing AJCC staging. The incidence of LOH
increased from 2 (25%) of 8 stage I-II patients to 5 (33%) of 15
stage III-IV patients. None of the early stage patients
demonstrated methylated DNA in their blood whereas 8 (53%) of 15
stage III-IV patients where positive for any one methylated marker
in their blood. Although, it appears that testing blood for
hypermethylation or LOH alone may be used to detect and diagnoses
prostate cancer, testing for both hypermethylation and LOH may
provide a higher sensitivity and accuracy in detection and
determination of the AJCC stage of prostate cancer.
[0166] Determining LOH and CpG Hypermethylation in Blood
(Serum/Plasma) of Colorectal Cancer Patients. To evaluate the
presence of circulating nucleic acids containing tumor-associated
genetic alterations in serum/plasma of colorectal cancer patients,
blood was collected from 33 colorectal cancer patients undergoing
surgical resection for their primary diagnosis. A panel of 11
microsatellite markers (D4S175, D4S1586, D5S299, D8S133, D8S264,
D15S127, TP53, D17S796, D17S1832, D18S58 and D18S61) on 6
chromosomes at loci demonstrating frequent LOH in primary
colorectal tumors suggestive of putative tumor suppressor gene
regions were utilized. In addition, methylation specific PCR and
capillary array electrophoresis was performed on the same samples
in 16 patients to evaluate the presence of CpG island
hypermethylation in the promoter regions of five known tumor
suppressor genes: MGMT, P16, APC, RASSF1A and RAR-.beta.. The
obtained results are shown in Tables 13 and 14 below.
TABLE-US-00013 TABLE 13 Frequency of Microsatellite Marker LOH in
Colorectal Cancer Patients' Blood D18S58 1/23 (4%) D18S61 1/32 (3%)
TP53 2/28 (7%) D17S1832 2/28 (7%) D5S299 5/25 (20%) D4S175 6/23
(26%) D4S1586 1/19 (5%) D8S133 3/19 (16%) D8S264 1/22 (5%) D15S127
2/29 (7%) D17S796 3/22 (14%)
TABLE-US-00014 TABLE 14 Frequency of Gene Promoter Methylation in
Colorectal Cancer Patients' Blood Methylation MGMT P16 RAR-.beta.
RASSF1A APC 2/16 (13%) 0/16 (0%) 0/16 (0%) 1/16 (6%) 4/16 (25%)
[0167] In this study LOH, for any one marker, was identified in 17
(52%) of 33 patients blood samples (Table 13) and promoter region
hypermethylation at any marker was detected in 6 (38%) of 16
patients (Table 14). The combination of hypermethylation and LOH
for assessing blood in those 16 patients evaluated using both
techniques identified a greater number of patients with colorectal
cancer, 15 (94%) of 16 as compared to either method alone:
methylation 6 (38%) or LOH 11 (69%) of 16 patients (Table 15).
TABLE-US-00015 TABLE 15 Frequency of Methylation, LOH, and Their
Combination in Colorectal Cancer Patients' Blood Assessed Using
Both Assays Methylation Total + - LOH LOH + 2 9 11 - 4 1 5 Total 6
10 Methylation
[0168] Detection of LOH and Methylation Markers in Primary Breast
Cancer Tumors to Predict Metastasis to Lymph Nodes and Disease
Outcome. Methylation PCR was carried out on primary tumors (151
patients) for the different markers. Patients were divided into
those that had no metastasis in their sentinel lymph node (first
tumor draining lymph node), occult or micrometastasis in their
lymph nodes, or had palpable (clinically detectable) metastasis in
their lymph nodes. Preliminary analysis demonstrated that
methylation of specific markers in the primary tumor can predict
the type of metastasis in the nodes. The results shown in Table 16
indicate that additional diagnostic data may be obtained from lymph
node metastasis samples.
TABLE-US-00016 TABLE 16 Methylation Status of Primary Tumor and
Lymph Node (LN) Metastasis (29 Pairs) Primary- LN met APC GSTP1
RASSF1A RAR-b E-cad TWIST* M-M 8 7 16 9 14 7 M-U 7 3 7 5 1 5 U-M 2
0 1 5 12 1 U-U 12 19 5 10 2 15 M stands for methylated and U stands
for unmethylated
[0169] Inventors believe that the same markers can be used to
analyze blood (plasma/serum) and BM samples of melanoma
patients.
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[0358] While the foregoing has been described in considerable
detail and in terms of preferred embodiments, these are not to be
construed as limitations on the disclosure or claims to follow.
Modifications and changes that are within the purview of those
skilled in the art are intended to fall within the scope of the
following claims. All literatures cited herein are incorporated by
reference in their entirety.
