Next-generation sequencing (NGS): applications and implications

 PCR: is based on DNA amplification

o In order to have PCR reaction you mix:
 Primers: you need them because you need to tell your reaction where you
want to amplificated
 You see the amplification by fluorescence
 With the amplificated DNA you can do a Sanger sequencing and translate
what you have in the reaction into letters (nucleotides) to determine if the area
amplificated is normal or not
 You can know this by comparing with a reference sequence
 If the sequence is normal  wild type
 If the sequence is abnormal  mutation
 Single mutation
 Deletion
 Insertion
 Translocation and amplification
 NGS:
o Massive sequencing (you can add as many primers as you want)
o It allows us to obtain several results
o Is a very robust technique
o Linked to personalized medicine
 Most of drugs that are being used targets mutation
 Why do I need to start thinking about NGS?
o Past: Molecular Biology to subclassify tumours and to target therapy - specific
mutation status (e.g. KRAS, BRAF, EGFR)
o Present: solid tumour and hematologic malignancies - genetically complex
o Future: Characterization of tumours  sequencing of multiple genes: panel of
selected genes, exome, transcriptome, or genome approach
 Morphological heterogeneity: non-genetic heterogeneity in normal tissues
o Phenotypic identities of normal cells  all the cells in an individual share the same
genotype
o There is a range of variations included in a normal tissue, it is important to establish
what is normal in order to be able to identify what is abnormal (cancer)
o Non-genetic heterogeneity
 Deterministic heterogeneity in normal tissues: the heterogeneity of cellular
phenotype that can be mapped to a tissue-specific differentiation hierarchy
 Stochastic heterogeneity: cellular phenotypes within given deterministic states
are not static: cell-to-cell variability emanating from the stochastic character
of biochemical processes within the cells
 Genetic heterogeneity: the initiation and progression of cancers are dependent on the
acquisition of multiples driver mutations that activate oncogenic pathways and that inactivate
tumour suppressors
 Determinants of phenotypic heterogeneity (in order to decide whether a sample is benign or
malignant)
o Deterministic heterogeneity
o Stochastic heterogeneity
o Genetic heterogeneity
 The cancer is formed by different diseases because of the heterogeneity of the different clones,
one clone is one disease. It changes the way you treat cancer.
o If the tumour start proliferating fast, there is a chance that something goes wrong
(mutations)
o The higher the proliferation rates of the tumour, the higher is the probability of
generating mutations.
o Each clone has different genetics, and every clone is a disease
o Every cancer starts with one cell that have a specific mutation that allows it to
proliferate in an abnormal way. Higher the proliferation rate, higher the mutation rate.
 Molecular genetics in Lung Cancer: adenocarcinoma, large-cell carcinoma, small-cell lung
cancer
o The genetics need to be described
 Independently of the histological subtypes, the genetics is much more
complex because there are different kinds of mutations in this cancer types
 Some tumours share the same mutations
 In the near future, we don’t care of the histology of the tumour because it will
not tell us if it will respond at KRAS inhibitors or EGFR inhibitors

 Tumour cell morphology

o Malignant cells: acceleration of the cell cycle; genomic alterations; invasive growth;
increased cell mobility; changes in the cellular surface; secretion of lytic factors, etc.
 Morphological characteristics: large nucleus, irregular size and shape,
prominent nucleoli, scarce cytoplasm - intensely coloured or pale
o Tumour assessment: estimation of tumour cell percentage for molecular testing
 Estimates of tumour cell percentages on HE stained slides are not accurate,
which could result in misinterpretation of test results
 You need to know the proportion of normal tissue and tumour tissues on order
to be your results reliable
 The ability to reliably detect mutations in samples consisting of a mixture of
tumour cells and normal cells, especially when the tumour content is very low
is the major challenge of any molecular test
 The minimally required percentage of tumour cells varies widely
 Approximately 25% tumour cells for direct sequencing
 Tumour cell estimate: the percentage of tumour cells relative to other cells
(eg, stromal cells, inflammatory infiltrate, and pre-existing epithelial cells)
 Before running a genetic experiment, you have to evaluate the proportion of
tumour cells present in the sample
 The NGS is performed in a chip with several wells, in each well you add nucleotides and if
this nucleotide is complementary to the sequence located in the well a hydrogen is released
giving a change in the pH. PH change is converted into voltage that is recorded in a computer.
 Allele frequencies in DNA pools: you must sequence with high coverage in order to detect
rare variants with lower frequencies (in order to check for mutations  my cancer genome)