Prognostic values ​​of biomarkers in patients with stable non-small cell lung cancer

Abstract

The present invention relates to an in vitro method for assessing the risk of non-small cell lung cancer (NSCLC) disease progression for a subject classified as having a stable condition under an ongoing NSCLC treatment regime. The method comprises: determining the level of CYFRA21-1 and / or the level of CA125 in a sample obtained from the subject; and (i) comparing the determined level of CYFRA21-1 with a CYFRA21-1 cut-off level, (ii) comparing the determined level of CA125 with a CA125 cut-off level, or (iii) comparing a score taking into account the determined level of CYFRA21-1 and / or the determined level of CA125 with a cut-off score. The method of the present invention further allows to evaluate whether the subject responds to the ongoing treatment and / or whether the treatment regime should be maintained or modified. The present invention also provides corresponding uses, computer-implemented methods, and computer program products.

Description

Technical Field

[0001] Field of the Invention The present invention relates to an in vitro method for assessing the risk of non - small cell lung cancer (NSCLC) disease progression for subjects classified as having a stable state during an ongoing NSCLC treatment regimen. The method comprises determining the level of CYFRA21 - 1 and / or the level of CA125 in a sample obtained from the subject; and (i) comparing the determined level of CYFRA21 - 1 with a CYFRA21 - 1 cut - off level, (ii) comparing the determined level of CA125 with a CA125 cut - off level, or (iii) comparing a score taking into account the determined level of CYFRA21 - 1 and / or the determined level of CA125 with a cut - off score. The method of the present invention further enables assessment of whether the subject will respond to the ongoing treatment and / or whether the treatment regimen should be maintained or modified. Also provided herein are corresponding uses, computer - implemented methods, and computer program products.

Background Art

[0002] Background of the Invention Lung cancer has the highest incidence rate among all cancer types worldwide (more than 2 million new cases were estimated in 2018), and is the second highest incidence rate in Central and Eastern Europe (GLOBOCAN 2018. 2018 ed.; International Agency for Research on Cancer. World Health Organization: 2018.). It is also the main cause of cancer mortality, with 131,000 deaths annually in Central and Eastern Europe and 1.8 million deaths annually worldwide (GLOBOCAN 2018. 2018 ed.; International Agency for Research on Cancer. World Health Organization: 2018.).

[0003] Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. The most common histological subtypes of NSCLC are adenocarcinoma (ADC; 40% of all cases) and squamous cell carcinoma (SCC; 35% of all cases) (Bender, E., Nature 2014, 513, S2-S3, doi:10.1038 / 513S2a.).

[0004] Lung cancer is mostly asymptomatic, and over 70% of all cases are diagnosed in the later stages (Molina, J. Ret al., Mayo Clinic Proceedings 2008, 83, 584-594, doi:10.4065 / 83.5.584). Treatment options include surgery, radiotherapy, chemotherapy, and immune checkpoint inhibitors. Adjuvant chemotherapy is recommended for patients with stage II and IIIA NSCLC (Postmus, PE et al., Annals of Oncology 2017, 28, iv1-iv21, doi:10.1093 / annonc / mdx222; NCCN Guidelines. Non-Small-Cell Lung Cancer. Version 4.2019.). Furthermore, small molecule tyrosine kinase inhibitors have been shown to improve survival and are recommended for patients with advanced NSCLC who have activating mutations in epidermal growth factor receptor, anaplastic lymphoma kinase, ROS1, or BRAF (NCCN Guidelines, Non-Small-Cell Lung Cancer, Version 4.2019; Planchard, D. et al., Annals of Oncology 2018, 29, iv192-iv237, doi:10.1093 / annonc / mdy275).

[0005] Computed tomography (CT) or positron emission tomography (PET) are widely used for clinical staging (Molina, J. Ret al., Mayo Clinic Proceedings 2008, 83, 584-594, doi:10.4065 / 83.5.584; Postmus, PE et al., Annals of Oncology 2017, 28, iv1-iv21, doi:10.1093 / annonc / mdx222; NCCN Guidelines. Non-Small-Cell Lung Cancer. Version 4.2019). Furthermore, response criteria based on CT imaging results have been successfully used in numerous randomized clinical trials, and CT is now the preferred method for evaluating treatment response (Eisenhauer, EA et al., Eur J Cancer 2009, 45, 228-247, doi:10.1016 / j.ejca.2008.10.026).

[0006] In patients undergoing cytotoxic chemotherapy, CT-based response criteria were developed as a means of evaluating the effectiveness of active agents based on the amount of tumor reduction they induced. WHO and subsequent RECIST criteria were adopted as the gold standard for evaluating treatment response in clinical trials, and bilateral and unilateral measurements remain central to response assessment in routine clinical practice. Nevertheless, objective tumor responses assessed based on imaging show an incomplete correlation with patient outcomes, and their usefulness has been questioned in recent studies evaluating non-chemotherapy treatments (Lee HY et al., Lung Cancer 2011, 73, 63-69; Hwang K and Kim H, Tuberc Respir Dis (Seoul) 2017, 80, 136-142, doi:10.4046 / trd.2017.80.2.136), particularly with checkpoint inhibitors (M. Tazdait et al., European Journal of Cancer, Volume 88, 2018, Pages 38-47; Wolchok, JD et al., Clin Cancer Res December 1 2009(15)(23) 7412-7420).

[0007] Several studies in lung cancer patients have demonstrated that lung cancer subtypes can be determined using cancer biomarkers, such as CYFRA21-1, CEA, ProGRP, and NSE, and that these can be correlated with tumor stage, prognosis, and response to treatment (Jing AGet al., BMC Cancer 2015, 15, doi:10.1186 / s12885-015-1403-x; Liu, L. et al., BioMed Research International 2017, 2017, 1-9, doi:10.1155 / 2017 / 2013989; Muley, T. et al., Lung Cancer 2018, 120, 46-53, doi:10.1016 / j.lungcan.2018.03.015; Wojcik E and Kulpa JK, Lung Cancer (Auckl) 2017;8:231-40). However, there is currently a lack of compelling use cases demonstrating the further value of biomarkers through vis-a-vis imaging approaches. In particular, it is unclear whether combining imaging and biomarker evaluation can provide further value for guiding treatment or prognosis.

[0008] The evaluation of treatment efficacy in advanced non-small cell lung cancer (NSCLC) is currently based on imaging using computed tomography (CT) scans. An established definition exists for evaluating the treatment response of solid tumors (RECIST), which classifies responses into four main categories: complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD), depending on the decrease / increase in tumor volume, the number of lesions, and new lesions / metastases (New response evaluation criteria in solid tumors: Revised RECIST Guidelines (Version 1.1), EAEisenhauer et al, European Journal of Cancer 45 (2009) 228-247). The decision to continue (or discontinue) treatment is typically based on these imaging results and is defined in the globally applicable treatment guidelines (NCCN Guidelines. Non-Small-Cell Lung Cancer. Version 4.2019.2019. Postmus PE, Kerr KM, Oudkerk M, Senan S, Waller DA, Vansteenkiste J, et al. Early and locally advanced non-small-cell lung cancer (NSCLC): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of Oncology 2017;28(suppl_4)).

[0009] In response categories of partial response (PR) and stable disease (SD), treatment is typically continued according to guideline recommendations. The SD group includes patients with measurable lesions that, according to RECIST 1.1 criteria, have neither sufficient tumor reduction to be considered a partial response (reduction of 30% or more in size) nor sufficient growth to be considered progressive disease (increase of 20% or more in size). In particular, the SD group, which covers a wide range of tumor sizes, remains highly uncertain regarding the response to ongoing treatment regimes and prognosis. However, there are currently no reliable means available to further characterize the treatment efficacy and / or prognosis of disease progression in this group of SD patients.

[0010] Wang and collaborators (Wang t al., 2002, Tumor, (25 Dec 2012) Vol.32, No.12, pp.1021-1024) investigated the clinical value of serum tumor marker expression levels when monitoring the response to targeted therapy with EGFR-TKIs (epidermal growth factor receptor tyrosine kinase inhibitors) in patients with advanced NSCLC. However, this study did not specifically address the prognosis of disease progression in the group of SD patients based on serum biomarkers. Furthermore, this study focuses only on changes in biomarker levels, which has the drawback of requiring at least two measurements using the same assay at different time points.

[0011] Hall C and collaborators (Hall C et al., Journal of Thoracic Oncology, (June 2011) Vol.6, No.6, Supp.SUPPL.2, pp.S979-S980) retrospectively evaluated the performance of decreased serum CYFRA21-1 levels for detecting disease progression in lung cancer patients. However, this study did not attempt to improve the prognosis of treatment response in SD patients and focused on measuring CYFRA21-1 levels over time.

[0012] The Iqbal Kashif study (Journal of Thoracic Oncology, (June 2011) Vol.6, No.6, Supp.SUPPL.2, pp.S1248-S1249) reported CEA and CYFRA21-1 as early predictors of response to first-line chemotherapy in advanced NSCLC. However, this study was again based on the cumbersome assessment of biomarker level changes, and no specific benefit was reported for the group of patients classified as SD by CT imaging.

[0013] Essink A. and collaborators (Essink et al., Journal of Thoracic Oncology, (April 2016) Vol.11, No.4, Supp.SUPPL.1, pp.S126) studied changes in serum tumor markers, including changes in CYFRA21-1, CEA, SCC, CEA, and NSE, in the context of response to immunotherapy in NSCLC patients. Here again, only changes in biomarker levels were evaluated, and specific benefits for the patient group classified as SD by CT imaging were not tested.

[0014] Another study evaluating the decline in CYFRA21-1 levels during chemotherapy in NSCLC patients was conducted by Alm El-Din Mohamed A and his collaborators (Alm El-Din Mohamed A et al., The International Journal of Biological Markers, (2012 Jul 19) Vol.27, No.2, pp.e139-46). This study focused on the decline in biomarker levels and therefore also included the assessment of two or more biomarkers at defined time points. Furthermore, this study did not report any specific benefit for evaluating disease progression and / or treatment response in SD patients.

[0015] Yang Liang et al. (Experimental and therapeutic medicine, (2012 Aug) Vol.4, No.2, pp.243-248) reported a decrease in serum CYFRA21-1 levels as a predictor of chemotherapy response in NSCLC patients. Again, only changes in biomarker levels were evaluated, and no specific benefits for SD patients were described.

[0016] Li Ling et al. investigated CEA and CYFRA21-1 levels as indicators of treatment efficacy in patients with advanced NSCLC. This study also focused on changes in biomarker levels, rather than absolute biomarker levels at defined time points.

[0017] Considering the above, there is a strong need to further improve the evaluation of treatment response and prognosis, especially in NSCLC patients classified as having a stable condition based on imaging. [Overview of the Initiative]

[0018] The above-mentioned needs are addressed by the present invention.

[0019] The present invention provides, in particular, the following items.

[0020] 1. An in vitro method for evaluating the risk of progression of non-small cell lung cancer (NSCLC) in subjects with non-small cell lung cancer (NSCLC) who are under an ongoing NSCLC treatment regime, a) Determine the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject; and b) (i) Compare the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, (ii) Compare the determined level of CA125 with the CA125 cutoff level, or (iii) Compare the score considering the determined level of CYFRA21-1 and / or the determined level of CA125 with the cutoff score. comprising, a method, wherein the subject is classified as having a stable state.

[0021] In 2.a), the level of CYFRA21-1 is determined, and the comparison in b) comprises or consists of (i) as described in b) of item 1, and the determined level of CYFRA 21-1 below the CYFRA21-1 cut-off level indicates a low risk of NSCLC disease progression, the method according to item 1.

[0022] In 3.a), the level of CYFRA21-1 is determined, and the comparison in b) comprises or consists of (i) as described in b) of item 1, and the determined level of CYFRA 21-1 higher than the CYFRA21-1 cut-off level indicates a high risk of NSCLC disease progression, the method according to item 1 or 2.

[0023] The method according to item 2 or 3, wherein the subject has NSCLC of the subtype adenocarcinoma.

[0024] In 5.a), the level of CA125 is determined, and the comparison in b) comprises or consists of (ii) as described in b) of item 1, and the determined level of CA125 lower than the CA125 cut-off level indicates a low risk of NSCLC disease progression, the method according to item 1.

[0025] In 6.a), the level of CA125 is determined, and the comparison in b) comprises or consists of (ii) as described in b) of item 1, and the determined level of CA125 higher than the CA125 cut-off level indicates a high risk of NSCLC disease progression, the method according to item 1 or 5.

[0026] The method according to item 5 or 6, wherein the subject has NSCLC of the subtype squamous cell carcinoma (SCC-NSCLC).

[0027] 8. a) In which the levels of CYFRA21-1 and / or CA125 are determined, and the comparison in b) comprises or consists of (iii) as defined in b) of item 1, and a determined score lower than the cut-off score indicates a low risk of NSCLC disease progression, the method according to item 1.

[0028] 9. a) In which the levels of CYFRA21-1 and / or CA125 are determined, and the comparison in b) comprises or consists of (iii) as defined in b) of item 1, and a determined score higher than the cut-off score indicates a high risk of NSCLC disease progression, the method according to item 1 or 8.

[0029] 10. a) In which the levels of CYFRA21-1 and CA125 in the sample are determined, and the score takes into account the determined levels of CYFRA21-1 and the determined level of CA125, the method according to item 8 or 9.

[0030] 11. The method according to any one of item 10, further comprising the step of determining said score.

[0031] 12. The risk of NSCLC disease progression is evaluated by comparison with the risk of NSCLC disease progression in a reference population, preferably the reference population on which each cut-off is based, the method according to any one of items 1 to 11

[0032] 13. The risk of progression of NSCLC disease comprises the risk of tumor growth (preferably evaluated according to the RECIST 1.1 criteria), the risk of formation of new lesions or metastases, or and / or the risk of death due to NSCLC, the method according to any one of items 1 to 12.

[0033] 14. An in vitro method for evaluating whether an ongoing NSCLC treatment regimen should be maintained or modified for a subject diagnosed with non-small cell lung cancer (NSCLC), a) Determine the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject; and b) (i) comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, (ii) comparing the determined level of CA125 with the CA125 cutoff level, or (iii) comparing a score that takes into account the determined level of CYFRA21-1 and / or the determined level of CA125 with the cutoff score. Includes; The subject of the above is a method that is classified as having a stable state.

[0034] 15.a) The method of item 14, wherein the level of CYFRA21-1 is determined in a) and the comparison in b) includes or consists of (i) as described in item 14 b), indicating that the determined level of CYFRA21-1 is below the CYFRA21-1 cutoff level, and the ongoing NSCLC treatment regime should be maintained.

[0035] 16.a) The method by which the level of CYFRA21-1 is determined in item 14 or 15, and the comparison in item 14 or 15 includes or consists of (i) as described in item 14 b), wherein the determined level of CYFRA21-1 is higher than the CYFRA21-1 cutoff level, indicating that the ongoing NSCLC treatment regime should be modified.

[0036] 17. The method described in item 15 or 16, wherein the subject has NSCLC, a subtype of adenocarcinoma.

[0037] 18.a) The method of item 14, wherein the level of CA125 is determined in a) and the comparison in b) includes or consists of (ii) as described in item 14 b), indicating that the determined level of CA125 lower than the CA125 cutoff level indicates that the ongoing NSCLC treatment regime should be maintained.

[0038] 19.a) The method of item 14 or 18, wherein the determined level of CA125 is determined in item 14, and the comparison in item 14, b) includes or consists of (ii) as described in item 14, b), and the determined level of CA125 being higher than the CA125 cutoff level indicates that the ongoing NSCLC treatment regime should be modified.

[0039] 20. The method described in item 18 or 19, for subjects who have NSCLC (SCC-NSCLC), a subtype of squamous cell carcinoma.

[0040] 21.a) The levels of CYFRA21-1 and CA125 are determined, and the comparison in b) includes or comprises the method of item 14, where a determined score lower than the cutoff score indicates that the ongoing NSCLC treatment regime should be maintained.

[0041] 22.a) The levels of CYFRA21-1 and CA125 are determined, and the comparison in b) includes or comprises the method of item 14 or 21, wherein a determined score higher than the cutoff score indicates that the ongoing NSCLC treatment regime should be modified.

[0042] The method according to item 21 or 22, wherein, in 23.a), the levels of CYFRA21-1 and CA125 in the sample are determined, and the score takes into account the determined levels of CYFRA21-1 and CA125.

[0043] 24. The method according to any one of items 21 to 23, further comprising the step of determining the score.

[0044] 25. An in vitro method for evaluating whether subjects diagnosed with non-small cell lung cancer (NSCLC) respond to an ongoing NSCLC treatment regime, a) Determine the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject; and b) (i) comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, (ii) comparing the determined level of CA125 with the CA125 cutoff level, or (iii) comparing a score that takes into account the determined level of CYFRA21-1 and / or the determined level of CA125 with the cutoff score. Includes, The subject of the above is a method that is classified as having a stable state.

[0045] 26.a) The method by which a level of CYFRA21-1 is determined in Item 25, and the comparison in Item 25 includes or consists of (i) as described in Item 25 b), indicating that the determined level of CYFRA21-1 below the CYFRA21-1 cutoff level indicates that the subject is responding to and / or will respond to the ongoing NSCLC treatment regime.

[0046] 27.a) The method by which a level of CYFRA21-1 is determined in Item 25 or 26, and the comparison in Item 25 or 26 includes or consists of (i) as described in Item 25 b), indicating that a determined level of CYFRA21-1 higher than the CYFRA21-1 cutoff level indicates that the subject is not responding to and / or will not respond to the ongoing NSCLC treatment regime.

[0047] 28. The method described in item 26 or 27, wherein the subject has NSCLC, a subtype of adenocarcinoma.

[0048] 29.a) The method of item 25, wherein the level of CA125 is determined in item 25, and the comparison in item 25 includes or consists of (i) as described in item 25 b), indicating that the determined level of CA125, which is lower than the CA125 cutoff level, indicates that the subject is responding to or will respond to the ongoing NSCLC treatment regime.

[0049] 30.a) The method of item 25 or 29, wherein the level of CA125 is determined in a) and the comparison in b) includes or consists of (i) as described in item 25 b), indicating that the determined level of CA125 higher than the CA125 cutoff level indicates that the subject is not responding to or will not respond to the ongoing NSCLC treatment regime.

[0050] 31. The method described in item 29 or 30, for subjects who have NSCLC (SCC-NSCLC), a subtype of squamous cell carcinoma.

[0051] 32.a) The levels of CYFRA21-1 and / or CA125 are determined, and the comparison in b) includes or comprises (iii) described in item 25 b), the method of item 25, wherein a determined combined score lower than the reference indicates that the subject is responding to the ongoing NSCLC treatment regime.

[0052] 33.a) The levels of CYFRA21-1 and CA125 are determined, and the comparison in b) includes or comprises (iii) as described in item 25 b), the method of item 25 or 32, wherein a determined combined score higher than the reference indicates that the subject is not responding to the ongoing NSCLC treatment regime.

[0053] 34. The method described in any one of items 1 to 33, further including the step of determining the level of CEA.

[0054] The comparison in 35.b) includes or consists of (iii) as described in item 1 b), item 14 b), or item 25 b), respectively, and the score further takes into account the determined level of CEA, as of item 34.

[0055] 36. The method described in any one of items 1 to 35, Preferably, this further includes obtaining information, based on histological data, on whether the subject has squamous cell carcinoma (SCC-NSCLC) or adenocarcinoma (ADC-NSCLC), a subtype of non-small cell lung cancer. A method in which the comparison in b) includes iii) described in item 1 b), item 14 b), or item 25 b), and the score further considers the NSCLC subtype by preferably using interaction terms, and more preferably by using interaction terms between information about the NSCLC subtype and biomarkers included (i.e., CYFRA21 and / or CA125, and optionally CEA).

[0056] 37. The method according to any one of items 1 to 36, wherein the CYFRA cutoff level, CA125 cutoff level and / or cutoff score are based on a reference population of subjects, preferably a reference population including subjects diagnosed with non-small cell lung cancer (NSCLC) who are in a stable state and under an ongoing treatment regime, and the cutoff level or score is determined based on the level or score after the second cycle of the treatment regime(s) has been applied to the reference population.

