WO2007107334A1

WO2007107334A1 - Ex vivo cancer diagnostic method

Info

Publication number: WO2007107334A1
Application number: PCT/EP2007/002461
Authority: WO
Inventors: Wassyl Nowicky; Askold Nowicky
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-03-20
Filing date: 2007-03-20
Publication date: 2007-09-27
Anticipated expiration: 2008-09-20

Abstract

The present invention relates to an ex vivo diagnostic method for the detection of malignant tumorigenic development or cancer in a human individual, the method being based on the determination of qualitative and/or quantitative alterations of at least one compound, preferably of an amino acid or an amino acid related compound, present in a bodily fluid of a diseased individual as opposed to a healthy control.

Description

EX VIVO CANCER DIAGNOSTIC METHOD

FIELD OF THE INVENTION

The present invention is in the field of medical diagnostics and relates to an ex vivo method of detection of a pathological condition, particularly of a malignant tumor, in a human or non-human mammalian individual.

BACKGROUND OF THE INVENTION

In the fight against cancer early and reliable detection of tumorigenic development and of the existence and/or distribution of cancer cells is of crucial importance. Currently, early detection programs, including screening and testing, are available for only a few of the most common cancers. For instance, the "American Cancer Society Guidelines for the Early Detection of Cancer, 2004" recommend screening for breast, cervical, colorectal, endometrial, prostate, and lung cancers. Almost all existing cancer screening tests are designed to detect just one specific form of cancer and cannot be used to indicate the presence of other cancers. Further, at least some of these tests are of low sensitivity even though fairly expensive.

It is known that various pathological conditions and diseases, and particularly cancer development, are typically associated with or even caused by defects in the immunological defense system. For instance, compounds affecting the protein synthesis in the cells of the immune system have significantly different functionality in healthy individuals as compared to cancer patients.

BRIEF DESCRIPTION OF THE INVENTION

In the attempt to find a reliable, ex vivo diagnostic method for the detection and/or confirmation of malignant tumorigenic development in an individual, it has been found that blood cells, particularly white blood cells, obtained from cancer patients differ significantly over their counterparts obtained from healthy blood donors as regards their physiological status and/or metabolic activity. For instance, the cells of diseased patients differ from the ones of healthy donors inter alia in their ability to take up nutritional compounds such as, e.g. amino acids, from their natural biological environment as well as from an artificial environment such as a suitable ex vivo nutrient medium. This effect was found to be even more pronounced in the presence of certain alkaloid derivatives in vitro, particularly in the presence of one or more alkaloid derivatives having a quaternary nitrogen atom such as the ones referred to in WO 2004/082698, which effect has already been reported in the co-pending patent application WO 2006/053680 (Appl.No.

PCT/EP2005/012083), the contents of which shall be entirely incorporated herein.

It has now been found, however, that such differences in the physiological status and/or metabolic activity of blood cells and, as the case may be, also of body tissues between healthy and diseased individuals can be monitored even in the absence of any exposure of such cells to external agents such as the above-mentioned nutrient or alkaloid compounds, namely by detecting and evaluating differences in the composition of body liquids obtained from healthy and diseased individuals.

Based on the exciting finding of this general principle a fully ex vivo diagnostic method for the determination of a pathological condition in an individual was established using the different cellular characteristics of body liquids, e.g. of red and/or white blood cells of diseased patients, as compared to corresponding body liquids, e.g. to corresponding red and/or white blood cells of healthy donors, as a measure for the prediction of the presence of a malignancy, particularly of a malignant tumorigenic activity and most particularly for the detection of a cancer disease in an afflicted individual. This ex vivo method is extremely patient-friendly as it does not require administration of any possibly hazardous agent to a patient solely for the purpose of cancer diagnostics.

DETAILED DESCRIPTION OF THE INVENTION

It was recognized that the concentration profiles of various compounds present in the blood plasma, in saliva or in sweat samples obtained from diseased and from healthy donors, differ significantly from each other and thus allow for a prediction of the presence of malignancy, particularly of tumorigenic development or an established cancer in a mammal, i.e. in an animal or human individual.

The recognition of this principle led to set up an ex vivo diagnostic method for the detection and/or confirmation of the presence of malignancy in a tested individual.

Additionally, it was found that the different physiological status and/or metabolic activity of blood cells is also detectable even without the addition of a cell culture medium or of nutritional compounds.

The term "amino acid" as used herein relates to any known L- or D-amino acid known to occur in human or animal individuals, including all essential and non-essential amino acids and all other naturally occurring amino acids.

