U.S. patent application number 14/925910 was filed with the patent office on 2016-05-05 for methods for personalizing cancer treatment.
The applicant listed for this patent is TOMA BIOSCIENCES, INC.. Invention is credited to Doug DOLGINOW, Vern NORVIEL, Dietrich STEPHAN, Janet WARRINGTON.
Application Number | 20160122830 14/925910 |
Document ID | / |
Family ID | 41797892 |
Filed Date | 2016-05-05 |
United States Patent
Application |
20160122830 |
Kind Code |
A1 |
STEPHAN; Dietrich ; et
al. |
May 5, 2016 |
METHODS FOR PERSONALIZING CANCER TREATMENT
Abstract
Personalized medicine involves the use of a patient's molecular
markers to guide treatment regimens for the patient. The scientific
literature provides multiple examples of correlations between drug
treatment efficacy and the presence or absence of molecular markers
in a patient sample. Methods are provided herein that permit
efficient dissemination of scientific findings regarding treatment
efficacy and molecular markers found in patient tumors to health
care providers.
Inventors: |
STEPHAN; Dietrich; (San
Francisco, CA) ; NORVIEL; Vern; (San Francisco,
CA) ; WARRINGTON; Janet; (Los Altos, CA) ;
DOLGINOW; Doug; (Basalt, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TOMA BIOSCIENCES, INC. |
FOSTER CITY |
CA |
US |
|
|
Family ID: |
41797892 |
Appl. No.: |
14/925910 |
Filed: |
October 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14075996 |
Nov 8, 2013 |
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14925910 |
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13060425 |
Jun 2, 2011 |
8583380 |
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PCT/US2009/056101 |
Sep 4, 2009 |
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14075996 |
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61231287 |
Aug 4, 2009 |
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61173179 |
Apr 27, 2009 |
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61155477 |
Feb 25, 2009 |
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61094855 |
Sep 5, 2008 |
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Current U.S.
Class: |
506/2 ;
506/9 |
Current CPC
Class: |
C12Q 2600/142 20130101;
C12Q 2600/112 20130101; G01N 33/57484 20130101; C12Q 2600/158
20130101; C12Q 2600/16 20130101; C12Q 2600/154 20130101; C12Q
2600/156 20130101; C12Q 1/6886 20130101; C12Q 2600/136 20130101;
C12Q 2600/106 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-75. (canceled)
76. A method of determining personalized cancer treatment options
for a subject, the method comprising: a. obtaining a sample from a
subject, wherein the sample comprises molecular markers from a
tumor cell; b. determining a status of the molecular markers,
wherein the molecular markers comprise at least four of the
molecular markers selected from the group consisting of c-kit,
Kras, BRAF, EGFR, microsatellite sequence, chromosome 18q,
thymidylate synthase, and Topo1; and c. stratifying one or more
cancer drug treatment options in a report based on the status of
the molecular markers.
77. The method of claim 76, wherein the at least four molecular
markers comprise c-kit, Kras, BRAF, and EGFR.
78. The method of claim 76, wherein the at least four molecular
markers comprise c-kit, and the c-kit comprises an activating
mutation.
79. The method of claim 78, wherein the activating mutation is in a
juxtamembrane domain of c-kit.
80. The method of claim 76, wherein the at least four molecular
markers comprise c-kit, and the c-kit is not expressed.
81. The method of claim 78, wherein the one or more cancer drug
treatment options comprise imatinib mesylate.
82. The method of claim 76, wherein the at least four molecular
markers comprise Kras, and wherein the Kras comprises a mutation in
exon 2.
83. The method of claim 82, wherein the mutation in exon 2 is in
codon 12 or 13.
84. The method of claim 82, wherein the one or more cancer drug
treatment options comprise cetuximab or panitumumab.
85. The method of claim 76, wherein the at least four molecular
markers comprise BRAF, and the BRAF encodes a V600E mutation.
86. The method of claim 85, wherein the one or more cancer drug
treatment options comprise cetuximab or panitumumab.
87. The method of claim 76, wherein the at least four molecular
markers comprise microsatellite sequence, and the microsatellite
sequence displays low-frequency microsatellite instability.
88. The method of claim 76, wherein the at least four molecular
markers comprise microsatellite sequence, and the microsatellite
sequence displays high-frequency microsatellite instability.
89. The method of claim 87, wherein the one or more cancer drug
treatment options comprise fluorouracil-based adjuvant
chemotherapy.
90. The method of claim 76, wherein the at least four molecular
markers comprise EGFR, and the EGFR comprises a copy number
increase relative to normal.
91. The method of claim 76, wherein the at least four molecular
markers comprise EGFR, and the EGFR comprises a mutation in exons
18-21.
92. The method of claim 90, wherein the one or more cancer drug
treatment options comprise cetuximab or panitumumab.
93. The method of claim 76, wherein the at least four molecular
markers comprise chromosome 18q, and the subject comprises
chromosome 18q allelic loss.
94. The method of claim 93, wherein the one or more cancer drug
treatment options comprise adjuvant therapy.
95. The method of claim 76, wherein the at least four molecular
markers comprise thymidylate synthase, and the thymidylate synthase
comprises levels that are low.
96. The method of claim 76, wherein the at least four molecular
markers comprise thymidylate synthase, and the thymidylate synthase
comprises levels that are high.
97. The method of claim 95, wherein the one or more cancer drug
treatment options comprise 5-FU.
98. The method of claim 76, wherein the at least four molecular
markers comprise Topo1, and the Topo1 comprises expression that is
moderate to high.
99. The method of claim 98, wherein the one or more cancer drug
treatment options comprise irinotecan.
100. The method of claim 76, wherein the one or more cancer drug
treatment options comprise imatinib mesylate, cetuximab,
panitumumab, bevacizumab, 5-FU, capecitabine, or irinotecan.
101. The method of claim 76, wherein the sample comprises a tumor
biopsy.
102. The method of claim 76, wherein the sample comprises a
cell-free sample.
103. The method of claim 76, wherein the tumor cell is a colon
cancer, a bone cancer, a breast cancer, a central nervous system
cancer, a gastric cancer, a cervical cancer, an esophageal cancer,
a head and neck cancer, a kidney cancer, a skin cancer, a lung
cancer, or a carcinoma.
104. The method of claim 103, wherein the tumor cell is a colon
cancer.
105. The method of claim 76, wherein the determining comprises
nucleic acid amplification, de novo DNA sequencing, fluorescent
in-situ hybridization (FISH), quantitative PCR (qPCR), digital PCR,
or immunohistochemistry (IHC).
106. The method of claim 105, wherein the determining comprises de
novo DNA sequencing.
107. The method of claim 106, wherein the de novo DNA sequencing
comprises use of reversibly terminating nucleotides.
108. The method of claim 105, wherein the determining comprises
digital PCR.
109. The method of claim 76, wherein the molecular markers comprise
nucleic acid sequence.
110. The method of claim 109, wherein the determining comprises
determining an absence of one or more mutations or a presence of
the one or more mutations in the molecular markers.
111. The method of claim 110, wherein the one or more mutations
comprise a de novo mutation, nonsense mutation, missense mutation,
silent mutation, frameshift mutation, insertion, substitution,
point mutation, deletion, rearrangement, amplification, chromosomal
translocation, interstitial deletion, chromosomal inversion, loss
of heterozygosity, loss of function mutation, gain of function
mutation, dominant negative mutation, or lethal mutation.
112. The method of claim 111, wherein the one or more mutations
comprise a substitution, insertion, deletion, amplification, or
rearrangement.
113. The method of claim 1, wherein the sample comprises greater
than 15% tumor.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 14/075,996, filed Nov. 8, 2013, which is a continuation of U.S.
application Ser. No. 13/060,425, now U.S. Pat. No. 8,583,380, which
was the National Stage of International Application Serial No.
PCT/US2009/056101, filed Sep. 4, 2009, which claims benefit of U.S.
Provisional Patent Application Ser. No. 61/094,855, filed Sep. 5,
2008, U.S. Provisional Patent Application Ser. No. 61/155,477,
filed Feb. 25, 2009, U.S. Provisional Patent Application Ser. No.
61/173,179, filed Apr. 27, 2009, and U.S. Patent Application Ser.
No. 61/231,287, filed Aug. 4, 2009, which applications are
incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] Approximately 50% of Americans will get cancer and
approximately 50% of those will die from cancer. Ten cancers made
up approximately 70% of the 1.4 million estimated new U.S. cases in
2008. The NIH estimated that the total costs of cancer in 2007 were
$219.2 billion, with direct medical costs at $89.0 billion (total
of all health expenditures), indirect morbidity costs at $18.2
billion (cost of lost productivity due to illness), and indirect
mortality costs at $112.0 billion (cost of lost productivity due to
premature death).
[0003] There are approximately 150 oncology drugs, and over 2000 in
development. Many drugs have known or measurable targets. However,
cancer patients are not routinely screened for the status of these
targets (or markers), and markers are not used to determine which
drug treatment options the patients receive. Rather, cancers are
often treated by organ. Furthermore, an integrated source of
knowledge regarding molecular markers and drug targets for
physicians or patients is lacking.
[0004] Personalized medicine involves the use of molecular markers
that characterize a patient's disease to direct the medical care
the patient receives. The scientific literature provides examples
where the efficacy of a cancer drug can be correlated with the
status of a molecular marker. There is a need to improve the
ability of health care providers to access and interpret
information in the scientific literature regarding connections
between molecular markers and drug efficacy. This information will
allow health care providers to use molecular markers to select
appropriate standard chemotherapy.
[0005] Another issue surrounding cancer treatment is the
reimbursement of fees associated with treatments. Medicare can
reimburse for off-label use of cancer drugs included in certain
medical compendia. Additionally, peer-reviewed medical journals can
provide guidance to Medicare contractors with respect to medically
appropriate off-label uses of cancer drugs supported by results of
clinical trials. There is a need to ensure the latest peer-reviewed
information regarding off-label use of cancer drugs and information
about experimental drugs supported by results of clinical trials is
readily accessible to physicians and patients when a cancer drug
treatment decision is made or when reimbursement from an insurance
company is being sought.
SUMMARY OF THE INVENTION
[0006] In general, in one aspect, a method for generating a
personalized cancer drug treatment option report is provided
including obtaining a sample from a subject, determining the status
of one or more molecular markers in the sample, stratifying one or
more cancer drug treatment options in the report based on the
status of the one or more molecular markers, and annotating the
report based on the status of the one or more molecular
markers.
[0007] The subject can be a cancer patient. The cancer patient can
be a colon cancer patient. The sample can be a tumor biopsy. The
sample can be preserved by formalin-fixing and paraffin-embedding.
The determining can include nucleic acid amplification, DNA
sequencing, FISH, qPCR, and/or IHC. The DNA sequencing can include
de novo DNA sequencing. The de novo DNA sequencing can include
bridge amplification of DNA. The sample can include genomic DNA
from an FFPE sample and the determining can include bridge
amplification and DNA sequencing.
[0008] The method can further include purifying cancer cells from
the sample. The purifying can include flow sorting or laser capture
microdissection. The sample can be a tumor biopsy obtained by fine
needle aspiration, the sample can be frozen, and the purifying can
include flow sorting.
[0009] The stratifying can include stratifying the drug treatment
options for a condition in the NCCN Clinical Practice Guidelines in
Oncology.TM. or the American Society of Clinical Oncology (ASCO)
clinical practice guidelines. The condition can be colon cancer.
The stratifying can be based on information in scientific
literature. The stratifying can take into account the status of one
or more molecular markers in drug absorption, distribution,
metabolism, or excretion genes in a sample from the subject. The
determining can include analyzing the sequence of UGT1A1 for one or
more SNPs. The stratifying can take into account whether the
subject is hypermetabolic. The stratifying can take into account
the CYP450 status of the subject. The stratifying and/or the
annotating can be based on clinical information for the subject.
The stratifying can include ranking drug treatment options with a
higher likelihood of efficacy higher than drug treatment options
with a lower likelihood of efficacy or for which no information
exists with regard to treating subjects with the determined status
of the one or more molecular markers. The stratifying can include
indicating on the report one or more drug treatment options for
which scientific information suggests the one or more drug
treatment options will be efficacious in a subject, based on the
status of one or more molecular markers in the sample from the
subject. The stratifying can include indicating on a report one or
more drug treatment options for which some scientific information
suggests the one or more drug treatment options will be efficacious
in the subject, and some scientific information suggests the one or
more drug treatment options will not be efficacious in the subject,
based on the status of one or more molecular markers in the sample
from the subject. The stratifying can include indicating on a
report one or more drug treatment options for which scientific
information indicates the one or more drug treatment options will
not be efficacious for the subject, based on the status of one or
more molecular markers in the sample from the subject. The
stratifying can include color coding the listed drug treatment
options on the report based on the rank of the predicted efficacy
of the drug treatment options.
[0010] The annotating can include annotating a report for a
condition in the NCCN Clinical Practice Guidelines in Oncology.TM.
or the American Society of Clinical Oncology (ASCO) clinical
practice guidelines. The annotating can include listing one or more
FDA-approved drugs for off-label use, one or more drugs listed in a
Centers for Medicare and Medicaid Services (CMS) anti-cancer
treatment compendia, and/or one or more experimental drugs found in
scientific literature, in the report. The annotating can include
connecting a listed drug treatment option to a reference containing
scientific information regarding the drug treatment option. The
scientific information can be from a peer-reviewed article from a
medical journal. The annotating can include using information
provided by Ingenuity.RTM. Systems. The annotating can include
providing a link to information on a clinical trial for a drug
treatment option in the report. The annotating can include
presenting information in a pop-up box or fly-over box near
provided drug treatment options in an electronic based report. The
annotating can include adding information to a report selected from
the group consisting of one or more drug treatment options,
scientific information concerning one or more drug treatment
options, one or more links to scientific information regarding one
or more drug treatment options, one or more links to citations for
scientific information regarding one or more drug treatment
options, and clinical trial information regarding one or more drug
treatment options.
[0011] The sample can include colon cancer cells, the one or more
molecular markers can include c-kit, and the one or more drug
treatment options can include imatinib mesylate.
[0012] The sample can include colon cancer cells, the one or more
molecular markers can include Kras, and the one or more drug
treatment options can include cetuximab, panitumumab, or
bevacizumab. If exon 2 of Kras does not have a mutation, the
stratifying and/or annotating can include recommending cetuximab or
panitumumab monotherapy for the subject. If exon 2 of Kras has a
mutation, the stratifying and/or annotating can include
recommending cetuximab or panitumumab monotherapy not be given to
the subject.
[0013] The sample can include colon cancer cells, the one or more
molecular markers can include BRAF, and the one or more drug
treatment options can include cetuximab or panitumumab. The
determining can include determining the presence or absence of the
BRAF V600E mutation. If BRAF sequence encodes a V600E mutation, the
stratifying and/or annotating can include providing a
recommendation in the report to the treat the subject with
sorafenib and cetuximab and/or panitumumab.
[0014] The determining can include determining the status of
microsatellite sequences. If the microsatellite sequences display
low-frequency microsatellite instability, and the sample is a stage
II or stage III colon cancer, the stratifying and/or annotating can
include recommending fluorouracil-based adjuvant chemotherapy for
the subject. If the microsatellite sequences display high-frequency
microsatellite instability, and the sample is a stage II or stage
III colon cancer, the stratifying and/or annotating can include
making a recommendation to not administer fluorouracil-based
adjuvant chemotherapy for the subject.
[0015] The determining can include determining EGFR copy number. If
EGFR copy number is increased relative to normal, the stratifying
and/or annotating can include recommending cetuximab or panitumumab
monotherapy to the subject.
[0016] The determining can include determining if the sample has
18q chromosome loss. If the subject is a stage II colorectal cancer
patient with chromosome 18q allelic loss the stratifying and/or
annotating can include recommending adjuvant therapy for the
subject.
[0017] The determining can include determining thymidylate synthase
levels. If thymidylate synthase levels are high, the stratifying
and/or annotating can include recommending adjuvant 5-FU-based
chemotherapy not be given to the subject in the report. If
thymidylate synthase levels are low, the stratifying and/or
annotating can include recommending to treat the subject with
adjuvant 5-FU-based chemotherapy.
[0018] The sample can include colon cancer cells, the one or more
molecular markers can include Topo1, and the one or more drug
treatment options can include 5-FU, irinotecan, or capecitabine. If
Topo1 expression is low, the stratifying and/or annotating can
include making a recommendation to not treat the subject with
irinotecan. If Topo1 expression is moderate to high, the
stratifying and/or annotating can include making a recommendation
to treat the subject with irinotecan.
[0019] The method can further include sending a kit to a health
care provider that provides health care to the subject. The kit can
include RNAlater.RTM..
[0020] The method can further include establishing an in vitro
culture using the sample. The method can further include
high-throughput screening of FDA approved off-label drugs or
experimental drugs using the in vitro culture.
[0021] The method can further include establishing a xenograft
model using the sample. The method can further include
high-throughput screening of FDA approved off-label drugs or
experimental drugs using the xenograft model. The sample can be a
tumor biopsy, the subject can be a cancer patient with end stage
cancer, and results of the high-throughput screening can be used to
determine an adjuvant therapy for administration to the cancer
patient.