[0057] 38. The method according to any one of items 1 to 37, wherein the ongoing NSCLC treatment regime is selected from the group consisting of chemotherapy, targeted therapy, immunotherapy, and combinations thereof.

[0058] 39. The method according to any one of items 1 to 38, wherein the subject is classified as having a stable state based on imaging data, preferably CT or PET CT data.

[0059] 40. The method according to any one of items 1 to 39, wherein the subject is classified as having a stable state based on RECIST, preferably RECIST 1.1 criteria.

[0060] 41. The method according to any one of items 1 to 40, wherein the sample is obtained 10 to 150 days after the start date of the ongoing treatment regime, preferably 20 to 120 days after the start date of the ongoing treatment regime, and most preferably 25 to 108 days after the start date of the ongoing treatment regime.

[0061] 42. The method according to any one of items 1 to 41, wherein the subject is classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained 10 to 150 days after the start of the ongoing treatment regime, preferably 20 to 120 days after the start of the ongoing treatment regime, and most preferably 35 to 108 days after the start of the ongoing treatment regime.

[0062] 43. The method according to any one of items 1 to 42, wherein the sample is obtained after the second cycle of a treatment regime (e.g., chemotherapy), and preferably the sample is obtained before the third cycle of the treatment regime.

[0063] 44. The method according to any one of items 1 to 43, wherein the sample is obtained within a specific time frame around the date of a health checkup (e.g., imaging such as CT), and the subject is ultimately classified as being in a stable state, preferably the specific time frame is from 35 days before to 35 days after the date of the health checkup, more preferably the specific time frame is from 30 days before to 30 days after the date of the health checkup, and most preferably the specific time frame is from 10 days before to 29 days after the date of the health checkup.

[0064] 45. The method described in any one of items 1 to 44, wherein the treatment regime is the first-line treatment regime.

[0065] 46. ​​The method described in any one of items 1 to 45, wherein the sample is a blood sample.

[0066] 47. The method described in item 46, wherein the blood sample is selected from the group consisting of whole blood, serum, or plasma.

[0067] 48. The method described in item 46, wherein the blood sample is serum or plasma.

[0068] 49. The method described in any one of items 46-48, wherein the blood sample is a venous blood sample.

[0069] 50. The method of any one of items 1 to 49, further comprising providing information on whether (i) the subject is at risk of NSCLC disease progression, (ii) whether the treatment regime should be maintained or modified, and / or (iii) whether the subject is responsive to the ongoing NSCLC treatment regime.

[0070] 51. A method for treating a subject diagnosed with NSCLC, comprising: (i) evaluating whether the subject is responding to an ongoing treatment regime described in any one of items 25-50; or (i)' evaluating whether the ongoing treatment regime for the subject should be maintained as described in any one of items 14-24 and 34-50; and (ii) continuing to treat the patient with the ongoing treatment regime, or modifying the treatment regime in accordance with the results of the evaluation of (i) or (i)'.

[0071] 52. (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Whether the ongoing NSCLC treatment regime should be maintained or modified for subjects diagnosed with NSCLC. The use of CYFRA21-1 and / or CA125 as biomarkers (multiple markers are possible) for evaluating, Uses in which the subject in (i), (ii), and (iii) is classified as having a stable state.

[0072] 53. Use of item 52, further including the use of CEA as a biomarker for (i), (ii) and / or (iii).

[0073] 54. The use described in item 52 or 53, preferably further comprising taking into account, based on histological data, information on whether the subject has squamous cell carcinoma (SCC-NSCLC) or adenocarcinoma (ADC-NSCLC) of the non-small cell lung cancer subtype.

[0074] 55. Use as described in any one of items 52-53, further including the steps described in any one of items 1-50.

[0075] 56. (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Whether the ongoing NSCLC treatment regime should be maintained or modified for subjects diagnosed with NSCLC. A computer implementation method for evaluating, The aforementioned computer implementation method a) Receiving data including the levels of CYFRA21-1, CA125 and / or CEA determined in any one of items 1-50, and optionally information regarding NSCLC subtypes; and b) Process the data and perform the comparison step described in b) of any one of items 1 to 50. A computer implementation method that includes, and the subject is classified as having a stable state.

[0076] 57. The method of item 56, further comprising outputting the evaluation (e.g., via a display).

[0077] 58. A computer program product that, when executed by a computer, contains instructions that cause the computer to perform the actions described in item 56 or 57.

[0078] 59. Computer-readable media containing instructions that, when executed by a computer, cause the computer to perform the actions described in item 56 or 57.

[0079] 60. A data processing system comprising a receiving unit configured to receive the data described in item 56 a); a processing unit configured to perform step b) described in item 56; and an output unit optionally configured to output evaluation results.

[0080] 61. A kit comprising a reagent or set of reagents for detecting the level of CYFRA21-1 in a sample obtained from a subject and / or a reagent or set of reagents for detecting CA125, The kit is, (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Whether the ongoing NSCLC treatment regime should be maintained or modified for subjects diagnosed with NSCLC. This is a kit for evaluating, A kit in which the subject(i), (ii), and (iii) is classified as having a stable state.

[0081] 62. A kit of item 61, further comprising a reagent or set of reagents for detecting the level of CEA in a sample obtained from a subject.

[0082] 63. A kit relating to item 61 or 62, wherein the kit includes a description of a method for providing the evaluation described in item 61(i), (ii), or (iii), the description including steps of the method described in any one of items 1 to 50 or a description of use described in any one of items 52 to 55, or referring to a computer implementation method of item 56 or 57, a computer program product of item 58, a computer-readable medium of item 59, or a data processing system of item 60.

[0083] 64. A kit as described in any one of items 61 to 63, including an accompanying document describing one or more of the purposes described in item 61(i) to (iii). The following figures are provided to aid in understanding the present invention, and its true scope is specified in the claims. It is understood that modifications to the described procedures can be made without departing from the spirit of the invention. [Brief explanation of the drawing]

[0084] [Figure 1-1] Progression-free survival (A) and overall survival (B) in patients with partial response or stable condition on the first CT scan after the second cycle, stratified by CT scan results. [Figure 1-2] Progression-free survival (A) and overall survival (B) in patients with partial response or stable condition on the first CT scan after the second cycle, stratified by CT scan results. [Figure 2-1] Progression-free survival in patients with stable status on the first CT scan after the second cycle, in patients with adenocarcinoma stratified by CYFRA21-1 (A), squamous cell carcinoma stratified by CA125 (B), adenocarcinoma or squamous cell carcinoma stratified by CYFRA21-1 and CA125 (C), and adenocarcinoma or squamous cell carcinoma stratified by CYFRA21-1, CA125 and CEA (D), above or below the median. The biomarker combinations by score in (C) and (D) (referred to as Pred in the figure) take into account the interaction terms between each biomarker and histology. Baseline correction of biomarker levels was not used. [Figure 2-2]Progression-free survival in patients with stable status on the first CT scan after the second cycle, in patients with adenocarcinoma stratified by CYFRA21-1 (A), squamous cell carcinoma stratified by CA125 (B), adenocarcinoma or squamous cell carcinoma stratified by CYFRA21-1 and CA125 (C), and adenocarcinoma or squamous cell carcinoma stratified by CYFRA21-1, CA125 and CEA (D), above or below the median. The biomarker combinations by score in (C) and (D) (referred to as Pred in the figure) take into account the interaction terms between each biomarker and histology. Baseline correction of biomarker levels was not used. [Figure 2-3] Progression-free survival in patients with stable status on the first CT scan after the second cycle, in patients with adenocarcinoma stratified by CYFRA21-1 (A), squamous cell carcinoma stratified by CA125 (B), adenocarcinoma or squamous cell carcinoma stratified by CYFRA21-1 and CA125 (C), and adenocarcinoma or squamous cell carcinoma stratified by CYFRA21-1, CA125 and CEA (D), above or below the median. The biomarker combinations by score in (C) and (D) (referred to as Pred in the figure) take into account the interaction terms between each biomarker and histology. Baseline correction of biomarker levels was not used. [Figure 2-4] Progression-free survival in patients with stable status on the first CT scan after the second cycle, in patients with adenocarcinoma stratified by CYFRA21-1 (A), squamous cell carcinoma stratified by CA125 (B), adenocarcinoma or squamous cell carcinoma stratified by CYFRA21-1 and CA125 (C), and adenocarcinoma or squamous cell carcinoma stratified by CYFRA21-1, CA125 and CEA (D), above or below the median. The biomarker combinations by score in (C) and (D) (referred to as Pred in the figure) take into account the interaction terms between each biomarker and histology. Baseline correction of biomarker levels was not used. [Figure 3-1]Progression-free survival (A) and overall survival (B) in patients with adenocarcinoma or squamous cell carcinoma stratified into high-risk and low-risk groups by CYFRA21-1, CA125, and CEA, with median values ​​above or below, who were stable on the first CT scan after the second cycle. The biomarker combination model took into account the interaction terms between each biomarker and histology. Baseline correction for biomarker levels was not used. [Figure 3-2] Progression-free survival (A) and overall survival (B) in patients with adenocarcinoma or squamous cell carcinoma stratified into high-risk and low-risk groups by CYFRA21-1, CA125, and CEA, with median values ​​above or below, who were stable on the first CT scan after the second cycle. The biomarker combination model took into account the interaction terms between each biomarker and histology. Baseline correction for biomarker levels was not used. [Figure 4-1] Progression-free survival (A) and overall survival (B) in patients with adenocarcinoma or squamous cell carcinoma stratified into high-risk and low-risk groups by CYFRA21-1, CA125, and CEA, above or below the optimized cutoff, who were stable on the first CT scan after the second cycle. The biomarker combination model took into account the interaction terms between each biomarker and histology. Baseline correction for biomarker levels was not used. [Figure 4-2] Progression-free survival (A) and overall survival (B) in patients with adenocarcinoma or squamous cell carcinoma stratified into high-risk and low-risk groups by CYFRA21-1, CA125, and CEA, above or below the optimized cutoff, who were stable on the first CT scan after the second cycle. The biomarker combination model took into account the interaction terms between each biomarker and histology. Baseline correction for biomarker levels was not used. [Modes for carrying out the invention]

[0085] Detailed description of the invention Surprisingly, in the context of the present invention, it has been found that by determining the protein levels of the biomarkers CYFRA21-1 and / or CA125 in a sample (e.g., serum or plasma sample), patients diagnosed with stable-state (SD) NSCLC (e.g., according to imaging and RECIST criteria) can be further divided into two groups: a first group with a low risk of disease progression under the ongoing treatment regime, and a second group with a high risk of disease progression under the ongoing treatment regime. This risk stratification can also provide an assessment of the likelihood of responding to the ongoing treatment regime and / or support for further treatment decisions, i.e., whether the ongoing treatment should be maintained or modified. A particularly surprising finding of the present invention was that the levels of CYFRA21-1 and / or CA125 at a single point in time (particularly at the first CT after the second treatment cycle), for example as absolute concentration or absolute volume (or its logarithmic transformation), performed far better in assessing the risk of disease progression than using changes in the levels of the same biomarkers compared to the start of treatment.

[0086] As is evident from the background section above, prior art studies, in contrast, focused on changes in biomarker levels to assess whether a patient would respond to treatment. Due to the known variability in the levels of the biomarkers used in the present invention among patients, it was entirely unexpected that risk stratification would be superior when the levels of each biomarker prior to treatment were not considered. There are also several other advantages to using a single biomarker level despite its better performance. For example, biomarker-based assessments are simplified because only a single biomarker measurement is required at a defined time point. In contrast to utilizing changes in biomarkers, the method of the present invention can be carried out independently of the availability of previous biomarker levels and is therefore available to an increased number of patients. Furthermore, the need to compare biomarker levels from measurements that may potentially involve two independent time points and different assays is eliminated by the present invention.

[0087] Using both biomarkers, CYFRA21-1 and CA125, the univariate marker performance for assessing NSCLC progression risk (e.g., as assessed by progression-free survival and / or overall survival) was significantly better than all other tumor biomarkers tested. CYFRA21-1 showed the best univariate performance in subjects with NSCLC of the histological subtype adenocarcinoma. CA125 showed the best univariate performance in subjects with NSCLC of the histological subtype squamous cell carcinoma (SCC-NSCLC).

[0088] The combination of CYFRA21-1 and CA125 levels in the score format further improved performance. In particular, the use of interaction terms that take into account the histological subtype (adenocarcinoma or SCC) for each biomarker further improved the performance for calculating the score.

[0089] Furthermore, it was found that including a third biomarker level, namely the level of CEA, in the score could further improve the prognosis of disease progression under an ongoing treatment regime.

[0090] In a first aspect, the present invention relates to an in vitro method for evaluating the risk of progression of non-small cell lung cancer (NSCLC) in a subject.

[0091] A method according to a first aspect of the present invention is: a) Determine the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject; and b) including (i) comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, (ii) comparing the determined level of CA125 with the CA125 cutoff level, or (iii) comparing a score that takes into account the determined level of CYFRA21-1 and / or the determined level of CA125 with the cutoff score.

[0092] The subjects from whom samples are obtained are those diagnosed with non-small cell lung cancer (NSCLC) and under an ongoing NSCLC treatment regime. In other words, the subjects from whom samples are obtained were diagnosed with NSCLC before the method according to the first embodiment was performed and are under an NSCLC treatment regime at the time the samples are obtained.

[0093] Furthermore, the sample obtained may be classified or diagnosed as being in a stable state by means other than those in the steps of this method. Preferably, the classification of the stable state is performed by imaging methods such as CT.

[0094] In this embodiment, the object from which the sample is obtained is diagnosed as being in a stable state before or at the time the sample is obtained.

[0095] In the embodiment, the sample is obtained within a specific time frame around the date of a health checkup (e.g., imaging such as CT) that leads to a diagnosis of a stable state. In the embodiment, the specific time frame may be from 35 days before to 35 days after the health checkup. In the preferred embodiment, the specific time frame may be from 30 days before to 30 days after the health checkup. In the even more preferred embodiment, the specific time frame may be from 10 days before to 29 days after the health checkup.

[0096] In one embodiment, a subject is classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained at least 10 days, preferably at least 20 days, more preferably at least 30 days, and most preferably at least 35 days after the start date of the ongoing treatment regime. In another embodiment, a subject is classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained up to 150 days, preferably up to 120 days, and most preferably up to 108 days, after the start date of the ongoing treatment regime. Therefore, a subject may be classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained 10 to 150 days after the start date of the ongoing treatment regime, preferably 20 to 120 days, and most preferably 35 to 108 days after the start date of the ongoing treatment regime.

[0097] In the embodiment, the sample is obtained from the subject at least 10 days, preferably at least 20 days, and most preferably at least 25 days, after the start date of the ongoing treatment regime. In the embodiment, the sample is obtained from the subject up to 150 days, preferably up to 120 days, and most preferably up to 108 days, after the start date of the ongoing treatment regime. Therefore, in the embodiment, the sample is obtained from the subject 10 to 150 days, preferably 20 to 120 days, and most preferably 25 to 108 days, after the start date of the ongoing treatment regime.

[0098] In this embodiment, subjects are classified as having a stable state based on data obtained 35 to 108 days after the start of an ongoing treatment regime (e.g., tumor imaging data), and the samples are obtained from the subjects 25 to 108 days after the start of an ongoing treatment regime.

[0099] The "start" or "start date" of an ongoing treatment regime is the date on which the first treatment of that regime was administered to the subject. For example, it is the date on which the drug or drug composition was first administered to the subject.

[0100] In embodiments of the first aspect of the method, the level of CYFRA21-1 may be determined in a), and the comparison in b) may include or consist of comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, where a determined level of CYFRA21-1 below the CYFRA21-1 cutoff level indicates a low risk of NSCLC disease progression.

[0101] In an embodiment of the method according to the first aspect, the level of CYFRA21-1 is determined in a), and the comparison in b) includes or consists of comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, wherein a determined level of CYFRA21-1 higher than the CYFRA21-1 cutoff level indicates a high risk of NSCLC disease progression.

[0102] Therefore, in a first embodiment of the present invention, a method for evaluating the risk of progression of non-small cell lung cancer (NSCLC) in a subject, a) Determine the level of CYFRA21-1 in the sample obtained from the subject; and b) Compare the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level. A method is provided which includes determining that a determined level of CYFRA21-1 below the CYFRA21-1 cutoff level indicates a low risk of NSCLC disease progression, and / or determining that a determined level of CYFRA21-1 above the CYFRA21-1 cutoff level indicates a high risk of NSCLC disease progression. Subjects from whom samples are obtained are diagnosed with non-small cell lung cancer (NSCLC), are under an ongoing NSCLC treatment regime, and have been previously classified (or diagnosed) as being in a stable stage. In this regard, it is even more preferable that subjects be diagnosed with NSCLC of the adenocarcinoma subtype (ADC-NSCLC).

[0103] In the attached examples, the performance of the biomarker CYFRA21-1 in assessing the risk of progression of non-small cell lung cancer (NSCLC) was demonstrated to be particularly good for subjects with NSCLC of the adenocarcinoma subtype. Therefore, in the embodiments for determining the biomarker CYFRA21-1, the subjects from whom samples were obtained may be, in particular, those diagnosed with NSCLC of the adenocarcinoma subtype.

[0104] In embodiments of the method according to the first aspect, the level of CA125 may be determined in a), and the comparison in b) may include or consist of comparing the determined level of CA125 with the CA125 cutoff level, where a determined level of CA125 below the CA125 cutoff level indicates a low risk of NSCLC disease progression.

[0105] In an embodiment of the method according to the first aspect, the level of CA125 is determined in a), and the comparison in b) includes or consists of comparing the determined level of CA125 with the CA125 cutoff level, wherein a determined level of CA125 higher than the CA125 cutoff level indicates a high risk of NSCLC disease progression.

[0106] Therefore, in a first embodiment of the present invention, a method for evaluating the risk of progression of non-small cell lung cancer (NSCLC) in a subject, a) Determine the level of CA125 in the sample obtained from the subject; and b) Compare the determined CA125 level with the CA125 cutoff level. A method is provided which includes determining that a determined level of CA125 below the CA125 cutoff level indicates a low risk of NSCLC disease progression, and / or determining that a determined level of CA125 above the CA125 cutoff level indicates a high risk of NSCLC disease progression. Subjects from whom samples are obtained are diagnosed with non-small cell lung cancer (NSCLC), are under an ongoing NSCLC treatment regime, and have been previously classified (or diagnosed) as being in a stable stage. In this regard, it is even more preferable that subjects be diagnosed with squamous cell carcinoma subtype NSCLC (SCC-NSCLC).

[0107] In the attached examples, the performance of the biomarker CA125 in assessing the risk of progression of non-small cell lung cancer (NSCLC) was demonstrated to be particularly good for subjects with the squamous cell carcinoma subtype (SCC). Therefore, in the embodiments for determining the biomarker CA125, the subjects from whom the samples were obtained may be those diagnosed with NSCLC of the squamous cell carcinoma subtype (SCC-NSCLC).

[0108] In a first embodiment of the present invention, the method may include determining a score taking into account the levels of CYFRA21-1 and / or CA125 determined in the sample. In these embodiments, the comparison in b) includes or consists of comparing the score taking into account the determined levels of CYFRA21-1 and / or CA125 with the cutoff score.

[0109] A score that takes into account the levels of CYFRA21-1 and / or CA125 in a sample can be determined by a weighted calculation using the determined levels of CYFRA21-1 and / or CA125. In some embodiments, the score may be based solely on CYFRA21-1 and / or CA125. In other embodiments, the score may take into account the presence or levels of one or more other biomarkers in the sample and one or more clinical parameters of the subject (e.g., histology, smoking status, disease stage, age, and / or sex). In certain embodiments, the score may take into account whether the histology is adenocarcinoma or SCC. In particular, histology may be considered by an interaction term with biomarker values. This allows for taking into account that the biomarkers CYFRA21-1 and CA125 exhibited slightly different performance depending on the histology.

[0110] Therefore, the method according to the first embodiment may be a method for evaluating the risk of progression of non-small cell lung cancer (NSCLC) in a subject, and the said method is a) Determine the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject; aa)a) The score is determined considering the level of CYFRA21-1 and / or the level of CA125 determined in aa); and b) Compare the score, taking into account the determined level of CYFRA21-1 and / or the determined level of CA125, with the cutoff score. Includes, The subjects from whom samples are obtained are those diagnosed with non-small cell lung cancer (NSCLC), under an ongoing NSCLC treatment regime, and previously classified (or diagnosed) as being in a stable stage.

