Methods of predicting risk of cancer post-surgical recurrence

Abstract

The present invention relates to a method of predicting the risk of recurrence of cancer after surgery, and its use in a method of predicting the survival of the subject; a method of prognosis of the subject; a method of administering adjuvant chemotherapy to the subject; and a method of preventing or reducing the risk of recurrence of cancer in the subject. The method comprises quantifying the level of adhesion of a population of cells cultured in the presence of a blood sample of a subject before and after surgical resection of the cancer.

Description

Technical Field

[0001] This invention relates to a method for predicting the risk of cancer recurrence after surgery and its application. Background Technology

[0002] Currently, the primary curative treatment strategy for most cancers still revolves around surgical removal of the tumor. In advanced stages of the disease, this approach is often accompanied by adjuvant therapy.

[0003] In recent years, thanks to advancements in screening programs, cancer treatment, standardized cancer care workflows, and surgical techniques, long-term patient outcomes have significantly improved. However, even after successful surgery, the recurrence rate remains high.

[0004] For example, about 30% of individuals who undergo surgery for colorectal cancer experience disease recurrence within five years of initial treatment, which is accompanied by an increased risk of death and increased morbidity.

[0005] Therefore, there is a need in the field to identify patients at risk of cancer recurrence after surgical treatment in order to provide such patients with the best possible care. Summary of the Invention

[0006] This disclosure relates to a newly identified method for predicting the risk of postoperative cancer recurrence in subjects.

[0007] Emerging preclinical evidence suggests that perioperative events can lead to disease recurrence by creating favorable conditions for pre-existing micrometastases or the spread of cancer cells during surgery. Here, the inventors disclose a method for assessing the risk of postoperative cancer recurrence by collecting blood samples from subjects before (referred to herein as “first sample”) and after (referred to herein as “second sample”), and culturing cell populations in the presence of these samples.

[0008] The risk of postoperative cancer recurrence increases when cell populations grown in the presence of a second blood sample exhibit higher adhesion levels to solid substrates than cell populations grown in the presence of a first blood sample.

[0009] Therefore, in the first aspect, this disclosure relates to a method for predicting the risk of postoperative cancer recurrence in a subject, comprising: a. Culture a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; b. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; c. Culture cell populations on a solid substrate in the presence of a second blood sample obtained from the subject after surgical removal of cancer; d. Quantify the adhesion level of cell populations to solid substrates in the presence of a second blood sample; e. Compare the adhesion levels of cell populations cultured in the presence of the first and second blood samples to solid substrates; The cell population in question is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level of the cell population in the second blood sample is higher than that in the first blood sample.

[0010] In another aspect, this disclosure relates to a method for predicting the survival of a subject after surgical removal of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in a subject using the methods described herein.

[0011] In another aspect, this disclosure relates to methods for assessing the prognosis of subjects after surgical resection of cancer, including: a) Predict the risk of postoperative recurrence of the cancer in a subject using the methods described herein.

[0012] In another aspect, this disclosure relates to a method for determining whether a subject should receive adjuvant chemotherapy and / or radiation therapy after surgical removal of cancer, comprising: a) Predicting the risk of postoperative recurrence of the aforementioned cancer in subjects using the methods described herein; and b) If the subject has a risk of recurrence of the aforementioned cancer after surgery, the conclusion is that the subject should receive adjuvant chemotherapy and / or radiotherapy.

[0013] In another aspect, this disclosure relates to a method for administering adjuvant chemotherapy and / or radiotherapy to a subject after surgical removal of cancer, comprising: a) Predicting the risk of postoperative recurrence of the cancer in subjects using the methods described herein; b) Administer adjuvant chemotherapy and / or radiation therapy to subjects who are predicted to have a risk of postoperative recurrence of the said cancer.

[0014] In another aspect, this disclosure relates to a method for administering adjuvant chemotherapy and / or radiotherapy to a subject after surgical resection of cancer, wherein the subject has been predicted to have a risk of postoperative cancer recurrence, and the prediction of the subject's risk of postoperative cancer recurrence has been performed by the method described herein.

[0015] In another aspect, this disclosure relates to a method for preventing or reducing the risk of cancer recurrence in a subject after surgical resection of cancer, comprising: a) Predicting the risk of postoperative recurrence of the cancer in subjects using the methods described herein; b) Administer adjuvant chemotherapy and / or radiotherapy to subjects predicted to have a risk of postoperative recurrence of the aforementioned cancer. This can help prevent or reduce the risk of cancer recurrence. Attached Figure Description

[0016] Figure 1 A: Adhesion of Caco-2-Luc cells seeded in preoperative patient serum as a measure of luciferase secretion, stratified according to recurrence status. p-value 0.4419. B: Adhesion of Caco-2-Luc cells seeded in postoperative patient serum, arranged by recurrence status. p-value 0.3238.

[0017] Figure 2 Differences in adhesion (adhesion score) of Caco-2-Luc cells cultured in preoperative and postoperative serum and classified according to recurrence status. Positive bars (gray) show higher adhesion of Caco-2-Luc cells in postoperative serum compared to preoperative serum. p-value 0.0293 RLU: Relative Light Unit.

[0018] Figure 3 A: ROC curve of adhesion score as a continuous variable. AUC 0.7302 (p < 0.0001). B: ROC curve of adhesion score as a categorical variable. AUC 0.7450 (p < 0.0001).

[0019] Figure 4 : A plot of adhesion scores for relapse-free survival estimation based on UICC staging. I: UICC stage 1, II: UICC stage 2, III: UICC stage 3, Blue: Increased perioperative adhesion (adhesion score +1 and +2), Red: Decreased perioperative adhesion (adhesion score -1 and -2), Gray: Neutral perioperative adhesion (adhesion score 0).

[0020] Detailed description

[0021] This disclosure describes how to assess the risk of postoperative cancer recurrence by collecting blood samples before (referred to herein as “first sample”) and after (referred to herein as “second sample”) a subject’s cancer is surgically removed, and culturing cell populations on a solid substrate in the presence of each sample.

[0022] The risk of postoperative cancer recurrence increases when cell populations grown in the presence of a second blood sample exhibit higher adhesion levels to solid substrates than cell populations grown in the presence of a first blood sample.

[0023] Therefore, in a first aspect of the invention, this disclosure relates to a method for predicting the risk of postoperative cancer recurrence in a subject, comprising: a. Culture a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; b. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; c. Culture cell populations on a solid substrate in the presence of a second blood sample obtained from the subject after surgical removal of cancer; d. Quantify the adhesion level of cell populations to solid substrates in the presence of a second blood sample; e. Compare the adhesion levels of cell populations cultured in the presence of the first and second blood samples to solid substrates; The cell population in question is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level of the cell population in the second blood sample is higher than that in the first blood sample.

