NZ617421B2 - Biomarkers for hedgehog inhibitor therapy - Google Patents
Biomarkers for hedgehog inhibitor therapy Download PDFInfo
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- NZ617421B2 NZ617421B2 NZ617421A NZ61742112A NZ617421B2 NZ 617421 B2 NZ617421 B2 NZ 617421B2 NZ 617421 A NZ617421 A NZ 617421A NZ 61742112 A NZ61742112 A NZ 61742112A NZ 617421 B2 NZ617421 B2 NZ 617421B2
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Abstract
Discloses a method of predicting response to treatment with a Hedgehog signalling inhibitor, the method comprising determining the level of expression of at least two biomarkers selected from the group consisting ofGLI-1, OTX-2, SHROOM2, PDLIM3, SPHK1, SFRP1, APBA2 and SP ATA20, in a biological sample derived from a subject having cancer, thereby to predict an increased likelihood of response to a Hedgehog signalling inhibitor. le derived from a subject having cancer, thereby to predict an increased likelihood of response to a Hedgehog signalling inhibitor.
Description
KERS FOR HEDGEHOG INHIBITOR THERAPY
FIELD OF THE INVENTION
The present invention relates to a method of personalized therapy.
BACKGROUND OF THE INVENTION
Hedgehog signaling is known to regulate a diverse range of biological processes, such as ar
proliferation, differentiation, and organ formation in a tissue specific and dose ent manner.
ly, Hedgehog signaling is strictly controlled during cellular proliferation, differentiation and
nic pattern formation. However, aberrant activity of the Hedgehog signaling pathway, due to
mutations that constitutively activate the pathway, for instance, may have pathological
consequences. By way of example, loss—of- on mutations of Patched are found in Gorlin's
syndrome (a hereditary syndrome with high risk of skin and brain cancers, also known as Basal Cell
Nevus Syndrome (BCNS)); and gain-of— function mutations of Smo and GIi are linked to basal cell
carcinoma and glioblastoma. Basal cell carcinoma (BCC) is the most common form of skin cancer,
affecting more than 90,000 Americans each year.
Constitutive activation of Hedgehog has been found to promote tumorigenesis in BCC,
medulloblastoma (the most common childhood brain tumor), rhabdomyosarcoma, pancreatic cancer,
small cell lung cancer, prostate cancer and breast cancer. Besides the roles in tumorigenesis,
Hedgehog signaling is also implicated in the metastasis of prostate cancer. Hedgehog signaling may
be involved in many additional types of tumor types and such links are expected to continue to be
discovered; this is an area of active research in many cancer s around the world.
SUMMARY OF THE INVENTION
The t invention is based on the finding that particular biomarkers can be used to select
individuals having cancer who are likely to respond to treatment with an inhibitor of the Hedgehog
signaling y. Specifically, it was found the level of expression of a ker listed in Table
l, e.g., the mRNA expression of a biomarker listed in Table 1 and/or the increased presence or
reduction in the amount of a protein product encoded by a biomarker listed in Table l in a sample
from an individual having cancer compared to-a control, can be used to predict whether that
individual will respond to treatment with an tor of the Hedgehog signaling pathway.
In a particular aspect, the present invention provides a method of predicting se to treatment
(followed by page la)
with a Hedgehog signaling inhibitor, the method comprising ining the level of expression of
at least two kers selected from the group consisting of GLI-l, OTX-2, SHROOMZ, PDLIM3,
SPHKl, SFRPI, APBA2 and SPATAZO, in a biological sample derived from a subject having
cancer, thereby to predict an increased likelihood of response to a Hedgehog signaling inhibitor.
In one aspect, the invention includes a method of selecting a subject having cancer for treatment with
a Hedgehog signaling inhibitor, the method ing determining the level of expression of at least
[FOLLOWED BY PAGE 2]
PCT/U82012/031993
one biomarker listed in Table 1, such as SHROOM 2 or SPHKl, in a biological sample derived from
the subject, thereby to predict an increased likelihood of response to a Hedgehog signaling tor.
In one embodiment, the ion es selecting at least two, at least three, at least four, at least
five, at least six, at least seven, or at least eight biomarkers in Table 1. In the method of the
ion, the hedgehog signaling inhibitor is amine, Iervine, GANT61, purmorphamine,
SAG, SANT—Z, tomatidine, zerumbone, GDC-0449; XL139, , IPI609 (IPIZ69609), or BMS-
833923x’XL139, or tives thereof. In one embodiment, the Hedgehog signaling inhibitor is
methyl—4'—trifluoromethoxy~biphenyl—3-carboxylic acid [6—(cis—2,6-dimethyl-morpholin—4—yl)—
pyridinyl]—amide (Compound I) or 2-[(R)(6—benzyl—4,5—dimethyl—pyridazin—3—yl)—2—methyl-
3,4,5,6—tetrahydr0—2H—[l,2']bipyrazinyl-5'-yl]-propan-2—ol Compound II. In the method of the
invention, the cancer can be BCC, Chronic myeloid leukemia (CML), bone sarcoma, soft tissue
sarcoma, medulloblastoma , myosarcoma, pancreatic cancer, small cell lung cancer, prostate
cancer, Gorlin syndrome, gastro—esophageal cancer, myeloproliferative neoplasia and acute
leukemias or breast cancer. In one embodiment, the biological sample is a tumor sample such as a
fresh frozen sample or a formalin fixed paraffin-embedded tissue sample.
In another aspect, the ion includes a method of selecting a subject having a tumor for treatment
with a hedgehog signaling inhibitor, the method comprising determining the level of expression of at
least one biomarker listed in Table l in a ical sample derived from the subject having
medullablastoma thereby to predict an increased likelihood of response to methyl—4'—
trifluoromethoxy—biphenyl—3—carboxylic acid [6-(cis-2,6-dimethyl-morpholin—4—yl)-pyridin-3—yl]-
amide.
The level of expression using the methods of the invention can include determining the level of
expression ofmRNA or determining protein level.
In another aspect, the invention includes a method of selecting a subject having cancer for treatment
with a hedgehog signaling inhibitor, the method including determining the level of expression of a
Hedgehog signaling biomarker listed in Table l in a biological sample derived from the t
having cancer; determining the level of mRNA of one or more reference/normalized genes listed in
Table 2; and normalizing the sion level of the hedgehog signaling biomarker to the reference
gene.
In another aspect, the ion includes a kit for determining if a tumor is sive to treatment
with a hedgehog signaling inhibitor sing providing one or more probes or primers for
detecting the expression level of at least one, at least two, at least three, at least four, at least five, at
C‘TTDCT‘T’T‘T T‘T‘U CLIUDT IDTTT 13 ’34\
W0 20121166241 PCT/U82012/031993
least six, at least seven, or at least eight biomarkers listed in Table I. The kit of the invention can
include a ity of agents for determining the level of one or more biomarkers listed in Table l
and optionally also include agents for determining the level of expression of one or more biomarkers
listed in Table 2 and ctions for use.
In one aspect, the kit of the ion can be a kit for predicting whether a subject with cancer would
benefit from treatment with a hedgehog signaling tor, the kit including: a plurality of agents for
determining the mRNA sion level of one or more biomarkers identified in Table l; and means
for analyzing the expression and ting a score to predict whether a patient would benefit from
treatment with a hedgehog signaling inhibitor. The agents for determining mRNA expression can
include an array of polynucleotides complementary to one or more kers identified in Table l.
The agents for measuring mRNA expression include a plurality of PCR probes and/or primers for
qRT-PCR.
