**sBLA**

**STN: 103792\\5008\\0**

\[Original BLA 98-0369\]

## **Trastuzumab**

# Herceptin

(rhuMAbHER2)

Date of Submission: April 2, 2001

Final Approval: Decision Pending

Sponsor: Genentech, Inc.

# 

# 

# Food and Drug Administration

### Center for Biologics Evaluation and Research 

**FDA/CBER Review Team**

Clinical Reviewer: Susan Jerian, MD

Statistical Reviewer: Chao Wang, PhD

BIMO Reviewer: Deborah Bower

**Table of Contents**

I.  Introduction 4

II. Background 5

III. FISH Studies Conducted 6

IV. Results of FISH Testing 12

V.  Conclusions 33

VI. Appendix A: Exploratory Analyses 35

VII. Appendix B: PathVysion Package Insert 45

VIII. Appendix C: HercepTest Package Insert 46

IX. Appendix D: Trastuzumab Package Insert 47

> **I.** **INTRODUCTION**

The sponsor of this supplemental biologic license application (sBLA),
Genentech, Inc., requests that information on use of an assay to detect
gene amplification, fluorescence in situ hybridization (FISH), be added
to the trastuzumab (Herceptin) package insert (PI). The original
licensing clinical trials upon which the approval of trastuzumab was
based, enrolled patients with HER2 protein overexpression as determined
by an immunohistochemistry assay (IHC). The trastuzumab approval was
necessarily linked to the approval of the diagnostic
immunohistochemistry (IHC) assay. FDA/CBER required that a diagnostic be
available to clinicians (commercially or otherwise) for instances where
the decision to treat a patient with a therapeutic product was dependent
upon using the diagnostic assay.

The approved assay for the detection of HER2 protein overexpression,
HercepTest manufactured by DAKO, was licensed at the same time as
trastuzumab. HercepTest was approved based upon a concordance study
comparing HercepTest to the Clinical Trial Assay (CTA). The CTA was the
immunohistochemistry assay used to determine patient eligibility for the
trastuzumab clinical trials; it employed two monoclonal antibodies: 4D5,
the parent antibody to trastuzumab, and CB11. HercepTest employed a
polyclonal antibody. IHC scores of 2+ and 3+ (on a scale of 0, 1+, 2+,
and 3+) were considered positive for overexpression in both assays.
HercepTest was not tested on tissue samples from the clinical trial,
because the only archived tissue samples available were pre-cut slides
upon which conduct of IHC was felt to be technically unreliable.

At the 1998 Oncologic Drugs Advisory Committee Meeting which reviewed
trastuzumab, exploratory analyses of the clinical trial data were
presented and strongly suggested that the clinical benefit of
trastuzumab was primarily seen in patients whose tumors were scored as
IHC 3+ using the CTA. While the committee members noted this as an
important finding, they also noted that there may be a subset of 2+
patients for whom therapy might be effective and they did not want the
PI to exclude 2+ patients from consideration for trastuzumab therapy.
FDA followed this advice by not restricting the indication to 3+
patients and included the data for these subgroups (2+ and 3+) in the
trastuzumab package insert to assist clinicians in treatment decisions.

Facing many unanswered questions regarding HER2 detection systems and
with the anticipation of further developments in the field, FDA asked
Genentech to make the following postmarketing commitment in 1998:

> "To assess the clinical outcome of patients selected for treatment on
> the basis of the DAKO test \[HercepTest\] and other HER2 diagnostics
> in the context of Herceptin clinical trials."
>
> While this supplemental biologic license application (sBLA)
> application does not fulfill this commitment in total, FDA recognizes
> the extensive use of FISH testing off label for the selection of
> patients to be treated with trastuzumab and feels it is in the
> interest of the public health to comment on the use of FISH in the
> trastuzumab label. FDA seeks the advice of the ODAC at this time
> regarding the type of information to include in the trastuzumab PI and
> what additional trial designs might provide useful data to enable more
> definitive recommendations to clinicians about HER testing.

##### II. BACKGROUND

###### Regulatory Timeline for the FISH sBLA

> In March 2000, Genentech informed CBER that they had conducted
> exploratory, retrospective concordance and clinical outcomes studies
> using fluorescence in situ hybridization (FISH) as a method for
> selecting HER2 amplified patients to be treated with trastuzumab.
> These studies were undertaken by Genentech, and not initially by the
> FISH assay manufacturer. They were not conducted under IND. The
> sponsor felt the results to be sufficiently provocative to warrant a
> proposal for a sBLA to allow FISH to be used to select patients for
> trastuzumab therapy. In light of developments in FISH assays and more
> extensive off label use of FISH to select patients for trastuzumab
> therapy, FDA agreed to review the initial proposal. Upon review it was
> found that the proposal was not acceptable, because there was a
> significant amount of missing data in the clinical outcome study and
> this missing data appeared to occur in a non-random fashion. FDA also
> advised the sponsor that in order to make predictive or comparative
> claims in the PI, a prospective trial would need to be conducted.
>
> In August 2000, FDA agreed with Genentech's proposal to try to
> minimize the amount of missing data by performing FISH on previously
> stained slides based on methods developed by Dr. Michael Press at the
> University of Southern California (USC). FDA advised the sponsor that
> even with this data, the clinical outcome data may at best be
> supportive and may not rise to the level of robustness to be included
> in the package insert. The sBLA proposing the use of FISH (PathVysion
> assay kit) as an aid in the selection of patients for trastuzumab
> therapy was received in April 2001. At approximately the same time, a
> Pre-Market Approval application (PMA) was also filed in the Center for
> Devices and Radiological Health (CDRH) by Vysis, the manufacturer of
> PathVysion.

###### B. HER2 Amplification as Detected by FISH

> There are currently two approved FISH assay kits (PathVysion and
> INFORM) for the detection of HER2 amplification, but neither is
> indicated as an aid in the selection of patients for treatment with
> trastuzumab. Genentech employed the PathVysion FISH assay in their
> studies. PathVysion by Vysis is presently indicated "as an adjunct to
> existing clinical and pathologic information currently used as
> prognostic factors in stage II, node-positive breast cancer patients"
> and "as an aid to predict disease-free and overall survival in
> patients with stage II, node positive breast cancer treated with
> adjuvant cyclophosphamide, doxorubicin, and 5-fluorouracil (CAF)
> chemotherapy." See Appendix B for PathVysion package insert.

###### C. Nature of the Data

FDA makes the following observations about the nature of the data
submitted in this application. They are exploratory, retrospective data
with provocative results and are, in part, appropriate for inclusion in
the trastuzumab label for the purpose of supplying clinicians with
information upon which they may base their decisions for individual
patient treatment or design of clinical trials. [The data are not from
prospective, randomized, double-blinded, controlled, multi-center trials
and can neither answer questions regarding the predictive capability of
FISH to select patients who would most benefit from trastuzumab therapy,
nor answer questions regarding a hypothesized comparative benefit of
FISH vs IHC. These data do not address the issue of the clinical utility
of administering trastuzumab therapy to patients whose tumors test as
FISH positive and IHC negative.]{.underline} As such, they do not rise
to the level for expansion of the indications in the trastuzumab label.

