RU2504785C1 - Diagnostic technique for breast cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a new diagnostic technique based on the detection of protein molecules being antibodies to a specific and sensitive combination of tumour associated glycans with underlying the patient's individual characteristics. The given invention can find application in immunology and medicine for the study and diagnosis of breast cancer. The diagnostic technique for breast cancer involves detecting the human blood antibodies to the following glycans: Manβ1-4GlcNAcβ, Galβ1-4G1cNAcβ1-6(Galβ1-3)GalNAcα, HOCH2(HOCH)4CH2NH2, GlcAβ, Galα, 6-O-Su-GlcNAcβ, GalNAcβ1-3Galβ, 6-O-Su-Galβ1-4(6-O-Su)Glcβ, GlcAβ1-SGa1β, Neu5Acα2-3Gaiβ1-4Glcβ, GlcNAcβ1-3Galβ1-3GalNAcα, Neu5Acα2-3Galβ1-3GalNAcα, Neu5Acα2-3Galβ1-3(Fucα1l-4)GlcNAcβ, Gaiβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Gaiβ1-4GlcNAcβ, Neu5Acα2-6(Galβ1-3)GlcNAcβ1-3Galβ1-4Glcβ with using a microchip with applied glycans; if the blood antibody level turns out to be higher than a threshold value to the above glycans simultaneously, breast cancer is diagnosed.

EFFECT: method enables improving the diagnostic sensitivity and specificity in breast cancer.

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Description

Technical field

The invention relates to medicine and immunology, and is a fundamentally new diagnostic method based on the determination of protein molecules - antibodies - to a specific and sensitive combination of tumor-associated glycans (signature) against the background of the individual characteristics of a particular patient. This invention may find application in immunology and medicine for the study and diagnosis of breast cancer.

State of the art

Breast cancer is one of the most significant diseases in women, characterized by a high incidence rate and is a leading cancer pathology in women older than 40 years. Early detection of breast cancer is a socially significant task of modern health care.

To date, the diagnosis of breast cancer is a comprehensive examination of the patient, which includes an individual medical examination, as well as instrumental research methods: mammography, ultrasound examination, biopsy. Unfortunately, in most cases, the disease is diagnosed at a rather late stage.

An important component of the diagnosis is the determination of tumor-associated antigens in the patient's blood serum. Numerous studies have revealed many antigens associated with human breast cancer and characterizing the biological characteristics of the tumor - proliferative activity, hormonal sensitivity, the severity of metastasis. However, no tumor specific marker has yet been found.

For example, CA15-3, is widely used to monitor the progress of the disease and the occurrence of relapses, which has established itself as a highly specific marker of distant metastases with a specificity of 96% (IP Tomlinson, A. Whyman, JA Barrett, JK Kremer. Tumour marker CA15-3: possible uses in the routine management of breast cancer Eur J Cancer. 1995 Jun; 31A (6): 899-902). However, it does not have sufficient sensitivity and specificity for diagnosis at an early stage of cancer (not more than 60% (M.L. Alekseeva E.V. Gusarova S.M. Mullabaeva T.S. Ponkratova. Oncomarkers, their characteristics and some aspects of clinical diagnostic use, Proprod 3, 65-79 (2005)). The group of oncofetal antigens - cancer embryonic antigen (CEA) and tissue polypeptide antigen (TPA) - are also not specific for tumor cells, their determination is used only to assess prognosis and early diagnosis of distant m The combined determination of the levels of CEA, TPA and CA15-3 in the blood of patients with breast cancer increases the sensitivity and specificity of evaluating the effectiveness of treatment and prognosis up to 90% (A. Nicolini, C. Colombini, L. Luciani, A. Carpi, L. Giuliani Evaluation of serum CA15-3 determination with CEA and TPA in the postoperative follow-up of breast cancer patients. Br. J. Cancer. 64 (1), 154-158 (1991)), but not diagnosis.

In clinical practice, cases are often observed when the level of tumor markers was within normal limits, despite the presence of a malignant tumor in the patient. This situation is not uncommon in the initial stages of tumor development. In this regard, tumor markers are usually used only for prophylactic studies of patients at risk of developing tumors, as well as for monitoring established treatment.

