Skin cancer prognosis prediction methods and their applications

Abstract

The present invention provides a technique for predicting the prognosis of skin cancer. The method includes: determining a correlation value associated with the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a skin cancer patient; and determining that the prognosis of the skin cancer is poorer when the correlation value is high compared to when the correlation value is low.

Description

Technical Field

[0001] The present disclosure relates to a method for predicting the prognosis of skin cancer. This application is based on Japanese application No. 2020-072636 filed on April 15, 2020, the contents of which are incorporated herein by reference. Background Art

[0002] In recent years, for various cancers represented by malignant melanoma, anti-tumor treatment using immune checkpoint inhibition, including inhibitors of PD-L1 (programmed cell death ligand 1) and its receptor PD-1 (programmed cell death 1, programmed cell death receptor 1), has achieved good results. For Merkel cell carcinoma, a type of skin cancer, clinical use of anti-PD-L1 antibody drugs has also begun. PD-L1 expressed in tumor cells helps the proliferation of tumors by binding to PD-1 expressed in T cells, thereby inhibiting the activation of T cells and immune response. Therefore, it is known that high expression of PD-L1 indicates a poor prognosis in most cancers. However, it is known that high expression of PD-L1 in Merkel cell carcinoma results in a good prognosis (for example, non-patent document 1).

[0003] Furthermore, PD-L1 is known to exhibit heterogeneity even within the same case, and the present inventors were the first to discover and report that PD-L1 also exhibits this heterogeneity in Merkel cell carcinoma (Non-Patent Document 2). Furthermore, the present inventors were the first to discover and report that there was no correlation between PD-L1 expression in primary Merkel cell carcinoma lesions and prognosis, while a strong correlation was observed between PD-L1 expression in skin metastases of Merkel cell carcinoma and prognosis (Non-Patent Document 3).

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: Lipson EJ, et al., Cancer Immunol. Res., 54-63, 2013

[0007] Non-patent literature 2: Nakamura M. et al., Br J Dermatol., 1228-1229, 2019

[0008] Non-patent literature 3: Nakamura M. et al., J Dermatol. Sci., 165-167, 2020 Summary of the Invention

[0009] According to non-patent document 2, the expression level of PD-L1 varies greatly in the same case depending on the period and site of resection. Therefore, it is difficult to use PD-L1 to predict the prognosis of Merkel cell carcinoma. In addition, according to non-patent document 3, although there is a strong correlation between the expression level of PD-L1 in the skin metastases of Merkel cell carcinoma and prognosis, if the prognosis can only be inferred after skin metastasis can be observed, it is difficult to use it clinically as a prognostic predictor. Therefore, other methods that can predict the prognosis of Merkel cell carcinoma are sought. It should be noted that such issues are not limited to Merkel cell carcinoma, but are also common issues in other skin cancers.

[0010] The present invention can be implemented as the following aspects.

[0011] (1) According to one embodiment of the present invention, a method for predicting the prognosis of skin cancer can be provided. This method for predicting the prognosis of skin cancer includes: a step of determining a correlation value that is correlated with the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer; and a step of determining that the prognosis of the skin cancer is poor when the correlation value is high compared to when the correlation value is low. This method for predicting the prognosis of skin cancer can predict the prognosis of skin cancer using the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer as an indicator.

[0012] (2) In the above-described method for predicting the prognosis of skin cancer, the skin cancer may be at least one selected from the group consisting of Merkel cell carcinoma, malignant melanoma, squamous cell carcinoma, extramammary Paget's disease, and cutaneous angiosarcoma.

[0013] (3) In the skin cancer prognosis prediction method according to the above-mentioned aspect, the skin cancer may include Merkel cell carcinoma.

[0014] (4) In the above-described method for predicting the prognosis of skin cancer, the step of determining the relevant amount may include the step of determining the expression level of the glucose-6-phosphate dehydrogenase by immunohistochemical staining of the sample. According to this method for predicting the prognosis of skin cancer, the prognosis of skin cancer can be easily predicted by performing immunohistochemical staining on a sample collected from a skin cancer patient to determine the expression level of glucose-6-phosphate dehydrogenase.

[0015] (5) In the above-described method for predicting the prognosis of skin cancer, the step of determining the relevant amount may include the step of determining the expression level of the mRNA encoding the glucose-6-phosphate dehydrogenase in the sample. According to this method for predicting the prognosis of skin cancer, the prognosis of skin cancer can be easily predicted by determining the expression level of the mRNA encoding the glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer.

[0016] (6) In the above-described method for predicting the prognosis of skin cancer, the step of determining the relevant amount may include the step of measuring the activity of the glucose-6-phosphate dehydrogenase in the blood or serum sample. According to this method for predicting the prognosis of skin cancer, the prognosis of skin cancer can be easily predicted by measuring the activity of the glucose-6-phosphate dehydrogenase in the blood or serum sample collected from a skin cancer patient.

[0017] (7) According to another embodiment of the present invention, a method for evaluating the effectiveness of an immune checkpoint inhibitor for skin cancer is provided. The evaluation method of this embodiment includes: a step of determining a correlation value, wherein the correlation value is correlated with the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer; and a step of evaluating that the effectiveness of the immune checkpoint inhibitor is high when the correlation value is small compared to when the correlation value is large. According to the evaluation method of this embodiment, the effectiveness of the immune checkpoint inhibitor can be evaluated using the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer as an indicator.

[0018] (8) According to another embodiment of the present invention, a method for determining whether to administer an immune checkpoint inhibitor to a patient with skin cancer can be provided. The method of this embodiment includes a method for evaluating the effectiveness of the immune checkpoint inhibitor for the above-mentioned skin cancer, including a step of determining whether to administer or continue administering the immune checkpoint inhibitor to the above-mentioned patient when the effectiveness of the immune checkpoint inhibitor is evaluated to be high. According to the method of this embodiment, the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer can be used as an indicator to determine whether to administer an immune checkpoint inhibitor.

[0019] (9) According to another aspect of the present invention, a method for evaluating the malignancy of skin cancer can be provided. The malignancy evaluation method of this aspect includes: a step of determining a correlation value that is correlated with the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer; and a step of determining that the malignancy of the skin cancer is higher when the correlation value is greater than when the correlation value is less. According to this aspect of the malignancy evaluation method, the malignancy of skin cancer can be evaluated using the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer as an indicator.

