Molecular markers for determining the efficacy of gemcitabine + S-1 therapy (NAC-GS) as neoadjuvant chemotherapy for pancreatic cancer, a kit for determining said efficacy, and a method for determining said efficacy.

Abstract

To determine the effectiveness of NAC-GS for resectable pancreatic cancer. [Solution] Protein ITGB1, protein PODXL, or a combination of protein ITGB1 and protein PODXL are used as molecular markers to indicate the effectiveness of NAC-GS therapy.

Description

Technical Field

[0001] The present invention relates to a molecular marker for determining the efficacy of gemcitabine + S-1 therapy (NAC-GS) as preoperative chemotherapy for pancreatic cancer, a kit for determining the efficacy, and a method for determining the efficacy.

Background Art

[0002] Pancreatic cancer is said to have the worst prognosis among cancers. The reasons include that it is difficult to detect pancreatic cancer at an early stage because it is a retroperitoneal organ, and because the motility of pancreatic cancer cells is extremely high, it quickly infiltrates surrounding blood vessels, the digestive tract, nerves, etc., and also metastasizes to nearby lymph nodes or distant metastases to the liver, etc. Pancreatic cancer is a representative of refractory cancers, and the only way to improve the survival rate is surgery by early detection.

[0003] Looking at all pancreatic cancer patients, the 5-year survival rate is extremely low, at 5-10%. On the other hand, in Reference Material 1, the 5-year survival rate of pancreatic cancer including postoperative cases is shown, which is Stage IA: 31-39%, Stage IB: 22-27%, Stage IIA: 16-25%, Stage IIB: 8-10%, Stage III: 0-7%, Stage IV: 0-4% (Reference Material 1: Lancet Oncol 2013; 14: pp. 476-85). Thus, it has been shown that the 5-year survival rate, that is, the prognosis of pancreatic cancer patients rapidly deteriorates across Stages IIA and IIB.

[0004] Incidentally, while biopsies are accurate for diagnosing cancer, they are painful for patients. Therefore, preliminary tests using cancer diagnostic markers are generally performed. Cancer diagnostic markers are substances that are specifically produced in the body by cancer, and the progression of cancer can be evaluated by measuring their amount in body fluids. For example, Patent Document 1 discloses a diagnostic marker for pancreatic cancer and intraductal papillary mucinous neoplasms characterized by containing one or more proteins selected from the group consisting of secretoglobin, Family 1D, Member 2, and podocalixin-like proteins. Non-Patent Document 1 also describes that PODXL can be used as a diagnostic tool for pancreatic ductal adenocarcinoma.

[0005] Under these circumstances, Patent Document 2 discloses a prognostic marker for pancreatic cancer that includes a combination of the protein ITGB1 and the protein PODXL and / or the protein BCL7B. The prognostic marker disclosed in Patent Document 2 can predict the "prognosis" of pancreatic cancer patients with high sensitivity and specificity. This makes it possible to further subdivide pancreatic cancer cases into good-prognosis and poor-prognosis groups, enabling the provision of more detailed medical care tailored to the patient's situation, and ultimately contributing to an improvement in the prognosis of pancreatic cancer patients.

[0006] Incidentally, the only curative treatment for pancreatic cancer is surgical resection. Resectability assessment determines whether surgery can be performed using standard surgical methods to ensure no residual cancer is macroscopically or histologically. For example, the National Comprehensive Cancer Network (NCCN) guidelines classify pancreatic cancer into three categories based on resectable criteria: "resectable," "borderline resectable," and "unresectable." In Japan, the "Pancreatic Cancer Treatment Guidelines (7th Edition)" classifies resecability into three categories based on the degree of local invasion and the presence or absence of distant metastasis: "resectable," "borderline resectable," and "unresectable."

[0007] According to the 2022 pancreatic cancer treatment guidelines, if surgical resection is possible, treatment should consist of surgical resection alone or a combination of surgical resection and drug therapy. The results of a randomized phase III trial (Prep-02 / JSAP05 trial) of neoadjuvant therapy using gemcitabine hydrochloride and S-1 for resectable pancreatic cancer have been reported, demonstrating the superiority of this neoadjuvant therapy. As a result, the 2022 pancreatic cancer treatment guidelines recommend neoadjuvant therapy using gemcitabine hydrochloride and S-1 (NAC-GS therapy) for resectable pancreatic cancer.

[0008] The most promising aspect of neoadjuvant therapy, including NAC-GS therapy, is its ability to control microscopic distant metastases. Chemotherapy can eliminate cancer cells in the liver, lymph nodes, and peritoneum, thereby reducing the frequency of postoperative recurrence and extending survival time. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 2017 / 098915 [Patent Document 2] Patent No. 7246731 [Non-patent literature]

[0010] [Non-Patent Document 1] TERESA J. et al., Podocalyxin-like protein 1 expression is useful to differentiate pancreatic ductal adenocarcinomas from adenocarcinomas of the biliary and gastrointestinal tracts, Hum. Pathol., 2017, Vol 38, pp. 359-364 [Overview of the project] [Problems that the invention aims to solve]

[0011] However, NAC-GS therapy for resectable pancreatic cancer generally takes several weeks or more, and if the treatment is ineffective, there is a risk that the pancreatic cancer may progress during the treatment period. In some cases, it may progress from a resectable stage to a borderline resectable stage, or even become unresectable. In other words, it cannot be denied that applying NAC-GS therapy carries the risk of missing the opportunity to resect the pancreatic cancer. This is the situation that must be avoided at all costs. Conversely, there is a certain percentage of cases that can be cured by surgery alone without NAC-GS therapy, and for these pancreatic cancer patients, preoperative treatment such as NAC-GS therapy is unnecessary. There are no biomarkers to select the group for which NAC-GS therapy is not expected to be effective and / or for which NAC-GS therapy is unnecessary, and the clinical application of biomarkers that can select these groups is an urgent issue.

[0012] Therefore, in view of the above circumstances, the present invention aims to provide a molecular marker for determining the effectiveness of gemcitabine + S-1 therapy (NAC-GS) as neoadjuvant chemotherapy for pancreatic cancer, a kit for determining said effectiveness, and a method for determining said effectiveness. [Means for solving the problem]

[0013] As a result of diligent research to achieve the above-mentioned objectives, the inventors of the present invention have found that when protein ITGB1 or protein PODXL is highly expressed, particularly when protein PODXL is highly expressed, or when both protein ITGB1 and protein PODXL are highly expressed, the efficacy of NAC-GS therapy is low, and that these proteins ITGB1 and / or PODXL function as molecular markers for determining the efficacy of gemcitabine + S-1 therapy (NAC-GS) as neoadjuvant chemotherapy for pancreatic cancer, thus completing the present invention.

[0014] This invention encompasses the following: [1] Protein ITGB1, or Protein PODXL, or The combination of protein ITGB1 and protein PODXL, A molecular marker that demonstrates the efficacy of gemcitabine and S-1(GS) therapy as neoadjuvant chemotherapy (NAC) for pancreatic cancer, characterized by containing [specific element].

[0015] [1-2] Protein PODXL, or The combination of protein ITGB1 and protein PODXL, A molecular marker as described in [1], including the molecular marker described in [1].

