The present invention relates to an index for prostate cancer diagnosis and a test method using the same.
Prostate cancer is a cancer specific for men and frequently occurred in the elderly, and about 90% or more of the patients are 60 years or older. With the increase of the elderly, the number of the patients is increasing. According to the projected cancer cases by part in male in 2017 in the “Cancer Registry and Statistics” by Cancer Information Service, National Cancer Center, the number of patients newly suffering from prostate cancer is 86,100, ranking third in the number of cases following stomach and lung cancers, and is predicted to increase further with aging in the future. Prostate cancer progresses relatively slowly in many cases, and therapies such as radiation therapy and endocrine (hormone) therapy, in addition to surgery, can be taken as effective measures. Thus, prostate cancer is treatable if detected early.
Prostate cancer has been diagnosed by measuring prostate specific antigen (PSA) in blood. PSA screening is a blood test, thus it is minimally invasive, and has high sensitivity. PSA screening has thus been incorporated and performed in medical checkup as a test for prostate cancer.
However, PSA is produced in prostate epithelium, and is overproduced in not only prostate cancer, but also other prostate diseases such as prostatic hypertrophy and prostatitis. Thus, false positives often occur in patient screenings with PSA, which is considered a problem. PSA has a cutoff value of 4 ng/ml. When PSA value in blood is greater than or equal to the value, prostate cancer is suspected, and diagnosis by transrectal prostate palpation, transrectal echo, or transperineal prostate needle biopsy becomes necessary. For the range referred to as a “gray zone” with a PSA value of 4 to 10 ng/ml, there is a result that about 70% of the subjects do not suffer from cancer, and 50% of the subjects do not suffer from cancer even their PSA values are 10 to 20 ng/ml (Non Patent Literature 1).
When the diagnosis is determined by transperineal prostate needle biopsy, it is necessary to perform 10 to 12 tissue samplings in the first biopsy after hospitalization and anesthesia, and it is accompanied with severe pain. Partly because of the high false positive rate, many patients with PSA values in the range of the gray zone do not have prostate biopsies even when they are recommended for secondary examination in medical checkup, so they often miss opportunities for early detection. In fact, there are statistics showing that only about 30 to 50% of those who were determined to be PSA positive at medical checkup centers receive prostate biopsy.
Conventionally, methods that increase specificity of PSA test, distinguish prostate cancer from prostatic hypertrophy and specifically detect prostate cancer have been proposed (Non Patent Literatures 2 to 4, Patent Literatures 1 to 8). As PSA tests, Food and Drug Administration (FDA) has already approved prostate health index (PHI) (Non Patent Literature 2) and prostate cancer antigen 3 (PCA3) examination (Non Patent Literatures 3, 4). Patent Literatures 1 to 5 describe methods of contacting PSA with lectin that recognizes specific binding sialic acids, and performing examination using the binding properties with lectin. Patent Literatures 6 to 8 disclose methods of analyzing sugar chain structures by mass spectrometry to detect prostate cancer.
All of the examination methods disclosed in the above literatures are, however, problematic in specificity or sensitivity, and any of them failed to specifically identify prostate cancer. PHI is high in sensitivity of 85%, but low in specificity of 45%, and cannot specifically identify prostate cancer (Non Patent Literature 5). PCA3 is an excellent examination method in that it is not affected by diseases other than cancer such as prostatic hypertrophy and prostatitis, but the results of four clinical tests showed that it is low in sensitivity, which is 53 to 84%, and insufficient to detect prostate cancer in the patients having PSA values of the gray zone (Non Patent Literature 6).
In the methods described in Patent Literatures 1 to 5, prostate cancer is diagnosed by analyzing saccharides modifying PSA by the binding properties to lectin. However, A problem is that lectin has low affinity compared to antibodies, leading to low sensitivity. Another problem is that lectin does not detect only saccharides modifying target PSA, and also binds to saccharides modifying other proteins. Thus, the methods using lectin cannot completely eliminate false positives.