It is also the perception of the FDA that there is considerable
confusion and misunderstanding on the part of the oncology community
regarding the conduct, performance characteristics, clinical application
and interpretation of IHC and FISH. These may be significant enough to
warrant general precautionary comment in the trastuzumab label for the
sake of clarification of these issues.

> FDA views both IHC and FISH as semi-quantitative if performed under
> ideal circumstances. Both methods require subjective interpretation of
> special stains.
>
> **III. FISH STUDIES CONDUCTED**

A.  **Overview**

> The FISH studies conducted on the archived clinical trial tissue
> samples proceeded in a progressive and complex fashion. The sponsor
> conducted retrospective concordance, clinical outcome, and validation
> studies. The first study undertaken was the concordance study
> comparing detection of gene amplification to protein overexpression.
> Gene amplification was assessed by LabCorp using a FISH assay kit
> (PathVysion). Protein overexpression was previously assessed by
> LabCorp using the CTA. Based upon the results of the concordance
> study, the clinical outcome study was initiated on tissues samples
> from patients for whom there were at least 2 unstained slides in
> storage. While the results of the clinical outcome study were of
> interest, there was a considerable amount of missing data due to the
> lack of sufficient number of unstained slides available and failure of
> the FISH assay in some cases where unstained slides were available.
> Efforts to fill in the missing data were made by employing the
> services of Dr. Michael Press who had experience with performing FISH
> on previously stained slides. The clinical outcome study was then
> expanded to include assessments at the lab of Dr. Press. In
> conjunction with this testing was a validation study to compare the
> results from the LabCorp testing to those with the Press lab testing.
> The validation study revealed potential systemic differences between
> the labs leading to further validation testing between LabCorp and
> Press. Below are descriptions of the various studies followed by flow
> diagrams outlining where tissue samples were assessed.

###### FISH assay used on all samples: PathVysion manufactured by Vysis. See package insert in Appendix B .

###### Laboratory sites: 

###### Commercial Laboratory site = Laboratory Corporation (LabCorp)

> Academic Laboratory site = Dr. Michael Press's laboratory at the
>
> University of Southern California (Press)

Retrospective studies conducted:

#### CTA vs FISH Concordance Study: samples from all screened patients, (IHC 

#### scores of 0, 1+, 2+, 3+)

> Clinical Outcome Study: samples from H0648g, H0649g, and
>
> H0650g (2+ and 3+ only)
>
> LabCorp vs Press Validation: samples from H0648g, H0649g, and
>
> H0650g (2+ and 3+ only)
>
> **Tissue source:** Archived pre-cut slides from previously conducted
> trastuzumab licensing clinical trials H0648g and H0649g, non-licensing
> Phase 2 trial H650g, and samples used to screen patients for
> trastuzumab clinical development program prior to approval were used.
> Slides may have been cut and unstained or cut and previously stained
> as IgG control slides. Slides used were from patients' tissue samples
> previously tested by LabCorp using CTA (IHC).

Description of clinical trials from which tissue samples were obtained:

> **H0648g:** Chemotherapy and Antibody Response Evaluation (CARE): A
> Phase III, Multinational, Randomized Study of Recombinant Humanized
> Anti-p185^HER2^ Monoclonal Antibody (rhuMAb HER2) Combined with
> Chemotherapy in Patients with HER2 Overexpression who have not
> Received Cytotoxic Chemotherapy for Metastatic Breast Cancer.
>
> Enrollment conducted from June 12, 1995 to March 7, 1997; patient
> follow up through October 1999.
>
> Multicenter, randomized, open label trial comparing chemotherapy in
> combination with trastuzumab vs chemotherapy alone. Depending on prior
> anthracycline use in the adjuvant setting, patients were either
> treated with standard AC (anthracycline plus cyclophosphamide) therapy
> if no prior anthracycline or Paclitaxel as the chemotherapy portion of
> the therapy.
>
> Enrolled population: Patients with metastatic breast cancer who had
> not received prior chemotherapy for their metastatic disease and whose
> tumor tissue tested as 2+ or 3+ by immunohistochemistry assay using
> CTA.
>
> The primary endpoint was median time to progression as determined by
> independent response evaluation committee with secondary endpoints of
> overall response rate, median duration of response, time to treatment
> failure, overall survival and quality of life.
>
> Results are as noted in the current package insert (Appendix D).
>
> **H0649g:** A Multinational, Open-Label Study of Recombinant Humanized
> Anti-p185^HER2^ Monoclonal Antibody (rhuMAb HER2) in Patients with
> HER2/neu Overexpression Who Have Relapsed Following One or Two
> Cytotoxic Chemotherapy Regimens for Metastatic Breast Cancer

######## Conducted April 24, 1995 to June 4, 1997

> Multicenter, single arm trial of trastuzumab as single agent therapy.
>
> Enrolled population: Patients with metastatic disease who had received
> treatment with one or two prior chemotherapy regimens and whose tumor
> tissue tested as 2+ or 3+ by immunohistochemistry assay using CTA.
>
> The primary endpoint was overall objective response rate (complete
> response plus partial response) as determined by independent response
> evaluation committee with secondary endpoints of median duration of
> response, median time to progression, median time to treatment
> failure, overall survival and quality of life.

Results are as noted in the current package insert (Appendix D).

#### H0650g: A Multinational, Randomized, Single-Blind Study of Recombinant Humanized Anti-p198HER2 Monoclonal Antibody (rhuMAb HER2) in Patients with HER2/neu Overexpression Who Have Not Received Prior Cytotoxic Chemotherapy for Metastatic Breast Cancer

> This trial enrolled patients who were not eligible to receive
> chemotherapy or who electively chose not to receive chemotherapy as
> front line therapy for their metastatic disease. All patients received
> trastuzumab as a single agent; randomization was to one of two dose
> levels.

Clinical efficacy data from this trial was not previously accepted or
reviewed by the FDA for inclusion in the package insert because the
trial was conducted in a subset of primary metastatic breast cancer
patients from which conclusions about use of trastuzumab as single agent
first line therapy could not be ascertained. Study H0659g data for this
FISH sBLA were reviewed by the FDA for concordance with CTA and for
comparisons of the two testing facilities, but not for clinical outcome.

