← Product Code NYQ · P050040

# SPOT-LIGHT HER2 CISH KIT (P050040)

_Invitrogen Corporation · NYQ · Jul 1, 2008 · Pathology · APWD_

**Canonical URL:** https://fda.innolitics.com/device/P050040

## Device Facts

- **Applicant:** Invitrogen Corporation
- **Product Code:** NYQ
- **Decision Date:** Jul 1, 2008
- **Decision:** APWD
- **Device Class:** Class 3
- **Review Panel:** Pathology

## Intended Use

The SPOT-Light® HER2 CISH Kit is intended to quantitatively determine HER2 gene amplification in formalin-fixed, paraffin-embedded (FFPE) breast carcinoma tissue sections using Chromogenic In Situ Hybridization (CISH) and brightfield microscopy. This test should be performed in a histopathology laboratory. The SPOT-Light® HER2 CISH Kit is indicated as an aid in the assessment of patients for whom Herceptin® (trastuzumab) treatment is being considered. The assay results are intended for use as an adjunct to the clinicopathological information currently being used as part of the management of breast cancer patients. Interpretation of test results must be made within the context of the patient’s clinical history by a qualified pathologist.

## Device Story

The SPOT-Light® HER2 CISH Kit is an in vitro diagnostic test for FFPE breast cancer tissue sections. It uses Subtraction Probe Technology (SPT) to create digoxigenin-labeled DNA probes specific to the HER2 gene locus (17q11.2-21), reducing repetitive sequence interference. The device processes tissue samples via heat pretreatment, pepsin digestion, hybridization, and immunodetection using a peroxidase-DAB reaction. Pathologists view the resulting chromogenic signals under a brightfield microscope. Amplification is identified by large DAB clusters or multiple dots (>5) per nucleus; non-amplified cells show ≤5 dots. The kit is used in histopathology laboratories to aid in assessing eligibility for Herceptin therapy. By enabling simultaneous visualization of gene amplification and tissue morphology, it assists pathologists in clinical decision-making, potentially identifying patients who may benefit from targeted treatment.

## Clinical Evidence

Pivotal clinical study (N=226) compared CISH to FISH (PathVysion) and IHC (HercepTest). Primary endpoint: total agreement between CISH and FISH. Consecutive cases showed 99.0% concordance between CISH and FISH (95% CI: 96.5%-99.9%). Positive agreement 94.4%, negative agreement 100%. Concordance with IHC (3+) was 95.1%. Study included subgroup analysis of IHC2+ (equivocal) and polysomy cases, demonstrating strong agreement across cohorts.

## Technological Characteristics

Chromogenic In Situ Hybridization (CISH) assay. Uses digoxigenin-labeled DNA probes generated via Subtraction Probe Technology (SPT) to target HER2 gene locus on chromosome 17q11.2-21. Detection via peroxidase-DAB reaction. Visualized using brightfield microscopy. Kit includes reagents, buffers, and FFPE control slides with amplified/non-amplified cell lines. Standardized for 4-6 µm FFPE tissue sections.

## Regulatory Identification

This device is intended to detect her2 gene amplification in formalin-fixed, paraffin-embedded breast carcinoma tissue sections using chromogenic in situ hybridization and brightfield microscopy.  Indicated as an aid in the assessment of patients for whom herceptin. (trastuzumab) treatment is being considered. Interpretation of test results must be made within the context of the patients clinical history by a qualified pathologist.

## Reference Devices

- Pathvysion™ HER-2 DNA Probe Kit ([P980024](/device/P980024.md))
- DAKO Herceptest™ ([P980018](/device/P980018.md))

## Submission Summary (Full Text)

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>
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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

## I. General Information

|  Device Generic Name: | In Vitro Diagnostic Test Kit for HER2 gene amplification in formalin-fixed, paraffin-embedded (FFPE) tissue sections using Chromogenic In Situ Hybridization (CISH)  |
| --- | --- |
|  Device Trade Name: | SPOT-Light® HER2 CISH Kit  |
|  Applicant’s Name and Address: | Invitrogen Corporation
5791 Van Allen Way
Carlsbad, CA 92008  |
|  Date of Panel Recommendation: | None  |
|  Premarket Approval Application (PMA) Number: | P050040  |
|  Date of FDA Notice of Approval: | July 1, 2008  |

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SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

## II. Indications for use

For In Vitro Diagnostic Use

The SPOT-Light® HER2 CISH Kit is intended to quantitatively determine HER2 gene amplification in formalin-fixed, paraffin-embedded (FFPE) breast carcinoma tissue sections using Chromogenic In Situ Hybridization (CISH) and brightfield microscopy. This test should be performed in a histopathology laboratory.

The SPOT-Light® HER2 CISH Kit is indicated as an aid in the assessment of patients for whom Herceptin® (trastuzumab) treatment is being considered. The assay results are intended for use as an adjunct to the clinicopathological information currently being used as part of the management of breast cancer patients. Interpretation of test results must be made within the context of the patient’s clinical history by a qualified pathologist.

## III. CONTRAINDICATIONS

None known

## IV. WARNINGS AND PRECAUTIONS

Refer to product labeling for a list of warnings and precautions.

## V. DEVICE DESCRIPTION

Invitrogen’s SPOT-Light® HER2 CISH Kit is intended for the quantitative detection of HER2 gene amplification in FFPE breast cancer tissue sections by Chromogenic In Situ Hybridization (CISH). CISH detects hybridization of labeled nucleic acid probes in situ to specific sections of complementary nucleic acid in the sample using conventional peroxidase-DAB reactions, which can be viewed under the brightfield microscope. This allows pathologists to view tissue morphology and gene aberrations simultaneously. The HER2 DNA probe in the SPOT-Light® HER2 CISH Kit is generated using Subtraction Probe Technology™ (SPT™), which creates specific probes by significantly reducing the repetitive sequences (e.g., Alu and LINE elements) found in human nucleic acids. Consequently, SPT™ probes are inherently specific and do not require repetitive sequence blocking, as required for traditional cytogenetic DNA probes.

Tumors with HER2 gene amplification typically appear as large DAB intra-nuclear clusters, as multiple DAB single dots (>5 single dots), or as a mixture of clusters and multiple dots. Tumors without HER2 gene amplification typically exhibit ≤5 single dots per nucleus.

The SPOT-Light® HER2 CISH Kit is a standardized kit containing 20 tests that includes all necessary reagents and buffers in a convenient ready-to-use format. The kit also includes a FFPE control slide with both HER2 non-amplified (negative) and amplified (positive)

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

cancer cell lines on each slide. A 4-color HER2 CISH Test Interpretation Guide is included to assist in the interpretation of HER2 CISH results.

The SPOT-Light® HER2 Probe (Reagent C) is a double-stranded DNA probe that has been labeled with digoxigenin. It is supplied in a liquid format in hybridization buffer. It has been demonstrated to contain the HER2 gene by PCR, and to bind specifically to the HER2 gene locus on chromosome 17q11.2-21 by metaphase FISH in normal lymphocytes. Repetitive nucleic acid sequences have been quantitatively removed from the probe by SPT™.

## VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are several HER2 fluorescence *in situ* hybridization (FISH) test kits commercially available for the determination of gene amplification in breast tissue. Additional procedures for detection of gene product overexpression in human breast tissue include immunohistochemical (IHC) and polymerase chain reaction (PCR) techniques.

## VII. MARKETING HISTORY

The SPOT-Light® HER2 CISH Kit was launched internationally in July 2004 and is CE marked for the EU. The product is available in the following countries:

### SPOT-Light® HER2 CISH Kit Marketing History

|  Argentina | Egypt | Italy | South Africa  |
| --- | --- | --- | --- |
|  Australia | Finland | Japan | Serbia  |
|  Austria | France | Korea | Singapore  |
|  Benelux | Germany | Kuwait | Spain  |
|  Brazil | Greece/Cyprus | Lithuania | Sweden  |
|  Canada | Hong Kong | Mexico | Taiwan  |
|  China | Ireland | Norway | Thailand  |
|  Denmark | Israel | Portugal | United Kingdom  |

The SPOT-Light® HER2 CISH Kit has not been withdrawn from any of these markets for any reason.

## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

As with any *in vitro* diagnostic test, the potential risks are associated with incorrect result interpretations. A false positive test result would likely assign patients to receive a more aggressive adjuvant therapy regimen than needed, possibly exposing the patient to serious side effects and, in rare cases, death. Alternatively, a false negative test result may exclude a patient who might benefit from therapy, potentially resulting in a poor outcome.

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

## IX. SUMMARY OF PRECLINICAL STUDIES

### A. Laboratory Studies :

**Key to Abbreviation for All Tables:**

NAM = Non-amplified
AM = Amplified
BC = Big Clusters
BL = Borderline
ACC = Acceptable
STD = Standard Deviation
%CV = Percent Coefficient of Variation
' = Minute
" = Second
ND = Not done

**1+ through 4+ refer to staining intensities**

1+: Difficult to see under 40X objective lens
2+: Difficult to see under 20X objective lens, but easy under 40X
3+: Difficult to see under 10X objective lens, but easy to see under 20X
4+: Easy to see under 10X objective lens

### 1. Non-Clinical Studies - Internal

#### a. Analytical Sensitivity

The analytical sensitivity study objective was to evaluate the hybridization efficiency and sensitivity of the SPOT-Light® HER2 CISH Kit when testing formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue sections of non-amplified and amplified HER2 status as well as the FFPE cell lines used in the control slides.

The samples under evaluation for the analytical sensitivity study included slides from two breast cancer tissue blocks: HER2 non-amplification (normal HER2), and HER2 amplification and four cell line blocks (two with HER2 non-amplification, and two with HER2 amplification). These cell lines are the cell lines used in the SPOT-Light® HER2 CISH Kit control slide.

The HER2 gene was detected as a single dot on the tissue section and cell block section with normal HER2 gene (Tissue 1, Cell Line 3, and Cell Line 4). The data for hybridization efficiency are shown in Table 1.

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Table 1. Hybridization efficiency

|  Sample Type | HER2 gene status | Total sections analyzed | Total cells analyzed | Total cells with CISH signal | Hybridization Efficiency  |
| --- | --- | --- | --- | --- | --- |
|  Tissue 1 | NAM | 10 | 300 | 284 | 94.7%  |
|  Tissue 2 | AM | 10 | 300 | 300 | 100%  |
|  Cell Line 1 | AM | 1 | 100 | 100 | 100%  |
|  Cell Line 2 | AM | 1 | 100 | 100 | 100%  |
|  Cell Line 3 | NAM | 1 | 300 | 285 | 95%  |
|  Cell Line 4 | NAM | 1 | 300 | 289 | 96.3%  |

## b. Analytical Specificity

The analytical specificity study objective was to evaluate the specificity of the HER2 probe when testing metaphase spreads from normal lymphocytes by fluorescence *in situ* hybridization (FISH), when testing polymerase chain reaction (PCR) with HER2 gene specific primer pair, and when sequencing both ends of the DNA probes.