Patent Literatures 6 to 8 disclose methods of identifying prostate cancer and prostatic hypertrophy by mass spectrometry. Patent Literature 6 describes a method for detecting prostate cancer by a ratio of fucose-bound sugar chains to fucose-unbound sugar chains, Patent Literature 7 describes that by an amount of sugar chains having LacdiNAc, and Patent Literature 8 describes that by the presence or absence of three or more sugar chains having LacdiNAc. However, none of them were enough to eliminate false positives because they are low in specificity.
PSA has been modified by sugar chains, and it has been reported that the type of modifying saccharides is correlates with prostate disease. However, accurate distinguishing between prostate cancer and prostatic hypertrophy has not achieved yet. An object of the present invention is to provide a method for accurately detecting prostate cancer even in a range of the gray zone in which a slight increase in PSA is observed, and to provide an index for identifying prostate cancer.
The present invention relates to the following method for testing prostate cancer and index for detecting prostate cancer. Furthermore, it relates to a marker for evaluating grade (malignancy).
(1) A test method of prostate cancer, comprising: analyzing a sugar chain modifying PSA in a specimen; and analyzing a multisialylated LacdiNAc structure.
(2) The test method of prostate cancer according to (1), comprising:
analyzing a sugar chain modifying PSA in a specimen; and analyzing relative abundance(s) of Glycan ID: 7512, Glycan ID: 7603, Glycan ID: 7612, and/or Glycan ID: 7613.
(3) The test method of prostate cancer according to (2), wherein the relative abundances of Glycan ID: 7512 and Glycan ID: 7603 are analyzed by logistic analysis.
(4) The test method of prostate cancer according to (2), comprising:
substituting the relative abundances of Glycan ID: 7512 and Glycan ID: 7603 into the following formula 1:
[Expression 1]
Loge(p/(1−p))=−5.85+4.72x1+0.80x2 (Formula 1)
wherein p represents a value of PSA G-index; x1 represents a relative abundance of Glycan ID: 7512, and x2 represents a relative abundance of Glycan ID: 7603.
(5) The test method of prostate cancer according to any one of (1) to (4), wherein the specimen is blood, serum, plasma, or urine.
(6) The test method of prostate cancer according to any one of (1) to (5), wherein the specimen is a specimen obtained from a patient having a PSA value of 4 to 10 ng/ml.
(7) The test method of prostate cancer according to any one of (1) to (6), wherein the sugar chain is analyzed using an oxonium monitoring method.
(8) An index for identifying prostate cancer, which is a PSA G-index determined by the following formula 1:
[Expression 2]
log(p/(1−p))=−5.85+4.72x1+0.80x2 (Formula 1)
wherein p represents a value of PSA G-index; x1 represents a relative abundance of Glycan ID: 7512, and x2 represents a relative abundance of Glycan ID: 7603.
(9) A method for testing a grade of prostate cancer, comprising:
analyzing a sugar chain modifying PSA in a specimen; and analyzing relative abundance(s) of at least one or more of Glycan ID: 7512, Glycan ID: 7603, Glycan ID: 3401, and/or Glycan ID: 5602.
(10) The test method according to (9), wherein the specimen is blood, serum, plasma, or urine.
(11) The test method according to (9) or (10), wherein the analysis uses an oxonium monitoring method.
(12) The test method of prostate cancer according to (1), (2), (5) or (6), wherein the analysis uses an antibody or lectin.
(13) The test method of prostate cancer according to (12), wherein the method uses an antibody or lectin that specifically recognizes a saccharide having a multisialylated LacdiNAc structure.
(14) A test kit of prostate cancer, comprising an antibody or lectin that specifically recognizes a saccharide having a multisialylated LacdiNAc structure.
(15) The test kit of prostate cancer according to (14), wherein the saccharide having a multisialylated LacdiNAc structure is Glycan ID: 7512, Glycan ID: 7603, Glycan ID: 3401, or Glycan ID: 5602.
The analysis by the present inventors has revealed that there is a significant increase in certain sugar chains having LacdiNAc (GalNAcβ1-4GlcNAc, N-acetylgalactosamine-N-acetylglucosamine) structure in patients suffering from prostate cancer. The analysis has been performed by mass spectrometry, but any method can be used as long as it is possible to recognize or analyze the multisialylated LacdiNAc structure or the specific sugar chain indicated by Glycan ID below.
