U.S. patent application number 14/718755 was filed with the patent office on 2015-11-26 for use of genetic modifications in human gene chk1 which codes for checkpoint kinase 1.
The applicant listed for this patent is Universitat Duisburg-Essen. Invention is credited to Kathrin Riemann, Winfried Siffert.
Application Number | 20150337389 14/718755 |
Document ID | / |
Family ID | 39402042 |
Filed Date | 2015-11-26 |
United States Patent
Application |
20150337389 |
Kind Code |
A1 |
Riemann; Kathrin ; et
al. |
November 26, 2015 |
Use Of Genetic Modifications In Human Gene CHK1 Which Codes For
Checkpoint Kinase 1
Abstract
The invention relates to an in vitro method for predicting
disease risks, progression of diseases, drug risks, success of
treatment and for finding drug targets by looking for one or more
genetic modifications in the promoter region of the CHK1 (CHEK1)
gene on human chromosome 11q23, the genetic modifications being a
substitution thymine for guanine in position -1143 in the promoter
of CHK1, of thymine for cytosine in position -1400, a substitution
of cytosine for thymine in position -1453 or an insertion of one
cytosine in position -1454 and the genetic modifications being
detected individually or in any combinations by way of known
methods.
Inventors: |
Riemann; Kathrin;
(Mittelbiberach, DE) ; Siffert; Winfried; (Essen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Universitat Duisburg-Essen |
Essen |
|
DE |
|
|
Family ID: |
39402042 |
Appl. No.: |
14/718755 |
Filed: |
May 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13666606 |
Nov 1, 2012 |
9074259 |
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14718755 |
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12312474 |
Jan 28, 2010 |
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PCT/EP2007/062519 |
Nov 19, 2007 |
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13666606 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/106 20130101; C12Q 2600/156 20130101; C12Q 1/6883
20130101; C12Q 2600/172 20130101; C12Q 2600/112 20130101; C12Q
2600/118 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 17, 2006 |
DE |
102006054292.4 |
Claims
1. An in vitro method for predicting disease risks, progression of
disease risks, progression of diseases, drug risks, success of
treatment and for finding drag targets, characterized by looking
for one or more genetic modifications in the promoter region of the
CHK1 (CHEK1) gene on human chromosome 11q23.
2. The method according to claim 1, characterized by looking for a
polymorphism (-1143) G>T in a patient sample.
3. The method according to claim 1, characterized by looking for a
polymorphism (-1400) C>T in a patient sample.
4. The method according to claim 1, characterized by looking for a
polymorphism (-1453) T>C in a patient sample.
5. The method according to claim 1, characterized by looking for a
polymorphism (-1454) insC in a patient sample.
6. The method according to claim 1, characterized by looking for
one, two, three, or four of the polymorphisms (-1143) G>T,
(-1400) C>T, (-1453) T>C and (-1154) insC in a patient
sample.
7. The method according to claim 1, characterized in that the
disease is a cancer.
8. The method according to claim 1, characterized in that the
treatment is one with cancer therapeutic agents or a physical
treatment of cancer.
9. The method according to claim 1, characterized in that the drugs
or cancer therapeutic agents are inhibitors of checkpoint kinase 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 13/666,606 filed Nov. 1, 2012, which was
granted as U.S. Pat. No. 9,074,259 on Jul. 7, 2015, which is a
continuation of U.S. application Ser. No. 12/312,474, filed Jan.
28, 2010, which is a national stage entry under 35 U.S.C. .sctn.371
of PCT International Application No. PCT/EP2007/062519, filed Nov.
19, 2007, which claims priority to German Patent Application No.
102006054292.4, filed Nov. 17, 2006, the disclosures of each of
which are incorporated by reference herein in their entireties.
TECHNICAL FIELD OF THE INVENTION
[0002] The invention relates to an in vitro method for predicting
disease risks, progression of diseases, drug risks and for finding
drug targets.
TECHNICOLOGICAL BACKGROUND OF THE INVENTION
[0003] Cancer cells are characterized by loss of contact inhibition
and uncontrolled cell growth. Such modifications are triggered
spontaneously or by noxae, co-called cancerogenes, which damage the
genetic make-up. Such noxae include many chemicals, tobacco smoke,
but also UV light. Besides that, genetic factors play a prominent
role in the formation of cancer. Characteristic for cancer cells,
beside their uninhibited growth, is also the tendency to form
metastases in other organs.
[0004] It is of exceedingly high medical relevance to define
prognosis factors for the progression of cancers, which provide
information about the response to certain forms of treatment or are
generally predictive for the occurrence of metastases, tumor
progression and survival. So far, prognosis factors generally known
to the person skilled in the art are used in medicine. These
include, for example, the size of the tumor, its penetration depth
into the surrounding tissue, cross-organ growth, the penetration
into blood or lymphatic vessels or into lymph nodes, as well as the
degree of differentiation of the tumor cells. In addition, some
relatively unspecific serological markers exist.
[0005] The cell cycle of eukaryotic cells is generally subdivided
into four phases: the G1-phase, in which the preparation for
replication takes place, the S-phase, in which the DNA is
synthesized and the actual cell functions take place, the G2-phase,
in which the preparation for mitosis takes place, and the M-phase,
the mitosis (FIG. 1). In addition, differentiated cells, which no
longer divide, are described as being in the G0-phase. This
organizational principle though is functional, but on closer
inspection it becomes clear that the cell cycle is far more
complex. Numerous processes must be initiated and activated,
individual components joined and various cascades coordinated. For
this reason, diverse control mechanisms exist, which ensure that
any processes within the cell cycle are completed correctly. These
control mechanisms are designated as "checkpoints.sup.1". These are
not fixedly defined points, as the word itself implies, but a
reaction cascade, which can be initiated under certain
circumstances. .sup.1 Original definition (according to Weinert et
al., The RAD9 gene controls the cell cycle response to DNA damage
in Saccharomyces cerevisiae. 1988, Science 241:317-22): If a
process B depends on the completion of a process A, then this
dependency is conditional on a checkpoint, unless a mutation can
eliminate the dependent relationship.
[0006] So far, several cell cycle checkpoints were characterized.
The best investigated checkpoints in mammals are shown in FIG. 1.
On the one hand, there is the DNA damage checkpoint, which can be
activated by a damage of the DNA in different cell cycle phases.
This damage can be caused by exogenous causes, like radiation, as
well as by endogenous processes, e.g. spontaneous mutations. On the
other hand, the replication checkpoint is activated by an
incomplete or defective replication of the DNA. The spindle
checkpoint monitors the formation of the bipolar spindle, the
attachment of the kinetochores and the new formation of the
centromere structures.
[0007] As long as these processes are not entirely completed or the
damage eliminated, the entrance of the cell into the next cell
cycle phase is inhibited to ensure that the genomic integrity of
the cell is maintained (Elledge, S. J., Cell cycle checkpoints:
preventing an identity crisis. 1996, Science 274:1664-72).