[0111] In these embodiments, a determined score lower than the cutoff score (considering the determined levels of CYFRA21-1 and / or CA125) indicates a low risk of NSCLC disease progression. In these embodiments, the score is configured to increase as the levels of CYFRA21-1 and / or CA125 increase.

[0112] In these embodiments, a determined score higher than the cutoff score (considering the determined levels of CYFRA21-1 and / or CA125) indicates a high risk of NSCLC disease progression. In these embodiments, the score is configured to increase as the levels of CYFRA21-1 and / or CA125 increase.

[0113] Those skilled in the art will understand that a score considering the determined levels of CYFRA21-1 and / or CA125 can be mathematically constructed in reverse as well; for example, a score higher than the cutoff score indicates a lower risk, and a score higher than the cutoff score indicates a higher risk. This can be achieved by constructing the score such that the higher the level of CYFRA21-1 and / or CA125, the lower the score. For example, this inversion of the score can be achieved by using a negative factor multiplied by the biomarker level in the formula for calculating the score.

[0114] In an embodiment of the first aspect of the method, both the levels of CYFRA21-1 and CA125 in the sample are determined, and the determined score takes into account both the determined levels of CYFRA21-1 and CA125.

[0115] Therefore, the method according to the first embodiment may be a method for evaluating the risk of progression of non-small cell lung cancer (NSCLC) in a subject, and the said method is a) Determine the levels of CYFRA21-1 and CA125 in the sample obtained from the subject; The score is determined considering the level of CYFRA21-1 and the level of CA125 determined in aa)a); and b) Compare the score, taking into account the determined levels of CYFRA21-1 and CA125, with the cutoff score. Includes, The subjects from whom samples are obtained are those diagnosed with non-small cell lung cancer (NSCLC), under an ongoing NSCLC treatment regime, and previously classified (or diagnosed) as being in a stable stage.

[0116] The score, taking into account the levels of CYFRA21-1 and CA125 in the sample, can be determined by a weighted calculation using the determined levels of CYFRA21-1 and / or CA125.

[0117] In embodiments, the score considering the levels of CYFRA21-1 and CA125 may be a two-component score, and the cutoff score may also be two-component. "Two-component" means that the score includes two values, for example, a first value (also called the CYFRA21-1 value) which is the level of CYFRA21-1 or a value derived therefrom, and a second value (also called the CA125 value) which is the level of CA125 or a value derived therefrom. The "derived value" may be a value obtained, for example, by mathematical calculation. The "derived value" is preferably directly proportional to each level. The value of the two-component cutoff score may be obtained by univariate analysis using each biomarker, i.e., in the same manner as the procedure for defining the cutoff of a single biomarker described herein.

[0118] Comparing a two-component score to a two-component cutoff score means comparing the first value of the determined two-component score to the first value of the cutoff two-component score, and comparing the second value of the determined two-component score to the second value of the cutoff two-component score. In embodiments, if both the first and second values ​​of the determined two-component score are higher than the first and second values ​​of the two-component reference score, respectively, this indicates a high risk of NSCLC disease progression in the subject from which the sample was obtained. In embodiments, if one or both of the first and second values ​​of the determined two-component score are lower than the first and second values ​​of the two-component reference score, respectively, this indicates a low risk of NSCLC disease progression in the subject from which the sample was obtained.

[0119] In this embodiment, a two-component score in which the determined CYFRA21-1 value exceeds the CYFRA21-1 cutoff value of the cutoff two-component score, and the determined CA125 value exceeds the CA125 cutoff value of the cutoff two-component score, indicates a high risk of NSCLC disease progression in the subject from which the sample was obtained.

[0120] In this embodiment, if the two-component scores of the determined two-component score, specifically the CYFRA21-1 value and / or CA125 value, are lower than the CYFRA21-1 cutoff value and / or CA125 cutoff value of the cutoff two-component score, it indicates a low risk of NSCLC disease progression in the subject from which the sample was obtained.

[0121] Therefore, the method according to the first embodiment may be a method for evaluating the risk of progression of non-small cell lung cancer (NSCLC) in a subject, and the said method is a) Determine the levels of CYFRA21-1 and CA125 in the sample obtained from the subject; aa) Determine a two-component score including a first value and a second value, taking into account the level of CYFRA21-1 determined in a) in the first value and the level of CA125 determined in a) in the second value; b) Comparing a two-component score that takes into account the determined levels of CYFRA21-1 and CA125 with a two-component cutoff score, wherein the two-component cutoff score includes the CYFRA21-1 cutoff value and the CA125 cutoff value, the first value of the determined two-component score being compared with the CYFRA21-1 cutoff value, and the second value of the determined two-component score being compared with the CA125 cutoff value; Based on the comparisons in c) and b), assess the risk of progression of non-small cell lung cancer (NSCLC) in the subjects. Includes, The subjects from whom samples are obtained are those diagnosed with non-small cell lung cancer (NSCLC), under an ongoing NSCLC treatment regime, and previously classified (or diagnosed) as being in a stable stage.

[0122] In embodiments of the first aspect of the method, the risk of NSCLC disease progression is assessed in comparison to the mean or median NSCLC disease progression risk of a reference population, such as the reference population on which each cutoff or cutoff score is based. The reference population may preferably be a group of subjects who have non-small cell lung cancer (NSCLC) and are classified / diagnosed as stable based on imaging (e.g., using CT). As will be shown in the accompanying examples, detecting CYFRA21-1 levels and / or CA125 levels or scores that take these levels into account results in further risk stratification within these subjects, making it possible to separate this patient population into patients with a lower disease progression risk comparable to subjects categorized as having a partial response (PR) and patients with a higher disease progression risk comparable to subjects with progressive disease (PD).

[0123] In one embodiment, a high disease progression risk may be comparable to the mean disease progression risk of a reference population consisting of subjects categorized as having progressive NSCLC disease by imaging (e.g., CT-based imaging).

[0124] In one embodiment, a low disease progression risk may be a disease progression risk comparable to the mean disease progression risk of a reference population consisting of subjects categorized as having a partial response to NSCLC treatment by imaging (e.g., CT-based imaging).

[0125] In embodiments, the high disease progression risk may be at least 1.5 times, preferably at least 2 times, higher than the low disease progression risk. In other words, the ratio between the high progression risk and the low progression risk may be at least 1.5, preferably at least 2. In embodiments, the ratio may be a hazard ratio.

[0126] In embodiments, the risk of progression of NSCLC disease includes the risk of tumor growth (preferably assessed by RECIST criteria), the risk of new lesion formation or metastasis, or / or the risk of death from NSCLC.

[0127] The method according to the first embodiment may further include determining the protein level of CEA in the sample. In these embodiments, the comparison step includes comparing a score that takes into account the determined levels of CYFRA21-1 and / or the determined levels of CA125 and CEA with a cutoff score.

[0128] Accordingly, this specification provides an in vitro method for assessing the risk of progression of non-small cell lung cancer (NSCLC) in subjects who have NSCLC and are under an ongoing NSCLC treatment regime, and the method is a) Determine the levels of CYFRA21-1, CA125, and CEA in the sample obtained from the subject; aa) Determine the score considering the levels of CYFRA21-1, CA125, and CEA in the sample; and b) Compare the score determined in aa) with the cutoff score. Includes, The subjects have been previously classified as being in a stable state (for example, by imaging such as CT).

[0129] In some embodiments, the method according to the first aspect may further include obtaining information on whether the subject has non-small cell lung cancer of the subtype squamous cell carcinoma (SCC-NSCLC) or the subtype adenocarcinoma (ADC-NSCLC). Preferably, the information obtained is based on histological data.

[0130] As shown in the attached examples, when using scores that take into account at least CYFRA21-1 and CA125 levels (and optionally CEA), further inclusion of information regarding the histological subtype of NSCLC (adenocarcinoma or SCC) by, for example, including an interaction term between the determined biomarker and histological type in the formula for calculating the score, further improved performance in assessing the disease progression risk in SD patients.

[0131] In an embodiment, the method of the first embodiment includes obtaining information on the histological subtype of NSCLC (adenocarcinoma or SCC), and the comparison step includes comparing a score that takes into account the level of the determined biomarkers (i.e., CYFRA21-1 and / or CA125, and optionally CEA) and the histological NSCLC subtype. In an embodiment, the score takes into account the histological subtype by one or more interaction terms between the histological NSCLC subtype and one or more of the determined biomarkers.

[0132] Therefore, in one embodiment of the first aspect, an in vitro method is provided for assessing the risk of progression of non-small cell lung cancer (NSCLC) in subjects who have NSCLC and are under an ongoing NSCLC treatment regime, the method being a) Determine the levels of CYFRA21-1 and CA125 in the sample obtained from the subject; aa) Obtain information on the histological NSCLC subtype to which the subject is affected. aaa)i) levels of CYFRA21-1 and CA125 in the sample; and ii) determining the score considering the histological NSCLC subtype affected by the subject (e.g., in the form of an interaction term between biomarker levels and histological subtype); and b) Compare the score determined in aaa) with the cutoff score. Includes, The subjects have been previously classified as being in a stable state (for example, by imaging such as CT).

[0133] In another embodiment of the first aspect, an in vitro method is provided for assessing the risk of progression of non-small cell lung cancer (NSCLC) in subjects who have NSCLC and are under an ongoing NSCLC treatment regime, the method being: a) Determine the levels of CYFRA21-1, CA125, and CEA in the sample obtained from the subject; aa) Obtain information on the histological NSCLC subtype to which the subject is affected. aaa)i) levels of CYFRA21-1, CA125, and CEA in the sample; and ii) determining the score considering the histological NSCLC subtype affected by the subject (e.g., in the form of an interaction term between biomarker levels and histological subtype); and b) Compare the score determined in aaa) with the cutoff score. Includes, The subjects have been previously classified as being in a stable state (for example, by imaging such as CT).

[0134] In embodiments of the first aspect of the method, the method may further include providing information to the subject and / or the physician regarding whether the subject is at high or low risk of disease progression. This information may then be used to assist in further treatment decisions, in that it provides additional valuable input for treatment responses beyond mere imaging results.

[0135] In a second aspect, the present invention relates to an in vitro method for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified in a subject diagnosed with non-small cell lung cancer (NSCLC), wherein the method is: a) Determine the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject; and b) (i) Compare the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, (ii) Compare the determined level of CA125 with the CA125 cutoff level, or (iii) Compare the score considering the determined level of CYFRA21-1 and / or the determined level of CA125 with the cutoff score. Includes, The subjects are those classified or diagnosed as being in a stable state (for example, by tumor imaging based on CT).

[0136] Therefore, the subjects from whom samples were obtained were diagnosed with non-small cell lung cancer (NSCLC) and were under an ongoing NSCLC treatment regime. In other words, the subjects from whom samples were obtained were diagnosed with NSCLC before the method according to the second embodiment was performed and were under an NSCLC treatment regime at the time the samples were obtained.

[0137] Furthermore, the sample obtained may be classified or diagnosed as being in a stable state by means other than those in the steps of this method. Preferably, the classification of the stable state is performed by imaging methods such as CT.

[0138] In this embodiment, the object from which the sample is obtained is diagnosed as being in a stable state before or at the time the sample is obtained.

[0139] In the embodiment, the sample is obtained within a specific time frame around the date of a health checkup (e.g., imaging such as CT) that leads to a diagnosis of a stable state. In the embodiment, the specific time frame may be from 35 days before to 35 days after the health checkup. In the preferred embodiment, the specific time frame may be from 30 days before to 30 days after the health checkup. In the even more preferred embodiment, the specific time frame may be from 10 days before to 29 days after the health checkup.

[0140] In one embodiment, a subject is classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained at least 10 days, preferably at least 20 days, more preferably at least 30 days, and most preferably at least 35 days after the start date of the ongoing treatment regime. In another embodiment, a subject is classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained up to 150 days, preferably up to 120 days, and most preferably up to 108 days, after the start date of the ongoing treatment regime. Therefore, a subject may be classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained 10 to 150 days after the start date of the ongoing treatment regime, preferably 20 to 120 days, and most preferably 35 to 108 days after the start date of the ongoing treatment regime.

[0141] In the embodiment, the sample is obtained from the subject at least 10 days, preferably at least 20 days, and most preferably at least 25 days, after the start date of the ongoing treatment regime. In the embodiment, the sample is obtained from the subject up to 150 days, preferably up to 120 days, and most preferably up to 108 days, after the start date of the ongoing treatment regime. Therefore, in the embodiment, the sample is obtained from the subject 10 to 150 days, preferably 20 to 120 days, and most preferably 25 to 108 days, after the start date of the ongoing treatment regime.

[0142] In this embodiment, subjects are classified as having a stable state based on data obtained 35 to 108 days after the start of an ongoing treatment regime (e.g., tumor imaging data), and the samples are obtained from the subjects 25 to 108 days after the start of an ongoing treatment regime.

[0143] The "start" or "start date" of an ongoing treatment regime is the date on which the first treatment of that regime was administered to the subject. For example, it is the date on which the drug or drug composition was first administered to the subject.

[0144] In embodiments of the method according to the second aspect, the level of CYFRA21-1 may be determined in a), and the comparison in b) may include or consist of comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, where a determined level of CYFRA21-1 below the CYFRA21-1 cutoff level indicates that the ongoing NSCLC treatment regime should be maintained.

[0145] In an embodiment of the method according to the second aspect, the level of CYFRA21-1 is determined in a), and the comparison in b) includes or consists of comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, where a determined level of CYFRA21-1 higher than the CYFRA21-1 cutoff level indicates that the ongoing NSCLC treatment regime should be modified.

[0146] Accordingly, in a second embodiment of the present invention, a method is provided for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified for a subject diagnosed with non-small cell lung cancer (NSCLC), the method being: a) Determine the level of CYFRA21-1 in the sample obtained from the subject; and b) Compare the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level. A determined level of CYFRA21-1 below the CYFRA21-1 cutoff level indicates that the ongoing NSCLC treatment regime should be maintained, and / or a determined level of CYFRA21-1 above the CYFRA21-1 cutoff level indicates that the ongoing NSCLC treatment regime should be modified, and the subject has been previously classified (or diagnosed) as having a stable condition (e.g., by CT-based imaging). In this regard, it is even more preferable that the subject be diagnosed with NSCLC of the adenocarcinoma subtype (ADC-NSCLC).

[0147] In the attached examples, the performance of the biomarker CYFRA21-1 in evaluating the risk of disease progression in non-small cell lung cancer (NSCLC) and the resulting treatment response was demonstrated to be particularly good for subjects with NSCLC of the adenocarcinoma subtype. Therefore, in embodiments for determining the biomarker CYFRA21-1, the subjects from whom samples are obtained may, in particular, be subjects previously diagnosed with NSCLC of the adenocarcinoma subtype.

[0148] In embodiments of the method according to the second aspect, the level of CA125 may be determined in a), and the comparison in b) may include or consist of comparing the determined level of CA125 with the CA125 cutoff level, where a determined level of CA125 below the CA125 cutoff level indicates that the ongoing NSCLC treatment regime should be maintained.

[0149] In an embodiment of the method according to the second aspect, the level of CA125 is determined in a), and the comparison in b) includes or consists of comparing the determined level of CA125 with the CA125 cutoff level, where a determined level of CA125 higher than the CA125 cutoff level indicates that the ongoing NSCLC treatment regime should be modified.

[0150] Accordingly, in a second embodiment of the present invention, a method is provided for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified for a subject diagnosed with non-small cell lung cancer (NSCLC), the method being: a) Determine the level of CA125 in the sample obtained from the subject; and b) Compare the determined CA125 level with the CA125 cutoff level. A determined CA125 level below the CA125 cutoff level indicates that the ongoing NSCLC treatment regime should be maintained, and / or a determined CA125 level above the CA125 cutoff level indicates that the ongoing NSCLC treatment regime should be modified. The subjects have been previously classified (or diagnosed) as having a stable stage. In this regard, it is even more preferable that the subjects be diagnosed with the subtype squamous cell carcinoma NSCLC (SCC-NSCLC).

[0151] In the attached examples, the performance of the biomarker CA125 in assessing the risk of progression of non-small cell lung cancer (NSCLC) was demonstrated to be particularly good for subjects with the squamous cell carcinoma subtype. Therefore, in embodiments for determining the biomarker CA125, subjects from whom samples may particularly originate may be those diagnosed with NSCLC of the squamous cell carcinoma subtype (SCC-NSCLC).

[0152] In a second embodiment of the present invention, the method may include determining a score taking into account the levels of CYFRA21-1 and / or CA125 determined in the sample. In these embodiments, the comparison in b) includes or consists of comparing the score taking into account the determined levels of CYFRA21-1 and / or CA125 with the cutoff score.

[0153] A score that takes into account the levels of CYFRA21-1 and / or CA125 in a sample can be determined by a weighted calculation using the determined levels of CYFRA21-1 and / or CA125. In some embodiments, the score may be based solely on CYFRA21-1 and / or CA125. In other embodiments, the score may take into account the presence or levels of one or more other biomarkers in the sample and one or more clinical parameters of the subject (e.g., histology, smoking status, disease stage, age, and / or sex). In certain embodiments, the score may take into account whether the histology is adenocarcinoma or SCC. In particular, histology may be considered by an interaction term with biomarker values. This allows for taking into account that the biomarkers CYFRA21-1 and CA125 exhibited slightly different performance depending on the histology.

[0154] Therefore, the method according to the second embodiment may be a method for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified for a subject diagnosed with non-small cell lung cancer (NSCLC), and the method is a) Determine the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject; aa)a) The score is determined considering the level of CYFRA21-1 and / or the level of CA125 determined in aa); and b) Compare the score, taking into account the determined level of CYFRA21-1 and / or the determined level of CA125, with the cutoff score. Includes, The subjects are those who have been previously classified (or diagnosed) as being in a stable stage.

[0155] In these embodiments, a determined score lower than the cutoff score (taking into account the determined levels of CYFRA21-1 and / or CA125) indicates that the ongoing NSCLC treatment regime should be maintained. In these embodiments, the score is configured to increase as the level of CYFRA21-1 and / or CA125 increases.

[0156] In these embodiments, a determined score higher than the cutoff score (taking into account the determined levels of CYFRA21-1 and / or CA125) indicates that the ongoing NSCLC treatment regime should be modified. In these embodiments, the score is configured to increase as the level of CYFRA21-1 and / or CA125 increases.

[0157] Those skilled in the art will understand that a score considering the determined levels of CYFRA21-1 and / or CA125 can be mathematically constructed in reverse as well; for example, a score higher than the cutoff score indicates a lower risk, and a score higher than the cutoff score indicates a higher risk. This can be achieved by constructing the score such that the higher the level of CYFRA21-1 and / or CA125, the lower the score. For example, this inversion of the score can be achieved by using a negative factor multiplied by the biomarker level in the formula for calculating the score.

[0158] In an embodiment of the second aspect of the method, both the levels of CYFRA21-1 and CA125 in the sample are determined, and the determined score takes into account both the determined levels of CYFRA21-1 and CA125.

[0159] Therefore, the method according to the second embodiment may be a method for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified for a subject diagnosed with non-small cell lung cancer (NSCLC), and the method is a) Determine the levels of CYFRA21-1 and CA125 in the sample obtained from the subject; The score is determined considering the level of CYFRA21-1 and the level of CA125 determined in aa)a); and b) Compare the score, taking into account the determined levels of CYFRA21-1 and CA125, with the cutoff score. Includes, The subjects are those who have been previously classified (or diagnosed) as being in a stable stage.

[0160] The score, taking into account the levels of CYFRA21-1 and CA125 in the sample, can be determined by a weighted calculation using the determined levels of CYFRA21-1 and / or CA125.

[0161] In embodiments, the score considering the levels of CYFRA21-1 and CA125 may be a two-component score, and the cutoff score may also be two-component. "Two-component" means that the score includes two values, for example, a first value (also called the CYFRA21-1 value) which is the level of CYFRA21-1 or a value derived therefrom, and a second value (also called the CA125 value) which is the level of CA125 or a value derived therefrom. The "derived value" may be a value obtained, for example, by mathematical calculation. The "derived value" is preferably directly proportional to each level. The value of the two-component cutoff score may be obtained by univariate analysis using each biomarker, i.e., in the same manner as the procedure for defining the cutoff of a single biomarker described herein.

