U.S. patent application number 10/077272 was filed with the patent office on 2003-03-27 for method and kit for the prognostication of breast cancer.
This patent application is currently assigned to Ventana Medical Systems, Inc.. Invention is credited to Flom, Kerry J., Jaffee, Deborah R..
Application Number | 20030059790 10/077272 |
Document ID | / |
Family ID | 26753513 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030059790 |
Kind Code |
A1 |
Jaffee, Deborah R. ; et
al. |
March 27, 2003 |
Method and kit for the prognostication of breast cancer
Abstract
This invention relates to a method, kit and controls for
detecting HER-2/neu gene amplification as a predictor of breast
cancer reoccurrence and patient survival. The method is a
fluorescent in-situ hybridization (FISH) assay using a labeled DNA
probe. By determining the genetic nature of the cancer cells,
appropriate treatment may be utilized. Control tumor cell lines
with predefined amounts of HER-2/neu gene amplification are also
disclosed.
Inventors: |
Jaffee, Deborah R.;
(Laytonsville, MD) ; Flom, Kerry J.; (Highland
Park, IL) |
Correspondence
Address: |
HESLIN ROTHENBERG FARLEY & MESITI PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
US
|
Assignee: |
Ventana Medical Systems,
Inc.
Tucson
AZ
|
Family ID: |
26753513 |
Appl. No.: |
10/077272 |
Filed: |
February 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10077272 |
Feb 15, 2002 |
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09237115 |
Jan 26, 1999 |
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6358682 |
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60072574 |
Jan 26, 1998 |
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Current U.S.
Class: |
435/6.14 ;
702/20 |
Current CPC
Class: |
Y02A 90/10 20180101;
Y02A 90/26 20180101; C12Q 1/6841 20130101; C12Q 1/6841 20130101;
C12Q 2545/113 20130101 |
Class at
Publication: |
435/6 ;
702/20 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50 |
Claims
We claim:
1. A method for determining the likelihood of cancer recurrence in
a patient comprising the steps of: (a) obtaining tumor cells from
the patient; (b) applying to each of said cells a detectable probe
capable of hybridizing to the HER-2/neu gene; (c) counting the
number of HER-2/neu genes per cell; and (d) determining the
likelihood of cancer recurrence wherein an average of about 10 or
more HER-2/neu genes per tumor cell indicates a high likelihood of
cancer recurrence and an average of about 3 or fewer BER-2/neu
genes per tumor cell indicates a low likelihood of cancer
recurrence.
2. The method according to claim 1 wherein a control cell line is
substantially simultaneously tested.
3. The method according to claim 1 wherein the number of cells
counted is about 40.
4. The method according to claim 1 wherein the number of cells
counted is about 20.
5. A method for determining the likelihood of cancer recurrence in
a patient comprising the steps of: (I) obtaining tumor cells from
the patient; (ii) applying to said cells a detectable probe capable
of hybridizing to the HER-2/neu gene; (iii) counting the number of
HER-2/neu genes in cells; and (iv) determining the likelihood of
cancer recurrence wherein an average of at least about 4 HER-2/neu
genes per tumor cell indicates a high likelihood of cancer
recurrence.
6. The method according to claim 5 wherein at least one control
cell line is substantially simultaneously tested.
7. The method according to claim 5 wherein the number of cells with
HER-2/neu genes counted therein is no more than about 20.
8. A kit of quality control cell lines for a HER-2/neu gene
amplification detection system comprising: (i) a non-amplified
control cell line that exhibits a mean of about 3 or fewer
HER-2/neu genes per cell; and (ii) an amplified control cell line
that exhibits a mean of about 10 or more HER-2/neu genes per
cell.
9. The kit according to claim 8 and further comprising a low
amplified control cell line that exhibits a mean of between about 3
and 10 HER-2/neu genes per cell.
10. A kit of quality control cell lines for use with a breast
cancer gene amplification detection system according to claim 9
comprising: (I) ATCC HTB 30 (SK-BR-3) cell line; (ii) ATCC HTB 132
(MDA-MB-468) cell line; and (iii) ATCC HTB 133 (T-47D) cell
line.
11. A kit for determining the number of copies of HER-2/neu genes
in a cell comprising; a) a detectable probe capable of hybridizing
to the HER-2/neu gene; and b) at least one control cell line
selected from the group consisting of one that exhibits a mean of
about 10 or more HER-2/neu genes per cell, one that exhibits a mean
of between about 3 and 10 HER-2/neu genes per cell, and one that
exhibits a mean of about 3 or fewer HER-2/neu genes per cell.
12. The kit according to claim 11 further comprising at least two
of the control cell lines.
13. The kit according to claim 12 further comprising all three of
the control cell lines.
14. A method for preparing a standardized control slide comprising
mixing cells of a cell line in a liquid to form a suspension,
solidifying the suspension, whereby the cells are dispersed
throughout the suspension to produce a solid form, cutting thin
sections from the solid form, and mounting the thin section onto a
slide.
15. The method according to claim 14 further comprising embedding
the solid form in a solid matrix.
16. The method according to claim 15 wherein the solid matrix is
paraffin.
17. The method according to claim 14 further comprising staining
the section.
18. The method according to claim 14 further comprising preparing
the section for binding an added ligand to a receptor in or on the
cells.
29. The method according to claim 14 further comprising preparing
the section so that nucleic acid in the cells is capable of
hybridizing to an added solution containing a complementary nucleic
acid probe.
20. A quality control material for use as a standardized control
comprising: (a) a first cell line having an amplified HER-2/neu
gene copy number; and (b) a second cell line having a non-amplified
HER-2/neu copy number; wherein all of the cells in the quality
control material are distributed throughout a solid medium in three
dimensions.
21. A thin section of a quality control material consisting
essentially of a thin section cut from the material of claim 20.
Description
[0001] This application is a continuation-in-part of U.S.
provisional patent application Serial No. 60/072,574, filed Jan.
26, 1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to determining the prognosis
of a patient with breast cancer by determining whether the
HER-2/neu gene is amplified in tumor cells.
[0004] 2. Description of Related Art
[0005] Breast cancer remains a major cause of illness and death
among women in the United States, with over 180,000 new cases and
44,000 deaths per year (American Cancer Society, 1997). Possibly
the most important predictor of clinical course in breast cancer is
the presence or absence of lymph node metastases. Many prognostic
indicators aid in evaluation of invasive cancers in addition to the
presence or absence of lymph node metastasis, including tumor size,
histologic type, tumor grade (differentiation reflected in extent
of gland formation), nuclear grade (extent of nuclear alteration
and frequency of mitosis), DNA content (ploidy), and hormone
receptor status. A reasonable and desirable approach would be the
use of prognostic factors to risk-stratify invasive breast cancer
patients into low-risk and high-risk groups in terms of the
probability of recurrence (McGuire, et al., 1990).
