U.S. patent application number 17/499284 was filed with the patent office on 2022-06-30 for methods and materials for assessing loss of heterozygosity.
This patent application is currently assigned to Myriad Genetics, Inc.. The applicant listed for this patent is Myriad Genetics, Inc.. Invention is credited to Victor Abkevich, Alexander Gutin, Jerry Lanchbury, Kirsten Timms.
Application Number | 20220205046 17/499284 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220205046 |
Kind Code |
A1 |
Abkevich; Victor ; et
al. |
June 30, 2022 |
METHODS AND MATERIALS FOR ASSESSING LOSS OF HETEROZYGOSITY
Abstract
This document provides methods and materials involved in
assessing samples (e.g., cancer cells) for the presence of a loss
of heterozygosity (LOH) signature. For example, methods and
materials for determining whether or not a cell (e.g., a cancer
cell) contains an LOH signature are provided. Materials and methods
for identifying cells (e.g., cancer cells) having a deficiency in
homology directed repair (HDR) as well as materials and methods for
identifying cancer patients likely to respond to a particular
cancer treatment regimen also are provided.
Inventors: |
Abkevich; Victor; (Salt Lake
City, UT) ; Gutin; Alexander; (Salt Lake City,
UT) ; Timms; Kirsten; (Salt Lake City, UT) ;
Lanchbury; Jerry; (Salt Lake City, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Myriad Genetics, Inc. |
Salt Lake City |
UT |
US |
|
|
Assignee: |
Myriad Genetics, Inc.
Salt Lake City
UT
|
Appl. No.: |
17/499284 |
Filed: |
October 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16691480 |
Nov 21, 2019 |
11174519 |
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17499284 |
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14554715 |
Nov 26, 2014 |
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16691480 |
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13164499 |
Jun 20, 2011 |
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14554715 |
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61356501 |
Jun 18, 2010 |
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International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; C12Q 1/6827 20060101 C12Q001/6827; G16B 20/00
20060101 G16B020/00; G16H 20/00 20060101 G16H020/00; G16B 20/20
20060101 G16B020/20; G16B 20/10 20060101 G16B020/10 |
Claims
1. A method of predicting a cancer patient's response to a cancer
treatment regimen comprising a DNA damaging agent, an
anthracycline, a topoisomerase I inhibitor, radiation, and/or a
PARP inhibitor, said method comprising: determining, in the cancer
cell, the total number of LOH regions in at least one pair of human
chromosomes of said cancer cell that are longer than a first length
but shorter than the length of the whole chromosome containing the
LOH region, wherein said at least one pair of human chromosomes is
not a human X/Y sex chromosome pair, wherein said first length is
about 15 or more megabases, wherein the cancer cell is selected
from the group consisting of breast cancer cells, ovarian cancer
cells, leukemia cancer cells, esophageal cancer cells, lung cancer
cells, and prostate cancer cells; and correlating said total number
that is greater than a reference number with an increased
likelihood that said cancer patient will respond to said cancer
treatment regimen.
2. The method of claim 1, wherein said LOH regions or Indicator LOH
Regions are determined in at least two, five, ten or 21 pairs of
human chromosomes.
3. The method of claim 1, wherein said total number of LOH regions
or Indicator LOH Regions is 9, 15, 20 or more.
4. The method of claim 1, wherein said reference number is 6, 7, 8,
9, 10, 11, 12 or 13 or greater.
5. The method of claim 1, wherein said at least one pair of human
chromosomes is not human chromosome 17.
6. The method of claim 1, wherein said DNA damaging agent is
cisplatin, carboplatin, oxalaplatin, or picoplatin, said
anthracycline is epirubicin or doxorubicin, said topoisomerase I
inhibitor is campothecin, topotecan, or irinotecan, or said PARP
inhibitor is iniparib, olaparib or veliparib.
7. A cancer treatment comprising one or more drugs chosen from the
group consisting of DNA damaging agents, anthracyclines,
topoisomerase I inhibitors, and PARP inhibitors for use in treating
cancer in a patient having an increased likelihood of response to
said treatment, wherein the determination of whether or not the
patient has an increased likelihood of response to said treatment
is carried out by the method of claim 1.
8. A method of predicting a cancer patient's response to a
treatment regimen including paclitaxel or docetaxel, comprising:
determining, in the cancer cell, the total number of LOH regions in
at least one pair of human chromosomes of said cancer cell that are
longer than a first length but shorter than the length of the whole
chromosome containing the LOH region, wherein said at least one
pair of human chromosomes is not a human X/Y sex chromosome pair,
wherein said first length is about 15 or more megabases, wherein
the cancer cell is selected from the group consisting of breast
cancer cells, ovarian cancer cells, leukemia cancer cells,
esophageal cancer cells, lung cancer cells, and prostate cancer
cells; and correlating said total number that is greater than a
reference number with an increased likelihood that said cancer
patient will not respond to a treatment regimen including
paclitaxel or docetaxel.
9. The method of claim 8, wherein said LOH regions or Indicator LOH
Regions are determined in at least two, five, ten or 21 pairs of
human chromosomes.
10. The method of claim 8, wherein said total number of LOH regions
or Indicator LOH Regions is 9, 15, 20 or more.
11. The method of claim 8, wherein said reference number is 6, 7,
8, 9, 10, 11, 12 or 13 or greater.
12. The method of claim 8, wherein said at least one pair of human
chromosomes is not human chromosome 17.
13. A cancer treatment comprising one or more drugs chosen from the
group consisting of DNA damaging agents, anthracyclines,
topoisomerase I inhibitors, and PARP inhibitors for use in treating
cancer in a patient having an increased likelihood of response to
said treatment, wherein the determination of whether or not the
patient has an increased likelihood of response to said treatment
is carried out by the method of claim 8.
14. A system for determining LOH status of a cancer cell of a
cancer patient, comprising: (a) a sample analyzer configured to
produce a plurality of signals about genomic DNA, wherein said
signals identify the homozygous or heterozygous nature of loci of
at least one pair of human chromosomes of said cancer cell, and
wherein the cancer cell is selected from the group consisting of
breast cancer cells, ovarian cancer cells, leukemia cancer cells,
esophageal cancer cells, lung cancer cells, and prostate cancer
cells, and (b) a computer sub-system programmed to calculate, based
on said plurality of signals, the number of Indicator LOH Regions
in said at least one pair of human chromosomes, wherein said
Indicator LOH Regions are LOH regions that are in a pair of human
chromosomes other than the human X/Y sex chromosome pair, and are
characterized by LOH with a length of about 15 or more megabases
but shorter than the length of the whole chromosome containing the
LOH regions, and wherein said computer sub-system is programmed to
compare said number of Indicator LOH Regions to a reference number
to determine a likelihood that said cancer patient will respond to
cancer treatment regimen comprising a DNA damaging agent, an
anthracycline, a topoisomerase I inhibitor, radiation, or a PARP
inhibitor.
15. The system of claim 14, wherein said LOH regions or Indicator
LOH Regions are determined in at least two, five, ten or 21 pairs
of human chromosomes.
16. The system of claim 14, wherein said total number of LOH
regions or Indicator LOH Regions is 9, 15, 20 or more.
17. The system of claim 14, wherein said reference number is 6, 7,
8, 9, 10, 11, 12 or 13 or greater.
18. The system of claim 14, wherein said at least one pair of human
chromosomes is not human chromosome 17.
19. The system of claim 14, wherein said Indicator LOH Regions are
not in human chromosome 17.
20. The system of claim 14, wherein said DNA damaging agent is a
platinum-based chemotherapy drug, said anthracycline is epirubicin
or doxorubicin, said topoisomerase I inhibitor is campothecin,
topotecan, or irinotecan, or said PARP inhibitor is iniparib,
olaparib or veliparib.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/691,480, filed Nov. 21, 2019, which is a
continuation of U.S. patent application Ser. No. 14/554,715, filed
Nov. 26, 2014, which is a continuation of U.S. patent application
Ser. No. 13/164,499, filed Jun. 20, 2011, which claims priority to
U.S. Provisional Application Ser. No. 61/356,501 filed Jun. 18,
2010, the entire contents of each of which are hereby incorporated
by reference.
BACKGROUND
1. Technical Field
[0002] This document relates to methods and materials involved in
assessing samples (e.g., cancer cells) for the presence of a loss
of heterozygosity (LOH) signature. For example, this document
provides methods and materials for determining whether or not a
cell (e.g., a cancer cell) contains an LOH signature. This document
also provides materials and methods for identifying cells (e.g.,
cancer cells) having a deficiency in homology directed repair (HDR)
as well as materials and methods for identifying cancer patients
likely to respond to a particular cancer treatment regimen.
2. Background Information
[0003] Cancer is a serious public health problem, with 562,340
people in the United States of America dying of cancer in 2009
alone. American Cancer Society, Cancer Facts & Figures 2009
(available at American Cancer Society website). One of the primary
challenges in cancer treatment is discovering relevant, clinically
useful characteristics of a patient's own cancer and then, based on
these characteristics, administering a treatment plan best suited
to the patient's cancer. While strides have been made in this field
of personalized medicine, there is still a significant need for
better molecular diagnostic tools to characterize patients'
cancers.
SUMMARY
[0004] This document provides methods and materials involved in
assessing samples (e.g. cancer cells) for the presence of a loss of
heterozygosity (LOH) signature. For example, this document provides
methods and materials for determining whether or not a cell (e.g.,
cancer cell) contains an LOH signature. An LOH signature as used
herein refers to the presence of five or more (e.g., six or more,
seven or more, eight or more, nine or more, ten or more, eleven or
more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more,
17 or more, 18 or more, 19 or more, or 20 or more) LOH regions that
are longer than about 1.5 megabases (e.g., longer than about 2,
2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30, 35, 40, 45, 50, 75, or 100 megabases (Mb)) and are less
than the length of the entire chromosome that contains that LOH
region. In general, all the chromosomes of a genome for a sample
(e.g., tumor biopsy) can be assessed for the presence of an LOH
signature. In some cases, all the chromosomes of a genome for a
sample with the exception of chromosome 17 can be assessed for the
presence of an LOH signature. For males only autosomal chromosomes
can be assessed for the presence of an LOH signature.
[0005] As described herein, cancer cells having a genome containing
an LOH signature can be identified as being likely to have a
deficiency in homology directed repair (HDR). In some cases, cancer
cells having a genome containing an LOH signature can be identified
as being likely to have a deficient status in one or more genes
involved in HDR. For example, cancer cells having a genome
containing an LOH signature can be identified as being likely to
have a deficient BRCA1 or BRCA2 status, and cells having a genome
lacking an LOH signature can be identified as being likely to have
an intact BRCA1 or BRCA2 status.
[0006] Determining whether a cell (e.g., a cancer cell) is likely
to have a deficiency in HDR or is likely to have a deficient status
in one or more genes involved in HDR can indicate that the mammal
(e.g., human) with that cell is likely to have one or more genetic
defects within the mammal's germline. Identifying humans with an
increased likelihood of having such a germline defect can allow the
human or clinicians to inform offspring of the possible inheritance
of such a germline defect. Such offspring can elect, based at least
in part on such information, to undergo genetic testing and
possible monitoring (e.g., early detection monitoring) for the
development of cancer.
[0007] As also described herein, cancer cells having a genome
containing an LOH signature can be identified as being likely to
respond to a particular cancer treatment regimen. For example,
patients having cancer cells with a genome containing an LOH
signature can be identified as being likely to respond to a cancer
treatment regimen that includes the use of a DNA damaging agent, a
PARP inhibitor, radiation, or a combination thereof. In some cases,
patients having cancer cells with a genome lacking an LOH signature
can be identified as being unlikely to respond to a cancer
treatment regimen designed to administer a single agent such as a
single DNA damaging agent, a single PARP inhibitor, or radiation
alone. In some cases, patients having cancer cells with a genome
lacking an LOH signature can be identified as being likely to
respond to a cancer treatment regimen that includes the use of a
standard cancer treatment agent not associated with HDR (e.g., a
taxol compound such as paclitaxel).
[0008] Determining whether or not cancer patients are likely to
respond to a particular cancer treatment regimen as described
herein can allow patients and clinicians to proceed with a
treatment regimen having an increased likelihood of treating cancer
(e.g., reducing the number of cancer cells within a patient). In
some cases, determining whether or not cancer patients are likely
to respond to a particular cancer treatment regimen as described
herein can allow patients and clinicians to select the most
effective initial cancer treatment regimen for that patient.
[0009] In general, one aspect of this document features a method
for assessing LOH in a cancer cell or genomic DNA thereof. The
method comprises, or consists essentially of, (a) detecting, in a
cancer cell or genomic DNA derived therefrom, LOH regions in at
least one pair of human chromosomes of the cancer cell, wherein the
at least one pair of human chromosomes is not a human X/Y sex
chromosome pair; and (b) determining the total number of LOH
regions, in the at least one pair of human chromosomes, that are
longer than a first length but shorter than the length of the whole
chromosome containing the LOH region, wherein the first length is
about 1.5 or more megabases.
[0010] In another aspect, this document features a method of
predicting the status of BRCA1 and BRCA2 genes in a cancer cell.
