Molecular Diagnostics in Neoplasia Flashcards

1
Q

Gene mutations

A

substitutions, deletions, insertions

tumor suppressor genes and oncogenes

histone/ epigenetic regulation genes

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2
Q
A

Sanger Sequencing

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3
Q

Size descrimination by capillary electrophoresis

A

Amplification of target regions of DNA

Run through capillary electrophoresis with control fragments of known size

good to look for insertion, deletion, expansion, contraction

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4
Q

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

A
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5
Q

KRAS codon 12 G12D mutation

A

Patient not eligible for anti EGFR therapy

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6
Q

Next generation sequencing AKA massively parallel sequencing

A

DNA fragmented

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

SNA hybridized to substrate (flow cell, bead)

Sequencing

Bioinformatics

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7
Q
A

Microsatellite instability

By fragment length analysis/capillary electrophoresis

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8
Q
A

Microsatellite instability via pyrosequencing

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9
Q

EFGR mutations in non-small cell lung cancer

A

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

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10
Q
A

HPV caqn turn off P53 as well as RB and P16

Causes cevervical dysplasia and can lead to head and neck dysplasia

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11
Q

What testin shoulde be used to ID lynch syndrome pts

A

microsatellate instability
Reflex MLH1 hypermethylatiohn
Relex BRAF mutation testing

MMR protein expression by IHC
MLH1 hypermethylation

Gene sequencing

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12
Q
A

PCR

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13
Q

Epigenetics/ Regulatory Alterations

A

DNA methylations

miRNA

viral effect

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14
Q

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

A
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15
Q
A
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16
Q

Pathways to Cancer

A

Gene Mutations

Rearrangements

Copy number variations

Gene expression

Epigenetics/ regulatory alterations

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17
Q

Labs for genetic testing need to be regulated by

A

CLIA

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18
Q

Pyrosequencing

A

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)

19
Q
A

Next generation sequencing, AKA massively parallel sequencing

20
Q

Reverse transcription PCR (RT-PCR)

A

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)

21
Q

Sanger Sequencing

A

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

22
Q

Mutations in what gene causes Lynch

A

MLH1

MSH2

MSH6

PMS2

23
Q
A

RT-PCR

24
Q

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?

A

Tumor develops resistance in Exon 20 (T790 resistance mutation)

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

25
Q

65 year old F with R breast mass

What additional testing is indicated

A

Immunohistochemistry

26
Q

What is lynch syndrome

A

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

27
Q
A

Microsatellite stability

28
Q

Tumor suppressor genes

A

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)

29
Q

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

A

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

30
Q
A

Sanger Sequencing

31
Q

Steps of PCR

A

1) denaturation
2) annealing
3) elongation

32
Q

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

A

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

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

33
Q

Oncogenes

A

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)

34
Q

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?

A

Consider 21 gene RT-PCR assay

35
Q

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

Is molecular testing indicated?

A

EFGR mutation testing

ALK testing

ROS1 testing

BRAF testing

(done through FISH/ next genertion sequencing)

PD-L1 testing

36
Q
A

Pyrosequencing

37
Q

Liquid biopsy

A

detection of mutation in tumor DNA in circulating blood

38
Q

What is lynch syndrome

A

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

39
Q

Spectrum of lynch syndrome

A

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

40
Q

Rearrangements

A

translocations

inversions

41
Q
A

Size descrimination by capillary electrophoresis

42
Q

Deletion in 3’ exon of what gene causes LYNCH

A

EPCAM gene. Causes methylation/inactivation of MSH2

43
Q

What are microsatellites

A

repeated DNA sequences 1-6bp in length