Sequence CWU 1
1
55119DNAArtificialSynthetic oligonucleotide 1gaacgcgagc gattcgagt
19219DNAArtificialSynthetic oligonucleotide 2gaccaatcca accgaaacg
19321DNAArtificialSynthetic oligonucleotide 3ggattgggat gttgagaatg
t 21421DNAArtificialSynthetic oligonucleotide 4caaccaatcc
aaccaaaaca a 21522DNAArtificialSynthetic oligonucleotide
5ttaggttaga gggttatcgc gt 22623DNAArtificialSynthetic
oligonucleotide 6taactaaaaa ttcacctacc gac
23725DNAArtificialSynthetic oligonucleotide 7taattttagg ttagagggtt
attgt 25820DNAArtificialSynthetic oligonucleotide 8cacaaccaat
caacaacaca 20918DNAArtificialSynthetic oligonucleotide 9tattgcggag
tgcgggtc 181018DNAArtificialSynthetic oligonucleotide 10tcgacgaact
cccgacga 181124DNAArtificialSynthetic oligonucleotide 11gtgttttatt
gtggagtgtg ggtt 241221DNAArtificialSynthetic oligonucleotide
12ccaatcacaa actcccaaca a 211320DNAArtificialSynthetic
oligonucleotide 13gtgttaacgc gttgcgtatc
201421DNAArtificialSynthetic oligonucleotide 14aaccccgcga
actaaaaacg a 211523DNAArtificialSynthetic oligonucleotide
15tttggttgga gtgtgttaat gtg 231623DNAArtificialSynthetic
oligonucleotide 16caaaccccac aaactaaaaa caa
231720DNAArtificialSynthetic oligonucleotide 17ttcggggtgt
agcggtcgtc 201822DNAArtificialSynthetic oligonucleotide
18gccccaatac taaatcacga cg 221924DNAArtificialSynthetic
oligonucleotide 19gatgtttggg gtgtagtggt tgtt
242024DNAArtificialSynthetic oligonucleotide 20ccaccccaat
actaaatcac aaca 242121DNAArtificialSynthetic oligonucleotide
21tttcggatgg ggttgttatc g 212220DNAArtificialSynthetic
oligonucleotide 22gacgaacgcg aaacgatttc
202320DNAArtificialSynthetic oligonucleotide 23ttggatgggg
ttgttattgt 202420DNAArtificialSynthetic oligonucleotide
24accttcctcc aacaaacaca 202528DNAArtificialSynthetic
oligonucleotide 25gggttatttg gtaaattaag gtatagag
282628DNAArtificialSynthetic oligonucleotide 26cacctaaaaa
taaaacaaaa actaccac 282723DNAArtificialSynthetic oligonucleotide
27gggagtttga gtttattgag ttg 232825DNAArtificialSynthetic
oligonucleotide 28ctctactcat ctataaccca aatac
252930DNAArtificialSynthetic oligonucleotide 29gtgtgataga
agtagtagga agtgagttgt 303029DNAArtificialSynthetic oligonucleotide
30actccatcaa actctacccc ttttttaac 293119DNAArtificialSynthetic
oligonucleotide 31gttgtyggag gattagggt 193221DNAArtificialSynthetic
oligonucleotide 32taccccttaa ctaccccttc c
213329DNAArtificialSynthetic oligonucleotide 33aatcataaat
tataacaaac aaaccaact 293428DNAArtificialSynthetic oligonucleotide
34cgtttcgtta ttttttgttt tcggtttc 283517DNAArtificialSynthetic
oligonucleotide 35ccgaaaaccc cgcctcg 173631DNAArtificialSynthetic
oligonucleotide 36ttttgttttg ttattttttg tttttggttt t
313720DNAArtificialSynthetic oligonucleotide 37cccccaaaaa
ccccacctca 203823DNAArtificialSynthetic oligonucleotide
38tttcgacgtt cgtaggtttt cgc 233922DNAArtificialSynthetic
oligonucleotide 39gcactcttcc gaaaacgaaa cg
224029DNAArtificialSynthetic oligonucleotide 40tttgtgtttt
gatgtttgta ggtttttgt 294127DNAArtificialSynthetic oligonucleotide
41aactccacac tcttccaaaa acaaaac 274224DNAArtificialSynthetic
oligonucleotide 42ggatagtcgg atcgagttaa cgtc
244316DNAArtificialSynthetic oligonucleotide 43ccctcccaaa cgccga
164427DNAArtificialSynthetic oligonucleotide 44ggaggatagt
tggattgagt taatgtt 274521DNAArtificialSynthetic oligonucleotide
45caaatccctc ccaaacacca a 214624DNAArtificialSynthetic
oligonucleotide 46ttattagagg gtggggcgga tcgc
244720DNAArtificialSynthetic oligonucleotide 47gacccgaacc
gcgaccgtaa 204824DNAArtificialSynthetic oligonucleotide
48ttattagagg gtggggtgga ttgt 244922DNAArtificialSynthetic
oligonucleotide 49caaccccaaa ccacaaccat aa
225023DNAArtificialSynthetic oligonucleotide 50ccaactccaa
atcccctctc tat 235130DNAArtificialSynthetic oligonucleotide
51tgattaattt agattgggtt tagagaagga 305226DNAArtificialSynthetic
oligonucleotide 52cccttcctat tcctaaatcc aaccta
265326DNAArtificialSynthetic oligonucleotide 53cgtttgcgat
ttggtgagtg tttggg 265433DNAArtificialSynthetic oligonucleotide
54caactaccgt ataaaattac acgcgatacc ccg 335526DNAArtificialSynthetic
oligonucleotide 55ccgaatacgt tccgaatcct accccg 26
* * * * *