[0024] It will be apparent to those skilled in the art that, in order to compare the adhesion levels of a first cell population cultured in the presence of a first blood sample with the adhesion levels of a second cell population cultured in the presence of a second blood sample, it is advantageous for the first and second cell populations to be similar. Therefore, in some embodiments, the first and second cell populations originate from the same cell population. Therefore, in some embodiments, the first and second cell populations originate from the same cell line. In some embodiments, the first and second populations originate from the same cancer. In some embodiments, the first and second populations originate from the same cellular tissue. In some embodiments, the first and second populations originate from the same subject. In some embodiments, the first and second populations originate from the same species of mammal.

[0025] In some implementations, cancer recurrence increases when the adhesion level of the cell population to the solid substrate in the second blood sample is at least 15%, such as 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 65%, higher than that in the first blood sample.

[0026] In one implementation, cancer recurrence increases when the adhesion level of the cell population to the solid substrate in the second blood sample is at least 40%, such as 45%, such as 50%, such as 55%, such as 65%, higher than the adhesion level of the cell population to the solid substrate in the first blood sample.

[0027] In one implementation, cancer recurrence is increased when the adhesion level of the cell population to the solid substrate in the second blood sample is at least 60%, for example, 65%, higher than the adhesion level of the cell population to the solid substrate in the first blood sample.

[0028] In some implementations, cancer recurrence increases when the adhesion level of the cell population to the solid substrate in the second blood sample is at least 65% higher than that in the first blood sample.

[0029] If the adhesion level of a cell population grown in the presence of a second blood sample is unchanged or decreased compared to the adhesion level of a cell population grown in the presence of a first blood sample, then the data in this paper indicate that the risk of cancer recurrence is not increased. Therefore, the methods disclosed herein can also be used to identify subjects who do not have a risk of cancer recurrence after surgery. Based on this knowledge, medical practitioners can avoid administering adjuvant chemotherapy and / or radiotherapy to subjects without a risk of recurrence, thereby avoiding unnecessary side effects for patients and reducing hospital costs.

[0030] Quantifying the adhesion level of solid substrates

[0031] As described in this article, the risk of postoperative cancer recurrence increases when cell populations grown in the presence of a second blood sample exhibit higher adhesion levels to solid substrates than cell populations grown in the presence of a first blood sample.

[0032] Those skilled in the art will understand that the quantification of the adhesion level of a solid substrate can be performed directly, for example, by directly counting the number of cells that adhere to the solid substrate; or indirectly, for example, by quantifying reporter genes and / or physiological parameters associated with the number of cells that adhere to the solid substrate.

[0033] Therefore, any method that allows a technician to estimate the number of cells that have adhered to a solid substrate under given conditions is suitable for the purposes of this method.

[0034] Therefore, in some embodiments, the cell population expresses a reporter gene. Therefore, in some embodiments, the cell population is a genetically engineered cell population.

[0035] In some embodiments, the reporter gene is selected from: luciferase reporter gene; fluorescent reporter gene; alkaline phosphatase reporter gene. In some embodiments, the reporter gene is luciferase.

[0036] In some implementations, the quantification of the adhesion level of a cell population to a solid substrate is performed by measuring the activity of reporter genes in the cells that are adhered to the substrate.

[0037] In some implementations, the quantification of the adhesion level of a cell population to a solid substrate is performed by counting the number of cells in the cell population that have adhered to the substrate; and / or by estimating the number of adhering cells by measuring the amount of protein and / or DNA and / or RNA and / or lipids in the adhering cells; and / or by measuring the metabolic activity of the adhering cells.

[0038] In some implementations, the quantification of the adhesion level of a cell population to a solid substrate is performed by counting the number of cells in the cell population that have adhered to the substrate and / or by estimating the number of adhering cells by measuring the amount of protein and / or DNA in the adhering cells.

[0039] In some implementations, the quantification of the adhesion level of a cell population to a solid substrate is performed by counting the number of cells in the cell population that have adhered to the substrate.

[0040] In some implementations, the quantification of the adhesion level of a cell population to a solid substrate is performed by estimating the number of adhering cells by measuring the amount of protein and / or DNA in the adhering cells.

[0041] In some implementations, the quantification of the adhesion level of a cell population to a solid substrate is performed by estimating the number of adherent cells by measuring the amount of protein in the adherent cells.

[0042] In some implementations, the quantification of the adhesion level of the cell population to the solid substrate is performed by estimating the number of adhesion cells by measuring the amount of DNA in the adhesion cells.

[0043] In some implementations, the quantification of the adhesion level of the cell population to the solid substrate is performed by estimating the number of adhesion cells by measuring the metabolic activity of the adhesion cells.

[0044] The quantification of adhesion levels of solid substrates can be performed manually or automatically, for example by a fully automated microscope / platform configured to perform adhesion level quantification in an automatic manner.

[0045] Therefore, in some embodiments, the quantification of the adhesion level of the solid substrate is performed manually. In other embodiments, the quantification of the adhesion level of the solid substrate is performed automatically.

[0046] Those skilled in the art will understand that, in order to perform the comparisons described herein, the number of cells cultured with the first blood sample and the number of cells cultured with the second sample are substantially the same.

[0047] In some implementations, the cell population is provided in the form of a suspension.

[0048] Suitable cell population

[0049] As described in this article, the risk of postoperative cancer recurrence increases when cell populations grown in the presence of a second blood sample exhibit higher adhesion levels to solid substrates than cell populations grown in the presence of a first blood sample.

[0050] Any mammalian cell population capable of adhering to a solid substrate during culture is suitable for the purpose of quantifying the adhesion level of a cell population to a solid substrate, as described herein. In some embodiments, the cell population is a human cell population. Extensively characterized cancer cell lines are particularly suitable because they are adapted to grow on artificial solid substrates. Therefore, in some embodiments, the cell population is a cancer cell population.

[0051] In some implementations, the cell population is selected from the following cell lines: Caco-2, HeLa, Jurkat, HEK293, MCF7, A549, U-2 OS, HepG2, PC-3, SH-SY5y, HCT-116, NIH / 3T3, SK-N-SH, RAW 264.7, THP-1, H1299, MDA-MB-231, COS-7, CHO, RPE-1, LS174T, SW680, HT29, MDA-MB-231, A549, H1299, HCT-116, SW480, PC-3, LNCaP, OVCAR-3, SK-OV-3, RL95-2, MIA PaCa-2, PANC-1, Hep3B, ACHN, 786-O, T24, RT4, KTC-1, B-CPAP, MKN-45, U-87 MG, U-251MG, A375, A431, U-2OS, HT-1080, A-204, OE33, KYSE-30, FaDu, CAL-27, GB-d1 , SW-1463, T24, TCam-2, NCI-H295R, SK-LMS-1, MSTO-211H, EL-4, SNU-16, BON-1 and H727.

