HC11: General principles: molecular diagnostics

Molecular diagnostics

Molecular diagnostics is a subspecialty of pathology that utilizes molecular biology techniques to:

• Detect normal and disease states
• Make a prognosis
• Identify druggable targets

Molecular biology techniques utilize DNA, RNA and enzymes that interact with nucleic acids to understand biology at a molecular level:

• KRAS mutations
• EGFR deletion
• EML4-ALK or ROS translocations in NSCLC
• Et cetera

Therapeutic targets

(Proto)oncogenes have several therapeutic targets based on the hallmarks of cancer:

• Growth factor
• Growth factor receptor
• Intracellular signal transduction pathway
• Cell cycle regulators
• Transcription factors
• Anti-apoptotic factors

Abnormal activation of these targets leads to cancer. This activation can be caused by:

• Dominant mutations at cellular level
• Single mutations → 1 allele is sufficient

Aneuploidy:

In a tumor cell, chromosomes become heavily dysregulated. This can even lead to entire chromosomes changing. When the chromosome number changes, it is called aneuploidy.

Type of mutations

Activating mutations can be divided into 3 categories:

• Translocation
• Point mutations
  • Missense mutations
    • An amino-acid is changed into another amino-acid
  • Nonsense mutations
    • A stop codon is introduced instead of an amino acid → truncated protein
  • Silent mutations
    • Even though a base pair is changed, it still codes for the same amino acid
  • Indel/frameshift mutations
    • A base is deleted and the next nucleotide is taken for translation
• Amplification
  • A normal gene becomes amplified and has multiple copies

Use:

Molecular diagnostics are useful to diagnose several things:

• Tumor phenotype
• Malignancy
• Origin of the tumor
• Heredity

Therapy:

Therapy can consist of:

• Personalized medicine
• Treatment of cancer
• Drugs targeting specific mutations/pathways

Material available for testing:

The vast majority of tissue is frozen and put into paraffine slides. This can be used for histological images → the location of tumor cells can be identified.  

Tumor heterogeneity

A tumor consists of:

• Parenchym
  • Real cancer cells/neoplastic cells
    • Have the actual mutations
    • Surrounded by macrophages, fibroblasts, etc.
• Stromal cells
  • Connective tissue and vessels
  • Different type of normal cells

Lots of lymphocytes are visible around tumor cells.

Detection of mutations

A tumor is a mix of neoplastic cells and supporting “normal” cells. The tumor cell percentage is the estimated % of tumor cells. Tumor cells are genetically instable and have a large variety. They can be distinguished as follows:

• Normal cell
  • 2 wild type alleles
• Tumor cell
  • 1 wild type allele
  • 1 mutation

Testing

There are 87 FDA approved drugs. For each drug, there is a molecular target which needs to be tested in the lab for 45 different disease indications. Every drug acts upon 1 hallmark of cancer.

BRAF:

A BRAF melanoma is caused by a BRAF mutation of the BRAF inhibitor. The following is found:

• Biopsy: neoplastic tissue with <1% tumor cells
• Microdissection: approximately 10% neoplastic cells
• 2,5-5% BRAF mutant allele

The melanoma can be treated with BRAF inhibitors:

• Dabrafenib
• Vemurafenib

EGFR pathway:

EGFR is a receptor which sits in the membrane of the cell. When it becomes phosphorylated, it activates the PI3K or KRAS/BRAF pathway. In the cell, it leads to proliferation and survival → oncogenic transformation. EGFR is mainly mutated in lung cancer → deletion/insertion on exon 19 will activate EGFR.

Treatment with TKI’s can inhibit the PI3K or KRAS/BRAF pathway. Patients treated with EGFR therapy which are mutation positive live longer in comparison to chemotherapy. The opposite happens if they are EGFR negative.

The first generation of TKI’s works as follows:

1. EGFR has a pocket where ATP binds → TKI’s bind in the same pocket
2. ATP can no longer bind to EGFR → cannot be activated

Examples of first generation TKI’s are erlotinib and gefitinib. After a while, EGFR can become resistant → a T790M mutation leads to a confirmation change where TKI can no longer bind. This can be solved by a third generation TKI → osimertinib can act regardless of the T790M. However, EGFR can again develop resistance against osimertinib.

HER2:

In breast cancer, HER2 amplification occurs frequently. If it is amplified, the pathway is activated. There is a HER2/neu amplification found in the breast of 20-30% of breast cancer cases. Pertuzumab blocks HER2 dimerization and signaling.

Detecting mutations

Mutations can be detected via:

• Small biopsies
• Formalin fixed paraffin
• Embedded

There are many tests:

• H&E/IHC
• FISH
  • 1 probe works on 1 cell
  • There isn’t enough material to apply this on every cell
• qPCR

However, the material is limited. It also is very labor intensive and expensive.

PCR:

PCR makes it possible to amplify 1 molecule from a cell and make billions of copies of it. This was a revolutionary discovery and is very useful for diagnostics.

Sanger sequencing method:

Using Sanger sequencing, the sequence of 1 molecule can be determined. The DNA code and mutations can be identified, from example deletions of nucleotides and subsequent frameshifts.

Next generation sequencing:

Next generation sequencing (NGS) is a form of massive parallel sequencing. Millions of nucleotides can be identified in 1 piece of tissue. Massive sequencing is done:

• Whole genome
  • Structural variants
  • Point mutations
  • Copy number variation
• Whole exome (1%)
  • Point mutations
  • Copy number variation
• PCR amplicon: only the relevant parts are sequenced
  • Point mutations
  • Deletions

Amplicon based NGS is done on a cancer hotspot gene panel, of which all genes are important to be tested for cancer. 1 target in 1 entity is developed into many targets in all entities. This can be rapidly tested in the lab.

A method of NGS is ion torrent sequencing:

1. A chip goes into a machine
2. There are tiny holes with molecules fixed on the machine
3. There are lots of repetitions to sequence the DNA
4. There is an enzyme which follows the DNA sequence and incorporates a nucleotide into it
5. Each time there is a match, a signal is given

Another method of NGS is illumina sequencing:

1. PCR is done on a chip
2. Every time a nucleotide is added a different colored signal is given.

Data analysis

Bioinformatics are necessary to make sense of the results of for example whole genome sequencing:

• Make sense out of the data
• Match millions of sequences to the genome
• Identify differences, but not mistakes