IE45284B1 - Tumour detection - Google Patents

Abstract

1520208 Detection of cancer F HOFFMANNLA ROCHE & CO A G 26 May 1977 [27 May 1976] 22283/77 Heading G1B Melanoma, bladder carcinoma, ovarian cancer, lung cancer, liver cancer, cervical cancer or sarcoma in humans are detected by a method comprising incubating a measured amount of patient leukocytes with aqueous basic extracts of the tumour of the cancer to be detected then determining the "leukocyte non-adherence index" (defined) by comparison with a control, for example by carrying out the incubation in glass tubes placed in a horizontal position then placing the tubes vertically and counting the number of cells not adhering to the walls of the tubes using a leucocytometer, the presence of a particular form of cancer being indicated when there is the greatest number of non-adherent cells. The tumour extracts may be diluted for use using "Medium 199" which is a (defined) composition containing amino acids, vitamins, inorganic salts and other components.

Description

The present invention relates to tumour detection. More particularly, the invention is concerned with a method for the detection of melanomas and other tumours in humans by evaluating antigen-induced leukocyte adherence inhibition caused by tumour specific cell mediated immunity.

Cell mediated tumour immunity has been demonstrated in experiments with transplanted tumours in mice and rats. For example, Halliday et al., Int. J. Cancer, Vol. 9, pages 477-483 (1972) established a simple assay of cell mediated immunity . called Leukocyte Adherence Inhibition (LAI). This assay was based on the findings that the adherence of peritoneal leukocytes to glass is specifically inhibited by the addition of tumour extracts if the peritoneal leukocytes are obtained from mice which have been presensitized against the specific tumour.

Holan et al., Cellular Immunology, Vol. 13, pages 107-115 (1974), using transplanted tumours in rats, mice and guinea pigs also demonstrated the same phenomenon with macrophage cells. Grosser et al., Cancer Research, Vol. 35, pages 2571-2579 (1975) established that breast cancer can be detected in humans by evaluating antigen-induced leukocyte adherence inhibition caused by tumour-specific cell mediated immunity. There has.been much research in attempts to use immunological principles for the detection and possible cure for various human cancers. Evidence for the existence of a host immune response to malignant melanoma has been developed recently by Lewis et al., British Medical Journal, Vol. 3, pages 547-552 (1969), Oren et al., -=3- <5 0 3.8 2 Clin. Exp. Immunol., Vol. 9, pages 45-46 (1971), Cocaraa et al., Lancet, Vol. 1, pages 1340-1341 (1972), de Vries etai., Int.

J. Cancer, Vol. S, pages 567-376 (1972), Hellstron etal., Int. J. Cancer, Vol. 11, pages 280-292 (1973), Heppner et al., Int-.

J. Cancer, Vol. 11, pages 2«5~260 (1973) and Holllnshead et al., Cancer, Vcl. 34, pages 1235-1243 (1S74).

While some success has been met its animal models having transplanted tumours, immunological methods developed for the defection of malignant melanomas have not been successful for mass screening. While it has been generally known that an immunological defence mechanism exists in the early stages of malignant diseases, and such defence mechanism has been demonstrated with malignant melanomas in humans, no assays using the principle have been developed which are suitable for large scale screening.

Ideally, a desirable test for malignant melanomas in humans would be a blood test which is simple, specific and reproducible and which detects the disease at a sufficiently early stage so that the disease is still curable.

In accordance with the present invention it has been found that the product of the immunological defence mechanism of the human body to malignant tumours (e.g. carcinomas, sarcomas and melanomas) can be detected at a stage of the disease which is sufficiently early so that the disease is still curable. The method used to detect the product of the immunological defence mechanism is a blood test which is simple to carry out, is specific to the malignant tumour, (e.g. melanoma) and gives reproducible results. As used herein, the term malignant melanoma” means a malignant tumour consisting of black masses of cells having a marked tendency to metastasis.

The present invention is based on the foregoing finding and is accordingly concerned with a method for detecting the presence of melanoma, bladder carcinoma, ovarian cancer, lung cancer, liver cancer, cervical cancer or sarcoma in huaians, which method comprises incubating a measured amount of a patient's blood leukocytes with aqueous basic extracts of the tumour of the cancer to be detected and then determining the leukocyte non-adherenee index by comparison with a control.

