JP6202362B2 - Diagnostic agents and methods for predicting local recurrence risk after breast-conserving breast cancer therapy - Google Patents

Description

  The present invention relates to a diagnostic agent and kit for use in predicting the risk of local recurrence after breast cancer breast-conserving therapy, and a method for predicting the local recurrence risk after breast cancer-preserving therapy.

  In recent years, breast conservation therapy (BCT) has been established as a widely approved standard treatment method for the treatment of early breast cancer (Non-Patent Documents 1-3). Breast-conserving therapy is a treatment method comprising tumorectomy followed by radiation therapy to the entire breast, and adjuvant therapies such as chemotherapy and hormone therapy. Breast-conserving therapy literally aims to preserve the breast as much as possible, taking into account the patient's quality of life. The treatment method is to remove only the visible lesion in the mammary gland by surgical operation, and confirm that there is no leftover of the lesion by tissue biopsy. It is based on the principle that micrometastasis that may have been eradicated by aging, and possibly other than breast, is eradicated by adjuvant therapy. However, even in breast cancer preserving therapy based on such a principle, 8.8-20% of patients have a local recurrence in the breast (Non-patent Documents 1-3).

  According to past reports, several factors such as age, Ki-67 antigen level, nuclear markers of cell proliferation and the like have been confirmed as risk factors for local recurrence after breast cancer preservation therapy (Non-patent Document 4). In addition, the five molecular subtypes of breast cancer (ie, “Luminal A-like”, “Luminal B-like”, “HER2-enriched”, “basal-like”) and categorized based on breast cancer gene expression signatures. It has also been reported that the frequency of recurrence after breast cancer-preserving therapy changes according to "normal-like") (Non-patent Documents 5 and 6). In addition, surgical margin status, nodal status, and tumor grade have also been reported to correlate with the frequency of local recurrence after breast cancer preservation therapy. (Non-Patent Documents 7 and 8).

  Furthermore, the breast cancer recurrence diagnostic method “MammaPrint” (Agendia) based on a gene expression signature consisting of a cluster of 70 genes, said to be useful for clinicopathological evaluation regarding the prediction of distant metastasis and overall survival of breast cancer. (Amsterdam, The Netherlands), and this diagnostic method has been approved by the US Food and Drug Administration (Non-Patent Documents 9, 10 and 11). On the other hand, "Mammaprint" also shows that local recurrence cannot be predicted (correct response rate 18%) (Kreike et al., Clin Cancer Res 15: 4181-4190, 2009).

  It has also been reported that wound-response signature (Non-Patent Document 12) is significantly correlated with local recurrence after breast cancer-preserving therapy by analyzing a gene expression profile data set (non-patent document 12). Patent Document 13). However, this signature has since been denied by the same research group (Kreike et al., Clin Cancer Res 15: 4181-4190, 2009).

Fisher B, Anderson S, Bryant J, et al: Twenty-year follow-up of a randomized trial comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer.N Engl J Med 347: 1233-41, 2002 Veronesi U, Cascinelli N, Mariani L, et al: Twenty-year follow-up of a randomized study comparing breast-conserving surgery with radical mastectomy for early breast cancer.N Engl J Med 347: 1227-32, 2002 Van Dongen JA, Voogd AC, Fentiman IS, et al: Long-term results of a randomized trial comparing breast-conserving therapy with mastectomy: European Organization for Research and Treatment of Cancer 10801 trial. J Natl Cancer Inst 92: 1143-50, 2000 Elkhuizen PH, Voogd AC, van den Broek LC, et al: Risk factors for local recurrence after breast-conserving therapy for invasive carcinomas: a case-control study of histological factors and alterations in oncogene expression.Int J Radiat Oncol Biol Phys 45: 73-83, 1999 Nguyen PL, Taghian AG, Katz MS, et al: Breast cancer subtype approximated by estrogen receptor, progesterone receptor, and HER-2 is associated with local and distant recurrence after breast-conserving therapy. J Clin Oncol 26: 2373-8, 2008 Millar EK, Graham PH, O'Toole SA, et al: Prediction of local recurrence, distant metastases, and death after breast-conserving therapy in early-stage invasive breast cancer using a five-biomarker panel.J Clin Oncol 27: 4701- 8, 2009 Jones HA, Antonini N, Hart AA, et al: Impact of pathological characteristics on local relapse after breast-conserving therapy: a subgroup analysis of the EORTC boost versus no boost trial.J Clin Oncol 27: 4939-47, 2009 Kunkler IH, Kerr GR, Thomas JS, et al: Impact of screening and risk factors for local recurrence and survival after conservative surgery and radiotherapy for early breast cancer: results from a large series with long-term follow-up.Int J Radiat Oncol Biol Phys 83: 829-38, 2012 van de Vijver MJ, He YD, van't Veer LJ, et al: A gene-expression signature as a predictor of survival in breast cancer.N Engl J Med 347: 1999-2009, 2002 van 't Veer LJ, Dai H, van de Vijver MJ, et al: Gene expression profiling predicts clinical outcome of breast cancer.Nature 415: 530-6, 2002 Buyse M, Loi S, van't Veer L, et al: Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer.J Natl Cancer Inst 98: 1183-92, 2006 Chang HY, Nuyten DS, Sneddon JB, et al: Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival.Proc Natl Acad Sci U S A 102: 3738-43, 2005 Nuyten DS, Kreike B, Hart AA, et al: Predicting a local recurrence after breast-conserving therapy by gene expression profiling.Breast Cancer Res 8: R62, 2006

  As described above, Non-Patent Documents 4 to 8 describe various risk factors related to local recurrence after breast cancer preservation therapy, but local recurrence after breast cancer preservation therapy that can be applied to clinical settings based on these risk factors. Risk could not be predicted (described above).