[0162] Comparing a two-component score to a two-component cutoff score involves comparing a first value of the determined two-component score to a first value of the cutoff two-component score, and comparing a second value of the determined two-component score to a second value of the cutoff two-component score. In embodiments, if both the first and second values ​​of the determined two-component score are higher than the first and second values ​​of the two-component reference score, respectively, this indicates that the ongoing NSCLC treatment regime should be modified. In embodiments, if one or both of the first and second values ​​of the determined two-component score are lower than the first and second values ​​of the two-component reference score, respectively, this indicates that the ongoing NSCLC treatment regime should be maintained.

[0163] In this embodiment, a two-component score in which the determined CYFRA21-1 value exceeds the CYFRA21-1 cutoff value of the cutoff two-component score, and the determined CA125 value exceeds the CA125 cutoff value of the cutoff two-component score, indicates that the ongoing NSCLC treatment regime should be modified.

[0164] In this embodiment, if the two-component scores of the determined two-component score, specifically the CYFRA21-1 value and / or CA125 value, are lower than the CYFRA21-1 cutoff value and / or CA125 cutoff value of the cutoff two-component score, it indicates that the ongoing NSCLC treatment regime should be maintained.

[0165] Therefore, the method according to the second embodiment may be a method for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified for a subject diagnosed with non-small cell lung cancer (NSCLC), and the method is a) Determine the levels of CYFRA21-1 and CA125 in the sample obtained from the subject; aa) Determine a two-component score including a first value and a second value, taking into account the level of CYFRA21-1 determined in a) in the first value and the level of CA125 determined in a) in the second value; b) Comparing a two-component score that takes into account the determined levels of CYFRA21-1 and CA125 with a two-component cutoff score, wherein the two-component cutoff score includes the CYFRA21-1 cutoff value and the CA125 cutoff value, the first value of the determined two-component score being compared with the CYFRA21-1 cutoff value, and the second value of the determined two-component score being compared with the CA125 cutoff value; Based on the comparisons in c) and b), evaluate whether the ongoing NSCLC treatment regime should be maintained or modified. Includes, The subjects from whom samples are obtained are those diagnosed with non-small cell lung cancer (NSCLC), under an ongoing NSCLC treatment regime, and previously classified (or diagnosed) as being in a stable stage.

[0166] A method according to a second embodiment may further include determining the level of CEA in the sample. In these embodiments, the comparison step includes comparing a score that takes into account the determined level of CYFRA21-1 and / or the determined level of CA125 and the determined level of CEA with a cutoff score.

[0167] Accordingly, in one embodiment of a second aspect of the present invention, an in vitro method is provided for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified for a subject diagnosed with non-small cell lung cancer (NSCLC), the method being: a) Determine the levels of CYFRA21-1, CA125, and CEA in the sample obtained from the subject; aa) Determine the score considering the levels of CYFRA21-1, CA125, and CEA in the sample; and b) Compare the score determined in aa) with the cutoff score. Includes, The subjects have been previously classified as being in a stable state (for example, by imaging such as CT).

[0168] In the embodiments, the method according to the second aspect may further include obtaining information on whether the subject has non-small cell lung cancer of the subtype squamous cell carcinoma (SCC-NSCLC) or the subtype adenocarcinoma (ADC-NSCLC). Preferably, the information obtained is based on histological data.

[0169] As shown in the attached example using scores that take into account CYFRA21-1 and CA125 levels (and optionally CEA), further inclusion of information about the histological subtype of NSCLC (adenocarcinoma or SCC) into the score, for example by including an interaction term between the determined biomarker and histological type in the formula for calculating the score, further improved performance in assessing the disease progression risk in SD patients.

[0170] In an embodiment, the method of the second embodiment includes obtaining information on the histological subtype of NSCLC (adenocarcinoma or SCC), and the comparison step includes comparing a score that takes into account the level of the determined biomarkers (i.e., CYFRA21-1 and / or CA125, and optionally CEA) and the histological NSCLC subtype. In an embodiment, the score takes into account the histological subtype by one or more interaction terms between the histological NSCLC subtype and one or more of the determined biomarkers.

[0171] Therefore, in one embodiment of the second aspect, an in vitro method is provided for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified for a subject diagnosed with non-small cell lung cancer (NSCLC), the method being: a) Determine the levels of CYFRA21-1 and CA125 in the sample obtained from the subject; aa) Obtain information on the histological NSCLC subtype to which the subject is affected; aaa)i) levels of CYFRA21-1 and CA125 in the sample; and ii) determining the score considering the histological NSCLC subtype affected by the subject (e.g., in the form of an interaction term between biomarker levels and histological subtype); and b) Compare the score determined in aaa) with the cutoff score. Includes, The subjects have been previously classified as being in a stable state (for example, by imaging such as CT).

[0172] In another embodiment of the second aspect, an in vitro method is provided for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified for a subject diagnosed with non-small cell lung cancer (NSCLC), the method being: a) Determine the levels of CYFRA21-1, CA125, and CEA in the sample obtained from the subject; aa) Obtain information on the histological NSCLC subtype to which the subject is affected; aaa)i) levels of CYFRA21-1, CA125, and CEA in the sample; and ii) determining the score considering the histological NSCLC subtype affected by the subject (e.g., in the form of an interaction term between biomarker levels and histological subtype); and b) Compare the score determined in aaa) with the cutoff score. Includes, The subjects have been previously classified as being in a stable state (for example, by imaging such as CT).

[0173] In embodiments of the second aspect of the method, the method may further include providing the subject and / or physician with information obtained by the method, namely whether the treatment should be maintained or modified. This information can then be used to assist in further treatment decisions, by providing additional valuable input for treatment response beyond mere imaging results.

[0174] In an embodiment of the method according to the second aspect, “maintaining the treatment regime” means that the ongoing treatment regime is continued, i.e., a further treatment cycle is carried out.

[0175] In embodiments of the method according to the second aspect, “modifying the treatment regime” includes, but is not limited to, (i) adjusting the dose of the treatment (e.g., increasing the dose of a drug), and (ii) changing the type of treatment to a different type of treatment (e.g., if the ongoing treatment is chemotherapy, modifying the treatment may mean changing the treatment to immunotherapy). In other words, “modifying the ongoing treatment regime” may be expressed as “adjusting the ongoing treatment regime” or “changing the ongoing treatment regime.”

[0176] In a third aspect, the present invention relates to an in vitro method for evaluating whether a subject with non-small cell lung cancer (NSCLC) responds to an ongoing NSCLC treatment regime, wherein the method is: a) Determine the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject; and b) (i) comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, (ii) comparing the determined level of CA125 with the CA125 cutoff level, or (iii) comparing a score that takes into account the determined level of CYFRA21-1 and / or the determined level of CA125 with the cutoff score. Includes.

[0177] In the context of this method, the subjects are those that have been classified or diagnosed as being in a stable state.

[0178] Therefore, the subjects from whom samples were obtained were diagnosed with non-small cell lung cancer (NSCLC) and were under an ongoing NSCLC treatment regime. In other words, the subjects from whom samples were obtained were diagnosed with NSCLC before the third embodiment of the method was performed and were under an NSCLC treatment regime at the time the samples were obtained.

[0179] Furthermore, the sample obtained may be classified or diagnosed as being in a stable state by means other than those in the steps of this method. Preferably, the classification of the stable state is performed by imaging methods such as CT.

[0180] In this embodiment, the object from which the sample is obtained is diagnosed as being in a stable state before or at the time the sample is obtained.

[0181] In the embodiment, the sample is obtained within a specific time frame around the date of a health checkup (e.g., imaging such as CT) that leads to a diagnosis of a stable state. In the embodiment, the specific time frame may be from 35 days before to 35 days after the health checkup. In the preferred embodiment, the specific time frame may be from 30 days before to 30 days after the health checkup. In the even more preferred embodiment, the specific time frame may be from 10 days before to 29 days after the health checkup.

[0182] In one embodiment, a subject is classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained at least 10 days, preferably at least 20 days, more preferably at least 30 days, and most preferably at least 35 days after the start date of the ongoing treatment regime. In another embodiment, a subject is classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained up to 150 days, preferably up to 120 days, and most preferably up to 108 days, after the start date of the ongoing treatment regime. Therefore, a subject may be classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained 10 to 150 days after the start date of the ongoing treatment regime, preferably 20 to 120 days, and most preferably 35 to 108 days after the start date of the ongoing treatment regime.

[0183] In the embodiment, the sample is obtained from the subject at least 10 days, preferably at least 20 days, and most preferably at least 25 days, after the start date of the ongoing treatment regime. In the embodiment, the sample is obtained from the subject up to 150 days, preferably up to 120 days, and most preferably up to 108 days, after the start date of the ongoing treatment regime. Therefore, in the embodiment, the sample is obtained from the subject 10 to 150 days, preferably 20 to 120 days, and most preferably 25 to 108 days, after the start date of the ongoing treatment regime.

[0184] In this embodiment, subjects are classified as having a stable state based on data obtained 35 to 108 days after the start of an ongoing treatment regime (e.g., tumor imaging data), and the samples are obtained from the subjects 25 to 108 days after the start of an ongoing treatment regime.

[0185] The "start" or "start date" of an ongoing treatment regime is the date on which the first treatment of that regime was administered to the subject. For example, it is the date on which the drug or drug composition was first administered to the subject.

[0186] In a third embodiment of the present invention, determining / indicating whether a subject responds to an ongoing treatment may be determining / indicating whether the subject responded to an ongoing treatment regime at the time the sample was obtained.

[0187] In a third embodiment of the present invention, determining / indicating whether a subject responds to an ongoing treatment may be determining / indicating whether a subject responds to an ongoing treatment regime (i.e., responds after the sample is obtained).

[0188] In an embodiment of the third aspect of the method, the level of CYFRA21-1 may be determined in a), and the comparison in b) may include or consist of comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, where a determined level of CYFRA21-1 below the CYFRA21-1 cutoff level indicates that the subject is responding to the ongoing NSCLC treatment regime.

[0189] In an embodiment of the third aspect of the method, the level of CYFRA21-1 is determined in a), and the comparison in b) includes or consists of comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, where a determined level of CYFRA21-1 higher than the CYFRA21-1 cutoff level indicates that the subject is not responding to the ongoing NSCLC treatment regime.

[0190] Accordingly, in a third embodiment of the present invention, the present invention provides an in vitro method for evaluating whether a subject with non-small cell lung cancer (NSCLC) responds to an ongoing NSCLC treatment regime, the method being: a) Determine the level of CYFRA21-1 in the sample obtained from the subject; and b) Compare the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level. Includes, A determined level of CYFRA21-1 below the CYFRA21-1 cutoff level indicates that the subject is responding to the ongoing NSCLC treatment regime, and / or a determined level of CYFRA21-1 above the CYFRA21-1 cutoff level indicates that the subject is not responding to the ongoing NSCLC treatment regime. In this regard, it is even more preferable that the subject be diagnosed with adenocarcinoma of NSCLC (ADC-NSCLC). The subjects have been previously classified (or diagnosed) as having a stable stage (e.g., by CT-based imaging). In this regard, it is even more preferable that the subjects be diagnosed with NSCLC of the squamous cell carcinoma subtype (ADC-NSCLC).

[0191] In the attached embodiments, the performance of the biomarker CYFRA21-1 in detecting the risk of disease progression in non-small cell lung cancer (NSCLC) and consequently in evaluating treatment response and prognosis was demonstrated to be particularly good for subjects with NSCLC of the adenocarcinoma subtype. Therefore, in embodiments for determining the biomarker CYFRA21-1, the subjects from whom samples are obtained may, in particular, be subjects previously diagnosed with NSCLC of the adenocarcinoma subtype.

[0192] In embodiments of the third aspect of the method, the level of CA125 may be determined in a), and the comparison in b) may include or consist of comparing the determined level of CA125 with a CA125 cutoff level, where a determined level of CA125 below the CA125 cutoff level indicates that the control is responding to the ongoing NSCLC treatment regime.

[0193] In an embodiment of the third aspect of the method, the level of CA125 is determined in a), and the comparison in b) includes or consists of comparing the determined level of CA125 with a CA125 cutoff level, where a determined level of CA125 higher than the CA125 cutoff level indicates that the subject is not responding to the ongoing NSCLC treatment regime.

[0194] Accordingly, in a third embodiment of the present invention, the present invention provides an in vitro method for evaluating whether a subject with non-small cell lung cancer (NSCLC) responds to an ongoing NSCLC treatment regime, the method being: a) Determine the level of CA125 in the sample obtained from the subject; and b) Compare the determined CA125 level with the CA125 cutoff level. Includes, A determined CA125 level below the CA125 cutoff level indicates that the ongoing NSCLC treatment regime should be maintained, and / or a determined CA125 level above the CA125 cutoff level indicates that the ongoing NSCLC treatment regime should be modified. The subjects have been previously classified (or diagnosed) as having a stable stage. In this regard, it is even more preferable that the subjects be diagnosed with the subtype squamous cell carcinoma NSCLC (SCC-NSCLC).

[0195] In the attached examples, the performance of the biomarker CA125 in assessing the risk of progression of non-small cell lung cancer (NSCLC) was demonstrated to be particularly good for subjects with the squamous cell carcinoma subtype. Therefore, in embodiments for determining the biomarker CA125, subjects from whom samples may particularly originate may be those diagnosed with NSCLC of the squamous cell carcinoma subtype (SCC-NSCLC).

[0196] In a third embodiment of the present invention, the method may include determining a score taking into account the levels of CYFRA21-1 and / or CA125 determined in the sample. In these embodiments, the comparison in b) includes or consists of comparing the score taking into account the determined levels of CYFRA21-1 and / or CA125 with the cutoff score.

[0197] A score that takes into account the levels of CYFRA21-1 and / or CA125 in a sample can be determined by a weighted calculation using the determined levels of CYFRA21-1 and / or CA125. In some embodiments, the score may be based solely on CYFRA21-1 and / or CA125. In other embodiments, the score may take into account the presence or levels of one or more other biomarkers in the sample and one or more clinical parameters of the subject (e.g., histology, smoking status, disease stage, age, and / or sex). In certain embodiments, the score may take into account whether the histology is adenocarcinoma or SCC. In particular, histology may be considered by an interaction term with biomarker values. This allows for taking into account that the biomarkers CYFRA21-1 and CA125 exhibited slightly different performance depending on the histology.

[0198] Therefore, the method according to the third aspect may be a method for evaluating whether a subject with non-small cell lung cancer (NSCLC) responds to an ongoing NSCLC treatment regime, and the method is a) Determine the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject; aa)a) The score is determined considering the level of CYFRA21-1 and / or the level of CA125 determined in aa); and b) Compare the score, taking into account the determined level of CYFRA21-1 and / or the determined level of CA125, with the cutoff score. Includes, The subjects are those who have been previously classified (or diagnosed) as being in a stable stage.

[0199] In these embodiments, a determined score lower than the cutoff score (considering the determined levels of CYFRA21-1 and / or CA125) indicates that the control is responding to the ongoing NSCLC treatment regime. In these embodiments, the score is configured to increase as the level of CYFRA21-1 and / or CA125 increases.

[0200] In these embodiments, a determined score higher than the cutoff score (considering the determined levels of CYFRA21-1 and / or CA125) indicates that the control is not responding to the ongoing NSCLC treatment regime. In these embodiments, the score is configured to increase as the level of CYFRA21-1 and / or CA125 increases.

[0201] Those skilled in the art will understand that a score considering the determined levels of CYFRA21-1 and / or CA125 can be mathematically constructed in reverse as well; for example, a score higher than the cutoff score indicates a lower risk, and a score higher than the cutoff score indicates a higher risk. This can be achieved by constructing the score such that the higher the level of CYFRA21-1 and / or CA125, the lower the score. For example, this inversion of the score can be achieved by using a negative factor multiplied by the biomarker level in the formula for calculating the score.

[0202] In an embodiment of the third aspect of the method, both the levels of CYFRA21-1 and CA125 in the sample are determined, and the determined score takes into account both the determined levels of CYFRA21-1 and CA125.

[0203] Therefore, the method according to the third aspect may be a method for evaluating whether a subject with non-small cell lung cancer (NSCLC) responds to an ongoing NSCLC treatment regime, and the method is a) Determine the levels of CYFRA21-1 and CA125 in the sample obtained from the subject; The score is determined considering the level of CYFRA21-1 and the level of CA125 determined in aa)a); and b) Compare the score, taking into account the determined levels of CYFRA21-1 and CA125, with the cutoff score. Includes, The subjects are those who have been previously classified (or diagnosed) as being in a stable stage.

[0204] The score, taking into account the levels of CYFRA21-1 and CA125 in the sample, can be determined by a weighted calculation using the determined levels of CYFRA21-1 and / or CA125.

[0205] In embodiments, the score considering the levels of CYFRA21-1 and CA125 may be a two-component score, and the cutoff score may also be two-component. "Two-component" means that the score includes two values, for example, a first value (also called the CYFRA21-1 value) which is the level of CYFRA21-1 or a value derived therefrom, and a second value (also called the CA125 value) which is the level of CA125 or a value derived therefrom. The "derived value" may be a value obtained, for example, by mathematical calculation. The "derived value" is preferably directly proportional to each level. The value of the two-component cutoff score may be obtained by univariate analysis using each biomarker, i.e., in the same manner as the procedure for defining the cutoff of a single biomarker described herein.

[0206] Comparing a two-component score to a two-component cutoff score involves comparing a first value of the determined two-component score to a first value of the cutoff two-component score, and comparing a second value of the determined two-component score to a second value of the cutoff two-component score. In embodiments, if both the first and second values ​​of the determined two-component score are higher than the first and second values ​​of the two-component reference score, respectively, this indicates that the subject is not responding to or will not respond to the ongoing NSCLC treatment regime. In embodiments, if one or both of the first and second values ​​of the determined two-component score are lower than the first and second values ​​of the two-component reference score, respectively, this indicates that the subject is responding to or will respond to the ongoing NSCLC treatment regime.

[0207] In this embodiment, a two-component score in which the determined two-component score's CYFRA21-1 value exceeds the CYFRA21-1 cutoff value of the cutoff two-component score, and the determined two-component score's CA125 value exceeds the CA125 cutoff value of the cutoff two-component score, indicates that the subject is not responding to or is unlikely to respond to the ongoing NSCLC treatment regime.

[0208] In this embodiment, if the two-component scores of the determined two-component score, specifically the CYFRA21-1 value and / or CA125 value, are lower than the CYFRA21-1 cutoff value and / or CA125 cutoff value of the cutoff two-component score, it indicates that the subject is responding to or will respond to the ongoing NSCLC treatment regime.

[0209] Therefore, the method according to the second embodiment may be a method for evaluating whether a subject with non-small cell lung cancer (NSCLC) is responding to or will respond to an ongoing NSCLC treatment regime: a) Determine the levels of CYFRA21-1 and CA125 in the sample obtained from the subject; aa) Determine a two-component score including a first value and a second value, taking into account the level of CYFRA21-1 determined in a) in the first value and the level of CA125 determined in a) in the second value; b) Comparing a two-component score that takes into account the determined levels of CYFRA21-1 and CA125 with a two-component cutoff score, wherein the two-component cutoff score includes the CYFRA21-1 cutoff value and the CA125 cutoff value, the first value of the determined two-component score being compared with the CYFRA21-1 cutoff value, and the second value of the determined two-component score being compared with the CA125 cutoff value; Based on the comparison in c) and b), assess whether the subject is responding to or will respond to the ongoing NSCLC treatment regime. Includes, The organisms from which the samples are obtained have been previously classified (or diagnosed) as having a stable state stage.

[0210] A third embodiment of the method may further include determining the level of CEA in the sample. In these embodiments, the comparison step includes comparing a score that takes into account the determined level of CYFRA21-1 and / or the determined level of CA125 and the determined level of CEA with a cutoff score.

[0211] Accordingly, in one embodiment of a third aspect of the present invention, the present invention provides an in vitro method for evaluating whether a subject with non-small cell lung cancer (NSCLC) responds to an ongoing NSCLC treatment regime, the method being: a) Determine the levels of CYFRA21-1, CA125, and CEA in the sample obtained from the subject; aa) Determine the score considering the levels of CYFRA21-1, CA125, and CEA in the sample; and b) Compare the score determined in aa) with the cutoff score. Includes, The subjects have been previously classified as being in a stable state (for example, by imaging such as CT).

