Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS OF DIAGNOSING AND TREATING PATIENTS WITH CUTANEOUS
SQUAMOUS CELL CARCINOMA
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
62/665,872,
filed on May 2,2018, and U.S. Provisional Application No. 62/737,863, filed on
September
27, 2018, both of which are incorporated by reference herein in their
entirety.
FIELD OF THE DISCLOSURE
The present disclosure relates to methods for predicting the risk of
recurrence and/or
metastasis in primary cutaneous squamous cell carcinoma (cSCC).
BACKGROUND
Cutaneous squamous cell carcinoma (cSCC) is rivaled only by basal cell
carcinoma as
the most common cancer in the U.S. Though most cases are cured by excision, a
subset recur
.. and become incurable with the number of deaths approximating melanoma
(Karia et
Am. Acad. Dermatol. 68(6): 957-66 (2013)). Despite overall good prognosis for
patients with
cSCC, a subset will develop local, regional, or distant recurrences following
complete
excision of the primary tumor. Those at high risk of recurrence are eligible
for adjuvant
treatment options. While specific clinical features are associated with
recurrence, they
collectively fail to identify 30-40% of all cSCC recurrences and many tumors
that possess
high risk features will not recur. Prediction models with increased positive
predictive values
are needed to prevent unnecessary procedures. To address the need for more
accurate
predictive factors and facilitate appropriate intervention strategies, gene
expression analysis
was used to determine a signature associated with recurrence in patients with
cSCC.
SUMMARY
There is a need in the art for a more objective method of predicting which
tumors
display aggressive metastatic activity. Development of an accurate molecular
footprint, such
as the gene expression profile assay disclosed herein, would be a significant
advance forward
.. for the field. A multi-center study using archived primary tissue samples
with extensive
capture of associated clinical data (subjects with pathologically confirmed
cSCC diagnosed
after 2006, minimum 2 years of follow-up, and two separate outcome measures:
nodal/distant
metastasis and local recurrence) was used to identify gene expression profiles
that accurately
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predict primary cSCC with a high risk of regional nodal/distant metastasis,
and primary cSCC
with high risk of local recurrence after complete surgical clearance.
In one embodiment, a method for treating a patient with a cutaneous squamous
cell
carcinoma (cSCC) tumor is disclosed herein, the method comprising: (a)
obtaining a
diagnosis identifying a risk of local recurrence, distant metastasis, or both,
in a cSCC tumor
sample from the patient, wherein the diagnosis was obtained by: (1)
determining the
expression level of at least 10 genes in a gene set; wherein the at least 10
genes in the gene
set are selected from: ACSBG1, AIM2, ALOX12, ANXA9, APOBEC3G, ARPC2,
ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1, Cl QL4, C21orf59, C3orf70, CCL27,
CD163, CEP76, CHI3L1, CHMP2B, CXCL10, CXCR4, CYP2D6 (L0C101929829), DARS,
DCT, DDAH1, DSS1, DUXAP8, EGFR, EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2,
HDDC3, HNRNPL, HOXA10 (HOXA9, MIR196B), HPGD, ID2, IL24, IL2RB, IL7R,
INHBA, IPO5P1, KIT, KLK5, KRT17, KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1
(ZGPAT), L0C100287896, L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916,
MKLN1, MMP1, MMP10, MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21,
MSANTD4, MYC, NEB, NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN,
PI3, PIG3, PIGBOS1, PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135,
RPL26L1, RPP38, RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3,
SLC25A11, SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B,
TNNC1, TUBB3, TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496,
ZNF839, and/or ZSCAN31; (2) comparing the expression levels of the at least 10
genes in
the gene set from the cSCC tumor sample to the expression levels of the at
least 10 genes in
the gene set from a predictive training set to generate a probability score of
the risk of local
recurrence, distant metastasis, or both, and; (3) providing an indication as
to whether the
cSCC tumor has a low risk to a high risk of local recurrence, distant
metastasis, or both,
based on the probability score generated in step (2); and (4) identifying that
the cSCC tumor
has a high risk of local recurrence, distant metastasis, or both, based on the
probability score
and diagnosing the cSCC tumor as having a high risk of local recurrence,
distant metastasis,
or both; (b) administering to the patient an aggressive treatment when the
determination is
made in the affirmative that the patient has a cSCC tumor with a high risk of
local recurrence,
distant metastasis, or both. In certain embodiments, the method further
comprises performing
a resection of the cSCC tumor when the determination is made in the
affirmative that the
patient has a cSCC tumor with a high risk of local recurrence, distant
metastasis, or both.
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In some embodiments, the expression level of each gene in the gene set is
determined
by reverse transcribing the isolated mRNA into cDNA and measuring a level of
fluorescence
for each gene in the gene set by a nucleic acid sequence detection system
following Real-
Time Polymerase Chain Reaction (RT-PCR). In certain embodiments, the cSCC
tumor
sample is obtained from formalin-fixed, paraffin embedded sample.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
.. NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 20 genes, 30 genes, or
40 genes
selected from the genes listed above.
In another embodiment, a method of treating a patient with a cutaneous
squamous cell
carcinoma (cSCC) tumor is disclosed herein, the method comprising
administering an
aggressive cancer treatment regimen to the patient, wherein the patient has a
cSCC tumor
with a probability score of between 0.500 and 1.00 as generated by comparing
the expression
levels of at least 10 genes selected from ACSBG1, AIM2, ALOX12, ANXA9,
APOBEC3G,
ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1, C1QL4, C21orf59, C3orf70,
CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10, CXCR4, CYP2D6
(L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR, EphB2, FCHSD1,
FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9, MIR196B), HPGD,
ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17, KRT18, KRT19, KRT6B,
LAMC2, LCE2B, LIME1 (ZGPAT), L0C100287896, L0C101927502, LOR, LRRC47,
MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10, MMP12, MMP13, MMP3,
MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB, NEFL, NFASC, NFIA, NFIB,
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NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1, PIM2, PLAU, PLS3, PTHLH,
PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38, RUNX3, S100A8, S100A9,
SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11, SNORD124, SPATA41, SPP1,
TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3, TUFM (MIR4721), TYRP1,
UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or ZSCAN31 from the cSCC
tumor with the expression levels of the same at least ten genes selected from
ACSBG1,
AIM2, ALOX12, ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9,
BLOC1S1, C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B,
CXCL10, CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8,
EGFR, EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10
(HOXA9, MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5,
KRT17, KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), L0C100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31 from a predictive training set. In one embodiment, the probability
score is
determined by a bimodal, two-class analysis, wherein a patient having a value
of between 0
and 0.499 is designated as class 1 with a low risk of local recurrence,
distant metastasis, or
both, and a patient having a value of between 0.500 and 1.00 is designated as
class 2 with an
increased risk of local recurrence, distant metastasis, or both.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
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PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 20 genes, 30 genes, or
40 genes
selected from the genes listed above.
In another embodiment, a kit comprising primer pairs suitable for the
detection and
quantification of nucleic acid expression of at least ten genes is disclosed
herein, wherein the
at least ten genes are selected from: ACSBG1, AIM2, ALOX12, ANXA9, APOBEC3G,
ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1, C1QL4, C21orf59, C3orf70,
CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10, CXCR4, CYP2D6
(L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR, EphB2, FCHSD1,
FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9, MIR196B), HPGD,
ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17, KRT18, KRT19, KRT6B,
LAMC2, LCE2B, LIME1 (ZGPAT), L0C100287896, L0C101927502, LOR, LRRC47,
MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10, MMP12, MMP13, MMP3,
MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB, NEFL, NFASC, NFIA, NFIB,
NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1, PIM2, PLAU, PLS3, PTHLH,
PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38, RUNX3, S100A8, S100A9,
SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11, SNORD124, SPATA41, SPP1,
TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3, TUFM (MIR4721), TYRP1,
UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or ZSCAN31.
In some embodiments, the primer pairs suitable for the detection and
quantification of
nucleic acid expression of at least ten genes are primer pairs for: ACSBG1,
AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
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RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the primer pairs comprise primer pairs for 10
genes, 20
genes, 30 genes, 40 genes, or more than 40 genes of the genes listed above.
In another embodiment, a method for predicting risk of recurrence, metastasis,
or
both, in a patient with a cutaneous squamous cell carcinoma (cSCC) tumor is
disclosed
herein, the method comprising: (a) obtaining a cSCC tumor sample from the
patient and
isolating mRNA from the sample; (b) determining the expression level of at
least 10 genes in
a gene set; wherein the at least ten genes in the gene set are selected from:
ACSBG1, AIM2,
ALOX12, ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9,
BLOC1S1, C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B,
CXCL10, CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8,
EGFR, EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10
.. (HOXA9, MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5,
KRT17, KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), L0C100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
.. PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31; (c) comparing the expression levels of the at least 10 genes in the
gene set from
the cSCC tumor sample to the expression levels of the at least 10 genes in the
gene set from a
predictive training set to generate a probability score of the risk of local
recurrence, distant
metastasis, or both; and (d) providing an indication as to whether the cSCC
tumor has a low
risk to a high risk of local recurrence, distant metastasis, or both, based on
the probability
score generated in step (c).
In some embodiments, the expression level of each gene in the gene set is
determined
by reverse transcribing the isolated mRNA into cDNA and measuring a level of
fluorescence
for each gene in the gene set by a nucleic acid sequence detection system
following Real-
Time Polymerase Chain Reaction (RT-PCR). In certain embodiments, the cSCC
tumor
sample is obtained from formalin-fixed, paraffin embedded sample. In one
embodiment, the
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method further comprises identifying the cSCC tumor as having a high risk of
local
recurrence, distant metastasis, or both, based on the probability score, and
administering to
the patient an aggressive tumor treatment.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 20 genes, 30 genes, or
40 genes
selected from the genes listed above.
In another embodiment, a method for predicting risk of recurrence, metastasis,
or
both, in a patient with a cutaneous squamous cell carcinoma (cSCC) tumor is
disclosed
herein, the method comprising: (a) obtaining a cSCC tumor sample from the
patient and
isolating mRNA from the sample; (b) determining the expression level of at
least 10 genes in
a gene set; wherein the at least ten genes in the gene set are selected from:
ACSBG1, AIM2,
ALOX12, ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9,
BLOC1S1, C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B,
CXCL10, CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8,
EGFR, EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10
(HOXA9, MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5,
KRT17, KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), L0C100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
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RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31; and (c) providing an indication as to whether the cSCC tumor has a
low risk to a
high risk of local recurrence, distant metastasis, or both, based on the
expression level of at
least 10 genes generated in step (b).
In some embodiments, the expression level of each gene in the gene set is
determined
by reverse transcribing the isolated mRNA into cDNA and measuring a level of
fluorescence
for each gene in the gene set by a nucleic acid sequence detection system
following Real-
Time Polymerase Chain Reaction (RT-PCR). In certain embodiments, the cSCC
tumor
sample is obtained from formalin-fixed, paraffin embedded sample.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 20 genes, 30 genes, or
40 genes
selected from the genes listed above.
In certain embodiments, the expression level of: ACSBG1 is decreased, AIM2 is
increased, ALOX12 is decreased, ANXA9 is decreased, APOBEC3G is increased,
ARPC2 is
decreased, ATP6AP1 is decreased, ATP6V0E2 is increased, BBC is increased,
BHLHB9 is
decreased, BLOC1S1 is decreased, Cl QL4 is increased, C21orf59 is increased,
C3orf70 is
increased, CCL27 is decreased, CD163 is increased, CEP76 is decreased, CHI3L1
is
increased, CHMP2B is decreased, CXCL10 is decreased, CXCR4 is increased,
CYP2D6
(L0C101929829) is decreased, DARS is decreased, DCT is decreased, DDAH1 is
decreased,
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DSS1 is decreased, DUXAP8 is increased, EGFR is increased, EphB2 is increased,
FCHSD1
is decreased, FDFT1 is decreased, FLG is decreased, FN1 is increased, GTPBP2
is decreased,
HDDC3 is increased, HNRNPL is decreased, HOXA10 (HOXA9, MIR196B) is decreased,
HPGD is decreased, ID2 is decreased, IL24 is increased, IL2RB is decreased,
IL7R is
increased, INHBA is increased, IPO5P1 is increased, KIT is increased, KLK5 is
decreased,
KRT17 is decreased, KRT18 is increased, KRT19 is decreased, KRT6B is
decreased,
LAMC2 is decreased, LCE2B is decreased, LIME1 (ZGPAT) is increased,
L0C100287896 is
increased, L0C101927502 is decreased, LOR is decreased, LRRC47 is increased,
MIER2 is
increased, MIR129-1 is increased, MIR3916 is increased, MKLN1 is increased,
MMP1 is
increased, MMP10 is decreased, MMP12 is increased, MMP13 is increased, MMP3 is
increased, MMP7 is increased, MMP9 is decreased, MRC1 is increased, MRPL21 is
increased, MSANTD4 is decreased, MYC is decreased, NEB is decreased, NEFL is
decreased, NFASC is decreased, NFIA is decreased, NFIB is decreased, NFIC is
decreased,
NOA1 is increased, PD1 is decreased, PDL1 is increased, PDPN is increased, PI3
is
decreased, PIG3 is decreased, PIGBOS1 is increased, PIM2 is increased, PLAU is
increased,
PLS3 is decreased, PTHLH is decreased, PTRHD1 is decreased, RBM33 is
increased,
RCHY1 is increased, RNF135 is increased, RPL26L1 is increased, RPP38 is
decreased,
RUNX3 is increased, S100A8 is decreased, S100A9 is decreased, SEPT3 is
decreased,
SERPINB2 is decreased, SERPINB4 is decreased, SLC1A3 is increased, SLC25A11 is
increased, SNORD124 is increased, SPATA41 is increased, SPP1 is increased,
TAF6L is
increased, TFAP2B is decreased, THYN1 is increased, TMEM41B is decreased,
TNNC1 is
decreased, TUBB3 is decreased, TUFM (MIR4721) is increased, TYRP1 is
decreased, UGP2
is decreased, USP7 is decreased, VIM is increased, YKT6 is increased, ZNF48 is
increased,
ZNF496 is increased, ZNF839 is increased, and/or ZSCAN31 is decreased. In
certain
embodiments, the increase or decrease in the expression level is the gene
level from a
recurrent tumor sample versus a non-recurrent tumor sample. In other
embodiments, the
increase or decrease in the expression level is the gene level from a
metastatic tumor sample
versus a non-metastatic tumor sample.
In another embodiment, a method for treating a patient with cutaneous squamous
cell
carcinoma (cSCC) tumor is disclosed herein, the method comprising: (a)
obtaining a cSCC
tumor sample from the patient and isolating mRNA from the sample; (b)
determining the
expression level of at least 10 genes in a gene set; wherein the at least ten
genes in the gene
set are selected from: ACSBG1, AIM2, ALOX12, ANXA9, APOBEC3G, ARPC2,
ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1, Cl QL4, C21orf59, C3orf70, CCL27,
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CD163, CEP76, CHI3L1, CHMP2B, CXCL10, CXCR4, CYP2D6 (L0C101929829), DARS,
DCT, DDAH1, DSS1, DUXAP8, EGFR, EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2,
HDDC3, HNRNPL, HOXA10 (HOXA9, MIR196B), HPGD, ID2, IL24, IL2RB, IL7R,
INHBA, IPO5P1, KIT, KLK5, KRT17, KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1
(ZGPAT), L0C100287896, L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916,
MKLN1, MMP1, MMP10, MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21,
MSANTD4, MYC, NEB, NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN,
PI3, PIG3, PIGBOS1, PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135,
RPL26L1, RPP38, RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3,
SLC25A11, SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B,
TNNC1, TUBB3, TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496,
ZNF839, and/or ZSCAN31; (c) providing an indication as to whether the cSCC
tumor has a
low risk to a high risk of local recurrence, distant metastasis, or both,
based on the expression
level of at least 10 genes generated in step (b); and (d) administering to the
patient an
aggressive treatment when the determination is made in the affirmative that
the patient has a
cSCC tumor with a high risk of local recurrence, distant metastasis, or both.
In some embodiments, the expression level of each gene in the gene set is
determined
by reverse transcribing the isolated mRNA into cDNA and measuring a level of
fluorescence
for each gene in the gene set by a nucleic acid sequence detection system
following Real-
Time Polymerase Chain Reaction (RT-PCR). In certain embodiments, the cSCC
tumor
sample is obtained from formalin-fixed, paraffin embedded sample.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
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TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 20 genes, 30 genes, or
40 genes
selected from the genes listed above.
Other aspects, embodiments, and implementations will become apparent from the
following detailed description and claims, with reference, where appropriate,
to the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the study design workflow.
FIG. 2 shows the differential expression of 18 genes found to be significantly
differentially
expressed between recurrent (Rec) and non-recurrent (NR) cSCC cases
FIG. 3 shows another exemplary study design workflow.
FIG. 4 shows a metastasis-free survival curve for low risk, Class 1, and high-
risk, Class 2,
tumors using the 20-1 gene set.
DETAILED DESCRIPTION
Despite overall good prognosis for patients with cSCC, a subset will develop
local,
regional, or distant recurrences following complete excision of the primary
tumor. Those at
high risk of recurrence are eligible for adjuvant treatment options. While
specific clinical
features are associated with recurrence, they collectively fail to identify 30-
40% of all cSCC
recurrences and many tumors that express high risk features will not recur. To
address the
need for more accurate predictive factors and facilitate appropriate
intervention strategies, a
gene expression analysis was used to determine a signature associated with
recurrence in
cSCC. In that analysis, 140 candidate genes were selected for evaluation of
gene expression
changes in recurrent and non-recurrent cases. A total of 230 primary cSCC
tumors were
collected under an IRB-approved, multi-center protocol and analyzed. After
quality filtering,
expression of the genes was assessed across 212 samples. Multiple subsets of
genes were
significantly differentially expressed between recurrent and non-recurrent
cases. The results
demonstrate that gene expression differences can distinguish between recurrent
and non-
recurrent cSCC. Such gene expression differences can help identify those
patients who might
benefit from additional therapeutic interventions and treatments.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as would be commonly understood by one of ordinary skill in the art to
which the
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claimed invention belongs. Although methods and materials similar or
equivalent to those
described herein can be used to practice the methods and kits disclosed or
claimed herein,
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 are not
intended to be limiting.
Other features and advantages of the claimed invention will be apparent from
the following
detailed description.
As used herein, the singular forms "a," "an," and "the" include plural
referents unless
the context clearly dictates otherwise. For example, reference to "a nucleic
acid" means one
or more nucleic acids.
It is noted that terms like "preferably," "commonly," and "typically" are not
utilized
herein to limit the scope of the claimed invention or to imply that certain
features are critical,
essential, or even important to the structure or function of the claimed
invention. Rather,
these terms are merely intended to highlight alternative or additional
features that can or
cannot be utilized in a particular embodiment disclosed or claimed herein.
As used herein, the terms "polynucleotide," "nucleotide," "oligonucleotide,"
and
"nucleic acid" can be used interchangeably to refer to nucleic acid comprising
DNA, cDNA,
RNA, derivatives thereof, or combinations thereof
In an embodiment, a method for treating a patient with a cutaneous squamous
cell
carcinoma (cSCC) tumor is disclosed herein, the method comprising: (a)
obtaining a
diagnosis identifying a risk of local recurrence, distant metastasis, or both,
in a cSCC tumor
sample from the patient, wherein the diagnosis was obtained by: (1)
determining the
expression level of at least 10 genes in a gene set; wherein the at least 10
genes in the gene
set are selected from: ACSBG1, AIM2, ALOX12, ANXA9, APOBEC3G, ARPC2,
ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1, Cl QL4, C21orf59, C3orf70, CCL27,
CD163, CEP76, CHI3L1, CHMP2B, CXCL10, CXCR4, CYP2D6 (L0C101929829), DARS,
DCT, DDAH1, DSS1, DUXAP8, EGFR, EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2,
HDDC3, HNRNPL, HOXA10 (HOXA9, MIR196B), HPGD, ID2, IL24, IL2RB, IL7R,
INHBA, IPO5P1, KIT, KLK5, KRT17, KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1
(ZGPAT), L0C100287896, L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916,
MKLN1, MMP1, MMP10, MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21,
MSANTD4, MYC, NEB, NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN,
PI3, PIG3, PIGBOS1, PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135,
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RPL26L1, RPP38, RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3,
SLC25A11, SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B,
TNNC1, TUBB3, TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496,
ZNF839, and/or ZSCAN31; (2) comparing the expression levels of the at least 10
genes in
.. the gene set from the cSCC tumor sample to the expression levels of the at
least 10 genes in
the gene set from a predictive training set to generate a probability score of
the risk of local
recurrence, distant metastasis, or both, and; (3) providing an indication as
to whether the
cSCC tumor has a low risk to a high risk of local recurrence, distant
metastasis, or both,
based on the probability score generated in step (2); and (4) identifying that
the cSCC tumor
has a high risk of local recurrence, distant metastasis, or both, based on the
probability score
and diagnosing the cSCC tumor as having a high risk of local recurrence,
distant metastasis,
or both; (b) administering to the patient an aggressive treatment when the
determination is
made in the affirmative that the patient has a cSCC tumor with a high risk of
local recurrence,
distant metastasis, or both. In certain embodiments, the method further
comprises performing
a resection of the cSCC tumor when the determination is made in the
affirmative that the
patient has a cSCC tumor with a high risk of local recurrence, distant
metastasis, or both.
