BM422 - Block A (NSCLC)

what is non small cell lung cancer?

any type of epithelial lung cancer other than small cell lung cancer (SCLC)

how does NSCLC compare to SCLC?

non:

= larger cells that grow more slowly

= more common

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small:

= smaller cells that grow/spread rapidly

= linked to smoking

what % of NSCLCs are squamous cell carcinomas?

30%

what % of NSCLCs are adenocarcinomas?

50%

in squamous cell carcinoma NSCLC, what driver mutations are commonly found?

- PIK3CA amplification (35%)

- FGFR1 (20%)

- unknown (20%)

- PIK3CA mutation (15%)

- PTEN (10%)

in adenocarcinoma NSCLC, what driver mutations are commonly found?

- unknown (40%)

- KRAS (30%)

- EGFR (15%)

- other (HER2/MEK, RET, etc) (1-5%)

which is more common in NSCLC, squamous cell carcinoma or adenocarcinoma?

adenocarcinoma

HER/MEK mutations are found in 2% of NSCLC adenocarcinomas while KRAS mutations are found in 30%: which is more likely to have a targeted treatment and why?

both are likely - medicines are developed regardless of mutation frequency

what is targeting driver mutations in cancer therapy an example of?

personalised medicine

what does imatinib target?

BCR-ABL

what type of drug is gefitinib?

EGFR inhibitor

what type of drug is trametinib and cobimetinib?

MEK inhibitors

what type of drug is sorafenib?

vascular EGFR inhibitor

what is targeted cancer therapy?

a type of cancer treatment that uses drugs which are targeted at pathways which are disrupted in cancer cells

what is the benefit of targeted cancer therapy?

avoids normal cells and goes directly to cancer cells

what does personalised cancer therapy involve?

molecular analysis to achieve optimum medical outcomes in the management of a patient's disease or disease pre-disposition

what are the three stages of personalised cancer therapy?

1 - molecular profiling

2 - identification of prognostic markers, markers predictive of drug sensitivity/resistance, markers predictive of adverse events

3 - treatment

what is meant by advanced stage NSCLC?

it is metastatic

as an example of clinical use of personalised medicine in cancer treatment, what treatment is now available for advanced stage NSCLC patients? (2) (general)

a number of targeted therapies aimed at particular driver mutations + several immunotherapies aimed at a patients own immune system

which driver mutation is most commonly targeted in NSCLC treatment?

EGFR

which driver mutations in NSCLC adenocarcinoma currently have FDA-approved targeted therapy drugs?

ALK

BRAF V600E

EGFR

KRAS

MET (exon 14 skipping)

NTRK

RET

ROS1

in addition to targeting driver mutations, what other drugs are approved for use in NSCLC treatment? (general category)

immune checkpoint inhibitors

in NSCLC treatment, what drug can target ALK?

alectinib, brigatinib, etc (inhibit ALK)

in NSCLC treatment, what drug can target BRAF V600E?

dabrafenib (BRAF inhibitor; given in combination with MEK inhibitor)

in NSCLC treatment, what drug can target EGFR?

gefitinib, afatinib, etc (EGFR inhibitors for exon 19 deletion or 21 substitution)

what is biomarker testing? what else is it known as?

looks for biological changes in genes or proteins (like EGFR etc) --- molecular testing

what samples are associated with biomarker testing? (type of sample)

a biopsy of the cancer

when is biomarker testing used diagnostically?

standard in lung cancer diagnosis

what process usually follows biomarker testing of biopsies?

grading/staging of tumours

in caucasians and asians with adenocarcinomas (NSCLC), how common is an activating EGFR mutation?

occurs in 15% of caucasians and 40% of asians --- majority of these are either exon 19 deletions or L858R point mutations in exon 21

what is a point mutation?

amino acid change

what tests/test strategies are used for EGFR(-TK (tyrosine kinase)) mutation testing? describe one example?

many

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DNA extraction from tissue biopsy (stored as formalin-fixed or paraffin-embedded tissue) >>> sanger sequencing >>> EGFR mutation kit >>> multiplex PCR

in EGFR-TK mutation testing, what EGFR mutation kit is used? what does it allow detection of?

COBAS EGFR mutation kit (Roche diagnostics) --- can detect 42 mutations in exons 18-21

in NSCLC EGFR mutations, what mutations can occur on exon 18 that are assessed for in multiplex PCR?

G719A/C/S

how do you interpret "G719A/C/S mutation"?

Glycine mutation, at position 719, to A, C, or S

in NSCLC EGFR mutations, what mutations can occur on exon 19 that are assessed for in multiplex PCR?

deletions and complex mutations

what is a complex mutation?

combination of a deletion and insertion

in NSCLC EGFR mutations, what mutations can occur on exon 20 that are assessed for in multiplex PCR?

S768I, T790M, insertions

in NSCLC EGFR mutations, what mutations can occur on exon 21 that are assessed for in multiplex PCR?

L858R and L861Q

what is meant by an L858R mutation?

leucine to arginine at position 858 (on exon 21 in EGFR)

in general, what reagents are required for real time PCR detecting EGFR-TK mutations?

complimentary primer pairs and fluorescently labelled probes (and prepared sample)

what issue exists that is trying to be addressed regarding EGFR TKI therapy?

often 9-14 months after EGFR TKI therapy, patients will develop further mutations in the EGFR gene (~60% of patients)

what secondary mutation (i.e., after EGFR TKI therapy) is the most common in EGFR?

