24 Colorectal cancer Flashcards
The most common diagnosed malignancies worldwide is which one?
colorectal cancer-CRC
Three distinct molecular pathways lead to the development of CRC
1) the conventional suppressor pathway or chromosomal instability (CIN) pathway;
2) the serrated pathway or CpG island methylator phenotype (CIMP);
3) the microsatellite instability (MSI) pathway
What does it mean if polyp is serrated?
Serrated polyps are a type of growth that stick out from the surface of the colon or rectum. The polyps are defined by their saw-toothed appearance under the microscope. They can often be hard to find. The only way to determine the types of polyps is by removing them and examining them under a microscope.
Comprehensive exome sequencing studies have demonstrated that individual CRCs harbor an average of 76 gene mutations.
Describe Conventional suppressor pathway or CIN pathway of CRC
1) CIN pathway demonstrates accelerated gains or losses of large portions or whole chromosomes and commonly results in gross chromosomal or karyotypic abnormalities.
2) The consequences of CIN is a high frequency of aneuploidy, genomic amplifications and loss of heterozygosity.
3) About 60% of all CRCs including FAP, follow this CIN pathway;
4) Poor prognosis;
5) Accumulations of mutations is specific oncogenes and tumor suppressor genes plus chromosomal abnormalities in CIN pathways, lead to initiation and progression of CRC over a period of years to decades.
Genes and signal pathways involved in conventional suppressor or CIN pathway of CRC and steps lead to CRC
1) CIN pathway is initiated by inactivation of the APC/β-catenin/Wnt signaling pathway;
2) typically by mutation of one copy of APC gene;
3) Then a second event leads to inactivation of the second APC allele through deletion or additional mutations;
4) APC genes alterations lead to the development of dysplasia in aberrant cryptic foci and early adenomas.
5) Sequential accumulations of other gene mutations: KRAS, DCC, SMAD4, TP53.
What is APC stands for?
1) adenomatous polyposis coli;
2) located on chr. 5q21;
3) a tumor suppressor gene
Role of APC in CRC
1) By negatively regulating canonical WNT signaling, APC counteracts proliferation, promotes differentiation, facilitates apoptosis and suppresses invasion and tumor progression.
2) APC further antagonizes canonical WNT signaling by interacting with and counteracting β-catenin in the nucleus.
% of all CRC have MSIs?
MSIs found in 15% of all CRCs
Why are MSIs prone to accumulation of mutations?
MSIs are prone to accumulation of mutations due to inefficient binding of DNA polymerases to these sequence motifs
What is MSI?
1) MSIs are stretches of short-tandem DNA sequence repeats, about 1- 6bp in length.
2) MSIs are found throughout the human genome.
Genes involved in MSI pathway of CRC?
1) The mismatch repair (MMR) system: MSH2, MLH1, MSH3, MSH6, PSM2;
2) As MSIs are present in the coding regions of key genes for regulation of cell growth and apoptosis, loss of MMR function may result in frameshift mutations due to expansion or contraction of these regions and create an environment of uncontrolled cell survival and carcinogenesis.
Functions of the mismatch repair (MMR) genes
1) MMR genes encode proteins for proper repair of DNA sequence mismatch errors missed by DNA polymerase;
2) Function to preserve the genomic integrity.
Effects of loss of MMR function
As MSIs are present in the coding regions of key genes for regulation of cell growth and apoptosis, loss of MMR function may result in frameshift mutations due to expansion or contraction of these regions and create an environment of uncontrolled cell survival and carcinogenesis.
1) frameshift mutations;
2) uncontrolled cell survival and carcinogenesis
TGF-βRII frameshift mutations found in how many % of CRC with MSIs?
TGF-βRII frameshift mutations reported in 90% of CRCs with MSI.
How does inactivation of MMR happen in Lynch Syndrome?
In Lynch Syndrome, MMR inactivation may be caused by a germline mutation in one of the four MMR genes:
MSH2,
MLH1,
MSH6,
PSM2
How does inactivation of MMR happen in sporadic CRC with MSIs?
