Method for predicting the risk of post-operative recurrence of a cancer

Abstract

The present invention relates to a method for predicting the risk of post-operative recurrence of a cancer, and applications thereof in: methods for predicting survival of said subject; methods of prognosis of said subject; methods to administer adjuvant chemotherapy to said subject; and methods to prevent or decrease the risk of recurrence of the cancer in said subject. The methods involve quantifying the level of adhesion of a cell population cultured in the presence of a blood sample from the subject prior to and after surgical resection of the cancer.

Description

[0001] Method for predicting the risk of post-operative recurrence of a cancer

[0002] Technical field

[0003] The present invention relates to a method for predicting the risk of post-operative recurrence of a cancer, and applications thereof.

[0004] Background

[0005] Presently, the primary curative treatment strategy for most cancers still revolves around surgically removing the tumor. In cases of advanced disease, this approach is often accompanied by adjuvant therapy.

[0006] In recent years, notable improvements in long-term patient outcomes have occurred, thanks to advancements in screening programs, oncological treatments, standardized cancer care workflows, and surgical techniques. However, even after successful surgery, the rates of postoperative recurrence remain high.

[0007] For example, approximately 30% of individuals who have undergone surgical therapy for colorectal cancer experience within five years of their initial treatment a recurrence of the disease, which is associated with an elevated risk of mortality and heightened morbidity.

[0008] Thus, there is a need in the art to be able to identify patients at risk of recurrence of cancer after surgical therapy in order to provide the best possible care for such patients.

[0009] Summary

[0010] The present disclosure relates to a newly identified method for predicting the risk of post-operative recurrence of a cancer in a subject.

[0011] Emerging preclinical evidence suggests that perioperative events cause disease recurrence by creating favourable conditions for preexisting micro-metastatic disease or cancer cells dissemination during surgery. Herein, the inventors disclose that the risk of post-operative recurrence of a cancer can be assessed by collecting a blood sample from the subject before (herein referred as “1stsample”) and after (herein referred as 2ndsample) surgical resection of the cancer, and by culturing a cell population in the presence of these samples.

[0012] The risk of post-operative recurrence of the cancer is increased when the level of adhesion to a solid substrate of the cell population grown in the presence of the second blood sample is higher than the level of adhesion to a solid substrate of the cell population grown in the presence the first blood sample.

[0013] Thus, in a first aspect, the present disclosure concerns a method for predicting the risk of post-operative recurrence of a cancer in a subject comprising a. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; b. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; c. culturing a cell population on a solid substrate in the presence of a second blood sample obtained from the subject after surgical resection of the cancer; d. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; e. comparing the level of adhesion to the solid substrate of the cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of postoperative recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is higher than the level of adhesion of the cell population in the first blood sample.

[0014] In another aspect, the present disclosure concerns a method for predicting survival of a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via the method described herein.

[0015] In another aspect, the present disclosure concerns a method of prognosis of a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via the method described herein.

[0016] In another aspect, the present disclosure concerns a method for determining if a subject should be treated with adjuvant chemotherapy and / or radiotherapy following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via the method described herein; and b) concluding that the subject should be treated with adjuvant chemotherapy and / or radiotherapy if the subject is at risk of post-operative recurrence of said cancer.

[0017] In another aspect, the present disclosure concerns a method of administering adjuvant chemotherapy and / or radiotherapy to a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via the method described herein; b) administering adjuvant chemotherapy and / or radiotherapy to the subject predicted as being at risk of post-operative recurrence of said cancer.

[0018] In another aspect, the present disclosure concerns a method of administering adjuvant chemotherapy and / or radiotherapy to a subject following surgical resection of a cancer, wherein the subject has been predicted of being at risk of post-operative recurrence of a cancer, wherein predicting the risk of post-operative recurrence of said cancer in the subject has been performed by the method described herein.

[0019] In another aspect, the present disclosure concerns a method of preventing or decreasing the risk of recurrence of a cancer in a subject following surgical resection of the cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via the method described herein; b) administering adjuvant chemotherapy and / or radiotherapy to the subject predicted as being at risk of post-operative recurrence of said cancer, thereby preventing or decreasing risk of recurrence of the cancer. Description of Drawings

[0020] Figure 1 : A: Adhesion as secreted luciferase measurements from the Caco-2-Luc cells seeded in preoperative patient serum is shown and 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

[0021] Figure 2: The difference in adhesion (AdhesionScore) for Caco-2-Luc cells cultured in pre- and postoperative serum and classified according to recurrence status. The positive bar (grey) shows higher adhesion of Caco-2-Luc cells in postoperative serum compared to preoperative serum. P-value 0.0293*. RLU: Relative Light Units.

[0022] Figure 3: A: ROC curve with AdhesionScore as a continuous variable. AUC 0.7302 (p- value <0.0001). B: ROC curve with AdhesionScore as a categorical variable. AUC 0.7450 (p-value <0.0001).

[0023] Figure 4: Estimated recurrence-free survival plot of AdhesionScore by UICC stage. I: UICC stage 1, II: UICC stage 2, III: UICC stage 3, Blue: Increased perioperative adhesion (AdhesionScore +1 and +2), Red: Decreased perioperative adhesion (AdhesionScore -1 and -2), Grey: Neutral perioperative adhesion (AdhesionScore 0).

[0024] Detailed description

[0025] The present disclosure describes that the risk of post-operative recurrence of a cancer can be assessed by collecting a blood sample from the subject before (herein referred as “1stsample”) and after (herein referred as 2ndsample) surgical resection of the cancer, and by culturing a cell population on a solid substrate in the presence of the respective samples.

