oncology

2. Introduction
• Cancer is, in essence, a genetic disease,and
that accumulation of molecular alterations in
the genome of somatic cells is the basis of
cancer progression.

3. • A genome is an organism’s complete set of DNA, including all
of its genes.
– Each genome contains all of the information needed to build and
maintain that organism.
– In humans, a copy of the entire genome—more than 3 billion DNA
base pairs—is contained in all cells that have a nucleus.
• The exome is the part of the genome formed by exons, the
sequences which when transcribed remain within the
mature RNA after introns are removed by RNA splicing.
• Transcriptome: all mRNAs
• Epigenome: change that leads genomic maintainance

6. • DNA sequencing is the process of determining
the precise order of nucleotides within
a DNA molecule. It includes any method or
technology that is used to determine the
order of the four bases—
adenine, guanine, cytosine, and thymine—in a
strand of DNA

7. CANCER GENES AND THEIR
MUTATIONS
Oncogenes
• car accelerator
– mutation in an
oncogene=accelerator
continuously pressed
• Mutations in oncogenes
– at specific hotspots, often
affecting the same codon or
clustered at neighboring
codons in different tumors.
– almost always missense,
– affect only one allele-
heterozygous.
Tumour supressor genes
• brakes,
– so that when they are not
mutated, inhibit tumorigenesis
• mutated throughout the gene
• a large number of the
mutations may truncate the
encoded protein and generally
affect both alleles, causing loss
of heterozygosity (LOH).

8. • Major types of somatic mutations present in
malignant tumors
– nucleotide substitutions,
– Small insertions and deletions (indels),
– chromosomal rearrangements,
– copy number alterations

9. • The first gene families to be completely sequenced
were those that involved protein and lipid
phosphorylation.
• The rationale for initially focusing on these gene
families was threefold:
– The corresponding proteins were already known to play a
pivotal role in the signaling and proliferation of normal and
cancerous cells.
– Multiple members of the protein kinases family had
already been linked to tumorigenesis.
– Kinases are clearly amenable to pharmacologic
inhibition,making them attractive drug targets.

11. Genome sequencing
• Genome sequencing is figuring out the order
of DNA nucleotides, or bases, in a genome—
the order of As, Cs, Gs, and Ts that make up an
organism's DNA.
• The human genome is made up of over 3
billion of these genetic letters

12. History of Sequencing
• Allan Maxam and Walter Gilbert developed an
important method of DNA sequencing in
1976-1977.
• This method of chemical modification of DNA
was technically complex and fallen out of
flavor due to the use of extensive hazardous
chemicals, and difficulties with scale-up.

13. History of Sequencing
• Sanger and his team developed the chain-
termination method of DNA sequencing in
1977.
• Only be used for fairly short strands (100 to
1000 base pairs) and longer sequences must
be subdivided into smaller fragments.
• After this, these small fragments subsequently
re-assembled to give the overall sequence

14. History of Sequencing
• Shotgun sequencing has been developed for
sequencing of large fragments of DNA in
1979.
• DNA is broken up randomly into numerous
small segments, which are sequenced using
the chain termination method and then short
reads have been produced.
• Shotgun sequencing was the initiative for full
genome sequencing.

15. Whole genome sequencing
• Information about coding and non coding part of an organism.
• To find out important pathways in microbes.
• For evolutionary study and species comparison.
• For more effective personalized medicine (why a drug works for person X
and not for Y).
• Identification of important secondary metabolite pathways (e.g. in plants).
• Disease-susceptibility prediction based on gene sequence variation.

16. Roche’s 454 FLX Ion torrentIllumina ABI’s Solid
Next generation sequencing
• Sequence full genome of an organism in a few days at a very low cost.
• Produce high throughput data in form of short reads.

17. Whole genome sequencing
• Complete sequencing of the genome of cancer
tissue at high redundancy, using germline DNA
sequence from the same patient as a
comparison:
– Powerful to discover the full range of genomic
alterations
– It requires high amount of sequencing
– It is the most costly.