[0008] The most important task of a cell is to maintain genomic
identity. Checkpoint kinase 1 is involved in essential control
mechanisms in the cell cycle, which ensure that the transfer of
defects to the daughter cell is minimized. The significant CHK1
reaction cascade at the G2/M checkpoint is shown in FIG. 2. The
activation of CHK1 takes place due to DNA damages, which are mainly
detected by the chromatin-bound Rad17 complex. Thereupon, the Rad17
complex recruits the Rad9-Hus1-Rad1 complex, which together with
the ATR-Atrip complex activates CHK1, which is partially present in
a chromatin-associated form, by phosphorylation. In that, ATR
(Ataxia-telangiectasia- and Rad3-related) represents the most
important activating component. It was shown that for the complete
activation of CHK1, phosphorylation by the protein Claspin is also
required. The activated CHK1 protein migrates from the cell nucleus
into the cytoplasm, where in its turn it activates CDC25C (cell
division cycle 25C) by phosphorylation. This process, on the other
hand, enables the 14-3-3 protein (tyrosine
3-monooxygenase/tryptophan 5-monooxygenase activation protein) to
bind to CHK1, so that it can return into the nucleus and remains
there. In this manner, CDC2 (cell division controller 2) as well as
the cyclin B complex are inhibited, which inhibits the entrance
into mitosis. Subsequently, the DNA repair system can be initiated
to eliminate the DNA damage (Jiang et al., Regulation of Chk1
includes chromatin association and 14-3-3 binding following
phosphorylation on Ser345. 2003, J. Biol. Chem. 278:25207-17; Jeong
et al, Phosphorylated claspin interacts with a phosphate-binding
site in the kinase domain of Chk1 during ATR-mediated activation.
2003, J. Biol. Chem. 278:46782-8).
[0009] For CHK1, involvement in a checkpoint in the S-phase could
also be verified. Here, upon defective replication, CHK1 is
activated by ATM (Ataxia-telangiectasia mutated) by
phosphorylation. For this checkpoint, too, additional activation by
Claspin is required. The completely activated CHK1 now activates
DNA protein kinases, together with which they phosphorylate p53 and
thus can increase its activity. CHK1 is likewise able to
phosphorylate TLK1 (tousled like kinase 1). This protein plays a
decisive role in chromatin condensation, which, however, is
inhibited by CHK1 to prevent progression in the cell cycle.
Furthermore, CHK1 phosphorylates CDC25A (cell division cycle 25A)
and thus initiates its degradation. As a consequence, the CDC
protein is no longer able to activate the Cyclin complexes, due to
which neither the S-phase can be advanced nor the M-phase
started.
SUMMARY OF THE INVENTION
[0010] The invention is therefore based on the object to provide a
means, which enables a better prognosis of the natural progression
of a cancer and the response to any form of treatment. In
particular, this means is to be able to detect those patients, in
which increased DNA repair mechanisms aggravate a cancer treatment.
The invention is further based on the object to provide a means to
generally predict disease risks and progression of diseases, since
DNA repair mechanisms are also very important for other
diseases.
[0011] In particular, [0012] (a) function-modifying genomic
polymorphisms in the promoter of the CHK1 gene are to be provided,
which either result in the modification of the protein expression
or in the modification of the expression of splicing variants, or
[0013] (b) which are suited to find and/or validate further
polymorphisms or haplotypes in the CHK1 gene, [0014] (c)
polymorphisms are to be provided, which are suited to generally
predict disease risks and progression, [0015] (d) polymorphisms are
to be provided, which are suited to generally predict the response
to pharmaceuticals and cancer treatments, in particular CHK1
inhibitors, and side-effects, [0016] (e) polymorphisms are to be
provided, which are suited to generally predict the effect of other
forms of treatment like radiation, warmth, heat, cold,
movement.
[0017] These objects are solved by an in vitro method for
predicting disease risks, progression of diseases, drug risks and
for finding drug targets by looking for one or more genetic
modifications in the promoter region of the CHK1 (CHEK1) gene on
human chromosome 11q23.
PREFERRED EMBODIMENTS OF THE INVENTION
[0018] Such polymorphisms are, for example, (-1143)G>T,
(-1400)C>T, (-1453)T>C, and (-1454)insC.
[0019] The human gene CHK1 is localized on chromosome 11q23
(Accession No. NM.sub.--033899 of the Gene Bank of the National
Center for Biotechnology information (NCBI)) and codes for a 54 kD
protein, which is expressed in the nucleus. At this point, it is to
be pointed out that the gene has the designation "CHK1" as well as
the designation "CHEK1". In the following, the designation "CHK1"
shall be used here. A schematic representation of the gene
structure is shown in FIG. 3. The active promoter region of CHK1
has already been characterized. The promoter sequence contains
numerous binding sites for the transcription factor E2F1, the
binding of which enhances the transcriptional activity. A positive
regulation of CHK1 is likewise observed by an isoform of the
p53-dependent p73 (Carrassa et al., Characterization of the
5'-flanking region of the human Chk1 gene. 2003, Cell Cycle
2:604-9).
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows a schematic representation of the cell cycle
with the most important checkpoints.
[0021] FIG. 2 shows a graphical representation of the reaction
cascade at the DNA damage checkpoint.
[0022] FIG. 3 shows the intron/exon structure of the human gene
CHK1 (not to scale).
[0023] FIG. 4 shows the structural relationship of some CHK1
inhibitors with staurosporine.
[0024] FIG. 5 shows a schematic representation of the polymorphisms
in the CHK1 gene (not to scale).
[0025] FIG. 6A-6C shows the coupling analyses of the promoter
polymorphisms of CHK1 with the program Haploview; A-Graphical
representation of the coupling of the polymorphisms among one
another. Black squares indicate r.sup.2=1, grey squares
r.sup.2<0.5 and light-grey squares r.sup.2<0.1; B-Frequencies
and coupling possibilities of the individual alleles; C-Frequencies
of the constructed haplotypes; alleles marked with a triangle are
designated so-called haplotype-tagging alleles, i.e. these alleles
must be determined to determine the respective haplotypes.
[0026] FIG. 7 shows putative binding sites for transcription
factors in the promoter of the CHK1 gene (SEQ ID NO: 1); the
numbers on the left-hand side represent the relation to the
ATG.
[0027] FIG. 8 shows the result of the Electrophoretic Mobility
Shift Assay (EMS A) with constructs containing the various alleles
of the -1143G>T polymorphism of CHK1. Following the addition of
cell nucleus extract, increased binding of core protein to the
"G-construct" can be detected. The binding is specifically
inhibited by the presence of a displacing oligonucleotide.
[0028] FIG. 9 shows the result of the Electrophoretic Mobility
Shift Assay (EMSA) with constructs containing the various alleles
of the -1400C>T polymorphism of CHK1. Following the addition of
cell nucleus extract, increased binding of core protein to the
"T-construct" can be detected. The binding is specifically
inhibited by the presence of a displacing oligonucleotide.
[0029] FIG. 10 shows constructs for measuring the
genotype-dependent regulatory activity of the promoter
polymorphisms after 24 h following the transfection of HELA cells
using secreted alkaline phosphatase (SEAP). The activity of the
1143G>T SNP is significantly higher than the activity of the
other SNPs. ***: p<0.001.
[0030] FIG. 11A-11C shows an expression of CHK1 mRNA in urinary
bladder carcinoma tissue depending on the alleles of the promoter
polymorphisms. Represented is the quotient CHK1/.beta.-actin mRNA.
A: -1143G>T, B: -1400C>T, C: -1453T>C and 1454insC; *:
p<0.05.
[0031] FIG. 12A-12C shows an expression of CHK1 mRNA in colorectal
carcinoma tissue depending on the alleles of the promoter
polymorphisms. Represented is the quotient CHK1/.beta.-actin mRNA.
A: -1143G>T, B: -1400C>T, C: 1453T>C and -1454insC; *:
p<0.05.