In sporadic CRC with MSIs, inactivation of MMR caused by aberrant epigenetic methylation of MLH1.
Which MMR genes have MSIs themselves?
1) MSH3 and MSH6.
2) MSH3 and MSH6 may be mutated in MSI-high (MSI-H) CRCs.
When compared to MSI-stable (MSS) CRCs, MSI-associated CRCs demonstrate a better prognosis at all stages, despite their known resistance to some chemotherapy regimens.
Function of DNA methylation
1) an epigenetic modification that regulates gene expression;
2) is essential for normal embryonic development and functions in X-chromosome inactivation and genomic imprinting;
3) The normal mammalian genome contains methylated CpG islands in non-promoter regions and promoter regions.
4) About half of CpG islands are located in promoter regions around the transcriptional start site, and are unmethlyated in normal cells.
5) Genes with these unmethlyated CpG islands will undergo normal transcription in the presence of transcriptional activators.
CIMP pathway in CRC
1) The CIMP pathway refers to widespread CpG island methylation within promoter regions of tumor suppressor genes.
2) In caner cells, hypermethylation of CpG islands within promoter regions leads to transcriptional silencing of tumor suppressor genes and loss of gene function, contributing to the tumorigenic process.
3) The CIMP positive phenotype accounts for about 35% of all CRCs.
4) Hypermethylation CpG islands–> gene silencing of tumor suppressor–> loss of gene function–>CRC
The most common CRC arising through CIMP pathway begin with sessile serrated adenomas (SSAs)
sessile serrated adenomas
1) SSAs;
2) prone to hypermethylation of a number of genes rich in CpG islands within the promoter regions.
Frequent activating mutation causes SSAs
BRAF mutations
% of CRC are MSI-stable and CIMP+
60%
% of CRC are MSI-High and CIMP+
40%
Most of sporadic MSI-H CRCs result from epigenetic silencing of which gene?
hMLH1 hypermethylation of CpG islands of promoter regions
Role of loss hMLH1 protein function in SSAs
1) leads to rapid accumulation of additional mutations in other genes: TGF-β, BAX;
2) then leads to cancer progression
Clinical features of SSAs with hMLH1 hypermethylation
1) cytologic dysplasia;
2) then rapid malignant transformation
CpG island hypermethylation may also occur in tumor suppressor genes other than hMLH1, resulting in CIMP+ MSS CRCs.
Hereditary colon cancer and polyposis syndromes are associated with a high risk of developing CRC. These account for how many % of all CRC?
<10%
Lynch Syndrome
1) The most common causes for developing hereditary CRC;
2) 2-7% of all CRCs;
3) Lynch syndrome is an autosomal dominant disorder that carries an increased risk for the development of CRC, endometrial cancer, and other cancers.
Lynch Syndrome is typically caused by a germline mutation in one of the MMR genes, what are these genes?
1) MLH1, MSH2, MSH6 and PSM2;
2) Loss of MMR function leads to development of CRC through the MSI pathway.
Which MMR genes mutations are involved in 90% of Lynch Syndrome?
MLH1, MSH2
What is the two-hit hypothesis of tumorigenesis of Lynch Syndrome?
Germline mutation in one copy of one MMR gene leads to somatic inactivation of the remaining wild-type allele.
Novel mechanisms identified in a subset of Lynch Syndrome
1) Hypermethylation of the MSH2 promoter without MMR gene mutations;
2) germline deletions in the 3’-region of the EPCAM (epithelial cell adhesion molecule)
What are the clinical guidelines used to evaluate the risk for Lynch Syndrome?
1) Amsterdam criteria;
2) Revised Bethesda guidelines - recommend MSI testing in CRCs
Tests used to diagnosis of Lynch Syndrome
1) Molecular testing for MSI or IHC;
2) Further differentiation of sporadic CRC from hereditary MSI-H CRC.
NCCN guidelines now recommend evaluation of MSI status by molecular or IHC on all resected CRCs, or CRCs diagnosed in patients <70-yrs old and in patient >70 meet the Bethesda.
