This document discusses stratified medicine approaches for cancer treatment. It describes how cellular pathologists classify gene mutations in cancer, including whether they are germline or somatic, synonymous or non-synonymous, activating or inactivating. Certain mutations can predict treatment response or resistance. Key driver mutations are discussed for lung cancer, including EGFR mutations and ALK translocations. EGFR mutant cancers may respond to EGFR tyrosine kinase inhibitors, while ALK rearrangements are targeted by crizotinib. Histopathology plays an important role in mutation detection and molecular testing to guide targeted therapies.

1. Presented By: Dr. Shubhi Saxena
Moderator: Dr. Surabhi Tyagi
STRATIFIED MEDICINE FOR CANCER:
The Role of Histopathologist

2. INTRODUCTION
• Cellular pathologists have been refining disease
classification for decades, with methods such as IHC and
in-situ hybridisation adopted to supplement morphological
assessment of tumours .
• Stratified Cancer Medicine involves predictive analysis:
The characterisation of tumours according to the presence
or absence of specific molecular abnormalities, to offer
appropriately targeted therapy and avoid unnecessary
treatment.

3. • Many cancer genomes have now been
sequenced and included as part of International
Cancer Genome Consortium.
• Cancer genome sequencing projects have
generated clinical and therapeutic relevance,
e.g. The identification of mutated BRAF
oncogene in half of malignant melanomas.

4. • The time from this discovery to the licensing of
the BRAF inhibitor Vemurafenib was
encouragingly short in drug development.
• Apart from the immuno-modulator
Ipilimumab, former represents the first
effective treatment for patients with advanced
melanomas.

6. CLASSIFICATION OF GENE MUTATIONS IN CANCER
• According to TIMING :
 Germline (Constitutional) - A mutation from
one parental gamete being transferred to all
cells by cell division.
Somatic (Acquired) - A mutation occuring
during DNA replication or division, present
only in a subset of cells.

8. • EFFECT ON PROTEIN CODING :
 Synonymous (Silent) – A nucleotide change resulting
in the same amino acid, due to redundancy in nucleotide
combinations coding for each amino acid, e.g. coding
DNA strands containing GTA and GTG would both
encode the amino acid valine.
 Non-synonymous – A nucleotide change leading to a
different amino acid, e.g. DNA sequence GTA codes for
valine but a change to GAA would result in glutamic acid.

9. • EFFECT ON PROTEIN FUNCTION :
 Activating (Gain of function) – A change leading to
enhanced effect of protein, e.g. The codon 600
BRAFV600E mutation leads to increased activity of
BRAF protein.
 Inactivating – A change leading to
nonfunctional/reduced function protein, e.g. Codon 594
mutation in BRAF gene cause loss of kinase activity.

10. • FUNCTIONAL EFFECT :
 Pathogenic – A mutation predicted to contribute to
initiation of tumor.
 Nonpathogenic – A genetic abnormality that does not
appear to contribute to initiation of tumor.

11. • PREDICTION OF TREATMENT RESPONSE :
 Sensitising – A mutation associated with treatment
response, e.g. L585R mutation in EGFR gene is predictive
of response to EGFR tyrosine kinase inhibitor drugs in
patients with Non small cell lung cancer (NSCLC).
 Resistance – A mutation associated with treatment
resistance, e.g. T790M mutation in EGFR gene predicts
lack of response to EGFR tyrosine kinase inhibitor drugs in
patients with NSCLC.

12. • IMPORTANCE IN CARCINOGENESIS :
 Driver – A mutation that is recurrent between different
tumours and is functionally linked to carcinogenesis, e.g.
KRAS mutation in colorectal cancers cause increased
activity of mutated KRAS protein leading to abnormal
cell proliferation.
 Passenger – A mutation that does not appear to play a
role in initiation of cancer and is likely to have occurred
as a bystander effect due to genetic instability.

13. • Major challenges of the stratified medicine
approach include :
 Tumour Heterogeneity
Treatment Resistance

14. Role of Histopathology also includes use of
specific molecular markers in specific
malignancies;
e.g. HER2 testing in breast cancer to
improve prognostic effect of Trastuzumab
(Herceptin, Roche).