Furthermore, breast cancer recurrence diagnosis methods described in Non-Patent Documents 9 to 11 are intended for general recurrence of breast cancer, and are not technologies that enable prediction of local recurrence after breast cancer preservation therapy.
Furthermore, since the techniques described in Non-Patent Documents 12 and 13 have not been investigated since then, clinical trials based on large-scale patient groups have not been made, and their usefulness is not clear.

  Therefore, an object of the present invention is to provide a predictive diagnostic agent and a prediction method capable of predicting a local recurrence risk after breast cancer preservation therapy.

  As a result of intensive studies to solve the above problems, the present inventors have found that the co-expression of at least two genes among the specific gene group belonging to the Arf6 pathway is significantly correlated with local recurrence after breast cancer preservation therapy. I found it. The present invention has been completed based on such knowledge.

  That is, the present invention relates to the following.

[1] A diagnostic agent for use in predicting the risk of local recurrence after breast-conserving breast cancer therapy,
A diagnostic agent comprising a protein and / or polypeptide capable of specifically recognizing a translation product of any one gene selected from the group consisting of AMAP1, GEP100, EBL5, and c-Met.
[2] The diagnostic agent according to [1], wherein the protein and / or polypeptide is an antibody and / or a fragment thereof.
[3] A kit for use in predicting the risk of local recurrence after breast cancer breast-conserving therapy, comprising the diagnostic agent according to [1] or [2].
[4] The diagnostic agent according to [3], wherein the diagnostic agent comprises two diagnostic agents capable of specifically detecting each of translation products of two genes of any of the combinations (i) to (ii) below: kit:
(I) AMAP1 and GEP100, and (ii) EBL5 and C-Met.
[5] (A) Determination of translation product expression levels of at least two genes selected from the group consisting of AMAP1, GEP100, EBL5, and c-Met in biological samples collected from patients who have received breast cancer breast-conserving therapy And (B) predicting the risk of local recurrence after breast cancer breast-conserving therapy for the patient based on the translation product expression level determined in step (A),
Predicting the risk of local recurrence after breast-conserving therapy for breast cancer.
[6] The method according to [5], wherein the at least two genes in step (A) are any of the following combinations (i) to (ii):
(I) AMAP1 and GEP100, and (ii) EBL5 and C-Met.
[7] The method according to [5] or [6], wherein the biological sample is breast tissue.
[8] In step (A), the translation product expression levels of the at least two genes are determined by an immunohistochemical method using a protein and / or polypeptide capable of specifically recognizing the translation products of the genes. The method according to any one of [5] to [7].
[9] The method according to [8], wherein the protein and / or polypeptide is an antibody and / or a fragment thereof.
[10] In step (A), the expression levels of the translation products of the at least two genes in the biological sample are determined using the expression levels of the translation products of the at least two genes in the non-cancerous breast tissue of the patient as controls. And in step (B), the method according to any one of [5] to [9], wherein the risk of local recurrence after breast cancer breast-conserving therapy of the patient is predicted according to the following criteria:
(I) if all of the translation product expression levels of the at least two genes determined in step (A) are higher than the expression levels of the corresponding controls, the patient is predicted to have a high risk of local recurrence of breast cancer Or (ii) if one of the translation product expression levels of the at least two genes determined in step (A) is lower than or equivalent to the expression level of the corresponding control, the patient is Predict that the risk of local recurrence of breast cancer is low.

  According to the present invention, it is possible to accurately predict the risk of local recurrence after breast cancer preservation therapy. As a result, it becomes possible to determine a treatment plan in consideration of the risk of local recurrence of breast cancer, and to effectively prevent local recurrence after breast cancer preservation therapy. Further, in conventional breast cancer preservation therapy, pathological diagnosis based on a biopsy tissue sample of a lesion site is performed. According to the present invention, it can be carried out by detecting the expression of a predetermined gene translation product in these biopsy tissue samples simultaneously with the pathological diagnosis. Therefore, according to the present invention, it is possible to predict the risk of local recurrence after breast cancer preserving therapy with little burden on the patient and easily.

It is a figure which shows the microscope picture of the immunohistochemical staining acquired in the Example. It is a figure which shows the microscope picture of the immunohistochemical staining acquired in the Example. It is a figure which shows the result of having investigated the correlation with co-expression of AMAP1 and GEP100, and the period from completion of breast cancer breast preservation therapy to local recurrence. It is a figure which shows the result of having investigated the correlation with co-expression of c-MET and EBL5, and the period from completion of breast cancer breast preservation therapy to local recurrence. It is a figure which shows the result of having investigated the correlation with various parameters and the period from completion of breast cancer breast preservation therapy to local recurrence.

Diagnostic Agent According to one aspect of the present invention, a diagnostic agent for use in predicting the risk of local recurrence after breast cancer breast-conserving therapy, selected from the group consisting of AMAP1, GEP100, EBL5, and c-Met A diagnostic agent containing a protein and / or polypeptide capable of specifically recognizing the translation product of any one gene is provided.

