JP2012181094A - Initial breast cancer detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an initial breast cancer detection method capable of detecting an initial breast cancer.SOLUTION: The initial breast cancer detection method includes measuring the expression level of at least one gene selected from a specific gene group, such as a Protein BEX1 gene, a Protein LETM2 mitochondrial precursor gene, and a Major urinary protein 6 precursor gene about a test sample separated from an organism.

Description

  The present invention relates to a method for detecting early breast cancer.

  The number of Japanese patients with breast cancer has been increasing year by year due to the westernization of lifestyle, and is expected to increase further in the future. Mortality is high and public interest is very high. Therefore, establishment and popularization of an effective early diagnosis method is required.

  In Japan, mammography and echo diagnosis have been introduced and some results have been achieved. However, the screening rate is only 20% due to exposure to exposure, anxiety about distress and shame. In addition, in November 2009, the US Government's Working Group on Preventive Medicine (USPSTF), a country that has developed breast cancer screening, will not recommend mammography diagnosis to young people under 50 years old (premenopausal women) who are not recommended. Announced. Thus, establishment of a simple and effective new early diagnosis method is demanded.

  On the other hand, CA 15-3 and CEA are used as existing breast cancer serum diagnostic markers (Non-Patent Document 1), but there are no effective early diagnostic markers.

  If an effective serum marker is obtained, a simple primary screening in a group health examination becomes possible, contributing to an improvement in the consultation rate. Furthermore, when combined with existing diagnostic imaging methods, it can be expected to improve the accuracy of early breast cancer detection. Serum marker exploration studies have focused on studies using human advanced breast cancer specimens because early breast cancer specimens are difficult to obtain. However, even if an early breast cancer specimen is used, it is difficult to find an effective marker because of the large amount of noise caused by various genetic backgrounds in human specimens.

JP2011-000054

Clinical Pathology Vol.43, No.7, 696-702, July 1995

  The objective of this invention is providing the detection method of an early stage breast cancer which can detect an early stage breast cancer.

  The present inventors have produced aPKC-mammary knockout mice that are cell polarity control proteins and are involved in the maintenance of stem / progenitor cells, which simulates early lesions of breast cancer, and abnormal growth of mammary tissue stem / progenitor cells (Patent Document 1). And, as specifically described in the examples described later, from the analysis of its molecular mechanism, ErbB2 (HER2) that is overexpressed in about 15 to 40% of human breast cancer and actually becomes a clinical diagnosis / treatment target We also found that aPKC negatively regulates the growth of mammary tissue stem / progenitor cells through molecular transcriptional control. Furthermore, analysis of 104 human breast cancer specimens confirmed abnormalities very similar to breast cancer early lesion model mice (hereinafter referred to as model mice) such as breast cancer stem cell marker, aPKC, ErbB2 abnormalities in 15.2% of human breast cancer specimens. These series of results mean that model mice can be used to develop diagnostic markers and treatments for breast cancer. Then, by measuring the amount of various polypeptides in the blood of this model mouse and healthy mice, identifying a polypeptide having a difference in blood concentration between the two, and identifying its gene, A gene serving as a marker was identified and the present invention was completed.