15. MOLECULAR MARKER DISEASE TREATMENT
t(15;17) translocation Acute Promyelocytic
Leukaemia (APML)
ATRA - all trans retinoic
acid.
t(9;22) translocation
(Philadelphia
chromosome); BCR-ABL
fusion
KIT/PDGFRA Mutation
Chronic Myeloid
Leukaemia
GIST
Imatinib (tyrosine-kinase
inhibitor)
HER2 gene amplification Breast cancer , Metastatic
Gastric cancer
(Transtuzumab only)
Trastuzumab
Pertuzumab

16. MOLECULAR MARKER DISEASE TREATMENT
KRAS wild type ( lack of
mutation)
Metastatic colorectal
carcinoma
Cetuximab
Panitumumab
EGFR mutation
ALK gene rearrangement
Non small cell lung
carcinoma
Geftinib
Erlotinib
Crizotinib
BRAF codon 600
mutation, V600E
Malignant melanoma Vemurafenib

17. MUTATION DETECTION
• Molecular analysis protocols are developed for formalin
fixed paraffin embedded (FFPE) tumour tissues.
• Materials on glass slides are provided along with a
matched H&E stained section, with the % tumor nuclei
content assessed and oulined on slide.
• Macrodissection performed on slide mounted sections to
isolate and enrich the material of tumour nuclei and avoid
analysis of adjacent non neoplastic tissue; However, with
the development of more sensitive analysis techniques this
method is not required.

18. • Macrodissection is mandatory if tumor content of
section is assessed as <20% by pathologist.
• For current methods, mutation analysis for mutation
hotspots in upto 5 genes can be performed on DNA
extracted from 5 micron paraffin section with tumour
cellularity >50%.
• Each section can be expected to yield at least 150 ng
DNA, with inputs as low as 10 ng.

19. • For assesing the tumour content, the pathologist should
note the specimen dependent factors that may influence
the success in subsequent analysis, such as Necrosis,
Melanin pigment in melanoma, which inhibits PCR.
• Specimen handling during preanalytical phase has an
important imapct on outcome of mutation analysis, such
as use of clean microtome blade to prevent cross
contamination of DNA and tissue.

20. • Detection of gene amplification or
translocations may be performed in thin
sections on glass slide using in-situ
hybridisation, whereas detection of gene
mutation or fusion trancripts requires
extraction of nucleic acids and analysis using
PCR and sequencing-based methods.

21. SANGER
SEQUENCING
PYRO-
SEQUENCING
NEXT
GENERATION
SEQUENCING
METHOD Amplification and
sequencing of PCR
products by chain-
terminating dideoxy
nucleotides during in
vitro DNA replication
Sequencing by
synthesis: detection of
luminescence released
when pyrophosphate-
labelled nucleotide
molecule is
incorporated during
DNA replication.
Massively parallel
sequencing of millions
of amplified DNA
molecules.
DNA INPUT Low Low High (~500 ng)
SENSTIVITY Lowest High High
LIMIT OF
DETECTION
10-20 % 5 % 10 %
ADVANTAGES Identification of
known variants
Targeted mutation
detection, Fast method
Highest throughput
technology, whole
exome analysis.
DISADVANTAGES labour intensive, miss
low level variant.
High sequencing error
rate
Larger quantities of
DNA required

22. High Resolution DNA
Melting Analysis (HRM)
Single Stranded
Conformational
Polymorphism Analysis
(SSCP/SSCA)
METHOD Comparison of melting curves
of PCR products against
normal samples.
Heat denatured PCR products
compared with known samples
using Capillary
Electrophoresis.
DNA INPUT Low (~100 ng) Low (~100 ng)
SENSTIVITY Low High
LIMIT OF DETECTION 5 % 1-10%
ADVANTAGES Quick melting products
identify exact abnormality
Established technique, low
cost
DISADVANTAGES Nonspecific amplification lead
to missed cells
Temp., gel composition must
be controlled.