[0026] The risk of post-operative recurrence of the cancer is increased when the level of adhesion to a solid substrate of the cell population grown in the presence of the second blood sample is higher than the level of adhesion to a solid substrate of the cell population grown in the presence the first blood sample. Thus, in a first aspect of the invention, the present disclosure concerns a method for predicting the risk of post-operative recurrence of a cancer in a subject comprising a. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; b. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; c. culturing a cell population on a solid substrate in the presence of a second blood sample obtained from the subject after surgical resection of the cancer; d. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; e. comparing the level of adhesion to the solid substrate of the cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of postoperative recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is higher than the level of adhesion of the cell population in the first blood sample.

[0027] It will be evident to the skilled person that, in order to compare the adhesion level of a first population of cells cultured in the presence of a first blood sample with the adhesion level of a second population of cells cultured in the presence of a second blood sample, it is advantageous that said first and second population of cells are similar. Thus, in some embodiments, said first and second population of cells are derived from the same cell population. Thus, in some embodiments, said first and second population of cells are derived from the same cell line. In some embodiments, said first and second population are derived from the same cancer. In some embodiments, said first and second population are derived from the same cell tissue. In some embodiments, said first and second population are derived from the same subject. In some embodiments, said first and second population are derived from mammals of the same species.

[0028] In some embodiments, the recurrence of the cancer is increased when the level of adhesion to the solid substrate of the cell population in the second blood sample is at least 15% higher, 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 level of adhesion to the solid substrate of the cell population in the first blood sample.

[0029] In one embodiment, the recurrence of the cancer is increased when the level of adhesion to the solid substrate of the cell population in the second blood sample is at least 40%, such as 45%, such as 50%, such as 55%, such as 65% higher than the level of adhesion to the solid substrate of the cell population in the first blood sample.

[0030] In one embodiment, the recurrence of the cancer is increased when the level of adhesion to the solid substrate of the cell population in the second blood sample is at least 60%, such as 65% higher than the level of adhesion to the solid substrate of the cell population in the first blood sample.

[0031] In some embodiments, the recurrence of the cancer is increased when the level of adhesion to the solid substrate of the cell population in the second blood sample is at least 65% higher than the level of adhesion to the solid substrate of the cell population in the first blood sample.

[0032] If there is no change in level of adhesion to the solid substrate of the cell population grown in the presence of the second blood sample, or a decrease in the level of adhesion to the solid substrate, compared to the level of adhesion to the solid substrate of the cell population grown in the presence the first blood sample, the data herein indicates that there is no increased risk of recurrence of the cancer. Accordingly, the methods of the present disclosure can also be used to identify subject which are not at risk of recurrence of cancer following surgery. With this knowledge, medical practitioners can avoid treating subjects who are not at risk of recurrence with adjuvant chemotherapy and / or radiotherapy, thereby avoiding unnecessary side effects for the patients and decreasing hospital costs.

[0033] Quantification of the level of adhesion to a solid substrate

[0034] As described herein, the risk of post-operative recurrence of the cancer is increased when the level of adhesion to a solid substrate of a cell population grown in the presence of the second blood sample is higher than the level of adhesion to a solid substrate of a cell population grown in the presence the first blood sample. The skilled person will understand that the quantification of the level of adhesion to a solid substrate can be performed directly, e.g., by directly counting the number of cells which adhere to the solid substrate; or indirectly, e.g., by quantifying a reporter gene and / or a physiological parameter that correlates with the number of cells which adhere to the solid substrate.

[0035] Thus, any method that allows a skilled person to estimate the number of cells which have adhered to a solid substrate in a given condition is suitable for the purpose of the present method.

[0036] Thus, in some embodiments, the cell population expresses a reporter gene. Thus, in some embodiments, the cell population is a genetically engineered cell population.

[0037] In some embodiments, the reporter gene is selected from the group consisting of: a luciferase reporter; a fluorescence reporter; an alkaline phosphatase reporter. In some embodiments, the reporter gene is luciferase.

[0038] In some embodiments, the quantification of the level of adhesion to the solid substrate of the cell population is performed by measuring the reporter gene activity in cells adhered to the substrate.

[0039] In some embodiments, the quantification of the level of adhesion to the solid substrate of the cell population is performed by counting the number of cells of the cell population that have adhered to the substrate; and / or by estimating the number of adhered cells by measuring the amount of protein and / or DNA and / or RNA and / or lipid in the adhered cells; and / or by measuring the metabolic activity of the adhered cells.

[0040] In some embodiments, the quantification of the level of adhesion to the solid substrate of the cell population is performed by counting the number of cells of the cell population that have adhered to the substrate and / or by estimating the number of adhered cells by measuring the amount of protein and / or DNA in the adhered cells.

[0041] In some embodiments, the quantification of the level of adhesion to the solid substrate of the cell population is performed by counting the number of cells of the cell population that have adhered to the substrate. In some embodiments, the quantification of the level of adhesion to the solid substrate of the cell population is performed by estimating the number of adhered cells by measuring the amount of protein and / or DNA in the adhered cells.

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

[0043] In some embodiments, the quantification of the level of adhesion to the solid substrate of the cell population is performed by estimating the number of adhered cells by measuring the amount of DNA in the adhered cells.

[0044] In some embodiments, the quantification of the level of adhesion to the solid substrate of the cell population is performed by estimating the number of adhered cells by measuring the metabolic activity of the adhered cells.

[0045] The quantification of the level of adhesion to a solid substrate can be performed manually, or automatically, such as for example by a high-throughput microscope / platform configured to perform the quantification of the level of adhesion in an automated way.