  In the present invention, “breast cancer breast-conserving therapy” is a treatment method including breast-conserving therapy tumor excision followed by radiation therapy to the entire breast. More specifically, breast cancer breast-conserving therapy is performed by surgically removing only a visible lesion in the mammary gland and confirming that there is no leftover by tissue biopsy. The therapy may be radiation therapy as a preventive treatment for the entire breast. In addition, breast cancer breast-conserving therapy can optionally include adjuvant therapies such as chemotherapy and hormone therapy. Breast cancer breast-conserving therapy is literally aimed at improving the patient's quality of life by preserving the breast, and is a standard that has been widely approved for early breast cancer in the art. It is a proper treatment method.

  In the present invention, the “local recurrence risk” means the risk that a patient who has received breast cancer breast-conserving therapy will relapse cancer in the breast after the treatment is completed. Local recurrence risk primarily means the risk of early recurrence, for example, within 5 years after receiving breast cancer breast-conserving therapy. However, it may include a medium-term recurrence risk of more than 5 years after receiving breast cancer breast-conserving therapy, or a recurrence risk of more than 10 years, or more than 10 years.

The method of “prediction of local recurrence risk” will be described later in “Prediction method”.
The therapeutic agent of the present invention can be used for the prediction of the risk of local recurrence after breast cancer breast-conserving therapy, as shown in Examples described later, a group of patients who have received breast cancer breast-conserving therapy and subsequently undergoes local recurrence of breast cancer In patients with breast tissue, the expression of the translation product of the specific gene combination is statistically compared to the patient with no expression of the translation product of the specific gene combination in the breast tissue, It is shown with statistical significance that the breast cancer relapses relatively early (for example, within 5 years). The specific gene combination is specifically at least two genes selected from the group consisting of AMAP1, GEP100, EBL5, and c-MET, and in particular, a combination of AMAP1 and GEP100, or EBL5 and c-Met. Patients with translation product expression in a combination of combinations have a high risk of local recurrence with statistical significance. That is, as described below for a method for predicting the risk of local recurrence after breast cancer breast-conserving therapy, if the expression of the translation product of the above gene combination is observed at a certain level in breast tissue, the patient has breast cancer after breast cancer-preserving therapy Predicted high risk of local recurrence early. Thus, in the present invention, “prediction of local recurrence risk after breast cancer breast-conserving therapy” is based on the premise that the expression of the translation product of the specific gene combination in breast tissue is detected. The diagnostic agent of the present invention to be used is specifically a reagent for use in detection of these gene translation products, and any one selected from the group consisting of AMAP1, GEP100, EBL5, and c-Met It contains a protein and / or polypeptide that can specifically recognize a translation product of a gene.

The gene AMAP1 (GeneID: 50807) is a downstream effector of Arf6 in cancer invasion.
Gene GEP100 (GeneID: 9922) is an Arf6 activator in cancer invasion.
The gene EPB41L5 (abbreviation EBL5) (GeneID: 57669) is a binding partner of AMAP1 in cancer invasion and is essential for invasion pseudopod formation.
The gene c-Met (GeneID: 4233) is a cell surface receptor that is transactivated by TGFβ1 in cancer invasion and activates GEP100. It is also directly activated by its own ligand, HGF, and promotes invasion.

  The protein and / or polypeptide capable of specifically recognizing the translation product of the gene is not particularly limited as long as it can specifically bind to the translation product of one of the genes. For example, an antibody against the translation product and / or a fragment thereof can be mentioned. Examples of antibodies and / or fragments thereof include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, humanized antibodies, and the like. Any method can be adopted as a method for preparing the protein and polypeptide capable of specifically recognizing the translation product of the gene as long as the specific binding to the gene translation product can be maintained. The desired polyclonal antibody, monoclonal antibody, single chain antibody, humanized antibody, etc. can be prepared by a known method using the protein and / or fragment thereof as an antigen.

  Furthermore, in the diagnostic agent of the present invention, the single diagnostic agent may contain only one protein or polypeptide that can specifically recognize the translation product of one of the above genes, Alternatively, two or more proteins and / or polypeptides capable of specifically recognizing each translation product of the gene may be included. In addition, the protein and / or polypeptide may be labeled with a labeling substance for the detection of gene translation products. Examples of the labeling substance include fluorescent substances (for example, fluorescent proteins such as GFP, and fluorescein). And the like). However, it is not intended to be limited to those having labels with these labeling substances. When the protein and / or polypeptide is not labeled in the diagnostic agent of the present invention, the final detection of the translation product of the gene involves the binding to the protein and / or polypeptide (primary antibody). A system using a secondary antibody can be used. Such a system using a secondary antibody includes a commercially available detection kit. For example, a secondary antibody against the primary antibody is labeled with peroxidase, and the primary antibody specifically bound to the target gene translation product in the sample. Furthermore, the above-mentioned secondary antibody can be specifically bound to 3,3'-diaminobenzidine tetrahydrochloride as a color substrate for peroxidase added to the sample, and the color development can be observed to detect the gene translation product. . Furthermore, in the present invention, when one diagnostic agent contains two or more proteins and / or polypeptides capable of specifically recognizing each of the translation products of the above genes, for example, two or more of them may be used. Each of the target gene translation products can be detected in the same sample by labeling these proteins and / or polypeptides with different fluorescent substances that emit fluorescence of different wavelengths.