[0052] In some implementations, the cell population is a genetically modified Caco-2 cell line.

[0053] Furthermore, using a cell population derived from the same type of cancer as the subject's cancer at risk of recurrence may be beneficial, as the cell line can exhibit a similar phenotype, thus better replicating the cancer's behavior. Therefore, in some implementations, the cell population is a population of cancer cells derived from the same type of cancer as the subject's cancer at risk of recurrence.

[0054] In some implementations, the cell population is obtained from the subject, for example, where the cell population originates from resected cancer. While isolating cells from a resected tumor can be laborious, it can provide greater insight into the risk of tumor recurrence in certain situations.

[0055] It will be apparent to those skilled in the art that a suitable cell population can be obtained from a subject at any time prior to performing the methods described herein.

[0056] As described herein, in some embodiments, the cell population is a genetically engineered cell population. Therefore, in some embodiments, the cell population has been genetically engineered to express the reporter gene as described herein.

[0057] cancer

[0058] As described in this article, the risk of postoperative cancer recurrence increases when cell populations grown in the presence of a second blood sample exhibit higher adhesion levels to solid substrates than cell populations grown in the presence of a first blood sample.

[0059] In some implementations, the subject has been diagnosed with stage II or higher cancer, such as stage III or higher cancer, such as stage IV cancer.

[0060] In some implementations, the subject has been diagnosed with stage III or higher cancer, such as stage IV cancer.

[0061] In some implementations, the subject has been diagnosed with stage II cancer. In some implementations, the subject has been diagnosed with stage III cancer. In some implementations, the subject has been diagnosed with stage IV cancer.

[0062] In some implementations, cancer is a solid tumor.

[0063] In some implementation schemes, the subject has been diagnosed with a cancer selected from the following: colorectal cancer; breast cancer; lung cancer; prostate cancer; ovarian cancer; uterine cancer; cervical cancer; pancreatic cancer; liver cancer; kidney cancer; bladder cancer; thyroid cancer; stomach cancer; brain cancer; skin cancer, such as melanoma, basal cell carcinoma, squamous cell carcinoma; bone cancer; sarcoma; esophageal cancer; head and neck cancer; gallbladder cancer; anal cancer; penile cancer; testicular cancer; adrenal cancer; soft tissue sarcoma; mesothelioma; thymic carcinoma; small bowel cancer; neuroendocrine carcinoma; and pituitary cancer.

[0064] In some implementations, the risk of postoperative cancer recurrence is defined as the risk of postoperative cancer recurrence within 5 years, such as 4 years, 3 years, 2 years, or 1 year after surgical removal of the cancer.

[0065] blood sample

[0066] As described in this article, the risk of postoperative cancer recurrence increases when cell populations grown in the presence of a second blood sample exhibit higher adhesion levels to solid substrates than cell populations grown in the presence of a first blood sample.

[0067] For the purposes of the methods described herein, it may be advantageous to remove red blood cells and other cellular components from blood samples.

[0068] Therefore, in some implementations, the first blood sample is a serum sample.

[0069] Therefore, in some implementations, the second blood sample is a serum sample.

[0070] In some implementations, the first and / or second blood sample may be a plasma sample.

[0071] In some implementations, the first and second blood samples are serum samples.

[0072] In some embodiments, the cell population is cultured in a culture medium containing at least 1%, for example at least 2%, for example at least 3%, for example at least 4%, for example at least 5%, for example at least 6%, for example at least 7%, for example at least 8%, for example at least 9%, for example at least 10% of a first or second blood sample.

[0073] In one embodiment, the cell population is cultured in a culture medium containing at least 5%, for example at least 6%, for example at least 7%, for example at least 8%, for example at least 9%, for example at least 10% of a first or second blood sample.

[0074] In one embodiment, the cell population is cultured in a culture medium containing approximately 10% of a first or second blood sample, respectively.

[0075] In some embodiments, the first blood sample is obtained from the subject within 7 days prior to surgical removal of the cancer, for example, within 6 days prior to surgical removal of the cancer, for example, within 5 days prior to surgical removal of the cancer, for example, within 4 days prior to surgical removal of the cancer, for example, within 3 days prior to surgical removal of the cancer, for example, within 2 days prior to surgical removal of the cancer, for example, within 1 day prior to surgical removal of the cancer. In some embodiments, the first blood sample is obtained from the subject within 3 days prior to surgical removal of the cancer. In some embodiments, the first blood sample is obtained from the subject within 1 day prior to surgical removal of the cancer.

[0076] In some implementations, the first blood sample is obtained from the subject at the time of diagnosis, which in some cases can be more than 7 days before surgical removal of the cancer.

[0077] In some embodiments, the second blood sample is obtained from the subject within 7 days after surgical removal of the cancer, for example, within 6 days after surgical removal of the cancer, for example, within 5 days after surgical removal of the cancer, for example, within 4 days after surgical removal of the cancer, for example, within 3 days after surgical removal of the cancer, for example, within 2 days after surgical removal of the cancer, for example, within 1 day after surgical removal of the cancer, preferably within 1 to 3 days after surgical removal of the cancer.

[0078] In some implementations, the cell population is cultured for a period of time in the presence of a first or second blood sample, during which time the cell population is allowed to adhere to the substrate.

[0079] In some implementations, the method includes removing unattached cells before quantifying the adhesion level of the cell population to the solid substrate.

[0080] In some implementations, the cell population is cultured in the presence of a first or second blood sample for at least 30 minutes, such as 60 minutes, 90 minutes, 120 minutes, 180 minutes, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or 24 hours.

[0081] In some implementations, cell populations cultured in the presence of a first blood sample and cell populations cultured in the presence of a second blood sample are cultured for the same amount of time before quantifying the adhesion level of each cell population to the solid substrate.

[0082] Adhesion score

[0083] To facilitate decision-making, a scoring system can be used to represent the difference in the level of adhesion to solid substrates between cell populations grown in the presence of the first blood sample and cell populations grown in the presence of the second blood sample.

[0084] An example of such a scoring system is the adhesion score described in Example 1 of this document.

[0085] application

[0086] As described in this article, the risk of postoperative cancer recurrence increases when cell populations grown in the presence of a second blood sample exhibit higher adhesion levels to solid substrates than cell populations grown in the presence of a first blood sample.

