molecular profiling of breast cancer dr dhanya a n sri siddhartha medical college, tumkur

1. Molecular profiling of breast
cancer
Presenter Dr Dhanya A N
Moderator Dr Niranjana Murthy B

2. Contents
• Introduction
• Gene expression profiling
– Immunohistochemistry (IHC)
– Fluorescent in situ hybridization (FISH)
– Reverse transcription PCR (RT- PCR)
– Microarray
– Next generation sequencing (NGS)
• Intrinsic subtypes of breast cancer

3. Contents
• Molecular profiling assays
– Oncotype DX
– Mamma print
– PAM50
– Breast cancer index
– Endopredict
– IHC4
– NGS
• Role of pathologist
• Conclusion

4. Introduction
• Breast cancer constitutes the most common
type of cancer
• Breast cancers are conventionally classified
into different types by
– morphological feature,
– histological features,
– tumor grade,
– proliferation status,
– lymphovascular invasion – prognostic variables

5. • Breast cancer is caused by heterogeneous
group of tumor cells whose behavior and
response to therapy depends on biological
features

6. Why do we need to do molecular
profiling ?

7. • Molecular testing in breast cancer is used to
– Classify tumor types,
– recognize hereditary implications (eg, BRCA1
mutations)
– identify appropriate therapeutic agents (eg, HER2+
disease or ER/PR + disease),
– determine the prognosis of the disease by giving the
risk score,
– identify biomarkers that can predict or monitor the
response to treatment
– To avoid unnecessary treatment to all cancer patients

8. • The first molecular classification system uses
only hormonal receptors and HER2 which
predicts response to hormonal therapy and anti
HER2 respectively.
• As the molecular technique evolve it is now
possible to analyze the expression of
thousands of gene in a single experiment in
order to predict the outcome of therapy

9. Gene expression profiling(GEP)
• It is the determination of the pattern of genes
expressed, at the level of transcription, under
specific circumstances or in a specific cell to give
a global picture of cellular function.
• Done by
– Immunohistochemistry (IHC)
– Fluorescent in situ hybridization (FISH)
– Reverse transcription Polymerase chain reaction
(RT-PCR)
– Gene Microarray
– Next generation sequencing (NGS)

10. IHC
• Description - Use of antibodies to detect
levels of a specific protein
• Detection - Protein expression levels
• Example assays - IHC4
• Sample requirement - Tissue sections -
– Formalin fixed paraffin embedded (FFPE) samples
– Frozen samples

12. IHC
Advantages
• Simple, inexpensive procedure
• Processed slides can be stored for years and reassessed
• Cell morphology can be viewed
Disadvantages
• Semiquantitative, subjective score
• Fixation time can affect results
• Results dependent on quality of antibody used to detect
the protein
• Usually only 1-2 proteins can be analyzed per section

13. FISH
• Description - Use of a fluorescently labeled
DNA probe to detect specific DNA sequences
in chromosome
• Detection –
– Gene number alterations,
– DNA rearrangements
• Example assays - HER2 FISH pharmDX Kit
• Sample requirement - Tissue sections - cut
from FFPE samples or fresh frozen

15. FISH
Advantages
• High sensitivity and specificity
• The resolution is better.
• Can be applied to both dividing and non-dividing
cells.
Disadvantages
• costly fluorescence microscope required;
• results must be captured and stored within a short
period (fluorescent signal decays within a few
weeks);
• Only 1-2 DNA regions analyzed per experiment

16. RT-PCR
• Description - Conversion of RNA to cDNA by
reverse transcriptase then quantification of
specific gene sequences using PCR
• Detection - Gene expression levels
• Example assays - Oncotype DX, PAM50,
Breast Cancer Index, EndoPredict
• Sample requirement –
– FFPE samples,
– fresh frozen specimens

17. RT-PCR

18. RT-PCR
Advantages
• Cost effective
• Rapid results,
• Sensitive
Disadvantages
• Requires knowledge of candidate genes

19. Gene Microarray technique
• Description - Detection of specific DNA
sequences or cDNAs (for RNA analysis) by
hybridization to an array of DNA probes
• Detection - Gene number alterations, DNA
rearrangements, gene expression levels, RNA
editing
• Example assays - MammaPrint,
• Sample requirement - As low as 75 ng DNA
from
– FFPE sample; or
– DNA from fresh frozen tissue specimens

20. Gene Microarray technique

21. Gene Microarray technique
Advantage
• Predict the disease behavior
Disadvantage
• Dependent on the sensitivity and specificity of
the probes
• Rare sequences not necessarily detected