[0032] FIG. 13 shows the Kaplan-Meier analysis on the survival of
patients with urinary bladder carcinoma depending on the genotype
of the -1143G>T polymorphism. A: all patients, B: only patients
older than 54 years; *: p<0.05, **: p<0.01
[0033] FIG. 14A-14B shows the Kaplan-Meier analysis on the survival
of patients with colorectal carcinoma depending on the genotype of
the promoter polymorphisms. A: Dependency on the -1143G>T SNP,
B: only patients with colon carcinoma depending on the -1400C>T
SNP, C: only patients with colon carcinoma depending on the
1453T>C SNP and -1454insC SNP; *: p<0.05, **:p<0.01
[0034] FIG. 15A-15C shows the Kaplan-Meier analysis on the survival
of patients with chronic lymphatic leukemia depending on the
genotype of the -1143G>T polymorphism.
[0035] FIG. 16 shows the Kaplan-Meier analysis of patients with
melanoma depending on the genotype of the -1400C>T promoter
polymorphism; A: Time from initial treatment to start of further
treatment; B: Survival; *: p<0.05.
[0036] FIG. 17A-17B shows the Kaplan-Meier analysis on the survival
of patients with cholangiocellular carcinoma depending on the
genotype of the -1400C>T polymorphism; *: p<0.05.
[0037] FIG. 18 shows a Kaplan-Meier analysis on the survival of
patients with cholangiocellular carcinoma depending on the genotype
of the -1400C>T polymorphism. *: p<0.05.
[0038] FIG. 19 shows a Kaplan-Meier analysis on the survival of
patients with mammary carcinoma depending on the genotype of the
-1143G>T polymorphism, *: p-0.05
[0039] CHK1 is considered a potential tumor suppressor gene, since
a defect in the regulation of the cell cycle results in cumulation
of defective DNA and an increased cellular proliferation rate, both
of which are characteristics of tumor cells. So far, somatic
mutations in this gene could be verified in some patients with
sporadic tumors, e.g. stomach and mammary carcinoma as well as
microsatellite-instable colorectal tumors showed alterations.
Unlike single nucleotide polymorphisms (SNPs), these mutations are,
for example, not found in peripheral blood cells in the respective
patients. Disease-specific associations for SNPs have not been
described yet.
[0040] Since checkpoints are involved in many regulatory cascades,
they are a suitable target for cancer therapeutic agents. Certain
characteristics of the checkpoint proteins contribute to that: (1)
the complex signal transduction system of checkpoints offers a
multitude of targets, (2) in healthy cells, some checkpoints seem
to be relatively insignificant, which highly reduces the toxicity
of the inhibitors, (3) the restoration of defective checkpoints
could result in a slow-down of the cell growth, (4) as a signal
transduction system, checkpoints are subject to adaption, which
could be interrupted, and (5) the restoration of affected
checkpoints could increase the apoptosis rate of cancer cells and
thus increase their sensitivity towards certain substances
(Hartwell et al., Cell cycle control and cancer. 1994, Science
266:1821-8).
[0041] Contrary to these points, which most likely can be realized
via a gene therapy approach, two further characteristics of
checkpoints represent simpler realizable targets. Cells with
defective checkpoints show a highly increased sensitivity towards
radiation and cytotoxic substances. Particularly the loss of CHK1
seems to predispose tumor cells for these types of treatment.
[0042] Diverse CHK1 inhibitors are already known or have already
been developed. Based on staurosporine, originally identified as
protein kinase C inhibitor, which likewise is a potent CHK1
inhibitor, various substances were derived. These include, for
example, the CHK1 inhibitors UCN-01 (7-hydroxy-staurosporine),
Go6976, SB-218078, ICP-1, XL844, and CEP-3891, which block the G2/M
checkpoint (FIG. 4). Furthermore, debromohymenialdisine and the
synthetic peptide TAT-S216A can inhibit CHK1 as well as a further
checkpoint protein, CHK2. It has to be assumed, that in the near
future even far more substances will be available, which inhibit
CHK1.
[0043] Among the CHK1 inhibitors available so far, UCN-01
(7-hydroxy-staurosporine) is the clinically best characterized
substance. UCN-01 has already passed through several clinical phase
I studies and is currently tested in phase II. The inhibition or
down regulation of CHK1 had a positive effect on the response of
cytostatics, like topoisomerase inhibitors, antimitotics and
antimetabolites, since due to the lack of CHK1, the toxicity of
these chemotherapeutic agents was potentiated especially in
aggressive tumors, which even following preceding conventional
treatment showed progressive growth. Furthermore, it could be
demonstrated that UCN-01 increases the sensitivity towards
radiation, whereby CHK1 inhibitors also have potential as
radio-sensitizers.
[0044] Due to the fundamental significance of CHK1 for the
maintenance of genomic integrity, such polymorphisms are suited to
generally predict disease risks or progression of diseases in tumor
diseases or to predict treatment responses/treatment failure or
undesired side-effects for all pharmaceuticals or
non-pharmacological treatments,
Verification of Polymorphisms in the Promoter of the CHK1 Gene
[0045] In the promoter region of CHK1, five polymorphisms are known
and can be found in generally accessible databases. With systematic
sequencing of human DNA samples, three polymorphisms were verified
and validated: -1143G>T (rs555752), -1400C>T (rs558351) and
-1453T>C (rs 1057733) (FIG. 5). For that, gene sequences of the
promoter area of CHK1 were amplified using PCR reaction and
sequenced with method according to Sanger. The person skilled in
the art is familiar with the methods required for that, e.g.
deriving primer pairs required for the PCR reaction and selecting
sequencing primers. In that, a new polymorphism was found, whereat
there is an insertion of one cytosine present (1454insC, no
database SNP identification present) at position 1454 in the
promoter region (FIG. 5). The numbering of these SNPs takes place
in a manner that nucleotide A of the start codon ATG is allocated
the number +1. Since according to the convention there is no number
0, the nucleotide located in front of the A of the start codon ATG
is allocated number -1.
[0046] The verification of these SNPs in terms of their use
according to the invention can be executed with any method the
person skilled in the art is familiar with, e.g. direct sequencing,
PCR with subsequent restriction analysis, reverse hybridization,
dot blot or slot blot methods, mass spectrometry, Taqman..RTM.. or
Light-Cycler..RTM.. technology, Pyrosequencing..RTM..,
Invader..RTM.. technology, Luminex methods, etc. Furthermore, these
gene polymorphisms can be defected simultaneously after multiplex
PCR and hybridization at one DNA chip.
[0047] The distribution of the -1143G>T, -1400C>T,
-1453T>C, and -1454insC polymorphisms, verification of
haplotypes and use of these genotypes were investigated for finding
further relevant polymorphisms and haplotypes.
