24 Colorectal cancer

Question 1

The most common diagnosed malignancies worldwide is which one?

Answer 1

colorectal cancer-CRC

Question 2

Three distinct molecular pathways lead to the development of CRC

Answer 2

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

Question 3

What does it mean if polyp is serrated?

Answer 3

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.

Question 4

Comprehensive exome sequencing studies have demonstrated that individual CRCs harbor an average of 76 gene mutations.

Question 5

Describe Conventional suppressor pathway or CIN pathway of CRC

Answer 5

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.

Question 6

Genes and signal pathways involved in conventional suppressor or CIN pathway of CRC and steps lead to CRC

Answer 6

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.

Question 7

What is APC stands for?

Answer 7

1) adenomatous polyposis coli;

2) located on chr. 5q21;

3) a tumor suppressor gene

Question 8

Role of APC in CRC

Answer 8

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.

Question 9

% of all CRC have MSIs?

Answer 9

MSIs found in 15% of all CRCs

Question 10

Why are MSIs prone to accumulation of mutations?

Answer 10

MSIs are prone to accumulation of mutations due to inefficient binding of DNA polymerases to these sequence motifs

Question 11

What is MSI?

Answer 11

1) MSIs are stretches of short-tandem DNA sequence repeats, about 1- 6bp in length.

2) MSIs are found throughout the human genome.

Question 12

Genes involved in MSI pathway of CRC?

Answer 12

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.

Question 13

Functions of the mismatch repair (MMR) genes

Answer 13

1) MMR genes encode proteins for proper repair of DNA sequence mismatch errors missed by DNA polymerase;

2) Function to preserve the genomic integrity.

Question 14

Effects of loss of MMR function

Answer 14

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

Question 15

TGF-βRII frameshift mutations found in how many % of CRC with MSIs?

Answer 15

TGF-βRII frameshift mutations reported in 90% of CRCs with MSI.

Question 16

How does inactivation of MMR happen in Lynch Syndrome?

Answer 16

In Lynch Syndrome, MMR inactivation may be caused by a germline mutation in one of the four MMR genes:
MSH2,
MLH1,
MSH6,
PSM2

Question 17

How does inactivation of MMR happen in sporadic CRC with MSIs?

Answer 17

In sporadic CRC with MSIs, inactivation of MMR caused by aberrant epigenetic methylation of MLH1.

Question 18

Which MMR genes have MSIs themselves?

Answer 18

1) MSH3 and MSH6.

2) MSH3 and MSH6 may be mutated in MSI-high (MSI-H) CRCs.

Question 19

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.

Question 20

Function of DNA methylation

Answer 20

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.

Question 21

CIMP pathway in CRC

Answer 21

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

Question 22

The most common CRC arising through CIMP pathway begin with sessile serrated adenomas (SSAs)

Question 23

sessile serrated adenomas

Answer 23

1) SSAs;

2) prone to hypermethylation of a number of genes rich in CpG islands within the promoter regions.

Question 24

Frequent activating mutation causes SSAs

Answer 24

BRAF mutations

Question 25

% of CRC are MSI-stable and CIMP+

Answer 25

60%

Question 26

% of CRC are MSI-High and CIMP+

Answer 26

40%

Question 27

Most of sporadic MSI-H CRCs result from epigenetic silencing of which gene?

Answer 27

hMLH1 hypermethylation of CpG islands of promoter regions

Question 28

Role of loss hMLH1 protein function in SSAs

Answer 28

1) leads to rapid accumulation of additional mutations in other genes: TGF-β, BAX; 2) then leads to cancer progression

Question 29

Clinical features of SSAs with hMLH1 hypermethylation

Answer 29

1) cytologic dysplasia; 2) then rapid malignant transformation

Question 30

CpG island hypermethylation may also occur in tumor suppressor genes other than hMLH1, resulting in CIMP+ MSS CRCs.

Question 31

Hereditary colon cancer and polyposis syndromes are associated with a high risk of developing CRC. These account for how many % of all CRC?

Answer 31

<10%

Question 32

Lynch Syndrome

Answer 32

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.

Question 33

Lynch Syndrome is typically caused by a germline mutation in one of the MMR genes, what are these genes?