18. • The major potential of whole-genome
sequencing for cancer:
– Discovery of chromosomal rearrangements
– Somatic mutations of non-coding regions
– rearrangements of repetitive elements

19. • The first solid cancer to undergo whole-genome
sequencing was a malignant melanoma that was
compared to a lymphoblastoid cell line from the
same individual.
• Most somatic base substitutions were C:G > T:A
transitions, and of the 510 dinucleotide
substitutions, 360 were CC.TT/GG.AA changes,
which is consistent with ultraviolet light exposure
mutation signatures previously reported in
melanoma.

20. • This study shows that a significant correlation
exists between the presence of a higher
proportion of C.A/G.T transitions in early
(82%) compared to late mutations (53%).
• cancer mutations are spread out unevenly
throughout the genome, with a lower
prevalence in regions of transcribed genes,
suggesting that DNA repair occurs mainly in
these areas.

21. • An interesting and pioneering example of the
power of whole genome sequencing in
interpretation of the mutation evolution in
carcinogenesis was seen in a study in which
– a basallike breast cancer tumor, a brain
metastasis, a tumor xenograft derived from the
primary tumor, and the peripheral blood from the
same patient were compared.

23. • Wide range of mutant allele frequencies in the
primary tumor, which was narrowed in the
metastasis and xenograft samples.
• This suggested that the primary tumor was
significantly more heterogeneous in its cell
populations compared to its matched
metastasis and xenograft samples because
these underwent selection processes whether
during metastasis or transplantation.

24. • The importance of performing whole-genome
sequencing has also been emphasized by the recent
identification of somatic mutations in regulatory
regions, which can also elicit tumorigenesis.
• In a study reviewing the noncoding mutations in 19
melanoma whole-genome samples,
– 2 recurrent mutations within the telomerase reverse
transcriptase (TERT) promoter region in 17 of the 19 cases.
– When these two mutations were investigated in an
extension of 51 additional tumors and their matched
normal tissues, it was observed that 33 tumors harbored 1
of the mutations and that the mutations occurred in a
mutually exclusive manner

25. • As the TERT promoter mutation discovery
shows, regions of the genome that do not
code for proteins are just as vital in our
understanding of the biology behind tumor
development and progression.
• Another class of non–protein-coding regions
in the genome are the noncoding RNAs.
• One class of noncoding RNAs are microRNAs
(miRNA)

26. • miRNAs
– known to be expressed in a tissue or developmentally
specific manner
– their expression can influence cellular growth and
differentiation along with cancer-related pathways such as
apoptosis or stress response.
• miRNAs do this through either
– overexpression,
• leading to the targeting and downregulation of tumor suppressor
genes,
– inversely through their own downregulation,
• leading to increased expression of their target oncogene.
• Extensively studied in gliomas and breast cancer.

27. • Recent analysis of whole genomes of many
different human tumors has demonstrated
that
– Multiple mutational processes are operative
during cancer development and progression, each
of which has the capacity to leave its particular
mutational signature on the genome.

28. • Kataegis -distinctive phenomenon of localized
hypermutation
• Chromothripsis implies a massive genomic
rearrangement acquired in a one-step
catastrophic event during cancer development
and has been detected in about 2% to 3% of
all tumors, but is present at high frequency in
some particular cases, such as bone cancers.

29. • Chromoplexy has been originally described in
prostate cancer
– involves many DNA translocations and deletions
that arise in a highly interdependent manner and
result in the coordinate disruption of multiple
cancer genes

30. Whole exome sequencing
• Exome sequencing (also known as Whole Exome
Sequencing, WES or WXS) is a technique for
sequencing all the expressed genes in a genome
(known as the exome).
• It consists of first selecting only the subset of
DNA that encodes proteins (known as exons), and
then sequencing that DNA using any high
throughput DNA sequencing technology.
• There are 180,000 exons, which constitute about
1% of the human genome, or approximately 30
million base pairs

