More focus on PCR and FISH

1. MOLECULAR METHODS IN
ONCOLOGY
Dr. Ankita Rungta Kapoor
Jupiter Hospital

2. INTRODUCTION
Diagnosis
Prognosis
Predicting response to particular therapies
Minimal residual disease monitoring
• Depends on detection or measurement of disease-specific molecular
biomarkers

3. Specimen Requirements
• Blood
• Bone marrow aspirates and biopsies
• Fluids
• Organ-specific fresh tissues in saline or tissue culture media such as
Roswell Park Memorial Institute (RPMI)
• FFPE tissues and cytology cell blocks

4. Molecular Diagnostics Testing Process

5. Molecular Methods in Oncology
1. Real-time PCR (q-PCR)
2. Allele-specific PCR (AS-PCR)
3. Reverse transcriptase PCR (RT-PCR)
4. Fragment analysis
5. FISH
6. High-resolution melting (HRM) curve analysis
7. Sanger sequencing
8. Pyrosequencing
9. Single nucleotide extension assay (SNaPshot)
10. Next-generation sequencing (NGS)
11. Genomic microarray

6. POLYMERASE CHAIN REACTION
• Rapid amplification of targeted DNA sequences
• Requirements:
• specimen template DNA
• forward and reverse primers (18 to 24 oligonucleotides long)
• Taq DNA polymerase
• each of the four nucleotides bases (dATP, dTTP, dCTP, dGTP)
• Selected genomic sequences undergo repetitive temperature cycling
(sequential heat and cooling)

7. Steps of PCR:
1. Denaturation of double-stranded DNA template
2. Annealing of the primers to the targeted complementary sequences
on the template
3. Extension of new strands of DNA by Taq polymerase from
nucleotides, using the primers as the starting point
• Each cycle doubles the copy number of PCR templates for next round
of polymerase activity
• PCR products (amplicons) detected by electrophoresis or in real-time
systems simultaneously to the amplification reaction

9. Real Time PCR
• Polymerase chain reaction performed with a PCR reporter
• Usually a fluorescent double-stranded DNA binding dye or a
fluorescent reporter probe
• Intensity of fluorescence produced at each amplification cycle
monitored in real time
• Both quantification and detection of targeted sequences
accomplished in the reaction tube
• Eg. JAK2 V617F mutation- found in several myeloproliferative
neoplasms (polycythemia vera, essential thrombocythemia, and
primary myelofibrosis)
• Eg. BRAFV600E- found in melanomas, thyroid and lung cancers

10. Allele specific PCR
 Performed as two PCR reactions:
• one employing a forward primer specific for the mutant sequence
• other using a forward primer specific for the correspondent wild-type
sequence
Eg. detection of JAK2 V617F and MPL mutations in myeloproliferative
neoplasms; BRAF V600E mutation; KIT D816V mutations in AML
Limitation: will not detect mutations other than those for which
specific primers are designed
Therefore utilized for highly recurrent mutations that occur at
specific locations within genes

11. RT-PCR
• Detection and quantification of RNA transcripts
• PCR is designed to work on DNA templates; RNA is not a suitable
substrate for Taq polymerase
• It can be adapted to RNA testing by including a reverse transcription
step to convert a RNA sequence into its cDNA sequence
• Eg. PML-RARA fusion detection in APML
• Eg. quantitative detection of BCR-ABL1 fusion transcript for
prognostication and minimal residual disease testing in CML

12. Advantages of PCR
• Flexible platforms that permit detection of a variety of conserved
hotspot mutations including nucleotide substitutions, small length
mutations (deletions, insertions) and translocations
• High sensitivity beneficial for residual disease testing and specimens
with limited tumor content
• Adaptable to quantitative assays
• Ability to amplify small amounts of low quality FFPE-derived DNA

13. Disadvantages of PCR
• Detects only specific targeted mutations/ chromosomal
translocations
• Not suitable for variable mutations
• May not determine the exact change in nucleotide sequence
• PCR reaction can be inhibited by heparin or melanin if present in the
extracted DNA, which may lead to assay failure
• Risk of false positives due to specimen or amplicon contamination

14. FLUORESCENT IN SITU HYBRIDIZATION
• Annealing of a large single-stranded fluorophore-labeled
oligonucleotide probe to complementary DNA target sequences
within a tissue or cell preparation
• Hybridization of the probe at the specific DNA region within a nucleus
is visible by direct detection using fluorescence microscopy
• Used for quantitative assessment of gene amplification or deletion
and for qualitative evaluation of gene rearrangements

16. 2 TYPES OF PROBES:
• locus specific probes - complementary to gene of interest
• centromeric probes - hybridize to alpha-satellite regions near
centromere of a specific chromosome and help in enumeration of
number of copies of that chromosome
USES:
• Detection of HER2gene amplification in breast cancer
• MYCN amplification in neuroblastoma
• 1p/19q codeletion in oligodendrogliomas
• EWSR1 translocation in soft tissue tumors
• ALK translocation in NSCLC

17. Dual Fusion Probes used in -
• IGH-BCL2 translocation that occurs in most follicular lymphomas and
a subset of diffuse large B-cell lymphomas
• For detection of IGH-CCND1 rearrangements in mantle cell
lymphomas

18. To detect Her2neu-
• Normal cell - 2 copies of the HER2/neu oncogene, 1 on each of its 2
copies of chromosome 17
• Using a centromeric chromosome 17 probe (green signal) in
combination with an allele-specific probe for the HER2/neu oncogene
(red signal)
• Visualization of 2 green and 2 red signals in each cell (ratio 1)
• In a breast cancer cell undergoing HER2/neu oncogene amplification,
4 or more red signals visualized in nuclei showing only 2 green signals
(ratio of 2 or more)

20. To detect t(9;22) Philadelphia chromosome
• Combination of 2 allele-specific probes to the breakpoint regions on
long arms of chromosome 9 (green signal) and 22 (red signal)
• Non-leukemic cell- 2 green and 2 red signals visualized
• t(9;22) - juxtapose 1 green and 1 red signal
• Appearance of a new yellow signal (color overlap) in addition to the
remaining 1 green and 1 red signal that did not participate in the
translocation

22. Advantages of FISH
• Applicable to a variety of specimen types, including FFPE tissue
• Because probes are hybridized to tissue in situ, the tumor
morphology is preserved (more accurate interpretation of
heterogeneous samples)
• Detects chromosomal translocation, gene amplification, and deletion

23. Disadvantages of FISH
• Targeted approach that will only detect specific alterations
• Because most probes are large (e.g., >100 kb), small deletions or
insertions will not be detected
• Poor tissue fixation, fixation artifacts, nuclear truncation on tissue
slides, and nuclear overlaps may hamper interpretation
• Some intra-chromosomal rearrangements (e.g., RET-PTC and EML4-
ALK) may be challenging to interpret by FISH due to subtle
rearrangements of the probe signals on the same chromosome arm

24. THANK YOU