Familial Adenomatous Polyposis (FAP)
1) FAP is the most common polyposis syndrome;
2) FAP is associated with about 0.5% of all CRC;
3) FAP is autosomal dominant but 25% of FAP are caused by de novo germline mutations
Clinical feature of FAP
1) characterized by the presence of hundreds to thousands of adenomas;
2) 100% of risk of development of CRC;
3) FAP median age for CRC development is 36-yrs old;
4) Classic FAP typically shows >100 colonic polyps and associated with other cancers
What is attenuated FAP (AFAP)?
1) Less severe form;
2) less than 100 adenomas, thus flat morphology;
3) 69% of risk of development of CRC
Gene mutations that causes FAP and AFAP
APC gene on chr. 5q21
The development of CRC in FAP/AFAP follows which pathway?
the conventional suppressor CIN pathway: mainly APC gene mutations
List the inherited adenomatous polyposis syndromes
1) FAP;
2) AFAP;
3) MAP (MUTYH-associated polyposis);
4) PPAP (polymerase proofreading associated polyposis);
5) HMPS (hereditary mixed polyposis syndrome)
MAP: MUTYH-associated polyposis
1) Autosomal recessive;
2) 0.5-1% of all CRCs;
3) MAP has a cumulative risk for CRC development of 80% by 70 years of age;
4) Caused by biallelic germline mutation in MUTYH
What is MUTYH gene?
1) a base-excision repair gene for oxidative DNA damage;
2) encodes MYH glycosylase
Who is recommended to test for MAP?
1) patients with more than 10 adenomatous polyps;
2) and with a family history of CRC consistent with recessive inheritance;
3) patient is negative for APC mutations
PPAP: polymerase proofreading associated polyposis
1) autosomal dominant;
2) characterized by the presence of multiple colorectal adenomas and early onset CRC
Germline mutated genes associated with PPAP
POLE and POLD1:
1) two DNA polymerases with exonuclease activity
2) germline mutations in the proofreading domains of POLE and POLD1
HMPS: hereditary mixed polyposis syndrome
1) autosomal dominant;
2) presents with polyps that display multiple and mixed morphologies: serrated polyps, Peutz-Jeghers polyps, juvenile polyps, and conventional adenomas
Gene mutations associated with HMPS
HMPS is associated with a 40 kb duplication present at the 3’ end of SCG5 gene and upstream of the GREM1 locus, leading to increased GREM1 expression.
What are the two major inherited hamartomatous polyposis syndrome?
1) Peutz-Jeghers syndrome (PJS);
2) juvenile polyposis syndrome (JPS);
3) PJS and JPS are both autosomal dominant;
4) both with increased risks of CRC, pancreatic and other GI cancers
Lifetime risk of PJS to develop CRC is?
39%
Lifetime risk of JPS to develop CRC is?
10-38%
Gene mutations associated with PJS
germline mutations or deletions in STK11:
1) STK11 is a serine-threonine kinase;
2) STK11 regulates TP53-mediated apoptosis
Gene mutations associated with JPS
SMADH4, BMPR1A, ENG:
all related to TGF-β/SMAD pathway
Transforming growth factor β functions
1) TGFβ functions as a tumor suppressor by mediating its antiproliferative effects in a large variety of cell types.
2) During early stages of tumorigenesis, TGFβ inhibits cell cycle promotion.
3) evasion of TGFβ-mediated antiproliferative effects is a prerequisite for advancement of tumor progression.
Types of hereditary CRC syndromes
1) nonpolyposis: Lynch Syndrome;
2) Adenomatous polyposis: FAP, AFAP, MAP, PPAP, HMPS;
3) Hamartomatous polyposis: PJS, JPS
EGFR signaling pathway
1) EGFR activation leads to autophosphorylation of its c-terminal tyrosine residues, which serve as the docking sites that bind to several intracellular proteins;
2) pathways activated are:
– RAS/RAF/MEK/MAPK
– PI3K/AKT1/mTOR;
3) these signaling pathways are involved in tumor cell proliferation, invasion, migration and inhibition of apoptosis
PI3K/AKT1/mTOR can be activated by several proteins
1) EGFR;
2) KRAS
% of CRCs with EGFR increased copy numbers
1) 85% of CRCs showed increased copy numbers of EGFR;
2) overexpression can be detected by IHC
Effects of EGFR overexpression on the response to therapy with anti-EGFR monoclonal antibodies (MoAbs) in CRC
1) no apparent correlation;
2) However, anti-EGFR MoAbs are effective in treating some metastatic CRC in clinical trials;
3) anti-EGFR MoAbs may play a role in inhibition of the downstream signaling such KRAS
anti-EGFR monoclonal antibodies (MoAbs) are?
