general oncology from devita presentation

1. DR RAJDEEP GUPTA
FIRST YEAR MEDICAL ONCOLOGY
DATE 27/10/2018
Tumor mutation burden and Next
generation sequence testing

2. Next-generation DNA sequencing (NGS)
• DNA sequencing technology that uses parallel
sequencing of multiple small fragments of
DNA to determine sequence.

3. • Types of testings :
1. Sanger ( first generation, conventional ,
traditional )
2. NGS ( second generation )
3. Third Generation

4. • Sanger sequencing:
• “Conventional" or "traditional" sequencing.
• Determines the sequence of large DNA fragments (up
to approximately 500 to 900 bases).
• Used clinically when the sequence of a specific gene is
being tested. As an example, to identify a mutation in
factor IX in a patient with suspected hemophilia B, to
cause hemophilia B.
• Cannot provide information about large portions of the
genome (eg, multiple genes) at a practical cost and
within a reasonable timeframe.
• One estimate predicted that sequencing an entire
human genome using Sanger sequencing would take
60 years.

5. • Next-generation sequencing (NGS) –
• Uses sequencing of multiple DNA fragments,
performed in parallel.
• High-throughput sequencing, deep sequencing,
second-generation sequencing.
• In contrast to Sanger sequencing, the speed of
sequencing and amounts of DNA sequence data
generated with NGS are exponentially greater,
and are produced at significantly reduced costs.

6. • Patient's DNA, which serves as the template, is
purified, amplified and fragmented, followed
by physical isolation of DNA fragments by
attachment to solid surfaces or small beads.
• Sequence data are generated on these small
fragments, and the electronic results are
computationally aligned against a "reference"
genome or sequence (ie, a previously
sequenced genome designated as a "normal"
reference).

7. Process of NGS

8. Difference between NGS and Sanger

9. • Third-generation sequencing –
• Third-generation sequencing uses parallel sequencing
similar to NGS, but unlike NGS, third-generation
sequencing uses single DNA molecules rather than
amplified DNA as a template.
• Thus, third-generation sequencing potentially
eliminates errors in DNA sequence introduced in the
laboratory during the DNA amplification process.
• Third-generation methods are under development and
generally are not clinically available.

10. • Source of DNA for NGS —
• Double-stranded nuclear DNA.
• DNA extracted from leukocytes in whole blood is a sterile
source of DNA used for most clinical testing.
• When DNA is derived from non-sterile sources (eg, buccal
swab, saliva sample), there is a potential risk for DNA
sequence from bacterial flora to be confused for host
sequence.
• However, non-sterile sources of DNA have the advantage of
not requiring a blood draw, and less-invasive means for
collecting DNA are increasingly used in large-scale genomic
research studies.

11. • If needed, DNA can also be extracted from fixed
tissues.
• For patients who have undergone hematopoietic cell
transplantation, leukocyte DNA from a blood sample is
likely to contain donor rather than recipient genetic
sequences.
• In this setting, DNA from a buccal swab, saliva, or hair
follicles can be used .
• Hair follicles are likely to be the most reliable source,
because buccal and saliva samples have been reported
to show chimerism for donor and recipient sequences.

12. • NGS can be used for :
• Whole genome panel ,
• Whole exome sequencing,
• Targeted gene panel

13. • Whole genome sequencing –
• Costlier than more limited sequencing.
• Because the whole genome is equivalent to
approximately 3.3 x 109 bases (3.3 gigabases
[Gb]).
• More information about the role of non-
coding DNA in human disease becomes
available.

14. • Whole Exome sequencing –
• The exome contains the portions of genes that
encode proteins.
• it represents only 1.5 to 2.0 percent of the
genome (ie, about 30 megabases [Mb]).
• Reasonable approach, because over 85 percent of
known disease-causing mutations are found in
exons.
• This approach substantially reduces cost and data
storage requirements compared with whole
genome sequencing.
• Exome sequencing also simplifies clinical
reporting, because the significance of variants in
exons is easier to interpret in most cases.

15. • The main disadvantage of exome sequencing
over whole genome sequencing is that exome
sequencing could potentially miss a
pathogenic variant(s) in a non-coding region
of the genome.
• Thus, whole genome sequencing may be used
in selected cases if initial exome sequencing is
not diagnostic.

16. • Targeted gene panels –
• Provide sequence data for a limited subset of genes
(typically 10 to 200 genes).
• Used in settings in which it would be appropriate to
sequence many genes to make a diagnosis.
• Targeted gene panels may be preferable to exome
sequencing due to
1. Considerable cost advantage,
2. Lower likelihood of identifying variants of unknown
significance.
3. Higher depth of coverage compared with whole exome
sequencing.
• Depth of coverage is important because it improves
sensitivity for individual genetic defects.