[0048] For that, different DNA samples of Caucasians (n=205) were
genotyped. The result is shown in the following table:
TABLE-US-00001 SNP Genotypes -1143G > T GG: 136 GT: 65 TT: 4
-1400C > T CC: 44 CT: 107 TT: 54 -1453T > C TT: 102 TC: 85
CC: 18 -1454insC ----: 102 --C: 85 CC: 18
[0049] Beside Caucasians, the genotype distributions in Black
Africans were likewise investigated:
TABLE-US-00002 SNP Genotypes -1143G > T GG: 57 GT: 40 TT: 5
-1400C > T CC: 61 CT: 38 TT: 2 -1453T > C TT: 54 TC: 38 CC: 4
-1454insC ----: 54 --C: 38 CC: 4
[0050] The genotype distributions in Chinese are shown in the
following table:
TABLE-US-00003 SNP Genotypes -1143G > T GG: 72 GT: 22 TT: 4
-1400C > T CC: 37 CT: 46 TT: 16 -1453T > C TT: 30 TC: 41 CC:
15 -1454insC ----: 30 --C: 41 CC: 15
[0051] A comparison of the genotype distributions in most cases
resulted in significant differences between the ethnic groups. Such
differences in the genotype distributions in different ethnic
groups normally point out, that associated phenotypes were
significant for evolution and provided the carriers with a certain
advantage. It is known to the person skilled in the art that
ethnically different genotype distributions are a reference to the
fact that even today, certain genotypes and haplotypes are
associated with certain diseases or physiological and
pathophysiological modes of reaction or responses to treatment,
e.g. with pharmaceuticals.
[0052] In one-hundred sequenced DNA samples of healthy Caucasians,
further analyses showed a coupling imbalance between certain
polymorphisms. Coupling imbalance means the occurrence of allele
combinations (haplotypes), which statistically clearly occur more
frequently or less frequently together, than this was to be
expected in relation to their frequency. In that, it turned out
that polymorphisms 1453T>C and 1454insC linkup completely.
Polymorphisms -1143G>T and -1440C>T, on the other hand, do
not link up, and they only restrictedly couple with the two other
variants (FIGS. 6A and B). The quality of the coupling is marked
with the values D' and r.sup.2. In that, D'=1 and r.sup.2=1 are
considered a significant coupling. The closer both values are to 1,
the narrower is the coupling imbalance. The calculation of the
haplotypes, which can be constructed from those four polymorphisms,
resulted in five different allele combinations. No preferential
haplotype exists, which results from these promoter variants (FIG.
6C). In order to determine any possible combinations, the
verification of at least three of the four polymorphisms is
necessary.
[0053] One subject of the invention is that these new polymorphisms
can be used to detect and validate further relevant genomic genetic
modifications in CHK1 or neighboring genes, which for example are
in coupling imbalance with genotypes in the CHK1 gene. These may
also be genes, which are likewise located on chromosome 11, but far
away from the CHK1 gene. For that, the procedure is as follows:
[0054] 1. For certain phenotypes (cellular characteristics,
diseases, progression of diseases, drug responses, etc.), an
association with the polymorphisms 1453T>C, 1454insC,
-1143G>T and -1400C>T is first established, whereat these
associations can be established for each genotype individually or
using all permutations of the haplotypes. [0055] 2. For newly
detected genetic modifications in CHK1 or neighboring genes it is
investigated, whether already existing associations are enhanced or
weakened using the genotypes or haplotypes described above.
Functional Significance of the Promoter Polymorphisms in the CHK1
Gene
[0056] It was investigated, which functional changes are to be
allocated to the promoter polymorphisms in the CHK1 gene.
Perceivable here are, for example, a correlation to alternative
splicing, tissue-specific expression or an over-expression of the
CHK1 protein depending on genotypes or haplotypes, respectively, of
the CHK1 promoter. For that, it was first investigated using a
computer program, whether the nucleotide exchanges found can
influence the binding of transcription factors. Transcription
factors bind to specific consensus sequences and can increase or
reduce the promoter activity, so that an enhanced or reduced
transcription of the gene results and thus the expression level of
the coded protein is increased or reduced. As shown in FIG. 7, all
promoter SNPs mentioned above are located in a consensus sequence
for binding sites of different transcription factors (e.g. E74A,
CF2-II or bZIP910), the binding of which can be effected by the
polymorphisms. The occurrence of certain genotypes results in an
omission of these binding sites by the modification of their
consensus sequences. For experimental investigation of this effect,
a so-called EMSA (electrophoretic mobility shift assay) is
performed. In this test, short nucleic acid sections, which include
the respective polymorphism, are incubated with cell nucleus
extracts. Transcription factor proteins present in these extracts
now bind to the nucleic acid sections with different intensity. The
binding to the DNA is finally made visible on the X-ray film. In
that, an intensive band results from a strong bond. FIG. 8 shows
the result of this test with specific constructs, which either
contain the G- or the T-allele of the -1143G>T polymorphism. The
presence of the G-construct band proves binding of a transcription
factor to this region. The T-construct has no band, which shows
that no transcription factor binds to this allele. The weakening of
the band intensity by a specific oligonucleotide shows, that the
binding transcription factor is a specific binding. FIG. 9 shows
the result of this test with specific constructs, which either
contain the C- or the T-allele of the -1400C>T polymorphism.
Only the T-construct results in the binding of a transcription
factor, while the C-construct shows no specific band, therefore
also no transcription factor binds to this allele. The displacement
of the band by a specific oligonucleotide shows that the binding
transcription factor is a specific binding.
[0057] For functional verification of a regulatory activity of
these promoter regions, depending on certain genotypes, different
fragments of the promoter were cloned into the vector pSEAP to
quantify the regulatory activity using a so-called reporter assay
following the expression of the vector in HELA cells, a cervix
carcinoma cell line (FIG. 10). For that, the constructs are cloned
in front of a gene, which codes for secreted alkaline phosphatase
(SEAP). If the construct has a gene-regulating activity, the
transcription of the SEAP gene is increased and the increased
secretion of alkaline phosphatase into the cell culture medium is
measurable. As shown in FIG. 10, the constructs with the alleles of
the -1143G>T polymorphism have a significantly higher activity
than the constructs of the other polymorphisms (p=0.0005). The
reporter activity of the individual alleles of this SNP is likewise
different. The T-allele shows a higher activity (3.82.+-0.0.6) than
the G-allele (2.36.+-0.0.3).
[0058] Since only the -1143G>T polymorphism of the CHK1 gene
shows a reporter activity, next it was investigated, how the
regulation in vivo takes place, because reporter assays are an
artificial cell system. For that, the expression of CHK1 at the
mRNA level was investigated using real-time PCR in human
tissue.
[0059] For that, mRNA was obtained from human surgery tissue from
urinary bladder and colon surgeries and transcribed into cDNA using
reverse transcriptase. The person skilled in the art is familiar
with this method. Subsequently, the expression level was determined
using real-time PCR (Taqman method) and matched with the expression
level of the housekeeping gene .beta.-actin. The results are shown
in FIGS. 11 and 12. It is shown in section 11 A, that the
GG-genotype of the -1143G>T SNP has a significantly higher mRNA
expression than the TT-genotype. The values of the heterozygote
genotype are located in-between, which indicates a gene dosage
effect. FIG. 12A, too, shows an increased mRNA expression for the
GG-genotype. The two other polymorphisms also show an
allele-dependent difference in the gene expression. As FIGS. 11B
and 12B illustrate, C-allele carriers of the -1400C>T
polymorphism have clearly more CHK1 mRNA than carriers of the
TT-genotype. The real-time PCR results for the SNPs -1453T>C and
1454insC are shown in FIGS. 11C and 12C. Carriers of the T-allele,
which do not have an additional insertion, show a clearly lower
mRNA expression.
[0060] Using this method, it was verified that there are genetic
modifications in the CHK1 gene, which effect a change of expression
of CHK1 in the carcinoma tissue. This can be the promoter
polymorphisms described above or polymorphisms in coupling
imbalance with these SNPs. One component of the invention described
here is thus also to quantify the expression of CHK1, to associate
it with known polymorphisms of CHK1 and to discover and validate
new, even better suitable polymorphisms.