[0046] Thus, in some embodiments, the quantification of the level of adhesion to the solid substrate is performed manually. In some embodiments, the quantification of the level of adhesion to the solid substrate is performed in an automated way.

[0047] The skilled person will understand that the in order to perform the comparison described herein the amount of cells cultured with the first blood sample and the amount of cells cultured with the second sample is about the same.

[0048] In some embodiments, the cell population is provided in a suspension.

[0049] Suitable cell populations

[0050] As described herein, the risk of post-operative recurrence of the cancer is increased when the level of adhesion to a solid substrate of a cell population grown in the presence of the second blood sample is higher than the level of adhesion to a solid substrate of a cell population grown in the presence the first blood sample.

[0051] Any mammalian cell population which, when cultured, is capable of adhering to a solid substrate is suitable for the purpose of quantifying the level of adhesion to a solid substrate of a cell population as described herein. In some embodiments, the cell population is a human cell population. Cancer cell lines which have been extensively characterized are particularly suitable as they have adapted to grow on artificial solid substrates. Thus, in some embodiments, the cell population is a cancer cell population.

[0052] In some embodiments, the cell population is cell line selected from the group consisting of: 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, 786-0, 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-211 H, EL-4, SNU-16, BON-1 and H727.

[0053] In some embodiments, the cell population is a genetically modified Caco-2 cell line.

[0054] Furthermore, it could be beneficial to use a cell population obtained from a cancer of the same type as the subject is at risk of recurrence, as this cell line may exhibit a similar phenotype and, consequently, better replicate the behaviour of the cancer. Thus, in some embodiments, the cell population is a cancer cell population derived from a cancer of the same type of cancer from which the subject is at risk of recurrence.

[0055] In some embodiments, the cell population has been obtained from the subject, such as wherein the cell population has been derived from the resected cancer. While isolating cells from the resected tumor can be laborious, in some cases, it can provide with a higher insight into the risk of recurrence of the tumor.

[0056] It will be evident to the skilled person that a suitable cell population can be obtained from a subject at any time prior to performing the method described herein. As described herein, in some embodiments, the cell population is a genetically engineered cell population. Thus, in some embodiments, the cell population has been genetically engineered to express a reported gene, as described herein.

[0057] The cancer

[0058] As described herein, the risk of post-operative recurrence of the cancer is increased when the level of adhesion to a solid substrate of a cell population grown in the presence of the second blood sample is higher than the level of adhesion to a solid substrate of a cell population grown in the presence the first blood sample.

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

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

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

[0062] In some embodiments, the cancer is a solid tumour.

[0063] In some embodiments, the subject has been diagnosed with a cancer selected from the group consisting of: 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; sarcomas; esophageal cancer; head and neck cancer; gallbladder cancer; anal cancer; penile cancer; testicular cancer; adrenal gland cancer; soft tissue sarcoma; mesothelioma; thymus cancer; small intestine cancer; neuroendocrine cancer; and pituitary cancer.

[0064] In some embodiments, the risk of post-operative recurrence of the cancer is the risk of post-operative recurrence of the cancer within a 5 year period, such as within a 4 year period, such as within a 3 year period, such as within a 2 year period, such as within 1 year following the surgical resection of the cancer.

[0065] Blood samples

[0066] As described herein, the risk of post-operative recurrence of the cancer is increased when the level of adhesion to a solid substrate of a cell population grown in the presence of the second blood sample is higher than the level of adhesion to a solid substrate of a cell population grown in the presence the first blood sample.

[0067] For the purpose of the method described herein, it may be advantageous to remove red blood cells and other cellular elements from the blood sample.

[0068] Thus, in some embodiments, the first blood sample is a serum sample.

[0069] Thus, in some embodiments, the second blood sample is a serum sample.

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

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

[0072] In some embodiments, the cell population is cultured in a medium which comprises at least 1%, such as at least 2%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as at least 9%, such as at least 10% of the first or second blood sample, respectively.

[0073] In one embodiment, the cell population is cultured in a medium which comprises at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as at least 9%, such as at least 10% of the first or second blood sample, respectively.

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

[0075] In some embodiments, the first blood sample has been obtained from the subject within 7 days before the surgical resection of the cancer, such as within 6 days before the surgical resection of the cancer, such as within 5 days before the surgical resection of the cancer, such as within 4 days before the surgical resection of the cancer, such within 3 days before the surgical resection of the cancer, such as within 2 days before the surgical resection of the cancer, such as within 1 day before the surgical resection of the cancer. In some embodiments, the first blood sample has been obtained from the subject within 3 days before the surgical resection of the cancer. In some embodiments, the first blood sample has been obtained from the subject within 1 day before the surgical resection of the cancer.

[0076] In some embodiments, the first blood sample has been obtained from the subject at the time of diagnosis of the subject, which may, in some instances, be more than 7 days before the surgical resection of the cancer.

[0077] In some embodiments, the second blood sample has been obtained from the subject within 7 days after the surgical resection of the cancer, such as within 6 days after the surgical resection of the cancer, such as within 5 days after the surgical resection of the cancer, such as within 4 days after the surgical resection of the cancer, such within 3 days after the surgical resection of the cancer, such as within 2 days after the surgical resection of the cancer, such as within 1 day after the surgical resection of the cancer, preferably within 1 to 3 days after the surgical resection of the cancer. In some embodiments, the second blood sample has been obtained from the subject within 1 to 3 days after the surgical resection of the cancer.

[0078] In some embodiments, the cell population is cultured in the presence of the first or the second blood sample for a time period that allows for adherence of the cell population to the substrate.

[0079] In some embodiments, the method comprises removing non-adhered cells prior to quantifying the level of adhesion to the solid substrate of the cell population.