[0087] Therefore, by comparing the adhesion level of a cell population grown in the presence of a second blood sample to a solid substrate with the adhesion level of a cell population grown in the presence of a first blood sample, the survival of a subject after surgical resection of cancer can be predicted.

[0088] Therefore, in one aspect, this disclosure relates to a method for predicting the survival of a subject after surgical removal of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in a subject using the methods described herein.

[0089] Therefore, in one aspect, this disclosure relates to a method for predicting the survival of a subject after surgical removal of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in a subject using a method that includes the following steps: i. Culture cell populations on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; ii. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; iii. Culture cell populations on solid substrates in the presence of a second blood sample obtained from the subject after surgical removal of cancer; iv. Quantifying the adhesion level of cell populations to solid substrates in the presence of a second blood sample; v. Compare the adhesion levels of cell populations cultured in the presence of first and second blood samples to solid substrates; The cell population in question is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level of the cell population in the second blood sample is higher than that in the first blood sample.

[0090] In some implementation schemes, the subjects' survival is defined as cancer-free survival.

[0091] In some implementations, the survival of the subject is 5 years after surgical removal of the cancer, such as 4 years, 3 years, 2 years, or 1 year.

[0092] Therefore, in one aspect, this disclosure relates to a method for assessing the prognosis of a subject after surgical resection of cancer, comprising: a) Predict the risk of postoperative cancer recurrence in subjects as described in this article.

[0093] Therefore, in one aspect, this disclosure relates to a method for assessing the prognosis of a subject after surgical resection of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in a subject using a method that includes the following steps: i. Culture cell populations on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; ii. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; iii. Culture cell populations on solid substrates in the presence of a second blood sample obtained from the subject after surgical removal of cancer; iv. Quantifying the adhesion level of cell populations to solid substrates in the presence of a second blood sample; v. Compare the adhesion levels of cell populations cultured in the presence of first and second blood samples to solid substrates; The cell population in question is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level of the cell population in the second blood sample is higher than that in the first blood sample.

[0094] If the adhesion level of a cell population grown in the presence of a second blood sample to a solid substrate is higher than that of a cell population grown in the presence of a first blood sample, then adjuvant chemotherapy and / or radiotherapy following surgical resection of the subject's cancer may be beneficial in preventing and / or delaying cancer recurrence.

[0095] Therefore, in one aspect, this disclosure relates to a method for determining whether a subject should receive adjuvant chemotherapy and / or radiotherapy after surgical resection of cancer, comprising: a) Predict the risk of postoperative cancer recurrence in subjects as described in this article. b) If the subject has a risk of recurrence of the aforementioned cancer after surgery, the conclusion is that the subject should receive adjuvant chemotherapy and / or radiotherapy.

[0096] Therefore, in one aspect, this disclosure relates to a method for determining whether a subject should receive adjuvant chemotherapy and / or radiotherapy after surgical resection of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in a subject using a method that includes the following steps: i. Culture cell populations on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; ii. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; iii. Culture cell populations on solid substrates in the presence of a second blood sample obtained from the subject after surgical removal of cancer; iv. Quantifying the adhesion level of cell populations to solid substrates in the presence of a second blood sample; v. Compare the adhesion levels of cell populations cultured in the presence of first and second blood samples to solid substrates; The cell population mentioned is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level of the cell population in the second blood sample is higher than that in the first blood sample. b) If the subject has a risk of recurrence of the aforementioned cancer after surgery, the conclusion is that the subject should receive adjuvant chemotherapy and / or radiotherapy.

[0097] In one aspect, this disclosure relates to a method of administering adjuvant chemotherapy and / or radiotherapy to a subject after surgical resection of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in the subject based on the methods described herein; b) Administer adjuvant chemotherapy and / or radiation therapy to subjects who are predicted to have a risk of postoperative recurrence of the said cancer.

[0098] Therefore, in one aspect, this disclosure relates to a method of administering adjuvant chemotherapy and / or radiotherapy to a subject after surgical resection of cancer, comprising: a) Predicting the risk of postoperative recurrence of the cancer in the subject using a method that includes the following steps: i. Culture cell populations on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; ii. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; iii. Culture cell populations on solid substrates in the presence of a second blood sample obtained from the subject after surgical removal of cancer; iv. Quantifying the adhesion level of cell populations to solid substrates in the presence of a second blood sample; v. Compare the adhesion levels of cell populations cultured in the presence of first and second blood samples to solid substrates; The cell population mentioned is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level of the cell population in the second blood sample is higher than that in the first blood sample. b) Administer adjuvant chemotherapy and / or radiotherapy to subjects who are predicted to have a risk of postoperative recurrence of the said cancer.

[0099] Therefore, in one aspect, this disclosure relates to a method of administering adjuvant chemotherapy and / or radiotherapy to a subject after surgical resection of cancer, wherein the subject has been predicted to have a risk of postoperative cancer recurrence, and the prediction of the subject's risk of postoperative cancer recurrence has been performed by the method described herein.

[0100] Therefore, in one aspect, this disclosure relates to a method of administering adjuvant chemotherapy and / or radiotherapy to a subject after surgical resection of cancer, wherein the subject has been predicted to have a risk of postoperative cancer recurrence, wherein predicting the subject's risk of postoperative cancer recurrence includes: i. Culture a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; ii. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; iii. Culture cell populations on solid substrates in the presence of a second blood sample obtained from the subject after surgical removal of cancer; iv. Quantifying the adhesion level of cell populations to solid substrates in the presence of a second blood sample; v. Compare the adhesion levels of cell populations cultured in the presence of first and second blood samples to solid substrates; The cell population in question is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level of the cell population in the second blood sample is higher than that in the first blood sample.

[0101] In one aspect, this disclosure relates to a method for preventing or reducing the risk of cancer recurrence in a subject after surgical removal of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in the subject as described herein; b) Administer adjuvant chemotherapy and / or radiation therapy to subjects predicted to have a risk of postoperative recurrence of the aforementioned cancer. This can prevent or reduce the risk of cancer recurrence.

[0102] Therefore, in one aspect, this disclosure relates to a method for preventing or reducing the risk of cancer recurrence in a subject after surgical removal of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in the subject by means of a method including the following steps: i. Culture cell populations on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; ii. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; iii. Culture cell populations on solid substrates in the presence of a second blood sample obtained from the subject after surgical removal of cancer; iv. Quantifying the adhesion level of cell populations to solid substrates in the presence of a second blood sample; v. Compare the adhesion levels of cell populations cultured in the presence of first and second blood samples to solid substrates; The cell population mentioned is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level of the cell population in the second blood sample is higher than that in the first blood sample. b) Administer adjuvant chemotherapy and / or radiation therapy to subjects predicted to have a risk of postoperative recurrence of the aforementioned cancer. This can help prevent or reduce the risk of cancer recurrence.