[0006] The HER-2/neu (ERBB2) gene is an oncogene which shares
significant homology to the epidermal growth factor receptor (EGFR)
gene (Yamamoto, et al, 1986) and the retroviral gene v-erbB. It was
first detected as a mutated transforming gene in chemically induced
rat neuronal tumors. It has been isolated from diverse sources,
including: rat neuroblastoma (Schechter et al, 1985); human tumor
lines from gastric cancer (Fukushige et al, 1986); salivary
adenocarcinoma (Semba et al, 1985); and a human breast cancer cell
line where HER-2/neu was identified in an amplified form (King et
al, 1985). The gene has been localized to 17q11.2q12 (Human Gene
Mapping 11, 1991), in a region where several genes relevant to
breast cancer are located, including BRCA1 estradiol-17.beta.
dehydrogenase, NM23 and RARA.
[0007] Current evidence indicates that HER-2/neu protein over
expression and gene amplification are indicative of poor patient
prognosis at all stages of breast tumor development. Amplification
appears early in tumor progression (Iglehart et al 1990 and Van de
Vijver et al 1988), and when present is homogeneously distributed
throughout the tumor (Press et al, 1994). Thus, it is a logical
choice as a prognostic marker when used as an adjunct with other
accepted prognostic indicators.
[0008] While such immunoassays for HER-2/neu protein have been
commercially available, interpreting results is somewhat difficult.
Protein denaturation or degradation during handling, staining,
embedding in paraffin and sectioning gives variable results,
including both false negatives and false positives. Additionally,
slightly different conditions during antibody-antigen binding
results in false positives and false negatives. Unacceptable
results have been reported for immunohistochemical detection of
HER-2/neu amplification. See Thor et al 1989; Richner et al, 1990;
O'Reilly et al, 1991; and Lovekin et al, 1991. By contrast,
counting the number of copies of the HER-2/neu gene in a cell
represents a more objective determination and involves DNA markers
which are less susceptible to degradation and provide less variable
results.
[0009] HER-2/neu gene amplification status is useful as an adjunct
in the evaluation of the prognosis of node negative breast cancer
patients and is also an independent marker of high risk in
node-negative patients. Amplification of HER-2/neu is indicative of
poor patient prognosis at all stages of breast cancer development
and correlates with relatively shorter disease-free and overall
survival.
[0010] Studies have shown positive correlation between HER-2/neu
gene amplification and other common indicators of poor prognosis in
breast cancer (Tsuda, et al., 1989 and Seshadri, et al., 1993 and
Slamon et al, U.S. Pat. No. 4,968,603). However, even strong breast
cancer prognostic factors, such as number of positive lymph nodes,
tumor size and histograde do not predict patient outcome
unfalteringly (Wright, et al, 1989 and Ro, et al., 1989). Current
evidence indicates that HER-2/neu protein over expression and gene
amplification are indicative of poor patient prognosis at all
stages of breast cancer development (Seshadri, et al., 1993,
Wright, et al., 1989 and Niehans et al., 1993). Because HER-2/neu
amplification appears early in breast cancer progression (Iglehart,
et al., 1990 and van de Vijver, et al, 1988) and, when present is
homogeneously distributed throughout the cancer (Inglehart, et al.,
1990 and Press, et al., 1994), it can serve as a prognostic marker
for this disease (when used as an adjunct with other accepted
prognostic indicators).
[0011] The use of Fluorescent In-Situ Hybridization (FISH) targeted
to the HER-2/neu gene, has successfully demonstrated gene
amplification in breast cancer cell lines and primary tumors, and
has shown that FISH results are concordant with other measures of
amplification (Kallioniemi, et al., 1992). [The gene has been
localized to 17q11.2-q12 (Human Gene Mapping 11, 1991), in a region
where several genes relevant to breast cancer are located,
including BRCA1, estradiol-17 dehydrogenase, NM23, and RARA.] FISH
technology combines the advantages of direct gene amplification
assessment with direct localization in morphologically identified
tumor cells. FISH is applicable to tumors of all sizes because
studies can be performed on sections from the original specimen
blocks used for diagnosis. In many samples, direct comparison can
be made with FISH assays on normal cells from the same preparation.
Further, if amplification were localized rather than diffusely
distributed within a tumor, it would be detectable by FISH but
could be diluted below detectable limits in extracted tumor DNA
required for other procedures.
[0012] When performing an assay of such importance to the patient,
it is critical to have appropriate controls. Sections of previously
tested tissue are somewhat undesirable as controls due to cell
variability, unclear boarders, necrotic tissue in the center of the
tumor, variable responses to protease digestions and finite source
material. Therefore, there is a need for quality control materials
which can be run with every test which lack the above mentioned
problems.
[0013] There is also a need for a set of statistical benchmarks to
allow the medical practicioner to stratify the patient according to
likelihood of cancer recurrence. This will aid the practioner and
patient in deciding whether agressive treatments (e.g.
chemotherapy, radiation and anti-HER-2/neu therapy) should be
empoloyed in lieu of a passive "watchful waiting" approach.
SUMMARY OF THE INVENTION
[0014] The present invention is directed to methods and reagents
which determine the number of copies of the HER-2/neu gene in a
breast cancer specimen. This method uses a FISH assay for HER-2/neu
in surgically removed breast cancer tissue. Determination of an
abnormally high copy number of the gene correlates with poor
prognosis and such patients should be treated aggressively.
[0015] The present invention is also directed to a set of control
slides, one of which has a normal copy number of the HER-2/neu
gene, one has a high copy number of the HER-2/neu gene and one has
a slightly elevated copy number of the HER-2/neu gene.
[0016] The present invention further includes the preparation of
control slides using cell lines instead of primary tumor tissue.
The preferred cell lines used for controls are one with high
amplification of the HER-2/neu gene, one with non-amplification and
one with low amplification.
[0017] The HER-2/neu gene detection system of the present invention
is a kit consisting of DNA probe and detection reagents that yields
a green fluorescent signal at the site of each HER-2/neu gene, on a
blue fluorescent background of stained nuclear DNA. The kit is
intended to be used with sections (4 .mu.m) of formalin-fixed,
paraffin-embedded human breast cancer tissue. The kit is untended
to include or recommend the use of another kit which includes the
control lines.
[0018] The HER-2/neu gene detection system of the present invention
is preferably a fluorescence In situ hybridization (FISH) DNA probe
assay that determines the qualitative presence of HER-2/neu gene
amplification on formalin-fixed, paraffin-embedded human breast
tissue as an aid to stratify breast cancer patients according to
risk for recurrence or disease-related death. It is indicated for
use as an adjunct to existing clinical and pathologic information
currently used as prognostic indicators in the risk stratification
of breast cancer in patients who have had a primary, invasive,
localized breast carcinoma and who are lymph node-negative.
[0019] A recent review and comparison is Ross et al, The Oncologist
3: 237-252 (1998).
BRIEF DESCRIPTION OF FIGURES
[0020] FIG. 1 is a survival curve of HER-2/neu Amplification Status
with the cumulative probability of early recurrence.
[0021] FIG. 2 is a survival curve of HER-2/neu Amplification Status
with the cumulative probability of disease-free survival.
[0022] FIG. 3 is a tumor size cumulative probability-1 cm overall
survival
[0023] FIG. 4 is a an interaction, without error bars, of HER-2/neu
amplification and tumor size cumulative probability of overall
survival HER-2/neu Amplification Status (amp+/amp-) and tumor size
(>1 cm/<1 cm)
DETAILED DESCRIPTION OF THE INVENTION
[0024] The HER-2/neu gene amplification detection system according
to the present invention is a fluorescence in situ hybridization
(FISH) DNA probe assay intended for formalin fixed,
paraffin-embedded human breast tissue as an aid in predicting risk
of breast cancer recurrence so that patient management decisions
can be improved. Post surgery lymph node negative patients with no
amplification may receive little or no further treatment whereas
patients with tumors having the HER-2/neu gene amplified may
receive more aggressive monitoring, chemotherapy and/or radiation.
