Tumor M2 Pyruvate Kinase (Tumor M2-PK)

ScheBo Biotech

Any new diagnostic test that could help to identify the presence of a tumour is to be welcomed. When clinical studies of the test show high levels of sensitivity and specificity, it needs to be taken very seriously.

Tumor M2-PK, a dimeric isoform of pyruvate kinase, has been identified as a very useful tumour metabolic marker by ScheBo Biotech. M2 - pyruvate kinase occurs in both a tetrameric form which shows a high affinity for the substrate phosphoenolpyruvate (PEP), and the dimeric form, which has a low affinity for PEP. The dimeric form predominates in tumours and was therefore named Tumor M2-PK by Eigenbrodt et al1.

Tumor M2-PK is a dimeric isoform of pyruvate kinase
Tumor M2-PK is a dimeric isoform of pyruvate kinase

This dimeric form is over-expressed by a wide range of different tumours, probably due to the different metabolic requirements shown by tumour cells - especially for increased nucleic acid, amino acid and phospholipid synthesis. ScheBo Biotech therefore developed a test to detect and monitor tumours by measuring the level of Tumor M2-PK in blood samples (exclusively EDTA-plasma samples are recommended). This is achieved by using monoclonal antibodies that are specific to the dimeric form. The company has also recently perfected a similar test for stool samples.

The Tumor M2-PK tests are both innovative and unique because they measure tumour metabolic activity, which in turn acts as a measure of tumour aggressiveness. Consequently, Tumor M2-PK can be very effective for monitoring the effects of therapy; detecting possible relapses or metastases; and providing useful supportive information in the diagnosis and detection of various tumours.

The term ‘tumour metabolome’ was coined by Mazurek and Eigenbrodt2 to indicate the metabolic characteristics of tumour cells. These include high glycolytic and glutaminolytic capacities, high phosphometabolite levels, and a high channelling of glucose carbons to synthetic processes. Tumor M2-PK is a key regulator of the tumour metabolome.

M2-PK stands at an important crossroads in tumour metabolism. Although different tissues (such as lungs or the brain) may have a completely different basic metabolism, they adopt the same metabolic phenotype during tumorigenesis. M2-PK can occur in the highly active tetrameric form and a nearly inactive dimeric form. In tumour cells, the dimeric form of M2-PK always predominates. M2-PK interacts directly with certain oncogenes, such as pp60v-src kinase, A - Raf kinase and HPV - 16 E7, which trigger a dimerization to Tumor M2-PK. In this dimeric state, energy is produced by glutaminolysis. (Visit www.metabolic-database.com for further details).

As previously mentioned, the new tumour marker test can be used with two types of material: EDTA-plasma samples and faecal samples.

The blood test
A study on the stability of different blood samples by Hugo et al3 discovered that EDTA-plasma samples are the most robust under typical daily laboratory conditions and handling. They give highly reproducible results - despite shaking or being kept at room temperature for several hours before centrifugation. However, heparin-plasma and serum samples showed increased levels of Tumor M2-PK when shaken. ScheBo Biotech therefore decided only to use EDTA-plasma samples for its new Tumor M2-PK test. Whole blood samples collected into EDTA tubes are centrifuged (e.g. 550g for 10 minutes) on the same day, and the supernatant EDTA-plasma should be refrigerated at 4°C (stable for three days) or frozen at -20°C (stable for one year) until the test is conducted.

Because Tumor M2-PK is a metabolic activity marker, it is over-expressed in the blood for a wide range of different tumours. To date, increased levels of Tumor M2-PK have been found in gastrointestinal, breast, lung, renal and prostate tumours. It is over-expressed in the blood of a large proportion of patients who suffer from any of these cancer types.

The Tumor M2-PK test developed by ScheBo Biotech uses a 96-well ELISA plate. This can be used for samples from up to 42 patients (in duplicate). The kit contains all the necessary reagents. The test is based on two monoclonal antibodies that react specifically with Tumor M2-PK and do not cross-react with other isoforms of pyruvate kinase. The end result is a colour reaction, with the optical density being measured using an ELISA plate-reader. This is then converted to a quantitative concentration of the tumour marker (U/ml).

The ScheBo Tumor M2-PK Stool Test
The ScheBo Tumor M2-PK Stool Test

Some of the earliest studies of the tumour marker were conducted on patients with renal cell carcinoma. Tests carried out by Oremek et al4 and Wechsel et al5 showed a clear difference between Tumor M2-PK values measured in patients with renal cell carcinoma and those in healthy control subjects. They also noted a significant correlation between the levels of Tumor M2-PK and the Robson score (tumour stage). A further study by Oremek et al6 also showed a highly significant discrimination between renal cell cancer and nephritis patients.