[0016] [2] ITGB1 detection means for detecting the protein ITGB1, or PODXL detection means for detecting the protein PODXL, or An ITGB1 detection means for detecting the protein ITGB1 and a PODXL detection means for detecting the protein PODXL, A kit for determining the efficacy of gemcitabine and S-1(GS) therapy as neoadjuvant chemotherapy (NAC) for pancreatic cancer, characterized by containing [specific ingredient / feature]. [2-1] PODXL detection means for detecting the protein PODXL, or ITGB1 detection means for detecting the protein ITGB1 and PODXL detection means for detecting the protein PODXL, The kit described in [2], including the kit described in [2]. [3] The kit according to [2], characterized in that the ITGB1 detection means and / or the PODXL detection means are an antibody against the protein ITGB1 and / or an antibody against the protein PODXL, respectively. [3-1] The kit according to [3], wherein the antibody described above comprises a polyclonal antibody or a monoclonal antibody. [4] The kit according to [2], characterized by comprising a reagent used in at least one method selected from immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and immunoaggregation. [5] The kit described in [2], wherein the above protein ITGB1 has one of the amino acid sequences (1) to (3) below. (1) The amino acid sequence described in Sequence ID No. 1; (2) It consists of an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added in the amino acid sequence described in SEQ ID NO: 1, and is an amino acid sequence of a protein that functions as an integrin β subunit; (3) It consists of an amino acid sequence having 90.0% or more homology to the amino acid sequence described in SEQ ID NO: 1, and is an amino acid sequence of a protein that functions as an integrin β subunit. [6] The kit according to [2], wherein the protein PODXL has any one of the following amino acid sequences (4) to (6). (4) The amino acid sequence described in SEQ ID NO: 2; (5) It consists of an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added in the amino acid sequence described in SEQ ID NO: 2, and is an amino acid sequence of a protein that functions as a podocalyxin-like protein; (6) It consists of an amino acid sequence having 90.0% or more homology to the amino acid sequence described in SEQ ID NO: 2, and has homology to an amino acid sequence of a protein that functions as a podocalyxin-like protein.

[0017] [7] A method for determining the efficacy of gemcitabine and S-1 (GS) therapy as neoadjuvant chemotherapy (NAC) for pancreatic cancer, which includes the step of measuring the expression of protein ITGB1, or the expression of protein PODXL, or the expression of protein ITGB1 and protein PODXL in a biological sample. [7-1] The method according to [7], wherein the step of measuring the expression of protein ITGB1, or the expression of protein PODXL, or the expression of protein ITGB1 and protein PODXL in a biological sample includes measuring the expression of protein PODXL, or the expression of protein ITGB1 and protein PODXL. [8] The method according to [7], characterized in that when the protein ITGB1 or the protein PODXL is highly expressed, or when the expression of the protein ITGB1 and the protein PODXL is highly expressed, it is determined that the efficacy is low. [9] The method according to [7], characterized in that when the above protein ITGB1 or the above protein PODXL is highly expressed, or when the expression of the above protein ITGB1 and the above protein PODXL is highly expressed, the above efficacy is judged to be low, and the indication for surgical treatment or the indication for other neoadjuvant therapy prior to surgical treatment is judged.

[10] The method according to [9], characterized in that the other neoadjuvant therapy includes at least one of gemcitabine / nab-paclitaxel (GnP) therapy, FOLFIRINOX therapy, chemoradiotherapy, and an immune checkpoint inhibitor. [Effects of the Invention]

[0018] The present invention provides a molecular marker that can be used to determine the effectiveness of gemcitabine and S-1(GS) therapy as neoadjuvant chemotherapy (NAC) for pancreatic cancer. By utilizing this molecular marker, it is possible to provide medical care based on the effectiveness of gemcitabine and S-1(GS) therapy as neoadjuvant chemotherapy (NAC) for pancreatic cancer. The present invention makes it possible to prevent situations in which surgery becomes impossible due to preoperative treatment being performed in cases where the effectiveness of NAC-GS therapy cannot be expected.

[0019] Furthermore, according to the present invention, it is possible to provide a kit for determining the effectiveness of gemcitabine and S-1 (GS) therapy as neoadjuvant chemotherapy (NAC) for pancreatic cancer, which detects the above molecular markers, and a method for determining the effectiveness. [Brief explanation of the drawing]

[0020] [Figure 1] This is a characteristic diagram showing the Kaplan-Meier curves for all cases, based on whether or not NAC-GS treatment was administered. [Figure 2] This is a characteristic diagram showing the Kaplan-Meier curves for resectable pancreatic cancer cases with and without NAC-GS treatment. [Figure 3] This is a characteristic diagram showing the Kaplan-Meier curve based on molecular marker scores for all cases. [Figure 4]This is a characteristic diagram showing Kaplan-Meier curves based on molecular marker scores for resectable pancreatic cancer cases. [Figure 5] This is a characteristic diagram showing Kaplan-Meier curves based on molecular marker scores for resectable pancreatic cancer cases that underwent NAC-GS therapy followed by surgical resection. [Figure 6] This is a characteristic diagram showing Kaplan-Meier curves based on molecular marker scores for cases of borderline resectable pancreatic cancer that underwent NAC-GS therapy followed by surgical resection. [Figure 7] This is a characteristic diagram showing Kaplan-Meier curves illustrating survival rates by stage for all cases. [Figure 8] This characteristic diagram shows Kaplan-Meier curves illustrating survival rates for resectable pancreatic cancer cases with and without NAC-GS therapy (preoperative treatment). [Figure 9] This characteristic diagram shows Kaplan-Meier curves illustrating survival rates for resectable pancreatic cancer cases, based on analysis results using monoclonal antibodies, comparing the group with high expression of ITGB1 and PODXL with the group without. [Figure 10] This characteristic diagram shows Kaplan-Meier curves illustrating survival rates for patients with resectable pancreatic cancer treated with NAC-GS therapy, comparing those with high expression of ITGB1 and PODXL (high expression of both) with those without. [Figure 11] This characteristic diagram shows Kaplan-Meier curves illustrating survival rates with and without preoperative treatment for patients with resectable pancreatic cancer who received NAC-GS therapy, specifically those who did not belong to the group expressing high levels of ITGB1 and PODXL, based on analysis results using monoclonal antibodies. [Figure 12] This characteristic diagram shows Kaplan-Meier curves illustrating survival rates for patients with resectable borderline pancreatic cancer, comparing those who highly express ITGB1 and PODXL with those who do not. [Figure 13]This characteristic diagram shows Kaplan-Meier curves illustrating survival rates for resectable pancreatic cancer cases, categorized by stage, based on analysis results using monoclonal antibodies, for a good prognosis group (groups that do not highly express both ITGB1 and PODXL) and a poor prognosis group (groups that highly express both ITGB1 and PODXL). [Figure 14A] This characteristic diagram shows Kaplan-Meier curves illustrating survival rates for all cases, categorized by stage into good-prognosis and poor-prognosis groups, based on analysis results using monoclonal antibodies. [Figure 14B] This characteristic diagram shows Kaplan-Meier curves illustrating survival rates for patients with resectable pancreatic cancer, categorized by stage into good-prognosis and poor-prognosis groups, based on analysis results using monoclonal antibodies. [Figure 15] This characteristic diagram shows the Kaplan-Meier curves illustrating the survival rates for all cases, categorized as resectable pancreatic cancer (R) and borderline resectable pancreatic cancer (BR). [Figure 16A] This characteristic diagram shows Kaplan-Meier curves indicating survival rates for all cases, based on tumor size, intrapancreatic location of pancreatic cancer, and CA19-9. [Figure 16B] This characteristic diagram shows Kaplan-Meier curves illustrating survival rates for patients with resectable pancreatic cancer, using tumor size, intrapancreatic location of the cancer, and CA19-9 as indicators. [Figure 17A] This characteristic diagram shows the survival rate curve obtained by performing CART analysis on eight factors for all cases: the combination of ITGB1 and PODXL, clinical stage, neoadjuvant chemotherapy, borderline resectable pancreatic cancer, NAC-GS, tumor size, intrapancreatic location of the pancreatic cancer, and CA19-9. [Figure 17B] This characteristic diagram shows the survival rate curve obtained by performing a CART analysis on eight factors for patients with resectable pancreatic cancer: the combination of ITGB1 and PODXL, clinical stage, neoadjuvant chemotherapy, borderline resectable pancreatic cancer, NAC-GS, tumor size, intrapancreatic location of the pancreatic cancer, and CA19-9. [Figure 18A]This characteristic diagram shows the survival rate curve obtained by performing CART analysis on all cases, considering factors such as tumor size, intrapancreatic location of pancreatic cancer, CA19-9 set, ITGB1 and PODXL combination, clinical stage, neoadjuvant chemotherapy, borderline resectable pancreatic cancer, and NAC-GS. [Figure 18B] This characteristic diagram shows the survival rate curve obtained by performing a CART analysis on patients with resectable pancreatic cancer, considering factors such as tumor size, intrapancreatic location of the pancreatic cancer, CA19-9 set, ITGB1 and PODXL combination, clinical stage, neoadjuvant chemotherapy, borderline resectable pancreatic cancer, and NAC-GS. [Figure 19] Figure 19A is a characteristic diagram showing Kaplan-Meier curves for all cases, based on analysis results using an anti-PODXL monoclonal antibody, illustrating the survival rates of the group that highly expresses PODXL and the group that does not. Figure 19B is a characteristic diagram showing Kaplan-Meier curves for resectable pancreatic cancer cases, based on analysis results using an anti-PODXL monoclonal antibody, illustrating the survival rates of the group that highly expresses PODXL and the group that does not. [Figure 20] This graph shows the results of measuring PODXL concentrations using ELISA with anti-PODXL monoclonal antibodies in serum from pancreatic cancer patients before treatment and serum from healthy individuals (controls). [Modes for carrying out the invention]

