U.S. patent application number 11/433722 was filed with the patent office on 2006-09-14 for pcr method and related apparatus.
Invention is credited to Sydney D. Finkelstein, Tony E. Godfrey, Lori A. Kelly, James D. Luketich, Siva Raja.
Application Number | 20060205006 11/433722 |
Document ID | / |
Family ID | 23043271 |
Filed Date | 2006-09-14 |
United States Patent
Application |
20060205006 |
Kind Code |
A1 |
Godfrey; Tony E. ; et
al. |
September 14, 2006 |
PCR method and related apparatus
Abstract
A method for balancing multiplexed PCR methods is provided. In
the method, two or more sequential temporal PCR stages are used to
effectively separate two or more PCR reactions in a single tube as
an alternative to primer limiting to modulate the relative rate of
production of a first amplicon by a first primer set and a second
amplicon by a second primer set during the first and second
amplification stages. Also provided are rapid RT-PCR methods that
find particular use in intraoperative diagnoses and prognoses, for
instance in diagnosing malignant esophageal adenocarcenoma by
determining expression levels of carcinoembryonic antigen (CEA) in
sentinel lymph nodes.
Inventors: |
Godfrey; Tony E.;
(Pittsburgh, PA) ; Luketich; James D.;
(Pittsburgh, PA) ; Raja; Siva; (Pittsburgh,
PA) ; Kelly; Lori A.; (Jeanette, PA) ;
Finkelstein; Sydney D.; (Pittsburgh, PA) |
Correspondence
Address: |
JESSE A. HIRSHMAN, ESQ.
1722 MURRAY AVENUE
PITTSBURGH
PA
15217
US
|
Family ID: |
23043271 |
Appl. No.: |
11/433722 |
Filed: |
May 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10090326 |
Mar 4, 2002 |
|
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11433722 |
May 12, 2006 |
|
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60273277 |
Mar 2, 2001 |
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Current U.S.
Class: |
435/6.11 ;
435/6.17; 435/91.2 |
Current CPC
Class: |
C12Q 2600/166 20130101;
C12Q 2537/143 20130101; C12Q 2527/107 20130101; C12Q 2561/101
20130101; C12Q 1/6886 20130101; C12Q 1/6851 20130101; C12Q 2600/158
20130101; C12Q 2600/16 20130101; C12Q 1/6851 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Goverment Interests
STATEMENT REGARDING FEDERAL SUPPORT
[0002] This invention was made with government support under Grant
No. CA90665-01, awarded by the National Institutes of Health. The
government has certain rights in this invention.
Claims
1-66. (canceled)
67. An oligonucleotide consisting of one of SEQ ID NOS: 6, 7, 13,
14, 16, 17, 19, 20 and 23-35, and a derivative thereof.
68-77. (canceled)
78. The oligonucleotide of claim 67, wherein the derivative is
capable of hybridizing in a PCR reaction to the same target
sequence as the oligonucleotides of SEQ ID NOS: 6, 7, 13, 14, 16,
17, 19, 20 and 23-35.
79. The oligonucleotide of claim 78, consisting of one of SEQ ID
NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
80. The oligonucleotide of claim 78, consisting of SEQ ID NO:
6.
81. The oligonucleotide of claim 78, consisting of SEQ ID NO:
7.
82. The oligonucleotide of claim 78, consisting of SEQ ID NO:
13.
83. The oligonucleotide of claim 78, consisting of SEQ ID NO:
14.
84. The oligonucleotide of claim 78, consisting of SEQ ID NO:
16.
85. The oligonucleotide of claim 78, consisting of SEQ ID NO:
17.
86. The oligonucleotide of claim 78, consisting of SEQ ID NO:
19.
87. The oligonucleotide of claim 78, consisting of SEQ ID NO:
20.
88. The oligonucleotide of claim 78, consisting of SEQ ID NO:
23.
89. The oligonucleotide of claim 78, consisting of SEQ ID NO:
24.
90. The oligonucleotide of claim 78, consisting of SEQ ID NO:
25.
91. The oligonucleotide of claim 78, consisting of SEQ ID NO:
26.
92. The oligonucleotide of claim 78, consisting of SEQ ID NO:
27.
93. The oligonucleotide of claim 78, consisting of SEQ ID NO:
28.
94. The oligonucleotide of claim 78, consisting of SEQ ID NO:
29.
95. The oligonucleotide of claim 78, consisting of SEQ ID NO:
30.
96. The oligonucleotide of claim 78, consisting of SEQ ID NO:
31.
97. The oligonucleotide of claim 78, consisting of SEQ ID NO:
32.
98. The oligonucleotide of claim 78, consisting of SEQ ID NO:
33.
99. The oligonucleotide of claim 78, consisting of SEQ ID NO:
34.
100. The oligonucleotide of claim 78, consisting of SEQ ID NO:
35.
101. A cartridge for use in a PCR system, the cartridge comprising
one or more compartments containing a first PCR primer set and a
second PCR primer set, wherein the first PCR primer set has a first
effective Tm and the second PCR primer set has a second effective
Tm that is different from the first effective Tm.
102. The cartridge of claim 101, further comprising a third
compartment including one of reverse transcription reagents, cell
lysis reagents and RNA purification reagents.
103. The cartridge of claim 102, comprising reverse transcriptase
and a reverse transcriptase primer.
104. The cartridge of claim 103, comprising random hexanucleotide
reverse transcriptase primers.
105. The cartridge of claim 103, comprising sequence-specific
reverse transcriptase primers.
106. The cartridge of claim 105, wherein the sequence-specific
reverse transcriptase primers are specific to one or more of
.beta.-glucuronidase mRNA, carcinoembryonic antigen mRNA,
tyrosinase mRNA and 18S ribosomal RNA.
107. The cartridge of claim 101, comprising a PCR primer set for
producing one or more of .beta.-glucuronidase, carcinoembryonic
antigen, tyrosinase mRNA and 18S ribosomal RNA-specific
amplicon.
108. The cartridge of claim 101, further comprising an internal
positive control nucleic acid.
109. The cartridge of claim 108, wherein the internal positive
control nucleic acid comprises one or more uracil residues.
110. The cartridge of claim 101, further comprising a fluorescent
reporter for indicating accumulation of a specific amplicon in a
PCR reaction.
111. The cartridge of claim 110, wherein the fluorescent reporter
is a probe for a fluorescent 5' nuclease assay.
112. The cartridge of claim 110, wherein the fluorescent reporter
is a molecular beacon probe.
113. The cartridge of claim 101, wherein the cartridge is
disposable after a single use.
114. The cartridge of claim 101, comprising an oligonucleotide
consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and
23-35 or a derivative thereof capable of hybridizing in a PCR
reaction to the same target sequence as the oligonucleotides of SEQ
ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
115. The cartridge of claim 114, comprising one or more
oligonucleotides consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16,
17, 19, 20 and 23-35.
116. The cartridge of claim 101, wherein the effective Tm of the
first primer set and the effective Tm of the second primer set
differ by at least 5.degree. C.
117. A kit comprising a first PCR primer set and a second PCR
primer set, wherein the first PCR primer set has a first effective
Tm and the second PCR primer set has a second effective Tm that is
different from the first effective Tm.
118. The kit of claim 117, comprising a cartridge for use in a PCR
system, the cartridge comprising one or more compartments
containing the first PCR primer set and the second PCR primer
set.
119. The kit of claim 117, further comprising one of reverse
transcription reagents, cell lysis reagents and RNA purification
reagents.
120. The kit of claim 119, comprising reverse transcriptase and a
reverse transcriptase primer.
121. The kit of claim 120, comprising random hexanucleotide reverse
transcriptase primers.
122. The kit of claim 120, comprising sequence-specific reverse
transcriptase primers.
123. The kit of claim 122, wherein the sequence-specific reverse
transcriptase primers are specific to one or more of
.beta.-glucuronidase mRNA, carcinoembryonic antigen mRNA,
tyrosinase mRNA and 18S ribosomal RNA.
124. The kit of claim 117, comprising a PCR primer set for
producing one or more of .beta.-glucuronidase, carcinoembryonic
antigen, tyrosinase mRNA and 18S ribosomal RNA-specific
amplicon.
125. The kit of claim 117, further comprising an internal positive
control nucleic acid.
126. The kit of claim 125, wherein the internal positive control
nucleic acid comprises one or more uracil residues.
127. The kit of claim 117, further comprising a fluorescent
reporter for indicating accumulation of a specific amplicon in a
PCR reaction.
128. The kit of claim 127, wherein the fluorescent reporter is a
probe for a fluorescent 5' nuclease assay.
129. The kit of claim 127, wherein the fluorescent reporter is a
molecular beacon probe.
130. The kit of claim 117, comprising an oligonucleotide consisting
of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35 or a
derivative thereof capable of hybridizing in a PCR reaction to the
same target sequence as the oligonucleotides of SEQ ID NOS: 6, 7,
13, 14, 16, 17, 19, 20 and 23-35.
131. The kit of claim 130, comprising one or more oligonucleotides
consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and
23-35.
132. The kit of claim 117, wherein the effective Tm of the first
primer set and the effective Tm of the second primer set differ by
at least 5.degree. C.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.120
to U.S. patent application Ser. No. 10/090,326, filed Mar. 4, 2002,
which, in turn, claims priority under 35 U.S.C. .sctn. 119(e) to
U.S. Application Ser. No. 60/273,277, filed Mar. 2, 2001, both of
which are incorporated herein by reference in their entirety.
BACKGROUND
[0003] This Application discloses a rapid PCR method, with
particular focus on a rapid multiplex QRT-PCR method and related
compositions and apparatus.
[0004] Polymerase chain reaction (PCR) is a powerful tool in the
field of molecular biology. This technique allows for
replicating/amplifying trace amounts DNA fragments into quantities
that can be analyzed in a meaningful way. As such this technology
has been adapted to molecular biological applications like DNA
sequencing, DNA fingerprinting, etc. Additionally, this method has
the ability to detect specific DNA fragments in samples, whose
presence may reflect a pathological state. Therefore, this method
is finding new applications in the field of molecular diagnostics.
Furthermore with the development of real-time quantitative PCR
(QPCR), this technology has become more reliable as well as
amenable to automation. Currently, this technology is used for the
detection of viral and bacterial pathogens in clinical samples and
for the detection of cancer cells in patients with a history of
leukemias (and other cancers such as those that arise in the
breast, lung, colon, esophagus and skin).
[0005] PCR in molecular diagnostics, despite its advantages, has
several shortcomings. Often, this is a technique that is dependent
on the technical expertise of the operator. Additionally, it is
also very prone to contamination. The combination of these two
above-mentioned factors results in false negative and false
positive results respectively. Therefore, there is a need to
incorporate internal controls along with the target of interest as
well as automate the process to ensure operation within a closed
system (to eliminate contamination). The incorporation of controls
involves the amplification of several different sets of DNA
fragments in addition to the target of interest. These controls
provide information about the quality of the samples being analyzed
as well as that of the assay in any given run. For technical
reasons, analyzing multiple DNA targets within a sample in the same
reaction tube through PCR (known as multiplexing) does not work
well when the targets are not present in similar abundance at the
beginning of the reaction. Historically, investigators have
attempted to overcome this by limiting the primers in the PCR
reaction. This approach is based on the idea that by limiting the
reagents in one reaction, it stops the reaction at a point after
adequate amplification has occurred for that target but before
inhibition of amplification of other sequences. Though this method
can increase the difference in initial abundance between the two
targets that will still result in the successful amplification of
both targets, it does not allow for the detection of a rare target
in the milieu of a second target that is several orders of
magnitude more abundant. Furthermore, when a rapid QPCR assay is
called for, decreasing the primer concentration also worsens
quantitation.
[0006] A further limitation of current PCR technologies is the time
it takes to perform PCR diagnoses. Typical PCR reactions take
hours, not minutes. As described below, decreasing the time it
takes to carry out a PCR reaction is desirable for many reasons.
Therefore, there is a need for an automated PCR based point of care
molecular diagnostic system, especially a rapid, multiplexed
(RT-)PCR assay.
SUMMARY OF THE INVENTION
[0007] Rapid and robust PCR and RT-PCR methods are provided that
permit execution of a complete PCR reaction in minutes, not hours,
permitting use of PCR in intraoperative diagnoses, for example, but
without limitation, for detecting micrometastases in sentinel lymph
nodes, as an superior alternative to, or in addition to typical
pathological methods such as histopathological examination of lymph
nodes.
[0008] Also provided is an alternative to primer limiting as a
method for balancing a multiplex PCR reaction, especially
quantitative PCR amplifications, with particular usefulness in
QRT-PCR reactions. This method finds particular use when one target
sequence to be amplified is far less prevalent than another to be
amplified in the same reaction mixture. The method comprises the
step of conducting a PCR amplification on a DNA sample in a PCR
reaction mixture in a first amplification stage and a second
amplification stage. The PCR amplification of the second
amplification stage is conducted under different reaction
conditions than the PCR amplification of the first amplification
stage to modulate the relative rate of production of a first
amplicon by a first primer set and a second amplicon by a second
primer set during the first and second amplification stages.
Additional amplification stages may be added.
[0009] Two non-limiting specific embodiments of this method are
disclosed. In the first embodiment, the second primer set is added
to the reaction mixture at the beginning of the second
amplification stage, thereby limiting the physical presence of the
second primer set during the first stage. In this method, the rarer
target sequence preferably is amplified before the less-rare
sequence which typically is a control, such as .beta.-gus or
18SrRNA sequences.
[0010] In the second embodiment, the PCR reaction mixture includes
the first primer set having a first effective Tm and the second
primer set having a second effective Tm different from the first
effective Tm. The relative rate of production of the first amplicon
by the first primer set and the second amplicon by the second
primer set during the first and second amplification stages is
modulated by conducting the annealing step of the first
amplification stage at a different temperature than the annealing
step of the second amplification stage. In this second embodiment,
the annealing temperature for the second amplification stage may be
higher or lower than the annealing temperature for the first
amplification stage.
[0011] Also provided is a rapid RT-PCR method that is based upon
the finding that the reverse transcription reaction of the RT-PCR
method need not be performed for longer than about 10 minutes, and
preferably only for about two minutes. This rapid step, when
coupled with a rapid PCR procedure conducted sequentially with the
RT reaction in the same reaction vessel as the RT reaction, permits
intraoperative use of the RT-PCR reaction, especially when the
entire process is automated.
[0012] Each of the above-described PCR and RT-PCR process find
special utility in their use in quantitative PCR methods, such as
QPCR and QRT-PCR, which are typically monitored during the PCR
amplification by the accumulation of, or loss of, a fluorescent
reporter, for instance by the use of TAQMAN and molecular beacon
probes.
[0013] The above-described methods may be automated in a
cartridge-based system, thereby reducing human error in the
methods, as well as the potential for contamination. In an
automated system, reagents for the various reactions are added
sequentially according to a programmed sequence. A cartridge, for
use in an automated system, for performing the described methods
also is provided.
[0014] Also provided are specific uses for the rapid PCR methods
described herein. In one embodiment, an intraoperative PCR
diagnostic method is provided that includes the steps of: obtaining
a tissue sample from a patient in an operation; analyzing the
sample according to one of the above-described PCR methods;
determining if expression of an indicator transcript exceeds a
threshold level; and continuing the operation in a manner dictated
by results of the analyzing step. In another embodiment, a method
for rapid detection of a malignancy is provided that includes the
steps of: obtaining nucleic acid from a tumor biopsy; performing a
PCR method specific to an indicator transcript on the nucleic acid
according to one of the above-described PCR methods; and
determining if expression of the indicator transcript exceeds a
threshold level, thereby indicating a malignancy.
[0015] In an additional embodiment, a method for rapid detection of
metastasized adenocarcenoma of the esophagus is provided. The
method includes the steps of: obtaining RNA from a sentinel lymph
node; performing a quantitative RT-PCR method specific to CEA on
the RNA according to any one of the above-described PCR methods;
and determining if expression of CEA exceeds a threshold level.
[0016] Lastly, also provided are specific novel oligonucleotide
primers useful in the detection of sequences specific to CEA and
tyrosinase genes, as well as .beta.-gus and 18SrRNA sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a schematic drawing of a cartridge according to
one embodiment of the present invention.
[0018] FIG. 2 shows two graphs comparing the sensitivity of the
one-tube RT-PCR with (B) and without (A) a wax separating the RT
and PCR reactions.
[0019] FIG. 3 is a photograph of an ethidium bromide-stained gel
comparing RT-PCR product prepared with and without use of a wax
layer to separate the RT and PCR reactions.
[0020] FIG. 4 provides four graphs showing the amplification of CEA
and 18SrRNA in a multiplexed reaction as described in Example 3.
Panels A and C show the results for 18S rRNA and CEA respectively
when run in a singleplex using optimal conditions. Panels B and D
show 18S rRNA and CEA multiplexed.
[0021] FIG. 5 provides the CEA nucleotide sequence, GenBank
Accession No. XM.sub.--13012777 (SEQ ID NO: 1).
[0022] FIG. 6 is a graph showing the relative CEA expression
measured in 10 esophageal tumors (light gray), 4 histologically
positive (N.sub.1) lymph nodes (white), and 10 benign lymph nodes
from patients without cancer (dark gray).
[0023] FIG. 7 shows the Relative CEA expression measured in
histologically negative lymph nodes from 30 esophageal cancer
patients. Graph shows the highest CEA level found for each patient.
Patients 1-20 (dark gray columns) did not recur, whereas patients
21-30 (light gray columns) did recur. The dotted line indicates the
most accurate cutoff value for predicting recurrence. Using this
cutoff, QRT-PCR correctly classified 90% of patients with respect
to disease recurrence at 3 years.
[0024] FIGS. 8A-D are Kaplan-Meier survival curves for disease-free
survival for patients classified by RT-PCR (8A and 8B) and QRT-PCR
(8C and 8D). Although log-rank tests indicate that both predictors
are statistically significant, the superior specificity of QRT-PCR
compared with RT-PCR leads to a greater ability to differentiate
patients on the basis of their risk of recurrence (P=0.0038 for
RT-PCR and <0.0001 for QRT-PCR).
[0025] FIG. 9 shows a SmartCycler analysis of CEA expression in
formalin-fixed lymph nodes from 30 pathologically node negative
esophagus cancer patients. Gray bars (21-30) indicate patients who
suffered recurrence, black bars (1-20) indicate patients who did
not suffer recurrence. Lymph nodes with no detectable CEA
expression have been arbitrarily plotted at a level of 0.02. The
dotted line indicates the most accurate CEA expression cut-off
value (0.183) for predicting disease recurrence.
[0026] FIG. 10 shows a Kaplan-Meier disease free survival curve for
30 pN.sub.0 esophagus cancer patients stratified as LN positive or
negative (above or below the cut-off value of 0.183) based on
Smartcycler QRT-PCR.
[0027] FIG. 11 shows Smartcycler analysis of CEA expression in
frozen lymph nodes. White (1-11)=nodes from patients without
cancer; black (12-17)=nodes determined on final pathology to be
negative for disease; gray (20-23)=nodes found to be positive for
disease; gray (18 and 19)=nodes that were negative by
intra-operative frozen section but positive on fixed tissue
histology; star=highly expressing node from a node negative patient
who suffered disease recurrence. The value of 0.001 was arbitrarily
assigned to all samples that had no detectable expression of
CEA.
[0028] FIGS. 12A-C show the optimization of the rapid QRT-PCR assay
for .beta.-gus and CEA. FIG. 12A compares the Ct values for assays
with different denaturation times and a 30 second extension time.
FIG. 12B compares the effect of different extension times on the Ct
value when the denaturation time is held constant at 10 seconds.
FIG. 12C demonstrates the effect of different RT times when the PCR
conditions are constant.
[0029] FIG. 13 shows Ct values for .beta.-gus and CEA in a 8-fold
serial dilution of CEA in a constant background of .beta.-gus. This
figure compares the slow conventional PCR (10 sec denaturation
followed by a 30 sec annealing/extension step) with rapid PCR (1
sec denaturation followed by a 6 second annealing/extension step).
.diamond-solid.=10, 30 GUS; .quadrature.=1, 6 GUS; .DELTA.=10, 30
CEA; and .circle-solid.=1, 6 CEA.
[0030] FIG. 14 is a RT-PCR run time comparison between a 15 min (2
min RT and 1/3 PCR), 17 min (2 min RT and 1/6 PCR), 20 min (5 min
RT, 1/6 PCR) and a 38 min (10 min RT and 5/15 PCR) assays.