In yet another aspect, the invention can include a microarray comprising polynncleotide probes
complementary and hybrdizable to at least one biomarker listed in Table I.
In yet another aspect, the invention can include a composition comprising a plurality of isolated
nucleic acid sequences, wherein each isolated nucleic acid sequence hybridizes to at least one RNA
products selected fiom the following genes GLI-l, OTX-2, SHROOMZ, PDLIM3, SPHKI, SFRPl,
APBA2 and SPATAZO, wherein the composition is used to measure the level of mRNA sion
of the genes.
In still yet another aspect, the invention can e a computer product for selecting a subject
having or suspected of having medullablastoma for treatment with a SMO inhibitor drug: means for
receiving data corresponding to the expression level of at least one gene in a sample from the subject
listed in Table 1; means for generating an expression value for each gene; and means for ting
a score based on inputting the expression value into a se comprising a reference expression
profile associated with ion, wherein score predicts whether the subject would benefit from
treatment with a SMO inhibitor. The computer product can be used in any of the methods described
above.
A "biomarker" is a le useful as an indicator of a biologic state in a subject. With reference to
the present subject matter, the biomarkers disclosed herein can be molecules that exhibit a change in
sion to predict whether a subject would benefit from receiving a treatment with a hedgehog
signaling inhibitor, e.g., a SMO tor.
CTTDC‘T‘T’T‘T T’T‘E‘ C‘U'E‘U'T‘ {DTTY U ’312\
2012/031993
"Hedgehog" refers generically to any of the mammalian homologs of the hila hedgehog
n, and includes at least Sonic hedgehog (SHedgehog), Desert hedgehog ehog) and
Indian hedgehog (Hedgehog).
"Hedgehog signaling pathway" as used herein refers to the ing cascade mediated by (or
downstream of) hedgehog and its receptors which results in changes of gene expression and other
phenotypic changes typical ofhedgehog activity. Activation of the signaling pathway can be due to
a Hedgehog signaling component that positively s the transmission ofthe Hedgehog signal,
i.e., stimulates downstream biological events when Hedgehog is present. Examples of such
components are Hedgehog, Ptch, Smo, and Gli. Hedgehog positive (Hh+) tumors are those tumors
having genetic alterations that results in tutive hedgehog y activation and this activation
of Hedgehog appears to be the major driver for tumor formation and growth.
DETAILED DESCRIPTION OF THE INVENTION
There is an increasing body of evidence that ts a patient’s genetic profile can be determinative
to a patient’s responsiveness to a therapeutic treatment. Given the numerous therapies available to
treat cancer, a determination of the genetic factors that influence, for example, response to a
particular drug, could be used to provide a patient with a personalized treatment regime. Such
personalized treatment regimes offer the potential to maximize therapeutic benefit to the t
while zing related side effects that can be associated with alternative treatment regimes. Thus,
there is a need to identify s which can be used to predict whether a patient is likely to respond
to a particular therapy.
To maximize the potential clinical benefit of a t ing a Hedgehog signaling inhibitor it is
important to be able to select those patients who have tumors that have an activated Hedgehog
signaling pathway. The methods described herein are based, in part, upon the identification of a
single or a ity of biomarkers listed in Table l, which can be used to determine a patient’s
likelihood of benefiting from treatment with a Hedgehog signaling inhibitor such as a SMO
inhibitor.
The biomarkers of the invention were purposefully optimized for use in Formalin—Fixed, Paraffin-
Embedded tissue (FFPE) specimens to make it applicable for routine clinical testing.
CTTDQ'T‘T’T‘T T’T‘E‘ C‘U‘E‘E‘T {DTTT 13 ”)fl\
W0 2012I166241 PCT/USZOlZ/031993
Hedgehog signaling inhibitors
Hedgehog signaling tors used in the present invention are agents known to inhibit the
Hedgehog signaling pathway. Such agents can be agents that inhibit aberrant growth states resulting
from ypes such as loss-of— function mutations in Ptch or Sufu, or gain—of-function mutations
in Hedgehog, Smoothened, or Gli. Hedgehog inhibitors are known in the art, and include for
example small molecule compounds, small peptides, antibodies, antisense oligonucleotides, siRNAs,
and the like.
In some embodiments, the hedgehog signaling inhibitor is a small molecule compound. In some
embodiments, the hedgehog signaling tor is a cyclopamine or derivative thereof. In some
embodiments, the hedgehog signaling inhibitor is Jervine, GANT61, purmorphamine, SAG, SANT-
2, tomatidine, zerumbone, or tives thereof. In some embodiments, the hedgehog signaling
inhibitor is GDC—0449 (available from ech and/or Curis); XL139, IPI926 (available from
Infinity Pharmaceuticals), IPI609 (IPI269609), BMS-S33923/XL139 (available from l—Myers
Squibb and/or Exelixis, TAK-44l (Millennium) or PF-04449913 (Pfizer) .
Additional og signaling inhibitors are provided in PCT/U82010/038568,
PCT/U32010/044168, , PCT/USZOO9/063696PCT/USZOO9/O39065, all of
which are herein incorporated by reference in their entireties.
'i’he og eignaiing irini‘oitors described herein can be the agents themseives, pharmaceutieaiiy
acee tabie salts thereni', pharmaceutieaiiy aeeeptabie esters thereof, as wet: as steroisontera
enantiomers, raeernic mixtures, and the hire. In one embodiment, the hedgehog signaiing inhibitor is
a SMO inhibitor such as 2—methy1—4'-trifluoromethoxy-biphenyl—3-carboxylic acid s-2,6-
dimethyl-morpholinyl)-pyridin—3 mide, also known as N-[6-(cis—2,6—dimethylmorpholin—4—
yl)pyridineyl]-2—methy1~4’-(trifluoromethoxy)[1, I ’—bipheny1]—3—carboxamide, which is disclosed
in International Patent Application Nos. 1201 and in W0 2008/154259, all of which are
herein orated by reference in their entireties. In r embodiment the hedgehog ing
inhibitor is 2—[(R)—4—(6-benzyl-4,5—dirnethyl-pyridazin—3—y1)—2-methyl-3,4,5,6—tetrahydro—2H-
bipyrazinyl-5’—yl]—propan—2—ol, disclosed in , all of which is herein
incorporated by reference in its entirety
The hedgehog signaling inhibitors includes a compound of Formula I:
C‘TTDQ’T‘T’TT T’T‘E‘ CUUU'T‘ 1131”” T3 ’7£\
WO 66241 PCT/U82012/031993
R5 0 R2
R4 R3
R6 0 R7
R8 0 /
in which
Y is selected from N and CR“); wherein R10 is selected from hydrogen, halo, C1.