ce-tu-xi-mab;
pani-tumu-mab
Are EGFR mutations common in CRCs?
No, rare
KRAS
1) KRAS is a small G protein that functions as a signal transducer;
2) downstream of EGFR
KRAS mutations effect in CRC
KRAS mutations leads to constitutively active RAS proteins that stimulates RAS/MAP pathway independently from EGFR signaling
90% of all KRAS mutations are located on which codons in sporadic CRC?
Codons 12 and 13
40-50% of sporadic CRC patients with KRAS mutations located on which KRAS codons?
Codons 12, 13 and 61
KRAS activating mutations are shown on which codons in all CRC?
Codons 12, 13 and 61 in sporadic CRC;
Codon 146 of exon 4: 1-6% of all CRCs;
Codon 117
NRAS activating mutations have been seen in 1-6% of CRCs
KRAS mutations have been shown in what types of CRCs?
1) MSS;
2) Sporadic MSI-H has lower of frequency of KRAS mutations;
3) HNPCC (Hereditary nonpolyposis colorectal cancer) CRCs
Sporadic MSI-H CRCs have a lower frequency of KRAS mutations but a higher frequency of BRAF mutations.
KRAS mutations effect on prognostic of CRC?
1) do no have a significant prognostic value;
2) KRAS and NRAS mutations have been shown to have poor response to anti-EGFR-MoAbs–> wild type of KRAS responded better
BRAF mutations in CRC
1) BRAF is a serine/threonine protein kinase;
2) BRAF is downstream of KRAS in the MAPK pathway;
3) BRAF mutations present in 5-22% of CRCs;
4) The most common BRAF mutation is V600E;
5) BRAF mutations is detected in high frequency in MSI-H than MSS, never detected in HNPCC
What gene mutation can differentiate HNPCC from sporadic MSI-H CRC?
BRAF mutations
Prognosis of CRC patients with BRAF mutations
1) poor prognosis associated with MSI-Low and MSS and BRAF mutations;
2) No effect in prognosis in MSI-H and BRAF mutations
Wild type BRAF is required for response to anti-EGFR-MoAbs in metastatic CRC.
NCCN guidelines state that BRAF mutations need to be tested in CRC patients with wild type KRAS.
BRAF inhibitor
1) vemurafenib;
2) enhance anti-tumor efficacy;
3) BRAF V600E showed only limited response to the inhibitor, likely due to rapid feedback activation of EGFR.
class 1A PI3K
1) class 1A PI3K are heterodimeric proteins composed of two subunits: p85 regulatory subunit and p110 catalytic subunit;
2) Three catalytic isoforms exist: PIK3CA, PIK3CB, PIK3CD
How are class 1A PI3Ks activated by?
1) G-protein coupled receptors;
2) receptor tyrosine kinases such as KRAS and EGFR;
3) certain oncoproteins such as RAS
PIK3CA mutations are the most common in PI3K pathway in CRC, what is % of all CRCs?
10-30% of CRCs
What exons of PIK3CA have the mutations in CRC? What is % for the each exon of all PIK3CA mutations?
1) Exon 9: 60-65% of all PIK3CA mutations;
2) Exon 20: 20-25% of all PIK3CA mutations
CRC prognosis associated with PIK3CA mutations
poor, unclear
PIK3CA exon 9 mutation is gain of function or loss of function?
gain of function
PIK3CA exon 9 gain of function mutation is RAS-dependent while the exon 20 mutation is not
Does PIK3CA exon gain of function mutation respond to anti-EGFR therapy?