Thanks to the work of Hakomori (S. Hakomori Tumor-associated carbohydrate antigens. Annu Rev Immunol. 2, 103-126 (1984)), oncologists are of practical interest in the class of glycan antigens known as tumor-associated carbohydrate antigens (TACAs) present on cell surfaces , tissue components and in the bloodstream. TACAs are detected on all malignant neoplasms, being the result of oncological transformation and playing the role of key molecules in metastasis. Anti-glycan antibodies play a significant role in oncotransformation (E.R. Smorodin, O.A. Kurtenkov, BL Sergeyev, KE Kodar, VI Chuzmarov, VP Afanasyev. Postoperative change of anti-Thomsen-Friedenreich and Tn IgG level: the follow -up study of gastrointestinal cancer patients. World J. Gastroenterol. 14 (27), 4352-4358 (2008), Schwartz-Albiez R. Naturally occurring antibodies directed against carbohydrate tumor antigens. Adv. Exp. Med. Biol. 750, 27 -43 (2012)). The main tool for studying anti-glycan antibodies is the glycochip or glycan erray (O. Blixt, et al .. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins Proc. Natl. Acad. Sci. USA. 101 (49), 17033 -17038 (2004)). Using this method, antibody profiles of healthy donors were studied (M.E. Huflejt, M. Vuskovic, D. Vasiliu, H. Xu, P. Obukhova, N. Shilova, A. Tuzikov, O. Galanina, B. Arun, K Lu, NV Bovin. Anti-carbohydrate antibodies of normal sera: findings, surprises and challenges. Mol. Immunol. 46 (15), 3037-3049 (2009)), as well as cancer patients, for example, Hodgkin's lymphoma (C. N. Lawrie, T. Marafloti, CS Hatton, S. Dimhofer, G. Roncador, P. Went, A. Tzankov, SA Pileri, K. Pulford, AH Banham. Cancer-associated carbohydrate identification in Hodgkin's lymphoma by carbohydrate array profiling. Int . J. Cancer. 118, 3161-3166 (2006)) and ovarian cancer (F. Jacob, DR Goldstein, NV Bovin, T. Pochechueva, M. Spengler, R. Caduff, D. Fink, M. Vuskovic, M. E. Huflejt, V. Heinzelmann-Schwarz. Serum antiglycan antibody detection of nonmucinous ovarian cancers by using a print ed glycan array. Int. J. Cancer. 130 (1), 138-146 (2012)). [one]. The approach described in article [1] is the closest analogue of the present invention.

According to the cited article [1], the search for a new generation of markers of nemucinous ovarian cancer was carried out using glycan erray containing 203 oligosaccharide structures. The study involved 24 healthy donors and 33 patients with ovarian cancer. Through cluster analysis, a pattern of antibodies to specific carbohydrate structures, consisting of 10 glycans, was identified. The found pattern allowed the authors [1] to reliably distinguish between healthy donors and cancer patients (sensitivity and specificity were 79.2 and 84.8%, respectively). However, this approach only slightly improved the characteristics of the classical test system based on the parallel determination of the tumor marker CA125 (enzyme-linked immunosorbent assay). Similar work in the field of breast cancer has not been conducted.

Disclosure of invention

The objective of the invention is to provide a specific and sensitive method for diagnosing breast cancer.

The technical result of the invention is a specific and sensitive combination of tumor-associated glycans.

The technical result is achieved by a method for the diagnosis of breast cancer, which consists in the fact that antibodies directed to the following glycans are detected in human blood: Manβ1-4GlcNAcβ, Galβ1-4GlcNAcβ1-6 (Galβ1-3) GalNAcα, LOSN ₂ (LOSN) ₄ CH ₂ NH ₂ , GlcAβ, Galα, 6-O-Su-GlcNAcβ, GalNAcβ1-3Galβ, 6-O-Su-Galβ1-4 (6-O-Su) Glcβ, GlcAβ1-3Galβ, Neu5Acα2-3Galβ1-4Glcβ, GlcNAcβ1-3Galβ1- 3GalNAcα, Neu5Acα2-3Galβ1-3GalNAcα, Neu5Acα2-3Galβ1-3 (Fucα1-4) GlcNAcβ, Galβ1-4GlcNAcβ1-3 (GlcNAcβ1-6) Galβ1-4GlcNAcβ, Neu5Acα2-6 (Galβc1g-3-3) with the indicated glycans deposited on it and, by the presence in the blood of an antibody level above the threshold, all are indicated simultaneously th glycans, is diagnosed with breast cancer.

The present invention provides a method for the diagnosis of breast cancer, based on the detection of carbohydrate-binding protein molecules - antibodies to a combination of carbohydrate ligands (glycans) using a microchip. These antibodies are detected in the studied serum samples simultaneously using secondary antibodies containing a fluorescent label, or any other imaging system. Ligands are oligosaccharides and / or polysaccharides and / or peptides and / or glycopeptides and / or biotin. One chip contains many individual ligands. Some of them serve as positive and negative controls for the operation of the microchip.