[0031] FIG. 15 shows four amplification plots for CEA singleplex
(panel A), CEA in the temperature-controlled multiplex (panel B),
CEA in the ordinary multiplex (panel C) and CEA in the conventional
primer limiting multiplex (panel D)
[0032] FIG. 16 shows a comparison of the singleplex PCR for CEA
with the temperature-controlled multiplex, ordinary multiplex and
the conventional primer limiting multiplex. The delta Ct values for
a serial dilution of CEA in constant background of .beta.-gus are
compared. Delta Ct of 25 is used to denote a dilution point that
failed to amplify.
[0033] FIG. 17 is a histogram of CEA expression relative to
.beta.-gus in lymph nodes. Bars in Black (1-3) correspond to lymph
nodes from patients with cancer with histological evidence of
cancer. Bars in Gray (4-8) correspond to lymph nodes from patients
without cancer. Samples 4 and 6 had no detectable CEA
expression.
[0034] FIG. 18 shows four representative amplification plots for
samples with the expression of CEA (A) and tyrosinase (C), and
samples without the expression of CEA (B) and tyrosinase (D). In
panels A and B, the fluorescence curves for .beta.-gus (+), CEA
(.diamond.) and CEA IC (-) are shown. Similarly in panels C and D,
the fluorescence curves for .beta.-gus (.diamond.), tyrosinase (-)
and tyrosinase IC (*) are shown.
DETAILED DESCRIPTION
[0035] Provided are improved PCR methods that permit rapid cycling
and/or improved sensitivity for PCR-based molecular diagnostics,
especially with respect to quantitative PCR methods, including
QRT-PCR. These improved methods permit PCR to be used
intraoperatively, and also are useful in detecting rare nucleic
acid species, even in multiplexed PCR reactions that concurrently
detect a more prevalent control nucleic acid.
[0036] A typical PCR reaction includes multiple amplification
steps, or cycles that selectively amplify a target nucleic acid
species. A full description of the PCR process, and common
variations thereof, such as quantitative PCR (QPCR), real-time
QPCR, reverse transcription PCR (RT-PCR) and quantitative reverse
transcription PCR (QRT-PCR) is beyond the scope of this disclosure
and these methods are well-described in the art and have been
broadly commercialized. A typical PCR reaction includes three
steps: a denaturing step in which a target nucleic acid is
denatured; an annealing step in which a set of PCR primers (forward
and backward primers) anneal to complementary DNA strands; and an
elongation step in which a thermostable DNA polymerase elongates
the primers. By repeating this step multiple times, a DNA fragment
is amplified to produce an amplicon, corresponding to the target
DNA sequence. Typical PCR reactions include 30 or more cycles of
denaturation, annealing and elongation. In many cases, the
annealing and elongation steps can be performed concurrently, in
which case the cycle contains only two steps.
[0037] The lengths of the denaturation, annealing and elongation
stages may be any desirable length of time. However, in attempting
to shorten the PCR amplification reaction to a time suitable for
intraoperative diagnosis, it has been found that the lengths of
these steps can be in the seconds range, rather than the minutes
range. Specifically, with certain new thermal cyclers being capable
of generating a thermal ramp rate (.DELTA.T) of at least about
5.degree. C. per second, PCR amplifications in 20 minutes are
possible. As used herein, the times provided for each step of the
PCR cycle does not include ramp times. The denaturation step may be
conducted for times of one second or less. In fact, some thermal
cyclers do not have settings for "0 seconds" which may be the
optimal duration of the denaturation step. That is, it is enough
that the thermal cycler reaches the denaturation temperature. The
annealing and elongation steps are optimally less than 10 seconds
each, and when conducted at the same temperature, the combination
annealing/elongation step may be less than 10 seconds.
[0038] As described herein, each cycle may be shortened
considerably without substantial deterioration of production of
amplicons by using substantially increased primer concentrations,
typically greater than about 400 nM, and often greater than about
800 nM, though the optimal concentration of primers will vary
somewhat from assay-to-assay. Sensitivity of RT-PCR assays may be
enhanced by the use of a sensitive Reverse Transcriptase enzyme
(described herein) and/or high concentrations of Reverse
Transcriptase primer to produce the initial target PCR
template.
[0039] The specificity of any given PCR reaction relies heavily,
but not exclusively, on the identity of the primer sets. The primer
sets are pairs of forward and reverse oligonucleotide primers that
anneal to a target DNA sequence to permit amplification of the
target sequence, thereby producing a target sequence-specific
amplicon. As used herein, a "derivative" of a specified
oligonucleotide is an oligonucleotide that binds to the same target
sequence as the specified oligonucleotide and amplifies the same
target sequence to produce essentially the same amplicon as the
specified oligonucleotide but for differences between the specified
oligonucleotide and the derivative. The derivative may differ from
the specified oligonucleotide by insertion, deletion and/or
substitution of any residue of the specified sequence so long as
the derivative substantially retains the characteristics of the
specified sequence in its use for the same purpose as the specified
sequence.
[0040] As used herein, "reagents" for any enzymatic reaction
mixture, such as a reverse transcription and PCR reaction mixture,
are any compound or composition that is added to the reaction
mixture including, without limitation, enzyme(s), nucleotides or
analogs thereof, primers and primer sets, buffers, salts and
co-factors. As used herein, unless expressed otherwise, "reaction
mixture" includes all necessary compounds and/or compositions
necessary to perform that enzymatic reaction, even if those
compounds or compositions are not expressly indicated.
[0041] Multiplexed PCR assays may be optimized, or balanced, by
time-shifting the production of amplicons, rather than by
manipulating primer concentrations. This may be achieved by using
two primer sets, each primer set having a different Tm so that a
two-stage PCR assay can be performed, with different annealing
and/or elongation temperatures for each stage to favor the
production of one amplicon over the other. This time and
temperature shifting method permits optimal balancing of the
multiplex reaction without the difficulties faced when manipulation
of primer concentrations is used to balance the reaction. This
technique is especially useful in a multiplex reaction where it is
desirable to amplify a rare cDNA along with a control cDNA, such as
the CEA/.beta.-GUS example shown below.
[0042] A quantitative reverse transcriptase polymerase chain
reaction (QRT-PCR) method is provided for rapidly and accurately
detecting low abundance RNA species in a population of RNA
molecules (for example, and without limitation, total RNA or mRNA),
including the steps of: a) incubating an RNA sample with a reverse
transcriptase and a high concentration of a target
sequence-specific reverse transcriptase primer under conditions
suitable to generate cDNA; b) subsequently adding suitable
polymerase chain reaction (PCR) reagents to the reverse
transcriptase reaction, including a high concentration of a PCR
primer set specific to the cDNA and a thermostable DNA polymerase
to the reverse transcriptase reaction, and c) cycling the PCR
reaction for a desired number of cycles and under suitable
conditions to generate PCR product ("amplicons") specific to the
cDNA. By temporally separating the reverse transcriptase and the
PCR reactions, and by using reverse transcriptase-optimized and
PCR-optimized primers, excellent specificity is obtained. The
reaction is conducted in a single tube (all tubes, containers,
vials, cells and the like in which a reaction is performed may be
referred to herein, from time to time, generically, as a "reaction
vessel"), removing a source of contamination typically found in
two-tube reactions. The high concentration primers permit very
rapid QRT-PCR reactions, typically on the order of 20 minutes from
the beginning of the reverse transcriptase reaction to the end of a
40 cycle PCR reaction. The realization of such a rapid QRT-PCR
experiment is assisted by the availability of thermal cycling
devices capable of generating a thermal ramp rate (.DELTA.T) of at
least about 5.degree. C. per second.
[0043] The reaction c) may be performed in the same tube as the
reverse transcriptase reaction by adding sufficient reagents to the
reverse transcriptase (RT) reaction to create good, or even optimal
conditions for the PCR reaction to proceed. A single tube may be
loaded, prior to the running of the reverse transcriptase reaction,
with: 1) the reverse transcriptase reaction mixture, and 2) the PCR
reaction mixture to be mixed with the cDNA mixture after the
reverse transcriptase reaction is completed. The reverse
transcriptase reaction mixture and the PCR reaction mixture may be
physically separated by a solid, or semi-solid (including
amorphous, glassy substances and waxy) barrier of a composition
that melts at a temperature greater than the incubation temperature
of the reverse transcriptase reaction, but below the denaturing
temperature of the PCR reaction. The barrier composition may be
hydrophobic in nature and forms a second phase with the RT and PCR
reaction mixtures when in liquid form. One example of such a
barrier composition is wax beads, commonly used in PCR reactions,
such as the AMPLIWAX PCR GEM products commercially available from
Applied Biosystems of Foster City, Calif. and the STRATASPHERE
Magnesium Wax Beads, commercially available from Stratagene of La
Jolla, Calif.
[0044] Alternatively, the separation of the reverse transcriptase
and the PCR reactions may be achieved by adding the PCR reagents,
including the PCR primer set and thermostable DNA polymerase, after
the reverse transcriptase reaction is completed. Preferably the PCR
reagents, are added mechanically by a robotic or fluidic means to
make sample contamination less likely and to remove human
error.
[0045] The products of the QRT-PCR process may be compared after a
fixed number of PCR cycles to determine the relative quantity of
the RNA species as compared to a given reporter gene. One method of
comparing the relative quantities of the products of the QRT-PCR
process is by gel electrophoresis, for instance, by running the
samples on a gel and detecting those samples by one of a number of
known methods including, without limitation, Southern blotting and
subsequent detection with a labeled probe, staining with ethidium
bromide and incorporating fluorescent or radioactive tags in the
amplicons.
[0046] However, the progress of the PCR reaction typically is
monitored by analyzing the relative rates of amplicon production
for each PCR primer set. Monitoring amplicons production may be
achieved by a number of processes, including without limitation,
fluorescent primers, fluorogenic probes and fluorescent dyes that
bind double-stranded DNA. A common method is the fluorescent 5'
nuclease assay. This method exploits the 5' nuclease activity of
certain thermostable DNA polymerases (such as Taq or Tfl DNA
polymerases) to cleave an oligomeric probe during the PCR process.
The oligomer is selected to anneal to the amplified target sequence
under elongation conditions. The probe typically has a fluorescent
reporter on its 5' end and a fluorescent quencher of the reporter
at the 3' end. So long as the oligomer is intact, the fluorescent
signal from the reporter is quenched. However, when the oligomer is
digested during the elongation process, the fluorescent reporter is
no longer in proximity to the quencher. The relative accumulation
of free fluorescent reporter for a given amplicon may be compared
to the accumulation of the same amplicons for a control sample
and/or to that of a control gene, such as, without limitation,
.beta.-gus, .beta.-actin or 18S rRNA to determine the relative
abundance of a given cDNA product of a given RNA in a RNA
population. Products and reagents for the fluorescent 5' nuclease
assay are readily available commercially, for instance from Applied
Biosystems.
[0047] Equipment and software also are readily available for
controlling and monitoring amplicon accumulation in PCR and QRT-PCR
according to the fluorescent 5' nuclease assay and other
QPCR/QRT-PCR procedures, including the Smart Cycler, commercially
available from Cepheid of Sunnyvale, Calif., the ABI Prism 7700
Sequence Detection System (TaqMan), commercially available from
Applied Biosystems. A cartridge-based sample preparation prototype
system (GenXpert) combines a thermal cycler and fluorescent
detection device having the capabilities of the Smart Cycler
product with fluid circuits and processing elements capable of
automatically extracting specific nucleic acids from a tissue
sample and performing QPCR or QRT-PCR on the nucleic acid. The
system uses disposable cartridges that can be configured and
pre-loaded with a broad variety of reagents. Such a system can be
configured to disrupt tissue and extract total RNA or mRNA from the
sample. The reverse transcriptase reaction components can be added
automatically to the RNA and the QPCR reaction components can be
added automatically upon completion of the reverse transcriptase
reaction.
[0048] Further, the PCR reaction may be monitored of production (or
loss) of a particular fluorochrome from the reaction. When the
fluorochrome levels reach (or fall to) a desired level, the
automated system will automatically alter the PCR conditions. In
one non-limiting example, this is particularly useful in the
multiplexed embodiment described above, where a more-abundant
(control) target species is amplified by the first, lower Tm,
primer set at a lower temperature than the less abundant species
amplified by the second, higher Tm, primer set. In the first stage
of the PCR amplification, the annealing step of the cycling
reaction is conducted at a temperature that permits amplification
of the more abundant target species. The annealing temperature then
is automatically raised to essentially stop amplification of the
more abundant target species.
[0049] In the above-described reactions, the amounts of certain
reverse transcriptase and the PCR reaction components typically are
atypical in order to take advantage of the faster ramp times of
some thermal cyclers. Specifically, the primer concentrations are
very high. Typical gene-specific primer concentrations for reverse
transcriptase reactions are less than about 20 nM. To achieve a
rapid reverse transcriptase reaction on the order of one to two
minutes, the reverse transcriptase primer concentration was raised
to greater than 20 nM, preferably at least about 50 nM, and
typically about 100 nM. Standard PCR primer concentrations range
from 100 nM to 300 nM. Higher concentrations may be used in
standard PCR reactions to compensate for Tm variations. However,
for purposes herein, the referenced primer concentrations are for
circumstances where no Tm compensation is needed. Proportionately
higher concentrations of primers may be empirically determined and
used if Tm compensation is necessary or desired. To achieve rapid
PCR reactions, the PCR primer concentrations typically are greater
than 200 nM, preferably greater than about 500 nM and typically
about 800 nM. Typically, the ratio of reverse transcriptase primer
to PCR primer is about 1 to 8 or more. The increase in primer
concentrations permitted PCR experiments of 40 cycles to be
conducted in less than 20 minutes, as described below in Example
2.
[0050] A sensitive reverse transcriptase may be preferred in
certain circumstances where either low amounts of RNA are present
or a target RNA is a low abundance RNA. By the term "sensitive
reverse transcriptase," it is meant a reverse transcriptase capable
of producing suitable PCR templates from low copy number
transcripts for use as PCR templates. The sensitivity of the
sensitive reverse transcriptase may derive from the physical nature
of the enzyme, or from specific reaction conditions of the reverse
transcriptase reaction mixture that produces the enhanced
sensitivity. One example of a sensitive reverse transcriptase is
SensiScript RT reverse transcriptase, commercially available from
Qiagen, Inc. of Valencia Calif. This reverse transcriptase is
optimized for the production of cDNA from RNA samples of <50 ng,
but also has the ability to produce PCR templates from low copy
number transcripts. In practice, in the assays described herein,
adequate results were obtained for samples of up to, and even in
excess of, about 400 ng RNA. Other sensitive reverse transcriptases
having substantially similar ability to reverse transcribe low copy
number transcripts would be equivalent sensitive reverse
transcriptase for the purposes described herein. Notwithstanding
the above, the ability of the sensitive reverse transcriptase to
produce cDNA from low quantities of RNA is secondary to the ability
of the enzyme, or enzyme reaction system to produce PCR templates
from low copy number sequences.
[0051] As discussed above, the procedures described herein also may
be used in multiplex QRT-PCR processes. In its broadest sense, a
multiplex PCR process involves production of two or more amplicons
in the same reaction vessel. Multiplex amplicons may be analyzed by
gel electrophoresis and detection of the amplicons by one of a
variety of methods, such as, without limitation ethidium bromide
staining, Southern blotting and hybridization to probes, or by
incorporating fluorescent or radioactive moieties into the
amplicons and subsequently viewing the product on a gel. However,
real-time monitoring of the production of two or more amplicons is
preferred. The fluorescent 5' nuclease assay is the most common
monitoring method. Equipment is now available (for example, the
above-described Smart Cycler and TaqMan products) that permits the
real-time monitoring of accumulation of two or more fluorescent
reporters in the same tube. For multiplex monitoring of the
fluorescent 5' nuclease assay, oligomers are provided corresponding
to each amplicon species to be detected. The oligomer probe for
each amplicon species has a fluorescent reporter with a different
peak emission wavelength than the oligomer probe(s) for each other
amplicons species. The accumulation of each unquenched fluorescent
reporter can be monitored to determine the relative amounts of the
target sequence corresponding to each amplicon.
[0052] In traditional multiplex QPCR and QRT-PCR procedures, the
selection of PCR primer sets having similar annealing and
elongation kinetics and similar sized amplicons are desirable. The
design and selection of appropriate PCR primer sets is a process
that is well known to a person skilled in the art. The process for
identifying optimal PCR primer sets, and respective ratios thereof
(primer limiting, that is, limiting the abundance of the PCR
primers for the more abundant RNA species in a multiplex PCR
experiment to permit the detection of less abundant species) to
achieve a balanced multiplex reaction also is known. By "balanced,"
it is meant that certain amplicon(s) do not out-compete the other
amplicon(s) for resources, such as dNTPs or enzyme. Equalization of
the Tm (melting temperature) for all PCR primer sets also is
encouraged. See, for instance, ABI PRISM 7700 Sequence Detection
System User Bulletin #5, "Multiplex PCR with TaqMan VIC Probes",
Applied Biosystems (1998/2001).
[0053] Despite the above, for very low copy number transcripts, it
is difficult to design accurate multiplex PCR experiments, even by
limiting the PCR primer sets for the more abundant control species.
One solution to this problem is to run the PCR reaction for the low
abundance RNA in a separate tube for the PCR reaction for the more
abundant species. However, that strategy does not take advantage of
the benefits of running a multiplex PCR experiment. A two-tube
process has several drawbacks, including cost, the addition of more
room for experimental error and the increased chance of sample
contamination, which is critical in PCR assays.
[0054] A method is therefore provided for performing a multiplex
PCR process, including QRT-PCR and QPCR, capable of detecting low
copy number nucleic acid species along with one or more higher copy
number species. The difference between low copy number and high
copy number nucleic acid species is relative, but is referred to
herein as a difference in the prevalence of a low (lower) copy
number species and a high (higher) copy number species of at least
about 30-fold, but more typically at least about 100-fold. For
purposes herein, the relative prevalence of two nucleic acid
species to be amplified is more salient than the relative
prevalence of the two nucleic acid species in relation to other
nucleic acid species in a given nucleic acid sample because other
nucleic acid species in the nucleic acid sample do not directly
compete with the species to be amplified for PCR resources.
[0055] As used herein, the prevalence of any given nucleic acid
species in a given nucleic acid sample, prior to testing, is
unknown. Thus, the "expected" number of copies of a given nucleic
acid species in an nucleic acid sample often is used herein and is
based on historical data on the prevalence of that species in
nucleic acid samples. For any given pair of nucleic acid species,
one would expect, based on previous determinations of the relative
prevalence of the two species in a sample, the prevalence of each
species to fall within a range. By determining these ranges one
would determine the -fold difference in the expected number of
target sequences for each species.
[0056] The multiplex PCR method involves performing a two- (or
more) stage PCR amplification, permitting modulation of the
relative rate of production of a first amplicon by a first primer
set and a second amplicon by a second primer set during the
respective amplification stages. The PCR amplification of the
second amplification stage is conducted under different reaction
conditions ("different reaction conditions" include, without
limitation, different temperatures for steps in the PCR cycle, such
as the annealing step, or differences in the reagents in the PCR
reaction mixture, such as differences in primers and/or primer
concentrations) than in the first amplification stage. By this
method, PCR amplifications to produce amplicons directed to a lower
abundance nucleic acid species are effectively "balanced" with PCR
amplifications to produce amplicons directed to a higher abundance
nucleic acid species. Separating the reaction into two or more
temporal stages may be achieved by omitting the PCR primer set for
any amplicons that are not to be produced in the first
amplification stage. The omitted PCR primer set may then be added
to the PCR reaction mixture at the beginning of the second
amplification stage. This is best achieved through use of automated
processes, such as the GenXpert prototype system described above.
Two or more separate amplification stages may be used to tailor and
balance multiplex assays, along with, or to the exclusion of
tailoring the concentration of the respective primer sets.
[0057] A second method for temporally separating the PCR
amplification process into two or more stages is to select PCR
primer sets with variation in their respective Tm. Two examples of
such a method are provided in Examples 3 and 6, below. In one
example, primers for a lower copy number nucleic acid species would
have a higher Tm (Tm.sub.1) than primers for a higher abundance
species (Tm.sub.2). In this process, the first stage of PCR
amplification is conducted for a predetermined number of cycles at
a temperature sufficiently high that there is substantially no
amplification of the higher abundance species. After the first
stage of amplification, the annealing and elongation steps of the
PCR reaction are conducted at a lower temperature, typically about
Tm.sub.2, so that both the lower abundance and the higher abundance
amplimers are amplified. It should be noted that Tm, as used herein
and unless otherwise noted, refers to "effective Tm," which is the
Tm for any given primer in a given reaction mix, which depends on
factors, including, without limitation, the nucleic acid sequence
of the primer and the primer concentration in the reaction
mixture.