[0103] Those skilled in the art will understand that any adjuvant chemotherapy suitable for treating cancer and / or said cancer recurrence can be used.

[0104] Therefore, in some implementations, adjuvant chemotherapy administered to a subject is known to prevent and / or delay cancer recurrence.

[0105] In some implementations, adjuvant chemotherapy includes administration of a biologic therapy and / or chemotherapeutic agents selected from the following: abiraterone, adriamycin, bleomycin, capecitabine, carboplatin, cisplatin, cyclophosphamide, dacarbazine, docetaxel, enzalutamide, etoposide, fluorouracil, and folinic acid. The following drugs are mentioned: acid, gemcitabine, irinotecan, ipilimumab, methotrexate, oxaliplatin, paclitaxel, pembrolizumab, prednisone, procarbazine, vinblastine, vincristine, vinorelbine, anti-EGFR therapy, and anti-VEGF therapy, or any combination thereof.

[0106] The radiotherapy described herein can be any type of radiotherapy known to those skilled in the art and considered suitable for preventing and / or delaying cancer recurrence after surgery.

[0107] project

[0108] 1. A method for predicting the risk of cancer recurrence after surgery in a subject, comprising: a. Culture a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; b. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; c. Culture cell populations on a solid substrate in the presence of a second blood sample obtained from the subject after surgical removal of cancer; d. Quantify the adhesion level of cell populations to solid substrates in the presence of a second blood sample; e. Compare the adhesion levels of cell populations cultured in the presence of the first and second blood samples to solid substrates; The cell population in question is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level of the cell population in the second blood sample is higher than that in the first blood sample.

[0109] 2. The method according to the preceding paragraph, wherein cancer recurrence increases when the adhesion level of the cell population in the second blood sample is at least 15%, such as 20%, such as 25%, such as 30%, such as 35%, such as 40%, such as 45%, such as 50%, such as 55%, such as 65%, higher than the adhesion level of the cell population in the first blood sample.

[0110] 3. The method according to any of the preceding claims, wherein the subject has been diagnosed with stage II or higher cancer, such as stage III or higher cancer, such as stage IV cancer.

[0111] 4. The method according to any of the preceding methods, wherein the subject has been diagnosed with stage II cancer.

[0112] 5. The method according to any of the preceding claims, wherein the subject has been diagnosed with stage III cancer.

[0113] 6. The method according to any of the preceding claims, wherein the subject has been diagnosed with stage IV cancer.

[0114] 7. The method according to any of the preceding claims, wherein the cancer is a solid tumor.

[0115] 8. The method according to any of the preceding claims, wherein the subject has been diagnosed with a cancer selected from: colorectal cancer; breast cancer; lung cancer; prostate cancer; ovarian cancer; uterine cancer; cervical cancer; pancreatic cancer; liver cancer; kidney cancer; bladder cancer; thyroid cancer; stomach cancer; brain cancer; skin cancer, such as melanoma, such as basal cell carcinoma, such as squamous cell carcinoma; bone cancer; sarcoma; esophageal cancer; head and neck cancer; gallbladder cancer; anal cancer; penile cancer; testicular cancer; adrenal cancer; soft tissue sarcoma; mesothelioma; thymic carcinoma; small bowel cancer; neuroendocrine carcinoma; and pituitary cancer.

[0116] 9. The method according to any of the preceding claims, wherein the risk of postoperative recurrence of cancer is the risk of postoperative recurrence of cancer within 5 years, for example 4 years, for example 3 years, for example 2 years, for example 1 year after surgical resection of cancer.

[0117] 10. The method according to any of the preceding claims, wherein the cell population is a cancer cell population.

[0118] 11. The method according to any of the preceding claims, wherein the cell population is a population of cancer cells derived from the same type of cancer as the cancer in which the subject has a risk of recurrence.

[0119] 12. The method according to any of the preceding claims, wherein the cell population is a genetically engineered cell population.

[0120] 13. The method according to any one of the preceding claims, wherein the cell population is selected from the following cell lines: Caco-2, HeLa, Jurkat, HEK293, MCF7, A549, U-2 OS, HepG2, PC-3, SH-SY5y, HCT-116, NIH / 3T3, SK-N-SH, RAW 264.7, THP-1, H1299, MDA-MB-231, COS-7, CHO, RPE-1, LS174T, SW680, HT29, MDA-MB-231, A549, H1299, HCT-116, SW480, PC-3, LNCaP, OVCAR-3, SK-OV-3, RL95-2, MIA PaCa-2, PANC-1, Hep3B, ACHN, 786-O, T24, RT4, KTC-1, B-CPAP, MKN-45, U-87 MG, U-251MG, A375, A431, U-2 OS, HT-1080, A-204, OE33, KYSE-30, FaDu, CAL-27, GB-d1, SW-1463, T24, TCam-2, NCI-H295R, SK-LMS-1, MSTO-211H, EL-4, SNU-16, BON-1 and H727.

[0121] 14. The method according to any of the preceding claims, wherein the cell population is a genetically modified Caco-2 cell line.

[0122] 15. The method according to any of the preceding claims, wherein the cell population has been obtained from the subject, for example, wherein the cell population is derived from resected cancer.

[0123] 16. The method according to any of the preceding claims, wherein the cell population expresses a reporter gene.

[0124] 17. The method according to any of the preceding claims, wherein the reporter gene is selected from: luciferase reporter gene; fluorescent reporter gene; alkaline phosphatase reporter gene.

[0125] 18. The method according to any of the preceding claims, wherein the reporter gene is a luciferase.

[0126] 19. The method according to any of the preceding claims, wherein the quantification of cell population adhesion levels is performed by measuring the activity of reporter genes in cells that adhere to the substrate.

[0127] 20. The method according to any of the preceding claims, wherein the quantification of the cell population adhesion level is performed by counting the number of cells in the cell population that have adhered to the substrate; and / or by estimating the number of adhering cells by measuring the amount of protein and / or DNA and / or RNA and / or lipids in the adhering cells; and / or by measuring the metabolic activity of the adhering cells.

[0128] 21. The method according to any of the preceding claims, wherein the quantification of the cell population adhesion level is performed by counting the number of cells in the cell population that have adhered to the substrate and / or by estimating the number of adhering cells by measuring the amount of protein and / or DNA in the adhering cells.

[0129] 22. The method according to any of the preceding claims, wherein the first blood sample is a serum sample.

