molecular pathogenesis of CRC approch to molecular diagnosis of CRC

2. Outline of Presentation
 Introduction
 Etiology
 Types of CRC
 Molecular pathogenesis of CRC
 Molecular diagnosis for colorectal cancer

3.  CRC accounts for about 9 percent of
all cancer deaths; ranks third in both
incidence and cause of cancer death
in both men and women.
 Approximately 782,000 new cases are
diagnosed worldwide each year, of
which 70% originate in the colon and
the rest in the rectum

4. Etiology
The only certain way to avoid cancer is not to be born, as to
live is to incur risk.
Genetic mutations
 inherited
 can be transmitted from parent to offspring
 occurs at or before fertilization
 Acquired
 occurs spontaneously in the sperm, ovum, or zygote -
future progeny may inherit such mutation
 somatic mutation - during the growth and/or
development
 Common in CRC (65%)

5. Types of CRC
 Sporadic = 70% of cases
 there is no family history, common above age of 50
 Inherited = 1 – 6% of cases
 Hereditary NonPolyposis CRC (HNPCC, Lynch syndrome)
 Multiple polyps CRC
 familial adenomatous polyposis (FAP)
 hamartomatous polyposis syndromes (e.g. Peutz-Jeghers, juvenile polyposis)
 MUTYH-associated polyposis (MAP)
 Familial CRC = 25% of cases
 have a family history of CRC, but the pattern is not consistent and the risk
is not as high as with the inherited syndromes.
 single affected first-degree relative = 1.7 ↑ed risk
 two affected first-degree relatives or if Dx before age 55 = Further ↑ risk

6. The adenoma-carcinoma sequence
 Most CRCs arise from adenomas
(adenomatous polyps) which are formed
when normal mechanisms regulating
epithelial renewal are disrupted.
 the accumulation of multiple germ-line
or somatic mutations determines the
behavior of a tumor
 clonal nature of tumors
 cancers result from the stepwise
accumulation of multiple somatic
mutations. Therefore, many CRCs
remain asymptomatic for years before
diagnosis.

7.  Comprehensive exome sequencing
has revealed that individual CRCs
harbor an average of 76 gene
mutations and the mutated genes in
the 2 tumors overlap to only a small
extent; a few genes such as APC are
mutated at high frequency, whereas a
much larger number of genes are
mutated at relatively low frequency.

8. MOLECULAR PATHOGENESIS
/Molecular tumorigenesis/
CRC can arise in more than one molecular
pathways.
1. Chromosomal instability (CIN),
2. mismatch repair pathway
3. epigenetic gene silencing

9. 1. The chromosomal instability (CIN/APC)
pathway
 encompasses 80% to 85% of all CRC and adenoma
 Result in abnormal karyotypes, gross chromosomal
abnormalities, such as aneuploidy, chromosome
rearrangement, oncogene activation and loss of
heterozygosity of tumor suppressor genes.
 Results "gain of function" mutations which may result in
 loss or mutation of tumor suppressor genes such as APC, TP53
 activation of oncogenes such as KRAS
 apoptotic pathways
 CRC caused by CIN usually have poor prognosis

10.  In 90% of CRCs inactivation of the Wnt signaling pathway
/APC or β-catenin gene/; usually by mutation of one copy
of the APC gene (70%).
 a tumor suppressor adenomatous polyposis coli gene
mutation + (allelic deletion or an additional mutation →
inactivation of the other allele) → development of dysplasia
in aberrant crypt foci and early adenomas → accumulation
of additional genetic mutations (KRAS, DCC, p53 and
others) → tumor progression

11. kRAS oncogene
 encode proteins that regulate cellular signal transduction of
extracellular growth signals(e.g., epidermal growth factors) to the
nucleus
 point mutations resulting in a constitutively active GTP-bound protein
and a continuous growth stimulus.
 The activation of RAS genes can promote cell survival and suppress
apoptosis
 RAS mutations are found in up to 50% of sporadic CRCs and 50% of
colonic adenomas larger than 1 cm; they are rarely seen in smaller
adenomas → acquired during later adenoma progression
 The presence of a RAS mutation in CRC is significantly associated
with the absence of response to agents targeting the epidermal growth
factor receptor (EGFR) such as cetuximab and panitumumab.