[0022] The method can further include monitoring tumor antigen for
recurrence detection.
[0023] The determining can include de novo DNA sequencing and the
stratifying can be based on analysis of a Kaplan-Meier survival
curve.
[0024] The method can further include charging a fee for generating
the report.
[0025] In another aspect, a method for generating a personalized
cancer drug treatment option report is provided including: a)
obtaining a sample from a subject; b) determining the status of one
or more molecular markers in the sample; c) stratifying drug
treatment options listed in a clinical practice guideline for a
condition based on the status of the one or more molecular markers
in the sample; and d) optionally annotating the report with
information comprising information regarding one or more additional
drug treatment options not listed in the clinical practice
guideline for the condition, wherein the information is included
based on the status of the one or more molecular markers in the
sample. The clinical practice guideline can be the NCCN Clinical
Practice Guidelines in Oncology or the American Society of Clinical
Oncology (ASCO) clinical practice guidelines. The one or more
additional drug treatment options can be described in National
Comprehensive Cancer Network (NCCN) Drugs and Biologics
Compendium.TM., Thomson Micromedex DrugDex.RTM., Elsevier Gold
Standard's Clinical Pharmacology, or American Hospital Formulary
Service--Drug Information Compendium. The one or more additional
drug treatment options can include a drug used in a clinical trial.
The one or more additional drug treatment options can include a
drug described in a scientific journal article.
[0026] The annotating can further include listing information in
the report referencing one or more scientific journal articles that
describe the use of one or more additional drug treatment
options.
[0027] The one or more additional drug treatment options can target
a molecular marker that is in a pathway for which the status of
another molecular marker indicates that targeting the pathway would
be efficacious for treating the subject. The stratifying can be
indicated by color coding drug treatment options listed on the
report. The color code can include a shade of green, a shade of
yellow, and a shade of red. The drug treatment options associated
with a green shade can be drugs that are recommended to the subject
based on the status of the one or more molecular markers. The drug
treatment options associated with a yellow shade can be drugs that
have some information that supports recommending the drug as a
treatment option to the subject based on the status of the one or
more molecular markers and at least one piece of information does
not support recommending the drug as a treatment option. The drug
treatment options associated with a red shade can be drugs for
which the status of the one or more molecular markers does not
support recommending the drug treatment option to the subject.
INCORPORATION BY REFERENCE
[0028] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0030] FIG. 1 is a flow diagram of a personalized medicine
service.
[0031] FIG. 2A-2C illustrate a physician accessing a report
provided by a personalized medicine business based on the NCCN
Clinical Practice Guidelines in Oncology.TM. on a computer and
finding the drug treatment options for colon cancer stratified
based on the status of a molecular marker present in the
cancer.
[0032] FIG. 3 illustrates a method for accessing a report provided
by a personalized medicine business based on the NCCN Clinical
Practice Guidelines in Oncology.TM. on a computer, selecting an
annotated drug treatment, accessing scientific information
regarding the drug treatment, and submitting the information to an
insurance company.
[0033] FIG. 4 illustrates a flow diagram for a personalized
medicine service for identifying (determining the status of)
molecular markers from a subject and reporting drug treatment
options to the subject.
[0034] FIG. 5 is a flow diagram for providing personalized
treatment options to a patient or physician.
[0035] FIG. 6 shows molecular markers or drug targets that can be
identified in a subject and drug treatment options that can be
recommended to the subject with the molecular marker, references,
and methods of testing the status of a molecular marker.
[0036] FIG. 7 shows an algorithm for determining which drug
treatment options to recommend to a subject with one or more
molecular markers of a particular status.
[0037] FIG. 8 illustrates an example of a report generated by a
method of the provided invention.
[0038] FIG. 9 illustrates a process for sequencing DNA from FFPE
samples.
DETAILED DESCRIPTION OF THE INVENTION
I. Overview
[0039] In general, methods are disclosed herein for a service that
provides personalized cancer drug treatment options based on the
status of one or more molecular markers in a patient's sample,
e.g., a tumor biopsy. The disclosed methods can include stratifying
cancer drug treatment options and/or annotating cancer drug
treatment options with scientific information on a report, e.g., a
printed form. The scientific information can be made accessible to
a patient or one or more health care providers and can be used for
insurance reimbursement purposes to aid selection of one or more
drug treatment options.
[0040] FIG. 1 provides an overview of one aspect of the provided
method. A cancer patient (100) visits a health care provider, and
the health care provider performs a tumor biopsy (102) and
potentially begins a first line drug treatment (104). The tumor
biopsy is then sent to a personalized medicine business for
analysis and testing. In one pathway (106), the tumor is used to
establish an in vitro culture and/or xenograft model for future
drug testing. In another pathway, the tumor cells can be isolated
from non-tumor cells in the biopsy (108) and used to identify
(determine the status of) molecular markers (110). For example, the
status of one or more molecular markers can be determined by
comparative genomic hybridization (CGH), high density expression,
de novo sequencing, or genotyping. The molecular markers can then
be used to stratify a list of cancer drug treatments (112). The
identified molecular markers can also be used as the basis for
annotating the drug treatment options by, for example, providing
links to scientific information for experimental drugs and
FDA-approved drugs for off-label use (114). The links to scientific
literature can be used to send literature to insurance companies
for reimbursement purposes. The stratified and annotated cancer
drug treatment options can then be provided in the form of a report
to the health care provider and/or patient (116), who can use the
information to select a second line drug treatment therapy, for
example, if the first line therapy fails. The stratified and
annotated drug treatment options can be tested in high-throughput
screens using the established in vitro cultures or xenograft models
(118). Results of the screens can be given to the physician (120),
who can use the information to select adjuncts for end stage
administration, for example. One or more fees can be charged by the
personalized medicine business in exchange for any of the
services.
[0041] In one aspect, a method is provided that includes a)
obtaining a sample from a subject, b) identifying (determining the
status of) one or more molecular markers in the sample, c)
stratifying one or more drug treatment options based on the status
of one or more molecular markers, and d) annotating drug treatment
options based on the status of one or more molecular markers.
[0042] In another aspect, a method is provided that provides
personalized cancer drug treatment options, including a) receiving
an order, b) obtaining a sample from a subject, c) purifying cancer
cells from the sample, d) identifying (determining the status of)
one or more molecular markers in the cancer cells, e) stratifying
one or more drug treatment options in a report, f) annotating one
or more drug treatment options in a report, and g) charging a fee
for one or more of steps (a)-(f).
[0043] In another aspect, a method for generating a personalized
cancer drug treatment option report is provided including a)
obtaining a sample from a subject; b) determining the status of one
or more molecular markers in the sample; c) stratifying drug
treatment options listed in a clinical practice guideline for a
condition based on the status of the one or more molecular markers
in the sample; and d) optionally annotating the report with
information comprising information regarding one or more additional
drug treatment options not listed in the clinical practice
guideline for the condition, wherein the information is included
based on the status of the one or more molecular markers in the
sample.
[0044] Another aspect of a method of the provided invention is
shown in FIG. 5, in which a patient or physician interfaces with a
personalized medicine business (500), a biopsy is performed (502),
a molecular analysis is performed (504), and information is
analyzed to provide recommendations to the patient or physician
(506) based on the status of one or more molecular markers. The
information can be scientific literature and/or clinical trial
information. The analysis can include annotating a clinical
practice guideline for a condition (e.g., NCCN Clinical Practice
Guidelines in Oncology) with information (e.g., references to
scientific journal articles; other drug treatment options). The
analysis can include analysis of molecular pathways that include
drug targets, e.g., drug targets not recommended in the clinical
practice guideline for the condition, that may be efficacious for
the patient based on the status of one or more molecular markers in
the biopsy.
II. Obtaining a Sample
[0045] The subject from whom a sample is taken can be a patient,
for example, a cancer patient or a patient suspected of having
cancer. The subject can be a mammal, e.g., a human, and can be male
or female. The sample can be a tumor biopsy. The biopsy can be
performed by, for example, a health care provider, including a
physician, physician assistant, nurse, veterinarian, dentist,
chiropractor, paramedic, dermatologist, oncologist,
gastroenterologist, or surgeon.
[0046] A. Receiving an Order
[0047] The methods of the provided invention can include a step in
which a personalized medicine business receives an order. The order
received by the personalized medicine business can be placed by a
health care provider, including, for example, a physician,
physician assistant, nurse, veterinarian, dentist, chiropractor,
paramedic, dermatologist, oncologist, gastroenterologist, or
surgeon. The order can be placed on behalf of a subject from whom a
sample is taken, e.g., a cancer patient. The order can be placed by
the subject from whom a sample has been or will be taken, and the
subject can place the order with the guidance or supervision of a
health care provider. The order can be submitted to the
personalized medicine business using any of a variety of means. For
example, an order can be placed using a computer website (a web
interface), by sending an email, a facsimile, or a text message, or
by placing a phone call to the personalized medicine business. If a
website or web interface is used, the website or web interface can
be maintained by the personalized medicine business. The person
placing the order can create or be given a username and/or
password. An order form can be downloaded from a website, sent
through the mail, sent using email, or sent by facsimile. The order
can be placed by filling out one or more forms and submitting it
(them) to the personalized medicine business using a computer
(e.g., web interface, email) or through a mail or other delivery
service, for example, the U.S. Postal Service, a courier service,
Federal-Express, DHL, or UPS.
[0048] A diagnostic biopsy of a primary tumor can be taken before
or after an order is placed. The status of one or more molecular
markers in a tumor of the subject can be identified (determined)
before or after an order is placed. The subject from whom the
sample is taken can be provided one or more therapies before or
after an order is placed.
[0049] The order can include information regarding a sample or
subject. The information can include a description of the sample,
e.g., the date the sample was taken, type of sample, or other
properties of the sample. The order can include information
regarding the subject from whom the sample is taken. For example,
this information can include height, weight, eye color, hair color,
age, ethnicity, gender; clinical information, e.g., blood pressure,
LDL cholesterol levels, HDL cholesterol levels, and triglyceride
levels, heart rate; personal medical history, including cancer
treatments already received; family medical history of the subject;
and information on molecular markers.
[0050] The order can include billing information and/or insurance
information. The order can contain a barcode that identifies the
order. The order can contain comments or notes about the sample
and/or the subject (e.g., patient). This information can be sent
with the sample to the personalized medicine business.
[0051] A customer service representative employed by the
personalized medicine business can process the order (e.g., enter
information in the order into a database operated by the
personalized medicine business). An employee of the personalized
medicine business, for example, a research nurse, can review and
process the clinical information. A fee can be charged by the
personalized medicine business in exchange for receiving and/or
processing the clinical information.
[0052] B. Kit for Processing Samples and/or Submitting Samples to a
Personalized Medicine Business
[0053] The personalized medicine business can provide a kit to a
health care provider (e.g., a surgeon, pathologist, oncologist)
involved in the care of a subject from whom a sample will be taken.
A kit can contain contents that can be used to process or transport
a sample from the subject.
[0054] A kit can include a reagent that protects RNA in fresh
specimens including, e.g., RNAlater.RTM. Tissue Collection: RNA
Stabilization Solution (Applied Biosystems) or RNASafer.TM. RNA
Stabilization Reagent (SABiosciences). Compositions for preserving
RNA in cells and tissues are described, for example, in U.S. Pat.
Nos. 6,204,375, 7,056,673, and 7,138,226. A kit of the provided
invention can contain instruments or tools for processing a
sample.
[0055] A kit can contain instructions for processing a sample. The
kit can contain instructions for performing a biopsy. For example,
the kit can contain instructions for performing fine needle
aspiration. The kit can include instructions and/or reagents for
preparing formalin-fixed, paraffin-embedded tissue or for freezing
(e.g., fresh frozen) a sample. The kit can contain dry ice (e.g.,
pelleted dry ice) A kit can contain one or more pre-paid shipping
labels. A kit can contain educational materials (e.g., a pamphlet
containing information about a specific cancer).
[0056] A kit of the provided invention can contain instructions for
shipping a sample, for example, to an entity that will analyze the
sample.
[0057] A kit can include one or more containers, e.g., an envelope,
a box, and/or a tube that can be used to transport one or more
samples to the personalized medicine business. The one or more
containers can be provided with identifying information, e.g.,
information identifying the subject from whom a sample is taken,
information identifying a health care provider that will
perform/has performed a biopsy, information identifying the sample
type to be sent to the personalized medicine business, and
information regarding a shipping address to which the sample can be
sent (e.g., address of the personalized medicine business). A kit
can contain logistics information; for example, the kit can contain
preprinted information that can be attached to the one or more
sample containers that can be returned to the personalized medicine
business. The shipping information can contain a barcode for
tracking the shipment or for identifying the sample. The preprinted
information can contain an adhesive that permits the information to
adhere to a container.
[0058] One or more logistics and operation personnel employed by
the personalized medicine business can send a kit to and/or receive
a kit from a health care provider. A fee can be charged by the
personalized medicine business in exchange for sending a kit.
Multiple individuals can be informed when a kit has been sent by
the personalized medicine business; for example, the subject from
whom the sample is taken (e.g., cancer patient) or an oncologist
that will review the subject's information, can be informed by the
personalized medicine business that a kit has been sent to a health
care provider.
[0059] A kit can be sent to a pathologist and/or pathology
laboratory for preparing a diagnostic paraffin primary biopsy, and
the subject (e.g., cancer patient) and an oncologist can be
notified that the kit has been sent to the pathologist. The
pathology laboratory can be a College of American Pathologists
(CAP) accredited laboratory. The laboratory can be a Clinical
Laboratory Improvement Amendments (CLIA) certified laboratory. A
kit for processing a biopsy can be sent to the person who will
perform the biopsy (e.g., surgeon), and the subject (e.g., patient)
and an oncologist can be notified that the kit was sent. A kit can
be sent to a person (e.g., a surgeon), for example, after surgery,
for processing a second biopsy.
[0060] C. Performing a Biopsy and Submitting a Sample
[0061] Any biopsy technique used by those skilled in the art can be
used for isolating a sample from a subject. The biopsy can be an
open biopsy, in which general anesthesia is used or a closed
biopsy, in which a smaller cut is made than in an open biopsy. The
biopsy can be a core or incisional biopsy, in which part of the
tissue is removed; an excisional biopsy, in which attempts to
remove an entire lesion are made; or a needle aspiration
(percutaneous) biopsy (fine needle aspiration biopsy), in which a
sample of tissue or fluid is removed with a needle. The needle can
be a thin, hollow needle, and it can be inserted into a mass to
extract cells from the mass. Tissue can be obtained by lumpectomy
or mastectomy. Tissue can be obtained by colectomy (e.g., a total
colectomy or partial colectomy). Tissue can be obtained by a
prostatectomy.
[0062] The biopsy can be performed by a health care provider,
including, for example, a physician, physician assistant, nurse,
veterinarian, dentist, chiropractor, paramedic, dermatologist,
oncologist, gastroenterologist, or surgeon.
[0063] The biopsy can be processed. For example, the biopsy can be
preserved by formalin-fixing and paraffin-embedding (FFPE) the
tissue. The biopsy can be processed into smaller pieces. The biopsy
can be treated to preserve RNA, e.g., with RNAlater.RTM. or
RNASafer.TM.. The biopsy can be stored on wet ice (approximately
4.degree. C.), at room temperature (approximately 25.degree. C.),
stored at approximately -20.degree. C., or at approximately
-80.degree. C., e.g., stored on dry ice, or frozen in liquid
nitrogen or a dry ice/alcohol slurry. The tissue can be frozen
within 0.5, 1, 5, 10, 15, 30, 60, 120, or 240 minutes of surgical
resection. Fixative agents that can be used on the biopsy tissue
include, for example, methanol-acetone, Carnoy's fixative (60%
ethanol, 30% chloroform, 10% glacial acetic acid), Bouin's
fixative, ethanol, acetone, formalin, methacarn (substitute 60%
methanol for the ethanol in Carnoy), UMFIX (universal molecular
fixative), Omnifix, and FINEfix.
[0064] The sample can be one or more cells or tissue from, e.g.,
liver, lung, colon, pancreas, bladder, brain, breast, cervix,
esophagus, eye, gallbladder, kidney, stomach, ovary, penis,
prostate, pituitary, salivary gland, skin, testicle, uterus, and
vagina. The sample can be, for example, blood or urine. The sample
can contain nucleic acid, for example, genomic DNA, amplified
genomic DNA, cDNA, amplified cDNA, RNA, amplified RNA, unspliced
RNA, spliced RNA, messenger RNA, or microRNA. The sample can be a
cell-free sample that contains nucleic acid, for example, DNA or
RNA.
[0065] The sample can be a bone marrow core or clot. The clot can
be decalcified. The sample can be one or more unstained slides from
an FFPE block. The one or more slides can be air-dried (unbaked).
The slides can be charged or uncharged. The sample can be a
pleural/ascitic fluid. The pleural/ascitic fluid can be formalin
fixed and parafilm-embedded in a cell block.