The method provided by this invention involves collecting the leukocytes from heparinised venous blood of the patient, suspending a measured amount of the leukocytes in a buffered solution and mixing the leukocytes with extracts of malignant melanoma tumours, there being used, as a control, tissue extracts from persons having benign breast disease, adenocarcinoma of the breast, various benign diseases or malignant tumours not being detected. After suitable incubation procedures, the number of cells in each case not adhering to the sides of the test tube are counted and the leukocyte adherence inhibition is measured as. the non-adherence index (NAI). This determines the presence or absence of malignant melanoma.

The non-adherence index is calculated according to the following formula η «_ί ii £-» a % non-adherent cell3 in presence of specific antigen % non-adherent cells in presence of non-specific antigens LOO % non-adherent cells in presence of non-specific antigen The leukocytes are collected and counted by conventional methods (e.g. a nemocytometer;. The heparinised blood sample is incubated at about 37 °C for about I hour and the plasma fraction is collected. The plasma fraction is rich in leukocytes. The leukocytes are separated by centrifugation and suspended in an isotonic buffer. Any erythrocytes remaining are lysed from the leukocyte suspension. The leukocytes are then separated by centrifugation and suspended in an appropriate 7 aqueous medium at a cell concentration of 1 x 10 /ml.

The tumour extracts are made by homogenising the tumour samples {or normal tissue samples), in an ice-cold buffered saline at pH about 7.3, centrifuging the homogenate and collecting the resulting supernatant. The buffer used is preferably a phosphate buffered saline (P3S). It is necessary to use a basic pH since an acid pH will destroy the antigenic activity.

For use in the present method, the extracts should be diluted with an appropriate aqueous medium, preferably Medium 199 (Microbiological Associates, Sethesda, Maryland). The optimum dilution is established for each extract by determining the concentration of tumour antigen which produces the largest increment between the specific and non-specific inhibition of leukocyte adherence. *& U w «The malignant melanoma tumour extract is the antigenic material which gives rise to the cell mediated immunity. The antigens which are suitable for use as the non-specific controls are tissue extracts of breast cancers, adenocarcinoma of the bowel, ovary and bladder and lung cancer and the like. The antigens used for non-specific controls as described herein can also be detected by the method of this invention by using the proper non-specific controls. Thus, ovary, bladder, cervical and lung cancers can also be detected.

The present method is carried out by mixing aliquots of a small amount of blood leukocyte suspension containing about x 10 leukocytes/ml with separate small amounts of buffer, malignant melanoma antigen or unrelated antigen in separate glass containers. Each mixture is then brought to a predetermined volume. Each mixture is then incubated in the glass container which is placed so that the contents cover at least three-fourths of the inner surfaces of the container. The incubation is carried out at about 37°C for a sufficient time to complete the reaction, if any, under a humidified atmosphere of 5% carbon dioxide/air. After completion of the incubation, the contents of the container are agitated and the number of non-adherent cells is counted.

I1 The aliquots of leukocyte suspension are conveniently very small, usually about 0.1 ml. The concentration of leukocytes is such that counting of the cells gives a meaningful result and the reaction with the antigen material can be readily assayed. The buffer used for dilution is Medium 199 which has a pH of 7.2-7.4. - 7 4 828 4 The amount of antigen extract used is conveniently about 0.1 ml of the diluted extract. The extract is diluted generally with Medium 199. The amount of dilution of the concentrated antigen extract on a volume basis can be between 1 part of antigen extract per 4 to 16 parts of diluent. Preferably a 1:4 dilution is used.

The final volume of the test mixture of leukocytes and antigen is conveniently about 0.5 ml, The diluent used to achieve this volume is Medium 199.

The glass containers used are of any convenient size and shape. for convenience of use and availability, 20 ml Pyrex (registered Trade Mark) or Kxmax (registered Trade Mark) (Fisher Scientific, Montreal, Canada) test tubes are preferred.

The incubation is carried out with the test tubes in a horizontal position in a holder which prevents their movement. This is critical since movement during the incubation can adversely affect the test results. The incubation is preferably carried out for about 2 hours. It can be carried out for longer, but this does not increase the accuracy of the method or the completeness of the reaction. . It can also be carried out for a shorter period, but this is not optimum.

Thus, incubation times of from about 1 to 3 hours are possible.

The non-adherent cells are counted in any convenient manner (e.g. using a hemocytometer).

The response of the leukocytes of a patient to malignant melanoma extracts does not depend upon the source of the extract. 4ΰ884 - 8 Extracts from'the patient who is being tested or extracts from other patient’s malignant melanomas give equivalent results.