1) Yes, with KRAS wild type;
2) Exon 20 PIK3CA mutation lack response to anti-EGFR therapy
PIK3CA mutated CRCs may also have either KRAS or BRAF mutations.
can’t be used as a single marker for predicting response to anti-EGFR therapy
PTEN
1) a tumor suppressor gene in the PI3K/AKT pathway;
2) negatively regulate the PI3K-AKT-mTOR pathway
Loss of PTEN function by what type of mutations?
1) mutations, deletions, promoter methylation;
2) PTEN mutations are seen in 20-40% of CRCs;
3) PTEN mutations lead to constitutive activation of PI3K-AKT-mTOR pathway
CRCs with inactivated PTEN may also have mutations of KRAS, BRAF, or PIK3CA.
Loss of PTEN have been shown to confer resistance to cetuximab in vitro
Prognosis of PTEN mutations in CRCs
lack of response and shorter survival in CRCs with WT KRAS metastatic CRCs treated with anti-EGFR therapy
Molecular diagnostic evaluation of sporadic CRCs commonly includes 8 genes:
MLH1, MSH2, MSH6, PMS2,
KRAS, BRAF, PIK3CA, PTEN
Molecular diagnostic evaluation of heretidary CRCs commonly includes 9 genes:
MLH1, MSH2, MSH6, PMS2,
APC, MUTYH, STK11, BMPR1A, SMAD4
Inherited CRCs typically arise from one gene although different families with the same disorder may have separate and distinct mutations in the implicated gene, except Lynch Syndrome
In CRC, the likelihood of finding the disease-causing mutation in the index patient may range from 50% to greater than 90% in a family.
Disadvantage in Sanger sequencing for genetic testing
failure to detect large deletions, low throughput and cost.
Sequencing methods for screening mutations
1) conformation-specific gel electrophoresis;
2) single-strand conformation polymorphism;
3) denaturing gradient gel electrophoresis
What is MLPA
1) Multiplex ligation-dependent probe amplification;
2) MLPA is used for large deletions or rearrangements within the gene;
3) used when mutations can’t be detected by Sanger
Confirmation testing used to confirm mutations after MLPA testing
Southern blot, PCR
Methods to detect gross chromosomal alterations caused by large deletions and gene rearrangements
1) Karyotyping;
2) fluorescent in situ hybridization
Protein truncation testing may be used to detect the presence of a truncated protein by gel electrophoresis.
Protein truncation usually caused by what kind of mutations
nonsense, frameshift
What is criteria for genetic testing of hereditary adenomatous polyposis syndrome?
individuals with more than 10 adenomas
Which gene needs to be screened for FAP and AFAP?
APC:
1) usually APC mutations are nonsense or frameshift, resulting in a termination codon and a truncated protein;
2) APC mutations are identified 95% times by sequencing
Which exons of APC that have mutations contribute to FAP typically?
Mutations between Exon 5 and the 5’ portion of exon 15 (codons 169-1393)
Where on the APC gene that have mutations contribute to AFAP typically?
APC mutations associated with AFAP typically occur at the 5’ or 3’ end of APC beyond codon 1595.
Linkage testing of APC may be performed if other molecular testing are unsuccessful.
What is linkage testing?
Linkage testing utilizes multiple DNA markers near or within the gene among multiple affected members of a family to correlate disease phenotype
How accurate is linkage testing for FAP?
> 98%
If a patient is suspected to have FAP or AFAP but APC mutations can’t be identified, what to do next?
Test for MAP: MUTYH gene mutations are found in 10-20% of these APC mutation negative patients with suspected FAP or AFAP.
80% suspected MAP case have which mutations?
1) MUTYH Y165C and G382D;
2) Both MUTYH alleles must be mutated to inactivate the gene and cause CRC.
MSI testing
1) PCR and/or IHC the expression of MLH1, MSH2, MSH6 and PMS2:
– IHC the loss of expression of all four proteins or just MSH6 and PMS2.