The analytical method by mass spectrometry is a method that can be examined with great sensitivity as shown below, but not limiting to mass spectrometry, the analysis can be performed using anything that recognizes a particular sugar chain, such as an antibody, lectin, or aptamer which recognizes a multisialylated LacdiNAc structure. When the detection is performed with an antibody, the detection may be performed by any method used in the art, such as ELISA or SPR. When the detection is performed with a lectin, the detection may be performed by any method such as lectin blot or capillary electrophoresis. As shown in the Examples below, lectins such as WFA or MAM does not specifically recognize a multisialylated LacdiNAc structure. However, a system capable of specifically detecting a multisialylated LacdiNAc structure can be created by using several lectins in combination.
The present invention can be also carried out not only by analysis of relative abundances of Glycan ID: 7512 and Glycan ID: 7603, but also by profile recognition on profiling data of saccharides containing a multisialylated LacdiNAc structure. Specifically, profiles of multiple sugar chains obtained from patients having diagnosis determined prostate cancer are machine-learned by a computer as training data, and used it as a diagnostic support system. Then, during the test, by inputting the profile obtained from the subject as it is, prostate cancer candidate data may be detected by pattern recognition.
As shown in the Examples below, as a result of analyzing sugar chain structures modifying PSA, it has been revealed that prostate cancer can be detected with high accuracy by using PSA G-index defined below. Examination with PSA G-index on patients of a gray zone in conventional PSA tests as a secondary screening makes it possible to detect patients suffering from prostate cancer in a non-invasive manner. Further details are described below with showing data.
First, whether sugar chains modifying PSA are able to be sensitively detected and quantified was confirmed with PSA standard. PSA obtained from human semen was dissolved in 8 M urea, 50 mM HEPES-NaOH, pH 8.0, reduced with 10 mM DTT (GE Healthcare Life Science), alkylated with 25 mM iodoacetamide (Sigma-Aldrich), and then digested with Trypsin/Lys-C mix (Promega Corporation). Glycoproteins were enriched by hydrophilic purification method (Non Patent Literature 7), and dried in vacuo.
The resulting glycoproteins were analyzed by oxonium ion monitoring method (Erexim method, Non Patent Literature 8, Patent Literature 9) with a triple quadrupole mass spectrometer LCMS-8060 (Shimadzu Corporation) coupled with a Prominence nanoflow liquid chromatogram to identify sugar chains.
The results of performing sugar chain structure analysis with 100 ng of PSA standard obtained from human semen are shown in
Oxonium ion monitoring method has been found to be not only a method with which the analysis was completed in a short time of 25 minutes of LS/MS operation time without the need for enzymatic separation of glycans or chemical modification, but also a very sensitive detection method. The saccharide of the highest content was 4[HexNAc]5[Hex]1[Fuc]2[Neu5Ac] (Glycan ID: 4512, 44.5±0.9%), and the saccharide of the lowest content was 6[Hex]7[NAcHex]0[Fuc]0[Neu5Ac] (Glycan ID: 6700, 0.01±0.001%). There has been previously no report of detecting such a very large number of types of saccharides as 67 types of glycans modifying PSA, or detecting a very small amount of saccharide as 0.01%. Thus, it has been shown that oxonium ion monitoring method is a very sensitive method.
Next, sugar chain analysis of PSA was performed using samples obtained from 15 prostate cancer patients and 15 prostatic hypertrophy patients having a PSA value in a gray zone, 4 to 10 ng/ml, to determine an index identifying prostate cancer, as a training set. Table 1 shows patient characteristics in the training set.
Analysis with clinical samples uses serum samples of prostate cancer patients and prostatic hypertrophy patients. It should be noted that final diagnosis is determined from histopathological diagnosis by prostate biopsy. Purification of PSA was performed as follows. To the serum was added 4-fold amount of wash solution (0.1% Tween-20 in PBS). The obtained solution was mixed with Protein G Sepharose to which anti-PSA monoclonal antibodies were immobilized (Fitzgerald), reacted overnight at 4° C., and washed. Then, PSA was eluted with 8 M urea, 50 mM HEPES-NaOH, pH 8.0, and purified. The eluted protein was reduced with 10 mM DTT, alkylated with 25 mM iodoacetamide, and then digested with Trypsin/Lys-C mix. Glycoproteins were enriched by hydrophilic purification method (Non Patent Literature 7), and dried in vacuo. It should be noted that, although serum samples were used here, not only blood derived samples but also urine can be used as samples.
