GB2530521A - Mass spectral analysis of urine and other bodily fluids for the detection of cancer biomarkers - Google Patents

Abstract

Mass spectral analysis of bodily fluids for cancer biomarker detection The invention relates to subjecting patient samples of bodily fluids preferably urine to direct mass spectrometry in order to detect cancer biomarkers. A particular marker of interest is human chorionic gonadotropin (hCG), the free subunits of which are frequently found in the urine of bladder cancer patients. Other potential markers of interest include Bence Jones protein and free kappa and lambda light chains. The spectral masses are compared with expected values in the range between 2,000 m/z and 100,000 m/z. The changes in spectral masses as compared to normal healthy controls are characteristic of cancer.

Description

Mass spectral analysis of urine and other bodily fluids for the detection of cancer biomarkers.

The invention relates to subjecting patient sample of bodily fluids to direct mass spectrometry and the analysis of spectral masses between 2,000 m/z and 100,000 m/z. The changes in spectral masses as compared to normal healthy controls are characteristic of cancer.

Background

Cancerous conditions are frequently associated with elevated levels of proteins or peptides, known as cancer markers, in one or more bodily fluids. The presence of these markers is commonly used to detect the presence of a tumour, and can also be indicative of the prognosis for long term survival.

Many germ cell and malignant epithelial tumours express human chorionic gonadotropin (hCGJ and or its free subunits; in particular malignant epithelial cancers express the free beta subunit of hCG (hCG). Human chorionic gonadotropin, alpha subunit, hCG and in particular the urinary metabolite of hCG beta-core fragment (hCGcfJ are found in the urine of patients with such tumours. A feature of malignancy is aberrant glycosylation which is also reflected in the variable masses of hCG and hCG-related molecules detected in urine and other bodily fluids.

Well-documented evidence for ectopic production of hCG/hCG by bladder tumors was not published until 1973. Subsequently, more case reports were reviewed in 1978 and again in 1983, whereupon ectopic hCG expression became a generally accepted phenomenon in cancer. in 1989 the free p-subunit of hCG was identified as a potential bladder tumor marker; at this time, the hCG-like product was determined to be hCG and not IICG. Bladder cancer is not unique in expressing hCG and over the past three decades, there have been many reports highlighting the ectopic production of hCG from non-germ-cell or non-placental origins; this has culminated in the latest wave of publications, linking hCG expression to poor prognosis in 93% of studies.

Since 1989, 57 significant studies on the detection of hCG in various epithelial cancers have been published. Positive detection ranges from 4% in prostate cancer to 93% in small cell (SC) lung cancer. in some cases 100% detection is observed when looking for the expression of "any" CGB gene; this is more likely to be due to the sensitivity of modern amplification techniques and not necessarily indicative of disease. There is considerable variation in positivity for hCG/CGB, not just between studies but between detection methods used in the same studies on the same sample set. Serum and urine levels rarely tally and there are even more clear differences when looking at CGB gene expression and protein profile.

More generally the overall incidence of hCG expression in epithelial cancer is likely to be around a third of all cases. We can see that in renal, prostate, vulval/vaginal, and neuroendocrine cases, the frequency is somewhat lower and in bladder, cervical, and pancreatic carcinoma, the frequency is a little higher. The prognostic significance of hCG3 expression has been noted and appears almost always to indicate poor prognosis: Aggressive tumors with lower response rates to radiotherapy and chemotherapy occur in patients where hCGj3 is elevated. In our own studies, survival analysis indicated a very strong association between early death and hCG expression.

In an analogous fashion B-cell malignancies such as multiple myeloma produce large levels of antibodies and fragments thereof termed "paraproteins", that can be found in blood and urine.

Myeloma is a malignancy of plasma cell or activated B-cells that produce antibodies (immunoglobulins). An anaplastic clones overproduction of a specific antibody results in a "spike" on the normal distribution of proteins within the gamma region of a serum (or urine) sample protein electrophoresis -an M spike (or monoclonal spike).

Detection of paraproteins in the urine or blood is most often associated with benign monoclonal gammopathy of undetermined significance (MGUS), where they remain "silent", and multiple myeloma.

Vastly excessive production of a monoclonal antibody or, more commonly, overproduction of immunoglobulin light chains are a feature of muftiple myeloma, which result in severe pathology as these free light chains are deposited in tissues? particularly the kidneys.