  Diagnostic agents can optionally include buffers, preservatives, cryoprotectants, and the like. Examples of the buffer include tris hydrochloride, potassium phosphate, sodium phosphate and the like. Examples of the preservative include sodium azide. Examples of the antifreezing agent include glycerol.

  The diagnostic agent of the present invention detects the translation product of a predetermined gene translation product in order to determine the expression level of the gene translation product in step (A) of the method for predicting the risk of local recurrence after breast cancer preservation therapy described later. Can be used.

Kit According to another aspect of the present invention, there is provided a kit for use in predicting the risk of local recurrence after breast cancer breast-conserving therapy, comprising the above-described diagnostic agent.

  The kit of the present invention provides components such as reagents and members that can be used in carrying out the following method for predicting the risk of local recurrence after breast cancer breast-conserving therapy, and includes the above-mentioned diagnostic agent It is characterized by.

  In the kit of the present invention, the diagnostic agent of the present invention is (1) a protein capable of specifically recognizing the translation product of one gene of AMAP1, GEP100, EBL5, and c-MET in a single diagnostic agent. Alternatively, it may be a diagnostic agent capable of detecting the translation product of one gene by including only one polypeptide, or (2) each of the translation products of each gene is specific in a single diagnostic agent. The single diagnostic agent may be a single diagnostic agent capable of detecting the plurality of gene translation products by including two or more recognizable proteins and / or polypeptides. Furthermore, in the kit of the present invention, a diagnostic agent capable of detecting the translation product of one gene of (1) above and / or a single agent capable of detecting a plurality of gene translation products of (2) above. Diagnostic agents may be included. Further, when the diagnostic agent contained in the kit enables detection of a plurality of gene translation products, whether the diagnostic agent contained in the kit of the present invention is one type or two types, the following (i) It is preferable that it is a diagnostic agent which can detect the translation product of the gene of any combination of-(ii).

(I) AMAP1 and GEP100, and (ii) EBL5 and c-Met.

  In addition to the above diagnostic agents, the kit of the present invention includes a blocking reagent, a labeled secondary antibody for use in detecting a protein and / or polypeptide (antibody) contained in the diagnostic agent, a coloring reagent, breast cancer breast-preserving therapy Instructions describing the experimental protocol regarding how to predict the risk of subsequent local recurrence may optionally be included. Examples of the blocking reagent include bovine serum albumin (BSA).

Prediction Method According to another aspect of the present invention, there is provided a method for predicting the risk of local recurrence after the following breast cancer-preserving therapy.
(A) determining a translation product expression level of at least two genes selected from the group consisting of AMAP1, GEP100, EBL5, and c-Met, respectively, in a biological sample collected from a patient who has received breast cancer breast-conserving therapy; And (B) predicting the risk of local recurrence after breast cancer breast-conserving therapy in the patient based on the translation product expression level determined in step (A),
Predicting the risk of local recurrence after breast-conserving therapy for breast cancer.

  In the method of the present invention, the definition of “breast cancer breast-conserving therapy” is as described above. In the method of the present invention, the patient is a patient who has received the breast cancer breast-conserving therapy described above.

  In the method of the present invention, the “biological sample” is any biological sample collected from a patient who has received the breast cancer breast preservation therapy. The biological sample is preferably breast tissue, and the breast tissue may be a tissue of a breast cancer lesion site removed from a patient at the time of tumor removal for breast cancer breast-conserving therapy, or a lesion site is present. It may be a nearby breast tissue that has been removed and taken after tumor removal. However, from the viewpoint of reducing the burden on the patient as much as possible and achieving an accurate prediction of the risk of local recurrence, the breast tissue is the tissue of the breast lesion site removed from the patient at the time of tumor removal for breast cancer breast preservation therapy. Preferably there is.

  In step (A) of the method of the present invention, translation product expression levels of at least two genes selected from the group consisting of AMAP1, GEP100, EBL5, and c-Met are determined in the biological sample. These genes are genes belonging to the Arf6 pathway. According to the results shown in the following examples, when a gene belonging to the Arf6 pathway is expressed at the tissue level in breast tissue of a breast cancer patient and the operation of the Arf6 pathway is estimated, the patient is relatively breast cancer breast. This confirms the high risk of local recurrence early after conservative therapy. Therefore, when the expression levels of the translation products of at least two of the genes belonging to the Arf6 pathway are determined and the expression of these two genes is confirmed, it is considered that local recurrence after breast cancer breast-conserving therapy is promoted. Therefore, in the step (A) of the method of the present invention, the patient is selected from the group consisting of genes belonging to the Arf pathway. The translation product expression levels of at least two genes to be determined are respectively determined.