Clearly, the appropriate use of the drug HERCEPTIN.RTM.(Genentech,
South San Frisco, Calif.), humanized monoclonal antibody to
HER-2/neu, is determinable by measuring HER-2/neu gene
amplification. In the few months since release of the commercial
assay it has become accepted standard practice to include the
HER-2/neu gene amplification detection system routinely on breast
cancer patients and particularly before the particular therapy
noted above.
[0025] The relationship between HER-2/neu gene amplification and
probability of remaining disease free and surviving is demonstrated
in the Figures. These clinical studies are based on breast cancer
patients who had excision of a primary, invasive, localized breast
tumor, who were node negative and who did not receive any adjuvant
therapy except in cases of disease recurrence. While remaining
disease-free is somewhat different from survival, both measures are
important even when the data does not exactly parallel.
[0026] While this specification is described with respect to breast
cancer, one skilled in the art will readily appreciate the
application of the techniques herein described of use with other
cancers where the HER-2/neu gene is amplified, such as ovarian,
prostate, endometrial and certain colon cancers. In such
situations, different control cell lines and different
amplification cut off numbers may be required but the need for
appropriate quality controls remains.
[0027] Briefly, the methodology is as follows. Sections of
formalin-fixed, paraffin-embedded breast cancer tissue mounted on
microscope slides are pretreated chemically (Pretreatment Step,
reduction of peptide disulfide bonds) and enzymatically (Protein
Digestion Step, digestion of proteins) to remove proteins that
block DNA access. The DNA in the sections is converted from double-
to single-strand by solution denaturation at 75.degree. C. using a
mixture of 20.times. SSC (saline sodium citrate) and formamide. A
hybridization solution, containing labeled DNA probe which is
complementary to the HER-2/neu gene sequence, is applied to the
tissue section, which is then incubated under conditions favorable
for annealing of probe DNA and genomic DNA sequences. Unannealed
probe is washed off using a mixture of 20.times. SSC and formamide.
The hybridized probe is detected using a fluorescently-tagged
ligand (fluorescein-labeled avidin) which binds to the label on the
DNA probe, thereby immobilizing the fluorescein at the site of the
HER-2/neu gene. The remainder of the DNA is then stained with an
intercalating fluorescent counterstain (DAPI in Antifade).
Excitement of fluorescein and DAPI by light from a mercury arc lamp
with appropriate filters in an epifluorescence microscope results
in the emission of green and blue light, respectively. The observer
selects for these two colors by using a microscope filter set
designed for simultaneous viewing of DAPI and fluorescein, and
scores nuclei in the tissue section for the number of green signals
on a blue background.
[0028] When performing such an assay of great importance to the
patient, it is always necessary to use the best available controls.
Tissue sections from an excised tumor are somewhat variable by
being a mixed population of cells, sometimes having unclear
boarders. Tissue sections can only be as good at the tumor itself
which may have a necrotic center, have blood vessels through it,
and contain a number of inflammatory response cells. The density of
cells in a primary tissue section (tumor or normal) may be high
with a 4 .mu.m thick section having only a small part of a cell. In
FISH assays, a protein digestion step is performed. The digestion
conditions differ between different tumors and normal cell types.
All of this requires skilled individuals to determine which cells
are appropriate tumor cells to be considered on the slide.
Additionally, tumor and normal sections represent a finite source
of controls as each new tissue block will require restandardization
before it can become a control standard.
[0029] By comparison, cell lines have the advantage of uniformity
in cell type with no chance of misidentifying the cells. The cell
concentration is regulatable so that the control slide will have
cells evenly distributed and clearly separated. Since the cell line
is uniform, the protein digestion conditions may be perfected, not
merely optimized. Uniformity also reduces interpretation mistakes
and permits use of less skilled, and less expensive, personnel.
Because cell lines are used, the exact cell type may be used
indefinitely as an permanent source of control cells without
further need to restandardize the cell line. Should doubt remain as
to the advantages of standardized controls over previously tested
samples, several subclasses of U.S. patents are devoted to
analytical clinical controls, their preparation and use.
[0030] In the present invention, the control slides include a slide
with a normal copy number of the HER-2/neu gene, a slide with a
highly amplified copy number of the HER-2/neu gene, and a slide
with a lowly amplified copy number of the HER-2/neu gene.
Representative examples are:
1 Level 1 Control ATCC HTB 132 (MDA-MB-468) non-amplification,
.ltoreq.3 copies per cell Level 2 Control ATCC HTB 133 (T-47D) low
amplification, 3-10 copies per cell Level 3 Control ATCC HTB 30
(SK-BR-3) high amplification, .gtoreq.10 copies per cell
[0031] The use of a control with a low level of amplification is
preferred as clinical samples with low levels of gene amplification
are the mostly likely to be miss detected. Such primary cancers may
be difficult to find and standardize, thus the use of such a cell
line has considerable benefits.
[0032] It will be appreciated that numerous other cell lines may be
used as controls provided that the number of copies of HER-2/neu
gene is adequately quantified and it is uniform in the cell line.
Cell line controls should fall into one of the three level control
ranges recited above. Once another cell line has been so
standardized, it may be used in lieu of the specific cell lines
recited above. It is preferred to use tumor cell lines originating
from the same tissue as being tested from the patient For example,
the three cell lines above originated from breast cancers.
[0033] Briefly, control slides are prepared by culturing the cell
lines, suspending a predetermined concentration of cells in plasma,
clotting the plasma, formalin fixing, embedding in paraffin,
sectioning and mounting on a slide. It should be noted that
additional and alternative steps of preparing the slide for FISH
may also be performed with a goal of preparing the control cell
line to resemble breast tumor tissue for comparative parallel
testing. Such sample preparation techniques are described for
example, in Diagnostic Molecular Pathology, Vol. 1, IRL Press,
NY.
[0034] While these steps are individually well known in the art,
numerous variations on the above procedure may be used. For
example, other solidifying materials may be used in the place of
plasma provided that they do not alter the cellular DNA. Examples
include agarose, gelatin, pectin, alginate, carrageenan, monomers,
polymers etc. where the gel is formed by cooling, adding ions
(calcium, potassium) adding a polymerizing or a cross linking
agent, etc. Other fixatives are known and may be used if any is
desired at all. Paraffin embedding may be standard but other
similar materials may be used and may even be optional. Likewise,
the thickness of the section cut from a block is variable and is
optimized depending on the microscope and assay conditions.
[0035] The relative sensitivity and specificity of the HER-2/neu
gene amplification detection system for measuring HER-2/neu gene
copy numbers was accessed. Breast cancer specimens with a known
HER-2/neu gene copy and expression levels were selected as archival
tissue specimens. Amplification was previously determined by
Southern Blot hybridization or dot blot using extracted DNA.