5alkyl, halosubstituted—C1.5alkyl, C1-6alkoxy, halosubstituted-C1.6alkoxy and ~0XNR10aR10b; Wherein
Rma and Run, are independently ed from hydrogen and C1_6alkyl;
R1 is selected from cyano, halo, C1_6alkyl, bstituted—Cl-6all(yl, C1_6alkoxy,
bstituted-C1_6all<oxy, 3134, dimethyl—amino, C1_6alkyl-sulfanyl and C3-3heterocycloalkyl
optionally substituted with up to 2 C1-5alkyl ls;
R2 and R5 are independently selected from en, cyano, halo, C1_6a1kyl,
halosubstituted—Cl.6all(yl, C1-6alkoxy, halosubstituted-C1-6alkoxy and dimethylamino;
R3 and R4 are independently selected from hydrogen, halo, cyano, C1-6alky1,
halosubstituted-C1_6a1ky1, C1_6alkoxy and halosubstituted-C1-6alkoxy; or either R1 and R2 or R1 and
R5 together with the phenyl to which they are both attached form C5_1oheter0aryl;
R5 and R7 are independently selected from hydrogen, C1_5alkyl, halosubstituted—C1_
salkyl, C1_5alkoxy and halosubstituted-C1-5alkoxy; with the proviso that R5 and R7 are not both
hydrogen;
R8 is selected from hydrogen, halo, C1-6alkyl, halosubstimted-C1.6alkyl, C1.6a1koxy
and halosubstituted—C1-5alkoxy;
R9 is selected from —S(O)2R11 and —R11; wherein R11 is selected from aryl, aryl,
cycloalkyl and heterocycloalkyl;
wherein said aryl, heteroaryl, cycloalkyl and heterocycloalkyl of R9 can be optionally
substituted with 1 to 3 radicals independently selected from Cmalkyl, halosubstimted-C1.6alky1, C1.
Galkoxy, halosubstituted—C1-6alkoxy, C6_1oaryl-C04alkyl, C5-1oheteroaryl-C04alkyl, C3_12cycloall<yl
and C3-gheterocycloalkyl;
wherein said aryl-alkyl substituent of R9 is optionally tuted with l to 3 radicals
independently selected from halo, C1_5all<yl, halosubstituted-C1-6alkyl, Cmalkoxy, bstituted—
C1_5alkoxy and methyl-piperazinyl;; and a pharmaceutically acceptable salts thereof.
QTTDC‘TT'TTT'T‘E‘ (‘U‘DU'T‘ IDTTI‘ E ’15‘\
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In another embodiment, the present invention includes a hedgehog ing inhibitors of
Formula 1(a):
R1 1<<—*_.. N /—\ N=N
\j"” N
)_/ \ /
(Ia)
or a pharmaceutically acceptable salt thereof, wherein
R11 is CH; alkyl, Cm alkenyl, €3-14 cycloalkyl, a €5.14 aryl group, a 5-14 ed heteroaryl
grOup, a 3—14 membered eteroalkyl group, Cm alkoxy, halo, NR13R14, C(O)OR13,
13R14, C1_ghaloalkyl, formyl, carbalkoxy, C1.galkyIOH, C(O)R13, SOgRl3, C(O)NHC1_
galkle13, 4, SOgNR13R14, OCF3, NHC(O)R13, CH20C(O)NR13R14, CH2NR13R14,
NHC(O)OR13, NHC(O)NR13R14, CHZNHSOZRB, CH2NHC(O)OR13, OC(O)R13, or
NHC(O)R13, which may be substituted or unsubstituted;
R12 is H, C1_3 alkyl, a €6.14 aryl group, C1_3 haloalkyl, Cm alkoxy, halo, NHz, CN, OCF3, OH,
C(O)NR13R14, C(O)R13, NR13R14, NHC(O)R13, SOzR13, SOgNRl3Rl4;
R13 and R14 are independently H, C1_g alkyl, C24; alkenyl, €3-14 lkyl, a €6-14 aryl group, a 5-14
membered heteroaryl group, a 3-14 membered cycloheteroalkyl group, loa1kyl, C1-g alkleH,
C1-8alkoxy, 01'R13 and R14 on one atom can form a heteroatom containing ring; and
Wherein R11, R13, and R14 can be unsubstituted or substituted by one or more of C1_g alkyl, €3-14
cycloalkyl, a C614 aryl group, a 5-14 membered heteroaryl group, a 3-14 membered cycloheteroalkyl
group, (31-3 alkleH, OH, oxo, CH; haloalkyl, carboxC1_g alkyl, or SOzCl_galkyl, halo, -OCH3, -
OCF3, -OH, -NH2.
Biomarker
The ker(s) of the invention includes one or more genes listed in Table 1 or their gene
products. By ing the expression level of one or more biomarkers identified in Table 1 it is
possible to select individuals having cancers in which the hedgehog pathway is activated and who
thus are likely to respond to treatment with an inhibitor of the Hedgehog signaling pathway, e. g., a
Smoothened (SMO) antagonist.
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The biomarkers ofthe invention include GLI-l, OTX—Z, SHROOM2, PDLIM3, SPHKl, SFRPl,
APBA2 and SPATAZO. GLI-l, SHROOM2, PDLIM3, SPHKl, SFRPl, and APBA2 are
upregulated (increase in mRNA expression) while OTX-2 and SPATAZO are downregulated
(decrease in mRNA expression). In one example, the expression profile can be a set of values
representing mRNA levels of one or more of the following genes GLI—l, OTX-Z, 2,
PDLIM3, SPHKl, SFRPl, APBA2 and SPATAZO. In another example, the expression profile can
be a set of values representing mRNA levels of one or more of the following genes GLI—l, OTX-2,
SHROOM2, PDLIM3 and SPHKl. In yet r example, the expression profile can include a set
of values representing one or more proteins or polypeptides encoded by GLI—l, OTX—2, SHROOM2,
PDLIM3, SPHKl, SFRPl, APBA2 and SPATAZO. The phrase “mRNA expression” can mean an
increase or decrease in the amount of mRNA expression in a sample from an individual having
cancer compared to a control , Typically, an increase or decrease in mRNA expression means
a 1.5 fold, or more (such as a 2, 3, 4, or 5 fold), difference in sion as compared with a control
(e.g., normal level as ined from a control).
Preparation at Samples
Any appropriate test sample of cells taken from an individual having a proliferative e can be
used. Generally, the test sample of cells or tissue sample will be obtained from the subject with
cancer by biopsy or surgical resection. A sample of cells, tissue, or fluid may be removed by needle
aspiration biopsy. For this, a fine needle ed to a syringe is inserted through the skin and into
the tissue of interest. The needle is typically guided to the region of interest using ultrasound 0r
computed aphy (CT) imaging. Once the needle is ed into the tissue, a vacuum is created
with the syringe such that cells or fluid may be sucked through the needle and collected in the
syringe. A sample of cells or tissue may also be removed by incisional or core . For this, a
cone, a cylinder, or a tiny bit of tissue is removed from the region of interest. CT imaging,
ultrasound, or an endoscope is generally used to guide this type of biopsy. More particularly, the
entire cancerous lesion may be removed by excisional biopsy or surgical resection. In the t
invention, the test sample is lly a sample of cells removed as part of surgical resection.
The test sample of, for example tissue, may also be stored in, e.g., RNAlater (Ambion; Austin Tex.)
or flash frozen and stored at 80%. for later use. The biopsied tissue sample may also be fixed with
a fixative, such as formaldehyde, paraformaldehyde, or acetic acid/ethanol. The fixed tissue sample
may be embedded in wax (paraffin) or a plastic resin. The ed tissue sample (or frozen tissue
sample) may be cut into thin sections. RNA or protein may also be extracted from a fixed or wax—
embedded tissue sample or a frozen tissue sample. Once a sample of cells or sample of tissue is
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removed from the subject with cancer, it may be sed for the isolation of RNA or protein using
techniques well known in the art and as described below.
An example of extraction of RNA from a biopsy taken from a patient with cancers can include, for
example, guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin, et al.,
Biochemistry 18:5294-5299, 1979). RNA from single cells may be obtained as described in
methods for preparing cDNA libraries from single cells (see, e.g., Dulac, Curr. Top. Dev. Biol.