  It has been reported that the Arf6 pathway including genes such as GEP100, Arf6, and AMAP1 plays an important role in promoting breast cancer invasion and metastasis. Among the genes belonging to the Arf6 pathway, GEP100 And EGFR, or the combination of Arf6 and AMAP1 have been shown to have high malignancy and invasion of breast cancer (Morishige M, Hashimoto S, Ogawa E, et al: GEP100 links epidermal growth factor receptor signaling to Arf6 activation to induce breast cancer invasion. Nat Cell Biol 10: 85-92, 2008; Hashimoto S, Onodera Y, Hashimoto A, et al: Requirement for Arf6 in breast cancer invasive activities.Proc Natl Acad Sci USA 101: 6647-52, 2004; Onodera Y, Hashimoto S, Hashimoto A, et al: Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities. EMBO J 24: 963-73, 2005). In addition, it is possible that high-level expression of genes belonging to the Arf6 pathway may be correlated with recurrence after breast cancer breast-conserving therapy (JMTO, Newsletter, September 2012, No. 14, p3-4; Fukui University School of Medicine Life Science Innovation Promotion Organization Seminar and 371st Seminar (Graduate Seminar) Summary: p53 mutation and TGFβ signaling culminate in cancer invasiveness via GEP100-Arf6-AMAP1 pathway), of which GEP100 and EGFR co-expression It was first examined whether it correlated with recurrence after breast-conserving therapy (R. Kinoshita et al., International Journal of Radiation Oncology / Biology / Physics, Volume 84, Number 3S, Supplement 2012 Poster Viewing Abstracts, S227, 2026: Co-overexpression of GEP100 with EGFR correlates with early recurrence after breast conservation therapy (BCT)). As a result, it was found that local recurrence after breast cancer breast-conserving therapy cannot be significantly predicted by the combined co-expression of GEP100 and EGFR, including detailed investigations thereafter, and other genes belonging to the Arf6 pathway are not To date, no significant correlation with local recurrence has been shown.

  In the step (A) of the method of the present invention, the method for determining the translation product expression level of the predetermined gene in the biological sample is not particularly limited, but specifically, the diagnostic agent of the present invention is described above. As described above, it is possible to determine the translation product expression level of the gene using a protein or polypeptide (diagnostic agent) that can specifically recognize the translation product of the gene to be detected. More specifically, since the expression level can be determined by accurately grasping the expression intensity and expression distribution of the gene translation product in a tissue section which is a biological sample, in particular, the protein and / or polypeptide (diagnostic agent) It is preferable to determine the expression level of the translation product of the gene by the immunohistochemical method used. Various known methods are known for immunohistochemical methods. In the present invention, those known methods may be used, or a newly developed method may be used, and the method is particularly limited as long as the expression level of the target gene translation product can be determined. It is not a thing.

  Furthermore, in the present invention, in order to ensure specific detection of a gene translation product, the protein and / or polypeptide is preferably an antibody against a gene translation product to be detected and / or a fragment thereof. .

  Furthermore, in the step (A) of the method of the present invention, in order to determine the expression level of translation products of at least two genes specified in the present invention, specifically, signals detected by an arbitrary protein detection method in a biological sample By comparing the intensity with a certain standard, the translation product expression level of a predetermined gene of the present invention can be determined. As an example of a certain standard, there is a form in which the expression level of the gene translation product of the present invention in a non-cancerous tissue (particularly non-cancerous breast tissue) collected from the same patient is used as a control. In this case, specifically, the signal intensity detected by the same protein detection method for the expression of the gene translation product of the present invention in the non-cancerous tissue (particularly non-cancerous breast tissue) collected from the same patient, and the target By comparing the signal intensities of the gene translation products of the present invention in biological samples, the expression levels of translation products of at least two genes of the present invention can be determined respectively. Furthermore, as a specific form of “determining the translation product expression level”, for example, a signal of translation product expression of at least two genes of the present invention in a subject biological sample as compared with the above-mentioned certain standard or control It can be determined that the intensity is “high”, “equivalent”, or “low”. Alternatively, for example, the signal intensity of the translation product expression of at least two genes of the present invention in the target biological sample in the target biological sample can be scored on the basis of the signal intensity in the certain standard or control. For scoring the signal intensity, for example, the signal intensity of the gene translation product in the control sample is set to 0, and the signal intensity is compared with the signal intensity of the gene translation product in the control biological sample. Can be scored with an integer.

  In step (B) of the method of the present invention, the risk of local recurrence after breast cancer breast-conserving therapy for the patient is predicted based on the translation product expression level determined in step (A).

  In order to predict the risk of local recurrence after breast cancer breast-conserving therapy in step (B), specifically, as described above with respect to step (A), the “high” obtained by comparison with the certain criteria or controls. Based on the “equivalent” or “low” or signal intensity scored value, the risk of local recurrence after breast cancer breast-conserving therapy can be predicted. More specifically, when it is determined that the expression level of the translation product of the at least two genes in the target biological sample is “high”, the patient is determined to have a high risk of local recurrence of breast cancer. When the expression level of translation products of the at least two genes is determined to be “equivalent” or “low”, the patient can be determined to have a low risk of local recurrence of breast cancer. Alternatively, when adopting a form in which the gene translation product expression level is scored as described above, a criterion such as “high”, “low” or “medium” is assigned to the risk of local recurrence in advance, The local recurrence risk of breast cancer may be determined as “high”, “low”, “medium”, or the like based on the score of the target biological sample obtained by the above scoring. In addition, the prediction of breast cancer local recurrence risk not only yields results such as “high”, “low” or “moderate”, but also the risk of developing breast cancer after breast cancer breast-conserving therapy is 50-90. It may also include determining how long a period that is% (eg, 50%, 60%, 70%, 80%, 90%, 95%, etc.).

Treatment or Prevention Methods Further, according to another aspect of the present invention, there is provided a method of treating or preventing breast cancer recurrence. Specifically, a method for treating or preventing local recurrence of breast cancer is a method for treating or preventing breast cancer recurrence based on a prediction result obtained by the method for predicting local recurrence risk after breast cancer-preserving therapy. It is.