Expression had been determined by Northern hybridization, Western
immunoblotting and/or immunohistochemistry using total RNA, total
protein or histologic sections from tumor tissue. Slamon et al,
1989 and Press et al, 1993. Gene amplification levels correlated
with gene expression levels in approximately 90% of the breast
cancers under research conditions. In a less standardized clinical
setting, the divergence may be higher. The comparison with FISH was
performed by the HER-2/neu gene amplification gene detection system
on 140 breast cancer specimens. Forty-nine were considered true
positive, 90 true negative, 0 false positive and 1 false negative.
This is a the relative sensitivity value of 98% and the relatively
specificity value of 100%.
[0036] The expected HER-2/neu gene detection system assay result in
normal breast tissue (non-cancerous) was estimated in a population
of 20 breast tissue samples from reduction mammoplasties. The
overall observed mean was 2.2 signals per nucleus with a range of
1.8-2.6 signals per nucleus. The target population for analysis
using the HER-2/neu gene detection system was patients with primary
node-negative, invasive breast carcinoma. The expected prevalence
of early recurrence within 2 years is 4 to 6%. The expected
prevalence of recurrence within 3 years is 2 to 10%. The expected
prevalence of disease-related death (within 3 years) is 10 to 15%
(Clinical Oncology, 1993, page 207).
[0037] A clinical study evaluated HER-2/neu gene amplification
status in 220 women with node negative invasive breast cancer whose
only course of treatment was surgery, unless diagnosed with disease
recurrence. For this study population HER-2/neu amplification was
shown to have predictive power independent of the other prognostic
markers evaluated (patient age at diagnosis, tumor size, tumor
grade, and estrogen receptor). HER-2/neu was shown to be the
strongest predictor for early recurrence (within 24 months),
recurrence and disease-related death.
[0038] The negative predictive value, probability of no disease
being present in women with HER-2/neu non amplified tumors, was
found to be high three years after diagnosis (93.3% based on a
prevalence of 10.4%). The probability of being alive three years
after diagnosis was 99.4%, based on a prevalence of 2.4%.
[0039] HER-2/neu was analyzed along with and controlling for the
above listed prognostic factors. The combined effect (interaction)
of tumor size and HER-2/neu amplification status is presented in
FIG. 4. One analysis used tumor size at 1 cm (FIG. 3) and
additional analysis looked at tumor size at 2 cm. In both sets of
analyses, tumor size is not significant (p>0.05) for predicting
recurrence and disease-related death when the tumor is HER-2/neu
amplified.
[0040] When the tumor is not amplified for the HER-2/neu gene,
tumor size is also an insignificant predictor of recurrence and
disease-related death within 3 years. With longer follow-up,
disease-related death was significantly predicted in a comparison
of tumors>1 cm. In this particular data set, there were no
disease-related deaths for HER-2/neu non-amplified tumors.
[0041] These data show that for this study tumor size failed to be
a good predictor of recurrence and disease-related death within 3
years. Tumor size is of little consequence in HER-2/neu positive
tumors and only becomes of value when evaluating disease-related
death, in HER-2/neu negative tumors . HER-2/neu amplification was
shown to have predictive power independent of all other prognostic
markers evaluated and to be the strongest predictor for recurrence
and disease-related death.
[0042] The following Examples utilized the commercially available
Oncor INFORM HER-2/neu Gene Detection System (Ventana Medical
Systems, Gaithersburg, Md., USA), Cat. No: S8000-KIT. The Procedure
and Interpretation Guide enclosed with the kit is expressly
incorporated by reference. This kit was the subject of a FDA PMA
No. P9400004, the public contents of which are expressly
incorporated by reference.
EXAMPLE I
Preparation of HER-2/neu Probes
[0043] Partial restriction enzyme digests of human Chromosome 17
DNA were prepared to create a library. Fragments were cloned into
BAM H1 restriction sites on a cosmid vector and grown in E. Coli HB
101. Positive clones were selected with Kanamycin containing
medium. The cosmid probe set represent overlapping segments with a
four member contig. A probe used to detect the cDNA is preparable
using primers 5'-CGGCCAAGATCCGGGAGTTGGT-3' and
5'-TCTTGATGCCAGCAGAAGTCAGGC-3'. Numerous publications exist
regarding the HER-2/neu gene and other probes may be prepared and
used.
[0044] Biotintylated HER-2/neu DNA probe was prepared containing a
biotin-labeled single-stranded DNA fragment derived from human
genomic DNA sequences, suspended in a solution of formamide, SSC
and blocking DNA. The probe DNA sequences are complementary to the
sample HER-2/neu (erb-b2) gene sequence and specifically bind to
them upon hybridization. The probe used below is the commercially
available HER-2/neu probe (Ventana Medical Systems, Gaithersburg,
Md.)
EXAMPLE II
Sample Preparation for Fish Assay for HER-2/neu
[0045] Slides were prepared by cutting paraffin embedded tissue
samples into 4 .mu.m thin sections and applying them to silanized
or positively charged slides. The slides were air dried and baked
at 65.degree. C..+-.5.degree. C. overnight. The slides were
deparaffinized in fresh xylene that has not been used for more than
one week and repeated through three changes of xylene for five
minutes each. The slides were then washed in fresh 100% ethanol
that has not been used for more than one week for two minutes. The
ethanol washing was repeated and the slides allowed to air dry.
[0046] The slides were pretreated by immersing slides in a coplin
jar containing 40 ml of pre-warmed 30% w/v sodium bisulfite
Pretreatment Solution in a 43.degree. C..+-.2.degree. C. water bath
for 15 minutes. This solution is designed to reduce disulfide
bonds, aid in protein digestion and improve probe penetration to
target DNA sequences. The slides were then washed in 40 ml of
2.times. SSC at room temperature for 1 minute and then washed twice
using fresh 2.times. SSC. The slides were then dehydrated through a
room-temperature graded series of ethanol solutions for 2 minutes
in each of 70%, 80%, 90% and 100% ethanol and allowed to air dry
inclined with label end down.
[0047] 40 ml of Protein Digesting Enzyme Working Solution was
freshly prepared by mixing 25 mg of proteinase K in 37.degree.
C..+-.2.degree. C. of prewarmed 2.times. SSC. Slides were immersed
in a coplin jar of prewarmed Protein Digesting Enzyme Working
Solution and incubated at 37.degree. C..+-.2.degree. C. for 40
minutes. This solution is needed to digest protein and improve
probe penetration. The slides were washed three times in 40 ml of
fresh 2.times. SSC at room temperature for 1 minute. The slides
were dehydrated through the room-temperature graded series of
ethanol solutions for 2 minutes in each grade of ethanol: 70%, 80%,
90% and 100% and allowed to air dry inclined with label end
down.
[0048] The slides were denatured by immersing them in a coplin jar
containing 40 ml of prewarmed Denaturation Solution (70%
formamide/2.times. SSC, pH 7.0) in a 75.degree. C. water bath for 8
minutes. The slides were immediately transferred to the pre-chilled
(-20.degree. C..+-.5.degree. C.) 70% ethanol and rinsed for 2
minutes. The rinse was repeated in pre-chilled (-20.degree.
C..+-.5.degree. C.) 80%, 90%, and 100% ethanol solutions,
successively and allowed to air dry inclined with label end
down.
