Principles and practice of Human Pathology – Lecture 5 + 6 (26-4-2018):
Molecular Pathology I
Part 1: Gene mutations (HC 1.5):
Actionable targets non small cell lung cancer:
e.g. EGFR, KRAS, BRAF, HER2, PI3K, etc.
The result of activation of these pathways is proliferation. The more these pathways
are activated, the more proliferation of cells occur.
By binding of the ligand, these pathways get activated.
Patients with mutations in these pathways (e.g. EGFR) can be treated by (specific
EGFR-) inhibitors inhibitors only affect the mutations/mutated cells, not healthy
cells benefit!
Genomic aberrations in cancer:
- By mutations, deletions, chromosomal translocations
Sanger sequencing:
First you do the sequencing/the synthesis by PCR with use of dNTPs, etc. and after
that you do the analysis to check what the sequence is.
Next generation sequencing (NGS): more sensitive than sanger sequencing,
sequencing by synthesis, you can analyze samples while synthesis takes place.
More DNA-fragments can be sequenced at the same time. As soon as the
nucleotides are incorporated, they can be detected.
Mutation detection by NGS, how?
Sample
1. Extract DNA
2. Generate library
3. Add linkers (barcoded)
4. Emulsion PCR
5. Sequence individual products
Ion-torrent:
One of the examples of an amplicon based NGS-technique.
- PCR of the exon
- Library preparation, end repair + annealing adapter primers (which are either
complementary to the ion sphere or which are used as sequencing primers)
Annealing of the adapter-flanked PCR amplicons:
You make a dilution of the library, in such a way that in one well there is only one
amplicon. This amplicon will then bind to an ion-sphere.
This allows clonal amplification amplify one fragment per ion sphere.
Then the sequencing reaction can take place in different wells.
, Ion-torrent works with pH. The ion-torrent technique detects the release of hydrogen-
atoms ‘’pH-meter’’.pH-meter’pH-meter’’.’pH-meter’’..
As soon as the complementary nucleotides are added to the strand
hydrogen and pyrophosphate are released, which can be measured with the
different NGS techniques. Ion torrent measures the release of this hydrogen!
During ion-torrent there will be several flows with fluid with ‘A’pH-meter’’.s , C’pH-meter’’.s, T’pH-meter’’.s and G’pH-meter’’.s
sequential flood.
And only when these are incorporated an H+ plus pyrophosphate will be released,
which can be read.
Only when the nucleotide is complementary, H+ will be released and can be
detected.
When one nucleotide is incorporated: only one H+ release
When two nucleotides are released: two H+’pH-meter’’.s are released, etc. This will result in a
higher peak.
BRAF-mutations cannot be detected by sanger sequencing, but it can be by NGS,
because it is a very ‘low’pH-meter’’. mutation.
Metastasis or a new primary tumor?:
Head & Neck squamous cell carcinoma (HNSCC): multiple primaries due to field
cancerization.
- Either progression of related fields (same field) metastasis
- Or progression of independent (other) field new (secondary) primary tumor
The treatment for both is totally different, so it is important to know which of the types
it is.
To check whether it is metastasis or a new primary tumor, you can use screening
techniques, in order to screen the mutation and then see if the mutations can be
found in the same region.
Part 2: Chromosomal abnormalities (HC 1.6) – (detection of) genomic
abberations:
Trastuzumab inhibits cell signaling of the HER2/ErbB2-receptor.
Fluorescent in situ hybridization (FISH):
FISH-technique can be used to detect genomic alterations:
- Dual color probe to detect ErbB2: part of the ErbB2-gene.
CEP17-probe colors the centrosome of chromosome 17, this is used as a numerical
control
There are two green spots, because: there are two chromosomes 17 (one from the
mother, one from the father) present.
The red spots mark the amplificated genes.
Molecular Pathology I
Part 1: Gene mutations (HC 1.5):
Actionable targets non small cell lung cancer:
e.g. EGFR, KRAS, BRAF, HER2, PI3K, etc.
The result of activation of these pathways is proliferation. The more these pathways
are activated, the more proliferation of cells occur.
By binding of the ligand, these pathways get activated.
Patients with mutations in these pathways (e.g. EGFR) can be treated by (specific
EGFR-) inhibitors inhibitors only affect the mutations/mutated cells, not healthy
cells benefit!
Genomic aberrations in cancer:
- By mutations, deletions, chromosomal translocations
Sanger sequencing:
First you do the sequencing/the synthesis by PCR with use of dNTPs, etc. and after
that you do the analysis to check what the sequence is.
Next generation sequencing (NGS): more sensitive than sanger sequencing,
sequencing by synthesis, you can analyze samples while synthesis takes place.
More DNA-fragments can be sequenced at the same time. As soon as the
nucleotides are incorporated, they can be detected.
Mutation detection by NGS, how?
Sample
1. Extract DNA
2. Generate library
3. Add linkers (barcoded)
4. Emulsion PCR
5. Sequence individual products
Ion-torrent:
One of the examples of an amplicon based NGS-technique.
- PCR of the exon
- Library preparation, end repair + annealing adapter primers (which are either
complementary to the ion sphere or which are used as sequencing primers)
Annealing of the adapter-flanked PCR amplicons:
You make a dilution of the library, in such a way that in one well there is only one
amplicon. This amplicon will then bind to an ion-sphere.
This allows clonal amplification amplify one fragment per ion sphere.
Then the sequencing reaction can take place in different wells.
, Ion-torrent works with pH. The ion-torrent technique detects the release of hydrogen-
atoms ‘’pH-meter’’.pH-meter’pH-meter’’.’pH-meter’’..
As soon as the complementary nucleotides are added to the strand
hydrogen and pyrophosphate are released, which can be measured with the
different NGS techniques. Ion torrent measures the release of this hydrogen!
During ion-torrent there will be several flows with fluid with ‘A’pH-meter’’.s , C’pH-meter’’.s, T’pH-meter’’.s and G’pH-meter’’.s
sequential flood.
And only when these are incorporated an H+ plus pyrophosphate will be released,
which can be read.
Only when the nucleotide is complementary, H+ will be released and can be
detected.
When one nucleotide is incorporated: only one H+ release
When two nucleotides are released: two H+’pH-meter’’.s are released, etc. This will result in a
higher peak.
BRAF-mutations cannot be detected by sanger sequencing, but it can be by NGS,
because it is a very ‘low’pH-meter’’. mutation.
Metastasis or a new primary tumor?:
Head & Neck squamous cell carcinoma (HNSCC): multiple primaries due to field
cancerization.
- Either progression of related fields (same field) metastasis
- Or progression of independent (other) field new (secondary) primary tumor
The treatment for both is totally different, so it is important to know which of the types
it is.
To check whether it is metastasis or a new primary tumor, you can use screening
techniques, in order to screen the mutation and then see if the mutations can be
found in the same region.
Part 2: Chromosomal abnormalities (HC 1.6) – (detection of) genomic
abberations:
Trastuzumab inhibits cell signaling of the HER2/ErbB2-receptor.
Fluorescent in situ hybridization (FISH):
FISH-technique can be used to detect genomic alterations:
- Dual color probe to detect ErbB2: part of the ErbB2-gene.
CEP17-probe colors the centrosome of chromosome 17, this is used as a numerical
control
There are two green spots, because: there are two chromosomes 17 (one from the
mother, one from the father) present.
The red spots mark the amplificated genes.