Human immunoglobulin molecules consist of two identical heavy chains which define immunoglobulin classes (lgG, IgA, 1gM, lgD and IgE] and identical light chains (kappa or lambda) that are covalently linked to a heavy chain. In healthy individuals, the majority of light chains in serum exist bound to heavy chain. However, low levels of free light chains (FLCs) are found in serum of normal individuals due to their excess production over heavy chains by mature B-cells. In serum, FLC kappa exists predominantly as a monomer with a molecular weight of 22.5 kDa and FLC lambda as a dimer with a molecular weight of 45 kDa. This size difference results in a differential glomerular filtration rate and, consequently, a ratio of FLC kappa to FLC lambda of 1:1.6 in serum.

FLCs are observed in urine too but filtration and reabsorption of low molecular proteins in the kidney strongly affects the FLC concentration so that urinary FLC level is low in healthy individuals.

FLC are a natural product of B lymphocytes and, as such, represent a unique biomarker of neoplastic and reactive B cell-related disorders. Increased FLCs are associated with malignant plasma dyscrasia and other lymphocyte related immuno-proliferative disorders.

The detection of paraproteins, such as the FLCs, is important diagnostic aid for a variety of monoclonal gammopathies, such as multiple myeloma, Waldenstrom macroglobulinemia, non-secretory myeloma, smoldering multiple myeloma, and MGUS. Accurate measurement of monoclonal free light chains in serum and/or urine is especially important in light-chain diseases, such as light-chain myeloma, primary systemic amyloidosis, and light chain-deposition disease. The ability to quantify monoclonal FLCs may be useful to monitor the disease. In patients with light chain myeloma, either of light chain, kappa (K) or lambda (A), is dominantly produced and resulting in marked changes of the PLC K/A ratio in the early phase of the disease. The detection of urinary monoclonal kappa or lambda free light chains of immunoglobulin, also know as Bence Jones proteins (BjP), are important for identifying and monitoring B-ce!! ma!ignancies.

Clinical efficacy needs to coupled with simple sampling rapid and affordable testing for a biomarker to be adopted as a routine service test by a health service.

The method describes rapid screening of patient samples of bodily fluids subjected to direct mass spectral ana!ysis using mass spectrometry, such as MALDI -ToP Mass spectrometry. Analysis may be carried out direct!y on a samp!e, post extraction or following dilution, for example in distilled deionised water, or other suitable diluent The resulting spectra is examined as charged ions at the Mass/charge range of 2,000 m/z -100,000m/z.

During direct mass spectral analysis) a spectra is generated using a matrix. Suitable matrix compounds include sinapinic acid (SA), ferulic acid (FA) and alpha 4-cyano hydroxycinnamic acid (CHCA). The intensity of the characteristic resolved mass peaks are measured as specific m/z values ranges or a ratio determines the relative abundance of specific m/z peaks.

The present application relates to a method of detecting a protein marker of cancer in a sample obtained from a patient using mass spectrometry.

The data generated from the mass spectrometry is used in the method, and not the inferred mass of the components present in the sample. Therefore the method involves direct mass spectral analysis Direct mass spectral analysis can be carried out by mass spectrometry. Suitable mass spectrometry techniques include fast atom bombardment (FABJ. chemical ionization (Cl), atmospheric-pressure chemical ionization (APCI), electrospray ionization (ESI), matrix-assisted!aser desorption/ionization (MALDI), Quadrupo!e mass analyzers, Triple Quadrapoles, ion traps such as quadrupole ion trap & orbitrap, Fourier transform ion cyclotron resonance mass spectrometry (F'TMS), Ion cyclotron resonance (ICR) or combinations of the above. Preferably, the direct mass spectral analysis can be carried out by MALDI -time of flight mass spectrometry (MALDI-TOF MS).

Cancers include anal, bile duct, bowel, breast, colon and rectal, eye, endocrine, head and neck, melanoma, small bowel, spinal cord, stomach, testicular, thymus, thyroid, endometrial, ovarian, kidney (renal), liver, brain, lung, bone, skin, epithelial cell cancer, small cell (SC) lung prostate, vulval/vaginal, neuroendocrine, bladder, cervical, pancreatic cancer, and blood cancers including leukemia, lymphoma, multiple myeloma, Waldenstrom macroglobulinemia, non-secretory myeloma, smoldering multiple myeloma, MGUS, light-chain myeloma, primary systemic amyloidosis, and light chain-deposition disease.