[0020] (10) According to another embodiment of the present invention, a biomarker for predicting the prognosis of skin cancer can be provided. The biomarker in this embodiment includes glucose-6-phosphate dehydrogenase in a sample collected from a patient with the skin cancer, and when the expression level of the biomarker in the sample is high, it indicates a poor prognosis for the skin cancer compared to when the expression level is low.

[0021] (11) According to another embodiment of the present invention, a biomarker for evaluating the effectiveness of an immune checkpoint inhibitor against skin cancer can be provided. The biomarker in this embodiment includes glucose-6-phosphate dehydrogenase in a sample collected from a patient with the skin cancer, and when the expression level of the biomarker in the sample is low, it indicates that the effectiveness of the immune checkpoint inhibitor is high compared to when the expression level is high.

[0022] (12) According to another embodiment of the present invention, a biomarker for evaluating the malignancy of skin cancer can be provided. The biomarker in this embodiment includes glucose-6-phosphate dehydrogenase in a sample collected from a patient with the skin cancer. When the expression level of the biomarker in the sample is high, it indicates a higher malignancy of the skin cancer than when the expression level is low.

[0023] (13) According to another embodiment of the present invention, a biomarker for predicting the recurrence of skin cancer can be provided. The biomarker in this embodiment includes glucose-6-phosphate dehydrogenase in a sample collected from a patient suffering from the skin cancer. When the expression level of the biomarker in the sample is high, the likelihood of recurrence of the skin cancer is higher than when the expression level is low.

[0024] (14) According to another embodiment of the present invention, a method for predicting the likelihood of developing grade 3 or higher immune-related adverse events caused by administering an immune checkpoint inhibitor to a patient with skin cancer can be provided. The method of this embodiment includes the following steps: when the effectiveness of the immune checkpoint inhibitor is evaluated to be high, evaluating the likelihood of developing grade 3 or higher immune-related adverse events caused by administering the immune checkpoint inhibitor to be high. According to the method of this embodiment, the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer is used as an indicator to predict the likelihood of developing grade 3 or higher immune-related adverse events caused by administering an immune checkpoint inhibitor.

[0025] (15) According to another embodiment of the present invention, a biomarker for predicting the likelihood of developing a grade 3 or higher immune-related adverse event resulting from the administration of an immune checkpoint inhibitor for skin cancer can be provided. The biomarker in this embodiment includes glucose-6-phosphate dehydrogenase in a sample collected from a patient with the skin cancer, and when the expression level of the biomarker in the sample is low, the likelihood of developing a grade 3 or higher immune-related adverse event resulting from the administration of the immune checkpoint inhibitor is higher than when the expression level is high.

[0026] (16) According to another embodiment of the present invention, a measurement kit can be provided. The measurement kit of this embodiment is used for at least one of the following: prediction of the prognosis of skin cancer, evaluation of the effectiveness of an immune checkpoint inhibitor for the above-mentioned skin cancer, evaluation of the malignancy of the above-mentioned skin cancer, prediction of the recurrence of the above-mentioned skin cancer, prediction of the possibility of onset of grade 3 or higher immune-related adverse events caused by the administration of the above-mentioned immune checkpoint inhibitor, and evaluation of the immune activity of the above-mentioned skin cancer. The above-mentioned measurement kit contains a substance capable of detecting a relevant amount, and the relevant amount is correlated with the expression amount of glucose-6-phosphate dehydrogenase in a sample collected from a patient with the above-mentioned skin cancer. According to the measurement kit of this embodiment, the relevant amount correlated with the expression amount of glucose-6-phosphate dehydrogenase in a sample collected from a patient with skin cancer can be easily detected.

[0027] (17) In the assay kit of the above-mentioned manner, the substance capable of detecting the relevant amount may include at least one selected from a substance capable of binding to the above-mentioned glucose-6-phosphate dehydrogenase or a fragment thereof, a substance capable of binding to a gene encoding the above-mentioned glucose-6-phosphate dehydrogenase, and glucose-6-phosphate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a graph showing the Kaplan-Meier curve of the G6PD mRNA expression level.

[0029] Figure 2 This is an explanatory diagram showing the results of G6PD expression analysis obtained by immunohistostaining.

[0030] Figure 3 It is a graph showing the Kaplan-Meier curve of the G6PD expression level.

[0031] Figure 4 It is a graph showing the Kaplan-Meier curve of the expression level of PD-L1.

[0032] Figure 5 It is a graph showing the results of correlation analysis of PD-L1 expression levels.

[0033] Figure 6 This is an explanatory diagram showing the transition of G6PD activity in Case 1.

[0034] Figure 7 This is an explanatory diagram showing the transition of G6PD activity in Case 2.

[0035] Figure 8 This is an explanatory diagram showing the transition of G6PD activity in Case 4.

[0036] Figure 9 This is an explanatory diagram showing the results of G6PD expression analysis obtained by immunohistochemical staining of malignant melanoma.

[0037] Figure 10 It is a graph showing the Kaplan-Meier curve of the G6PD expression level in malignant melanoma.

[0038] Figure 11 This is an explanatory diagram showing the relationship between the presence or absence of grade 3 or higher immune-related adverse events in malignant melanoma and the expression level of G6PD.

[0039] Figure 12 This is an explanatory diagram showing the results of G6PD expression analysis obtained by immunohistostaining of cutaneous angiosarcoma.

[0040] Figure 13 This is an explanatory diagram showing the results of G6PD expression analysis obtained by next-generation sequencing analysis of cutaneous angiosarcoma.

[0041] Figure 14 This is an explanatory diagram showing the results of GSEA analysis of a group with a high G6PD expression level in cutaneous angiosarcoma.

[0042] Figure 15 This is an explanatory diagram showing the results of GSEA analysis of a group with a high G6PD expression level in cutaneous angiosarcoma. DETAILED DESCRIPTION

[0043] According to one embodiment of the present disclosure, a method for predicting the prognosis of skin cancer can be provided. The types of skin cancer are not particularly limited, and examples thereof include Merkel cell carcinoma, malignant melanoma, squamous cell carcinoma, extramammary Paget's disease, and cutaneous angiosarcoma. The patient with skin cancer can be a patient with multiple skin cancers concurrently, or any patient before or after treatment. More specifically, the patient with skin cancer can be any patient before or after surgery, or any patient before or after immunotherapy, or any patient before or after chemotherapy, or any patient before or after radiotherapy. In addition, the sex and age of the patient with skin cancer are not particularly limited. The animal species of the patient in this embodiment are mainly mammals. The above-mentioned mammals are not particularly limited, and examples thereof include humans, primates such as chimpanzees, dogs, cats, rabbits, and the like.

