TUMOUR LIBERATED PROTEIN (TLP):

ITS POTENTIAL FOR DIAGNOSIS AND THERAPY

GIULIO TARRO

in <<Anticancer Research>> 19: 1755-1758 (1999) 

"D. Cotugno" Hospital

Via G. Quagliariello, 54 - 80131 Naples, Italy

Phone & Fax +39/81/546.32.22

Key words: TLP, tumor marker.

Running title: TLP in diagnosis and treatment

Experimental study

Date: 13 Ottobre 1998

Abstract

Our earlier studies led to the purification of a similar antigen from several types of human tumours which had the following properties: It provoked a delayed reaction in certain cancer patients as could be demonstrated both in vivo and in vitro (1). In contrast healthy individuals did not react with this antigen. Subsequent studies led to a higher degree of purity of tumour antigen which enabled a partial sequence (2). The eight aminoacid sequence obtained led to the synthesis of the corresponding peptide.

This synthetic peptide was then used to raise specific antibodies in rabbits. The availability of such antibodies enabled to undertake a range of studies. Those investigations revealed that the antibodies were tumour specific because they reacted only with certain types of tumours but not with normal cells nor with other types of tumour tissues. We decided to name this tumour antigen as TLP for tumour liberated protein.

Background and current state of TLP Research

In the previous studies we purified a tumour associated antigen (TAA) which was extracted from a human kidney carcinoma tissue (3) and we found it to be similar to a glycoprotein which is induced in herpes simplex virus infected cells (4). The specific antibodies which were raised against this antigen reacted also with the HSV-TAA in primary cervical carcinoma (5). However, those antibodies failed to react with TLP. Since TLP was found in several common malignancies we decided to concentrate our studies on this antigen in order to try and establish whether it was tumour specific.

A reasonably high level of purification of TLP enabled us to obtain an initial aminoacid sequence which in turn led to the synthesis of the following three specific peptides from lung and colon cancer: ArgThrAsnLysGluAlaSerIle; GlySerAlaXPheThrAsn; AsnGlnArgAsnArgAsp. A fourth peptide GlyProProGluValGlnAsnAlaAsn was obtained from TLP which was purified from a urogenital tumour.

Recent studies with rabbit antibody which was raised against the eight aminoacid peptide from lung and colon cancer revealed that TLP is produced in the cytoplasm of the malignant cells (Table I, Fig.). Since it was not detected in normal tissues we concluded that it was a tumour specific antigen (6). These preliminary observations need further confirmation with additional studies using a larger number of patients and a wider range of tumour tissues.

Detection of TLP in sera of cancer patients

The TLP specific antibodies which were raised in rabbits against one of the peptides ArgThrAsnLysGluAlaSerIle enabled us to screen human sera for the presence of this antigen in the blood of cancer patients. Using the Elisa method, 64 sera samples from patients with non-small cell lung cancer were tested and 34 (53%) gave a positive reaction. In contrast none of the 30 samples obtained from patients with other forms of malignancies reacted with the antibodies (7). A larger study of well defined tumour types and their stage of disease is likely to reveal a pattern of distribution for the presence of TLP in the blood of corresponding patients and might correlate to the stage of disease.

Detection of antibodies to TLP

The same eight amino acid peptide from TLP of lung cancer was used also to screen for specific antibodies in a range of human sera. Of the 25 samples obtained from patients with epidermoid carcinoma, nine gave positive reaction. Likewise four out of eight samples from adenocarcinoma reacted as well as one out of five with large cell carcinoma. Sixty-four sera samples from normal and patients without malignancies gave negative reaction (8).

Discussion and Conclusion

To date, the best available studies of tumour markers in lung cancer patients are CEA (9), TPA (10) and Cyfra21-1 (11). Several studies have been reported on the sensitivity of those markers with a range of 66%-95% specificity (9,11). However, since the level of these antigens was very low they were not useful in determining the tumour load or the degree of malignancy in the corresponding patients. Our work on TLP indicates that this protein appears to be a new tumour marker with potential clinical application. Using Western blot analysis, we were able to show that TLP was expressed in all lung cancer derived cell lines, breast cancer derived cell lines and in colo-rectal derived cell lines that were studied (6). Since lung and intestinal tissues have a common embryonic origin, it will not be unreasonable to suggest that TLP might be tumour specific antigen for at least some of the malignancies that arise from tissues of epithelial origin. Since we were not able to detect TLP in normal lung tissues nor in the osteosarcoma cell lines SAOS-2 (6) it supports the notion that TLP is indeed a tumour specific antigen. On the basis of the immunohistochemical studies which revealed a large amount of TLP in the columnar epithelial cells and lumen (secreted TLP) as illustrated in the figure, it appears that TLP is a cytoplasmic antigen which is specific for epithelial derived neoplasms.

Further studies with a larger number of patients and a wider range of malignant tissues is needed in order to be in a position to confirm that TLP is a tumour specific protein.

Present studies attempt to achieve the complete sequence of TLP. This in turn should enable in vitro preparations of large quantities of TLP by genetic engineering. The availability of such antigen preparations will facilitate future studies on the role of TLP in human malignancy. It could also enable the preparation of an assay for early diagnosis of the corresponding tumours and might even be useful in the generation of a specific anti cancer vaccine.

Table I

REACTIVITY OF RABBIT ANTISERUM TO THE TLP DERIVED PEPTIDE

(RTNKEASIC)

ORGAN ORIGIN OF TISSUE PEROXIDASE

POSITIVITY

1 LUNG NORMAL ADULT 0

2 LUNG FOETUS 18 WEEK 0

3 LUNG INTERSTITIAL INFLAMMATION 0

4 LUNG BRONCHIOLO-ALVEOLAR ADENOCA. 3+

5 LUNG ACINAR ADENOCARCINOMA 3+

6 LUNG PAPILLARY ADENOCARCINOMA 3+

7 LUNG EPIDERMOID CA (DIFFERENTIATED) 2+

8 LUNG EPIDERMOID CA NON KERATINIZING 0

9 LUNG SMALL CELL CARCINOMA 0

10 LUNG SARCOMA 0

11 LUNG MALIGNANT MESOTHELIOMA 0

12 SKIN MALIGNANT MELANOMA 0

13 BREAST INFILTRATING DUCT CARCINOMA 0

14 BREAST INTRADUCTAL ADENOCARCINOMA 0

15 THYMUS MALIGNANT THYMOMA 0

16 ENDOMETRIUM ADENOCARCINOMA 0

17 BLADDER PAPILLARY UROTHELIAL CARCINOMA 0

18 COLON ADENOCARCINOMA 2+

Figure: Histological section of a colo-rectal carcinoma which was incubated with the rabbit antiserum to the TLP peptide. The red staining represents the distribution of TLP in this malignant tissue.