The protein marker of cancer is a protein, polypeptide or peptide which is expressed at elevated amount by a tumour cell and is indicative of the presence of a tumour. The terms "protein", polypeptide" and "peptide" are used interchangeably herein, and refer to a polymer of amino acids. A peptide is a polymer of at least S amino acids, preferably at least 10, 20, 30, 50 or more amino acids. The protein or peptide marker may be an intact protein, subunit of a protein, variant forms of the protein, or a truncation thereof, such as a peptide fragment. Variant forms include modified proteins and well as afternate expression products formed by differential splicing of the mRNA. Modifications include glycosylation including hyperglycosylation, lipid attachment, hydroxylation, sulfation, y-carboxylation of glutamic acid residues, and ADP-ribosylation. The proteins may also include any post-translation modifications normally carried out in the cell. Truncations refer to the deletion of one or more amino acids at one or both ends of the protein. The proteins may also be modified by the insertion or deletion of one or more amino acids within the sequence as compared to the normal wild type protein produced by the cell. The proteins may be linear or branched.

Preferably the protein or peptide marker is human chorionic gonadotrophin, hCG.

hCG' as used herein refers to hCG beta subunit, hCG whole protein, hCG alpha subunit, splice variants of hCG, proteins formed by the expression of any of the hCG genes, including CGB1 and CGB2, and glycosylated forms thereof. Preferably the cancer detected and/or monitored utilizing hCG proteins is selected from epithelial, small cell [SC) lung, renal, prostate, vulval/vaginal, and neuroendocrine, bladder, S cervical, and pancreatic cancer.

Alternatively the protein or peptide marker can be a paraprotein such as a Bence Jones protein (BJP) i.e. kappa or lambda free light chains, or fragments thereof Preferably the ratio of lambda to kappa free light chains is determined. Preferably the cancer detected and/or monitored utilizing these proteins is selected from multiple myeloma, Waldenstrom macroglobulinemia, non-secretory myeloma, smoldering multiple myeloma, MGUS, light-chain myeloma, primary systemic amyloidosis, and light chain-deposition disease.

The sample obtained from the patient is a bodily fluid sample. Bodily fluids include cerebrospinal fluid, seminal fluids, vaginal fluids, interstitial fluids, tissue aspirates, saliva, urine, blood and serum. The sample may be a spot card sample wherein the sample is applied to filter paper or other capture material, allowed to dry and stored for future analysis. The blood sample can be a whole blood sample collected using conventional phlebotomy methods. For example, the sample can be obtained through venupuncture or as a pin prick sample, such as a finger-stick or heel prick. The blood sample may be a dried blood spot captured on filter paper or other suitable blood spot capture material. Preferably the sample is a urine sample or a serum sample.

The sample may be a neat sample. Alternatively, the sample may be diluted or processed (concentrated, filtered, etc.).

Preferably the sample is diluted or extracted. The sample may be diluted 1/2 (i.e. one part sample in 1 part diluent), 1/5, 1/10, 1/100, 1/200, 1/500, 1/1000, 1/2500 or more. Most preferably the sample is diluted 1/1000 i.e. one part sample in 1000 parts diluent. Preferably the diluent is water or 0.1% trifluoroacetic acid (TFA) in distilled deionised H20, more preferably distilled deionized water.

If the sample is stored on a spot card or blood spot capture materiaL it can be reconstituted using a suitable buffer, or a diluent. Preferably the diluent is water or 0.1% trifluoroacetic acid [TFA) in distilled deionised H20, more preferably distilled deionized water.

Preferably the sample is not processed prior to dilution. Such processing includes concentrating the proteins of interest e.g. hCG; isolating hCG by for example HPLC or treatment with a chemical agent to disrupt or break intramolecular bonds. In particular, the sample is preferably not treated with a reducing agenL More preferably the sample is not treated with dithiothrietol (DTT).

During direct mass spectral analysis, a spectra is generated using a matrix. Suitable matrix compounds include sinapinic acid, ferulic acid [FA] and alpha 4-cyano hydroxycinnamic acid (CHCA). The intensity of the characteristic resolved mass peaks are measured as specific m/z values ranges or a ratio determines the relative abundance of specific m/z peaks. The spectra is analysed over the range of 2000 m/z to 100,000 m/z. Preferred ranges include 2,000 m/z -14,000 m/z; 20,000 m/z - 50,000 m/z; 13,000 m/z -14,000 m/z; 23,000 m/z -25,000 m/z; 40,000 m/z - 44,000 m/z; 25,000 m/z -28,000 m/z; and 40,000 m/z -46,000 m/z.

Clusters of peaks are usually seen in urine at: 2,000 m/z to 6,000m/z -predominately, but not exclusively, arising from metabolites of trypsin inhibitors.

6,000 m/z to 14,000m/z -predominately, but not exclusively, arising from the beta core metabolite of human chorionic gonadotropin.