In some embodiments, the expression level of each gene in the gene set is
determined
by reverse transcribing the isolated mRNA into cDNA and measuring a level of
fluorescence
for each gene in the gene set by a nucleic acid sequence detection system
following Real-
Time Polymerase Chain Reaction (RT-PCR). In certain embodiments, the cSCC
tumor
sample is obtained from formalin-fixed, paraffin embedded sample.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
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TUFM (MIR4721), TYRPI, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 20 genes, 30 genes, or
40 genes
selected from the genes listed above.
In an embodiment, a method of treating a patient with a cutaneous squamous
cell
.. carcinoma (cSCC) tumor is disclosed herein, the method comprising
administering an
aggressive cancer treatment regimen to the patient, wherein the patient has a
cSCC tumor
with a probability score of between 0.500 and 1.00 as generated by comparing
the expression
levels of at least 10 genes selected from ACSBG1, AIM2, ALOX12, ANXA9,
APOBEC3G,
ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1, C1QL4, C21orf59, C3orf70,
CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10, CXCR4, CYP2D6
(L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR, EphB2, FCHSD1,
FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9, MIR196B), HPGD,
ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17, KRT18, KRT19, KRT6B,
LAMC2, LCE2B, LIME1 (ZGPAT), L0C100287896, L0C101927502, LOR, LRRC47,
MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10, MMP12, MMP13, MMP3,
MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB, NEFL, NFASC, NFIA, NFIB,
NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1, PIM2, PLAU, PLS3, PTHLH,
PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38, RUNX3, S100A8, S100A9,
SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11, SNORD124, SPATA41, SPP1,
TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3, TUFM (MIR4721), TYRP1,
UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or ZSCAN31 from the cSCC
tumor with the expression levels of the same at least ten genes selected from
ACSBG1,
AIM2, ALOX12, ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9,
BLOC1S1, C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B,
CXCL10, CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8,
EGFR, EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10
(HOXA9, MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5,
KRT17, KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), L0C100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
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TUFM (MIR4721), TYRPI, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31 from a predictive training set. In one embodiment, the probability
score is
determined by a bimodal, two-class analysis, wherein a patient having a value
of between 0
and 0.499 is designated as class 1 with a low risk of local recurrence,
distant metastasis, or
both, and a patient having a value of between 0.500 and 1.00 is designated as
class 2 with an
increased risk of local recurrence, distant metastasis, or both.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
.. MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 20 genes, 30 genes, or
40 genes
selected from the genes listed above.
As used herein, the term "recurrence" refers to the recurrence or disease
progression
that may occur locally (such as local recurrence and in transit disease),
regionally (such as
nodal micrometastasis or macrometastasis), or distally (such as brain, lung
and other tissues).
Risk, as used herein, includes low-risk or high-risk of metastasis according
to any of the
statistical methods disclosed herein. In one embodiment, risk of recurrence or
metastasis for
cSCC can be classified from a low risk to a high risk (for example, the cSCC
tumor has a
graduated risk from low risk to high risk or high risk to low risk of local
recurrence,
.. locoregional recurrence, or distant metastasis). In other embodiments, low
risk refers to a 5-
year relapse-free survival rate, a 5-year metastasis free survival rate, or a
5-year disease
specific survival rate of greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%,
95%, or more than 95%, and high risk refers to a 5-year relapse-free survival
rate, a 5-year
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metastasis free survival rate, or a 5-year disease specific survival rate of
less than 50%, 45%,
40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than 5%.
In certain embodiments, risk stratifications may be binned, for example a
group with
an arbitrary designation Class A may be selected based on recurrence risk of
less than 25%,
20%, 15%, 10%, 5%, or less than 5%. A group with arbitrary designation Class B
may be
selected based on a risk of 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%,
25%, or
any number in between. A group with arbitrary designation Class C may be
selected based
on a risk of 75%, 80%, 85%, 90%, 95%, or higher than 95%. These class
designations may
comprise more than three groups or as few as two groups depending on the
separation
characteristics of the predictive algorithm. A person familiar with the art
will be able to
determine the optimal binning strategy depending on the distributions of class
probability
scores developed by modeling.
As used herein, the term "metastasis" refers to the recurrence or disease
progression
that may occur locally, regionally (such as nodal metastasis), or distally
(such as distant
metastasis to the brain, lung and other tissues). Class 1 or class 2 of
metastasis, as used
herein, includes low-risk (class 1; for example, having a 5-year relapse-free
survival rate, a 5-
year metastasis free survival rate, or a 5-year disease specific survival rate
of greater than
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%) or high-
risk
(class 2; for example, having a 5-year metastasis free survival rate, or a 5-
year disease
.. specific survival rate of less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,
10%, 5%, or
less than 5%) of metastasis according to any of the statistical methods
disclosed herein. Class
A, Class B, or Class C of metastasis, as used herein, includes low-risk (class
A; for example
having a recurrence risk of less than 25%, 20%, 15%, 10%, 5%, or less than
5%),
intermediated risk (class B; for example having a recurrence risk of 75%, 70%,
65%, 60%,
55%, 50%, 45%, 40%, 35%, 30%, 25%, or any number in between) or high-risk
(class C; for
example, having a recurrence risk of 75%, 80%, 85%, 90%, 95%, or higher than
95%) of
metastasis according to any of the statistical methods disclosed herein. The
term "distant
metastasis" as used herein, refers to metastases from a primary cSCC tumor
that are
disseminated widely. Patients with distant metastases require aggressive
treatments, which
.. can eradicate metastatic cSCC, prolong life, and/or cure some patients.
As used herein, the terms "locoregional recurrence" and "local recurrence" can
be
used interchangeably and refer to cancer cells that have spread to tissue
immediately
surrounding the primary cSCC tumor or were not completely ablated or removed
by previous
treatment or surgical resection. Locoregional recurrences are typically
resistant to
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chemotherapy and radiation therapy. Locoregional recurrence can be difficult
to control
and/or treat if: (1) the primary cSCC tumor is located or involves a vital
organ or structure
that limits the potential for treatment; (2) recurrence after surgery or other
therapy occurs,
because while likely not a result from metastasis, high rates of recurrence
indicate an
advanced cSCC tumor; and (3) presence of lymph node metastases, while rare in
cSCC,
indicate advanced disease.
In some embodiments, the methods described herein can comprise determining
that
the cSCC tumor has an increased risk of metastasis or decreased overall
survival by
combining with clinical staging factors recommended by, for example, the
American Joint
Committee on Cancer (AJCC), the Brigham Women's Hospital (BWH), or the
National
Comprehensive Cancer Network (NCCN), to stage the primary cSCC tumor, or other
histological features associated with risk of cSCC tumor metastasis or disease-
related death.
As used herein, the terms "cutaneous squamous cell carcinoma" or "cSCC" or
"SCC"
refer to any cutaneous squamous cell carcinoma, regardless of tumor size, in
patients without
clinical or histologic evidence of regional or distant metastatic disease. A
cutaneous
squamous cell carcinoma sample may be obtained through a variety of sampling
methods
such as punch biopsy, shave biopsy, surgical excision, core needle biopsy,
incisional biopsy,
endoscope ultrasound (EUS) guided-fine needle aspirate (FNA) biopsy,
percutaneous biopsy,
and other means of extracting RNA from the primary cSCC tumor. A carcinoma is
a type of
cancer that develops from epithelial cells. Specifically, a carcinoma is a
cancer that begins in
a tissue that lines the inner or outer surfaces of the body, and that arises
from cells originating
in the endodermal, mesodermal, and ectodermal germ layer during embryogenesis.
Squamous cell carcinomas have observable features and characteristics
indicative of
squamous differentiation (e.g., intercellular bridges, keratinization,
squamous pearls). The
most recognized risk factor for cSCC is exposure to sunlight; thus, most cSCC
tumors
develop on sun-exposed skin sites, for example, the head or neck area. They
can also be
found on the face, ears, lips, trunk, arms, legs, hands, or feet. Squamous
cell carcinoma is the
second most common skin cancer.
As used herein, "overall survival" (OS) refers to the percentage of people in
a study or
treatment group who are still alive for a certain period of time after they
were diagnosed with
or started treatment for a disease, such as cancer. The overall survival rate
is often stated as a
five-year survival rate, which is the percentage of people in a study or
treatment group who
are alive five years after their diagnosis or the start of treatment.
The phrase "measuring the gene-expression levels" or "determining the gene-
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expression levels," as used herein, refers to determining or quantifying RNA
or proteins
expressed by the gene or genes. The term "RNA" includes mRNA transcripts,
and/or specific
spliced variants of mRNA. The term "RNA product of the gene," as used herein,
refers to
RNA transcripts transcribed from the gene and/or specific spliced variants. In
some
embodiments, mRNA is converted to cDNA before the gene expression levels are
measured.
With respect to proteins, gene expression refers to proteins translated from
the RNA
transcripts transcribed from the gene. The term "protein product of the gene"
refers to
proteins translated from RNA products of the gene. A number of methods can be
used to
detect or quantify the level of RNA products of the gene or genes within a
sample, including
microarrays, Real-Time PCR (RT-PCR; including quantitative RT-PCR), nuclease
protection
assays, RNA-sequencing, and Northern blot analyses. In one embodiment, the
assay uses the
APPLIED BIOSYSTEMSTm HT7900 fast Real-Time PCR system. In addition, a person
skilled in the art will appreciate that a number of methods can be used to
determine the
amount of a protein product of a gene of the methods disclosed herein,
including
immunoassays such as Western blots, ELISA, and immunoprecipitation followed by
SDS-
PAGE and immunocytochemistry. In certain embodiments, the expression level of
each gene
in the gene set is determined by reverse transcribing the isolated mRNA into
cDNA and
measuring a level of fluorescence for each gene in the gene set by a nucleic
acid sequence
detection system following Real-Time Polymerase Chain Reaction (RT-PCR).
A person skilled in the art will appreciate that a number of detection agents
can be
used to determine gene expression. For example, to detect RNA products of the
biomarkers,
probes, primers, complementary nucleotide sequences, or nucleotide sequences
that hybridize
to the RNA products can be used. In another example, to detect cDNA products
of the
biomarkers, probes, primers, complementary nucleotide sequences, or nucleotide
sequences
that hybridize to the cDNA products can be used. To detect protein products of
the
biomarkers, ligands or antibodies that specifically bind to the protein
products can be used.
As used herein, the term "hybridize" refers to the sequence specific non-
covalent
binding interaction with a complementary nucleic acid. In one embodiment, the
hybridization is under high stringency conditions. Appropriate stringency
conditions that
promote hybridization are known to those skilled in the art.
As used herein, the terms "probe" and "primer" refer to a nucleic acid
sequence that
will hybridize to a nucleic acid target sequence. In one example, the probe
and/or primer
hybridizes to an RNA product of the gene or a complementary nucleic acid
sequence. In
another example, the probe and/or primer hybridizes to a cDNA product. The
length of probe
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or primer depends on the hybridizing conditions and the sequences of the probe
or primer and
nucleic acid target sequence. In one embodiment, the probe or primer is at
least 8, 10, 15, 20,
25, 50, 75, 100, 150, 200, 250, 400, 500, or more than 500 nucleotides in
length. Probes
and/or primers may include one or more label. Probes and/or primers may be
commercially
.. sourced from various providers (e.g., ThermoFisher Scientific). In certain
embodiments, a
label may be any substance capable of aiding a machine, detector, sensor,
device, or
enhanced or unenhanced human eye from differentiating a labeled composition
from an
unlabeled composition. Examples of labels include, but are not limited to: a
radioactive
isotope or chelate thereof, dye (fluorescent or non-fluorescent), stain,
enzyme, or
nonradioactive metal. Specific examples include, but are not limited to:
fluorescein, biotin,
digoxigenin, alkaline phosphates, biotin, streptavidin, 3H, 14c,32P, S, or any
other
compound capable of emitting radiation, rhodamine, 4-(4'-dimethylamino-
phenylazo)benzoic
acid; 4-(4'-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride); 5-((2-
aminoethyl)-
amino)-naphtalene-1-sulfonic acid; Psoralene derivatives, haptens, cyanines,
acridines,
fluorescent rhodol derivatives, cholesterol derivatives; ethylene-diamine-
tetra-acetic acid and
derivatives thereof, or any other compound that may be differentially
detected. The label
may also include one or more fluorescent dyes. Examples of dyes include, but
are not limited
to: CAL-Fluor Red 610, CAL-Fluor Orange 560, dR110, 5-FAM, 6FAM, dR6G, JOE,
HEX,
VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540, and LIZ.
As used herein, a "sequence detection system" is any computational method in
the art
that can be used to analyze the results of a PCR reaction. One example is the
APPLIED
BIOSYSTEMSTm HT7900 fast Real-Time PCR system. In certain embodiments, gene
expression can be analyzed using, e.g., direct DNA expression in microarray,
Sanger
sequencing analysis, Northern blot, the NANOSTRINGO technology, serial
analysis of gene
expression (SAGE), RNA-seq, tissue microarray, or protein expression with
immunohistochemistry or western blot technique. PCR generally involves the
mixing of a
nucleic acid sample, two or more primers that are designed to recognize the
template DNA, a
DNA polymerase, which may be a thermostable DNA polymerase such as Taq or Pfu,
and
deoxyribose nucleoside triphosphates (dNTP's). Reverse transcription PCR,
quantitative
reverse transcription PCR, and quantitative real time reverse transcription
PCR are other
specific examples of PCR. In real-time PCR analysis, additional reagents,
methods, optical
detection systems, and devices known in the art are used that allow a
measurement of the
magnitude of fluorescence in proportion to concentration of amplified DNA. In
such
analyses, incorporation of fluorescent dye into the amplified strands may be
detected or
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measured. In one embodiment, the expression level of each gene in the gene set
is
determined by reverse transcribing the isolated mRNA into cDNA and measuring a
level of
fluorescence for each gene in the gene set by a nucleic acid sequence
detection system
following Real-Time Polymerase Chain Reaction (RT-PCR).
As used herein, the terms "differentially expressed" or "differential
expression" refer
to a difference in the level of expression of the genes that can be assayed by
measuring the
level of expression of the products of the genes, such as the difference in
level of messenger
RNA transcript expressed (or converted cDNA) or proteins expressed of the
genes. In one
embodiment, the difference can be statistically significant. The term
"difference in the level
of expression" refers to an increase or decrease in the measurable expression
level of a given
gene as measured by the amount of messenger RNA transcript (or converted cDNA)
and/or
the amount of protein in a sample as compared with the measurable expression
level of a
given gene in a control, or control gene or genes in the same sample (for
example, a non-
recurrence sample). In another embodiment, the differential expression can be
compared
using the ratio of the level of expression of a given gene or genes as
compared with the
expression level of the given gene or genes of a control, wherein the ratio is
not equal to 1Ø
For example, an RNA, cDNA, or protein is differentially expressed if the ratio
of the level of
expression in a first sample as compared with a second sample is greater than
or less than 1Ø
For example, a ratio of greater than 1, 1.2, 1.5, 1.7, 2, 3, 3, 5, 10, 15, 20,
or more than 20, or a
ratio less than 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.001, or less than 0.0001.
In yet another
embodiment, the differential expression is measured using p-value. For
instance, when using
p-value, a biomarker is identified as being differentially expressed as
between a first sample
and a second sample when the p-value is less than 0.1, less than 0.05, less
than 0.01, less than
0.005, or less than 0.001.
The terms "increased expression" or "decreased expression," as used herein,
refer to
an expression level of one or more genes, or prognostic RNA transcripts, or
their
corresponding cDNAs, or their expression products that has been found to be
differentially
expressed in recurrent versus non-recurrent cSCC tumors. The higher the
expression level of
a gene that predominantly has increased expression in tumors of patients who
had recurrence,
the higher is the likelihood that the patient suffering from this tumor is
expected to have a
poor clinical outcome (i.e., higher risk of recurrence, metastasis, or both).
In contrast, the
lower the expression level of a gene that predominantly has increased
expressed in tumors of
patients who have recurrent tumors, the higher is the likelihood that the
patient suffering from
this tumor is expected to have a promising clinical outcome (i.e., decreased
risk of
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recurrence, metastasis, or both). The lower the expression level of a gene
that predominantly
has decreased expression in tumors of patients who had recurrence, the higher
is the
likelihood that the patient suffering from this tumor is expected to have a
poor clinical
outcome (i.e., higher risk of recurrence, metastasis, or both). In contrast,
the higher the
expression level of a gene that predominantly has decreased expressed in
tumors of patients
who have recurrent tumors, the higher is the likelihood that the patient
suffering from this
tumor is expected to have a promising clinical outcome (i.e., decreased risk
of recurrence,
metastasis, or both).
References herein to the "same" level of biomarker indicate that the level of
biomarker measured in each sample is identical (i.e., when compared to the
selected
reference). References herein to a "similar" level of biomarker indicate that
levels are not
identical but the difference between them is not statistically significant
(i.e., the levels have
comparable quantities).
As used herein, the terms "control" and "standard" refer to a specific value
that one
can use to determine the value obtained from the sample. In one embodiment, a
dataset may
be obtained from samples from a group of subjects known to have a cutaneous
squamous cell
carcinoma or subtype. The expression data of the genes in the dataset can be
used to create a
control (standard) value that is used in testing samples from new subjects. In
such an
embodiment, the "control" or "standard" is a predetermined value for each gene
or set of
genes obtained from subjects with a cutaneous squamous cell carcinoma whose
gene
expression values and tumor types are known. In certain embodiments of the
methods
disclosed herein, non-limiting examples of control genes can include, but are
not limited to,
BAG6 (probe ID: Hs00190383), KMT2D/MLL2 (probe ID: Hs00912419 ml), MDM2
(probe ID: Hs00540450 sl), FXR1 (probe ID: Hs01096876_g1), KMT2C (probe ID:
Hs01005521 ml), MDM4 (probe ID: Hs00967238 ml), VIM, and NF1B. In some
embodiments, a control population may comprise healthy individuals,
individuals with
cancer, or a mixed population of individuals with or without cancer. In
certain embodiments,
a control population may comprise individuals with non-metastatic cancer or
cancer that did
not recur.
As used herein, the term "normal" when used with respect to a sample
population
refers to an individual or group of individuals that does/do not have a
particular disease or
condition (e.g., cSCC or recurrent cSCC) and is also not suspected of having
or being at risk
for developing the disease or condition. The term "normal" is also used herein
to qualify a
biological specimen or sample (e.g., a biological fluid) isolated from a
normal or healthy
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individual or subject (or group of such subjects), for example, a "normal
control sample."
The "normal" level of expression of a marker is the level of expression of the
marker in cells
in a similar environment or response situation, in a patient not afflicted
with cancer. A
normal level of expression of a marker may also refer to the level of
expression of a
"reference sample" (e.g., a sample from a healthy subject not having the
marker associated
disease). A reference sample expression may be comprised of an expression
level of one or
more markers from a reference database. Alternatively, a "normal" level of
expression of a
marker is the level of expression of the marker in non-tumor cells in a
similar environment or
response situation from the same patient that the tumor is derived from.
As used herein, the terms "gene-expression profile," "GEP," or "gene-
expression
profile signature" refer to any combination of genes, the measured messenger
RNA transcript
expression levels, cDNA levels, or direct DNA expression levels, or
immunohistochemistry
levels of which can be used to distinguish between two biologically different
corporal tissues
and/or cells and/or cellular changes. In certain embodiments, a gene-
expression profile is
comprised of the gene-expression levels of at least 140, 139, 138, 137, 136,
135, 134, 133,
132,131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117,
116, 115, 114,
113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98,
97, 96, 95, 94,
93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75,
74, 73, 72, 71, 70, 69,
68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50,
49, 48, 47, 46, 45, 44,
43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25,
24, 23, 22, 21, 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, or 10 genes, or less than 10 genes. In one
embodiment, the
gene-expression profile is comprised of 56 genes. In another embodiment, the
gene-
expression profile is comprised of 40 genes. In another embodiment, the gene-
expression
profile is comprised of 30 genes. In another embodiment, the gene-expression
profile is
comprised of 20 genes. In certain embodiments, the genes selected are: ACSBG1,
AIM2,
ALOX12, ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9,
BLOC1S1, C1QL4, C2lorf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B,
CXCL10, CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8,
EGFR, EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10
(HOXA9, MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5,
KRT17, KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), L0C100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
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PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 20 genes, 30 genes, or
40 genes
selected from the genes listed above. In some embodiments, the gene set
further comprises
control genes or normalization genes selected from: BAG6 (probe ID:
Hs00190383),
KMT2D/MLL2 (probe ID: Hs00912419 ml), MDM2 (probe ID: Hs00540450 sl), FXR1
(probe ID: Hs01096876 gl), KMT2C (probe ID: Hs01005521 ml), MDM4 (probe ID:
Hs00967238 ml), VIM, and NF1B.
As used herein, the term "predictive training set" refers to a cohort of cSCC
tumors
with known clinical outcome for local recurrence, distant metastasis, or both
and known
genetic expression profile, used to define or establish all other cSCC tumors,
based upon the
genetic expression profile of each, as a low-risk, class 1 tumor type or a
high-risk, class 2
tumor type. Additionally, included in the predictive training set is the
definition of "threshold
points," which are points at which a classification of metastatic risk is
determined, specific to
each individual gene expression level.