[0066] The sample can contain greater than 10% tumor, greater than
15% tumor, greater than 20% tumor, greater than 25% tumor, greater
than 30% tumor, greater than 35% tumor, greater than 40% tumor,
greater than 45% tumor, greater than 50% tumor, greater than 55%
tumor, greater than 60% tumor, greater than 65% tumor, greater than
70% tumor, greater than 75% tumor, greater than 80% tumor, greater
than 85% tumor, greater than 90% tumor, greater than 95% tumor, or
greater than 99% tumor. The sample can contain 100% tumor. The type
of sample submitted to a personalized medicine business can depend
on the type of molecular analysis to be performed on the sample
(e.g., DNA microarray, DNA sequencing, IHC).
[0067] The sample (e.g., biopsy) can be obtained by a personalized
medicine business by receiving it from, for example, the health
care provider who removed the sample from the subject. The sample
can be obtained by a personalized medicine business through a mail
or delivery service; for example, a physician can perform a tumor
biopsy and arrange to send the tumor biopsy. Parafilm-embedded
tissue can be sent to a personalized medicine business in an
envelope. The paraffin embedded sample can be a diagnostic primary
biopsy. A fresh-frozen sample can be sent to a personalized
medicine business in a container, and the container can include dry
ice. A blood sample can be sent in a container, e.g., a tube. A
sample can be sent in a container with an ice-water mixture. A
sample can be sent to a personalized medicine business in a
container provided by the personalized medicine business.
[0068] One or more employees of a personalized medicine business
can review the samples that are obtained by the personalized
medicine business. The samples can be reviewed by a pathologist
employed by the personalized medicine business. The personalized
medicine business can request that one or more additional samples
be sent to the personalized medicine business, for example, after
receiving an initial sample from the subject.
[0069] D. Purifying Cancer Cells
[0070] The methods of the provided invention can include a step in
which primary tumor cells are separated/purified from non-tumor
cells in a sample, e.g., a biopsy sample Any technique used by
those skilled in the art can be appropriate for isolating primary
tumor cells from non-tumor cells in the tumor biopsy. For example,
the tumor cells can be isolated using techniques that include
optical cell sorting, flow-cytometry, flow-sorting, fluorescence
activated cell sorting (FACS), magnetic cell sorting (MACS; e.g.,
using antibody coated magnetic particles), size-based separation
(e.g., using a sieve, an array of obstacles, a filter), sorting in
a microfluidics device, antibody-based separation, sedimentation,
affinity adsorption, affinity extraction, or density gradient
centrifugation. Fresh-frozen or FFPE samples can be used for
flow-sorting. Sorting can be based on cell size, morphology, or
intracellular or extracellular markers. Methods for isolating or
sorting tumor cells are described, for example, in Nagrath S. et
al. (2007) Nature 450:1235-1239; U.S. Pat. Nos. 6,008,002,
7,232,653, and 7,332,288; PCT Publication No. WO2008157220A1; and
US Patent Application Nos. US20080138805A1 and US20090186065; and
Rosenberg R. et al. (2002) Cytometry 49:150-158, each of which is
herein incorporated by reference in their entireties.
[0071] Fluorescence-activated cell sorting (FACS) uses light
scattering and fluorescent characteristics to sort cells. A
fluorescent property can be imparted on a cell using, e.g., nucleic
acid probes or antibodies conjugated to a fluorescent dye. A cell
suspension can form a stream of flowing liquid. The stream of cells
forms drops that contain approximately one cell per drop. Before
the stream forms drops, a fluorescent characteristic of each cell
is measured. A charge is placed on an electrical charging ring
prior to fluorescence intensity measurement and the opposite charge
is carried on the drop as it breaks from the stream. The charged
drops pass through two high voltage deflection plates that divert
drops into different containers based upon their charge. The charge
can be directly applied to the stream and the drop breaking off
retains the charge of the same sign as the stream. The stream is
then returned to neutral after the drop breaks off.
[0072] Direct or indirect immunofluorescence can be used in FACS.
In direct immunofluorescence, an antibody is directly conjugated to
a fluorescent dye. In indirect immunofluorescence, the primary
antibody is not labeled, and a secondary antibody is conjugated to
a fluorescent dye.
[0073] Different types of cancer cells can be sorted based on one
or more different molecular markers, and these markers can be on
the surface of the cancer cells. Cells, e.g., circulating tumor
cells, can be isolated from a sample, e.g., blood, using technology
from, e.g., CELLective Dx Corporation.
[0074] A sample can be obtained from a subject, e.g., a colon
cancer patient, by fine needle aspiration biopsy, the sample can be
stored fresh-frozen, and the cancer cells in the sample can be
isolated by flow-sorting.
[0075] Cancer cells can be purified by laser capture
microdissection. In laser capture microdissection, a transparent
transfer film is applied on the surface of a tissue section. Cells
to be removed are identified using a microscope, and a near
infrared (IR) laser diode is activated. The laser beam fuses the
transfer film to the underlying cells. The film can then be removed
with the desired cells attached. Fresh-frozen and paraffin embedded
tissue may be used for laser capture microdissection.
[0076] Cancer cells can be purified using Panomics
(Affymetrix.RTM.) Cancer Cell Isolation Kit (Catalog number CI0002,
CI0004, or CI0010). Cancer cells can be isolated using
CytoSelect.TM. Clonogenic Tumor Cell Isolation Kit (Catalog number
CBA-155 or CBA-155-5) from Cell Biolabs, Inc.
[0077] A fee can be charged by a personalized medicine business in
exchange for purifying cancer cells from a sample.
III. Molecular Markers
[0078] A. Techniques for Determining the Status of Molecular
Markers
[0079] The methods of the provided invention can include analyzing
a sample for the status of one or more molecular markers. The
sample can be analyzed for one or more molecular markers that can
include, for example, nucleic acids, including DNA and RNA,
proteins, including antibodies, autoantibodies, and cell surface
receptors, gene or protein expression profiles, carbohydrates, and
lipids. The status of one or more molecular markers in the sample
can be identified (determined) using techniques that include, for
example, comparative genomic hybridization (CGH) or chromosomal
microarray analysis (CMA), in which copy number changes of DNA in a
tumor are detected, expression profiling, DNA microarray,
high-density oligonucleotide microarray, whole-genome RNA
expression array, peptide microarray, enzyme-linked immunosorbent
assay (ELISA), genome sequencing, de novo sequencing, 454
sequencing, pyrosequencing, Helicos True Single Molecule
Sequencing, SOLiD.TM. sequencing, SOLEXA sequencing,
nanosequencing, chemical-sensitive field effect transistor
(chemFET) array sequencing, polony sequencing, copy number
variation (CNV) analysis sequencing, small nucleotide polymorphism
(SNP) analysis, immunohistochemistry (IHC), immunocytochemistry
(ICC), mass spectrometry, tandem mass spectrometry, matrix-assisted
laser desorption ionization time of flight mass spectrometry
(MALDI-TOF MS), in-situ hybridization, fluorescent in-situ
hybridization (FISH), chromogenic in-situ hybridization (CISH),
silver in situ hybridization (SISH), polymerase chain reaction
(PCR), digital PCR (dPCR), reverse transcription PCR, quantitative
PCR (Q-PCR), single marker qPCR, real-time PCR, nCounter Analysis
(Nanostring technology), Western blotting, Southern blotting,
SDS-PAGE, gel electrophoresis, and Northern blotting.
[0080] The status of one or more molecular markers corresponding to
genetic polymorphisms between members of a population can be
detected by numerous methods well-established in the art e.g.,
PCR-based sequence specific amplification, restriction fragment
length polymorphisms (RFLPs), isozyme markers, northern analysis,
allele specific hybridization (ASH), array based hybridization,
amplified variable sequences of the genome, self-sustained sequence
replication, simple sequence repeat (SSR), single nucleotide
polymorphism (SNP), random amplified polymorphic DNA ("RAPD") or
amplified fragment length polymorphisms (AFLP).
[0081] Techniques for determining the status of one or more
molecular markers can use hybridization of a probe nucleic acid to
nucleic acids corresponding to the molecular marker (e.g.,
amplified nucleic acids produced using genomic DNA as a template).
Hybridization formats for allele detection can include, for
example, solution phase, solid phase, mixed phase, or in situ
hybridization assays. A guide to the hybridization of nucleic acids
is found in Tijssen (1993) Laboratory Techniques in Biochemistry
and Molecular Biology--Hybridization with Nucleic Acid Probes
Elsevier, N.Y.
[0082] The status of a molecular marker can include, for example,
gene expression level (e.g., messenger RNA level) or protein
expression level. The expression level can be, for example, higher
than normal, normal, or below normal. The status of a molecular
marker can include absence of a mutation (e.g., wild-type) or
presence of one or more mutations (e.g., de novo mutation, nonsense
mutation, missense mutation, silent mutation, frameshift mutation,
insertion, substitution, point mutation, single nucleotide
polymorphism (SNP), deletion, rearrangement, amplification,
chromosomal translocation, interstitial deletion, chromosomal
inversion, loss of heterozygosity, loss of function, gain of
function, dominant negative, or lethal); nucleic acid modification
(e.g., methylation); or presence or absence of a post-translational
modification on a protein (e.g., acetylation, alkylation,
amidation, biotinylation, glutamylation, glycosylation, glycation,
glycylation, hydroxylation, iodination, isoprenylation,
lipoylation, prenylation, myristoylation, farnesylation,
geranylgeranylation, ADP-ribosylation, oxdiation, palmitoylation,
pegylation, phosphatidylinositol addition,
phosphopantetheinylation, phosphorylation, polysialyation,
pyroglutamate formation, arginylation, sulfation, or
selenoylation).
[0083] B. De Novo DNA Sequencing Techniques
[0084] The methods of the provided invention can use de novo
sequencing to determine the status of one or more molecular markers
in a sample, e.g., a sample from a colon cancer patient. De novo
sequencing techniques include, for example, Helicos True Single
Molecule Sequencing (tSMS) (Harris T. D. et al. (2008) Science
320:106-109). In the tSMS technique, a DNA sample is cleaved into
strands of approximately 100 to 200 nucleotides, and a polyA
sequence is added to the 3' end of each DNA strand. Each strand is
labeled by the addition of a fluorescently labeled adenosine
nucleotide. The DNA strands are then hybridized to a flow cell,
which contains millions of oligo-T capture sites that are
immobilized to flow cell surface. The templates can be at a density
of about 100 million templates/cm.sup.2. The flow cell is then
loaded into an instrument, e.g., HeliScope.TM. sequencer, and a
laser illuminates the surface of the flow cell, revealing the
position of each template. A CCD camera can map the position of the
templates on the flow cell surface. The template fluorescent label
is then cleaved and washed away. The sequencing reaction begins by
introducing a DNA polymerase and a fluorescently labeled
nucleotide. The oligo-T nucleic acid serves as a primer. The
polymerase incorporates the labeled nucleotides to the primer in a
template directed manner. The polymerase and unincorporated
nucleotides are removed. The templates that have directed
incorporation of the fluorescently labeled nucleotide are detected
by imaging the flow cell surface. After imaging, a cleavage step
removes the fluorescent label, and the process is repeated with
other fluorescently labeled nucleotides until the desired read
length is achieved. Sequence information is collected with each
nucleotide addition step.
[0085] Another example of a de novo DNA sequencing technique that
can be used to determine the status of one or more molecular
markers in a sample is 454 sequencing (Roche) (Margulies, M et al.
2005, Nature, 437, 376-380). 454 sequencing involves two steps. In
the first step, DNA is sheared into fragments of approximately
300-800 base pairs, and the fragments are blunt ended.
Oligonucleotide adaptors are then ligated to the ends of the
fragments. The adaptors serve as primers for amplification and
sequencing of the fragments. The fragments can be attached to DNA
capture beads, e.g., streptavidin-coated beads using, e.g., Adaptor
B, which contains 5'-biotin tag. The fragments attached to the
beads are PCR amplified within droplets of an oil-water emulsion.
The result is multiple copies of clonally amplified DNA fragments
on each bead. In the second step, the beads are captured in wells
(pico-liter sized). Pyrosequencing is performed on each DNA
fragment in parallel. Addition of one or more nucleotides generates
a light signal that is recorded by a CCD camera in a sequencing
instrument. The signal strength is proportional to the number of
nucleotides incorporated.
[0086] Pyrosequencing makes use of pyrophosphate (PPi) which is
released upon nucleotide addition. PPi is converted to ATP by ATP
sulfurylase in the presence of adenosine 5' phosphosulfate.
Luciferase uses ATP to convert luciferin to oxyluciferin, and this
reaction generates light that is detected and analyzed.
[0087] Another example of a de novo DNA sequencing technique that
can be used to determine the status of one or more molecular
markers in a sample is SOLiD technology (Applied Biosystems). In
SOLID sequencing, genomic DNA is sheared into fragments, and
adaptors are attached to the 5' and 3' ends of the fragments to
generate a fragment library. Alternatively, internal adaptors can
be introduced by ligating adaptors to the 5' and 3' ends of the
fragments, circularizing the fragments, digesting the circularized
fragment to generate an internal adaptor, and attaching adaptors to
the 5' and 3' ends of the resulting fragments to generate a
mate-paired library. Next, clonal bead populations are prepared in
microreactors containing beads, primers, template, and PCR
components. Following PCR, the templates are denatured and beads
are enriched to separate the beads with extended templates.
Templates on the selected beads are subjected to a 3' modification
that permits bonding to a glass slide.
[0088] The sequence can be determined by sequential hybridization
and ligation of partially random oligonucleotides with a central
determined base (or pair of bases) that is identified by a specific
fluorophore. After a color is recorded, the ligated oligonucleotide
is cleaved and removed and the process is then repeated.
[0089] Another example of a de novo sequencing technology that can
be used to determine the status of one or more molecular markers in
a sample is SOLEXA sequencing (Illumina). SOLEXA sequencing is
based on the amplification of DNA on a solid surface using
fold-back PCR and anchored primers. Genomic DNA is fragmented, and
adapters are added to the 5' and 3' ends of the fragments. DNA
fragments that are attached to the surface of flow cell channels
are extended and bridge amplified. The fragments become double
stranded, and the double stranded molecules are denatured. Multiple
cycles of the solid-phase amplification followed by denaturation
can create several million clusters of approximately 1,000 copies
of single-stranded DNA molecules of the same template in each
channel of the flow cell. Primers, DNA polymerase and four
fluorophore-labeled, reversibly terminating nucleotides are used to
perform sequential sequencing. After nucleotide incorporation, a
laser is used to excite the fluorophores, and an image is captured
and the identity of the first base is recorded. The 3' terminators
and fluorophores from each incorporated base are removed and the
incorporation, detection and identification steps are repeated.
[0090] Another example of de novo sequencing technology that can be
used to determine the status of one or more molecular markers in a
sample is the single molecule, real-time (SMRT.TM.) technology of
Pacific Biosciences. In SMRT, each of the four DNA bases is
attached to one of four different fluorescent dyes. These dyes are
phospholinked. A single DNA polymerase is immobilized with a single
molecule of template single stranded DNA at the bottom of a
zero-mode waveguide (ZMW). A ZMW is a confinement structure which
enables observation of incorporation of a single nucleotide by DNA
polymerase against the background of fluorescent nucleotides that
rapidly diffuse in an out of the ZMW (in microseconds). It takes
several milliseconds to incorporate a nucleotide into a growing
strand. During this time, the fluorescent label is excited and
produces a fluorescent signal, and the fluorescent tag is cleaved
off. Detection of the corresponding fluorescence of the dye
indicates which base was incorporated. The process is repeated.
[0091] Another example of de novo sequencing that can be used to
determine the status of one or more molecular markers in a sample
is nanopore sequencing (Soni G V and Meller A. (2007) Clin Chem 53:
1996-2001). A nanopore is a small hole, of the order of 1 nanometer
in diameter. Immersion of a nanopore in a conducting fluid and
application of a potential across it results in a slight electrical
current due to conduction of ions through the nanopore. The amount
of current which flows is sensitive to the size of the nanopore. As
a DNA molecule passes through a nanopore, each nucleotide on the
DNA molecule obstructs the nanopore to a different degree. Thus,
the change in the current passing through the nanopore as the DNA
molecule passes through the nanopore represents a reading of the
DNA sequence.
[0092] Another example of de novo sequencing that can be used to
determine the status of one or more molecular markers in a sample
involves using a chemical-sensitive field effect transistor
(chemFET) array to sequence DNA (as described in US Patent
Application Publication No. 20090026082). In one example of the
technique, DNA molecules can be placed into reaction chambers, and
the template molecules can be hybridized to a sequencing primer
bound to a polymerase. Incorporation of one or more triphosphates
into a new nucleic acid strand at the 3' end of the sequencing
primer can be detected by a change in current by a chemFET. An
array can have multiple chemFET sensors. In another example, single
nucleic acids can be attached to beads, and the nucleic acids can
be amplified on the bead, and the individual beads can be
transferred to individual reaction chambers on a chemFET array,
with each chamber having a chemFET sensor, and the nucleic acids
can be sequenced.
[0093] Any one of the de novo sequencing techniques described
herein can be used to determine the status of one or more molecular
markers in the methods of the provided invention.
