Application and method of metabolic marker

Abstract

The invention discloses application and a method of a metabolic marker. Comprising an application of the metabolic marker in preparing a medicine for detecting the effectiveness of liver cancer immunotherapy and a method for detecting the effect of liver cancer immunotherapy by using the metabolic marker. The metabolic marker with high specificity, universality and reliability is used as a marker, animal experiments prove that the specific inhibition of the metabolic marker can enhance the curative effect of liver cancer immunotherapy, and the metabolic marker has clinical transformation potential.

Description

Technical Field

[0001] The present invention relates to a metabolic marker, and in particular to an application and method of a metabolic marker. Background Art

[0002] The diagnosis of liver cancer relies on imaging examinations (such as ultrasound, CT, MRI) and serum marker tests (such as alpha-fetoprotein AFP). Pathological puncture biopsy can also be used as a means of diagnosis. The treatment of liver cancer depends on the progression of the cancer, mainly including surgical resection, liver transplantation, local ablation therapy (such as radiofrequency ablation, microwave ablation), transarterial chemoembolization (TACE), molecular targeted therapy and immunotherapy. In recent years, many treatment methods have emerged for liver cancer in the late stage that has lost the opportunity for surgery, including photothermal therapy, targeted drugs and immunotherapy.

[0003] Liver cancer has a strong immune escape ability. It can inhibit the activity of immune cells by changing the tumor microenvironment, thus protecting tumor cells from the attack of the immune system. The goal of immunotherapy is to activate or enhance the immune system so that it can effectively identify and eliminate tumor cells. Immune checkpoint inhibitors block the binding of inhibitory immune checkpoint molecules (such as PD-1, PD-L1, and CTLA-4) to their ligands, thereby relieving the inhibitory effect on T cells and restoring the killing function of T cells against tumors. Clinical trials have shown that immune checkpoint inhibitors can significantly prolong the overall survival of patients with liver cancer, and some patients have a lasting response to treatment. However, there are still some patients who do not respond to immunotherapy, and even accelerate the progression of liver cancer. Therefore, in order to improve the success rate of immunotherapy, it is of great significance to develop new predictive markers to evaluate which patients can benefit from immunotherapy.

[0004] Sphingolipid metabolism plays an important role in the occurrence and development of cancer, especially in regulating multiple physiological processes such as cell proliferation, apoptosis, differentiation and autophagy. As a bioactive lipid, sphingolipids are closely related to tumor proliferation, metastasis and chemotherapy resistance in various malignant tumors such as liver cancer. In liver cancer, the imbalance of sphingolipid metabolism usually promotes the survival of tumor cells. For example, certain sphingosine and its derivatives, such as sphingosine-1-phosphate (S1P), can promote tumor proliferation and anti-apoptosis ability by activating growth signaling pathways. Therefore, metabolic markers of sphingolipid metabolism have great potential value in predicting the efficacy of cancer immunotherapy and predicting patient survival prognosis. Summary of the invention

[0005] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide an application and method of a metabolic marker S1P. In order to achieve the above purpose and other related purposes, the present invention has demonstrated at the clinical case, animal and molecular levels that the metabolic marker S1P in the serum and tissues of liver cancer patients who do not respond to immunotherapy is significantly increased. Therefore, the level of the metabolic marker S1P in the in vitro liver cancer serum sample solution can be detected to detect whether immunotherapy is effective for liver cancer, providing a new way to detect the effectiveness of liver cancer immunotherapy. The first aspect of the present invention provides an application of a metabolic marker. The second aspect of the present invention provides a method for detecting the effect of liver cancer immunotherapy.

[0006] The technical solution adopted by the present invention is:

[0007] 1. Application of a metabolic marker

[0008] The metabolic marker is used in the preparation of a drug for detecting the effectiveness of liver cancer immunotherapy.

[0009] The liver cancer immunotherapy adopts transarterial chemoembolization combined with programmed death receptor-1 (TACE+PD-1) monoclonal antibody treatment.

[0010] The metabolic marker is a sphingolipid metabolite.

[0011] The metabolite marker is sphingolipid metabolite sphingosine-1-phosphate.

[0012] The medicine comprises a detection reagent and a detection tool for detecting the expression level of a metabolic marker.

