PHARMACODIAGNOSTIC ®

HER2  Proven Diagnostic Accuracy Now Available for Gastric Cancer

FROM CERTAINTY COMES TRUST

Since September 1998, laboratories worldwide have relied on HercepTest™, the first test kit developed exclusively for a specific pharmaceutical treatment Dako has more than 40 years of experience providing accurate, consistent and reliable staining results. More than 5 million patients have been tested for HER2 status using HercepTest™; your guarantee of a proven assay. Data from a new clinical trial (ToGA) has shown that HercepTest™ and HER2 FISH pharmDx™ Kit accurately identify patients with gastric cancer who may benefit from trastuzumab treatment. These breakthrough results have led to a new indication in gastric cancer for these assays.

HER2 and gastric cancer Gastric cancer is the second most common cancer-related death in the world (1). The diagnosis of advanced gastric cancer is associated with poor prognosis with current available therapies (1, 2).

There is a clear and urgent need for better treatment of gastric cancer.

Overexpression of HER2 is involved in gastric cancer (3). Testing for HER2 status with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods has established that overexpression of HER2 or amplification of the HER2 gene is observed about as frequently in advanced gastric cancer as in breast cancer, approximately 22% (3). HER2 status has been shown to have important implications for the treatment of gastric cancer (4).

HER2 is overexpressed in approximately 22% of gastric cancers.

The ToGA trial ToGA is a randomized, open-label, multicenter, phase III study where advanced gastric cancer patients were randomized to receive chemotherapy or chemotherapy plus trastuzumab.

Setting the standards in HER2 testing: HercepTest™ continues to be the preferred choice of assay in clinical trials.

The results from the ToGA trial clearly indicate that use of trastuzumab in gastric cancer in combination with chemotherapy is an effective treatment which significantly improves overall survival to over 13 months (Figure 1, 2) (4).

HercepTest™ and HER2 FISH pharmDx™ were exclusively used in the ToGA trial for evaluating the benefit of trastuzumab treatment in HER2-positive gastric cancer patients. This is your guarantee for proven correlation between the test and clinical evidence.

20

Probability of survival

1.0

CT CT + T

0.8

OS (months) 16.0

15

13.5 11.1

0.6

CT CT+T

11.8

10

0.4 5 0.2

11.1

0.0 0

2

4

6

13.5

8 10 12 14 16 18 20 22 24 26 28 30 32 34 Time (months)

4. Van Cutsem E, et al. J Clin Oncol 2009; 27: 18s, (suppl; ASCO abstr LBA2409).

Figure 1: Efficacy results from the ToGA trial showing that the median overall survival was significantly improved with trastuzumab in combination with chemotherapy (CT + T) compared to chemotherapy alone (CT) : 13.5 vs. 11.1 months, respectively (p=0.0048; HR 0.74; 95% CI 0.60, 0.91).

0 IHC3+ or FISH+ (n=584)

IHC3+ and IHC2+/FISH+ (n=446)

4. Van Cutsem E, et al. J Clin Oncol 2009; 27: 18s, (suppl; ASCO abstr LBA2409).

Figure 2: Efficacy results from the ToGA trial showing median overall survival (OS) for two different groups of patients after treatment with trastuzumab (T) in addition to chemotherapy (CT) or chemotherapy alone.  When the primary analysis was repeated for the high HER2 expressing group, the median OS for the group of patients who had received chemotherapy plus trastuzumab increased to 16.0 months compared to 11.8 months.

Trastuzumab n

 umanized monoclonal antibody that targets the H HER2 receptor

n

 roven to be a beneficial treatment in breast cancer P with HER2 overexpression

n

 atients with gastric cancer overexpressing HER2 P may benefit from trastuzumab therapy

n

 rastuzumab therapy is associated with a small risk T of serious cardiac toxicity and reliable HER2 test results are essential for responsible patient care

HER2 test algorithm for gastric cancer Trastuzumab is associated with a small risk of serious cardiac toxicity (5). It is therefore important to be able to reliably identify patients who would benefit the most from this treatment. The outcome of the ToGA trial has resulted in new test algorithms for HER2 testing in gastric cancer determining the HER2-positive patients who should receive trastuzumab treatment. You may already be familiar with HER2 scoring in breast cancer but there are important differences in the scoring system for gastric cancer. The suggested algorithm specifies that all cases are diagnosed using IHC as the primary method. Those cases that are scored as equivocal 2+ are then subsequently tested using the FISH technique to ensure that appropriate assessment of patients for whom trastuzumab treatment is being considered, is adhered to (Figure 3).

HercepTest™ and HER2 FISH pharmDx™ Kit were exclusively used in the ToGA trial.