17. • Accuracy of Sequencing methods —
• 92 to 95 percent for whole genome and
exome sequencing NGS.
• Accuracy for targeted NGS gene panels is
higher, since sequencing a smaller region of
the genome allows for a greater degree of
probe-template overlap (also called "probe
tiling").
• Sanger sequencing - >99.99 percent accuracy

18. • Results of NGS :
• "Pathogenic" – variants previously reported in patients with
disease and/or are strongly suspected of being pathogenic based on
preclinical studies.
• "Likely pathogenic" – those with sequence features that are likely to be
implicated in disease pathogenesis but for which conclusive evidence of
pathogenicity is not available.
• "Likely benign" – those for which weak data in the medical literature
supporting pathogenicity may be available, but for which the majority of
evidence suggests the effect of the variant is benign.
• "Benign" – genetic variants not predicted to alter gene expression or
function.
• "Unknown clinical significance" – some features suggestive of possible
functional consequence, but for which there is insufficient evidence for
either a pathogenic or benign role.

19. • Indications for NGS:
• Diagnosis of complex diseases —
• In individuals for whom sequencing of a single gene is
unlikely to provide a diagnosis.
• Examples :
• More than one potential genes may be responsible.
• Obvious candidate genes have been tested and were
found to be normal.
• It would be less costly and more efficient to sequence
the entire genome, exome, or gene panel than to
sequence individual candidate genes sequentially.

20. • Cancer screening and management —
• Genomic information about the tumor (somatic changes), germline
changes in inherited cancer genes (eg, BRCA1 and BRCA2), and
germline changes in genetic modifiers.
• Targeted gene panels have shown expanded usefulness across
many cancer types, especially those for which more than one
genetic variant may be responsible.
• More efficient and cost-effective.
• Using a cancer gene panel that includes BRCA1 and BRCA2 if there
is personal or family history of prostate and/or pancreatic cancer,
even in the absence of breast or ovarian cancer.
• Other genes for which pathogenic variants may be associated
increased breast cancer risk
include ATM, CDH1, CHEK2, NF1, PALB2, and TP53.

21. • Screening for inherited causes of gastrointestinal cancers (eg,
panels-
APC, BMPR1A, EPCAM, MLH1, MSH2, MSH6, MUTYH, PMS2, PTEN,
SMAD4, STK11, TP53, BLM, CHEK2, GALNT12, GREM1, POLD1, and/
or POLE).
• Identification of familial acute leukemia syndromes.
• Categorization of prognostic groups in acute myeloid leukemia.
• Classification of certain inherited bone marrow failure syndromes.
• Analysis of tumor tissue to identify genetic abnormalities that may
potentially match molecularly targeted therapies.
• As an example, a polyadenosine diphosphate-ribose polymerase
(PARP) inhibitor could be considered in a patient with an
identified BRCA1 or BRCA2mutation.

22. • Children —
• One of the most common medical indications
for whole genome sequencing or whole
exome sequencing
• Evaluation of severe intellectual disability or
developmental delay believed to have a
genetic etiology in a child with a negative
initial evaluation.
• In some cases, evaluation of an affected child
and both parents ("trio sequencing").

23. • Adults —
• NGS is also being incorporated into the
National Institutes of Health "Undiagnosed
diseases program" (UDP), which evaluates
patients who have a longstanding medical
condition that eludes diagnosis

24. • Diagnosis of infections —
• NGS might be helpful in identifying an
infectious pathogen when usual microbial or
serologic testing is unrevealing,

25. • Healthy people —
• For proactive genetic screening
• To determine increased disease risks,
pharmacogenomic variants, and nonmedical
information (eg, ancestry).

26. • Limitations —
• May not be as accurate as other methods for detecting specific
types of mutations.
• As an example- detection of chromosomal copy number
changes and/or large gains, losses, or translocations by NGS is
problematic due to the short DNA sequence read lengths.
• These may result in failure to detect chromosomal deletions or
insertions.
• Traditional Sanger sequencing shares some of the same limitations
but, theoretically, to a lesser degree with its longer read lengths.
• In general, when large chromosomal aberrations are suspected,
alternative platforms are usually preferred over NGS such as
comparative genomic hybridization (CGH) microarray, multiplex
ligation-dependent probe amplification (MLPA), fluorescence in situ
hybridization (FISH), or cytogenetics.

27. Tumor mutation burden

28. • TMB is a quantitative measure of the total
number of mutations per coding area of a
tumor genome.
• Tumors that have higher levels of TMB are
believed to express more neoantigens – a type
of cancer-specific antigen – that may allow for
a more robust immune response and
therefore a more durable response to
immunotherapy.

