Molecular profiling of breast cancer

Presenter Dr Dhanya A N Moderator Dr Niranjana Murthy B

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

Contents

• Molecular profiling assays – Oncotype DX– Mamma print– PAM50– Breast cancer index– Endopredict – IHC4– NGS

• Role of pathologist • Conclusion

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

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

Why do we need to do molecular profiling ?

• 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

• 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

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)

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

IHCAdvantages• Simple, inexpensive procedure• Processed slides can be stored for years and reassessed• Cell morphology can be viewedDisadvantages• 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

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

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

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

RT-PCR

RT-PCR

Advantages• Cost effective • Rapid results, • SensitiveDisadvantages• Requires knowledge of candidate genes

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

Gene Microarray technique

Gene Microarray technique

Advantage • Predict the disease behavior Disadvantage• Dependent on the sensitivity and specificity of

the probes• Rare sequences not necessarily detected

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

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

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)

Scoring Her-2/neu

Staining pattern Her-2/neu protein overexpression

0 No reactivity seen Negative1 Weak incomplete staining in any proportion of

tumour cellsNegative

2 Non uniform or weak to moderate complete membranous reactivity in >10% of the tumour cells ORIntense complete staining of <30% of the invasive tumour cells.

Equivocal

3 Uniform, intense, complete membranous reactivity in >30% of the invasive tumour cells.

Positive

IHC HER2

HER2 immunohistochemical staining with a score of 3+

• 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

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.

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

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.

IHC for ER/PR

Intrinsic Breast Cancer Subtypes

1) Luminal-like Breast Cancer Types– Luminal A – Luminal B

2) HER2 enriched breast cancer subtype3) Basal-like breast cancer subtype4) Claudin-low breast cancer subtype.

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

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.

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

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

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.

Gene for EMT transition1. cell communication genes, eg, chemokine 2. extracellular matrix formation genes, eg

vimentin and fibroblast growth factor 7 genes3. cell differentiation genes, eg Krüppel-like factor 4. cell migration genes, eg integrin a5 5. angiogenesis genes, eg vascular endothelial

growth factor6. immunerelated genes, eg CD79b, CD147. stem-cell like genes, eg CD44+/CD24-

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

Luminal A Luminal B Her-2/neu Basal-like Luminal B tends to be

higher histological grade than luminal A

TP53 mutation commonMore likely to be high grade and node positive.

TP 53 mutation common; BRCA-1 dysfunction (germline sporadic)

Histological correlation

Tubular carcinomaCribriform carcinomaLow grade IDC (NOS)lobular carcinoma

IDC (NOS)Micropapillary carcinoma.

High grade IDC (NOS) High grade IDC (NOS) Metaplastic carcinomaMedullary 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

Molecular profiling assays

• 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.

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

Molecular profiling assays

• Oncotype DX• Mammaprint • PAM50• Breast cancer index• Endopridict index • Next generation sequencing

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.

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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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