This indicates that the leukocytes of a patient with malignant melanoma exhibit cell mediated immunity which is specific to all malignant melanoma of the same type and is not affected by the source of the antigen. 2 S d The following Examples illustrate the present inventions Example 1 Tumour Extracts Malignant melanoma tumour samples were received at operation and placed in a sterile container. Eat and fibrous tissue were dissected away and the specimen was finely minced with sharp scissors -n ice-cold phosphate buffered saline (PBS), (0.15-M phosphate buffer, 0.15-M saline) at pH 7.3. The resulting material was homogenised for 10-15 minutes in five volumes of BBS at 40,000 revolutions per minute in a VirTis 45 homogeniser. The homogenate was centrifuged at 20,000 times gravity for 30 minutes and the supernatants were collected and stored at -40°C in 2 ml aliquots. Extracts of breast cancer, bladder cancer and other tumours such as ovary and lung cancers were prepared in an identical manner and were used as the controls. The protein concentrations of the stock extracts were 6-3.5 mg/ml. ?or use, the extracts were thawed and diluted to 1:4 by volume with Medium 199. Medium 199 is marketed by Microbiological Associates, Bethesda, Maryland and has the following composition: Components mg/litre Amino Acids L-Alanine 25.0 1-Arginine hydrochloride 70.0 L-Aspartic acid 30.0 L-Cysteine hydrochloride 0.1 L-Cystine .0 ¢5284 Components mg/litre Amino Acids (continued) L-Glutamic acid 57.0 L-Glutamine 100.0 Glycine 50.0 L-Histidine hydrochloride hydrate 22.0 Hydroxy-L-proline 10.0 L-Isoleucine 20.0 L-Leueine 60.0 L-Lysine hydrochloride 70.0 L-Methionine 15.0 L-Phenylalanine 25.0 L-Proline 40.0 L-Serine 25.0 L-Threonine 30.0 L-Tryptophan 10.0 L-Tyrosine 40.0 L-Valine 25.0 Vitamins g-Aminobenzoic acid 0.050 Ascorbic acid 0.050 D-Biotin 0.010 Calciferol 0.100 D-Calcium pantothenate 0.010 Cholesterol 0.200 Choline chloride 0.500 Folic acid 0.010 i-Inositol 0.050 Menadione 0.010 d ίί 2 8 Components mg/litre Vitamins ;continued) Nicotinamide 0.025 Nicotinic acid 0.025 Pyridoxal hydrochloride 0.025 Pyridoxine hydrochloride 0.025 Riboflavin 0.010 Thiamine hydrochloride 0.010 OL-a-Tocopherti phosphate (disodium) 0.010 Tween 80* 5.000 Vitamin A acetate 0.140 Other Components Adenine hydrochloride dihydrate 12.10 Adenosine-51-monoclespheric acid, dihydrate (AMP) (Muscle Adenylic Acid) 0,20 Adenosine-5‘-triphosphate disodium tetrahydrate (ATP) 1.08 Deoxyribose 0.50 Dextrose 1000.00 Glutathione (reduced) 0,05 Guanine hydrochloride hydrate 0.33 Hypoxanthine 0.30 Phenol red 20.00 Ribose 0.50 Sodium acetate trihydrate 83.00 Thymine 0.30 Uracil 0.30 Xanthine 0.34 Components mg/litre Inorganic Salts Calcium chloride dihydrate 186.0 Ferric nitrate nonahvdrate 0.7 Potassium chloride Potassium dihydrogen phosphate Magnesium sulphate heptahydrate Sodium chloride 400.0 60.0 200.0 8000.0 Sodium hydrogen carbonate 1400.0 Disodium hydrogen phosphate heptahydrate SO.O Vialed at pH 7.2 to 7.4.

••‘Trade Mark of Atlas Powder Company; polyoxyethylene-20-sorbitan monooleate.

Example 2 Preparation of the Leukocyte Reagent Slood was taken from patients having malignant melanoma and from control subjects and immediately stored at 4°C. After retraction of the clot overnight, the serum was separated and stored at -40°C. These blood samples were heparinised blood samples obtained from patients having malignant melanoma and from control subjects having bladder or breast cancer and a variety of non-malignant diseases or other unrelated malignancies. The diagnosis of. malignant melanoma in the so-designated patients was confirmed histologically. The clinical diagnosis Of all the controls and unrelated malignancies was confirmed by histological examination of a surgical specimen. 20 ml samples of the heparinised blood (i.e. venous blood) were obtained in two 10 ml tubes and placed vertically in glass universal bottles δ 5 3 8 -ΰ at 37°C for 1 hour. The resulting leukocyte-rich plasma fraction was aspirated and centrifuged at 200 times gravity for 15 minutes. The cell-free plasma was removed and discarded.