[0130] 23. The method according to any of the preceding claims, wherein the second blood sample is a serum sample.

[0131] 24. The method according to any of the preceding claims, wherein the first and second blood samples are serum samples.

[0132] 25. The method according to any one of the preceding claims, wherein the cell population is cultured in a culture medium containing at least 1%, for example at least 2%, for example at least 3%, for example at least 4%, for example at least 5%, for example at least 6%, for example at least 7%, for example at least 8%, for example at least 9%, for example at least 10% of a first or second blood sample.

[0133] 26. The method according to any of the preceding claims, wherein the first blood sample is obtained from the subject within 7 days prior to surgical removal of the cancer, for example within 6 days prior to surgical removal of the cancer, for example within 5 days prior to surgical removal of the cancer, for example within 4 days prior to surgical removal of the cancer, for example within 3 days prior to surgical removal of the cancer, for example within 2 days prior to surgical removal of the cancer, for example within 1 day prior to surgical removal of the cancer.

[0134] 27. The method according to any of the preceding claims, wherein the second blood sample is obtained from the subject within 7 days after surgical resection of the cancer, for example within 6 days after surgical resection of the cancer, for example within 5 days after surgical resection of the cancer, for example within 4 days after surgical resection of the cancer, for example within 3 days after surgical resection of the cancer, for example within 2 days after surgical resection of the cancer, for example within 1 day after surgical resection of the cancer, preferably within 1 to 3 days after surgical resection of the cancer.

[0135] 28. The method according to any of the preceding claims, wherein the cell population is cultured for a period of time in the presence of a first or second blood sample, during which time the cell population is allowed to adhere to the substrate.

[0136] 29. The method according to any of the preceding claims, wherein the method includes removing non-adhered cells before quantifying the cell population adhesion level.

[0137] 30. The method according to any of the preceding claims, wherein the cell population is cultured in the presence of a first or second blood sample for at least 30 minutes, such as 60 minutes, 90 minutes, 120 minutes, 180 minutes, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or 24 hours.

[0138] 31. The method according to any of the preceding claims, wherein the cell population cultured in the presence of the first blood sample and the cell population cultured in the presence of the second blood sample are cultured for the same amount of time before quantifying the adhesion level of each population.

[0139] 32. A method for predicting the survival of a subject after surgical removal of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in a subject by means of the method described in any of the preceding methods.

[0140] 33. Methods for assessing the prognosis of subjects after surgical resection of cancer, including: a) Predict the risk of postoperative recurrence of the cancer in a subject by means of the method described in any of the preceding methods.

[0141] 34. A method for determining whether a subject should receive adjuvant chemotherapy and / or radiation therapy after surgical removal of cancer, comprising: a) Predicting the risk of postoperative recurrence of the cancer in a subject according to the method described in any of the preceding claims; and b) If the subject has a risk of recurrence of the aforementioned cancer after surgery, the conclusion is that the subject should receive adjuvant chemotherapy and / or radiotherapy.

[0142] 35. A method for administering adjuvant chemotherapy and / or radiation therapy to a subject after surgical removal of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in the subject according to the method described in any of the preceding methods; b) Administer adjuvant chemotherapy and / or radiation therapy to subjects who are predicted to have a risk of postoperative recurrence of the said cancer.

[0143] 36. A method for administering adjuvant chemotherapy and / or radiotherapy to a subject after surgical resection of cancer, wherein the subject has been predicted to have a risk of postoperative cancer recurrence, wherein the prediction of the subject's risk of postoperative cancer recurrence has been performed by the method according to any one of the preceding claims.

[0144] 37. A method for preventing or reducing the risk of cancer recurrence in a subject after surgical removal of cancer, comprising: a) Predict the risk of postoperative recurrence of the cancer in the subject based on any of the foregoing criteria; b) Administer adjuvant chemotherapy and / or radiation therapy to subjects predicted to have a risk of postoperative recurrence of the aforementioned cancer. This can help prevent or reduce the risk of cancer recurrence.

[0145] 38. The method according to any of the preceding claims, wherein adjuvant chemotherapy comprises administering a biological therapy and / or chemotherapeutic agent selected from: abiraterone, doxorubicin, bleomycin, capecitabine, carboplatin, cisplatin, cyclophosphamide, dacarbazine, docetaxel, enzalutamide, etoposide, fluorouracil, leucovorin, gemcitabine, irinotecan, ipilimumab, methotrexate, oxaliplatin, paclitaxel, pembrolizumab, prednisone, procarbazine, vinblastine, vincristine, vinorelbine, anti-EGFR therapy, and anti-VEGF therapy, or any combination thereof.

[0146] Example

[0147] Example 1: Materials and Methods

[0148] Participants and Settings

[0149] This disclosure is based on clinical information and perioperative blood samples prospectively collected in the REBECCA Biomarker Study in Denmark (“Biomarkers in patients with colorectal cancer – Can they provide new information on diagnosis, treatment efficacy, side effects, and prognosis?”). Between July 15, 2014, and March 31, 2019, patients with histologically confirmed UICC stage I-III colorectal cancer scheduled for their first radical surgery at Copenhagen University Hospitals (Herlev and Gentofte) were eligible for enrollment. The study cohort included 467 adult patients who underwent their first radical surgery for colorectal adenocarcinoma and had available preoperative and postoperative blood samples. Patients who underwent local resection, emergency surgery, or palliative surgery were excluded. Patients who received neoadjuvant therapy were also excluded. All patients obtained informed consent prior to enrollment. Patients were monitored until August 2022.

[0150] The surgical procedure was performed in accordance with Herlev Hospital's institutional policy and, when technically feasible, as a minimally invasive procedure. The Danish Colorectal Cancer Group provides standardized guidelines regarding preoperative staging, adjuvant therapy, and follow-up. The Danish National Guidelines for Elective Colorectal Cancer Surgery recommend a preoperative staging strategy comprised of colonoscopy, histopathology, computed tomography (CT) scans, and preoperative evaluation by a multidisciplinary institutional team.

[0151] This study was conducted in accordance with the Declaration of Helsinki. The REBECCA study protocol (VEK j.nr. H-2-2013-078) was approved by the Regional Ethics Committee of the Capital Region and the Danish Data Protection Authority in Copenhagen, Denmark (j. No. HEH-2014-044, I-suite No. 02771 and PACTIUS P-2019-614).

[0152] Data collection and processing

[0153] Following standard procedures, blood samples were collected the day before surgery and approximately 24 hours after surgery. Samples were collected in serum separation tubing via standardized venipuncture procedure. Samples were allowed to coagulate at room temperature for 30 minutes, then centrifuged at 2300 g for 10 min at 4°C over 3 hours. Serum was transferred to Eppendorf tubes and stored at -80°C.