The method comprises, or consists essentially of, determining, in
the cancer cell, the total number of LOH regions in at least one
pair of human chromosomes of the cancer cell that are longer than a
first length but shorter than the length of the whole chromosome
containing the LOH region, wherein the at least one pair of human
chromosomes is not a human X/Y sex chromosome pair, wherein the
first length is about 1.5 or more megabases; and correlating the
total number that is greater than a reference number with an
increased likelihood of a deficiency in the BRCA1 or BRCA2
gene.
[0011] In another aspect, this document features a method of
predicting the status of HDR in a cancer cell. The method
comprises, or consists essentially of, determining, in the cancer
cell, the total number of LOH regions in at least one pair of human
chromosomes of the cancer cell that are longer than a first length
but shorter than the length of the whole chromosome containing the
LOH region, wherein the at least one pair of human chromosomes is
not a human X/Y sex chromosome pair, wherein the first length is
about 1.5 or more megabases; and correlating the total number that
is greater than a reference number with an increased likelihood of
a deficiency in HDR.
[0012] In another aspect, this document features a method of
predicting a cancer patient's response to a cancer treatment
regimen comprising a DNA damaging agent, an anthracycline, a
topoisomerase I inhibitor, radiation, and/or a PARP inhibitor. The
method comprises, or consists essentially of, determining, in a
cancer cell from the cancer patient, the number of LOH regions in
at least one pair of human chromosomes of a cancer cell of the
cancer patient that are longer than a first length but shorter than
the length of the whole chromosome containing the LOH region,
wherein the at least one pair of human chromosomes is not a human
X/Y sex chromosome pair, wherein the first length is about 1.5 or
more megabases; and correlating the total number that is greater
than a reference number with an increased likelihood that the
cancer patient will respond to the cancer treatment regimen.
[0013] In another aspect, this document features a method of
predicting a cancer patient's response to a treatment regimen. The
method comprises, or consists essentially of, determining, in a
cancer cell from the cancer patient, the total number of LOH
regions in at least one pair of human chromosomes of a cancer cell
of the cancer patient that are longer than a first length but
shorter than the length of the whole chromosome containing the LOH
region, wherein the at least one pair of human chromosomes is not a
human X/Y sex chromosome pair, wherein the first length is about
1.5 or more megabases; and correlating the total number that is
greater than a reference number with an increased likelihood that
the cancer patient will not respond to a treatment regimen
including paclitaxel or docetaxel.
[0014] In another aspect, this document features a method of
treating cancer. The method comprises, or consists essentially of,
(a) determining, in a cancer cell from a cancer patient or genomic
DNA obtained therefrom, the total number of LOH regions in at least
one pair of human chromosomes of the cancer cell that are longer
than a first length but shorter than the length of the whole
chromosome containing the LOH region, wherein the at least one pair
of human chromosomes is not a human X/Y sex chromosome pair,
wherein the first length is about 1.5 or more megabases; and (b)
administering to the cancer patient a cancer treatment regimen
comprising one or more drugs chosen from the group consisting of
DNA damaging agents, anthracyclines, topoisomerase I inhibitors,
and PARP inhibitors, if the total number of LOH regions is greater
than a reference number.
[0015] For any one or more of the methods described in the
preceding six paragraphs, any one or more of the following can be
applied as appropriate. The LOH regions can be determined in at
least two, five, ten, or 21 pairs of human chromosomes. The cancer
cell can be an ovarian, breast, or esophageal cancer cell. The
total number of LOH regions can be 9, 15, 20 or more. The first
length can be about 6, 12, or 15 or more megabases. The reference
number can be 6, 7, 8, 9, 10, 11, 12, 13, or greater. The at least
one pair of human chromosomes can exclude human chromosome 17. The
DNA damaging agent can be cisplatin, carboplatin, oxalaplatin, or
picoplatin, the anthracycline can be epirubincin or doxorubicin,
the topoisomerase I inhibitor can be campothecin, topotecan, or
irinotecan, or the PARP inhibitor can be iniparib, olaparib or
velapirib.
[0016] In another aspect, this document features the use of one or
more drugs selected from the group consisting of DNA damaging
agents, anthracyclines, topoisomerase I inhibitors, and PARP
inhibitors, in the manufacture of a medicament useful for treating
a cancer in a patient identified as having a cancer cell determined
to have a total of 5 or more Indicator LOH Regions. The Indicator
LOH Regions can be determined in at least two, five, ten, or 21
pairs of human chromosomes. The cancer cell can be an ovarian,
breast, or esophageal cancer cell. The total number of Indicator
LOH Regions can be 9, 15, 20 or more. The Indicator LOH Regions can
have a length of about 6, 12, or 15 or more megabases. The
Indicator LOH Regions can be present on a chromosome other than
human chromosome 17. The DNA damaging agent can be a platinum-based
chemotherapy drug, the anthracycline can be epirubincin or
doxorubicin, the topoisomerase I inhibitor can be campothecin,
topotecan, or irinotecan, or the PARP inhibitor can be iniparib,
olaparib or velapirib.
[0017] In another aspect, this document features the use of a
plurality of oligonucleotides capable of hybridizing to a plurality
of polymorphic regions of human genomic DNA, in the manufacture of
a diagnostic kit useful for determining the total number of
Indicator LOH Regions in at least a chromosome pair of a human
cancer cell obtained from a cancer patient, and for detecting (a)
an increased likelihood of a deficiency in the BRCA1 or BRCA2 gene
in the cancer cell, (b) an increased likelihood of a deficiency in
HDR in the cancer cell, or (c) an increased likelihood that the
cancer patient will respond to cancer treatment regimen comprising
a DNA damaging agent, an anthracycline, a topoisomerase I
inhibitor, radiation, or a PARP inhibitor. The Indicator LOH
Regions can be determined in at least two, five, ten, or 21 pairs
of human chromosomes. The cancer cell can be an ovarian, breast, or
esophageal cancer cell. The total number of Indicator LOH Regions
can be 9, 15, 20 or more. The Indicator LOH Regions can have a
length of about 6, 12, or 15 or more megabases. The Indicator LOH
Regions can be present on a chromosome other than human chromosome
17.
[0018] In another aspect, this document features a system for
determining LOH status of a cancer cell of a cancer patient. The
system comprises, or consists essentially of, (a) a sample analyzer
configured to produce a plurality of signals about genomic DNA of
at least one pair of human chromosomes of the cancer cell, and (b)
a computer sub-system programmed to calculate, based on the
plurality of signals, the number of Indicator LOH Regions in the at
least one pair of human chromosomes. The computer sub-system can be
programmed to compare the number of Indicator LOH Regions to a
reference number to determine (a) a likelihood of a deficiency in
BRCA1 and/or BRCA2 genes in the cancer cell, (b) a likelihood of a
deficiency in HDR in the cancer cell, or (c) a likelihood that the
cancer patient will respond to cancer treatment regimen comprising
a DNA damaging agent, an anthracycline, a topoisomerase I
inhibitor, radiation, or a PARP inhibitor. The system can comprise
an output module configured to display the likelihood of (a), (b),
or (c). The system can comprise an output module configured to
display a recommendation for the use of the cancer treatment
regimen. The Indicator LOH Regions can be determined in at least
two, five, ten, or 21 pairs of human chromosomes. The cancer cell
can be an ovarian, breast, or esophageal cancer cell. The total
number of Indicator LOH Regions can be 9, 15, 20, or more. The
Indicator LOH Regions can have a length of about 6, 12, or 15 or
more megabases. The Indicator LOH Regions can be present on
chromosomes other than a human chromosome 17. The DNA damaging
agent can be a platinum-based chemotherapy drug, the anthracycline
can be epirubincin or doxorubicin, the topoisomerase I inhibitor
can be campothecin, topotecan, or irinotecan, or the PARP inhibitor
can be iniparib, olaparib or velapirib.
[0019] In another aspect, this document features a computer program
product embodied in a computer readable medium that, when executing
on a computer, provides instructions for detecting the presence or
absence of any LOH region along one or more of human chromosomes
other than the human X and Y sex chromosomes, and the LOH region
having a length of about 1.5 or more megabases but shorter than the
length of the whole chromosome containing the LOH region; and
determining the total number of the LOH region in the one or more
chromosome pairs. The computer program product can include other
instructions. The Indicator LOH Regions can be determined in at
least two, five, ten or 21 pairs of human chromosomes. The cancer
cell can be an ovarian, breast, or esophageal cancer cell. The
total number of Indicator LOH Regions can be 9, 15, 20, or more.
The Indicator LOH Regions can have a length of about 6, 12, or 15
or more megabases. The Indicator LOH Regions can be present on
chromosomes other than a human chromosome 17. The DNA damaging
agent can be a platinum-based chemotherapy drug, the anthracycline
can be epirubincin or doxorubicin, the topoisomerase I inhibitor
can be campothecin, topotecan, or irinotecan, or the PARP inhibitor
can be iniparib, olaparib or velapirib.
[0020] In another aspect, this document features a diagnostic kit.
The kit comprises, or consists essentially of, at least 500
oligonucleotides capable of hybridizing to a plurality of
polymorphic regions of human genomic DNA; and a computer program
product provided herein. The computer program product can be
embodied in a computer readable medium that, when executing on a
computer, provides instructions for detecting the presence or
absence of any LOH region along one or more of human chromosomes
other than the human X and Y sex chromosomes, and the LOH region
having a length of about 1.5 or more megabases but shorter than the
length of the whole chromosome containing the LOH region; and
determining the total number of the LOH region in the one or more
chromosome pairs. The computer program product can include other
instructions. In another aspect, this document features a method
for assessing cancer cells of a patient for the presence of an LOH
signature. The method comprises, or consists essentially of, (a)
detecting the presence of more than a reference number of LOH
regions in at least one pair of human chromosomes of a cancer cell
of the cancer patient that are longer than a first length but
shorter than the length of the whole chromosome containing the LOH
region, wherein the at least one pair of human chromosomes is not a
human X/Y sex chromosome pair, wherein the first length is about
1.5 or more megabases, and (b) identifying the patient as having
cancer cells with the LOH signature.
[0021] In another aspect, this document features a method for
assessing cancer cells of a patient for the presence of an HDR
deficient status. The method comprises, or consists essentially of,
(a) detecting the presence of more than a reference number of LOH
regions in at least one pair of human chromosomes of a cancer cell
of the cancer patient that are longer than a first length but
shorter than the length of the whole chromosome containing the LOH
region, wherein the at least one pair of human chromosomes is not a
human X/Y sex chromosome pair, wherein the first length is about
1.5 or more megabases, and (b) identifying the patient as having
cancer cells with the HDR deficient status.
[0022] In another aspect, this document features a method for
assessing cancer cells of a patient for the presence of a genetic
mutation within a gene from an HDR pathway. The method comprises,
or consists essentially of, (a) detecting the presence of more than
a reference number of LOH regions in at least one pair of human
chromosomes of a cancer cell of the cancer patient that are longer
than a first length but shorter than the length of the whole
chromosome containing the LOH region, wherein the at least one pair
of human chromosomes is not a human X/Y sex chromosome pair,
wherein the first length is about 1.5 or more megabases, and (b)
identifying the patient as having cancer cells with the genetic
mutation.
[0023] In another aspect, this document features a method for
determining if a patient is likely to respond to a cancer treatment
regimen comprising administering radiation or a drug selected from
the group consisting of DNA damaging agents, anthracyclines,
topoisomerase I inhibitors, and PARP inhibitors. The method
comprises, or consists essentially of, (a) detecting the presence
of more than a reference number of LOH regions in at least one pair
of human chromosomes of a cancer cell of the cancer patient that
are longer than a first length but shorter than the length of the
whole chromosome containing the LOH region, wherein the at least
one pair of human chromosomes is not a human X/Y sex chromosome
pair, wherein the first length is about 1.5 or more megabases, and
(b) identifying the patient as being likely to respond to the
cancer treatment regimen.
[0024] In another aspect, this document features a method for
assessing a patient. The method comprises, or consists essentially
of, (a) determining that the patient comprises cancer cells having
an LOH signature, wherein the presence of more than a reference
number of LOH regions in at least one pair of human chromosomes of
a cancer cell of the cancer patient that are longer than a first
length but shorter than the length of the whole chromosome
containing the LOH region indicates that the cancer cells have the
LOH signature, wherein the at least one pair of human chromosomes
is not a human X/Y sex chromosome pair, wherein the first length is
about 1.5 or more megabases, and (b) diagnosing the patient as
having cancer cells with the LOH signature.
[0025] In another aspect, this document features a method for
assessing a patient. The method comprises, or consists essentially
of, (a) determining that the patient comprises cancer cells having
an HDR deficiency status, wherein the presence of more than a
reference number of LOH regions in at least one pair of human
chromosomes of a cancer cell of the cancer patient that are longer
than a first length but shorter than the length of the whole
chromosome containing the LOH region indicates that the cancer
cells have the HDR deficiency status, wherein the at least one pair
of human chromosomes is not a human X/Y sex chromosome pair,
wherein the first length is about 1.5 or more megabases, and (b)
diagnosing the patient as having cancer cells with the HDR
deficient status.