The term "amino acid related compound" as used herein mainly relates to modified amino acids, amino acid derivatives and metabolites, and amino acid-based compounds such as smaller peptides naturally occurring in the animal or human body.

The term "compound fraction" as used herein relates to an amino acid fraction, a fraction comprising an amino acid related compound or any other compound. The present invention therefore in its first aspect relates to a method of detecting a pathological condition, particularly the presence of a malignancy, more particularly of a malignant tumor or a cancer disease, in an individual, the method comprising: - obtaining a sample of a bodily fluid from said individual;

- obtaining a cell-free liquid fraction of said sample;

- subjecting said cell-free fraction to analytical determination of at least one amino acid and/or amino acid related compound, preferably of a plurality of amino acids and/or amino acid related compounds, present in said fraction; and

- evaluating qualitative and/or quantitative results of said analytical determination against corresponding values obtained from control samples derived from healthy blood donors, wherein statistically significant qualitative and/or quantitative differences in the values of said individual as compared to the controls indicates a pathological condition, particularly the presence of a malignancy, more particularly of a malignant tumor or of cancer cells or tissues.

In a further aspect the invention relates to such a method, wherein said body liquid sample is selected from the group consisting of a whole blood sample, typically a heparinised whole blood sample, a saliva sample, and a sweat sample.

In a further aspect the invention relates to such a method, wherein said method comprises separating said cell-free liquid fraction into single compound fractions by liquid chromatography, preferably by HPLC, and detecting at least one, preferably a plurality of compound fractions, present in said cell-free fraction, using a suitable detection system.

In a further aspect the invention relates to such a method, which comprises detecting in the control sample at least one compound fraction which is not detected, or only at a significantly lower concentration, in the sample obtained from a said individual.

This significantly lower concentration is typically in a range of equal or less than 20 %, preferably equal or less than 10%, of the corresponding values of a said compound fraction in a pooled control.

In a further aspect the invention relates to such a method, wherein under the analytical conditions referred to in Example 1 at least one of said compound fractions present in the control sample but not or at a significantly lower concentration in the sample of a said individual elutes at a retention time prior to aspartic acid.

While samples obtained from patients suffering from manifest cancers were typically void of said particular compound fraction eluting at a retention time prior to the corresponding retention time of aspartic acid, some samples obtained from early stage cancer patients exhibited very low concentrations of said amino acid or related compound, i.e. concentrations of equal or less than 20 %, mostly of equal or less than 10% and typically of less than 5% of the corresponding values of the control.

Subsequent analysis revealed that said compound fraction which may qualify as a "cancer marker compound faction" elutes at the same retention time as glutathione.

In a further aspect the invention relates to such a method, wherein under the analytical conditions referred to in Example 1 one of said compound fractions present in the control sample but not or at a significantly lower concentration in the sample of said individual elutes at a retention time of about 2.5 min, e.g. at 2.57 min, and/or another one at about 3 min, e.g. at 3.07 min, and/or another one at about 3.7 min, e.g. at 3.79 min, while apartic acid elutes at about 4.3 min, e.g. at 4.34 min. In a further aspect the invention relates to such a method which is suitable for early prognosis on the risk of cancer development in a said individual.

In a further aspect the invention relates to such a method, wherein the detection of a compound fraction present in the sample of a said individual at a concentration significantly lower than in a pooled control is indicative of an increased risk of developing cancer or of the presence of an early stage cancer in said individual.

In a further aspect the invention relates to such a method, wherein a failure to detect in the sample of a said individual a compound fraction present in a pooled control is indicative of the presence of a malignant tumor or of cancer cells or tissues in said individual.

In a further aspect the invention relates to such a method, wherein said compound fraction comprises a compound that elutes prior to aspartic acid under the analytical conditions referred to in Example 1 .

In a further aspect the invention relates to such a method which comprises detecting at least one compound fraction present in the experimental sample and not in a pooled control sample.

In a further aspect the invention relates to such a method, wherein under the analytical conditions referred to in Example 1 at least one of said compound fractions present in the experimental sample and not in the control sample elutes at a retention time later than that of alanine.

In a further aspect the invention relates to such a method, wherein under the analytical conditions referred to in Example 1 one compound fraction present in the experimental sample and not in the control sample elutes at a retention time of about 50 min and/or another one compound fraction present in the experimental sample and not in the control sample elutes at a retention time of about 70 min.