T790M in exon 20 (~60% of patients develop)

what is the preferred material for EGFR mutation analysis?

biopsy

what other material can be used for mutation analysis? (i.e., other than biopsy)

liquid biopsy - cell free tumour DNA (cfDNA) extracted from peripheral blood

why is biopsy preferred over liquid biopsy (cfDNA)? why?

liquid biopsy has reduced sensitivity - a negative result cannot excluse the presence of a mutation (hence biopsy is strongly recommended if clinically appropriate)

what is cell free DNA?

DNA released by dying cells (dead tumour sheds DNA into blood > can isolate DNA from blood sample)

what is osimertinib?

EGFR inhibitor (first line treatment for EGFR mutation)

on what basis are liquid biopsies conducted? why?

case-by-case basis - they can be more sensitive to some mutations (and so would be useful for those)

what mutation are liquid biopsies sensitive to?

T790M

what is meant by T790M?

threonine to methionine at position 790

how common are KRAS mutations in caucasian and asian NSCLC patients?

found in 30% of NSCLC caucasians and 15% of asians

what type of patient (i.e., what mutation do they have) is unlikely to respond to EGFR therapy?

those with EGFR-sensitising mutation and/or KRAS mutation

why are patients with EGFR sensitising mutation/KRAS mutation unlikely to respond to EGFR therapy?

in these patients, even with EGFR blocked, the pathway will still occur (because now KRAS is the issue (it is the first intracellular step in the pathway))

what specific mutation can be targeted in NSCLC patients with KRAS mutation?

G12C mutation

G12C mutations are UV-specific; what does this mean? (KRAS/NSCLC)

the mutation is directly linked to UV-induced DNA damage

what technique is used for KRAS mutation analysis?

PCR followed by pyrosequencing or next gen sequencing

what specific mutations are assessed in KRAS mutation analysis?

mutations in exons 2, 3, or 4

what is the ALK gene?

anaplastic lymphoma kinase - tyrosine kinase normally expressed in neuronal cells

what association exists between ALK and NSCLC? how common is this?

ALK can form a fusion protein (by deletion or translocation) which is abnormally expressed in 2-5% of NSCLC patients

what can the presence of ALK gene rearrangement predict in relation to NSCLC treatment?

response to ALK inhibitor therapy for cancer treatment

what biochemical pathway is ALK associated with?

MAPK

what is the most common ALK fusion protein? what does this fusion protein achieve?

EML4-ALK --- makes the receptor always active

what technique is used to assess ALK mutation/expression?

immunohistochemistry - FISH (fluorescence in-situ hybridisation)

why is PCR not used for ALK mutation analysis when it is used for KRAS and EGFR?

ALK mutation = gene rearrangements

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detecting gene fusions would require primers designed for each possible fusion event - too many to possibly predict

in terms of mutation analysis, what is the drawback of FISH for ALK?

it cannot identify specific mutations - can only tell you about presence/location of ALK protein

you are presented with two pathology slides - the left shows regions of pink with lots of distinct purple cells, the right shows an indistinguishable brown stain - you were trying to analyse ALK, what is your interpretation of each slide?

left = no/very little ALK

right = lots of ALK

why is it sufficient to just look at the presence/location of ALK in cancer therapy?

regardless of the specific mutation, ALK should only be present in low amounts - any increase in presence indicates mutations/tumour

when using FISH for ALK detection - what is considered a negative result?

negative = unlikely to have mutation --- if <5 of 50 cells are positive

when using FISH for ALK detection - what is considered a positive result?

negative = has mutation --- if >25 of 50 cells are positive

what type of probe is used in ALK FISH? what does this type of probe do?

break apart probe --- targets two regions of the gene (typically each end) (typically one green and one red) --- allows visualisation of the gene fusing/being broken apart

in the context of ALK FISH, when are cells considered rearrangement positive?

when at least one set of red and green signals are two or more signal diameters apart

in the context of ALK FISH, when are cells considered fusion positive?

when green and red have separated (indicates break in gene)

what is ROS1?

a receptor tyrosine kinase

how frequent are ROS1 rearrangements in NSCLC patients?

occur in ~1% of patients

what technique is used to assess ROS1 gene mutations?

IHC (to assess expression of protein)

in general, what does a ROS1 mutation involve?

deletion or translocation leading to fusion protein (not always with same protein) - switches receptor in "on" position

where can the ROS1 protein be present?

plasma membrane, in cytosol, golgi appratus

what type of signals will be observed in ROS1 FISH if the ROS1 has mutated?

fused and split signals (from 3' probe and 5' probe movement)

what therapy is considered for patients with evidence of ROS1 rearrangement?

ROS1 inhibitor therapy

in summary, what are patients diagnosed with NSCLC adenocarcinoma routinely tested for?

mutations in EGFR and KRAS, and ALK and ROS1 gene rearrangement

in summary, what is the purpose of biomarker testing?

can be used to decide which treatment a patient is likely to respond to