[0026] In another aspect, this document features a method for
assessing a patient. The method comprises, or consists essentially
of, (a) determining that the patient comprises cancer cells having
a genetic mutation within a gene from an HDR pathway, wherein the
presence of more than a reference number of LOH regions in at least
one pair of human chromosomes of a cancer cell of the cancer
patient that are longer than a first length but shorter than the
length of the whole chromosome containing the LOH region indicates
that the cancer cells have the genetic mutation, wherein the at
least one pair of human chromosomes is not a human X/Y sex
chromosome pair, wherein the first length is about 1.5 or more
megabases, and (b) diagnosing the patient as having cancer cells
with the genetic mutation.
[0027] In another aspect, this document features a method for
assessing a patient for a likelihood to respond to a cancer
treatment regimen comprising administering radiation or a drug
selected from the group consisting of DNA damaging agents,
anthracyclines, topoisomerase I inhibitors, and PARP inhibitors.
The method comprises, or consists essentially of, (a) determining
that the patient comprises cancer cells having an LOH signature,
wherein the presence of more than a reference number of LOH regions
in at least one pair of human chromosomes of a cancer cell of the
cancer patient that are longer than a first length but shorter than
the length of the whole chromosome containing the LOH region
indicates that the cancer cells have the LOH signature, wherein the
at least one pair of human chromosomes is not a human X/Y sex
chromosome pair, wherein the first length is about 1.5 or more
megabases, and (b) diagnosing, based at least in part on the
presence of the LOH signature, the patient as being likely to
respond to the cancer treatment regimen.
[0028] In another aspect, this document features a method for
performing a diagnostic analysis of a cancer cell of a patient. The
method comprises, or consists essentially of, (a) detecting the
presence of more than a reference number of LOH regions in at least
one pair of human chromosomes of the cancer cell that are longer
than a first length but shorter than the length of the whole
chromosome containing the LOH region, wherein the at least one pair
of human chromosomes is not a human X/Y sex chromosome pair,
wherein the first length is about 1.5 or more megabases, and (b)
identifying the patient as having cancer cells with an LOH
signature.
[0029] In another aspect, this document features a method for
performing a diagnostic analysis of a cancer cell of a patient. The
method comprises, or consists essentially of, (a) detecting the
presence of more than a reference number of LOH regions in at least
one pair of human chromosomes of the cancer cell that are longer
than a first length but shorter than the length of the whole
chromosome containing the LOH region, wherein the at least one pair
of human chromosomes is not a human X/Y sex chromosome pair,
wherein the first length is about 1.5 or more megabases, and (b)
identifying the patient as having cancer cells with a HDR deficient
status.
[0030] In another aspect, this document features a method for
performing a diagnostic analysis of a cancer cell of a patient. The
method comprises, or consists essentially of, (a) detecting the
presence of more than a reference number of LOH regions in at least
one pair of human chromosomes of the cancer cell that are longer
than a first length but shorter than the length of the whole
chromosome containing the LOH region, wherein the at least one pair
of human chromosomes is not a human X/Y sex chromosome pair,
wherein the first length is about 1.5 or more megabases, and (b)
identifying the patient as having cancer cells with a genetic
mutation within a gene from an HDR pathway.
[0031] In another aspect, this document features a method for
performing a diagnostic analysis of a cancer cell of a patient to
determine if the cancer patient is likely to respond to a cancer
treatment regimen comprising administering radiation or a drug
selected from the group consisting of DNA damaging agents,
anthracyclines, topoisomerase I inhibitors, and PARP inhibitors.
The method comprises, or consists essentially of, (a) detecting the
presence of more than a reference number of LOH regions in at least
one pair of human chromosomes of the cancer cell that are longer
than a first length but shorter than the length of the whole
chromosome containing the LOH region, wherein the at least one pair
of human chromosomes is not a human X/Y sex chromosome pair,
wherein the first length is about 1.5 or more megabases, and (b)
identifying the patient as being likely to respond to the cancer
treatment regimen.
[0032] In another aspect, this document features a method for
diagnosing a patient as having cancer cells having an LOH
signature. The method comprises, or consists essentially of, (a)
determining that the patient comprises cancer cells having the LOH
signature, wherein the presence of more than a reference number of
LOH regions in at least one pair of human chromosomes of a cancer
cell of the cancer patient that are longer than a first length but
shorter than the length of the whole chromosome containing the LOH
region indicates that the cancer cells have the LOH signature,
wherein the at least one pair of human chromosomes is not a human
X/Y sex chromosome pair, wherein the first length is about 1.5 or
more megabases, and (b) diagnosing the patient as having cancer
cells with the LOH signature.
[0033] In another aspect, this document features a method for
diagnosing a patient as having cancer cells with an HDR deficient
status. The method comprises, or consists essentially of, (a)
determining that the patient comprises cancer cells having the HDR
deficiency status, wherein the presence of more than a reference
number of LOH regions in at least one pair of human chromosomes of
a cancer cell of the cancer patient that are longer than a first
length but shorter than the length of the whole chromosome
containing the LOH region indicates that the cancer cells have the
HDR deficiency status, wherein the at least one pair of human
chromosomes is not a human X/Y sex chromosome pair, wherein the
first length is about 1.5 or more megabases, and (b) diagnosing the
patient as having cancer cells with the HDR deficient status.
[0034] In another aspect, this document features a method for
diagnosing a patient as having cancer cells with a genetic mutation
within a gene from an HDR pathway. The method comprises, or
consists essentially of, (a) determining that the patient comprises
cancer cells having the genetic mutation, wherein the presence of
more than a reference number of LOH regions in at least one pair of
human chromosomes of a cancer cell of the cancer patient that are
longer than a first length but shorter than the length of the whole
chromosome containing the LOH region indicates that the cancer
cells have the genetic mutation, wherein the at least one pair of
human chromosomes is not a human X/Y sex chromosome pair, wherein
the first length is about 1.5 or more megabases, and (b) diagnosing
the patient as having cancer cells with the genetic mutation.
[0035] In another aspect, this document features a method for
diagnosing a patient as being a candidate for a cancer treatment
regimen comprising administering radiation or a drug selected from
the group consisting of DNA damaging agents, anthracyclines,
topoisomerase I inhibitors, and PARP inhibitors. The method
comprises, or consists essentially of, (a) determining that the
patient comprises cancer cells having an LOH signature, wherein the
presence of more than a reference number of LOH regions in at least
one pair of human chromosomes of a cancer cell of the cancer
patient that are longer than a first length but shorter than the
length of the whole chromosome containing the LOH region indicates
that the cancer cells have the LOH signature, wherein the at least
one pair of human chromosomes is not a human X/Y sex chromosome
pair, wherein the first length is about 1.5 or more megabases, and
(b) diagnosing, based at least in part on the presence of the LOH
signature, the patient as being likely to respond to the cancer
treatment regimen.
[0036] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0037] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a graph plotting allele dosages of breast cancer
cells from a breast cancer patient along chromosome 1 as determined
using a SNP array. The arrow indicates a transition between a
region of heterozygosity and an LOH region.
[0039] FIG. 2 is a graph plotting allele dosages of breast cancer
cells for the same breast cancer patient as on FIG. 1 along
chromosome 1 as determined using high-throughput sequencing. The
arrow indicates a transition between a region of heterozygosity and
an LOH region.
[0040] FIG. 3 is a flow chart of an example process for assessing
the genome of a cell (e.g., a cancer cell) for an LOH
signature.
[0041] FIG. 4 is a diagram of an example of a computer device and a
mobile computer device that can be used to implement the techniques
described herein.
[0042] FIG. 5 is a graph plotting the length distribution of LOH
regions detected in ovarian cancer cells from 62 human patients.
The adjusted length refers to the fraction of chromosomes arms
covered by LOH regions.
[0043] FIG. 6 is a graph plotting the number of LOH regions longer
than 15 Mb and shorter than the entire chromosome for a training
set of ovarian cancer cell samples with intact or deficient BRCA1
and BRCA2 genes. The size of the circles is proportional to the
number of samples with such number of LOH regions.
[0044] FIG. 7 is a graph plotting the number of LOH regions longer
than 15 Mb and shorter than the entire chromosome for a training
and validation sets of ovarian cancer cell samples with intact or
deficient BRCA1 and BRCA2 genes. The size of the circles is
proportional to the number of samples with such number of LOH
regions.
[0045] FIG. 8 is a graph plotting the number of LOH regions longer
than 15 Mb and shorter than the entire chromosome for ovarian
cancer cell samples with somatic BRCA mutations, with germline BRCA
mutations, with low BRCA1 expression, or with intact BRCA (BRCA
normal). The size of the circles is proportional to the number of
samples with such number of LOH regions.
[0046] FIG. 9 is a table showing the percent of ovarian cancer
samples that are BRCA deficient, HDR deficient/BRCA intact, and HDR
intact.
[0047] FIG. 10 is a graph plotting the number of LOH regions longer
than 15 Mb and shorter than the entire chromosome for cancer cell
lines for the indicated cancers. The size of the circles is
proportional to the number of samples with such number of LOH
regions.
[0048] FIG. 11 is a graph plotting the number of LOH regions longer
than 15 Mb and shorter than the entire chromosome for lung cancer
samples.
[0049] FIG. 12 is a graph plotting the percentage of the indicated
cancers or cancer cell lines having an HDR deficiency.
[0050] FIG. 13 contains graphs plotting the IC.sub.50 values
(Log.sub.10(IC.sub.50) of camptothecin, as well as averaged
Log.sub.10(IC.sub.50) values for platinum compounds (oxaliplatin,
cisplatin, and carboplatin), or anthracyclines (doxorubicin and
epirubicin) when exposed to 29 breast cancer cell lines having the
indicated number of LOH regions longer than 15 Mb and shorter than
the entire chromosome or the IC.sub.50 values
(Log.sub.10(IC.sub.50)) of paclitaxel when exposed to 27 ovarian
cancer cell lines having the indicated number of LOH regions longer
than 15 Mb and shorter than the entire chromosome. The dashed lines
place a threshold number at nine.
[0051] FIG. 14 is a labeled version of a graph from FIG. 13 that
plots the averaged Log.sub.10(IC.sub.50) values of platinum
compounds (oxaliplatin, cisplatin, and carboplatin) when exposed to
29 breast cancer cell lines having the indicated number of LOH
regions longer than 15 Mb and shorter than the entire
chromosome.
[0052] FIG. 15 is a flow chart of an example computational process
for identifying LOH loci and regions.
DETAILED DESCRIPTION
[0053] This document provides methods and materials involved in
assessing samples (e.g., cancer cells) for the presence of an LOH
signature. For example, this document provides methods and
materials for determining whether or not a cell (e.g., a cancer
cell) contains an LOH signature.
[0054] In general, a comparison of sequences present at the same
locus on each chromosome (each autosomal chromosome for males) can
reveal whether that particular locus is homozygous or heterozygous
within the genome of a cell. Polymorphic loci within the human
genome are generally heterozygous within an individual since that
individual typically receives one copy from the biological father
and one copy from the biological mother. In some cases, a
polymorphic locus or a string of polymorphic loci within an
individual are homozygous as a result in inheriting identical
copies from both biological parents.
[0055] Loss of heterozygosity (LOH) may result from several
mechanisms. For example, in some cases, a region of one chromosome
can be deleted in a somatic cell. The region that remains present
on the other chromosome (the other non-sex chromosome for males) is
an LOH region as there is only one copy (instead of two copies) of
that region present within the genome of the affected cells. This
LOH region can be any length (e.g., from a length less than about
1.5 Mb up to a length equal to the entire length of the
chromosome). This type of LOH event results in a copy number
reduction. In other cases, a region of one chromosome (one non-sex
chromosome for males) in a somatic cell can be replaced with a copy
of that region from the other chromosome, thereby eliminating any
heterozygosity that may have been present within the replaced
region. In such cases, the region that remains present on each
chromosome is an LOH region and can be referred to as a copy
neutral LOH region. Copy neutral LOH regions can be any length
(e.g., from a length less than about 1.5 Mb up to a length equal to
the entire length of the chromosome).
[0056] As described herein, a cellular sample (e.g., cancer cell
sample) can be identified as having a positive LOH signature status
if the genome of the cells being assessed contains five or more
(e.g., six or more, seven or more, eight or more, nine or more, ten
or more, eleven or more, 12 or more, 13 or more, 14 or more, 15 or
more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or
more) LOH regions that are (a) longer than about 1.5 megabases
(e.g., longer than about 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100
megabases (Mb)) and (b) less than the length of the entire
chromosome that contains that LOH region. In some cases, a cancer
cell sample can be identified as having a positive LOH signature
status if the genome of the cells being assessed contains nine or
more LOH regions that are (a) longer than about 15 Mb and (b) less
than the length of the entire chromosome that contains that LOH
region. Unless otherwise defined, the term "Indicator LOH Region"
refers to an LOH region that is in a pair of human chromosomes
other than the human X/Y sex chromosome pair, and that is
characterized by loss of heterozygosity with a length of about 1.5
or more megabases but shorter than the length of the whole
chromosome containing the LOH region. The length of the whole
chromosome containing an LOH region may be determined by examining
the length of the shorter chromosome of the corresponding
chromosome pair in a germline cell or a non-tumor somatic cell. In
some embodiments, an Indicator LOH Region is any LOH region about
2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 75, or 100 megabases (Mb)) or more
and less than the length of the whole chromosome that contains that
LOH region.