362245, 1998; Jena, et al., J. l. s 1902199, 1996). In one embodiment, the RNA
population may be enriched for sequences of interest, as detailed in Tables 1 and 2. Enrichment may
be accomplished, for example, by random hexamers and primer-specific cDNA synthesis, or
multiple rounds of linear amplification based on cDNA synthesis and template—directed in vitro
transcription (see, e.g., Wang, et al., Proc. Natl. Acad. Sci. USA 8629717, 1989; Dulac, et al., supra;
Jena, et al., supra). Other methods of isolating RNA from a sample are known in the art and e
Trizol (lnvitrogen), Guanidinium thiocyanate—phenol—chloroform extraction, PureLink Micro-to-
Midi Total RNA Purification System (invitrogen), RNeasy kit (Qiagen), Oligotex kit (Qiagen),
PureYieldTM RNA Midiprep (Promega), PolyATtract System 1000 ga), l(R) 16
System (Promega), SV Total RNA Isolation (Promega), TOTALLY RNATM Kit (Ambion),
Poiy(A)PuristTM Kit (Ambion) and any other methods. Methods for extracting and analysing an
RNA sample are disclosed in lar Cloning, A Laboratory Manual ook and Russell
(ed), 3rd edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,
USA.
The og expression profile can be performed on a biopsy taken fiom a subject such as fresh
tissue, frozen tissue, tissue processed in formalin (FFPE) or other fixatives. In particular, where the
sample is an FFPE sample, RNA is extracted from FFPE sections using the Qiagen RNeasy FFPE
extraction kit n), and reverse transcribed to cDNA using random hexamers and ABI’s High
ty cDNA archive kit (Applied Biosystems, Foster City, CA).
The subject with a tumor or cancer will lly be a mammalian subject such as a primate. In an
exemplary embodiment, the subject is a human. As used herein the terms patient and subject are
synonymous.
Any cancer or tumor can be screened according to the methods of the invention and include, but are
not limited to, colon cancer, lung cancer, pancreatic cancer, gastric cancer, prostate cancer, and
hepatocellular carcinoma, basal cell oma, breast cancer, bone sarcoma, soft tissue a,
chronic d leukemia, acute myeloid leukemia, hematological cancer, medulloblastoma,
QTTDC‘TTTTTTU (‘U‘E‘UW‘ IDTTT ‘E‘ ’hfi\
rhabdomyosaracoma, neuroblastoma, pancreatic cancer, breast carcinoma, meningioma,
astoma, ytoma, melanoma, stomach cancer, esophageal cancer, y tract cancer,
prostate cancer, small cell lung cancer, non-small cell lung cancer, glial cell cancer, le
myeloma, colon cancer, neuroectodermal tumor, neuroendocrine tumor, mastocytoma and Gorlin
syndrome, glioma, colorectal cancer, GIST, —esophageal , myeloproliferative neoplasia
and an acute leukemia.
ion 0t expression at the biomarker
In one example, the method includes determining expression of one or more of the genes GLI—l,
OTX-2, SHROOMZ, PDLIM3, SPHKl, SFRP1,APBA2 and SPATA20. The gene sequences of
interest can be detected using agents that can be used to specifically detect the gene, for example,
RNA transcribed from the gene or ptides encoded by the gene.
In one embodiment, the method includes: ing a nucleic acid probe comprising a nucleotide
sequence, for example, at least 10, 15, 25 or 40 tides, and up to all or nearly all of the coding
sequence which is complementary to a portion of the coding sequence of a nucleic acid ce of
GLI-l, OTX-Z, SHROOM2, PDLIM3, SPHKl, SFRPl, APBA2 and SPATA20; obtaining a tissue
sample from a mammal having a cancerous cell; contacting the nucleic acid probe under stringent
ions with RNA ed from a biopsy taken from a patient with medulablastoma (e. g., in a
Northern blot, in situ hybridization assay, PCR etc); and determining the amount of hybridization of
the probe with RNA. Nucleic acids may be labeled during or after enrichment and/0r amplification
of RNAs.
The biomarkers GLI—l, OTX-2, SHROOMZ, PDLIM3, SPHKl, SFRPl, APBA2 and SPATA20 are
intended to also include naturally occurring sequences including allelic variants and other family
members. The biomarkers ofthe invention also include sequences that are complementary to those
listed sequences resulting from the degeneracy of the code and also ces that are sufficiently
homologous and sequences which hybridize under stringent conditions to the genes of the invention.
Conditions for hybridization are known to those skilled in the art and can be found in Current
Protocols in Molecular Biology, John Wiley and Sons, NY. (1989), 63.1-63.6. A preferred, non-
limiting example of highly stringent hybridization conditions are hybridization in 6 X sodium
chloride/sodium citrate (SSC) at about 45 degrees centigrade followed by one or more washes in 0.2
X SSC, 0.1 percent SDS at 50-65 degrees centigrade. By "sufficiently homologous" it is meant a
amino acid or nucleotide sequence of a biomarker which contains a sufficient or m number
of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid
CTT‘DQ'T‘T’T‘T T'T‘U GUD‘D'T‘ IDTTT 1: ”FAN
residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second
amino acid or nucleotide sequences share common structural domains or motifs and/or a common
functional activity. For example, amino acid or nucleotide sequences which share common ural
domains have at least about 50 percent homology, at least about 60 percent homology, at least about
70 percent, at least about 80 percent, and at least about 90-95 percent gy across the amino
acid sequences of the domains are defined herein as sufficiently gous. Furthermore, amino
acid or nucleotide sequences at least about 50 t homology, at least about 60-70 percent
homology, at least about 70—80 percent, at least about 80—90 percent, and at least about 90—95 percent
and share a common functional activity are defined herein as sufficiently homologous.
The comparison of sequences and determination of percent homology between two sequences can be
accomplished using a atical algorithim. A red, non—limiting example of a mathematical
algorithim utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 872264-68, d as in Karlin and Altschul (1993) Proc. Natl. Acad.
Sci. USA 90:5873 —77. Such an algorithm is incorporated into the NBLAST and XBLAST programs
(version 2.0) of Altschul, et a1. (1990) J. Mol. Biol. 3—10. BLAST nucleotide searches can be
performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences
homologous to TRL nucleic acid molecules ofthe invention. BLAST protein searches can be
performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid ces
homologous to the protein sequences encoded by the genes/oligonucleotides listed in Table 1, 2
and/or Table 3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et a1., (1997) Nucleic Acids Research 25(17):3389—3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs
(e.g., XBLAST and ) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred,
non-limiting example of a mathematical algorithim utilized for the comparison of sequences is the
ALIGN algorithm of Myers and Miller, CABIOS (1989). When utilizing the ALIGN program for
comparing amino acid ces, a PAMl 20 weight residue table, a gap length penalty of 12, and a
gap penalty of 4 can be used.
The present invention includes ing the expression of one or more genes GLI—l, OTX-Z,
SHROOM2, PDLIM3, SPHKI, SFRPl, APBAZ and SPATA20 in a tumor biopsy taken from a
subject suffering from cancer due to hedgehog pathway activation. The sion levels can be
analyzed and used to generate a score which can be used to differentiate those patients having a
tumor exhibiting og pathway activation versus those who do not.
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In one embodiment, the method ofthe invention includes ing any one of GLI—l, OTX—2,
SHROOM2, PDLIM3, SPHKI, SFRPl, APBA2 and SPATA2O listed in Table 1. In another
embodiment, the method of the invention includes measuring at least two, at least three, at least four,
at least five, at least six, at least seven, or at least eight genes from Table 1.