Answer 33

1) MLH1, MSH2, MSH6 and PSM2; 2) Loss of MMR function leads to development of CRC through the MSI pathway.

Question 34

Which MMR genes mutations are involved in 90% of Lynch Syndrome?

Answer 34

MLH1, MSH2

Question 35

What is the two-hit hypothesis of tumorigenesis of Lynch Syndrome?

Answer 35

Germline mutation in one copy of one MMR gene leads to somatic inactivation of the remaining wild-type allele.

Question 36

Novel mechanisms identified in a subset of Lynch Syndrome

Answer 36

1) Hypermethylation of the MSH2 promoter without MMR gene mutations; 2) germline deletions in the 3'-region of the EPCAM (epithelial cell adhesion molecule)

Question 37

What are the clinical guidelines used to evaluate the risk for Lynch Syndrome?

Answer 37

1) Amsterdam criteria; 2) Revised Bethesda guidelines - recommend MSI testing in CRCs

Question 38

Tests used to diagnosis of Lynch Syndrome

Answer 38

1) Molecular testing for MSI or IHC; 2) Further differentiation of sporadic CRC from hereditary MSI-H CRC.

Question 39

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.

Question 40

Familial Adenomatous Polyposis (FAP)

Answer 40

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

Question 41

Clinical feature of FAP

Answer 41

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

Question 42

What is attenuated FAP (AFAP)?

Answer 42

1) Less severe form; 2) less than 100 adenomas, thus flat morphology; 3) 69% of risk of development of CRC

Question 43

Gene mutations that causes FAP and AFAP

Answer 43

APC gene on chr. 5q21

Question 44

The development of CRC in FAP/AFAP follows which pathway?

Answer 44

the conventional suppressor CIN pathway: mainly APC gene mutations

Question 45

List the inherited adenomatous polyposis syndromes

Answer 45

1) FAP; 2) AFAP; 3) MAP (MUTYH-associated polyposis); 4) PPAP (polymerase proofreading associated polyposis); 5) HMPS (hereditary mixed polyposis syndrome)

Question 46

MAP: MUTYH-associated polyposis

Answer 46

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

Question 47

What is MUTYH gene?

Answer 47

1) a base-excision repair gene for oxidative DNA damage; 2) encodes MYH glycosylase

Question 48

Who is recommended to test for MAP?

Answer 48

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

Question 49

PPAP: polymerase proofreading associated polyposis

Answer 49

1) autosomal dominant; 2) characterized by the presence of multiple colorectal adenomas and early onset CRC

Question 50

Germline mutated genes associated with PPAP

Answer 50

POLE and POLD1: 1) two DNA polymerases with exonuclease activity 2) germline mutations in the proofreading domains of POLE and POLD1

Question 51

HMPS: hereditary mixed polyposis syndrome

Answer 51

1) autosomal dominant; 2) presents with polyps that display multiple and mixed morphologies: serrated polyps, Peutz-Jeghers polyps, juvenile polyps, and conventional adenomas

Question 52

Gene mutations associated with HMPS

Answer 52

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.

Question 53

What are the two major inherited hamartomatous polyposis syndrome?

Answer 53

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

Question 54

Lifetime risk of PJS to develop CRC is?

Answer 54

39%

Question 55

Lifetime risk of JPS to develop CRC is?

Answer 55

10-38%

Question 56

Gene mutations associated with PJS

Answer 56

germline mutations or deletions in STK11: 1) STK11 is a serine-threonine kinase; 2) STK11 regulates TP53-mediated apoptosis

Question 57

Gene mutations associated with JPS

Answer 57

SMADH4, BMPR1A, ENG: all related to TGF-β/SMAD pathway

Question 58

Transforming growth factor β functions

Answer 58

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.

Question 59

Types of hereditary CRC syndromes

Answer 59

1) nonpolyposis: Lynch Syndrome; 2) Adenomatous polyposis: FAP, AFAP, MAP, PPAP, HMPS; 3) Hamartomatous polyposis: PJS, JPS

Question 60

EGFR signaling pathway

Answer 60

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

Question 61

PI3K/AKT1/mTOR can be activated by several proteins

Answer 61

1) EGFR; 2) KRAS

Question 62

% of CRCs with EGFR increased copy numbers

Answer 62

1) 85% of CRCs showed increased copy numbers of EGFR; 2) overexpression can be detected by IHC

Question 63

Effects of EGFR overexpression on the response to therapy with anti-EGFR monoclonal antibodies (MoAbs) in CRC

Answer 63

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

Question 64

anti-EGFR monoclonal antibodies (MoAbs) are?