[0057] Cells (e.g., cancer cells) identified as having a positive
LOH signature status can be classified as having an increased
likelihood of having an HDR deficiency and/or as having an
increased likelihood of having a deficient status in one or more
genes in the HDR pathway. For example, cancer cells identified as
having a positive LOH signature status can be classified as having
an increased likelihood of having an HDR deficient status. In some
cases, cancer cells identified as having a positive LOH signature
status can be classified as having an increased likelihood of
having a deficient status for one or more genes in the HDR pathway.
As used herein, deficient status for a gene means the sequence,
structure, expression and/or activity of the gene or its product
is/are deficient as compared to normal. Examples include, but are
not limited to, low or no mRNA or protein expression, deleterious
mutations, hypermethylation, attenuated activity (e.g., enzymatic
activity, ability to bind to another biomolecule), etc. As used
herein, deficient status for a pathway (e.g., HDR pathway) means at
least one gene in that pathway (e.g., BRCA1) has a deficient
status. Examples of highly deleterious mutations include frameshift
mutations, stop codon mutations, and mutations that lead to altered
RNA splicing. Deficient status in a gene in the HDR pathway may
result in deficiency or reduced activity in homology directed
repair in the cancer cells. Examples of genes in the HDR pathway
include, without limitation, the genes listed in Table 1.
TABLE-US-00001 TABLE 1 Selected HDR Pathway Genes Entrez Gene Gene
Symbol (if Entrez Name different) Gene Id BLM BLM 641 BRCA1 BRCA1
672 BRCA2 BRCA2 675 CtIP RBBP8 5932 DNA POLD1 5424 polymerase POLD2
5424 delta POLD3 10714 POLD4 57804 DNA POLH 5429 polymerase eta
DNA2 DNA2 1763 EME1 EME1 146956 ERCC1 ERCC1 2067 EXO1 EXO1 9156
FANCM FANCM 57697 GEN1 GEN1 348654 MRE11 MRE11A 4361 MUS81 MUS81
80198 NBS1 NBN 4683 PALB2 PALB2 79728 PCNA PCNA 5111 RAD50 RAD50
10111 RAD51 RAD51 5888 RAD51AP1 RAD51AP1 10635 RAD51B RAD51L1 5890
RAD51C RAD51C 5889 RAD51D RAD51L3 5892 RAD54 ATRX 546 RAD54B RAD54B
25788 RMI1 RMI1 80010 RMI2 C16orf75 116028 RPA RPA1 6117 RTEL1
RTEL1 51750 SLX1 SLX2 SLX4 SLX4 84464 TOP2A TOP2A 7153 XPF ERCC4
2072 XRCC2 XRCC2 7516 XRCC3 XRCC3 7517
[0058] Examples of genetic mutations that can be present within a
gene of the HDR pathway include, without limitation, those listed
in Table 2.
TABLE-US-00002 TABLE 2 Possible genetic mutations within selected
genes of the HDR pathway. Gene Mutation Entrez Gene ID BRCA1 C24F
672 BRCA1 E29X 672 BRCA2 R3052W 675 BRCA2 2881delG 675 RAD51C G125V
5889 RAD51C L138F 5889 RAD51C Y75XfsX0 5889
[0059] In some cases, a cellular sample (e.g., cancer cell sample)
can be identified as having an increased number of LOH regions
(e.g., at least 7, 8, 9, 10, or more LOH regions) that cover the
whole chromosome. Cells (e.g., cancer cells) identified as having
an increased number of LOH regions that cover the whole chromosome
can be classified as having an increased likelihood of having
intact genes in the HDR pathway. For example, cancer cells
identified as having an increased number of LOH regions that cover
the whole chromosome can be classified as being more likely to have
intact BRCA1 and BRCA2 genes.
[0060] As described herein, identifying LOH loci (as well as the
size and number of LOH regions) can include, first, determining the
genotype of a sample at various genomic loci (e.g., SNP loci,
individual bases in large sequencing) and, second, determining
whether homozygous loci are due to LOH events. Any appropriate
technique can be used to determine genotypes at loci of interest
within the genome of a cell. For example, single nucleotide
polymorphisms (SNP) arrays (e.g., human genome-wide SNP arrays),
targeted sequencing of loci of interest (e.g., sequencing SNP loci
and their surrounding sequences), and even untargeted sequencing
(e.g., whole exome, transcriptome, or genome sequencing) can be
used to identify loci as being homozygous or heterozygous. In some
cases, an analysis of the homozygous or heterozygous nature of loci
over a length of a chromosome can be performed to determine the
length of regions of homozygosity or heterozygosity. For example, a
stretch of SNP locations that are spaced apart (e.g., spaced about
25 kb to about 100 kb apart) along a chromosome can be evaluated
using SNP array results to determine not only the presence of a
region of homozygosity along a chromosome but also the length of
that region. Results from a SNP array can be used to generate a
graph that plots allele dosages along a chromosome. Allele dosage
di for SNP i can be calculated from adjusted signal intensities of
two alleles (A.sub.i and B.sub.i):
d.sub.i=A.sub.i/(A.sub.i+B.sub.i). An example of such a graph is
presented in FIG. 1.
[0061] Once a sample's genotype has been determined for a plurality
of loci (e.g., SNPs), common techniques can be used to identify
loci and regions of LOH. One way to determine whether homozygosity
is due to LOH is to compare the somatic genotype to the germline.
For example, the genotype for a plurality of loci (e.g., SNPs) can
be determined in both a germline (e.g., blood) sample and a somatic
(e.g., tumor) sample. The genotypes for each sample can be compared
(typically computationally) to determine where the genome of the
germline cell was heterozygous and the genome of the somatic cell
is homozygous. Such loci are LOH loci and regions of such loci are
LOH regions.
[0062] Computational techniques can also be used to determine
whether homozygosity is due to LOH. Such techniques are
particularly useful when a germline sample is not available for
analysis and comparison. For example, algorithms such as those
described elsewhere can be used to detect LOH regions using
information from SNP arrays (Nannya et al., CANCER RES. (2005)
65:6071-6079). Typically these algorithms do not explicitly take
into account contamination of tumor samples with benign tissue. Cf.
International Application No. PCT/US2011/026098 to Abkevish et al.;
Goransson et al., PLoS ONE (2009) 4(6):e6057. This contamination is
often high enough to make the detection of LOH regions challenging.
Improved analytical methods according to the present invention for
identifying LOH, even in spite of contamination, include those
embodied in computer software products as described below.
[0063] The following is one example. If the observed ratio of the
signals of two alleles, A and B, is two to one, there are two
possibilities. The first possibility is that cancer cells have LOH
with deletion of allele B in a sample with 50% contamination with
normal cells. The second possibility is that there is no LOH but
allele A is duplicated in a sample with no contamination with
normal cells. An algorithm can be implemented as a computer program
as described herein to reconstruct LOH regions based on genotype
(e.g., SNP genotype) data. One point of the algorithm is to first
reconstruct allele specific copy numbers (ASCN) at each locus
(e.g., SNP). ASCNs are the numbers of copies of both paternal and
maternal alleles. An LOH region is then determined as a stretch of
SNPs with one of the ASCNs (paternal or maternal) being zero. The
algorithm can be based on maximizing a likelihood function and can
be conceptually akin to a previously described algorithm designed
to reconstruct total copy number (rather than ASCN) at each locus
(e.g., SNP). See International Application No. PCT/US2011/026098 to
Abkevish et al. The likelihood function can be maximized over ASCN
of all loci, level of contamination with benign tissue, total copy
number averaged over the whole genome, and sample specific noise
level. The input data for the algorithm can include or consist of
(1) sample-specific normalized signal intensities for both allele
of each locus and (2) assay-specific (specific for different SNP
arrays and for sequence based approach) set of parameters defined
based on analysis of large number of samples with known ASCN
profiles.
[0064] In some cases, nucleic acid sequencing techniques can be
used to identify loci as being homozygous or heterozygous. For
example, genomic DNA from a cell sample (e.g., a cancer cell
sample) can be extracted and fragmented. Any appropriate method can
be used to extract and fragment genomic nucleic acid including,
without limitation, commercial kits such as QIAamp DNA Mini Kit
(Qiagen), MagNA Pure DNA Isolation Kit (Roche Applied Science) and
GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). Once
extracted and fragmented, either targeted or untargeted sequencing
can be done to determine the sample's genotypes at loci. For
example, whole genome, whole transcriptome, or whole exome
sequencing can be done to determine genotypes at millions or even
billions of base pairs (i.e., base pairs can be "loci" to be
evaluated).
[0065] In some cases, targeted sequencing of known polymorphic loci
(e.g., SNPs and surrounding sequences) can be done as an
alternative to microarray analysis. For example, the genomic DNA
can be enriched for those fragments containing a locus (e.g., SNP
location) to be analyzed using kits designed for this purpose
(e.g., Agilent SureSelect, Illumina TruSeq Capture, and Nimblegen
SeqCap EZ Choice). For example, genomic DNA containing the loci to
be analyzed can be hybridized to biotinylated capture RNA fragments
to form biotinylated RNA/genomic DNA complexes. Alternatively, DNA
capture probes may be utilized resulting in the formation of
biotinylated DNA/genomic DNA hybrids. Streptavidin coated magnetic
beads and a magnetic force can be used to separate the biotinylated
RNA/genomic DNA complexes from those genomic DNA fragments not
present within a biotinylated RNA/genomic DNA complex. The obtained
biotinylated RNA/genomic DNA complexes can be treated to remove the
captured RNA from the magnetic beads, thereby leaving intact
genomic DNA fragments containing a locus to be analyzed. These
intact genomic DNA fragments containing the loci to be analyzed can
be amplified using, for example, PCR techniques. The amplified
genomic DNA fragments can be sequenced using a high-throughput
sequencing technology or a next-generation sequencing technology
such as Illumina HiSeq, Illumina MiSeq, Life Technologies SoLID, or
Roche's 454.
[0066] The sequencing results from the genomic DNA fragments can be
used to identify loci as being homozygous or heterozygous,
analogous to the microarray analysis described herein. In some
cases, an analysis of the homozygous or heterozygous nature of loci
over a length of a chromosome can be performed to determine the
length of regions of homozygosity or heterozygosity. For example, a
stretch of SNP locations that are spaced apart (e.g., spaced about
25 kb to about 100 kb apart) along a chromosome can be evaluated by
sequencing, and the sequencing results used to determine not only
the presence of a region of homozygosity along a chromosome but
also the length of that LOH region. Obtained sequencing results can
be used to generate a graph that plots allele dosages along a
chromosome. Allele dosage di for SNP i can be calculated from
adjusted number of captured probes for two alleles (A.sub.i and
B.sub.i): d.sub.i=.A.sub.i/(A.sub.i+B.sub.i). An example of such a
graph is presented in FIG. 2. Determining whether homozygosity is
due to LOH (as opposed to homozygosity in the germline) can be
performed as described herein.
[0067] In some cases, a selection process can be used to select
loci (e.g., SNP loci) to be evaluated using an assay configured to
identify loci as being homozygous or heterozygous (e.g., SNP
array-based assays and sequencing-based assays). For example, any
human SNP location can be selected for inclusion in a SNP
array-based assay or a sequencing-based assay configured to
identify loci as being homozygous or heterozygous within the genome
of cells. In some cases, 0.5, 1.0, 1.5, 2.0, 2.5 million or more
SNP locations present within the human genome can be evaluated to
identify those SNPs that (a) are not present on the Y chromosome,
(b) are not mitochondrial SNPs, (c) have a minor allele frequency
of at least about five percent in Caucasians, (d) have a minor
allele frequency of at least about one percent in three races other
than Caucasians (e.g., Chinese, Japanese, and Yoruba), and/or (e)
do not have a significant deviation from Hardy Weinberg equilibrium
in any of the four races. In some cases, more than 100,000,
150,000, or 200,000 human SNPs can be selected that meet criteria
(a) through (e). Of the human SNPs meeting criteria (a) through
(e), a group of SNPs (e.g., top 110,000 SNPs) can be selected such
that the SNPs have a high degree of allele frequency in Caucasians,
cover the human genome in a somewhat evenly spaced manner (e.g., at
least one SNP every about 25 kb to about 500 kb), and are not in
linkage disequilibrium with another selected SNP for in any of the
four races. In some cases, about 40, 50, 60, 70, 80, 90, 100, 110,
120, 130 thousand or more SNPs can be selected as meeting each of
these criteria and included in an assay configured to identify LOH
regions across a human genome. For example, between about 70,000
and about 90,000 (e.g., about 80,000) SNPs can be selected for
analysis with a SNP array-based assay, and between about 45,000 and
about 55,000 (e.g., about 54,000) SNPs can be selected for analysis
with a sequencing-based assay.