[0058] It should be noted that PCR amplification is a dynamic
process. Without limitation, when using temperature to modulate the
respective PCR reactions in a multiplex PCR reaction, the higher
temperature annealing stage may be carried out at any temperature
typically ranging from just above the lower Tm to just above the
higher Tm, so long as the reaction favors production of the
amplicon by the higher Tm primer set. Similarly, without
limitation, the annealing for the lower temperature reaction
typically is at any temperature below the higher Tm of the low
temperature primer set that will allow sufficient amplification
efficiency by the lower Tm primer set.
[0059] In the example provided above, in the higher temperature
stage, the amplicon for the low abundance RNA is amplified at a
rate faster than the amplicon for the higher abundance RNA (and
preferably to the substantial exclusion of production of the second
amplicon), so that, prior to the second amplification stage, where
it is desirable that amplification of all amplicons proceeds in a
substantially balanced manner, the amplicon for the lower abundance
RNA is of sufficient abundance that the amplification of the higher
abundance RNA does not interfere with the amplification of the
amplicon for the lower abundance RNA. In the first stage of
amplification, when the amplicon for the low abundance nucleic acid
is preferentially amplified, the annealing and elongation steps may
be performed above the higher Tm to gain specificity over
efficiency (during the second stage of the amplification, since
there is a relatively large number of low abundance nucleic acid
amplicons, selectivity no longer is a significant issue, and
efficiency of amplicon production is preferred). It, therefore,
should be noted that although favorable in many instances, the
temperature variations may not necessarily result in the complete
shutdown of one amplification reaction over another.
[0060] In another embodiment, a first primer set with a first Tm
may target a more-abundant template sequence (for instance, a
control template sequence) and a second primer set with a higher Tm
may target a less-abundant template sequence. In this case, the
more-abundant template and the less-abundant template may both be
amplified in a first stage at a temperature sufficient to allow
sufficient amplification with the lower Tm primer set, typically at
or above the Tm of the first, lower Tm, primer set. When a
threshold amount of amplicon corresponding to the more abundant
template is reached, the annealing and/or elongation temperature of
the reaction is raised to effectively shut down amplification of
the more abundant template.
[0061] Selection of three or more sets of PCR primer sets having
three or more different Tms (for instance,
Tm.sub.1>Tm.sub.2>Tm.sub.3) can be used to amplify sequences
of varying abundance in a stepwise manner, so long as the
differences in the Tms are sufficiently large to permit
preferential amplification of desired sequences to the substantial
exclusion of undesired sequences for a desired number of cycles. In
one process, the lowest abundance sequences are amplified in a
first stage for a predetermined number of cycles. Next, the lowest
abundance and the lesser abundance sequences are amplified in a
second stage for a predetermined number of cycles. Lastly, all
sequences are amplified in a third stage. As with the two-stage
reaction described above, the annealing temperature for each stage
may vary, depending on the relative efficiencies of each single
amplification reaction of the multiplex reaction. It should be
recognized that two or more amplimers may have substantially the
same Tm, to permit amplification of more than one species of
similar abundance at any stage of the amplification process. As
with the two-stage reaction, the three-stage reaction may also
proceed stepwise from amplification of the most abundant nucleic
acid species at the lowest annealing temperature to amplification
of the least abundant species at the highest annealing
temperature.
[0062] By this sequential amplification method, an additional tool
is provided for the "balancing" of multiplex PCR reactions besides
the matching of Tms and using limiting amounts of one or more PCR
primer sets. The exploitation of PCR primer sets with different Tms
as a method for sequentially amplifying different amplicons may be
preferred in certain circumstances to the sequential addition of
additional primer sets. However, the use of temperature-dependent
sequencing of multiplex PCR reactions may be coupled with the
sequential physical addition of primer sets to a single reaction
mixture.
[0063] Also provided is an internal positive control that confirms
the operation of a particular amplification reaction for a negative
result. The internal positive controls (IPC) are DNA
oligonucleotides that have the same primer sequences as the target
gene (CEA or tyrosinase) but have a different internal probe
sequence. Selected sites in the IPCs optionally may be synthesized
with uracil instead of thymine so that contamination with the
highly concentrated mimic could be controlled using uracil DNA
glycosylase, if required. The IPCs may be added to any PCR reaction
mastermix in amounts that are determined empirically to give Ct
values typically greater than the Ct values of the endogenous
target of the primer set. The PCR assays are then performed
according to standard protocols, and even when there is no
endogenous target for the primer set, the IPC would be amplified,
thereby verifying that the failure to amplify the target endogenous
DNA is not a failure of the PCR reagents in the mastermix. In this
embodiment, the IPC probe fluoresces differently than the probe for
the endogenous sequences. A variation of this for use in RT-PCR
reactions is where the IPC is an RNA and the RNA includes an RT
primer sequence. In this embodiment, the IPC verifies function of
both the RT and PCR reactions. Both RNA and DNA IPCs (with
different corresponding probes) may also be employed to
differentiate difficulties in the RT and PCR reactions.
[0064] The methods described herein are generally applicable to
quantitative PCR and RT-PCR methods. Described herein and the
attached manuscripts are methods for the detection of
carcinoembryonic antigen (CEA) and prognosis of adenocarcinomas of
the esophagus. The methods described herein are methods that are
equally applicable to the identification of other micrometastases,
including occult micrometastases, in a variety of other tumor
types. The rapid protocols described herein may be run in about 20
minutes. This short time period permits the assay to be run
intraoperatively so that a surgeon can decide on a surgical course
during a single operation, rather than requiring a second
operation, or requiring the surgeon to perform unneeded or overly
broad prophylactic procedures. For instance, in the surgical
evaluation of certain cancers, including breast cancer and
melanoma, sentinel lymph nodes are removed in a first operation.
The sentinel nodes are later evaluated for micrometastases, and,
when micrometastases are detected in a patient's sentinel lymph
node, the patient will need a second operation, thereby increasing
the patient's surgical risks and patient discomfort associated with
multiple operations. In the case of lung or esophageal cancer,
intraoperative analysis of lymph nodes can be used to determine the
extent of resection required and/or the need for neoadjuvant
chemotherapy. With the ability to determine the expression levels
of certain tumor-specific markers, such as, without limitation CEA,
MUC-1, CK-19, tyrosinase and MART-1, in less than 30 minutes with
increased accuracy, a physician can make an immediate decision on
how to proceed. The rapid test also is applicable to needle
biopsies taken in a physician's office. A patient need not wait for
days to get the results of a biopsy (such as a needle biopsy of a
tumor or lymph node), but can now get more accurate results in a
very short time. The methods described herein are applicable to
detect, without limitation, a variety of expressed RNAs, whether
normal or abnormal, DNA rearrangements or the presence of
additional or abnormal nucleic acids, such as viral nucleic
acids.
[0065] In the commercialization of the above-described methods for
multiplexed and non-multiplexed QRT-PCR and/or QPCR, certain kits
for detection of specific nucleic acids will be particularly
useful. One example of such a kit would include reagents necessary
for the one-tube QRT-PCR process described above. In one example,
the kit would include the above-described reagents, including
reverse transcriptase, a reverse transcriptase primer, a
corresponding PCR primer set, a thermostable DNA polymerase, such
as Taq polymerase, and a suitable fluorescent reporter, such as,
without limitation, a probe for a fluorescent 5' nuclease assay, a
molecular beacon probe, a single dye primer or a fluorescent dye
specific to double-stranded DNA, such as ethidium bromide. The
primers may be present in quantities that would yield the high
concentrations described above. Thermostable DNA polymerases are
commonly and commercially available from a variety of
manufacturers. Additional materials in the kit may include:
suitable reaction tubes or vials, a barrier composition, typically
a wax bead, optionally including magnesium; reaction mixtures
(typically 10.times.) for the reverse transcriptase and the PCR
stages, including necessary buffers and reagents such as dNTPs;
nuclease- or RNase-free water; RNase inhibitor; control nucleic
acid(s) and/or any additional buffers, compounds, co-factors, ionic
constituents, proteins and enzymes, polymers, and the like that may
be used in reverse transcriptase and/or PCR stages of QRT-PCR
reactions.
[0066] A second kit is specific to the above-described multiplex
PCR procedure. The kit may include, without limitation, a first PCR
primer set for a low abundance nucleic acid, having a first Tm and
a second PCR primer set for a more abundant nucleic acid, having a
second Tm. The relative Tms of the primer sets are selected for
their ability to balance a multiplex PCR reaction according to the
methods described herein. In a kit for QRT-PCR, the kit also may
include: any suitable reverse transcriptase, reverse transcriptase
primers specific to the nucleic acids to be amplified, a barrier
composition, such as a wax bead, a thermostable DNA polymerase
and/or a suitable fluorescent reporter, such as, without
limitation, a probe for a fluorescent 5' nuclease assay, a
molecular beacon probe, a single dye primer or a fluorescent dye
specific to double-stranded DNA, such as ethidium bromide. The kit
may include a sensitive reverse transcriptase for the detection of
low abundance RNAs. As above, additional materials in the kit may
include: suitable reaction tubes or vials, a barrier, typically a
wax bead, optionally including magnesium; reaction mixtures
(typically 10.times.) for the reverse transcriptase and the PCR
stages, including necessary buffers and reagents such as dNTPs;
nuclease- or RNase-free water; RNase inhibitor; control nucleic
acid(s) and/or any additional buffers, compounds, co-factors, ionic
constituents, proteins and enzymes, polymers, and the like that may
be used in reverse transcriptase and/or PCR stages of QRT-PCR
reactions.
[0067] The above-described kits or cartridges also may include
reagents and mechanical components suitable for the manual or
automated extraction of nucleic acid from tissue samples. These
reagents are known to those skilled in the art and typically are a
matter of design choice. For instance, in an automated process,
tissue may be disrupted ultrasonically in a suitable lysis solution
provided in the kit or cartridge. The resultant lysate solution may
then be filtered and RNA may be bound to RNA-binding magnetic beads
also provided in the kit or cartridge. The beads/RNA may be washed,
and the RNA eluted prior to the reverse transcriptase reaction. In
the case of automated nucleic acid extraction, the choice of
reagents and their mode of packaging (for instance disposable
single-use cartridges) typically are dictated by the physical
configuration of the robotics and fluidics of the specific
extraction system, such as Cepheid's GenXpert prototype system.
[0068] The constituents of the kits may be packaged together or
separately, and each constituent may be presented in one or more
tubes or vials, or in cartridge form (a modular unit containing one
or more reagents for use in an automated device), as is
appropriate. The constituents, independently or together, may be
packaged in a variety of states, including without limitation, in
lyophilized, glassified, aqueous or other forms as is
appropriate.
[0069] FIG. 1 is a schematic cross-section diagram of a cartridge
10 for use in the above-described automated methods. Cartridge 10
includes compartments 20 in which any desired reagent may be stored
for use. Compartments 20 are separated by walls 25. Cartridge 10
includes multiple passageways 30 fluidly connected to a common
passageway 40. A valve member 50 is shown within common passageway
40. Valve member 50 controls flow of reagent from individual
compartments 20 into common passageway 40. Common passageway 40 is
fluidly connected to a reaction vessel (not shown) into which
reagents from compartments 20 are transferred. The reagents
contained within compartments 20 may include, without limitation,
reagents for cell lysis, for nucleic acid purification, for reverse
transcription or for PCR reactions. FIG. 1 shows one of many
possible permutations of a cartridge device useful in automating
molecular purifications and assays. The cartridge and compartments
may have any desired shape and size, as dictated by empirical
factors as well as by designer preference. The choice of and
configuration of fluid connections and valves also is a matter of
design choice, and may vary greatly.
EXAMPLES
Example 1
One-Tube QRT-PCR
[0070] With the introduction of real-time, fluorescence-based 5'
nuclease PCR (Gibson, U. E., C. A. Heid and P. M. Williams. 1996. A
novel method for real time quantitative RT PCR. Genome Res.
6:995-1001; Heid, C. A., J. Stevens, K. J. Livak and P. M.
Williams. 1996. Real time quantitative PCR. Genome Res. 6:986-994)
and instruments such as the ABI PRISMT.TM. 7700 (TaqMan.RTM.)
sequence detector (Applied Biosystems, Foster City, Calif., USA),
quantitative RT-PCR is now a widely accepted method for measuring
gene expression levels. Quantitative RT-PCR is a sensitive
technique and is particularly useful for the analysis of samples
containing limited amounts of nucleic acids, such as in clinical
tissues (Collins, C., J. M. Rommens, D. Kowbel, T. Godrey, M.
Tanner, S. I. Hwang, D. Polikoff, G. Nonet et al. 1998. Positional
cloning of ZNF217 and NABC1: genes amplified at 20q13.2 and
overexpressed in breast carcinoma. Proc. Natl. Acad. Sci. USA
95:8703-8708). When quantitating these small amounts of RNA and/or
very low-abundance mRNA species, obtaining maximum sensitivity from
a quantitative RT-PCR is extremely important. While consecutive
rounds of nested PCR are often used to obtain maximum sensitivity,
this is difficult to achieve and still maintain accurate
quantitation. Furthermore, multiple rounds of PCR increase the risk
of contamination, a serious problem when working at the desired
sensitivity levels. One-tube RT-PCR (RT and PCR in the same tube
using the reverse PCR primer for the RT) reduces the risk of
contamination when using the ABI PRISM 7700 because the reaction
tubes are never opened.
[0071] Theoretically, a one-tube procedure should have the same
sensitivity as a two-step approach (separate RT followed by PCR),
but in practice this is not the case (Battaglia, M., P. Pedrazzoli,
B. Palermo, A. Lanza, F. Bertolini, N. Gibelli, G. A. Da Prada, A.
Zambelli et al. 1998. Epithelial tumour cell detection and the
unsolved problems of nested RT-PCR: a new sensitive one step method
without false positive results. Bone Marrow Transplant 22:693-698).
It has been found that the sensitivity of one-tube RT-PCR is
limited by the relative nonspecificity of the RT step. This
nonspecificity arises from the fact that the RT is carried out at
relatively low temperature and without a hot start, thus allowing
nonspecific priming by both the desired RT "reverse" primer and
also from the "forward" PCR primer. As the amount of target
decreases in the input RNA sample, priming artifacts from the
cold-start RT process can compete with, and reduce the efficiency
of, PCR amplification of the desired target sequence. Thus, as RNA
input decreases in a one-tube procedure, nonspecific side reactions
eventually out-compete the desired reaction, and no specific
product is generated. In a two-step or nested RT-PCR procedure,
specificity can be achieved with the use of hot-start PCR and a
primer set 5' upstream from the RT primer. However, this is not the
case in a one-tube procedure unless one is willing to open the
reaction tube to add new primers (thus making it a one-tube but
two-step procedure). It has been hypothesized that by using an
external RT primer and keeping the RT and PCR primers separated
during the RT step, PCR specificity and therefore sensitivity in a
one-tube RT-PCR should be maintainable. Here, a modified one-tube
RT-PCR assay that greatly increases sensitivity and can be used for
quantitative RT-PCR on the ABI PRISM 7700 is presented.
[0072] Standard one-tube reactions were set up for
.beta.-glucuronidase (.beta.-gus) mRNA in 50 .mu.L volumes with the
following final concentrations: 10 nM .beta.-gus RT primer
(5'-TTTGGTTGT-CTCTGCCGAGT-3') (SEQ ID NO: 2), 100 nM each
.beta.-gus PCR primer (GUS-F, 5'-C-TCATTTGGAATTTTGCCGATT-3')(SEQ ID
NO: 3); GUS-R, (5'-CCGAGTGAAGATCCC-CTTTTTA-3')(SEQ ID NO: 4), 100
nM .beta.-gus probe(5'-6-fam-TGAACAGTCACCGACG-AGAGTGCTGG-tamra-3')
(SEQ ID NO: 5), 1.times. TaqMan reaction buffer (Applied
Biosystems), 5.5 mM MgCl.sub.2, 300 .mu.M each dNTP, 20 U Rnase
inhibitor, 62.5 U SUPERSCRIPT II.TM. reverse transcriptase (Life
Technologies, Rockville, Md., USA) and 1.25 U AmpliTaq Gold.RTM.
(Applied Biosystems).
[0073] In the modified procedure, physical separation between the
RT reaction mixture and the PCR primers was achieved by the use of
AmpliWax.RTM. PCR gem 50 (Applied Biosystems). First, the
.beta.-gus PCR primers were pipetted into the PCR plate in a final
5.0-.mu.L volume. One PCR gem 50 was placed in each well, the wells
were capped and the plate was centrifuged briefly to avoid the
adherence of reagents to the tube wall above the wax barrier. The
plate was then heated to 80.degree. C. for 2 min and cooled to
4.degree. C. to produce a wax barrier. A 45 .mu.L upper layer was
then pipetted into each well. This mixture contained the .beta.-gus
RT primer, the RNA, Rnase inhibitor and SUPERSCRIPT II reverse
transcriptase. Both layers were formulated to contain all of the
remaining reaction components (buffer, nucleotides, MgCl.sub.2) at
the concentrations described above. The presence of AmpliTaq Gold
in the RT layer is inconsequential because this enzyme is inactive
until heated to 95.degree. C.
[0074] All reactions were carried out on the ABI PRISM 7700 with
the following thermocycler conditions: 48.degree. C. hold for 30
min, 95.degree. C. hold for 12 min, followed by 40 cycles of
95.degree. C. for 20 s and 60.degree. C. for 1 min. The wax layer
remained intact for the RT step at 48.degree. C. but was melted
during the 12-min, 95.degree. C. AmpliTaq Gold activation step,
thus allowing the two layers to mix before the PCR begins. Data
were analyzed with Applied Biosystems' sequence detection
software.
[0075] First, the effect of the wax layer on the fluorescence
detection in the TaqMan assay was evaluated to determine the extent
of fluorescence quenching by the wax. Using randomly primed cDNA
from a lung adenocarcinoma cell line (A549), 20 replicates of PCR
for .beta.-gus with and without the wax layer were performed. The
results showed no decrease in the overall fluorescence (P=0.935)
and no change in the cycle threshold value (P=0.55) between the two
groups when compared by the independent samples t-test.
[0076] To compare the sensitivity of the one-tube RT-PCR with and
without the wax, serial dilutions of spleen total RNA (Clontech
Laboratories, Palo Alto, Calif., USA) from 5 ng to 10 pg were used.
The results of the RT-PCR without the wax layer showed that the
fluorescence (.DELTA.Rn) was weak even at 5 ng RNA input and
decreased further by an average factor of 75% every dilution (FIG.
2A). As a result, the 200-pg sample fell below the threshold for
detection. However, with the use of the wax layer, the .DELTA.Rn
remained essentially the same down to the 40-pg dilution, and only
at 10 pg was there a 60% drop in the .DELTA.Rn (FIG. 2B). Thus,
this modified procedure resulted in at least a 20-fold increase in
sensitivity. The efficiency (E) of the RT-PCR (as calculated by the
formula E.times.10.sup.(1/-s)-1, where "s" is the slope of the
standard curve from the dilutions) (PE Applied Biosystems User
Bulletin #2. 1997. Relative quantitation of gene expression.
Applied Biosystems, Foster City, Calif.) was also improved by the
use of the wax (67% without wax and 77% with wax). A 10 .mu.L
aliquot of each reaction was run out on a 10% non-denaturing
polyacrylamide gel and stained with ethidium bromide (FIG. 3). With
the wax layer, the 81-bp product, corresponding to the expected
size, was visible in all but the 10-pg RNA dilution (demonstrating
the extra sensitivity of TaqMan detection versus ethidium bromide).
In the reactions without wax, however, no reactions produced a
clean signal at 81 bp. Instead, there was a smear of nonspecific
products at all RNA concentrations.