[0094] DNA can be extracted from an FFPE sample and the DNA can be
bridge amplified and sequenced. The sequences generated can be used
to determine the status (e.g., wild-type or mutant) of one or more
molecular markers (e.g., genes). An example of the process is
illustrated in FIG. 9. Greater than 1 .mu.g of DNA can be extracted
(902) from an FFPE sample (900). Adaptor sequences can be ligated
to the DNA in an in vitro ligation reaction (904). The extracted
DNA can be bridge amplified to generate a library of single
molecules that are spatially clustered (906). The bridge amplified
DNA can then be sequenced by serial extension of primed templates
(908). The accuracy of sequence reads can be up to 35 bp (910). The
sequences can be used to determine the status (e.g., wild-type or
mutant) of one or more genes in the sample.
[0095] DNA from an FFPE tissue sample, including a historic FFPE
tissue sample, can be sequenced, for example using 454 sequencing,
to determine the status of one or more molecular markers (Thomas R
K et al. (2006) Nature Medicine 12:852-855). Sequences from one or
more historic samples can be compared to sequences from a sample
from a subject, e.g., a cancer patient. The status of one or more
molecular markers in a historic sample can be correlated with one
or more treatment outcomes, and the correlation of a treatment
outcome with molecular marker status in one or more historic
samples can be used to predict treatment outcomes for the subject,
e.g., a cancer patient. These predictions can be the basis for
determining whether or not to recommend a drug treatment option to
the subject.
[0096] The status of one or more molecular markers can be
determined by de novo DNA sequencing of samples from cancer
patients, and cancer patients can be grouped based on the status of
the one or more molecular markers. For instance, cancer patients
with wild-type Kras can be grouped together, and patients with
mutant Kras can be grouped together. The survival of untreated
patients in each group can be followed over time, and patient
survival can be plotted to generate a Kaplan-Meier curve. A
Kaplan-Meier plot can be used to illustrate the fraction of
patients alive after a certain amount of time. The survival of
cancer patients in each group after treatment with a drug can be
followed over time, and patient survival can be plotted to generate
a Kaplan-Meier curve. Analysis of a previously generated
Kaplan-Meier curve can be used to recommend one or more drug
treatment options to a subject (e.g., a cancer patient), based on
the status of one or more molecular markers in a sample from the
subject (e.g., a cancer patient).
[0097] C. Business Considerations
[0098] A personalized medicine business can outsource or
sub-contract the determining of the status of one or more molecular
markers to a Clinical Laboratory Improvement Amendments (CLIA)
certified laboratory. The personalized medicine business can
outsource or sub-contract the determining of the status of one or
more molecular markers, for example, whole genome analysis, to
another company, for example, Illumia.RTM. or Affymetrix.RTM.. Mass
spectrometry (e.g., matrix-assisted laser desorption ionization
time of flight mass spectrometry (MALDI-TOF MS)) can be performed
at, e.g., Biodesix. Only a portion of the whole genome analysis
data can be reported to a patient and/or health care provider, and
the rest of the data can be stored by the personalized medicine
business. The stored data can be sent to the subject and/or health
care provider at a later date.
[0099] Other in vitro or in vivo tests can be performed on a
sample. The personalized medicine business can outsource or
sub-contract another company to perform the in vitro or in vivo
tests.
[0100] A fee can be charged by the personalized medicine business
in exchange for performing the techniques and/or identifying
(determining the status of) one or more molecular markers. The
molecular marker analysis can be performed by one or more employees
of the personalized medicine business, for example, one or more
laboratory technicians.
IV. Drug Metabolism
[0101] The methods of the provided invention can include analyzing
a sample from a subject for the status of one more markers related
to drug absorption, distribution, metabolism, and excretion. The
sample to be analyzed can be, for example, a tumor biopsy, blood,
cheek (buccal) swab, or other fluid or tissue taken from the
subject. Any test known by those skilled in the art to investigate
drug absorption, distribution, metabolism, or excretion genes can
be used. For example, the Affymetrix.RTM. Drug Metabolizing Enzymes
and Transporters (DMET)-Early Access Solution can be used to
analyze drug metabolism biomarkers. CYP450 status can be assessed
using the Roche.RTM. AmpliChip CYP450 Test, which tests for gene
variations in CYP2D6 and CYP2C19. The Human1M BeadChip from
Illumina.RTM., Human1M-Duo DNA Analysis BeadChip from
Illumina.RTM., and HumanExon510S-Duo DNA Analysis BeadChip from
Illumina.RTM. can be used to identify SNPs in ADME (absorption,
distribution, metabolism, and excretion) genes. The status of ADME
associated genes can be investigated using DTEx.TM. Gene Expression
Analysis from NoAb BioDiscoveries.
[0102] A sample can be analyzed for one or more SNPs in the UGT1A1
gene, which encodes UDP-glucuronosyltransferase. Identification of
UGT1A1 promoter polymorphisms can be used to predict the toxicity
of treatment with CPT-111 (irinotecan). Irinotecan is a
topoisomerase I inhibitor that can be used for treating colon
cancer. Irinotecan is activated by hydrolysis to SN-38, and SN-38
is inactivated by glucuronidation by UGT1A1. Subjects with the *28
variant ((TA).sub.7 allele) of UGT1A1 express reduced amounts of
UGT1A1 in their liver, and they do not clear irinotecan as rapidly
as others. This can correspond to increased rates of severe
diarrhea and neutropenia. Patients with polymorphisms in the UGT1A1
gene (e.g., *28 variant) have been recommended by the FDA to
receive reduced doses of irinotecan. If a sample from a subject,
e.g., a cancer patient, has a *28 variant UGT1A1 allele, a
recommendation can be made in a report to treat the subject with a
reduced amount of irinotecan.
[0103] A fee can be charged by the personalized medicine business
for analyzing one or more molecular markers related to absorption,
distribution, metabolism, or excretion of one or more drugs. The
analysis of one or more molecular markers related to drug
absorption, distribution, metabolism, or excretion can be performed
by one or more laboratory technicians employed by the personalized
medicine business.
[0104] Stratifying drug treatment options and annotating drug
treatment options in a report regarding the use of a drug treatment
option can be done based on the status of one or more molecular
markers that regulate drug absorption, distribution, metabolism, or
excretion in a sample from a subject.
V. Stratifying Drug Treatment Options
[0105] The methods of the provided invention can include using the
status of one or more molecular markers identified in a sample to
stratify (rank) drug treatment options for the subject from whom
the sample was taken (target-drug approach). The stratifying of
drug treatments can be based on scientific information regarding
the molecular markers. For example, the scientific information can
be data from one or more studies published in one or more
scientific journals (e.g., New England Journal of Medicine (NEJM),
Lancet, etc.). The scientific information can be data provided in a
commercial database (e.g., data stored in a database provided by
Ingenuity.RTM. Systems). One or more pieces of scientific
information can be used to stratify the treatments.
[0106] A. Classes of Drugs
[0107] Drug treatment options can be stratified into classes based
on the status of one or more molecular markers in a sample. For
example, a first class of drug treatment options can be those for
which scientific information predicts a drug will be efficacious
for a subject whose sample has one or more molecular markers of a
particular status. Drugs in this first class can be a recommended
drug treatment option for a subject.
[0108] A second class of drug treatment options can be those for
which some scientific information predicts a drug will be
efficacious for a subject with one or more molecular markers of a
particular status, and some scientific information does not support
use of the drug for the subject, based on one or more molecular
markers of a particular status in a sample from a subject. For
example, a sample may contain a marker whose status indicates the
drug will be efficacious in the subject and another marker (e.g., a
mutant version of a drug metabolism gene) that indicates the drug
would also have a toxic affect on the subject. Up to 9 molecular
markers in a sample from a patient can indicate that a drug is
likely to be efficacious for treating a subject from whom the
sample was taken, and one molecular marker, for instance a drug
metabolism marker, can indicate that the drug would have a high
level of toxicity in the subject.
[0109] This second class can also include drugs for which there is
indirect scientific support for drug efficacy in a subject (e.g.,
the drug targets a protein that is in the same molecular pathway as
a molecular marker in a sample). For example, a drug in this class
could target a kinase that functions downstream of an overexpressed
cell surface receptor that is known to be targeted by an
efficacious drug. A drug in this second class can be a recommended
drug treatment option for a subject.
[0110] A third class of drugs can be those for which scientific
information indicates the drug will not be efficacious in the
subject based on the status of one or more molecular markers in a
sample from the subject. For example, a drug that targets a cell
surface receptor may not display efficacy if a downstream kinase is
mutated. It can be recommended that a subject not be treated with a
drug in this third class.
[0111] The drug treatment options can be stratified using an
algorithm-based approach. An example of an algorithm for selecting
a marker is shown in FIG. 7. The status of one or more molecular
markers in a patient sample is determined (700). The scientific
literature is analyzed for information related to the status of the
molecular marker and the efficacy of one or more different drugs
(702, 704). If the status of a molecular marker correlates with
efficacy of a drug, then a recommendation can be made to treat the
subject with that drug (706). If the status of a molecular marker
does not correlate with efficacy of a drug, then a recommendation
can be made not to treat a subject with the drug (708). A computer
and computer readable medium can be used to stratify the drug
treatment options.
[0112] A list of stratified drug treatment options can be presented
in the form of a report. The stratification of drug treatment
options can be indicated by color coding. For example, drugs in the
first class can be color coded in green, drugs in the second class
can be color coded in yellow, and drugs in the third class can be
color coded in red.
[0113] The recommendation of a drug treatment option for a subject
can be based on the stage of the cancer of the subject, e.g., a
late stage cancer. Drug treatment options can also be stratified
based on other factors, e.g., the type of cancer, age of the
subject, status of drug metabolism genes (genes involved in
absorption, distribution, metabolism, and excretion), efficacy of
other drugs the patient has received, clinical information
regarding the subject, and family medical history.
[0114] A fee can be charged by the personalized medicine business
in exchange for stratifying the drug treatment options. The drug
treatment options can be stratified by one or more employees of the
personalized medicine business, for example, a molecular
pathologist and research nurse.
[0115] B. Examples of Molecular Marker/Drug Relationships and Drugs
Used for Cancer Treatment
[0116] Drug treatment options can be stratified based on a
relationship between the status of one or more molecular markers in
the sample and the efficacy of a drug treatment used on other
subjects with the one or more molecular markers of a particular
status. The scientific literature contains multiple examples of
drugs that have efficacy in treating subjects with tumors with
certain molecular markers.
[0117] The status of c-kit (also known as CD117, KIT, PBT, SCFR) in
a sample from a subject can be determined by, e.g., expression or
DNA sequencing, and can be used to determine whether to recommend
imatinib mesylate (Gleevec) as a drug treatment option for the
subject, e.g., a gastrointestinal tumor patient with a metastatic
and/or unresectable malignant tumor (NCCN Clinical Practice
Guidelines in Oncology; Henrich M C et al. (2003) J. Clin. Oncol.
21:4342-4349). C-kit is a receptor for cytokine stem cell factor
(SCF, steel factor, or c-kit ligand). When c-kit binds to SCF it
forms a dimer which activates signaling through second messengers.
Signaling through c-kit plays a role in cell survival,
proliferation, and differentiation. Imatinib mesylate (Gleevec) is
an inhibitor of receptor tyrosine kinases. If a sample from a
subject with a metastatic and/or unresectable malignant
gastrointestinal stromal tumor expresses c-kit or c-kit with
activating mutations, a recommendation can be made to treat the
subject with imatinib mesylate (Gleevec). If a sample from a
subject does not express c-kit or c-kit with activating mutations,
a recommendation can be made to not treat the subject with imatinib
mesylate (Gleevec). Imatinib mesylate (Gleevec) is efficacious in
GIST patients with activating mutations in the juxtamembrane (JM)
domain of c-kit.
[0118] The presence of Bcr-Ab1 in a sample from a subject can be
used to determine whether to recommend imatinib mesylate (Gleevec)
as a drug treatment option to the subject, e.g., a chronic myeloid
leukemia (CML) patient. For example, a patient with CML with the
Philadelphia chromosome (Philadelphia translocation) that produces
the Bcr-Ab1 protein, a tyrosine kinase, can be recommended to be
treated with imatinib mesylate (Gleevec). Approximately 95% of
people with CML have the Philadelphia chromosome. Imatinib mesylate
(Gleevec) can be recommended as an initial therapy for a CML
patient. Imatinib mesylate (Gleevec) can be recommended for use
while a CML patient is in the first phase of CML (the chronic
phase) (Kantarjian H. et al. (2002) NEJM 356:645-652). Imatinib
mesylate (Gleevec) can be recommended to a CML patient if
interferon alpha therapy fails (Druker B J et al. (2001) NEJM
344:1031-1037).
[0119] The status of PDGFR in a sample can be used to determine
whether to recommend imatinib mesylate (Gleevec) as a drug
treatment option to a subject, e.g., a cancer patient. Cancer cells
overexpressing platelet derived growth factor receptor (PDGFR), a
tyrosine kinase, or with activating mutations in PDGFR can be
targeted with imatinib mesylate (Gleevec) (Heinrich M C et al.
(2003) J. Clin. Oncol. 21:4342-4349). If a sample from a subject
overexpresses PDGFR, or expresses PDGFR with activating mutations,
a recommendation can be made to treat the subject with imatinib
mesylate (Gleevec). If a sample from a subject does not overexpress
PDGFR, a recommendation can be made to not treat the subject with
imatinib mesylate (Gleevec).
[0120] A subject with renal cell carinoma (RCC) or
imatinib-resistant GIST that expresses the marker PDGFR can be
treated with Sutent (Sunitib or SU11248), a receptor tyrosine
kinase inhibitor. If a sample from a subject with RCC or
imatinib-resistant GIST expresses PDGFR, a recommendation can be
made to treat the subject with Sutent (Sunitib).
[0121] The status of secreted protein acidic and rich in cysteine
(SPARC; also known as ON, osteonectin) in a sample can be used to
determine whether to recommend Abraxane as a drug treatment option
for a subject, e.g., a metastatic breast cancer patient. SPARC is a
matrix-associated protein that can elicit alterations in cell
shape, inhibit cell-cycle progression, and influence the synthesis
of the extracellular matrix (ECM). Pre-clinical studies indicate
that a patient with a tumor expressing SPARC, which binds albumin,
can be targeted with a paclitaxel albumin-stabilized nanoparticle
formulation (Abraxane) and that Abranxe may have a higher level of
efficacy in these patients compared with patients whose tumors do
not express SPARC. If a sample from a subject expresses SPARC, a
recommendation can be made to treat the subject with Abraxane. If a
sample from a subject does not express SPARC, a recommendation can
be made to not treat the subject with Abraxane. Abraxane is
indicated for the treatment of breast cancer after failure of
combination chemotherapy for metastatic disease or relapse within 6
months of adjuvant chemotherapy.
[0122] The status of HSP90 (also known as HSPN; LAP2; HSP86; HSPC1;
HSPCA; Hsp89; HSP89A; HSP90A; HSP90N; HSPCAL1; HSPCAL4; FLJ31884;
HSP90AA1) in a sample can be used to determine whether to recommend
CNF2024 (BIIB021) as a drug treatment option for a subject. Hsp90
is a molecular chaperone that can facilitate the maturation and
stabilization of mutated, overexpressed and constitutively or
persistently active oncoproteins (Taldone T, Curr Opin Pharmacol.
(2008) August; 8(4):370-374). A patient with GIST expressing heat
shock protein 90 (HSP90) can be targeted with CNF2024 (BIIB021), an
oral inhibitor of HSP90. If a sample from a subject expresses
HSP90, a recommendation can be made to treat the subject with
CNF2024 (BIIB021). If a sample from a subject does not express
HSP90, a recommendation can be made to not treat the subject with
CNF2024 (BIIB021).
[0123] The status of MGMT (O-6-methylguanine-DNA methyltransferase)
promoter methylation in a sample can be used to determine whether
to recommend temozolomide (Temodar, Temodal) as a drug treatment
option for a subject, e.g., a glioblastoma patient. The MGMT gene
is located on chromosome 10q26. MGMT encodes a DNA-repair protein
that removes alkyl groups from the O6 position of guanine, a site
of DNA alkylation. O6-methylguanine lesions induced by chemotherapy
that remain unrepaired can trigger cytotoxicity and apoptosis. High
levels of MGMT activity in cancer cells can reduce the therapeutic
effect of an alkylating agent. Loss of MGMT expression and reduced
DNA-repair activity are associated with epigenetic silencing of the
MGMT gene by promoter methylation. Temozolomide (Temodar, Temodal)
is an alkylating agent. Data suggest that MGMT promoter methylation
is associated with a favorable outcome after temozolomide
chemotherapy in patients with recently diagnosed glioblastoma (Hegi
M E et al. (2005) NEJM 352:997-1003). If a sample from a subject
has MGMT promoter methylation, a recommendation can be made to
treat the subject with temozolomide. If a sample from a subject
does not have MGMT promoter methylation, a recommendation can be
made to not treat the subject with temozolomide.
