Molecular Diagnostics in Neoplasia

Question 1

Gene mutations

Answer 1

substitutions, deletions, insertions

tumor suppressor genes and oncogenes

histone/ epigenetic regulation genes

Question 2

Answer 2

Sanger Sequencing

Question 3

Size descrimination by capillary electrophoresis

Answer 3

Amplification of target regions of DNA

Run through capillary electrophoresis with control fragments of known size

good to look for insertion, deletion, expansion, contraction

Question 4

Increased progression free survival and overall survival in pts with wild type RAS (KRAS and NRAS) and BRAF with MAB+ standard chemo in pts with advanced dz

Question 5

KRAS codon 12 G12D mutation

Answer 5

Patient not eligible for anti EGFR therapy

Question 6

Next generation sequencing AKA massively parallel sequencing

Answer 6

DNA fragmented

DNA ligated to unique uniquely coded sequenced (primer site, hybridization site, unique identifier)

SNA hybridized to substrate (flow cell, bead)

Sequencing

Bioinformatics

Question 7

Answer 7

Microsatellite instability

By fragment length analysis/capillary electrophoresis

Question 8

Answer 8

Microsatellite instability via pyrosequencing

Question 9

EFGR mutations in non-small cell lung cancer

Answer 9

EFGR mutatios in exons 18,19,20,21 (kinase domain)
Rarely foind in association with other mutations (KRAS, ALK, ect)

Priamrily in adenocarcinoma histology, never smokers, females, asians
10% of US pts (35% of asians)

Pt with eligible mutations for targeted FDA approved small molecular inhibitors
testing for EFGR mutations part of NCCN guidelines

Some mutations confer resistance to FDA approved small molecular inhibitors

Question 10

Answer 10

HPV caqn turn off P53 as well as RB and P16

Causes cevervical dysplasia and can lead to head and neck dysplasia

Question 11

What testin shoulde be used to ID lynch syndrome pts

Answer 11

microsatellate instability
Reflex MLH1 hypermethylatiohn
Relex BRAF mutation testing

MMR protein expression by IHC
MLH1 hypermethylation

Gene sequencing

Question 12

Answer 12

PCR

Question 13

Epigenetics/ Regulatory Alterations

Answer 13

DNA methylations

miRNA

viral effect

Question 14

Patient specimen (FFPE) sent for KRAS, NRAS, BRAF, and P13KCA testing by next generation sequencing

Question 16

Pathways to Cancer

Answer 16

Gene Mutations

Rearrangements

Copy number variations

Gene expression

Epigenetics/ regulatory alterations

Question 17

Labs for genetic testing need to be regulated by

Answer 17

CLIA

Question 18

Pyrosequencing

Answer 18

Sequencing by synthesis

Use of Luciferase, which interacts with ATP released when dNTP is added to elongation chain

Sensativity 5%

Detect base pair substitutions (not good for insertion/ deletion)

Question 19

Answer 19

Next generation sequencing, AKA massively parallel sequencing

Question 20

Reverse transcription PCR (RT-PCR)

Answer 20

RNA translated into cDNA (complementary) then DNA amplified by PCR (fusion transcripts (translocations), gene expression)

Not to be confused with Real Tume PCR (qPCR) (often used to quantitate amount of DNA or RNA) Can be coupled with RT-PCR)

Question 21

Sanger Sequencing

Answer 21

Use of dye-labeled ddNTPs in elongation phase

run through capillary electrophoresis

Can detect base pair substitution, as well as insertions and delestions

Sensativity 10-20%

will NOT detect large rearrangements

Question 22

Mutations in what gene causes Lynch

Answer 22

MLH1

MSH2

MSH6

PMS2

Question 23

Answer 23

RT-PCR

Question 24

Pt (lung ca) is stable on erlotinib for 3 months, but then imaging shows increased lung nodules

His performance also deteriorates, with an increased risk of performing another lung biopsy

What may be happening?

What options for testing in leu of biopsy?