[0013] 2. A method for detecting the effect of immunotherapy for liver cancer

[0014] The method includes: obtaining an in vitro liver cancer serum sample solution, detecting the expression level of a metabolic marker in the liver cancer serum sample solution, determining whether the expression level is greater than a preset expression threshold, and then predicting the effectiveness of liver cancer immunotherapy: if the expression level is greater than the expression threshold, it is predicted that the liver cancer immunotherapy is ineffective; if the expression level is less than or equal to the expression threshold, it is predicted that the liver cancer immunotherapy is effective.

[0015] Experimental tests have shown that the metabolic marker sphingosine-1-phosphate (S1P) in the serum and liver cancer tissue lysate of liver cancer patients were significantly higher than those in the benign controls of healthy humans.

[0016] Generally speaking, after TACE+PD-1 monoclonal antibody treatment, the CT images of liver cancer patients who respond to the treatment show tumor shrinkage, while the CT images of non-responders show that the tumor size remains unchanged or increases.

[0017] The present invention found in the experiment that the metabolic difference analysis of the serum of liver cancer patients who were unresponsive and responsive to treatment showed that the sphingolipid signaling pathway was significantly enriched in the serum metabolites of patients who were unresponsive to treatment.

[0018] Specifically, in patients treated with TACE+PD-1 monoclonal antibody, the metabolite marker S1P was significantly higher in the serum of patients who did not respond to the treatment than in the serum of patients who responded to the treatment.

[0019] There was no significant difference in the serum level of the metabolic marker sphingosine (sphingosine is a precursor of sphingosine 1-phosphate, and sphingosine kinase catalyzes the phosphorylation of sphingosine to produce S1P) in patients who underwent TACE+PD-1 monoclonal antibody treatment, between those who did not respond to treatment and those who responded to treatment.

[0020] There was no significant difference in the serum levels of the metabolic marker sphingomyelin (sphingomyelin is the main component of cell membranes, and sphingomyelin produces S1P under the catalysis of multiple kinases) in patients who underwent TACE+PD-1 monoclonal antibody treatment, between patients who did not respond to treatment and those who responded to treatment.

[0021] Experiments showed that the area under the curve (ROC) of the metabolic marker S1P in predicting the effectiveness of TACE+PD-1 monoclonal antibody treatment was 0.9167, indicating that the metabolic marker S1P has high sensitivity and specificity as a predictive factor.

[0022] The metabolite marker is sphingosine-1-phosphate, a sphingolipid metabolite.

[0023] The expression levels of the metabolic markers were quantitatively detected using enzyme-linked immunosorbent assay (ELISA).

[0024] The method further includes: the liver cancer serum sample solution comes from a liver cancer human body treated with transarterial chemoembolization combined with programmed death receptor-1 monoclonal antibody.

[0025] By judging whether the expression level is greater than the preset expression threshold, the response type of the liver cancer patient in the monoclonal antibody treatment is judged: if the expression level is greater than the expression threshold, it is predicted that the liver cancer patient is a non-responsive type to the transarterial chemoembolization combined with programmed death receptor-1 monoclonal antibody treatment; if the expression level is less than or equal to the expression threshold, it is predicted that the liver cancer patient is a responder to the transarterial chemoembolization combined with programmed death receptor-1 monoclonal antibody treatment.

[0026] The metabolite marker S1P is significantly positively correlated with the tumor immunosuppressive components tumor-associated macrophages (TAMs) and Tregs, and negatively correlated with the immune activation component CD8+T cells. The above correlation is used to reflect the tumor status and immunotherapy status in human liver cancer patients through the metabolite marker S1P.

[0027] The method further includes: judging whether the expression level is greater than a preset expression threshold and then judging the prognosis of liver cancer immunotherapy: if the expression level is greater than the expression threshold, the recurrence-free survival and overall survival are shorter, and the prognosis is poor; if the expression level is less than or equal to the expression threshold, the recurrence-free survival and overall survival are longer, and the prognosis is better.