In one example, the level of expression of one gene, e.g., GLI-l, from Table 1 is measured. In
another example, the level of expression oftwo genes, e. g., GLI—l, OTX—2, from Table l is
measured. In yet another example, the level of expression of three genes GLI—l, OTX—2, and
SHROOM2 from Table l is measured. In yet another e, the level of expression of four genes
GLI—l, OTX—2, SHROOM2 or PDLIM3 from Table l is measured. In yet another example, the level
of expression of five genes GLI-l, OTX—2, SHROOM2, PDLIM3 and SPHKl from Table l is
measured.
Gene Name Accession # Uni Gene ID
GLI—l L'GID:2139596
OTX-2 L'GID: 178376
SHROOM2 L'GID: 1782725
PDL1M3 L'GID:237739
SPHK] UGID: 139253
SFRPI UGID2164788
APBA2 087123
SPATA20 UGID: 143 13 1
Table 1
The kers of the invention also include any combination of genes identified in Table 1 whose
level of expression or gene t serves as a predictive biomarker. The biomarkers of the
invention, including their gene sequence, are known in the art (as provided above) or, for example,
for GLI-l ((GLI family zinc finger 1; Nature 332:371-374(l988)), for OTX—2 (Orthodenticle
homeobox 2; BBQ 1. 12:2735—2747(1993)), for SHROOM2 (Shroom family member 2; Hum. Mol.
Genet. 4:373-382(1995)), for PDL1M3 (PDZ and LIM domain 3; J. Cell Biol. 139:507-5 15(1997)),
for SPHKl (sphingosine kinase 1; Gene -26(2000)), for SFRPl (secreted frizzled-related
n 1; Proc. Natl. Acad. Sci. USA. 94:6770-6775(1997)), for APBA2 (amyloid beta (A4)
precursor protein-binding, family A; J. Biol. Chem. 272:31459—31464(l997)), for SPATA20
(spermatogenesis associated 20; Proc. Natl. Acad. Sci. USA. 99 (26), 16899-16903 ).
QT‘IDQT‘T’T‘TT’T‘E‘ QUUU'T‘ f'DTlT 13 ’LC\
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In the method of the invention the level of expression of one or more genes as described in Table 1 is
measured and analyzed and compared to a l. The control for comparison can be determined
by one skilled in the art. In one e, the control is determined by choosing a value that serves as
a cut-off value. For example, the value can be a value that differentiates between e.g., those test
samples that have hedgehog activation (hedgehog +) from those that do not show hedgehog
activation (hedgehog -). In another example, the gene expression profile of a biomarker of the
invention is compared to a control (presence of expression of the biomarker in a sample taken from a
healthy person or a tumor that is og—activated).
In a particular embodiment of the invention, the control is predetermined and a score is generated
which can be used to select those subjects having a tumor due to hedgehog pathway activation. The
expression threshold can be used to select for those individuals who have will respond to a og
signaling inhibitor.
In another example, the control can include one or more ized genes (such as described below)
which are genes that exhibit a vely constant level of gene expression. The normalized genes
correct for (normalize away) both differences in the amount ofRNA assayed and variability in the
quality of the RNA used. Therefore, the assay typically measures and incorporates the expression of
certain normalizing genes such as those normalized genes shown in Table 2.
‘ Gene Name Accession #
‘ HUWEl UGID: 150675
LARPl UGlD: 179374
UGID: 23 8951
UGID: 714304
Table 2
In one of the methods of the invention, the expression of one or more of the genes of Table 1 (e.g.,
two, three, four, five, six, seven, or eight genes) is ed and typically will be converted into an
expression value after normalization by the expression level of the single control gene or the average
of 4 or 3 or 2 control genes described in Table 2. These expression values then will be used to
te a score which is then compared against a cut-off to select which subjects have a Hedgehog—
activated tumor and ore are likely to benefit from treatment with a og signaling
inhibitor. In one example, the mRNA level of at last two, three, four or all five genes of GLI-l,
OTX-2, SHROOMZ, PDLIM3 and SPHKl are measured and the mRNA values are ted into
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an expression value after normalization by the expression level ofthe a control gene or the average
of 4 or 3 or 2 control genes described in Table 2. The normalized genes of the ion UBA and
WWE domain containing 1 (HUWEI), La ribonucleoprotein domain family, member 1 (LARPl),
Superoxide dismutase l, soluble (SODl), of the invention, YMEl-like 1 (YMElLl) including their
gene sequence, are known in the art, for example, gene sequence accession numbers as provided by
the NCBI are provided above.
The biomarkers of the invention can be ed using any method known in the art such as reverse
Transcriptase PCR (RT—PCR). The method includes isolating mRNA using any technique known in
the art and described above, e.g., by using a purification kit, buffer set and protease from
commercial cturers, such as Qiagen. The reverse transcription step is typically primed using
specific primers, random hexamers, or oligo—dT primers, depending on the circumstances and the
goal of expression profiling and the cDNA derived can then be used as a template in the subsequent
PCR reaction. TaqMan(R) RT-PCR can then be performed using available
, e.g., commercially
equipment.
The isolated mRNA can then be further analyzed using any method known in the art such as
microarray analysis, tative ('real-time') PCR, northern blotting, and nuclease protection assay.
In one e, real time quantitative PCR is used which es PCR product accumulation
through a dual—labeled fluorigenic probe (e.g., using TaqMan(R) probe). Real time PCR is
compatible both with quantitative competitive PCR, where al competitor for each target
sequence is used for normalization, and with quantitative comparative PCR using a ization
gene contained within the sample, or a housekeeping gene for RT—PCR. For further details see, c. g.
Held et al, Genome Research 6:986-994 (1996). In a real time PCR assay a positive reaction is
detected by accumulation of a fluorescent signal. The Ct (cycle threshold) is defined as the number
of cycles required for the fluorescent signal to cross the threshold (i.e. s background level). Ct
levels are ely proportional to the amount of target nucleic acid in the sample (i.e. the lower the
Ct level the greater the amount of target nucleic acid in the sample). Most real time assays undergo
40 cycles of amplification.
In another example, microariays are used which include one or more probes corresponding to one or
more of genes GLI-l, OTX-2, SHROOMZ, PDLIMS, SPHKl, SFRPl, APBA2 and SPATAZO. Use
of a microarray results in the tion of ization patterns of labeled target nucleic acids on
the array surface. The resultant hybridization patterns of labeled nucleic acids may be visualized or
detected in a y ofways, with the particular manner of detection selected based on the particular
label of the target nucleic acid. Representative detection means include scintillation counting,
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2012/031993
autoradiography, fluorescence measurement, calorimetric measurement, light emission
measurement, light scattering, and the like.
In another e, a TaqMan® Low Density Array ( TLDA) card can be used which can include
one or more probes corresponding to one or more of genes GLI-l, OTX—2, SHROOM2, PDLIM3,
SPHKI, SFRP], APBA2 and SPATA20. This method uses a microfluidic card that performs
simultaneous real time PCR reactions.
In one example, the method of detection utilizes an array scanner that is commercially ble
(Affymetrix, Santa Clara, Calif), for example, the 417.TM. Arrayer, the 418.TM. Array Scanner, or
the t GeneArray.TM. Scanner. This scanner is controlled from a system computer with an
interface and o-use software tools. The output may be directly imported into or directly read by
a variety of software applications. Scanning devices are described in, for e, US. Pat. Nos.
,143,854 and 5,424,186.