[0049] The HER-2/neu DNA probe was prewarmed at 37.degree.
C..+-.2.degree. C. for 5 minutes, vortexed for 1 minute and
centrifuged for 2 to 3 seconds to collect contents in the bottom of
the tube. 10 .mu.l of probe solution was pipetted onto the
denatured tissue section and covered gently with a 25 mm.times.25
mm glass coverslip. Larger tissue sections may require up to 20
.mu.l of probe and larger glass coverslips. The slides were
incubated at 37.degree. C..+-.2.degree. C. for 12 to 16 hours in a
humidified chamber.
[0050] The coverslips were then removed by sliding it to the side
and lifting the overhanging edge with forceps. The slides were
washed in a coplin jar containing 40 ml of pre-warmed
Post-Hybridization Wash Solution (50% Formamide/2.times. SSC, pH
7.0) in the 43.degree. C..+-..degree.C. water bath for 15 minutes.
The slides were then rinsed in a coplin jar containing 40 ml of
pre-warmed 2.times. SSC in the 37.degree. C. water bath with
frequent agitation for 10 minutes and repeated with fresh 2.times.
SSC and placed in a coplin jar containing 40 ml of 1.times. PBD
(phosphate buffered detergent) at 18.degree. C. to 25.degree.
C.
[0051] 60 .mu.l of Blocking Reagent One (0.05 g nonfat dry milk,
Nonidet P40, phosphate buffer and sodium azide) was added to each
slide, a plastic coverslip placed over the solution and incubated 5
minutes in a humidified chamber at room temperature. The plastic
coverslip was pealed off and blotted dry for re-use. The slide was
tilted to allow fluid to drain briefly. This reagent contains
salts, detergent, proteins and sodium azide (preservative) which
aid in reducing non-specific binding of fluorescein-labeled avidin
to the hybridized and washed tissue section.
[0052] 60 .mu.l of Detection Reagent (fluorescein labeled avidin in
sodium azide preservative) was added to each slide and the plastic
coverslip replaced over the solution. The slide was incubated 20
minutes in a humidified chamber at room temperature. After 10
minutes of the 20 minute incubation, one lifts and replaces the
plastic coverslip to ensure even fluid distribution. This reagent
detects hybridized probe DNA by binding to the biotin conjugated to
the probe.
[0053] The plastic coverslip was then pealed off and discarded. The
slides were washed in a coplin jar containing 40 ml of 1.times. PBD
at room temperature for 2 minutes and the wash repeated 2 times
using fresh 1.times. PBD.
[0054] The slides were removed from 1.times. PBD, tilted to allow
fluid to drain briefly, then excess fluid was briefly blotted from
the edge.
[0055] 60 .mu.l of Blocking Reagent Two (0.05 ml goat serum,
Nonidet P40, phosphate buffer and sodium azide) was added to each
slide and a fresh plastic coverslip placed over the solution. The
slides were incubated 5 minutes in a humidified chamber at room
temperature, the plastic coverslip pealed off and blotted dry for
reuse and the slide tilted to allow fluid to drain briefly. This
reagent is a mixture of salts, detergent and proteins in a sodium
azide preservative which reduce non-specific binding of the
Anti-Avidin Antibody to the hybridized ans washed tissue section
during the signal amplification phase of detection.
[0056] 60 .mu.l of Biotin-labeled Anti-Avidin Antibody was added to
each slide and the plastic coverslip replaced over the solution.
Incubation was for 20 minutes in a humidified chamber at room
temperature. At 10 minutes of incubation, the plastic coverslip was
lifted and replaced to ensure even fluid distribution. This reagent
binds to fluorescein-labeled avidin what has previously bound to
the hybridized probe and allows amplification of the fluorescent
signal by providing multiple additional biotin moieties for binding
by fluorescein-labeled avidin for each one originally bound with
probe. The reagent contains sodium azide as a preservative.
[0057] The plastic coverslip was removed and discarded. Slides were
washed in a coplin jar containing 40 .mu.l of 1.times. PBD at room
temperature for 2 minutes. This wash was repeated 2 times using
fresh 1.times. PBD.
[0058] 60 .mu.l of Blocking Reagent One was applied to each slide
and a fresh plastic coverslip placed over the solution. Incubation
was for 5 minutes in a humidified chamber at room temperature.
After that the plastic coverslip was pealed and blotted dry for
reuse while the slide was tilted to allow fluid to drain
briefly.
[0059] 60 .mu.l of Detection Reagent was applied to each slide and
the plastic coverslip replaced over the solution. Incubation was
for 20 minutes in a humidified chamber at room temperature. After
10 minutes of incubation, the plastic coverslip was lifted and
replaced to ensure even fluid distribution.
[0060] The plastic coverslip was pealed and discarded and the
slides washed in a coplin jar containing 40 ml of 1.times. PBD at
room temperature for 2 minutes. The wash was repeated 2 times using
fresh 1.times. PBD.
[0061] The cell nuclei were counterstained by adding 20 .mu.l of
DAPI/Antifade (DAPI, glycerol, P-phenylene diamine dihydrochloride,
sodium bicarbonate, sodium hydroxide in phosphate buffered saline)
to each slide and covered with a 24.times.50 mm glass coverslip.
Stained slides may be stored in the dark at -15.degree. C. to
-25.degree. C. for up to five days before analysis. This reagent is
a mixture of a blue-fluorescing DNA-intercalating dye and a
chemical which reduces photo bleaching. This is used to
counterstain nuclear DNA blue to prolong probe signal
fluorescence.
[0062] If the tissue section was insufficiently digested under the
designated digestion conditions and is determined to interfere with
interpretation of assay results, an extended protein digestion may
be used as follows. The coverslip was removed by gently wiping off
the immersion oil with tissue paper and soaking the slide in 40 ml
2.times. SSC, pH 7.0 in a coplin jar at room temperature until the
coverslip falls off. The slide was placed in a coplin jar
containing fresh 2.times. SSC, pH 7.0 for several minutes to clean
off any residual DAPI/Antifade.
[0063] The slides were placed in prewarmed Protein Digestion Enzyme
Working Solution at 37.degree. C..+-.2.degree. C. The effect of
this protein digestion and the initial digestion is cumulative.
Twenty (20) additional minutes of digestion might be an appropriate
starting time for tissue that seems very undigested after the
initial 40 minute digestion.
[0064] The slides were washed in 40 ml 2.times. SSC, pH 7.0 room
temperature with agitation for 10 seconds, then dehydrated in 70%,
80%, 90%, and 100% ethanol at room temperature for 1 minute each
and allowed to air dry. The process above is then repeated.
