Trial Information

Study to Assess the Effectiveness of the CAAb Test With Ovarian Cancer Patients

General Information

Antibodies are a specific response to any "foreign" antigen, and they are usually detectable
in the serum within 5-7 days after the initial exposure to it. However, there are some cases
where there is a suppression of the specific immune response, such as in the case of a
tumor. To date, there are no reports on serum antibodies that are associated to cancer. Some
tumors, when surgically removed and studied have been found to have both infiltrates of T
lymphocytes and antibodies bound to the tumor cells. These antibodies (and other specific
immune responses) are assumed to be formed at later stages of the tumor growth, however,
even in those cases, serum antibodies have not been reported (probably due to them being
mostly "absorbed" by the tumor mass).

Since the mutations of a normal cell that lead to a malignant cell mass involves changes in
the structure and function of the cells, it could be assumed that the immune system has
recognized some of these changes as antigenic determinants that should be responded against;
if so, than the suppression caused by the tumor would be the reason for the lack of
antibodies.

If this immune suppression could be overcome, in vitro, it could lead to the formation of
antibodies that are not detectable in the serum (we call this process Cimmunology).
Identifying the structures that the antibodies bind to, and looking for a correlation
between the tumor pathology and the antibodies' specificity – could provide additional
information about the tumor – via a blood test.

Future CAAb Test

The Cimmunology based test is comprised of an in-vitro stimulation step and an antibody
detection step.

The assay will be used to find Cancer Associated Antibodies in ovarian cancer patients.

Identification of ovarian cancer associated antibodies could provide the clinician with
additional immunological and antigenic information regarding the patient's condition or
medical status.

The Cimmunology process is comprised of a blood collection and processing step and an
incubation (in a special stimulation "tube" called CimTube) period in a humid, 37oC
incubator with 5% CO2.

The test for the detection of specific antibodies is ELISA based, and includes different
potentially interesting antigens.

The relevant medical and pathological information, to be collected from the patient's file,
will enable a more detailed analysis of the test results.

STUDY OBJECTIVES

Primary objective:

- To find Cancer Associated Antibodies

Effectiveness:

- To determine the ability of the Cimmunology process to lead to in vitro antibody
production, the ability of the ELISA assays to detect any of those antibodies, and to
establish the relationship between the ELISA results and the clinical/pathological
status of the patient. The statistical significance of the CAAb test results will be
determined.

STUDY POPULATIONS AND PATIENT SELECTION

Ovarian cancer patients

The study population will include subjects that are suspected and going to be treated for
ovarian cancer, prior to any surgical procedure or any other anti-cancer treatment. The
clinical suspicion includes one of the following: Pelvic mass (by pelvic examination or
other imaging techniques), or high CA-125 levels in post menopausal women, ascites, or due
to incidental finding of distant metastasis. Final analysis will be done in relation to
pathology only.

Control group

The control group will include patients, at the gynecology department (or any other
appropriate care unit), with no prior cancer history.

STUDY DESIGN

Overall Description

The current study is a comparative observational two-arm study, involving all consecutive
ovarian cancer patients during the study duration, and age matched control subjects. The
purpose of the study is to assess the effectiveness of the CAAb test in detecting ovarian
cancer associated antibodies.

Subjects will be screened for potential participation in the study, according to the
inclusion and exclusion criteria. Patients recruited will be asked to sign an informed
consent form.

The data on the patients will include parameters of the clinical and pathological state of
the patients, results of CimTube culture step and the experimental kits. The effectiveness
of the CAAb test will be assessed by evaluating the statistical significance of the CAAb
test used.

The study will utilize a three step group-sequential design with two interim and one final
analysis using the O'Brian-Fleming' boundaries. The data collection may be stopped after the
first or second interim analysis if the bounds are reached. This could substantially
decrease the number of patients and the time of the study, while just slightly increasing
the maximum number of patients. (See Sample size considerations below).

We will use 1:2 ratio between cancer and control patients to decrease the necessary number
of cancer patients which is the main limiting factor.

The sponsor will conduct an interim analysis, following the first third and second third of
ovarian cancer cases and the sample size will be recalculated according to the obtained
estimates of the mean and variance of the test in the two groups.

Since the data gathered to date was from epithelial cell carcinoma only, our study will also
focus on epithelial cell carcinoma. Therefore stromal and germ cell tumors, which comprise
less than 10% of OC cases will be dropped based on the final pathology analysis. In
addition, non ovarian cases that were mistaken for ovarian carcinoma comprise approximately
10% of women undergoing surgery for OC. These will also be dropped based on the final
pathology.

Therefore, at least 60 patients will be enrolled in the study, in order to obtain the
statistically projected needed sample size of 50 ovarian cancer samples. The control group
will be 120 patients.

Study Procedure

In the hospital:

First, an identified study patient has to sign an informed consent form, and her Eligibility
Form filled; both should be bar-coded.