[0021] This application claims priority to Japanese Patent Application No. 2024-188719, filed on October 28, 2024.

[0022] The molecular marker according to the present invention is a marker related to the effectiveness of gemcitabine and S-1(GS) therapy (hereinafter referred to as NAC-GS therapy) as neoadjuvant chemotherapy (NAC) for pancreatic cancer. By detecting the molecular marker according to the present invention, it is possible to predict that the effectiveness of NAC-GS therapy will be low in pancreatic cancer patients who are to be treated surgically for pancreatic cancer.

[0023] 1. Molecular markers The molecular markers according to the present invention are intended to predict the effectiveness of NAC-GS therapy in pancreatic cancer patients who are to be treated surgically. If the effectiveness of NAC-GS therapy in pancreatic cancer patients who are to be treated surgically can be predicted, it will be possible to select a more effective treatment method without missing the optimal timing for pancreatic cancer resection.

[0024] The inventors' research revealed that molecular markers, including protein ITGB1 alone, protein PODXL alone, or a combination of protein ITGB1 and protein PODXL, can surprisingly predict the effectiveness of NAC-GS therapy in pancreatic cancer patients who are undergoing surgical treatment for pancreatic cancer.

[0025] In Japanese Patent No. 7246731, it was shown that the proteins ITGB1 and PODXL could be used as prognostic markers capable of predicting the prognosis of pancreatic cancer patients with high sensitivity and specificity. The fact that these proteins, ITGB1, PODXL, or a combination of ITGB1 and PODXL, can predict the effectiveness of NAC-GS therapy in pancreatic cancer patients undergoing surgical treatment is surprising and contradicts conventional technological understanding.

[0026] In other words, the molecular marker according to the present invention is the protein ITGB1 (hereinafter abbreviated as "ITGB1"), the protein PODXL (hereinafter abbreviated as "PODXL"), or a combination of ITGB1 and PODXL. By detecting this protein, it becomes possible to predict the effectiveness of NAC-GS therapy in pancreatic cancer patients who are to be treated surgically for pancreatic cancer.

[0027] In this invention, "pancreatic cancer patients to whom surgical treatment for pancreatic cancer is applicable" is determined for each pancreatic cancer patient in accordance with the Pancreatic Cancer Treatment Guidelines and Pancreatic Cancer Clinical Practice Guidelines compiled by the Japan Pancreas Society, the National Comprehensive Cancer Information Network (NCCN) Guidelines, etc. "Pancreatic cancer patients to whom surgical treatment for pancreatic cancer is applicable" means pancreatic cancer patients other than those classified as unresectable or borderline resectable in the resectability classification. The resectability classification is determined, for example, in the above-mentioned Pancreatic Cancer Treatment Guidelines, based on pancreatic dynamic CT images, and the criteria are classified into "Resectable (R)", "Borderline resectable (BR)", and "Unresectable (UR)" from the perspective that R0 resection without residual cancer is possible macroscopically and histologically by standard surgery.

[0028] Specifically, "resectable: R" is defined as a tumor in which there is no contact with the superior mesenteric vein or portal vein, or contact / invasion is observed at less than 180 degrees but no occlusion is present, and clear adipose tissue is observed between the superior mesenteric artery, celiac artery, and common hepatic artery and the tumor, with no contact / invasion.

[0029] Furthermore, the "resectable boundary (BR)" is further subdivided depending on whether the invasion is into the portal venous system or the arterial system. BR with only portal venous system invasion (BR-PV) is defined as a case where there is no tumor contact or invasion into the superior mesenteric artery, celiac artery, or common hepatic artery, but there is contact, invasion, or occlusion of the superior mesenteric vein or portal vein at an angle of 180 degrees or more, and the extent of this does not extend beyond the lower edge of the duodenum. BR with arterial system invasion (BR-A) is defined as a case where there is contact or invasion of the tumor with the superior mesenteric artery or celiac artery at an angle of less than 180 degrees, but no stenosis or deformation is observed, or where there is tumor contact or invasion into the common hepatic artery, but no contact or invasion into the proper hepatic artery or celiac artery.

[0030] Furthermore, "unresectable (UR)" is further classified according to the presence or absence of distant metastasis. Locally advanced UR without distant metastasis (UR-LA) is defined as cases where there is contact, invasion, or occlusion of the superior mesenteric vein or portal vein at an angle of 180 degrees or more with the tumor, and the extent of this extends beyond the lower edge of the duodenum; cases where there is contact or invasion of the tumor at an angle of 180 degrees or more with the tumor with the superior mesenteric artery or celiac artery; cases where there is contact or invasion of the tumor with the tumor with the common hepatic artery, and the contact or invasion extends to the proper hepatic artery and celiac artery; or cases where there is contact or invasion of the tumor with the aorta. UR with distant metastasis (UR-M) includes cases with distant metastasis (including metastasis to lymph nodes other than regional lymph nodes), and lymph node metastasis beyond group 3 that does not fall into the group classification is also considered distant metastasis.

[0031] In this specification, "NAC-GS therapy" refers to adjuvant chemotherapy using gemcitabine hydrochloride and S-1 (also called TS-1) administered before surgery to pancreatic cancer patients who are eligible for surgical treatment of pancreatic cancer. S-1 is a combination drug containing tegafur, gimeracil, and oteracil potassium. The method and dosage of administration of gemcitabine hydrochloride and S-1 are not particularly limited, but for example, gemcitabine hydrochloride may be administered for 2 weeks with a 1-week rest period, and S-1 may be administered twice a day (after breakfast and dinner) for 7 days from the day of gemcitabine hydrochloride administration, resulting in a total of 4 administrations of gemcitabine hydrochloride and 4 weeks of oral S-1 within 8 weeks. Alternatively, two courses can be performed, each consisting of 3 weeks, where gemcitabine hydrochloride is administered intravenously on days 1 and 8, S-1 is administered orally twice a day (after breakfast and dinner) from day 1 to day 15, and there is a rest period from day 16 to day 22.