### Analytical Specificity Results by FISH

The sample under evaluation for the analytical specificity study by FISH consisted of eight cytogenetically prepared slides with metaphase spreads from a normal lymphocyte cell line. There were a total of 132 metaphases in the eight cytogenetically prepared slides. All 132 metaphases showed that the green signal (HER2) and the red signal (chromosome 17 centromere) are co-localized on the same chromosome, while the green signal is located on the long arm of Chromosome 17 band 11.2-21. No cross-hybridization to other chromosome loci was observed in any of 132 metaphase examples and hybridization was limited to the intended target regions of the two probes. Results demonstrated that the DNA probe was bound specifically to the HER2 gene locus on chromosome band 17q11.2-21. In addition, chromosome localization was confirmed by metaphase FISH in normal lymphocytes.

### Analytical Specificity Results by PCR

The sample under evaluation for the analytical specificity study by PCR included DNA from the BAC clones, which were used to prepare the HER2 DNA probe. The HER2 SPT™ DNA template has been shown to contain the HER2 gene sequence by PCR with HER2 specific primers (Schneeberger et al., 1996. Anticancer Res, 16:849-852).

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

PCR Primers:

HER2-1F: 5' GAT GTA TTT GAT GGT GAC CT 3'

HER2-2R: 5' ATC TGG CTG GTT CAC ATA TT 3'

## PCR Condition and Electrophoresis

The PCR reaction was performed in a volume of 25 µl containing HER2 DNA template, 20 pmols of each primer, 1 x KlenTag DNA polymerase, and 200 µm of each dNTPs. The PCR was performed for 30 cycles at 94°C for 5 seconds, 50°C for 30 seconds and 72°C for 45 seconds. The 1% gel electrophoresis PCR product showed correct size for HER2 specific primers.

## Analytical Specificity Result by DNA Sequencing

After sequencing both ends of the HER2 BAC clones, the HER2 probe size is 180 kb.

## Analytical Sensitivity and Specificity Results and Conclusions

i. All CISH slides showed strong CISH signal, and good morphology.
ii. Hybridization efficiency: 95 - 100%.
iii. A single HER2 gene copy in a FFPE control cell block or in a breast cancer tissue section can be detected; the analytical sensitivity is 1 dot.
iv. The HER2 DNA probe is located on the long arm of chromosome 17q11.2-21 and contains the HER2 gene sequence.

## c. Repeatability and Reproducibility Studies on Consecutive Tissue Sections and Various Tissue Thickness

The repeatability and reproducibility studies were conducted to evaluate the SPOT-Light® HER2 CISH Kit repeatability and reproducibility when testing consecutive non-amplified, borderline, and amplified breast cancer tissues of varying thicknesses.

The samples under evaluation included slides from three breast cancer tissue blocks: HER2 non-amplification (normal), HER2 borderline amplification, and HER2 amplification. Ten samples per block of consecutive sections of 4 µm thickness were processed and tested according to standard procedures (control condition), and samples of different thicknesses (range 2-8 µm) from each block were similarly processed and tested in duplicate according to standard procedures.

Results are shown in Tables 2-10.

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Table 2. Average HER2 CISH dots per cell in consecutive sections at 4 μm (breast cancer with normal HER2)

|   | Section Number  |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10  |
|  Average HER2 CISH dots/nucleus | 1.8 | 2.0 | 1.9 | 2.0 | 1.6 | 1.7 | 1.7 | 1.6 | 1.7 | 2.0  |
|  Average HER2 | 1.8  |   |   |   |   |   |   |   |   |   |
|  STD | 0.16  |   |   |   |   |   |   |   |   |   |
|  %CV | 8.9  |   |   |   |   |   |   |   |   |   |

Table 3. Average HER2 CISH dots per cell in consecutive sections at 4 μm (breast cancer with borderline amplification of HER2)

|   | Section Number  |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10  |
|  Average HER2 CISH dots/nucleus | 5.4 | 5.5 | 5.3 | 5.8 | 5.2 | 6.2 | 5.7 | 5.4 | 5.5 | 5.8  |
|  Average HER2 | 5.6  |   |   |   |   |   |   |   |   |   |
|  STD | 0.30  |   |   |   |   |   |   |   |   |   |
|  %CV | 5.4  |   |   |   |   |   |   |   |   |   |

Table 4. Average HER2 CISH dots per cell in consecutive sections at 4 μm (breast cancer with amplified HER2)

|   | Section Number  |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10  |
|  Average HER2 CISH dots/nucleus | BC | BC | BC | BC | BC | BC | BC | BC | BC | BC  |

Table 5. Average HER2 CISH dots per cell in consecutive sections of different thickness (breast cancer with normal HER2)

|   | Thickness of Section (microns)  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  2 | 2 | 4 | 4 | 6 | 6 | 8 | 8  |
|  Average HER2 CISH dots/nucleus | 1.5 | 1.4 | 1.7 | 1.6 | 2 | 2 | 2.1 | 2.2  |

Data from 8 consecutive tissue sections from 2-8 μm thickness:
Average HER2 1.8
STD 0.30
%CV 17.0

Data from 6 consecutive tissue sections from 4-8 μm thickness:
Average HER2 1.9
STD 0.23
%CV 12.0

Data from 6 consecutive tissue sections from 2-6 μm thickness:
Average HER2 1.7
STD 0.25
%CV 14.7

Data from 4 consecutive tissue sections from 4-6 μm thickness:
Average HER2 1.8
STD 0.21
%CV 12.0

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

Table 6. Average HER2 CISH dots per cell in consecutive sections of different thickness (breast cancer with borderline amplification of HER2)

|   | Thickness of Section (microns)  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  2 | 2 | 4 | 4 | 6 | 6 | 8 | 8  |
|  Average HER2 CISH dots /nucleus | 4.5 | 4.0 | 5.2 | 5.6 | 6.3 | 6.7 | 7.8 | 8.0  |

Data from 8 consecutive tissue sections from 2-8 µm thickness:
Average HER2 6.0
STD 1.46
%CV 24.0

Data from 6 consecutive tissue sections from 4-8 µm thickness:
Average HER2 6.6
STD 1.14
%CV 17.0

Data from 6 consecutive tissue sections from 2-6 µm thickness:
Average HER2 5.4
STD 1.03
%CV 19.1

Data from 4 consecutive tissue sections from 2-8 µm thickness:
Average HER2 6.0
STD 0.68
%CV 11.0

Table 7. Average HER2 CISH dots per cell in consecutive sections of different thicknesses (breast cancer with amplification of HER2)

|   | Thickness of Section (microns)  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  2 | 2 | 4 | 4 | 6 | 6 | 8 | 8  |
|  Average HER2 CISH dots/nucleus | BC | BC | BC | BC | BC | BC | BC | BC  |

Data from 8 consecutive tissue sections from 2-8 µm thickness: amplified with Big Clusters

Table 8. Average HER2 CISH dots per cell in consecutive sections of different thickness (breast cancer with normal HER2)

|   | Thickness of Section (microns)  |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |  3 | 3 | 4 | 4 | 5 | 5  |
|  Average HER2 CISH dots/nucleus | 1.7 | 1.7 | 1.8 | 1.7 | 2.1 | 2.0  |

Data from 6 consecutive tissue sections of 3-5 µm thickness:
Average HER2 1.8
STD 0.18
%CV 10.0

Data from 4 consecutive tissue sections of 4-5 µm thickness:
Average HER2 1.9
STD 0.18
%CV 9.4

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

Table 9. Average HER2 CISH dots per cell in consecutive sections of different thickness (breast cancer with borderline amplification of HER2)

|   | Thickness of Section (microns)  |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |  3 | 3 | 4 | 4 | 5 | 5  |
|  Average HER2 CISH dots/nucleus | 4.8 | 4.3 | 5.7 | 5.3 | 6.2 | 6.8  |

Data from 6 consecutive tissue sections of 3-5 µm thickness:
Average HER2 5.5
STD 0.92
%CV 17.0

Data from 4 consecutive tissue sections of 2-4 µm thickness:
Average HER2 5.0
STD 0.61
%CV 12.2

Data from 4 consecutive tissue sections of 4-5 µm thickness:
Average HER2 6.0
STD 0.65
%CV 10.8

Table 10. Average HER2 CISH dots per cell in consecutive sections of different thickness (breast cancer with amplification of HER2)

|   | Thickness of Section (microns)  |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |  3 | 3 | 4 | 4 | 5 | 5  |
|  Average HER2 CISH dots/nucleus | BC | BC | BC | BC | BC | BC  |

Data from 6 consecutive tissue sections of 3-5 µm thickness: amplified with Big Clusters

## Repeatability and Reproducibility Results and Conclusions

i. The %CV for the control condition of 4 µm with the non-amplification sample is 8.9%, and the %CV for the borderline amplification sample was 5.4%.
ii. There was no misclassification for HER2 amplification and HER2 non-amplification.
iii. Misclassification occurred for the borderline amplification when the tissue section was <4 µm (2 and 3 µm) thickness.
iv. Misclassification did not occur for HER2 amplification and non-amplification when the tissue section was ≥4 µm.
v. %CV for the borderline amplification was >15% (17%) when the tissue section was 4-8 µm. %CV for the low amplification was <15% (11%) when the tissue section was 4-6 µm.

The results from the repeatability testing with consecutive tissue sections with the same thicknesses of breast cancers with normal HER2 gene, HER2 gene borderline amplification, and HER2 gene amplification indicated an acceptable degree of repeatability of the HER2 CISH assay.

The results from reproducibility testing with different thicknesses (2-8 µm) of breast cancers with normal HER2 gene, HER2 gene borderline amplification, and HER2 gene amplification indicated an acceptable degree of reproducibility of the HER2 CISH assay when using tissue sections with thicknesses between 4-6 µm.

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The 4-5 µm thickness is the recommendation in the product labeling, with 6 µm as the outside limit.

## d. Reproducibility Studies

### i. Day-to-Day Reproducibility

The day-to-day reproducibility of the SPOT-Light® HER2 CISH Kit was evaluated with slides from three breast cancer tissue blocks: HER2 non-amplification (normal), HER2 borderline amplification, and HER2 amplification, and the kit control slide that includes positive and negative cell lines. The samples were processed and tested in duplicate for four days according to standard procedure.