12.  Causes mutation of serine/threonine protein
kinase which is involved with that acts as a
downstream effector of the KRAS gene.
 substitution of valine forglutamate
 Not detected in any LS/HNPCC tumors
 occurs in approximately 12% of all CRC,
mutually exclusive of KRAS mutation
 a prognostic and predictive marker to predict
resistance to anti-EGFR therapy
BRAF gene mutation

13. Role and Expression Patternof Epidermal GrowthFactor
Receptor in Colon Cancer
• EGFR is expressed in normal
colon epithelium and in 80-
100%of colorectal cancers
(by IHC)
• Up to 40%response rate to
anti-EGFR in wild type tumors
• BRAF, NRAS, and PIK3CA exon
20 mutations are significantly
associated with a low
response rate

14. P53
 DNA damage → ↑ p53 protein levels intracellular → up
regulation of cyclin dependent cell cycle inhibitor
p21Waf1/Cip1→ cell cycle arrest → cells undergo DNA
repair
 if the cell is unable to repair the DNA injury, p53 leads
to an increase in the levels of the proapoptotic protein
BAX and the cell undergoes apoptosis.
 p53 may also promote apoptosis by baseline
suppression of the inhibitor of apoptosis (IAP)

15.  Somatic mutations in both alleles are present in 80 percent
of sporadic CRCs, and a single germ-line mutation in this
gene is responsible for FAP
 Loss of heterozygosity (LOH) can be defined as the
manifestation that results from the loss of large genomic
regions.
 Lack of amplification of a particular segment of DNA can be
identified by PCR.
 Amplification of two to ten segments of interest is performed.
Loss of heterozygosity (LOH)

16. DCC (deleted in colorectal cancer)
 Tumor suppressor genes, affected as a result of 18q
mutations
 DCC is an adhesion molecule of the immunoglobulin
family, responsible for homotypic binding between cells.
 DCC protein loss can be examined by immunohistochemistry.

17. 2. mismatch repair pathway
/The mutator phenotype /
 accounts for 15% of CRC
 Mismatch repair corrects errors made when DNA is
copied. For example, a C could be inserted opposite an
A, or the polymerase could slip or stutter and insert two
to five extra unpaired bases.
 If a mismatch or small loop is found, endonuclease (MSH2
protein recognizes, MSH6 or MSH3) cuts the strand bearing
the mutation and exonuclease (MLH1 and PMS2) then
digests this strand.
 Finally it will be filled in by normal cellular enzymes.

18.  microsatellites are short,
sequences of 1 to 6 nucleotide base
pairs which are repeated dozens to
hundred times throughout the
genome.
 aka simple sequences or short tandem
repeats
 are a ubiquitous component of the
genome of higher organisms.
 The most common microsatellite in
humans is a dinucleotide repeat of
the nucleotides C and A, which
occurs tens of thousands of times
across the genome.
 microsatellite loci with many
repeats are rarely detected in the
genome
Microsatellites

19. Microsatellites…
 due to their repetitive nature
they are liable for backward
slippage during DNA
replication so that the same
short sequence is copied
twice
 a higher number of repeats
causes a higher mutation rates
single-stranded loop or
insertion/deletion loops

21. Microsatellite instability (MSI)
 due to the presence of the mismatch repair system, in vivo
microsatellite mutation rates range from 10-6 to 10-2 per
generation.
 Mutation of both alleles of mismatch repair (MMR) system
→ DNA strand (microsatellites) might be displaced, and
realigns out of register creating small loop of unpaired DNA
→ unable to remove the loops → microsatellite increases
or decrease in size due to either insertion or deletion of
repeating units when compared to the normal cell’s =
Microsatellite instability (MSI)
 MSI is implicated in 50–60% of inherited condition
HNPCC(Lynch syndrome) → germline mutations