[0077] The same experiments were performed using Sensiscript
RT.RTM. (Qiagen, Valencia, Calif., USA). With this enzyme, PCR
product was just detectable down to the 40-pg dilution even without
the use of the wax layer. However, the total fluorescence continued
to drop with each consecutive dilution. With the addition of the
wax, the .DELTA.Rn remained constant, and detection was easily
achieved down to 10 pg. Notably, the efficiency of this one-tube
reaction was near 100% (measured using the methods described
above). Thus, the sensitivity of the one-tube RT-PCR for the 5'
fluorogenic assay in the ABI PRISM 7700 is significantly enhanced
by the use of the AmpliWax PCR gem 50. PCR gems were originally
designed to facilitate hot start PCR, but this is no longer
necessary with new enzymes for automatic hot start. Here, it is
shown that these same AmpliWax PCR gems are beneficial in the
context of a one-step quantitative RT-PCR. Furthermore, by
eliminating the need to open the PCR tubes the occurrence of cDNA
or PCR product contamination was minimazed. Finally, the
preservation of PCR specifically facilitates the amplification of
the desired product, and as such, is relevant even in
non-quantitative end-point assays.
Example 2
Quantitative RT-PCR in Less than Twenty Minutes
[0078] The following is a one-tube two-step assay for quantitative
reverse transcription followed by polymerase chain reaction
(QRT-PCR), which can be completed in less than twenty minutes using
Cepheid's Smart Cycler. Current methods of QRT-PCR for the 5'
fluorogenic assay in the Applied Biosystem's 7700 require more than
two hours. By altering primer and probe concentrations and
utilizing the fast ramping ability of the Smart Cycler, the reverse
transcriptase reaction time was reduced to 2 minutes and the PCR
time was reduced to 16 minutes using a 1 second denaturation and a
6 second extension for 40 cycles.
[0079] PCR reactions were designed for .beta.-glucuronidase
(.beta.-gus) and carcinoembryonic antigen (CEA) cDNA respectively
in 25 .mu.l volumes with the following final concentrations: 400 nM
each .beta.-gus PCR primer (GUS-F, 5'-CTC ATT TGG AAT TTT GCC GAT
T-3' (SEQ ID NO: 3); (GUS-R, 5'-CCG AGT GAA GAT CCC CTT TTT
A-3')(SEQ ID NO: 4) or CEA primer (CEA-F, 5'-AGA CAA TCA CAG TCT
CTG CGG A-3')(SEQ ID NO: 6); (CEA-R, 5'-ATC CTT GTC CTC CAC GGG
TT-3') (SEQ ID NO: 7), 200 nM .beta.-gus probe
(5'-6-fam-TGAACAGTCACCGACGAGAGTGCTGG-tamra-3) (SEQ ID NO: 5) or 200
nM CEA probe (5'-6-fam-CAA GCC CTC CAT CTC CAG CAA CAA CT-tamra-3)
(SEQ ID NO: 8), 1.times. Platinum Taq reaction buffer, 4.5 mM
MgCl.sub.2, 300 .mu.M each dNTP, 0.06 U/.mu.l Platinum Taq DNA
polymerase (GIBCO BRL).
[0080] Tests were run using MgCl.sub.2 at 1.5, 2.5, 3.5 and 4.5 mM
concentrations and it was determined that 4.5 mM is optimal for
this assay. The cDNA for these reactions was generated from gene
specific reverse transcriptase reactions for GUS and CEA using a
250 ng input of RNA from an A549 cell line (GUS) and fresh lymph
node RNA that was positive for CEA.
[0081] RT-PCR reactions were designed for .beta.-gus mRNA and CEA
mRNA in 25 .mu.l volumes with the same PCR concentrations as above
and the following reverse transcriptase concentrations: 60 nM
.beta.-gus reverse transcriptase primer
(5'-TTTGGTTGTCTCTGCCGAGT-3') (SEQ ID NO: 2) or CEA reverse
transcriptase primer (5'-GTG AAG GCC ACA GCA T-3') (SEQ ID NO: 9),
1 .mu.l Sensiscript and 0.8 U/.mu.l RNase inhibitor. The RNA input
for the RT-PCR was 400 ng A549 and 25 ng lymph node.
[0082] The denaturation step was first optimized by comparing 1, 2,
5, 7 and 10 second denaturation at 95.degree. C. in combination
with a 30 second extension for 40 cycles. Platinum Taq activation
was done at 95.degree. C. for 30 seconds. The results of this
testing show no significant loss of sensitivity between a 1 second
denaturation versus a 10 second denaturation for either gene. Next,
the extension step was optimized by comparing 3, 5, 7, 10, 13, 15
and 30 second extension at 64.degree. C. in combination with a 15
second denaturation for 40 cycles. Platinum Taq activation was done
at 95.degree. C. for 30 seconds.
[0083] The results of this testing show minimal loss of sensitivity
of approximately one and a half cycles from 30 second extension to
3 second extension for GUS. For CEA, no significant loss is seen
from 30 second extension to 3 second extension.
[0084] Next, the combined effect of altering denaturation/extension
time was evaluated by comparing a 1/3 second PCR to a 2/15 second
PCR over 40 cycles. The results show a 2.2 and 1.1 cycle loss in
sensitivity for GUS and CEA, respectively. A 2/15 second PCR
requires 22 minutes while a 1/3 second PCR requires 16 minutes,
thus this insignificant loss in sensitivity is well worth cutting
the reaction time by 6 minutes.
[0085] In an attempt to reduce the ramping time from denaturation
to extension, the effect of decreasing the denaturation temperature
from 95.degree. C. to 90.degree. C. to 85.degree. C. was evaluated.
For GUS, there is no significant loss in sensitivity when
denaturation is done at 95.degree. C. or 85.degree. C. For CEA, the
reaction failed when denaturation was done at 85.degree. C. but no
significant loss in sensitivity was seen from denaturation at
95.degree. C. to 90.degree. C. The amount of time gained by doing a
90.degree. C. versus a 95.degree. C. denaturation is about 1.5
minutes over 40 cycles.
[0086] Taq activation time was evaluated and no significant loss in
sensitivity for either gene by decreasing Taq activation from 30
seconds to 10 seconds was found.
[0087] After optimizing the PCR conditions, the reverse
transcriptase reaction was optimized. The reverse transcriptase
reaction was done in a total volume of 15 .mu.l. After completion
of the reaction, the mixture was held at 70.degree. C. at which
time the PCR components (total volume, 10 .mu.l) were added.
Reverse transcriptase reaction times of 2, 3, 5, 7 and 10 minutes
were compared. The reverse transcriptase reactions were combined
with PCR conditions that included a 1 second denaturation at
95.degree. C. and a 5 second extension at 64.degree. C. for 40
cycles for both genes.
[0088] The results of these reverse transcriptase reaction time
trials show a loss in sensitivity of 1.1 cycle for GUS and 1.81
cycles for CEA from a 10 minute reverse transcriptase reaction to a
2 minute reverse transcriptase reaction. Next, the total effect of
decreasing the RT-PCR times on the sensitivity of the assay was
evaluated by comparing the following RT-PCR conditions: 1) a "gold
standard" with a 10 minute reverse transcriptase reaction followed
by 10 second denaturation and 15 second extension for 40 cycles,
total run time of 38 minutes, 2) a 5 minute reverse transcriptase
reaction followed by optimized PCR conditions of 1 second
denaturation and 5 second extension for 40 cycles, total run time
of 20 minutes, 3) a 2 minute reverse transcriptase reaction
followed by optimized PCR conditions, total run time of 17 minutes,
and 4) a "quick" RT-PCR with a 2 minute reverse transcriptase
reaction followed by 1 second denaturation and 3 second extension
for 40 cycles, total run time of 15 minutes. For Gus, the "gold
standard" RT-PCR had a Ct (number of cycles required to reach a
predetermined threshold, reference fluorescence level) of
25.88.+-.0.78 while the 2 min reverse transcriptase reaction with
optimized PCR conditions had a Ct of 29.42.+-.0.7 showing a total
cycle difference of 3.54. For CEA, the "gold standard" RT-PCR had a
Ct of 29.94.+-.2.2 while the 2 min RT with optimized PCR conditions
had a Ct of 34.92.+-.0.5 showing a total cycle difference of
4.98.
[0089] In an attempt to increase the sensitivity of the shorter
protocol, the effects of increasing the primer concentrations for
Gus and CEA were evaluated. The experiment described above was
repeated with an increase of the concentration of the RT primer
from 60 nM to 100 nM and the F/R PCR primers from 400 nM to 800 nM.
The results of this test show a 2.3 cycle difference (4.98 with low
primer concentrations) between the gold standard versus the 2 min
optimal protocol for CEA and a 1.63 (3.54 with low primer
concentrations) cycle difference for Gus. This small loss in
sensitivity is insignificant considering that the total RT-PCR time
was reduced from 38 minutes to 17 minutes.
[0090] The PCR efficiency for CEA was evaluated using the optimized
conditions of 1 second denaturation and 6 second extension for 40
cycles on a dilution series of the fresh lymph node cDNA. The
correlation coefficient of this assay is 0.9974 indicating
excellent reproducibility. The PCR efficiency can be calculated as
follows: E=10.sup.(1/-S)-1,
[0091] where S equals the slope of a standard curve of a serial
dilution of template, for which Ct is plotted versus the log DNA
concentration. Therefore, the PCR efficiency for this assay is
96.7%.
[0092] Next, the RT-PCR efficiency of the assay was evaluated using
a 2 min reverse transcriptase reaction followed by 40 cycles of PCR
using the optimized conditions. A fresh lymph node RNA
2.times.dilution series was prepared in 400 ng spleen RNA. RT-PCR
for both CEA and GUS were performed. The mean GUS Ct was
28.39.+-.1.36. The efficiency for this assay was greater than 100
percent. The same assay was done using an equal mixture of
Superscript II and Sensiscript rather than only Sensiscript. The
efficiency of this assay was closer to 100 percent.
Example 3
Rapid QRT-PCR: Multiplexed Assay
[0093] The Smart Cycler is currently capable of 4-color
fluorescence detection and therefore allows multiplexing of QRT-PCR
reactions. One goal is to multiplex internal controls for RT-PCR,
an endogenous reference gene control to correct for RNA input, and
the target gene (for example CEA) all in one tube. Initial tests
multiplexing .beta.GUS and CEA worked well at moderate CEA mRNA
levels but failed when very low levels of CEA were present. Thus,
the sensitivity of this reaction was not adequate for
micrometastasis detection. One method to overcome this is to limit
the amount of PCR primer used for the endogenous control gene.
Theoretically this allows the rare CEA mRNA species to more
effectively compete for PCR reagents, especially in later cycles.
Attempts to do this with .beta.-Gus, or a second endogenous control
gene (18s ribosomal RNA), also failed to give adequate
sensitivity.
[0094] It was hypothesized that the problem lay in the initial
cycles, when competition between the two PCR reactions was most
critical. To overcome this, but still carry out the assay in a
single tube with no extra handling, the 18S rRNA (and .beta.GUS)
endogenous control primers were redesigned so that the annealing
temperature was 10.degree. C. below that of the CEA primers (all
primers used in the QRT-PCR reactions described herein are listed
below in Table 1). PCR was then carried out in two 20-cycle steps,
the first with an anneal/extend temperature of 64.degree. C. and
the second with an anneal-extend temperature of 53.degree. C. The
multiplex reaction reagent concentrations were the same as those
used in the singleplex reaction with the following modifications.
The target gene primer concentrations were at 400 nM while that of
the endogenous controls' were at 100 nM, the reverse transcriptase
primer concentrations were at 60 nM each and the cycling conditions
were modified as previously mentioned. Theoretically the 18S rRNA
primers would not function in the first 20 cycles and the CEA
amplification could proceed without competition. In the next 20
cycles, CEA PCR product would already be boosted to the point that
it could compete efficiently with the 18S rRNA PCR. The results of
this experiment are shown in FIG. 4. Panels A and C show the
results for 18S rRNA and CEA respectively when run in a singleplex
using optimal conditions. Panels B and D show 18S rRNA and CEA
multiplexed. Note that while these reactions were multiplexed in
the same tube, with different fluorescent dyes, the software does
not allow two dyes to be plotted on the same graph. In the
singleplex reaction, 18SrRNA crossed the 30-fluorescence unit
threshold at 10 cycles (Panel A). Using the new PCR primers, and
modified protocol, the 18S rRNA PCR reaction did not cross
threshold until 26 cycles (Panel B), 6 cycles after the
anneal/extend temperature was dropped to 53.degree. C. This
reaction was expected to cross threshold at 30 cycles (20+10), thus
it appears that there is some 18S rRNA PCR amplification occurring
during the first 20 cycles, even at 64.degree. C. In the CEA
reactions the singleplex reached threshold at 33.5 cycles (Panel C)
while the multiplexed CEA reached threshold at 34.5 cycles. Thus
only one cycle sensitivity was lost in this reaction.
TABLE-US-00001 TABLE 1 Name Sequence CEA probe CAA GCC CTC CAT CTC
CAG CAA CAA CT (SEQ ID NO: 8) CEA RT GTG AAG GCC ACA GCA T (SEQ ID
NO: 9) CEA-F77 AGA CAA TCA CAG TCT CTG CGG A (SEQ ID NO: 6) CEA-R77
ATC CTT GTC CTC CAC GGG TT (SEQ ID NO: 7) 18S probe TGC TGG CAC CAG
ACT TGC CCT C (SEQ ID NO: 10) 18S-F89 CCC TGT AAT TGG AAT GAG TCC
AC (SEQ ID NO: 11) 18S-R89 GCT GGA ATT ACC GCG GCT (SEQ ID NO: 12)
18S-F2- CCC TGT AAT TGG AAT GAG T low Temp (SEQ ID NO: 13) 18S-R2-
GCT GGA ATT ACC GCG low Temp (SEQ ID NO: 14) Gus probe TGA ACA GTC
ACC GAC GAG AGT GCT GG (SEQ ID NO: 5) GUS RT TGG TTG TCT CTG CCG A
(SEQ ID NO: 15) Gus 81F CTC ATT TGG AAT TTT GCC GAT T (SEQ ID NO:
3) Gus 81R CCG AGT GAA GAT CCC CTT TTT A (SEQ ID NO: 4) Gus 80F-
CTC ATT TGG AAT TTT GCC low Temp (SEQ ID NO: 16) Gus 80R- CG AGT
GAA GAT CCC CTT low Temp (SEQ ID NO: 17)
[0095] Of note, the above-described CEA primers were designed to
span the junction between exons 6 and 7 of the CEA mRNA. The
primers also amplify sequences spanning the junction between exons
2-3 of the CEA mRNA. This primer set was selected to yield superior
selectivity to certain earlier described primer sets. The addition
of one or more flanking nucleotides of the CEA sequence (FIG. 5,
GenBank Accession No. XM.sub.--012777) to the 5' or 3' ends of
either of the CEA primers would not appreciably affect the
above-described assays, except with respect to expected changes in
the Tm of the primer set. Other CEA-specific primers may be
selected from the same general regions (exon 2-3 and exon 6-7
junctions) that the above-described CEA forward and reverse primers
are selected to the same or similar effect as the above-described
CEA primers. Preferably, any selected primer sets will yield an
amplicon of less than about 100 bases, which adds to the ability to
conduct a rapid QRT-PCR assay.
Example 4
Prognostic Value of Quantitative Reverse Transcription-Polymerase
Chain Reaction in Node-Negative Esophageal Cancer Patients
Introduction
[0096] The incidence of adenocarcinoma of the esophagus is
increasing at an alarming rate, exceeding that of any other solid
tumor. Up to 50% of patients present with advanced disease,
yielding an overall 5-year survival of 10-13%. As with other tumor
types, survival of esophageal cancer patients is strongly predicted
by histological evidence of lymph node involvement. Although
current histological methods for lymph node staging provide
reliable information about populations of patients, they cannot
predict individual patient outcome within that population. For
example, 30-50% of histologically node-negative esophageal cancer
patients will suffer disease recurrence within 5 years, despite a
potentially curative resection. There is a circumstantial body of
evidence indicating that this primary treatment failure is
attributable to micrometastatic spread of the tumor that went
undetected by routine histological evaluation. Thus, there is a
clear need for more sensitive detection of lymph node
micrometastases, thereby allowing more individualized prognosis and
treatment planning of esophageal cancer patients.
[0097] The main problems with current lymph node evaluation are
sampling error and poor sensitivity for detecting individual tumor
cells or small tumor foci. Histological examination only samples a
very small percentage of each lymph node, and it has been
calculated that a pathologist has only a 1% chance of detecting a
micrometastatic focus of three tumor cell diameter.
Immunohistochemical staining for tumor markers improves the
sensitivity of micrometastasis detection and, when combined with
serial sectioning to reduce the sampling error, results in
upstaging of a significant number of patients. This technique has
been used in esophagus cancer, and 17% of histologically negative
nodes were found to contain micrometastatic disease. In this
report, IHC3-positive lymph nodes correlated with disease
recurrence, but these findings were questioned in a later study in
which IHC did not show any prognostic value. Other studies have
used molecular methods such as RT-PCR to detect micrometastases.
RT-PCR is capable of detecting the mRNA for tumor markers, such as
CEA, cytokeratin 19, cytokeratin 20, and others, in a variety of
tissues, including blood, bone marrow, and lymph nodes, that are
histologically cancer free. In esophageal cancer, RT-PCR has been
used on several small series of patients, but the clinical
significance of RT-PCR-positive nodes is not known because little
clinical follow-up has been reported. In other tumor types, studies
have shown that RT-PCR improves sensitivity, but poor specificity
and false-positive results in control lymph nodes from patients
without cancer have made the clinical application of this
information difficult. False positives are, at least in part,
attributable to the previously described phenomenon of ectopic gene
expression, which results in very low background levels of
expression of most genes in all tissue types. Thus, although
previous studies have used qualitative, gel-based RT-PCR methods,
it is now becoming apparent that this simple plus/minus method for
detection of tumor markers is not always a reliable sign of
micrometastases. With the introduction of the fluorescent 5'
nuclease assay, QRT-PCR is now a relatively simple technique. It
was therefore hypothesized that a quantitative analysis would offer
significant benefits over gel-based RT-PCR and would allow accurate
prediction of disease recurrence in histologically node-negative
esophageal cancer patients. The objectives of the present were
3-fold: (a) to determined the ability of QRT-PCR to accurately
distinguish between background gene expression of CEA in lymph
nodes and clinically relevant levels that are diagnostic of true
micrometastasis; (b) to use real-time QRT-PCR to analyze lymph
nodes from 30 node-negative esophageal cancer patients and
correlated the results with disease recurrence; and (c) to compare
QRT-PCR with standard gel-based RT-PCR on the same samples. It was
found that QRT-PCR can easily discriminate background expression
from true metastatic disease, QRT-PCR is both sensitive and
specific for predicting disease recurrence in nodenegative
esophageal cancer patients, and QRT-PCR has greater specificity
than gel-based RT-PCR. From these results, it is believed that
quantitation addresses all of the problems that have kept RT-PCR
from becoming a useful clinical test for micrometastatic
disease.
Materials and Methods
[0098] Patients: Tissue from 140 paraffin blocks containing 387
lymph nodes were studied from 30 patients who underwent curative
resection for histologically lymph node-negative esophageal cancer.
The Section of Thoracic Surgery performed all surgeries at the
University of Pittsburgh Medical Center between 1991 and 1998.
Vital status and recurrence information was obtained from a
combination of medical record review, personal contact with the
Primary Care Physician, and death certificates. Follow-up data were
confirmed for all patients as of August 2001. The median follow-up
time for surviving patients was 49 months (range, 28-91 months).
Demographics and clinical characteristics were collected (Table 2).
Tissue blocks from 10 primary tumors (8 adenocarcinoma and 2
squamous cell carcinoma) and 4 histologically positive lymph nodes
were obtained as positive controls. Negative control (benign) lymph
nodes were obtained from patients who underwent esophageal surgery
for causes unrelated to cancer (hernia repairs and antireflux
procedures) and whose lymph nodes were removed incidentally.