[0032] Furthermore, surgical treatment for pancreatic cancer after completion of NAC-GS therapy refers to pancreatectomy, and surgical approaches include open surgery, laparoscopic-assisted surgery, laparoscopic surgery, and robot-assisted surgery. Standard pancreatectomy procedures include pancreaticoduodenectomy (pylorus-preserving or subtotal stomach-preserving) and distal pancreatectomy. Other less invasive procedures include duodenum-preserving pancreaticodontectomy, pancreaticoduodenectomy II, and distal pancreatectomy. Extended procedures include pancreaticoduodenectomy with proper hepatic artery and portal vein, and distal pancreatectomy with celiac artery. In addition to pancreatectomy, surgical treatment for pancreatic cancer after completion of NAC-GS therapy also includes reconstructive surgery and lymph node dissection. Other pancreatectomy procedures for low-grade pancreatic tumors include central pancreatectomy, partial pancreatectomy, and pancreatic enucleation.

[0033] Here, ITGB1, one of the molecular markers, is an integrin β subunit that functions as a cell adhesion receptor on the cell surface. ITGB1 links the extracellular matrix and the intracellular skeletal structure and is involved in cell-to-cell adhesion, but its specific function in cancer cells, including pancreatic cancer, remains largely unknown.

[0034] PODXL is a glycoprotein belonging to the CD34-related family that functions as a podocalixin-like protein. It is involved in the development and differentiation of blood cells, cell adhesion and morphogenesis, as well as the regulation of carcinogenesis. Podocalixin has also been reported to increase the malignant phenotype of breast cancer cells and prostate cancer cells, and some reports suggest it could be used as a marker of cancer progression.

[0035] The above-mentioned ITGB1 has the amino acid sequence of SEQ ID NO: 1, and the above-mentioned PODXL has the amino acid sequence of SEQ ID NO: 2. It should be noted that in some individuals, ITGB1 and PODXL may have various variants due to mutations known as single nucleotide polymorphisms, and such variants also serve as molecular markers according to the present invention. In other words, the molecular markers ITGB1 and PODXL according to the present invention are not limited to the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, but also include variants containing amino acid sequences different from those sequences.

[0036] For example, ITGB1 can be a protein consisting of any of the following amino acid sequences (1) to (3): (1) The amino acid sequence described in Sequence ID No. 1; (2) An amino acid sequence of a protein that functions as an integrin β subunit, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence described in Sequence ID No. 1; (3) An amino acid sequence of a protein that has 90.0% or more homology to the amino acid sequence described in Sequence ID No. 1 and functions as an integrin β subunit.

[0037] Here, "multiple" in (2) can be 80, which corresponds to approximately 10% of the total 798 amino acids in the sequence number 1, and can be 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2. Also, the homology value in (3) may exceed 90.0%, for example, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, or 99.8%.

[0038] These variants of ITGB1 specified in (2) and (3) have different amino acid sequences from the amino acid sequence of SEQ ID NO: 1, but have the same function as the protein ITGB1 consisting of the amino acid sequence of SEQ ID NO: 1. Having the same function as the protein ITGB1 means, as mentioned above, that they are integrin β subunits and function as cell adhesion receptors on the cell surface.

[0039] Furthermore, for example, PODXL can be a protein consisting of any of the following amino acid sequences (4) to (6): (4) The amino acid sequence described in Sequence ID No. 2; (5) An amino acid sequence of a protein that functions as a podocalixin-like protein, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence described in Sequence ID No. 2; (6) The amino acid sequence of a protein that has 90.0% or more homology to the amino acid sequence described in Sequence ID No. 2 and functions as a podocalixin-like protein.

[0040] Here, "multiple" in (5) can be 60, which corresponds to approximately 10% of the total 558 amino acids in the sequence number 2, and can be 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2. Also, the homology value in (6) may exceed 90.0%, for example, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, or 99.8%.

[0041] These variants of PODXL specified in (4) and (5) have different amino acid sequences from the amino acid sequence of SEQ ID NO: 2, but have the same function as the protein PODXL consisting of the amino acid sequence of SEQ ID NO: 2. Having the same function as the protein PODXL means, as described above, that it functions as a podocalixin-like protein.

[0042] Furthermore, the presence and location of deletions, substitutions, and / or additions in amino acid sequences (2), (3), (5), and (6), as well as sequence identity, can be analyzed by direct comparison of the sequences. Specifically, this can be done using commercially available sequence analysis software.

[0043] 2. Method for determining the effectiveness of NAC-GS therapy Next, we will explain, step by step, the method for determining the effectiveness of NAC-GS therapy using the above molecular markers as the target of detection.

[0044] (1) Process for obtaining biological samples In this process, biological samples are obtained from the subject. Here, biological samples refer to pancreatic cancer cells and pancreatic cancer tissue fragments collected by biopsy, as well as the subject's bodily fluids such as blood, lymph, and urine, in addition to serum and plasma, which are collected bodily fluids that have been processed. Surgically removed pancreatic cancer tissue is also included. In one embodiment, the biological sample used in this process is a pancreatic cancer tissue fragment removed during pathological diagnosis. For example, pancreatic cancer cells and pancreatic cancer tissue fragments can be obtained by biopsy such as endoscopic ultrasonography-fine needle aspiration (EUS-FNA), ERCP-guided pancreatic fluid cytology, and abdominal ultrasound-guided fine needle biopsy. In another embodiment, the biological sample used in this process is blood, such as whole blood, serum, or plasma.

[0045] (2) Measurement process for molecular markers In this process, ITGB1 and / or PODXL, which are molecular markers indicating the efficacy of NAC-GS therapy, are detected in a biological sample. Here, detecting the molecular markers ITGB1 and / or PODXL includes both measuring the ITGB1 protein and / or PODXL protein contained in the biological sample, and measuring the mRNA encoding the ITGB1 protein and / or PODXL protein contained in the biological sample.

[0046] To detect molecular marker proteins, substances that can specifically bind to the target protein, such as ligands or antibodies, are used. The term "antibody" includes not only immunoglobulins such as polyclonal antibodies, monoclonal antibodies, and chimeric antibodies, but also fragments containing variable regions, such as Fab fragments, F(ab')2 fragments, Fv fragments, minibodies, scFv-Fc, scFv, diabodies, triabodies, and tetrabodies.

[0047] Furthermore, the molecular marker protein can be measured by detecting the label of the primary antibody against the protein, or by detecting the binding of a secondary antibody or reagent to the primary antibody.

[0048] To detect mRNA encoding a molecular marker protein, nucleic acid probes that can specifically hybridize to the target mRNA or reverse-transcribed cDNA, and PCR primers that can specifically amplify the target mRNA are used. Furthermore, reagents used for hybridization with nucleic acid probes and nucleic acid amplification reactions are also employed to detect mRNA encoding a molecular marker protein.

[0049] In one embodiment, it is preferable to use a pancreatic cancer tissue sample excised during pathological diagnosis as a biological sample and detect and measure molecular markers by immunohistochemical staining. For example, molecular markers can be detected and measured by measuring the concentration as a staining score based on the results of immunohistochemical staining. The measurement method is not particularly limited, but in addition to staining scores obtained by immunohistochemical staining, spectrophotometric analysis methods such as ultraviolet absorption spectroscopy, Bradford method, Lowry method, and BCA method can also be used. In another embodiment, molecular markers can be detected and measured by enzyme-linked immunosorbent assay (ELISA) using antibodies that specifically bind to ITGB1 and antibodies that specifically bind to PODXL. In yet another embodiment, molecular markers can be detected and measured by immunoassay (e.g., latex agglutination) using antibodies that specifically bind to ITGB1 and antibodies that specifically bind to PODXL.

[0050] (3) Judgment process In the above process, the effectiveness of NAC-GS therapy is determined based on the detection and measurement results of molecular markers. Specifically, if the molecular markers ITGB1 or PODXL are highly expressed in biological samples such as pancreatic cancer tissue fragments or blood, particularly if PODXL is highly expressed, or if both ITGB1 and PODXL are highly expressed, the effectiveness of NAC-GS therapy is judged to be low. Here, "high expression" is not particularly limited, but it can be defined as an expression level that is significantly higher compared to the expression levels of ITGB1 and / or PODXL in normal samples or samples from healthy individuals (such as normal pancreatic tissue fragments or blood from healthy individuals) that do not have pancreatic cancer. It also applies if, in the same case, the staining score of pancreatic cancer cells in pancreatic cancer tissue fragments is higher than that of normal pancreatic ducts or islets of Langerhans.