Patient demographic and clinical information acquired from the patient’s medical file,
including age, country of origin, medical history and results of tests done leading to the
diagnostic evaluation of ovarian cancer (not relevant in the control group) will be recorded
on the appropriate, bar-coded, pre-study case report forms. The reports will be either
electronic (a dedicated and secured internet site) or via a hard copy. A hard copy of the
records will be kept in the department. The study is anonymous and the Department will keep
the name of the patient without revealing it to investigators and to the study sponsor.

The Study Sponsor will provide the tubes and the barcode labels for monitoring. The
doctor/nurse/phlebotomist will collect three heparin vacuum tubes (20-24 ml), label
(bar-code) them, and fill up the initial step in the "Sample follow-up form" (SFF). The
tubes with the blood will be packaged in a double sealed container (will be provided by the
sponsor). The department will notify the designated shipper to collect the blood up to 1
hour from the time that it has been sampled.

For each OC sample two age matched controls (+/- 3 years) will be recruited too.

Transportation from the Hospital to the Handling Lab:

The blood will be transported at room temperature (18-250C), according to the relevant
regulations, to the laboratory. Transportation time will be not more than 6 hours. Times to
be recorded in SFF.

In the blood handling laboratory:

The blood handling laboratory (to be located no further than 200km from the collection site)
will isolate PBMC (Peripheral blood mononuclear cells) and put them into Cimmunology culture
within 20 hours from the sampling time (fill up time in SFF).

The detailed protocol and the Cimmunology media will be provided by Lab Discoveries to the
participating laboratories (where applicable). All hospitals in Israel will send the blood
samples to the laboratory at Lab Discoveries. After the culture step, the culture fluid will
be collected and frozen at -80, in properly labeled aliquots. The frozen samples will be
sent to the diagnostic laboratory for antibody tests.

In the diagnostic laboratory:

Antibody tests, such as ELISA, will be prepared using different antigens. The samples will
be tested for response to the two antigens that have been identified in the past, and for
additional new ones. All the data will be permanently recorded directly into a dedicated
computer. For each sample all antibody results will be put into the coded patient file. The
results will be analyzed by a statistician with expertise in cancer population studies.

At the Hospital, post surgery:

2-3 weeks after the surgery, the CRO will collect from the patient's file the results from
the pathology lab and any other relevant information as to the nature and state of the tumor
that was removed during surgery. These results will be recorded on the dedicated and secured
internet site and a printed bar-coded CRF-OC will be kept in the Department.

STATISTICAL CONSIDERATIONS

The present study is a comparative observational two-arm study, involving all consecutive
ovarian cancer patients during the study duration, and age matched control subjects. The
main purpose of the study is to assess the effectiveness of the CAAb tests. This
effectiveness will be measured by the Fisher' distance (often called “effect size”) d
defined as d = /m1-m2//2 Where m1 is the mean value of the test in the controls, m2 is the
mean value in the ovarian cancer patients, /m1-m2/ denotes the absolute value of the
difference (m1-m2), and s is the common estimate of the standard deviation of the
distribution of the test in one group (control or ovarian cancer).

Statistical Hypothesis

Null Hypothesis: There is no relationship between the presence or absence of OC and the CAAb
i.e. d=0

Alternative Hypothesis: The expectation of the CAAb in the cancer population differs from
that of the control population, i.e. m1 is not equal to m2. Since the sign of the difference
is not important, the test will be two-sided.

General Considerations

According to our previous results, we assume that

1. The distributions of the test results in each group are close to Normal or may be
transformed to the Normal by log-transformation.

2. The variance of the distributions in the two groups are approximately equal

3. Having in mind that this is a pilot study we will consider the results as significant
if the two-sided p-value is 0.1 or less. The bound 0.1 instead of 0.05 was chosen to
decrease the chances of random exclusion of some potentially informative tests.

When deciding the continuation of the study no correction for multiple comparisons is
assumed. However, the results will be presented without and with the correction using the
FDR of Benjamini & Hochberg approach (Benjamini, Hochberg 1995).

Where confidence limits are appropriate, the confidence level will be 95% .

Sample Size

The sample size was calculated using relevant information from our previous study on
Peptides P3 and P1 and ovarian cancer. There is no information in the literature regarding
cancer associated antibodies.

The sample size is calculated for a three step sequential group design with two sided
significance 0.1 and 0.05, power 0.8, for cancer: control ratio 1:2, for D of 0.5. The
calculations used the standard formulas for sample size necessary for comparison mean values
in two independent groups by t-test. The correction for a group-sequential procedure with
O’Bien and Fleming’s test increases the numbers by 1.017 and 1.027 for significance values
0.05 and 0.1, that is negligible for our sample sizes. (see C. Jennison, B.W.Turnbill
Group Sequential Methods with Applications to Clinical Trials Chapmann & Hall (2000) Boca
Raton FL US pp30 table 2.4).

The calculations were done using NCSS-PASS software.

Endpoint Evaluation

Effectiveness Analysis

A calculated D between 0.3 and 0.5 will mark a test as non effective to be used alone.
However, it may be important in combination with other tests as a part of the multivariate
test procedure. Tests with d < 0.3 will be considered as non-effective. A test, or a
combination of tests, with d > 0.5 will mark the CAAb test as effective.