22. How does faulty mismatch repair result in
colon cancer?
 tri- (and hexa-) nucleotide repeats are overrepresented
in coding sequences.
 If the mutation do NOT encompassed a codon triplet →
frameshift mutations → affecting critical areas of cell
growth regulation genes → promotion of tumorigenesis.
 These kinds of tumors are diploid

23. 3. epigenetic gene silencing
(Hypermethylation phenotype (CIMP+)) pathway
 DNA methylation is involved in normal cellular control of gene
expression
 (CpG island) are usually found in the regions close to promoters
 characterized by methylation of a number of genes rich in
CpG islands → silencing of multiple genes or promoter region
→ loss of gene function or transcriptional inactivation (e.g.
tumor-suppressor genes, APC)
 If MMR system is silenced→ MSI
 CRCs with high frequency of methylation of some CpG
islands are referred to as CpG island methylation phenotype
(CIMP) positive tumors

24. 3. epigenetic gene silencing…
 encompasses 35%-40% of sporadic CRC
 begin with serrated polyps, especially sessile serrated
adenomas (SSAs) bearing an activating mutation in the
BRAF gene.
 Epigenetic changes are potentially reversible by drugs.
 CIMP+ CRCs
 microsatellite stable (MSS) cancer (60% of CIMP+ ) or
 MSI-H (40% of CIMP+)
 Methylation occur in hMLH1
 methylation may occur in tumor suppressor genes

26. FAP
 FAP is the most common polyposis syndrome.
 the risk of CRC by the age of 40 years is almost 100%.
 It is autosomal dominant and is caused by de novo germline
mutations.
 The presence of FAP can be diagnosed by direct sequencing
of the germ-line mutations in APC gene on chromosome
5q21.
 Somatic APC mutations are also present in most sporadic
colorectal adenomas and cancers.

27. HNPCC (Lynch syndrome)
 The presence of HNPCC is defined as the presence of germ-line
mutation found in one of the four MMR genes, namely
MLH1, MSH2, MSH6 and PMS2.
 HNPCC is the most common hereditary colon cancer syndrome.
 It is autosomal dominant.
 2-hit hypothesis = germline mutation in 1 copy of 1 MMR gene
represents the “first hit,” and somatic inactivation of the wild
type allele the “second hit.”
 The BRAF gene is almost never mutated in Lynch syndrome–
associated CRCs; however, KRAS and p53 mutations can be
present.

28. 90%

29. Bethesda guidelines
 Bethesda guidelines can be used as a screening tool for
HNPCC.
(1) CRC in a patient younger than 50 years of age;
(2) synchronous or metachronous colorectal or other HNPCC-
related tumors,
(3) CRC with histologic features associated with MSI-H status in a
patient younger than 60 years of age;
(4) CRC in 1 or more first-degree relatives with an HNPCC-
associated tumor, with one of the patients being diagnosed
before age 50 years; and
(5) CRC in 2 or more first- or second-degree relatives with
HNPCC-related tumors, regardless of age.

30.  Molecular testing of CRC
 Screening
 Diagnosis
 Prognosis
 Targeted drug therapy (personalized cancer care)
 Follow up
Molecular Diagnosis for CRC

31. Screening of CRC
 The risk of recurrence and subsequent death due to CRC is closely
related to the stage of the disease at the time of the first diagnosis.
 A good marker helps the detection of disease at earlier stage so
that diseases can be cured effectively.
Screening of CRC
1. Fecal occult blood testing (FOBT)
 is simple, non-invasive and inexpensive
 false positive results might be yield by diet and medication.
2. Immunological FOBT
 detects human haemoglobin
 Specific but low sensitivity at detecting adenomas and CRC
3. Colonoscopy and sigmoidoscopy
 more effective and sensitive in screening
 invasive, high cost and inconvenience (privacy, extensive bowel preparation…)

32. Screening of CRC…
4. molecular markers testing
 Following the better understanding in the molecular basis
of CRC, Molecular markers that detect gene mutation in
the early stages of CRC can be used as non-invasive
screening tests for early detection of CRC, followed by
invasive confirmatory tests such as colonoscopy for
individuals with positive results.
 e.g. Carcinoembryonic antigen (CEA) and carbohydrate
antigen (CA)