31. • It increases sequence coverage of regions of
the coding exons of genes at lower cost and
higher throughput compared with WGS
• Powerful and effective approach to focus on
the coding genes of greatest interest in many
cancer studies
– Somatic mutions
– Damaging mutations
– Allow large data set analysis at lower cost

32. • Target-enrichment strategies
– Target-enrichment methods allow one to
selectively capture genomic regions of interest
from a DNA sample prior to sequencing.
• PCR
• MIP(molecular inversion probes)
• Hybrid capture
• In-solution capture

33. • Limitations:
– uneven capture efficiency across exons
– some off-target hybridization can occur

34. 34

35. Structural
Genomics
• Determination of genome sequence-virus,
bacteria,yeast,plants,mammals
• Determination of genome variability-intra species
polymorphisms
• Determination of genomic evolution-inter species
polymorphisms
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36. Structural genomics aims and techniques
• Automated, high throughput
fluorescent sequencing
(size-dependent time)
• micro-array-based resequencing
(size independent time)
• single nucleotide polymorphism
detection techniques
• ultra-microscope-based sequencing (SNP)
• direct polymerase output sequencing
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37. Structural genomics current impact on clinics
• More thorough assessment of cancer-linked
predispositions
• Discovery of genes linked to neuro-
degenerative conditions
• microsatellite polymorphism markers
• chromosomal painting
• Identification of age-linked gene loci
• amelioration and speeding up of molecular
diagnostics
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38. Comparative Genomics
• Analyzing DNA sequence patterns of different organisms
side by side
• to identify genes and determine functions
• Looking for similar genes in different species to determine
possible relationships and genomic variations
• Identify evolutionary changes such as genomic additions or
deletions
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39. Functional Genomics
• Beginning to understand what the sequence codes
for…
• Transcriptomics
• Genomic Variation
• Proteomics
• Comparative genomics
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40. Transcriptomics
Large-scale analysis of mRNA
Gene Chip
Microarray
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41. 41

42. THE DNA CHIP
• A single microarray (or DNA chip) can be used to
screen 100,000 SNPs in a patient's genome in a
matter of hours
• Southern blotting is the principle
• Unknown "target" nucleic acid [IN SOLUTION] is
discerned by its complementarity with the nucleic
acid "probe" (oligonucleotide) [STATIONARY] of
known sequence
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43. 43

44. 44

45. Competitive Hybridization
• Two probes are hybridized to the microarray at the
same time to quantitatively measure and compare
the gene expression in both samples at the same
time
• Target -DNA molecule spotted on the slide
• Probe -Complementary cDNA molecule labeled
with fluorescent dye that binds to the target cDNA
spotted on the slide
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46. 46

47. Microarray
• Used to evaluate genes and their expression
• Interpret when a particular gene is turned on or
turned off
• Look for genes that may play a role in causing or
suppressing diseases and cancers
• Answer questions about complex processes within
organisms
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48. Biomedical Applications
• DNA microarrays have been used to
understand and diagnose cancer
• Microarrays have also been used to
understand how medications work and
how to improve their effectiveness
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49. SOMATIC ALTERATION CLASSES
DETECTED BY CANCER GENOME
ANALY SIS
• Nucleotide substitutions - the most frequent
somatic mutations.
• Human malignancies have 1 nucleotide change
per million bases,
– but melanomas reach mutational rates 10-fold higher,
– tumors with mutator phenotype caused by DNA
mismatch repair deficiencies may accumulate tens of
mutations per million nucleotides.
– tumors of hematopoietic origin have less than one
base substitution per million.

50. • For years, the main focus of cancer genome
analyses has been on identifying coding
mutations that cause a change in the amino acid
sequence of a gene
• any mutation that creates a novel protein or
truncates an essential protein has the potential to
drastically change the cellular environment(BRAF
KRAS)
• advancements in next-generation sequencing- to
detect mutations occurring in the cancer genome
at a lower frequency.