Answer 64

ce-tu-xi-mab; pani-tumu-mab

Question 65

Are EGFR mutations common in CRCs?

Answer 65

No, rare

Question 66

KRAS

Answer 66

1) KRAS is a small G protein that functions as a signal transducer; 2) downstream of EGFR

Question 67

KRAS mutations effect in CRC

Answer 67

KRAS mutations leads to constitutively active RAS proteins that stimulates RAS/MAP pathway independently from EGFR signaling

Question 68

90% of all KRAS mutations are located on which codons in sporadic CRC?

Answer 68

Codons 12 and 13

Question 69

40-50% of sporadic CRC patients with KRAS mutations located on which KRAS codons?

Answer 69

Codons 12, 13 and 61

Question 70

KRAS activating mutations are shown on which codons in all CRC?

Answer 70

Codons 12, 13 and 61 in sporadic CRC; Codon 146 of exon 4: 1-6% of all CRCs; Codon 117

Question 71

NRAS activating mutations have been seen in 1-6% of CRCs

Question 72

KRAS mutations have been shown in what types of CRCs?

Answer 72

1) MSS; 2) Sporadic MSI-H has lower of frequency of KRAS mutations; 3) HNPCC (Hereditary nonpolyposis colorectal cancer) CRCs

Question 73

Sporadic MSI-H CRCs have a lower frequency of KRAS mutations but a higher frequency of BRAF mutations.

Question 74

KRAS mutations effect on prognostic of CRC?

Answer 74

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

Question 75

BRAF mutations in CRC

Answer 75

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

Question 76

What gene mutation can differentiate HNPCC from sporadic MSI-H CRC?

Answer 76

BRAF mutations

Question 77

Prognosis of CRC patients with BRAF mutations

Answer 77

1) poor prognosis associated with MSI-Low and MSS and BRAF mutations; 2) No effect in prognosis in MSI-H and BRAF mutations

Question 78

Wild type BRAF is required for response to anti-EGFR-MoAbs in metastatic CRC.

Question 79

NCCN guidelines state that BRAF mutations need to be tested in CRC patients with wild type KRAS.

Question 80

BRAF inhibitor

Answer 80

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.

Question 81

class 1A PI3K

Answer 81

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

Question 82

How are class 1A PI3Ks activated by?

Answer 82

1) G-protein coupled receptors; 2) receptor tyrosine kinases such as KRAS and EGFR; 3) certain oncoproteins such as RAS

Question 83

PIK3CA mutations are the most common in PI3K pathway in CRC, what is % of all CRCs?

Answer 83

10-30% of CRCs

Question 84

What exons of PIK3CA have the mutations in CRC? What is % for the each exon of all PIK3CA mutations?

Answer 84

1) Exon 9: 60-65% of all PIK3CA mutations; 2) Exon 20: 20-25% of all PIK3CA mutations

Question 85

CRC prognosis associated with PIK3CA mutations

Answer 85

poor, unclear

Question 86

PIK3CA exon 9 mutation is gain of function or loss of function?

Answer 86

gain of function

Question 87

PIK3CA exon 9 gain of function mutation is RAS-dependent while the exon 20 mutation is not

Question 88

Does PIK3CA exon gain of function mutation respond to anti-EGFR therapy?

Answer 88

1) Yes, with KRAS wild type; 2) Exon 20 PIK3CA mutation lack response to anti-EGFR therapy

Question 89

PIK3CA mutated CRCs may also have either KRAS or BRAF mutations.

Answer 89

can't be used as a single marker for predicting response to anti-EGFR therapy

Question 90

PTEN

Answer 90

1) a tumor suppressor gene in the PI3K/AKT pathway; 2) negatively regulate the PI3K-AKT-mTOR pathway

Question 91

Loss of PTEN function by what type of mutations?

Answer 91

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

Question 92

CRCs with inactivated PTEN may also have mutations of KRAS, BRAF, or PIK3CA.