  Specifically, this method includes means capable of treating or preventing any recurrence of breast cancer, such as chemotherapy using anticancer agents, hormonal therapy, radiation therapy, surgical treatment, diet therapy, lifestyle guidance, and the like.

  This method is not particularly limited as long as it treats or prevents the recurrence of breast cancer based on the prediction result obtained by the method for predicting the risk of local recurrence after the breast cancer preservation therapy. The method steps for predicting the risk of recurrence may or may not be included.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not particularly limited by the following examples.

<Materials and methods>
(Materials and methods)
(1) Patient population From 1988 to 2008, 479 breast cancer patients who underwent breast-conserving surgery at Hokkaido University Hospital (Hokkaido, Japan) and subsequently received radiation therapy to the entire breast. The breasts were analyzed retrospectively. By May 2010, a total of 20 recurrences were observed in 20 patients with a median follow-up of 54 months. Of these 20 patients, 19 histopathology specimens were available for analysis. Only one had a recurrence in the breast as the first site of failure. In the remaining one, local lymph nodes recurred in the first 4 months after completion of radiation therapy, and breasts recurred in 18 months. The time of recurrence in the breast was used for analysis in 19 patients.
This study was approved by the Hokkaido University Clinical Trial Review Board.

(2) Treatment method Eight patients underwent massectomy, and eight patients underwent breast 4/1 resection. Of the 19 patients, 16 (84%) had axillary lymph node dissection and the other 3 (16%) had a sentinel lymph node biopsy. One patient with a TNM classification (UICC 5th edition) of cT3N1M0 had received preoperative chemotherapy including trastuzumab and was also given trastuzumab as an adjuvant therapy after surgery. One patient was receiving chemotherapy during surgery. One patient was receiving chemotherapy during and after surgery. Three patients received chemotherapy after radiation therapy. Five patients received hormone therapy after radiation therapy. All patients had full breast tangent irradiation on the affected breast. One patient had been irradiated at the sites of parasternal and supraclavicular lymph nodes. The prescription dose for patients who have undergone complete resection by microscopic examination is 45 Gy after 18 irradiations, and patients who have undergone incomplete resection by microscopic examination [positive margin or close margin (close margin) ( The prescription dose of less than 5 mm) was 50 Gy after 20 irradiations. Since July 2005, patients under the age of 50 have received 50 Gy of irradiation in 20 doses, regardless of the condition of the resected stump.

(3) Tissue specimens All 19 pathological specimens were surgical specimens obtained by extensive excision. One patient had received neoadjuvant chemotherapy prior to surgery. The pathological features and tumor-free state of the resected stump were examined blindly by a pathologist who did not grasp the disease parameters in this study.

The state of the resected stump was defined as follows and evaluated by a pathologist.
(I) Resection stump if tumor [invasive ductal carcinoma (IDC) or ductal carcinoma in situ (DCIS)] is observed at the inked edge of resection Positive;
(Ii) Close margin where tumor (IDC or DCIS) is observed 5 mm or less from the resected margin; and (iii) Stumps that are more than 5 mm from the resected margin and are tumor-free are negative.

(4) Immunohistochemistry (IHC)
Antibodies against AMAP1 and GEP100 have been reported previously (Onodera et al., 2006; Morishige et al., 2008). The EPB41L5 antibody was obtained by immunizing rabbits with a peptide containing amino acids 558 to 733 of the human protein as an antigen, collecting antisera by standard techniques, and affinity-purifying with the antigen. The antibody against c-MET was purchased from Abcam, the EGFR antibody from Nichirei, and the antibody against HER2 from DAKO.

  Immunohistochemical staining was performed using formalin-fixed, paraffin-embedded serial sections with a thickness of 4 μm as follows. All slides were first deparaffinized in xylene, dehydrated in graded alcohol, then sections were tris-buffered saline (TBS: 25 mM Tris-HCl (pH 7.4), 137 mM NaCl) , 2.7 mM KCl).

Then, the antigen activation process of the section | slice was performed about each antigen on the following conditions.
(I) HER2 or EPB41L5 (EBL5): incubation with citrate buffer (pH 6.0) at 95 ° C. for 40 minutes;
(Ii) EGFR: Incubate with pepsin solution at 37 ° C. for 10 minutes;
(Iii) AMAP1 or GEP100: incubation with an antigen activation solution (EDTA solution, pH 9, purchased from Nichirei) at 95 ° C. for 40 minutes;
(Iv) Incubate at 95 ° C. for 40 minutes with c-MET :: antigen activation solution (EDTA solution, pH 9, purchased from Nichirei).

The endogenous peroxidase was then blocked by incubation in 0.3% H ₂ O ₂ / methanol at room temperature. After washing the sections with TBS, EGFR (1:50), HER2 (1: 200), AMAP1 (1: 500), GEP100 (1: 100), EPB41L5 (EBL5; 1: 1000), or c-MET ( 1:50) with the primary antibody (the ratio in parentheses is the dilution factor) for 30 minutes at room temperature. After further washing with TBS, each primary antibody was detected using EnVision (trademark) (DAKO, Japan) as a secondary antibody, and color was developed using diaminobenzidine. Each section was counterstained with hematoxylin, washed with water, and then subjected to penetration treatment and encapsulation treatment according to a routine method.