TABLE-US-00002 TABLE 2 Clinical Characteristics of the study
population QRT-PCR Result Patients Negative Positive Characteristic
(n = 30) (n = 19) (n = 11) Gender Male 22 15 7 Female 8 4 4 Months
of follow up Median 36.0 44.6 28.0 Range 5-90.6 5-90.6 6.3-57.4
Mean Age at Diagnosis 68.3 68.8 67.5 Adjuvant Therapy Chemotherapy
16 7 9 Radiation 9 3 6 Lymphadenopathy on scan 8 3 5 Tumor Type
Adenocarcinoma 26 18 8 Squamous cell 4 1 3 pT category* pT1 10 8 2
pT2 5 4 1 pT3 10 5 5 Stage* I 10 8 2 IIA 15 9 6 Mean Number of
Nodes 12.5 12 15 examined (range) (2-31) (2-31) (3-23) *Four
patients who received chemotherapy had no tumor at time of
surgery
[0099] Tissue and RNA Isolation: All tissues used in the study were
formalin-fixed, paraffin-embedded archival specimens obtained from
the Pathology tissue banks. H&E-stained slides were also
retrieved for each tissue block and were examined to confirm the
original node-negative diagnosis. Tissue blocks were mounted on a
microtome, and 5-15 5.0 cM sections were cut and placed in 2.0 ml
of RNase-free tubes. At the same time, 2 5.0 .mu.M sections were
cut (first and last cuts) and mounted on microscope slides for
immunohistochemical staining with antibodies against CEA. RNA was
isolated using methods described previously (Godfrey, T. E., Kim,
S.-H., Chavira, M., Ruff, D. W., Warren, R. S., Gray, J. W., and
Jensen, R. H. Quantitative mRNA expression analysis from
formalin-fixed, paraffin-embedded tissues using 5' nuclease
quantitative RT-PCR. J. Mol. Diagn., 2: 84-91, 2000) and stored in
RNA secure resuspension solution (Ambion, Austin, Tex.). The RNA
was DNase treated with the DNA free kit (Ambion) and quantitated
spectrophotometrically.
[0100] QRT-PCR: QRT-PCR was carried out using the 5' nuclease assay
and an Applied Biosystems 7700 Sequence Detection Instrument
(TaqMan). CEA expression was measured relative to the endogenous
control gene, .beta.-gus, using the comparative CT method described
previously (Godfrey, T. E., Kim, S.-H., Chavira, M., Ruff, D. W.,
Warren, R. S., Gray, J. W., and Jensen, R. H. Quantitative mRNA
expression analysis from formalin-fixed, paraffin-embedded tissues
using 5' nuclease quantitative RT-PCR. J. Mol. Diagn., 2: 84-91,
2000; Tassone, F., Hagerman, R. J., Taylor, A. K., Gane, L. W.,
Godfrey, T. E., and Hagerman, P. J. Elevated Levels of FMR1 mRNA in
carrier males: a new mechanism of involvement in the fragile-X
syndrome. Am. J. Hum. Genet., 66: 6-15, 2000). All QRT-PCR assays
were carried out at two RNA inputs, 400 and 100 ng, and duplicate
reactions were set up for each concentration. Thus, the reported
CEA expression levels are an average of four independent QRT-PCR
reactions. RT-negative controls were run for all samples using 400
ng of RNA but omitting the reverse transcriptase. Template-negative
controls were also run on each PCR plate. A calibrator RNA sample
was amplified in parallel on all plates to allow comparison of
samples run at different times (Godfrey, T. E., Kim, S.-H.,
Chavira, M., Ruff, D. W., Warren, R. S., Gray, J. W., and Jensen,
R. H. Quantitative mRNA expression analysis from formalin-fixed,
paraffin-embedded tissues using 5' nuclease quantitative RT-PCR. J.
Mol. Diagn., 2: 84-91, 2000; PE Applied Biosystems user bulletin
#2. Relative quantitation of gene expression, Perkin-Elmer, Corp.,
Norwalk, Conn., 1997.).
[0101] For maximum sensitivity and to eliminate the risk of cross
contamination, a single-tube QRT-PCR procedure described previously
was used (Raja, S., Luketich, J. D., Ruff, D. W., Kelly, L. A., and
Godfrey, T. E. A Method for increased sensitivity of one-step
quantitative RT-PCR. Biotechniques, 29: 702-705, 2000). In this
procedure, physical separation of the reverse transcription
reaction mixture (RT) and the PCR primers using a wax layer results
in a more specific and sensitive RT-PCR. The PCR primers were
pipetted into the PCR plate in a 10 .mu.l volume. One Ampliwax PCR
gem 50 (Applied Biosystems) was then placed in each well, and the
plate was heated to 80.degree. C. for 2 min and cooled to 4.degree.
C. to produce the wax barrier. A 40 .mu.l upper layer was then
pipetted into each well. The final concentrations of the reaction
components were as follows: 1.times.PCR buffer A, 300 nM each
deoxynucleotide triphosphate, 3.5 mM MgCl.sub.2, 0.4 unit/.mu.l
RNase Inhibitor, 1.25 units/.mu.l Superscript II reverse
transcriptase (Life Technologies, Inc., Gaithersburg, Md.), 0.06
unit/.mu.l Amplitaq Gold (Applied Biosystems), 20 nM reverse
transcriptase primer, 200 nM of each PCR primer, 200 nM probe
(.beta.-Gus primers and probe were at 100 nM), and 100 or 400 ng
total RNA. The oligonucleotide sequences used are shown in Table 3.
All RT-PCR reactions were carried out on the ABI 7700 with the
following thermocycler conditions: 48.degree. C. hold for 40 min,
95.degree. C. hold for 12 min followed by 45 cycles of 95.degree.
C. for 15 s, and 64.degree. C. (60.degree. C. for .beta.-gus) for 1
min. Data were analyzed using Sequence Detection Software (Applied
Biosystems) with thresholds set at 0.03 for CEA and 0.045 for
.beta.-gus. TABLE-US-00003 TABLE 3 Oligonucleotide sequences used
for CEA and-Gus RT-PCR Oligo- nucleotide B-Gus CEA Forward
CTCATTTGGAATTTTGCCGATT AGACAATCACAGTCTCTGCGG primer (SEQ ID NO: 3)
(SEQ ID NO: 6) Reverse CCGAGTGAAGATCCCCTTTTTA ATCCTTGTCCTCCACGGGTT
primer (SEQ ID NO: 4) (SEQ ID NO: 7) RT primer TGGTTGTCTCTGCCGA
GTGAAGGCCACAGCAT (SEQ ID NO: 15) (SEQ ID NO: 9) Probe*
TGAACAGTCACCGACGAGAGTGCTGG CAAGCCCTCCATCTCCAGCAACAACT (SEQ ID NO:
5) (SEQ ID NO: 8) *TaqMan probes were labeled with 5'
6-carboxyfluorescein and 3' 6-carboxytetramethylrhodamine.
[0102] Gel-based RT-PCR Analysis: To avoid the possibility of PCR
product contamination, all PCR plates from QRT-PCR runs were stored
unopened until the quantitative analyses were complete. PCR
products from the two 400 ng RNA input reactions and the 400 ng
No-RT control were then separated on a 4% agarose gel, stained with
ethidium bromide, and visualized on a gel documentation system.
Patients were classified as RT-PCR positive if a correctly sized
band was observed in both of the duplicate reactions but not in the
No-RT control.
[0103] Statistical Analysis: Comparisons of CEA levels in control
tissues samples (FIG. 6) were conducted with the Mann-Whitney U
Test. For pathologically negative lymph nodes from esophageal
cancer patients, the primary end point was disease recurrence
measured from the time of surgery to the time of diagnosed
recurrence or last date of follow-up. The highest CEA levels from
each patient's tissue blocks were used to construct a ROC curve
using recurrence as the gold standard. The CEA expression level
cutoff value was identified that produced the most accurate
classification, and that level was used to classify patients as
QRT-PCR positive or negative for risk of recurrence. Because a
second set of patients was not available for prospective validation
of the cutoff, the cutoff selection procedure was evaluated
statistically by cross validation, and the SDs of ROC curve
statistics were calculated by bootstrap resampling (Davison, A. C.,
and Hinkley, D. V. Bootstrap Methods and Their Applications.
Cambridge, United Kingdom: Cambridge University Press, 1997).
Kaplan-Meier disease-free and overall survival curves were plotted
for clinical and pathological factors including standard RT-PCR and
QRT-PCR results. The analysis of disease-free survival was
conducted by log-rank tests, and multivariate analysis was
performed by constructing Cox proportionate hazards models. The
list of predictors included categorical factors gender, tumor
pathology, classification by RT-PCR and QRT-PCR, preoperative
chemotherapy and/or radiotherapy, and pathological T stage, as well
as quantitative factors of CEA expression level, age, and number of
nodes removed for examination. All comparisons between models were
based on differences between likelihood ratio tests for nested
models. The adequacy of the proportional assumption was checked two
ways: by plotting the log cumulative hazard by log time and by
plotting Schoenfeld residuals and estimating the correlation
between the regression coefficient and time.
[0104] RESULTS--Characteristics of Node-negative Patients: TNM
classification of the 30 patients with node-negative esophageal
cancer included T.sub.1N.sub.0M.sub.0(n=10), T.sub.2N.sub.0M.sub.0
(n=5), and T.sub.3N.sub.0M.sub.0 (n=10). One patient with biopsy
diagnosed cancer had no evidence of cancer on the resection
specimen, and four patients who received neoadjuvant therapy had no
detectable tumor remaining at the time of resection. The location
of the primary tumor included 25 lower third, 5 mid, and 0 upper
esophageal sites. There were 26 adeno and 4 squamous cell cancers.
There were 22 males and 8 females, and the median age of the
patients at diagnosis was 70 years. Seventeen of the 30 patients
have died, 10 from their cancers and 7 from other causes. For
surviving patients, the median follow-up was 49 months (range,
28-91 months). The median overall survival was 36 months. A full
breakdown of patient characteristics is provided in Table 4,
below.
[0105] Quantitative Analysis of CEA Expression: Initially, RNA was
isolated and analyzed from three sources, distinct from the
node-negative study group, and included primary esophageal tumors,
lymph nodes that were histologically positive for metastases
(N.sub.1), and benign lymph nodes from patients without cancer. CEA
expression was detected in all tumors, and N.sub.1 nodes and in 50%
of benign lymph nodes (FIG. 6). Individual, pairwise comparisons
were carried out using a Mann-Whitney U test, and expression levels
in both tumor and N.sub.1 nodes were found to be significantly
higher than in benign lymph nodes (P=0.002 and 0.0021,
respectively). CEA expression was slightly higher in tumor samples
than in N.sub.1 lymph nodes, but this was not statistically
significant (P=0.171).
[0106] Analysis of histologically negative (N.sub.0) lymph nodes
from the study group demonstrated a wide range of CEA expression.
Expression ranged from undetectable to one node with CEA expression
equal to that seen in histologically positive lymph nodes. Data
from the most highly expressing node from each patient, in
conjunction with disease recurrence information, were analyzed
using a ROC curve analysis. From this analysis, an expression level
cutoff that most accurately predicted recurrence was determined
(FIG. 7). The area under the ROC curve was 0.88 with a 95%
confidence of 0.71 to 0.97, indicating that classification accuracy
is significantly better than chance alone. Of the 140 tissue blocks
analyzed, 12 (9%) had CEA expression levels above the cutoff point,
and a total of 11 patients (34%) had one or more blocks with
expression above the cutoff. These 11 patients were classified as
having QRT-PCR evidence of occult micrometastases. Sections from
all but 1 (patient 28) of the "most positive" tissue blocks were
negative for immunohistochemical staining with antibodies against
CEA.
[0107] Gel-based Analysis of CEA Expression: A total of 25 (18%)
tissue blocks were positive for CEA expression using the gel-based
assay, and 15 (50%) patients had at least one positive block in
this analysis. In 2 of the 10 lymph nodes from patients with benign
disorders, a CEA PCR product was present on the gel-based assays. A
comparison of the quantitative and gelbased assays showed that all
samples positive on gels also gave signals in the TaqMan assay.
[0108] Occult Metastases and Recurrence: Of the 30 patients
studied, 10 suffered disease recurrence and died by the end of the
study. Of the remaining 20 patients, 7 died from other causes and
13 remain alive with no evidence of recurrent disease. One patient
suffered an early anastomatic recurrence that was treated with
photodynamic therapy and radiotherapy. This patient was later
diagnosed with, and died of, small cell lung cancer. Using the most
accurate cutoff for the quantitative assay, a total of 11 patients
were classified as QRT-PCR positive, and 9 of these suffered
recurrence. The same 9 patients were identified as RT-PCR positive
using the gel-based assay, along with 6 other patients who did not
recur. Sensitivity and specificity for predicting disease
recurrence using the QRT-PCR assay was 90 and 90%, with a positive
predictive value of 82%. Using the gel-based assay, the sensitivity
and specificity were 90 and 70% with a positive predictive value of
60%. FIG. 8 shows disease-free survival for the patient cohort
classified by RT-PCR (A) and QRT-PCR(C) as well as overall survival
(B and D). Disease-free survival of patients who were RT-PCR
negative was 94% when using either assay. Patients with a positive
classification had poorer prognosis by either method. The log-rank
test indicates that both QRT-PCR(P=0001) and gel-based
RT-PCR(P=0038) are significant predictors of recurrence in
node-negative esophageal cancer patients. Overall survival was also
worse in QRT-PCR- or RT-PCR-positive patients (P=0.0006 and 0.106,
respectively).
[0109] Besides QRT-PCR and RT-PCR, the only other clinical,
pathological, or treatment factors found to predict disease
recurrence in this cohort were pathological T stage (log-rank test,
P=0.0777) and preoperative chemotherapy and/or radiotherapy (log
rank test, P=0.0307). In a multivariate analysis, the likelihood
ratio statistics from Cox regression models showed that the
classification of CEA as a binary variable, QRT-PCR positive or
negative, was a significant and independent predictor of recurrence
when compared with pathological T stage and preoperative
chemotherapy or radiotherapy.
Discussion
[0110] Lymph node involvement is the strongest prognostic factor in
many solid tumors, and detection of lymph node micrometastases has
received much interest in the recent literature. Current lymph node
evaluation involves microscopic examination of H&E-stained
tissue sections and suffers from two major limitations: (a) single
tumor cells, or small foci of cells, are easily missed; and (b)
only one or two tissue sections are studied, and thus the vast
majority of each node is left unexamined. Serial sectioning can
overcome the issue of sampling error, and IHC can identify
individual tumor cells. The combination of these methods, however,
is too costly and time consuming for routine analysis and is
limited to special cases such as sentinel lymph node examination.
RT-PCR overcomes the problem of sampling error because larger
amounts of tissue can be analyzed, and several reports indicate
that RT-PCR identifies more positive lymph nodes than IHC. This was
also the case in the present study, where only one of the
QRT-PCR-positive lymph node blocks was positive by IHC
analysis.
[0111] Recent studies also show that non-QRT-PCR positivity
correlates with disease recurrence, but the specificity reported in
these studies is low. In one study by Liefers et al. (Liefers, G.
J., Cleton-Jansen, A. M., van de Velde, C. J., Hermans, J., van
Krieken, J. H., Cornelisse, C. J., and Tollenaar, R. A.
Micrometastases and survival in stage II colorectal cancer [see
comments]. N. Engl. J. Med., 339: 223-228, 1998.), 14 of 26
histologically N.sub.0 colon cancer patients had evidence of
micrometastatic disease using RT-PCR, and the remaining 12 were
RT-PCR negative. Of the 12 RT-PCR N.sub.0 patients, only 1 recurred
during the 6-year follow-up period, whereas of the 14 RT-PCR
N.sub.1 patients, 7 suffered recurrence. Thus, using recurrence as
an end point, this study achieved a sensitivity of 88%. The
specificity, however, was only 61%, because 7 patients with
RT-PCR-positive nodes did not recur. Other studies have shown a
similar low specificity for non-QRT-PCR in melanoma patients.
Shivers et al. (Shivers, S. C., Wang, X., Li, W., Joseph, E.,
Messina, J., Glass, L. F., DeConti, R., Cruse, C. W., Berman, C.,
Fenske, N. A., Lyman, G. H., and Reintgen, D. S. Molecular staging
of malignant melanoma: correlation with clinical outcome. JAMA,
280: 1410-1415, 1998) achieved 86% sensitivity and 51% specificity,
and Bostick et al. (Bostick, P. J., Morton, D. L., Turner, R. R.,
Huynh, K. T., Wang, H. J., Elashoff, R., Essner, R., and Hoon, D.
S. Prognostic significance of occult metastases detected by
sentinel lymphadenectomy and reverse transcriptase-polymerase chain
reaction in early-stage melanoma patients. J. Clin. Oncol., 17:
3238-3244, 1999) reported 100% sensitivity and 67% specificity.
This low specificity, along with an inability to accurately control
for inter-run variability, has limited the potential of RT-PCR in a
clinical setting.
[0112] In the current study, the use of TaqMan RT-PCR to
quantitatively assay CEA expression and detect occult
micrometastases in lymph nodes of histologically N.sub.0 esophageal
cancer patients was evaluated. This quantitative analysis resulted
in the definition of a clinically relevant CEA expression level
cutoff and the overcoming of the problems associated with
background, or ectopic, CEA expression reported by others. Using
this approach, 11 of 30 patients were identified as being QRT-PCR
positive, and 9 of these patients suffered disease recurrence. Only
1 of the 19 patients who were QRT-PCR negative suffered recurrence
during the course of this study. Interestingly, of the 20 patients
who did not recur, 11 had detectable CEA expression higher than
that seen in control lymph nodes (higher than background levels),
and two were above the cutoff level for predicting disease
recurrence. In some cases, this could indicate the presence of
limited nodal disease that was cured by surgery, because even
pN.sub.1 patients can have an expected 5-year survival
of.about.20%. CEA expression in the remaining samples could
possibly be a result of either individual disseminated tumor cells
that are unable to survive and are possibly undergoing apoptosis,
cell-free RNA in the lymph system as a result of tumor cell
apoptosis at the primary site, or contaminating cells introduced
inadvertently by the surgeon.
[0113] Both disease-free survival and overall survival were
significantly higher in the QRT-PCR-negative patients compared with
QRT-PCR-positive patients. Furthermore, disease-free survival in
the QRT-PCR-positive group was only 27% at 3 years, indicating that
micrometastatic disease may be as clinically significant as
histological N.sub.1 disease. With the exception of one patient,
only one positive tissue block was identified per patient,
indicating limited disease spread. All positive lymph nodes were
locoregional and would therefore confer N.sub.1 status, rather than
M.sub.1a, as defined by celiac or cervical lymph node involvement.
The limited nodal involvement in these histologically N.sub.0
patients emphasizes the need for adequate lymph node sampling, from
different nodal stations, during staging procedures.
[0114] Although the subset of patients with node-negative disease
has been relatively small in the past, the dramatic increase in
esophageal cancer has led to surveillance programs that are
identifying patients with early stage disease more frequently.
Methods for accurate staging of these patients will thus become
even more important. Comparison herein of the quantitative data
with gel-based analysis of the same samples showed that QRT-PCR
improves the test specificity while maintaining the same high
sensitivity. QRT-PCR also gives objective results, which are
amenable to automated, hands-free analysis, with minimal risk of
PCR product cross-contamination. Such automation will be essential
if QRT-PCR is to become a routine clinical assay.
[0115] In addition, the quantitative procedure allows for use of
rigorous controls to confirm the accuracy and reliability of the
assay from run to run. For example, in the described experiments, a
calibrator sample was run on all PCR plates to correct for day to
day variability. Using this methodology, reproducibility tests
indicate that the 95% confidence limits on measurements are 0.511
cycles (data not shown). The ability to accurately assess the
reproducibility of the quantitative assay will be essential if
RT-PCR is to be used in a clinical setting. In a gel-based assay,
this level of assay verification is not attainable. It is
acknowledged that the predictive ability of classifying patients by
their CEA levels determined by QRT-PCR will be optimistic because
the cutoff value was evaluated in the same patients in whom it was
determined. It is likely that if the same procedure for the most
accurate cutoff were applied to a second set of patients, the
classification would be less successful. To this end, a reanalysis
of the sensitivity, specificity, and classification accuracy was
conducted by cross-validation (n=10000). The cross-validation
estimates of specificity were 0.82 and accuracy of 0.82 (compared
with 0.90 and 0.90, respectively, in the original sample). Thus,
the classification success of the original sample was slightly
overstated, but even after correction for this optimism,
classification by QRT-PCR remains an improvement over the gel-based
assay.
[0116] In conclusion, these data demonstrate that QRT-PCR can
detect, with high specificity, micrometastatic disease in
histologically negative lymph nodes of esophagus cancer patients.
It also has been shown that the presence of micrometastatic disease
is a strong, independent predictor of cancer recurrence and that
quantitation is superior to standard RT-PCR assays. Quantitative
RT-PCR should be able to identify which patients with early stage
esophageal cancer are at high risk for recurrence and who might
benefit from additional therapy. Finally, this quantitative
approach should result in similar benefits in other tumor types.
