Predictors of HER2 FISH amplification Predictors of HER2 FISH amplification in immunohistochemistry score 2+ in immunohistochemistry score 2+

infiltrating breast cancer: infiltrating breast cancer: a single institution analysisa single institution analysis

Maria Vittoria Dieci1, Elena Barbieri1, Stefania Bettelli², Federico Piacentini1, Guido Ficarra², Sara Balduzzi1, Massimo Dominici1,

PierFranco Conte1, Valentina Guarneri1

1Department of Oncology, Hematology and Respiratory Diseases, and ²Department of Pathology, University Hospital, Modena, Italy

Background Background

Herbst. Int J Radiat Oncol Biol Phys. 2004;59(suppl):21; Roskoski. Biochem Biophys Res Commun. 2004;319:1; Rowinsky. Annu Rev Med. 2004;55:433

Background (cont’d)Background (cont’d)

HER2 amplification is the primary mechanism for overexpression

HER2 is amplified in 18-20% of infiltrating breast cancers

HER2 overexpression is associated with higher risk of recurrence and

death, relative resistance to endocrine therapy, apparent lesser benefit

from certain chemotherapeutic regimens

The MoAb antiHER2 Trastuzumab was FDA-approved in 1998 for

treatment of metastatic disease

Adjuvant trastuzumab reduces the risk of recurrence and death by 1/2

and 1/3, respectively, in patients with early stage, high-risk HER2

positive breast cancers

One year trastuzumab costs: $70,000-$110,000

The Need For Accurate Testing The Need For Accurate Testing

Various strategies to determine or confirm HER2 expression have been used in clinical trials

Correct HER2 assessment is critical for patients and clinicians since anti-HER2 therapies are effective inin HER2+ disease only

IHC Images by Kornstein, MD, Medical College of Virginia

Abnormal 2+ Abnormal 3+Normal 0

Normal

Normal 1+

Normal Abnormal lowamplification

Abnormal highamplification

HER2 expressionHER2 expression

From FDA to ASCO/CAPFrom FDA to ASCO/CAP For IHCIHC: the cut-off for 3+ score was raised from 10% to

30% of invasive tumor cells with uniform and intense membrane staining

For FISH amplificationFISH amplification: a HER2/CEP17 ratio greater than 2.2, rather than equal or greater than 2.0

Recent studies have shown that the concordance between FISH and IHC is higher when the cut-off level to define HER2 3+ score is > 30%

Mass. Proc Am Soc Clin Oncol. 2000; Shah, Hum Pathol 2010

As a consequence: a higher proportion of breast cancer specimens are scored IHC 2+ but only 24% of the IHC 2+ tumors have gene amplification when tested by FISH the request for FISH assay is increasing

Endpoint Endpoint

To evaluate if routinely assessed pathologic parameters such as tumor grade, hormone

receptor status and Ki67 are able to predict FISH results in patients with an

equivocal IHC HER2 score 2+

Tissue specimensTissue specimens

• 480 infiltrating breast cancer with IHC 2+ and evaluable HER2/CEP17 ratio

ANTIBODY: Novocastra Laboratories, clone CB11 until June 2008 and Ventana, clone 4B5 from July 2008

PROBE: PathVysion HER-2 DNA Probe Kit (Vysis Inc., Downers Grove, IL)

• 96% were primary surgical samples or core biopsy for patients undergoing neoadjuvant therapy

• 4% were samples from bilateral breast cancer or metastases biopsy

Tumor characteristicsTumor characteristicsSamples 480

Hormone Receptor expression Negative (ER and PgR)Positive (ER and/or PgR)Not evaluable

44433

3

9.2%90,2%0.6%

Estrogen Receptor expression Negative (<10%)Positive (>10%)Not evaluable

56420

4

11.7%87.5%0.8%

Progesteron Receptor expression Negative (<10%)Positive (>10%)Not evaluable

133345

2

27.7%71.9%0.4%

Ki67 expressionLow proliferation (<15%)High proliferation (>15%)Not evaluable

Median Ki67 expression (range)

15631113

23.5% (0-98)

32.5%64.8%2.7%

Histologic GradeG1-2G3Not evaluable

15326859

31.9%55.8%12.3%

Distribution of FISH resultsDistribution of FISH results

Negative Negative (<1.8)(<1.8)

Borderline Borderline (1.8-2.2)(1.8-2.2)

Positive Positive (>2.2)(>2.2)

ASCO/CAPASCO/CAP332

(69.2%)

48

(10.0%)

100

(20.8%)

Negative Negative (<2.0)(<2.0)

Positive Positive ((>>2.0)2.0)

FDAFDA348

(72.5%)

132

(27.5%)

Distribution of FISH results by G, HR and Ki67Distribution of FISH results by G, HR and Ki67 (ASCO/CAP criteria) (ASCO/CAP criteria)

NNNegative Negative

(<1.8)(<1.8)Borderline Borderline

(1.8-2.2)(1.8-2.2)Positive Positive

(>2.2)(>2.2)p p

valuevalue

G1-2G1-2 153126

(82%)

13

(9%)

14

(9%)0.000

G3G3 268166

(62%)

30

(11%)

72

(27%)

HR -veHR -ve 4433

(75%)

3

(6.8%)

8

(18%)0.638

HR +veHR +ve 433297

(68.6%)

45

(10.4%)

91

(21%)

Low Ki67Low Ki67 156117

(75%)

20

(13%)

19

(12%)0.003

High Ki67High Ki67 311206

(66%)

26

(8%)

79

(25%)

Distribution of FISH results by G, HR and Ki67Distribution of FISH results by G, HR and Ki67 (FDA criteria) (FDA criteria)

NNNegative Negative

(<2.0)(<2.0)Positive Positive

((>>2.0)2.0)p p

valuevalue

G1-2G1-2 153126

(82%)

27

(18%)0.000

G3G3 268178

(66%)

90

(34%)

HR –veHR –ve 4435

(79.5%)

9

(20.5%)0.274

HR +veHR +ve 433311

(72%)

122

(28%)

Low Ki67Low Ki67 156123

(79%)

33

(21%)0.022

High Ki67High Ki67 311214

(69%)

97

(31%)

OR for FISH positive test OR for FISH positive test ASCO/CAP and FDA criteriaASCO/CAP and FDA criteria

ASCO/CAPASCO/CAPOR for OR for FISH FISH

positivitypositivity95% CI95% CI p p

valuevalue

G1-2G1-2 Ref.

1.97-6.72 0.000

G3G3 3.64

Low Ki67Low Ki67Ref.

1.42-4.22 0.001

High Ki67High Ki67 2.45

FDAFDAOR for OR for FISH FISH

positivitypositivity95% CI95% CI p p

valuevalue

G1-2G1-2 Ref.

1.45-3.83 0.001

G3G3 2.35

Low Ki67Low Ki67Ref.

1.07-2.65 0.023

High Ki67High Ki67 1.68

Discussion & ConclusionsDiscussion & Conclusions While the association between HER2 status and pathological and

molecular characteristics has been largely documented in literature, the possibility to predict HER2 amplification in HER2 equivocal (IHC 2+) cases has been less extensively studied

This is the largest single-institution series of HER2 IHC equivocal breast cancer specimens tested by FISH and analyzed to identify potential predictors for FISH amplification

This is the first report on the role of Ki67 index in such contest

On a series of 480 HER2 equivocal samples, both tumor grade and proliferation were found to be significantly associated with HER2 FISH amplification, according to the FDA and ASCO/CAP guidelines