Detecting expression of the biomarker gene product
Detecting for the presence of a protein t encoded by one or more of GLI-l, OTX-2,
SHROOMZ, PDLIM3, SPHKI, SFRPl, APBA2 and SPATA20 can be done by using any
appropriate method known in the art. For example, an agent of interest that can be used to detect a
particular protein of interest, for example using an dy. The method for producing polyclonal
and/or monoclonal antibodies that specifically bind to polypeptides useful in the present ion is
known to those of skill in the art and may be found in, for example, Dymecki, et al., (J. Biol. Chem.
26724815, 1992); Boersma and Van Leeuwen, (J. Neurosci. Methods 51:317, 1994); Green, et al.,
(Cell 28:477, 1982); and Arnheiter, et al., (Nature 8, 1981). In one embodiment, an
immunoassay can be used to quantitate the levels of proteins in cell samples. The invention is not
limited to a particular assay procedure, and therefore, is intended to include both homogeneous and
heterogeneous procedures.
Exemplary immunoassays that may be conducted according to the invention include fluorescence
polarization immunoassay (FPIA)5 fluorescence immunoassay (FIA), enzyme immunoassay (EIA),
nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and
mmunoassay (RIA). An indicator moiety, or label group, may be ed to the subject
antibodies and is selected so as to meet the needs of various uses of the method that are often
dictated by the availability of assay ent and compatible immunoassay procedures. General
techniques to be used in performing the various immunoassays noted above are known to those of
ordinary skill in the art.
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Alternatively other methods can be used such as Western blot analysis that includes
electrophoretically ting proteins on a polyacrylamide gel, and after staining the separated
proteins, the relative amount of each protein can be quantified by assessing its optical density.
Alternatively, other methods such as dot-blot assays, FACS or immunohistochemistry can be used.
Typically, antibodies generated against the kers of the invention can be used for visualizing
for the presence of a protein of interest can be labeled, for example, using a reporter molecule such
as fluorophores, s, biotin, chemiluminescent molecules, inescent molecules,
digoxigenin, avidin, streptavidin or radioisotopes.
In yet another embodiment, the invention contemplates using a panel of antibodies that are ted
against the marker polypeptides of this invention.
Data analvsis
To tate the sample is operation, the data obtained by the reader from the device may be
analyzed using a digital computer. lly, the computer will be appropriately programmed for
receipt and storage of the data from the device, as well as for analysis and reporting of the data
gathered, for example, subtraction of the background, verifying that controls have performed
properly, normalizing the signals, interpreting fluorescence data to determine the amount of
hybridized , normalization of background, and the like.
Kits
The invention r provides kits for determining the expression level of the biomarkers described
herein. The kits may be useful for determining who will benefit from treatment with a Hedgehog
signaling inhibitor. A kit can se probes of genes identified in Table 1 can be used to measure
gene expression of a test sample. In one embodiment, the kit comprises a computer readable medium
which includes expression profile analysis software capable of being loaded into the memory of a
computer system and which can t the measured expression values into a risk score. A kit may
fuither comprise nucleic acid controls, s, and instructions for use.
Administration
The hedgehog signaling inhibitors described herein can be selectively administered in therapeutically
effective amounts via any of the usual and acceptable modes known in the art, either singly or in
combination with one or more therapeutic agents based on the individual having been determined to
have an ted hedgehog signaling pathway as described herein. A therapeutically ive
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amount may vary widely ing on the severity ofthe disease, the age and relative health of the
subject, the potency of the compound used and other factors.
One skilled in the art will recognize many methods and materials similar or equivalent to those
described herein, which could be used in the ce of the present invention. Indeed, the present
invention is in no way limited to the s and materials described. For purposes of the present
invention, the following terms are defined below.
Examples
Example 1
The multi—gene Hedgehog signature was initially developed by analyzing 40 FFPE medulloblastoma
(MB) specimens. The matching fresh frozen specimens from the same cases were usly
profiled as described by Y—J Cho and colleagues (Y-J Cho et a1, JCO, 2010) and each individual case
was determined to be either Hedgehog—activated or non—Hedgehog—activated based on its gene
expression profile. A panel of 32 ate genes that are differentially expressed in hedgehog
positive (Hh+) versus hedgehog negative (Hh-) tumors and another 22 potential normalization genes
were selected from a combined dataset of ble profiling studies. All candidate genes that
demonstrated differential Hh+ versus Hh— expression were consistent in all profiling studies
evaluated. The RT—PCR assays for these ate genes was developed and optimized for use in
FFPE specimens. Candidate genes that did not show robust sion in FFPE en type were
eliminated from further use. Assays with robust mance in FFPE for 10 up—regulated and 8
down-regulated genes in Hedgehog+ versus Hedgehog- tumors plus 5 control genes, were further
selected and assembled onto a TLDA card which offers more economic tissue consumption
compared to the single assay format. This panel of 23 candidate genes was then analyzed in the 40
FFPE specimens with known Hedgehog activation .
The optimal model that has the minimal error in predicting the Hedgehog tion status and
involves the least number of genes was selected using the Elastic Net method (J. Friedman, T.
Hastie, and R. Tibshirani, J. of Statistical Software, 2008). Two optimal , one with a S-gene
signature and the other with an 8-gene signature, showed similar performance in a 5-fold cross
validation. These models allow computation of a probability score of being og+ for a given
sample based on the expression levels of either 5 or 8 genes, respectively. A cut—offwas established
based on the probability score to make determination of the Hedgehog+ versus Hedgehog— status for
tested samples.
(‘TTDC'T'TT‘T T'T‘D C‘TJ‘E‘U'I" IDTTT U ’Mfi\
PCT/U82012/031993
In addition, it was explored to use one single gene (SPHKI, OTXZ, SFRPl, PDLIM3, or
SHROOMZ) to make the prediction of the Hedgehog activation status and a cut-off for each of these
genes for making Hedgehog+ versus Hedgehog- calls was established using this set of 40
medulloblastoma tumors.
Both the 5— and 8-gene models as well as the single gene models were further externally validated in
an independent sample set of 25 medulloblastoma tumors. A11 25 patients had pathologically
confirmed diagnosis including 13 with c histology, 9 with nodular/desmoplastic ogy, and
2 with large cell/anaplastic ogy. Among the 25 patients, 13 were male and 12 female. Tissue
specimens from 24 patients were collected at diagnosis and one patient collected after chemotherapy
treatment. The median age of these 25 patients at diagnosis is 3 years-01d with a range between 6
months and 16 years. The FFPE specimens from these 25 medulloblastoma cases were analyzed by
the method of the invention to determine the og activation status by the S-gene and the 8-
gene models. The ng fresh frozen tumor tissue specimens from the same 25 patients ted
at the same time points as the FFPE samples were subjected to gene expression profiling using the
GeneChip human genome U133 Plus 2.0 array (Affymetric, Santa Clara, CA). The 25 ts were
classified to Subgroup c3 (Hedgehog activated sub-class) or bgroup 03 tumors based on gene
profile-based molecular sub-classification of medullobastoma as described by Y-J Cho and his
colleagues (YJ Cho et al. JCO, 2010).
Eight out of the 25 patients were determined to have a up c3, Hedgehog-activated tumor,
while 17 with non—Hedgehog—activated tumors. This molecular classification of the 25
medulloblastoma ts was performed prior to the analysis of the FFPE tumor specimens by the
method of the invention. Therefore, the FFPE specimens that were ed by the method of the
invention were considered to have known Hedgehog activation status. Based on the expression
levels of the 8 genes (GLI—l, OTX-Z, SHROOM2, PDLIM3, SPHKI, SFRP], APBA2 and
SPATAZO), a predictive model was used to compute a probability score of being Hedgehog+ (0—
100%) for a given sample. Based on a pre—specified probability cut-off, each specimen was
determined to be either Hedgehog-activated or non—Hedgehog—activated. Eight specimens were
called Hedgehog-activated and the remaining 17 were called non-Hedgehog—activated,
demonstrating 100% agreement with the known Hedgehog activation status of these s. In
addition, the median probability score for 17 non-Hedgehog-activated tumors is 0.9% with a range
between 0.2% and 3.1%. The median probability score for 8 Hedgehog-activated tumors is 87.0%
ranging from 70.9% to 96.6%. The considerable ence in the median probability score between
the negative and positive cases suggested the robustness of the Hedgehog+ versus og-
determination by the 8—gene model.