[0068] As described herein, a cell sample can be assessed to
determine if the genome of cells of the sample contains an LOH
signature, lacks an LOH signature, has an increased number of LOH
regions that cover the whole chromosome, or lacks an increased
number of LOH regions that cover the whole chromosome. Any
appropriate type of sample can be assessed. For example, a sample
containing cancer cells can be assessed to determine if the genome
of the cancer cells contains an LOH signature, lacks an LOH
signature, has an increased number of LOH regions that cover the
whole chromosome, or lacks an increased number of LOH regions that
cover the whole chromosome. Examples of samples containing cancer
cells that can be assessed as described herein include, without
limitation, tumor biopsy samples (e.g., breast tumor biopsy
samples), formalin-fixed, paraffin-embedded tissue samples
containing cancer cells, core needle biopsies, fine needle
aspirates, and samples containing cancer cells shed from a tumor
(e.g., blood, urine or other bodily fluids). For formalin-fixed,
paraffin-embedded tissue samples, the sample can be prepared by DNA
extraction using a genomic DNA extraction kit optimized for FFPE
tissue, including but not limited to those described above (e.g.,
QuickExtract FFPE DNA Extraction Kit (Epicentre), and QIAamp DNA
FFPE Tissue Kit (Qiagen)).
[0069] In some cases, laser dissection techniques can be performed
on a tissue sample to minimize the number of non-cancer cells
within a cancer cell sample to be assessed. In some cases, antibody
based purification methods can be used to enrich for cancer cells
and/or deplete non-cancer cells. Examples of antibodies that could
be used for cancer cell enrichment include, without limitation,
anti-EpCAM, anti-TROP-2, anti-c-Met, anti-Folate binding protein,
anti-N-Cadherin, anti-CD318, anti-antimesencymal stem cell antigen,
anti-Her2, anti-MUC1, anti-EGFR, anti-cytokeratins (e.g.,
cytokeratin 7, cytokeratin 20, etc.), anti-Caveolin-1, anti-PSA,
anti-CA125, and anti-surfactant protein antibodies.
[0070] Any type of cancer cell can be assessed using the methods
and materials described herein. For example, breast cancer cells,
ovarian cancer cells, liver cancer cells, esophageal cancer cells,
lung cancer cells, head and neck cancer cells, prostate cancer
cells, colon, rectal, or colorectal cancer cells, and pancreatic
cancer cells can be assessed to determine if the genome of the
cancer cells contains an LOH signature, lacks an LOH signature, has
an increased number of LOH regions that cover the whole chromosome,
or lacks an increased number of LOH regions that cover the whole
chromosome.
[0071] When assessing the genome of cancer cells for the presence
or absence of an LOH signature, one or more (e.g., one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, or 23) pairs of chromosomes can be
assessed. In some cases, the genome of cancer cells is assessed for
the presence or absence of an LOH signature using one or more
(e.g., one, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) pairs
of chromosomes.
[0072] In some cases, it can be helpful to exclude certain
chromosomes from this analysis. For example, in the case of
females, a pair to be assessed can include the pair of X sex
chromosomes; whereas, in the case of males, a pair of any autosomal
chromosomes (i.e., any pair other than the pair of X and Y sex
chromosomes) can be assessed. As another example, in some cases the
chromosome number 17 pair may be excluded from the analysis. It has
been determined that certain chromosomes carry unusually high
levels of LOH in certain cancers and, thus, it can be helpful to
exclude such chromosomes when analyzing samples as described herein
from patients having these cancers. In some cases, the sample is
from a patient having ovarian cancer, and the chromosome to be
excluded is chromosome 17.
[0073] When assessing the genome of cancer cells for the presence
or absence of an increased number of LOH regions that cover the
whole chromosome, 10 or more (e.g., 13, 16, 19 or 23) pairs of
chromosomes can be assessed. In the case of females, a pair to be
assessed can include the pair of X sex chromosomes; whereas, in the
case of males, a pair of any autosomal chromosomes (i.e., any pair
other than the pair of X and Y sex chromosomes) can be assessed. In
some cases, the chromosome number 17 pair may be excluded from the
analysis. In some cases, the sample is from a patient having
ovarian cancer, and the chromosome to be excluded is chromosome 17.
In some cases, the genome of cancer cells is assessed for the
presence or absence of an increased number of LOH regions that
cover the whole chromosome using 10 or more (e.g., 13, 16 19, or
23) pairs of chromosomes.
[0074] As described herein, patients having cancer cells identified
as having a positive LOH signature status can be classified, based
at least in part on a positive LOH signature status, as being
likely to respond to a particular cancer treatment regimen. For
example, patients having cancer cells with a genome containing an
LOH signature can be classified, based at least in part on a
positive LOH signature status, as being likely to respond to a
cancer treatment regimen that includes the use of a DNA damaging
agent, a PARP inhibitor, radiation, or a combination thereof.
Examples of DNA damaging agents include, without limitation,
platinum-based chemotherapy drugs (e.g., cisplatin, carboplatin,
oxaliplatin, and picoplatin), anthracyclines (e.g., epirubicin and
doxorubicin), topoisomerase I inhibitors (e.g., campothecin,
topotecan, and irinotecan), and triazene compounds (e.g.,
dacarbazine and temozolomide). Examples of PARP inhibitors include,
without limitation, olaparib, iniparib, and veliparib. Examples of
information that can be used in addition to a positive LOH
signature status to base a classification of being likely to
respond to a particular cancer treatment regimen include, without
limitation, previous treatment results, germline or somatic DNA
mutations, gene or protein expression profiling (e.g., ER/PR/HER2
status, PSA levels), tumor histology (e.g., adenocarcinoma,
squamous cell carcinoma, papillary serous carcinoma, mucinous
carcinoma, invasive ductal carcinoma, ductal carcinoma insitu
(non-invasive), etc.), disease stage, tumor or cancer grade (e.g.,
well, moderately, or poorly differentiated (e.g., Gleason, modified
Bloom Richardson), etc.), number of previous courses of treatment,
etc.
[0075] Once classified as being likely to respond to a particular
cancer treatment regimen (e.g., a cancer treatment regimen that
includes the use of a DNA damaging agent, a PARP inhibitor,
radiation, or a combination thereof), the cancer patient can be
treated with such a cancer treatment regimen. Any appropriate
method for treating the cancer at issue can be used to treat a
cancer patient identified as having cancer cells having a positive
LOH signature status. For example, platinum-based chemotherapy
drugs or a combination of platinum-based chemotherapy drugs can be
used to treat cancer as described elsewhere (see, e.g., U.S. Pat.
Nos. 3,892,790, 3,904,663, 7,759,510, 7,759,488 and 7,754,684. In
some cases, anthracyclines or a combination of anthracyclines can
be used to treat cancer as described elsewhere (see, e.g., U.S.
Pat. Nos. 3,590,028, 4,138,480, 4,950,738, 6,087,340, 7,868,040,
and 7,485,707. In some cases, topoisomerase I inhibitors or a
combination of topoisomerase I inhibitors can be used to treat
cancer as described elsewhere (see, e.g., U.S. Pat. Nos. 5,633,016
and 6,403,563. In some cases, PARP inhibitors or a combination of
PARP inhibitors can be used to treat cancer as described elsewhere
(see, e.g., U.S. Pat. Nos. 5,177,075, 7,915,280, and 7,351,701. In
some cases, radiation can be used to treat cancer as described
elsewhere (see, e.g., U.S. Pat. No. 5,295,944). In some cases, a
combination comprising different agents (e.g., a combination
comprising any of platinum-based chemotherapy drugs,
anthracyclines, topoisomerase I inhibitors, and/or PARP inhibitors)
with or without radiation treatments can be used to treat cancer.
In some cases, a combination treatment may comprise any of the
above agents or treatments (e.g., a DNA damaging agent, a PARP
inhibitor, radiation, or a combination thereof) together with
another agent or treatment--e.g., a taxane agent (e.g., doxetaxel,
paclitaxel, abraxane), a growth factor or growth factor receptor
inhibitor (e.g., erlotinib, gefitinib, lapatinib, sunitinib,
bevacizumab, cetuximab, trastuzumab, panitumumab), and/or an
antimetabolite (e.g., 5-flourouracil, methotrexate).
[0076] In some cases, patients identified as having cancer cells
with a genome lacking an LOH signature can be classified, based at
least in part on a negative LOH signature status, as being less
likely to respond to a treatment regimen that includes a DNA
damaging agent, a PARP inhibitor, radiation, or a combination
thereof. In turn, such a patient can be classified as likely to
respond to a cancer treatment regimen that includes the use of one
or more cancer treatment agents not associated with HDR, such as a
taxane agent (e.g., doxetaxel, paclitaxel, abraxane), a growth
factor or growth factor receptor inhibitor (e.g., erlotinib,
gefitinib, lapatinib, sunitinib, bevacizumab, cetuximab,
trastuzumab, panitumumab), and/or an antimetabolite agent (e.g.,
5-flourouracil, methotrexate). Once classified as being likely to
respond to a particular cancer treatment regimen (e.g., a cancer
treatment regimen that includes the use of a cancer treatment agent
not associated with HDR), the cancer patient can be treated with
such a cancer treatment regimen. Any appropriate method for the
cancer being treated can be used to treat a cancer patient
identified as having cancer cells having a negative LOH signature
status. Examples of information that can be used in addition to a
negative LOH signature status to base a classification of being
likely to respond to a particular cancer treatment regimen include,
without limitation, previous treatment results, germline or somatic
DNA mutations, gene or protein expression profiling (e.g.,
ER/PR/HER2 status, PSA levels), tumor histology (e.g.,
adenocarcinoma, squamous cell carcinoma, papillary serous
carcinoma, mucinous carcinoma, invasive ductal carcinoma, ductal
carcinoma in situ (non-invasive), etc.), disease stage, tumor or
cancer grade (e.g., well, moderately, or poorly differentiated
(e.g., Gleason, modified Bloom Richardson), etc.), number of
previous courses of treatment, etc.
[0077] Once treated for a particular period of time (e.g., between
one to six months), the patient can be assessed to determine
whether or not the treatment regimen has an effect. If a beneficial
effect is detected, the patient can continue with the same or a
similar cancer treatment regimen. If a minimal or no beneficial
effect is detected, then adjustments to the cancer treatment
regimen can be made. For example, the dose, frequency of
administration, or duration of treatment can be increased. In some
cases, additional anti-cancer agents can be added to the treatment
regimen or a particular anti-cancer agent can be replaced with one
or more different anti-cancer agents. The patient being treated can
continue to be monitored as appropriate, and changes can be made to
the cancer treatment regimen as appropriate.
[0078] As described herein, this document provides methods for
assessing patients for cells (e.g., cancer cells) having a genome
containing an LOH signature. For example, one or more clinicians or
medical professionals can determine if a patient contains cancer
cells having a genome containing an LOH signature. In some cases,
one or more clinicians or medical professionals can determine if a
patient contains cancer cells having a genome containing an LOH
signature by obtaining a cancer cell sample from the patient and
assessing the genome of cancer cells of the cancer cell sample to
determine the presence or absence of an LOH signature as described
herein.
[0079] In some cases, one or more clinicians or medical
professionals can obtain a cancer cell sample from a patient and
provide that sample to a testing laboratory having the ability to
assess the genome of cancer cells of the cancer cell sample to
provide an indication about the presence or absence of an LOH
signature as described herein. In such cases, the one or more
clinicians or medical professionals can determine if a patient
contains cancer cells having a genome containing an LOH signature
by receiving information about the presence or absence of an LOH
signature directly or indirectly from the testing laboratory. For
example, a testing laboratory, after assessing the genome of cancer
cells for presence or absence of an LOH signature as described
herein, can provide a clinician or medical professional with, or
access to, a written, electronic, or oral report or medical record
that provides an indication about the presence or absence of an LOH
signature for a particular patient being assessed. Such a written,
electronic, or oral report or medical record can allow the one or
more clinicians or medical professionals to determine if a
particular patient being assessed contains cancer cells having a
genome containing an LOH signature.
[0080] Once a clinician or medical professional or group of
clinicians or medical professionals determines that a particular
patient being assessed contains cancer cells having a genome
containing an LOH signature, the clinician or medical professional
(or group) can classify that patient as having cancer cells whose
genome contains the presence of an LOH signature. In some cases, a
clinician or medical professional or group of clinicians or medical
professionals can diagnose a patient determined to have cancer
cells whose genome contains the presence of an LOH signature as
having cancer cells likely to be deficient in HDR. Such a diagnosis
can be based solely on a determination that a particular patient
being assessed contains cancer cells having a genome containing an
LOH signature or can be based at least in part on a determination
that a particular patient being assessed contains cancer cells
having a genome containing an LOH signature. For example, a patient
determined to have cancer cells whose genome contains the presence
of an LOH signature can be diagnosed as likely to be deficient in
HDR based on the combination of a positive LOH signature status and
deficient status in one or more tumor suppressor genes (e.g.,
BRCA1/2, RAD51), a family history of cancer, or the presence of
behavioral risk factors (e.g., smoking).