The normal HER2 sample and the amplified HER2 sample demonstrated the expected “<5 dots” and “big clusters” interpretations, respectively, 100% of the time (8/8 cases per sample). The borderline HER2 amplification sample demonstrated the correct borderline quantities, with an average dot count of 5.4, a range of 5.1 to 5.9, and a %CV of 5.7%. The control samples also demonstrated the expected results 100% of the time. Results from the testing of the clinical specimens are found in Table 11, and results from the control testing are found in Table 12.

Table 11. Day-to-day reproducibility for breast cancer tissue sections (over a single lot/technologist/and reader)

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Day 1 |   | Day 2 |   | Day 3 |   | Day 4  |   |
|   |  Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2  |
|  Tissue 1 (NAM) | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+  |
|   |  <5 | <5 | <5 | <5 | <5 | <5 | <5 | <5  |
|   |  NAM | NAM | NAM | NAM | NAM | NAM | NAM | NAM  |
|  Tissue 2 (borderline) | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+  |
|   |  5.2 | 5.4 | 5.8 | 5.1 | 5.3 | 5.1 | 5.9 | 5.6  |
|   |  AM | AM | AM | AM | AM | AM | AM | AM  |
|  Tissue 3 (AM) | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+  |
|   |  BC | BC | BC | BC | BC | BC | BC | BC  |
|   |  AM | AM | AM | AM | AM | AM | AM | AM  |

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Table 12. Day-to-day reproducibility on control cell line block sections

|  Cell Line Name | CISH Signal, Average HER2 CISH dots/cell, and HER2 Gene Status  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Day 1 |   | Day 2 |   | Day 3 |   | Day 4  |   |
|   |  Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2  |
|  Cell Line 1 (AM) | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+  |
|   |  BC | BC | BC | BC | BC | BC | BC | BC  |
|   |  AM | AM | AM | AM | AM | AM | AM | AM  |
|  Cell Line 2 (NAM) | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+  |
|   |  <5 | <5 | <5 | <5 | <5 | <5 | <5 | <5  |
|   |  NAM | NAM | NAM | NAM | NAM | NAM | NAM | NAM  |

On eight consecutive tissue sections from one breast cancer specimen with borderline amplification, the day-to-day data (4 different days) showed:

Average HER2 5.4
STD 0.31
%CV 5.7

## ii. Lot-to-Lot Reproducibility

The lot-to-lot reproducibility of the SPOT-Light® HER2 CISH Kit was shown with slides from three breast cancer tissue blocks: HER2 non-amplification (normal), HER2 borderline amplification, and HER2 amplification, and two sets of the kit control slide that includes positive and negative cell lines. The samples were processed and tested in duplicate with three distinct lots, plus a fourth lot that included a different HER2 DNA probe. All testing was done according to standard procedure.

The normal HER2 sample and the amplified HER2 sample demonstrated the expected “&lt;5 dots” and “big clusters” interpretations, respectively, 100% of the time (16 slides, 2 slides per sample, per lot). The borderline HER2 amplification sample demonstrated the correct borderline results, with an average dot count of 5.7, a range of 5.1 to 6.2, and a %CV of 7.7%. The control samples also demonstrated the expected results 100% of the time. Results from the testing of the clinical specimens are found in Table 13, and results from the control testing are found in Table 14.

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Table 13. Lot-to-lot reproducibility of three breast cancer tissue specimens

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell, and HER2 Gene Status  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Lot 1 |   | Lot 2 |   | Lot 3 |   | Lot 4  |   |
|   |  Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2  |
|  Tissue 1 (NAM) | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM  |
|  Tissue 2 (borderline) | 3+
5.1
AM | 3+
6.0
AM | 3+
5.9
AM | 3+
5.2
AM | 3+
6.1
AM | 3+
5.9
AM | 3+
6.2
AM | 3+
5.2
AM  |
|  Tissue 3 (AM) | 4+
BC
AM | 4+
BC
AM | 4+
BC
AM | 4+
BC
AM | 4+
BC
AM | 4+
BC
AM | 4+
BC
AM | 4+
BC
AM  |

Table 14. Lot-to-lot reproducibility of two sets of control cell line block sections

|  Cell Line Name | CISH Signal, Average HER2 CISH dots/cell, and HER2 Gene Status  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Lot 1 |   | Lot 2 |   | Lot 3 |   | Lot 4  |   |
|   |  Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2  |
|  Cell Line 1 (AM) | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+  |
|   |  BC | BC | BC | BC | BC | BC | BC | BC  |
|   |  AM | AM | AM | AM | AM | AM | AM | AM  |
|  Cell Line 2 (NAM) | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+  |
|   |  <5 | <5 | <5 | <5 | <5 | <5 | <5 | <5  |
|   |  NAM | NAM | NAM | NAM | NAM | NAM | NAM | NAM  |
|  Cell Line 3 (AM) | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+  |
|   |  BC | BC | BC | BC | BC | BC | BC | BC  |
|   |  AM | AM | AM | AM | AM | AM | AM | AM  |
|  Cell Line 4 (NAM) | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+  |
|   |  <5 | <5 | <5 | <5 | <5 | <5 | <5 | <5  |
|   |  NAM | NAM | NAM | NAM | NAM | NAM | NAM | NAM  |

On eight consecutive tissue sections from the HER2 borderline amplification breast cancer specimen, the lot-to-lot data showed:

Average HER2 5.7

STD 0.44

% CV 7.7%

iii. Inter-run, Day-to-day

The inter-run, day-to-day reproducibility of the SPOT-Light® HER2 CISH Kit was shown using archived breast cancer tissue blocks: HER2 non-amplified (1-2 dots per cell), HER2 non-amplified with polysomy (3-5 dots per cell), and HER2 amplified (&gt;5 dots per cell). The polysomy specimens were verified as polysomy by assessing the copy number of chromosome 17 centromere. The samples were processed and tested in triplicate over three separate days.

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All slides demonstrated a strong CISH signal and good morphology. The inter-day data represents the average, standard deviation, and correlation variance determined for the pair of slides tested on each day. There is no significant difference in the variability with the HER2 status of the specimen. The average %CV for HER2 non-amplified, HER2 non-amplified with polysomy, and amplified HER2 specimens is 3%, 4%, and 8%, respectively.

The inter-day %CV and standard deviation are listed in Tables 15, 16, and 17 for each specimen for each day. Average, standard deviation, and %CV is calculated for each specimen for three different days. Intra-day average, standard deviation, and %CV are also provided. The overall inter-day %CV for all specimens tested was 5%.

Table 15. Inter-run, day-to-day reproducibility for breast cancer tissue sections for HER2 Non-amplified breast cancer archived specimens

|   | Breast Cancer HER2 Non-amplified  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Specimen 1 |   |   | Specimen 2 |   |   | Specimen 3  |   |   |
|   |  AVG | STD | %CV | AVG | STD | %CV | AVG | STD | %CV  |
|  Day 1 | 1.90 | 0.05 | 2% | 1.83 | 0.00 | 0% | 1.75 | 0.02 | 1%  |
|  Day 2 | 1.77 | 0.05 | 3% | 1.78 | 0.16 | 9% | 1.77 | 0.05 | 3%  |
|  Day 3 | 1.85 | 0.07 | 4% | 1.75 | 0.02 | 1% | 1.73 | 0.00 | 0%  |
|   | Inter-day, Summary  |   |   |   |   |   |   |   |   |
|  AVG | 1.84 |   |   | 1.79 |   |   | 1.75  |   |   |
|  STD | 0.07 |   |   | 0.04 |   |   | 0.02  |   |   |
|  %CV | 4% |   |   | 2% |   |   | 1%  |   |   |
|  Average % CV | 3%  |   |   |   |   |   |   |   |   |

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Table 16. Inter-run, day-to-day reproducibility for breast cancer tissue sections for HER2 Non-amplified polysomy breast cancer archived specimens

|   | Breast Cancer HER2 Non-amplified, Polysomy  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Specimen 1 |   |   | Specimen 2 |   |   | Specimen 3  |   |   |
|   |  AVG | STDV | %CV | AVG | STDV | %CV | AVG | STDV | %CV  |
|  Day 1 | 3.27 | 0.28 | 9% | 3.83 | 0.05 | 1% | 3.1 | 0.05 | 2%  |
|  Day 2 | 3.4 | 0.24 | 7% | 3.95 | 0.05 | 1% | 3.13 | 0.00 | 0%  |
|  Day 3 | 3.63 | 0.8 | 10% | 3.7 | 0.09 | 3% | 3.03 | 0.0 | 0%  |
|   | Inter-day, Summary  |   |   |   |   |   |   |   |   |
|  AVG | 3.43 |   |   | 3.83 |   |   | 3.09  |   |   |
|  STDV | 0.19 |   |   | 0.13 |   |   | 0.05  |   |   |
|  %CV | 5% |   |   | 3% |   |   | 2%  |   |   |
|  Average % CV | 4%  |   |   |   |   |   |   |   |   |

Table 17. Inter-run, day-to-day reproducibility for breast cancer tissue sections for HER2 amplified breast cancer archived specimens

|   | Breast Cancer HER2 Amplified  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Specimen 1 |   |   | Specimen 2 |   |   | Specimen 3  |   |   |
|   |  AVG | STDV | %CV | AVG | STDV | %CV | AVG | STDV | %CV  |
|  Day 1 | 25.65 | 0.68 | 3% | 17.68 | 0.5 | 2% | 15.88 | 1.11 | 7%  |
|  Day 2 | 24.60 | 0.42 | 2% | 18.77 | 0.19 | 1% | 15.93 | 0.5 | 5%  |
|  Day 3 | 27.37 | 6.51 | 24% | 19.97 | 4.1 | 21% | 16.13 | 1.37 | 8%  |
|   | Inter-day, Summary  |   |   |   |   |   |   |   |   |
|  AVG | 25.87 |   |   | 18.81 |   |   | 15.98  |   |   |
|  STDV | 1.40 |   |   | 1.14 |   |   | 0.13  |   |   |
|  %CV | 5% |   |   | 6% |   |   | 1%  |   |   |
|  Average % CV | 8%  |   |   |   |   |   |   |   |   |

## e. Assay Robustness Studies

### i. Pretreatment conditions

The study objective was to determine the tolerance limits of the pretreatment condition of the SPOT-Light® HER2 CISH Kit when testing breast cancer tissue sections of normal (non-amplified), borderline, and amplified HER2 status.