[0044] As shown in the Examples described below, the inventors of the present application have found that in skin cancer, when the expression level of glucose-6-phosphate dehydrogenase is high, the prognosis tends to be worse compared to when the expression level of the enzyme is low.

[0045] The method for predicting the prognosis of skin cancer according to the present embodiment includes (I) a step of determining a correlation value, wherein the correlation value is correlated with the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a skin cancer patient (hereinafter also referred to as step (I)); and (II) a step of determining that the prognosis of the skin cancer is poor when the correlation value is large compared to when the correlation value is small (hereinafter also referred to as step (II)).

[0046] As the sample used in step (I), a tissue containing a tumor isolated from a skin cancer patient, blood or serum collected from a skin cancer patient, etc. can be used. The tissue is not particularly limited, and examples thereof include formalin-fixed paraffin-embedded (FFPE) specimens, at least a portion of a tumor removed by surgery, etc., and a sample containing a plurality of tissue structural cells collected by biopsy, etc.

[0047] In this embodiment, glucose-6-phosphate dehydrogenase refers to an enzyme with enzyme number EC 1.1.1.49. Glucose-6-phosphate dehydrogenase (hereinafter also referred to as "G6PD") catalyzes the reaction in which the substrate glucose-6-phosphate is oxidized in the presence of coenzymes such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) to produce glucono-1,5-lactone-6-phosphate and reduced NAD (NADH) or reduced NADP (NADPH), or the reverse reaction thereof.

[0048] In this embodiment, the correlation amount related to the expression level of G6PD refers to a correlation amount that is positively correlated with the expression level of G6PD. This correlation amount is not particularly limited, and examples thereof include the expression level of G6PD, the expression level of mRNA encoding G6PD, and the activity of G6PD.

[0049] The expression level of G6PD can be obtained, for example, by Western blotting, dot blot, immunoprecipitation, ELISA, immunohistochemical staining, etc. based on antigen-antibody reaction using anti-G6PD antibodies (Anti-G6PD-antibody) or fragments thereof. The expression level of mRNA encoding G6PD can be obtained, for example, by next-generation sequencing analysis, Northern hybridization, dot hybridization, RT-PCR, real-time PCR, etc. The activity of G6PD can be measured, for example, using a kit for determining the activity of G6PD using glucose-6-phosphate as a matrix. The expression level of G6PD obtained by immunostaining has little variation in the case and is excellent as a biomarker. In addition, the sensitivity is high when the expression level of G6PD is obtained by serum testing using serum, and is therefore suitable for monitoring.

[0050] In step (II), for example, (i) when the expression level of G6PD or the expression level of mRNA encoding G6PD is high, the prognosis of skin cancer can be determined to be poor compared to when the expression level is low, and (ii) when the activity of G6PD is high, the prognosis of skin cancer can be determined to be poor compared to when the activity of G6PD is low. The expression level or activity level can be compared with a predetermined baseline value or with values from samples collected from the same patient at different time points. In this embodiment, "poor prognosis" means that the skin cancer has a high malignancy and a tendency for symptoms to worsen. In other words, a poor prognosis indicates the possibility of metastasis and recurrence.

[0051] For example, if G6PD expression levels are high in a resected specimen, the patient is considered to be at high risk for skin cancer malignancy, and postoperative radiotherapy, etc., can be added. Alternatively, if G6PD expression levels are low in a resected specimen, the patient is considered to be at low risk for skin cancer malignancy, and postoperative radiotherapy, etc., can be added to monitor the patient's course. Thus, the skin cancer prognosis prediction method of this embodiment can be used as a method to assist in determining a treatment strategy for skin cancer. It should be noted that the skin cancer prognosis prediction method can be alternatively referred to as a method for predicting the prognosis of skin cancer.

[0052] According to other embodiments of the present disclosure, a method for evaluating the effectiveness of immune checkpoint inhibitors for skin cancer can be provided. In this embodiment, an immune checkpoint inhibitor refers to an agent that hinders the signal transmission based on the immune checkpoint by hindering the binding of the immune checkpoint and its ligand. As immune checkpoint inhibitors, for example, anti-PD-L1 antibody drugs, anti-PD-1 antibody drugs, etc. can be cited. Anti-PD-L1 antibody drugs are composed of human monoclonal antibodies and bind to PD-L1 (programmed cell death ligand 1) expressed in tumor cells, thereby hindering PD-1 (programmed cell death 1, programmed death receptor 1) expressed in T cells from binding to PD-L1, thereby maintaining the activation of T cells. Anti-PD-1 antibody drugs bind to PD-1, hindering the binding of PD-1 to PD-L1, and maintaining the activation of T cells.

[0053] As shown in the Examples below, the inventors of the present application have discovered that G6PD expression in skin cancer is negatively correlated with PD-L1 expression, with higher G6PD expression associated with lower PD-L1 expression. Higher G6PD expression, and therefore lower PD-L1 expression, suggests a higher likelihood of a tumor with low immunological activity.

[0054] In this embodiment, the method for evaluating the effectiveness of an immune checkpoint inhibitor against skin cancer includes the above-mentioned step (I), i.e., the step of determining a correlation value correlated with the expression level of glucose-6-phosphate dehydrogenase in a sample collected from a patient; and (III), when the correlation value is small, the step of evaluating that the effectiveness of the immune checkpoint inhibitor is higher than when the correlation value is large (hereinafter also referred to as step (III)).

[0055] In step (III), for example, (i) when the expression level of G6PD or the expression level of mRNA encoding G6PD is low, the effectiveness of the immune checkpoint inhibitor can be determined to be higher than when the expression level is high, and (ii) when the activity of G6PD is low, the effectiveness of the immune checkpoint inhibitor can be evaluated to be higher than when the activity of G6PD is high. The expression level and activity can be compared with a predetermined reference value, or the values of samples collected from the same patient at different time periods can be compared.