As used herein, the term "altered in a predictive manner" refers to changes in
genetic
expression profile that predict local recurrence, distant metastasis,
metastatic risk, or predict
overall survival. Predictive modeling risk assessment can be measured as: 1) a
binary
outcome having risk of metastasis or overall survival that is classified as
low risk (e.g.,
termed Class 1 herein) vs. high risk (e.g., termed Class 2 herein); and/or 2)
a linear outcome
based upon a probability score from 0 to 1 that reflects the correlation of
the genetic
expression profile of a cSCC tumor with the genetic expression profile of the
samples that
.. comprise the training set used to predict risk outcome. Within the
probability score range
from 0 to 1, a probability score, for example, of less than 0.5 reflects a
tumor sample with a
low risk of local recurrence, metastasis, or death from disease, while a
probability score, for
example, of greater than 0.5 reflects a tumor sample with a high risk of local
recurrence,
metastasis, or death from disease. The increasing probability score from 0 to
1 reflects
incrementally declining metastasis free survival. In one embodiment, the
probability score is
a bimodal, two-class analysis, wherein a patient having a value of between 0
and 0.499 is
designated as class 1 (low risk; for example, having a 5-year relapse-free
survival rate, a 5-
year metastasis free survival rate, or a 5-year disease specific survival rate
of greater than
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%) and a
patient
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having a value of between 0.500 and 1.00 is designated as class 2 (high risk;
for example,
having a 5-year metastasis free survival rate, or a 5-year disease specific
survival rate of less
than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than 5%).
In certain embodiments, the probability score is a tri-modal, three-class
analysis,
wherein patients are designated as class A (low risk; for example having a
recurrence risk of
less than 25%, 20%, 15%, 10%, 5%, or less than 5%), class B (intermediate
risk; for example
having a recurrence risk of 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%,
25%, or
any number in between), or class C (high risk; for example, having a
recurrence risk of 75%,
80%, 85%, 90%, 95%, or higher than 95%). To develop a ternary, or three-class
system of
risk assessment, with Class A having a low risk of metastasis or death from
disease, Class B
having an intermediate risk, and Class C having a high risk, the median
probability score
value for all low risk or high risk tumor samples in the training set was
determined, and one
standard deviation from the median was established as a numerical boundary to
define low or
high risk. For example, low risk cSCC tumors within the ternary classification
system can
have a 5-year metastasis free survival of 100% (e.g., Class A; with a
probability score of 0-
0.337), compared to high risk (e.g., Class C; with a probability score of
0.673-1) cSCC
tumors which can have a 20% 5-year metastasis free survival. Cases falling
outside of one
standard deviation from the median low or high risk probability scores have an
intermediate
risk, and intermediate risk (Class B; with a probability score of 0.338-0.672)
cSCC tumors
can have a 55% 5-year metastasis free survival rate.
The TNM (Tumor-Node-Metastasis) status system is the most widely used cancer
staging system among clinicians and is maintained by the American Joint
Committee on
Cancer (AJCC) and the International Union for Cancer Control (UICC). Cancer
staging
systems codify the extent of cancer to provide clinicians and patients with
the means to
quantify prognosis for individual patients and to compare groups of patients
in clinical trials
and who receive standard care around the world.
Local recurrence rates for cSCC have been reported to be 1-10%, but can be as
high
as 47% in patients who have cSCCs with high-risk clinical features. While the
overall rate of
metastasis is -5%, this rate increases up to -45% in patients with high-risk
clinical features
or who have already experienced a recurrence. After regional or distant
metastasis occurs,
prognosis is usually poor, with 5-year survival rates ranging from 26-34% and
10-year
survival rates of 16%. Although the overall percentages of patients who die
from cSCC
(-1%) are low, the absolute number of deaths are estimated to be equal to or
greater than
those attributed to melanoma, due to the large number of yearly cSCC diagnoses
(400,000-
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700,000 patients), and account for the majority of NMSC-related deaths. In
effect, local and
regional recurrence from primary cSCC tumors remains a significant health
burden.
Cutaneous squamous cell carcinoma stems from interfollicular epidermal
keratinocytes and can arise from precancerous lesions, the most common of
which are actinic
keratoses. Once the malignant cells enter the dermis, the cSCC becomes
invasive.
Squamous cell carcinoma can present as smooth or hyperkeratinized lesions that
are pink or
skin-colored. They can exhibit ulceration and bleed when traumatized. Risk
factors that
contribute to the development of cSCC include exposures to ultraviolet
radiation, ionizing
radiation, and chemicals, as well as increased age and male gender.
Immunosuppressed
individuals, those with a history of non-Hodgkin lymphoma, including chronic
lymphocytic
leukemia, those with certain genetic skin conditions, and those who have had
organ
transplants are at a significantly increased risk for developing cSCC. In
fact, the latter group
has risk up to 100 times that of the normal population. Some drugs used to
treat other types
of skin cancer (e.g., basal cell carcinoma (BCC), melanoma), including
hedgehog, BRAF,
and MET inhibitors, can also increase the propensity for cSCC. Small, low-risk
lesions can
be treated with cryosurgery, curettage and electrodessication, or surgery,
while larger, higher
risk lesions are generally treated with surgical excision or Mohs surgery.
Radiotherapy can
be used in conjunction with surgery if margins are not cleared surgically or
if there is
perineural invasion. If regional recurrence occurs, the lymph nodes are the
primary site of
involvement, accounting for ¨80-85% of cSCC recurrences, while distant
metastasis occurs
in ¨15-20% of patients.
Because the risk for local recurrence in clinically or pathologically high
risk
squamous cell carcinoma lesions can approach 45%, and the risk of metastasis
then increases
upon local recurrence, there has been an increased interest in more accurately
identifying
such lesions. As such, the National Comprehensive Cancer Network (NCCN) and
American
Joint Committee on Cancer (AJCC) have recently proposed parameters to
distinguish high
risk lesions and follow-up measures for these lesions. These high-risk
features include tumor
size and location ("mask" areas of the face and/or ear and non-glabrous lip),
increased
thickness or Clark's level, immunosuppression, recurrent lesions, sites of
chronic
inflammation or previous radiation, poor differentiation, and perineural
invasion. However,
high-risk cSCC definitions from different groups are discordant, with the AJCC
classifying a
majority of lesions as low-risk and NCCN classifying a majority as high-risk.
Such
discrepancies, especially in the T2a and T2b groups, have led to the proposal
of alternative
staging criteria that can better elucidate high risk cSCC cases. In effect,
there is a clinically
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unmet need for better markers to identify high-risk lesions, particularly
molecular biomarkers
that can be objectively evaluated. The validated prognostic gene expression
profiles
disclosed herein could inform clinical decision-making on, for example: (1)
preoperative
surgical staging, based on shave biopsy; (2) adjuvant radiation, nodal
staging, adjuvant
systemic therapy to reduce regional/distant metastasis; and (3) improving
identification of
patients with cSCC who can benefit from surgical, radiation and immunotherapy
interventions.
Squamous cell carcinoma that is predicted to have an increased risk of
recurrence,
progression, or metastasis can be treated with an aggressive cancer treatment
regimen.
Advanced cSCC may be defined under two headings: (1) locoregional disease;
and/or (2)
distant metastases. Locoregional disease can be difficult to control and/or
treat if: (1) the
primary cSCC has invaded into neuronal or vascular structures; (2) there is
presence of lymph
node metastases, which indicate advanced disease; or (3) distant metastases
have been
detected.
In an embodiment, a method for predicting risk of recurrence, metastasis, or
both, in a
patient with a cutaneous squamous cell carcinoma (cSCC) tumor is disclosed
herein, the
method comprising: (a) obtaining a cSCC tumor sample from the patient and
isolating mRNA
from the sample; (b) determining the expression level of at least 10 genes in
a gene set;
wherein the at least ten genes in the gene set are selected from: ACSBG1,
AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, 5100A8, 5100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31; (c) comparing the expression levels of the at least 10 genes in the
gene set from
the cSCC tumor sample to the expression levels of the at least 10 genes in the
gene set from a
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predictive training set to generate a probability score of the risk of local
recurrence, distant
metastasis, or both; and (d) providing an indication as to whether the cSCC
tumor has a low
risk to a high risk of local recurrence, distant metastasis, or both, based on
the probability
score generated in step (c).
In some embodiments, the expression level of each gene in the gene set is
determined
by reverse transcribing the isolated mRNA into cDNA and measuring a level of
fluorescence
for each gene in the gene set by a nucleic acid sequence detection system
following Real-
Time Polymerase Chain Reaction (RT-PCR). In certain embodiments, the cSCC
tumor
sample is obtained from formalin-fixed, paraffin embedded sample. In one
embodiment, the
method further comprises identifying the cSCC tumor as having a high risk of
local
recurrence, distant metastasis, or both, based on the probability score, and
administering to
the patient an aggressive tumor treatment.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 10 genes, 20 genes, 30
genes, or
40 genes selected from the genes listed above.
In an embodiment, a method for predicting risk of recurrence, metastasis, or
both, in a
patient with a cutaneous squamous cell carcinoma (cSCC) tumor is disclosed
herein, the
method comprising: (a) obtaining a cSCC tumor sample from the patient and
isolating mRNA
from the sample; (b) determining the expression level of at least 10 genes in
a gene set;
wherein the at least ten genes in the gene set are selected from: ACSBG1,
AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
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C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31; and (c) providing an indication as to whether the cSCC tumor has a
low risk to a
high risk of local recurrence, distant metastasis, or both, based on the
expression level of at
least 10 genes generated in step (b).
In some embodiments, the expression level of each gene in the gene set is
determined
by reverse transcribing the isolated mRNA into cDNA and measuring a level of
fluorescence
for each gene in the gene set by a nucleic acid sequence detection system
following Real-
Time Polymerase Chain Reaction (RT-PCR). In certain embodiments, the cSCC
tumor
sample is obtained from formalin-fixed, paraffin embedded sample.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
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ZSCAN31. In other embodiments, the gene set comprises 10 genes, 20 genes, 30
genes, or
40 genes selected from the genes listed above.
In certain embodiments, the expression level of: ACSBG1 is decreased, AIM2 is
increased, ALOX12 is decreased, ANXA9 is decreased, APOBEC3G is increased,
ARPC2 is
.. decreased, ATP6AP1 is decreased, ATP6V0E2 is increased, BBC is increased,
BHLHB9 is
decreased, BLOC1S1 is decreased, Cl QL4 is increased, C21orf59 is increased,
C3orf70 is
increased, CCL27 is decreased, CD163 is increased, CEP76 is decreased, CHI3L1
is
increased, CHMP2B is decreased, CXCL10 is decreased, CXCR4 is increased,
CYP2D6
(L0C101929829) is decreased, DARS is decreased, DCT is decreased, DDAH1 is
decreased,
.. DSS1 is decreased, DUXAP8 is increased, EGFR is increased, EphB2 is
increased, FCHSD1
is decreased, FDFT1 is decreased, FLG is decreased, FN1 is increased, GTPBP2
is decreased,
HDDC3 is increased, HNRNPL is decreased, HOXA10 (HOXA9, MIR196B) is decreased,
HPGD is decreased, ID2 is decreased, IL24 is increased, IL2RB is decreased,
IL7R is
increased, INHBA is increased, IPO5P1 is increased, KIT is increased, KLK5 is
decreased,
KRT17 is decreased, KRT18 is increased, KRT19 is decreased, KRT6B is
decreased,
LAMC2 is decreased, LCE2B is decreased, LIME1 (ZGPAT) is increased,
L0C100287896 is
increased, L0C101927502 is decreased, LOR is decreased, LRRC47 is increased,
MIER2 is
increased, MIR129-1 is increased, MIR3916 is increased, MKLN1 is increased,
MMP1 is
increased, MMP10 is decreased, MMP12 is increased, MMP13 is increased, MMP3 is
increased, MMP7 is increased, MMP9 is decreased, MRC1 is increased, MRPL21 is
increased, MSANTD4 is decreased, MYC is decreased, NEB is decreased, NEFL is
decreased, NFASC is decreased, NFIA is decreased, NFIB is decreased, NFIC is
decreased,
NOA1 is increased, PD1 is decreased, PDL1 is increased, PDPN is increased, PI3
is
decreased, PIG3 is decreased, PIGBOS1 is increased, PIM2 is increased, PLAU is
increased,
PLS3 is decreased, PTHLH is decreased, PTRHD1 is decreased, RBM33 is
increased,
RCHY1 is increased, RNF135 is increased, RPL26L1 is increased, RPP38 is
decreased,
RUNX3 is increased, S100A8 is decreased, S100A9 is decreased, SEPT3 is
decreased,
SERPINB2 is decreased, SERPINB4 is decreased, SLC1A3 is increased, SLC25A11 is
increased, SNORD124 is increased, SPATA41 is increased, SPP1 is increased,
TAF6L is
increased, TFAP2B is decreased, THYN1 is increased, TMEM41B is decreased,
TNNC1 is
decreased, TUBB3 is decreased, TUFM (MIR4721) is increased, TYRP1 is
decreased, UGP2
is decreased, USP7 is decreased, VIM is increased, YKT6 is increased, ZNF48 is
increased,
ZNF496 is increased, ZNF839 is increased, and/or ZSCAN31 is decreased. In
certain
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embodiments, the increase or decrease in the expression level is the gene
level from a
recurrent tumor sample versus a non-recurrent tumor sample.
In an embodiment, a method for treating a patient with cutaneous squamous cell
carcinoma (cSCC) tumor is disclosed herein, the method comprising: (a)
obtaining a cSCC
tumor sample from the patient and isolating mRNA from the sample; (b)
determining the
expression level of at least 10 genes in a gene set; wherein the at least ten
genes in the gene
set are selected from: ACSBG1, AIM2, ALOX12, ANXA9, APOBEC3G, ARPC2,
ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1, Cl QL4, C21orf59, C3orf70, CCL27,
CD163, CEP76, CHI3L1, CHMP2B, CXCL10, CXCR4, CYP2D6 (L0C101929829), DARS,
DCT, DDAH1, DSS1, DUXAP8, EGFR, EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2,
HDDC3, HNRNPL, HOXA10 (HOXA9, MIR196B), HPGD, ID2, IL24, IL2RB, IL7R,
INHBA, IPO5P1, KIT, KLK5, KRT17, KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1
(ZGPAT), L0C100287896, L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916,
MKLN1, MMP1, MMP10, MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21,
MSANTD4, MYC, NEB, NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN,
PI3, PIG3, PIGBOS1, PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135,
RPL26L1, RPP38, RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3,
SLC25A11, SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B,
TNNC1, TUBB3, TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496,
ZNF839, and/or ZSCAN31; (c) providing an indication as to whether the cSCC
tumor has a
low risk to a high risk of local recurrence, distant metastasis, or both,
based on the expression
level of at least 10 genes generated in step (b); and (d) administering to the
patient an
aggressive treatment when the determination is made in the affirmative that
the patient has a
cSCC tumor with a high risk of local recurrence, distant metastasis, or both.
In some embodiments, the expression level of each gene in the gene set is
determined
by reverse transcribing the isolated mRNA into cDNA and measuring a level of
fluorescence
for each gene in the gene set by a nucleic acid sequence detection system
following Real-
Time Polymerase Chain Reaction (RT-PCR). In certain embodiments, the cSCC
tumor
sample is obtained from formalin-fixed, paraffin embedded sample.
In another embodiment, the gene set comprises the genes ACSBG1, AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
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MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31. In other embodiments, the gene set comprises 20 genes, 30 genes, or
40 genes
selected from the genes listed above.
As used herein, the terms "treatment," "treat," or "treating" refer to a
method of
reducing the effects of a disease or condition or symptom of the disease or
condition. Thus,
in the methods disclosed herein, treatment can refer to a 5%, 10%, 20%, 30%,
40%, 50%,
60%, 70%, 80%, 90%, or 100% reduction in the severity of an established
disease or
condition or symptom of the disease or condition. For example, a method of
treating a
disease is considered to be a treatment if there is a 5% reduction in one or
more symptoms of
the disease in a subject as compared to a control. Thus, the reduction can be
a 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percent reduction between
5%
and 100% as compared to native or control levels. It is understood that
treatment does not
necessarily refer to a cure or complete ablation of the disease, condition, or
symptoms of the
disease or condition. After a cSCC is found and staged, a medical professional
or team of
medical professionals will recommend one or several treatment options. In
determining a
treatment plan, factors to consider include the type, location, and stage of
the cancer, as well
as the patient's overall physical health. Prior to the initiation of treatment
and or therapy, all
patients should be evaluated and managed by a multidisciplinary team with
expertise and
experience in cSCC. Patients with cSCC typically have a multidisciplinary
health care team
made up of doctors from different specialties, such as: a dermatologist (in
particular, a
dermatologist who specializes in Mohs micrographic surgery), an orthopedic
surgeon (in
particular, a surgeon who specializes in diseases of the bones, muscles, and
joints), a surgical
oncologist, a thoracic surgeon, a medical oncologist, a radiation oncologist,
and/or a
physiatrist (or rehabilitation doctor). After a cSCC is found and staged, a
medical
professional or team of medical professionals will typically recommend one or
several
treatment options including one or more of surgery, radiation, chemotherapy,
and targeted
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therapy.
Low risk cSCC tumors as defined in the NCCN Guidelines involve: (1) an area
of
less than 20 mm (for truck and extremities) or less than 10 mm for the cheeks,
forehead,
scalp, neck and pretibial; (2) well defined borders; (3) primary cSCC tumor;
(4) not rapidly
growing; (5) from a patient who has no neurologic symptoms and is not
considered
immunosuppressed; (6) from a site free of chronic inflammation; (7) well or
moderately
differentiated; (8) free of acantholytic, adenosquamous, desmoplastic, or
metaplastic
subtypes; (9) depths of less than 2 mm; and (10) free of perineural,
lymphatic, or vascular
involvement.
High risk cSCC tumors as defined in the NCCN Guidelines involve: (1) an area
of
greater than 20 mm (for truck and extremities), greater than 10 mm for the
cheeks, forehead,
scalp, neck and pretibial, or any cSCC involving the "mask areas" (such as
central face,
eyelids, eyebrows, periorbital, nose, lips, chin, mandible, temple or ear),
genitalia, hands and
feet; (2) poorly defined borders; (3) recurrent cSCC tumor; (4) rapidly
growing; (5) from a
patient who has neurologic symptoms or is considered immunosuppressed; (6)
from a site
with chronic inflammation; (7) poorly differentiated; (8) presence of
acantholytic,
adenosquamous, desmoplastic, or metaplastic subtypes; (9) depths of greater
than or equal 2
mm; and (10) presence of perineural, lymphatic, or vascular involvement.
As used herein, the term "aggressive cancer treatment regimen" refers to a
treatment
regimen that is determined by a medical professional or team of medical
professionals and
can be specific to each patient. In certain embodiments, a cSCC tumor
predicted to have a
high-risk of recurrence or a high-risk of metastasis, or a decreased chance of
survival using
the methods and kits disclosed herein, would be treated using an aggressive
cancer treatment
regimen. Whether a treatment is considered to be aggressive will generally
depend on the
cancer-type, the age of the patient, and other factors known to those of skill
in the art. For
example, in breast cancer, adjuvant chemotherapy is a common aggressive
treatment given to
complement the less aggressive standards of surgery and hormonal therapy.
Those skilled in
the art are familiar with various other aggressive and less aggressive
treatments for each type
of cancer. An aggressive cancer treatment regimen is defined by the National
Comprehensive Cancer Network (NCCN), and has been defined in the NCCN
Guidelines
as including one or more of: 1) imaging (CT scan, PET/CT, MRI, chest X-ray),
2) discussion
and/or offering of tumor resection if a tumor is determined to be resectable
(e.g., by Mohs
micrographic surgery or resection with complete circumferential margin
assessment), 3)
radiation therapy (RT), 4) chemoradiation, 5) chemotherapy, 6) regional limb
therapy, 7)
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palliative surgery, 8) systemic therapy, 9) immunotherapy, and 10) inclusion
in ongoing
clinical trials. Guidelines for clinical practice are published in the
National Comprehensive
Cancer Network (NCCN Guidelines Squamous Cell Skin Cancer Version 2.2018,
updated
October 5, 2017, available on the World Wide Web at NCCN.org).
Additional therapeutic options may include, but are not limited to: 1)
combination
regimens such as: AD (doxorubicin, dacarbazine); AIM (doxorubicin, ifosfamide,
mesna);
MAID (mesna, doxorubicin, ifosfamide, dacarbazine); ifosfamide, epirubicin,
mesna;
gemcitabine and docetaxel; gemcitabine and vinorelbine; gemcitabine and
dacarbazine;
doxorubicin and olaratumab ; methotrexate and vinblastine; tamoxifen and
sulindac;
vincristine, dactinomycin, cylclophosphamide; vincristine, doxorubicin,
cyclophosphamide;
vincristine, doxorubicin, cyclophosphamide with ifosfamide and etoposide;
vincristine,
doxorubicin, ifosfamide; cyclophosphamide topotecan; or ifosfamide,
doxorubicin; and/or 2)
single agents, such as: cisplatin or other metallic compounds, 5-
FU/capecitabine (Xeloda0),
cetuximab (Erbitux0), pembrolizumab (MK-3475), panitumumab (Vectibix0),
dacomitinib
.. (PF-00299804), gefitinib (ZD1839, Iressa), doxorubicin, ifosfamide,
epirubicin, gemcitabine,
dacarbazine, temozolomide, vinorelbine, eribulin, trabectedin, pazopanib,
imatinib, sunitinib,
regorafenib, sorafenib, nilotinib, dasatinib, interferon, toremifene,
methotrexate, irinotecan,
topotecan, paclitaxel, nab-paclitaxel (abraxane), docetaxel, bevacizumab,
temozolomide,
sirolimus (Rapamune0), everolimus, temsirolimus, crizotinib, ceritinib, or
palbociclib.