[0028] The beneficial effects of the present invention are:

[0029] The present invention provides an application and method of a metabolite marker S1P, and establishes a practical, sensitive, and specific immunological detection method for the serum metabolite marker S1P. The research of the present invention shows that the metabolite marker S1P is a metabolite marker for detecting the effect of immunotherapy for liver cancer with high specificity, versatility, and reliability, and is easy to popularize and popularize; moreover, compared with obtaining cancer tissue samples by biopsy to detect the expression of the metabolite marker S1P, in vitro serum samples are easier to obtain, which is more convenient for detecting the level of the metabolite marker S1P, and the detection method of the metabolite marker S1P is simpler and easier to promote and apply in clinical or primary hospitals; in addition, the method for detecting the effect of immunotherapy for liver cancer using the metabolite marker S1P established by the present invention provides a reliable biological detection basis for liver cancer, which provides help for the current detection method based on clinical data. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram for stratifying patients into those who respond to treatment and those who do not;

[0031] Figure 2 Schematic diagram of obtaining serum for metabolomics sequencing and enrichment analysis;

[0032] Figure 3 Schematic diagram for comparing the differences in the levels of sphingolipid metabolism-related markers in different groups;

[0033] Figure 4 Schematic diagram for evaluating the sensitivity and specificity of the metabolic marker S1P in predicting TACE+PD-1 monoclonal antibody treatment;

[0034] Figure 5 This is a schematic diagram showing that the content of the metabolic marker S1P is significantly higher than that of the benign liver disease control group;

[0035] Figure 6A schematic diagram showing a trend that the recurrence-free survival of the group with a high level of the metabolic marker S1P is shorter than that of the group with a low level of the metabolic marker S1P;

[0036] Figure 7 A schematic diagram showing that the metabolic marker S1P in serum and tissues of the advanced liver cancer group was significantly higher than that of the early group;

[0037] Figure 8 A schematic diagram showing that the overall survival of the group with high S1P level is significantly shorter than that of the group with low S1P level;

[0038] Fig. 9 A schematic diagram showing that the level of the metabolic marker S1P in tissues is positively correlated with the number of tumor-associated macrophages and Tregs, which are anti-tumor immune suppressive components, and negatively correlated with the number of CD8+T cells, which are anti-tumor immune activating components;

[0039] Fig.10 A schematic diagram showing the feasibility of combining the metabolic marker S1P inhibitor Fingolimod with PD-1 monoclonal antibody in an animal model of liver cancer;

[0040] Fig.11 Schematic diagram showing that the use of PD-1 monoclonal antibody alone did not significantly inhibit the progression of liver cancer, the use of the metabolic marker S1P inhibitor Fingolimod alone could slightly inhibit the progression of liver cancer, and the combination of the metabolic marker S1P inhibitor and PD-1 monoclonal antibody significantly inhibited the progression of liver cancer and significantly prolonged the survival of mice;

[0041] Fig.12 This is a schematic diagram showing that the combination of metabolic marker S1P inhibitor and PD-1 monoclonal antibody has certain safety.

[0042] Fig.13 Schematic diagram showing that the spleen size and mass of mice in each group did not change significantly;

[0043] Fig.14 This is a schematic diagram showing that the metabolic marker S1P in serum can be used as a metabolic marker for detecting the effectiveness of immunotherapy. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Embodiments of the present invention are as follows:

[0046] According to the principles of medical ethics, the present invention collects CT imaging data of patients in clinical trials of TACE+PD-1 monoclonal antibody treatment and divides the patients into those who respond to treatment and those who do not respond (see Figure 1 ), the tumor lesions of those who responded to the treatment were significantly reduced after treatment, while the tumor lesions of those who did not respond did not reduce or even increased.

[0047] First, serum was collected from patients for metabolomics sequencing and enrichment analysis (see Figure 2 ) and compared the levels of sphingolipid metabolism-related markers in different groups (see Figure 3 ), the results showed that the sphingolipid metabolism pathway was the most significantly enriched pathway.

[0048] The area under the receiver operating curve (AUC) was then used to evaluate the sensitivity and specificity of the metabolic marker S1P in predicting response to TACE+PD-1 monoclonal antibody treatment (see Figure 4 ), the results showed that AUC = 0.9167, p < 0.0001, indicating that the predictive sensitivity and specificity of the metabolic marker S1P were high.