[0124] The status of HER2 (also known as ERBB2, NEU, NGL, TKR1,
CD340, HER-2, HER-2/neu) in a sample can be determined by, e.g.,
IHC, PCR, SISH/CISH/FISH, and can be used to determine whether to
recommend trastuzumab (Herceptin) as a drug treatment option for a
subject, e.g., a breast cancer patient. HER2 is an epidermal growth
factor receptor. HER2 can bind to other ligand-bound EGF receptor
family members to form a heterodimer. This binding can stabilize
ligand binding and enhance kinase-mediated activation of downstream
signaling pathways, such as those involving mitogen-activated
protein kinase and phosphatidylinositol-3 kinase. Amplification
and/or overexpression of HER2 is reported in numerous cancers,
including breast and ovarian tumors. Breast cancer patients that
overexpress the HER2 receptor can be treated with trastuzumab
(Herceptin), a monoclonal antibody that can bind to the domain IV
of the extracellular segment of HER2. Herceptin is approved for
adjuvant treatment of HER2-overexpressing metastatic breast cancer.
As a single agent, Herceptin is approved for treatment of
HER2-overexpressing breast cancer in patients who have received one
or more chemotherapy regimens for metastatic disease. If a sample
from a subject (e.g., a breast cancer patient) overexpresses HER2,
a recommendation can be made to treat the subject with trastuzumab
(Herceptin). If a sample from a subject (e.g., a breast cancer
patient) does not overexpress HER2, a recommendation can be made to
not treat the subject with trastuzumab (Herceptin).
[0125] The status of human epidermal growth factor receptor 1 (also
known as HER1, EGFR, ERBB, mENA, ERBB1, PIG61) in a sample can be
used to determine whether to recommend Erlotinib (Tarceva) as a
drug treatment option for a subject. EGFR is a transmembrane
glycoprotein receptor for epidermal growth factor family members.
Binding of the EGFR to a ligand induces receptor dimerization and
tyrosine autophosphorylation and leads to cell proliferation.
Mutations in HER1 are associated with lung cancer. Erlotinib
(Tarceva) binds to the ATP binding site of the EGFR tyrosine
kinase. Tarceva monotherapy is indicated for the treatment of
patients with locally advanced or metastatic non-small cell lung
cancer after failure of at least one prior chemotherapy regimen.
Tarceva in combination with gemcitabine is indicated for the
first-line treatment of patients with locally advanced,
unresectable or metastatic pancreatic cancer. Erlotinib (Tarceva)
is efficacious in patients with pancreatic cancer and non-small
cell lung cancer that are positive by immunohistochemistry (IHC)
for EGFR. If a sample from a subject expresses EGFR, erlotinib
(Tarceva) can be recommended as a drug treatment option for the
subject. If a sample from a subject does not express EGFR, a
recommendation can be made to not treat the subject with erlotinib
(Tarceva).
[0126] The status of vascular endothelial growth factor (VEGF) can
be used to determine whether to recommend Bevacizumab (Avastin) as
a drug treatment option. VEGF is a glycosylated mitogen that can
act on endothelial cells. VEGF can mediate increased vascular
permeability, inducing angiogenesis, vasculogenesis and endothelial
cell growth, promote cell migration, and inhibit apoptosis.
Bevacizumab (Avastin) is a recombinant humanized monoclonal
antibody directed to VEGF. Bevacizumab (Avastin) can block tumor
growth by inhibiting the formation of new blood vessels.
Bevacizumab (Avastin) is indicated for first or second-line
treatment of patients with metastatic carcinoma of the colon or
rectum in combination with intravenous 5-fluorouracil-based
chemotherapy. Bevacizumab (Avastin) is also indicated for the
first-line treatment of unresectable, locally advanced, recurrent
or metastatic non-squamous non-small cell lung cancer in
combination with carboplatin and paclitaxel. Bevacizumab (Avastin)
is also indicated for the treatment of patients who have not
received chemotherapy for metastatic HER2-negative breast cancer in
combination with paclitaxel. Bevacizumab (Avastin) is indicated for
the treatment of metastatic renal cell carcinoma in combination
with interferon alpha. If a sample from a subject expresses VEGF,
Bevacizumab (Avastin) can be recommended as a drug treatment
option. If a sample from a subject does not express VEGF, a
recommendation can be made to not treat the subject with
Bevacizumab (Avastin).
[0127] The status of ER (also known as estrogen receptor; ESR; Era;
ESRA; NR3A1; DKFZp686N23123; ESR1) in a sample from a subject can
be determined by, e.g., IHC, and can be used to determine whether
to recommend hormonal therapeutics to the subject, e.g., a subject
with breast cancer. ER is a ligand-activated transcription factor
that includes domains for hormone binding, DNA binding, and
activation of transcription. ER can localize to the nucleus and can
form a homodimer or a heterodimer with estrogen receptor 2. If a
sample, e.g., a biopsy from a breast cancer patient, is analyzed by
IHC and receives a score that establishes it as ER-positive, a
recommendation can be made to treat the subject with hormonal
therapeutics. If the sample from a subject (e.g., a breast cancer
patient) is determined to be ER-negative, a recommendation can be
made to not treat the subject with hormonal therapeutics.
[0128] The status of PR (also known as progesterone receptor;
NR3C3; PGR) in a sample from a subject can be determined by, e.g.,
IHC, and can be used to determine whether to recommend hormonal
therapeutics to the subject, e.g., a subject with breast cancer. PR
is a member of the steroid receptor superfamily and mediates the
physiological effects of progesterone. If a sample, e.g., a biopsy
from a breast cancer patient, is analyzed by IHC and receives a
score that establishes it as PR-positive, a recommendation can be
made to treat the subject with hormonal therapeutics. If the sample
from a subject (e.g., a breast cancer patient) is determined to be
PR-negative (by, e.g., IHC), a recommendation can be made to not
treat the subject with hormonal therapeutics.
[0129] The expression status of 21 genes in a sample from a subject
(e.g., a breast cancer patient) can be determined using the
Oncotype Dx assay (Genomic Health Inc.), which involves reverse
transcription followed by RT-PCR of FFPE samples from primary
breast cancer. The levels of expression are used to calculate an RS
(Recurrence Score.RTM.), which can be used to assign a patient to a
treatment group. The score generated from the analysis can be used
to determine whether to recommend tamoxifen as a drug treatment
option for a breast cancer patient. Tamoxifen is a selective
estrogen receptor modulator (SERM). The Oncotype Dx test can be
used after surgery (e.g., lumpectomy or mastectomy) but before a
decision on adjuvant therapy is made.
[0130] The status of the PAM 50 expression signature in sample from
a subject can be determined by, e.g., RT-PCR, and can be used to
determine whether to recommend endocrine therapy for the subject,
e.g., a breast cancer patient (Parker J S et al. (2009) J Clin
Oncol. 27:1160-1167). The 50-gene PAM50 subtype predictor can be
used to assign intrinsic subtypes to tumor samples and can be used
to assess the likelihood of efficacy from neoadjuvant
chemotherapy.
[0131] The status of vras and Kras in a sample from a subject can
be used to determine whether to recommend bevacizumab (Avastin) as
a drug treatment option to the subject, e.g., a colon cancer
patient or a patient with metastatic colon cancer. (Grothey A and
Turja J H (2008) http://cme.medscape.com/viewarticle/577606).
[0132] Capecitabine (Xeloda) can be recommended as an adjuvant to a
subject with colorectal cancer Stage III (Dukes' C) colon cancer
(Twelves C. et al. (2005) NEJM 352:2696-2704) or as a first-line
monotherapy for metastatic colorectal cancer. Capecitabine (Xeloda)
is a prodrug that can be converted to 5-fluorouracil (5-FU) in a
tumor. 5-FU inhibits DNA synthesis, thereby slowing tumor
growth.
[0133] The status of a VeriStrat.RTM. (Biodesix) serum protein
signature in a sample from a subject can be determined by, e.g.,
matrix-assisted laser desorption ionization time of flight mass
spectrometry (MALDI-TOF MS), and can be used to determine whether
to recommend cetuximab (Erbitux) as a drug treatment option for the
subject, e.g., a colon cancer patient. The VeriStrat.RTM. test can
predict the response to EGFR tyrosine kinase inhibitors (e.g.,
gefitinib or erlotinib) for patients with non-small cell lung
cancer (NSCLC), and can be used to determine whether to recommend
erlobtinib (Tarceva) or gefitinib (Iressa) to a subject, e.g., a
metastatic lung cancer patient, non-small-cell lung cancer patient,
adenocarinoma (ADC) patient, or squamous cell carcinoma (SCC)
patient. Erlotinib (Tarceva) can target the epidermal growth factor
receptor (EGFR) tyrosine kinase.
[0134] The status of EGFR expression in a sample from a subject can
be used to determine whether to recommend cetuximab (Erbitux) as a
drug treatment option for a subject, e.g., a colon cancer patient
with an irinotecan resistant tumor (irinotecan (Camptosar) is a
topoisomerase 1 inhibitor). For example, if a sample from a subject
is determined to be an EGFR-expressing, metastatic colorectal
carcinoma and the subject is refractory to irinotecan-based
chemotherapy, a recommendation can be made to treat the subject
with cetuximab (Erbitux).
[0135] The status of TOPO1 (also known as DNA topoisomerase; TOPI;
TOP1) expression in a sample from a subject can be determined by,
e.g., IHC, and can be used to determine whether to recommend
fluorouracil (5-FU; F5U; Adrucil) with or without irinotecan or
oxaliplatin for the subject, e.g., a colon cancer patient (Braun M.
S. et al. (2008) J Clin Oncol. 26:2690-2698). TOPO1 can alter the
topologic states of DNA by catalyzing the transient breaking and
rejoining of a single strand of DNA which allows the strands to
pass through one another. Fluorouracil (Adrucil) is a pyrimidine
analog that can act as an inhibitor of thymidylate synthase, which
makes thymidine for DNA replication. If a biopsy from a colon
cancer patient is determined to have low TOPO1 expression by, e.g.,
IHC, 5-FU (Adrucil) can be recommended as a drug treatment option
without irinotecan or oxaliplatin. If a biopsy from a colon cancer
patient is determined to have moderate or high TOPO1 expression by,
e.g., IHC, 5-FU (Adrucil) treatment with irinotecan or oxaliplatin
can be recommended as a drug treatment option for the colon cancer
patient.
[0136] The status of TOPO1 in a sample from a subject can be used
to determine whether to recommend irinotecan (Camptosar) as a drug
treatment option for the subject, e.g., a metastatic colon cancer
patient. Irinotecan can be recommended to a metastatic colon cancer
patient after recurrence of colon cancer or progression after 5-FU
therapy.
[0137] The status of Phosphatase and Tensin Homolog (PTEN) in a
sample from a subject can be used to determine whether to recommend
cetuximab (Erbitux) or panitumumab (Vectibix) for the subject,
e.g., a metastatic colorectal cancer patient (Frattini M. et al.
(2007) Br J Cancer 97:1139-1145). PTEN can regulate the cell cycle.
PTEN dephosphorylates phosphoinositide substrates. PTEN can
regulate the intracellular levels of
phosphatidylinositol-3,4,5-triphosphate and can act as a tumor
suppressor by negatively regulating the Akt/PKB signalling pathway.
If a sample from a subject expresses PTEN, a recommendation can be
made to treat the subject with cetuximab (Erbitux) or panitumumab
(Vectibix). If a sample from a subject does not express PTEN, a
recommendation can be made to not treat the subject with cetuximab
(Erbitux) or panitumumab (Vectibix).
[0138] The status of PIK3CA in a sample from a subject can be used
to determine whether to recommend cetuximab (Erbitux) or
panitumumab (Vectibix) for the subject, e.g., a metastatic
colorectal cancer patient (Satore-Bianchi A et al. (2009) Cancer
Res 69:1851-1857). PIK3CA encodes the 110 kDa catalytic subunit or
phosphatidylinositol 3-kinase (which is also composed of an 85 kDa
regulatory subunit). The catalytic subunit can phosphorylate
PtdIns, PtdIns4P and PtdIns(4,5)P2. Membrane localization of PIK3CA
inhibits PTEN and promotes AKT1 phosphorylation. If a sample from a
subject has wild-type PIK3CA, a recommendation can be made to treat
the subject with cetuximab (Erbitux) or panitumumab (Vectibix). If
a sample from a subject has mutations in PIK3CA, a recommendation
can be made to not treat the subject with cetuximab (Erbitux) or
panitumumab (Vectibix).
[0139] Leucovorin (Wellcovorin, folinic acid) can be recommended as
a drug treatment option for a subject, e.g., a colon cancer
patient, in combination with 5-FU to prolong survival and promote
palliative care. Leucovorin enhances the binding of 5-FU to
thymidylate synthase and as a result prolongs the life span of
5-FU. This results in an anti-cancer effect of 5-FU.
[0140] Levamisole (Ergamisol) can be recommended as a drug
treatment option for a subject, e.g., a patient with Dukes' Stage C
colon cancer, in combination with 5-FU after surgical
resection.
[0141] Oxaliplatin (Eloxatin) can be recommended as a drug
treatment option for a subject, e.g., a patient with metastatic
colon cancer, in combination with 5-FU and leucovorin (LV).
Oxaliplatin (Eloxatin) is a platinum-based chemotherapy drug
thought to inhibit DNA synthesis. Oxaliplatin (Eloxatin) used in
combination with infusional 5-FU/LV is indicated for adjuvant
treatment of stage III colon cancer patients who have undergone
complete resection of the primary tumor and for treatment of
advanced carcinoma of the colon or rectum.
[0142] Celecoxib (Celebrex) can be recommended as a drug treatment
option for a subject, e.g., a colon cancer patient, a patient with
familial adenomatous polyposis (FAP), a patient with colonic
polyps, or a patient with colonic polyposis syndrome (FAPC)
(Bertagnolli M M et al. (2006) NEJM 355:873-884; Arber N et al.
(2006) NEJM 355:885-895). Celecoxib (Celebrex) is a nonsteroidal
anti-inflammatory drug (NSAID) that is a specific COX-2
inhibitor.
[0143] The status of Kras (also known as v-Ki-ras2 Kirsten rat
sarcoma viral oncogene homolog; NS3; KRAS1; KRAS2; RASK2; KI-RAS;
C-K-RAS; K-RAS2A; K-RAS2B; K-RAS4A; K-RAS4B) in sample from a
subject can be used to determine whether to recommend cetuximab
(Erbitux) as a drug treatment option to the subject, e.g., a colon
or rectal cancer patient with stage IV disease (COL-5, COL-9,
COL-10) (NCCN Clinical Practice Guidelines in Oncology). Kras is a
member of the small GTPase superfamily. Cetuximab (Erbitux), a
monoclonal antibody that targets the epidermal growth factor
receptor (EGFR), is efficacious in colorectal cancer patients with
wild-type Kras (Karapetis C S et al. NEJM 359, 1757-1765).
Cetuximab is indicated for treatment of locally or regionally
advanced squamous cell carcinoma of the head and neck in
combination with radiation therapy and recurrent or metastatic
squamous cell carcinoma of the head and neck progressing after
platinum-based therapy. If a sample from a subject has wild-type
Kras, a recommendation can be made to treat the subject with
cetuximab (Erbitux). If a sample from a subject has mutated Kras, a
recommendation can be made to not treat the subject with cetuximab
(Erbitux). If a sample from a subject has mutations in Kras in
codon 12 or 13, a recommendation can be made to not treat the
subject with cetuximab (Erbitux). A recommendation can be made to
treat a subject, e.g., a colon or rectal cancer patient, with
cetuximab (Erbitux) if the patient is not able to tolerate
cetuximab and irinotecan.
[0144] Irinotecan (Camptosar) can be recommended as a drug
treatment option for a subject, e.g., a colon or rectal cancer
patient (NCCN Clinical Practice Guidelines in Oncology). Irinotecan
(Camptosar) is indicated as a component of first-line therapy in
combination with 5-fluorouracil and leucovorin for patients with
metastatic carcinoma of the colon or rectum. Irinotecan (Camptosar)
is also indicated for patients with metastatic carcinoma of the
colon or rectum whose disease has recurred or progressed following
initial fluorouracil-based therapy.
[0145] The status of epidermal growth factor receptor (EGFR) can be
used to determine whether to recommend panitumumab (Vectibix) as a
drug treatment option to a subject, e.g., a metastatic colon or
rectal cancer patient. Panitumumab (Vectibix) is a human monoclonal
antibody that can target epidermal growth factor receptor (EGFR).
Panitumumab (Vectibix) is indicated as a single agent for the
treatment of EGFR-expressing metastatic colorectal carcinoma (mCRC)
with disease progression on or following fluoropyrimidine-,
oxaliplatin-, and irinotecan-containing chemotherapy regimens. If a
sample from a subject is determined to be an EGFR-expressing
metastatic colorectal cancer, panitumumab (Vectibix) can be
recommended as a drug treatment option for the subject. If a sample
from a subject is not an EGFR-expressing metastatic colorectal
cancer, a recommendation can be made to not treat the subject with
panitumumab (Vectibix).