Answer 24

Tumor develops resistance in Exon 20 (T790 resistance mutation)

We can do a liquid biopsy- detection of mutations in tumor DNA ciculating in blood

Question 25

65 year old F with R breast mass

What additional testing is indicated

Answer 25

Immunohistochemistry

Question 26

What is lynch syndrome

Answer 26

Microsatellite instability is characteristif of lynch syndrome cancers

15-20% of all sporadic are also microsatellite unstable

MSI in sporadic tumors caused by hypermethylation of MLH1, not byt germline mutations in MMR genes

Question 27

Answer 27

Microsatellite stability

Question 28

Tumor suppressor genes

Answer 28

Inhibit cell proliferation ( cell cycle inhibitors, DNA damage repair, promote apoptosis, cell adhesion. Eg RB, TP53, P16)

Loss of 2 alleles > development of neoplasia (2 hit hypothesis) (mutations cause loss of function. Loss of one allele raises risk of loss of second allele, often through loss of heteozygosity)

Accounts for vast majority of familial cancer syndromes (Inheritance of one bad copy of gene, easier (and sooner) to sevelop second mutation. Eg Lynch syndrome, Li Frumeni, BRCA1/2)

Large variety of mutation types seen (single base substitutions, insertions, deletions, hypermethylation. Multiple foci, occur through gene)

Question 29

60 year old M presents with fatigue.

Cytogenetic is sent and FISH for t(9;22)

Fish confirms t(9;22) = CML

Is genetic testing still necessary even though we know his diagnosis

Answer 29

Yes! It is important to continue testing. Monitering status of CA. Monitering response to therapy.

Can do this by RT-PCR followd by real time PCR

Question 30

Answer 30

Sanger Sequencing

Question 31

Steps of PCR

Answer 31

1) denaturation
2) annealing
3) elongation

Question 32

65 year old M with mass in sigmoid colon, likely adenocarcinoma. Imaging shows a mass in the liver suspicous for metastatic disease.

What molecular testing is indicated and why? Is screening for lynch syndrome indicated

Answer 32

Determination of tumor gene status for RAS (KRAS and NRAS) and BRAF

Determination of tumor MMR or MSI status (if not done previously)

Question 33

Oncogenes

Answer 33

Activation of cell cycle/ cell proliferation (tyrosine kinases/ pathway (EGFR/ KRAS), growth factors, transcription factors)

Usually specific mutations (activating point mutations), gene amplification, or chromosomal trqanslocation/ rearrangement (lead to constituative activation (gain of function). “mutation hotspots”)

Often therapeutic agents

Familial syndromes are rare (KIT/PDGFR-A, MET, RASopathies)

Question 34

On imaging there are no suspicous lymph nodes

Patient proceeds to lumpecctomy with sentinel lymph node biopsy

Final stage pT1c N0 (between 1 and 2 cm and no lymph node involvement)

Any additional molecular testing?

Answer 34

Consider 21 gene RT-PCR assay

Question 35

64 year old M hx smoking with lung nodule. Mult pleural nodules biopsied. Consistant with metastatic dz.

Is molecular testing indicated?

Answer 35

EFGR mutation testing

ALK testing

ROS1 testing

BRAF testing

(done through FISH/ next genertion sequencing)

PD-L1 testing

Question 36

Answer 36

Pyrosequencing

Question 37

Liquid biopsy

Answer 37

detection of mutation in tumor DNA in circulating blood

Question 38

What is lynch syndrome

Answer 38

Hereditary cancer syndrome caused in 1 of 4 DNA mismatch repair proteins (form of tumor supressor gene)

MLH1

MSH2

MSH6

PMS2

Deletion in 3’exons of EPCAM gene causing methylation/inacti ation of adjacent MSH2

BRAF mutations almost never found in lynch syndrome but can occasionally see mutations in RAS genes

Question 39

Spectrum of lynch syndrome

Answer 39

Hereditary Non-polyposis Colorectal Cancer (HNPCC)
2-7% all colon cancer

Endometrial Cancer
Lynch accounts for 2-3% of all endometrial cancers
Women with lynch syndrome have 40-60% lifetime risk of developing endometrial cancer, average age if 48 years dx

Increased risk ovarian, gastric, small intestine, hepatobiliary, upper GU, brain, and skin cancers

Question 40

Rearrangements

Answer 40

translocations

inversions

Question 41

Answer 41

Size descrimination by capillary electrophoresis

Question 42

Deletion in 3’ exon of what gene causes LYNCH

Answer 42

EPCAM gene. Causes methylation/inactivation of MSH2

Question 43

What are microsatellites

Answer 43

repeated DNA sequences 1-6bp in length