The remaining cell button was suspended in an ice-cold isotonic Tris-buffered ammonium chloride solution by repeated pipetting and left for IS minutes at 4*0 in order to lyse any remaining erythrocytes. This procedure was terminated by the addition of 3 ml cf Medium 199 and the cells were centrifuged at 200 times gravity for 15 minutes. The resulting supernatant was removed and discarded and the remaining cells were then washed twice with 10 ml portions of Medium 199 tach time. Aliquots of the leukocytes were made at a concentration of 1 x 10 cells per ml using Medium 199 as the diluent.

Example 3 IAX Assay Aliquots of 0.1 Mi of blood leukocytes suspensions containing 1 x 10' cells per ml were placed in 20 ml 16 mm by 150 m glass test tubes. 0.1 mi of extracts of unrelated tumour cell derived preparations or 0.1 ml of extracts of malignant melanoma tumours were added to these test tubes.

Various concentrations of tumour extracts were used to determine the optimum concentration. The nixcure of leukocytes and test ceils were brought tc a final volume of 0.5 mi by the addition of Medium 199. The mixture was then stirred and the tubes were placed in a horizontal position so that the contents of the tubes covered 2/4 cf the surface of the tubes. The horizontal tubes were then incubated at 37°C in a humidified atmosphere of 5% φ ΰ 3 θ 4 carbon dioxide/air for 2 hours. The tubes were not disturbed or moved during the incubation period. After the incubation, the tubes were placed vertically and the contents were agitated with a pasteur pipette. The number of non-adherent cells per ml was counted in a hemocytometer. All assays were carried out in triplicate. The results expressed as the non-adherence index (KAI) is shown in Table I hereinafter: '28 ί Table I Antigen-Mediated Leukocytes Adherence Inhibition in Glass Tubes by Malignant Melanoma Tumour Extract Diagnosis in clxnic T**' .......................................... ............ % of Non-adherent cells in presence of Malignant Breast „ melanoma cancer __ antigen antigen anfciJeil Non-adherence index malignant3 melanoma Melanoma 39% 22% 8% 77 Melanoma 43% 23% 5% 100 1 ; Melanoma J - Λ 21% 5% 95 Breast cancer 20% 29% 10% NC Breast cancer 35% 60% 13% NC Bowel cancer 27% 28% 6% -3 Lung cancer 32% 27% 9% 14 Ovarian cancer 39% 3s% 18% 8 Bladder cancer 31% 24% 13% 29 Cholecystitis ' 24% 24% 9% 0 Inguinal hernia 17% 18% 2% _ -6 aNon-adherence index (KAX) to malignant melanoma = Number of non-adherent Number of non-adherent cells in presence of - cells in presence of malignant melanoma extract Unrelated antigen __— ------——- x poo Number of non-adherent cells in presence of unrelated antigen NC » not calculated’ The results in Table X indicate that the patients having malignant melanoma have a significantly higher NAI when compared v.’ith other patisnts when the dilution of the melanoma tumour extract was 1:8. Peripheral blood leukocytes from malignant 4! ΰ 2 8 4 - 16 melanoma patients incubated with melanoma tumour extract had leukocyte non-adherence ranging from 35-65%, whereas the same cells incubated with breast cancer extract had a 15-39% leukocyte non-adherence. Control subjects showed a leukocyte non-adherence in the range of 15-39% and the responses to the malignant melanoma and control tumour extracts were the same.

The NAI for the control subjects was less than 30 and NAI for patients with malignant melanoma was greater than 30.

When blood leukocytes from malignant melanoma patients or 10 control subjects are incubated in glass tubes without added antigen for 2 hours, about 10% non-adherent cells were found.