[0146] The status of Kras in a sample from a subject can be used to
determine whether to recommend panitumumab (Vectibix) as a drug
treatment option for the subject, e.g., a metastatic colon or
rectal cancer patient with stage IV disease (COL-5, COL-9, COL-10)
(NCCN Clinical Practice Guidelines in Oncology). If a metastatic
colorectal cancer sample from a subject has wild-type Kras, a
recommendation can be made to treat the subject with panitumumab
(Vectibix). If a metastatic colorectal cancer sample from a subject
has mutated Kras, a recommendation can be made not to treat the
subject with panitumumab (Vectibix). If a metastatic colorectal
cancer sample from a subject has mutations in Kras in codons 12 or
13, a recommendation can be made to not treat the subject with
panitumumab (Vectibix). A recommendation can be made to treat a
subject, e.g., a colon or rectal cancer patient, with panitumumab
(Vectibix) if the subject is not able to tolerate cetuximab and
irinotecan.
[0147] Mitomycin C (Mitozytrex) can be recommended as a drug
treatment option for a subject, e.g., a gastrointestinal cancer
patient, for palliative treatment of disseminated adenocarcinoma of
the stomach or pancreas. Mitomycin C is a DNA crosslinker.
[0148] Cyclophosphamide (Neosar, Cytoxan) can be recommended as a
drug treatment option for treating a subject, e.g., a lung cancer
patient. Cyclophosphamide is a nitrogen mustard alkylating
agent.
[0149] The status of Nrf2 (also known as nuclear factor
(erythroid-derived 2)-like 2; NFE2L2) in a sample from a subject
can be used to determine whether to recommend doxorubicin
(Adriamycin) as a drug treatment option for the subject, e.g., a
lung cancer patient (Wang et al. (2008) Carcinogenesis
29:1235-1243). Overexpression of Nrf2 resulted in enhanced
resistance of cancer cells to doxorubicin (Adriamycin). NFE2,
NFE2L1, and NFE2L2 comprise a family of human genes encoding basic
leucine zipper (bZIP) transcription factors. Doxorubicin
(Adriamycin) can intercalate DNA. If a sample from a subject
overexpresses Nrf2, a recommendation can be made to not treat the
subject with doxorubicin (Adriamycin). If a sample from a subject
does not overexpress Nrf2, a recommendation can be made to treat
the subject with doxorubicin (Adriamycin).
[0150] The status of DPD (also known as dihydropyrimidine
dehydrogenase; DHP; DHPDHASE; MGC70799; MGC132008; DPYD) in a
sample from a subject can be determined by, e.g., tandem mass
spectrometry, and can be used to determine whether to recommend
fluorouracil (5-FU) as a drug treatment option for the subject,
e.g., a lung cancer patient or a non-small-cell lung cancer patient
(Nakano J et al. (2006) British Journal of Cancer 95:607-615). DPD
protein is a pyrimidine catabolic enzyme and the initial and
rate-limiting factor in the pathway of uracil and thymidine
catabolism. Mutations in DPD can result in dihydropyrimidine
dehydrogenase deficiency, an error in pyrimidine metabolism
associated with thymine-uraciluria and an increased risk of
toxicity in cancer patients receiving 5-fluorouracil chemotherapy.
If a sample from a subject is negative for DPD expression, a
recommendation can be made to treat the subject with 5-FU. If a
sample from a subject is positive for DPD expression, a
recommendation can be made to not treat the subject with 5-FU. 5-FU
is a pyrimidine analog that can act as a thymidylate synthase
inhibitor. A dose of 5-FU can be recommended based on the level of
DPD expression in a sample from a subject.
[0151] The status of OPRT (also known as uridine monophosphate
synthetase; UMPS uridine monophosphate synthase; OPRtase;
OMPdecase; UMP synthase; orotidine 5'-phosphate decarboxylase;
orotate phosphoribosyltransferase phosphoribosyltransferase;
orotate phosphoribosyl transferase; orotidine-5'-decarboxylase) in
a sample from a subject can be used to determine whether to
recommend 5-FU as a drug treatment option for the subject, e.g., a
lung cancer patient or a non-small-cell lung cancer patient (Nakano
J et al. (2006) British Journal of Cancer 95:607-615). For example,
if a sample from a subject is positive for OPRT expression, a
recommendation can be made to treat the subject with 5-FU. If a
sample from a subject is negative for OPRT expression, a
recommendation can be made to not treat the subject with 5-FU.
[0152] The status of TS (also known as thymidylate synthetase; TMS;
TSase; HsT422; MGC88736; TYMS) in a sample from a subject can be
used to determine whether to recommend 5-FU as a drug treatment
option for the subject, e.g., a lung cancer patient or a
non-small-cell lung cancer patient (Nakano J et al. (2006) British
Journal of Cancer 95:607-615; Cascinu S et al. (2001) Ann Oncol
2:239-244). Thymidylate synthase catalyzes the methylation of
deoxyuridylate to deoxythymidylate using
5,10-methylenetetrahydrofolate (methylene-THF) as a cofactor. This
function maintains the dTMP (thymidine-5-prime monophosphate) pool
critical for DNA replication and repair. TS is considered to be the
primary site of action for 5-fluorouracil,
5-fluoro-2-prime-deoxyuridine, and some folate analogs. If a sample
from a subject is negative for TS expression (or has low TS
expression), a recommendation can be made to treat the subject with
5-FU. If a sample from a subject is positive for TS expression (or
has high TS expression), a recommendation can be made to not treat
the subject with 5-FU.
[0153] Bevacizumab (Avastin) can be recommended as a drug treatment
option for a subject, e.g., a lung or NSCLC patient.
[0154] Docetaxel (Taxotere) can be recommended as a drug treatment
option for a subject, e.g., a metastatic lung cancer patient, a
non-small-cell lung cancer patient, a breast cancer patient, a
prostate cancer patient, a gastric cancer patient, or a head and
neck cancer patient. Docetaxel (Taxotere) an anti-mitotic that
stabilizes microtubules. Docetaxel (Taxotere) is indicated for the
treatment of patients with locally advanced or metastatic breast
cancer after failure of prior chemotherapy, and it is indicated in
combination with doxorubicin and cyclophosphamide for the adjuvant
treatment of patients with operable node-positive breast cancer.
Docetaxel (Taxotere) as a single agent is indicated for the
treatment of patients with locally advanced or metastatic non-small
cell lung cancer (NSCLC) after failure of prior platinum-based
chemotherapy. Docetaxel (Taxotere) in combination with cisplatin is
indicated for the treatment of patients with unresectable, locally
advanced or metastatic NSCLC who have not previously received
chemotherapy for this condition. Docetaxel (Taxotere) in
combination with prednisone is indicated for the treatment of
patients with androgen independent (hormone refractory) metastatic
prostate cancer. Docetaxel (Taxotere) in combination with cisplatin
and fluorouracil is indicated for the treatment of patients with
advanced gastric adenocarcinoma, including adenocarcinoma of the
gastroesophageal junction, who have not received prior chemotherapy
for advanced disease. Docetaxel (Taxotere) in combination with
cisplatin and fluorouracil is indicated for the induction treatment
of patients with locally advanced squamous cell carcinoma of the
head and neck (SCCHN).
[0155] The status of Kras in a sample from a subject can be used to
determine whether to recommend gefitinib (Iressa) as a drug
treatment option for the subject, e.g., a lung cancer patient or a
non-small-cell lung cancer patient (Carbone D P (2004) Nature
Clinical Practice Oncology 1:66-67). For example, if a sample from
a subject expresses wild-type Kras, a recommendation can be made to
treat the subject with gefitinib (Iressa). If a sample from a
subject expresses mutated Kras, a recommendation can be made to not
treat the subject with gefitinib (Iressa). If a sample from a
subject has mutations in Kras in exon 2, a recommendation can be
made to not treat the subject with gefitinib (Iressa) (Pao W et al.
(2005) PLoS Med 2(1): e17.doi:10.1371/journal.pmed.0020017).
Gefitinib (Iressa) is an anticancer drug that can inhibit tyrosine
kinase activity of EGFR.
[0156] Gemcitabine (Gemzar) can be recommended as a drug treatment
option for a subject, e.g., a lung cancer patient, a ovarian cancer
patient, a pancreatic cancer patient, or a NSCLC patient.
Gemcitabine (Gemzar) is a nucleoside analog in which the hydrogen
atoms on the 2' carbons of deoxycytidine are replaced by fluorine
atoms. Gemcitabine (Gemzar) can be recommended as a first line drug
treatment option for inoperable stage IIIA, IIIB, or metastatic
stage IV lung cancer. Gemcitabine (Gemzar) is indicated in
combination with carboplatin for a patient with ovarian cancer that
has returned at least 6 months after the patient had finished
platinum-based therapy. Gemcitabine (Gemzar) is indicated in
combination with cisplatin for the first-line treatment of patients
with locally advanced (Stage IIIA or Stage IIIB) or metastatic
(Stage IV or cancer that has spread) non-small cell lung cancer for
whom surgery is not possible. Gemcitabine (Gemzar) in combination
with paclitaxel is approved by the FDA for the first-line treatment
of patients with metastatic breast cancer after they have received
anthracycline, unless their medical condition did not allow them to
receive an anthracycline. Gemcitabine (Gemzar) is indicated as a
single agent first-line treatment for patients with locally
advanced (Stage II or Stage III when surgery is not an option) or
metastatic (Stage IV) adenocarcinoma of the pancreas. Gemcitabine
(Gemzar) is also indicated for pancreatic cancer patients
previously treated with 5-FU.
[0157] Vinorelbine (Navelbine) can be recommended as a drug
treatment option for a subject, e.g., a lung cancer patient, a
breast cancer patient, or NSCLC patient with and unresectable
advanced tumor. Vinorelbine (Navelbine) can be recommended as a
drug treatment option alone or in combination with cisplatin.
Vinorelbine (Navelbine) can be recommended as a drug treatment
option for a patient with Stage III or Stage IV lung cancer or
NSCLC. Vinorelbine (Navelbine) is a 5'NOR semi-synthetic vinca
alkaloid that can interfere with microtubule assembly.
[0158] The status of Kras or EGFR in a sample from a subject can be
used to determine whether to recommend erlotinib (Tarceva) as a
drug treatment option for the subject, e.g., a lung cancer patient,
non-small-cell lung cancer patient, adenocarinoma (ADC) patient, or
squamous cell carcinoma (SCC) patient (Pao W. et al. (2005) PLOS
Medicine 2(1):e17). For example if a sample has wild-type Kras, a
recommendation can be made to treat the subject with erlotinib
(Tarceva). If a sample has mutated Kras, a recommendation can be
made to not treat the subject with erlotinib (Tarceva). If a sample
has mutated EGFR, a recommendation can be made to treat the subject
with erlotinib (Tarceva). If a sample from a subject has mutations
in exons 18-21 of EGFR, a recommendation can be made to treat the
subject with erlotinib (Tarceva). If a sample has wild-type EGFR, a
recommendation can be made to not treat the subject with erlotinib
(Tarceva). Erlotinib (Tarceva) can be recommended as a first line
therapy.
[0159] A recommendation can be made to administer topotecan
(Hycamtin) to a subject, e.g., a lung cancer patient or a small
cell lung cancer patient, that has failed a first line of therapy
or has cancer progression after an initial response to chemotherapy
(O'Brien M E et al. (2006) J Clin Oncol 24:5441-5447). Topotecan
(Hycamtin) is a topoisomerase 1 inhibitor.
[0160] Pemetrexed disodium (Alimta) can be recommended as a drug
treatment option for a subject, e.g., a lung cancer patient or
non-small-cell lung cancer patient that has locally advanced or
metastatic cancer. Pemetrexed disodium is a member of the folate
antimetabolite class of chemotherapy drugs. Pemetrexed can inhibit
three enzymes used in purine and pyrimidine synthesis: thymidylate
synthase (TS), dihydrofolate reductase (DHFR), and glycinamide
ribonucleotide formyltransferase (GARFT). Pemetrexed for injection
(Alimta) is approved by the FDA in combination with cisplatin for
the initial treatment of advanced nonsquamous non-small cell lung
cancer (NSCLC). Pemetrexed for injection (Alimta) as a single agent
is approved for maintaining the initial treatment effect of
chemotherapy in patients with advanced nonsquamous non-small cell
lung cancer whose disease has not worsened after initial therapy.
Pemetrexed for injection (Alimta) is approved by the FDA as a
single agent for the treatment of patients with advanced
nonsquamous non-small cell lung cancer (NSCLC) after prior
chemotherapy. Pemetrexed for injection (Alimta) is a treatment for
malignant pleural mesothelioma (MPM), a cancer that affects the
inside lining of the chest cavity. Pemetrexed for injection
(Alimta) can be given with cisplatin when surgery is not an
option.
[0161] Porfimer sodium (Photofrin) can be recommended as a drug
treatment option for a subject, e.g., a lung cancer patient or
non-small-cell lung cancer patient who is undergoing photodynamic
therapy. Porfimer sodium is a sensitizer that can be administered
intravenously for use in photodynamic therapy. Photofrin is
indicated for palliation of patients with completely obstructing
esophageal cancer, or of patients with partially obstructing
esophageal cancer who, in the opinion of their physician, cannot be
satisfactorily treated with Nd:YAG laser therapy. Photofrin is
indicated for treatment of microinvasive endobronchial
non-small-cell lung cancer (NSCLC) in patients for whom surgery and
radiotherapy are not indicated. Photofrin is indicated for
reduction of obstruction and palliation of symptoms in patients
with completely or partially obstructing endobronchial NSCLC.
[0162] Paclitaxel (Taxol) can be recommended as a drug treatment
option for a subject, e.g., a lung cancer patient or non-small-cell
lung cancer patient, as a first line therapy and/or in combination
with cisplatin. Paclitaxel is a mitotic inhibitor that stabilizes
microtubules.
[0163] Bleomycin (Blenoxane) can be recommended as a drug treatment
option for a subject, e.g., a patient with pleural effusion or
malignant pleural effusion (MPE), the accumulation of fluid in the
pleural space that can result from cancer metastasis. Bleomycin
(Blenoxane) can be recommended to be used in the management of the
following conditions as either a single agent or in combination
with other chemotherapeutic agents: squamous cell carcinoma (head
and neck, including: mouth, tongue, tonsil, nasopharynx,
oropharynx, sinus, palate, lip, buccal mucosa, gingivae,
epiglottis, skin, larynx; penis, cervix, and vulva); Hodgkin's
Disease and non-Hodgkin's lymphoma; and testicular carcinoma
(embryonal cell, choriocarcinoma, and teratocarcinoma). Bleomycin
(Blenoxane) is a glycopeptide that can induce DNA strand
breaks.
[0164] Leucovorin (folinic acid, Wellcovorin) can be recommended as
a drug treatment option for a subject, e.g., a prostate cancer
patient, in combination with 5-FU to prolong survival or provide
palliative care.
[0165] The status of EGFR can be determined by, e.g., sequencing or
detection of expression, and be used to determine whether to
recommend lapatinib as a drug treatment option (Trowe T. et al.
(2008) Clinical Cancer Research 14:2465-2475). Lapatinib is a
tyrosine kinase inhibitor. If a sample overexpresses EGFR, a
recommendation can be made to treat the subject with lapatinib. If
a sample does not overexpress EGFR, a recommendation can be made to
not treat the subject with lapatinib.
[0166] The status of ErbB2 (also known as HER2, NEU, NGL, TKR1,
CD340, HER-2, HER-2/neu) can be used to determine whether to
recommend leucovorin as a drug treatment option for a subject,
e.g., a cancer patient (NCCN).
[0167] The status of TOPO1 can be determined by, e.g., IHC, and can
be used to determine whether to recommend oxaliplatin as a drug
treatment option for a subject, e.g., a colon cancer patient (Braun
M. S. et al. (2008) J Clin Oncol 26:2690-2698). Oxaliplatin is
thought to inhibit DNA synthesis.
[0168] Etoposide phosphate (Etopophos) can be recommended as a drug
treatment option for a subject, e.g., a lung cancer or small cell
lung cancer patient. Etoposide phosphate is an inhibitor of
topoisomerase II.
[0169] In the methods of the provided invention, one or more
recommendations in a report for treating or not treating a subject
with one or more drugs can be made based on one or more
correlations between one or more molecular markers of a particular
status and one or more drugs. A recommendation in a report (e.g.,
treat with a drug or do not treat with a drug) can be made based on
the subject's type of cancer.
[0170] C. Examples of Marker/Drug Combinations for Colon Cancer
[0171] In the methods of the provided invention a drug can be
recommended as a treatment option (first class or second class) or
recommended not to be used as a treatment option (third class)
based on the status of one or more molecular markers in a sample in
a subject, e.g., a colon cancer patient.
[0172] The status of c-kit (CD117) in a sample from a colon cancer
patient can be determined by, e.g., flow cytometry and/or
sequencing, and imatinib mesylate (Gleevec) can be recommended for
the subject based on the status of c-kit. For example, if flow
cytometry indicates the expression of c-kit in the sample, and the
protein sequence has key mutations, imatinib mesylate (Gleevec) can
be recommended for the subject as a drug treatment option. If a
sample from a colon cancer patient does not express c-kit or have
c-kit with key mutations, a recommendation can be made to not treat
the subject with imatinib mesylate (Gleevec).
