Principles and practice of Human Pathology – Lecture 7 + 8 (18-5-2018):
Molecular Pathology II
Mainly lymphoid malignancies will be discussed:
Malignant lymphoma:
Tumors of B/T cells in the lymph nodes and/or extranodal sites.
The B-cells are located in the ‘germinal center’, surrounded by T-cells.
B-cells produce immunoglobulins (antibodies).
B-cells are generated in the bone marrow.
The B-cells will go to the lymph nodes and will be stimulated by their antigen (for the
first time)
B-cells can be transformed malignant lymphoma
There are several malignant types of B-cells: lymphomas
In the blood: leukaemia.
e.g. Burkitt lymphoma
e.g. Follicular lymphoma
Molecular diagnostics lymphoma:
Diagnosis based on:
- Morphology
- Immunohistochemistry (marker expression)
- Chromosomal aberrations
- Clonality detection (next lecture)
IGH is a target gene (Immunoglobulin heavy chain) that is frequently involved in
chromosomal aberrations in lymphomas.
Chromosomal translocations:
*In Sarcoma there is a fusion gene
In Lymphoma:
Gene A will be chromosomally rearranged and becomes under the control of gene B
(via promoter or enhancer)
Effects:
- Tissue specificity is altered
- Extent and timing of gene (A) expression is altered.
Follicular lymphoma:
*Chromosomal translocation
Breakpoints: MBR (major breakpoints region)
Breakpoints occur downstream of the coding DNA in BCL2. In IGH breakpoint occur
mainly in the J genes.
Enhancers promote transcription: either downstream or upstream.
, Enhancer promotes the transcription of BCL2, so by translocation: there is no fusion
transcript, no fusion protein, it is the same protein, but it is expressed in excess due
to the enhancer which is present in IGH.
BCL2 is an apoptosis-inhibitor.
*Important to remember: in sarcomas chromosomal translocations work differently
than in lymphomas.
Detection of chromosomal aberrations for follicular lymphoma:
In two ways:
1. PCR
2. FISH
a. Split probes
b. Break apart probes
- DNA extraction
- Quality assessment of extracted DNA PCR-sizeladder
- Specific PCRs and readout on agarose gel this will show the break points.
o MBR/IGH junction
When there is a translocation, the primers will give rise to a PCR-product
indicating that there is a true translocation, otherwise no product.
No band and good DNA quality no translocation present.
Fluorescence in Situ hybridization (FISH):
In order to detect breakpoints, using probes for BCL2 with different colors.
Example 1: Split signal (break apart) probe
One probe downstream (green), one probe upstream (red).
- When there is no translocation: green + red close to each other yellow color
is expressed.
- When there is a translocation: breaks occur green + red dot will arise
individually, showing that translocation has happened.
BCL2-break is associated with follicular lymphoma.
Example 2: IGH/BCL2 fusion probe
- No translocation: both green and red
- Translocation: green + red will combine resulting in yellow.
PCR vs. FISH:
PCR:
- Detection of PCR product = presence of translocation
- PCR: primers flanking the different breakpoints
- Added value of PCR: cases with recurrent disease/relapse: information on the
exact breakpoint
Molecular Pathology II
Mainly lymphoid malignancies will be discussed:
Malignant lymphoma:
Tumors of B/T cells in the lymph nodes and/or extranodal sites.
The B-cells are located in the ‘germinal center’, surrounded by T-cells.
B-cells produce immunoglobulins (antibodies).
B-cells are generated in the bone marrow.
The B-cells will go to the lymph nodes and will be stimulated by their antigen (for the
first time)
B-cells can be transformed malignant lymphoma
There are several malignant types of B-cells: lymphomas
In the blood: leukaemia.
e.g. Burkitt lymphoma
e.g. Follicular lymphoma
Molecular diagnostics lymphoma:
Diagnosis based on:
- Morphology
- Immunohistochemistry (marker expression)
- Chromosomal aberrations
- Clonality detection (next lecture)
IGH is a target gene (Immunoglobulin heavy chain) that is frequently involved in
chromosomal aberrations in lymphomas.
Chromosomal translocations:
*In Sarcoma there is a fusion gene
In Lymphoma:
Gene A will be chromosomally rearranged and becomes under the control of gene B
(via promoter or enhancer)
Effects:
- Tissue specificity is altered
- Extent and timing of gene (A) expression is altered.
Follicular lymphoma:
*Chromosomal translocation
Breakpoints: MBR (major breakpoints region)
Breakpoints occur downstream of the coding DNA in BCL2. In IGH breakpoint occur
mainly in the J genes.
Enhancers promote transcription: either downstream or upstream.
, Enhancer promotes the transcription of BCL2, so by translocation: there is no fusion
transcript, no fusion protein, it is the same protein, but it is expressed in excess due
to the enhancer which is present in IGH.
BCL2 is an apoptosis-inhibitor.
*Important to remember: in sarcomas chromosomal translocations work differently
than in lymphomas.
Detection of chromosomal aberrations for follicular lymphoma:
In two ways:
1. PCR
2. FISH
a. Split probes
b. Break apart probes
- DNA extraction
- Quality assessment of extracted DNA PCR-sizeladder
- Specific PCRs and readout on agarose gel this will show the break points.
o MBR/IGH junction
When there is a translocation, the primers will give rise to a PCR-product
indicating that there is a true translocation, otherwise no product.
No band and good DNA quality no translocation present.
Fluorescence in Situ hybridization (FISH):
In order to detect breakpoints, using probes for BCL2 with different colors.
Example 1: Split signal (break apart) probe
One probe downstream (green), one probe upstream (red).
- When there is no translocation: green + red close to each other yellow color
is expressed.
- When there is a translocation: breaks occur green + red dot will arise
individually, showing that translocation has happened.
BCL2-break is associated with follicular lymphoma.
Example 2: IGH/BCL2 fusion probe
- No translocation: both green and red
- Translocation: green + red will combine resulting in yellow.
PCR vs. FISH:
PCR:
- Detection of PCR product = presence of translocation
- PCR: primers flanking the different breakpoints
- Added value of PCR: cases with recurrent disease/relapse: information on the
exact breakpoint