[0081] In some cases, a clinician or medical professional or group
of clinicians or medical professionals can diagnose a patient
determined to have cancer cells whose genome contains the presence
of an LOH signature as having cancer cells likely to contain
genetic mutations in one or more genes in the HDR pathway. Such a
diagnosis can be based solely on a determination that a particular
patient being assessed contains cancer cells having a genome
containing an LOH signature or can be based at least in part on a
determination that a particular patient being assessed contains
cancer cells having a genome containing an LOH signature. For
example, a patient determined to have cancer cells whose genome
contains the presence of an LOH signature can be diagnosed as
having cancer cells likely to contain genetic mutations in one or
more genes in the HDR pathway based on the combination of a
positive LOH positive status and a family history of cancer, or the
presence of behavioral risk factors (e.g., smoking).
[0082] In some cases, a clinician or medical professional or group
of clinicians or medical professionals can diagnose a patient
determined to have cancer cells whose genome contains the presence
of an LOH signature as having cancer cells likely to respond to a
particular cancer treatment regimen. Such a diagnosis can be based
solely on a determination that a particular patient being assessed
contains cancer cells having a genome containing an LOH signature
or can be based at least in part on a determination that a
particular patient being assessed contains cancer cells having a
genome containing an LOH signature. For example, a patient
determined to have cancer cells whose genome contains the presence
of an LOH signature can be diagnosed as being likely to respond to
a particular cancer treatment regimen based on the combination of a
positive LOH signature status and deficient status in one or more
tumor suppressor genes (e.g., BRCA1/2, RAD51), a family history of
cancer, or the presence of behavioral risk factors (e.g., smoking).
As described herein, a patient determined to have cancer cells
whose genome contains the presence of an LOH signature can be
diagnosed as likely to respond to a cancer treatment regimen that
includes the use of a platinum-based chemotherapy drug such as
cisplatin, carboplatin, oxaliplatin, or picoplatin, an
anthracycline such as epirubicin or doxorubicin, a topoisomerase I
inhibitor such as campothecin, topotecan, or irinotecan, a PARP
inhibitor, radiation, a combination thereof, or a combination of
any of the preceding with another anti-cancer agent.
[0083] Once a clinician or medical professional or group of
clinicians or medical professionals determines that a particular
patient being assessed contains cancer cells having a genome
lacking an LOH signature, the clinician or medical professional (or
group) can classify that patient as having cancer cells whose
genome contains an absence of an LOH signature. In some cases, a
clinician or medical professional or group of clinicians or medical
professionals can diagnose a patient determined to have cancer
cells containing a genome that lacks the presence of an LOH
signature as having cancer cells likely to have functional HDR. In
some cases, a clinician or medical professional or group of
clinicians or medical professionals can diagnose a patient
determined to have cancer cells containing a genome that lacks the
presence of an LOH signature as having cancer cells that do not
likely contain genetic mutations in one or more genes in the HDR
pathway. In some cases, a clinician or medical professional or
group of clinicians or medical professionals can diagnose a patient
determined to have cancer cells containing a genome that lacks the
presence of an LOH signature or contains an increased number of LOH
regions that cover the whole chromosome as having cancer cells that
are less likely to respond to a platinum-based chemotherapy drug
such as cisplatin, carboplatin, oxalaplatin, or picoplatin, an
anthracycline such as epirubincin or doxorubicin, a topoisomerase I
inhibitor such as campothecin, topotecan, or irinotecan, a PARP
inhibitor, or radiation and/or more likely to respond to a cancer
treatment regimen that includes the use of a cancer treatment agent
not associated with HDR such as one or more taxane agents, growth
factor or growth factor receptor inhibitors, anti-metabolite
agents, etc.
[0084] As described herein, this document also provides methods for
performing a diagnostic analysis of a nucleic acid sample (e.g., a
genomic nucleic acid sample or amplified genomic nucleic acid
sample) of a cancer patient to determine if cancer cells within the
patient have a genome containing an LOH signature and/or an
increased number of LOH regions that cover the whole chromosome.
For example, one or more laboratory technicians or laboratory
professionals can detect the presence or absence of an LOH
signature in the genome of cancer cells of the patient or the
presence or absence of an increased number of LOH regions that
cover the whole chromosome in the genome of cancer cells of the
patient. In some cases, one or more laboratory technicians or
laboratory professionals can detect the presence or absence of an
LOH signature or the presence or absence of an increased number of
LOH regions that cover the whole chromosome in the genome of cancer
cells of the patient by (a) receiving a cancer cell sample obtained
from the patient, receiving a genomic nucleic acid sample obtained
from cancer cells obtained from the patient, or receiving an
enriched and/or amplified genomic nucleic acid sample obtained from
cancer cells obtained from the patient and (b) performing an
analysis (e.g., a SNP array-based assay or a sequencing-based
assay) using the received material to detect the presence or
absence of an LOH signature or the presence or absence of an
increased number of LOH regions that cover the whole chromosome as
described herein. In some cases, one or more laboratory technicians
or laboratory professionals can receive a sample to be analyzed
(e.g., a cancer cell sample obtained from the patient, a genomic
nucleic acid sample obtained from cancer cells obtained from the
patient, or an enriched and/or amplified genomic nucleic acid
sample obtained from cancer cells obtained from the patient)
directly or indirectly from a clinician or medical
professional.
[0085] Once a laboratory technician or laboratory professional or
group of laboratory technicians or laboratory professionals detects
the presence of an LOH signature as described herein, the
laboratory technician or laboratory professional (or group) can
identify the patient whose cancer cells were detected as having an
LOH signature as having cancer cells with a positive LOH signature
status. For example, one or more laboratory technicians or
laboratory professionals can identify a patient having cancer cells
that were detected to have an LOH signature as having cancer cells
with a positive LOH signature status by associating that positive
LOH signature status or the result (or results or a summary of
results) of the performed diagnostic analysis with the
corresponding patient's name, medical record, symbolic/numerical
identifier, or a combination thereof. In some cases, a laboratory
technician or laboratory professional or group of laboratory
technicians or laboratory professionals can identify a patient
having cancer cells that were detected to have an LOH signature as
having cancer cells potentially deficient in HDR by associating the
positive LOH signature status, the potentially deficient in HDR
status, or the result (or results or a summary of results) of the
performed diagnostic analysis with the corresponding patient's
name, medical record, symbolic/numerical identifier, or a
combination thereof. Such identification can be based solely on
detecting the presence of an LOH signature or can be based at least
in part on detecting the presence of an LOH signature. For example,
a laboratory technician or laboratory professional can identify a
patient having cancer cells that were detected to have an LOH
signature as having cancer cells potentially deficient in HDR based
on a combination of a positive LOH signature status and the results
of other genetic and biochemical tests performed at the testing
laboratory.
[0086] In some cases, a laboratory technician or laboratory
professional or group of laboratory technicians or laboratory
professionals can identify a patient having cancer cells that were
detected to have an LOH signature as having cancer cells
potentially containing a genetic mutation in one or more genes in
the HDR pathway by associating the positive LOH signature status,
the potential presence of a genetic mutation in one or more genes
in the HDR pathway, or the result (or results or a summary of
results) of the performed diagnostic analysis with the
corresponding patient's name, medical record, symbolic/numerical
identifier, or a combination thereof. Such identification can be
based solely on detecting the presence of an LOH signature or can
be based at least in part on detecting the presence of an LOH
signature. For example, a laboratory technician or laboratory
professional can identify a patient having cancer cells that were
detected to have an LOH signature as having cancer cells
potentially containing a genetic mutation in one or more genes in
the HDR pathway based on a combination of a positive LOH signature
status and the results of other genetic and biochemical tests
performed at the testing laboratory.
[0087] In some cases, a laboratory technician or laboratory
professional or group of laboratory technicians or laboratory
professionals can identify a patient having cancer cells that were
detected to have an LOH signature as having cancer cells likely to
respond to a particular cancer treatment regimen by associating the
positive LOH signature status, a potentially deficient HDR status,
a potential presence of a deficient status in one or more genes in
the HDR pathway, or the result (or results or a summary of results)
of the performed diagnostic analysis with the corresponding
patient's name, medical record, symbolic/numerical identifier, or a
combination thereof. Such identification can be based solely on
detecting the presence of an LOH signature or can be based at least
in part on detecting the presence of an LOH signature. For example,
a laboratory technician or laboratory professional can identify a
patient having cancer cells that were detected to have an LOH
signature as having cancer cells likely to respond to a particular
cancer treatment regimen based on a combination of a positive LOH
signature status and the results of other genetic and biochemical
tests performed at the testing laboratory.
[0088] Once a laboratory technician or laboratory professional or
group of laboratory technicians or laboratory professionals detects
the absence of an LOH signature, the laboratory technician or
laboratory professional (or group) can identify the patient whose
cancer cells were detected as lacking an LOH signature as having
cancer cells with a negative LOH signature status. For example, one
or more laboratory technicians or laboratory professionals can
identify a patient having cancer cells that were detected to lack
an LOH signature as having cancer cells with a negative LOH
signature status by associating that negative LOH signature status
or the result (or results or a summary of results) of the performed
diagnostic analysis with the corresponding patient's name, medical
record, symbolic/numerical identifier, or a combination thereof. In
some cases, a laboratory technician or laboratory professional or
group of laboratory technicians or laboratory professionals can
identify a patient having cancer cells that were detected to lack
an LOH signature as having cancer cells with potentially intact HDR
by associating the negative LOH signature status, the potentially
intact HDR status, or the result (or results or a summary of
results) of the performed diagnostic analysis with the
corresponding patient's name, medical record, symbolic/numerical
identifier, or a combination thereof.
[0089] In some cases, a laboratory technician or laboratory
professional or group of laboratory technicians or laboratory
professionals can identify a patient having cancer cells that were
detected to lack an LOH signature as having cancer cells with
potentially intact genes of the HDR pathway by associating the
negative LOH signature status, the potential absence of genetic
mutations in genes of the HDR pathway, or the result (or results or
a summary of results) of the performed diagnostic analysis with the
corresponding patient's name, medical record, symbolic/numerical
identifier, or a combination thereof.
[0090] In some cases, a laboratory technician or laboratory
professional or group of laboratory technicians or laboratory
professionals can identify a patient having cancer cells that were
detected to lack an LOH signature as having cancer cells as less
likely to respond to one particular treatment (e.g., a
platinum-based chemotherapy drug such as cisplatin, carboplatin,
oxalaplatin, or picoplatin, an anthracycline such as epirubincin or
doxorubicin, a topoisomerase I inhibitor such as campothecin,
topotecan, or irinotecan, a PARP inhibitor such as iniparib,
olaparib, or velapirib, or radiation) and/or more likely to respond
to a particular cancer treatment regimen (e.g., a cancer treatment
regimen that includes the use of a cancer treatment agent not
associated with HDR) by associating the negative LOH signature
status, a potentially intact HDR status, a potential absence of
genetic mutations in genes of the HDR pathway, or the result (or
results or a summary of results) of the performed diagnostic
analysis with the corresponding patient's name, medical record,
symbolic/numerical identifier, or a combination thereof.
[0091] Once a laboratory technician or laboratory professional or
group of laboratory technicians or laboratory professionals detects
the presence of an increased number of LOH regions that cover the
whole chromosome, the laboratory technician or laboratory
professional (or group) can identify the patient whose cancer cells
were detected as having an increased number of LOH regions that
cover the whole chromosome as likely having cancer cells with an
intact BRCA1 and BRCA2 status. For example, one or more laboratory
technicians or laboratory professionals can identify a patient
having cancer cells that were detected to have an increased number
of LOH regions that cover the whole chromosome as likely having
cancer cells with an intact BRCA1 and BRCA2 status by associating
the presence of an increased number of LOH regions that cover the
whole chromosome or the result (or results or a summary of results)
of the performed diagnostic analysis with the corresponding
patient's name, medical record, symbolic/numerical identifier, or a
combination thereof.
[0092] FIG. 15 shows an exemplary process by which a computing
system (or a computer program (e.g., software) containing
computer-executable instructions) can identify LOH loci or regions
from genotype data as described herein. If the observed ratio of
the signals of two alleles, A and B, is two to one, there are two
possibilities. The first possibility is that cancer cells have LOH
with deletion of allele B in a sample with 50% contamination with
normal cells. The second possibility is that there is no LOH but
allele A is duplicated in a sample with no contamination with
normal cells. The process begins at box 1500, where the following
data are collected by the computing system; (1) sample-specific
normalized signal intensities for both alleles of each locus and
(2) assay-specific (specific for different SNP arrays and for
sequence based approach) set of parameters defined based on
analysis of large number of samples with known ASCN profiles. As
described herein, any appropriate assay such as a SNP array-based
assay or sequencing-based assay can be used to assess loci along a
chromosome for homozygosity or heterozygosity. In some cases, a
system including a signal detector and a computer can be used to
collect data (e.g., fluorescent signals or sequencing results)
regarding the homozygous or heterozygous nature of the plurality of
loci (e.g., sample-specific normalized signal intensities for both
alleles of each locus). At box 1510, allele specific copy numbers
(ASCN) are reconstructed at each locus (e.g., each SNP). ASCNs are
the numbers of copies of both paternal and maternal alleles. At box
1530, a likelihood function is used to determine whether a
homozygous locus or region of homozygous loci is due to LOH. This
can be conceptually analogous to a previously described algorithm
designed to reconstruct total copy number (rather than ASCN) at
each locus (e.g., SNP). See International Application No.