[0173] The status of the Kras in a sample from a subject can be
determined by, e.g., sequencing, and cetuximab (Erbitux) or
panitumumab (Vectibix) monotherapy can be recommended for the
subject based on the status of the Kras sequence. For example, if
the sample from a colon cancer patient has a wild type Kras
sequence (e.g., as determined by exon 2 genotyping or sequencing),
cetuximab (Erbitux) or panitumumab (Vectibix) monotherapy can be
recommended as a drug treatment option for the colon cancer
patient. If the sample from a colon cancer patient has a mutant
Kras sequence (e.g., as determined by exon 2 genotyping or
sequencing), a recommendation can be made not to treat the colon
cancer patient with cetuximab (Erbitux) or panitumumab (Vectibix)
monotherapy.
[0174] The status of BRAF in a sample from a colon cancer patient
can be determined by, e.g., sequencing, and cetuximab (Erbitux) or
panitumumab (Vectibix) can be recommended to a subject based on the
status of BRAF (Nicolantonio D et al. (2008) J. Clin. Oncol.
26:5705-5712; Cappuzzo F et al. (2008) Br J Cancer 99:83-89). For
example, if a subject does not have a V600E mutation in its BRAF
sequence (e.g., as determined by V600E genotyping or sequencing),
cetuximab (Erbitux) or panitumumab (Vectibix) can be recommended as
a drug treatment option. If a subject has a V600E mutation in its
BRAF sequence (e.g., as determined by V600E genotyping or
sequencing), then a recommendation can be made not to treat the
patient with cetuximab (Erbitux) or Panitumumab (Vectibix).
Treatment of subjects with the BRAF V600E mutation with the BRAF
inhibitor sorafenib can restore sensitivity to cetuximab (Erbitux)
or panitumumab (Vectibix) monotherapy. If a sample from a subject
has a V600E mutation in its BRAF sequence, then a recommendation
can be made to treat the subject with sorafenib and cetuximab
(Erbitux) and/or panitumumab (Vectibix).
[0175] The presence or absence of microsatellite stability in a
sample from a subject, e.g., a colon cancer patient, can be used to
determine whether or not to recommend fluorouracil-based adjuvant
chemotherapy to the subject. Fluorouracil-based adjuvant
chemotherapy benefited patients with stage II or stage III colon
cancer with microsatellite-stable tumors or tumors exhibiting
low-frequency microsatellite instability but not those with tumors
exhibiting high-frequency microsatellite instability (Ribic C M et
al. (2003) NEJM 349:247-257). If a sample from a subject with stage
II or stage III colon cancer has a microsatellite-stable tumor or a
tumor exhibiting low-frequency microsatellite instability, a
recommendation can be made to treat the subject with
fluorouracil-based adjuvant chemotherapy. If a subject with stage
II or stage III colon cancer has a tumor that exhibits
high-frequency microsatellite instability, a recommendation can be
made not to treat the subject with fluorouracil-based adjuvant
chemotherapy. Microsatellite stability can be determined by DNA
sequencing.
[0176] The status of EGFR copy number in a sample from a subject,
e.g. a colon cancer patient, can be determined by, e.g., FISH or
qPCR, and can be used to determine whether to recommend cetuximab
(Erbitux) or panitumumab (Vectibix) to the subject. An increased
copy number of EGFR correlates with a good response to cetuximab
(Erbitux) or panitumumab (Vectibix) monotherapy (Cappuzzo F et al.
(2007) Annals of Oncology 19:717-723). If a sample from a subject
has an increased copy number of EGFR, a recommendation can be made
to treat the subject with cetuximab (Erbitux) or panitumumab
(Vectibix) monotherapy.
[0177] The status of the 18q chromosome in a sample from a subject,
e.g., a colon cancer patient, can be determined by, e.g., qPCR, and
can be used to determine whether to treat the subject with adjuvant
therapy (Jen J. et al. (1994) NEJM 331:213-221). The status of
chromosome 18q has prognostic value in patients with stage II
colorectal cancer. Stage II colorectal cancer patients with
chromosome 18q allelic loss have a prognosis that is similar to
that in patients with stage III cancer, who are thought to benefit
from adjuvant therapy. In contrast, stage II colorectal cancer
patients who do not have chromosome 18q allelic loss in their tumor
have a survival rate similar to that of patients with stage I
disease and may not require additional therapy. If a sample from a
subject (e.g., a stage II colon cancer patient) has chromosome 18q
allelic loss, then a recommendation can be made to treat the
subject with adjuvant therapy as if the patient were a stage III
cancer patient. If a sample from a subject (e.g., a stage II colon
cancer patient) does not have chromosome 18q allelic loss, a
recommendation can be made to not treat the subject with adjuvant
therapy.
[0178] The status of thymidylate synthase levels in a sample from a
subject, e.g., a colon cancer patient, can be determined by, e.g.,
IHC, and can be used to determine whether to treat the subject with
5-FU-based chemotherapy (Elder D. (2002) J. Clin. Oncol.
20:1721-1728; Cascinu S et al. (2001) Ann Oncol 2:239-244;
Ciaparrone M. et al. (2006) Oncology 70:366-377).
[0179] If a sample from a subject (e.g., a colon cancer patient)
has a high TS level (as determined by, e.g., IHC), a recommendation
can be made to treat or to not treat the subject with adjuvant
5-FU-based chemotherapy. If a sample from a subject (e.g., a colon
cancer patient) has a low TS level, a recommendation can be made to
treat or to not treat the subject with adjuvant 5-FU-based
chemotherapy.
[0180] The status of Topo1 expression levels in a sample from a
subject, e.g., a colon cancer patient, can be determined by, e.g.,
IHC, and can be used to determine whether to treat the subject with
irinotecan. Progression-free survival (PFS) was not improved in
patients with low Topo1 by the addition of irinotecan, but patients
with moderate/high Topo1 benefited from the addition of irinotecan
(Braun M S et al. (2008) J. Clin. Oncol. 26:2690-2698). If a sample
from a subject has low Topo1 expression, then a recommendation can
be made to not treat the subject with irinotecan. If a sample from
a subject has moderate to high Topo1 expression, then a
recommendation can be made to treat the subject with
irinotecan.
[0181] The status of Kras in a sample from a colon cancer patient
can be determined by, e.g., sequencing, and bevacizumab (Avastin)
can be recommended as a drug treatment option based on the status
of Kras.
[0182] The status of TOPO1 in a sample from a colon cancer patient
can be determined by, e.g., immunohistochemistry (IHC) and/or
sequencing, and can be used to determine whether to recommend 5-FU
or capecitabine (Xeloda) as a drug treatment option. For example,
if TOPO1 expression can be detected by IHC or if TOPO1 has a
certain sequence, then capecitabine (Xeloda), or fluorouracil
(5-FU) with or without irinotecan (Camptosar), can be recommended
as a drug treatment option.
[0183] Combinations of markers/targets, drug/therapeutics, and
cancers can be found in FIG. 6.
[0184] D. Drugs with Inhibitors of DNA Synthesis
[0185] Other cancer drug therapies that can be recommended to a
subject include, for example, FOLFOX. FOLFOX is a chemotherapy
regimen that can be used to treat colorectal cancer. The FOLFOX
regimen includes folinic acid (leucovorin), fluorouracil (5-FU),
and oxaliplatin (Eloxatin). 5-FU can act as an inhibitor of
thymidylate synthase, which can block synthesis of thymidine, which
can affect DNA replication. Capecitabine (Xeloda) is a prodrug that
can be converted enzymatically to 5-FU.
[0186] Capecitabine (Xeloda) can be used to treat metastatic breast
cancer and colorectal cancer. Irinotecan (Camptosar) is an
inhibitor of topoisomerase 1 that can be used for treatment of
colon cancer. FOLFIRI is a chemotherapy regimen that can be used to
treat colorectal cancer. The FOLFIRI regimen includes folinic acid
(leucovorin), fluorouracil (5-FU), irinotecan (Camptosar). CapeOx
is a chemotherapy regimen that includes capecitabine (Xeloda) and
oxaliplatin (Eloxatin).
[0187] E. Patient Metabolism
[0188] The stratifying of cancer drug treatments can take into
account the role of genes that affect drug absorption,
distribution, metabolism, and excretion (the pharmacokinetics and
pharmacodynamics of the drug treatment). The stratifying of drug
treatments can take into account whether the subject or patient is
hypermetabolic. The stratifying of drug treatments can take into
account the cytochrome P450 (CYP450) status of said subject or
patient. The CYP450 status of a subject or a patient can be
determined, for example, from assessments made by genotyping genes
encoding CYP450 (e.g., CYP2D6, CYP2C19, and CYP2C9). For example,
the CYP450 status can be assessed using the Roche.RTM. AmpliChip
CYP450 Test, which tests for gene variations in CYP2D6 and CYP2C19.
The stratifying of one or more drug treatment options can be based
on tests for defects in the enzyme thiopurine methyltransferase
(TMPT), which can prevent metabolism of the anti-cancer drug
6-mercaptopurine (6MP). Results from Affymetrix.RTM. Drug
Metabolizing Enzymes and Transporters (DMET)--Early Access
Solution, The Human1M BeadChip from Illumina.RTM., Human1M-Duo DNA
Analysis BeadChip from Illumina.RTM., and HumanExon510S-Duo DNA
Analysis BeadChip from Illumina.RTM., can be used to stratify the
cancer drug treatment options.
[0189] F. Sources of Drug Treatment Options
[0190] The list of drug treatments to be stratified can be compiled
from information sources available to those skilled in the art. The
drugs can be described in scientific literature, and the drugs can
be those used in unpublished clinical trials
(http://clinicaltrials.gov/). The drugs can be listed in the
Centers for Medicare and Medicaid Services (CMS) anti-cancer
treatment compendia for determining which drugs may be covered
under Medicare Part B to treat cancer patients, including the
National Comprehensive Cancer Network (NCCN) Drugs and Biologics
Compendium.TM., Thomson Micromedex DrugDex.RTM., and Elsevier Gold
Standard's Clinical Pharmacology compendium, and American Hospital
Formulary Service--Drug Information Compendium. The drugs can be
those listed for a specific cancer on the NCCN Clinical Practice
Guidelines in Oncology.TM. website or in the American Society of
Clinical Oncology (ASCO) clinical practice guidelines.
[0191] G. Types of Cancers
[0192] The NCCN Clinical Practice Guidelines in Oncology.TM.
website provides clinical practice guidelines for treating a
variety of cancers. These clinical practice guidelines can be
downloaded. The guidelines provide information regarding care
options that take into account risks and benefits associated with a
procedure.
[0193] The condition for which one or more drug treatment options
can be stratified and/or annotated can include a cancer listed on
the NCCN website. The conditions or cancers can include, for
example, acute myeloid leukemia; bladder cancer, including upper
tract tumors and urothelial carcinoma of the prostate; bone cancer,
including chondrosarcoma, Ewing's sarcoma, and osteosarcoma; breast
cancer, including noninvasive, invasive, phyllodes tumor, Paget's
disease, and breast cancer during pregnancy; central nervous system
cancers, adult low-grade infiltrative supratentorial
astrocytoma/oligodendroglioma, adult intracranial ependymoma,
anaplastic astrocytoma/anaplastic oligodendroglioma/glioblastoma
multiforme, limited (1-3) metastatic lesions, multiple (>3)
metastatic lesions, carcinomatous lymphomatous meningitis,
nonimmunosuppressed primary CNS lymphoma, and metastatic spine
tumors; cervical cancer; chronic myelogenous leukemia (CML); colon
cancer, rectal cancer, anal carcinoma; esophageal cancer; gastric
(stomach) cancer; head and neck cancers, including ethmoid sinus
tumors, maxillary sinus tumors, salivary gland tumors, cancer of
the lip, cancer of the oral cavity, cancer of the oropharynx,
cancer of the hypopharynx, occult primary, cancer of the glottic
larynx, cancer of the supraglottic larynx, cancer of the
nasopharynx, and advanced head and neck cancer; hepatobiliary
cancers, including hepatocellular carcinoma, gallbladder cancer,
intrahepatic cholangiocarcinoma, and extrahepatic
cholangiocarcinoma; Hodgkin disease/lymphoma; kidney cancer;
melanoma; multiple myeloma, systemic light chain amyloidosis,
Waldenstrom's macroglobulinemia; myelodysplastic syndromes;
neuroendocrine tumors, including multiple endocrine neoplasia, type
1, multiple endocrine neoplasia, type 2, carcinoid tumors, islet
cell tumors, pheochromocytoma, poorly differentiated/small
cell/atypical lung carcinoids; Non-Hodgkin's Lymphomas, including
chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular
lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse
large B-Cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma,
AIDS-Related B-Cell lymphoma, peripheral T-Cell lymphoma, and
mycosis fungoides/Sezary Syndrome; non-melanoma skin cancers,
including basal and squamous cell skin cancers, dermatofibrosarcoma
protuberans, Merkel cell carcinoma; non-small cell lung cancer
(NSCLC), including thymic malignancies; occult primary; ovarian
cancer, including epithelial ovarian cancer, borderline epithelial
ovarian cancer (Low Malignant Potential), and less common ovarian
histologies; pancreatic adenocarcinoma; prostate cancer; small cell
lung cancer and lung neuroendocrine tumors; soft tissue sarcoma,
including soft-tissue extremity, retroperitoneal, intra-abdominal
sarcoma, and desmoid; testicular cancer; thymic malignancies,
including thyroid carcinoma, nodule evaluation, papillary
carcinoma, follicular carcinoma, Hirthle cell neoplasm, medullary
carcinoma, and anaplastic carcinoma; uterine neoplasms, including
endometrial cancer and uterine sarcoma.
[0194] Medical information regarding cancers can be found at
websites, for example, www.cancer.gov, www.nexcura.cancer.com,
www.asco.org, and http://nccn.org/professionals/physician.
[0195] H. How Stratification is Indicated on a Report
[0196] The stratifying of drug treatment options can be indicated
by markings on a form downloaded from a website, for example, the
NCCN Clinical Practice Guidelines in Oncology.TM. or in the
American Society of Clinical Oncology (ASCO) clinical practice
guidelines, or on a printed report. For example, color coding of
drug treatment options can be used to indicate stratification of
drug treatment options on information downloaded or printed, for
example, from the NCCN Clinical Practice Guidelines in Oncology.TM.
website. The drug treatment options that can be stratified include
those found in the American Society of Clinical Oncology (ASCO)
clinical practice guidelines. The drug treatment options can be
stratified such that drug treatment options most likely to be
efficacious (first class; recommend the subject be treated with the
drug), based on the status of one of more molecular markers of the
sample or tumor, are highlighted or written in green or a shade of
green; drugs that may have some efficacy are highlighted or written
in yellow or a shade of yellow (second class; recommend the subject
be treated with the drug with some caution or caveat), and drugs
for which no information is available or for which the scientific
literature indicates the drug will not be efficacious or will be
toxic in light of the status of the subject's one or more molecular
markers are highlighted or written in red or a shade of red (third
class; recommend the subject not be treated with the drug). FIG.
2A-2C illustrate an example in which (FIG. 2A) a health care
provider accesses a report on a computer based on (FIG. 2B) the
NCCN Clinical Practice Guidelines in Oncology related to colon
cancer, and (FIG. 2C) drugs treatment options are classified
(stratified) by colors. FIG. 8 illustrates a printed hard copy
example of a report in which drug, target (molecular marker), and
status of a target (molecular marker) are indicated. Other
identifiers, such as number ranking, separate groups, asterisks,
etc., can be used to indicate stratification of the drug treatment
options for a condition.
[0197] The report can contain other information, including, e.g.,
type of assay or technique used for determining the status of a
molecular marker, different type of molecular marker status (e.g.,
wild-type or mutant sequence; e.g., DNA, RNA, or protein sequence;
high expression or low expression levels, e.g., mRNA expression or
protein expression; etc.). The report can contain information on a
drug dosing recommendation based on the status of one or molecular
markers.
[0198] The report with the stratified treatment options can be
provided by the personalized medicine business to a health care
professional (e.g., oncologist) and/or the subject for whom the
report is prepared. The report can be made available online or be
delivered in hardcopy form by a delivery service. An alert, such as
an email, text message, phone call, facsimile, etc. can be sent to
a subject or health care provider indicating that the report is
available. A fee can be charged by the personalized medicine
business in exchange for preparing and/or sending the report.
[0199] I. Validation of Tumor Markers
[0200] The methods of the provided invention can be used to
validate molecular markers. A molecular marker to be validated for
a particular cancer can be selected by analyzing the literature.
Stored samples from subjects whose treatment outcome is known
(retrospective sample) can be used to validate a molecular marker
of a condition, e.g., a cancer. Validating a tumor marker can
include determining a DNA sequence variation and correlating the
variation to RNA expression. Hundreds of markers can be validated
in parallel. Quantitative cellular heterogeneity can be used as a
measure of survival. A validation study can be a FFPE retrospective
validation study. A validation study can be a prospective
validation study. A validation study can be a multi-marker
retrospective correlation study.
V. Annotating Cancer Drug Treatment Options
[0201] A. Annotating with Information Regarding Experimental Drugs
and FDA-Approved Drugs for Off-Label Use
[0202] The methods of the provided invention can include obtaining
a sample, determining the status of one or more molecular markers,
stratifying one or more drug treatment options based on the status
of the one or more molecular markers, and annotating drug treatment
options in a report based on the status of the one or more
molecular markers. The annotated information can be used by a
health care provider to select other drug treatment options and/or
provide information about drug treatment options to an insurance
company. The method can include annotating the drug treatment
options for a condition in, for example, the NCCN Clinical Practice
Guidelines in Oncology.TM. or the American Society of Clinical
Oncology (ASCO) clinical practice guidelines.