The samples under evaluation included slides from four breast cancer tissue blocks (HER2 non-amplification, borderline, and two HER2 amplification specimens).

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Samples were processed and tested in duplicate for different heat pretreatment times (5, 10, 15, 20, and 30 minutes) at $100^{\circ}\mathrm{C}$ (Table 18), different heat pretreatment temperatures $(93^{\circ}\mathrm{C}, 97^{\circ}\mathrm{C}, 98^{\circ}\mathrm{C}, 99^{\circ}\mathrm{C},$ and $100^{\circ}\mathrm{C})$ for 15 minutes (Table 19), and different pepsin incubation times (4, 7, 8, 10, 12, 13, and 16 minutes) at room temperature (Table 20), according to standard procedure.

Table 18. CISH results on three breast cancer tissue using different heat pretreatment times at $100^{\circ}\mathrm{C}$

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |
| --- | --- | --- | --- | --- | --- |
|   |  5 Minute | 10 Minute | 15 Minute | 20 Minute | 30 Minute  |
|   |  Slide 1 and 2 | Slide 1 and 2 | Slide 1 and 2 | Slide 1 and 2 | Slide 1 and 2  |
|  Tissue 1 (NAM) | 2+, local 3+
< 5
NAM | 3+
< 5
NAM | 3+
< 5
NAM | 3+
< 5
NAM | 3+*
< 5
NAM  |
|  Tissue 2 (borderline) | † | 3+
6.2, 5.8
AM | 3+
5.8, 5.2
AM | 3+
5.5, 5.3
AM | †  |
|  Tissue 3 (AM) | 3+, local 4+
BC
AM | 4+
BC
AM | 4+
BC
AM | 4+
BC
AM | 4+*
BC
AM  |

* Tissue partly damaged
† Results showed weak staining or tissue damage

Table 19. CISH results on four breast cancer tissue using different heat pretreatment temperatures for 15 minutes

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |
| --- | --- | --- | --- | --- | --- |
|   |  93°C | 97°C | 98°C | 99°C | 100°C  |
|   |  Slide 1 and 2 | Slide 1 and 2 | Slide 1 and 2 | Slide 1 and 2 | Slide 1 and 2  |
|  Tissue 1 (NAM) | 2+, some 3+
< 5
NAM | 3+
< 5
NAM | 2-3+ | 3+
< 5
NAM | 3+
< 5
NAM  |
|  Tissue 2 (borderline) | ND | 2-3+ | 2-3+ | 3+
5.4, 6.0
AM | ND  |
|  Tissue 3 (AM) | 3+
BC
AM | 4+
BC
AM | ND | NA | 4+
BC
AM  |
|  Tissue 4 (AM) | ND | ND | 3-4 | 4+
BC
AM | ND  |

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Table 20. CISH results on four breast cancer tissue specimens using different pepsin pretreatment times at room temperature

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |  4 Min | 7 Min | 8 Min | 10 Min | 12 Min | 13 Min | 16 Min  |
|  Tissue 1 (NAM) | 1-2+
<5
NAM | 2-3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 2+
<5
NAM | 2, local
3+
<5
NAM  |
|  Tissue 2 (borderline) | ND | ND | 3+
5.4, 6.0
AM | 3+
5.4, 6.0
AM | 3+
5.4, 6.0
AM | ND | ND  |
|  Tissue 3 (AM) | 3+, local
4+
BC
AM | 3+, local
4+
BC
AM | ND | 4+
BC
AM | ND | 3+, local
4+
BC
AM | 3+, local
4+
BC
AM  |
|  Tissue 4 (AM) | ND | ND | 4+
BC
AM | 4+
BC
AM | 4+
BC
AM | ND | ND  |

## Additional Pepsin Digestion Studies

Additional pepsin digestion studies were performed to determine the suitable pepsin digestion time for a set of archival tissue samples. Tissue sections from three types of HER2 gene status were selected based on gene copy number: HER2 non-amplified (1-2 dots per cell), HER2 non-amplified with polysomy (3-5 dots per cell), and HER2 amplified (&gt; 5 dots per cell). The polysomy cases were confirmed using a Cep-17 probe prior to the study. For each type of tissue samples, the maximum number of average CISH signals per cell was determined for the entire digestion series. The percent of total slides with at least 50% of the maximum number of CISH signals per cell was also determined for each digestion time. The predominant result used to determine the optimal pepsin digestion time was the average CISH signal per cell, although the cellular morphology and CISH signal intensity were also considered. The correlation of pepsin digestion time with average CISH signal per cell is summarized in Figure 1.

There is no difference between the suitable pepsin digestion time for all three categories of HER2 gene status. Pepsin digestion for any length of time between 2 and 20 minutes resulted in greater than 80% of specimens having at least 50% of the maximum number of CISH signals per cell for any set of tissues. About 90% of the samples demonstrated at least 50% of the maximum CISH signals per cell when digested for 4 to 6 minutes. For all samples that failed the criterion pepsin digestion for 10 minutes, resulted in at least 50% of the maximum CISH signals per cell.

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![img-0.jpeg](img-0.jpeg)
Figure 1. Effect of Pepsin Digestion Time on CISH™ Signals per Cell.

For each tissue, the maximum number of average CISH signals per cell was determined for each tissue of the entire digestion series. The percent of total slides with at least 50% of the maximum number of CISH signals per cell was determined for each digestion time.

The correlation between pepsin digestion time with cellular morphology is summarized in Figure 2. For the purposes of this study, a numerical system for quantifying cellular morphology was adopted to reduce observer variability. The cellular morphology score ranged from 0 to 8. A score of 0 was defined as “dark nuclear counterstaining with most cells lacking chromogenic signals” in which the CISH signal was significantly obscured by the nuclear counterstaining. The maximum score of 8 was defined as “nearly complete loss of CISH signals from many cells or “severe loss of nuclear morphology and staining.” The percent of total samples, which have pepsin cellular morphology numerical values between 2 and 6, was determined for each pepsin digestion time.

Pepsin digestion times between 4 and 14 minutes (Figure 2) resulted in at least 80% of the tissue samples having optimal pepsin cellular morphology (cellular morphology score between 2 and 6). Based on these results, the same range of pepsin digestion time can be applied across all three categories of HER2 gene status.

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![img-1.jpeg](img-1.jpeg)
Figure 2. The Effect of Pepsin Digestion Time on the Percent of samples with digestion morphology numerical values between 2 and 6.

An initial pepsin digestion time of 5 minutes is recommended. If 5 minutes of pepsin digestion is inadequate, the test should be repeated using a 10 minute digestion time. Based on this study, 100% of the samples will demonstrate at least 50% of the maximum CISH signals per cell using this approach.

## Pretreatment Condition Results and Conclusions

(a) The heat pretreatment temperature range is &gt;98.0-100°C for 15 minutes.
(b) The heat pretreatment time at 100°C is 10-20 minutes.
(c) The pepsin pretreatment time is 5 minutes.
(d) Under the pretreatment conditions described above, all slides from each specimen demonstrated the strong (≥3+) CISH signal, and good morphology.
(e) The normal HER2 sample and the amplified HER2 sample demonstrated the correct “&lt;5” and “big clusters” interpretations, respectively, in all sections tested.
(f) The borderline sample demonstrated X-bar of HER2 copies 5.67, STD 0.33, and %CV 5.8% (&lt;15%).
(g) There was no misclassification for HER2 amplification, borderline, or HER2 non-amplification.

## ii. Denaturization conditions

The denaturization stringency study was conducted to determine the tolerance limits for denaturization using the SPOT-Light® HER2 CISH Kit when testing breast cancer tissue section of normal, borderline, and amplified HER2 status.

The samples under evaluation included slides from four breast cancer tissue blocks (HER2 non-amplification, borderline, and two HER2 amplification specimens). Samples were processed and tested in duplicate at different denaturing temperatures (75°C, 85°C, 90°C, 93°C, 95°C, and 98°C) and different times (2, 5, and 8 minutes) using PCR themalcycler according to standard procedure. The results from this testing appear in Tables 21 and 22.

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Table 21. CISH results on two breast cancer tissue using different denaturing temperatures for 2-8 minutes

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  75°C |   |   | 85°C |   |   | 90°C  |   |   |
|   |  2min | 5min | 8min | 2min | 5min | 8min | 2min | 5min | 8min  |
|  Tissue 1 (NAM) | 2+ | 2+ | 2+ | 2-3+ | 2-3+ | 2-3+ | 2-3+ | 2-3+ | 2-3+  |
|   |  <5 | <5 | <5 | <5 | <5 | <5 | <5 | <5 | <5  |
|   |  NAM | NAM | NAM | NAM | NAM | NAM | NAM | NAM | NAM  |
|  Tissue 3 (AM) | 3+ | 3+ | 3+ | 3+ | 3-4+ | 3-4+ | 4+ | 4+ | 4+  |
|   |  BC | BC | BC | BC | BC | BC | BC | BC | BC  |
|   |  AM | AM | AM | AM | AM | AM | AM | AM | AM  |

Table 22. CISH results on four breast cancer tissue using different denaturing temperatures for 2-8 minutes

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  93°C |   |   | 95°C |   |   | 98°C  |   |   |
|   |  2min | 5min | 8min | 2min | 5min | 8min | 2min | 5min | 8min  |
|  Tissue 1 (NAM) | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+  |
|   |  <5 | <5 | <5 | <5 | <5 | <5 | <5 | <5 | <5  |
|   |  NAM | NAM | NAM | NAM | NAM | NAM | NAM | NAM | NAM  |
|  Tissue 2 (borderline) | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 3+  |
|   |  5.6, 5.5 | 5.3, 5.7 | 6.1, 5.9 | 5.8, 5.6 | 5.3, 5.3 | 5.4, 5.5 | 5.6, 6.4 | 5.3, 6.0 | 5.7, 6.0  |
|   |  AM | AM | AM | AM | AM | AM | AM | AM | AM  |
|  Tissue 3 (AM) | ND | ND | ND | 4+ | 4+ | 4+ | 4+ | 4+ | 4+  |
|   |   |   |   |  BC | BC | BC | BC | BC | BC  |
|   |   |   |   |  AM | AM | AM | AM | AM | AM  |
|  Tissue 4 (AM) | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+  |
|   |  BC | BC | BC | BC | BC | BC | BC | BC | BC  |
|   |  AM | AM | AM | AM | AM | AM | AM | AM | AM  |

## Denaturization Condition Results and Conclusions

(a) The recommended temperature and time range for denaturing when using the PCR thermalcycler is $95(\pm 1^{\circ}\mathrm{C})$ for $5\mathrm{min}$, followed by overnight incubation (10-18 hrs) at $37^{\circ}\mathrm{C}(\pm 1^{\circ}\mathrm{C})$.
(b) If using a heating block and humidity chamber with $37^{\circ}\mathrm{C}$ incubator: Denature at $95^{\circ}\mathrm{C} \left( \pm 1^{\circ}\mathrm{C} \right)$ for $5\mathrm{min}$, followed by overnight incubation (10-18 hrs) at $37^{\circ}\mathrm{C} \left( \pm 1^{\circ}\mathrm{C} \right)$.
(c) Under the pretreatment conditions described above, all slides from each specimen demonstrated a strong $(\geq 3+)$ CISH signal, and good morphology.