While surgical excision remains the mainstay for treating operable (Stage I-
III) cSCC
patients, for Stage I patients, en bloc resection with negative margins is
generally considered
sufficient for long-term local control. For those with incomplete excision
margins and/or
other unfavorable pathologic features, pre- or post-operative chemotherapy
and/or radiation
treatment can be recommended. No therapy has shown consistent efficacy for the
treatment
of excised cSCC, and treatment options for unresectable or advanced cSCC are
limited.
Immunotherapy using an anti-PD1 inhibitor has shown promising results in early
phase studies with cSCC patients. Examples of immunotherapies (that can be
used alone or
in combination with any one or more of tumor resection if a tumor is
determined to be
resectable, radiation therapy, chemoradiation, chemotherapy, regional limb
therapy, palliative
surgery, systemic therapy, additional immunotherapeutic, or inclusion in
ongoing clinical
trials), can include, for example, pembrolizumab (Keytruda0) and nivolumab
(Opdivo0),
cemiplimab (REGN2810; a fully human monoclonal antibody to Programmed Death-
1).
PD-1 is a protein on T-cells that normally help keep T-cells from attacking
other cells in the
body. By blocking PD-1, these drugs can boost the immune response against
cancer cells.
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CTLA-4 inhibitors (for example, ipilimumab (Yervoy0)) are another class of
drugs that can
boost the immune response. In some instances, cytokine therapy (such as,
interferon-alpha
and interleukin-2) can be used to boost the immune system. Examples of
interferon and
interleukin-based treatments can include, but are not limited to, aldesleukin
(proleukin0),
interferon alpha-2b (INTRONO), and pegylated interferon alpha-2b (Sylvatron0;
PEG-
INTRONO, PEGASYS). In another embodiment, oncolytic virus therapy can be used.
Along with killing the cells directly, the oncolytic viruses can also alert
the immune system to
attack the cancer cells. For example, talimogene laherparepvec (Imlygic0),
also known as T-
VEC, is an oncolytic virus that can be used to treat melanomas. Additional
immunotherapies
may include CV8102.
Additionally, targeted therapies may be used to treat patients with cSCC. For
example, targeted therapies can include, but are not limited to, vemurafenib
(Zelboraf0),
dabrafenib (Tafinlar0), trametinib (Mekinist0), CLL442, and cobimetinib
(Cotellic0).
These drugs target common genetic mutations, such as the BRAFV600 mutation,
that may be
found in a subset of cSCC patients.
In some embodiments, the cSCC tumor is a frozen sample. In another embodiment,
the cSCC sample is formalin-fixed and paraffin embedded. In certain
embodiments, the
cSCC sample is taken from a formalin-fixed, paraffin embedded wide local
excision sample.
In another embodiment, the cSCC tumor is taken from a formalin-fixed, paraffin
embedded
primary biopsy sample. In some embodiments, the cSCC sample can be from image
guided
surgical biopsy, shave biopsy, wide excision, or a lymph node dissection.
In certain embodiments, analysis of genetic expression and determination of
outcome
is carried out using radial basis machine and/or partial least squares
analysis (PLS), partition
tree analysis, logistic regression analysis (LRA), K-nearest neighbor, neural
networks,
ensemble learners, voting algorithms, or other algorithmic approach. These
analysis
techniques take into account the large number of samples required to generate
a training set
that will enable accurate prediction of outcomes as a result of cut-points
established with an
in-process training set or cut-points defined for non-algorithmic analysis,
but that any number
of linear and nonlinear approaches can produce a statistically significant and
clinically
.. significant result. As used herein, the term "Kaplan-Meier survival
analysis" is understood in
the art to be also known as the product limit estimator, which is used to
estimate the survival
function from lifetime data. In medical research, it is often used to measure
the fraction of
patients living for a certain amount of time after treatment. JMP GENOMICSO,
R, Python
libraries including SciPy, SciKit, and numpy software or systems such as
TensorFlow
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provides an interface for utilizing each of the predictive modeling methods
disclosed herein,
and should not limit the claims to methods performed only with JMP GENOMICSO,
R,
Python, or TensorFlow software.
In an embodiment, a kit comprising primer pairs suitable for the detection and
.. quantification of nucleic acid expression of at least ten genes is
disclosed herein, wherein the
at least ten genes are selected from: ACSBG1, AIM2, ALOX12, ANXA9, APOBEC3G,
ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1, C1QL4, C21orf59, C3orf70,
CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10, CXCR4, CYP2D6
(L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR, EphB2, FCHSD1,
FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9, MIR196B), HPGD,
ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17, KRT18, KRT19, KRT6B,
LAMC2, LCE2B, LIME1 (ZGPAT), L0C100287896, L0C101927502, LOR, LRRC47,
MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10, MMP12, MMP13, MMP3,
MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB, NEFL, NFASC, NFIA, NFIB,
NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1, PIM2, PLAU, PLS3, PTHLH,
PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38, RUNX3, S100A8, S100A9,
SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11, SNORD124, SPATA41, SPP1,
TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3, TUFM (MIR4721), TYRP1,
UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or ZSCAN31.
In some embodiments, the primer pairs suitable for the detection and
quantification of
nucleic acid expression of at least ten genes are primer pairs for: ACSBG1,
AIM2, ALOX12,
ANXA9, APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1,
C1QL4, C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10,
CXCR4, CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR,
EphB2, FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
.. NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
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ZSCAN31. In one embodiment, the primer pairs comprise primer pairs for 10
genes, 20
genes, 30 genes, 40 genes or more.
In another aspect, this disclosure relates to kits to be used in assessing the
expression
of a gene or set of genes in a cSCC sample or biological sample from a subject
to assess the
risk of developing recurrence, metastasis, or both. In one embodiment, the
disclosure relates
to a kit comprising primer pairs suitable for the detection and quantification
of nucleic acid
expression of at least ten genes selected from: ACSBG1, AIM2, ALOX12, ANXA9,
APOBEC3G, ARPC2, ATP6AP1, ATP6V0E2, BBC, BHLHB9, BLOC1S1, Cl QL4,
C21orf59, C3orf70, CCL27, CD163, CEP76, CHI3L1, CHMP2B, CXCL10, CXCR4,
CYP2D6 (L0C101929829), DARS, DCT, DDAH1, DSS1, DUXAP8, EGFR, EphB2,
FCHSD1, FDFT1, FLG, FN1, GTPBP2, HDDC3, HNRNPL, HOXA10 (HOXA9,
MIR196B), HPGD, ID2, IL24, IL2RB, IL7R, INHBA, IPO5P1, KIT, KLK5, KRT17,
KRT18, KRT19, KRT6B, LAMC2, LCE2B, LIME1 (ZGPAT), LOC100287896,
L0C101927502, LOR, LRRC47, MIER2, MIR129-1, MIR3916, MKLN1, MMP1, MMP10,
.. MMP12, MMP13, MMP3, MMP7, MMP9, MRC1, MRPL21, MSANTD4, MYC, NEB,
NEFL, NFASC, NFIA, NFIB, NFIC, NOA1, PD1, PDL1, PDPN, PI3, PIG3, PIGBOS1,
PIM2, PLAU, PLS3, PTHLH, PTRHD1, RBM33, RCHY1, RNF135, RPL26L1, RPP38,
RUNX3, S100A8, S100A9, SEPT3, SERPINB2, SERPINB4, SLC1A3, SLC25A11,
SNORD124, SPATA41, SPP1, TAF6L, TFAP2B, THYN1, TMEM41B, TNNC1, TUBB3,
TUFM (MIR4721), TYRP1, UGP2, USP7, VIM, YKT6, ZNF48, ZNF496, ZNF839, and/or
ZSCAN31.
Kits can include any combination of components that facilitates the
performance of an
assay. A kit that facilitates assessing the expression of the gene or genes
may include
suitable nucleic acid-based and/or immunological reagents as well as suitable
buffers, control
reagents, and printed protocols. A "kit" is any article of manufacture (e.g.,
a package or
container) comprising at least one reagent, e.g., a probe or primer set, for
specifically
detecting a marker or set of markers used in the methods disclosed herein. The
article of
manufacture may be promoted, distributed, sold, or offered for sale as a unit
for performing
the methods disclosed herein. The reagents included in such a kit comprise
probes, primers,
or antibodies for use in detecting one or more of the genes and/or gene sets
disclosed herein
and demonstrated to be useful for predicting recurrence, metastasis, or both,
in patients with
cSCC. Kits that facilitate nucleic acid based methods may further include one
or more of the
following: specific nucleic acids such as oligonucleotides, labeling reagents,
enzymes
including PCR amplification reagents such as Taq or Pfu, reverse
transcriptase, or other,
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and/or reagents that facilitate hybridization. In addition, the kits disclosed
herein may
preferably contain instructions which describe a suitable detection assay.
Such kits can be
conveniently used, e.g., in clinical settings, to diagnose and evaluate
patients exhibiting
symptoms of cancer, in particular patients exhibiting the possible presence of
a cutaneous
squamous cell carcinoma.
EXAMPLES
The Examples that follow are illustrative of specific embodiments of the
claimed
invention, and various uses thereof They are set forth for explanatory
purposes only, and
should not be construed as limiting the scope of the claimed invention in any
way.
Example 1: cSSC tumor sample preparation and expression analysis
a. cSCC tumor sample preparation and RNA isolation
Formalin-fixed paraffin embedded (FFPE) primary squamous cell carcinoma tumor
specimens arranged in 5 p.m sections on microscope slides were acquired from
multiple
institutions under Institutional Review Board (IRB) approved protocols. All
tissue was
reviewed by a pathologist. Tissue was marked and tumor tissue was dissected
from the slide
using a sterile disposable scalpel, collected into a microcentrifuge tube, and
deparaffinized
using xylene. RNA was isolated from each specimen using the QIAGEN QIAsymphony
RNA kit (Hilden, Germany) on the QIAGEN QIAsymphony SP sample preparation
automated extractor. RNA quantity was assessed using the NanoDropTM 8000
system.
b. cDNA generation and RT-PCR analysis
RNA isolated from FFPE samples was converted to cDNA using the Applied
Biosystems High Capacity cDNA Reverse Transcription Kit (Life Technologies
Corporation,
Grand Island, NY). Prior to performing the RT-PCR assay, each cDNA sample
underwent a
14-cycle pre-amplification step. Pre-amplified cDNA samples were diluted 20-
fold in TE
buffer. 7.5 pL of each diluted sample was mixed with 7.5 pL of TaqMan
OpenArray Real-
Time Mastermix, and the solution was loaded to a custom high throughput
microfluidics
OpenArray card containing primers specific for the genes. Each sample was run
in triplicate.
The gene expression profile test was performed on a ThermoFisher
QuantStudio12k Flex
Real-Time PCR system (Life Technologies Corporation, Grand Island, NY).
c. Expression analysis and class assignment
Mean Ct values were calculated for triplicate sample sets, and AC t values
were
calculated by subtracting the mean Ct of each discriminating gene from the
geometric mean
of the mean Ct values of all endogenous control genes. AC t values were
standardized
according to the mean of the expression of all discriminant genes with a scale
equivalent to
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the standard deviation. Various predictive modeling methods, including radial
basis machine,
k-nearest neighbor, partition tree, logistic regression, discriminant analysis
and distance
scoring, and neural network analysis were performed using R version 3.3.2.
Example 2: cSCC metastatic risk genetic signature and biomarker expression
The study design workflow is shown in Figure 1. First, in order to develop the
gene
expression profile for cSCC prognostication, cases with annotated clinical
data and sufficient
follow-up were used as a development set. Pre-specified bins of patients
within the recurrent
and non-recurrent group were created, including immunocompromised,
immunocompetent,
those with a certain number of high risk features and low risk cases. The goal
was to satisfy
the pre-specified number of cases in each bin for development. Predictive
modeling was
performed on gene expression data from the development cohort. The predictive
model was
then validated. 221 cases were included in the development set (see Table 1).
Table 1 shows
the demographics for the cohort of 221 cases used in this study. They are also
stratified by
non-recurrence or recurrence. Recurrence is defined as any recurrence ¨ local
nodal
(satellitosis through regional nodes and distant metastasis). Note that cases
with R1 or R2
and local recurrence in the scar or contiguous to the scar were embargoed from
this analysis.
Characteristics that are associated with higher risk tumors (such as male sex,
compromised
immune system, head and neck primary tumor, poor differentiation or
undifferentiated,
higher Clark Level, perineural invasion, and invasion into subcutaneous fat)
are features
included. This is after embargoing cases that have not yet had data monitoring
and did not
meet very stringent gene expression data requirements.
Table 1: Demographics for the cohort of 221 cases used in Examples 2 and 3
All Non- With
Feature Recurrence Recurrence p-value
(n=221)
(n=196) (n=25)
Age: Median years (range) 74 (43-97) 74 (45-97) 69 (43-91)
n.s.
Mean +/- SD 72.8 +/- 11.2 73.3 +/- 10.8 68.6 +/-
13.2
Definitive surgery: Mohs 181 (82%) 1 161 (82%) 20 (80%)
n.s.
WLE 39 (18%) 34 (17%) 5 (20%)
Male sex 164 (74%) 143 (73%) 21(84%) n.s.
Patient immunocompromised 30 (14%) 20 (10%) 10 (40%) p<0.001
Located on head or neck 146 (66%) 129 (66%) 17 (68%) n.s.
Tumor diameter:
p<0.001
Median cm (range) 1.4 (0-28) I 1.15 (0-8.8) 2.9 (0.25-28)
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Mean +/- SD 1.88 +/- 2.34 1.62 +/- 1.47 3.89 +/-
5.27
Differentiation Status:
Poor / Undifferentiated p<0.001
Clark Level IV / V 73 (33%) 67 (34%) 6 (24%) p<0.001
Perineural invasion present 12 (5%) 9 (5%) 3 (12%) p<0.001
Invasion into subcutaneous fat 22 (10%) 19 (10%) 3 (12%) P=0.015
Gene expression differences (RT-PCR data from 73 genes) between recurrent and
non-recurrent cSCC cases were evaluated. Using the gene expression data,
several control
genes were identified that had stable expression across all of the samples.
These control
genes were then used to normalize the expression of the remaining genes. Gene
expression
differences between recurrent and non-recurrent cases were investigated to
find the genes that
are significant. Significant gene expression differences that were associated
with local
recurrences, regional metastases, and distant metastases were also evaluated.
Table 2 below
shows genes associated with regional/distant metastases. Genetic expression of
the
discriminant genes in the signature (Table 2) was assessed in a cohort of 240
cSCC samples
using RT-PCR, 18 of these were independently significant to a p-value of
p<0.05 (see Figure
2). As shown in Table 3 below, of the 63 discriminating genes, 18 were altered
in metastatic
cSCC tumors compared to nonmetastatic tumors with a p-value of p<0.05.
Table 2: 63 candidate genes for the GEP signature to predict metastatic risk
and/recurrence in cSCC tumors
Gene symbol p-value mean - Recurrence mean - Non-Recurrence
LOR 0.000 0.310 2.935
KRT18 0.000 0.454 -1.081
LCE2B 0.000 0.447 2.490
EphB2 0.001 -1.846 -2.648
FLG 0.001 -4.104 -1.594
DCT 0.001 -5.696 -3.003
TFAP2B 0.001 -6.571 -3.836
NEB 0.002 -3.495 -2.807
TYRP1 0.006 -5.869 -3.709
MMP3 0.006 -2.887 -5.159
MMP7 0.010 -3.915 -5.568
MMP1 0.014 2.290 0.970
INHB A 0.016 -1.203 -2.222
ACSBG1 0.024 -3.613 -2.489
USP7 0.029 0.689 1.076
APOBEC3G 0.035 -3.800 -4.260
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NFIB 0.036 -2.623 -2.385
ANXA9 0.050 -8.007 -7.059
RCHY1 0.055 -2.418 -2.665
PDPN 0.056 1.672 1.251
ALOX12 0.066 -4.112 -3.688
YKT6 0.070 1.449 1.140
PLAU 0.091 1.873 1.913
ID2 0.110 -0.921 -0.550
MMP10 0.119 -0.067 -1.164
HPGD 0.141 -6.830 -5.781
FN1 0.147 1.594 1.186
HNRNPL 0.156 -0.036 0.085
AIM2 0.159 -4.093 -4.610
MMP13 0.178 -7.096 -8.365
BBC 0.179 -7.464 -7.738
EGFR 0.189 0.908 0.705
SPP1 0.200 -0.144 -1.332
SERPINB4 0.251 -11.815 -10.852
NEFL 0.292 -2.876 -1.321
NFASC 0.301 -3.832 -3.738
P13 0.324 4.686 4.847
PIG3 0.333 -3.420 -3.698
LAMC2 0.350 0.480 0.196
ARPC2 0.353 -0.053 -0.006
AADAC 0.379 -16.094 -15.629
IL24 0.387 -3.969 -4.629
S100A8 0.388 3.838 3.936
CCL27 0.398 -12.922 -13.230
PTHLH 0.401 0.777 0.907
S100A9 0.457 7.525 7.534
DDA1-11 0.461 -5.660 -5.216
PDL1 0.471 -3.067 -3.124
DSS1 0.477 -5.722 -5.442
KRT19 0.486 -2.982 -3.853
KIT 0.529 -2.391 -2.415
TUBB3 0.599 -4.541 -5.167
MYC 0.631 -3.816 -3.749
CHI3L1 0.653 -0.029 0.025
MMP9 0.684 1.649 1.733
CXCR4 0.742 -8.733 -8.858
ATP6V0E2 0.750 -8.562 -8.519
CXCL10 0.785 -3.039 -3.184
PD1 0.825 -1.878 -1.870
IL7R 0.872 -8.524 -8.094
MMP12 0.919 -2.958 -3.225
CEP76 0.981 -4.361 -4.656
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Table 3: 18 Genes included in a GEP signature able to predict recurrence in
cSCC
Gene mean -
symbol p-value Recurrence mean - Non-Recurrence
ACSBG1 0.024 -3.613 -2.489
ANXA9 0.050 -8.007 -7.059
APOBEC3G 0.035 -3.800 -4.260
DCT 0.001 -5.696 -3.003
EphB2 0.001 -1.846 -2.648
FLG 0.001 -4.104 -1.594
INHBA 0.016 -1.203 -2.222
KRT18 0.000 0.454 -1.081
LCE2B 0.000 0.447 2.490
LOR 0.000 0.310 2.935
MMP 1 0.014 2.290 0.970
MMP3 0.006 -2.887 -5.159
MMP7 0.010 -3.915 -5.568
NEB 0.002 -3.495 -2.807
NFIB 0.036 -2.623 -2.385
TFAP2B 0.001 -6.571 -3.836
TYRP1 0.006 -5.869 -3.709
USP7 0.029 0.689 1.076
Example 3: Initial training set development studies and comparison to
validation
cohort
R version 3.3.2 was used to train multiple predictive models (e.g., multiple
machine-
learning methods such as, neural networks, gradient boosting machine,
generalized linear
model boost, radial basis function, rule-based classification, decision tree
classification,
and/or regularized linear discriminant analysis) against the normalized Ct
values obtained
from RT-PCR analysis in 181 cSCC cases selected at random from the 240 cases
in the
combined set. The average of the top predictive models was more sensitive than
either the
Brigham and Women's Hospital (BWH) or American Joint Committee on Cancer
(AJCC)
models with minimal loss of specificity. These results show that recurrent and
non-recurrent
cSCC can be identified through gene expression profiling and gene expression
can be used to
identify cSCC patients with a higher risk of recurrence. A validated
prognostic test could
inform clinical decision-making on preoperative surgical staging (for example,
based on
shave biopsy), surgical approach (SLNB) or adjuvant radiation to reduce local
recurrence,
and adjuvant radiation, nodal staging, adjuvant systemic therapy to reduce
regional/distant
metastasis. Such a test could improve such intervention decisions and help
determine which
patients may benefit from additional therapeutic modalities.
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Table 4: Predictive modeling - local recurrence
Local Recurrence GEP BWH AJCCv7 AJCCv8
Example 2
Sensitivity 75% 17% 0% 39%
Specificity 92% 90% 99.5% 79%
negative predictive value
98% 92% 92% 94%
(NPV)
positive predictive value
50% 13% 94% 14%
(PPV)
Table 5: Predictive modeling - metastasis
Regional/Distant GEP BWH AJCCv7 AJCCv8
Metastasis Example 2
Sensitivity 83% 23% 0% 46%
Specificity 95% 90% 100% 79%
negative predictive value
99% 95% 94% 96%
(NPV)
positive predictive value
53% 13% 0% 12%
(PPV)
Example 4: Prognostic gene expression profile test in cSCC in patients with
one or
more high-risk features
To identify a gene expression profile that accurately predicts: (1) primary
cSCC with
a high risk of regional nodal/distant metastasis; and (2) primary cSCC with
high risk of local
recurrence after complete surgical clearance, a multi-center study was
performed using
archived primary tissue samples with extensive capture of associated clinical
data. The
approach uses targeted candidate genes from the literature combined with genes
from a
global approach microarray screen. Samples are from subjects with
pathologically confirmed
cSCC diagnosed after 2006, minimum 3 years of follow-up or event (see Tables 6
and 7).