29. • In metastatic NSCLC, checkpoint inhibitors
were first approved in the second-line setting.
• Nivolumab (hazard ratio [HR], 0.72),
pembrolizumab (HR, 0.71), and atezolizumab
(HR, 0.73) all demonstrate improved survival
over docetaxel in patients who experience
treatment failure with platinum doublet
therapy.
• J Clin Oncol 35:3924-3933, 2017
• Lancet 387:1540-1550, 2016
• Lancet 387:1837-1846, 2016

30. • Recently, pembrolizumab was approved for
first-line therapy in patients with NSCLC
whose tumors have > 50% PD-L1 expression,
with an impressive 30.2-month median overall
survival (OS) compared with 14.2 months for
chemotherapy
• J Thorac Oncol 12:S1793-S1794, 2017

31. • Despite the overall efficacy demonstrated by the immune
checkpoint inhibitors in NSCLC, use of these drugs in the clinic
remains imprecise, with a limited ability to identify patients who
will benefit from treatment.
• PD-L1 expression is the only predictive biomarker currently used
for patient selection, but it is an imperfect biomarker with several
limitations.
• Therefore, the development of biomarkers, such as TMB, to aid
patient selection continues to be a major focus of ongoing research
efforts.
• In melanoma, Snyder et al noted that TMB by whole-exome
sequencing (WES) is a predictor of increased survival for patients
who receive ipilimumab or tremelimumab.
• N Engl J Med 371:2189-2199, 2014

32. • Tumors with higher TMB have been hypothesized to
have more neoantigens that can be recognized by the
immune system in response to checkpoint inhibition.
• In a separate study, WES was performed in 34 patients
with NSCLC who received pembrolizumab, and
improved overall response rates (ORRs), progression-
free survival (PFS), and durable clinical benefit in
patients with high somatic nonsynonymous mutation
burden were observed.
• (Science 348:124-128,2015)
• Recently, TMB was examined as part of an exploratory
analysis of the Checkmate 026 study, which compared
nivolumab with platinum doublet chemotherapy in
firstline metastatic NSCLC.

33. • For patients with a high TMB, the response rate
was higher in those who received nivolumab
versus chemotherapy (47%v 28%), and PFS was
improved (9.7 v 5.8months).
• Patients with high TMB and high PD-L1 had the
best outcomes, and those who were negative for
both did the worst.
• In all these studies, TMB was assessed by WES,
which is mainly used for research purposes in
select centers.
• N Engl J Med 376:2415-2426, 2017

34. • NGS is available in many academic centers and
made available by several commercial entities.
• Therefore, the ability to use NGS to obtain
important molecular data as well as to assess
TMB is of great practical relevance.
• In addition, NGS is less expensive than WES..

35. • Rizvi et al used the :
• MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation
Profiling of Actionable Cancer Targets)
• (Recently authorized by the Food and Drug Administration)
• To evaluate TMB in 240 patients with advanced NSCLC who
received an immune checkpoint inhibitor.
• The median TMB was 7.4 single-nucleotide variants/megabase
(Mb), and patients with clinical benefit from immune checkpoint
inhibition had a higher TMB (median, 8.5 v 6.6 single-nucleotide
variants/ Mb; P = 0.0062).
• In addition, as the TMB increased, the durable clinical benefit and
PFS rates and ORRs all improved.
• (J Clin Oncol 36:633-641, 2018)

36. • Kowanetz et al, 454 patients treated with
Atezolizumab were assessed for TMB from a 315-gene
NGS panel run on pretreatment tumor specimens.
• With a median mutational load of 9.9/Mb, >75th
percentile was classified as high TMB.
• With high TMB, ORR, PFS, and OS were all significantly
improved for patients who received atezolizumab and
seemed to be independent of PD-L1 status.
( European Society for Medical Oncology 2016, October
7-11, 2016)

37. • Rizvi et al is the correlation between TMB
determined by NGS versus WES in an
admittedly small cohort of 49 patient samples.
• Good correlation in clinical outcomes with
checkpoint inhibition on the basis of TMB
determination by NGS or WES. (P < .001).
• Oncotarget 6:34221-34227, 2015

38. • Limiting factors :
1. Tissue specimen availability,
2. Wide genomic heterogeneity of tumors,
3. Varying testing platforms,
4. The relatively longer turnaround time,
5. Cost.
• Recently, assessment of TMB in cell-free DNA in
peripheral blood was shown to be predictive of
benefit from immune checkpoint inhibition.
• Ann Oncol 28:v460-v496, 2017(suppl 5)

39. Thanking You.