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

[0081] AdhesionScore

[0082] In order to facilitate decision making, a score system might be employed to represent the difference in the level of adhesion to the solid substrate between a cell population grown in the presence of the first blood sample and one grown in the presence of the second blood sample.

[0083] An example of such score system is the AdhesionScore described herein in Example 1.

[0084] Applications

[0085] As described herein, the risk of post-operative recurrence of the cancer is increased when the level of adhesion to a solid substrate of a cell population grown in the presence of the second blood sample is higher than the level of adhesion to a solid substrate of a cell population grown in the presence the first blood sample.

[0086] Thus, by comparing the level of adhesion to a solid substrate of a cell population grown in the presence of the second blood sample to the level of adhesion to a solid substrate of a cell population grown in the presence of the first blood sample, it is possible to predict the survival of the subject following surgical resection of a cancer.

[0087] Thus, in an aspect the present disclosure relates to a method for predicting survival of a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via the method as described herein.

[0088] Thus, in an aspect the present disclosure relates to a method for predicting survival of a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via a method comprising the following steps: i. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; ii. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; iii. culturing a cell population on a solid substrate in the presence of a second blood sample obtained from the subject after surgical resection of the cancer; iv. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; v. comparing the level of adhesion to the solid substrate of the cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of post-operative recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is higher than the level of adhesion of the cell population in the first blood sample.

[0089] In some embodiments, the survival of the subject is cancer-free survival.

[0090] In some embodiments, the survival of the subject is 5 years following the surgical resection of the cancer, such as 4 years, such as 3 years, such as 2 years, such as 1 year following the surgical resection of the cancer.

[0091] Thus, in an aspect the present disclosure relates to a method of prognosis of a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject as described herein.

[0092] Thus, in an aspect the present disclosure relates to a method of prognosis of a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via a method comprising the following steps: i. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; ii. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; iii. culturing a cell population on a solid substrate in the presence of a second blood sample obtained from the subject after surgical resection of the cancer; iv. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; v. comparing the level of adhesion to the solid substrate of the cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of post-operative recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is higher than the level of adhesion of the cell population in the first blood sample.

[0093] If the level of adhesion to a solid substrate of a cell population grown in the presence of the second blood sample is higher than the level of adhesion to a solid substrate of the cell population grown in the presence the first blood sample, it might advantageous to administer adjuvant chemotherapy and / or radiotherapy following surgical resection of the cancer from the subject in order to prevent and / or delay the cancer recurrence.

[0094] Thus, in an aspect the present disclosure relates to a method for determining if a subject should be treated with adjuvant chemotherapy and / or radiotherapy following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject as described herein, b) concluding that the subject should be treated with adjuvant chemotherapy and / or radiotherapy if the subject is at risk of post-operative recurrence of said cancer. Thus, in an aspect the present disclosure relates to a method for determining if a subject should be treated with adjuvant chemotherapy and / or radiotherapy following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via a method comprising the following steps: i. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; ii. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; iii. culturing a cell population on a solid substrate in the presence of a second blood obtained from the subject after surgical resection of the cancer; iv. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; v. comparing the level of adhesion to the solid substrate of the cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of post-operative recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is higher than the level of adhesion of the cell population in the first blood sample; b) concluding that the subject should be treated with adjuvant chemotherapy and / or radiotherapy if the subject is at risk of post-operative recurrence of said cancer.

[0095] In an aspect the present disclosure relates to a method of administering adjuvant chemotherapy and / or radiotherapy to a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject according to the method described herein; b) administering adjuvant chemotherapy and / or radiotherapy to the subject predicted as being at risk of post-operative recurrence of said cancer. Thus, in an aspect the present disclosure relates to a method of administering adjuvant chemotherapy and / or radiotherapy to a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via a method comprising the following steps: i. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; ii. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; iii. culturing a cell population on a solid substrate in the presence of a second blood sample obtained from the subject after surgical resection of the cancer; iv. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; v. comparing the level of adhesion to the solid substrate of the cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of post-operative recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is higher than the level of adhesion of the cell population in the first blood sample; b) administering adjuvant chemotherapy and / or radiotherapy to the subject predicted as being at risk of post-operative recurrence of said cancer.

[0096] Thus, in an aspect the present disclosure relates to a method of administering adjuvant chemotherapy and / or radiotherapy to a subject following surgical resection of a cancer, wherein the subject has been predicted of being at risk of post-operative recurrence of a cancer, wherein predicting the risk of post-operative recurrence of said cancer in the subject has been performed by the method as described herein.

[0097] Thus, in an aspect the present disclosure relates to a method of administering adjuvant chemotherapy and / or radiotherapy to a subject following surgical resection of a cancer, wherein the subject has been predicted of being at risk of post-operative recurrence of a cancer, wherein predicting the risk of post-operative recurrence of said cancer in the subject comprises: i. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; ii. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; iii. culturing a cell population on a solid substrate in the presence of a second blood sample obtained from the subject after surgical resection of the cancer; iv. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; v. comparing the level of adhesion to the solid substrate of the cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of post-operative recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is higher than the level of adhesion of the cell population in the first blood sample.

[0098] In an aspect the present disclosure relates to a method of preventing or decreasing the risk of recurrence of a cancer in a subject following surgical resection of the cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject as described herein; b) administering adjuvant chemotherapy and / or radiotherapy to the subject predicted as being at risk of post-operative recurrence of said cancer, thereby preventing or decreasing risk of recurrence of the cancer.