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

(d) The normal HER2 sample and the amplified HER2 sample demonstrated the correct “&lt;5” and “big clusters” interpretations, respectively, in all sections tested.

(e) The borderline sample demonstrated x-bar of HER2 copies 5.61, STD 0.32, and %CV 5.7% (&lt;15%).

(f) There was no misclassification for HER2 amplification, borderline, or HER2 non-amplification.

(g) All sample duplicate slides showed similar staining intensity.

## iii. Hybridization conditions

The hybridization stringency study evaluated the hybridization tolerance limits of the SPOT-Light® HER2 CISH Kit when testing breast cancer tissue sections of HER2 non-amplified, borderline, and amplified HER2 status.

Samples under evaluation included slides from four breast cancer tissue blocks (HER2 non-amplification, borderline, and two HER2 amplification specimens). Samples were processed and tested in duplicate at different hybridization temperatures (27°C, 32°C, 37°C, 39°C, 40°C, and 42°C) for 15 hours, and different hybridization times (10 hours, 14 hours, 18 hours, and 22 hours) at 37°C according to standard procedure. The results from this study are presented in Tables 23 and 24.

Table 23. CISH results on two breast cancer tissues using different hybridization temperatures for 15 hours

|  Tissue Name | CISII Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  27°C |   | 32°C |   | 37°C |   | 39°C |   | 40°C |   | 42°C  |   |
|   |  Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2  |
|  Tissue 1 (NAM) | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 2-3+ | 2-3+ | 2+
<5
NAM | 2+
<5
NAM  |
|  Tissue 2 (borderline) | ND | ND | ND | ND | ND | ND | 3+
5.5
AM | 3+
5.2
AM | 2-3+ | 2-3+ | NA | NA  |
|  Tissue 3 (AM) | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM | ND | ND | ND | ND | 3-4+
BC
AM | 3+
BC
AM  |
|  Tissue 4 (AM) | ND | ND | ND | ND | ND | ND | 4+
BC
AM | 4+
BC
AM | 3-4+ | 3-4+ | ND | ND  |

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Table 24. CISH results on two breast cancer tissue specimens using different hybridization times at 37°C

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |
| --- | --- | --- | --- | --- |
|   |  10 Hours | 14 Hours | 18 Hours | 22 Hours  |
|   |  Slide 1 and 2 | Slide 1 and 2 | Slide 1 and 2 | Slide 1 and 2  |
|  Tissue 1 (NAM) | 3+ | 3+ | 3+ | 3+  |
|   |  <5 | <5 | <5 | <5  |
|   |  NAM | NAM | NAM | NAM  |
|  Tissue 3 (AM) | 3-4+ | 3-4+ | 3-4+ | 3-4+*  |
|   |  BC | BC | BC | BC  |
|   |  AM | AM | AM | AM  |

*some cytoplasmic background staining

Additionally, the test for hybridization temperature (27°C and 39°C) was performed for different hybridization times (10, 15, and 18 hours) according to standard procedure. Samples under evaluation included slides from three breast cancer tissue blocks (HER2 non-amplification, borderline, and HER2 amplification specimens). The results from this study are presented in Tables 25 and 26.

Table 25. CISH results on three breast cancer tissues using hybridization temperatures 27°C for different time

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |  10 hours |   | 15 hours |   | 18 hours  |   |
|   |  Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2  |
|  Tissue 1 (NAM) | ND | ND | ND | ND | ND | ND  |
|  Tissue 2 (borderline) | 2-3+ | 2-3+ | 3+ | 3+ | 3+ | 3+  |
|   |   |   |  5.6 | 5.7 | 5.1 | 5.5  |
|   |   |   |  AM | AM | AM | AM  |
|  Tissue 4 (AM) | ND | ND | ND | ND | ND | ND  |

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Table 26. CISH results on three breast cancer tissues using hybridization temperatures 39°C for different time

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |
| --- | --- | --- | --- |
|   |  10 hours | 15 hours | 18 hours  |
|   |  Slide 1, 2 | Slide 1, 2 | Slide 1, 2  |
|  Tissue 1 (NAM) | 3+ | 3+ | 3+  |
|   |  < 5 | < 5 | < 5  |
|   |  NAM | NAM | NAM  |
|  Tissue 2 (borderline) | 3+ | 3+ | 3+  |
|   |  4.9, 4.8 | 5.4, 5.3 | 4.9, 5.1  |
|   |  NAM | AM | NAM, AM  |
|  Tissue 4 (AM) | 4+ | 4+ | 4+  |
|   |  BC | BC | BC  |
|   |  AM | AM | AM  |

## Hybridization Condition Results and Conclusions

(a) The CISH denaturization conditions are 30-39°C and 10-18 hours with a recommendation of 37°C (±1°C) over 10-18 hours as a start.
(b) Under the pretreatment conditions described above, all slides from each specimen demonstrated a strong (≥3+) CISH signal, and good morphology.
(c) The normal (non-amplified) HER2 sample and the amplified HER2 sample demonstrated the correct “&lt;5” and “big clusters” interpretations, respectively, in all sections tested.
(d) The borderline sample demonstrated X-bar of HER2 copies 5.29, STD 0.30 and %CV 5.6% (&lt;15%).
(e) There was no misclassification for HER2 amplification, borderline, or HER2 non-amplification.

## iv. Stringent wash

The stringent wash study identified the tolerance limits of the stringent wash conditions for the SPOT-Light® HER2 CISH kit when testing breast cancer tissue sections of normal and amplified HER2 status.

The samples under evaluation included slides from three breast cancer tissue blocks (HER2 non-amplification, borderline, and HER2 amplification). Samples were processed and tested in duplicate at different stringent wash temperatures for 5 minutes and three different temperatures for different times according to standard operating procedures (Table 27 and 28). Samples were also processed and tested at different stringent wash temperatures for 5 minutes (Table 29).

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Table 27. CISH results using different stringent wash temperature and time on three breast cancer tissue

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  66°C | 69°C | 72°C | 75°C | 78°C |   |   | 80°C |   |   | 83°C  |   |   |
|   |  5min | 5min | 5min | 5min | 2 min | 5min | 8min | 2 min | 5 min | 8min | 2min | 5min | 8min  |
|  Tissue 1 (NAM) | 3+ < 5 NAM | 3+ < 5 NAM | 3+ < 5 NAM | 3+ < 5 NAM | ND | 3+ < 5 NAM | ND | 3+ < 5 NAM | 3+ < 5 NAM | 3+ < 5 NAM | 2-3+ < 5 NAM | 2+ < 5 NAM | 2+ < 5 NAM  |
|  Tissue 2 (borderline) | ND | ND | ND | ND | 3+ 5.6, 5.3 AM | ND | 3+ 4.8, 5.3 NAM, AM | 2-3+ | 3+ | 3+ | ND | ND | ND  |
|  Tissue 3 (AM) | 4+ BC AM | 4+ BC AM | 4+ BC AM | 4+ BC AM | ND | 4+ BC AM | ND | 4+ BC AM | 4+ BC AM | 4+ BC AM | 3-4+ BC AM | 3-4+ BC AM | 3+ BC AM  |

Table 28. CISH results using different stringent wash temperatures and constant time on three breast cancer tissue

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |  37°C | 45°C | 55°C | 60°C  |   |   |
|   |  5min | 5min | 5min | 2min | 5min | 8min  |
|  Tissue 1 (NAM) | 3+* < 5 NAM | 3+* < 5 NAM | 3+* < 5 NAM | ND | 3+ < 5 NAM | ND  |
|  Tissue 2 (borderline) | ND | ND | ND | 3+ 5.4, 5.4 AM | 3+ 5.1, 5.9 AM | 3+ 5.3, 5.7 AM  |
|  Tissue 3 (AM) | 4+* BC AM | 4+* BC AM | 4+* BC AM | ND | 4+ BC AM | ND  |

*some background staining

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Table 29. CISH results using three different stringent wash temperatures and constant time (5 minutes) on eight breast cancer tissue, one normal breast and two FFPE cell line blocks

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |
| --- | --- | --- | --- |
|   |  55°C* | 65°C | 75°C  |
|  Normal breast 1
(NAM) | 3+ | 3+ | 3+  |
|   |  2 | 2 | 2  |
|   |  NAM | NAM | NAM  |
|  Breast cancer 1
(AM) | 4+ | 3-4+ | 3-4+  |
|   |  BC | BC | BC  |
|   |  AM | AM | AM  |
|  Breast cancer 2
(AM) | 4+ | 4+ | 4+  |
|   |  BC | BC | BC  |
|   |  AM | AM | AM  |
|  Breast cancer 3
(NAM) | 3+ | 3+ | 3+  |
|   |  2 | BC | BC  |
|   |  NAM | NAM | NAM  |
|  Breast cancer 4
(NAM) | 3+ | 3+ | 3+  |
|   |  3-5 | 3-5 | 3-5  |
|   |  NAM | NAM | NAM  |
|  Breast cancer 5
(NAM) | 3+ | 3+ | 3+  |
|   |  2 | 2 | 2  |
|   |  NAM | NAM | NAM  |
|  Breast cancer 6
(NAM) | 3+ | 3+ | 3+  |
|   |  2 | 2 | 2  |
|   |  NAM | NAM | NAM  |
|  Breast cancer 7
(NAM) | 3+ | 3+ | 3+  |
|   |  2 | 2 | 2  |
|   |  NAM | NAM | NAM  |
|  Breast cancer 8
(NAM) | 3-4+ | 3-4+ | 3-4+  |
|   |  2 | 2 | 2  |
|   |  NAM | NAM | NAM  |
|  FFPE Cell line block 1
(NAM) | 3+ | 3+ | 3+  |
|   |  2 | 2 | 2  |
|   |  NAM | NAM | NAM  |
|  FFPE Cell line block 2
(AM) | 3-4+ | 3-4+ | 3-4+  |
|   |  BC | BC | BC  |
|   |  AM | AM | AM  |

*some background staining

## Stringent Wash Conclusions

(a) The recommended time and temperature are 60-78°C for 2 – 8 minutes, with a recommended stringent wash at 70°C at 5 minutes.