23. AMPLIFICATION
REFRACTORY MUTATION
SYSTEM
FRAGMENT LENGTH
ANALYSIS
METHOD Selective amplification of
sequences containing defined
mutation
DNA fragment length analysed
against size to detect deletions
and insertions
DNA INPUT High (~500 ng) Low (~100 ng)
SENSTIVITY Highest High
LIMIT OF DETECTION <1-8 % 1-2 %
ADVANTAGES Fast and sensitive Fast and sensitive
DISADVANTAGES Detects predefined hotspot
mutations
Cannot be used to detect point
mutations

24. REQUIREMENT OF MOLECULAR PATHOLOGY
REPORT
• Dates
• Patient information
• Information about request - nature of sample, tissue and
tumour type, percentage content tumour nuclei as assessed by
referring pathologist, clinical indication for analysis.
• Information about analysis - Technique used, scope of
test, senstivity/limit of detection.
• Results
• Contact information

25. DISEASE
APPLICATIONS

26. LUNG CANCER
 Two major types of key driver mutations in pulmonary
adenocarcinoma :
• EGFR mutations
• ALK translocations
 Novel targets also identified and linked to drugs in
development includes KIF5B-RET and ROS.
 Progress in pulmonary squamous cell carcinoma is not
so promising.

28. • EGFR IN LUNG CANCER :
- Epidermal growth factor receptor gene (ERBB1
or HER1)
- Responsible for cell membrane bound epidermal
growth receptor.
- Mutations in above determines response to
tyrosine kinase inhibitors (TKIs) erlotinib and
gefitinib in non small cell lung cancer (NSCLC).

29. - 90% EGFR mutations are located in tyrosine
kinase-binding domain (Exons 18-21).
- The mutant EGFR protein activates cellular
pathways implicated in cancer cell growth,
survival and migration.
- The most common ACTIVATING mutations
are exon 19 deletions and codon 858 missense
mutation in exon 21 (L585R).

31. - The most common RESISTANCE mutations is
EGFR T790M, others include amplification or
overexpression of MET, PIK3CA mutation and
transformation.
- Histologically features are adenocarcinoma of any
growth pattern esp. papillary or micropapillary
pattern, but with exception of Mucinous carcinoma.
- Less common occur in neuroendocrine,
mucoepidermoid and adenoid cystic carcinoma.

33. • Mutant EGF receptors are targeted using
small molecule inhibitor drugs, which act
inside the cell against the internal
tyrosine kinase domain of receptor called
as tyrosine kinase inhibitors like
erlotinib (Tarceva) and gefitinib (Iressa).

34. • EML4-ALK IN LUNG CANCER :
- It is a fusion gene derived from inversion
affecting chromosome 2 and leading to fusion
of EML4 (echinoderm microtubule-associated
protein-like 4) gene with ALK (anaplastic
lymphoma kinase) gene.
- Fusion gene is found in adenocarcinoma more
commonly than squamous cell carcinoma.

35. - Alternative ALK fusion partners are TRK-fused
gene TFG, nucleophosmin gene NPM1 and
kinesin family member 5B gene, KIF5B.
- ALK gene rearrangements generally occurs of
EGFR or KRAS mutations.
- Fusion gene encodes a fusion protein leading to
ALK signaling which causes increased cell
proliferation.

37. • More frequent in –
 Never Smokers
 Younger age at onset of disease
 Adenocarcinoma
 Females

38. Crizotinib (Xalkori, Pfizer)
• ALK/MET inhibitor
• Multitargeted small molecule TKI
• Administered orally
• Inhibits ALK phosphorylation and signal transduction.
• Licensed for use in NSCLC by FDA in 2011.
• Also used as :
– Treatment of aggressive and resistant forms of
anaplastic large cell lymphoma carrying t(2;5)
translocation involving NPM1 and ALK.
– Neuroblastoma in pediatric population.

40. BREAST CANCER
• Ascertaining the status of HER2 in invasive
breast cancer has now become the standard of
care.
• It is achieved using immuno-histochemical
assessment of protein expression.
• In-situ hybridization (confirmation of gene
amplificarion) is reserved only in case of
equivocal immuno-histochemistry results.