Example III
Preparation of Control Slides
[0065] The control slides are prepared using the following cell
lines:
[0066] Level 1 Control ATCC HTB 132 (MDA-MB-468) cell line
[0067] Level 2 Control ATCC HTB 133 (T-47D) cell line
[0068] Level 3 Control ATCC HTB 30 (SK-BR-3) cell line
[0069] Each cell line is available from the American Type Culture
Collection, Manassas, Va., USA and grown using standard media and
techniques to produce approximately 1.5.times.10.sup.8 cells. The
cell growth is divided into approximately 30 5.times.10.sup.6
cells. Pellets of the cultured cells were suspended in a
plasma/thrombin matrix and clotted. Fibrin clotted blocks were then
formalin fixed. Each block is paraflin embedded, cut into 4 .mu.m
sections and mounted on a silanized glass slide. The HER-2/neu
Control Slides were stored at 18.degree. C. to 25.degree. C. prior
to processing and at -15.degree. C. to -25.degree. C. after
processing
EXAMPLE IV
Scoring of Control Slides
[0070] A non-amplified sample has a mean HER-2/neu signal per
nucleus less than or equal to (.ltoreq.) 4. Specimens with a mean
signal per nucleus greater than (>) 4 are amplified for the
HER-2/neu gene. Control Slides are divided into three (3)
categories. These are listed below and should not be confused with
the amplification cut-off value of 4.
[0071] Level 1 (0 to 3 Signals/Nucleus) Control Slides
[0072] A Level 1 control has a mean signal per nucleus value of
less than or equal to 3. This range of assay scores (0 to 3) is
defined as non-amplified for the HER-2/neu gene. In most patient
specimens, an internal non-amplified control is present in the form
of cells that are identifiably non-cancerous by the criteria of
histopathologic morphology.
[0073] Level 1 control slides are 4.mu.m sections of a
formalin-fixed, paraffin-embedded human breast cancer tissue
culture cell line on silanized slides. The preferred cell line is
MDA-MB-468 (ATCC # HTB 132).
[0074] Level 2 (>3 to <10 Signals/Nucleus) Control Slides
[0075] A Level 2 control has a mean signal per nucleus value of
greater than 3 to less than 10. This range of assay scores (>3
to <10) is defined as low amplified HER-2/neu gene
amplification. Level 2 control slides are 4.mu.m sections of a
formalin-fixed, paraffin-embedded human breast cancer tissue
culture cell line on silanized slides. The preferred cell line is
T-47D (ATCC # HTB 133).
[0076] Level 3 (Symbol.gtoreq.10 Signals/Nucleus) Control
Slides
[0077] Level 3 control specimens represent a highly amplified
specimen. A Level 3 control has a mean signal per nucleus value
equal to or greater than 10. This range of assay scores
(.gtoreq.10) is well above the cutoff of >4 signals per nucleus.
Level 3 control slides are 4 .mu.m sections of a formalin-fixed,
paraffin-embedded human breast cancer tissue culture cell line on
silanized slides. The preferred cell line is SK-BR-3 (ATCC # HTB
30).
[0078] Level 1, Level 2 and Level 3 controls should be run and
evaluated with each run of the HBER-2/neu gene detection system
assay. Paraffin-embedded human breast cancer cell lines are run
simultaneously with each run of samples. The control slides are
read by scoring 20 cells from each of two (2) randomly selected
areas of the slide (total of 40 nuclei) and the results interpreted
as described below. Scoring criteria for invasive cancer do not
apply to the paraffin-embedded cell line controls.
[0079] Because breast cancer cell nuclei are often considerably
thicker than the 4 .mu.m sections of tissue required to perform the
assay, the control tissue nuclei are frequently not intact. This
effect of sectioning will result in the observation of fewer
HER-2/neu signals than are actually contained in an intact
nucleus.
[0080] The mean signal per nucleus of a Level 1 control must be
less than the mean signal per nucleus of a Level 2 control for a
processing run to be considered valid. For cell line controls
acceptance ranges see the list below:
[0081] Based on 393 observations (40 nuclei scored per observation)
of 4 .mu.m sections of the Level I control cell line a mean of 2.4
(standard deviation=0.25) HER-2/neu signals per nucleus was
determined.
[0082] Based on 102 observations (40 nuclei scored per observation)
of 4 .mu.m sections of the Level 2 control cell line, a mean of 3.5
(standard deviation=0.71) HER-2/neu signals per nucleus was
determined.
[0083] Based on 338 observations (40 nuclei scored per observation)
of 4 .mu.m sections of the recommended Level 3 control cell line,
an acceptance range of 15.8 to 20.0 HER-2/neu signals per nucleus
was determined (determined by non-parametric analysis).
[0084] In addition to these HER-2/neu Control Slides, controls may
also take the form of 4 .mu.m tissue sections from invasive breast
cancers that have been previously identified to have specific
levels of HER-2/neu gene amplification by fluorescence in situ
hybridization (FISH). Use of breast cancer tissue as control
material requires qualification and validation by the user
laboratory according to the laboratory's established procedures.
While these controls may be useful to classify or group tissues
according to HER-2/neu amplification status such controls are time
consuming to prepare and generally considered inferior to
standardized controls. Additionally, controls from cell lines are
homogeneous and reproducible, neither quality can be attributed to
surgically removed tumors and their sections
[0085] For determination of HER-2/neu gene amplification level in
tissue specimens, 40 nuclei were scored from specimens processed
with two (2) lots of the control cell lines. Multiple observers
were used (3 or 4) to achieve accurate estimates of the mean and
standard deviation (SD). Acceptance ranges were calculated from the
mean plus and minus three (3) standard deviations. The results of
six (6) Level 1 tissue specimens are summarized below.
2TABLE 1 Examples of Level 1 Tissue Specimen Means and Acceptance
Ranges Specimen Mean .+-. SD, ( no. of observations) Acceptance
Range 1. 1.96 .+-. 0.37, (N = 42) 0.85-3.07 2 2.14 .+-. 0.79, (N =
42) 0-4.51 3 2.01 .+-. 0.41, (N = 42) 0.78-3.24 4 2.08 .+-. 0.64,
(N = 42) 0.16-4.00 5 1.70 .+-. 0.31, (N = 36) 0.77-2.63 6 2.01 .+-.
0.29, (N = 36) 1.14-2.88
[0086] In general, a 4 .mu.m section of a Level 2 control tissue
will exhibit a mean of greater than 3 to less than 10 (>3 to
<10) HER-2/neu signals per nucleus (40 nuclei scored) when
assayed with the HER-2/neu gene detection system of the present
invention.
[0087] For determination of HER-2/neu gene amplification level in
tissue specimens, 40 nuclei were scored from specimens processed
with two (2) lots of the control slides. Multiple observers were
used (3 or 4) to achieve accurate estimates of the mean and
standard deviation (SD). Acceptance ranges were calculated from the
mean plus and minus two (2) standard deviations. The results of ten
(10) Level 2 tissue specimens are summarized below.
3TABLE 2 Examples of Level 2 Tissue Specimen Means and Acceptance
Ranges Specimen Mean .+-. SD, ( no. of observations) Acceptance
Range 1 3.93 .+-. 0.74, (N = 36) 2.46-5.41 2 5.69 .+-. 1.31, (N =
36) 3.07-8.31 3 3.97 .+-. 1.65, (N = 36) 0.67-7.27 4 3.56 .+-.
0.47, (N = 36) 2.62-4.50 5 3.05 .+-. 0.41, (N = 36) 2.23-3.87 6
6.35 .+-. 1.06, (N = 51) 4.23-8.47 7 8.07 .+-. 2.08, (N = 53)
3.91-12.23 8 6.17 .+-. 2.19, (N = 36) 1.79-10.55 9 5.52 .+-. 1.6,
(N = 36) 2.32-5.52 10 8.21 .+-. 2.47, (N = 30) 3.27-13.51
[0088] In general, a 4 .mu.m section of a Level 3 control tissue
will exhibit a mean of greater than or equal to 10 HER-2/neu
signals per nucleus (40 nuclei scored) when assayed with the
HER-2/neu gene detection system.