###### B. CTA vs FISH Concordance Study

#### LabCorp Protocol: Retrospective Study

> [Primary Endpoint]{.underline}: Determine the level of concordance
> between two methods of measuring HER2 overexpression in patients with
> metastatic breast cancer: 1) Clinical Trial Assay (CTA)
> immunohistochemistry (IHC) utilized in the trastuzumab clinical trials
> and 2) Fluorescence *in situ* Hybridization (FISH). Concordance of \>
> 75% acceptable.
>
> [Study Design]{.underline}: Retrospective, single center, single blind
> study of 600 randomly selected specimens. The specimens were from
> patients *screened* (i.e. not necessarily enrolled and treated) for
> the trastuzumab trials. The ratio of CTA positive and negative samples
> will be in a forced 1:1 ratio. All pathologists and technicians
> performing FISH will be blinded to results from previous CTA
> assessments.
>
> [Specimens]{.underline}: Archived slides from patients previously
> screened for trastuzumab studies will be retrieved and accessioned for
> testing by non-laboratory staff. The number of slides per patient will
> be tracked. Demographic information will accompany the specimens
> including patient initials, ID number, date of collection and date of
> birth.
>
> [Blinding]{.underline}: Specimen management staff, the project manager
> and the project monitor will have password restricted access to the
> database which contains specimen numbers form previous protocol (CTA)
> along with newly assigned specimen numbers for this protocol.
>
> [Assays]{.underline}:
>
> The CTA is an avidin-biotin method of IHC using both the 4D5 and CB11
> antibodies. Results were reported on a 0-3+ scale with "positive"
> meaning more than 10% of the cells have weak to moderate complete
> membrane staining stain (2+) or moderate to strong staining (3+).
>
> The FISH assay is PathVysion HER-2/neu DNA Probe manufactured by Vysis
> or INFORM HER2 manufactured by Ventana \[INFORM was ultimately not
> used in this study.\] The probe is a 190 Kb SpectrumOrange directly
> labeled fluorescent DNA probe specific for the HER-2/neu gene locus at
> 17q11.2-q12. The CEP 17 DNA probe is a 5.4 Kb Spectrum Green directly
> labeled specific for the alpha satellite DNA sequence at the
> centromeric region of chromosome 17 at 17p11.1-q11.1. The scoring is
> based on the ratio of the number of HER-2/neu to CEP 17 signals per
> nucleus. A signal ration of 2.0 is considered abnormal amplification.
> Assessment will be of 60 interphase tumor cell nuclei per target.
>
> [Data collection]{.underline}: Results of FISH determination will be
> recorded manually and entered into a secure database. Test result data
> will be supplied in electronic format to Genentech upon completion of
> each of the Vysis and Ventana assays or more often as requested.
>
> [Data analysis]{.underline}: The proportion of samples sharing
> agreement between the CTA and FISH will be calculated and the
> confidence intervals will be obtained. This will be called the level
> of concordance between the two assays. Data will be presented in a 2x2
> table \[FISH (+) and FISH (-) vs IHC (+) and IHC (-) where IHC (+) =
> score of 2+ or 3+\]. A one-sided test on proportion will be conducted
> on the concordance rate at the 5% significance level. The secondary
> analysis will extrapolate the results to the population using the
> population distribution of 0, 1+, 2+ and 3+ scores. The point estimate
> of the concordance level of the population will be calculated
> accordingly with the target being 85%-90% range. The Kappa statistic
> for the 2x2 table will be calculated. Exploratory analysis to identify
> and optimal cut point for FISH ratio denoting positivity will be
> assessed using 1.5, 2.0 and 2.5 as possible cut points. Additional cut
> offs may be assessed.

##### C. Clinical Outcome Study

> There was no prospective protocol for the clinical outcome study.
> There were only standard operating procedures (SOPs) written by
> LabCorp and Press for running assays on tissue samples. At the time of
> the writing of this document FDA is awaiting receipt of these SOPs
> from the sponsor.

Press Protocol: Laboratory Testing for HER-2/neu Gene Amplification:
Retrospective Study of Genentech Herceptin Clinical Trial Specimens.

> [Primary Endpoint]{.underline}: Test approximately 345
> paraffin-embedded breast cancer tissue specimens for HER-2/neu gene
> amplification using FISH and report results to Genentech, Inc. for
> comparison with clinical outcome data already collected in three
> clinical trials (H0648g, H0649g, and H0650g).
>
> Study Design: Retrospective, single center study to test tissue
> samples for HER-2/neu gene amplification.
>
> Specimens: Paraffin-embedded tissue specimens currently in storage at
> LabCorp, Inc.
>
> Assays: not described
>
> Data collection: not described
>
> Data analysis: not described

##### D. LabCorp vs Press Validation Study

> Not included in application. Awaiting protocol outline from sponsor.
>
> **IV. RESULTS OF FISH TESTING ON ARCHIVED TISSUE SAMPLES FROM CLINICAL
> TRIALS**
>
> Data submitted were assessed for the following items:

-   Difference in methods between the two FISH testing laboratories
    (LabCorp and Press)

-   Failure rate of FISH (i.e. the relative number of times a FISH
    result could not be obtained when tissue was available for testing)

-   Difference in performance of FISH between the two FISH testing
    laboratories

-   Concordance between FISH results and previously conducted CTA
    results

-   Degree of agreement between LabCorp and Press laboratories

-   Retrospective clinical outcome assessment by FISH result and IHC
    results

##### A. Methodological Differences Between LabCorp and Press

Due to a variety of tissue specific factors (e.g. storage, age of
samples, previous staining), both laboratories conducted the FISH assays
with some modifications to the PathVysion package insert instructions.
Table 1 identifies the methodologic procedures for slide preparation and
staining which differed between each lab and that recommended in the
PathVysion package insert.

> Table 1. Differences in methodology between two laboratories
> performing FISH.