Specimen

HER2 IHC

0 Negative

1+ Negative

2+ Equivocal

3+ Positive

HER2 FISH Report to Oncologist for Trastuzumab consideration

Negative Non-amplified

Figure 3: HER2 test algorithm for gastric cancer.

Positive Amplified

HER2 testing in gastric cancer is different to that of breast cancer Gastric cancer is generally more heterogeneous than breast cancer (6). It is therefore essential to find the representative tissue for scoring.

B A

In addition, membranous HER2 staining in gastric cancer is commonly incomplete, which would give a negative score in breast cancer. The incomplete HER2 membrane staining is caused by glandular formations (6). A basolateral staining is a staining without luminal staining (making the membrane appear ”U” shaped). A lateral membrane staining is a staining without luminal and basal staining (making the membrane appear ”II” shaped) (Figure 4).

Scoring HercepTest™ in gastric cancer tissue HercepTest™ scoring and HER2 FISH pharmDx™ interpretation should only be performed by professionals experienced in interpreting IHC stains and FISH slides respectively. Table 1 describes how surgical and biopsy specimens are scored differently for gastric cancer using IHC. The criterion of at least 10% stained tumor cells is restricted to surgical specimens. For interpretation of biopsies, a cluster of at least five tumor cells is recommended (Table 1).

C

Figure 4: Gastric cancer. Score 3+. HercepTest™ stains showing complete (A), basolateral (B) and lateral (C) HER2 membrane staining.

Table 1. HercepTest™ scoring for gastric cancer HER2 protein Score to report overexpression assessment 0

1+

2+

3+

Surgical specimen staining pattern

Biopsy specimen staining pattern

Negative

No reactivity or membranous reactivity in < 10% of tumor cell

No reactivity or no membranous reactivity in any tumor cell

Negative

Faint/barely perceptible membranous reactivity in ≥ 10% of tumor cells; cells are reactive only in part of their membrane

Tumor cell cluster with a faint/barely perceptible membranous reactivity irrespective of percentage of tumor cells stained

Equivocal

Weak to moderate complete, basolateral or lateral membranous reactivity in ≥ 10% of tumor cells

Tumor cell cluster with a weak to moderate complete, basolateral or lateral membranous reactivity irrespective of percentage of tumor cells stained

Positive

Strong complete, basolateral or lateral membranous reactivity in ≥ 10% of tumor cells

Tumor cell cluster with a strong complete, basolateral or lateral membranous reactivity irrespective of percentage of tumor cells stained

Guidelines based on Hofmann M, et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopath 2008; 52:797-805.

Step-by-step evaluation of HercepTest™ stained gastric cancer specimens Evaluation of the sections stained for HER2 protein should be done at low power magnification first. The majority of positive cases will be obvious at low power magnification. For 2+ cases 10x-20x objective magnification is recommended to

verify membrane staining and for 1+ cases 40x objective magnification should be used to verify membrane staining. Figure 5 has been developed as a practical tool in the scoring procedure using the microscope magnification levels as a guidance.

HER2 stained biopsy/ surgical specimen Yes No Specific HER2 staining

Yes HER2 score 0

No

No membrane staining (at 40x)

No

Membrane staining of tumor cells (distinct)

Yes Membrane staining intensity and pattern of tumor cells

Exclude from scoring Exclude from scoring n Intestinal metaplasia n

 egenerative changes R (e.g. near ulceration)

n

Basal staining

n

Luminal staining only

n

Cytoplasmic staining

n

Edge artifacts

n

Crush artifacts

n

Retraction artifacts

Faint/barely perceptible membrane staining (at 40x)

Weak to moderate complete, basolateral or lateral membrane staining (at 10x-20x)

Strong complete, basolateral or lateral membrane staining (at 2.5x-5x)

HER2 score 1+*

HER2 score 2+*

HER2 score 3+*

* Must at least be a cluster of five stained tumor cells for biopsy specimens and at least 10% stained tumor cells for surgical specimens.

Figure 5: Step-by step evaluation of HercepTestTM stained gastric cancer specimens.

Scoring HER2 gene amplification in gastric cancer The analysis of gene amplification is to a large extent carried out analogous to the HER2 FISH guidelines for breast cancer. For the enumeration of FISH signals in gastric cancer the HER2 (Texas Red) and CEN-17 (FITC) signals (Figure 6A, 6B) should be counted in 20 nuclei in representative tumor areas. Then the HER2/CEN-17 ratio is calculated by dividing the total number of HER2 signals by the total number of CEN-17 signals and the specimen is scored according to Table 3.

Dako continues to set the standard for HER2 testing with HercepTest™ and now also HER2 FISH pharmDx™ Kit for both breast and gastric cancer.

Table 3. Determination of HER2 gene status from HER2/CEN-17 signal ratio Ratio of HER2 /CEN-17 signals

HER2 gene status

Result