Two separate outcomes were measured: (1) nodal/distant metastasis; and (2)
local
recurrence. Accuracy metrics demonstrate that the gene expression signature
has prognostic
value for in an independent cohort (see Table 8 and Figure 4). The prognostic
test could
inform clinical decision-making on: (1) preoperative surgical staging, based
on shave biopsy;
and (2) adjuvant radiation, nodal staging, adjuvant systemic therapy to reduce
regional/distant
metastasis.
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Table 6: Demographics for development stage of Example 4.
All Non-Metastatic Regional/distant
Feature
(n=122) (n=108) metastasis (n=14)
Age: Median years (range) 74 (49-97) 74 (50-97) 74.5 (49-
91)
# #
Definitive surgery: Mohs 99 (82%) 88 (82%) 11(79%)
Male sex 94 (77%) 81(75%) 13 (93%)
Patient immunocompromised 17 (14%) 13 (12%) 4 (29%)
Located on head or neck 87 (71%) 77 (71%) 10 (71%)
Tumor diameter: Mean +/- SD 2.0 +/- 2.9 1.5 +/- 1.3 5.8 +/-
6.7***
Differentiation Status:
Poorly differentiated
5 (4%) 4 (4%) 1 (7%)
Clark Level IV / V 45 (37%) 40 (37%) 5 (36%)
Perineural invasion present 7 (6%) 6 (6%) 1 (7%)
Invasion into subcutaneous fat 7 (6%) 4 (4%) 3 (21%) **
#
1 case with unknown surgery type; Wilcoxon F or Chi-square test p **<0.01
***<0.001
Table 7: Demographics for validation stage of Example 4.
All Non-Met Regional/distant
met
Feature
(n=107) (n=90) (n=17)
Age: Median years (range) 72 (30-93) 72.5 (45-93) 72 (30-88)
Definitive surgery: Mohs 86 (81%)# 76 (84%) 10 (63%)#*
Male sex 78 (73%) 64 (71%) 14 (82%)
Patient immunocompromised 12 (11%) 10 (11%) 2 (12%)
Located on head or neck 76 (71%) 62 (69%) 14 (82%)
Tumor diameter: Mean +/- SD 1.9 +/- 1.7 1.9 +/- 1.2 3.3 +/-
2.6**
Differentiation Status:
Poorly differentiated 13 (12%) 6 (7%) 7 (42%)***
Clark Level IV / V 32 (30%) 25 (28%) 7 (41%)
Perineural invasion present 9 (8%) 3 (3%) 6 (35%)***
Invasion into subcutaneous fat 17 (16%) 11(12%) 6 (35%)*
#
1 case with unknown surgery type; Wilcoxon F or Chi-square test p *<0.05
**<0.01 ***<0.001
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Table 8: Predictive modeling
Metric GEP AJCC 8 BWH
Example 4
Sensitivity 53% 53% 41%
Specificity 93% 87% 88%
negative predictive value
91% 91% 89%
(NPV)
positive predictive value
60% 43% 39%
(PPV)
Example 5: Prognostic gene expression signature for risk assessment in cSCC
with a
subanalysis in the head and neck region
To identify a gene expression profile that accurately predicts: (1) primary
cSCC with
a high risk of regional nodal/distant metastasis; and (2) primary cSCC with
high risk of local
recurrence after complete surgical clearance, a multi-center study was
performed using
archived primary tissue samples with extensive capture of associated clinical
data. The
approach uses targeted candidate genes from the literature combined with genes
from a
global approach microarray screen. Samples are from subjects with
pathologically confirmed
cSCC diagnosed after 2006, minimum 3 years of follow-up or event (see Table
9). Two
separate outcomes were measured: (1) nodal/distant metastasis; and (2) local
recurrence.
Accuracy metrics accuracy metrics for all and head and neck cSCC cases suggest
that gene
expression signature has prognostic value in an independent cohort (see Table
10). The
prognostic signature with a robust PPV for high-risk disease will improve
identification of
patients with cSCC who can benefit from surgical, radiation and immunotherapy
interventions.
Table 9: Demographics - head and neck subanalysis
Non-
Metastasis
Feature of head and neck case Metastatic
(n=34) (n=9)
Age: Median years (range) 75 (49-89) 77 (49-89)
Definitive surgery: Mohs 33 (97%) 8 (89%)
Male sex 31(91%) 8 (89%)
Tumor diameter: Mean cm +/- SD 2.52 +/- 1.35 5.89 +/-
8.36
Differentiation Status:
Poor / Undifferentiated 2 (6%) 2 (22%)
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Clark Level IV / V 4 (12%) 1(11%)
Perineural invasion present 4 (12%) 0 (0%)
Invasion into subcutaneous fat 7 (21%) 1(11%)
Table 10: Predictive modeling - head and neck subanalysis
All (n = 107) H&N (n = 76)
Metric GEP BWH GEP BWH
Example 5 this study
Sensitivity 53% 41% 43% 43%
Specificity 93% 88% 94% 89%
negative predictive value
91% 89% 88% 87%
(NPV)
positive predictive value
60% 39% 60% 46%
(PPV)
Table 11: Genes included in the gene sets that are able to predict risk of
recurrence and/or metastasis
Gene name Probe Identifier median median delta p-
value
(ThermoFisher) Recurrent Non- median *
Recurrent
KRT6B Hs00745492_s1 5.522 7.091 -1.569 0.000070
LOR Hs01894962_s1 1.970 4.492 -2.522 0.000265
FLG Hs00856927_gl -2.724 0.303 -3.027 0.000291
LCE2B Hs04194422_s1 1.153 3.665 -2.512 0.000809
PLS3 Hs00543973_m1 -0.416 0.080 -0.497 0.001048
SERPINB2 Hs01010736_m1 0.304 1.455 -1.150 0.001277
KLK5 Hs00202752_m1 1.170 3.239 -2.069 0.001468
KRT18 Hs01920599_gH 0.975 -0.238 1.213 0.002094
BBC Hs00248075_m1 -4.614 -5.334 0.720 0.002663
M1R3916 Hs04232205_s1 -0.709 -1.334 0.625 0.002734
L0C100287896 Hs01931732_s1 -2.224 -2.796 0.572 0.003547
TFAP2B Hs01560931_m1 -4.288 -2.456 -1.832 0.004135
HPGD Hs00960591_m1 -5.491 -3.113 -2.378 0.007656
CHMP2B Hs00387770_m1 -3.117 -2.591 -0.526 0.008827
ANXA9 Hs01070154_m1 -5.583 -4.284 -1.299 0.009038
ID2 Hs00747379_m1 -0.345 0.493 -0.838 0.009695
EphB2 Hs00362096 ml -1.124 -1.614 0.491 0.012203
NEB Hs00189880_m1 -2.611 -1.904 -0.706 0.014937
FDFT1 Hs00926053_m1 -1.589 -0.657 -0.932 0.017046
USP7 Hs00931763_m1 1.509 1.960 -0.452 0.017046
TAF6L Hs01008033_m1 -0.699 -0.961 0.262 0.018195
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Gene name Probe Identifier median median delta p-value
(ThermoFisher) Recurrent Non- median *
Recurrent
ACSBG1 Hs01025572_m1 -2.992 -1.336 -1.657 0.026077
HNRNPL Hs00704853_s1 0.776 0.980 -0.204 0.031337
ARPC2 Hs01031740_m 1 0.715 1.147 -0.432 0.031337
DUXAP8 Hs04942686_m1 -6.816 -9.507 2.691 0.039746
PIM2 Hs01546752_g 1 -1.160 -1.752 0.592 0.050944
KRT17 Hs00356958_m1 6.944 7.254 -0.310 0.053874
APOBEC3G Hs00222415_m1 -2.574 -3.024 0.450 0.056942
DSS1 Hs00428732_m 1 -4.131 -3.182 -0.949 0.056942
EGFR Hs01076090_m 1 1.598 1.332 0.266 0.069464
SERPINB4 Hs01691258_g 1 -12.838 -8.116 -4.722 0.070706
UGP2 Hs00900510_ml -1.783 -1.437 -0.346 0.073246
SPATA41 Hs03028557_sl -12.073 -13.333 1.261 0.077195
SN0RD124 Hs03464469_sl -2.848 -2.958 0.110 0.082729
P13 Hs00964384_gl 5.550 6.140 -0.589 0.085614
LIME1-ZGPAT Hs00738791_gl -4.044 -4.312 0.267 0.090094
MMP3 Hs00968305_m 1 -1.478 -2.397 0.919 0.099619
S100A8 Hs00374264_gl 4.237 5.014 -0.777 0.104673
PTRHD1 Hs00415546_m 1 -1.338 -1.216 -0.122 0.109930
MMP7 Hs01042796_m 1 -2.399 -3.937 1.538 0.115392
TMEM41B Hs01379134_m 1 -1.979 -1.562 -0.417 0.119151
SPP1 Hs00959010_m 1 1.650 0.427 1.224 0.121066
RBM33 Hs00997579_ml 1.600 1.349 0.251 0.152768
NFIB Hs01029174_m 1 -1.757 -1.633 -0.124 0.159806
NEFL Hs00196245_m 1 -0.069 0.561 -0.631 0.162206
NFIC Hs00232157_m 1 -0.500 -0.300 -0.200 0.167086
DCT Hs01098278_ml -3.033 -1.300 -1.733 0.174613
RCHY1 Hs00996236_ml -1.807 -2.038 0.231 0.177178
ZSCAN31 Hs00372831_g 1 -3.639 -2.926 -0.713 0.179770
IPO5P1 Hs05052601_sl -2.927 -3.231 0.303 0.179770
RUNX3 Hs00231709_ml -0.927 -1.342 0.415 0.204381
MKLN1 Hs00992679_ml -0.787 -0.930 0.144 0.204381
ATP6V0E2 Hs04189864_ml -5.596 -6.247 0.651 0.207260
YKT6 Hs00559914_m 1 2.007 1.788 0.220 0.210168
FCHSD1 Hs00703025_sl -6.048 -5.195 -0.854 0.216073
MMP1 Hs00899658_m 1 3.156 2.381 0.774 0.225153
CEP76 Hs00950371_m 1 -3.743 -3.455 -0.288 0.225153
TUFM-M1R4721 Hs00944507_gl 2.465 2.281 0.184 0.228239
AIM2 Hs00915710_m 1 -2.525 -2.720 0.195 0.244123
PTHLH Hs00174969_ml 0.986 1.833 -0.848 0.264188
BHLHB9 Hs01089557_s 1 -14.090 -12.657 -1.433 0.264188
CD163 Hs00174705_m 1 -0.829 -1.156 0.327 0.307655
ZNF839 Hs00901350_g 1 -1.060 -1.316 0.256 0.307655
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Gene name Probe Identifier median median delta p-value
(ThermoFisher) Recurrent Non- median *
Recurrent
BLOC1S1 Hs00155241_m1 -1.061 -0.787 -0.273 0.311480
HDDC3 Hs00826827_gl -1.299 -1.567 0.267 0.319223
TNNC1 Hs00896999_gl -7.015 -5.911 -1.105 0.323141
S100A9 Hs00610058_m1 8.071 8.385 -0.314 0.327091
TUBB3 Hs00801390_s1 -3.190 -2.711 -0.479 0.331071
KIT Hs00174029_m1 -1.168 -1.574 0.406 0.351443
FN1 Hs01549976_m1 2.302 1.859 0.443 0.364039
INHBA Hs01081598_m1 -1.107 -1.166 0.060 0.368299
PIGBOS1 Hs05036222_s1 -0.970 -1.132 0.162 0.372591
THYN1 Hs01553775_gl 0.011 -0.219 0.230 0.376913
HOXA10- Hs00365956_m1 -3.574 -2.521 -1.053 0.412594
HOXA9-
MIR196B
MYC Hs00153408_m1 -2.553 -2.337 -0.215 0.440624
IL24 Hs01114274_m1 -3.039 -3.394 0.355 0.455038
NFIA Hs00379134_m1 -0.852 -0.709 -0.143 0.499836
RPL26L1 Hs01631495_s1 -6.405 -6.603 0.198 0.504954
ZNF48 Hs00399035_m1 -3.340 -3.577 0.237 0.520473
MIER2 Hs00380101_m1 -0.275 -0.382 0.108 0.530953
MMP13 Hs00942584_m1 -4.547 -5.058 0.511 0.536233
TYRP1 Hs00167051_m1 -2.547 -2.510 -0.037 0.546872
VIM Hs00958111_m1 4.763 4.373 0.390 0.552231
LRRC47 Hs00975850_m1 0.130 0.070 0.060 0.552231
ALOX12 Hs00167524_m1 -3.032 -2.563 -0.469 0.590445
PLAU Hs01547054_m1 3.212 2.870 0.342 0.612814
IL7R Hs00902334_m1 -4.480 -4.820 0.340 0.624137
DARS Hs00962398_m1 2.314 2.486 -0.172 0.624137
L0C101927502 Hs05033260_s1 -8.529 -8.227 -0.302 0.624137
M1R129-1 Hs03302824_pri -12.122 -13.033 0.910 0.647050
PD1 Hs00240906_m1 -1.233 -1.099 -0.134 0.652832
CYP2D6- Hs03043789_gl -5.343 -5.128 -0.215 0.676166
LOC101929829
GTPBP2 Hs01051445_gl -2.289 -2.127 -0.163 0.687952
CXCL10 Hs00171042_m1 -1.850 -1.595 -0.255 0.693874
SLC1A3 Hs00904817_m1 -2.518 -2.534 0.016 0.699815
RNF135 Hs00260480_m1 -0.694 -0.725 0.030 0.711752
NOA1 Hs00260452_m1 -2.426 -2.528 0.102 0.747977
ZNF496 Hs00262107_m1 -1.484 -1.549 0.065 0.760181
MMP12 Hs00159178_m1 -2.301 -2.567 0.266 0.772445
C3orf70 Hs01395177_m1 -4.175 -4.227 0.052 0.784767
LAMC2 Hs01043717_m1 0.874 1.000 -0.126 0.797143
MMP10 Hs00233987_m1 -0.255 0.441 -0.696 0.803350
C1QL4 Hs00884853 sl -10.397 -10.511 0.113 0.822045
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Gene name Probe Identifier median median delta p-value
(ThermoFisher) Recurrent Non- median *
Recurrent
C21orf59 Hs00937509_m1 0.903 0.829 0.074 0.822045
KRT19 Hs01051611_gH -3.414 -2.626 -0.788 0.828299
PDL1 Hs00204257_m1 -2.051 -2.218 0.166 0.847127
SLC25A11 Hs01087946_gl 0.664 0.641 0.024 0.847127
MRC1 Hs00267207_m1 -5.005 -5.020 0.015 0.853423
PIG3 Hs00936519_m1 -3.104 -2.896 -0.207 0.853423
IL2RB Hs00386692_m1 -1.702 -1.592 -0.110 0.878697
ATP6AP 1 Hs05016463_s1 0.173 0.183 -0.011 0.878697
MSANTD4 Hs00411188_gl -3.627 -3.612 -0.015 0.929591
MRPL21 Hs00698959_m1 0.665 0.664 0.002 0.929591
CXCR4 Hs00607978_s1 -5.555 -5.868 0.313 0.935977
RPP38 Hs00705626_s1 -4.839 -4.719 -0.120 0.935977
SEPT3 Hs00251883_m1 -5.165 -5.094 -0.071 0.942368
PDPN Hs00366766_m1 1.995 1.893 0.102 0.948762
CCL27 Hs00171157_m1 -12.962 -11.407 -1.555 0.967963
CHI3L1 Hs01072228_m1 0.794 0.689 0.105 0.974368
DDAH1 Hs00201707_m1 -3.775 -3.568 -0.207 0.980774
MMP9 Hs00957562_m1 2.233 2.368 -0.135 0.987182
NFASC Hs00978280_m1 -2.781 -2.716 -0.066 0.993591
* Positive values indicate an INCREASE in gene expression in recurrent cancer
when compared to
non-recurrent control; and negative values indicate a DECREASE in gene
expression in recurrent
cancer when compared to non-recurrent control.
Table 12: Accuracy of gene sets used to predict risk of recurrence and/or
metastasis
Gene Sensitivity Specificity PPV NPV AUC Kappa
set
20-1 0.4958 0.9481 0.5931 0.9370 0.8604 0.4537
20-2 0.5208 0.9333 0.5869 0.9385 0.8101 0.4524
20-3 0.4438 0.9472 0.5829 0.9292 0.8131 0.4147
20-4 0.4708 0.9324 0.5688 0.9318 0.8766 0.4084
20-5 0.4833 0.9324 0.4990 0.9335 0.8242 0.4033
20-6 0.4542 0.9389 0.5722 0.9306 0.8275 0.3991
20-7 0.5396 0.9065 0.4634 0.9395 0.8384 0.3934
20-8 0.3917 0.9537 0.5922 0.9238 0.7275 0.3783
20-9 0.4396 0.9259 0.5132 0.9274 0.8220 0.3673
20-10 0.3708 0.9556 0.5888 0.9228 0.7970 0.3621
20-11 0.4542 0.9241 0.4625 0.9299 0.7701 0.3615
20-12 0.4292 0.9324 0.4984 0.9280 0.7876 0.3613
20-13 0.3896 0.9472 0.5367 0.9234 0.8228 0.3605
20-14 0.4146 0.9343 0.5150 0.9254 0.7698 0.3600
20-15 0.4417 0.9278 0.4798 0.9299 0.7799 0.3553
20-16 0.4271 0.9278 0.4667 0.9262 0.7650 0.3506
20-17 0.4146 0.9287 0.4673 0.9248 0.7613 0.3506
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20-18 0.4563 0.9139 0.4518 0.9297 0.8198 0.3491
20-19 0.4188 0.9315 0.5352 0.9270 0.8132 0.3489
20-20 0.4229 0.9296 0.4484 0.9264 0.7674 0.3438
20-21 0.4396 0.9231 0.4449 0.9290 0.8336 0.3420
20-22 0.4354 0.9194 0.4282 0.9268 0.8127 0.3418
20-23 0.3563 0.9537 0.5608 0.9213 0.7605 0.3379
20-24 0.3896 0.9296 0.4846 0.9221 0.7662 0.3357
20-25 0.3896 0.9370 0.4919 0.9218 0.8326 0.3354
30-1 0.4021 0.9648 0.6285 0.9275 0.8091 0.3893
30-2 0.4771 0.9204 0.4672 0.9335 0.8005 0.3739
30-3 0.4438 0.9287 0.4984 0.9287 0.8083 0.3685
30-4 0.4208 0.9306 0.5525 0.9270 0.8064 0.3613
30-5 0.4000 0.9407 0.5432 0.9255 0.7804 0.3513
30-6 0.4542 0.9167 0.4574 0.9288 0.7920 0.3480
30-7 0.3875 0.9407 0.5049 0.9240 0.8209 0.3378
30-8 0.3792 0.9454 0.5218 0.9225 0.7739 0.3375
30-9 0.3542 0.9593 0.5714 0.9207 0.6822 0.3347
30-10 0.4458 0.9157 0.4271 0.9281 0.7544 0.3339
30-11 0.4167 0.9213 0.4288 0.9245 0.7915 0.3329
30-12 0.3813 0.9380 0.4732 0.9216 0.7236 0.3318
30-13 0.3229 0.9565 0.6146 0.9170 0.7093 0.3243
30-14 0.3729 0.9361 0.4768 0.9222 0.7127 0.3187
30-15 0.4042 0.9176 0.3988 0.9225 0.7716 0.3103
30-16 0.3667 0.9306 0.4688 0.9195 0.7127 0.3052
30-17 0.3375 0.9426 0.4744 0.9178 0.6708 0.3025
30-18 0.3813 0.9213 0.4323 0.9205 0.8029 0.2996
30-19 0.4146 0.9102 0.3787 0.9241 0.7652 0.2984
30-20 0.3667 0.9296 0.4427 0.9199 0.7452 0.2954
30-21 0.3583 0.9306 0.4480 0.9197 0.7475 0.2900
30-22 0.3625 0.9241 0.4685 0.9181 0.7671 0.2897
30-23 0.3833 0.9194 0.3956 0.9199 0.7480 0.2891
30-24 0.3417 0.9380 0.4473 0.9187 0.7222 0.2885
30-25 0.3979 0.9120 0.3898 0.9221 0.7419 0.2868
40-1 0.4688 0.9481 0.6105 0.9334 0.8198 0.4340
40-2 0.4021 0.9435 0.5360 0.9242 0.7960 0.3565
40-3 0.3792 0.9426 0.5311 0.9230 0.7486 0.3354
40-4 0.3563 0.9509 0.5030 0.9200 0.7427 0.3325
40-5 0.4125 0.9278 0.4898 0.9257 0.8127 0.3300
40-6 0.3896 0.9361 0.4924 0.9235 0.7824 0.3294
40-7 0.3854 0.9324 0.4662 0.9219 0.7421 0.3248
40-8 0.3646 0.9398 0.5220 0.9212 0.7262 0.3228
40-9 0.3583 0.9380 0.5303 0.9199 0.7621 0.3189
40-10 0.3500 0.9472 0.4906 0.9201 0.7059 0.3161
40-11 0.3938 0.9222 0.4707 0.9225 0.7623 0.3143
40-12 0.3417 0.9500 0.5070 0.9188 0.7769 0.3115
40-13 0.3896 0.9296 0.4195 0.9233 0.7851 0.3047
40-14 0.3479 0.9407 0.4750 0.9178 0.8177 0.3036
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40-15 0.3729 0.9269 0.4124 0.9206 0.6769 -- 0.3034
40-16 0.3646 0.9343 0.4224 0.9200 0.7467 0.2989
40-17 0.3792 0.9222 0.4296 0.9204 0.7539 0.2983
40-18 0.3208 0.9435 0.5381 0.9152 0.6774 -- 0.2980
40-19 0.3688 0.9194 0.4660 0.9192 0.6873 0.2940
40-20 0.3854 0.9204 0.4162 0.9224 0.8275 0.2939
40-21 0.3833 0.9167 0.3896 0.9215 0.7007 0.2904
40-22 0.3625 0.9185 0.4270 0.9177 0.6769 0.2904
40-23 0.3313 0.9343 0.4227 0.9160 0.6716 0.2836
40-24 0.3438 0.9407 0.4236 0.9193 0.6736 0.2799
40-25 0.3250 0.9389 0.4582 0.9160 0.6687 -- 0.2774
Table 13: Exemplary gene sets used to predict risk of recurrence and/or
metastasis
Gene Probe identifiers used for each gene set (probe identifiers from
ThermoFisher
set Scientific).