+----------------+-----------------+----------------+-----------------+
| **Parameter**  | **LabCorp**     | **Press Lab**  | **PathVysion    |
|                |                 |                | PI**            |
+----------------+-----------------+----------------+-----------------+
| Specimen       | Formalin-fixed, | F              | Formalin-fixed, |
| fixation       | paraffin        | ormalin-fixed, | paraffin        |
|                | embedded        | paraffin       | embedded        |
|                |                 | embedded       |                 |
+----------------+-----------------+----------------+-----------------+
| Specimen slide | 4-6 μm sections | 4-6 μm         | 4-6 μm sections |
| prep           | not             | sections with  |                 |
|                | coverslipped    | Cytoseal       |                 |
|                |                 | mounting and   |                 |
|                |                 | coverslip      |                 |
+----------------+-----------------+----------------+-----------------+
| Specimen       | 2.5 years       | 2.5 years      | Length not      |
| storage        |                 |                | specified       |
+----------------+-----------------+----------------+-----------------+
| Removal of     | Not applicable  | Xylene 24-48hr | Not specified   |
| coverslip      |                 | followed by    |                 |
|                |                 | EtOH to remove |                 |
|                |                 | xylene         |                 |
+----------------+-----------------+----------------+-----------------+
| Chaotrope      | 1M NaSCN 80^o^C | 1M NaSCN       | 1M NaSCN 80^o^C |
| Treatment      | for 60 min      | 80^o^C for 30  | for 30 min      |
| (part of       |                 | min            |                 |
| depa           |                 |                |                 |
| raffinization) |                 |                |                 |
+----------------+-----------------+----------------+-----------------+
| Processing     | VP 2000 (80%)   | VP 2000 (100%) | Not addressed   |
|                | and manual      |                |                 |
|                | (20%)           |                |                 |
+----------------+-----------------+----------------+-----------------+
| Probe          | 10-20μl for 24  | 10 μl probe    | 10 μl probe for |
| application    | x 40 coverslip; | for 22x22      | 22x22 coverslip |
|                | amount of probe | coverslip;     |                 |
|                | sometimes       | amount of      |                 |
|                | increased for   | probe doubled  |                 |
|                | larger          | for larger     |                 |
|                | coverslip       | coverslip      |                 |
+----------------+-----------------+----------------+-----------------+
| Counterstain   | 10μl DAPI for   | 10 μl DAPI for | 10 μl DAPI for  |
|                | 24 x 40         | 22x22          | 22x22 coverslip |
|                | coverslip       | coverslip      |                 |
+----------------+-----------------+----------------+-----------------+
| Microscope     | DAPI/Or/Gr v.2  | DAPI/Or/Gr v.2 | DAPI/9-Or       |
| filters        |                 |                |                 |
|                | DAPI/Or v.2     | DAPI/Or v.2    | DAPI/Gr         |
|                |                 |                |                 |
|                | Green single    | DAPI/Gr        | DAPI/Or/Gr      |
+----------------+-----------------+----------------+-----------------+
| Microscope     | 10x, 40x, 100x  | 5x, 10x, 20x,  | 40x, 63x, 100x  |
| Objectives     | oil             | oil:40x, 63x,  |                 |
|                |                 | 100x           |                 |
+----------------+-----------------+----------------+-----------------+
| Number of      | 40              | 60             | 60              |
| nuclei scored  |                 |                |                 |
|                | if ratio        |                |                 |
|                | 1.8-2.2 then 60 |                |                 |
+----------------+-----------------+----------------+-----------------+
| Signal         | Not specified   | Per PI         | Guidelines      |
| enumeration    |                 |                | provided        |
+----------------+-----------------+----------------+-----------------+
| Ratio          | Total           | Average        | Not specified   |
| calculation    | HER2/Total CEP  | HER2/Average   |                 |
| (HER2:CEP17)   | 17; for high    | CEP17          |                 |
|                | number of       |                |                 |
|                | signals a mid   |                |                 |
|                | range chart was |                |                 |
|                | used.           |                |                 |
+----------------+-----------------+----------------+-----------------+
| Control slides | Formalin fixed, | Internal       | ProbeChek       |
|                | pa              | control        | control slides  |
|                | raffin-embedded | (benign and    |                 |
|                | breast cancer   | normal cells   |                 |
|                | tissue          | and            |                 |
|                | previously      | non-amplified  |                 |
|                | identified as   | and amplified  |                 |
|                | HER2 positive   | cell lines. If |                 |
|                | by IHC or FISH. | negative       |                 |
|                | Performance     | control failed |                 |
|                | characteristics | normal         |                 |
|                | not specified.  | lymphocytes    |                 |
|                |                 | used.          |                 |
|                |                 | ProbeChek      |                 |
|                |                 | HER2. At least |                 |
|                |                 | 2 controls     |                 |
|                |                 | with           |                 |
|                |                 | successful     |                 |
|                |                 | assay results  |                 |
+----------------+-----------------+----------------+-----------------+

##### B. Success/Failure Rates for Tissue Samples tested by FISH

###### Summary of Sample Testing

###### 

###### For the CTA vs FISH concordance study, the prospective plan was to test 600 out of 5251 unique patients with available tissue from 5998 patients screened. Selection of samples was random, but in a forced 1:1 ratio. Instead, 623 samples were tested of which 49% were 0-1+ and 52% were 2-3+ by CTA. Of the 623 samples, a FISH result was obtained on 529 samples for a testing failure rate of 15% by LabCorp (Diagram 1).

###### 

###### For the Clinical Outcome study, the plan was to test all available samples (784 out of 799 patients enrolled). Patients with at least 2 unstained slides numbered 618 and of these, a result was obtained on 540 samples for a testing failure rate of 13% by LabCorp. The total missing data for the clinical trials was 32%. To minimize the amount of missing data, Press tested 244 samples for which there were \< 2 unstained slides or no FISH result. Of these, a result was obtained on 225 samples for a testing failure rate of 8% by Press. (Diagram 2)

###### 

###### For the LabCorp vs Press Validation study, the plan was to test 90 samples in a forced 1:1 ratio of FISH (+) vs (-). Due to poor concordance with LabCorp negative specimens, additional samples were tested: 52 known (+) and 108 (-). Of the 250 samples tested by Press, a result was obtained on 223 samples for a testing failure rate of 11% by Press. (Diagram 3) 

######### Diagram 1: Sample testing flow diagram for CTA vs FISH Concordance Study

> **5998**
>
> Samples screened by CTA for trastuzumab clinical trials
>
> 68% scored 0-1+
>
> 32% scored 2-3+
>
> **5251 (88%)**
>
> samples available
>
> **623**
>
> samples randomly selected in 1:1 ratio (0-1+ vs 2-3+)
>
> **306 CTA Score 0-1+** **317 CTA Score 2-3+**
>
> No FISH Result FISH Result FISH Result No FISH Result
>
> **62** **244** **285** **32**
>
> (20%) (80%) (90%) (10%)

####### 529 Samples with result

> Prospectively identified number needed = 600

####### Percent of intended obtained = 88%

####### Testing failure rate = 15%

> **Diagram 2: Sample testing flow diagram for Clinical Outcome Study**
>
> **799**
>
> Patients enrolled in H0648g, H0649g, H0650g

**15**

Original CTA testing not done at

LabCorp; slides not available.

> **784**
>
> Slides available
>
> **166**
>
> \< 2 unstained slides
>
> **618**
>
> 2 unstained slides
>
> **78 292**
>
> No FISH result (14%) Not tested by Press
>
> **540**
>
> Obtained a FISH result by LabCorp
>
> 68% of patients on clinical trials
>
> **244 250**
>
> Run by Press Lab Validation testing by Press
>
> **19 225**
>
> No FISH result Obtained FISH result **448**
>
> (8%)
>
> Obtained FISH result by Press
>
> 56% of patients on clinical trials
>
> **765**
>
> Total with FISH result from both labs together
>
> 96% of patients on clinical trials

###### Diagram 3: Sample testing flow diagram for LabCorp vs Press Validation Study

**540**

Obtained FISH result at LabCorp

**290** not tested by Press

**250**

Tested by Press

**90** **160**

Validation Additional Validation

By Press Testing by Press

> 52 random known (+) and
>
> 108 (-)

1

Specimen missing

**7** **19**

No result No result

(8%) (12%)

**82** **141**

FISH Result FISH Result

> **223**

FISH result for validation LabCorp vs Press

###### Diagram 4: Sample testing flow diagram for Press lab

###### 

**799**

Patients enrolled

> **784** slides available **540** **292**
>
> LabCorp results Not tested by Press

**244** **90** **160**

Unavailable for LabCorp testing Original validation Further Testing

Tested by Press Testing by Press Testing by Press

**494**

Tested by Press

**448**

Obtained result by Press

Testing Failure rate = 9%

###### 

###### 

###### 

######  

###### 

###### C. Comparison of FISH Results Between LabCorp and Press Laboratories

###### 

###### FISH scoring required the counting of nuclei for CEP 17 staining (centromere of Chromosome 17) and HER2 staining. The ratio of the HER2:CEP 17 was the final score. Scores 2.0 were considered positive for HER2 amplification.

