Adenoma to Colonic Carcinoma
transformation
Dr.Vikram Prabhakar (DCP,DNB)

p53
• Guardian gene, located on short arm of
chromosome 17 (17p13.1)

WNT pathway

EGFR-KRAS
• 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

CpG Island

CpG island
• Region of the genome with high frequency of
CpG sites than the rest of the genome.
• Formal Definition - CpG island is a region with
at least 200 bp, and a GC percentage that is
greater than 50 % .
• CpG is shorthand for “—C—phosphate—G—
that is, cytosine and guanine separated by
only one phosphate

Genome ~ 3 billion characters. Find
gene ?

Importance of CpG Islands
• CpG island acts as a proxy to identify a gene.
• They often occur at the start of the gene.

• 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

Microsatellites…
• due to their repetitive nature
they are liable for backward
slippage during DNA replication,
if miss-match repair genes are
defective, 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

• 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

Etiology
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%)

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

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
• cancers result from the stepwise
accumulation of multiple somatic
mutations. Therefore, many CRCs remain
asymptomatic for years before diagnosis.

• Comprehensive 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.

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

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

• 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

kRAS oncogene
• 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.

– Causes mutation of serine/threonine protein
kinase which is involved with that acts as a
downstream effector of the KRAS gene.
– substitution of valinefor glutamate
– Not detected inany LS/HNPCCtumors
– 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

Roleand ExpressionPatternofEpidermal GrowthFactor
Receptor inColonCancer
• EGFRisexpressed innormal
colon epitheliumand in80-100%
of colorectal cancers (by IHC)
• Up to 40%response rate to anti-
EGFRinwild type tumors
• BRAF,NRAS,and PIK3CA
exon 20mutationsare
significantly associated with a
low response rate

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.

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

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)
• methylation phenotype (CIMP) positive tumors

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

• If MMR system is silenced→ MSI
• CRCs with high frequency of methylation of
some CpG islands are referred to as CpG island

Molecular Pathologic classification of colorectal
cancer

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.

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.

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