The effectiveness of using a combination of markers was shown in (R. Radpour, Z. Barekati, C. Kohler, Q. Lv, N. Bürki, C. Diesch, J. Bitzer, H. Zheng, S. Schmid, XY Zhong. Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer. PLoS One. 6 (1), el 6080. (2011)) when developing an epigenetic test for breast cancer in the blood of patients. The test is based on recording changes in the methylation of areas of tumor suppressor genes for breast cancer. The authors showed that the sensitivity and specificity of diagnosis increases significantly, if you use a combination of 8 genes instead of one, you can increase the sensitivity and specificity to a level of more than 90%. However, as noted by the authors (Van De Voorde L, Speeckaert R, Van Gestel D, Bracke M, De Neve W, Delanghe J, Speeckaert M. DNA methylation-based biomarkers in serum of patients with breast cancer. Mutat. Res. 751 ( 2), 304-325 (2012)), it should be cautious about epigenetic markers, such as DNA methylation, since they are quite strongly influenced by normal physiological processes, such as, for example, aging.

In the present invention, the same principle of combining several markers into a diagnostic combination of ligands (signature) is applied for the diagnosis of breast cancer. Compared with the above approach [1], such a combination has fundamentally improved the sensitivity and specificity of diagnostics, since the existing biomarkers are recommended for use only to assess the effectiveness of treatment and preventive studies, but not for diagnosis.

Further, the invention is illustrated by reference to the drawings, as well as examples that are provided solely to illustrate and explain the essence of the claimed invention, but which are not intended to limit the scope of claims.

List of abbreviations used

ROC analysis - analysis of the interaction of sensitivity and representativeness

AUC - area under the ROC curve, an indicator of the accuracy of the diagnostic test;

CMG - diglycyl-N-carboxymethylglycine;

BovSlOG - Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-NH ₂ ;

IFB - 0.1 M isotonic phosphate buffer (0.01 M Na ₂ HPO ₄ , 0.01 M NaH ₂ PO ₄ , 0.138 M NaCl and 0.0027 M KCl, pH 7.4).

IFB - 1%, 0.1%, 0.05% - IFB containing 1, 0.1 and and 0.05% Tween20, respectively;

BSA - bovine serum albumin;

Ig (G + M + A) - immunoglobulins of classes G, M, A.

Figure 1 shows the results of a ROC analysis of the effectiveness of the diagnosis of breast cancer based on the use of the signature of 15 glycans. Next to the curve is the value of the threshold value with the corresponding indicators of specificity and sensitivity, as well as the value of AUC with confidence intervals.

The implementation of the invention

The present invention provides a method for diagnosing breast cancer based on the determination of protein molecules - antibodies - to a specific and sensitive combination of tumor-associated glycans (signature) against the background of the individual characteristics of a particular patient.

The method for the diagnosis of breast cancer according to this invention consists in the simultaneous detection of carbohydrate-binding protein molecules - antibodies D in the test samples of blood serum to a signature of carbohydrate ligands specific for breast cancer using microarrays. Antibodies are detected simultaneously using secondary antibodies containing a fluorescent label, or any other antibody imaging system.

The microchip ligands used are amino-spacer oligosaccharides, as well as polysaccharides (the presence of a spacer in the latter is not necessary), as well as non-carbohydrate substances such as biotin and peptides. Ligands not included in the signature are on the chip as positive and negative controls.

The selection of glycans to be included in the signature was based on the non-parametric Wilcoxon-Mann-Whitney (WMW) test to discriminate between groups of healthy donors and patients with breast cancer. Based on this test, glycans were ranked and then sequentially summed into the signature; in this case, each subsequent addition of glycan should not impair the accuracy of the test.

When using only one glycan as a marker - Manβ1-4GlcNAcβ - the test accuracy was only average (AUC = 0.693), when combining two glycans, the test accuracy increased to 0.737, which is already a good indicator. When using a signature of 15 glycans, the AUC value was 0.888, which corresponds to a very good test accuracy.