[0154] Serum samples and clinical data from all patients were retrieved and linked using unique patient IDs. Viewing each patient's electronic medical record provided information on relapse and mortality status.

[0155] Statistical analysis

[0156] The primary outcome is postoperative recurrence of colorectal cancer, defined as any postoperative tumor mass confirmed by clinical, histological, or radiological evidence at any time after the initial surgical procedure.

[0157] The statistical differences in cell adhesion in preoperative or postoperative serum samples, stratified according to recurrence, were analyzed using the Mann-Whitney test or unpaired t-test. The statistical difference in adhesion scores between relapsed patients and controls was validated using the unpaired t-test.

[0158] The relationship between adhesion scores and postoperative recurrence was assessed using multivariate logistic regression and Cox proportional hazards regression analysis. All models were adjusted for UICC staging, ASA score, and adjuvant chemotherapy based on clinical inference. Multivariate logistic regression analysis was tested using the Hosmer-Lemeshow test, the adjusted Akaike Information Criterion (AIC), the area under the ROC curve (AUC), and multicollinearity.

[0159] Scaled Schoenfeld residuals were plotted over time to assess the Cox proportional hazards hypothesis. The model was further evaluated using the Wald test. Patients lost to follow-up or still alive and relapse-free at the end of the current follow-up period were censored.

[0160] Adhesion scores were analyzed as both continuous and categorical variables. The cutoff values ​​for adhesion scores as categorical variables are shown in the table below.

[0161]

[0162] The p-cutoff value for statistical significance was set at < 0.05. p-value < 0.01 p-value < 0.001 Statistical analysis and visualization were performed using GraphPad Prism 9 and Microsoft Excel.

[0163] Example 2: Demographic data of patients undergoing colorectal cancer surgery

[0164] Overall, blood samples and clinical data from 467 patients were retrieved from the REBECCA study biobank. After exclusion, 434 patients were enrolled in this study. 33 patients were excluded due to disease severity, surgical approach, unclear tumor localization, and neoadjuvant therapy. Among the enrolled patients, 55.8% were male. The median age was 70 years (IQR 64–75), and the median BMI was 25 (IQR 23–28). Patients were diagnosed with UICC stage I–III, ASA score range I–III, and 32% received adjuvant chemotherapy. Patient demographics are shown in the table below. The median follow-up period was 4.8 years. During the study, 55 patients (12.6%) experienced colorectal cancer recurrence, and 93 (21.4%) died. A total of 56.4% of patients experiencing recurrence died during the follow-up period. Within 3 months post-surgery, 4 patients (0.9%) experienced recurrence events, and 6 (1.4%) died. Overall, 434 patients had available preoperative serum samples, while 225 patients had samples collected both before and after the surgical procedure. Of the 225 patients with both samples, 29 experienced recurrence.

[0165]

[0166] Example 3: Increased adhesion levels to solid substrates predict relapse risk

[0167] Caco 2-Luc (a genetically modified polyclonal colon cancer cell line that secretes luciferase into the culture medium) was selected to quantify adhesion by measuring luciferase activity in the culture medium. The Caco-2-Luc cell line was cultured in medium supplemented with preoperative serum from each of 434 patients. Results showed no significant difference in preoperative adhesion based on the occurrence of recurrence or the absence of stratification (p=0.4419). Figure 1 A). No significant differences were found when Caco-2-Luc cell lines were cultured in postoperative serum and stratified according to recurrence status (p=0.3238). Figure 1 B). For example Figure 2 As shown, when investigating the differences in adhesion (adhesion score) of Caco-2-Luc cells inoculated in serum before and after surgery, a significant difference was observed between patients who experienced relapse and those who did not (p=0.0293).

[0168] Example 4: Adhesion Scoring

[0169] To establish a model of the relationship between postoperative recurrence of colorectal cancer and adhesion score as a predictor, multivariate logistic regression was performed, adjusted for ASA score, UICC stage, and adjuvant chemotherapy. Results showed that adhesion score was associated with colorectal cancer recurrence (p=0.0155) (Table 3). The results also showed that when stratified by adhesion level using categorical predictors, patients with the highest perioperative increase in adhesion (adhesion score +2) had a significantly higher probability of recurrence (p=0.0175).

[0170]

[0171] As shown in the table above and Figure 3 As shown, both models exhibited significant AUC (p < 0.0001), which, combined with the additional statistical tests described in the Methods section, supports the quality of these models as predictors of postoperative recurrence.

[0172] In the adhesion score +2 category, 31.25% of patients experienced recurrence. Among patients with an adhesion score +2, the distribution of UICC stage was 3 (19%) UICC 1, 6 (37.5%) UICC 2, and 7 (43.5%) UICC 3, with ASA score rankings from I (50%) to II (50%). Furthermore, all patients with an adhesion score +2 who experienced recurrence had perioperative increases in adhesion exceeding 100,000 RLU, placing them in the top 50% of this category with the highest perioperative increase in adhesion.

[0173] To assess the relative risk of postoperative recurrence, Cox proportional hazards regression was used. The recurrence time model using adhesion score as a predictor is shown in the table below. The model was adjusted for ASA score, UICC stage, and adjuvant chemotherapy. Results showed that adhesion score was statistically significantly associated with recurrence risk (p = 0.0126). When stratified by adhesion level using categorical predictors, our results showed that patients with the highest perioperative adhesion increase (adhesion score +2) had a significantly increased risk of recurrence (HR = 7, 95% CI 1.6–37.9, p = 0.0130).

[0174]

[0175] Estimated recurrence-free survival maps were generated for visualization and graphical assessment of the impact of UICC staging based on perioperative decreased, neutral, and increased adhesion stratification on recurrence. Figure 4An increased adhesion score was associated with an increased risk of recurrence in UICC stages 2 and 3. No risk was associated with UICC stage 1 regardless of the adhesion score. For a neutral adhesion score, a slightly increased risk of recurrence was observed according to UICC staging. A decreased adhesion score showed a slightly increased risk compared to the corresponding UICC stage with a neutral adhesion score. As shown, increased perioperative adhesion was associated with an increased risk of recurrence after radical colorectal cancer surgery, independent of UICC staging.

[0176] Example 5: Correlating the adhesion score with the relative increase in the level of adhesion to solid substrates.

[0177] To correlate different categories of adhesion scores with differences in adhesion levels between cells grown in the presence of the first or second blood sample, the percentage change in mean RLU values ​​from preoperative to postoperative was calculated.