[0061] The findings of a genotype-dependent expression of CHK1 in
human carcinoma tissue shown here are exceedingly significant,
since a lower activity of CHK1 can cause microsatellite and
chromosomal instability, which both are included in the
characteristics of genomic instability and thus favor oncogenesis
and have a negative effect on tumor progression (Durkin et. al.,
Depletion of CHK1, but not CHK2, induces chromosomal instability
and breaks at common fragile sites. 2007, Oncogene 25:4381-8;
Furlan et al, Genetic progression in sporadic endometrial and
gastrointestinal cancers with high microsatellite instability.
2002, J. Pathol. 197:603-9). Furthermore, this genotype-dependent
gene expression of CHK1 can also affect the response to treatment
with CHK1 inhibitors. It has to be expected that a low gene
expression predisposed by a certain genotype, e.g. the TT-genotype
of the -1143G>T polymorphism, responds stronger to CHK1
inhibitors than other genotypes. Thus, genetic modifications in the
CHK1 gene can be used to predict the response to a cancer treatment
to discriminate, for example, responder versus non-responder. These
genetic modifications can also be used for dosage finding or for
predicting the occurrence of undesired drug effects, respectively.
Such cancer treatments can take place as drug treatments in the
broadest sense, i.e. by supplying substances into the body, or
these cancer therapeutic agents can have a physical effect
(radiation, warmth, cold).
[0062] We thus expect an influence on the progression of diseases,
in particular in case of tumor diseases, as well as a changed
response to substances, which influence the regulation cascade of
CHK1, or substances, which directly inhibit CHK1.
Use of Genetic Modifications in CHK1 for Predicting Disease Risks
and Progression of Diseases
[0063] Due to the key function of checkpoint kinase 1 for the
regulation of the cell cycle, it is an essential component of the
invention that using genetic modifications in CHK1, disease risks
and progression of diseases can be generally predicted.
[0064] The multistep development of cancer reflects the
accumulation of genetic modifications, which result in the
transformation of normal cells into cancer cells and of normal
tissue to benign and possibly malignant, invasive tumors. The
accumulation of alterations in tumor suppressor genes and
proto-oncogenes accelerates tumorgenesis and can influence radio-as
well as chemotherapy. However, it becomes more and more clear that
disturbed DNA repair mechanisms as well as checkpoints are the
reason for the increased genomic instability of tumors (Hoeijmaker
J. H., Genome maintenance mechanisms for preventing cancer. 2001
Nature, 411:366-74; Khanna et al., DNA double-strand breaks:
signaling, repair and the cancer connection, 2001, Nat. Gent.
27:247-54). Since checkpoints play a central role in the
maintenance of genomic integrity, it has to be directly expected
that the progression of varied and completely different tumor
diseases with a genetically determined, reduced activatability is
influenced by checkpoints. That means that with changes in the
expression of proteins, which are expressed in all human body cells
and protect the cell from DNA damage, cell functions are regulated,
which decisively influence or at least modulate all physiological
and pathophysiological processes. Besides that, responses to
pharmaceuticals are also influenced in a particular manner. This
affects desired, but also undesired drug effects.
[0065] It was repeatedly postulated in the scientific literature
that functional modifications of checkpoint proteins have a
sustained influence on varied diseases or on the progression of
varied diseases, respectively, since these are phylogenetically
highly conserved pathways. Such genetic modifications can be
structure-modifying mutations in the checkpoint proteins, which,
for example, reduce the activation of the proteins by
phosphorylation or the substrate selectivity. Furthermore, the
expression level can be modified, whereby the initiation of the
subsequent reaction cascades, which e.g. induce apoptosis, is
reduced, or splicing variants with a changed function can occur.
All these modifications are considered a genetic predisposition for
cancer.
[0066] The following results from the examples stated: [0067] 1.
Genetic modifications in genes coding for ubiquitarily expressed
proteins influence varied diseases or cause varied disease risks,
respectively. [0068] 2. Checkpoint proteins are part of the complex
network for maintaining the genomic integrity in the human
body.
[0069] Diseases accompanied by a genetic modification in CHK1 and
determined, for example, by a changed level of expression of the
CHK1 protein, are benign neoplasias of any tissue of origin and
malignant neoplasias of any tissue of origin.
[0070] Such neoplasias comprise, for example, tumor diseases like
tumors of the urogenital tract: [0071] urinary bladder carcinoma,
kidney cell carcinoma, prostate carcinoma and seminoma; [0072]
tumors of the female genitals: mammary carcinoma, corpus carcinoma,
ovarian carcinoma, cervix carcinoma; [0073] tumors of the
gastrointestinal tract: oral cavity carcinoma, esophagus carcinoma,
stomach carcinoma, liver carcinoma, bile duct carcinoma, pancreas
carcinoma, colon carcinoma, rectum carcinoma; [0074] tumors of the
respiratory tract: larynx carcinoma, bronchial carcinoma; [0075]
tumors of the skin: malignant melanoma, basalioma, T-cell lymphoma;
[0076] tumor diseases of the hematopoietic system: Hodgkin and
non-Hodgkin lymphomas, acute and chronic leukemias, plasmocytoma;
[0077] tumor diseases of the brain or the nerve tissue,
respectively: glioblastoma, neuroblastoma, medulloblastoma,
meningeal sarcoma, astrocytoma; [0078] soft tissue tumors: for
example sarcomas and head-neck tumors.
Use of Genetic Modifications in the CHK1 Gene for Predicting
Progression of Diseases and Response to Treatment
[0079] Since the essential functions of CHK1 are known, genetic
modifications in the CHK1 gene can increase the risk for tumor
diseases or influence the progression of diseases. It is generally
impossible to investigate all human tumor diseases and their
progression. However, we have demonstrated this here by way of
example for five different carcinomas: urinary bladder carcinoma,
colorectal carcinoma, chronic lymphatic leukemia, malignant
melanoma and cholangiocellular carcinoma. These data clearly prove
the usability of genetic modifications in the CHK1 gene for the
purpose described here. These diseases are a priori not associated
at all.
The Significance of Checkpoint Kinase 1 for Chemotherapeutic Agents
and Radiation
[0080] Genetic instability is a characteristic of all tumors and
also plays a role in oncogenesis, progression and the development
of resistances against pharmaceuticals (Hartwell L., Defects in a
cell cycle checkpoint may be responsible for the genomic
instability of cancer cells. 1992, Cell 71:543-6). Most tumor cells
have a defective G1-S checkpoint, which gives them a survival
advantage. This defect, however, causes the tumor cells to depend
on the G2 checkpoint very much, if stimuli are present, which
threaten the genomic integrity. The inhibition of the G2 checkpoint
by administration of DNA-damaging substances can result in a
so-called "mitotic catastrophe", i.e. cell death. CHK1 is
responsible for the maintenance of the G2 checkpoint, if DNA damage
occurs. Thus, the inhibition of CHK1 by the omission of the G2
checkpoint offers the possibility, that DNA damages and
modifications caused by genotoxic substances and radiation can
accumulate and that the tumor cell dies of it. This, however,
requires that the inhibition of CHK1 does not promote somatic cell
death, which would mean general cellular toxicity and little tumor
specificity. The use of CHK1 siRNA in vitro has shown that CHK1
inhibition has only little influence on the cell cycle and the
survival of the cell, as long as no DNA-damaging substances are
present. Upon using these substances, however, the G2 checkpoint is
securely inhibited and the apoptosis increased. Since the discovery
and development of the CHK1 inhibitors, it could be verified, that
by using them, the effect of chemo- and radio-therapeutic measures
could be increased.