(5) Evaluation All samples were evaluated independently by two pathologists in a blinded manner.

  For EGFR, the degree of positivity was evaluated for each sample based on a signal intensity of 0 to 2+, with a normal (non-cancerous) duct in the same patient as control 1+.

  For HER2, each sample was evaluated based on the signal intensity, the degree of staining of the cell membrane, and the ratio of the number of cells. When tumor cells with strong membrane staining are more than 30%, the value is 3+. When tumor cells with “weak” to “moderate” membrane staining are 10% to 30%, or strong membrane staining is observed 2+ if tumor cells are less than 10%, 1+ if less than 10% of tumor cells are observed with slight membrane staining, tumor with no membrane staining or less than 10% membrane staining When it was observed only in cells, it was set to 0.

  For AMAP1, normal background staining was 1+, and when staining was observed in both cell membrane and cytoplasm, it was evaluated as 2+.

  For GEP100, each sample was evaluated at 1+ and 2+ based on signal intensity. The normal gland cytoplasmic staining was determined as 1+.

  For c-MET, a control (normal non-cancerous) duct sample with a lower signal intensity was evaluated as 0, and a signal intensity equivalent to that of a normal duct sample was 1+. Those having higher signal intensity were evaluated as 2+.

  Regarding EPB41L5 (EBL5), first, those having no staining in the cytoplasm, that is, those having a staining signal intensity equivalent to that of a normal normal duct sample as a control, are evaluated as 0, and are moderate compared with normal duct samples Those having a high staining signal intensity were evaluated as 1+, and those having a significantly high staining signal intensity compared to normal breast duct samples were evaluated as 2+.

(6) Statistical analysis To determine whether factors that have been considered predictive factors, such as age and resection margin, have a significant impact on the period from completion of radiotherapy to recurrence, Kaplan A Meyer curve was determined. Using a stepwise multiple regression analysis (InstituteSas IS: JMPR 10 Modeling and Multivariate Methods, Cary, NC: SAS Institute Inc, 2012 pp. 139-160), the natural logarithm and expression of the period from completion of radiation therapy to recurrence were Correlated markers were identified. Predictor variables that are candidates for stepwise multiple regression analysis are EGFR, HER2, AMAP1, GEP100, EPB4IL5 (EBL5), c-MET, and their interaction terms. The p-value threshold used to enter or leave the factor is 0.05. Each subgroup was determined according to the selected marker combination. Logarithmic time to recurrence was compared between subgroups by analysis of variance using the Tukey-Kramer HSD (honestly significant difference) test. A Kaplan-Meier curve was calculated for the time to the subgroup event. A p value less than 0.05 was judged to be statistically significant. These analyzes were performed using JMP (registered trademark) (Version 10, SAS Institute Inc., NC.).

<Result>
(1) Clinical characteristics and pathological characteristics of patients The characteristics and treatment methods of patients are shown in Table 1 below. In addition, Table 2 shows the pathological characteristics of the patient, as well as the time period after completion of radiation therapy to recurrence. Furthermore, the immunohistochemical score for each marker is shown in Table 3.

  For pathological tumor stage, 7 patients were Tis, 9 patients were T1, and 3 patients were T2. One of the seven DCIS patients had received chemotherapy prior to surgery and his biopsy sample had been diagnosed with invasive ductal carcinoma with a clinical tumor stage of T3. 15 patients were negative for lymph node metastasis and 4 patients were positive for lymph node metastasis. Twelve patients were diagnosed with negative margins, while three patients were diagnosed with proximal margins and four were positive margins. For these 19 patients, the median period from completion of radiation therapy to local recurrence was 38 months (distributed in the range of 8-179 months).

(2) Clinical factor and period until local recurrence First, it was investigated whether the state of the resected stump, age, ER positivity, or PgR positivity was correlated with the period until local recurrence after BCT. As a result, patients with negative margins showed a median of 40 months for survival without local recurrence after BCT (FIG. 5A). In contrast, patients who were near or positive at the resection end showed a median value of 38 months (FIG. 5A). Similarly, patients younger than 50 years showed a median of 35 months, while patients over 50 years of age also showed a median of 35 months (FIG. 5B). ER positive patients showed a median of 35 months, whereas ER negative patients showed a median of 31.5 months (FIG. 5C). PgR positive patients showed a median of 35 months, whereas PgR negative patients showed a median of 20.5 months (FIG. 5D). As determined by the log rank test, the resected stump (p = 0.48), age (p = 0.97), ER (p = 0.58), and PgR (p = 0.32) There was no statistical difference in the median period of local recurrence after BCT.

(3) Expression of gene translation product in GEP100-Arf6-AMAP1 pathway It was found that high level co-expression of GEP100 and AMAP1 correlates with local recurrence. The GEP100-Arf6-AMAP1 pathway can be activated by several receptor tyrosine kinases such as EGFR and Her2 described above. Therefore, protein expression of AMP1 and GEP100, and EGFR and Her2 was analyzed.