Question 93

Loss of PTEN have been shown to confer resistance to cetuximab in vitro

Question 94

Prognosis of PTEN mutations in CRCs

Answer 94

lack of response and shorter survival in CRCs with WT KRAS metastatic CRCs treated with anti-EGFR therapy

Question 95

Molecular diagnostic evaluation of sporadic CRCs commonly includes 8 genes:

Answer 95

MLH1, MSH2, MSH6, PMS2, KRAS, BRAF, PIK3CA, PTEN

Question 96

Molecular diagnostic evaluation of heretidary CRCs commonly includes 9 genes:

Answer 96

MLH1, MSH2, MSH6, PMS2, APC, MUTYH, STK11, BMPR1A, SMAD4

Question 97

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

Question 98

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.

Question 99

Disadvantage in Sanger sequencing for genetic testing

Answer 99

failure to detect large deletions, low throughput and cost.

Question 100

Sequencing methods for screening mutations

Answer 100

1) conformation-specific gel electrophoresis; 2) single-strand conformation polymorphism; 3) denaturing gradient gel electrophoresis

Question 101

What is MLPA

Answer 101

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

Question 102

Confirmation testing used to confirm mutations after MLPA testing

Answer 102

Southern blot, PCR

Question 103

Methods to detect gross chromosomal alterations caused by large deletions and gene rearrangements

Answer 103

1) Karyotyping; 2) fluorescent in situ hybridization

Question 104

Protein truncation testing may be used to detect the presence of a truncated protein by gel electrophoresis.

Question 105

Protein truncation usually caused by what kind of mutations

Answer 105

nonsense, frameshift

Question 106

What is criteria for genetic testing of hereditary adenomatous polyposis syndrome?

Answer 106

individuals with more than 10 adenomas

Question 107

Which gene needs to be screened for FAP and AFAP?

Answer 107

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

Question 108

Which exons of APC that have mutations contribute to FAP typically?

Answer 108

Mutations between Exon 5 and the 5' portion of exon 15 (codons 169-1393)

Question 109

Where on the APC gene that have mutations contribute to AFAP typically?

Answer 109

APC mutations associated with AFAP typically occur at the 5' or 3' end of APC beyond codon 1595.

Question 110

Linkage testing of APC may be performed if other molecular testing are unsuccessful.

Question 111

What is linkage testing?

Answer 111

Linkage testing utilizes multiple DNA markers near or within the gene among multiple affected members of a family to correlate disease phenotype

Question 112

How accurate is linkage testing for FAP?

Answer 112

>98%

Question 113

If a patient is suspected to have FAP or AFAP but APC mutations can't be identified, what to do next?

Answer 113

Test for MAP: MUTYH gene mutations are found in 10-20% of these APC mutation negative patients with suspected FAP or AFAP.

Question 114

80% suspected MAP case have which mutations?

Answer 114

1) MUTYH Y165C and G382D; 2) Both MUTYH alleles must be mutated to inactivate the gene and cause CRC.

Question 115

MSI testing

Answer 115

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.

Question 116

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.

Answer 116

Therefor, positive IHC staining can't rule out Lynch Syndrome completely. Same situation apply to other MMR protein IHC.

Question 117

What can cause false MMR protein IHC staining in CRC?1)

Answer 117

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.

Question 118

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.

Question 119

Loss of MLH1 expression due to the result of promoter methylation is in seen in which type of CRCs?

Answer 119

Sporadic MSI-H CRC; Lynch Syndrome

Question 120

Loss of MSH2/MSH6 expression almost always indicated Lynch Syndrome.

Question 121

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.

Question 122

What method is used for MSI testing?

Answer 122

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.

Question 123

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.

Question 124

Traditional Bethesda guidelines for MSI testing for individuals with HNPCC.

Answer 124

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.

Question 125

Revised Bethesda guidelines for MSI testing for individuals with HNPCC

Answer 125

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.

Question 126

What is the commercially available kit for MSI PCR testing?

Answer 126

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.

Question 127

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)

Question 128

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.

Question 129

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.

Question 130

The vast majority of gene mutations in CRCs are SNVs.

Question 131

The V600E BRAF mutation confers a worse prognosis to stage II and stage III colon cancer patients independently of disease stage and therapy