[0212] In some embodiments, the method according to the third aspect may further include obtaining information on whether the subject has non-small cell lung cancer of the subtype squamous cell carcinoma (SCC-NSCLC) or the subtype adenocarcinoma (ADC-NSCLC). Preferably, the information obtained is based on histological data.

[0213] As shown in the attached examples using scores that take CYFRA21-1 and CA125 levels (and optionally CEA) into account, further inclusion of information regarding the histological subtype of NSCLC (adenocarcinoma or SCC) into the score, for example by including an interaction term between the determined biomarker and histological type in the formula for calculating the score, further improved performance in assessing the risk of disease progression in SD patients. As a result, performance in assessing treatment response also improved.

[0214] In an embodiment, the method of the third embodiment includes obtaining information on the histological subtype of NSCLC (adenocarcinoma or SCC), and the comparison step includes comparing a score that takes into account the level of the determined biomarkers (i.e., CYFRA21-1 and / or CA125, and optionally CEA) and the histological NSCLC subtype. In an embodiment, the score takes into account the histological subtype by one or more interaction terms between the histological NSCLC subtype and one or more of the determined biomarkers.

[0215] Accordingly, in one embodiment of the third aspect, the present invention provides an in vitro method for evaluating whether a subject with non-small cell lung cancer (NSCLC) responds to an ongoing NSCLC treatment regime, the method being: a) Determine the levels of CYFRA21-1 and CA125 in the sample obtained from the subject; aa) Obtain information on the histological NSCLC subtype to which the subject is affected; aaa)i) levels of CYFRA21-1 and CA125 in the sample; and ii) determining the score considering the histological NSCLC subtype affected by the subject (e.g., in the form of an interaction term between biomarker levels and histological subtype); and b) Compare the score determined in aaa) with the cutoff score. Includes, The subjects have been previously classified as being in a stable state (for example, by imaging such as CT).

[0216] In another embodiment of a third aspect, the present invention provides an in vitro method for evaluating whether a subject with non-small cell lung cancer (NSCLC) responds to an ongoing NSCLC treatment regime, the method being: a) Determine the levels of CYFRA21-1, CA125, and CEA in the sample obtained from the subject; aa) Obtain information on the histological NSCLC subtype to which the subject is affected; aaa)i) levels of CYFRA21-1, CA125, and CEA in the sample; and ii) determining the score considering the histological NSCLC subtype affected by the subject (e.g., in the form of an interaction term between biomarker levels and histological subtype); and b) Compare the score determined in aaa) with the cutoff score. Includes, The subjects have been previously classified as being in a stable state (for example, by imaging such as CT).

[0217] In embodiments of the third aspect of the method, the method may further include providing the subject and / or physician with information obtained by the method, namely whether the subject is responding to the ongoing treatment regime. This information may be used to assist in further treatment decisions, for example, to maintain or modify the ongoing treatment regime.

[0218] In a fourth aspect, the present invention provides a method for treating a subject suffering from NSCLC. The treatment method preferably comprises one step of a method according to a first, second, or third aspect of the present invention. All embodiments of the methods according to the first, second, and third aspects disclosed elsewhere herein are applied to the method according to the fourth aspect with necessary modifications. The treatment method according to the fourth aspect of the present invention further comprises continuing to treat the patient in an ongoing treatment regime or modifying the treatment regime based on the results of an evaluation according to a method according to a first, second, or third aspect.

[0219] Accordingly, a method according to a fourth aspect of the present invention may include (i) evaluating whether a subject with non-small cell lung cancer (NSCLC) responds to an ongoing treatment regime by the method described in any one embodiment of the third aspect of the present invention; or (i)' evaluating whether the ongoing treatment regime of the subject should be maintained or modified, as described in any one embodiment of the second aspect of the present invention; or (ii) continuing treatment for the patient with the ongoing treatment regime, or modifying the treatment regime based on the results of the evaluation in (i) or (i)'.

[0220] In embodiments of the fourth aspect of the method, “continuing the treatment regime” means that the ongoing treatment regime is maintained, i.e., that further treatment cycles are carried out as planned or recommended by the treatment guidelines.

[0221] The ongoing treatment regime can, in principle, be any treatment regime for NSCLC. Guidelines for NSCLC are publicly known in the art (S3-Leitlinie Patientopathies, Diagnostics, Therapies and Aftercares for the Language Disease; and NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines® for NSCLC in the latest version as of the filing date)). In embodiments, the “ongoing treatment regime” may be selected from the group consisting of chemotherapy, targeted therapy, immunotherapy, radiotherapy, and any combination thereof.

[0222] The attached examples demonstrate that responses to various different treatment regimes can be successfully evaluated using the methods and uses described herein.

[0223] In certain embodiments, the “ongoing treatment regime” may be a chemotherapy treatment regime.

[0224] Specific but non-limiting, but exemplary ongoing treatment regimes are disclosed in the attached examples, specifically in Tables 1a, 1b, and 1c.

[0225] In embodiments, chemotherapy can be selected from an unrestricted group of platinum-based chemotherapy and platinum-free chemotherapy.

[0226] Platinum-based chemotherapy is a treatment comprising the administration of a pharmaceutical composition containing a platinum-based chemotherapeutic agent. The platinum-based chemotherapeutic agent may be selected from cisplatin, carboplatin, oxaplatin, nedaplatin, triplatin nitrate, phenatriplatin, picoplatin, satraplatin, or any combination thereof. In certain embodiments, the platinum-based chemotherapeutic agent may be selected from carboplatin and cisplatin.

[0227] Platinum-free chemotherapy is a treatment that administers only chemotherapeutic agents other than platinum-based chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents other than platinum-based chemotherapeutic drugs are etoposide, docetaxel, gemcitabine, paclitaxel, pemetrexed, vinorelbine, and vincristine.

[0228] Targeted therapy is a type of cancer treatment that specifically targets proteins that control the ways of cancer cell growth, division, and / or expansion. Therefore, these treatments typically target cancer cells preferentially.

[0229] An exemplary but non-limiting targeted therapy is treatment using tyrosine kinase inhibitors (also referred to herein as TKIs). Exemplary but non-limiting examples of tyrosine kinase inhibitors are afatinib, alectinib, crizotinib, erlotinib, and osimertinib.

[0230] Immunotherapy is treatment with antibodies (monospecific or multispecific), antibody fragments, or antibody-like molecules. ​​​​​​​​​​Radiation therapy, also often abbreviated as RT, RTx, or XRT, is a treatment that uses ionizing radiation. Radiation therapy may be given together with chemotherapy and may be used for curative purposes in those with NSCLC who are not eligible for surgery. This form of high-intensity radiation therapy is called radical radiation therapy. An improvement in this technique is continuous hyperfractionated accelerated radiotherapy (CHART), in which high doses of radiation therapy are given over a short period. Postoperative chest radiation therapy generally should not be used for curative purposes after surgery for NSCLC. When the growth of cancer blocks a short segment of the bronchus, brachytherapy (local radiation therapy) can be directly applied inside the airway to open the passage. Compared with external beam radiation therapy, brachytherapy allows for a reduction in treatment time and radiation exposure to medical staff. However, the evidence for brachytherapy is less than that for external beam radiation therapy.

[0234] In an embodiment of the method according to the fourth aspect, "modifying the treatment regimen" includes, but is not limited to, the following: (i) adjusting the dose of the treatment (e.g., increasing the dose of a drug), (ii) changing the type of treatment to another type of treatment (e.g., if the ongoing treatment is chemotherapy, modifying the treatment could be changing the treatment to immunotherapy). In particular, modifying the ongoing treatment regimen can be changing from a specific ongoing treatment regimen to any one of the above examples of an "ongoing treatment regimen". In other words, the term "modifying the ongoing treatment regimen" can be "adjusting the ongoing treatment regimen" or "changing the ongoing treatment regimen".

[0235] In a fifth aspect, the invention (i) the risk of NSCLC disease progression under an ongoing NSCLC treatment regimen for a subject diagnosed with NSCLC; (ii) whether a subject diagnosed with NSCLC responds to the ongoing NSCLC treatment regimen; and / or (iii) for a subject diagnosed with NSCLC, whether the ongoing NSCLC treatment regimen should be maintained or modified Regarding the use of CYFRA21-1 and / or CA125 as biomarkers(s) for evaluating [the condition].

[0236] The subjects to be evaluated are those classified or diagnosed as having stable NSCLC under an ongoing treatment regime.

[0237] The fifth aspect of use may include any of the embodiments described in the first, second, and third aspects of the present invention, with necessary modifications.

[0238] For example, use in the fifth aspect is, (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Whether the ongoing NSCLC treatment regime should be maintained or modified for subjects diagnosed with NSCLC. This may further include using CEA as a biomarker to evaluate [the condition].

[0239] In the embodiments, use may further include taking into account, preferably based on histological data, information on whether the subject has squamous cell carcinoma (SCC-NSCLC) or adenocarcinoma (ADC-NSCLC) of the non-small cell lung cancer subtype.

[0240] In a sixth aspect, the present invention is (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Whether the ongoing NSCLC treatment regime should be maintained or modified for subjects diagnosed with NSCLC. A computer implementation method for evaluating, (i), (ii), and (iii) pertain to computer implementation methods that are classified as having a stable state.

[0241] Simply put, a computer implementation method may be a computer implementation variation of any of the methods and uses described above in the present invention, except that the computer implementation method does not necessarily involve determining the levels of CYFRA21-1, CA125, and / or CEA, and any other information or data that may be taken into consideration in calculating the score.

[0242] The computer implementation method is, a) Receiving data including the levels of CYFRA21-1 and / or CA125 in the sample obtained from the subject, and optionally the level of CEA, and optionally information regarding the NSCLC subtype of the subject; and b) The data may be processed and any of the comparison steps defined in the embodiments of the above-described aspects may be performed.

[0243] The embodiments described above, and all embodiments described below in this specification, are applicable with necessary modifications.

[0244] Those skilled in the art know of persons who establish and configure such computer implementation methods using the everyday methods described in the art.

[0245] In the embodiment, the computer implementation method according to the sixth aspect outputs the evaluation results, that is, (i) Whether the subject is at high or low risk of NSCLC disease progression under an ongoing NSCLC treatment regime; (ii) Whether the subject is responding to the ongoing NSCLC treatment regime; and / or (iii) Whether the regime in question should be maintained or modified. It may also include outputting the following:

[0246] The output may be achieved by a display.

[0247] In a seventh aspect, the present invention relates to a computer program product including instructions that cause a computer to execute any one of the methods and uses defined in the embodiments according to the first, second, third, and fifth aspects of the present invention when the program is executed by the computer.

[0248] For the foregoing aspects, all embodiments described herein and described below are applied with necessary modifications.

[0249] In an eighth aspect, the present invention provides a computer-readable medium including instructions that cause a computer to execute any one of the methods and uses defined in the embodiments according to the first, second, third, and fifth aspects of the present invention when the program is executed by the computer.

[0250] For the foregoing aspects, all embodiments described herein and described below are applied with necessary modifications.

[0251] In a ninth aspect, the present invention (i) a receiving unit configured to receive data including the level of CYFRA21-1 and / or the level of CA125 in a sample obtained from a subject, and optionally the level of CEA, and optionally information regarding the NSCLC subtype of the subject; (ii) a processing unit configured to execute any of the comparing steps defined in the embodiments of the foregoing aspects; and (iii) optionally, an output unit configured to output an evaluation result and provides a data processing system including the same.

[0252] The evaluation result is (i) whether the subject has a high or low risk of NSCLC disease progression under an ongoing NSCLC treatment regimen; (ii) whether the subject responds to the ongoing NSCLC treatment regimen; and / or (iii) Whether the regime in question should be maintained or modified. Information about this would also be acceptable.

[0253] The embodiments described above and all embodiments described below are applicable to a data processing system according to a ninth aspect of the present invention, with necessary modifications.

[0254] In a tenth embodiment, the present invention is This relates to a reagent or set of reagents for detecting the level of CYFRA21-1 in a sample obtained from a subject, and / or a kit containing a reagent or set of reagents for detecting CA125.

[0255] The embodiments described above and all embodiments described below are applicable to a kit according to a ninth aspect of the present invention, with necessary modifications.

[0256] The kit is preferably, (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Whether the ongoing NSCLC treatment regime should be maintained or modified for subjects diagnosed with NSCLC. This is a kit for evaluating, The subjects in (i), (ii), and (iii) are those diagnosed as being in a stable state (for example, by CT-based imaging).

[0257] In certain embodiments, the kit includes a reagent or set of reagents for detecting the level of CYFRA21-1 in a sample obtained from a subject and a reagent or set of reagents for detecting CA125.

[0258] In the embodiment, the kit further includes a reagent or set of reagents for detecting the level of CEA in a sample obtained from a subject.

[0259] Reagents or sets of reagents for detecting CYFRA21-1 are well known in the art. Exemplary reagents are antibodies or antibody pairs specifically targeted to each biomarker. An example of such reagents is the reagent used in the Elecsys® assay used in the accompanying examples.

[0260] In embodiments, the kit may include instructions for a method of evaluating, based on detected levels of CYFRA21-1, CA125, and optionally CEA, and / or information regarding the subtype of NSCLC (i.e., adenocarcinoma or SCC), (i) the risk of NSCLC disease progression under an ongoing NSCLC treatment regime for subjects diagnosed with NSCLC; (ii) whether subjects diagnosed with NSCLC will respond to an ongoing NSCLC treatment regime; and / or (iii) whether an ongoing NSCLC treatment regime should be maintained or modified for subjects diagnosed with NSCLC.

[0261] These instructions may include cutoffs where levels need to be compared. In embodiments, the instructions may include information on the calculation or construction of scores (in particular, as described elsewhere in this specification) based on biomarkers (i.e., CYFRA21-1, CA125, and / or CEA) in which a detection agent or set of detection agents is included in the kit.

[0262] In embodiments, the description may include references to computer implementation methods (e.g., via web links), computer program products, computer-readable media, or data processing systems described herein.

[0263] In one embodiment, the kit is (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Whether the ongoing NSCLC treatment regime should be maintained or modified for subjects diagnosed with NSCLC. The accompanying documentation may include a statement that it is a kit for evaluating, The subjects in (i), (ii), and (iii) are those diagnosed as being in a stable state (for example, by CT-based imaging).

[0264] The following definitions and embodiments apply to all aspects of the present invention described herein.

[0265] Non-small cell lung carcinoma (NSCLC) is a histological type of lung cancer. Lung cancer, also known as cancer of the lung or pulmonary cancer, is a malignant lung tumor characterized by uncontrolled cell proliferation in lung tissue. If left untreated, this proliferation can spread beyond the lungs through the process of metastasis to nearby tissues or other parts of the body. Most cancers that occur in the lungs are known as primary lung cancers and are cancers that originate from epithelial cells. The four main histological types of lung cancer are squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and small cell carcinoma (SCLC). The first three subtypes are commonly referred to as non-small cell carcinoma (NSCLC) and account for about 80% of lung cancers. Diagnosis of lung tumors is generally based on imaging techniques and analysis of biopsy specimens. The 2004 World Health Organization (WHO) schema of lung tumors forms the basis of lung cancer classification. This has incorporated several developments, including the recognition of heterogeneity in lung cancer, the introduction of diagnostic immunohistochemistry (IHC) techniques for the routine diagnosis of several neuroendocrine tumors, and the recognition of newly described entities such as fetal adenocarcinoma, cystic mucinous neoplasm, and large cell neuroendocrine carcinoma. In 2011, a multidisciplinary expert panel representing the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS), and the European Respiratory Society (ERS) proposed a major revision of the classification system. These changes primarily affect the classification of adenocarcinoma and its distinction from squamous cell carcinoma. The current international standard for tumor classification by oncologists and pathologists is provided by "WHO Classification of Tumors of the Lung, Pleura, Thymus and Heart" (Travis et al, 2015, WHO Classification of Tumors, Volume 7, fourth edition). Where there is doubt in the context of this invention, the above criteria should be applied to define NSCLC and its histological subtypes.

[0266] Squamous cell lung cancer (SCC) is a type of non-small cell lung cancer formed from round cells that replace injured or damaged cells in the inner wall of the bronchi, the main airways of the lung. Squamous cell tumors usually occur in the central part of the lung or in one of the main airway bifurcations. As these tumors grow to a large size, they can form cavities in the lung. Squamous cell carcinoma accounts for 25–30% of all lung cancers and can spread to the bones, adrenal glands, liver, small intestine, or brain. The prognosis for this type of lung cancer is poor as it progresses. However, the 5-year survival rate for people with localized lung cancer identified and removed in the early stages can be as high as 35–40%. These 5-year survival rates approach 85% for patients under 30 years of age. This type of cancer is almost always caused by smoking. Secondary risk factors include age, family history, and exposure to secondhand smoke, mineral and metal dust, asbestos, or radon.

[0267] The histological diagnosis of squamous cell carcinoma is predicted based on the presence of keratin production by tumor cells and / or intercellular desmosomes (called "intercellular crosslinks"). Some tumors that are primarily spindle-shaped (spindle cell variants of pleomorphic carcinoma) or have a characteristic pattern of peripheral myelination can also be classified as squamous cell carcinoma. Historically, most squamous cell carcinomas (60–80%) have occurred in the proximal parts of the tracheobronchial tree via squamous metaplasia, dysplasia, or in situ carcinoma (in situ squamous cell carcinoma). A small number of cases occur peripherally and may be associated with bronchiectatic cavities or scarring. Central and peripheral squamous cell carcinomas can show extensive central necrosis, resulting in cavitation. A small subset of central, well-differentiated squamous cell carcinomas arises as laterally proliferating intrabronchial papillary lesions. Patients with this unusual variant of squamous cell carcinoma typically present with persistent cough due to airway obstruction, recurrent hemoptysis, or recurrent lung infections.

[0268] Adenocarcinoma is the most common type of lung cancer in the modern series, accounting for about half of all lung cancer cases. It is a type of lung cancer that forms in the mucinous glands throughout the body. Adenocarcinoma is usually found in the outer part of the lungs, tends to grow more slowly than other types of lung cancer, and is more likely to be seen before it spreads outside the lungs. It occurs mainly in current or former smokers, but is also the most common type of lung cancer seen in non-smokers. It is more common in women than in men and is more likely to occur in younger people than other types of lung cancer. The increase in the incidence of adenocarcinoma is thought to be due to the introduction of low-tar filtered cigarettes in the 1960s, but such a causal relationship has not been proven.

[0269] The subjects for which evaluation is provided in the context of the present invention preferably have NSCLC of the subtype adenocarcinoma and / or squamous cell lung cancer.

[0270] The ongoing treatment regime can, in principle, be any treatment regime for NSCLC. Guidelines for NSCLC are publicly known in the art (S3-Leitlinie Pathovention, Diagnostik, Therapie und Nachsorge des Lungenkarzinoms Langversion 1.0-February 2018 AWMF-Registernummer:020 / 007OL; and NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for NSCLC, Version 7.2015). In embodiments, the “ongoing treatment regime” may be selected from the group consisting of chemotherapy, targeted therapy, immunotherapy, radiotherapy, and any combination thereof.

[0271] The attached examples demonstrate that responses to various different treatment regimes can be successfully evaluated using the methods and uses described herein.

[0272] In certain embodiments, the “ongoing treatment regime” may be a chemotherapy treatment regime.

[0273] Specific but non-limiting, but exemplary ongoing treatment regimes are disclosed in the attached examples, specifically in Tables 1a, 1b, and 1c.

[0274] In embodiments, chemotherapy can be selected from an unrestricted group of platinum-based chemotherapy and platinum-free chemotherapy.

[0275] Platinum-based chemotherapy is a treatment comprising the administration of a pharmaceutical composition containing a platinum-based chemotherapeutic agent. The platinum-based chemotherapeutic agent may be selected from cisplatin, carboplatin, oxaplatin, nedaplatin, triplatin nitrate, phenatriplatin, picoplatin, satraplatin, or any combination thereof. In certain embodiments, the platinum-based chemotherapeutic agent may be selected from carboplatin and cisplatin.