The addition of any tumour extract to non-sensitised peripheral blood leukocytes inhibited adherence of 18-38% of the leukocytes In order to achieve the optimum assay conditions, that is 15 to say, the maximum specific inhibition of leukocyte adherence with the least non-specific inhibition, the antigen extracts were tested at different concentrations with leukocytes from control subjects then with leukocytes from patients having malignant melanoma. As shown in Table II hereinafter, the NAI falls with increasing dilutions. In this instance, however, a dilution of 1/8 'of the breast cancer extract and malignant melanoma extract gave a high per cent leukocyte non-adherence value for the patient having malignant melanoma and a low per cent leukocyte non-adherence value to malignant melanoma for both the control and patient having breast cancer. Therefore, for the purpose of this invention, a dilution of 1/8 of the antigen, that is to say, one part of antigen extract to 8 parts of diluent, is preferred. Good results are obtained, however, with dilutions from 1:4 to as high as 1:IS.

Table XI Effect of Diluting Antigen on Leukocyte Adherence inhibition % Leukocyte non1 -adherence to melanoma i tumour extract Diagnosis ! Dilution of antigen 1 Cholelithiasis 1:4 42% 1:6 i 37% i 1:8 ί 30% I 1:16 j 20% Hernia 1:4 38% 1:6 35% 1:S 23% 1:16 21% Breast cancer 1:4 39% 1:6 36% 1:8 29% 1:16 25% ’ ! Malignant melanoma 1:4 47% 1:6 47% 1:8 46% ί 1:16 39% Cholelithiasis and hernia patients were the controls, 25 breast cancer patients provided the non-specific antigen and malignant melanoma patients provided the specific antigen.

Tabla III hereinafter shows that the KAI was similar whether the peripheral blood leukocytes of patients having malignant melanoma were exposed to allogeneic or autochthonous melanoma extracts. The only patient who showed a low reactivity to autochthonous tumour extract had an advance case of malignant melanoma and the patient's KAI accordingly had decreased. 32 8 4 - 18 Table III Non-Adherence Index of Leukocytes of Malignant Melanoma Patients to Pour Different Extracts of Malignant Melanoma Patient diagnosis Control NAIa to extract of malignant melanoma from McK LC DC 10 McK 100 85 — 72 LC 50 68 30 57 DC 40 106 86 70 10 185 157 57 33 27 -15 -5 0 “NAI value greater than 30 is considered-to be significant When other tumours were used as the non-specific control antigen, the results were similar to those with the breast cancer extract as the non-speeifie control. When a bladder cancer extract was tested against malignant melanoma and bladder cancer patients and control subject, the leukocytes of the melanoma patients were reactive with the melanoma tumour extract and the leukocytes from patients having bladder cancer were reactive with the bladder cancer extract. Neither reacted with the non-specific antigen or control. This indicates that the method provided by this invention is applicable to assaying for bladder cancer as well as for malignant melanoma. Bladder cancer is a carcinoma. The foregoing is summarised in Table IV hereinafter: Table IV Leukocyte Adherence Inhibition In Test Tubes. to.. Malignant Melanoma with Bladder Cancer Extract as tha Non-Specific Antigen Patient diagnosis No. tested No. positive Γ NAI Mean to melanoma3 Range Malignant meiancma 6 6 85 41-133 Bladder cancer 15 0 -22 25-(-47)b Benign disease 4 0 9 0-15 j 1 Other malignancy 2 0 15 14-18 j 1C NaI calculated with melanoma tumour as the specific antigen and bladder cancer as the non-specific antigen NAI calculated with bladder extract as the specific antigen and melanoma tumour extract as the non-specific antigen showed that 9 out of 15 patients having bladder cancer had a NAI greater than 30 tc bladder cancer extract and a mean NAI of 38 and a range cf 90- (-20) ·.

Leukocytes from two malignant melanoma which were reactive to malignant melanoma extract showed no evidence of cross reactivity to tumour extracts of lung, bladder or ovary when assayed by the leukocyte adherence inhibition assay provided by this invention.

In 33 patients having histologically proven malignant melanoma, leukocyte adherence inhibition occurred with peripheral blood leukocytes from 22 malignant melanoma patients. In this series of patients and with these tumour extracts, a NAI greater than 30 was selected to indicate significant melanoma patients and control subjects. Table V hereinafter shows that, of 475 control subjects, 21 had a NAI value greater than 30. Upon re-testing, only 2 continued to have a NAI value greater than .