In another aspect, the present invention relates to the above described method which further comprises quantitative evaluation of the concentration of the amino acid or amino acid related compound corresponding to said product peak appearing ahead of the aspartic acid peak, for early prognosis on the risk of cancer development in said individual. If preliminary results hold true, it can be inferred from this method that if in a said individual a concentration level of said amino acid or amino acid related compound is detected of less than 20 %, particularly of less than 10 %, of the concentration level of a pooled control such low concentration is indicative of an increased risk of developing cancer or of the presence of an early stage cancer in said individual.

In order that the invention described herein may be more fully understood, the following examples are set forth. They are for illustrative purposes only and shall not be construed as limiting this invention in any respect.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 represents a concentration profile of amino acids and amino acid related compounds determined by HPLC and fluorescence detection from a healthy volunteer (control sample); ordinate = light units, abscissa time in minutes.

Table 1 : Le end of Fi . 1

n.d.: not determined

Fig. 2 represents a concentration profile of amino acids and amino acid related compounds determined by HPLC and fluorescence detection from a blood plasma sample of a colon cancer patient; ordinate = light units, abscissa = time in minutes.

n.d.: not determined

Fig. 3 represents an overlay of the concentration profiles of Fig. 1 and Fig. 2; A = Fig. 1 , B = Fig. 2; ordinate = light units, abscissa = time in minutes. A detailed legend for the concentration profiles A and B is found in Table 1 and Table 2, respectively.

From Figures 1 and 2 and especially from overlay in Fig. 3 it can easily be derived that two product peaks (peaks 1 and 4) appear in the control sample of a healthy volunteer, whereas the same product peaks are absent from the sample of a cancer patient. Apparently, metabolic products present in the blood of the healthy control have been utilized or further decomposed by the organism of the cancer patient and thus do not show up in the LC or HPLC chromatograms of the experimental samples. Based on this recognition the present inventor has developed an ex vivo cancer diagnostic method as herein described and claimed.

Fig. 4 represents a profile of amino acids and amino acid related compounds determined by HPLC and fluorescence detection of blood plasma from a healthy volunteer (control sample); ordinate = light units, abscissa = time in minutes.

Table 3: Legend of Fig. 4

n.d. : not determined

Fig. 5 represents a profile of amino acids and amino acid related compounds determined by HPLC and fluorescence detection of blood plasma of a stomach cancer patient (experimental sample); ordinate = light units, abscissa = time in minutes.

Table 4: Legend of Fig. 5

n.d.: not determined

Fig. 6 represents an overlay of the profiles of Fig. 4 and Fig. 5; A

= Fig. 4, B = Fig. 5; ordinate = light units, abscissa = time in minutes. A detailed legend for the profiles A and B is found in Table 3 and Table 4, respectively.

Fig. 7 represents an enlarged and stretched view of a part of Fig.6.

From Figures 4 and 5 and especially from the overlay in Fig. 6 and Fig. 7 it can be derived that one product peak appears in the control sample of a healthy volunteer but not in the sample of a cancer patient (peak 2). More specifically, under the conditions applied herein an amino acid or amino acid related compound elutes under the chosen HPLC conditions shortly before aspartic acid (peak 3), which compound appears to be very much decreased in its concentration or is entirely absent (i.e. not detectable by this HPLC method) in the sample of the stomach cancer patient (experimental sample).

Moreover, a comparison of the chromatogram of the stomach cancer patient with the one of the healthy control represents that the peaks occurring at 5.35 min (peak 4), 10.35 min (peak 1 1 ), 23.23 min (peak 19; arginine), 33.99 min (peak 21 ; β-alanine) and 38.58 min (peak 24) are very low or entirely missing in the plot of the control while they are rather prominent in the plot of the cancer patient.

Also, it can be seen that in the chromatogram of the healthy control (Fig.4) peaks show up at a retention time of approximately 3.8 min (peak 4) and 7.3 min (peak 7) which are absent from the chromatogram of the cancer patient (Fig.5). The compound eluting at approx. 3.8 min under the given HPLC conditions is believed to be glutathione, as subsequent runs under identical HPLC conditions using inter alia glutathione as an amino acid marker showed that glutathione elutes at a retention time of approx 3.8.

Additionally, it was found that the concentration of asparagine (peak 6), glutamine (peak 1 2) and histidine (peak 1 3) tends to be lower in the sample of cancer patients as compared to healthy controls.

Example 1 : Determination of metabolite profiles in blood samples of cancer patients and healthy donors

Method:

5ml venous blood were taken from a lymphoma or prostate cancer patient and transferred to a heparinized glass vial. 1 ml thereof was transferred into an Eppendorf microcentrifuge glass container and centrifuged for 1 5 min at 1 500 g.