TABLE-US-00004 TABLE 4 Survival and RT-PCR data of individual
patients Outcome Survival CEA status Patient T Adjuvant therapy
Vital Disease Disease QRT- RT- ID stage Chemotherapy Radiation
Status Status.sup.a Overall Free PCR PCR 1 2 None None Alive NED
43.9 43.9 - - 2 1 None None Alive NED 28.4 28.4 - - 3.sup.b 1 Post
Post Alive NED 30.8 30.8 - - 4 1 Pre None Dead Other 17.8 17.8 - -
5 1 None None Alive NED 90.6 90.6 - - 6 3 Pre None Alive NED 60.6
60.6 - + 7 1 None None Alive NED 50.5 50.5 - + 8 2 None None Alive
NED 75.6 75.6 - - 9 1 None None Dead Other 38.1 38.1 - - 10 3 Pre
None Alive NED 69.1 69.1 - + 11 2 None None Alive NED 44.6 44.6 - -
12 3 Pre Pre Dead Other 31.7 31.7 - - 13 1 None None Dead Other
63.1 63.1 - - 14.sup.b 0.sup.c Pre Pre Alive NED 35.0 35.0 + +
15.sup.b 1 None None Dead Other 31.8 31.8 + + 16 0.sup.d None None
Alive NED 49.4 49.4 - - 17 2 None None Alive NED 36.9 36.9 - - 18 1
None None Alive NED 65.2 65.2 - - 19 0.sup.c Pre None Dead Other
56.3 15.8 - + 20 3 Pre Pre Dead Other 32.2 32.2 - - 21 3 None None
Dead NED 5.0 5.0 - - 22 3 Pre None Dead Recurred 24.8 24.3 + + 23 1
Pre None Dead Recurred 57.4 53.0 + + 24 3 Pre None Dead Recurred
28.0 7.9 + + 25.sup.b 3 None None Dead Recurred* 6.3 5.0 + + 26 3
Pre Pre Dead Recurred 11.1 10.6 + + 27 2 Pre Pre Dead Recurred 14.6
10.4 + + 28 0.sup.c Pre Pre Dead Recurred 46.4 8.3 + + 29 3 Pre Pre
Dead Recurred 26.3 18.7 + + 30 0.sup.c Pre Pre Dead Recurred 31.9
27.0 + + .sup.aNED, no evidence of disease. .sup.bPatients with
squamous cell carcinoma. .sup.cPatients who had no residual tumor
after chemotherapy. .sup.dPatient with cancer on biopsy, but not at
resection.
Example 5
Intra-Operative Quantitative RT-PCR Detects Nodal Metastasis in
Patients with Esophageal Cancer
Introduction
[0117] Surgical decisions in esophageal cancer and other
malignancies are often based on intra-operative frozen section
analysis of lymph nodes. The 5-year survival of patients with
esophageal cancer remains poor at 5-10% due to the presence of
advanced disease in many patients at the time of initial
presentation. If local and regional lymph nodes are histologically
negative however, there is a dramatic improvement in the 5-year
survival. Nevertheless, 30-50% of histologically node negative
patients suffer disease recurrence. This is most likely due to the
limitations of current staging techniques in the detection of
micrometastases. As a result, other techniques such as
immunohistochemistry or reverse transcriptase-polymerase chain
reaction (RT-PCR) have been used in attempts to detect
histologically occult micrometastases.
[0118] Studies on esophagus, colon, melanoma and breast cancers
have shown that RT-PCR can detect histologically occult
micrometastases, and may predict recurrence. These data have been
criticized however, due to the occurrence of false positive results
in control samples and the subsequent low specificity and positive
predictive value of the RT-PCR assay. It has been shown that
Quantitative RT-PCR (QRT-PCR) allows the distinction of background,
ectopic, gene expression from true micrometases and can therefore
avoid false positives and increase specificity for predicting
disease recurrence. The next goal is to be able to provide the
surgeon with this critical information at a time when important
surgical decisions are made, intra-operatively.
[0119] Described in this Example is the development and testing of
an extremely rapid QRT-PCR assay that can be carried out in less
than 30 minutes. To validate the rapid assay, it is compared to
standard QRT-PCR and to clinical outcomes in patients with
esophageal cancer.
Materials and Methods
Patients
[0120] For the retrospective analysis, lymph nodes were studied
from 30 patients (Table 6) who underwent curative resection for
histologically lymph node-negative esophageal cancer. All surgeries
were performed in the Division of Thoracic Surgery at the
University of Pittsburgh Medical Center between 1991 and 1998.
Clinical follow up was obtained from the medical records and was
confirmed for all patients as of August 2001. The median clinical
follow up time for all patients was 36 months and for the surviving
patients was 49 months (range 28-91 months). TABLE-US-00005 TABLE 6
Clinical Characteristics of the study population. QRT-PCR Result
Patients Negative Positive Characteristic (n = 30) (n = 17) (n =
13) Gender Male 22 14 8 Female 8 3 5 Months follow up Median 36
43.9 31.8 Range 5-90.5 5-90.5 6.3-75.6 Mean Age at Diagnosis 68.3
68.4 68.2 Tumor Type Adenocarcinoma 26 16 10 Squamous cell 4 1 3
Stage* 0\ 1 1 0 I 10 8 2 IIA 15 8 7 Median Number of Nodes 12.5 12
15 Examined *4 patients who received chemotherapy had no tumor at
time of surgery. \1 patient had cancer on biopsy but not at
resection.
[0121] Prospective analysis, lymph node sample were obtained from
12 patients undergoing either staging or resection for esophageal
cancer identified by the Section of Thoracic Surgery at the
University of Pittsburgh Medical Center from 1999 to 2000 (Table
7). Control nodes from patients without cancer were obtained
incidentally from patients undergoing abdominal surgery for
anti-reflux procedures. At the time of excision, one half of each
lymph node was frozen in liquid nitrogen for analysis and the
remainder was sent for routine pathological analysis. The exercise
tissue was part of the routine clinical course at our institutional
and, as such, no additional tissue was removed for any purely
research purpose. All tissues were collected as part of an ongoing,
TRB approved, Esophagus Cancer Risk Registry protocol at the
University of Pittsburgh. TABLE-US-00006 TABLE 7 Tumor stage and
histology for patients in the rapid QRT-PCR study on fresh frozen
lymph nodes. Patient stage Tumor Type 12 IIA adeno 13 IIA adeno 14
I adeno 15 IIA adeno 16 IIA adeno 17 IIA squamous 18 III adeno 19
III adeno 20 III adeno 21 III adeno 22 III adeno 23 III adeno
Tissue and RNA Isolation
[0122] All tissues used in the retrospective study were
foramalin-fixed, paraffin-embedded archival specimens obtained from
the Pathology tissue banks. Hematoxylin and eosin stained slides
were also retrieved for each tissue block and were examined to
confirm the original node-negative diagnosis. Tissue blocks were
mounted on a microtome and 5-15, 5.0 .mu.M sections were cut and
placed in 2.0 ml RNase free tubes. At the same time, 2, 5.0 .mu.M
sections were cut (first and last sections) and mounted on
microscope slides for H&E staining as well as
immunohistochemical staining with antibodies against CEA. RNA was
isolated using previously described methods (Godfrey T E, Kim S-H,
Chavira M, Ruff D W, Warren R S, Gray J W et al. Quantitative mRNA
expression analysis from formalin-fixed, paraffin-embedded tissues
using 5' nuclease quantitative RT-PCR. J. of Molecular Diagnostics
2000; 2(2):84-91) and quantitated spectrophotometrically.
[0123] The fresh frozen lymph node tissues were embedded in OCT
compound and 10-15, 4.0 .mu.M sections were cut. At the same time,
2, 4.0 .mu.M sections were cut (first and last sections) and
mounted on microscope slides for H&E staining. The remaining
sections were placed in 1.5 ml RNase free tubes with the lysis
buffer from the RNeasy Mini kit (Qiagen, Valencia, Calif.). RNA was
extracted using the manufacturers recommended protocol with the
following modifications. Centrifugation times over 1 minute were
reduced to 1 minute, and the RNA was reconstituted in 60 .mu.l RNA
secure resuspension solution (Ambion, Austin, Tex.). Although most
of the samples were processed together, five samples were processed
individually to determine the median extraction time per sample.
The RNA yield and purity was determined spectrophotometrically for
quality control purposes.
[0124] Quantitative Reverse Transcription-PCR--QRT-PCR was carried
out using the 5' nuclease assay. Rapid QRT-PCR was carried out on
the Cepheid Smart Cycler.RTM. (CSC) real-time DNA amplification and
detection system as described below. Standard QRT-PCR was carried
out on the Applied Biosystems 7700 Sequence Detection Instrument
(TaqMan.RTM.) using a one-tube QRT-PCR procedure described
herein.
[0125] Rapid QRT-PCR--CEA expression was measured relative to the
endogenous control gene, .beta.-glucuronidase (.beta.-GUS) using
the comparative C.sub.T method described previously (Godfrey T E,
Kim S-H, Chavira M, Ruff D W, Warren R S, Gray J W et al.
Quantitative mRNA expression analysis from formalin-fixed,
paraffin-embedded tissues using 5' nuclease quantitative RT-PCR. J.
Molecular Diagnostics 2000; 2(2):84-91; Tassone F, Hagerman R J,
Taylor A K, Gane L W, Godfrey T E, Hagerman P J. Elevated levels of
FMR1 mRNA in carrier males: a new mechanism of involvement in the
fragile X syndrome. Am. J. Hum. Genet. 2000; 66(1):6-15.). All
QRT-PCR assays were carried out on 400 ng of total RNA in
triplicate. Both CEA and .beta.-GUS PCR primers were designed and
tested not to amplify genomic DNA. However, control reactions were
still run using RNA without reverse transcription (NO-RT control)
and water (no-template control) in place of cDNA as PCR template.
Together these control reactions rule out the possibility that
signal is generated from either genomic DNA contamination of the
RNA, or PCR product contamination of the reagents. In addition, a
calibrator RNA sample was amplified in parallel on all runs to
allow comparison of samples run at different times (Godfrey et al.
(2000); PE Applied Biosystems User Bulletin #2. ABI Prism 7700
Sequence Detection System: Relative Quantitaion of Gene expression.
1997. Norwalk, Conn., Perkin Elmer Corp.), and to determine
reproducibility of the assay.
[0126] The final concentrations of the reaction components were as
follows: 1.times.PCR Platinum Taq buffer, 300 nM each dNTP, 4.5 mM
MgCl.sub.2, 0.8 U/.mu.l RNase Inhibitor, 1.25 .mu.l Sensiscript
reverse transcriptase (Qiagen, Valencia, Calif.), 0.06 U/.mu.l
Platinum Taq (Gibco, Gaithersburg, Md.), 60 nM RT primer, 400 nM
each PCR primer, 200 nM probe and 400 ng of total RNA. The total
RNA input for the fresh tissue analysis was 5 .mu.l of the sample.
For the fixed tissue analysis, 5 .mu.l of an 80 ng/.mu.l dilution
of the RNA stock was used. In the rapid assay, the RT reaction was
carried out without the PCR primers or probe, then the tube was
opened and the primers and probe were added. This was necessary
because a standard one-step QRT-PCR lacked the sensitivity to
detect rare messages. The oligonucleotide sequences used were as
follows: .beta.-GUS RT primer 5' TGG TTG TCT CTG CCG A 3' (SEQ ID
NO: 15), .beta.-GUS forward PCR primer 5' CTC ATT TGG AAT TTT GCC
GAT T 3' (SEQ ID NO: 3), .beta.-GUS reverse PCR primer 5' CCG AGT
GAA GAT CCC CTT TTT A 3' (SEQ ID NO: 4), .beta.-GUS probe 5'-Vic
TGA ACA GTC ACC GAC GAG AGT GCT GG 3' (SEQ ID NO: 5), CEA RT primer
5' GTG AAG GCC ACA GCA T 3' (SEQ ID NO: 9), CEA forward PCR primer
5' AGA CAA TCA CAG TCT CTG CGG A 3' (SEQ ID NO: 6), CEA reverse PCR
primer 5' ATC CTT GTC CTC CAC GGG TT 3' (SEQ ID NO: 7) and CEA
probe 5'-Fam CAA GCC CTC CAT CTC CAG CAA CAA CT 3' (SEQ ID NO: 8).
Rapid QRT-PCR reactions were carried out on the CSC with the
following thermocycler conditions; 48.degree. C. hold for 5
minutes, 70.degree. C. for 60 seconds (for the addition of the PCR
primers and probe), 95.degree. C. hold for 30 seconds (for Platinum
Taq activation) followed by 45 cycles of 95.degree. C. for 2
seconds and 64.degree. C. for 15 seconds. Data was analyzed with
SmartCycler Software (Ver 1.0) using the second derivative method
for determining the threshold. For the fixed tissue analysis, a 30
minute RT reaction was required because of the reduced sensitivity
associated with the degradation inherent in fixed tissue RNA
samples.
[0127] Statistical Analysis--The predictive validity of rapid
QRT-PCR determination of CEA expression level was evaluated by
proportionate hazards regression for disease-free survival.
Disease-free survival was defined as the time from surgery to the
time of diagnosed recurrence of esophageal cancer. Deaths due to
other causes and patients alive without disease as of Aug. 1, 2001
were censored. CEA expression level was also used to classify
patients as either QRT-PCR positive or negative. The cutoff level
for classification, determined by ROC curve analysis (DeLong E R,
DeLong D M, Clarke-Pearson D L. Comparing the areas under two or
more correlated receiver operating characteristic curves: a
nonparametric approach. Biometrics 1988; 44(3):837-845; Heagerty P
J, Lumley T, Pepe M S. Time-dependent ROC curves for censored
survival data and a diagnostic marker. Biometrics 2000;
56(2):337-344), was defined as the CEA expression level value that
produced the most accurate classification using disease recurrence
as the standard. Sensitivity and specificity of QRT-PCR results
were calculated for diagnosing occult metastasis. Patients
classified as QRT-PCR positive or negative based on the
classification method were tested for differences in disease-free
survival with the log rank test.
Results
Archived Tissue Analysis
[0128] Initially, archived, histologically negative (N.sub.0) lymph
nodes from 30 patients with esophageal cancer were analyzed (FIG.
9). Of the 30 tissue blocks analyzed, 13 had CEA expression higher
than the ROC curve determined cutoff (CEA expression greater than
0.183). In this group, nine patients suffered disease recurrence
and died by the end of the study, 2 patients have died from other
causes and 2 are alive without disease. Of the remaining 17 QRT-PCR
negative patients, 1 suffered disease recurrence at 5 months and
died by the end of this study, 5 have died from other causes and 11
remain alive and disease free (Table 8). Using the rapid QRT-PCR
assay, the sensitivity and specificity for predicting disease
recurrence were 90% and 80% respectively and 83% of patients in the
cohort were classified correctly. FIG. 10 shows the Kaplan-Meier
disease-free survival curves for QRT-PCR positive and negative
patients. Survival of patients who were QRT-PCR negative and
positive was 94% and 20% respectively at 5 years. The survival
functions for these two groups were significantly different
(p=0.001, log rank test). TABLE-US-00007 TABLE 8 Rapid QRT-PCR
results on 30 pN.sub.0 esophagus cancer patients using a CEA
expression cut-off of 0.183. Sensitivity was 90% (9/10) and
specificity was 80%. Recurrence (true +ve) No recurrence (true -ve)
QRT-PCR positive 9 4 QRT-PCR negative 1 16
[0129] Using the proportionate hazards model for disease free
survival, the relative risk (RR) of an increase of one unit of CEA
expression was 1.52 (1.11-2.09, p-0.027) and that of having CEA
expression greater than 0.183 was 3.78 (1.34-10.7, p=0.0007)
compared to CEA expression less than or equal to 0.183.
Fresh Tissue Analysis
[0130] Fresh frozen sections of lymph nodes from patients
undergoing minimally invasive staging for esophageal cancer, or
benign esophageal disorders, were analyzed. In the 11 lymph nodes
from the benign cases, rapid QRT-PCR detected very low levels of
CEA expression in only 2 lymph nodes (patients 8 and 11, FIG. 11).
This low level in the benign cases presumably represented
background, ectopic CEA expression. The highest level of CEA
expression in any benign node was 34-fold lower than the lowest
expressing positive node (FIG. 11), which had only a small focus of
tumor involvement.
[0131] Of the 26 lymph nodes from 12 patients with esophageal
cancer, 12 (6 patients) were ultimately shown to be positive on
final pathology. In 2 (2 patients) of these lymph nodes however,
intra-operative frozen section analysis was negative and N.sub.1
status was determined post-operatively. Rapid QRT-PCR showed CEA
expression levels in these 2 nodes to be in the range of the
histologically positive nodes (patient 18 and 19, FIG. 11). In the
6 patients who had histologically node negative esophageal cancer,
only one had a single node with increased CEA expression by rapid
QRT-PCR (patient 15, FIG. 11). This patient had a clinical
recurrence of his esophageal cancer at a follow-up of 16.5
months.
[0132] Analysis of the calibrator samples on each run revealed
that, over 13 separate runs, the standard deviation of the delta Ct
value was 0.14 cycles (.about.10% standard deviation of relative
expression measurements). Additionally, the QRT-PCR required
approximately twenty-five minutes for all 40-cycle runs. With an
RNA isolation time of 7 minutes, the entire assay takes around 30
minutes. Therefore, this is a very rapid and reproducible
assay.
Discussion
[0133] In many malignancies, as in esophagus cancer, TNM staging
yields the best prognostic information. The absence of nodal
involvement is associated with a more localized lesion suggesting
that a cure may be obtained by surgical resection alone. Nodal
metastases however, are indicators of tumor spread beyond the site
of the primary tumor and, as such, necessitate systemic treatment.
Although the role of chemotherapy in esophageal cancer remains
controversial, some trials have shown trends indicating the benefit
of pre-operative chemotherapy. One advantage of this approach is
that more patients are able to complete the chemotherapy protocol,
than would otherwise do so if they were to receive treatment
following a very morbid surgical procedure. Ongoing prospective,
randomized clinical trials should resolve the utility of
pre-operative chemotherapy in esophagus cancer patients in the near
future. With the advent of minimally invasive surgical approaches
to staging, there exists the potential to stage prior to resection
and offer pre-operative chemotherapy when appropriate. Therefore,
the accurate determination of nodal stage prior to attempted
curative resection could become very important in the
decision-making process for patients with esophageal cancer.
[0134] Currently, intra-operative nodal staging is performed by
frozen sectioning and H&E staining. While this methodology has
at least a 93% correlation with the gold standard of formalin
fixed, H&E stained tissue examination, both methods suffer
significant limitations. Specifically, 30-50% of histologically
node negative esophagus cancer patients suffer disease recurrence
despite potentially curative resection. This finding is most likely
due to sampling error that is inherent in the conventional
methodologies, as well as a lack of sensitivity for detecting
micrometastases. Single tumor cells, or small foci of cells, are
easily missed since only one or two 4 .mu.M sections are studied
and the vast majority of each node is left unexamined. Serial
sectioning of lymph nodes can reduce the sampling error and thus
minimize this limitation. For example, in studies on breast cancer,
serial sectioning of lymph nodes, combined with
immunohistochemistry, led to an upstaging in an additional 10-23%
of nodes that were negative by routine histopathologic evaluation.
This method is not routinely used however due to the significant
time and labor involved in serial sectioning of numerous lymph
nodes from each case.