[0051] In this process, the effectiveness of NAC-GS therapy is assessed as described above. If the effectiveness of NAC-GS therapy is deemed low, the surgical treatment described above can be selected without undergoing NAC-GS therapy. Alternatively, if the effectiveness of NAC-GS therapy is deemed low, a different preoperative therapy can be selected prior to surgical treatment.

[0052] Other neoadjuvant therapies available besides NAC-GS therapy are not limited to any particular category and include any therapy that has shown efficacy for resectable pancreatic cancer. These non-NAC-GS neoadjuvant therapies also include novel treatment methods currently in clinical trials. For example, gemcitabine / nab-paclitaxel therapy (Gem / nab-PTX therapy, GnP therapy), whose efficacy as a neoadjuvant therapy is being investigated in the ongoing Phase II clinical trial of neoadjuvant gemcitabine + nab-paclitaxel therapy for resectable pancreatic cancer (https: / / jrct.niph.go.jp / latest-detail / jRCT1011180032) and the randomized controlled Phase III trial comparing neoadjuvant gemcitabine + S-1 therapy and neoadjuvant gemcitabine + nab-paclitaxel therapy for elderly patients with resectable pancreatic cancer (https: / / rctportal.niph.go.jp / s / detail / um?trial_id=jRCTs031220351), can also be used as a neoadjuvant therapy other than NAC-GS therapy. Furthermore, a multi-drug combination therapy known as FOLFIRINOX therapy, which includes three anticancer drugs—5-FU, irinotecan, and oxaliplatin—plus levofolinate, a 5-FU enhancer, can also be used as a neoadjuvant therapy other than NAC-GS therapy. Regarding clinical trials using gemcitabine / nab-paclitaxel therapy and FOLFIRINOX therapy as neoadjuvant therapy, the NUPAT-01 trial (Ann Surg 2022;275(6):1043-1049) was conducted for resectable borderline pancreatic cancer. The results showed that using gemcitabine / nab-paclitaxel therapy or FOLFIRINOX therapy as neoadjuvant therapy extended post-resection survival in patients with resectable borderline pancreatic cancer. Other options include combinations of radiotherapy and chemotherapy, such as chemoradiotherapy with GnP therapy or chemoradiotherapy with S-1. Furthermore, the ongoing Phase III trial, JCOG1908E, is investigating the efficacy of S-1 combination chemoradiotherapy plus the immune checkpoint inhibitor nivolumab (Opdivo) in pancreatic cancer patients. The trial also includes combination therapies of existing pancreatic cancer treatments such as gemcitabine and nab-paclitaxel with immune checkpoint inhibitors.Treatment options that include immune checkpoint inhibitors are also available as neoadjuvant therapies.

[0053] Furthermore, in this process, if the expression levels of either or both ITGB1 and / or PODXL exceed a predetermined threshold, it can be determined that the effectiveness of NAC-GS therapy is extremely low. The predetermined threshold can be, for example, the reference value used when dividing cases with "high expression" as defined above into two groups according to their expression levels. Thus, if the expression levels of ITGB1 and / or PODXL exceed a predetermined threshold, "high expression" can be selected as neoadjuvant therapy, including gemcitabine / nab-paclitaxel (GnP) therapy (also known as NAC-GnP), FOLFIRINOX therapy, chemoradiotherapy, or immune checkpoint inhibitors.

[0054] The method according to the present invention may further use other markers (such as CA19-9) to determine the effectiveness of NAC-GS therapy, or may be used in combination with other diagnostic methods (e.g., diagnosis based on the location of pancreatic cancer in the pancreas, diagnosis based on tumor size, etc.). For example, as shown in Example 2(7), combining multiple factors enables a more detailed and reliable evaluation. This allows for a more accurate determination of the effectiveness of NAC-GS therapy.

[0055] 3. NAC-GS Therapy Efficacy Assessment Kit The NAC-GS therapy effectiveness determination kit according to the present invention is a kit that realizes the method for determining the effectiveness of NAC-GS therapy described above. The NAC-GS therapy effectiveness determination kit includes at least an ITGB1 detection means for detecting the protein ITGB1, or a PODXL detection means for detecting the protein PODXL, or an ITGB1 detection means for detecting the protein ITGB1 and a PODXL detection means for detecting the protein PODXL. This NAC-GS therapy effectiveness determination kit detects the protein itself, which is a molecular marker in a biological sample, or the mRNA encoding the protein. Therefore, the ITGB1 detection means means substances such as antibodies that detect the protein ITGB1, and reagents for detecting the mRNA encoding the protein ITGB1. Similarly, the PODXL detection means means substances such as antibodies that detect the protein PODXL, and reagents for detecting the mRNA encoding the protein PODXL. [Examples]

[0056] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0057] [Example 1] (1) Selection of subjects This study included 63 cases of pancreatic cancer resected between 2019 and 2021 at Kochi University Hospital (Kochi Prefecture), Chikamori Hospital (Kochi Prefecture), Tokyo Metropolitan Tama General Medical Center (Tokyo), and Kanagawa Cancer Center (Kanagawa Prefecture) (Table 1). Patients were registered after obtaining written consent using explanatory documents approved by the ethics committees of Kochi University Hospital, Chikamori Hospital, Tama General Medical Center, and Kanagawa Cancer Center. In this study, residual cadavers of pancreatic cancer tissue biopsied before treatment by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) for diagnostic purposes were used. The characteristics of the 63 subjects are summarized in Table 1.

[0058] [Table 1]

[0059] As shown in Table 1, this example included 56 cases of resectable pancreatic cancer and 7 cases of borderline resectable pancreatic cancer. 82.5% of the patients received NAC-GS therapy.

[0060] (2) Staining test In this example, polyclonal antibodies against the ITGB1 protein and polyclonal antibodies against the PODXL protein were produced. Immunohistochemical staining was performed on residual tissue samples from pancreatic cancer biopsies taken from subjects using these polyclonal antibodies (conducted in 2022), and the staining results were scored based on the following criteria. Specifically, the score was calculated by adding the scores for the extent of positive cells in the tumor tissue on the section ("1" < 50%; "2" 50-80%; "3" > 80%) and the intensity of the staining ("1" weaker than normal pancreatic duct; "2" about the same as normal pancreatic duct; "3" stronger than normal pancreatic duct). A score of 4 or higher was judged as "high expression," and a score of 3 or lower was judged as "low expression." The polyclonal antibodies used in this example were produced by the inventors according to standard methods.

[0061] Staining tests revealed that 48 cases showed high expression of ITGB1, 29 cases showed high expression of PODXL, 23 cases showed high expression of both ITGB1 and PODXL, and 40 cases were in the remaining group.

[0062] (3) Case Analysis 1 First, for all cases shown in Table 1, we calculated survival curves using the Kaplan-Meier method, showing the difference between cases with and without NAC-GS therapy. The results are shown in Figure 1. As can be seen from Figure 1, the survival rate was significantly higher in cases that received NAC-GS therapy compared to cases that did not, confirming the prognostic improvement effect of NAC-GS therapy.

[0063] Furthermore, for patients with resectable pancreatic cancer [n=56], we calculated survival curves using the Kaplan-Meier method based on whether or not they received NAC-GS therapy. The results are shown in Figure 2. As can be seen from Figure 2, in cases of resectable pancreatic cancer treated with NAC-GS therapy, the survival rate was significantly higher compared to cases that did not receive NAC-GS therapy, confirming that NAC-GS therapy is extremely effective in improving the prognosis of patients with resectable pancreatic cancer.