That is, the present invention provides a method for detecting early-stage breast cancer, comprising measuring the expression level of at least one gene selected from the following gene groups (1) to (34) for a test sample separated from a living body: To do.
(1) Protein BEX1
(2) Protein LETM2 mitochondrial precursor
(3) Major urinary protein 6 precursor
(4) Vacuolar protein sorting-associated protein 18 homolog
(5) Apolipoprotein E precursor
(6) Uncharacterized protein C10orf132 homolog
(7) Apolipoprotein CI precursor
(8) YEATS domain-containing protein 2
(9) Peroxisome proliferator-activated receptor gamma coactivator-related protein 1
(10) Beta-taxilin
(11) Regulator of G-protein signaling 11
(12) Centrosomal protein of 290 kDa
(13) Sterile alpha motif domain-containing protein 9-like
(14) ATP-dependent RNA helicase DDX18
(15) Cathepsin G precursor
(16) 182 kDa tankyrase 1-binding protein
(17) Beta, beta-carotene 9 ', 10'-dioxygenase
(18) Cyclin-dependent kinase inhibitor 2A isoform 3
(19) Centromere protein C 1
(20) Dynamin-binding protein
(21) Receptor tyrosine-protein kinase erbB-2 precursor
(22) Extended synaptotagmin-2
(23) Latent-transforming growth factor beta-binding protein 2 precursor
(24) Latexin
(25) Mastermind-like domain
(26) Serine / threonine-protein kinase PCTAIRE-3
(27) Transcription elongation regulator 1
(28) Zinc finger CCHC domain-containing protein 4
(29) Superkiller viralicidic activity 2-like 2
(30) Piwi-like protein 4
(31) Keratin type I cytoskeletal 10
(32) Keratin type I cytoskeletal 14
(33) Leukemia inhibitory factor receptor
(34) Transcription elongation factor SPT5

  According to the present invention, it is possible to detect early breast cancer that is difficult to detect by conventional methods. Therefore, the present invention is expected to greatly contribute to early treatment and prevention of breast cancer.

It is a figure which shows the result of the immuno-staining of the human breast cancer sample performed in the following Example. It is a figure which shows the result of the immunofluorescence analysis of the human breast cancer sample performed in the following Example.

  As described above, the method of the present invention includes measuring the expression level of at least one gene selected from the gene groups (1) to (34) described above. Each gene of (1) to (34) is known, and its nucleotide sequence (genomic), cDNA sequence, and amino acid sequence of the polypeptide encoded thereby can be found in UniProtKB / Swiss-Prot and / or NCBI (GenBank). It is registered. Even those registered in different databases can be skewered from the NCBI (GenBank) website (http://www.ncbi.nlm.nih.gov/genbank/index.html). The accession numbers of these genes in mice and humans are as shown in Table 1 below. Further, the cDNA sequence of each gene and the amino acid sequence of the polypeptide encoded by the gene are shown in the odd numbers of SEQ ID NOs: 1 to 162. Those obtained by extracting only the amino acid sequence from them are shown in the even numbers of SEQ ID NOs: 1 to 162. In addition, as shown in Table 1 below, some cDNA isoforms exist even in the same gene.

  These genes were picked up from genes encoding polypeptides with an early breast cancer model mouse / control (healthy) mouse ratio of 1.2 or more and 0.75 or less, as specifically described in the Examples below. Among these genes, (1) to (13) are statistically significant differences (P <0.05). In (14) to (34), the number of polypeptides that could be identified was small and statistical processing could not be performed.

  Examples of the test sample include body fluid samples such as blood samples (including serum samples and plasma samples), biopsy samples from cancer tissues, etc., but body fluid samples with a low burden on patients, particularly blood samples are preferred. . The blood concentration is also measured in the following examples.

  The gene expression level can be measured by various known methods. For example, by measuring the concentration of the protein of the gene product of each gene or a fragment thereof (the amino acid sequence of each polypeptide measured in the following examples is described in the examples) by the method specifically described in the following examples. It can be carried out. In addition, since the amino acid sequences of the proteins of the gene products of each gene or fragments thereof are known, specific antibodies against these proteins or fragments thereof can be prepared by conventional methods. The protein or fragment thereof of the gene product of each gene can also be quantified by measurement. It can also be performed by measuring the amount of mRNA of each gene in the test sample (in the case of measuring the amount of mRNA, the test sample is preferably a breast biopsy sample). For the measurement of the amount of mRNA, cDNA is prepared from mRNA by a conventional method, and the amount is quantitatively determined by PCR such as real-time detection PCR using a primer set characteristic of each gene (preferably about 18 to 50 bases). It can be measured by a gene amplification method, or by a method in which each cDNA is labeled and reacted with a DNA chip on which the genes (1) to (34) are immobilized to measure the amount of binding. These methods are well-known methods in the field of genetic testing, and necessary kits are also available on the market. Therefore, those skilled in the art can easily carry out a gene whose base sequence is known.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Quantification of various polypeptides in the blood of model mice and control mice
(1) Analysis method of serum sugar chain protein fractionation using a sugar chain affinity column (WGA) Sugar chain affinity column (more specifically Wheat Germ Agglutinin (WGA) column, trade name Glycoprotein Isolation Kit, WGA) The concentration of each polypeptide in the blood of an early breast cancer model mouse described in Patent Document 1 was measured by the following operation. In the following description, “KO mouse” is a model mouse described in Patent Document 1.