22. Next generation DNA sequencing
• Description - Sequencing of thousands or
millions of DNA sequences in 1 reaction
• Detection –
– DNA amplification
– DNA rearrangements,
– DNA mutations
– DNA deletion
– RNA editing
• Sample required - > 100 ng DNA/RNA from
fresh frozen specimens more ideal or FFPE

24. Next generation DNA sequencing
Advantages
• Whole genome sequencing
• Targeted genome sequencing
• Facilitate the sequencing at a greater depth (at base pair
level)
• Enable the detection of rare gene sequences
• Large panels (a few hundred) of cancer-specific genes
are selectively sequenced at a time.
Disadvantage
• Costly
• complicated data analysis
• length of time (weeks) for results
• High-tech lab, not routinely done

25. Immunohistochemistry and
Cytogenetics
• Hormonal receptors (HR) and HER2 are
prognostic marker and therapeutic target for
breast cancer
• The techniques for identifying HER2 & HR
– Immunohistochemistry (IHC) and
– Fluorescence in situ hybridization (FISH) - lack of
morphologic details
– Chromogenic ISH (CISH)

26. Scoring
Her-
2/neu
Staining pattern Her-2/neu protein
overexpression
0 No reactivity seen Negative
1 Weak incomplete staining in any proportion of
tumour cells
Negative
2 Non uniform or weak to moderate complete
membranous reactivity in >10% of the tumour
cells
OR
Intense complete staining of <30% of the invasive
tumour cells.
Equivocal
3 Uniform, intense, complete membranous
reactivity in >30% of the invasive tumour cells.
Positive

27. IHC HER2
HER2 immunohistochemical staining with a score of 3+

29. • There are two types of bright-field chromogenic
HER2 ISH assays:
1) single color ISH for the HER2 gene only –
– six or > HER2 positive,
– four-six HER2 signals considered equivocal and
– less than four signals considered HER2 negative
2) Dual color ISH for the HER2 gene and CEN17
(chromosome 17 centromere). - the ratio of HER2
gene copy numbers to CEN17 copy numbers
– negative: HER2/CEN17 ratio <1.8;
– equivocal: HER2/CEN17 ratio 1.8-2.2;
– positive: HER2/CEN17 ratio >2.2

30. Normal HER2 gene status is observed with 1-2 copies of HER2
gene (black dots) and CEN17 (red dots) targets in each nucleus.
B) Amplified HER2 gene status is observed with multiple HER2
gene copies.

31. HER2 Testing using fluorescence in-situ hybridization (FISH).

32. Scoring system for ER/PR
Score for propotion Score for intensity
0= No staining 0= No staining
1<1% staining 1=Weak staining
2=(1-10)% staining 2= Moderate staining
3=(11-33)% staining 3=Strong staining
4=(34-66)% staining
5=(67-100)% staining
Total score ranges from 0 to 8.
Tumors scoring ≤2 are regarded as ER negative and have a negligible chance of response.

33. IHC for ER/PR

34. Intrinsic Breast Cancer Subtypes
1) Luminal-like Breast Cancer Types
– Luminal A
– Luminal B
2) HER2 enriched breast cancer subtype
3) Basal-like breast cancer subtype
4) Claudin-low breast cancer subtype.

35. Luminal A
• Derives its name from its similarity to the expression
profile of normal luminal breast epithelium.
• Overexpression of ER-regulated genes
• Underexpression of an HER2 gene cluster
• Underexpression of proliferation-related genes.
• Sensitive to endocrine manipulation( hormonal
therapy).
• Less sensitive to cytotoxic agents in both the
neoadjuvant and metastatic settings.
• Approximately 40% of all breast cancers are classified
as luminal A.
• They have favorable prognosis

36. Luminal B
• Have lower expression of ER-related genes
• Variable expression of an HER2 cluster of genes,
• Relatively higher expression of proliferation-
related genes.
• They represent about 20% of breast cancers.
• They also been shown to have genomic
instability, and to harbor mutations in TP53.
• less sensitive to cytotoxic chemotherapy, sensitive
to hormonal therapy
• Associated with a relatively higher risk of relapse.