33. Screening of CRC…
4.1 Carcinoembryonic antigen (CEA)
 glycoproteins initially found in embryonic tissue and colon
malignancies.
 may not be detected until the cancer is in advanced stages
 found in patients with IBD, malignancies of breast, pancreas,
stomach and lung.
 So, better used as prognostic markers and for follow up (Half
life of CEA is approximately 2 weeks) rather than screening

34. Screening of CRC…
4.2 Blood samples
 apoptotic release of DNA from tumor cells into the
bloodstream → elevated level of circulating methylated
SEPT9 DNA (oncogen) sequences was found.

35. Screening of CRC…
4.3 Fecal DNA Tests (Cologuard™)
 during apoptosis of normal colonic cell some DNA fragments of 180-
200 bps long is released into stool.
 However, CRC cells show a decreased rate of apoptosis and DNA
sequences with 1800-2400 bp are detected in stool samples
 CRC cells gene assay for
 APC (mutated in up to 70% of specimens),
 Kras (mutated in up to 60% of specimens) and
 p53 (mutated in up to 60% of specimens).
 for screening
 specificity 95%; sensitivity 60 - 90%
 not currently used – expensive, low sensitivity

36. Detection of gene mutations with known positions and
base changes
o Sanger Sequencing
o Allele Specific PCR

37. Name/Metho
d Target
Intended Use Detected
Property
Source
Material
Molecular Method Use
/Availability
KRAS therapeutic decision EGFR
targeted therapy
KRAS mutations FFPE or snap
frozen tissue
Sequencing clinical routine
BRAF chemotherapeutic
susceptibility
BRAF mutations >> >> sequencing, Real-time PCR clinical routine
BRAF chemotherapeutic
susceptibility
BRAF mutations >> >> Digital PCR, COLD-PCR studies
MSI status PCR chemotherapeutic
susceptibility
MSI status >> >> PCR clinical routine
MSI status IHC chemotherapeutic
susceptibility
MSI status >> >> IHC clinical routine
TP53 mutation screening p53 mutation
analysis
>> >> sequencing clinical routine
TP53 mutation screening >> >> >> >> oligonucleotide microarray studies
CIMP probable screening
/staging
methylation >> >> methylation microarray studies
miRNA assay for
blood/stool
screening miRNA expression
level
plasma, stool micro array studies
Multitarget stool
DNA test
screening, increasing
sensitivity
for colonoscopy
KRAS mutation,
NDRG4, BMP3
methylation,
hemoglobin
immunoassay
stool mutation and methylation
analysis, immunoassay
under approval
for clinical use
Epi proColon
early detection
assay
screening, increasing
sensitivity for colonoscopy
Septin 9 DNA
methylation assay
blood plasma Real-time PCR available for
clinical use
CIMP = CpG island methylator phenotype, FFPE = formalin fixed paraffin embedded, IHC = immunohistochemistry, miRNA = micro
ribonucleic acid,MSI = microsatellite instability, PCR = polymerase chain reaction. ARMS= amplification refractory mutation
system

38. Sanger sequencing
 uses dideoxy nucleotides to terminate
DNA synthesis, yielding a series of DNA
fragments whose sizes can be measured
by electrophoresis. The last base in each
of these fragments is known, because we
know which dideoxy nucleotide was used
to terminate each reaction.
 directly detects nucleotide sequences of
regions of interest.
 Base substitution, insertion and deletion
mutations, could be detected
 long turnover time and high running cost

39. Allele Specific PCR
 allele-specific PCR, also known as amplification refractory
mutation system (ARMS) that detects specific known mutated
form of a gene i.e. primers bind to mutant DNA but not WT DNA
so that you only get amplification if the mutant allele is present..
 could be adopted to a real-time PCR platform in order to
increase the speed and accuracy of the detection
 Employs probe consists of a fluorophore and a quencher attached
covalently on both ends; that is specific to the targeted DNA sequence.
 As this probe is broken down by the 5’ to 3’ exonuclease activity of DNA
polymerase, a fluorescent tag is separated from a quenching tag, so
fluorescence increases, and this increase can be measured in real time in
a fluorimeter.

