Table of Contents

1) The RECAF™ cancer marker

2) RECAF™ in companion animals

3) Download RECAF™ scientific information

4) RECAF™ uses

5) Sample collection and shipping

6) Price and Payment

7) Ordering information

8) Warranty

1) The RECAF™ cancer marker

Introduction:

Herein, we present data related to a new cancer marker, named RECAF™, which exhibits high sensitivity and specificity on tissue sections and serum of patients with diverse types of malignancies.

RECAF Biology:

RECAF is a receptor present on the surface and in the cytoplasm of fetal cells. It binds and internalizes circulating Alpha-Fetoprotein (AFP), a protein that behaves much like albumin in the early stages of prenatal life. Thus its name (RECeptor for AFp). RECAF and AFP should not be confused; the latter is a liver and testicular cancer marker known since 1963 whereas RECAF is an emerging broad-spectrum cancer marker. The two are completely different in structure and behavior.

Much like albumin, AFP binds and transports smaller molecules, such as long chain, poly-unsaturated fatty acids (PUFA). Experiments have shown that following binding to RECAF, AFP penetrates the cell via coated-pits, releases its load of fatty acids and then leaves the cell immunologically intact, probably ready to cross again the placenta and fetch another fatty acid molecule from the mother.

The uptake of AFP and hence the expression of RECAF is related to the degree of cell differentiation: When a given fetal organ or tissue has reached a certain maturity, AFP is no longer taken up.

Since cancer cells are poorly differentiated, they re-express RECAF, which behaves as an oncofetal antigen (these are molecules expressed by fetal and cancer cells but not by normal adult cells. AFP and CEA [Carcino Embryonic Antigen] are typical oncofetal antigens).

During fetal life, AFP uptake occurs in most organs and tissues and therefore RECAF, which mediates the uptake, is re-expressed in many types of cancers.

The early work on the AFP receptor was done in a variety of species, including chicken, mice, rats, baboons and humans, all of which showed a similar mechanism of AFP internalization. Moreover, it was also shown, that the AFP from one species such as chicken binds and is taken up by the AFP receptor of another species as far away - evolutionary speaking - as mice thus suggesting that the molecule(s) are highly conserved throughout the zoological scale. While this poses a problem at the time of generating antibodies (RECAF from a different species is still seen as ‘self’ by the immunized animal), once a monoclonal antibody is developed against the AFP binding site on RECAF, it will recognize RECAF from many other species. Our parent company, BioCurex Inc. has developed such an antibody, which has been used to stain cancer cells on tissue sections and in an immunoassay, to measure RECAF in thousands of human serum samples.

The results indicate that such a test can detect the presence of cancer with approximately 90% sensitivity and 95% specificity (see figures on the right and tables below). The tables below show the sensitivity, the number and the efficiency for several types of cancer when compared to normal sera, using two different cutoff values:

*103 normal control sera were used throughout this study.

* 97 cancer samples were compared to 22 benign tumor samples.
** 62 cancer samples were compared to 77 benign prostate hyperplasias (BPH).

Studies carried out on prostate and breast cancer serum samples also indicate that the test can detect very early lesions (stages I & II) with high sensitivity and specificity.

Summary of results in human patients

Click the image to see it full size.

Click the image to see it full size.

Click the image to see it full size.

ROC analysis of 103 normal individuals and 333 assorted cancer samples. The area under the curve is 0.988 (perfect discrimination = 1.0). Overall sensitivity was 94% with 95% specificity or 91% with 99% specificity. The better the discrimination, the closer the graph to a right angle (no discrimination results in the points being arranged in a diagonal line).

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Different tissues stained with an anti-RECAF™ antibody.(A) Normal stomach 40X; (B) stomach cancer 100X; (C) Prostate adenoma, 40X; (D) Prostate adenocarcinoma, 100X; (E) Normal colon 200X; (F) Colon carcinoma 100x; (G) Normal lung 40X; (H) Lung cancer 40X. Positive cells are stained brown.

Click the image to see it full size.

2) RECAF™ in companion animals

The early work on the AFP receptor was done in a variety of species, including chicken, mice, rats, baboons and humans, all of which showed a similar mechanism of AFP internalization. Moreover, it was also shown, that the AFP from one species - such as chicken - binds and is taken up by the AFP receptor of another species as distant as the mouse thus suggesting that the molecule(s) is highly conserved throughout the zoological scale. The anti-RECAF monoclonal antibody was selected to recognize the same RECAF epitope as AFP and therefore, the antibody behaves in a similar way to AFP, recognizing mouse, rat, human, porcine, feline and canine RECAF. We have concentrated our efforts on developing a canine RECAF serum test for cancer and we have found results that closely follow those obtained with human samples; the sensitivity was 85% at 95% specificity (n = 300). The figure on the right shows the distribution of results from samples taken from dogs with cancer and from healthy donors.