[0276] Platinum-free chemotherapy is a treatment that involves administering only chemotherapy agents other than platinum-based chemotherapy agents. Non-specific examples of chemotherapy agents other than platinum-based chemotherapy agents include etoposide, dogemcitabine, cetaxel, paclitaxel, permetrexed, vinorelbine, and vincristine.

[0277] Targeted therapy is a type of cancer treatment that specifically targets proteins that control how cancer cells proliferate, divide, and / or expand. Therefore, these treatments typically preferentially target cancer cells.

[0278] Exemplary but non-limiting targeted therapies include treatment with tyrosine kinase inhibitors (also known herein as TKIs). Exemplary but non-limiting examples of tyrosine kinase inhibitors include afatinib, alectinib, crizotinib, erlotinib, and osimertinib.

[0279] Immunotherapy involves treatment with antibodies (monospecific or multispecific), antibody fragments, or antibody-like molecules.

[0280] Exemplary but non-limiting immunotherapeutic agents include: atezolizumab (Tecentriq®), durvalumab (Imfinzi®), nivolumab (Opdivo®), and pembrolizumab (Keytruda®). In embodiments, treatment with an immunotherapeutic agent may be an immunotherapeutic agent selected from atezolizumab (Tecentriq®), nivolumab (Opdivo®), and pembrolizumab (Keytruda®).

[0281] In some embodiments, immunotherapy may be a checkpoint inhibitor such as a PD-1 or PD-L1 blocker.

[0282] Radiation therapy, often abbreviated as RT, RTx, or XRT, is a treatment that uses ionizing radiation. Radiation therapy may be given in conjunction with chemotherapy and may be used for curative purposes in individuals with NSCLC who are not eligible for surgery. This form of high-intensity radiation therapy is called curative radiation therapy. An improvement on this technique is sequential multifractionated accelerated radiation therapy (CHART), in which high doses of radiation are given over a short period of time. Postoperative thoracic radiation therapy should generally not be used after curative surgery for NSCLC. If cancer growth blocks a short portion of the bronchus, close-range radiotherapy (local radiation therapy) can be administered directly into the airway to open the passage. Compared to external beam radiation therapy, close-range radiotherapy allows for shorter treatment times and reduced radiation exposure to medical staff. However, the evidence for close-range radiotherapy is less than that for external beam radiation therapy.

[0283] In a preferred embodiment of the present invention, the ongoing treatment regime may consist of a first-choice treatment (i.e., a first treatment received by the subject).

[0284] As used herein, “modifying an NSCLC treatment regime” may include, but is not limited to, (i) adjusting the dosage of the treatment (e.g., increasing the dosage of a drug), or (ii) changing the type of treatment to a different type of treatment (e.g., if the ongoing treatment is chemotherapy, modifying the treatment may mean changing the treatment to immunotherapy). In particular, modifying an ongoing treatment regime may mean changing from a particular ongoing treatment regime to any one of the above-mentioned examples of “ongoing treatment regimes.” In other words, the term “modifying an ongoing treatment regime” may mean “adjusting an ongoing treatment regime” or “changing an ongoing treatment regime.” In a preferred embodiment, modifying an ongoing treatment regime may mean changing from a first-line therapy to a second-line therapy.

[0285] The treatment regimes described herein include several treatment cycles. A treatment cycle includes a period of treatment, depending on the treatment type, followed by rest periods (no treatment) repeated on a regular schedule. For example, one treatment cycle consists of one week of treatment followed by three weeks of rest. If this cycle is repeated multiple times on a regular schedule, it constitutes a treatment regime including several cycles. Preferably, in the context of the present invention, a treatment regime includes three to six treatment cycles (optionally followed by maintenance therapy). Exemplarily, a treatment regime may include four treatment cycles. The preferred length of a treatment cycle is 15 to 27 days, preferably 18 to 24 days, and most preferably 21 days.

[0286] In the context of all aspects of the present invention, the expression “treatment regime in progress” refers to the treatment regime administered to the subject at the time the sample was obtained. The treatment regime itself does not explicitly form part of any of the methods and uses according to the first, second, third, fifth, and sixth aspects of the present invention. The term “in progress” means only that the sample subjected to the method of the present invention was obtained at a time when the subject was under a treatment regime. Therefore, the methods and uses of the present invention, with the exception of the method according to the fourth aspect, are not performed in humans and are merely “in vitro” methods and uses.

[0287] In preferred embodiments of the present invention, the response to a treatment regime means that the subject benefits from the treatment regime. In the context of the present invention, this may specifically mean that the risk of disease progression (tumor growth, new lesions, metastasis formation, and / or death from NSCLC) is delayed or avoided.

[0288] NSCLC is typically classified into stages I to IV. In the context of all embodiments of the present invention, the subject from which the sample is obtained may be in any of these stages. In preferred embodiments of the present invention, the subject may have stage III or IV NSCLC.

[0289] Currently, the staging of NSCLC is based on "The Revised International System for Staging Lung Cancer." Lung cancer is classified based on information from a clinical database of over 5,000 patients and was adopted in 2010 by the American Joint Committee on Cancer (AJCC) and the Union Internationale Contre le Cancer.

[0290] The T (primary tumor) classification is as follows: T1 = Tumor size ≤ 3cm T2 = Tumor size > 3-7cm T3 = Tumor size > 7cm or multiple tumor nodules within the same lung lobe T4 = Multiple tumor nodules located in the same lung but in different lobes.

[0291] The N / M (transfer) classification is as follows: N0 = No lymph node metastasis N1 = Metastasis in the ipsilateral peribronchial and / or ipsilateral hilar lymph nodes and intrapulmonary lymph nodes, including involvement by direct extension. N2 = Metastasis in the ipsilateral mediastinal and / or tracheal bifurcation lymph nodes (multiple nodes possible). N3 = Metastasis in the contralateral mediastinum, contralateral pulmonary hilum, ipsilateral or contralateral oblique line, or supraclavicular lymph nodes (multiple may be present). M0T No distant transfer M1 = Distant Transfer

[0292] Tumor stages I-IV are classified as follows according to the TNM categories above: [Table 1]

[0293] Further detailed information regarding disease staging can be found in "The IASLC Lung Cancer Staging Project: Proposal for Revision of the TNM Stage Groupings in the Forthcoming eighth edition of the TNM Classification for Lung Cancer" (Goldstraw et al JTO 2016;11:39-51).

[0294] The ECOG performance status scale is a measure of the impact of disease on a subject's daily living abilities (known to physicians and researchers as the patient's performance status). It describes a patient's level of function in terms of their ability to care for themselves, daily activities, and physical abilities (walking, working, etc.).

[0295] This scale was developed by the Eastern Cooperative Oncology Group (ECOG), now part of the ECOG-ACRIN Cancer Research Group, and published in 1982 (Oken M, et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982;5:649-655).

[0296] According to this reference, the ECOG scale is as follows: [Table 2]

[0297] In the context of the present invention, there are no specific limitations on the ECOG score that the subject being evaluated (i.e., from which the sample is derived) may have. In certain embodiments of the present invention, the subject may have an ECOG score of 0 to 2.

[0298] NSCLC patients in an ongoing treatment regime are typically monitored by imaging-based methods such as computed tomography (CT). Based on this imaging monitoring, the tumor response to treatment can be evaluated. Based on tumor imaging results, patients are typically classified into one of the following categories: complete response (CR), partial response (PR), progressive disease (PD), and stable state (SD). Standard criteria for this classification, such as RECIST 1.1, are available. In the context of this invention, the definitions of the categories are preferably as follows: Partial response (PR) is assigned to a reduction of 30% or more in tumor size. Progressive disease (PD) is assigned to an increase of 20% or more in tumor size. In such cases, a stable state is assigned in the sense that neither a size reduction sufficient to be considered a PR nor growth sufficient to be considered a PD is found (i.e., a size reduction of less than 30% to a size increase of less than 20%). CR is defined as the disappearance of all target lesions. This classification is based on response evaluation criteria in solid tumors (see RECIST 1.1; Eisenhauer et al., Eur J Cancer 2009, 45, 228-247, doi:10.1016 / j.ejca.2008.10.026).

[0299] In the context of the present invention, subjects may be classified as having a stable state. Classification is preferably by imaging methods such as CT according to RECIST 1.1 as defined above or RECIST-based criteria as defined in RECIST 1.1 (see Eisenhauer et al., Eur J Cancer 2009, 45, 228-247, doi:10.1016 / j.ejca.2008.10.026, which is incorporated in its entirety). In case of conflict, the RECIST 1.1 criteria take precedence.

[0300] As used herein, the expressions “classified as having a stable state” or “diagnosed as having a stable state” mean that the subject’s response to an ongoing treatment regime has been evaluated, preferably by imaging (e.g., CT or PET CT), and classified as having a stable state according to the above criteria (preferably RECIST 1.1). In embodiments of the present invention (including all aspects of the present invention), a subject may be classified as having a stable state after a second treatment cycle of an ongoing treatment regime. In particular, a subject may be classified as having a stable state within 100 days, preferably 80 days, preferably 70 days, preferably 60 days, after the second treatment cycle (e.g., within 100 days, preferably 80 days, preferably 70 days, preferably 60 days, after the drug / treatment administration of the second treatment cycle). In preferred embodiments, a subject is classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained at least 10 days, preferably at least 20 days, more preferably at least 30 days, and most preferably at least 35 days, after the start date of the ongoing treatment regime. In this embodiment, subjects are classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained up to 150 days, preferably up to 120 days, and most preferably up to 108 days, from the start date of the ongoing treatment regime. Therefore, subjects can be classified as having a stable state based on data (e.g., tumor imaging data such as CT data) obtained 10 to 150 days, preferably 20 to 120 days, and most preferably 35 to 108 days, from the start date of the ongoing treatment regime.

[0301] The "start" or "start date" of an ongoing treatment regime is the date on which the first treatment of that regime was administered to the subject. For example, it is the date on which the drug or drug composition was first administered to the subject.

[0302] In the context of the present invention, a sample is a sample obtained from a subject (or, as referred herein, an “individual”). The subject according to the present invention may be any human or non-human animal, in particular mammals. Accordingly, the methods and uses described herein are applicable to both human and veterinary diseases. Obviously, non-human mammals of particular interest include livestock, pets and animals with commercial value (e.g., horses) or personal value (e.g., dogs, cats). This method is particularly preferred for human subjects in which the diagnostic method is commonly used. Accordingly, in a particularly preferred embodiment, the subject from which the sample was obtained is human.

[0303] As used in the context of the present invention, “sample” can be any sample suitable for measuring the biomarkers according to the present invention (i.e., CYFRA21-1 and / or CA125, and optionally CEA), and refers to a biological sample obtained for the purpose of in vitro evaluation. It can be particularly relevant to an individual and may include material from which specific information about the individual can be determined, calculated, or inferred. A sample may consist of whole or in part of a patient-derived biomaterial (e.g., a solid tissue sample obtained from a lung biopsy). A sample may also be material that has come into contact with a patient in a manner that allows tests providing information about the individual to be performed on the sample. A sample may preferably include any body fluid. Exemplary test samples include blood, serum, plasma, urine, saliva, and fluids from the lung (e.g., endothelial fluid), obtained, for example, by bronchoscopy or bronchonutrition. A sample may be taken from a subject and used or processed immediately before determination / measurement. Processing may include purification (e.g., separation such as centrifugation), concentration, dilution, lysis of cellular components, freezing, acidification, storage, etc. Preferred samples are whole blood, serum, and plasma. In a particularly preferred embodiment, the sample is serum or plasma.

[0304] Typically, blood-related samples are preferred test samples for use in the context of the present invention. These samples may be obtained by collecting blood from a vein, usually from the inside of the elbow or the back of the hand. In particular, in infants, the skin may be punctured using a sharp instrument called a lancet to induce bleeding. Thus, the blood may be venous blood. The blood may be collected, for example, in a pipette, or on a slide or test strip. Thus, in preferred embodiments of the present invention, the sample obtained from an individual is a blood sample selected from the group consisting particularly of serum, plasma, and whole blood. Most preferably, the sample is a blood sample selected from serum or plasma.

[0305] In the context of the present invention, a sample obtained from a subject being evaluated may be a sample obtained at a defined point in time within the ongoing NSCLC treatment regime the subject is undergoing. In embodiments, the sample is a sample obtained within a specific time frame around the date of a health checkup (e.g., imaging such as CT) that ultimately leads to a diagnosis of stable state. In embodiments, the specific time frame may be from 30 days before to 30 days after the health checkup. In preferred embodiments, the specific time frame may be from 20 days before to 30 days after the health checkup. In even more preferred embodiments, the specific time frame may be from 10 days before to 29 days after the health checkup. In embodiments, the sample is a sample obtained after the treatment administration stage of the second cycle of the treatment regime. In preferred embodiments, the sample is a sample obtained from the subject at least 10 days, preferably at least 20 days, and most preferably at least 25 days after the start date of the ongoing treatment regime. In one embodiment, the sample is obtained from the subject up to 150 days, preferably up to 120 days, and most preferably up to 108 days, after the start date of the ongoing treatment regime. Therefore, in one embodiment, the sample is obtained from the subject 10 to 150 days, preferably 20 to 120 days, and most preferably 25 to 108 days, after the start date of the ongoing treatment regime. The "start" or "start date" of the ongoing treatment regime is the day the first treatment of this treatment regime was administered to the subject. For example, the day the drug or drug composition was first administered to the subject.

[0306] This invention utilizes the biomarkers CYFRA21-1 and / or CA125. Optionally, CEA may also be used.

[0307] CYFRA21-1 belongs to the cytokeratin family. Cytokeratins are structural proteins that form subunits of epithelial intermediate filaments, which are major components of the cell cytoskeleton. Twenty different cytokeratin polypeptides with molecular weights ranging from 40 to 70 kilodaltons (kD) have been identified to date. The type of cytokeratin synthesized by cells is also influenced by growth and differentiation rates. Due to their specific distribution patterns, they are very suitable for use as differentiation markers in tumor pathology. CYFRA21-1 is a fragment of cytokeratin 19, which is part of the cytoskeleton of epithelial cells, and can be found overexpressed in tumors of epithelial origin. While intact cytokeratin polypeptides are poorly soluble, soluble fragments can be detected in serum (Bodenmueller et al., 1994, Int. J. Biol. Markers 9:75-81).

[0308] CA125 (also known as mucin 16 or MUC16, or cancer antigen 125, or carbohydrate antigen 125) is a protein encoded by the MUC16 gene in humans. MUC16 is a member of the mucin family of glycoproteins. CA125 is a membrane-bound mucin with a single transmembrane domain. A distinctive feature of CA125 is its large size. CA125 is more than twice as long as MUC1 and MUC4, contains approximately 22,000 amino acids, and is the largest membrane-bound mucin. CA125 has been shown to play a role in tumorigenesis and tumor growth progression through several different mechanisms.

[0309] CEA is a monomeric glycoprotein (molecular weight approximately 180,000 daltons) with a variable carbohydrate component of approximately 45-60%. Like AFP, CEA belongs to the group of carcinoembryonic antigens produced during the embryonic and fetal periods. The CEA gene family consists of approximately 17 active genes in two subgroups. The first group contains CEA and nonspecific cross-reactive antigens (NCAs), while the second group contains pregnancy-specific glycoproteins (PSGs). CEA is mainly found in the fetal gastrointestinal tract and fetal serum. It also occurs in small amounts in the intestinal, pancreatic, and liver tissues of healthy adults.

[0310] In the context of this invention, when CYFRA21-1, CA125, and CEA are mentioned, they refer to the respective proteins, not the nucleic acids that encode them.

[0311] Methods and uses of the present invention may include the step of determining the level of a specific biomarker protein. Specifically, the levels of CYFRA21-1 and / or CA125, and optionally CEA, may be determined. Determining the level may also be expressed as "measuring the level of a biomarker."

[0312] Various methods for measuring protein biomarker molecules (particularly CYFRA21-1, CA125, or CEA) are known in the art, and any of these can be used.

[0313] Preferably, the biomarker(s) are specifically measured from the liquid sample by the use of a specific binder.

[0314] The specific binding agent is, for example, a receptor for the biomarker or an antibody against the marker. In the context of the present invention, the biomarker molecule (i.e., CYFRA21-1, CA125, and / or CEA) is measured at the protein level.

[0315] The determination of a protein as a binding partner for a marker polypeptide can be carried out using one of several known methods for identifying and obtaining proteins that specifically interact with a protein or polypeptide, for example, a yeast two-hybrid screening system as described in U.S. Patent Nos. 5,283,173 and U.S. Patent Nos. 5,468,614 or equivalent. The specific binder has an affinity for its target molecule, preferably at least 10⁸ l / mol, or more preferably at least 10⁹ l / mol. Those skilled in the art will understand that the term "specific" is used to indicate that other biomolecules present in the sample do not significantly bind to the marker-specific binder. Preferably, the binding level to biomolecules other than the target molecule results in a binding affinity of only 10% or less, more preferably only 5%, of the affinity to the target molecule. A preferred specific binder satisfies both of the above minimum criteria for affinity and specificity.

[0316] The specific binder is preferably an antibody that is reactive with a biomarker, particularly CYFRA21-1, CA125, or CEA. The term antibody refers to polyclonal antibodies, monoclonal antibodies, antigen-binding fragments of such antibodies, single-chain antibodies, and gene constructs containing the binding domain of an antibody.

[0317] The term "antibody" includes polyclonal antibodies, monoclonal antibodies, their fragments such as F(ab')2, and Fab fragments, as well as any naturally occurring or recombinantly produced binding partners that are molecules that specifically bind to CYFRA21-1, CA125, or CEA polypeptides. Any antibody fragment that meets the above criteria for specific binding agents can be used. Antibodies are produced by state-of-the-art procedures, for example, as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays, Elsevier Science Publishers BV, Amsterdam (1990), entire book, especially pp. 43-78). In addition, those skilled in the art are well aware of immunosorbent-based methods that can be used for the specific isolation of antibodies. These means can improve the quality of polyclonal antibodies and, therefore, their performance in immunoassays (Tijssen, P., ibid., pp. 108-115).

[0318] For the results disclosed in this invention, polyclonal antibodies and monoclonal antibodies produced in animals such as goats, rats, rabbits, or guinea pigs can be used. Monoclonal antibodies are ideal tools in the development of assays for clinical routines because they can be produced in any required amount with specific properties.

[0319] To determine the levels of CYFRA21-1, CA125, CEA, or any other protein biomarker, a sample obtained from an organism may be incubated with a specific binder for the marker in question under conditions suitable for the formation of a binder-marker complex. Such conditions do not need to be explicitly stated, as those skilled in the art can readily identify them without any inventive effort. The amount of the binder-marker complex is measured and used in the method and use of the present invention. As those skilled in the art will understand, many methods exist for measuring the amount of a specific binder-marker complex, all of which are described in detail in relevant textbooks (see, for example, Tijssen P., (above) or Diamandis, EP and Christopoulos, TK (eds.), Immunoassay, Academic Press, Boston (1996)).

[0320] In particular, monoclonal antibodies against markers (CYFRA21-1, CA125, and CEA) can be used in quantitative immunoassays (where the amount or concentration of the marker is determined).

[0321] Preferably, the marker in question is detected in a sandwich assay format. In such an assay, a first specific binder is used to capture the marker in question on one side, and a second specific binder (e.g., a second antibody) is used on the other side, labeled directly or indirectly to be detectable. The second specific binder may include a detectable reporter moiety or label, such as an enzyme, dye, radionuclide, luminescent group, fluorescent group or biotin, or an electrochemiluminescent label (e.g., a ruthenium-based electrochemiluminescent label). Any reporter moiety or label can be used with the methods disclosed herein, insofar as such a signal is directly related to or proportional to the amount of binder remaining on the support after washing. The use of an electrochemiluminescent label (e.g., a ruthenium-based electrochemiluminescent label) is preferred. The amount of the second binder remaining bound to the solid support is then determined using a method suitable for the specific detectable reporter moiety or label. For radioactive groups, scintillation counting or autoradiography is generally suitable. Antibody-enzyme conjugates can be prepared using various coupling techniques (see, for example, Scouten, WH, Methods in Enzymology 135:30-65, 1987 for a review). Spectroscopy can be used to detect dyes (e.g., including colorimetric products of enzymatic reactions), luminescent groups, and fluorescent groups. Biotin can be detected by conjugating it to different reporter groups (usually radioactive or fluorescent groups or enzymes) using avidin or streptavidin. Enzyme reporter groups can generally be detected by substrate addition (generally for a specific period), followed by spectroscopic analysis, spectrophotometric analysis, or other analysis of the reaction product. Using standards and standard additions, the level of antigen in a sample can be determined using well-known techniques.