[0099] Thus, in an aspect the present disclosure relates to a method of preventing or decreasing the risk of recurrence of a cancer in a subject following surgical resection of the cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via a method comprising the following steps: i. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; ii. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; iii. culturing a cell population on a solid substrate in the presence of a second blood sample obtained from the subject after surgical resection of the cancer; iv. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; v. comparing the level of adhesion to the solid substrate of cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of post-operative recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is higher than the level of adhesion of the cell population in the first blood sample; b) administering adjuvant chemotherapy and / or radiotherapy to the subject predicted as being at risk of post-operative recurrence of said cancer, thereby preventing or decreasing risk of recurrence of the cancer.

[0100] The skilled person will understand that any adjuvant chemotherapy suitable for treating the cancer and / or the recurrence of said cancer can be employed.

[0101] Thus, in some embodiments, the adjuvant chemotherapy administered to the subject is known to prevent and / or delay recurrence of the cancer.

[0102] In some embodiments, the adjuvant chemotherapy comprises administering a biologic therapy and / or a chemotherapeutic agent selected from the group consisting of: abiraterone, adriamycin, bleomycin, capecitabine, carboplatin, cisplatin, cyclophosphamide, dacarbazine, docetaxel, enzalutamide, etoposide, fluorouracil, folinic acid, gemcitabine, irinotecan, ipilimumab, methotrexate, oxaliplatin, paclitaxel, pembrolizumab, prednisone, procarbazine, vinblastine, vincristine, vinorelbine, anti- EGFR therapy and anti-VEGF therapy or any combination thereof. The radiotherapy herein may be any type of radiotherapy which is known to the person of skill and considered suitable for prevention and / or delaying recurrence of a cancer following surgery.

[0103] Items

[0104] 1. A method for predicting the risk of post-operative recurrence of a cancer in a subject comprising a. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; b. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; c. culturing a cell population on a solid substrate in the presence of a second blood sample obtained from the subject after surgical resection of the cancer; d. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; e. comparing the level of adhesion to the solid substrate of the cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of postoperative recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is higher than the level of adhesion of the cell population in the first blood sample.

[0105] 2. The method according to the preceding item, where the recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is at least 15% higher, 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 level of adhesion of the cell population in the first blood sample.

[0106] 3. The method according to any one of the preceding items, wherein the subject has been diagnosed with a cancer at stage II or higher, such as a cancer at stage III or higher, such as a cancer at stage IV. 4. The method according to any one of the preceding items, wherein the subject has been diagnosed with a cancer at stage II.

[0107] 5. The method according to any one of the preceding items, wherein the subject has been diagnosed with a cancer at stage III.

[0108] 6. The method according to any one of the preceding items, wherein the subject has been diagnosed with a cancer at stage IV.

[0109] 7. The method according to any one of the preceding items, wherein the cancer is a solid tumour.

[0110] 8. The method according to any one of the preceding items, wherein the subject has been diagnosed with a cancer selected from the group consisting of: 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; sarcomas; esophageal cancer; head and neck cancer; gallbladder cancer; anal cancer; penile cancer; testicular cancer; adrenal gland cancer; soft tissue sarcoma; mesothelioma; thymus cancer; small intestine cancer; neuroendocrine cancer; and pituitary cancer.

[0111] 9. The method according to any one of the preceding items, wherein the risk of postoperative recurrence of the cancer is the risk of post-operative recurrence of the cancer within a 5 year period, such as within a 4 year period, such as within a 3 year period, such as within a 2 year period, such as within 1 year following the surgical resection of the cancer.

[0112] 10. The method according to any one of the preceding items, wherein the cell population is a cancer cell population. 11. The method according to any one of the preceding items, wherein the cell population is a cancer cell population derived from a cancer of the same type of cancer from which the subject is at risk of recurrence.

[0113] 12. The method according to any one of the preceding items, wherein the cell population is a genetically engineered cell population.

[0114] 13. The method according to any one of the preceding items, wherein the cell population is cell line selected from the group consisting of: 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, 786-0, 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-211 H, EL-4, SNU-16, BON-1 and H727.

[0115] 14. The method according to any one of the preceding items, wherein the cell population is a genetically modified Caco-2 cell line.

[0116] 15. The method according to any one of the preceding items, wherein the cell population has been obtained from the subject, such as wherein the cell population has been derived from the resected cancer.

[0117] 16. The method according to any one of the preceding items, wherein the cell population expresses a reporter gene.

[0118] 17. The method according to any one of the preceding items, wherein the reporter gene is selected from the group consisting of: a luciferase reporter; a fluorescence reporter; an alkaline phosphatase reporter.

[0119] 18. The method according to any one of the preceding items, wherein the reporter gene is luciferase. 19. The method according to any one of the preceding items, wherein the quantification of the level of adhesion of the cell population is performed by measuring the reporter gene activity in cells adhered to the substrate.

[0120] 20. The method according to any one of the preceding items, wherein the quantification of the level of adhesion of the cell population is performed by counting the number of cells of the cell population that have adhered to the substrate; and / or by estimating the number of adhered cells by measuring the amount of protein and / or DNA and / or RNA and / or lipid in the adhered cells; and / or by measuring the metabolic activity of the adhered cells.

[0121] 21. The method according to any one of the preceding items, wherein the quantification of the level of adhesion of the cell population is performed by counting the number of cells of the cell population that have adhered to the substrate and / or by estimating the number of adhered cells by measuring the amount of protein and / or DNA in the adhered cells.

[0122] 22. The method according to any one of the preceding items, wherein the first blood sample is a serum sample.

[0123] 23. The method according to any one of the preceding items, wherein the second blood sample is a serum sample.