14,000 m/z to 100,000 m/z predominately, but not exclusively, molecular variants of hCG [36,000 m/z to 40,000 m/z); the alpha subunit of hCG (13000 m/z to 14000m/zJ; the beta subunit of hCG [23,000 m/z to 25000 m/zJ and also hCGbeta-beta dimers (40000 m/z to 44000m/z); Immunoglobulin Kappa produces peaks in the range about 25000 m/z to 28000 m/z and immunoglobulin Lambda dimeric chains produces peaks in the range about 40000 m/z to 46000m/z Methods of generating mass spectra, such as mass spectrometry, are commonly not quantitative techniques. For example the Y axis in these spectra is an indicator of "relative strength" of mass peak within the spectra, but not between mass peaks in one sample versus another samp'e. In order to overcome this, normalisation needs to render Y axis value comparable between sample spectra. Thus the spectra obtained from the direct mass spectral analysis is preferably normalised. The spectra is subjected to data processing which results in a normalised statistically determined index of relative proportion of mass spectra. This converts the qualitative mass spectra into a quantitative value. Normalization is the process of producing a data structure to reduce repetition and inconsistencies of data. Several normalisation techniques are possible. Typical normalisation methods include percentage of total area at a given point, Square difference and ratio of differences. The percentage difference is calculated as Percentage difference=[Y1-Yref/ Y ref X 100%) Wherein Y ref is the minimum Y value of the spectra, and Yl is Y value for each point.

The square difference is calculated as Square Difference= [Yl -Y ret]2 The ratio difference is calculated as Ratio Difference= [Ratio 1-Ratio 2) Thus the data from the mass spectra is manipulated in order to provide a quantitative measure of the qualitative change shown on the spectra.

Preferably, the spectral model is created by a method of data processing which results in a normalised statistically determined index of relative proportion of mass

B

spectra within a set range. This renders all spectra comparable such that the median and centile variability at any given mass value can be modelled.

A normalised statistically determined index of relative proportion of mass spectra within a given range can be calculated from using the total area under the curve of the mass spectra in a defined area of interest e.g. 2000 m/z -6000m/z; 6000 m/z - 14,000m/z; 25,000 m/z -46,000 m/z and 14,000 m/z to 100,000/mz. This can then be used to calculate the relative intensity of mass regions that alter in samples from patients with particular disorders.

The area under the curve of mass spectra is calculated by dividing the mass spectra into a plurality of bins of a given number of m/z. As used herein "Bin" has its usual statistical meaning) for example, of being one of a series of ranges of numerical value into which data are sorted in statistical analysis. For example the bins can be lOOm/z, SOm/z, 25m/z, lOm/z or Sm/z in size. The smaller the size of the bin used, the more refined the method.

The relative intensity (Y Axis value] can be calculated by the "square of difference" method and therefore a comparable Y value given for every bin. In this method, the minimum Y value of the spectra (Y ret) was subtracted from the Y value at every bin and the difference was squared. The formula used to calculate square of difference =(yl-yref]2 and the calculated square of difference was then named as "relative intensity".

The relative intensity at each mass bin in a sample can be captured using commercially available statistical tests such as MATLAB ®, Stats DirectTM and Origin 31M Preferably, each sample is compared against a reference spectral model. The "reference spectral model" is the expected mass within a set range, determined from statistical analysis of a collection of samples obtained for normal healthy controls. As used herein a "normal healthy" control is a subject who does not have cancer.

Preferably the reference spectral model of expected mass is determined from statistical analysis of a collection of samples at matched age. Any changes, such as a change in mass, an increase or decrease in the relative intensity, or a change in the ratio of relative intensity between two or more peaks in the sample spectra as compared to the reference spectra maybe indicative of cancer. For example, an increase in the relative intensity of a peak associated with a cancer marker may be indicative of cancer, or a tumour being present.

Preferably the range is between about 500 m/z -100,000m/z, for example 1,000 m/z -75,000 m/z, 2,500 m/z -50,000 m/z, 5,000 m/z -25,000m/z, 6,000 m/z -14,000 m/z, or 25,000 m/z -45,000 m/z. Most preferably the range is 6,000 m/z -14,000 m/z. Preferably the spectral model of expected mass in the same range, e.g. between about 500 m/z -100,000m/z, is determined from statistical analysis of a collection of samples at matched age.

Preferably, the spectral model is created by a method of data processing which results in a normalised statistically determined index of relative proportion of mass spectra within a set range. This renders all spectra comparable such that the median and centile variability at any given mass value can be modelled.