[0064] Next, for the cases shown in Table 1, survival rates were examined by dividing them into a group with high expression of ITGB1 and PODXL and a group without. Survival curves were obtained for all cases using the Kaplan-Meier method. The results are shown in Figure 3. As can be seen from Figure 3, as disclosed in Japanese Patent No. 7246731, the group with high expression of ITGB1 and PODXL (high expression of both) had a poorer prognosis compared to the other group. Furthermore, survival rates were also examined for patients with resectable pancreatic cancer, dividing them into a group with high expression of ITGB1 and PODXL and a group without. Figure 4 shows the results of obtaining survival curves using the Kaplan-Meier method for patients with resectable pancreatic cancer. As can be seen from Figure 4, the group of patients with resectable pancreatic cancer who high expression of ITGB1 and PODXL (high expression of both) had a poorer prognosis compared to the other group.

[0065] Next, we examined the survival rates of patients with resectable pancreatic cancer who received NAC-GS therapy, dividing them into two groups: those with high expression of ITGB1 and PODXL (high expression of both) and those without. Survival curves were calculated for all cases using the Kaplan-Meier method. The results are shown in Figure 5. Surprisingly, as shown in Figure 5, the group with high expression of ITGB1 and PODXL had a poorer prognosis compared to the other group, and was found to have a prognosis similar to that of patients with resectable pancreatic cancer who did not receive NAC-GS therapy (see Figure 2).

[0066] These results demonstrate that the expression levels of ITGB1 and PODXL can predict the effectiveness of NAC-GS therapy in patients with resectable pancreatic cancer.

[0067] (4) Case Analysis 2 In this example, the analysis data was further validated using a COX proportional hazards model. R version 3.3.1, 'survival' and 'party' packages were used as the statistical analysis software.

[0068] <Key Evaluation Criteria> The variables used for the number of trials were the period from the biopsy date to the final observation date and the patient's survival status.

[0069] <Predictors> The eight variables were age, sex, clinical stage, whether or not CA19-9, ITGB1, ITGB1 and PODXL were highly expressed (both highly expressed), whether or not NAC-GS therapy was administered, and whether the pancreatic cancer was resectable or borderline resectable.

[0070] <Result> We confirmed the absence of multicollinearity using the VIF (Variance Inflation Factor) criterion. Furthermore, based on the AIC (Akaike Information Criteria), we selected four variables, as shown in Table 2.

[0071] [Table 2]

[0072] As shown in Table 2, the combination of ITGB1 and PODXL was found to exhibit a high hazard ratio.

[0073] (5) Analysis of cases 3 Among the subjects listed in Table 1, patients with borderline resectable pancreatic cancer (all receiving NAC-GS therapy) were divided into two groups: one with high expression of ITGB1 and PODXL, and the other group. Survival rates were then examined. Survival curves were calculated for all cases using the Kaplan-Meier method. The results are shown in Figure 6. As can be seen from Figure 6, there was no significant difference in overall survival between the group with high expression of ITGB1 and PODXL and the other group. This result indicates that the expression levels of ITGB1 and PODXL can predict the effectiveness of NAC-GS therapy in patients with resectable pancreatic cancer, but not in patients with borderline resectable pancreatic cancer.

[0074] (6) Consideration of other neoadjuvant chemotherapy As an exploratory study, we investigated the association between ITGB1 and PODXL expression levels and GnP therapy or FOLFIRINOX therapy using surgically resected specimens from two cases of locally advanced pancreatic cancer. GnP therapy and FOLFIRINOX therapy are approved for locally advanced pancreatic cancer that is more advanced than borderline resectable pancreatic cancer.

[0075] In the first case, infiltration was observed from the celiac artery to the common hepatic artery, making surgery unsuitable. Therefore, chemotherapy with GnP therapy was administered for 6 months. Following GnP therapy, the tumor shrank, and surgical resection was performed with the aim of curative resection. Histological examination of the surgically resected specimen showed no high expression of PODXL or ITGB1, and the survival time from EUS-FNA to death was 26 months.

[0076] In the second case, invasion into the superior mesenteric artery, aorta, and portal vein was observed, making surgery unsuitable. Chemotherapy with GnP was administered for 3 months, but the tumor did not shrink. Subsequently, FOLFIRINOX therapy was administered for 3 months, resulting in tumor shrinkage, and surgical resection was performed with the aim of curative resection. The patient recurred with liver metastasis, and the survival time from EUS-FNA to death was 30 months. Histological examination of the surgically resected specimen showed no high expression of PODXL and ITGB1.

[0077] The average survival time after resection for locally advanced pancreatic cancer is approximately 6 months, and both cases showed an extension of survival time. For locally advanced pancreatic cancer cases where PODXL and ITGB1 are not highly expressed, GnP therapy or FOLFIRINOX therapy may reduce tumor size and make surgery possible. These results suggest that GnP therapy or FOLFIRINOX therapy may also be effective for resectable borderline pancreatic cancer where NAC-GS therapy was not effective, and for resectable pancreatic cancer with high expression of PODXL and ITGB1. It is thought that using PODXL and ITGB1 as molecular markers can provide useful information for selecting appropriate neoadjuvant chemotherapy in the current treatment system where NAC-GS therapy is preferentially adopted for resectable and borderline resectable pancreatic cancer.

[0078] [Example 2] (1) Immunohistochemical staining using monoclonal antibodies In this example, the 63 pancreatic cancer cases used in Example 1 were evaluated by changing the method of immunohistochemistry in the staining test. Although the analysis using polyclonal antibodies in Example 1 was performed in 2022, prognosis follow-up was possible while the monoclonal antibodies were being produced, and the analysis using monoclonal antibodies was performed in 2024.

[0079] Specifically, residual tissue samples from pancreatic cancer biopsies derived from subjects, as shown in Table 3, were subjected to immunohistochemistry using anti-PODXL monoclonal antibody (manufactured by Salus Science Co., Ltd., sold by Cosmo Bio Inc.) and anti-ITGB1 monoclonal antibody (manufactured by Salus Science Co., Ltd., sold by Cosmo Bio Inc.) as primary antibodies (dilution ratio: ×100). Following the manufacturer's protocol, the samples were heat-treated at 100°C for 28 minutes, the primary antibody reaction for 32 minutes, and the secondary antibody reaction for 8 minutes, followed by immunohistochemistry. A Ventana Discovery XT automated stainer (Roche Diagnostics, Rotkreuz, Switzerland) was used as the instrument. The staining results were scored in the same manner as in Example 1.

[0080] Staining tests revealed that 46 cases highly expressed ITGB1, 32 cases highly expressed PODXL, 28 cases highly expressed both ITGB1 and PODXL (high expression of both), and 35 cases remained in the other group. Therefore, the group expressing both was assessed as 44.4%.

[0081] [Table 3]

[0082] (2) Case Analysis 1 First, based on the analysis results using polyclonal antibodies, similar to Example 1, survival curves using the Kaplan-Meier method were obtained for all cases shown in Table 1, categorized by stage. The results are shown in Figure 7. As shown in Figure 7, a tendency toward poorer prognosis was observed with increasing stage progression, particularly in Stage IIB. The graph in Figure 7 is consistent with the general trend.

[0083] Furthermore, similar to Example 1, we calculated survival curves using the Kaplan-Meier method for resectable pancreatic cancer cases with and without NAC-GS therapy (preoperative treatment). The results are shown in Figure 8. As can be seen from Figure 8, the survival rate up to 4 years was significantly higher in cases that received NAC-GS therapy compared to cases that did not, confirming the prognostic improvement effect of NAC-GS therapy.