In the same manner as described above, 52 sugar chain structures on PSA could be quantified (
Among the 52 types of sugar chains, sugar chains containing a multisialylated structure, specifically di-/tri-sialylated LacdiNAc (GalNAcβ1-4GlcNAc), were significantly increased in the prostate cancer patient group compared to the prostatic hypertrophy patient group (
Based on these findings, we aimed to establish a novel diagnostic algorithm that complements the specificity of PSA test and can reliably reduce false positive rates. Two sugar chain structures specific for prostate cancer, Glycan ID: 7512 (p=9.91×10−8) and Glycan ID: 7603 (p=1.66×10−5), which showed significant differences between the prostate cancer patient group and the prostatic hypertrophy patient group in the training set, were selected, and the relative abundance thereof were plotted.
Based on these results, a diagnostic model (PSA G-index) based on logistic regression was established. In formula 1, p represents a value of PSA G-index; x1 represents a relative abundance of Glycan ID: 7512; and x2 represents a relative abundance of Glycan ID: 7603.
[Expression 3]
loge(p/(1−p)=−5.85+4.72x1 0.80x2 (Formula 1)
When a cutoff value of PSA G-index was set to 0.5, the sensitivity and specificity of the training set were 93.3% and 100%, respectively (
Although all the saccharides modifying PSA are analyzed and their relative amounts are determined here, analysis may be performed of only a particular saccharide, such as a saccharide having a multisialylated LacdiNAc structure. In addition, among the peptides of PSA, peptides in the region that is not glycosylated may be taken as a reference, and a ratio of PSA modified by a particular saccharide to total PSA may then be determined to use for analysis. Although the relative values of saccharides described above differ depending on the saccharide used as a reference or the amount of PSA, the amounts of the saccharides are significantly different in prostate cancer and other prostate diseases, and thus it is possible to distinguish prostate cancer and other prostate diseases with high sensitivity by logistic analysis.
The PSA G-index was then evaluated using the validation sample set of Table 1 (
Next, whether sugar chain structures on PSA in serum reflect the grade (malignancy) of prostate cancer was analyzed. Using serum from 77 patients of different grades shown in Table 2, sugar chain analysis by oxonium ion monitoring method was performed.
As a result, the frequency of Glycan ID: 7512 (
Furthermore, Glycan ID: 3401 (
To identify the cause of the increase of multisialylated LacdiNAc structure in serum PSA, lectin histochemical staining was performed using a tissue microarray (US Biomax) containing 9, 31, and 111 prostate tissues of normal, prostatic hypertrophy patients, and prostate cancer patients, respectively. Histochemical staining was performed using WFA, a lectin that detects a LacdiNAc structure, or MAM, a lectin that detects a Siaa2-3Gal structure. Biotinylated lectins were used for both WFA and MAM, and the analysis was performed using a Vectastain Elite ABC kit (Vector Laboratories). As a result, cytoplasmic and protoplasmic membranes of cancer cells were significantly stained (
WFA and MAM lectins specifically recognize GalNAc structures containing a LacdiNAc structure and a Neu5Acα2-3Gal structure, respectively. The above results indicate that prostate cancer tissue tends to strongly express glycoproteins containing LacdiNAc and Neu5Ac structures. These histological findings were consistent with the results of PSA sugar chain structure analysis by mass spectrometry. Accordingly, it is possible to determine prostate cancer, and even the stage of prostate cancer, by analyzing sugar chains of PSA in blood without performing tissue biopsy.
Test using the diagnostic marker, PSA G-index, shown in the Examples, performed as a secondary screening of patients diagnosed as having a suspected prostate cancer from the PSA value, can identify prostate cancer with good specificity. The test can identify even prostate cancer at an early stage with good specificity, which makes it possible to lead to early treatment.
Number | Date | Country | Kind |
---|---|---|---|
2018-131774 | Jul 2018 | JP | national |
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/JP2019/026858 | 7/5/2019 | WO | 00 |