[0049] Tissues and serum of liver cancer patients and benign liver disease controls were collected, and the level of the metabolic marker S1P was detected by ELISA. The results showed that the content of the metabolic marker S1P in the serum and cancer tissues of liver cancer patients was significantly higher than that in benign liver disease controls (see Figure 5 ), and in the clinical cohort of patients with liver cancer, the recurrence-free survival of the group with high S1P levels tended to be shorter than that of the group with low S1P levels (p = 0.06, see Figure 6 ), the overall survival of the group with high S1P level was significantly shorter than that of the group with low S1P level (p<0.05, see Figure 8 ), which indicates that the metabolic marker S1P in serum can be used as a metabolic marker to detect after treatment.

[0050] Liver cancer patients were divided into early stage group (I and II stage) and late stage group (III and IV stage). The expression level of the metabolic marker S1P in serum and cancer tissues of the two groups was detected by ELISA. The results showed that the metabolic marker S1P in serum and tissues of the late stage liver cancer group was significantly higher than that of the early stage group (see Figure 7 ), which indicates that the metabolic marker S1P in serum can be used as a metabolic marker for liver cancer staging.

[0051] Multiple immunohistochemical staining was used to stain tumor-associated macrophages, Tregs, and CD8+T cells in liver cancer tissues. The results showed that the level of the metabolic marker S1P in the tissue was positively correlated with the number of tumor-associated macrophages and Tregs, which are anti-tumor immune inhibitory components, and negatively correlated with the number of CD8+T cells, which are anti-tumor immune activation components (see Fig. 9), which indicates that the metabolic marker S1P in serum can be used as a metabolic marker to detect the effectiveness of immunotherapy.

[0052] The feasibility of combining the metabolic marker S1P inhibitor fingolimod with PD-1 monoclonal antibody was verified in an animal model of liver cancer (see Fig.10 ), the use of PD-1 monoclonal antibody alone did not significantly inhibit the progression of liver cancer, the use of the metabolic marker S1P inhibitor Fingolimod alone could slightly inhibit the progression of liver cancer, and the combination of the metabolic marker S1P inhibitor and PD-1 monoclonal antibody significantly inhibited the progression of liver cancer and significantly prolonged the survival of mice (see Fig.11 ). A quantitative analysis of liver cancer was then performed, and the results showed that the combination of the metabolic marker S1P inhibitor and PD-1 monoclonal antibody significantly reduced the number of liver cancers in the mouse model, while the body weight of the mice in each group did not change significantly, indicating that the combination of the metabolic marker S1P inhibitor and PD-1 monoclonal antibody has a certain safety (see Fig.12 ).

[0053] To further evaluate the safety of the combination of metabolic marker S1P inhibitor and PD-1 monoclonal antibody, the spleens of mice in each intervention group were compared. The results showed that the spleen size and mass of mice in each group did not change significantly (see Fig.13 ).

[0054] In order to further evaluate the effectiveness of the combination of metabolic marker S1P inhibitor and PD-1 monoclonal antibody, flow cytometry analysis was performed on liver cancer tissues of mouse models. The results showed that the combination of metabolic marker S1P inhibitor and PD-1 monoclonal antibody reduced the number of M2 tumor-associated macrophages, reduced the number of Tregs cells, increased the number of CD8+T cells, and enhanced the function of CD8+T cells, which was manifested by upregulation of GZMB of CD8+T cells. This suggests that the metabolic marker S1P in serum can be used as a metabolic marker for detecting the effectiveness of immunotherapy (see Fig.14 ).

[0055] The present invention first discovered that in the serum of liver cancer patients, the metabolite marker S1P in patients who did not respond to immunotherapy was higher than that in patients who responded, proving that the metabolite marker S1P can be used as a biomarker to predict the effectiveness of liver cancer immunotherapy. Further, animal experiments verified that the increase in the metabolite S1P metabolite discovered by the present invention is associated with a poor prognosis, and that inhibiting the metabolite marker S1P in combination with PD-1 monoclonal antibodies can effectively inhibit the progression of liver cancer, which provides potential clinical use value for immunotherapy of liver cancer.