###### 

#### Case Report Form Review

###### All case report forms (CRF's) for samples from study H0648g which were tested at both laboratory sites were reviewed. Case report forms were reviewed for the number of readers per laboratory site, accuracy of calculations, and for comparison of centromere CEP 17 and HER2 scores between LabCorp and Press. It was noted that at LabCorp, there were scores sheets completed by 3 different individuals, however, 90% of these were completed by one of the three. At the Press lab, all scores sheets were completed by Dr. Press together with one of two other individuals in his laboratory. Approximately 5% of CRF's were reviewed for accuracy of calculations and all were accurate. Each lab used a different method for calculating the final FISH score.

Mean centromere counts and HER2 counts were calculated by the reviewer
from LabCorp CRF's; mean centromere counts and HER2 counts were entered
on Press CRF's. Calculated ratios of HER:centromere were entered on both
sets of CRF's. Comparisons were made between the scoring of patient
samples. The data were not provided as SAS tables and therefore, paired
correlation analyses could not be performed within the time frame of
this review.

There was a consistent trend for the scores at LabCorp to be lower
overall than those done by the Press lab. Of the 198 patient cases
reviewed, a result was obtained by both labs for 141 cases. The
remaining 57 cases had a failed result at one or both labs. Mean numeric
scores were lower overall at LabCorp than at the Press lab for
centromere and particularly for HER2 scores: centromere 2.4 vs 2.5 and
HER2 5.2 vs 11.9, LabCorp vs Press respectively. As a result the ratio
scores were also lower at LabCorp 83% (117 out of 141) of the time with
mean values of 2.4 vs 4.7, LabCorp vs Press respectively.

There were 23 cases in which both labs obtained discordant results
(16%). Of these, in only one case was the LabCorp result positive while
the Press result was negative; the other 22 cases were negative by
LabCorp and positive by Press. The mean value comparisons for these 23
cases are as follows: centromere 3.5 vs 3.2, HER2 5.8 vs 15.2, and ratio
1.7 vs 5.1, LabCorp vs Press respectively.

Exploratory analyses of the concordance data appear in Appendix A. These
include a concordance analysis of LabCorp vs. Press, exploration of
alternative cut off points for FISH score, and concordance of numeric
FISH with numeric IHC.

**D. CTA vs FISH Concordance Study**

FISH results were compared with those obtained using the Clinical Trial
Assay IHC testing. The pre-specified comparison was FISH (+) and FISH
(-) vs IHC (+) and IHC (-) where IHC positive included scores of 2+ and
3+ (Table 2). The current package insert for trastuzumab, however,
states that "Data from both efficacy trials suggest that the beneficial
treatment effects were largely limited to patients with the highest
level of HER2 protein overexpression (3+)." For that reason FDA also
undertook an exploratory analysis where IHC (+) was defined as 3+ only
(Table 2). It is important to consider the outcomes for patients scored
as 2+ vs 3+ juxtaposed to those scored as FISH (+) vs FISH (-). The
interpretation of IHC 2+ has been a problematic area because the
distinction between 1+ vs 2+ and in some cases between 2+ vs 3+ is very
difficult and subject to technical differences between laboratories and
to reader interpretation. It has also been a matter of debate whether a
score of 2+ represents protein overexpression.

Please note that for purposes of the primary analysis, the value
obtained at LabCorp took precedence over that obtained at Press in cases
where a result at both labs was determined. This was consistent with the
prospective plan, since the Press results were collected for the purpose
of filling in missing data and not initially for validation of LabCorp
results.

The concordance was moderate for the primary analysis (Kappa = 0.64) and
stronger when 2+ by IHC was defined as negative (Kappa = 0.80). FISH
testing missed 11% of the 3+ samples and selected 4% of the 0-1+ samples
as positive. Twenty-four percent of the 2+ samples were FISH (+).

> Table 2. Concordance of FISH (LabCorp and Press pooled with LabCorp
> result taking precedence over Press) vs CTA for screened samples.\*

  -------------- -------------- -------------- -------------- --------------
                 CTA = 0        CTA = 1+       CTA = 2+       CTA +3+

  FISH (+)       7              2              21             176

  FISH (-)       207            28             67             21

  Unknown        57             5              8              24
  -------------- -------------- -------------- -------------- --------------

\*Analysis performed with assumption that CTA 2+ and 3+ are a positive
result and CTA 0 and 1+ are a negative result: **Kappa = 0.64 with 95%
CI (0.58, 0.70) and**

> **Concordance 82% with 95% CI (78%, 85%).** This is in agreement with
> the sponsor assessment.
>
> \*Analysis performed with assumption that CTA 3+ is positive result
> and CTA 0, 1+, and 2+ are negative results: **Kappa = 0.80 with 95% CI
> (0.74, 0.85) and**
>
> **Concordance = 90% with 95% CI (88%, 93%).** Not performed by
> sponsor.
>
> Another exploratory analysis undertaken by FDA was to look at
> concordance of the CTA with FISH for all samples assessed with a FISH
> result from patients enrolled on the three clinical trials conducted.
> These data do not assess concordance with 0 and 1+ samples. For this
> analysis an IHC positive score was defined as 3+ only (Table 3). Of
> those patients tested by CTA as 3+, FISH testing missed 13% (75 out of
> 559 which were 3+). Of those which were 2+, FISH was positive in 34%
> (65 out of 190 which were 2+). The level of concordance (kappa = 0.52)
> is not as good when looking at this selected population of patients.
>
> Table 3. Concordance in clinical trial samples of enrolled patients
> from all studies (h0648, 649 and 650g) with pooled FISH data.\*

  -------------- ------------- -------------- ----- -------------- ----- --------------
                 CTA = 0       CTA = 1+             CTA = 2+             CTA +3+

  FISH (+)                                    65                   484   

  FISH (-)                     1              125                  75    
  -------------- ------------- -------------- ----- -------------- ----- --------------

> \*Analysis performed with assumption that CTA 3+ is positive and CTA
> 0, 1+, and 2+ are negative result: Kappa = 0.52 with 95% CI (0.45,
> 0.59) and Concordance = 81% with 95% CI (79%, 84%).
>
> The HercepTest package insert provides additional information which
> should be considered along with these results. Concordance testing
> comparing HercepTest to CTA was conducted with a separate bank of
> tumor specimens (i.e. not from the trastuzumab clinical trials). In
> another study, gene amplification was compared to HercepTest results
> on tumor specimens previously characterized using Northern, Southern,
> and Western blotting, FISH, and IHC. Please refer to HercepTest
> package insert in Appendix C.
>
> **E. Clinical Outcome Study**

The clinical outcome data discussion will be limited to samples tested
from studies H0648g and H0649g. The H0650g study was not accepted by FDA
for efficacy analysis because the design of the trial was felt to be an
inappropriate representation of patients with primary metastatic breast
cancer since it enrolled patients who either refused chemotherapy or
could not receive chemotherapy. Because the efficacy data have not been
rigorously reviewed by FDA the clinical outcome data for purposes of
FISH analysis are not represented here.