[0089] In a study for determination of HER-2/neu gene amplification
level in tissue specimens, 40 nuclei were scored from specimens
processed with two (2) lots of the control slides. Multiple
observers were used (3 or 4) to achieve accurate estimates of the
mean. Acceptance ranges were calculated from the mean plus and
minus two (2) standard deviations with the upper limit truncated at
20. The results of eight (8) Level 3 tissue specimens are
summarized below.
4TABLE 3 Examples of Level 3 Tissue Specimen Means and Acceptance
Ranges Specimen Mean ( no. of observations) Acceptance Range 1
17.72 (N = 36) 12.34-17.72 2 17.46 (N = 36) 11.66-20.00* 3 15.95 (N
= 36) 11.15-20.00 4 10.85 (N = 44) 6.25-15.45 5 14.54 (N = 42)
8.84-20.00 6 10.73 (N = 42) 6.16-15.31 7 15.09 (N = 36) 6.19-20.00
8 12.20 (N = 36) 4.46-19.94 *Acceptance ranges >20 have been set
to 20 as discussed in the Interpretation Section of this Procedure
and Interpretation Guide.
EXAMPLE V
Fish Assay for HER-2/neu Amplification on Clinical Samples
[0090] Slides were viewed with an epifluorescence microscope
equipped with a DAPI filter set and a DAPI/FITC/Texas Red triple
band pass filter set (a filter set capable of simultaneously
passing FITC and DAPI fluorescence). A FITC/Texas Red dual band
pass filter set (a filter set that allows visualization of the FITC
signal but not the DAPI counterstain) is helpful in resolving
background from true signal. The microscope may be equipped with
10.times., 40.times. (optional for viewing hematoxylin and eosin
stained sections) and 100.times. objectives and a 100 watt mercury
arc light source. Scoring should be performed in a darkened room
with excessive light leaking from microscopes minimized.
[0091] Using the DAPI filter set and the same low power objective
used to view hematoxylin and eosin stained sections, it was
confirmed that the tissue section contains areas of invasion as
previously identified in hematoxylin and eosin stained sections.
Areas of invasion are scored; carcinoma in situ should not be
scored.
[0092] Using the DAPI/FITC/Texas Red triple band pass filter set
and a 100.times. oil objective, the FITC signal was present in
approximately 3/4 or more of the cancer cell nuclei in the area to
be scored.
[0093] It should be noted that non-cancerous cell nuclei (e.g. from
normal epithelium) may be more resistant to protein digestion and
may show lower levels of hybridization than tumor cell nuclei;
therefore, these non-cancerous cell nuclei are not a reliable gauge
of hybridization efficiency for the cancerous cell nuclei. The
hybridization signals to be scored within a cancer cell nucleus
will be of similar size and intensity, whether separated or
clustered.
[0094] With the DAPI filter set and a 100.times. oil objective,
individual cancer nuclei were selected for scoring. Only cancer
nuclei that are non-overlapping are selected. Severely truncated
cancer nuclei were excluded. Cancer nuclei that are less than 1/3
the diameter of the average cancer cell nucleus are not selected.
Overdigested and mechanically damaged cancer cell nuclei are not
selected. Only cancer cell nuclei that have relatively well-defined
borders are selected to be scored.
[0095] Using the DAPI/FITC/Texas Red triple band pass filter set
and a 100.times. oil objective, probe signals were differentiated
from background if present. FITC stain appearing over cytoplasm or
in the extra-cellular matrix is considered background. Background
confined to the cancer nucleus is more difficult to interpret and
could interfere with counting, but the background is generally much
smaller and more diffuse than true probe signal.
[0096] Using the DAPI/FITC/Texas Red triple band pass filter set
and a 100.times. oil objective, the number of FITC signals present
in each of 20 randomly-selected cancer nuclei that meet all the
above mentioned criteria were counted. In FISH analysis, signals
are often in different planes of focus within the tumor cell
nucleus. Focusing up and down through the section to find all of
the signals present in the cancer cell nucleus was used. If the
signal count is greater than 20 per cancer cell nucleus, it was
recorded as 20+ and not grouped with any other counts.
[0097] Scoring in a second area of invasive breast cancer was
repeated following all steps above. The two areas examined were
separate, distinct microscopic areas within a single section. The
total number of cancer nuclei scored was 40 from 2 distinct areas
of the same lesion in one section the mean number of HER-2/neu
signals per nucleus was determined.
[0098] If more than 5% of the fluorescein signals (those of similar
size and intensity to true signal within invasive tumor nuclei) are
located over the cytoplasmic compartment or extra cellular matrix
and all troubleshooting methods have been exhausted, the background
is excessive and the assay repeated.
[0099] When the positive or negative control results fall outside
the expected values, then the specimen results are unreliable and
the assay repeated.
[0100] When 40 non-overlapping nuclei cannot be identified, then
the sample is inadequate and the assay repeated on a new slide.
[0101] When signal intensity varies widely after all
troubleshooting methods have been exhausted, then the specimen
results is unreliable and the assay repeated.
[0102] Values at or near the cut-off (3.5 to 4.5 mean
signals/nucleus) are expected to occur in approximately 3.6% of the
patient population. Scoring of borderline specimens should be
repeated by another qualified user or the test should be repeated
using a new tissue section. If the value of 3.5 to 4.5 persists,
then the borderline results should be interpreted with caution and
increased emphasis should be given to the other clinical and
prognostic information available to the practitioner.
[0103] A retrospective study of 220 node-negative breast cancer
patient specimens were collected from multiple sources and analyzed
at two clinical sites in the United States. This combined data set
was used to determine the association of HER-2/neu gene
amplification, using the HER-2/neu gene detection system of the
present invention, to the clinical outcomes; early recurrence
(within 24 month of diagnosis), recurrences, and death, due to
breast cancer.
[0104] The clinical performance characteristics of the HER-2/neu
gene detection system are described with amplification defined as
>4 signals per nucleus and non-amplification defined as <4
signals per nucleus.
[0105] The HER-2/neu gene detection system was used to
retrospectively identify the risk of recurrence and death for
node-negative breast cancer patients meeting the following
criteria:
[0106] 1) Diagnosis of invasive breast cancer;
[0107] 2) Available formalin-fixed, paraffin-embedded tissue for
HER-2/neu analysis;
[0108] 3) Primary treatment surgery only;
[0109] 4) Clinical follow up for at least 2 years for early
recurrence, 3 years for recurrence and death.
[0110] The safety and effectiveness of the HER-2/neu Gene Detection
System was evaluated in a population of 220 node-negative, invasive
breast cancer patients for early recurrence within two years. Two
hundred twelve (212) of the 220 specimens were eligible for
evaluation of recurrence at anytime and 210 of the 220 specimens
were eligible for evaluation of disease-related death. Eight (8)
subjects did not recur and were lost to follow-up before 36 months;
ten (10) subjects did not die of their disease and were lost to
follow-up before 36 months.) The relationship of the HER-2/neu gene
detection system assay result to the probability of remaining
recurrence-free (disease-free survival) in lymph node negative
breast cancer is presented in Table 4. The relationship of the
assay results to the probability of surviving (overall survival) is
shown in Table 5.