(b) The non-amplified HER2 sample and the amplified HER2 sample demonstrated the correct “&lt;5” and “big clusters” interpretations, respectively, in all sections tested.

(c) The borderline sample demonstrated x-bar of HER2 copies 5.39, STD 0.30, and %CV 5.6% (&lt;15%).

(d) There was no misclassification for HER2 amplification, borderline, or HER2 non-amplification.

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(e) All sample duplicate slides showed similar staining intensity.

## v. Immunodetection

The immunodetection study was conducted to determine an acceptable range of incubation times for the major components in the SPOT-Light® HER2 CISH Kit detection step when testing breast cancer tissue sections of normal, borderline, and amplified HER2 status.

Samples under evaluation included slides from four breast cancer tissue blocks: HER2 non-amplification (normal), borderline, and two HER2 amplification specimens. Samples were processed and tested in duplicate using different incubation times for the major components in the detection step (mouse anti-DIG, Polymer HRP conjugated goat anti-mouse), (15, 20, 25, 30, and 60 minutes), and different incubation times for DAB, (15, 30, 45, and 60 minutes), according to standard procedures. Results are shown in Tables 30 and 31 below.

Table 30. Result of CISH performance using different incubation times of mouse-anti DIG, polymer HRP conjugate goat anti-mouse, and DAB chromogen on two breast cancer tissue

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  15 minute |   | 20 minute |   | 25 minute |   | 30 minute |   | 60 minute  |   |
|   |  Slide1 | Slide 2 | Slide1 | Slide 2 | Slide1 | Slide 2 | Slide1 | Slide 2 | Slide 1 | Slide 2  |
|  Tissue 1 (NAM) | 2 -3+
< 5
NAM | 2 -3+
< 5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM  |
|  Tissue 2 (borderline) | ND | ND | 3+ | 3+ | 3+
5.2
AM | 3+
5.3
AM | ND | ND | ND | ND  |
|  Tissue 3 (AM) | 3+
BC
AM | 3+
BC
AM | ND | ND | ND | ND | 4+
BC
AM | *
BC
AM | 4+
BC
AM | 4+
BC
AM  |
|  Tissue 4 (AM) | ND | ND | 3-4+
BC
AM | 3-4+
BC
AM | 4+
BC
AM | 4+
BC
AM | ND | ND | ND | ND  |

* one of the steps was omitted; therefore no signal was recorded.

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Table 31. Result of CISH performance using different incubation times for DAB on three breast cancer tissue

|  Tissue Name | CISH Signal, Average HER2 CISH dots/cell and HER2 Gene Status  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  15 minute |   | 30 minute |   | 45 minute |   | 60 minute  |   |
|   |  Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2 | Slide 1 | Slide 2  |
|  Tissue 1 (NAM) | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM | 3+
<5
NAM  |
|  Tissue 2 (borderline) | 3+
5.4, 5.1
AM | 3+
5.1, 5.1
AM | ND | ND | ND | ND | 3+
5.1
AM | 3+
5.1
AM  |
|  Tissue 3 (AM) | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM | 3-4+
BC
AM  |

## Immunodetection Conclusions

(a) The incubation time range for mouse anti-DIG, Polymer HRP conjugated goat anti-mouse in the SPOT-Light® HER2 CISH Kit was 15 to 60 minutes. There were no differences seen in the range of 25 to 60 minutes, while the 20 minute incubation time showed weaker staining intensity (Table 30).

(b) The incubation range of DAB in the SPOT-Light® HER2 CISH Kit was from 15 to 60 minutes. There were no differences in the range of 15 to 60 minutes (Table 31). The non-amplified sample and the amplified HER2 sample demonstrated the correct &lt;5 and big clusters interpretations, respectively, in all sections tested. The borderline sample demonstrated X-bar of HER2 copies 5.18, STD 0.15, and %CV 2.9% (&lt;15%).

(c) The results from the immunodetection stringency study indicated that the recommended incubation time for mouse anti-DIG, Polymer HRP conjugated goat anti-mouse, and for DAB, is 30 minutes.

## vi. Hematoxylin counterstaining conditions

The hematoxylin counterstaining stringency study was conducted to evaluate the counterstaining conditions and tolerance limits when testing breast cancer tissue sections of normal (non-amplified), borderline, and amplified HER2 status with the SPOT-Light® HER2 CISH Kit.

The samples under evaluation included slides from seven breast cancer tissue blocks (three with HER2 non-amplification, three with HER2 amplification, and one borderline), and one cell block containing one cell line with HER2 amplification and one cell line with HER2 non-amplification. Samples were processed and tested at different hematoxylin counterstaining times (5, 10, 20, 30, and 40 seconds) according to standard procedure. The results from this testing appear in Table 32.

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Table 32. CISH results on seven breast cancer tissue and one cell block section using different hematoxylin counterstaining times

|  Tissue or cell block name | HER2 CISH Signal |   |   |   |   | Hematoxylin Counterstaining  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  5sec | 10sec | 20sec | 30sec | 40sec | 5sec | 10sec | 20sec | 30sec | 40sec  |
|  Tissue 1 (AM) | 4+ AM | 4+ AM | 4+ AM | 4+ AM | 4+ AM | AM light | ACC | ACC | ACC | ACC  |
|  Tissue 2 (AM) | 4+ AM | 4+ AM | 4+ AM | 3-4+ AM | 3-4+ AM | AM light | ACC | ACC | ACC | ACC  |
|  Tissue 3 (NAM) | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | AM light | ACC | ACC | ACC | ACC  |
|  Tissue 4 (NAM) | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | AM light | ACC | ACC | ACC | ACC  |
|  Tissue 5 (NAM) | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | AM light | ACC | ACC | ACC | ACC  |
|  Tissue 6 (NAM) | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | AM light | ACC | ACC | NAM too dark | NAM too dark  |
|  Tissue 7 (borderline) | ND | 3+ 5.5, 5.7 AM | 3+ 5.6, 4.9 AM | 3+ 5.3, 6.0 AM | ND | ND | ND | ND | ND | ND  |
|  FFPE Cell line 1 (AM) | 3-4+ AM | 3-4+ AM | 3-4+ AM | 3-4+ AM | 3-4+ AM | AM light | ACC | ACC | ACC | ACC  |
|  FFPE Cell line 2 (NAM) | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | 3+ NAM | AM light | ACC | ACC | ACC | ACC  |

## Hematoxylin Counterstaining Condition Results/Conclusion

(a) The hematoxylin counterstaining time ranges from 5-30 seconds in this study. In this range, the counterstaining was not too light for tissue morphology, nor too dark to obscure positive staining signals. The final counterstaining depends on the pathologist’s preferences. To avoid any accidental over-counterstaining that may obscure CISH signal, 3-5 seconds of counterstaining time is recommended as a start. Additional 3-5 seconds or more may be added to adjust the intensity of the counterstaining based on the individual preference.

(b) Under these hematoxylin counterstaining times, all slides from each specimen demonstrated a high (≥3+) CISH signal, and good morphology.

(c) The HER2 non-amplification sample and the HER2 amplified sample demonstrated the correct “&lt;5” and “big clusters” interpretations, respectively, in all sections tested. The borderline sample demonstrated X-bar of HER2 copies 5.5, STD 0.37, and %CV 6.8% (&lt;15%).

(d) There was no misclassification for HER2 amplification, borderline, or HER2 non-amplification.

## f. Kit Stability

### Kits Manufactured at the South San Francisco, CA Site

Real time stability testing was done for each component separately prior to the HER2 CISH Kit configuration. The stability of the SPOT-Light® HER2 CISH™ Kit was demonstrated via both accelerated stability testing at an elevated temperature, and real-time stability testing at 2-8°C.

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i. Real time stability testing using one lot indicated that DIG-labeled HER2 DNA probe (ready-to-use) is stable at $-20^{\circ}\mathrm{C}$ for at least 4.5 years. Real time stability testing using one lot indicated that DIG-labeled HER2 DNA probe is stable at $2 - 8^{\circ}$ for 2 years.

ii. The QC data from different lots of ready-to-use DIG-labeled DNA probe made from two distinct concentrated DIG-labeled DNA probe demonstrated that the concentrated format of the probe is stable for at least 2 years.

iii. Real time stability testing indicated that the CISH Polymer Detection Kit is stable at $2 - 8^{\circ}$ for at least 1 year.

The accelerated model consisted of incubations at $37^{\circ}\mathrm{C}$ for the following time points: Weeks 1, 2, 3, and 4 (one lot), and additionally Weeks 5, 6, 7, and 8 for the second lot. At each time point, retained material was removed from the stress condition, and tested for conformance to the release specifications of the controlled product. The results from this testing showed that the testing probe had a strong CISH signal, and that the signal intensity was equal to that of the control probe through Week 7. At Week 8, the testing probe showed a little weaker staining than that of the control probe (see Table 33). Based on experience with the accelerated model, it is estimated that one week at $37^{\circ}\mathrm{C}$ corresponds to approximately six months at $2 - 8^{\circ}\mathrm{C}$, and therefore the real time stability should be just under four years. The product now in use is labeled with one-year stability from the data of manufacture.

Table 33. Summarized data from accelerated stability testing Kits Manufactured at South San Francisco, CA Manufacturing Site

|  Testing Material | Lot Number | Note | Result  |
| --- | --- | --- | --- |
|  84-0100, SPOT-Light® HER2 DNA Probe | 01062294 | Accelerated stability 1-4 weeks at 37°C | Pass  |
|  84-0100, SPOT-Light® HER2 DNA Probe | 30778586 | Accelerated stability 5-8 weeks at 37°C | Pass 5-7 weeks
Fail 8th week  |

To support the one-year labeling claim, stability data from three CISH Kit lots manufactured at the Zymed South San Francisco site were generated. Results from samples retained at $2 - 8^{\circ}\mathrm{C}$ for 1-year have passed, and thus support the 1-year expiry dating. These results are contained in Table 34.