[0056] Considering the mechanism of immune checkpoint inhibitors such as anti-PD-L1 antibody drugs, it is estimated that the effect of immune checkpoint inhibitors such as anti-PD-L1 antibody drugs is low when the expression level of PD-L1 is low, and it is estimated that the effect of immune checkpoint inhibitors such as anti-PD-L1 antibody drugs is high when the expression level of PD-L1 is high. Therefore, in the above step (III), when the expression level of G6PD is low, that is, when the expression level of PD-L1 is high based on the correlation between G6PD and PD-L1, the effectiveness of immune checkpoint inhibitors such as anti-PD-L1 antibody drugs is evaluated as being higher than when the expression level of G6PD is high, that is, when the expression level of PD-L1 is low based on the correlation between G6PD and PD-L1.

[0057] For example, when the expression level of G6PD in the sample is low and the expression level of PD-L1 is low, it is expected that the expression level of PD-L1 will temporarily decrease due to the heterogeneity of PD-L1. In such a case, since the expression level of G6PD is low, it can be evaluated that the effectiveness of immune checkpoint inhibitors such as anti-PD-L1 antibody drugs is high. Since G6PD has a small fluctuation in the value in the same case, it is an excellent evaluation indicator for the effectiveness of immune checkpoint inhibitors, etc., compared with PD-L1, which has a large fluctuation in the value in the same case. The evaluation method for evaluating the effectiveness of immune checkpoint inhibitors for skin cancer of this embodiment can be used as a method for assisting in determining the administration of immune checkpoint inhibitors, etc., and determining the treatment policy for skin cancer.

[0058] According to another embodiment of the present disclosure, a method for determining whether to administer an immune checkpoint inhibitor to a patient with skin cancer can be provided. This method includes, in step (III) of the above-mentioned method for evaluating the effectiveness of an immune checkpoint inhibitor for skin cancer, a step (IV) of determining whether to administer or continue administering the immune checkpoint inhibitor to the patient if the effectiveness of the immune checkpoint inhibitor is assessed to be high (hereinafter also referred to as step (IV)).

[0059] In step (IV), it may be determined whether to newly administer an immune checkpoint inhibitor to a patient who has not been administered an immune checkpoint inhibitor, or it may be determined whether to continue administering an immune checkpoint inhibitor to a patient who has already been administered an immune checkpoint inhibitor.

[0060] For example, when the expression level of G6PD in the sample is high and the expression level of PD-L1 is low, it is evaluated that the effect of immune checkpoint inhibitors such as anti-PD-L1 antibody drugs is low, so surgical treatments such as lymph node resection can be selected instead of the administration of immune checkpoint inhibitors. In addition, for example, by administering immune checkpoint inhibitors such as anti-PD-L1 antibody drugs, when the G6PD value in the blood remains low, it is evaluated that the effectiveness of the immune checkpoint inhibitor is high, so the immune checkpoint inhibitor can be continued to be administered. In addition, for example, after the administration of immune checkpoint inhibitors, when the G6PD value in the blood tends to rise, it is evaluated that the effectiveness of the immune checkpoint inhibitor is low, so additional treatments such as radiotherapy can be studied. In addition, for example, when the expression level of G6PD in the sample is low and the inferred expression level of PD-L1 is high, it is considered to be a tumor with high immune activity, so immune checkpoint inhibitors can be introduced while paying full attention to side effects.

[0061] As described above, the inventors of the present application have discovered that the higher the G6PD expression level, that is, the lower the estimated PD-L1 expression level, the higher the likelihood of the tumor having low immunological activity. Therefore, the G6PD expression level can be used as an indicator to evaluate the immune activity of skin cancer. In other words, as another embodiment of the present disclosure, a method for evaluating the immune activity of skin cancer is provided. This evaluation method includes: a step of determining a correlation value related to the G6PD expression level in a sample collected from a skin cancer patient; and a step of determining that the immune activity of the skin cancer is high when the correlation value is high compared to when the correlation value is low.

[0062] According to other embodiments of the present disclosure, a biomarker may be provided. The biomarker comprises G6PD in a sample taken from a patient with skin cancer. The biomarker can be used to predict the prognosis of skin cancer, to evaluate the effectiveness of immune checkpoint inhibitors for skin cancer, to evaluate the malignancy of skin cancer, and to predict the recurrence of skin cancer. In addition, the biomarker can predict the likelihood of developing grade 3 or higher immune-related adverse events caused by the administration of immune checkpoint inhibitors for skin cancer, and therefore can be used to evaluate the immune activity of skin cancer. In addition, according to the present disclosure, it is conceivable to use it as a biomarker of G6PD in a sample taken from a patient with skin cancer. More specifically, it is conceivable to use it as a biomarker of G6PD in a sample taken from a patient with skin cancer for at least one of the following: prediction of the prognosis of skin cancer, evaluation of the effectiveness of immune checkpoint inhibitors for skin cancer, evaluation of the malignancy of skin cancer, prediction of the recurrence of skin cancer, prediction of the likelihood of developing grade 3 or higher immune-related adverse events caused by the administration of immune checkpoint inhibitors, and evaluation of the immune activity of skin cancer.

[0063] When a biomarker for predicting the prognosis of skin cancer is expressed at a high level, it indicates a poor prognosis for the skin cancer compared to when the expression level is low. When a biomarker for evaluating the effectiveness of an immune checkpoint inhibitor for skin cancer is expressed at a high level, it indicates a high effectiveness of the immune checkpoint inhibitor compared to when the expression level is low. When a biomarker for evaluating the malignancy of skin cancer is expressed at a high level, it indicates a high malignancy of the skin cancer compared to when the expression level is low.

[0064] Biomarkers used to predict skin cancer recurrence indicate a higher likelihood of skin cancer recurrence when the biomarker is expressed at higher levels in samples collected from patients with skin cancer than when it is expressed at lower levels. For example, if blood G6PD levels increase during postoperative observation, this suggests a likelihood of skin cancer recurrence and can be evaluated through imaging.

[0065] When a biomarker for evaluating immune activity against skin cancer is expressed at a high level in a sample collected from a patient with skin cancer, it indicates higher immune activity against skin cancer than when the expression level is low.