[0111] The survival curves presented in the figures are graphical
representations of the probability of early recurrence-free
survival (no recurrence within 24 months) (FIG. 1), recurrence-free
survival (no recurrence at anytime) (FIG. 2), and overall survival
for subjects with and without HER-2/neu amplification. Error bars
show the standard error around the values.
5TABLE 4 Probability of disease-free survival of breast cancer
patients with non-amplified and amplified lesions. Time from
Probability of Remaining Disease-Free* Surgery Non-Amplified
Amplified (in Years) (95% CI).dagger. N** (95% CI).dagger. N** 0.5
100% (100.0% to 100.0%) 179 93.8% (85.7% to 100.0%) 31 1.0 98.3%
(96.4% to 100.0%) 176 81.8% (68.7% to 95.0%) 27 1.5 96.7% (94.1% to
99.2%) 173 75.8% (61.1% to 90.5%) 25 2.0 94.4% (91.1% to 97.7%) 169
75.8% (61.1% to 90.5%) 25 2.5 93.9% (90.3% to 97.4%) 168 72.7%
(57.4% to 88.0%) 24 3.0 93.3% (89.6% to 97.0%) 167 69.7% (54.0% to
85.4%) 23 5.0 85.9% (80.6% to 91.2%) 121 66.7% (50.6% to 82.7%) 19
10.0 70.5% (60.3% to 80.7%) 23 61.9% (44.5% to 79.3%) 4
Non-Amplified Amplified Time from Cumulative Probability of
Cumulative Probability of Surgery Cumulative No. Cases Remaining
Cumulative No. Cases Remaining (in years) N** No. Events Censored
Disease Free N** No. Events Censored Disease Free 0.5 179 0 0
100.0% 31 2 0 93.9% 1.0 176 3 0 98.3% 27 6 0 81.8% 1.5 173 6 0
96.7% 25 8 0 75.8% 2.0 169 10 0 94.4% 25 8 0 75.8% 2.5 168 11 0
93.9% 24 9 0 72.7% 3.0 167 12 0 93.3% 23 10 0 69.7% 5.0 121 24 34
85.9% 19 11 3 66.7% 10.0 23 35 121 70.5% 4 12 17 61.9% *Point
estimate generated from the Kaplan Meier Statistic (Kaplan, E.L.,
and Meier, P., 1958) .dagger.95% Confidence Interval (C.I.)
generated from the Greenwood estimate of standard error (Greenwood,
M., 1926) **Number of Cases = number of cases at risk remaining in
analyses at the time interval specified. The N values decrease with
time due to patients experiencing an event (death or recurrence) or
being censored (lost to follow-up).
[0112]
6TABLE 5 Probability of Overall Survival Tumor Size large (>1
cm) / small (<1 cm) and HER-2/neu Amplification Status
Probability of overall survival of breast cancer patients with
large/small and non- amplified/amplified tumors. Probability of
Survival* Small (.ltoreq.1 cm); Small (.ltoreq.1 cm); Time from
Surgery Non-Amplified (.ltoreq.4) Amplified (>4) (in Years) (95%
CI).dagger. N** (95% CI).dagger. N** 0.5 100% (100.0% to 100.0%) 39
100% (100.0% to 100.0%) 5 1.0 100% (100.0% to 100.0%) 39 100%
(100.0% to 100.0%) 5 1.5 100% (100.0% to 100.0%) 39 100% (100.0% to
100.0%) 5 2.0 100% (100.0% to 100.0%) 39 80.0% (44.9% to 100.0%) 4
2.5 100% (100.0% to 100.0%) 39 80.0% (44.9% to 100.0%) 4 3.0 100%
(100.0% to 100.0%) 39 80.0% (44.9% to 100.0%) 4 5.0 100% (100.0% to
100.0%) 29 60.0% (17.1% to 100.0%) 2 10.0 100% (100.0% to 100.0%) 6
60.0% (17.1% to 100.0%) 1 Probability of Survival* Large (>1
cm;) Large (>1 cm); Time from Surgery Non-Amplified (.ltoreq.4)
Amplified (>4) (in Years) (95% CI).dagger. N** (95% CI).dagger.
N** 0.5 100% (100.0% to 100.0%) 117 100% (100.0% to 100.0%) 25 1.0
100% (100.0% to 100.0%) 117 100% (100.0% to 100.0%) 25 1.5 100%
(100.0% to 100.0%) 117 96.0% (88.4% to 100.0%) 24 2.0 100% (100.0%
to 100.0%) 117 96.0% (88.4% to 100.0%) 24 2.5 99.2% (97.4% to
100.0%) 116 96.0% (88.4% to 100.0%) 24 3.0 99.2% (97.4% to 100.0%)
116 88.0% (75.3% to 100.0%) 22 5.0 96.4% (92.9% to 99.9%) 89 68.0%
(49.8% to 86.2%) 15 10.0 83.4% (74.2% to 92.6%) 21 51.0% (19.1% to
82.9%) 3 *Point estimate generated from the Kaplan Meier Statistic
(Kaplan, E.L., and Meier, P., 1958). .dagger.95% Confidence
Interval (C.I.) generated from the Greenwood estimate of standard
error (Greenwood, M., 1926) **Number of Cases = number of cases at
risk remaining in analyses at the time interval specified. Tumor
size was available for 186 specimens out of the 210 specimens in
the "disease-related death" database. The table above is calculated
from these 186 specimens. The N values decrease with time due to
patients experiencing an event # (death or recurrence) or being
lost to follow-up.
EXAMPLE VI
Multi-Tiered Cutoffs for HER-2/Neu Gene Copy
[0113] The data from the above testing was analyzed to determine
the effect for using a .ltoreq.3 cutoff and a .ltoreq.10 cutoff on
early recurrence (within 24 months), recurrence anytime and disease
related death at any time. The relative hazard for each was
calculated buth unadjusted and adjusted for estrogen receptor,
tumor size, patient age, study site and tumor grade. The results
are in Tables 6.
7TABLE 6 Relative Risk Unadjusted Adjusted .ltoreq.3 cutoff
.ltoreq.10 cutoff .gtoreq.10 .ltoreq.3 cutoff .ltoreq.10 cutoff
.gtoreq.10 Early Recurrence 4.8 6.6 7.8 4.3 5.5 8.3 Recurrence 2.0
3.4 3.4 2.0 3.8 4.3 Death 4.7 5.8 6.9 4.5 7.3 11.0
[0114] As can be seen from this data, the higher the average
HER-2/neu gene copy number per cell, the greater the risk to the
patient.
[0115] Although preferred embodiments are specifically described
herein, it will be appreciated that many modifications and
variations of the present invention are possible in light of the
above teachings and within the purview of the appended claims
without departing from the spirit and intended scope of the
invention. Other and further embodiments will be apparent to those
in the art from the preceding description and examples. No
unreasonable limitations or the like are to be drawn therefrom in
interpreting the following claims.
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[0217] All references mentioned above are herein incorporated by
reference.
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