[0178]

Claims

1. A method for predicting the risk of cancer recurrence after surgery in a subject, including a. Culture a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical removal of cancer; b. Quantify the adhesion level of cell populations to solid substrates in the presence of a first blood sample; c. Culture cell populations on a solid substrate in the presence of a second blood sample obtained from the subject after surgical removal of cancer; d. Quantify the adhesion level of cell populations to solid substrates in the presence of a second blood sample; e. Compare the adhesion levels of cell populations cultured in the presence of the first and second blood samples to solid substrates; The cell population mentioned is a mammalian cell population, and the risk of postoperative cancer recurrence increases when the adhesion level in the second blood sample is higher than that in the first blood sample.

2. The method according to any one of the preceding claims, wherein cancer recurrence increases when the adhesion level of the cell population in the second blood sample is at least 15%, such as 20%, such as 25%, such as 30%, such as 35%, such as 40%, such as 45%, such as 50%, such as 55%, such as 65% higher than the adhesion level of the cell population in the first blood sample.

3. The method according to any of the preceding claims, wherein the subject has been diagnosed with stage II or higher cancer, such as stage III or higher cancer, such as stage IV cancer.

4. The method according to any one of the preceding claims, wherein the subject has been diagnosed with cancer selected from: colorectal cancer; breast cancer; lung cancer; prostate cancer; ovarian cancer; uterine cancer; cervical cancer; pancreatic cancer; liver cancer; kidney cancer; bladder cancer; thyroid cancer; stomach cancer; brain cancer; skin cancer, such as melanoma, such as basal cell carcinoma, such as squamous cell carcinoma; bone cancer; sarcoma; esophageal cancer; head and neck cancer; gallbladder cancer; anal cancer; penile cancer; testicular cancer; adrenal cancer; soft tissue sarcoma; mesothelioma; thymic carcinoma; small bowel cancer; neuroendocrine carcinoma; and pituitary cancer.

5. The method according to any one of the preceding claims, wherein the cell population is a population of cancer cells derived from the same type of cancer as the cancer at risk of recurrence in the subject, optionally wherein the cell population has been obtained from the subject, for example wherein the cell population is derived from resected cancer.

6. The method according to any one of the preceding claims, wherein the cell population is selected from the cell lines: Caco-2, HeLa, Jurkat, HEK293, MCF7, A549, U-2 OS, HepG2, PC-3, SH-SY5y, HCT-116, NIH / 3T3, SK-N-SH, RAW 264.7, THP-1, H1299, MDA-MB-231, COS-7, CHO, RPE-1, LS174T, SW680, HT29, MDA-MB-231, A549, H1299, HCT-116, SW480, PC-3, LNCaP, OVCAR-3, SK-OV-3, RL95-2, MIA PaCa-2, PANC-1, Hep3B, ACHN, 786-O, T24, RT4, KTC-1, B-CPAP, MKN-45, U-87 MG, U-251 MG, A375, A431, U-2 OS, HT-1080, A-204, OE33, KYSE-30, FaDu, CAL-27, GB-d1, SW-1463, T24, TCam-2, NCI-H295R, SK-LMS-1, MSTO-211H, EL-4, SNU-16, BON-1 and H727.

7. The method according to any one of the preceding claims, wherein the cell population expresses a reporter gene, such as a reporter gene selected from: luciferase reporter gene; fluorescent reporter gene; alkaline phosphatase reporter gene, and wherein the quantification of the cell population adhesion level is performed by measuring the reporter gene activity in cells adhered to the substrate.

8. The method according to any one of the preceding claims, wherein the quantification of the cell population adhesion level is performed by counting the number of cells in the cell population that have adhered to the substrate; and / or by estimating the number of adhering cells by measuring the amount of protein and / or DNA and / or RNA and / or lipids in the adhering cells; and / or by measuring the metabolic activity of the adhering cells.

9. The method according to any one of the preceding claims, wherein the first and / or second blood sample is a serum sample.

10. The method according to any one of the preceding claims, wherein the cell population is cultured in a culture medium containing at least 1%, for example at least 2%, for example at least 3%, for example at least 4%, for example at least 5%, for example at least 6%, for example at least 7%, for example at least 8%, for example at least 9%, for example at least 10% of a first or second blood sample.

11. The method according to any of the preceding claims, wherein the first blood sample was obtained from the subject within 7 days prior to surgical removal of the cancer, for example within 6 days prior to surgical removal of the cancer, for example within 5 days prior to surgical removal of the cancer, for example within 4 days prior to surgical removal of the cancer, for example within 3 days prior to surgical removal of the cancer, for example within 2 days prior to surgical removal of the cancer, for example within 1 day prior to surgical removal of the cancer.

12. The method according to any one of the preceding claims, wherein the second blood sample was obtained from the subject within 7 days after surgical resection of the cancer, for example within 6 days after surgical resection of the cancer, for example within 5 days after surgical resection of the cancer, for example within 4 days after surgical resection of the cancer, for example within 3 days after surgical resection of the cancer, for example within 2 days after surgical resection of the cancer, for example within 1 day after surgical resection of the cancer, preferably within 1 to 3 days after surgical resection of the cancer.

13. The method according to any of the preceding claims, wherein the cell population is cultured in the presence of a first or second blood sample for at least 30 minutes, for example about 60 minutes, for example about 90 minutes, for example about 120 minutes, for example about 180 minutes, for example about 3 hours, for example about 4 hours, for example about 5 hours, for example about 6 hours, for example about 7 hours, for example about 8 hours, for example about 9 hours, for example about 10 hours, for example about 11 hours, for example about 12 hours, for example about 24 hours.

14. A method for determining whether a subject should receive adjuvant chemotherapy and / or radiation therapy after surgical removal of cancer, comprising: a) To predict the risk of postoperative recurrence of the cancer in the subject according to any of the preceding claims; as well as b) If the subject has a risk of recurrence of the aforementioned cancer after surgery, the conclusion is that the subject should receive adjuvant chemotherapy and / or radiotherapy.

15. The method according to any one of the preceding claims, wherein adjuvant chemotherapy comprises administration of a biological therapy and / or chemotherapeutic agent selected from: abiraterone, doxorubicin, bleomycin, capecitabine, carboplatin, cisplatin, cyclophosphamide, dacarbazine, docetaxel, enzalutamide, etoposide, fluorouracil, leucovorin, gemcitabine, irinotecan, ipilimumab, methotrexate, oxaliplatin, paclitaxel, pembrolizumab, prednisone, procarbazine, vinblastine, vincristine, vinorelbine, anti-EGFR therapy, and anti-VEGF therapy, or any combination thereof.

Images