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Table 34. Summarized Kit Stability Results for Kits Manufactured at South San Francisco, CA Manufacturing Site

|  Lot Designation | Testing Material | Lot Number | Condition | Result  |
| --- | --- | --- | --- | --- |
|  1^{st} | SPOT-Light® HER2 CISH™ Kit 84-0146 | 41281186 | Real time stability 1 year at 2-8°C | Pass  |
|  2^{nd} | SPOT-Light® HER2 CISH™ Kit 84-0146 | 50481599 | Real time stability 1 year at 2-8°C | Pass  |
|  3^{rd} | SPOT-Light® HER2 CISH™ Kit 84-0146 | 50681715 | Real time stability 1 year at 2-8°C | Pass  |

## Kits Manufactured at the Camarillo, CA Manufacturing Site

In support of the CISH manufacturing site transfer from the South San Francisco, CA site to the Camarillo, CA site, additional stability data have been generated on three CISH Kit lots manufactured at the Camarillo, CA manufacturing site. To date, stability data is available for time periods up to and including twelve months, on three lots of product manufactured at the Camarillo facility using the same analytical methods as the initial stability program. The stability test results for three lots manufactured at Camarillo, CA are included in the following Table 35.

Table 35: Kit Stability Results for Kits Manufactured at the Invitrogen Camarillo, CA Manufacturing Site

|  Lot Designation | Testing Material | Lot Number | Condition | Result  |
| --- | --- | --- | --- | --- |
|  1^{st} | SPOT-Light® HER2 CISH™ Kit 84-0146 | 1402569 | Real time stability 3-month at 2-8°C | Pass  |
|   |   |   |  Real time stability 6-month at 2-8°C | Pass  |
|   |   |   |  Real time stability 9-month at 2-8°C | Pass  |
|   |   |   |  Real time stability 12-month at 2-8°C | Pass  |
|  2^{nd} | SPOT-Light® HER2 CISH™ Kit 84-0146 | 1402570 | Real time stability 3-month at 2-8°C | Pass  |
|   |   |   |  Real time stability 6-month at 2-8°C | Pass  |
|   |   |   |  Real time stability 9-month at 2-8°C | Pass  |
|   |   |   |  Real time stability 12-month at 2-8°C | Pass  |
|  3^{rd} | SPOT-Light® HER2 CISH™ Kit 84-0146 | 1413291 | Real time stability 3-month at 2-8°C | Pass  |
|   |   |   |  Real time stability 6-month at 2-8°C | Pass  |
|   |   |   |  Real time stability 9-month at 2-8°C | Pass  |
|   |   |   |  Real time stability 12-month at 2-8°C | Pass  |

The 12-month stability results for the SPOT-Light HER2 CISH Kit continue to support the previously established one-year expiry dating when stored according to instructions for use.

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## 2. Non-Clinical Studies- External

### a. Site-to Site Reproducibility

The objective of the site-to-site reproducibility study was to additionally evaluate the reproducibility of the SPOT-Light® HER2 CISH Kit results at different sites in the U.S. with different personnel performing the assay. Trained histotechnologists performed the CISH assay, and trained pathologists performed the interpretations.

The samples used in this study included slides from three breast cancer tissue blocks (non-amplified, amplified, and borderline) and one cell block containing a positive cell line (amplified) and a negative cell line (not amplified). All samples were identified only by an ID number, and therefore the histotechnologists were blinded to the correct results. The 14 slides (12 samples and 2 control cell line slides from the kit) were stained at each external site, and after the samples were stained, they were given to the pathologists. The results appear in Tables 36-39.

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Table 36. Site-to-site reproducibility

|  Slide | Cell line or Tissue | Site 1 | Site 2 | Site 3  |
| --- | --- | --- | --- | --- |
|  1 | Cell line A (NAM)
Cell line B (AM) | <5 dots, NAM
>5 dots, AM | Not Evaluable
Not Evaluable | <5 dots, NAM
>5 dots, AM  |
|  2 | Cell line A (NAM)
Cell line B (AM) | <5 dots, NAM
>5 dots, AM | Not Evaluable
Not Evaluable | <5 dots, NAM
>5 dots, AM  |
|  3 | Tissue 1 (NAM) | NAM (not counted) | NAM (not counted) | NAM (not counted)  |
|  4 | Tissue 1 (NAM) | NAM (not counted) | NAM (not counted) | NAM (not counted)  |
|  5 | Tissue 1: NAM | NAM (not counted) | NAM (not counted) | NAM (not counted)  |
|  6 | Tissue 2: AM | AM
Mixture multiple dots & large clusters | AM
Mixture multiple dots & large clusters | AM
Mixture multiple dots & large clusters  |
|  7 | Tissue 2: AM | AM
Mixture multiple dots & large clusters | AM
Mixture multiple dots & large clusters | AM
Mixture multiple dots & large clusters  |
|  8 | Tissue 2: AM | AM
Mixture multiple dots & large clusters | AM
Mixture multiple dots & large clusters | AM
Mixture multiple dots & large clusters  |
|  9 | Tissue 3: BL | AM
5.1 (counted 60 cells) | NAM
3.6 (counted 30 cells) | AM
5.6 (counted 30 cells)  |
|  10 | Tissue 3: BL | AM
6.1 (counted 30 cells) | NAM
3.4 (counted 30 cells) | NAM
4.6 (counted 30 cells)  |
|  11 | Tissue 3: BL | AM
6.0 (counted 30 cells) | NAM
1-5 (not counted) | AM
5.0 (counted 60 cells)  |
|  12 | Cell line L
Cell line R | AM
Mixture multiple dots and large clusters
NAM (not counted) | Not Evaluable | AM
Mixture multiple dots and large clusters
NAM (not counted)  |
|  13 | Cell line L
Cell line R | AM
Mixture multiple dots and large clusters
NAM (not counted) | Not Evaluable | AM
Mixture multiple dots and large clusters
NAM (not counted)  |
|  14 | Cell line L
Cell line R | AM
Mixture multiple dots and large clusters
NAM (not counted) | Not Evaluable | AM
Mixture multiple dots and large clusters
NAM (not counted)  |

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Table 37. Results from Sites 1 and 3 (not including borderline cases)

|  Number of
Samples Tested
For Each Site | Number Agree |   | Number Discordant | % Agreement  |
| --- | --- | --- | --- | --- |
|   |  Amplified | Non-Amplified  |   |   |
|  16 | 8 | 8 | 0 | 100%  |

Table 38. Samples with Evaluable Results from All Three Sites (not including borderline cases)

|  Number of
Samples Tested
For Each Site | Number Agree |   | Number
Discordant | % Agreement  |
| --- | --- | --- | --- | --- |
|   |  Amplified | Non-Amplified  |   |   |
|  6 | 3 | 3 | 0 | 100%  |

Table 39. Borderline Cases

|  Number of
Samples Tested
For Each Site | Number Site 1 and 3 agree
Amplified, Site 2 disagree | Number Site 2 and 3 agree Non-
Amplified, Site 1 disagree  |
| --- | --- | --- |
|  3 | 2 | 1  |

Due to a technician error at Site 2, non-evaluable results were recorded. After further investigation, it was determined that the cells were excessively digested during the pepsin digestion step.

## b. Observer-to-Observer Reproducibility

The objective of the observer-to-observer reproducibility study was to evaluate the reproducibility of the SPOT-Light® HER2 CISH Kit results at different sites with different personnel interpreting the slide results. The slides (n=8) were all stained at Invitrogen, and were represented by: two non-amplified cases, two amplified cases, and four non-amplified cases with polysomy.

A total of three pathologists at three different sites interpreted and reported the results of a set of stained slides. Each pathologist reported the results in the Sample Reporting Worksheet and returned them to Invitrogen. The results appear in Table 40.

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Table 40. Observer-to-observer reproducibility

|  Slide ID | Slide # | CISH Signal, Average HER2 CISH dots/cell, and HER2 Gene Status  |   |   |
| --- | --- | --- | --- | --- |
|   |   |  Observer 1 | Observer 2 | Observer 3  |
|  1 | Sample 1 (NAM) | 2 dots NAM | 1-5 dots NAM | 1-2 NAM  |
|  2 | Sample 2 (AM) | Large Cluster + multiple dots AM | Large Cluster + dots AM | >10 AM  |
|  3 | Sample 3 (NAM) | 2-5 NAM | 1-5 NAM | 3-5 NAM  |
|  4 | Sample 4 (NAM) | 2-4 NAM | 2.8 NAM | 3-5 NAM  |
|  5 | Sample 5 (NAM) | 2 NAM | 1-5 NAM | 1-2 NAM  |
|  6 | Sample 6 (NAM) | 2-5 NAM | 1-5 NAM | 4 NAM  |
|  7 | Sample 7 (AM) | Large cluster + multiple dots AM | Large cluster AM | Large cluster + multiple dots AM  |
|  8 | Sample 8 (NAM) | 2-5 NAM | 3.4 NAM | 4.3 NAM  |

The correct interpretations were achieved 100% of the time.

B. Animal Studies: None

C. Additional Studies: None

X. Summary of clinical studies

A. Study design

The safety and effectiveness of the SPOT-Light® HER2 CISH™ Kit has been evaluated in the Invitrogen sponsored pivotal clinical study (Clinical Report 30266CA: SPOT-Light® HER2 CISH™ Kit for the Evaluation of HER-2/neu Gene Status in Human Breast Tissue). This study provides comparative data between the SPOT-Light® HER2 CISH™ Kit method and Pathvysion™ HER-2 DNA Probe Kit, an FDA-approved, commercially-available fluorescence in-situ hybridization (FISH) method (P980024), along with data from DAKO Herceptest™ a standardized and FDA-approved (P980018) immunohistochemistry (IHC) method for the detection HER2 protein expressed on the surface of tumor cells (P980018). This Invitrogen

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sponsored clinical study provides the pivotal clinical data for assessing the safety and effectiveness of the SPOT- Light® HER2 CISH™ Kit.

## Invitrogen Pivotal Clinical Study (Clinical Report 30266CA): SPOT-Light® HER2 CISH™ Kit for the Evaluation of HER-2/neu Gene Status in Human Breast Tissue

The primary study objective was to evaluate the concordance of the SPOT-Light® HER2 CISH™ Kit to PathVysion™ HER-2 DNA Probe kit for the detection of HER2/neu gene amplification status in human breast tissue. The secondary objective was to evaluate concordance of the SPOT-Light® HER2 CISH™ Kit to HercepTest™, across three IHC classes of protein expression (0, 1+: negative HER2 expression, 2+: weakly positive, and 3+: strongly positive), with a detailed analysis in Equivocal Cases of IHC2+ staining. The study was conducted at three study sites: Department of Pathology at MD Anderson Cancer Center, Jilab, Inc. Tampere University, and Invitrogen Corporation.