[0124] 24. The method according to any one of the preceding items, wherein the first and the second blood sample are serum samples.

[0125] 25. The method according to any one of the preceding items, wherein the cell population is cultured in a medium which comprises at least 1%, such as at least 2%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as at least 9%, such as at least 10% of the first or second blood sample, respectively.

[0126] 26. The method according to any one of the preceding items, wherein the first blood sample has been obtained from the subject within 7 days before the surgical resection of the cancer, such as within 6 days before the surgical resection of the cancer, such as within 5 days before the surgical resection of the cancer, such as within 4 days before the surgical resection of the cancer, such within 3 days before the surgical resection of the cancer, such as within 2 days before the surgical resection of the cancer, such as within 1 day before the surgical resection of the cancer.

[0127] 27. The method according to any one of the preceding items, wherein the second blood sample has been obtained from the subject within 7 days after the surgical resection of the cancer, such as within 6 days after the surgical resection of the cancer, such as within 5 days after the surgical resection of the cancer, such as within 4 days after the surgical resection of the cancer, such within 3 days after the surgical resection of the cancer, such as within 2 days after the surgical resection of the cancer, such as within 1 day after the surgical resection of the cancer, preferably within 1 to 3 days after the surgical resection of the cancer.

[0128] 28. The method according to any one of the preceding items, wherein the cell population is cultured in the presence of the first or the second blood sample for a time period that allows for adherence of the cell population to the substrate.

[0129] 29. The method according to any one of the preceding items, wherein the method comprises removing non-adhered cells prior to quantifying the level of adhesion of the cell population.

[0130] 30. The method according to any one of the preceding items, wherein the cell population is cultured in the presence of first or the second blood sample for at least 30 minutes, such as 60 minutes, such as 90 minutes, such as 120 minutes, such as 180 minutes, such as 3 hours, such as 4 hours, such as 5 hours, such as 6 hours, such as 7 hours, such as 8 hours, such as 9 hours, such as 10 hours, such as 11 hours, such as 12 hours, such as 24 hours.

[0131] 31. The method according to any one of the preceding items, 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 level of adhesion of each population. 32. A method for predicting survival of a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via the method according to any one of the preceding items.

[0132] 33. A method of prognosis of a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject via the method according to any one of the preceding items.

[0133] 34. A method for determining if a subject should be treated with adjuvant chemotherapy and / or radiotherapy following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject according to any one of the preceding items; and b) concluding that the subject should be treated with adjuvant chemotherapy and / or radiotherapy if the subject is at risk of post-operative recurrence of said cancer.

[0134] 35. A method of administering adjuvant chemotherapy and / or radiotherapy to a subject following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject according to any one of the preceding items; b) administering adjuvant chemotherapy and / or radiotherapy to the subject predicted as being at risk of post-operative recurrence of said cancer.

[0135] 36. A method of administering adjuvant chemotherapy and / or radiotherapy to a subject following surgical resection of a cancer, wherein the subject has been predicted of being at risk of post-operative recurrence of a cancer, wherein predicting the risk of post-operative recurrence of said cancer in the subject has been performed by the method according to any one of the preceding items.

[0136] 37. A method of preventing or decreasing the risk of recurrence of a cancer in a subject following surgical resection of the cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject according to any one of the preceding items; b) administering adjuvant chemotherapy and / or radiotherapy to the subject predicted as being at risk of post-operative recurrence of said cancer, thereby preventing or decreasing risk of recurrence of the cancer. The method according to any one of the preceding items, wherein the adjuvant chemotherapy comprises administering a biologic therapy and / or a chemotherapeutic agent selected from the group consisting of: abiraterone, adriamycin, bleomycin, capecitabine, carboplatin, cisplatin, cyclophosphamide, dacarbazine, docetaxel, enzalutamide, etoposide, fluorouracil, folinic acid, gemcitabine, irinotecan, ipilimumab, methotrexate, oxaliplatin, paclitaxel, pembrolizumab, prednisone, procarbazine, vinblastine, vincristine, vinorelbine, anti- EGFR therapy and anti-VEGF therapy, or any combination thereof.

[0137] Examples

[0138] Example 1: Material and Methods

[0139] Participants and setting

[0140] The examples of the present disclosure are based on clinical information and perioperative blood samples collected prospectively in the Danish REBECCA biomarker study (“Biomarkers in patients with colorectal cancer - can they provide new information on the diagnosis, treatment efficacy, adverse effects and prognosis?”). Between July 15, 2014, and March 31, 2019, patients at Copenhagen University Hospital - Herlev and Gentofte who were scheduled for primary curative intended surgery with a histologically confirmed colon or rectum cancer UICC stage l-lll were eligible for inclusion. This study cohort included 467 adult patients who underwent primary curative surgery for adenocarcinoma of the colon or rectum, where pre- and postoperative blood samples were available. Patients undergoing local resection, acute surgery as well as palliative surgery were excluded. Patients receiving neoadjuvant treatment were also excluded. All patients provided informed consent prior to enrolment. Patients were monitored until August 2022.

[0141] The surgical procedure was performed according to the institutional policies at Herlev Hospital and executed as minimally invasive surgery when it was technically possible. Standardized guidelines were provided by the Danish Colorectal Cancer Group concerning preoperative staging, adjuvant therapy as well as follow-up. A preoperative staging strategy consisting of colonoscopy, histopathology, computed tomography (CT) scan as well as preoperative evaluation by the multidisciplinary institutional team was recommended by the Danish national guidelines for elective colorectal cancer surgery. 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 Region Ethical Committee of the Capital Region and the Danish Data Protection Agency in Copenhagen, Denmark (j. No. HEH-2014-044, l-suite No. 02771 and PACTIUS P- 2019-614).