– Two antibodies (MSH6 and PMS2) panel is used in IHC since loss of MLH1 and MSH2 causes concurrent loss of MSH6 and PMS2.
– intact expression of all four proteins indicating the MMR protein present but does not rule of Lynch Syndrome.
2) Testing of human genome for microsatellites.
Example, missense mutations in MLH1 are present in 5-8% Lynch Syndrome, and may lead to nonfunctional proteins with retained antigenicity thus + IHC staining for MLH1.
Therefor, positive IHC staining can’t rule out Lynch Syndrome completely.
Same situation apply to other MMR protein IHC.
What can cause false MMR protein IHC staining in CRC?1)
1) Neoadjuvant treatments such as chemotherapy and radiation therapy:
— may reduce MMR protein expression and lead to a false-negative IHC–> positive for Lynch Syndrome;
2) Loss of MLH1 expression may be the result of promoter methylation;
3) Loss of MSH2/MSH6 expression almost always indicates Lynch Syndrome.
In the gastrointestinal tract, loss of MMR protein expression has been reported in 25–35% of nonneoplastic colonic and small bowel crypts of Lynch syndrome patients, a subset of which also demonstrated MSI by PCR.
Loss of MLH1 expression due to the result of promoter methylation is in seen in which type of CRCs?
Sporadic MSI-H CRC;
Lynch Syndrome
Loss of MSH2/MSH6 expression almost always indicated Lynch Syndrome.
As microsatellites vary in size between individuals, DNA is typically extracted from both normal and cancer tissue for evaluation.
Either fresh tissues or FFPE are used.
What method is used for MSI testing?
1) florescent multiplex PCR amplification of the selected microsatellites, the size of PCR products obtained from normal and cancer tissue are compared.
2) MSI is considered to be present when the PCR products size changes observed.
— the size could be increased or decreased.
3) current MSI PCR test detect 5-7 microsatellites markers.
4) following PCR–>fluorescently-labeled PCR products are sized by capillary electrophoresis.
5) low cancer cellularity contributes to false-negative PCR-based testing.
The mechanism of MSI generation is generally believed to be DNA slippage in the process of replication, or mismatch of the basic group of slippage strand and complementary strand in the process of DNA replication and repair, resulting in one or more of the repeating units missing or insert.
Traditional Bethesda guidelines for MSI testing for individuals with HNPCC.
1) test a panel of five mononucleotide or dinucleotide microsatellites.
2) CRCs are classified as MSI-H if two or more microsatellites instability is present.
3) CRCs are classified as MSI-L if instability is present at one locus.
3) CRCs are classified as MSS if no instability observed.
Revised Bethesda guidelines for MSI testing for individuals with HNPCC
1) It recommends a secondary MSI panel of mononucleotide markers to be used in MSI-L cases in which only a dinucleotide marker is mutated.
2) This is because mononucleotide markers are more sensitive than dinucleotide or trinucleotide markers.
What is the commercially available kit for MSI PCR testing?
1) a kit uses 5 mononucleotide markers for detection of MSI:
— BAT-25, BAT-26, MON0-27, NR-21, and NR-24;
2) this kit also uses 2 pentanucleotide markers:
— Penta C and Penta D;
— these are for specimen identification to
ensure that cancer and normal samples
are from the same patient.
Somatic mutation of BRAF V600E may be indicated for CRCs that show MSI-H or loss of MLH1 expression, as this mutation is present in sporadic MSI-H cancers but not in HNPCC associated cancers. (HNPCC=Hereditary Non-polyposis Colorectal Cancer)
If BRAF is wild type, methylation analysis of the hMLH1 promoter may be performed using methylation-specific MLPA or methylation-specific PCR testing, as the hMLH1 promoter is rarely methylated in HNPCCs.
Germline mutation analysis is required in MSI-H CRCs that are BRAF WT and lack hMLH1 promoter methylation due to the high probability of Lynch Syndrome in these cases.
The vast majority of gene mutations in CRCs are SNVs.
The V600E BRAF mutation confers a worse prognosis to stage II and stage III colon cancer patients independently of disease stage and therapy