Subsequent studies have underlined the powerful potential of the Tumor M2-PK blood test. As well as its use as an independent tumour marker to support cancer detection, there are two further key ways in which it can be employed. The first is in combination with other tumour markers, to increase specificity without the substantial loss of sensitivity that could occur if two markers with a similar conventional mode of action are used together. Secondly, it can be used to monitor patients following therapy, as changes in the level of Tumor M2-PK reflect the changes that are happening to the tumour.

These different uses of the tumour marker have been borne out by various recent studies. For instance, research by Lueftner et al7 showed that the combined use of Tumor M2-PK and serum CA27.29 gives valuable additional information about patients with advanced breast cancer. The former highlights the disease activity whilst the latter indicates the tumour burden. Two years later, Hoopmann et al8 reported that Tumor M2-PK determination in the plasma of patients with metastasized breast cancer could be a helpful tool for monitoring therapeutic success.

Meanwhile, in three separate studies, Schneider et al9,10,11 discovered that Tumor M2-PK could be a valuable diagnostic tool for the detection and monitoring of lung cancer and for the detection of its progression.

Tumor markers in a patient with lung cancer
Tumor markers in a patient with lung cancer

This was reinforced by work carried out by Sapoutzis and Oremek12. The test can therefore provide important information that will help a clinician to tailor the duration, dose and choice of a patient’s therapy so that it meets their specific needs.

In the case of surgical intervention, Wechsel et al4 found that Tumour M2-PK levels returned to normal in the majority of renal cell carcinoma patients within six weeks (maximum 11 weeks) of successful surgery, but remained high or increased in the case of tumour relapse or metastasis.

One of the key uses of the new tumour marker is for the detection of gastrointestinal (GI) cancer - including colorectal, gastric, pancreatic and oesophageal tumours. Schneider and Schulze13 found that Tumor M2-PK was significantly more sensitive than CEA in 250 colorectal cancer patients. It also performed well in gastric and oesophageal cancer patients.

These findings have been reinforced by Zhang et al14, whose recent study showed that Tumor M2PK is more sensitive than CEA for colorectal cancer, and more sensitive that CA72-4 for gastric cancer.

In an earlier study, Schulze15 reported Tumor M2-PK’s superior overall sensitivity for colorectal, gastric and oesophageal cancers when compared with CEA, CA19-9 or CA72-4. This study also showed “a remarkable increase in the sensitivities” when Tumor M2-PK is combined with the appropriate traditional marker. For example, the sensitivity for the combination of Tumor M2-PK with CEA in colorectal cancer rose to 67% (Tumor M2-PK alone: 50%). When it was combined with CA 72-4 in gastric cancer, and with CA 19-9 in pancreatic or oesophageal cancers, the sensitivity was 82%, 96% and 65% respectively (compared with 67%, 73% and 59% for Tumor M2-PK on its own).

In a group of patients with various gastrointestinal cancers, Hardt et al16 found Tumor M2-PK was much more sensitive overall than CEA or CA19-9. They concluded that “Tumor M2-PK might be a valuable marker in gastrointestinal cancer.”

The stool test
The potential for Tumor M2-PK is even more exciting when ScheBo Biotech’s new stool test is taken into account. This is primarily intended for the screening and detection of colorectal cancer (bowel cancer). The latest studies show that the tumour marker is significantly more sensitive than current laboratory tests.

The Tumor M2-PK stool test only requires a single, random faecal sample, about the size of a large grape. This remains stable at 250C for two days, so can be sent to the laboratory through the post if necessary - there is no urgency to carry out the test immediately. Unlike conventional tests, there are no dietary restrictions for the patient.

The particular strength of the new tumour marker in detecting colorectal cancers is due to the fact that these occur almost exclusively as tumours along the gut wall. Stool samples can therefore be used to measure levels of Tumor M2-PK from cells shed into the gut lumen. The new test is independent of blood in the faeces, and can therefore also detect non-bleeding lesions.

An extensive clinical study conducted by Hardt et al17 at the Giessen University Hospital has evaluated the usefulness of the Tumor M2-PK stool test. The study was carried out in secondary care gastroenterology clinics, and included 60 patients with colorectal cancer, who were compared with a control group of 144 subjects who had undergone colonoscopy without any pathological findings of cancer.