  For EGFR, expression of score 1+ was observed in 5 cases (27.7%), 13 cases (72.2%) were negative (score 0), and one case was not applicable. For HER2, 5 cases (26.3%), 8 cases (42.1%), 4 cases (21.0%), and 2 cases (10.5%) were 3+, 2+, respectively. Scored as 1+ and 0. For AMAP1, 5 cases (26.3%) were scored as 2+ and 14 cases (73.7%) were scored as 1+. For GEP100, 8 cases (42.1%) were scored as 2+ and 11 cases (57.9%) were scored as 1+. For c-Met, 7 cases (36.8%) are scored 2+, 6 cases (31.6%) are scored 1+, and 6 cases (31.6%) are 0. Scored. For EPB41L5 (EBL5), 2 cases (10.5%) are scored as 3+, 4 cases (21.1%) are scored as 2+, and 5 cases (26.3%) are 1+ And 8 cases (42.1%) were scored as 0. (Table 3). Furthermore, examples of photomicrographs of immunohistochemical staining are shown in FIGS.

  Gene translation in combination with GEP100 and AMAP1 by analysis using stepwise multiple regression analysis (InstituteSas IS: JMP (registered trademark) 10 Modeling and Multivariate Methods, Cary, NC: SAS Institute Inc, 2012 pp. 139-160) In cases where the product is co-expressed at a high level, there is a significant correlation with the short period from completion of BCT to local recurrence (P <0.001), and the interaction effect is also short in the same period. (P = 0.018).

  FIG. 3 and FIG. 4 show the results of Kaplan-Meier curve analysis showing how many patients escape from recurrence over time after completion of BCT. In FIG. 3, “Homo” indicates a case where AMAP1 and GEP100 are co-expressed, “Wild / Hetero” indicates a case where only one of AMAP1 or GEP100 is expressed, and both of these genes are expressed. Indicates a case that was not. In FIG. 4, “Homo” indicates a case where c-MET and EBL5 are co-expressed, “Wild / Hetero” indicates a case where only one of c-MET or EBL5 is expressed, and any of these genes In this case, no case is expressed.

  According to the Kaplan-Meier curve of FIG. 3, there was a statistically significant difference between the “Homo” group and “Wild / Hetero” (P = 0.0001) (FIG. 3). On the other hand, high level expression of GEP100 or AMAP1 itself did not show such superiority (FIG. 3). Furthermore, according to the Kaplan-Meier curve in FIG. 4, there was a statistically significant difference between the “Homo” group and “Wild / Hetero” (P = 0.0005) (FIG. 4). On the other hand, high level expression of c-MET or EBL5 itself did not show such superiority (FIG. 4).

  In addition, none of EGFR or HER2 itself, or GEP100 or AMAP1 co-expression with EGFR or HER2 showed any superiority to the rapidity of local recurrence.

  Breast-conserving therapy (BCT) for the early stages of invasive and non-invasive ductal carcinoma provides excellent local control rates and patient survival. However, there is still a significant population of patients who experience local recurrence after BCT. In this example, as a result of paying attention to the population who experienced local recurrence after BCT, the state and age of the resection stump were not correlated with the short period until local recurrence, but GEP100 protein and AMAP1 protein It was found that each of the co-expressions, as well as the co-expression with the combination of c-MET and EBL5, correlated statistically predominantly with the short time to local recurrence. On the other hand, the expression of any one of GEP100, AMAP1, c-MET, and EBL5 was not correlated with the short period until local recurrence. These results are consistent with the hypothesis that GEP100, AMAP1, c-MET and EBL5 are components of the Arf6 pathway, that is, expression of only one of these genes constitutes an active Arf6 pathway. Will not. The expression of any of EGFR, HER2, ER or PgR itself was not correlated with the short time to local recurrence as previously reported (Millar EK, Graham PH, O'Toole SA, et al: Prediction of local recurrence, distant metastases, and death after breast-conserving therapy in early-stage invasive breast cancer using a five-biomarker panel. J Clin Oncol 27: 4701-8, 2009).

Claims (7)

A kit for predicting the risk of local recurrence after breast-conserving breast cancer therapy,
A kit comprising two diagnostic agents containing a protein and / or polypeptide capable of specifically detecting each of the translation products of two genes of any of the following combinations (i) to (ii):
(I) AMAP1 and GEP100, and
(Ii) EBL5 and C-Met . The kit according to claim 1, wherein the protein and / or polypeptide is an antibody and / or a fragment thereof. To predict a patient's risk of local recurrence after breast-conserving therapy,
In a biological sample collected from a patient who has received breast cancer breast-conserving therapy, the translation product expression levels of any two genes of the following combinations (i) to (ii) are respectively determined:
Methods for predicting the risk of local recurrence after breast-conserving breast cancer therapy , including :
(I) AMAP1 and GEP100, and
(Ii) EBL5 and C-Met . The method of claim 3 , wherein the biological sample is breast tissue. The two gene translation product expression levels are determined the translation product thereof genes by each capable of specifically recognizing the protein and / or polypeptide immunohistochemical method using, according to claim 3 or 4 Method. The method according to claim 5 , wherein the protein and / or polypeptide is an antibody and / or a fragment thereof. The method according to any one of claims 3 to 6 , wherein the criteria used to predict the risk of local recurrence after breast cancer breast-conserving therapy for the patient are the following (i) and (ii) :
(I) all determined above two genes translation product expression levels is higher than the corresponding control level of expression, the patient local recurrence risk of breast cancer is not high;
(Ii) in one of the determined above-mentioned two gene translation product expression levels, when or lower than the corresponding control level of expression, or are equivalent, the patient local recurrence risk of breast cancer have low .