Thus, the proposed method allows to diagnose breast cancer using a signature of 15 glycans with an accuracy of 88.8% (confidence interval 82.4-95.2%) and sensitivity and specificity of 80 and 88%, respectively. The corresponding results of the ROC analysis are shown in figure 1. The recorded value - “signature response” - is calculated as the sum of the signals from the developed biochip from the 15 glycans listed below (the summation sign is shown after each glycan) converted as described in Example: 1. Manβ1-4GlcNAcβ (+), 2. Galβ1-4GlcNAcβ1- 6 (Galβ1-3) GalNAcα (+), 3. LOCH ₂ (LOC) ₄ CH ₂ NH ₂ (+), 4. GlcAβ (-), 5. Galα (+), 6. 6-O-Su-GlcNAcβ (-), 7. GalNAcβ1-3Galβ (-), 8. 6-O-Su-Galβ1-4 (6-O-Su) Glcβ (+), 9. GlcAβ1-3Galβ (+), 10. Neu5Acα2-3Galβ1 -4Glcβ (+), 11. GlcNAcβ1-3Galβ1-3GalNAcα (-), 12. Neu5Acα2-3Galβ1-3GalNAcα (-), 13. Neu5Acα2-3Galβ1-3 (Fucα1-4) GlcNAcβ (+), 14. Galβ1-4GlcNAcβ1 -3 (GlcNAcβ1-6) Galβ1-4GlcNAcβ (+), 15. Neu5Acα2-6 (Galβ1-3) GlcNAcβ1-3Galβ1-4Glcβ (+).

The effectiveness of the found signatures was tested on a group of 50 donors (“No disease”) and 60 patients with breast cancer (“There is a disease”). For all patients, the diagnosis was previously confirmed by surgical methods. The results are shown in the Table. As can be seen from the Table, some of the results are false positive (donors for whom the test showed a positive result), and some are false negative (patients for whom the test showed a negative result), which, however, is not critical and corresponds to the very good accuracy of the developed diagnostic test .

Table Validated Diagnostic Test Disease is present absent Breast Cancer Diagnosis The test is positive (there is cancer) 49 9 The test is negative (no cancer) eleven 41 Total 60 fifty

The following example is not intended to limit the claims, but solely to illustrate individual embodiments of the present invention.

Example. The use of a microchip for the diagnosis of breast cancer.

The following glycans and other ligands were used to construct the chip.

1. Spacer-bonded oligosaccharides: Manβ1-4GlcNAcβ, Galβ1-4GlcNAcβ1-6 (Galβ1-3) GalNAcα, НОСН ₂ (НОСН) ₄ CH ₂ NH ₂ , GlcAβ, Galα, 6-O-Su-GlcNAcβ, GalNAcβ1-3Galβ, 6- O-Su-Galβ1-4 (6-O-Su) Glcβ, GlcAβ1-3Galβ, Neu5Acα2-3Galβ1-4Glcβ, GlcNAcβ1-3Galβ1-3GalNAcα, Neu5Acα2-3Galβ1-3GalNAcα, Neu5Acα2-3Galβ1-3 Galβ1-4GlcNAcβ1-3 (GlcNAcβ1-6) Galβ1-4GlcNAcβ, Neu5Acα2-6 (Galβ1-3) GlcNAcβ1-3Galβ1-4Glcβ.

2. The glycopeptide GlcNAcβ1- [HOOC (CH ₃ ) CH] -3-O-GlcNAcβ-L-alanyl-D-isoglutaminyl-L-lysine.

3. Polysaccharides: Escherichia coli 052, 058, 073.

4. Biotin in the form of a derivative with a 6-aminohexane spacer.

5. Peptides BovS10G and CMG.

Spacer-coated glycans or other ligands were dissolved in 300 mM phosphate buffer, pH 8.0, containing 0.005% Tween 20, at a concentration of 50 μM and applied to the surface of N-hydroxysuccinimide-activated H slides (Schott Nextenon, Germany) using a contact device OmniGrid II prints (Digilab, USA) as described in (Blixt, O., et al., Printed covalent glycan array for ligand profiling of diverse glycan binding proteins, Proc. Natl. Acad. Sci. USA, 2004, v. 101 (49), p. 17033-17038). After sequential application of all ligands, the chip was kept for 1 h at room temperature and relative humidity of 80%, after which the remaining unreacted activated surface groups were blocked with a 100 mM boric acid solution (Helikon, Russia) containing 25 mM ethanolamine (Merck, USA) and 0.2% Tween-20, pH 8.5. Blocked microchips were washed with double-distilled water and dried by centrifugation.

The study involved 50 healthy donors and 60 patients with surgically confirmed breast cancer.