20-1 "Hs00705626 sl" "Hs00248075 ml" "Hs01560931 ml" "Hs00167524 ml"
"Hs00366766 ml" "Hs01051445 gl" "Hs00996236 ml" "Hs01089557 sl"
"Hs00262107 ml" "Hs01931732 sl" "Hs00399035 ml" "Hs00231709 ml"
"Hs00411188 gl" "Hs00978280 ml" "Hs00826827 gl" "Hs00232157 ml"
"Hs00747379 ml" "Hs00233987 ml" "Hs01008033 ml" "Hs04194422 sl"
20-2 "Hs00884853 sl" "Hs01920599_gH" "Hs00996236 ml" "Hs00248075 ml"
"Hs00167524 ml" "Hs00747379 ml" "Hs00942584 ml" "Hs01042796 ml"
"Hs00964384 gl" "Hs05052601 sl" "Hs00356958 ml" "Hs00901350_gl"
"Hs01691258 gl" "Hs00992679 ml" "Hs01051611 gH" "Hs04194422 sl"
"Hs01089557 sl" "Hs01087946_gl" "Hs05036222 sl" "Hs00856927 gl"
20-3 "Hs00248075 ml" "Hs00251883 ml" "Hs01089557 sl" "Hs00356958 ml"
"Hs00856927 gl" "Hs00202752 ml" "Hs00950371 ml" "Hs00899658 ml"
"Hs00362096 ml" "Hs01043717 ml" "Hs01560931 ml" "Hs00826827_gl"
"Hs01010736 ml" "Hs00167524 ml" "Hs01031740 ml" "Hs01920599_gH"
"Hs00201707 ml" "Hs00738791 gl" "Hs00962398 ml" "Hs00543973 ml"
20-4 "Hs00962398 ml" "Hs01920599_gH" "Hs01025572 ml" "Hs00159178 ml"
"Hs01089557 sl" "Hs00167524 ml" "Hs00248075 ml" "Hs00386692 ml"
"Hs00856927 gl" "Hs00996236 ml" "Hs01031740 ml" "Hs01010736 ml"
"Hs00900510 ml" "Hs00826827 gl" "Hs01008033 ml" "Hs00415546 ml"
"Hs04942686 ml" "Hs00801390 sl" "Hs01072228 ml" "Hs01547054 ml"
20-5 "Hs00411188 gl" "Hs00248075 ml" "Hs00202752 ml" "Hs00747379 ml"
"Hs01042796 ml" "Hs01920599_gH" "Hs01114274 ml" "Hs00942584 ml"
"Hs00996236 ml" "Hs00167524 ml" "Hs00978280 ml" "Hs00543973 ml"
"Hs00826827 gl" "Hs01560931 ml" "Hs00931763 ml" "Hs01089557 sl"
"Hs00174029 ml" "Hs01029174 ml" "Hs00415546 ml" "Hs00964384_gl"
20-6 "Hs00975850 ml" "Hs04942686 ml" "Hs00202752 ml" "Hs00233987 ml"
"Hs00926053 ml" "Hs00856927 gl" "Hs00992679 ml" "Hs00251883 ml"
"Hs00415546 ml" "Hs00960591 ml" "Hs00901350_gl" "Hs00747379 ml"
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs01089557 sl" "Hs00936519 ml" "Hs03043789 gl" "Hs01560931 ml"
"Hs00232157 ml" "Hs00957562 ml" "Hs00248075 ml" "Hs01549976 ml"
20-7 "Hs04194422 s1' "Hs00262107 ml" "Hs01546752 gl" "Hs01920599_gH"
"Hs04189864 ml" "Hs01089557 sl" "Hs01560931 ml" "Hs00705626 s 1"
"Hs01043717 ml" "Hs00747379 ml" "Hs00248075 ml" "Hs00856927_g1"
"Hs01029174 ml" "Hs00543973 ml" "Hs01395177 ml" "Hs00260480 ml"
"Hs00174029 ml" "Hs00387770 ml" "Hs01894962 sl" "Hs00745492 sl"
20-8 "Hs01560931 ml" "Hs00738791 gl" "Hs00856927 gl" "Hs00362096 ml"
"Hs00826827 gl" "Hs01098278 ml" "Hs00975850 ml" "Hs00167524 ml"
"Hs00260452 ml" "Hs04194422 sl" "Hs01043717 ml" "Hs00233987 ml"
"Hs00703025 sl" "Hs00896999_gl" "Hs00167051 ml" "Hs00942584 ml"
"Hs01087946 gl" "Hs00411188 gl" "Hs00747379 ml" "Hs05016463 s 1"
20-9 "Hs00362096 ml" "Hs00942584 ml" "Hs01560931 ml" "Hs00167524 ml"
"Hs00884853 sl" "Hs00248075 ml" "Hs01920599 gH" "Hs00996236 ml"
"Hs00747379 ml" "Hs01089557 sl" "Hs00959010 ml" "Hs00372831_gl"
"Hs04194422 sl" "Hs01043717 ml" "Hs00399035 ml" "Hs01051611_gH"
"Hs01042796 ml" "Hs00968305 ml" "Hs00260452 ml" "Hs01031740 ml"
20-10 "Hs05033260 sl" "Hs00233987 ml" "Hs04194422 sl" "Hs00992679 ml"
"Hs00926053 ml" "Hs00167524 ml" "Hs00202752 ml" "Hs01549976 ml"
"Hs00415546 ml" "Hs01072228 ml" "Hs01691258_gl" "Hs00387770 ml"
"Hs00380101 ml" "Hs00231709 ml" "Hs01920599 gH" "Hs00543973 ml"
"Hs00386692 ml" "Hs00705626 sl" "Hs00196245 ml" "Hs01081598 ml"
20-11 "Hs01114274 ml" "Hs01560931 ml" "Hs00738791_gl" "Hs00931763 ml"
"Hs00996236 ml" "Hs00362096 ml" "Hs00747379 ml" "Hs00411188_gl"
"Hs00900510 ml" "Hs01098278 ml" "Hs00233987 ml" "Hs04194422 s 1"
"Hs00826827 gl" "Hs00856927 gl" "Hs00232157 ml" "Hs01010736 ml"
"Hs00704853 sl" "Hs00959010 ml" "Hs00260480 ml" "Hs00915710 ml"
20-12 "Hs00992679 ml" "Hs00159178 ml" "Hs00167524 ml" "Hs00958111 ml"
"Hs00901350 gl" "Hs00931763 ml" "Hs00233987 ml" "Hs01549976 ml"
"Hs01894962 sl" "Hs01089557 sl" "Hs00171157 ml" "Hs00153408 ml"
"Hs00248075 ml" "Hs03464469 sl" "Hs04194422 sl" "Hs00745492 sl"
"Hs00366766 ml" "Hs00856927 gl" "Hs00957562 ml" "Hs01025572 ml"
20-13 "Hs01379134 ml" "Hs00362096 ml" "Hs05052601 sl" "Hs00959010 ml"
"Hs00251883 ml" "Hs01089557 sl" "Hs00856927 gl" "Hs00167524 ml"
"Hs00159178 ml" "Hs04942686 ml" "Hs00356958 ml" "Hs01042796 ml"
"Hs03464469 sl" "Hs01029174 ml" "Hs00248075 ml" "Hs00610058 ml"
"Hs01070154 ml" "Hs00703025 sl" "Hs00964384 gl" "Hs00705626 sl"
20-14 "Hs00362096 ml" "Hs00996236 ml" "Hs00931763 ml" "Hs01081598 ml"
"Hs01560931 ml" "Hs00167524 ml" "Hs00543973 ml" "Hs01098278 ml"
"Hs00856927 gl" "Hs03043789 gl" "Hs01089557 sl" "Hs01051445 gl"
"Hs00747379 ml" "Hs01114274 ml" "Hs00826827_gl" "Hs00936519 ml"
"Hs00960591 ml" "Hs00201707 ml" "Hs00899658 ml" "Hs04189864 ml"
20-15 "Hs00856927 gl" "Hs04942686 ml" "Hs00248075 ml" "Hs01029174 ml"
51
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs00992679 ml" "Hs00975850 ml" "Hs03464469 sl" "Hs00167524 ml"
"Hs01691258 gl" "Hs00960591 ml" "Hs01089557 sl" "Hs04194422 sl"
"Hs01114274 ml" "Hs00901350 gl" "Hs00936519 ml" "Hs00233987 ml"
"Hs00411188 gl" "Hs00900510 ml" "Hs00174969 ml" "Hs01070154 ml"
20-16 "Hs00826827 gl" "Hs00738791 gl" "Hs00698959 ml" "Hs00153408 ml"
"Hs00167051 ml" "Hs00365956 ml" "Hs04194422 sl" "Hs00167524 ml"
"Hs01560931 ml" "Hs00610058 ml" "Hs00232157 ml" "Hs00996236 ml"
"Hs00705626 sl" "Hs00362096 ml" "Hs01098278 ml" "Hs00997579 ml"
"Hs00559914 ml" "Hs00856927 gl" "Hs00944507 gl" "Hs00379134 ml"
20-17 "Hs00232157 ml" "Hs00543973 ml" "Hs05052601 sl" "Hs00196245 ml"
"Hs01042796 ml" "Hs00411188 gl" "Hs00899658 ml" "Hs00374264_gl"
"Hs01894962 sl" "Hs04194422 sl" "Hs03464469 sl" "Hs00992679 ml"
"Hs00901350 gl" "Hs00362096 ml" "Hs00856927 gl" "Hs00167524 ml"
"Hs01089557 sl" "Hs01931732 sl" "Hs01549976 ml" "Hs01395177 ml"
20-18 "Hs00884853 sl" "Hs00167524 ml" "Hs00978280 ml" "Hs00747379 ml"
"Hs01931732 sl" "Hs00931763 ml" "Hs04942686 ml" "Hs04194422 sl"
"Hs00856927 gl" "Hs00248075 ml" "Hs00901350 gl" "Hs00415546 ml"
"Hs01089557 sl" "Hs01025572 ml" "Hs00231709 ml" "Hs00386692 ml"
"Hs01920599 gH" "Hs00202752 ml" "Hs01029174 ml" "Hs00942584 ml"
20-19 "Hs01547054 ml" "Hs00960591 ml" "Hs03464469 sl" "Hs01560931 ml"
"Hs00153408 ml" "Hs00233987 ml" "Hs01089557 sl" "Hs00366766 ml"
"Hs00248075 ml" "Hs01010736 ml" "Hs00251883 ml" "Hs00996236 ml"
"Hs00610058 ml" "Hs00900510 ml" "Hs01008033 ml" "Hs00978280 ml"
"Hs00260452 ml" "Hs04194422 sl" "Hs00196245 ml" "Hs05052601 sl"
20-20 "Hs01098278 ml" "Hs01072228 ml" "Hs01691258_gl" "Hs00387770 ml"
"Hs00543973 ml" "Hs01920599_gH" "Hs04189864 ml" "Hs05033260 sl"
"Hs00856927 gl" "Hs00366766 ml" "Hs03043789 gl" "Hs04194422 sl"
"Hs00202752 ml" "Hs00936519 ml" "Hs01560931 ml" "Hs00958111 ml"
"Hs00251883 ml" "Hs00704853 sl" "Hs00738791 gl" "Hs00962398 ml"
20-21 "Hs01042796 ml" "Hs01560931 ml" "Hs00884853 sl" "Hs00958111 ml"
"Hs00411188 gl" "Hs05052601 sl" "Hs01920599 gH" "Hs00248075 ml"
"Hs00747379 ml" "Hs00996236 ml" "Hs00251883 ml" "Hs00202752 ml"
"Hs01008033 ml" "Hs00201707 ml" "Hs01051445_gl" "Hs00950371 ml"
"Hs01029174 ml" "Hs00232157 ml" "Hs01087946_gl" "Hs00267207 ml"
20-22 "Hs00559914 ml" "Hs00856927 gl" "Hs00978280 ml" "Hs01920599_gH"
"Hs01560931 ml" "Hs00365956 ml" "Hs00610058 ml" "Hs01008033 ml"
"Hs04194422 sl" "Hs00975850 ml" "Hs00204257 ml" "Hs00950371 ml"
"Hs00705626 sl" "Hs01089557 sl" "Hs00196245 ml" "Hs01042796 ml"
"Hs00174029 ml" "Hs01546752 gl" "Hs01098278 ml" "Hs00231709 ml"
20-23 "Hs04194422 sl" "Hs01089557 sl" "Hs00155241 ml" "Hs00942584 ml"
"Hs05033260 sl" "Hs01546752_gl" "Hs01920599 gH" "Hs01560931 ml"
"Hs00387770 ml" "Hs00399035 ml" "Hs00232157 ml" "Hs00931763 ml"
"Hs00231709 ml" "Hs00856927 gl" "Hs00233987 ml" "Hs01098278 ml"
52
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs00978280 ml" "Hs04942686 ml" "Hs00411188_g1" "Hs00201707 ml"
20-24 "Hs00232157 ml" "Hs01010736 ml" "Hs00704853 sl" "Hs00167051 ml"
"Hs00738791 gl" "Hs00975850 ml" "Hs00362096 ml" "Hs00826827_gl"
"Hs00233987 ml" "Hs00950371 ml" "Hs00204257 ml" "Hs01560931 ml"
"Hs00196245 ml" "Hs01042796 ml" "Hs00174029 ml" "Hs00705626 sl"
"Hs00856927 gl" "Hs01051445 gl" "Hs00399035 ml" "Hs05036222 sl"
20-25 "Hs00233987 ml" "Hs00380101 ml" "Hs00167524 ml" "Hs04189864 ml"
"Hs00543973 ml" "Hs01920599_gH" "Hs00856927_gl" "Hs00155241 ml"
"Hs01098278 ml" "Hs00703025 sl" "Hs00231709 ml" "Hs00365956 ml"
"Hs00204257 ml" "Hs01395177 ml" "Hs00968305 ml" "Hs00374264_gl"
"Hs00248075 ml" "Hs00202752 ml" "Hs00801390 sl" "Hs03464469 sl"
30-1 "Hs00387770 ml" "Hs00155241 ml" "Hs00251883 ml" "Hs01560931 ml"
"Hs00428732 ml" "Hs00992679 ml" "Hs00705626 sl" "Hs00356958 ml"
"Hs00960591 ml" "Hs00171157 ml" "Hs01072228 ml" "Hs01051445_gl"
"Hs00232157 ml" "Hs00233987 ml" "Hs04232205 sl" "Hs00559914 ml"
"Hs00931763 ml" "Hs00167524 ml" "Hs00171042 ml" "Hs01089557 sl"
"Hs04194422 sl" "Hs03464469 sl" "Hs00944507_gl" "Hs00196245 ml"
"Hs00950371 ml" "Hs01395177 ml" "Hs01070154 ml" "Hs01051611_gH"
"Hs00399035 ml" "Hs00703025 sl"
30-2 "Hs00937509 ml" "Hs00379134 ml" "Hs00899658 ml" "Hs00248075 ml"
"Hs01920599 gH" "Hs04194422 sl" "Hs03043789 gl" "Hs00610058 ml"
"Hs01089557 sl" "Hs00705626 sl" "Hs00362096 ml" "Hs01560931 ml"
"Hs00233987 ml" "Hs00372831 gl" "Hs01072228 ml" "Hs01379134 ml"
"Hs01076090 ml" "Hs04189864 ml" "Hs00957562 ml" "Hs01931732 sl"
"Hs00856927 gl" "Hs01546752 gl" "Hs00171157 ml" "Hs00992679 ml"
"Hs00931763 ml" "Hs00411188 gl" "Hs01008033 ml" "Hs00387770 ml"
"Hs00996236 ml" "Hs00231709 ml"
30-3 "Hs00411188 gl" "Hs01395177 ml" "Hs01560931 ml" "Hs00379134 ml"
"Hs00960591 ml" "Hs00745492 sl" "Hs00232157 ml" "Hs00899658 ml"
"Hs00248075 ml" "Hs01010736 ml" "Hs00167524 ml" "Hs01691258_gl"
"Hs00260452 ml" "Hs01070154 ml" "Hs00196245 ml" "Hs00747379 ml"
"Hs01547054 ml" "Hs00950371 ml" "Hs00962398 ml" "Hs01087946_gl"
"Hs00262107 ml" "Hs00260480 ml" "Hs00978280 ml" "Hs00428732 ml"
"Hs00944507 gl" "Hs00387770 ml" "Hs00399035 ml" "Hs00738791_gl"
"Hs00698959 ml" "Hs01549976 ml"
30-4 "Hs00415546 ml" "Hs00705626 sl" "Hs00204257 ml" "Hs00174969 ml"
"Hs00745492 sl" "Hs01920599_gH" "Hs00387770 ml" "Hs01560931 ml"
"Hs00958111 ml" "Hs01076090 ml" "Hs00379134 ml" "Hs00196245 ml"
"Hs00232157 ml" "Hs01549976 ml" "Hs00801390 sl" "Hs00399035 ml"
"Hs01089557 sl" "Hs00607978 sl" "Hs00960591 ml" "Hs00171157 ml"
"Hs05033260 sl" "Hs00233987 ml" "Hs00904817 ml" "Hs01031740 ml"
"Hs05016463 sl" "Hs00153408 ml" "Hs00968305 ml" "Hs00174029 ml"
"Hs00944507 gl" "Hs00248075 ml"
53
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
30-5 "Hs05033260 sl" "Hs00362096 ml" "Hs00559914 ml" "Hs01560931 ml"
"Hs00968305 ml" "Hs00931763 ml" "Hs04942686 ml" "Hs01920599_gH"
"Hs00899658 ml" "Hs00248075 ml" "Hs00958111 ml" "Hs05016463 sl"
"Hs03464469 sl" "Hs01931732 sl" "Hs01076090 ml" "Hs00992679 ml"
"Hs00901350 gl" "Hs00937509 ml" "Hs00960591 ml" "Hs01114274 ml"
"Hs01043717 ml" "Hs00167524 ml" "Hs01089557 sl" "Hs00204257 ml"
"Hs00703025 sl" "Hs01549976 ml" "Hs01031740 ml" "Hs00262107 ml"
"Hs00957562 ml" "Hs01029174 ml"
30-6 "Hs00958111 ml" "Hs00411188 gl" "Hs05036222 sl" "Hs00386692 ml"
"Hs00745492 sl" "Hs01010736 ml" "Hs00997579 ml" "Hs00222415 ml"
"Hs03464469 sl" "Hs00233987 ml" "Hs01547054 ml" "Hs00202752 ml"
"Hs01025572 ml" "Hs00379134 ml" "Hs04194422 sl" "Hs00926053 ml"
"Hs01560931 ml" "Hs00705626 sl" "Hs01920599 gH" "Hs00171042 ml"
"Hs03043789 gl" "Hs00610058 ml" "Hs01089557 sl" "Hs00362096 ml"
"Hs00380101 ml" "Hs00899658 ml" "Hs00801390 sl" "Hs00374264_gl"
"Hs00964384 gl" "Hs00240906 ml"
30-7 "Hs00704853 sl" "Hs01560931 ml" "Hs01379134 ml" "Hs00958111 ml"
"Hs00386692 ml" "Hs04942686 ml" "Hs00747379 ml" "Hs00167524 ml"
"Hs03302824_pri" "Hs00248075 ml" "Hs00387770 ml" "Hs00978280 ml"
"Hs00379134 ml" "Hs00233987 ml" "Hs00992679 ml" "Hs01931732 sl"
"Hs00380101 ml" "Hs00705626 sl" "Hs00155241 ml" "Hs05033260 sl"
"Hs01089557 sl" "Hs00171157 ml" "Hs00745492 sl" "Hs00937509 ml"
"Hs00374264 gl" "Hs00240906 ml" "Hs01098278 ml" "Hs00174705 ml"
"Hs00260480 ml" "Hs00372831_gl"
30-8 "Hs00202752 ml" "Hs00231709 ml" "Hs00366766 ml" "Hs00201707 ml"
"Hs00826827 gl" "Hs00856927 gl" "Hs00174969 ml" "Hs00387770 ml"
"Hs00167524 ml" "Hs01031740 ml" "Hs03464469 sl" "Hs01025572 ml"
"Hs00996236 ml" "Hs00950371 ml" "Hs00610058 ml" "Hs01920599_gH"
"Hs01051445 gl" "Hs00975850 ml" "Hs00978280 ml" "Hs00167051 ml"
"Hs01560931 ml" "Hs00704853 sl" "Hs00902334 ml" "Hs01553775_gl"
"Hs00962398 ml" "Hs01098278 ml" "Hs00356958 ml" "Hs01087946_gl"
"Hs01029174 ml" "Hs00747379 ml"
30-9 "Hs00362096 ml" "Hs01560931 ml" "Hs04942686 ml" "Hs05033260 sl"
"Hs00958111 ml" "Hs00232157 ml" "Hs00899658 ml" "Hs00607978 sl"
"Hs01549976 ml" "Hs00262107 ml" "Hs00968305 ml" "Hs00233987 ml"