[0135] In the last decade, many studies have used RT-PCR to detect
tumor related mRNAs in lymph nodes in attempts to improve the
sensitivity of micrometastasis detection. Studies on several cancer
types have reported that RT-PCR is a reasonable prognostic
indicator, but despite this, RT-PCR has not made its way into
clinical practice. The main reasons for this are poor specificity
(40-60%), false positive results in control nodes from patients
without cancer and the lack of standardized assays for multi-center
trials. In other work, it has been shown that a quantitative
approach to RT-PCR (QRT-PCR) can overcome false positives due to
background or ectopic gene expression, and can increase the
specificity for predicting disease recurrence Godfrey T E, Raja S,
Finkelstein S D, Kelly L A, Gooding W, Luketich J D. Quantitative
RT-PCR Predicts Disease Recurrence in lymph Node-Negative Esophagus
Cancer Patients. Proceedings from the 2001 Annual Meeting of the
American Association of Cancer Researchers 42. Mar. 1, 2001. Due to
the techniques used in that study, however, the RNA isolation and
RT-PCR assay requires 4-6 hours to complete. As a result, that
method can only be utilized post-operatively. Intraoperative
QRT-PCR can be a reality only if it is performed rapidly enough to
yield a result in a time frame comparable to intra-operative frozen
section analysis (approximately 20-30 minutes). Along with rapid
RNA isolation and reverse transcription protocols, this requires
extremely fast PCR ramping times such as those attainable with the
Roche Light Cycler.RTM. or the Cepheid Smart Cycler.RTM.. The
Cepheid instrument was used herein due to the relative ease of use
(no glass capillaries), independent control of each reaction site,
availability of four color multiplexing and the potential for
automated sample preparation and QRT-PCR. With this instrument it
has been shown that a QRT-PCR assay can be performed on OCT
embedded tissue sections within 30 minutes from RNA extraction to
result. With modifications to the protocol reported here, QRT-PCR
can be carried out in 18.5 minutes (data not shown). Using our
rapid assay, a retrospective analysis of RNA from archival tissues
with a median patient follow-up of 36 months was performed. Data
showed that the rapid assay predicted disease recurrence with a
sensitivity and specificity of 90% and 80% respectively. This data
was comparable with the slower, and more traditional, TaqMan based
analysis (90% and 90%). In the fresh tissue analysis, it was
possible to distinguish all the histologically positive nodes from
the benign control nodes in under 30 minutes. Using the expression
level of CEA, it was also possible to characterize pathologically
negative nodes as having an expression pattern that resembled
either benign or pathologically positive nodes.
[0136] From this data, it appears that rapid QRT-PCR is at least as
sensitive as fixed tissue examination and is more sensitive than
intraoperative frozen section examination. This is demonstrated by
the 2 positive nodes that were deferred or misdiagnosed during the
intraoperative examination. To confirm this data, study of a much
larger number of fresh lymph nodes is planned with the object to
correlate the results with pathologic staging. As the data set
matures, QRT-PCR will be correlated with with recurrence. This will
permit determination of an optimal CEA expression cut-off value in
the fresh tissue data set since this may be different from the
cut-off for archived tissues. Due to the preliminary fresh tissue
data, and problems inherent in analysis of archived tissues, it is
possible that specificity and positive predictive values will
improve with analysis of fresh tissues.
[0137] Further development of this technology will be necessary to
bring intraoperative QRT-PCR from bench to bedside. In a clinical
setting, the QRT-PCR assay has to be a simple and automated process
with minimal handling. Towards this end, the goal is to develop a
cartridge based processor for automated analysis. It is envisioned
that the end product will be a single-use, disposable cartridge
capable of both RNA isolation and quantitative RT-PCR set up. When
integrated with a rapid cycling, real-time quantitative thermal
cycler, this system will provide a QRT-PCR result from frozen
sections in less than 25 minutes. Upon its development, and the
identification of new and accurate markers, this technology could
also be used for molecular analysis of surgical margins as well as
an adjunct to fine needle aspirate cytology. Thus, molecular
information regarding micrometastases and completeness of resection
could, for the first time, be made available intraoperatively to
the surgeon. Furthermore, the availability of an automated and
reproducible QRT-PCR format will allow the true molecular
diagnostic value of QRT-PCR to be evaluated in standardized
multi-center trials.
Example 6
Description of a Novel Method for Multiplexing Polymerase Chain
Reaction.
Introduction
[0138] Despite the enormous power and flexibility of the Polymerase
Chain Reaction (PCR), this technology has been slow to find its way
into clinical diagnostic laboratories. In large part, this is due
to the fact that the PCR process (from sample processing to
reaction set-up and data analysis) is labor intensive, prone to
contamination and technically quite demanding. As a result, false
positive and false negative results are frequent and constitute a
major concern in the clinical setting. Even so, most major clinical
diagnostic laboratories are still running a handful of internally
validated, PCR-based assays for a variety of applications such as
viral detection and minimal residual disease detection in leukemia
patients. Technological advances that simplify and, if possible,
automate PCR procedures would greatly enhance the reproducibility
and reliability of these assays.
[0139] One such technological advance, the introduction of rapid
cycling, quantitative PCR instruments, is opening up new potential
uses for molecular testing. The ability to complete PCR assays in
less than 30 minutes now makes it possible to carry out time
dependent, point of care molecular diagnostics such as testing for
group B streptococcus in pregnant women and nosocomial agents in
immunocompromised patients. In cancer diagnostics, such rapid tests
bring the possibility of primary cancer diagnosis on core biopsies
or fine needle aspirates (FNA) while the patient is still in the
clinic, tumor profiling at the time of surgery to determine
response to chemotherapy and intraoperative testing of lymph nodes
and surgical margins to determine extent of disease and treatment
options. For example, Reverse Transcription-PCR (RT-PCR) has been
shown in many studies to improve the sensitivity of cancer cell
detection in lymph nodes of cancer patients otherwise staged as
node negative. These patients are at higher risk for disease
recurrence and may benefit from more aggressive therapy. In the
case of lung cancer for example, patients with mediastinal lymph
node involvement may benefit from neoadjuvant chemotherapy but
lymph node status needs to be determined prior to major surgical
intervention. This can be achieved through minimally invasive
surgery or even ultrasound guided FNA. Accurate intraoperative
lymph node diagnosis would allow negative patients to undergo
complete tumor resection while surgery would be halted in node
positive patients to allow administration of chemotherapy. A
similar scenario exists in patients with breast cancer or melanoma.
In both diseases, sentinel lymph node positivity results in a
complete lymph node dissection.
[0140] Since current intraoperative lymph node analysis methods are
not very sensitive (.about.70% in breast cancer (Weiser M R,
Montgomery L L, Susnik B, Tan L K, Borgen P I, Cody H S. Is routine
intraoperative frozen-section examination of sentinel lymph nodes
in breast cancer worthwhile? Ann Surg Oncol. 2000;7:651-5) and 47%
in melanoma (Fitzgerald R C, Triadafilopoulos G. Recent
developments in the molecular characterization of Barrett's
esophagus. Dig. Dis. 1998;16:63-80.)) many patients are not
diagnosed as lymph node positive until after completion of the
surgery. These patients then have to undergo a second surgical
procedure to complete the lymph node dissection. A more sensitive,
intraoperative lymph node assessment would clearly benefit these
patients. This example illustrates just one potential use of rapid
PCR-based assays but many more are likely to be forthcoming in the
near future. Once again however, advances are needed in PCR
technology to make these exciting possibilities practical in the
clinical diagnostic laboratory.
[0141] The recent introduction of fluorescence-based PCR, and
RT-PCR, has greatly simplified data analysis steps by eliminating
the need for post-PCR processing and gel electrophoresis. Added
benefits of this technology include reduced assay contamination
(since PCR tubes are never opened) and, of course, the ability to
obtain highly accurate and reproducible quantitative results.
Quantitation not only enhances the clinical utility of many
potential diagnostic tests but also provides the ability to verify
assay sensitivity and reproducibility from test to test with the
use of external quality control standards. Internal quality
controls are also required, including amplification of endogenous
control sequences to check for quality and quantity of the template
DNA or RNA, and internal positive controls to verify that the assay
worked in the case of a negative result. While these `internal`
controls can actually be set up in separate tubes, it would be
advantageous if all assays could be run (multiplexed) in the same
PCR tube, taking advantage of different colored fluorogenic probes
to distinguish the PCR products. Unfortunately, standard multiplex
PCR does not work well when the amplification targets are not
present in similar abundance at the beginning of the reaction and
it is especially difficult to maintain accurate quantitation over a
wide range of target concentrations. The dynamic range of a
quantitative multiplex can be improved to some degree by severely
limiting the PCR primer concentration for the more abundant gene in
the PCR reaction. While this approach works reasonably well for
standard RTPCR assays, the concept of primer limiting goes against
the requirements of a rapid PCR assay, where higher primer
concentrations are necessary to maintain amplification. Here, a
method is described for multiplex PCR that has the same sensitivity
and quantitative dynamic range as a singleplex PCR, and that can
also be performed in a rapid RT-PCR reaction. It is demonstrated
herein that this methodology is applicable to multiple different
amplification targets and that, with the addition of a synthetic
oligonucleotide mimic, it provides for an internally controlled,
rapid, multiplex QRT-PCR. The value of this method in cancer
diagnostics is demonstrated here by the detection of tumor cells in
lymph nodes of esophageal cancer and melanoma patients. This rapid,
intraoperative, QRT-PCR may eventually be used for more accurate
intraoperative cancer staging and will therefore result in more
appropriate and timely treatment of cancer patients.
Materials and Methods
[0142] Tissues and RNA isolation: All patient tissue samples were
obtained through University of Pittsburgh IRB approved protocols.
The lymph nodes from patients with esophagus cancer, and the benign
nodes (obtained incidentally from patients undergoing anti-reflux
procedures) were snap-frozen in liquid nitrogen and RNA was
extracted using the RNeasy Mini kit (Qiagen, Valencia Calif.)
following the manufacturer's protocol. The histologically positive
and negative lymph nodes from melanoma patients were obtained as
formalin-fixed and paraffin embedded archived samples and RNA was
extracted using previously described protocols (Godfrey T E, Kim S
H, Chavira M, Ruff D W, Warren R S, Gray J W, Jensen R H.
Quantitative mRNA expression analysis from formalin-fixed,
paraffin-embedded tissues using 5' nuclease quantitative reverse
transcription-polymerase chain reaction. J Mol Diagn.
2000;2:84-91.).
[0143] Development of the Rapid PCR Assay: Rapid PCR was initially
tested using cDNA synthesized from human colon total RNA (Ambion,
Austin Tex.) as template. PCR primers and probes for
.beta.-glucuronidase (.beta.-gus) and carcinoembryonic antigen
(CEA) were designed to be cDNA specific with amplicon sizes of 81
and 77 base pairs, respectively. Known pseudogenes for CEA were
avoided and control PCR on 200 ng of genomic DNA was negative. PCR
was performed in 25 .mu.L reactions containing 800 nmol/L of each
primer, 200 nmol/L of each probe (primer and probe sequences are
shown in Table 9) and the PCR master mix [1.times. Platinum Taq
reaction buffer, 4.5 mmol/L MgCl.sub.2, 300 .mu.mol/L of each dNTP
and 0.09 U/.mu.L Platinum Taq DNA polymerase (Invitrogen, Carlsbad
Calif.)]. Assays were performed on the Cepheid Smartcycler.TM.
(Cepheid, Sunnyvale, Calif.) and analyzed with Smartcycler software
v1.2b. PCR denaturation (95.degree. C.) times of 10, 7, 5, 2 and 1
second, and annealing/extension (64.degree. C.) times of 30, 15,
10, 7, 5 and 3 seconds were initially tested on a single cDNA
input. The optimal rapid PCR cycling conditions (1 sec denaturation
followed by a 6 sec annealing/extension) were then used to compare
the rapid PCR protocol with more conventional cycling times of 10
sec denaturation and 30 sec annealing/extension. This was carried
out on a serially diluted CEA standard curve to determine any
effect of the rapid PCR protocol over a wide range of CEA inputs.
The CEA cDNA was serially diluted (8.times.) in a constant
background of .beta.-gus template (purified .beta.-gus PCR product)
in amounts that were empirically determined to give constant
.beta.-gus cycle threshold (Ct) values between 19 and 20 cycles.
Similar experiments were carried out to optimize and validate the
tyrosinase assay. TABLE-US-00008 TABLE 9 Primer and probe sequences
Name Sequence (5'.fwdarw.3') CEA-F AGACAATCACAGTCTCTGCGGA (SEQ ID
NO: 6) CEA-R ATCCTTGTCCTCCACGGGTT (SEQ ID NO: 7) CEA probe-FAM
FAM-AGCUGCCCAAGCCCU-BHQ1* (SEQ ID NO: 18) CEA RT GTGAAGGCCACAGCAT
(SEQ ID NO: 9) Gus 81F CTCATTTGGAATTTTGCCGATT (SEQ ID NO: 3) Gus
81R CCGAGTGAAGATCCCCTTTTTA (SEQ ID NO: 4) Gus 80F
CTCATTTGGAATTTTGCC (SEQ ID NO: 16) Gus 80R CGAGTGAAGATCCGCTT (SEQ
ID NO: 17) Gus probe Texas Red- TGAACAGTCACCGACGAGAGTGCTGG-BHQ2
(SEQ ID NO: 5) Gus RT TTTGGTTGTCTCTGCCGAGT (SEQ ID NO: 2)
Tyrosinase-F ACTTACTCAGCCCAGCATCATTC (SEQ ID NO: 19) Tyrosinase-R
ACTGATGGCTGTTGTACTCCTCG (SEQ ID NO: 20) Tyrosinase FAM- probe
TCTCCTCTTGGCAGATTGTCTGTAGCCGA BHQ1 (SEQ ID NO: 21) Tyrosinase RT
CGTTCCATTGCATAAG (SEQ ID NO: 22) Internal Alexa 546- Control
AGCATCATCCTCTGCATGGTCAGGTCAT-BHQ1 Probe (SEQ ID NO: 23) *C and U
were changed to their propyne pyrimidine counterparts
[0144] Development of the rapid reverse transcription assay. The
RNA input for the RT-PCR was a mixture of 400 ng obtained from the
A549 lung adenocarcinoma cell line and 25 ng from a lymph node with
metastatic esophageal adenocarcinoma. RT-PCR reactions were
performed for .beta.-gus and CEA mRNA in 25 .mu.L containing the
PCR master mix described above plus 60 nmol/L .beta.-gus and CEA RT
primers, 1 .mu.L SensiScript reverse transcriptase (Qiagen) and 0.8
U/.mu.L RNase inhibitor. RT reaction was carried out at 48.degree.
C. in a 20.5 .mu.L volume and the PCR primers and probes were added
to the reaction mixture in a 4.5 .mu.L volume during a 70.degree.
C. hold following the RT. This was done in order to maintain PCR
specificity and sensitivity. RT was tested using 30, 10, 7, 5, 3
and 2 minutes to determine the fastest RT possible without loss of
sensitivity.
[0145] Multiplex PCR Assays: In the temperature-controlled
multiplex, both primer and probe sets for CEA and .beta.-gus were
added to the reaction mixture. In this case however, the .beta.-gus
primers (.beta.-gus-80, 400 nmol/L) were redesigned to have a Tm of
50.degree. C. compared to the CEA primers which have a Tm of
60.degree. C. PCR was performed with a one second denaturation, a
four second hold at 53.degree. C. for annealing and a six second
hold at 64.degree. C. for extension and optical read (total
anneal/extend time of ten seconds). The 53.degree. C. anneal step
was eliminated one cycle after the .beta.-gus amplification plot
reached threshold (determined beforehand in a separate singleplex
reaction) and subsequent cycles were carried out with only the
64.degree. C. anneal/extend as in the optimal, rapid PCR singleplex
assay. Simple multiplexing was performed using 800 nM primers for
both genes and utilized our standard .beta.-gus-81 (Tm 60.degree.
C.) primer set. The conventional primer-limited multiplex was
carried out using 300 nmol/L .beta.-gus-81 primers since this was
determined to be the lowest concentration that did not result in a
significant increase (>1 cycle) in the .beta.-gus Ct values in a
rapid PCR. The first 20 PCR cycles using the conventional
methodologies were performed with an anneal/extend time of 10
seconds at 64.degree. C. followed by a change to 6 seconds for the
remaining cycles. This was done in order to better simulate the
conditions used in the temperature-controlled multiplex and allow
direct comparison of the methods. All three multiplex methods were
tested on serial dilutions of CEA cDNA (as described above) in
order to determine the accuracy and dynamic range compared with
singleplex reactions.
[0146] Internal positive controls. The internal positive controls
(IPC) are DNA oligonucleotides that have the same primer sequences
as the target gene (CEA or tyrosinase) but have a different
internal probe sequence (Table 10). Selected sites in the IPC's
were synthesized with uracil instead of thymine so that
contamination with the highly concentrated mimic could be
controlled using uracil DNA glycosylase if required. The IPCs were
added to the reaction mastermix in amounts that were determined
empirically to give Ct values of 36-37 cycles. The rapid triplex
assays were then performed as described for the primer-limited
multiplex (duplex) above with the addition of the internal control
(IC) probe at a concentration of 200 nmol/L. TABLE-US-00009 TABLE
10 CEA and Tyrosinase internal positive control mimics. CEA mimic:
(SEQ ID NO: 24) AGACAATCACAGTCTCTGCGGAAGCATCATCCTCTGCATGGTCAGGTCAT
AACTCCAAACCCGTGGAGGACAAGGAT Tyrosinase mimic: (SEQ ID NO: 25)
ACTTACUCAGCCCAGCAUCATTCTAGCATCATCCTCTGCATGGTCAGGTC
ATTTGGAGGAGUACAACAGCCAUCAGT
Italic=gene specific primer sites. Bold=probe sequence from
bacterial .beta.-galactosidase gene.
[0147] Lymph node assays. Histologically positive and negative
lymph nodes from esophageal cancer and melanoma patients were
tested using rapid, multiplexed QRT-PCR in order to demonstrate the
utility of this technique. Lymph node RNA was analyzed using a
triplex QRT-PCR (.beta.-gus, CEA or Tyrosinase and appropriate
mimic), with the temperature controlled primer limiting technique.
Reverse transcription was carried out as described above with a 5
minute RT followed by a 30 second hold at 70.degree. C. for
addition of PCR primers. Cycle threshold values for .beta.-gus were
determined in a prior, singleplex run so that the temperature
change could be initiated at the appropriate cycle in the multiplex
reaction.
Results
[0148] Rapid PCR development: Rapid PCR assays were developed
separately for all genes in singleplex assays prior to using them
in a multiplex reaction. For development of the rapid PCR assays,
the effect of reducing both the denaturation time (10, 7, 5, 2, 1
seconds at 95.degree. C.) and the anneal/extend time (30, 15, 13,
10, 7, 6, 3 seconds at 64.degree. C.) was evaluated to determine
any observed changes in Ct values. For the denaturation time test
the anneal/extend time was kept constant at 30 seconds and for the
anneal/extension time test the denaturation time was kept constant
at 10 seconds. It was found that reducing the denaturation time to
1 second had no effect on the Ct values for either CEA or
.beta.-gus genes (FIG. 12, panel A). Similarly, reducing the
annealing/extension time had minimal effect (.about.0.5 cycles) on
the Ct values for both genes down to 5 seconds while a 3 second
anneal/extend resulted in a Ct increase of 1.1 cycles for CEA and
1.8 cycles for .beta.-gus when compared with the 30 second
anneal/extend control (FIG. 12, panel B). Thus, for further testing
a 1 second denaturation and a 6 second anneal extend were chosen.
To evaluate the effect of the rapid PCR assay on sensitivity and
quantitation over a wide range of CEA input amounts a serially
diluted standard curve of CEA cDNA in a constant background of
.beta.-gus PCR target was generated. Using this standard curve, the
rapid assay was compared with a more conventional PCR with 10
second denaturation and 30 second anneal/extension (FIG. 13). The
rapid assay resulted in an average increase in Ct value of 1.6
cycles for CEA and 0.2 cycles for .beta.-gus. These increases
appear to be consistent from point to point, and as a result, the
quantitative ability of the rapid assay is equal to that of the
slower PCR. The total PCR time to run 40 cycles using the rapid
assay was 15 minutes versus 48 minutes for the slower PCR
protocol.
[0149] Rapid reverse transcription: Rapid analysis of gene
expression by RT-PCR requires that both the reverse transcription
and PCR components of the assay are rapid. For this reason the
effect of reducing the reverse transcription time from 30 minutes
down to 10, 7, 5, 3 and 2 minutes (FIG. 12, panel C) was evaluated.
In this assay, the PCR times were held constant at 2 second
denaturation and 15 second anneal/extension. The results showed an
increase in Ct value of only 1.1 and 0.6 cycles for .beta.-gus and
CEA respectively with a 5 minute reverse transcription and 1.1 and
1.8 cycles with a 2 minute reverse transcription when compared with
30 minutes. While none of these differences were significant for
individual time point comparisons, there does appear to be a clear
trend towards increased Ct with reduced time as would be expected.