(‘TTDOTT'FT T'T‘U C‘U‘DE‘T {DTTT 13 ")£\
PCT/U52012/031993
Based on the expression levels of the 5 genes (GLl—l, OTX-2, SHROOMZ, PDLIM3, SPHKI) and
the associated predictive model, a probability score was calculated for each of the 25 specimens and
compared to a pre-specified cut-off. Hedgehog activation status was then determined for each
tumor, again in 100% agreement with the known Hedgehog tion status. The median
probability score for the 17 non—Hedgehog—activated tumors is 0.7% ranging from 0.1% to 3.0% and
for 8 Hedgehog-activated tumors 87.9% ranging from 69.1% to 97.6%, demonstrating the similar
robustness of Hedgehog status determination as the S-gene model.
Based on the expression level of a single gene, SPHKl, OTX2, SFRPl, PDLIM3, or SHROOMZ, a
Hedgehog+ or Hedgehog— call was made to the 25 medulloblastoma cases based on a pre—specified
threshold for each of the genes. The Hedgehog calls made by SPHKl, OTX2, or SFRPl for each of
the 25 tumors were in te agreement with the known Hedgehog tion status with 8
Hedgehog+ calls and 17 Hedgehog- calls. Based on PDLIM3, 24/25 correct calls were made. One
Hedgehog+ tumor was wrongly called Hedgehog, ing in 87.5% sensitivity and 100%
specificity of the tion. The prediction made based on SHROOM2 demonstrated 75%
sensitivity and 94% city with 22/25 correct calls. Two Hedgehog+ tumors were wrongly
called Hedgehog- and one Hedgehog- tumor called Hedgehog+. Therefore, in addition to the multi—
gene models that rely on composite measurement of involved genes, single genes within the multi-
gene Hedgehog signature demonstrated significant predictive power by itself in determining
og activation status for medulloblastoma. The present study provides a tion that each
of these genes in the method of the invention can be used to determine Hedgehog activation status in
medulloblastoma.
The control genes HUWEl, LAPRl, SODl and YMElLl were selected to normalize the expression
of the genes that fied the medulloblastoma (MB) tumors based on hedgehog activation status.
Ideally, the control gene should be expressed stably and at a similar level in all tumor tissues under
investigation. Several studies have suggested that even widely used control genes such as n
and GAPDH are unsuitable in certain situations. In addition, in a combined dataset from available
external profiling studies of fresh frozen MB tumor ens these widely used control genes
showed a very high expression levels of greater than lg2>11 as compared to the og pathway
genes with expression levels around lg2>8.. Thus the controls genes with similar sion levels
as the hedgehog pathway genes were selected. Any probesets that had an expression level of lg2<8
were not successfully detected by RT—PCR analysis in our study.
An important criterion for the selection of the controls is primarily based on the invariance of the
genes across all the data sets analyzed. The percent ient of variance was set to be less than 4
C‘TTDC'TW'T‘T T’T‘E‘ Q'LI'E‘E‘T {DTTT 12‘ ”UC\
W0 2012/‘166241 PCT/U82012/031993
t The normalization genes were further selected based on whether it is le to design an
assay with <80 bp amplicon size and then whether robust expression could be demonstrated in FFPE
samples with the optimized assay.
Exemplary probes are available as shown in Table 3.
exon Function in
Gene ABI Assay ID ReqSeq boundary Signature
GLII HsOOl71790_ml NM_005269.2 llandlZ Up
PDLIM3 H301062534_ml NM_014476.3 Up
SHROOM2 Hs01113636_ml NM_001649.2 9and10 Up
SPHKI H300184211fiml NMw1829652- Up
orxz Hs00222238_ml NM_021728.2 4and5 Down
5385_m1 NM_005503.3 Up
Table 3
Exemplary probes for SFRPl include a forward primer 5’-CCAATGCCACCGAAGCC—3’ (SEQ ID
N021) and reverse primer 5~TCACAGGGAGGACACACCG-3’ (SEQ ID N02) and FAM.
Exemplary probes for SPATA20 e a forward primer 5’—CAAGGCCAGGAAGGAAAACA-3’
(SEQ ID NO:3) and reverse primer 5’—CACCAGTGGCAGGTGGAGTA3’ (SEQ ID NO:4) and
FAM.
Example 2
Adult cancer patients including patients with medulloblastoma, were ed into an ongoing phase
I, dose escalation trial of —4'—trifluoromethoxy-biphenylcarb0xylic acid [6-(cis—2,6-
dimethyl—morpholin—4—y1)-pyridin—3 —y1]—amide to evaluate safety and tolerability of methyl-4'-
romethoxy-bipheny1carboxylic acid [6-(cis-2,6-dimethyl-morpholin-4—y1)-pyridinyl]—
amide and assess maximally tolerated dose of methyl—4'-trifluoromethoxy-biphenylcarboxylic
acid [6-(cis—2,6-dimethyl—morpholinyl)—pyridin—3—yl]—amide for adult patients. Archival FFPE
CTTDQTT'T‘TTTU OUU‘D‘T‘ (BTW I? 0£\
PCT/U82012/031993
tumor specimens collected prior to the start of the trial are ble for analysis by the method of the
invention from 3 enrolled medulloblastoma patients in this trial. One patient treated with a daily
dose of200 mg of methyl-4’-trifluoromethoxy-biphenylcarboxylic acid [6-(cis-2,6-dimethyl-
morpholin~4-yl)—pyridin-3 -yl]—amide achieved a partial se (PR) by the RECIST criteria after 2
months of treatment and the response maintained for 4 months before disease progression. The other
metastatic medulloblastoma patient without a measurable lesion was treated with a 1500 mg daily
dose and demonstrated lic partial se measured by positron emission tomography (PET)
which lasted for 7 months before disease relapse. The 3rd patient progressed rapidly after only 52
days of methyl—4'—trifluoromethoxy—biphenylcarboxylic acid [6-(cis—2,6—dimethyl—morpholin—4-
yl)—pyridin-3—yl]-amide treatment at a daily dose of 200 mg.
Based on the 8—gene and S—gene predictive model, the method of the invention was used to determine
the og activation status in tumor for these 3 ts. The two responders were ined to
have Hedgehog-activated tumor while the tumor from the non-responder was called non—Hedgehog-
activated. In addition, the single genes, SPHKl, OTX2, SFRPl, PDLIM3, or SHROOM2 were
each used to determine the og activation status based on the pie-established cut-off for each
of the genes. By each ofthe single gene, the same Hedgehog—activated and non—Hedgehog—activated
calls were made to the two ders and one non-responder, respectively. Among the 3 ts,
only one patient had adequate tumor tissue samples available for additional mutational analysis of
the PTCHl and SMO gene. This patient who achieved lic partial response and was called
Hedgehog+ by the method of the invention was found to have a somatic mutation in PTCHl gene.