"Hs00904817 ml" "Hs00356958 ml" "Hs00900510 ml" "Hs00174969 ml"
"Hs04194422 sl" "Hs01043717 ml" "Hs00745492 sl" "Hs00171042 ml"
"Hs00379134 ml" "Hs00559914 ml" "Hs04232205 sl" "Hs00957562 ml"
"Hs00167051 ml" "Hs05052601 sl" "Hs00380101 ml" "Hs01546752_gl"
"Hs00801390 sl" "Hs01070154 ml"
30-10 "Hs00380101 ml" "Hs03464469 sl" "Hs00372831 gl" "Hs05052601 sl"
"Hs04189864 ml" "Hs00248075 ml" "Hs00968305 ml" "Hs04942686 ml"
"Hs00159178 ml" "Hs00386692 ml" "Hs00937509 ml" "Hs00960591 ml"
54
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs00543973 ml" "Hs01894962 sl" "Hs00233987 ml" "Hs01051445_gl"
"Hs01560931 ml" "Hs01089557 sl" "Hs00232157 ml" "Hs00387770 ml"
"Hs01025572 ml" "Hs01395177 ml" "Hs00801390 sl" "Hs00884853 sl"
"Hs01072228 ml" "Hs00201707 ml" "Hs00996236 ml" "Hs01114274 ml"
"Hs00747379 ml" "Hs00167051 ml"
30-11 "Hs00801390 sl" "Hs01920599_gH" "Hs00931763 ml" "Hs00262107 ml"
"Hs04942686 ml" "Hs00233987 ml" "Hs00399035 ml" "Hs00248075 ml"
"Hs00171157 ml" "Hs00167524 ml" "Hs00899658 ml" "Hs00232157 ml"
"Hs00362096 ml" "Hs00915710 ml" "Hs00174969 ml" "Hs00900510 ml"
"Hs00387770 ml" "Hs00428732 ml" "Hs00607978 sl" "Hs00380101 ml"
"Hs00260480 ml" "Hs00996236 ml" "Hs00958111 ml" "Hs01070154 ml"
"Hs00543973 ml" "Hs00937509 ml" "Hs01089557 sl" "Hs00968305 ml"
"Hs01072228 ml" "Hs01042796 ml"
30-12 "Hs00745492 sl" "Hs00950371 ml" "Hs00233987 ml" "Hs01560931 ml"
"Hs00379134 ml" "Hs00167524 ml" "Hs00826827_gl" "Hs00978280 ml"
"Hs00904817 ml" "Hs00856927_gl" "Hs00899658 ml" "Hs01008033 ml"
"Hs00931763 ml" "Hs00365956 ml" "Hs03302824_pri" "Hs00428732 ml"
"Hs00975850 ml" "Hs01894962 sl" "Hs00703025 sl" "Hs01072228 ml"
"Hs01098278 ml" "Hs00958111 ml" "Hs00944507_gl" "Hs01010736 ml"
"Hs00996236 ml" "Hs01920599_gH" "Hs00387770 ml" "Hs01547054 ml"
"Hs00610058 ml" "Hs01087946_gl"
30-13 "Hs00856927 gl" "Hs00915710 ml" "Hs00379134 ml" "Hs00233987 ml"
"Hs01008033 ml" "Hs00167524 ml" "Hs00899658 ml" "Hs00559914 ml"
"Hs00904817 ml" "Hs04942686 ml" "Hs00386692 ml" "Hs00704853 sl"
"Hs00978280 ml" "Hs00415546 ml" "Hs00399035 ml" "Hs00374264 gl"
"Hs01560931 ml" "Hs01546752 gl" "Hs00231709 ml" "Hs00387770 ml"
"Hs00964384 gl" "Hs00698959 ml" "Hs00196245 ml" "Hs01395177 ml"
"Hs01076090 ml" "Hs00201707 ml" "Hs03302824_pri" "Hs00703025 sl"
"Hs01114274 ml" "Hs01631495 sl"
30-14 "Hs00380101 ml" "Hs00232157 ml" "Hs00428732 ml" "Hs03302824_pri"
"Hs01031740 ml" "Hs00171042 ml" "Hs01549976 ml" "Hs00260452 ml"
"Hs00745492 sl" "Hs00411188_gl" "Hs00167524 ml" "Hs01560931 ml"
"Hs00356958 ml" "Hs00559914 ml" "Hs00262107 ml" "Hs03464469 sl"
"Hs00801390 sl" "Hs00202752 ml" "Hs00233987 ml" "Hs00196245 ml"
"Hs00960591 ml" "Hs01546752_gl" "Hs01087946_gl" "Hs01547054 ml"
"Hs00260480 ml" "Hs01070154 ml" "Hs00267207 ml" "Hs00174705 ml"
"Hs00747379 ml" "Hs00387770 ml"
30-15 "Hs00960591 ml" "Hs00231709 ml" "Hs00251883 ml" "Hs00387770 ml"
"Hs00356958 ml" "Hs00196245 ml" "Hs00174969 ml" "Hs00996236 ml"
"Hs00559914 ml" "Hs00962398 ml" "Hs00856927_gl" "Hs00826827_gl"
"Hs01025572 ml" "Hs00801390 sl" "Hs03043789 gl" "Hs00950371 ml"
"Hs00233987 ml" "Hs00202752 ml" "Hs00260480 ml" "Hs00944507_gl"
"Hs00978280 ml" "Hs01894962 sl" "Hs00204257 ml" "Hs00174029 ml"
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs00703025 sl" "Hs01051611_gH" "Hs00958111 ml" "Hs00262107 ml"
"Hs00167524 ml" "Hs01098278 ml"
30-16 "Hs00944507 gl" "Hs05016463 sl" "Hs03464469 sl" "Hs00167051 ml"
"Hs00900510 ml" "Hs00904817 ml" "Hs04232205 sl" "Hs00356958 ml"
"Hs00957562 ml" "Hs00745492 sl" "Hs00931763 ml" "Hs01043717 ml"
"Hs00167524 ml" "Hs00856927 gl" "Hs00992679 ml" "Hs00380101 ml"
"Hs01549976 ml" "Hs00559914 ml" "Hs01560931 ml" "Hs01098278 ml"
"Hs00703025 sl" "Hs00248075 ml" "Hs01081598 ml" "Hs00428732 ml"
"Hs00968305 ml" "Hs00705626 sl" "Hs00386692 ml" "Hs00174705 ml"
"Hs00251883 ml" "Hs00415546 ml"
30-17 "Hs00543973 ml" "Hs00159178 ml" "Hs01098278 ml" "Hs00399035 ml"
"Hs01029174 ml" "Hs00705626 sl" "Hs03302824_pri" "Hs01051611 gH"
"Hs05033260 sl" "Hs01560931 ml" "Hs00167524 ml" "Hs01042796 ml"
"Hs05052601 sl" "Hs00944507_gl" "Hs00978280 ml" "Hs00747379 ml"
"Hs00171042 ml" "Hs00251883 ml" "Hs00960591 ml" "Hs01072228 ml"
"Hs03043789 gl" "Hs00233987 ml" "Hs04942686 ml" "Hs00801390 sl"
"Hs00992679 ml" "Hs01010736 ml" "Hs00232157 ml" "Hs00896999 gl"
"Hs00826827 gl" "Hs00231709 ml"
30-18 "Hs01920599_gH" "Hs00248075 ml" "Hs00411188_gl" "Hs01560931 ml"
"Hs00900510 ml" "Hs00996236 ml" "Hs00745492 sl" "Hs00202752 ml"
"Hs00233987 ml" "Hs00222415 ml" "Hs01089557 sl" "Hs05052601 sl"
"Hs00960591 ml" "Hs00958111 ml" "Hs01379134 ml" "Hs00380101 ml"
"Hs00856927 gl" "Hs04189864 ml" "Hs01031740 ml" "Hs01042796 ml"
"Hs01098278 ml" "Hs01395177 ml" "Hs00950371 ml" "Hs01691258_gl"
"Hs00899658 ml" "Hs00962398 ml" "Hs00240906 ml" "Hs00399035 ml"
"Hs00428732 ml" "Hs04942686 ml"
30-19 "Hs00428732 ml" "Hs01560931 ml" "Hs01025572 ml" "Hs00232157 ml"
"Hs01089557 sl" "Hs01043717 ml" "Hs00705626 sl" "Hs00884853 sl"
"Hs00543973 ml" "Hs00992679 ml" "Hs00899658 ml" "Hs00233987 ml"
"Hs00745492 sl" "Hs01031740 ml" "Hs01076090 ml" "Hs00260480 ml"
"Hs00856927 gl" "Hs00189880 ml" "Hs00231709 ml" "Hs01920599_gH"
"Hs00171042 ml" "Hs00415546 ml" "Hs01894962 sl" "Hs04189864 ml"
"Hs00174969 ml" "Hs00610058 ml" "Hs00153408 ml" "Hs01379134 ml"
"Hs00915710 ml" "Hs01395177 ml"
30-20 "Hs00267207 ml" "Hs00366766 ml" "Hs04942686 ml" "Hs00231709 ml"
"Hs01098278 ml" "Hs05016463 sl" "Hs00155241 ml" "Hs00900510 ml"
"Hs00975850 ml" "Hs04194422 sl" "Hs01114274 ml" "Hs01072228 ml"
"Hs01089557 sl" "Hs00747379 ml" "Hs00944507 gl" "Hs00705626 sl"
"Hs00202752 ml" "Hs00978280 ml" "Hs01920599 gH" "Hs00196245 ml"
"Hs04189864 ml" "Hs01025572 ml" "Hs05052601 sl" "Hs00380101 ml"
"Hs01081598 ml" "Hs00372831 gl" "Hs00167051 ml" "Hs00232157 ml"
"Hs01553775 gl" "Hs00738791 gl"
30-21 "Hs00174705 ml" "Hs01042796 ml" "Hs00856927_gl" "Hs00372831_gl"
56
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs03302824_pri" "Hs05033260 sl" "Hs04189864 ml" "Hs00992679 ml"
"Hs01560931 ml" "Hs00159178 ml" "Hs00196245 ml" "Hs05052601 sl"
"Hs01031740 ml" "Hs00233987 ml" "Hs00975850 ml" "Hs01098278 ml"
"Hs00248075 ml" "Hs00167524 ml" "Hs00171042 ml" "Hs01920599 gH"
"Hs00380101 ml" "Hs00559914 ml" "Hs04942686 ml" "Hs00202752 ml"
"Hs00937509 ml" "Hs00978280 ml" "Hs00936519 ml" "Hs00222415 ml"
"Hs00365956 ml" "Hs00379134 ml"
30-22 "Hs00380101 ml" "Hs00233987 ml" "Hs00171042 ml" "Hs00411188_gl"
"Hs00248075 ml" "Hs00559914 ml" "Hs00826827_gl" "Hs01894962 sl"
"Hs01098278 ml" "Hs00232157 ml" "Hs01920599 gH" "Hs00975850 ml"
"Hs00356958 ml" "Hs00543973 ml" "Hs01029174 ml" "Hs00159178 ml"
"Hs05052601 sl" "Hs04194422 sl" "Hs01025572 ml" "Hs00950371 ml"
"Hs00167524 ml" "Hs01072228 ml" "Hs00900510 ml" "Hs00705626 sl"
"Hs01114274 ml" "Hs00904817 ml" "Hs00231709 ml" "Hs00745492 sl"
"Hs00155241 ml" "Hs01081598 ml"
30-23 "Hs00964384 gl" "Hs00899658 ml" "Hs00374264 gl" "Hs00399035 ml"
"Hs00703025 sl" "Hs00415546 ml" "Hs00232157 ml" "Hs00387770 ml"
"Hs00826827 gl" "Hs01395177 ml" "Hs00202752 ml" "Hs00171042 ml"
"Hs00996236 ml" "Hs00937509 ml" "Hs05016463 sl" "Hs00233987 ml"
"Hs01114274 ml" "Hs01042796 ml" "Hs00248075 ml" "Hs00957562 ml"
"Hs00196245 ml" "Hs01560931 ml" "Hs01089557 sl" "Hs00222415 ml"
"Hs04194422 sl" "Hs01931732 sl" "Hs00884853 sl" "Hs00978280 ml"
"Hs00959010 ml" "Hs00559914 ml"
30-24 "Hs01042796 ml" "Hs00801390 sl" "Hs00960591 ml" "Hs00992679 ml"
"Hs00978280 ml" "Hs00937509 ml" "Hs01931732 sl" "Hs01560931 ml"
"Hs05016463 sl" "Hs00171157 ml" "Hs00167524 ml" "Hs00260452 ml"
"Hs01089557 sl" "Hs00232157 ml" "Hs00899658 ml" "Hs00904817 ml"
"Hs00202752 ml" "Hs00958111 ml" "Hs00968305 ml" "Hs00248075 ml"
"Hs00607978 sl" "Hs04942686 ml" "Hs01920599 gH" "Hs00365956 ml"
"Hs00944507 gl" "Hs01043717 ml" "Hs00745492 sl" "Hs00704853 sl"
"Hs00610058 ml" "Hs04194422 sl"
30-25 "Hs00738791 gl" "Hs04232205 sl" "Hs00362096 ml" "Hs00260452 ml"
"Hs01547054 ml" "Hs00196245 ml" "Hs01560931 ml" "Hs00964384_gl"
"Hs00415546 ml" "Hs00202752 ml" "Hs03043789_gl" "Hs00997579 ml"
"Hs00745492 sl" "Hs00174029 ml" "Hs04194422 sl" "Hs00899658 ml"
"Hs00251883 ml" "Hs00915710 ml" "Hs01042796 ml" "Hs04942686 ml"
"Hs00248075 ml" "Hs00222415 ml" "Hs01081598 ml" "Hs01089557 sl"
"Hs00705626 sl" "Hs00356958 ml" "Hs00204257 ml" "Hs00926053 ml"
"Hs05052601 sl" "Hs00372831_gl"
40-1 "Hs00262107 ml" "Hs01560931 ml" "Hs00159178 ml" "Hs00975850 ml"
"Hs00366766 ml" "Hs00543973 ml" "Hs00231709 ml" "Hs00372831_gl"
"Hs00745492 sl" "Hs01072228 ml" "Hs04194422 sl" "Hs00174029 ml"
"Hs00171157 ml" "Hs01029174 ml" "Hs01089557 sl" "Hs00801390 sl"
57
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs00958111 ml" "Hs01098278 ml" "Hs01920599 gH" "Hs00233987 ml"
"Hs00374264 gl" "Hs04189864 ml" "Hs00155241 ml" "Hs00610058 ml"
"Hs03464469 sl" "Hs00386692 ml" "Hs00964384 gl" "Hs01025572 ml"
"Hs01546752 gl" "Hs01395177 ml" "Hs00248075 ml" "Hs00937509 ml"
"Hs00704853 sl" "Hs03028557 sl" "Hs00747379 ml" "Hs00904817 ml"
"Hs00362096 ml" "Hs01087946 gl" "Hs01031740 ml" "Hs00978280 ml"
40-2 "Hs00231709 ml" "Hs01098278 ml" "Hs00174969 ml" "Hs01029174 ml"
"Hs05036222 sl" "Hs00262107 ml" "Hs00856927 gl" "Hs05052601 sl"
"Hs00233987 ml" "Hs00559914 ml" "Hs01931732 sl" "Hs01560931 ml"
"Hs00167524 ml" "Hs00543973 ml" "Hs01010736 ml" "Hs00174029 ml"
"Hs00260480 ml" "Hs00996236 ml" "Hs00745492 sl" "Hs00204257 ml"
"Hs00374264 gl" "Hs04942686 ml" "Hs00411188 gl" "Hs00380101 ml"
"Hs01051611 gH" "Hs01089557 sl" "Hs00901350 gl" "Hs01081598 ml"
"Hs00738791 gl" "Hs01025572 ml" "Hs00248075 ml" "Hs01894962 sl"
"Hs00957562 ml" "Hs00189880 ml" "Hs00937509 ml" "Hs00362096 ml"
"Hs00962398 ml" "Hs00171042 ml" "Hs01076090 ml" "Hs00801390 sl"
40-3 "Hs00978280 ml" "Hs00900510 ml" "Hs00411188_gl" "Hs00174969 ml"
"Hs00171157 ml" "Hs01546752_gl" "Hs04194422 sl" "Hs00826827_gl"
"Hs01010736 ml" "Hs01043717 ml" "Hs00931763 ml" "Hs01089557 sl"
"Hs00703025 sl" "Hs00559914 ml" "Hs03302824_pri" "Hs01087946 gl"
"Hs00950371 ml" "Hs01051445_gl" "Hs00202752 ml" "Hs00380101 ml"
"Hs01553775 gl" "Hs00232157 ml" "Hs00698959 ml" "Hs01098278 ml"
"Hs03464469 sl" "Hs00884853 sl" "Hs00926053 ml" "Hs00936519 ml"
"Hs00745492 sl" "Hs01560931 ml" "Hs00362096 ml" "Hs00204257 ml"
"Hs00240906 ml" "Hs00366766 ml" "Hs00260480 ml" "Hs01549976 ml"
"Hs00997579 ml" "Hs00738791 gl" "Hs01114274 ml" "Hs03043789_gl"
40-4 "Hs00899658 ml" "Hs00543973 ml" "Hs00260452 ml" "Hs00233987 ml"
"Hs03302824_pri" "Hs04189864 ml" "Hs01089557 sl" "Hs05036222 sl"
"Hs00428732 ml" "Hs00374264_gl" "Hs00958111 ml" "Hs00167524 ml"
"Hs00856927 gl" "Hs00964384 gl" "Hs00900510 ml" "Hs00747379 ml"
"Hs01087946 gl" "Hs05033260 sl" "Hs00607978 sl" "Hs04942686 ml"
"Hs00937509 ml" "Hs00380101 ml" "Hs00386692 ml" "Hs00155241 ml"
"Hs01114274 ml" "Hs03028557 sl" "Hs00240906 ml" "Hs01546752_gl"
"Hs00975850 ml" "Hs00610058 ml" "Hs01631495 sl" "Hs00174029 ml"
"Hs00411188 gl" "Hs00559914 ml" "Hs03043789 gl" "Hs00698959 ml"
"Hs00703025 sl" "Hs00745492 sl" "Hs00387770 ml" "Hs01008033 ml"
40-5 "Hs00607978 sl" "Hs01031740 ml" "Hs00174969 ml" "Hs01114274 ml"
"Hs04942686 ml" "Hs00931763 ml" "Hs00996236 ml" "Hs01395177 ml"
"Hs01098278 ml" "Hs00387770 ml" "Hs04194422 sl" "Hs00248075 ml"
"Hs01546752 gl" "Hs00705626 sl" "Hs01920599 gH" "Hs00801390 sl"
"Hs05052601 sl" "Hs00960591 ml" "Hs01076090 ml" "Hs00232157 ml"
"Hs00262107 ml" "Hs00944507 gl" "Hs00174705 ml" "Hs01089557 sl"
"Hs01029174 ml" "Hs01051445 gl" "Hs00362096 ml" "Hs00399035 ml"
58
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs00745492 sl" "Hs00978280 ml" "Hs01043717 ml" "Hs03028557 sl"
"Hs00233987 ml" "Hs00704853 sl" "Hs01008033 ml" "Hs00543973 ml"
"Hs00251883 ml" "Hs01691258 gl" "Hs00155241 ml" "Hs01042796 ml"
40-6 "Hs01931732 sl" "Hs00174705 ml" "Hs00248075 ml" "Hs00174969 ml"
"Hs00950371 ml" "Hs00374264 gl" "Hs00232157 ml" "Hs00747379 ml"
"Hs00884853 sl" "Hs00411188_gl" "Hs01089557 sl" "Hs00196245 ml"
"Hs01029174 ml" "Hs01560931 ml" "Hs00379134 ml" "Hs04942686 ml"
"Hs00738791 gl" "Hs00960591 ml" "Hs00171042 ml" "Hs05016463 sl"
"Hs01025572 ml" "Hs00260480 ml" "Hs00428732 ml" "Hs00705626 sl"
"Hs01114274 ml" "Hs00153408 ml" "Hs00944507_gl" "Hs00171157 ml"
"Hs00962398 ml" "Hs00975850 ml" "Hs00997579 ml" "Hs00931763 ml"
"Hs01379134 ml" "Hs04194422 sl" "Hs00703025 sl" "Hs01553775 gl"
"Hs00365956 ml" "Hs01098278 ml" "Hs00559914 ml" "Hs00992679 ml"
40-7 "Hs00387770 ml" "Hs01031740 ml" "Hs00356958 ml" "Hs00959010 ml"
"Hs01395177 ml" "Hs00705626 sl" "Hs01010736 ml" "Hs00703025 sl"
"Hs00801390 sl" "Hs01894962 sl" "Hs00233987 ml" "Hs00174029 ml"
"Hs00196245 ml" "Hs01051445_gl" "Hs00153408 ml" "Hs01081598 ml"
