Class 12 DNA sequencing and cancer
DNA pol error rate ~10-9 per base copied
How many errors in a “typical” somatic cell?
Most errors don’t have detectable effects
But some errors do: oncogenesN – dominant if “activated”tumor suppressor genesN – recessive
2-hit hypothesis in inherited cancer syndromes, BRCA, FAP
loss of heterozygosity in tumor DNACancer nowadays viewed in molecular-genetic terms
Implications for therapy
Can inhibit some overactive oncogenes with small molecule inhibitors (imatinib, etc) often act intracellularly or with antibodies to cell surface receptors
(panitumimab, etc) that act in pathwaysthat stimulate intracellular oncogenes
But can’t replace function of inactive suppressors
Example of pathway activating oncogenesExtracellular ligand(epidermal growth factor,EGF) binds EGF receptor,which binds another protein, which causes cytoplasmic tail of EGFR to get phosphorylated,which activates otherproteins (here including Ras oncogene)…which turn on other genesthat stimulate cell growth. Antibody to EGFR may stop process, but if Ras is mutated and constitutively active, Ab to EGFR won’t work because Ras is “downstream”Image from Google search “egfr kras signaling pathway”
Kras mutated and constitutively active in ~40% ofcolon cancers
Large effort has gone into whole genomesequencing of tumors and comparisonto non-tumor DNA from same patient
What are main results?several hundred oncogenesseveral hundred tumor suppressor genesorganized in at least tens of pathways
Tumors are “clonal” but continue to acquire mutations
When you sequence a tumor, do you get sequence ofmajority of cells or of individual cells, withunique mutations?
What are “driver” vs. “passenger” mutations?
What are some clues to identifying driver mutations?
occurrence in multiple tumorsmutated in inherited cancer syndromes
Do you think there a more tumor suppressor mutationsor oncogene mutations driving tumors? Why?
How fast do tumors grow?cell birth rate b (# divisions/day, ~1/few days)
balanced by cell death rate dcell doubling rate k, N(t)=N02kt
k related to b-d
More ways to inactivate a gene (stop codon nearly anywhere) than to make it overactive, so suppressor mutations should exceedactivating oncogene mutations, but need toinactivate both copies of a suppressor, soanswer not obvious
Types of cancer therapysurgery – curative intent or for palliationradiationchemo to kill rapidly dividing cells
-> toxicity from killing normal rapidly dividingcells in gut, bone marrow, skin
drugs or antibodies that target oncogenescould be more specificbut still often have major side-effectsexamples – antibody to EGFR (drug names
ending in “ab” are antibodies) small drug inhibitors (drug names
ending in “ib” are inhibitors)
Problem of “development” of resistance to chemo
Roles of DNA sequencing
Research – find what genes are involved in cancerbig challenge – interpreting changespassenger vs driver mutationsare mutations in non-coding regions (98.5% of total)
important?which mutations in coding regions are relevant?
Patient carewhich genes are mutated in a specific tumor?is whole genome seq. necessary or would seq. of ~hundred known oncogenes and suppressors do?
Patient care – cont’d.
diagnostics – circulating tumor DNA akin topre-natal dx from circ. fetal DNA
? useful for screening or just dx of already ill? use to follow treatment – ? more sensitive
than other biomarkers, e.g. CEA, PSAdo genetic assays need to be specific for individual
patient’s mutations or are mutationssufficiently common that “generic” tests ok?
“Beaming” assay emulsion pcr for particular oncogenes
-> copies single templates on beadsbreak emulsion, hybridize flourescent oligo probes
to beads, different colors for oligosmatching wt, mutant, and common seq.
determine bead color with flow cytometry
http://openwetware.org/wiki/Image:Flow_cytometry
pre-op day 3
day 48 day 244
What is plotted?What do #sin quadrantsindicate?
Beaming assay for Kras mutations from Vogelstein
How sensitive is assay to mutations occurringin fraction of tumor cells as tumors evolve?
What fraction of circulating DNA is from tumor? How many beads can you assay?