[0322] As mentioned above, there are various methods for measuring CYFRA21-1 levels. The CYFRA21-1 assay specifically measures soluble fragments of cytokeratin 19 present in circulation. Measurement of CYFRA21-1 is typically based on two monoclonal antibodies (Bodenmueller et al., 1994, Int. J. Biol. Markers 9:75-81). Commercially available products for measuring CYFRA21-1 include Roche Diagnostics' Elecsys® CYFRA21-1 assay, the Cytokeratin 19 Fragment (CYFRA21-1) Immunoradiometric Assay Kit (Cisbo Assays, Codolet, France), the ELISA Kit for Cytokeratin Fragment Antigen 21-1 (Wuhan USCN Business Co., Ltd., China), and the ARCHITECT CYFRA21-1 assay (Abbott, Wiesbaden, Germany). The CYFRA21-1 assay from Roche Diagnostics (Germany) uses two specific monoclonal antibodies (KS 19.1 and BM 19.21) to measure soluble fragments of cytokeratin 19 with a molecular weight of approximately 30,000 daltons. Preferably, CYFRA21-1 levels are measured using Elecsys® with Roche product number 11820966122 according to the manufacturer's instructions.

[0323] Various commercially available assays exist for measuring CA125 levels. Examples of commercially available products for measuring CA125 levels include the Elecsys® CA125 II assay and the CA 125 II assay (Siemens Healthineers, Erlangen, Germany) from Roche Diagnostics. Preferably, CA125 levels are measured using the Elecsys® CA125 II assay (product number 11776223322).

[0324] Various assays exist for measuring CEA levels. Commercially available products for measuring CEA include the ADVIA Centaur® CEA immunoassay (Siemens Healthineers, Erlangen, Germany) and the ARCHITECT CEA assay (Abbott, Wiesbaden, Germany). Preferably, CEA is measured using the electrochemiluminescence immunoassay (ECLIA) for the quantification of CEA, "Elecsys® CEA" (material number: 11731629322, Roche Diagnostics, Ltd, Rotkreuz, Switzerland).

[0325] The step of determining the marker level can be carried out as follows: The sample, and optionally calibrators and / or controls, can be brought into contact with a binder (which can be immobilized, for example, on a solid phase) under conditions that allow the drug to bind to the marker. Unbound binders can be removed by a separation step (e.g., one or more washing steps). A second drug (e.g., a labeling agent) may be added to detect bound binders and enable their binding and quantification. Unbound second drugs can be removed. The amount of the second binder, proportional to the amount of the marker, can be quantified, for example, based on labeling. Quantification can be performed, for example, based on a calibration curve constructed for each assay by plotting the measured values ​​against the concentrations of each calibrator. The concentration or amount of the marker in the sample can then be read from the calibration curve.

[0326] Determining the "level" of a biomarker (e.g., CYFRA21-1, CA125, or CEA) in a sample means determining the amount or concentration of each biomarker molecule in the sample. The amount of a substance can be an absolute amount (e.g., giving mass) or a standardized, defined quantity that measures the size of an aggregate of fundamental entities such as atoms, molecules, electrons, and other particles. It is sometimes called a stoichiometric quantity. The International System of Units (SI) defines the amount of a substance as being proportional to the number of fundamental entities present. The SI unit for the amount of substance is the mole, with the unit symbol mol. The concentration of a substance is the amount of a component divided by the total volume of the mixture. Several types of mathematical descriptions can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration. The term concentration can be applied to any kind of chemical mixture, but most frequently refers to the solute and solvent in a solution. Molar concentration has variations such as ordinary concentration and osmotic concentration.

[0327] As used herein, “biomarker level” refers to the level of the biomarker based on a measurement of the amount or concentration in the subject sample at a specified point in time during an ongoing treatment. As used herein, “biomarker level” does not specifically refer to a change in the biomarker level over two point in time. Surprisingly, the gist of the present invention is that a single biomarker value yields a better assessment of disease progression risk (and consequently, treatment response / prognosis) than previously used changes in biomarker levels.

[0328] In the context of the present invention, a “score considering…” may be determined. This means that the individual parameters to be considered are mathematically combined, for example, by weighting calculations. Alternatively, a two-component score can be constructed as described above herein. The individual parameters to be considered may be determined CYFRA21-1 levels and / or determined CA125 levels in the sample. Optionally, CEA levels in the sample may also be considered. The levels may be used as is or mathematically transformed (e.g., by logarithmic transformations such as log2 or log10 transformations). In a preferred embodiment, the biomarkers CYFRA21-1 and / or CA125, and optionally the CEA levels, may be logarithmically transformed (i.e., log2 or log10 values ​​are calculated) to determine the score. Optionally, information on whether the subject has NSCLC of the histological subtype adenocarcinoma or SCC may also be considered, preferably by including an interaction term between the biomarkers and histological subtypes.

[0329] In embodiments of the present invention, the score can be obtained by weighting the amount or concentration of marker molecules(s) in the sample. This means that markers may be given different weights than other markers. For example, if the score takes into account the levels (amount or concentration) of CYFRA21-1 ([CYFRA21-1]) and CA125 ([CA125]) in the sample, the score may be calculated by the following formula: Score = a * [CYFRA 21-1]+b * [CA 125], (wherein a and b represent weighting coefficients). Preferably, the weighting coefficients are obtained by analyzing a reference group (e.g., any reference group defined in the context of the following cutoff values). Appropriate procedures are described in the examples.

[0330] In embodiments of the present invention, the score may also take into account information regarding the histological type of NSCLC (i.e., adenocarcinoma or SCC) in addition to the biomarkers. Information regarding tumor histology is preferably taken into account by using the interaction term between tumor histology information and each biomarker (Vatcheva, KP, et al, Epidemiology (Sunnyvale, Calif.) 6.1, 2015) to take into account the relationship between histology and each biomarker.

[0331] An example of a model equation including three biomarkers and an interaction term between the biomarkers and the histology of Cox regression is as follows:

number

[0332] In patients without SCC, setting SCC to 0 reduces the formula:

number

[0333] In embodiments, the score considering the levels of CYFRA21-1 and CA125 may be a two-component score, and the cutoff score may also be two-component. "Two-component" means that the score includes two values, for example, a first value (also called the CYFRA21-1 value) which is the level of CYFRA21-1 or a value derived therefrom, and a second value (also called the CA125 value) which is the level of CA125 or a value derived therefrom. The "derived value" may be a value obtained, for example, by mathematical calculation. The "derived value" is preferably directly proportional to each level. The value of the two-component cutoff score may be obtained by univariate analysis using each biomarker, i.e., in the same manner as the procedure for defining the cutoff of a single biomarker described herein. Comparing the two-component score to the two-component cutoff score includes comparing the first value of the determined two-component score to the first value of the cutoff two-component score, and comparing the second value of the determined two-component score to the second value of the cutoff two-component score.

[0334] Those skilled in the art will recognize that the score and the corresponding cutoff score can be optimized based on a reference set (e.g., any one as defined in the context of the cutoff determination below).

[0335] For example, a score can be determined for each sample in the reference population, and then the median of the combined values ​​or an appropriate cutoff can be selected. In a suitable cohort, a Cox proportional hazards regression model can be computed to evaluate the effects of CYFRA21-1 and / or CA125 as predictors of survival, and optionally CEA.

[0336] Next, using regression coefficients obtained by a Cox proportional hazards regression model for CYFRA21-1 and / or CA125, and optionally CEA, and optionally the interaction term between biomarkers and histological information, the score for each patient in the cohort can be calculated as a weighted combination of biomarkers and potential histological information.

[0337] Since Cox's proportional hazards model is a relative risk model, the prediction for each patient is calculated relative to the sample mean (e.g., based on a reference population specified elsewhere in this specification). This means that the parameter levels for each patient are adjusted by the population mean. The score can then be calculated as follows:

number

[0338] Regarding the score, the cutoff can be calculated using a reference population, for example, by determining the median score or an optimized cutoff score (see below).

[0339] Next, for illustrative purposes, risk groups can be compared using Kaplan-Meier curves.

[0340] Cox's proportional hazards regression (Cox, David R 1972 Journal of the Royal Statistical Society, Breslow, NE 1975 International Statistical Review) is a typical method in survival analysis for investigating the relationship between covariates and survival distributions. The Cox model is typically described by the hazard model equation shown below. This model represents the hazard at time t for an individual with a given designation of a set of explanatory variables denoted by X. X represents the set of predictor variables modeled to predict an individual's hazard. The Cox model equation shows that the hazard at time t is the product of two quantities. h0(t) is called the baseline hazard function. The second quantity is β i X i e is the exponential representation of a linear sum of x and x, where the sum spans p explanatory variables X (Gail, M. et al 1996 Statistics for Biology and Health).

number

[0341] The Kaplan-Meier estimation method is a statistical method that uses several estimators to approximate its variance. One of the most common such estimation methods is Greenwood's formula:

number

number

[0342] In some cases, it may be desirable to compare different Kaplan-Meier curves. This can be done using several methods, including the log-rank test or the Cox proportional hazards test.

[0343] The expressions "comparing the determined level of CA125 to the CA 125 cutoff value," "comparing the determined level of CA125 to the CA125 cutoff value," and "comparing the score taking... into account to the cutoff score" are merely used to further explain something that is obvious to those skilled in the art.

[0344] According to the present invention, each cutoff can be defined in particular by using a reference population. Preferably, the reference population may consist of NSCLC patients under an ongoing treatment regime (as defined elsewhere herein) and may be classified as having a stable state (as defined elsewhere herein). The reference population preferably includes SD patients at high risk of disease progression and SD patients at low risk of disease progression. In embodiments, patients in the reference population may have NSCLC of the subtype adenocarcinoma or SCC. In embodiments, patients in the reference population may have NSCLC of stage III or IV. In embodiments, the ECOG of patients in the reference population may be 0 to 2. In one embodiment, the reference population may be the group of patients defined in the “Test Population” section of the appended Examples. Disease progression (i.e., survival time and time at which disease progression was detected) must be known for patients in the reference population. Similarly, relevant parameters for which the cutoff is defined, namely CYFRA21 levels, CA125 levels and / or scores in the sample, must be known for patients in the reference population. Subsequently, each cutoff can be defined as a score that best divides the CYFRA21-1, CA125 level and / or reference population of SD patients into two groups (i.e., a first group with a high risk of disease progression and a further group with a lower risk of progression). Each cutoff can be selected to best achieve the desired separation between the two groups. The quality of separation can be measured, for example, by hazard ratios. For example, marker levels (quantity or concentration) in patient samples can be compared to levels known to be associated with high-risk or low-risk patients. Selecting an appropriate cutoff or cutoff value for the markers or scores herein is within the realm of those skilled in the art. As will be apparent to those skilled in the art, the levels of each biomarker established in the control depend on the assay used. Preferably, the cutoff is established using samples from 100 or more well-characterized individuals from a suitable reference population. A preferred reference population may also be selected to consist of at least 20, 30, 50, 100, 200, 500, or 1000 individuals.Preferably, the biomarker level is obtained at the time the sample of subject to be analyzed by the present invention (as defined elsewhere in this specification) is obtained.

[0345] In embodiments of the present invention, the levels of CYFRA21-1, CA125, or the score according to the present invention, corresponding to the median level of each measurement in the reference population, can be selected as the cutoff.

[0346] In a preferred embodiment of the present invention, each cutoff may be an optimized cutoff. Such an optimized cutoff may be determined by testing all quantiles from 0.2 to 0.8 on a 0.05 scale for biomarker or score values, for their ability to divide patients into two groups, and calculating the hazard ratio and log-rank p-value for each division. The quantile having the lowest log-rank p-value (highest hazard ratio) may then be selected as the optimized cutoff. The optimized cutoff value may also be selected by an alternative measure of risk identification between high-risk and low-risk patient groups, thereby allowing a suitable measure to be selected by those skilled in the art.

[0347] In general, this disclosure is not limited to the specific methodologies, protocols, and reagents described herein, because they may vary. Furthermore, the terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of this disclosure. Where used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context otherwise explicitly indicates otherwise. Similarly, the words “comprise,” “contain,” and “encompass” are interpreted inclusively, not exclusively.

[0348] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by those skilled in the art of this disclosure.

[0349] It should be understood that the word "comprise," as well as variations such as "comprises" and "comprising," means the inclusion of a given integer or step, or a group of integers or steps, but not the exclusion of any other integer or step, or a group of integers or steps.

[0350] As used herein and in the appended claims, the singular forms "a," "an," and "the" also include individual plural terms unless otherwise explicitly indicated.

[0351] The following figures and examples are provided to aid in understanding the present invention, and the true scope of the invention is described in the appended claims. It is understood that modifications to the described procedures can be made without departing from the spirit of the invention. [Examples]

[0352] The following embodiments are provided to aid in understanding the present invention, and its true scope is specified in the claims. It is understood that modifications to the prescribed procedures may be made without departing from the spirit of the invention.

[0353] Example 1: Evaluation of prognostic values ​​of serum-based biomarkers in NSCLC patients To investigate whether blood-based tumor biomarkers have predictive value in monitoring treatment success and disease progression prognosis, either on their own or in addition to imaging-based analysis, we conducted a clinical study involving parallel, state-of-the-art imaging-based tumor staging / treatment monitoring and parallel measurement of seven pre-selected biomarker candidates (CEA, ProGRP, NSE, CYFRA21-1, SCC, CA15-3, and CA125).

[0354] research group This clinical study cohort includes patients aged 18 years or older with previously untreated stage III or IV NSCLC (histology of adenocarcinoma or squamous cell carcinoma (SCC)) and an Eastern Cooperative Oncology Group (ECOG) performance score of 0–2.

[0355] Of the 387 NSCLC patients, 265 received first-line treatment, had no progressive disease prior to treatment cycle 2 (cycle 2), and had available computed tomography (CT) data between 25 and 108 days (median 44 days) after the start of treatment.

[0356] In the first CT scan after Cycle 2, 230 patients were disease-free (100 patients had stable disease [SD] and 130 had partial response [PR]). Complete biomarker results, namely levels of CEA, ProGRP, NSE, CYFRA21-1, SCC, CA15-3, and CA125, were available for 228 of these patients (100 with SD and 128 with PR).

[0357] Exclusion criteria included inability to obtain a blood sample, a history of secondary malignancies, serious comorbidities, prior treatment for any type of tumor (some patients had received first-line treatment defined by radiotherapy), pregnancy, or lactation.

[0358] Patients were administered, at the discretion of the treating physician, as first-line chemotherapy (75.1% of all patients), tyrosine kinase inhibitors, and / or immune checkpoint inhibitors.

[0359] NSCLC (adenocarcinoma or SCC) patients after the second treatment cycle of first-line therapy and available CT scan data were used for biomarker analysis (see below). Patients in whom disease progression was demonstrated before the first CT scan were excluded from the analysis using biomarker data.

[0360] The patient demographics and treatment schemes of the analysis population are summarized in Tables 1a, 1b, and 1c below. [Table 3] [Table 4] [Table 5]

[0361] Research purpose The primary objective was to evaluate the predictive value of serum biomarkers in monitoring treatment success and the prognosis of disease progression. Specifically, we assessed whether patients with stable disease (SD) based on CT could be further differentiated into a risk group (e.g., respondents with a favorable prognosis) based on serum biomarker levels. This analysis was performed after the first two cycles of treatment. The measures used for evaluation were overall survival (OS) and progression-free survival (PFS), which were analyzed separately.

[0362] Evaluation of treatment effectiveness using the latest CT imaging technology. The first CT scan after cycle 2 of the first-line treatment was set as the analysis point for all patients (the maximum time between the second cycle of treatment and the first CT after cycle 2 was 60 days). Tumor response on CT was defined according to response evaluation criteria for solid tumors (see RECIST 1.1; Eisenhauer et al., Eur J Cancer 2009, 45, 228-247, doi:10.1016 / j.ejca.2008.10.026). Partial response (PR) was assigned to a size reduction of 30% or more. Progressive disease (PD) was assigned to a size increase of 20% or more. Stable state was assigned to such cases, but neither a size reduction sufficient to be considered a PR nor growth sufficient to be considered a PD was found (i.e., a size reduction of less than 30% to a size increase of less than 20%).

[0363] Progression-free survival (PFS) and overall survival (OS) were calculated from the start of treatment. PFS was defined as follows: Progression = The patient has observed at least one progression (i.e., PD according to RECIST) or has died; No progression = The patient did not progress and was alive at the end of the study; PFS time for patients with advanced disease = Days of progression (detection) - Day of treatment initiation + 1 (day); PFS time for patients who died during the study = Date of death - Date of treatment initiation + 1 (day); For patients without progression events, the PFS time is calculated as: last contact date - treatment start date + 1 day.

[0364] OS time was defined as follows: The overall survival time (OS) of patients who died during the study was calculated as: date of death - date of treatment initiation + 1 day. The operating time (OS) of a patient who is still alive after the last contact is calculated as: date of last contact - date of treatment commencement + 1 day.

[0365] Sample collection and biomarker evaluation Venous blood samples were collected from each patient at baseline (i.e., before the start of treatment) and, if possible, at each routine visit (typically each treatment cycle, each approximately 21 days). Samples for analysis on the first CT after the second treatment cycle were collected between 10 days before and 29 days after the CT. Samples were collected 25 to 108 days after the start of treatment. Serum samples were stored as 500 μl aliquots at below -70°C.

[0366] Biomarker levels were measured at a later time point in these serum samples. Specifically, electrochemiluminescence immunoassays (ECLIAs) of the in vitro diagnostic protein biomarkers CEA, ProGRP, NSE, CYFRA21-1, SCC, CA15-3, and CA125 were performed using a cobas® system from Roche Diagnostics Centralized and Point of Care Solutions (CPS), in accordance with the manufacturer's guidelines.

[0367] statistical analysis Patient demographics and disease characteristics were summarized using descriptive statistics. Risk prediction for disease progression (PFS or OS) was compared between CT response and biomarker values ​​using Cox regression models and Kaplan-Meier curves.

[0368] The prognostic models were based on the response (PR vs. SD) on the first CT after cycle 2, the biomarker value on the first CT after cycle 2, and the change in biomarker between baseline and the first CT after cycle 2 (baseline corrected), respectively. Biomarker values ​​were log2 transformed. For univariate models (containing only one biomarker), a cutoff based on the biomarker value was used to divide patients into low-risk and high-risk groups, for example, using the median of the biomarker. For illustrative purposes, the cutoff for the univariate models was inversely transformed to the original biomarker scale for the Kaplan-Meier curve. For models containing more than one biomarker, scores were constructed by a weighted linear combination based on the linear predictive values ​​of the Cox regression model. To divide patients into low-risk and high-risk groups, for example, the median of this score was used equal to the univariate biomarker.

[0369] Furthermore, as an alternative, for models containing more than one biomarker, a two-component score was constructed based on the combination of risk groups in the univariate model for each included biomarker. Briefly, a cutoff was defined based on the individual value of each biomarker, similar to the univariate model (containing one biomarker) (e.g., the median biomarker level). If both biomarker levels were above their respective biomarker cutoffs, which form part of the two-component score cutoff, the patient was classified as high-risk. If a single biomarker was below its respective cutoff; i.e., if one of the levels in the two-component score fell below the corresponding cutoff value in the two-component score cutoff, the patient was classified as low-risk.

[0370] The prognostic model was evaluated using hazard ratios (HR) and the C index. The C index is a nonparametric estimate of the proportion of all patient pairs where the model prediction and the observed outcome match, and is therefore an overall evaluation criterion for the Cox regression model. A C index of 1 corresponds to the best model prediction, and a C index of 0.5 represents a random prediction. The hazard ratio represents the risk between patient groups; for example, a hazard ratio of 2 means that patients in the high-risk group have twice the risk compared to patients in the low-risk group.

