Molecular Genetics - HER2 in Breast Cancer (VII)

Question 1

Isoforms of HER2

Activation and effect?

Answer 1

HER1-4

Human Epidermal Growth Factor Receptor gene ERBB2

Transmembrane protein activated by epidermal growth factor > dimerizes > Tyrosine kinase domain for downstream signaling

Question 2

Associated risks of HER2 expression in breast cancer

Answer 2

Higher risk of:

• Recurrence and mortality
• Relative resistance to endocrine treatment
• Less benefit from some chemotherapy

Question 3

2 drugs and MoA for HER2 +ve breast cancer

Answer 3

1) Tratuzumab (Herceptin) - Anti-HER2 therapy interferes with signal transduction cascade initiated by HER2
2) Lapatinib - Inhibitor of tyrosine kinase activity of HER2 and EGFR

Question 4

Benefit and drawbacks of Herceptin

Answer 4

Indicate for early HER2 +ve breast cancer

• Reduce recurrences by 1/2
• Reduce risk of mortality by 1/3

Expensive
Cardiotoxicity

Question 5

Which type of breast CA needs HER2 testing

Answer 5

All Invasive breast carcinoma

especially in-situ carcinoma

Question 6

2 assays for HER2 testing

Answer 6

1) IHC for protein overexpression

2) In situ hybridization
- Fluorescent ISH (FISH)
- Bright field ISH
a) CISH - chromosome
b) SISH - Silver
c) DISH - Dual (both silver and chromosome)

Question 7

Categorize HER2 IHC scores from 0 to 3+

Answer 7

Score 0 = no stain
Score 1+ = Incomplete membrane, weak staining of >10% invasive cells
Score 2+ = Circumferential membrane, weak/ moderate staining of >10% invasive cells
Score 3+ = Circumferential membrane, complete/ intense staining of > 10% invasive cells

Question 8

Indication of ISH based on IHC score of HER2 status

Answer 8

Score 0, 1+ with incomplete staining of membrane&raquo_space;> Negative

Score 2+ with weak to moderate staining&raquo_space;> repeat IHC or proceed with ISH

Score 3+ with intense, complete staining&raquo_space;> Positive, no need to do ISH

Question 9

4 modalities to visualize probes in In-situ hybridization

Answer 9

1) Radioactive label

2) Non-radioactive labels
- Chromogenic
- Fluorescence
- Metallographic

Question 10

FISH or IHC

Which is better for HER2 status confirmation in breast CA?

Answer 10

FISH is better than IHC

• FISH correlates better with response to Trastuzumab or lapatinib
• FISH has no issue with non-standardized formalin fixation of samples

Question 11

3 requirements for optimal tissue handling for HER2 testing

Answer 11

Under 1 hour cold ischemic time

Sufficient neutral buffered formalin

Minimum 6 hours fixation

Question 12

Difference between FISH, CISH and SISH

Answer 12

FISH = fluorescent probe to HER2 gene and ch17 centromere

DISH = Digoxigenin-labeled DNA probe to HER2 gene, detect with mouse anti-digoxigenin antibody

SISH: Dinitrophenol-labeled DNA probe to HER2

Question 13

FISH procedure?

Answer 13

Specimen DNA denatured to single strand

Hybridize with probes

Unbound probes is washed

Nuclei counterstained with DAPI

Signal enumeration

Question 14

2 metrics used to determine the HER2 expression after FISH?

Answer 14

Signal enumeration: Count number of HER2 dots and Centromere17 dots in 20 nuclei

1) Ratio of total HER2 dots: Centromere17 dots
Higher ratio = amplified HER2 status

2) Average HER2 signal per cell

Question 15

Define the HER2/CEP17 ratio and Average HER2 copy per cell for ISH POSITIVE result

Answer 15

HER/CEP17 ratio >=2.0

Average HER2 copy number >= 4.0 signals/ cell

Question 16

Define the HER2/CEP17 ratio and Average HER2 copy per cell for ISH negative result

Answer 16

HER/CEP17 ratio <2.0

Average HER2 copy number <4 signal/ cell

Question 17

Sample limitations for HER2 FISH assay on breast cancer?

Answer 17

• Only done on invasive carcinoma, only IHC grade 3+ tumours usually have HER2 +ve
• Good prognosis subtypes don’t usually have HER2 +ve, but some might have
• Tissue fixed for more than 72 hours

Question 18

initial HER2 test on core needle biopsy of breast CA is negative
Criteria for ordering a repeat HER2 test?

Answer 18

• Grade 3+ Tumour
• Core biopsy sample amount is too small
• High grade CA cells present in excision specimen
• Biopsy sample is mishandled
• Core biopsy result ambiguous for HER2 after ISH and IHC

Question 19

What to do before counting cells on an ISH slide for HER2 amplification in breast CA?

Answer 19

Scan the whole ISH slide before counting
Find areas of HER2 amplification

Areas with increased signaling need separate counting

Question 20

What to do if histopathological result is discordant with HER2 test results on breast CA tissue?

Answer 20

Reflex testing with alternative assays

Question 21

What to do if HER2 testing IHC score is 3+ and ISH is amplified?

Answer 21

Targeted therapy for HER2 +ve breast cancer