[0142] Data collection and processing

[0143] Following regular procedures, blood samples were collected the day prior to surgery and approximately 24 h after surgery. Samples were collected by standardized venipuncture procedure in serum separation gel tubes. The samples were allowed to clot at room temperature for 30 minutes and then centrifuged within 3 hours at 2300 g at 4 °C for 10 min. The serum was transferred to Eppendorf tubes and stored at -80 °C. Serum samples, and clinical data, were retrieved for all patients and linked using a unique patient ID. Reviewing each patient's electronic medical record provided information on recurrence and death status.

[0144] Statistical analysis

[0145] The primary outcome was postoperative colorectal cancer recurrence and defined as any postoperative tumor mass confirmed clinically, histologically, or by radiographic evidence at any time after the initial surgical procedure.

[0146] The statistical differences in the adhesion of cells cultured in pre- or postoperative serum samples, stratified according to recurrence, were analysed using the Mann- Whitney test or unpaired t-test, respectively. The statistical difference in AdhesionScore for patients having recurrence compared to the controls was verified using the unpaired t-test.

[0147] By using multiple logistic regression and cox proportional hazard regression analysis, the relationship between AdhesionScore values and postoperative recurrence was evaluated. All models were adjusted for IIICC stage, ASA score, and adjuvant chemotherapy based on clinical reasoning. The multiple logistic regression analysis was tested by Hosmer-Lemeshow, corrected Akaike Information Criterion (AIC), Area under the ROC curve (AUC), and multicollinearity.

[0148] Scaled Schoenfeld residuals versus time were plotted to evaluate the Cox proportional hazard assumption. The models were further assessed by the Wald test. Patients who were lost to follow-up or were still alive and recurrence-free at the end of the current follow-up period were censored.

[0149] The AdhesionScore was analyzed as continuous and categorical variables. The cut-off for AdhesionScore as a categorical variable was set as displayed in the table below.

[0150] The p-value cutoff for statistical significance was set at < 0.05. (p-value<0.05*, p- value<0.01**, p-value<0.001***). GraphPad Prism 9 and Microsoft Excel were used for statistical analysis and visualization.

[0151] Example 2: Demographics for patients undergoing colorectal cancer surgery

[0152] Overall, blood samples and clinical data from 467 patients were retrieved from the REBECCA study biobank, and after the exclusion, 434 patients were enrolled in this study. Thirty-three patients were excluded due to extent of disease, surgical access, unknown tumor localization as well as neoadjuvant therapy. Of the patients enrolled, 55.8% were male. The median population was 70 years (IQR 64 - 75), with a median BMI of 25 (IQR 23 - 28). The patients were diagnosed within IIICC stage l-lll, with an ASA score ranging from l-lll, and 32% received adjuvant chemotherapy. The patient demographics are shown in the table below. The median period of follow-up was 4.8 years. During the course of the study, 55 (12.6%) patients had colorectal cancer recurrences, and 93 (21.4%) died. In total, 56.4% of the patients who experienced recurrence died within the follow-up period. In the 3 months following surgery, 4 patients (0.9%) had a recurrence event, and 6 (1.4%) died. Overall, 434 patients had preoperative serum samples available, whereas 225 patients had samples taken before and after the surgical procedure. Within the 225 patients having both samples, 29 patients experienced recurrence.

[0153] Example 3: An increase in level of adhesion to a solid substrate predicts the risk of recurrence Caco 2-Luc, a genetically modified polyclonal colon cancer cell line secreting luciferase to the growth media, was chosen to quantify adhesion by measuring luciferase activity in the media. The Caco-2-Luc cell line was cultured in media supplemented with preoperative serum from each of the 434 patients. The results showed no significant difference in preoperative adhesion stratified according to the occurrence or absence of a recurrence event (p=0.4419) (Figure 1A). When culturing the Caco-2-Luc cell line in postoperative serum, no significant difference was found when stratified according to recurrence status (p=0.3238) (Figure 1B). As shown in Figure 2, when investigating the differences in adhesion (AdhesionScore) of Caco-2-Luc cells seeded in pre- and postoperative serum, a significant difference between patients experiencing a recurrence and those who did not was observed (p=0.0293).

[0154] Example 4: AdhesionScore

[0155] For modeling the relationship between colorectal cancer postoperative recurrence and AdhesionScore as a predictor variable, a multiple logistic regression was performed and adjusted for ASA score, IIICC stage, and adjuvant chemotherapy. The results revealed that AdhesionScore was associated with colorectal cancer recurrence (p=0.0155) (Table 3). The results showed that patients with the highest level of increased perioperative adhesion (AdhesionScore +2) had a considerably higher probability of recurrence event when stratified by the level of adhesion using a categorical predictor variable (p=0.0175).

[0156] Variable Continuous Z-score P-value

[0157] AdhesionScore 2.419 0.0155 *

[0158] Area under the ROC Area 95 % Cl P-value curve

[0159] 0 7302 0.6 to 0.8 <0.0001

[0160] Variable Categorical Odds Ratio 95 % Cl P-value

[0161] AdhesionScore -2 1.6 0.4-8.5 0.5377

[0162] AdhesionScore -1 2.0 0.5-10.3 0.3735

[0163] AdhesionScore +1 1.8 0.4-9.9 0.4741

[0164] AdhesionScore +2 8.8 1.5-60.2 0.0173 *

[0165] Area under the ROC Area 95 % Cl P-value curve

[0166] 0.7450 0.6-0.8 <0.0001 ****

[0167] As shown in the table above and Figure 3, both models showed significant AUC (p- value <0.0001), supporting the quality of the models as predictors of postoperative recurrence combined with additional statistical tests as described in the methods section.