Preferably, a parallel "disease" model, as generated above from normalised statistically determined index of relative proportion of mass spectra within a set range is created from samples obtained from a cancer patient The spectra from a sample can then be compared to the disease model. The presence of a peak associated with a protein cancer marker may be indicative of cancer.

Once the spectra has undergone a method of data processing which results in a normalised statistically determined index of relative proportion of mass spectra any significant changes in mass can be attributed to a given disorder or diagnostic utility.

Also described is a method of detecting a protein or peptide marker of cancer comprising a] obtaining a sample from a subject b] subjecting the sample to direct mass spectral analysis using mass spectrometry; and c) comparing the spectra resulting from said analysis to mass spectral spectra obtained from a sample from a normal healthy control to determine whether said spectra from said sample from said subject is indicative of cancer.

Preferably the method provides a prognosis of cancer. The presence of hCG is indicative of an aggressive form of cancer) and poor prognosis e.g. short expected survival time.

The progress of a cancerous state may also be monitored, as increased levels of the protein or peptide cancer marker may be indicative of progression of the disease.

In this specification, the verb "comprise" has its normal dictionary meaning to denote non-exclusive inclusion. That is, use of the word "comprise" (or any of its derivatives] to include one feature or more, does not exclude the possibility of also including further features. The word "preferable" (or any of its derivatives] indicates one feature or more that is preferred but not essential.

All or any of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all or any of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings], may be replaced by alternative features serving the same, equivalent or similar purpose) unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

The application will now be described in the examples below which refer to the following figure: Figure 1 shows the MALDI ToF Mass Spectra indicating mass charge shifts, and peak distributions within the spectra for Control, Normal and Cancer samples. In particular the proportion of dimer to monomer between Normal and Cancer spectra can clearly be seen.

Example

Culture media was taken from confluent 75cm2 culture flasks of bladder cancer cell line SCaBER grown in serum free culture media -PCi -for 72hours. This was subject to direct MALDI ToF mass spectrometry as described above and labeled as in the figure as "cancer".

The figure shows the resultant spectra in the region of 19500 to 40500m/z overlaid with spectra generated culture media "control" (not exposed to cells) and culture media spiked with recombinant beta-subunit hCG (labeled "normal"). The control spectra revealed no clear discernable peaks. The "Normal" spectra (media spiked with recombinant beta-subunit of hCG) showed an abundant broad mass peak centering at approximately 26500 m/z and a minor broad peak centering at 46600m/z, labeled monomer and dimer respectively.

The "cancer" sample revealed a clear but minor broad peak centering at 23500m/z and an abundant broad peak at approximately 47,700 m/z.

Bladder cancer cell line SCaBER is known to express and secrete the free beta-subunit of hCG. This spectra implies it is predominantly as a homo-dimer of approximately 47,700 m/z. Therefore, a change in the ratio of relative intensity between two peaks, such as that for the free beta-subunit of hCG and that for the homo-dimer in the sample spectra, as compared to the reference spectra, may be indicative of a cancer. A shift in the increased prevalence of the homo-dimer as compared to the free beta-subunit can be seen, and used to diagnose cancer.

Claims (10)

1. CLAIMS1. A method of detecting a protein marker of cancer in a sample obtained from a patient using mass spectrometry.
2. 2. The method of claim 1 wherein the sample is selected from urine, blood or serum sample.
3. 3. The method of claim 1 or claim 2 wherein the protein marker is human chorionic gonadotrophin (hCG), Bence Jones proteins, kappa free light chain, or lambda free light chain.
4. 4. The method of any preceding claim wherein the ratio of kappa free light chain to lambda free light chain is measured.
5. S. The method of any preceding claim wherein the sample is not diluted prior to direct mass spectral analysis.
6. 6. A method according to any of the preceding daims, wherein the spectra obtained from the direct mass spectral analysis is normalised.
7. 7. A method according to any of the preceding claims, wherein each sample is compared against a reference spectral model of expected mass between about 500 m/z -100,000 m/z determined from statistical analysis of a collection of samples obtained from normal healthy controls.
8. 8. A method according to any of the preceding claims, wherein each sample is compared against a disease model of expected mass between about 500 m/z - 100,000 m/z determined from statistical analysis of a collection of samples obtained from cancer patients.
9. 9. A method according to any of the preceding claims, wherein the mass spectral analysis carried out by mass spectrometry
10. 1O.A method according to Claim 9, wherein the mass spectrometry is matrix- assisted laser desorption/ ionization time of-flight mass spectrometry (MALD-ToF MS).