[0371] In the biomarker-based risk prediction model, patients in the SD group were separated into two risk groups using either the median biomarker / score as a cutoff or an optimized cutoff. The optimized cutoff was determined by testing all quantiles from 0.2 to 0.8, using a 0.05 step in biomarker / score values, for their ability to divide patients into two groups, and calculating the hazard ratio and log-rank p-value for each division. The quantile with the lowest log-rank p-value was selected as the optimized cutoff.

[0372] The Cox proportional hazards model is based on a single biomarker or a combination of two or three biomarkers. For the combined population of adenocarcinoma and SCC (histological information), the model also includes an interaction term between the histological information and each biomarker to explain the relationship between histology and each biomarker (Vatcheva, KP, et al, Epidemiology (Sunnyvale, Calif.) 6.1, 2015). An example of a model equation including three biomarkers and the interaction term between the biomarkers and histology in Cox regression is as follows:

number

number

[0373] Similar to models incorporating multiple biomarkers, we constructed scores based on the risk predictions of Cox regression models.

[0374] Research results a) Prognostic value of the first frontal CT scan after the second treatment cycle First, we evaluated the prognostic value of the first CT scan after the second treatment cycle in predicting progression-free survival (PFS) or overall survival (OS). CT monitoring is currently the most advanced technique for evaluating NSCLC progression and treatment response.

[0375] Patients identified with PR or SD on the initial CT scan had a similar risk of progression (see Figure 1A), suggesting that the initial CT scan was associated with poor PFS performance in patients who were clinically benefiting from their ongoing treatment regime, i.e., the PR and SD groups.

[0376] The specific results regarding PFS are as follows: Adenocarcinoma or SCC: Hazard ratio (HR) = 1.326 (p-value = 0.055), C index = 0.579. Adenocarcinoma: HR=1.482 (p value=0.025), C index: 0.616. SCC: HR=0.992 (p value=0.978), C index: 0.507.

[0377] The initial CT scan also showed poor prognostic performance for overall survival (OS), with the OS rate being almost the same in patients with partial response (PR) and stable disease (SD) (Figure 1B).

[0378] The specific results regarding the OS are as follows: Adenocarcinoma or SCC: HR=1.517 (p value=0.012), C index=0.608. Adenocarcinoma: HR=1.784 (p value=0.005), C index 0.656 SCC: 0.924 (p-value = 0.786), C index 0.527

[0379] b) Prognostic values ​​of cancer biomarkers using a non-baseline corrected single measure The prognostic values ​​of individual cancer biomarkers (CA125, CA15-3, CEA, CYFRA21-1, NSE, SCC, and proGRP) or combinations of cancer biomarkers for predicting PFS or OS were evaluated in SD patients using serum single-protein biomarker levels without baseline adjustment (i.e., absolute biomarker levels that do not take into account pre-treatment biomarker concentrations).

[0380] Analysis based on the median cutoff of biomarkers In the first series of analyses, biomarker data (unadjusted for baseline) were analyzed by dividing SD patients into two groups based on the median value for each biomarker, i.e., by using either the median absolute biomarker value or the median combined biomarker value (score) as the cutoff. The performance of single biomarkers (univariate analysis) and several biomarkers combined with scores (multivariate analysis) was evaluated.

[0381] The results of the progression-free survival period read out are summarized in Table 2 below. [Table 6]

[0382] Of the single biomarkers tested, CYFRA21-1 had the highest prognostic value for adenocarcinoma in SD patients, as indicated by the C index and HR (see Table 2 and Figure 2A). The combination of CYFRA21-1 and CA125 showed improved performance in differentiating between high-risk and low-risk groups for disease progression, as indicated by a higher HR. The best performance was achieved by a combination analysis (score) including CYFRA21-1, CA125, and CEA.

[0383] For SCC patients, CA125 had the highest prognostic value in patients with SD as a single biomarker, as indicated by the C index and HR (see Table 2 and Figure 2B). The combination of CYFRA21-1 and CA125 showed improved performance in the C index and HR. The best performance was achieved by a combination analysis (score) including CYFRA21-1, CA125, and CEA.

[0384] In the combined population of adenocarcinoma and SCC, the combination of CYFRA21-1 and CA125 (using the interaction term) was associated with a higher prognosis in RECIST-diagnosed SD patients than either biomarker alone (see Table 2 and Figure 2C). By constructing a score that takes into account CYFRA21-1, CA125, CEA, and further interaction terms between biomarkers and histology (SCC or adenocarcinoma), the prognostic performance of the combination of adenocarcinoma and SCC in the patient cohort with SD NSCLC could be further improved (see Table 2):

[0385] Notably, patients with SD and those with a lower risk of progression or death based on combination models including the respective biomarkers CYFRA21-1 or CA125, or CYFRA21-1, CA125 and CEA (and optionally interaction terms) had similar probabilities of progression compared to patients with PR, as indicated by hazard ratios (HRs) (Figure 3, Table 3). To calculate these hazard ratios, we created the variables "PR" for patients with PR, "low risk" for SD patients in the low-risk group, and "high risk" for SD patients in the high-risk group. Further Cox models including these covariates were fitted to obtain HRs between each combination of the three groups ("low risk" vs. "high risk", "low risk" vs. PR, and "high risk" vs. PR). [Table 7]

[0386] Regarding PFS, the hazard ratio (HR) for high-risk versus low-risk patients (based on scores considering CYFRA21-1 CA125, CEA, and histological interaction items) reached 2.372 (p<0.001), indicating that the risk of disease progression is significantly higher in high-risk patients according to the biomarker model (see Table 2). Based on this score, the HR for PR versus SD in low-risk patients reached 1.107 (p<0.594), indicating that SD patients categorized as low-risk for disease progression according to the biomarker model have a similar risk of disease progression compared to PR patients based on imaging (Figure 3A and Table 3). A similar analysis performed for the first readout PFS was also performed for the second readout OS.

[0387] The data related to the OS (see Table 4) was confirmed based on findings from PFS readouts.

[0388] Here too, based on the same biomarkers and scores, we were able to achieve robust prognosis differentiation between patients at high risk of disease progression / death and those at low risk.

[0389] Regarding overall survival (OS), the hazard ratio (HR) for high-risk patients versus low-risk patients (based on scores considering CYFRA21-1 CA125, CEA, and histological interaction items) reached 2.091 (p-value 0.002), confirming that the biomarker model effectively separates high-risk and low-risk patients (see Table 4). Based on this score, the HR for partial response (PR) versus low-risk patients reached 0.960 (p-value 0.853), confirming that the low-risk group identified by the biomarker model had a similar level of risk as the PR patient group (see Table 5). The HR for progressive disease (PD) versus high-risk patients was 1.760 (Figure 3B). [Table 8] [Table 9]

[0390] In summary, the results above demonstrate that the absolute levels of CYFRA21-1 and CA125 themselves, combined scores of the absolute levels of the two biomarkers, and especially scores considering the absolute levels of CYFRA21-1, CA125, and CEA, can provide additional guidance for the first CT scan after a second treatment cycle in patients with an uncertain CT response by differentiating patients with SD into high-risk and low-risk groups. This was validated by using PFS and OS. Furthermore, the inventors demonstrated that biomarker scores can be further improved by considering interaction terms based on tumor histology (SCC or adenocarcinoma) and optimizing the cutoff.

[0391] Analysis-based optimized biomarker cutoff We used models / scores based on CYFRA21-1, CA125, and CEA, and repeated analyses with interaction terms using optimized cutoffs. Specifically, we optimized the cutoffs to obtain the maximum risk difference between biomarker groups by preserving 20% ​​of patients in each of the two risk groups (high-risk and low-risk).

[0392] With the optimized cutoff, patients with high-risk SD according to the biomarker model had a similar survival probability to patients with PD for OS, and patients with low-risk SD according to the biomarker model had a similar survival probability to patients with PR for PFS and OS (Figure 4). Cutoff optimization can further increase the hazard ratios for high-risk and low-risk groups, highlighting the improvements in prognosis that can be made by using biomarker scores.

[0393] For PFS, the hazard ratio (HR) for high-risk versus low-risk in a model including CYFRA21-1, CA125, CEA, and interaction terms was 3.241. The HR for PR versus low-risk was 1.023 (Figure 4A).

[0394] For overall survival (OS), the hazard ratio (HR) for high-risk versus low-risk in a model including CYFRA21-1, CA125, CEA, and interaction terms was 4.206. The HR for partial response (PR) versus low-risk was 0.924. The HR for progressive disease (PD) versus high-risk was 0.940 (Figure 4B).

[0395] Analysis based on combinations of individual biomarker splits (two-component score) Analysis using models / scores based on CYFRA21-1 and CA125 with optimized cutoffs was repeated by applying the optimized cutoffs (as described above) to a single biomarker, separating all patients into low-risk and high-risk SD groups based on the single marker, and then combining the splits of the two biomarkers. Briefly, a two-component score was constructed containing the cutoffs for CYFRA21-1 and CA125, respectively, and the CYFRA21-1 and CA125 levels were compared to their respective cutoffs. If the biomarker level was higher than the cutoff, each component of the two-component score was defined as high. If the biomarker level was below the cutoff, each component of the two-component score was defined as low. Next, the groups for which two-component scores were obtained based on the combination of individual splits were further summarized into high-risk groups (CYFRA21-1 and CA125 above the cutoff) versus low-risk groups (CYFRA21-1 and CA125 below the cutoff, CYFRA21-1 below the cutoff and CA125 above the cutoff, and CYFRA21-1 above the cutoff and CA125 below the cutoff).

[0396] With the optimized cutoff, patients with high-risk SD according to the biomarker model had a similar survival probability to patients with PD for overall survival (OS), while patients with low-risk SD according to the biomarker model had a similar survival probability to patients with PR for both progression-free survival (PFS) and overall survival (OS).

[0397] For PFS based on all patients, the hazard ratio (HR) for high-risk versus low-risk in the model including CYFRA21-1 and CA125 items was 3.430. The HR for PR versus low-risk was 0.856.

[0398] For overall survival (OS) based on all patients, the hazard ratio (HR) for high-risk versus low-risk in the model including CYFRA21-1 and CA125 was 3.341. The HR for partial response (PR) versus low-risk was 0.796. The HR for progressive disease (PD) versus high-risk was 0.913.

[0399] Therefore, the two-component score yields similar results to the score using a linear combination.

[0400] c) Prognostic values ​​of cancer biomarkers using baseline adjustment of biomarker levels based on pre-treatment biomarker levels. The results obtained from the analysis of progression-free survival using baseline-adjusted biomarker levels (i.e., the ratio of biomarker levels on the first CT after the second treatment cycle to the initial biomarker concentration before treatment) are summarized in Table 6 below. Baseline-adjusted biomarker levels were log2 transformed. [Table 10]

[0401] The results surprisingly show that baseline-uncorrected data, which depend on the absolute level of the biomarker (see Table 2), are far better at distinguishing the SD patient group into high-risk and low-risk patients for PFS and OS than using changes in biomarker levels (see Table 6). The fact that risk assessment, and therefore the determination of whether a patient will benefit from the treatment, can be based on a single biomarker measurement, without requiring a baseline measurement, is a true advantage and a surprising finding. Even more surprising is the performance of single biomarker measurements and the scores derived from them, which are far superior to those based on changes in the level(s) of the same biomarker(s) compared to baseline (before treatment).

[0402] conclusion CT is currently the preferred method for evaluating the response to treatment in lung cancer. However, in this study, patients with a partial response (PR) on the first CT scan after the second treatment cycle had a similar risk of progression and survival as patients with stable disease (SD). Furthermore, CT scans and RECIST categorical criteria do not allow for risk identification within SD patients.

[0403] Of the seven biomarkers tested, the optimal prognostic biomarker varied depending on the tumor histology; CYFRA21-1 had the highest prognostic value for adenocarcinoma NSCLC, while CA125 had the highest value for SCC NSCLC.

[0404] Patients with SD could be divided into two prognostic groups: high-risk and low-risk, based on a combination of CYFRA21-1, CA125, and CEA (scores using interaction terms that optionally consider the histologically relevant performance of biomarkers CYFRA21-1 and CA125 and CEA) that were above or below the median. When patients were separated based on an optimized cutoff, the prognostic performance of a triple combination (scores using interaction terms that optionally consider the histologically dependent performance of biomarkers CYFRA21-1 and CA125 and CEA) increased, suggesting that patients with high-risk SD outcomes had outcomes comparable to those with PD for OS, and patients with low-risk SD outcomes had outcomes comparable to those with PR for OS and PFS.

[0405] The inventors have demonstrated herein that the absolute levels of CYFRA21-1 and CA125 themselves, a combined score of the absolute levels of the two biomarkers, and in particular a score considering the absolute levels of CYFRA21-1, CA125, and CEA, can provide additional guidance for the first CT scan after a second treatment cycle in patients with an uncertain CT response by differentiating patients with SD into high-risk and low-risk groups for PFS and OS. Furthermore, the inventors have demonstrated that the biomarker score can be further improved by considering interaction terms based on tumor histology (SCC or adenocarcinoma) and optimizing the cutoff.

[0406] A particularly surprising finding of this study was that biomarker levels based on a single measurement after the first CT scan following the second treatment cycle performed far better in stratifying SD patients at high risk of disease progression from those at low risk of disease progression than changes in the levels of the same biomarker measured before treatment. This finding indicates that a single biomarker measurement, without the cumbersome requirement of continuous monitoring of biomarker levels, is sufficient, and even the best, to predict disease progression. Furthermore, the sufficiency of a single measurement opens up this risk assessment to patients who have not undergone continuous biomarker surveys and avoids any potential problems associated with using different methods for measurement at different points in treatment.

[0407] These findings can help physicians identify patients who are more or less likely to respond to treatment early (after cycle 2). The ability to predict this already at the time of the first CT scan in SD patients can provide additional guidance in a stable state, and therefore provide uncertain radiological results that can help guide further action decisions (e.g., monitoring for possible adjustments to treatment).

Claims

1. An in vitro method for evaluating the risk of progression of non-small cell lung cancer (NSLC) in subjects with non-small cell lung cancer (NSLC) who are under an ongoing NSCLC treatment regime, wherein the subjects are classified as being in a stable state by imaging, and the method is a) Determining the level of CYFRA21-1 and / or CA125 in the sample obtained from the subject; and b) (i) comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, (ii) comparing the determined level of CA125 with the CA125 cutoff level, or (iii) comparing a score that takes into account the determined level of CYFRA21-1 and / or the determined level of CA125 with the cutoff score. Includes, A method wherein the sample is a blood sample.

2. a) The level of CYFRA21-1 is determined, and b) The comparison includes or consists of (i) as described in claim 1 b), A determined level of CYFRA21-1 below the CYFRA21-1 cutoff level indicates a low risk of NSCLC disease progression; and / or The method according to claim 1, wherein a determined level of CYFRA21-1 higher than the CYFRA21-1 cutoff level indicates a high risk of NSCLC disease progression.

3. The method according to claim 2, wherein the subject is suffering from NSCLC of the adenocarcinoma subtype (ADC-NSLC).

4. a) The level of CA125 is determined, and b) The comparison includes or consists of (ii) as described in claim 1 b), A determined level of CA125 lower than the CA125 cutoff level indicates a low risk of NSCLC disease progression; and / or The method according to claim 1, wherein a determined level of CA125 higher than the CA125 cutoff level indicates a high risk of NSCLC disease progression.

5. The method according to claim 4, wherein the subject is suffering from NSCLC (SCC-NSLC), a subtype of squamous cell carcinoma.

6. a) The levels of CYFRA21-1 and / or CA125 are determined, and b) the comparison includes or consists of (iii) as described in claim 1 b), wherein a determined score lower than the cutoff score indicates a low risk of NSCLC disease progression; and / or The method according to claim 1, wherein a determined score higher than the cutoff score indicates a high risk of NSCLC disease progression.

7. An in vitro method for evaluating whether an ongoing NSCLC treatment regime should be maintained or modified in a subject diagnosed with non-small cell lung cancer (NSLC), wherein the subject is classified as stable by imaging, and the method is a) Determining the level of CYFRA21-1 and / or CA125 in the sample obtained from the subject; and b) (i) comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, (ii) comparing the determined level of CA125 with the CA125 cutoff level, or (iii) comparing a score that takes into account the determined level of CYFRA21-1 and / or the determined level of CA125 with the cutoff score. Includes, A method wherein the sample is a blood sample.

8. An in vitro method for evaluating whether a subject diagnosed with non-small cell lung cancer (NSLCC) responds to an ongoing NSCLC treatment regime, wherein the subject is classified as stable by imaging, and the method is a) Determining the level of CYFRA21-1 and / or CA125 in the sample obtained from the subject; and b) (i) comparing the determined level of CYFRA21-1 with the CYFRA21-1 cutoff level, (ii) comparing the determined level of CA125 with the CA125 cutoff level, or (iii) comparing a score that takes into account the determined level of CYFRA21-1 and / or the determined level of CA125 with the cutoff score. Includes, A method wherein the sample is a blood sample.

9. The method according to any one of claims 1 to 8, wherein the method further comprises determining a level of CEA, and the comparison in b) comprises or comprises (iii) as described in claim 1 b), claim 7 b) or claim 8 b), the score further takes into account the determined level of CEA.

10. This further includes obtaining information on whether the subject has squamous cell carcinoma (SCC-NSCLC) or adenocarcinoma (ADC-NSCLC), a subtype of non-small cell lung cancer. The method according to any one of claims 1 to 9, wherein the comparison in b) includes iii) as described in claim 1 b), claim 7 b), or claim 8 b), and the score further considers the NSCLC subtype.

11. The method according to claim 10, wherein the score further considers the NSCLC subtype by using an interaction term between the information relating to the NSCLC subtype and the level of CYFRA21 and / or the level of CA125 in the sample.

12. The method according to claim 10, wherein the score further considers the NSCLC subtype by using the information relating to the NSCLC subtype and an interaction term between the level of CYFRA21 and / or the level of CA125 and the level of CEA in the sample.

13. The method according to any one of claims 1 to 12, wherein the sample is a sample obtained 10 to 150 days after the start date of the ongoing treatment regime, a sample obtained 20 to 120 days after the start date of the ongoing treatment regime, or a sample obtained 25 to 108 days after the start date of the ongoing treatment regime.

14. The method according to any one of claims 1 to 13, wherein the blood sample is selected from the group consisting of whole blood, serum, and plasma.

15. (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Should the ongoing NSCLC treatment regime be maintained or modified for subjects diagnosed with NSCLC? The use of CYFRA21-1 and / or CA125 as biomarkers(s) for evaluating the subject(i), (ii), and (iii) which is classified as having a stable state by imaging.

16. The use according to claim 15, further comprising the use of CEA as a biomarker.

17. (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Should the ongoing NSCLC treatment regime be maintained or modified for subjects diagnosed with NSCLC? A computer implementation method for evaluating, The objects in (i), (ii), and (iii) are classified as having a stable state by imaging, and the computer implementation method is a) receiving data including the levels of CYFRA21-1, CA125 and / or CEA determined in any one of claims 1 to 14; and b) Processing the data to perform the comparison described in b) of any one of claims 1 to 14. Computer implementation methods, including those mentioned above.

18. The computer implementation method according to claim 17, wherein, in a), information relating to the NSCLC subtype is further received.

19. A computer program product or computer-readable medium wherein, when the program is executed by a computer, the program includes instructions that cause the computer to perform the method described in claim 17 or 18.

20. A data processing system comprising: a receiving unit configured to receive the data described in claim 17 or 18 a); and a processing unit configured to perform step b) described in claim 17 or 18.

21. The system according to claim 20, further comprising an output unit configured to output evaluation results.

22. A kit comprising a reagent or set of reagents for detecting the level of CYFRA21-1 in a sample obtained from a target and / or a reagent or set of reagents for detecting CA125, The aforementioned kit is (i) Risk of NSCLC disease progression in subjects diagnosed with NSCLC under an ongoing NSCLC treatment regime; (ii) Whether the subject diagnosed with NSCLC is responding to the ongoing NSCLC treatment regime; and / or (iii) Should the ongoing NSCLC treatment regime be maintained or modified for subjects diagnosed with NSCLC? This is a kit for evaluating, The aforementioned objects in (i), (ii), and (iii) are classified as having a stable state by imaging, A kit in which the sample is a blood sample.

23. The kit according to claim 22, further comprising a reagent or set of reagents for detecting CEA.

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