51. • Small insertions and deletions (indels) represent a
second category of somatic mutations that can be
discovered by whole-genome sequencing of
cancer specimens.
– 10-fold less frequent than nucleotide substitutions,
but may also have an obvious impact in cancer
progression
• identification of large chromosomal
rearrangements in cancer genomes represents
one of the most successful applications of next-
generation sequencing methodologies

52. • a combination of bioinformatics and functional methods
has allowed for the finding of recurrent translocations in
solid epithelial tumors such as TMPRSS2–ERG in prostate
cancer and EML4–ALK in non–small cell lung cancer.
• By using a next-generation sequencing analysis of genomes
and transcriptomes, it is possible to systematically search
for both intrachromosomal and interchromosomal
rearrangements occurring in cancer specimens.(e.g.
discovery of recurrent translocations involving genes of the
RAF kinase pathway in prostate and gastric cancers and in
melanomas)

53. • next-generation sequencing approaches have
also demonstrated their feasibility
– to analyze the pattern of copy number alterations
in cancer,
• because they allow researchers to count the number of
reads in both tumor and normal samples at any given
genomic region and then to evaluate the tumor-to-
normal copy number ratio at this particular region.

54. INTEGRATIVE ANALY SIS OF CANCER
GENOMICS
• Integration of genomic, epigenomic, transcriptomic,
and proteomic landscapes from tumor samples.
• AML, glioblastoma, medulloblastoma, and renal cell,
colorectal, ovarian, endometrial, prostate, and breast
carcinomas.
• In these cases, the integration of whole exome and
whole-genome sequencing with studies involving
genomic DNA copy number arrays, DNA methylation,
transcriptomic arrays, miRNA sequencing, and
proteomic profiling has contributed to improving the
molecular classification of complex and heterogeneous
tumors

55. THE CANCER GENOME AND THE NEW
TAXONOMY OF TUMORS
• Until the genomic revolution, tumors had been
classified based on two criteria:
– their localization (site of occurrence)
– their appearance (histology).
• For many decades, it has been known that
patients with histologically similar tumors have
different clinical outcomes.
• Furthermore, tumors that cannot be
distinguished based on an histologic analysis can
respond very differently to identical therapies.

56. • Frequency and distribution of mutations- used to
redefine the histology-based taxonomy of a given
tumor type.
• Activating mutations in the receptor tyrosine
kinase EGFR in lung adenocarcinomas.
– Defines a subtype of non–small-cell lung cancers
(NSCLC) that occur mainly in nonsmoking women
– Better prognosis,
– Typically respond to epidermal growth factor
receptor(EGFR)-targeted therapies

57. • Recent discovery of the EML4-ALK fusion
identifies yet another subset of NSCLC that is
clearly distinct from those that harbor EGFR
mutations that respond to ALK inhibitors
• Another example: KRAS & BRAF mutations in
CRC.

58. • KRAS and BRAF mutations-mutually exclusive.
• Prognosis of CRC pts with wild type
KRAS/BRAF – more favourable compared pts
with these genes mutations.
• Mutations in these genes-impair
responsiveness to the anti-EGFR monoclonal
antibodies therapies in CRC patients

59. • Cancers are heterogeneous,
– intratumoral heterogeneity - with different areas
of the same tumor showing different genetic
profiles
– likewise, heterogeneity exists between metastases
within the same patient (i.e.,
intermetastaticheterogeneity).
• Biopsy from one part of a solitary tumor will
miss the molecular intratumoral as well as
intermetastatic heterogeneity.

60. Which tissue to study for DNA
analysis?
• In 1948, the publication of a manuscript describing the
presence of cell-free circulating DNA (cfDNA) in the
blood of humans- probably without realizing
unprecedented opportunities in this area.
• Several groups have reported that the analysis of
circulating tumor DNA can provide the same genetic
information obtained from tumor tissue.
• The levels of cfDNA are typically higher in cancer
patients than healthy individuals, indicating that it is
possible to screen for the presence of disease through
a simple blood test.