The such obtained plasma was deproteinized by admixing an equal volume of 1 M perchloric acid (HCIO₄) and centrifuging the mixture for 20 minutes at 1 2000 g. As a control, pooled venous blood from healthy donors was used and was subjected to the same procedure described above . As an internal standard, homotaurine (Sigma Aldrich) was used. The protein- free extracts obtained by this procedure were stored at -20⁰C and kept for subsequent analysis.

Various compounds including a number of amino acids were determined qualitatively and quantitatively by HPLC (Agilent 1 1 00), applying isocratic elution from a Diasorb 1 30 C₁₆ T, 6 micrometer, 3x250 nm column (Elsiko, Russia); mobile phase: 0. 1 M sodium acetate buffer, ph 5.71 - acetonitryhwater ( 1 : 1 ) 65:35; flow rate 0.6 ml/min, 30⁰C. As a standard, a commercially available amino acid mixture (L-Amino Acid Kit, Sigma-Aldrich) was applied. The statistical evaluation was done using the software Statistica 6.0. Results:

Table 5: Presence/absence of low molecular weight compounds in blood plasma of lymphoma and prostate cancer patients

n.d. = not determined

Conclusion:

The concentration values of some low molecular weight compounds, more specifically of some amino acids or amino acid related compounds determined from the plasma fraction of said blood samples exhibit significant differences in samples from healthy probands as compared to those from diseased patients, e.g. cancer patients. More specifically, whereas a product peak appears at a retention time of 2.57 min in the pooled plasma samples of the control group of healthy volunteers and not in those of the experimental group, two other product peaks appear at retention times of 57 and 70 min in the experimental samples (Table 5).

It seems that the peaks may not be attributed to any of the twenty natural amino acids usually present in a human body, but instead are assumed to be amino acid derivatives or related amino compounds, e.g. polar metabolic reaction products bearing at least one amino group. The concentration profile, i.e. the absence or presence of compounds at certain retention times under the given analytical conditions, obtained from the experimental samples as compared to the concentration profile obtained from the controls gives statistically relevant information as to the presence of malignancy in an individual.

Example 2: Determination of concentration profiles of selected amino acids and amino acid related compounds in blood samples of cancer patients and healthy donors Blood samples of a colon cancer patient and of a healthy donor have been prepared according to Example 1 . The plasma fractions obtained therefrom have been analysed according to Example 1 .

Results:

Table 6: Concentration profiles of selected amino acids and amino acid related compounds in blood plasma of a colon cancer patient and a healthy donor.

n.d. = not determined

Conclusion:

The concentration values of selected amino acids and amino acid related compounds determined from the plasma fraction of said blood samples exhibit significant differences in the control samples of the healthy proband as compared to the experimental samples of the diseased patient, i.e. cancer patient. Whereas some of the determined compounds have been found to have reduced levels in the experimental plasma sample as compared to the sample of the healthy volunteer, other compounds have been found to have increased levels, as can be seen in Table 6 and Figures 1 to 3. More specifically, whereas two peaks appear at retention times of 3.07 min (peak 1 ) and 3.79 min (peak 4) in the control samples, these product peaks are absent from the experimental samples as can be seen in Figures 1 to 3. The concentration profile and/or the absence of distinct product peaks from the experimental samples gives statistically relevant information as to the presence of malignancy in the tested individual. Example 3: Determination of profiles of amino acids and amino acid related compounds in blood samples of cancer patients and healthy donors

Starting from the premise that tumor growth is accompanied by certain shifts in the formation of pools of amino acids and/or amino acid related compounds it was undertaken to monitor the changes in the content of amino acids and amino acid related compounds for cancer detection. 5ml venous blood were taken from 29 patients with different types of cancer (stomach, rectal, lung, breast, bladder, prostate, and leukemia). Also 3 patients with benign processes and 10 healthy donors were included. All patients were investigated before treatment. Heparinized blood samples of patients were incubated for 2.5 h at 37⁰C in Eppendorf tubes without and with 100 μ\ of Ukrain^® , diluted to 1 :5, 1 : 10, 1 : 100 or 1 : 1000, followed by a centrifugation step for 15 min at 1500 g in an Eppendorf microcentrifuge. The such obtained plasma was deproteinized by admixing an equal volume of 1 M perchloric acid (HCIO₄). Homotaurine (Sigma) was used as an internal standard. The protein-free extracts obtained by this procedure were stored at -20⁰C and kept for subsequent analysis according to Example 1 .