[0203] The drug treatment options that are stratified in a report
can be annotated in the report by listing additional drug treatment
options. An additional drug treatment can be an FDA-approved drug
for an off-label use. A provision in the 1993 Omnibus Budget
Reconciliation Act (OBRA) requires Medicare to cover off-label uses
of anticancer drugs that are included in standard medical
compendia. The drugs used for annotating lists can be found in CMS
approved compendia, including the National Comprehensive Cancer
Network (NCCN) Drugs and Biologics Compendium.TM., Thomson
Micromedex DrugDex.RTM., Elsevier Gold Standard's Clinical
Pharmacology compendium, and American Hospital Formulary
Service--Drug Information Compendium.RTM..
[0204] The drug treatment options can be annotated by listing an
experimental drug that may be useful in treating a cancer with one
or more molecular markers of a particular status. The experimental
drug can be a drug for which in vitro data, in vivo data, animal
model data, pre-clinical trial data, or clinical-trial data are
available. The data can be published in peer-reviewed medical
literature found in journals listed in the CMS Medicare Benefit
Policy Manual, including, for example, American Journal of
Medicine, Annals of Internal Medicine, Annals of Oncology, Annals
of Surgical Oncology, Biology of Blood and Marrow Transplantation,
Blood, Bone Marrow Transplantation, British Journal of Cancer,
British Journal of Hematology, British Medical Journal, Cancer,
Clinical Cancer Research, Drugs, European Journal of Cancer
(formerly the European Journal of Cancer and Clinical Oncology),
Gynecologic Oncology, International Journal of Radiation, Oncology,
Biology, and Physics, The Journal of the American Medical
Association, Journal of Clinical Oncology, Journal of the National
Cancer Institute, Journal of the National Comprehensive Cancer
Network (NCCN), Journal of Urology, Lancet, Lancet Oncology,
Leukemia, The New England Journal of Medicine, and Radiation
Oncology.
[0205] B. Annotating with Scientific Information about the
Drugs
[0206] The drug treatment options can be annotated by providing a
link on an electronic based report connecting a listed drug to
scientific information regarding the drug. For example, a link can
be provided to information regarding a clinical trial for a drug
(http://clinicaltrials.gov). If the report is provided via a
computer or computer website, the link can be a footnote, a
hyperlink to a website, a pop-up box, or a fly-over box with
information, etc. The annotated information can become available
when a computer user clicks on a condition or a treatment option on
the downloaded and annotated Clinical Practice Guidelines in
Oncology.TM. (FIG. 3). The report and the annotated information can
be provided on a printed form, and the annotations can be, for
example, a footnote to a reference.
[0207] The information for annotating one or more drug treatment
options in a report can be provided by a commercial entity that
stores scientific information, for example, Ingenuity.RTM. Systems.
A health care provider can treat a subject, such as a cancer
patient, with an experimental drug listed in the annotated
information, and the health care provider can access the annotated
drug treatment option, retrieve the scientific information (e.g.,
print a medical journal article) and submit it (e.g., a printed
journal article) to an insurance company along with a request for
reimbursement for providing the drug treatment. Physicians can use
any of a variety of Diagnosis-related group (DRG) codes to enable
reimbursement.
[0208] A drug treatment option in a report can also be annotated
with information regarding other molecular components in a pathway
that a drug affects (e.g., information on a drug that targets a
kinase downstream of a cell-surface receptor that is a drug
target). The drug treatment option can be annotated with
information on drugs that target one or more other molecular
pathway components. The identification and/or annotation of
information related to pathways can be outsourced or subcontracted
to another company, for example Ingenuity.RTM..
[0209] The annotated information can be, for example, a drug name
(e.g., an FDA approved drug for off-label use; a drug found in a
CMS approved compendium, and/or a drug described in a scientific
(medical) journal article), scientific information concerning one
or more drug treatment options, one or more links to scientific
information regarding one or more drugs, clinical trial information
regarding one or more drugs (e.g., information from
http://clinicaltrials.gov/), one or more links to citations for
scientific information regarding drugs, etc.
[0210] The annotated information can be inserted into any location
in a report. Annotated information can be inserted in multiple
locations on a report. Annotated information can be inserted in a
report near a section on stratified drug treatment options.
Annotated information can be inserted into a report on a separate
page from stratified drug treatment options. A report that does not
contain stratified drug treatment options can be annotated with
information.
VI. Screening Drugs Using Xenograft Models and In Vitro
Cultures
[0211] The provided methods can also include means for
investigating the effects of drugs on sample (e.g. tumor cells)
isolated from a subject (e.g. cancer patient). An in vitro culture
using a tumor from a cancer patient can be established using
techniques known to those skilled in the art.
[0212] The provided method can also include establishing a
xenograft model using said sample. The sample can be a tumor biopsy
from a human subject. Cells from tumor biopsy can be transplanted
to a species that includes, for example, a pig, mouse, severe
combined immunodeficiency (SCID) mouse, rat, nude rat, etc.
[0213] The provided method can also include high-throughput
screening of FDA approved off-label drugs or experimental drugs
using said in vitro culture and/or xenograft model.
[0214] The provided method can also include monitoring tumor
antigen for recurrence detection.
[0215] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
EXAMPLES
Prophetic Example 1
Stratifying Colon Cancer Drug Treatment Options
[0216] A colon cancer patient visits a physician, and the physician
performs a tumor biopsy. The physician arranges to submit the tumor
biopsy to a personalized medicine business for characterization of
molecular markers. In the meantime, the physician prescribes a
first line drug treatment for the cancer patient selected from
those disclosed in the NCCN Clinical Practice Guidelines in
Oncology.TM. guidelines for Colon Cancer.
[0217] The tumor biopsy is obtained by the personalized medicine
business, and primary tumor cells are separated from non-tumor
cells by flow-sorting. The isolated primary tumor cells are tested
for the status of one or more molecular marker(s) by comparative
genomic hydridization (CGH), DNA sequencing, and high density
expression. The status of the one or more molecular markers is used
to analyze scientific publications for literature related to
responses of tumors with molecular markers of a similar status to
drug treatment options. The analysis is completed by one or more
individuals with knowledge or expertise in colon cancer treatment
employed by the personalized medicine business or outsourced or
subcontracted to another company. The treatment options listed in
the NCCN Clinical Practice Guidelines in Oncology.TM. guidelines
for Colon Cancer are evaluated in light of the status of the one or
more molecular markers identified in the tumor cells and in light
of available scientific information regarding those markers and
colon or other cancer drug treatment options. An individual or
teams ranks or classifies the drug treatment options based on
predicted efficacy given the status of the molecular markers in the
cancer patient's sample. Those drug treatment options predicted to
have the highest efficacy are highlighted in green, those predicted
to have lower efficacy are highlighted in yellow, and those for
which a marker indicates a drug will not be efficacious or will be
toxic are indicated in red.
[0218] In addition, scientific literature and clinical trial
information are reviewed for other drug treatment options not
listed in the NCCN Clinical Practice Guidelines in Oncology.TM.
guidelines for Colon Cancer. Based on these information sources, a
report containing the NCCN Clinical Practice Guidelines in
Oncology.TM. guidelines for Colon Cancer is annotated by listing
other drug treatment options that include FDA-approved drugs for an
off-label use and/or experimental drugs. The downloaded version of
the guidelines lists the annotated drug, and a link is provided
from each listed drug treatment option to scientific information
related to the use of that drug.
[0219] The stratified drugs treatment options and annotated drug
treatment options are shared with the patient and/or the physician
in the form of an electronic or hard copy report. The physician
and/or patient make a second line treatment decision to use an
experimental drug based on the molecular marker data. The health
care provide can access the modified NCCN Clinical Practice
Guidelines in Oncology.TM. guidelines for Colon Cancer on a
computer, point to the annotated drug, which brings up a pop-box
with a link to a journal article concerning the experimental drug.
The health care provider can submit the journal article to an
insurance provider to seek reimbursement for the treatment.
Prophetic Example 2
Xenograft and In Vitro Cultures
[0220] A colon cancer patient visits a physician, and the physician
performs a tumor biopsy. The physician submits the biopsy to a
personalized medicine business for molecular characterization. In
addition, an in vitro culture and nude rat xenograft model are
established. A series of experimental drugs are identified as
described in Prophetic Example 1 that may be helpful for the
patient. While the patient is in a first line drug therapy, a
high-throughput screen is performed with the experimental drugs and
the in vitro culture and xenograft models. Results of the
high-throughput screen are reported to the patient and the
physician, who use the information in deciding on a second-line
therapy if the first line therapy fails or an adjunct in the end
stage administration if appropriate.
Prophetic Example 3
Annotating Treatment Options on the NCCN Website
[0221] The scientific literature lists a treatment X for cancer A
and cancer B. Treatment Y is useful in treating cancer B, but the
affect of treatment Y on cancer A, however, is not known or
studied. A report with the NCCN Clinical Practice Guidelines in
Oncology.TM. on a website is annotated with information describing
the relationship between treatment Y and cancer B and treatment X
and cancers A and B, and a note is added that treatment Y may be
effective for off-label use in treating cancer A.
Prophetic Example 4
Physician Use of NCCN Website with Stratified Treatment Options
[0222] A physician (FIG. 2A) accesses a NCCN Clinical Practice
Guidelines in Oncology.TM. file on Colon Cancer (FIG. 2B) in which
a patient's treatment options have been stratified based on the
status of the patient's molecular markers (FIG. 2C). The highest
ranked drug treatment option for a patient with marker X and is
marked in green (Drug A). The next ranked drug is marked in yellow
(Drug B). The drug marked in red (Drug C) does not display efficacy
based on the status of the molecular marker X.
Prophetic Example 5
Physician Use of NCCN Website with Annotated Treatment Options
[0223] A physician accesses a NCCN Clinical Practice Guidelines in
Oncology.TM. file on Colon Cancer that has been annotated with an
additional drug treatment option D (FIG. 3; 300). Option D is an
experimental drug with published data in vitro on treating a tumor
with a marker whose status is shared with the physician's patient.
The physician treats the patient with drug D (302). The physician
follows the link on the page to a medical journal article on the
use of drug D in the in vitro study (304) and submits the article
to an insurance company with a request for reimbursement (306).
[0224] FIG. 8 depicts a report with annotated treatment options.
The markers (targets), status of the markers (targets), and drugs
are listed. Annotated information appears in multiple places on the
report.
Prophetic Example 6
Stratification and Annotation of a Combination Regimen Drug
Treatment Option
[0225] An oncologist submits an order to a personalized medicine
business on behalf of a colon cancer patient. The oncologist
submits a tumor biopsy from the colon cancer patient to the
personalized medicine business, and the personalized medicine
business identifies molecular markers in the sample. FOLFOX is a
chemotherapy regimen that can be used to treat colorectal cancer.
The FOLFOX treatment regimen includes the drugs folinic acid
(leucovorin), fluorouracil (5-FU), and oxaliplatin (Eloxatin). The
molecular marker information in the sample from a colon cancer
patient indicates that two of the drugs in FOLFOX will be
efficacious, while the molecular marker information indicates that
one of the drugs will not. The personalized medicine business
highlights the drug in yellow in the list of drug treatment
options, and annotates the report with scientific information. A
computer downloadable report is prepared by the personalized
medicine business and transmitted to the subject and the subject's
oncologist with the FOLFOX drug listing highlighted in yellow. When
the subject or the oncologist click on the FOLFOX drug listing, a
pop-up box appears that indicates the two drugs in the FOLFOX
regimen that are likely to be efficacious and the one drug that is
not likely to be efficacious in treating the subject.
Prophetic Example 7
Platinum-Based Drug Treatment Options
[0226] Platinum-based drug treatment options include cisplatin,
caroplatin, picoplatin, satraplatin, oxaliplatin. The efficacy or
resistance of some platinum-based therapies can be correlated with
certain expression profiles. A platinum-based drug is listed as a
drug treatment option in a report and is annotated to indicate
which expression profiles are correlated with efficacy or
resistance.
Prophetic Example 8
An Aspect of a Method of the Provided Invention
[0227] A health care provider performs a diagnostic biopsy of a
primary tumor in a subject. A therapy is begun, and the tumor is
tested for the status of one or more molecular markers (402). An
order is placed to the personalized medicine business (404). A
physician places the order, or assists the subject (e.g., cancer
patient) in placing the order. The order can contain clinical
information and molecular information regarding the subject and the
tumor of the subject at the time the order is placed (404). The
order can be placed on a website (404). The clinical and/or
molecular information is transmitted to the personalized medicine
business via one or more paper documents, emails, or facsimiles. A
customer service representative employed by the personalized
medicine business processes the order and a research nurse
processes the clinical and molecular information (404).
[0228] One or more kits are sent by the personalized medicine
business before the second biopsy (406). The patient and an
oncologist are notified that a kit is sent to a pathologist, and
the patient and the oncologist are notified that a kit is sent to a
surgeon (406). The kit sent to the pathologist contains
instructions for preparing a diagnostic paraffin primary biopsy, an
envelope for sending the sample to the personalized medicine
business, and identifier information (406). The kit sent to the
surgeon contains RNAlater.RTM. and instructions for freezing the
sample (406). A kit is presented to the pathologist after the
surgery (406). A logistics and operations team employed by the
personalized medicine business sends the kits (406), and a fee is
charged for sending the kits (406).
[0229] A biopsy of the sample is performed by a surgeon and the
sample is frozen (408). The frozen sample is sent by FedEx in a box
with dry ice (406). The frozen tissue is sent in a tube, and a
blood sample from the patient is sent to the personalized medicine
business for analyzing metabolism genes (406). Specific directions
are provided for sending the sample (406). Parafilm embedded,
frozen, and/or fine needle biopsies are sent to the personalized
medicine business (406).
[0230] A pathologist reviews or blocks the samples (410). Cancer
cells are purified from a sample that is sent (412). The cancer
cells are purified by flow sorting (using markers and/or ploidy) or
by laser capture microscopy (412). Two full-time employees (FTE) of
the personalized medicine business are available to purify the
cancer cells. A fee is charged in exchange for purifying the cancer
cells.
[0231] Cells, e.g., circulating tumor cells, can be isolated from a
sample, e.g., blood, using technology from, e.g., CELLective Dx
Corporation.
[0232] The purified cancer cells are analyzed by a variety of
assays by the personalized medicine business. The personalized
medicine business performs targeted assays dependent on the tumor
type (414). A logistics and operations employee and two laboratory
technicians perform these services (414). A fee is charged for the
services (414). Some of the targeted assays are outsourced
(414).
[0233] The personalized medicine business performs whole genome
analysis assays (416). These assays include comparative genomic
hybridization (CGH), high density expression, analysis for small
nucleotide polymorphisms (SNPs), proteins assays, and sequencing
(416). Up to six laboratory technicians perform these tests, and a
fee is charged in exchange for performing these tests.
[0234] Molecular tests are performed to analyze drug metabolism
genes (418). These tests include the Affymetrix.RTM. Drug
Metabolizing Enzymes and Transporters (DMET)--Early Access
Solution, Human1M BeadChip from Illumina.RTM., Human1M-Duo DNA
Analysis BeadChip from Illumina.RTM., HumanExon510S-Duo DNA
Analysis BeadChip from Illumina.RTM., and the Roche.RTM. AmpliChip
CYP450 Test (418). Laboratory technicians employed by the
personalized medicine business perform the tests. The personalized
medicine business charges a fee in exchange for performing the
tests.
[0235] Other in vitro and in vivo assays are outsourced by the
personalized medicine business to another company (420).
[0236] The information from the targeted assays, whole genome
assays, drug metabolism tests, and/or in vitro and in vivo assays
are provided to Ingenuity.RTM.. Ingenuity.RTM. as well as
bioinformatics employees of the personalized medicine business
identify scientific information related to cancer drugs and the
status of the molecular markers in the patient's sample(s) (422). A
molecular pathologist and research nurse annotate the drug
information with links to appropriate scientific literature (424).
A report is sent to an oncologist (426).
Prophetic Example 9
Drug Treatment Recommendations
[0237] A cancer patient contacts a personalized medicine business
to obtain a report regarding personalized drug treatment options
based on the status of molecular markers in his tumor. The cancer
patient submits a sample to the personalized medicine business. DNA
is extracted from the sample and is subjected to massively parallel
DNA sequencing. The sample is also subjected to qPCR. The sequence
of Kras is determined and EGFR copy number is determined. The Kras
sequence is determined to be wild-type, and EGFR copy number is
determined to be higher than normal. A report is prepared in which
cetuximab (Erbitux) and panitumumab (Vectibix) are recommended for
the colon cancer patient. The drug treatment option names are
highlighted in green on the report. Each drug treatment option is
annotated with a number for a footnote, which list references that
support the use of cetuximab (Erbitux) and panitumumab (Vectibix)
for cancer patients with tumors with wild-type Kras and increased
EGFR copy number.
* * * * *
References