PCT/US2011/026098 to Abkevish et al. The likelihood function can be
maximized over ASCN of all loci, level of contamination with benign
tissue, total copy number averaged over the whole genome, and
sample specific noise level. At box 1540, an LOH region is
determined as a stretch of SNPs with one of the ASCNs (paternal or
maternal) being zero.
[0093] FIG. 3 shows an exemplary process by which a computing
system can determine the presence or absence of an LOH signature.
The process begins at box 300, where data regarding the homozygous
or heterozygous nature of a plurality of loci along a chromosome is
collected by the computing system. As described herein, any
appropriate assay such as a SNP array-based assay or
sequencing-based assay can be used to assess loci along a
chromosome for homozygosity or heterozygosity. In some cases, a
system including a signal detector and a computer can be used to
collect data (e.g., fluorescent signals or sequencing results)
regarding the homozygous or heterozygous nature of the plurality of
loci. At box 310, data regarding the homozygous or heterozygous
nature of a plurality of loci as well as the location or spatial
relationship of each locus is assessed by the computing system to
determine the length of any LOH regions present along a chromosome.
At box 320, data regarding the number of LOH regions detected and
the length of each detected LOH region is assessed by the computing
system to determine the number of LOH regions that have a length
(a) greater than or equal to a preset number of Mb (e.g., 15 Mb)
and (b) less than the entire length of the chromosome containing
that LOH region. At box 330, the computing system formats an output
providing an indication of the presence or absence of an LOH
signature. Once formatted, the computing system can present the
output to a user (e.g., a laboratory technician, clinician, or
medical professional). As described herein, the presence or absence
of an LOH signature can be used to provide an indication about a
patient's likely HDR status, an indication about the likely
presence or absence of genetic mutations in genes of the HDR
pathway, and/or an indication about possible cancer treatment
regimens.
[0094] FIG. 4 is a diagram of an example of a computer device 1400
and a mobile computer device 1450, which may be used with the
techniques described herein. Computing device 1400 is intended to
represent various forms of digital computers, such as laptops,
desktops, workstations, personal digital assistants, servers, blade
servers, mainframes, and other appropriate computers. Computing
device 1450 is intended to represent various forms of mobile
devices, such as personal digital assistants, cellular telephones,
smart phones, and other similar computing devices. The components
shown here, their connections and relationships, and their
functions, are meant to be exemplary only, and are not meant to
limit implementations of the inventions described and/or claimed in
this document.
[0095] Computing device 1400 includes a processor 1402, memory
1404, a storage device 1406, a high-speed interface 1408 connecting
to memory 1404 and high-speed expansion ports 1410, and a low speed
interface 1415 connecting to low speed bus 1414 and storage device
1406. Each of the components 1402, 1404, 1406, 1408, 1410, and
1415, are interconnected using various busses, and may be mounted
on a common motherboard or in other manners as appropriate. The
processor 1402 can process instructions for execution within the
computing device 1400, including instructions stored in the memory
1404 or on the storage device 1406 to display graphical information
for a GUI on an external input/output device, such as display 1416
coupled to high speed interface 1408. In other implementations,
multiple processors and/or multiple buses may be used, as
appropriate, along with multiple memories and types of memory.
Also, multiple computing devices 1400 may be connected, with each
device providing portions of the necessary operations (e.g., as a
server bank, a group of blade servers, or a multi-processor
system).
[0096] The memory 1404 stores information within the computing
device 1400. In one implementation, the memory 1404 is a volatile
memory unit or units. In another implementation, the memory 1404 is
a non-volatile memory unit or units. The memory 1404 may also be
another form of computer-readable medium, such as a magnetic or
optical disk.
[0097] The storage device 1406 is capable of providing mass storage
for the computing device 1400. In one implementation, the storage
device 1406 may be or contain a computer-readable medium, such as a
floppy disk device, a hard disk device, an optical disk device, or
a tape device, a flash memory or other similar solid state memory
device, or an array of devices, including devices in a storage area
network or other configurations. A computer program product can be
tangibly embodied in an information carrier. The computer program
product may also contain instructions that, when executed, perform
one or more methods, such as those described herein. The
information carrier is a computer- or machine-readable medium, such
as the memory 1404, the storage device 1406, memory on processor
1402, or a propagated signal.
[0098] The high speed controller 1408 manages bandwidth-intensive
operations for the computing device 1400, while the low speed
controller 1415 manages lower bandwidth-intensive operations. Such
allocation of functions is exemplary only. In one implementation,
the high-speed controller 1408 is coupled to memory 1404, display
1416 (e.g., through a graphics processor or accelerator), and to
high-speed expansion ports 1410, which may accept various expansion
cards (not shown). In the implementation, low-speed controller 1415
is coupled to storage device 1406 and low-speed expansion port
1414. The low-speed expansion port, which may include various
communication ports (e.g., USB, Bluetooth, Ethernet, or wireless
Ethernet) may be coupled to one or more input/output devices, such
as a keyboard, a pointing device, a scanner, an optical reader, a
fluorescent signal detector, or a networking device such as a
switch or router, e.g., through a network adapter.
[0099] The computing device 1400 may be implemented in a number of
different forms, as shown in the figure. For example, it may be
implemented as a standard server 1420, or multiple times in a group
of such servers. It may also be implemented as part of a rack
server system 1424. In addition, it may be implemented in a
personal computer such as a laptop computer 1422. Alternatively,
components from computing device 1400 may be combined with other
components in a mobile device (not shown), such as device 1450.
Each of such devices may contain one or more of computing device
1400, 1450, and an entire system may be made up of multiple
computing devices 1400, 1450 communicating with each other.
[0100] Computing device 1450 includes a processor 1452, memory
1464, an input/output device such as a display 1454, a
communication interface 1466, and a transceiver 1468, among other
components (e.g., a scanner, an optical reader, a fluorescent
signal detector). The device 1450 may also be provided with a
storage device, such as a microdrive or other device, to provide
additional storage. Each of the components 1450, 1452, 1464, 1454,
1466, and 1468, are interconnected using various buses, and several
of the components may be mounted on a common motherboard or in
other manners as appropriate.
[0101] The processor 1452 can execute instructions within the
computing device 1450, including instructions stored in the memory
1464. The processor may be implemented as a chipset of chips that
include separate and multiple analog and digital processors. The
processor may provide, for example, for coordination of the other
components of the device 1450, such as control of user interfaces,
applications run by device 1450, and wireless communication by
device 1450.
[0102] Processor 1452 may communicate with a user through control
interface 1458 and display interface 1456 coupled to a display
1454. The display 1454 may be, for example, a TFT LCD
(Thin-Film-Transistor Liquid Crystal Display) or an OLED (Organic
Light Emitting Diode) display, or other appropriate display
technology. The display interface 1456 may comprise appropriate
circuitry for driving the display 1454 to present graphical and
other information to a user. The control interface 1458 may receive
commands from a user and convert them for submission to the
processor 1452. In addition, an external interface 1462 may be
provide in communication with processor 1452, so as to enable near
area communication of device 1450 with other devices. External
interface 1462 may provide, for example, for wired communication in
some implementations, or for wireless communication in other
implementations, and multiple interfaces may also be used.
[0103] The memory 1464 stores information within the computing
device 1450. The memory 1464 can be implemented as one or more of a
computer-readable medium or media, a volatile memory unit or units,
or a non-volatile memory unit or units. Expansion memory 1474 may
also be provided and connected to device 1450 through expansion
interface 1472, which may include, for example, a SIMM (Single In
Line Memory Module) card interface. Such expansion memory 1474 may
provide extra storage space for device 1450, or may also store
applications or other information for device 1450. For example,
expansion memory 1474 may include instructions to carry out or
supplement the processes described herein, and may include secure
information also. Thus, for example, expansion memory 1474 may be
provide as a security module for device 1450, and may be programmed
with instructions that permit secure use of device 1450. In
addition, secure applications may be provided via the SIMM cards,
along with additional information, such as placing identifying
information on the SIMM card in a non-hackable manner.
[0104] The memory may include, for example, flash memory and/or
NVRAM memory, as discussed below. In one implementation, a computer
program product is tangibly embodied in an information carrier. The
computer program product contains instructions that, when executed,
perform one or more methods, such as those described herein. The
information carrier is a computer- or machine-readable medium, such
as the memory 1464, expansion memory 1474, memory on processor
1452, or a propagated signal that may be received, for example,
over transceiver 1468 or external interface 1462.
[0105] Device 1450 may communicate wirelessly through communication
interface 1466, which may include digital signal processing
circuitry where necessary. Communication interface 1466 may provide
for communications under various modes or protocols, such as GSM
voice calls, SMS, EMS, or MMS messaging, CDMA, TDMA, PDC, WCDMA,
CDMA2000, or GPRS, among others. Such communication may occur, for
example, through radio-frequency transceiver 1468. In addition,
short-range communication may occur, such as using a Bluetooth,
WiFi, or other such transceiver (not shown). In addition, GPS
(Global Positioning System) receiver module 1470 may provide
additional navigation- and location-related wireless data to device
1450, which may be used as appropriate by applications running on
device 1450.
[0106] Device 1450 may also communicate audibly using audio codec
1460, which may receive spoken information from a user and convert
it to usable digital information. Audio codec 1460 may likewise
generate audible sound for a user, such as through a speaker, e.g.,
in a handset of device 1450. Such sound may include sound from
voice telephone calls, may include recorded sound (e.g., voice
messages, music files, etc.) and may also include sound generated
by applications operating on device 1450.
[0107] The computing device 1450 may be implemented in a number of
different forms, as shown in the figure. For example, it may be
implemented as a cellular telephone 1480. It may also be
implemented as part of a smartphone 1482, personal digital
assistant, or other similar mobile device.
[0108] Various implementations of the systems and techniques
described herein can be realized in digital electronic circuitry,
integrated circuitry, specially designed ASICs (application
specific integrated circuits), computer hardware, firmware,
software, and/or combinations thereof. These various
implementations can include implementation in one or more computer
programs that are executable and/or interpretable on a programmable
system including at least one programmable processor, which may be
special or general purpose, coupled to receive data and
instructions from, and to transmit data and instructions to, a
storage system, at least one input device, and at least one output
device.
[0109] These computer programs (also known as programs, software,
software applications or code) include machine instructions for a
programmable processor, and can be implemented in a high-level
procedural and/or object-oriented programming language, and/or in
assembly/machine language. As used herein, the terms
"machine-readable medium" and "computer-readable medium" refer to
any computer program product, apparatus and/or device (e.g.,
magnetic discs, optical disks, memory, and Programmable Logic
Devices (PLDs)) used to provide machine instructions and/or data to
a programmable processor, including a machine-readable medium that
receives machine instructions as a machine-readable signal. The
term "machine-readable signal" refers to any signal used to provide
machine instructions and/or data to a programmable processor.
[0110] To provide for interaction with a user, the systems and
techniques described herein can be implemented on a computer having
a display device (e.g., a CRT (cathode ray tube) or LCD (liquid
crystal display) monitor) for displaying information to the user
and a keyboard and a pointing device (e.g., a mouse or a trackball)
by which the user can provide input to the computer. Other kinds of
devices can be used to provide for interaction with a user as well;
for example, feedback provided to the user can be any form of
sensory feedback (e.g., visual feedback, auditory feedback, or
tactile feedback); and input from the user can be received in any
form, including acoustic, speech, or tactile input.
[0111] The systems and techniques described herein can be
implemented in a computing system that includes a back end
component (e.g., as a data server), or that includes a middleware
component (e.g., an application server), or that includes a front
end component (e.g., a client computer having a graphical user
interface or a Web browser through which a user can interact with
an implementation of the systems and techniques described herein),
or any combination of such back end, middleware, or front end
components. The components of the system can be interconnected by
any form or medium of digital data communication (e.g., a
communication network). Examples of communication networks include
a local area network ("LAN"), a wide area network ("WAN"), and the
Internet.
[0112] The computing system can include clients and servers. A
client and server are generally remote from each other and
typically interact through a communication network. The
relationship of client and server arises by virtue of computer
programs running on the respective computers and having a
client-server relationship to each other.
[0113] In some cases, a computing system provided herein can be
configured to include one or more sample analyzers. A sample
analyzer can be configured to produce a plurality of signals about
genomic DNA of at least one pair of human chromosomes of a cancer
cell. For example, a sample analyzer can produce signals that are
capable of being interpreted in a manner that identifies the
homozygous or heterozygous nature of loci along a chromosome. In
some cases, a sample analyzer can be configured to carry out one or
more steps of a SNP array-based assay or sequencing-based assay and
can be configured to produce and/or capture signals from such
assays. In some cases, a computing system provided herein can be
configured to include a computing device. In such cases, the
computing device can be configured to receive signals from a sample
analyzer. The computing device can include computer-executable
instructions or a computer program (e.g., software) containing
computer-executable instructions for carrying out one or more of
the methods or steps described herein. In some cases, such
computer-executable instructions can instruct a computing device to
analyze signals from a sample analyzer, from another computing
device, from a SNP array-based assay, or from a sequencing-based
assay. The analysis of such signals can be carried out to determine
genotypes, homozygosity at certain loci, regions of homozygosity,
the number of LOH regions, to determine the size of LOH regions, to
determine the number of LOH regions having a particular size or
range of sizes, to determine whether or not a sample is positive
for an LOH signature, to determine the number of Indicator LOH
Regions in at least one pair of human chromosomes, to determine a
likelihood of a deficiency in BRCA1 and/or BRCA2 genes, to
determine a likelihood of a deficiency in HDR, to determine a
likelihood that a cancer patient will respond to a particular
cancer treatment regimen (e.g., a regimen that includes a DNA
damaging agent, an anthracycline, a topoisomerase I inhibitor,
radiation, a PARP inhibitor, or a combination thereof), or to
determine a combination of these items.