40.  the amount of target DNA
molecules can be measured.
 turnover time is shorter than that
of Sanger sequencing
 Compared to conventional PCR,
real-time PCR is quantitative, fast
and sensitive.
 As no post-PCR manipulation,
such as gel or capillary
electrophoresis, is required, the
risk of laboratory cross-
contamination can be minimized.

41. Identification of MSI-H CRC
 microsatellites are marker of choice
 are highly liable for mutation
 high variability in size and number of repeats from one individual to another,
 co-dominance and distribution over the euchromatic genome
 MSI status determination could be done by
 immunohistochemical staining for MMR protein expression
 PCR (A fluorescent multiplex PCR-based method, RT-PCR)

42. TESTING MSI IN TUMORS PROTOCOL
1. Unstained sections from formalin fixed paraffin
embedded tissue (5-7 micron); overlap with H&E
stained section
• From same patient select one or two tissue blocks
containing
o Viable Tumor
o Noneoplastic tissue
• Scrape selected areas for DNA
extraction at least 1cm2
o Tumor Area (T); Nonneoplastic tissue (N)

43. PCR Identification of MSI-H …
2. A tumor tissue specimen and normal tissue specimen are
amplified using PCR for 5 to 7 microsatellite markers
3. Fluorescently labeled products are sized by capillary
electrophoresis
mononucleotide loci
dinucleotide loci

44. PCR Identification of MSI-H …
4. Analyze 5 loci (2 mononucleotide repeats and 3
dinucleotide repeats) for change of any length
1. microsatellite-high(MSI-H) = instability ≥2 loci
 5-fluorouracil is not effective in treating
 betterprognostic factor
 sporadic (~12%) or Lynch syndrome–associated (~3%)
2. microsatellite-low (MSI-L) = instability at 1 locus
 If that locus is dinucleotide → additional analysis of one
mononucleotide repeat with BAT-40 ) is recommended
3. microsatellite-stable (MSS) = no microsatellite
mutations

45. DNA hypermethylation detection
 DNA hypermethylation can be detected in primary colorectal
carcinomas using bisulfite conversion of DNA samples
followed by methylation-specific PCR.
 Methylation-Specific PCR (MSP), which is based on a
chemical reaction of sodium bisulfite with DNA that converts
unmethylated cytosines of CpG dinucleotides to uracil or
UpG, followed by traditional PCR. However, methylated
cytosines will not be converted in this process, and primers
are designed to overlap the CpG site of interest, which allows
one to determine methylation status as methylated or
unmethylated.

46. Microarray Analysis
 is a technique that permits the study of up to thousands of genes
concurrently in a single experiment. Multiplex lab on chip
 Genes are represented by 15–20 different 25mer oligonucleotides on
GeneChip® that serve as unique, sequence–sequence detectors
 Evolved from southern blotting

47.  Is collection of microscopic DNA
spots attached to solid surface
(glass or silcon chip).
 The identity of the the feature is
known by its position
 Each DNA spot contain 10-12
moles of specific DNA probs
which is used to hybridize c DNA
cRNA
 Prob target hybridization is
detected & quantified by
fluorophore labled targets

49. microRNAs in CRC
 are a class of small non-coding single-stranded RNAs with
important posttranscriptional regulatory functions by binding
to their target mRNAs.
 E.g. involvement of miRNA-133a in regulating the EGFR
pathway

50. Personalized Cancer Medicine
/Targeted therapy/

51.  http://www.en.wikipedia.org/wiki/Microsatellite_instability
 http://www.gradworks.umi.com/33/30/3330991.html
 http://www.wjgnet.com/1007-9327/pdf/v20/i14/3847.pdf
 UpToDate 21.2
 Harper's Illustrated Biochemistry - Robert K. Murray
 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing
Group / www.els.net
 www.medscape.com/viewarticle/575850
 Arch Pathol Lab Med—Vol 135, May 2011