[0084] Next, based on the analysis results using the monoclonal antibody of this embodiment, survival rates were examined for patients with resectable pancreatic cancer, divided into a group that highly expressed ITGB1 and PODXL and a group that did not. Figure 9 shows the survival curves obtained using the Kaplan-Meier method for patients with resectable pancreatic cancer. As can be seen from Figure 9, the group of patients with resectable pancreatic cancer who highly expressed ITGB1 and PODXL (high expression of both) had a poorer prognosis compared to the other group, while approximately 70% of the other group survived, indicating a good prognosis.

[0085] Furthermore, survival rates were examined for patients with resectable pancreatic cancer who received NAC-GS therapy, divided into a group with high expression of both ITGB1 and PODXL (high expression of both) and a group without high expression. Survival curves were obtained using the Kaplan-Meier method. The results are shown in Figure 10. As shown in Figure 10, the group with high expression of both ITGB1 and PODXL had a poorer prognosis compared to the other group, and it was found that their prognosis was similar to that of patients with resectable pancreatic cancer who did not receive NAC-GS therapy (patients who underwent surgery only) (see Figure 2). Currently, there are no markers to evaluate the therapeutic effect of NAC-GS therapy, so NAC-GS therapy is being administered even to the poor-prognosis group with high expression of both ITGB1 and PODXL.

[0086] For patients with resectable pancreatic cancer who received NAC-GS therapy, survival curves using the Kaplan-Meier method were calculated for cases that did not belong to the group that highly expressed ITGB1 and PODXL (good prognosis group), with and without preoperative treatment. The results are shown in Figure 11. As shown in Figure 11, in patients with resectable pancreatic cancer who did not belong to the group that highly expressed ITGB1 and PODXL, NAC-GS therapy improved the prognosis up to 4 years postoperatively.

[0087] These results demonstrate that the expression levels of ITGB1 and PODXL can predict the effectiveness of NAC-GS therapy in patients with resectable pancreatic cancer.

[0088] (3) Case Analysis 2 Similar to Example 1, based on the analysis results using polyclonal antibodies, the survival rates of patients with resectable borderline pancreatic cancer (all 7 cases received NAC-GS therapy and surgery) from the subjects listed in Table 1 were divided into a group that highly expressed ITGB1 and PODXL and a group that did not, and survival curves were obtained using the Kaplan-Meier method. The results are shown in Figure 12. As can be seen from Figure 12, there was no significant difference in overall survival between the group that highly expressed ITGB1 and PODXL and the other group compared to patients with resectable pancreatic cancer. Survival rates tended to be shorter compared to resectable pancreatic cancer, and the trend was almost the same as that of the poor prognosis group of resectable pancreatic cancer based on ITGB1 and PODXL.

[0089] (4) Case Analysis 3 Similar to Example 1, multivariate analysis was performed on the results of the monoclonal antibody analysis using the COX proportional hazards model. Specifically, the absence of multicollinearity was confirmed using the VIF (Variance Inflation Factor) criteria for eight variables: age, sex, clinical stage, high expression of ITGB1 and PODXL (both high expression), neoadjuvant chemotherapy, borderline resectable pancreatic cancer, and CA19-9. Furthermore, variable selection was performed based on the AIC (Akaike Information Criteria). The results are shown in Table 4.

[0090] [Table 4]

[0091] As shown in Table 4, the combination of ITGB1 and PODXL was most strongly associated with the prognosis of NAC-GS therapy plus surgery.

[0092] (5) Analysis of cases 4 Based on the results of analysis using monoclonal antibodies, survival curves using the Kaplan-Meier method were obtained for resectable pancreatic cancer cases among the cases shown in Table 3, separated by stage into a poor prognosis group (groups with high expression of ITGB1 and PODXL) and a good prognosis group (other groups, i.e., groups that do not highly express both ITGB1 and PODXL). The results are shown in Figure 13. As shown in Figure 13, in the good prognosis group, postoperative prognosis improved in both Stage I and Stage II, with a particularly significant improvement in survival rates of 2 years or more in Stage II. On the other hand, in the poor prognosis group, even in Stage I, there was a significant decrease in survival rates of 3 years or more.

[0093] Furthermore, based on the analysis results using monoclonal antibodies, survival curves using the Kaplan-Meier method were calculated for all cases shown in Table 3, categorized by stage for the good prognosis group and the poor prognosis group. The results are shown in Figure 14. As can be seen from Figure 14, the good prognosis group shows a significant improvement in survival rates for two years or more in Stage II. On the other hand, in the poor prognosis group, even in Stage I, there is a significant decrease in survival rates for three years or more, and the survival rate is lower than that of Stage II in the good prognosis group.

[0094] Furthermore, based on the analysis results using polyclonal antibodies, similar to Example 1, Kaplan-Meier survival curves were obtained for all cases, showing the survival rates of resectable pancreatic cancer (R) and borderline resectable pancreatic cancer (BR). The results are shown in Figure 15. From Figure 15, it can be seen that there is a need to improve the prognosis of resectable pancreatic cancer (R), that regimens for the poor-prognosis group of resectable pancreatic cancer are a future challenge, and that it is necessary to consider treating the poor-prognosis group with regimens for borderline resectable pancreatic cancer (BR).

[0095] (6) Analysis of Cases 5 For all cases shown in Table 3, survival curves were calculated using the Kaplan-Meier method, with tumor size, intrapancreatic location of pancreatic cancer, and CA19-9 as indicators. Here, a tumor size of 2 cm or more in diameter, intrapancreatic location of pancreatic cancer in the pancreatic head, and CA19-9 of 200 or more were considered applicable. The results are shown in Figure 16. As shown in Figure 16, in cases where none of these three factors were present (score=0), cases where one of the three factors was present (score=1), cases where two of the three factors were present (score=2), and cases where all three factors were present (score=3), the prognosis worsened as the score increased. The effectiveness of conventional predictive factors was confirmed.

[0096] (7) Analysis of cases 6 For all cases and patients with resectable pancreatic cancer shown in Table 3, CART (classification and regression tree) analysis was performed using eight factors: combination of ITGB1 and PODXL, clinical stage, neoadjuvant chemotherapy, borderline resectable pancreatic cancer, NAC-GS, tumor size, intrapancreatic location of the pancreatic cancer, and CA19-9. Survival curves were then obtained. Briefly, CART analysis is a decision tree analysis method that creates rules to branch out like a tree based on the characteristics (explanatory variables) of the data in order to predict and classify a certain outcome (dependent variable). The results are shown in Figures 17A (all cases) and 17B (patients with resectable pancreatic cancer). As shown in Figure 16, the effectiveness of conventional predictive factors was confirmed. However, as shown in Figures 17A and 17B, in the CART analysis for all cases and patients with resectable pancreases, only the combination of ITGB1 and PODXL remained as an explanatory variable. Cases with high expression of both (right-hand Kaplan-Meier curve) and cases with no high expression of both (left-hand Kaplan-Meier curve) were more associated with the prognosis of patients undergoing NAC-GS+ surgery than all other variables. Similar to the results in Table 4, this biomarker was most strongly associated with the prognosis of NAC-GS+ surgery, and it was shown that cases with high expression of both may have a poor prognosis even after undergoing NAC-GS.