37. HER2 enriched breast cancer
subtype
• It is characterized by high expression of
– HER2
– Proliferation genes and
• low expression of luminal clusters.
• Constitute 20% to 30% of all breast tumors.
• Clinically, they are associated with a poorer
prognosis

38. Basal-like breast cancer subtype
• Constitute about 15% of invasive ductal breast
cancers.
• Its name is derived from shared gene expression
patterns with normal basal epithelial cells.
• They are considered ER/PR and HER2 negative
(“triple negative”)
• This subtype is also characterized by relatively
high frequency of BRCA1 mutations, increased
genomic instability, high expression of the
proliferation cluster of genes, and a high
histologic grade

39. Claudin-low breast cancer subtype
• Is characterized by overexpression of genes
associated with epithelial-to-mesenchymal
(EMT) transition.
• Have no expression of luminal differentiation
markers, are HER2 and hormone-receptor-
negative by IHC
• Frequently exhibit metaplastic and medullary
differentiation, and are often part of the basal
intrinsic subgroup.

40. Gene for EMT transition
1. cell communication genes, eg, chemokine
2. extracellular matrix formation genes, eg
vimentin and fibroblast growth factor 7 genes
3. cell differentiation genes, eg Krüppel-like
factor
4. cell migration genes, eg integrin a5
5. angiogenesis genes, eg vascular endothelial
growth factor
6. immunerelated genes, eg CD79b, CD14
7. stem-cell like genes, eg CD44+/CD24-

41. Luminal A Luminal B Her-2/neu Basal-like
Gene
expression
pattern
Expression(LMW)
cytokeratins, and high
expression of HR’s and
associated genes
Expression (LMW)
cytokeratins, and
moderate to weak
expression of HR’s
and associated
genes.
High expression of
Her-2/neu .
Low expression of
ER and associated
genes.
High expression of
basal epithelial
genes, basal
cytokeratins. Low
expression of ER and
Her-2/neu
associated genes.
Clinical ~ 50% of invasive breast
cancer
~20% of invasive
breast cancers
~15% of invasive
breast cancers
~15% of invasive
breast cancers
ER/PR status ER/PR positive ER/PR positive ER/PR negative Most ER/PR negative
Her-2/neu
status
Her-2/neu negative Her-2/neu
expression variable
(+/-)
Her-2/neu positive
(by definition)
Her-2/neu
negative(“triple
negative”)
Biological
features
High proliferation
than luminal A
High proliferation High proliferation

42. Luminal A Luminal B Her-2/neu Basal-like
Luminal B tends to be
higher histological
grade than luminal A
TP53 mutation
common
More likely to be high
grade and node
positive.
TP 53 mutation
common; BRCA-1
dysfunction (germline
sporadic)
Histological
correlation
Tubular carcinoma
Cribriform carcinoma
Low grade IDC (NOS)
lobular carcinoma
IDC (NOS)
Micropapillary
carcinoma.
High grade IDC (NOS) High grade IDC (NOS)
Metaplastic carcinoma
Medullary carcinoma
Treatment Respond to endocrine
therapy
Respond to endocrine
therapy (tamoxifen&
aromatase inhibitors)
Respond to
trastuzumab
No response to
Endocrine therapy and
trastuzumab
Response to
chemotherapy
variable variable (> in luminal
A)
Good
(anthracycline based
chemotherapy)
Good
(platinum based
chemotherapy )
Prognosis Good prognosis Prognosis not as good
as for luminal A
Generally poor
prognosis
Generally poor
prognosis

43. Molecular profiling assays

44. • Gene signature - is a group of genes in a cell
whose combined expression pattern is uniquely
characteristic of a biological phenotype or
medical condition.
• Molecular profiling - is a method of testing that
looks at each person's cancer tumor and studies
the genetic characteristics as well as any unique
biomarkers. The information gathered is used to
identify and create targeted therapies that are
designed to work better for a specific cancer
tumor profile.

45. Molecular profiling
• Many gene signature is been identified which
will predict the response to specific therapies
• The assays of those genes also gives the risk
score and give the recurrence free survival rate
for the patients
• These assays done on patients
– Who were diagnosed with early-stage (stage I-III)
breast cancer
– HR positive and
– HER2-negative tumors

46. Molecular profiling assays
• Oncotype DX
• Mammaprint
• PAM50
• Breast cancer index
• Endopridict index
• Next generation sequencing

47. Oncotype DX
• Based on RT-PCR
• Measures the expression of 21 genes (16
cancer-related genes and 5 reference genes that
serve as internal controls).
• The cancer-related genes include
– Estrogen group gene
– Her2 group gene
– Proliferation gene
– invasion groups genes.