[0066] Biomarkers for predicting the likelihood of developing grade 3 or higher immune-related adverse events (irAEs) caused by the administration of immune checkpoint inhibitors for skin cancer indicate a higher likelihood of developing grade 3 or higher irAEs due to the administration of immune checkpoint inhibitors when the expression level of the biomarker in samples collected from skin cancer patients is low compared to when the expression level is high. As shown in the examples described below, when the expression level of G6PD is low, the frequency of severe irAEs due to the administration of immune checkpoint inhibitors is confirmed to be higher than when the expression level of G6PD is high.

[0067] According to other aspects of the present disclosure, there is provided a method for predicting the likelihood of developing an immune-related adverse event (irAE) of level 3 or higher caused by administering an immune checkpoint inhibitor to a patient with skin cancer. The method is included in the step (III) of the above-mentioned evaluation method for evaluating the effectiveness of an immune checkpoint inhibitor for skin cancer, and when the effectiveness of the immune checkpoint inhibitor is evaluated to be high, (V) evaluates the likelihood of developing an irAE of level 3 or higher caused by the administration of the immune checkpoint inhibitor (hereinafter also referred to as step (V)). In addition, the method for predicting the likelihood of developing an irAE of level 3 or higher caused by administering an immune checkpoint inhibitor to a patient with skin cancer may be a method comprising the above-mentioned step (I), i.e., (I) obtaining a correlation amount related to the expression amount of G6PD in a sample taken from a patient; (VI) when the correlation amount is small, a method for evaluating the likelihood of developing an irAE of level 3 or higher caused by the administration of the immune checkpoint inhibitor (hereinafter also referred to as step (VI)) is high compared to a case where the correlation amount is large.

[0068] For example, when the expression level of G6PD in the sample is low and the inferred expression level of PD-L1 is high, it is considered to be a tumor with high immune activity, so the possibility of high effectiveness of immune checkpoint inhibitors is high. In such tumors with high immune activity, the possibility of the onset of irAEs of grade 3 or above is high due to the administration of immune checkpoint inhibitors, so when administering immune checkpoint inhibitors, it is set that full attention should be paid to irAEs. In this way, the method of this embodiment for predicting the possibility of the onset of irAEs of grade 3 or above caused by the administration of immune checkpoint inhibitors to patients with skin cancer can be used as a method for assisting in the determination of treatment guidelines for skin cancer, such as determinations related to the administration of immune checkpoint inhibitors.

[0069] According to other aspects of the present disclosure, a determination kit may be provided. The determination kit may be used in at least one of the following: prediction of the prognosis of skin cancer, evaluation of the effectiveness of immune checkpoint inhibitors for skin cancer, evaluation of the malignancy of skin cancer, prediction of the recurrence of skin cancer, prediction of the likelihood of onset of immune-related adverse events of grade 3 or higher caused by the administration of immune checkpoint inhibitors, and evaluation of the immune activity of skin cancer, and includes a substance capable of detecting a related amount of the expression level of G6PD in a sample taken from a patient with skin cancer. In other words, the determination kit includes a companion diagnostic drug that can be used for the companion diagnosis of skin cancer. The substance capable of detecting a related amount of the expression level of G6PD may include a substance selected from a substance capable of binding to G6PD or a fragment of G6PD, a substance capable of binding to a gene encoding G6PD, and at least one of glucose-6-phosphate.

[0070] Examples of substances capable of binding to G6PD or G6PD fragments include anti-G6PD antibodies and fragments thereof. Furthermore, examples of substances capable of binding to the gene encoding G6PD include primer sets capable of amplifying the G6PD gene and probes that specifically hybridize to the G6PD gene. Furthermore, glucose-6-phosphate can be included in an assay kit as a substrate for measuring G6PD activity.

[0071] The form of the assay kit is not particularly limited, and for example, it may be in a dry state, or in a state where the above-mentioned substance solvent is in a solution in an amount that is capable of detecting the relevant amount of the G6PD expression level. In addition, the above-mentioned substance may be labeled with a marker such as a secondary antibody, a fluorescent substance, or a radioisotope, or may be fixed to a support such as a microarray substrate, a microtiter plate, or a resin or metal bead. In addition, in addition to the above-mentioned substances, the assay kit may include various components such as reverse transcriptase, DNA polymerase, dNTP, oligonucleotide dT primer, random primer, RNase inhibitor, RNaseH, labeling substance, and buffer. Furthermore, the assay kit may include various machines, instructions for use, and the like that can be used to determine the expression level of G6PD.

[0072] Example

[0073] Hereinafter, the present invention will be further described in detail with reference to examples, but the present invention is not limited to the following examples.

[0074] Merkel cell carcinoma

[0075] 1. Specimen

[0076] Formalin-fixed paraffin-embedded (FFPE) specimens and serum from Merkel cell carcinoma patients who underwent histopathological examination and had a defined postoperative course were used as samples. FFPE specimens comprised 90 specimens from 71 patients, and serum specimens comprised 50 specimens from 19 patients. The age of patients with FFPE specimens ranged from 40 to 98 years, with a mean age of 77.27 years, and a male to female ratio of 26:45. Tumor locations in these cases were the head and neck in 66.2% (42 cases on the face, 2 cases on the neck, 1 case on the ear, and 2 cases on the head), the limbs in 29.6% (4 cases on the upper arm, 3 cases on the forearm, 3 cases on the finger, 6 cases on the thigh, 3 cases on the calf, and 2 cases on the foot), and the trunk in 2.8% (2 cases on the buttocks). Spontaneous regression occurred in 6 of these cases.

[0077] 2. Exploration of biomarkers believed to be associated with PD-L1

[0078] 2-1. RNA Extraction

[0079] Unstained slides prepared from FFPE specimens were macrodissected using an 18G needle from the tumor and surrounding inflammatory cell-infiltrating areas, and RNA was extracted using the AllPrep DNA / RNA FFPE Kit (Qiagen). RIN values and DV200 (median value of RNA fragment size) were measured using a bioanalyzer (Agilent 2100, Agilent). Forty-four samples with a DV200 of 30% or greater were selected.

[0080] 2-2. Next-generation sequencing analysis

[0081] Next-generation sequencing (NGS) analysis was performed on 44 samples using the MiniSeq (Illumina) system and the Immune Response Panel (Ampliseq). The resulting data were analyzed using BaseSpace (registered trademark) Sequence Hub (Illumina).