Results:

In the plasma samples of cancer patients (see Fig. 5) as compared with the plasma samples of healthy controls (see Fig. 4) it was observed that in the samples that were not subjected to UKRAIN® treatment the plasma levels of histidine, asparagine and glutamine (peaks 6, 12 and 13, respectively) tended to be lower in cancer patients than in healthy subjects, while the plasma levels of arginine and β-alanine (peaks 19 and 21 , respectively) tended to be higher in cancer patients than in the healthy controls. A few unidentified peaks were found under the applied HPLC conditions which occurred only in cancer patients and not in healthy controls, while one peak occurred in healthy subjects and not, or at very low levels only, in cancer patients, which latter peak (peak 2) typically showed up shortly before the aspartic acid peak as can be seen in Tables 3 and 4 and in Figures 4 to 7. Subsequent tests using additional marker compounds have elucidated (data not shown herein) that under the same HPLC conditions glutathione elutes at identical or nearly identical retention times thus indicating that the compound present in the healthy control and absent or reduced in the cancer patients may very likely be glutathione.

Further useful information can be derived from the plots in Figures 1 to 7.

Since the experiments showed that the area beneath said "cancer marker" peak ahead of the aspartic acid peak may vary among the healthy controls such finding raises the question whether quantitative evaluation of said peak could be used for very early prognosis of a potential risk of an individual for developing cancer. Should this prove true everyone having a genetic disposition of developing malign tumors or cancer could, at a very early stage already, start monitoring his or her risk of falling ill of cancer by checking for the presence and size of said "cancer marker" peak at regular intervals.

While the above experiments have been carried out using whole blood samples it is within the scope of the present invention to retrieve analogous information from individuals by taking and using samples of other bodily fluids such as, for example saliva or sweat samples.

Claims

1 . Method of detecting a pathological condition, particularly the presence of a malignancy, more particularly of a malignant tumor or a cancer disease, in an individual, the method comprising:

- obtaining a sample of a bodily fluid from said individual;

- obtaining a cell-free liquid fraction of said sample;

2. The method according to claim 1 , wherein said sample is selected from the group consisting of a whole blood sample, a saliva sample, and a sweat sample.

3. The method according to claim 1 or 2, comprising separating said cell- free liquid fraction into single compound fractions by liquid chromatography, preferably by HPLC, and detecting at least one, preferably a plurality of compound fractions, present in said cell-free fraction, using a suitable detection system.

4. The method according to claim 3, which comprises detecting in the control sample at least one compound fraction which is not detected, or only at a significantly lower concentration, in the sample obtained from said individual.

5. The method according to claim 4, wherein said significantly lower concentration is in a range of equal or less than 20 %, preferably equal or less than 10%, of a pooled control.

6. The method according to claim 4 or 5, wherein under the analytical conditions referred to in Example 1 at least one of said compound fractions present in the control sample but not or at a significantly lower concentration in the sample of said individual elutes at a retention time prior to aspartic acid.

7. The method according to claim 6, wherein under the analytical conditions referred to in Example 1 one of said compound fractions present in the control sample but not or at a significantly lower concentration in the sample of said individual elutes at a retention time of about 2.5 min and/or another one at about 3 min and/or another one at about 3.7 min,, while apartic acid elutes at about 4.3 min.

8. The method according to any one of claims 1 to 7, for early prognosis on the risk of cancer development in a said individual.

9. The method according to any one of claims 4 to 7, wherein the detection of a compound fraction present in the sample of a said individual at a concentration significantly lower than in a pooled control is indicative of an increased risk of developing cancer or of the presence of an early stage cancer in said individual.

10. The method according to any one of claims 4 to 7, wherein a failure to detect in the sample of a said individual a compound fraction present in a pooled control is indicative of the presence of a malignant tumor or of cancer cells or tissues in said individual.

1 1 . The method of claims 9 and 10, wherein said compound fraction comprises a compound that elutes prior to aspartic acid under the analytical conditions referred to in Example 1 .

12. The method according to claim 1 , which comprises detecting at least one compound fraction present in the experimental sample and not in a pooled control sample.

13. The method according to claim 12, wherein under the analytical conditions referred to in Example 1 at least one of said compound fractions present in the experimental sample and not in the control sample elutes at a retention time later than that of alanine.

14. The method according to claim 13, wherein under the analytical conditions referred to in Example 1 one compound fraction present in the experimental sample and not in the control sample elutes at a retention time of about 50 min and/or another one compound fraction present in the experimental sample and not in the control sample elutes at a retention time of about 70 min.