[0081] If genetic modifications occur in CHK1, which influence the
gene expression, then this has an impact on the effectiveness of
these CHK1 inhibitors. It has to be expected, that patients with a
genotype-dependent lower CHK1 expression respond better to the
inhibitors than patients with a higher CHK1 expression.
Additionally, it means that the combined treatment of CHK1
inhibitors with chemo- and immunotherapeutic agents and/or
radiation can be influenced. From this results the possibility of
individual diagnostics of the general responsiveness to these
cancer therapeutic agents and therapy measures as well as an
individual prediction of the risk of undesired effects by these
therapies.
[0082] Genotype-Dependent Diagnostics of the Expression of CHK1
Enables General Diagnostics of the Effectiveness of
Chemotherapeutic Agents and Radiation, Their Optimal Dosage and the
Occurrence of Side-Effects.
[0083] Chemotherapy uses such substances, which exert their
damaging effect on certain cancer cells as targeted as possible and
kill them or inhibit them in their growth. A certain cytostatics
dosage can always only kill a certain portion of the target cells,
which remains the same with proceeding treatment. Therefore,
chemotherapy must not be reduced within the course of the
treatment, even if the tumor is not even detectable anymore. It
rather has to be assumed, that with a weak treatment, especially
the resistant tumor cell clones are selected. Chemotherapy is
applied in fast succession, and almost always two or more
cytostatics are combined to increase effectiveness. The therapy
thus also causes side-effects, which are classified according to
the common toxicity criteria. These criteria include: number of
leukocytes and thrombocytes, sickness, vomiting, diarrhea and
stomatitis.
[0084] Radiotherapy means the use of ionizing high-energy radiation
to heal malignant tumor diseases. Such malignant tumors are often
also treated in combination with chemo- and radiotherapy. A
multitude of tumor diseases can thus also be healed in advanced
stages. In order to keep the side-effects low, the radiation is
divided into many daily single doses and administered over several
weeks. Still, side-effects like redness, sickness, diarrhea, or
hair loss occur, depending on the dosage, penetration depth and
number of single doses. The invention is now based on the fact that
a method has been developed, which is generally suited for
diagnostics of the activatability of checkpoint kinase 1 and,
associated with it, the G2 checkpoint. For that, one or more
polymorphisms in the CHK1 gene are investigated. With high
expression, there predictably is an increased activatability of the
G2 checkpoint and thus sufficient time to perform repair mechanisms
in the DNA after damaging of the same. With low CHK1 expression,
the G2 checkpoint is less activatable and DNA damage is not or not
sufficiently repaired.
[0085] In order to verify by way of experiments, that there is a
connection between CHK1 polymorphisms and the activity of the G2
checkpoint, and thus also with DNA repair mechanisms, lymphocytes
of healthy subjects were cultivated and stimulated for cleavage.
After 72 hours, these cells were radiated with a dose of 1 Gy and
subsequently arrested in the M-phase by the mitosis inhibitor
colchicine. From these cells, chromosomes were prepared using
methods the person skilled in the art is familiar with and,
depending on the -1143G>T polymorphism, evaluated for damage by
radiation in 50 metaphases each. In this manner, only those cells
were included into the evaluation, which at the time of radiation
were in the G2-phase and until the chromosome preparation reached
the M-phase. Thus, with this method, the activity of the G2/M
checkpoint can be assessed. As shown in FIG. 13, the average number
of chromosome breaks per metaphase for the GG-genotype was 2.7, for
the GT-genotype 4.1, and for the TT-genotype 4.9 (p=0.031). Thus it
could be demonstrated, that with the GG-genotype, which forms the
most mRNA, the G2/M checkpoint is the most active and the
respective DNA repair mechanisms could work best. The weak
checkpoint for the TT-genotype, on the other hand, allows only few
repair mechanisms to work, and more damage can accumulate.
[0086] Thus, a determination of the presence of polymorphisms in
CHK1 allows for diagnostics of the effectiveness and undesired
effects of drugs, in particular cytostatics, as well as other forms
of treatment, which damage the genetic make-up of the tumor cells,
e.g. radiation. Besides that, such polymorphisms in CHK1 can be
used to diagnose the effects of pharmaceuticals used in combination
with a CHK1 inhibitor. Additionally, the diagnostics of the allele
or haplotype status in CHK1 can be used to determine the
individually optimal and tolerated dosage of drugs.
[0087] For diagnostics of an increased or reduced activatability of
checkpoint kinase 1 and the G2 checkpoint serves in particular the
verification of the CHK1 promoter polymorphisms described here,
either alone or in any perceivable combinations.
[0088] Besides that, any further genetic modifications in CHK1 can
be used for diagnostics, which are in a coupling imbalance to these
polymorphisms and/or additionally promote or inhibit the
alternative splicing process or the expression.
[0089] These genetic modifications can be verified with the methods
described above, which the person skilled in the art is familiar
with. Furthermore, these gene polymorphisms can be simultaneously
detected after multiplex PCR and hybridization to a DNA chip.
Besides that, other methods may also be used for diagnostics, which
allow for the direct verification of the expression level of CHK1
or splicing variants of CHK1.
[0090] The method stated is particularly suited for diagnostics of
the effect of substances, which damage the DNA of the tumor cells.
These substances include oxaliplatin, 5-fluorouracil, folinic acid,
irinotecan, capecitabine and cisplatin, whereat, the list could be
randomly extended. Besides that, the effects of immunotherapeutic
agents (e.g. interferons or interleukins) or inhibitors of signal
transduction in tumor cells, respectively, can be predicted.
[0091] Furthermore predicted can be the effects of
radio-therapeutic measures, like gamma radiation, X-ray radiation,
electrons, neutrons, protons and carbon ions, whereat the list
could be randomly extended. In the broader sense, radiation therapy
also implies the medical application of microwaves and heat rays,
light and UV therapy as well as the treatment with ultrasound
radiation.
[0092] A proof for the general usability of the CHK1 polymorphisms
for predicting drug effects results from the genotypes observed and
their dependent progression of diseases in the examples stated
above. Patients, the tumors of which were intensively treated with
chemo- or radiotherapy, respectively, show a more favorable
progression of the disease, if, genotype-dependent, they show less
CHK1 expression. With the lower quantify of CHK1 protein, the G2
checkpoint is less activatable and cytostatics or radiation,
respectively, can have a more effective effect. On the other hand,
the disease progresses more favorable in tumor patients, who
received other therapeutic measures, if they, depending on the
genotype, show more CHK1 expression. With the higher quantity of
CHK1 protein, the G2 checkpoint is more active and can thus
contribute to DNA repair mechanisms and limit the genomic
instability of the tumor.
[0093] A substantial subject of the invention is the provision of
diagnostically relevant genetic modifications in the CHK1 gene as
prognosis factor for all human tumor diseases. Naturally, not all
tumor diseases can be described in that. The principle will
therefore be further explained in selected examples, which
demonstrate general usability without restricting the scope of
patent to the exemplary embodiments.
EXAMPLES
Example 1
Urinary Bladder Carcinoma
[0094] Bladder cancer is a malignant tumor of the mucous membrane
of the urinary bladder and most frequently occurs between the age
of 80 and 70. Men are affected by it three times as often as women.