41. • In last few years, there is rapid increase in use
of gene expression profiling in breast cancer to
provide risk stratification in addition to
traditional histopathological parameters like
grade, vascular invasion and lymph node
involvement.

44. MELANOMA
• BRAF gene encodes a serine/threonine kinase, an enzyme
activated by phosphorylation which is responsible for
transferring phosphate groups to other proteins to
modulate their functions that is part of RAS kinase
family.
• 90% mutation are in codon 600 (V600E).
• BRIM3 trials shows that untreated melanoma stages
IIIC/IV with V600E mutations have overall improved
progression free survival with vemurafenib as compared
to dacarbazine therapy.

45. • Unexpected finding was increased risk of
cutaneous squamous cell carcinoma in patients
receiving vemurafenib due to paradoxical
stimulation of cellular pathway in epidermal
cells containing wild type BRAF.
• The MEK pathway also shows overactivity in
melanomas harbouring BRAF mutations.

47. CHALLENGES OF THE APPROACH
• Involved with molecular analysis of FFPE
material leads to cross-linking and degradation of
DNA into fragments >200 base pairs in length
due to over-fixation and under-fixation of
specimens.
• There is a need for optimal standardized sample
handling protocols in the pre analytical phase to
extract maximum diagnostic potential from DNA
extracted from FFPE tumor samples.

48. • Today’s chemotherapy like kinase inhibitors halt
tumor growth rather than killing existing cells.
• Therefore, patients experience a temporary
response to therapy with subsequent development
of resistance.
• This increases the need to develop a different
treatment strategy.

49. • Due to tumor heterogeneity, there is increased
requirement to track mutational profile using
longitudinal tissue sampling i.e. to assess
mutational status of multiple genes at the same
time rather than consecutive manner.

50. • Recent retrospective study of pre and post
treatment biopsies in EGFR mutation positive
NSCLC patients, demonstrated new molecular
abnormalities like resistance mutation of T790M,
conferring in post treatment biopsy samples.

51. • Pathology departments must be adequately
resourced for sample preparations,
macrodissection and any testing performed
within the cellular pathology department.
• Equally important is the reliability of output of
testing.

52. PROGRESS
• DNA based predictive analysis of solid tumors
is currently carried out on individual genes in a
sequential manner for particular licensed
therapy.
• Cancer research UK in 2011- phase 1 is dealing
with routine delivery of mutational analysis of
4-5 key genes in different solid tumors like
breast, colorectal, lung, ovarian, prostate and
malignant melanoma.

53. CONCLUSION
• The characterisation of tumor genomes to
identify oncogenic drivers and therapeutic
targets is having a significant in management
of people having cancers with important
implications for histopathologists.

54. • Mutation testing performed on nucleic acids
extracted from tumor has focused on minimal
invasive techniques for treatment like LIQUID
BIPOSIES, using free circulating tumor DNA
fragments isolated from plasma.

55. • There is a need to move from a single gene
mutation test to simultaneous assessment of a
panel of inter related biomarkers like
assessment of EGFR and EML4-ALK provide
information about two different licensed
treatment options.

56. • Since Histopathology is the gold standard for
classification of disease, predictive molecular
analysis must become fully integrated in
cancer diagnosis and prognostication in
histopathology.

57. KEY POINTS
1. The characterisation of tumor genomes by
histopathologists, to identify oncogenic drivers and
therapeutic targets is having significant impact in
management of cancer.
2. Some DNA based tests linked to licensed treatment are :
HER2 amplification in breast cancer, KRAS mutation in
metastatic colorectal cancer, BRAF mutation in
advanced malignant melanoma, EGFR mutation and
ALK translocation in pulmonary adenocarcinoma.

58. 3. Molecular analysis can be performed in FFPE
tumor tissue.
4. Detection of gene amplification or translocation
may be performed in thin sections on glass slide
using in-situ hybridization, whereas detection of
gene mutation requires extraction of nucleic acids
and analysis using PCR and sequencing method.

59. 5. Major challenges of the stratified medicine
include tumor heterogeneity and tumor
resistance.
6. Higher throughput next generation sequencing
technology is enabling a broader scope of
analysis.