> It is important to note that [there are no clinical outcome data for
> patients who were IHC 0-1+ and FISH (+) or FISH (-)]{.underline}
> because by definition all patients were 2+ or 3+ in order to be
> eligible for these trials.
>
> For purposes of discussion, a variety of subgroups will be discussed
> in the clinical outcome results. Terminology will be defined as
> follows:
>
> 3+ = Patients whose tumors scored as 3+ on IHC testing using CTA
>
> 2+ = Patients whose tumors scored as 2+ on IHC testing using CTA
>
> FISH (+) = Patients whose tumors scored as FISH 2.0 using PathVysion
>
> FISH (-) = Patients whose tumors scored as FISH \< 2.0 using
> PathVysion
>
> FISH+/3+ = Patients whose tumors scored as 3+ and FISH 2.0
>
> FISH+/2+ = Patients whose tumors scored as 2+ and FISH 2.0
>
> FISH-/3+ = Patients whose tumors scored as 3+ and FISH \<2.0
>
> FISH-/2+ = Patients whose tumors scored as 2+ and FISH \<2.0
>
> A prospective analysis plan did not exist for this study. All analyses
> presented are considered exploratory. The clinical outcome study
> analysis was also limited by the fact that for the H0648g study
> treatment subgroups (anthracycline with or without trastuzumab,
> paclitaxel with or without trastuzumab) the number of patients was
> very small. For that reason, only the pooled groups (chemotherapy with
> or without trastuzumab) are presented. Even with that, some of the
> subgroups such as FISH+/2+ are very small making interpretation of
> results difficult and very limited.
>
> Finally, data analyses of 3+ vs 2+ are also included here for
> comparison. As noted in the trastuzumab label, "Data from both
> efficacy trials suggest that the beneficial treatment effects were
> largely limited to patients with the highest level of HER2 protein
> overexpression (3+)."
>
> Presented here are clinical outcome data for H0648g and H0649g. The
> primary endpoints for the two clinical trials were median time to
> progression on the H0648g study and overall response rate for the
> H0649g study. The secondary endpoints for the H0648g study presented
> here include overall response rate, and median overall survival.
> Because the H0649g study was a single arm study, time to progression
> and overall survival will not be presented here as there is no
> comparative arm.
>
> **Study H0648g**

###### Time to Progression

> Time to progression Kaplan-Meier analysis of H0648g appears below for
> patients whose tumors tested as FISH(+), FISH(-), 3+, and 2+ (Figures
> 1-4). For additional exploratory analyses of subgroups (e.g. FISH+/3+,
> FISH-/3+, etc) please see Appendix A.
>
> Figure 1. **Time to Progression, FISH (+)** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.44 (0.34,
> 0.57) and the p value \< 0.001. This is in agreement with the sponsor
> assessment.
>
> ![](media/image1.png){width="5.998611111111111in"
> height="3.895138888888889in"}
>
> Figure 2. **Time to Progression, FISH (-)** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.66 (0.45,
> 0.99) and the p value is 0.043. This is in agreement with the sponsor
> assessment.
>
> ![](media/image2.png){width="5.996527777777778in" height="3.9625in"}
>
> Figure 3. **Time to Progression, 3+** samples (regardless of FISH
> status) for patients enrolled in clinical trial H0648g. Comparison of
> trastuzumab plus chemotherapy vs chemotherapy alone. The relative risk
> is 0.42 (0.33, 0.55) and the p value is \< 0.001.
>
> ![](media/image3.png){width="5.99375in" height="3.732638888888889in"}
>
> Figure 4. **Time to Progression, 2+** samples (regardless of FISH
> status) for patients enrolled in clinical trial H0648g. Comparison of
> trastuzumab plus chemotherapy vs chemotherapy alone. The relative risk
> is 0.82 (0.54, 1.24) and the p value is 0.34.
>
> ![](media/image4.png){width="5.992361111111111in"
> height="3.870138888888889in"}
>
> Reviewer comment: For time to progression, FISH (+) patients
> (regardless of IHC score) and 3+ patients (regardless of FISH score)
> both had greater clinical benefit from the addition of trastuzumab to
> chemotherapy than did FISH (-) or IHC 2+ patients respectively.
> However, FISH (-) patients did appear to experience clinical benefit,
> though not to as large a degree. This is likely due to inclusion of
> some FISH-/3+ patients (see exploratory analyses in Appendix A).

###### Overall Response Rate

> Overall response rate was defined as objective complete plus partial
> responses sustained fro at least 4 weeks. Below are the analyses for
> response rate by FISH result, IHC result and various subgroups. These
> numbers differ slightly from those in the package insert (e.g. for
> 2+/3+;T+C, the ORR for patients with FISH result is 114 out of 226 or
> 50% which is higher than the 45% listed in the PI). The values in the
> package insert were based on the FDA assessment of responder status
> and were slightly lower than the sponsor's original report of
> response. We have not included the FDA assessed values for response
> here for the following reasons: 1) the data are not markedly different
> from what appears below, 2) these are all retrospective subgroup
> analyses from which firm conclusions will be difficult to justify.
>
> Table 4. H0648g: Overall Response Rate by FISH results (+) vs (-)
> (Response is defined as complete plus partial response sustained for 4
> weeks.) Sponsor analysis.

+---------+----------+---------+---------+---------+--------+--------+
|         | 2+/3+    | 2+/3+   | 3+      | 3+      | 2+     | 2+     |
|         |          |         |         |         |        |        |
|         | T+C      | C       | T+C     | C       | T+C    | C      |
|         |          |         |         |         |        |        |
|         | n = 226  | n = 225 | n = 169 | n = 167 | n = 57 | n = 58 |
+---------+----------+---------+---------+---------+--------+--------+
| FISH    | **54%**  | **30%** | 55%     | 28%     | 50%    | 56%    |
| (+)     |          |         |         |         |        |        |
|         | **(8     | **(49   | (       | (       | (8/16) | (9/16) |
| n = 325 | 9/164)** | /161)** | 81/148) | 40/145) |        |        |
+---------+----------+---------+---------+---------+--------+--------+
| FISH    | **40%**  | **38%** | 62%     | 55%     | 29%    | 29%    |
| (--)    |          |         |         |         |        |        |
|         | **(      | **(2    | (13/21) | (12/22) | (      | (      |
| n = 126 | 25/62)** | 4/64)** |         |         | 12/41) | 12/42) |
+---------+----------+---------+---------+---------+--------+--------+

> T+C = Trastuzumab plus chemo
>
> C = Chemo alone

Table 5. H0648g: Overall Response Rate by IHC score 2+ vs 3+ from the
trastuzumab package insert. (Response is defined as complete plus
partial response sustained for 4 weeks.)