[0082] Comparing the high and low PD-L1 expression groups, the only factor associated with PD-L1 with a q value (p-adjust) of 0.05 or less was glucose-6-phosphate dehydrogenase (G6PD). When PD-L1 was low, G6PD was significantly higher (p=0.00011, q=0.040). This indicates that G6PD exhibits a negative correlation with PD-L1.

[0083] 3. Study on the correlation between G6PD mRNA expression and prognosis

[0084] The correlation between the mRNA expression level of G6PD and the metastasis during the course of the disease in patients with skin cancer was studied. The results showed that the mRNA expression level of G6PD was correlated with the presence or absence of metastasis during the course of the disease in patients with skin cancer. In cases with high mRNA expression levels of G6PD, the proportion of lymph node metastasis and distant metastasis was significantly increased during the course of the disease (p = 0.00016, q = 0.018). An ROC curve was drawn for the presence or absence of metastasis, and 1071 CPM (counts per million) or more calculated as the benchmark point was used as a high mRNA expression level of G6PD, and 1071 CPM or less was used as a low mRNA expression level of G6PD.

[0085] Figure 1 It is a graph showing the Kaplan-Meier curve of the G6PD mRNA expression level. Figure 1In the figure, Kaplan-Meier curves of the survival rates of the group with high G6PD mRNA expression level and the group with low G6PD mRNA expression level are shown. Figure 1 In the figure, the solid line indicates the group with high G6PD mRNA expression level, and the dotted line indicates the group with low G6PD mRNA expression level. Figure 1 The vertical axis shows the overall survival rate, the horizontal axis shows the number of years, and the P value represents the log rank test. Figure 1 As shown, when the G6PD mRNA expression level was high (high), the survival rate was lower than when the G6PD mRNA expression level was low (low), that is, a clear correlation with poor prognosis was observed (P=0.036).

[0086] 4. Immunohistochemical Staining

[0087] 4-1. Immunohistochemical staining of G6PD

[0088] Immunohistochemical staining for G6PD was performed using slides prepared from FFPE specimens. Anti-G6PD antibody (HPA000247, manufactured by Sigma-Ardrich) was used. Photography and analysis were performed using an integrated fluorescence microscope BZ-X800 (manufactured by KEYENCE). The staining results were digitized, and the number of positive cells and expression intensity were numerically evaluated.

[0089] Figure 2 This is an explanatory diagram showing the results of G6PD expression analysis obtained by immunohistostaining. Figure 2 In the figure, an example of an immunohistochemical staining image of a group showing high G6PD expression is shown on the left side of the paper, and an example of an immunohistochemical staining image of a group showing low G6PD expression is shown on the right side of the paper. It should be noted that the evaluation of G6PD expression levels by immunohistochemical staining was performed by visual observation of the stained specimen under a microscope, with a staining rate of 50% or more being considered high (high) and less than 50% being considered low (low).

[0090] Figure 3 It is a graph showing the Kaplan-Meier curve of the G6PD expression level. Figure 3 In the figure, Kaplan-Meier curves of the survival rates of the high G6PD expression group and the low G6PD expression group are shown. Figure 3 In the figure, the dotted line shows the group with high G6PD expression, and the solid line shows the group with low G6PD expression. Figure 3 In the figure, the vertical axis represents the overall survival rate, and the horizontal axis represents the overall survival period (OS). Figure 3As shown in the figures, when the G6PD expression level was high, the survival rate was lower than when the G6PD expression level was low, that is, a clear correlation with poor prognosis was observed (P=0.036).

[0091] 4-2. Immunohistochemical staining of PD-L1 in primary tumors

[0092] As a comparative example, immunohistochemical staining for PD-L1 was performed using slides prepared from FFPE specimens of primary tumors. Anti-PD-L1 antibodies (28-8, ab205921, manufactured by Abcam) were used for imaging and analysis using an integrated fluorescence microscope, the BZ-X800 (manufactured by KEYENCE). The staining results were digitized, and the number of positive cells and expression intensity was numerically evaluated.

[0093] Figure 4 It is a graph showing the Kaplan-Meier curve of the expression level of PD-L1. Figure 5 It is a graph showing the results of correlation analysis of PD-L1 expression levels. Figure 4 Kaplan-Meier curves for the survival rates of the group with high PD-L1 expression and the group with low PD-L1 expression are shown. Figure 4 In the figure, the solid line represents the group with high PD-L1 expression, and the dotted line represents the group with low G6PD expression. Figure 4 The vertical axis represents the overall survival rate, and the horizontal axis represents the number of years. Figure 5 The vertical axis represents the expression level of PD-L1 (pixel value), and the horizontal axis represents the number of months. Figure 4 and Figure 5 As shown, no correlation with the survival rate was found in the comparison between the group showing high PD-L1 expression and the group showing low PD-L1 expression (r = 0.068, CI [from -0.19 to 0.31], P = 0.59).

[0094] 4-3. Examination of immunohistochemical staining

[0095] The results of immunohistochemical staining for G6PD and PD-L1 indicate that PD-L1 expression has no correlation with prognosis, whereas G6PD expression does. This suggests that G6PD can be used as a better prognostic marker than PD-L1.

[0096] 5.G6PD activity

[0097] 5-1. Measurement of G6PD activity using serum

[0098] G6PD activity was measured using a G6PD assay kit (Abcam, ab102529) in 50 serum samples from 19 patients. The average activity value of the 50 samples was 11.45 mU / ml.

[0099] 5-2. Relationship between G6PD activity and disease course

[0100] Among the 50 specimens, 3 specimens (cases 1 to 3) with particularly high G6PD activity (19 mU / ml or more) confirmed the progression of Merkel cell carcinoma. In addition, the progression of Merkel cell carcinoma was confirmed in the case (case 4) where G6PD activity was reduced. Figures 6-8 In the figure, the vertical axis represents G6PD activity (mU / ml), and the horizontal axis represents the period.

[0101] (1) Case 1

[0102] Figure 6 This diagram illustrates the evolution of G6PD activity in Case 1. Case 1 was a Stage IV case, with the primary tumor located in the right lower jaw. Surgical resection and radiotherapy were performed, but metastasis to lymph nodes throughout the body was observed approximately nine months after resection. Combination therapy with CBDGB and VP-16 was subsequently attempted, but no therapeutic effect was observed. Therefore, treatment with avelumab, an immune checkpoint inhibitor, was initiated, but the patient succumbed to the underlying disease several months later. High G6PD activity was observed at the time of the patient's succumb to the underlying disease, reaching 21.84 mU / ml.