Use of sequence info in therapy
possibly to identify unexpected mutations (e.g. uncommon in patient’s tumor type) that might suggest use of different drug – this is hypothetical
identify drugs unlikely to be effective – e.g. Ab to EGFRin pts with oncogenic Kras mutations
Use of sequence info in therapy
relevance to patients – avoid (often severe) toxicityin patients in whom drug won’t work(panitumimab has lots of toxic skin, gut effects)
relevance to payors - @$1000’s/dose, cheaper to genetest everyone to avoid use when predictably ineffective = “companion diagnostics”
relevance to pharmaceutical companies – use in resistant patients weakens evidence for efficacy, lack of efficacy is major cause of failure to get FDA approval
Questions from this paper
How fast do tumors (cells resistant to chemo) grow?How sensitive are tests for tumor mutations?What is normal mutation rate?What is probability that particular oncogene mutation
has occurred?How many mutations -> drug resistance?Do resistance mutations pre-exist in tumors, explaining
usual drug failure after few months?Implications for multi-drug therapy
How would you describe the patients in this study?
What is progression-free vs.overall survival?
Does prior Kras mutation predict poor response?How long before progression in those w/acquired
Kras mutations?
What do panels show?
Do mutationsor CEA ortumor sizeassays predict treatment failure sooner?
What is doubling rate?
patient 1 patient 2
If doubling time t is ~10d and progression time T is ~150 weeks
how much has mutant cell # increased in time T?
N/N0 = 2T/t = 215 = 3*104
How much circulating DNA?How many cell equivalents in 1ml @6pg/cell? What fraction f is from tumor cells vs. normal cells?
What are these plots?
wtmutant
How many dots?What is the lowest % (or number) mutant detectable?Suppose 1 mutant dot is reliable and 105 dots ->min fraction of mut. tumor cells detectable = 1/(f*105)If f = 0.1%, 1% tumor cells is min detectable
How many tumor cells in a 100mm2 (x-ray) tumor
Tumor vol = (area)3/2 = 1000mm3
Cell vol ~ (10mm)3 => 109 tumor cells
If 1% are mutant when mutation first detected, howmany were there before panatumumab was started?
107/(3*104) = 3*102
Is this consistent with expectation if DNA polmakes 1 base error every generation andyou have 109 cells => 109 genomes copied?
-> ~1 error in every position
If 42 positions confer resistance to panitumumab(their estimate), expect ~40 mutant cells to pre-exist; not too far off estimate of 300 givenlarge variance in rates of doubling, etc.
If 40 (or 300) mutant cells are expected to bepresent, on average, by chance in small tumor,what is probability that a tumor has no such cells?
Poisson distribution pi=e-mmi/i!pi = probability of a tumor having i
when average number/tumor = mp0 = e-m = e-40 or e-300 = 10-18 or 10-131
What is chance that at least 1 cell in tumor with 109 cellshas oncogene mutations conferring resistance to 2 different drugs, if the mutations do not overlap and changes at 40 positions confer resistance to each drug?
(40/109) * (40/109) * 109cells @ 10-6
Implication – multidrug therapy might avoidoutgrowth of resistant mutants
Main ideas
Mutations in cancer cells drive growthgain of function = oncogenesloss of function = tumor suppressor genes
Some drugs target oncogenes by binding to themor their partners in cell signaling cascades
Mutations conferring resistance to individual drugs likely preexist in tumors becausethey contain large numbers of cellsharboring mutations just on basisof DNA pol error rate
Multidrug therapy targeting different oncogenes/pathways might overcome these resistancemechanisms, but …
DNA sequencing has been important for discoveryof different mutations driving cancer
Often difficult to determine if individual mutationsare drivers or passengers
Genotyping specific genes in patient tumor DNAs tosee if most tumor cells already carry resistance-causing mutations can prevent futile use of expensive toxic drugs
Not clear if routine sequencing of exons orwhole tumor genomes is useful clinicallyat present, as opposed to targetedgenotyping or sequencing
“Beaming” is nice use of emulsion pcr and flowcytometry to detect not too rare mutations intumor cells
HAPPY THANKSGIVING – work on picking a topic forstudent presentations beginning 11/30