U 2 8 4 - 20 Control patients having cancers other than malignant melanoma did not show LAI when tested with a melanoma tumour extract. The malignant melanoma patients tested prior to surgical excision of the melanoma tumour had a mean NAI of 61, 103 and 32 for Stage I, stage II and Stage III malignant melanomas respectively. This is statistically significantly different from the control group (p greater than 0.001 by unpaired t-test). u!a38d Table V Summary of Patients Tested by LAI In Test Tubes for Reactivity to Melanoma Tumour Extract Patients studied j Total Positive8 Mean NAI Malignant ί Stage I 13 11 61 Stage II 7 7 103 Stage III s 2 32 Disease free 1 year δ 2 22 Halo nevus 1 1 31 Control subjects Presurgicai benign disease 162 5 -2 Benign breast disease 119 4 Healthy West Indian Negroes 8 0 5 Unrelated cancer Breast cancer 153 10 -21 Lung 6 0 BOWel 5 1 Bladder 15 0 Bladder 15 0 -4 Ovary 5 0 Thyroid 2 I ^AI greater than 30 was regarded as being positive Stage I - a localised primary malignant melanoma confined to the skin or eye Stage II - regional lymph node metastasis or local recurrence of tumour in skin or lymph nodes.

Stage III - distant metastasis Depending on the stage of the cancer, the peripheral blood leukocytes of the malignant melanoma patients had different I degrees of reactivity in the LAI assay. Of the 13 patients having stage 2 malignant melanoma in situ, 11 showed reactivity in the LAI assay although many had small cutaneous lesions.

Their mean NAI was 61. Two patients had a KAI below 30. One patient had a 3 mm by 5 mm malignant melanoma of the conjunctiva without any invasion and the other patient had a 1 mm by 1.2 cm lesion of the leg. All patients having Stage II malignant melanoma showed LAI reactivity and their mean NAI of 103 was the highest of all the stages.

By contrast, three of the five patients having Stage III malignant melanoma did not show LAI reactivity. Moreover, the positive NAI of one of the patients became negative as the tumour burden increased. The other Stage III patient having a positive NAI had a solitary liver metastasis resected and the patient was clinically cancer-free after 16 months. The mean NAI of 32 of the Stage III patients was the lowest for patients having active cancer.

Finally, eight patients, all of.whom had previously had Stage I or II malignant melanoma resected at least 1 year previously and were clinically free of cancer, were assayed by LAI in test tubes. Six of the eight patients had a NAI of 30 or less and the remaining two patients had a NAI of 38 and 40.

These patients with a history of malignant melanoma who were clinically free of cancer had a mean NAI of 22. « ΰ 2 8,5 Ascitic fluid from a patient having malignant melanoma metastasis in the abdominal cavity showed than the leukocytes of a reactive melanoma patient reacted to the ascitic fluid and to the melanoma extract, indicating the ascitic fluid contains melanoma tumour antigen.

Prom the foregoing, it will be appreciated that the method provided by the present invention is a simple and quantitative method for measuring cell-mediated immunity to malignant melanoma tumour antigens and bladder carcinoma tumour antigens as well as other tumour antigens (e.g. cervix, ovarian, liver carcinomas and sarcomas). The method is immunologioally specific and reproducible and can be used with allogenic and autologous tumour extracts.

Claims (9)

1. cmsMo 1) A method for detecting the presence of melanoma, bladder carcinoma, ovarian cancer, lung cancer, liver cancer, cervical cancer or sarcoma in humans, which method comprises incubating a measured amount of a patient's blood leukocytes with aqueous 5 basic extracts of the tumour of the cancer to be detected and then determining the leukocyte non-adherence index by comparison with a control.

2. ) A method according to claim 1, wherein the cancer to be detected is malignant melanoma. 10

3. ) A method according to claim 1, wherein the cancer to be detected is bladder carcinoma. {

4. ) A method according to claim 1, wherein the cancer to be detected is ovarian cancer.

5. ) A method according to claim 1, wherein the cancer to be 15 detected is cervical cancer.

6. ) A method according to any one of claims 1 to 5 inclusive, wherein the tumour extract is from a tumour of the patient being tested.

7. ) A method according to any one of claims 1 to 5 inclusive, 20 wherein the tumour extract is from a tumour of a patient not being tested.

8. ) A method according to any one of claims 1 to 7 inclusive, wherein the malignant melanoma tumour extract is an aqueous extract at pH 7.3.

9. ) A method for detecting the presence of melanoma, bladder 5 carcinoma, ovarian cancer, lung cancer, liver cancer, cervical cancer or sarcoma in humans, substantially as hereinbefore described with reference to the foregoing Examples.