"Hs00411188_gl" "Hs01089557 sl" "Hs01920599_gH" "Hs00901350 gl"
"Hs00950371 ml" "Hs00997579 ml" "Hs00415546 ml" "Hs00884853 sl"
"Hs00155241 ml" "Hs00915710 ml" "Hs03302824_pri" "Hs01025572 ml"
"Hs00698959 ml" "Hs00167524 ml" "Hs00936519 ml" "Hs00992679 ml"
"Hs00704853 sl" "Hs00960591 ml" "Hs00968305 ml" "Hs00380101 ml"
"Hs00745492 sl" "Hs00201707 ml" "Hs00996236 ml" "Hs00366766 ml"
40-8 "Hs00155241 ml" "Hs01025572 ml" "Hs01087946_gl" "Hs00204257 ml"
"Hs01395177 ml" "Hs05052601 sl" "Hs00962398 ml" "Hs03028557 sl"
"Hs05033260 sl" "Hs00610058 ml" "Hs00174029 ml" "Hs05036222 sl"
"Hs04189864 ml" "Hs00543973 ml" "Hs01081598 ml" "Hs01894962 sl"
"Hs00738791 gl" "Hs00171042 ml" "Hs00957562 ml" "Hs01560931 ml"
"Hs00231709 ml" "Hs01098278 ml" "Hs00703025 sl" "Hs01089557 sl"
"Hs00374264 gl" "Hs01010736 ml" "Hs00901350 gl" "Hs00174969 ml"
"Hs00260480 ml" "Hs00996236 ml" "Hs05016463 sl" "Hs00415546 ml"
"Hs00362096 ml" "Hs00704853 sl" "Hs00975850 ml" "Hs00174705 ml"
"Hs00251883 ml" "Hs00196245 ml" "Hs00222415 ml" "Hs01931732 sl"
40-9 "Hs00267207 ml" "Hs00904817 ml" "Hs01087946_gl" "Hs00233987 ml"
"Hs04194422 sl" "Hs00196245 ml" "Hs03302824_pri" "Hs01089557 sl"
"Hs00936519 ml" "Hs01560931 ml" "Hs00559914 ml" "Hs00171157 ml"
"Hs00900510 ml" "Hs00174705 ml" "Hs00856927_gl" "Hs00950371 ml"
"Hs01043717 ml" "Hs00960591 ml" "Hs01546752_gl" "Hs00957562 ml"
"Hs00372831 gl" "Hs01029174 ml" "Hs00937509 ml" "Hs00411188_gl"
"Hs05016463 sl" "Hs00705626 sl" "Hs00232157 ml" "Hs00174029 ml"
"Hs00978280 ml" "Hs05052601 sl" "Hs05033260 sl" "Hs01920599 gH"
"Hs00231709 ml" "Hs01098278 ml" "Hs01081598 ml" "Hs00997579 ml"
"Hs01051445 gl" "Hs04232205 sl" "Hs00884853 sl" "Hs00738791_gl"
59
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
40-10 "Hs01076090 ml" "Hs00387770 ml" "Hs01031740 ml" "Hs00196245 ml"
"Hs03464469 sl" "Hs04232205 sl" "Hs00884853 sl" "Hs00174969 ml"
"Hs00607978 sl" "Hs04942686 ml" "Hs00931763 ml" "Hs01098278 ml"
"Hs00996236 ml" "Hs00960591 ml" "Hs01920599 gH" "Hs01546752 gl"
"Hs00997579 ml" "Hs01087946 gl" "Hs01560931 ml" "Hs00950371 ml"
"Hs01549976 ml" "Hs00747379 ml" "Hs01081598 ml" "Hs01691258_gl"
"Hs00231709 ml" "Hs00399035 ml" "Hs00428732 ml" "Hs00543973 ml"
"Hs00240906 ml" "Hs00372831 gl" "Hs00703025 sl" "Hs00171042 ml"
"Hs01043717 ml" "Hs01025572 ml" "Hs00222415 ml" "Hs00937509 ml"
"Hs00267207 ml" "Hs00915710 ml" "Hs00366766 ml" "Hs00610058 ml"
40-11 "Hs00610058 ml" "Hs00745492 sl" "Hs00901350_gl" "Hs01051611_gH"
"Hs01087946 gl" "Hs05052601 sl" "Hs03028557 sl" "Hs00248075 ml"
"Hs01089557 sl" "Hs00738791 gl" "Hs00374264_gl" "Hs00543973 ml"
"Hs04189864 ml" "Hs00698959 ml" "Hs05036222 sl" "Hs00962398 ml"
"Hs00362096 ml" "Hs03043789_gl" "Hs03302824_pri" "Hs00380101 ml"
"Hs00232157 ml" "Hs01553775_gl" "Hs01547054 ml" "Hs00174705 ml"
"Hs00936519 ml" "Hs00386692 ml" "Hs00926053 ml" "Hs00996236 ml"
"Hs00960591 ml" "Hs01098278 ml" "Hs01560931 ml" "Hs00202752 ml"
"Hs04942686 ml" "Hs00428732 ml" "Hs01072228 ml" "Hs00884853 sl"
"Hs00931763 ml" "Hs00964384_gl" "Hs00826827_gl" "Hs00155241 ml"
40-12 "Hs00174705 ml" "Hs00698959 ml" "Hs01546752_gl" "Hs00262107 ml"
"Hs03464469 sl" "Hs00996236 ml" "Hs00960591 ml" "Hs00387770 ml"
"Hs00204257 ml" "Hs00801390 sl" "Hs00610058 ml" "Hs01631495 sl"
"Hs01081598 ml" "Hs00240906 ml" "Hs01553775_gl" "Hs00362096 ml"
"Hs00899658 ml" "Hs00248075 ml" "Hs01894962 sl" "Hs01089557 sl"
"Hs00747379 ml" "Hs01560931 ml" "Hs00267207 ml" "Hs01010736 ml"
"Hs00901350 gl" "Hs03302824_pri" "Hs01547054 ml" "Hs00386692 ml"
"Hs01098278 ml" "Hs00399035 ml" "Hs00189880 ml" "Hs00232157 ml"
"Hs00356958 ml" "Hs00167524 ml" "Hs00380101 ml" "Hs04942686 ml"
"Hs00174969 ml" "Hs00968305 ml" "Hs00745492 sl" "Hs00366766 ml"
40-13 "Hs01546752 gl" "Hs01920599 gH" "Hs00801390 sl" "Hs01560931 ml"
"Hs04194422 sl" "Hs01081598 ml" "Hs00251883 ml" "Hs00559914 ml"
"Hs00262107 ml" "Hs00374264_gl" "Hs00543973 ml" "Hs00171157 ml"
"Hs00937509 ml" "Hs01029174 ml" "Hs00174969 ml" "Hs00232157 ml"
"Hs00705626 sl" "Hs05016463 sl" "Hs01553775 gl" "Hs00704853 sl"
"Hs01691258 gl" "Hs00167524 ml" "Hs00962398 ml" "Hs00978280 ml"
"Hs00248075 ml" "Hs00386692 ml" "Hs00959010 ml" "Hs01098278 ml"
"Hs00884853 sl" "Hs00174705 ml" "Hs00996236 ml" "Hs00379134 ml"
"Hs04942686 ml" "Hs00202752 ml" "Hs00189880 ml" "Hs00365956 ml"
"Hs03302824_pri" "Hs00926053 ml" "Hs01395177 ml" "Hs04189864 ml"
40-14 "Hs00747379 ml" "Hs00362096 ml" "Hs00386692 ml" "Hs01089557 sl"
"Hs01029174 ml" "Hs00960591 ml" "Hs00248075 ml" "Hs01547054 ml"
"Hs01631495 sl" "Hs00189880 ml" "Hs00167524 ml" "Hs01098278 ml"
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs00996236 ml" "Hs00240906 ml" "Hs01087946_g1" "Hs00884853 sl"
"Hs00899658 ml" "Hs00380101 ml" "Hs04942686 ml" "Hs00936519 ml"
"Hs00856927 gl" "Hs01043717 ml" "Hs00915710 ml" "Hs00231709 ml"
"Hs00964384 gl" "Hs00387770 ml" "Hs00155241 ml" "Hs01081598 ml"
"Hs00926053 ml" "Hs00366766 ml" "Hs00171042 ml" "Hs01031740 ml"
"Hs00959010 ml" "Hs01546752 gl" "Hs00233987 ml" "Hs01042796 ml"
"Hs00896999 gl" "Hs00374264 gl" "Hs04232205 sl" "Hs00201707 ml"
40-15 "Hs00703025 sl" "Hs01087946_gl" "Hs05016463 sl" "Hs00167524 ml"
"Hs00543973 ml" "Hs01010736 ml" "Hs05052601 sl" "Hs00957562 ml"
"Hs00856927 gl" "Hs04232205 sl" "Hs01029174 ml" "Hs00251883 ml"
"Hs01931732 sl" "Hs00262107 ml" "Hs00374264 gl" "Hs01560931 ml"
"Hs00174969 ml" "Hs00937509 ml" "Hs00222415 ml" "Hs00801390 sl"
"Hs00959010 ml" "Hs00231709 ml" "Hs00747379 ml" "Hs00411188_gl"
"Hs01051445 gl" "Hs00174029 ml" "Hs01553775 gl" "Hs01072228 ml"
"Hs01089557 sl" "Hs00415546 ml" "Hs00705626 sl" "Hs00387770 ml"
"Hs00745492 sl" "Hs00153408 ml" "Hs00196245 ml" "Hs00884853 sl"
"Hs04189864 ml" "Hs00936519 ml" "Hs00958111 ml" "Hs00607978 sl"
40-16 "Hs01920599 gH" "Hs04189864 ml" "Hs01114274 ml" "Hs01395177 ml"
"Hs00374264 gl" "Hs00174029 ml" "Hs00958111 ml" "Hs03028557 sl"
"Hs00856927 gl" "Hs00153408 ml" "Hs01089557 sl" "Hs00607978 sl"
"Hs00201707 ml" "Hs00155241 ml" "Hs00233987 ml" "Hs00745492 sl"
"Hs00610058 ml" "Hs00964384 gl" "Hs04942686 ml" "Hs00996236 ml"
"Hs00174969 ml" "Hs00944507 gl" "Hs00248075 ml" "Hs00362096 ml"
"Hs00174705 ml" "Hs01043717 ml" "Hs00260452 ml" "Hs00167524 ml"
"Hs00380101 ml" "Hs00559914 ml" "Hs00159178 ml" "Hs04194422 sl"
"Hs00399035 ml" "Hs01931732 sl" "Hs00703025 sl" "Hs00738791 gl"
"Hs00826827 gl" "Hs00997579 ml" "Hs00204257 ml" "Hs01070154 ml"
40-17 "Hs00978280 ml" "Hs01553775 gl" "Hs01029174 ml" "Hs00996236 ml"
"Hs00386692 ml" "Hs00738791 gl" "Hs01089557 sl" "Hs00365956 ml"
"Hs00607978 sl" "Hs00248075 ml" "Hs01098278 ml" "Hs00962398 ml"
"Hs00196245 ml" "Hs00232157 ml" "Hs00167524 ml" "Hs04942686 ml"
"Hs00201707 ml" "Hs00915710 ml" "Hs01549976 ml" "Hs00901350_gl"
"Hs04194422 sl" "Hs00997579 ml" "Hs01031740 ml" "Hs00904817 ml"
"Hs01631495 sl" "Hs00153408 ml" "Hs00975850 ml" "Hs00428732 ml"
"Hs00366766 ml" "Hs04232205 sl" "Hs01547054 ml" "Hs00900510 ml"
"Hs01560931 ml" "Hs00747379 ml" "Hs00356958 ml" "Hs00899658 ml"
"Hs00415546 ml" "Hs00362096 ml" "Hs00937509 ml" "Hs00958111 ml"
40-18 "Hs00201707 ml" "Hs04942686 ml" "Hs04194422 sl" "Hs00899658 ml"
"Hs00260452 ml" "Hs01043717 ml" "Hs00262107 ml" "Hs01070154 ml"
"Hs01029174 ml" "Hs00856927 gl" "Hs01098278 ml" "Hs00996236 ml"
"Hs00944507 gl" "Hs00372831 gl" "Hs00356958 ml" "Hs01051611_gH"
"Hs00380101 ml" "Hs01076090 ml" "Hs00703025 sl" "Hs00251883 ml"
"Hs01560931 ml" "Hs00171157 ml" "Hs00386692 ml" "Hs05016463 sl"
61
CA 03098152 2020-10-22
WO 2019/213321
PCT/US2019/030282
"Hs00167524 ml" "Hs01089557 sl" "Hs00171042 ml" "Hs00745492 sl"
"Hs00610058 ml" "Hs00884853 sl" "Hs00958111 ml" "Hs04189864 ml"
"Hs00704853 sl" "Hs00543973 ml" "Hs00374264 gl" "Hs01087946_gl"
"Hs00415546 ml" "Hs05036222 sl" "Hs00904817 ml" "Hs01072228 ml"
40-19 "Hs01072228 ml" "Hs00915710 ml" "Hs00174705 ml" "Hs00356958 ml"
"Hs00174029 ml" "Hs00958111 ml" "Hs00248075 ml" "Hs01553775_gl"
"Hs01098278 ml" "Hs00957562 ml" "Hs00937509 ml" "Hs00960591 ml"
"Hs00747379 ml" "Hs00387770 ml" "Hs01547054 ml" "Hs00698959 ml"
"Hs00399035 ml" "Hs00607978 sl" "Hs04942686 ml" "Hs00240906 ml"
"Hs00962398 ml" "Hs00232157 ml" "Hs00267207 ml" "Hs00996236 ml"
"Hs00704853 sl" "Hs00365956 ml" "Hs00196245 ml" "Hs00959010 ml"
"Hs00884853 sl" "Hs01691258_gl" "Hs00950371 ml" "Hs00260480 ml"
"Hs00174969 ml" "Hs01560931 ml" "Hs00964384_gl" "Hs00738791_gl"
"Hs00543973 ml" "Hs00171042 ml" "Hs04189864 ml" "Hs00415546 ml"
40-20 "Hs00559914 ml" "Hs00856927_gl" "Hs00232157 ml" "Hs01029174 ml"
"Hs00978280 ml" "Hs01051445_gl" "Hs01031740 ml" "Hs00899658 ml"
"Hs00174705 ml" "Hs00153408 ml" "Hs00362096 ml" "Hs00944507 gl"
"Hs00260452 ml" "Hs00937509 ml" "Hs00826827_gl" "Hs00248075 ml"
"Hs01043717 ml" "Hs01070154 ml" "Hs01076090 ml" "Hs01560931 ml"
"Hs01920599 gH" "Hs00543973 ml" "Hs00380101 ml" "Hs00997579 ml"
"Hs03043789 gl" "Hs00901350 gl" "Hs03302824_pri" "Hs00399035 ml"
"Hs00747379 ml" "Hs01114274 ml" "Hs04232205 sl" "Hs05016463 sl"
"Hs00387770 ml" "Hs00366766 ml" "Hs04194422 sl" "Hs01008033 ml"
"Hs01395177 ml" "Hs00904817 ml" "Hs03464469 sl" "Hs01894962 sl"
40-21 "Hs00826827 gl" "Hs00899658 ml" "Hs01076090 ml" "Hs00411188_gl"
"Hs05036222 sl" "Hs00171042 ml" "Hs00926053 ml" "Hs00233987 ml"
"Hs00174029 ml" "Hs00902334 ml" "Hs00958111 ml" "Hs00153408 ml"
"Hs01081598 ml" "Hs01051445 gl" "Hs04189864 ml" "Hs00962398 ml"
"Hs00155241 ml" "Hs00380101 ml" "Hs01560931 ml" "Hs00167524 ml"
"Hs00171157 ml" "Hs00260480 ml" "Hs00174969 ml" "Hs01114274 ml"
"Hs00705626 sl" "Hs01920599_gH" "Hs00428732 ml" "Hs01031740 ml"
"Hs05016463 sl" "Hs01549976 ml" "Hs00931763 ml" "Hs00703025 sl"
"Hs00960591 ml" "Hs05052601 sl" "Hs00937509 ml" "Hs01098278 ml"
"Hs00968305 ml" "Hs01087946 gl" "Hs00959010 ml" "Hs01089557 sl"
40-22 "Hs00904817 ml" "Hs00260480 ml" "Hs00856927_gl" "Hs01894962 sl"
"Hs00171042 ml" "Hs00171157 ml" "Hs01098278 ml" "Hs00202752 ml"
"Hs01076090 ml" "Hs00964384 gl" "Hs00155241 ml" "Hs00950371 ml"
"Hs01042796 ml" "Hs00826827 gl" "Hs00958111 ml" "Hs00899658 ml"
"Hs00233987 ml" "Hs00926053 ml" "Hs01029174 ml" "Hs00174029 ml"
"Hs01081598 ml" "Hs00159178 ml" "Hs00975850 ml" "Hs00232157 ml"
"Hs00251883 ml" "Hs00372831 gl" "Hs00957562 ml" "Hs00610058 ml"
"Hs01043717 ml" "Hs05033260 sl" "Hs01920599 gH" "Hs00543973 ml"
"Hs01560931 ml" "Hs00901350 gl" "Hs00705626 sl" "Hs05016463 sl"
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CA 03098152 2020-10-22
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"Hs00260452 ml" "Hs01553775 gl" "Hs00153408 ml" "Hs00962398 ml"
40-23 "Hs00801390 sl" "Hs00174969 ml" "Hs00959010 ml" "Hs00968305 ml"
"Hs01547054 ml" "Hs01072228 ml" "Hs00262107 ml" "Hs01114274 ml"
"Hs00937509 ml" "Hs01051611_gH" "Hs01920599 gH" "Hs01031740 ml"
"Hs00399035 ml" "Hs00962398 ml" "Hs00233987 ml" "Hs00167524 ml"
"Hs00826827 gl" "Hs01010736 ml" "Hs03302824_pri" "Hs01549976 ml"
"Hs00901350 gl" "Hs00975850 ml" "Hs00904817 ml" "Hs00380101 ml"
"Hs00387770 ml" "Hs04232205 sl" "Hs00240906 ml" "Hs00997579 ml"
"Hs01560931 ml" "Hs01631495 sl" "Hs05052601 sl" "Hs00738791 gl"
"Hs00171157 ml" "Hs00232157 ml" "Hs00978280 ml" "Hs00372831 gl"
"Hs00704853 sl" "Hs00745492 sl" "Hs00936519 ml" "Hs00204257 ml"
40-24 "Hs01549976 ml" "Hs00260480 ml" "Hs01031740 ml" "Hs05052601 sl"
"Hs05016463 sl" "Hs00196245 ml" "Hs01025572 ml" "Hs01042796 ml"
"Hs00944507 gl" "Hs00171157 ml" "Hs00167524 ml" "Hs00962398 ml"
"Hs00428732 ml" "Hs00543973 ml" "Hs01560931 ml" "Hs01087946 gl"
"Hs00960591 ml" "Hs04189864 ml" "Hs00826827_gl" "Hs00380101 ml"
"Hs01691258 gl" "Hs00386692 ml" "Hs00374264_gl" "Hs00745492 sl"
"Hs00884853 sl" "Hs00411188_gl" "Hs00936519 ml" "Hs00240906 ml"
"Hs00232157 ml" "Hs01098278 ml" "Hs03302824_pri" "Hs00248075 ml"
"Hs00801390 sl" "Hs01553775_gl" "Hs03464469 sl" "Hs00738791 gl"
"Hs01631495 sl" "Hs00703025 sl" "Hs00896999 gl" "Hs01010736 ml"
40-25 "Hs04189864 ml" "Hs00386692 ml" "Hs00926053 ml" "Hs00260452 ml"
"Hs00610058 ml" "Hs00958111 ml" "Hs00704853 sl" "Hs00380101 ml"
"Hs00543973 ml" "Hs00167524 ml" "Hs00747379 ml" "Hs00428732 ml"
"Hs00559914 ml" "Hs00738791 gl" "Hs00260480 ml" "Hs00703025 sl"
"Hs00975850 ml" "Hs01025572 ml" "Hs01098278 ml" "Hs01560931 ml"
"Hs00745492 sl" "Hs00233987 ml" "Hs00232157 ml" "Hs05016463 sl"
"Hs00607978 sl" "Hs00171042 ml" "Hs00884853 sl" "Hs01691258 gl"
"Hs00196245 ml" "Hs01081598 ml" "Hs00251883 ml" "Hs00904817 ml"
"Hs01114274 ml" "Hs00222415 ml" "Hs01051445_gl" "Hs01042796 ml"
"Hs00356958 ml" "Hs00944507 gl" "Hs00379134 ml" "Hs00705626 sl"
See Table 11 for gene name associated with each probe ID.
All references cited in this application are expressly incorporated by
reference herein.
63