[0056] The present invention also conducted biological analysis and clinical performance evaluation:

[0057] 1) Collect serum and tissues from liver cancer patients and other patients who received TACE+PD-1 monoclonal antibody treatment in a hospital, and conduct the following biological analysis (metabolome sequencing technology, enrichment analysis, enzyme-linked immunosorbent assay (ELISA) and animal experiments) and clinical manifestation evaluation (clinical CT image comparison); other patients are patients with benign diseases, and blood is also required for follow-up testing during the testing process.

[0058] 2) Use metabolome sequencing technology to process patient samples that respond and do not respond to TACE+PD-1 monoclonal antibody treatment, and perform enrichment analysis to obtain metabolome differential pathways;

[0059] 3) Measure the level of metabolic marker S1P in serum and tissues of HCC patients using ELISA;

[0060] 4) Use multiplex immunostaining to quantify the number of cells in each immune component in HCC patient tissues.

[0061] Through the above steps, the correlation between the levels of the metabolic marker S1P in the serum and tissues of liver cancer patients and the effectiveness of TACE+PD-1 monoclonal antibody treatment was analyzed. The results showed that the metabolic marker S1P was highly expressed in the serum and tissues of the group that did not respond to TACE+PD-1 monoclonal antibody treatment, and increased with the increase of liver cancer stage (see Figure 7 ).like Fig. 9 As shown, the level of the metabolite marker S1P is positively correlated with the anti-tumor immune suppression components TAMs and Tregs, and negatively correlated with the anti-tumor immune activation component CD8+T cells. This proves that the metabolite marker S1P can be used as a metabolic marker to predict the effectiveness of immunotherapy for liver cancer.

[0062] The present invention also conducted an animal experiment, dividing several liver cancer model C57 mice into four groups, namely, a negative control group without intervention, a group treated only with the metabolic marker S1P inhibitor Fingolimod, a group treated only with PD-1 monoclonal antibody, and a group treated with a combination of the metabolic marker S1P inhibitor Fingolimod and PD-1 monoclonal antibody; the blood, liver tissue, and spleen of the liver cancer model C57 mice were respectively taken for analysis:

[0063] a) extracting serum and cancer tissues from mice in each group, and detecting the levels of the metabolic marker S1P in the four groups using ELISA;

[0064] b) extracting cancer tissues from each group of mice and dispersing them into single cells, and using tissue flow cytometry to detect the composition of the immune microenvironment in the cancer tissues of each of the four groups;

[0065] c) extracting the spleen of each group of mice, observing the morphology and size of the spleen, and weighing and analyzing its mass;

[0066] Through the above-mentioned animal experiments, the results showed that the liver cancer of mice in the group treated with the combined treatment of the metabolic marker S1P inhibitor Fingolimod and PD-1 monoclonal antibody was significantly inhibited, and the survival time of mice in the group treated with the combined treatment of the metabolic marker S1P inhibitor Fingolimod and PD-1 monoclonal antibody was the longest.

[0067] Among them, in b) above, after using tissue technology, it was found that the number of M2 tumor-associated macrophages, an anti-tumor immunosuppressive component, significantly decreased in the mice in the group treated with the metabolic marker S1P inhibitor Fingolimod and PD-1 monoclonal antibody, the number of Tregs cells, an anti-tumor immunosuppressive component, significantly decreased, and the number of CD8+T cells, an anti-tumor immune activation component, significantly increased, and GZMB, a marker related to the anti-tumor function of CD8+T cells, was also significantly upregulated in CD8+T cells. In c) above, after removing the spleens of mice in each group, there was no significant difference in the spleens of mice in each group by naked eye observation, and the results of spleen mass analysis showed that there was no significant difference in the spleen mass of mice in each group.

[0068] In the present invention, the metabolite marker S1P is used as a metabolite marker in detecting the effect of liver cancer immunotherapy and judging the prognosis of liver cancer immunotherapy as follows:

[0069] Detection of the effect of immunotherapy for liver cancer: It was found that liver cancer patients with high levels of the metabolic marker S1P in their serum tended not to benefit from TACE+PD-1 monoclonal antibody immunotherapy. The serum of the patients was collected and the level of the metabolic marker S1P in the serum was detected by ELISA. Based on the level of the metabolic marker S1P in the serum, it was predicted whether the patients could benefit from TACE+PD-1 monoclonal antibody immunotherapy.