Samples pooled 5μL each (control, KO)
2) Pair of Control KO 14.6w, 21w, 40w ↓ Use 20ml of each sample Remove high-concentration serum protein with an antibody column (Agilent Multiple Affinity Removal Spin Cartridge for mouse)
↓
Adjusted to 75mg / 60ml protein concentration after desalting and concentration ↓
Label each sample with iTRAQ (trade name) reagent of different mass and mix ↓
Using a sugar chain antibody column (PIERCE, WGA), fractionation between glycoprotein and non-sugar-bound protein (separated into adsorbed fraction and non-adsorbed fraction)
↓
After digestion with trypsin, it is divided into 8 fractions with 2D-LC (SCX and C18).
↓
Mass spectrometry analysis
MALDI TOF-TOF: 4800 (ABI) was used. Protein Pilot Software 2.0 analysis was performed.

  As a result, the genes (1) to (30) were identified as genes encoding polypeptides having a model mouse / control mouse ratio of 1.2 or more and 0.75 or less. The model mouse / control mouse ratio for each gene and the SEQ ID NOs of the amino acid sequences of the quantified polypeptide fragments are shown in Table 2 below.

(2) SDS-PAGE analysis
Samples were taken by pooling 4 μL each (total 44 μL).
↓
Remove high-concentration serum proteins with antibody column (ProteoPrep (trade name) 20 Plasma Immunodepletion Kit (SIGMA) or ProteomeLab (trade name) IgY-R7 SC (BECKMAN COULTER)
↓
Approximately 200-250mg of protein is recovered after desalting and concentration ↓
Control and KO samples are labeled with different mass of iTRAQ (trade name) reagent and mixed ↓ (i) Separation fraction by HPLC or (ii) SDS-PAGE
(ii) Separation fraction by HPLC
A 20 μg sample labeled with iTRAQ (trade name) reagent was subjected to separation fractionation using SMART (trade name) System (GE Healthcare). As the column, a gel filtration column (Superose 6) was used.
(iii) Separation fraction by SDS-PAGE
A 20 μg sample labeled with iTRAQ (trade name) reagent was electrophoresed on a 10% SDS polyacrylamide gel, and the lane gel was divided into 20 gels for each molecular weight.
↓
Mass spectrometry analysis
Analysis was performed using ESI LIT-TOF: NanoFrontier LD (Hitachi High-Tech), ESI Q-TOF: Q-Tof micro (Waters), and ESI Q-TOF: SYNAPT MS (Waters). Proteins were identified by Mascot MS / MS ion search analysis.

  As a result, the genes (31) to (34) were identified as genes encoding peptides having a model mouse / control mouse ratio of 1.2 or more and 0.75 or less. The model mouse / control mouse ratio for each gene and the SEQ ID NOs of the amino acid sequences of the quantified peptide fragments are shown in Table 3 below. The gene numbers (31) and (32) have the same quantified amino acid sequence but are derived from different polypeptides. The grounds for this were experimental methods that could not be quantified, but a large number of (mouse) peptides specific to (31) and (32) were identified (data not shown).

2. Immunohistochemical analysis of human breast cancer specimens
(1) Antibody The antibodies used in this experiment were as follows. Anti-aPKC (λ) mouse monoclonal antibody (BD Biosciences), anti-ALDH mouse monoclonal antibody (BD Biosciences), anti-ALDH rabbit monoclonal antibody (Abcam) and anti-HER2 (ErbB2) rat monoclonal antibody (Abcam) ).