[0103] (2) Case 2

[0104] Figure 7 This diagram illustrates the transition of G6PD activity in Case 2, a Stage IV case with the primary tumor located in the left calf. Despite surgical resection, SLNB, and radiotherapy, local recurrence was observed approximately one year after excision and skin grafting. High G6PD activity was observed at the time of local recurrence, reaching 19.63 mU / ml.

[0105] (3) Case 3

[0106] Case 3 is a stage II case, with the primary tumor located in the anterior chest. Although surgical resection and radiotherapy were performed, Case 3 passed away from another illness approximately two months after the completion of radiotherapy. Elevated G6PD activity was observed before the patient's death, at 19.37 mU / ml.

[0107] (4) Case 4

[0108] Figure 8 This is an explanatory diagram showing the progression of G6PD activity in Case 4. Case 4 is a stage III case, with the primary lesion located at the root of the nose. In Case 4, after surgical resection, nodules were observed in the right submandibular lymph node on PET / CT, so administration of the immune checkpoint inhibitor avelumab was started. Since enhancement of the nodules was observed on PET / CT 3 months later, IMRT (intensity-modulated radiation therapy) was used in combination, resulting in a CR (complete remission). G6PD activity was significantly reduced by the combination of avelumab and IMRT.

[0109] 6. Summary

[0110] According to the above results, in Merkel cell carcinoma, which is a skin cancer, the expression level of G6PD shows a negative correlation with PD-L1, and the more G6PD is expressed, the less PD-L1 is expressed. In addition, when the expression level of G6PD is high, a correlation with poor prognosis is observed compared to when the expression level of G6PD is low. In addition, when the activity of G6PD in serum is high and the expression level of G6PD is high, compared to when the activity of G6PD is low and the expression level of G6PD is low, a high possibility of recurrence of skin cancer is given. In other words, when the expression level of G6PD is high, compared to when the expression level of G6PD is low, a high malignancy of skin cancer and a tendency of worsening of the disease are given. Therefore, it can be seen that G6PD is effective as a biomarker for predicting the prognosis of skin cancer, a biomarker for evaluating the malignancy of skin cancer, and a biomarker for predicting the recurrence of skin cancer. Therefore, it was found that using G6PD expression as an indicator can predict the prognosis of skin cancer, evaluate the effectiveness of immune checkpoint inhibitors, determine whether to administer immune checkpoint inhibitors, and assess the malignancy of skin cancer. Furthermore, since higher G6PD expression correlates with lower PD-L1 expression, this suggests that using G6PD expression as an indicator can be used to assess immune activity in skin cancer.

[0111] Malignant melanoma

[0112] 1. Specimen

[0113] Samples from 30 primary lesions of malignant melanoma patients receiving immune checkpoint therapy were used. Immune checkpoint therapy refers to the administration of immune checkpoint inhibitors. The RNA extraction method, next-generation sequencing analysis method, G6PD immunohistochemical staining method, and G6PD expression evaluation criteria were the same as those described in the Merkel cell carcinoma example.

[0114] 2. Immunohistochemical Staining

[0115] Figure 9 This is an explanatory diagram showing the results of G6PD expression analysis obtained by immunohistochemical staining of malignant melanoma. Figure 9 An example of an immunohistochemical staining image of a group showing high G6PD expression is shown on the left side of the paper, and an example of an immunohistochemical staining image of a group showing low G6PD expression is shown on the right side of the paper. The results of immunohistochemical staining of G6PD samples from 30 cases showed high G6PD expression in 12 cases and low G6PD expression in 18 cases. It should be noted that the evaluation of G6PD expression levels by immunohistochemical staining was performed by visual observation of the stained specimens under a microscope, with a staining rate of 50% or more being considered high (high expression) and a staining rate of less than 50% being considered low (low expression).

[0116] 3. Correlation between G6PD mRNA expression and prognosis

[0117] Seventeen samples from patients in whom no grade 3 or higher immune-related adverse events (irAEs) were observed after administration of immune checkpoint inhibitors were used as subjects, and survival curves were created using the Kaplan-Meier method.

[0118] Figure 10 It is a graph showing the Kaplan-Meier curve of the G6PD expression level in malignant melanoma. Figure 10 In the figure, Kaplan-Meier curves of the survival rates of the high G6PD expression group and the low G6PD expression group are shown. Figure 10 In the figure, the thin line indicates the group with high G6PD expression level (high), and the thick line indicates the group with low G6PD expression level (low). Figure 10 In the data, the vertical axis represents progression-free survival, and the horizontal axis represents the number of years after ICI start. Figure 10 The results show that the survival rate of the group with low G6PD expression is higher than that of the group with high G6PD expression, which indicates a good prognosis. From this, it can be seen that even in the case where the type of skin cancer is malignant melanoma, it is believed that there is a trend similar to that in the case where the type of skin cancer is Merkel cell carcinoma. Therefore, for malignant melanoma, the expression level of G6PD is used as an indicator in the same way as in the case of Merkel cell carcinoma, which provides the following inspiration: it is possible to predict the prognosis of skin cancer, to evaluate the effectiveness of immune checkpoint inhibitors, to determine whether to give immune checkpoint inhibitors, to evaluate the malignancy of skin cancer, to predict the possibility of recurrence of skin cancer, and to evaluate the immune activity of skin cancer.

[0119] 4. Relationship between G6PD expression and immune-related adverse events

[0120] The relationship between G6PD expression and immune-related adverse events was analyzed. Whether grade 3 or higher immune-related adverse events (irAEs) were observed after administration of immune checkpoint inhibitors was analyzed in groups with high and low G6PD expression.

[0121] Figure 11 This is an explanatory diagram showing the relationship between the presence or absence of grade 3 or higher immune-related adverse events in malignant melanoma and the expression level of G6PD. Figure 11 The figure on the right side of the paper is a 2×2 split table of Fisher's test that correlates the expression level of G6PD with the presence or absence of irAEs of grade 3 or higher. Figure 11 The figure on the left side of the paper is a chart made together with the 2×2 partitioning table. Figure 11 "irAE+" means irAEs of grade 3 or higher were observed, and "irAE-" means no irAEs of grade 3 or higher were observed. Figure 11 The results shown show that in the group with low G6PD expression, significantly more cases of irAEs of grade 3 or above were observed (p = 0.019). That is, when the expression of G6PD is low, compared with the case where the expression of G6PD is high, the administration of immune checkpoint inhibitors clearly shows a high correlation with the possibility of developing irAEs of grade 3 or above. Therefore, it can be seen that using the expression of G6PD as an indicator, the possibility of developing irAEs of grade 3 or above due to the administration of immune checkpoint inhibitors to patients with skin cancer can be predicted.