[0114] In some cases, a computing system provided herein can
include computer-executable instructions or a computer program
(e.g., software) containing computer-executable instructions for
formatting an output providing an indication about the number of
LOH regions, the size of LOH regions, the number of LOH regions
having a particular size or range of sizes, whether or not a sample
is positive for an LOH signature, the number of Indicator LOH
Regions in at least one pair of human chromosomes, a likelihood of
a deficiency in BRCA1 and/or BRCA2 genes, a likelihood of a
deficiency in HDR, a likelihood that a cancer patient will respond
to a particular cancer treatment regimen (e.g., a regimen that
includes a DNA damaging agent, an anthracycline, a topoisomerase I
inhibitor, radiation, a PARP inhibitor, or a combination thereof),
or a combination of these items. In some cases, a computing system
provided herein can include computer-executable instructions or a
computer program (e.g., software) containing computer-executable
instructions for determining a desired cancer treatment regimen for
a particular patient based at least in part on the presence or
absence of an LOH signature or on the number of Indicator LOH
Regions.
[0115] In some cases, a computing system provided herein can
include a pre-processing device configured to process a sample
(e.g., cancer cells) such that a SNP array-based assay or
sequencing-based assay can be performed. Examples of pre-processing
devices include, without limitation, devices configured to enrich
cell populations for cancer cells as opposed to non-cancer cells,
devices configured to lyse cells and/or extract genomic nucleic
acid, and devices configured to enrich a sample for particular
genomic DNA fragments.
[0116] This document also provides kits for assessing samples
(e.g., cancer cells) as described herein. For example, this
document provides kits for assessing cancer cells for the presence
of an LOH signature or to determine the number of Indicator LOH
Regions in at least one pair of human chromosomes. A kit provided
herein can include either SNP probes (e.g., an array of SNP probes
for carrying out a SNP array-based assay described herein) or
primers (e.g., primers designed for sequencing SNP regions via a
sequencing-based assay) in combination with a computer program
product containing computer-executable instructions for carrying
out one or more of the methods or steps described herein (e.g.,
computer-executable instructions for determining the number of LOH
regions having a particular size or range of sizes). In some cases,
a kit provided herein can include at least 500, 1000, 10,000,
25,000, or 50,000 SNP probes capable of hybridizing to polymorphic
regions of human genomic DNA. In some cases, a kit provided herein
can include at least 500, 1000, 10,000, 25,000, or 50,000 primers
capable of sequencing polymorphic regions of human genomic DNA. In
some cases, a kit provided herein can include one or more other
ingredients for performing a SNP array-based assay or a
sequencing-based assay. Examples of such other ingredients include,
without limitation, buffers, sequencing nucleotides, enzymes (e.g.,
polymerases), etc. This document also provides the use of any
appropriate number of the materials provided herein in the
manufacture of a kit for carrying out one or more of the methods or
steps described herein. For example, this document provides the use
of a collection of SNP probes (e.g., a collection of 10,000 to
100,000 SNP probes) and a computer program product provided herein
in the manufacture of a kit for assessing cancer cells for the
presence of an LOH signature. As another example, this document
provides the use of a collection of primers (e.g., a collection of
10,000 to 100,000 primers for sequencing SNP regions) and a
computer program product provided herein in the manufacture of a
kit for assessing cancer cells for the presence of an LOH
signature.
[0117] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1--Assessing LOH Regions and HDR
[0118] Two sets of tumors were used from advanced ovarian cancer
patients. The first set of 94 tumors (training set) was used to
derive a candidate signature, and the second set of 40 tumors
(validation set) was used to validate the signature. All coding
regions of BRCA1 and BRCA2 genes were sequenced to detect germ line
and somatic mutations. Levels of BRCA1 and BRCA2 mRNA expression
were measured, and Affymetrix SNP microarrays were performed.
[0119] A computer program was used to reconstruct LOH signature
status based on allele intensities derived from the microarray
data. An algorithm was developed and implemented as a computer
program to reconstruct LOH regions based on genotype (e.g., SNP
genotype) data.
[0120] One point of the algorithm was to first reconstruct allele
specific copy numbers (ASCN) at each locus (e.g., SNP). ASCNs are
the numbers of copies of both paternal and maternal alleles. An LOH
region was then determined as a stretch of SNPs with one of the
ASCNs (paternal or maternal) being zero. The algorithm was based on
maximizing a likelihood function and was conceptually analogous to
a previously described algorithm designed to reconstruct total copy
number (rather than ASCN) at each locus (e.g., SNP). See
International Application No. PCT/US2011/026098 to Abkevish et al.
The likelihood function was maximized over ASCN of all loci, level
of contamination with benign tissue, total copy number averaged
over the whole genome, and sample specific noise level. The input
data for the algorithm included (1) sample-specific normalized
signal intensities for both allele of each locus and (2)
assay-specific (specific for different SNP arrays and for sequence
based approach) set of parameters defined based on analysis of
large number of samples with known ASCN profiles.
[0121] Tumors were defined as being HDR deficient for the purpose
of this analysis if they either had one or more deleterious
mutations in BRCA1 and/or BRCA2 genes or if they had low expression
of BRCA1 mRNA. The rest of the tumors were defined as likely HDR
non-deficient for the purpose of this analysis.
[0122] The distribution of the lengths of LOH regions was
investigated (FIG. 5). Three categories of LOH regions were used:
(1) LOH affecting a whole chromosome; (2) large LOH regions
(greater than about 15 Mb), which typically affect a part of a
chromosomal arm or the whole chromosomal arm; and (3) multiple
short LOH regions (less than about 15 Mb). Second, using the
training set only, the number of LOH regions of one of these three
categories was assessed for possible correlations with HDR
deficiency. It was discovered that (1) the number of short LOH
regions did not significantly correlate with HDR deficiency
(p>0.05); (2) LOH covering an entire chromosome correlated
weakly with HDR deficiency (p=0.0011); and (3) the number of large
LOH regions correlated significantly with HDR deficiency
(p=1.9e-8). More specifically, it was discovered that all HDR
deficient tumors had a high number of large LOH regions (e.g., nine
or more), while the majority of tumors likely to be HDR
non-deficient had a small number of large LOH regions (FIGS. 6-8).
It was probable that tumors likely to be HDR non-deficient were in
fact HDR deficient due to other genetic alterations, excluding
BRCA1 and BRCA2 mutations and low mRNA expression. In addition to
the number of large LOH regions, the total length of these regions
also correlated significantly with HDR deficiency.
[0123] These results were confirmed with the validation set: (1)
the number of short LOH regions did not significantly correlate
with HDR deficiency (p>0.05); (2) LOH covering an entire
chromosome correlated weakly with HDR deficiency (p=0.05); and (3)
the number of large LOH regions correlated significantly with HDR
deficiency (p=3.9e-6).
[0124] The 134 tumors were divided from combined training and
validation data sets into three groups: (1) BRCA deficient if they
either had one or more deleterious mutations in BRCA1 and/or BRCA2
genes or if they had low expression of BRCA1 mRNA; (2) HDR
deficient/BRCA intact if they have 9 or more large LOH regions
(greater than 15 Mb but less than the length of the entire
chromosome); (3) HDR intact if they have less than 9 large LOH
regions (greater than 15 Mb but less than the length of the entire
chromosome). Results of this analysis are presented in FIG. 9. It
shows a high frequency of BRCA deficiency as well as HDR deficiency
that is not due to BRCA deficiency among ovarian tumors.
[0125] FIG. 10 shows the distribution of large LOH regions (greater
than 15 Mb but less than the length of the entire chromosome) for
different types of cancer cell lines. The size of the circles is
proportional to the number of samples with such number of large LOH
regions. Frequency of HDR deficiency (cell lines with at least 9 of
such large LOH regions) is the highest among breast and esophagus
cancer cell lines. No HDR deficiency was observed among colon
cancer cell lines. Validating the previous findings for ovarian
tumors, all BRCA deficient cell lines were found to be HDR
deficient as well.
[0126] FIG. 11 shows the distribution of large LOH regions (greater
than 15 Mb but less than the length of the entire chromosome) for
publicly available lung tumor data set (GSE19399 from Gene
Expression Omnibus). It was observed that frequency of HDR
deficiency (defined as having at least 9 large LOH regions) is
quite large among lung tumors (39%).
[0127] In FIG. 12 the results of analysis of different tumors and
cell lines are summarized. Frequency of HDR deficiency defined as
fraction of samples with at least 9 large LOH regions (greater than
15 Mb but less than the length of the entire chromosome) is
presented for several tumors and cell lines. This frequency is as
high as 50% among ovarian tumors and was not observed at all among
brain and colon cell lines. Thus it appears that HDR deficiency
plays an important role for the majority of cancers.
Example 2--Chemo Toxicity Responses
[0128] In preparation of chemo toxicity response experiments, all
cell lines were grown at 37.degree. C. plus 5% CO.sub.2 in 75
cm.sup.2 tissue culture flasks (VWR International, Inc. Cat
#353136) and the recommended growth medium. Before performing each
experiment, each cell line was trypsinized (Invitrogen Corporation
Cat #25200-056), counted, and seeded in Advanced RPMI 1640
(Invitrogen Corporation Cat #12633-020), 3% FBS, 1%
penicillin/streptomycin (Invitrogen Corporation Cat #15140-122) at
2500 cells or 5000 cells in 100 .mu.L media per well from columns
2-12 of 96-well polystyrene microplates with clear bottom (Perkin
Elmer Cat #6005181), leaving column 1 with 100 .mu.L per well of
media only. The cell-seeded plates were then incubated at
37.degree. C. plus 5% CO.sub.2 overnight.
[0129] Two different final drug concentration working stocks were
prepared. In cases where 100% DMSO was required for drug
solubility, Advanced RPMI 1640 was used as the diluent for the
highest concentration. Advanced RPMI 1640 plus a predetermined
amount of DMSO equal to the total DMSO in the high concentration
working stock was used for the low concentration, with a maximum of
60% DMSO used for the lowest concentration. This was done to keep
the DMSO concentrations equal in every well and prevent
non-specific cell death as a result of DMSO. The lower of the two
drug concentrations was placed in a 96-well, thin-wall PCR cycle
plate (Robbins Scientific Cat #1055-00-0) in rows A-D, column 12,
while the higher concentration was placed in rows E-H, column 12,
of the same plate. Serial dilutions of 1:2 or 1:3 were performed in
a descending manner from column 12 to 3, leaving columns 1 and 2 to
be used for no cell/no drug and no drug controls. This allowed for
quadruplet data points for each drug concentration. Once dilutions
were complete, 5 .mu.L was transferred from the dilution plate to
the corresponding well of the seeded cell plate. Plates receiving
drugs were then incubated at 37.degree. C. plus 5% CO.sub.2 for
either 3 days or 6 days.
[0130] Following a 3-day or 6-day dose regimen, ATPlite assays
(Perkin Elmer cat #6016941) were run on each well of each plate
according to the ATPLite Assay protocol. The luminescence was then
read on a FUSION machine and saved as a .CSV file. For each
cell-line and drug combination, the four replicates of the no-drug
control were averaged and divided by 100 to create a "normalization
factor" used to calculate a normalized percent survival. The
normalized percent survival for the no-drug controls was 100%. The
four replicates of the cell-plus-drug wells were averaged and
divided by the normalization factor for each drug concentration.
The percent survival for each drug concentration, starting with a
concentration equal to 0, was used to calculate an IC.sub.50 using
proprietary software.
[0131] FIG. 13 shows response to chemotherapy for breast and
ovarian cancer cell lines. On y-axis are indicated values of
Log.sub.10(IC.sub.50) for different chemotherapy drugs
(camptothecin, as well as averaged results for platinum compounds
(oxaliplatin, cisplatin, and carboplatin) or anthracyclines
(doxorubicin and epirubicin)) when exposed to 29 breast cancer cell
lines as well as Log.sub.10(IC.sub.50) of paclitaxel when exposed
to 27 ovarian cancer cell lines. On the x-axis the number of large
LOH regions longer than 15 Mb and shorter than the entire
chromosome are indicated for these cell lines. The dashed lines
place a threshold number at nine.
[0132] FIG. 14 is a version of a graph from FIG. 13 that indicates
specificity and sensitivity among responders and non-responders to
treatment with platinum compounds (oxaliplatin, cisplatin, and
carboplatin) when exposed to 29 breast cancer cell lines. The
dashed lines place a threshold number of large LOH regions longer
than 15 Mb and shorter than the entire chromosome at nine. The
solid line divides cell lines into responders and
non-responders.
OTHER EMBODIMENTS
[0133] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *