Lecture 7 - Molecular Diagnostics in Hitopatholgy

Question 1

What is the fundamental principle that key molecular techniques in histopathology rely on?

Answer 1

Key molecular techniques in histopathology rely on the fundamental base-pairing of DNA molecules.

Question 2

What is the reversible process involved in separating and reforming polynucleotide strands in DNA within cells, and why is it essential for molecular diagnostics?

Answer 2

The reversible process is DNA annealing/hybridization. It is essential as it allows the identification of specific DNA or RNA sequences.

Question 3

Describe the main applications of in-situ hybridization in histopathology.

Answer 3

The main applications include identifying viruses, cancer diagnosis, predicting the response of some cancers to therapy, and detecting abnormal genes/gene expression.

Question 4

What are the key steps in the methodology of in-situ hybridization?

Answer 4

The steps include tissue section attachment, revealing target DNA/RNA, denaturation and hybridization, removal of unbound probe, and visualization of the probe binding site.

Question 5

Why is rapid fixation crucial in the preparation of sections for in-situ hybridization and frozen sections?

Answer 5

Rapid fixation prevents autolysis and the release of nucleases. For frozen sections, fixation is necessary to prevent nucleases in tissue from destroying target DNA during thawing.

Question 6

How is target DNA/RNA revealed in the in-situ hybridization process, and why is this step necessary?

Answer 6

Proteinase K (PK) treatment is used to partially permeabilize the cross-linked protein mesh formed during formalin fixation, allowing access to the probe.

Question 7

Explain the denaturation and hybridization steps in in-situ hybridization, including the role of formamide.

Answer 7

Denaturation and hybridization involve incubation in a hybridization buffer, with formamide lowering the denaturing temperature. This step is crucial for probe binding specificity.

Question 8

What are the types of probes used in in-situ hybridization, and what are the characteristics of oligonucleotide probes?

Answer 8

Probes include oligonucleotide, ssDNA, dsDNA, and RNA. Oligonucleotide probes are 20-50 nucleotides, allowing good tissue penetration, and are resistant to RNAase.

Question 9

How does probe labelling occur in in-situ hybridization, and what are the advantages of direct probe labelling?

Answer 9

Probe labelling involves direct or indirect methods. Direct labelling allows direct visualization, and advantages include simple detection and less background noise.

Question 10

Explain the visualisation methods used for in-situ hybridization with directly labelled probes.

Answer 10

For radiolabelled probes, exposure to film (RISH); for fluorescently labelled probes, visualization using a fluorescence microscope (FISH).

Question 11

What is the role of secondary detection systems in in-situ hybridization with indirectly labelled probes, and what is CISH?

Answer 11

Secondary detection systems are used for indirect labelling. CISH uses a chromogenic substrate for detection and involves an avidin-biotin complex for biotin-labelled probes.

Question 11

What is the principle of signal amplification in in-situ hybridization, and how does CARD work?

Answer 11

Signal amplification involves increasing target DNA/RNA. CARD (Catalysed Reporter Deposition) uses HRP to catalyze biotinylated tyramide, depositing biotin close to the hybridization site.

Question 12

How is signal amplification achieved in PISH and RT-PISH in in-situ hybridization?

Answer 12

Signal amplification involves applying PCR reagents for genomic targets or reverse transcription PCR for mRNA targets, followed by detection using anti-DIG antibodies.

Question 13

In cancer diagnosis, how can in-situ hybridization be used to identify patients who would benefit from therapy, particularly in NSCLC?

Answer 13

In NSCLC, in-situ hybridization can identify patients with EML4-ALK translocations who would benefit from ALK kinase inhibitors.

Question 14

Describe the utility of break-apart probes and dual fusion probes in in-situ hybridization for cancer diagnosis.

Answer 14

Break-apart probes detect separation of sequences, and dual fusion probes detect joining of sequences, aiding in the diagnosis of cancer-related translocations.

Question 15

In chronic myelogenous leukemia, what genetic event leads to the BCR-ABL oncogenic fusion, and how does in-situ hybridization contribute to treatment decisions with imatinib?

Answer 15

The reciprocal translocation between chromosomes 22 and 9 causes the BCR-ABL oncogenic fusion. In-situ hybridization helps determine if imatinib treatment will be effective.

Question 16

What does PISH stand for, and how is it applied to maximize signal production in in-situ hybridization?

Answer 16

PISH stands for PCR In-Situ Hybridization. It involves applying PCR reagents directly to tissue sections, followed by amplification using 5’-labelled PCR primers.

Question 17

How does in-situ hybridization contribute to the identification of abnormal genes and gene expression?

Answer 17

In-situ hybridization helps detect abnormal genes and gene expression by using labelled probes to identify specific DNA or RNA sequences in cells or tissue.

Question 18

What are the potential consequences of overfixation or slow fixation in the preparation of sections for in-situ hybridization?

Answer 18

Overfixation may result in decreased access to the probe, while slow fixation could lead to autolysis and the release of nucleases, which could digest the target DNA.

Question 19

Provide examples of molecular events that can be identified through in-situ hybridization, contributing to cancer diagnosis and therapy decisions.

Answer 19

Examples include identifying translocations (e.g., EML4-ALK), determining responses to therapy, and predicting the effectiveness of targeted treatments in various cancers.