+---------+----------+---------+---------+---------+--------+--------+
|         | 2+/3+    | 2+/3+   | 3+      | 3+      | 2+     | 2+     |
|         |          |         |         |         |        |        |
|         | T+C      | C       | T+C     | C       | T+C    | C      |
|         |          |         |         |         |        |        |
|         | n = 235  | n = 234 | n = 176 | n = 173 | n = 59 | n = 61 |
+---------+----------+---------+---------+---------+--------+--------+
| Any     | 45%      | 29%     | **49%** | **27%** | *      | *      |
| FISH    |          |         |         |         | *32%** | *34%** |
|         | (        | (       | **(87   | **(46   |        |        |
| N = 469 | 106/235) | 67/234) | /176)** | /173)** | **(19  | **(21  |
|         |          |         |         |         | /59)** | /61)** |
+---------+----------+---------+---------+---------+--------+--------+

> T+C = Trastuzumab plus chemo
>
> C = Chemo alone
>
> Reviewer comment: For overall response rate, FISH (+) patients
> (regardless of IHC score) and 3+ patients (regardless of FISH score)
> both had greater clinical benefit from the addition of trastuzumab to
> chemotherapy than did FISH (-) or IHC 2+ patients respectively.
> Patients in the FISH-/3+ group may have also benefited, though the
> number of patients is too small to draw firm conclusions.

###### Overall Survival

> Overall Survival Kaplan-Meier analysis of H0648g appears below for
> patients whose tumors tested as FISH(+), FISH(-), 3+, and 2+ (Figures
> 5-8). For additional exploratory analyses of subgroups (e.g. FISH+/3+,
> FISH-/3+, etc) please see Appendix A .
>
> Figure 5. **Overall Survival, FISH (+)** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.69 (0.53,
> 0.91) and the p value is 0.007. This is in agreement with the sponsor
> assessment.
>
> ![](media/image5.png){width="5.99375in" height="3.7958333333333334in"}
>
> Figure 6. **Overall Survival, FISH (-)** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 1.07 (0.70,
> 1.63) and the p value is 0.76. This is in agreement with the sponsor
> assessment.
>
> ![](media/image6.png){width="5.992361111111111in" height="3.8875in"}
>
> Figure 7. **Overall Survival, 3+** samples (regardless of FISH status)
> for patients enrolled in clinical trial H0648g. Comparison of
> trastuzumab plus chemotherapy vs chemotherapy alone. The relative risk
> is 0.70 (0.54, 0.92) and the p value is \< 0.010.
>
> ![](media/image7.png){width="5.995138888888889in"
> height="3.845138888888889in"}
>
> Figure 8. **Overall Survival, 2+** samples (regardless of FISH status)
> for patients enrolled in clinical trial H0648g. Comparison of
> trastuzumab plus chemotherapy vs chemotherapy alone. The relative risk
> is 1.09 (0.71, 1.58) and the p value is 0.70.
>
> ![](media/image8.png){width="5.99375in" height="3.8319444444444444in"}
>
> Reviewer comment: For overall survival, FISH (+) patients (regardless
> of IHC score) and 3+ patients (regardless of FISH score) both had
> greater clinical benefit from the addition of trastuzumab to
> chemotherapy than did FISH (-) or IHC 2+ patients respectively.
>
> **Study H0649g**

###### Overall Response Rate

> Table 6. H0649g: Overall Response Rate by FISH results (+) vs (-)
> (Response is defined as complete plus partial response sustained for 4
> weeks.) Sponsor analysis.

  ----------------- ----------------- ----------------- -----------------
                    CTA 3+            CTA 2+            ALL

  FISH (+)          22% (30/136)      11% (3/27)        20% (33/163)

  FISH (-)          0% (0/21)         0% (0/25)         0% (0/46)

  ALL               19% (30/157)      6% (3/52)         
  ----------------- ----------------- ----------------- -----------------

> Reviewer comment: For overall response rate, FISH (+) patients
> (regardless of IHC score) and 3+ patients (regardless of FISH score)
> both had greater clinical benefit from the addition of trastuzumab to
> chemotherapy than did FISH (-) or IHC 2+ patients respectively. Given
> the small number of patients in the CTA 2+ subgroup, it is difficult
> to conclude differences between FISH-/2+ and FISH+/2+ patients.

##### V. CONCLUSIONS

A.  FISH and IHC testing are both semiquantitative assays in the most
    optimal circumstances. Even in this setting, there can be
    considerable variability in laboratory methodology as evidence by
    systemic differences between the two laboratories conducting the
    FISH assays for these studies. LabCorp FISH scores were consistently
    lower than those of the Press lab, leading to less confidence in
    scores surrounding the cutoff of 2.0.

B.  There is moderate concordance (82% \[78%, 85%\] with Kappa statistic
    = 0.64) between the original CTA assay conducted at LabCorp and the
    FISH assay conducted at LabCorp using the PathVysion HER2 gene
    amplification FISH kit. In exploratory analyses, when the CTA
    positive definition was changed to 3+ only, instead of 3+ and 2+,
    the agreement between the CTA and FISH remained moderate to good.
    This was upheld when looking at the agreement between FISH and the
    original CTA value of enrolled patients.

C.  The level of concordance between CTA and FISH in this study is
    similar to that observed between HercepTest and CTA in a previous
    study.

D.  The clinical outcome data does not provide information on the
    clinical outcome for patients whose tumors score IHC 0 or 1+ and
    FISH (+). For this reason, there are insufficient data to describe
    the predictability for response to trastuzumab based upon FISH test
    results.

E.  The clinical outcome data were not obtained in a fashion to allow
    direct comparison with IHC, therefore, comparative claims of
    equivalence cannot be made, and claims of superiority cannot be
    made. It may be possible to note that trends proceed in a similar
    fashion for IHC 3+ and FISH(+) subgroups.

F.  The trend toward beneficial effects of trastuzumab in the clinical
    outcome study are prominent in the FISH (+) subgroup. A smaller
    clinical benefit was also seen in the FISH (-) subgroup as evidenced
    in the analysis of time to progression and overall response rate;
    this did not translate into increased survival, however.

G.  The general magnitude of the beneficial effects of trastuzumab
    therapy in the FISH (+) subgroup is similar to that in the ICH 3+
    subgroup.

H.  There is insufficient information to draw conclusions regarding
    potential sequencing of HER2 detection assays. For example, there is
    insufficient data to claim that if IHC is performed first and the
    score is 2+, then FISH should be performed next.

I.  There is insufficient information to draw conclusions regarding the
    use of FISH and IHC simultaneously. For example, should patients
    whose tumors are FISH+/2+ receive trastuzumab therapy? Or
    FISH+/0-1+?

J.  Given the subjective nature of both FISH and IHC testing and given
    the relative lack of knowledge regarding the functioning and
    detection of the trastuzumab target, the obtaining of clinical
    outcome data remains the only gold standard for determination of the
    ability of a HER2 detection methodology to optimally select patients
    for trastuzumab therapy.

K.  These retrospective examinations of archived clinical trial samples
    are useful for hypothesis generation for future studies: Will
    FISH-/3+ or FISH+/2+ patients benefit from trastuzumab therapy? Is
    3+ or FISH + a better predictor of clinical outcome with trastuzumab
    therapy? Will FISH+/0-1+ patients benefit from trastuzumab therapy?