[0070] Determining the prognosis of liver cancer immunotherapy:

[0071] (1) The level of metabolic marker S1P was detected by enzyme-linked immunosorbent assay (ELISA).

[0072] (2) For patients who have been confirmed to have liver cancer and are suitable for immunotherapy, serum should be collected and sent to the laboratory for testing.

[0073] (3) The laboratory uses the enzyme-linked immunosorbent assay (ELISA) method to detect the level of the metabolic marker S1P and provides feedback on the detection results of the metabolic marker S1P.

[0074] (4) Based on the level of the metabolic marker S1P in the serum, the efficacy of immunotherapy in patients with liver cancer can be predicted and evaluated.

[0075] The present invention demonstrates at the animal and human levels that liver cancer patients who do not respond to TACE+PD-1 monoclonal antibody treatment have high levels of the metabolic marker S1P, and verifies the feasibility of using the metabolic marker S1P as a metabolic marker to predict the effectiveness of immunotherapy in liver cancer patients; through clinical sample information, it is verified that the metabolic marker S1P in serum can be used to predict the effectiveness of immunotherapy for liver cancer, and the clinical value of serum metabolic marker S1P as a prognostic assessment marker is evaluated; therefore, the level of the metabolic marker S1P in the serum of individual liver cancer subjects can be detected to predict the effectiveness of immunotherapy for liver cancer, providing a new approach for the comprehensive treatment of liver cancer.

[0076] The present invention provides a metabolic marker S1P in serum as a metabolic marker for predicting the effectiveness of liver cancer immunotherapy and judging the prognosis of liver cancer immunotherapy.

[0077] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. An application of a metabolic marker, characterized in that: The metabolic marker is used in the preparation of a drug for detecting the effectiveness of liver cancer immunotherapy.

2. The use of a metabolic marker according to claim 1, characterized in that: The metabolic marker is a sphingolipid metabolite.

3. The use of a metabolic marker according to claim 1 or 2, characterized in that: The metabolic marker is sphingosine-1-phosphate.

4. The use of a metabolic marker according to claim 1, characterized in that: The medicine comprises a detection reagent and a detection tool for detecting the expression level of a metabolic marker.

5. A method for detecting the effect of immunotherapy for liver cancer, characterized in that: Methods include: Obtain an in vitro liver cancer serum sample solution, detect the expression level of the metabolic markers in the liver cancer serum sample solution, and determine whether the expression level is greater than a preset expression threshold, thereby predicting the effectiveness of liver cancer immunotherapy: If the expression level is greater than the expression threshold, it is predicted that liver cancer immunotherapy is ineffective; If the expression level is less than or equal to the expression threshold, it is predicted that liver cancer immunotherapy is effective.

6. A method for detecting the effect of liver cancer immunotherapy according to claim 5, characterized in that: The metabolite marker is sphingosine-1-phosphate, a sphingolipid metabolite.

7. A method for detecting the effect of liver cancer immunotherapy according to claim 5, characterized in that: The expression levels of the metabolic markers were quantitatively detected using enzyme-linked immunosorbent assay (ELISA).

8. The method for detecting the effect of liver cancer immunotherapy according to claim 5, characterized in that: The method further comprises: By judging whether the expression level is greater than the preset expression threshold, we can determine the response type of liver cancer patients to monoclonal antibody treatment: If the expression level is greater than the expression threshold, it is predicted that the liver cancer patient is unresponsive to transarterial chemoembolization combined with programmed death receptor-1 monoclonal antibody therapy; If the expression level is less than or equal to the expression threshold, it is predicted that the liver cancer patient is a responsive type to transarterial chemoembolization combined with programmed death receptor-1 monoclonal antibody treatment.

9. A method for detecting the effect of liver cancer immunotherapy according to claim 4, characterized in that: The method further comprises: The prognosis of liver cancer immunotherapy can be determined by judging whether the expression level is greater than the preset expression threshold: If the expression level is greater than the expression threshold, the recurrence-free survival and overall survival are shorter and the prognosis is poorer; If the expression level is less than or equal to the expression threshold, the recurrence-free survival and overall survival are longer and the prognosis is better.

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