(2) Immunostaining Human breast cancer specimens were provided by Kanagawa Cancer Clinical Research and Information Agency (KCRIA). The research protocol used in this experiment was approved by the Yokohama City University and the Ethics Committee of Kanagawa Cancer Research and Information Organization. Informed consent was obtained in advance from all patients for use of tissue samples for research purposes. A frozen section (4 μm) of human breast cancer tissue was fixed with 10% formalin. The above antibody was used as a primary antibody. The labeled antigen was visualized with DAB plus (trade name, manufactured by DAKO), and the sections were counterstained with hematoxylin.

Specifically, the above operation was performed as follows.
Primary antibody (A) anti-aPKCiota / lambda (TDL) (mouse monoclo) 1 / 500dil.
(B) anti-ALDH (TDL) (mouse monoclo) 1/50 dil.
(i) Fix frozen section to 10% formalin, on ice, 5 min
(ii) H ₂ O, 5min
(iii) PBS, 5min.
(iv) 0.3% hydrogen peroxide / PBS, RT, 30min.
(v) PBS, 5min
(vi) Blocking with 10% normal rabbit serum / PBS, 37 ℃, 15min
(vii) Primary antibody 4 ℃, O / N
(viii) PBS 5min x 3 times
(ix) Biotinylated anti-mouse secondary antibody 10 min (hereinafter, Histofine Kit, Nichirei Code: 426032)
(x) PBS 5min x 3 times
(xi) Peroxidase-labeled streptavidin 5min (Nichirei Code: 426062)
(xii) PBS 5min x 3 times
(xiii) DAB plus (DAKO Code: K3468)
(xiv) Nuclear dye Dehydration Toru

  The results are shown in FIG. In FIG. 1, a and b are representative immunostained images of aPKCλ in a human breast cancer specimen. aPKCλ positive (a), negative (b). c shows an immunostained image of ALDH of aPKCλ-negative cancer. Arrows indicate ALDH positive cancer cells. The scale bar is 100 μm.

(3) Immunofluorescence
Cy3-conjugated anti-rabbit antibody (Amersham), Cy5-conjugated anti-rat antibody (Amersham) and Alexa 488 anti-mouse antibody (Invitrogen) were used. DAPI (trade name, manufactured by Roche) was used for nuclear staining. The stained section was embedded in Prolong gold antifade reagent (trade name, manufactured by Invitrogen).

Specifically, the above operation was performed as follows.
Primary antibody (A) anti-aPKCiota / lambda (TDL) (mouse monoclo) 1 / 500dil.
(B) anti-ALDH (Abcam) (rabbit monoclo) 1/200 dil.
(C) anti-HER2 (Abcam) (rat monoclo) 1 / 50dil.
(i) Fix frozen section to 10% formalin, on ice, 5 min
(ii) H ₂ O, 5min
(iii) PBS, 5min.
(iv) Blocking with 10% normal rabbit serum / TBST, RT, 30 min
(v) Primary antibody 4 ℃, O / N
(vi) TBST 5min x 3 times
(vii) Fluorescently labeled secondary antibody (1/500 each; anti-mouse Alexa488, anti-rabbit cy3, anti-rat cy5) + DAPI
(viii) TBST 5min × 3 times
(ix) Embedded in Prolong gold antifade (Invitrogen).

  The results are shown in FIG. In FIG. 2, a to c are aPKCλ-positive breast cancer cases, d to i are aPKCλ-negative breast cancer cases-1, and j to o are further aPKCλ-negative breast cancer cases-2. aPKCλ (a, d, j) staining and DAPI (b, e, k) staining and aPKCλ and DAPI integrated image (c, f, i). (i, o) is an integrated image of triple staining of aPKCλ, ALDH, and HER2. Scale bar is 10μm. In these two breast cancer cases, most cells did not express aPKCλ and expressed both ALDH and HER2.