Inactivating PTCHl mutations have been identified as a major ism of constitutively
activating Hedgehog pathway in medulloblastoma. Therefore, identification ofthis PTCHl
mutation provides a mechanistic basis for Hedgehog y activation and observed al
activity against Hedgehog signaling inhibitor in this patient and further validates the finding by the
method of the invention.
Example 3
The method of the invention was used to verify the Hedgehog activation status for another panel of
40 medulloblastomas with pathologically confirmed diagnosis. Among the 40 cases, 26 had classic
medulloblastoma histology, 12 nodular/desmoplastic histology, and 1 large cell/anaplastic histology.
Other demographic data associated with these cases were not available. The Hedgehog tion
status of these tumors was previously characterized by the affymetric gene expression profiling
method as described by Y—J Cho and his colleagues (YJ Cho et al. 1C0, 2010). The Hedgehog-
activated call in 15 cases and the non—Hedgehog—activated call in the remaining 25 cases were
CTTDQ'T‘T'T‘T T’T‘E‘ OUTPUT {DTTT 13‘ ’35‘s
verified in FFPE tumor specimens by the method of the ion based on the 8—gene model, the 5-
gene model and each of the single genes SPHKl, OTXZ, SFRPl, PDLIM3, or SHROOMZ.
Example 4
In addition to the Hedgehog—activated medulloblastoma, basal cell carcinoma (BCC) is another
cancer type that is mostly driven by mutations or genetic lesions of the Hedgehog pathway genes
that lead to constitutive activation of the Hedgehog pathway. Over 90% of the patients with BCC,
the most prevalent skin cancer, were found to either have inactivating mutations in the PTCHl gene
or less frequently genetic lesions of other Hedgehog pathway genes such as activating mutations of
SMO. In an ongoing phase 1, dose escalation trial of methyl—4'—trifluoromethoxy-biphenyl
carboxylic acid s-2,6—dimethyl—morpholin—4—yl)—pyridinyl]—amide, an og signaling
tor antagonizing SMO, adult BCC patients were enrolled among patients with other solid
tumors. Mutational analysis of PTCHl and SMO by directly sequencing was performed to the
archival tumor ens collected at the time of diagnosis from the enrolled BCC patients. Six
BCC patients were found to have ons in either PTCH or SMO, including two Gorlin
Syndrome patients where a germline PTCHl mutation was identified. The method of the invention
was used to determine the Hedgehog activation status of BCC tumors from these patients. With both
the S-gene and 8—gene models, the probability score of all 6 cases were above the pre—specified cut-
off and therefore all were called Hedgehog—activated. Among these 6 patients, 2 achieved confirmed
partial response on methyl—4'—trifluoromethoxy~biphenyl~3 ~carboxylic acid [6—(cis-2,6—dimethyl—
morpholin-4—yl)—pyridin—3~yl]~amide ent, 2 had stable disease for more than 6 month, and the
other 2 patients discontinued methyl-4'—trifluoromethoxy—biphenyl—3 —carboxylic acid [6—(cis—2,6—
dimethyl—morpholin—4-yl)—pyridin-3 —yl]—amide due to adverse events after less than 2 months of
therapy which might be too brief to e any clinical activities. The consistency demonstrated in
the positive mutation results in PTCHl/SMO and the Hedgehog+ calls made by the method of the
invention, although in another cancer type, r validates the method of the invention. The
method of the invention can be used for identifying other types of tumor with the same underlining
Hedgehog-mutation driven etiology that might preferentially benefit from a Hedgehog signaling
inhibitor y.
Example 5
Immunohistochemistiy (THC) analysis of GLI—l and SFRP~l expression at the n level was
described in PA Northcott et al., JCO, 2010). It was suggested that the presence of the positive IHC
CTTDC‘T‘T'T‘TT’T‘E‘ QUDUT {DTTT 'E.‘ ’)£\
staining from these two n markers could be used as a mean of identifying Hh+
medulloblastoma. GLI-l and SFRP—l are both individual genes described in the method of
invention. Using antibodies against the protein product of GLI-l (Cell Signaling, Beverly, MA) and
SFRP-l (Abcam, Cambridge, MA), 40 medulloblastoma FFPE tissue specimens with Hh activation
status determined by the method of invention were subjected to the IHC analysis. ens with
any levels of either nuclear staining of Gli—l or cytoplasmic staining of SFRP-l within the tumor
content on slides were called Hh positive. Specimens with no staining from both markers in the
appropriate ar location were called Hh negative. The Hh activation status determined by the
IHC method and by the method of invention was consistent in all cases tested, resulting in 100%
agreement between the two methods. In addition, high degree of dance between the Gli-l and
SFRP-l IHC staining was observed. This data not only further validated the method of invention but
also confirm that the individual markers described in the method of invention can be expressed not
only at the transcript levels but also at the protein level.
‘T‘T'FT T’T‘E‘ QIIE‘E‘T {DTTT T1 "Jfl‘v.
Claims (11)
- l. A method icting response to treatment with a Hedgehog signaling inhibitor, the method comprising determining the level of expression of at least two biomarkers selected from the group consisting of GLl-l, OTX-Z, SHROOMZ, PDLIMS, SPHKI, SFRPl, APBAZ and SPATAZO, in a biological sample derived from a subject having cancer, thereby to predict an sed likelihood of response to a Hedgehog signaling inhibitor.
- 2. The method according to claim 1, comprising determining the level of at least three biomarkers.
- 3. The method according to claim 1 sing determining the level of at least four biomarkers.
- 4. The method according to claim 1 sing determining the level of at least five biomarkers.
- 5. The method according to any one of claims 1—4, wherein the hedgehog signaling inhibitor is methyl-4‘-trifluoromethoxy—biphenylcarboxylic acid [6—(cis-2,6-dimethyl-morpholinyl)- pyridin-3 -yl]-amide or 2-[(R)(6-benzyl-4,5—dimethyl-pyridazin—3—yl)—2—methyl-3,4,5,6-tetrahydro- 2H-[1,2']bipyrazinyl-5'-yl]-propan-2—ol.
- 6. The method according to any one of claims 1-5, wherein the determining is determining the level of expression ofmRNA.
- 7. The method of any one of claims 1—6, wherein the cancer is BCC, medulloblastoma, rhabdomyosarcoma, CML, bone sarcoma, soft tissue sarcoma, pancreatic cancer, small cell lung cancer, prostate cancer Gorlin syndrome, or breast .
- 8. The method of claim 7, wherein the cancer is medulloblastoma.
- 9. The method ing to any one of claims 1—8, wherein the inhibitor is methyl-4'— trifluoromethoxy—biphenyl-3~carboxylic acid [6-(cis-Z,6—dimethyl-morpholin—4-yl)-pyridin—3-yl]- amide.
- 10. The method according to any one of claims 1—8, wherein the inhibitor is 2-[(R)(6-benzyl~4,5- yl-pyridazinyl)methyl—3 ,4,5,6-tetrahydro-2H—[ l ,2']bipyrazinyl-5'-yl]—propanol.
- 11. The method according to any one of claims 1-10, substantially as herein described with reference to any one of the Examples thereof. S4673 Seq List.txt
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161492603P | 2011-06-02 | 2011-06-02 | |
US61/492,603 | 2011-06-02 | ||
PCT/US2012/031993 WO2012166241A1 (en) | 2011-06-02 | 2012-04-03 | Biomarkers for hedgehog inhibitor therapy |
Publications (2)
Publication Number | Publication Date |
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NZ617421A NZ617421A (en) | 2016-04-29 |
NZ617421B2 true NZ617421B2 (en) | 2016-08-02 |
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