Analysis of blood serum of donors and patients was carried out as follows:

The chips were kept for 15 min in an IFB containing 0.1% Tween-20 (IFB 0.1%), then 200 μl of the test blood serum of the donor or patient was diluted 15 times with IFB 1% containing 1 % BSA and kept on a shaker at a relative humidity of 80% for 1 h at 37 ° C. Then, the chips were washed with IFB-0.05% and 200 μl of a solution of biotinylated goat antibodies against human immunoglobulins Ig (G + M + A) (ThermoSci, USA) was added, 1: 200 dilution in IFB - 0.1% containing 1% BSA . After incubation on a shaker at a relative humidity of 80% for 1 h at 37 ° C, the chip was washed with an IFB of 0.05% and a solution of streptavidin labeled with fluorescent dye Alexa555 was added, a dilution of 1: 1000 in IFB was 0.1%. After the chips were incubated for 40 min at room temperature in the absence of light, they were washed first with IFB — 0.05%, then with bidistilled water and dried by centrifugation. Images were obtained using a ProScanArray Gx confocal scanner (PerkinElmer, USA) with a resolution of 5 μm (laser power 90%). The resulting files were viewed and processed using ScanArray Express 3.0 software and Microsoft Excel.

To calculate the response of the signal, the signals received from the chips — relative fluorescent units — were first normalized using the formulas below for linear normalization:

и x_ij - необработанные и нормализованные значения интенсивностей сигнала для пациента i и гликана j, а l_i и s_i - параметры положения и масштабирования.Where

and x _ij are the raw and normalized values of the signal intensities for patient i and glycan j, and l _i and s _i are the position and scaling parameters.

The parameters l _i and s _{i were} determined for each patient (donor) independently:

where J is the set of column indices that correspond to glycans, as well as control compounds.

After that, the data was subjected to the Box-Cox normalizing transformation procedure (Box GEP, Cox DR, An analysis of transformations. JR Stat Soc, 1964. In 26: p.211-252) taking into account negative data values (according to the recommendations of the authors (John JA, Draper NR, An alternative family of transformations. Appl Stat, 1980.29: p. 190-197)):

where λ is the degree of the transformation parameter equal to 0.2.

The signature response was calculated as the sum of the converted signals from the 15 glycans listed below (the summation sign is shown after each glycan): 11. Manβ1-4GlcNAcβ (+), 2. Galβ1-4GlcNAcβ1-6 (Galβ1-3) GalNAcα (+), 3. LOSN ₂ (LOSN) ₄ CH ₂ NH ₂ (+), 4. GlcAβ (-), 5. Galα (+), 6. 6-O-Su-GlcNAcβ (-), 7. GalNAcβ1-3Galβ (-), 8 .6-O-Su-Galβ1-4 (6-O-Su) Glcβ (+), 9. GlcAβ1-3Galβ (+), 10. Neu5Acα2-3Galβ1-4Glcβ (+), 11. GlcNAcβ1-3Galβ1-3GalNAcα ( -), 12. Neu5Acα2-3Galβ1-3GalNAcα (-), 13. Neu5Acα2-3Galβ1-3 (Fucα1-4) GlcNAcβ (+), 14. Galβ1-4GlcNAcβ1-3 (GlcNAcβ1-6) Galβ1-4GlcNAcβ (+), 15. Neu5Acα2-6 (Galβ1-3) GlcNAcβ1-3Galβ1-4Glcβ (+).

If the obtained value was higher than the threshold value of -2.3, then the result of the analysis was considered positive (there is cancer), if lower, the result was negative (no cancer).

Claims (1)

A method for the diagnosis of breast cancer, which consists in the fact that antibodies directed to the following glycans are detected in human blood: Manβ1-4GlcNAcβ, Galβ1-4GlcNAcβ1-6 (Galβ1-3) GalNAcα, HOSN ₂ (HOSN) ₄ CH ₂ NH ₂ , GlcAβ , Galα, 6-O-Su-GlcNAcβ, GalNAcβ1-3Galβ, 6-O-Su-Galβ1-4 (6-O-Su) Glcβ, GlcAβ1-3Galβ, Neu5Acα2-3Galβ1-4Glcβ, GlcNAcβ1-3Galβ1-3GalNAcα, Neu5A -3Galβ1-3GalNAcα, Neu5Acα2-3Galβ1-3 (Fucα1-4) GlcNAcβ, Galβ1-4GlcNAcβ1-3 (GlcNAcβ1-6) Galβ1-4GlcNAcβ, Neu5Acα2-6 (Galβ1-3) GlcNAcβ1-3Galβ1-4 with the indicated glycans and by the presence in the blood of a level of antibodies above the threshold, at the same time, all the indicated glycans are diagnosed with breast cancer th gland.

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