[0122] Cutaneous angiosarcoma

[0123] 1. Specimen

[0124] Fourteen specimens from seven cases of cutaneous angiosarcoma were used as samples. The RNA extraction method, next-generation sequencing analysis method, G6PD immunohistochemical staining method, and G6PD expression evaluation criteria were the same as those described in the Merkel cell carcinoma example.

[0125] (1) Case A

[0126] In Case A, the primary tumor was located in the head and was treated with wPTX and radiotherapy. Despite wPTX, Case A's performance status (PS) worsened, leading to a switch to Best Supportive Care (BSC). Lung metastasis was subsequently observed, and the patient died from the primary disease. Case A's overall survival (OS) was 389 days.

[0127] (2) Case B

[0128] Case B, where the primary tumor was located in the head, was treated with wPTX, radiotherapy, and eribulin. During wPTX, metastasis to the parotid gland lymph nodes was observed in Case B, leading to a switch to eribulin, which resulted in pneumothorax (PD). The overall survival (OS) in Case B was 488 days.

[0129] (3) Case C

[0130] Case C, whose primary tumor was located in the head, underwent surgery, IL-2 immunotherapy, wDTX therapy, wPTX therapy, and radiotherapy. In Case C, wDTX administration caused pneumothorax, so treatment was switched to wPTX. At the patient's request, chemotherapy was discontinued, and initiation of chemotherapy is under consideration. Case C's overall survival (OS) was over 4826 days.

[0131] 2. Immunohistochemical Staining

[0132] Figure 12 This is an explanatory diagram showing the results of G6PD expression analysis obtained by immunohistostaining of cutaneous angiosarcoma. Figure 12 In the figure, immunohistochemical staining images of cases A, B, and C of cutaneous angiosarcoma are shown on the upper side of the paper, and magnified images are shown on the lower side of the paper. Cases A and B show the results of biochemical test specimens used as samples, and case C shows the results of surgical specimens used as samples. Figure 12 The results shown confirm that G6PD is highly expressed in Case A and Case B, and G6PD is lowly expressed in Case C. In addition, based on the relationship between the expression level of G6PD and prognosis, it is suggested that when the expression level of G6PD is low, it indicates a good prognosis, and when the expression level of G6PD is high, there is a tendency for poor prognosis. Thus, in the case where the type of skin cancer is cutaneous angiosarcoma, the same trend as when the type of skin cancer is Merkel cell carcinoma can be observed. Therefore, for cutaneous angiosarcoma, similar to the case of Merkel cell carcinoma, the following suggestions are given: using the expression level of G6PD as an indicator, the prognosis of skin cancer can be predicted, the effectiveness of immune checkpoint inhibitors can be evaluated, whether immune checkpoint inhibitors can be given, the malignancy of skin cancer can be evaluated, the possibility of recurrence of skin cancer can be predicted, and the immune activity of skin cancer can be evaluated.

[0133] 3. Next-generation sequencing analysis

[0134] Figure 13This figure illustrates the results of G6PD expression analysis obtained through next-generation sequencing of cutaneous angiosarcoma. Of the 14 specimens used, 10 were G6PD-positive and 4 were G6PD-negative. G6PD-positive indicates high G6PD expression, with a staining rate of 50% or more in tumor cells as determined by immunohistochemical staining. G6PD-negative indicates low G6PD expression, with a staining rate of less than 50% in tumor cells as determined by immunohistochemical staining.

[0135] 4.GSEA analysis

[0136] Gene set enrichment analysis (GSEA) was performed using GSEA software (https: / / www.gsea-msigdb.org / gsea / ) to analyze the gene sets and gene lists with high expression levels in the G6PD high-expression group. The c5 Gene Ontology (GO) gene set collection provided by MSigDB (Molecular Signatures Database) was used as the gene set.

[0137] Figure 14 and Figure 15 This is an explanatory diagram showing the results of GSEA analysis of the group with high G6PD expression in cutaneous angiosarcoma. Figure 14 and Figure 15 The results shown show that the group with high G6PD RNA expression showed abnormalities in antibody production. Therefore, the group with high G6PD expression may have decreased immunity.

[0138] Based on the results of the examples described above, it can be considered that the same trend exists regardless of the type of skin cancer. Therefore, the following implications can be obtained: regardless of the type of skin cancer, by using the expression level of G6PD as an indicator, the prognosis of skin cancer can be predicted, the effectiveness of immune checkpoint inhibitors can be evaluated, the administration of immune checkpoint inhibitors can be determined, the malignancy of skin cancer can be evaluated, the possibility of recurrence of skin cancer can be predicted, the immune activity of skin cancer can be evaluated, and the possibility of the onset of grade 3 or higher irAEs caused by the administration of immune checkpoint inhibitors can be predicted.

[0139] The present invention is not limited by the above-mentioned embodiments and examples, and various modifications are encompassed by the present invention within the scope that can be easily conceived by a person skilled in the art without departing from the scope of the claims.

Claims

1. Use of glucose-6-phosphate dehydrogenase in the manufacture of a kit for predicting the prognosis of Merkel cell carcinoma or cutaneous angiosarcoma.

2. Use of glucose-6-phosphate dehydrogenase in the manufacture of a kit for evaluating the effectiveness of immune checkpoint inhibitors against Merkel cell carcinoma or cutaneous angiosarcoma.

3. Use of glucose-6-phosphate dehydrogenase in the manufacture of a kit for evaluating the malignancy of Merkel cell carcinoma or cutaneous angiosarcoma.

4. Use of glucose-6-phosphate dehydrogenase in the manufacture of a kit for predicting the recurrence of Merkel cell carcinoma or cutaneous angiosarcoma.

5. Use of glucose-6-phosphate dehydrogenase in the manufacture of a kit for evaluating the immunoreactivity of Merkel cell carcinoma or cutaneous angiosarcoma.

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