This recent research reported a sensitivity of 73% for the Tumor M2-PK stool test, combined with a specificity of 78%. These results show that the stool test could be an extremely useful screening tool, with positive results then being followed up by colonoscopy.

This initial study has been extended to cover more patients, and the results have been reported by Toepler et al18. These show an even higher specificity of 81% and a sensitivity of 80% for faecal Tumor M2-PK.

Tumor M2-PK levels in stool samples from patients with colon cancer, rectal cancer and control subjects
Tumor M2-PK levels in stool samples from patients with colon cancer, rectal cancer and control subjects

A number of clinical studies have been completed with the exciting new tumour metabolic marker, Tumor M2-PK. These have shown encouraging results for both the EDTA-plasma test and the newer stool test. It is perhaps just a matter of time before this new marker test becomes accepted as a standard diagnostic and monitoring tool for a range of cancers. It certainly appears to provide a practical, simple and yet highly effective test.

Phil Allcock
Phil Allcock is a professional freelance copywriter and a Chartered Biologist, based in Southampton, UK. He has worked for a wide variety of international, national, and local companies. Please visit www.phila.co.uk for further details.

1. Eigenbrodt, E et al. (1992). A double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells. In Critical Reviews in Oncogenesis, pp 91 -115 [M Perucho, editor]. Boca Raton, FL: CRC Press

2. Mazurek, S and Eigenbrodt, E (2003). The Tumor Metabolome. Anticancer Research 23: 1149-1154.

3. Hugo, F et al. (1999). Quantitative Detection of Tumor M2-PK in Serum and Plasma. Anticancer Research 19: 2753-2758.

4. Oremek, GM et al. (1999). The Pyruvate Kinase Isoenzyme Tumor M2 (Tu M2-PK) as a Tumor Marker for Renal Carcinoma. Anticancer Research 19: 2599-2602.

5. Wechsel, HW et al. (1999). Marker for Renal Cell Carcinoma (RCC): The Dimeric Form of Pyruvate Kinase Type M2 (Tu M2-PK). Anticancer Research 19: 2583-2590.

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8. Hoopmann, M et al. (2002) Tumor M2 pyruvate kinase - determination in breast cancer patients receiving trastuzumab therapy. Cancer Letters 187: 223-228.

9. Schneider, J et al. (2000). Quantitative Detection of Tumor M2-Pyruvate Kinase in Plasma of Patients with Lung Cancer in Comparison to Other Lung Diseases. Cancer Detection and Prevention 24: 531-535.

10. Schneider, J et al. (2002). Tumor M2-Pyruvate Kinase in Lung Cancer Patients: Immunohistochemical Detection and Disease Monitoring. Anticancer Research 22: 311-318.

11. Schneider, J et al. (2003). Fuzzy Logic-based Tumor Marker Profiles Including a New Marker Tumor M2-PK Improved Sensitivity to the Detection of Progression in Lung Cancer Patients. Anticancer Research 23: 899-906.

12. Sapoutzis, N and Oremek, GM (2002). Evaluation of Tumor M2 Pyruvatekinase Values in Patients with Lung Diseases. 8th Central European Lung Cancer Conference: 75-80.

13. Schneider, J and Schulze, G (2003). Comparison of Tumor M2-Pyruvate Kinase (Tumor M2-PK), Carcinoembryonic Antigen (CEA), Carbohydrate Antigens CA 19-9 and CA 72-4 in the Diagnosis of Gastrointestinal Cancer. Anticancer Research 23: 5089-5094.

14. Zhang, B et al. (2004). Tumor type M2 pyruvate kinase expression in gastric cancer, colorectal cancer and controls. World Journal of Gastroenterology 10 (11): 1643-1646.

15. Schulze, G. (2000). The Tumor Marker Tumor M2-PK: An Application in the Diagnosis of Gastrointestinal Cancer. Anticancer Research 20: 4961-4964.

16. Hardt, PD et al. (2000). Tumor M2-PK- Pyruvate Kinase: A Promising Tumor Marker in the Diagnosis of Gastro-intestinal Cancer. Anticancer Research 20: 4965-4968.

17. Hardt, PD et al. (2004). Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer. British Journal of Cancer 91: 980-984

18. Toepler, M et al. (2004). Tumor M2-PK Stool Test: A screening tool for colorectal cancer. Poster from the 32nd Meeting of the International Society for Oncodevelopmental Biology and Medicine (ISOBM).

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