48. Oncotype DX
• A Recurrence Score scale range from 0 to 100
• Kaplan-Meier estimates of the rates of distant
recurrence at 10 years in patients with score
– < 17 (low risk) is 7%
– >31 (high risk) is 31%
– 17 to 31 (intermediate risk) is 14%
• Scoring is done by measuring the different gene
expression and multiplying by sets of multiplication
factors and adding the total value.
• In summary, a low level of ER expression and a high
level of proliferation/invasion gene expression and/or
HER2 expression predict a higher risk of recurrence

49. MammaPrint
• Microarray based
• Uses 70 gene expression to asses the prognosis of
breast tumor
• The biological functions of the 70 genes are
– regulating cell cycle,
– invasion, metastasis,
– proliferation,
– survival in circulation,
– extravasation,
– adaptation to the micro-environment as well as
angiogenesis

50. Conti..
• Risk assess
– low- risk - 10% will recur within 10 years without any
additional adjuvant treatment
– high-risk - 29% will recur within 10 years without any
additional adjuvant treatment
• Tumors are ranked according to their correlation with
the previously determined average profile in tumors
from patients with a good prognosis
• A patient with a correlation coefficient of more than 0.4
are grouped with a good-prognosis signature, and all
other patients are grouped with a poor-prognosis
signature

51. Red indicates a high level of expression of messenger RNA (mRNA) in
the tumor, as compared with the reference level of mRNA, and green
indicates a low level of expression The yellow line is the previously
determined threshold between a good-prognosis signature and a poor-
prognosis signature

52. PAM50
• Commercially it is called as Prosigna kit
• Based on RT-PCR
• 50-gene expression is assessed
– cell cycle regulating genes
– gene for proliferation
• Developed to provide
– breast cancer classification into the intrinsic
subtypes
– to give risk of recurrence score

53. Conti..
• Results are reported as a risk of recurrence (ROR)
score from 0 to 100 in two ways, and tells distant
recurrence-free survival at 10 years
1. node-negative cancers are classified as
– low (0-40),
– intermediate (41-60),
– high (61-100) risk
2. node-positive cancers are classified as
– low (0-40)
– high (41-100) risk

54. Conti..
• Score is calculated using coefficients from a
Cox model, a proliferation score, and gross
tumor size.
• The test variables are multiplied by the
corresponding coefficients from the Cox
Model to generate the score,
• Which is then adjusted to a 1-100 scale based
on coefficients generated

55. Breast Cancer Index Test
• It is based on RT-PCR
• This assay includes combination of gene
signature
– the ratio of HOXB13:IL17BR (a homeo domain–
containing protein and interleukin 17 receptor B )
– Molecular grade index which analyze the
expression of 5 gene which involves in
proliferation and cell cycle

56. Conti..
• Cox model and Kaplan-Meier analysis were
used to examine the associations between gene
expression indices and relapse-free survival for
10 years and the score is given
• Scores range from 0 to 10
• BCI risk categories
– Low 0-5
– High 5.1-10

57. EndoPredict Test
• Based on RT PCR
• Assay measures the expression of eight cancer
genes and three housekeeping control genes
• Risk score is been combined with clinical
variables like
– LN status
– Tumor size
• Gives the risk of recurrence at 10 years
• Risk is given
– High risk
– Low risk

58. IHC4 assay
• Uses IHC technique
• FFPE tissues
• Based on the assessment of ER, PR, HER2, Ki67
• Ki67
– Low risk <15%
– High risk >15%
• It uses the mathematical formula that weighs the
semiquantitative expression values and combines
these into a single risk score using cox model
• Risk score
– High risk
– Low risk

59. Next generation sequencing
• NGS-based assays that can detect gene mutations from
small amounts of DNA are also in development;
– DNA from fine needle aspirates or
– circulating tumor DNA (ctDNA) from blood samples
• Several studies have shown a high degree of
concordance between mutations in ctDNA (detected
using NGS techniques) and mutations from the primary
tumors
• NGS panels gene sets are available in companies such
as Foundation Medicine, Life Technologies, and
Illumina etc.

60. Role of pathologist
• To classify the tumor on the basis of molecular
profiling
• To suggest for appropriate investigations that a
patients should undergo
• Testing for specific molecular markers
• To suggest for appropriate therapy and
management for the patients

61. Conclusion
• Molecular testing is becoming increasingly
important in the precise classification,
diagnosis and treatment of breast cancer
• In spite of so many assays the molecular
profiling experiments still evolving
• With the increasing use of molecular profiling
sequencing, the identification of novel
therapeutic targets is possible

62. Genomic test in India
• Done in onquest superspeciality lab, New
Delhi, India
• Oncotype DX, MammaPrint, BRACA 1, 2
mutation, NGS are done in that lab
• Cost of IHC will be 500 to 800 rps
• Cost of genetic test will be approximately 1.5
to 2 lakhs in rps.

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