The combined effect of decreasing both the RT and PCR times on the
sensitivity of the assay was evaluated using 4 different RT and PCR
parameter combinations (FIG. 14). Once again a trend was observed
towards higher Ct's with shorter RT-PCR protocols but with a 20
minute total RT-PCR time (5 minute RT with a 1/6 PCR) the Ct
difference was only 1.4 cycles for both genes when compared with a
38 minute total RT-PCR (10 minute RT, 10/15 PCR).
[0150] Multiplex PCR assay: Testing of different multiplexing
methods was carried out on the same dilution series of CEA in
constant background of .beta.-gus that was used for the rapid PCR
development. This dilution series spanned approximately 12 cycles,
corresponding to a 4,096 fold difference in the starting CEA
abundance between the first and the last points on the curve (FIG.
15, panel A). The dilution series was used to compare singleplex
reactions for each gene with a simple multiplex PCR (FIG. 15, panel
C), a conventional primer limited multiplex PCR (FIG. 15, panel D)
and our temperature controlled, primer limited multiplex PCR (FIG.
15, panel B). When a simple multiplex reaction was carried out for
both CEA and .beta.-gus only the first three of five points from
the series reached threshold for CEA while the reaction for the
more abundant .beta.-gus amplified consistently (not shown).
Similar results were observed for the conventional primer limited
reaction while the temperature controlled primer limiting produced
results almost identical to the singleplex reactions.
[0151] Perhaps, more relevant than the range of CEA concentrations
is the range of delta Ct (difference in Ct value between the more
abundant .beta.-gus gene and the CEA target gene) that can be
achieved with the different techniques. In these experiments the
.beta.-gus Ct was constant for all assays between 19 and 20 cycles
and thus the largest delta Ct in this dilution series with a
singleplex assay was almost 15 cycles (.about.32,000 fold
difference in abundance). FIG. 16 shows the delta Ct's plotted for
each of the three multiplexing methods as well as the singleplex
data. In the simple multiplex the third point in the series had a
Ct value of 38.5 cycles compared with 29.0 for the same point in
the singleplex reaction, while subsequent points failed to amplify
at all. Thus the simple multiplex demonstrates a lack of
quantitation with a true delta Ct of.about.8 cycles, as well as a
very limited dynamic range. The conventional primer limited
multiplex worked a little better (delta Ct for the third dilution
point was 4.0 cycles higher than the singleplex) but once again,
dilution points four and five failed to amplify. Thus it seems that
despite its feasibility in standard PCR reactions, conventional
primer limiting is not amenable to rapid assays where increased
primer concentrations are required. In comparison, the temperature
controlled multiplex amplified all points on the dilution series
and no difference was observed in the delta Ct until the last
point. These results clearly demonstrate that the temperature
controlled primer limit was comparable to singleplex reactions in
the rapid QPCR assay while all other multiplexing approaches
remained inadequate. Furthermore, this technique has been utilized
on other targets, such as tyrosinase, and the method appears to be
easily adaptable to new targets.
[0152] Lymph node analysis using rapid, multiplex RT-PCR: For lymph
node analysis internal positive control mimics for CEA and
tyrosinase were included in the multiplex reactions. This
internally controlled, rapid RT-PCR multiplex was used to evaluate
lymph nodes from patients with esophageal cancer and with melanoma
as well as benign lymph nodes from patients without cancer. In
three histologically positive esophageal cancer lymph nodes, high
levels of CEA expression were detected. In lymph nodes from
non-cancer patients, 2 of 5 nodes had no detectable levels of CEA
expression and the remaining three had low levels of CEA expression
(FIG. 17). Furthermore, in cases where CEA signal was detected, the
internal positive control was not amplified (FIG. 18, panel A).
However, in CEA-negative samples, there was amplification of the
CEA internal positive control confirming that negative results were
indeed due to the absence of CEA expression and not a failed CEA
PCR (FIG. 18, panel B). Similar results were obtained for melanoma
lymph nodes using tyrosinase mRNA as the cancer marker. Five lymph
nodes that were histologically positive for melanoma and 5 lymph
nodes without any evidence of cancer were tested. Tyrosinase was
detected at high levels in the all histologically positive nodes
and at very low levels in only one of the five negative nodes (data
not shown). In the positive nodes, the expression of tyrosinase was
higher than that of .beta.-gus and as a result only the tyrosinase
gene amplification was seen. In all the five negative nodes the
amplification of .beta.-gus and the IPC was seen (FIG. 18, panels C
and D).
Discussion
[0153] The introduction of rapid cycling PCR instruments capable of
real time fluorescence-based quantitation has opened the door for
application of molecular diagnostic assays in clinical situations
where time is limited or where a rapid result would be of
psychological or physical benefit to the patient. One case where
rapid molecular assays may be advantageous is in intra-operative
lymph node staging of malignancies such as melanoma, breast and
lung cancer. Conventional analysis of frozen tissue sections is
very quick (approximately 20 minutes in most institutions) but the
sensitivity of this technique for detecting small foci of tumor is
relatively low. Furthermore, many patients who are staged as lymph
node negative by histologic examination suffer recurrence of their
disease, probably as a result of occult disseminated tumor cells
that were missed by routine examination. There is much evidence
that RT-PCR, and particularly quantitative RT-PCR, can detect these
occult cells and identify patients at high risk for recurrence. If
this information can be obtained intraoperatively, surgeons will be
able to make more informed treatment decisions regarding the need
for adjuvant therapy and extent of resection.
[0154] It has been shown that it is feasible to carry out QRT-PCR
in an intraoperative time frame and that this technique can be
superior to frozen sectioning and, at least, comparable to formalin
fixed histology. In the present Example, these rapid QRT-PCR
protocols are tested and it is shown that this complete assay can
be carried out in under 20 minutes. While this time frame is
adequate for intra-operative testing there are still several
practical and technical hurdles that need to be addressed before
rapid QRT-PCR can be considered feasible in the clinical diagnostic
setting. It is believed that the assays must be standardized to
allow data comparison between sites in multi-center trials,
automated (including RNA isolation) to eliminate operator dependent
variability and contamination and controlled for all aspects of the
assay including internal endogenous and exogenous positive
controls.
[0155] The latter of these requirements, quality controls,
necessitates the incorporation of multiplexing into the rapid
QRT-PCR. At least two control reactions are required, an endogenous
control gene to verify presence and adequate quantity and quality
of RNA and an exogenous positive control to verify that the target
gene QRT-PCR functioned with adequate sensitivity in samples that
are negative for target gene expression. Together these two
controls ensure that a negative result is truly negative and not a
result of failed RT-PCR. Thus, at least a triplex PCR reaction is
needed. Unfortunately, maintaining quantitation in a multiplex PCR
has not been easy even in a standard, slow PCR assay. This is
because accumulation of one PCR product (from the most abundant
target gene) inhibits amplification of other targets.
[0156] Initially this inhibition is seen as a shift of the cycle
threshold value to a higher cycle number, and a drop in the total
fluorescence, when compared with a singleplex reaction (FIG. 15
panels A, C and D). If the difference in abundance of the two genes
is too great however, the less abundant gene will fail to amplify
completely. Thus the typical multiplex reaction lacks quantitation
and suffers from a poor dynamic range. Inhibition of the less
abundant target sequence is probably a consequence of several
factors including the accumulation of pyrophosphate released from
the addition of nucleotides during DNA synthesis and
inhibition/sequestration of the Taq enzyme by accumulating PCR
product. One way to overcome this inhibition is to somehow stop
amplification of the more abundant gene soon after signal has been
detected and before pyrophosphate and PCR product have accumulated
enough to cause inhibition. Conventional methods for this rely on
limiting the primer concentration for the more abundant target gene
such that the primers are used up, and PCR of that specific target
stops, before inhibition can occur. While this method works
reasonably well in a slow PCR assay, reducing primer concentration
in a rapid assay results in a lower PCR efficiency, and eventually,
complete PCR failure. In our studies the lowest primer
concentration that did not change the cycle threshold value for
.beta.-gus was determined and tested in a conventional primer
limited assay using our rapid PCR protocol. While this proved
slightly better than multiplexing without a primer limit,
quantitation and dynamic range were still poor compared with
singleplex reactions. In the method described herein, the melting
temperature (Tm) of the primers for the more abundant species
(.beta.-gus) are designed to function at a lower temperature than
those for the target gene (CEA or Tyrosinase). The PCR itself is
carried out in two stages. The annealing step for the first stage
of the PCR is performed at a relatively low annealing temperature
(53.degree. C. in this example) until the amplification curve of
the more abundant species reaches detection threshold. Following
this, the second stage of the PCR is carried out with an annealing
temperature that is at least 10.degree. C. higher than the Tm of
the low temperature primers (64.degree. C. for this example). The
higher temperature of the second stage shifts the binding state of
the low temperature .beta.-gus PCR primers to favor the single
stranded state, effectively terminating amplification of .beta.-gus
while amplification of the CEA target continues uninhibited. Thus,
it is possible to maintain the high primer concentration needed for
rapid PCR of the more abundant control gene and still maintain
quantitation over a large dynamic range of the target gene.
[0157] In addition to developing the rapid multiplexing technology,
an internal positive control mimic is incorporated that can be used
to prove that adequate RT-PCR sensitivity was achieved in samples
that are negative for target gene expression. This mimic utilizes
the same primers as the target gene and therefore controls for the
function of these oligonucleotides. The DNA mimic might be replaced
with an RNA mimic that also includes the reverse transcription
primer site. This will then control for both the RT and PCR steps
of target gene amplification. By adding this mimic to samples at
concentrations that are low enough to produce Ct values greater
than 35 cycles, one can be sure that the RNA isolation and RT-PCR
reaction not only worked but that it worked well enough to detect a
very low abundance of target gene if it were present. In cases
where the target gene is highly expressed, the internal mimic may
fail to amplify. This is acceptable because the mimic is only
intended as a control for negative reactions.
[0158] Therefore, the potential use of rapid QRT-PCR for detection
of metastatic tumor cells in lymph nodes of cancer patients has
been shown. While this is a particularly exciting use of rapid
molecular diagnostics it is only one potential application. As new
cancer markers are developed it is possible that rapid QRT-PCR
could also be used intraoperatively for analysis of surgical
margins, in the clinic for analysis of biopsies or fine needle
aspirates or for analysis of primary tumors to predict response to
chemotherapy or new biotherapies such as tyrosine kinase
inhibitors. Although the dual temperature PCR cycling required for
the temperature controlled multiplex adds approximately 2 minutes
to the overall reaction time, a complete, multiplexed QRT-PCR assay
can still be carried out in under 22 minutes. With the addition of
external cooling to the SmartCycler.TM., or its future generations,
it is believe that an extra 3-4 minutes can be saved. Also, in the
current work, the cycle number at which the temperature change
needed to be initiated was pre-determined. Ultimately, a minor
modification in the instrument's software will automatically
increase the PCR annealing temperature after the endogenous control
gene fluorescence reaches threshold. Since all PCR sites are
independently controlled, this will allow multiple reactions to be
run at the same time with unknown amounts of starting RNA template.
Finally, rapid QRT-PCR is now becoming feasible in a clinical
setting. Using technology currently under development, RNA
isolation can also be carried out very quickly in an automated
process. To this end, certain fully integrated RNA isolation,
reverse transcription and quantitative PCR instruments, such as
Cepheid's GenXpert.TM., will automate, according to a series of
programming steps, all of these processes and provide a QRT-PCR
result in under 30 minutes. The combination of the rapid,
internally controlled multiplex technology described herein with an
automated sample processing platform will allow for accurate and
yet expeditious measurements in a closed environment. The
simplicity and sensitivity of this integrated system should enable
many new applications of molecular assays in cancer
diagnostics.
[0159] The use of numerical values in the various ranges specified
in this application, unless expressly indicated otherwise, are
stated as approximations as though the minimum and maximum values
within the stated ranges were both preceded by the word "about". In
this manner, slight variations above and below the stated ranges
can be used to achieve substantially the same results as values
within the ranges. Also, the disclosure of these ranges is intended
as a continuous range including every value between the minimum and
maximum values.
Sequence CWU 1
1
25 1 2975 DNA Homo sapiens 1 ctcagggcag agggaggaag gacagcagac
cagacagtca cagcagcctt gacaaaacgt 60 tcctggaact caagctcttc
tccacagagg aggacagagc agacagcaga gaccatggag 120 tctccctcgg
cccctcccca cagatggtgc atcccctggc agaggctcct gctcacagcc 180
tcacttctaa ccttctggaa cccgcccacc actgccaagc tcactattga atccacgccg
240 ttcaatgtcg cagaggggaa ggaggtgctt ctacttgtcc acaatctgcc
ccagcatctt 300 tttggctaca gctggtacaa aggtgaaaga gtggatggca
accgtcaaat tataggatat 360 gtaataggaa ctcaacaagc taccccaggg
cccgcataca gtggtcgaga gataatatac 420 cccaatgcat ccctgctgat
ccagaacatc atccagaatg acacaggatt ctacacccta 480 cacgtcataa
agtcagatct tgtgaatgaa gaagcaactg gccagttccg ggtatacccg 540
gagctgccca agccctccat ctccagcaac aactccaaac ccgtggagga caaggatgct
600 gtggccttca cctgtgaacc tgagactcag gacgcaacct acctgtggtg
ggtaaacaat 660 cagagcctcc cggtcagtcc caggctgcag ctgtccaatg
gcaacaggac cctcactcta 720 ttcaatgtca caagaaatga cacagcaagc
tacaaatgtg aaacccagaa cccagtgagt 780 gccaggcgca gtgattcagt
catcctgaat gtcctctatg gcccggatgc ccccaccatt 840 tcccctctaa
acacatctta cagatcaggg gaaaatctga acctctcctg ccacgcagcc 900
tctaacccac ctgcacagta ctcttggttt gtcaatggga ctttccagca atccacccaa
960 gagctcttta tccccaacat cactgtgaat aatagtggat cctatacgtg
ccaagcccat 1020 aactcagaca ctggcctcaa taggaccaca gtcacgacga
tcacagtcta tgcagagcca 1080 cccaaaccct tcatcaccag caacaactcc
aaccccgtgg aggatgagga tgctgtagcc 1140 ttaacctgtg aacctgagat
tcagaacaca acctacctgt ggtgggtaaa taatcagagc 1200 ctcccggtca
gtcccaggct gcagctgtcc aatgacaaca ggaccctcac tctactcagt 1260
gtcacaagga atgatgtagg accctatgag tgtggaatcc agaacaaatt aagtgttgac
1320 cacagcgacc cagtcatcct gaatgtcctc tatggcccag acgaccccac
catttccccc 1380 tcatacacct attaccgtcc aggggtgaac ctcagcctct
cctgccatgc agcctctaac 1440 ccacctgcac agtattcttg gctgattgat
gggaacatcc agcaacacac acaagagctc 1500 tttatctcca acatcactga
gaagaacagc ggactctata cctgccaggc caataactca 1560 gccagtggcc
acagcaggac tacagtcaag acaatcacag tctctgcgga gctgcccaag 1620
ccctccatct ccagcaacaa ctccaaaccc gtggaggaca aggatgctgt ggccttcacc
1680 tgtgaacctg aggctcagaa cacaacctac ctgtggtggg taaatggtca
gagcctccca 1740 gtcagtccca ggctgcagct gtccaatggc aacaggaccc
tcactctatt caatgtcaca 1800 agaaatgacg caagagccta tgtatgtgga
atccagaact cagtgagtgc aaaccgcagt 1860 gacccagtca ccctggatgt
cctctatggg ccggacaccc ccatcatttc ccccccagac 1920 tcgtcttacc
tttcgggagc gaacctcaac ctctcctgcc actcggcctc taacccatcc 1980
ccgcagtatt cttggcgtat caatgggata ccgcagcaac acacacaagt tctctttatc
2040 gccaaaatca cgccaaataa taacgggacc tatgcctgtt ttgtctctaa
cttggctact 2100 ggccgcaata attccatagt caagagcatc acagtctctg
catctggaac ttctcctggt 2160 ctctcagctg gggccactgt cggcatcatg
attggagtgc tggttggggt tgctctgata 2220 tagcagccct ggtgtagttt
cttcatttca ggaagactga cagttgtttt gcttcttcct 2280 taaagcattt
gcaacagcta cagtctaaaa ttgcttcttt accaaggata tttacagaaa 2340
agactctgac cagagatcga gaccatccta gccaacatcg tgaaacccca tctctactaa
2400 aaatacaaaa atgagctggg cttggtggcg cgcacctgta gtcccagtta
ctcgggaggc 2460 tgaggcagga gaatcgcttg aacccgggag gtggagattg
cagtgagccc agatcgcacc 2520 actgcactcc agtctggcaa cagagcaaga
ctccatctca aaaagaaaag aaaagaagac 2580 tctgacctgt actcttgaat
acaagtttct gataccactg cactgtctga gaatttccaa 2640 aactttaatg
aactaactga cagcttcatg aaactgtcca ccaagatcaa gcagagaaaa 2700
taattaattt catgggacta aatgaactaa tgaggattgc tgattcttta aatgtcttgt
2760 ttcccagatt tcaggaaact ttttttcttt taagctatcc acagcttaca
gcaatttgat 2820 aaaatatact tttgtgaaca aaaattgaga catttacatt
ttctccctat gtggtcgctc 2880 cagacttggg aaactattca tgaatattta
tattgtatgg taatatagtt attgcacaag 2940 ttcaataaaa atctgctctt
tgtatgacag aatac 2975 2 20 DNA Artificial Sequence B-gus RT primer
2 tttggttgtc tctgccgagt 20 3 22 DNA Artificial Sequence B-gus
forward PCR primer 3 ctcatttgga attttgccga tt 22 4 22 DNA
Artificial Sequence B-gus Reverse PCR primer 4 ccgagtgaag
atcccctttt ta 22 5 26 DNA Artificial Sequence B-gus taqman probe 5
tgaacagtca ccgacgagag tgctgg 26 6 22 DNA Artificial Sequence CEA
Forward PCR primer 6 agacaatcac agtctctgcg ga 22 7 20 DNA
Artificial Sequence CEA Reverse PCR Primer 7 atccttgtcc tccacgggtt
20 8 26 DNA Artificial Sequence CEA Taqman probe 8 caagccctcc
atctccagca acaact 26 9 16 DNA Artificial Sequence CEA RT primer 9
gtgaaggcca cagcat 16 10 22 DNA Artificial Sequence 18SrRNA Taqman
probe 10 tgctggcacc agacttgccc tc 22 11 23 DNA Artificial Sequence
18SrRNA forward PCR primer 11 ccctgtaatt ggaatgagtc cac 23 12 18
DNA Artificial Sequence 18SrRNA Reverse PCR primer 12 gctggaatta
ccgcggct 18 13 19 DNA Artificial Sequence 18SrRNA Forward - low
temp -PCR primer 13 ccctgtaatt ggaatgagt 19 14 15 DNA Artificial
Sequence 18SrRNA Reverse - low temp PCR primer 14 gctggaatta ccgcg
15 15 16 DNA Artificial Sequence B-gus RT primer2 15 tggttgtctc
tgccga 16 16 18 DNA Artificial Sequence B-gus Forward PCR Primer -
low temp 16 ctcatttgga attttgcc 18 17 17 DNA Artificial Sequence
B-gus Reverse Primer - Low temp 17 cgagtgaaga tcccctt 17 18 15 DNA
Artificial Sequence CEA Taqman Probe2 18 agcngcccaa gcccn 15 19 23
DNA Artificial Sequence Tyrosinase Forward PCR primer 19 acttactcag
cccagcatca ttc 23 20 23 DNA Artificial Sequence Tyrosinase Reverse
PCR Primer 20 actgatggct gttgtactcc tcc 23 21 29 DNA Artificial
Sequence Tyrosinase Taqman probe 21 tctcctcttg gcagattgtc tgtagccga
29 22 17 DNA Artificial Sequence Tyrosinase RT primer 22 cgttccattg
cataaag 17 23 28 DNA Artificial Sequence Internal control probe 23
agcatcatcc tctgcatggt caggtcat 28 24 77 DNA Artificial Sequence CEA
mimic - Internal control 24 agacaatcac agtctctgcg gaagcatcat
cctctgcatg gtcaggtcat aactccaaac 60 ccgtggagga caaggat 77 25 77 DNA
Artificial Sequence Tyrosinase mimic - Internal Control Probe 25
acttacncag cccagcanca ttctagcatc atcctctgca tggtcaggtc atttggagga
60 gnacaacagc cancagt 77
* * * * *