## 1. Clinical Inclusion and Exclusion Criteria

a. The Inclusion Criteria for cases selected for the clinical study included the following:

i. Confirmed pathology diagnosis of primary invasive breast cancer
ii. Adequate tissue sample for the entire study.

b. The Exclusion Criteria for cases selected for the clinical study included any one or more of the following:

i. The case is missing the clinically relevant data such as pathology description of the tumor.
ii. The case has inadequate or no existing tissue sample for the entire clinical study or the samples are not readily available at the Study Site. Such situations may include the referral cases where the existing tissue samples will be difficult to obtain, or samples obtained through biopsy and/or fine needle aspirate where such samples are inherently inadequate for further analysis.
iii. The case has missing tissue sample or medical records.
iv. The tissue for the case is from a core biopsy.
v. A case may be excluded from some or all of the analysis, if the case has adequate tissue sample but it fails CISH, FISH, or IHC staining after two attempts or after no additional slides are available to complete staining.

## 2. Follow-up schedule – Not applicable

## 3. Clinical endpoints

The primary endpoint was the total agreement rate between CISH and FISH. The positive (amplified) and negative (not amplified) CISH and FISH outcomes were defined as follows:

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

FISH: PathVysion™ (Vysis Inc., Downer’s Grove, IL)

Not amplified: HER2:CEP17 ratio &lt; 2.0

Amplification: HER2:CEP17 ratio ≥ 2.0

CISH: SPOT-Light® HER2 CISH Kit, (Invitrogen Corporation, Camarillo, CA)

Not amplified: 1-5 signals (dots)/nucleus in tumor cells

Amplification: &gt; 5 signal/nucleus, or clusters of amplified spots/nucleus in 30 tumor cells

The percentage of cases with agreed CISH and FISH outcomes (i.e. amplified from both tests or non-amplified from both tests) was calculated and the corresponding 95% confidence interval was calculated using normal distribution approximation. Cohen’s kappa was also used to evaluate the agreement between CISH and FISH and between CISH and IHC.

## B. Study population demographics, baseline parameters, PMA cohort accountability, Safety and Effectiveness Results

This study included two sets of cases, from Consecutive Cases and Supplemental Cases that had met the inclusion and exclusion criteria as stated in the Clinical Study Protocol. The Consecutive Cases were selected in reverse chronological order, starting with Consecutive Cases identified in December 2006, and working backward in time until the desired number of Consecutive Cases was selected at both the MD Anderson and Tampere sites. Table 41 shows the distribution of the specimen characteristics for Consecutive Cases from the MD Anderson and Tampere sites as reported in the pathology reports.

Table 41: Specimen Accountability Consecutive Cases

|   | MD Anderson | Tampere | Total  |
| --- | --- | --- | --- |
|  Total Cases^{1} | N = 110 | N = 116 | N = 226  |
|  Primary | 103 | 116 | 219  |
|  Secondary | 7 | 0 | 7  |
|  Fisher's Exact Test | 0.006  |   |   |
|  Ductal | 86 | 89 | 175  |
|  Lobular | 6 | 20 | 26  |
|  Other histologic type | 18 | 7 | 25  |
|  Fisher's Exact Test | 0.002  |   |   |
|  # of Positive Nodes |   |   |   |
|  N^{2} | 106 | See Table 42 | 106  |
|  Mean (SD) | 1.41 (4.06) | See Table 42 | 1.41 (4.06)  |
|  Range | 0.00, 25.0 | See Table 42 | 0.00, 25.0  |
|  Tumor Size |   |   |   |
|  N^{2} | 110 | See Table 42 | 110  |

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

|  Mean (SD) | 1.75 (1.56) | See Table 42 | 1.75 (1.56)  |
| --- | --- | --- | --- |
|  Range | 0.15, 10.5 | See Table 42 | 0.15, 10.5  |

1 Number of consecutive cases provided by the corresponding Study Sites.
2 Smaller total number of subjects for each parameter indicates missing values.

Table 42: Specimen Accountability Consecutive Cases; supplemental information from Tampere

|  Summary of Tumor Size |   |   | Summary of Nodal Status  |   |   |
| --- | --- | --- | --- | --- | --- |
|  No data available | 1 | 0.86% | No data available | 3 | 2.59%  |
|  T1 | 78 | 67.24% | 0 | 69 | 59.48%  |
|  T2 | 27 | 23.28% | 1 | 33 | 28.45%  |
|  T3 | 5 | 4.31% | 2 | 8 | 6.90%  |
|  T4 | 5 | 4.31% | 3 | 3 | 2.59%  |

Table 42 shows one of the 116 (0.86%) consecutive cases from the Tampere supplemental information had no data on tumor size. Three of the 116 cases (2.59%) have no nodal status in the reports. One of these 3 cases (B-010) was reported to have distant metastasis.

The MD Anderson Cancer Center also selected 60 Supplemental Cases that showed an IHC2+ score for IHC testing (antibody AB8, Neomarkers) during patient care at the MD Anderson Cancer Center. A summary of specimen characteristics for the Supplemental Cases for this study are shown in Table 43.

Table 43: Specimen Accountability Supplemental Cases from MD Anderson

|   | MD Anderson  |
| --- | --- |
|  Total Cases^{1} | N = 60  |
|  Primary | 56  |
|  Secondary | 4  |
|  Ductal | 52  |
|  Lobular | 4  |
|  Other histologic type | 4  |
|  # of Positive Nodes |   |
|  N^{2} | 58  |
|  Mean (SD) | 0.81 (2.57)  |
|  Range | 0.00, 18.00  |
|  Tumor Size |   |
|  N^{2} | 59  |
|  Mean (SD) | 1.75 (1.53)  |
|  Range | 0.15, 7.50  |

1 Number of supplemental cases provided by the corresponding Study Site.
2 Smaller total number of subjects for each parameter indicates missing values.

## 1. Consecutive Cases

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

Table 44, summarizes the staining distribution of all consecutive cases for the concordance analyses. The invalid cases were the result of failed staining twice during test sample preparation or no invasive tumor in the tissue section.

Table 44: Accountability of IHC, FISH, and CISH Tests Consecutive Cases Examined by the Study Sites

|   | Consecutive Case Series  |   |
| --- | --- | --- |
|   |  # Cases | Percent  |
|  IHC test successfully completed | 221 | 97.8%  |
|  IHC test invalid | 5 | 2.2%  |
|  Test Not Available | 0 | 0.0%  |
|  Total (N) | 226 | 100.0%  |
|  FISH test successfully completed | 220 | 97.3%  |
|  FISH test invalid | 6 | 2.7%  |
|  Test Not Available | 0 | 0.0%  |
|  Total (N) | 226 | 100.0%  |
|  CISH test successfully completed | 209 | 92.5%  |
|  CISH test invalid | 17 | 7.5%  |
|  Test Not Available | 0 | 0.0%  |
|  Total (N) | 226 | 100.0%  |

A total of 226 cases were available for IHC, FISH, and CISH tests. IHC was successfully completed for 221 (97.8%), FISH for 220 (97.3%), and CISH for 209 (92.5%) of 226 cases. A summary of the reasons the tests were invalid are provided in Table 45.

Table 45: Reasons for Invalid IHC, FISH, and CISH Tests

|   | # IHC | # FISH | # CISH  |
| --- | --- | --- | --- |
|  Failed staining twice | 0 | 2 | 14  |
|  No invasive tumor | 4 | 4 | 3  |
|  Morphology, signal intensity, or nuclear morphology is poor | 1 | 0 | 0  |
|  Missing cases, no slides | 0 | 0 | 0  |

A summary of the distribution of CISH and FISH test results in relation to the HercepTest™ scores, based on the manufacturers' guidelines, are included in Table 46.

Table 46: Results of IHC, FISH, and CISH

|  Protein Expression | 0 | 1 | 2 | 3 | Total  |
| --- | --- | --- | --- | --- | --- |
|  IHC, HercepTest™ Score (N) | 141 | 19 | 21 | 40 | 221  |
|  (%)¹ | 63.8% | 8.6% | 9.5% | 18.1% | 100%  |
|  Gene Ratio with FISH HER2 status |  |   |   |   |   |
|  Number of valid FISH cases² | 140 | 19 | 21 | 38 | 218  |
|  Amplified (n,%)³ | 1 (0.5) | 0 (0.0) | 5 (2.3) | 31 (14.2) | 37  |
|  Non-amplified (n,%)³ | 139 (63.8) | 19 (8.7) | 16 (7.3) | 7 (3.2) | 181  |

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# SUMMARY OF SAFETY and EFFECTIVENESS (SSED)

|  Gene Copies with CISH HER2 status |  |   |   |   |   |
| --- | --- | --- | --- | --- | --- |
|  Number of valid CISH cases² | 132 | 17 | 19 | 38 | 206  |
|  Amplified (n,%)³ | 1 (0.5) | 0 (0.0) | 3 (1.5) | 32 (15.5) | 36  |
|  Non-amplified (n,%)³ | 131 (63.6) | 17 (8.3) | 16 (7.8) | 6 (2.9) | 170  |

¹ % = N ÷ Total N × 100%
² Number of valid FISH (or CISH) cases for valid IHC cases with the corresponding IHC grade.
³ % = n ÷ Total number of valid FISH (or CISH) cases × 100%

## a. Comparison study with the HercepTest™

Table 47: Agreement between CISH and IHC

|  CISH Result | IHC Result |   | Total  |
| --- | --- | --- | --- |
|   |  Positive (3+) | Negative (<3+)  |   |
|  Amplified | 32 | 4 | 36  |
|  Non-amplified | 6 | 164 | 170  |
|  Total | 38 | 168 | 206  |

20 cases were reported with either missing or invalid IHC or CISH test outcomes and were excluded from the table.

Positive agreement = 32/38 = 84.2% (95% CI: 68.8%, 94.0%)
Negative agreement = 164/168 = 97.6% (95% CI: 94.0%, 99.4%)
Total percentage agreement = (32+164)/206 = 95.1% (95% CI: 91.3%, 97.7%)

## b. Comparison study with the PathVysion™ HER2 DNA Probe Kit

Table 48: Agreement between FISH and IHC

|  FISH Result | IHC Result |   | Total  |
| --- | --- | --- | --- |
|   |  Positive (3+) | Negative (<3+)  |   |
|  Amplified | 31 | 6 | 37  |
|  Non-amplified | 7 | 174 | 181  |
|  Total | 38 | 180 | 218  |

8 cases were reported with either missing or invalid IHC or FISH test outcomes and were…

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