[0097] To investigate whether combining this biomarker with other predictive factors enables highly accurate prognosis prediction, CART analysis was performed on all cases and patients with resectable pancreatic cancer using the following factors: tumor size, intrapancreatic location of pancreatic cancer, CA19-9 set, ITGB1 and PODXL combination, clinical stage, neoadjuvant chemotherapy, borderline resectable pancreatic cancer, and NAC-GS. Survival curves were then obtained. The results are shown in Figures 18A (all cases) and 18B (patients with resectable pancreatic cancer). As shown in Figures 18A and 18B, tumor size, intrapancreatic location of pancreatic cancer, CA19-9 set, and this biomarker remained as explanatory factors associated with the prognosis of NAC-GS + surgery. By combining this biomarker with other factor sets (tumor size, intrapancreatic location of pancreatic cancer, CA19-9), it is possible to differentiate cases likely to recur from the good prognosis group. In the analysis of all cases, the group without tumor size, intrapancreatic location of pancreatic cancer, or CA19-9 (far left) and the poor prognosis group (far right) were extracted as separate groups. The group with the best prognosis was the group that did not meet any of the following criteria: tumor size, intrapancreatic location of pancreatic cancer, and CA19-9 (leftmost figure in Figure 18A). The group with the worst prognosis was the group that met at least one of these criteria and both were highly expressed (rightmost figure). In the analysis of resectable pancreatic cancers, the group with the best prognosis was the group that did not meet any of the following criteria: tumor size, intrapancreatic location of pancreatic cancer, and CA19-9 (leftmost figure in Figure 18B). The good prognosis group was not divided into these three groups (center figure). The group with the worst prognosis was the group that met at least one of these criteria and both were highly expressed (rightmost figure). Cases without these three factors had a good prognosis, while cases with high expression of both of these biomarkers had a poor prognosis.

[0098] The results of the CART (Classification and Regression Tree) analysis described above revealed that the combination of PODXL and ITGB1 was the most accurate predictor of patient prognosis among the various factors examined. In cases where both were highly expressed, improvement in prognosis could not be expected even with NAC-GS followed by surgery. On the other hand, in other cases, improvement in prognosis was observed with NAC-GS, suggesting that it may be an important indicator in determining treatment strategy. CART analysis is a method that divides data into a tree structure to explore the degree of influence of each factor on prognosis and treatment efficacy, and to find the optimal threshold. This analysis showed that the expression levels of ITGB1 and PODXL are strongly associated with prognosis and the therapeutic effect of NAC-GS, and that treatment success / failure can be clearly stratified based on the presence or absence and amount of their expression. Furthermore, combining this biomarker with other predictive factors will enable more detailed and accurate prognostic prediction. In the population where all variables—tumor size, intrapancreatic location of pancreatic cancer, and CA19-9—are not met, NAC-GS followed by surgery can be expected to improve prognosis. Conversely, in cases where both of these biomarkers are highly expressed, the presence of one or more of these three factors further increases the likelihood that NAC-GS followed by surgery will not improve prognosis.

[0099] [Example 3] In this study, we evaluated whether PODXL alone could serve as a biomarker for NAC-GS therapy in pancreatic cancer patients.

[0100] (1) Case Analysis 1 Similar to Example 2, PODXL levels were measured in 63 pancreatic cancer cases using immunohistochemistry with an anti-PODXL monoclonal antibody.

[0101] For all cases, survival curves using the Kaplan-Meier method were calculated for both the group with high PODXL expression and the group without. Similarly, for resectable pancreatic cancer cases, survival curves using the Kaplan-Meier method were calculated for both the group with high PODXL expression and the group without. The results are shown in Figures 19A and 19B. As can be seen from Figure 19, cases with high PODXL expression had a significantly worse prognosis compared to cases with low expression, indicating that the effect of NAC-GS was not fully realized.

[0102] (2) Case Analysis 2 PODXL concentrations were measured using ELISA in pre-treatment serum from 59 pancreatic cancer patients (stages 0 to IV) and serum from 18 healthy individuals (controls). Specifically, serum samples and the detection antibody were reacted on a plate immobilized with anti-PODXL antibody (manufactured by Salus Science Co., Ltd., sold by Cosmo Bio Co., Ltd.), sandwiching the PODXL in the serum. The mixture was then color-developed by an enzymatic reaction, absorbance was measured, and the serum PODXL concentration was quantified from a standard curve.

[0103] The results are shown in Figure 20 and Table 5 below. Serum PODXL concentrations were significantly higher in pancreatic cancer patients, and the AUC for stages 0 and IIA, which correspond to resectable pancreatic cancer, was higher than that for stages III and IV. This suggests that serum PODXL may be useful as a diagnostic marker for pancreatic cancer from stage 0 to IIA. Furthermore, since the prognosis after NAC-GS+ surgery is poor in resectable pancreatic cancer patients with high PODXL expression in pancreatic cancer biopsy tissue, it can be predicted that the prognosis after NAC-GS+ surgery is poor in pancreatic cancer patients from stage 0 to IIA with high serum PODXL levels.

[0104] [Table 5] [Industrial applicability]

[0105] The molecular markers according to the present invention can provide useful guidance in clinical practice for deciding whether to administer NAC-GS therapy to patients with resectable pancreatic cancer, whether to perform pancreatic cancer resection surgery without NAC-GS therapy, or whether to administer chemotherapy other than NAC-GS therapy. Therefore, using the molecular markers according to the present invention has the advantage of allowing for surgical intervention before the stage of resectable pancreatic cancer progresses to borderline resectable pancreatic cancer, or even to unresectable pancreatic cancer, during NAC-GS therapy, which is not expected to be effective.

Claims

1. Protein ITGB1, or Protein PODXL, or The combination of protein ITGB1 and protein PODXL, A molecular marker that indicates the efficacy of gemcitabine and S-1 (GS) therapy as neoadjuvant chemotherapy (NAC) for pancreatic cancer, characterized by containing [specific element].

2. Protein PODXL, or The combination of protein ITGB1 and protein PODXL, A molecular marker according to claim 1, comprising:

3. ITGB1 detection means for detecting the protein ITGB1, or PODXL detection means for detecting the protein PODXL, or ITGB1 detection means for detecting the protein ITGB1 and PODXL detection means for detecting the protein PODXL, A kit for determining the efficacy of gemcitabine and S-1 (GS) therapy as neoadjuvant chemotherapy (NAC) for pancreatic cancer, characterized by containing [specific ingredient / feature].

4. PODXL detection means for detecting the protein PODXL, or ITGB1 detection means for detecting the protein ITGB1 and PODXL detection means for detecting the protein PODXL, The kit according to claim 3, including the following:

5. The kit according to claim 3, characterized in that the ITGB1 detection means and / or the PODXL detection means are, respectively, an antibody against the protein ITGB1 and / or an antibody against the protein PODXL.

6. The kit according to claim 5, wherein the above antibody comprises a polyclonal antibody or a monoclonal antibody.

7. The kit according to claim 3, characterized in that it includes a reagent used in at least one method selected from immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and immunoaggregation assay.

8. A method for determining the effectiveness of gemcitabine and S-1 (GS) therapy as neoadjuvant chemotherapy (NAC) for pancreatic cancer, A method characterized by comprising the step of measuring the expression of protein ITGB1, or the expression of protein PODXL, or the expression of both protein ITGB1 and protein PODXL in a biological sample.

9. The method according to claim 8, wherein the step of measuring the expression of protein ITGB1, or the expression of protein PODXL, or the expression of protein ITGB1 and protein PODXL in a biological sample includes measuring the expression of protein PODXL, or the expression of protein ITGB1 and protein PODXL.

10. The method according to claim 8, characterized in that if the above-mentioned protein ITGB1 or the above-mentioned protein PODXL is highly expressed, or if the expression of both the above-mentioned protein ITGB1 and the above-mentioned protein PODXL is highly expressed, the efficacy is determined to be low.

11. The method according to claim 8, characterized in that if the above-mentioned protein ITGB1 or the above-mentioned protein PODXL is highly expressed, or if the expression of the above-mentioned protein ITGB1 and the above-mentioned protein PODXL is highly expressed, the efficacy is judged to be low, and the indication for surgical treatment or the indication for other neoadjuvant therapy prior to surgical treatment is determined.

12. The method according to claim 11, characterized in that the above-mentioned other preoperative therapies include at least one of the following: gemcitabine / nab-paclitaxel (GnP) therapy, FOLFIRINOX therapy, chemoradiotherapy, and immune checkpoint inhibitors.

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