[0168] In the AdhesionScore +2 category, 31.25% of patients experienced recurrence. Among patients in AdhesionScore + 2, the IIICC stage was distributed accordingly 3 (19%) UICC 1, 6 (37.5%) UICC 2 and 7 (43.5%) UICC 3 and ASA scores ranking from I (50%) to II (50%). In addition, all patients with a recurrent event in AdhesionScore +2 had a perioperative increase in adhesion above 100.000 RLU, placing them in the top 50% within the category, with the highest increase in perioperative adhesion.

[0169] For evaluating the relative risk of postoperative recurrence, a Cox proportional hazard regression was used. Modeling time to recurrence with AdhesionScore as a predictor variable is shown in the table below. The model was adjusted for ASA score, UICC stage, and adjuvant chemotherapy. The results demonstrated that AdhesionScore was statistically significantly associated with the risk of recurrence (p= 0.0126). When stratifying by the level of adhesion using a categorical predictor variable, our results revealed a significantly increased risk of recurrence in patients having the highest increase (AdhesionScore +2) in perioperative adhesion (HR=7, 95% Cl 1.6-37.9, p=0.0130).

[0170] Variable Continuous Z-score P-value

[0171] AdhesionScore 2.5 0.0126 *

[0172] Variable Categorical Hazard Ratio 95 % Cl P-value

[0173] AdhesionScore -2 1.6 0.4-7.8 0.5028

[0174] AdhesionScore -1 1.9 0.5-9.1 0.3692

[0175] AdhesionScore +1 1.7 0.4-8.7 0.4783

[0176] AdhesionScore +2 7.0 1.6-37.8 0.0130 *

[0177] An estimated recurrence-free survival plot was generated for visualization and a graphical assessment of the impact of UICC stages on recurrence stratified according to decreased-, neutral- and increased perioperative adhesion (Figure 4). An increased AdhesionScore was associated with an increased risk of recurrence in UICC stage 2 and 3. No risks were associated with IIICC stage 1 , regardless of AdhesionScore. For neutral AdhesionScores, a minor increase in the risk of recurrence was seen according to the IIICC stage. Decreased AdhesionScore showed slightly elevated risk compared to the corresponding IIICC stage with neutral AdhesionScore. As displayed, increased perioperative adhesion was associated with an increased risk of recurrence after curatively intended colorectal cancer surgery independent of IIICC stage.

[0178] Example 5: Correlating Adhesion Score with the relative increase in the level of adhesion to a solid substrate.

[0179] In order to correlate the different categories of AdhesionScore with a difference in the level of adhesion between cells grown in the presence of the first or second blood sample, the percentage change from pre-operative to post-operative average RLU values was calculated.

Claims

Claims1. A method for predicting the risk of post-operative recurrence of a cancer in a subject comprising a. culturing a cell population on a solid substrate in the presence of a first blood sample obtained from the subject prior to surgical resection of the cancer; b. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the first blood sample; c. culturing a cell population on a solid substrate in the presence of a second blood sample obtained from the subject after surgical resection of the cancer; d. quantifying the level of adhesion to the solid substrate of the cell population cultured in the presence of the second blood sample; e. comparing the level of adhesion to the solid substrate of the cell populations cultured in the presence of the first and the second blood sample, respectively; wherein the cell population is a mammalian cell population and the risk of postoperative recurrence of the cancer is increased when the level of adhesion in the second blood sample is higher than the level of adhesion in the first blood sample.

2. The method according to any one of the preceding claims, where the recurrence of the cancer is increased when the level of adhesion of the cell population in the second blood sample is at least 15% higher, 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 level of adhesion of the cell population in the first blood sample.

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

4. The method according to any one of the preceding claims, wherein the subject has been diagnosed with a cancer selected from the group consisting of: colorectalcancer; 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; sarcomas; esophageal cancer; head and neck cancer; gallbladder cancer; anal cancer; penile cancer; testicular cancer; adrenal gland cancer; soft tissue sarcoma; mesothelioma; thymus cancer; small intestine cancer; neuroendocrine cancer; and pituitary cancer.

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

6. The method according to any one of the preceding claims, wherein the cell population is a cell line selected from the group consisting of: 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, 786-0, 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-211 H, 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 the group consisting of: a luciferase reporter; a fluorescence reporter; an alkaline phosphatase reporter, and wherein the quantification of the level of adhesion of the cell population 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 level of adhesion of the cell population is performed by counting the number of cells of the cell population that have adhered to thesubstrate; and / or by estimating the number of adhered cells by measuring the amount of protein and / or DNA and / or RNA and / or lipid in the adhered cells; and / or by measuring the metabolic activity of the adhered cells.

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

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

11. The method according to any one of the preceding claims, wherein the first blood sample has been obtained from the subject within 7 days before the surgical resection of the cancer, such as within 6 days before the surgical resection of the cancer, such as within 5 days before the surgical resection of the cancer, such as within 4 days before the surgical resection of the cancer, such within 3 days before the surgical resection of the cancer, such as within 2 days before the surgical resection of the cancer, such as within 1 day before the surgical resection of the cancer.

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

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

14. A method for determining if a subject should be treated with adjuvant chemotherapy and / or radiotherapy following surgical resection of a cancer comprising: a) predicting the risk of post-operative recurrence of said cancer in the subject according to any one of the preceding claims; and b) concluding that the subject should be treated with adjuvant chemotherapy and / or radiotherapy if the subject is at risk of post-operative recurrence of said cancer.

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

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