Download the complete canine study as well as previous posters and presentations made at congresses.

Very preliminary results suggest that the test can be adapted for use in cats and we welcome professionals interested in collaborating with us to advance these studies.

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3) Download RECAF™ scientific information

Review of the original basic research work describing RECAF

Review of tissue staining and RECAF serum tests on human samples

Independent article describing the presence of RECAF in human stomach cancer

Complete canine study as well as previous posters and presentations made at congresses

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4) RECAF™ uses

There are three main applications where the OncoPet RECAF™ test can be useful:

A) In the diagnostic process.

B) In the follow up of dogs already diagnosed with cancer, as an alert to a possible recurrence or metastatic disease as well as to monitor the effect of therapy.

C) For screening.

A) In the diagnostic process: The veterinarian can incorporate the result of a RECAF test as an additional piece of information to determine whether or not an animal has cancer. Initially, along with the RECAF value, we shall provide the cutoff values at 90%, 95% and 99% specificity (as the cutoff value increases, so does the specificity, but at the expense of the sensitivity). This information can provide the professional with a rough idea of how likely it is that an animal has the disease. As we collect and test more samples - with your collaboration in letting us know the final diagnosis - we shall add an easier way to evaluate the situation by providing the statistical chance that the tested animal has cancer.

B) For follow up: We have not yet investigated the curve of circulating RECAF in treated animals. However, from the information gathered in the use of other markers in humans, it is anticipated that following therapy/surgery, the circulating RECAF values will increase in those cases with recurrence or metastases.

C) For screening: This application requires high specificity because only a few dogs in the screened population will have cancer and if the specificity is not high, then the few true positive cases could get buried within the number of healthy dogs who just happen to have a normal high RECAF value. Using a high cutoff value ensures that a positive test in indicative of cancer but has the drawback that a significant number of cancers might be missed. This means that both the professional and the animal caregiver need to be aware that when the 99% specificity cutoff is used, the animal might still have cancer and be considered as negative (it would be a false negative). With that in mind, the situation for a false negative dog is then no different than what would have been if it had not received a RECAF test: The cancer would grow until it became clinically evident. It is up to the caregiver and the veterinarian to decide if the money spent on the test is too much to bear for the benefit of having a 50%-65% chance of detecting an unknown malignancy earlier than it would otherwise manifest itself clinically with the obvious prognostic disadvantage for the animal. Since we also report the 95% cutoff value, the veterinarian could choose to use that cutoff level and further investigate animals testing positive, or to keep a vigilant eye on the animal and repeat the test a few weeks later. An increase in serum RECAF on the second test would be indicative of disease.

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5) Sample collection and shipping

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6) Price and Payment:

If you bought the test from a distributor, you only need to ship the sample. If you ordered directly from us, you need to enclose a cheque payable to Oncopet Diagnostics Inc. (credit card payment coming up soon!).

The price per test is $59.00 + shipping.

Due to volume, the price offered by our distributors is likely to be lower than ours.

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7) Ordering information

Orders are best placed online. This reduces potential errors and allows tracking samples in transit. This method also reduces data input for you, the customer, since data is only entered once rather than each time you place an order.

Notes on ordering online:

- Leaving your mouse on a text box or a button for a few seconds brings up a help box.

- Set your browser to accept cookies and Javascript.

- Depending on your browser security level settings, you might be asked for permission to access our pages. Please grant permission when required since we have eliminated any potential threats to your computer.

How to place an order step by step:

a. Sign in: You set up your own user name and password. If you are part of a group of clinics with the same name, your password will differentiate your location from the other clinics in the group.

b. The first time you sign in, you will be requested to input your clinic’s information and the names and email addresses of the veterinarians that will order the test. You may use a separate email address for each veterinarian, in which case we will send the results directly to the professional who ordered the test. You can also have the same email for all veterinarians in your clinic (e.g. info@yourclinic.com) and then all test results will be emailed to the same address, with the name of the veterinarian requesting the test. At a minimum, there should one name and one email address.

c. Click "Continue".

d. Fill in the order form. For database integrity, you cannot type or modify a veterinarian's name directly. Instead, you must click on the name list. To add, delete or change the veterinarians information you should click ‘Update clinic’s information’, change it as needed and return to the order page, to finish your order.

e. Clicking ‘Submit order’ takes you to a confirmation page displaying all the pertinent information. The order has now been created and a unique order number has been issued. This order number is your reference for tracking down an order in case you need to contact us.

f. Print the confirmation page (print only one copy; the system automatically prints a second copy for your records). Send the confirmation page along with the sample.

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8) Warranty:

OncoPet Diagnostics will reimburse you for any false positives or false negatives (at our recommended cutoff values) in the form of a test free of charge. To claim the warranty, please send us a copy of the pathology report.

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