> **VI. Appendix A**
>
> **Exploratory Analyses of Concordance Data**

**Concordance between LabCorp and Press results**

The sponsor conducted a validation study to compare the results between
the two laboratories. After the first 90 samples were assessed, there
appeared to be underscoring at LabCorp which led to the decision to test
additional samples. The effect persisted. Concordance between LabCorp
and Press results were assessed by FDA and appear in Table 7. There was
poor concordance between LabCorp and Press FISH (+) results. Of the
samples testing positive at the Press lab, 32% (37/116) were negative at
LabCorp.

Table 7. Concordance between LabCorp and Press Lab Results (H0648g,
H0649g, H0650g samples)

  -------------- --------------- -------------- -------------- ------------
                 Missing data    Press FISH (+) Press FISH (-) Total

  Missing data   0               176            50             226

  LabCorp FISH   308             **79**         **2**          81
  (+)                                                          

  LabCorp FISH   14              **37**         **105**        142
  (-)                                                          

  Total                          116            107            223
  -------------- --------------- -------------- -------------- ------------

**Cut off point for FISH positivity**

Given the consistently lower scoring by LabCorp, it was hypothesized by
the reviewer that samples scored as negative by LabCorp with a result in
the range of 1.0 to 2.0 would have a high rate of positivity by Press
laboratory scoring. The reviewer examined all results from all studies
for which there was both a LabCorp result and a Press result. For scores
in the range of 1.0 to 2.0 by LabCorp, 38% tested positive by Press. For
scores in the range of 1.0 to 2.0 by Press, 1% tested positive by
LabCorp.

The reviewer also examined the H0648g study data and the CTA vs FISH
concordance data separately. For the H0648g study, 35% of negative
LabCorp samples in the 1.0-2.0 range were positive by Press (most of
these were 3+ by CTA). For the concordance trial (2+ and 3+ samples
only), 29% of 1.0-2.0 LabCorp samples were 3+ by CTA. The effect seemed
to be consistent across studies. For the concordance study 0-3+ samples,
10% of 1.0-2.0 LabCorp samples were 3+ by CTA. We estimate that for CTA
2+ and 3+ patients, LabCorp values in the range of 1.0 to 2.0 will miss
at least 30% of patients who would benefit from trastuzumab therapy.
Likewise for all CTA vales, LabCorp values in the range of 1.0-2.0 will
miss 10% of patients who would benefit from trastuzumab therapy based on
an IHC score of 3+.

A separate exploratory analysis to explore alternate cut off points for
FISH positivity demonstrated 1.0 to be a better cut point than 2.0 in
terms of time to progression clinical outcome data for all three
clinical trials. Cut points tested were 1.0, 1.5, 2.0, 2.5, 3.0, and
4.0. The relative risk was best for 1.0 (RR = 0.50) as compared to 2.0
(RR = 0.69), 3.0 (RR = 0.74), and 4.0 (RR = 0.84). This is most likely
explained by the consistent lower scoring of the LabCorp slides. While
there are many confounding factors in these studies, it is concerning
that scores surrounding the cut off and in the range of 1.0 -- 2.0 are
problematic and may not be as reliable. It is not clear that increasing
the number of nuclei counted will overcome this problem. It also does
not make biologic sense to have a cut off \< 2.0. Therefore,
extrapolation of this analysis is problematic.

**Numeric FISH score vs CTA result**

While it is recognized that the numeric value for the FISH score cannot
be interpreted as a quantitative value, we felt that an analysis of
scores vs CTA results would be of interest. The data from the
concordance study of CTA vs FISH using tissue samples from the screened
patient pool run by LabCorp were used in the analysis. The results
appear in Table 8. From this distribution, there is a trend for
magnitude of CTA score to correlate with magnitude of FISH numeric
score. The greatest ambiguity exists for CTA 2+ cases; only 24% of these
tested FISH (+) with varying degrees of amplification. High levels of
amplification (FISH score \> 4) were rare in CTA 0-1+ cases, though the
rate of FISH positives in the IHC 0-1+ population (4%) cannot be
ignored. Cases which were CTA 3+ were more likely to have high levels of
amplification. Any conclusions beyond trend analysis should not be drawn
from these data as neither IHC nor FISH assay is meant to be interpreted
quantitatively.

Table 8. Concordance analysis of numeric FISH score from LabCorp vs CTA
results for samples from screened patient pool.

![](media/image9.png){width="5.9944444444444445in"
height="3.457638888888889in"}

> **Exploratory analyses of Clinical Outcome Data**

###### Study H0648g

###### Time Progression

> Table 9. Median Time to Progression (months) for H0648g subgroups

  ----------- --------------- ------------- ------------- ---------------
              3+ ; H+C        3+; C         2+; H+C       2+ ; C

  FISH +      7.3             4.6           9.4           5.6

                                                          

  FISH -      11.3            6.3           5.3           4.5

                                                          
  ----------- --------------- ------------- ------------- ---------------

> Figure 9. **Time to Progression, FISH+/3+** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.42 (0.32,
> 0.55).
>
> ![](media/image10.png){width="5.99375in"
> height="3.7243055555555555in"}
>
> Figure 10. **Time to Progression, FISH-/3+** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.40 (0.19,
> 0.87).
>
> ![](media/image11.png){width="5.995833333333334in"
> height="3.8958333333333335in"}
>
> Figure 11. **Time to Progression, FISH+/2+** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.72 (0.31,
> 1.64).
>
> ![](media/image12.png){width="5.998611111111111in"
> height="3.9034722222222222in"}
>
> Figure 12. **Time to Progression, FISH-/2+** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.86 (0.53,
> 1.38).
>
> ![](media/image13.png){width="5.99375in"
> height="3.7604166666666665in"}
>
> **Overall Survival**
>
> Table 10. Median Overall Survival (months) for H0648g subgroups

  ----------- --------------- ------------- ------------- ---------------
              3+ ; H+C        3+; C         2+; H+C       2+ ; C

  FISH +      26.3            18.8          21.4          24.5

                                                          

  FISH -      29.1            29.7          19.3          18.4

                                                          
  ----------- --------------- ------------- ------------- ---------------

> Figure 13. **Overall Survival, FISH+/3+** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.57 (0.51,
> 0.89).
>
> ![](media/image14.png){width="5.99375in" height="3.779861111111111in"}
>
> Figure 14. **Overall Survival, FISH-/3+** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.94 (0.42,
> 2.11).
>
> ![](media/image15.png){width="5.995138888888889in"
> height="3.888888888888889in"}
>
> Figure 15. **Overall Survival, FISH+/2+** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 0.98 (0.41,
> 2.35).
>
> ![](media/image16.png){width="5.99375in" height="3.7625in"}
>
> Figure 16. **Overall Survival, FISH-/2+** samples, for patients
> enrolled in clinical trial H0648g. Comparison of trastuzumab plus
> chemotherapy vs chemotherapy alone. The relative risk is 1.15 (0.70,
> 1.89).
>
> ![](media/image17.png){width="5.995138888888889in"
> height="3.8534722222222224in"}
>
> **VII. Appendix B**
>
> **PathVysion Current Package Insert**

#### VIII. Appendix C

> **HercepTest current package insert**

IX. **Appendix D**

> **Trastuzumab Package Insert**