(4) Statistical processing All statistical analyzes were performed using PASW statistics 18 (trade name, manufactured by SPSS Inc.). A P value <0.05 was considered significant. The clinicopathological parameters used in this experiment are shown in Table 4 below. To analyze the correlation between clinicopathological features and aPKCλ expression, the χ ² test was used. When the number of cases was less than 5, Fisher's exact test was applied. Residual analysis was performed when the P value was less than 0.05 in both the χ ² test and Fisher's exact test. When the absolute value of the prepared residual was greater than 1.96, it was considered that the P value was less than 0.05.

  Table 4 below lists the clinicopathological parameters and some histochemical parameters of the 104 breast cancer cases examined in this experiment. Frozen tissue sections were used to assess ER, PgR, HER2, ALDH and aPKCλ expression. ER and PgR were defined as positive when immunohistochemical reactivity was greater than 10%. For HER2, +2 and +3 were defined as positive by Hercep test. ALDH was positive when 5% of the tumor cells showed immunohistochemical reactivity. aPKCλ was defined as negative when less than 20% of the tumor cells showed immunohistochemical reactivity.

Table 5 below shows the correlation between clinicopathological parameters and aPKCλ expression. To analyze the correlation between clinicopathological features (described in Table 4 above) and aPKCλ expression, a χ ² test was used. When the number of cases was less than 5, Fisher's exact test was applied.

The correlation between HER2 and ALDH in aPKCλ-negative breast cancer was also analyzed by Fisher's exact test. Residual analysis was performed when the P value was less than 0.05 in both the χ ² test and Fisher's exact test. When the absolute value of the prepared residual was greater than 1.96, it was considered that the P value was less than 0.05. The results are shown in Table 6 below.

As shown in Table 6, ALDH was positively correlated with HER2 in aPKCλ-negative breast cancer cases (n = 40) (P = 0.015, Fisher exact test). The value of the prepared residual of ALDH ⁺ / HER2 ⁺ was +2.5.

Claims (6)

An early breast cancer detection method comprising measuring an expression level of at least one gene selected from the following gene groups (1) to (34) for a test sample separated from a living body.
(1) Protein BEX1
(2) Protein LETM2 mitochondrial precursor
(3) Major urinary protein 6 precursor
(4) Vacuolar protein sorting-associated protein 18 homolog
(5) Apolipoprotein E precursor
(6) Uncharacterized protein C10orf132 homolog
(7) Apolipoprotein CI precursor
(8) YEATS domain-containing protein 2
(9) Peroxisome proliferator-activated receptor gamma coactivator-related protein 1
(10) Beta-taxilin
(11) Regulator of G-protein signaling 11
(12) Centrosomal protein of 290 kDa
(13) Sterile alpha motif domain-containing protein 9-like
(14) ATP-dependent RNA helicase DDX18
(15) Cathepsin G precursor
(16) 182 kDa tankyrase 1-binding protein
(17) Beta, beta-carotene 9 ', 10'-dioxygenase
(18) Cyclin-dependent kinase inhibitor 2A isoform 3
(19) Centromere protein C 1
(20) Dynamin-binding protein
(21) Receptor tyrosine-protein kinase erbB-2 precursor
(22) Extended synaptotagmin-2
(23) Latent-transforming growth factor beta-binding protein 2 precursor
(24) Latexin
(25) Mastermind-like domain
(26) Serine / threonine-protein kinase PCTAIRE-3
(27) Transcription elongation regulator 1
(28) Zinc finger CCHC domain-containing protein 4
(29) Superkiller viralicidic activity 2-like 2
(30) Piwi-like protein 4
(31) Keratin type I cytoskeletal 10
(32) Keratin type I cytoskeletal 14
(33) Leukemia inhibitory factor receptor
(34) Transcription elongation factor SPT5   The method according to claim 1, comprising measuring the expression level of at least one gene selected from the gene group of (1), (2), (4) to (34).   The method according to claim 1, comprising measuring the expression level of at least one gene selected from the gene group of (1) to (13).   4. The method according to claim 3, comprising measuring the expression level of at least one gene selected from the gene group of (1), (2), (4) to (13).   The method according to claim 1, wherein the test sample is a blood sample.   The method according to any one of claims 1 to 5, wherein the gene expression level is measured based on a concentration of a gene product contained in the test sample.