In men, bladder cancer is the third most frequent type of cancer
after lung and prostate cancer. Bladder cancer can be caused by
external influences. The risk factors include smoking, permanent
strain on the organism by chemicals, like for example colorants or
analgesic misuse. In many patients, the examinations show that it
is a superficial tumor. This can be removed by surgery using a
cystoscope. More than 70% of the patients treated because of a
superficial bladder carcinoma show tumor rescrudescence in
progression. In that, in more than half the patients,
rescrudescence tumors with non-muscle-invasive disease occur. These
can be curatively treated or controlled, respectively, by
transurethral resection. It is therefore important to detect these
lesions early and to provide regular and closely monitored
aftercare for the patients. At regular intervals, excretion
urograms serve to control possible tumor manifestations in the
renal pelvis and ureters. So far, there are hardly any valid
markers predictive for the further progression of the disease.
Therefore, currently the classic factors like penetration depth,
degree of differentiation, formation of metastases, involvement of
the lymph nodes, etc, are used for prognosis. Genetic markers for
probability of survival and therapy response would substantially
improve the care for patients with urinary bladder carcinoma. It is
the object of the invention to demonstrate that the use of genetic
modifications in CHK1 is suited to predict the further progression
of the disease.
[0095] FIG. 14A shows the survival depending on the -1143G>T
SNP. In that, the risk of dying in patients with the TT-genotype is
increased by approx. the 2-fold (p=0.042). The median time until
death is only 48 months for carriers of the TT-genotype, while for
G-allele carriers, no median time can be stated, since up to the
end of the study, less than half of these patients died. A similar
relation is found, if only the survival of the older patients is
investigated (FIG. 14B). The progression of the curve is
significantly different for the genotypes (p=0.004), whereat the
G-allele carriers are allocated the more favorable progression. The
median time of survival is 87 months (GG-genotype) or 50 months
(GT-genotype), respectively, in patients with the G-allele; for the
homozygous T-allele carriers, on the other hand, only 33
months.
Example 2
Colorectal Carcinoma
[0096] The colorectal carcinoma is the most frequent type of tumor
in the gastrointestinal tract and one of the main causes for
tumor-related death worldwide (12-15% of the total cancer
mortality). In Germany, the incidence is about 51,000 new cases per
year. The average 5-year survival rate after tumor resection is
only approx. 50%. Eating habits, cancer-promoting metabolites,
exogenous carcinogens and certain predisposing diseases are
included in the risk factors for the formation of a colorectal
carcinoma. The standard method for predicting the progression of
the disease is the TNM or UICC stage system, respectively. Patients
with UICC stages III or IV generally have a worse prognosis than
patients with UICC stages I or II. An adjuvant chemotherapy is
performed for metastasized colorectal carcinomas (UICC stages III
and IV) and can enhance the local effect of radiation therapy. A
majority of these patients develops recrudescences or metastases,
which makes intensive aftercare necessary. Thus, it is important to
identify and establish molecular markers, which can predict the
further progression of the disease. A further component of the
invention consists in using genetic modifications in CHK1 to
predict the further progression of the colorectal carcinoma.
[0097] FIG. 15A shows a significant difference in regards to
survival depending on the -1143G>T polymorphism (p=0.026).
Patients with the GG-genotype in median survive 26 months, whereas
in the T-allele carriers, less than half the patients died during
the observation period. Additionally, a gene dosage effect is
detectable, since carriers of the heterozygous genotype have a
higher risk than the TT-genotype and a lower risk than the
GG-genotype. FIG. 15B shows, depending on the -1400C>T SNP, the
survival of the patients with tumor localization in the colon.
Carriers of the TT-genotype survive significantly longer than
carriers of the C-allele (p=0.033). While the median survival for
the TT-genotype is 70 months, it is only 34 months for the
CT-genotype and only 26 months for the CC-genotype. Depending on
the genotypes of the polymorphisms 1453T>C and 1454insC, a
significant difference in regards to survival can likewise be
detected (FIG. 15C). Patients, who have the TT-genotype at position
1453 and have no insertion at position 1454 survive longer than
patients, who have the 1454C allele and the insertion (p=0.007).
The median survival is 70 months for the TT-genotype without
insertion compared to 21 and 15 months for the other genotypes.
[0098] Above all interesting here is the observation, that patients
with a low CHK1 mRNA expression, since they are carriers of the
TT-genotype, survive the longest. Since patients with UICC stages
III and IV, who constitute the biggest portion of this collective,
received intensive treatment against their tumors, the circumstance
to survive longer, if one has little CHK1 protein, is of high
significance. Reduced CHK1 may be considered a predisposition for
tumor diseases; however, it also makes the tumor cells more
susceptible to therapeutic measures. This is confirmed by the
curves of survival shown here.
Example 3
Chronic Lymphatic Leukemia
[0099] Chronic lymphatic leukemia (CLL) is a chronic form of
leukemia. Characteristic for the disease is a high number of
abnormal lymphocytes. A total of 30 percent of all leukemic
diseases are chronic lymphatic leukemias. The median disease age is
65 years. A CLL can be benign for up to 20 years, i.e. the patients
show no symptoms except for enlarged lymph nodes, tiredness and
lack of appetite. The treatment only starts, if the number of
lymphocytes highly increases, the portion of erythrocytes and
thrombocytes decreases, or other complications occur. An early
treatment has no influence on the progression of the disease. The
most important therapeutic measure is chemotherapy. The further the
disease has progressed, the higher are the disturbances of health
by the modification of the organ system. Depending on the Binet
stage of the disease, the doctor can estimate the prognosis. The
stage of a CLL is, among others, characterized by how many
lymphocytes are present in blood and bone marrow, how large spleen
and liver are and whether an anemia is present. A CLL results in
modifications in the immune system, so that humans suffering from
this disease have a higher risk of developing other types of
cancer. At the same Binet stage, however, patients show a
completely different progression of the disease. It is an object of
the invention to demonstrate that genetic modifications in the CHK1
gene are suited to predict the progression of CLL.
[0100] For that, patients with CLL were genotyped in regards to the
described genetic modifications in CHK1 and the gene status was
associated with survival. FIG. 16 shows the survival depending on
the -1143G>T genotype. Patients, who are carriers of the
T-allele, survive longer than patients, who are homozygous GG. For
the GG-genotype, the median survival is 146 months; on the other
hand, however, less than half the patients carrying the T-allele
died during the observation period. Here, too, if shows that under
intensive treatment, a genotype, which results in a low CHK1
expression, is most favorable for survival.
Example 4
Malignant Melanoma
[0101] The malignant melanoma is a malignant abnormality of the
melanocytes (pigment cells of the skin), which is why it is also
called "black skin cancer". This type of tumor tends to spread
metastases via the blood and lymph streams very early. The
incidence of malignant melanoma is increasing, it doubles every 15
years. Particularly at risk are persons with low pigmentation. The
risk factors include, above all, intensive sun exposure and a
sunburn anamnesis of 5 or more episodes in the youth. In the
Western world, malignant melanoma is the most frequent cancer in
women between the age of 20 and 40. Criteria for prognosis and
therapy are provided by the stages of the TNM classification, the
tumor thickness according to Breslow, the penetration depth
according to Clark, the differentiation by subtypes and
localization. With early diagnosis and treatment, the prognosis
still is good. With late diagnosed melanomas with lymph node
metastases, the 5-year survival rate lies at approx. 30%; if remote
metastases are present already, it is only 0-5%. There are no
molecular markers for the progression of this disease and responses
to treatment. The identification of such markers would highly
improve the pre- and aftercare of the patients.