61. Challanges to detection of ctDNA
• Discrimination between DNA released from
tumor cells (ctDNA) and circulating normal DNA
• Discerning ctDNA from normal cfDNA is aided by
the fact that tumor DNA is defined by the
presence of mutations.
• These somatic mutations,
– commonly single base pair substitutions, are present
only in the genomes of cancer cells or precancerous
cells and are absent in the DNA of normal cells of the
same individual

62. • Next-generation sequencing strategies and
digital PCR
– define rare mutant variants in complex mixtures of
DNA.
• It is possible to detect
– point mutations
– Rearrangements
– gene copy number changes in individual genes
from a few milliliters of plasma

63. • The detection of tumor-specific genetic alterations in
patients’ blood (liquid biopsies)
• Applications:
– Analyses of cfDNA can be used to genotype tumors when a
tissue sample is not available or is difficult to obtain.
• Circulating tumor DNA fragments contain the identical
genetic defects as the tumor themselves,
– thus the blood can reveal tumor point mutations (EGFR,
KRAS, BRAF, PIK3CA),
– rearrangements(e.g., EML4-ALK),
– tumor amplifications(MET).

64. • Useful in monitoring tumor burden
• Another application of ctDNA is the detection of
minimal residual disease following surgery or therapy
with curative intent.
• Finally, liquid biopsies can be used to monitor the
genomic drift (clonal evolution) of tumors upon
treatment.
• In this setting, the analysis of ctDNA in plasma samples
obtained pretreatment, during, and posttreatment can
lead to an understanding of the mechanisms of
primary and, especially, acquired resistance to
therapies.

65. CANCER GENOMICS AND DRUG
RESISTANCE
• In most tumor types, a fraction of patients’
tumors are refractory to therapies (intrinsic
resistance).
• Even if an initial response to therapies is
obtained, the vast majority of tumors
subsequently become refractory (i.e., acquired
resistance),
• And patients eventually succumb to disease
progression.
• Therefore, secondary resistance should be
regarded as a key obstacle to treatment progress.

66. • Gliomas that recur after temozolomide treatment have
been shown to harbor large numbers of mutations with
a signature typical of a DNA alkylating agent.
• Although temozolomide has limited efficacy, almost all
of the cells in a glioma respond to the drug.
• However, a single cell that was resistant to the
chemotherapy proliferated and formed a cell clone.
• Later genomic analyses of the cell clone allowed for the
identification of the underlying mutated resistance
genes

67. • Single-molecule–targeted therapy is almost
always followed by acquired drug resistance.
• Despite the effectiveness of gefitinib and
erlotinib in EGFR mutant cases of NSCLC drug
resistance develops within 6 to 12 months
after the initiation of therapy.
• The underlying reason for this resistance was
identified as a secondary mutation in EGFR
exon.

68. • Some studies have shown the mutation to be
present before the patient was treated with
the drug.
• A recent elegant study, which also represents
the use of genomics in understanding drug-
resistance mechanisms, focused on the
inhibition of activating BRAF (V600E)
mutations, which occur in 7% of human
malignancies and in 60% of melanomas.

69. • 9 Clinical trials using PLX4032, a novel class I RAF-
selective inhibitor, showed an 80% antitumor
response rate in melanoma patients with BRAF
(V600E) mutations; however, cases of drug
resistance were observed.
• The use of microarray and sequencing
technologies showed that, in this case, the
resistance was not due to secondary mutations in
BRAF, but due rather to either upregulation of
PDGFRB or NRAS mutations.

70. Human Genome Project (HGP)
• A 13 year project where all of the genes in the
human DNA were discovered
• Determined the sequences of approximately 3
billion chemical base pairs that make up the human
DNA
• Completed in 2003 by the U.S. Department of
Energy and the National Institutes of Health with
help from many other countries including Japan,
Germany and France
70

71. What We Know About The Human
genome
• 3 billion bases ; 30,000 –35,000 genes
• 3000 nucleotides/gene (on the average)
• 99 % nucleotide similarity to chimpanzees
• Less than 2% codes for proteins
• Chromosome 1 has the most genes
• Chromosome Y has the fewest genes 71

72. Applications of the HGP
• Able to determine the molecular cause of
disease
• Identify new drugs
• Compare human genome with other
organisms to understand diversity
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