[0102] FIG. 17A shows the genotype-dependent difference of the
-1400C>T SNP from the time of initial diagnosis and initial
treatment up to the time of requirement of further treatment.
Patients carrying at least one C-allele, start continuative
treatment significantly later than patients with the TT-genotype
(p=0.033). For the heterozygous or homozygous, respectively,
C-allele carrier, treatment becomes necessary in median after 71 or
57 months, respectively, and for the TT-genotype after 45 months
already. Contrary to the curve progressions discussed so far, here
it shows that without medical measures, those genotypes are
advantaged, which have a high CHK1 expression and thus very well
working checkpoints. FIG. 17B represents the survival of all
patients. Here, too, it can be recognized that patients carrying at
least one C-allele survive longer than patients with the
TT-genotype (p=0.013). The median survival for the TT-genotype is
only 69 months, for the CC-genotype, on the other hand, 101 months,
and the heterozygous genotype does not even reach the median
survival time during the observation period. For this type of
tumor, the treatment of the primary tumor is a completely surgical
one. After the excision of the tumor, further treatment is
considered. In 76.5% of the patients, there was no further
treatment, 11.2% received an immune therapy with
interferon-.alpha., 3.4% a hyper-thermal extremities perfusion, in
5.4%, a re-resection was necessary and the remaining 3.5% were
subjected to other therapeutic measures. Since only 1.6% of the
patients received a chemo- and/or radiotherapy, which would be
favored by a low CHK1 expression, here those genotypes are
advantaged, which have a strong CHK1 expression (real-time PCR
results from FIG. 12B: CC- and CT-genotype 0.009.+-.0.002,
TT-genotype 0.004.+-0.0.002, p=0.049) with a strongly working
checkpoint kinase 1.
Example 5
Cholangiocellular Carcinoma
[0103] The cholangiocellular carcinoma (CCC) is a malignant tumor
of the bile ducts of the liver. Compared to Asia and Africa, where
it represents the most frequent type of tumor with 20-30% of the
malignant tumors, it is relatively seldom in Central Europe (<1%
of all malignant diseases). The risk factors include colitis
ulcerosa, chronic bile duct inflammations and viral hepatitides.
The curative treatment of a CCC is a partial liver resection or a
total hepatectomy with liver transplantation. The recrudescence
rate is very high. The prognosis for CCC is therefore very
unfavorable, in particular in patients with non-resectable tumors,
who have a 5-year survival rate of <10%. The median survival
period in patients with non-resectable CCC is 6 to 10 months. So
far, no molecular markers are known for the progression of this
disease, however, would substantially improve the care for patients
with CCC. It is therefore a component of the invention to
demonstrate that the use of genetic modifications in CHK1 is suited
to predict the further progression of the disease.
[0104] FIG. 18 shows the survival of CCC patients. Patients with
the CC- and CT-genotypes survive significantly longer than patients
with the TT-genotype (p=0.036). The median survival of the
TT-genotype is 5 months, that of the other genotypes 9 months.
Since the probability of survival of the CCC patients is only very
low, the time for an intensive treatment after the surgery is very
short. Due to the mostly pre-damaged cirrhotic liver, the
indication for radiotherapy is given only rarely; additionally, the
liver is extremely radiosensitive. Thus, until achieving a
tumoricidal dose, the liver might already be destroyed. So far,
systemic chemotherapy with numerous cytostatics and combinations
has not shown lasting effectiveness. The remission rates are 20 to
30%, the median duration of remission is 4 to 6 months.
Alternatively, chemoembolizations or targeted intratumoral alcohol
injections can be applied. Since for CCC neither radio-nor
chemotherapy can be used to a sufficient extent, which for a low
CHK1 level would show advantages, for this type of tumor, it is the
genotypes showing an increased CHK1 expression, which show longer
survival.
Example 6
Mammary Carcinoma
[0105] The mammary carcinoma is the most frequent tumor of the
female population in Europe and the USA. It affects 7-10% of all
women and accounts for 25% of the total female cancer mortality.
The etiology of the mammary carcinoma is still unknown, however,
risk factors have been described, like a family disposition,
radiation exposure or estrogen influence. In most patients, the
examinations show that it is an invasive carcinoma. With a few
exceptions, any operable mammary carcinoma even with verified
remote metastatization is treated surgically. The differently
radical initial surgical treatment results in variations of the
locoregional recrudescence rate, but not the long-term chance for
survival. Furthermore, recrudescences or remote metastases quite
often can become manifest after 5 or even 10 years only. It is
therefore important to detect these lesions early and to closely
monitor the patients in aftercare. Aftercare examinations are
performed at regular intervals, with an interim suspicion even up
to 10 years after surgery. So far, there are hardly any valid
markers predictive for the further progression of the disease.
Therefore, currently the classic factors like tumor size,
metastatization, involvement of lymph nodes, hormone receptor
status, etc. are used for prognosis. Genetic markers for
probability of survival and therapy responses would substantially
improve the care for patients with mammary carcinoma. It is an
object of the invention to demonstrate that the use of genetic
modifications in CHK1 is suited to predict the further progression
of the disease.
[0106] FIG. 19 shows a significant difference in regards to the
survival depending on the -1143G>T polymorphism (p=0.017).
Patients with the GG-genotype survived in median 87 months, whereas
for the patients with the GT-genotype the median survival time is
101 months. Contrary to that, in homozygous T-allele carriers, less
than half the patients died during the observation period.
[0107] Here, above ail the observation is interesting again, that
patients with a low CHK1-mRNA expression, since they are carriers
of the TT-genotype, survive longest. Besides a possible hormone
therapy, patients with mammary carcinoma in most cases receive an
adjuvant radio- and/or chemotherapy. That means that a reduced CHK1
protein quantity in association with such a treatment strategy
results in longer survival of the patients.
Other Diseases
[0108] While the examples represented here exclusively relate to
cancer diseases, if still has to foe emphasized that proliferation
processes, apoptosis and cellular modification occur with all human
diseases and that thus DNA repair mechanisms and DNA checkpoints
play an important role in these processes. For example, a heart
attack develops on the basis of vascular changes of the coronary
arteries, in which proliferative processes play an important role.
The recovery of the myocardium, too, requires such modification
processes after the infarct. The same applies to the brain after an
ischemic infarct. Insofar, one or more of the genetic modifications
described here can thus be used to predict the progression of
cardiovascular diseases. For infectious diseases, like e.g. with
the hepatitis virus, such proliferation processes take place as
well, which, for example, can result in cirrhosis in the liver.
Insofar, one or more of the genetic modifications described here
can thus be used to predict the progression of infectious diseases.
For neurodegenerative diseases, e.g. Alzheimer disease or multiple
sclerosis, too, growth and cellular modification processes play an
important role.
Sequence CWU 1
1
11350DNAHomo sapiens 1aaaaagaccg ggctgaagta aagcattgtt ttggagctgg
ttcacagaaa aaaggcaaaa 60ctggttatcc tgacttcaag ctccaacata aactgctcgc
tttctccggg aaacttgccc 120cgccacacac acttgactgc gtggccagtt
ctttcgaagc ctctcgctcc caacacggag 180ttcctcccat ttcttcacag
agtcctgtcc ggtggcctca cgcaggtggc ggtgcagcct 240ttcaggccca
gagcggccag gagcgaagcc cgcagccccg cctggaagcg cagcgcggtc
300ggtcgcgcgc ccctgaggct tggaggcctg ggcttccccc agcagcgctc 350
* * * * *